Characterization of hepatic subfractions from phenobarbital induced cattle and their use in the study of the metabolic fate of boldenone and androsta-1,4-diene-3,17-dione

Despite the EU ban (1), anabolic steroids such as boldenone (Bol) are currently illicitly used as growth promoters for cattle fattening. Monitoring of the ban requires further knowledge about biotransformation processes to identify a possible biomarker of illegal treatment. In this study hepatic subfractions from phenobarbital (PB)-induced or untreated cattle were first characterized as to their cytochrome P450 (CYP) expression and then used to investigate the metabolic pathways of Bol and its important metabolite/precursor androsta-1,4-diene-3,17-dione (ADD).Results: As expected, the main isoforms involved in PB induction were CYP2B, CYP2C and CYP3A, showing 1.5 to 15 fold increase in enzyme activity and 2 to 3 fold increase in protein levels. Incubations with 17β-Bol revealed ADD as the major biotransformation product in both C and PB subfractions. Moreover, three hydroxylated metabolites (OH-Bol) were identified and the amount of one of them was 4 times higher in PB than in C group. Incubations with ADD yielded 17α- and 17β-Bol: both in C and PB fractions the β epimer was present to a far greater extent than the α epimer (about 20 fold). Also in this case the amount of one of the 3 OH-Bol formed was about 6 times higher in PB than in C group. Conclusions: Only the enzymes responsible for OH-Bol generation would appear to be induced by PB; moreover this study demonstrate for the first time the in vitro production of 17α-Bol as the result of ADD incubation.