Testosterone oxidation by rat liver microsomes: Effects of phenobarbital pretreatment and the detection of seven metabolites by HPLC

Genetically engineered V79 chinese hamster cell expression of purified cytochrome P‐450iib1 monooxygenase activity

“…For our purposes, photometric detection and quantification were well suited, com- bining satisfactory sensitivity with experimental convenience. Incubation of testosterone with the microsomal liver fraction of untreated male Sprague-Dawley rats resulted in the formation of 2a-, 16a-OHT and 17-OT as main oxidated metabolites followed by 7a-and 60-OHT and very small amounts of 6a-, 160-, and 20-OHT (Table 1) in accordance with previous reports (23,30,31,37). The conversion to these metabolites proceeded proportionally to the amount of microsoma1 protein (up to 1.5 mg) and to the incubation time (up to 15 min) (data not shown).…”

“…With the exception of 2a-OHT, all other metabolites contributed to this increase, but to strikingly different extents; most pronounced was the increase of 160-OHT by a factor of 26 (Table 1). This result, which corresponds well with the literature (31,37) is the consequence of the induction of cytochromes P-450b and e in the liver of phenobarbital-treated rats (42). Both isozymes catalyze the formation of 16a-, 160-OHT, and 17-OT (14,16,17,19).…”

“…The oxidative metabolism of several A*-3-keto steroids, that is, progesterone (20,24), testosterone (13-20,25-41), 4-androsten-3,17-dione (14,15,20,25) has been used to characterize the cytochrome P-450-dependent monooxygenases in microsomal fractions of different organs (19,28,38,40), after pretreatment with drugs (25,32,(34)(35)(36) and enzyme inducers (25)(26)(27)(28)(29)(30)(31)33,36,37,39,41) and to distinguish between isozymes of cytochrome P-450 (13-20). Since the most extensive information has been accumulated on the catalytic activity of cytochrome P-450 isozymes toward the regio-and stereoselective conversion of testosterone to hydroxylated metabolites (Figure l), we chose this steroid for the characterization of the monooxygenase expressed by SD1 cells.…”

“…For the separation of the metabolites, paper chromatography (25), thin-layer chromatography (15,(28)(29)(30)33), and HPLC (14,15,23,31,36) were applied followed by radiometric (15,25,29,33) or photometric quantification (14,23,30,31,36). For the chromatographic separation of hydroxytestosterones, we used the HPLC system described by van der Hoeven (23) that was modified by taking a binary instead of a ternary solvent delivery system (23).…”

“…Both isozymes catalyze the formation of 16a-, 160-OHT, and 17-OT (14,16,17,19). In hepatic microsomes of phenobarbital-treated male rats, these three metabolites were indeed the most prominent products of oxidative testosterone metabolism among other hydroxytestosterones, that is, 7a-, 60-, 2a-, 20-, and 6a-OHT (Table 1) (14,19,31). The strongest indication for the induction of cytochromes P-450b and e, however, is the occurrence of 16P-OHT as one of the main metabolites.…”