Probing the mechanism of the carcinogenic activation of N-nitrosodiethanolamine with deuterium isotope effects: in vivo induction of DNA single-strand breaks and related in vitro assays.
A series of bioassays, including in vivo induction of DNA single-strand breaks (SSB) and cytotoxicity in cytochrome P450 2E1-transfected cells, were utilized with N-nitrosodiethanolamine (NDELA), its deuterated isotopomers (alpha-D4NDELA and beta-D4NDELA), N-nitroso-2-hydroxymorpholine (NHMOR), and two of its deuterated isotopomers (2-D-NHMOR and 5,5-D2-NHMOR) to probe the mechanism of carcinogenic activation of NDELA and the role of its metabolite NHMOR. DNA samples, taken from the livers of male Wistar rats 4 h after the administration of NDELA, exhibited dose-dependent DNA SSB levels over the range of 0.08-0.75 mmol/kg (body weight), with the greatest SSB level at the highest dose. Deuterium isotope effects on DNA SSB levels were inversely dependent on dose: alpha-D4NDELA, 3. 22-1.37; and beta-D4NDELA, 1.38-0.79. At the lowest dose of 0.15 mmol/kg (body weight), 5,5-D2-NHMOR gave an isotope effect for DNA SSB of 2.8 while that for 2-D-NHMOR was 0.7. NDELA and beta-D4NDELA were equally cytotoxic to human P450 2E1-transfected V79 Chinese hamster cells, while alpha-D4NDELA was not. Significant DNA SSB levels were observed in these cells for NDELA and beta-D4NDELA but not for alpha-D4NDELA. A kinetic deuterium isotope effect of 2.6 for Vmax/Km was observed for the horse liver alcohol dehydrogenase-mediated oxidation of beta-D4NDELA to NHMOR, while kH/kD for alpha-D4NDELA was 1.05. These data provide the first definitive evidence for the activation of NDELA by a pathway involving the scission of the alpha-CH bond and are consistent with P450 2E1-mediated alpha-hydroxylation of NDELA producing the corresponding reactive alpha-hydroxynitrosamine.
Loeppky
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RN
Department of Chemistry, University of Missouri, Columbia, Missouri 65211, USA.
Fuchs
A
A
Janzowski
C
C
Humberd
C
C
Goelzer
P
P
Schneider
H
H
Eisenbrand
G
G
D000818:Animals; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry*; D003903:Deuterium; D004052:Diethylnitrosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects; D008297:Male; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D013431:Sulfates / Q000737:chemistry; D014162:Transfection
D002273:Carcinogens; D004277:DNA, Single-Stranded; D013431:Sulfates; D009243:NAD; C014129:N-nitrosodiethanolamine; D004052:Diethylnitrosamine; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium