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.

Figure 1

Catalytic activity of V79 cells expressing wild-type and mutant (a) CYP3A28, (b) CYP3A38 and (c) CYP3A48. After incubation of transfected V79 cells with 25 µM TST or 5 µM NIF for 3 or 1.5 hours, the medium was subjected to LC-MS/MS analysis as reported in the Material and Methods. The catalytic activity of wild-type and mutant CYP3A28, CYP3A38 and CYP3A48 proteins was calculated dividing the amount of metabolite formed by the incubation time and the total protein content (nmoles min⁻¹ mg⁻¹ protein). Variant CYP3A specific activities were normalized to that of the wild-type CYP3A isoform (set at 1.00). Four independent experiments were performed. Unpaired T-test was used for statistical analysis (*p < 0.05; ***p < 0.001). AU: Arbitrary Units.

Figure 2

6β-OH TST production in liver microsomes isolated from Piedmontese bulls genotyped for CYP3A28_7 Gly197Ser. The catalytic activity was calculated in nmoles min⁻¹ mg⁻¹ protein. ANOVA followed by Tukey’s post-test was used for statistical analysis (*p < 0.05; **p < 0.01). Black and dotted lines represent median values and quartiles, respectively.

Figure 3

The docking of TST to bCYP3A enzymes. (a) The location of the substrate pocket in wild-type CYP3A28 (grey cloud), CYP3A38 (green cloud) and CYP3A48 (magenta cloud) isoforms. (b–d) Close-up view of interactions of TST (magenta) in the substrate pockets of (b) CYP3A28, (c) CYP3A38 and (d) CYP3A48. Only the most important pocket residues (green) are shown for clarity. Black dashes indicate hydrogen bonding, heme is in blue and CYP backbone in grey ribbon.

Figure 4

NIF-amino acid interactions in the CYP3A48 substrate pocket. Residues that line the substrate-binding pocket are highlighted by purple, the heme group is shown by blue and the CYP backbone by grey ribbon. Black dashes indicate hydrogen bonding, green dashes salt bridges and magenta dashes π-cation interactions.