Hepatocyte primary cultures (HPCS) as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.

Introduction. Cattle HPCs have been successfully used to investigate the metabolism and bioactivity of illicit steroids and prohormones (ISAPs). Nevertheless, their usefulness as a multi-parametric screening bioassay has never been investigated so far. Thus, cattle HPCs were incubated with known ISAPs and submitted to investigations aiming to characterize: (a) their effects on mRNA levels of drug metabolizing enzymes (DMEs) and related transcription factors (TFs); (b) on CYP3A28 apoprotein and catalytic activity; (c) the ISAPs metabolite profiling. Materials and Methods. HPCs were incubated with 100 μM boldenone (BOLD), its precursor androsta-l,4-diene-3,17-dione (ADD), dehydroepiandrosterone (DHEA) or an association of BOLD:ADD (90:10 µM). ISAPs effects on 16 DMEs and 5 TFs mRNAs were measured, after 6 hrs of incubation, by qPCR; their effect on CYP3A28 apoprotein and catalytic activity, after 24 hrs of incubation, by immunoblotting and a testosterone (TST) HPLC assay. Finally, the time-dependent (0, 3, 6 and 24 hrs) metabolite profiling of ADD, BOLD and ADD:BOLD was characterized by using a LC-HRMS technique. Results. Cells exposed to DHEA showed a significant up-regulation of CYP2C9, GSTA1, DHEA-ST, 17βHSDII, CAR, PXR, RXRα and PPARα mRNAs. In contrast, ISAPs did not alter mRNA levels in BOLD-, ADD- or BOLD:ADD-exposed cells, except for a significant reduction of CYP1A1 mRNA in ADD-incubated hepatocytes. No effect of ISAPs was ever noticed on CYP3A28 gene and protein expression, while a significant inhibition of 6β-, 2β and 16β-OHTSTase was observed in cells exposed to ADD and BOLD. Finally, the production of ADD and α-BOLD from BOLD as well as of α-BOLD and β-BOLD from ADD as main metabolites was demonstrated. Furthermore, several OH-ADD derivatives were detected, and 16-OH-ADD epimers were more abundant than 6-OH-ADD ones. As observed with other anabolic steroids, four mono-hydroxylated BOLD derivatives were isolated and tentatively identified. Conclusions. Cattle HPCs represent an useful in vitro model to study species-specific differences in xenobiotic metabolism and molecular mechanisms involved in DMEs expression and regulation, as well as a complementary tool for the screening of ISAPs abuse in cattle. If liver tissue collected at the slaughterhouse was useful to obtain viable HPCs, this in vitro model might reduce the use of experimental animals, according to the 3R’s philosophy.