Synthesis, characterization, and comparative ³²P-postlabeling efficiencies of 2,6-dimethylaniline-DNA adducts

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-7,8,9,10 -tetrahydrobenzo[α]pyrene 3′-phosphate, the major adduct derived from the environmental pollutant benzo[α]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[α]pyrene were both labeled with higher efficiencies than the C8 adduct derived from 2,6-diMeA, with the benzo[α]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.