Sensitive Radioimmunoassay for Detection of O6-Ethyldeoxyguanosine in D N A Exposed to the Carcinogen Ethylnitrosourea in vivo or in vitro

Abstract

O8-ethyl-2′-deoxyguanosine (O8-EtdGuo) is a major premutational product formed in both in­tracellular DNA and in purified DNA in vitro, after exposure to the potent alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Antibodies directed against O8-EtdGuo were obtained by immunizing rabbits with a conjungate of O6-EtGuo and bovine serum albumin. In a competitive radioimmuno­assay (RIA), with O8-Et[8,5′·3H]dGuo as a tracer and various alkylated and natural nucleic acid components as inhibitors, these antibodies show high specificity for O6-EtdGuo and detect this product at a level of 0.3 picomol (antibody association constant, 7×108l/mol). In a sample of 130 μg of hydrolyzed DNA, O6-EtdGuo can thus be measured at a molar ratio of O8-EtdGuo/2′- deoxyguanosine of about 3×10-8, i. e., about 5×103 O8-EtdGuo molecules per diploid cell. Exam­ples are given for the quantitation of Oe-EtdGuo in DNA exposed to EtNU in vivo or in vitro.