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Oxidation of Vinyl Carbamate and Formation of 1,N⁶-Ethenodeoxyadenosine in Murine Lung

Departments of Anatomy and Cell Biology (P.-G.F., G.B., K.C., A.S.), Biochemistry (M.K., G.J.), and Chemistry (R.B.), Queen's University, Kingston, Ontario, Canada; and Food Research Division, Bureau of Chemical Safety, Health Canada, Ottawa, Ontario, Canada (H.C.)

Vinyl carbamate (VC) is derived from ethyl carbamate, a carcinogen formed in fermentation of food and alcoholic products. We have undertaken studies to test the hypothesis that an epoxide generated from VC oxidation leads to formation of 1,N⁶-ethenodeoxyadenosine (dAS). We have developed approaches using liquid chromatography-mass spectrometry and liquid chromatographytandem mass spectrometry for identification and quantitation of dAS. Scanning and fragment ion analyses confirmed the identity of dAS based on the molecular ion [M + H]⁺ m/z 276 and the specific fragment ion m/z 160. Chemical oxidation of VC in reactions containing 2'-deoxyadenosine produced dAS with ¹H NMR, chromatographic, and mass spectral characteristics identical to those of the authentic dAS, suggesting DNA alkylation by the VC epoxide. Subsequent studies evaluated formation of dAS in incubations of murine lung microsomes or recombinant CYP2E1 with VC. The formation of dAS in incubations of lung microsomes or recombinant CYP2E1 with VC was dependent on protein concentrations, CYP2E1 enzyme levels, and incubation time. The rates of dAS formation were highly correlated with VC concentrations. Peak rates were produced by lung microsomes and recombinant CYP2E1 at 3.0 and 2.5 mM VC, respectively. In inhibitory studies, incubations of VC were performed using lung microsomes from mice treated with the CYP2E1 inhibitor diallyl sulfone (100 mg/kg, p.o.). Results from these studies showed significantly decreased dAS formation in microsomes incubated with VC, with an inhibition of 70% at 3.0 mM. These findings suggested that CYP2E1 is a major enzyme mediating VC oxidation, leading to the formation of a metabolite that alkylates DNA to form the dAS adduct.

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