The effects of diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxamide (4-ma) and (4r)-5,10-SECO-19-norpregna-4,5-diene-3,10,20-trione (SECO) on androgen biosynthesis in the rat testis and epididymis

Abstract

We have investigated the effects of two 4-ene-steroid 5α-reductase inhibitors, diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxamide (4-MA) and (4R)-5,10-seco-19-norpregna-4,5-diene-3, 10,20-trione (SECO), on testicular and epididymal androgen biosynthesis. Kinetic analyses revealed that both compounds inhibited epididymal DHT biosynthesis. 4-MA was a competitive inhibitor of epididymal nuclear and microsomal 4-ene-steroid 5α-reductases (3-oxo-5α -steroid: NADP 4-ene-oxidoreductase EC 1.3.1.22) with K i app values of 12.8 and 15.1 nmol/1 compared to the respective K m app values of 185 and 240 nmol/l. Values for the V max app were always within 70–130% of the control. SECO at 1.0 μmol/l, also inhibited epididymal nuclear and microsomal 4-ene-steroid-5α-reductases, causing respectively 2.9 and 5.2-fold increases in K m app . The V V app values were unchanged. However, SECO concentrations of 5 and 25 μmol/l abolished 4-ene-steroid 5α-reductase activity at all testosterone concentrations. To examine the specificity of these compounds, we investigated their effects on the enzymes that convert pregnenolone to testosterone. Rat testis microsomes converted pregnenolone to testosterone via the 4-ene-3-oxo pathway, with the major metabolites being progesterone, 17-hydroxyprogesterone, 4-androstenedione and testosterone; some 17-hydroxypregnenolone was also formed. Very small amounts of dehydroepiandrosterone (DHA) and 5-androstenediol were detected. SECO, at a concentration that completely inhibited epididymal 4-ene-steroid 5α-reductase activity, did not alter the metabolic profile of pregnenolone metabolism. However, 4-MA prevented the appearance of 4-ene steroids, and large quantities of 17-hydroxypregnenolone and DHA accumulated, suggesting that inhibition of the 3β-hydroxysteroid: NAD(P) + oxidoreductase (EC 1.1.1.51) and 3-oxosteroid 5-ene-4-ene-isomerase (EC 5.3.3.1) [3β-hydroxysteroid dehydrogenase-isomerase] was occurring. Optimal conditions for the microsomal conversion of DHA to 4-androstenedione were determined; kinetic analyses of the 3β -hydroxysteroid dehydrogenase-isomerase activity revealed that 4-MA inhibited this reaction non-competitively, reducing V max app values to 25% of the control. The K i app determined from the intercept replot, was 121 nmol/1, and the V m app was always between 90 and 130% of the control value. It is concluded that SECO is more specific than 4-MA in its effects on androgen biosynthesis in the testis and epididymis and that both these drugs should provide useful tools in assessments of the relative contributions of 5α-reduced androgens to androgen dependent processes.