Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos

CYP3A4 is the most abundant isoform of cytochrome P450 (CYP) in adult human liver. It metabolizes numerous clinically, physiologically, and toxicologi cally important compounds. The expression of CYP3A4 varies 40-fold in indiv idual human livers, and metabolism of CYP3A4 substrates varies at least 10- fold in vivo. Single nucleotide polymorphisms (SNPs) in CYP3A4 were identif ied by direct sequencing of genomic DNA in 72 individuals from three differ ent ethnic groups, including Caucasians, Blacks (African-Americans and Afri can pygmies), and Asians. A total of 28 SNPs were identified, including fiv e which produced coding changes M445T (CYP3A4*3), R162Q (CYP3A4*15), F189S (CYP3A4*17), L293P (CYP3A4*18), and P467S (CYP3A4*19). The latter four repr esent new alleic variants. Racial variability was observed for the frequenc y of individual SNPs. CYP3A R162Q was identified only in Black populations with an allelic frequency of 4%. CYP3A4 F189S and CYP3A4 M445T were identif ied in Caucasians with allelic frequencies 2% and 4%, respectively. L293P a nd P467S were only observed in Asians at allelic frequencies of 2%. The cDN As for the F189S, L293P, M445T, and P467S mutant alleles were constructed b y site-directed mutagenesis and expressed in an Escherichia coli expression system. Testosterone and the insecticide chlorpyrifos were used to assess the catalytic activities of the most common CYP3A4 allele (CYP3A4*1) and it s allelic variants. CYP3A4 F189S exhibited lower turnover numbers for testo sterone and chlorpyrifos, while CYP3A4 L293P had higher turnover numbers fo r both substrates. The turnover numbers of the CYP3A4 M445T and P467S allel es to metabolize these compounds were not significantly different from thos e of wild-type CYP3A4.