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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., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.

Cytochrome P450 (P450) enzyme expression patterns were determined for a panel of 60 human tumor cell lines, representing nine tumor tissue types, used by the National Cancer Institute (NCI) Anticancer Drug Screening Program. All 60 tumor cell lines displayed significant P450 activity, as well as P450 reductase activity, as determined using the general P450 substrate 7-benzyloxyresorufin. Cell line-specific P450 enzyme patterns were observed using three other P450 substrates, 7-ethoxycoumarin, coumarin, and 7-ethoxyresorufin, each of which was metabolized at a low rate. Using a pattern-matching computer program, COMPARE, correlative relationships were investigated between the arrays of P450 activities and the patterns of cytotoxicity exhibited by a large group of anticancer agents of proven or potential clinical utility. Significant negative correlations between the patterns of P450-dependent 7-benzyloxyresorufin metabolism activity and cell line chemosensitivity were observed for 10 standard anticancer agents (including 6 alkylating agents) and 55 investigational compounds, suggesting a role for P450 metabolism in the inactivation of these agents. Negative correlations between 7-ethoxycoumarin O-deethylation and cell line chemosensitivity to a group of topoisomerase inhibitors were also seen, again suggesting P450-dependent drug inactivation. P450 enzyme profiling may thus aid in interpreting the patterns of drug sensitivity and resistance in the NCI tumor cell panel, and may facilitate the identification of anticancer agents whose activity can be altered via cytochrome P450 metabolism.

CYP2D16 is expressed at high levels in the zona reticularis (ZR) of guinea pig adrenal glands and contributes to adrenal metabolism of xenobiotics. Studies were done to evaluate the effects of age and gender on adrenal CYP2D16 expression and xenobiotic metabolism. In both male and female guinea pigs at 1, 7, 14, or 30 weeks of age, in situ hybridization and immunohistochemistry confirmed that CYP2D16 was highly localized to the ZR of the adrenal gland. The steroidogenic P450 isozyme, CYP17, by contrast, was expressed in both the zona fasciculata and ZR. The intensity of CYP2D16 staining was not age- or gender-dependent. However, the proportion of each adrenal gland comprised by ZR and thus expressing CYP2D16 increased with aging in both sexes and was greater in males than in females. The rates of metabolism of bufuralol, a CYP2D-selective substrate, by adrenal microsomal preparations generally correlated with the amount of ZR (and CYP2D16) in the gland. Thus, adrenal xenobiotic-metabolizing activities were greater in males than in females at all ages and increased with aging in males. However, the rates of bufuralol metabolism declined in sexually mature females (14 weeks) from the levels found in prepubertal females (7 weeks) and then increased markedly in retired breeders (30 weeks), suggesting an inhibitory effect of estrogens on enzyme activity. The results indicate that the age and gender differences in adrenal CYP2D16 content are largely determined by differences in the size of the ZR rather than the concentrations of CYP2D16 within cells of the ZR. However, adrenal xenobiotic-metabolizing activities in females seem to be further modulated by an inhibitory effect of estrogens.

Kinetic parameters were determined for the hydroxylation of N'-nitrosonornicotine (NNN), N-nitrosobenzylmethylamine (NBzMA), coumarin, and ethoxycoumarin catalyzed by rat nasal mucosa microsomes. NNN is a tobacco-specific nitrosamine that, in rats, causes tumors in the nasal cavity and esophagus, whereas NBzMA induces tumors in rat esophagus. Both nitrosamines require alpha-hydroxylation to exert their carcinogenic effects. NNN, NBzMA, coumarin, and ethoxycoumarin were all extensively hydroxylated by rat nasal mucosa microsomes. The KM values for the hydroxylation of each substrate were low, ranging between 2 and 5 microM. 2'- and 5'-Hydroxylation of NNN were catalyzed to a similar extent. NBzMA was metabolized predominantly to benzaldehyde, the product of debenzylation, or methylene hydroxylation. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), NNN, and NBzMA were inhibitors of coumarin and ethoxycoumarin hydroxylation. NNN hydroxylation by nasal mucosa microsomes was inhibited by coumarin, ethoxycoumarin, NNK, and NBzMA but not by N-nitrosodimethylamine. 8-Methoxypsoralen, a potent inhibitor of P450 2A6- and 2a5-dependent coumarin hydroxylation in human and mouse liver microsomes, also significantly inhibited NNN activation. The results of this study suggest that the four substrates examined are hydroxylated by closely related P450 enzymes in rat nasal mucosa and that a coumarin hydroxylase metabolizes both NNN and NBzMA.

Short-chain saturated halocarbons, including isoflurane and the chlorofluorocarbon substitute HCFC-123, can strongly potentiate the cytochrome P450-dependent oxidation of gaseous haloethenes, such as 2-chloro-1,1-difluoroethene (CDE) and vinyl chloride, in vivo and in vitro. P450 isozyme specificity in this effect is suggested by the fact that the interaction is pronounced in microsomes from rats treated with phenobarbital, but does not occur in microsomes of isoniazid- or beta-naphthoflavone-treated animals. We examined the effect of isoflurane on CDE defluorination in liver microsomes from 10 human organ donors to determine whether saturated halocarbon/haloethene interactions also occur in humans and, if so, to determine the cytochromes P450 involved. Three of the samples exhibited isoflurane-stimulated increases (24, 32, and 41%) in CDE defluorination; isoflurane either inhibited or had no effect on CDE metabolism in the other seven samples. Two samples in which isoflurane potentiated CDE metabolism to the greatest rates had higher coumarin 7-hydroxylase (indicative of CYP2A6), 7-ethoxycoumarin O-deethylase (CYP2B6), and nifedipine oxidase (CYP3A4) activities than the other eight samples. However, all 10 subjects had similar rates of phenacetin O-deethylation (CYP1A2) and chlorzoxazone 6-hydroxylation (CYP2E1). In microsomes from cells transfected with cDNAs coding for individual human P450s, CDE metabolism by CYP2B6 was stimulated (216%) by isoflurane, whereas isoflurane did not stimulate CDE metabolism by human CYP2A6, CYP3A4, CYP2D6, or CYP2E1. Isoflurane highly increased CDE defluorination in purified rat CYP2B1 (470%).(ABSTRACT TRUNCATED AT 250 WORDS)

The activities of NAD(P)H-dependent quinone reductase (QR) and the cytochrome P-450 monooxygenases 7-ethoxycoumarin O-deethylase (7-ECD) and 7-ethoxyresorufin O-deethylase (7-ERD) were measured in four subpopulations of murine epidermal keratinocytes (MKs) that differed in their stages of differentiation. Noninduced per cell 7-ECD and 7-ERD activities were the lowest in basal cell MKs and progressively increased as the MKs underwent differentiation. In contrast, noninduced per cell QR activities in the three less differentiated MK subpopulations were very similar to one another and greater than the activities measured in the most differentiated subpopulation. Treatment of dorsal skin with 100 nmol of dibenz[a,c]anthracene (DB[a,c]A) increased CYPIA1 mRNA abundance and elevated 7-ERD activities to similar per cell levels in all MK subpopulations. This was achieved by differential inductions (200- to greater than or equal to 1850-fold) of 7-ERD in the different subpopulations. In contrast, QR induction by DB[a,c]A was similar (less than 3-fold) in all MK subpopulations. Consequently, the expressions of noninduced QR and 7-ERD activities in skin are regulated as a function of MK differentiation. However, the distributions of the noninduced activities of these two enzymes in MK subpopulations are the exact opposite. Furthermore, the relative inducibility of 7-ERD, but not QR, in skin is also regulated as a function of epidermal differentiation.