Print Email Facebook Twitter Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH- DNA adducts Title Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH- DNA adducts Author Randerath, K. Sriram, P. Moorthy, B. Aston, J.P. Baan, R.A. van den Berg, P.T.M. Booth, E.D. Watson, W.P. Centraal Instituut voor Voedingsonderzoek TNO Publication year 1998 Abstract 32P-postlabelling analysis for detecting DNA adducts formed by polycyclic 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 analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)- DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ((±)-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene- 10t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained 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 DMBA-modified DNA or with 32P4abelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed annuals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts; efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH- DNA adducts. Subject Nutrition32P-postlabellingDNA adductsImmunoaffinityNuclease P1PAHNucleasePhosphorus 32Polycyclic aromatic hydrocarbonAdsorptionAnimal cellColumn chromatographyContaminationDna adductEnzyme analysisEnzyme specificityGenotoxicityImmunoaffinity chromatographyMutagenicityNonhumanProtein modification9,10-Dimethyl-1,2-benzanthraceneAnimalsAntibodies, MonoclonalAspergillus Nuclease S1Benzo(a)pyreneCarcinogensChromatography, AffinityChrysenesDNA AdductsFluorescent Antibody TechniqueIsotope LabelingMiceMutagenicity TestsMutagensPhosphorus RadioisotopesPolycyclic Hydrocarbons, AromaticSkinAnimalia To reference this document use: http://resolver.tudelft.nl/uuid:6c3d624b-37cb-4d33-b7ac-b7cac45bd856 DOI https://doi.org/10.1016/s0009-2797(98)00003-9 TNO identifier 234401 ISSN 0009-2797 Source Chemico-Biological Interactions, 110 (1-2), 85-102 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.