COMPARISON OF IMMUNOAFFINITY CHROMATOGRAPHY ENRICHMENT AND NUCLEASE P1 PROCEDURES FOR P-32 POSTLABELING ANALYSIS OF PAH-DNA ADDUCTS

P-32-postlabelling analysis for detecting DNA adducts formed by polycy clic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the anal ysis is an enrichment procedure for adducts prior to the radiolabellin g step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure deve loped for class specific recognition for polycyclic aromatic hydrocarb on (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was c arried out with skin DNA from mice treated topically with benzo[a]pyre ne, 7,12-dimethyl-benz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurall y similar to the BPDE-I-deoxyguanosine adduct etrahydrobenzo[a]pyrene- 10t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was o btained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA -modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of D MBA-modified DNA or with P-32-labelled DMBA adducts. I-compounds (endo genous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by th e immunoaffinity procedure. Compared to the simple nuclease P1 enhance ment procedure, the immunoaffinity methods were lengthier and more lab our intensive. Advantages of the immunoaffinity procedure include: spe cificity, allowing the selective detection of a certain class of adduc ts; efficient adduct enrichment, providing a viable alternative to oth er enrichment procedures; adequate sensitivity for model studies and t he potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nu clease P1 procedure was viewed as the initial method of choice since i t was capable of detecting a wider range of PAH-DNA adducts. (C) 1998 Published by Elsevier Science Ireland Ltd. All rights reserved.