Functional impact of cytochrome P450 3A (CYP3A) exonic polymorphisms in cattle

Background. The human cytochrome P450 3A (hCYP3A) subfamily is the most important subfamily involved in human xenobiotic metabolism, on account of its major role in the metabolism of ~30% of clinically used drugs1. Current increasing evidences show that hCYP3A4 and hCYP3A5 genetic variants significantly contribute to inter-individual variability in drug metabolism2. No information are actually available for bovine CYP3A (bCYP3A), despite the need of improving the safe use of veterinary drugs and, in the meantime, avoid the presence of harmful xenobiotic residues in cattle food products. Within this scenario, the aim of this study was to identify the exonic variants of bCYP3A subfamily members known so far3 in Piedmontese cattle breed, and evaluate their potential functional impact in terms of catalytic activity towards marker substrates. Methods. bCYP3A28, 3A38 and 3A48 exonic variants were identified, through a targeted Illumina sequencing approach, on 16 DNA pools obtained from a total of 300 Piedmontese beef cattle liver samples. Then, each individual genotype was determined using the melting curve genotyping analysis. Afterwards, wild type and mutated sequences were cloned and heterologously expressed in V79 cells together with CYP oxido-reductase. Their metabolite profiling, following the incubation with two CYP3A marker substrates, i.e. testosterone (TST) and nifedipine (NIF), was assessed by using mass spectrometry. To appraise the functional impact of aforementioned variants in the Piedmontese cattle population, the individual genotype was finally combined with the corresponding CYP3A-dependent catalytic activity (TST hydroxylation) in liver subcellular fractions4. Results. Thirteen exonic variants were identified in bCYP3A cluster (3, 3 and 7 for bCYP3A28, 3A38 and 3A48, respectively) and validated using HybProbe probes. Five variants out of 13 showed differences in enzyme activity. In particular, all bCYP3A28 variants significantly reduced (-50%) the production of 6β-OH TST; however, one variant of bCYP3A38 and bCYP3A48 increased ~3-fold 16β-OH TST hydroxylation and NIF oxidation, respectively. In Piedmontese cattle breed population, individuals homozygous for rs384467435 SNP (one of identified bCYP3A28 variants), showed a reduced pattern of TST 6β-hydroxylation in liver subcellular fractions. Conclusion. Overall, these findings suggest that variation in bCYP3A genotype might have a functional relevance, providing evidence of the possible consequences either on animal (kinetics, clinical efficacy/side effects and toxicity) than on human (presence of harmful residues) health.