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 O^e-EtdGuo in DNA exposed to EtNU in vivo or in vitro.