Nitrosonornicotine Research Articles

Insight into the Interaction Mechanism of Nicotine, NNK, and NNN with Cytochrome P450 2A13 Based on Molecular Dynamics Simulation.

Tobacco smoke contains various cancer-causing toxic substances, including nicotine and nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). The cytochrome 2A13 is involved in nicotine metabolism and in the activation of the pro-carcinogenic agents NNK and NNN, by means of α-hydroxylation reactions. Despite the significance of cytochrome 2A13 in the biotransformation of these molecules, its conformational mechanism and the molecular basis involved in the process are not fully understood. In this study, we used molecular dynamics and principal component analysis simulations for an in-depth analysis of the essential protein motions involved in the interaction of cytochrome 2A13 with its substrates. We also evaluated the interaction of these substrates with the amino acid residues in the binding pocket of cytochrome 2A13. Furthermore, we quantified the nature of these chemical interactions from free energy calculations using the Molecular Mechanics/Generalized Born Surface Area method. The ligands remained favorably oriented toward compound I (cytochrome P450 O═FeIV state), to undergo α-hydroxylation. The hydrogen bond with asparagine 297 was essential to maintaining the substrates in a favorable catalytic orientation. The plot of first principal motion vs second principal motion revealed that the enzyme's interaction with nicotine and NNK involved different conformational subgroups, whereas the conformational subgroups in the interaction with NNN are more similar. These results provide new mechanistic insights into the mode of interaction of the substrates with the active site of cytochrome 2A13, in the presence of compound I, which is essential for α-hydroxylation.

ES11.06 Toxicology of Tobacco and Metabolites, and Impact on Cancer

ES11.06 Toxicology of Tobacco and Metabolites, and Impact on Cancer

Effects of the Tobacco Carcinogens N'-Nitrosonornicotine and Dibenzo[a,l]pyrene Individually and in Combination on DNA Damage in Human Oral Leukoplakia and on Mutagenicity and Mutation Profiles in lacI Mouse Tongue.

In previous studies, we showed that the topical application of dibenzo[a,l]pyrene (DB[a,l]P), also known as dibenzo[def,p]chrysene, to the oral cavity of mice induced oral squamous cell carcinoma. We also showed that dA and dG adducts likely account for most of the mutagenic activity of DB[a,l]P in the oral tissues in vivo. Here we report for the first time that the oral treatment of lacI mice with a combination of tobacco smoke carcinogens, DB[a,l]P and N'-nitrosonornicotine (NNN), induces a higher fraction of mutations than expected from a simple sum of their induced individual mutation fractions, and a change in the mutational profile compared with that expected from the sum of the individual agents. The mutational profile of the combination of agents resembled that of the P53 gene in human head and neck cancers more than that ofeither of the individual agents, in that the percentage of the major class of mutations (GC > AT transitions) is similar to that seen in the P53 gene. A preliminary study was performed to understand the origin of the unexpected mutagenesis observations by measuring specific DNA adducts produced by both NNN and DB[a,l]P in human oral leukoplakia cells. No significant differences in the expected and observed major adduct levels from either agent were observed between individual or combined treatments, suggesting that additional adducts are important in mutagenesis induced by the mixture. Taken together, the above observations support the use of this animal model not only to investigate tobacco smoke-induced oral cancer but also to study chemoprevention.

Cytochrome P450 Enzyme Contributions to the N‐oxide Detoxification Pathway of Tobacco‐Specific Nitrosamines; a Possible Role in Tobacco‐Related Cancer Risk

Tobacco‐specific nitrosamines (TSNAs) including 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) are among the most potent carcinogens found in tobacco products. The major mode of metabolism of NNK is by reduction to form 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), which, like other TSNAs including N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), and N′‐nitrosoanabatine (NAT), is detoxified primarily by UDP‐glucuronosyltransferases (UGTs) to their glucuronide products, but also by N‐oxidation of the common pyridine ring. TSNA‐N‐oxides were identified previously in the urine of rodents exposed to TSNAs, and NNAL‐N‐oxide was found in the urine of smokers. The enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors (furafylline, tranylcypromine, clopidrogrel, montelukast, sulfaphenazole, quinidine, chlomethiazole, and ketoconazole) of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in oxidation reactions using pooled human liver microsomes. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors of CYPs 2A6, 2C8, 2C19, 2E1 and 3A4; no detectable levels of NNN‐, NAT‐ or NAB‐N‐oxide formation were observed in human liver microsomes. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation were examined using microsomal fractions from CYP over‐expressing HEK293 cell lines. CYPs 2A13, 2C8 and 2E1 exhibited the highest oxidation activity against NNK, with Vmax/KM values of 675, 610 and 559 nL•min−1•mg−1, respectively, and against NNAL, with Vmax/KM values of 105, 67 and 86 nL•min−1•mg−1, respectively. These data suggest that several cytochrome P450 enzymes play an important role in the in situ detoxification of NNK and NNAL and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk assessment.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program (to Y. Perez‐Paramo), and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Use of autoclave extraction-supercritical fluid chromatography/tandem mass spectrometry to analyze 4-(methylintrosamino)-1-(3-pyridyl)-1-butanone and N’-nitrosonornicotine in tobacco

4-(methylintrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) are the most prevalent and toxic tobacco specific nitrosamines (TSNAs). Due to their carcinogenicity, knowledge of the composition of NNK and NNN in tobacco is necessary. Herein, a sensitive and rapid method, which employs autoclave extraction-supercritical fluid chromatography/tandem mass spectrometry (SFC-MS/MS), has been developed for the analysis of NNK and NNN in tobacco. Both water-soluble and matrix-bound NNK and NNN were extracted with 100 mM ammonium acetate in an autoclave (130 °C, 4 h), and the aqueous extract was subjected to solvent replacement prior to SFC-MS/MS analysis. NNK and NNN were effectively separated within 5 min by using supercritical CO2 as the main mobile phase coupled with a co-solvent of methanol. Excellent linearity was obtained with coefficients of determination (R2) greater than 0.9997 in the range of 1–160 ng/mL and 5–800 ng/mL for NNK and NNN, respectively. The recoveries were in the range of 92.5–110.0% at different spiked levels of real samples. 12 tobacco samples which include 3 typical tobacco varieties of burley, flue-cured, and oriental tobaccos had been analyzed, and the fraction of matrix-bound NNK was determined as well. In addition, a comparison between the proposed SFC-MS/MS method and a validated liquid chromatography tandem mass spectrometry (LC–MS/MS) internal standard method was conducted. Both techniques exhibit comparable analysis results, but peak splitting of NNN was observed by LC-MSMS due to the existence of E/Z isomers, while SFC-MS/MS offers great improvement through elution condition optimization, demonstrating the applicability of SFC-MS/MS as an alternative tool for NNK and NNN analysis.

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.

Impacts of texture properties and airborne particles on accumulation of tobacco-derived chemicals in fabrics.

Vapor-phase constituents of tobacco smoke are known to accumulate on clothing surfaces; however, the significance of texture properties, such as specific surface area, porosity, and surface roughness, and airborne particles to the sorption capacity of fabrics has not been adequately addressed. In the present study, cotton (t-shirt) and polyester (pajama and lab coat) fabrics were exposed to cigarette smoke containing gaseous and particulate tobacco-derived compounds (e.g., N-nitrosamines). Fabric-air distribution coefficients and particle deposition fluxes were then determined to evaluate the accumulation of the target analytes. Appreciable amounts of N'-nitrosoanabasine (NAB) and 4'-(nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) were detected in all three fabric types although particle-bound NAB and NNK were found only in cigarette smoke. In addition, the root mean square surface roughness heights for three types of clothes were within the same order of magnitude. As such, the deposition fluxes of particle-bound N'-nitrosonornicotine (NNN) and NNK to fabric surface may have contributed to 6-20% and 56-100% of total NNN and NNK in fabrics, respectively, estimated based on the assumed deposition velocity of 0.65 m h-1. Apparently, the sorption capacity of fabrics can be greatly influenced by particle-bound compounds on clothing surfaces, resulting in either over- or under-estimation of fabric-air distribution/partitioning coefficients.

Metabolic Activation and Carcinogenesis of Tobacco-Specific Nitrosamine N'-Nitrosonornicotine (NNN): A Density Function Theory and Molecular Docking Study.

N’-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines (TSNAs) that exists widely in smoke and smokeless tobacco products. NNN can induce tumors in various laboratory animal models and has been identified by International Agency for Research on Cancer (IARC) as a human carcinogen. Metabolic activation of NNN is primarily initiated by cytochrome P450 enzymes (CYP450s) via 2′-hydroxylation or 5′-hydroxylation. Subsequently, the hydroxylating intermediates undergo spontaneous decomposition to generate diazohydroxides, which can be further converted to alkyldiazonium ions, followed by attacking DNA to form various DNA damages, such as pyridyloxobutyl (POB)-DNA adducts and pyridyl-N-pyrrolidinyl (py-py)-DNA adducts. If not repaired correctly, these lesions would lead to tumor formation. In the present study, we performed density functional theory (DFT) computations and molecular docking studies to understand the mechanism of metabolic activation and carcinogenesis of NNN. DFT calculations were performed to explore the 2′- or 5′- hydroxylation reaction of (R)-NNN and (S)-NNN. The results indicated that NNN catalyzed by the ferric porphyrin (Compound I, Cpd I) at the active center of CYP450 included two steps, hydrogen abstraction and rebound reactions. The free energy barriers of the 2′- and 5′-hydroxylation of NNN are 9.82/8.44 kcal/mol (R/S) and 7.99/9.19 kcal/mol (R/S), respectively, suggesting that the 2′-(S) and 5′-(R) pathways have a slight advantage. The free energy barriers of the decomposition occurred at the 2′-position and 5′-position of NNN are 18.04/18.02 kcal/mol (R/S) and 18.33/19.53 kcal/mol (R/S), respectively. Moreover, we calculated the alkylation reactions occurred at ten DNA base sites induced by the 2′-hydroxylation product of NNN, generating the free energy barriers ranging from 0.86 to 4.72 kcal/mol, which indicated that these reactions occurred easily. The docking study showed that (S)-NNN had better affinity with CYP450s than that of (R)-NNN, which was consistent with the experimental results. Overall, the combined results of the DFT calculations and the docking obtained in this study provide an insight into the understanding of the carcinogenesis of NNN and other TSNAs.

Analytical Method for Measurement of Tobacco-Specific Nitrosamines in E-Cigarette Liquid and Aerosol

An experimental method was developed and validated for the collection and analysis of tobacco-specific nitrosamines (TSNAs) that are present in electronic cigarette (EC) liquid or are released from aerosol samples using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. As part of this study, the relative recovery of four target TSNAs was assessed by spiking standards in a mixture of propylene glycol and vegetable glycerin. Recovery was assessed against two major variables: (1) the chemical media (solution) selected for sample dilution (acetonitrile [ACN] vs. ammonium acetate [AA]) and (2) the type of sampling filter used (Cambridge filter pad [CFP] vs. quartz wool [QW] tube). The average recovery of TSNAs in terms of variable 1 was 134 ± 22.1% for ACN and 92.6 ± 8.27% for AA. The average recovery in terms of variable 2 was 83.4 ± 7.33% for QW and 58.5 ± 12.9% for CFP. Based on these conditions, the detection limits of N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine (NAT), and N′-nitrosoanabasine (NAB) were calculated as 4.40, 4.47, 3.71, and 3.28 ng mL−1, respectively. The concentration of TSNAs in liquid and aerosol samples of six commercial EC solutions was measured as below the detection limit.

Grass roots approach to control levels of carcinogenic nitrosamines, NNN and NNK in smokeless tobacco products

Due to the extensive ban on public smoking, tobacco companies focused their business on new smokeless tobacco (SLT) products promoting them as a harm reduction strategy and a safer alternative to cigarettes. Two nitrosamines, N′-nitrosonornicotine (NNN) and nicotine-derived nitrosamine ketone (NNK), present in SLT, listed as group 1 human carcinogens by the International Agency for Research on Cancer, are found to be the prime agents for most of cancers in SLT users. This review illustrates the mechanism of cancer induction by NNN and NNK in humans along with factors influencing the formation of NNN and NNK at various stages of tobacco manufacturing. It reveals the high levels and wide variations of NNN and NNK found among the diverse variety of SLT products sold worldwide. According to a recent report by FDA- Centre for Tobacco Products, reducing levels of nitrosamines in SLT products could greatly enhance the quality of life by reducing mortality, morbidity and medical expenditures due to cancer. For the first time, grass root approaches to minimize the levels of NNN and NNK in tobacco, from plant growth to the finished products, have been systematically compiled as they have the potential to contribute to reducing tobacco related disease burden.

Sharing in the INTERVALS platform study details and data from the assessment of the impact of aerosol from the carbon-heated tobacco product (CHTP) 1.2, a potential modified risk tobacco product, on human organotypic gingival cultures

Tobacco-specific nitrosamines (TSNA) levels in tobacco cut filler and cigarette smoke were measured in more than 1000 commercially available cigarettes sampled between 2008 and 2014. Relative contributions to their transfer from tobacco to the mainstream smoke in terms of direct transfer by distillation, pyrorelease, and pyrosynthesis were evaluated on the basis of the comparison with the transfer of nicotine from tobacco to smoke. N′-nitrosonornicotine (NNN) was transferred essentially by distillation, while N′-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-bipyridyl)-1-butanone (NNK) and N′-nitrosoanabasine (NAB) were transferred by pyrorelease or pyrosynthesis as well. In the case of the Tobacco Heating System 2.2, the transfer of nicotine from tobacco to the aerosol was similar to that observed for cigarettes, while the % transfer of TSNAs from tobacco to THS 2.2 aerosol was 2–3 times lower than in cigarettes. This difference is due to the fact that the tobacco is heated instead of burnt resulting in a lower direct transfer by distillation and a lower if any contribution of pyrosynthesis or pyrorelease.

Presence of the Carcinogen N'-Nitrosonornicotine in Saliva of E-cigarette Users.

Many harmful constituents are present in e-cigarettes at much lower levels than in cigarette smoke, and the results of analysis of urinary biomarkers in e-cigarette users are consistent with these findings. However, understanding the health effects of chronic exposures to e-cigarette aerosols may require thinking beyond these comparisons. In this study, we investigated the endogenous formation of the tobacco-specific oral and esophageal carcinogen N'-nitrosonornicotine (NNN) in e-cigarette users. Salivary NNN, nornicotine, and nicotine as well as urinary tobacco biomarkers, including total NNN, were analyzed in 20 e-cigarette users, 20 smokers, and 19 nonsmokers. Nornicotine and NNN levels in e-cigarettes used by the study participants were also analyzed. The mean of NNN in saliva of e-cigarette users was 14.6 (±23.1) pg/mL, ranging from nonquantifiable (below the limit of quantitation, LOQ) to 76.0 pg/mL. In smokers, salivary NNN ranged from below LOQ to 739pg/mL, with 80% of smokers having salivary NNN in the range of levels found in e-cigarette users. Consistent with a previous report, very low levels of urinary total NNN were present in only 5 out of 20 e-cigarette users (ranging from 0.001 to 0.01 pmol/mL urine). Only trace levels of NNN were found in e-cigarette liquids. Together, our findings demonstrate that NNN is formed endogenously in e-cigarette users. While the overall exposure to NNN in e-cigarette users is dramatically lower than in smokers, the known carcinogenic potency of NNN warrants further investigations into the potential consequences of its endogenous formation. Salivary NNN, rather than urinary total NNN, which accounts for only 1-3% of the NNN dose, should be used to monitor e-cigarette users' exposure to this carcinogen.

New shape-selectivity discovered on graphene-based materials in catching tobacco specific nitrosamines

New shape-selectivity of graphene-based materials was discovered on this article. To explore the new selectivity, the structure and surface state of graphene and carbon nanotube were examined firstly, and their specific selectivity was verified and was compared with that of ZSM-5 zeolite in aqueous solutions of tobacco specific nitrosamines (TSNA) along with dyes. These two adsorbents trapped about 55% and 70% of 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) but only 3% of N’-nitrosonornicotine (NNN) in solution, having an obvious selectivity for the former, due to its stronger interaction with graphene. NNK on graphene sheet obtained more electrons (0.015 e) and owned larger adsorption energy (15.63 kcal mol−1) than that of NNN (0.003 e, 9.19 kcal mol−1), according to theoretical calculation and FTIR results. More 95 or 136 mg g -1 acid red 88 than methyl orange was captured by graphene or carbon nanotube, demonstrating this special and abnormal selectivity again. With new selectivity, graphene showed a higher capacity (6.9%) and shorter adorption equilibrium time (5 min) for TSNA than the typical selecive sorbent ZSM-5 zeolite (1.7% and 20 min) in tobacco solution but kept the similar selctivity to NNK, paving a new way to control the carcinogens like TSNA in environment.

Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cells

The tobacco-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are known human carcinogens. Following metabolic activation, NNK and NNN can induce a number of DNA lesions, including several 4-(3-pyridyl)-4-oxobut-1-yl (POB) adducts. However, it remains unclear to what extent these lesions affect the efficiency and accuracy of DNA replication and how their replicative bypass is influenced by translesion synthesis (TLS) DNA polymerases. In this study, we investigated the effects of three stable POB DNA adducts (O2-POB-dT, O4-POB-dT, and O6-POB-dG) on the efficiency and fidelity of DNA replication in HEK293T human cells. We found that, when situated in a double-stranded plasmid, O2-POB-dT and O4-POB-dT moderately blocked DNA replication and induced exclusively T→A (∼14.9%) and T→C (∼35.2%) mutations, respectively. On the other hand, O6-POB-dG slightly impeded DNA replication, and this lesion elicited primarily the G→A transition (∼75%) together with a low frequency of the G→T transversion (∼3%). By conducting replication studies in isogenic cells in which specific TLS DNA polymerases (Pols) were deleted by CRISPR-Cas9 genome editing, we observed that multiple TLS Pols, especially Pol η and Pol ζ, are involved in bypassing these lesions. Our findings reveal the cytotoxic and mutagenic properties of specific POB DNA adducts and unravel the roles of several TLS polymerases in the replicative bypass of these adducts in human cells. Together, these results provide important new knowledge about the biological consequences of POB adducts.

Investigation and comparison of the transfer of TSNA from tobacco to cigarette mainstream smoke and to the aerosol of a heated tobacco product, THS2.2

Tobacco-specific nitrosamines (TSNA) levels in tobacco cut filler and cigarette smoke were measured in more than 1000 commercially available cigarettes sampled between 2008 and 2014. Relative contributions to their transfer from tobacco to the mainstream smoke in terms of direct transfer by distillation, pyrorelease, and pyrosynthesis were evaluated on the basis of the comparison with the transfer of nicotine from tobacco to smoke. N′-nitrosonornicotine (NNN) was transferred essentially by distillation, while N′-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-bipyridyl)-1-butanone (NNK) and N′-nitrosoanabasine (NAB) were transferred by pyrorelease or pyrosynthesis as well. In the case of the Tobacco Heating System 2.2, the transfer of nicotine from tobacco to the aerosol was similar to that observed for cigarettes, while the % transfer of TSNAs from tobacco to THS 2.2 aerosol was 2–3 times lower than in cigarettes. This difference is due to the fact that the tobacco is heated instead of burnt resulting in a lower direct transfer by distillation and a lower if any contribution of pyrosynthesis or pyrorelease.

Identification of the enzyme isoforms responsible for the N‐oxide detoxification pathway in tobacco specific nitrosamines (TSNAs)

Tobacco‐specific nitrosamines (TSNAs), are among the most potent carcinogens found in tobacco products. Among them are N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), N′‐nitrosoanabatine (NAT), 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and 4‐ (methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), a major metabolite of NNK. TSNAs are detoxified by a number of metabolic pathways, including glucuronidation by the UDP‐glucuronosyltransferase (UGT) family of enzymes, as well as N‐oxidation by an as yet unidentified enzyme or family of enzymes. TSNAs share a pyridine ring in their chemical structure that can be further metabolized to a detoxification N‐oxide product. TSNA‐N‐oxides have been identified in the urine of rodents exposed to TSNAs and NNAL‐N‐oxide was found in the urine of smokers, however, the enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors for the major hepatic cytochrome P450 (CYP) enzymes (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in oxidation reactions with pooled human liver microsomes, including phenacetin, coumarin, bupropion, amodiaquine, diclofenac, omeprazole, dextrometrphan, chlorzoxazone and midazolam. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors for CYPs 1A2, 2A6, 2E1, 2C19 and 3A4. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation was examined using microsomal fractions from CYP over‐expressing HEK293 cells. Microsomes from cells over‐expressing CYPs 1A2, 2C19, 2E1 and 3A4 isoforms exhibited TSNA‐N‐oxide formation. The isoforms 2C19 and 2E1 presented an approximate two‐fold higher activity than the other active isoforms. These data suggest that several cytochrome P450 enzymes may play an important role in the N‐oxidation of TSNAs, and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk.Support or Funding Information National Institutes of Health, National Institutes of Environmental Health Sciences [Grant R01‐ES025460] Health Sciences and Services Authority of Spokane, WA [Grant WSU002292] This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simultaneous Determination of Tobacco Alkaloids, Tobacco-Specific Nitrosamines, and Solanesol in Consumer Products Using UPLC–ESI-MS/MS

The US Alcohol and Tobacco Tax and Trade Bureau (TTB) is responsible for collecting Federal excise taxes on tobacco products. Tobacco products in the USA may fall into several taxable categories including cigars, cigarettes, snuff, chewing tobacco, pipe tobacco, and roll-your-own. The existence of these taxable categories means that the TTB is also responsible for the determination of proper tax classification. Not only does proper classification determine the amount of tax owed, but comprehensive classification procedures must also determine if a consumer product is subject to the tobacco excise tax. Since a product must contain tobacco to be subject to the excise tax, laboratory methods that test for the presence of tobacco can provide useful information to ascertain the taxable status of a product. To test for the presence of chemical markers associated with tobacco, an analytical method was developed that permits the simultaneous determination of nicotine and related alkaloids, tobacco-specific nitrosamines (TSNA), and solanesol in methanolic extracts of tobacco. The method utilizes ultra performance liquid chromatography with electrospray ionization–tandem mass spectrometric detection (UPLC–ESI-MS/MS) and was optimized for the analysis of nicotine, cotinine, nornicotine, anatabine, myosmine, anabasine, isonicoteine, nornicotyrine, nicotyrine, N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN) and solanesol. The analytical method was designed to attenuate the instrument response of nicotine, which is overwhelming, to permit simultaneous analysis of all analytes.

Liquid Chromatography-Tandem Mass Spectrometry for the Quantification of Tobacco-Specific Nitrosamine-Induced DNA Adducts in Mammalian Cells.

Quantification of DNA lesions constitutes one of the main tasks in toxicology and in assessing health risks accompanied by exposure to carcinogens. Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) can undergo metabolic transformation to give a reactive intermediate that pyridyloxobutylates nucleobases and phosphate backbone of DNA. Here, we reported a highly sensitive method, relying on the use of nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS), for the simultaneous quantifications of O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POBdG) as well as O2- and O4-[4-(3-pyridyl)-4-oxobut-1-yl]-thymidine (O2-POBdT and O4-POBdT). By using this method, we measured the levels of the three DNA adducts with the use of 10 μg of DNA isolated from cultured mammalian cells exposed to a model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). Our results demonstrated, for the first time, the formation of O4-POBdT in naked DNA and in genomic DNA of cultured mammalian cells exposed with NNKOAc. We also revealed that the levels of the three lesions increased with the dose of NNKOAc and that O2-POBdT and O4-POBdT could be subjected to repair by the nucleotide excision repair (NER) pathway. The method reported here will be useful for investigations about the involvement of other DNA repair pathways in the removal of these lesions and for human toxicological studies in the future.

Trends Over Time in Tobacco-Specific Nitrosamines (TSNAs) in Whole Tobacco and Smoke Emissions From Cigarettes Sold in Canada.

Tobacco-specific nitrosamines (TSNAs) are potent carcinogens. Levels of TSNAs can be modified through manufacturing practices. In the 2000s, TSNA levels in cigarettes sold in Canada were reduced by changes in tobacco curing processes. The current study examined TSNA levels over the following decade to examine trends over time. Data submitted to Health Canada under the Tobacco Reporting Regulations were used to examine whole tobacco constituents for 1809 brands and mainstream smoke emissions for 191 brands manufactured by Canada's three leading cigarette companies from 2005 through 2011/12 using one-way analysis of variances (ANOVAs) and linear regression models. Levels of N-nitrosoanatabine (NAT) (p < .001) and 4-(methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) (p < .001) in whole tobacco showed significant differences over time, decreasing between 2005 and 2007, and generally increasing from 2007 through 2012. Levels of all TSNAs in mainstream smoke emissions reflected a similar pattern: N-nitrosoanabasine (NAB) (p < .001), NAT (p < .001), NNK (p < .001), and N'-nitrosonornicotine (NNN) (p = .021). Linear regression analyses showed that TSNA levels varied by manufacturer over time in whole tobacco for NAT, NNK, and NNN (p < .001 for all), and in smoke emissions for NAB, NAT, NNK, and NNN (p < .001 for all). The findings indicate that levels of TSNAs in whole tobacco and smoke emissions of cigarettes sold in Canada increased from 2007 through 2011/12, following initial reductions over the previous 2 years. Differences in TSNA levels between companies raise questions about manufacturing practices that may be responsible for these changes. Although increased levels of carcinogenic TSNAs may be alarming, it remains unclear whether these differences translate into differences in health risk. The wide variation of TSNAs within the Canadian market across time and across cigarette companies demonstrates the feasibility of reducing the levels of these potent carcinogens. Although it is unclear whether changes made to levels of TSNAs will result in less tobacco-related disease, the tobacco industry bears a responsibility to minimize the harm from smoking to the fullest extent possible.

Impact of proton: Capturing tobacco specific N-nitrosamines (TSNA) with HZSM-5 zeolite

HZSM-5 zeolite was used to conquer the challenge of liquid adsorption of tobacco specific N-nitrosamines (TSNA), either in aqueous solution or tobacco extract solution, offering a candidate with an optimal Si/Al ratio and channel structure to capture the carcinogens. It could trap 84% of N′-nitrosonornicotine (NNN) and 99% of 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) in aqueous solution, much better than that by NaZSM-5 zeolite. Both FTIR and TG/MS technologies were employed to study the adsorption and catalytic degradation of NNN and NNK by HZSM-5 zeolite. The N–N bond of NNK was broken in the range of 423–573K and the pyridine fragments appeared at 630K due to the breakage of C–C bond between pyridine and carbonyl groups. Besides, HZSM-5 zeolite was sprayed on the rod of cigarette and smoked with different manners for the first time, in order to assess its function of reducing TSNA content in mainstream smoke and compared with that of liquid adsorption.