QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: dmd.108.024273v1 37/2/268    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sakai, N. Articles by Fujita, S. Search for Related Content PubMed PubMed Citation Articles by Sakai, N. Articles by Fujita, S.

SHORT COMMUNICATION

Genetic Basis of Inter- and Intrastrain Differences in Diazepam p-Hydroxylation in Rats

Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

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.