Studies on Squamous Metaplasia in Rat Bladder II . Effects of Estradiol and Estradiol Plus Hexestrol*T

Studies on Squamous Metaplasia in Rat Bladder II . Effects of Estradiol and Estradiol plus Hexestrol*t

A. ANGRIST, P. CAPURRO, AND B. MOUMGIS

(Department of Pathology, Albert Einstein College of Medicine of Yeshiva University, New York 61, N.Y.)

SUMMARY The effects of estrogens were studied with and without foreign body (rough glass beads and paraffin pellets) on the metaplasia of the bladder of rats on stock main tenance diet and on a vitamin A-deficient diet. Estradiol increased the degree of metaplasia in the bladder of rats when combined with vitamin A deficiency and/or foreign body stimulation. Estradiol affected bladder epithelium already made squamous more effectively than it did the normal transitional uroepithelium. A high dose of hexestrol, when added to estradiol, showed no enhance ment of the degree of metaplasi.a by estradiol benzoate in the bladder of the rat. The combination of vitamin A deficiency, foreign body in situ, and estrogenadminis tration was an effective means of obtaining keratinizing squamous metaplasia in the urinary bladder for studies of its developmental and reversal changes.

In a previous presentation (4) the relation of The animals were divided into the following different forms of foreign-body irritation and of groups (the number of rats surviving with tissue vitamin A deficiency to squamous metaplasia in for study and the total number in each group mi the bladders of rats was reported. It is also known tinily are given following each group): that estrogens will cause squamous metaplasia. I. Stock diet + estradiol (6 survivals/lI rats) The metaplasia following estrogen administration II. Stock diet, estradiol + hexestrol (4/9) has been described in various organs of the genito Ill. Stock diet, rough glass bead + estradiol urinary system in different species (SO). It has (@/4) been found in the uterus (@,17, 19), and the mets IV. Stock diet, paraffin pellet + estradiol (8/7) plastic effect of estrogens in the male has also been V. Stock diet, paraffin pellet, estradiol + described for the prostate (14). hexestrol (6/16) In this paper we are concerned with the effects VI. Vitamin A-deficient diet + estradiol of estradiol and the combination of estradiol and (11/15) the synthetic estrogen hexestrol with vitamin A VII. Vitamin A-deficient diet, estradiol + deficiency and irritative. foreign body stimulation hexestrol (18/@5) on metaplasia in the bladder. VIII. Vitamin A-deficient diet, rough glass bead + estradiol (13/19) MATERIALS AND METHODS IX. Vitamin A-deficient diet, rough glass Young rats of the Sprague-Dawley strain were bead, estradiol + hexestrol (10/17) used. Some of the rats were feJ regular stock x. Vitamin A-deficient diet, paraffin pellet + Rockland rat diet (complete) and the others were estradiol (4/8) fed a vitamin A-deficient diet and given a small XI. Vitamin A-deficient diet, paraffin pellet, vitamin A supplement to prevent early mortality estradiol + hexestrol (6/16) (4). The same procedure described earlier for in Injections of estrogenic agents were begun 1â€”@ sertion of foreign bodies in the bladder was used. weeks after the implantation of foreign bodies,

* Presented before the 50th Annual Meeting of the Amen unless otherwise noted, in Groups Ill, IV, V. VIII, can Association for Cancer Research in Atlantic City, April IX, X, and XI. In groups without foreign bodies 19-12, 1959. (I, II, VI, and VII), the estrogen treatment was t This work has beensupportedby GrantNo. RG-4572 begun 8â€”4weeks after Groups VI and VII devel (C1-C2) from the U.S. Public Health Service. oped vitamin A deficiency. The dose in oil of es Received for publication December 7, 1959. tradiol benzoate (Progynon benzoate, Schering 568

Corp.) was 0.05 mg. 3 times a week. The dose of 36 weeks on estradiol, and one after 38 weeks. The meso-3,4-di-(p-hydroxyphenyl)-in hexane (Hexes bladders of the males showed @+changes. A single trol, Wm. S. Merrell Co.) was 0.5 mg. S times a surviving female showed a 4+ change after 36 week. Injections were continued until sacrifice or weeks of estradiol. All showed prominent hyper death of the animals (see Charts 1 and @).The plasia of the epithelium, one with active mitosis. bladders were fixed in neutral calcium formol and In Group V, three males and three females were stained with hematoxylin and eosin, periodic acid examined between @5and 39 weeks after implan Schiff (PAS), and Alcian Blue (4). tation of the paraffin pellet and @3â€”37weeks of The changes in the bladders have been classified estrogen plus hexestrol administration. Three (two as before (4) : 1+, having loss of the PAS-positive males and one female) of the six showed only 1+ material in the superficial layer; @+, featuring modification of the bladder mucosa after such pro parakeratosis; 3+, showing mild epidermidaliza longed treatment with the combined estrogens, tion with no granular layer; and 4+, showing and three rats showed some minimal metaplastic marked whitish thickening, often with opaque alteration but less than 1+ change. Four of six keratinized papillary projections and the keratin showed epithelial hyperplasia, with one male layer with stratum granulosum histologically. showing some papillary hyperplastic mucosa. In Group VI there were five males and six RESULTS females on vitamin A-deficient diet plus estradiol. The mucosal changes are tabulated, with the groups subdivided by time periods of up to 7 REG. DIET weeks, 8â€”@0weeks, and @0â€”40+weeks after the GRAMS ESTRADIOL 450 + HEXESTROL beginning of estrogen treatment, in Table 1. 400 dl A high early mortality prevailed in Groups I 350 II, Ill, IV, V, X, and XI. In Group I, on estradiol vii: A DEF. 300 only, three animals were examined after 10 days, 4â€•EST. + HEXESt. 250 200 REG. DIET GRAMS REG DICT ESTRADIOL 150 400 1@ESTROGEN + HEXESTROL 350 I 00 300 V1TADEE REG.DIET 50 250 @FESTROGEN +EST. ,@:1II@ 0 IIII IIII 200 ?VIT.A DEE I 2 3 4 5 6 7 8 150 + ESTROGEN MONTHS 100 CHART 2.â€”Weight gain of animals on stock (reg.) and vita 50 mm-deficient diet with estradiol and hexestrol. 0 IIII 1 2 3 4 5 6 7 8 MONTHS Two females showed 3â€”4+ change after 10 weeks CHART 1.â€”Weight gain of animals on stock (reg.) and vita of vitamin deficiency and 1@and @4weeks of es mm-deficient diet with estradiol. tradiol (Fig. 5), respectively. Another female showed @+to 3+ change after 1@weeks of vita three after 9 months. No significant metaplasia mm deficiency and 8 weeks of estradiol (Fig. 1). was found, though one female showed hyperplastic A fourth female showed @+change after 16 weeks thickening of the mucosa and another female had of vitamin depletion and 1@weeks of estradiol. The some focal hyperplasia of the deeper layers of the remaining two females showed a 1+ grade of epithelium. metaplasia after 17@ weeks. Three male rats on In Group II, given estradiol and hexestrol in estradiol for 1@weeks, 3 weeks, and 5@weeks, re combination, four animals were examined after spectively, showed a 1 + change. One of the two @ @7â€”37weeks.The bladders showed no metaplastic remaining male rats received estradiol for weeks epithelial changes; one male showed some focal and showed minimal, i.e., less than 1+, change, hyperplasia. and the other showed @+to 8+ change after only In Group III, only two bladders were obtained 1@weeks of estradiol. for examination, both at 7 weeks after the glass In Group VII there were eight males and five @ bead was implanted but only weeks after estro females, vitamin-deficient, treated with both es gen administration began. Both showed no mets tradiol and hexestrol. Only one of four males on plastic alterations and only minimal focal epi the vitamin-deficient diet for 11 weeks, and 6 thelial hyperplasia. weeks of administration of estradiol and hexestrol, In Group IV, two animals were examined after showed a @+ change, three showing minimal

0 O@

.@.@:@+

.@+ -@

(.5 @ 2 2 @. @ u 0 @ V 4? @ c@cJ@C

C @.E.E z lIE @I

cI@ z @ ::

@ *@ .;Z -l-+++ ++++ @@@@ Â©@@4r:@@ .:.:

0 .@ +z @5 @ut-@@ *

changes (0â€”1+) (Fig. 6). One female, with 11 weeks. All six rats showed marked 4+ changes, weeks of both estrogens, showed a 8 + to 4+ alter one female as early as 7@weeks after administra ation in the bladder; another female showed a 3+ tion of the drugs, and the remaining female and @ change after weeks of such administration. two males after 16 weeks (Fig. 10). Maximal Both showed calculi in the bladder. Two males changes were present in all with invagination of showed 1+ change after 9 weeks and 15@ weeks epithelium and epidermoid cyst formation in two of combined estrogen, and two females showed females and one male. In this series a high mor this degree of change after 8@ and 11 weeks. The tality prevailed early. remainder of the group (two males and one fe A separate group of rats was studied with vita male) showed very minimal changes after periods mm A deficiency, with paraffin pellets in situ in of both estrogens varying from 9 to 17 weeks. the bladder, and with subcutaneous diethylstil Keratinization was found in the rats with calculi besterol pellets of 0.5 mg. for continuous slow and was not present in the other rats. absorption. In 30 such rats @4showed 4+ meta In Group VIII there were five males and eight plasia in periods varying from 3 to 8 weeks. These females. This group was kept on a vitamin-defi findings are not included in the tabulation be cient diet for an average of 3â€”4weeks and than cause of the different form of the extrogen used. had a rough glass bead inserted ; after periods Bladders stained with PAS and Alcian Blue averaging 4â€”5weeks post-operatively, estradiol showed an increase of staining in the stroma of was administered. All animals, with one exception, the bladder when estrogen was given to animals showed a 4+ change in the bladder after periods with foreign bodies in situ, whether or not the ani @ ranging from week to 11 weeks of such adminis mals were vitamin A deficient. In the bladders of tration. the animals receiving hexestrol the staining was Group IX consisted of six males and four f e not as intense. males. The rats were placed on a vitamin-deficient diet for 3â€”4weeks and then had a rough glass bead DISCUSSION inserted. In this group estradiol and hexestrol were Vitamin A deficiency and the presence of a for @ administered beginning weeks post-operatively. eign body individually have been shown to yield Only one male and one female were carried on keratinization in the bladder of rats; when the two estradiol and hexestrol for 10 weeks and 7@weeks, treatments were combined, earlier and more respectively, and showed a 4+ change (Fig. @) marked metaplasia occurred (4). and a 1 + change. Four rats were carried on both Estradiol at the dose level employed enhanced drugs for 5â€”6 weeks. One of the two females the metaplasia produced by these means. How showed a 4 + alteration, and the remaining female ever, it failed to yield metaplasia of the bladder showed a 3+ change. Two males showed a 3+ epithelium of rats on the stock diet without a for change with some papillary areas of the mucosa in eign body in 8itU, although others have found some one showing a focal 4+ alteration (Figs. 3 and 8). such changes (@0). The estrogens apparently are The other rats were on the drugs for less than 3 most effective on the epithelium of the bladder al weeks. One male at 3 weeks had a 3â€”4+ change, ready altered by previous stimuli known to pro one female had a @+ change, and two males mote metaplasia, suggesting that squamous, rath showed a 1+ alteration. er than transitional, epithelium in the bladder is In Group X there were only two males and two the more susceptible target tissue. When estradiol females that survived for study. These animals was administered to rats with implanted paraffin were placed on a vitamin-deficient diet for 4 weeks pellets on a stock diet (Group IV) keratinization and then had a paraffin pellet inserted; the adinin appeared, while practically none was observed in istration of estradiol began 1 week post-operative such rats without estrogen (4). In vitamin A-defi ly. One male showed a 8+ change after 3 weeks of cient animals with foreign bodies in situ, estrogen estradiol administration. The remaining three also increased the metaplastic modification of the showed marked 4+ changes, one male after 14 bladder epithelium (Groups VIII and X). weeks of the drug (Fig. 9) and the two females Hexestrol in the dosage used seemed to have a after 18 weeks. The keratinization was marked and surprising effect of failing to increase the meta diffuse, and epidermoid cysts were present. plasia of the other estrogen, estradiol, on the blad In Group XI, two males and four females sur der epithelium. In some instances it seemed to vived. The conditions in this group were identical diminish it. No valid comparison is permissible for with those in Group X, except that estradiol and the inhibiting effect of hexestrol in Groups IV and hexestrol were both administered 1 week post V because of the high mortality. In comparing operatively after a paraffin pellet was inserted in Groups VI and VII some inhibiting effect of rats that had been depleted of vitamin A for 4 hexestrol was noted. However, in two of the rats

in Group VII with a 3+ change, calculi were 4. CAPURRO,P.;ANGRIST,A.;Bi..&cic,J.;and Mou@sois,B. present. Hexestrol and estradiol together were Studies in Squamous Metaplasia in Rat Bladder: I. Ef fects of ITypovitaminosis A, Foreign Bodies and Methyl less effective in producing metaplasia than estra cholanthrene. Cancer Research, 20:563â€”67, 1960. diol alone, despite the discrepancy in over-all dos 5. ENOLF.,E. T.; KRAKOWER,C.A.; and HAAGENSEN,C.D. @gein Group IX as compared with Group VIII. Estrogen Administration to Aged Female Monkeys with In Groups X and XI, a comparison of this differ No Resultant Tumors. Cancer Research, 3:858-66, 1943. @entialeffect cannot be made because, in addition 6. ENOLE, E. T., and Sstn,H, P. E. Some Uterine Effects Oh tamed in Female Monkeys during Continued Oestrin Ad to the small number of animals, the bladders were ministration, with Especial Reference to the Cervix Uteri. .examined after a prolonged period, and the majori Anat. Rec., 61:471â€”83, 1935. ty of results in both groups were all 4+. 7. GRANGAtTD,R., and CONQUT, T. Effets de In progesterone It is generally accepted that synthetic estrogens administrÃ©o on injections a iz femelle due rat blanc like hexestrol (such as stilbestrol) induce keratini carancÃ©e en vitamine A. Compt. Rend. Aced. Sc. (Paris), 246:3274-77, 1958. zation of the vaginal and cervical epithelium in 8. HARTMAN, C. G.; GESCHICETER, G. F.; and SPESET, H. monkeys and in rats (3, 5, 6, 8, 9, 1@â€”15,17),and Effects of Continuous Estrogen Administration in Very squamous metaplasia of the prostate (1, 10, @0). Large Doses. Anat. Rae., 79:31, Suppl. 2, 1941. No reference to an antagonistic effect of stilbestrol 9. HISAW, F. L.; Gaasp, R. 0.; and Fzvow, H. L. Effects of Oestnin-Progestin Combinations on the Endometrium, @nthe metaplasia produced by estradiol or other Vagina and Sexual Skin of Monkeys. Am. J. Aunt., 61: natural estrogens could be found in the literature. 488â€”596,1937. Estrogens induce adrenal enlargement (18) and 10. Huooias, C., and Ci@.&ax,P. J. Quantitative Studies of may increase glucocorticoid output. Such steroids Prostatic Secretion: Effect of Castration and of Estrogen may offset the metaplastic effect of estrogens, Injection on the Normal and on the Hyperplastic Prostate Glands of Dogs. J. Exper. Med., 72:747â€”62, 1940. much as glucocorticoids inhibit growth and heal 11. LOWE,J. S., and Moai@oN,R. A. Some Aspects of Vitamin ing processes. This failure to obtain a summation A Metabolism.Vitaminsand Hormones,14:97â€”132,1956. @effectof hexestrol and estradiol may therefore rep 12. OVERR0LSER,M.D., and Au@ss@,E.Atypical Growth In resent a dosage phenomenon, for the dosage of duced in Cervical Epithelium of the Monkey by Prolonged Injections of Ovarian Hormone Combined with Chronic hexestrol used was rather large, so that the Trauma. Sung. Gyn. & Obst., 60: 129â€”36,1935. amount of glucocorticoid produced may have been 13. Przia@rza, C. A., and Azmi, E. Attempts To Produce sufficient to offset the keratinizing influence of es Cancer in Rhesus Monkeys with Carcinogenic Hydro trogens. Alternatively, the stimulated adrenal may carbons and Estrogens. Cancer Research, 8:97â€”127, 1948. put out amounts of progesterone (16, 18) sufficient 14. Razas, P. R.; C4wzsur@r,V. 0.; and RIMER,H. B. The to offset the influence of estrogens. In the vagina, Metaplastic Effects of Estrogen and Vitamin A Deficiency in Certain Pathological States of the Prostate and the @atleast, keratinization by estrogen is inhibited by Liver. Jackson Mem. Hosp. Bull., 5(2):50â€”86, 1951. progesterone (7). 15. SCoTT, R. B@,and WHARTON, L. R. The Effects of Exces The combination of vitamin A deficiency, for sive Amounts of DiethyLstilbestrol on Experimental Endo @eigubody in situ, and administration of estrogens metniosis in Monkeys. Am. J. Obst. & Gynecol., 69:573-91, 1955. is an effective means of obtaining keratinizing 16. SELTE, H., and Coiup, J. B. Fundamental Factors in the squamous met.aplasia in the urinary bladder for a Interpretation of Stimuli Influencing Endocrine Glands. study of the finer morphological alterations. Endocrinology, 20:667â€”72, 1936. 17. SELYE, H.; Tnoasaoi@, D. L.; and Coi.up, J. B. Metaplasia REFERENCES of Uterine Epitheium Produced by Chronic Oestnin Ad 1. BERG, 0. A. The Effect of Stilboestrol on the PrOstate ministration. Nature, 135:65â€”66, 1985. Gland in Normal Puppies and Adult Dogs. Acta Endo 18. Tzppznaws, J.; ENGEL,F. L.; and LONG,C. N. H. A Re crinol., 27: 155â€”69,1958. view of Adrenal Cortical Hypertrophy. Endocrinology, @.Bo, W. J. The Origin and Development of Estrogen-In 32:373â€”402, 1943. duced Uterine Metaplasia. A.M.A. Arch. Path., 64:595- 19. ZONDEK,B.The Effect of Long-Continued Large Doses of 600, 1957. Follicle Hormones upon the Uterus of the Rat. Am. J. S. Bua.wx, E.; Woura, J. M., and Wiuowr, A. W. Effects of Obst. &Gynecol.,33:979-88, 1937. Administration of Estrogen on Connective Tissues of the 20. ZUCKERMAN,S.The Histogenesis of Changes in Tissues GenitalTract ofthe Rat. Endocrinology, 30:385â€”342, 1942. Sensitive to Oestrogens. Biol. Rev., 15:281â€”71,1940.

FIG. 1.â€”GroupVI. Gross appearance of bladder of a female male rat on vitamin A-deficient diet for 10 weeks, with rough rat on vitamin A-deficient diet for 12 weeks and on estradiol for glass bead for 7 weeks and estradiol plus stilbestrolfor 5 weeks. 8 weeks. Linear zones of elevated keratinization (2â€”3+ The metaplastic changes are limited to this papillary hera @change)and opacity of the mucosa (k) are seen centrally. tinized protuberant area only (8-4+ change). The remainder FIG. 2.â€”Group IX. Gross appearance of bladder of a male ofthe bladder surface (b)showssomecobble-stonegranularity. rat on vitamin A-deficientdiet for 15 weekswith rough glass FIG. 4.â€”Group VIII. Gross appearance of th, bladder of bead for 12 weeks and estradiol plus stilbestrol for 10 weeks. male rat on vitamin A-deficient diet for 15 weeks, with rough The bladder shows folded thickened mucosa with opaque white glass bead for 12 weeks and estradiol for 10 weeks. Maximal keratinized crests (8+ change). changes are present (4+ change, papillary growths). (Corn Fio. 3.â€”Group IX. Gross appearance of the bladder of a pare with Fig. 2.)

_.@fr@' -----

@ @L\ J

,@ :@â€˜@â€˜@â€˜

@I@;:5l )@

: â€˜;@

@ I@ . b

â€˜.1t,@

4@)@(

â€˜I @,-

[4J

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. All sections stained with hematoxylin and eosin. Fm. 5.â€”Group VI. Bladder of a female rat on vitamin A- (leficient diet for 10 weeks with estradiol administration for @24 weeks (3â€”4+ change) . Most of the keratinized area lacks a granular zone, present at (g). X180. FIG. 6.â€”Group VII. Bladder of a male rat on vitamin A- deficient diet for 11 weeks and estradiol plus stilbesterol admin istration for 6 weeks, showing no metaplastic changes. Epi theliurn cut tangentially at (t). X 150. FIG. 7.â€”Group VIII. Bladder of male rat on vitamin A- deficient diet for 15 weeks with rough glass bead in situ for H weeks and estradiol for 10 weeks (4+ change). Papillary, keratinized squamous epithelium with sharply delimited eleidin granular zone (g). Downgrowth of epithelium not included in photograph. X360. FIG. 8.â€”Group IX. This is the microscopic section of the papillary formation in Figure 3. The keratin has been detached; a granular layer is not present ; some inflammatory cells are seen in the underlying stroma (st). Some hyperplasia of basal layer is seen at upper margin of photograph (b). X150. FIG. 9.â€”Group X. Bladder of a male rat on vitamin A- deficient diet for 194 weeks with paraffin pellet in situ for 15 weeks and estradiol administration for 14 weeks (4+ changes@. Numerous cysts, small cells and atrophy (a) of viable epi thelium. X@40. FIG. 10.â€”Group XI. Bladder of a male rat on vitamin A- deficient diet for @14weeks with paraffin pellet in situ for 17 weeks and estradiol and stilbestrol for 16 weeks (4+ change). Wide basal, intermediate, and parakeratotic zones, with little keratin. (Compare with Fig. 9.) XI@40.

@ :s5I@@@ :@: @@ i1â€•.@' .. . - , .â€˜\!,@ .% â€¢ @ @â€˜. .- .. @â€˜@:

@@@ - -@!.@;â€˜@: @-

I __ @@@ .. â€˜-;-%b.. S

@ .--.@ S..' 3@@@,

@@ @:â€˜@ :1d@ @ @:sÃ¢ , @@j@:r@t'-,@-

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. Studies on Squamous Metaplasia in Rat Bladder: II. Effects of Estradiol and Estradiol plus Hexestrol

A. Angrist, P. Capurro and B. Moumgis

Cancer Res 1960;20:568-572.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/20/4/568

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/20/4/568. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.