³²P-postlabeling of N-(deoxyguanosin-8-yl)arylamine adducts: A comparative study of labeling efficiencies

³²P-Postlabeling is an extremely powerful technique for the detection of DNA adducts. Typically, the quantitation of DNA adducts by ³²P- postlabeling is achieved by relative adduct labeling, via comparison of the radioactivity incorporated into the adducts to that associated with the normal nucleotides. This approach is based on a number of assumptions, the foremost being that normal and adducted nucleotide 3'-phosphates are converted to 3',5'-bisphosphates with similar efficiencies. To evaluate labeling efficiencies for specific DNA adducts, we conducted a comparative study of the kinetics of phosphorylation by T₄ polynucleotide kinase using 2'-deoxyguanosine 3'-phosphate (dG3'p) and a series of N-(deoxyguanosin-8- yl)arylamine 3'-phosphate adduct standards (dG3'p-C8-Ar, Ar being 4- aminobiphenyl, 3- and 4-methylaniline, and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3'p and the dG3'p-C8-Ar adducts followed Michaelis- Menten kinetics. The apparent turnover numbers were 40-240-fold lower when labeling dG3'p-C8-Ar adducts compared to that when labeling dG3'p. The apparent specificity constant calculated for dG3'p-C8-4-aminobiphenyl (1.4 μM^-1 min^-1) was approximately 4-fold lower than that (5.4 μM^-1 min^{- 1}) found for dG3'p. Apparent specificity constants for the monoarylamine adducts were even lower (0.043-0.23 μM^-1 min^-1) and decreased in the following order: 4-methylaniline > 3,4-dimethylaniline > 3-methylaniline > 2,4-dimethylaniline. Similar experiments conducted with dG3'p-C8-Ar standards for 2-methylaniline and 2,3-dimethylaniline produced very poor and irreproducible labeling. These results indicate that ³²P-postlabeling of dG3'p-C8-Ar adducts is less efficient than that of dG3'p and suggest that normal nucleotides will be labeled preferentially to the arylamine adducts under kinetically controlled conditions. The data also indicate a further decrease in labeling efficiency upon substitution ortho to the amino group (e.g., 2,4-dimethylaniline). In addition, the ATP concentrations required for optimal labeling were found to be substantially higher than those used in typical ³²P-postlabeling assays. Since the high specific activity of carrier-free [γ-³²P]-ATP precludes increasing the ATP concentration to a significant extent, these data emphasize the need for using highly efficient adduct enrichment procedures when conducting ³²P-postlabeling analyses of DNA adducts.