Europ. J. Cancer Vol. 12, pp. 363-370. Pergamon Press 1976. Printed in Great Britain

The Effects of Dexamethasone on Mouse Skin Initiation and Aryl Hydrocarbon Hydroxylase*

SARA THOMPSON and THOMAS J. SLAGA

Fred Hutchinson Cancer Research Center, 1102 Columbia Street, Seattle, Washington 98104, U.S.A.

Abstract--Mouse epidermis contains a NADPH dependent aryl hydrocarbon hydroxy- lase (AHH), which is inducible by polycydic hydrocarbons. The effects of dexametha- sone, an anti-inflammatory steroid, on skin carcinogenesis and on mouse epidermal AHH were investigated. Dexamethasone was found to reduce tumor initiation by both 3-methyl- cholanthrene and 7,12-dimethylbenz(a)anthracene. Dexamethason~ applied topically to mice at a dose level of 25 or 75 pg slightly stimulated the AHH activity in mouse epidermis with peaks of 150 or 180% of controls, respectively, at 2 hr and a return to control level by 4 hr. This was followed by a weak inhibition between 4 and 8 hr after treatment with either dose of dexamethasone. Dexamethasone did not inhibit benz(a)- anthracene-induced AHH activity in vitro when added to the assay, even at concentra- tions greater than equimolar amounts to benzo(a)pyrene. However, i f dexamethasone or fluocinolone acetonide were applied topically with 3-methyeholanthrene (MC), they inhibited the MC induction of AHH by at least 50% between 4 and 18 hr after simul- taneous treatment. 5"he more potent anti-inflammatory steroids, fluocinolone acetonide and fluclorolone acetonide, had a greater inhibitory effect on MC induction of AHH after simultaneous t~,eatment. The mouse skin tumor promoters, croton oil and 12-0- tetradecanoylphorbol-13-acetate, had only a weak stimulatory effect on the AHH activity. The mechanism by which the anti-inflammatory steroids inhibit tumor initiation may be related to their ability to inhibit the metabolism of polycydic hydrocarbons to their ultimate carcinogenic form(s).

INTRODUCTION

IT HAS been shown by Belman and Troll [1] that a series of steroidal anti-inflammatory agents inhibited, in a dose dependent manner, tumor promotion by croton oil in mouse skin (dexamethasone > Schering No. 11572 > pred- nesolone > hydrocortisone > cortisone) which correlated with their anti-inflammatory ac- tivities in mouse skin. They also showed that dexamethasone inhibited croton oil induced hyperplasia. Nakai [2] also showed that MC- induced sarcomas in mice was inhibited by steroids in the order dexamethasone > triamci- nolone > methyl prednesolone > hydrocorti- sone > cortisone and that this correlated with their anti-inflaramatory potencies. We later showed that dexamethasone could completely

Accepted 22 October 1975. *This work was supported in part by grant CA-13155 from the National[ Cancer Institute of NIH.

363

suppress tumor promotion by 12-0-tetradeca- noylphorbol-13-acetate (TPA) for 6 months with a judicious choice of doses [3]. Recently, dexamethasone was found to reduce tumor initiation and complete carcinogenesis by MC- [4]. It also inhibited mouse epidermal DNA synthesis [5] and binding of radioactive phorbol esters to a cytosol protein and chromatin [6]. Dexamethasone decreased TPA-induced hyper- plasia and also reduced several protein fractions of epidermal cytosol as revealed by polyacryl- amide gels which were greatly enhanced after TPA treatment [6].

If a common mechanism exists for the inhibitory effect of dexamethasone on tumor initiation, promotion and complete carcino- genesis, it may be related to an alteration in the metabolism of the carcinogen and tumor promoter by the mixed-function oxidase system. In order to test this possibility, the effects of dexamethasone on epidermal aryl hydrocarbon hydroxylase (AHH) were investigated.

364 Sara Thompson and Thomas J. Slaga

M A T E R I A L A N D M E T H O D S

Animals

Female Charles River CD-1 mice were purchased from Charles River Mouse Farms, North Wilmington, Mass. Mice 7-9 weeks old were carefully shaved with surgical clippers 2 days before treatment and only those mice in the resting phase of the hair cycle were used in biochemical and tumor experiments. Groups of 30 mice were used in the tumor experiments. The incidence of both papillomas and carci- nomas were recorded weekly and at random papillomas and carcinomas were removed for histological verification.

Chemicals

Benz(a) anthracene (BA), benz(a) pyrene (BP) and 7,8-benzoflavone (7,8-BF) were obtained from Aldrich Chemical Company, Inc., Milwaukee, Wisconsin. 3-Methycholan- threne (MC) was purchased from J. T. Baker~ Philipsburg, New Jersey, and dexamethasone and 7,12-dimethylbenz (a) anthracene (DMBA) from Sigma Chemical Company, St. Louis, Missouri. Croton oil was obtained from S. B. Penick and Company, New York, New York. 12-0-Tetradecanoylphorbol-13-acetate (TPA) was prepared as previously described [7] and purified by preparative thin layer chromato- graphy. 3-Hydroxybenzo(a) pyrene was a generous gift from Dr. H. V. Gelboin, National Cancer Institute, Bethesda, Maryland. The anti-inflammatory steroids, fluocinolone aceto- nide and fluclorolone acetonide* were generous- ly supplied by Syntex Corporation, Palo Alto, California. All compounds were applied topically in acetone. In experiments where both a carcinogen and dexamethasone were given together, we applied them simultaneously in acetone. Similar results were obtained if dexamethasone was given 5 min before the carcinogen.

AHH enzyme assay

A modification of an assay described by Bowden et al. was used [8]. Three mice were used for each enzyme assay. Mice were killed between 8:00 and 10:00 a.m. by cervical dis-

*Fluocinolone acetonide, 6a¢, 9~-difluoro-llp, 16~, 17, 21-tetrahydroxypregnan-l,4-diene-3, 20-dione, eyelle

16,17-aeetal; fluelorolone acetonide, 6~-fluoro-9~, llfl-diehloro-16~, 17~, 21-triol-pregnan-l,4-diene-3, 20-dione~ cyclic 16,17-acetal.

location, and Nudit cream (generously supplied by Helena Rubinstein, Inc.) was applied to the shaved area of the back. After 5 rain the Nudit cream was thoroughly washed off under cold running water and the skins were removed and placed on ice. The whole skin was placed dermis side down on a cold glass plate and the epidermis was scraped off with fifteen strokes of a razor blade and placed in 1 ml of a 0"25M sucrose-0.05M Tris buffer pH 7-5. The epider- mal material from three mice was homogenized with a Polytron PT10 homogenizer for 45 see at setting "6". This epidermal homogenate was the source of enzyme for the majority of AHH assays.

The assay was performed in semi-darkness in a total volume of 1 ml which contained 50 /~moles Tris-HC1 buffer pH 7.5, 0.5 mg of NADPH, 3/~moles MgC12, 0.2 ml skin homo- genate (containing 2-4 mg protein) and 100 nmoles of BP (added in 40 ttl of acetone just prior to starting the incubation). The reaction mixture was incubated in the air at 37°C for 30 min. The reaction was stopped by adding one ml of acetone and placing the reaction flask on ice. Next, 3-25 ml of n-hexane was added to the flask. The flasks were returned to the 37°C incubator for 10 min with vigorous shaking and then a 2"5 ml aliquot of the organic phase was extracted with 2"5 ml of 1.0N NaOH by vigorous vortexing for 1 rain. The concen- tration of the extracted phenolic derivatives in the alkaline phase was determined spectro- photofluorometrically with excitation at 396 nm and emission at 522 nm in a Turner spectrofluorometer. The spectrofluorometer was calibrated with an authentic sample of 3- hydroxybenzo(a)pyrene. The enzyme units are defined as the pmoles equivalent to the fluo- rescence of 3-hydroxybenzo (a) pyrene formed in 30 min of incubation. Enzyme activity was determined in duplicate and compared with a blank control flask to which acetone had been added to incubation. The protein content of the homogenates was determined by the Lowry method using bovine serum albumin as a standard.

The specific activity is expressed as pmoles of 3-hydroxybenzo(a)pyrene formed in 30 min of incubation per mg of protein. In most cases, the specific activities are expressed in the charts as a percentage of the acetone control groups and each time point tested represents an average of two to five groups containing three mice each. The average percentage standard devia- tion for the control groups for each experiment is plotted on each chart to indicate the extent of variation in the data.

Dexamethasone and Aty l Hydrocarbon Hydroxylase 365

R E S U L T S

Isolated epidermis has 4-5 times the AHH activity as the remaining dermis and about twice that of whole skin.* It was found that 15 strokes of a razor blade, if preceded by Nudit cream depilatory agent, was sufficient to remove all the epidermal layer without much dermal contamination. Additional scraping only added dermal material to the preparation. Without Nudit cream, no consistent separation of the epidermis from the dermis could be obtained.

Figure 1 shows the effect of a single topical application of dexamethasone on tumor initia-

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I I I I I I I 12 16 20 24 28 32 36 40 44

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Fig. 1. Inhibition of MC tumor initiation by 75 I~g of dexamethasone. Each group consisted of 30 mice. All animals were initiated with 13.4 #g MC and promoted with 10/lg TPA twice weekly. Dexamethasone was given once simulta- neously with MC initiation. Control (MC) 0 ©;

dexamethasone + MC • • .

tion by MC. Dexamethasone caused at least a 50% reduction in both the percentage of animals with tumors and the number of papillomas per mouse. In the control mice, initiated and promoted only, 43% of the surviving mice developed carcinomas after 44 weeks, whereas only one out of 30 mice devel- oped a carcinoma in the dexamethasone treated mice alter 44 weeks. Figure 2 shows a similar inhibitory effect when a single topical

*S. Thompson and T. J. Slaga: Mouse Epidermal Aryl Hydrocarbon Hydroxylase. Submitted to J. Investigative Dermatology.

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= 50

6 I0 14 18 22 26 30

W e e k s of p r o m o t i o n

Fig. 2. Inhibition of DMBA tumor initiation by 75 ltg of dexamethasone. Each group consisted of 30 mice. All animals were initiated with 2.5 pg DMBA and promoted with 10 pg TPA twice weekly. Dexamethasone was given once simulta- neously with DMBA initiation. Control (DMBA) • • ;

dexamethasone + DMBA © - - - - O.

application of dexamethasone was given with DMBA initiation.

Since dexamethasone is so effective in inhibiting polycyclic hydrocarbon carcinogen- esis, as well as phorbol ester promotion in mouse skin, it was decided to study the effect of dexamethasone on AHH activity. Figure 3 shows that dexamethasone, when applied topically to mice, slightly stimulated the AHH

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Fig. 3. Time course of A H H activity in mouse epidermis after a single topical application of either 25 (O) or 75 pg ( 0 ) of dexamethasone. The epidermis was isolated by 15 strokes of a razor blade. The specific activity is defined as pmoles of 3-OH-BP[mg protein~30 rain. The specific activity is expressed as a percentage of the acetone control groups and each time point tested represents an average of 2-5 groups containing 3 mice each. The average percentage standard deviation for the control groups (dashed line) is plotted on the

figure to indicate the extent of variation in the data.

366 Sara Thompson and Thomas J . Slaga

specific activity in mouse epidermis with a peak of 180% of controls at 2 hr for the higher dose and then re turning to control level by 4 hr. A similar response was observed when dexa- methasone was applied topically at a dose of 25 pg. The in vitro effect of various concentra- tions of dexamethasone and fluocinolone acetonide on BA-induced mouse epidermal A H H activity was also determined. Dexame- thasone did not inhibit the in vitro A H H activity when added directly to the incubat ion tubes even at concentrat ions greater than equimolar amounts to the substrate, BP (Table 1).

Table 1. In vitro effect of dexamethasone, fluocinolone acetonide and 7,8-BF on A H H activity from mouse epi- dermis. The mice were pretreated topically with 0'5 mg

of BA 17 hr before being sacrificed. *

In vitro BA-Induced addition]" Concentration (SA % of Controls)

Dexamethasone 2.55 x 10-6 93 2.55 x 10 -5 99 6.37 x 10-s 105 1.27 x 10 -4 96 2-55 x 10 -4 90

Fluocinolone 10 - 4 74 aeetonide 2 x 10 .4 56

7,8-BF 10- 4 8 2 x 10 -4 4

*The enzyme source for the assays was epidermal homogenate and the substrate concentration was 10-4 M benzo(a)pyrene (BP). The average SA for the BA-treated mice was 356pmoles of 3-OH-BP formed/rag of protein.

"['The compounds in acetone were added directly to the incubation tubes at the concentrations as indicated. Each value represents an average of at least 2 experi- ments with duplicate determinations for each experiment.

Similar in vitro results were obtained with dexamethasone when mouse epidermal A H H was not induced or was induced by MC. Fluocinolone acetonide had a mild inhibi tory effect on the in vitro A H H activity.

However , 7,8-BF is a very effective inhibitor o f A H H activity (96% inhibition) when added to the incubat ion mixture as was also reported by others [8, 9]. Table 2 depicts the in vitro effect of various concentrat ions o f steroids wi thout ant i - inf lammatory ability on BA- induced mouse epidermal A H H activity. All o f the steroids tested inhibited to various degrees the in vitro A H H activity when added directly to the incubat ion tubes. Estradiol

Table 2. In vitro effect of a series of steroids on A H H activity from mouse epidermis. The mice were pretreated topically with 0'5 mg of BA 17 hr before being sacrificed.*

In v i t ro Concentration BA-induced additionJ" (M) (SA % of Control)

estrone

estradiol

estriol

testosterone

progesterone

10 -4 8.9 2 x 10 -4 11.9

10 - s 8-8 10 -4 6.0

2 x 10 -4 4-8 10 -4 19.1

2 x 10 -4 12.1 10- s 60"0 10 -4 48.2

2 x 10 -4 25.0 10 -4 52.1

2 x 10 -4 30.0

*The enzyme source for the assays was epidermal homogenate and the substrate concentration was 10 -4 M benzo(a)pyrene (BP). The average SA for the BA-treated mice was 356 pmoles of 3-OH-BP formed/rag of protein.

J'The steroids in acetone were added directly to the incubation tubes.

inhibited the in vitro A H H activity to a com- parable level as shown for 7,8-BF in Tab le 1.

Figure 4 reveals the interesting results that

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Fig. 4. The effect of simultaneous treatment of 20pg estradiol, 20lag testosterone, or 2"7lag 7,8-BF on DMBA tumor initiation. Each group consisted of 30 mice and all animals were initiated with 2.56 lag of DMBA and promoted with 10lag of TPA twice weekly. Control (DMBA) 0 0 ; DMBA + 7,8-BF 0 - - 0 ; DMBA +estradiol

A Zk ; and DMBA + testosterone • • .

Dexamethasone and Aryl Hydrocarbon Hydroxylase 367

when either estradiol or testosterone were given simultaneously with DMBA they did not have any effect on DMBA tumor initiation, whereas, 7,8-BF at a concentration less than that used for the steroids was a potent inhibitor of DMBA tumor initiation.

Figure 5 depicts the inducibility of epidermal A H H by 200 nmoles of MC applied topically

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Fig. 5. Time coun'e of A H H activity in mouse epidermis after a single topical application of either 200 nmoles of MG ( 0 ) or 200 nmoles of MCplus 75 ltg of dexamethasone ( 0 ) . See Fig. 3 for details. Each time point tested represents an average of 3-5 groups containing 3 mice each. The average percentage standard deviation for the control groups (dashed line) is plotted on ttw figure to indicate the extent of variation

in the data.

and the effect of simultaneous dexamethasone treatment on A H H induction by MC. By 8 hr after MC treatment , there was about a 13-fold increase in A H H activity and greater than a 4-fold increase 36 hr after MC. Simultaneous treatment of 75 pg of dexamethasone in vivo inhibited the MC induction of A H H by more

than 50% for the first 18 hr after treatment. Dexamethasone at a dose level of 75 #g had a much greater inhibitory effect on MC induc- tion of A H H by 50 nmoles which was the same dose used in the tumor initiation experiments. Similar results were obtained when dexame- thasone was given simultaneously with 200 nmoles DMBA in vivo. However, DMBA had less of an inducing effect on epidermal A H H than MC. We found that 10 gg of fluocinolone acetonide also inhibited MC-induced A H H activity, but for a longer period than 75 #g of dexamethasone (Table 3). Fluclorolone aceto- nide and fluocinolone acetonide at dose levels of 10 and 1 pg, respectively, also had an inhibitory action on MC-induced epidermal A H H activity, but less than that of 10 gg of fluocinolone acetonide (Table 3). Recently, fluocinolone acetonide was found to be at least 10 times more active in inhibiting TPA- induced hyperplasia and TPA tumor promotion than dexamethasone.*

The possibility was entertained that croton oil and tumor promoting phorbol esters may have an inducing effect on A H H activity and that dexamethasone may have a counteracting effect on the induction. Figure 6 reveals that croton oil and TPA had a minor effect on A H H activity in mouse epidermis when com- pared to that of polycyclic hydrocarbons. Both compounds gave a reproducible but small increase in epidermal A H H specific activity 12 and 24 hr after treatment. Because of the minor

*The inducibility of epidermal A H H by 200 nmoles of MC appears different when comparing the results in Fig. 5 and Table 3 because of the way we expressed the data. In Fig. 5, the A H H specific activity is expressed as a percentage of the acetone controls, whereas, in Table 3 it is not.

Table 3. The effect of steroidal anti-inflammatory agents on the induction of aryl hydrocarbon hydroxylase by 200 nmoles of 3-methylcholanthrene ( MC) in mouse epidermis

Compounds given simultaneously with MC (Specific Activity expressed as percent of MC treated mice.)

Time after SA after Treatment (hr) MC only* 10/tg F.A. 1 pg F.A. 10 pg F.C.

2 44 206 6 126 83 115 82

12 349 39 75 79 18 274 71 79 55 24 353 50 112 115 36 253 58 73

*The specific activity is expressed as pmoles of 3 - - O H - - B P formed/rag of epi- dermal protein/30 rain. The average specific activity of un-induced epidermis is 40.8.

368 Sara Thompson and Thomas J. Slaga

inducing effect of croton oil and TPA on epidermal A H H activity, the effect of dexame- thasone on the induction would be without meaning.

24C

20C

16C

I 1 4 8

V I I I I I 1 I

12 16 20 24 28 32.

hr after treatment

Fig. 6. Time course of A H H activity in mouse epidermis after a single topical application of either 1 mg of croton oil ( 0 ) or 5 ltg of TPA ( O). See legend to Fig. 3 for details. Each time point tested represents an average of 2 groups containing 3 mice each. The average percentage standard deviation for the control groups (dashed line) is plotted on the figure to indicate the extent of variation in the data.

D I S C U S S I O N

Our results show that dexamethasone, a potent inhibitor of chemical carcinogenesis in mouse skin, has only a slight inducing effect on epidermal AHH activity, which occurs at 2 hr. Most polycyclic hydrocarbon carcinogens in- duce a several fold increase in AHH activity which lasts for at least 24 hr [8, 10]. Dexa- methasone did not inhibit the in vitro AHH activity when added directly to the incubation tubes even at concentrations greater than equimolar amounts to the substrate, BP. Briggs and Briggs [11] also showed that several synthetic steroids did not inhibit the epidermal AHH activity when added to the in vitro assay at concentrations equimolar to BP. Hydro- cortisone, cortisone and dexamethasone were reported to have no effect on the AHH activity in hamster fetal cell culture [12]. However, Briggs and Briggs [11] reported that certain synthetic corticosteroids applied topically in ointment twice daily for 7 days induced AHH activity in mouse epidermis comparable to that of MC. There are several differences in experimental design that could account for the lack of agreement in results. First, they applied their steroids in ointment, whereas we applied ours in acetone. Second, they applied the steroids twice daily for 7 days with a total dose ranging from approximately 2 to 35 mg, which

may have had a systemic effect, whereas, we applied dexamethasone once at a dose of 25 or 75 pg. Finally, we used female mice in our experiments and they used male mice.

Several investigators have shown that steroid- al anti-inflammatory agents can reduce the tumor response to mouse skin promoters [1, 13, 14]. With a judicious choice of doses, Scribner and Slaga [3] reported a complete suppression of TPA tumor promotion by dexamethasone for 6 months. Another report by the above authors showed that dexamethasone reduced tumor initiation and complete carcinogenesis by MC [4]. In this report, both DMBA and MC tumor initiation were inhibited by dexa- methasone. Cortisone also was reported to reduce mouse skin tumors by the complete carcinogens, BP [13], MC [15] and DMBA [16]. A recent study showed that certain non- steroidal anti-inflammatory agents were partially effective in decreasing the promoting ability of TPA [4]. However, there was no correlation between their effects on tumorigene- sis and on inflammation-hyperplasia [5, 17].

The slight induction of epidermal AHH by dexamethasone in vivo and the lack of inhibition by dexamethasone when added directly to the in vitro A H H assay was at first discouraging because we hypothesized that the inhibitory effect of dexamethasone on polycyclic hydro- carbon carcinogenesis was through its effect on the induction of AHH in mouse epidermis. However, we found that dexamethasone if applied simultaneously with MC inhibits the MC induction of AHH by approximately 61% at 12hr after treatment (Fig. 4). Similar results were obtained with two new anti- in fammatory steroids (Table 3). It is possible that simultaneous dexamethasone treatment inhibits the metabolism of MC to an ultimate carcinogenic form; such a mechanism would explain its action on polycyclic hydrocarbon initiation and complete carcinogenesis. In a preliminary experiment, we found that micro- some dependent covalent binding of MC to DNA in vitro was decreased when epidermal microsomes were isolated from mice pretreated with dexamethasone and MC 12 hr before sacrificing as compared to MC pretreated mice.

We considered the possibility that croton oil and tumor promoting phorbol esters may have an inducing effect on AHH activity and that dexamethasone may have an inhibitory effect on the induction. Figure 5 reveals that the tumor promoting agents, croton oil and TPA, had a minor but reproducible stimulatory effect on AHH activity in mouse epidermis. Since croton oil and TPA had such a slight

Dexamethasone and Aryl Hydrocarbon Hydroxylase 369

effect on A H H activity, the effect of dexa- methasone on the induction of A H H by tumor promoters would be without meaning. The possibility still exists that esterase activity could be affected by dexamethasone which could explain the inhibition of tumor promotion and the bindi~ag of tumor promoters to a cytosol receptor protein and chromatin by simultaneous dexamethasone treatment [3, 6]. Kreibich et al. [18, 19] reported that tumor promoting phorbol esters are poorly metabol- ized by mouse skin and Hela ceils, but are metabolized to a considerable extent in cultures of L-cells where phorbol esters do not block DNA synthesis or stimulate choline incorpora- tion into phospholipids which showed an excellent correlation with the promoting ability of a series of phorbol esters [20, 21].

The mechanism by which dexamethasone and other glucocorticoids inhibit mouse skin tumor promotion and complete carcinogenesis may be related to their ability to inhibit DNA synthesis which consequently counteracts the induction of hyperplasia induced by tumor promoters and complete carcinogens [5]. We recently found that dexamethasone also inhi- bits TPA-induced increase in DNA synthesis. In preliminary experiments, we have found that fluocinolone acetonide is at least 10 times more active than dexamethasone in inhibiting DNA synthesis, counteracting TPA-induced increase in DNA synthesis and epidermal hyper- plasia and in inhibiting the induction of epidermal A H H by MC. Also, our results indicate that there is excellent correlation

between the promoting ability of a series of phorbol esters and their ability to induce epidermal hyperplasia. The correlation com- pletely fails when other compounds are com- pared outside the phorbol ester series [22]. Furthermore, the anti-inflammatory ability of a series of steroids was correlated with their ability to inhibit mouse skin tumor promotion by TPA and their ability to counteract TPA- induced hyperplasia (fluocinolone acetonide > fluocinonide ~ dexamethasone >> cortisol) [23]. Once again, the correlation fails when com- paring non-steroidal anti-inflammatory agents [24]; Belman and Troll [1] also showed that a series of steroidal anti-inflammatory agents inhibited tumor promotion by croton oil in mouse skin. They also showed that the steroids they tested were truly anti-inflammatory to mouse skin, which we also showed for the steroids we tested using their criteria of inflam- mation [1, 24].

The effect of dexamethasone on tumor initiation by polycyclic hydrocarbons [4] is another point at issue. Low initiating doses of polycyclic hydrocarbons have been shown to inhibit DNA synthesis without subsequent inflammation or hyperplasia [5]. As stated earlier, we have found that dexamethasone and other powerful anti-inflammatory steroids if applied simultaneously with MC, inhibit the MC induction of AHH. The major effect of the anti-inflammatory steroids on tumor initiation may be related to their inhibiting the metabolism of MC to an ultimate carcinogenic form, which we are currently investigating.

REFERENCES

l. S. BELMAN and W. TROLL, The inhibition of croton oil-promoted mouse skin tumorigenesis by steroid hormones. Cancer Res. 32, 450 (1972).

2. T. NAKm, Influences of small doses of various corticosteroids on the incidence of chemically induced subcutaneous sarcomas in mice. Cancer Res. 21, 221 (1961).

3. J .D. SCRmNER and T. J. SLAOA, Multiple effects ofdexamethasone on protein synthesis and hyperplasia caused by a tumor promoter. Cancer Res. 33~ 542 (1973).

4. T.J . SLAOA and J. D. SCRmNER, Inhibition of tumor initiation and promotion by anti-inflammatory agents. J. nat. Cancer Inst. 51, 1723 (1973).

5. T . J . SLAOA, S. THOMPSON and E. A. SMUCKLER, Prolonged inhibition of mouse epidermal DNA synthesis by dexamethasone. 3". nat. Cancer Inst. 54, 931 (1975).

6. T.J . SLAOA, J. D. 8CRIENER, J. M. RICE, S. B. DAS and S. THOMPSON, Inhibi- tion by dexamethasone of intracellular binding of phorbol esters to mouse skin. J. nat. Cancer Inst. 52, 1611 (1974).

7. W . M . BAIRD and R. K. BOUTWELL, Tumor-promoting activity of phorbol and four diesters ofphorbol in mouse skin. Cancer Res. 31, 1074 (1971).

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370 Sara Thompson and Thomas J. Slaga

9. H . V . GELBOIN, F. J. WIEBEL and L. DIAMOND, Dimethylbenzanthracene tumorigenesis and aryl hydrocarbon hydroxylase in mouse skin. Inhibition by 7,8-benzoflavone. Science 170, 169 (1970).

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11. M . H . BRIGGS and M. BRIOGS, Induction by topical corticosteroids of skin enzymes metabolizing carcinogenic hydrocarbons. Brit. J. Dermatol 88, 75 (1973).

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14. N. TRAININ, Adrenal imbalances in mouse skin carcinogenesis. Cancer Res. 23, 415 (1963).

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19. G. KREIBICH, R. Sfiss and V. KINZ~L, On the biochemical mechanism of tumorigenesis in mouse skin V. Studies of the metabolism of tumor promoting phorbol derivatives in vivo and in vitro. Z. Krebsforsch 81, 135 (1974).

20. R. Stiss, V. KINSFL and G. KREIBICH, Cocarcinogenic croton oil factor A. Stimulates lipid synthesis in cell cultures. Experientia (Basel) 27, 46 (197 I).

21. lZ. Sfiss, G. KREmlCH and V. KINSF.L, Phorbol esters as a tool in cell research. Europ. or. Cancer 8, 299 (1972).

22. T . J . SLAOA, G. T. BOWDEN and R. K. BOUTW~LL, Acetic acid, a potent stimulator of mouse epidermal macromolecular synthesis and hyperplasia but with weak tumor-promoting ability, or. nat. Cancer Inst. (In press).

23. T . J . SLAOA, S. THOMPSON and J. A. SCHWARZ, unpublished results. 24. T . J . SLAGA, Inflammation, hyperplasia and tumor promotion, to be sub-

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