The genetic bases of inter- and intrastrain differences in CYP2D-dependent drug metabolism in rats

The rat is an important model for understanding human physiology and disease. The rat contributes to the biomedical researches of human cardiovascular disease, diabetes, arthritis and many behavioral disorders. In particular, the rat has frequently been used in the fields of pharmacology and toxicology. Thus, pharmaceutical companies use rats for a large proportion of their mandatory toxicity testing. However, inter- and intrastrain differences in drug response are often observed among rat strains, and are an important issue to be overcome. Still, the fundamental mechanism of inter- and intrastrain differences regarding drug metabolizing enzymes have been overlooked. In this thesis, I focused on the CYP2D subfamily that is a key enzyme involved in individual differences in drug metabolism. CYP2D2 enzyme is known to metabolize the majority of typical substrates of the human CYP2D6 enzyme. Despite its impact on drug metabolism in rats, the transcriptional regulation of CYP2D2 remained to be elucidated. In chapter 1, I clarified the molecular mechanism of CYP2D2 gene expression. The CYP2D2 gene was positively regulated by the poly(C)-binding protein hnRNP K through a transcriptional regulatory element located in the 5'-flanking region from -94 to -113. Acting as a docking platform, the hnRNP K protein is known to be implicated in the regulation of transcription as an activator or a repressor, as a translational repressor, and as a participant in a variety of signaling systems. Although it has only been reported that hnRNP A1 protein, one of the hnRNPs, involves in the stabilization of the CYP2A5 and CYP2A6 mRNA, nothing is known about the potential role of hnRNP K in P450 gene regulation. Thus, this is the first report that hnRNP K protein is involved in CYP2D2 gene regulation. Furthermore, I elucidated the genetic basis of the extremely low expression of CYP2D2 mRNA in DA rats. Due to its relative low abundance, DA rats have been frequently used for the study of CYP2D substrate metabolism as the animal model of PM phenotype for CYP2D6 in comparison to SD rats as an EM phenotype. To take full advantage of physiological variation among rat strains, it is essential to further unravel the mechanism of low expression of CYP2D2 mRNA. I found a single substitution within the transcriptional regulatory element of the CYP2D2 gene in DA rats. The mutation was detected in the polypyrimidine sequence that is the preferred binding site for hnRNP K protein. Indeed, the mutation within the transcriptional regulatory element abolished the binding of hnRNP K protein. Thus, I conclude that decreased recruitment of hnRNP K protein to the mutated sequence causes the low expression of CYP2D2 mRNA in DA rats. Inter- and intrastrain differences in drug response in rats are a difficult problem for basic research as well as drug discovery as described above. Currently, information on genetic variation in laboratory rat strains is rapidly accumulating as a set of microsatellite markers, simple sequence length polymorphism markers and a variety of single nucleotide polymorphism in coding regions. Nevertheless, little is known about the differences in their drug metabolism characteristics that are ascribed to genetic variation. In chapter 2, I clarified the mechanism underlying inter- and intrastrain differences in diazepam p-hydroxylation among rat strains. The recent studies demonstrated that the pharmacokinetics of diazepam, which is one of the benzodiazepines, were quite different among rat strains because of its metabolic polymorphisms in diazepam p-hydroxylation. Although diazepam p-hydroxylation is a major metabolic pathway, SD and BN rats had 300-fold higher diazepam p-hydroxylation activity than DA rats at low concentration of diazepam. And Wistar rats showed 200-fold intrastrain differences in diazepam p-hydroxylation activity (EM-W > PM-W). In addition, it was suggested that CYP2D subfamily was involved in this activity. Based on these observations, I separated the specific protein expressed in liver microsomes of SD, BN and EM-W, and identified the specific protein to be CYP2D3. Then, I confirmed that only CYP2D3 but no other CYP2D isoforms had a diazepam p-hydroxylation activity. To date, there is little information about the catalytic specificity of CYP2D3. Thus, I demonstrated that CYP2D3 was involved in diazepam p-hydroxylation. Moreover, I analyzed the genetic polymorphism of the CYP2D3 gene among rat strains. I found a single insertion in exon 8 in DA and PM-W rats. A premature termination codon created by this frameshift consequently deleted the heme-binding region that is essential to maintain proper heme binding and active P450 enzymes. Therefore, I conclude that the deficiency of a functional CYP2D3 protein must be the cause of the significantly low diazepam p-hydroxylation in DA and PM-W rats. Additionally, the genotype frequency of CYP2D3 polymorphism is in good agreement with the phenotype frequency among rat strains. In this thesis, I clarified the genetic bases of inter- and intrastrain differences in CYP2D-dependent drug metabolism in rats. Clarification of inter- and intrastrain differences in rats will be further useful for predicting variability in human pharmacokinetics. Consequently, it is worthwhile to fully characterize animals used in pharmacokinetics studies from the point of view of the genetic expression of metabolic enzymes. Therefore, my work will strongly support the strain consideration in selection of a rat strain for drug metabolisms.