Cytochrome P-450 3A4 (nifedipine oxidase) is responsible for the C-oxidative metabolism of 1-nitropyrene in human liver microsomal samples.

The nitrated polycyclic aromatic hydrocarbon 1-nitropyrene is a ubiquitous environmental pollutant. The role of cytochromes P-450 in the human metabolism of [3H]-1-nitropyrene was investigated using human liver microsomes. The range of microsomal metabolism from 16 individual liver specimens was 0.13 to 0.99 nmol/min/mg protein. Using 3 microsomal samples exhibiting different maximal velocities, the Km of 1-nitropyrene metabolism was 3.3 +/- 0.5 microM, indicating that perhaps a single or similar cytochromes P-450 was involved in the metabolism of 1-nitropyrene in these samples. The P-450 3A inhibitor triacetyloleandomycin inhibited 86 +/- 8% of the microsomal metabolism of 1-nitropyrene. Further evidence for the role of P-450 3A in human microsomal metabolism of 1-nitropyrene was gained using inhibitory anti-P-450 3A antibodies. Using 3 separate microsomal samples, antibody conditions that inhibited approximately 90% of the metabolism of the P-450 3A4-specific substrate nifedipine inhibited approximately 60-70% of the metabolism of 1-nitropyrene. Human liver microsomes demonstrated a preference for 1-nitropyren-3-ol formation over 1-nitropyren-6-ol or 1-nitropyren-8-ol, which is in contrast to that noted in rodents where the 6-ol and 8-ol are preferentially formed over the 3-ol, yet in agreement with earlier studies on the metabolism of 1-nitropyrene using Vaccinia-expressed human cytochromes P-450. These results indicate that the human hepatic metabolism of 1-nitropyrene is carried out by at least two or more P-450s including those in the P-450 3A subfamily. These studies also suggest that the metabolism of this compound by humans may differ from that in rodents in both the cytochromes that are involved and the specific metabolites that are formed.

Role of nitroreductases but not cytochromes P450 in the metabolic activation of 1-nitropyrene in the HepG2 human hepatoblastoma cell line.

1-Nitropyrene is an environmental contaminant that is mutagenic in many prokaryotic and eukaryotic systems, including the hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) locus in the human hepatoma cell line HepG2. Metabolism and DNA adduct formation of [3H]1-nitropyrene in the HepG2 were quantified to understand the role of nitroreduction and/or cytochrome P450-mediated C-oxidation of 1-nitropyrene in DNA adduct formation and mutagenicity. In uninduced HepG2 cells, 10 microM [3H]1-nitropyrene was metabolized principally by nitroreduction to 1-aminopyrene (516 pmol/24 hr/10(6) cells), and by cytochrome P450-mediated C-oxidation to K-region trans-dihydrodiols (37 pmol/24 hr/10(6) cells), 1-nitropyren-3-ol (51 pmol/24 hr/10(6) cells), and 1-nitropyren-6-ol and 1-nitropyren-8-ol (77 pmol/24 hr/10(6) cells). Pretreatment of the HepG2 cells for 24 hr with 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a complete change in the metabolism of [3H]1-nitropyrene, with 1-nitropyren-6-ol and 1-nitropyren-8-ol formation (449 pmol/24 hr/10(6) cells) being 80-fold greater than 1-aminopyrene formation (6 pmol/24 hr/10(6) cells). This increase in C-oxidation of 1-nitropyrene was consistent with increased levels of cytochrome P450 1A. The only DNA adduct detected using the 32P-postlabeling assay in the HepG2 cells administered 1-nitropyrene was N-(2'-deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP). Induction of C-oxidative metabolism through TCDD treatment resulted in a concomitant decrease in dG-C8-AP formation. DNA adducts for oxidized 1-nitropyrene metabolites were not detected in the TCDD-treated HepG2 cells administered 1-nitropyrene, which indicates that cytochrome P450-mediated C-oxidative pathways are detoxification pathways in HepG2 cells.

Pathways for the mutagenesis of 1-nitropyrene and dinitropyrenes in the human hepatoma cell line HepG2.

The mutagenicity, metabolism, DNA adduction and induction of unscheduled DNA synthesis (UDS) of 1-nitropyrene and 1,8-dinitropyrene were investigated in the human hepatoma cell line HepG2. Previous results had demonstrated that 1-nitropyrene was both mutagenic at the hgprt locus and induced UDS in these cells. In the present study, we find that the dinitropyrenes, although highly mutagenic in Salmonella typhimurium, are not mutagenic and do not induce UDS in the HepG2. Although the rate of 1,8-dinitropyrene nitroreduction was less than that of 1-nitropyrene nitroreduction, this did not explain the lack of mutagenicity and UDS induction by the dinitropyrenes. Therefore, it is proposed that the arylhydroxylamine O-esterificase is not expressed in these cells. Since cytochrome P450-mediated C-oxidation is the predominant metabolic pathway in vivo, we sought to determine if an increase in the ratio of cytochrome P450-mediated C-oxidation over nitroreduction would result in increased or decreased DNA adducts in the HepG2. The administration of 2.5 microM 3-methylcholanthrene to the HepG2 increased the ratio of C-oxidation/nitroreduction from 2.8 +/- 1.9 to 50.4 +/- 46.1. This was accompanied by a decrease in the C8-guanyl adduct of 1-nitropyrene (via nitroreduction) from 18.7 +/- 7.0 to 4.8 +/- 1.7 fmoles/micrograms DNA, without any further increase in other 1-nitropyrene DNA adducts. These results suggest that the cytochrome P450-mediated metabolism of 1-nitropyrene to epoxides, phenols, and dihydrodiols is not an activation pathway in the HepG2 cells, and may explain the weak carcinogenicity of 1-nitropyrene in vivo, where cytochrome P450-mediated C-oxidation predominates.