Chemical models for cytochrome P450 as a biomimetic metabolic activation system in mutation assays

Abstract

DNA damage is a critical factor in carcinogenesis. The Ames assay is a short-term test that screens for DNA-damaging agents. To be detected in the assay, most carcinogens require oxidation by cytochrome P450, a component of the liver homogenate preparation (S9 mix) that is traditionally used to metabolize promutagens to an active form in vitro. A combination of iron(III) porphyrin plus an oxidant activates many promutagens by mimicking cytochrome P450 metabolism. We previously reported that the mutagenicity of the N-nitrosodialkylamines was detected following reaction with tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (Fe(F5P)Cl) plus tert-butyl hydroperoxide (t-BuOOH), which yielded the same alcohols and aldehydes as the enzymatic reaction. In the present study, to extend the scope of biomimetic models, we tested the mutagenicity of other carcinogens exposed to chemical oxidation systems.We investigated the optimal assay conditions for the models in Salmonella typhimurium TA1538, a strain sensitive to frame-shift mutagens. We activated 2-aminofluorene (AF), benzo[a]pyrene (B[a]P), a tryptophane pyrolysate 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), and 2-acetylaminofluorene (AAF) with Fe(F5P)Cl plus an oxidant—t-BuOOH, m-chloroperoxybenzoic acid (mCPBA), or magnesium monoperoxyphthalate (MPPT)—and we noted the effect of three solvents—acetonitrile (CH3CN),1,4-dioxane, and N,N-dimethylformamide (DMF)—on AF activation.All the promutagens became mutagenic in the presence of Fe(F5P)Cl plus an oxidant, with the effectiveness of the oxidant varying with the chemical. Aromatic amines, for example, showed the strongest mutagenicity with t-BuOOH whereas polycyclic hydrocarbons showed the strongest mutagenicity with mCPBA. All the promutagens were mutagenic in the presence of Fe(F5P)Cl plus MPPT. For AF activation, the order of effectiveness of the solvents was CH3CN>1,4-dioxane>DMF. The results suggested that these systems would serve as useful models for microsomal activating systems.