DNA adducts from N-nitrosodiethanolamine and related beta-oxidized nitrosamines in vivo: (32)P-postlabeling methods for glyoxal- and O(6)-hydroxyethyldeoxyguanosine adducts.
The mechanism by which environmentally prevalent N-nitrosodiethanolamine (NDELA) and related 2-hydroxyethyl- or other beta-oxidized nitrosamines initiate the carcinogenic process has remained obscure. (32)P-Postlabeling assays for the pH sensitive glyoxal-deoxyguanosine (gdG) and the O(6)-2-hydroxyethyldeoxyguanosine (OHEdG) DNA adducts have been developed as probes in this mechanistic investigation and used in both in vitro and in vivo experiments. The ready cleavage of the glyoxal fragment from gdG at pH 7 and greater has required methods of optimization in order to achieve a detection limit of 0.05 micromol/mol of DNA. Nuclease P1 treatment enhances the detection of gdG adducts but does not increase the detection limit for OHEdG. For OHEdG, best results were achieved using fraction collection from HPLC (0.3 micromol/mol of DNA). Using radiochemical methods, both adducts could be detected either by HPLC or 2D TLC. NDELA, N-nitrosomorpholine (NMOR), N-nitrosomethyethanolamine (NMELA), and N-nitrosoethylethanolamine (NEELA) all produce both gdG and OHEdG adducts in rat liver DNA in vivo and are called bident carcinogens because fragments from both chains of the nitrosamine are incorporated into DNA. N-Nitroso-2-hydroxymorpholine (NHMOR), a metabolite of NDELA and NMOR, generates gdG in DNA in vitro and in vivo. gdG DNA adducts were found in the range 1.1-6.5 micromol/mol of DNA. OHEdG DNA adducts were produced from equimolar amounts of nitrosamines in rat liver in vivo over the range 4-25 micromol/mol of DNA and in the order NMELA > NEELA > NDELA > NMOR. Deuterated isotopomers of NDELA showed a marked isotope effect on DNA OHEdG adduct formation. alpha-Deuteration markedly decreased OHEdG adduct formation while beta-deuteration had the opposite effect. These data support the hypothesis that NDELA and related nitrosamines are activated by both enzyme mediated alpha-hydroxylation and beta-oxidation. The formation of OHEdG adducts from NDELA requires alpha-hydroxylation of the 2-hydroxyethyl chain, and formation of gdG necessitates a beta-oxidation as well. The bident nature of these carcinogens may explain why they are relatively potent carcinogens despite the fact that major proportions of doses are excreted unchanged.
Loeppky
Richard N
RN
Department of Chemistry, University of Missouri, Columbia, Missouri 65211, USA. LoeppkyR@missouri.edu
Ye
Qiuping
Q
Goelzer
Petra
P
Chen
Yuen
Y
D000818:Animals; D001345:Autoradiography; D002273:Carcinogens / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D018736:DNA Adducts / Q000032:analysis / Q000096:biosynthesis*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D004052:Diethylnitrosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D006037:Glyoxal / Q000737:chemistry / Q000378:metabolism; D009602:Nitrosamines / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D051381:Rats
D002273:Carcinogens; D018736:DNA Adducts; D009602:Nitrosamines; D010761:Phosphorus Radioisotopes; C054529:O(6)-(2-hydroxyethyl)-2'-deoxyguanosine; C014129:N-nitrosodiethanolamine; D004052:Diethylnitrosamine; D006037:Glyoxal; C043883:N-nitroso-2-hydroxymorpholine; D003849:Deoxyguanosine