Characterization and mapping of DNA damage induced by reactive metabolitesof 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its comp lex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modif ied DNA, makes DNA damage difficult to characterize and quantify. Therefore , we use the NNK analogue 4-[(acetoxymethyl)nitrosa mino]-1-(3-pyridyl)-1-b utanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nuctide resolution using ligation-mediated polymera se chain reaction and terminal transferase-dependent polymerase chain react ions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentrat ion-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosyl ase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease Ill, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible on ly when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine > aden ine > cytosine > thymine. In contrast to NNKOAc damage distribution pattern s, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic m eans that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' te rmini of the NNKOAc induced single-strand breaks using a P-32-post-labeling assay or a nucleotide exchange reaction at the 3`-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indica te that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydroly sis leading to strand breaks in DNA. The POB-damage could be mutagenic beca use NNKOAc produces single-strand breaks with the products being a 5'-hydro xyl group and a 3'-blocking group and strand breaks. These results represen t the first step in determining if NNK pyridyloxobutylates DNA with sequenc e specificity similar to those observed with other model compounds. (C) 200 1 Academic Press.