Phytoestrogens Are Partial Estrogen Agonists in the Adult Male Mouse Sari Makela,' Risto Santti,' Leena Salo,1 and John A. McLachlan2 1lnstitute of Biomedicine and Medicity Research Laboratory, University of Turku, Turku, Finland; 2Tulane/Xavier Center for Bioenvironmental Research, Tulane University, New Orleans, Louisiana The intake, as well as serum and urinary concentrations, of phytoestrogens is high in countries where incidence of prostate cancer is low, suggesting a chemopreventive role for phytoestrogens. Their significance could be explained by the ability to antagonize the action of more potent endogenous estrogens in initiation or promotion of tumor formation. We have studied estrogenicity and antiestrogenicity of dietary soy and two phytoestrogens, coumestrol and daidzein, in our neoDES mouse model for the study of prostatic neoplasia. Soy was chosen because it is rich in phytoestrogens, is widely used in Oriental diets, and has antiestrogenic and anticarcinogenic properties in the neoDES mouse when given from fertilization onward. In short-term tests with adult animals, no evidence for estrogenicity or antiestrogenicity (capability to antagonize the action of 1 7,B-estradiol) of soy was found when development of epithelial metaplasia and expression of c-fos protooncogene in prostate were used as end points of estrogen action. Estrogenic activity of coumestrol and daidzein on c-fos expression was subtle. Coumestrol, either given alone or in combination with 17,B-estradiol, had no effect on development of epithelial metaplasia. These marginal or missing effects in adult males could be interpreted by assuming that the neonatal period is more critical for estrogenic or antiestrogenic action of soy and phytoestrogens. Once initiated, estrogen-related lesions would develop spontaneously. Alternatively, the chemopreventive action of soy is not due to antiestrogenicity of soyderived phytoestrogens. - Environ Health Perspect 103(Suppl 7):123-127 (1995) Key words: accessory sex glands, c-fos, coumestrol, daidzein, estrogens, male, mouse, phytoestrogens, prostate, soy Introduction Dietary soy has chemopreventive properties in several animal models for cancers (1-3). There is also suggestive evidence that dietary soy (tofu) is chemopreventive in human prostate cancer (4). Using the developmentally estrogenized mouse model (5,6), we have shown that the estrogenrelated inhibition of prostatic growth is reduced and the development of dysplastic changes is delayed in the prostate when the animals are kept on soy-containing feed from fertilization onward (7). Both the number of animals showing dysplasia and This paper was presented at the Symposium on Estrogens in the Environment, III: Global Health Implications held 9-11 January 1994 in Washington, DC. Manuscript received: March 15, 1995; manuscript accepted: April 4, 1995. Hanna Laine, Leena Simola, Silja Simola, and Tuula Tanner are acknowledged for their skilful technical assistance. This work was financially supported by Yrjo Jahnsson Foundation, Turku University Foundation,and Emil Aaltonen Foundation. Address correspondence to Dr. Sari Makela, University of Turku, Institute of Biomedicine, Department of Anatomy, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Telephone: 358-21-633 7361. Fax: 358-21-633 7352. Abbreviations used: DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; bw, body weight; CG, coagulating gland; DES, diethylstilbestrol; DLP, dorsolateral prostate (lobe); DP, dorsal prostate (lobe); LP, lateral prostate (lobe); neoDES mouse, neonatally DES-treated mouse; PC, prostatic cancer; PIN, prostatic intraepithelial neoplasia; SV, seminal vesicle; T, testis; ER, estrogen receptor; E2, 71 W estradiol. Environmental Health Perspectives also the severity of the alterations in dysplastic epithelium were lower in animals given soy. Morphologically the dysplastic lesions in the prostate of neoDES animals are similar to prostatic intraepithelial neoplasia (PIN) in human prostate (8). Although no progression to carcinomas with invasion of surrounding tissues or metastasis could be demonstrated, the morphological changes and increased expression of protooncogenes in developmentally estrogenized mice suggest an increased potential for benign and malignant growth (Table 1). Soy is rich in phytoestrogens, weakly estrogenic nonsteroidal compounds. Their urinary excretion (particularly that of isoflavones) correlates with the ingestion of soy in our animal experiments (7). In humans, urinary excretion and serum concentrations of phytoestrogens are higher in countries where the incidence of prostate cancer is low (9,10). This suggests that phytoestrogens may account for the chemopreventive action of soy in prostate carcinogenesis. The mechanisms of the possible chemopreventive action of phytoestrogens are not known. Their significance could be explained by their ability to antagonize the action of more potent endogenous estrogens in initiation or promotion of carcinogenesis, although currently no direct evidence for this action is available. The mouse model based on neonatal diethylstilbestrol treatment (neoDES model) is particularly suitable for testing this hypothesis. In neoDES mice the histological response to 17p-estradiol in terms of metaplastic transformation and the 17p-estradiol-induced expression of c-fos protooncogene are greatly enhanced. Based on these end points of estrogen action, we have tested the estrogenicity and antiestrogenicity of dietary soy and two structurally different phytoestrogens, coumestrol and daidzein, both known to be present in soy. Materials and Methods Animals and Diets Outbred Han-NMRI mice (produced by the Animal Quarters, Institute of Biomedicine, University of Turku, Turku, Finland) were used throughout the study. The test protocols were approved by the Turku University Committee on the Laboratory Animal Center. The animals were given free access to feed and tap water. A soyfree diet, where soy is substituted with casein, was purchased from Finnewos (Helsinki, Finland) and Special Diet Services, (Witham, Essex, United Kingdom). A standard soy-containing laboratory feed for mice (Ewos R3) containing cereals, wheat germ, wheat middlings, roasted soy meal (7%), fish protein concentrate, fodder yeast, minerals, animal and vegetable fat, vitamin concentrate, 123 MAKELA ETAL. Table 1. Effects of neonatal estrogenization in mouse urethroprostatic complex (2 pg of diethylstilbestrol per day on days 1 to 3 after birth). Prostatic growth Permanent growth inhibition of all lobes (5) Tissue composition Increase in the relative volume of prostatic interacinar stroma (5) Increased amount of periurethral glands (5) Epithelial structure Hyperplasia and dysplasia in posterior periurethral region and collecting ducts (sites adjacent to estrogen receptor-positive stroma) (6) Expression of estrogenPermanent increase in c-myc and c-fos expression [(6); S Makel3, unpublished responsive protooncogenes results] Response to secondary Enhanced estrogen sensitivity (estimated by the development of epithelial estrogen treatment metaplasia in collecting ducts and expression of c-fos oncogene after treatment with 17p-estradiol) [(6); S Makela, unpublished results] Voiding function Altered voiding pattern (increased voiding frequency, decreased voiding volumes) (S Makela, unpublished results) Decreased ratio of urinary flow to bladder pressure (S Makela, unpublished results) Immune system Inflammatory changes (5) intensities of autoradiographic films were scanned with the Microcomputer Imaging Device (MCID) by using M4, version 2.1 software program (Imaging Research Inc., Ontario, Canada). The intensity values of c-fos were corrected by the corresponding intensity values obtained after hybridization with mouse 28S ribosomal RNA probe. The 4.8 kb SalI-EcoRI fragment of mouse 28S ribosomal RNA cDNA was 32P-dCTP labeled by random priming. Resufts Effects of Dietary Soy and Coumestrol on Development of Squamous Epithelial Metaplasia and trace element concentrate was prepared by Finnewos, Helsinki, Finland. The animals were kept on soyfree diet, and the soy-containing diet was used only in the experiments. Neonatal Estrogenization with sections were carefully studied for the presence of squamous epithelial metaplasia in the periurethral collecting ducts and proximal parts of coagulating gland, dorsolateral prostate, and seminal vesicles. diethylstilbestrol (DES) in 20 pl of corn oil per day for the first 3 days after birth. and, after 7 days, they were injected sc with 17,B-estradiol (100 pg/animal), coumestrol (200 pg/animal), or daidzein (200 pg/animal) diluted in corn oil (100 pl/animal). The controls received the vehicle only. In the soy experiment, the animals were kept on the soy diet from castration onward. Animals were sacrificed 3, 6, or 12 hr after injection and urethroprostatic blocks were removed. Tissues were transferred to a petri dish containing phosphate-buffered saline (PBS) and prostatic lobes, seminal vesicles, and prostatic urethra were dissected under a microscope. Tissue samples were immediately frozen in liquid nitrogen and stored at -70°C. Total RNAs were extracted with the single step method (11). Fifteen-microgram aliquots of total RNA Induction of c-Jbs Expression were size-fractionated in 1% agarose/for- 17J-Estradiol induced the expression of cmaldehyde gels and blotted onto nylon fos in neoDES mice given a soy-free diet. membrane (GeneScreen, DuPont, NEN, After the sc injection of 170-estradiol Boston, MA). The filters were hybridized (100 pg/animal) the increased expression and washed as suggested by the manufac- was evident in the prostatic urethra and turer. The 32P-labeled c-fos antisense RNA coagulating glands at 3 to 12 hr after probe was synthesized from the insert in a injection (Figure 2). pGEM vector (Promega, Madison, WI) Soy given to neoDES animals for 1 according to the manufacturers directions, week before 17,B-estradiol injection had no using Sp6 RNA polymerase; the radiola- effect on the expression. Further, coumebeled probe was added directly to the pre- strol and daidzein in a dose of 200 pg/anihybridization mixture. The c-fos probe was mal sc showed weak estrogenic action kindly provided by George Stancel (Figure 2), but there is no evidence that (University of Texas, Houston TX). This soy acts as an antiestrogen based on the mouse c-fos probe was originally obtained induction of c-fos expression. by digestion and subcloning of mouse pcfos-3 (12). For quantitation, the signal Induction ofc-bs Expression Diethyistilbestrol (neoDES Treatnent) Adult (3-5 months of age) neoDES mice Male pups were injected sc with 2 pg of were castrated under barbiturate anesthesia Induction ofMetaplastic Reaction Adult neoDES mice (3-5 months of age) were castrated under barbiturate anesthesia. In soy experiments the animals were divided into four groups: group 1 continued on the soyfree diet; group 2 was transferred to the soy-containing diet; group 3 received an sc implant with 50 pg estradiol and continued on the soyfree diet; and group 4 received an sc implant with 50 pg estradiol and was transferred to the soycontaining diet. In experiments with coumestrol, the animals were divided into four groups: group 1 received the vehicle, 20 pl of dimethyl sulfoxide (DMSO) per day sc; group 2 received 25 pg of estradiol in DMSO sc per day; group 3 received 50 pg of coumestrol in DMSO sc per day; and group 4 received both estradiol and coumestrol. After 10 days (soy experiment) and 7 days (coumestrol experiment) the animals were sacrificed; the urethroprostatic blocks were removed and used for histologic preparations. Tissue blocks were fixed whole in Bouin's fixative, dehydrated, and embedded in paraffin. Serial horizontal 6-pm sections with 200-pm intervals were cut through the tissue block, from lower urethra up to the upper half of the urinary bladder. The sections were stained with routine hematoxylin and eosin, dehydrated, and mounted with Permount. The 124 An extensive squamous metaplasia was observed in the periurethral collecting ducts, as well as in the periurethral parts of coagulating glands, when castrated neoDES animals fed a soy-free diet were treated with estrogen pellets (50 pg of 17pestradiol per pellet) for 10 days or with daily injections (25 pg of 17p-estradiol per day) for 7 days (Figure 1A,B) When adult neoDES animals were kept on a soy-containing diet for 10 or 21 days after castration, no signs of squamous epithelial metaplasia could be observed. Soy feeding did not prevent the metaplastic reaction induced by 17 3-estradiol implants (Figure 1C,D). Coumestrol (50 pg per day for 7 days given sc) did not induce squamous metaplasia in neoDES animals. Neither did it inhibit the metaplastic reaction induced by 1 71B-estradiol injections (Figure 1 E,F). Thus, based on squamous epithelial metaplasia, there is no evidence that either coumestrol or dietary soy are estrogenic or antiestrogenic. Environmental Health Perspectives PHYTOESTROGENACTION IN MOUSE PROSTATE A 150 *i 125- .s 100 75 Oil Figure 1. Microscopic structure of prostatic collecting ducts in the posterior periurethral region of an adult castrated neoDES mouse kept on soyfree diet. (A) treated with an sc implant containing 50 pg estradiol and kept on soyfree diet for 7 days; (B) given a soy-containing diet for 10 days postcastration; (C) treated with an sc implant with 50 pg estradiol and kept on a soy-containing diet for 10 days postcastration; (D) treated with 50 pg coumestrol in DMSO sc per day for 7 days postcastration; (E) treated sc with 25 pg estradiol; and (F) 50 pg coumestrol in DMSO for 7 days postcastration. Discussion glands were reduced but the differences Despite the greater estrogen sensitivity, no were not statistically significant (7). evidence for the estrogenicity of dietary soy Coumestrol, one of the most potent (diet with 7% of roasted soy meal) was phytoestrogens, was also incapable of found in the prostate of neonatally estroge- inducing metaplastic transformation in nized, adult castrated male mice when neonatally estrogenized mice; as docujudged on the basis of development of mented earlier, it did not inhibit the prosquamous epithelial metaplasia. The lack of static growth when administered to normal the estrogenicity of soy is in contrast to the adult rats (13). However, when the inducfindings in the immature female mouse in tion of the expression of estrogen-responwhich the estrogenicity of diet with 7% of sive gene, c-fos (one of the immediate early roasted soy meal was confirmed by the uter- genes in mitosis) in adult neoDES mice ine growth response (7). The estrogenlike was used as an end point, both coumestrol effect by dietary soy was also demonstrable and daidzein showed weak estrogenlike on the prostatic growth in the male rat activity. Also in rat uterus the antiestrogen when exposed to dietary soy from fertiliza- tamoxifen was shown to induce a weak tion onward: the size of the ventral prostate response in c-fos expression (14). This was reduced at 2 months of age (13). In the weak estrogenlike (or antiestrogenlike) corresponding feeding experiment with the effect is induced by phytoestrogen doses male mouse, the sizes of the sex accessory comparable to the amounts ingested by Volume 103, Supplement 7, October 1995 E2 Cou Dai inject-on Figure 2. Effects of 171-estradiol, coumestrol, daidzein, and soy diet on c-fos in the prostatic urethra and coagulating gland of castrated neoDES male mice. Abbreviations: Oil, vehicle only; E2, 17f-estradiol (100 pg sc); Cou, coumestrol (200 pg sc); Dai, daidzein (200 pg sc). (A) Effect of 17)3-estradiol, coumestrol, daidzein on the expression of c-fos in the prostatic urethra of castrated neoDES mice. The bars show the combined data from all experiments. Values are expressed as percentages of expression after 3-hr E2-treatment (using the corrected scanning units of c-fos relative to 28S). Each bar represents the expression in three to nine animals. (B) A representative Northern blot from one experiment. Lane 1-vehicle only (oil), 3 hr; lane 2-E2, 3 hr; lane 3-E2, 6 hr; lane 4-E2, 12 hr; lane 5-coumestrol, 3 hr; lane 6-coumestrol, 6 hr; lane 7-coumestrol, 12 hr; lane 8-daidzein, 3 hr; lane 9-daidzein, 6 hr; lane 10-daidzein, 12 hr. Each sample consists of mRNA from three animals. (C) Effect of dietary soy on the estradiolinduced expression of c-fos in the coagulating gland of castrated neoDES mice. Lane 1, soyfree diet from castration onward plus treatment with vehicle only; lane 2, soyfree diet from castration onward plus treatment with 17p3-estradiol (100 pg sc); lane 3, soy diet from castration onward plus treatment with 17j3-estradiol (100 pg sc). Injections were given on day 7 postcastration and all samples were taken 3 hr after injection. Corresponding ethidium bromide (E + Br) staining is shown below c-fos. 125 MAKELA ETAL. laboratory rodents in daily soy-based feed (coumestrol, daidzein, genistein) are clearly (15) and may therefore be of significance estrogen agonists in breast cancer cells in for the effects associated with the high vitro and act through the estrogen receptor (ER)-mediated mechanisms (21,22). intake of dietary soy. The weak estrogenicity of coumestrol Phytoestrogens with high binding affinities seen in the adult male is again contradic- for estrogen receptor are also most active tory with the findings in female rodents. It biologically (e.g., they enhance cell prolifis very well documented that coumestrol is eration) (23). Phytoestrogens are supposed a potent estrogen in the developing female to act as antiestrogens by competing with reproductive tract. It had DES-like effects more potent endogenous estrogens for the in the neonatal female mouse (16) and binding to ER. Dietary estrogens reprepromoted uterine growth in immature rats senting three structurally different groups when given in doses (per body weight) (coumestans, isoflavonoids, and resorcyclic similar to those we used (15,17-19). In acid lactones) had additive effects with adult ovariectomized rats, coumestrol 17p-estradiol in the presence of the conshowed partial estrogen agonism; it was centration giving submaximal stimulation, uterotrophic but did not induce ovum and none of the phytoestrogens we studied implantation in mated, ovariectomized (at concentrations below 1 PM) reduced gestagen-maintained animals (20). The the proliferation rate of breast cancer cells, conflicting findings on the hormonal i.e., had antiestrogenic effects in the prespotency of soy or coumestrol cannot yet be ence of 17p-estradiol (22). In addition to the interaction with ER, explained, but they could be due to sex- or age-related differences in the uptake or dietary estrogens or structurally related metabolism of phytoestrogens. compounds might compete with endogeIt is also intriguing that in adult male nous estrogens for the active site of the mice soy did not block the estrogen- estrogen-biosynthesizing and estrogeninduced metaplastic transformation or metabolizing enzymes and thus reduce the expression of c-fos protooncogene, and concentration of biologically active coumestrol did not inhibit the development endogenous estrogens. Coumestrol and of metaplasia in 17p-estradiol-treated ani- genistein have been shown to inhibit the mals. This is in conflict with the idea of the reduction of estrone to 170-estradiol by antiestrogenicity of soy seen as reduction of estrogen-specific 17p-hydroxysteroid oxithe growth inhibition of the prostate and doreductase type 1 (EC 1.1.1.62, also prevention of dysplastic development after known as 17,B-hydroxysteroid dehydrogeneonatal estrogenization and the reduction nase type 1, 17P-HSD type 1) (24). This of the estrogen-induced growth of imma- enzyme is expressed in steroidogenic cells ture uterus (7). One could interpret these such as ovarian granulosa cells and placenfindings by assuming that in males the tal trophoblasts, as well as in some target neonatal or prepubertal period would be tissues of estrogen action, such as normal more critical for estrogen and antiestrogen and malignant breast and endometrium. actions. Once initiated, the estrogen-related The antibody against estrogen-specific 17phydroxysteroid oxidoreductase stains the lesions would develop spontaneously. It is not easily concievable how soy or urethral epithelium in the mouse (25) as phytoestrogens could antagonize the estro- well as in man (26). The immunostaining gen action at the target cells. Phytoestrogens of epithelium extends to the periurethral parts of the dorsolateral lobes, coagulating glands, and seminal vesicles in the mouse. These are also the sites where metaplastic epithelium and most dysplastic lesions are found. Changes in the 17p-oxidoreduction status of endogenous estrogens (estradiol and estrone) may considerably modify the biological activities of these hormones. This would have biological significance if continuous estrogen stimulation were needed for the development of dysplasia. At present, there is no direct evidence to support this hypothesis. Moreover, the possibility remains that the chemopreventive action of soy is not due to the antiestrogenicity of soy-derived phytoestrogens. At very high concentrations, phytoestrogens are reported to have effects not related to estrogen action. Genistein, an isoflavonoid phytoestrogen, is a potent inhibitor of both estrogen receptor negative and positive breast cancer cells (27) and of tyrosine protein kinase activity of several growth factor receptors and oncogenes that may be associated with tumor cell growth (28). Further, the inhibition of DNA topoisomerase II has been suggested as an alternative mechanism for the action of isoflavones (29). Genistein is also an inhibitor of angiogenesis, which may partly explain the possible antitumor activity of phytoestrogens (30). The relevance of these mechanisms for understanding the possible antiestrogenic action of soy or soy-derived estrogens in the male mouse is not known. It is noteworthy that soy had no general inhibitory effect on prostatic growth in neonatally untreated animals (7). In addition to phytoestrogens, soybeans contain several other potential anticarcinogenic agents such as protease inhibitors, phytosterols, saponins, and inositol hexaphosphate (1,31); their possible roles as chemopreventive agents cannot be excluded in the present model. REFERENCES 1. Barnes S, Grubbs C, Setchell KDR, Carlson J. Soybeans inhibit mammary tumors in models of breast cancer. In: Mutagens and Carcinogens in Diet (Pariza MW, Aeschbacher H-U, Felton JS, Sato S, eds). New York:Wiley-Liss, 1990;239-253. 2. Hirono I, Funahashi M, Kaneko C, Ogino H, Ito M, Yoshida A. Gastric lesions in rats fed salted food materials commonly eaten by Japanese. Nutr Cancer 14:127-132 (1990). 3. Nagahara A, Benjamin H, Storkson J, Krewson J, Sheng K, Liu W, Pariza MW. Inhibition of benzo[a]pyrene-induced mouse forestomach neoplasia by a principal flavor component of Japanese-style fermented soy sauce. Cancer Res 52:1754-1756 (1992). 4. Severson RK, Nomura AMY, Grove JS, Stemmermann GN. A 126 5. 6. 7. 8. prospective study of demographics, diet, and prostate cancer among men of Japanese ancestry in Hawaii. Cancer Res 49:1857-1860 (1989). Pylkkanen L, Santti R, Newbold R, McLachlan JA. Regional differences in the prostate of the neonatally estrogenized mouse. Prostate 18:117-129 (1991). Pylkkinen L, Mdkeld S, Valve E, Harkonen P, Santti R. Prostatic dysplasia associated with increased expression of c-myc in neonatally estrogenized mice. J Urol 149:1593-1601 (1993). Mdkeld S, Pylkkinen L, Santti R, Adlercreutz H. Dietary soybean may be antiestrogenic in male mouse. J Nutr 125:437-445 (1995). Sakr WA, Haas GP, Cassin BF, Pontes JE, Crissman JD. The Environmental Health Perspectives PHYTOESTROGEN ACTION IN MOUSE PROSTATE 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. frequency of carcinoma and intraepithelial neoplasia of the prostate in young male patients. J Urol 150:379-385 (1993). Adlercreutz H, Honjo H, Higashi A, Fotsis T, Hamaildinen E, Hasegawa T, Okada H. Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am J Clin Nutr 54:1093-1100 (1991). Adlercreutz H, Markkanen H, Watanabe S. Plasma concentrations of phyto-oestrogens in Japanese men. Lancet 342:1209-1210 (1993). Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-plienol-chloroform extraction. Anal Biochem 162:156-159 (1987). Loose-Mitchell DS, Chiapetta C, Stancel GM. Estrogen regulation of c-fos messenger ribonucleic acid. Mol Endocrinol 2:946-951 (1988). MAkela S, Santti R, Martikainen P. Nienstedt W, Paranko J. The influence of steroidal and nonsteroidal estrogens on the 5a-reduction of testosterone by the ventral prostate of the rat. J Steroid Biochem 35:249-256 (1990). Kirkland JL, Murthy L, Stancel GM. Tamoxifen stimulates expression of the c-fos protooncogene in rodent uterus. Mol Pharmacol 43:709-714 (1993). Whitten PL, Naftolin F. Effects of a phytoestrogen diet on estrogen-dependent reproductive processes in immature female rats. Steroids 57:56-61 (1992). Burroughs CD, Mills KT, Bern HA. Reproductive abnormalities in female mice exposed neonatally to various doses of coumestrol. J Toxicol Environ Health 30:105-122 (1990). Folman Y, Pope GS. The interaction in the immature mouse of potent oestrogens with coumestrol, genistein and other uterovaginotrophic compounds of low potency. J Endocrinol 34:215-225 (1966). Folman Y, Pope GS. Effect of norethisterone acetate, dimethylstilboestrol, genistein and coumestrol on uptake of [3H]oestradiol by uterus, vagina and skeletal muscle of immature mice. J Endocrinol 44:213-218 (1969). Whitten PL, Russell E, Naftolin F. Effects of a normal, humanconcentration, phytoestrogen diet on rat uterine growth. Steroids 57:98-106 (1992). Perel E, Lindner HR. Dissociation of uterotrophic action from implantation-inducing activity in two non-steroidal oestrogens (coumestrol and genistein). J Reprod Fertil 21:171-175 (1970). Volume 103, Supplement 7, October 1995 View publication stats 21. Mayr U, Butsch A, Schnieder S. Validation of two in vitro test systems for estrogenic activities with zearalenone, phytoestrogens and cereal extracts. Toxicology 74:135-149 (1992). 22. Mdkela S, Davis VL, Tally WC, Korkman J, Salo L, Vihko R, Santti R, Korach K. Dietary estrogens act through estrogen receptor mediated processes and show no antiestrogenicity in cultured breast cancer cells. Environ Health Perspect 102:572-578 (1994). 23. Martin PM, Horwitz KB, Ryan DS, McGuire WL. Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinology 103:1860-1867 (1978). 24. Mikela S, Poutanen M, Lehtimiki J, Kostian ML, Santti R, Vihko R. Estrogen-specific 170-hydroxysteroid oxidoreductase type 1 (E.C. 1.1.1.62) as a possible target for the action of phytoestrogens. Proc Soc Exp Biol Med 208:51-59 (1995). 25. Pylkkanen L, Santti R, Maentausta 0, Vihko R. Distribution of estradiol-17P hydroxysteroid oxidoreductase in the urogenital tract of control and neonatally estrogenized male mice: immunohistochemical, enzymehistochemical, and biochemical study. Prostate 20:59-72 (1992). 26. Pylkkanen L, Santti R, Salo L, Maentausta 0, Vihko R, Nurmi M. Immunohistochemical localization of estrogen-specific 170hydroxysteroid oxidoreductase in the human and mouse prostate. Prostate 25:292-300 (1994). 27. Peterson G, Barnes S. Genistein inhibition of the growth of human breast cancer cells - independence from estrogen receptors and multidrug resistance gene. Biochem Biophys Res Comm 179:661-667 (1991). 28. Akiyama T, Ishida J, Nakagawa S, Ogawara S, Watanabe S, Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262:5592-5595 (1987). 29. Markovitz J, Linassier C, Fosse P, Couprie J, Jaquemin-Sablon A, Pecq JB, Larson AK. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 49:5111-5117 (1987). 30. Fotsis T, Pepper M, Adlercreutz H, Fleischmann G, Hase T, Montesano R, Schweigerer L. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc Natl Acad Sci USA 90:2690-2694 (1993). 31. Messina M, Barnes S. The role of soy products in reducing risk of cancer. J Natl Cancer Inst 83:541-546 (1991). 127