Quantification of Bleomycin Pulmonary Toxicity in Mice by Changes in LungHydroxyproline Content and Morphometric Histopathology
Branimir Ivan Sikic,1 David M. Young, Edward G. Mimnaugh, and Theodore E. Gram
Laboratory of Toxicology, National Cancer Institute, NIH, Bethesda, Maryland 20014
ABSTRACT
Bleomycin treatment produced dose-dependent changesin lung collagen content and in several measurable histo-pathological parameters. NIH/Swiss mice were treatedtwice weekly for 6 weeks with bleomycin, 0, 1, 20, or 40mg/kg s.c. The two highest doses produced mortalities of35 and 100%, respectively, as well as loss of body weightand increase in lung wet weight. Lung hydroxyprolinecontent, an index of collagen, increased to 40 to 50%above control levels at 6 and 8 weeks after initiation oftreatment with bleomycin 20 mg/kg. Morphometric analysis was applied to the following parameters at lightmicroscopy: number of intraalveolar macrophages andleukocytes, total pulmonary cell count, alveolar wall thickness, and percentage of consolidation of lung parenchyma. The two highest doses produced increases in allof these parameters as compared to controls. The mostmarked changes occurred in the number of intraalveolarcells, which in the group given 20 mg/kg rose to 150,190,and 210% of controls at 4, 6, and 8 weeks. The lowestdose of bleomycin, 1 mg/kg twice weekly for 6 weeks,evoked no pulmonary or other toxicity by the parametersexamined. This model of chronic pulmonary toxicity maybe useful in analog development, in testing potentialantidotes, and in examining the effects of other factorsthat might modify the pulmonary toxicity of bleomycin.
INTRODUCTION
BLM,2 a mixture of antibiotic and antineoplastic glyco-
peptides, produces only minor hematological and gastrointestinal toxicity but is limited in its clinical usefulness by adose-related pulmonary toxicity (5). This drug-induced interstitial pneumonitis and subsequent pulmonary fibrosishave been described extensively in patients and in severalanimal species, including monkeys, dogs, rats, mice, andpheasants (1-4, 8, 9, 12, 13, 17, 19-22). The incidence andseverity of BLM lung damage have been related to totaldrug dose, age of patient, and prior chest radiotherapy (5).However, systematic studies of the effects of other treatments or drugs, potential antidotes, or modifications ofdosage schedule on the development of the lesion havebeen hampered by the lack of sensitive, measurable parameters of chronic pneumotoxicity. Previous reports havebeen largely subjective descriptions of lung histopathologywithout application of morphometric techniques, such as
1 To whom requests for reprints should be addressed, at Laboratory of
Toxicology, National Cancer Institute, Building 37/5822, Bethesda, Md.20014.
' The abbreviations used are: BLM, bleomycin; OH-Pro, hydroxyproline.Received October 18, 1977; accepted December 12, 1977.
described by Weibel (23), to derive quantifiable histopatho-logical characteristics for analysis. We have been interestedin delineating and correlating such histopathological andbiochemical parameters, which could be used as indices ofBLM lung toxicity.
Previous descriptions of the sequential development ofthis lesion in mice provided the basis for our studies (1-2).Since interstitial and intraalveolar fibrosis is a prominenthistological finding after BLM treatment, we estimated totallung collagen by measuring its OH-Pro content. This wascorrelated in each animal at several time points and dosesof BLM with morphometric analysis of the following features with light microscopy: number of intraalveolar macrophages and leukocytes, total pulmonary cell density,mean alveolar wall thickness, and percentage of consolidation of lung parenchyma.
MATERIALS AND METHODS
Animals. Male NIH/Swiss albino mice weighing 20 to 25g were obtained from the NIH colony and provided withPurina rodent chow and water ad libitum.
Drug Doses and Administration. BLM was obtained fromNippon Kayaku, Inc., Lot U11AS, through the courtesy ofDr. John Douros of the Natural Products Section, Developmental Therapeutics Program, National Cancer Institute.This material is known to be a mixture of 13 relatedglycopeptides elaborated by the actinomycete Strepto-myces verticillus. BLM was administered s.c. at doses of 1,20, and 40 mg/kg twice weekly for up to 6 weeks. Controlanimals received 0.9% NaCI solution s.c. Six mice/groupwere sacrificed 2, 4, 6, and 8 weeks after initiation oftreatment for OH-Pro assay of the right lung and histopathological studies of the left lung.
OH-Pro Assay. Since lung OH-Pro is almost exclusivelyderived from collagen (7), whole lung collagen content wasestimated by measuring OH-Pro levels as described byWoessner (25). Mice were sacrificed by craniocervical dislocation. The thorax was opened, the right hilum wasligated, and the 3 right pulmonary lobes were dissected freefrom their bronchi and blood vessels and removed. The wetweights of the whole right lungs were recorded. The lungswere transferred to Pyrex hydrolysis tubes (Preiser Scientific, Inc., Charleston, W. Va.), and 5 volumes of 6-N HCIwere added. The tubes were then sealed and heated in anoven for 16 hr at 110°.The hydrolysate was titrated to pH7.0 with NaOH and diluted with glass-distilled water to afinal volume of 400 times the tissue wet weight. The dilutedhydrolysate was filtered, and 2.0-ml aliquots were assayedin duplicate for OH-Pro content. Tissue blanks consisted ofhydrolysate to which perchloric acid was added beforechloramine T.
las were connected via plastic i.v. tubing to a reservoir offixative at 30 cm of fluid pressure, as recommended byDungworth ef al. (10). A manifold attachment of 3-way
stopcocks allowed 7 mouse lungs to be perfused simultaneously with fixative. This procedure resulted in consistentexpansion and fixation of the left anterior lobe of each lungfor 1 hr at 30 cm of fluid pressure. A modified Karnovsky's
fixative, suitable for both light and electron microscopy,was used (10). After immersion in fixative for at least 48 hr,the left anterior lobes were processed, embedded in paraffin, serially sectioned (6 /¿m)in the anterior-posterior plane,
mounted on glass slides, and stained with hematoxylin andeosin, van Gieson's, and Masson's trichrome stains.
Morphometric Analysis. Based on previous descriptionsof BLM toxicity in mice (1, 2) and on our own studies, thefollowing parameters were chosen for morphometry at thelight microscopic level: number of intraalveolar macrophages and leukocytes, total pulmonary cell density, meanalveolar wall thickness, and percentage of consolidation oflung parenchyma. Each slide was projected onto a rectangular grid that represented 0.033 sq mm of actual lung areawith a Leitz-Wetzlar projection microscope, Model 396, that
produced x1100 image magnification. Three representativeareas in each lung were selected by the following criteria:only lung parenchyma with alveoli and smaller alveolarducts was included within the counting grid; larger bloodvessels, bronchioles and bronchi, and areas of dense consolidation were carefully excluded; and 1 grid was examined in each of the anterior, middle, and posterior thirds ofthe left anterior lobe. Only this lobe of the left lung wasmeasured to further standardize the measurement procedure. The width of 10 alveolar walls in each of the 3projected grids was measured so that the mean alveolarwall thickness represents the average of 30 such measurements in each lobe.
All intraalveolar macrophages and leukocytes within eachgrid and all cell nuclei in the same area, as an estimate oftotal parenchymal cell density, were counted. Because BLMdoes not produce a uniformly distributed lesion throughoutthe lung, counting 3 separate sites in each lobe resulted inless sampling error. Percentage of consolidation of eachleft anterior lobe was estimated by projecting a low-powerx70 image of each slide onto a point-counting grid system
to determine the total area of each specimen and the areaof consolidation (23). Consolidation was defined as the areaof lung parenchyma in which alveolar air space was replaced by cellular infiltrate, fluid exúdate, and/or connective tissue.
All slides were coded, randomized, and read by 2 observers without knowledge of type or duration of treatment. Thestatistical significance of the results was calculated byStudent's i test.
RESULTS
Overall Toxicity. The highest BLM dose, 40 mg/kg, pro
duced 100% mortality before the end of the 6-week treat
ment period (Chart 1). The intermediate dose, 20 mg/kg,produced 35% mortality during the 8-week course of the
experiment, while 1 mg/kg resulted in no deaths. Bodyweights show a similar pattern of no evident toxicity in thelow-dose group and progressive losses of body weight inthe 2 higher-dose groups (Chart 2).
Mice in the 2 highest-dose groups exhibited typical der-
100
50
1 mg/kg
BLEOMYCIN INJECTIONS
t t t t t t t t 1 It
0 10 20 30 40 50 60
DAYS
Chart 1. Percentage of survival of mice treated with 3 different doses ofBLM S.C., twice weekly for 6 weeks.
30
Io
20
CONTROL
1mg/kg
» 20 mg/kg
40 mg/kg
BLEOMYCIN INJECTIONSt t t t t t t t t t t t
12345678
WEEKS
Chart 2. Body weights of mice treated with 3 different doses of BLM for6 weeks. *, significantly different from control (p < 0.05).
matological toxicity, with sclerodermatous changes, patchyalopecia, and brittle nails leading to onychoptosis. Grossevaluation of tissues at necropsy revealed normal-appearing livers, kidneys, and intestinal tract in the 2 higher-dosegroups. Histological examination after 3 weeks of high-dose treatment revealed normal liver and kidneys, someflattening of small intestinal epithelium, and relatively normal bone marrow and spleen. As expected, the lungs werethe site of major pathological changes.
Lung Collagen. Wet weight of the lungs increased at 4weeks in the groups given 20 and 40 mg/kg and rose toalmost twice the control levels in surviving animals at 6 and8 weeks (Chart 3). Whole lung collagen, as measured byOH-Pro content, increased above controls at 6 and 8 weeksin the group given 20 mg/kg (Chart 4). Histological examination of lungs with van Gieson's and Masson's stains inanimals with increased lung OH-Pro content clearly revealed increased numbers of collagen bundles, most prominent in areas of consolidation. In animals with increasedlung wet weights prior to Week 6, consolidated areasconsisted mainly of interstitial and intraalveolar edema anda cellular exúdatewith macrophages and leukocytes.
Morphometric Analysis. Areas of consolidation werefound in the 20 and 40 mg/kg groups beginning at 6 and at4 weeks, respectively (Table 1). These areas ranged fromfocal lesions, frequently subpleural, to almost completeloss of recognizable alveolar architecture in moribund animals (Fig. 1). The transition from consolidated areas toareas of normal-appearing lung was characteristically abrupt, as illustrated in Fig. 3. Consolidation in the 40 mg/kggroup at 4 weeks exhibited the features of an acute inflammatory response, with large amounts of intraalveolar fluidexúdate and leukocytes, including neutrophils, lympho-
200
200
U 150
Io
0 100
1
occ
50
2468
WEEKS
Chart 3. Wet weights of whole right lungs of mice during BLM treatment.A, controls; O, 1 mg/kg; •.20 mg/kg; D, 40 mg/kg. ', significantly differentfrom control (p < 0.05). Mean ±S.E., n = 3 to 6.
oz
HIOOC
150
100
SO
2468WE:KS
Chart 4. OH-Pro contents of whole right lungs during BLM treatment. A,controls; O, 1 mg/kg; •.20 mg/kg; D, 40 mg/kg. *, significantly differentfrom control (p < 0.05). Mean ±S.E., n = 3 to 6.
Table 1Percentage ol consolidation of mouse lung after various doses of
BLMBLM was injected s.c. twice weekly for up to 6 weeks. Percent
age of consolidation was measured by projecting light microscopicslides of the left upper lobe of each animal at low power (x70)magnification onto a point-counting grid. The number of pointsincluded in consolidated areas was divided by the total number ofpoints encompassed in each lobe.
Percentage of consolidation
Week"24
68Control0
0111
mg/kg0
00020
mg/kg0
125"37»40
mg/kg2
366
" Animals were sacrificed 2, 4, 6, and 8 weeks after the initiation
of treatments.b Significantly different from controls, p < 0.05. Mean of N = 6.
cytes, and plasma cells, as well as foamy macrophages(Fig. 5). In the intermediate-dose group, 20 mg/kg, consolidation did not appear until the sixth week, with fewerneutrophils, the appearance of fibroblasts, and evidence ofscattered collagen bundles on Masson's and van Gieson's
stains.The number of intraalveolar macrophages and leukocytes
per sq mm of lung area was elevated significantly to morethan 3 times control values at 4 weeks in the high-dosegroup (Chart 5). Increases in intraalveolar cells also werepresent in the 20 mg/kg group at this time and progressedat 6 weeks. Data for 2 animals in the high-dose group thatsurvived until Week 5 are included in Charts 5 to 7. Alveolarmacrophages in toxic animals, which had abundant cytoplasm and the granular, foamy appearance of actively
phagocytosing cells (Fig. 4), were qualitatively as well asquantitatively different from macrophages of controls (Fig.
2).The total pulmonary cell count was increased in the 2
higher-dose groups, although this was significant at only 2time points because of greater variability in this parameter
aoo
55
S600
<< 400
m5
200
4
WEEKS
Chart 5. Number of intraalveolar cells per sq mm of lung tissue. Threenonconsolidated areas totaling 0.1 sq mm were counted in each left anteriorlobe. A, control; O. 1 mg/kg; •,20 mg/kg; D, 40 mg/kg. *. significantlydifferent from control (p < 0.05). Mean ±S.E., n = 3 to 6.
6000
5000z
I
< 4000
o
3000
2000
(N = 2)
WEEKS
Chart 6. Total pulmonary cell count during BLM treatment. A, control;O, 1 mg/kg; •,20 mg/kg; D, 40 mg/kg. *, significantly different, fromcontrol (p < 0.05). Mean ±S.E., n = 3 to 6.
22-
2.0
c/f 1-8
x 1.6
j u
2> 1.2
1.0-
034
WEEKS
Chart 7. Alveolar wall thickness during BLM treatment. Thirty alveolarwalls were counted in each left anterior lobe. A, control; O, 1 mg/kg; •,20mg/kg; D, 40 mg/kg. *, significantly different from control (p < 0.05). Mean
±S.E.,0 = 3 to 6.
(Chart 6). This and the other morphometric parametersexamined at the high-power x1100 magnification weremeasured in nonconsolidated areas of lung, since consolidation was considered as a separate index of toxicity (Table1). This selection of nonconsolidated areas is a measure ofpulmonary toxicity in areas where gross toxicity was notevident at low power and thus is a conservative estimate oftotal pulmonary and intraalveolar cells in animals withconsolidation.
Finally, alveolar wall thickness was significantly increased only at the most distant time point, 8 weeks, inanimals receiving BLM, 20 mg/kg. The low-dose group,which received BLM, 1 mg/kg s.c., twice weekly for 6weeks, did not differ from control animals in any of theparameters studied.
DISCUSSION
Although BLM pulmonary toxicity has been extensivelyreported and described (1-5, 8, 9, 12, 13, 17, 19-22), it hasbeen difficult to quantify in both patients and animalmodels. In this report, we have derived quantifiable indicesfor both biochemical and histopathological parameters inan experimental model of BLM lung toxicity. Animals receiving the lowest BLM dose, 1 mg/kg, demonstrated noabnormalities by toxicological, biochemical, and histopathological criteria. An intermediate dose, 20 mg/kg, resultedin the morphological appearance of chronic pulmonarytoxicity, with significant increases in lung collagen contentby 6 and 8 weeks. The high-dose group exhibited a moreacute toxicity, with changes in morphometry, but 100%lethality occurred before significant increases in lung collagen were detectable.
For reproducible development of chronic BLM lung toxicity in mice, our data suggest an optimal dose of 10 to 20
mg/kg s.c. twice weekly for 4 to 6 weeks. The s.c. route ispreferred because there is no appreciable local toxicity andbecause intraintestinal injection of BLM may occur in 10 to20% of i.p. injections of BLM in mice, with subsequentdigestion of the glycopeptide (unpublished data).
Several other biochemical approaches to evaluation ofpneumotoxicity have been attempted with pulmonary toxinsother than BLM, including studies of nucleic acid, protein,and phospholipid synthesis; mixed-function oxidation; intermediary metabolism; and lipid peroxidation (24). Elevation of lung collagen content has been described previouslywith silica administration (14), hyperbaric oxygen (6), radiation (18), and paraquat (15) but not with BLM. BLM hasbeen reported to produce increases in total phospholipidsand disaturated lecithin in the alveolar washings of miceafter 1 and 2 weeks of daily administration, but a dose-response effect and a relationship to possible chronictoxicity were not studied (2).
Morphometric studies of lung have been reported withoxygen toxicity and experimental pulmonary calcinosis (11,16). Unless techniques of automated image analysis become more available on a routine basis, quantitative evaluations will remain rather painstakingly slow and tedious.Maintenance of stringently controlled conditions in preparation of tissues is also essential, since the type of fixative,the degree of lung expansion, and variation in thickness oftissue sections may drastically alter measurements of numbers of cells and anatomical architecture.
Studies are currently underway in our laboratory on theeffects of schedule of administration of BLM, as well as onpotential antidotes and modifiers, with the parameters presented in this paper as indices of lung toxicity.
ACKNOWLEDGMENTS
We thank Randall Waring, Tammy Schanwald, and Chris Armstrong fortheir enthusiastic and capable technical support and Gail Johnston forassistance in preparation of the paper.
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Fig. 1. Low-power views of cross-sections of the left anterior lobes of mouse lungs, as used for measurement of percentage of consolidation byprojection on a point grid, a, control animal, 0% consolidation; b. BLM, 20 mg/kg, for 6 weeks, 21%; c, BLM, 20 mg/kg, for 8 weeks, 5%; d, BLM, 40 mg/kg,for 4 weeks, 89%. H & E, x 8.
Fig. 2. Normal lung architecture including intraalveolar macrophages (arrows) from a control animal. H & E, x 350.Fig. 3. Transition from an area of consolidation to an area of relatively normal lung in a mouse receiving BLM, 20 mg/kg, for 6 weeks. Significant
increases in numbers of intraalveolar macrophages and leukocytes were present in nonconsolidated areas at this time point, 6 weeks. H & E, x 54.Fig. 4. Area of consolidation in an animal receiving BLM, 20 mg/kg, for 6 weeks, showing large numbers of intraalveolar macrophages (arrow) and
leukocytes and proteinaceous exúdate.H & E, x 350.Fig. 5. Area of consolidation in an animal receiving BLM, 40 mg/kg, for 4 weeks, showing almost complete loss of alveolar architecture with large
amounts of fluid and inflammatory cell exúdate.H & E, x 54.
1978;38:787-792. Cancer Res Branimir Ivan Sikic, David M. Young, Edward G. Mimnaugh, et al. HistopathologyChanges in Lung Hydroxyproline Content and Morphometric Quantification of Bleomycin Pulmonary Toxicity in Mice by
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