Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols.

Abstract

The aim of this project was to devise and test improved protocols of the 32P-postlabelling assay for the detection of carcinogen-DNA adducts. The intention was to reverse the drift of different investigators using increasingly divergent experimental conditions. This would lead to a more standardized assay that can be used in future applications by different investigators for the monitoring of human exposure to genotoxic agents, permitting more meaningful comparisons between different studies or between different participants in the same study. As part of this process, there was perceived to be a need for carcinogen-modified DNA standards of known levels of adducts for use as positive controls, as standards for normalization of results with unknown samples and to assist interlaboratory comparisons. The preparation of characterized DNA standards modified by benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), 4-aminobiphenyl (ABP), an aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine, and N-methyl-N-nitrosourea (MNU), a methylating agent yielding DNA containing O6-methylguanine, was carried out. A critical appraisal of all aspects of the 32P-postlabelling procedure and investigations to examine the influence of a number of key variations on the assay were conducted. There followed testing of a consensus protocol in a first interlaboratory trial involving 25 participants in Europe and the USA, conducted on the prepared synthetic DNA standards, the assessment of interlaboratory variability and the reasons for it. Revision of the protocols was followed by further testing in a second interlaboratory trial in which liver DNA from mice treated with BaP or ABP were assayed together with the synthetic DNA standards. Adduct levels were found to be significantly lower by 32P-postlabelling than by 3H incorporation. A recommended set of procedures has been developed for the detection and quantitation of DNA adducts formed by PAHs, aromatic amines and methylating agents. These trials have led to a much clearer idea as to what are the critical features and procedures of the 32P-postlabelling assay and there is a set of standard DNA samples for use in quality control and against which biological samples can be normalized. Use of these standards and procedures has reduced interlaboratory variability in quantitation of DNA adducts.