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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (commonly known as NNK) is one of the most prevalent and potent pulmonary carcinogens in tobacco products that increases the human lung cancer risk. Kava has the potential to reduce NNK and tobacco smoke-induced lung cancer risk by enhancing urinary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major metabolite of NNK) and thus reducing NNK-induced DNA damage. In this study, we quantified N -glucuronidated NNAL (NNAL- N -gluc), O -glucuronidated NNAL (NNAL- O -gluc), and free NNAL in the urine samples collected before and after 1-week kava dietary supplementation. The results showed that kava increased both NNAL-N-glucuronidation and O-glucuronidation. Since NNAL-N-glucuronidation is dominantly catalyzed by UGT2B10, its representative single-nucleotide polymorphisms (SNPs) were analyzed among the clinical trial participants. Individuals with any of the four analyzed SNPs appear to have a reduced basal capacity in NNAL-N-glucuronidation. Among these individuals, kava also resulted in a smaller extent of increases in NNAL-N-glucuronidation, suggesting that participants with those UGT 2 B 10 SNPs may not benefit as much from kava with respect to enhancing NNAL-N-glucuronidation. In summary, our results provide further evidence that kava enhances NNAL urinary detoxification via an increase in both N-glucuronidation and O-glucuronidation. UGT2B10 genetic status has not only the potential to predict the basal capacity of the participants in NNAL-N-glucuronidation but also potentially the extent of kava benefits.

Menthol, which creates mint flavor and scent, is often added to tobacco in both menthol and nonmenthol cigarettes. A potent tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is extensively metabolized to its equally carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) as ( R )- or ( S )-NNAL enantiomers. NNAL is detoxified by UDP-glucuronosyltransferase (UGT) enzymes, with glucuronidation occurring on either NNAL's pyridine ring nitrogen (NNAL- N -Gluc) or the chiral alcohol [( R )- or ( S )-NNAL- O -Gluc]. To characterize a potential effect by menthol on NNAL glucuronidation, in vitro menthol glucuronidation assays and menthol inhibition of NNAL-Gluc formation assays were performed. Additionally, NNAL and menthol glucuronides (MG) were measured in the urine of smokers ( n = 100) from the Southern Community Cohort Study. UGTs 1A9, 1A10, 2A1, 2A2, 2A3, 2B4, 2B7, and 2B17 all exhibited glucuronidating activity against both l- and d-menthol. In human liver microsomes, both l- and d-menthol inhibited the formation of each NNAL-Gluc, with a stereospecific difference observed between the formation of ( R )-NNAL- O -Gluc and ( S )-NNAL- O -Gluc in the presence of d-menthol but not l-menthol. With the exception of three nonmenthol cigarette smokers, urinary MG was detected in all menthol and nonmenthol smokers, with l-MG comprising >98% of total urinary MG. Levels of urinary NNAL- N -Gluc were significantly ( P < 0.05) lower among subjects with high levels of total urinary MG; no significant changes in free NNAL were observed. These data suggest that the presence of menthol could lead to increases in alternative, activating metabolic pathways of NNAL in tobacco target tissues, increasing the opportunity for NNAL to damage DNA and lead to the development of tobacco-related cancers. SIGNIFICANCE STATEMENT: High levels of the major menthol metabolite, menthol-glucuronide, was observed in the urine of smokers of either menthol or nonmenthol cigarettes. The fact that a significant inverse correlation was observed between the levels of urinary menthol-glucuronide and NNAL-N-glucuronide, a major detoxification metabolite of the tobacco carcinogen, NNK, suggests that menthol may inhibit clearance of this important tobacco carcinogen., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Given the diverse cigar market and limited data on biomarker patterns by cigar type, we compared biomarkers of nicotine and tobacco toxicants among cigar smokers and other groups., Methods: Using Wave 1 urinary biomarker data from 5,604 adults in the Population Assessment of Tobacco and Health (PATH) Study, we compared geometric mean concentrations among cigar-only smokers (all cigars and separately for traditional, cigarillo, and filtered cigars), cigarette-only smokers, dual cigar/cigarette smokers, and never users of tobacco. We calculated geometric mean ratios comparing groups with never users adjusting for sex, age, race/ethnicity, education and creatinine., Results: Some day cigar-only smokers had lower biomarker concentrations than every day cigar-only smokers, but higher than never users. Every day cigar-only smokers ( n = 61) had lower TNE-2 (cotinine+trans-3'-hydroxycotinine) compared to every day cigarette-only ( n = 2217; P < 0.0001) and dual cigar/cigarette smokers ( n = 601; P < 0.0001). Several biomarkers, including NNAL (NNK metabolite) and CYMA (metabolite of acrylonitrile), were comparable in these groups. In exploratory analyses, every day filtered cigar-only ( n = 7) smokers had higher biomarker concentrations compared with every day traditional cigar-only smokers ( n = 12) and cigarillo-only smokers ( n = 24). Every day smokers of each cigar type were similar to exclusive cigarette smokers. For some biomarkers, particularly for every day filtered cigar-only smokers, concentrations were higher., Conclusions: For some biomarkers, every day cigar-only smokers were comparable with every day cigarette-only smokers. Exploratory analyses suggest that biomarkers vary by cigar type with every day filtered cigar-only smokers having the highest concentrations., Impact: High exposure to harmful constituents among cigar smokers is a continuing health issue., (©2019 American Association for Cancer Research.)

Background: How carcinogen exposure varies across users of different, particularly noncigarette, tobacco products remains poorly understood., Methods: We randomly selected 165 participants of the Golestan Cohort Study from northeastern Iran: 60 never users of any tobacco, 35 exclusive cigarette, 40 exclusive (78% daily) waterpipe, and 30 exclusive smokeless tobacco (nass) users. We measured concentrations of 39 biomarkers of exposure in 4 chemical classes in baseline urine samples: tobacco alkaloids, tobacco-specific nitrosamines (TSNA), polycyclic aromatic hydrocarbons (PAH), and volatile organic compounds (VOC). We also quantified the same biomarkers in a second urine sample, obtained 5 years later, among continuing cigarette smokers and never tobacco users., Results: Nass users had the highest concentrations of tobacco alkaloids. All tobacco users had elevated TSNA concentrations, which correlated with nicotine dose. In both cigarette and waterpipe smokers, PAH and VOC biomarkers were higher than never tobacco users and nass users, and highly correlated with nicotine dose. PAH biomarkers of phenanthrene and pyrene and two VOC metabolites (phenylmercapturic acid and phenylglyoxylic acid) were higher in waterpipe smokers than in all other groups. PAH biomarkers among Golestan never tobacco users were comparable to those in U.S. cigarette smokers. All biomarkers had moderate to good correlations over 5 years, particularly in continuing cigarette smokers., Conclusions: We observed two patterns of exposure biomarkers that differentiated the use of the combustible products (cigarettes and waterpipe) from the smokeless product. Environmental exposure from nontobacco sources appeared to contribute to the presence of high levels of PAH metabolites in the Golestan Cohort., Impact: Most of these biomarkers would be useful for exposure assessment in a longitudinal study., (©2019 American Association for Cancer Research.)

Background: Increased cigarette costs have inadvertently strengthened the appeal of discounted brands to price-sensitive smokers. Although smokers perceive discounted brands as having poorer quality, little is known about their delivery of toxic tobacco smoke constituents compared with premium-branded tobacco products. Methods: We investigated the differences between discount and premium brand smokers using the National Health and Nutrition Examination Survey 2011-2012 Special Smoker Sample. Our analyses focused on demographic differences and 27 biomarkers of harmful and potentially harmful constituents (HPHC) listed by the FDA, including volatile organic compounds, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide; reported as total NNAL (tNNAL)], metals, and polycyclic aromatic hydrocarbons (PAHs). Data were analyzed using linear regression models adjusting for potential confounders. Results: A total of 976 non-tobacco users and 578 recent cigarette smokers were eligible for analysis, of which 141 (26.0% weighted) smoked discount brand cigarettes and 437 (74.0% weighted) smoked premium. Discount brand smokers were older, predominantly non-Hispanic white, and had higher serum cotinine. Discount brand smokers had significantly higher levels of 13 smoking-related biomarkers, including tNNAL, uranium, styrene, xylene, and biomarkers of exposure to PAHs (naphthalene, fluorene, and phenanthrene), compared with premium brand smokers. Conclusions: These findings suggest that discount cigarette use is associated with higher exposure to several carcinogenic and toxic HPHCs. Impact: These results may have important regulatory implications for product standards, as higher exposures could lead to a greater degree of harm. Cancer Epidemiol Biomarkers Prev; 27(5); 601-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Tobacco-specific nitrosamines (TSNAs) are a class of carcinogens found in tobacco products, whose levels can vary considerably depending on tobacco blends and manufacturing processes. The current study examined whether recent increases in levels of the TSNA NNK [4-(methylnitrosamino-1-(3-pyridyl)-1-butanone] in Canadian cigarettes translated into differences in exposure among Canadian tobacco users. Methods: Nationally representative data from the Canadian Health Measures Survey (CHMS) were used to measure levels of total urinary NNAL [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol], a metabolite of the TSNA NNK, among tobacco users. Data from CHMS Cycle 3 (2012-13) were used to examine NNAL, and linear regression was used to examine predictors. Data from CHMS Cycle 1 (2007-09) and Cycle 3 (2012-13) were used to examine changes in NNAL over time. Results: From 2007-2009 through 2012-2013, levels of creatinine-corrected NNAL increased by 64% ( P < 0.0001). Levels of NNAL in 2012-2013 were higher among older respondents ( P = 0.04), among females ( P = 0.03), among respondents identifying as "white" and "Aboriginal" ( P < 0.0001), and among those with greater daily cigarette consumption ( P < 0.001), as well as greater levels of urinary free cotinine ( P < 0.0001) and urinary creatinine ( P < 0.0001). Conclusions: The findings indicate that exposure to the TSNA NNK among Canadian tobacco users has increased considerably from 2007-2009 through 2012-2013, in parallel to changes in TSNA levels in Canadian cigarettes. In the absence of epidemiologic data, it is unclear whether this change translates into increased risk. Impact: The study findings have potential implications for tobacco manufacturers, who bear a responsibility to reduce levels of tobacco carcinogens to the full extent possible. Cancer Epidemiol Biomarkers Prev; 27(3); 262-7. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Tobacco smoking and exposure to tobacco secondhand smoke (SHS) can cause lung cancer. We determined uptake of NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone), a tobacco specific potent pulmonary carcinogen, in hookah smokers and non-smokers exposed to hookah tobacco SHS. We analyzed data from a community-based convenience sample of 201 of adult (aged ≥18 years) exclusive hookah smokers (n = 99) and non-smokers (n = 102) residing in San Diego County, California. Participants spent an average of three consecutive hours indoors, in hookah lounges or private homes, where hookah tobacco was smoked exclusively. Total NNAL [the sum of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides], the major metabolites of NNK, were quantified in spot urine samples provided the morning of and the morning after attending a hookah event. Among hookah smokers urinary NNAL increased significantly (p<0.001) following a hookah social event; the geometric mean doubled, from 1.97 to 4.16 pg/mg. Among non-smokers the increase was not significant (p = 0.059). Post hookah event urinary NNAL levels were highest in daily hookah smokers, and significantly higher than in non-daily smokers or non-smokers (GM: 14.96 pg/mg vs. 3.13 pg/mg and 0.67 pg/mg, respectively). For both hookah smokers and non-smokers, pre-to-post event change in urinary NNAL was not significantly different between hookah lounges and homes. We suggest posting health warning signs inside hookah lounges, and encouraging voluntary bans of smoking hookah tobacco in private homes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Smoking is a major risk factor for the development of bladder cancer; however, the functional consequences of the carcinogens in tobacco smoke and bladder cancer-associated metabolic alterations remain poorly defined. We assessed the metabolic profiles in bladder cancer smokers and non-smokers and identified the key alterations in their metabolism. LC/MS and bioinformatic analysis were performed to determine the metabolome associated with bladder cancer smokers and were further validated in cell line models. Smokers with bladder cancer were found to have elevated levels of methylated metabolites, polycyclic aromatic hydrocarbons, DNA adducts, and DNA damage. DNA methyltransferase 1 (DNMT1) expression was significantly higher in smokers than non-smokers with bladder cancer. An integromics approach, using multiple patient cohorts, revealed strong associations between smokers and high-grade bladder cancer. In vitro exposure to the tobacco smoke carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (BaP) led to increase in levels of methylated metabolites, DNA adducts, and extensive DNA damage in bladder cancer cells. Cotreatment of bladder cancer cells with these carcinogens and the methylation inhibitor 5-aza-2'-deoxycytidine rewired the methylated metabolites, DNA adducts, and DNA damage. These findings were confirmed through the isotopic-labeled metabolic flux analysis. Screens using smoke-associated metabolites and DNA adducts could provide robust biomarkers and improve individual risk prediction in bladder cancer smokers. Noninvasive predictive biomarkers that can stratify the risk of developing bladder cancer in smokers could aid in early detection and treatment. Cancer Prev Res; 10(10); 588-97. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: 1,3-Butadiene (BD) is an important carcinogen in tobacco smoke that undergoes metabolic activation to DNA-reactive epoxides. These species can be detoxified via glutathione conjugation and excreted in urine as the corresponding N-acetylcysteine conjugates. We hypothesize that single nucleotide polymorphisms (SNPs) in BD-metabolizing genes may change the balance of BD bioactivation and detoxification in White, Japanese American, and African American smokers, potentially contributing to ethnic differences in lung cancer risk. Methods: We measured the levels of BD metabolites, 1- and 2-( N -acetyl-L-cysteine-S-yl)-1-hydroxybut-3-ene (MHBMA) and N -acetyl- S -(3,4-dihydroxybutyl)-L-cysteine (DHBMA), in urine samples from a total of 1,072 White, Japanese American, and African American smokers and adjusted these values for body mass index, age, batch, and total nicotine equivalents. We also conducted a genome-wide association study to identify genetic determinants of BD metabolism. Results: We found that mean urinary MHBMA concentrations differed significantly by ethnicity ( P = 4.0 × 10 -25 ). African Americans excreted the highest levels of MHBMA followed by Whites and Japanese Americans. MHBMA levels were affected by GSTT1 gene copy number ( P < 0.0001); conditional on GSTT1 , no other polymorphisms showed a significant association. Urinary DHBMA levels also differed between ethnic groups ( P = 3.3 × 10 -4 ), but were not affected by GSTT1 copy number ( P = 0.226). Conclusions: GSTT1 gene deletion has a strong effect on urinary MHBMA levels, and therefore BD metabolism, in smokers. Impact: Our results show that the order of MHBMA levels among ethnic groups is consistent with their respective lung cancer risk and can be partially explained by GSTT1 genotype. Cancer Epidemiol Biomarkers Prev; 26(7); 1034-42. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Introduction: The high prevalence of smoking and smokeless tobacco (ST) use during pregnancy in Alaska Native (AN) women is concerning due to the detrimental effects of these products to the mother and the developing fetus. We sought to correlate maternal cotinine levels with fetal exposure to a tobacco-specific carcinogen to incorporate in a biomarker feedback intervention to motivate tobacco cessation during pregnancy., Methods: Demographic and tobacco use data were collected from a convenience sample of pregnant AN smokers, ST users, and non-users. Maternal and neonatal urine were collected at delivery. Maternal urine cotinine and neonatal urine total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, a tobacco-specific carcinogen) levels in smokers and ST users were analyzed and their correlations determined by Spearman correlation coefficients., Results: During 2012-2014, we enrolled 64 non-users, 54 smokers, and 30 ST (20 homemade iqmik; 10 commercial ST) users (n = 148). Analyses of paired maternal-infant urine samples obtained for 36 smokers demonstrated a moderate to strong correlation (r = 0.73, P < .001) between maternal cotinine and infant NNAL levels. The correlation was not significant for 25 iqmik users (r = 0.36, P = .17) or 9 commercial ST users (r = 0.60, P = .09). No analysis was conducted for 55 non-users with cotinine and NNAL levels < limits of quantification., Conclusions: There is a moderate to strong correlation between maternal smoking and fetal exposure to the tobacco-specific carcinogen NNAL., Implications: The correlation between maternal smoking and fetal carcinogen exposure may provide an education tool to help motivate smoking cessation among pregnant AN women. Further investigation is warranted to determine correlations between maternal commercial ST and iqmik use and neonatal NNAL., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The most abundant and potent carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In vivo, NNK is rapidly metabolized to both the (R)- and (S)-enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which possesses similar carcinogenic properties as NNK. The major detoxification pathway for both NNAL enantiomers is glucuronidation by UDP-glucuronosyltransferase (UGT) enzymes including UGT2B10 and UGT2B17. The goal of the present study was to directly examine the role of UGT genotypes on urinary levels of NNAL glucuronides in smokers., Methods: NNAL-N-Gluc, (R)-NNAL-O-Gluc, (S)-NNAL-O-Gluc, and free NNAL were simultaneously and directly quantified in the urine of smokers by LC/MS analysis. Genotypes were determined by TaqMan assay using genomic DNA., Results: The functional knockout polymorphism in the UGT2B10 gene at codon 67 (Asp>Tyr) was significantly (P < 0.0001) associated with a 93% decrease in creatinine-adjusted NNAL-N-Gluc. The polymorphic whole-gene deletion of the UGT2B17 gene was associated with significant (P = 0.0048) decreases in the levels of creatinine-adjusted (R)-NNAL-O-Gluc, with a 32% decrease in the levels of urinary (R)-NNAL-O-Gluc/(S)-NNAL-O-Gluc among subjects with the UGT2B17 (*2/*2) genotype as compared to subjects with the UGT2B17 (*1/*1) genotype., Conclusions: These results suggest that functional polymorphisms in UGT2B10 and UGT2B17 are associated with a reduced detoxification capacity against NNAL and may therefore affect individual cancer risk upon exposure to tobacco., Impact: This is the first report to clearly demonstrate strong genotype-phenotype associations between both the UGT2B10 codon 67 Asp

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., (©2016 American Association for Cancer Research.)

Background: Differences in carcinogen exposure from different cigarette products could contribute to differences in smoking-associated cancer incidence among Chinese compared with US smokers., Methods: Urine concentrations of metabolites of nicotine, the tobacco-specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and polycyclic aromatic hydrocarbon metabolites (PAHs) were compared in 238 Chinese and 203 US daily smokers., Results: Comparing Chinese versus US smokers, daily nicotine intake and nicotine intake per cigarette smoked were found to be similar. When normalised for cigarettes per day, urine NNAL excretion was fourfold higher in US smokers, while the excretion of urine metabolites of the PAHs fluorene, phenanthrene and pyrene metabolites was 50% to fourfold higher in Chinese smokers (all, p<0.0001). Similar results were seen when NNAL and PAHs excretion was normalised for daily nicotine intake., Conclusions: Patterns of carcinogen exposure differ, with lower exposure to TSNA and higher exposure to PAHs in Chinese compared with US smokers. These results most likely reflect country differences in cigarette tobacco blends and manufacturing processes, as well as different environmental exposures., Trial Registration Number: NCT00264342., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Introduction: Electronic cigarettes (e-cigarettes) are rapidly increasing in popularity but little information is available on their potential toxic or carcinogenic effects., Methods: Twenty-eight e-cigarette smokers who had not smoked tobacco cigarettes for at least 2 months provided urine samples which were analyzed by validated methods for a suite of toxicant and carcinogen metabolites including 1-hydroxypyrene (1-HOP), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), 3-hydroxypropylmercapturic acid (3-HPMA), 2-hydroxypropylmercapturic acid (2-HPMA), 3-hydroxy-1-methylpropylmercapturic acid (HMPMA), S-phenylmercapturic acid (SPMA), nicotine, and cotinine. Levels of these compounds were compared to those found in cigarette smokers from three previous studies., Results: Levels of 1-HOP, total NNAL, 3-HPMA, 2-HPMA, HMPMA, and SPMA were significantly lower in the urine of e-cigarette users compared to cigarette smokers. Levels of nicotine and cotinine were significantly lower in e-cigarette users compared to cigarette smokers in one study but not in another., Conclusions: With respect to the compounds analyzed here, e-cigarettes have a more favorable toxicity profile than tobacco cigarettes., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acrylamide (AA), a rodent carcinogen, is widely used in industry and present in cigarette smoke as well as in foods processed at high temperatures. The metabolic activation of AA to glycidamide (GA) could be critical for AA carcinogenicity since GA causes DNA adduct formation in vivo. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct of AA, is subjected to spontaneous and enzymatic depurination and excreted through urine. Urinary N7-GAG analysis can confirm AA genotoxicity and identify active species of AA metabolites in humans, thereby serving as a risk-associated biomarker for molecular epidemiology studies. This study aimed to develop an isotope-dilution solid-phase extraction liquid chromatography tandem mass spectrometry method to comparatively analyze urinary N7-GAG levels in nonsmokers and smokers. Urinary N-acetyl-S-(propionamide)-cysteine (AAMA), a metabolite of AA, was also analyzed as a biomarker for current AA exposure. Urinary N7-GAG was quantified by monitoring m/z 239 → 152 for N7-GAG and m/z 242 → 152 for (13)C3-labeled N7-GAG under positive electron spray ionization and multiple reaction mode. The median urinary N7-GAG level was 0.93 μg/g creatinine in nonsmokers (n = 33) and 1.41 μg/g creatinine in smokers (n = 30). Multiple linear regression analysis of data revealed that N7-GAG levels were only significantly associated with AAMA levels. These results demonstrate that urinary N7-GAG of nonsmokers and smokers is significantly associated with a very low level of dietary AA intake, assessed by analyzing urinary AAMA.

An accidental exposure of six workers to ethylene oxide (EO) provided the rationale for a biomonitoring and follow-up study, whose aim was to analyse protein adduct kinetics and examine the differentiation between accidental and environmental exposure, e.g., from tobacco smoke. For this purpose, the decrease in the concentration of the haemoglobin adduct N-2-hydroxyethylvaline (HEV) was followed during a five-month period after the accident, together with N-2-cyanoethylvaline (CEV) and urinary cotinine, two well-established biomarkers for smoking. The follow-up study showed that EO adduct concentrations significantly increased after a short but presumably high exposure. Initial biomonitoring revealed HEV levels above 500 pmol g(-1) globin in all cases, with a maximum of about 2,400 pmol g(-1) globin. This compares to a German EKA value (exposure equivalent for carcinogenic substances) for a daily 8-h-exposure to 1 ppm EO of 90 μg L(-1) blood (~3,900 pmol g(-1) globin). The adduct levels dropped in accordance with the expected zero-order kinetics for a single exposure. After the five-month observation interval, the HEV concentrations in blood reflected the individual background from tobacco smoking. The results of this study show that even a short exposure to ethylene oxide may result in a significant rise in haemoglobin adduct levels. Although protein adducts and their occupational-medical assessment values are considered for long-term exposure surveillance, they can also be used for monitoring accidental exposures. In these cases, the calculation of daily 'ppm-equivalents' may provide a means for a comparison with the existing assessment values.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an efficient biomarker of tobacco-specific carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The ability to monitor biomarker concentrations is very important in understanding potential cancer risk. An analytical method using molecularly imprinted polymer (MIP) column coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the determination of total NNAL in human urine was developed and validated. The combination of MIP column extraction and LC-MS/MS can provide a high sensitive and relatively simple analytical method. The limit of detection (LOD) was 0.30 pg/ml and analysis time was 6min. The method has been applied to urine samples of 36 nonsmokers and 207 smokers. NNAL was found to be significantly higher in the urine of smokers compared with nonsmokers. Compared with smokers with blended cigarettes, Chinese virginia cigarettes smokers had low urinary NNAL levels. There was a direct association between the 24-h mouth-level exposure of carcinogen NNK from cigarette smoking and the concentration of NNAL in the urine of smokers. However, there was not a positive correlation between urinary total NNAL levels in 24 h and tar. Total urinary NNAL is a valuable biomarker for monitoring exposure to carcinogenic NNK in smokers and in nonsmokers. A prediction model of cigarette smoke NNK and urinary average NNAL levels in 24 h was established (y=2.8987x-245.38, r²=0.9952, n=204)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

1,3-Butadiene (BD) is a known human carcinogen present in cigarette smoke and in automobile exhaust, leading to widespread exposure of human populations. BD requires cytochrome P450-mediated metabolic activation to electrophilic species, e.g. 3,4-epoxy-1-butene (EB), hydroxymethyl vinyl ketone (HMVK), and 3,4-epoxy-1,2-diol (EBD), which form covalent adducts with DNA. EB, HMVK, and EBD can be conjugated with glutathione and ultimately excreted in urine as monohydroxybutenyl mercapturic acid (MHBMA), dihydroxybutyl mercapturic acid (DHBMA), and trihydroxybutyl mercapturic acid (THBMA), respectively, which can serve as biomarkers of BD exposure and metabolic processing. While MHBMA and DHBMA have been found in smokers and nonsmokers, THBMA has not been previously detected in humans. In the present work, an isotope dilution HPLC-ESI(-)-MS/MS methodology was developed and employed to quantify THBMA in urine of known smokers and nonsmokers (19-27 per group). The new method has excellent sensitivity (LOQ, 1 ng/mL urine) and achieves accurate quantitation using a small sample volume (100 μL). Mean urinary THBMA concentrations in smokers and nonsmokers were found to be 21.6 and 13.7 ng/mg creatinine, respectively, suggesting that there are sources of THBMA other than exposure to tobacco smoke in humans, as is also the case for DHBMA. However, THBMA concentrations are significantly greater in urine of smokers than that of nonsmokers (p < 0.01). Furthermore, THBMA amounts in human urine declined 25-50% following smoking cessation, suggesting that smoking is an important source of this metabolite in humans. The HPLC-ESI(-)-MS/MS methodology developed in the present work will be useful for future epidemiological studies of BD exposure and metabolism.

In recent years the number of waterpipe smokers has increased substantially worldwide. Here we report on the concentrations of tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) in waterpipe smoke and the analysis of selected biomarkers indicative for the body burden in waterpipe users. We further identify high amounts of unburned humectants (glycerol and propylene glycol) in the waterpipe smoke as main part of the so-called "tar" fraction. These results give cause for serious concern. For standardization we applied a machine smoking protocol. Smoke was collected on glass fiber filters and analyzed for nicotine, water, humectants, TSNAs, and PAHs. In addition, we determined carbon monoxide and found high amounts in the smoke being causative for high levels of carboxyhemoglobin (COHb) in the blood of smokers. In comparison to the reference cigarette 3R4F, the nicotine contents were 10-times higher, but TSNA levels were found lower in waterpipe smoke. This finding explained the low levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol detected in the urine of waterpipe smokers. Finally, the levels of benzo[a]pyrene were three times higher in waterpipe smoke compared to the reference cigarette. Altogether, the data presented in this study point to the health hazards associated with the consumption of waterpipes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol plus its glucuronides (total NNAL), metabolites of the lung carcinogen NNK, and total cotinine, metabolites of nicotine, are biomarkers of active and passive cigarette smoking. We calculated the total NNAL:total cotinine (×10(3)) ratio in 408 passive (infants, children, and adults) and 1088 active smokers. The weighted averages were 0.73 (95% confidence interval 0.71, 0.76) for passive smokers and 0.07 (0.06, 0.08) for active smokers (p < 0.0001). These results demonstrate that cotinine measurements may underestimate exposure of passive smokers to the lung carcinogen NNK in second-hand cigarette smoke. The total NNAL:total cotinine (×10(3)) ratio may provide an improved biomarker for evaluating the health effects of passive smoking.

Objectives: The possibly carcinogenic properties of naphthalene are, regarding to its ubiquitary presence, of environmental-medical and occupational-medical importance. Seven isomeric dihydroxynaphthalenes (DHN) were examined for their suitability as biomarkers in human biomonitoring and to get insights in human naphthalene metabolism., Methods: We developed a GC-MS-method for the quantification of 1,2-, 1,4-, 1,5-, 1,6-, 1,7-, 2,6- and 2,7-DHN after solid phase extraction and derivatization with BSA/TMCS. The internal burden of DHN after exposure to naphthalene was determined by measuring urine collected from smokers and non-smokers among the general population and among occupationally exposed persons., Results: The elaborated method can be regarded as specific and sensitive procedure to quantify the seven different DHN. In human urine, we detected 1,2-DHN as main metabolite in 54 of the 55 analysed samples. Median 1,2-DHN values (range) were 1012 μg/L (22-6477 μg/L) for workers and 8 μg/L (

Polycyclic aromatic hydrocarbons (PAH) are well established carcinogens that are likely to play a role in causing some human cancers. One accepted pathway of PAH metabolic activation is the formation of bay region diol epoxides. Some individuals may be particularly susceptible to PAH carcinogenesis because they metabolically activate PAH more effectively than others. We have used the measurement of urinary phenanthrene tetraols (Phe-tetraols) as a biomarker of PAH exposure plus metabolic activation since bay region diol epoxides are hydrolyzed to tetraols. Because of stereoselectivity in Phe metabolism, Phe-(1R,2S,3R,4S)-tetraol (4) results mainly from the bay region diol epoxide pathway, and Phe-(1S,2R,3S,4R)-tetraol (7) is formed mainly from the reverse diol epoxide pathway, not generally associated with carcinogenicity. The latter pathway accounts for more than 95% of human urinary Phe-tetraol. In most previous studies, Phe-tetraol was quantified without enantiomeric resolution, using a relatively rapid and practical method, applicable to large studies. It was not clear, however, whether measurement of overall unresolved Phe-tetraol would accurately represent the bay region diol epoxide metabolic activation pathway. Therefore, in this study we specifically quantified Phe-(1R,2S,3R,4S)-tetraol (4) by supplementing our usual analysis with chiral HPLC separations and using [(13)C(6)]Phe-(1R,2S,3R,4S)-tetraol as internal standard. We then investigated the relationship of urinary levels of 4 to those of Phe-tetraols (4 + 7), quantified without enantiomeric resolution. We applied these methods to urine samples from cigarette smokers and highly PAH-exposed creosote workers. The results were also compared to levels of benzo[a]pyrene-7,8,9,10-tetraol and 1-hydroxypyrene in the same samples. Levels of 4 were highly correlated with those of 4 + 7 (r > 0.9, P < 0.0001) in both types of urine samples. Strong correlations of 4 and 4 + 7 with benzo[a]pyrene-7,8,9,10-tetraol and 1-hydroxypyrene were also observed. The results of this study demonstrate therefore that practical and convenient measurement of overall Phe-tetraols (4 + 7) in human urine, without enantiomeric resolution, is an excellent indicator of PAH exposure and metabolism by the bay region diol epoxide metabolic activation pathway.

Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of cancers in humans. Benzo[a]pyrene (BaP) is a prototypic carcinogenic PAH, which requires metabolic activation to elicit its detrimental effects. The major end product of its diol epoxide metabolic activation pathway is r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans, anti-BaPT). Individual differences in exposure to, and metabolic activation of, carcinogenic PAH may influence cancer risk. Measurement of PAH metabolites in human urine could provide a direct way to assess individual differences in susceptibility to PAH-related cancer. In this article, we describe a sensitive and reliable method for the quantitation of trans, anti-BaPT in human urine using gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). [(13)C(6)] trans, anti-BaPT was used as the internal standard. The urine was treated with β-glucuronidase and sulfatase, and then trans, anti-BaPT was enriched by solid-phase extraction with polymeric reversed phase and phenylboronic acid cartridges. The sample was silylated and analyzed by GC-NICI-MS/MS with selected reaction monitoring (SRM) for the trimethylsilyl (TMS) derivatives of trans, anti-BaPT (m/z 446 → m/z 255) and [(13)C(6)]trans, anti-BaPT (m/z 452 → m/z 261). The mean assay recovery was 44%. The instrumental on-column detection limit was about 20 amol of trans, anti-BaPT (as BaPT-TMS). trans, anti-BaPT was readily detected in all urine samples analyzed including those of 30 smokers (0.71 ± 0.64 fmol/mg creatinine) and 30 nonsmokers (0.34 ± 0.2 fmol/mg creatinine) (P = 0.0036). The results of this study demonstrate a highly sensitive and selective method for the quantitation of trans, anti-BaPT in human urine. This is to our knowledge the first study to show that smokers have significantly higher levels of trans, anti-BaPT in their urine than do nonsmokers. This method may be useful as a direct phenotyping approach to assess individual differences in uptake and metabolic activation of carcinogenic PAH.

Background: The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its reduction product in the body, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent pulmonary carcinogens. We have measured total NNAL in the U.S. population of tobacco users and nonsmokers exposed to secondhand smoke., Methods: We measured total urinary NNAL (free NNAL plus its glucuronides following hydrolysis) by using a sensitive and specific high-performance liquid chromatography/tandem mass spectrometry method. We calculated the percentage above the limit of detection, the 50th through 95th percentiles, and in some cases, geometric means for groups classified by age, gender, and race/ethnicity., Results: Total urinary NNAL was measureable at or above its limit of detection (0.6 pg/mL) in 55% of the study participants, including 41% of nonsmokers. The population distribution of urinary NNAL included smoker and nonsmoker regions similar to the bimodal distribution of serum cotinine, and serum cotinine and total urinary NNAL were strongly correlated (r = 0.92; P < 0.001). Among nonsmokers, children had significantly higher concentrations of NNAL than did adults with the age of ≥20 years (P < 0.001)., Conclusions: Among National Health and Nutrition Examination Survey participants, total NNAL was found at measurable levels in the urine of 41% of nonsmokers and in 87.5% of those with substantial secondhand-smoke exposure (with serum cotinine concentrations of 0.1-10 ng/mL). Children with the age of 6 to 11 years had the highest NNAL concentrations among all nonsmokers., Impact: We describe for the first time the distribution of total urinary NNAL in the entire U.S. population, including smokers and nonsmokers. NNAL was detected in 41% of all nonsmokers., (©2010 AACR.)

Background: Low-tar cigarette smoking is gaining popularity in China. The China National Tobacco Corporation (CNTC) promotes low-tar cigarettes as safer than regular cigarettes., Methods: A total of 543 male smokers smoking cigarettes with different tar yields (15 mg, regular cigarettes, 10-13 mg low-tar cigarettes and < 10 mg low-tar cigarettes) were recruited in Shanghai, China, who then completed a questionnaire on smoking behaviour and provided a urine sample for analysis of the nicotine metabolites cotinine and trans-3'-hydroxycotinine. A total of 177 urine samples were selected at random for the analysis of the carcinogens polycyclic aromatic hydrocarbon metabolites (PAHs) (1-hydroxypyrene, naphthols, hydroxyfluorenes and hydroxyphenanthrenes) and the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) metabolites, 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNAL) and NNAL-glucuronide. Values were normalised by creatinine to correct for possible distortions introduced by dilution or concentration of the urine., Results: Smokers of low-tar cigarettes smoked fewer cigarettes per day (p=0.001) compared to smokers of regular cigarettes. Despite this lower reported consumption, levels of cotinine, trans-3'-hydroxycotinine and PAHs in urine of people smoking low-tar cigarettes were not correlated with nominal tar delivery of the cigarettes they smoked. Urine concentrations of NNAL were higher in smokers of lower tar than higher tar cigarettes., Conclusions: Chinese low-tar cigarettes do not deliver lower doses of nicotine and carcinogens than regular cigarettes, therefore it is unlikely that there would be any reduction in harm. CNTC's promotion of low-tar cigarettes as 'less harmful' is a violation of the World Health Organization Framework Convention on Tobacco Control, which China ratified in 2005.

N'-nitrosonornicotine (NNN) is a strong carcinogen present in unburned tobacco and cigarette smoke. We here analyze data obtained in two studies, in which a biomarker of exposure to NNN--the sum of NNN and its pyridine-N-glucuronide, called total NNN--was quantified in the urine of people who had stopped smoking and used various nicotine replacement therapy (NRT) products. In 13 of 34 nicotine gum or lozenge users from both studies, total NNN at one or more time points after biochemically confirmed smoking cessation was comparable with, or considerably higher than, the baseline levels. For most of the subjects who used the nicotine patch as a smoking cessation aid, urinary total NNN at all post-quit time points was <37% of their mean baseline levels. These results indicate that endogenous formation of significant amounts of NNN may occur sporadically in some users of oral NRT. Given the carcinogenicity of NNN and the frequent use of nicotine gum as a smoking cessation aid, further studies are needed so that preventive measures can be developed.

The tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) is carcinogenic to humans (IARC Group 1). Assessing the tobacco smoke-related exposure to NNN by suitable biomarkers is of interest for risk evaluation. Recently, NNN and NNN-N-glucuronide have been quantified in urine of smokers. However, it is unknown what percentage of the absorbed dose of NNN is excreted as total NNN (sum of free and conjugated NNN) in urine of smokers. We developed a sensitive method based on liquid chromatography with tandem mass spectrometry with deuterium-labeled internal standard for the determination of total NNN in human urine. The limit of quantitation of the method was 2 pg/mL with a calibration line linear up to 256 pg/mL. In a study with 16 smokers in which the respiratory retention of NNN was measured through controlled smoking, we found that on average about 1% of the pulmonary NNN dose was excreted in 24 h urine as total NNN.

We determined the persistence at various times (3, 7, 14, 21, 28, 42, and 56 days) of eight tobacco smoke carcinogen and toxicant biomarkers in the urine of 17 smokers who stopped smoking. The biomarkers were 1-hydroxy-2-(N-acetylcysteinyl)-3-butene (1) and 1-(N-acetylcysteinyl)-2-hydroxy-3-butene (2) [collectively called MHBMA for monohydroxybutyl mercapturic acid] and 1,2-dihydroxy-4-(N-acetylcysteinyl)butane (3) [DHBMA for dihydroxybutyl mercapturic acid], metabolites of 1,3-butadiene; 1-(N-acetylcysteinyl)-propan-3-ol (4, HPMA for 3-hydroxypropyl mercapturic acid), a metabolite of acrolein; 2-(N-acetylcysteinyl)butan-4-ol (5, HBMA for 4-hydroxybut-2-yl mercapturic acid), a metabolite of crotonaldehyde; (N-acetylcysteinyl)benzene (6, SPMA for S-phenyl mercapturic acid), a metabolite of benzene; (N-acetylcysteinyl)ethanol (7, HEMA for 2-hydroxyethyl mercapturic acid), a metabolite of ethylene oxide; 1-hydroxypyrene (8) and its glucuronides (1-HOP), metabolites of pyrene; and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (9) and its glucuronides (total NNAL), a biomarker of exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These biomarkers represent some of the major carcinogens and toxicants in cigarette smoke: 1,3-butadiene, acrolein, crotonaldehyde, benzene, ethylene oxide, polycyclic aromatic hydrocarbons (PAH), and NNK. With the exception of DHBMA, levels of which did not change after cessation of smoking, all other biomarkers decreased significantly after 3 days of cessation (P < 0.001). The decreases in MHBMA, HPMA, HBMA, SPMA, and HEMA were rapid, nearly reaching their ultimate levels (81-91% reduction) after 3 days. The decrease in total NNAL was gradual, reaching 92% after 42 days, while reduction in 1-HOP was variable among subjects to about 50% of baseline. Since DHBMA did not change upon smoking cessation, there appear to be sources of this metabolite other than 1,3-butadiene. The results of this study demonstrate that the tobacco smoke carcinogen/toxicant biomarkers MHBMA, HPMA, HBMA, SPMA, HEMA, 1-HOP, and NNAL are related to smoking and are good indicators of the impact of smoking on human exposure to 1,3-butadiene, acrolein, crotonaldehyde, benzene, ethylene oxide, PAH, and NNK.

Introduction: Nitrosation of nicotine or its metabolites in the human body could lead to formation of the 2 carcinogenic tobacco-specific nitrosamines-N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)., Methods: We investigated the possibility of endogenous formation of NNN in people who had stopped smoking and used the 21-mg nicotine patch for 6 months. We quantified urinary biomarkers of exposure to NNN-the sum of NNN and its pyridine-N-glucuronide, referred to as total NNN. Also measured were NNK metabolites-the sum of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its N- and O-glucuronides, referred to as total NNAL., Results: The average decline of urinary total NNN was less drastic than that of total NNAL: 22% of baseline total NNN and 7.3% of baseline total NNAL were detected in urine 24 weeks after smoking cessation and patch use (p = .02). The average ratio of total NNN to total NNAL in the same urine samples increased from 0.14 in baseline urine to 0.38 after 24 weeks of nicotine patch use., Discussion: Overall, these results demonstrate that endogenous formation of NNN may occur in nicotine patch users. However, the levels of urinary total NNN during patch use were generally extremely low. Moreover, in 10 of 20 subjects analyzed here, the rate of decline in total NNN was similar to that in total NNAL, indicating that endogenous formation of NNN is virtually nonexistent in these subjects. Supplementation with ascorbic acid could be a simple approach to block possible NNN formation in nicotine patch users.

There may contain a great many carcinogens in cigarette smoke. Various carcinogens and their metabolites were quantified in urine of smokers, including trans, trans-muconic acid, S-phenylmercapturic acid, 4-(methylnitrosamino)-1-(3-pyridyl )-1-butanol and its glucuronides and so on. Quantification of carcinogens and their metabolites in urine of smokers could provide information on carcinogen exposure, which may be helpful to the research for mechanisms of carcinogen and in the exploitation of new harmless cigarettes. The study advances on carcinogens and their metabolites in urine of smokers were reviewed.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in both unburned tobacco and cigarette smoke. The sum of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, referred to as total NNAL, is an established urinary biomarker of human NNK uptake. Metabolic activation of NNK to DNA adducts proceeds via alpha-hydroxylation pathways, and 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid) are the principal end products of these pathways in rodents and primates. The purpose of this study was to determine NNK metabolic activation in smokers, as measured by the sum of keto acid and hydroxy acid, relative to total NNAL. To specifically identify NNK-derived keto acid and hydroxy acid, which are also formed from nicotine, we added [pyridine-D(4)]NNK to cigarettes that were originally low in NNK, and measured the deuterium-labeled metabolites in the urine of people who smoked these cigarettes. The total amount of [pyridine-D(4)]keto acid plus [pyridine-D(4)]hydroxy acid averaged 4.00 +/- 2.49 nmol/24 h, whereas the average amount of total [pyridine-D(4)]NNAL was 0.511 +/- 0.368 nmol/24 h. The results of this study show for the first time that NNK metabolic activation is a quantitatively significant pathway in smokers, accounting for approximately 86% of total urinary excretion of NNK metabolites. The large interindividual variation in the excreted [pyridine-D(4)]keto acid and [pyridine-D(4)]hydroxy acid among 20 smokers strongly supports our hypothesis that some smokers activate NNK more extensively than others and that the ratio between biomarkers of metabolic activation and detoxification at a given dose of NNK could be a potential indicator of cancer risk.

Carcinogenic heterocyclic aromatic amines (HAA) are formed in cooked meats, poultry, and fish and arise in tobacco smoke. We measured the concentrations of four prevalent HAAs in spot urine samples collected at baseline from 170 participants of the Shanghai Cohort study, a population-based cohort study of adult men recruited during 1986 to 1989 in Shanghai, China. Sixteen (18.6%) of 86 nonsmokers were positive for urinary 2-amino-9H-pyrido[2,3-b]indole (AalphaC) versus 41 (48.8%) of 84 cigarette smokers; the difference was statistically significant (P < 0.001). The number of cigarettes smoked per day was positively and significantly related to urinary levels of AalphaC in study subjects (P < 0.001); the mean level among nonsmokers was 2.54 ng/g creatinine, whereas the means for light (1-19 cigarettes per day) and heavy (20+ cigarettes per day) smokers were 7.50 and 11.92 ng/g creatinine, respectively. 2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline was undetected in the urine of the 170 subjects. Only 5 (2.9%) and 6 (3.5%) subjects, respectively, showed detectable levels of urinary 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and smoking status was unrelated to levels of either HAA. Quantitative measurements of HAAs in commonly eaten pork and chicken dishes in Shanghai showed low concentrations of HAAs (<1 ng/g meat). Our data indicate that AalphaC represents a major HAA exposure in adult men of Shanghai, China, and that tobacco smoke is an important point source of their AalphaC exposure.

Smokeless tobacco has been proposed as a reduced risk substitute for smoking, but no large studies have investigated exposure to the powerful carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokeless tobacco users versus smokers. The purpose of this study was to carry out such a comparison. Levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), a biomarker of NNK exposure, and cotinine, a biomarker of nicotine exposure, were quantified in the urine of 420 smokers and 182 smokeless tobacco users who were participants in studies designed to reduce their use of these products. The measurements were taken at baseline, before intervention. Levels of total NNAL per milliliter of urine were significantly higher in smokeless tobacco users than in smokers (P < 0.0001). When adjusted for age and gender, levels of total NNAL per milligram of creatinine were also significantly higher in smokeless tobacco users than in smokers (P < 0.001). Levels of cotinine per milliliter of urine and per milligram of creatinine were significantly higher in smokeless tobacco users than in smokers (P < 0.001). These results show similar exposures to the potent tobacco-specific carcinogen NNK in smokeless tobacco users and smokers. These findings do not support the use of smokeless tobacco as a safe substitute for smoking.

Mutagenic/carcinogenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)] is formed from norharman and aniline in the presence of cytochrome P450 3A4/1A2. Because both precursors are widely distributed in the environment, human exposure is unavoidable. To clarify APNH formation in the human body, amounts of the compound in 24-h human urine collected from smokers and nonsmokers, eating a normal diet, were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry. In addition, norharman and aniline were also analyzed by high-performance liquid chromatography and gas chromatography, respectively. APNH could be detected in all urine samples at levels 49 to 449 pg for smokers and 21 to 594 pg for nonsmokers per 24-h urine, respectively. The amounts of norharman and aniline were 46 to 185 ng and 0.70 to 8.10 microg for smokers and 52 to 447 ng and 0.49 to 5.72 microg for nonsmokers, respectively, per 24-h urine (none of the levels differing significantly between smokers and nonsmokers). To exclude exogenous exposure to norharman and aniline, we analyzed the levels of APNH, norharman, and aniline in urine samples collected from inpatients receiving parenteral alimentation. Similar to the healthy volunteers, all urine samples contained 12 to 338 pg of APNH, 6 to 75 ng of norharman, and 0.33 to 1.86 microg of aniline per 24-h urine. These results suggest that APNH should be considered as a novel endogenous mutagen/carcinogen; thus, it is very important to determine the biological significance of this carcinogen for human cancer development.

The analysis of protein adducts is a valuable tool for the biomonitoring of humans exposed to alkylating compounds. In case of single or intermittent exposures, however, the significance and validity of adduct analysis is limited due to the typically small increase in adduct levels and a nonlinear adduct elimination kinetic. This issue was addressed in a follow-up observation of four workers accidentally exposed to acrylonitrile (ACN) in a train depot. N-2-Cyanoethylvaline (CEV) in hemoglobin was analyzed in blood samples of the workers and of seven rescue team and hospital members, approximately, 25 days after the accident. Of the 11 potentially exposed persons, only the cleaning workers revealed adduct levels significantly above the general background. Further blood samples of the workers were analyzed after 85, 115 and 175 days, respectively. In these cases, the adduct concentrations declined to background levels associated with individual smoking habits. A linear regression analysis of the data points to a total elimination interval of 148 days, 2 weeks longer than the standard lifespan of erythrocytes and possibly due to a period of acrylonitrile circulation in the blood stream or release from unstable intermediates. The data allow a rough estimate of the initial protein adduct concentration and an evaluation of the internal dose on the basis of biological exposure equivalents. In conclusion, adduct analyses offer valuable information even in the case of short-term exposures.

Tobacco-specific nitrosamines are believed to play a significant role as causes of cancer in people who use tobacco products. Whereas the uptake of one tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, has been shown by analysis of its metabolites in urine, there are no published studies on urinary levels of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), and N'-nitrosoanabasine (NAB) or their metabolites in human urine. We developed a method for quantitation of NNN, NAT, NAB, and their pyridine-N-glucuronides NNN-N-Gluc, NAT-N-Gluc, and NAB-N-Gluc in human urine. Total NNN (NNN plus NNN-N-Gluc) was assayed using 5-methyl-N'-nitrosonornicotine as internal standard. Urine was treated with beta-glucuronidase. Following solvent partitioning and solid-phase extraction, total NNN was determined using gas chromatography with nitrosamine-selective detection. Total NAT and total NAB were quantified in the same samples. Separate quantitation of NNN and NNN-N-Gluc was accomplished by extraction of the urine with ethyl acetate before beta-glucuronidase hydrolysis; NNN was analyzed in the ethyl acetate extract, and after enzyme treatment, NNN released from NNN-N-Gluc was quantified in the extracted urine. Separate analyses of NAT, NAT-N-Gluc, NAB, and NAB-N-Gluc proceeded similarly. Analyte identities were confirmed by gas chromatography-tandem mass spectrometry. Mean levels of total NNN, NAT, and NAB in the urine of 14 smokers were (pmol/mg creatinine) 0.18 +/- 0.22, 0.19 +/- 0.20, and 0.040 +/- 0.039, respectively, whereas the corresponding amounts in the urine of 11 smokeless tobacco users were 0.64 +/- 0.44, 1.43 +/- 1.10, and 0.23 +/- 0.19, respectively. Pyridine-N-glucuronides accounted for 59% to 90% of total NNN, NAT, and NAB. The results of this study show the presence of NNN, NAT, NAB, and their pyridine-N-glucuronides in human urine and provide a quantitative method for application in mechanistic and epidemiologic studies of the role of tobacco-specific nitrosamines in human cancer.

Vegetable consumption, including cruciferous vegetables, is protective against lung cancer, but the mechanisms are poorly understood. The purpose of this study was to investigate the effects of cruciferous vegetable consumption on the metabolism of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers. The study was carried out in Singapore Chinese, whose mean daily intake of cruciferous vegetables is three times greater than that of people in the United States. Eighty-four smokers provided urine samples and were interviewed about dietary habits using a structured questionnaire, which included questions on consumption of nine commonly consumed cruciferous vegetables. Samples of these vegetables obtained in Singapore markets at three different times of year were analyzed for glucosinolates. Urine was analyzed for metabolites of NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides (NNAL-Glucs). Glucobrassicins, which release indole-3-carbinols on chewing, were the major glucosinolates in seven of the nine cruciferous vegetables, accounting for 70.0% to 93.2% of all glucosinolates in these vegetables. There was a significant correlation (P = 0.01) between increased consumption of glucobrassicins and decreased levels of NNAL in urine after adjustment for number of cigarettes smoked per day; similar trends were observed for NNAL-Glucs (P = 0.08) and NNAL plus NNAL-Glucs (P = 0.03). These results are consistent with those of previous studies, which demonstrate that indole-3-carbinol decreases levels of urinary NNAL probably by inducing hepatic metabolism of NNK. The results are discussed with respect to the known chemopreventive activity of indole-3-carbinol against lung tumorigenesis by NNK in mice and the effects of isothiocyanates, which are also formed on consumption of cruciferous vegetables, on NNK metabolism. The results of this study demonstrate the complexities in assessing effects of cruciferous vegetables on carcinogen metabolism.

Measurement of human urinary carcinogen metabolites is a practical approach for obtaining important information about tobacco and cancer. This review presents currently available methods and evaluates their utility. Carcinogens and their metabolites and related compounds that have been quantified in the urine of smokers or non-smokers exposed to environmental tobacco smoke (ETS) include trans,trans-muconic acid (tt-MA) and S-phenylmercapturic acid (metabolites of benzene), 1- and 2-naphthol, hydroxyphenanthrenes and phenanthrene dihydrodiols, 1-hydroxypyrene (1-HOP), metabolites of benzo[a]pyrene, aromatic amines and heterocyclic aromatic amines, N-nitrosoproline, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (NNAL and NNAL-Gluc), 8-oxodeoxyguanosine, thioethers, mercapturic acids, and alkyladenines. Nitrosamines and their metabolites have also been quantified in the urine of smokeless tobacco users. The utility of these assays to provide information about carcinogen dose, delineation of exposed vs. non-exposed individuals, and carcinogen metabolism in humans is discussed. NNAL and NNAL-Gluc are exceptionally useful biomarkers because they are derived from a carcinogen- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- that is specific to tobacco products. The NNAL assay has high sensitivity and specificity, which are particularly important for studies on ETS exposure. Other useful assays that have been widely applied involve quantitation of 1-HOP and tt-MA. Urinary carcinogen metabolite biomarkers will be critical components of future studies on tobacco and human cancer, particularly with respect to new tobacco products and strategies for harm reduction, the role of metabolic polymorphisms in cancer, and further evaluation of human carcinogen exposure from ETS.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific lung carcinogen which may play an important role as a cause of lung cancer in smokers. NNK is extensively metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which like NNK is a potent pulmonary carcinogen. NNAL in turn is glucuronidated, and both NNAL and its glucuronides are excreted in human urine. Previous studies have clearly demonstrated the presence in human urine of 4-(methylnitrosamino)-1-(3-pyridyl)-1-(O-beta-D-glucopyranuronosyl)butane (NNAL-O-Gluc), but did not exclude the presence of 4-(methylnitrosamino)-1-(3-pyridyl-N-beta-D-glucopyranuronosyl)-1-butanolonium inner salt (NNAL-N-Gluc). In this study, we quantified NNAL, NNAL-N-Gluc, and NNAL-O-Gluc in the urine of smokers, snuff-dippers, and people who used the oral tobacco product "toombak". The presence of NNAL-N-Gluc in the urine of toombak users was confirmed by LC-ESI-MS/MS. In smokers' urine, NNAL-N-Gluc, NNAL-O-Gluc, and NNAL comprised (mean +/- SD) 26.5 +/- 6.2, 32.1 +/- 17.6, and 41.4 +/- 16.6%, respectively, of total NNAL. In snuff-dippers' urine, the corresponding figures were 13.6 +/- 5.1, 46.6 +/- 11.7, and 36.6 +/- 9.3%. NNAL-N-Gluc comprised 50 +/- 25% of total glucuronidated NNAL in smokers and 24 +/- 12% in snuff-dippers. This difference was significant (P = 0.01), suggesting that smoking induces glucuronidation of NNAL. The results of this study demonstrate that NNAL-N-Gluc contributes substantially to NNAL-glucuronides in human urine. These results are important for a clearer understanding of mechanisms of detoxification of NNK in humans.

A new, rapid method for selective extraction of hydroxylated polycyclic aromatic hydrocarbons metabolites (OH-PAHs) in human urine was developed using an immunosorbent of anti-pyrene antibodies which were encapsulated in a sol-gel glass (SGG) matrix. Resulting chromatograms after immunoextraction of urine samples and HPLC analysis of the extracts were free from matrix interferences. The LODs for the determination of OH-PAHs in these difficult samples were in the low-ppt range (1-16 ng/L). In addition to its high selectivity, the immunosorbent proved to be robust and reusable. Obtained recoveries in spiked urine samples ranged from 83 to 107% for the hydroxyphenanthrene and hydroxypyrene compounds under investigation, while recovery for 3-hydroxybenzo[a]pyrene was only 45-62%. In a biomonitoring study, the SGG immunosorbent was successfully used for trace-level analysis of OH-PAHs in 20 human urine samples. Results were compared to data obtained by an independent reference analysis method and revealed good correlation between both methods.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen, is believed to be important as a causative agent for lung cancer in smokers. NNK is extensively metabolized to its carbonyl reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which, in turn, can be glucuronidated, producing [4-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic+ ++ acid (NNAL-Gluc). Metabolism of NNK to NNAL produces a chiral center. A recent study demonstrated that (R)-NNAL is more tumorigenic in mice than (S)-NNAL and that these enantiomers have substantially different metabolic pathways. Therefore, it is important to determine the stereochemistry of NNAL and NNAL-Gluc in smokers. In this study, we used chiral stationary phase-gas chromatography-nitrosamine-selective detection with confirmation by liquid chromatography-tandem mass spectrometry to determine the stereochemistry of NNAL and NNAL-Gluc in smokers' urine. The two methods agreed well. The results of analyses of urine samples from 30 smokers demonstrated that the enantiomeric distribution of NNAL in urine was 54% (R) and 46% (S) +/- 7.0 (SD), whereas the diastereomeric distribution of NNAL-Gluc was 68% (R) and 32% (S) +/- 8.1. These results conclusively demonstrate that both (R)- and (S)-NNAL are formed metabolically from NNK in smokers. These data are essential for furthering our understanding of the role of NNK as a cause of lung cancer in smokers.

N-acetyl-S-(2-hydroxyethyl)-L-cysteine (2-hydroxyethyl mercapturic acid, HEMA) is a urinary metabolite of several hazardous chemicals, including vinyl chloride (VC), ethylene oxide (EO), and ethylene dibromide (EDB). Information about the levels of HEMA in the general population is useful for assessing human exposures to HEMA parent compounds, including VC, EO, and EDB. To establish reference range concentrations for HEMA, we analyzed urine samples from 412 adult participants in the Third National Health and Nutrition Examination Survey (NHANES II) by using isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). HEMA was detected in 71% of the samples examined. Creatinine-corrected concentrations ranged from less than 0.68 microg/g creatinine to 58.7 microg/g creatinine; the 95th percentile concentration was 11.2 microg/g creatinine; and the geometric mean and median creatinine-corrected concentrations were both 1.6 microg/g creatinine. We observed a statistically significant difference (P=0.0001) in the creatinine-corrected geometric mean concentration values of HEMA between smokers (2.8 microg/g creatinine) and nonsmokers (1.1 microg/g creatinine). The high levels of HEMA seen among smokers likely originated from HEMA-producing chemicals known to be present in tobacco smoke.

To investigate the ways in which different levels of exposure affect the metabolic activation pathways of benzene in humans, and to examine the relationship between urinary metabolites and other biological markers, we have developed two sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for quantitation of the benzene metabolites trans,transmuconic acid (t,t-MA), S-phenylmercapturic acid (S-PMA), hydroquinone (HQ), catechol (CAT), and for estimation of 1,2,4-trihydroxybenzene (BT). In our first assay, urinary S-PMA and t,t-MA were measured simultaneously by liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM) in the negative ionization mode. In this assay, the metabolites [13C6]-S-PMA and [13C6]-t,t-MA were used as internal standards. The efficacy of this specific assay was evaluated in human urine specimens from 28 smokers and 18 nonsmokers serving as the benzene-exposed and nonexposed groups, respectively. The coefficient of variation (CV) of analyses on different days (n = 8) for S-PMA was 7% for samples containing 9.4 micrograms/L urine, and for t,t-MA was 10% for samples containing 0.07 mg/L. The mean levels of S-PMA and t,t-MA in smokers were 1.9-fold (p = 0.02) and 2.1-fold (p = 0.03) higher, respectively, than those in nonsmokers.

Four smokers were chosen for their different smoking habits, and their declared cigarette consumption confirmed by urinary measurement of nicotine and its metabolites. The promutagenicity of their urine was evaluated by the Ames test, modified according to Kado et al. (Mutation Res., 31 (1983)25-32) after extraction on XAD2 Amberlite resin. The different Salmonella typhimurium strains TA 98, YG 1021 and YG 1024 were compared to determine the presence of amino aromatic compounds in the urine of smokers of blond and black tobacco. The strain YG 1024 shows higher mutagenicity than TA 98 for extracts from the smoker's urine and more particularly from black tobacco smokers. In addition, the pretreatment of urine by external enzymatic systems (beta-glucuronidase or arylsulfatase) reveals the presence in the urine of glucurono- and sulfoconjugated forms of promutagens, including amino aromatic compounds.

We examined the mutagenicity of cigarette smoker's urine in 32 healthy male cigarette smoker and 37 healthy male non-smoker. Twenty-four-hour urine specimens were subjected to blue rayon extraction which selectively adsorb polycyclic compounds, after which the elutions were fractionated by carboxymethyl cellulose column chromatography for removing antimutagenic compounds. The mutagens were measured by using an S9-mediated Salmonella mutagenicity test on strain TA98. Compared with those with non-smokers, smokers' urine showed a significantly higher urinary level of mutagens in the acid-elutable and in the sum of all chromatography fractions. A similar tendency was also seen in the alkali-elutable fraction. The subjects were classified into three groups according to the number of smoked cigarettes. Heavy smokers, who smoked more than 20 cigarettes per day, showed a significantly higher urinary level of mutagens than both non-smokers and light smokers especially in the acid-elutable and in the sum of all chromatography fractions. Our findings suggest that smokers are exposed to a great amount of polycyclic carcinogens and mutagens by cigarette smoking. These results also suggest that urinary level of mutagens measured by using blue rayon extraction combined with carboxymethyl cellulose chromatography could be a good index for estimating the exposure to carcinogens and mutagens such as polycyclic compounds.

Bladder cancer is associated with smoking. Among the tobacco-derived carcinogens suspected of being involved in initiating the disease are nitrosamines found in urine. In this study a nitrosamine found in the urine of smokers was tested using a tissue culture model of normal human urothelium. Explant cultures were established from ureters and exposed to 5 ng/ml of the derivative. This level had been demonstrated previously to induce a variety of changes associated with initiation of carcinogenesis. Proliferation of the cultures was increased following exposure to the carcinogen, and the gap junction intercellular communication was reversibly inhibited. Examination of the connexin 43 protein and message status showed that the mRNA was unaffected, but the protein was not detectable using anti-connexin 43 antibody. The expression of the protein recovered within 24 h of removal of the carcinogen, indicating that the continued presence of the agent was necessary. Given the roles of cell proliferation and cell communication in carcinogenesis, the results may suggest a mechanism involving pre- or post-initiation deregulation of cell communication systems. Whether the enhanced growth is a separate effect or a consequence of reduced communication is an intriguing question.

This study describes quantitation in smokers' urine of two metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The metabolites are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen, and its O-glucuronide (NNAL-Gluc), a presumed detoxification product of NNK. Using updated methodology, levels of NNAL, NNAL-Gluc, and cotinine were determined in the urine of 61 smokers. The NNAL-Gluc: NNAL ratio, a potential marker for NNK detoxification potential, varied 16-fold in this group. Two phenotypes of this ratio were apparent; one ranging from 0 to 6 and found in 85% of the smokers and a second ranging from 6 to 11. The short-term and long-term consistency of the ratio was investigated. Studies carried out over a 4-5 day period indicated that the NNAL-Gluc: NNAL ratio was reasonably stable. Subjects who donated urine samples on two occasions separated by 4-16 months were classified in the same group (ratio range, 0-6 or 6-11) each time. Different urine collection protocols appeared to have little influence on the NNAL-Gluc: ratio. Thus, intraindividual differences in the NNAL-Gluc:NNAL ratio were generally small, whereas interindividual differences were large. Amounts of NNAL, NNAL-Gluc, and cotinine excreted by smokers were constant in 24-h samples obtained over a 3-day period of constant cigarette intake and controlled diet. Levels of NNAL, NNAL-Gluc, and NNAL plus NNAL-Gluc correlated with cotinine in a study of 61 smokers without controlled diet or smoking (r = 0.58; P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the genotypes of two polymorphic genes involved in the detoxification of several mutagenic and carcinogenic compounds in relation to tobacco smoking-associated urinary mutagenicity. The genes studied were the glutathione S-transferase-encoding GSTM1 gene and acetyltransferase-encoding NAT2 gene. Smokers with no GSTM1 gene (n = 7) had urine that was several times more mutagenic than that of smokers with the gene (n = 10). The mean level of urinary mutagenicity in presence of metabolic activation was 2527 +/- 958 revertants/100 ml urine for GSTM1-smokers compared to 766 +/- 560 revertants/100 ml for GSTM1+ smokers (P < 0.001) using the bacterial strain YG1024. The corresponding values using the TA98 strain were 336 +/- 124 and 123 +/- 75 (P < 0.001). In contrast, we failed to show any difference in the level of urinary mutagenicity between slow-acetylator and fast-acetylator NAT2 genotypes among smokers (n = 17) or non-smokers (n = 35). Our results offer one explanation for the recent findings that GSTM1 polymorphism is a risk modifier in smoking-related cancers, especially bladder cancer.

The potential endogenous nitrosation of nicotine and/or nicotine metabolites has led to speculation on the possible formation of 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso-NNAC) in smokers. A gas chromatographic method with thermal energy analytical detection is described for the determination of iso-NNAC in tobacco, tobacco smoke and urine. Sample pre-concentration is performed using C18 extraction cartridges prior to esterification of iso-NNAC using ethereal diazomethane solution. Sample clean-up includes chromatography on aluminum and silica, and fractionation using high-performance liquid chromatography. The detection limits for iso-NNAC in tobacco, tobacco smoke and urine are 2 ng/g tobacco, 0.1 ng/cigarette and 20 ng/l urine, respectively.