Functional impact of cytochrome P450 3A (CYP3A) missense variants in cattle

Abstract

Cytochrome P450 3A is the most important CYP subfamily in humans, and CYP3A4/CYP3A5 genetic variants contribute to inter-individual variability in drug metabolism. However, no information is available for bovine CYP3A (bCYP3A). Here we described bCYP3A missense single nucleotide variants (SNVs) and evaluated their functional effects. CYP3A28, CYP3A38 and CYP3A48 missense SNVs were identified in 300 bulls of Piedmontese breed through targeted sequencing. Wild-type and mutant bCYP3A cDNAs were cloned and expressed in V79 cells. CYP3A-dependent oxidative metabolism of testosterone (TST) and nifedipine (NIF) was assessed by LC-MS/MS. Finally, SNVs functional impact on TST hydroxylation was measured ex vivo in liver microsomes from individually genotyped animals. Thirteen missense SNVs were identified and validated. Five variants showed differences in CYP3A catalytic activity: three CYP3A28 SNVs reduced TST 6β-hydroxylation; one CYP3A38 variant increased TST 16β-hydroxylation, while a CYP3A48 SNV showed enhanced NIF oxidation. Individuals homozygous for rs384467435 SNV showed a reduced TST 6β-hydroxylation. Molecular modelling showed that most of SNVs were distal to CYP3A active site, suggesting indirect effects on the catalytic activity. Collectively, these findings demonstrate the importance of pharmacogenetics studies in veterinary species and suggest bCYP3A genotype variation might affect the fate of xenobiotics in food-producing species such as cattle.

Midazolam oxidation in cattle liver microsomes: The role of cytochrome P450 3A

“…Finally, the 4‐OHMDZ production (expressed as nmol/min/mg protein) was individually measured (in duplicates) in 300 Piedmontese cattle liver microsomes. A correlation analysis was made between these results and data referring to 6β‐hydroxytestosterone (6β‐OHTST) and 16β‐OHTST, obtained previously in the same cohort of samples (Giantin et al, 2019).…”

“…In bovines, the substrates most commonly used to evaluate CYP3A‐dependent catalytic activity metabolism are testosterone (TST; hydroxylation), nifedipine (oxidation), erythromycin, ethylmorphine, and triacetyl‐oleandomycin ( N ‐demethylation) (Giantin et al, 2008; Giantin et al, 2019; Maté et al, 2015; Nebbia et al, 2001; Pegolo et al, 2010b; Szotáková et al, 2004). Among these ones, TST 6β‐hydroxylation is the only catalytic activity proved specific for bovine CYP3A4/5‐ like by chemical and antibody‐mediated inhibition studies (Pegolo et al, 2010a).…”

“…In bovine liver, CYP3A28 gene is poorly expressed; however, CYP3A38 is the most abundant gene, followed by CYP3A48 (Zancanella et al, 2014). The coded proteins show a different behavior toward TST: CYP3A28 produces exclusively 6β‐OHTST, whereas CYP3A38 yields both 6β‐OH and 16β‐OH TST derivatives; conversely, CYP3A48 only marginally participates to TST metabolism, but it is involved in nifedipine oxidation (Giantin et al, 2019).…”

“…Microsomal fractions were isolated by ultracentrifugation from the liver of 300 purebred Piedmontese young bulls regularly slaughtered (mean body weight ~550 kg), according to Pegolo et al (2010b), while microsomal protein concentrations were determined as reported in Giantin et al (2019).…”

“…Control reactions were carried out using an equal volume of nonimmune rabbit serum (XenoTech LLC, Lenexa, KS, USA). The antibody cross‐reactivity toward bovine CYP3A protein was assessed using an immunoblotting protocol and according to Giantin et al (2019) (Figure ).…”