Monkey P450 Research Articles

Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey ( Callithrix jacchus)

Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo- p-dioxin or tetrabromodibenzo- p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYPIA1, CYPIA2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYPIA2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYPIAI is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYPIAI and CYPIA2 in liver, but only CYPIA1 in lung. No induction of CYPIA1 or CYPIA2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYPIA2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYPIA2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a K i of 10 μM. In the lung the induction of CYPIAI was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufm O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYPIAI does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.

Development of in VitroVmax and K m Values for the Metabolism of Isofenphos by P-450 Liver Enzymes in Animals and Human

The rate of metabolism of [ 14C]isofenphos (IFP) to isofenphos oxon (IFP-oxon), des N-isofenphos (d- N-IFP), and des N-isofenphos oxon (d-N-IFP-oxon) by rat, guinea pig, monkey, dog, and human liver microsomal P-450 enzymes was studied to obtain V max and K m values for Michaelis-Menten kinetics. The monkey had the highest V max value for the conversion of IFP to IFP-oxon (desulfuration), 162 nmol isofenphos hr −1 per 1.3 nanomoles P-450, followed by guinea pig (98), rat (66), dog (43), and human (14). The K m values for the desulfuration of isofenphos were 19.2, 7.4, 14.1, 23.3, and 18.4 μM, respectively, for the monkey, guinea pig, rat, dog, and human. The V max values for the dealkylation process (conversion of IFP to d- N-IFP) were 64.6, 17.2, 9.7, and 7.3 nmol isofenphos hr −1 per 1.3 nanomoles P-450 for the monkey, rat, dog, and human, respectively. For the dealkylation process, monkey had the highest K m value, 16.3 μM IFP, followed by human (11.2), rat (9.9), and dog (9.3). The rate of metabolism of IFP-oxon and d- N-IFP to d- N-IFP-oxon were separately studied. The V max and K m values obtained in this study for animal and human liver P-450 enzymes will be used to develop a PB-PK/PB-PD model to predict the fate and toxicity of isofenphos in animals and man.

Molecular cloning of monkey liver cytochrome P-450 cDNAs : Similarity of the primary sequences to human cytochromes P-450

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a λgt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs

Molecular cloning of monkey P450 1A1 cDNA and expression in yeast

Monkey P450 1A1 cDNA (MKah1) was isolated from the λ;gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey using a dog P450 1A1 cDNA fragment as a probe. MKah1 was 2453 bp long and contained an entire coding region for a polypeptide of 512 residues. The nucleotide and deduced amino acid sequences of MKah1 displayed 95% and 94% identity with those of the human P450 1A1 gene, respectively. Even in the 3′ noncoding region, MKah1 showed 94% homology with human P450 1A1, whereas it showed less than 69% homology with other mammalian P450 1A1. Monkey P450 1A1 mRNA was not detectable in untreated livers, but was induced by polychlorinated biphenyl and 3MC. The expression plasmid (designated as pMKC-1) was constructed by introduction of the coding region of MKah1 into a yeast expression vector (pAM82) containing the promoter of acid phosphatase (APase). Northern blot analysis revealed that monkey P450 1A1 mRNA was expressed in yeast under the control of the APase promoter. Microsomes from yeast transformed by pMKC-1 catalyzed 7-ethoxycoumarin O-deethylation, benzo(a)pyrene hydroxylation and the mutagenic activation of 2-amino-3-methyl-imidazo[4,5- f]quinoline (IQ), 3-amino-1-methyl-5H-pyrido(4,3- b)-indole acetate (Trp-P-2) and 2-amino-6-methyldipyrido(1,2- a : 3′,2′- d)imidazole acetate (Glu-P-1).

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogenous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448 1 and MC P-448 2) catalyzed 7-ethoxycoumarin O-deethylation activities.