Oxidation of vinyl carbamate and formation of 1,N6-ethenodeoxyadenosine in murine lung.
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 (epsilon dAS). We have developed approaches using liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry for identification and quantitation of epsilon dAS. Scanning and fragment ion analyses confirmed the identity of epsilon 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 epsilon dAS with 1H NMR, chromatographic, and mass spectral characteristics identical to those of the authentic epsilon dAS, suggesting DNA alkylation by the VC epoxide. Subsequent studies evaluated formation of epsilon dAS in incubations of murine lung microsomes or recombinant CYP2E1 with VC. The formation of epsilon 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 epsilon 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 epsilon 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 epsilon dAS adduct.
Forkert
Poh-Gek
PG
Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. forkertp@post.queensu.ca
Kaufmann
Martin
M
Black
Gordon
G
Bowers
Raymond
R
Chen
Heidi
H
Collins
Kathy
K
Sharma
Ashish
A
Jones
Glenville
G
D000241:Adenosine / Q000737:chemistry / Q000378:metabolism; D000284:Administration, Oral; D000478:Alkylation / Q000187:drug effects; D000498:Allyl Compounds / Q000008:administration & dosage / Q000494:pharmacology; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D003839:Deoxyadenosines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D008168:Lung / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013450:Sulfones / Q000008:administration & dosage / Q000494:pharmacology; D053719:Tandem Mass Spectrometry; D014520:Urethane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000498:Allyl Compounds; D003839:Deoxyadenosines; D011994:Recombinant Proteins; D013450:Sulfones; C068327:diallyl sulfone; D014520:Urethane; D009249:NADP; C032221:1,N(6)-ethenodeoxyadenosine; C017963:vinyl carbamate; D019392:Cytochrome P-450 CYP2E1; D000241:Adenosine; C058118:2'-deoxyadenosine