A yeast screen system for aromatase inhibitors and ligands for androgen receptor: Yeast cells transformed with aromatase and androgen receptor

Endocrine disrupters are hormone mimics that modify hormonal action in huma ns and animals. It is thought that some endocrine disrupters modify estroge n and androgen action in humans and animals by suppressing aromatase activi ty. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed at novel aromatase inhibitor screening method that allows us to identify antiaromatase activit y of various environmental chemicals. The screen was developed by coexpress ing the human aromatase and the mouse androgen receptor in yeast cells, whi ch carry the androgen-responsive beta-galactosidase reporter plasmid. Funct ional expression of aromatase in yeast has been demonstrated using the [H-3 ]-water release assay with intact cells as well as with yeast microsomes. T he aromatase activity could be blocked by known aromatase inhibitors such a s aminoglutethimide (AG). Yeast-produced androgen receptors were able to tr ansactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responde d to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotest osterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, trans criptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and andros tenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphth olflavone, are inhibitors of aromatase. Thus, this yeast system allows us t o develop a high-throughput screening method, without using radioactive sub strate, to identify aromatase inhibitors as well as new ligands (nonaromati zable androgen mimics) for the androgen receptors. In addition, this screen ing 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 activi ty and for their interaction with androgen receptor.