Evidence for an unstable DNA adduct from N-nitroso-N-methylaniline

Abstract

N-Nitroso-N-methylaniline (NMA) is an esophageal carcinogen in F344 rats. Attempts to detect binding of NMA to DNA or RNA have not been successful. NMA is not mutagenic in the standard Ames bacterial assay, and it did not induce sister chromatid exchanges in mammalian cells. NMA forms the benzenediazonium ion (BDI) during metabolism. This ion has been known to react with aromatic amines, such as adenine, to form triazene coupling products. The purpose of this research was to demonstrate that a triazene adduct, which would be expected to be hydrolytically unstable, was formed by coupling with the adenine residues in DNA. Liver DNA from a rat treated with NMA or from in vitro reactions of BDI with DNA was treated with sodium borohydride. This reaction was shown to result in the reduction of 6-(1-phenyltriazeno)purine to 6-hydrazinopurine (N6-aminoadenine). The hydrolysate of the DNA, presumably containing the hydrazine, was treated with 4-(dimethylamino)naphthaldehyde, and the resulting hydrazone was isolated by reverse-phase HPLC using fluorescence detection. The identity of the adduct was demonstrated by high-resolution mass spectrometry. These data suggest strongly that NMA forms an unstable triazene adduct with adenine in DNA both in vitro and in vivo.