Tobacco-specific Lung Carcinogen Research Articles

LncRNA XIST modulates miR-328-3p ectopic expression in lung injury induced by tobacco-specific lung carcinogen NNK both in vitro and in vivo.

It is well acknowledged that tobacco-derived lung carcinogens can induce lung injury and even lung cancer through a complex mechanism. MicroRNAs (MiRNAs) are differentially expressed in tobacco-derived carcinogen nicotine-derived nitrosamine ketone (NNK)-treated A/J mice. RNA sequencing was used to detect the level of long non-coding RNAs (lncRNAs). Murine and human lung normal and cancer cells were used to evaluate the function of lncRNA XIST and miR-328-3p in vitro, and NNK-treated A/J mice were used to test their function in vivo. In vivo levels of miR-328-3p and lncRNA XIST were analysed, using in situ hybridization. miR-328-3p agomir and lncRNA XIST-specific siRNA were used to manipulate in vivo levels of miR-328-3p and lncRNA XIST in A/J mice. LncRNA XIST was up-regulated in NNK-induced lung injury and dominated the NNK-induced ectopic miRNA expression in NNK-induced lung injury both in vitro and in vivo. Either lncRNA XIST silencing or miR-328-3p overexpression exerted opposing effects in lung normal and cancer cells regarding cell migration. LncRNA XIST down-regulated miR-328-3p levels as a miRNA sponge, and miR-328-3p targeted the 3'-UTR of FZD7 mRNA, which is ectopically overexpressed in lung cancer patients. Both in vivo lncRNA XIST silencing and miR-328 overexpression could rescue NNK-induced lung injury and aberrant overexpression of the lung cancer biomarker CK19 in NNK-treated A/J mice. Our results highlight the promotive effect of lncRNA XIST in NNK-induced lung injury and elucidate its post-transcriptional mechanisms, indicating that targeting lncRNA XIST/miR-328-3p could be a potential therapeutic strategy to prevent tobacco carcinogen-induced lung injury in vivo.

Association between Second-hand Smoke Exposure and Urinary NNAL Level in Korean Adolescents.

BackgroundThe 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a metabolite of tobacco-specific lung carcinogen that can be found in both smokers and non-smokers. Particularly, NNAL levels of children with a history of exposure to second-hand smoke (SHS) are higher than those of adults. Thus, we aimed to investigate the association between SHS exposure and urine NNAL levels in Korean adolescents.MethodsThis cross-sectional study used data from the Korea National Health and Nutrition Examination Survey VII. Overall, 648 never-smoking adolescents (425 boys and 223 girls) aged 12 to 18 were included in this study. Logistic regression analyses identified the relationship between SHS exposure and elevated urine NNAL levels.ResultsThe mean urine NNAL levels of the no exposure and exposure group in boys were 1.39 and 2.26 ng/mL, respectively, whereas they were 1.01 and 2.45 ng/mL in girls, respectively (P < 0.001). Among the adolescents exposed to SHS, the confounder-adjusted odds ratio (95% confidence intervals) for elevated urine NNAL levels according to exposure area as overall, home, and public area were 2.68 (1.58–4.53), 31.02 (9.46–101.74), and 1.89 (1.12–3.17) in boys; and 6.50 (3.22–13.11), 20.09 (7.08–57.04), and 3.94 (1.98–7.77) in girls, respectively.ConclusionSHS exposure was significantly associated with elevated urine NNAL levels in Korean adolescents, particularly in female adolescents and in those with home exposure. These findings remind us of the need to protect adolescents from SHS.

Targeted High Resolution LC/MS3 Adductomics Method for the Characterization of Endogenous DNA Damage.

DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.

The relationship between nicotine metabolism and nicotine and carcinogen exposure among American Indian commercial cigarette smokers and electronic nicotine delivery system users

BackgroundIn American Indian (AI) tobacco users from the southern plains region of the US, we examined the relationship between nicotine and carcinogen exposure and nicotine metabolism. MethodsSmokers (n = 27), electronic nicotine delivery system (ENDS) users (n = 21), and dual users (n = 25) of AI descent were recruited from a southern plains state. Urinary biomarkers of nicotine metabolism (nicotine metabolite ratio [NMR]), nicotine dose (total nicotine equivalents [TNE]), and a tobacco-specific lung carcinogen (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides [total NNAL] were measured. ResultsThe geometric mean of NMR was 3.35 (95% Confidence Interval(CI): 2.42, 4.65), 4.67 (95% CI: 3.39, 6.43), and 3.26 (95% CI: 2.44, 4.37) among smokers, ENDS users, and dual users. Each of the three user groups had relatively low levels of TNE, indicative of light tobacco use. Among smokers, there were inverse relationships between NMR and TNE (r = −0.45) and between NMR and NNAL (r = −0.50). Among dual users, NMR and TNE, and NMR and NNAL were not associated. Among ENDS users, NMR and TNE were not associated. ConclusionsAI tobacco users with higher NMR did not have higher TNE or NNAL exposure than those with lower NMR. This supports prior work among light tobacco users who do not alter their tobacco consumption to account for nicotine metabolism. ImpactThe high prevalences of smoking and ENDS among AI in the southern plains may not be related to nicotine metabolism. Environmental and social cues may play a more important role in light tobacco users and this may be particularly true among AI light tobacco users who have strong cultural ties.

Influence of UGT2B10 Genotype on Urinary Excretion of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol- N-glucuronide by African American Smokers.

At similar smoking levels, African American's lung cancer risk is as much as twice that of whites. We hypothesized that racial/ethnic differences in UDP-glucuronosyltransferase (UGT)-catalyzed glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a detoxication pathway for the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) may contribute to this variable risk. UGT2B10 catalyzes NNAL- N-glucuronidation, and a UGT2B10 splice variant is common among African Americans. Smokers from two independent studies were genotyped for this variant (rs116294140) and an Asp67Tyr variant (rs61750900), and urinary NNAL and NNAL-glucuronide concentrations were quantified. In the first, no significant differences in NNAL- N-glucuronidation between African Americans ( n = 257) and whites ( n = 354) or between homozygous carriers of UGT2B10 variants (genetic score 2) and noncarriers (score 0) were detected. However, total NNAL glucuronidation by score 2 compared to score 0 smokers was lower (68.9 vs 71.2%, p < 0.0001). For NNAL- N-glucuronide to be more precisely quantified in a second study, a sensitive high-resolution LC-MS/MS-based method, which separated NNAL, NNAL- O-glucuronide, and NNAL- N-glucuronide prior to analysis, was developed. In this study, the excretion of total NNAL (free plus glucuronides) by African American ( n = 52) and white ( n = 54) smokers was not different; however, total NNAL glucuronidation by African Americans (64.0%) was slightly less than by whites (68.3%, p = 0.05). The mean NNAL- N-glucuronidation by African Americans was much lower than for whites (14 vs 24.9%, p < 0.00001), but the NNAL- O-glucuronidation was greater (50.0 vs 43.3%, p = 0.013). UGT2B10 genotype influenced NNAL- N-glucuronidation; the geometric mean percentage N-glucuronidation was 22.5% for smokers with genetic score 0 ( n = 57) and 11.2% for score 2 ( n = 11). In summary, the high prevalence of a UGT2B10 splice variant among African Americans results in lower NNAL- N-glucuronidation but only a small decrease in total NNAL glucuronidation. Therefore, despite the significant contribution of UGT2B10 to NNAL- N-glucuronidation, the UGT2B10 genotype does not play a large role in NNAL detoxication. Any decrease in N-glucuronidation was accompanied by a parallel increase in O-glucuronidation.

Pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate adduct formation in rats treated chronically with enantiomers of the tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol.

The tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically converted to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reaction which is both stereoselective and reversible. NNAL is also a lung carcinogen, with both (R)-NNAL and (S)-NNAL inducing a high incidence of lung tumours in rats. Both NNAL and NNK undergo metabolic activation to intermediates which react with DNA to form pyridylhydroxybutyl and pyridyloxobutyl DNA adducts, respectively. DNA adduct formation by NNAL and NNK is an important step in their mechanisms of carcinogenesis. In this study, we quantified both pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate adducts in the lung of rats treated with 5 ppm of (R)-NNAL or (S)-NNAL in drinking water for 10, 30, 50 and 70 weeks. In (R)-NNAL-treated rats, the pyridylhydroxybutyl and pyridyloxobutyl phosphate adducts were 4530-6920 fmol/mg DNA and 46-175 fmol/mg DNA, accounting for 45-51% and 0.3-1% of the total measured DNA phosphate and base adducts, respectively. In (S)-NNAL-treated rats, the two types of phosphate adducts were 3480-4180 fmol/mg DNA and 1180-4650 fmol/mg DNA, accounting for 30-36% and 11-38% of the total adducts, respectively. Distinct patterns of adduct formation were observed, with higher levels of NNAL-derived pyridylhydroxybutyl phosphate adducts and lower levels of NNK-derived pyridyloxobutyl phosphate adducts in the (R)-NNAL treatment group than the (S)-NNAL group. The persistence and increase over time of certain pyridylhydroxybutyl phosphate adducts over the course of the study suggest that these adducts could be useful biomarkers of chronic exposure to NNAL and NNK. The results of this study provide important new information regarding DNA damage by NNAL and NNK, and contribute to understanding mechanisms of tobacco-related carcinogenesis.

CYP2A6 genetic polymorphisms and biomarkers of tobacco smoke constituents in relation to risk of lung cancer in the Singapore Chinese Health Study.

Cytochrome P450 2A6 (CYP2A6) catalyzes the metabolism of nicotine and the tobacco-specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Genetic variation in CYP2A6 may affect smoking behavior and contribute to lung cancer risk. A nested case-control study of 197 lung cancer cases and 197 matched controls was conducted within a prospective cohort of 63 257 Chinese men and women in Singapore. Quantified were five genetic variants of CYP2A6 (*1A, *4, *7, *9 and *12) and urinary metabolites of nicotine [total nicotine, total cotinine, total trans-3'-hydroxycotinine (3HC)] and NNK (total NNAL, free NNAL, NNAL-glucuronide, NNAL-N-glucuronide, and NNAL-O-glucuronide). Higher urinary metabolites of nicotine and NNK were significantly associated with a 2- to 3-fold increased risk of lung cancer after adjustment for smoking intensity and duration. Lower CYP2A6-determined nicotine metabolizer status was significantly associated with a lower ratio of total 3HC over total cotinine, lower total nicotine equivalent and reduced risk of developing lung cancer (all Ptrend < 0.001). Compared with normal metabolizers, odds ratios (95% confidence intervals) of developing lung cancer for intermediate, slow and poor metabolizers determined by CYP2A6 genotypes were 0.85 (0.41-1.77), 0.55 (0.28-1.08) and 0.32 (0.15-0.70), respectively, after adjustment for smoking intensity and duration and urinary total nicotine equivalents. Thus the reduced risk of lung cancer in smokers with lower CYP2A6 activity may be explained by lower consumption of cigarettes, less intense smoking and reduced CYP2A6-catalyzed activation of the tobacco-specific lung carcinogen NNK.

P3.01-052 DNA Adductomics to Identify the Role of Inflammation in NNK-Induced Lung Carcinogenesis: Topic: Models of Lung Cancer

The association between pulmonary inflammation and lung cancer is well established. Smokers with chronic obstructive pulmonary disease (COPD) have higher risk of developing lung cancer than smokers without this condition. However, the molecular events underlying the association between inflammation and cancer in the lung are poorly understood. To better understand this association, an A/J mouse model was recently developed which combines exposure to the tobacco specific lung carcinogen NNK and the pro-inflammatory agent LPS. Using an innovative mass spectrometry based DNA adductomic approach, we plan to measure the DNA damage resulting from these exposures in this model. Both NNK and LPS can induce DNA modifications (DNA adducts), if not eliminated or repaired these adducts can result in miscoding events that can lead to misregulation of normal cellular growth control mechanisms and ultimately may result in cancer formation. Traditionally, the standard LC-MS methodology used for DNA adduct measurement focuses on the investigation of small numbers of anticipated DNA adducts based on a priori assumptions regarding their formation from specific exposures or chemicals. This approach does not account for the complexity of in vivo DNA adduct formation resulting from endogenous sources such as oxidative stress, lipid peroxidation or aberrant metabolism, or as a result of exposure to complex mixtures of chemicals which cannot be completely anticipated or predicted. To address this limitation, we have developed a high resolution/accurate mass data dependent-constant neutral loss-MS3 methodology for DNA adductomics using ion trap-orbital trap technology to screen for all DNA modifications simultaneously. We have successfully tested our method on mixtures of standards and applied it to lung DNA samples collected from mice exposed to NNK and to LPS. Our method allowed for the detection of the expected DNA adducts resulting from NNK as well as a number of endogenous DNA adducts resulting from lipid peroxidation, oxidative stress and aberrant metabolism resulting from the LPS induced inflammatory process. These results confirm the ability of our DNA adductomic approach to characterize the DNA damage deriving from these exposures. Our comprehensive DNA adductomic approach contributes to the development of new tools needed to investigate lung carcinogenesis, to elucidate its mechanisms and dissect the molecular pathways involved in inflammation-driven lung cancer, with the ultimate goal of identifying preventive and therapeutic strategies.

Abstract 4347: A randomized phase 2 clinical trial of PEITC on detoxification of tobacco-specific and non-specific carcinogens and toxicants

Abstract Abstract Background: Cigarette smoke contains the tobacco-specific lung carcinogen NNK and other carcinogens and toxicants. 2-Phenethyl isothiocyanate (PEITC), a natural product found as a conjugate in cruciferous vegetables, can inhibit the metabolic activation and lung carcinogenicity of NNK in rodents. Intake of a broccoli sprout beverage containing high levels of sulforaphane can increase urinary excretion of benzene and acrolein metaboites. Glutathione S-transferases (GST) catalyze the conjugation of carcinogens and toxicants with glutathione enhancing their urinary excretion, thus functional GST genotypes may modify the effect of PEITC. Methods. The goal of this randomized phase 2 clinical trial with a crossover was to determine if PEITC inhibits the metabolic activation of NNK and enhances the detoxification of benzene, acrolein and crotonaldehyde. Eighty-two smokers were completed the trial. The smokers smoked cigarettes containing deuterium labeled [pyridine-D4]NNK to allow measurement of metabolic activation. The subjects were randomly assigned to one of two arms: PEITC-placebo, or placebo-PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 ml of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. Three 24h urine samples were collected during each of the 2 treatment periods. The NNK metabolic activation ratio: [pyridine-D4]hydroxy acid/total [pyridine-D4]NNAL, and the mercapturic acids of benzene, acrolein and crotonaldehye in urine were quantified using validated LC-ESI-MS/MS methods. The linear mixed model with random effect was used to examine the PEITC effect. Results. Overall, PEITC treatment significantly reduced the NNK metabolic activation ratio by 7.7% (P = 0.023), and increased the detoxification metabolites of benzene by 24.6% (P = 0.002) and acrolein by 15.1% (P = 0.005), but had no effect on crotonaldehyde (5.5%, P = 0.148). PEITC had stronger effects in subjects with GST null genotype. PEITC reduced the NNK metabolic activation ratio by 15.6% (P = 0.039), and increased detoxification metabolites of benzene by 95.4% (P &amp;lt;0.001), acrolein by 32.7% (P = 0.034), and crotonaldehyde by 29.8% (P = 0.006) among the subjects with null genotype of both GSTM1 and GSTT1, but had no effect in those possessing both genes (Pinteraction &amp;lt; 0.05). Conclusion. The results of this trial demonstrate that PEITC inhibits the metabolic activation of NNK in smokers, and has a stronger effect on the detoxification of environmental carcinogens and toxicants such as benzene, acrolein, and crotonaldehyde. A more pronounced effect of PEITC in subjects lacking both GSTM1 and GSTT1 genes supports the epidemiological findings of stronger protection of dietary isothiocyanates against the development of lung cancer in such individuals. [Grant support: NCI R01CA122244]. Citation Format: Jian-Min Yuan, Jian-Min Yuan, Irina Stepanov, Sharon E. Murphy, Sharon E. Murphy, Renwei Wang, Steven G. Carmella, Heather H. Nelson, Heather H. Nelson, Dorothy Hatsukami, Stephen S. Hecht. A randomized phase 2 clinical trial of PEITC on detoxification of tobacco-specific and non-specific carcinogens and toxicants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4347.

Clinical Trial of 2-Phenethyl Isothiocyanate as an Inhibitor of Metabolic Activation of a Tobacco-Specific Lung Carcinogen in Cigarette Smokers.

2-Phenethyl isothiocyanate (PEITC), a natural product found as a conjugate in watercress and other cruciferous vegetables, is an inhibitor of the metabolic activation and lung carcinogenicity of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats and A/J mice. We carried out a clinical trial to determine whether PEITC also inhibits the metabolic activation of NNK in smokers. Cigarette smokers were recruited and asked to smoke cigarettes containing deuterium-labeled [pyridine-D4]NNK for an acclimation period of at least 1 week. Then subjects were randomly assigned to one of two arms: PEITC followed by placebo, or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. The NNK metabolic activation ratio [pyridine-D4]hydroxy acid/total [pyridine-D4]NNAL was measured in urine samples to test the hypothesis that PEITC treatment modified NNK metabolism. Eighty-two smokers completed the study and were included in the analysis. Overall, the NNK metabolic activation ratio was reduced by 7.7% with PEITC treatment (P = 0.023). The results of this trial, while modest in effect size, provide a basis for further investigation of PEITC as an inhibitor of lung carcinogenesis by NNK in smokers. Cancer Prev Res; 9(5); 396-405. ©2016 AACR.

Abstract IA02: Genetic, environmental, and biological contributions to racial disparities in lung cancer incidence

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States (1). In addition, there are significant disparities in lung cancer across populations. Compared with European Americans, African Americans are disproportionally affected by lung cancer, both in terms of incidence and survival (2,3). Lung adenocarcinoma among African Americans is 1.30 (1.10 - 1.90) times higher compared with European Americans; squamous cell carcinoma 1.80 (95% C.I. 1.1 - 2.3) times higher (4). There are also differences in lung cancer outcomes between European Americans and African Americans (2,5), some of which may be due to differences in cell biology between these two populations. However, studies have generally shown that differences in access to care may explain most of the variance in survival times (5-7). In addition, the gains in survival over the past 20 years among lung cancer patients have been similar in both European Americans and African Americans. Thus, the causal factors – genetic, environmental or otherwise – that lead to a greater incidence of lung cancer in African Americans are largely unknown. Smoking is the strongest risk factor for lung cancer development. However, several studies suggest that smoking prevalence and behaviors do not fully explain the disparity in lung cancer incidence. For example, African Americans have a lower smoking prevalence than European Americans and only 5.8% of African Americans are heavy smokers, compared with 19.2% of European Americans (8). In fact, when one compares smoking rates across ethnic groups matched for equal levels of smoking, African Americans still experience a higher burden of lung cancer incidence (8) - although this is attenuated with higher numbers of cigarettes smoked per day. This has led some to suggest that African Americans are more susceptible to the carcinogenic effects of tobacco at lower doses. However, this has not been empirically tested. It is possible that there are differences in nicotine and tobacco carcinogen metabolism between European Americans and European Americans that could account for the differences in cancer incidence between. On average, 72% of nicotine is converted to cotinine, the primary proximate metabolite of nicotine (9). Racial and ethnic differences in urinary (10,11) and blood cotinine (11-13) concentration per cigarette smoked have been well established. In addition, studies have found that levels of NNK metabolites (11,14) and thiocyanate (11) are higher in African Americans compared with European Americans. However, no clear genes or pathways have yet been identified that explain the disparity. And, given the strong relationship between smoking and lung cancer, it is hard to completely discount smoking as a cause of incidence disparities (8). Other risk factors to explain higher lung cancer incidence among African Americans include genetics, SES, smoking, BMI, and/or alcohol consumption. Recent studies show that the same loci associated with risk of lung cancer in European American smokers, i.e., chr15q25, are also associated with lung cancer in African Americans (15-18). Disparities in lung cancer incidence could be driven by the environment. For example, a recent study found that African Americans were more likely to reside in areas with the highest levels of air toxin exposure (19). Studies of breast and prostate cancer (20,21) have shown that whatever the cause of cancer health disparities, the consequences of these disparities manifest themselves as differences in the molecular and cell biology of the cancer. In lung cancer, we have explored the biological differences between lung cancer in European Americans and African Americans. These studies show that while some of the perturbed pathways in lung cancer remain the same for European Americans and African Americans, there are specific differences distinct to African Americans. Continued research efforts in the field are needed to determine the contribution of smoking, and indeed other factors, to lung cancer disparities. This work needs to be complemented with research that dissects the consequences of these disparities and the translation of these discoveries into improved patient care.

Genetic determinants of cytochrome P450 2A6 activity and biomarkers of tobacco smoke exposure in relation to risk of lung cancer development in the Shanghai cohort study.

Cytochrome P450 2A6 (CYP2A6) catalyzes nicotine metabolism and contributes to the metabolism of the tobacco-specific lung carcinogen, NNK. Genetic variation in CYP2A6 may affect smoking behavior and contribute to lung cancer risk. A nested case-control study of 325 lung cancer cases and 356 controls was conducted within a prospective cohort of 18,244 Chinese men in Shanghai, China. Quantified were 4 allelic variants of CYP2A6 [*1(+51A), *4, *7, and *9] and urinary total nicotine, total cotinine, total trans-3'-hydroxycotinine (3HC) and total NNAL (an NNK metabolite). Calculated were total nicotine equivalents (TNE), the sum of total nicotine, total cotinine and total 3HC and the total 3HC:total cotinine ratio as a measure of CYP2A6 activity. The nicotine metabolizer status (normal, intermediate, slow and poor) was determined by CYP2A6 genotypes. The smoking-adjusted odds ratios (95% confidence intervals) of lung cancer for the highest vs lowest quartile of total nicotine, total cotinine, total 3HC, TNE and total NNAL were 3.03 (1.80-5.10), 4.70 (2.61-8.46), 4.26 (2.37-7.68), 4.71 (2.61-8.52), and 3.15 (1.86-5.33) (all Ptrend < 0.001), respectively. Among controls CYP2A6 poor metabolizers had a 78% lower total 3HC:total cotinine ratio and 72% higher total nicotine (Ptrend ≤ 0.002). Poor metabolizers had an odds ratio of 0.64 (95% confidence interval = 0.43-0.97) for lung cancer, which was statistically nonsignificant (odds ratio = 0.74, 95% confidence interval = 0.48-1.15) after adjustment for urinary TNE and smoking intensity and duration. The lower lung cancer risk observed in CYP2A6 poor metabolizers is partially explained by the strong influence of CYP2A6 genetic polymorphisms on nicotine uptake and metabolism.

Exposure and Metabolic Activation Biomarkers of Carcinogenic Tobacco-Specific Nitrosamines.

Lung cancer is the leading cause of cancer death in the world, and cigarette smoking is its main cause. Oral cavity cancer is another debilitating and often fatal cancer closely linked to tobacco product use. While great strides have been made in decreasing tobacco use in the United States and some other countries, there are still an estimated 1 billion men and 250 million women in the world who are cigarette smokers and there are hundreds of millions of smokeless tobacco users, all at risk for cancer. Worldwide, lung cancer kills about three people per minute. This Account focuses on metabolites and biomarkers of two powerful tobacco-specific nitrosamine carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), considered to be among the main causes of lung cancer and oral cavity cancer in people who use tobacco products. Three properties of NNK and NNN are critical for successful biomarker studies: they are present in all tobacco products, they are tobacco-specific and are not found in any other product, and they are strong carcinogens. NNK and NNN are converted in humans to urinary metabolites that can be quantified by mass spectrometry as biomarkers of exposure to these carcinogens. They are also metabolized to diazonium ions and related electrophiles that react with DNA to form addition products that can be detected and quantified by mass spectrometry. These urinary metabolites and DNA addition products can serve as biomarkers of exposure and metabolic activation, respectively. The biomarkers of exposure, in particular the urinary NNK metabolites 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, have been extensively applied to document tobacco-specific lung carcinogen uptake in smokers and nonsmokers exposed to secondhand tobacco smoke. Highly sensitive mass spectrometric methods have been developed for quantitative analysis of these NNK metabolites as well as metabolites of NNN in human urine, blood, and toenails. Urinary and serum NNAL have been related to lung cancer risk, and urinary NNN has been related to esophageal cancer risk in prospective epidemiology studies. These results are consistent with carcinogenicity studies of NNK, NNAL, and NNN in rats, which show that NNK and NNAL induce mainly lung tumors, while NNN causes tumors of the esophagus and oral cavity. Biomarkers of metabolic activation of NNK and NNN applied in human studies include the metabolism of deuterium labeled substrates to distinguish NNK and NNN metabolism from that of nicotine and the determination of DNA and hemoglobin adducts in tissues, blood, and oral cells from people exposed to tobacco products. As these methods are continually improved in parallel with the ever increasing sensitivity and selectivity of mass spectrometers, development of a comprehensive biomarker panel for identifying tobacco users at high risk for cancer appears to be a realistic goal. Targeting high risk individuals for smoking cessation and cancer surveillance can potentially decrease the risk of developing fatal cancers.

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.

Liquid chromatography-electrospray ionization-tandem mass spectrometry quantitation of urinary [pyridine-D4]4-hydroxy-4-(3-pyridyl)butanoic acid, a biomarker of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolic activation in smokers.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) is a potent tobacco-specific lung carcinogen believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK to DNA damaging reactive intermediates proceeds via α-hydroxylation pathways. The end products of these pathways are excreted in the urine of smokers as 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 3) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid, 4). The sum of these biomarkers (after NaBH4 treatment), referred to as total hydroxy acid, could potentially be used to measure the extent of NNK metabolic activation in smokers. However, the same metabolites are formed from nicotine; therefore, there is a need to distinguish the NNK- and nicotine-derived keto and hydroxy acid in smokers’ urine. We previously developed a unique methodology based on the use of [pyridine-D4]NNK ([D4]1), which metabolizes to the correspondingly labeled biomarkers. In this study, we developed a sensitive and reproducible assay for the detection and quantitation of total [pyridine-D4]hydroxy acid ([D4]4) in human urine. A two-step derivatization approach was used to convert [D4]4 to [pyridine-D4]methyl 4-hexanoyl-4-(3-pyridyl)butanoate ([D4]6), and an LC-ESI-MS/MS method was developed for the analysis of this derivative with excellent sensitivity, accuracy, and precision. The robustness and reproducibility of the assay was further confirmed by its application for the analysis of urine samples from 87 smokers who smoked [D4]1-containing cigarettes for 1 week. The measured level averaged 130 fmol/mL urine. The developed assay can be used in future studies that may require evaluation of the relative efficiency of NNK metabolic activation in humans.

Children's exposure to secondhand and thirdhand smoke carcinogens and toxicants in homes of hookah smokers.

We examined homes of hookah-only smokers and nonsmokers for levels of indoor air nicotine (a marker of secondhand smoke) and indoor surface nicotine (a marker of thirdhand smoke), child uptake of nicotine, the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and the toxicant acrolein by analyzing their corresponding metabolites cotinine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-glucuronides (total NNAL) and 3-hydroxypropylmercapturic acid. Data were collected at 3 home visits during a 7-day study period from a convenience sample of 24 households with a child 5 years or younger. Three child urine samples and 2 air and surface samples from the living room and the child bedroom were taken in homes of nonsmokers (n = 5) and hookah-only smokers (n = 19) comprised of daily hookah smokers (n = 8) and weekly/monthly hookah smokers (n = 11). Nicotine levels in indoor air and on surfaces in the child bedrooms in homes of daily hookah smokers were significantly higher than in homes of nonsmokers. Uptake of nicotine, NNK, and acrolein in children living in daily hookah smoker homes was significantly higher than in children living in nonsmoker homes. Uptake of nicotine and NNK in children living in weekly/monthly hookah smoker homes was significantly higher than in children living in nonsmoker homes. Our data provide the first evidence for uptake of nicotine, the tobacco-specific lung carcinogen NNK, and the ciliatoxic and cardiotoxic agent acrolein in children living in homes of hookah smokers. Our findings suggest that daily and occasional hookah use in homes present a serious, emerging threat to children's long-term health.

Thirdhand tobacco smoke: a tobacco-specific lung carcinogen on surfaces in smokers' homes.

Thirdhand tobacco smoke consists of substances remaining on the surfaces or in the dust of areas where people have smoked. While previous studies have demonstrated the presence of nicotine and various other constituents of tobacco smoke on surfaces in smokers' homes, none has investigated the presence of tobacco-specific carcinogens. We used liquid chromatography-tandem mass spectrometry to analyze surface dust samples from both the homes of smokers and nonsmokers for the powerful tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). We positively identified NNK on surfaces in 33 of 37 smokers' homes (700±788 pg/100cm(2) [range, not detected-3,500 pg/100cm(2)]), but only in 3 of 19 nonsmokers' homes (235±176 pg/100cm(2) in the homes where NNK was detected [range, not detected-435 pg/100cm(2)]). The differences in occurrence and levels of NNK in the homes of smokers and nonsmokers were significant (p < .0001). The powerful tobacco-specific lung carcinogen NNK is present on surfaces in most homes occupied by smokers. Potential renters or buyers of apartments or homes should be notified if previous residents were smokers in order to avoid unnecessary exposure of their families to a potent lung carcinogen.

DNA Adducts in Aldehyde Dehydrogenase-Positive Lung Stem Cells of A/J Mice Treated with the Tobacco Specific Lung Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Lung cancer is the leading cause of cancer death in the world. Evidence suggests that lung cancer could originate from mutations accumulating in a subpopulation of self-renewing cells, lung stem cells. Aldehyde dehydrogenase (ALDH) is a marker of stem cells. To investigate the presence of DNA modifications in these cells, we isolated ALDH-positive lung cells from A/J mice exposed to the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Using LC-NSI-HRMS/MS-PRM, O(6)-methyl-G, 7-POB-G, and O(2)-POB-dT were positively identified in ALDH-positive cell DNA. This is the first example of detection of carcinogen-DNA adducts in lung stem cells, supporting the hypothesis of their role in lung carcinogenesis.

Abstract PL04-02: Genetics and health care disparities caused by smoking.

Abstract In the U.S., racial/ethnic differences are seen in smoking behavior and related disease risk. Compared to smokers of European ancestry, African Americans smoke fewer cigarettes per day, but take in more nicotine and smoke per cigarette and appear to be more highly dependent. Asians also smoke fewer cigarettes per day put appear to be less dependent than smokers of European ancestry. Lung cancer incidence normalized by cigarettes smoked per day is higher for African Americans and lower in Asians compared to smokers of European Ancestry. Genetic variations in two genes are most strongly associated with smoking behavior and lung cancer risks, and these are found in different frequencies across races. CYP2A6 is the enzyme that is primarily responsible for nicotine metabolism, and is also involved in the bioactivation of the tobacco-specific nitrosamine and lung carcinogen NNK. There are many reduced function variants of the CYP2A6 gene, and these are more frequent in Asians and African Americans compared to those of European ancestry. The presence of reduced function CYP2A6 variants is associated with smoking fewer cigarettes per day and with lower dependence, the latter evidenced by more successful quitting. Among smokers of European ancestry and Asians, reduced function CYP2A6 variants are associated with lower lung cancer risk. However, for unknown reasons, despite having a higher prevalence of reduced function CYP2A6 alleles, African Americans have higher risks for lung cancer. Genes in the nicotine receptor subunit gene cluster CHRNA5-A3-B4 have been associated with lung cancer risk in a number of studies, and have also been associated with nicotine intake and with risk of chronic obstructive lung disease and peripheral arterial disease. It is that believed that the α5 nicotinic receptor subunit influences sensitivity to nicotine; and reduced function of the subunit results in a greater level of nicotine and smoke intake, and therefore a higher level of dependence. Relatively little is known about the effects of this gene cluster on smoking behavior or cancer risk in African Americans or Asians. Among European Ancestry smokers, the CYP2A6 gene and CHRNA5-A3-B4 gene cluster are additively associated with cigarette consumption, nicotine dependence and lung cancer risk. Genetic risk factors are important determinants of racial differences in smoking behavior and lung cancer risk, but explain only a relatively small amount of variation in risk among smokers. Citation Format: Neal L. Benowitz. Genetics and health care disparities caused by smoking. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL04-02.

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol

Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (β-nicotyrine) and (−)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (-)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2μM and 110μM, respectively). β-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17μM). Neither menthol nor β-nicotyrine was an inactivator of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106μM, kinact was 0.61min−1). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that β-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.