| A Yeast Screen System for Aromatase Inhibitors and Ligands for Androgen Receptor: Yeast Cells Transformed with Aromatase and Androgen Receptor Paul Mak,1 Fernando Dela Cruz,2 and Shiuan Chen3 1Wyeth-Ayerst Research, Pearl River, New York, USA
2American Cyanamid Company, Princeton, New Jersey, USA
3Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California, USA Abstract
Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ß-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG) . Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 -dihydrotestosterone (5-DHT) . In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and -naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast screening method will be useful to screen environmental chemicals for their antiaromatase activity and for their interaction with androgen receptor. Key words: androgen receptor, aromatase, cytochrome P450, endocrine disruptors, yeast screening method. Environ Health Perspect 107:855-860 (1999) . [Online September 28, 1999] http://ehpnet1.niehs.nih.gov/docs/1999/107p855-860mak/ abstract.html Address correspondence to S. Chen, Division of Immunology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte, CA 91910 USA. Telephone: (626) 359-8111, ext. 2601. Fax: (626) 301-8186. E-mail: schen@smtplink.coh.org S.C. was supported by NIEHS grant ES08258. Received 26 February 1999 ; accepted 17 June 1999.
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