Characterization of (+/-)-bufuralol hydroxylation activities in liver microsomes of Japanese and Caucasian subjects genotyped for CYP2D6.

Twenty-four genetic polymorphisms in the CYP2D6 gene were analysed in liver DNA samples of 39 Japanese and 44 Caucasians and compared with CYP2D6 protein levels and bufuralol 1'- and 6-hydroxylation activities in liver microsomes of these human samples. We detected 13 types of CYP2D6 genetic polymorphisms and classified these into 20 genotypes; nine types were found in Japanese and 14 types in Caucasian samples. CYP2D610B, but not CYP2D610A, was the most frequent (34.6%) in Japanese. In Caucasians, several CYP2D6 polymorphisms including CYP2D6*4, *4D, *4E, *4L, *3, *9, *5 and 2E (frequencies of 6.8, 3.4, 4.5, 9.1, 1.1, 2.3, 2.3 and 4.5%, respectively) were detected. A Caucasian having CYP2D63/5 had a protein with slower gel mobility (immunoblotting with anti-CYP2D6 antibody) and very low activity for bufuralol 1'-hydroxylation. Five Caucasian samples (CYP2D64/*4, *4/*4L, or *4D/4L) had no measurable CYP2D6 protein and very low bufuralol 1'-hydroxylation activities. Seven Japanese subjects with CYP2D610B/10B had CYP2D6 protein at levels of approximately 20% of those present in humans with CYP2D61 and 2 and catalysed bufuralol 1'-hydroxylation at low rates. Kinetic analysis of bufuralol 1'- and 6-hydroxylation indicates that (i) the Km values for 1'-hydroxylation were lower in individuals with CYP2D61/*1, *1/*2, *1/*2X2, and *2/2 than those with CYP2D64/*4, *4/*4L, *4D/*4L, or *10B/*10B and Vmax values tended to be higher in the former groups (*1, 2), and (ii) individuals with heterozygous CYP2D61/*4D, *1/*4L, and *1/5 had relatively high Vmax/Km ratios, whereas individuals with heterozygous CYP2D61/*9, *2/4D, *2/*5, *2/*10B, *2E/*4E, *3/*5, *4L/*9, and 10B/39 had lower Vmax/Km ratios for bufuralol 1'-hydroxylation. Quinidine inhibited bufuralol 1'-hydroxylation in liver microsomes, particularly at low substrate concentrations, in individuals with CYP2D61/1, and 1/12, but not those with CYP2D64/4 and very slightly in individuals with CYP2D610B/10B. The latter two groups were found to be more sensitive to alpha-naphthoflavone than the former groups, indicative of the contribution of CYP1A2. These results support the view that CYP2D63, *4, *4D, and 4L are major genotypes producing poor metabolizer phenotypes in CYP2D6 in Caucasians, whereas CYP2D610B is a major factor in decreased CYP2D6 protein expression and catalytic activities in Japanese.