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

Mery Giantin,Lorena Lucatello,Marianna Pauletto,Francesca Pezzato,Rosa M Lopparelli,Mauro Dacasto,Paolo Carnier,Roberta Merlanti,Francesca Capolongo,Minna Rahnasto-Rilla,Roberta Tolosi,Giorgia Guerra,Paavo Honkakoski

doi:10.1038/s41598-019-56271-8

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.

Highlights

Cytochrome P450 (CYP) enzymes are heme-containing proteins responsible for oxidation of drugs, environmental pollutants, food constituents as well as important endogenous compounds such as steroids and bile acids[1]
The mean coverage measured in the Bovine CYP3A (bCYP3A) cluster was 105X
Alignment files (.bam) with reads mapped in the bCYP3A cluster have been deposited in the Sequence Read Archive (SRA) with the accession numbers SRR7353738 - SRR7353753

Summary

Introduction

Cytochrome P450 (CYP) enzymes are heme-containing proteins responsible for oxidation of drugs, environmental pollutants, food constituents as well as important endogenous compounds such as steroids and bile acids[1]. Increasing evidence shows that human CYP3A genetic variants may contribute to inter-individual variability in their metabolic activity, explaining adverse effects or unusual drug pharmacokinetics. Our study aims to fill this scientific gap of knowledge by identifying missense mutations that could modify bCYP3A activity, with potential consequences on drug kinetics, therapeutic or adverse effects as well as on the levels of harmful residues in foodstuff. To this purpose, a pure Piedmontese cattle breed with precise individual pedigree information was selected. Testosterone (TST) hydroxylation was determined in liver microsomes isolated from genotyped Piedmontese bulls

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Conclusion