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
Nutrition
32P-postlabelling
DNA adducts
Immunoaffinity
Nuclease P1
PAH
Nuclease
Phosphorus 32
Polycyclic aromatic hydrocarbon
Adsorption
Animal cell
Column chromatography
Contamination
Dna adduct
Enzyme analysis
Enzyme specificity
Genotoxicity
Immunoaffinity chromatography
Mutagenicity
Nonhuman
Protein modification
9,10-Dimethyl-1,2-benzanthracene
Animals
Antibodies, Monoclonal
Aspergillus Nuclease S1
Benzo(a)pyrene
Carcinogens
Chromatography, Affinity
Chrysenes
DNA Adducts
Fluorescent Antibody Technique
Isotope Labeling
Mice
Mutagenicity Tests
Mutagens
Phosphorus Radioisotopes
Polycyclic Hydrocarbons, Aromatic
Skin
Animalia
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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