Synthesis, Characterization, and Comparative32P-Postlabeling Efficiencies of 2,6-Dimethylaniline−DNA Adducts

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Abstract
2,6-Dimethylaniline (2,6-diMeA) is a ubiquitous environmental pollutant that is used in industry as a synthetic intermediate. It is also found in tobacco smoke and as a major metabolite of lidocaine. Although the potential carcinogenicity of 2,6-diMeA in humans is presently uncertain, this aromatic amine has been classified as a rodent carcinogen. In addition, it is known to form hemoglobin adducts in humans, which indicates a profile of metabolic activation similar to that of typical arylamine carcinogens. Like other aromatic amines, 2,6-diMeA has been shown to yield N-(deoxyguanosin-8-yl)-2,6-dimethylaniline (dG-C8-2,6-diMeA) as a major DNA adduct in vitro. In this study, we show that 2,6-diMeA yields an unusual pattern of DNA adducts. In addition to dG-C8-2,6-diMeA, we have isolated two new adducts, 4-(deoxyguanosin-N2-yl)-2,6-dimethylaniline (dG-N2-2,6-diMeA) and 4-(deoxyguanosin-O6-yl)-2,6-dimethylaniline (dG-O6-2,6-diMeA), from the reaction of N-acetoxy-2,6-dimethylaniline with deoxyguanosine. A similar reaction conducted with deoxyadenosine yielded 4-(deoxyadenosin-N6-yl)-2,6-dimethylaniline (dA-N6-2,6-diMeA). All four adducts were detected in DNA reacted with N-acetoxy-2,6-dimethylaniline, with the relative yields being 46% for dA-N6-2,6-diMeA, 22% for dG-N2-2,6-diMeA, 20% for dG-O6-2,6-diMeA, and 12% for dG-C8-2,6-diMeA. This product profile contrasts markedly with the usual pattern of adducts obtained with aromatic amines, where C8-substituted deoxyguanosine products typically predominate. We further analyzed the kinetics of the T4 polynucleotide kinase (PNK)-catalyzed phosphorylation of the C8 and N2 deoxyguanosine 3‘-phosphate adducts from 2,6-diMeA. The kinetic parameters obtained with these two structurally different adducts are compared to those determined with the parent nucleotide (dG3‘p), and with (±)-anti-10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10tetrahydrobenzo[a]pyrene 3‘-phosphate, the major adduct derived from the environmental pollutant benzo[a]pyrene. The results indicate that all the adducts were labeled with lower efficiencies than dG3‘p, stressing the likely underestimation of adduct levels in typical 32P-postlabeling protocols. Nonetheless, the N2 adducts derived from 2,6-diMeA and benzo[a]pyrene were both labeled with higher efficiencies than the C8 adduct derived from 2,6-diMeA, with the benzo[a]pyrene adduct being the best substrate for PNK. Thus, the data suggest that N2 adducts from dG3‘p are intrinsically better substrates than their C8 analogues for PNK, and that bulkier aromatic fragments may favor the enzyme−substrate interaction during the labeling step.

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