Logistics of defense: The contribution of endomembranes to plant innate immunity.

Phytopathogens cause plant diseases that threaten food security. Unlike mammals, plants lack an adaptive immune system and rely on their innate immune system to recognize and respond to pathogens. Plant response to a pathogen attack requires precise coordination of intracellular traffic and signaling. Spatial and/or temporal defects in coordinating signals and cargo can lead to detrimental effects on cell development. The role of intracellular traffic comes into a critical focus when the cell sustains biotic stress. In this review, we discuss the current understanding of the post-immune activation logistics of plant defense. Specifically, we focus on packaging and shipping of defense-related cargo, rerouting of intracellular traffic, the players enabling defense-related traffic, and pathogen-mediated subversion of these pathways. We highlight the roles of the cytoskeleton, cytoskeleton-organelle bridging proteins, and secretory vesicles in maintaining pathways of exocytic defense, acting as sentinels during pathogen attack, and the necessary elements for building the cell wall as a barrier to pathogens. We also identify points of convergence between mammalian and plant trafficking pathways during defense and highlight plant unique responses to illustrate evolutionary adaptations that plants have undergone to resist biotic stress.

Influence of Half-life and Smoking/Nonsmoking Ratio on Biomarker Consistency between Waves 1 and 2 of the Population Assessment of Tobacco and Health Study.

BACKGROUND: Biomarkers of exposure are tools for understanding the impact of tobacco use on health outcomes if confounders like demographics, use behavior, biological half-life, and other sources of exposure are accounted for in the analysis. METHODS: We performed multiple regression analysis of longitudinal measures of urinary biomarkers of alkaloids, tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, volatile organic compounds (VOC), and metals to examine the sample-to-sample consistency in Waves 1 and 2 of the Population Assessment of Tobacco and Health (PATH) Study including demographic characteristics and use behavior variables of persons who smoked exclusively. Regression coefficients, within- and between-person variance, and intra-class correlation coefficients (ICC) were compared with biomarker smoking/nonsmoking population mean ratios and biological half-lives. RESULTS: Most biomarkers were similarly associated with sex, age, race/ethnicity, and product use behavior. The biomarkers with larger smoking/nonsmoking population mean ratios had greater regression coefficients related to recency of exposure. For VOC and alkaloid metabolites, longer biological half-life was associated with lower within-person variance. For each chemical class studied, there were biomarkers that demonstrated good ICCs. CONCLUSIONS: For most of the biomarkers of exposure reported in the PATH Study, for people who smoke cigarettes exclusively, associations are similar between urinary biomarkers of exposure and demographic and use behavior covariates. Biomarkers of exposure within-subject consistency is likely associated with nontobacco sources of exposure and biological half-life. IMPACT: Biomarkers measured in the PATH Study provide consistent sample-to-sample measures from which to investigate the association of adverse health outcomes with the characteristics of cigarettes and their use.

Exposure to polycyclic aromatic hydrocarbons, volatile organic compounds, and tobacco-specific nitrosamines and incidence of esophageal cancer.

BACKGROUND: Studying carcinogens in tobacco and nontobacco sources may be key to understanding the pathogenesis and geographic distribution of esophageal cancer. METHODS: The Golestan Cohort Study has been conducted since 2004 in a region with high rates of esophageal squamous cell carcinoma. For this nested study, the cases comprised of all incident cases by January 1, 2018; controls were matched to the case by age, sex, residence, time in cohort, and tobacco use. We measured urinary concentrations of 33 exposure biomarkers of nicotine, polycyclic aromatic hydrocarbons, volatile organic compounds, and tobacco-specific nitrosamines. We used conditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals for associations between the 90th vs the 10th percentiles of the biomarker concentrations and incident esophageal squamous cell carcinoma. RESULTS: Among individuals who did not currently use tobacco (148 cases and 163 controls), 2 acrolein metabolites, 2 acrylonitrile metabolites, 1 propylene oxide metabolite, and one 1,3-butadiene metabolite were significantly associated with incident esophageal squamous cell carcinoma (adjusted odds ratios between 1.8 and 4.3). Among tobacco users (57 cases and 63 controls), metabolites of 2 other volatile organic compounds (styrene and xylene) were associated with esophageal squamous cell carcinoma (OR = 6.2 and 9.0, respectively). In tobacco users, 2 tobacco-specific nitrosamines (NNN and N'-Nitrosoanatabine) were also associated with esophageal squamous cell carcinoma. Suggestive associations were seen with some polycyclic aromatic hydrocarbons (especially 2-hydroxynaphthalene) in nonusers of tobacco products and other tobacco-specific nitrosamines in tobacco users. CONCLUSION: These novel associations based on individual-level data and samples collected many years before cancer diagnosis, from a population without occupational exposure, have important public health implications.

Perioperative exposure to volatile organic compounds in neonates undergoing cardiac surgery.

OBJECTIVE: Volatile organic compounds (VOCs) are used in the sterilization and manufacture of medical equipment. These compounds have high vapor pressures with low water solubility and are emitted as gases from solids or liquids. They can be mutagenic, neurotoxic, genotoxic, and/or carcinogenic. Safe limits of exposure are not known for neonates. This study examined determinants of exposure in newborns undergoing cardiac surgery. METHODS: Twenty metabolites of 16 VOCs (eg, xylene, cyanide, acrolein, acrylonitrile, N, N-dimethylformamide, 1,3-butadiene, styrene, and benzene) were measured as metabolites in daily urine samples collected from 10 neonates undergoing cardiac operations (n = 150 samples). Metabolites were quantified using reversed-phase ultra-high performance liquid chromatography and electrospray ionization tandem mass spectrometry. Repeated measures analysis of covariance was performed for each metabolite to examine associations with use of medical devices. RESULTS: At least 3 metabolites were detected in every sample. The median number of metabolites detected in each sample was 14 (range, 3-15). In a model controlling for other factors, the use of extracorporeal membrane oxygenation was associated with significantly (P ≤ .05) greater metabolite levels of acrolein, acrylonitrile, ethylene oxide, propylene oxide, styrene, and ethylbenzene. Patients breathing ambient air had greater levels of metabolites of acrolein, xylene, N,N-dimethylformamide, methyl isocyanate, cyanide, 1,3-butadiene (all P ≤ .05). CONCLUSIONS: Exposure to volatile organic compounds is pervasive in newborns undergoing cardiac surgery. Sources of exposure likely include medical devices and inhalation from the air in the intensive care unit. The contribution of VOC exposure during cardiac surgery in newborns to adverse outcomes warrants further evaluation.

Smoke exposure associated with higher urinary benzene biomarker muconic acid (MUCA) in Golestan Cohort Study participants.

Background. Benzene is a known human carcinogen. Human exposure to benzene can be assessed by measuring trans, trans-muconic acid (MUCA) in urine. Golestan Province in northeastern Iran has been reported to have high incidence of esophageal cancer linked to the use of tobacco products. This manuscript evaluates the urinary MUCA concentrations among the participants of the Golestan Cohort Study (GCS).Methods. We analyzed MUCA concentration in 177 GCS participants' urine samples and performed nonparametric pairwise multiple comparisons to determine statistically significant difference among six different product use groups. Mixed effects model was fitted on 22 participants who exclusively smoked cigarette and 51 participants who were classified as nonusers. The urinary MUCA data were collected at the baseline and approximately five years later, and intraclass correlation coefficient (ICC) was calculated from the model.Results. Compared with nonusers, tobacco smoking was associated with higher urinary MUCA concentrations. Based on the nonparametric test of pairwise multiple comparisons, MUCA concentrations among participants who smoked combusted tobacco products were statistically significantly higher compared to nonusers. Urinary MUCA collected five years apart from the same individuals showed moderate reliability (ICC = 0.41), which was expected given the relatively short half-life (∼6 h) of MUCA.Conclusion. Our study revealed that tobacco smoke was positively associated with increased levels of urinary MUCA concentration, indicating that it is a significant source of benzene exposure among GCS participants.

Defense against phytopathogens relies on efficient antimicrobial protein secretion mediated by the microtubule-binding protein TGNap1.

Plant immunity depends on the secretion of antimicrobial proteins, which occurs through yet-largely unknown mechanisms. The trans-Golgi network (TGN), a hub for intracellular and extracellular trafficking pathways, and the cytoskeleton, which is required for antimicrobial protein secretion, are emerging as pathogen targets to dampen plant immunity. In this work, we demonstrate that tgnap1-2, a loss-of-function mutant of Arabidopsis TGNap1, a TGN-associated and microtubule (MT)-binding protein, is susceptible to Pseudomonas syringae (Pst DC3000). Pst DC3000 infected tgnap1-2 is capable of mobilizing defense pathways, accumulating salicylic acid (SA), and expressing antimicrobial proteins. The susceptibility of tgnap1-2 is due to a failure to efficiently transport antimicrobial proteins to the apoplast in a partially MT-dependent pathway but independent from SA and is additive to the pathogen-antagonizing MIN7, a TGN-associated ARF-GEF protein. Therefore, our data demonstrate that plant immunity relies on TGNap1 for secretion of antimicrobial proteins, and that TGNap1 is a key immunity element that functionally links secretion and cytoskeleton in SA-independent pathogen responses.

Assessment of urinary 6-hydroxy-2,4-cyclohexadienyl mercapturic acid as a novel biomarker of benzene exposure.

Assessing benzene exposure is a public health priority due to its deleterious health effects and ubiquitous industrial and environmental sources of exposure. Phenyl mercapturic acid (PhMA) is a commonly used urinary biomarker to assess benzene exposure. However, recent work has identified significant interlaboratory variation in urinary PhMA concentrations related to methodological differences. In this study, we present urinary 6-hydroxy-2,4-cyclohexadienyl mercapturic acid (pre-PhMA), a metabolite that undergoes acid-catalyzed dehydration to form PhMA, as a novel and specific urinary biomarker for assessing benzene exposure. We developed and validated the first quantitative liquid chromatography-tandem mass spectrometry assay for measuring urinary concentrations of pre-PhMA. The pH effect on the method of ruggedness testing determined that pre-PhMA is stable across the normal human urine pH range and that neutral conditions must be maintained throughout quantification for robust and accurate measurement of urinary pre-PhMA concentrations. The method exhibited below 2 ng/mL sensitivity for pre-PhMA, linearity over three orders of magnitude, and precision and accuracy within 10%. Urinary pre-PhMA concentrations were assessed in 369 human urine samples. Smoking individuals exhibited elevated levels of pre-PhMA compared to non-smoking individuals. Furthermore, the relationship between benzene exposure and urinary pre-PhMA levels was explored by examining the correlation of pre-PhMA with 2-cyanoethyl mercapturic acid, a smoke exposure biomarker. The urinary biomarkers exhibited a positive correlation (r = 0.720), indicating that pre-PhMA levels increased with benzene exposure. The results of this study demonstrate that urinary pre-PhMA is a rugged and effective novel biomarker of benzene exposure that can be widely implemented for future biomonitoring studies.

Immune activation during Pseudomonas infection causes local cell wall remodeling and alters AGP accumulation.

The plant cell boundary generally comprises constituents of the primary and secondary cell wall (CW) that are deposited sequentially during development. Although it is known that the CW acts as a barrier against phytopathogens and undergoes modifications to limit their invasion, the extent, sequence, and requirements of the pathogen-induced modifications of the CW components are still largely unknown, especially at the level of the polysaccharide fraction. To address this significant knowledge gap, we adopted the compatible Pseudomonas syringae-Arabidopsis thaliana system. We found that, despite systemic signaling actuation, Pseudomonas infection leads only to local CW modifications. Furthermore, by utilizing a combination of CW and immune signaling-deficient mutants infected with virulent or non-virulent bacteria, we demonstrated that the pathogen-induced changes in CW polysaccharides depend on the combination of pathogen virulence and the host's ability to mount an immune response. This results in a pathogen-driven accumulation of CW hexoses, such as galactose, and an immune signaling-dependent increase in CW pentoses, mainly arabinose, and xylose. Our analyses of CW changes during disease progression also revealed a distinct spatiotemporal pattern of arabinogalactan protein (AGP) deposition and significant modifications of rhamnogalacturonan sidechains. Furthermore, genetic analyses demonstrated a critical role of AGPs, specifically of the Arabinoxylan Pectin Arabinogalactan Protein1, in limiting pathogen growth. Collectively, our results provide evidence for the actuation of significant remodeling of CW polysaccharides in a compatible host-pathogen interaction, and, by identifying AGPs as critical elements of the CW in plant defense, they pinpoint opportunities to improve plants against diverse pathogens.

Evaluating Exposure to VOCs and Naphthalene for Firefighters Wearing Different PPE Configurations through Measures in Air, Exhaled Breath, and Urine.

Firefighters are at an increased risk of cancer due to their occupational exposure to combustion byproducts, especially when those compounds penetrate the firefighter personal protective equipment (PPE) ensemble. This has led to questions about the impact of base layers (i.e., shorts vs. pants) under PPE ensembles. This study asked 23 firefighters to perform firefighting activities while wearing one of three different PPE ensembles with varying degrees of protection. Additionally, half of the firefighters unzipped their jackets after the scenario while the other half kept their jackets zipped for five additional minutes. Several volatile organic compound (VOC) and naphthalene air concentrations outside and inside of hoods, turnout jackets, and turnout pants were evaluated; biological (urinary and exhaled breath) samples were also collected. VOCs and naphthalene penetrated the three sampling areas (hoods, jackets, pants). Significant (p-value < 0.05) increases from pre- to post-fire for some metabolites of VOCs (e.g., benzene, toluene) and naphthalene were found. Firefighters wearing shorts and short sleeves absorbed higher amounts of certain compounds (p-value < 0.05), and the PPE designed with enhanced interface control features appeared to provide more protection from some compounds. These results suggest that firefighters can dermally absorb VOCs and naphthalene that penetrate the PPE ensemble.

Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Arabidopsis Epoxide Hydrolase Related to Phytohormone Signaling.

Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.

Distinguishing Exposure to Secondhand and Thirdhand Tobacco Smoke among U.S. Children Using Machine Learning: NHANES 2013-2016.

While the thirdhand smoke (THS) residue from tobacco smoke has been recognized as a distinct public health hazard, there are currently no gold standard biomarkers to differentiate THS from secondhand smoke (SHS) exposure. This study used machine learning algorithms to assess which combinations of biomarkers and reported tobacco smoke exposure measures best differentiate children into three groups: no/minimal tobacco smoke exposure (NEG); predominant THS exposure (TEG); and mixed SHS and THS exposure (MEG). Participants were 4485 nonsmoking 3-17-year-olds from the National Health and Nutrition Examination Survey 2013-2016. We fitted and tested random forest models, and the majority (76%) of children were classified in NEG, 16% were classified in TEG, and 8% were classified in MEG. The final classification model based on reported exposure, biomarker, and biomarker ratio variables had a prediction accuracy of 95%. This final model had prediction accuracies of 100% for NEG, 88% for TEG, followed by 71% for MEG. The most important predictors were the reported number of household smokers, serum cotinine, serum hydroxycotinine, and urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In the absence of validated biomarkers specific to THS, comprehensive biomarker and questionnaire data for tobacco smoke exposure can distinguish children exposed to SHS and THS with high accuracy.

Mitigating Matrix Effects in LC-ESI-MS-MS Analysis of a Urinary Biomarker of Xylenes Exposure.

Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) with stable isotope-labeled internal standards (SIL-ISs) is the gold standard for quantitative analysis of drugs and metabolites in complex biological samples. Significant isotopic effects associated with deuterium labeling often cause the deuterated IS to elute at a different retention time from the target analyte, diminishing its capability to compensate for matrix effects. In this study, we systematically compared the analytical performance of deuterated (2H) SIL-IS to non-deuterated (13C and 15N) SIL-ISs for quantifying urinary 2-methylhippuric acid (2MHA) and 4-methylhippuric acid (4MHA), biomarkers of xylenes exposure, with an LC-ESI-MS-MS assay. Analytical method comparison between ISs demonstrated a quantitative bias for urinary 2MHA results, with concentrations generated with 2MHA-[2H7] on average 59.2% lower than concentrations generated with 2MHA-[13C6]. Spike accuracy, measured by quantifying the analyte-spiked urine matrix and comparing the result to the known spike concentration, determined that 2MHA-[2H7] generated negatively biased urinary results of -38.4%, whereas no significant bias was observed for 2MHA-[13C6]. Post-column infusion demonstrated that ion suppression experienced by 2MHA and 2MHA-[13C6] was not equally experienced by 2MHA-[2H7], explaining the negatively biased 2MHA results. The quantitation of urinary 4MHA results between ISs exhibited no significant quantitative bias. These results underscore the importance of the careful selection of ISs for targeted quantitative analysis in complex biological samples.

Firefighters' urinary concentrations of VOC metabolites after controlled-residential and training fire responses.

INTRODUCTION: Firefighters are exposed to volatile organic compounds (VOCs) during structural fire responses and training fires, several of which (e.g., benzene, acrolein, styrene) are known or probable carcinogens. Exposure studies have found that firefighters can absorb chemicals like benzene even when self-contained breathing apparatus (SCBA) are worn, suggesting that dermal absorption contributes to potentially harmful exposures. However, few studies have characterized VOC metabolites in urine from firefighters. OBJECTIVES: We quantified VOC metabolites in firefighters' urine following live firefighting activity across two field studies. METHODS: In two separate controlled field studies, spot urine was collected before and 3 h after firefighters and firefighter students responded to simulated residential and training fires. Urine was also collected from instructors from the training fire study before the first and 3 h after the last training scenario for each day (instructors led three training scenarios per day). Samples were analyzed for metabolites of VOCs to which firefighters may be exposed. RESULTS: In the residential fire study, urinary metabolites of xylenes (2MHA), toluene (BzMA), and styrene (MADA) increased significantly (at 0.05 level) from pre- to post-fire. In the training fire study, MADA concentrations increased significantly from pre- to post-fire for both firefighter students and instructors. Urinary concentrations of benzene metabolites (MUCA and PhMA) increased significantly from pre- to post-fire for instructors, while metabolites of xylenes (3MHA+4MHA) and acrolein (3HPMA) increased significantly for firefighter students. The two highest MUCA concentrations measured post-shift from instructors exceeded the BEI of 500 µg/g creatinine. CONCLUSIONS: Some of the metabolites that were significantly elevated post-fire are known or probable human carcinogens (benzene, styrene, acrolein); thus, exposure to these compounds should be eliminated or reduced as much as possible through the hierarchy of controls. Given stringent use of SCBA, it appears that dermal exposure contributes in part to the levels measured here.

Cavity surface residues of PAD4 and SAG101 contribute to EDS1 dimer signaling specificity in plant immunity.

Arabidopsis pathogen effector-triggered immunity (ETI) is controlled by a family of three lipase-like proteins (EDS1, PAD4, and SAG101) and two subfamilies of HET-S/LOB-B (HeLo)-domain "helper" nucleotide-binding/leucine-rich repeats (ADR1s and NRG1s). EDS1-PAD4 dimers cooperate with ADR1s, and EDS1-SAG101 dimers with NRG1s, in two separate defense-promoting modules. EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 complexes were detected in immune-activated leaf extracts but the molecular determinants for specific complex formation and function remain unknown. EDS1 signaling is mediated by a C-terminal EP domain (EPD) surface surrounding a cavity formed by the heterodimer. Here we investigated whether the EPDs of PAD4 and SAG101 contribute to EDS1 dimer functions. Using a structure-guided approach, we undertook a comprehensive mutational analysis of Arabidopsis PAD4. We identify two conserved residues (Arg314 and Lys380) lining the PAD4 EPD cavity that are essential for EDS1-PAD4-mediated pathogen resistance, but are dispensable for the PAD4-mediated restriction of green peach aphid infestation. Positionally equivalent Met304 and Arg373 at the SAG101 EPD cavity are required for EDS1-SAG101 promotion of ETI-related cell death. In a PAD4 and SAG101 interactome analysis of ETI-activated tissues, PAD4R314A and SAG101M304R EPD variants maintain interaction with EDS1 but lose association, respectively, with helper nucleotide-binding/leucine-rich repeats ADR1-L1 and NRG1.1, and other immune-related proteins. Our data reveal a fundamental contribution of similar but non-identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity.

Changes in Biomarkers of Tobacco Exposure among Cigarette Smokers Transitioning to ENDS Use: The Population Assessment of Tobacco and Health Study, 2013-2015.

Limited data are available for how biomarkers of tobacco exposure (BOE) change when cigarette smokers transition to using electronic nicotine delivery systems (ENDS). Using biomarker data from Waves 1 (2013-2014) and 2 (2014-2015) of the PATH Study, we examined how mean BOE concentrations, including metabolites of nicotine, tobacco-specific nitrosamines (TSNA), polycyclic aromatic hydrocarbons (PAH), and volatile organic compounds (VOC) and metals, changed when 2475 adult smokers transitioned to using ENDS or quit tobacco products. Exclusive smokers who transitioned to dual use had a significant decrease in NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol), but not nicotine metabolites, most PAHs, metals, or VOCs. Exclusive smokers who became dual users had significant reductions in total nicotine equivalents, NNAL, and 2CyEMA (acrylonitrile metabolite), but only in those who reduced cigarettes per day (CPD) by >=50%. Smokers who transitioned to exclusive ENDS use had significant reductions in most TSNAs, PAHs, and VOCs; however, nicotine metabolites did not decrease in dual users who became exclusive ENDS users. Smokers who quit tobacco use had significant decreases in nicotine metabolites, all TSNAs, most PAHs, and most VOCs. Cigarette smokers who became dual users did not experience significant reductions in most BOEs. Reductions were impacted by changes in CPD. However, transitioning from smoking to no tobacco or exclusive ENDS use was associated with reduced exposure to most BOEs measured. Future analyses could incorporate additional waves of PATH data and examine changes in biomarker exposure by ENDS device type and CPD.

Cigarette smoking is associated with acrylamide exposure among the U.S. population: NHANES 2011-2016.

2-carbamoylethyl mercapturic acid (2CaEMA, N-Acetyl-S-carbamoylethyl-L-cysteine) is a urinary metabolite and exposure biomarker of acrylamide, which is a harmful volatile organic compound found in cigarette smoke and in some foods. The goal of this study was to determine the association between cigarette smoking and urinary 2CaEMA concentrations among the U.S. population while considering potential dietary sources of acrylamide intake and demographics. We measured 2CaEMA concentrations in urine specimens collected during the National Health and Nutrition Examination Survey 2011-2012, 2013-2014, and 2015-2016 cycles from eligible participants 18 years and older (n = 5443) using liquid chromatography/tandem mass spectrometry. We developed multiple regression models with urinary 2CaEMA concentrations as the dependent variable and sex, age, race/Hispanic origin, reported primary sources of dietary acrylamide intake, and cigarette smoke exposure as independent variables. This study demonstrates that cigarette smoking is strongly associated with urinary 2CaEMA, suggests that cigarette smoking is likely a primary source of acrylamide exposure, and provides a baseline measure for 2CaEMA in the U.S. population.

Optimal Cutoff Concentration of Urinary Cyanoethyl Mercapturic Acid for Differentiating Cigarette Smokers From Nonsmokers.

INTRODUCTION: Cotinine is a widely used biomarker for classifying cigarette smoking status. However, cotinine does not differentiate between the use of combustible and noncombustible tobacco products. The increasing use of noncombustible tobacco drives the need for a complementary biomarker for distinguishing cigarette smokers from users of noncombustible tobacco products. AIMS AND METHODS: We evaluated the urinary acrylonitrile metabolite, 2CyEMA, as a biomarker of exposure to cigarette smoke in the US population-representative data from the National Health and Nutritional Examination Survey (NHANES). Smoking status was categorized based on the recent tobacco use questionnaire. The receiver operating characteristic (ROC) curve analysis was performed to identify optimal cutoff concentrations by maximizing Youden's J index. The area under the curve (AUC) was used to compare 2CyEMA effectiveness with respect to serum cotinine. RESULTS: The overall cutoff concentration for the classification of cigarette smokers from nonsmokers was 7.32 ng/ml with high sensitivity and specificity (≥0.925). When stratified by demographic variables, the cutoff concentrations varied among subgroups based on age, sex, and race/Hispanic origin. Non-Hispanic Blacks had the highest cutoff concentration (15.3 ng/ml), and Hispanics had the lowest (4.63 ng/ml). Females had higher cutoff concentrations (8.80 ng/ml) compared to males (6.10 ng/ml). Among different age groups, the cutoff concentrations varied between 4.63 ng/ml (21-39 years old) and 10.6 ng/ml (for ≥60 years old). We also explored the creatinine adjusted cutoff values. CONCLUSIONS: 2CyEMA is an effective biomarker for distinguishing cigarette smokers from nonsmokers (users of noncombustible tobacco products or nonusers). IMPLICATIONS: Distinguishes smokers from noncombustible tobacco product users.

Characterization of the association between cigarette smoking intensity and urinary concentrations of 2-hydroxyethyl mercapturic acid among exclusive cigarette smokers in the National Health and Nutrition Examination Survey (NHANES) 2011-2016.

BACKGROUND: 2-Hydroxyethyl mercapturic acid (2HEMA, N-acetyl-S-(2-hydroxyethyl)-L-cysteine) is a urinary metabolite of several volatile organic compounds including acrylonitrile and ethylene oxide, which are found in cigarette smoke. METHODS: We measured 2HEMA concentrations in urine specimens collected during the National Health and Nutrition Examination Survey (2011-2016) from eligible participants aged >12 years (N = 7,416). We developed two multiple linear regression models to characterize the association between cigarette smoking and 2HEMA concentrations wherein the dependent variable was 2HEMA concentrations among participants who exclusively smoked cigarettes at the time of specimen collection and the independent variables included sex, age, race/ethnicity, creatinine, diet, and either cigarettes smoked per day (CPD) or serum cotinine. RESULTS: We detected 2HEMA in 85% of samples tested among exclusive cigarette smokers, and only 40% of specimens from non-smokers. When compared to exclusive cigarette smokers who smoked 1-9 CPD, smoking 10-19 CPD was associated with 36% higher 2HEMA (p < 0.0001) and smoking >19 CPD was associated with 61% higher 2HEMA (p < 0.0001). Additionally, 2HEMA was positively associated with serum cotinine. CONCLUSIONS: This study demonstrates that cigarette smoking intensity is associated with higher urinary 2HEMA concentrations and is likely a major source of acrylonitrile and/or ethylene oxide exposure.

Associations between Biomarkers of Exposure and Lung Cancer Risk among Exclusive Cigarette Smokers in the Golestan Cohort Study.

Biomarkers of tobacco exposure are known to be associated with disease risk but previous studies are limited in number and restricted to certain regions. We conducted a nested case-control study examining baseline levels and subsequent lung cancer incidence among current male exclusive cigarette smokers in the Golestan Cohort Study in Iran. We calculated geometric mean biomarker concentrations for 28 matched cases and 52 controls for the correlation of biomarker levels among controls and for adjusted odds' ratios (ORs) for lung cancer incidence by biomarker concentration, accounting for demographic characteristics, smoking quantity and duration, and opium use. Lung cancer cases had higher average levels of most biomarkers including total nicotine equivalents (TNE-2), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 3-hydroxyfluorene (3-FLU). Many biomarkers correlated highly with one another including TNE-2 with NNAL and N-Acetyl-S-(2-cyanoethyl)-L-cysteine (2CYEMA), and N-Acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine (t4HBEMA) with N-Acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (3HMPMA) and N-Acetyl-S-(4-hydroxy-2-methyl-2-buten-1-yl)-L-cysteine (4HMBEMA). Lung cancer risk increased with concentration for several biomarkers, including TNE-2 (OR = 2.22, 95% CI = 1.03, 4.78) and NNN (OR = 2.44, 95% CI = 1.13, 5.27), and estimates were significant after further adjustment for demographic and smoking characteristics for 2CYEMA (OR = 2.17, 95% CI = 1.03, 4.55), N-Acetyl-S-(2-carbamoylethyl)-L-cysteine (2CAEMA) (OR = 2.14, 95% CI = 1.01, 4.55), and N-Acetyl-S-(2-hydroxypropyl)-L-cysteine (2HPMA) (OR = 2.85, 95% CI = 1.04, 7.81). Estimates were not significant with adjustment for opium use. Concentrations of many biomarkers were higher at the baseline for participants who subsequently developed lung cancer than among the matched controls. Odds of lung cancer were higher for several biomarkers including with adjustment for smoking exposure for some but not with adjustment for opium use.