The dietary mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are activated to genotoxins by rat and human liver cytochrome P450 (P450) 1A1- and 1A2-mediated N-oxidation. Immunoquantitation of 51 human liver samples revealed a wide range in P450 1A2 expression (10-250 pmol/mg of microsomal protein, median 71 pmol/mg), with 39% of the livers containing >100 pmol/mg of protein. There was no evidence for expression of P450 1A1 (<1 pmol/mg of protein). P450 1A2 levels were correlated to MeIQx and PhIP N-oxidation rates (r = 0.83, 0.73, respectively). In male Fischer-344 and Sprague-Dawley rats, hepatic P450 1A2 ranged from 5 to 35 pmol/mg of protein, while P450 1A1 was <1 pmol/mg. Animal pretreatment with 3-methylcholanthrene, beta-naphthoflavone, or polychlorinated biphenyls (PCB) resulted inasmuch as 340-fold and >1000-fold induction of P450 1A2 and 1A1, respectively, and a 220-fold increase in N-oxidation activity. Approximately 20% of the human samples were as active in N-oxidation and conversion of MeIQx to bacterial mutagens as microsomes of PCB-pretreated rats [3-4 nmol of NHOH-MeIQx formed min-1 (mg of protein)-1]. In contrast, microsomes from PCB-treated rats displayed higher rates of PhIP N-oxidation and activation to mutagens than the most active human liver microsomes [8-24 vs 2-4 nmol of HNOH-PhIP formed min-1 (mg of protein)-1]. Recombinant human P450 1A2 showed catalytic efficiencies of MeIQx and PhIP N-oxidation that were 10-19-fold higher than purified rat P450 1A2. Cytochrome P450 1A2 expression in rodent and human liver tissue varies greatly and there are considerable differences between the enzymes in the two species in the activation of some heterocyclic aromatic amines, which must be considered when assessing human health risk.
A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP. The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end. This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA. The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase [Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., & Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354], and this cDNA, like the rabbit clone, appears to be part of a multigene family. At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence. The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.
ESR spin trapping was used to investigate the reaction of rabbit cytochrome P450 (P450) 1A2 with cumene hydroperoxide. Cumene hydroperoxide-derived peroxyl, alkoxyl, and carbon-centered radicals were formed and trapped during the reaction. The relative contributions of each radical adduct to the composite ESR spectrum were influenced by the concentration of the spin trap. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide (DMPO) concentrations was used to quantitate the contributions of each radical adduct to the composite ESR spectrum. The alkoxyl radical was the initial radical produced during the reaction. Experiments with 2-methyl-2-nitrosopropane identified the carbon-centered adducts as those of the methyl radical, hydroxymethyl radical, and a secondary carbon-centered radical. The reaction did not require NADPH-cytochrome P450 reductase or NADPH. It is concluded that the reaction involves the initial homolytic scission of the peroxide O-O bond to produce the cumoxyl radical. Methyl radicals were produced from the beta-scission of the cumoxyl radical. The peroxyl adduct was not observed in the absence of molecular oxygen. We conclude that the DMPO peroxyl radical adduct detected in the presence of oxygen was due to the methylperoxyl radical formed by the reaction of the methyl radical with oxygen. At a higher P450 concentration, a protein-derived radical adduct was also detected.
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