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Substrate Selectivities and Catalytic Activities of Marmoset Liver Cytochrome P450 2A6 Differed from Those of Human P450 2A6.

Drug metabolism and disposition: the biological fate of chemicals (2015-04-11)
Shotaro Uehara, Yasuhiro Uno, Takashi Inoue, Erika Sasaki, Hiroshi Yamazaki
ABSTRACT

The common marmoset (Callithrix jacchus), a New World primate species, is potentially a useful animal model for preclinical studies in drug development. However, cytochrome P450 (P450) enzymes have not been fully identified and characterized in marmosets. In this study, we identified P450 2A6 cDNA with the sequence highly identical (91-94%) to human P450 2A6, 2A7, and 2A13 cDNA and cynomolgus monkey P450 2A23, 2A24, and 2A26 cDNA. Among the tissue types examined, marmoset P450 2A6 mRNA was most abundantly expressed in livers where P450 2A6 protein was also detected by immunoblotting. Phylogenetic analysis showed that marmoset P450 2A6 was more closely clustered with human and cynomolgus monkey P450 2As than P450 2As of dog, rat, and mouse (the species also used in drug metabolism). Marmoset P450 2A6 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation, similar to human P450 2A6 and 2A13 and cynomolgus monkey P450 2A23, 2A24, and 2A26, but much less effectively coumarin 7-hydroxylation, showing some difference as well. Interestingly, marmoset P450 2A6 and cynomolgus monkey P450 2A23 catalyzed phenacetin O-deethylation, which is catalyzed by human P450 1A2 and 2A13, but not by P450 2A6. Marmoset P450 2A6 also exhibited catalytic activity toward testosterone by the multiple sites, but not rat P450 2A-specific testosterone 7α-hydroxylation activity. These results indicated that marmoset P450 2A6 had functional characteristics different from those of human and cynomolgus monkey P450 2As in terms of partially different substrate specificities and catalytic activities, indicating its importance of further studies for P450 2A-dependent drug metabolism in marmosets.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Coumarin, ≥98%
Sigma-Aldrich
Phenacetin, ≥98.0% (HPLC)
Sigma-Aldrich
Testosterone, purum, ≥99.0% (HPLC)
Sigma-Aldrich
7-Ethoxycoumarin
Sigma-Aldrich
Coumarin, ≥99% (HPLC)
Sigma-Aldrich
Testosterone, ≥98%
Supelco
Testosterone solution, 1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®
Sigma-Aldrich
Umbelliferone, 99%
Sigma-Aldrich
Acetaminophen, BioXtra, ≥99.0%
Sigma-Aldrich
Acetaminophen, meets USP testing specifications, 98.0-102.0%, powder
Sigma-Aldrich
Acetaminophen, analytical standard
Sigma-Aldrich
Umbelliferone, suitable for fluorescence indicator, ≥98.0% (HPLC)
Supelco
Acetaminophen solution, 1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®