Skip to main content
PMC home page
Environmental Health Perspectives logoLink to Environmental Health Perspectives
. 2001 Jul;109(7):691–697. doi: 10.1289/ehp.01109691

Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder.

P De Boever 1, W Demaré 1, E Vanderperren 1, K Cooreman 1, P Bossier 1, W Verstraete 1
PMCID: PMC1240372  PMID: 11485867

Abstract

Here we describe a redesigned protocol of the yeast estrogen screen developed by Routledge and Sumpter. The redesigned test comprises two steps. First, a large amount of yeast with estrogenic compounds is incubated for 24 hr. Subsequently, a mixture of cycloheximide and the chromogenic substrate chlorophenol red-beta-d-galactopyranoside (CPRG) is added. The cycloheximide stops protein synthesis and allows for an end-point measurement of beta-galactosidase activity generated during the first 24 hr. CPRG is converted to chlorophenol red and reflects beta-galactosidase activity, which is indicative of the estrogenic activity. The modifications shorten the duration of the assay at least 1 day and avoid interference of the estrogenic CPRG or chlorophenol red. The redesigned and the original protocol were used to study the estrogenic activity of bisphenol A, methoxychlor, p,p'-DDT, and isoflavones (genistein, daidzein, and glycitein). Bisphenol A, methoxychlor, and genistein triggered higher levels of beta-galactosidase activity in the redesigned protocol. Estrogenic activity of p,p'-DDT could only be demonstrated with the redesigned protocol. Glycitein and daidzein failed to give a response with both protocols. We also studied deconjugation of beta-glycosidic isoflavones present in soygerm powder. Treatment of the soygerm powder with beta-glycosidase released isoflavones. The estrogenic response of the samples was confirmed with the redesigned protocol and correlated with the amount of genistein present. The release of isoflavones under conditions prevailing in the intestines was studied. Bacterial beta-glycosidase present in the large intestine released isoflavones, and moderate estrogenic activity could be demonstrated.

Full Text

The Full Text of this article is available as a PDF (114.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anthony M. S., Clarkson T. B., Williams J. K. Effects of soy isoflavones on atherosclerosis: potential mechanisms. Am J Clin Nutr. 1998 Dec;68(6 Suppl):1390S–1393S. doi: 10.1093/ajcn/68.6.1390S. [DOI] [PubMed] [Google Scholar]
  2. Arnold S. F., Collins B. M., Robinson M. K., Guillette L. J., Jr, McLachlan J. A. Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen. Steroids. 1996 Nov;61(11):642–646. doi: 10.1016/s0039-128x(96)00183-3. [DOI] [PubMed] [Google Scholar]
  3. Arnold S. F., Robinson M. K., Notides A. C., Guillette L. J., Jr, McLachlan J. A. A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens. Environ Health Perspect. 1996 May;104(5):544–548. doi: 10.1289/ehp.96104544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beresford N., Routledge E. J., Harris C. A., Sumpter J. P. Issues arising when interpreting results from an in vitro assay for estrogenic activity. Toxicol Appl Pharmacol. 2000 Jan 1;162(1):22–33. doi: 10.1006/taap.1999.8817. [DOI] [PubMed] [Google Scholar]
  5. Berg J. O., Nord C. E., Wadström T. Formation of glycosidases in batch and continuous culture of Bacteroides fragilis. Appl Environ Microbiol. 1978 Feb;35(2):269–273. doi: 10.1128/aem.35.2.269-273.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brandi M. L. Natural and synthetic isoflavones in the prevention and treatment of chronic diseases. Calcif Tissue Int. 1997;61 (Suppl 1):S5–S8. doi: 10.1007/s002239900376. [DOI] [PubMed] [Google Scholar]
  7. Breithofer A., Graumann K., Scicchitano M. S., Karathanasis S. K., Butt T. R., Jungbauer A. Regulation of human estrogen receptor by phytoestrogens in yeast and human cells. J Steroid Biochem Mol Biol. 1998 Dec;67(5-6):421–429. doi: 10.1016/s0960-0760(98)00139-3. [DOI] [PubMed] [Google Scholar]
  8. Brzezinski A., Debi A. Phytoestrogens: the "natural" selective estrogen receptor modulators? Eur J Obstet Gynecol Reprod Biol. 1999 Jul;85(1):47–51. doi: 10.1016/s0301-2115(98)00281-4. [DOI] [PubMed] [Google Scholar]
  9. Chen C. W., Hurd C., Vorojeikina D. P., Arnold S. F., Notides A. C. Transcriptional activation of the human estrogen receptor by DDT isomers and metabolites in yeast and MCF-7 cells. Biochem Pharmacol. 1997 Apr 25;53(8):1161–1172. doi: 10.1016/s0006-2952(97)00097-x. [DOI] [PubMed] [Google Scholar]
  10. Coldham N. G., Dave M., Sivapathasundaram S., McDonnell D. P., Connor C., Sauer M. J. Evaluation of a recombinant yeast cell estrogen screening assay. Environ Health Perspect. 1997 Jul;105(7):734–742. doi: 10.1289/ehp.97105734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Collins B. M., McLachlan J. A., Arnold S. F. The estrogenic and antiestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast. Steroids. 1997 Apr;62(4):365–372. doi: 10.1016/s0039-128x(96)00246-2. [DOI] [PubMed] [Google Scholar]
  12. Cooper R. L., Kavlock R. J. Endocrine disruptors and reproductive development: a weight-of-evidence overview. J Endocrinol. 1997 Feb;152(2):159–166. doi: 10.1677/joe.0.1520159. [DOI] [PubMed] [Google Scholar]
  13. Cooper T. G., Bossinger J. Selective inhibition of protein synthesis initiation in Saccharomyces cerevisiae by low concentrations of cycloheximide. J Biol Chem. 1976 Nov 25;251(22):7278–7280. [PubMed] [Google Scholar]
  14. Daston G. P., Gooch J. W., Breslin W. J., Shuey D. L., Nikiforov A. I., Fico T. A., Gorsuch J. W. Environmental estrogens and reproductive health: a discussion of the human and environmental data. Reprod Toxicol. 1997 Jul-Aug;11(4):465–481. doi: 10.1016/s0890-6238(97)00014-2. [DOI] [PubMed] [Google Scholar]
  15. Davis D. L., Bradlow H. L. Can environmental estrogens cause breast cancer? Sci Am. 1995 Oct;273(4):167–172. [PubMed] [Google Scholar]
  16. De Boever P, Verstraete W. Bile salt deconjugation by lactobacillus plantarum 80 and its implication for bacterial toxicity. J Appl Microbiol. 1999 Sep;87(3):345–352. doi: 10.1046/j.1365-2672.1999.00019.x. [DOI] [PubMed] [Google Scholar]
  17. De Boever P., Wouters R., Verschaeve L., Berckmans P., Schoeters G., Verstraete W. Protective effect of the bile salt hydrolase-active Lactobacillus reuteri against bile salt cytotoxicity. Appl Microbiol Biotechnol. 2000 Jun;53(6):709–714. doi: 10.1007/s002530000330. [DOI] [PubMed] [Google Scholar]
  18. Decottignies A., Goffeau A. Complete inventory of the yeast ABC proteins. Nat Genet. 1997 Feb;15(2):137–145. doi: 10.1038/ng0297-137. [DOI] [PubMed] [Google Scholar]
  19. Fioravanti L., Cappelletti V., Miodini P., Ronchi E., Brivio M., Di Fronzo G. Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro. Cancer Lett. 1998 Aug 14;130(1-2):143–152. doi: 10.1016/s0304-3835(98)00130-x. [DOI] [PubMed] [Google Scholar]
  20. Gaido K. W., Leonard L. S., Lovell S., Gould J. C., Babaï D., Portier C. J., McDonnell D. P. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol Appl Pharmacol. 1997 Mar;143(1):205–212. doi: 10.1006/taap.1996.8069. [DOI] [PubMed] [Google Scholar]
  21. Goffeau A., Park J., Paulsen I. T., Jonniaux J. L., Dinh T., Mordant P., Saier M. H., Jr Multidrug-resistant transport proteins in yeast: complete inventory and phylogenetic characterization of yeast open reading frames with the major facilitator superfamily. Yeast. 1997 Jan;13(1):43–54. doi: 10.1002/(SICI)1097-0061(199701)13:1<43::AID-YEA56>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  22. Graumann K., Breithofer A., Jungbauer A. Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds? Sci Total Environ. 1999 Jan 12;225(1-2):69–79. doi: 10.1016/s0048-9697(99)80018-7. [DOI] [PubMed] [Google Scholar]
  23. Gustafsson J. A. Estrogen receptor beta--a new dimension in estrogen mechanism of action. J Endocrinol. 1999 Dec;163(3):379–383. doi: 10.1677/joe.0.1630379. [DOI] [PubMed] [Google Scholar]
  24. Heuman D. M. Bile salt-membrane interactions and the physico-chemical mechanisms of bile salt toxicity. Ital J Gastroenterol. 1995 Sep;27(7):372–375. [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. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Lichtenstein A. H. Soy protein, isoflavones and cardiovascular disease risk. J Nutr. 1998 Oct;128(10):1589–1592. doi: 10.1093/jn/128.10.1589. [DOI] [PubMed] [Google Scholar]
  28. Nilsson S., Kuiper G., Gustafsson J. A. ERbeta: a novel estrogen receptor offers the potential for new drug development. Trends Endocrinol Metab. 1998 Dec;9(10):387–395. doi: 10.1016/s1043-2760(98)00096-4. [DOI] [PubMed] [Google Scholar]
  29. Pazzi P., Puviani A. C., Dalla Libera M., Guerra G., Ricci D., Gullini S., Ottolenghi C. Bile salt-induced cytotoxicity and ursodeoxycholate cytoprotection: in-vitro study in perifused rat hepatocytes. Eur J Gastroenterol Hepatol. 1997 Jul;9(7):703–709. doi: 10.1097/00042737-199707000-00011. [DOI] [PubMed] [Google Scholar]
  30. Sathyamoorthy N., Wang T. T. Differential effects of dietary phyto-oestrogens daidzein and equol on human breast cancer MCF-7 cells. Eur J Cancer. 1997 Dec;33(14):2384–2389. doi: 10.1016/s0959-8049(97)00303-1. [DOI] [PubMed] [Google Scholar]
  31. Setchell K. D., Cassidy A. Dietary isoflavones: biological effects and relevance to human health. J Nutr. 1999 Mar;129(3):758S–767S. doi: 10.1093/jn/129.3.758S. [DOI] [PubMed] [Google Scholar]
  32. Setchell K. D. Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. Am J Clin Nutr. 1998 Dec;68(6 Suppl):1333S–1346S. doi: 10.1093/ajcn/68.6.1333S. [DOI] [PubMed] [Google Scholar]
  33. Sharpe R. M., Skakkebaek N. E. Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet. 1993 May 29;341(8857):1392–1395. doi: 10.1016/0140-6736(93)90953-e. [DOI] [PubMed] [Google Scholar]
  34. Sharpe R. M. The roles of oestrogen in the male. Trends Endocrinol Metab. 1998 Nov;9(9):371–377. doi: 10.1016/s1043-2760(98)00089-7. [DOI] [PubMed] [Google Scholar]
  35. Sohoni P., Sumpter J. P. Several environmental oestrogens are also anti-androgens. J Endocrinol. 1998 Sep;158(3):327–339. doi: 10.1677/joe.0.1580327. [DOI] [PubMed] [Google Scholar]
  36. Song T. T., Hendrich S., Murphy P. A. Estrogenic activity of glycitein, a soy isoflavone. J Agric Food Chem. 1999 Apr;47(4):1607–1610. doi: 10.1021/jf981054j. [DOI] [PubMed] [Google Scholar]
  37. Zhang Y., Song T. T., Cunnick J. E., Murphy P. A., Hendrich S. Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations. J Nutr. 1999 Feb;129(2):399–405. doi: 10.1093/jn/129.2.399. [DOI] [PubMed] [Google Scholar]
  38. Zhang Y., Wang G. J., Song T. T., Murphy P. A., Hendrich S. Urinary disposition of the soybean isoflavones daidzein, genistein and glycitein differs among humans with moderate fecal isoflavone degradation activity. J Nutr. 1999 May;129(5):957–962. doi: 10.1093/jn/129.5.957. [DOI] [PubMed] [Google Scholar]

Articles from Environmental Health Perspectives are provided here courtesy of National Institute of Environmental Health Sciences

RESOURCES