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.