Aromatization of 7 alpha-methyl-19-nortestosterone by human placental microsomes in vitro.
Part of the biological effects of testosterone (T) are mediated by its enzymatic reduction to 5 alpha-dihydrotestosterone (DHT) or aromatization to estradiol (E2). 7 alpha-Methyl-19-nortestosterone (MENT) is a synthetic androgen that is considerably more potent than T. Previous studies have shown that MENT is not 5 alpha-reduced. The studies reported here were undertaken to determine whether MENT undergoes enzymatic aromatization in vitro. Human placental microsomes were used as the source of the aromatase. Radioactive or nonradioactive T or MENT was incubated with the microsomes in the presence of NADPH and the metabolites extracted out with ethyl ether. Following evaporation of ether, the residue was dissolved in benzene-petroleum ether and extracted with 0.4 N NaOH which selectively removes phenolic metabolites of the androgens. When either radioactive T or MENT was incubated with the aromatase in the presence of NADPH, there was a 20-fold increase in the amount of radioactivity extracted with NaOH. In contrast, if the incubation was carried out in the absence of NADPH or in the presence of R76713, an aromatase inhibitor, most of the radioactivity remained in the benzene-petroleum ether phase. To further identify the enzymatic reaction products, thin layer chromatography (TLC) was performed. The Rf value for MENT was 0.22 while that of the major reaction product was 0.34, which corresponded with the RF value of the estrogen, 7 alpha-methyl-estradiol (MeE2). This was further verified by using a second solvent system for the chromatographic separation. In an effort to ascertain whether the metabolites bind to estrogen receptors (ER), rat uterine cytosol was used. NaOH extracts of medium following incubation of nonradioactive MENT with microsomes showed competitive inhibition of [3H]E2 binding to rat uterine ER. Furthermore, after [3H]MENT was incubated with microsomes, the radioactive metabolite extracted in NaOH showed specific binding to the ER which could readily be displaced with E2 or MeE2. These results indicate that like T, MENT undergoes enzymatic aromatization.
LaMorte
A
A
Center for Biomedical Research, Population Council, New York, NY 10021.
Kumar
N
N
Bardin
C W
CW
Sundaram
K
K
D001141:Aromatase / Q000378:metabolism*; D001667:Binding, Competitive; D002855:Chromatography, Thin Layer; D005260:Female; D006801:Humans; D008861:Microsomes / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D009277:Nandrolone / Q000031:analogs & derivatives* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology* / Q000648:ultrastructure; D011247:Pregnancy; D011960:Receptors, Estrogen / Q000378:metabolism; D012997:Solvents; D013739:Testosterone / Q000378:metabolism
D011960:Receptors, Estrogen; D012997:Solvents; D013739:Testosterone; C064709:trestolone; D009249:NADP; D009277:Nandrolone; D001141:Aromatase