Abstract
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 (ϵdAS). We have developed approaches using liquid chromatography-mass spectrometry and liquid chromatographytandem mass spectrometry for identification and quantitation of ϵdAS. Scanning and fragment ion analyses confirmed the identity of ϵdAS 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 ϵdAS with 1H NMR, chromatographic, and mass spectral characteristics identical to those of the authentic ϵdAS, suggesting DNA alkylation by the VC epoxide. Subsequent studies evaluated formation of ϵdAS in incubations of murine lung microsomes or recombinant CYP2E1 with VC. The formation of ϵdAS in incubations of lung microsomes or recombinant CYP2E1 with VC was dependent on protein concentrations, CYP2E1 enzyme levels, and incubation time. The rates of ϵdAS 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 ϵdAS 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 ϵdAS adduct.
Footnotes
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This study was funded by the Canadian Cancer Society through Grant 014061 from the National Cancer Institute of Canada (to P.G.F.). Funding for the mass spectrometer (LC-MS/MS) and for the Service Contract on the LC-MS/MS was provided by Grant MA-9475 and Grant MMA-69106, respectively, from the Canadian Institutes of Health Research (to G.J.). M.K. was supported by an Ontario Graduate Scholarship. A.S. was supported by postdoctoral fellowships from the Cancer Research Society and Queen's University Principal's Development Fund.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.013805.
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ABBREVIATIONS: VC, vinyl carbamate; dAS, 2′-deoxyadenosine; DDAS, 2′-deoxy-2-deuteroethenoadenosine; DASO2, diallyl sulfone; ϵdAS, 1,N6-ethenodeoxyadenosine; EC, ethyl carbamate; HPLC, high performance liquid chromatography; LC-MS, high performance liquid chromatography-mass spectrometry; LC-MS/MS, high performance liquid chromatography-tandem mass spectrometry; mCPBA, m-chloroperoxybenzoic acid; MRM, multiple reaction monitoring; P450, cytochrome P450; PNP, p-nitrophenol; r, recombinant; VCO, vinyl carbamate epoxide.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Received November 9, 2006.
- Accepted February 9, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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