title: Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA.
abstract: The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, 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 only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means 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' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate 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 hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.
authors.affiliation: Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Quebec City, G1K 7P4, Canada.
authors.forename: J F
authors.initials: JF
authors.lastname: Cloutier
authors.forename: R
authors.initials: R
authors.lastname: Drouin
authors.forename: M
authors.initials: M
authors.lastname: Weinfeld
authors.forename: T R
authors.initials: TR
authors.lastname: O'Connor
authors.forename: A
authors.initials: A
authors.lastname: Castonguay
mesh_terms: D000478:Alkylation; D001483:Base Sequence; D002273:Carcinogens / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005091:Exons / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009602:Nitrosamines / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012907:Smoking / Q000009:adverse effects; D013379:Substrate Specificity
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D004277:DNA, Single-Stranded; D009602:Nitrosamines; D009711:Nucleotides; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I