Delshanee Kotandeniya

Evaluation of Toxicant and Carcinogen Metabolites in the Urine of E-Cigarette Users Versus Cigarette Smokers

INTRODUCTION Electronic cigarettes (e-cigarettes) are rapidly increasing in popularity but little information is available on their potential toxic or carcinogenic effects. METHODS Twenty-eight e-cigarette smokers who had not smoked tobacco cigarettes for at least 2 months provided urine samples which were analyzed by validated methods for a suite of toxicant and carcinogen metabolites including 1-hydroxypyrene (1-HOP), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), 3-hydroxypropylmercapturic acid (3-HPMA), 2-hydroxypropylmercapturic acid (2-HPMA), 3-hydroxy-1-methylpropylmercapturic acid (HMPMA), S-phenylmercapturic acid (SPMA), nicotine, and cotinine. Levels of these compounds were compared to those found in cigarette smokers from three previous studies. RESULTS Levels of 1-HOP, total NNAL, 3-HPMA, 2-HPMA, HMPMA, and SPMA were significantly lower in the urine of e-cigarette users compared to cigarette smokers. Levels of nicotine and cotinine were significantly lower in e-cigarette users compared to cigarette smokers in one study but not in another. CONCLUSIONS With respect to the compounds analyzed here, e-cigarettes have a more favorable toxicity profile than tobacco cigarettes.

APOBEC3B is an enzymatic source of mutation in breast cancer

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.

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.

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, 1) 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, 2), 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 in vivo 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 O6-Pyridyloxobutyl-2′-deoxyguanosine Repair by Human O6-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 O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine (O(6)-POB-dG) lesions. If not repaired, O(6)-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that O(6)-POB-dG can be directly repaired by O(6)-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from O(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 O(6)-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific O(6)-POB-dG adducts within K-ras and p53 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 O(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 O(6)-POB-dG was 2-7 times slower than that of O(6)-Me-dG adducts. To evaluate the contribution of AGT to O(6)-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and O(6)-POB-dG adduct repair over time was monitored by HPLC-ESI(+)-MS/MS. We found that HBEC cells were capable of removing O(6)-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (O(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 O(6)-POB-dG at a specific sequences contributes to mutational spectra observed in smoking-induced lung cancer.

Combined analysis of the tobacco metabolites cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine.

Two of the most widely measured compounds in the urine of people who use tobacco products are cotinine, a major metabolite of the addictive constituent nicotine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of the powerful lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Thousands of analyses have been reported in the literature, carried out exclusively, to the best of our knowledge, by separate methods. In the study reported here, we have developed a sensitive, accurate, and precise liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring method for the combined analysis of total cotinine (the sum of cotinine and its glucuronide) and total NNAL (the sum of NNAL and its glucuronide). The new method quantifies naturally occurring [13C]cotinine to minimize problems associated with the vast differences in concentration of total cotinine and total NNAL in urine. This method should greatly facilitate future determinations of these important compounds.

Maintenance DNA Methyltransferase Activity in the Presence of Oxidized Forms of 5-Methylcytosine: Structural Basis for Ten Eleven Translocation-Mediated DNA Demethylation

A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten eleven translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. In this study, we investigated the catalytic activity of human DNMT1 in the presence of oxidized forms of mC. A mass spectrometry-based assay was employed to study the kinetics of DNMT1-mediated cytosine methylation in CG dinucleotides containing C, mC, hmC, fC, or caC across from the target cytosine. Homology modeling, coupled with molecular dynamics simulations, was used to explore the structural consequences of mC oxidation with regard to the geometry of protein-DNA complexes. The DNMT1 enzymatic activity was strongly affected by the oxidation status of mC, with the catalytic efficiency decreasing in the following order: mC > hmC > fC > caC. Molecular dynamics simulations revealed that DNMT1 forms an unproductive complex with DNA duplexes containing oxidized forms of mC as a consequence of altered interactions of the target recognition domain of the protein with the C-5 substituent on cytosine. Our results provide new structural and mechanistic insight into TET-mediated DNA demethylation.

Corrigendum: APOBEC3B is an enzymatic source of mutation in breast cancer

This corrects the article DOI: 10.1038/nature11881

Abstract 1697: A study on the reactivity of activated polycyclic aromatic hydrocarbons (PAH) with guanines base pared to C-5 substituted cytosines

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC All CG dinucleotides within exons 5-8 of the human p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). Guanine residues within these sites (e.g. codons 157, 158, 245, 248, and 273) are the major mutational hotspots for smoking induced lung cancer, suggesting that MeC mediates the reactivity of neighboring guanine bases towards tobacco carcinogens. Previous studies have shown that the reactivity of a carcinogenic diol epoxide metabolite of the human carcinogen, benzo[a]pyrene (B[a]P), towards the exocyclic amino group of guanine, is increased when guanine is base paired to MeC as compared to unmethylated cytosine. In the present study, the structural basis for the enhanced reactivity of BPDE towards MeC: G base pairs was investigated using a stable isotope labeling approach and a series of MeC structural analogs, including 5-ethyl-dC, 5-propyl-dC, N4-ethyl-dC, 5-chloro-dC, 5-bromo-dC, 5-iodo-dC, 5-propynyl-dC, difluorotoluene, pyrrolo-dC, phenylpyrrolo-dC, and diaminonaphthyl-derived nucleoside. Synthetic DNA duplexes derived from the frequently mutated region of the p53 tumor suppressor gene (5′-CCCGGCACCCGC[15N3, 13C1-G]TCCGCG-3′, from exon 5) were prepared containing [15N3, 13C1]-labeled guanine opposite C, MeC, or nucleobase analogs. Circular dichroism (CD) and UV melting studies have shown that C-5 substituents on cytosine do not disrupt the structure and stability of the DNA duplex. Following treatment with (±)-anti-benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide [(±)-anti-BPDE], (-)-anti-benzo[a]pyrene-s-7,t-8-dihydrodiol-t-9,10-epoxide [(-)-anti-BPDE], or related PAH diol epoxides, 5-methyl chrysene diol epoxide, benzo[c]phenanthrene diol epoxide, benzo[g]chrysene diol epoxide, and benzo[a,l]pyrene diol epoxide, and enzymatic hydrolysis of the adducted DNA to 2′-deoxynucleosides, the amounts of stereoisomeric N2-guanine adducts formed at the labeled site were determined by capillary HPLC-ESI+-MS/MS. We found that the presence of 5-methylcytosine and nucleobases with extended aromatic systems increases the reactivity of the partner guanine towards BPDE and other PAH diolepoxides, while 5-fluoro-dC and 5-iodo-dC lead to a decreased reactivity. Furthermore, the presence of C-5-cytosine analog modifies the stereoisomeric composition of the resulting adducts. Low temperature fluorescence and molecular docking studies reveal that the presence of MeC and unnatural base analogs with extended aromatic systems facilitate the formation of the pre-covalent BPDE-DNA complexes which place BPDE in a favorable orientation for trans attack by the N2 position of guanine. These results provide a mechanistic insight into the origins of increased reactivity of PAHs towards MeCG dinucleotides. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1697.