Characterization of adductomic totality of NNK, (R)-NNAL and (S)-NNAL in A/J mice, and their correlations with distinct lung carcinogenicity.

Lung cancer is the leading cause of cancer-related deaths. While tobacco use is the main cause, only 10-20% of smokers eventually develop clinical lung cancer. Thus, the ability of lung cancer risk prediction among smokers could transform lung cancer management with early preventive interventions. Given that DNA damage by tobacco carcinogens is the potential root cause of lung carcinogenesis, we characterized the adductomic totality of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a potent lung carcinogen in tobacco, commonly known as NNK) in the target lung tissues, the liver tissues and the peripheral serum samples in a single-dose NNK-induced lung carcinogenesis A/J mouse model. We also characterized these adductomic totalities from the two enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major in vivo metabolite of NNK) given their distinct carcinogenicity in A/J mice. With these adductomic data, we demonstrated that tissue protein adductomics have the highest abundance. We also identified that the adductomic levels at the 8 h time point after carcinogen exposure were among the highest. More importantly, the relationships among these adductomics were characterized with overall strong positive linear correlations, demonstrating the potential of using peripheral serum protein adductomics to reflect DNA adductomics in the target lung tissues. Lastly, we explored the relationships of these adductomics with lung tumor status in A/J mice, providing preliminary but promising evidence of the feasibility of lung cancer risk prediction using peripheral adductomic profiling.

Investigation of 2'-Deoxyadenosine-Derived Adducts Specifically Formed in Rat Liver and Lung DNA by N'-Nitrosonornicotine Metabolism.

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans" (Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2'-hydroxylation and 5'-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5'-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5'-hydroxylation in rat tissues are 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N6-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5'-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N6-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine (N6-Py-THF-dAdo) and 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2'-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5'-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N6-Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH3CN led to the formation of Py-Py-dN both in vitro and in vivo, which was confirmed by its isotopically labeled internal standard [pyridine-d4]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5'-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.

Coexposure to Inhaled Aldehydes or Carbon Dioxide Enhances the Carcinogenic Properties of the Tobacco-Specific Nitrosamine 4-Methylnitrosamino-1-(3-pyridyl)-1-butanone in the A/J Mouse Lung.

Tobacco smoke is a complex mixture of chemicals, many of which are toxic and carcinogenic. Hazard assessments of tobacco smoke exposure have predominantly focused on either single chemical exposures or the more complex mixtures of tobacco smoke or its fractions. There are fewer studies exploring interactions between specific tobacco smoke chemicals. Aldehydes such as formaldehyde and acetaldehyde were hypothesized to enhance the carcinogenic properties of the human carcinogen, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) through a variety of mechanisms. This hypothesis was tested in the established NNK-induced A/J mouse lung tumor model. A/J mice were exposed to NNK (intraperitoneal injection, 0, 2.5, or 7.5 µmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 17 ppmv) for 3 h in a nose-only inhalation chamber, and lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had more adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase the severity of NNK-induced lung adenomas. The aldehyde coexposure did not affect the levels of NNK-derived DNA adduct levels. Similar studies tested the ability of a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure to influence lung adenoma formation by NNK. While carbon dioxide alone was not carcinogenic, it significantly increased the number of NNK-derived lung adenomas without affecting NNK-derived DNA damage. These studies indicate that the chemicals in tobacco smoke work together to form a potent lung carcinogenic mixture.

Modulation of the PD-1/PD-L1 immune checkpoint axis during inflammation-associated lung tumorigenesis.

Chronic obstructive pulmonary disease (COPD) is a significant risk factor for lung cancer. One potential mechanism through which COPD contributes to lung cancer development could be through generation of an immunosuppressive microenvironment that allows tumor formation and progression. In this study, we compared the status of immune cells and immune checkpoint proteins in lung tumors induced by the tobacco smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or NNK + lipopolysaccharide (LPS), a model for COPD-associated lung tumors. Compared with NNK-induced lung tumors, NNK+LPS-induced lung tumors exhibited an immunosuppressive microenvironment characterized by higher relative abundances of PD-1+ tumor-associated macrophages, PD-L1+ tumor cells, PD-1+ CD4 and CD8 T lymphocytes and FOXP3+ CD4 and CD8 T lymphocytes. Also, these markers were more abundant in the tumor tissue than in the surrounding 'normal' lung tissue of NNK+LPS-induced lung tumors. PD-L1 expression in lung tumors was associated with IFNγ/STAT1/STAT3 signaling axis. In cell line models, PD-L1 expression was found to be significantly enhanced in phorbol-12-myristate 13-acetate activated THP-1 human monocytes (macrophages) treated with LPS or incubated in conditioned media (CM) generated by non-small cell lung cancer (NSCLC) cells. Similarly, when NSCLC cells were incubated in CM generated by activated THP-1 cells, PD-L1 expression was upregulated in EGFR- and ERK-dependent manner. Overall, our observations indicate that COPD-like chronic inflammation creates a favorable immunosuppressive microenvironment for tumor development and COPD-associated lung tumors might show a better response to immune checkpoint therapies.

Mass Spectrometric Quantitation of Apurinic/Apyrimidinic Sites in Tissue DNA of Rats Exposed to Tobacco-Specific Nitrosamines and in Lung and Leukocyte DNA of Cigarette Smokers and Nonsmokers.

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N7POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 108 nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and nonsmokers. Rats treated with 5 or 21 mg/kg bw NNK for 4 days by s.c. injection had 2-6 and 2-17 times more AP sites than controls in liver and lung DNA (p < 0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks (p < 0.05) but not for 50 and 70 weeks to 5 ppm of NNK in their drinking water. Levels of N7POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm (S)-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA (p < 0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to nonsmokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals and to evaluate AP sites in DNA of smokers and nonsmokers.

Oral Dosing of Dihydromethysticin Ahead of Tobacco Carcinogen NNK Effectively Prevents Lung Tumorigenesis in A/J Mice.

Our early studies demonstrated an impressive chemopreventive efficacy of dihydromethysticin (DHM), unique in kava, against tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice in which DHM was supplemented in the diet. The current work was carried out to validate the efficacy, optimize the dosing schedule, and further elucidate the mechanisms using oral bolus dosing of DHM. The results demonstrated a dose-dependent chemopreventive efficacy of DHM (orally administered 1 h before each of the two NNK intraperitoneal injections, 1 week apart) against NNK-induced lung adenoma formation. Temporally, DHM at 0.8 mg per dose (∼32 mg per kg body weight) exhibited 100% lung adenoma inhibition when given 3 and 8 h before each NNK injection and attained >93% inhibition when dosed at either 1 or 16 h before each NNK injection. The simultaneous treatment (0 h) or 40 h pretreatment (-40 h) decreased lung adenoma burden by 49.8% and 52.1%, respectively. However, post-NNK administration of DHM (1-8 h after each NNK injection) was ineffective against lung tumor formation. In short-term experiments for mechanistic exploration, DHM treatment reduced the formation of NNK-induced O6-methylguanine (O6-mG, a carcinogenic DNA adduct in A/J mice) in the target lung tissue and increased the urinary excretion of NNK detoxification metabolites as judged by the ratio of urinary NNAL-O-gluc to free NNAL, generally in synchrony with the tumor prevention efficacy outcomes in the dose scheduling time-course experiment. Overall, these results suggest DHM as a potential chemopreventive agent against lung tumorigenesis in smokers, with O6-mG and NNAL detoxification as possible surrogate biomarkers.

Hyaluronan-CD44/RHAMM interaction-dependent cell proliferation and survival in lung cancer cells.

Although members of the hyaluronan (HA)-CD44/HA-mediated motility receptor (RHAMM) signaling pathway have been shown to be overexpressed in lung cancer, their role in lung tumorigenesis is unclear. In the present study, we first determined levels of HA and its receptors CD44 and RHAMM in human non-small cell lung cancer (NSCLC) cells and stromal cells as well as mouse lung tumors. Subsequently, we examined the role of HA-CD44/RHAMM signaling pathway in mediating the proliferation and survival of NSCLC cells and the cross-talk between NSCLC cells and normal human lung fibroblasts (NHLFs)/lung cancer-associated fibroblasts (LCAFs). The highest levels of HA and CD44 were observed in NHLFs/LCAFs followed by NSCLC cells, whereas THP-1 monocytes/macrophages showed negligible levels of both HA and CD44. Simultaneous silencing of HA synthase 2 (HAS2) and HAS3 or CD44 and RHAMM suppressed cell proliferation and survival as well as the EGFR/AKT/ERK signaling pathway. Exogenous HA partially rescued the defect in cell proliferation and survival. Moreover, conditioned media (CM) generated by NHLFs/LCAFs enhanced the proliferation of NSCLC cells in a HA-dependent manner as treatment of NHLFs and LCAFs with HAS2 siRNA, 4-methylumbelliferone, an inhibitor of HASs, LY2228820, an inhibitor of p38MAPK, or treatment of A549 cells with CD44 blocking antibody suppressed the effects of the CM. Upon incubation in CM generated by A549 cells or THP-1 macrophages, NHLFs/LCAFs secreted higher concentrations of HA. Overall, our findings indicate that targeting the HA-CD44/RHAMM signaling pathway could be a promising approach for the prevention and therapy of lung cancer.

Mass Spectrometric Quantitation of Pyridyloxobutyl DNA Phosphate Adducts in Rats Chronically Treated with N'-Nitrosonornicotine.

The tobacco-specific carcinogens N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) require metabolic activation to exert their carcinogenicity. NNN and NNK are metabolized to the same reactive diazonium ions, which alkylate DNA forming pyridyloxobutyl (POB) DNA base and phosphate adducts. We have characterized the formation of both POB DNA base and phosphate adducts in NNK-treated rats and the formation of POB DNA base adducts in NNN-treated rats. However, POB DNA phosphate adducts in NNN-treated rats are still uncharacterized. In this study, we quantified the levels of POB DNA phosphate adducts in tissues of rats chronically treated with ( S)-NNN or ( R)-NNN for 10, 30, 50, and 70 weeks during a carcinogenicity study. The highest amounts of POB DNA phosphate adducts were observed in the esophagus of the ( S)-NNN-treated rats, with a maximum level of 5400 ± 317 fmol/mg DNA at 50 weeks. The abundance of POB DNA phosphate adducts in the esophagus was consistent with the results of the carcinogenicity study showing that the esophagus was the primary site of tumor formation from treatment with ( S)-NNN. Compared to the ( R)-NNN group, the levels of POB DNA phosphate adducts were higher in the oral mucosa, esophagus, and liver, while lower in the nasal mucosa of the ( S)-NNN-treated rats. Among 10 combinations of all isomers of POB DNA phosphate adducts, Ap(POB)C and combinations with thymidine predominated across all the rat tissues examined. In the primary target tissue, esophageal mucosa, Ap(POB)C accounted for ∼20% of total phosphate adducts in the ( S)-NNN treatment group throughout the 70 weeks, with levels ranging from 780 ± 194 to 1010 ± 700 fmol/mg DNA. The results of this study showed that POB DNA phosphate adducts were present in high levels and persisted in target tissues of rats chronically treated with ( S)- or ( R)-NNN. These results improve our understanding of DNA damage during NNN-induced carcinogenesis. The predominant POB DNA phosphate isomers observed, such as Ap(POB)C, may serve as biomarkers for monitoring chronic exposure of tobacco-specific nitrosamines in humans.

Strain-specific altered nicotine metabolism in 3,3'-diindolylmethane (DIM) exposed mice.

Two indole compounds, indole-3-carbinol (I3C) and its acid condensation product, 3,3'-diindolymethane (DIM), have been shown to suppress the expression of flavin-containing monooxygenases (FMO) and to induce some hepatic cytochrome P450s (CYPs) in rats. In liver microsomes prepared from rats fed I3C or DIM, FMO-mediated nicotine N-oxygenation was decreased, whereas CYP-mediated nicotine metabolism to nicotine iminium and subsequently to cotinine was unchanged. Therefore, it was hypothesized that in mice DIM would also suppress nicotine N-oxygenation without affecting CYP-mediated nicotine metabolism. Liver microsomes were produced from male and female C57BL/6 J and CD1 mice fed 2500 parts per million (ppm) DIM for 14 days. In liver microsomes from DIM-fed mice, FMO-mediated nicotine N-oxygenation did not differ from the controls, but CYP-mediated nicotine metabolism was significantly increased, with results varying by sex and strain. To confirm the effects of DIM in vivo, control and DIM-fed CD1 male mice were injected subcutaneously with nicotine, and the plasma concentrations of nicotine, cotinine and nicotine-N-oxide were measured over 30 minutes. The DIM-fed mice showed greater cotinine concentrations compared with the controls 10 minutes following injection. It is concluded that the effects of DIM on nicotine metabolism in vitro and in vivo differ between mice and rats and between mouse strains, and that DIM is an effective inducer of CYP-mediated nicotine metabolism in commonly studied mouse strains.

Nitric oxide-donating aspirin (NO-Aspirin) suppresses lung tumorigenesis in vitro and in vivo and these effects are associated with modulation of the EGFR signaling pathway.

Although regular aspirin use has been shown to lower the risk of colorectal cancer, its efficacy against lung cancer is weak or inconsistent. Moreover, aspirin use increases the risk of ulcers and stomach bleeding. In this study, we determined the efficacy of nitric oxide-donating aspirin (NO-Aspirin), a safer form of aspirin in which the parent drug is linked to a nitric oxide-releasing moiety through a spacer, to suppress lung tumorigenesis. Under in vitro conditions, NO-Aspirin significantly reduced the proliferation and survival of tumorigenic bronchial cell line (1170) and non-small cell lung cancer (NSCLC) cell lines (A549, H1650, H1975 and HCC827) and colony formation by NSCLC cells at sub- or low micromolar concentrations (≤1 µM for 1170 cells and ≤6 µM for NSCLC cells) in a COX-2 independent manner. These effects were paralleled by suppression of phospho-epidermal growth factor receptor (EGFR), -STAT3, -Akt and -ERK and enhanced caspase 3 and PARP cleavage. Among NSCLC cells, EGFR mutant cells (H1650, H1975 and HCC827) were more sensitive than cells expressing wild-type EGFR (A549) and H1650 cells were the most sensitive. Moreover, NO-Aspirin sensitized H1650 and H1975 cells to the antiproliferative effects of erlotinib, a tyrosine kinase inhibitor. In in vivo studies using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) + lipopolysaccharide (LPS)-induced model of lung tumorigenesis, NO-Aspirin significantly reduced the number and size of lung tumors, expression of phospho-EGFR and -Akt as well as the pro-inflammatory molecules TNF-α and interferon-gamma. Overall, these results indicate the potential of NO-Aspirin for the chemoprevention of lung cancer in high risk populations.

Identification of more than 100 structurally unique DNA-phosphate adducts formed during rat lung carcinogenesis by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in people who use tobacco products. NNK undergoes metabolic activation-a critical step in its mechanism of carcinogenesis-to an intermediate which reacts with DNA to form pyridyloxobutyl DNA base and phosphate adducts. Another important metabolic pathway of NNK is its conversion to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which similarly forms pyridylhydroxybutyl DNA base adducts that have been characterized previously. In this study, we investigated the potential formation of pyridylhydroxybutyl DNA phosphate adducts. We report the characterization and quantitation of 107 structurally unique pyridylhydroxybutyl DNA phosphate adducts in the lungs of rats treated chronically with a carcinogenic dose of 5 ppm of NNK in their drinking water for up to 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Our findings demonstrate that pyridylhydroxybutyl phosphate adducts account for 38-55 and 34-40% of all the measured pyridine-containing DNA adducts in rat lung and liver, respectively, upon treatment with NNK. Some of the pyridylhydroxybutyl DNA phosphate adducts persisted in both tissues for over 70 weeks, suggesting that they could be potential biomarkers of chronic exposure to NNK and NNAL. This study provides comprehensive characterization and relative quantitation of a panel of NNK/NNAL-derived DNA phosphate adducts, thus identifying NNK as the source of the most structurally diverse set of DNA adducts identified to date from any carcinogen.

In Vivo Stable-Isotope Labeling and Mass-Spectrometry-Based Metabolic Profiling of a Potent Tobacco-Specific Carcinogen in Rats.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen that exerts its carcinogenic effects upon metabolic activation. The identification and quantitation of NNK metabolites could identify potential biomarkers of bioactivation and detoxification of this potent carcinogen and may be used to predict lung cancer susceptibility among smokers. Here, we used in vivo isotope-labeling and high-resolution-mass-spectrometry-based methods for the comprehensive profiling of all known and unknown NNK metabolites. The sample-enrichment, LC-MS, and data-analysis workflow, including a custom script for automated d0- d4- m/ z-pair-peak detection, enabled unbiased identification of numerous NNK metabolites. The structures of the metabolites were confirmed using targeted LC-MS2 with retention-time ( tR) and MS2-fragmentation comparisons to those of standards when possible. Eleven known metabolites and unchanged NNK were identified simultaneously. More importantly, our workflow revealed novel NNK metabolites, including 1,3-Diol (13), α-OH-methyl-NNAL-Gluc (14), nitro-NK- N-oxide (15), nitro-NAL- N-oxide (16), γ-OH NNAL (17), and three N-acetylcysteine (NAC) metabolites (18a-c). We measured the differences in the relative distributions of a panel of nitroso-containing NNK-specific metabolites in rats before and after phenobarbital (PB) treatment, and this served as a demonstration of a general strategy for the detection of metabolic differences in animal and cell systems. Lastly, we generated a d4-labeled NNK-metabolite mixture to be used as internal standards ( d4-rat urine) for the relative quantitation of NNK metabolites in humans, and this new strategy will be used to assess carcinogen exposure and ultimately to evaluate lung-cancer risk and susceptibility in smokers.

Analysis and Identification of 2'-Deoxyadenosine-Derived Adducts in Lung and Liver DNA of F-344 Rats Treated with the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are carcinogenic in animal models and are believed to play an important role in human lung carcinogenesis for cigarette smokers. Cytochrome P450-mediated metabolism of these tobacco-specific nitrosamines produces reactive species that alkylate DNA in the form of pyridyloxobutyl (POB)- or pyridylhydroxybutyl (PHB)-DNA adducts. Understanding the formation mechanism and overall levels of these adducts can potentially enhance cancer prevention methods through the identification of particularly susceptible smokers. Previous studies have identified and measured a panel of POB- and PHB-DNA base adducts of dGuo, dCyd, and Thd; however, dAdo adducts have yet to be determined. In this study, we complete this DNA adduct panel by identifying and quantifying levels of NNK- and NNAL-derived dAdo adducts in vitro and in vivo. To accomplish this, we synthesized standards for expected dAdo-derived DNA adducts and used isotope-dilution LC-ESI+-MS/MS to identify POB adducts formed in vitro from the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) with calf thymus DNA. Adduct levels were then quantified in lung and liver DNA of rats chronically treated with NNK or NNAL for 50 weeks using similar LC-MS detection methods. The in vitro studies identified N6-POB-dAdo and N1-POB-dIno as products of the reaction of NNKOAc with DNA, which supports our proposed mechanism of formation. Though both N6-dAdo and N1-dIno adducts were found in vitro, only N6-dAdo adducts were found in vivo, implying possible intervention by DNA repair mechanisms. Analogous to previous studies, levels of N6-POB-dAdo and N6-PHB-dAdo varied both with tissue and treatment type. Despite the adduct levels being relatively modest compared to most other POB- and PHB-DNA adducts, they may play a biological role and could be used in future studies as NNK- and NNAL-specific DNA damage biomarkers.

Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL, or (R)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290-4510, 872-1120, and 763-1430 fmol/mg DNA, accounting for 15-38%, 8%, and 5-9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents.

Multiclass Carcinogenic DNA Adduct Quantification in Formalin-Fixed Paraffin-Embedded Tissues by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals with cancer risk.

Transcriptome profiling in oral cavity and esophagus tissues from (S)-N'-nitrosonornicotine-treated rats reveals candidate genes involved in human oral cavity and esophageal carcinogenesis.

Recently, we have shown that (S)-N'-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 wk and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate P-value < 0.05 and fold-change ≥2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4, and CD80), inflammation (Ephx2 and Inpp5d) and cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1, and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80, and Dgkg) are also altered in esophageal and head and neck tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and, therefore, could contribute to the development of biomarkers for the early detection and prevention of (S)-NNN-associated oral and esophageal cancers. © 2016 Wiley Periodicals, Inc.

Dietary Dihydromethysticin Increases Glucuronidation of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol in A/J Mice, Potentially Enhancing Its Detoxification.

Effective chemopreventive agents are needed against lung cancer, the leading cause of cancer death. Results from our previous work showed that dietary dihydromethysticin (DHM) effectively blocked initiation of lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice, and it preferentially reduced 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-derived DNA adducts in lung. This study explored the mechanism(s) responsible for DHM's differential effects on NNK/NNAL-derived DNA damage by quantifying their metabolites in A/J mice. The results showed that dietary DHM had no effect on NNK or NNAL abundance in vivo, indicating that DHM does not affect NNAL formation from NNK. DHM had a minimal effect on cytochrome P450 2A5 (CYP2A5, which catalyzes NNK and NNAL bioactivation in A/J mouse lung), suggesting that it does not inhibit NNAL bioactivation. Dietary DHM significantly increased O-glucuronidated NNAL (NNAL-O-gluc) in A/J mice. Lung and liver microsomes from dietary DHM-treated mice showed enhanced activities for NNAL O-glucuronidation. These results overall support the notion that dietary DHM treatment increases NNAL detoxification, potentially accounting for its chemopreventive efficacy against NNK-induced lung tumorigenesis in A/J mice. The ratio of urinary NNAL-O-gluc and free NNAL may serve as a biomarker to facilitate the clinical evaluation of DHM-based lung cancer chemopreventive agents.

DNA Adduct Formation from Metabolic 5'-Hydroxylation of the Tobacco-Specific Carcinogen N'-Nitrosonornicotine in Human Enzyme Systems and in Rats.

N'-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2'-hydroxylation and 5'-hydroxylation. While most previous studies have focused on 2'-hydroxylation in target tissues of rats, available evidence suggests that 5'-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5'-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2'-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7-500 ppm), py-py-dI was the major DNA adduct resulting from 5'-hydroxylation of NNN in vivo. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (S)-NNN or (R)-NNN, the ratios of formation of (R)-py-py-dI to (S)-py-py-dI were not the expected mirror image, suggesting that there may be a carrier for one of the unstable intermediates formed upon 5'-hydroxylation of NNN. Rat hepatocytes treated with (S)- or (R)-NNN or (2'S)- or (2'R)-5'-acetoxyNNN exhibited a pattern of adduct formation similar to that of live rats. In vitro studies with human liver S9 fraction or human hepatocytes incubated with NNN (2-500 µM) demonstrated that py-py-dI formation was greater than the formation of pyridyloxobutyl-DNA adducts resulting from 2'-hydroxylation of NNN. (S)-NNN formed more total py-py-dI adducts than (R)-NNN in human liver enzyme systems, which is consistent with the critical role of CYP2A6 in the 5'-hydroxylation of NNN in human liver. The results of this study demonstrate that the major DNA adduct resulting from NNN metabolism by human enzymes is py-py-dI and provide potentially important new insights into the metabolic activation of NNN in rodents and humans.

Evaluation of Nitrosamide Formation in the Cytochrome P450-Mediated Metabolism of Tobacco-Specific Nitrosamines.

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are carcinogenic tobacco-specific nitrosamines believed to play a vital role in the initiation of tobacco-related cancers. For their carcinogenicities to be exhibited, both NNN and NNK must be metabolically activated by cytochrome P450s, specifically P450 2A6 and P450 2A13, respectively. Prior research has focused on α-hydroxylation, which leads to the formation of several DNA adducts that have been identified and quantified in vivo. However, some studies indicate that P450s can retain substrates within their active sites and perform processive oxidation. For nitrosamines, this would oxidize the highly unstable α-hydroxynitrosamines to potentially more stable nitrosamides, which could also alkylate DNA. Thus, we hypothesized that both NNN and NNK are processively oxidized in vitro to nitrosamides by P450 2A6 and P450 2A13, respectively. To test this hypothesis, we synthesized the NNN- and NNK-derived nitrosamides, determined their half-lives at pH 7.4 and 37 °C, and monitored for nitrosamide formation in an in vitro P450 system with product analysis by LC/NSI+-HRMS/MS. Half-lives of the nitrosamides were determined by HPLC-UV and ranged from 7-35 min, which is more than 40 times longer than the corresponding α-hydroxynitrosamines. Incubation of NNN in the P450 2A6 system resulted in the formation of the nitrosamide N'-nitrosonorcotinine (NNC) at low levels. Similarly, the nitrosamide 4-(methylnitrosamino)-1-(3-pyridyl)-1,4-butanedione (CH2-oxo-NNK) was detected in low amounts in the incubation of NNK with the P450 2A13 system. The other possible NNK-derived nitrosamide, 4-(nitrosoformamido)-1-(3-pyridyl)-1-butanone (CH3-oxo-NNK), was not observed in the P450 2A13 reactions. CH2-oxo-NNK readily formed O6meGua in reactions with dGuo and calf thymus DNA. These results demonstrate that NNC and CH2-oxo-NNK are novel metabolites of NNN and NNK, respectively. Though low-forming, their increased stability may allow for mutagenic DNA damage in vivo. More broadly, this study provides the first account of a cytochrome P450-mediated conversion of nitrosamines to nitrosamides, which warrants further studies to determine how general this phenomenon is in nitrosamine metabolism.

Analysis of O(6)-[4-(3-Pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine and Other DNA Adducts in Rats Treated with Enantiomeric or Racemic N'-Nitrosonornicotine.

(S)-N'-Nitrosonornicotine [(S)-NNN] and racemic NNN are powerful oral and esophageal carcinogens in the F344 rat, whereas (R)-NNN has only weak activity. Tumor formation in these tissues of rats treated with racemic NNN was far greater than the sum of the activities of the individual enantiomers. We hypothesized that metabolites of (R)-NNN enhanced levels of DNA adducts produced by (S)-NNN. A test of that hypothesis necessitated the development of a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for the analysis of O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo), a highly mutagenic DNA adduct not previously quantified in rats treated with NNN. The new method, with a limit of detection of 6.5 amol for diluted standard and 100 amol for DNA samples, was applied in this study. Groups of nine F344 rats were treated with doses as follows: 7 ppm (R)-NNN, 7 ppm (S)-NNN, and 14 ppm racemic NNN; 14 ppm (R)-NNN, 14 ppm (S)-NNN, and 28 ppm racemic NNN; or 28 ppm (R)-NNN, 28 ppm (S)-NNN, and 56 ppm racemic NNN for 5 weeks, and tissues were analyzed for DNA adducts. We found statistically significant, but modest, synergistic enhancement of levels of O(6)-POB-dGuo in the esophagus but not the oral cavity of rats treated with racemic NNN (low and median doses only) compared to the sum of the amounts formed in these tissues of rats treated with (S)-NNN or (R)-NNN. There was no synergy in the formation of other POB-DNA adducts of NNN in oral cavity and esophagus, nor was there any evidence for synergy in nasal respiratory and olfactory epithelium, lung, or liver. Our results provide the first quantitation of O(6)-POB-dGuo in DNA from tissues of rats treated with NNN and evidence for synergy in DNA adduct formation as one possible mechanism by which (R)-NNN enhances the carcinogenicity of (S)-NNN in rats.