Functional characterization and tissue expression of marmoset cytochrome P450 2E1

Abstract

Common marmosets (Callithrix jacchus) have attracted increasing attention as a useful small non-human primate model in preclinical research. However, studies on marmoset cytochrome P450 (P450) 2E enzyme have scarcely been conducted. In this study, the full-length cDNA encoding P450 2E1 enzyme was isolated from marmoset livers by reverse transcription (RT)-polymerase chain reaction (PCR). Marmoset P450 2E1 amino acid sequences were highly identical (>88%) to those of cynomolgus monkey and human P450 2E1 enzymes. Phylogenetic analysis indicated a close evolutionary relationship among marmoset, cynomolgus monkey, and human P450 2E1 enzymes. The tissue expression pattern analyzed by real-time RT-PCR and immunoblotting demonstrated that marmoset P450 2E1 mRNA and proteins were predominantly expressed in livers. Marmoset P450 2E1 enzyme heterologously expressed in Escherichia coli catalyzed the hydroxylation of p-nitrophenol, chlorzoxazone, and theophylline, similar to cynomolgus monkey and human P450 2E1 enzymes. By kinetic analyses, those P450 2E1 enzymes catalyzed p-nitrophenol hydroxylation with similar affinities and relatively high intrinsic clearance efficiencies. These results indicated that tissue distribution and enzyme-substrate specificity of marmoset P450 2E1 were similar to cynomolgus monkey and human P450 2E1 enzymes, suggesting that marmosets are a suitable primate model for P450 2E1-dependent drug and xenobiotic metabolism.

Survey of Drug Oxidation Activities in Hepatic and Intestinal Microsomes of Individual Common Marmosets, a New Nonhuman Primate Animal Model

“…Recombinant human and marmoset P450 2D6 enzymes effectively catalyzed R -metoprolol O -demethylation, consistent with the activities of human and marmoset hepatic microsomes (Table 2 ). Investigations of typical human P450 2E1 substrates showed that hepatic microsomes from humans, cynomolgus monkeys, and marmosets catalyzed p -nitrophenol 2-hydroxylation, chlorzoxazone 6-hydroxylation, and theophylline 8-hydroxylation at similar rates [26]. Recombinant human, cynomolgus monkey, and marmoset P450 2E1 catalyzed these drug oxidation reactions, suggesting that P450 2E1 plays a similar role in drug-related oxidation reactions in human and marmoset livers (Table 2 ).…”

“…Numbers in parentheses are the positions of drug oxidations. The substrate concentrations were: ethoxyresorufin (2.0 µM) [22], 7-ethoxycoumarin (20 µM) [30], coumarin (100 µM), pentoxyresorufin (10 µM) [30], propofol (20 µM) [30], efavirenz (20 µM) [30], paclitaxel (100 µM) [12], tolbutamide (100 µM) [12], flurbiprofen (20 µM) [12], diclofenac (10 µM for 4′-hydroxylation and 100 µM for 5-hydroxylation) [12], R/S -warfarin (10 µM) [41], R/S -omeprazole (10 µM) [12], bufuralol (20 µM) [24], R/S -metoprolol (10 µM) [27], chlorzoxazone (50 µM) [26], p -nitrophenol (50 µM) [26], theophylline (500 µM) [26], midazolam (20 µM) [24], nifedipine (50 µM) [24], testosterone (100 µM) [24], progesterone (100 µM) [24], terfenadine (2.0 µM) [23], and ebastine (20 µM) [19].…”

“…Data are taken from a Uehara et al [22]; b Oshio et al [30]; c Uehara et al [12] ; d Uno et al [40]; e Hosoi et al [41]; f Uehara et al [21]; g Uehara et al [24]; h Uehara et al [27]; i Uehara et al [26], j Uehara et al [23]; and k Uehara et al [19]. …”