Abstract

2-Amino-alpha-carboline (A alpha C) is a mutagenic and carcinogenic heterocyclic amine that is formed as a pyrolysis product during the high temperature cooking of food and the burning of tobacco. Human, rat, and mouse hepatic microsomes each catalyzed the NADPH-dependent oxidation of A alpha C to form six products separable by HPLC. The two major metabolites, which together accounted for approximately 85% of the total metabolism, were characterized by UV, fluorescence, proton magnetic resonance, and mass spectral analyses as 3-hydroxy-A alpha C and 6-hydroxy-A alpha C. The remaining 15% were judged to be N-hydroxy-A alpha C and its oxidation products, based on chromatographic and spectral comparisons with a standard, whose synthesis and characterization are also described. Although the proportions of each metabolite were similar across species and individuals, the overall rate of metabolism of A alpha C by human hepatic microsomes showed a wide interindividual variation (37-fold), with a mean activity that was comparable with that observed with rat or mouse liver microsomes. alpha-Naphthoflavone, a selective inhibitor for cytochromes P4501A1 and P4501A2, strongly inhibited formation of both ring-hydroxylation and N-oxidation products by human, rat, or mouse liver hepatic microsomes. In addition, A alpha C oxidation was strongly correlated (r = 0.98; p < 0.001) with the oxidation of 4-aminobiphenyl, a known selective substrate for human and rodent cytochromes P4501A2. Immunoblot analyses confirmed the presence of cytochromes P4501A2, and not P4501A1, in human liver microsomes. Additional studies using recombinant human cytochromes P450 show that high catalytic activity for A alpha C metabolism was associated with human cytochrome P4501A2. Lower, but significant activity was also noted for P4501A1 and P4502C10, which could have important implications for the metabolic activation of A alpha C extrahepatic tissues. Neither A alpha C metabolism nor immunoreactive cytochrome P4501A2 (or P4501A1) was detected in human pancreatic microsomes. Although further carcinogenicity and biomarker studies for A alpha C are needed, the high rate of A alpha C metabolism by human liver cytochrome P4501A2 suggests that humans with the rapid P4501A2 phenotype with may be more susceptible than rodents to this heterocyclic amine carcinogen.