RT Journal Article SR Electronic T1 Genetic Basis of Inter- and Intrastrain Differences in Diazepam p-Hydroxylation in Rats JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP 268 OP 271 DO 10.1124/dmd.108.024273 VO 37 IS 2 A1 Noriaki Sakai A1 Konomu Saito A1 Kentaro Q. Sakamoto A1 Mayumi Ishizuka A1 Shoichi Fujita YR 2009 UL http://dmd.aspetjournals.org/content/37/2/268.abstract AB Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) is widely used as a sedative, hypnotic, and anti-anxiety drug. At low diazepam concentrations, p-hydroxylation is the major metabolic pathway in rat liver microsomes. However, there are marked (∼300-fold) inter- and intrastrain differences in the activity among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. In our previous study, we determined that a deficiency of CYP2D3 protein, not CYP2D2, was responsible for the inter- and intrastrain differences in diazepam p-hydroxylation (Drug Metab Dispos 33:1657–1660, 2005). Quantitative real-time polymerase chain reaction (PCR) did not provide enough evidence to explain the inter- and intrastrain differences in the expression of CYP2D3 protein. Nucleotide sequence analysis revealed the insertion of a thymine in exon 8 of the CYP2D3 gene in the poor diazepam metabolizers. This single nucleotide mutation caused a shift in the reading frame and introduced a premature termination signal. It is noteworthy that the heme binding region, which is essential to maintain proper heme binding and active cytochrome P450 enzymes, was consequently deleted by the premature termination signal. In contrast, no mutation was detected in the CYP2D3 gene of extensive metabolizers. Thus, the truncated CYP2D3 must be a nonfunctional enzyme in poor metabolizers. In addition, we developed a convenient and specific genotyping assay using PCR-restriction, fragment-length polymorphism to distinguish homozygotes from heterozygotes. The genotyping gave results fully consistent with those of the inter- and intrastrain differences in diazepam p-hydroxylation. The American Society for Pharmacology and Experimental Therapeutics