Molecular basis of the Dark Agouti rat drug oxidation polymorphism: importance of CYP2D1 and CYP2D2

ABSTRACT:In rats, six cytochrome P450 (P450) 2D isoforms have been genetically identified. Nonetheless, there is little evidence of catalytic properties of each CYP2D isoform. In this study, using recombinant CYP2D isoforms (rat CYP2D1, CYP2D2, CYP2D3, and CYP2D4 and human CYP2D6) or hepatic microsomes, we investigated the catalytic specificity toward bufuralol, debrisoquine, and propranolol, which are frequently used as CYP2D substrates. Bufuralol was oxidized to three metabolites by rat and human hepatic microsomes. 1-Hydroxybufuralol was the major metabolite. 12-Ethenylbufuralol, one of the others, was identified as a novel metabolite. The formation of 1-hydroxybufuralol and 12-ethenylbufuralol in hepatic microsomes was inhibited by anti-CYP2D antibody, suggesting that these metabolites were formed by CYP2D isoforms. All rat and human recombinant CYP2D isoforms possessed activity for the 1-hydroxylation of bufuralol, indicating that this catalytic property was common to all CYP2D isoforms. However, the 12-ethenylation of bufuralol was catalyzed only by rat CYP2D4 and human CYP2D6. Debrisoquine was oxidized to two metabolites, 3-hydroxydebrisoquine, and 4-hydroxydebrisoquine, by hepatic microsomes. Recombinant CYP2D2 and CYP2D6 had very high levels of activity for the 4-hydroxylation of debrisoquine with low K m values. Only CYP2D1 had a higher level of 3-hydroxylation than 4-hydroxylation activity. Propranolol 4-hydroxylation was catalyzed by CYP2D2, CYP2D4, and CYP2D6. The 7-hydroxylation of propranolol was catalyzed only by CYP2D2. In conclusion, in rats, bufuralol 12-ethenylation activity was specific to CYP2D4 and debrisoquine 4-hydroxylation and propranolol 7-hydroxylation activities were specific to CYP2D2. These catalytic activities are useful as a probe for rat CYP2D isoforms.

Section: Hiroi Et Alsupporting

confidence: 60%

Catalytic Specificity of CYP2D Isoforms in Rat and Human

Hiroi

Chow²,

Imaoka³

et al. 2002

“…The lack of definitive knowledge about their origin has led to speculation that DA rats might have been mixed with the Wistar strain before the completion of inbreeding. In addition to CYP2D3, due to the low level of CYP2D2 mRNA expression, DA rats have been proposed as a model for human debrisoquine 4-hydroxylase polymorphism (Yamamoto et al, 1998). Moreover, we reported that DA rats have a high level of expression of …”

Section: Genetic Polymorphism Of Cyp2d3 In Ratsmentioning

confidence: 96%

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

Sakai

Saito²,

Sakamoto³

et al. 2009

Self Cite

ABSTRACT:Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) is widely used as a sedative, hypnotic, and antianxiety drug. At low diazepam concentrations, p-hydroxylation is the major metabolic pathway in rat liver microsomes. However, there are marked (ϳ300-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 PCRrestriction, 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.

“…CYP2D1 had a low diazepam N-desmethylation activity, whereas CYP2D2 had no activity toward diazepam metabolism. Although CYP2D1 and CYP2D2 play an important role in the metabolism of numerous drugs such as debrisoquine (Kobayashi et al, 1989), bunitrolol (Suzuki et al, 1992;Yamamoto et al, 1996Yamamoto et al, , 1998, and propranolol (Fujita et al, 1993), it seems that they do not participate in diazepam metabolism as much.…”

Section: Role Of Cyp2d3 In Polymorphism Of Diazepam P-hydroxylationmentioning

confidence: 99%

“…Moreover, the patterns of debrisoquine 4-hydroxylation activity, which is a CYP2D2-dependent activity, did not coincide with those of diazepam p-hydroxylation activity in the liver of four rat strains. Then, we concluded that diazepam p-hydroxylation was catalyzed by neither CYP2D1 nor CYP2D2, which contributed to the metabolism of debrisoquine, bunitrolol, and bufuralol (Boobis et al, 1986;Gonzalez et al, 1987;Suzuki et al, 1992;Yamamoto et al, 1996Yamamoto et al, , 1998. The enzyme that catalyzes diazepam p-hydroxylation is still not clear.…”

mentioning

confidence: 99%

Importance of Cyp2d3 in Polymorphism of Diazepam P-Hydroxylation in Rats

Sakai

Saito²,

Kim³

et al. 2005

Self Cite

ABSTRACT:Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter-or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked (ϳ300 fold) inter-or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam phydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. Nterminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter-or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.