The catalytic oxidation of lipophilic molecules by cytochrome P450 enzymes to produce genotoxic metabolites is a major toxicity pathway. Bioactivated metabolites induce DNA damage by forming covalently bound DNA adducts, which may initiate complex processes that lead to mutagenesis and carcinogenesis. We have developed a method that mimics the toxicity pathway in mammalian liver and which can be used as a screening technique for genotoxic chemicals. We assembled myoglobin (Mb) and DNA layer-by-layer on top of a PDDA layer (PDDA = polydiallyldimethylammonium chloride) on carbon cloth. Styrene was used as a test molecule that is metabolized to genotoxic styrene oxide (SO). PDDA/DNA/(Mb/DNA)2 films were incubated with styrene and H2O2 for different lengths of time. H2O2 activates Mb on the film, and the activated Mb converts styrene to SO, mimicking the action of cytochrome P450s in the liver. The in situ generated SO then reacts with DNA on the film. After incubation, the film is subjected to neutral thermal hydrolysis to release guanine adducts alklyated at N7 and adenine adducts alkylated at N3. The hydrolysate is analyzed for N7-GSO using CapLC-MS/MS with trapping column. MRM for the transition m/z 272 &rarr 152 (GSO &rarr G) was used for the analyses. GSO was detected in films incubated in styrene and H2O2 for as short as three minutes. The method developed is simple, yet highly sensitive for genotoxicity screening by measurement of rates of DNA damage with high chemical information afforded by the tandem mass spectrometry.
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