title: Non-steroidal inhibition of granulosa cell aromatase activity in vitro.
abstract: Treatment of cyclic rats with the substituted triazole R151885 (1,1-di (4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol causes delayed ovulation with suppressed blood oestradiol levels. To determine if R151885 can exert a direct action on ovarian oestrogen biosynthesis, we studied its effect on steroidogenesis in granulosa cell cultures from prepubertal rat ovaries. The cells were incubated for 48 h in medium containing 100 ng human FSH/ml and 10(-7) M testosterone to induce steroidogenic enzymes. When R151885 was also present in the culture medium, there was a marked and concentration-dependent reduction in granulosa cell oestradiol production. Inhibition was half-maximal at approx 3 X 10(-7) M and almost complete at 10(-5) M R151885. Progesterone and 20 alpha-hydroxy-4-pregnen-3-one production were unaffected except by the highest concentration of the substituted triazole (36% inhibition at 10(-5) M). Direct assessment of aromatase activity in the 48-h cultured monolayers (oestradiol formation during a 3-h incubation with 10(-7) M testosterone) was made to determine if the inhibitory effect of R151885 was due to reduced aromatase induction/activation. This was not the case, since cells cultured in the presence of 10(-6) or 10(-5) M R151885 had levels of aromatase up to 60% higher than those cultured in its absence. To determine acute effects of R151885 on testosterone (10(-7) M) aromatization, 3-h incubations were carried out using granulosa cell suspensions with high extant aromatase activity due to stimulation by ovine FSH (100 micrograms sc, twice daily for 2 days) in vivo. The triazole acted as an apparent competitive aromatase inhibitor (apparent Km for testosterone 2.5 X 10(-8) M in the absence of R151885 rising to 4.4 X 10(-8) M in the presence of 10(-7) M R151885). Its potency as an aromatase inhibitor was approximately 10 times greater than that of the naturally occurring steroidal aromatase inhibitor 5 alpha-dihydrotestosterone. Various structurally related substances proved to be even more potent aromatase inhibitors than R151885. The most active were also substituted 4,4'-difluorophenyl derivatives containing an imidazolyl or pyridyl moiety instead of the 1,2,4-triazolyl substituent in R151885. This study has identified a novel series of nonsteroidal substances which have the characteristics of potent and specific inhibitors of testosterone aromatization by rat granulosa cells in vitro.
mesh_terms: D000818:Animals; D047072:Aromatase Inhibitors; D001667:Binding, Competitive; D002478:Cells, Cultured; D013196:Dihydrotestosterone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D011372:Progestins / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D013739:Testosterone / Q000378:metabolism / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology*
chemical_list: D047072:Aromatase Inhibitors; D011372:Progestins; D014230:Triazoles; D013196:Dihydrotestosterone; D013739:Testosterone; D004958:Estradiol; C048318:R 151885; D005640:Follicle Stimulating Hormone