INTRODUCTION:The tobacco-specific nitrosamines (TSNAs) are an important group of carcinogens found in tobacco and tobacco smoke. To describe and characterize the levels of TSNAs in the Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014), we present four biomarkers of TSNA exposure: N'-nitrosonornicotine, N'-nitrosoanabasine, N'-nitrosoanatabine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which is the primary urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. METHODS:We measured total TSNAs in 11 522 adults who provided urine using automated solid-phase extraction coupled to isotope dilution liquid chromatography-tandem mass spectrometry. After exclusions in this current analysis, we selected 11 004 NNAL results, 10 753 N'-nitrosonornicotine results, 10 919 N'-nitrosoanatabine results, and 10 996 N'-nitrosoanabasine results for data analysis. Geometric means and correlations were calculated using SAS and SUDAAN. RESULTS:TSNA concentrations were associated with choice of tobacco product and frequency of use. Among established, every day, exclusive tobacco product users, the geometric mean urinary NNAL concentration was highest for smokeless tobacco users (993.3; 95% confidence interval [CI: 839.2, 1147.3] ng/g creatinine), followed by all types of combustible tobacco product users (285.4; 95% CI: [267.9, 303.0] ng/g creatinine), poly tobacco users (278.6; 95% CI: [254.9, 302.2] ng/g creatinine), and e-cigarette product users (6.3; 95% CI: [4.7, 7.9] ng/g creatinine). TSNA concentrations were higher in every day users than in intermittent users for all the tobacco product groups. Among single product users, exposure to TSNAs differed by sex, age, race/ethnicity, and education. Urinary TSNAs and nicotine metabolite biomarkers were also highly correlated. CONCLUSIONS:We have provided PATH Study estimates of TSNA exposure among US adult users of a variety of tobacco products. These data can inform future tobacco product and human exposure evaluations and related regulatory activities.

Background: Heterocyclic aromatic amines (HAA) are a group of hazardous substances produced during combustion of tobacco or high-temperature cooking of meats. 2-Amino-9H-pyrido[2,3-b]indole (A alpha C) is a major carcinogenic HAA in tobacco smoke. Methods: Urinary A alpha C, used as a marker of A alpha C exposure, was analyzed on spot urine samples from adult participants of the 2013-2014 cycle of the National Health and Nutrition Examination Survey (N = 1,792). A alpha C was measured using isotope-dilution liquid chromatography-tandem mass spectrometry. Exclusive combusted tobacco smokers were differentiated from nonusers of tobacco products through both self-report and serum cotinine data. Results: Among exclusive smokers, sample-weighted median urinary A alpha C was 40 times higher than nonusers. Sample-weighted regression models showed that urinary A alpha C increased significantly with serum cotinine among both exclusive tobacco users and nonusers with secondhand smoke exposure. Among nonusers, eating beef cooked at high temperature was associated with a significant increase in urinary A alpha C, whereas consuming vegetables was associated with decreased A alpha C. In addition, smoking one-half pack of cigarettes per day was associated with a significant increase of 23.6 pg A alpha C/mL calculated at geometric mean of A alpha C, controlling for potential confounders. In comparison, increase in A alpha C attributable to consuming the 99th percentile of beef cooked at high temperature was 0.99 pg A alpha C/mL. Conclusions: Both exclusive smokers and nonusers of tobacco in the general U.S. population are exposed to A alpha C from tobacco smoke, with additional, lesser contributions from certain dietary components. Impact: A alpha C is an important biomarker that is associated with tobacco smoke exposure.

Formamidine pesticides are widely used in pest control for crops and livestock, creating a potential risk in animal-origin food safety. This Application Note presents a reliable and robust method using QuEChERS extraction followed by Agilent Captiva EMR—Lipid cleanup in the residue analysis of amitraz, chlordimeform, and their metabolites in pork and porcine liver. The method delivers good accuracy (>60.0 %) and precision (RSD<12 %) for the four analytes at all levels, providing a fast and effective analysis in high-fat samples.

Heterocyclic aromatic amines (HCAA) are listed by the US Food and Drug Administration (FDA) as harmful or potentially harmful constituents of tobacco smoke. However, quantifying HCAA exposure is challenging. In this study, we developed a sensitive, precise, and accurate isotope dilution, liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify urinary HCAAs in smokers and nonsmokers. The high-throughput robotic sample preparation system could handle a throughput of over 300 samples per day, while maintaining intra-day and inter-day imprecision and bias ≤10 %. The limits of detection of carcinogenic HCAAs ranged from 0.31 to 0.83 pg/mL. The validated method was applied to measure HCAAs in urine collected from smokers and non-smokers. This sensitive and efficient analytical method is ideal to support large-scale biomonitoring studies of HCAA exposure.

Background: Marijuana is seeing increased therapeutic use, and is the world's third most-popular recreational drug following alcohol and tobacco. This widening use poses increased exposure to potentially toxic combustion by-products from marijuana smoke and the potential for public health concerns.Objectives: To compare urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs) among self-reported recent marijuana users and nonusers, while accounting for tobacco smoke exposure.Methods: Measurements of PAH and VOC metabolites in urine samples were combined with questionnaire data collected from participants in the National Health and Nutrition Examination Surveys (NHANES) from 2005 to 2012 in order to categorize participants (>= 18 years) into exclusive recent marijuana users and nonusers. Adjusted geometric means (GMs) of urinary concentrations were computed for these groups using multiple regression analyses to adjust for potential confounders.Results: Adjusted GMs of many individual monohydroxy PAHs (OH-PAHs) were significantly higher in recent marijuana users than in nonusers (p < 0.05). Urinary thiocyanate (p < 0.001) and urinary concentrations of many VOC metabolites, including metabolites of acrylonitrile (p < 0.001) and acrylamide (p < 0.001), were significantly higher in recent marijuana users than in nonusers.Conclusions: We found elevated levels of biomarkers for potentially harmful chemicals among self-identified, recent marijuana users compared with nonusers. These findings suggest that further studies are needed to evaluate the potential health risks to humans from the exposure to these agents when smoking marijuana. Published by Elsevier Ltd.

INTRODUCTION:Smoking prevalence is high among men in China. One result is that a large number of nonsmoking Chinese women may be exposed daily to secondhand smoke (SHS). Exposure is particularly problematic for pregnant women because of potential adverse reproductive effects. To determine the extent of this exposure and to summarize existing intervention studies designed to reduce SHS exposure in China, a systematic review of the literature published from 1995 through 2012 was conducted. METHODS:We searched the PubMed and Wanfang databases for studies published from 1995 through 2012 using various search terms including SHS, pregnant women, and China. Only articles on prevalence of SHS exposure and interventions to reduce exposure to SHS were selected. RESULTS:We identified 132 studies during the initial searches. Eight of 13 eligible studies reported the prevalence of SHS exposure among pregnant women; estimates ranged from 38.9% to 75.1%. Few SHS prevention interventions among pregnant women in China have been studied; we found only 5 such studies. The interventions primarily focused on changing husbands' smoking behaviors; some interventions focused on women's avoidance behaviors. CONCLUSION:Prevalence of exposure to SHS among pregnant women is high in China. Information is limited on effective interventions to protect pregnant women from exposure. The results of this review can provide the basis for the design and evaluation of interventions to help pregnant women avoid SHS exposure.

Background: It has been suggested that smokeless tobacco users have high nicotine and toxicant exposure, but studies with nationally representative data have been limited. Methods: We analyzed biomarkers of tobacco exposure for 23,684 adult participants from the National Health and Nutrition Examination Survey from 1999 to 2012. The biomarkers analyzed were serum cotinine, urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), blood lead, blood cadmium, blood mercury, urinary arsenic, and urinary N-acetyl-S-(2-cyanoethyl)-L-cysteine. We calculated geometric mean concentrations for each biomarker by tobacco use category and geometric mean ratios adjusting for demographic factors. Results: Exclusive smokeless tobacco users had higher geometric mean concentrations of serum cotinine [178.9 ng/mL, 95% confidence interval (CI), 145.5–220.0] and NNAL (583.0 pg/mg creatinine, 95% CI, 445.2–763.5) than exclusive cigarette smokers (130.6 ng/mL, 95% CI, 122.3–139.6 and 217.6 pg/mg creatinine, 95% CI, 193.0–245.2, respectively). Smokeless tobacco users also had higher concentrations of blood lead compared with nontobacco users (adjusted geometric mean ratio = 1.30, 95% CI, 1.21–1.38). Based on limited sample sizes, NNAL concentrations for smokeless tobacco users appear to have declined from 2007 to 2008 (geometric mean = 1013.7 pg/mg creatinine, 95% CI, 738.9–1390.8) to 2011 to 2012 (geometric mean = 325.7 pg/mg creatinine, 95% CI, 159.6–664.9). Conclusions: Exclusive smokeless tobacco users have higher observed levels of exposure to nicotine and carcinogenic tobacco-specific nitrosamines, as measured by cotinine and NNAL biomarker concentrations, than exclusive cigarette smokers. These patterns in NNAL levels for smokeless tobacco users may be changing over time. Impact: High exposure to harmful constituents among smokeless tobacco users is a continuing health issue. Cancer Epidemiol Biomarkers Prev; 24(12); 1829–37. ©2015 AACR.

The Dahongpao Zanthoxylum bungeanum Maximfrom Hancheng City is subjected to steam distillation extraction(HD),solvent free microwave extraction(SFME)and ultrasonic-assisted extraction(UAE)in order to obtain volatile oil.The chemical components are analyzed by GCMS/AMDIS,and they are similar.62 compounds identified from volatile oils are extracted by three different methods.In volatile oils obtained by UAE,the relative content of main compounds in Zanthoxylum bungeanum Maxim including linalool,α-terpineol,β-myrcene,1,8-cineole,limonene,geraniol is 64.27%,the relative content of linalool is 34.79%.The result shows that the volatile oils extracted by UAE have higher relative content of aroma components.

BACKGROUND:Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was measured in all participants aged 6 years and older from the Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey 2007-2008. The suitability of using creatinine or specific gravity for urinary NNAL correction in exposure assessment is examined in this study. METHODS:Effects of both specific gravity and creatinine correction on urinary NNAL among smokers were investigated with multiple linear regression models using either normalization or the fitting of creatinine and specific gravity in the model as covariates. RESULTS:When log-scaled NNAL was normalized by either creatinine or specific gravity, R(2) was slightly higher for creatinine than for specific gravity (R(2) = 0.1694 and 0.1439, for creatinine and specific gravity, respectively). When log-scaled NNAL was normalized by both factors, the R(2) was improved (R(2) = 0.2068). When specific gravity or creatinine was included as a covariate separately in the models, they were highly significant factors (P < 0.001, R(2) = 0.2226 and 0.1681 for creatinine and specific gravity, respectively). However, when both were included in the model as covariates, creatinine remained highly significant (P < 0.001), whereas the significance of specific gravity was eliminated (P = 0.4294). CONCLUSION:This study confirms significant relationships between NNAL concentrations and both urine creatinine and specific gravity. We conclude that creatinine is the more influential and preferred variable to account for urine dilution in tobacco-specific nitrosamine exposure assessment.

The migration of non-phthalate plasticizer, di(isononyl) cyclohexane-1,2-dicarboxylate(DINCH) in plastic food packaging materials was evaluated by GC-MS. Distilled water, 3%(V/V) acetic acid aqueous solution and 15%(V/V) ethanol a queous solution were used as aqueous food stimulants and isooctane as a fatty food stimulant. The aqueous food stimulants were extracted with n-hexane after food packaging materials were immersed in each of them. After being used to immerse samples, the fatty food stimulant was blown to dryness by nitrogen and then re-dissolved and diltetedin n-hexane direct injection. The results showed that the linear range was between 10 μg/mL and 400 μg/mL(r = 0.9999), and the recoveries of DINCH ranged from 95.2% to 103.4% with relative standard deviation(RSD) of less than 5% in four stimulants. The developed method is accurate and repeatable. It can be used for the evaluation of DINCH migration in p lastic food packaging materials.

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of five total tobacco-specific N-nitrosamines (TSNA), including free and conjugated forms in urine. The limits of detection for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, N'-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N'-nitrosoanatabine and N'-nitrosoanabasine were 0.6, 0.6, 10.0, 0.4 and 0.4 pg/mL, respectively, with a linear calibration range of up to 20,000 pg/mL. Intra- and inter-day precision for TSNA measurements ranged from 0.82 to 3.67% and from 2.04 to 7.73% respectively. For total TSNAs, the β-glucuronidase amount was optimized for hydrolysis time and yield. Different liquid chromatography columns and mobile phases with different pH conditions were evaluated. The validated method was then applied to 50 smoker and 30 nonsmoker urine samples. Our results suggest that this sensitive and relatively simple analytical method is suitable for application to epidemiological investigations of health risks associated with the exposure to tobacco smoke or secondhand smoke in both smokers and nonsmokers.

BACKGROUND:With an increase in indoor smoking bans, many smokers smoke outside establishments and near their entrances, which has become a public health concern.OBJECTIVES:We characterized the exposure of nonsmokers to secondhand smoke (SHS) outside a restaurant and bar in Athens, Georgia, where indoor smoking is banned, using salivary cotinine and urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL).METHODS:In a crossover study, we assigned 28 participants to outdoor patios of a restaurant and a bar and an open-air site with no smokers on three weekend days; participants visited each site once and stayed for 3 hr. We collected saliva and urine samples immediately before and after the visits (postexposure) and on the following morning and analyzed samples for cotinine and total NNAL, respectively. Regression models were fitted and changes in biomarkers were contrasted between locations.RESULTS:Postexposure and preexposure geometric mean salivary cotinine concentrations differed by 0.115 ng/mL [95% confidence interval (CI): 0.105, 0.126)] and by 0.030 ng/mL (95% CI: 0.028, 0.031) for bar and restaurant visits, respectively. There were no significant post- and preexposure differences in cotinine levels after control site visits, and changes after bar and restaurant site visits were significantly different from changes after control site visits (p < 0.001). Results comparing next-day and preexposure salivary cotinine levels were similar. Next-day creatinine-corrected urinary NNAL concentrations also were higher than preexposure levels following bar and restaurant visits [1.858 pg/mg creatinine higher (95% CI: 0.897, 3.758) and 0.615 pg/mg creatinine higher (95% CI: 0.210, 1.761), respectively], and were significantly different from changes after the control visits (p = 0.005).CONCLUSION:Salivary cotinine and urinary NNAL increased significantly in nonsmokers after outdoor SHS exposure. Our findings indicate that such exposures may increase risks of health effects associated with tobacco carcinogens.

Objectives Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease. In this study, we tried to investigate the effects of saturated and unsaturated fatty acids on hepatoma cell apoptosis. Methods H4IIE liver cells were treated with palmitic acid, linoleic acid, or both with or without the calcium-specific chelator BAPTA-AM after which the expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels, and calcium flux were measured. Results Palmitic or linoleic acid (250 μM) induced H4IIE cell apoptosis, which required calcium flux but not caspase-3. Apoptosis was not observed when cells were co-treated with linoleic acid (125 μM) and palmitic acid (250 μM). Importantly, the release of cytochrome C from mitochondria into cytoplasm during cell apoptosis was specifically detected only when linoleic acid (125 μM), but not palmitic acid (250 μM), was added to the cells. Depletion of intracellular calcium flux by the calcium-specific chelator, BAPTA-AM, abolished linoleic acid-induced apoptosis. Moreover, in the presence of BAPTA-AM, expression of the unfolded protein response (UPR)-associated genes, CHOP, GRP78, and GRP94, was induced by linoleic acid, but not palmitic acid. Conclusions The results suggest that linoleic acid promotes cell apoptosis through the release of cytochrome C, only if the intracellular calcium flux is unperturbed and intact. These results confirm that ER stress contributes to fatty acid-induced liver cell apoptosis.

BACKGROUND:Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of saturated and unsaturated fatty acids (FA) on apoptosis in primary rat hepatocytes.METHODS:The primary rat hepatocytes were treated with palmitic acid and/or α-linolenic acid in vitro. The expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels were detected after the treatment.RESULTS:The treatment with palmitic acid produced a significant increase in cell death. The unfolded protein response (UPR)-associated genes CHOP, GRP78, and GRP94 were induced to higher expression levels by palmitic acid. Co-treatment with α-linolenic acid reversed the apoptotic effect and levels of all three indicators of ER stress exerted by palmitic acid. Tunicamycin, which induces ER stress produced similar effects to those obtained using palmitic acid; its effects were also reversed by α-linolenic acid.CONCLUSIONS:α-Linolenic acid may provide a useful strategy to avoid the lipotoxicity of dietary palmitic acid and nutrient overload accompanied with obesity and NAFLD.

Nicotine (NIC), cotinine (COT) and trans-3'-hydroxycotinine (OHCOT) are the most prevalent and abundant tobacco biomarkers in meconium. We have developed and validated an accurate and precise method for the measurement of these analytes in meconium in which potassium hydroxide is used to digest the meconium sample, followed by solid phase extraction from the liquified sample. The precision of OHCOT, COT and NIC measurements (intra-day and inter-day) were 4.8-10.6%, 3.4-11.6% and 9.3-15.8%, respectively. Evaluation of accuracy indicated bias of -4.0, 2.0 and 0.8% for OHCOT at concentrations of 0.5, 2.5 and 7.5 ng/g. The accuracy estimates for COT at concentrations of 0.5, 2.5 and 7.5 ng/g are 4.0, 4.0 and 5.7%, respectively. For NIC at 2, 10 and 30 ng/g the accuracy was calculated to be 3.0, 5.0 and 5.1%, respectively. The linear range of standard solutions was 0.125-37.5 ng/mL for OHCOT and COT, and 0.75-150 ng/mL for NIC. This method was applied to the analysis of 374 meconium samples from infants of both smoking and nonsmoking mothers. Positive correlations with r(2)≥0.63 were observed between NIC and COT, COT and OHCOT, NIC and OHCOT, and NIC and (OHCOT+COT) in these samples.

Background: Smokers are exposed to significant doses of carcinogens, including tobacco-specific nitrosamines (TSNA). Previous studies have shown significant global differences in the levels of TSNAs in cigarette smoke because of the variation in tobacco blending and curing practices around the world. Methods: Mouth-level exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) measured in cigarette butts and urinary concentrations of its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) were examined among 126 daily smokers in four countries over a 24-hour study period. Results: As mouth-level exposure of NNK increased, the urinary NNAL increased even after adjustment for other covariates (β = 0.46, P = 0.004). The relationship between mouth-level exposure to nicotine and its salivary metabolite, cotinine, was not statistically significant (β = 0.29, P = 0.057), likely because of the very limited range of differences in mouth-level nicotine exposure in this population. Conclusions: We have shown a direct association between the 24-hour mouth-level exposure of NNK resulting from cigarette smoking and the concentration of its primary metabolite, NNAL, in the urine of smokers. Internal dose concentrations of urinary NNAL are significantly lower in smokers in countries that have lower TSNA levels in cigarettes such as Canada and Australia in contrast to countries that have high levels of these carcinogens in cigarettes, such as the United States. Impact: Lowering the levels of NNK in the mainstream smoke of cigarettes through the use of specific tobacco types and known curing practices can significantly affect the exposure of smokers to this known carcinogen. Cancer Epidemiol Biomarkers Prev; 19(6); 1389–98. ©2010 AACR.

ownloade kground: The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and uction product in the body, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent pulmoarcinogens. We have measured total NNAL in the U.S. population of tobacco users and nonsmokers d to secondhand smoke. thods: We measured total urinary NNAL (free NNAL plus its glucuronides following hydrolysis) by a sensitive and specific high-performance liquid chromatography/tandem mass spectrometry method. lculated the percentage above the limit of detection, the 50th through 95th percentiles, and in some geometric means for groups classified by age, gender, and race/ethnicity. ults: Total urinary NNAL was measureable at or above its limit of detection (0.6 pg/mL) in 55% of the participants, including 41% of nonsmokers. The population distribution of urinary NNAL included r and nonsmoker regions similar to the bimodal distribution of serum cotinine, and serum cotinine tal urinary NNAL were strongly correlated (r = 0.92; P < 0.001). Among nonsmokers, children had cantly higher concentrations of NNAL than did adults with the age of ≥20 years (P < 0.001). clusions: Among National Health and Nutrition Examination Survey participants, total NNAL was at measurable levels in the urine of 41% of nonsmokers and in 87.5% of those with substantial hand-smoke exposure (with serum cotinine concentrations of 0.1-10 ng/mL). Children with the 6 to 11 years had the highest NNAL concentrations among all nonsmokers. act: We describe for the first time the distribution of total urinary NNAL in the entire U.S. population, Imp including smokers and nonsmokers. NNAL was detected in 41% of all nonsmokers. Cancer Epidemiol Biomarkers Prev; 19(11); 2969–77. ©2010 AACR.

Urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-glucuronide, which are metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are valuable biomarkers of tobacco exposure. In epidemiologic investigations, it is often necessary for samples to be collected and stored for various periods of time prior to analysis, and as a consequence, it is important to determine the stability of these analytes during storage. In this study, two urine pools were stored at six different temperatures ranging from -70 degrees C to 55 degrees C. Free NNAL and NNAL-glucuronide were measured at scheduled time intervals for 28 days, after which samples stored at -20 degrees C and -70 degrees C were further monitored for an additional four years. NNAL-glucuronide was not completely stable at room temperature (ca. 22 degrees C) or above. At those temperatures, a gradual loss of NNAL-glucuronide with concomitant increases in free NNAL was observed. However, both free NNAL and NNAL-glucuronide appeared to be stable for at least four weeks when stored in the refrigerator (4 degrees C) and for at least four years at -20 degrees C and -70 degrees C, respectively. These results suggest that valid NNAL concentrations can be measured in frozen urine samples that have been stored for an extended period of time prior to analysis.