the influence of stilbestrol on skin tumors induced in ... · memorial institute, 1 was a smoke...

The Influence of Stilbestrol on Skin Tumors Induced in Mice with Tobacco Tar and Methylcholanthrene*

DONALD J. SVOBODA

(Department of Pathology and Oncology, University o] Kansas Medical Center, Kansas City, Kan.)

SUMMARY Experiments were carried out on 400 Swiss female mice to determine the effect of

continuous, long-term absorption of stilbestrol on the incidence of skin tumors induced with 3-methylcholanthrene and the neutral fraction of tobacco tar. The incidence of carcinoma in the methylcholanthrene-plus-stilbestrol group was 39 per cent, whereas in the methylcholanthrene-only group the incidence was 17 per cent. Induction times in the two groups were similar. There was no enhancement of malignant tumor formation by stilbestrol among the animals painted with tobacco tar. After 15 months of application there were nine carcinomas among mice given tobacco tar only. Four papillomas occurred in the tobacco tar-plus-stilbestrol group. Keratoacanthomas were observed in several animals.

Evidence for the controversial reasons for sex differences in human neoplasms, such as epider- moid carcinomas of skin and lung, has come largely from statistical observations. I t is of interest, therefore, to study with controlled experiments one possible factor, female sex hormones, and their relation to experimental skin tumor formation.

The work of Paletta and Max (18), Gilmour (10), and Marchant (14) indicates enhancement of induction of skin tumors in mice with various estrogenic substances. In contrast, the studies of Perry and Gintzon (19), Segaloff (20), Smith, Wells, and D'Amour (21), and Stewart ( ~ ) in- dicate no enhancement by estrogens of carcinogenic agents.

Since methylcholanthrene is a strong carcinogen, it was felt that the anti- or co-carcinogenic effect of stilbestrol might not be apparent unless it is" quite marked. Therefore, the comparatively weaker carcinogen, the neutral fraction of tobacco tar, was also used in this study. The experience with tobacco tar, in relation to sex hormones, has been somewhat limited.

Enge lbre th-Holm (6) and Wynder (26) found the incidence of skin tumors in mice treated with tobacco tar to be virtually equal in both sexes.

* This study was supported by U.S.P.H.S. grant nos. CRT- 5006 and CS.9441.

Received for publication January 13, 1961.

MATERIALS AND M E T H O D S Five hundred Swiss female mice, 10-12 weeks

old, were divided and treated as follows: (a) 100 with methylcholanthrene, (b) 100 with methylcholanthrene plus stilbestrol, (c) 100 with tobacco tar, (d) 100 with tobacco tar plus stilbestrol, and (e) 100 untreated controls, The animals were obtained from the Dan Rolfsmeyer Co. of Madison, Wisconsin. They were individually identified by ear puncture.

Methylcholanthrene was applied to the backs of mice once a week for 5 weeks as a 0.6 per cent solution in benzene. This concentration and the solvent have been shown by Stowell and Cramer to evoke a tumor response in a significant percentage of Swiss mice by the 19th week after application (24). The estrogen was administered as 4-rag. pellets of 25 per cent stilbestrol in choles- terol, inserted subcutaneously in the left axilla. Preliminary experiments with subcutaneous in- jections of stilbestrol in oil demonstrated that this method of hormone administration was unsatis- factory because it was impossible to prevent leak- age of small amounts of the mixture from the needle puncture site. Furthermore, with subcutaneous injection of the hormone in oil, there was a 40 per cent mortality within the first 3 months of the experiment. This high death rate may have been due to too rapid or erratic absorption of stil-

993

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994 Cancer Research Vol. s S e p t e m b e r 1961

bestrol from the subcutaneous site. The degree of vaginal cornification as seen on serial Papanico- laou smears was used as an indication of continuous estrogen effect. In addition, squamous meta- plasia of the endometrium was noted at autopsy. To insure tha t the pellets were still hormonally active at the conclusion of the experiment, they were removed from the original mice and re-inserted into the left axillae of oophorectomized animals. Repeated Papanicolaou smears from the second host females revealed a consistent preponderance of cornified cells.

The tobacco tar, obtained from Roswell Park Memorial Institute, 1 was a smoke condensate pre- pared from three popular brands of 70-mm. cig- arettes. The smoking machine, from which this tar was obtained, was designed to take ten puffs from each cigarette at 1-minute intervals. Each puff was of ~ seconds' duration and ~ ml. in vol- ume, drawn by a pressure differential of 11-~ inches of water. The but t lengths were ~ mm. long. The smoke was collected in a glass trap sys- teln cooled to - 8 0 ~ C. The condensate was collected by washing the traps with acetone and con- densing the acetone solution under reduced pressure. The condensate was redissolved in acetone a n d treated with 1 N H2SO4 until a few drops of solution in a small amount of water gave a pH of less than 4. The aqueous-acetone solution was extracted twice with benzene. The combined benzene extracts were washed with water until the washings showed no further acid reaction. The benzene solution was then condensed under reduced pressure. The nicotine-free, neutral fraction was used approximately 1 week after its production and was administered as a 50 per cent solution in benzene applied 3 times per week for 1~ months to the backs of mice in approximately 40-mg. doses. Between paintings, tar was stored in brown bottles in a refrigerator at 4 ~ C. With the tar preparation used, 1 gm. of tar represented the extract from approximately one carton of ciga- rettes.

Both methylcholanthrene and tobacco tar were applied with a no. 5 camel's hair brush. Several brush strokes were applied from nape to tail and over the entire width of the back. Animals were shaved with electric clippers at the beginning of the experiment and at ter subsequent regrowth of hair.

Because of the sex differences in skin of mice (3) (epidermal thickness and collagen content) probably not directly related only to sex hormones, only females were used. Animals were not oophorectomized, since only normal estrogen to hyper- estrogen states were compared.

1 Personal communication.

The tumor incidence in the methylcholanthrene treated animals was constant after 5 months. Tumor-free animals were observed for an addi- tional 7 months, during which time no new tumors appeared, nor did already formed carcinomas re- gress. The animals treated with tobacco tar were observed for a total of 15 months. In all groups, animals without tumors were autopsied at the end of the period of observation. Tumors were diag- nosed grossly as malignant when they became ulcerated and fixed to underlying tissues and were no longer superficial papillomatous growths. All tumor designations were confirmed histologically. Preliminary tumor induction experiments with methylcholanthrene were performed to study gross and microscopic features of skin tumors from the time of earliest appearance to the inception of malignancy. The gross characteristics of skin tumors, particularly changes indicating malignant transformation, were assessed by two observers and correlated with histologic study.

RESULTS

There was no mortali ty from stilbestrol or tobacco tar preparations.

The number of tumors in both groups of animals receiving methylcholanthrene was tabulated at weekly intervals until ~10 days after the first painting. At 154 days, the number of animals with tumors, both benign and malignant, was constant and remained so thereafter. The percentage of animals bearing tumors--papi l loma and carci- n o m a - i n the methylcholanthrene-plus stilbestrol and the methylcholanthrene-only group was approximately 70 per cent and 80 per cent, respectively (S.D. = 5.3) (Chart 1).

Animals with papillomas were approximately twice as numerous in the group treated only with methylcholanthrene, in which the incidence was 65 per cent, compared with 36 per cent of the animals of the methylcholanthrene-plus-stilbestrol group (S.D. = 3.5) (Chart ~). In contrast, the incidence of animals with carcinoma in the stilbestrol-treated group was approximately twice tha t of the methylcholanthrene-only group--39 and 17, respectively (S.D. = 1.7).

The number of animals with tumors in the groups of animals receiving tobacco tar was re- corded at monthly intervals until 15 months after the beginning of the experiment. The results of painting tobacco tar were in marked contrast to the studies with methylcholanthrene (Chart 3). In the tobacco tar groups, the first papillomas appeared at 7 months, and the number of mice with tumors increased to the maximum, sixteen, at 15 months. The first carcinoma arose at 11 months, and the number of mice with tumors in-



SvoBoDA--Stilbestrol and Skin Tumors in Mice 9 9 5

creased to a maximum, nine, at 15 months. Com- pared with a total of 150 mice with tumors in the methylcholanthrene-treated groups, there were 39 animals with tumors in the tobacco tar-treated groups (S.D.=0.67). Of these ~9 tumor-bearing animals among the tobacco tar-treated groups, there were sixteen mice with papillomas in the group which received only tobacco tar and four mice with papillomas in the group which received tobacco tar plus stilbestrol (S.D.=0.56). Nine carcinomas appeared in the tobacco tar-onlygroup; there were no carcinomas in the group painted with tobacco tar and given stilbestrol (S.D.= 0.3~). Approximately 70 per cent of carcinomas arose from pre-existing papillomas; the remainder arose from ulcerated lesions.

WcY

U.I

Z

8~ f 70 60

50

30 20 I0

II TOTAL TUMOR IN MCA 81 STILBESTROL GROUP

|TOTAL TUMOR IN MGA GROUP

89 97 105 115 121 129 157 145 154 DAYS AFTER FIRST P A I N T I N G

W I T H M C A CHART 1.--Comparison, up to 154 days, of the percentage

of animals bearing tumors, papillomas and carcinomas, in the methylcholanthrene-plus-stilbestrol and the methylcholanthrene-only groups. There were 100 animals in each group.

The carcinomas in the tobacco tar group arose in the 11th, l$th, and 15th months of the experiment after application of an average of 5-7 gin. of tar to the skin of each host mouse. I t has been shown that, after 13 months' exposure to tobacco tar, the carcinogenic action persists and leads to later carcinomas after painting has been discon- tinued (11). Although it is possible that, in this experiment, more tumors would have been noted with longer observation, it is also possible that the stilbestrol effect, considered over an equally extended period, would not have altered the ratio of benign to malignant tumors in the two groups. In the early induction of carcinomas by tobacco tar the same changes described by Croninger and Suntzeff (5) were seen--namely, the regression and disappearance of the initial tumors grossly, leaving a scar with later reappearance of a pro- gressive invasive tumor.

There were no significant differences among the treated groups in the percentage of animals with more than one malignant tumor.

MORPHOLOGIC CONSIDERATIONS In the present experiment, mast cells were found

in both tobacco tar- and methylcholanthrene-induced carcinomas and were situated about blood vessels of dermal papillae (Fig. 1). Since heparin, a mast-cell constituent, has been shown to have

~ 6 0 r MCA + ~.[]CARCINOMA STI LB ESTROL")| PA PI LLO M A

" ~ 5 0 0 MCA ONLY =PAPILLOMA ~ 40 ~GARGINOMA II

J 89 97 105 115 121 129 157 145 154

DAYS AFTER FIRST P A I N T I N G WITH M C A

C~ART ~.--The percentage of animals with papillomas compared with the percentage with carcinomas in the methylcholanthrene-plus-stilbestrol and methylcholanthrene-only groups.

~ 1 6

:~EI 4 I -

ra. 12

I... ~. I0 Z , ~ :

=:~I, 6

a . , ~ 4

z 2

PAPILLOMA ~ITOBACCO TAR ONLY CARCINOMA

PAPILLOMA II TOBACCO TAR -t- ESTROGEN

LL LIJLJLISI[ LJ 7 "8 9 I0 I I 12 15 14 15

MONTHS OF APPLICATION OF TOBACCO TARS

CHXaT 3.--Comparison of the percentage of animals with papillomas and carcinomas in the group treated with tobacco tar only and those treated with tobacco tar and given stilbestrol pellets. There were 100 animals in each group.

an inhibitory effect on the in vitro growth of both normal and malignant cells (7) and an antihya- luronidase effect (15), one possible explanation for the presence of mast cells in the carcinomas of mouse skin is tha t they represent an a t tempt to counteract the spread of hyaluronidase-producing growths by restoring to normal the increased per- meability of connective tissue.

A second point of interest is the similarity in appearance of several of the tumors induced both with tobacco tar and methylcholanthrene to the tumor designated keratoacanthoma in man (16) (Figs. 3, 3).



996 Cancer Research Vol. ~1, S e p t e m b e r 1961

The present experiment demonstrates that tobacco tar, like other chemical carcinogens, is cap- able of inducing keratoacanthomas in mice. More- over, as Ghadially (8, 9) contended, unlike the usual behavior of the tumor in man, the keratoacanthoma in mice frequently metastasizes to regional lymph nodes

Multiple pulmonary adenomas (~3) were noted in several of the mice at autopsy. Among the two groups of methylcholanthrene-treated animals, the percentage of animals with pulmonary adenomas was 98 per cent at 210 days, compared with a spontaneous incidence of 11 per cent at 8-14 months. There was no discernible increase in pulmonary adenomas due to tobacco tar nor exog- enous stilbestrol administration.

DISCUSSION

I t is possible that, by producing epidermal and dermal atrophy (1~), estrogens may expedite the strictly carcinogenic effect of methylcholanthrene. Normally, the mitotic frequency decreases sudden- ly after ovulation. I t might be hypothesized that constant estrogen absorption, and the resultant constant estrus, prevents normal postovulatory decline in mitoses and, by maintaining abnormally increased mitotic frequency, promotes eventual tumor formation.

The reason for the difference in observed effect of stilbestrol on methylcholanthrene-induced tumors compared with tobacco tar-induced tumors may lie in the composition or mode of action of the neutral fraction of tobacco tar. I t is not only a slowly acting, weak carcinogen but also a chem- ically complex one (13, 17, 25, ~7). I t is possible that, among the unidentified constituents, some which are antagonistic to stilbestrol may reside.

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2. Bv~ou~, W. S., and VA~ OOaD~, G. J. The Mitogenie Actions of Testosterone Propionate aad of Oestrone on the Epidermis of the Adult Male Mouse. Acta Endocrinol., 4: ~91-305, 1950.

3. COWDRV, E. V. Epidermal Carcinogenesis. In: J. P. GREE~STEIN and A. HAl)VOW (eds.), Advances in Cancer Research, 1:51-10I. New York: Academic Press, 1958.

4. Cnx~ZR, W., and S ~ o ~ , W. ~ Maat Cells in :Experi- mental Skin Carcinogenesis. Cancer Research, 4: 601-16, 1944.

5. C R O N ~ E ~ A. B., and SV~TZEFF, V. The Malignancy of a Cigarette Tar Carcinoma. VI. A Description of I(Y2 Suc- cessive Transplantations and of Metastasis to Lung and Node. Cancer Research, 12:731-34, 1959.

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i



FIG. 1 . - -Focal accumulation of mast cells in the dermis ad- jacent to a tobacco tar-induced squamous-cell carcinoma. Toluidine blue, X 1,0.

FIG. ~. - -Kera toacanthoma of mouse skin after 1~ months of application of tobacco tar.

Fro. 3 . - -Kera toacan thoma of mouse skin after 1~2 months of application of tobacco tar showing a bud-shaped, papillary lesion with a well demarcated dermal-epidermal junction. The tumor later metastasized to an axillary lymph node. H. & E., X40.



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