INTRODUCTION Within cytochromes P450 (CYP) superfamily, CYP3A is one of the most important subfamily involved in xenobiotic metabolism. Recently, an increasing evidence showed that human CYP3A4 and 3A5 genetic variants significantly contribute to interindividual variability in drug metabolism. No information are actually available for cattle, despite the need of improving the safe use of veterinary drugs. Therefore, the aim of this study was to identify exonic CYP3As variants in Piedmontese cattle breed and understand their functional significance. MATERIALS AND METHODS Liver aliquots from 300 certified male Piedmontese cattle were collected at the slaughterhouse, to extract DNA and obtain microsomal subcellular fractions. DNA extracts were pooled into 16 samples according to the fatherhood. Illumina pairedend multiplexed libraries were prepared and enriched by using the Agilent SureSelectXT Target Enrichment Kit and subsequently processed on Illumina Hiseq2000 platform. Exonic single nucleotide polymorphisms (SNPs) identified on CYP3A cluster (bovine chromosome 25), and affecting the protein sequence, were validated through melting curve genotyping and specific FRET assays. The potential functional significance of each SNP was finally evaluated combining the individual genotype with the corresponding CYP3A-dependent catalytic activity (testosterone hydroxylation), measured by using a HPLC method. RESULTS AND CONCLUSIONS Next generation sequencing identified 3 SNPs for CYP3A28, 3 for CYP3A38 and 4 for CYP3A48 resulting in protein changes, as well as 2 adjacent SNPs in CYP3A28 alternative splicing sites. These SNPs were all analyzed through FRET assays. The effects of the genotype of 300 individuals on the corresponding catalytic activity pointed out a significant reduction of 6b- and 2b-hydroxytestosterone production (56% and 55%, respectively) in individuals homozygous for rs384467435 SNP, and a significant decrease (72%) of androstenedione production in cattle heterozygous for a new SNP identified in exon 7 of CYP3A48. These findings suggest that variation of CYP3A genotype might result in interindividual differences in the corresponding phenotype also in cattle, providing something to chew on the safe use of veterinary drugs in cattle farming. A confirmatory step using heterologous expression vectors might be considered in perspective. ACKNOWLEDGEMENTS Project supported by MIUR (2009ZE5HJP) and University of Padua (60A08-2874/14).
Exonic polymorphisms of cytochrome P450 3As (CYP3As) in Piedmontese cattle breed.
GIANTIN, MERY;LOPPARELLI, ROSA MARIA;ZANCANELLA, VANESSA;MERLANTI, ROBERTA;CAPOLONGO, FRANCESCA;DACASTO, MAURO
2015
Abstract
INTRODUCTION Within cytochromes P450 (CYP) superfamily, CYP3A is one of the most important subfamily involved in xenobiotic metabolism. Recently, an increasing evidence showed that human CYP3A4 and 3A5 genetic variants significantly contribute to interindividual variability in drug metabolism. No information are actually available for cattle, despite the need of improving the safe use of veterinary drugs. Therefore, the aim of this study was to identify exonic CYP3As variants in Piedmontese cattle breed and understand their functional significance. MATERIALS AND METHODS Liver aliquots from 300 certified male Piedmontese cattle were collected at the slaughterhouse, to extract DNA and obtain microsomal subcellular fractions. DNA extracts were pooled into 16 samples according to the fatherhood. Illumina pairedend multiplexed libraries were prepared and enriched by using the Agilent SureSelectXT Target Enrichment Kit and subsequently processed on Illumina Hiseq2000 platform. Exonic single nucleotide polymorphisms (SNPs) identified on CYP3A cluster (bovine chromosome 25), and affecting the protein sequence, were validated through melting curve genotyping and specific FRET assays. The potential functional significance of each SNP was finally evaluated combining the individual genotype with the corresponding CYP3A-dependent catalytic activity (testosterone hydroxylation), measured by using a HPLC method. RESULTS AND CONCLUSIONS Next generation sequencing identified 3 SNPs for CYP3A28, 3 for CYP3A38 and 4 for CYP3A48 resulting in protein changes, as well as 2 adjacent SNPs in CYP3A28 alternative splicing sites. These SNPs were all analyzed through FRET assays. The effects of the genotype of 300 individuals on the corresponding catalytic activity pointed out a significant reduction of 6b- and 2b-hydroxytestosterone production (56% and 55%, respectively) in individuals homozygous for rs384467435 SNP, and a significant decrease (72%) of androstenedione production in cattle heterozygous for a new SNP identified in exon 7 of CYP3A48. These findings suggest that variation of CYP3A genotype might result in interindividual differences in the corresponding phenotype also in cattle, providing something to chew on the safe use of veterinary drugs in cattle farming. A confirmatory step using heterologous expression vectors might be considered in perspective. ACKNOWLEDGEMENTS Project supported by MIUR (2009ZE5HJP) and University of Padua (60A08-2874/14).
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