Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA

Endogenous 5-methylcytosine (MeC) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational ‘hotspots' for smoking induced lung cancer. MeC enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5′-CCCGGCACCC GC[15N3,13C1-G]TCCGCG-3′, + strand) were prepared containing [15N3, 13C1]-guanine opposite unsubstituted cytosine, MeC, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2′-deoxynucleosides, N2-BPDE-dG adducts formed at the [15N3, 13C1]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N2-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N2 position of guanin

Oxidative DNA base damage by the antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine

Abstract-Tirapazamine is a bioreductively activated DNA-damaging agent that selectively kills the hypoxic cells found in solid tumors. In this work, base excision repair enzymes were used to provide evidence that tirapazamine causes significant amounts of damage to both purine and pyrimidine residues in double-stranded DNA.

Comprehensive High-Resolution Mass Spectrometric Analysis of DNA Phosphate Adducts Formed by the Tobacco-Specific Lung Carcinogen 4‑(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, <b>1</b>) is a potent lung carcinogen in laboratory animals and is believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK leads to the formation of pyridyloxobutyl DNA adducts, a critical step in its mechanism of carcinogenesis. In addition to DNA nucleobase adducts, DNA phosphate adducts can be formed by pyridyloxobutylation of the oxygen atoms of the internucleotidic phosphodiester linkages. We report the use of a liquid chromatography–nanoelectrospray ionization–high-resolution tandem mass spectrometry technique to characterize 30 novel pyridyloxobutyl DNA phosphate adducts in calf thymus DNA (CT-DNA) treated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, <b>2</b>), a regiochemically activated form of NNK. A ¹⁵N₃-labeled internal standard was synthesized for one of the most abundant phosphate adducts, dCp­[4-oxo-4-(3-pyridyl)­butyl]­dC (CpopC), and this standard was used to quantify CpopC and to estimate the levels of other adducts in the NNKOAc-treated CT-DNA. Formation of DNA phosphate adducts by NNK <i>in vivo</i> was further investigated in rats treated with NNK acutely (0.1 mmol/kg once daily for 4 days by subcutaneous injection) and chronically (5 ppm in drinking water for 10, 30, 50, and 70 weeks). This study provides the first comprehensive structural identification and quantitation of a panel of DNA phosphate adducts of a structurally complex carcinogen and chemical support for future mechanistic studies of tobacco carcinogenesis in humans

Kinetics of <i>O</i>⁶‑Pyridyloxobutyl-2′-deoxyguanosine Repair by Human <i>O</i>⁶‑alkylguanine DNA Alkyltransferase

Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed to contribute to the development of lung tumors in smokers. NNK and NNN are metabolized to DNA-reactive species that form a range of nucleobase adducts, including bulky <i>O</i>⁶-[4-oxo-4-(3-pyridyl)­but-1-yl]­deoxyguanosine (<i>O</i>⁶-POB-dG) lesions. If not repaired, <i>O</i>⁶-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that <i>O</i>⁶-POB-dG can be directly repaired by <i>O</i>⁶-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from <i>O</i>⁶-alkylguanines in DNA to an active site cysteine residue within the protein. In the present study, we investigated the influence of DNA sequence context and endogenous cytosine methylation on the kinetics of AGT-dependent repair of <i>O</i>⁶-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific <i>O</i>⁶-POB-dG adducts within <i>K-ras</i> and <i>p53</i> gene-derived DNA sequences were incubated with recombinant human AGT protein, and the kinetics of POB group transfer was monitored by isotope dilution HPLC-ESI⁺-MS/MS analysis of <i>O</i>⁶-POB-dG remaining in DNA over time. We found that the second-order rates of AGT-mediated repair were influenced by DNA sequence context (10-fold differences) but were only weakly affected by the methylation status of neighboring cytosines. Overall, AGT-mediated repair of <i>O</i>⁶-POB-dG was 2–7 times slower than that of <i>O</i>⁶-Me-dG adducts. To evaluate the contribution of AGT to <i>O</i>⁶-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and <i>O</i>⁶-POB-dG adduct repair over time was monitored by HPLC-ESI⁺-MS/MS. We found that HBEC cells were capable of removing <i>O</i>⁶-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (<i>O</i>⁶-benzylguanine). Taken together, our results suggest that AGT plays an important role in protecting human lung against tobacco nitrosamine-mediated DNA damage and that inefficient AGT repair of <i>O</i>⁶-POB-dG at a specific sequences contributes to mutational spectra observed in smoking-induced lung cancer

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA

Endogenous 5-methylcytosine (MeC) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational ‘hotspots’ for smoking induced lung cancer. MeC enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5′-CCCGGCACCC GC[15N3,13C1-G]TCCGCG-3′, + strand) were prepared containing [15N3, 13C1]-guanine opposite unsubstituted cytosine, MeC, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2′-deoxynucleosides, N2-BPDE-dG adducts formed at the [15N3, 13C1]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N2-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE–DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N2 position of guanine.ISSN:1362-4962ISSN:0301-561

Inhalation exposure to cigarette smoke and inflammatory agents induces epigenetic changes in the lung

Smoking-related lung tumors are characterized by profound epigenetic changes including scrambled patterns of DNA methylation, deregulated histone acetylation, altered gene expression levels, distorted microRNA profiles, and a global loss of cytosine hydroxymethylation marks. Here, we employed an enhanced version of bisulfite sequencing (RRBS/oxRRBS) followed by next generation sequencing to separately map DNA epigenetic marks 5-methyl-dC and 5-hydroxymethyl-dC in genomic DNA isolated from lungs of A/J mice exposed whole-body to environmental cigarette smoke for 10 weeks. Exposure to cigarette smoke significantly affected the patterns of cytosine methylation and hydroxymethylation in the lungs. Differentially hydroxymethylated regions were associated with inflammatory response/disease, organismal injury, and respiratory diseases and were involved in regulation of cellular development, function, growth, and proliferation. To identify epigenetic changes in the lung associated with exposure to tobacco carcinogens and inflammation, A/J mice were intranasally treated with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the inflammatory agent lipopolysaccharide (LPS), or both. NNK alone caused minimal epigenetic alterations, while exposure either to LPS or NNK/LPS in combination led to increased levels of global cytosine methylation and formylation, reduced cytosine hydroxymethylation, decreased histone acetylation, and altered expression levels of multiple genes. Our results suggest that inflammatory processes are responsible for epigenetic changes contributing to lung cancer development