Novel Biomarkers Of Exposure Research Articles

Unmasking metabolic disruptors: The NEMESIS project's quest for Novel Biomarkers, Evidence on Adverse Effects, and Efficient Methodologies

Metabolism disrupting chemicals (MDCs) elicit negative effects on metabolically active organs such as the liver and the pancreas, altering normal metabolic processes. Chemicals that are known, or suspected MDCs include compounds found in everyday consumer products and food, making low-dose, continuous exposure inevitable for humans. Through the discovery of chemically induced metabolic disruption, a concern has surfaced whether and how MDCs impact human health and the development of metabolic diseases. This has accelerated research around the topic, and it has been found that exposure to MDCs is linked to increased incidence of metabolic diseases including obesity and liver steatosis. Effective regulatory action is hindered by the lack of accurate methods to identify MDCs. The NEMESIS project addresses this regulatory gap by investigating the mechanisms through which MDCs cause metabolic disruption. The project aims at identifying novel biomarkers of exposure and link exposure to disease outcomes. As chemical toxicity testing is rapidly moving towards new approach methodologies (NAMs), NEMESIS promotes non-animal methodologies by employing state-of-the-art in vitro methods, epidemiological data, systems biology approaches, and seeks to replace mammalian in vivo experiments with alternative models. By understanding mechanisms of MDC-induced metabolic health effects, and through the development of reliable effect biomarkers and testing strategies, the NEMESIS project aims to facilitate more effective regulatory measures to improve and protect the health and well-being of EU citizens. The project is particularly focused on maximizing its impact through effective dissemination and communication efforts, to ensure that the project’s message and results reach a broad audience and are tailored to different population groups. These actions will improve the risk assessment of MDCs and ensure that the EU citizens are informed and protected from the harmful effects of MDCs and can adapt their consumer patterns and behaviors to prevent exposure.

Mercaptans in malodorants break disulfide bridges in human serum albumin and form adducts suitable as biomarkers of exposure in vitro.

Malodorants comprise notoriously smelling mercaptans and might be applied for crowd control. Because exposure to malodorants may lead to irritation of the respiratory system, choking, and coma, bioanalytical verification of poisoning might be required in a medical and forensic context. We herein present the detection and identification of novel biomarkers of exposure to ethyl mercaptan, n-butyl mercaptan, tert-butyl mercaptan, and iso-amyl mercaptan. These alkyl thiol compounds were found to form disulfide adducts in human serum albumin (HSA) in plasma in vitro with the only non-disulfide-bridged Cys34 residue and with other residues being part of the disulfide-bridged pattern in HSA. After proteinase K-catalyzed proteolysis, adducts of all mercaptans were detected simultaneously as the tripeptide Cys34*ProPhe and the dipeptides Cys369*Tyr, ValCys316*, and Cysx*Ala (x denominates either Positions 91, 200, 253, 361, and/or 448) by a sensitive micro-liquid chromatography-electrospray ionization tandem mass spectrometry (μLC-ESI MS/MS) method working in the scheduled multiple reaction monitoring (sMRM) mode. Time- and concentration-dependent adduct formations while exposure and proteolysis were investigated and the suitability of adducts as biomarkers of exposure was elaborated. Adducts at Cys34 showed the lowest limits of identification (LOIs, 6nM to 1.2μM mercaptan in plasma) and superior stability in plasma at 37°C. Therefore, Cys34*ProPhe appears as the most promising target to prove exposure to mercaptans at least in vitro.

Air Pollution and microRNAs: The Role of Association in Airway Inflammation.

Air pollution exposure plays a key role in the alteration of gene expression profiles, which can be regulated by microRNAs, inducing the development of various diseases. Moreover, there is also evidence of sensitivity of miRNAs to environmental factors, including tobacco smoke. Various diseases are related to specific microRNA signatures, suggesting their potential role in pathophysiological processes; considering their association with environmental pollutants, they could become novel biomarkers of exposure. Therefore, the aim of the present work is to analyse data reported in the literature on the role of environmental stressors on microRNA alterations and, in particular, to identify specific alterations that might be related to the development of airway diseases so as to propose future preventive, diagnostic, and therapeutic strategies.

Realistic biomarkers from plasma extracellular vesicles for detection of beryllium exposure

PurposeExposures related to beryllium (Be) are an enduring concern among workers in the nuclear weapons and other high-tech industries, calling for regular and rigorous biological monitoring. Conventional biomonitoring of Be in urine is not informative of cumulative exposure nor health outcomes. Biomarkers of exposure to Be based on non-invasive biomonitoring could help refine disease risk assessment. In a cohort of workers with Be exposure, we employed blood plasma extracellular vesicles (EVs) to discover novel biomarkers of exposure to Be.MethodsEVs were isolated from plasma using size-exclusion chromatography and subjected to mass spectrometry-based proteomics. A protein-based classifier was developed using LASSO regression and validated by ELISA.ResultsWe discovered a dual biomarker signature comprising zymogen granule protein 16B and putative protein FAM10A4 that differentiated between Be-exposed and -unexposed subjects. ELISA-based quantification of the biomarkers in an independent cohort of samples confirmed higher expression of the signature in the Be-exposed group, displaying high predictive accuracy (AUROC = 0.919). Furthermore, the biomarkers efficiently discriminated high- and low-exposure groups (AUROC = 0.749).ConclusionsThis is the first report of EV biomarkers associated with Be exposure and exposure levels. The biomarkers could be implemented in resource-limited settings for Be exposure assessment.

DNA Methylation in Peripheral Blood: Providing Novel Biomarkers of Exposure and Immunity to Examine Cancer Risk.

DNA methylation is an epigenetic phenomenon that can alter and control gene expression. Because methylation plays a key role in cell differentiation, methylation markers have been identified that are unique to a given cell type; these markers are stable and can be measured in tissue or whole blood. The article by Katzke and colleagues, published in this issue, uses methylation markers to estimate proportions of immune cell subtypes in peripheral blood samples that were collected prior to diagnosis, thus allowing them to directly examine associations with pancreatic cancer risk. Given that immune-cell counts cannot be measured from archived blood, and that retrospective case-control studies rely on blood that is collected after cancer diagnosis, few studies have been able to examine the role of the systemic immune response in cancer risk. Measurement of DNA methylation in peripheral blood, primarily through development of whole-genome approaches, has also opened new doors to examining cancer etiology.See related article by Katzke et al., p. 2179.

The exposome in practice: an exploratory panel study of biomarkers of air pollutant exposure in Chinese people aged 60-69 years (China BAPE Study).

The exposome overhauls conventional environmental health impact research paradigms and provides a novel methodological framework that comprehensively addresses the complex, highly dynamic interplays of exogenous exposures, endogenous exposures, and modifiable factors in humans. Holistic assessments of the adverse health effects and systematic elucidation of the mechanisms underlying environmental exposures are major scientific challenges with widespread societal implications. However, to date, few studies have comprehensively and simultaneously measured airborne pollutant exposures and explored the associated biomarkers in susceptible healthy elderly subjects, potentially resulting in the suboptimal assessment and management of health risks. To demonstrate the exposome paradigm, we describe the rationale and design of a comprehensive biomarker and biomonitoring panel study to systematically explore the association between individual airborne exposure and adverse health outcomes. We used a combination of personal monitoring for airborne pollutants, extensive human biomonitoring, advanced omics analysis, confounding information, and statistical methods. We established an exploratory panel study of Biomarkers of Air Pollutant Exposure in Chinese people aged 60-69years (China BAPE), which included 76 healthy residents from a representative community in Jinan City, Shandong Province. During the period between September 2018 and January 2019, we conducted prospective longitudinal monitoring with a 3-day assessment every month. This project: (1) leveraged advanced tools for personal airborne exposure monitoring (external exposures); (2) comprehensively characterized biological samples for exogenous and endogenous compounds (e.g., targeted and untargeted monitoring) and multi-omics scale measurements to explore potential biomarkers and putative toxicity pathways; and (3) systematically evaluated the relationships between personal exposure to air pollutants, and novel biomarkers of exposures and effects using exposome-wide association study approaches. These findings will contribute to our understanding of the mechanisms underlying the adverse health impacts of air pollution exposures and identify potential adverse clinical outcomes that can facilitate the development of effective prevention and targeted intervention techniques.

1,2-Propylene Glycol: A Biomarker of Exposure Specific to e-Cigarette Consumption

Over the past decade, new emerging tobacco and nicotine-delivery products have changed the tobacco landscape. Especially, electronic cigarettes (ECs) have been suggested to be considered for tobacco harm reduction, reinforcing the need to identify novel biomarkers of exposure (BoE) specific to the EC use as this would complement exposure assessment and product compliance monitoring. Therefore, a sensitive LC-MS/MS method for the quantification of 1,2-propylene glycol (PG) and glycerol (G), the main e-liquid constituents, was established. PG and G were analyzed in plasma and urine samples from a clinical study comparing five nicotine product user groups, users of combustible cigarettes (CC), electronic cigarettes (EC), heated tobacco products (HTP), oral tobacco (OT), and oral/dermal nicotine delivery products (used for nicotine replacement therapy, NRT) with a control group of non-users (NU). Data demonstrate significantly elevated PG levels in urine and plasma in EC users compared to users of CC, HTP, NRT, OT as well as NU. In addition, PG in plasma and urine of vapers significantly correlated with nicotine (plasma) and total nicotine equivalents (urine), biomarkers reflecting product consumption, emphasizing the high specificity of PG as a BoE for EC consumption. We therefore suggest the use of PG as BoE in urine and/or plasma in order to monitor EC use compliance in exposure assessments.

Urinary carboxylic acid metabolites as possible novel biomarkers of exposures to alkylated polycyclic aromatic hydrocarbons

Previous studies have found that alkylated polycyclic aromatic hydrocarbons (alkyl-PAHs) were more abundant in petrogenic sources (e.g., crude oil and its refined products) than pyrogenic sources of incomplete combustion. While urinary hydroxylated metabolites of unsubstituted PAHs have been widely used as biomarkers of PAHs exposures, little information is available as to the occurrence of alkyl-PAH metabolites. In this study, we have detected carboxylic acid metabolites of alkyl-naphthalene (2-NAPCA) and alkyl-phenanthrene (2-PHECA) in 314 urine samples repeatedly collected from 45 Los Angeles residents before, during, and after they spent ten weeks in Beijing in summers of 2014–2017. We found that traveling from Los Angeles to Beijing led to 348% (95% CI: 243 to 485%) and 209% (95% CI: 149 to 282%) increases in 2-NAPCA and 2-PHECA concentrations, respectively, which returned to baseline levels after participants came back to Los Angeles. The concentration ratio between 2-PHECA and hydroxy-phenanthrenes was significantly (p < 0.05) lower in Beijing (median: 0.40, IQR: 0.27–0.53) than in Los Angeles (median: 0.51, IQR: 0.32–0.77), where more than 5,000 active gas and oil wells were located. From 2014 to 2017, the concentration ratio of 2-PHECA to hydroxy-phenanthrenes increased by 28.7 (95%CI: 12.3 to 47.6) %/yr in Los Angeles and 18.6 (95%CI: 7.9 to 30.3) %/yr in Beijing, likely resulted from both cities’ efforts to reduce pyrogenic emissions (e.g. vehicle exhaust). These results provided indirect evidence supporting the use of 2-PHECA to hydroxy-phenanthrene ratio as an index to reflect the relative exposure contributions from petrogenic and pyrogenic sources. While our study suggested that urinary PAHCAs may be novel biomarkers of exposure to alkyl-PAHs, future studies with external exposure characterization are warranted to further validate these biomarkers.

Identification of creatine kinase and alpha-1 antitrypsin as protein targets of alkylation by sulfur mustard.

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two-dimensional-thiol-differences in gel electrophoresis (2D-thiol-DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared-maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide-thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D-thiol-DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha-1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys282 of CK and Cys232 of A1AT were unambiguously identified by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl-(HETE)-moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.

Regulation of Hepatic Long Noncoding RNAs by Pregnane X Receptor and Constitutive Androstane Receptor Agonists in Mouse Liver.

Altered expression of long noncoding RNAs (lncRNAs) by environmental chemicals modulates the expression of xenobiotic biotransformation-related genes and may serve as therapeutic targets and novel biomarkers of exposure. The pregnane X receptor (PXR/NR1I2) is a critical xenobiotic-sensing nuclear receptor that regulates the expression of many drug-processing genes, and it has similar target-gene profiles and DNA-binding motifs with another xenobiotic-sensing nuclear receptor, namely, constitutive andronstrane receptor (CAR/Nr1i3). To test our hypothesis that lncRNAs are regulated by PXR in concert with protein-coding genes (PCGs) and to compare the PXR-targeted lncRNAs with CAR-targeted lncRNAs, RNA-Seq was performed from livers of adult male C57BL/6 mice treated with corn oil, the PXR agonist PCN, or the CAR agonist 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Among 125,680 known lncRNAs, 3843 were expressed in liver, and 193 were differentially regulated by PXR (among which 40% were also regulated by CAR). Most PXR- or CAR-regulated lncRNAs were mapped to the introns and 3'-untranslated regions (UTRs) of PCGs, as well as intergenic regions. Combining the RNA-Seq data with a published PXR chromatin immunoprecipitation coupled with high-throughput sequencing; cytochrome P450 (P450; ChIP-Seq) data set, we identified 774 expressed lncRNAs with direct PXR-DNA binding sites, and 26.8% of differentially expressed lncRNAs had changes in PXR-DNA binding after PCN exposure. De novo motif analysis identified colocalization of PXR with liver receptor homolog (LRH-1), which regulates bile acid synthesis after PCN exposure. There was limited overlap of PXR binding with an epigenetic mark for transcriptional activation (histone-H3K4-di-methylation, H3K4me2) but no overlap with epigenetic marks for transcriptional silencing [H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation]. Among differentially expressed lncRNAs, 264 were in proximity of PCGs, and the lncRNA-PCG pairs displayed a high coregulatory pattern by PXR and CAR activation. This study was among the first to demonstrate that lncRNAs are regulated by PXR and CAR activation and that they may be important regulators of PCGs involved in xenobiotic metabolism.

Serum miR-375-3p increase in mice exposed to a high dose of ionizing radiation

Exposure to high-doses of ionizing radiation (IR) leads to development of a strong acute radiation syndrome (ARS) in mammals. ARS manifests after a latency period and it is important to develop fast prognostic biomarkers for its early detection and assessment. Analysis of chromosomal aberrations in peripheral blood lymphocytes is the gold standard of biological dosimetry, but it fails after high doses of IR. Therefore, it is important to establish novel biomarkers of exposure that are fast and reliable also in the high dose range. Here, we investigated the applicability of miRNA levels in mouse serum. We found significantly increased levels of miR-375-3p following whole body exposure to 7 Gy of X-rays. In addition, we analyzed their levels in various organs of control mice and found them to be especially abundant in the pancreas and the intestine. Following a dose of 7 Gy, extensive cell death occurred in these tissues and this correlated negatively with the levels of miR-375-3p in the organs. We conclude that high expressing tissues of miR-375-3p may secrete this miRNA in serum following exposure to 7 Gy. Therefore, elevated miR-375-3p in serum may be a predictor of tissue damage induced by exposure to a high radiation dose.

Application of cDNA-AFLP to biomarker exploration in a non-model species Grandidierella japonica

Biomarkers of exposure can be used to identify specific contaminants that are adversely affecting aquatic organisms. However, it remains prohibitively costly to investigate multiple novel biomarkers of exposure in a non-model species, despite the development of next-generation sequencing technology. In this study, we focused on the use of cDNA-amplified fragment length polymorphism (AFLP) as a cost-effective biomarker discovery tool to test whether it could identify biomarkers of exposure in the non-model amphipod species Grandidierella japonica. Loci were identified that were differentially expressed in amphipods exposed to reference chemicals (Cu, Zn, and nicotine) and to an environmental sample (road dust) at sublethal concentrations. Eight loci were shown to respond consistently to nicotine at different concentrations, but not to Cu or Zn. Some of the loci also responded to an environmental road dust sample containing nicotine. These findings suggest that loci identified using cDNA-AFLP could be used as biomarkers of nicotine exposure in environmental samples with complex matrices. Further studies with other organisms and toxicants are needed, but we have demonstrated that the use of cDNA-AFLP to identify biomarkers for ecotoxicological studies of non-model species is at least feasible.

The use of omics-based approaches in regulatory toxicology: an alternative approach to assess the no observed transcriptional effect level

The evaluation of chemical hazard is based on the identification of the quality and the quantity of adverse effects as a consequence of exposure. The adverse effects that do not involve genetic damage are often related to the chemical dose or concentration. The adverse outcome is the consequence of a row of key events, each targeting a different biological trait. The identification of these key events at molecular and cellular level would provide novel biomarkers of exposure and risk. The application of toxicogenomics approaches to experimental models of chemical exposure allows the detection of gene pathways involved in response to low doses of the chemical as an early endpoint of adversity. The use of toxicogenomics would improve the knowledge on the dose-response relationship, linking the environmental exposure to the effect on the population and allowing a better refinement of the quantitative risk assessment. In this context, the gene modulation data can be used to calculate a No Observed Transcriptional Effect Level (NOTEL).In this paper we present a method for evaluating the NOTEL based on anomaly detection: a classifier is built that discriminates between target class instances, i.e., normal cases, and anomalies, i.e., samples with significant transcriptional effects. The strength of this method is that (i) it can be applied to cases in which few samples are available and the space dimension is high and (ii) it makes use of the complete gene expression profiles.

Biomarker responses to estrogen and androgen exposure in the brook stickleback (Culaea inconstans): A new bioindicator species for endocrine disrupting compounds

Small-bodied freshwater fish are commonly used in regulatory testing for endocrine disrupting compounds (EDCs) but most lack a sensitive and quantifiable androgen-specific biomarker. Brook stickleback (Culaea inconstans) are a North American freshwater fish whose males produce an androgen-regulated glycoprotein in the kidney called spiggin. Although spiggin induction in females has been used as an androgen-specific biomarker of exposure in other stickleback species it has not been characterized in brook stickleback. Therefore, our objective was to develop a bioassay using brook stickleback to measure estrogenic and androgenic responses and establish the sensitivity of traditional and novel biomarkers of exposure. We first developed and optimized a qPCR assay to measure spiggin and vitellogenin transcript levels in kidney and liver tissue, respectively. Basal levels were differentially expressed in mature wild-caught male and female brook stickleback. To determine their sensitivity to EDCs, fish were exposed to nominal concentrations of 1, 10 and 100ng/L of 17α-methyltestosterone (MT) or 17α-ethinylestradiol (EE2) for 21days (sampled at 7 and 21days) under semi-static renewal conditions. MT and EE2 exposure induced spiggin and vitellogenin transcripts in female kidneys and male livers, respectively. Exposure to EE2 also increased hepatosomatic index in both sexes and decreased gonadosomatic index in females. Histopathological alterations were observed in the kidney of EE2-exposed fish and an increase in kidney epithelium cell height occurred in MT-exposed females. Given the sensitivity of these endpoints, the brook stickleback is a promising new freshwater fish model for EDC evaluation and a potential bioindicator for EDCs in North American freshwater environments.

A cytogenetic study of hospital workers occupationally exposed to radionuclides in Serbia: premature centromere division as novel biomarker of exposure?

The health risk of chronic exposure to radionuclides includes changes in the genome (e.g., chromosomal aberrations and micronuclei) that increase chromosomal instability. There are also other phenomena, which seem to appear more frequently in metaphases of exposed persons (such as premature centromere division). The aim of this study was to discover whether or not there is correlation between incidence of named cytogenetic changes in persons occupationally exposed to radionuclides in comparison with unexposed control group, and if significant correlation is determined, can premature centromere division be consider as a biomarker of radiation exposure? The exposed group comprised 50 individuals occupationally exposed to radionuclides. The reference control group consisted of 40 unexposed individuals. Chromosomal aberrations, micronuclei and premature centromere division were analyzed according to a standard International Atomic Energy Agency protocol. Statistical analyses were performed using SPSS 17.0 statistics. The means for analyzed cytogenetic changes were significantly higher in the exposed group. Positive correlation between them was found in exposed group. Premature centromere division parameter PCD5-10 was selected as particularly suitable for separating groups (exposed/unexposed). Identification of other phenomena related to radionuclide exposure, beside well known, may clarify recent problems in radiobiology concerning the biological response to low doses of ionizing radiation and its consequences.

The EPA/NIEHS Children’s Environmental Health and Disease Prevention Research Centers: More Than 15 Years of Innovative Research on Important Challenges in Children’s Environmental Health

The EPA/NIEHS Children’s Environmental Health and Disease Prevention Research Centers: More Than 15 Years of Innovative Research on Important Challenges in Children’s Environmental HealthAbstract Number:2798 Kimberly Gray* and Nica Louie* Kimberly Gray* U.S. Environmental Protection Agency, United States, E-mail Address: [email protected] Search for more papers by this author and Nica Louie* U.S. Environmental Protection Agency, United States, E-mail Address: [email protected] Search for more papers by this author AbstractBackground: The U.S. Environmental Protection Agency (EPA) and the National Institute of Environmental Health Sciences (NIEHS) have jointly supported the Children's Environmental Health and Disease Prevention Research Centers (‘Children’s Centers’) program since 1998, forming a highly successful and collaborative, interdisciplinary research network.Methods: These multidisciplinary, translational research centers are investigating the role of a wide range of environmental exposures in adverse children's health outcomes and how to protect children's health. Studies include how exposure to chemicals such as ambient air pollutants, arsenic in water and food, endocrine-disrupting compounds (EDCs) including bisphenol A (BPA), manganese, organophosphate pesticides and polybrominated flame retardants may, in combination with other factors such as social and behavioral factors and genetic susceptibility, result in adverse birth and health outcomes including asthma, autism, childhood leukemia, changes in epigenetics/gene expression, changes in neurodevelopment and immune system function -- and how to prevent adverse health outcomes. The Children's Centers are using approaches including longitudinal cohort and case-control studies and environmental epidemiology in conjunction with laboratory-based studies to find novel biomarkers of exposure, early developmental and pubertal effects and gene-environment interactions. Community engagement is a key part of the program.Results: This presentation will review some of the important findings from the program over the past 15 years and summarize some of the research currently being funded.Conclusion: Program grantees have published over 1,000 peer-reviewed publications, some highly cited in high-impact journals and cited in environmental policy. Research from the EPA/NIEHS Children’s Centers program is having a significant impact on the field of children’s environmental health and in protecting children's health.

Application of CE-MS to a metabonomics study of human urine from cigarette smokers and non-smokers.

Novel biomarkers of exposure and early adverse effects are needed for comparative studies of combustible and non-combustible tobacco products for regulatory authority evaluation. Metabolic biomarkers reflect both gene and environmental effects. CE-MS has been applied to human urine samples from non-smokers and smokers of cigarettes at two tar levels. Validated chemometric models were able to separate smokers from non-smokers, with discrimination mainly based on the presence of nicotine metabolites. With these removed, it still proved possible to discriminate smokers from non-smokers with models now based on endogenous metabolites. The biochemical relevance of these biomarkers is discussed. This proof-of-principle metabonomics study illustrates the potential of CE-MS to discover novel biomarkers in urine from tobacco users.

Urine Menthol as a Biomarker of Mentholated Cigarette Smoking

Menthol cigarettes are smoked by 27% of U.S. smokers, and there are concerns that menthol might enhance toxicity of cigarette smoking by increasing systemic absorption of smoke toxins. We measured urine menthol concentrations in relation to biomarkers of exposure to nicotine and tobacco carcinogens. Concentrations of menthol glucuronide (using a novel analytical method), nicotine plus metabolites (nicotine equivalents, NE), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and polycyclic aromatic hydrocarbon (PAH) metabolites were measured in the urine of 60 menthol and 67 regular cigarette smokers. Urine menthol was measurable in 82% of menthol and 54% in regular cigarette smokers. Among menthol smokers, urine menthol was highly correlated with NE, NNAL, and PAHs. In a multiple regression model NE but not menthol was significantly associated with NNAL and PAHs. Urine menthol concentration is a novel biomarker of exposure in menthol cigarette smokers, and is highly correlated with exposure to nicotine and carcinogens. Menthol is not independently associated with carcinogen exposure when nicotine intake is considered.

Glutathione pathway in ethylbenzene metabolism: Novel biomarkers of exposure in the rat

Glutathione pathway was specifically studied in rats exposed by inhalation to a range of ethylbenzene vapours (5–2000 ppm). Urines were collected during exposure (6 h) and over the 18 h following the exposure. The potential metabolites coming from either side-chain or ring oxidation were synthesized: 1-, 2-phenylethylmercapturic acids (1-, and 2-PEMA) and 2-, 3- and 4-ethylphenylmercapturic acids (2-, 3-, and 4-EPMA). Their synthesis was fully described and the molecules characterized. Urine samples were analysed using a selective HPLC–fluorescence method. Among the five metabolites, 2-PEMA was never observed in any urine sample. By contrast, 1-PEMA was discovered in its two diastereomeric forms, and it was shown that one of them was mainly present. 2-EPMA, 3-EPMA and 4-EPMA (in the ratio 1:2:6) were also found, and their combined excretion levels were similar to that of 1-PEMA. The atmospheric concentrations and urinary excretions yielded very close correlations which allow us to consider these mercapturic acids as novel ethylbenzene exposure biomarkers.

Repeated Measures of Validated and Novel Biomarkers of Exposure to Polycyclic Aromatic Hydrocarbons in Individuals Living Near an Aluminum Plant in Quebec, Canada

A longitudinal biomonitoring study was conducted to assess exposure to polycyclic aromatic hydrocarbons (PAH) in non-occupationally exposed nonsmoking adults living in the vicinity of an aluminum plant. Metabolites of several PAH (pyrene, naphthalene, chrysene, fluoranthene, benz[a]anthracene) were measured in the urine of the participants, including 1-hydroxypyrene (1-OHP) as a validated biomarker and pyrene diones as novel biomarkers. In total, 73 individuals living about 1 km away from the plant (taken as the exposed group) were compared repeatedly with 71 individuals living at least 11 km from the smelter (used as the control group). Complete first morning voids were collected twice, at a 2-wk interval, in the fall of 2005 and twice weekly for 2 consecutive weeks in the spring of 2006. Urinary biomarker concentrations were then measured by an ultra-performance liquid chromatography (UPLC) method with time-of-flight mass spectrometry detection (MS-TOF) (UPLC-MS-TOF). For most sampling days, individuals living near the plant showed significantly higher excretion values of both 1-OHP and pyrene diones (mean ratio up to 2- and 2.4-fold, respectively) than individuals living further from the plant. In the group living near the plant, geometric mean concentrations of 1-OHP varied from 0.047 to 0.058 μmol/mol creatinine, depending on the sampling day, as compared to 0.025 to 0.04 μmol/mol creatinine in the reference group. Corresponding mean values for pyrene diones were 0.017–0.056 μmol/mol creatinine and 0.014–0.039 μmol/mol creatinine, respectively. Urinary 1- and 2-naphthols were also measured as a reference and showed no significant differences between the two groups for most sampling days; metabolite concentrations of the other monitored PAH (chrysene, fluoranthene, benz[a]anthracene) were mostly below the analytical limit of detection of 0.005 to 0.01 μg/L, depending on the metabolite, with a detection rate varying from 0 to at most 21%. Individuals living near the aluminum plant thus appeared to be repeatedly exposed to higher pyrene levels than the control group, on the basis of both 1-OHP and pyrene dione excretions. However, 1-OHP concentrations observed in this first group were similar to those of other reference populations of nonsmokers studied in the past. Uptake of the other PAH associated with plant emissions was too small to significantly increase the excretion of their metabolites.