Carcinogen-DNA adducts are DNA damage resulting from the interaction of carcinogen exposure and inherited capacity for metabolism and DNA repair. Our current methods for the detection of carcinogen-DNA adducts using the 32P-postlabeling assay are labor intensive, such that we have only performed small studies (i.e., less than 100 study subjects). To address this limitation, and to develop corroborative methods, we are collaborating with Gerald Wogan to incorporate the ADAM procedure (adduct detection by acylation with 35S-methionine) into our studies. This method is a postlabeling procedure that relies upon chemical acylation of the ribose sugar in nucleosides and HPLC detection, with excellent sensitivity and specificity. Its advantages over the 32P- postlabeling assay is that it is a chemical postlabeling method (rather than enzymatic) so that it is more reproducible and adaptable to many adducts. It also substitutes HPLC for TLC, which has better resolution, is more rapid and reproducible and is much less labor intensive. Because it uses 35S-methionine, it is safer than 32P-radionuclides. We are currently developing standards and validating the method for the detection of 4-aminobiphenyl DNA adducts. Thus far, standard reagents have been synthesized and optimal labeling conditions are being determined. Among the most important accomplishments to date is the development of genotyping assays from archived paraffin-embedded tissues and serum. These procedures required modification of both the conventional phenol- based methods and the use of nested-PCR strategies. Current methods allow us to detect polymorphisms in CYP1A1, GSTM1, CYP2E1, and NAT2. This method is important because we can now test hypotheses from archived tissue banks (i.e., performing nested case-control studies from prospectively established cohorts where serum has been stored). We can therefore, save money and rapidly test hypotheses, without having to wait for new field studies.
