Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes: Gas chromatographic–mass spectrometric determination of metabolites

Abstract

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17α-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17α-methyltestosterone, produced 6β-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography–mass spectrometry (GC–MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6β-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6β-hydroxyl metabolites with testosterone and 17α-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6β-hydroxylation.