1,N-6-ETHENODEOXYADENOSINE AND 3,N-4-ETHENODEOXYCYTIDINE IN LIVER DNAFROM HUMANS AND UNTREATED RODENTS DETECTED BY IMMUNOAFFINITY P-32 POSTLABELING

The etheno-bridged exocyclic DNA adducts 1,N-6-ethenodeoxyadenosine (e psilon dA) and epsilon,N-4-ethenadeoxycytine (epsilon dC) can be forme d by several structurally diverse carcinogens and mutagens that includ e vinyl chloride and urethane. In order to investigate the occurrence and persistence of these adducts in rodents exposed to such DNA-damagi ng agents, an ultra-sensitive detection method has been developed. It is based on immunoaffinity purification of the etheno adducts and subs equent P-32-postlabelling followed by separation as 5'-monophosphates on polyethyleneimine-cellulose-coated thin-layer plates. Normal nucleo tides in the DNA samples were quantitated by HPLC. Optimal conditions for enzymatic hydrolysis of DNA are described: deoxyuridine 3'-monopho sphate was used as internal standard to correct for labelling efficien cy of the etheno adducts. The method had a detection limit of 25 amol of epsilon dA and epsilon dC for a 50 mu g DNA sample. Using this tech nique, analysis of liver DNA from humans with unknown exposure reveale d the presence of epsilon dA and epsilon dC residues in the range of 0 -27 adducts per 10(9) parent bases. Liver DNA obtained from untreated mice and rats was also shown to contain similar low but variable level s of these etheno adducts. In vitro studies indicated that these promu tagenic DNA lesions could arise from endogenously formed lipid peroxid ation products.