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Volume III

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JESSE P. GREENSTEIN NationaE Cancer Institute, National Institutes of Health,

U.S. Public Health Service, Bethesda, Maryland

ALEXANDER HADDOW Chester Beatty Research Institute, Royal

Cancer Hospital, London, England

Volume 1 I I



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125 East 23rd Street, New York 10, N.Y.

All Rights Reserved No part of this book may be reproduced in any form, by photostat, microfilm, or any other means without written permission from

the publishers.

Library of Congress Catalog Card Number: 52-13360


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RICHARD DOLL, Statistical Research ['?zit, The Medical Research Council, London School of Hygiene and Tropical Medicine, London, England

HAROLD P. MORRIS, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

A. PULLMAX, Institut du Radium, Paris, France

R. PULLMAX, Institut d u Radium, Paris, France

P. ROSDONI, Cancer Institute, Milan, Italy

MICHAEL B. SHIMKIN, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

SIDNEY WEINHOUSE, The Lankenau Hospital Research Institute and The Instal iite jor Cancer Research, Philadelphia, Pennsylcania

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CONTRIBUTORS TO VOLUME 111 . . . . . . . . . . . . . . . . . . . . . . v

Etiology of Lung Cancer BY RICHARD DOLL. Statistical Research Crnit. The Medical Research Council. London

S c h l of Hygiene and Tropical Medicine. London. England 1 . Introdurtion . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

I1 . Increase in Incidence . . . . . . . . . . . . . . . . . . . . . . . 2 I11 . Etiological Factors . . . . . . . . . . . . . . . . . . . . . . . . 8 IV . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

The Experimental Development and Metabolism of Thyroid Gland Tumors

BY HAROLD P . MORRIS. IAhoratory of Biochemistry. National Cancer Institute. National Institutes of Health. Belhesda. Maryland

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 53 56 58 60

Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GO VII . Goitrogen-Induced Thyroid Gland Tumors in hlice . . . . . . . . . . 62

62 65 68

cinogem . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 81

XI11 . Biochemistry of Thyroid Gland Tumors in Mice . . . . . . . . . . . 80 XIV . Thyroid Gland Cancer in %Ian . . . . . . . . . . . . . . . . . . . 102

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

I1 . Thyroid Gland Biochemistry and Metabolism . . . . . . . . . . . . .

I V . Effect of Goitrogens and Iodine on Body Weight . and Thyroid Weight . . . V . Induction of Thyroid Gland Cancer by Chemical Carcinogens . . . . . .

I11 . Spontaneous Thyroid Tumors . . . . . . . . . . . . . . . . . . . .

V I . Dietary Iodine Deficiency and Experimental Development of Thyroid

VIII . Transplantability of Thyroid Gland Tumors in Mice . . . . . . . . . .

X . Effects of Ionizing Rndintions in Thyroid Gland Carcinogenesis . . . . .

XI1 . Transplantability and Xlrtnbolism of Rat Thyroid Gland Tumors . . . .

XV . Summary nnd Conclusions . . . . . . . . . . . . . . . . . . . . . 109

IX . Thyroid-Pituitary Interrelationships . . . . . . . . . . . . . . . . .

XI . Experiment.al Thyroid Tumors in Rats Produced by Goitrogens and Car-

Electronic Structure and Carcinogenic Activity and Aromatic Molecules New Developments

BY A . PULLMAN A N D I3 . PULLMAS. Institut du Radium. Paris. France 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

I1 . The Localization Theory of Chemical Reactions . . . . . . . . . . . . 118 111 . Electronic Structure and Carcinogenic Artivity of Unsuhstituted Polynu-

clear Hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . 122 IV . Extension of the Theory to Substituted Ilerivatives of Polycylic Hydro-

carbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 1‘. General Conclusions and Suggestions . . . . . . . . . . . . . . . . 156

Appendix . The Metabolic Renetivity of Carcinogenic Hydrocarbons . . . 161 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 167


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Some Aspects of Carcinogenesis BY P . RONDONI. Cancer Institute. Milan. Italy

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 I1 . Cancer as a Regressive Process in General Pathology . . . . . . . . . 172

I11 . The Energy Changes in Carcinogenesis (The Concept of Entropy in Pathol- ogy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

IV . The Supposed Significance of Derangements of Oxidative Metabolism in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . 180

V . The Interaction between Some Carcinogenic Agents and Cell Constituents 185 194

VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

VI . Cancer as a Problem of Protein Chemistry . . . . . . . . . . . . . .

Pulmonary Tumors in Experimental Animals BY MICHAEL B . SHIMKIN. National Cancer Institute. National Institutes of Health.

Bethesda. Maryland I . Historical Introduction . . . . . . . . . . . . . . . . . . . . . . . 223

I1 . Frequency and Distribution of Pulmonary Tumors in Mice . . . . . . . 225 I11 . Pulmonary Tumors in Other Animals . . . . . . . . . . . . . . . . 227 IV . Morphology and Biochemistry of Pulmonary Tumors in Mice . . . . . . 229 V . Histogenesis of Pulmonary Tumors in Mice . . . . . . . . . . . . . . 233

VI . Influence of Heredity in Pulmonary Tumors in Mice . . . . . . . . . . 235 VII . Polycyclic Hydrocarbons and Related Compounds . . . . . . . . . . . 237

VIII . Urethane and Related Compounds . . . . . . . . . . . . . . . . . 242 I X . Other Chemical and Physical Agents, Including Inhalants . . . . . . . 244 X . Factors Affecting Pulmonary Tumor Induction in Mice . . . . . . . . 248

XI . Mechanism of Induction of Pulmonary Tumors in Mice . . . . . . . . 252 XI1 . Pulmonary Tumors in Man and General Discussion . . . . . . . . . . 256

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

Oxidative Metabolism of Neoplastic Tissues BY SIDNEY WEINHOUSE. The Lankenau Hospital Research Institute and The Institute

for Cancer Research. Philadelphia. Pennsylvania I . The Concepts of Warburg . . . . . . . . . . . . . . . . . . . . . 270

11 . The Pasteur Effect . . . . . . . . . . . . . . . . . . . . . . . . 274 111 . Present Concept of Carbohydrate Oxidation . . . . . . . . . . . . . 278 IV . &Oxidation of Fatty Acids . . . . . . . . . . . . . . . . . . . . . 282 V . Mechanisms of Glycolysis in Tumors . . . . . . . . . . . . . . . . 283

VI . Electron Transport in Tumors . . . . . . . . . . . . . . . . . . . 288 VII . Oxidation in Tumor Homogenates . . . . . . . . . . . . . . . . . . 315

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . 327

SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . 339

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Etiology of Lung Cancer



Statistical Research Unit of the Medical Research Council, London School of Hygiene and Tropical Medicine, London, England

Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f Louvain Symposium.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Increase in Incidence.. . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . 1. Extent of Increase.. . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Changes in Sex Distribution.. . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . 3. Changes in Age Distribution.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Changes in Histological Distribution. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.II. Etiological Factors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Tobacco _ . . . . . . . , . . . . , , . , . . , . . . . . . , . . . . , . . . . . . . , . . . . . . . . . . . . . . . .

A. Retrospective Inquiries. , . . . . . . . . , , , . , , , . . , . , . . . . , . . . . . . . . . . . . . . B. Prospective Inquiries. . , . . . . . . . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Method of Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Extent of Risk.. , . . . , , . . . , , . . , , . . . . . . . . . . . . . E. Difference between Histological Types. . . . . . . . . . . F. Vital Statistics and Tobacco Consumption. . . . . . . . G. Identification of Carcinogenic Agent, . . . . . . , . . . . . . . . . . . . . . . . . . . . . H. Various Criticisms. . . . . . . . . . . . . . . . . . . . . . . . . .

2. Industrial Hazards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Atmospheric Pollution. . . . . . . - . . . . . . . . . . . . . . . . .

A. Mortality in Town B. Pollution of Town Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Differences in Urban an D. Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. Atmospheric Radioacti . . . . . . . . . . . . . . . . . . . . 5. Previous Respiratory I . . . . . . . . . . . . . . . . . . . . . . . .

IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . . . . . . _ .

. . . . . . .

I. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 2 5 5 7 8 8 8

11 15 16 19 20 23 29 32 33 36 36 38 40 41 41 44 46 47


1. Conclusions of Louvain Symposium

Knowledge of the causes of lung cancer was reviewed a t an interna- tional symposium on the “ Endemiology of Lung Cancer” held at Louvain in 1952 (Council for International Organizations of Medical Sciences, 1953). The members of the symposium were unable to decide what factors were responsible for the majority of cases, but important conclusions


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were reached on more limited problems. Firstly, it was agreed that ‘(a significant part” of the increase in mortality which had been reported from many countries “is absolute and represents a real increase in the number of people suffering from primary cancer of the lung”; secondly, “that there is now evidence of an association between cigarette smoking and cancer of the lung, and that this association is in general proportional to the total consumption”; and thirdly, that “occupational hazards giving rise to lung carcinoma have been demonstrated in a number of industries, in particular, in the handling of asbestos and chromates, in gas-works, in a factory refining nickel and in certain mines bearing radio-active ores.”

Other possible etiological factors were considered-in particular, atmospheric pollution by effluvia and smoke from factories and domestic chimneys and by exhaust fumes from petrol and diesel engines. The possibility that carcinogenic agents might be absorbed through ingestion or skin contact was reviewed, as was the possibility that individuals might vary in their susceptibility to the environmental influences to which they were exposed. No positive conclusions were reached with regard to these latter problems.

In the last two years, however, much new evidence has been obtained, and it is now possible to give a more complete picture of the etiology of the disease.

11. INCREASE IN INCIDENCE I. Extent of Increase

The highest death rate from lung cancer is recorded in Britain, where, in 1953, it was 342 per million persons. For both sexes taken together, lung cancer was the commonest type of fatal cancer, accounting for 17% of all cancer deaths; it accounted for 5% of male deaths from all causes a t all ages and, in the age group 45 to 64 years, for 10% of all male deaths. In other countries for which detailed statistics are available the rate varies from a seventh to approximately two-thirds the British rate (Table I).* The disparity between the rates is mainly due to a disparity between the rates for men; with the exception of England and Wales, Scotland, and Finland the female rates are similar, varying only between 34 and 47 per million women. Each of the countries listed has experienced an increase in the mortality attributed to lung cancer in the last half century, and the increase appears to be still continuing (Fig. 1).

* I n Table I, the figures for England and Wales and for Scotland are shown separately. The Scottish rate for all persons has usually been lower than the English and Welsh rate, but in 1953 the rate was slightly higher-346 per million against 342 per million.

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I n England and Wales the rate of increase has slackened in the last five years, and the death rate among men under the age of 50 years is now steady. On the assumption that the rates a t the younger ages remain steady and that the rates a t the older ages continue to increase until the age distribution of deaths from lung cancer resembles that of other extra- genital epithelial cancers, Mackenzie (personal communication) estimates that the male death rate may increase to approximately 1350 per million men, ie., to more than twice its present level of 602 per million, before it stabilizes. By a similar method, Clemmesen, Nielsen, and Jensen (1953)

TABLE I Crude Death Rate from Lung Cancer in Various Countries

Crude Death Rate per 1,000,000

Country Year Men Women Persons

England and Wales 1951 530 91 303 Scotland 1951 470 104 279 Fioland 1950 353 61 20 1 Holland 1951 27 1 45 158 Switzerland 1950 252 38 136 U.S.A. 1951 214 45 129 Denmark 1951 185 46 115 Australia 1951-52 173 37 106 Canada 1951 154 34 95 France 1950 161 47 87 Sweden 1951 111 43 77 Norway 1951 81 39 60

- 42 Iceland 1950 -

Rates have been shown for 1951, whenever possible, as data were available for the greatest number of countries around that year.

estimate that the death rate among men in Copenhagen may become even greater (ie., 2200 per million).

Much of the recorded increase is due to the advancing average age of the population. This factor can, however, be allowed for. I n England and Wales, for example, the recorded death rate from lung cancer rose from 8 per million in 1900 to 342 per million in 1953; ie., 43 times. But if the sex and age-specific death rates of 1953 had occurred in a population with the sex and age distribution characteristic of the population a t the beginning of the century, the total death rate would have been only 188 per million. The extent of the recorded increase after allowing for demo- graphic changes is, therefore, 24 times, or little more than half the figure given by the comparison of the crude rates. Similar conclusions apply to the increases recorded in other countries.

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How much of the increase "is absolute and represents a real increase in the number of people suffering from primary cancer of the lung" and how much is merely due to better diagnosis is uncertain. It is doubtful if the nature of the data concerned will ever permit a precise answer to be given. Rigdon and Kirchoff (1953) still maintain that the whole in- crease may be spurious, but in this opinion they are almost alone.

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E N G L A N D 6 WALES ( S C O T L A N D )

( H o L L A N D) S W I T Z E R L A N D / ' p'y'

1920 1925 19'30 1935 1940 1945 1950 1953 Date

FIG. 1. Increase in crude death rate from lung cancer in various countries, 1920- 1953. The trend of the death rate in those countries shown in parentheses has been similar to that in the countries against which they are placed.

Clemmesen, Nielsen, and Jensen (1953) in Denmark; Doll (1953a) and Stocks (1953a) in England; Kreyberg (1954b) in Norway; and Dorn (1954) in the United States have recently cited the reasons for believing that part of the increase is real. Three of the reasons are based on observa- tions which are of special significance for the etiology of the disease. The observations are that the increase has fallen unevenly on:

1. The two sexes. 2. Different age groups. 3. Different histological types.

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2. Changes in Sex Distribution

National mortality statistics and autopsy series both agree that the change in incidence of the disease has been accompanied by an increasing preponderance of male cases. Figure 2 shows how in different countries

L L0:l 2.0:I 3.011 4.011 5.O:I 6.0:l 7.O:l


FIG. 2. Increase in ratio of male to female death rates with the increase in the crude lung cancer mortality in various countries.

the proportion of male to female deaths has become progressively greater as the total mortality has arisen. The reality and implication of this change are too well recognized to warrant further comment.

3. Changes in Age Distribution

It has long been noted that the age distribution of lung cancer in men differs from that in women and from that of other extragenital epithelial tumors, in that, in countries with a high incidence, the male mortality rises to a maximum comparatively early and falls off rapidly in the later age groups. The increase in mortality over the last 50 years did not affect all ages equally; a t first the younger age groups were principally affected

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and the maximum mortality came to be between the ages of GO and 64 years; recently the increase has been most marked in the older age groups and the age of maximum mortality has risen. Korteweg (1951) has pointed out that these trends can be understood if comparisons are made between the age-specific death rates of groups of men all of whom were born a t a given period, rather than between groups of men living a t a given date, as is the normal custom. By this method of “cohort analysis” similar results have been obtained in Australia (Lancaster, personal communica- tion), Denmark (Clemmesen, Kielsen, and Jensen, 1953), England and

0 10 20 30 40 50 60 70 80 90

Age FIG. 3. Male death rates from lung cancer in the U.S.A. by age in 1914, 1930-32,

1939-41, and 1949-50, showing (heavy lines) the increase in mortality with age for men born in 1850,1860, 1870, 1880, and 1890. (Reproduced from a paper by Dr. H. F. Dorn in Industrial Medicine and Surgery 23, 253-257, 1954.)

Wales (Korteweg, 1951, and-in a modified form-Stocks, 1953a), and the United States (Dorn, 1954). Dorn’s data are reproduced in Fig. 3. The dotted lines indicate the pattern of age-specific death rates when studied a t different dates (1914, 1930-32, etc.); the solid lines indicate the pattern when men who were born a t a given period (1850-59,1860-69, etc.) are followed throughout their lifetime. It is seen that for each (‘ cohort” the mortality increases continuously with age, but that the later “cohorts” have a progressively higher mortality a t each age than the earlier ones. The changes in the shape of the customary age distribu- tion curve for lung cancer can, therefore, be understood if it is postulated (1) that groups of men born a t each period suffer a mortality which

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increases in a way similar to that observed for other forms of extragenital epithelial cancer and (2) that men born a t successive periods were in- creasingly exposed to an environmental carcinogen. On the other hand, the observed changes cannot be explained, as Clemmesen (1954) has pointed out, if men of all age groups were equally exposed to a new agent a t the same time.

4. Changes in Histological Distribution

With the increase in lung cancer the proportions recorded as belonging to the various histological types have altered ; adenocarcinoma has be- come relatively less common, and its incidence must, theref ore, be pre- sumed to have increased less than that of other types. In conformity with this and with the comparatively small increase of lung cancer in women, the sex ratio for adenocarcinoma has remained close to equality, whereas that for other types has shown a marked male predominance. Moreover, adenocarcinoma was not observed among the industrial tumors from which the Schneeberg and Joachimstal miners suffered (Schmorl, 1928; Hueper, 1942; Sikl, 1950). For these and other reasons, Womack and Graham (1938, 194l), Lickint (1953), and Kreyberg (1954a,b,c,d) have concluded that lung cancer may be divided into two essentially different types-endogenous and exogenous in origin.

Kreyberg’s papers are particularly important because the data have been collected in a country where the total lung cancer mortality is low and during a period when changes similar to those which took place in Britain and the United States 20 to 30 years ago are only beginning to appear. Kreyberg classified his cases into two main groups: group I con- sisting of squamous and large- and small-cell carcinomas, and group I1 of adenocarcinomas, bronchiolar cell carcinomas, and benign and malig- nant adenomas and salivary gland type tumors. The group I tumors were predominantly male (273 M to 31 F) and, when related to the size of the Norwegian population in 1950, showed an age distribution similar t o that observed for all lung cancer in countries with a high incidence- save only that the characteristics of the distribution were more pro- nounced, ie., the “incidence” had an earlier peak (50 to 59 years) and fell off more sharply in the older age groups. The group I1 tumors were found almost equally often in each sex (81 M to 76 F) and showed an (‘incidence” which increased steadily with age in the case of adenocar- cinoma and was approximately evenly distributed throughout the range of adult ages in the case of the adenomas and the salivary gland type tumors. When the cases were subdivided accordirlg to their date of occur- rence, Kreyberg found that there had been no increase in the proportion of group I t o group I1 cases among women over the whole period 1925

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t o 1953, despite the fact that the standardized mortality rate for women increased four and a half times. On the other hand, group I tumors became relatively much more frequent among men compared with group I1 tumors, while the standardized male mortality rate increased seven- fold. Despite the considerable difference in the total mortality experience of the two sexes, the sex ratio for the group I1 tumors remained close t o equality.

It is easy to criticize Kreyberg’s material on the grounds that i t was heterogeneous in origin (part collected from clinical and part from autopsy series) and that the relative amounts collected in the different ways varied over the period studied. Moreover, i t is likely that his cases provided a larger sample of those occurring in the younger age groups than in the older groups. Nevertheless, the characteristics of the histological types varied so markedly and the observations agree so well with the trend of the data obtained in other countries, that i t would be unreasonable to dismiss the material because i t falls short of perfection. Kreyberg interpreted his findings to mean that the group I tumors were largely the result of the introduction of some new carcinogenic agent into the environment, t o which men were more exposed than women, whereas the adenocarcinomas “are probably caused by comparatively weak carcinogenic influenccs, evenly distributed over large areas, well established in the society and striking both sexes with equal force.” The recorded increase in mortality in women in Norway may, he suggests, indicate the extent of the increase due to better diagnosis, and the total mortality in women (including a small proportion due to group I tumors) may, with present knowledge, be regarded as “unavoidable” cancer. In contrast, the increased mortality in men additional to that recorded in women and attributed solely to group I tumors can be regarded as “avoidable” cancer. It may well prove that these conclusions are of general significance and also apply to many countries other than the one in which the data were collected.


I. Tobacco

A. Retrospective Inquir ies . When the Louvain symposium concluded (‘that there is now evidence of an association between cigarette smoking and cancer of the lung,” it did so on the basis of evidence which was de- rived entirely from retrospective studies of patient’s histories. I n these studies the histories given by patients with lung cancer had been com- pared with the histories given by patients without lung cancer who, in one or other way, had been selected as ‘(controls.” Many studies of this general type have been reported, and the principal results obtained from

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them are summarized in Table 11. All agree in showing that there are more heavy smokers and fewer nonsmokers among patients with lung cancer than among patients with other diseases. With one exception (the differ- ence between the proportions of nonsmokers found by McConnell,

TABLE I1 Principal Characteristics of Smoking Histories of Men with and without Lung Cancer,

Reported by Various Authors

Percentage of Percentage of “Nonsmokers” “Heavy Smokers”

Numbcr of Men among Men among Men

With Without With Without With Without Lung Lung Lung Lung Lung Lung

Author Date Cancer Cancer Cancer Cancer Cancer Cancer

hliiller 1939 86 86 3.5 Schairer and Schoniger 1943 93 270 3 .2 Wassink 1948 134 100 4 . 5 Schrek et al. 1950 82 522 14.6 Mills and Porter 1950 444 430 7 Levin et al. 1950 236 481 15.3 Wynder and Graham 1950 GO5 780 1.3 McConnell et al. 1952 93 186 5.4 Doll and Hill 1952 1357 1357 0.5 Sadowsky et al. 1953 477 615 3 . 8 Wynder and Cornfield 1953 63 133 4 . 1 Koulumies 1953 812 300 0.6 Lickint 1953 224 1000 1.8 Breslow et al. 1954 518 518 3 . 7 Watson and Conte 1954 265 277 1 .9 Gsell 1954 135 135 0.7 Randig 1954 415 381 1 . 2

16.3 15.9 19.0 23.9 31 21.7 14.6 6.5 4.5

13.2 20.6 18.0 16.0 10.8 9 . 7

1F.7 5 . 8

65 36 52 27 55 19 18 9

- - 51 19 35 22 25 13

68 29 66 31 74 29 74 42 73 57 86 33 34 18

- -

Note. It has not been possible to make all the figures in this Table completely comparable. Some series include, for example, a few women; in others the proportions of heavy smokers are based on totals which are different from those used to calculate the proportion of nonsmokers. One series excludes adenocarcinoma. The individual papers should be referred to before any detailed use is made of the figures.

Gordon, and Jones), the differences are large enough to be important. More detailed results of two of the investigations are shown in Tables 111 and IV. From these it is seen (1) that there is a steady increase in the relative proportions of lung cancer to control patients as the amount smoked daily increases, and (2) that the difference in smoking habits between persons with and without the disease is more marked for men than for women.

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TABLE I11 Average Amount of Tobacco Smoked Daily: Lung Carcinoma Patients and Control

Patients with Other Diseases*

% Smoking a Daily Average for 20 Years of:

No. of % Non- ___ Sex Disease Group Patients smokerst 1 g.- 10 g.- 16 g.- 21 g.- 35 g.+

Lung Carcinoma 605 (squamous or (100.1%) 1 . 8 2 . 3 10.1 35.2 3 0 . 9 20.3

M undifferentiated) Other Diseasest 780

(99.8%) 14 .6 11.5 19 .0 35 .6 11 .5 7 . 6

Lung Carcinoma 25 (squamousor (100.0%) 40.0 4 .0 16 .0 2 4 . 0 8 . 0 8 . 0

F undifferentiated) Other Diseases$ 522

(IOO.IoJ,) 79 .6 9 . 2 6 . 9 3 . 2 0 . 6 0 . 6

* After Wynder and Graham, 1950. t Nonsmokers defined as persons smoking an average of less than I cigarette a day (or its equivalent

in pipe tobacco or cigars) over the previous 20 years. $ The age distributions of the control patients were different from those of the lung carcinonla

patients; the percentages quoted were therefore obtained by weighting the age groups so as to make them have the same relative importance as they had in the group of 605 men with squamou.8 cancer.

TABLE IV Average Amount of Tobacco Smoked Daily: Lung Carcinoma Patients and Control

Patients with Other Diseases* ~

% Smoking a Daily Average for 10 Years of:

No. of % Non- Sex Disease Group Patients smokerst < 5 g. 5 g.- 15 g.- 25 g.- 50 g. +

Lung Carcinoma 1357

Other Diseases1 1357 (99.9%) 0 . 5 4 . 0 3 6 . 0 35 .0 21 .6 2 . 8

(100.0%) 4 . 5 9 . 5 4 2 . 0 31 .8 11 .3 0 . 9

Lung Carcinoma 108

Other Diseases1 108 (100.0%) 37.0 14 .8 2 2 . 2 13.0 13.0 0 . 0

(100.0%) 54 .6 23.1 16 .7 5 . 6 0 . 0 0.0

* After Doll and Hill, 1952. t Nonsmokers defined as persons who had never consistently smoked as much as 1 g. of tobacco a

day for as long as one year. $ Patients with other diseases matched to be within the same five-year age group and to be in haspi-

tals of the same type and in the same region at approximately the same tiineasthelungcarcinomapatients.

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The conclusions to be drawn from these investigations depend on whether the comparisons between the smoking histories of the various groups of lung cancer and control patients are valid and on the extent to which the control patients were representative of the populations from which the lung cancer patients were drawn. In some of the earlier in- vestigations there were reasons for doubting whether the comparisons were valid-for example, when the histories of the two groups of patients were recorded by different methods. Other investigations, in which the patients were interviewed by the same persons and by the same methods throughout and in which the control patients were chosen to “match” the lung cancer patients with regard to sex and age, the date of interview, and the hospital in which they were treated, were not open to objection on this score. Nevertheless, there was the possibility that bias of one or another sort could have entered into the selection of the patients or the recording of the results. The various types of bias which might have oc- curred were considered in detail by Doll and Hill (1950, 1952). They concluded that bias could not be responsible for their results and that the only logical explanation was that the observed association between the smoking of tobacco and the development of lung cancer was real.

Further important evidence has been obtained from the preliminary results of two “prospective” inquiries. These inquiries have been con- ducted on a different principle and are not subjected to the types of bias which might theoretically have occurred in the retrospective studies. Since they lead to the same conclusion, i t is not now necessary to give further detailed consideration to the evidence from which it was deduced that the association shown by the retrospective studies was real.

B. Prospective Inquiries. I n the prospective inquiries, the smoking habits of large numbers of “normal” persons have been recorded, and the subjects have subsequently been watched to see what diseases they developed. Preliminary results of studies of this type have been reported by Doll and Hill (1954a) and by Hammond and Horn (1954).

I n Hammond and Horn’s inquiry, a large number of people who volunteered to help the American Cancer Society were each asked to interview approximately 10 white men, aged between 50 and 69 years, to be chosen from among acquaintances with whom they expected to remain in contact for several years. The smoking histories obtained a t the interview were recorded on a standard questionnaire. Subsequently, on the 1st of November each year, the interviewers filled in a follow-up form stating whether the men were alive or dead or had been lost sight of. The State Health Department was then asked to supply an abstract of the death certificate of each man reported to have died. When cancer was certified as the cause of death, an attempt was made to obtain further

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details from the certifying physician. A total of 204,547 questionnaires was collected, of which 14,413 were eliminated because they referred to inappropriate subjects or to subjects who were interviewed outside the specified period 1.1.52 to 31.5.52, or because they were inadequately completed. Of the subjects corresponding to the remaining questionnaires, 187,766 (98.8%) were successfully traced a t 1.11.53. Altogether, 4854

TABLE V Lung Cancer Death Rates among Men by Type of Smoking and by Amount


Microscopically All Cases Proved

Reported as Lung Cancer Primary (Excluding

Lung Cancer Adenocarcinoma)

No. of Death No. of Death Type of Smoking Population Deaths Rates Deaths Rates

Never smoked or occasional

Cigar and/or pipe smoking only 44,091 12 27.2 4 9 . 1

but never smoked cigarettes regularly 35,853 12 35.5 3 8 .4

smoking 107,822 143 132.6 45 41.7 History of regular cigarette

Total 187,766 167 88 .9 52 27.7

Regular cigarette smoking; less than 1 pack a day at time of questioning 54,799 62 113.1 17 3 1 . 0

Regular cigarette smoking; 1 pack or more a day a t time of questioning 25,497 61 239.2 24 94 .1


* Reproduced from the Journal of the American Medical Association (IIammond and Horn, 19.54).

men were reported to have died, and the certified cause of death was ob- tained for 4710 (ie., in 97%). Cancer of the lung was certified as the cause in 167 instances. According to the authors, “The evidence at present at hand does not warrant presenting the findings in any greater detail than is shown in Table 13 (reproduced above as Table V). The lung cancer death rate was higher among men with a history of regular ciga- rette smoking than among men who had never smoked regularly and even higher among men who currently smoked one pack or more of ciga- rettes a day a t the time of questioning. The differences are statistically

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significant (P = 0.002 or less). In fact, even the men smoking less than one pack of cigarettes daily have significantly higher death rates from lung cancer than those who have never smoked regularly ( P = 0.03 or less). The best estimate that can be made a t the present time (at the 5% level of confidence) is that lung cancer deaths are from 3 to 9 times as common among men with a history of cigarette smoking as among men who have never smoked regularly and that lung cancer deaths are from 5 to 16 times as common among men who smoke one pack or more per day.”

Differences in the age distributions of the men in the different cate- gories have not been allowed for in Table V. Such differences cannot, however, be responsible for the results, because Hammond and Horn have also shown that the proportion of nonsmokers is greater and the proportion of cigarette smokers is smaller in the older age groups in which lung cancer is more common. If, therefore, an allowance for age differences is made it will be found that the real difference in mortality between cigarette smokers and nonsmokers is, in fact, even greater than would appear from the above data.

The investigation reported by Doll and Hill (1954a) was on a smaller scale and was organized differently, but the trend of the results is similar. A postal questionnaire was sent to nearly 60,000 men and women on the British Medical Register. Just over 40,000 replied, giving details of their smoking habits. Subsequently the national offices for the registration of deaths notified the causes of death of all doctors, and clinical details of the deaths attributed to lung cancer were obtained through the physicians who had signed the death certificates. In the first 29 months following the date when the questionnaires were sent out, 789 deaths occurred among the 24,389 male doctors, aged 35 years and above, whose smoking habits had previously been recorded and classified. The numbers of deaths from lung cancer which occurred among men in the different smoking categories are shown in Table VI, in comparison with the numbers which would

TABLE VI Number of Deaths from Lung Cancer, Observed and Expected, among Doctors

Smoking Different Amounts of Tobacco *

Most Recent Amount Smoked Daily t 0 1-14g. 15-24g. 25 g.f

No. of Observed Deaths 0 12 14 13 No. of Expected Deaths 3.77 14.20 10.73 7.33

Observed as Percentage of Expected 0 85 130 177

* After Doll and Hill, 1954a. t Defined as the amount smoked a t the time of completing the questionnaire or, if smoking had been

stopped, immediately before stopping.

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have been expected to occur if smoking had been unrelated to the disease. (As in the American investigation, the proportions of nonsmokers, of pipe smokers, and of light cigarette smokers were greatest in the oldest age groups, so that the expected numbers had to be calculated separately for each age group and added for all ages.) The number of cases so far studied is small, but there is a steady and striking increase in the ratio between the numbers of cases observed and expected in each smoking category as the amount smoked increases. When this biologically im- portant trend is taken into account, the differences are statistically highly significant (P < 0.01). The similarity of the quantitative relationships between smoking and mortality which have been estimated from the retrospective and the prospective inquiries is also striking (Doll and Hill, 1952, 1954a). The mortality rates estimated by the two methods for each of the smoking categories have been expressed as percentages of the un- weighted averages of the four rates, and a comparison of the relationships between them is shown in Fig. 4. The slopes of the two graphs are almost identical.

Four explanations are theoretically possible. 1. Doll and Hill’s results might have been produced if heavy smokers

who suspected that they had lung cancer had replied to the questionnaire more readily than nonsmokers or lighter smokers in a similar situation. If this had been so, the effect would necessarily wear off as the duration of time increased between the completion of the questionnaire and death. In fact, no such diminution in the strength of the relationship occurred over the first 29 months of the inquiry. It is, in any case, unlikely that a similar form of selection could have entered into the choice of subjects for interview in Hammond and Horn’s inquiry.

2. Certification of the cause of death may have been biased by knowl- edge of the subject’s smoking history. If, however, there was a tendency to diagnose lung cancer more readily in heavy smokers, the death rate attributed to other causes among men in this category would be expected to be proportionately less than average, and this was not so in either investigation. I n fact, by no means all doctors are convinced of the reality of the association-as was shown, for example, in response to a ques- tionnaire sent t o Massachusetts physicians by Snegireff and Lombard (1954) ; bias in diagnosis, if i t existed a t all, may well have operated in the opposite direction.

3. Smoking may be associated with lung cancer only indirectly, being linked with another factor which is associated with it directly. Such an indirect link may, perhaps, account for some of the association found by Hammond and Horn, since both smoking and the disease may be com- moner in certain social and occupational groups within the population,

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Such factors are unlikely to have contributed to Doll and Hill’s results, since the population studied was entirely composed of doctors and was, therefore, comparatively homogeneous. In both inquiries an indirect link may have arisen because cigarette smoking and lung cancer are both commoner in towns than in the countryside. But this cannot account for


Retro-spedive inquiry X-x Pro-specllve lnqulry @---a 200-



Y 125-

c C U



Non- Light Moderate Heavy smokers Smokers Smokers Smokers

( I - 149 (15-249. ( 259. or a day) a day) more a Qy)

FIG. 4. Standardized death rate from lung cancer among men smoking four differ- ent amounts of tobacco, expressed as a percentage of the unweighted average of the four rates: ( I ) estimated from a retrospective inquiry into patients smoking histories (Doll and Hill, 1952), and (2) observed during a prospective inquiry into the mortality of doctors (Doll and Hill, 1954a).

much of the observed differences, since the association between smoking habits and place of residence (Doll and Hill, 1952; Hammond and Horn, 1954) is much weaker than the association between smoking and the disease.

4. There remains, therefore, the possibility that the association is real and direct.

C. Method of Smoking. The evidence suggests that all forms of smoking are not equally associated with the disease. Pipe smoking and cigar smok-

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ing are less closely associated with it than cigarette smoking (Wynder and Graham, 1950; Levin et al., 1950; Schrek et al., 1950; Doll and Hill, 1952,1954a; Sadowsky, Gilliam, and Cornfield, 1953; Breslow et al., 1954; Watson and Conte, 1954; and Hammond and Horn, 1954). Only McCon- nell, Gordon, and Jones (1952) and Randig (1954) failed to find any dis- tinction between the various methods of consumption of tobacco. From the mortality rates estimated by Doll and Hill (1952) it would appear that the risk among “pure pipe smokers” may be as much as two-thirds the risk among ‘ I pure cigarette smokers,” but they hesitated to draw any precise conclusion because of the variation in the average amounts of tobacco smoked by the different types of smoker and because of the difficulty of separating with certainty a group of smokers who had never smoked cigarettes a t all. Sadowsky, Gilliam, and Cornfield (1953) found a greater difference between pipe and cigarette smokers, and Hammond and Horn (1954) found that the mortality among smokers who had never smoked cigarettes was practically identical with that among nonsmokers (see Table V). Hammond and Horn’s evidence is particularly important because it was obtained from a prospective inquiry in which great care had been taken to eliminate from the pipe and cigar group all men who had ever smoked as much as ten packs of cigarettes in their entire lives. The investigation was on such a scale that even with this definition i t was still possible to secure a large group for study, and these results are likely to be more reliable than those of other workers who defined the categories of smokers less strictly.

Few people (outside South Africa) have smoked filter-tipped ciga- rettes or used cigarette holders regularly for any length of time, and i t has, therefore, been difficult t o obtain evidence regarding the possible protective effect of these methods of smoking. The data obtained by Doll and Hill (1952) are shown in Table VII. A smaller proportion of the lung cancer patients than of the control patients had used holders and a smaller proportion had smoked filter-tipped cigarettes, but the numbers are small and it would be unwise to draw any positive conclusions from this very limited evidence. It might be that the use of cigarette holders and of filter-tipped cigarettes are both associated with light smoking, but this did not appear to be the explanation among the patients referred to above.

D. Extent of Risk. The results of the two prospective inquiries which have been reported do not as yet permit direct measurements to be made of the full extent of the lung cancer mortality among smokers of different quantities of tobacco. Persons who were seriously ill when the inquiries were started are relatively unlikely to have been included in the initial population, and consequently the mortality rates recorded in the first year

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or two of follow-up are almost certainly too low. Until a few more years have elapsed, estimates of the risks to which the different categories of smokers are exposed must, therefore, still be based on the data derived from the retrospective studies.

Estimates have been made by Doll and Hill (1952), Heady and Barley (1953), Sadowsky, Gilliam, and Cornfield (1953), and Wynder and Corn- field (1953). The results obtained in Britain and the United States have

TABLE VI I Use of Cigarette Holders and of Filter-Tipped Cigarettes: Male Lung Cancer Patients

and Matched Control Patients*

Male Lung hfale Cancer Patients Control Patients

Type of Smoker No. % No. %

Use of Cigarette Holders Regularly Occasionally Never

10 2 . 0 27 5 . 8 15 3 .0 27 5 . 8

479 95.0 413 88.4

Total Cigarette Smokers 504 100.0 467 100.0

Use of Filter-Tipped Cigarettes Ever regularly Never regularly

3 0 . 6 15 3 . 2 501 99.4 452 96.8

Total Cigarette Smokers 504 100.0 467 100.0

Sniokers FVho Had Never Smoked Cigarettes 15 30 Nonsmokers 4 26

Total men 523 t 523 t

_~ -

__ - -- --

* After Doll and Hill, 1052 t The total numbers of men are diffeient froin the numbers shown in Table IV , because questions

about the use of cigarette holders and filter-tipped cigarettes were only Introduced in the last part of the inquiry.

been compared by the two latter groups of authors and, in a more detailed fashion, by Cutler and Loveland (1954). Cutler and Loveland’s estimates are summarized in Table VIII. From the table i t appears that for a man aged 40 years who smokes 20 or more cigarettes a day (1) the risk of dying of lung cancer before the age of 80 years is of the order of 8% and that (2) this risk is some 6+ to 30 times as high as that among nonsmokers.

The reliability of these estimates depends on the validity of certain

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assumptions which had to be made before the rates could be calculated. These are:

1. That the deaths recorded nationally as being due to lung cancer provide a fair estimate of the actual number of deaths due to the disease.

2. That the smoking habits recorded by patients with lung cancer were, at each age and in each sex, typical of all those persons who died of the disease during the period of the survey.

3. That the smoking habits recorded by the ((control” patients with- out lung cancer were similarly representative of those of all members of the population from which the lung cancer patients were drawn.

TABLE VIII Risk of Developing Lung Cancer among Men Smoking Different Amounts of Tobacco,

Estimated from the Results of Three Groups of Investigators*

Estimated Risk of Developing Lung Cancer by the Age of 80 Years, per 1000 Men Aged 40 Years

Sadowsky, Wynder Doll Gilliam, and and

Amount and Cornfield Graham Hill Combined Smoked (1953) (1950) (1952) Results

Nonsmokers 10 3 5 6 Smokers, of under 10 g.

a day 22 19 34 25 Smokers of l(f20 g. a

Smokers of more than day 46 52 48 49

20 g. a day 65 90 86 80

* After Cutler and Loveland, 1954.

Further assumptions are also required about the trend of future changes in mortality in order to present the risks in the form chosen by Heady and Barley (1953) and by Cutler and Loveland (1954), but these are of minor importance in that they have little effect on the relative sizes of the risks for the different smoking categories.

Whether the assumptions are justified is impossible to say with cer- tainty, and the rates must be regarded as provisional. In view, however, of the conformity of the estimates calculated from data from independent investigations in different countries and the further confirmation of the relative sizes of the risks by the preliminary results of the prospective inquiries, it is unlikely that the estimated rates are seriously in error. The correspondence between the results of Doll and Hill’s two investiga- tions has been shown previously in Fig. 4, and Hammond and Horn’s

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“best estimate . . . that lung cancer deaths are from 5 to 16 times as common among men who smoke one pack or more per day” as among nonsmokers largely overlaps the estimates made by Cutler and Loveland (Table VIII). On the other hand, the fact that smoking habits are not invariable and that the amounts which have been related to mortality have been recorded only a t one point in time must have blurred the differ- ences between the smoking categories, so that the estimated rates for light smokers are probably somewhat overestimated, whereas the rates for heavy smokers are likely to have been underestimated.

The estimated rate for nonsmokers is low but it is not intrinsically unreasonable. At ages 45 to 74 the rates calculated from the English data are slightly lower than the rates which actually occurred among women in rural areas in England (Doll, 1953b), and they are similar t o the rates now recorded among women in Denmark and several other countries (see Table I). If these rates represent the mortality risk in the absence of smoking, then the number of deaths from lung cancer attribut- able to causes other than smoking among persons aged 25 to 74 years in England and Wales in 1950 would have been about one-fifth of the total number actually recorded.

E. Diference between Histological T y p e s . In the foregoing discussion no consideration has been given to the possibility that the different his- tological types of carcinoma of the lung may have different etiological relationships to smoking. It has, however, been suggested that the rela- tionship with smoking holds only for squamous, oat-cell, and anaplastic carcinomas. This qualification makes little difference to the conclusions which have already been drawn, because, wherever lung cancer is com- mon, the great majority of cases are of the squamous, oat-cell, or ana- plastic types. The distinction is, however, of considerable theoretical interest.

Three reports have paid special attention to histological differences. Wynder and Graham (1950) found that the smoking habits of 39 men and 15 women with adenocarcinoma were closely similar t o those of con- trol patients with diseases other than lung cancer. Doll and Hill (1952) reported that there was “no statistically significant difference between the amounts smoked by patients in the different histological groups in either sex. The number of cases of adenocarcinoma is, however, too small (33 male and 10 female) to conclude that no difference exists. There were, in fact, relatively more non-smokers and very light smokers . . . among the patients with adenocarcinoma in both sexes.’’ Breslow et al. (1954) noted that “Six out of 46 (13 per cent) of the cases of adenocar- cinoma did not smoke cigarettes; whereas only 28 out of 472 (6 per cent) of the patients with other types of carcinoma . . . did not smoke ciga-

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rettes.” The interpretation of these data is complicated by the inclusion of men and women in a single series. When, however, the sexes are sepa- rated, the distinction still persists (Breslow, personal communication).

The most striking evidence has, however, been obtained by Kreyberg (personal communication), and the author is indebted to him for permis- sion to cite his results obtained up to the end of 1954. Smoking histories were taken from patients in the wards of the Rikshospitalet, Oslo, before operation, and the histological typing of the tumors was made inde- pendently without knowledge of the patient’s history, sex, or age. The most recent amount of tobacco smoked daily by men whose tumors were classified as belonging to the two main histological groups (see page 7) was as follows:

No. of No. of Men Smoking Daily: Total No. Type of Tumor Nonsmokers 1-9 g. 10-19 g. 20 g. + of Men

Group 1 3 40 98 52 193 Group 2 2 11 14 5 32 Ratio of group 1 to group

2 tumors 1.5/1 3.6/1 7.0/1 10.4/1 6 0/1

Finally, Wynder (1954) has studied the problem by collecting details of the histology of the cases of lung cancer which are reported to have occurred among male nonsmokers. Twenty nine per cent (i.e., 14 out of 48) were adenocarcinomatous, whereas only 5% (i.e., 54 out of 1019) were adenocarcinomatous among male smokers with lung cancer in his personal series.

It must, therefore, be concluded that adenocarcinoma of the lung is less closely related to smoking habits than are the squamous, oat-cell, and undifferentiated types of cancer, and i t may well prove that smoking plays no part a t all in its production.

F. Vital Statistics and Tobacco Consumption. The sharp increase in the number of deaths attributed to lung cancer during a period when tobacco consumption was also increasing has been cited as one reason for believing that tobacco is a cause of the disease. In fact, correlations in time may be-and often are-entirely irrelevant, so that they are of no value in proving the existence of a causal relationship. On the other hand, if a relationship can be demonstrated by other means, i t is reason- able to test its significance by seeing if i t is consistent with such temporal changes as are observed to occur.

The changes which have taken place in tobacco and cigarette con- sumption and in lung cancer mortality in England and Wales in the last 70 years are illustrated in Fig. 5 and Table IX. Whether the correlation between cigarette consumption and mortality is as close as would be expected if cigarettes were one of the principal causes of the disease is

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.---. 0......0

P 3 x-*


I P ) 1920 1930 1940 1950

FIG. 5. Crude death rate from lung cancer in England and Wales and per capita consumption of cigarettes and of all tobacco products in Great Britain, 1900-1953.

TABLE IX Crude Lung Cancer Death Rate and Consumption of Cigarettes and Other Tobacco

Products for Men and Women Separately in England and Wales, 1881-1950

Annual Consumption, Lbs. per Adult (aged 15 years +)

Lung Cancer Death Rate

per 1,000,000 Persons Men Women (aged 15 years $)

Period Cigarettes Other Tobacco Cigarettes Date Men Women

1881-90 0.006 6 . 1 0.0 1891-1900 0 . 4 6 . 2 0 .0 1901-10 1 . 8 4 . 9 0.0 19 11-20 3 . 8 4 . 3 0.0 1920 25 13 192 1-30 5 . 1 3 . 7 0 . 2 1930 74 27 1931-40 6 . 9 2 . 7 0 . 8 1940 256 68 1941-50 8 . 3 2 . 4 2 . 4 1950 624 111

Note. The figures shown in this table are derived from a different source from those shown in Fig. 5 and differ in that the estimates of tobacco consumption exclude the amounts consumed duty-free in the Merchant Navy and in the Armed Forces abroad. Except in wartime these amounts are negligible. The figures also differ in that the table shows rates per adult (or per 1,000,000 adults) and the figure shows rates per person (or per million persons).

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uncertain, because many of the relevant facts are unknown. It is, for example, not known:

1. What proportion of the increase in recorded mortality is real. 2. What are the relative risks attached to the smoking of tobacco

3. What is the biological relationship between the dose of cigarette

On the basis of present knowledge one may, perhaps, suggest more or less reasonable solutions to the first two problems; although it must be admitted that no estimate of the extent of the real increase in mortality can be more than an intelligent guess. Save, however, that the evidence indicates that mortality varies in direct arithmetical proportion with the amount smoked at a given time, we are completely ignorant of the third. We neither know the induction time of the tumor nor the relative effects of the same dose a t different periods of life; and different hypotheses about either of these must lead to gross differences in the temporal relationship between consumption and mortality. If, for example, it is postulated that the mechanism of carcinogenesis is of the type suggested by Nordling (1953), Stocks (1953b), and Ambrose (1954), i t might well be that the effect of a dose of cigarette smoke a t a given time is pro- portional to the fourth or fifth power of the time elapsing after its admin- istration (Armitage and Doll, 1954). On such a hypothesis a reasonable agreement between the figures in Table IX can be demonstrated. In the present state of ignorance, however, it is probably better not to attempt any exact correlation; but to note only that if smoking is a major cause of lung cancer i t will be difficult to account for the figures unless there is also a considerable difference in the relative effects of smoking cigarettes and pipes.

It is almost equally difficult to decide whether differences in smoking habits are adequate to account for the difference in mortality observed in men and women. At first sight i t would seem unlikely that they were, since women have been responsible for an increasing proportion of the total amount smoked and, in all countries, the preponderance of men among subjects of the disease has become more marked. The difficulty, however, is the same as was encountered previously; that is, we do not know the induction time of the disease nor the relative importance of smoking a t different periods of life. From the figures which are available for Britain (Table IX) it would seem that so long as the effect of smoking does not reach its maximum till after 20 years, differences in smoking habits could readily account for a large and still increasing difference in the mortality of men and women. I n fact, it may well be that the cases of

in cigarettes and in other forms.

smoke and the development of the disease.

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lung cancer among women still include only a small proportion specifically related to tobacco and that the major increase in female mortality is to come.

An alternative approach is to estimate the mortality among male and female nonsmokers. According to Doll (1953b) the proportions of men and women found among the nonsmokers with lung cancer in Doll and Hill’s (1952) series are consistent with the hypothesis that in the absence of smoking (and exposure to certain industrial carcinogens) the death rates are equal in the two sexes.

The attempt to compare mortality and tobacco consumption in dif- ferent countries is even more hazardous, for not only do standards of death certification vary but so do methods of smoking. It is said, for example, that few Europeans throw away as large an unsmoked butt as is commonly discarded in the United States. It is, however, of interest to make the comparisons, provided that the deficiencies of the data are recognized. Statistics for 8 countries have been collected by Nielsen and Clemmesen (1955), and these have largely been drawn on, in the prepara- tion of Figs. 6 and 7. In Fig. 6, the male death rate from lung cancer in 11 countries in 1950 (or in the nearest year for which the information is available) has been plotted against the annual consumption of all tobacco products per head of the population 20 years earlier; in Fig. 7 i t has been plotted against the annual consumption of cigarettes per head 20 years earlier. In fact, nearly all the tobacco consumed in 1930 was consumed by men (even in the Scandinavian countries the amount of tobacco smoked by women a t that period was small) so that i t is not unreasonable to compare the male death rate with the per capita con- sumption. In nearly all the countries the consumption per man in 1930 is likely to have been approximately double the consumption per person. Figure 6 fails to show any relationship between lung cancer mortality and total tobacco consumption in the various countries, but from Fig. 7 i t would appear that the data (with the exception of those from the United States) are not inconsistent with the existence of a relationship with cigarette consumption. To a small extent the anomalous position of the United States can be explained by the high proportion of young people in its population; whether the sort of consideration which has been referred to above can account for the rest is a matter for conjecture. What is cer- tain is that the observed facts fit the hypothesis of a relationship between the disease and cigarettes much better than the hypothesis of a relation- ship with tobacco generally.

G. Identification of Carcinogenic Agent. Numerous attempts have been made to induce cancer with tobacco products in animals. A significantly increased incidence of the common pulmonary adenoma of mice was ob-

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served on one occasion by exposing animals with a high spontaneous incidence of the tumor to strong concentrations of cigarette smoke (Essen- berg, 1952), but no change, or very little change, in incidence has been observed by others (Passey, 1929; Campbell, 1936; Loren2 et al., 1943).

I I I I I I 1 1.0 2.0 3 .0

Annual tobacco consumption, kg. per person (1930)


FIG. 6. Crude male death rate from lung cancer in 1950 and per capita consumption of all tobacco products in 1930 in various countries: (1) Great Britain, (2) Finland, (3) Switzerland (tobacco consumption estimated from data published by Gsell, 1951, and Nielsen and Clernmesen, 1954), (4) Holland, (5) U.S.A., (6 ) Australia (death rate for 1951-52), (7) Denmark, (8) Canada, (9) Sweden, and (10) Norway. Coescient of correlation between death rate and tobacco consumption, 0.10 k 0.31.

Wright (1955), in particular, exposed 80 mice of the “Strong A” strain to an average concentration of 0.08 mg. of smoke per liter for 20 hours a day for 5 days a week. There were many deaths in the early weeks of exposure, and Wright doubts if a stronger concentration could be used successfully. The average number of tumors in the 34 mice which survived

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from 3 to 15 months exposure was higher in the experimental group than in a control group of paired 1it)ter mates killed a t the same ages (1.15 against 0.76), but the number of tumor-bearing mice was less (44 % against 56%). Neither difference is statistically significant. According to Moore

4 0 0 t

Annual cigarette consumption c ige re t tes

FIG. 7. Crude male death rate from lung cancer in 1950 and per capita consumption of cigarettes in 1930 in various countries: key as in Fig. 6, with the addition of (11) Iceland (death rate estimated from cancer notification rate). Coeflcient of correlation between death rate and cigarette consumption, 0.73 f 0.30.

per person(1930)

(1953) Graham produced “what appeared to be one small papilloma in the bronchus of a dog” by painting tar from cigarette smoke through a fistula onto the bronchial mucosa; but the report proved to be erroneous and no tumor has appeared after three years (Graham, personal com- munication). Tumors comparable to bronchial carcinoma as i t appears in man have not been produced by tobacco products by any method.

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Occasional tumors on the skin of mice and rabbits have, on the other hand, been produced by several workers. The literature is reviewed by Wynder, Graham, and Croninger (1953), who have themselves been able to produce tumors in a high proportion of treated animals. They ob- tained tar from cigarettes smoked mechanically under conditions which approximated to the physical conditions of normal smoking and applied it three times a week to the skin of mice, until a carcinoma appeared or the animal died. To avoid toxic reactions from the nicotine content of the tar, the dose given initially was small and it was increased gradually over the following two months. The first papilloma appeared after eight months of painting: the first carcinoma, after one year. Of 81 mice ini- tially included in the series, 62 survived for a year or more and 36 of them (Le . , 58 %) finally developed cancer. Passey (personal communica- tion) has, however, pointed out that with the method of combustion used “Temperatures up to 966°C. were obtained” and that this is appreciably higher than the combustion temperatures in normal smoking (see page 27). Tar obtained from cigarettes burnt at temperatures not exceeding 750°C. has, in the hands of Passey et al. (1955) not reproduced Wynder, Graham, and Croninger’s results.

Three potentially carcinogenic substances have been distihguished in tobacco smoke : arsenic, benzpyrene, and radioactive potassium. Arsenic is present in many tobaccos, probably because of its use as an insecticide. It is present in greatest amounts in tobacco of American origin and is completely, or almost completely, absent from Oriental types. Daff and Kennaway (1950) estimated that an ordinary “Virginian” cigarette as smoked in England, contains about 50 pg. expressed as AszOa and that approximately 15% is volatilized in smoking. Smoking 10 cigarettes a day means, therefore, that as much arsenic as is present in one maximum official dose of Fowler’s solution is volatilized in 10 weeks. Arsenic is believed to be capable of inducing bronchial carcinoma in man (see page 33) but it is unlikely to be the responsible agent in tobacco smoke, since (1) the amount to which smokers are exposed is very small compared with the amounts encountered industrially, and (2) bronchial carcinoma forms a high proportion of cancer cases found at necropsy in Istanbul (Schwartz, reported by Daff, Doll, and Kennaway, 1951) and arsenic is almost completely absent from Turkish tobacco.

The presence of polycyclic hydrocarbons in tobacco smoke has long been suspected, but no individual substances were identified until Cooper and Lindsey (1953) and Commins, Cooper, and Lindsey (1954) reported the presence of anthracene and pyrene in tar obtained from cigarette smoke. Subsequently Cooper, Lindsey, and Waller (1954) reported that they had also distinguished the presence of 3,4-benzpyrene. To obtain

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the tar, the cigarettes were smoked mechanically but care was taken to make the physical conditions of combustion correspond closely to those which occur in normal smoking. The substances were detected by means of chromatography followed by absorption spectrophotometry ; 10.2 pg. of anthracene, 9.0 pg. of pyrene, and 1.0 pg. of 3,4-benzpyrene were estimated to be present in the tar collected from the smoke of 100 cigarettes.

The amount of benzpyrene is small and i t is necessary to smoke 200 cigarettes to obtain enough to produce, by local injection, a sarcoma in a mouse. Quantitatively it is less important than the amount in town air, since i t would be necessary to smoke 50 cigarettes in order t o inspire as much as is inspired from the air in one day by a “standard man” in an average English industrial town (Waller, 1952; Blacklock et al., 1954). On the other hand, the benzpyrene in cigarette smoke, being dissolved in the form of a fine suspension in a solvent material, may well be more active than the atmospheric benzpyrene, which is largely adsorbed on carbon particles and is, in this state, relatively inactive (Steiner, 1954). Part of the atmospheric benzpyrene is also likely to be filtered off by the nose.

Benzpyrene has also been identified in the tar collected from the smoke of “cigarettes” made entirely of paper (Cooper and Lindsey, 1954; Lefemine, 1954). The quantity present was, however, such that the combustion of the paper could account for only about 5% of the benzpyrene present in the smoke from ordinary tobacco cigarettes. Lindsey (1954 and personal communication) has, moreover, also found that benzpyrene is present in the smoke from tobacco burnt in a pipe in the same order of quantity, weight for weight of tobacco, as was obtained from cigarette smoke.

Commins, Cooper, and Lindsey (1954) suggest that the polycyclic hydrocarbons may be formed by the pyrolysis of acetylene. Kennaway (1924, 1925) has shown that strong heating of acetylene and other un- saturated materials produces carcinogenic tars a t temperatures of 700°C. and above, and Fishel and Haskins (1949) showed that acetylene was present in tobacco smoke. The temperature of combustion in ordinary cigarettes, in paper “cigarettes,” in a pipe, and in a cigar, were, according to Lindsey (1954 and personal communication), as follows:

Ordinary cigarette Paper “cigarette ” Pipe Cigar (Havana)

Quiescent Suction Combustion Combustion Surface

Temperature Temperature Temperature 650°C. 700°C. 900°C. + Varies 655°C. 900°C. + Varies 470°C. 700°C. +

400”-500”C. 560°C. 800°C. +

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Closely similar temperatures in burning cigarettes have been re- corded by Wynder, Graham, and Croninger (1953) and by Hamer (1954), and a similar temperature for combustion in pipes was recorded by Cooper et al. (1932). If Lindsey’s observations on the presence of benz- pyrene in the smoke from tobacco burnt in pipes are confirmed, it would seem likely that polycyclic compounds can be formed a t lower tempera- tures than have, hitherto, been thought to be necessary. If benzpyrene is the active agent responsible for tobacco cancer, the findidg of its pres- ence in pipe smoke will accord with the high incidence of cancer of the lip and buccal cavity, known to occur among pipe smokers. In this case i t will be necessary to explain the considerable difference in the incidence of lung cancer which is found between pipe and cigarette smokers on the basis of differences in the physical dispersal of the smoke (and of particles and droplets in the smoke), associated with the two methods of smoking- for example, in the proportion of smokers who inhale.

It will, however, be recalled that although benzpyrene is a strong carcinogen to which man and animals are both susceptible, i t has yet to be directly established that i t has any such action on the bronchial mucosa. It is possible that the active agent is, in fact, some substance hitherto not recognized as being carcinogenic. On the other hand, the high mortality from lung cancer among gasworkers, who are specifically exposed to large quantities of benzpyrene in the course of their work (see page 3P), supports the hypothesis that benzpyrene is also carcino- genic in the human bronchus.

Mulvaney (1953) suggested that radioactive potassium, present in tobacco as a naturally occurring isotope, might be an effective carcinogen. Swinbank (personal communication) found that a typical cigarette con- tained 24 mg. of potassium and that the radioactivity corresponded to 2 f 1.7 mg. more. The errors of the experiment were likely to have been greater than the statistical error, so that there was probably no excess activity a t all. Even if there were, and i t were all due to the presence of the most dangerous substance, radium, the amount estimated would not be biologically important. Swinbank estimates that, if the whole potas- sium content of cigarettes were inspired and if 20 cigarettes were smoked per day for 50 years, it is unlikely that the total dose would amount to more than the maximum permissible weekly dose recommended by the International Commission on Radiological Protection. Spiers (1954) found, however, that such radioactivity as is present in cigarettes remains almost entirely in the ash, and he was able to detect only the equivalent of 6 fig. of potassium in the smoke. It is, therefore, not possible to attribute any significant carcinogenic effect to radioactivity in tobacco.

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The further possibility remains that cigarette smoke is not in itself carcinogenic but that i t acts as an activator or a co-carcinogen to sub- stances already present in the air from other sources. This hypothesis is less attractive than i t was, now that benzpyrene has been found to be present in appreciable quantities in cigarette smoke, but it is possible that the effect of tobacco is enhanced by the presence of solvents in the smoke such as pyridine and pyrrole which could, theoretically, elute the benzpyrene adsorbed on to carbon particles in the inspired town air and so render the atmospheric benzpyrene more active. Such a secondary effect can readily be envisaged as being responsible for some of the differ- ences in mortality in urban and rural areas referred to below.

H. Various Criticisms. The conclusion that cigarette smoking is a cause of lung cancer has not been uniformly accepted. It has not, to the author’s knowledge, been argued that the basic data are factually er- roneous (this would hardly be possible, since all who have investigated the subject have found the same general trends) but i t has been suggested that the wrong interpretation has been put on the results-an interpreta- tion incompatible with all the known facts. At first the principal objec- tions were (I) that the retrospective study of patients’ histories provided too many opportunities of bias for the results to be relied on (Hammond and Horn, 1953; Shapiro, 1954) and (2) that no known carcinogen had been identified in tobacco smoke. In the light of the follow-up studies on men of known smoking habits and of the recent biological and chemical stud- ies on cigarette smoke, these criticisms are now only of historical interest.

The current objections may be considered under five main heads. 1. That the evidence i s purely “statistical” and that i t has not been

possible to produce the disease in laboratory animals by means of tobacco smoke (Shapiro, 1954). A similar type of objection could have been made to Snow’s conclusion that cholera was a water-borne disease or to Pott’s conclusion that employment as a chimney sweep in childhood led to cancer of the scrotum. It is difficult t o see what can be more relevant to the etiology of human cancer than observations on the extent of human mortality under different environmental conditions and, in this instance, nature has performed the appropriate experiment, in which the amount smoked has been varied while, so far as can be seen, other variables have been kept constant. In view of the variation in animal susceptibility and the impracticability of reproducing the exact conditions of human smoking in animal experiments, the failure to reproduce the human type of bronchial carcinoma by exposing animals to tobacco smoke cannot outweigh the extensive positive evidence obtained from direct observa- tions on men.

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2. That the data recorded in the various investigations are not wholly consistent. Although the general trend of all the results has been consistent, there have been inconsistencies in the reports of the relative risks attached to cigarette, cigar, and pipe smoking and in regard to the significance of inhaling (Hueper, 1954). The inconsistencies reported in relation to the different methods of smoking have already been considered (see page 16), but no reference has yet been made to inhaling. It would commonly be expected that any effect of cigarette smoking would be most noticeable among persons who inhaled the smoke, and both Lickint (1953) and Breslow and his co-workers (1954) found that a higher proportion of patients with lung cancer than of control patients said they inhaled. On the other hand, Doll and Hill (1952) found no difference in the pro- portion of inhalers in the two groups, although there was a suggestion that inhaling might be commoner among men with peripheral growths and even less common than among the controls in men with central growths. The explanation of these conflicting reports is unknown. It may, perhaps, derive from a failure on the part of the patients t o under- stand correctly the import of the questions.

3. That the interpretation put on the results i s incompatible with the evidence from vital statistics. Several of the principal criticisms of this type have already been discussed and they will, therefore, only be listed here.

a. That although the consumption of tobacco has undoubtedly in- creased, i t has not yet been shown satisfactorily that there has been any real increase in the incidence of the disease (Rigdon and Kirch- off, 1953; Shapiro, 1954). See page 4.

b. That (on the contrary) the increase in tobacco consumption has not been great enough to account for the increase in the incidence of the disease (Todd, 1954). See page 22.

c. That the correlation between mortality and cigarette consumption as recorded in a number of countries is imperfect (Hueper, 1954) and that the data from Britain and the United States are, in par- ticular, incompatible (Russ, 1954). See page 23.

d. That the male predominance among cases of the disease persists despite the fact that the increase in cigarette smoking has been relatively greater in women (Hueper, 1954). See page 22.

Other objections which have not been referred to previously are:

e. According to Hueper (1954) the fact that the sex ratio of cases of the disease was practically equal in Norway until about 1930, and that since then an increase has begun to be recorded which is most marked in men and in towns, weighs against the view that

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smoking is an important cause of the disease. He considers that special factors must have influenced the epidemiological behavior of lung cancer in Norway, whereas Norwegians smoke the same type of cigarette as that smoked in the United States. He omits, however, to take into consideration the very small quantity of cigarettes which were consumed in Norway before 1930, and the Norwegian experience may, with greater justification, be cited in support of the view that cigarettes are one of the principal causes of the disease.

f . Several authors have pointed out that the mucosa of the lip, mouth, and larynx also comes into contact with cigarette smoke and yet the mortality from cancer of these sites, in contrast with the mortality from lung cancer, has remained stationary or has fallen (Hueper, 1954; Passey, 1954; Maxwell, 1955). Many in- vestigators have, however, shown that cancer of the lip and mouth is, if anything, associated with the smoking of cigars and pipes (Levin et al., 1950; Sadowsky et al., 1953), so that a reduction in mortality from cancer a t these sites would have been expected to occur as smoking habits were switched from cigars and pipes to cigarettes. The evidence with regard to laryngeal cancer is more conflicting. There is no a priori reason to suppose that it is neces- sarily produced by the same factors as produce lung cancer, and, in fact, none of the known industrial causes of lung cancer are known to cause laryngeal cancer. On the other hand, there is evidence that laryngeal cancer is associated with cigarette smoking (Levin et al., 1950; Sadowsky et al., 1953). It is possible that the lack of any marked increase in the recorded mortality from laryn- geal cancer results partly from improvements in the treatment of intrinsic cancer of the larynx and partly from confusion by the classification under one head of cancer of the extrinsic and intrinsic larnyx.

4. That an association i s not necessarily causal and that both lung cancer and smoking may be the end results of a third common factor. It is not pos- sible to give any conclusiye answer to this type of criticism and, indeed, a similar hypothesis always provides an alternative explanation to any scientific theory. The principle of Occam’s razor has, however, proved of value to the development of scientific thought in the past, and it would seem reasonable to adhere to it now, and to work on the basis of the most economical hypothesis-unless or until some conclusive reason is shown for abandoning it. The possibility that lung cancer and smoking may both be end results of a third common cause is as much applicable to the results

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of the prospective studies on mortality among smokers and nonsmokers as i t was to the results of the retrospective studies among patients. There is, however, no evidence to suggest that i t is the explanation of them. Some of the theoretically possible common factors have been considered above (see page 14) ; another which has been suggested is that persons of a particular physical constitution might be prone to lung cancer and to heavy smoking (Parnell, 1951). There is no evidence of such a physical constitution characteristic of patients with lung cancer; and if one did exist, we should still have to find some environmental factor to account for the increase in the incidence of the disease.

5. T h a t the e$ect of smoking i s limited to determining the site of the growth in persons previously destined to develop cancer (Fairweather, 1954 ; Loxton, 1954). This objection derives from Cramer’s (1936) hypothesis that the total incidence of cancer in a population is constant and that environmental and hormonal factors exert their effect by determining the site a t which the cancers develop. In this general form, it can readily be demonstrated to be not true (Case, 1954). I n the particular case of cigarette smoking it can also be shown to be untrue; for, if i t were true, i t wouId follow that cancer of sites other than the lung would have to be relatively more common among nonsmokers and light smokers than among heavy smokers. Several reports of the smoking habits of persons with cancer in other sites have been made. Except for a report by Gilliam (1954) none has, in fact, shown a negative association between smoking and the type of cancer investigated, though several have suggested the possibility of other positive associations, e.g., between cigarette smoking and cancer of the larynx and between pipe smoking and cancer of the lip (Levin et al., 1950; Doll and Hill, 1950, 1954a,b; Sadowsky et al., 1953; Hammond and Horn, 1954). Gilliam added details of the data on cancer of the skin to the data previously reported in conjunction with Sadowsky and Cornfield, and these showed a greater prevalence among nonsmokers than among cigarette smokers. The excess prevalence of skin cancer among nonsmokers could conceivably result if i t were found that there were fewer smokers in the South of the United States than in the North.

I . Conclusion. In a review of the evidence relating lung cancer to smoking, Gilliam (1954) concludes that:

“Proof in the mathematical sense is unobtainable in dealing with medical prob- lems. Direct experimental verification in humans ‘is possible to conceive but impossi- ble to conduct.’ Indirect experimental verification in humans, through country-wide discontinuance of smoking and subsequent determination of trends of the disease, could be practically accomplished only by informing the public that the disease is caused by cigarettes. If this is true, the procedure is unnecessary as an experiment.

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Production of the disease in experimental animals, under conditions simulating human smoking, would strengthen though not establish the hypothesis, but inability to do so could in no circumferences justify its rejection. . . .

‘ I We are left for ‘proof,’ therefore, with indirect and circumstantial evidence derived largely from considerations of the pathogenesis of the disease in individuals and its observed distribution in human populations: in short, with epidemiological evidence. It is a matter of opinion how many and what facts must be consistent before this hypothesis may justifiably be accepted or rejected.”

I n the author’s opinion, taking into consideration the philosophical principle of Occam’s razor which has already been referred to, the facts are such that the hypothesis that cigarette smoking is a cause of the main histological types of lung cancer should be accepted. They also, in his opinion, justify a strong presumption that the smoking of pipes and cigars is, in this respect, relatively innocuous. The discovery that a known and powerful carcinogen is present in tobacco smoke in significant quan- tity strengthens the credibility of the conclusion, but i t has yet to be shown experimentally that the substance concerned has a direct action on the bronchial mucosa.

The great majority of the observed facts accord with the hypothesis, but the picture is not yet complete. We need to know, in particular, why the mortality from the disease in the United States is so low relative to the past consumption of cigarettes; and why the association which ap- pears t o exist between cancer of the larynx and cigarette smoking has not been reflected in an increase in the incidence of cancer of the larynx com- parable to that believed to have occurred with cancer of the lung. The data on the significance of inhaling are also conflicting, and i t is uncertain whether the difference between the effects of smoking tobacco in the form of cigarettes and in a pipe can be attributed to differences in the extent to which the smoke is usually inhaled or whether it is necessary to postu- late some other mechanism. These fields of uncertainty are, however, small in relation to the extent of established knowledge and do not justify throwing doubt on the main conclusion.

2. Industrial Hazards

When Smith (1953) presented his report to the Louvain symposium, five industrial processes (the mining of certain radioactive ores, the refining of nickel, and the manufacture of asbestos, chromates, and coal gas) had been recognized as involving a special risk of lung cancer; and there was fairly strong evidence to suggest that exposure to heavy con- centrations of arsenic in the air-of up to 1000 pg. per cubic meter- might also produce the disease (Hill and Faning, 1948; Perry et al., 1948). Bonser (1955) has now suggested that hematite miners should be added t o the list. During the last 20 years, 17 cases of lung cancer were

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observed a t autopsy among 192 hematite miners (8.9 %), whereas the same pathologist found only 44 lung cancers among 2378 autopsies on men over 20 years old from the same area (1.9%). The evidence is sug- gestive, but it is not conclusive, since i t is possible that miners with chest symptoms may have been more likely to come to autopsy than men with similar symptoms in nondusty occupations.

The extent of the risk has now been defined more closely in the case of asbestos, chromates, and coal gas. Brinton, Frasier, and Koven (1952) extended the initial observations of Machle and Gregorius (1948) and compared the sickness and mortality experience of insured workers in the seven chromate-producing plants in the United States with the whole sickness data obtained by the U.S. Public Health Service and the death rates for the U.S. population. They found that, over the period 1940 to 1948, the mortality from lung cancer among white males employed by the plants was 14 times the expected; and among colored males it was 80 times the expected. In view of the small number of cases, these esti- mates must be liable to considerable error, but i t is clear that employees of the industry were exposed to a risk which was many times the normal. The physical conditions to which the workers were exposed have been investigated by the Division of Occupational Health of the U.S. Public Health Service (Federal Security Agency, 1953), but it has not, as yet, been possible to define which of the substances involved in the manu- facturing process are carcinogenic. The authors suggest that acid-soluble- water-insoluble compounds found principally in the residue from the leaching tanks may be responsible. If this were so, i t might explain why the hazard has been less apparent in British factories, where the residue is discarded (Bidstrup, 1951). The British industry has, however, only recently come under observation and the possibility that a considerable risk exists has not yet been excluded.

Doll (1952) studied the causes of death among 2071 male pensioners of a London gas company and found that the number of deaths from lung cancer was approximately double that expected by comparison with male inhabitants of London of the same age distribution (25 deaths against 13.8)-that is, showed practically the same excess as had been estimated by Kennaway and Kennaway (1947) from study of the national mortality statistics. In both cases the gasworkers covered a multiplicity of occupa- tions, and it is possible that the risk may have been greater for those most closely concerned with the production process. I n a more detailed study, Sutherland (personal communication) found an incidence of respiratory cancer among the employees and pensioners of a Canadian gas company who had worked in the retort house, which was several times higher than that recorded in the general population of the district. The excess was

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apparent only among men who had worked a t a particular station where the gas was manufactured in horizontal retorts. No excess was observed among employees who had never worked in the retort houses. According to the earlier reports of Kuroda and Kawahata (1936) the risk experienced by men employed in generator gas plants of a Japanese steel mill is likely to have been greater still (21 cases of lung cancer occurred in a 6-year period among 100 workers who had been employed for more than 10 years).

The risk to which asbestos workers were exposed has been defined more fully by a study of the mortality among 113 men who had been exposed to the dust for 20 or more years (Doll, 1955). Eleven of the thirty- nine men who had died were found to have asbestosis and cancer of the lung a t autopsy, whereas the number of deaths expected to have been due to lung cancer was estimated as less than one (0.8). Since the mor- tality was considerably less among men who had been employed for less than 10 years in the conditions which existed before 1932, when measures were taken to reduce the amount of dust in the atmosphere, i t must be presumed that the risk had a t one time been appreciably greater than the average estimated over the whole period. The occurrence of lung cancer in 14 out of 72 subjects found to have asbestosis a t autopsy has been reported by Bonser (1955).

The number of men employed in all these occupations taken together constitutes only a small fraction of the total number of men employed in industry, and the number of cases of lung cancer due to these special hazards can have contributed only a very small proportion to the total number of cases. Some of the occupations are, however, of particular interest since the carcinogenic agents which are presumed to be respon- sible for the added risks also have a more general distribution, i.e., radio- active substances and benzpyrene. It is also of interest, as was pointed out by Smith (1953), that the majority of the specific industrial risks appear to be related to inorganic substances, whereas occupational tumors of other organs have usually been traced to organic compounds.

Numerous other occupations have been suggested as possibly giving rise to specific risks. The evidence has been fully reviewed by Hueper (1951, 1952), but in no other instance is i t adequate to justify a positive conclusion.* Recent studies by Wynder and Graham (1951), Doll (1953a), and Breslow et al. (1954) have compared the occupational histories of men with lung cancer with the histories of comparable groups of men with other diseases, and Kreyberg (1954a) has compared the occupational histories of patients with his “group I ” tumors with the histories of

* Nor is the recent evidence adduced by Dunner and Hicks (1953) with regard to boiler scalers and grain dockers.

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patients with ‘ I group I1 ” tumors and with the occupational distribution of the population of Norway, as shown by the Census. The most interest- ing finding has been the negative one, that workers who were particularly exposed to the fumes of motor exhausts (road transport drivers, etc.) were not disproportionately represented among the lung cancer patients in any of the series.

3. Atmospheric Pollution

A. Mortal i ty in T o w n and Country. The principal reason for thinking that atmospheric pollution may be responsible for some cases of lung

4000 r

3000- I- 1 Men

- - c 9



o- - - -. Women - L W

01 c

>100,000 popn. 2 2000- 5 01

73 - m < 100,000 popn. S S

I Rural districts

Conurbations /Other towns

,r-Towns c />100,000 popn.

#++- ’I=&=-* < ~ O O , O O O popn ‘Rursl districts



0 : i * + e d c 15 25 35 45 55 65 75 85

Age in years FIG. 8. Male and female death rates from lung cancer in England and Wales in

1953, by age and place of residence.

cancer is that the mortality has consistently been recorded as being higher in urban than in rural areas (Stocks, 1947, 1952; Clemmesen, Nielsen, and Jensen, 1953 ; McKinlay, 1953 ; Curwen, Kennaway and Kennaway, 1954; Hoffman and Gilliam, 1954; Kreyberg, 1954c; Sax&, 1955). The difference has not been great-the mortality in Copenhagen, London, Oslo, and American and Finnish cities being some one and a half t o four times the rate in the corresponding countryside-but in each country for which detailed data are available the mortality has increased steadily with the degree of urbanization. Data for England and Wales for 1953 are illustrated in Figure 8. Particularly striking was the correla-

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tion demonstrated by Stocks (1952) between lung cancer mortality in men and the number of occupied houses in 84 large towns (Table X) and the finding that a similar correlation between density of population and male lung cancer mortality also holds for rural areas (Table XI, Curwen, Kennaway, and Kennaway, 1954).

Three explanations of these findings are possible: (1) that they are artifacts due to a greater efficiency of diagnosis in the areas of greater

TABLE X Standardized Mortality Ratios Due to Lung Cancer for Men in 84 Towns of England

and Wales (1946-1949) Compared with No. of Occupied Dwellings and Density of Population per Acre*



Standardized Mortality

No. of Persons Ratio for Occupied per Lung Cancer Dwellings Acre in Men

(1931) (1951) (1946-49)

tlondon, Croydon, East Ham, and West Ham t Birmingham, Smethwick, Walsall, and West

t Manchester, Salford, and Stockport t Liverpool, Bootle, Birkenhead, and Wallasey t Leeds, Bradford, and Halifax Sheffield t Newcastle on Tyne and Gateshead Average of 6 towns Average of 3 towns Average of 12 towns Average of 13 towns Average of 29 towns



297,600 259,900 242,400 237,700 123,800 86,700 66,700 43,600 33,100 24,000 14,700


20 25 25 11 13 26 19 16 16 16 11



134 159 162 132 135 114 113 106 105 101 89

~ ~~~

* After Stocks, 1952. Some of the mortality ratios shown differ very slightly from those quoted by

t Groups of adjacent towns treated as one unit. Stocks, because the data for the last 5 group8 are, for simplicity of tabulation, shown as averages.

population density, (2) that they result from the presence of a carcino- genic pollutant in town air, and (3) that they result from differences in the way of life of individuals in town and country.

The first possibility cannot be entirely excluded. Bonser and Thomas (1955) found, for example, that during the years 1950 to 1952 the number of cases diagnosed in hospital in a largely rural region of Scotland was 21% less than the number of persons recorded as having died of the dis- ease, whereas in Leeds the deficiency was only 8%. Clemmesen, Nielsen, and Jensen (1953) found a similar difference between the proportion of cases not admitted to hospital in the rural districts of Denmark and in

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S.M.R. Male S.M.R. Female

Copenhagen (25% against 8 %). There has, therefore, clearly been less ready access to hospital for patients in rural areas, and the possibility must be admitted that some of the difference in mortality may be spuri- ous. Doll and Hill (1952), on the other hand, found that the proportion of lung cancer patients who had lived for 10 or more years in the country was less than that among a matched group of control patients-irrespec- tive of the place of residence a t the time of interview. The differences were small and were not statistically significant, but they were all in the same direction and provide some support for the belief that the risk of developing lung cancer has been, in fact, lower in the countryside.

69 63 76 60 60 65 55 50 58 55 45 (22t) (77) 96 (84) 67 61 (61) (65) 72 (60) (84)

TABLE XI Standardized Mortality Ratios Due to Lung Cancer in the Rural Districts of 11

Geographical Regions of England and Wales (1946-1949) Compared with the Average Population per 100 Acres*

Density of Population, Persons per 100 Acres

39 38 36 32 27 24 22 20 19 13 12

Coefficient of correlation between density of population and male

Coescient of correlation between density of population and female S.M.R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.81, S.E. 0.30

S.M.R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -0.11, S.E. 0.30 ~~~~ ~ ~ ~ ~

*After Curwen, Kennaway, and Kennaway, 1954. t Based on 17 deaths; other figures in parentheses on 25-100 deaths and all others on more than

100 deaths.

B. Pollution of Town Air. Three known carcinogens have been de- tected among the pollutants of town air: arsenic, 3,4-benzpyrene1 and radium. The amount of arsenic was, however, of the order of 6 pg. As203 per 100 m.3 (Goulden, Kennaway, and Urquhart, 1952) and was minute in comparison with the amount to which men have been exposed in industry, without the apparent production of any great excess of lung cancer (see page 33). Waller (1952), Kotin, Falk, Mader, and Thomas (1954), and Kotin, Falk, and Thomas (1954) reported that 3,4- benzpyrene was present in town smoke and in the exhaust fumes of cars, but if, as is likely, i t occurs adsorbed onto particles of carbon it must be presumed to be biologically inactive (Steiner, 1954). The extent to which the radium (and radon) content of coal smoke may be significant is uncertain (Anderson, Mayneord, and Turner, 1954) (see page 42).

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The chemical findings do not, as yet, give material support to the suggestion that the increased mortality from lung cancer in urban areas may be due to pollution of the atmosphere, but i t is possible that further work may show the pollutants to be effectively carcinogenic. For ex- ample, the petrol vapors present with benzpyrene in motor exhaust fumes may themselves act as adequate eluents (Kotin, Falk, Mader, and Thomas, 1954). In the present state of knowledge conclusions about the role of atmospheric pollution must be based on the epidemiological findings.

State asphalt highways'

' Motor fuel consumption

400 consumption

200 -

Coal consumption

1900 1910 1920 1930 1940 1950 1960 Year

FIG. 9. Trends in prevalence of selected environmental factors expressed as a per- centage of the prevalence in 1926, U.S.A. 1900-1953. (Reproduced from a paper by Dr. E. C. Hammond, in Cancer 7, 1100-1109, 1954.)

From this point of view, i t is difficult t o believe that the increase in mortality from lung cancer can have been directly due to pollution with chimney smoke or with petrol or oil fumes. On the one hand, the amount of coal consumed in the industrialized countries has not increased greatly, and the total amount of smoke pollution has probably decreased because of greater efficiency of combustion (in Britain, for example, coal con- sumption increased from about 165 million tons in 1900 to 206 million tons in 1953, but the amount burnt in gasworks and electricity-generating stations increased from 23 million tons in 1921* to 64 million tons in 1953. The pattern of coal consumption in the U.S.A. is shown in Fig. 9). On the other hand, there has been a marked increase in the amount of petrol and oil burnt (Hammond, 1954, see Fig. 9), but men who have

* Earlier figures not available.

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had special occupational exposure to the fumes do not appear to have suffered any abnormally high mortality from the disease (see page 35). Diesel fumes are a special case in that oil-burning engines have not been in general use on the roads for a sufficient length of time-to judge from the induction time of industrial cancers-to have exerted any significant carcinogenic effect.

I n England and Wales the excess urban mortality has, until recently, increased pari passu with the total lung cancer mortality; in Denmark i t has increased even more rapidly. Unless, therefore, i t is postulated that the whole increase in mortality is due to improved diagnosis (which is contrary to the general opinion, see page 4), it must be concluded either that atmospheric pollution acts as a co-carcinogen to some other sub- stance which has increased in prevalence (for example, in cigarette smoke) or that i t cannot be responsible for more than a relatively small and constant part of the specific urban mortality. In either of these cases a number of apparently anomalous findings will have to be accounted for:

1. The difference between urban and rural mortality is greater for Copenhagen and the Danish countryside (4 to 1) than i t is for large English towns and the English countryside (2 to l), although the smoke pollution of Copenhagen air-judged colorimetrically-is only about one- tenth of the level of a typical English industrial town (Kennaway and Wilkins, personal communications).

2 . A general atmospheric pollutant could be expected to affect both sexes equally, whereas, in fact, (a) urban areas with a high mortality show a greater predominance of male cases than rural areas with a low mortality (Stocks, 1952, Clemmesen, n’ielsen, and Jensen, 1953) and ( b ) the correlation between mortality and density of population in rural regions which Curwen, Kennaway, and Kennaway (1954) found to be strong for men was absent for women (see Table XI).

3. No consistent difference in lung cancer mortality has been found among nonsmokers in areas of different population densities (Doll, 1953b).

C. Diferences in Urban and Rural Habits. The urban excess may also be due to differences in the personal habits of townsmen and country- men. Kreyberg (1954c, 1954d) came to the conclusion that this was the most probable explanation from comparing the place of residence and the occupation of patients with his “group I ” and “group 11” tumors. He found that the ratio was greater for towns than for rural areas, but that it was unaffected by the degree of industrialization of the town or by the extent to which the town was exposed to wind from the sea.

One possibly relevant difference to have been recorded is a difference in smoking habits. Doll and Hill (1952) found that townsmen tended to

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smoke more than countrymen and that a higher proportion of townsmen who smoked, smoked cigarettes. Moreover the difference was greater for men in big towns than for men in small towns. Stocks (1954)) Hammond and Horn (1954), and Kreyberg (personal communication) have reported similar differences. The differences are not, themselves, great enough to account for more than a small part of the excess urban mortality. Present habits are, however, unlikely to be relevant to present mortality, and i t is possible that the differences may have been greater 20 or 30 years ago. Unfortunately, precise data about prewar differences are not available.

D. Conclusion. The present evidence is inadequate to allow an ex- planation of the urban-rural difference in mortality to be given with confidence. Several considerations weigh against the suggestion that i t is primarily due to atmospheric pollution with chimney smoke or motor exhaust fumes; but the possibility has not been excluded that chimney smoke may be responsible for a proportion of cases-perhaps as a conse- quence of its radium content-or that it may act as a co-carcinogen with, say, tobacco. The urban-rural difference can be partly accounted for by (1) geographical differences in the efficiency of diagnosis and (2) dif- ferences in the past smoking habits of townsmen and countrymen. The effect of these factors is likely to diminish, and i t may, therefore, be anticipated that the differences will also gradually diminish. Postwar experience in England and Wales suggests that this may, in fact, have begun to happen (Waller, personal communication).

Standardized male D.R. * in Greater London Standardized male D.R. as a percentage of the

1950 1953 667 permillion 782 per million

rate in Greater London Greater London 100 100 Other conurbations 87 90 Towns more than 100,000 popn. 82 83 Towns 50,000-100,000 popn. 64 72 Towns less than 50,000 popn. 58 64 Rural districts 47 49 * standardized on the age distribution of the male population of England and Wales in 1950.

4. Atmospheric Radioactivity

Uranium and thorium are widely distributed throughout the earth’s crust; both materials decay through radioactive series, one member of which is gaseous. The radioactive gases, i.e., radon and thoron, escape into the atmosphere, where their respective decay products eventually attach themselves to dust particles.* In addition a small amount of radon and

* The small amount of radioactivity attributable to thoron is not distinguished from that attributable to radon in the remainder of this discussion.

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of radium is released into the atmosphere by the combustion of coal. Minute quantities of the radioactive isotopes of the common elements (e.g., potassium and carbon) are also present, but their effect would be insignificant in comparison with the effect of members of the uranium and thorium series.

Whether atmospheric radioactivity is a cause of any cases of lung cancer is uncertain. It cannot have been responsible for the increase in incidence which took place before 1954, and there is no evident reason why i t should affect men more than women. There is, however, a possi- bility that i t might contribute to the increased incidence in towns, because of the presence of radium in coal smoke (Anderson, Msyneord, and Turner, 1954). During conditions of fog both coal smoke and the radon naturally diffusing from buildings and the soil are likely to be retained near the surface of the earth, and a considerable increase in radioactivity may be observed. On the first day of the London smog of December, 1952, Anderson, Mayneord, and Turner found a level 400 times that previously recorded on a clear sunny day. Dawson (1952) found that radioactivity indoors was approximately double that in the open air, and in a closed cellar i t was increased a hundredfold. On the other hand, he could not find any appreciable difference between the average radioactivity of the air in towns and country. The large day-to- day variations which occurred in all districts were chiefly related to meteorological conditions.

The average amount of radioactivity present was estimated by Daw- son (1952) to be of the order of 5 X lo-“ pc. per milliliter, and this agrees fairly well with estimates made earlier in the century in England, Canada, and the United States (2 X lo-“ to 2 X 10-lo pc. per milliliter). Dawson’s estimates were, however, made by drawing air through filter papers and deducing the radon content of the air samples by assuming that the radon was in equilibrium with the radium A, B, and C on the retained particles. Anderson, Mayneord, and Turner (1954) suggest, however, that the method may underestimate the amount when the suspended particles are small. By measuring whole air samples in an ion- chamber apparatus, they obtained values 10 to 100 times greater than with the filter paper method; and the average value for air on the roof of the Institute of Cancer Research, London, was found to be 2 to 3 x 10-~ pc. per milliliter.

The “tolerance concentration” of radon is at present set a t lo-’ pc. per milliliter; but it is not possible to determine whether atmospheric radioactivity can ever be carcinogenic by reference to such an arbitrary standard, since the standard has been set in relation to the safety of individuals. The only way a t present available of testing whether a given

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level might produce an incidence of, say, 1 in 100,000 in a large popula- tion is by comparison with the effects produced by the known levels in the highly radioactive mines in Schneeberg and Jachymov and in other similar areas.

According to Evans (1950) the mean concentration of radon in the air of the mines was equivalent t o an activity of 3 X 10W pc. per milliliter. The content of the air varied in different parts of the mines, and other estimates have set the average value 10 times higher (Mitchell, personal communication). Evans calculates that an activity of 3 X lop6 pc. per milliliter would have delivered a dose of approximately 0.5 r.e.m. per working day to the epithelium of the larger bronchi. The average induc- tion time for the development of the tumors was 17 years (Sikl, 1950), so that the total dose received mould, in this case, have been of the order of 3000 r.e.m. Shapiro (1954) and Anderson, Mayneord, and Turner (1955) point out that Evans ignored the effect of the particulate matter in the air bearing the radioactive breakdown products of radon and they esti- mate that the total dose received by some areas of the bronchi is likely to have been 70 times higher. Since persons suffering from chronic radium poisoning who developed bone sarcoma are estimated to have received local doses of about 35,000 r.e.m. (Evans, 1950), the physical data may be considered reasonably consistent with the hypothesis that the Jachymov cancers were due to exposure to radon in the air.

If it is assumed that there is a linear relationship between strength of dose and cancer incidence-and the assumption is not necessarily justifiable, particularly for very small doses-it is possible to estimate the incidence of lung cancer which may be produced by normal atmos- pheric radiation. According to Sikl (1950) the mortality among the miners of Jachymov was approximately 1 % per year, so that exposure to normal atmospheric radioactivity for the length of time the miners were exposed to the air of the mines, might be expected to produce

'O-" X 1 % (the ratio of the mini- an annual mortality of between

mum estimate of normal atmospheric radioactivity and the maximum estimate of the radioactivity of the air in the mines, times the mortality

among the miners) and 2'5 - lo-' X 1 % (the ratio of the maximum

estimate of normal atmospheric radioactivity and the minimum estimate of the radioactivity of the air in the mines, times the mortality among the miners) ie., between 0.017 and 8.333 per million.

Evans assumed that the miners were exposed for 12 hours out of the 24, whereas people are exposed to normal atmospheric radiation through- out the day. More importantly, people are normally exposed from birth,

3 x 10-5

3 x 10-6

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whereas the miners were exposed, on the average, for 17 years from the age of 33 years. If the effect were proportional to the total dose irrespec- tive of the period of life a t which it was administered the expected annual mortality would be between 0.017 X 2 X 3 and 8.333 X 2 X 3 per million, i.e., between 0.1 and 50.0 per million.

In England and Wales the annual mortality from lung cancer among men aged 25 t o 74 was 912 per million in 1953, but much of this appeared to be attributable to smoking. Estimates of the rates among nonsmokers have been made by Doll (1953b), from which i t can be calculated that the mortality in this agegroup attributable to causes other thansmoking may be of the order of 69 per million. The Jachymov population cannot have con- tained as high a proportion of old people as does the adult population of England and Wales, so that the comparable mortality due to causes other than smoking is certainly much less than 69 per million-perhaps as little as 30 or 40 per million.*

On the basis of these calculations, it seems that atmospheric radiation might well be a significant cause of lung cancer in Britain. This conclusion is strengthened if it is considered, from analogy with other types of cancer, that the total dose of the carcinogenic agent is not the only determinant of the incidence of the disease. As with other types of cancer there appears to have been an appreciable induction time between initial exposure and the appearance of the Jachymov cancers, and i t is possible that exposure to a given dose of radiation early in life may have a greater effect a t the age of 50 years than exposure to the same dose at the age of, say, 35 years. I n these circumstances, the expected mortality due to atmospheric radiation may be many times greater than the annual rate estimated above. On the other hand, the fact that the Jachymov and Schneeberg cancers were almost invariably squamous, oat-cell, or undifferentiated cancers (Schmorl, 1928; Hueper, 1942; &kl, 1950), whereas an important part of the nontobacco cancers appears to consist of adenocarcinomas, weighs against the concept that radioactivity could account for all the cancers not attributable to smoking or to specific industrial hazards.

6. Previous Respiratory Infections

Previous inflammation and the formation of scar tissue in the lungs have long been thought to be possible precursors of lung cancer, but there is little firm evidence t o implicate them. Woodruff and Nahas (1951) and Woodruff et al. (1952) found that large calcified foci-larger than in any other part of the lung-were present in the same lobe as the tumor,

* If the entire population of miners and retired miners observed by gikl is assumed to have been aged 25 to 44 years, the comparable mortality among nonsmokers would be 20 per million.

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or in the tracheobronchial nodes draining the lobe, in 27 out of 40 cases of squamous and anaplastic bronchial cancer. They suggested that calci- fied foci might increase the susceptibility of the neighboring bronchial mucosa to carcinogenic substances reaching i t from the inspired air, or that bronchiectasis following primary tuberculosis might be a predis- posing factor. A similar type of conclusion was suggested by Schwartz (1950), who described cases of bronchial carcinoma in association with lesions of the bronchial wall brought about by neighboring tuberculous lymph nodes.

Raeburn and Spencer (1953) reported a close histological association between the site of origin of cancer and lung fibrosis and bronchiectasis. They sectioned the whole of both lungs a t autopsy and removed all suspicious nodules and scars for microscopy. In 750 autopsies, they found 9 unsuspected microscopic cancers in association with scars in the periph- ery of the lung and one unsuspected small carcinoma in a large bronchus. The authors acknowledged that I ‘ great difficulty has been experienced in determining the borderline between innocent reparative proliferation and true malignant change,’’ but they were satisfied that “only cases which have shown obvious malignant change have been included in the series.” If the lesions were, in fact, true cancers, i t must be postulated that their evolution into clinical malignancy would have taken many years, since otherwise their incidence was much greater than could be explained by the known rate of cancer mortality. The observation em- phasizes the need for a long-term study of the end results of respiratory infection.

It has often been noted that a long-standing bronchitis is a common complaint of persons with lung cancer (e.g., Bryson and Spencer, 1951), but there have been few studies of the frequency of its occurrence in comparable control series. Doll and Hill (1952) compared the history of previous respiratory disease in 1465 patients with carcinoma of the lung and in 853 patients with cancer in other sites. After making allow- ance for the age and sex of the patients they found that the proportions of patients complaining of attacks of respiratory tuberculosis, pleural effusion, asthma, or chronic nasal catarrh more than five years previously were practically the same in both groups, but that the proportions com- plaining of chronic bronchitis or of pneumonia more than five years pre- viously were significantly greater in the lung carcinoma group. When, however, the lung carcinoma patients were compared with another group of 335 patients who had been thought to have lung cancer a t the time they were interviewed but who were finally proved not to have it, no significant difference was detected. This latter group, however, contained a high proportion of patients with other respiratory diseases and may not

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have been a suitable control group. All that could be concluded was that either chronic bronchitis and pneumonia predispose to a whole group of respiratory disorders, including bronchial carcinoma, or that patients with respiratory disorders recall previous chronic bronchitis and pneu- monia more readily than do patients with diseases in other systems.

Lea (1952) compared the incidence of long-standing pulmonary symp- toms in men with different histological types of lung cancer and found that it was significantly higher in men with squamous carcinoma than in men with oat-cell carcinoma or adenocarcinoma (20 out of 91 against 33 out of 303). He did not, however, allow for the greater average age of the pa- tients with squamous carcinoma, and this may have accounted for some of the difference.

Direct evidence implicating chronic bronchitis has recently been ob- tained by Case and Lea (1955). I n a large group of chronic bronchitics who were followed for more than 30 years, they found that the mortality from cancer in sites other than the lung was close to the expected mor- tality, whereas the mortality from lung cancer was about double what they had calculated it should be. A result of this type might be accounted for if the development of bronchitis was itself closely related to smoking habits. According to Palmer (1954) bronchitis is commoner among smokers than among nonsmokers, and its incidence increases with the amount smoked; the data are, however, insufficient to exclude the possi- bility that the association between bronchitis and lung cancer may be, at least in part, independent and direct. On the other hand, bronchitis cannot be the effective intermediate stage in the carcinogenic process initiated by smoking, since the relationship between smoking and cancer is closer than the relationship between cancer and bronchitis.

IV. CONCLUSION From the work which has been reviewed in the preceding sections,

a fairly distinct picture of the etiology of the disease is beginning to appear.

Firstly, there is the rise in incidence which has taken place in many countries and which has principally affected men. Corresponding to this rise, it must be postulated that there has been an increased prevalence of one or more causal factors in the environment.

Secondly, there is the evidence that cigarette smoking is an important factor in the production of squamous, oat-cell, and undifferentiated lung cancer, and that a few individual cases result from exposure to five or more independent industrial processes. Whether the increase in cigarette consumption and the growth of the specific industries can together ac- count for the real increase in mortality and for the extent of the male

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preponderance, cannot be seen with certainty. Knowledge of the true extent of the change in mortality and of the fundamental mechanisms of carcinogenesis is, unfortunately, insufficient t o permit the preparation of a precise balance sheet. There is, however, no direct evidence to implicate those other environmental factors which are also known to have increased in prevalence in the last four or five decades; and i t is a reasonable pre- sumption that the changes which have taken place in tobacco consump- tion (in amount and in method) are responsible for the major part of the real increase in mortality. Whether the action of cigarette smoke is due to its 3,4-benzpyrene content or t o some other substance, and why i t should be different from that of smoke from pipes and cigars, is unknown.

Thirdly, there is a group of cases, of relatively stable incidence and occurring almost equally in men and women, which is characterized histologically by the inclusion of a high proportion of adenocarcinomas. Some of these cases-though perhaps not the adenocarcinomas-may be due to atmospheric radioactivity; others may conceivably result from long-standing respiratory infections.

Two other factors have a t times received considerable prominence, namely, atmospheric pollution and hereditary susceptibility. The evidence concerning the former permits no definite conclusion, save only that i t is not independently responsible for a large proportion of cases nor for the recent increase in mortality. There is no evidence concerning the latter, though doubtless susceptibility t o inspired carcinogens varies as does susceptibility t o other environmental stimuli.

Perhaps the most striking conclusion is the wide range of substances -several of them inorganic-which can induce cancer in the bronchial mucosa. Whether there may be a common mechanism, through which each exerts its effect, remains one of the principal problems for future research.


I am most grateful to Prof. A. Bradford Hill for his advice in the preparation of this paper, and to Dr. P. Bidstrup, Mr. W. Binks, Dr. G. Bonser, Dr. R. A. M. Case, Dr. J. Clemmesen, Dr. H. F. Dorn, Dr. E. A. Graham, Sir Ernest Kennaway, Prof. L. Kreyberg, Dr. H. 0. Lancaster, Dr. A. J. Lea, Dr. A. J. Lindsey, Dr. A. XlcKenzie, Prof. J. S. Mitchell, Prof. R. D. Passey, Dr. R. B. Sutherland, Mr. P. Swinbank, Dr. R. C . Turner, Dr. N. Veall, Mr. R. E. Waller, Dr. E. T. Wilkins, and Dr. B. M. Wright for allowing me to see unpublished data and for their helpful comments.

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The Experimental Development and Metabolism of Thyroid Gland Tumors


Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

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. . . GO

Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GO

11. Thyroid Gland Biochemistry and Metabolism . . . . . . . . . . . . . . . . . 53 111. Spontaneous Thyroid Tumors . . . . . . . . . . . . . . . . . IV. Effect of Goitrogens and Iodine on Body Weight and Thyroid Weight., .

VI. Dietary Iodine Deficiency and Experimental Development of Thyroid

. . . . . . . , . , . . .

V. Induction of Thyroid Gland Cancer by Chemical Carcinogens

VII. Goitrogen-Induced Thyroid Gland Tumors in Mice.. . . . , . . VIII. Transplantability of Thyroid Gland Tumors in Mice. . . . . . .

I S . Thyroid-Pituitary Interrelationships, . , , , , , . , , . , . . , . . . . , . , . . . . . . . . . . , S. Effects of Ionizing Radiations in Thyroid Gland Carcinogenesis.. . . . . . .

XI. Experimental Thyroid Tumors in Rats Produced by Goitrogens and

65 68

Carcinogens. . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 1. Carcinogens and Goitroge . . . . . . . . . . . . . . . . . . . . . . . . . . 75 2. Malignant Tumors in Rats Given Goitrogens.. . . . . . . . . . . . . . . . . . . . . . 76

MI. Transplantability and Metabolism of Rat Thyroid Gland Tumors.. . . . . . 81 1. Transplantability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 2. Tumor Dependency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . , . _ . . . . . . 82 3. Hormonal Imbalance and Pituitary Cytology.. . , . . , . . . . . . . , . . . . , . . 83 4. Metabolism of Goitrogen-Produced Tumors in Rats . . . , . . . . . . . . , , . . . 84

XIII. Biochemistry of Thyroid Gland Tumors in Mice. . . . 1. Interrelationships between Thyroid Hormone (

Stimulating Hormone (TSH) . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 86 2. Biochemical and Histological Comparisons. . . . . . . . . . . . . . . . . . . . . . . . . 89 3. Competition of Host Thyroid and Transplantable Tumor for Radio-

91 4. Radioactive Iodine Uptake per Unit of Tissue.. . . . . . . . . . . . . . . . . . . . . 96 5. Factors Altering the Thyroid/Serum Radioiodide Ratio (T/S) of the

Thyroid Gland and Thyroid Gland Tumors.. . . . , . . . . , . . . . . . . , , . . . , 97 6. Effect of Thiouracil and Thyrotropic Hormone on Tumor Weights.. . . 100 7 . Miscellaneous Observations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

XIV. Thyroid Gland Cancer in Man. . . . . , , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 1. Frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 2. Histological Patterns in Benign and Malignant Thyroid Gland Tumors 103 3. Some Metabolic Characteristics of Human Thyroid Gland Cancer

. . . . . . . . . . . . . . 104 4. Use of Radioactive Iodine and Goitrogens. . . . . . . . . . . . . . . . . . . . . . . . . 104

active Iodine. . . . . , . . . . . . . . .

Tissue. . . . . . . . . . . . . . . . . . . . . . .


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Page 5. Possible Consequences of Thyroid Hormone Deficiency. . . . . . . . . . . . . . 108

XV. Summary and Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

I. INTRODUCTION The discovery of specific and extremely potent goitrogenic drugs dur-

ing the last decade provided a new and exceedingly powerful tool with which to study functional and growth potentialities of the thyroid gland. These goitrogenic drugs are believed to act directly on the thyroid gland to block biochemical reactions involved in the synthesis of thyroid hor- mone. The ensuing decreased level of thyroid hormone in the circulation is thought t o provoke an increased output of thyrotropic hormone (TSH) by the anterior lobe of the hypophysis which acts on the secretory epi- thelium of the thyroid producing hypertrophy and hyperplasia. The ingestion of these goitrogenic drugs by animals for prolonged periods has far-reaching effects on thyroid tissue-effects which could not be produced or even thought of by the technique of surgical thyroidectomy.

The ingestion of iodine-deficient diets by animals for prolonged periods also results in a comparable thyroid hormone deficit. Depending upon the severity and duration of the iodine deficiency, conditions appear t o be produced in the thyroid gland not too unlike those occurring after pro- longed goitrogen administration.

The very great affinity of the thyroid gland for iodine and the avail- ability of radioactive iodine has placed another unique tool in the hands of the investigator, making possible studies of normal and abnormal thyroid gland metabolism not hitherto attainable. Whereas this affinity of the thyroid gland for iodine is notably affected by a variety of factors, conditions can be selected so that thyroid-lethal doses of ionizing radia- tions can be delivered to the thyroid with what is thought to be only slight chance of damage to other organs or tissues. On the other hand, tracer doses of radioactive iodine can be used which are thought not t o interfere with the normal pathways of iodine metabolism in the animal. Such tracer doses are making possible studies of metabolic pathways of this chemical which had heretofore been beyond the grasp of the experimentalist.

This review is primarily concerned with data obtained during the last decade utilizing these newer techniques or various combinations of them which give insight into the mechanism of thyroid carcinogenesis and thyroid tumor metabolism. Since animal experiments merely serve as guideposts t o the study of the disease in man, this review concludes with brief comments on thyroid cancer frequency and thyroid cancer metab- olism and treatment in man; attempts have been made to compare some thyroid gland responses in man to similar responses occurring in

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laboratory animals where experimental conditions can be more precisely controlled.

11. THYROID GLAND BIOCHEMISTRY AND METABOLISM Some acquaintance with the biochemistry of the more important,

known functions of the thyroid gland appears quite necessary for a better understanding of the metabolism of the tumors of the thyroid gland, only a brief outline of which can be presented here. Recent compre- hensive reviews may be consulted for more complete details: Roche and Michel (1951); Means (1951) ; Barker (1951); Albert (1952) ; Gross and Pitt-Rivers (1952, 1953).

The thyroid gland functions as the principal organ in the body for the collection of iodine from the plasma, for the synthesis and storage of the organically bound iodine found in the thyroglobulin, and for the release of the thyroid hormone(s) into the circulation. Much remains to be learned about these unique biochemical properties of the thyroid gland as they relate t o the changes which occur in thyroid tissue during the process of thyroid gland carcinogenesis. A better understanding of thyroid gland biochemistry is especially important since most malignant tumors of the thyroid gland lose partially or completely their ability t o concentrate iodide. If such biochemical processes were more fully ap- prehended, they might not only illuminate the mechanisms involved in the development of thyroid gland malignancy but also provide more logical and effective therapy of thyroid tumors in man.

One of the long-known and well-established functions of the thyroid gland is its ability far exceeding that of all other organs to concentrate iodide from the circulation. VanderLaan and Greer (1950) found the thyroid of the rat able to increase the iodide concentration over that in serum by a factor of 25, and Taurog et al. (1951) report a concentration of iodide in the thyroid 500 times greater than that in the plasma. Wollman and Scow (1953a,b) found values of 91/1-250/1 for the mouse, and Lipner et al. (1954) observed values of 200/1 in the mouse receiving a low-iodine diet. The addition of iodide to the low-iodine diet depressed the ratio for both rats and mice (Lipner et al., 1954) to values of 30/1 and 72/1, respectively.

VanderLaan and Greer (1950) report that the degree of concentration in the thyroid is dependent on the rate of secretion of thyrotropic hor- mone by the pituitary gland. Some of the factors that may be responsible for the large variation in the ability of the thyroid to concentrate iodide are: (1) variation in hypophyseal activity of the animals used in the experiment; (2) variation in the dietary iodine intake of the animal, which may indirectly influence the hypophyseal activity of the animal;

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and (3) the possible influence of light on the thyroid or pituitary gland (Lipner et al., 1954). Although the complete mechanism of the thyroid in concentrating iodine is not known, the concentration of iodide consti- tutes the first stage in the binding of iodine by the thyroid gland. The investigations of Taurog et al. (1947), VanderLaan and VanderLaan (1947), Raben and Astwood (1949), and others indicate that the iodide concentrating power of the thyroid is not affected by goitrogenic drugs of the thiocarbamide type, but i t is inhibited in the intact animal by thiocyanate (Barker, 1936), perchlorates, and other ions (Wyngaarden et al., 1950). This concentrating ability appears t o be largely controlled by the thyrotropin secretion (Leblond et al., 1940; VanderLaan and Greer, 1950; Randall and Albert, 1951; and others). The first step in the incorporation of iodine into the bound form according to Fawcett and Kirkwood (1953a) takes place in cell-free preparations of thyroid tissue by the oxidation of iodide to iodine in the presence of cupric ion (ferric ion and HzOz were also effective oxidants), and the combination of the free iodine with free t,yrosine by an enzyme, tyrosine iodinase, to form monoiodotyrosine, although Weiss (1953) interpreted his observations on the copper requirement in thyroxine synthesis as indicating that a copper- containing enzyme was directly involved.

Taurog et al., 1955, on the other hand, found that neither copper nor tyrosine need be added to thyroid homogenates or thyroid particulate fractions in order to effect organic iodine formation because they found their cell free preparations, not reinforced with substrates such as copper or tyrosine, converted appreciable quantities of added iodide131 to mono- iodotyrosine which was bound to the thyroid protein, and was released only after hydrolysis of that protein. This latter observation, therefore, does not sustain the view that copper plays an essential role in the forma- tion of organic iodine by the thyroid gland but rather supports a prior observation (Tong et al., 1951) that iodination of tyrosine takes place within the protein molecule. The question of whether tyrosine is iodinated in the thyroid gland as the free amino acid or as part of the thyroid pro- tein thus requires further study. The next step in organic binding may occur through the process of nonenzymatic iodination of protein (Reineke, 1949) in which diiodotyrosine would be formed from monoiodotyrosine. The aromatic antithyroid substances such as p-aminobenzoic acid, sul- fanilamide, and phenol probably exert their goitrogenic effect by com- petitively inhibiting the action of the enzyme tyrosine iodinase (Fawcett and Kirkwood, 1953b).

It is now generally accepted that diiodotyrosine is the precursor of thyroxine in the thyroid gland. This synthesis could occur by the coupling of two molecules of diiodotyrosine with the loss of one three-carbon side

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chain-a suggestion first made by Harington and Barger (1927). It has been supported by the in v i t m experiments of von Mutzenbecher (1939), and Mann et al. (1942), and by the in vivo experiments of Taurog and Chaikoff (1947), but the mechanism of the in vivo incorporation is still uncertain. It appears unlikely that free thyroxine in the thyroid gland is the precursor of thyroglobulin, which is the storage form of bound iodine of the thyroid (Leblond and Gross, 1948; Tong et al., 1951). It is believed this organic binding is largely controlled by the pituitary gland because radiodiiodotyrosine conversion to radiothyroxine is depressed in the hypophysectomized animal (Morton et al., 1942), but hypophysectomy does not completely abolish the conversion (Albert and Lorenx, 1951), although it does reduce the rate. It is unknown a t the present time whether the pituitary actually controls organic fixation of iodine or merely accelerates the reactions involved in the process.

Small amounts of monoiodotyrosine, diiodotyrosine, thyroxine, and triiodothyronine existing in the thyroid gland are presumably formed by proteolysis of thyroglobulin (Gross et al., 1950). Thyroid slices contain an enzyme which is able to deiodinate both mono- and diiodotyrosine and thyroxine only slightly if a t all (Roche et al., 1951a,b). This deiodinating enzyme is inactive toward iodinated tyrosine bound in thyroglobulin. At the present time i t seems that 3,5,3’-triiodothyronine may be con- tinuously liberated from peptide linkage together with thyroxine by proteolytic enzymes present in the gland. Although i t is possible that some circulating thyroxine may be deiodinated resulting in the formation of a t least iodide and possibly triiodothyronine (Gross and Pitt-Rivers, 1952, 1953), the generally accepted view that thyroxine alone is the circulating thyroid hormone (Taurog and Chaikoff , 1948; Laidlaw, 1949) has had to be revised by the work of Gross et al. (1950) to include triiodothyronine in the term “thyroid hormone.”

The appearance of the thyroid hormone in the blood could follow the following scheme: (1) synthesis of thyroxine and triiodothyronine in the thyroid, (2) proteolytic liberation of thyroxine and triiodothyronine from thyroglobulin, and (3) the passage of thyroxine and triiodothyronine into the circulation (Gross and Pitt-Rivers, 1953). The plasma organic bound iodine in thyroxine which is loosely bound to a protein having a mobility similar to that of al-globulin (Gordon et al., 1952) probably comprises most of the plasma-bound iodine. Triiodothyronine does not appear t o be specifically associated with this protein (Gross and Pitt- Rivers, 1953). Both thyroxine and triiodothyronine are present in the circulation and tissues, and the future still holds the solution as t o whether thyroxine itself is the active hormone or whether its whole effect is due t o its conversion to triiodothyronine.

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111. SPONTANEOUS THYROID TUMORS The spontaneous development of tumors of the thyroid gland in

several species having variable incidences of the disease will be presented in this section as well as some of the predisposing factors for these tumors. Goiter has freqently been observed in dogs; and many students have commented on the relative susceptibility of dogs to thyroid cancer. Sticker (1902) reported 32 thyroid carcinomas among 766 cases of pri- mary carcinoma of dogs-an incidence of 4.1 %. Wegelin (1928) mentioned that malignant neoplasms usually developed in the goitrous gland of the dog. Schlotthauer et al. (1930) reported 54 goitrous thyroids in 234dog thyroids in animals from a goiter endemic area of Minnesota; 3.7% were carcinomatous with metastases.

According to Ivy (1947), 10 dogs of 2000 autopsied in Chicago between 1918-1925 showed evidence of local invasiveness with thyroid metastases to the lungs. He was unable to produce lung tumors in any of these tumor-bearing dogs by inserting small pieces of the diseased thyroid tissue into the external jugular vein. This failure could have arisen be- cause of the heterologous nature of the tissues used. Ivy reports an inci- dence of goiter in dogs in Chicago during the above-mentioned period of 98% and of “cancer” of the thyroid with gross pulmonary metastases of 1.6%. Goiter completely disappeared (Ivy, 1947) from the dogs in the Chicago area about 1925 following the more general use of iodized salt. Whether or not cancer of the thyroid in the dogs of this area also disappeared is unknown from the evidence available.

According to Feldman (1932), carcinoma of the thyroid appears rarely in the horse. Simple adenomas are present in the thyroid glands in a con- siderable percentage of older horses, but epithelial malignancy is rare. Woundenberg (1909) reported four cases with metastases in older animals. Wegelin (1928) was of the opinion that malignant neoplasms of the thy- roid gland observed in the horse developed in a previously goitrous gland.

Among laboratory mice Slye et al. (1926) found only 12 malignant thyroid tumors in more than 51,000 autopsies of animals averaging about 14 months of age. Demonstrable metastases were found in only 2 of the 12 tumors. I n addition, Slye et al. (1926) observed only 5 simple goiters in the same stock of mice. From the review of spontaneous rat tumors by Bullock and Rohdenburg (1917) it would seem that spontaneous tumors of the thyroid in the rat are about as infrequent as those for mice. More recently Bullock and Curtis (1930) state: “Of the many thousands of rats which have been examined post-mortem only two had developed thyroid tumors.” Both tumors occurred in female rats. A thyroid car-

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cinoma had developed in one rat 948 days of age. An adenoma developed in the other between 427 and 856 days of age.

The thyroid glands of 16 very old albino rats 801 to 906 days of age comprising six males and ten females were examined by Van Dyke (1944), who detected eight neoplasms in the females and one in the males. He was of the opinion that spontaneous adenomas exhibiting both cyst for- mation and papilliferous structure occur rather frequently in the atrophic thyroid gland of very old rats and originate from atypical glandlike cells derived from the epithelium of ultimobranchial cysts.

Because of the infrequency of occurrence of thyroid gland tumors in both the rat and the mouse these species appear t o be suitable subjects for use in the induction of experimental cancer of the thyroid gland.

Wegelin (1928) concluded that in both man and animals there is a close' relationship between endemic goiter and malignant tumors of the thyroid gland. It was his belief that invariably malignant changes proceed from benign tissue proliferation; i.e., the transition from adenomas to carcinomas occurred through a gradual alteration in the biological char- acter of the tumor cell. Wegelin believed that iodine deficiency was one very important factor simply because the thyroid gland can not store the active hormone when the iodine supply is insufficient for long periods. He agreed with the view of Marine and Lenhart (1911) that simple thyroid gland hypertrophy in the Salmonidae which can be cured by the addition of iodine represents only a simple goiter, but he also believed that the Salmonidae may develop malignant tumors as well; these tumors show infiltrative and destructive invasion of the muscles, cartilages, bones, vessels, and skin. The occurrence of such tumors might be typical instances of the development of malignant tumors from an initial benign goitrous hyperplasia. Wegelin (1928) reported that he had observed in a partially nodular stroma of an old rat, a spindle-cell sarcoma which had metastasized to the lung, myocardium, and pericardium. On the basis of the incomplete data a t that time, Wegelin (1928) believed that in all prob- ability malignant tumors of the thyroid in the animal occurred more frequently in endemic goiter districts than in goiter-free areas. He also thought i t possible to trace the histological transition from benign prolif- eration to frankly malignant tumors. Epithelial proliferation in the thyroid gland in man was recognized by Dunhill (1931-1932) as a charac- teristic response t o stimulation. He held that such proliferation may progress spontaneously to form a benign tumor or form a tumor which invades and disseminates. Histologically, according to Dunhill (1931- 1932), these stages merge into one another by insensible gradations, causing difficulty in deciding just when a tumor has become malignant.

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Although he believed that the cause of the carcinoma of the thyroid was stimulation, he apparently was unaware of the role now believed to be possessed by the pituitary in causing such stimulation.


The influence of propylthiouracil (PTU), PTU plus iodide (I), and PTU plus dried thyroid (Th) in rats of both sexes of the Wistar strain was studied by Sellers et al. (1953). The basal diet per gram contained 2 pg. of iodine. The rats were sacrificed after 15 months. The body weight of the animals receiving PTU + T h was almost equal to the weight of the controls, in contrast with animals receiving PTU alone or PTU + I, whose weight was approximately half that of the controls. This effect of a goitrogen in depressing body weight, also observed in mice of two strains by Morris et al. (1951) and Dubnik et al. (1950) and in rats by Goldberg and Chaikoff (1952), is probably not due to a direct inhibitory effect of the goitrogen, but is more likely caused by an insufficiency of the thyroid hormone. This conclusion is supported by the observations of Griesbach (1951) that in rats given thiouracil or surgically thyroid- ectomized the skeletal growth and weight gain ceased about three weeks after thyroxine secretion had ceased. Complete degrada t ion of acidophil cells of the pituitary had occurred by this time. The administration of small amounts of thyroxine to the growth-checked thyroxine-deficient animals showed a close relationship between renewed growth of bone and regranulation of the pituitary acidophil cells. The “acidophil test,” therefore, can be used to detect the presence of small amounts of thyroxine in the rat because the presence of only a few intact thyroid acini are sufficient t o maintain some acidophil granulation (Purves and Griesbach, 1946b, 1951; Goldberg and Chaikoff, 1949).

The pituitary ‘‘ basophil test ”-the appearance of numerous baso- philic staining cells of the anterior pituitary-is, on the other hand, a valuable criterion for detecting slight thyroxine deficiency in the animal (Griesbach et al., 1949, Griesbach, 1951), because when such cells are present abnormally increased secretion of thyroid-stimulating hormone (TSH) may reasonably be assumed to occur.

The increase in thyroid weight which takes place either during an absolute dietary iodine deficiency or during goitrogen administration is illustrated in Table I. Average values 100 times normal (per 100 g. body weight) were obtained by Goldberg and Chaikoff (1952) with rats given PTU, whereas values of only 3 to 6 times normal weight on an absolute iodine deficiency were observed for young growing animals by Remington et al. (1937), and for old rats by Bielschowsky (1953).

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Iodine Content of Diet


No. of Rats

300 PTU (26-32 mo.)


Age (months)

34.2 (Deficient)

960 7600

100 300


Deficient Deficient Deficient

TABLE I Relation of Thyroid Weight to Body Weight of Rats under Various Treatments

Daily Iodine Intake


1-2 for 24 days 3-6 for 24 days

15 for 24 days

- -

(+ KI for 12 weeks before autopsy)

26-32 ;: 1 33*5

- 8 8

14 - -

I 25 a Benign adenomas were noted deep within the gland. b Goitrous.

Body Wt. (g. )

350-450 134-165

284 252 276

293 250 292

Thyroid Wt.


30-42 306-1510

26.9 * 1.7 20.0 * 1.2 20.0 f 1.1

82 127.6 25.2

Fresh Thyroid Wt. (mg./100 g.

body wt.)

9 .0 230-915O

8 . 8 f 4

25.2 * l . g b 8.0 f .32 8.3 k .22

9 .5 8 .0 7 .3

28. Oc 51.0" 8.6"

U m 3 Source E! v +d

Goldberg and Chaikoff E (1952) 3

+ z U

E B Remington, et al. a L (1937)

Taurog and Chaikoff (1946) 0

r @

Bielschowsky (1953) 4 z U


C Adenomas accompanied by hyperplasia and basophilic adenomas of pituitary comprised of neoplastic thyrotrophs. Thyroid glands weighing 7-10 mg./100 g. body weight Pc-ere considered normal.

01 co

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CHEMICAL CARCINOGENS The first experimental induction of thyroid tumors by direct-acting

chemical carcinogens was apparently obtained by the application of 10 mg. of methylcholanthrene to the thyroids of rats by Esmarch (1942) and resulted in the induction of six malignant tumors in ten treated rats. Three sarcomas and three squamous epitheliomas developed in from three to eight and one-half months after application of the carcinogen.


One of the earliest reports of experimental development of thyroid adenomas in the rat by dietary procedures was that of Hellwig (1935). He fed a cooked diet of corn meal and rolled oats containing 2% calcium chloride to adult rats for a period of 140 days. Control animals received a daily supplement of 2 pg. of KI. The average weight of the thyroids of stock diet control rats was 26.3 mg., whereas the average weight of those kept on the iodine-low diet was 73.8 mg. and that of those suppplemented with KI was 40.9 mg. Two large, well-circumscribed nodules 4.5 and 3.0 mm. in diameter and one smaller adenoma were found in 16 experimental rats. Hellwig also noted that epithelial cells in the smaller nodule were higher than in the surrounding tissue and formed even papillae protrud- ing into the lumen of many acini, which suggested to him that this lesion originated in a proliferative process in which increased secretory activity had occurred. Hellwig thought the high-calcium chloride low-iodine diet caused the development of parenchymatous aden omas within local- ized areas. He apparently was unaware of the possible role of the secretion from the anterior lobe of the pituitary in furnishing the stimulation for the thyroid enlargement.

Bielschowsky (1953) has observed thyroid tumors in old rats of two strains. Simultaneous occurrence of basophilic adcnomas of the pituitary were noted which were thought t o be true tumors formed by neoplastic thyrotrophs. It seems most likely, in accord with Bielschowsky’s views, that the thyroid tumors were due to the elaboration of excessive amounts of thyrotropic hormone. The prolonged deficiency of dietary iodine in thc rats observed by Bielschowsky is thought to have resulted in the synthesis of insufficient amounts of thyroxine, the ensuing thyroxine deficiency providing the stimulus for increased anterior pituitary TSH secretion which eventually resulted in the pituitary neoplasms. Two instances of tumors were reported by Bielschowsky (1953) in resting-type thyroid glands. The low-iodine diet of those animals had been supplemented with

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KI for several weeks previous to autopsy. (See Table I.) These two tumors were considered evidence of prior prolonged stimulation. Morphologically the epithelium of these two neoplasms was rather low and contained prominent cystic follicles filled with colloid. Six instances of thyroid neoplasms in stimulated-type glands of old rats were described by Biel- schowsky (1953). The thyroids appeared hyperemic with loss of colloid, and the glandular epithelium within the adenomas was increased in height compared to the epithelium of the remainder of the gland. The patho- genesis and structure of these thyroid adenomas seemed similar to that of thyroid tumors induced in rodents by goitrogens which are known to act by blocking the synthesis of thyroxine. The degree, duration, and intensity of the stimulus were important factors in the development of these spontaneous neoplasms.

Thyroid adenomas in two rats, one an adenocarcinoma, were recorded by Fischer (1926) in a colony of rats in whichgoiter wasendemic. Both rats also had pituitary neoplasms. Wegelin (cited from Fischer) had observed only 3 adenomas of the thyroid in about 150 examinations of rat thyroid glands.

The development of thyroid tumors in a high percentage of Sherman strain female rats fed a low-iodine diet has been reported by Axelrad and Leblond (1953). The tumors developed in animals that survived one year or more, whether or not a 0.03 % level of 2-acetylaminofluorene was included in their diet either a t the beginning or after four months on the low-iodine diet. The features observed histologically in the thyroid glands of the iodine-depleted rats were classified as follows by Axelrad and Leblond (1954) :

Type 01. Hypertrophied polarized cells lining large follicles with de- creased colloid content.

Type p. Polarized cells with hyperchromatic nuclei which form nodules consisting of cysts and tubules showing varying degrees of papillary infolding.

Type y. Unpolarized light-staining cells which form solid nodules.

After supplementing the diet of rats maintained in iodine deficiency for 1; years with 21 pg. of iodide daily for 8 to 32 days the previously enlarged thyroid glands appeared normal, or were slightly enlarged with only an occasional protruding nodule. The histologically a! type cells had returned to normal and contained dense-staining colloid; 0 type nodules consisted of large colloid masses within follicular structures re- sembling, according to the authors, “colloid nodules” in man. Such nodules appeared different from surrounding tissue. Type y cells were unaffected by iodide supplementation, although follicles within y type nodules accumulated colloid. Type 0 and y tissues were considered true

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thyroid gland neoplasms by Axelrad and Leblond (1954) because of their structural distinctiveness, focal nature, and irreversibility after removing the stimulus responsible for their development.

VII. GOITROGEN-INDUCED THYROID GLAND TUMORS IN MICE The presence of thyroid gland tissue metastases in the lungs of C3H

mice was observed in 1945 by Morris et al. (1946) and Dalton et al. (1 948). Other investigators verified the presence of lung metastases in mice of (Gorbman, 1946, 1947) two inbred strains, A and C57 Black, as well as in several F1 hybrids. All the mice in the above experiments had received goitrogens for prolonged periods. Both groups of investigators interpreted their initial observations with caution, Morris et al. (1946) because the metastatic lesion resembled the hyperplastic thyroid tissue of the host after prolonged treatment with thiourea, Gorbman (1946) be- cause the resumption of a normal diet in mice exposed to the effects of the goitrogen for long periods resulted in involution of the activated epi- thelium. The presence of pulmonary metastases in mice without evidence of a primary tumor in the thyroid (Gorbman, 1947; Dalton, et al., 1948) was not seen in the rats observed by the New Zealand workers, Hall and Bielschowsky (1949), since lung metastases in rats treated for prolonged periods with goitrogens always showed signs of neoplasia. The morpho- logical evidences of malignancy in mice noted above still did not prove that a state of autonomy had been attained or that such tumors could grow in the absence of increased thyrotropic hormone stimulation pro- duced by ingestion of a goitrogen. The initial studies of Morris et al. (1946), and Dalton et al. (1948) were subsequently extended to include longer periods of thiouracil treatment in the belief that prolonged stimula- tion of thyroid tissue would result in the development of thyroid gland tumors (Morris and Green, 1951). From morphological evidence Moore et al. (1953) noted carcinoma of the thyroid in four strain A mice fed a commercial diet containing 0.8 % propylthiouracil for 18 months. There was evidence of invasion by thyroid tissue of veins in all four mice, of strap muscle in one mouse, and of the lymphatics in another. Two mice developed metastases of thyroid tissue in the lungs. These observations not only substantiated the observations of Gorbman (1946) and of Morris et al. (1951) on the role of thiourea and thiouracil in the development of carcinoma in the mouse but extended the observations to still another goitrogenic compound.

VIII. TRANSPLANTABILITY OF THYROID GLAND TUMORS IN MICE Thyroid gland tissue or lung metastatic thyroid gland tissue of strain

C3H mice ingesting a diet containing thiouracil for 16 to 20 months was

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successfully transplanted subcutaneously to younger mice also ingest,ing thiouracil (Morris and Green, 1951). Such transplanted thyroid tissue grafts were maintained by subsequent transplantation from 4 to IG serial transfers and for a maximum period of 51 to 74 months, two to three times the normal life span of the mouse. The smaller size of mouse thyroids even in goitrogen-treated animals compared to the thyroids of goitrogen- treated rats did not provide as much material for study and transfer as was available to Purves et al. (1951) in transplanting thyroid gland tissue of rats treated with methylthiouracil (CH3TU).

The experiments of Morris and Green (1951) were not carried out in such a way as to determine the earliest transfer a t which these thyroid tissue grafts would grow without the host being maintained in a state of thyroid deficiency by a goitrogen. This was bec,ause of (1) the small size of the initial inoculum used, (2) the small size of grafts a t subsequent transfers, (3) the lack of knowledge of the growth potential of such trans- plants, and (4) the lack of information on the degree of thyroid deficieiicy in the host which was essential for continued growth of the grafts. Since it was not known initially how long stimulated thyroid tissue would con- tinue to survive and grow as subcutaneous or intramuscular grafts, the ability of the tissue to grow in the absence of augmented amounts of thyrotropic hormone was not tested as carefully in the early phases of the experiments of Morris and Green (1951) as would have been desirable. Neither a t first was the importance of the iodine content of the basal food fully recognized. Even today few studies have been carried out with diets adequate except for iodine content (Bielschowsky, 1953; Axelrad and Leblond, 1953,1954; Wegelin, 1928). Although thyroid gland tumors have been produced in rats deficient in iodine, such tumors have not yet been successfully transplanted to heterologous hosts. Yet, if such grafts can be transplanted to new hosts ingesting a low-iodine diet, they might also later grow successfully in animals fed diets adequate in iodine. The pellet diet used by Morris (1944) which contains approximately 1 pg. of iodine per gram would appear t o be adequate for testing growth autonomy. The use of stock meal diet (Morris, 1944; Lipner et al., 1954) with an iodine content of 0.2 pg. per gram of diet may be even more favorable for studies of growth autonomy. Commercial stock diets vary greatly in iodine content and have a tremendous effect on iodine metabolism (Gorb- man, 1950). One explanation of the more successful transplantation of functional thyroid gland tumors to normal mice than to rats is that it may have been due to the keeping of such tissue for longer periods in a continuous state of stimulation as well as to the more highly inbred strains of animals used to develop the tumors.

Several lines of transplantable thyroid gland tumor tissue of diverse

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origin have been maintained in this laboratory (Morris, 1954) for ten years through successive serial transfers. The four main tumor lines de- scribed by Morris and Green (1951) (numbers 177, 180, 183, and 199) have been transferred through 10, 19, 19, and 12 serial transfers, respec- tively. Line 177, the first one to be transplanted, grew much slower, never grew independently of TSH stimulation indirectly augmented by thioura- cil ingestion by the host, and became extinct in the 11th transfer through failure to grow. Subline 1, derived from line 180 (Morris et al., 1951) through transplantation of a metastatic lung thyroid tissue nodule, was the most rapidly growing of any transplantable line developed thus far in this laboratory. The morphological structure of subline 1 was constant throughout 19 transfers. Histologically it appeared as an adenomatous simple tortuous cordlike arrangement of cells (Morris et al., 1951). The follicles of the adenoma were lined by a layer of simple columnar epi- thelium which appeared to contain little if any colloid. This subline re- tained little if any ability to concentrate (Wollman et al., 1951), frequently metastasized to the lungs, and was first found growing as an independent tumor in the 7th serial transfer (Morris et al., 1951). It was inadvertently lost through failure to transplant the tumor soon enough from the 19th serial generation. Other sublines of line 180 have been some- what slower growing, having been transferred through only 17 transfers a t this writing (Morris, 1954). The host animal from which tumor line 183 arose had received acetylaminofluorene (2-AAF) in its diet for 16 months prior t o the initial transfer. Tumor line 183 developed ability to grow independently of the goitrogen in the 3rd transfer generation and was the first of the above tumor lines to develop growth independence. It is still unknown whether this growth independence was due to the carcinogenic action of 2-AAF or to a more adequate test of growth inde- pendence. Tumor line 183 (Morris and Green, 1951) has been transferred through 19 serial transfers (Morris, 1954). The metabolism of a number of sublines derived from lines 183 and 180 has been studied (Wollman et al., 1951, 1953a,b), and a number of their functional characteristics have been noted which are described below.

Line 199 developed from a lung thyroid tissue metastatic nodule (Morris and Green, 1951). It has been grown through 12 serial transfers, and the functional activity in one subline has been studied (Lipner et al., 1954). Complete growth independence from augmented TSH stimu- lation during TU ingestion was first noted in line 199 in the 5th serial transfer.

Grafts from two other lines were started a t the same time as lines 177 and 180 (Morris and Green, 1951). One of these was maintained through 7 and the other through 9 serial transfers. Both were slower growing than

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any of the others mentioned above. During 1953 three other lines of grafts were started in the same way as the ones originally described by Morris and Green (1951) except that PTU was used as the goitrogen. At this writing these new grafts are still in the 1st transfer generation and appear t o be growing satisfactorily in approximately 75% of the inocu- lated animals. No other published account of transplantable tumors of the thyroid gland in mice has come to the author's attention as of this date. The experience in our own laboratory with these thyroid gland neoplasms leaves no room for doubt as to the ease of their development and propaga- tion, their progressive growth, their ability t o kill the host, their rather low and variable metastatic activity, and their ability t o occasionally invade muscle.

IX. THYROID-PITUITARY INTERRELATIONSHIPS The possible development of thyroid gland tumors after partial beta-

ray destruction of the gland was studied by Gorbman (1949) after sub- cutaneous injections of I 1 3 l in four strains of mice. The single subcutaneous dosages ranged between 10 and 1000 pc. per mouse. These ranges in dosage on a weight basis were lower, higher, and equivalent to the dosage given to rats (Goldberg and Chaikoff, 1952) which resulted in rat thyroid gland tumor development. The higher dosages of in mice as used by Gorbman resulted in the development of tumorous growths of the an- terior lobe of the pituitary and tracheal tumors, but no thyroidal tumorous growths were found.

Radiation administered to young three-day-old nursing mice following a single injection of 1 1 3 1 to the nursing mother has been investigated by Rugh (1951a). The maximum radiation dose to the nursling mouse did not exceed 35 pc. (Rugh, 1951b), most of which was concentrated in the more-radiation-sensitive thyroid gland of the nursling. Even the maxi- mum exposure did not impede body growth, yet ten months later damage to the thyroid was directly proportional t o the dose which had been ad- ministered to the mother. Rugh found the three-day-old-irradiated mouse thyroid gland incapable of enlargement by increased TSH secre- tion. The thyroid hormone level, however, most certainly was depressed, and at ten months enlargement of the anterior lobe of the pituitary had occurred, forming what appeared to be basophilic adenomas. No tumors of the thyroid gland were reported.

The development of pituitary tumors in almost every mouse whose thyroid had been destroyed by if survival was longer than 13 months has been reported by Gadsden and Furth (1953) and Furth and Burnett (1952). The thyroidicidal dose of to mice on a moderate iodine- containing diet as used by Gorbman (1950), who first noted these mouse

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pituitary tumors (Gorbman, 1949), was 200 or more pc. per mouse. That radiothyroidectomy, per se, would not lead to the development of hypophyseal tumors was suggested by Gorbman (1952). He failed to obtain pituitary tumors after complete or almost complete destruction of the thyroid, as indicated by morphological criteria, by administering 30 pc. of t o C5, mice fed a low-iodine diet for 10 days prior to, and 25 days after, irradiation. Irradiation doses of 200 pc. of t o other mice on the same dietary regimen, however, did result in the development of pituitary tumors. Gorbman (1952) concluded that radiation thyroid- ectomy was a necessary, but not the sole, factor leading to the develop- ment of the pituitary tumors in I131-treated mice, because thyroid im- plants or adequate thyroxine dosages prevented the appearance of the pituitary tumors following doses of sufficient to result otherwise in pituitary tumor development.

Some doubt about the validity of Gorbman’s (1950) suggestion was raised by Moore et al. (1953), who noted that strain A mice given PTU in their drinking water for 18 months developed chromophobe adenomas of the anterior lobe of the pituitary. Dalton et al. (1948) have also noted that some strain C3H mice developed adenoma-like structures of the pituitary after more than 15 months ingestion of a low-iodine diet con- taining TU. Mice in Gorbman’s (1952) experiments that received the low-iodine diet and 30 pc. of did not show any increase in pituitary weight, suggesting that morphological evidence may be insufficient t o detect complete ablation of the thyroid. A functional test in the form of a tracer dose of might give a more adequate estimate of the destructive action of ionizing radiations from Thus six weeks after the adminis- tration of 30 pc. of to C3H mice maintained on a low-iodine diet, Lipner, Morris, and Wagner (1954) found a nearly 50% depression of

uptake by the thyroid gland. Although there was incomplete de- struction of the thyroid six weeks after such treatment, the pituitaries were increased in weight by 25% to 35%. The administration of exoge- nous TSH daily for four weeks produced no increase in the avidity for

of two transplantable thyroid gland tumor sublines, although the tumors of one subline, 3C, averaged a 100% increase in weight.

The depressing effect of two transplantable thyroid gland tumors on the weight of the host’s thyroid gland during chronic PTU ingestion has been observed by Lipner et al. (1954). This effect is shown in Fig. 1. The weight of the host’s thyroid gland was not depressed when the tumors were extremely small, but as the tumors grew larger a much greater de- pression of thyroid gland weight was noted. This finding suggests that these thyroid gland tumors are capable of inactivating a portion of the increased amounts of endogenous TSH elaborated during prolonged

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goitrogen ingestion. The effect of the very large subline 6 tumor a t ten weeks after inoculation is especially noteworthy (Fig. 1). Rawson (1949) has observed the TSH inactivating properties of explants of normal thyroid tissue. According to Rawson the thyroid-tissue-inactivated-TSH could be reactivated by certain reducing goitrogenic agents such as thiouracil or 2-thiol.


0 3 6 9 I2 15 I 2 3 6 91.7 4 7 1 0





1060 ~

c 3

560 0 4


0 I

4 0 0 2 3 9






FIG. 1. The effect of two transplantable thyroid gland tumors, subline 3C and 6 , upon the weight of the host’s thyroid during chronic PTU ingestion. A-weight of thyroid glands in PTU-treated non-tumor-bearing mice; B-weight of thyroid glands in mice carrying tumor 3C; C-weight of thyroid glands in mice carrying tumor 6; D-weight of thyroid glands of control non-PTU-treated non-tumor-bearing mice.

Rasmussen and Nelson (1938) describe two cases of basophil pituitary adenomas in man-one in a 77-year-old male and the other in a 55-year- old female. Both individuals also had atrophic thyroids containing in one case a cystic adenoma and in the other an adenoma of mixed type in one lobe and an adenoma of undescribed morphology in the other lobe. The significance of pituitary adenomas in the development of thyroid cancer in man of the two cases observed by Rasmussen and Nelson is not apparent a t this time, but in view of the close interrelationship of the

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thyroid and the pituitary tumors noted in the rodent, it is at least sug- gestive and worthy of further study to ascertain if a similar close rela- tionship may exist in man.


The unique property of the thyroid gland in concentrating iodine to a level many times greater than that of the blood or other organs has been utilized as a means of delivering to that gland varying amounts of ionizing radiations from radioactive iodine. This uptake and retention of by thyroid tissue and the subsequent effects of such ionizing radia- tions on the thyroid and indirect effects on other organs have been ex- tensively studied during the last decade. Various effects on the thyroid glands of rats have been noted; thus atypical thyroid cells, single and in groups, were observed in adult Long-Evans strain rats five to eight months after a single injection of 525 pc. (Goldberg et al., 1950). These atypical cells showed some resemblance to the so-called Hurthle cells found in certain thyroid lesions in man. Regeneration to normal occurred with a 300 pc. radiation dose, but no thyroid epithelial elements were found in rats eight months after a single injection of 875 FC. 1131 .

The development of 8 malignant and 10 benign thyroid tumors in 9 of 25 treated rats 1+ to 2 years after a single injection of 400 pc. of 1 1 3 1

per rat (Goldberg and Chaikoff, 1952) illustrates some profound and pro- longed after-effects of treatment. This single intraperitoneal injection of was made into 3-months-old male Long-Evans strain rats that had been raised and maintained on a stock diet containing approximately 3 pg. of iodine per gram. These tumors were classified according t o their resemblance to four types of malignant human thyroid neoplasms as follows : (1) alveolar adenocarcinoma, (2) papillary adenocarcinoma, (3) small-cell carcinoma; and (4) spindle-cell carcinoma. One to nine single adenomas and nine follicular adenomas were also described.

The alveolar adenocarcinoma type appearing in rats was described as composed of closely packed large, pale, polyhedral cells only occasionally arranged in acini devoid of colloid. Pale-staining, chromatin-poor nuclei with a low mitotic activity that showed considerable pleomorphism were noted, and a metastasis was found in the lung of one rat. One tumor was found which closely resembled the papillary adenocarcinoma seen in man. Hyperchromatic nuclei showing little pleomorphism were found in the eosinophilic neoplastic columnar cells. The cervical lymph nodes con- tained a metastasis, but neither the primary tumor nor the metastasis con- centrated injected A highly anaplastic small-cell type of carcinoma was found in three rats. This neoplasm was composed of small tightly

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packed cells seldom arranged in acinar fashion. Pleomorphic hyper- chromatic nuclei showing high mitotic activity were present. Vascular invasion was noted in all three rats. Metastases were noted in the lungs, the sternum, the adrenal gland, and subcutaneous tissues. The authors report that radioautographs from sections of the pulmonary and sub- cutaneous metastases caused blackening above background only in the few acini that had redifferentiated. The major portion of the cells in one tumor resembled the spindle-cell type seen in man, and it was classified as a spindle-cell carcinoma. Acinous formation was quite rare in this tumor, and i t gave no evidence of concentrating 1131, although it did have a large metastasis in the lung. Ten benign tumors were described by Goldberg and Chaikoff (195%) under two general types: (1) fetal, and (2) follicular adenomas. Histologically these benign tumors resembled those described by several investigators in the rat (Griesbach et al., 1945; Money and Rawson, 1947; Lacquer, 1949). In the rats that de- veloped cancerous thyroids, the thyroid epithelium did not appear un- usually active as indicated by cell height, vascularity, and colloid content (Goldberg and Chaikoff, 1952). Such tissue was atypical only in contain- ing yellow pigment granules, and considerable amounts of appareiiJly viable thyroid tissue still remained. According to their interpretation no unduly high levels of thyrotropic hormone secretion occurred in the rats from the large single dose of They were of the opinion that the ioniz- ing radiation from was the carcinogenic agent responsible for the induction of these thyroid gland tumors. Subcutaneous injections of

in four strains of mice (Gorbman, 1949) in dosage ranges on a weight basis higher, lower, and equivalent t o the dosage given to rats by Goldberg and Chaikoff (1952) resulted in no induction of thyroid gland tumors. Tumorous enlargements were found in the anterior lobe of the hypophysis when doses of radiation were given sufficient t o destroy all or most of the thyroid gland tissue.

A series of eight experiments were designed and carried out by Doniach (1950) to test the influence of 1131 and other substances in inducing tumors and adenomas of the thyroid gland in the rat. He studied 1131, methyl- thiouracil (CH3TU), 2-acetylaminofluorene (2-AAF), and different com- binations of these three chemicals on Lister strain hooded rats. The

was administered intraperitoneally in two doses each of 16 p c . given 59 months apart. The period of observation extended from 10 to 16 months. A summary of the data presented in Table I1 shows that only two histologically defined tumors of the thyroid gland developed in rats receiving CH3TU + I l 3 I , and no tumors developed in the group receiving only It must be remembered that the dosage of Ilal was extremely low, although it had a striking effect on thyroid weight, reduc-

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Gland in Rats (Doniach, 1950) Summary of Effect of and Other Agents on Development of Tumors of Thyroid

Expt Treatment

Controls Controls I131


Methylthio- uracil (CHBTU)


+ 1131

Acetyl- amino- flu o r e n e (AAF)

AAF + 1131 AAF



CHITU AAF + 1131

Treat- ment Avg.


13 15 13 14

14 16


10 14 12


12 12

No. and Sex

8 M 7 F 6 M .O F

9M 8 F

3 F

4M 8 F 6 M


OF 6 M -

Mean Thyroid wt.


20.6 f 5.7 14.4 f 4.3 9.0 f 1.6 8.6 f 2.6

96.6 4 23.7 82.7 rt 29.4

32.3 f 17.laa

30.0 5 8 . 0 17.5 f 5.2 10.1 f 1.9

03.6 f 32.2

04.4 & 21.0 33.2 rt 5.8a,

Adenomatous Changes in Thyroid

t S i I - + + + :an- cer -


1 -

0 = no adenomas. + = 1 adenoma per gland. + + = 2-3 adenomas per gland. + + + = multiple adenomas. + + + + = adenomatous replacement of most of the gland. a = Thyroids of rats developing cancer excluded because of greatly increased weight.

= The weight of cancerous thyroid in group 4 was 364 mg. 0 = In group 8 the cancerous thyroid weight waa 99 mg.

ing it to approximately one-third that attained by the rats ingesting the goitrogen. The largest number of adenomas were noted in all rats receiv- ing and 2-AAF + CH,TU. Feller et aE. (1949) have reported that 10 days after the injection of 30 FC. I'31 into rats there was no interference with the ability of the thyroid to concentrate a second dose of no histological changes, and no alteration of thyroid function.

The thyroid glands of the rats used by Doniach (1950) were exposed

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t o a roughly estimated dose of radiation of 15,000 rep (roentgen equiva- lent physical) contrasted to 28,000 rep over a 10-day interval for the rats used by Feller et al. (1949). The report of Doniach (1950) supports the conclusion that radiation damage occurred in the rats exposed to I'31. The strain of rat used by the two different laboratories differed as well as did the diet, and especially the iodine content of the diet. The evidence, however, does not as yet permit a clear-cut explanation for the differences noted in rats receiving roughly comparable doses of in the two laboratories.

The total radiation dose to the rat's thyroid, which at best can be only roughly estimated, has been calculated (Doniach, 1953) by following the uptake of radioactive iodine into the thyroid and its discharge from the gland. A conversion factor for rep as calculated by Evans (1947) according to which 1 pc., per mg. tissue delivers 8.5 rep per minute, has been utilized by Maloof et al. (1952) with doses varying from 1 t o 300 p c . and by Doniach (1953) with doses varying from 5 t o 100 pc. 1131 , for estimating radiation exposure to the rat thyroid. The presence of thyroid carcinomas as reported by Doniach (1953) 15 months following treatment with 30 pc. of + CH,TU (Table 111) and the absence of carcinomas in animals treated only with CH3TU suggests an additive carcinogenic action of antithyroid drugs on the thyroid following irradia- tion with The most effective estimated range of radiation dosage for carcinogenesis was found to lie between 2270 and 16,200 rep (Doniach, 1953). The ineffectiveness for thyroid carcinogenesis of higher radiation exposures to the thyroid may be due to the greatly diminished capacity of the more heavily irradiation-damaged gland to undergo hyperplasia (Maloof et al., 1952; Doniach, 1953).

Irradiation alone a t the 5 pc. and 30 pc. levels produced adenomas in rats having a morphology similar t o the adenomas produced in rats by antithyroid drugs (Purves and Griesbach, 1946a). The irradiation dosages at 5 and 30 pc. levels of administration were also within the range found by Maloof e t al. (1952) t o interfere with thyroid function. Such an inter- ference in thyroxine secretion probably lowers circulating thyroid hor- mone and would be expected to lead to stimulation of thyroid epithelium by an increased output of thyrotropic hormone.

A dosage of 100 pc. 1 1 3 1 did not affect body weight of rats over a period of 15 months (Doniach, 1953), and a good compliment of acidophil cells was found in the pituitaries of such treated rats. The maintenance of acidophil cells has been shown to be dependent on the presence of thyrox- ine (Purves and Griesbach, 1946). These two latter observations support the view that a certain amount of thyroxine was being synthesized and released by the thyroids of the rats given the 100 pc. of Doniach

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ThB1,I: IT1 Sonic Eflects of IIJ1 and Tliiouracil ( T U ) on the Thyroid (;land and the Devdopment

of Thyroid Tuiiiora i n the Rat

Trcxatnient and Source

Controls (Dj

j Estimated ' Total

Irradiation Dosage to Thyroid G1:incl ( r w )









2 , tioo-90, om I 97 ,000

Tliyroid \Vt, (mg./100 g. body n-t.)

Std. 1 Io- Diet dinc ' 30

Diet" Daysu

15 , 0"




5 . :3

3 .8*


Histological ObscrvationsCJ

Hooded rats Listcr strain 10 weeks old used (fed re- search rat cubes). Moder- ate no. of micro adenomas in i of 9 rats (D).

3Iotlerate no. of adenomas in I9 of 20 rats (D).

Thyroid gland normal 1 yr. post-irradiation, Sprague- Daa-ley rats 100-125 g. in weight used (XI).

3Iodcmte no. of adcnonias in 3 of (i rats (D).

1 yr. post-irradiation celhilnr hypcrtrophy, swollcn cell cytoplasm, wrriation in size of cell nuclei; after 1: yr. 1 adenomn obsrrvcd (M).

A t 48 days post irradiation enlarged nucleus in thy- roid; at 1 yr. cellular hyper- trophy, colloid s p r s c , ntypicbal arid bizarre nuclei (11).

SIotlerate no. of admomas in 7 of 1 4 rats (D).

48 days post-irradiation en- larged nuclei at 1 yr. sinii- lar to 20 pc. groiip (M).

htlenonias alwiit i n 7 of 7 rats (I)).

IS days post-irradintion en- larged nuclei more numer- ous, some irregular in shape; increascd amount of connective tissue at 1 yr. Similar to 50 pc. group but more pronounced (M).

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Std. Diet




Treatment and Source Low-

IO- dine Diet"





300 pc. 1"' (M)

5 p ~ . 1131 + 30 p ~ . 1131 +

100 1131 +




TABLE I11 (Continued)

Estimated Total

Irradiation Dosage to Thyroid Gland (rep)





Thyroid Wt. (mg./100 g. body wt.)


TU for 30



-~~ ~

Histological Observationscsd

$8 days post-irradiation thy- roids virtually obliterated; replaced by dense scar tis- sue; occasional clusters of irregular hyperchromatic nuclei without cytoplasm at 1 yr. Hardly a vestige of gland tissue present (M).

nomas in 17 of 17 rats (D).

nomas in 20 of 20 rats; also 5 carcinomas of thyroid (D).

nomas in 4 of 7 rats (D).

Very numerous large ade-

Very numerous large ade-

Moderate number of ade-

(D) = Doniach (1953). (M) = Maloof el al. (1052); fed Purina Fox Chow diet post-irradiation. 0 60 days after irradiation: 11 days on low-iodine diet pre-irradiation. b 95 days after irradiation; 30 days of TU treatment: moderate iodine diet. e Doniach histological observations 15 months after irradiation. d Maloof et d. (1952); histological observations at 48, 64, 90 days, 1 and I+ yr. post-irradiation; diet

adequate in iodine following irradiation.

(1953) suggested that the diminished capacity of such irradiated thyroid glands to regenerate prevented their return to a quantitatively normal thyroxine synthesis, and thus indirectly increased elaboration of thyro- tropic hormone, which then induced thyroid cell hypertrophy without evidence of thyroid hyperplasia. The reduction in number of dividing cells following irradiation might account for the absence of thyroid adenomas in the 100 pc. treated group.

The development of neoplastic thyroid cells following irradiation is likely to be closely related to the number of post-irradiation mitoses which occur, since neoplasms generally arise in tissues comprised of actively dividing cells. The presence of a few adenomas in the CHaTU-post- irradiation-100 pc. 1 1 3 1 treated group may indicate there was additional stimulation to the few remaining viable thyroid cells by the augmented

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secretion of thyrotropin. X longer observation period in this group might well have led to the development of thyroid cancers such as were reported by Goldberg and Chaikoff (1932). This development of malignant thyroid iieoplasms of low functional activity following a single massive dose of 1131 (Goldberg and Chaikoff, 1952) may be due to the complete destruc- tion of cells that carry on normal thyroid function, namely, cells unable to concentrate iodine or to synthesize iodine-containing intermediates required in the synthesis of thyroid hormone(s) while leaving some thyroid tissue cells still capable of responding to thyrotropin stimulation. Such altered cells still may be able to utilize or inactivate thyrotropin (Rawson, 1919) elaborated by the Il3l-'' completely "-thyroidectomized animal.

Some evidence (as noted abovc in Fig. 1) in support of Rawson's (1939) observations that thyroid tissue can inactivate thyrotropin was found by a decreased thyroid size in mice carrying transplantable thyroid gland tumors while being maintained on a PTU-containing diet (Lipner et al., 1954). These tumors had a very low avidity for but apparently could inactivate some thyrotropin. This inactivation seemed to be greater in the larger tumors. Although the tumors grew in mice in the absence of thyrotropin stimulation induced by the goitrogenic drug, they grew more rapidly in animals receiving PTU and presumably subjected to increased amounts of endogenous TSH (Table 111). A much higher total thyroid radiation was obtained by Maloof et al. (1952) than by Doniach (11353) for any given dosage because of the prior iodine depletion of the thyroid glands obtained by feeding an iodine-deficient diet during an 11-day period just before injection. This treatment greatly increased thyroid I L S 1 concentration.

With observations made 60 days post irradiation in rats fed the stand- ard diet lllaloof et nl. (1952) found a 4% t o 6% #&hour uptake of a tracer dose of in all groups irradiated with 1 to 100 p~c. I13'. After 11 days on a low-iodine diet the animals that receivcd only 1 pc. of 1 1 3 1

took up 53 % of a tracer dose in 48 hours. Animals irradiated with 5: 20, and 50 p c . , respectively, took up 26% to 30% and with 100 p c . the animals took up in 18 hours only 14%. The effect on thyroid weight of the various amounts of irradiation 60 days later for the standard diet and the low-iodine diet groups is recorded in Table 111. No indication of enlargement of the thyroid mas observed in the groups irradiated with 20, 50, and 100 pc. , but there mas possibly a slight impairment in the group receiving 5 pc. and 110 impairment in the 1 pc. group. I t will be noted, too, that as little as 5 pc. administered to rats on a low-iodine diet interfered with thyroid function, yet only one adenoma of the thyroid gland was reported 13 years post irradiation.

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1. Carcinogens and Goitrogens

The experimental production of goiter in rats ingesting brassica seed diets (Kennedy and Purvis, 1941 ; Griesbach, 1941) and the decisive role of the pituitary in the pathogenesis of goiters were important basic ob- servations preceding many recent investigations dealing with the experi- mental development of thyroid and pituitary tumors. The hypothesis advanced by Kennedy (1942) that a urea derivative was the substance causing the goitrogenic brassica seed effect and his observation that allylthiourea produced goiter in rats provided a focal point for attempts to induce benign and malignant tumors of the thyroid by the simul- taneous treatment of rats with allylthiourea and 2-acetylaminofluorene (2-AAF) by Bielschowsky (1944) when neither compound alone appeared to be sufficient t o induce the neoplastic process. Bielschowsky (1944) noted that the goitrous gland was susceptible to the action of the car- cinogen, whereas the carcinogen did not affect the unstimulated thyroid; a t the same time he suggested another alternative, namely, that the carcinogenic agent only hastened a process which may occur spontane- ously in a hyperplastic gland. Bielschowsky (1945) later obtained nodular goiter in rats fed 2-AAF followed by allylthiourea. He noted that animals receiving only the goitrogen developed single adenomas, whereas those receiving both compounds developed multiple adenomas provided the goitrogen stimulation lasted more than ten weeks. The tumors induced by the goitrogen alone were considered to be benign because no metas- tases were found upon histological examination, whereas tumors develop- ing in two animals receiving both agents were considered malignant because pulmonary metastases were found (Bielschowsky, 1945). Gries- bach et al. (1945) described the induction of thyroid adenomas in rats after the long-term ingestion of a diet containing 45% brassica or rape seed. The development of such benign tumors was thought to be due to the long-continued stimulation of the thyroid by the thyrotropic hormone rather than to any neoplasia-inducing properties of the rape seed. Mul- tiple adenomas of the thyroid were obtained by feeding smaller doses of 2-AAF than Bielschowsky had used followed by CH,TU. Hall thought the active stimulus was the thyrotropic hormone acting on latent tumor cells of the thyroid induced by 2-AAF. Paschkis et al. (1948) using 2-AAF and thiouracil independently confirmed the observations of Bielschowsky (1944, 1945) and Hall (1 948).

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2. Xalignant Tumors in Rats Giwn Goitrogens

The development of malignant tumors of the thyroid in 2 of 30 Wistar strain rats after prolonged ingestion of thiourea was described by Purves arid Griesbach (194Ga). These tumors tended to invade veilis, and the observed presence of tumor cell emboli in branches of the pulmonary vein could explain the occurrence of lung metastases. The development of these lung metastases occurred after approsimately two years, which was a further indication of the low degree of malignancy of such goitrogen- induced tumors. The administration of thyroid powder after two years of thiourea ingestion to one rat three weeks before sacrifice appeared to produce an involution of metastatic lesious in the presence of colloid, indicating that thc adenocarcinomas of low-grade malignancy were still responsive to t hyrotropic hormone (Purves and Griesbach, 104Ga).

Thyroid tumors developed in 22 of 25 Wistar strain rats which received thiourea for 12 or more months (Purves and Griesbach, 1947). These tumors were of three histological types, namely, ( I ) benign adenomas, ( 2 ) adenocarcinomas, and (3) fetal adenomas. The latter were dcscaribed as nodules of solid cellular growth.” Mitotic figures wcrc observed but no iiivasioii of blood vessels nor lung metastases of fetal adenoma struc- turc were found. The tumors appeared after prolonged treatment with the goitrogen, but once developed they grew rapidly. S o certainty of difference between the two sexes in susceptibility to thyroid tumor de- velopment was discovered. Purves and Griesbach (1947) usually found nialignant tumors to be relatively large and associated with smaller tumors of benign histological appearance.

Prompt thyroid cell involution and storage of colloid occurred fol- ion-ing inhibition of pituitary thyrotropic secretion by administration of thyroid active material. There was a prompt cellular hypertrophy and signs of colloid resorption wheii thyrotropic stimulation was resumed. Thc adenocarcinomas also showed full cell involution after withdrawal of t hyrotropic hormone, arid stored colloid in acinar structures when they were present. Cellular involution without colloid storage took place on withdrawal of thyrotropic hormone in those tumors possessing only a trabecular appearance. Purves and Griesbach (1947) noted that thyroid tumors developing in rats treated with goitrogenic agents were not thc result of any carcinogenic action of the goitrogen, but were due to the long-continued intense simulation of thyroid gland tissue by the thyro- tropic hormone. The absence of neoplasia in tissues other than the thy- roid after prolonged thiourea administration was also considered hy Purves and Griesbach (19-17) as evidence against any direct carcinogenic action of thiourea. The observaiice of similar tumors by Wegelin (1928)

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and Hellwig (1935) in the thyroids of rats ingesting iodine-deficient diets also supports this view.

The effect of thiouracil administered to adult male rats of the Sprague- Dawley stock for periods extending from 10 days to 18 months was studied by Money and Rawson (1947). The well-known diffused hyper- plasia was the early change noted, but two other types of changes de- scribed as neoplastic were observed in the thyroid gland after long periods of thiouracil administration. Histologically, one type of growth resembled fetal thyroid tissue of the more differentiated type, a type also noted by Purves and Griesbach (1946a). The other type of growth observed re- sembled the papillary cystadenomas found in some cases of human thyroid disease. These papillary cystadenomas were described histo- logically as developing in four more or less well-defined stages as follows: atrophy and hyperchromatism of the acinar epithelium, localized papil- lary adenomas, hemorrhage into the adenomas with subsequent reorgani- zation, and colloid cysts which developed in some of the adenomas oc- casionally sufficiently extended as to replace most of the thyroid tissue. No histological signs of malignancy were found, and no metastases were found in the lung, kidney, or liver.

Transplants of the thyroid tissue to young rats and to the inner chamber of the guinea pig eye failed to grow. These tumors, however, could be found in the experimental animals for as long as nine months after the thiouracil had been removed. The experiments of Money and Rawson (1947) lend support t o the view that thyroid hyperplasia is a precursor of neoplasia and that it results from a prolonged and intensified action of thyroid-stimulating hormone.

The relationship between duration of exposure to thiouracil and the production of tumors of the thyroid gland was studied by Money et al. (1953). Sprague-Dawley rats maintained on a commercial diet of mod- erate iodine content were used in their experiments. Some of the results are illustrated in Fig. 2.

This diagram indicates that 25 ’% of the rats developed histologically recognizable tumors after 100 days of treatment, but 100% of the animals developed tumors of the thyroid gland only after a much longer period of treatment which approximated 500 or more days.

The histological changes produced by prolonged, indirect stimulation of the thyroid gland of the rat with dietary thiouracil and dibenzanthra- cene (DBA) (Money and Rawson, 1950) suggested that DBA had an anticarcinogenic effect in contrast to the early enhancing effects noted for 2-AAF (Bielschowsky, 1944; Hall, 1948; Hall and Bielschowsky, 1949) on goitrogen-stimulated tumors. No additional evidence was obtained by Money and Rawson (1950) that such tumors were malignant.

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In a suhsequent study, Hall and Bielschowsky (1949) administered a small dose of 15 mg. of 2--4iAF during the first week and methylthiouracil (CHaTI') throughout the experiment. The thyroids of rats receiving the c>arcinogen during the first week of the esperiment had larger and more iiunierous adrnomas up to the 64th week than did animals receiving only the goitrogen, but after 18 months of CH,TU treatment, the thyroids of both groups appeared similar. Metastases, invasion of neigh- boring structures, and penetration of the capsule were the morphological criteria used to determine malignancy. The presence of cancers in both groups at about the same time indicated that the change to malignancy occurred independently of the initiating effect of the carcinogen, Paschkis

FIG. 2. Thr r~lationship betneen the length of T U treatment and the percentage of rats developing thyroid gland tumors (Money et al. , 1953).

ct af. (194S), on the other hand, found that the simultaneous administra- tion of thioiiracil and 2--hA\F to rats not only hastened the development of tumors of the thyroid gland but increased their incidencc.

Tn T,ong-Evans strain rats fed a basic stock diet containing 3 pg. of iodine per gram and 0.2% PTT' for 18 to 32 months, progressive increase in thyroid weight with time was noted by Goldberg and Chaikoff (1952). Some thyroids increased to 50 times the size of the glands of control animals not receiving PTC, yet none of the goitrous animals appeared to develop neoplastic lesions of the thyroid other than adenomas.

d refractoriness of thyroid cells in rats treated with 2-AAF and C'H3TL- was noted by Bielschoivsky and Griesbach (1950) who found that rats injected with 5.0 pg. of DL-thyroxine per 100 g. body weight daily for the last three weeks of the experiment reacted to the hormone

Page 88: .


treatment with complete reversal of thyroid hyperplasia and pituitary basophilia. A dose of 2.5 pg. of DL-thyroxine equivalent to the normal daily requirement of their rats, on the other hand, resulted in significant dif- ferences in response between animals receiving 2-AAF plus CH3TU and those on CHaTU alone. Most of the 2-AAF-treated females receiving 2.5 pg. DL-thyroxine daily had active thyroids in contrast to the controls, but the pituitaries appeared normal. Many of the animals still had large goiters in which the epithelium was almost as high as in animals not receiving thyroxine, indicating that the pituitary had not been inhibited in most of the 2-AAF-CH3TU-2.5 pg. daily-thyroxine-treated rats. The 2-AAF, therefore, seemed to have effected some change in the thyroid not restored by the usual maintenance amounts of DL-thyroxine. The nature of the change is a t present unknown.

The prolonged ingestion of PTU combined with a relatively large amount of iodide (1.5 mg. NaI per rat per day) reduced the goitrogenic effect of the drug (Sellers et al., 1953). The average metabolic rate of the rats after 15 months was decreased more in the PTU + I group than in the PTU groups. Thyroid powder almost completely restored the meta- bolic rate. The plasma protein-bound iodide, however, in the group given PTU + I was above the value of the group receiving only PTU, and the protein-bound iodide of the group given PTU + Th was slightly below the PTU-treated controls. This supports the view of Sellers et al. (1953), that the amount of thyroid given was inadequate to replace that normally supplied by the normal gland.

The 24-hour uptake of in rats treated with PTU for 15 months, as expected, was diminished per unit of thyroid tissue. The uptake was less in animals fed PTU + T h and least for animals fed PTU + I, but when calculated on the total weight of thyroid tissue no significant dif- ference existed. Twenty-four-hour radioautographs of the thyroids showed little or no concentration of 1131 in the three PTU-treated groups and no apparent concentration of iodine in the deeply stained colloid of the adenomas.

The thyroids of all treated groups varied greatly not only in weight, as will be noted in Table IV, but also in morphology. Large areas com- prising most of the gland showed hypertrophy and hyperplasia of the follicular cells and mitotic figures to such an extent that the normal architecture of the gland was lost. Glands which weighed double or more than control glands had frequent adenomas resembling papillary cystad- enomas of human thyroids. Metastases in the lungs were found in a few PTU- and PTU + Th-treated animals. Thyroid follicles were found invading the capsule of the gland and muscle venules adjacent t o the gland; they were also found in the submucosa of the esophagus, in the

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Thyroid \\-eight i Pituitary (I%.) (mg.)

I---- -~

pulmonary artery, and in numerous alveolar vessels. The metastasizing thyroid tissue resemliled the normal gland in some iiistaiiws, whereas sometimes hyperchromatism, stratification, and frequent mitotic figures were seen. The tremendous enlargement of the thyroid in PTU + Th- treated rats was unexplained, but the suggestion was made that during prolonged treatment the amount of thyroxine present in the dried thyroid powder was insufficient t o inhibit the pituitary. The thyroid powder contained considerably more iodine than the maximum allowed

Iodine Content of Thyroid

bg./100 mg. urct wt.)

TABLE IV Observation on Rats after Prolonged Treatment with PTZi

(from Sellers et al., 1953)

36.0 27.4- 47.4! 11.8 9.2- 18.0 28.5 20.2- 39.0; 14.5 10.8- 35.7


130,4 88,4-196.4 Control



PTU + Th

I 97.7 44.6- 395.2 12.2 7.8- 17.0 79.8 40.2- 568.0 15.0 9.0- 22.4

50.1 27.6- 143.4 11.6 8.2- 15.6 48.6 32.0- 94.0' 15.3 7.8-500.0

517.1 363.8- 674.1 20.9 10.0-164.0

2 , 4 o,4- 11.4

23.7 8.3- 39,9

5,6 2,6-

Range dian Me- 1 .\re-

Range Range d i m 1 diari

PTU = Proyyl tliiouracil. I = KI. Th = Thyroid r,owder.

by the T7.S.P. for such preparations. The great enlargement of the thy- roid n-it h the development of metastasizing aderiomas makes i t appear likely that such adenomas, if not actually malignant, were potentially so, and probably the important causal factor in their formation was the continued stimulation of the thyroid by the pituitary.

A relative increase in the ehromophobic cells of the pituitary of treated rats was noted by Sellers et al. (1933), who thought such cells might have originated from chromophil cells, but in the large hemorrhagic tumors chromophobic cells were present interspered with mast cells. The pitui- taries of PTU + Th-treated rats were also significantly heavier (Table

Page 90: .


IV) than those of glands of other treated or control groups. The develop- ment of chromophobe adenomas after prolonged PTU treatment in both rats (Sellers et al., 1953) and mice (Moore et d., 1953) suggests the pro- longed depression of the circulating hormone of the thyroid as the likely causative factor.

The thyroid powder used by Sellers et al. (1953) had a higher iodine content than that of the U.S.P. product, and their experimental evidence would suggest that one explanation of the perplexing observations was that an insufficient amount of thyroid hormone(s) was administered in the thyroid powder to inhibit the TSH of the pituitary. The additional iodine intake of the animal via the thyroid powder further complicated an interpretation of the effects observed. These complex results need further clarification.

Although rats treated with 2-AAF alone for periods up to seven months do not develop tumors of the thyroid gland (Bielschowsky, 1949), i t would appear from the observations of Hall (1948) that neoplastic cells of the thyroid gland were created earlier by exposure to this car- cinogen, and that such neoplastic cells remained latent for considerable periods. The subsequent stimulation of the gland by the use of a goitrogen after 2-AAF treatment appears to cause thyroid gland tumors to develop more rapidly than they do by the effects of goitrogen stimulation alone. Partial thyroidectomy was found by Bielschowsky (1949) also to be an effective means of providing sufficient stimulation from the pituitary to result in the induction of thyroid adenomas in the thyroid remnants of rats treated for several weeks with 2-AAF.


1. Transplantability

The transplantability of tumor tissues constitutes one useful criterion for determining the degree of malignancy of a neoplasm. Unsuccessful attempts made by Bielschowsky et al. (1949) to transplant thyroid gland tumors into normal rats regardless of whether the original tumor was induced by CH3TU alone, or in combination with 2-AAF, or whether it had metastasized were good indications of the low degree of malignancy of such tumors. The tumors could be transplanted successfully, however, to thyroid-deficient hosts where hormonal conditions were similar to those existing during the development of the original neoplasms. Even though these rat thyroid tumors (Bielschowsky et al., 1949) showed evi- dences of invasive growth, ability to metastasize, and a close histological resemblance to malignant thyroid neoplasms in man, they still appeared

Page 91: .


to have a low degree of malignancy aiid were still dependent tumors, lacking complete autonomy.

The de\.elopment of a transplantable tumor of the thyroid gland from a rat \\-hich had ingested C'HxTU for 23 mouths was obtained by Purves el al. (1!)51). Part of the same inoculum placed in each of two rats rereiv- ing C'HS'I"' comprised the first transplant generation. Histological examination of the first generation grafts indicated the tumor to be an adenoma with well-developed variable acini filled with colloid. The tumor was g r o w for several transplant generations, and by the fourth genera- tion three different histological types were observed; namely, (1) micro- follirular adenoma, (2) a lightly staining tissue having a well-developed aciiiar struvture closely resembling normal hyperplastic thyroid and sur- rounded by adenomatoiis tissue, aiid (3) anaplastic carcinomatous tissue devoid of acinar structure.

The anaplastic carcinoma, (3) above (designated as TB'L), varied widely in rate of growth in different animals, but growth of the graft was much more rapid than the growth of the original transplantable tumor. Giant cells with variable numbers of nuclei were a constant fea- ture of this tumor, which Purves et al. (1951) classed as a giant cell car- cinoma of the thyroid. This tumor usually killed its host within three ivecks, although some tumor-hearing animals survived several months. These transplantable tumors attained weights of 30 to 50 g., whereas the host weight was less after removal of these enormous tumors than before inoculation, suggesting that withdrawal of nutrient from the host had omirrcd for the use of the tumor. TZe transplantable tumors were found to grow equally well in normal animals as in animals ingesting CI-I sTI', shoning that they were 110 longer dependent on increased amounts of endogenous TSH for continued growth.

2. 7' imor Depeiidericg

The transplantable tumors of the thyroid gland of the rat which devcJloped after proloiiged C'H,TU intake were dependent upon high thyrotropic hormone levels for their continued growth. Such tumors either in the thyroid gland or as grafted tumors disappeared upon the withdrawal of the C'H3TI- aiid could not be made to reappear by thc subsequent readmiiiistration of the goitrogen (Purves et al., 1931). The view was held that the goitrogeri had no influence on the formation of the ncoplastic cells which Purves el al. (1951) believed arose spontane- ously in the nornial rat thyroid. Yisible neoplasms occurred only when conditions were suitable for the growth of such neoplastic cells. The suggestion was made (Purves et a!., 1951) that the carcinogen 2-AAF acted by accelerating the formation of neoplastic cells of the thyroid

Page 92: .


gland, and the almost total absence of spontaneous tumors of the gland in normal rats could then be explained by the usual amounts of secretion of thyrotropic hormone being insufficient for the growth of the preformed cells. The opinion was advanced (Purves et al., 1951) that the continuous growth of thyroid gland tumors or their propagation by transplantation occurs by the natural selection of fast-growing types which would lead to an increase in the malignancy of such tumors. The slow-growing benign type of structure by virtue of its slow growth and low metastasizing power could not be selectively propagated by transplantation. The truly invasive and metastasizing carcinoma once it appears (Purves et al., 1951) would invariably supplant the benign type entirely after a few transfers.

3. Hormonal Imbalance and Pituitary Cytologg

The existence of a hormonal imbalance for a prolonged period could provide the stimulus (Purves et al., 1951) for the growth of a primary benign tumor from which a malignant variant could be derived possessing complete independence of the original hormonal stimulus causing its development. Such a condition, if it exists, might have considerable im- portance in human thyroid gland cancer, provided i t became possible to recognize precancerous states which could be altered by variation in hormonal levels during the precancerous period. Would i t be possible to develop a TSH assay procedure sufficiently sensitive to detect dangerous levels of circulating thyrotropic hormone which could be used to recog- nize the existence of potentially precancerous states? Additional work needs to be done on assaying circulating levels of TSH in order to estab- lish its role in the thyroid precancerous and cancerous processes. Some studies of “ thyroidectomy ” cells (Griesbach, 1951) which develop in the center of the pituitary in thyroxine-deficient rats and are derived from the “thyrotropic” cells may help to clarify our knowledge in this area. This cytological method of distinguishing sugar-containing hormones of the pituitary by means of the periodic acid-leocofuchsin test in the rat, dog, sheep, and rabbit has been used by Griesbach (1951) to show that pituitary cells which lose their hormone content and staining properties in thyroxine overdosage are the cells which hypertrophy in response to thyroxine deficiency. The basophil cells (Griesbach, 1951) are the only cell units in the pituitary which contain glycoprotein. The Gomori (Gomori, 1950) elastin tissue stain has been shown by Purves and Gries- bach (1951) to stain cells in the rat pituitary which are identical to those “basophils” to which thyrotropic function has been ascribed on the basis of correlation of glycoprotein content with content of thyrotropic hor- mone. The Gomori positive granules are discharged during thyroxine

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8-1 H.4ROLD 1’. MORRIS

defic*ienr.y, and according to Purves and Griesbach (1951) such stained granules are the storage form of thyrotropic hormone.

4. dletabolism of Goitrogen-Produced Tumors in Rats

Jlctaholism studies with transplantable thyroid gland tumor Tzz (Purvcs et al., 1951) showed that the tumor concentrated only half as much IIs’ as did the plasma 24 hours after administration. The iodine metabolism of dependent transplantable thyroid gland tumor TI in the 211d transplant generation was found to be approximately one-fifth that of normal rat thyroid. Tl tumors did not grow continually but seemed to reach a maximum weight of about 1 g. Second generation TI tumors also appeared to secrete almost sufficient amounts of thyroxine to meet the requirements of the host, although the tumor growth appeared to he depc~rideiit on the presence of unnaturally large aniouiits of TSH. The fact that such tumors reached a growth stasis when wvighing about 1 g.

TAkBI,E V Inflriencc. of TI Rat Thyroid Tuntor Sccrction 011 Pituitary Crll Composition

(i’nrvcs el nf., 16351)

Siiiiihcr .Xcidophils narophils Chromophohcs of .ktimnls (70) (%‘I ( % I

Sormal 1 0 51 2 8 i 30 1 Thyroidectoni y 4 0 1 13 3 86 7 Thyroiclrrtomy + turnor graft 3 e3 5 11 I 25 4

suggests that an equilibrium may have heen cstahlishcd twtween the tumor thyroxine elaboration and the host thyrotropic hormone secretion. The increase in acidophil and decrease in chromophobe cells of the pituitary in thyroidectomized rats bearing Tl tumor grafts (Table V) supports such a view. Functional activity indicated by radioautography in one of three types of tumors of the thyroid gland in rats was also ob- served by Money and Rawson (1950) after prolonged treatment with thiouracil.

There appeared to be no gross morphological correlation between fol- livular diameter, cell height, or colloid staining and concentration of I l o r

in tumors of the thyroid gland in rats after prolonged ingestion of the thiouracil (Money et al., 1933). Marked differences in concentration rvcrc frequently observed in identical-appearing adjacent follicles. TTn- differentiated masses of embryonic-looking cells containing a few follicles and surrounded by typical goitrogen-stimulated cells were not observed to take up in radioautographic sections. Money et al. (1953) were unable to state whether this type of tissue collects I’31.

Page 94: .


Tumors comprised of cordlike tubular arrangement of follicles with relatively little colloid present in the lumen of the follicles were observed radioautographically by Money et al. (1953) in a few instances to take up some Ii31 within the cells that make up the tubular follicles, but in no case were they found to concentrate radioactivity to the extent that macrofollicular areas did. A somewhat similar condition was noted in a transplantable malignant thyroid gland tumor, subline 1, of the mouse, which was characterized histologically by a tortuous cordlike arrangement of cells (Morris et al., 1951) and took up only n \ a t h as much in 5 hours after administration of as was taken up by the host's thyroid gland (Wollman et al., 1951).

Radioautographs of involuted follicles scattered throughout areas of hyperplasia were found by Money et al. (1953) to develop intense black- ened areas indicative of concentration, which was roughly propor- tional to the size of the colloid follicle even though some of the follicles appeared to be completely surrounded by hyperplastic tissue. The greatest uptake of 113' in tumors indicated by radioautographs were in lesions showing abundant colloid (Money et al., 1953). Heavy concenbra- tion of isotope was roughly correlated to the duration of goitrogen admin- istration. Dalton et al. (1948) noted after more than 12 months of thiou- racil treatment in C3H mice a tendency for colloid-containing follicles t o reappear in the hyperplastic thyroid gland. Their observation may have some similarity to the abundant colloid-containing lesions of rats given thiouracil for 13 to 21 months (Money et al., 1953). Massive follicular areas of tumors, however, were found unable to collect 1131, although small follicles containing colloid within the same lobe showed some functional activity. The opinion was advanced (Money et al., 1953) that many areas including single follicles and obvious primary tumors differed from normal thyroids in their response to thiouracil because such areas concentrated significant amounts of I'31 as indicated by radioautographs. The inorganic

should have been almost completely removed during histological sectioning.

The measurements of collected I'31 24 hours after administration as presented at the bottom of Table VI for three rats show in one of three animals the uptake per milligram thyroid to be five times that of rats on thiouracil for only ten days (Money et al., 1953). The other two animals, although showing a high uptake per gland, did not have any greater up- take per unit of thyroid tissue than controls treated with thiouracil for ten days (last column, 3rd line from top).

1131 in thyroid gland tumors and abnormal follicles in goitrogen- treated rats, possibly occurring as organically bound radioactive iodine, may be present in much higher concentration in localized areas than is

Page 95: .


possible in the gland as a whole. If such were the case, the possibility arises that some parts of the tissue may have escaped the thiouracil block. Some explanations for such a condition (Money et aZ., 1953; Purves et al., 1931) might include: (1) that the amount of thiouracil necessary to block 1 l 3 l concentration may differ in long treated compared to normal glands, (2) that thiouracil may not reach the cells in abnormal areas- this possibility seems unlikely because of the great vascularity of thyroid glands of rats ingesting thiouracil, and (3) that the iodinating mechanism of such thyroid glands is abnormal. Nuch additional work will have to be (wried out to adequately explain the mechanisms of the observed changes.


(Money el al., 1053) 24-Hour Collection of by Rat Thyroids

Thyroid Kcight Per Cent 1 1 3 1 / Per Cent

Trestmen t (mg.) Gland 1131/1ng.

Controls Low-iodine dirt Thioriracil

Long-term thiouracil

1 1 4 10 67 0 98 12 0 40 00 3 38

0 67 0 03 0 B i 0 01

59 80 0 16 16 88 0 01

XIII. BIOCHEMISTRY OF THYROID GLAXD TCMORS IN MICE 1 . Inferrelalionships between Thyroid Hormone ( T H ) and

Thyroid-Stimulating Hormone ( T S H )

X more detailed knowledge of the metabolism of the normal thyroid gland and thyroid gland neoplasms is needed to clarify the marly factors which are involved in the biosynthesis of TH by t’w normal thyroid gland and the partial or complete loss of avidity for iodine which occurs in many thyroid gland tumors. The variety of procedures available today for the production of thyroid gland tumors in animals provides a unique opportunity to study merhanisms of thyroid gland carcinogenesis. The direct chemical incorporation of radioiodine into the secretion(s) of the thyroid gland provides a precise isotopic label for following the functional activities of that gland during a sequence of gradual morphological changes which occur usually ill portions of the thyroid gland during the development of thyroid gland neoplasms in rats and mice. These gradual changes may best be described as a sequence of changes from the normal

Page 96: .


through hyperplasia to conditioned or dependent neoplasia and finally to autonomous or independent growth.

The reciprocal relationship between T H and TSH of the pituitary, as formulated by Salter (1940); Stanley and Astwood (1949); D’Angelo and Gordon (1949); D’Angelo et al. (1951); and others, is illustrated dia- grammatically in Fig. 3 (Kracht, 1952; Furth, 1953). Although this rela- tionship has been quite generally accepted, Goldberg (1954) suggested that the level of plasma protein-bound iodine may not be the only factor governing the output of TSH by the pituitary gland.

1 Blood thyroid hormone (TH)

(thyroxine and/or triiodothyronine)

High i Low I

1. Anterior pituitary:

1 Inhibition of TSH output

1 Thyroid:

J Anterior pituitary:

Augmented TSH output 1

J- Thyroid:

Decreased protease activity

,-Increased thyroblogulin storage

Increased protease activity 1

Hydrolysis of thyroglogulin 1

Diminished iodide uptake Diminished T H synthesis

1 -Release of T H (thyroxine)

Increased iodide uptake Increased synthesis of thyroglobulin

(triiodo thyronine)

J Declining -- Blood TH ----+ Rising

FIG. 3. Influence of blood thyroid hormone level on thyroid and pituitary gland

The most convenient experimental methods now available for the production of thyroid gland tumors exert their effect by goitrogenic inter- ference with the hormonal balance between the thyroid and pituitary glands which seemingly is initiated by a low circulating level of thyroid hormone(s). Figure 3 shows some of the events which take place when either a low or a high blood TH level occurs. The maintenance of an imbalance for prolonged periods appears necessary to implement thyroid tumor development. Pituitary hyperplasia, which may be followed later by pituitary adenomas and eventually pituitary tumors, also appears to result from the prolonged imbalance.

It appears that this thyroid-pituitary imbalance in mice can be pro- duced in different ways, (1) surgical thyroidectomy (Dent et d., 1955),


Page 97: .


(2) prolonged treatment with goitrogens (Dalton et al., 1948; Moore et al., 1953), (3) irradiation (Gorbman, 1949; Gadsden and Furth, 1953; Furth and Burnett, 1952; Dent et al., 1955). Evidence that surgical thyroidectomy in mice was about as effective in the induction of pituitary tumors as a single injection of 50 gc. of was noted by Dent et al. (1955) when they found micro tumors and other pretumorous changes in the pituitaries of surgically thyroidectomized mice which had received neither Il3' nor pituitary tumor implants.

Most of the thyroid remnants of surgically thyroidectomized mice including several bearing transplantable pituitary tumors were composed of a few typically formed follicles \vith greatly thickened epithelium con- taining much intracellular colloid. Other remnants were described by Dent et al. (1955) as sharply circumscribed nodular structures containing irregularly shaped follicles and considerable numbers of interstitial cells which were held to be thyroid adenomas not unlike those described by Morris et al. (1951) for the mouse and Money and Rawson (1950) for the rat.

Surgical depression of the thyroid was also shown by Dent et al. (1955) to be as effective as radiological depression in the conditioning of hosts to carry grafts of dependent pituitary tumors. The development of pituitary tumors in surgically thyroidectomized mice retaining thyroid remnants as well as in mice injected ni th single doses of 25 or 50 pc. of (Dent ( 7 1 al. , 1955) clearly indicate complete destruction of the thyroid is unneressary for their deyelopment or growth. The precise level of thyroid function, howewr, in mice or other species to prevent pituitary tumor formation is unknown. A long latent period follou-ing depression of thyroid actix ity is essential preceding tumor formation. Some support was gireii (Dent et al., 1955) that the latent period varies inversely with the extent of thyroid depression, but insufficient information was obtained to express such a relation in quantitative terms.

Although the precise conditions required for the induct,ion of thyroid and pituitary tumors by goitrogens in mice are undetermined (Dent et al., 1955; Moore ct nl., 1953), the deciding factor appears to be the degree of block of synthesis of TSH-a partial block yielding thyroid tumors; a more complete block pituitary and thyroid tumors; and the complete destruction of the thyroid epithelium, pituitary tumors only (Furth, 1%4). Tumorigenesis by derangement of the usual hormonal equilibrium between thyroid and pituitary might serve as an attractive model for research 011 neoplastic growths of other cells because of the relative ease and precision of quantitatively assaying the two opposing stimulating agents, TH and TSH.

Autonomous pituitary tumors produced in mice, radiothyroidwto-

Page 98: .


mized by large doses of when grafted into normal mice cause thyroid stimulation (Furth, 1954). The degree of thyroid enlargement and of thyroid adenoma formation was directly related to the length of the tumor-bearing period, and indicated excessive thyrotropin production by the pituitary graft (Furth, 1954). The stimulated thyroid glands of pituitary tumor-bearing mice were characterized microscopically by resorption of colloid, enlargement of epithelial cells, cytoplasmic colloid masses, vacuolization of the cells, and, after prolonged periods of stimula- tion, formation of increasing numbers of adenomas some of which were papillary. Radioautographs of such glands (Furth, 1954) indicated a great variability of 113’ uptake in stimulated follicles, but an almost complete lack of uptake in the thyroid adenomas.

Invasion of blood vessels by thyroid tissue and metastasis of thyroid adenomas in regional lymph nodes were rare findings, but no special search was made by Furth (1954) to detect the spread of thyroid ade- nomas. Pulmonary metastases, however, of the thyroid adenomas were not observed. The adenomas were grafted in muscle of mice bearing autonomous pituitary tumors or by intravenous injection of minute thyroid particles. Disseminated “tumor ’’ nodules developed in the lungs which exhibited the same degree of stimulation as the animal’s own thyroid gland, but Furth (1954) did not observe malignant transforma- tion thus indicating that these thyroid adenomas are still dependent tumors. If the observations of Morris et al. (1951) that successive grafts of thiouracil-induced thyroid adenomas become carcinomas can be used as a criterion the malignant transformation of thyroid adenomas induced by pituitary grafts might also be achieved by successive grafts of such adenomas in mice bearing autonomous thyrotropin-secreting tumors. Should such dependent growths (thyroid and pituitary) be called true neoplasms? Any answer i t is believed to this question will remain aca- demic until more complete knowledge has been obtained of the alterations in the hosts and in the cells which accompany formation of dependent and autonomous growths.

2. Biochemical and Histological Comparisons

Transplantable thyroid gland tumors of the mouse (Morris and Green, 1951) possess a number of important biochemical as well as histological characteristics. Some of these characteristics as investigated by Wollman, Morris, and Green (1951) have been summarized in Table VII.

The histological characteristics of the generation (Table VII) of trans- planted grafts immediately preceding the uptake measurements were characterized as follows:

Page 99: .


Sllh- linc

Avg. Wt'

Tumor (mg.)

1 2 3 4 -


Admn. and


1 1 3 1

T irn t b

Turnor- I h r i n g

IIost Given

TlJ-Con- taining Diet


131 140 50 71

57 86 98 98

5 17 18 18

TABLE VLI Summary of Characteristics of Four Transplantable Mouse Thyroid Gland Tumor Sublines

(From Wollman el al., 1051)

Off TU


14 20 20 20

Growth of

Tumor I)c-


Goitro- grri




Gener- ation

11 5 5 7

kvg. Wt Thyroid Gland (mg.1

9.9 10.2 17.6 8 . 8



I 1 3 1

Admn. Uosc in Thyroid Gland

(avg. %)

15.7 f 2.f 0 . 6 f 6.1 18 3 f 3.1 2 . 1 k 2.8


I 1 3 1

Admn. Dose

in Tumor (avg. %J)

1.21 5 0 .12 b.06 f 1 . 8 0 . 8 f 0.35 4 .4 f 6 . 5

Ratio in Thyroid to 118 ' in Tumor

202 f 75 0.1 15 ..55 k 0.23 .31 f 0.08:

1 1 8 1

Conccn- trating Power


roid Gland

to Thy-


TBh 1/10.3 112.16


% 1'31

as Thy- roxine in Tumor r

U Relative to Thy- ?'

mid Gland 3 5

E m -

0 .14 0 .57 1.10

Page 100: .


Subline 1 .-Adenocarcinoma-invasion of muscle, long tortuous tubules,

Subline 2.-Adenomatous, colloid in tissue space. Subline 3.-Adenocarcinoma, lung metastases, variable-sized follicles,

Subline 4.-Adenoma, numerous small follicles.

absence of colloid.

ligh t-s taining colloid.

These four tumor sublines were functionally different, as shown by the data in Table VII. The most rapidly growing tumor, subline 1, had prac- tically no ability to concentrate I'31. The uptake of tumor sublines 2 and 3 was & to as effective as that of the thyroid glands of the host, whereas subline 4 tumors possessed 8 the ability to concentrate 1131 as their host's thyroid. The per cent of 113' measured as the thyroxine- like fraction in tumors of sublines 2, 3, and 4 was 0.14, 0.57, and 1.1 that found in the respective thyroid glands of the tumor-bearing host. The thyroid glands of the tumor-bearing mice were intermediate in size, com- pared with those of non-tumor-bearing mice given thiouracil (TU) for three months (Dalton et al., 1948). This smaller size may in part have been due to the fact that the mice were deprived of goitrogen for two or three weeks before sacrifice, but the tumors showed progressive growth averaging from 3 to 14 times the size of the thyroid glands together with less ability to concentrate

Sublines 2 and 3 derived from the same initial thyroid tissue appeared strikingly different after five generations of transplantation-an observa- tion not in accord with the view of Purves et al. (1951) that the propaga- tion of rat thyroid gland tumors occurred through the natural selection of fast-growing types which were more malignant than the slower growing types. The mouse thyroid gland tumor sublines 2 and 3 grew a t approxi- mately equal rates, i.e., 5th generation in both cases, but they differed in both functional and histological characteristics.

3. Competition of Host Thyroid and Transplantable Tumor for Radioactive Iodine

A later and more extensive investigation of the radioiodine uptake in transplantable tumors of the thyroid gland after 9 to 11 generations of serial transplantation was carried out by Wollman et al. (1953a). Two groups, descendants of the original subline 4, were studied in the 10th and 11th transplant generations. One of these sublines, 4F, had become independent, whereas the other subline, 4HJK, was still dependent on thiouracil ingestion by the host for continued growth. Subline 3C, an independent subline-descendant of the original subline 3, was examined in the 9th and 10th transplant generations. The 10th transplant genera-

Page 101: .


tion of subline G was examined for ability to concentrate for the first time. Among the first of the many problems considered was whether any of these transplantable mouse thyroid gland tumors actively competed with the thyroid gland for radioactive iodine. A high negative correlation existed in uptake of 113' between the thyroid gland of the tumor-bearing host and that of the tumor in dependent tumor subline 4HJIC. This cor- relation is illustrated by Fig. 4, which shows that a high uptake of IL3' by the tumor depressed uptake by the thyroid gland, although there appears to be no correlation between the weight of the host's thyroid and the size of the tumor in this subline (Fig. 5 ) .


FIG. 4. T h r relation bet\\ ern tltc pcr cent of adniinistcrcd IL3' in thtt thyroid gland and that in the dcpmdcnt thyroid gland tumor, subline 4HJK, in thc same host at Levera1 intwvals after 113' administration (\Vollnian el nl., 1953a).

The influence of thyroid gland tumor subline 4HJK in depressing uptake by the thyroid may he due in part to the higher avidity of this

tumor for radioiodide. The removal of a tumor as active as this one could be expected to affect both the iodide calearance by the thyroid gland and the host's blood 1 1 3 ' concentration (Wollman, 19.53). The observed results as shown in Fig. 4 for the subline 4HJIC tumors suggest that re- moval of a tumor collecting 70% of an administered dose of I'31 would ultimately increase thyroid uptake from the low value of 5% to 70";, provided all the administered became freely available to the thyroid gland and kidney. However, according to the kinetics of clearance as described by Wollman (19.53), the expected thyroid uptake would be

Page 102: .


increased by a factor of 1 + 3 = 3.3. This obvious discrepancy is not clearly explained by the experimental observations (Wollman et al., 1953a) and must await further study for clarification.

On the other hand, an inactive independent tumor, subline 6, showed no correlation in 113' uptake between the thyroid gland of the tumor- bearing host and the tumor, as shown a t intervals of 2, 6, and 25 hours after administration of a tracer dose of (Fig. 6) (Wollman et al., 1953a). The variation in clearance of in one organ would not be ex- pected to affect uptake of the other as noted (Fig. 6) where the clearance

I I I I I I I 1 c I

w (3


* * a

** a

a TUMOR LINE 4 H J K Chronic Th iouroc i l * O f 1 Thiouroci l 5 Days 0011 Thiouraci l I9 Days


2 I- 0; 2b0 A 0 6 b O A 0 l , b O l , b O ,100 '

TOTAL TUMOR WEIGHT MG. FIG. 5. The relation between the weight of the thyroid gland and the weight of the

dependent transplantable thyroid gland tumor, subline 4HJK, in the same host (Wollman et al., 1953a).

by thyroid gland and tumor are both small with respect to kidney clear- ance (Wollman, 1953). The uptake of expressed as the percentage of the administered dose, however, increased in each transplantable tumor line studied (Wollman et al., 1953a) with increasing weight of the tumor. This increase occurred either with or without thiouracil administration to the host, as illustrated by Figs. 7, 8, and 9. This correlation appeared to hold for periods of 2,6, and 25 hours after was given. One dependent tumor line investigated, 4HJK, had a proportionately greater uptake in the smaller tumors than in the larger tumors. This is illustrated by Fig. 9. This decrease in uptake per milligram tumor when uptakes were above 30% has been described by Wollman (1953) as a kind of saturation phenomenon.

Page 103: .



b J 0 COZ

0 - J 2 0 - (3

o a


O f l I 5 g z 2: c+ 10 w

5 -



r r I I 1 r I I 1

TUMOR LINE 6 No T h i ~ u i ~ c ~ l - m 2 noVr% qtt.r

a 0 6 . ' * A2S ' - *



21 +tour, A

A a

. a 6 H o u r i

a c - . 2 nour* S

I I I I I I I i

0 200 400 600 800 1000 1200 1400 1600


FIG. 7 . The relation between 1131 content and the weight of transplantable inactive independent thyroid gland tumor, subline 6, in a low-iodine diet containing TU. The arabic number near some of the points indicates the per cent of the administered dose of I la l in the thyroid gland of the host hearing the tumor (Wollman et al., 1953a).

FIG. 6 . The relation between the per eent of administered 113' in the thyroid gland and that in an inactive independent transplantalile thyroid gland tumor, siibline 6, in the same host at several intervals after administration (\VolIman et al., 1953a).

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e - - 0




4 - -




I I 0 200 400 600 800 1000 1200 1400 I600 I800

TUMOR LINE 6 No Thlourasll e Hour$ oiter I"'

m 6 - * - A 2 5 . . .


TUMOR WEIGHT MG. FIG. 8. The relation between the content and the tumor weight of inactive

transplantable thyroid gland tumor, subline 6, in mice fed a diet adequate in iodine (Wollman et al., 1953a).


TUMOR LINE 4HJK Chronic ThiwrocI I

rn I naUr 1'''

0 5 " " * m 2 5 " : 0 4 9 -

, I I I I I I 200 400 600 800 1000 1200 1400

TUMOR WEIGHT MG. FIG. 9. The relation between the 1 1 8 1 content and the tumor weight in a dependent

transplantable thyroid gland tumor, subline 4HJK (Wollman et al., 1953a).

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4. Radioactive Ioditie I'ptake per Un i t of Tissue

Much of the variation in uptake of 1 1 3 1 in these transplantable thyroid gland tumors was removed when the uptake of radioactive iodine was expressed in terms of unit weight of tissue (Wollman et al., 1953a) (Fig. 10). I t was also rioted that the Ii31 uptake per milligram of thyroid gland usually continued to increase after administration of the isotope, but the uptake in animals ingesting TU was a t a lower level. It was further

I I I I 1 1


1 I I I I 1 0 I0 2 0 30 0 10 2 0


FIG. 10. The per rent of acln1iIiistert.d T l a ' per milligram tissue in thyroid gland and in transplantahle thyroid gland turnor, subline 6, a t several time intervals after administration. The wrtical line through each point represents the standard error. The scale For thyroid gland is on ttir Irft; the srnlc f o r thr tumor is on the right (Wollman r t a!., 1953a).

observed that the maximum thyroid content of TI--treated animals was reached prior to 6 hours after administration, whereas untreated mice had not reavhed maximum I i 3 I content by 24 hours. The uptake per milligram of tissue in the more active thyroid gland tumors, 4H,JIC, and 3C', more nearly paralleled the corresponding values for the thyroid gland but at a lowered concentration (Fig. 11) . Some evidences of binding of 113 ' by tumor, subline 6, was shown by the increase in the coilcentration of the radioiodirie up to 24 hours after administration (Fig. 10). Further indication of binding in subline 6 n-as shown 25 hours after administra- tion of the isotope by the 40-fold greater Ii31 content per unit of tissue in the tumor compared to the liver. A suggestion indicating failure of bind-

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z w a C n J 00

0 LL-

n 3.0


- - D D m m;o a J 0

- 3.0~ lo-' IT z z

TUMOR LINE 4 H J K Chrome Thiouraol OThyroid *Tumor

- 9 4


c -


n I I I I I 0

2.0 2 q

1.0 ;o g 3 00




ing, however, was the insignificant increase in concentration per milli- gram of tumor compared to the concentration in the host's liver which was noted in subline 4F for several time intervals.

was always less for the tumor than for the thyroid gland in each mouse when the uptake per milligram of thyroid tissue was compared to the uptake per milligram of thyroid tumor. The expression of the uptake values per milligram of tissue was found to be a more pre- cise way of describing the ability of the tumors to take up than was the total uptake, even though the total uptake in the more active lines was many times greater than the total 113' uptake of the thyroid gland.

The uptake of

FIG. 11. The per cent of administered 1 1 3 1 per milligram tissue in the thyroid gland and in dependent transplantable thyroid gland tumors, subline 4HJK, a t several time intervals after I L 3 1 administration. The vertical line through each point represents the standard error (Wollrnan et al., 1953a).

The ability of a dependent tumor subline, 4HJK, to take up 25 hours after administration of the isotope was also studied after the re- moval of TU from the diet of the host. There was a relative increase of the content per milligram tumor at 19 days compared to 5 days, yet during the same time interval there was a decreased activity per milligram of thyroid so that the ratio uptake per milligram tumor/uptake per milligram thyroid had increased from 0.091 f 0.006 at 5 days to 0.193 f 0.022 at 19 days.

5. Factors Altering the Thyroid lSerum Radioiodide Ratio ( T / S ) of the Thyroid Gland and Thyroid Gland Tumors

Two functions of the thyroid gland, (1) ability to concentrate iodide, and (2) ability to bind the collected iodide, can be measured inde- pendently; first, by carrying out the tests under conditions in which

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organic binding of radioiodide is blocked by the administration of an appropriate goitrogen such as PTC, and secondly, by permitting binding to occur. The first type of test measures the iodide-concentrating capacity of the t>issue over that of the blood, whereas the second measures the capacity of the tissue both to concentrate and organically bind iodide. If the radioiodide concentration of the thyroid gland tumors to that of serum (T/S) is greater than 1, then that tissue possesses ability to con- centrate radioiodide. The above procedure thus becomes a useful means of studying two separate processes of thyroid function. As pointed out elsewhere in this review, many factors affecting the concentrating or trapping proclivities of the thyroid gland or thyroid gland tumors require evaluation so that a more complete picture of thyroid gland tumor development and metabolism may be obtained. A beginning of such an evaluation has been made (Wollman et al., 1953a) in the study of the radioiodide-concentrating ability of several different sublines of transplantable tumors of the thyroid gland in mice. The T/S of thyroid glands in tumor-bearing animals measured when organic binding was Mocked varied betn-een 90 and 250. The factors causing this wide varia- tion were not completely resolved. The T/’S obtained for tumors varied from 0.4 to 60. The ratio was always significantly lower for the tumor than for the thyroid gland in the same animal. The T/S was significantly less in the independent tumor lines than in dependent lines, and the concentration of radioiodide was not much higher in the independent tumors studied than i t was in the serum. Transplantable thyroid gland tumor tissue of the mouse, when i t becomes independent, apparently loses much of its ability to perform biochemical functions characteristic of normal thyroid gland tissue, and types of tissue may arise which possess different biochemical functions. Three such types of tumor tissue of the mouse have been noted (Wollman et al., 1953b) which possess (1) ability tlo eoucentrate and organically bind iodide, (2) inability to perform either of the functions under ( l ) , and (3) inability to concentrate iodide, but ability to organirally bind iodine. Tumor tissues possessing ability to concentrate and bind iodide are the most similar qualitatively to thyroid gland tissue. That the loss in ability to concentrate iodide may occur during the transition from a dependent to an independent tumor received some support (Wollman et al., 1953b) when i t was observed that the T S ratio of the dependent tumor was higher than that of the independent tumor. The iiiterference tvith the binding mechanism in the biosynthesis of T H by the prolonged ingestion of goitrogens would be expected to reduce the blood T H to a minimum value. The feeding of the tumor- bearing animal with a low-iodine diet following such prolonged periods of TV ingestion was found after 21 days to significantly lower the T/S

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ratio of the thyroid gland below that observed in animals given a low- iodine diet for only 7 days, but the T/S ratio of the dependent tumor, subline 4J, was unaffected by the low dietary iodine regimen.

Since one of the principal regulating factors governing the biosynthesis of TH is TSH elaborated by the anterior lobe of the hypophysis, the surgical ablation of the latter gland would also be expected to have a profound effect on the concentration of iodide by the thyroid gland. It has been shown that hypophysectomy reduced the T/S ratio of the thyroid in the tumor-free mouse to a value somewhat above 50 (Wollman and Scow, 1953a,b). An almost equal depression of the T/S of the thyroid occurs, however, by administration of small amounts of iodide to mice (Lipner et al., 1954). More attention, therefore, needs to be given to the iodine intake of the hypophysectomized animal. Goldberg et al. (1953) in studying the effect of iodine intake after hypophysectomy have noted increased efficiency of extraction of iodide from the blood by the thyroid gland of the rat. Although all the factors which affect the T/S ratio are not known, it has been found (Wollman et al., 1953a) that depression of the T/S ratio occurred in a dependent thyroid gland tumor, subline 4H, after hypophysectomy of the host. The host mice had ingested TU for a prolonged period before hypophysectomy, and were fed a low-iodine diet preceding and following the operation. Although a doubling of the T/S value has been noted for the thyroid gland of tumor-bearing mice after ingestion of PTU in a low-iodine diet for 14 days, the T/S ratio of a thyroid gland tumor, subline 3C, no longer possessing ability to concentrate iodide, was unaffected. Neither was there a significant alteration in the T/S ratio value of the thyroid gland of mice ingesting TU for long periods with or without transplantable dependent tumors of the thyroid gland. It appears that the presence of some dependent transplantable tumors of the thyroid gland in mice does not affect the T/S ratio of the thyroid gland of the tumor-bearing animal.

The T/S of hyperplastic thyroids of tumor-bearing mice in which organic binding of iodide could take place was 15 to 20. The T/S of 12.8 of the transplantable tumor in one dependent tumor, subline 4H, was almost equal to the T/S ratio of 15 for the thyroid gland in the same host. The T/S ratio for several independent thyroid gland tumor sublines was significantIy less for the tumor than for the host’s thyroid when organic binding of iodide could occur. The inability to maintain a concentration of iodide above that in the serum, as found in two independent tumor sublines 3C and 6 (Wollman et al., 1953b), suggests that the iodide in such tumors is largely extracellular. Several nonthyroid tissues also lack ability to concentrate iodide (Stevens et al., 1950). Although it was also found that six different transplantable tumor sublines in mice in the ninth

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to eleventh transplant generations incorporated iodide into organic com- bination, the amount of bound per milligram of tissue was in each subline less than that in the thyroid gland. The concentration of I 1 3 l in the thyroid gland tumors of dependent tumor line 4H, when binding could occur was approximately equal to that occurring in the thyroid gland, but the organic binding in the tumor of this line was less than in the thyroid per unit 15 eight of tissue. This could conceivably indicate an impairment in the binding mechanism. The lower concentration in the tumor compared to the thyroid in several other tumor sublines in- vestigated may esplain a t least a part of the impairment in binding.

6. E$ect of l'hiozcracil and Tkyrotropic Hormone on Tumor Weights

Several different factors may affect the weight of transplantable thyroid gland tumors of the mouse (Morris, 1952; Wollman et a,?., 1953a; Lipner el al., 1954). Some of the data are presented in Table VIII. Tumor grafts of sublines 4 and 4F grew to a larger size in mice ingesting thiouracil than they did in control groups not ingesting the drug. The weight of subline 6 tumors, however, did not appear to be influenced by thiouracil ingestion. These observations raised the question as to whether or not the growth of some tumor sublines was increased by TSH. Obviously, although exogenous TSH obtained from another animal species was not the best material to use to test such an effect, i t was the only way pres- ently available and, therefore, was used by Lipner et al. (1954) to study the effect of the hormone in two sublines. The results as shown in Table VIII mere equivocal. When the thyroid gland was partially ablated by 1 1 3 1 prior to tumor inoculation and then followed by exogenous TSH administration, similar results for the same sublines were obtained (Table VIII). The results to date suggest that some transplantable thy- roid gland tumors i n mice are stimulated, while others are not, by the pituitary thyrotropic hormone. Additional work must be done on factors affecting the growth of thyroid gland tumors before arriving a t final conclusions.

7 . rlliscellaneoids Observations

The frequently observed decreased ability of thyroid gland tumors iii man and ariinials to concentrate iodide could mean that the pathway of synthesis had been altered in the tumors. One test on transplanted thyroid gland tumors in mice has been reported (Morris, 1952) in which the same compounds were qualitatively identified from the mouse thyroid gland tumors that were present in the host's thyroid glands. Th;s was true in one dependent and one independent subline. It is still too early to predict, however, that the pathway of synthesis of iodinated compounds in the thyroid and the thyroid gland tumors may qualitatively be the same.

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TABLE VIII Average Weight of Transplantable Mouse Thyroid Gland Tumors after Various


Tumor Subline Average Weight of Tumors (mg.)

No. of No. of Tumors Thiouracil Tumors No Thiouracil Source

6 30 405 k 373 41 554 f 460 Wollman et al. (1953a) 4F 15 188 f 10 14 87 f 92 Wollman et al. (1953a) 4 10 641 & 115 15 260 f 38 Morris (1952)


TSH Injections for 33 days No TSH

4JW 7 10.4 f 3 . 1 7 19 .4 f 5.4 Lipner et al. (1954) 3 c 7 23.7 f 6.7 7 10.6 f 2.1 Lipner et al. (1954)

- Partial Partial

Thyroidectomy Thyroidectom y + TSH - 33 days No TSH

4JW 7 14.2 2.5 7 30.1 f 11.9 Lipner et al. (1954) 3 c 7 20.3 f 4 . 8 7 10.0 f 2.0 Lipner et al. (1954)

Does the removal of thyroid gland tissue from its normal site by autotransplantation alone alter the iodide-concentrating properties of the transplanted tissue? This question was studied by Wollman and Scow (Wollman et al., 1953a), who report the uptake in a subcutaneous autotransplanted lobe of the thyroid gland of the rat to be only 70% of that present in the intact lobe. Lipner et al. (1954), on the other hand, found no significant difference in uptake in the mouse between the intact lobe and that portion of the lobe transplanted to a subcutaneous site for various periods up to three months after transplantation (Table IX). The mice received PTU in a low-iodine diet during the above- mentioned periods so that the experimental conditions were similar to those in which thyroid gland tumors develop (Morris and Green, 1951). In a subsequent report Wollman and Scow (1955) found no significant difference in the radioiodide clearance from the blood by thyroid lobes of the rat in situ and autotransplanted. Neither did they find any signifi- cant difference in response of such thyroid tissue to hypophysectomy or to ingestion of PTU.

The decreased ability of thyroid gland tumors to concentrate iodide compared to the capacity of the thyroid gland to concentrate iodide may represent an actual loss in the capability of the tumor to maintain a con- centration gradient much higher than that of the serum iodide. If such a

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TABLE I S Effect of Xutotransplantation on the Functional Status of Thyroid Tissue in the


?iumtwr Period after IVeight Intact Weight of T/S T/S

Animals (months) (w. 1 (mg. 1 Lobe plant of Transplantation Lobe Autotransplant Intact Autotrans-

4 1 12.8 k 2.0 1 .6 rf: 0.9 179 i- 11 170 f 18 5 2 1 4 . 4 rf: 1 . G 3.0 C 0 . 8 198 k 10 177 rf: 23 !I 3 17.2 rf: 2 . 3 8 . 8 k 1 .5 195 i- G 212 & 9

condition exists, it might also decrease the rate of organic binding of iodine.

These few studies on the metabolism of transplantable thyroid gland tumors in mire serve to emphasize the great biochemical differences in surh tumor tissues. Further elucidation of the causes of these biochemical differences must await future investigations.


1, Freqiwncy

Cancer of the thyroid gland, although comprising a relatively small percentage of human cancer, occupies a unique position for the study of mechanisms of carcinogenesis hot h in man and animals. The enormous capacity of the normal thyroid gland tissue to concentrate iodine and to utilize that iodine in metabolic functions provides a tissue wherein not only anatomical but also extremely sensitive functional changes can be detected and studied simultaneously. Many of these functional changes may he studied by the use of tracer amounts of radioactive iodine. Larger or caiiceroriclal doses of irradiation, on the other hand, can be delivered to those thyroid gland cancers in man which possess sufficient avidity for iodine. Such large doses of 113* provide a means of administering internal ionizing radiations to specific areas with what is thought to be minimal danger of injury to other body tissues.

In a study of frequency of cancer in man, i t should be pointed out that as a c’ause of morbidity and mortality, the position of cancer has changed greatly during the first half of the twentieth century and is still changing. The ratio of cancer of a given site to those of all sites has changed rela- tively little, however (Sokal, 1953). It is possible by the use of such a ratio, therefore, to compare statistics of different years and to some extent for diverse population groups, whereas incidence and mortality rates are less suitable (Table X). The conclusions arrived a t by the study of the

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TABLE X Frequency of Thyroid Cancer in Man as a Percentage of All Cancer

Summary by Sokal (1953)

Clinical Frequency Cause of Death

Range Average Range Average (%) (%I (%) (%I

Clinical data 0.3-0.8 0.56 Mortality statistics 0.4-0.6 0.41 Autopsy statistics 0.0-1.3 0.47 Aggregate 0.56 0.44

statistics of the occurrence of cancer of the thyroid gland in terms of what might be expected in a “typical” community of the United States comprising a population of one million afford a convenient illustration. In such a community according to Sokal (1953) there would be 25 pa- tients with thyroid cancer with one new case appearing every month; there would be one death bimonthly; one or two cases out of six would be studied at autopsy. Certain surgical statistics indicate a high incidence of nontoxic nodular goiters to be malignant a t operation. Such statistics appear to be in disagreement with figures presented by Sokal, but they may be explained by the fact that nontoxic goiters reaching the operating table comprise a highly selected group not representative of the popula- tion a t large-a point emphasized by Crile and Dempsey (1949). Accord- ing to Sokal less than 1% of unselected nodular goiters are malignant.

2. Histological Patterns in Benign and Malignant Thyroid Gland Tumors

Meissner and McManus (1952) have classified histologically 500 thy- roid gland adenomas and 200 thyroid gland cancers. It will be noted from their tabulations (Table XI) that the follicular group of benign tumors is common, whereas the group having a papillary structure is rare (ratio 17/1). The reverse is true in the malignant tumors (ratio 1/2). The age a t which benign and malignant tumors arise was not statistically sig- nificantly different. There is no demonstratable relation between nodular goiter and the common papillary cancer of the thyroid (Crile, 1953); this supports the idea expressed by Crile and Dempsey (1949) and by Crile (1953) that in man most thyroid gland cancers are malignant from their onset and do not arise from a preexisting benign phase. Such a view was not held by earlier investigators (Wegelin, 1928; Dunhill, 1931-1932) and is a t variance with most animal studies on the experimental induction of thyroid gland tumors.

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T.IRI,E XI Benign and Malignant Thyroid Tumors in Man

Benign Adenomas Carcinomas

Description (%) (%)

Follicular Embryonal Fetal Simple Colloid Hurthle-cell

Papillary Unclassified


86.6 (13.0) (52.3) (12.2)

( 3 . 8 ) ( 5 . 2 ) 4.8 8 . 6


2 7 . 0 (3 .5 )

(16.0) ( 2 . 5 ) (0) (5.0) 50.0 2 3 . 0


3. )Come lllrfabolic Characteristics of H u m a n Thyroid Gland Cancer Tissue

Little correlation was found between malignancy and transaminating activity of human thyroid tumors by Awapara (1952), who noted in the same hpecimen a tendency for tumor tissue to have more transaminating activity than normal tissue. The number and concentration of free amino acids as well as the transaminating enzyme were higher in samples having more ~11s .

S o hiJtologica1 changes in the thyroid tissue of man were noted by Freedberg el nl. (1932) 7 days after doses of 17 and 20 mc. of Central destrurtion of the gland was noted 14 and 24 days, respectively, after the administration of 39 and 26 mc. of I131. Up to periods of one year thyroid glands showed increasing amounts of fibrous tissue after dosage. The amount of fibrodis depended on the length of time following treatment. I t is be l i e id more studies on thc metabolic rharacteristics of human thyroid gland cancer tissue would give valuable aid in treating the disease in man.

$. I'se of Radioactive Iodine and Goitrogens

C'nncw of the thyroid gland in man as noted above is not high in com- parison with other types of caticer with which man is afflicted, and only a small proportion of those patients with malignant thyroid cancer benefit from treatment with ionizing radiations from radioactive iodine. Although the effert thus far produced by the introduction of this method of treat- ment on the general problem of the management of patients with cancer is probably insignificant (Smithers, 19531, nevertheless i t has an interest out of all proportion to its success as a tool in rancer treatment because it permits achievements in therapy never before possible. Those few

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patients who are now alive, and apparently well, who would otherwise have died are living testimonials to the importance of the use of radio- active iodine in treating the disease.

Radioactive iodine is not needed for the treatment of the less malig- nant localized tumors whose structures and function approach more nearly that of the normal thyroid. It has been found ineffective for the more malignant undifferentiated types (Smithers, 1953). The present value of radioiodine in the treatment of cancer of the thyroid gland lies in its use in treating a few patients having well-differentiated tumors which have advanced locally to the point of inoperability or which have metastasized. The future use of this new weapon in the fight against cancer in man would appear to depend on the discovery of ways of in- creasing the avidity of thyroid gland tumors for I'31. Removal of func- tional tissue has been tried by Dobyns and Maloof (1951); Freedberg et al. (1951); Seidlin et al. (1948); Rawson et al. (1948); Rawson et al. (1951); and others in attempts to increase collection by thyroid tumors in man. Dobyns and Maloof (1951) tried to increase uptake by thiouracil; Rall ef al. (1951) by a combination of total thyroidectomy and thiouracil or by thyrotropic hormone administration (Trunnell et al., 1949). These attempts have, more often than not, been unsuccessful in significantly increasing the number of cases materially benefited. The theories underlying the use of thiouracil were: (1) that the patient may be reduced to a state of iodine deficiency so that the avidity for iodine of any tissue remotely capable of functioning as thyroid tissue would be greatly enhanced; (2) the goitrogen may augment endogenous TSH elaboration, which would increase functional activity of the cancerous thyroid tissue; and (3) the action of endogenous TSH on the tumor may be augmented. The thyroid gland in man appears to function like that of the rat when thyroid hormone synthesis is inhibited by antithyroid metabolites, since the human thyroid gland is able to collect appreciable quantities of radioactive iodine after virtually complete inhibition of thyroid hormone synthesis by antithyroid medication (Stanley and Astwood, 1948). It is further believed that the collected iodide is in the form of iodide ion because the administration of thiocyanate ion or large doses of iodide promptly discharges the collected iodide from the thyroid gland.

was injected into rats and mice point to the possibility that the use of in treating thyrotoxicosis in man could eventually prove carcinogenic. Treated human patients probably would need to be followed for 15 to 25 years, however, to substantiate the danger and to determine whether or not a higher proportion of such patients receiving 1 1 3 1 develop thyroid carcinoma.

Animal experiments in which

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Precautions need to be taken in the use of radioactive iodine in the treatment of thyroid gland cancer in man. Some of the precautions advo- cated (Pochin, 1932; Wayne, 1932; Doniach, 1953; Ramson et al., 1953) inclutle: (1) treatment of thyrotoxic patients under 45 years of age with 1 1 3 ' only when other methods of treatment are contraindicated; (2) the use of a minimal dose of to produce remission only after a period of 1015- iodine intake; (3) treatment only of cases in which tracer doses of I131 indicate tumor uptakes sufficient to produce therapeutic effects; (4) iiistitution and maintenance of thyroxine medication after thyro- toxic symptoms have been relieved; (5) avoidance of doses so large that serious body irradiation occurs; and ( G ) avoidance of use of goitrogenic drugs a t any time after radioiodine therapy.

It is suggested that a number of procedures to improve the uptake of 1 ' 3 1 by tlie thyroid might he explored with the intention of decreasing the minimal effective dose of II3' required to obtain a therapeutic effect. .imong these are drastic reduction of iodine intake accomplished by low food iodine lei-el for several weeks followed by a 24-hour fast just prior to administration, the use of cyanate or chlorate ions to discharge inorganic iodide, and the use of hesperidine methyl chalcone (Reilly el al., 19.52).

It frequently happens (Egmark et aZ., 1953) after total thyroidectomy, without any suhstitution therapy, that a hormonal balance takes place in man which sometimes lasts for several years with no evidence of mysedenia. I t is presumed that the metastatic lesions in such cases take over the functional activity of the thyroid, and in some subjects appear capable not only of taking up iodine but of converting it to thyroxine. One of the first steps followed by Rawson et al. (1953) in treating pa- tients irhose tumors exhibit a minimal or no thyroidal function is to subject suc*h patients to total thyroidectomy either surgically or with l I 3 ' . .ipprosimately 5 0 7 of their patients after such treatment were ob- served to develop a significant capacity to concentrate radioactive iodine in one or more metastases within six months. Some patients after total thyroidectomy have been treated with thiouracil, and the subsequent increase in thyroidal function observed has been attributed (Rawson et aZ., 1953) to an increased secretion and availability of thyrotropic hor- mone. One third of 1.3 patients treated with exogenous TSH have been observed to increase or develop significant function in one or more meta- static lesions. After more than ten years experience with the use of I I 3 l in the treatment of thyroid canter in man, Rawson et al. (1953) recom- mended its use only in those patients whose lesions cannot be removed hy a competent surgeon, and then only when a tracer dose of indi- cates the cancer would concentrate a cancerocidal amount of without

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serious damage to extrathyroidal tissues. Patients with metastatic car- cinoma of the thyroid who are accepted in some clinics (Rawson et al., 1953) have their tumors evaluated for ability to concentrate radioactive iodine by use of tracer doses of The amount of the tracer dose ex- creted after 48 and 96 hours is measured, in vivo, over the gland and over the metastatic lesions. The normal gland is ablated, followed by tracer doses every four to six weeks for three to six months. At that time thiouracil is administered in doses of 1 to 1.5 g. daily. Radio-tracer studies are made every six to eight weeks 48 hours after stopping thiouracil administration. If less than 40% of the administered dose is excreted in 96 hours, the patient becomes a candidate for radioiodine treatment,. A second but larger test dose of I I 3 ' is given. Daily urinary excretion and


(Rawson et al., 1953) Some Results of Therapy in Man

No. Patients % Comment

10 28.5 Showing sustained objective improvement 10 28.5 Showing transient objective improvement 8 23.0 Failures 7 20.0 Treatment too recent for evaluation - -

Total 35 100.0

blood levels are determined, as well as in vivo measurements of radio- activity a t 48 hours over the known tumor area. From a graphic plot of blood levels and retained there is calculated the amount of radiation that will be delivered to the hemopoietic system and other extrathyroidal tissues. Attempts are made to deliver less than 400 rep to the blood (using a factor of 10.2 rep per gram per day). If the calculations and estimated size of the tumor indicate that a cancerocidal dose of 1131

can be delivered to the tumor without causing the patient serious harm, the maximum safe dose of radioactive iodine is administered.

The same determinations of urinary, blood, and tumor radioactivity are made after a therapeutic dose as before. Careful checks are made on the peripheral blood for three weeks after 113' therapy for evidences of injury. Using the above rather rigid criteria in selecting and treating cancer of the thyroid with radioactive iodine, Rawson et al. (1953) report that (Table XII) about one half of these cases showed sustained or tran- sient improvement-an indication that certain cancers of the thyroid

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gland can be a t least partially destroyed with The opinion was ex- pressed (Rawson ~1 al., 1953) that a large percciitage of the thyroid gland ('anrers treated by the procedures described above had been made to assume some functions of normal thyroid tissue.

5. Possible Consequeiices of Thyroid Hormone Deficiency The report of IIerrmann (19.51) of two patients with exophthalmic

goiter who had received methylthiouracil medication over a long period (3 to 4 years) arid who developed aderiopapillomatous carcinoma and hemarigioenciothelioma, respectively, deserves careful consideration. The two patients observed by Herrmann came from an endemic goiter area. and therefore, may have lived for many years on insufficient iodine. which probably resulted in a prolonged thyroxine deficit,. The thyroxine deficit would also have continued during goitrogen adminis- tration. Herrmaiin (1951) was of the opinion that the occurrence of the adenomas resulted from excess elaboration of thyrotropic hormone. Two rases also of neoplastic changes in the thyroid glands in man after pro- longed treatment with thiouracil \yere reported by Money and Rawson (1947). The histological examination of the thyroid of one patient after ten months of the thiouracil treatment for Graves' disease showed a dis- crete nodule which proved to have localized changes similar to some of those observed in thiouracil-treated rats. The changes observed consisted of intrafollicular growth of undiff ereiitiated tissue projecting into the lumina of several follicles.

Thyroxine deficiency may also occur if sufficiently large doses of 1 1 3 1

are given so as to almost completely ablate the thyroid gland. Unless such treatment is followed by an adequate thyroxine medication, the thyroxine deficiency could possibly result in an excessive secretion of thyrotropic hormone.

Since the effective dose range in rats appears to be comparable with the therapeutic dose range in the thyrotoxic thyroid gland in man ill so far as its biological effects in reducing thyroid size without actual gland destruction or gross decrease in thyroxine synthesis is concerned, if the dosage of 113* used in the treatment of exophthalmic goiter in man (Doiiiach, 1953) rendered the patient euthyroid without producing an irreversible interference with thyroxine synthesis, the carcinogenic danger from its use would likely be very slight. If, on the other hand, future thyrotropic hormone studies show an augmented and persistent thyro- tropiii secretion above normal after radioiodine treatment, the carcino- genic danger may be much greater. From the evidence available i t would appear reasonable to believe that any dosage of in man which suffi- ciently interferes with thyroxine secretion so as to lower the circulation

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level of this hormone below normal might well augment secretion of TSH unless followed by adequate thyroxine medication.


The occurrence of spontaneous tumors of the thyroid gland in rats and mice is exceedingly rare. Thyroid gland tumors, however, have been produced in these laboratory animals by several different methods. There is ample indirect proof that such tumors occur when the thyroid gland tissue is subjected to continuous prolonged stimulation by increased amounts of thyrotropin evoked by a deficiency of circulating thyroid hormone. Such neoplasms in rats and mice appear to develop through a series of gradual changes (Dubin, 1953) which include (1) hyperplasia, (2) local areas of proliferation of altered cells, (3) formation of adenomas, and finally (4) development of carcinomas. The amount of thyrotropin required to produce the necessary stimulation is unknown.

The essential techniques used to produce experimental tumors of the thyroid gland include (1) chronic ingestion of a thiocarbamide type goitrogen by both rats and mice, (2) the direct irradiation of the thyroid gland (of rats only) by a single massive dose of radioiodine, (3) the chronic feeding of an iodine-deficient diet to rats, (4) a combination of a goitrogen with low doses of radiation in rats, and ( 5 ) a combination of the carcinogen 2-acetylaminofluorene with a goitrogen. The fifth proce- dure has been found unnecessary but may serve to accelerate the process in the earlier stages.

The initial tumors produced by goitrogens depend on thyroid hor- mone deficiency for continued growth. After a variable number of serial transfers to TH-deficient hosts some, although not all, of the transplant- able dependent tumors become independent of the stimulus causing their development. Numerous attempts to transplant experimentally induced thyroid gland neoplasms of the rat have frequently been unsuccessful except when made into thyroid-hormone-deficient hosts, although ex- ceptions to this general experience were those obtained (Purves et al., 1951) in methylthiouracil-induced thyroid gland tumors. A partial ex- planation of the frequent failure to obtain independent transplantable tumors of the thyroid gland in rats may be due to the use of noninbred stocks of animals. The production of thyroid gland neoplasms in the mouse following prolonged chronic ingestion of goitrogens has received ample verification, although transplantation has been obtained only in the CSH strain. Nevertheless, i t is believed other strains of inbred mice when adequately tested will also prove capable of carrying transplantable thyroid gland tumors. Evidence also has been obtained that thyroid

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gland tumors develop in mice from the direct stimulation from TSH p r o d u d by functional pituitary tumors.

The conditions most farorahle to the development of independent or autoiromous thyroid gland tumors have not yet been completely appre- hended. -1vailable studies support, the view that much variation occurs in experimentally produced thyroid tumorsjn both rats and mice. A long bu t8 also 1-ariable induction period exists. Once developed, such tumors show progressive growth, ability to kill the host, rather low and variable metastastic activity, occasional invasion of surrounding tissues, and a reduced capacity to concentrate iodine compared to that of the normal thyroid gland tissue.

have produced malignant thyroid neoplasms in rats. 111 such neoplasms the induction period was long, and thyroid gland epithelium did not appear unusually active, indicating the absence of unduly high levels of thyrotropic hormone secretion. 7'hc i o n i h g radiation from 1131, therefore, appeared to be the dire(-t initiating mrcinogenic agent responsible for the formation of the thyroid neoplasms. Dosages of ionizing radiation lower, higher, and equivalent on a weight basis to the cancerogenic dosages of ionizing radiation administered to rats did not induce thyroid gland tumors in mice. Administration of sufficient radiation to mice to destroy all or most of the thyroid gland tissue, however, did produce tumorous en- largements of the anterior lobe of the hypophysis.

Low doscs of Ill1 (30 pc.) plus the goitrogen niethylthiouracil pro- duced thyroid gland carcinomas in rats, whereas such neoplasms were absent in rats receiving the goitrogen alone. The stimulation to thyroid tissue by increased thyrotropic hormone secretion produced during goitrogen ingestion appears to have an additive carcinogenic effect on the rats' thyroid gland when combined with low doses of IL3' irradiation. Such a low range of irradiation has been found to interfere with thyroid function ; one explanation may be that if the interference is sufficient to lower circulating TH, the increased secretion of endogenous TSH which might he expected to occur would lead to stimulation of thyroid epithelium, thus resulting in conditions conducive to thyroid gland cwvinogcnesis.

The direct chemical incorporation of radioiodine into the secretion of the thyroid gland or of functional thyroid gland tumors provides very precise labels adaptable to the correlation of the functional activities of the gland and of its tumors simultaneously with histological changes k n o ~ v n to take place during the transition of the thyroid gland to a thyroid gland neoplasm. Several transplantable thyroid gland tumors varied from practically no ability to concentrate radioiodine in some

Single doses of ionizing radiations from

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independent tumors to about one-half the concentrating capacity of the host's thyroid gland in some dependent tumors. A high uptake of in some dependent tumors depressed 113' uptake by the thyroid gland, although the thyroid gland weight in such instances was not related to tumor size. Some almost completely functionally inactive independent tumors showed no correlation in uptake between the thyroid gland of the host and the tumor. The uptake of 113' increased in several trans- plantable tumor lines with increasing weight of the tumor, and some dependent tumors had a proportionately greater uptake for smaller tumors than for larger ones. The uptake per unit of tumor tissue was much less variable than the total uptake because of the large differences in tumor size. The uptake per unit weight of tumor tissue of the most active tumors also more nearly paralleled uptake of the thyroid gland than did less active tumors, but all tumors studied in both rats and mice possessed less capacity to concentrate than did the thyroid gland.

Ability of the thyroid gland and the tumor to concentrate and or- ganically bind the collected iodide has been demonstrated in several transplantable tumors of the thyroid gland in mice. The thyroid to serum radioiodide ratio (T/S) was always significantly lower for the tumor than for'the thyroid gland in the same animal. The T/S was also significantly less in the independent tumor lines than!in dependent lines, and the radio- iodide concentration in the tumor of several independent tumor lines was not much above that in the serum. The evidence a t present indicates that transplantable thyroid gland tumor tissue of the mouse and rat when it becomes independent loses much of its ability to perform certain bio- chemical functions characteristic of normal thyroid gland tissue. Some mouse tumors possess ability to both concentrate and organically bind radioiodide; other tumors could bind but lacked ability to concentrate the iodide; whereas still others lacked both functions. Organic binding of

in several transplantable thyroid gland tumors was always less per milligram of tumor tissue than that in the thyroid gland. One dependent tumor possessed approximately equal ability with the thyroid gland to organically bind Of several factors studied hypophysectomy pro- duced the greatest depression of the iodide-trapping capacity in both the thyroid gland and the tumor.

Pituitary thyrotropic hormone stimulated the growth of some trans- plantable thyroid gland tumors of the mouse, whereas the growth of others was unaffected by the hormone. The pathway of synthesis of thyroid hormone intermediates appeared unchanged in some thyroid gland tumors from that occurring in the normal thyroid gland. The autotransplantation of thyroid gland tissue of the mouse did not per se

Page 121: .


aff wt its capacity to concentrate I 1 3 * . Heterologous transplantation should not, therefore, be the primary cause for the loss in concentrating capacity observed in most transplantable neoplasms.

Although the reasons for the decreased capacity to collect and bind iodine which occurs in experimentally produced thyroid gland tumors remain largely unknown, and since most cancers of the thyroid gland in man have also lost much of their ability to collect iodine compared to that of the normal thyroid gland, it seems reasonable to conclude that quite similiar explanations may exist for t,hese functional changes of thyroid gland neoplasms which occur during thyroid gland carcinogenesis in both animals and man. The experimental development of transplant- able dependent and independent thyroid gland tumors in both the rat and the mouse, therefore, should contribute their share to the solution of some of the many thyroid gland neoplasm enigmas. These problems pose unique and promising challenges for future solution.

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146, 288-290.

Rugh, R. 1951b. J. Morphol. 89, 323-365.

ASSOC. 76, 811-819.

Research 10, 155-161.

R. w. 1949. J . C h . Endocrinol. 9, 1138-1152.

Inst. 13, 785-805.


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Electronic Structure and Carcinogenic Activity of Aromatic Molecules

New Developments


Znstitut du Radium, Paris, France Page

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 11. The Localization Theory of Chemical Reactions.. . . . . . .

1. Basic Refined Theoretical Indexes. . . . . . . . . . . . . . . . . A. Carbon Localization Energies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 B. Bond Localization Energy.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 C. Para Localization'Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

2. Complex Indexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 111. Electronic Structure and Carcinogenic Activity of Unsubstituted Poly-

nuclear Hydrocarbons . . 122 1. Resume of Biologic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

A. K and L Regions. Fundamental Propositions . . . . . 127 2. Theoretical Correlations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

B. The Principle of the Theoretical Verification.. . . . . . . . . . . . . . . . . . . . . 128 C. Numerical Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 D. Technical References E. Discussion of the Re . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

3. Correlations with Chemical Rea . . . . . . . . . . . . . . . . . . . . . . . . . 141 A. The Reactivity of the L Reg . . . . . . . . . . . . . . . . . . . . . . . . . . 141 .B. The Reactivity of the K Region.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 C. The Reactivity in Substitution Reactions.. . . . . . . . . . . . . . . . . . . . . . . . 144 D. Miscellaneous Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 E. Conclusion and Discussion. . . . . . . . . . . I46

carbons.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

2. The Influence of the Substituents on th? L Region.. . . . . . . . . . . . 3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

V. General Conclusions and 1. General Conclusions.. 2. Suggestions.. . . . . . . . . . Appendix. The Metabolic References. . . . . . . . . . . . . . . . . . . . .

IV. Extension of the Theory to Substituted Deri

1. The Influence of the Substituents on the K Region.. . . . . . . . . . .

I. INTRODUCTION A general account of the principal results of the theoretical investiga-

tions of a possible relationship between electronic structure and carcino- 117

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genic activity of aromatic molecules has been given recently in Advances ip2 ('auctv- Kedearch hy ~'oulsoii (19.53). However, since the publication of this account new results have been obtained which seem to constitute an important step forward and even to represent practically a new stage of the whole work. ,Is a matter of fact the calculations which lcd to these net\ results have heen goiiig on for some time now, but the results have only become available since the publication of Coulson's review. It is the aim of the preseiit paper to give an account of these new developments.

Thest developments are of t x o kinds: 1. The introduction of refined theoretical quantities for the descrip-

tion of tlir properties of conjugated molecules. 2 . The tle\~elopmeiit of the theory itself and the extension of its field

of applicat ion. The major part of this review will deal with the second of these

developments, which is by far the most interesting for biochemists. We shall say only a few words about the first, which is rather technical. Scverthelehs, the principles of these technical refinements may be easily unclerstood, and they will necessarily help in uiiderstandirig the theory itself.

11. THE I,OC.~LIZATIOS THEORY OF CHEMICAL REACTIONS I n our earlier theory (.I. Pullman, 1945, 1946, 1947; A. Pullman arid

B. Pullman, 1946, 1948) we have been using for the description of the electronic structure of conjugated molecules indexes such as: bond orders, free valences, and total charges. The feature common to all these quantities, such as they were defined and calculated in this work, is that they refer to the isolated niolecdc, that is, a molecule which is not affected by any esterual perturbation. .Is is well k n o ~ n , these quantities, though they do not take into account the polarization of the molecule by ap- proaching reagents, have nevertheless been very useful for the interpreta- tion of the chemical reactivity of conjugated molecules, so that their appiication to the study of the relationship between electronic structure and carcinogenic activity was quite justified.

Iii recent years a new set of electronic indexes has been introduced by diflereiit authors and widely accepted, n-hich presents over the old one the uiidoubted advantage of describing not the isolated molecule but the reacting one-the molecule ready to be engaged in an activated complex. Referring thus more closely to the transition state of a reaction it has effectively been found to be more convenient for the description of chemi- cal reactivity than was the old set of indexes, which referred rather to its initial state. The theory of chemical reactivity based on these indexes has been named the localization theory (for a general account see B. Pullman and A. Pullman, 1932; Brown, 1932; B. Pullman, 1954b). It is this new

Page 128: .


set of indexes which has now been adopted for the study of the relation. between structure and carcinogenic activity.

1. Basic Refined Theoretical Indexes

The set is composed of three main quantities: A. Carbon Localization Energies (Wheland, 1942). The transition

state of a reaction such as the nitration of naphthalene is usually repre- sented by a set of mesomeric formulas of the type shown in Fig. 1. The attacking reagent being a positive ion, it is obvious that the formation of a bond between the reagent and the molecule, postulated by this representation, requires that a couple of ?r electrons must

The formation of the activated complex will be the easier, the easier i t is to obtain such a localization of a pair of ?r

electrons. The name carbon localization energy (C.L.E.) has been given to the difference in resonance energy between the initial unperturbed hydrocarbon and the hydrocarbon polarized in such a way that a pair of its ?r electrons is localized a t a given carbon atom and consequently no longer takes part in the conjugation. In the case of naphthalene eight ?r electrons will be left which conjugate Over the nine remaining carbon atoms. Different amounts of energy will be needed to localize a pair of ?r electrons at an a or a p carbon of naphthalene because the residual conjugated fragments of the molecule (Fig. 2a and 2b) will not be identical and thus will not have the same energy content in the

be provided by the conjugating system a t the point of attack. H, ,No1 a +

FIG. 1

@J .. .. . . . ._... . ..... . (4 (4

FIG. 2

two cases. Calculations by the usual valence bond or molecular orbital methods show that less energy is required for the localization of two ?r

electrons a t the CY than a t the p position, and this result explains the preferential reactivity of the a carbon of naphthalene in nitration.

The example of nitration illustrates what happens in the case of electrophilic substitution. The theory may immediately be extended to substitution by nucleophilic and radical reagents; for such reactions no electrons or one electron, respectively, is located a t the attacked position. The remaining ten or nine electrons are allowed to conjugate over the nine remaining carbons, and calculations proceed in the same way. A fundamental theorem developed by Wheland (1942) states that in polynuclear benzenoid hydrocarbons the localization energy of any carbon

Page 129: .


is independent of the nature of the localization assumed. This means that the same amount of energy is needed to localize a pair of electrons, one elettroii, or no electrons, at a given carbon atom belonging to this type of conjugated system. I t follows from this theorem that in such a molecwle the same carbon should be the most reactive in all types of sub- stitutions. This conclusion is verified by experiment in the cases (e.g., naphthalene) in which all the necessary data are available.

R. Bond Localization E'nergy (Iiooyman and Ketelaar, 1946; Brown, 193Ob, 1951). This is the amount of energy which is needed in order t o perturb the electronic structure of a conjugated molecule in such a way

as to localize a pair of A electrons (a double bond) between two adjacent carbon atoms. The bond Zocnlization energy (B.L.E.) is thus the difference in resonance energy between the initial molecule and the conjugated frag- ment of it which remains when the bond in question is eliminated from conjugation. Thus, the localization energy of the a-0 bond of naphthalene will be equal to the difference in resonance energy between naphthalene and styrene (Fig. 3a), and the localization energy of its 0-0 bond, to the difference in resonance energy between naphthalene and ortho-quino- dimethane (Fig. 3 b ) .

C. Para Localization Energy (Brown, 1950a). This is the amount of energy needed in order to perturb the electroiiic structure of a conjugated molecule in such a way as to localize simultaneously two electrons a t para

posit,ions, one with respect t o the other. The para localization energy (P.L.E.) for t3he 1-4 carbons of naphthalene mill thus be the difference in resonance energy between naphthalene and benzene (Fig. 4a) ; that for the 9-10 carbons in anthracene mill be the difference in resonance energy between anthracene on the one hand and two separated benzene rings on the other (Fig. 4b).

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I?. Complex Indexes Current theory tends to admit that the bond and para localization

energies are quantities suitable for measuring the ease of addition reac- tions occurring respectively a t a bond and a t 1-4 positions of a conjugated system. Though such is frequently the case (especially when different bonds or para positions within the same molecule are considered), a detailed examination of known facts shows that these simple indexes are frequently insufficient, especially for the comparison of reactivities of bonds and para positions in different molecules. We may illustrate this state of affairs by two striking examples:

1. Calculations show (Pullman and Baudet, 1953) that the localiza- tion energies of the 3-4 bond of 1,2-benzanthracene (Fig. 5a) and the 6-7 bond of 3,4-benzpyrene (Fig. 5b) are practically the same. Nevertheless,

& \ / / a & \ / / 1 4 6

(4 (b) FIG. 5

the addition of osmium tetroxide, which takes place in these two molecules just at the bonds indicated, is much more rapid in benzpyrene than in benzanthracene (Badger, 1949a,b).

2. The substitution of a methyl group for one of the hydrogens of, say, benzanthracene or the replacement of one of its carbon atoms by a nitrogen has a similar effect on the localization energy of the 3-4 bond of this molecule. Each increases the localization energy and should thus diminish the reactivity towards Os04. Experimentally i t is found that, whereas the introduction of nitrogen effectively brings about this result, methyl substitution, on the contrary, increases this reactivity (Badger and Lynn, 1950).

The readers who have gone carefully through Coulson’s (1953) paper in Advances in Cancer Research will realize immediately that the difficul- ties encountered with these new indexes are to a large extent similar to those which existed with the old ones; the examples just quoted remind us of the difficulties which manifest themselves when one tries to relate the ease of bond addition reactions to such simple indexes as the bond order. The way out of this difficulty will be largely the same in both cases. It is shown that agreement with experiment is greatly improved when the simple basic indexes are replaced by more complex ones, which generally consist of a combination of these simple ones. Such is the case for instance,

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122 A . P U L L M h S AND B. PULLMAN

as regards the localization energies, e.g., for bond additions, if instead of using only the 13.L.E we use a combination of this quantity with the localization energies of the carbon atoms which form the bond. A par- ticularly suitable quantity for the study of bond additions, which gives an excelleiit agreement with chemical experience, e.g., in the two previ- ously quoted examples, is the sum of B.L.E and the smallest of the two possible C‘.I,.E’s (C.L.En,Ln). A similar index, P.L.E and C.L.E ,,,, proves adequate for para addition reactions ( A . Pullman, l(353d).

Consequently, though these complex indexes are to some extent ad hoe quantitic.;, their success in the interpretation of chemical reactivity is definite and entirely justifies their practical use in this field. For these reasons wc shall use them also for the interpretation of carcinogenic activity.

The significance of such complex indexes may be the following: additions are most prolinbl) tno-step reactions. this rule concerning either additions of two separate reagents. or of one complex reagent (for particularly significant discussions of this iiiatter as concerns bond additions. s e r Badger, 1952: Sixms and Wibaut, 1952: SixniR, 1!153). The first step, the original attack. is then determined mostly by the C.I,.l<,,,,,. The stability of thc interinedinte complex thus formed and the ease of the second attack which conipletes the addition are governed for the greater part by the I3 L.E. or the P.I,.l<. or by factors n h05e influence is directly related to thcse quantities.


1. ResiimB of Biological Results

111 our earlier theory principal attention has been given to the study of series of related compounds, deriving from the same basic hydrocar- hons, e.g., the family of methylated benzanthracenes and benzacridines. This was the kind of study to which the theory was best adapted. Direct comparison between different unsubstitnted hydrocarbons was limited to some of the tetracyclic polynuclear compounds, and we have stressed already the difficulties of the problem (Pullman, Berthier, and Pullman, I W)). l‘ i ic ~ f n n d a m e n t a l development proposed now i s the extension of the / h i or!/ to corer all (lie basic polyrz iiclear hydrocarbons which hnve been tested jor carcirioycti ir acfii-ity.

There are about 30 such hydrocarbons. It may perhaps not have been realized clearly enough that only a very limited number of these basic structures are carc’inogenic. It will be useful t o recall here the principal biological results. (Data taken mostly, unless explicit references are given, from Hartwell’s (1951) compilation. For useful reviews see also Badger (19-&8), Haddon- and Kon (1943, Haddow (1947), Lacassagne (1946, 1948), Truhaut (1947), and Wolf (1 932) .)

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Benzene Naphthalene Anthracene 1 I1 I11

3,4-Benzphenanthrene V

Chrysene VI I I

\ / /

3,4-Benzpyrene X I

1,2-Benzanthracene V I

Pyrene IX

1,2,5,6-Dibenzanthracene XI1

Phenanthrene IV

a /

Naphtlisccne VII

($ / \

Tripheny leno X

1,2,5,6-Dibenzphenanthrene XI11

0 ‘2

\ / /

1,2,3,4-Dibenzphenanthrene l,Z,S,8-DibenzantIiracene XIV xv

Compounds of small dimensions, composed of less than four condensed benzene rings (benzene (I), naphthalene (XI), anthracene (111), and phenanthrene (IV)) are inactive.

Of the six possible tetracyclic hydrocarbons (V to X) only 3,4- benzphenanthrene (V) has been shown to possess definite activity. Re- cently Steiner and collaborators (1951, 1952) have observed tumors with 1,2-benzanthracene (VI), and even, to a very small extent, with chrysene

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(YIII) . The particularly important case of benzarithracene undoubtedly deserves further investigation.

-4rnoiig the fifteen possible pent acyclic aromatic hydrocarbons, which have all been tested for carcinogenic activity, five only have been shown

1 ,Z-Renzpyrene 1,2-Renznaphthacene Pentacene XVI XVII XVIII

Pentaphene 1,2,3,4-Dibcnzanthraccne XIX XX

Pieene XXI

3,4,5,6-Dibenzphenanthrene 2,3,7,S-Dibenzphenanthrene XXII XXIII

@ / \

/ \

\ / \ /

2,3,~,6-Dibeiizi~11e1i~iit hrene Perylene XXIV xxv

to be definitely active. These are, approximately in the order of decreasing activity: 3,4-benzpyrene (XI) , very active; 1 ,Z,ri,G-dibenzatithraceiie (XII), moderately active; 1,2,5,6- arid 1,2,3,-Ldibenzphenanthrene (XIII) and (XIV), slightly active; and 1,2,’7,8-dibenzanthracene (XV), very slightly active. (For the grading of carcinogenic potency, see Badger, 1948.)

,411 the remaining pentacyclic isomers XV to XXIV are inactive.

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/ \ \ 0 \ 4 / /

lJ2,3,4-Dibenzpyrene XXVI

3,4,6,i-Dibenzpyrene XXVIII

Anthanthrene XXXI

3,4,8,9-Dibenzpyrene XXVII

1,2,6,i-Dibenzpyrene 2’,3’-Kaphtho-3,4-pyrene XXIX xxx

1,2,3,4,5,6-Tribenzanthracene 1,2,7,8-Dibenznaplithacene XXXII XXXIII

\ / / /

1,2,9,1O-Dibenznaphthacene XXXIV

2’,1 ‘-Anthra- 1 ,Z-anthracene XXXVI

1’,2’-Anlhra-l,2-anthracene xxxv

2’,3’-Plienanthr~11,2-anthracene XXXVII

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Only a limited number of the possible hesacyclic isomeric hydrocar- bons hah heell tested. Among the cornpounds studied, two only have been proved to be carcinogenic; these are the 1,2,3,4- and the 3,4,8,9-dibenz- pyrcnes (XXYI) and (XXVII), the latter being particularly potent.

The case of 3,-t,6,7-dibenzp3-reite (XXj’III) is not clear. In 1938, 1)omagk (see HartIvell, 1951) tested a compound which he considered to I)cl the 2,3,10,11-Di-(1’,2’-naphtho)-peryleiie (XXXVIII). The com- pound was found to be inactive. r’ery recently, however, Zinlie and his collaborators (Zinke and Zimmer, 1950; Zinke, 1931) have shown that this so-called dinaphthoperylene ivas in fact the 3,4,6,7-diheiizpyrene (NST’III). It follows from this result that this last compound is inactive. Srycrtheless, practically a t the same time, Arbuzov aiid Grechkin (1952) claimed that the ~,-1,6,’7-dibeiizpyreiie was markedly active. T‘nfortu- ]lately they do not give in their work, which describes only the chemical

preparation of the compound, any indication concerning the biological experiments which led to this statement. Coiisecluently the case of this compound needs to be clarified.

Only a fen- compounds containing more than six condensed benzene rings have heen tested for pathological potency. S o n e of them has been found act iw.

Thus, zt is seeti that of nil the hydrocarbons studied, eight only are un- rlouDtedly carcinogeriic. These active compounds may be classified in three g”oI1ps:

1 . 3,-i-Rcnzphenanthreiie (V) and the tm-o related dibenzphea- :inthrenes ( S I I I ) and (XI\-).

2. The two dihe~izanthracenrs (NIT) and (XIr). 3. 3,4-Henzpyrene (XI) and the two related dibenzpyrenes (XXVI)

and (XXI-I I ) . If the activity of 1,2-benzanthracene (YI) aiid 3,3,G77-dibenzpyrene

(XX\.III) was definitely confirmed, these two compounds lvould lielong to groups 2 and 3, respectively.

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2. Theoretical Correlations

A. I< and L Regions. Fundamental Propositions. The quantum me- chanical study of the electronic structure of the polycyclic aromatic hydrocarbons, whether carried out by the valency bond or molecular orbital method, whether concerned with the indexes of the isolated mole- cule or of the polarized one, invariably shows (B. Pullman and A. Pull- man, 1952) that the majority of these compounds contain two regions which are of particular importance for their chemical behavior (some of them contain only one of these regions). These are the regions of the type of the 9-10 bond in phenanthrene and the 9-10 carbons in anthracene, which we shall call respectively the K and L regions (Fig. 6).

In the isolated molecule approximation, these were the regions which contained, respectively, the bond which had the highest mobile order and the carbons which had the highest free valencies. In the localization theory these are the regions formed, respectively, of the bond which has the smallest B.L.E (or the smallest corresponding complex index,


L region 4 K region

FIQ. 6

B.L.E + C.L.E,i,) and of the carbons which have the smallest C.L.E’s and a t the same time the smallest P.L.E (or the smallest corresponding complex index, P.L.E + C.L.Emi,).

In agreement with these theoretical results these two types of regions are we11 known to be the prirlcipal reactive centers of polynuclear hydro- carbons. A detailed discussion of this aspect of the question in relation to the problem treated in this paper will be given in Section II1,3. The interesting point for the moment is that the same two regions seem also to be of predominant importance for their carcinogenic activity.

The search for a relationship between the electronic structure and the carcinogenic activity of polynuclear hydrocarbons has, in fact, led to the two following fundamental propositions (A. Pullman, 1954) :

1. The appearance of carcinogenic activity in aromatic hydrocarbons i s determined by the existence of a n active K region.

2. I f , however, the molecule contains also a n L region, a supplementary condition requires that this region should be rather inactive.

We shall specify shortly the exact meaning of the expressions “active l 1

or “inactive” regions. For the time being we may content ourselves with the intuitive meaning of these words and consider ‘(active” as being

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equivalent to “reactive.” I-nder these conditions the previous proposi- tions have the following meaning: we suppose that one of the essential steps in carcinogenesis, probably one of the first, consists in a “reaction” between the carcinogenic molecule and tlhe cellular receiver. Without any a prioi-i idea about the nature of this readion, at least for the time being, we postulate that it takes place a t or through the K region of the molecule. In order that the reaction should be able to take place, the region has to be sufficiently active. This is a necessary, hut not, however, a sufficient, condition. Particularly i t appears that this reaction can manifest itself only if the molecule does not rontain a too reactive I, region; this, when present, may probably engage the compound in a different type of reac- tivity which does not lead to carcinogenesis. It may be noted (for details, sce Section 111,s) that though a direct comparison of the reactivities of the two regions is not possible because they are generally engaged in reac- tions of different types, the L region seems, nevertheless, t o be the most reactive of the two in the sense that the most common and frequent in ritro reactions generally occur a t this region. The K region takes part only in some more specific reactions. It seems possible that the same happens also i n uizio. The necessity for an inactive L region therefore entails a certain stability enabling the molecule to resist any fundamental changes till the Ii region is able to manifest its specific reactivity.

Our two basic propositions may be verified in two ways: 1. By quantitative theoretical calculation. 2. By comparison with available chemical data. The comparison with chemical experience mill be treated in Sec-

tion III,3. In the present chapter we shall describe only the theoretical verification.

W. T h e Principle of the Theoretical I‘erijcalion. This consists in ex- pressing the “activities” of the I< and L regions in terms of suitable, precise electronic indexes and in esamining the values of these indexes in all the hydrocarbons which have been tested for carcinogenic activity.

The proper indeses are of course those which confer 011 each of these regions the specific characteristics. The I< region will, thus, be suitably described by its H.L.E., or still better, by the complex index B.L.E. and C.L.E,,,, and the L region by its P.L.E., or still better, by the complex index P.L.E. and C.L.E,,,. Table I contains the results of detailed cal- culations of these simple and complex indexes for all the hydrocarbons previously quoted. For the L region we have also included in the table the sum of the localization energies of the two carbons which form the region ( 2 : C.L.E.), these data being useful in the discussion of the chemical reactivity of the region. All the calculations have been carried out uni- formly by the molecular orbital method (for a general account of the

Page 138: .


method see B. Pullman and A. Pullman, 1952, and for technical details see Section 111,2,D) and the results are expressed in terms of the usual resonance integral p (-20 kcal/mole). Let us remind the reader that a region or an atom is the more active, the smaller the corresponding localization energies.

C. Numerical Results. (See Table 5, pages 130-137.) D. Technical References. The evaluation of the (B.L.E.)k and (P.L.E.)L requires a

knowledge of the resonance energies of the hydrocarbons themselves and of the residual conjugated fragments which result from the proper localizations. These residual frag- ments may consist either of condensed-ring hydrocarbons of smaller dimensions or of hydrocarbons with conjugated rings.

The resonance energies of hydrocarbons I, 11, 111, IV, V, VI, VII, VIII, IX, X, XI, XII, XV, XVIII, XIX, XX, XXI, XXV, XXVII, and XXXI have been cal- culated rigorously by the L.C.A.O. method (Berthier et al., 1948; Baldock et al., 1949; B. Pullman and Baudet, 1953; Scrocco and Chiorboli, 1950a,b). Resonance energies of the remaining hydrocarbons of Table I have been calculated by Brown’s (1950~) approximation procedure.

The resonance energies of hydrocarbons with conjugated rings have been cal- culated rigorously by the L.C.A.O. method for small compounds, biphenyl, phenyl- naphthalenes (B. Pullman and A. Pullman, 1952), and dinaphthyls (Bonnemay, 1950), and by the approximation method of Coulson and Longuet-Higgins (1948) for larger molecules.

The C.L.E’s may be calculated rigorously by Wheland’s (1942) method, which is long and laborious, or by an approximation method developed by Dewar (1951a,b,c), which is much more rapid. Unfortunately Dewar’s method, though it gives a self- consistent set of C.L.E’s, represents a particular procedure and leads to results appre- ciably different in absolute values from the rigorous ones. Nevertheless, A. Pullman (1953d) has shown that the two sets of values are parallel and has traced a reference curve which makes it possible to transform Dewar’s values of C.L.E’s into Wheland’s values. The C.L.E. values listed in Table I are values corresponding to Wheland’s procedure but evaluated mostly indirectly with the help of this curve from Dewar’s corresponding values.

It would, of course, be useful to have one day the precise direct Wheland’s values of these quantities but there is practically no possible doubt that such a refinement will not alter to any important extent the results obtained in the simplified way.

E. Discussion of the Results. In the presence of the numerical results listed in Table I our two fundamental propositions may be given the following quantitative form:

1. The appearance of carcinogenic activity in aromatic hydrocarbons i s determined by the existence of a K region whose complex index, B.L.E. + C.L.E.mi,, i s equal to or smaller than 3.31p.

2. If , however, the molecule contains also an L region, the complex index of this region P.L.E. + C.L.E.mi,, should be equal to, or greater than, 5.66p.

These two numerical limits are those which characterize 1,2,7,8-di- benzanthracene (XV), considered as the weakest carcinogenic hydro- carbon.

Page 139: .



Electronic Indexes of the K and L Regions of the Aromatic Hydrocarbons (Parentheses around figures mean that the corresponding regions are not true K or L regions, hut are only similar t o them)

K region L region Carcino-

S O . Compound Bond B.L.E. C.L.E.,i., C.L.E.,i.. bons P.L.E. C.L.E.,;., C.L.E.,i,. 2C.L.E. activity B.L.E. + Car- P.L.E. + genic








(1-2) (1.26) (2.30) 4

0 1 a2 (1-2) (1.20) (2.33)







(1-4) (4) (2.54) (6.54)

(1-4) (3.68) (2.30) (5.98)

9-10 3 .31 2.07 5.38

? cd (5.08) -

z (4.60) - *

2: U


+ -

Page 140: .























I 0

* 9 m




cv 9


cv n






". n




c.l v






I 3


I I I I I I 00


". W

cv cv

cv 3


cv 3

I I I I I I m






W 9


cv 4

I 1 I I I I 3

09 n


cv d:

00 M


cv I 4

+ + + + I I I I I n

c! n


cv c!

m 9


t- I W

I-\ Y

x x






& \ \ / 3 3-4

10 4

fl 1-2



@ 1-2

\ /

X I 6-7

\ / I 5 6



1 .12

1 .06





2 .26

2 .27





3 .38




9-10 3.42 2.11

6-11 3.25 2.00

5 .53 4.26 ? E : 3

rj m 0


5.25 4.00 -

c cu c

Page 141: .

TABLE I (Confinued)

K region L region . Carcino-

B.L.E. + Car- P.L.E. + genic No. Compound Bond B.L.E. C.L.E.,in. C.L.Emin. bons P.L.E. C.L.E.,i,. C.L.E.,i., 2C.L.E. activity

XI1 @&l 3-4 1.045 2 . 2 6 3.305 9-10 3.51 2 .18 5 .69 4 .36 4-4-





q \ - - - - 9-10 1 .15 2 .27 3 . 4 2 - +

3-4 1 .04 2 .27 3 .31 9-10 3 .51 2 .15 5 .66 4 . 3 8 + 10 4

Page 142: .

























r 0,

CJ c? r- (D

I t.





o? m 9






I (D

o? 0



Q, i



0 i

* I M

=\ /”

w \/ r(


I 0

1- M


m 9


“9 3




2 I









CJ 9














t. (D

* *


c? (0




‘9 Y


3 3









2 I


2 I





c? Y





c? h


c.l o? v

E x x x




6 4



6-7 1.08 2.29

3-4 1.01 2.19

(1-2) (1.18) (2.09)

6-7 1.01 2 .22

(5-6) (1.24) (2.27)




3 .23


- -

6-11 3.27

6-13 3.18

5-14 3.45

9-10 3.49






5.25 3.97 -

5.03 3.70 -

5.56 4.27 -

5.67 4.36 -

Page 143: .

TABLE I (Continued)

K region L region Carcino-

NO. Compound Bond B.L.E. C.L.E.,,,. C.L.E.,,,. bons P.L.E. C.IA.E.min. C.L.E.,i,. z C.L.E. activity B.L.E. + Car- P.L.E. + genic

F W !&

- - - - XXI 8 11-12 1 .11 2 . 2 6 3 .37 - -



XXIII 8 9-10 1.09 2 .18 3 .27 1-4 3 .39 2 . 0 8 5 .47 4 .20 -

\ / 10 9


XXIV 0 / \ 9-10 1.07 2 . 2 3 3 .30 1-4 3 .39 2 .09 5 . 4 8 4 .19 -

\ / 10 9

> z U

Page 144: .

























+ + + + + + +

I c.







I * m




N. I


N. I


.n t- 9



I m




I rl


I (D





-;. \/




E >


x sc




5 x

x sc k x xxv









2.22 3.24 - -

2.10 3.175 - -

- 2.21 3.345 -

Page 145: .

c cu 0

TABLE I (Continued)

K region L region Carcino-

N O . Compound Bond B.L.E. C.L.E.,i,. C.L.E.,i.. bons P.L.E. C.L.E.,i,. C.L.Emi,. ZC.L.E. activity B.L.E. + Car- P.L.E. + genic






& \ \ / f 7 6-7 1 .00

4' G

4-5 1.03

/ / 4 G 5

2 .14 3 . 1 4

2 .17 3 . 2 0

2 .27 3 .33

2 .21 3 . 2 4

1'-4' 3 .34

- -

9-10 3 . 5 8

5-12 3 .37

cd - 1.96 5.30 4 . 0 1 s E 2



2 .05 5 . 4 2 4.10 -

Page 146: .

I I 2

3 3

* *









c1 63









s I m



Ir) 0



c? c!












* I * m




I b

* 3

* m



N 9


M 1

s A * N M






* I m

* m Y








5 i$ U

5 1






















x sc



Page 147: .


A detailed study of the data contained i n Table I leads then to the folloning principal conclusions:

1. Compounds I11 and YII are inactive both because of the absence of a suitable B region and because of the presence of an unfavorable L region.

2 . C'ompounds I, 11, IV, YIII, IX, X, XYI, XXI, XXII , XXV, XXTIII , SXIX, and XXXII are inactive because of the absence of a suitable Ii region.

3. C'ompounds T'I, XVII, XTTII, XIX, XX, SXIII, XXIV, XXX, X X S I I I , XXXIV, XXXV, XXXYI, and XXXVII are inactive because of the presence of an unfavorable L region.

1. The two dibeiizarithracciies (XI1 and XV) carcinogenic, obey the two limiting conditions. I t i s the fus ion of the lateral rings to the anthracene system uhich has the double eflect of iucreasing the reactivitg of the K regions and clecrcasing that 0.f the L regions, thus favoring conditions suitable f o r carcinogenic ac f ic i fy . It may be useful to stress the simple way in which the theory esplaiiis the inactivity of closely related compounds; 1,2-benz- anthraeene (1.1) is inactive (or perhaps slightly active) because the fusion of only one lateral ring to anthracene leaves an I, region which is still slightly too reactive; 1,2,3,4-dibeiizaiithrace1~e (XX), because it is devoid of a I< region; tribenzanthracene (XXXII) , because the fusion of another lateral ring diminishes the reactivity of the I< region with respect to that of the dit~enzanthracciies (XI1 and XIr) ; the two dibenznaphtha- cenes ( S X S I I I and XXXIY), because the lengthening of the linear chain creates too reactive L regions; etc.

5. The actif-ity of 3,4-benzpyrene (XI) and of the two dibenzpyrenes (XX1.1 and XXJ*II), all very carcinogenic, is immediately explained by the theory: their I< regioii is very reactive and they are devoid of an L region. I u fact the 3,J-benzpyrene may be considered as 1,2-6enzantkracene in which thc I, rcgion has bceu suppressed. Again we may note the simple way in which the theory esplaiiis the absence of activity in some closely related compounds: e.g., the 1,2,6,i-dibenzpyrene (XXIX) is inactive because it does not possess a li region, the 3,4-naphthopyrene (XXX), because it possesses a too reactive I, region, etc.

6. The 3,4-benzphenanthrene (Y) and the two related dihenzphen- antlirenes (XI11 and XIT) , carcinogenic compounds, which are devoid of an I, region, but whose Ii region is, in principle, insuficiently reactive, constitute apparent exccptioiis to the theory. In fact their case is some- what special; in contrast t o the other compounds of Table I which are planar and nonpolar, 3,4-benzpheiianthreiie has been shown, by X-rays (Ilerhstrin and Schmidt, 19.34) and by 1*.1-. absorption spectra (Clar and S tmar t , 1952) to be nonplanar and to possess a small dipole moment

Page 148: .


(0.7 D) (Bergmann et al., 1951). The phenomenon is probably due to steric interference between positions 1 and 5 and is certainly present also in the dibenzphenanthrenes. It creates in the molecule conditions which have not been taken into account in the calculation and which may well have the effect of increasing the reactivity of the K region of these compounds beyond the calculated value. It may be useful to note that the carcinogenic activity of the compounds of this group presents some peculiarities (e.g., Badger, 1948), which may not be unrelated to these structural anomalies.

7. Another exception is anthranthrene (XXXI), which according to the calculations should be carcinogenic, but which is inactive. Further studies about this compound are thus required. It may be that its inac- tivity is due to some physical causes. It may also be related to the fact that though it is devoid of an L region, it has two very reactive carbons (11 and 12) situated relatively near each other. But for the moment nothing can be decided. $a / \ / / \

\ /

/ I


Likewise, if the activity of the 3,4,6,7-dibenzpyrene (XXVIII) was confirmed, this compound, which is devoid of an L region but whose K region is not sufficiently active, would be another exception to the theory. A possible explanation of this case will be given in Section V,1.

8. The results obtained for pentacyclic and hexacyclic compounds explain the absence of activity in larger compounds. These will in most cases possess a too reactive L region (such will be the case of stretched compounds of the type of 4,5,10,1 l-di(l’,2’-naphtho)-chrysene (XXXIX) and will sometimes be devoid of a sufficiently active K region) e.g., highly condensed compounds, like dibenzoperylenes of the type XL or naphtho- dianthrene (XLI). It is difficult to imagine large molecules, obeying our two fundamental conditions (for some interesting predictions, however, see Section V,2).

9. In conclusion, i t thus seems that we have arrived at a precise relation- ship between well-dejined and quantitatively calculated electronic character- istics of aromatic hydrocarbons and their carcinogenic activity. The theory i s completely consistent and apart f rom three compounds in which there exist

Page 149: .


particular steric e$ects, leaves only very f e w isolated exceptions. It m a y be added that for the active hydrocarbons deooid of steric effects the theory re- produ6e.s quantitatively lhe order of magnitude of their carcinogenic potency.

Before finishing this paragraph a few complementary remarks may be useful.

1. In the preceding discussion we have supposed that lJ2-benz- anthracene was inactive. -4s quoted earlier, recent work by Steiner and collaborators (1951, 1952) indicates that it may be active. If such be the case, the threshold value for the localization energy of the L region would have to be modified so as to include this compound among the active ones and fixed at 5.53p instead of 5.66@. It would follow from such a change that two other compounds, considered until now as inactive, must also be active. These are 2',3'-phenanthra-l,Z-anthracene (XXXVII) and especially pentaphene (XIX) .

Sew biological assays of these compounds would be useful. * 2. In the preceding discussion we have adopted as electronic charac-

teristics of the K and L regions of aromatic hydrocarbons, which may be related to their carcinogenic activity, the complex indexes defined in the first chapter of this review. The reason for this was that these indexes give in fact the best interpretation of the chemical reactivity of these regions in the same compounds. I t may, nevertheless, be useful to know what would be the modifications of the theory if we adopted the simple indexes: B.L.E. for the I< region and P.L.E. for the L region. It is easily seen that the effect of such a simplification would not be very important. The principal effect would be that a number of compounds which are considered inactive because they possess a too reactive L region would now appear inactive both for this reason and also because they would not possess a sufficiently active I< region. Xevertheless, the use of the simple indexes would also lead to the conclusion that 3,4,8,9-dibenxpyrene (XXVII) is inactive, contrary toexperimental rcsultsand to the predictions of the refined theory using comples indexes, and would also not reproduce correctly the order of magnitude of the carcinogenic potency of the active compounds devoid of steric effects. Thus, the introduction of the complex indexes leads here, just as in problems of chemical reactivity, to greater agreement between theory and experiment.

3. The above discussion clearly shows that it is possible to establish a purely electronic theory of carcinogenic activity which does not make any explicit appeal t o factors such as geometrical shape and size. Espe- cially it follows that the inactivity of large molecules, generally attributed precisely to their dimensions, may probably be explained on purely elec-

* Before the work of Steiner and co-workers, nearly twenty investigators declared 1,2 benzanthracene inactive (see Hartwell, 1951).

Page 150: .


tronic grounds. Nevertheless, this does not mean that we can in fact dismiss completely the role that may be played by this factor. In the first place there is of course an internal correlation between geometrical shape and electronic structure, the second being a direct function of the first. The partial success of different attempts a t classification of car- cinogenic hydrocarbons according to their shape (e.g., Hewett, 1940; Bergmann, 1942; Haddow, 1943) is probably due to such a correlation. But it may also be that the form and dimensions of the molecule have a more direct influence. This should then be detected most easily, as pre- viously supposed, by studying the biological action of appropriate large molecules. We shall indicate in the last chapter of this review a number of compounds which seem to us to be the best suited for such a study.

3. Correlations with Chemical Reactivitv

The second way of verifying the basic propositions of the theory is by establishing correlations between the previously quoted results and the chemical reactivity of carcinogenic hydrocarbons. There is, of course, no warrant that the behavior of these hydrocarbons in vivo is identical t o their behavior in experiments carried out in the laboratory.* It seems, nevertheless, reasonable to suppose that the potential possibilities of these compounds are the same in both cases. Consequently if a consistent correlation could be shown to exist between some types of chemical reac- tivity of aromatic compounds and their carcinogenic activity, it would have necessarily to be considered as extremely significant for the process of carcinogenesis itself. It will now be shown that such a correlation effectively exists and that it corresponds precisely to what might have been deduced from the theory just developed.

A. The Reactivity of the L Region. From the chemical point of view the reactivity of a region is measured by its ability to undergo additions. The L region may undergo additions of two types:

1. Cyclic additions by a single complex reagent of suitable geometric dimensions.

2. Ordinary additions by two identical or different reagents attacking separately the two carbons under consideration.

Among additions of the first type the best known for the compounds in which we are interested are the fixation of maleic anhydride and photo- oxidation. In fact these reactions (and this remark holds good for prac- tically all the chemical reactions which have been investigated in aro- matic hydrocarbons) have been studied only for a very limited number of compounds which are considered here. Nevertheless, the compounds effectively studied are the most representative ones, and the data are

* See Section V and the Appendix.

Page 151: .


sufficient to enable sure predictions to be made about the behavior of remaining compounds in similar reactions.

Thus the fixation of maleic anhydride arid the photooxidation take place in aromatic hydrocarbons a t their L regions (Figs. 7 and 8).

Following our theory which requires that carcinogenic compounds should have a relatively unreactivc L region, this type of reactivity should be abbelit or very much reduced in the active compounds.* This prediction is entirely substantiated by experiment. Thus the reactivity

0 //

0 C CH ~


\ \ P



HC/c\ a f II ,o - HC, \ /

c\ H 0

FIG. 7

towards mnleic anhydride decreases in the series anthracene > 1,2- henzanthracene > 1,2,j,6-dibenzanthracene, and is practically non- esistcnt in 3,4-benzpyrene (Bachmann and Iiloetzel, 1938; Fieser, 1938; .Jones et al . , 1948). t Higher acenes like naphthacene and pentacene, which

=+a- FIG. 8

may he considered as typical noncarcinogenic compounds, are very reac- tive towards this reagent, more reactive than is anthracene (Clar, 1943; 1952). So is also the noncarcinogenic naphthopyrene XXX (Cook and Ilewet t , 1933).

Similarly. the photoosidatioii which takes place very easily in acenes, such as anthracene and naphthacene (Clar, 1943, 1952; Etieniie, 1949) is becoming much more difficult on the fusion of lateral benzene rings, so that 1.2,3,6-dibenzanthraceiie is entirely unreactive (Velluz, 1938). Complete

* I t follo\\s from a basic theorem pnt forward by Wheland, which we have quoted 111 Section 11. 1.1. that in iinsribstitiited hydrocarbons the relative ability of a given I epion to undergo additions shoutd in principle bc comparable for all tkpes of addition J\ hich thir region may undergo This conclusion should he borne in mind also in the disrtis-ion of the reactivity of the K region and mutatis mulandis in sribstitution rcartions.

t Some noncnrcinogtnic hydrocarbons devoid of an L region (e.g., phenanthrene, p? rtne, cahrysenr 1 are also unreactive towards this reagent. Their lack of carcinogenic act i r i t y 13 evitlentlj due to tile absence of a suitable K region, the ahsence of a reactive 1, region being a necesqary, hut not a sufficient, condition for the existencc of pathologi- cal potency).

Page 152: .


absence of reactivity towards photooxidation was also observed with 3,4-benzpyrene (Cook et al., 1939).

The interpretation of ordinary additions, consisting of the fixation of two separate (identical or different) reagents, is slightly more awkward owing to the lack of information about the real mechanism of these reac- tions. Thus, Fieser and Putnam (1947a) have shown that the first step of the oxidation of anthracene with lead tetraacetate consists of an addition of acetoxyl radicals to the L region of this hydrocarbon. However, it seems that in other related hydrocarbons, like 1,2-benzanthracenel the same reaction may consist rather of a substitution (Fieser and Putnam, 1947b). Anyway, such is undoubtedly the case of 3,4-benzpyrene1 which is devoid of an L region and which has only one isolated reactive center (carbon 5, whose localization energy is very small, equal t o 2.0lp).

Similarly, Kooyman and Farenhorst (1952, 1953 ; see also Kooyman and Heringa, 1952), who have studied the reactivity of carcinogenic hydrocarbons towards CC13 radicals, consider that the reaction involves an addition. In fact, Bickel and Kooyman (1952) have postulated that the reactivity of aromatic hydrocarbons towards free radicals proceeds generally through an addition to their L region. Though it seems possible that the mechanism of the CCl3 reaction involves an addition in some instances, e.g., anthracene, it certainly consists in a direct substitution in cases such as 3,4-benzpyrene.

Fortunately i t so happens that in the hydrocarbons which possess an L region, and which are the only ones in which the mechanism of the reaction may be open to discussion, it is of no importance whatever, as far as our problem is concerned, whether this mechanism consists of an addition or a substitution. The reason for this, as will be shown in detail in Section 111,3,C, concerned with the substitution reactions is that both these types of reactions should proceed in a similar way in the same com- pounds. The behavior of hydrocarbons possessing an L region in the pre- viously quoted reactions confirms again our basic statement that the L region of the carcinogenic ones must be rather unreactive. The order of decreasing reactivity towards Pb(0Ac) is: anthracene > l12-benz- anthracene > 1,2,5,6-dibenzanthracene = 0. The order of decreasing reactivity towards CC13 radicals is: naphthacene > l12-benzanthracene > anthracene > l12,5,6-dibenzanthracene.

B. The Reactivity of the K Region. This work has to some extent been developed under the influence of the theory which pointed out the need of such a study. A number of reagents are known which seem to have the property of adding preferentially to reactive bonds (see Badger, 1950b, 1951). Among those most suited for the study of polycyclic hydrocarbons in which we are interested seems to be Griegee’s reagent: osmium tetroxide

Page 153: .


in the presence of pyridine. This reagent adds to reactive bonds to give microcrystalline known complexes which on mild hydrolysis yield the corresponding cis-diols:

--CH --CIE-0 --CHOH

osos, C&,X i \ ; 1 + i ,,

--CH --CH-0 --CHOW

In polycyclic hydrocarbons the reaction, I\ hen it occurs, takes place invariably at the I.( region (Cook and Schoental, 1948a,b; Cook, 1950; Badger and iiecd, 1948; Badger, 1949a, 195Oa). A quantitative study by Badger (1!)49a, 1950a) has shown the existence of the following order of in- creasing reactivity in a series of representative compounds: phenanthrene < pyrene < 1,2-beiizaiithraceiie < 1,2,3,G-dihenza1ithracene < 3,4-benx- pyreiie. The order runs parallel to the increasing order of carcinogenic activity. It may be predicted that a number of noncarcinogenic com- pounds, e.g., pentaphcne, will also be rather reactive towards this reagent, their pathological inactivity being due to a too reactive L region. But the important point is that this type of reactivity exists in all categories of carcinogenic hydrocarbons (at least in those devoid of steric effects).

C‘. The Reaciicity in Subsli t i t t ion Reaclions. The reactivity towards substitution does not depend upon the properties of active regions, but upoii those of active cenlcrs, that is t o say. of different carbons considered alone. The examiliation of this type of reactivity seems nevertheless par- ticularly important for t he problem discussed here.

It call be s h o w that there is a distinction to be made between two cases (-1. Pullman, 1053~) :

1. The hydrocarbons possess two (or a fern pairs of) active carbons, arranged i n such a way that they form one (or a few) L regions. This is the case, e.g., in 1,3,3,G-dibeiizanthracene, whose carttons 9 and 10 con- stitute both its L region and its reactive centers. I t may be seen from the data included in Table I that in such cases a parallelism, which is prac- tically absolute, esihts between the P.L.E. of the I, region and its C.L.E.’s, the observation being true whether we consider each C.L.E. separately or their sum. * Carcinogenic hydrocarbons having necessarily ’a high P.I,.E. of their I, region, it follon-s immediately that active compounds of this type niu.st also have high C.L.E.’s of this region and consequently that such compouids must have only a very reduced reactivity, if any, towards substitution, reactivity i n any case much smaller than that of the corresponding noncarcinogenic isomers. This prediction is again entirely verified by experiment : 1 ,“,j,G-dibenzaiithracene manifests only

corresponding quantities of the I< region. * The parallelism is much more roinplete than that which exists between the

Page 154: .


a very reduced reactivity, if any, in diazo-coupling (Fieser and Campbell, 1938; Fieser, 1938), in thiocyanation (Wood and Fieser, 1941), towards methylformanilide (Fieser and Hershberg, 1938), sulfur monochloride (Fieser and Wood, 1940), benzoylperoxide (Roitt and Waters, 1952), etc. The acenes, noncarcinogenic, and particularly anthracene, to which most attention has been paid, are much more reactive towards all these reagents, and 1,Z-benzanthracene, almost or slightly carcinogenic, has in general an intermediate reactivity.

2. The hydrocarbon possesses only one isolated reactive center (or a few dispersed reactive centers, which do not form an L region). This is the case, e.g., of 3,4-benzpyrene1 which has an isolated reactive center a t carbon 5 , a t which all its substitution reactions take place. The car- cinogenic activity of this type of hydrocarbon (devoid of an L region) depending only, following our theory, on the properties of its K region, should then be independent of the properties of this reactive center and consequently of the ability of the molecule to undergo substitutions. If the reactive center has a relatively high C.L.E., the molecule will be only slightly reactive in substitutions; if, on the contrary, its C.L.E. is rela- tively low, the molecule should be very reactive in substitutions. Both possibilities should be without direct relationship to its carcinogenicity. It so happens that in practice the second possibility occurs much more frequently. Thus, the localization energy of carbon 5 of 3,4-benzpyrene has the relatively low value of 2.01p. Consequently this hydrocarbon is, in contrast to l12,5,6-dibenzanthracene, very reactive in all the previously quoted substitution reactions : diazo-coupling, thiocyanation, action of methylformanilide, sulfur monochloride, benzoylperoxide, etc. 3,4-Benz- pyrene is also very reactive towards lead tetraacetate and CC13 radicals which in this compound undoubtedly give substitutions.

It follows from the existence of these two different cases (compounds containing an L region and compounds devoid of such a region) that, contrary to what takes place for addition reaction, no general consistent relation may be expected to exist, and in fact does not seem to exist, between the carcinogenic activity of aromatic molecules and their ability to undergo substitutions. Consequently this type of reactivity cannot be essential for pathological activity. This conclusion confirms one of our basic hypotheses, namely, that carcinogenic activity must be related to properties of reactive regions and not to those of reactive centers.

D. Miscellaneous Reactions. The absence of a general correlation with carcinogenic activity concerns not only substitution reactions but also some other reactions which depend upon, reactive centers. Such is the case of the influence of carcinogenic and related noncarcinogenic polycyclic hydrocarbons on the autoxidation of benzaldehyde and other autoxidiz-

Page 155: .


able systems (Rusch and Wasley, 1942; Lisle, 1951 ; Dunn et al., 1954), or their influence on the speed of thermal polymerization of styrene (Magat and BonGme, 1951). It can also be predicted that no general relationship should exist between carcinogenic activity and the basicity of conjugated hydrocarbons, as it can be shown that this last property should develop in a way closely parallel to the aptitude of these hydrocarbons towards substitutions (see, e.g., Gold and Tye, 1952).*

Two other interesting reactions of polycyclic hydrocarbons have been studied with a view to correlation with their carcinogenic activity: the action of perbenzoic acid (Eckhardt, 1940; Roitt and Waters, 1949; see also Boyland, 1949, 1950a) and the action of Milas's reagent: hydrogen peroxide in tert-butanol, catalyzed by Os04 (Cook and Schoental, 1949, 1950; Cook, 1950). S o general relationship of any kind whatever seems to exist with either of these reagents. This is not surprising because these reagents are not specific and may attack both reactive centers or reactive bonds. The course of these reactions is thus rather complex. (For details, see A. Pullman and B. Pullman, 1955.)

E. Conclusion and Discussion. Figure 9 summarizes schematically the behavior of four particularly representative molecules in the principal activities considered here. It may lie useful to emphasize that the theory developed in this paper not only explains each of these activities sepa- rately in terms of the proper electronic properties of the molecules con- sidered, but that i t establishes also the correlations which exist between these different activities and especially between carcinogenic activity and the different types of possible chemical reactivity. It thus becomes obvious that, whereas definite relationships (parallelism or antiparallelism) seem to exist between carcinogenic activity and the ability of hydro- carbons to undergo different types of additions, no general relationship seems to exist, on the contrary, between carcinogenic activity and the ability of the molecules to undergo substitutions. This last conclusion seems particularly important, as Fieser and his collaborators (Fieser,

* -1 relationship Iictween basicity and carcinogenic activity has been postulated b y PagCs-Flon et al. (1953), based on a partial correlation observed in methylbene- acrictines. Though these are substituted derivatives, o w prediction extends to them also, as \\ill be realized from the next chapter. The observation of PagBs-Flon et al., that in carh scrics of methyl benzacridines the active molecules are thc most basic, is cluc to thc fact that in the compounds which they have studied it so happcns that the artivation of the I< region towarcts OsOI by inethylation is approximately parallel to tlrr activation of the nitrogen atom towards proton fixation. The evidence that the rrlationship is fragmentary and without any general significance to the problem of carcinogenesis is found in the fact that the niethylated derivatives of the 5,6-bene- acridine are on the whole more basic than the corresponding derivatives of the 7,s- benzacridine, though the last derivatives are in general, more carcinogenic.

Page 156: .


1938, 1914) have strongly advocated a causative association between carcinogenic activity and precisely the reactivity of polycyclic hydro- carbons in some substitution reactions. This conception was based essen- tially on the observation that some of the most potent carcinogens such as 3,4-benzpyrene or methylcholanthrene (a substituted derivative, but which can nevertheless be included without difficulty in the present dis- cussion) are particularly reactive, more reactive than any other com- pounds which they have studied, in some substitution reactions, such as diazo-coupling (Fieser and Campbell, 1938) or oxidation by lead tetra- acetate (Fieser and Putnam, 1947b). The foregoing discussion of the relationship between chemical reactivity and carcinogenic potency points

:- 0 -

.--... \ .-=.. \

--... . . -...

_-_---- 1 :2 :5:6-Dibenzanthracene

3 :4-Benzpyrene Anthmccne I

1 :2- Benzanthraccne

Chemical reactivity' of the I< region

Reactivity towards :. substitutions

/... /.. Carcinogenic

r. . I /

. a ..' . 0 activity

Chemical reactivity 1 of the L region

FIG. 9

to the reason why Fieser's suggestions may require some degree of modi- fication. The problems to be considered in this connection are as follows:

1. I n the first place it is desirable to lay emphasis on the fact that other carcinogenic, and even highly carcinogenic, hydrocarbons are totally unreactive in these specific reactions (e.g., 1,2,5,6-dibenzanthra- cene or lO-methy1-1,2-benzanthracene were totally unreactive in diazo- coupling, and the first of these hydrocarbons was also totally unreactive towards lead tetraacetate). It seems obvious to the reviewers that a chemical reaction which is to be related to carcinogenic activity must be present a t least in the very great majority of active compounds, if not in all.

2. Secondly, it is equally desirable to point out that some of the noncarcinogenic hydrocarbons, such as anthracene, are also reactive in these specific reactions. Of course, the fact that noncarcinogenic molecules exhibit the same specific reaction as the carcinogenic ones is

Page 157: .

148 A. PULLMIX . i S D B . PCLLM.4N

a less serious objection to the theory than the fact that some active com- pourids are inert in these reactions. The absciire of pathologicai activity may he due to other reasons. Hut of course, it is essential in such cases t o have some indication of these reasons. S o such indication has been given in thr literature.

3. Finally, and this is perhaps the most important objection, Fieser seems to consider that the most active carcinogens, such as 3,4-benzpyrene or methylcholanthrene, are also the most reactive of all known hydro- carttons in the specific substitutions previously quoted. This is certainly an erroneous conception owing to the fact that the reaction has riot been applied to suitable noncarcinogenic hydrocarbons comparable in dimen- sions to the active ones. Thus, nnthracene is already moderately active in these substitutions. aiid it can be predicted that higher acenes such as iiaphthacene or pentacene, which are typical noncarcinogenic hydro- carbons, will be much more reactive still in the same substitutions, prob- ably considtmbly more reactive even than benzpyrene or methylchol- anthrenP.* A confirmation of this prediction may be found in the work of Iiooyman arid Farciihorst (1952, 1033) on the reactivity of polycyclic hydrocarbons towards CCl, radicals. This reactivity is very similar to that studied by I”ieser iii diazo-coupling and towards lead tetraacctate. liooyman and Fareiihorst find in fact that bcnzpyrene and methyl- cholatithreiie arc estremcly reactive towards CC’13 radicals, much more so thaii aiithracene or 1 .2,j,G-di}jen/,aiithl.aceiie, but they also find that, iiaphthacene is far more reactive still towards this reagent than are bcnzpyrene or mcthylcholanthreiie.

It therefore seems obvious that thew suggestions that carcinogenic activity may be related to the :xbility of polycytlic hydrocarbons to undergo some substitution reactions ha1 e limited validity. From the rriticism of this conception, presented above, it can be concluded that the mere fact that some of the very potent carcinogens are also very reactivc in some definite reactions cannot tic c-onsidered as sofe basis for a theory relating carcinogenic activity t o chemical reactivity. A more complete set of ctonditions must be fulfilled. The princaipal conditions sewn to bc. (1) The reaction must occ~ir in all carciiiogeiiic compounds, or a t least in the great majority of them; ( 2 ) if the reaction occurs also in rionc~arcinogenic compounds, an esplaiiatioii of the absence of car- cinogenic activity should be gi\Ten. Partial correlations, useful as they are, must lie cotisiderrd with much caution, iinlcsr they can be included in a geiieral scheme covering the majority of active and inactive compounds. The absei1c.e of reactivity towards some reagents or in some types of

* .t similar predic*tion concci ns tlic iioiicnrvinogrnlc tiaphthopyrcne (S-LS), n hoso cnrl)on 1’ should he very reactive ton ards substitutions.

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reaction may sometimes, as has been shown, be more significant for such a scheme than the presence of high reactivity.


The extension of the present theory to substituted derivatives of polycyclic hydrocarbons, and especially to their methylated and aza- derivatives, assumes two aspects corresponding to the effect of the sub- stituents or heteroatoms on the properties of the K and L regions, respectively.

1. The InJluence of the Substituents on the K Region

As far as this problem is concerned, we simply fall back on our old theory (A. Pullman, 1945, 1946, 1947; A. Pullman arid B. Pullman, 1946, 1948), which can be applied again, practically without any important modifications. Thus the activating effect of a methyl group and the de- activating effect of a heterocyclic nitrogen on carcinogenesis may be related to the effects of these substituents on the ease of electrophilic additions to the K region. The effect depends greatly upon the position of the substituent on the molecular periphery. Our own, valency bond, calculations have been verified by Greenwood (1951) by the molecular orbital method. The general agreement between the two procedures, though not complete, is nevertheless satisfactory (Coulson, 1953). On the other hand, the theoretically predicted effects of the substituents on the reactivity of the K region have also been largely confirmed by Badger (1949a,b) and Badger and Lynn (1950), who have studied the reactivity of a number of substituted, especially methylated, benz- anthracenes and benzacridines towards osmium tetroxide. The correla- tion between the theoretically evaluated activation or deactivation or the chemically determined activation or deactivation of the K region and the corresponding variations of carcinogenic activity, though it leaves a number of exceptions, is nevertheless again satisfactory.

It must be noted that both our own valency bond calculations and Greenwood’s molecular orbital calculations of the effects of substituents on the electronic properties of the K region were carried out in the isolated molecule approximation. This means that the calculations for the sub- stituted molecules provide us only with quantities such as electrical charges and bond orders, which to some extent are less reliable than the refined localization energies which we have been able to evaluate for the unsubstituted ones. It is certainly desirable that refined calculations should also be carried out for the substituted derivatives, but in fact it may be predicted that such refinements will not modify the previous

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results to a large extent. This prediction is supported by preliminary calculatioiis by an approximate method (A. Pullman et al., 1953). More refined calculations are in progress in our laboratory.

2 . The InJluencP of the Substitrients on the L Region

This problem appears a t first sight to be particularly arduous. In the first place, no detailed calculations are available in this field (though this, in fact, is not an important handicap, as will be realized shortly). Consequently it is in the field of chemical reactivity that we shall look for indications which might help towards the solution of the problem. The strict correlations established between carcinogenic activity and the different types of chemical reactivity of unsubstituted hydrocarbons entirely justify such an expectation.

In the second place an apparent complication arises from the fact that whereas only one type of reaction, namely, a dieriophile cyclic addition, has to be considered for the I< region, the number of reactions to be taken into consideration for the L region is much greater. Sow, while all these different reac.tions of the I, region developed in a similar may in a series of unsuhstituted hydrocarbons, an entirely different situation occurs in the substituted oues. I t should, in fact, be borne in mind that Wheland’s fundamental rule stating that i n polycyclic berizenoid hydrocarbons the carbon localization energies are indcpendent of the nature of the localiza- tion assumed, is no longer valid in the substituted derivatives. In these compounds orientation and activation effects come into play, and bring about a distinction between different types of reagents and reactions (see, r.g. , 13. Pullmaii and =i. Pullman, 1952).

We have centered our attention on the particularly important group of methylatcd derivatives of hydrocarbons. There exists in fact a con- siderable amount of information concerning the reactivity of this type of derivative$, especially in the l~enzanthracene series. The examination of the available data, as regards the problem of the 1, region, leads to the following principal assertions:

1. Methyl substitution, and especially methyl substitution a t the L region itself, produces a marked activation of the reactivity of the I, region towards cyclic dienophile additions. Thus, to quote a very striking example, 9,lO-dimethylbenzanthracene (XLII) is much more reactive than the parent hydrocarbon towards maleic anhydride (Bachmann and Chemerda, 1938) and towards photo- oxidation (Cook et al., 1939).

2. On the other hand, methyl substitution may also, in other reac- tions, produce a marked deactivation of the L region. This deac- tivation may be brought about by different mechanisms:

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a. Direct etectronic deactivation. Such seems to be the case as re- gards reactivity towards CCla radicals. Thus, Kooyman and Farenhorst (1952, 1953) have shown that both in the 9,lO- dimethylbenzanthracene (XLII) and in the 2’,7-dimethyl- benzanthracene (XLIII) the reactivity of the L region towards CC13 radicals is much less than in benzanthracene itself.

@ \ / / CH3& \ / / & \ / /


b. Displacement of the point of attack. This is in fact one of the most frequent ways in which a reaction which takes place a t the L region in the unsubstituted hydrocarbon, no longer affects this region in the substituted derivative. For example, the attack on anthracene and perhaps also on its higher homologs by lead tetraacetate proceeds through an addition a t the L region, but the attack on 10-methylbeneanthracene (XLIV) by the same reagent no longer takes place at the nuclear carbons of the L region but at the aliphatic carbon of the methyl (Fieser and



Putnam, 1947a,b). A t the same time the reaction which involved probably an addition in the unsubstituted hydrocarbon becomes certainly a substitution in the methylated derivative. The same displacement of the point of attack from the nuclear carbons of the L region to the external aliphatic carbons is also observed in the action of lead tetraacetate on methylcholan- threne (XLV). In this compound the attack occurs on the ali- phatic carbon linked to the 10 carbon of benzanthracene, as indicated by the arrow. It may be added that an absolutely similar displacement of the point of attack occurs also in a num- ber of substitution reactions such as diazo-coupling, in which it

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occurs quite generally (Fieser, 1938; Fieser and Campbell, 1938), or in thiocyanation, where i t takes place in the case of methyl- cholarithrene (\F700d and Fieser, 1941).

Whatever may be the mechanism of these displacements of the points of attack, which we shall not discuss here (see A. Pullman arid B. Pullman, 1955), the important result is that in practice a number of reactions which take place in the unsub- stitutecl hydrocarhons at the L region no longer take place at this region in the substituted derivatives.

c. Deactivation b y steric eflects. In some cases a complementary deactivation of the I, region may be brought about by the steric hiiidrance dur to the presence of the substituents a t the region or iii proximity to the region. Such seems to be in par- ticular the case of compouiids of the cholaiithrene group. Thus, methylcholanthrene (XLV) not only very frequently manifests a displacement of the point of attack from the L region towards an external aliphatic carbon (e.g., in reaction with lead tetra- awtatc, in thiocyanation, probably in the reaction with CCls radicals) but also show a very reduced reactivity, if any, towards cyclic dieiiophile additions (in contrast t o the marked reactivity of the closely related methyl benzanthracenes). Thus, methylcholant hreiic is even less reactive than benzanthracene towards maleic aiihydridc (Jones et al., 1948) and is totally un- reactive in photooxidation (T’elluz, 1938). This state of affairs is undoubtedly due to steric factors, probably to the difficulty of pushing the dimethylenic bridge out of the plane of the mole- cule-a coiiditioii which must be fulfilled in order that the at- tarking reagent could approach the 1, region. The cholanthrenes arc just a particularly p r i i d c y c d group of compounds in which the L wgioti s e ~ m s to bc absolutely protected from attack by any type of reagents.

3. Oiseiission

The principal conclusions which may be drawn from this survey of experimental chemical results seem to be the following:

1. It is possible to account to a large extent, though not completely, for the carcinogenic activity of substituted and in the first place methyl- ated and aza-derivatives of polycyclic hydrocarbons, merely by consid- ering the irifliierice of the substituent on the reactivity of the K region towards cyclic dieiiophile additions of the type of osmium tetroxide fixa- tion. In this type of compouiid, there does not seem to be any absolute necessity to take into consideration the properties of the L region.

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2. The presence of a substituent and particularly of a methyl group on the molecular periphery has the effect of increasing the reactivity of the L region towards some types of reagent (e.g., maleic anhydride) and of decreasing or even destroying its reactivity, by a number of means, towards other types of reagent (especially free radicals like CCL or OAc).

In the presence of these results it seems natural to suppose that the unknown reaction, which, when occurring in vivo a t the L region of a hydrocarbon prevents it from being carcinogenic, belongs to the second type. As a matter of fact this seems highly plausible, an attack by some free radicals or related reagents being more probable, in vivo, than an attack by a complex dienophile reagent of the maleic anhydride type. The effect of the substituent would then consist partly in protecting the L region from these attacks, by one of the mechanisms previously indi- cated: direct electronic deactivation of the region, displacement of the point of attack to carbons situated outside this region, steric hindrance, etc. The L region thus neutralized, the carcinogenic activity of the molecule will then depend, according to our ideas, only on the properties of the K region, This may be the reason why in the substituted deriva- tives the properties of this region alone seem to determine largely the pathological behavior of the compound. It is clear, of course, that if the neutralization of the L region is only partial, the carcinogenic activity will depend partially upon the properties of this region also and will be a compromise between the opposing tendencies of the K and L regions. In this respect, as already mentioned, the cholanthrenes seem to consti- tute a particularly privileged group of compounds because in them the L region is particularly well deactivated. It may be that the very high carcinogenic potency of these molecules is due to this fact to a greater extent than previously realized. Thus, Badger (1948) has shown, with the help of the OsOc reaction, that the K region of methylcholanthrene, though very strongly activated in comparison with benzanthracene, is nevertheless, chemically, less activated than the K region of some related polymethylbenzanthracenes of similar or even slightly lower carcinogenic activity. It may be that the very high carcinogenic activity of methyl- cholanthrene is due to the fact that in this compound the L region does not interfere a t all with carcinogenesis, whereas some interference may persist in the methylated benzanthracenes.

Another point which deserves to be stressed is the following: We have seen that a number of reactions (additions or substitutions) which take place in the unsubstiiuted polycyclic hydrocarbons at the L region, no longer take place a t this region in the substituted derivatives. It follows from the principles of our theory that while the reactivity of carcinogenic unsubstituted hydrocarbons possessing an L region must necessarily be

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very reduced towards both such additions and substitutions, no limita- tion of this type applies t o the substituted derivatives of such compounds. Consequently it may be predicted that most probably no consistent gen- eral relation exists between these reactions and the carcinogenic activity of substituted molecules. That such is effectively the case can be easily shown by the following particularly significant examples.

1. 20-12lethylcholanthrene (XLV) , which is highly carcinogenic, and 20-methyl-I ,2,3,1,11,14-hexahydrocholanthrene (XLVI) which is non- carcinogenic. are hoth very reactive towards lead tetraacetate (Fieser and Puttiam, 1947b).

2. Although the carcinogenic activity of 10-alkylbenzanthracenes diminishes strongly with the increase in the length of the alkyl group, the reactivity of the same compounds towards lead tetraacetate remains practically unchanged (Fieser and Putnam, 1917b).

3. Whrreas 9,lO-dimethylbenzanthracene is, as already mentioned, practically inert towards CCls radicals, methylcholanthrene, on the rontrary, is highly reactive towards this reagent (Kooyman and Faren- horst, 1952, 1953) (the reaction consisting most probably in a substitu- tion on one of the aliphatic carbons of the dimethylene bridge).

1. Again, whereas methylcholanthrene is very reactive in diazo- coupling, 10-methyl- or 5,10-dimethylbenzaiithracenes, all appreciably carcinogenic, are totally inartive towards this reagent. On the other hand, 9-methylbenzanthracene moderately carcinogenic is slightly reactive (Fieser and Campbell, 1938).

N'ithout entering into a detailed discussion of the mechanism of these reactions in each particular case (for such a discussion see A. Pullman and B. Pullman, 1955) it is obvious from these fern examples that once they cease to affect the L region, the different reactions just quoted are without any apparent relationship to carcinogenic activity. This illus- tration confirms our basic proposition, that only reactions affecting the I< or L regions may be correlated with carcinogenic activity. It also suggests modification of the concept of a linkage between these types of substitutions and carcinogenic activity.

It must be acknowledged that the extension of the theory to substi- tuted derivatives of polycyclic hydrocarbons is at present far from having achieved a completely consistent and satisfactory form. Apart from the fact that it has not been given such a quantitative form as the theory of unsubstituted hydrocarbons, many aspects of the problem, such as the existence of some apparent exceptions or the influence of all the substit- uents tested, have not been considered. Further work is thus urgently needed in this field. It nevertheless appears t o the reviewers that the general conceptions presented in this chapter, which are a development

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of the more firmly established conclusions concerning unsubstituted hydrocarbons, form a suitable basis for such work. If verified and con- firmed, they will yield a consistent theory covering all groups of car- cinogenic polycyclic hydrocarbons. It must nevertheless be realized that the complexity of the problem renders theoretical calculations in this field particularly difficult. It seems highly probable that much more rapid advances could be made through proper chemical experiment. We shall indicate in the next section a few hints for some such experiments which seem to us to be particularly useful.

The great complexity of the problem, which surely need not be stressed, may nevertheless be illustrated by the following example. We suppose that methyl sub- stitution generally has the effect of inhibiting, by different means depending upon conditions peculiar to each case, the reactivity of the L region towards the unknown reaction which prevents the K region from developing its activity leading to carcino- genesis. Nevertheless, it seems extremely tempting to admit that in some cases the effect of methyl substitution may be just the reverse. Let us quote an example. It is extremely puzzling to observe that, whereas 3,4-benzpyrene (XLVII) and its

4 ' 5 6


5,6,8,9 and 4' methyl derivatives are all more or less potent carcinogens, the 2' and 3' methyl derivatives are totally inactive. Unless purely geometrical reasons (unverified and at present unverifiable) are invoked, it is hard to find an explanation of this puzzling phenomenon. I t seem8 tempting to consider the possibility that in these last two derivatives the substituents have the effect of creating, a t the 1'-4' carbons, a n L region, absent in benzpyrene and in the other methylated derivatives. The 2' and 3' methylbenzpyrenes would thus bear some analogy to the inactive naphthopyrene (XXX). (This analogy has been indicated already by Bergmann, 1942.) The same reasons might be invoked to explain the inactivity of 6,7-dimethyl-3,4-benzphen- anthrene (XLVIII) and 4,5-dimethylchrysene (XLIX) analogous to the inactive


2,3,5,6- and 2,3,7,%dibenzphenanthrenes (XXIV and XXIII). Whatever may be the value of these suggestions, it is obvious that they may be more easily verified by proper chemical experiment than by theoretical calculations.

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1. General Conclusions

The correlations established between electronic characteristics, chemi- cal reactivity, and carcinogenic activity of aromatic molecules point t o the conclusion that the mechanism of action of carcinogenic molecules involves, at, least at one of its stages, a chemical reaction between the carcinogen a i d a cellular receiver. The reaction probably consists in the formation of an addition product or complex, and it is one of the funda- mental assumptions of this work that this formation takes place through the I< region of the carcinogens. The cellular receiver is probably of an electrophilic nature. On the other hand, in order that the I< region should he able to develop its activity leading to carcinogenesis, the molecule must be devoid of an active L region. The reaction which, when i t occurs at the L region, prevents the development. of carcinogenic activity con- ' ts probably in ail attack by radical reagents. It seems probable that

the incompatibility lxtween carcinogenic activity and marked ability to undergo additions at the L region is due to the fact that such a reac- tion produwh a disruption of the conjugated system, bends the molecule along the nxi3 of the L region, and thus robs i t of a large part of its aro- matic properties.

The formation of an addition complex hetween the carcinogen and the living cell has recently been demonstrated by a number of workers (Boyland, 1948, 1949, 193Oa,b,c, 1952; Ileidelberger, 1953; Miller and Miller, 1953). The present theory goes, or at least tries t o go, a step further by inclicating which are the probable molecular regions involved i n the formation of such a complex and what may be the probable type of the reaction which occurs.

The theory lags down that the regions important in carcinogenesis are also those which constitute in eitro the principal reactive atoms or bonds of the molecule. In this respect i t may be useful to recall the following argument advanced in favor of this hypothesis by Boylarid (1948, 1949, 1950~) . It is well known that metabolic oxidation of carcinogenic hydro- carboiis leads, through dehydration of intermediate dihydroxydihydro dcrivates, to hydroxy derivatives (Boyland and Weigert, 1947; for the mechanism of the dehydration see Badger, 1949b). The interesting point is that this metabolic transformation does not take place at any of the reactive regions of the carcinogens but a t points of secondary reactivity. In fact the reaction seems to occur nearly always at positions adjacent to the K region, as is shown by the isolation of the following metabolic products of the principal hydrocarbons:

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Boyland suggested that this phenomenon could be explained by the hypothesis that metabolic perhydroxylation takes place a t positions other than the reactive K bond because this bond i s already engaged in a diferent reaction with the cell:

Metabolic perhydroxylation

‘ 2 9 The cell

FIG. 10

We shall call the positions reactive in metabolic perhydroxylation, the M region.

The validity of this hypothesis would be enhanced if it could be shown that such a formation of a carcinogen-cell complex through the K region would effectively increase the reactivity of the positions attacked in metabolic perhydroxylation. It has been suggested (Daudel and Daudel, 1949) that such would be the case if it were supposed that the formation of the complex results in the elimination from conjugation of the pair of T electrons associated with the K region, but this explanation is erroneous. In effect, if we adopt such a simplified picture of the complex, the remain- ing conjugated fragment of the carcinogen would be equivalent, in the case of, say, benzanthracene, to p-phenylnaphthalene (see Fig. 10). Now it is obvious that the reactive centers of p-phenylnaphthalene are situated a t the naphthalene nucleus rather than at the phenyl group (B. Pullman and Baudet, 1954). Consequently it is obvious that a much more refined picture has to be adopted for the carcinogen-cell addition complex, if the course of metabolic perhydroxylation is to be interpreted within the framework of Boyland’s hypothesis. Calculations are being carried out at this very moment in our laboratory on this problem.*

The following point deserves to be mentioned in relation to the question of meta- bolic transformation. The calculations indicate that in 3,4-benzpyrene only the 6-7 bond should be considered as a K region. Now it is found experimentally that 3,4-benz- pyrene gives two different metabolic products which are the compounds (LII) and

*See Appendix.

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(LIII). If Boyland’s hypothesis is correct, this would suggest that, for undetermined reasons, the 1-2 hond of 3,4-henzpyrene niay also art as a K region. This conclusion may be of intercst: thus, if the carcinogenic activity of S.~,G,7-dibenzpyrene (XXVIII) Rere confirmed it might be nttrit)utrd to the existence i:i this compound of a n unpre- dicted hut effective I< region, situat.eti at the 1-2 bond.



2 . S irggrst ions

I t is not our intention to suggest in this paragraph anything which might be considered as a scheme for future work. Such a suggestion would shojv far too much conceit on the part of theoreticians. In fact we have sufficient confidence in the ingenuity of our experimentalist friends to be stire that thry will quickly devise some mrans of demonstrating the insufficiencies of the theory. We should just like to suggest some hints for research which would br particulnrly intrrehting and assist the develop- ment of the theoretical investigatiolis.

111 the first place, it seems useful to test the theory by verifying some predictions to which it may lead. The following two predictions seem particularly interesting:

1. There are five possible isomeric dibenzpyrenes, four of which have already been tested for carcinogenic activity. The 3,1,9,10-dibenzpyrene (LIV) is the only one which has not yet been tested. I t may be predicted


that this compound would be avtive, as it is devoid of an L region (in fact it may be considered as a derivative of pentaphene, in which the introduction of a complementary ring has suppressed the two L regions) and as the complex index of its I< region is equal to 3.1613.

2. Following our theory naphthopyrene (XXX) is iiiactive because it has a too reactive L region (the same thing happens in the inactive phenanthrapyrene ( IJT*) , the complex indexes of its T< and L regions heing, respectively, 3.178 and 5.4@). Sow, two isomeric naphthopyrenes may

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be imagined, LVI and LVII, in which this troublesome region is no longer present. The complex indexes of the K regions of these two compounds being, respectively, 3.258 and 3.248, they should both be carcinogenic.


It is always dangerous to make predictions about the possible activity of large molecules, as it is not impossible that steric factors may play an important part in their case. As a matter of fact it would be most useful to have some definite evidence, which at present seems to us com- pletely lacking, as to the part played by these factors. A particularly suitable case for demonstrating the part played by these factors might be provided by the study of compounds of the type LVIII and LIX and


eventually their higher homologs. Though no calculations have been carried out for these compounds it seems probable, by analogy with similar compounds actually studied, such as 3,4,8,9-dibenzpyrene (XXVII), that from the purely electronic point of view they should be carcinogenic. Consequently, if these compounds were inactive, the steric factor would thus be demonstrated (care must of course be taken to compare their physical properties, such as solubilities, etc.).

In the field of biological experiment it would also be most useful to settle the problem of the carcinogenic activity of the 3,4,6,7-dibenzpyrene (XXVIII) and, following the discovery of carcinogenic activity in 1,2- benzanthracene, to reinvestigate some other compounds such as an- thanthrene and pentaphene. Generally speaking interest in the biological investigation of new compounds seems to have decreased recently. It is our opinion that such investigations should be actively continued. A de- tailed biological investigation of the different possible methylated deriva- tives of benznaphthacene (XVII), comparable with that which has been carried out for benzanthracene, would for instance be extremely useful.

In view of Boyland’s hypothesis previously discussed it would also be interesting to have some results on the metabolism of large noncar- cinogenic hydrocarbons comparable in dimensions to the carcinogenic

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ones. I t should in fact be remembered that whereas carcinogenic hydro- carbons undergo metabolic perhydrosylation exclusively a t positions adjacent to the Ii region, the noncarcinogenic phenanthrene undergoes in some cases perhydroxylation at the I< region itself (Boyland and Wolf, l%O). It might be useful to knoir whethcr such a property is found also in other inactive compounds. Care should be taken to investigate different types of iiinctive hydrocarbons: those whose inactivity is due to the absence of a suitable I< region, those whosc inactivity is due to the pres- ence of an unfavorable L region, etc.

So much for biological investigation. We feel that the present state of the theory also suggcsts a iiuniber of lines of chemical research. It seems in fact ohvious from the theory that ail extremely close correlation esists hct\\-een carcinogetiic. activity aiid chemicd reactiT,ity of aromatic molecules .is a matter of fact it cari el-en he said that the correlation is so close that the theory developed in this paper could hake been based simply 011 tlic htiidy of the chemical reactivity of the molecules involved. It \\-odd trot then be as quantitative and would not rover such a great nunil)er of c*ompouncls, but would be practically identical. Nevertheless the Jiurnlwr of tw ic unsubst ituted carcinogenic and noncarcinogenic hydrocarbons ivhose reactivity is \I ell kno~rn is very limited, and prob- ably oiic of the first things to do is to verify the fuudamental predictions of the theory relative to the reactivities of the L and K regions. It should I)e iiotccl that i n the unsubstituted compounds the choice of the reagents is of secondary importance; OsO, for the I< region and maleic anhydride for thc I, region seem quite suitahie. Particular attention should be paid to the study of anthanthreiie and similar exceptional compounds.

The study of chemical reactivity should, of course, be continued in- tensely for thc suht i tuted dcrivativeb of the hydrocarbons also. Spccial attention should he paid to the aspect of the theory concerned with the role of the 1, regioii in these compounds, and one of the reactions which i t ivould be mod useful to study carefully i n relation to this problem is the action of the CC13 radicals. This reaction seems in effect to give a direct measure of the deactivation of the L region, by the substituents present 011 the molecule, towards free radical addition. There seems to be no complication, as is often the case in other similar reactions, arising from the appearance of new centers of attack.

Finally, the study of substituted derivatives poses, among other prob- lems. one of general chemical importance and of particular importance for carcinogenic activity, whose significance has not yet been clearly realized. The problem is that of the orientation and activalion of c$ects oJ si&stif writs for addition reactions, especially cyclic additions. During the last few decades detailed rules have been established which determine

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the orientation and activation effects of different types of substituents for a second substitution. It should be realized that (apart from some individual cases) no such rules exist which determine the orientation and activation effects of substituents for an addition reaction. Most frequently the orientation and activation rules for substitution reactions are im- plicitly adopted also for the interpretation or prediction of the orientation and activation effects in addition reactions. That such a conception is erroneous may be seen, for instance, from the preliminary studies by Badger and Lynn (1950) on the effect of different substituents on the rate of addition of oso4 to the K region of l12-benzanthracene. Let us quote from this work two particularly striking examples: (1) Although the methoxy group is an ortho-para directing substituent, strongly ac- tivating a second electrophilic substitution (much more than does a methyl group), it has practically no effect on the rate of addition of OsOc to the K region of bensanthracene (whereas a methyl substituent increases this rate to marked extent); (2) The acetoxy group, which is usually regarded as a slightly activating group for a second electrophilic substitution, seems t o act as a deactivating substituent in the oso4 reaction.

It will immediately be realized that this problem is of basic impor- tance, both to general chemistry and to the understanding of the action of carcinogenic compounds. Thus, for instance, Badger’s results might perhaps explain why a hydroxy substituent is unfavorable for carcinogenic activity-a fact which could not be deduced from its directing and ac- tivating effect in substitution reactions. The reactions important in carcinogenic activity being most probably, as previously shown, addition reactions, this problem of the orientation and activation effect of sub- stituents upon additions should then be most carefully examined. Theoretical investigations of it are being carried out a t present in our laboratory.


Since the calculations concerning the metabolic reactivity of carcino- genic hydrocarbons, announced in Section V, were finished before this paper was sent to press, it seemed useful to include them in the present review. (For a more detailed discussion see A. Pullman and B. Pullman, 1954.) .

The first problem considered was the following : whereas both diols and phenols have been isolated from small hydrocarbons (naphthalene, anthracene, and phenanthrene) , only phenols have been isolated from higher hydrocarbons, including some carcinogenic ones, and from benzene

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TABLE I1 Resonance Energy of Metabolites

Conjugated fragment of the diol

Biphenyl: 4.388

8-Vinylnnphthalene: 4.108

Styrene: 2.428

1 -Vinylphenanthrene : 5.898

Fully aromatic system

dOH Phenanthrene: 5.458

Anthracene: 5.318

Naphthalene: 3.688


Chrysene: 7.198

Gain of resonance energy upon dehydration


5 F F

2 * 2 U


1.218 m


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/ /

m-Vinylnaphthalene : 4.128

5-Vinylbenzanthra- cene: 7.548

1-Vinylanthracene: 5.7513

2-Methylenebenzan- threne: 6.778

Butadiene: 0.478

1'-Methylenebenz- anthrene: 6.678

&,OH Phenanthrene: 5.458


1,2,5,6-Dibenzan- thracene: 8.888


1,2-Benzanthracene: '7.100

\ / /


3,4Benzpyrene: 8.228

Benzene: 2.008

woH \ / / 3,CBenzpyrene: 8.228


F M d c3 m 0 1.3413 3 m

el m M

2 1.358 c

+ Z U

1.458 d


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(which gives also trans-trans muconic acid) (see, e.g., Boyland, 1950b; Wolf, lY.32) diols are easily dehydrated to phenols, i t is nevertheless generally admitted that diols are probably intermediates in the metab- olism of all aromatic hydrocarbons.

111 support of such a hypothesis it may be shown (B. Pullman, 1954) that the tendency of the diols towards dehydration should in general be effectively more pronounced in carcinogenic hydrocarbons than in the small ones (benzene excepted), the reason for this being that the gain of resonance energy due to the recovering of a fully aromatic structure would be more pronounced in the first case.

Thus, if we neglect the increment of resonance energy due to the eonjugatioii of the hydrosy groups in the phenols, this gain would be equal t o the tlifference in resonance energy tictween the conjugated frag- meiit of the d i d and the correspondiiig fully aromatic system. The results of an evalunt ion of such cliffereiices for the principal experimentally studied nieta1)olites are givw in Table 11.

It may be beell that ui th the esccption of an inversion between the 1 ,'L-diol of phenanthrene and the diol of chrysene, the tendency fowards deh!jrlr.diotz shottld e.feclirely be more pronoiorccd i n compounds for which otily a phenol has becn isolated (benzene incliitlcd). The inversion quoted may probahly be attributed to the theoretical underestimation of the gain of resonance energy corresponding to dehydration of chrysene, due to overestimation of the resonance energy of l-vinylphenanthrene ; in fact, thtb cxperimental conjugating power of phenanthrenyl radicals and the experimental chemical reactivity of the 1 carbon of phenanthrene are frequently smaller than would be expected from theoretical calcula- tions (see, e.g., Braude and Fan-cett, 1950).

It niay 1)r slionn that the gcneral aspcct of the rrsiilts of Table I1 reiliains prac- tically tinchonjird if avconnt IS takcii of the complementary increasc of thc gain of resonance etirigy upon dehydration due to the conjugation of the OH groups with the nroiiiatic s ~ s t t m s (U. I'ullman. 1951: A. Prlllrnnn arid B. Pullma11, 1954, 1955).

The secwnd problem is t o account for the fact that metabolic per- hydrosylation does iiot take place either a t the L or a t the I< region, which are the usual reactive centers in chemical reactivity, but a t posi- tions quite insignificant in the original molecule, though similarly placed in all molecules, and .i\.hicli we have called the d l region. As already men- tioned, Boyland (1948, 1949) suggested that this phenomenon may lie due to the fact that the I< regioii is blocked because of a primary reaction with the cell and that in the addition complex which is thus formed the $1 region is activated towards the metabolic transformation.

The calculations (B. Pullman and Baudet, 1954) have been carried out in order to verify the possibility of such an activation. The model

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adopted for the complex between the cell and the hydrocarbon consisted in admitting a spreading out of the T electrons of the hydrocarbon into the bonds which unite i t to the cell. These bonds would then become par- tially double, the whole electronic cloud of the hydrocarbon assuming a n approximately quinonoic configuration (Fig. 11). As a matter of fact the calculations have been

of the hydrocarbon, once the addition complex (yf& _...... ,..- ..._ ..... ... carried out by considering the electronic cloud

with the cell i s established, as being equivalent to an ortho-quinone. The results of such calcula- tions for 1,Zbenzanthracene are given inFig. 12.

It is immediately seen that in such a system the greatest concentration of electrical charge is on the 3' carbon (binuclear carbons excepted), that is, at the M region. Though the interpre- tation of the reactivity of heterocyclic systems, based on such diagrams, is not absolutely sure yet (B. Pullman and A. Pullman, 1952), it seems nevertheless highly probable that this carbon should effectively form the reactive center for electrophilic attacks.


.....,.... . ..,,..... . .,., , 1 .. : %.

' Cell

FIG. 11

0.997 1.001



0.627 0.313 0.604 0.61 1



FIG. 12

These results not only give a physical base for Boyland's hypothesis but seem to be able to throw some light on the possible mechanism of the metabolic reactivity. Thus, with the metabolic diols having the trans configuration, the most probable mechanism for their formation is either a direct attack by free OH radicals or a hydrolysis of an intermediate

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epoxide (Boyland, 195011). The calculations exclude perhydroxylation by OH radicals because such a reaction would most probably take place a t the L region of the hydrocarbon, as this region conserves in the ortho- quinone the highest free valencies. On the contrary, the calculations sup- port pcrhydroxylation through an intermediate formation of an epoxide, as such a formation may be brought about by the action of electrophilic reagents such as peracids. The primary attack of such reagents occurring at the 3’ carbon, the epoxide will be formed a t the 3’-4‘ bond and not at the 3‘-2’ bond of the quinonic system because the 3’-4‘ bond has the higher bond order (B. Pullman and A. Pullman, 1952). The hydrolysis of the epoxide (which has to be enzymatic in order to account for the op- tical activity of the diols) will yield the trans-diol. Dehydration of the diol Lvill then lcad t o the 4’ hydroxy derivative, the 4’ carbon having in the initial molecule a free valence higher than the 3’ carbon (following the mechanism indicated by Badger, 194Db; see also A. Pullman and B. Pullman, 1934, 19%; B. Pullman and A. Pullman, 1052). The course of the metabolic reactivity of carcinogenic hydrocarbons can thus be schematized in the follo\ving way:

--+ -cell -

This conception establishes a bridge between the chemical and the metaholic reactivity of carcinogenic hydrocarbons and enables us to include them both in a homogeneous theory. An experimental study of the reactivity of ortho-quinones would be useful in order t o verify the sugges- tions of the calculations.

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ACKNOWLEDGMENT This paper is an elaborated version of a lecture given by the authors at the Chester

Beatty Research Institute, Royal Cancer Hospital, London, at the meeting of the European Section of the Union Internationale contre le Cancer, in October 1953.

We wish to thank all those who, by taking part in the discussion, helped to clarify a number of ideas and put forward useful suggestions. Our particular thanks are due to Professors Haddow and Boyland for having kindly agreed to read the manuscript, for a number of corrections, and for useful discussions. At the same time we are sure that all those who were present a t the London meeting will join with us in expressing sincere thanks to Professor Haddow and his colleagues for the most perfect organiza- tion that has ever been given to a meeting.

We wish also to thank Professor Lacassagne for his constant interest in this work and his guidance in the fields of biological facts.

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Bickel, A. F., and Kooyman, E. C. 1952. Rec. trav. chim. 71, 1137-1144. Bonnemay, A. 1950. Compt rend. 231, 1147-1149. Boyland, E. 1948. Yale J . Biol. and Med. 20, 321-341. Boyland, E. 1949. Ann. Rev. Biochem , pp. 217-242. Boyland, E. 1950a. Biochim. et Biophys. Acta. 4, 293-300. Boyland, E. 1950b. Biochem. SOC. Symposia (Camhridge, Engl.) NO. 6 , 40-54. Boyland, E. 1950c. J . chim. phys. 47, 942-952. Boyland, E. 1952. Cancer Research 12, 77-87. Boyland, E., and Weigert, F. 1947. Brit. Med. Bull. 4, 354-359. Boyland, E., and Wolf, G. 1950. Biochem. J . 47, 64-69. Braude, E., and Fawcett, J. S. 1950. J . Chem. SOC., pp. 800-808. Brown, R. D. 1950a. J . Chem. SOC., pp. 691-697. Brown, R. D. 1950b. J . Chem. Soc., pp. 3249-3254. Brown, R. D. 1950c. Trans. Faruday SOC. 46, 1013-1018. Brown, R. D. 1951. J . Chem. Soc., pp. 1950-1955. Brown, R. D. 1952. Quart. Revs. (London) 6, 63-99. Clar, E. 1943. Die Chemie 66, 293-304.

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Clnr, E. 1952. “ Aromatischc Kohlenwasserstoff e,” 2nd ed. Springer Verlag, Berlin. Clar, E.. and Stewart, D. G. 1952. J. Ant. Chem. Soc. 74, 6235-6238. Cook, J. JV. 1050. J . Chetn. Soc., pp. 1210-1219. Cook, J. If’., and IIewett, C. L. 1933. J . Chem. Soc., pp. 403-406. Cook, J. IT., Martin, R., and Roe, E. M. F. 1939. Nulure 143, 1020. Cook, -1. IT., and Schoental, R . 1948a. Nature 161,237-238. Cook, J . W., and Schoental, R. 1948b. J. Chem. Soc., p p . 170-173. Cook, J. I\*., and Schoental, R. 1949. Bull. soc. chirn. b i d . 31, 358-364. Cook, J. W.: and Schoental? R. 1950. J . Chetn. Soc., pp. 47-54. Coulson, C. .\. 1953. Adranees in Caricer Research 1, 1-56. Coulson, C. A, , and Longuet-Higgins, €1. C. 1948. Proc. Roy. Soc. (London) A196

Daudel, l’., and Daudcl, It. 1949. B7111. soc. chitti. bio?. 31, 349-356. Dewar, 11. J. S. 195la. J. Arn. Chetn. Soc. 74, 3341-3345. Dcn-ar. A f . J. S. 1951h. J . Ant. Chetri. Soc. 74, 33-15-3349. Dcxar, 11. J . S. 195lc. J . A t t i . Chetn. Soc. 74, 3357-3363. Dunn, J. H . , \Vatem, \V. A, , and Roitt., E’. hl. 1954. J . Chcttt. Soc., pp. 580-586. Eckhard, H. J. 1940. Ber. deiit. chew Ges. 73B, 13-15. Etiennc, A. 19-19. “Traite de Chimic organiquc de Grignard,” 1‘01. SVII/2, pp. 1298-

Fiescr, I,. F. 1938. A t t i . J . Cancer 34, 3T-12-1. Fieser, L. 12. 19-14. Research Confcrcnce on Cancer, publ. A m . Assoc. Advance. Sci . ,

Fiesrr, I,. F., and Campbell, \T. P. 1938. J. Am. Chem. Soc. 60, 1142-1145. Ficscr. I,. F., and Hcrshberg, E. B. 1938. J. Atn. Chetn. Soc. 60, 25-22-2548. Fiesvr, I,. F., and I’utnam, S. T. 1947a. J . Am. Chetn. Soc. 69, 1038-1041. Ficwr, I,. F., and I’utnam, S. T. 1947b. J. Atn. Chettt. Soc. 69, 1041-1046. E’icscr, L. I;., and Wood, J. L. 1940. J. A r ~ t . C h e w Soc. 62, 2674-2681. Gold, I-., and Tye, F. L. 1952. J. Chefti. Soc., pp. 2184-2188. Greenwood, 13. H. 1961. Brit. J . Cancer 6, 441-455. IIaddow, A. 1943. Endeacour 2, 27-33. Haddon-, A. 19-17, Brit. 3 r d . Bull. 4, 331-342. Haddon-, A,, and lion, G. A. It. 1947. Brit. N e d . Bull. 7, 314-326. Hartwell, J. I,. 1951. “Survey of Compounds \Thich Have Been Tested for Car-

cinogenic Activity,” 2nd ed. Federal Security Sgency, Public Health Service, Bethcsda. 1Iaryl:md.

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pp. 108-116.

Heidellxrger, C:. 1953. :I dcances in Cancer Research 1, 273-338. Herlistcin, F. Ff., and Schmidt, G. JI. J. 1954. J. Chetti.. Soc., pp. 3302-3313. Ffewett. C. L. 1940. J. C‘hertt. Soc., p p . 293-303. Jones, R. 1.. Gogck, C. J., and Sharpe. R. W. 19-18. Can. J. Research B26, 719-727.

C., and Farenhorst, E. 1952. Xature 169, 153-15-1. C’,, arid Farenhorst, I:. 1!)53. Trans. Faraday Soc. 49, 58-67. (:., rind IIrringa, J. ]I-. 1952. Nalicre 170, 661-662. (I., and Kctelaar, J. A. X. 1946. Rec. frail. chini. 66, 859-864.

I,:icnssagnc:, A. 19-16, “Les cancers produits par lcs substances chimiques endogenes.”

Lncnssagnc, .I. 19-18. “Lcs cancers produits par lcs substances chimihues endogGnes.”

Lislr, E. B. 1951. Cancer Rescarch 11, 153-156. AIagat, >I., and l30n6nie, R. 1951. Conipt. rend. 232, 1657-1659.

Hcrniann, Paris.

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Miller, J. A., and Miller, E. C. 1953. Advances in Cancer Research 1, 339-396. Pag6s-Flon, M., Buu-Hol, N. P., and Daudel, R. 1953. Compt. rend. 236, 2182-2184. Pullman, A. 1945. Compt. rend. 221, 140-142. Pullman, A. 1946. Bull. Cancer 33, 120-130. Pullman, A. 1947. Ann. chim. (Par is ) 2, 5-71. Pullman, A. 1953a. Compt. rend. 236, 2318-2320, Pullman, A. 195313. Compt. rend. 236, 2508-2510. Pullman, A. 1953c. Compt. rend. 237, 173-l75. Pullman, A. 1953d. J . chim. phys. 60, 548-550. Pullman, A. 1954. Bull. soc. chim. (France) 21, 595-603. Pullman, A., Berthier, G., and Pullman, B. 1950. Acta u n i o n intern. contre cancer 7,

Pullman, A., and Pullman, B. 1946. Rev. sci. 84, 145-158. Pullman, A., and Pullman, B. 1948. Acta r ~ n i o n intern. contre cancer 6, 57-63. Pullman, A., and Pullman, B. 1954. Bull. SOC. chzm. (France) 21, 1097-1104. Pullman, A., and Pullman, B. 1955. “La structure nioleculaire e t la cancerisation par

le substances chimiques.” Masson, Paris. Pullman, A., Pullman, B., and Berthier, G. 1953. Cornpt. rend. 236, 2037-2069. Pullman, B. 1954a. Compt. rend. 238, 1935-1937. Pullman, B. 1954b. Cahiers phys. 48, 42-66. Pullman, B., and Baudet, J. 1953. Compt. rend. 237, 986-988. Pullman, B., and Baudet, J. 1954. Compl. rend. 238, 964-966. Pullman, B., and Pullman, A. 1952. “Les theories Clectroniques de la Chimie or-

Roitt, I. M., and Waters, W. A. 1949. J . Chene. Soc., pp. 3060-3062. Roitt, I. M., and Waters, W. A. 1952. J . Chem. Soc., pp. 2695-2705. Rusch, H. P., and Wasley, W. L. 1942. Cancer Research 2, 422-426. Scrocco, E., and Chiorboli, P. 1950a. -4nn. chzm. (Rome) 40, 395-399. Scrocco, E., and Chiorboli, P. 1950b. Ann. chim. (Rome) 40, G60-661. Sixma, F. L. J. 1953. Rec. trav. chim. 72, 273-284. Sixma, F. L. J., and Wibaut, J. P. 1952. Rec. trav. chin^. 71, 473-481. Steiner, P. E., and Edgcomb, J. H. 1952. Cancer Research 12, 657-659. Steiner, P. E., and Falk, H. L. 1951. Cancer Research 11, 56-63. Truhaut, R. 1947. Biol. m a . (Paris) 38, 1-47. Velluz, R. 1938. Compt. rend. 206, 1514-1516. Wheland, G. W. 1942. J . Am. Chem. Soc. 64, 900-908. Wolf, G. 1952. “Chemical Induction of Cancer.” Cassell & Co., London. Wood, J. L., and Fieser, L. F. 1941. J . Am. Chem. Soc. 63, 2323-2331. Zinke, A. 1951. Monatsh. Chem. 82, 387-388. Zinke, A., and Zimmer, W. 1950. Monatsh. Chem. 81, 783-786.


ganique.” Masson, Paris.

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Some Aspects of Carcinogenesis


Cancer Institute, Milan, Italy Page

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 172 XI. Cancer as a Regressive Process in General Pathology. . . . . . . . . .

111. The Energy Changes in Carcinogenesis (The Concept of Entropy in

IV. The Supposed Oxidative Metabolism in

nts and Cell Constituents 185

Pathology). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Lipoidolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

V. The Interaction betwc 1 . The Localization of Carcinogenic Hydrocarbons in Cells. The Concept of

2. The Reaction of Some h Proteins. The Interaction between Carcinogenic Hydrocarbons and SH-Groups. . . . . . . . . . . . . . . . . 188

3 . Some Aspects of the Electronic Theory of Carcinogencsis . . . . . . . . . . VI. Cancer as a Problem of Protein Chemistry., . . . . . . . . . . . . . . . . . . . . . . . .

1. Protein Biosynthesis and Its Deviations (Antibody Formation, Virus

2. Some Observations on Proteins in Cancer Tissue.. . . . . . . . . . . . . . . . . . . . 201 3. Infrared Absorption Spectra of Cancer Tissue.. . . . . . . . . . . . 4. Ultrastructural and Immunological Considera 5. A Comparison of the h’eoplastic Transformation of the Cell with a

Multiplication, Carcinogenesis) . . . . . . . . . . . . . . . . . . . . . . . 194

Process of Protein Denaturation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 VII. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214


This contribution reviews somc problems of carcinogenesis, with the aim of illustrating certain special aspects which have been specially con- sidered by us, and may be of some interest even now, when cancer re- search has attained an enormous development and almost every related subject has been deeply examined and discussed. Of course speculative points of view are often associated with experimental work; and personal or other Italian contributions are set forth in the framework of modern researches in general. Hence, some sections of this article may be a partial repetition of the work of other contributors. The problem of carcinogenesis is so complex and has so much stimulated the interest of biologists and medical men of late, that the elaboration of original ideas


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is growing more and more difficult. It is surprising how many precursors of modern conceptions can be found, e.g., in the five volumes of the pondcrous work of Wolff (1907-1928) published mostly before World War I.

The fundamental question of the nature of the malignant transforma- tion of cells still remains the most obscure point in cancer research; and any new consideration may represent a spark which can kindle a future more important fire.


It has long been disputed whether malignant growth is due to some systemic influence or rather to an intrinsic change in the proliferating cells. At the present time the latter opinion tends to prevail, and is brilliantly supported by experiments demonstrating the malignant trans- formation, under certain conditions, of normal cells growing in vitro (Earle and Settleship, 1943; Earle et al., 1950; Gey et al., 1919; Goldblatt and Cameron, 1953). Of course systemic influences on tumor growth occur; but the starting point of malignancy is to be looked for in one or a few cells directly affected by a carcinogenic agent. The cancer problem is a problem of cellular pathology. Cancer means the synthesis of a sort of living matter which, according to an expression of Graffi (1940), is deaf to the correlating and differentiating stimulations of the body. The classic general pathology serves to distinguish regressive and progressive changes of cells: the former are metabolic disorders, damaging the physiological functions more or less and ha\+ig their highest degree in the death of the cell (necrosis); the latter are adaptive and regenerative processes, aiming to restore the organs or tissues to their normal condition, and generally involve an increase of physiological activity as well as a synthesis of living matter by means of cellular hypertrophy or cell multiplication. By some, tumors were considered as progressive processes because of the cell proliferation and new formation of living matter; but such matter is in no way includrd in the organization of the body and is either a useless pro- duction (henign tumors) or a dangerous, aggressive outgrowth (malignant tumors). So the malignant tumor may be looked upon rather as a regres- sive process, coming near to other regressive (degenerative) changes, with diminution or loss of physiological specialization of the cells. We know that very often the malignant growth represents the outcome of a regener- ative or hyperplastic process, prolonged and in some way disturbed; we see here a real progressive change turning into a regressive one involving a degradation of the cell life. We may perhaps quote here the view of A. Fischer and R. C. Parker (1939), and of A. Fischer (1935), that in can- cer the high lability of the cells is the primary change, and vigorous prolif-

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eration should be considered as an obvious consequence, i.e., an attempt to regenerate the tissue possessing an abnormally high death rate of the cells (cancer being “ a never-healing wound”). On the other hand, we cannot but remark that the prelethal condition of many tumor cells and the well-known necrotic alterations in cancer tissue may be largely due also to the deficiencies of blood supply and therefore may be secondary in nature, not representing a primary feature of the malignant cells. At any rate the abnormal relationship between blood supply and neoplastic growth may itself be considered as an expression of the low and defective level of organization, and as a proof of the disordered character of the process of growth as a whole. Such growth according to some authors should indeed be considered from a unitarian point of view, as consisting of parenchyma and stroma, both affected by the neoplastic transforma- tion, the latter not being merely an accessory production as is usually believed. We owe a valuable concept of this subject to Hueck, in relation to mesenchymatous (1939) and epithelial tumors (1941), including some carcinomas, where the stroma is not simply a support for vessels but participates in the neoplastic process and contributes to the tumor type. This organoid conception may help to explain the beneficent effect of sex hormones in the treatment of cancer of the prostate and breast; a modification of the abnormal hormonal background and a direct action on the malignant cells are not sufficient to account for the result; and an indirect effect through an activation of the stroma may also be sus- pected (Sirtori and Grattarola, 1947; Meythaler and Handel, 1952; Emerson et al., 1953). Of course, we may not generalize an organoid conception of tumors; and in any event in malignant tumors the paren- chymal cell is still the determining and leading feature of the morbid process.

The findings of both earlier and more recent investigators point to a structural disorder of the cancer cell, affecting chiefly the inner organizers, e.g., the nucleolus-heterochromatin system (Caspersson and Santesson, 1942). Barigozzi et al. (1946, 1948, 1950) remark on the importance of studying the nuclei of resting, nondividing cells and lay stress on the increase of chromocenters in the resting nuclei of tumors-a feature which may have diagnostic significance.

I have earlier pointed out (1939a) that phytopathology allows a specially clear understanding of the regressive character of certain growth processes in plants. Thus, according to Ni5mec (1939) two processes may be considered as regressive in plants: those where the cell structure is simplified, the cell content is diminished, and the cell undergoes degenera- tion; and those where transformation into embryonic meristems occurs. Such meristems can undergo uncontrolled growth.

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A living organism is an open steady system, exchanging matter and energy with the environment. It takes up food (substances having high contents of free energy), which is employed for many different functional activities and undergoes many transfers through chemical systems. The organism is not a heat machine but a chemical one, where most of the energy is used t o charge colloidal systems, whence i t is released by means of adequate weak stimulation. A part of the energy is employed in the synthesis of the very complex constituents of the body; this part is ob- viously larger during the growth of the organism and must be taken into particular consideration in every pathological growth process. At the end, the energy is liberated from the organism as mechanical work or heat, eventually as electric or light energy. There is no reasonable doubt that the first law of thermodynamics has its full validity in biological sys- tems (intake of energy equals output). There has long been discussion con- cerning the application of the second law, which is a statistical one and which is known as the principle of increase of entropy in natural (irre- versible) phenomena (for a full but clear discussion, see Gray, 1931 ; Schro- dinger, 1945; von Bertalanffy, 1951). We may remember here that when energy is supplied to a system, only a portion of i t can be taken up and used as free energy; whereas another portion is degraded to heat energy, i.e., converted into heat motion of the molecules and therefore unable to be recovered as free, useful energy. Such bound energy is measured by a physical quantity called entropy. At the absolute zero point of tempera- ture (roughly -273°C.) the entropy is zero, while the heat motion of molecules and atoms is abolished. If a substance is brought into any other state by small reversible steps, the entropy increases by an amount which is computed by dividing each small portion of heat by the absolute tem- perature a t which i t was supplied, and summing up all these small con- tributions. Therefore the unit in which the entropy can be measured is caloric per Oh’. (where OK. means absolute degrees). A clearer concept of entropy can be gained by a consideration from the point of view of probability according to Boltzmann ; the second law of thermodynamics states that in every transfer of energy there is a tendency towards the distribution of energy and of particles a t random. In natural events in inanimate systems a certain state of matter and energy can transform only into a more probable state, i.e., a state more approaching uniformity of distribution, which is, of course, more probable than a condition of differentiation and inhomogeneity. The Boltzmann formula is expressed as S = K In P , where S = entropy, P = probability, and K is a constant

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(Boltzmann’s constant). The entropy is to be regarded as proportional to the logarithm of the probability. It means that S increases when the degree of random distribution of the constituents of a given system in- creases. Such random distribution also corresponds to disorder in the system; and the second law is consequently also named the law of in- creasing disorder in natural events, which have a unique tendency towards maximum entropy (thermodynamic equilibrium). We may remark that the word “disorder” for the physicist means uniformity, lack of energy gradients, and lack of orderly distribution ; therefore entropy may be expressed as the logarithm of disorder (Schrodinger, 1945). The concept of entropy may be illustrated very well, in almost popular form, by the following words of Gray: “We may imagine a bag of soot and a bag of flour. If we empty them carefully into a barrel, the two powders are segre- gated into two heaps in contact with each other. If we begin to shake the barrel the particles of soot and flour begin to mix together-and the longer we shake the more the powders are mixed up: short of picking out each individual particle or using some method which discriminates be- tween a grain of soot and a grain of flour, we cannot separate them. If we replace the flour and soot by molecules, half of which have a higher degree of thermal agitation than the other, then the longer they are left in contact the more uniformly will this energy be distributed throughout the whole mass. The degree of complete distribution of energy at random throughout the system is known as the entropy of the system. Since the most probable state is that condition where the energy is distributed completely at random, the most probable state is that of maximum entropy. Once this condition is reached in a macroscopic system of mole- cules i t is highly improbable but not impossible that the process can reverse itself spontaneously, just as it is possible but highly improbable that by shaking the flour and the soot long enough the two types will again be completely segregated from each other. It will be noted that the law of progression towards maximum entropy breaks down in small systems, since the entropy of the system is a function of the probability of state.”

Now in living organisms we find a quite different sequence of events. There occur of course some processes obeying the second law of thermo- dynamics, i.e., with increase of entropy: as such may be considered the catabolic processes, liberating energy (mechanical work, heat, etc.) and involving breakdown of highly complicated, unstable compounds. But the most characteristic processes of life are the anabolic ones, whereby from more simple compounds very complex molecular structures are synthesized, i.e., very improbable states are originated from more prob- able ones, in opposition to the statistical law regulating the events in the inorganic world. The organism concentrates order in itself (Schrodinger,

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1915), working against the law of increasing disorder. I n life we have, a t least as far as synthetic processes are concerned, a negative variation of entropy. In this way the organism is prevented from falling into decay as inorganic bodies do; and death means a suppression of the antientropic efficiency of life and therefore the restitution of statistical law, directing events towards thermodynamic equilibrium. Bertalanffy (1951) says that in the organism a principle dominates which is really foreign to theclassi- cal energetivs of the closed system; but that of course the second law resumes its significance if the organism is considered as an open system (organism + environmeiit). We do not, want to give here an exhaustive discussioii on such \-pry difficult, topics. It may he sufficient to assert that life is a very unlikely event from the physicomathematical point of view. The existence of a protein or :in enzymc molecule could be considered as nearly impossible hy a physicist. or mathematician quite ignorant of biology (Imomte du Koiiy, 1939). If we consider again the example adopted by Gray, and look on the.different particles as belonging to a living system, we then sce the particles segregating from each other spon- taneously and in very orderly fashion. During embryonic development more and more complicated structures appear; in every cell me find differentiations, sometimes polarities, adaptations, functional specializa- tions. Schrodinger suggests that organisms feed on negative entropy in order t o counterbalance the positive entropy produced.

The growth and shape of organisms and their parts are the true expres- sions of life, demonstrating the quite peculiar position (Eigenst&uiiqleeit) of living beings in the world (Buchner, 1950). When the inanimate system of proteins, fat, and salts, found in the yolk of an egg, is converted into a living embryo, the process occurs with little or no loss of energy in the form of heat and yet the final product has more free energy than the origi- nal system, as Gray points out. Such considcrations may indeed be ap- plied to all growth processes, t o normal as well as t o pathological ones.

After these general remarks we may divide all pathological changes of cells on the basis of the entropy concept (Rondoni, 1939a, 19-12, 1943-44) : the regressive processes are associated with a positive variation of entropy in so far as there is more or less considerable breakdown of cellular com- ponents with diminution of free energy. The progressive processes consist chiefly in the building or rebuilding of complex protoplasmic components, with accumulation of free energy and therefore negative variation of entropy. In latter processes, new structures, differentiations, and energy gradients are evolved; i e . , less probable states are organized from more probable, and the biological level is elevated.

To turn now to tumors, their position as a regressive event in cell life may be easily recognized. There is in neoplastic tissue a more or less

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intense synthesis of protoplasmic components, namely, an inhibition of the mechanism controlling protein synthesis, so that cellular proteins are built up at an increased rate. But such components generally remain a t a lower degree of differentiation and adaptation, the polarities of the parent normal cells are often lost, and the organization of the tissue is simplified. Not only is the microscopic structure modified, but very likely the ultra- structure and the chemical structure too have partly lost their normal high degree of specialization; one may speak (Rondoni, 1933) of a chemical dedigerentiation paralleling the histological one. An excellent example of such chemical dedifferentiation may be found in the extensive enzymatic studies of Greenstein (1949, 1954) , demonstrating an enzyme pattern common to many tumors with loss of specialized activity. Therefore the synthesis of neoplastic living matter requires a positive variation of free energy which nevertheless remains at a lower level in comparison with the synthesis of normal protoplasm. In other words, the living system of cancer cells, possessing a higher degree of random disposition of its con- stituents (expressed by dedifferentiation), with loss or diminution of energy gradients, may be considered as having less negative entropy or more positive entropy than the related normal living system. If we say that entropy is proportional to the logarithm of probability or disorder, we recognize that in the neoplastic transformation of cells an increase of disorder takes place, i.e., of entropy. It may sound strange when we say that the cancerous condition is more probable than the normal one; but the former implies a more or less pronounced simplification of some struc- tures, a reversion to a less specialized function-a state of the living system requiring a minor exertion of the synthesizing activities. If such activities were compared, in the normal cell, with the elevation of a heavy body as far as 100 (of any given length unit), they could be said to cor- respond, in the cancer cell, to an elevation, for instance, as far as 70. In the change from the normal to the malignant condition, therefore, there is a fall of the energy level from 100 to 70, i.e., a real degradation of energy. Tumor growth, as well as every form of growth, involves a struggle for life against the second law of thermodynamics; but in the malignant tumor the struggle is less effective.

It is known that some undoubtedly malignant tumors show a high degree of differentiation, going further than that of the parent normal cells (prosoplasia: e.g. , intense keratinization of epidermoidal carcinomas) ; or show qualitatively abnormal differentiation (e .g . , mucoid secretion of mammary carcinomas). But these facts cannot contradict the general principle : such specialized productions are the expression of degeneration with severe metabolic disorders. Let us consider, for example, the strong keratinization occurring in some epidermoidal cancers; here the inter-

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mitotic cells transform into postmitotic differentiating cells (Cowdry, 1942), as in normal epidermis, but the differentiation goes beyond the normal degree and the functional needs, representing a form of precocious death of the cells. Many degenerations have their normal prototypes, with physiological significance; and on the other hand, every true functional and morphological differentiation brings in itself an impairment or limita- tion of some other activities. Sirtori (1952) has called such overdifferen- tiated cells or strains of cells which can be found in some tumors “ D cells,’, in addition to the A and B cells of Caspersson and Santesson (1942). The presence of such cells does not invalidate the malignancy of a tumor. As a matter of fact,, we find in most cancers a variety of cell strains, very likely with different degrees of viability and growth vigor. A malignant tumor is a community of individual cells, having on the whole a faulty organiza- tion. A study of Davids (1953) on eye melanomas may be quoted here; various cellular strains or races, possessing different amounts of a tumor- specific substrate (Nothdurft, 19-18, 1949) may be present in every tumor, involving different degrees of differentiation, pigment formation, etc. According to Barigozzi (1950), genomic and chromosomal mutations may occur in every growing tumor, producing secondary changes in the genetic constitution of the cells.

In benign tumors the defects of organization and differentiation are slight; such tumors are often more or less correlated with abnormal pro- gressive processes or with embryonic malformations.

We may suppose that a carcinogenic reaction is comparable to an exergonic reaction, by which a system possessing a given level of energy goes over to another state with a lower energy content. The transition from the initial state of the system to the final involves the overcoming of an energy threshold or potential barrier, according to general thermo- dynamic theory. If we consider the reaction AB + MN -+ AM + NB, the rate of the reaction depends upon the number of impacts between the molecules. Rut only a relatively small number of impacts is efficient, i .e., those which happen between molecules possessing an energy level higher than the mean level of the initial system. Such molecules endowed with high energy are called active, in opposition to the normal ones. The activa- tion can be produced by photons (photochemical reactions) or by heat, which supply the energy necessary for the increase of the number of re- acting molecules up to the point of the effective reaction. Such a point represents the critical energy, and corresponds to the above-mentioned threshold. The difference between the critical energy and the initial energy level of the system is called the relative critical energy or activation heat. Boretti (1943) has described the energy levels of the initial and final state, i .e. , of the system of reacting molecules (AB + MN) and of the

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reaction products (AM + NB), with the potential barrier which must be overcome. The activation heat is measured by the height between the starting level and the peak of the curve, and can be calculated by the reaction speed a t different absolute temperatures.

A positive catalyst may be considered as an agent capable of lowering the relative critical energy, i.e., of reducing the height of the potential barrier or energy threshold. Enzymes, as organic catalysts, have such a function, themselves being compounds with a low activation heat.

In cancer research such an estimation has been attempted by Boretti (1943) in the case of a peptidase from normal and neoplastic tissues (rat liver and subcutis, rat benzpyrene sarcomas), using leucylglycylglycine as a substrate; some differences were detected, but more work is required before this important point can be considered as settled.

In general we may conclude that the carcinogenic reaction can be started by a supply of energy (by radiations) or by agents which are in some way able to lower a potential barrier and t o permit (as in mutations) the quantum jump needed by that molecular rearrangement in living mat- ter which we call carcinogenesis. According to 0. Schmidt (l940,1941a,b) carcinogenic compounds have a low heat of activation and therefore act as catalysts; in other words an excitation state, sufficient for the produc- tion of the carcinogenic reaction, can be reached in their presence by a relatively small energy supply (see also Section V,3).

A law may be established as to the i n t ens i t y of the carcinogenic stimu- lation (by chemical as well as by physical agents) : the stimulation must exert on cells a damaging action comprised in a certain range of intensity (‘( zonale Schudigung,” Rondoni, 1938b). A too strong stimulation brings about a necrotic lesion, whereas a weak stimulation produces only tran- sient reversible changes. The cell cancerization is a response to an ade- quate degree of cellular damaging stimulation. A clear demonstration of such a law may be found in some recent observations of Shubik and Ritchie (1953); Swiss female mice received either one, two, or three skin applications of a 0.2% solution (in mineral oil) of 9,10-dimethyl-l, 2-benzanthracene followed by croton oil repeatedly. A decreasing yield of tumors was recorded with increase in the number of hydrocarbon appli- cations, and the authors discuss the necrotizing action of the carcinogenic hydrocarbons. The intensity range within which a given agent can induce cancerization is very likely different for every animal species and every cell type.

A much more extensive study of quantitative conditions in carcino- genesis is due to Druckrey and Kupfmuller (1948), who discuss differ- ences between mutation and carcinogenesis, the absence of effect of dose fractionation, ete.

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For many years there has been a strong tendency among investiga- tors to consider the malignant transformation as etiologically correlated with a particular type (glycolytic type) of the metabolism of carbo- hydrates.

As regards the present state of oxidative metabolism (chiefly of car- bohydrate) in tumors, we may refer to the conclusions reached by C. Ileidelberger (1953) on the basis of application of radioisotopes. Such conclusions agree with the results obtained by Wennec et al. (1952). It has definitely been established that the oxidation of substrates in tumors takes place by means of the Krebs cycle. There appears to be no qualita- tive difference in the over-all oxidative metabolism of normal and tumor tissues, and tumors appear able to carry out the oxidations necessary to satisfy the energy requirements for the synthetic processes that are constantly demanded by a rapidly growing tissue. However, there is evi- dence of some quantitative differences between some tumors and the more metabolically active of the normal tissues. But we must be very careful in generalizations from particular experiments. As an example of the difficulty of assessing the real changes in enzymatic equipment corre- sponding to carc*inogenesis, we may quote Table I in an article by Potter (1!)31), where the contents of succinosidase in tumors (homogenates) of rats and mice (including hcpatomas), and in normal tissues, are given: the valucs for tumors are much lower. But certain data of Carruthers and Suntzeff (1947) are in contrast, normal skin epithelium of mice showing a lower succinosidase content than the samc epithelium after canceriza- tion by mcthylcholanthrene. Different tumors may therefore give different results; and the influence of necrosis should not be forgotten (see below). Furthermore, the results obtained by different techniques (slices, homoge- nates, animal experiments) are not at all comparable (Potter, 1951). It is believed that the enzymes of the Krebs cycle are contained in the mitochondria1 fraction of the cells. R’enner and Weinhouse (1952, 1953) found that mitochondria from neoplastic tissues, when fortified by addi- tion of diphosphopyridine nucleotide (DPN) , could readily oxidize pyru- vate and the components of the citric acid cycle; such a DPN requirement of tumor mitochondria could be attributed to a loosening of the binding of DPS, which is released from the labile mitochondria1 structure, rather than to an original deficiency of the nucleotide in the tumor mitochondria. The mitochondria of neoplastic cells have essentially the same content of oxidative enzymes as their normal counterpart. Differences of results may be due to various degrees of inactivation during preparation of the

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Absolute Values in mml. Relative Values

Normal Tarred Normal Tarred Organ Organ Organ Organ

-~~ -

0 2 COZ 0 2 and 0 2 COI 02 CO? cot


49.24 43.45418.7304.8 100 850.3701.5 232.1 166.6 333.0164.3 100 143.5 98 .6

31.21 - 394.6282.2 100 1264.5 - 159.7 241.2 638.6529.1 100 399.8219.3 41.02 - 307.1100.7 100 748.7 - 79.66 68.27281.8251.5 100 353.7368.4 32.55 32.52266.8257.8 100 819.7792.7

Mean values 100 513.4436.1

_ _ - - 353.5263.2 -

homogenates, slices, or extracts in relation to differences in fragility of the mitochondria. The suggestion that mitochondria of tumor cells are more fragile than those of liver and other normal organs may bevery fruitful, giving some support to the conception of the neoplastic process as a regressive cellular change. We know that mitochondria1 lesions char- acterize the more severe degenerative changes known as cloudy swelling and vacuolization, involving enzyme defects as well as structural altera- tions (Ciaranfi, 1953). Of course, further work is required before all these

TABLE I O? Consumption and C 0 2 Production in Normal and Tarred Ear Auricles in Rabbits

9 9 0 8 3

g F I

a 2


0.88 0 .73 0 .72 0.49


1.51 0.82

0 .86 0.89 0.999 0.966

- - 0.715

- -

4 5 6 7 9 10 12 13


1 6 1 7 3 2 10 10


C02/0? O2 Consumption and C 0 2 Production per 100 g. Organ in 1 Minute


points can be definitely settled. However, the idea that enzymatic activity may depend not only on purely quantitative factors but also on factors of distribution and binding of enzymes in the structure and ultrastructure of cells, may a t any rate be considered. On the other hand, experiments i n vivo would surely give much more convincing information on the real behavior of tumors in regard to oxidative metabolism than all the number- less experiments in vitro. Such experiments are very scanty; therefore it may be of interest to mention here some old experiments carried out by Deotto (1936) on the over-all respiration of tissues during carcinogenesis. Deotto tarred one ear auricle in rabbits, leaving the other as untreated control; at different times after starting the treatment the estimation of

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O2 and C0.r content i n arterial blood (a. femoralis) and in the venous blood from both auricles was undertaken by means of the Barcroft ap- paratus. The blood flow (volume per minute) i n normal arid that in tarred organs, as well as their wet and dry weight, were determined. This very careful experimental work allowed a precise calculation of the gaseous exchanges and of the oxidative metabolism in the tarred and in the normal organ of every animal. Histological examination of the tarred skin was made in every case, in order t o establish the presence and the stage of the precancerous and cancerous lesions induced by tar. Table I summarizes the main results of Deotto's experiments.

Xn enormous increase of O2 consumption and COZ production, ie., a marked elevation of the respiratory process, was an obvious arid regular finding in the tarred ear. Furthermore, Deotto studied in rabbits, tarred as above, the in zdtro dehydrogenase activity (m-dinitrophenol or methyl- ene blue as acceptors) of the tarred skiii and of control skin. Higher activity was observed in skin showing an atypical and invasive growth of the epidermal epithelium.

This increase of osidative metabolism lasted longer than the merely inflammatory changes, and hence may be considered as at least in part dependent upon the growth processes going on in the skin.

This study, carried out, at a time n-hen in ziilro experiments on tissue slices were the usual procedure for investigating metabolic changes, pro- vides some support to an osybiotic conception of growth. It may be mentioned that Bori (1936), studying by the same technique the respira- tion of the ear auricle after subcutaneous injection of scarlet-red dissolved in olive oil, found no increase or only a very weak increasc in O2 consump- tion. Such a finding, when compared with the results of Deotto, indicates that the epithelial, noiimalignant proliferation due to the azo dye (Fischer's phenomenon) involves a much less corisiderablc elevation of the tissue respiration ; and strengthens the conclusions that increase of the oxidation processes is associated with malignant growth.

Dickens (1936) has demonstrated that some thiazine and phenazine dyes like thionine and pyocyaniiie act, as respiratory catalysts, enhancing respiration and inhibiting glycolysis. Beltrami (1938) found that thionine promotes the growth of cultures of embryonic chicken fibroblasts; and Soresina (1938) found the same phenomenon with pyocyanirie in the earlier period of culture grou-th. Piemonte (1938) observed earlier development of warts on the skin of mice painted with 3,4-benzpyrene when treatment with thionine (painting) was added; and Beltrami (1940) found that similar treatment with pyocyanine during benzpyrene painting induced a more precocious occurrence not only of warts but also of car- cinomas on the skin. It may be inferred that these respiratory catalysts

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can favor autonomous and atypical growth. Furthermore, the same sub- stances accelerate the development of fertilized eggs of the sea urchin (Deotto, 1939a) and promote the regeneration of parts of the body in coelenterates (Deotto, 193913). From these observations and from many other data (reviewed by Rondoni, 1940, 1946a, 1949b) the fundamental importance of oxidation as a source of energy for growth may be inferred. Indeed, the most actively proliferating cells of tumors (Caspersson’s A cells) are found in the neighborhood of blood vessels, where oxygen uptake may be supposed larger and the oxidative metabolism more intense. On the other hand, a frequent and often prominent feature of tumor metabo- lism is the anaerobic and aerobic glycolysis, demonstrated by the studies of 0. Warburg (1926), working with manometric methods on tissue slices. Although the fundamental observations are doubtless correct, it was soon recognized that aerobic glycolysis is not a t all specific for tumors (Penti- malli, 1927, and others) and that some tumors in fact possess a high respiration rate. Among the many researches on this subject may be cited the work of Berenblum el al. (1940), in which the behavior of the oxidoreductive metabolism during cell proliferation was carefully fol- lowed; measurements were made of the oxygen uptake, aerobic and anaerobic glycolysis, and respiratory quotient of normal skin epithelium and of the Shope papilloma. The values obtained for normal epithelium were found to be almost identical with those of the Shope papilloma and similar to the values for many squamous carcinomas quoted in the litera- ture. It was concluded, theref ore, that aerobic glycolysis and a low respira- tory quotient of a glycolyzing tissue are both normal physiological proc- esses and do not represent a pathological disturbance characteristic either of tumor growth or any other lesion.

Attempts have more recently been made to correlate glycolysis with other modern observations; for example, Wenner and Weinhouse (1953) suggest that the high level of coenzyme DPN in the soluble portion of the cytoplasm (supra) may account for the high glycolytic rate of intact tumor cells. Many years ago (Rondoni, 1933, 1936a,b) I expressed the opinion that glycolysis represents simply an emergency measure for cells whose structure and ultrastructure are damaged in such a way that more exacting respiratory process cannot be performed. Whereas respiration is more sensitive to changes of the colloidal protoplasmic systems, glycolysis can remain active in a damaged cell and be adopted as the main source of energy despite its minor energetic efficiency. Glycolysis is con- sequently a secondary adaptation of little significance for malignant growth; it is rather the functional aspect of the regressive changes occur- ring in tumor cells. All cells, when submitted to damaging agents, can show aerobic glycolysis, very often after a period of increased O2 uptake

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(respiration) due to increased membrane permeability (see Druckrey, 1935, 1936; and Brock et al., 1938, 1939). The oxidoreductive metabolism of tumors is chiefly a metabolism of tissues submitted to damage of various types; such metabolism may be fixed as an hereditary character of the malignant cell race (Druckrey, 1935). The glycolytic type of metabolism must therefore be considered as a result, rather than a cause, of malignancy. An important study of Olson (1951), comparing normal liver and hepatoma, asserts that no qualitative differences exist between these tissues in regard to carbohydrate metabolism; but some quantitative differences may be very evident: the liver largely utilizes glucose for synthesis of glycogen, whereas the hepatoma diverts the glucose uptake into the glycolytic pathway. Here the glucose carbon may become avail- able for synthesis of fatty acids, amino acids, purines, etc., or for further oxidation to COe. In this way glycolysis in tumors may facilitate certain synthetic processes of growth, whereas the physiological synthesis and the storage of glycogen (characteristic for liver) may be impaired. The neoplastic liver betsomes an extrahepatic tissue (Olson, 1951). We may see here a case of true chemical dedifferentiation (Rondoni, 1933).

It is known that the energy produced by oxidation and glycolysis is employed to phosphorylate adenosinediphosphoric acid (ADP) and to produce adenosinetriphosphoric acid (ATP), a compound containing high-energy phosphate bonds, which can transfer phosphoric acid radicals to other acceptors and supply energy for chemical work, e.g., for synthesis. In other words, the generation of such energy-rich phosphate bonds (phosphorylation) must be coupled to energy-yielding reactions (e.g., oxidation of a pyridine nucleotide by molecular oxygen via the flavin and the cytochrome systems). As Fruton and Simmonds (1953) recognize in an excellent review of the subject, relatively little definite information is available about the enzymatic mechanism involved. The exact mecha- nism of the coupling of phosphorylation to oxidation is “one of the chal- lenging unsolved problems of modern biochemistry” (Zoc. cit., p. 486). These authors emphasize the view that the mechanisms of oxidative phosphorylation are largely associated with the particulate matter of cells; the experiments of Lehninger and Smith (1949) may provide evi- dence for the significance of mitochondria in the reaction.

I n view of the incompleteness of our physiological knowledge it is obvious that the behavior of cancer cells in regard to phosphorylation is quite obscure. The regressive changes occurring in such cells, including also alteration of colloidal systems and modification of the particulate condition, may give rise to some disturbance of the phosphorylating processes and of their coupling to oxidation. On the other hand, phos- phorylation of fatty acids and amino acids is a well-known prerequisite

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for the introduction of such compounds into the higher molecular com- plexes of lipids and proteins, i.e., for the synthetic processes of the proto- plasm, which are particularly enhanced in cancer cells; and the generation of energy-rich phosphate bonds is likely to be particularly active in a growing tissue. It seems that some substances disrupting the links be- tween oxidation and phosphorylation have an inhibitory effect on the growth of some tumors and act also as mitotic poisons; thus Cudkowicz (1954a) found that sodium azide exerts a certain inhibitory function on epithelioma induced in mice by 3,4-benzpyrene. The Italian acridine derivative Italchina corresponding to the antimalaria drug Atebrin showed, according to Rondoni and Necco (1951), a colchicin-like action on embryonic chicken fibroblasts cultivated in vitro; the acridine deriva- tive enhances the stathmokinetic action of colchicine (Rondoni and Necco, 1953). Now Atebrin seems to interfere with the coupling of phosphorylation to oxidation (Loomis and Lipmann, 1948) ; Loomis (1950) also found that the antibiotic aureomycin may have a similar action. But much more work will have to be done before we are able to assess these points. We may conclude that as yet no special etiological significance attaches to such changes in the oxidoreductive metabolism as occur in malignant degeneration of cells. It seems that it is not the energy-yielding reactions but rather the utilization of energy for growth which is altered in cancer.


CELL CONSTITUENTS 1. The Localization of Carcinogenic Hydrocarbons in Cells. The

Concept of Lipoidolysis

An approach to the problem of cancerous degeneration of the cell may result from the study of the mechanism of entrance of chemical carcino- gens into the cell, and of their primary localization in structural elements and their further distribution. This has been studied chiefly with regard to the powerful carcinogenic hydrocarbons, which can be easily followed by optical methods, although it must be kept in mind that other types of carcinogenesis may have their own and quite different laws. Thus Graffi (1940), after painting the skin of mice with 3,4-benzpyrene1 studied the distribution of the hydrocarbon in the epidermal epithelium by fluores- cence microscopy and found its highest concentration in the mitochondria. According to Graffi every type of cell concentrates benzpyrene in the chondriomes, and the utilization of this substance is indeed suggested for the demonstration of mitochondria in living cells of any kind. Hamperl and Graffi (1941) and Hamperl el al. (1942) confirmed these observations; the benzpyrene is probably dissolved by the lipids of the cytoplaam and

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could therefore disorganize lipoprotein structures such as the chondriomes and Golgi apparatus. The nucleus seems primarily unaffected; but, when during mitosis the nuclear membrane disintegrates, the nucleus may then be exposed to the hydrocarbon, which we know to be capable of inducing mitotic abnormalities in many cell types; Prada (1946), for instance, saw such abnormalities in regenerating skin epithelium of Triton after treatment with benzpyrene. The recent researches of Calcutt and Payne (1953) do not support the prevailing concept of fixation of the hydro- carbon i n the mitochondria. These authors developed a technique for separating both mitochondria and nuclei from liver cells by centrifuga- tion, and applied i t to the fractionation of mouse liver after intraperi- toneal injection of 3,4-benzpyrene. Examination of the resulting fractions shelved the persistence of unchanged hydrocarbon in mitochondria for up to 5 days, and in nuclei up to 21 weeks after injection. KO trace of benzpyrene was found in the supernatant fraction.

These different kinds of experiment are not quite comparable. The centrifugation method of the British authors segregates the cell organelles rather violently and may generate artificial surfaces which could then adsorb the hydrocarbon and retain i t more tenaciously than in the sur- faces of the natural, undisturbed polyphasic system of the living cell. The hydrocarbons possess a high surface activity and stick tenaciously to many suspensoids (Brock et al., 1938). On the other hand, the fluorcs- eence microscopy adopted by the German authors accounts very well for the primary concentration of the foreign substance; but i t gives no information as to subsequent events up t o the shape of malignant degen- eration. It should also be noted that the cells investigated by Calcutt and Payne (19.53) were liver cells, which seem to be very little responsive to the carcinogenic action of benzpyrene, whereas Hamper1 and associ- ates studied the highly sensitive skin epithelia.

At any rate it is difficult to dismiss thr likelihood that hydrocarbons, soluble in fat, nearly iiisoluble in water, find their way into the cell through the lipoid components of the cytoplasm and that the mito- chondria, rich in lipids, are able to localize them. We know that complex lipids and sterols play an important role in the maintenance of the struc- tural and ultrastructural organization of protoplasm, and a stabilizing function of cholesterol for protoplasm has been suggested by G. Pfeiffer (1931).

I t is probable that the penetration of large, rigid molecules of a hydro- carbon into lipoid and sterol structures may be able to alter such struc- tures and to modify their association with proteins. Thus Clowes et al. (1939) studied the interaction bet ween hydrocarbons and various sterols in vitro, the orientation of the latter a t interfaces being modified by the

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hydrocarbons. Such compounds as lipoids and sterols contribute largely to the constitution of limiting layers and membranes, and their molecules have a given orientation, resulting from the distribution of polar and apolar groups. In the molecular network of protoplasm (Frey-Wyssling, 1948) the lipoids occupy definite loci, and every change in their position or the resolution of their bonds with the peptide chains of the network can induce significant modifications in the network itself, such as the establishment of new bonds and an extensive rearrangement of the com- ponents of the whole system. Thus, Runnstrom (1942) supposed that in egg fertilization a lipoid modification could free certain groups and allow new bonds between the peptide chains, inducing a stabilization of the colloidal system and in such a manner facilitating cell division. Rondoni (1931a,b) suggested a process of lipoidolysis as a preliminary change occurring under the influence of carcinogenic agents, by lipoidolysis being meant more a physical than a chemical change in the structural lipids of the cell, so leading to increased dispersion of colloidal lipid constituents, disintegration or loosening of limiting layers, resolution of lipid-protein bonds, and a new orientation of lipid molecules at interfaces. This concept however still remains speculative and requires further experimental investigation, especially through studies of birefringence.

It is of course possible that all polycyclic hydrocarbons, noncarcino- genic as well as carcinogenic, have the same property, after penetration into the cell, of inducing a rearrangement of lipoids, and the clue to the specific carcinogenic action may not reside only in lipoid modification. Abnormalities of lipid metabolism of course occur in tumors themselves and (like respiratory inadequacy and glycolysis) are an expression of metabolic deficiency; some older data were summarized by Rondoni (1935). A recent interesting result is due to Medes et aZ. (1953). Although tumors can synthesize the carbon chains of fatty acids, the process is too slow to supply the lipid needs of a rapid growing tumor, so that the tumor must obtain some of its lipids from the host and hence may con- stitute a drain on its lipid resources. An abnormal balance of lipid con- stituents or the “unmasking” of some lipoid could have an influence on proteolytic cell enzymes (Rondoni, 1932) and so interfere with protein metabolism. A particular aggregation or configuration of lipoid compo- nents in tumors has been suggested by some investigators. Thus Ascoli et al. (1934) and Ascoli (1935) described a condition of particular looseness of the bonds of certain lipoids which could be extracted more easily than from normal tissues.

The question of specific lipoid antigens in tumors has also been widely discussed, e.g., by the school of H. Sachs (1932, 19371, by Hoyle (1940), and by others, although in general with little in the way of positive results.

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Immunological conceptions (antilipid immunity) also suggested some work by Rondoni (1926, 1928, 1930, 1931a,b, 1937) which indicated a nonspecific increase of resistance against transplantable tumors, induced in mice by lipoid fractions when injected together with a heterologous serum. The influence of alcohol extracts from different tissues on the growth of transplantable tumors was also investigated by Carminati (1938). A new development of the investigations on lipoidal antigens in cancer is due to the work of Penn (1952) who claims to have obtained specific serodiagnostic reactions with toluene extracts from cancer tissues. Also the attempts of Guidetti (1953) to cure cancer with lipoidal extracts may be mentioned. Here, as in many other sections of cancer research, more experience is needed, but a rearrangement of lipidic and sterolic components may likely be associated, in a specific or not specific manner, with carcinogenesis or precarcinogenesis.

2. The Reaction of Some Carcinogens with Proteins. The Interaction between Carcinogenic Hydrocarbons and SHGroups

Evidence is increasing that carcinogenic agents must in some way interfere with cell proteins in order to exert t,heir biological function. The work of E. C. Miller (1951) on the formation of protein bonds with derivatives of 3,4-benzpyrene in the epithelium of mouse skin, and of E. C. Miller and J. A. Miller (1952), reporting observations on protein- azo dye compounds in the liver of rats and including an interesting dis- cussion of the subject, may be quoted. Still more recently the investiga- tions of Wiest and Heidelberger (1953), using hydrocarbons labeled with C14, demonstrate a chemical binding (not a surface adsorption) of 1,2,5,6- dibenxanthracene-9, 10-C14 to the proteins of a skin fraction (epidermis and part of the dermis). The binding occurs only in vivo, between the hydrocarbon or its metabolites and nucleoproteins, or particulate or solu- ble proteins, but not with nucleic acids alone. These findings appear contrary to the opinion, advanced by Boyland (1952), of a direct action of carcinogens on nucleic acid of chromosomes. It is, however, possible that different carcinogens find their primary targets in different cell components; for instance, the nitrogen mustards studied by the school of Haddow might well react primarily with nucleic acids and so bring about the well-recognized chromosomal abnormalities which these substances induce.

As regards the hydrocarbons, an interaction with SH-groups of cell proteins, and particularly of enzyme-proteins, has also been considered. Reimann and Hall (1936) suggested an interference between sulfhydryl compounds and the carcinogenic hydrocarbons. Crabtree (1944) found that the local application of bromobenzene to the skin of mice inhibits

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and sometimes prevents the carcinogenic action of 3,4-benzpyrene; later (1945) he also found that maleic and citraconic anhydrides have a similar action. Such compounds bind SH-groups and depress sulfur metabolism. It is therefore supposed that they inhibit the action of the hydrocarbon in so far as the first stage of carcinogenesis involves a fixation of the carcinogen through an S-linkage to cellular enzyme. Lusky et aE. (1947) found that treatment of mice with British Antilewisite (BAL), during painting with 3,4-benzpyrene1 reduced the incidence of tumors.

Many papers deal with the influence of hydrocarbons on SH-activated enzymes in vitro; if the hydrocarbon is found to inhibit enzymatic func- tion, a blocking of the SH-groups may reasonably be supposed. After a few observations of Pozzi (1935) and of Rondoni and Beltrami (1937) suggesting a slight and not quite regular inhibition of autolysis in tissue extracts by 3,4-benzpyrene1 Gaetani (1946) demonstrated a partial in- hibition of papain by benzpyrene and methylcholanthrene, but not by noncarcinogenic hydrocarbons. This investigation was thereafter ex- tended by Rondoni and Bassi (1948)) Rondoni (1949a), and Rondoni and Barbieri (1950). A larger series of carcinogenic and noncarcinogenic com- pounds was tested on the proteolytic cell enzymes (cathepsins) of horse liver, a few carcinogens other than the hydrocarbon also being included. Representative findings are shown in Table 11.

From the data tabulated, there would appear to be some concordance between carcinogenic potency and inhibitory activity on autolytic enzymes, such activity also being indicated by the capacity to counteract the activating function of cysteine or British antilewisite (BAL). Certain discrepancies were ascribed by Rondoni to solubility differences and to other factors not clearly defined. However, earlier work by Feigenbaum had given negative results, and Mueller and Rusch (1943), describing the influence of benzpyrene on urease, showed that freshly prepared aqueous solutions of the hydrocarbon (with caffeine) are relatively nontoxic to this thiol-activated enzyme. However, irradiation of the hydrocarbon solution with ultraviolet light produced a t least two types of substance inhibiting urease activity; one of these agents could be detected after prolonged irradiation and may be considered as derived from the benz- pyrene, and the second inhibitory substance is very likely hydrogen peroxide. These investigators believe that benzpyrene acts as a photosen- sitizing catalyst in the production of HzOz during ultraviolet irradiation.

Later, irregular or negative results were obtained by Rondoni himself (unpublished) ; and the question whether the polycyclic hydrocarbons are able to react with SH-groups and to inhibit SH-activated enzymes re- mained unsettled, till a clear explanation of such contradictory results was provided more recently by the work of Mills and Wood (1953) on

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TABLE I1 Autoproteolysis in Liver Extracts

Effert on Biological Function Hydrocarbons or Other Substances Proteolysis

Benzpyrene . . . . . . . . . . . . . . . . . . . . .

I’yrene . . . . . . . . . . . . . . . . . . . . . . . Anthracerie . . . . . . . . . . . . . . . . . . . . . Phennnthrene . . . . . . . . . . . . . . . . . . . Perylene . . . . . . . . . . . . . . . . . . . . . . 1,2,5.Ci-IM,enzncridine . . . . . . . . . 1,2.5,0-Dibenzanthracene. . . . . . . . 3,4,5.6-Dibenzphenanthrene. . . . . . 1,2-I~irnethylchrysene. . . . . . . . . . . 1.2.5,6Dibenzfluoren~. . . . . . . . . .

9.10-Din~ethyl-1,2-henzantlirarene ,icenaphthanthracene, . . . . . . . . . . . Acet ylaminofluorene . . . . . . . . . . . . . o-Aniinoazotoluene . . . . . . . . . . . . .

Methylcholanthrene . . . . . . . . . . . . . ++ ++ 0 0 0 0 0 ++ 0 + -+ -+ -+ ++ -+

ca ca

Non-ca Non-ca Non-ca Non-ca SI. ca

ca Non-ca 81. ca

ca Xon-ca/Sl. ca

ca ca



Effecl on proleolysis: Bzological juncfion: + + = evident inhibition + = slight inhibition S1. ca = slightly carcinogenic - + = inhibition of cysteine activation orily

0 = no inhibition

ca = carcinogenic

Non-ca = noncarcinogenic

urease inactivation. These authors found that benzpyrene which had been stored in the dark had no effect upon the activity of urease; whereas benzpyrene which had been exposed in the solid state to ordinary light, inhibited urease. The inhibitory effect disappeared after prolonged storage in the dark arid mas prevented by cysteine. The treatment of the enzyme with increasing amounts of p-chloromercuribenzoate (blocking the SH-groups of the enzyme molecule), brought about an increasing inacti- vation, which was reinforced by benzpyrene. It was concluded that the hydrocarbon “covers” the SH-groups of the enzyme, although the chemi- cal nature of such a “covering reaction’’ still needs to be established. A further investigation of the interaction between benzpyrene and the SH-groups of protoplasm in zizto was attempted by Rondoni and Boretti (1947), who studied the content of SH-groups in a water-soluble protein fraction of various rat organs after injection of benzpyrene intravenously. The SH-groups were estimated in a water extract of the organs 1 to 21 days after the injection (by the modified method of Mirsky-Anson). Comparative determinations on normal nontreated rats were also made. The results were not, however, striking, and any decrease of SH-groups

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in liver proteins or in kidney proteins after benzpyrene injection must be treated with reserve (Table 111).


SH-Groups Expressed as mg. Cysteine for 1 mg. N Precipit. by Trichloroacetic Acid

Liver Kidney Muscle Lung

Mean values for 5 control rats 0.045 0.038 0.043 0.025 Mean values for 6 benzpyrene injected rats 0.035 0.030 0.041 0,021 ~~ ~ ~~

However, the liver is the organ where the greatest amount of injected benzpyrene is stored (Boyland and Weigert, 1947), and some blocking of the SH-groups of a soluble protein fraction in liver might reasonably be expected. According to Wood and Kraynak (1953) intravenous injec- tions of aqueous colloidal dispersions of 3,4-benzpyrene in rabbits and dogs produced a significant decrease of the serum and plasma sulfhydryl content which persisted for a considerable period of time after the dis- appearance of the benzpyrene colloid from the circulation. This effect was not produced by molecularly equivalent amounts of anthracene ad- ministered in the same manner. These authors believe that their results favor the theory that carcinogenic hydrocarbons can affect SH-groups, and this opinion is supported by the observation of Calcutt and New- house (1948) concerning the inhibition exerted by cysteine on the photo- dynamic action of benzpyrene on Paramecium: in this case the toxic action of the hydrocarbon on the protozoon in the presence of light is partially prevented by a sulfhydryl compound. We owe to Calcutt (1949) a study of the conditions under which benzpyrene can react with the SH-groups of certain compounds. The reaction products are isolated and described; on the basis of physical properties and of fluorescence spectra they are shown to be similar to derivatives of benzpyrene obtained from the animal body. Calcutt (1950) suggests that the oxidation of the hydrocarbon in presence of autoxidizing thiols is due to hydrogen peroxide formation.

We may conclude that an interaction of carcinogenic hydrocarbons with SH-groups of some protein fractions of protoplasm is very likely; and that such an interaction perhaps plays some as yet undefined role in carcinogenesis, at least in so far as hydrocarbons and related agents are concerned. A thorough study of the influence of benzpyrene and methyl- cholanthrene on some enzymes of isolated mitochondria from normal organs is due to Dianzani (1953).

Mitotic poisons, too, as substances interfering like carcinogens with the genetic structures of cells, exert some modifications of enzymatic

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functions (Rernelli-Zazzera, 1951a,b) and a mitotic poison, sodium cyanate, exerts an inhibition of papain, which is partly reversible by cysteine and is accounted for by a reaction of the poison with the SH- groups of the enzyme (Bernelli-Zazzera, 1950).

Perhaps further support for the significance of sulfur metabolism in carcinogenesis may be seen in the researches of Ghosh and Lardy (1952), who try to demonstrate the relationship between inhibition of the Pasteur effect in tumors and alterations in S-containing proteins. They obtained from tumors of various origin extracts which were able to inhibit the Pasteur effect in baker’s yeast; similar extracts from normal tissues were ineffective (except tests and semen). The active principle was demon- strated to be elemental sulfur. As the total sulfur content of tumors and that of normal tissues did not differ much from each other, i t is supposed that a structural difference exists between the S-containing proteins of tumors and normal tissues, involving a greater lability of sulfur in tumors. According to these authors, many facts implicate the ‘(strategic involve- ment ” of S-rich proteins in the phenomena of cell division.

3. Some Aspects of the Electronic Theory of Carcinogenesis

A physical interaction between chemical carcinogens and protoplasmic components was postulated by 0. Schmidt (1940, 1941a,b), who sug- gested a relation between electronic configuration and the carcinogenic activity of organic molecules. The school of Lacassagne, whose work has been reviewed by him (1949) and by A. and B. Pullman (1946, 1948), has greatly developed the electronic theory; further contributions and some modifications are due to Badger (1948), Greenwood (1951), and others. For a full review on this important theory we may refer to the recent article of Coulson (1953).

Further insight may be obtained by studying certain physical prop- erties of carcinogenic compounds which are known to be strictly depend- ent upon the electronic structure (e .g . , distribution of u-electrons in aromatic and other cyclic compounds). Of such properties, an important one is the behavior in a magnetic field, diamagnetism, which is related to the electronic orbits. Pacault (1941) compared the experimental values of the magnetic susceptibility ( x ) with the values that can be calculated on the grounds of the distribution of double bonds in aromatic com- pounds according to the KekulB formula as well as the (abnormal) Dewar formula; and found the experimental and theoretical (calculated) values to be consistent. Rondoni et al. (1949a) studied the magnetic suscepti- bility of some aromatic hydrocarbons and of a few other compounds, working with a magnetic pendulum of the Weiss type modified by Mayr (194445). All measurements were related to water ( x = 0.72 X

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Experimental results and values calculated on the assumption of a normal electronic structure were compared (see Table IV).

TABLE IV Magnetic Susceptibility ( x )


Methy lcholanthrene . . . . . . . 3,4 Benzpyrene . . . . . . . . . . . . Acenaphthanthracene.. . . . .

Acetylaminofluorene . . . . . . . Perylene . . . . . . . . . . . . . . . . .

3,4,5,6-Dibenzphenanthrene o- Aminoaeotoluene . . . . . . . .

1,2,5,6-Dibenzofluorene . . . .

Calcu- lated


-0.75 -0.73 -0.73

-0.66 -0.68 -0.73 -0.72 -0.64

Found Values

-0.68 f 0.04 -0.59 f 0.06 -0.73 f 0.03

-0.63 f 0.02 -0.69 f 0.03 -0.69 f 0.03 -0.73 f 0.03 -0.61 f 0.02


No. of Meas- ure- nents

48 36 48

48 42 42 48 36

Biological Function

carcinogenic carcinogenic Non-carcinogenic or

carcinogenic Non-carcinogenic carcinogenic Non-carcinogenic carcinogenic

slowly carcinogenic

While noncarcinogenic or very slightly carcinogenic compounds show a concordance of the two sets of values, in the case of carcinogenic sub- stances the experimental value of remains more or less below those calcu- lated. The difference is more striking for 3,4-benzpyrene than for methyl- cholanthrene and is less evident for weak carcinogens. The behavior of x in carcinogenic compounds may express an abnormal distribution of electrons, brought about for example by some electron-attracting or electron-repelling atomic groups, with formation of highly charged centers in the molecule, which are very apt to transfer energy on cell components. Rondoni et al. (1949b) investigated also the diamagnetism of some syn- thetic steroid hormones. Here the calculation of the theoretic values ac- cording to known magnetochemical data is difficult because of the molecu- lar complexity; and the comparative evaluation of experimental results remains more uncertain than in the case of hydrocarbons. In general the values of x found were approximately coincident with those calculated. Only for a powerful estrogenic compound, the methylester of dihydro- doisynolic acid, was there a clear difference between calculated and experi- mental values. It is well known that estrogens may be considered as conditionally carcinogenic substances (Butenandt, 1951). Mayr and Gallic0 (19504 also studied the diamagnetism of some estrogenic stilbene derivatives, finding an anomalous behavior in some of them. The same investigators (1950b) also demonstrated magnetic anisotropy in some

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aromatic hydrocarbons, without any clear difference between carcino- genic and noncarcinogenic compounds. This property has, consequently, no direct importance for biological function. However, i t may express a molecular dyssymmetry, on the basis of a particular orientation of orbital and spin movements of electrons; and a disturbance of some molecular systems of the cell may be facilitated by the entrance of such foreign, anisotropic molecules into the protoplasm. We have here, as in the case of solubility in lipids (see Paragraph l), a general property of a class of compounds (aromatic hydrocarbons) which perhaps contributes to ex- plain why this class, when other more subtle and specific properties coexist, includes many carcinogenic compounds. The chemical carcinogens are so numerous and belong to so many different chemical classes that it would be unwise to claim any general validity for the electronic theory. Rut some influence on the electronic behavior in cell proteins seems very likely i n certain cases, if we take into consideration that in proteins not only single atom groupings and bonds can determine the functional state, h i t also the cooperation of the whole molecule, and intermolecular inter- actions, can account for function. Szent-Gyorgyi (1917) admits molecu- lar orbits of electrons in proteins; and Evans and Gergely (1949) speak of unitarian eiicrgetic levels in proteins and of electronic interactions be twcn protein molecules. Compounds having centers of high energy levels may be partirularly apt to provoke long-range disturbances in protein systems, with rearrangements of components and functional consequences.

YI. CANCER AS A PROBLEM OF PROTEIN CHEMISTRY I. Protein Biosynthesis arid I t s Deviations (Ant ibody Formation,

V i r u s *If dt ip l ica t ion , Carcinogenesis)

It has been suggested by some investigators (Haurowitz, 1950; Haurowitz and Crampton, 1952a,b) that protein synthesis takes place in two phases: (1) formation of an expanded monomolecular layer, with reproductioii of the specific amino acid pattern in the chains; (2) folding of such a two-dimensional film in order to replicate the specific tridimen- sional shape and inner configuration of the molecule, determined by the position and manner of folding of the peptide chains. We must therefore distinguish two types of specificity in proteins: the specificity of the a m i n o m i d pattern (nature, number, and sequence along the chains); and the specijicity of folding; i.e., a purely chemical specificity and an architectural specificity.

Xow the first phase, according to the views developed by Herzfeld and Klinger (1920), by Rondoni (1936b, 1938a, 1940, 1949b), and others, and especially by Haurowitz (1950), consists of a preferential adsorption

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of each amino acid by the identical amino acid residue of the pre-existing molecular film, functioning as a two-dimensional template. Thus the same amino acid pattern is reproduced selectively by means of short- range intermolecular electrostatic forces, directing every molecule of amino acid on the identical one, as in the process of crystallization. After orderly deposition of the amino acids on the protein template, the enzymes catalyze the establishment of the peptide bonds. Hence, the enzymes cannot be considered as the organizers of synthesis. Even if we admit that synthesis is not simply the reversal of proteolysis and recog- nize (Fruton, 1949-50) the importance of replacement reactions (trans- peptidation), the specificity of enzymes is insufficient to explain the high specificity of cellular proteins. The enzymes operate chiefly by binding the amino acids after their deposition according to a definite pattern. Haurowitz thinks that nucleic acid plays a chiefly mechanical role, main- taining the protein two-dimensional template in an expanded state. But we know that nucleic acids have high specificity (Chargaff, 1950), and it may be considered whether the two-dimensional replica is a positive one (Haurowitz) or whether a negative replica is first organized consisting of a film of nucleic acid, whose negative acidic groups combine with the positive basic group of the protein template, determining thereafter a new positive protein replica, according to the hypothesis of Friedrich- Freksa (1940), adopted also by Schramm (1951) to explain virus multipli- cation. We are led to consider the lattice of the short-range intermolecular polar forces deriving from the pre-existing protein film as the eEcient mold, inducing Pepetition of the specific chemical structure.

The second step, inducing the tridimensional conititution of globular proteins (as those of blood plasma) and of the molecular network (Frey- Wyssling, 1948) of cell proteins, involves also the molding action of some template imposing an identical type of folding and binding to the peptide chains and so inducing a complementary print (negative replica). The nature of such templates and their mode of action is unknown, and left as yet to speculation. Haurowitz states that the lipids, carbohydrates, or proteins could be the molding template ; but pre-existing proteins or nucleoproteins are the most probable. We may quote here the opinion of Tyler (1947) that antigen-antibody-like systems of complementary substances are normally formed in the cell. I n any event the final result should be the identical repetition of the uniquely defined tridimensional configuration of the protein system. The biosynthesis of protein, as well as of other highly polymeric organic compounds, takes place according to the principle of a starter or primer, providing a specific template for further neoformation of living matter. Of course, in growth with differen- tiation (embryonic development), the organizers are represented by the

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complex system of nucleo- and plasma-genes; and the problem is still more obscure.

Sorthrop rt al. (1948) developed a different conception of protein synthesis: from an undifferentiated primordial protein (proteinogen), the different specific proteins n-ould arise through an autocatalytic process, without further supply of energy. But the existence of a proteinogen is very doubtful.

As regards the localization of protein synthesis in the cell, some in- vestigators emphasize the importance of the nucleus (Caspersson, 1950), whereas others consider the cytoplasmic granules (microsomes) as the main centers of synthesis (Brachet and Jeener, 1944; Brachet, 1952). Haurowitz and Crampton (1932a,b), on the basis of results from re- searches on antibody formation considered as a modified protein synthesis, suggest that the cytoplasm is the site of such metabolic events. However, they take into consideration the possibility of the nucleus being involved in the first phase of synthesis. According to Dounce (1952) protein metabolism occurs within the nucleus, as indicated by the considerable uptake of radioactive lysine by the histone fraction of rabbit liver nuclei after the injection of radioactive lysine together with other nonradioactive amino acids into the animal, as well as by the rapid uptake of radioactive phosphate by a nuclear fraction of pentose-nucleic acid. Metabolism requires the presence of enzymes; hence the conclusion is drawn by Dounce that the nucleus is an actively metabolizing cell particulate con- taining enzymes rather than an inert sac of nucleoprotein functioning only during mitosis. Xovikoff (1952) was able to demonstrate a large range of enzymes in the nucleus. The presence of cathepsins in the nucleus of normal and regenerating rat liver cells, as well as of hepatoma cells, has been demonstrated also by &laver and Greco (1951) and by Maver et al. (1952) and gives support t o nuclear participation in a t least the first enzymatic phase of protein synthesis.

I t is well known that nucleic acids are generally associated with the synthesis of proteins. Their mode of action has been evaluated from dif- ferent points of view: mechanical support for the peptide chains and protein films during their evolution; supply of energy-rich phosphate bonds; supply of split products, as sugars (energy supply) and N-bases, which could be utilized for building coenzymes, ie., active groups of im- portant enzymes operating in oxidoreductive metabolism (Rondoni, 1940).

Recently Deotto (1952a,b) has reported work on the influence of nucleic acids on proliferating cells, taking into consideration different bio- logical sys t em : (1) intermitotic vegetative elements (cultures of em- bryonic chicken fibroblasts as normal elements, Ehrlich tumor growing

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in vivo as pathological elements) ; (2) intermitotic differentiating cells (sea-urchin fertilized eggs and chicken embryo). Thymus DNA and yeast RNA in 1/6000 to 1/60000 concentration brought about severe damage and often cytolysis of chicken fibroblasts in vitro (Deotto and Necco, 1952). RNA or DNA from the Ehrlich carcinoma and DNA from thymus inhibited the growth of the Ehrlich tumor, while thymus DNA gave rise to an increase in size of the cancer cells (average size 3.3 times larger than in control tumors (Deotto, 195%)). Thymus DNA and a DNA from sperm in concentration 1 X promoted marked development of the fertilized eggs of “ Sphaerechinus microtuberculatus,” giving pre- cocious formation of plutei, while the controls remained a t the blastula or gastrula stage; higher concentrations of the same nucleic acids as well as concentrations 1 X of RNA and thymus DNA produced an in- hibition of development (Deotto, 1952b). The injection of nucleic acids into the allantois of chicken embryos brought about a rather high death rate, but no definite results were obtained; only a larger size of the liver cells of the embryos and of the newborn 10-day-old chicken were observed. A diminution in the mitotic rate of regenerating rat liver seemed to be produced by DNA from liver, but not by thymus DNA (Deotto, 1954). The conclusion may be reached that different sorts of nucleic acid exert a different action on cell multiplication.

Now we may enumerate three cases where protein synthesis does not replicate the structural pattern of pre-existing cell proteins, but is molded on an abnormal template which imposes itself and gives rise to a more or less modified protein type (Rondoni, 1938a, 1940, 1946a, 1949b): (1) antibody formation; (2) virus multiplication; (3) carcinogenesis.

As regards antibody formation, a majority of investigators considers i t as a modification of the synthesis of some fractions of the plasma pro- teins (very likely also of some cell proteins: sessile antibodies or desmo- antibodies, in cellular immunity, besides the lyo-antibodies in humoral immunity). It seems that the antibody-globulins have the same amino acid composition as normal plasma globulins; and that the sequence of the amino acids along the chains is not changed, at least in the chain sections explored by Porter (1950). It may well be that the antigen acts as a foreign template in the second phase of synthesis, inducing a new type of folding of already synthesized peptide chains and giving rise to a shape of the globulin molecule which in some point of its surface carries a complementary print of the determinant group of the antigen.

The second case of synthesis on an abnormal template is represented by virus multiplication; this subject has been extensively discussed of late and need not be dealt with here in detail. The mechanism of identical reproduction suggested by Schramm (1951) gives a full account of the

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esperimental facts. There is not a division (fission) of the molecule of a virus-protein, but a new and identical synthesis on the template provided by the pre-[xisting virus particles, diverting the synthetic activities of the host cell from the normal path (synthesis of normal cell proteins) towards the pattern of the virus protein itself. We may quote the conclu- sions of Kozloff (1952), who studied the fate of virus particles (bacte- riophagrsj in the bacterial cell: “The molecular processes involved in the replication process do not require the physical transfer of parent virus material. I t is the form, not the matter, that is important, according to a truism of geneticists. Although viruses cannot be equated with genes, i t is plausible that a similar situation holds for the reduplication of genes. Although it has been shown that the bacteriophage particle is disrupted during the reproductive process, i t must be assumed that the genetic portion must survive sufficiently long to pass on its inheritable character- istics. However, the problem remains as to how this virus, or rather a por- tion of it, is able to induce i n the host. a process by which the original particle. not merely the effective portion, is replicated in an exact fashion. One is left with the possibility that the specificity of the replication process is due to an alteration or distortion of the normal processes in the host, possibly for the production of host genes, with the virus particle destroy- ing and displacing the host genes as the controlling patterns.”

Whereas i n the preceding cases an abnormal template is imposed to the cell from outside, in the third case, the malignant transformation of the cell, a new template is originated in the cell itself and is transmitted in the cell generation as an essentially permanent new organizer, directing the synthetic processes towards the formation of a degraded living matter (see Section 11). The abnormal mold may arise in this case with the most different estrinsic factors (carcinogenic agents) disturbing the synthetic metabolism. Genotypic factors may also facilitate such a shift of synthesis from the normal pattern to an abnormal one.

The normal synthetic processes are so regulated as t o produce a given type of protein for every organism and every tissue; but i t is probable that some slight deviations from the fundamental pattern may occur under different conditions of life. Schenk and Wollschitt (1933) found in rats that different amounts of some amino acids (tryptophan) can be in- corporated into the organ proteins according to nutritional conditions and that this shift in the protein composition is increased by some damag- ing agents. In bacteria a large range of variation in metabolism with accompanying structural changes is well known, producing the dissocia- tion forms S and R and, in some bacterial species, further variants as well (Deotto, 1942) ; synthesis for every bacterial species takes place according t o a fundamental pattern, around which certain fluctuations are permis-

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sible. The important investigations of Schoenheimer (1946) and co- workers emphasized the dynamic state of the body constituents, i.e., a continuous flow of atomic groups through the cell structures during life. An analogy, which may be taken as an incomplete illustration of this concept of living matter, can be drawn with a military regiment, having a steady organization but a continuous incoming and outgoing of individuals.

With these facts in mind, i t is not surprising that under different con- ditions errors different in degree and significance can occur, affecting the chemical composition or the inner configuration or intermolecular asso- ciation of proteins. Some of such errors may have no important bearing on functional activity, whereas others can induce functional disturbance. The neoplastic transformation must obviously belong to the latter type.

There are certain more or less evident links between such cases. Thus antibodies are considered by Jordan (1940) as autocatalytically self-reproducing molecules; and in fact it is entirely possible that the globulins carrying the specific print can act as a secondary mold, inducing in the cells of the R.E.S. the formation of similarly modified globulins. This can very well explain the so called arnnestic reactions in immunity. Many students of virus biology are now inclined to accept an intracellular origin of some or of all viruses. Acqua (1935), on the basis of experiments on polyhedral-diseases of silk worms, suggested that the virus is a product of cells of the organism affected by metabolic disturbances due to nu- tritional factors. An endogenous origin of herpes-virus also seems likely (Doerr and Hallauer, 1938-39). A general endogenous theory, embracing all viruses (at least the euviruses or true protein-viruses), is supported by Darlington (1948), Troll (1951), and others. The cell constituents which under certain circumstances can become autonomous and behave as viruses can be equated with the plasmagenes or other particulates of the protoplasm. Moriyama and Ohashi (1938) think that viruses are proteins with a strong tendency to appear as particles; the viruses should be considered as denaturing agents (denaturases) , inducing in cells the formation of new virus radicals which are released and repeat the same process in other cells. Yamafuji and associates in a long series of papers (1944 to 1952) claim to be able to bring about the formation of some viruses in animal and plant tissues by treating the organisms with catalase poisons (hydroxylamine, etc.), capable of producing an accumulation of hydrogen peroxide in the tissues. Such metabolites, derived from oxida- tive metabolism and not destroyed on account of the poison, might thus exert a polymerizing action on some proteins and produce the formation of virus particles. Certainly some attempt to verify the claims of the Japanese workers, or otherwise, is much to be desired.

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As regards tumor-producing viruses, the likelihood of an intrinsic origin has been widely supported for many years. Graffi (1940) has even thought t o define the nature of the cell constituents which are able to undergo transformation into Rous-virus particles under certain condi- tions, as the mitochondria of mesenchymal chicken cells, and Haddow (1944) likens the Rous-sarcoma virus to the plasmagene. But recent speculation has gone beyond the consideration of a few filterable tumor agents and has tried to include all tumors in a general conception; thus particulate cell constituents, e .g . , plasmagenes (Darlington, 1948) or cytoplasmic duplicants (Korthdurft, 19-18, 1949; Druckrey and Kupf- muller, 1948; Butenandt, 1951), may be transformed into a kind of malignant entity, remaining inside the cell and multiplying synchronously with i t (as in most mammalian tumors), or more or less separable from i t (as in the filterable tumor agents). This entity must be something like a self-reproducing enzyme or an endogenous virus (Potter, 1943). These two cases could parallel the well-known distinction between desmo- enzymes and lyoenzymes. Such working hypotheses are combined with or opposed to the mutation theories, which concern either the nuclear genes or the plasmagenes. The endogenous virus theory may be recon- ciled with the mutation theory if we admit that the new formed principle in the cell behaves as an intruder in the genic system, i . e . , as a new organ- izer (Rondoni, 1939b), directing the synthesis on a faulty pathway.

I t is difficult and perhaps as yet fruitless to discuss whether the ab- normal organizer is produced in t.he nucleus or in the cytoplasm. We have seen that very likely normal protein synthesis involves the cooperation of both; and the defect in the synthetic processes may therefore arise from different parts of the complex system of inner cell oryanizers (nucleo- and plasma-genes, nucleolus-heterochromatin system, etc.). In fact there is a strict interaction between nucleus and cytoplasm in differentiation, a defect of which is the basic process in carcinogenesis. A brilliant article by Haddow (1944) gives a full account of such interrelations in biological phenomena, which can throw some light on the cancer problem; and more recently Schultz (1952) and Hogeboom et al. (1953) have thoroughly dis- cussed the functional relationship between cell constituents. According to the latter authors, who analyzed the localization and integration of cellular functions, the cell is neither a bag of enzymes arranged in a haphazard fashion nor a collection of mitochondria but rather a compli- cated mosaic of structural units, mutually dependent on one another in their contribution to the cell metabolism.

As a fruitful example of an inductive function exerted by a definite compound on synthetic processes we may quote also the bacterial trans- forming agents discovered by Avery et al. (1944) and by McCarty and

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Avery (1946) in Pneumococcus and afterwards demonstrated in other bacteria (Austrian, 1952). According to Hotchkiss (1952) “a given agent, determining an immunologically specific type of capsule, displaces from the cell or at least precludes the action of alternative capsular determi- nants, so that an individual cell produces only a given capsule with a particular specificity and contains only the corresponding type of capsular transforming agent. The capsular types of transforming agents are thought of as mutually replacing each other a t some unknown locus or region of the bacterial cell, much as allelic determinants in nuclei can be thought of as replacing one another. . . . ” We may note the similarity of this phenomenon with the process of carcinogenesis, where a new template displaces the normal one and maintains itself in the new cell strain. It is possible that the neoplastic transformation of cells also affects the cell surface, modifying those specific structures which according to Tyler (1947) permit the adhesion of cells to each other acting as an antigen-antibody-like system; the disruption of specific intercellular ad- hesion in tissues may contribute to explain the tendency to infiltration and metastasis.

2. Some Observations on Proteins in Cancer Tissue

An excellent review on this subject has been given by Toennies (1947). Among more recent work, electrophoretic investigations have been made by Sorof and Cohen (1951) on extracts of azo-dye-induced-and other tumors: a decrease in the amount of proteins with slow electrophoretic motility, and an increase in faster components, were observed. No such change was found in extracts of livers of normal or fasting rats. Sorof et al. (1951) confirmed these earlier results. However, Hoffman and Schechtman (1952) observed similar changes in the protein composition of livers of very young rats and of rats fed a noncarcinogenic azo com- pound, and question the relation of the observed changes with carcino- genesis. Eldredge and Luck (1952) found, like Sorof and co-workers, an increase of protein of higher motility in azo-dye-induced tumors. Elec- trophoresis alone is perhaps not sufficient to disclose the more subtle differences which doubtless occur in carcinogenesis.

A simple method for the study of the difficult question of protein modification was devised by Rondoni (1941). An extract obtained from tissues by treatment with 10% NaCl aqueous solution was heated a t dif- ferent temperatures and a flocculation curve established by a nephelo- metric technique. Certain differences were observed in the flocculation of extracts of normal and tumor tissues. Such studies were taken up again by Bassi and Bernelli-Zazzera (1954) on a larger scale; the same salt-soluble protein fraction (10% NaCl), with constant N-content, ;vas

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used from normal rat livers? regenerating livers, and the livers of rats fed different diet,s with or without the addition of dimethylaminoazo- benzene (DAB). The experiments were continued until the appearance of hepatomas in the rats fed DXB. The nephelometric curve was estab- lished by progressive heating of the extracts t o 43.5"C. or by separate heating a t different temperatures. A regular decrease in heat precipita- tion (lower nephelometric curves) was found in livers of rats fed DAB; the change occurs early (15th day) and lasts for many months (see Table 1'). In hepatomas the change may persist, but sometimes diminishes or

TABLE v Extracts of Livers of Rats Fed DAB Added to a Rice arid Carrot Diet

Values of relative turbidity after heating (Stufen-photometer) (incan values of groups of four rats earh).

~~ ~ _____

Final Values Values after in Progres- Heating a t sive Henting Constant from 3i"C. Tempera-

Diet to 56OC. P tures P

Controls (nornial diets) After 8 days DAB feeding

I ( 15 '( h i

" 30 " "

" 60 " I '

" 50 i ( "

I . 120 ' ( I '

'' 150 " "

' < L

After 10 days

f = fitatistiral probability ,i 20

565.7 559.0 265 5 403.5 427.2 381.4 311.5 339.2

501.4 545.7

306 6 0 Y > P > 0 8 2 6 1 0

1> < 0 01 167 7 I' < 0 01 319 9 P < 0 01 303 5 P < 0 0 1 202 8 P < O O 1 192 7 I> < 0 01 224 G

DAB Feeding Stopped 0 1 > P > 0 0 5 246 I 0 7 > P > O G 2 6 1 0

0.05 > P > 0.02 I' < 0.01

0 . 8 > I' > 0.7 P > 0 .9

0 . 1 > P > 0.05 1' < 0.01 1' < 0.01

0 .05 > P > 0.02 0 .3 > P > 0.02

disappears (the necrotic alterations may play a role). The greater heat stability of the investigated protein-nucleoprotein fraction may be due to the azo compound bound by the protein and so exerting protection against heat coagulation of the protein itself. Possibly the protection exerted by thymus nucleate on the heat coagulation of proteins (Carter and Creenstein, 1946) may be mentioned. If the administration of DAB is terminated, even after five months, the flocculation curve again resumes its normal character. On the other hand the administration of riboflavin (which is well known to be capable of retarding the carcinogenic action of D.4B) more or less inhibits the development of the flocculation change.

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In regenerating liver (after partial hepatectomy) a change in an opposite sense is observed 4 to 5 days after operation; higher turbidity curves are an expression of increased heat coagulability.

It is difficult to assess the significance of this change in heat floccula- tion of a protein fraction, and especially to determine whether it is simply related to the presence of protein-bound azo compound or has some rela- tion with the carcinogenic process itself.

Several studies on the amino acid composition of tumor tissues have been recently undertaken; whereas Schweigert et al. (1949) found differ- ences in comparison with normal tissues, the majority of investigators obtained no significant results, for example, Zamecnik et al. (1949), Sauberlich and Baumann (1951), and Biserte (1953), in accordance with the older work of Greenstein et al. (1941b). Similarly, Bassi and Bernelli- Zazzera (1954) found (using bidimensional paper chromatography) no difference in amino acid composition between acidic hydrolyzates from normal rat livers and hydrolyzates from livers of rats fed DAB to the time of hepatoma development. On the whole, there is no clear evidence of a specific amino acid pattern in tumors. Some difference in the sequence of the amino acids along the peptide chains may be suspected (Biserte) ; but is obviously difficult to demonstrate. Much work has, however, indicated very active amino acid and synthetic protein metabolism in tumors, which have accordingly been defined as " nitrogen traps l f

(Mider, 1951); Boulanger (1953), on the basis of experiments with radio- active glycine, advances the opinion that amino acids can penetrate the cellular membranes more readily in tumor cells, with, in addition, a more active interchange between different amino acids.

An attempt to establish a specific pattern of amino acids and peptides in tumor tissue was made by A. Fischer (1950), using a biological method; embryonic chicken fibroblasts were grown in hydrolyzates from normal chicken muscles and in similarly prepared hydrolyzates from a methyl- cholanthrene-induced fowl sarcoma. The growth was more vigorous in the former medium than in the latter; and this author believes that the peptides from normal cells may be more easily utilized by the normal fibroblasts because of their physiological composition. It is, however, possible that the tumor hydrolyzates exert a toxic action because of those secondary necrotic changes which are seldom absent. Indeed, some slight deviations from the normal pattern of amino acids may be accounted for in malignant tumors by such degenerative and necrotic changes, which involve autolytic processes. The split products of proteins in the necrotic patches may be absorbed a t a different extent, blood plasma pro- teins may infiltrate the necrotic areas, and differing amounts of stroma may also hinder the comparison of tumor with normal tissue.

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204 P. RONDON1

3. Infrared Absorption Spectra of Cancer Tissue

h study of proteins and of tissues by infrared absorption spectrography has been attempted by some investigators. The infrared absorption of proteins as well as of other organic compounds is due to the absorption of certain atomic groups a t characteristic frequencies, and the vibration and rotation spectra (Mellon, 1950) inform us of (1) the moment of inertia of the molecule about the axis of rotation, giving the molecular dimension; (2) the bond strength which is deduced from the fundamental frequency and the mass of the molecule; and (3) the configuration of the molecule. The study of protein solutions is particularly difficult as a result of molecular complexity and the infrared absorption of water. Neverthe- less, much work of biological interest has already been conducted, as, for example, that of Lenormant and co-workers (1950-1953), who introduced the study of proteins treated with heavy water, so that the hydrogen of the imino-group in the peptide bond is substituted by deuterium ( ‘ I deuterisation des proteines ”). Lenormant (1952~) has also studied proteins in living cells and the denaturation process (1953). Further there are the important contributions of Darmon and Sutherland (1919), Ambrose et al. (1919), Mizoushima et al. (1950), and Elliot et al. (1950). As regards tumors, the paper of Blout and Mellors (1949) includes data on the infrared spectra of blood, normal tissues (e.g., breast gland), and neoplastic tissues (mammary cancer). Here the observations were made on fixed and paraffin-imbedded tissues and on dried and frozen tissues, differences being noted according to the method of treatment. An absorption spectrum was recorded in tumor material corresponding to an increased concentration of nucleic acid. Woernley (1952) gives many absorption curves of normal and neoplastic tissues, remarking that, owing to the complexity of the material under investigation, each ab- sorption is probably a composite structure resulting from several different types of vibrations. The curves cover the spectral region of 1 to 8 p

as well as the region of greater interest 8 to 15 p. The absorption in the former region depends on the distribution of some atomic groups; while above 8 p the skeletal vibrations generally mask the bond type. In a great number of tissues there is a more or less pronounced band at 8.1 p which may be assigned to rocking, wagging, deformation, bending, or twisting vibrations of CH, CHI, or CHa structures. For some tissues there is a band at approximately 9.2 p ; for others there are two or more bands a t 8.8 to 10 p. Definite bands are also observed at about 10.2 p. Neoplastic tissues (human and mouse tumors) show a characteristic absorption pattern in the region between 8.0 and 11 p (bands approximately a t 8.1 p, 9.2 p, 10.25 p) . In order to explain such findings absorption data

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were obtained for tissue constituents, for nuclei and cytoplasm separately, in normal and neoplastic materials, these results being correlated with the chemical composition of the samples. It was concluded that the absorp- tion in the region 8 to 11 p is mainly due to nucleic acids; therefore highly cellular neoplastic and normal tissues, rich in nucleic acids (RNA as well as DNA), produce a strong and characteristic absorption in this region.

More recently Ceselli and Guzd (1954) have investigated the infrared absorption spectra of the livers of 30 rats fed for 15 to 180 days on a rice- carrots-cod-liver oil diet with addition of dimethylaminoaxobenzene, as well as of livers of normal rats, using paraffin-imbedded tissues. They fol- lowed those changes occurring in the precancerous stages, of greatest significance for the malignant transformation. During treatment the tissues showed an increasing reduction of the bands between 8 p and 11 p,

which are considered as due to the nucleic acids. Also, livers affected by precancerous lesions gave spectral curves with an evident diminution of absorption a t 9.25 and 9.70 p (the latter being more characteristic for DNA). Only in livers of some rats submitted to a more prolonged treat- ment with carcinogen did the band a t 9.70 p again appear evident. The findings of these authors are in contrast with those of others mentioned above, who studied human or animal tumors but not the earlier stages of the carcinogenic process. It may be that the DAB or some metabolic derivative combines with the tissue components and so modifies the spectra in the region 8 to 11 p ; or that in the initial phases a disorganiza- tion of high polymers, including NA, takes place, as a prelude to a rear- rangement corresponding to definite malignancy, when the concentration of such compounds increases in association with vigorous cell multiplica- tion. On the other hand, i t is also possible that in the initial stages the NA undergoes further polymerization or new combinations with different protein fractions, involving a shift of absorption towards other regions of the spectra where the enormous complexity of the tissue components prevents a clear evaluation.

On the whole, the infrared absorption study demonstrates the occur- rence of rather complex modifications of the NA behavior in tissues during carcinogenesis and in tumors. The opinion of a rearrangement a t the level of macromolecular constituents may be generally confirmed, but no evidence can be given about the distribution of atomic groups or their bond strength.

4. Ultrastructural and Immunological Considerations

From the admittedly scanty data so far available, the opinion may nevertheless be held that cell transformation in malignancy affects either the architectural and folding specificity of certain protein constit-

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iients or the state of protein aggregation. Hence, the change may reside a t a higher level than that of chemical composition and may consist of Some abnormal configuration in the three-dimensional molecular network of protoplasm, with distortion of peptide chains, new interrelations bettveen the chains, and the generation of a new field of polar forces. We have therefore to deal with an abnormal tridimensional template hence- forth dominating the synthetic processes. The change would fundamen- tally concern the u2trastructzrre of the protoplasm (Rondoni, 1946a,b), i . e . , the structural level above that of purely chemical composition. The fundamental significance of an ultrastructural change in carcinogenesis has also been suggested by Powell (1944, 1946, 1947), who concludes that “ an irreversible or long persisting disorganization of protoplasm atid especially the inm-ordination of the components of the cytoskel- eta1 framework results in an anaplastic transformation of the affected cells. It is possible that the majority of tumors arise in this way. I n such tumors the initiation of the disorganization or inco-ordination may be regardd as the proximate cause of the anaplastic state since i t can produce most of the main features of tumor cells and is the root, cause of those properties which together constitute the facies of the anaplastic state. Protoplasmic disorganization or cytoskcletnl inco-ordination need iiot necessarily he the primary cause of anaplastic growth but could be I)rought about by, for example, viruses or gene mutations. On the other hand, inco-ordination of the protein microfibrils may be both a primary and proxiniltte intracellular cause in many instances. In view of the diversity of mrcinogenic agents, which also have other effects on cells, and the complexity of protoplasm, it seems that the competence to re- spond t o such agents by the development of the anaplastic state is deter- mined hy a (-ommoil effect on some ubiquitous sul)stratum in protoplasm. There is abundant evidence that carcinogenic agents affect the fibrous proteins of protoplasm; for example, hlottram (1942) observed that carcinogens changr the viscosity of cytoplasm, and cytological aberrations consequent upon alterations in viscosity occur in cells within a few hours of treatment (Pullinger, 1940). Summation experiments with different carcinogenic agents indicate that a common cellular substratum is in- volved in carcinogenesis.” ,J . Seedham (193Ga,b), who considers tumors as a product of abnormal embryonic organizers, also suggested a dis- organization of the tridimensional structure of the protoplasm.

If such hypothesis is correct, then carcinogenesis should occupy the same cellular level of organization as antibody formation, of course with a much more extensive involvement of cellular mechanisms. The carcinogenic agent in most cases is not a template in itself, as are antigens; but it induces that modification in cell constituents (proteins, nucleic

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acids, and perhaps some lipoids) which brings about the generation of the abnormal template. Indeed, the most powerful chemical carcinogens (polycyclic hydrocarbons) are represented by large, rigid molecules, which, bound to a protein carrier, could exert antigenic activity (Creech, 1952). But there is no need for a carcinogen to be an antigenic itself, and i t may induce changes in proteins which may themselves be antago- nized by the process of antibody formation, for example, in the experi- ments of Michle and Emde (1944). These authors found a decrease in the incidence of sarcomas in mice injected with benzpyrene and subjected to parenteral treatment with various antigens. It is possible that antibody formation, as well as carcinogenesis, interferes with some similar aspect of protein metabolism. Pauling’s views (1948) concerning the self-dupli- cation of viruses and genes and the formation of antibodies are relevant here; he believes that “it is molecular size and shape, on the atomic scale, that are of primary importance in these phenomena, rather than the ordinary chemical properties of the substances, involving their power of entering into reactions in which ordinary chemical bonds are broken and formed.”

We know that the largest part of the cell proteins, perhaps nearly the whole, consists of enzyme proteins (Meyerhof, 1949) : “What was for- merly regarded as protoplasm, containing the machinery of the life proc- esses, must now be regarded as a multiple of distinct enzymes which are coordinated in their function by chemical cycles, by the influence of the cell structures and by the effect of hormones.” In these words of Meyer- hof the assumption is expressed that the proteins, some containing a dis- sociable effective group and some not, are in strict interrelation to each other, forming a complex system, where intermolecular association and ultrastructural conditions play an important role for enzymatic activity and consequently for all the processes of life.

Villa et al. (1953) consider contraction of the chromosomes and an increase in the viscosity of DNA as characteristic features of leukemic leucocytes in comparison with normal ones. This finding might also indi- cate a higher degree of polymerization of DNA in leukemic cells, and therefore an alteration chiefly concerning the ultrastructure. It may be recalled here that Boretti (1946) investigated the enzymes controlling the metabolism of RNA in extracts from human mammary gland and from breast cancer, finding those enzymes which catalyze the lower steps of RNA metabolism (nucleotidases) to be more active in cancer tissue, while the enzymes operating in the higher metabolic processes (poly- nucleotidases) are almost as active in the tumor as in the normal organ. This finding does not conflict with the results of Villa and others, since it concerns RNA only and is based on experiments in vitro.

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An abnormal protein system (nucleoprotein or lipoprotein), charac- terized by a new intramolecular configuration or by some new inter- molecular arrangement, could involve immunological specificity ; hence the search for antigens in tumors, more or less distinguishable from the species and normal organ antigens, is justified. There is a large number of papers on this subject; and for recent work we may refer to the review of Hauschka (1952). Others include the older work of Witebsky (1930) on a specific globulin in certain tumors; the observations of Maculla (1947-1948) on quantitative serological differences between adult and embryonic organs and tumors of mice; and the work of Malmgren et al. (1951), as well as of Weiler (1952), who investigated the particulate com- ponents of the protoplasm of normal liver and of hepatomas of rats and found clear serological quantitative differences in comparison with the normal organ. Fink et a l . (1953) present data which demonstrate that inbred mice are capable of producing antibodies to a lyophilized prepara- tion of their homologous tumor, as evidenced by the in vivo immunologic reaction of anaphylactic shock; the tail inoculation of a tumor produces an immunity to a subsequent inoculation. The immunological differences between cat,hepsins of normal and tumor tissue described by Maver and Barrett (1943-44) and Maver and Thomson (1944) are noteworthy in this respect. We may agree with the conclusions of Hauschka: '' . . . in retrospect, the serological studies of tumors, though extensive, were seldom controlled in a way which (except in the case of some viruses) would allow unequivocal isolation of specific neoplastic antigens. Where claims for specificity have been made, the genetic gap between tumor and host has generally been objectionable. The evidence for quantitative if not qualitative differences in the antigenic components of normal and malignant tissues is, however, convincing . . . " (see also Section V,l).

5. r l Comparison of the Neoplastic Transformation of the Cell with a Process of Protein Denaturation

It can be postulated that the basic change responsible for malignancy is the constitution of a new and abnormal organization center overcoming the normal organizing system and inducing the formation of a low-grade protoplasm. Such protoplasm can be regarded as devoid of receptors for regulation and differentiation, and the word " despecialization '' used by Powell (1946, 1947) may express the nature of the protoplasmic change.

A very common in vilro phenomenon shows some similarity with the carcinogenic reaction (Rondoni, 1938a, 19-10, 1946a,b, 1949b), namely, that of protein denaturation. Fruton and Simmonds (1953) state that a denatured protein consists of disorganized peptide chains : the denatura-

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tion brings about a change in the shape of the protein molecules, and there is generally an increase in asymmetry. There is also an increase of re- activity of certain groups, such as the sulfhydryl groups of cysteine, the disulfide groups of cystine, and the phenolic groups of tyrosine, owing to the unmasking of these groups by the uncoiling of the chains. The de- natured proteins cannot be crystallized because of the disorganization of the internal structure. Here therefore are some of the changes, which may equally occur in the proteins of the cell subjected to malignant transformation. An increase of entropy takes place in the process of denaturation (Mirsky and Pauling, 1936; Anson, 1938) just as it most probably does in carcinogenesis (see Section 111). Indeed, the protein molecule from a specific, uniquely defined configuration passes after denaturation into a less orderly structure, i.e., from a less probable con- dition to a more probable one. The word “despecialization” may be applied here as also in the case of the neoplastic transformation. Some specific functions are lost in denaturation, but new functions might be acquired as a result of the molecular rearrangements permitting new bonds and new reactivities. Also, the sensitivity to enzymes is modified.

Denaturation, involving the breaking of many weak bonds between the peptide chains, has a high value of heat of activation; the same seems to be true for the carcinogenic reaction, where an energy threshold must be overcome according to the considerations set forth in Section 111.

Carcinogenesis is a new event in the life history of the cell, just as is denaturation in the in vitro condition of a protein; both processes are caused by many agents, physical and chemical, very different in nature and having a common issue in the despecialization of a protein system. Protein denaturation was first considered to be an irreversible process; but we now know that there are some types of denaturation which are reversible (Anson and Mirsky, 1931, 1934). It is, however, difficult to reproduce the folding specificity of a protein, i .e., to regenerate the normal protein configuration destroyed by a denaturing agent, and equally car- cinogenesis itself appears to be an irreversible process.

The work of A. Fischer (1936a,b) and of Rondoni (1938~) demon- strates that the heat denaturation of some protein systems is a partly transmissible process; a small amount of the already denatured protein induces an acceleration of heat denaturation in the homologous protein. The denaturation process seems to behave like a chain reaction. Of course the chain very soon breaks down in the in vitro reaction, whereas in vivo it may be easily perpetuated through the continuous turnover of the cell constituents. The protein molecules are rather rigid atomic structures, which notwithstanding can be very easily molded and submitted to deformation. A given deformation can be transmitted from one molecule

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to another; Astbury et al. (1935) spoke of “cannibalism” exerted amongst protein molecules.

The abnormal organizer might be compared to a denaturation product able autocatalytically to induce a further denaturation. The opinion of 0. Schmidt (1940, 1941a,b) is similar: carcinogenic agents react with some protein system of the protoplasm and induce the formation of a more stable, denaturation product, with release of energy which might be employed in the uncontrolled cell multiplication. Perhaps too much stress has been laid by rancer workers on the rapid growth of tumors and on the frequency of cell division. But such processes are in most tumors less pronounced than in the cmbryo or in the physiological regeneration of tissues. The outstanding characteristic of malignancy consists rather of a permanent error of differentiation, with reversion of the cell to a kind of autonomy. If the proteins in cancer have a simplified structure. the synthesizing forces are very likely able to build larger amounts of living matter than in the case of the more specialized and energetically more exacting proteins of normal cells. Whenever rapid protein synthesis takes place, a simplified protein type is usually found (histories and protamines in germinal tissues; histone combined with DXh in euchromatin). The presence in the cancer cell of a stabilized protein fraction acting as an abnormal template is not inconsistent with the well-known lability of such cells. The abnormal structural and ultra- structural substrate is largely masked by the normal constituents and therefore difficult to identify even by serological procedures.

The physical and chemical carcinogenic agents should be considered as denaturing factors, producing in some protein system related with the inner cell organization, and with differentiation, a particular type of deiiaturation or disorganization consistent with cell life and multiplica- tion. Some viruses also should be able to interfere in such a way with cell organization. Reference has been made to a concept of viruses as L 4 denatitrasea.” The introduction of heterologous proteins (or of proteins in any way foreign to the blood) could bring about i n some cells, like those of the reticuloeiidothelial system, a derangement in protein synthesis and cause the formation of abnormal, immature cell strains; thus Pentimalli (1953) obtained, in rabbit blood and organs, pictures suggesting a leu- kemic or Ieukemia-like change, by means of repeated injections of foreign proteins, and pointed to a mechanism involving derangement of protein synthesis.

Denaturation might be started by some masking process or by the binding of a chemical group (see Section IV), so inducing a shift in the dynamic equilibrium of the living system. A denaturation-like modifica- tion of the aggregation state arid of the stability of a protein system may

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be produced by treatment with small quantities of hydrogen peroxide (Rondoni and Pozzi, 1933, 1935; Rondoni, 193%; Rondoni and Bassi, 1951). In complex protein systems (e.g., serum or organ extracts) the addition of HzOz in certain concentrations brings about an increase of the trichloroacetic-acid-precipitable N-fraction. This phenomenon was attributed to a physical aggregation of protein and peptide molecules induced by the oxidative destruction of peripheral polar hydrophilic groups, and to oxidation of SH-groups with formation of intermolecu- lar -S-S- bridges. It is also, very likely, the same phenomenon as was observed by Yamafuji and co-workers (see Paragraph 1 of this section), and considered by them to be a true polymerization with significance for the origin of virus particles. The “aggregation efect” of HzOZ.was sub- stantially confirmed by Blagowestschenski and Korman (1934). (For a full discussion of this subject, see Rondoni, 1949b.)

We know from the work of Greenstein et al. (1941a), and of v. Euler and Heller (1949) and others, that one of the most striking features of the enzymatic pattern of tumors is an enormous decrease of catalase. According to Greenstein the catalase activity of rat hepatomas and normal adult rat livers may be in the ratio 1/1000, whereas differences affecting other enzymes of the oxidative metabolism are much smaller. Regenerating liver has nearly the same catalase activity as normal liver. Rondoni (1952) advanced the hypothesis that the marked reduction in catalase activity might have some bearing on the protein rearrangement in carcinogenesis, producing an accumulation of H202 and consequently an aggregating effect on certain of the protein fractions. Indeed, Rondoni and Cudkowicz (1953) found a high content of HzOz in transplantable tumors of rats and mice, more or less approximating the content of organs with a highly active oxidative metabolism. It must be recognized that the chemical estimation of hydrogen peroxide in tissue is difficult; and the method then adopted (colorimetric estimation after reaction with titanium sulfate) was later modified by Cudkowicz (195410). The absolute values are therefore doubtful; however, the relative values may be justi- fied. Cudkowicz (1955) reported with more reliable techniques the H20z content of the livers of rats fed for some months on a rice-carrot- cod-liver oil diet with or without dimethylaminoazobenzene (DAB) (before appearance of hepatomas in the DAB series), and has found a regular increase in concentration in the livers of rats fed the special diet known to facilitate neoplastic degeneration, in comparison with the livers of rats fed a standard (Coward) diet: addition of the azo compound had no further effect on the HzOz content (see Table VI). Catalase activity also increased during administration of the cancer-promoting diet. The provisional opinion may theref ore be advanced that some cancer-pro-

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TABLE TI H Z 0 2 Content (g. per mg. Dry Weight) in 1,i.c.ers of Rats Fed Different Diets:

I. Kormal (Coward diet) 11. Promoting diet (rice-carrot-cod-liver oil)

111. As in 11, plus p-dimethylaminoazobenzene (DAB) IV. A s in I plus DAB

Duration of Treatment, Days I I1 I11 IV

15 6 . 5 10 9 4 7 8 8 6 5 . 5 7 5 . 5 6 . 5

30 7 12 15 5 . 5 1 11 12 6 8 10 .5 12 5

60 5 13 12 6 7 12 15 6 . 5 1 . 5 8 12 5

90 16 10 14 12 12 .5 12

120 15 13 14 10 9 11

150 12 12 .5 13 14 13 9

The dejicieni diet i n II and I l l and the administration of D A B i n 111 snd ZV discontinued and normal (Coward) diet given to all groups of rats

165 8 7 7 6 . 5 7 8

180 6 . 5 6 7 7 5 7

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moting or cancer-inducing agents can increase the formation of H20z in the exposed organ, and that an increase of the HzOz-splitting enzyme catalase may occur, as a regulatory attempt which later fails to operate when neoplastic degeneration sets in. Then there occurs a marked depres- sion of activity and an increase in HzOz concentration.

According to Cudkowicz (1952), the carcinogenic hydrocarbons 20- methylcholanthrene and 3,4-benzpyrene when dissolved in caffeine solu- tion exert a partial inhibition of liver catalase in vitro, whereas noncar- cinogenic hydrocarbons are devoid of such action. However, aqueous suspensions of these hydrocarbons exert no such inhibition. That the accumulation of peroxide may play a role in the suggested protein rear- rangement, interfering with the intermolecular relationship, remains of course a working hypothesis. The accumulation of H20f should bring about the abnormal protein arrangement or molecular deformation when it affects cells proliferating under influence of different stimulations, i.e., labilized protoplasmic systems. Such a hypothesis might, however, be supported by the fact that ionizing radiations, a well-known carcinogenic and mutagenic agent, operate by forming OH-radicals from tissue water, i.e., by a chemical mechanism involving oxidation reactions and very likely some kind of denaturation process in proteins. Abnormal growth of bacteria can also be associated with decrease of catalase activity, and Pontieri (1953) found such a decrease in the abnormal growth forms of Escherichia coli produced by urethane. The bacteriological literature con- tains many other observations on the production of atypical growth by accumulation of H202 and other peroxides in the culture medium under the influence of radiations.


Malignant tumors belong to the regressive processes of classical pathology, involving the growth of a more or less despecialized living matter. In the neoplastic transformation of a normal tissue we see a transition of the living system from a less probable condition to a more probable one, i.e., an increase of entropy, as in all degenerative and pre- lethal changes. Some features of the oxidative metabolism of the cancer cell are the result and not the cause of neoplastic degeneration, and depend chiefly upon the necrobiotic changes in many malignant tumors. The carcinogenic aromatic hydrocarbons enter the cell through the lipoid con- stituents, and some disorganization of lipoids (lipoidolysis) is probably associated with the initial steps of the carcinogenic reaction chain; but the decisive attack affects some complex protein system. The binding or covering of the SH-groups of protoplasm by the carcinogenic hydrocarbons is discussed. The diamagnetic behavior of the hydrocarbons and of some

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steroids may give support to the electronic theory. Recent work on the electrophoresis of proteins from neoplastic tissues, on amino acids pat- tern, and on the heat coagulation of protein fractions, is reviewed and discussed, with studies on infrared absorption spectra. I n general, the purely chemical composition of proteins from cancer tissue does not seem to differ from that of the normal parent tissue. It is therefore supposed that the main change concerns not the composition specificity but the Joldiny speci’icity, z.e., the shape and internal configuration of the mole- cules as well as their association. The change may reside chiefly at the ultrastructural level and concerns a faulty step in protein synthesis, occurring a t the same level (generation of shape specificity) as is involved in antibody formation. As a result of this interference with synthesis an abnormal tridimensional template is produced, which henceforth controls the synthetic processes and inducps the generation of a despecialized protoplasm. .\ comparison is made of neoplastic change with protein denaturation and the hypothesis is advanced that an abnormal accumu- lation of hydrogen peroside may play a role in denaturation, on the basis o f t he hydrogen peroxide content of neoplastic and preneoplastic tissues.

I< EFEK EX C ES A q u a , C . 1945. d t t i accad. nazl. Lincei, Rend., Classe sci. 5s. mat. e nat. 21, 593-599. Ain1)rose. 1.;. J.. Elliot, A., and Temple, lt. B. 1949. :\iatrtre 163, 859-862. Anson, 11. L. 1938. In “Chemistry of the -\mino .kids and Proteins” (L. A. Schmidt,

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Yamafuji, K., Kawakami, T., and Shinohara, K. 1952-53. Enzymologia 16, 199-203. Yamafuji, K., Kondo, H., and Omura, H. 1950-51. Enzymologia 14, 153-156. Yamafuji, K., and Omura, H. 1950-51. Enzymologia 14, 107-111. Yamafuji, K., Omura, H., and Yoshihara, F. 1952-53. Enzymologia 16, 28-30. Yamafuji, K., and Rosa, Y. 1944. Biochem. 2. 317, 81-86. Yamafuji, K., and Shirozu, Y. 1944. Biochem. 2. 317, 94-98. Yamafuji, K., Tokuyasu, K., and Wada, K. 1952-53. Enzymologia 16, 31-32. Yamafuji, K., Wada, K., a,nd Sato, M. 1952-53. Enzymologia 16, 130-133. Yamafuji, K., and Yoshihara, F. 1944. Biochem. 2. 317, 87-93. Yamafuji, K., and Yoshihara, F. 1950-51. Enzymologia 14, 124-127. Yamafuji, K., and Yoshihara, F. 1952-53. Enzymologia 16, 10-13. Yamafuji, K., and Yoshihara, F. 1952-53. Enzymologia 16, 321-326. Yamafuji, K., Yoshihara, F., and Sato, M. 1952-53. Enzymologia 16, 204-206. Yamafuji, K., Yoshihara, F., and Kondo, H. 1950-51. Enzymologia 14, 30-38. Yamafuji, K., Yoshihara, F., and Omura, H. 195%53. Enzymologia 16, 182-186. Yamafuji, K., Yoshihara, F., and Wada, K. 1950-51. Enzymologia 14, 170-176. Zamecnik, P. C., Frantz, I. D., Stephenson, M. L. 1949. Cancer Research 9, 612-613. Zollinger, H. U. 1948. Ezperientia 4, 312-314.

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Pulmonary Tumors in Experimental Animals

MICHAEL B. SHIMKIN National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Page I. Historical Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

11. Frequency and Distribution of Pulmonary T u . . . . . . . . . . . . 225 111. Pulmonary Tumors in Other Animals.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 IV. Morphology and Biochemistry of Pulmonary Tumors in .Mice. . . . . . . . . . . 229

1. Gross and Microscopic Appearance.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 2. Biochemical Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V. IIistogenesis of Pulmonary Tumors in LVice.. . . . . . . . . . . . . . . . . . . . . . . . . . 233 VI. Influence of Heredity in Pulmonary Tumors in Micc.. . . . . . . . . . . . . . . . . . 235

VII. Polycyclic Hydrocarbons and Related Compounds. . . . . . . . . . . . . . . . . . . . . 237 VIII. Urethane and Related Compounds.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

S. Factors Affecting Pulmonary Tumor Induction in Mice.. . . . . . . . . . . . . . . 248 1. Sex, Age, Diet, and Inflammation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 2. Pulmonary Tumors in Embryo Mice.. . . . . . . . . . . . . . . . . . .

XI. Mechanism of Induction of Pulmonary Tumors in Mice.. . . . . 1. Mode of Action of the Carcinogen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mode of Reaction of the Lung. . . . . . . . . . . . . . 254

XII. Pulmonary Tumors in Man and General Discussion.. . . . . . . . . . . . . . . . . . . 256 1. Bronchogenic Carcinoma, . . . . . . . . . . . . . . . 2. Other Pulmonary Tumors.. . . . . . . . . . . . . . . . . . . . . . . . . 258 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

IX. Other Chemical and Physical Agents, Including Inhalants. . . . . 1. Miscellaneous Agents.. . 2. Exposure to Inhalants.. . . . . . . . . . . . . . . . . . . . . . . . . . .


. . . . . . . . . . . 256

I. HISTORICAL INTRODUCTION This review summarizes investigations concerning primary pulmonary

neoplasms in animals, and selectively covers the literature from 1896 to 1953. The terms of reference exclude metastatic neoplasms of the lung, and more generalized neoplasms with incidental localization in the lung, such as lymphosarcoma or hemangioendothelioma.

Most of the experimental work has been performed with the adenoma- tous pulmonary tumor of the mouse, and most observations and con- clusions perforce must be limited to mice. Recent reports on the induction of pulmonary tumors in rats and in guinea pigs allow the extension of the investigations to other species. Reports on pulmonary tumors in larger animals are limited to descriptions of individual cases and a few, mostly unsuccessful, attempts to induce the tumors.


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Cancer research, as all biological research, is dependent upon the availability of proper materials and techniques for its work. The chief material has been the mouse, and the chief techniques have been the transplantation and the induction of neoplasms. On this basis, a large portion of the history of cancer research can be divided into three main periods. The period 1890 to 1918 can be designated as the morphology- transplantation era. The period of 1918 to 1935 can be designated as the genetics-tar tumor era. The modern period, since 1935, dates from the general use of homozygous mice and the galaxy of defined chemical agents for the induction of experimental neoplasms.

Studies on pulmonary tumors in mice reflect these three periods of research on cancer. The first report of a primary pulmonary tumor in a mouse is usually attributed to Livingood (116) in 1896. During the next fifteen years, a large number of cases were collected and described by Murray (1 43), Tyzzer (1 96) , Jobling (93), and Haaland (63). Slye, Holmes, and Wells (183) in 1914 published the findings on the first six thousand autopsies of mice in their colony. Among these were 160 mice with pulmonary tumors, of which 63 were considered histologically malig- nant and 4 had metastasized.

During the tar-painting era of cancer research, Murphy and Sturm (144) in 1925 first clearly demonstrated the induction of primary pul- monary tumors with tar, at the same time avoiding the appearance of skin carcinomas. With the availability of inbred strains of mice, Lynch (124) in 1926 showed that different strains had markedly different fre- quencies of pulmonary tumors and that susceptibility to tar-induced pulmonary tumors paralleled the spontaneous frequency. She applied the material to genetic studies on induced and spontaneous neoplasms.

With the advent of carcinogenic polycyclic hydrocarbons, it was soon reported that the injection of these compounds into mice also markedly increased the frequency of pulmonary tumors. Andervont (1-18) in 1935 initiated extensive studies, and his important contributions dealt with a comparison of strain susceptibility, the effects of heredity, various car- cinogens and modes of administration, and the successful serial trans- plantation of the tumors. Shimkin (171) in 1940 introduced more exact quantitative methods by considering the number of nodules in the lungs. Grady and Stewart (56) in 1940 rlarified the problem of the histogenesis of the neoplasm. Heston (69-72) in 1940 began his gene-linkage studies on the tumor, which remain among the better contributions to experimental work on the inheritance of cancer. Nettleship, Henshaw, and Meyer (145) in 1943 stimulated wide interest in their discovery that urethane induced pulmonary tumors in mice. The work was extended along chemi- cal lines by Larsen (102-104) and to the induction of pulmonary tumors

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in rats by Jaff6 (91, 92). The recent work of W. E. Smith (185, 187) on pulmonary tumors induced in embryonic lungs of mice is certain to be of continuing importance.

In the investigations of pulmonary tumors in mice, three groups in three institutions have made particularly noteworthy and continually productive advances. The Imperial Cancer Research Fund in England laid the morphological foundations. The Rockefeller Institute for Medical Research in New York initiated the studies on induction and inheritance of the tumors. The National Cancer Institute in Bethesda has been most strongly represented in the advances since 1937.


The original investigators using mice noted that mammary tumors and primary pulmonary tumors were the most frequent types of neo- plasms in the species. Nevertheless, the frequency of pulmonary tumors is relatively low in unselected, nonhomozygous mouse populations. Twort and Twort (195), in a study of 60,000 mice, stated that the frequency was approximately 1%. Wells, Slye, and Holmes (20l), in 147,132 autopsies of mice, found pulmonary tumors in 2865, or 2%. Andervont (14) studied 34 wild house mice and their progeny and found 6 with pulmonary tumors, a t the age of 20 to 23 months.

Tumors of the lung are seldom found in noninbred mice under one year of age and occur in the same frequency among males and females. The presence of pulmonary tumors is not associated with parasitic in- festations, nor is there any evidence of communicability or infectious transmission between animals.

With the development and general use of inbred mice, it was found that various strains had markedly different frequencies of pulmonary tumors. Since inbreeding and selection of strains toward homogeneity were made without particular reference to the occurrence of pulmonary tumors, the segregation of this characteristic in specific strains was unpremeditated. In retrospect, therefore, there must have been connection between certain definite genic characters and the susceptibility to pul- monary tumors.

Since 1935, data have been accumulating on the frequency and dis- tribution of pulmonary tumors in the various homozygous strains of mice that are used in cancer research. It is notable that the frequency of pulmonary tumors in a strain appears to be more constant, as reported by various laboratories, than the frequency of mammary or hepatic tumors. This would indicate that pulmonary tumors are less under the influence of various environmental factors than many other neoplasms

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of the mouse. It is now also clear that the frequency of pulmonary tumors involves the factors of time of appearance and multiplicity; i.e., in more susceptible strains, the tumors appear a t an earlier age and a higher pro- portion of animals have multiple tumors, whereas in the resistant strains, only rare single tumors appear in animals that are 18 months of age or older.

The classical most susceptible strain of mice relative to pulmonary tumors is strain A, established by Strong (192) in 1921. Pulmonary tumors that are recognizable grossly are seen in mice as young as 3

2 4 6 8 10 12 14 16 18 20 22 24 Age in months

FIG. 1. Frequency of pulnionary tumors in three inbred strains of mice, a t different ages. Data for strain A mice from Shimkin ( l i l) , Heston (71), and Bittner (24); for strain C (Bagg alb C) from -4ndervont (13); for strain CJEI from Andervont (12).

months, and the frequency rises steadily to approximately 90% by 18 months (Fig. 1). Two or more tumors, but usually not more than five, begin to appear in animals 8 months of age. The Swiss albino mice are apparently as susceptible to pulmonary tumors as strain A (57, 152). Bagg albino C, I, Y, and C3H are of intermediate susceptibility in the approximate order given, showiiig pulmonary tumors in 10% to 30% of the animals over a year old (7, 12, 13); tumors are not observed in mice under 6 months of age and multiple tumors are infrequent. Strains C57 black and ( ‘57 leaden (L or 31) are most resistant, having practically no pulmoriary tumors even when the mice are two years of age. Little ct uZ. (115) recorded one pulmonary tumor in 742 mice of the C57 black strain.

Table I presents data on the frequencies of pulmonary and of mam- mary tumors in nine commonly used strains of mice. There is no relation- ship between the frequencies of these two types of iteoplasm, and there

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appears to be no relationship of frequency or of susceptibility to induction of pulmonary tumors to any other spontaneous or induced neoplastic process, such as hepatoma, lymphoma, subcutaneous sarcoma, or cu- taneous carcinoma.

The essential identity of spontaneous and of induced pulmonary t.umors is further attested by the fact that the frequency, time of appear- ance, and multiplicity of induced tumors are in parallel with the spon- taneous occurrence of the neoplasms. To all induction procedures, strain

TABLE I Frequency of Pulmonary and Mammary Tumors in Eight Strains of Mice&

Pulmonary Tumors Mammary Tumors Per Cent of Animals

Strain 12-18 Months Old Breeding Females Per Cent of

A Swiss B alb C (C) I Y C,H dba C57 leaden (L or M) Cg7 black

70-90 40-50 15-25 10-20 10-20 5-1 5

5 < 1 < I

70-85 20 5 1 5

75-100 55-75 <1 < 1

0 Modified from Heston (73).

A mice have proved to be most susceptible, and strains CW black and C6, leaden the least susceptible, with the intermediate strains retaining their relative position. This observation has greatly facilitated many investigations in that observations can be made within a few weeks with induced tumors instead of within the 18 to 24 months required for studies on spontaneous tumors. It is important to note that Shapiro and Kirschbaum (167), in a study of seven strains of mice, recorded that the NH strain, stated to have a very low spontaneous frequency of pul- monary tumors, was susceptible to the induction of such tumors with urethane. Since the actual frequency of spontaneous pulmonary tumors in the NH strain at various ages has not been published, this seeming exception should be considered sub judice at the present time.


It is a commonly accepted impression that primary pulmonary tumors are rare except in the mouse and in the human being. The conclusion regarding mice is accentuated by the high frequency observed in some inbred strains. In unselected populations it is approximately 2 % when

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the animals are maintained for their whole life span. In man, the fre- quency among those dying of all causes does not exceed 2% (198) even considering the marked rise in frequency among males during the past 30 years.

Pulmonary tumors in laboratory animals other than the mouse are stated to he rare. McCoy (13-1) found no pulmonary tumors among autopsies on 100,000 wild rats, a population in which rats of advanced age undoubtedly were exceptional. Saxton et al. (156) observed two pul- monary adenomas among 498 rats of the Osborne-Mendel strain-a fre- quency of 0.4%. Horn and Stewart (87) reported a pulmonary tumor, histologically identical with the mouse neoplasm, in a one-year-old rat of Marshall strain 520 as the only example of a spontaneous pulmonary tumor in a rat at the Xational Cancer Institute. In regard to pulmonary tumors in rats, it should be noted that lungs of old rats often have areas of inflammation and bronchial metaplasia that can be readily confused with neoplastic reactions (149).

Only individual cases of pulmonary tumors have been recorded in guinea pigs, and this species is supposed to have very few spontaneous neoplasms of any type. Goldberg (55) described a single adenocarcinoma of the lung in a guinea pig, and Norris (146) published an occurrence of pulmonary adenomatosis in one animal. One pulmonary tumor was found by Heston and Deringer (81) in a guinea pig of inbred strain 2; the tumor resembled the pulmonary tumor of the mouse. Lorenz et aZ. (120a) re- cently reported three cases of pulmonary tumors in 19 guinea pigs three to six years of age.

Polson (150) recorded one carcinoma of the lung among 66 instances of neoplasms in rabbits. Sjolte (181) included one adenocarcinoma of the lung in one rabbit. Schinz (162, 163) reported two instances of adeno- carcinoma of the lung in rabbits. One had received 0.1 g. of cobalt into the thigh four years previously, and the other was a female that five years before had been injected with methylcholanthrene into the pregnant uterus. Whether these are instances of induced or of spontaneous neo- plasms is anyone’s guess.

The only example of a pulmonary tumor in a fowl was the case of Apperly (19), in a Black Orpington over one year of age, that had an adenocarcinoma of the lung with metastases to the liver.

Although i t is impossible not to conclude that rats, guinea pigs, rab- bits, and fowl develop pulmonary tumors much less frequently than mice, one cannot but wonder to what extent the age of the animals at the termination of most experiments, the preponderant interest of the investi- gators in other neoplasms, and lack of care in the autopsy examination contribute to this rarity.

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Sticker (191) in 1902 tabulated extensive data regarding carcinomas of larger domestic animals, primarily from veterinary and abattoir sources in Germany from 1858 to 1900. He included 13 of the lung among 311 carcinomas of the horse, 3 of the lung among 73 carcinomas in bo- vines, 10 of the lung among 766 carcinomas in dogs, 3 of the lung among 121 carcinomas of the cat, and 1 carcinoma of the lung among 7 car- cinomas in sheep. Feldman (52) in his monograph which gives many primary sources added 3 pulmonary carcinomas among 41 carcinomas in sheep, and 1 in a kangaroo. Sjolte (181) reviewed the subject of primary pulmonary cancer in animals and presented 23 cases from Copenhagen, of which 10 were in the dog, 4 in the horse, 7 in cattle, and 1 in a jaguar. Ten of the neoplasms were adenocarcinomas, 6 were solid carcinomas, 3 were epidermoid carcinomas, and 3 contained sarcomatous elements and were designated as adenocarcinosarcoma. Metastases of the tumors were stated to follow the same distribution as was to be anticipated in man.

It is difficult, if not impossible, to indicate the true frequency of pulmonary neoplasms in various species of animals, especially when in- formation is lacking regarding the age distribution of the population coming to autopsy. It can certainly be concluded that pulmonary neo- plasms are to be found in a wide variety of domesticated and wild animals but that the frequency is probably considerably lower than among mice and human beings. For example, Feldman (52) estimated that 8% to 10% of the older dogs are affected with neoplasms, of which carcinoma repre- sents 40% to 50%. If we take 10 pulmonary carcinomas among 766 canine carcinomas, or 1.3% of carcinomas from Sticker’s (191) data as representative, this would indicate the frequency of the tumor among older dogs at approximately 0.05%, or less than one-twentieth of that found in the murine and the human species.


1. Gross and Microscopic Appearance

Primary adenomatous pulmonary tumors in mice have an extremely uniform gross and microscopic appearance. In the gross, or after fixation, the tumors are pearly white, glistening, discrete round nodules, often situated just below the visceral pleura (Fig. 2). There is no predilection €or side or lobe. The tumors are sharply contrasted against the normal tissue of the lung and have a rubbery consistence. With practice, the tumors can be correctly identified with the naked eye or under a dissecting microscope when they are a fraction of a millimeter in diameter. The

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FIG. 2. .\Iultiple induced tumors of the lung of mouse x 3.8. From Stewart (189).

FIG. 3. Primary pulmonary tumor of the mouse x 60. From Stewart (189).

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presence of a superimposed pneumonic consolidation may obscure the appearance of small tumors, and the presence of an obvious tumor else- where may make distinction difficult between primary pulmonary tumor and metastasis.

Under the microscope, the tumor is devoid of a capsule and infiltrates and compresses the surrounding pulmonary tissue, with the intact some- what thickened pleura over the mass (Fig. 3). The tumor is usually of

FIG. 4. Primary pulmonary tumor of the mouse X 200. From Stewart (189).

a uniform adenomatous pattern, consisting of closely packed columns of cuboidal or columnar cells (Fig. 4). Papillary formation is frequent in larger tumors. The cells are uniform in size and shape, with a homogenous, acidophilic cytoplasm and round, hyperchromatic nucleus of moderate size. Cilia are not encountered. Mitotic figures are rare. The sparse stroma is composed of adult-appearing fibroblasts, and there are few blood vessels. The margins of the tumor are usually completely devoid of inflammatory reaction, lymphocytic infiltration, or increase in fibrous elements. Occasional small areas of necrosis and small cysts may be encountered in the larger masses.

Well over 95% of all pulmonary tumors in mice present this appear-

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ance. There seems to be no morphological difference between the neo- plasms seen in iioniribred mice and in mice of various homozygous strains (151, 170). The tumors are the same whether they are spontaneous or induced by carcinogenic agents, except, that the latter are multiple, whereas most spontaneous tumors are single.

In large series of pulmonary tumors in mice, a few unusual forms have been encountered. Tyzzer (196) described one epidermoid carcinoma, and in this animal unidentified crystals were observed in the pulmonary parenchyma and bronchioles. Such crystals have been noted also by Green (57) in mice of three strains, but apparently are not related to the occurrence of pulmonary tumors. Wells, Slye, and Holmes (201) added seven epidermoid tumors among 2865 mice with primary pulmonary neoplasms.

Horn et al. (86) have described another type of pulmonary tumor in tiyo mice, in which a single layer of columnar cells containing mucus lined the pulmonary alveoli, with papillary tufted projections. The authors believed that these were cases of pulmonary adenomatosis in the mouse. An illustration published by Twort and Twort (195) in 1932 also pre- sented a similar pulmonary tumor.

Primary pulmonary tumors are usually seen in older mice, unless the neoplasms have been induced. The induced tumors seem to appear rather suddenly, grow to a size of 3 to 6 mm. in diameter, and then prog- ress slowly, eventually coalescing with neighboring nodules. Relatively few mire dying with pulmonary tumors can be stated to have died of the neoplasm, although in some cases i t might replace half of the thoracic space and invade the diaphragm or chest wall.

Metastasis of pulmonary tumors is infrequent and late. Wells, Slye, and Holmes (201) found metastases in 104 of 2865 mice with pulmonary tumors, or 3.6%. All had metastases to the mediastinal lymph nodes, and ten to distant organs, including five to the kidney, three to the heart, and one each to the seminal vesicle and skull. One-third of the metas- tases were sarromatous in appearance, even when the primary neoplasm lacked sarcomatous elements. Campbell (32) found distant metastases in 3 of 192 mice with pulmonary tumors; two were to the kidney and one to the heart. In a group of 60 mice with pulmonary tumors induced by oral administrations of 1,2,5,6-dibenzanthracene, Magnus (132) found distant metastases in two, of which one was to the liver and one to a suprarenal gland. Thus, induced as well as spontaneous pulmonary tumors can develop distant metastases, in approximately 3 % of the cases.

The monotonous adenomatous pattern, the uniformity in the size and shape of individual cells, and the infrequent mitotic figures in small pulmonary tumors suggest that the neoplasms undergo a ((benign ” stage

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when they may be designated as adenomas. That all these tumors either originally or eventually are malignant is indicated by their lack of en- capsulation, invasion of pulmonary tissues, progressive growth, metas- tasis, and transplantability to the subcutaneous tissue of genetically homologous mice. The terms that have been applied to these tumors include adenoma, adenocarcinoma, and papillary cystadenoma. Stewart (189) recently suggested “alveologenic carcinoma,” on the basis of histogenesis. It would appear to me that a purely descriptive and non- committal name such as “primary adenomatous pulmonary tumor” for the common variety of these neoplasms still remains preferable.

2. Biochemical Properties

Biochemical comparisons between normal lungs and pulmonary tu- mors in mice are limited to the study of one transplantable pulmonary tumor. This is Andervont’s (4) Lung Tumor F, that arose in a strain A mouse and has maintained its adenomatous pattern for well over 100 passages. Greenstein (58) in his monograph summarized data on eleven comparable measures of enzymatic activity of this tumor and normal mouse lungs. The tumor was higher in xanthine dehydrogenase, and con- siderably lower in alkaline phosphatase and in esterase. The same range of activity as in normal lung was found in respect to arginase, acid phos- phatase, ribonucleodepolymerase, deoxyribonucleodepolymerase, ribo- nucleodeaminase, and deoxyribonucleodeaminase. These characteristics are in each instance in conformity with findings on a wide variety of transplanted tumors in mice and in rats. The biotin content of Lung Tumor F is approximately half that of normal mouse lung (202). Schneider (164) reported that Lung Tumor F contained approximately three times the ribosenucleic acid and twice the deoxyribosenucleic acid content of normal lung tissue of mice.

Thus, in biochemical terms, at least one transplantable pulmonary tumor has the general characteristics of malignant neoplasms.

V. HISTOGENESIS OF PULMONARY TUMORS IN MICE The earlier workers on pulmonary tumors in mice could not come to a

conclusion regarding the histogenesis of the tumor. There was constant involvement of the alveoli, and no tumors appeared to be limited to the bronchioles, although they were sometimes involved by the neoplastic process. In general, the tumors were considered to arise from the alveolar cells or from the bronchial epithelium.

The rapid production of pulmonary tumors in susceptible mice with carcinogenic hydrocarbons allowed a systematic investigation of their histogenesis by Grady and Stewart (56). Strain A mice were injected

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subcutaneously with 1,2,5,G-di benzanthracene or 20-methylc holant hrene and killed a t frequent intervals, and a lobe of the lung was serially sec- tioned. C’onsecutive morphological changes in three dimensions could thus he follou-ed. Jlostofi and Larsen (141) recently repeated the study using urethane, and recorded a practically identical sequence of events.

The initial morphological effect, seen during the first two weeks, is a proliferation of alveolar cells, particularly prominent in the subpleural alveoli remote from t hc bronchioles. .%mong the alveolar cells appear individual or small groups of enlargcd cells which sonietimcs project into the a\-eolar lumen. Ih r ing the third and fourth weeks there are islands of such cells, shon-ing increased numbers of mitotic figures, and by the sixth week these masses arc recognizable tumors. The bronchial epithe- lium shows no hyperplasia or proliferation, and there are no inflammatory changes. Only a t later stages are the tumors seen in close proximity or invading the lumen of the bronchioles.

These studies coiiclusivdy demonstrated that the usual pulmonary tumors i n mice arise from what appear to be the alveolar lining cells. Orr’s (148) opinion that pulmonary tumors in mice and in rats following urethane arise from bronchi within areas of inflammation and atalectasis appears to be erroneoub and attributable to the examination of larger tumors at later stages of their growth.

The nature of the cell from nhich pulmonary tumors in mice are derived is ohscure and relates to the general problem of the nature of the alveolar cell i n the lung. Whether these are epithelial cells or mesenchymal cells, or a combination of both, has heen the subject of considerable con- troversy obviously involving semantics as well as embryology. The reader is referred to papers by Bell (23) and by Geever ct al. (54) for further discussion.

An interesting problem that is related to the topic of the nature of the alveolar cell and tumors derived therefrom is the behavior of metastases and of transplants of the tumors. I t has already been rioted that one-third of metastases are sarciomatous i n appearance (201). Andervont (4), who i n 1937 first recorded the subcutaneous transplantation of pulmonary tumors, also obserl-ed that 3 of 7 such transplants changed into sarcomas in subsequent passages. The study was repeated (10) on 20 spontaneous and induced pulmonary tumors from three strains of mice, and 5 became sarcomas. Stewart, Grady, and Andervont (190) more recently reviewed and estended the investigation, using more precise histologic techniques, with exactly the same conclusions. The authors indicated a number of interesting postulations regarding the finding. It would seem t o this reviewer that two possibilities are most likely. One is that the alveolar lining cell is mesenchymal in origin and, on the one hand, possesses the

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property to appear epithelial and give rise to adenomatous epithelial-like tumors and, on the other, to revert to its mesenchymal nature and pro- duce sarcomas. The other explanation is that the primary pulmonary tumors are mixed tumors containing both epithelial and mesenchymal elements. Breedis et al. (29), in a study of two transplanted pulmonary tumors, one of which altered in appearance on passage, concluded that this represented a modification in the appearance of the tumor cells and not an overgrowth of sarcomatous elements present in the original tumor. Newer methods of histochemistry and cytochemistry may yet yield additional information on these points.


The characteristic of different frequencies of pulmonary tumors in different strains of mice was appiied to genetic studies by Lynch (124-130) in 1926. Matings between a low-tumor strain 1194 of which 7 % developed pulmonary tumors, and a high-tumor strain Bagg albino, of which 40% developed pulmonary tumors, resulted in the F1 generation that resem- bled the high-tumor strain, and backcross generations resembled the strain to which the F, mice were mated. Lynch employed pulmonary tumors induced by tar-painting and injection of l12,5,6-dibenzanthracene as well as spontaneous tumors.

Andervont (3, 6, S), using spontaneous and induced tumors, and Bittner (24, 25) using spontaneous tumors in strains A and C57 black, obtained F1 and backcross ratios that also were compatible with an interpretation of single-dominant-factor inheritance of susceptibility. Extension of the work to crosses between other strains (11) indicated that although susceptibility was inherited in a dominant manner, multiple genetic factors or modifying factors had to be involved.

Heston (69-73), and Heston and Deringer (78-82) have carried out extensive, exact studies on the genetic linkages between susceptibility to pulmonary tumors and known genes of mice. Most of the investigations have been performed with tumors induced by means of a single intra- venous dose of 0.5 mg. of 1,2,5,6-dibenzanthracene, but observations on spontaneous tumors thus far have reiterated the conclusions reached on induced neoplasms. The use of induced tumors not only allowed more rapid observations, but made it possible to consider latent time and num- ber of tumors as well as the frequency of occurrence (Figs. 5 and 6).

Outcrosses of strain A mice to three low-tumor strains showed that the response was different in each group, and indicated multiple genetic factors with cumulative effects. Linkage studies demonstrated an associa- tion between susceptibility to pulmonary tumors with a t least five genes. The lethal yellow gene (Au) increased susceptibility, although in the cross

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:t 25


0 S T R A I N - L

. STRAIN - A - _cr__LG.

FIG. 5. Frequency of pulmonary tumors in strain A and strain L (C6, brown) mice and their F,. F,, and hackcross hybrids killed a t intervals following intravenous injection of 0.5 mg. 1,2,5,6-dibenzanthracene. Open circles represent groups of 20 to 108 mice; closed circles, groups of 15 to 18 mice. From Heston (71, 73).

0) 0 L ,,t ' 2 5 1

25 t 50

n Fl


L-BACKCROSS b 0 20 40 60 80 100

Number of nodules

FIG. 6. Frequency polygons for number of tumor nodules in strains A and L mice and their F1, F2, and backcross hybrids at 16 weeks following intravenous injection of 0.5 mg. 1,2,5,6-dibenzanthracene. From Heston (71, 73).

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employed i t was introduced from the resistant strain. Flexed tail (f), hairless (hr), and the linked genes shaker-2 (sh-2) and waved 2 (wa-2) all were associated with a decrease in susceptibility. No association was found between susceptibility and eight genes, the leaden (In), piebald (s), dilution (d), agouti (a), brown ( b ) , waltzing ( v ) , waved-1 (wa-l), and pink-eye ( p ) genes.

In 1942, Heston (71) concluded that a minimum of four pairs of sus- ceptibility genetic factors were involved in the difference between strains A and L, and that in the total variance in susceptibility 86% were due to genetic factors and 14% to nongenetic factors. I n a later review (73) the opinion was modified to stating that the number of genes involved was not known, and that i t was not clear whether susceptibility is carried on the chromosomes with the marker genes or is an added effect of the identified genes. Later complete analysis (82) showed that the association between susceptibility to pulmonary tumors and the flexed tail and lethal yellow genes was attributable to the action of the genes per se. There was no association between these genes and susceptibility to mammary tumors or hepatomas.

Burdette (30) studied the genic linkage of urethane-induced tumors, and found such association with shaker-2 and waved-2 genes, but not with the flexed-tail gene. This finding indicates that the linkage of suscepti- bility genes and certain marker genes might not be the same for tumors induced with urethane and with carcinogenic hydrocarbons. Additional complications are also introduced by the recent work of Deringer and Heston (43), in which i t was shown that the Swiss (SWR) mice developed a greater mean number of pulmonary tumors than the strain A mice, and that in two crosses of strains the males developed more tumors than the females. Thus, it suggests that the number of nodules induced with intravenously injected 1,2,5,6-dibenzanthracene is not necessarily con- trolled by factors identical with those controlling frequency of spon- taneous tumors in all strains, and that factors connected with sex may exert an influence in certain crosses of strains.

The reader is referred to the reviews of Heston (73, 75, 76) for a more extensive presentation of this line of investigation, and its relationship to heredity factors in other neoplasms.


During the second decade of the century, several workers (45, 98, 200) recorded that pulmonary tumors were found in mice painted with tar. The interest was in cutaneous carcinomas, however, and the distinction between primary pulmonary tumors and metastatic deposits was not clearly made in the publications. Murphy and Sturm (144) in 1925 first

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clearly demonstrated the carcinogenic effect of tar upon pulmonary tissue of mice by rotating the applications of tar over different areas of the skin and thus avoiding the appearance of ski11 cancers. Of 10 mice painted in this manner that survived for sis months, 85 % had multiple pulmonary tumors, whereas none of 38 untreated controls of the same stock had tumors. The possihility of the tar or some constituent thereof reaching the lungs was mentioned but discarded in favor of a postulation that tar- painting altered the body state so that tumors occurred at points of incidental irritation.

The findings of Murphy and Sturm (144) were confirmed and extended in a number of laboratories (27, 121, 157). Schabad (157-161) exposed mice to tar by several routes, including subcutaneous injection, intra- vaginal and rectal introduction, and intraperitoneal injection, and elicited pulmonary tumors. The descendants of mice that developed pulmonary tumors were reported to be more susceptible to pulmonary tumors than descendants of mice that did not; he postulated (158) that this was due to the transmission of the carcinogen to the progeny.

\\.'ithin a few years after the publication of the report on the carcino- genic property of 1,2,5,fj-dibenzanthracene, a number of workers (1 11, 128, 159) found that a single subcutaneous injection of approximately 1 mg. of the chemical produced pulmonary tumors in mice. Andervont ( 2 ) reported that multiple tumors arose in 25 of 26 strain A mice within three months of the injection and before any had developed subcutaneous sarcomas, suggesting that the lungs of these mice mere a more delicate test object than the subcutaneous tissue.

Preparation by Lorenz (1 18, 122) of carcinogenic hydrocarbon disper- sions and adsorbed on charcoal allowed investigation of the effects of various routes of administration and of the physical state of the chemicals. Intravenous injection was the most effective route, producing the greatest number of tumors within a few weeks. Dibenzanthracene adsorbed on charcoal, and thus maintained at the site of injection, produced local sarcomas and few, if any, pulmonary tumors, 1vhere:ts the same prepara- tion administered intravenously induced multiple tumors of the lung (15, 16). Contact of the lung with inserted threads coated with dibenz- anthracene evoked not only the usual adenomatous humors but epider- nioid carcinomas ill mice of strains A and ('3H (5, 9).

In 1938, Andervont (7) published a study on the comparative suscepti- bility of eight inbred strains of mice to the spontaneous occurrence and induction of pulmonary tumors following subcutaneous administration of 1,2,5,G-dibenzanthracene. Susceptibility t o induced tumors paralleled the spontaneous frequency of the tumors; i.e., mice most susceptible to spontaneous tumors were also most susceptible to the induction of pul-

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monary tumors with this carcinogen. Even mice of strains most resistant to spontaneous tumors, such as Cb7 black, developed some pulmonary tumors within 38 weeks following intravenous injection of 0.5 mg. of the carcinogen (10). Shimkin (170) extended the observations with intra- venously injected 20-methylcholanthrene, with essentially identical

/ 8 14 20

Time in weeks

FIG. 7 . Response of lungs of strain A mice to a single intravenous injection of approximately 0.25 mg. of nine compounds dispersed in 0.25 cc. of water. Carcinogenic index is the per cent of mice with pulmonary tumors times the mean number of pul- monary tumors in positive animals. The broken line represents untreated control mice. A, 1,2,5,6-dibenzanthracene; B, 3,4,5,6-dibenzcarbazole; C, 3,4-benzpyrene; D, 15,lG- benzdehydrocholanthrene; E, 1,2,5,G-dibenzacridine; F, Zmethyl-3,l-benzphenan- threne; G, 4’-methyl-3,4-benzpyrene; H , 1,2-benzanthrscene; I, 3-methoxy-10- propyl-1,2-benzanthracene. All but three points represent groups of 10 to 20 mice. From Andervont and Shimkin (17).

results. A detailed study (171) of the dose-time-response relationships of the two carcinogens in strain A mice indicated that this was a rapid, sensitive, and reliable biological system for the quantitative testing of carcinogenic properties of a wide variety of chemical materials, as well as for other experimental applications.

The procedure of pulmonary tumor induction as a test for carcinogenic properties of a number of chemicals was elaborated by Andervont and Shimkin (17). Figure 7 presents the results on nine compounds admin-

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istered intravenously in single doses of approximately 0.25 mg. By con- sideration of the per cent of animals developing tumors and of the number of tumors at specific periods after injection, the quantitative carcinogenic potency of a wide range of chemicals for specific tissue can be derived, and most tests completed in four months. Perhaps the greatest disadvantage of the method is the lack of a clear end-point, since strain A mice develop pulmonary tumors spontaneously at an early age. Since all strains of mice develop the neoplasm if observed for a sufficient length of time, and since the susceptibility of the strains to spontaneous tumors parallels susceptibility to induction of such tumors, this objection cannot be overcome by the use of more resistant mice. Reliable conclusions can be derived only by having comparable control groups of sufficient num- ber that are sacrificed at the same time as the experimental animals. With strain A mice, four months is a desirable time to kill the first groups of animals. It is more difficult to establish whether pulmonary tumors are produced in the course of an experiment in which the animals are per- mitted to live out their life span. When the lungs of such mice are studded with multiple tumors, it is probable that the tumors were induced. On this basis, 8.9-dimethyl-1 ,Zbenzanthracene, as reported by Shear (168), and several compounds included i n the tests of Badger et al. (20), of Dunlap and Warren (48), and of Law and Lewisohn (108) would be in- cluded as carcinogenic for the pulmonary tissue of mice.

The investigations of Andervont and Shimkin (17) showed that there was no complete parallelism between the ability of some compounds to produce pulmonary tumors in strain A mice and their carcinogenicity as revealed by the induction of sarcomas following subcutaneous injection or of carcinomas following percutaneous applications. Badger et al. (20) and Iiennaway et al. (95) pointed out a number of polycyclic hydrocarbons that were of low but positive carcinogenic potency in producing cutaneous carcinomas in mice and were inactive in eliciting sarcomas upon sub- cutaneous injection. By the same token, no exact parallelism or even the same qualitative response should be anticipated in results obtained by the pulmonary-induction technique and in those obtained by the sub- cutaneous or percutaneous methods. The procedures are ancillary, and each contributes independent information.

The statistical aspects of the reaction in strain A mice, including the number of animals to be considered in relation to the desired accuracy and levels of statistical significance, have been calculated by Shimkin and McClelland (179). Heston and Schneiderman (85) also presented statistical considerations of the response. Rogers (152), in using the size of nodules as well as their number, added another measure by which the actual mass of neoplastic tissue produced by carcinogenic stimuli

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can be determined with some degree of accuracy (Fig. 8). The application of quantitative differential techniques, such as devised by Chalkley (36), would make it feasible to determine the number of cells and their mass appearing at various periods under the stimulus of various doses of carcinogenic agents. The recent report of Telford and Jeffery (194a) is a step in this direction.

25 125

ail 15 74


10 50

J 70

Mouse wt. Lung wt.

‘hmope pep mouse

Total tumors

+ at injection (weeks)

FIG. 8. The relationships between age, weight of lungs and body weight, and the induction of pulmonary tumors in Swiss mice with ethyl carbamate (urethane). Mice received single intraperitoneal injections of 1 mg. urethane per gram mouse and were killed seven weeks later. Each point represents the mean value of 25 animals. From Rogers (152).

No specific reports have been noted on the induction of pulmonary tumors in rats with carcinogenic polycyclic hydrocarbons. Even such an extensive investigation as that of Dunning, Curtis, and Bullock (49) , who injected subcutaneously 1,2,5,6-dibenzanthracene and 3,4-benzypyrene into 688 rats of six strains, made no mention of the presence or absence of pulmonary tumors, but the paper reveals that the interest was limited to the subcutaneous neoplasms. That pulmonary tumors can be induced in rats following such procedure is indicated by Lewis and King (112), who comment upon the presence of “lung neoplasms’’ in the King strain of

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rats following the sucutaneous injection of 1 to 4 mg. of dibenzantkracene, benzpyrene, or methylcholanthrene. Apparently no pulmonary masses were seen in 14 other strains used by the authors, and the lesions in the King strain are not descrihed or illustrated. Following the intravenous injection of 5 mg. of methylcholanthrene into a few rats of the Wistar, C'olumbia, and Buffalo strains, Shimkin (172) obtained a single pulmonary tumor in one of five Buffalo rats a t 6 months, and definite multiple pul- monary tumors in another Buffalo rat killed a t l l months. These tumors were histologically indist iiiguishable from the adenomatous pulmonary tumor of the mouse.

In guinea pigs no pulmonary tumors were observed following sub- cutaneous injection of 20 t o 40 mg. of methylcholanthrene, although sub- cwtaneouc; sarcomas were elicited in 29 of 34 animals (180). Heston and Deringer (S l ) , howevcr, injected 10 to 30 mg. of methylcholanthrene intravenously and obtained pulmonary tumors in 17 of 51 guinea pigs of strain 2 and in 13 of GO animals of strain 13. These neoplasms appeared to be of thr same histologic type as the mouse tumors and were found to occur in greater multiplicity among female than among male atiirnals.


In the course of in\wtigating the effects of exposures to roentgen rays. Srtt leship, Henshan., and JIeyer (145) in 1043 encountered un- expected multiple pulmonary tumors in their experimental mice of strain C'J-I. -Analysis of the observation and futher studies established that thc anesthetic used. ethyl carbamate (urethane), was the causative agent. IIenshaw arid Meyer (68) reported that singlc intraperitoneal doses of 1 mg. of iirethane per gram body weight in strain A mice produced an average of 9.5 pulmonary tumors in all of 18 mirr hy four months, and the response was linearly increased to an average of over 30 modules when four or five weekly injections \\-ere given. Subcutaneous injection and oral aclniinistratioiis also produced pulmoiiary tumors, but no other neoplastic reactions (67, 166). I t was thus established that urethane was a potent carcinogen for the pulmonary tissue of mice, and apparently did not produce neoplasms a t the site of injection or other distant sites.

There \\-as immediate and wide interest in the observations, since urethane is an old, well-known chemical, of simple molecular structure, water-soluble, and obviously more applicable to, and convenient in, many studies than the carcinogenic hydrocarbons. The morphology and histo- genesis of the pulmonary tumors are identical with spontaneous neo- plasms or those induced with the hydrocarbons. The response of mice to pulmonary tumor induction with urethane is parallel to their suscepti-

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bility to spontaneous neoplasms of the lung (40), although Shapiro and Kirschbaum (167) reported that the NH strain of mice may be an excep- tion to this conclusion.

Larsen (100-104) carried out a series of systematic studies on the relationship of chemical structure and biologic activity of urethane and related materials. He showed that the anesthetic action of urethane was not involved in its ability to evoke pulmonary tumors (100). Twelve barbituric acid derivatives and nine miscellaneous hypnotics including paraldehyde, ethanol, and chloral hydrate did not produce pulmonary tumors in strain A mice. Orr (147) also found that three hypnotic agents, including nembutal, were negative for pulmonary carcinogenic activity.

An examination by Larsen (102) of various esters of carbamic acid showed ethyl carbamate to be a t least 20 times more active than any of the esters tested. Isopropyl, n-propyl, and trichloroethyl carbamates had some activity, whereas methyl, n-butyl, isoamyl, and chloroethyl esters were negative. Study of nitrogen-alkylated derivatives of ethyl carbamate demonstrated (103) that n-isopropyl urethane, and methylene and ethyli- dene duirethanes a t 0.5 mg. per gram mouse per week for 13 weeks were active, but less so than ethyl carbamate. This suggested that the car- cinogenic activity of alkylated urethanes may have been due to pre- liminary dealkylation to ethyl carbamate. Possible degradation products of urethane, including ammonium carbamate, sodium bicarbonate, am- monium chloride, and potassium cyanate, had no effect upon the fre- quency of pulmonary tumors in strain A mice (104).

The effects of urethane were explored in other species. No pulmonary tumors were obtained by Cowen (42) in guinea pigs or in chickens. Gross et al. (61) reported that white-footed deer mice (Peromyscus leucopus noueboracensis) were completely resistant t o lung tumors following 16 weekly intraperitoneal injections of urethane.

Jafft5 and JaffB (91, 92) first recorded that pulmonary tumors could be induced with urethane in rats. Rats were maintained on 0.15% urethane in the diet, or received 30 intraperitoneal injections of approxi- mately 100 mg. each during three months. Of 38 animals surviving for one year, 8 had primary pulmonary tumors. Four hepatomas were also found, and the authors believed they were induced by urethane-a point that requires confirmation. Guyer and Claus (62) administered three to five intraperitoneal injections of 1 cc. of a 10% solution of urethane, and elicited pulmonary tumors in 66 of 91 rats within eight to ten months. Mostofi and Larsen (140) also produced pulmonary tumors with urethane in the Wistar strain of rats. The histologic appearance and the histogenesis of pulmonary tumors in rats are apparently identical to the tumors evoked in mouse. It should be noted, however, that metastasis and trans-

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plantation of the rat tumors still remain to be described.' The relative susceptibility of various strains of rats t o urethane-induced neoplasms also has not been investigated.

The addition of the rat to experimental animals in which pulmonary tumors can be readily induced is of obvious importance and makes feasi- ble a number of physiological and biochemical approaches that would be much more difficult to carry out on the mouse.


1. Miscellaneous Agents

hlorosenskaya (137, 138) and -4ndervont (10) established that o-amino-5-azotoluene, when injected subcutaneously or intravenously or administered in the diet, significantly raised the frequency of pulmonary tumors in susceptible mice. The agent also induced hepatomas and hemangioendotheliomas, and the chief interest has been directed toward these neoplastic reactions. The dose is greater than necessary with poly- cyclic hydrocarbons, 10 to 100 mg. usually being given over a period of some weeks. In the extensive investigations of carcinogenic azo dyes in rats, no mention is made of the presence of primary pulmonary tumors, suggesting that the compounds are not carcinogenic for the pulmonary tissue of the rat.

Bielschowsky (23) reported the induction of pulmonary tumors in 11 of 104 rats with 2 amino- and 2-acetylaminofluorene. The agents were given in the diet, about 4 mg. per day for 30 weeks. The pulmonary tumors that were produced were apparently of the same adenomatous type as seen following the administration of urethane, although squa- mous metaplasia of the bronchi appeared to be more common (149). The great number of other neoplastic reactions that is evoked by the fluorenes obscures the relatively weak effect that is demonstrated in the lung. Morris et al. (139) reported no pulmonary tumors in rats of the Minnesota, Wistar, and Buffalo strains maintained on diets containing 2-nitro-2-amino-2-acetyl- and 2-diacetyl aminofluorene, although two rats developed lymphosarcoma of the lung. -4 progress report by Harding and Green (64) stated that multiple pulmonary tumors were induced in cats that mere maintained on diets containing 2-acetyl aminofluorene, but no complete report of this interesting finding has become available.

Law (107) in a single study injected sodium deoxycholate and cholic 1 A urethsne-induced pulmonary tumor of the M-520 strain rat was successfully

transplanted by Larsen in 1951 and is being maintained at the National Cancer Institute (187a).

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acid intravenously in strain A mice. Of 20 mice that received deoxy- cholate, 16 developed pulmonary tumors by six months, a t a time when 18 of 38 untreated controls had such tumors. The difference is significant, but the experiment bears repetition before it is concluded that sodium deoxycholate is carcinogenic for the pulmonary tissue of mice.

Heston (74, 77) has shown that the nitrogen and sulfur mustards are carcinogenic for the pulmonary tissue of mice. Two to four intravenous injections of approximately 0.025 mg. of methyl bis (0-chloroethyl) amine hydrophloride (HN,) produced an average of 3.5 pulmonary tumors in all strain A mice in four months. Positive effects were also ob- tained by the intravenous injection and inhalation (77) of bis (0-chloro- ethyl) sulfide. Nitrogen mustard, as urethane, is used in the clinical management of leukemia and lymphoma. Shimkin et aZ. (175) reported that a related compound, trisethylene-imino-s-triazine (TEM) also pro- duced pulmonary tumors following one or two intraperitoneal doses of 0.05 mg. in strain A mice. An investigation (174) of other agents used clinically for the lymphomas was negative ; this included potassium arsenite, 1,4-dimethylsulfonyl butane (myleran), 4,4’diamidinostilbene (stilbamidine), 4-aminopteroylglutamic acid (aminopterin), and 17- hydroxy-1 l-dehydrocorticosterone (cortisone).

The effect of ionizing radiations upon pulmonary carcinogenesis in mice was investigated by Lorenz et al. (120). Mice of strain A were ex- posed to total body irradiation from a radium source for 8 hours daily for nine months, receiving a total dose of approximately 2500 ry. Of 43 control animals, 47 % had pulmonary tumors, whereas among 55 irradi- ated mice 77 % had tumors and a higher proportion of these were multiple. The difference is statistically significant, but certainly the effect is not marked, and apparently quite slow in comparison with such agents at the carcinogenic hydrocarbons, urethane, or nitrogen mustard. An inter- esting question remains as to whether the effect is mediated through the same mechanisms. Heston and Lorenz (84) indicate that the combina- tion of exposure to x-ray and nitrogen mustard does not enhance the induction of pulmonary tumors, at least not under the conditions of their experiment. This might suggest the possibility of different modes of ac- tion for the two agents, although further work is necessary to establish the point.

Intravenous injection of colloidal thorium dioxide did not increase the number of pulmonary tumors in strain A mice (18). Lorenz et al. (120a) found 18 pulmonary tumors, of which 10 were multiple, in 42 guinea pigs exposed to long-continued total body gamma irradiation that survived for three to six years. Among 19 unexposed animals, 3 had single pulmonary tumors.

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A detailed analysis was made hy Blum (26) of the effect of ultraviolet radiation upon the frequency of pulmonary tumors in strain A mice, a high proportion of which developed skin carcinoma and subcutaneous sarcoma. Xot only was there no increase in the frequency of pulmonary tumors, but actually the experimental mice had somewhat fewer tumors. Thus, this form of carcinogenic modality is apparently localized to the site of contact, and its carcinogenic effect is prohnbly not mediated through the formation of hydrocarbon-like carcinogens from body con- stituents such as rholesterol.

Copeland and Salmon (38, 50) have published interesting observa- tions on the development of neoplasms i n rats on prolonged diets deficient in choline. I t wis reported that 385; of the rats developed pulmonary tumors, and 3 0 5 had aderiocarcinoma of thc liver. The neoplastic nature of the pulmonary lesions has bccn questioned, and the finding is con- sidered to be sub j i tdice a t this time.

The wide variety of chemical and physical agents that have been shown to produce pulmonary tumors i n mice and rats provides an irre- futable demonstration of the relative nature of the concept of carcino- genicity. Table I1 presents the observations on six types of carcinogenic

T;IBLE I1 Carcinogenic Properties of Six Carcinogens upon Four Tissues in MiceY

Pulmonary Cutaneous Suhcuta~ieous Adenomatous

Carcinogen Carcinoma Sarcoma Hepatoina Tumor

++ 20-~lethylcholanthrenc ++ ++ + If, 10-l)iitiethylanthrncerie + 3,~,5,C-I~ihmzcarhazole ++ ++ ++ ++ o-Amino-5-azotoliiene - + +-I- + ++ Ethyl ralhnniatr - - -

Ultra\ iolet rad13tion ++ +

- - -

- -

0 I’lrrs signs indicate the iniluctiun of neoplasnrs; iuinus signs indicate that such tuniors have not been elicitcd.

stimuli in mice. On the one hand, there are chemicals such as 3,4,5,6- dibenzcarbazole, which induces cutaueous carcinoma and subcutaneous sarcoma a t the site of application, and hepatomas and pulmonary tumors a t distant sites. I-ltraviolet radiation, on the other hand, produces tumors a t the site of contact in the skin or immediate subcutaneous tissue, and has 110 distant carcinogenic effects. Aminoazotoluene produces hepa- tomas, pulmonary tumors, hemangioendotheliomas and sarcomas in mice, and hepatomas in rats. These tremendous differences in effects,

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relative to species, tissue, and type of neoplasm induced, should certainly indicate that an over-all explanation of carcinogenic reaction is hardly to be anticipated.

2. Exposure to Inhalants

In attempting to demonstrate that inhalation of environmental dusts and fumes might have a causal connection with the development of pulmonary carcinoma in man, a number of investigations have been performed on the effect of such inhalants in animals, particularly mice.

As far back as 1923, Kimura (96) claimed that one adenocarcinoma was produced in a guinea pig following the intratracheal introduction of coal tar, but his description and illustration are not convincing. Valade (197) failed to elicit tumors in rats by the intratracheal injection of methylcholanthrene. An increase in pulmonary tumors in strain A mice by the intratracheal introduction of methylcholanthrene was produced by Shimkin (169).

R. E. Smith (184), in 1928, kept mice for 6 hours daily and then three times a week in atmospheres containing coal tar fumes and fumes from an automobile engine. No increase in pulmonary tumors was observed.

Campbell (31-35), in an extensive series of investigations from 1934 to 1943, exposed mice to various dusts over protracted periods. In 100 mice exposed to dust from tarred roads, 71 developed pulmonary tumors and 59 developed skin carcinomas, as compared with 90 controls, of which 7 had pulmonary tumors and no skin cancers. Exposure of mice to motor exhaust gas, carbon monoxide, and cigarette smoke did not result in significantly greater frequency of pulmonary tumors. Dusts from various sources were also examined, and a number of them increased the frequency of pulmonary tumors. In the summary article, Campbell (35) reluctantly concluded that these effects were of a ‘ I prolonged chemical nature, although it is not possible a t the moment to exclude entirely some effect of prolonged mechanical irritation. l 1

Seelig and Benignus (165) performed a similar experiment, exposing mice to soot from coal smoke, and obtained eight tumors of the lung among 100 Buffalo strain mice, whereas one tumor was found in 50 con- trols. McDonald and Woodhouse (135) also increased the frequency of pulmonary tumors in mice that inhaled dust which on spectrographic analysis contained benzpyrene. Leiter et a2. (109, 110) showed that ben- zene extracts of atmospheric dusts gathered in a number of cities in the United States produced sarcomas at the site of injection in 30 of 372 mice.

These studies demonstrate that dusts and other atmospheric con- taminants that may be expected to contain chemical agents of the polycyclic hydrocarbon type, are carcinogenic for the pulmonary tissue

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of mice when introduced as inhalants, and for the cutaneous and sub- cutaneous tissues of mice upon suitable application.

Studies with tobacco fumes are somewhat more controversial. Loren2 et al. (123) failed to elicit pulmonary tumors in strain A mice exposed to tobacco fumes. Essenberg (51) , however, succeeded in demonstrating that cigarette fumes increased the number of such tumors in mice. Strain A mice were exposed to cigarette fumes for one year, and 21 of 23 animals developed pulmonary tumors, as compared with a frequency of 59 % among 32 unexposed controls. In this connection, the carcinogenic effects of the products of tobacco has been described by Koffo (153), who ob- tained carcinomas of the ears of rabbits painted with tars from tobacco, and by Flory (53), who obtained papillomas and two carcinomas in mice. The recent publication of Wynder el a2. (209) summarizes the subject of experimental production of carcinoma in mice with cigarette tar, and reports the induction of epidermoid carcinoma in 44% of 81 mice painted with condensates obtained from cigarettes. The evidence seems convinc- ing that under certain conditions tobacco fumes as well as coal fumes may contain carcinogenic materials.

In a short abstract] Lisco and Finkel (114) mention that rats exposed to inhalations of radioactive cerium developed metaplastic changes in the bronchial epithelium and malignant tumors arising therefrom. Com- pletc descriptions would be useful, particularly since epidermoid car- cinoma of the lung has been produced but rarely ( 5 , 88) in experimental animals. r'orwald (I 99) has reported the appearance of pulmonary tumors in rats exposed to inhalations of beryllium sulfate aerosol.


1. Sex, Age, Diet, and Inflammation

It has already been noted that pulmonary tumors in mice, either of spontaneous occurrence or induced with various carcinogens, appear t o be less dependent upon and less affected by different physiological states and environmental factors than many other types of neoplastic growth, par- ticularly the mammary carcinoma and the hepatoma.

The orcurrence of pulmonary tumors and their induction are the same in the male arid the female mice, although some differences have been reported in crosses between inbred strains (43). That the tumor is inde- pendent of hormonal factors is also indicated by the lack of effect of breeding, of endogenous injections of estrogens or androgens, of deoxy- corticosterone (176), or of cortisone (174 .

Foster-nursing, i.e., the presence or absence of the Bittner milk agent necessary for the appearance of most mammary tumors in mice, has no effect upon the frequency of pulmonary tumors (21, 178).

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The investigations on carcinogenesis in embryonic lungs and in young mice (152, 185, 189) prove that these rapidly growing tissues are more susceptible to the effects of carcinogens than lungs of adult mice. Age after maturity, however, does not have a definite role, since little if any difference in response is seen in mice that are between 2 months and 11 months in age (152, 178).

Restriction in diet is one of the few environmental conditions that has been demonstrated to affect the occurrence of pulmonary tumors in mice. Tannenbaum (193, 194), in his systematic study of the subject, showed that the frequency of spontaneous pulmonary tumors was reduced by approximately 50% in Swiss mice on restricted diets. A decrease in the frequency of pulmonary tumors by some 30% by underfeeding was also demonstrated in strain A mice by Larsen and Heston (105). This appears t o be an over-all effect related to caloric restriction and body weight, and is not attributable to more specific restrictions in fat, car- b oh ydr a t es, or cys tine.

Special mention must be made of the role of nonspecific chronic or acute inflammation and irritation upon the initiation of pulmonary tumors in mice. Many colonies of laboratory mice are plagued by the constant or intermittent occurrence of parasites and other infections, and i t is not surprising that connections are sometimes drawn between such infections and neoplasia. The early morphologists soon discarded such postulations, and studies on the histogenesis of pulmonary tumors defi- nitely fail to connect any morphological evidence of inflammation with the initiation of the tumors.

An investigation was carried out by Shimkin and Leiter (177) which further dissociates inflammation from the spontaneous or the induced neoplastic reaction. Strain A mice were injected intravenously with finely ground arsenopyrite, chromite, thorite, or quartz. Despite the presence of the ore particles in the lungs and histologic evidence of chronic irrita- tion, the frequency of pulmonary tumors was not increased. The injec- tion of 0.1 mg. of methylcholanthrene concurrently with the ores resulted in no increase in the number of induced nodules as compared with mice that received only the carcinogen (Fig. 9). Soot from a chimney burning soft coal, a benzene extract of which induced subcutaneous sarcomas in C,H mice, increased the frequency of pulmonary tumors. Lorenz and Andervont (119) raised mice in special dust-free chambers in which the atmosphere contained only 3% of the dust that was found in the sur- rounding laboratory. This reduction in atmospheric dust did not influence the development of pulmonary tumors when the mice were injected sub- cutaneously with 1,2,5,6-dibenzanthracene.

Steiner and Loosli (188) infected mice with influenza Type A. Prolifer- ation of the bronchial epithelial cells typical of the infection was observed,

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but the surviving mice did not shorn any increase in the frequency of pulmonary tumors. In fact, there were only 20 such tumors among 250 virus-infected strain A mice, whereas among 105 controls, 38 pulmonary tumors were found-a significantly higher frequency. No pulmonary tumors were elicited in the C57 black mice. Glover and Jennings (Ma) showed that infection of mice with the grey-lung virus did not affect the

3 4.5 6 Time in months


FIG. 9. Response of lungs of strain A mice to intravenously injected ores and soot. A , single injection of 5.0 mg. of arsenopyrite, chromite, or thorite, or 1.0 mg. of quartz. R , untreated controls. C , single injection of 2.5 mg. of soot. D, injection of ores as in A, 0.1 mg. methylcholanthrene a week later. E , single injection of 0.1 mg. methylchol- anthrene. Carcinogenic index is the per cent of mice developing pulmonary tumors times the mean number of tumors per positive animal. Each point represents from 8 to 60 mice. From Shinikin and Leiter (177).

production of pulmonary tumors in mice with urethane. “Reticulo- endothelial blockade” by the injection of trypan blue had no effect upon the induction of pulmonary tumors in mice (178).

The available evidence clearly dissociates nonspecific irritation and inflammation from the occurrence of pulmonary tumors in mice. The dissociation seems to hold also, on histologic evidence, in pulmonary tumors induced in rats with urethane (140, 154) and in guinea pigs with intravenous methylcholanthrene (81). Whether this holds for human beings or any other species should not be adduced without reservation. The increase of pulmonary tumors in mice following exposure, inhalation, or injection with a material is good evidence of a presence of a carcino-

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genic agent in the material, an agent carcinogenic for the pulmonary tissue of mice.

2. Pulmonary Tumors in Embryo Mice

Law (106) in 1940 injected 0.125 mg. of 1,2,5,6-dibenzanthracene into the aminotic fluid of mice three to six days pre-partum, into mice 24 hours old, and into two-month old mice. Six to eight months later, 80% of the mice which were exposed to the carcinogen in utero, and 100% of those injected within a day after birth had multiple pulmonary nodules, whereas of 29 animals injected at the age of two months only 2 had tumors. This proved that embryonic lung was responsive to the carcinogen and sug- gested that it was more susceptible than adult lung. Larsen (101) and Klein (97) reported a high frequency of pulmonary tumors in the off- spring of mice injected intravenously with urethane during pregnancy, especially if the urethane was administered during the last day of par- turition. This established the transplacental effect of the compound, and that the penetration was increased just before parturition.

W. E. Smith (185), by the ingenious method of exposing strain C mouse embryo lungs to methylcholanthrene in vitro and then transplant- ing them to the subcutaneous tissue of adult strain C mice, was able to produce adenocarcinomas at the site of implantation within three weeks. Since pulmonary tumors in adult strain C mice following 0.5 mg. of methylcholanthrene arise between 13 and 20 weeks, and certainly not as early as 6 weeks, the observation suggested that embryonic lung was more susceptible to carcinogenesis than adult tissue. Studies on the injec- tion of pregnant strain C mice with urethane revealed that adenomatous growths were present in the offspring three and ten days old (187). Thus, the carcinogenic reaction was shown to occur much faster in the em- bryonic lungs than in adult tissue. It is of interest that mitoses were abundant in the tumors thus induced for the first two months, after which cell division almost ceased, indicating that the carcinogenic reac- tion to urethane, as to methylcholanthrene, is a self-limited one. Rogers (152) recently extended the studies in Swiss mice. He found that urethane, given in doses per weight of the animals, produced more pulmonary tumors in rapidly growing mice, up to six weeks in age, than in adult mice. Urethane injected into mice who already had pulmonary tumors induced with previous treatment with urethane produced an increased number of nodules, but the size of the nodules was not increased, indicat- ing that the role of urethane was to initiate neoplastic change and that urethane did not lead neoplastic cells to multiply. In this connection, colchicine had no effect upon the frequency of spontaneous pulmonary tumors, nor upon the induction of such tumors with urethane.

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The mechanism of induction of pulmonary tumors in mice can be divided into two main considerations: (1) the mode of action of the carcinogen; and (2) the mode of response of the organism or tissue to the stimulus.

1 . Mode of Action of the Carcinogen

The appearance of pulmonary tumors in mice injected with carcino- genic hydrocarbons and other carcinogens can be postulated to be due either to the local action of the carcinogens on the pulmonary tissue, to a general systemic action of the compound resulting in a lowered resistance to tumor development, or to a combination of the two.

With the polycyclic hydrocarbons, the most efficacious method of inducing primary pulmonary tumors in mice, both from the standpoint of the shortest latent period and the greatest response as measured by the number of discrete tumors, is by intravenous injection. This route produces the maximum contact of the carcinogen with the lungs. Moreover, more lung tumors are produced by dispersions containing larger particles, of which 607, become lodged in the lung, than with smaller particles, of which only 10% are retained in the lung (178). This shows that the num- ber of pulmonary tumors induced in strain A mice is a function of the dose of the hydrocarbon that reaches and lodges in the lungs, rather than of the dose injected into the whole organism.

Pulmonary tumors are also induced when threads coated with 1,2,5,6- dibenzanthracene are placed through the lungs of mice, or when disper- sions of the compound are introduced into the trachea. Experiments with charcoal-adsorbed dibenzanthracene also yield important support to the belief that actual contact of the carcinogen and the lung is necessary for the neoplastic reaction. In general, more pulmonary tumors are evoked when a carcinogen is injected in media that leave the site of injection than in menstrua that are retained at the site of injection.

After subcutaneous or oral administration of large amounts of 1,2,5,6- dibenzanthracene, absorption spectrum analysis fails to reveal the pres- ence of the compound or of its derivatives in the lungs, although multiple pulmonary tumors are induced. That the agent or some derivatives reach the lungs is suggested by the presence of photodynamic activity in sus- pensions of lungs of mice injected subcutaneously with 3,4-benzpyrene (142). With 1,2,5,6-dibenzanthracene labeled with radioactive carbon a t positions 9 and 10, and injected intravenously as an aqueous colloid in doses of 0.5 mg., Heidelberger and Jones (65) recovered only 0.4% of the dose in the lungs & hour and 24 hours later. Less than 1 % of 0.45 mg. of 3,4-benzpyrene labeled at the 5 position was recovered in the lungs (66).

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No radioactivity could be detected in the respiratory carbon dioxide. These findings are in strange contrast, perhaps explainable on the basis of the size of the particles in the dispersions, with the recovery of 50% of 0.5 mg. of 20-methylcholanthrene from the lungs of mice 30 minutes following intravenous injection-the detection being accomplished by absorption spectrum analysis of Lorenz and Shimkin (121). The amounts of the carcinogen in the lung decreased to one-tenth in four days, and extracts of lungs three days after injection induced sarcomas a t the site of subcutaneous injection in recipient mice, confirming biologically the presence of a carcinogen.

The distribution and elimination of urethane labeled with radiocarbon a t the carbonyl and the ethoxy positions has been published by Skipper et al. (182). After an equilibration of the injected material during the first 30 minutes, approximately 7% of the activity is excreted per hour via the respiratory route. At 24 hours, a t least 97% of the administered dose can be accounted for in the’expired air. Malmgren and Saxen (133) also demonstrated biologically that the carcinogenic effect of urethane is confined to the first 24 hours after injection.

All evidence would appear t o indicate that the carcinogenic reaction in the induction of pulmonary tumors is the result of a direct contact of the carcinogen with the lung. The reaction is modified by the dose of the carcinogen, the route of administration, and the physical state of the agent.

With the polycyclic hydrocarbons, urethane, nitrogen mustard, or trisethylene melamine, one injection is sufficient to evoke the neoplastic reaction. Whether this applied to roentgen rays and other stimuli remains to be established. The division of the dose of methylcholanthrene (178) or of urethane (152) over a period of several days evokes no more pul- monary tumors than the same dose administered in a single injection. This would suggest that the reaction is an acute one and that prolonged exposure of the animal or of the tissue to the carcinogenic agent is not required. The reaction, once invoked, apparently proceeds without the necessity of the presence of the initial stimulus.

Studies (179) on the quantitative aspects of dose-time response to methylcholanthrene have yielded some additional information. Gross observations of induced pulmonary tumors suggested that they appeared suddenly, grew rapidly for a number of weeks, and then increased only slowly in size. Figure 10 shows that the number of nodules produced a t various intravenous doses of methylcholanthrene increased markedly between the eighth and thirteenth weeks, and hardly a t all during the subsequent five weeks. The reaction is thus apparently a self-limited one. Rogers (152) also reached a similar conclusion in his fine studies on urethane-induced tumors in mice.

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0.0625 0.125 0.25 0.5 1.0 Methylcholanthrene in milligrams

FIG. 10. Dose-time-response relationships to strain A mice injected intravenously with methylcholanthrene. The mean number of pulmonary nodules per mouse is plotted against dose on a log-log scale. Each point is represented by groups of 25 to 30 mice. The response a t 8, 13, and 18 weeks after injection is indicated, showing the increase in number between 8 and 13 weeks, and practically no increase between 13 and 18 weeks. Data from Shimkin and McClelland (179).

2. Mode of Reaction of the Lung

The susceptibility of mice of various homozygous strains to induced pulmonary tumors is parallel to the susceptibility of the strains to the spontaneous development of this neoplasm. This suggests that the car- cinogens, whether they be polycylic hydrocarbons or urethane, are accelerators of some process inherent in the animal.

That the locus of the reaction and that the susceptibility factors are in the lungs and not mediated through physiological changes in the whole orgallism has been demonstrated beautifully in two studies. Smith (185) and Horning (88) have shown that carcinogenesis can be induced in tissues exposed to carcinogens in vitro and then transplanted subcutaneously into compatible animals.

Shapiro and Kirschbaum (167) obtained F1 mice from a cross between Bagg albino, a pulmonary-tumor-susceptible strain, and dba, a pul- monary-tumor-resistant strain. Transplants of lungs from one-day-old mice from either parent strains into the subcutaneous tissue of the ear

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were shown to survive in these hybrids. In mice implanted with lung tissue from Bagg albino, and then injected intraperitoneally with ure- thane, pulmonary tumors were produced in 12 of 17 transplants. When the transplanted lung was derived from dba mice, only one pulmonary tumor was produced in 17 transplants. Heston and Dunn (83) trans- planted lungs of a resistant strain (CU leaden or L) on one side and lungs of a susceptible strain (A) into the other flank of LAF, mice, who were then injected intravenously with l72,5,6-dibenxanthracene. Pulmonary tumors developed in 40% of the transplants from strain A donors and in 4% of transplants from strain L.

What occurs in the lungs when the carcinogen reaches the susceptible tissue and what determines the differences in susceptibility to the neo- plastic reaction in mice of different genetic backgrounds are questions that remain as unanswered for pulmonary tumors as for any other type of neoplastic transformation. As such, all theories that may be applicable to other forms of neoplasia are also relevant to pulmonary tumors. Analogies to the localization of intravenously injected Shope papilloma virus in the tarred ears of rabbits can be drawn, but no evidence exists that a viral agent is involved in the pulmonary tumors of mice, or that nonspecific chronic irritation in the lungs of mice enhances the neoplastic reaction. From quantitative dose-response relationships Heston and Schneiderman (85) have suggested that the reaction is a single, one-step change in the cell. As the authors carefully state, if this were a genic change it would be assumed to be a dominant mutation. The evidence is obviously insufficient to establish that the locus of reaction is a gene, and the term “somatic mutation” has validity only if it is used in the general sense of a change rather than in its more precise genetic meaning.

Except for the studies on the decrease in frequency of pulmonary tumors in mice maintained on restricted diets, relatively little attention has been directed toward influences that may inhibit the appearance of the tumors. It has been noted that mice surviving infection with influenza had fewer pulmonary tumors than the normal controls (188), and that mice developing cutaneous carcinoma following ultraviolet irradiation had fewer pulmonary tumors than similar mice that did not develop skin tumors (26). Cowen (41) reported that the induction of pulmonary tumors with urethane was inhibited by injecting the mice with pentose nucleotides. Rogers (152a) also stated that intraperitoneal injection of a deoxyribonucleate inhibited the formation of pulmonary tumors with urethane.

Only one attempt was found to follow specific biochemical changes in the lung as it undergoes carcinogenesis. Alkaline phosphatase was studied by Greenstein and Shimkin (59) in the lungs of strain A mice following

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intravenous injection of methylcholanthrene. The phosphatase activity remained completely normal, although the tumors themselves showed a decrease to about 25% as compared with normal lung. Thus, there was either an abrupt change, or the chemical analyses were not sufficiently sensitive to reveal subtle changes in the tissues. A study was carried out by Lorenz and Shimkin (121) on the elimination of methylcholanthrene from the lungs and bodies of strain A and strain Cb7 black mice, postulat- ing that the difference in susceptibility to the carcinogen may be reflected in the ability of the animals to conjugate, detoxify, and eliminate the chemical, but no differences in the rates were observed. It would be of interest to determine whether a protein-carcinogen binding occurs in the lungs of mice and whether it is different in strains of different suscepti- bilities, analogous to the important studies on the azo-liver relationships reported by Miller and Miller (136).

At the present time, the conclusions must still be restricted to the ones reached by Shimkin and Lorenz (178) in 1942: (1) carcinogens act directly upon the pulmonary tissue, (2) the pulmonary tissue is the locus of a process, present in varying degrees in all strains, that under normal conditions eventuates in the development of spontaneous tumors; (3) the carcinogen accelerates this process, both in regard to time of appear- ance of the tumors and in the number of discrete sites of carcinogenesis, and (4) the acceleration of the process is rapid and apparently the in- creased pace continues without further stimulation by the chemical agent.


I . Bronchogenic Carcinoma

The great majority of primary epithelial tumors of the lung in man are bronchogenic carcinomas, which may be undifferentiated, adenocarcino- matous, or epidermoid in pattern. There seems to be little question but that practically all arise from the basal cell layer of the bronchi, and some three-fourths from the epithelium of the larger bronchi (113, 203). It is one of the more malignant of the neoplasms of man, as manifested by its rapid invasion of surrounding tissues, its early, wide metastatic spread which may involve almost any organ or tissue, and its ability to kill the host within a relatively short period after the diagnosis has been estab- lished. The neoplasm is usually single and occurs several times more fre- quently among males than among females.

The marked increase in frequency of bronchogenic carcinoma among males in the United States and in Europe during the past 30 years has created a problem of great interest and concern to clinicians and public health officers. The evidence (37, 94, 99, 206) is unequivocal and cannot

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be explained by changes in the age of the population, diagnostic improve- ments, or statistical inaccuracy.

Epidemiological studies (192a) that have been carried out in the United States, England, Denmark, and Holland now allow a number of conclusions regarding this increase in the frequency of bronchogenic car- cinoma. The increase cannot be due to changes in the hereditary com- position of the population and must therefore be caused by changes in environmental influences. Cancer of the lung as an occupational disease would not be expected to affect appreciably the mortality figures as a whole. Pneumoconiosis has no established relationship to bronchogenic carcinoma. In this connection, careful review of available evidence on the high frequency of pulmonary cancer in the pitchblende miners of Joa- chimsthal and Schneeberg does not allow conclusions of causal connection to radioactivity (1 17). Exposure among workers in chromate industries, however, is related to markedly greater frequency of pulmonary cancer (131). No acceptable demonstration is available that specific infections, such as tuberculosis or influenza, have an influence on the development of pulmonary neoplasms.

Among environmental influences that may play a role in the steadily increasing frequency of bronchogenic carcinoma in man, two have gained particular attention. The first are the various atmospheric contaminants from industrial smokes and fumes, exhaust fumes and dusts created by individual inhabitants, and dusts from tarred roads. Urban populations have a higher frequency of bronchogenic carcinoma than rural popula- tions (165, 207). There is also ample laboratory evidence (31-35, 109, 110, 135) that atmospheric contaminants may contain substances that are carcinogenic when introduced into mice by injection or by inhalation. The second area of suspicion has been focused upon the smoking of tobacco. The reports of Doll and Hill (44), Doll (43a), Wynder and Graham (208), Korteweg (99), Sadowsky et al. (155), and others make it difficult, if not impossible, to deny that there is an association between prolonged, excessive smoking, particularly of cigarettes, and the occur- rence of bronchogenic carcinoma, particularly of the epidermoid and undifferentiated types. The laboratory investigations as summarized by Wynder et al. (209) indicate that materials carcinogenic in rabbits and in mice can be demonstrated in some tobacco products.

The present status of this important problem is well stated by one of the resolutions passed by a conference on the Endemiology of Lung Cancer (192a), held in July 1952 in Louvain under the auspices of the Council for the International Organizations of Medical Science: “The smoking of tobacco-especially cigarettes-has often been regarded as a causal factor of cancer of the lung. While it would be impossible to accept

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tobacco smoking as the only cause of cancer of the lung, there is now evidence of an association between cigarette smoking and cancer of the lung, and that this association is in general proportional to the total consumption. Further research on this subject is imperative.”

The demonstration of the relationship of smoking and of other en- vironmental factors and pulmonary cancer must be derived from direct epidemiologic studies on human populations. The follow-up studies on this problem now in progress in the United States (63a) and in England should provide the necessary definitive information. The variation in response among different species, to different chemicals, routes, and methods of administration should make it obvious that studies on man and studies on laboratory animals do not necessarily parallel. Even superficial com- parison of the human bronchogenic carcinoma and the adenomatous pulmonary tumor of the mouse, rat, and guinea pig makes it evident that biologically and morphologically they are quite different. Nevertheless, such animal studies are most useful in clarifying which agents or combina- tion of agents in the atmosphere or in tobacco are carcinogenic under laboratory conditions and therefore of possible significance in the human population. In this concept of the relationship of laboratory and clinical investigations, exact duplication of the human lesion in animals is not an essential; the skin of the rabbit, for example, may be as informative as the pulmonary tissue of strain A mice.

In my opinion, however, the data already available on the epi- demiology of human cancer of the lung and from the laboratory seem sufficiently impressive to urge the initiation of public education and re- lated public health measures toward the reduction in the individual con- sumption of tobacco, particularly in the form of cigarettes, and toward the control of atmospheric pollution that is becoming an ever-increasing problem in many urban centers.

The reader is directed to the important papers of Wynder (206), W. E. Smith (186), Hueper (go), and particularly to the reports of the Louvain conference (192a) for recent discussions and surveys of this topic, and for further bibliographic references.

2. Other Pulmonary Tumors

Two other pulmonary neoplasms of man should be mentioned in connection with the comparative pathology of human and animal tumors. The first is the bronchial adenoma, a slowly growing neoplasm that is found in approximately the same frequency in females as in males (113, 203). Bronchial adenomas show local invasion, and metastases can occur to the regional lymph nodes and even to distant organs. During the past

*Added in proof. See Hammond, E. C. and Horn, D., 1954. J. Am. Med. Assoc. 166, 1316-1328.

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few years a well-founded tendency has developed to classify this growth as a low-grade carcinoma. The fact remains, however, that histologically and biologically it represents a somewhat different neoplasm from the usual bronchogenic carcinoma.

The second, less frequent neoplasm or allied disease in man is pul- m,onary adenomatosis, or bronchiolar carcinoma (1 13), a multicentric process manifested by cuboidal or columnar cells containing mucus lining the alveoli and projecting in papillary structures. One of the synonyms of the disease is alveolar-cell carcinoma, and the disagreement regarding the nature of the alveolar lining cell is recapitulated in discussions of this topic (60, 205). The tumor grows by extension, but metastasis to regional lymph nodes and to distant organs does occur and cannot be facilely circumvented by rediagnosing these cases.

Pulmonary adenomatosis in man is morphologically indistinguishable from a disease of sheep that has been named Jagziekte, Montana Progres- sive Pneumonia, and Verminous Pneumonia. Its frequency has reached epizootic proportions in Iceland and in South Africa. Cowdry (39) in 1925 did not consider it a neoplasm but an “alveolar proliferation,” and at that time no metastases had been described. Bonne (28) on the other hand, referred to it as a “carcinosis caused by an infectious agent.” Dungal (47), in his extensive studies, stated that he had observed one irrefutable metastasis in a lymph node. Inoculations of filtered or un- filtered material into healthy sheep produced very few results, but the exposure of healthy sheep to the exhaled breath of sick sheep or the injection of such excretions was stated to transmit the disease readily after an incubation period of six to eight months. He concluded that the disease was probably caused by a pneumotropic virus, but the data cannot be said to have established the conclusion. Transmission to man has not been observed, and the presence of lung worms has no role in the disease in sheep. It seems reasonable to agree with Dungal’s (46) earlier statement, in that a discussion of whether Jagziekte is neoplastic or infectious in origin is rather unprofitable so long as we do not know the cause of either.

The question of the relationship of pulmonary adenomatosis of man to Jagziekte of sheep, and of the resemblance of both to the pulmonary adenomatous tumor in the mouse, is as wide as the whole intriguing problem of cancer. What are the relationships between proliferative cellular processes, metaplasia, and “true” neoplasia? Are they contin- uous, over-lapping, or entirely different reactions of tissue? Does the dem- onstration of the presence of an infectious agent ips0 fact0 place an ana- plastic cellular process that metastasizes into a different group of diseases?

Examination of the experiences in cancer research soon reveals that no sharp division exists between certain infectious processes and neo-

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TABLE I11 Comparative Fcaktrrs of Some Pulmonary Neoplasms and Related Lesions in Man and Animals

Autonomy Anaplasia Transmission ___- ____-

Trans- Cell-frce Pulmonary Cell of planta- trans- Communi-

Lesion Species Origin Mass Invasion Metastases Pattern Cell Mitosis tion mission rability 8 31 P M


- Bronchogenic Man I3ronchial ++ ++ ++ ++ ++ ++ ? ?

Bronchial Man Bronchial + f T - 3 ? ? carcinoma epithelium r

- - adenoma epithelium

7 - T ? - - Adenomatosis Man Alveolar ++ + T

Pulmonary Mouse Alveolar + + T T * + Pulmonary Rat Alveolar + + ? - - i + ? -

Pulmonary Guinea Alveolar + + ? - - T ? -

Jagziekte Sheep Alveolar ++ + T

cell? - - -

tumor cell

tumor cell

cell tumor Pig



3 -

- + + + + - -

rn X

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plasia, nor between so-called benign and malignant neoplasms. The conclusions maintained at present are reflections of authoritative insist- ence rather than of demonstrable fact. One is reminded of a statement purported to have been made by James Ewing during one of the recurrent arguments as to whether Rous sarcoma of fowl is a neoplasm or a pro- liferative response to an infection. His remark, in essence, was that since neoplasms were defined as new growths of unknown etiology, and since the etiologic agent in Rous sarcoma was known, Rous sarcoma by defini- tion could not be a neoplasm!

In a field that is as much a mystery as cancer is at present, the most rigid considerations must be applied to define any specific disease as belonging to the group of neoplastic diseases. Such specificity of concept immediately precludes any but the most tenuous extrapolations from one neoplastic disease to another, or conclusions of identity between mor- phologically similar diseases in different species of animals. At the same time, it must not remove from consideration certain related conditions and pathological processes simply because they do not fit neatly under the self-imposed criteria. One wonders, for example, why the intriguing neoplasm, for so it must be labeled on the basis of factual evidence, the transmissible lymphosarcoma of the dog (204), is no longer a subject of interest in cancer research. In Table 111 are indicated seven types of pulmonary neoplasms and allied reactions, and some of their character- istics. It is best, at this time, to consider these as separate entities, and at the same time of course to draw tentative analogies as a basis for further experimentation.

Some years ago, it was proposed (173) that a “vertical” approach to the cancer problem, in which a specific neoplasm is studied from as many aspects and scientific disciplines as possible, may be more rewarding than the usual attack, in which many types of neoplasms are dealt with from the standpoint of the particular scientific discipline in which the investigator is trained. Experiences and the record of cancer research during the intervening eight years have reinforced this view. If this is more generally accepted, and long-term support of cancer research as a thirty-years’ war and not as a five-year skirmish continues, the primary adenomatous pulmonary tumors of the mouse, rat, and guinea pig would be strongly represented among the experimental neoplasms, and the bronchogenic carcinoma of man among the neoplastic diseases particu- larly suitable for epideminologic studies and preventive public health measures.

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Oxidative Metabolism of Neoplastic Tissues

SIDNEY WEINHOUSE The Lankenau Hospital Research Institute and The Institute for Cancer Research,

Philadelphia, Pennsylvania Page

I. The Concepts of Warburg.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 1. Glycolysisin ~ i v o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

11. The Pasteur Effect.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 1. Respiratory Quotient Data of Dickens.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

111. Present Concept of Carbohydrate Oxidation. . . . . . . . . . . . . . . . . . . . . . . . . . . 278 1. Catabolism of Glucose.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

IV. p-Oxidation of Fatty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

VI. Electron Transport in Tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 1. Cytochrome c and Cytochrome Oxidase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

2. Pyruvate Oxidatio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

V. Mechanisms of Glycolysis in Tumors.. . . . . . . . . . . . . . . 283

2. Dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Respiration in the Intact Tumor Cell.. .................... 4. Nature of Substrates for Respiration of Tumor Cells. . . . . . . . 5. Isotope Tracer Studies on Tumor Respiration-Glucose Oxidat 6. Oxidation of Fatty Acids in Neoplastic Tissues.. . . . . . . . . . . . . . . . . . . . . 305 7. Oxidation of Acetoacetate in Liver and Tumor Slices.. . . . . . . . . . . . . . . . 309 8. The Citric Acid Cycle.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 9. Isotopic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

10. The “Condensin e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 VII. Oxidation in Tumor Homogenates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

1. Citric Acid Cycle Intermediates. . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 2. The DPN+ Effect.. . . . . . . . . . 317 3. Effects of Phosphorylation and Dephosphorylation on Oxidation in

Tumor Homogenates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 4. Possible Causes of High Glycolysis in Tumors . . . . 321 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

The biochemist concerned with the cancer problem is guided and probably also motivated by the belief that the uncontrolled growth of the cancer cell has its origin in some metabolic or enzymatic peculiarity- a point of departure from the normal cell-which might provide a rational basis for the control or annihilation of this disease. The invasive growth of cancer cells, depending as it does on a high synthetic capacity, has directed much attention to the mechanisms by which energy is made available for anabolic processes, and since the main source of such energy is the oxidation of fats and carbohydrates, it is in this field that bio-


. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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chemical exploration of the cancer cell has probed most deeply. Many attempts have been made to formulate differences between normal and neoplastic tissues on the basis of differences in metabolism, particularly of glucose. Stimulated originally by the pioneering efforts of Warburg, a concept of tumor metabolism has arisen which maintains as a funda- mental thesis that the neoplastic process is somehow associated with dis- turbances or peculiarities of oxidative metabolism. It is the object of this report to review the evidence for this concept and to examine i t against our present knowledge of intermediary cell metabolism.


Biochemical thought concerning oxidative metabolism of tumor cells has been dominated by Otto Warburg, whose pioneering work in the metabolism of cancer tissue extended over some eight years of intensive work up to 1930 and has continued sporadically to the present time. So completely has Warburg’s approach to the subject captured the imagina- tion of biochemists that no serious discussion of this problem is complete without some description of \Tarburg’s results and ideas. These may be found i n extenso in a collection of the researches of Warburg and his col- leagues ( l ) , and in a lucid review and interpretation of Warburg’s work on tumor metabolism by Dean Burk (2).

Warburg’s important contributions to the biochemistry of cancer stem from the development of techniques for measurement of gas ex- changes, made possible by the manometric apparatus which bears his name. I-sing these techniques, he and his colleagues measured the con- sumption of oxygen and at the same time measured the production of lactic acid by these tissues, either in oxygen (aerobic glycolysis) or in nitrogen (anaerobic glycolysis).

T ii t hesc studies Warburg discovered a metabolic characteristic of tumor tissues, which to this day represents perhaps their most outstand- ing hiochemical feature, namely, a high aerobic and anaerobic glycolysis. Dean Hurk has summarized the data of Warburg and other early workers in a series of tables which not o d y reveal the experimental results of the early investigations but also give an idea of the type of information upon which the various concepts were built. For a detailed description these should be consulted in the original. For the purpose of the present dis- cussion, these data have beem drastically condensed and are presented in Table I. As shown in this table, lactic acid formation in tumor slices under nitrogen, averaging 25.6, is over three times as high as in slices of nongrowing normal tissues. Glycolysis persists in the absence of oxygen for long periods, for days in fact, without diminution in rate. The process has a pH optimum of i . 3 and a temperature optimum of 37°C. It occurs

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in the presence of other sugars, such as mannose, fructose, and galactose, but not nearly so rapidly, and is optimal a t a glucose concentration of 0.2%, which is in the range of its physiological concentration in body fluids in the postabsorptive state.

TABLE I Condensed Tumor Metabolism Data of Warburg and Others from the Review

by Burk (2)

Normal, Nongrowinga Malignantb Growing"

Respiration, Qo, 9 . 3 (3-21) 11.8 (5.3-19.8) 9 . 7 (4-14) Aerobic glycolysis, Q L O ~ 2 . 1 (0-10) 14.0 (4.7-24.6) 7 (0-15) Anaerobic glycolysis, Q L N ~ 7 . 2 (2-19) 25.6 (14.0-34.8) 20 (13-28) Absolute Pasteur Effect 5 . 1 (1-16.5) 11.6 (6.3-17.8) 12 (4-19)


~ O O ( Q L ~ ~ - Q L O ~ ) / Q L ~ ~

( Q L ~ ~ - &LO,) /%Qo,

Per cent Pasteur Effect 78 (12-100) 46 (23-70) 64 (28-100)

Meyerhof oxidation quotient 2 . 1 (0.2-4.5) 3 . 2 (1.4-4.3) 4 . 1 (1.7-6.0)

The Q notation refers t o microliters of gas corresponding to the product in question consumed or produced per milligram dry weight of tissue per hour.

(1 Averages and ranges of 14 different tissues of various animals. t, These are meam and ranges of mean values obtained with 15 different tumor types, but they are

@ Individual values on 7 tiesues of 3 tissue types: chicken embryo, and rat placenta and embryo. not weighted means.

More striking is the difference between neoplastic and nonneoplastic tissue sliceq in the presence of oxygen. Aerobically, glycolysis is on the average seven times as high in tumor slices as in nongrowing tissues; in fact, aerobic glycolysis in tumor slices is about twice as high on the aver- age as anaerobic glycolysis in normal tissue slices. Warburg pointed out (1) that such rates are equivalent t o a lactic acid production of as much as 12% of their dry weight per hour; under the same conditions red blood cells produce only 0.1%) and frog muscle 0.06% a t rest and 1.5% doing maximum work.

1. Glycolysis in Vivo That glycolysis is high in the intact tumor growing in situ in its host

as well as in slices of the excised tissue was first demonstrated by Cori and Cori (3). They showed that tissues of .the fasting, tumor-bearing mouse had a low lactic acid content, ranging from about 0.01 % to 0.1 % in tumors, liver, and muscle. On administration of glucose, the lactic acid content was considerably increased in the tumor but not in the liver. Later these investigators (4), in comparing the glucose and lactic acid content of the blood from the axillary veins of a chicken carrying a Rous

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sarcoma in one wing, found 23 mg. less glucose and 16 mg. more lactic acid per 100 ml. in the vein draining the tumor. A similar experiment with a human patient carrying an axillary tumor gave similar results. War- burg, Wind, and Negelein (5) also observed large differences in lactic acid and glucose content between arterial and venous blood of rats bearing the Jensen sarcoma; these findings again pointed to a rapid utilization of glucose by the tumor, with production of lactic acid.

Similar, though somewhat less direct, evidence for lactic acid produc- tion in cico by tumors has been advanced by Voegtlin et al. (6) and by Kahler and Robinson (7), who found that the intercellular pH of a rat hepatoma decreased in response to glucose administration, whereas the pH of liver did not change. Despite many subsequent in vitro studies of tumor metabolism, the author has not found any exceptions to the high glycolysis of tumor tissues. This appears t o be a distinct metabolic feature of tumor cells, regardless of type or host. The force of this conclusion is blunted somewhat by the fact that glycolysis is not an exclusive feature of neoplastic cells. Apparently all cells glycolyze under certain conditions, more so in the absence of oxygen; and as seen in the last column of Table I, actively growing tissues have a considerably higher glycolysis than adult, nongrowing cells. As seen in Table I, the glycolysis pattern of embryonic tissue is not far different from that of neoplastic cells (ex- tended discussions of glycolysis in various tissues will be found in reviews by Burk ( 2 ) arid Dickeris (8) and in the monographs by Greenstein (9), and Stern and Willheim (lo)). It can therefore be assumed that high glycolysis is a general phenomenon of growing cells, whether it is the physiological, orderly development of the embryo or the pathological invasive growth of the tumor.

It thus appears, as Warburg pointed out, that whereas normal tissues display predominantly an oxidative type of metabolism, tumor slices pre- dominantly ferment glucose. This difference can easily be seen by com- paring data for normal tissue with those for malignant tissue in Table I. Since the oxidation of one molecule of glucose requires six molecules of oxygen, we can calculate that under aerobic conditions the normal tis- sues on the average osidize 9.3,'6 = 1.5 molecules of glucose while they glycolyze 2.1/2 = 1 molecule of glucose. The tumor slices, on the other hand, osidize 11.8/6 = 2.0 molecules of glucose, on the average, while they glycolyze 14.0/2 = 7 molecules of glucose. Thus, the ratio, glucose fermented/glucose oxidized is 0.7 for normal tissue and 3.5 for tumors. It is important t o note that the high aerobic and anaerobic glycolysis of tumor tissues is not associated with any noticeable derangement in oxygen consumption, since the averages and ranges of oxygen consumption cor- respond closely in all three tissue types. This fact should be kept firmly

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in mind, since, as we shall see, many ideas, even of present-day investiga- tors in this field, have been predicated on a supposed quantitative impair- ment in the oxygen consumption of tumor slices.

Warburg’s interpretations of his findings can perhaps be best expressed in his own words, taken from the preface of the English Edition of his book (1) :

“Aerobic glycolysis results if the respiration of growing cells is injured, whether by diminishing its extent or by interfering with the relationship which holds between respiration and fermentation (gly- colysis). . . . Interference with the respiration in growing cells is, from the standpoint of the physiology of metabolism, the cause of tumors. If the respiration of a growing cell is disturbed, as a rule the cell dies. If it does not die, a tumor cell results. This is no theory, but a comprehensive summary of all the measurements a t present available.”

This categorical statement by Warburg with its somewhat mystical connotations, and carrying the authority of a recognized leader in the field, directed the course of many years of subsequent biochemical re- search in tumor metabolism. In many instances the Warburg concept became distorted and misinterpreted by subsequent investigators, and though a great mass of additional information accumulated in the twenty odd years since Warburg’s book on tumor metabolism appeared, no decisive advances were recorded and the Warburg view generally pre- vailed, namely, that a disturbance in respiration was a characteristic feature of tumor metabolism.

If the experimental observations used by Warburg in support of this point of view are examined critically, it is seen that they offer relatively little support to his idea. Originally Warburg’s choice of tissue was the Flexner-Jobling carcinoma and various human tumors, all of which had low respiration. It was easy to see, therefore, how an apparent association of high aerobic and anaerobic glycolysis with a low oxygen consumption could lead to the conclusion that these phenomena are causally inter- related. Subsequent studies with tumors (no less malignant) with high Q0,% led Warburg to modify his original view that there is a quantitatively lower respiration in tumors.

Though Warburg relinquished the idea that respiration may be quan- titatively disturbed in tumor cells, he still insisted that there was a dis- turbance in the relationship between respiration and fermentation. In justification of this idea Warburg pointed to the fact that in normal tissues the respiration is able to abolish glycolysis; that is, in normal cells aerobic glycolysis is zero or close to zero, whereas in tumor cells the

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aerobic glycolysis is high. In his words (1, p. 327), ‘(Whether the respira- tion of the tumor cell is large or small, aerobic glycolysis is present in every case. The respiration is always disturbed, inasmuch as it is incap- able of causing the disappearance of the fermentation (i.e. glycolysis) .’J

This insistence on a disturbance of respiration as being the cause of aerobic glycolysis is not only not justified by the high respiration of tumor cells; it is also inconsistent with Warburg’s own thoughts expressed elsewhere in this book (1, p. 139). But before discussing this matter further it will be necessary to digress to say a few words about the rela- tionship between respiration and fermentation, which Warburg termed the Pasteur Effect.

11. THE PASTEUR EFFECT The fact that living cells carry out a slower rate of fermentation in the

presence than in the absence of air stems from an observation originally made by Pasteur. His words (11, see p. 276) are:

“Free oxygen imparts to yeast an increased vital activity. . . . If we supply yeast with a sufficient quantity of free oxygen for the necessities of life, nutrition and respiratory combustion, it ceases to be a ferment, that is, the ratio between the weight of the plant developed and that of the sugar decomposed is similar in amount to that in the case of fungi. On the other hand, if we deprive the yeast of air entirely it will multiply just as if air were present, although with less activity, and under these circumstances its fermentative character will be most marked ; under these circumstances, moreover, we shall find the greatest disproportion, all other conditions being the same, between the weight of yeast formed and the weight of sugar decomposed . . . if free oxygen occurs in varying quantities, the ferment power of yeast may pass through all the degrees comprehended between the two extreme limits of which me have just spoken. It must be borne in mind that the equation of a fermentation varies essentially with the conditions under which that fermentation is accomplished, and that a statement of this equation is a problem no loss complicated than that of a living being.”

It was logical to assume, of course, that this effect was due merely to the removal of fermentation product or some intermediary thereof by oxidation. By measuring simultaneously oxygen consumption, glucose utilization, and fermentation product appearance (alcohol in the case of yeast, lactic acid in the case of muscle), Meyerhof (12, 13) demonstrated that such a theory was untenable. He observed that t<he decrease in cleav- age product (alcohol or lactic acid) brought about by the presence of oxygen was far higher than could be accounted for if the effect of oxygen

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was merely to oxidize away the cleavage product. Since in the case of lactic acid formation three molecules of oxygen are required to oxidize a molecule of lactic acid, the decrease in lactic acid divided by one- third of the oxygen consumption should equal unity if the only effect of oxygen is to remove lactic acid by oxidation. However, Meyerhof found that the decrease ranged from three to six times the amount which could have been oxidized by the oxygen consumed. Put in terms of experi- mentally definable quantities.

This decrease in fermentation brought about by oxygen was termed by Warburg the ((Pasteur Effect” and the above ratio, the “Meyerhof Quotient.” Warburg noted that, by and large, tumor tissues had the same ( ( Meyerhof Quotient ” as normal tissues. In Warburg’s words (1, p. 139) :

“We determined the Meyerhof quotient for carcinoma tissue, lactic acid bacteria, embryonic tissue and a number of other glycolyzing tissues, and as a rule obtained the same mean values as Meyerhof. As a rule 1 mol. of breathed oxygen, just as in muscle, causes the dis- appearance of 1-2 mol. lactic acid. This result . . . proves that the influence of the respiration on the cleavage metabolism in the car- cinoma-cell is normal. . . . Although in the tumor every oxygen molecule breathed is just as effective as in muscle-the Meyerhof Quotient is equal in the two cases-yet the respiration does not cause the glycolysis to disappear. The respiration of the carcinoma tissue is too small in comparison with its glycolytic power.”

Nowhere in this statement is there any mention of a quantitatively disturbed respiration or any other respiratory disturbance, nor is there any mention of any disturbance in the relationship between respiration and glycolysis-in fact, the opposite is explicitly stated. It is difficult to recognize in this statement any similarity to the categorical dictum quoted earlier. It is true, of course, that because of the high aerobic glycolysis the decrease in glycolysis due to oxygen is lower percentagewise in tumors than in most normal tissues (Table I). For this reason a low Pasteur Effect has been mistakenly attributed to cancer cells.

Examination of the data of Table I reveals that if we measure the Pasteur Effect, most simply and directly, as the differences between glycolysis in air and glycolysis in nitrogen (absolute Pasteur Effect, Table I), the values for tumor slices are on the average over twice as

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high as those for normal tissues. Here we must be on guard to avoid the equally wrong but opposite conclusion, namely, that the Pasteur Effect is greater in tumor tissues. The reason why the Pasteur Effect is smaller in most normal tissues is that it is limited by the low rate of anaerobic gly- colysis. Obviously tissues such as kidney or liver, which have an anaerobic glycolysis of three, cannot have a greater absolute Pasteur Effect than three. However, rat brain cortex, which has an anaerobic glycolysis of 19, displays a Pasteur Effect of 16.5, which is in the range exhibited by tumor slices.

For the same reasons it is obvious why the percentage Pasteur Effect gives an entirely erroneous impression of the magnitude of this quantity. From Table I it may be seen that whereas glycolysis in normal tissues is decreased 78% by oxygen, glycolysis in tumors is lowered only 46% on the average. Warburg stated: “The respiration is always disturbed, inas- much as it is incapable of causing the disappearance of the fermentation.” It would have been more accurate to state that the anaerobic glycolysis of tumor slices is so high that a normal respiration and a normal Pasteur Effect are incapable of eliminating it. This latter statement places the emphasis on the high glycolysis of tumor slices rather than on the respira- tion or on the absolute Pasteur Effect, neither of which are quantitatively diminished in neoplastic cells.

1. Respiratory Quotient Data of Dickens

In the various quantitative expressions employed by Warburg, Meyerhof, and others, the possibility of the metabolism of other food- stuffs was ignored, and it was tacitly assumed that under the conditions of the experiments with tissue slices, i.e., in the presence of glucose, only sugar was being metabolized. Dickens and Simer (14) recognized the possibility that disturbances in carbohydrate metabolism may not be manifested in quantitative changes in respiration, since it is possible that other foodstuffs such as fats, etc., might be undergoing catabolism and thus contribute to the respiratory activity. Dickens and his colleagues accordingly developed methods which made i t possible to measure glycol- ysis, oxygen consumption, and carbon dioxide production simultaneously in citro with tissue slices, and embarked on a study of the respiratory quotients (R.Q.) of various normal and neoplastic tissues. These studies revealed an interesting relationship between glycolysis and R.Q. Refer- ence to Table 11, in which data are taken from the work of Dickens and Simer, reveals that normal tissues fall into two main groups. The first, containing all of the resting tissues except brain and retina, has a low R.Q., which is closer to the theoretical value of 0.7 for fat oxidation, and a low anaerobic glycolysis, indicating that these tissues predominantly

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oxidize fat despite the presence of glucose in the medium. The second group, which includes brain and retina in addition to growing tissues such as embryo and chorion, displays a high glycolysis and R.Q. values characteristic of the oxidation of carbohydrate.

TABLE I1 Mean Values of R.Q. and Anaerobic Glycolysis of Normal and Neoplastic Tissuesa (14)

Tissue R.Q. Q c ~ ~ ~ z Animal Tissue R.Q. Q c o , ~ ~

Liver Kidney Intestinal mucosa Submaxillary

Spleen Testis Embryo Embryo, chicken Brain cortex Chorion


0.79 3 0.85 3 0.85 4 0.87 7

0.89 8 0.94 8 1.04 8 1.00 18 0.99 19 1.02 32

Retina 1.00 88

Rat Rat Chicken Mouse

Mouse Mouse Mouse Mouse Mouse Human


Jensen sarcoma Slow sarcoma Rous sarcoma Spindle-cell tar tumor

Tar carcinoma 2146 Crocker sarcoma Sarcoma 37 S Spontaneous tumor I Spontaneous tumor I1 Papillary carcinoma

Carcinoma of breast


of bladder

0.82 34 0.92 18 0.93 30 0.91 21

0.87 22 0.89 22 0.86 27 0.91 20 0.87 (16)a 0.86 (3.4)b

0.84 (7.1)b

a Of rat unless otherwise stated. Mixed tumors; much connective tiaaue.

Thus in normal tissues a low value for R.Q. is associated with low ability to form lactate anaerobically. There appears to be a gradual transition to a type which displays high glycolysis and exclusive carbo- hydrate respiration. In these tissues carbohydrate can be recognized as the main fuel for respiration.

In contrast with these data for normal tissues, the data for tumor slices reveal a third metabolic pattern; here a low R.Q. is associated with a high rate of glycolysis. All of the tumors studied displayed R.Q. values which are distinctly below the value characteristic of total carbo- hydrate oxidation, while giving high values for Q C o n N 2 . Dickens and Simer reasoned that in normal, resting cells carbohydrate metabolism is limited by a low rate of formation of the substrate for respiration, whereas in tumors carbohydrate oxidation is limited by a defective mechanism for oxidation of glycolysis products. These conclusions represent an impor- tant extension of Warburg’s ideas in that they agree fundamentally that there is a defective respiration in tumor cells, and they extend his ideas by “pinpointing” the defect in the oxidation of the glycolysis product.

There are several reasons why this hypothesis may not be entirely acceptable. Dickens and Simer themselves point out that the R.Q. of

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tumor slices is increased in the presence of pyruvate t o values close to the theoretical for pyruvate oxidation. It is obvious that this finding is not in accord with a disturbance in oxidative metabolism of carbohydrate unless the assumption is made that the defect is between lactate and pyruvate, an assumption which seems rather unlikely. This theory has been criticized on other grounds. Boyland (15) showed that high rates of glycolysis and low respiratory quotients are not necessarily character- istic of tumors, and Berenblum et al. (16) pointed out that the tumors, whose metabolism had been studied by Dickens and Simer (14), were de- rived from tissues which have the same types of metabolism as their neo- plastic counterparts, e.g., skin, intestinal epithelium, and fibroblasts. These investigators found (1 7), using a specially constructed microrespirom- eter, that Shope papilloma has a glycolytic and respiratory pattern very similar to normal skin epithelium (see Table 111).

TABLE I11 Mean Values for Oxygen IJptake, Aerobic and Anaerobic Glycolysis, and Respiratory

Quotient of Normal Skin Epithelium and Shope Papilloma (17)”


Sormal skin cpithelium 0 9 0 45 1 . 3 0 . 7 Shope papilloma 0 6 0 3 1 25 0.6

a The PQ values represent cu. mm of gas per pg. of nucleic acid phosphorus content of tissue per hour, as distinguished from the usual Q values. which represent cu. mm. of gas per mg. dry weight of tissue per hour.


hfter thus presenting the background of early information leading to the concept of an impaired oxidative metabolism in tumor cells, it will be our task to try to interpret these findings in terms of our present knowledge of cellular metabolism. The terms glycolysis, respiration, and Pasteur Effect were used by Warburg to describe phenomena which had relatively little meaning with respect to detailed reaction processes. At the time IVarburg was carrying out his pioneering studies of glycolysis in tumors, little was known of the reaction pathways by which glucose is converted to lactic acid. The adenine and pyridine nucleotides were yet, undiscovered. Investigators at that time knew little more than Pasteur concerning the processes by which carbon compounds became converted to carbon dioxide. It is not surprising, therefore, that Warburg and others regarded the Pasteur Effect in terms of what Warburg called a “Pasteur reaction,” or that investigators even much later sought what they called a “Pasteur Enzyme” (2). Though such a view no doubt seemed eminently reasonable a t the time, our present-day picture of the complicated multi-

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step processes of glycolysis and respiration with their numerous cross- linkages through electron transport factors, and the interlinkages with fat ty acid and amino acid metabolism, leaves no provision for a single reaction by which respiration (or oxygen) can influence glycolysis. It will probably be worth while a t this point to review briefly our present concepts of the intermediary metabolism of carbohydrate in animal cells.

Though there still remains much to be learned before the complete picture of energy metabolism is brought into sharp focus, biochemistry has now reached a stage where a broad outline of intermediary metabolism is visible. Pathways can now be envisioned by which the major metabolic fuels, such as the carbohydrates and the fatty acids, are interconverted or are converted to COz or t o the constituents of proteins and other cell components. In many instances chemical equations for these reactions can be written with confidence that they are representative of occurrences in the intact cell; in addition, knowledge of electron transport and oxida- tive phosphorylation has increased to such an extent that we now have a t least a rudimentary idea of how oxidative energy production may be coupled with the processes of synthesis which underlie all growth phenomena.

I . Catabolism of Glucose

As illustrated in Fig. 1, glucose is activated by conversion to glucose- &phosphate, from which two routes diverge for the further catabolism of the sugar. One of these processes comprises a series of isomerizations, transphosphorylations, and an oxidation and a reduction step, resulting in the conversion of one molecule of glucose to two molecules of lactic acid.



Fructose-6-Phosphate 6-Phosphogluconic Acid

Fructose-1,6-Diphosphate Ribulose-5-Phosphate

1 1 1 1

J - 1 J1 Dihydroxyacetone Phosphate $ Glyceraldehyde-3-Phosphate + Glycolaldehyde

I .L

3-Phosphoglyceric Acid

2-Phosphoglyceric Acid

Phosphoenolpyruvic Acid

1 1 1

Lactic Acid +ri Pyruvi.: Acid

FIG 1.

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This is the so-called pathway of Embden and Meyerhof. The other mechanism branches off with the oxidation of glucose-6-phosphate to 6-phosphogluconic acid. The latter substance is then presumed to undergo decarboxylation and a further oxidation to yield a pentose-bphosphate, which is probably the ketopentose, ribulose-5-phophate (18, 19), but which ultimately yields ribose-5-phosphate. The further metabolism of this substance is not entirely certain, but from the recent studies of Racker (20) it would appear that it undergoes an ((aldolase” type of split to triose phosphate and a “diose,” possibly glycolaldehyde. This so-called ‘( oxidative ” or ( ( hexose monophosphate shunt ” mechanism of glucose breakdown has been studied primarily in microorganisms, and little information is yet available concerning its extent in animal tissues. According to Bloom, Stetten, and Stetten (21) the shunt pathway does not occur to an appreciable extent in the normal, intact rat. This con- clusion was based on an isotope tracer procedure, involving measurement of the relative extents of conversion to COZ of variously labeled glucoses, viz., glucose-1, and 6-CI4, and uniformly labeled glucose; and of the three C14-labeled lactates. Application of the same method to tissue slices re- vealed that the shunt was not operative in kidney and diaphragm but occurred t o a major extent in liver (22). Similar conclusions were drawn by Lewis et al. (23), using a more direct method based on comparison of the specific activity of lactates and acetoacetates arising when slices of rat tissues were incubated with glucose-l-Cl4 or uniformly labeled glucose. With the esception of liver, therefore, whose low rate of glucose catab- olism is overshadowed by that in the musculature, the hexose mono- phosphate shunt does not appear to occupy a prominent quantitative position in glucose catabolism in the normal intact animal. It is generally assumed that the glycolysis which occurs anaerobically proceeds via the Embden-Neyerhof pathway. Anaerobic glycolysis via the Embden- Meyerhof process results in the quantitative conversion of glucose to lactic acid, whereas the so-called shunt mechanism gives a t most one lactic acid molecule per molecule of glucose consumed. Negelein (24) found that the Flexner-Jobling carcinoma gave two molecules of lactic acid per molecule of glucose disappearing from the medium, indicating the major occurrence of the E.M. process in this tumor; and the isotopic tracer method of Lewis et al. (23) indicated that the shunt is not an important pathway in the mouse hepatoma 98/15. On the other hand, preliminary data of Lewis et al. (23) indicate that the shunt operates to the extent of 40% to 50% in the mouse sarcoma 37 and in an ascites form of the T.43 carcinoma. It would thus appear that the shunt mechanism is of quantitative significance a t least in some tumor tissues; however, the matter requires further investigation and extension to more tumor

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types. The pathway of glucose breakdown via 6-phosphogluconate has been designated the " oxidative pathway "-the implication of course being that this mechanism may come into play only in presence of oxygen. The possibility that such a mechanism may account for the Pasteur Effect has been entertained (8). Such a mechanism might be favored in the presence of oxygen, since in contrast with the balanced oxidoreduc- tions of ordinary glycolysis, the shunt requires dehydrogenations equiva- lent to the consumption of a mole of oxygen per mole of glucose, as shown in Eq. 1.

CsHlzOe + Oz 4 C3H603 + CzH,02 + COz + HzO (1)

There is, however, no compelling reason why the shunt mechanism would be restricted to oxidative conditions. It is conceivable that in an intact cell these dehydrogenations may be coupled with other reductive processes, and it is therefore possible that the shunt reaction can also occur anaerobically, just as other oxidative processes may be coupled, through common coenzymes, with reductive reactions.

2. Pyruvate Oxidation

Whatever the mechanism by which it is formed, triose phosphate is oxidized to pyruvic acid, and the further oxidation of this substance results in the formation of carbon dioxide and an active acetyl derivative. Evidence of recent years has shed considerable light on the mechanism of this hitherto obscure reaction. In addition to the apoenzymes, at least four cofactors are apparently necessary : coenzyme A, diphosphopyridine nucleotide, diphosphothiamine, and a-lipoic (thioctic) acid. The over-all course of events is formulated by Korkes et al. (25) as in Eq. 2.

Pyruvate + DPN+ + CoA + Acetyl CoA + DPNH + H+ (2)

According t o Gunsalus (26) the process of pyruvate decarboxylation to yield acetyl CoA in various microbial species can be represented in the following four equations.

CHaCOCOO- + DPT+ -+ CHaC0:DPT + COz (3) CHaCO : DPT + S-R-S (4) II

CHaCO: S-R-S- + COASH -+ CHaCOSCoA + HSRS- (5) HSRS- + DPN+ -+ S-R-S + DPNH (6)

The first step, Eq. 3, is a straight, diphosphothiamine-catalyzed de- carboxylation to COZ and an acetaldehyde-diphosphothiamine complex. In the second step, Eq. 4, the acetaldehyde is transferred to a sulfur of

+ CHaCO : S-R-S- + DPT+


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a-lipoic acid. This cyclic disulfide of 6,8-dimercapto-octanoic acid (desig- nated in the equation by S-R-S) undergoes a reductive cleavage to

form acetyl lipoate, and in the third step, Eq. 5 , the acetJyl group is transferred to the thiol group of coenzyme A, yielding acetyl SCoA and reduced lipoic acid. The latter is reoxidized via DPN+ to continue the cycle of reactions. Further details of this process will be found in several recent reviews (27, 28).


IJ-. P-OXIDATIOX OF F a w Y ACIDS Present conceptions of fatty acid osidation envision this process as

occurring by successive removal of units of acetyl CoA to complete degradation of the carbon chain. Summaries of recent findings with respect to the individual enzymatic steps will be found in reviews by Mahler (291, Lyncii (30), and JVeinhouse (27 ) . The initial step is the formation of an acyl V0- i ester, either by transacylation from a natural lipid such as a phospholipid to CoASH or by activation of the acid with Coh and adenosirietriphosphate (AXTI'), as shown in Eq. 7 . The succeeding steps shoivn i n Eqs. 8 to 11 are: a,p-dehydrogeiiation, by a flnvoprotein enzyme; hydration to the 0-hydroxy-acid ; dehydrogenation to the corresponding 8-keto arid: atid finally, a "thiolytic" split of the P-keto acid to one mole- cule of ncrtyl SC'oA and the COX ester of the next lower homologous fatty acid. The process continues to complete conversion of the fatty acid carbon chain to acetyl SCoA.

RCII,C'H-('OOH + Co.4SH + ATP ----t RCH?CH?COSCoA + A3IY + inorganic pyrophosphate (7 )


The nest step in the catabolism of acetyl COX is its entry into the citric acid cycle by condensation with oxalacetate to yield citric acid plus coenzyme -1 (31). There then ensues the now well-established stepwise reaction sequence resulting in the complete oxidation of a molecule of acetate arid the regeneration of osalacetate to carry 011 the cycle. During the course of the complete oxidation of a molecule of glucose, 23 electrons are transferred to oxygen, 12 for each triose molecule, and these come off in pairs of 2 a t the following stages: triose phosphate, pyruvate, isocitrate, a-ketoglutarate, succinate, and malate. Before reaching oxygen, the electrons pass through the pyridine and flavoprotein enzymes and probably all funnel through the cytochrome system.

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From the foregoing discussion, it is possible to consider various over- all influences which may be exerted on the intermediary formation of lactic acid. First, lactic acid will accumulate if the glycolytic reactions occur too rapidly for the pathways of carbon or electron transport t o dispose of pyruvic acid. The accumulation of pyruvate would then result in its competing with the flavoproteins for the electrons taken up by the pyridine nucleotides, and would result therefore in the accumulation of lactic acid. Similarly, any defect in carbon transport through the citric acid cycle or in one of the electron transport steps could also con- ceivably result in the reduction of pyruvate. Evidence bearing on these possibilities will be discussed in subsequent sections.

V. MECHANISMS OF GLYCOLYSIS IN TUMORS Ever since the delineation of the route of sugar catabolism in muscle

by Embden and Meyerhof (13), wide interest has been manifested in the possible existence of other pathways, particularly one not involving the usual phosphorylated intermediates. For the earlier history of these studies the reader is referred to the excellent review of Dorfman (32). No attempt will be made to cover the voluminous literature related to glycolysis in tumor cells. Only a few examples have been chosen which illustrate progress in our conceptions of the mechanisms of this process.

Stimulated by Warburg’s findings, Barr, Ronzoni, and Glaser (33) observed that an extract of pancreas inhibited glycolysis in slices of the Rous sarcoma; and in contrast with the behavior of muscle, they ob- served in tumor tissues a diminished glycolytic activity on mincing, grind- ing, or freezing. They concluded that the characteristic phosphorylative glycolysis of normal tissues was not occurring in tumor tissues. This view was supported in experiments conducted by Scharles et al. (34). It was found that although glycolysis from glucose was completely inhibited by cell destruction in both normal and tumor tissues (a finding previously made also by Warburg), this process did occur in tissue extracts in the presence of hexose diphosphate. They found, however, that glycolysis was not affected by fluoride in tumors as it was in normal tissues; it was less susceptible to inhibition by iodoacetate, and it did not require any “ coenzyme” (which a t that time meant adenosinetriphosphate) .

At the same time a systematic study by Boyland and Boyland (35) revealed that some of these differences could be attributed to the fact that adenosinetriphosphate (ATP) was rapidly destroyed by frozen malignant tissues. They found that tumor extracts would glycolyze hexose diphosphate (but not glucose) if supplied with relatively high concentrations of ATP, and they also observed the presence of zymo- hexase (a mixture of aldolase and triose phosphate isomerase) in these

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tumor extracts. Though the rates of glycolysis from hexose diphosphate were only about one-fourth those from glucose in slices, and considerably lower than those in muscle extracts, in view of the losses of enzymes by extraction and by the action of adenylpyrophosphatase, these investi- gators saw no reason to assume that hexose diphosphate was not an intermediate of glycolysis in the intact cell, or that the mechanism of glycolysis differed in any significant manner in normal and neoplastic cells. Following upon the finding of Meyerhof and Ohlmeyer (36) that cozymase (DPX) was required for glycolysis in muscle, Boyland et al. (37) found that in extracts of frozen Crocker sarcoma 180 the addition of cozymase raised the levels of glycolysis to virtually that of slices. They also found that glycolysis from glucose, fructose, and glycogen occurred in these extracts and was enhanced by the addition of adenylic acid.

The recent exhaustive studies of LePage and his colleagues have brought forward overwhelming support to the idea that, qualitatively, glycolysis is similar in tumor and other tissue types. LePage (38), in 1948, developed a medium in which suspended tissue homogenates could carry out glycolysis of hexose diphosphate at a high rate. The system contained phosphate, magnesium, and bicarbonate ions, ATP and Dl" as cofactors, hexose diphosphate as substrate, and nicotinamide as an inhibitor of DPN breakdown. The system contained fluoride ions to inhibit dephosphorylation reactions, and since this inhibited the further reactions of phosphoglycerate, pyruvate was added t,o couple the oxida- tion of triose phosphate to lactic acid production. In this system, homoge- nates of the Flexner-Jobling rat carcinoma actively glycolyzed hexose diphosphate (and glucose when hexose diphosphate was present in low concentration) and maintained organic phosphate a t a high level, indicat- ing that phosphorylation was a t least as rapid as organic phosphate breakdown. Cnder optimal conditions lactic acid formation occurred at a rate of 10 micromoles per 30 mg. of tissue in 40 minutes, which corresponds to a Q (lartic) of over 50. No " Pasteur EfTect " was displayed by these homogenates, the rate of lactate formation being as high in air as in nitrogen. A further study by Novikoff, Potter, and LePage (39) extended these results to the Jensen and Walker 256 tumors. The authors concluded, on the basis of their detailed study, that the Embden-Meyer- hof scheme of phosphorylative glycolysis operates in tumors. This con- clusion was further substantiated by a detailed analysis, by LePage (40), of a large number of phosphorylated intermediates, and cofactors of the Embden-Meyerhof scheme. Table IV shows that all of the intermediates found in normal, resting tissues were present also in a number of primary and transplanted tumors.

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TABLE IV Analyses of Glycolysis Intermediates and Factors of Normal and Neoplastic Tissues of Rat (40)

(Values are in micromoles per 100 g. tissue.)


Lactic acid Glycogen” Acid-soluble phosphorus Inorganic phosphorus Organic phosphorus Phosphocreatine Adenylic acid Adenosine diphosphate Adenosine triphosphate Glucose-1-phosphate Glucose-6-phosphate Fructose-&phosphate Hexose diphosphate Phosphoglyceric acid “Coenzymes” Free pentose phosphate Per cent of organic

phosphate accounted for

Primary 0

Primary Liver Human Flexner- Walker Mouse 9 Mouse Carci- Breast Jobling 256 Ear D Carci- noma Carci- Carci- Carcino- Jensen Carci- 7

Brain Muscle Liver Kidney Heart noma (rat) noma noma sarcoma Sarcoma noma M

E 141 188 230 155 578 833 590 1458 862 824 637 704

2390 5070 3040 2530 3200 1830 2810 ,2030 2650 2430 2130 2950 495 748 417 497 730 723 828 382 1035 622 580 726

1895 4322 2623 2033 2470 1108 1983 1649 1615 1808 1550 2224

151 155 144 213 329 97 278 137 131 171 183 251 46 135 3

179 542 8 138 105 93 54 67 106 152 142 161 $

185 250 423 264 249 335 555 470 278 454 393 500 5

531 3480 28450 81 2460 232 468 1620 67 65 43 56 w

311 1630 274 116 219 88 0 46 92 116 78 94 g 27 59 330 48 65 12 133 51 49 25

61 80 42 42 175 47 100 101 104 130 106 156

30 33 24 17 53 11 34 16 7 14 17 37 s 6 7 17 4 7 5 21 15 5 6 5 11

98 140 183 102 209 167 116 161 119 148 98 165 d M 17 17 35 16 25

42 22 48 72 50 m

80 81 70 68 65 95 78 77 72 86 91 89

0 As herose.

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LePage and Schneider (41) found, using the differential centrifugation technique, that the enzymes which carry out glycolysis, both in rabbit liver and Flexner-Jobling carcinoma cells, are concentrated in the solu- ble portions of the cytoplasm. Though the presence of other cell com- ponents, principally the small granules (microsomes), considerably en- hances the glycolytic activity of the supernatant fraction, none of the other fractions by themselves were nearly as active as the supernatant alone (see Table V).

TABLE V Glycolysis Obtained in 40 Minutes with 30 mg. of Tissue or Fraction Obtained

Therefrom (41)

Flesner-Jobling Carcinonia Rabbit Liver __.____ - _ _ _ _ . _ _ _ _ ~

Lactic acid Lactic acid produced S e t P bptake producccl Net P uptake per flask per flask per flask per flask

Tissue Fraction (m icromoles) (micromoles)

Homogenatc i . 3 5 3.62 6 .26 0 .23 Nuclei 1 . 3 1 1.23 0 . 7 9 0 .26 Mitochondria 0 -0.13 0 0.05

Supernatant fluid 2 .69 2.07 3 . 3 0 0.42 hIicrosomes 0.05 -0. 76 0 .17 -0 .31

Some of the differences between tumor tissues and normal tissues in their metabolism of glucose ha\-e been brought out more clearly by LePage (-12) in a further study of glycolysis in whole homogenates. As shown in Table TI, all tissues glycolyzed rapidly in the basic medium containing hexose diphosphate, but only brain and Flexner-Jobling car- cinoma displayed an increased glycolysis when glucose was added to the medium. ,4s shown in Table VII, however, tissues which were apparently unable to utilize glucose, such as diaphragm, kidney, and liver, were able to utilize glucose-6- and fructose-6-phosphate. LePage therefore was able to pinpoint the limiting step in glucose utilization for glycolysis to the hesokinase reaction by which glucose and ATP react to give glucose-6- phosphate. He suggested that this step remains under hormonal inhibition even i n the dissected, surviving tissue. However, attempts to relieve this inhibition i n zdro or to demonstrate hormonal effects on glycolysis were i n general not successful. A highly significant feature of LePage’s results is that potential rates of glycolysis of normal tissues are as great as, or greater than, those of tumor tissues, and that the low rates of glycolysis displayed by normal tissue slices are not due to lack of enzymes for the reactions involved.

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TABLE VI Anaerobic Glycolysis with Homogenates of Tissues from Normal Intact Rats (42)

(40-Minute incubation with 30 mg. wet weight of tissue except in the case of diaphragm, where 15 mg. was used. Each figure represents the average

of 15-20 experiments except those for heart and skeletal muscle, where only three experiments were available.)

Basic Medium Basic Medium Minus Glucose with Glucose

Lactic acid Net P Lactic acid Net P production uptake production uptake

Tissue (micromoles) (micromoles)

Brain 6 .0 0 . 4 5 9 . 8 6 . 5 Flexner-Jobling carcinoma 6 .2 0 9 . 5 4 . 5 Liver 6 . 4 -1 .5 6.8 - 1 . 3 Kidney 6 . 5 - 2 . 5 6.8 -2.3 Diaphragm muscle 5 . 5 -1 .0 5 .6 -0.65 Heart 9 . 1 - 1 . 9 9 . 9 -0 .39 Skeletal muscle 9 . 0 -2.2 8 . 7 - 5 . 3

TABLE VII Anaerobic Glycolysis with Glucose and Phosphorylated Sugars by Homogenates of

Rat Tissues (42) (40-Minute incubation in each case, with 30 mg. wet weight of tissue except in the

case of diaphragm (20 mg.). Reaction mixture contained cofactors as listed in the basic medium.)

(Values are in micromoles lactic acid.)

Substrate Added in Addition to Glucose Micromoles Brain Diaphragm Kidney Liver

None Hexose diphosphate Hexose diphospbate Glucose Hexose phosphate Glucose-6-phosphate Hexose diphosphate Fructose-6-phosphate

0 0 . 1 0 . 1 0 . 1 0 .1 6 6 . 8 6 . 2 6 . 0 7 . 7 6

30 9 . 1 6.6 6 . 0 7 . 6 6 4 .5 8 . 6 8 . 9 8 . 3 9 . 0 6 4 . 5 9 . 0 9 . 5 8.2 9 . 6

Experiments by Meyerhof and collaborators (43, 44) have in general confirmed the idea that over-all processes of lactic acid formation are a t least qualitatively similar in normal and neoplastic cells. Their data emphasize the probability that quantitative differences observed in homogenates, extracts, or other broken cell preparations are more likely due to differences in the intracellular distribution or to differences in

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breakdown rates of coenzymes or other factors than to actual differences in types or amounts of enzymes. In seeking an explanation for the fact that glycolysis from glucose occurred readily in extracts of brain, but not in homogenates, Meyerhof and Geliazkowa (13) found that most of the ATPase of brain cells was in the particles removed on low-speed centri- fugation. Consequently the level of ATP could be maintained readily in extracts, from which these particles were removed, hut not in homoge- nates. Subsequently, Meyerhof and Wilson (44) found that most of the ATPase of tumor cells was in soluble form, not removed by centrifuga- tion, and they reasoned that the glycolytic inactivity of tumor extracts or homogenates was due t o excessively rapid breakdown of ATP. In con- firmation of this hypothesis they found that if ATPase was inhibited by addition of agents like octyl alcohol or toluene, or if yeast hexokinase was added, steady and high glycolysis from free glucose could be achieved in tumor extracts and homogenates.

There is no evidence known which would indicate that the catabolism of glucose to lactic acid occurs in tumors via channels not present in normal cells. From the foregoing data it is clear that careful, critical studies offer no suggestion of a nonphosphorylative pathway, but, on the contrary, point t o the Embden-Meyerhof pathway as the major mechanism. The possible occurrence of the oxidative hexose monophos- phate shunt remains for further exploration; indeed preliminary data already discussed (23) make i t probable that this process may be of considerable significance in the carbohydrate metabolism of tumor cells.

JrI. ELECTROX TRANSPORT IN TUMORS .4 great deal of evidence has been advanced in favor of the plausible

idea that the high aerobic glycolysis of tumor tissues might have its origin in a disturbance in the pathways by which electrons are transported to oxygen. Shortly after the role of the pyridine nucleotides in respiration was recognized, Euler and colleagues (45) compared a number of normal and neoplastic tissues of the rat and found the diphosphopyridine nucleotide (DPK+) and triphosphopyridine nucleotide (TPNf) content of the Jensen tumor to be ats about the same level as in muscle or liver. An interesting observation of these authors was a markedly higher pro- portion of reduced DPN in the Jensen sarcoma. The ratio DPNH/DPN+ was 8.5 and 6.2 in two assays on the tumor, whereas rat muscle had approximately equal amounts of each form, the ratio ranging from 0.56 to 1.0 in three analyses cited. Somewhat later Kensler et al. (46) reported a lowering in the DPK+ content of rat liver from 1390 pg./g. fresh weight to 500 pg. in the “precancerous” liver after long-term feeding of butter yellow. The tumors thus induced had a still much lower DPNf content,

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namely, 150 pg./g. fresh weight. Bernheim and Felsovanyi (47) reported DPN+ plus TPN+ values of 71 pg./g. of Walker 256 tumor as compared with values of about 500 pg./g. for muscle, spleen, kidney, and liver.

More recently, Fisher and Schlenk (48) reported results of analyses of several tumors for their content of the oxidized and reduced forms of DPN+. Their values for DPN+, as shown in Table VIII, ranged from 9 to 165 pg./g. fresh tissue, whereas the DPNH values ranged from 21 to 91 pg. In contrast with the previously cited study of Euler et al. (45), the content of reduced DPN was generally lower than that of the oxidized form. The authors concluded that neoplastic tissues have a lower total DPN content than normal tissues, but their comparison was based only on data for normal rat liver, whose DPN content was in the vicinity of 500 pg./g. tissue.

TABLE VIII Content of Reduced and Oxidized Forms of DPN in Tumor Tissues (48)

Nucleotide Content No. of (pg./g. fresh tissue)

Determina- Tissue tions DPN+ DPNH Total

Rat sarcoma AH 8 49-165 41-91 98-240 Rat tumor (methylcholanthrene) 2 9, 31 36, 21 49,45 Mouse breast carcinoma (C3H) 2 75, 123 42, 57 124,189 Rat fetus 2 41, 31 25, 25 61,56

For reasons to be discussed in a later section a comparison of normal and neoplastic tissues with respect to their content of DPN+ and DPNH was carried out in the author’s laboratory by L. Jedeikin. It was found that both DPN+ and DPNH are highly susceptible both to enzymatic and nonenzymatic destruction, and their recovery from tissues re- quires rigid control of experimental conditions. Using conditions for extraction which ensured a predictable recovery of both forms of the nucleotide, and a highly specific enzymatic method of assay, with crystal- line alcohol dehydrogenase, a generally much lower content of total pyridine nucleotide was found in neoplastic than in normal mouse and rat tissues. In both tissue types, however, the same pattern of reduced and oxidized forms was observed, the latter being greatly preponderant. A summary of data secured thus far is given in Table IX.

Carruthers and Suntzeff (49) developed a polarographic procedure for determination of total pyridine nucleotides (DPN+ and TPN+). Their values for normal tissues ranged from 110 pg./g. fresh tissue for mouse epidermis up to 540 pg. for mouse liver. Comparable values for a series

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TABLE I S Content of DPS+ and DPXH in Various TissuesD (Values are in micrograms per gram fresh tissue.)

hlormal Tissues Liverb Kidney b

Heartb Brain< Spleenc Muscle (skeletal)' Muscle (pigeon breast)

Seoplastic Tissues (Mouse) Hepatoma 98/15 Rhabdom yosarcoma Sarcoma 37 Mammary carcinoma Ascites sarcoma 37 .4scites carcinoma (Ehrliclr)


No. of No. of Analyses Range Analyses Range

- ___

13 6 6 2 2 4 2

2 2 2 2 1 1

338-592 13 206-435 7 334-477 6 102,204 2 162,184 2 344-370 3 530,630 2

92,114 2 99,118 2 89, 121 2 99,111 2

206 1 297 1

57-372 137-264 41-272 80, 114 37) 44 16-38 27,38

35) 38 19,29 33, 51 37, 41

38 0

0 Unpublished date of Jedeikin and Weinhouse.

c Of rat and mouse. Of rat, mouse, rabbit, pigeon.

TABLE 9 B Vitaniins in Human, Rat, and Mouse Seoplasms (51)

(B vitamin levels pg./g. moist tissue.)

Human Human Cancer/ Rat Rat Cancer/ Xormal Cancer Sormal Sormal Cancer Normal

Vitamin Tissues Tissues ( %,) Tissues Tissues ( %)

Thiamine 1.80 Riboflavin 8.10 Nicotinic acid 31.2 Pantothenic acid 10.3 Pyridoxine 0.52 Biotin 0.18 Inositol 632 Folk acid 1 . 4

1.28 2.35

5.51 0.11 0.04



8 i 7

50 29 75 54 21 21

138 200

3.7 9.6

87.0 20.4 0.87 0.22

3.7 924

1.54 42 3.4 35

23.6 27 7.7 32 0.196 22 0.05 23

516 56 3.51 100

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of transplanted tumors ranged from 60 to 202 pg./g. The tumors displayed values in the range of such normal tissues as lung, spleen, pancreas, and epidermis. Similar results have been reported in abstract form by Strength and Seibert (50).

That tumors may have a relatively low content of factors concerned in electron transport is suggested also by an investigation of the B vitamin content of human and rat tumors, carried out by Pollock, Taylor, and Williams (51). A summary of their extensive data is given in Table X. All of the vitamins except inositol and folic acid were lower in human and rat tumors than in normal tissue of the same species. Especially note- worthy for this discussion is the lowered riboflavin and nicotinic acid content.

1. Cytochrome c and Cytochrome Oxidase

Evidence for a relative deficiency of cytochrome c in neoplastic cells is available from a number of studies. DuBois and Potter (52) , using a

TABLE XI Cytochrome c Content of Normal and Neoplastic Tissues of Rat

Data of DuBois and Potter (52)

Cyt. c Cyt. c ( a l p . Normal ( a l g . fresh tissues, fresh

Tumors tissue) rat tissue)

Flexner-Jobling carcinoma

Walker 256 carcinoma Jensen sarcoma Yale #1 mouse tumor Ultraviolet ear tumor Rous chicken sarcoma Rat liver tumor Tumorous rat liver

12 Heart 371

9 Kidney 247 12 Skeletal muscle 97 16 Liver 90 11 Brain 50 12 Spleen 43 20 Lung 21 61

Data of Rosenthal and Drabkin (53)

Cyt. c (l.4g.k. fresh

Tissue tissue)

Kidney cortex Liver Brain cortex Submaxillary Colon mucosa Mammary gland Lung Spontaneous mam-

mary adenoma Transplanted adeno-

carcinoma Walker 256 carcinoma

1430 607 375 378 136 32 24 51



spectrophotometric assay procedure, found that the cytochrome c con- tent (based on an estimated molecular weight of 16,500) ranged from 9 to 61 pg./g. of fresh tissue for a series of transplanted tumors as com-

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pared with values ranging from 21 to 371 pg./g. for a variety of normal rat tissues (Table XI). These authors conclude that their results lend weight t o It-arburg’s idea that tumor tissue may have an anaerobic pat- tern of metabolism. In a similar study, Rosenthal and Drabkin (53) found that liver, kidney, and brain cortex (in descending order) had highest contents of cytochrome c, and a series of tumors had only about 2% of the cytochrome c content of kidney cortex (Table XI). These differences are not due to ‘‘ cellularity ’’ differences, since the relative cytochrome c content does not change materially when calculated on the basis of protein phosphorus content. Though a low cytochrome c content was displayed by tumor tissues, this is not a distinguishing feature, since similar lorn cytochrome c levels were found in such normal tissues as colon mucosa, mammary gland, and lung.

Shack (54) measured relative cytochrome oxidase activity of various tumors and normal tissues, using oxygen uptake, measured manomet- rically in the presence of excess phenylenediamine and cytochrome c, as a measure of cytochrome oxidase activity. Cytochrome oxidase activity was four to five times higher in liver than in hepatoma or in other neo- plastic cells. h tenfold higher D-amino acid oxidase content was also found in liver than in hepatoma or other tumors.

Extensive investigations of the cytochrome oxidase activity of normal and neoplastic tissues have been carried out by Elliott and Greig (55) and Schneider and Potter (56), and the pertinent information is collected in Table XII. Though the values given by Schneider and Potter are far higher than those obtained by Elliott and Greig, both studies are in agreement in indicating a rather wide range in variation of cytochrome oxidase activity in normal tissues, and a much narrower range of activities in neoplastic tissues, of a magnitude similar to that of the less active normal tissues. It can be seen, from Schneider and Potter’s data, that the cytochrome oxidase activity ranged from 92 t o 974, whereas that of a variety of tumors of the rat, mouse, and chicken ranged from 44 to 136. Schneider and Potter concluded that oxidative metabolism is deficient in tumors, but that succinic dehydrogenase was not the weakest link in the electron transport chain.

Despite its low activity in tumors i t would appear that the cytochrome oxidase activity is not a limiting factor in electron transport. Greenstein et al. (57) have shown that a t least one factor which is lower in activity in a variety of rat and human tumors is cytochrome c.

These investigators noted, in agreement with others, that in the pres- ence of p-phenylenediamine the addition of cytochrome c to a fixed amount of cytochrome oxidase preparation produced an increased velocity of oxygen consumption up to a point beyond which further additions are

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TABLE XI1 The Succinic Dehydrogenases (Q8) and Cytochrome Oxidase (QJ Content of Normal

and Cancer Tissues (56)

Average Q. Average QOx


Tissue Etiology S & P a E & G b S & P E & G & .

Rat heart Normal 219 62 974 506 4 . 4 Rat kidney Normal 195 112 549 288 2 . 8 Rat liver Normal 87.7 66 392 167 4 .5 Rat brain Normal 48.7 18 420 134 8 .6 Rat muscle Normal 35.5 6 . 6 180 38 5 . 1 Rat spleen Normal 23.3 0 .5 195 32 8 .4 Rat lung Normal 17.9 7.5 92.3 31 5 . 2 Rat liver tumor Orally ingested BYd 26.3 - 134 - 5 . 1 Rat liver tumorC Orally ingested BYd 25 .O - 67.2 - 2.7

6 . 3 Hepatoma 31 rat Originally oral BYd 21.7 - 136 Transplantable hepa-

6 .9 toma Originally oral BYd 18.1 - I24 Walker 256 rat

carcinosarcoma Originally spontaneous 9 .4 - 61.5 - 6.6 Walker 256c rat

carcinosarcoma Originally spontaneous 12,3 0 ,6 77,8 15 6.3 Flexner-Jobling rat

Flexner-Jobling rat carcinomac Originally spontaneous 14.8 12.8 75.9 28 5 .2

Yale No. 1 mouse 5.2 tumor Estrin 20.2 - 106

Yale No. 1 mouse tumorc Estrin 19.0 - 87.5 - 4.8

Mouse ear tumorc Ultraviolet irradiation 19.1 - 64.0 - 3 . 3 Rous chicken sarcomaC Virus 11.1 - 44.4 - 4.0 Mouse mammary

tumorsc Spontaneous 27.7 0 . 5 87.8 - 3 . 2 Jensen rat sarcoma Originally spontaneous 17.8 13 129 43 7 . 2



carcinoma Originally spontaneous 15.5 - 91.3 - 5.9


a Schneider and Potter. a Elliott and Greig. 4 These tissues were homogenized in 0.033 If phosphate buffer at pH 7.4. -411 other tissues were

d BY = p-dimethylaminoazobenzene. homogenized in glass-redistilled water.

without effect. This maximum velocity represents a measure of the cyto- chrome oxidase activity under conditions in which the enzyme is operating a t highest efficiency. Since calculation of the Michaelis constant for cytochrome oxidase in tissue suspensions gave the same value obtained by others for preparations of this enzyme, this author concluded that the respiratory activity of the tumor suspension truly represents cytochrome

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osidase activity. From the Pllichaelis-Menten expression

V m a x S ’ = K F S

where 2: = velocity of the reaction without added cytochrome c, T’,,, = maximal velocity with escess cytochrome c,

S = cytochrome c concentration of the tissues, i t should be possible to calculate values for u from the cytochrome c content of tissues and from the difference between T’,,, and 1’.

It was found that the observed values corresponded quite closely with those calculated by the Jlichaelis-Menten equation. It was also found, using the observed values for t i , that the calculated values for the cytochrome c content corresponded reasonably well with observed values. The authors reasoned, therefore, that the difference between the observed oxygen consumption rates, with and without added cytochrome c, represents the disparity between the cytochrome oxidase activity and

the cytochrome c content. The value (””” ’~- ’) 100, called the per cent

response to cytochrome c addition, was calculated for the tissues studied, and it was found to fall into four categories.

C’ategory 1, with per cent responses ranging from 100 to 400, included only normal tissues, viz., heart, muscle, liver, kidney, and brain.

Categories 2 and 3, with per cent responses ranging from 600 to 1200, included some normal and some neoplastic tissues.

(’ategory 4, with per cent responses ranging from 1500 to 6,000 in- cluded only neoplastic tissues.

It mas concluded that normal tissues are characterized by generally high values for cytochrome oxidase and cytochrome c, whereas neoplastic tissues have a generally low activity of cytochrome oxidase with a great disparity between cytochrome osidase activity and cytochrome c content.

The earlier studies of the cytochrome system in tumors failed to take into account the question of intracellular distribution of the oxida- tive enzymes and cofactors. The more recent results of Schneider and Hogeboom (58) on the intracellular distribution of cytochrome oxidase activities are of particular interest. These authors have compared these activities in four well-defined fractions of the transplantable mouse hepatoma 98/15 and the liver of its host; their data are in Table XIII. Despite generally much low\.er activities in the tumor fractions the dis- tribution was remarkably similar. The very high specific activity of the mitochondria indicates that essentially all of the cytochrome oxidase activity is in this fraction. Such comparisons between normal and neo- plastic cells with respect to intracellular distribution of enzyme activities

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C3H Mouse Liver Cytochrome oxidase activity

are as yet few in number but promise to yield a rich harvest in our under- standing of relationships between over-all metabolic patterns and indi- vidual enzyme activities.

Since all of these published data are in agreement with respect to a relative deficiency of cytochrome c and cytochrome oxidase in a wide variety of tumor cells, it might be thought that this would definitely represent a distinct characteristic of tumor cells, lending weight to the idea that respiratory activity may be impaired in tumors. A recent paper by Chance and Castor (59), however, indicates that the whole question of cytochrome deficiency will probably need reinvestigation. Using sensitive

TABLE XI11 Cytochrome Oxidase Activities of Mouse Liver and Hepatoma Fractions (58)

(The total values reported are for 100 mg. of fresh tissue or an equivalent amount of each fraction, and each figure represents the average of 3 experiments.)

Hepatoma 98/15 Cytochrome oxidase activity

Tissue Fraction

Per cent of Total total

(mm.3 0 2 homogenate per hr.) activity

Qo, (mm.3 0 2

per hr. per mg.


Homogenate Nuclei Mitochondria Microsomes Supernatant

6860 (100) 1360 19.8 5390 78.6 292 4 . 1

0 0

2060 2440 6460 351


Total (mm.3 0 2

per hr.)

1520 195 964 247


Q o ~ Per cent of (mm.3 0 2

total per hr. homogenate per mg.

activity nitrogen)

(100) 633 12.8 379 63.4 3300 16.3 624 0 0

spectrophotometric methods which allow the measurement of light ab- sorption through whole cells, these investigators measured the changes in optical density a t various wave lengths corresponding to reduction of the cytochromes a, b, c, and a3, in several ascites tumor cells, when respira- tion is stopped by exhaustion of oxygen in solution. A comparison of the behavior of three ascites tumors with that of other cell-types is given in Table XIV. The quantity, K , measures the rate of removal of oxygen from solution and therefore gives an indication of the intensity a t which cyto- chrome oxidase is operating. The values for the ascites tumor cells are within the range of values for the normal cells, not as high as yeast, but higher than the sarcosomes of rabbit heart or fly muscle. The patterns of cytochrome distribution are rather similar except that cytochrome b is apparently lower in the tumors than in the normal cells (it was undetect- able in the Ehrlich and Krebs 2 tumors). The most significant result with

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respect t o the present discussion is the high relative cytochrome c content of the ascites tumors, actually exceeding that of the highly respiring yeast cells. Another point of interest is that there is a wider disparity between cytochromes c and a3 (cytochrome oxidase) in the normal cells than in the tumor cells. In commenting on these discrepancies with pre- vious reports, Chance states the belief that the direct spectrophotometric determination in freely suspended, living cells is probably more decisive than the various indirect assay methods that have led t o the earlier conclusions.

TABLE XIV Comparison of the Pattern of Respiratory Pigments of Tumor and Other Cells (59)

k‘= pJI 02/sec. Relative optical density change

D115-180 Cytochrornes Material (25°C.) a b C a3 DPNH

Keilin and Hartrw heart muscle preparation 49 1 0.6 0.8 9

Rabbit heart sarcosomes 14 1 0 . 8 0 . 9 11 - Fly muscle sarcosomes 12 1 0 .6 1 . 5 9 Bakers’ yeast cells 160 1 1.7 2.7 11 60



Ehrlich ascites Krehs 2 ascites dba thymoma

- 37 1 0 . 5 4 12 40 1 0.5 4 6 13 32 1 0.4 2 6 11

Relatively little attention has been given to studies of other factors involved in electron transport. Lenta and Riehl ( G O ) have recently made a study of the enzyme systems involved in the transfer of electrons from reduced diphosphopyridine nucleotide to oxygen. The source of the ac- tivities measured was a phosphate buffer extract of homogenized tissues, precipitated by acetate at pH 4.6, and taken up in phosphate buffer a t pH 8.5. Over-all DPS-oxidase activity was determined by spectrophoto- metric measurement of the oxidation of reduced DPN by 2,6-dichloro- phenol indophenol; diaphorase activity was measured by oxidation of reduced DPN in the presence of methylene blue, and cyanide to prevent functioning of the cytochrome system; and cytochrome c reductase activity was determined by spectrometric measurement of the reduction of cytochrome c by reduced DPN in the presence of cyanide. The relative activities are designated in Table XV. Over-all coenzyme I oxidase activity was present in the three tumors studied but was on the low side of the normal tissues. This behavior could not be specifically localized

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in one particular step, since low activities were observed generally in the tumor cells, except for cytochrome c reductase, which appeared in one tumor at least to be as high as in the highly active normal tissues. The high activity of DPN-cytochrome c reductase in the hepatoma 98/15 is in accord with a study of the intracellular distribution of this enzyme by Hogeboom and Schneider (61)) who previously found a somewhat higher content of this enzyme in the hepatoma than in the C3H mouse

TABLE XV General Summary of the Coenzyme I Oxidase System and Its Components in Normal

and Tumor Tissue Extracts (60)

Cyto- Coenzyme I chrome c Cyto- Cytochrome

Oxidase Diaphorase Reductase chrome c Oxidase

Liver Kidney Heart Brain Muscle 5-37 Adenocarcinoma Hepatoma 98/15

+++ ++++ ++++ ++++ +++++ ++++ ++ ++ + + + + + + + +++

+++++ ++ ++++ +++ ++++ ++++ +++ + +? + + +? ++ + ++ + +++++ +

+++ ++++ ++++ +++ +++ + + ++ liver. In contrast with cytochrome oxidase, cytochrome c reductase was present in all cell fractions, but highest specific activity was observed in the microsomes. In a previous study Rhian and Potter (62) found a low activity of cytochrome c reductase in tumors, but the manometric pro- cedure used leaves open the possibility that some factor other than the enzyme per se may have been limiting the rate of reaction.

2. Dehydrogenases

Many efforts have been made to relate the high glycolytic activity of tumor cells to some peculiarity of lactic dehydrogenase, and though widely divergent results have been reported with respect to the occur- rence of this enzyme in neoplastic tissues, a recent study by Meister (63) of a very wide variety of tumor tissues failed to reveal any consistent differences among tumors, tissues of origin, or various normal tissues in their content of this enzyme. A highly condensed summary of his results is given in Table XVI. Similar results have been reported for a number of mouse and rat tissues by Wenner et al. (64) (Table XVII). Both of these studies employed spectrophotometric measurement of the oxidation of reduced DPN in the presence of pyruvate as substrate, and hence the

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TABLE XVI Data of JIeister on Lactic Dehydrogenase A4ctivity of Various Normal and

Seoplastic Tissues (63)


Mouse liver Liver of tumor-bearing miw llouse kidney Kidney of tumor-bearing mice Skeletal muscle, mouse Skeletal muscle of tumor-bearing mice Other tissues of mouse Primary mousc tumors Transplanted mouse tumors Adult rat liver Fetal rat liver Primary hepatoma Transplanted fibrosarcoma

Kurnber of Samples

Activityo Range

12 13 13 13 13 13 54 19 57 5 4 3 3

36 1-490 390-523 284-426 296-405 900- 1040 940-1100 78-770

172-642 133-565 367-47 1 5 9 9 - 6 7 9 476-516 390-444

Activity is expressed as moles X 10-8 pyrurate reduced per nig. total nitrogen per minute.

TABLE SVII Ilehydrogenase Assays in .4cetone Powder Extracts (64)

Deh ydrogenase (units/mg. acetone powder)

a-Ketoglutaric Tissue Lactic Nalic Isocitric Qo2

Normal : Heart (rat) l iver (mouse) Kidney (rat) Nusclc (mouse)

320 383 56 0 200 256 10 8 7.7 104 I i 3 ti6 0 4 . 0 ,520 330 15 6

Neoplastic: Rhabdomyosarcoma (mouse) 160,220 148, 208 6 7 3 3 Mammary tumor (mouse) 168 380 16 0 3 2 Hepatoma (rat) 238 540 12 5 1 2 8" 2 4 Hepatoma (mouse) 108 288 14 8 2 1 Xscites (mouse) 165 200 4 8

n Range of three determinations

enzyme assays were independent of the presence of other electron trans- port factors.

Because of the question, t o be discussed later, of the participation of the citric acid cycle in oxidation processes in tumors, assays of various citric acid cycle enzymes mere made by Wenner et al. (64). Among these

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were malic, isocitric, and a-ketoglutaric dehydrogenases (Table XVII) . All three were found to be present in a variety of transplanted mouse tumors, though the last was present in rather low activity when com- pared with several normal tissues. Malic and isocitric dehydrogenases were assayed by spectrophotometric observation of the oxidation or re- duction of the pyridine nucleotide coenzyme, and the results were there- fore independent of other electron transport factors. It is of interest that when such specific assay methods were employed, as was the case with lactic dehydrogenase (63) and DPN-cytochrome c reductase (61), high values were obtained for the enzyme activities in tumors, whereas, when manometric methods were employed, as for example for DPN- cytochrome c reductase (62) or for malic dehydrogenase (65), invariably low results were obtained. These findings suggest that some factor in- volved in electron transport from the substrates in question to oxygen is either diminished in tumor cells or is destroyed more rapidly in such tissues during preparation of the enzyme. As we shall see later, this sub- stance is probably DPNf itself.

To summarize the present status of electron transport, it seems cer- tain that the same factors, namely, the pyridine and flavin nucleotides and the cytochromes, are involved in oxidative reactions in both normal and neoplastic tissue types. No evidence is available to indicate that other means of electron transport are employed in the cancer cell. Al- though quantitative differences in some of these enzymes and cofactors have been observed, and some of these seem so marked as to suggest that they may account for some of the peculiarities of metabolism exhibited by tumors, no decisive information is available to justify any definite statements. Although the low cytochrome activity of tumor cells can be cited in support of the idea that respiration may be deficient in tumor cells, such a deficiency if it exists is not manifested in any marked quan- titative diminution in oxygen consumption of such cells in vitro. The situation is further clouded by the above-cited spectroscopic data of Chance and Castor (59), which suggest that perhaps the earlier assays may not have measured the total cytochrome content of neoplastic tissues.

3. Respiration in the Intact Tumor Cell

Although, as we have already seen, Warburg, before 1930, abandoned his earlier idea that respiration was quantitatively impaired in tumor slices, this view has tenaciously persisted and is still often expressed or implied. A careful exploration of the available literature convincingly refutes such a concept. Studies of oxygen consumption of tumor slices by Crabtree (66), Murphy and Hawkins (67), and Warburg et al. (68), which

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are included in the data of Table I, clearly demonstrated that respiration of tumor slices is about as high as that of various representative normal tissues; and a large body of data to be found in the monographs of Greenstein ((3) and Stern and Willheim (10) have, without exception, confirmed and extended these findings. Elliott, whose work will be dis- cussed presently, has stated (69) : " . . . Most cancer tissues show only a moderately high respiration rate, a moderately low respiratory quotient,

and Dean Burk, in comparisons of various liver tumors with homologous normal, embryonic, aged, cirrhotic, and regenerating liver (70), has left no doubt that the neoplastic transformation of the liver cell is not asso- ciated with any regular, significant., qunntitativc diminution of oxygen consumption. There thus appears to be no basis in fact for the proposition that, when shaken in a saline medium, slices of tumor tissue consume appreciably less oxygen than do normal tissues similarly handled. Un- fortunately this represents the only manner which a t present we have to measure respiratory activity of tumor cells, and it is, of course, possible that these methods do not convey an accurate picture of the behavior of tumor tissues in sitzi. By the same token, however, there is no evidence that the studies in vitro portray a false picture of the respiratory capability of living cells.

and a definitely high, sustained aerobic and anaerobic glycolysis . . . ? 9 . ,

4. Nature of Substrates for Respiration of Tumor Cells

Prior to the use of isotopically labeled substrates, relatively few cri- teria could be applied to determine whether or not a substance underwent oxidation by tissues. Perhaps the most direct means a t the disposal of earlier investigators was to test whether addition of a substance to respir- ing tissue slices in vitro brought about an increase in the rate of oxygen consumption. Virtually all animal tissue slices display a rather high endogenous oxygen Consumption. When the addition of a metabolite increased the oxygen consumption, the evidence was clear and un- equivocal; when, as often happened, oxygen consumption was not in- creased, it could not be stated definitely that the added substance was inert, since it was conceivable that its oxidation replaced that of some metabolite endogenous to the tissue slice. Even when oxygen consump- tion is increased on the addition of a subst.rate, it cannot easily be as- certained from oxygen consumption data alone to what extent the added substance may be replacing the endogenous tissue metabolites.

It is sometimes possible, by means other than oxygen consumption measurements, t o obtain the desired information indirectly. For example, Quastel and Wheatley (71) and Leloir and hIunoz (72) demonstrated the ability of liver slices to oxidize fatty acids by measuring fatty acid disap-

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pearance and ketone body formation, even though increases in oxygen consumption were minimal. These studies paved the way for later demon- stration of fatty acid oxidation in homogenates and mitochondria. Other indirect criteria of the type of substrate undergoing oxidation may also be applied; for example, the respiratory quotient may indicate whether the oxidation is predominantly that of fatty acid or carbohy- drate; or the appearance of ammonia or urea may disclose the oxidation of protein or amino acids. In general, however, this type of approach has provided no definite answers to the fundamental question of what types of substrates are oxidized by tumor tissues.

Another approach to this question is the study of known or presumed intermediates. The Thunberg technique (73) has shown that tissues con- tain dehydrogenases for many substances; on the assumption that sub- stances are not entirely foreign to cells which can rapidly carry out their oxidation, this technique has been instrumental in tracing reaction sequences in carbohydrate metabolism. This procedure can be extended further to imply that tissues which oxidize such intermediates also utilize carbohydrate for oxidation. The usefulness of this type of procedure is well demonstrated by the fact that such reasoning applied to oxidation of various intermediates in pigeon breast muscle supplied some of the basic information from which Krebs (74) formulated the citric acid cycle.

So far, however, this type of approach has not yielded any decisive data which would allow definite conclusions concerning the types of foodstuffs oxidized by tumor cells. Such evidence as is available is rather indirect. We have already referred to Dickens’ studies of the respiratory quotient which suggested that glucose did not undergo oxidation readily in tumor cells. In line with this conclusion is the finding that oxygen con- sumption of tumor slices is not increased in the presence of glucose (66, 75).

Somewhat later, Elliott, Benoy, and Baker (76) observed that whereas the addition of lactate, pyruvate, succinate, fumarate, or malate in- creased the oxygen consumption of rat kidney cortex slices, they had no effect on the oxygen uptake of slices of Philadelphia number 1 sarcoma or Walker 256 carcinoma (Table XVIII). Though these investigators carefully refrained from drawing the obvious conclusion that carbohy- drate oxidation is impaired in these tumors, this paper has often been cited in support of such a thesis.

A more detailed investigation of the problem of respiration in tumor slices was undertaken by Salter and his colleagues, some of which has already been discussed in connection with electron transport. Craig, Bassett, and Salter (77) made an interesting observation which formed the basis of a detailed investigation. It was found (Table XIX) that

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whereas various normal tissues such as liver, muscle, and “ benign” granulation tissue exhibited large increases in oxygen consumption on addition of sucrinate to the medium, the oxidative response of the cor- responding malignant counterpart was far lower. This phenomenon appeared to be rather general, being noted also in a comparison of a

TABLE XVIII &o, Values in Presence of Various Substrates (76)

Suhstl-ate Rat Kidney Cortex Phila. Sarcoma Walker 256 Carcinoma

Xone 21 5 13 6 11 5 m-Lact a t e 32 1 13 2 11 5 Pyruvat e 33 6 12 8 13 5 None 19 9 14 1 11 7 Succinatc 32 8 13 8 11 0 Fumarate 24 5 12 2 12 0 h l fa la te 21 0 13 3 10 8

TABLE XIX Effect of Succinatp on Oxygen Consumption of Tissue Slices (77)

(Succinate when present was 0.02 M . Each value is the mean of approximately 10 observations.)

&or Succinate: Per Cent

Absent Present Change

Sormal liver 10.6 25 2 137 Hepatoma 10.5 12 2 17 Muscle 7 0 2 0 9 242 Rhabdom yosarcoma 8 7 1 2 2 41 Granulation tissue (bcnign) 1 52 4 14 192 Sarcoma 180 8 2 1 0 2 27 “Benign” sarcoma 180

(presumably slow growing) 6 4 8 6 37

variety of normal and neoplastic human tissues (78). The oxidative re- sponse of the normal tissues ranged from 100% to 158%, whereas 22 dif- ferent tumors displayed responses ranging from -25% to +76%. A similar effect was found in the “precancerous” liver of the rat fed butter yellow. With increasing periods of feeding of the dye, there was a steady diminution in the Oxidative response, ultimately reaching values charac- teristic of neoplastic tissues. Since p-phenylenediamine exhibited a similar behavior when used as a substrate, and since both p-phenylenediamine and succinic acid transfer electrons t o cytochrome c, these results were interpreted, in the light of their lowered cytochrome c, already discussed,

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to indicate that the tumor cell has a lower “oxidative reserve”; that is, its oxidative capacity is sufficiently high to maintain a high respiratory rate, but it cannot respond by increased oxygen consumption to the stress of higher substrate concentrations. These findings provide further support for the idea that a possible bottleneck in electron transport in tumors is the cytochrome level.

5. Isotope Tracer Studies on Tumor Respiration-Glucose Oxidation

The value of the tracer technique is perhaps nowhere better exempli- fied than in its application to the question of the metabolic fuel of the cancer cell. The availability of carbon-14-labeled glucose has finally made it possible to establish, without uncertainty or equivocation, that the neoplastic cell can convert glucose to carbon dioxide. Using this method, the appearance of radioactivity in the respiratory carbon dioxide affords a simple, direct, and reliable criterion for the oxidation of a substrate. By carrying out such experiments with tissue slices in Warburg vessels it is possible to correlate oxygen consumption and other respiratory data with the extent of oxidation determined quantitatively by measurement of the incorporation of isotopic carbon in the COz. In 1949 Olson and Stare (79) reported that glucose, labeled uniformly with C14 in all of its carbon atoms, was oxidized to CO2 in slices of primary tumor more rapidly than in the adjacent normal liver, and that both the carboxyl and the CY carbons of pyruvic acid likewise were oxidized by the hepatoma slices. A more detailed report of these studies was given at a symposium on carbohydrate metabolism in tumors, held under the auspices of the American Association for Cancer Research (80). Since most of the data were presented graphically, their reproduction is somewhat inconvenient. Briefly summarized, the data showed that glucose, D( -) and L( +) lac- tate, acetate, succinate, and pyruvate are oxidized both by hepatoma and liver cells; and though quantitative differences were observed between the two tissue types, no uniformity in response was exhibited. Thus, although glucose was oxidized far more rapidly by hepatoma slices, rates of oxidation of methyl- and carboxyl-labeled acetates, uniformly labeled L ( + ) and D( -) lactate, and 1- and 2-C14 pyruvate were about the same in liver and hepatoma slices, and succinate (carboxyl-labeled) was oxi- dized more readily by liver than by hepatoma. The results of Olson will be discussed later in other connections.

In a parallel study of a variety of transplanted rat and mouse tumors, Weinhouse (81) and Weinhouse et al. (82) made similar findings. As shown in Table XX the oxidation of glucose in slices of such representative nor- mal tissues as kidney, brain, liver, and muscle was compared with that dis- played by slices of four transplanted tumors: a mouse and rat hepatoma,

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a rhabdomyosarcoma, and a mammary adenocarcinoma. The relative specific activity of the respiratory carbon dioxide revealed that an appre- ciable percentage of the respiration of all of the tissues studied arose from the oxidation of C'4-labeled glucose added to the medium. This ranged from 34% for brain down to 8.5% for liver. Though somewhat lower than those of brain and heart, the relative specific activities of the respiratory C 0 2 produced by the tumor slices were in the range of those of kidney and heart and decidedly higher than the values displayed by skeletal

TABLE XX CL4-Glurose Oxidation by Sormal and Kcoplastic Tissue Slices (82)


Respiratory CO, 0.C.b R.S.4.a (microatoms

(per cent.) carbon)

Sormal, rat: Kidney Brain (homogenate) Heart Liver Skeletal niusrle (homogenate)

19.9 111 3 4 . 2 93 26 .8 87

8 . 5 23 10.0 7 . 0

Neoplastic: Hepatoma (mouse) 1 6 . 5 4 3 . 8 Hepatoma (rat) 17 .0 38.3

Maminary adrnocarciuorns (~iiouse) 1 7 . 0 25 .5 Rhahdornyosarcoma (mouse) 23 .7 35

0 The relative sperific artivity is the ratio of activity of COz to substrate ( X 100) and represents the

b 0.C. (Oxidative Capacity) represents the microatoms of substrate carbon oxidized to CO, per relative nmount of substrate carbon in the cnrhon of Cot .

grain dry tissue per hour at 38'C. at a substrate concentration of 0.005 M.

muscle and liver. Thus, there is no suggestion from these data of any quaiititative impairment of glucose oxidation in the tumors studied. -4 similar picture emerges from a comparison of the oxidative capacities given i n the last column (O.C.). This value is defined as the microatoms of labeled substrate carbon which have been converted to COZ per gram of tissue per hour. It is calculated from the relative specific activity and quantity of respiratory COT. This value applies only to the conditions of these experiments. I t may \*ary greatly, particularly with concentration of the substrate, but is of value for comparative purposes when conditions of experiments are similar. In this table me see that the oxidative capacity of the neoplastic tissues is within the range of values exhibited by the normal slices.

Essentially the same conclusions were reached with respect to the

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oxidation of lactic acid (Table XXI). Normal tissues showed a very wide range of oxidative capacity toward lactate, and the tumors studied gave values which fell within the normal range-lower than the most active, but higher than the less active, normal tissues. Here again, the data pro- vide no confirmation of a respiratory disturbance in tumor cells which leads to quantitative impairment in the oxidation of lactic acid. These data are of limited value, since they were carried out with lactate labeled


(Substrate concentration, 0.005 M.) Lactic Acid Oxidation by Normal and Tumor Tissues (82)

Normal Tissue 0.c.a Neoplastic Tissue . 0.c.a

Rat liver Rat kidney Rat heart Rat muscle Rat brain Rat spleen Mouse liver Mouse kidney Mouse heart Mouse muscle

44 Rat hepatoma 61 152 Mouse hepatoma 87 148 Mouse Andervont hepatoma 48

3 Mouse Sarcoma 37 10.1 112 Mouse rhabdomyosarcoma 72 130 Mouse mammary adenocarcinoma 65 32 Ehrlich ascites 116

440 108


a O.C. = oxidative capacity = microatoms substrate carbon converted t o CO, per gram dry tissue per hour.

only in the carboxyl carbon and, therefore, give no indication of the rate of Oxidation of the a! and P carbons (the acetyl moiety) ; however, they supplement and extend the studies of Olson (80), which were carried out with other types of labeled lactate and pyruvate but which were limited to a comparison of liver with primary hepatoma.

6. Oxidation of Fatty Acids in Neoplastic Tissues

For want of any specific information to the contrary, it has been gen- erally assumed that the major fuel for the tumor cell is glucose. We have already referred to the studies of Dickens and Simer (14) in which the low respiratory quotient of tumor slices was interpreted as indicative that fat may supply, a t least partially, the energy needs of the cancer cell. Direct studies of the capacity of neoplastic cells to oxidize fatty acids are relatively few. Kirsch (83) found that none of the fatty acids ranging from formate to octanoate increased the respiration of the Jensen sarcoma. Ciaranfi (84) attempted a manometric study of the effect of fatty acids on oxygen consumption by slices of a variety of human and animal tumors. None of the acids tested, ranging in chain length from 4

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to 18 carbons, gave any significant increase in oxygen upt,ake or the slightest indication of the presence of ketone bodies. The only effect observed was a slight increase displayed by the Ehrlich adenocarcinoma in the presence of /3-hydroxybutyric acid, in which case acetoacetic acid was detected as the oxidation product. In a brief note (85) amplified in a more detailed study (86) the same investigator, however, reported that both normal and neoplastic tissues slices readily oxidized methyl esters of fatty acids ranging from one to eight carbons. He found also that various normal tissues oxidized the methyl esters a t considerably greater speed than they oxidized the sodium salts.

Dickens and Weil-Malherbe (87) in a comparison of the metabolism of hepatomas with their tissue of origin found that the liver tumor arising in rats by butter yellow feeding largely lost the capacity for acetoacetate formation from caproic acid. A decrease was also noted for spontaneous mouse hepatoma, but in these experiments the tumors retained to a con- siderable degree their ketogenic ability. A study by Baker and Meister (88) revealed that homogenates of various mouse and rat hepatomas largely lost the capacity of the tissue of origin for oxidation of short- chain fatty acids. Oxygen consumption values in the presence of octanoic and hexanoic acids ranged from 0% t o 14% of the values obtained with normal liver. It appeared in this study that the neoplastic tissue contained some inhibitor of fatty acid oxidation, since the addition of the hepatoma homogenate decreased fatty acid oxidation in the liver homogenate. In similar experiments with leukemic infiltrated liver homogenates, Vestling et al. (89) observed a marked diminution in octanoic acid oxidation, which was greater proportionately than the extent of leukemic infiltration.

Though these data suggest that fatty acid oxidation may be impaired in the neoplastic cell, they provide no basis for a categorical answer. ;\lore recently the application of the isotope tracer method has left no doubt that tumor cells can oxidize fatty acids, but the possibility still remains that there may be a quantitative diminution in fatty acid oxidation in neoplastic as compared with normal cells.

Olson (80) found a somewhat lower capacity for oxidation of carboxyl- and methyl-labeled acetate by slices of primary rat hepatoma in compari- son with normal tissue of origin; similar findings have been reported by Pardee, Heidelberger, and Potter (90). These investigators found that COOH-labeled acetate was oxidized much less rapidly by slices of Flexner-Jobling and Walker 236 tumors than by kidney, liver, or lung slices. A more extensive study of the oxidation of C'J-labeled fatty acids by tumor slices was carried out in the author's laboratory (82). As shown i n Table S X I I , C'3-carboxyl-labeled palmitic acid is oxidized about as readily by slices of four tumor types as by slices of liver and kidney. A

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TABLE XXII Oxidation of 1 4 - 1 4 Palmitate by Slices of Normal and Neoplastic Rat Tissues (91)

(Substrate Concn. = 0.0005 M.)


Normal Rat Tissue Kidney Liver (fasted) Liver (fed) Liver of tumor-bearing animal

Hepatoma (mouse) Hepatoma (rat) Mammary adenocarcinoma (mouse) Rhabdomyosarcoma (mouse)

Neoplastic Tissue

3 9 . 8 18 .6 10.0 8 . 7

2 0 . 3 13 .6 11 .0 16.5

O.C. represent8 the number of microatoma of substrate carbon oxidized to carbon dioxide per gram dry tiesue weight per hour.

further investigation of fatty acid oxidation in neoplastic tissue (91) was devoted to the following questions: (1) Does the hepatoma, the neoplastic counterpart of liver, display the same specialized features of fatty acid metabolism characteristic of normal liver? (2) Does the hepa- toma resemble other neoplastic tissue types in its fatty acid metabolism? (3) Does the liver of the tumor-bearing host show any deviations from the normal liver?

TABLE XXIII Oxidation of Fatty Acids by Liver and Tumor Slices of the Mouse (91)

Oxidative Capacity

Acetic Butyric Octanoic Palmitic

Normal liver 58 59 43 11

Host liver Hepatoma 98/15

49 83 104 17 19 42 45 11

Host liver 82 54 90 16 Mammary adenocarcinoma 6 12 0 . 6 5

Host liver Sarcoma 37

Host liver Rhabdomyosarcoma

40 54 63 13 6 10 0 . 2 9

67 30 72 15 7 16 0 . 6 11

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To summarize the data shown in Table XXIII, oxidation of all four fatty acids is more rapid in the liver slices than in the tumor slices, the differences being more marked with the short-chain acids than with palmitate. I t appears that the apparently lower oxidative capacity of the

TABLE XXIV Conversion of 1-C-14 Butyrate to Acetoacetate by Liver and Hepatoma Slices (91)


Kesp. COZ Total B COOH C.C.b C.C. C.C. C.C.

pstoms patoms patorns patoms

n’ornial liver

Host liver Hepat oma

59.5 122 56 66

82 .8 87 .5 36 52 42.0 4 . 4 -0.4 3 . 9

Host liver 53.5 125 55 70 1Iammary adenorarcirioma 12 0 3 . 1 -1 -2

Host liver Sarcoma 37

Tumor lix-er Rhabdomyovnrcoma

Host liver, rat Hepatoma, rata

Host liver. rat Hepatoma, rat0

- - - 53.8 9 . 8 1 . 2 -0 -1

30.2 90 .3 46 44 15.8 6 . 1 -1 -5

159 152 58 94 19.3 4 . 5 -1 N 4

190 164 47 117 180 -5 -2 -3

0 Pool of unlabeled acetoacetate added. * C.C. = conversion capacity, represents the number of microatoms of substrate carbon converted

to the product in question per gram dry tissue per hour.

tumor tissues for the short-chain acids is due less t o ail inability to oxidize these substances than to powerful inhibitory effects exerted by the fatty acids on the tumor cells. When octanoate was used as a substrate in lower concentration than the levels used in the experiments shown in Table XXIII, its oxidation by various tumor slices was of the same mag- nitude as was displayed by liver slices. Thus, although a case could be made for a lowered fatty acid oxidation in tumor slices as compared with liver, 011 the whole it seems fair t o conclude that by and large fatty acid oxidation is not markedly impaired in neoplastic cells. It also seems

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fair to state, on the basis of rather scant evidence, i t is true, that the liver of the tumor-bearing animal has no impairment in its pattern of fatty acid metabolism.

Some interesting differences were disclosed, however, in ketogenesis and in acetoacetate oxidation. Inasmuch as the liver has a much greater ketogenic capacity than nonhepatic tissues, it was of interest t o test whether the hepatoma would resemble liver or nonhepatic tissue in this respect. I n contrast with liver slices ketogenesis was very low in neo- plastic tissue slices. Even with butyrate, the most ketogenic of the fatty acids, as the substrate (Table XXIV), none of the neoplastic slices, including the hepatoma, showed a degree of ketogenesis approaching that of liver slices. The possibility was considered that acetoacetate was being produced by tumor cells but was being rapidly metabolized. To check on this possibility a “trapping” procedure was employed in which the isotopic butyrate was oxidized in the presence of a pool of unlabeled acetoacetate. Under these conditions i t was expected that any labeled metabolic acetoacetate would mix with the unlabeled material and would be detected by radioactivity of the recovered acetoacetate. As shown in the last two experiments of Table XXIV, no appreciable activity was trapped by this procedure, and, therefore, the conclusion seems war- ranted that ketogenesis is of low or questionable occurrence in these tumor cells. Lack of ketogenesis in the hepatoma is of particular interest in that i t represents a marked deviation from the metabolism of the normal liver cell.

Y . Oxidation of Acetoacetate in Liver and Tumor Slices

Since tumors, including the hepatoma, appeared to display a non- hepatic metabolic pattern with respect to ketogenesis, it was of further interest to compare the rates a t which these tissues oxidize acetoacetate. The results, as shown in Table XXV, leave no doubt that tumor slices can oxidize acetoacetate. A considerable range of variation in the ability to oxidize acetoacetate was displayed by liver slices, the C.C. values ranging from 6.7 to 26.8. In every experiment except one, however, higher values than these were observed for the tumor slices. This is particularly significant, since in every instance the over-all respiratory rate, as shown in columns 2 and 3, was considerably lower in the tumor than in the liver slices. This may be more clearly seen in a comparison of the respective R.S.A.’s (column 4). The respiratory COa samples from the tumor slices contained much more acetoacetate carbon than did the COZ derived from the liver slices. Here again, tumor cells, including the hepatoma, displayed a metabolic pattern which resembles that of nonhepatic rather than liver cells.

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The results of this investigation leave no doubt that neoplastic cells can carry out the oxidation of fatty acids to COZ, sharing this property with hepatic and nonhepatic normal cells. Though the results with short- chain acids provide some justification for the belief that oxidation of fatty acids may not proceed as readily in tumor cells, such a conclusion is probably unwarranted in view of various uncertainties of the in vitro

TABLE XXV Oxidation of 2.4-C-11 Acetoacetate by Liver and Yeoplastic Tissues (01 )

(Substrate concentration = 0.002 M.)

O2 uptake Respiratory COz

Amt. Amt. R.S.A. C. C. (pmoles) (crmoles) (per cent) (patoms)

Host liver Hepatonla

Host liver Mnmmary adenocarcinoma

Host liver Sarcoma 37

Host liver Rhahdom yosarcoma

Host liver Hepatoma, rat

Host liver. rat Hepatoma, rat

Normal mousr lirrr

455 36-1

482 136

392 217

382 219

3-19 132

272 142


371 310

319 155

312 216

268 I70

287 161

310 165

26 1

7 . 2 2 6 . 8 1 6 . 2 55.1

5 . 7 18 .1 1 3 . 0 2 0 . 2

5 . 6 17.5 1.5.2 3 2 . 8

3 . 3 8 . 8 12 .0 2 0 . 2

6 . 3 18.2 7 . 8 12 .6

2 . 2 6 . 7 8 . 1 13 .3

7 . 8 2 0 . 4

experiments. It is noteworthy that with respect t o acetoacetate formation and utilization the neoplastic transformation of the liver cell is associated with a metabolic transformation to a pattern characteristic of non- hepatic tissue. The loss in the ability of the neoplastic liver cell t o produce acetoacetate from fatty acids is in line with many examples of the loss of specific metabolic function when normal cells become neoplastic (9). It is of particular interest, in this connection that the neoplastic trans- formation of the liver cell is associated also with an enhancement in a metabolic function, namely, acetoacetate oxidation-an observation which merits further exploration.

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8. The Citric Acid Cycle When the studies of Krebs and others led to the formulation of the

citric acid cycle as the mechanism by which pyruvic acid is completely oxidized to COZ, it was natural to assume that the alleged defect in respiration of tumor cells might have its origin in their inability to carry out certain reactions of the citric acid cycle. An observation which pointed in this direction was made by Potter et al. (92). These investigators found that whole homogenates of tumor tissues failed to carry out the oxidation of oxalacetate under conditions which resulted in its rapid oxidation by homogenate‘s of normal tissues. Since oxidation processes in tissue homoge- nates require the maintenance of a high level of organic phosphate, and since tumors have been found to contain very active dephosphorylating enzymes, the possibility was considered that the inactivity of oxalacetate in tumor homogenates may have been due to inadequacy of oxidative reactions to maintain the organic phosphate level. In a direct test of this assumption Potter and LePage (93) found that there was no significant disappearance of oxalacetate in a Flexner-Jobling tumor homogenate, in which the ATP level was being maintained by glycolysis. This obser- vation led them to conclude that a “bottleneck” existed somewhere on the pat,h of oxalacetate oxidation, presumably the introductory step of the citric acid cycle. The interesting hypothesis was advanced that oxal- acetate, blocked in its oxidative path via the citric acid cycIe, may foIlow alternate pathways leading to synthesis of building blocks for cell com- ponents, such as pyrimidines.

Another observation was made by Potter and Busch (94)) also pointing to a defect of the citric acid cycle in tumors. Buffa and Peters found that when fluoracetate was given to rats in toxic doses, citric acid accumulated in heart, kidney, and brain. It has already been amply demonstrated that fluoracetate inhibits certain of the reactions of the citric acid cycle (now known to be a t the aconitase stage (95)). The accumulation of citrate thus results whenever its rate of formation exceeds its rate of dis- appearance. Utilizing this observation as a means of testing for citric acid formation in tissues, Potter and Busch (94) injected fluoracetate into tumor-bearing rats and determined the citric acid content of the tissues one hour later. From the results shown in Table XXVI most tissues responded with a large increase in citrate content, the notable exceptions being liver, testis, and tumor. Since liver is known to display high citric acid cycle activity, its failure to accumulate this acid was explained on the basis of an alternate metabolic pathway, namely, aceto- acetate formation. The lack of citrate accumulation in the tumors was

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regarded as “strong evidence that the ability of tumor tissues to form citrate is very low in comparison with a variety of normal tissues.”

Recognizing the possibility that the fluoracetate technique may be subject to uncertainty in interpretation, and taking into consideration other evidence to be presented later for the occurrence of the citric acid cycle in tumors, Busch and Potter (96) developed another technique for

TABLE XXVI Meet of Fluoracetate on the Citrate Content of Korinal Tissues in Normal and

Tumor-Bearing Rats (94) (Values are presentcd in micrograms per gram wet weight of tissues.)


Brain Heart Lung Thymus Liver Kidney Spleen Testis Blood 31 llscle Panweas

Lninjected .4nimals

so. of Samples Average Range

Tumor-Bearing Animals 1 Hour after Injection

s o . of Samples Average Range

5 5 5 5 5 5 5 5 5 4 3

57 49 i 5 55 4 i 56 59 73 54 31 53

3tk 65 24- 73 40-114 24- 79 20- 86 16-123 28- 87 58-1 13 35-114 25- 38 29- 78

5 5 5 5 5 5 5 4 5 2

1 GO 448 206 275 39

714 514 7 9 79 42

134- 234 289- 638 133- 295 230- 337

6- 77 445-1039 306- 754

49- 119 48- 102 41- 42

Walker 256 Tumor 4 49 38- 61 4 42 31- 52 Flexrier-Jobling 4 121 92-144 4 90 61- 119 Jenseri 4 85 53-141 3 6G 40- 116 Hepatoma, primary 2 95 89-110 2 60 52- 67

testing the occurrcnce of the citric acid cycle in viuo. This method con- sisted in injecting malonate into tumor-bearing rats. Since malonate inhibits succinic dehydrogenase, its presence in tissues should lead to the accumulation of succinate in those tissues in which succinat,e is a metab- olite (i.e., those which carry on the reactions of the citric acid cycle). With the use of column chromatography for isolation of citric acid cycle components, the succinate contents of the tissues of malonate-poisoned rats were determined. Despite a wide range of levels, all tissues except brain, muscle, and blood accumulated succinate, including five tumors. The low content in the blood discounts the possibility of a migration from one tissue to another and thus provides support for the idea that suc-

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cinate represents an intermediary metabolite in those tissues in which it accumulates. The authors caution against the obvious conclusion that these data can be interpreted only in favor of the occurrence of the citric acid cycle. In discussing the uncertainties of in vivo techniques they point out that succinate may arise from sources other than citrate, for example, from glutamate and related compounds.

9. Isotopic Studies

When isotopic studies conducted by the author and his colleagues (82) demonstrated the complete oxidation of glucose and fatty acids by tumor slices, it was of interest to ascertain whether citric acid was an intermediate. This was successfully shown by a “trapping” procedure. Slices of tissue were incubated with labeled substrates, under conditions which result in their oxidation to COO, together with a “pool” of un- labeled citrate. It was expected that any metabolic citrate would mix

TABLE XXVII Radioactive Citrate from Tumor Oxidations (82)



Quinidine Citrate

Specific Specific Activity Activity

Substrate (counts/min.) (counts/min.)

Normal, mouse: Heart Liver Kidney

Neoplastic, mouse: Hepatoma Mammary tumor Rhabdomyosarcoma Ehrlich ascites Mammary tumor Mammary tumor Hepatoma Rhabdomyosarcoma

Glucose Glucose Glucose

Glucose Glucose Glucose Glucose Acetate Palmitate Palmitate Palmitate

5 . 5 x 105 5 . 5 x 105 5 . 5 x 10‘

1.37 X 106 1.37 X 108 5 . 5 x 106 5 . 5 x 106 1 . 4 X 10’ 66,200 66,200 66,200

150 126


1225 1750 1000 910 293 74

138 83

with the large quantity of added citrate and its further oxidation be prevented thereby. By isolating and assaying the citric acid remaining at the close of the experiment, it was found that appreciable activity had been converted to citric acid. From Table XXVII it is seen that glucose, acetate, and palmitate all yielded citkate. Though the data are

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regarded as only qualitative, giving no indication whether citrate is a major or minor metabolite, there is also, by the same token, no indication from these radioactivity data that citrate is less important quantitatively as an intermediate in the tumor cells than in the normal ones.

Further evidence for the intermediary formation of citric acid was obtained by a study of the effect of trans-aconitate on the respiration of tumor slices (82). Saffran and Prado (97) showed that trans-aconitate is an inhibitor of aconitase; when this acid is added to tissue slices, respiration is impaired and citrate accumulates. Similar treatment of tumor slices also resulted in lowering of respiration and accumulation of citrate. hgain, these results must be regarded as primarily qualitative, demonstrating only that citric acid is an intermediary metabolite in tumor cells, without indicating the magnitude of its formation.

10. The “Condensing” Enzyme

One of the most decisive points i n favor of the operation of the citric acid cycle is the presence in tumors of the