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. 2002 Aug;110(8):743–748. doi: 10.1289/ehp.02110743

Dual effects of phytoestrogens result in u-shaped dose-response curves.

Kristian Almstrup 1, Mariana F Fernández 1, Jørgen H Petersen 1, Nicolas Olea 1, Niels E Skakkebaek 1, Henrik Leffers 1
PMCID: PMC1240943  PMID: 12153753

Abstract

Endocrine disruptors can affect the endocrine system without directly interacting with receptors, for example, by interfering with the synthesis or metabolism of steroid hormones. The aromatase that converts testosterone to 17beta-estradiol is a possible target. In this paper we describe an assay that simultaneously detects aromatase inhibition and estrogenicity. The principle is similar to that of other MCF-7 estrogenicity assays, but with a fixed amount of testosterone added. The endogenous aromatase activity in MCF-7 cells converts some of the testosterone to 17beta-estradiol, which is assayed by quantifying differences in the expression level of the estrogen-induced pS2 mRNA. Potential aromatase inhibitors can be identified by a dose-dependent reduction in the pS2 mRNA expression level after exposure to testosterone and the test compound. Using this assay, we have investigated several compounds, including synthetic chemicals and phytoestrogens, for aromatase inhibition. The phytoestrogens, except genistein, were aromatase inhibitors at low concentrations (< 1 micro M) but estrogenic at higher concentrations (greater than or equal to 1 micro M), resulting in U-shaped dose-response curves. None of the tested synthetic chemicals were aromatase inhibitors. The low-dose aromatase inhibition distinguished phytoestrogens from other estrogenic compounds and may partly explain reports about antiestrogenic properties of phytoestrogens. Aromatase inhibition may play an important role in the protective effects of phytoestrogens against breast cancer.

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Selected References

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  1. Adlercreutz H., Bannwart C., Wähälä K., Mäkelä T., Brunow G., Hase T., Arosemena P. J., Kellis J. T., Jr, Vickery L. E. Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochem Mol Biol. 1993 Feb;44(2):147–153. doi: 10.1016/0960-0760(93)90022-o. [DOI] [PubMed] [Google Scholar]
  2. Arai Y., Uehara M., Sato Y., Kimira M., Eboshida A., Adlercreutz H., Watanabe S. Comparison of isoflavones among dietary intake, plasma concentration and urinary excretion for accurate estimation of phytoestrogen intake. J Epidemiol. 2000 Mar;10(2):127–135. doi: 10.2188/jea.10.127. [DOI] [PubMed] [Google Scholar]
  3. Belaid B., Richard-Mercier N., Pieau C., Dorizzi M. Sex reversal and aromatase in the European pond turtle: treatment with letrozole after the thermosensitive period for sex determination. J Exp Zool. 2001 Sep 15;290(5):490–497. doi: 10.1002/jez.1092. [DOI] [PubMed] [Google Scholar]
  4. Brodie A., Lu Q., Liu Y., Long B. Aromatase inhibitors and their antitumor effects in model systems. Endocr Relat Cancer. 1999 Jun;6(2):205–210. doi: 10.1677/erc.0.0060205. [DOI] [PubMed] [Google Scholar]
  5. Burak W. E., Jr, Quinn A. L., Farrar W. B., Brueggemeier R. W. Androgens influence estrogen-induced responses in human breast carcinoma cells through cytochrome P450 aromatase. Breast Cancer Res Treat. 1997 May;44(1):57–64. doi: 10.1023/a:1005782311558. [DOI] [PubMed] [Google Scholar]
  6. Burow M. E., Boue S. M., Collins-Burow B. M., Melnik L. I., Duong B. N., Carter-Wientjes C. H., Li S., Wiese T. E., Cleveland T. E., McLachlan J. A. Phytochemical glyceollins, isolated from soy, mediate antihormonal effects through estrogen receptor alpha and beta. J Clin Endocrinol Metab. 2001 Apr;86(4):1750–1758. doi: 10.1210/jcem.86.4.7430. [DOI] [PubMed] [Google Scholar]
  7. Campbell D. R., Kurzer M. S. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes. J Steroid Biochem Mol Biol. 1993 Sep;46(3):381–388. doi: 10.1016/0960-0760(93)90228-o. [DOI] [PubMed] [Google Scholar]
  8. Davis D. L., Axelrod D., Bailey L., Gaynor M., Sasco A. J. Rethinking breast cancer risk and the environment: the case for the precautionary principle. Environ Health Perspect. 1998 Sep;106(9):523–529. doi: 10.1289/ehp.98106523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dowsett M., Lee K., Macaulay V. M., Detre S., Rowlands M., Grimshaw R. The control and biological importance of intratumoural aromatase in breast cancer. J Steroid Biochem Mol Biol. 1996 Jan;56(1-6):145–150. doi: 10.1016/0960-0760(95)00231-6. [DOI] [PubMed] [Google Scholar]
  10. Dukes M., Edwards P. N., Large M., Smith I. K., Boyle T. The preclinical pharmacology of "Arimidex" (anastrozole; ZD1033)--a potent, selective aromatase inhibitor. J Steroid Biochem Mol Biol. 1996 Jul;58(4):439–445. doi: 10.1016/0960-0760(96)00064-7. [DOI] [PubMed] [Google Scholar]
  11. Gray S. A., Mannan M. A., O'Shaughnessy P. J. Development of cytochrome P450 aromatase mRNA levels and enzyme activity in ovaries of normal and hypogonadal (hpg) mice. J Mol Endocrinol. 1995 Jun;14(3):295–301. doi: 10.1677/jme.0.0140295. [DOI] [PubMed] [Google Scholar]
  12. Hargreaves D. F., Potten C. S., Harding C., Shaw L. E., Morton M. S., Roberts S. A., Howell A., Bundred N. J. Two-week dietary soy supplementation has an estrogenic effect on normal premenopausal breast. J Clin Endocrinol Metab. 1999 Nov;84(11):4017–4024. doi: 10.1210/jcem.84.11.6152. [DOI] [PubMed] [Google Scholar]
  13. Hilakivi-Clarke L., Cho E., Clarke R. Maternal genistein exposure mimics the effects of estrogen on mammary gland development in female mouse offspring. Oncol Rep. 1998 May-Jun;5(3):609–616. doi: 10.3892/or.5.3.609. [DOI] [PubMed] [Google Scholar]
  14. Hilakivi-Clarke L., Cho E., Onojafe I., Raygada M., Clarke R. Maternal exposure to genistein during pregnancy increases carcinogen-induced mammary tumorigenesis in female rat offspring. Oncol Rep. 1999 Sep-Oct;6(5):1089–1095. doi: 10.3892/or.6.5.1089. [DOI] [PubMed] [Google Scholar]
  15. Humfrey C. D. Phytoestrogens and human health effects: weighing up the current evidence. Nat Toxins. 1998;6(2):51–59. doi: 10.1002/(sici)1522-7189(199804)6:2<51::aid-nt11>3.0.co;2-9. [DOI] [PubMed] [Google Scholar]
  16. Ito K., Fukabori Y., Shibata Y., Suzuki K., Mieda M., Gotanda K., Honma S., Yamanaka H. Effects of a new steroidal aromatase inhibitor, TZA-2237, and/or chlormadinone acetate on hormone-induced and spontaneous canine benign prostatic hyperplasia. Eur J Endocrinol. 2000 Oct;143(4):543–554. doi: 10.1530/eje.0.1430543. [DOI] [PubMed] [Google Scholar]
  17. Jakowlew S. B., Breathnach R., Jeltsch J. M., Masiakowski P., Chambon P. Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7. Nucleic Acids Res. 1984 Mar 26;12(6):2861–2878. doi: 10.1093/nar/12.6.2861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jørgensen M., Bévort M., Kledal T. S., Hansen B. V., Dalgaard M., Leffers H. Differential display competitive polymerase chain reaction: an optimal tool for assaying gene expression. Electrophoresis. 1999 Feb;20(2):230–240. doi: 10.1002/(SICI)1522-2683(19990201)20:2<230::AID-ELPS230>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  19. Jørgensen M., Vendelbo B., Skakkebaek N. E., Leffers H. Assaying estrogenicity by quantitating the expression levels of endogenous estrogen-regulated genes. Environ Health Perspect. 2000 May;108(5):403–412. doi: 10.1289/ehp.108-1638061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kao Y. C., Zhou C., Sherman M., Laughton C. A., Chen S. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study. Environ Health Perspect. 1998 Feb;106(2):85–92. doi: 10.1289/ehp.9810685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kavlock R. J., Daston G. P., DeRosa C., Fenner-Crisp P., Gray L. E., Kaattari S., Lucier G., Luster M., Mac M. J., Maczka C. Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop. Environ Health Perspect. 1996 Aug;104 (Suppl 4):715–740. doi: 10.1289/ehp.96104s4715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kellis J. T., Jr, Vickery L. E. Inhibition of human estrogen synthetase (aromatase) by flavones. Science. 1984 Sep 7;225(4666):1032–1034. doi: 10.1126/science.6474163. [DOI] [PubMed] [Google Scholar]
  23. Kitawaki J., Kim T., Kanno H., Noguchi T., Yamamoto T., Okada H. Growth suppression of MCF-7 human breast cancer cells by aromatase inhibitors: a new system for aromatase inhibitor screening. J Steroid Biochem Mol Biol. 1993 Mar;44(4-6):667–670. doi: 10.1016/0960-0760(93)90277-4. [DOI] [PubMed] [Google Scholar]
  24. Knight D. C., Eden J. A. A review of the clinical effects of phytoestrogens. Obstet Gynecol. 1996 May;87(5 Pt 2):897–904. [PubMed] [Google Scholar]
  25. Kurzer M. S., Xu X. Dietary phytoestrogens. Annu Rev Nutr. 1997;17:353–381. doi: 10.1146/annurev.nutr.17.1.353. [DOI] [PubMed] [Google Scholar]
  26. Labrie F., Luu-The V., Lin S. X., Simard J., Labrie C., El-Alfy M., Pelletier G., Bélanger A. Intracrinology: role of the family of 17 beta-hydroxysteroid dehydrogenases in human physiology and disease. J Mol Endocrinol. 2000 Aug;25(1):1–16. doi: 10.1677/jme.0.0250001. [DOI] [PubMed] [Google Scholar]
  27. Le Bail J. C., Champavier Y., Chulia A. J., Habrioux G. Effects of phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer cells. Life Sci. 2000 Feb 25;66(14):1281–1291. doi: 10.1016/s0024-3205(00)00435-5. [DOI] [PubMed] [Google Scholar]
  28. Leffers H., Naesby M., Vendelbo B., Skakkebaek N. E., Jørgensen M. Oestrogenic potencies of Zeranol, oestradiol, diethylstilboestrol, Bisphenol-A and genistein: implications for exposure assessment of potential endocrine disrupters. Hum Reprod. 2001 May;16(5):1037–1045. doi: 10.1093/humrep/16.5.1037. [DOI] [PubMed] [Google Scholar]
  29. Lu Q., Yue W., Wang J., Liu Y., Long B., Brodie A. The effects of aromatase inhibitors and antiestrogens in the nude mouse model. Breast Cancer Res Treat. 1998 Jul;50(1):63–71. doi: 10.1023/a:1006004930930. [DOI] [PubMed] [Google Scholar]
  30. Matzkin H., Soloway M. S. Immunohistochemical evidence of the existence and localization of aromatase in human prostatic tissues. Prostate. 1992;21(4):309–314. doi: 10.1002/pros.2990210407. [DOI] [PubMed] [Google Scholar]
  31. Mäkelä S. I., Pylkkänen L. H., Santti R. S., Adlercreutz H. Dietary soybean may be antiestrogenic in male mice. J Nutr. 1995 Mar;125(3):437–445. doi: 10.1093/jn/125.3.437. [DOI] [PubMed] [Google Scholar]
  32. Nabholtz J. M., Buzdar A., Pollak M., Harwin W., Burton G., Mangalik A., Steinberg M., Webster A., von Euler M. Anastrozole is superior to tamoxifen as first-line therapy for advanced breast cancer in postmenopausal women: results of a North American multicenter randomized trial. Arimidex Study Group. J Clin Oncol. 2000 Nov 15;18(22):3758–3767. doi: 10.1200/JCO.2000.18.22.3758. [DOI] [PubMed] [Google Scholar]
  33. Nathan L., Shi W., Dinh H., Mukherjee T. K., Wang X., Lusis A. J., Chaudhuri G. Testosterone inhibits early atherogenesis by conversion to estradiol: critical role of aromatase. Proc Natl Acad Sci U S A. 2001 Mar 6;98(6):3589–3593. doi: 10.1073/pnas.051003698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Newbold R. R., Banks E. P., Bullock B., Jefferson W. N. Uterine adenocarcinoma in mice treated neonatally with genistein. Cancer Res. 2001 Jun 1;61(11):4325–4328. [PubMed] [Google Scholar]
  35. North K., Golding J. A maternal vegetarian diet in pregnancy is associated with hypospadias. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood. BJU Int. 2000 Jan;85(1):107–113. doi: 10.1046/j.1464-410x.2000.00436.x. [DOI] [PubMed] [Google Scholar]
  36. Oz O. K., Zerwekh J. E., Fisher C., Graves K., Nanu L., Millsaps R., Simpson E. R. Bone has a sexually dimorphic response to aromatase deficiency. J Bone Miner Res. 2000 Mar;15(3):507–514. doi: 10.1359/jbmr.2000.15.3.507. [DOI] [PubMed] [Google Scholar]
  37. Pelissero C., Lenczowski M. J., Chinzi D., Davail-Cuisset B., Sumpter J. P., Fostier A. Effects of flavonoids on aromatase activity, an in vitro study. J Steroid Biochem Mol Biol. 1996 Feb;57(3-4):215–223. doi: 10.1016/0960-0760(95)00261-8. [DOI] [PubMed] [Google Scholar]
  38. Qian Y., Deng C., Song W. C. Expression of estrogen sulfotransferase in MCF-7 cells by cDNA transfection suppresses the estrogen response: potential role of the enzyme in regulating estrogen-dependent growth of breast epithelial cells. J Pharmacol Exp Ther. 1998 Jul;286(1):555–560. [PubMed] [Google Scholar]
  39. Ruh M. F., Zacharewski T., Connor K., Howell J., Chen I., Safe S. Naringenin: a weakly estrogenic bioflavonoid that exhibits antiestrogenic activity. Biochem Pharmacol. 1995 Oct 26;50(9):1485–1493. doi: 10.1016/0006-2952(95)02061-6. [DOI] [PubMed] [Google Scholar]
  40. Sadekova S. I., Tan L., Chow T. Y. Identification of the aromatase in the breast carcinoma cell lines T47D and MCF-7. Anticancer Res. 1994 Mar-Apr;14(2A):507–511. [PubMed] [Google Scholar]
  41. Schmitt M., Klinga K., Schnarr B., Morfin R., Mayer D. Dehydroepiandrosterone stimulates proliferation and gene expression in MCF-7 cells after conversion to estradiol. Mol Cell Endocrinol. 2001 Feb 28;173(1-2):1–13. doi: 10.1016/s0303-7207(00)00442-1. [DOI] [PubMed] [Google Scholar]
  42. Soto A. M., Sonnenschein C., Chung K. L., Fernandez M. F., Olea N., Serrano F. O. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect. 1995 Oct;103 (Suppl 7):113–122. doi: 10.1289/ehp.95103s7113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tham D. M., Gardner C. D., Haskell W. L. Clinical review 97: Potential health benefits of dietary phytoestrogens: a review of the clinical, epidemiological, and mechanistic evidence. J Clin Endocrinol Metab. 1998 Jul;83(7):2223–2235. doi: 10.1210/jcem.83.7.4752. [DOI] [PubMed] [Google Scholar]
  44. Toppari J., Larsen J. C., Christiansen P., Giwercman A., Grandjean P., Guillette L. J., Jr, Jégou B., Jensen T. K., Jouannet P., Keiding N. Male reproductive health and environmental xenoestrogens. Environ Health Perspect. 1996 Aug;104 (Suppl 4):741–803. doi: 10.1289/ehp.96104s4741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wickman S., Sipilä I., Ankarberg-Lindgren C., Norjavaara E., Dunkel L. A specific aromatase inhibitor and potential increase in adult height in boys with delayed puberty: a randomised controlled trial. Lancet. 2001 Jun 2;357(9270):1743–1748. doi: 10.1016/S0140-6736(00)04895-9. [DOI] [PubMed] [Google Scholar]
  46. Yue W., Wang J. P., Hamilton C. J., Demers L. M., Santen R. J. In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res. 1998 Mar 1;58(5):927–932. [PubMed] [Google Scholar]

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