Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/ umu assay

Abstract

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3 μM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3–30 μM) was similar (∼300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/ O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10 μM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/ O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.