CYP2A6 genetic polymorphisms and liver microsomal coumarin and nicotine oxidation activities in Japanese and Caucasians

Abstract

Genotypes of CYP2A6, namely CYP2A6 ^* 1 (wild-type), CYP2A6 ^* 2, and CYP2A6 ^* 3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6 ^* 2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6 ^* 2- and CYP2A6 ^* 3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6 ^* 3-type polymorphism in the samples examined in this study, and no cases of CYP2A6 ^* 2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6 ^* 1/ ^* 2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6 ^* 1/ ^* 2 individuals had a very low ratio of V _max to K _m for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6 ^* 1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6 ^* 1/ ^* 2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.