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Mycotoxins are a heterogeneous group of toxins produced by fungi that can grow in staple crops (e.g., maize, cereals), resulting in health risks due to widespread exposure from human consumption and inhalation. Dried blood spot (DBS), dried serum spot (DSS), and volumetric tip microsampling (VTS) assays were developed and validated for several important mycotoxins. This review summarizes studies that have developed these assays to monitor mycotoxin exposures in human biological samples and highlights future directions to facilitate minimally invasive sampling techniques as global public health tools. A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted. Key assay performance metrics were extracted to provide a critical review of the available methods. This search identified 11 published reports related to measuring mycotoxins (ochratoxins, aflatoxins, and fumonisins) using DBS/DSS and VTS assays. Multimycotoxin assays adapted for DBS/DSS and VTS have undergone sufficient laboratory validation for applications in large-scale population health and human biomonitoring studies. Future work should expand the number of mycotoxins that can be measured in multimycotoxin assays, continue to improve multimycotoxin assay sensitivities of several biomarkers with low detection rates, and validate multimycotoxin assays across diverse populations with varying exposure levels. Validated low-cost and ultrasensitive minimally invasive sampling methods should be deployed in human biomonitoring and public health surveillance studies to guide policy interventions to reduce inequities in global mycotoxin exposures.

Background: Dried blood spot (DBS) sampling is a simple, cost-effective, and minimally invasive alternative to venipuncture for measuring exposure biomarkers in public health and epidemiological research. DBS sampling provides advantages in field-based studies conducted in low-resource settings and in studies involving infants and children. In addition, DBS samples are routinely collected from newborns after birth (i.e., newborn dried blood spots, NDBS), with many states in the United States permitting access to archived NDBS samples for research purposes., Objectives: We review the state of the science for analyzing exposure biomarkers in DBS samples, both archived and newly collected, and provide guidance on sample collection, storage, and blood volume requirements associated with individual DBS assays. We discuss recent progress regarding analytical methods, analytical sensitivity, and specificity, sample volume requirements, contamination considerations, estimating extracted blood volumes, assessing stability and analyte recovery, and hematocrit effects., Methods: A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted in March 2022. DBS method development and application studies were divided into three main chemical classes: environmental tobacco smoke, trace elements (including lead, mercury, cadmium, and arsenic), and industrial chemicals (including endocrine-disrupting chemicals and persistent organic pollutants). DBS method development and validation studies were scored on key quality-control and performance parameters by two members of the review team., Results: Our search identified 47 published reports related to measuring environmental exposure biomarkers in human DBS samples. A total of 28 reports (37 total studies) were on methods development and validation and 19 reports were primarily the application of previously developed DBS assays. High-performing DBS methods have been developed, validated, and applied for detecting environmental exposures to tobacco smoke, trace elements, and several important endocrine-disrupting chemicals and persistent organic pollutants. Additional work is needed for measuring cadmium, arsenic, inorganic mercury, and bisphenol A in DBS and NDBS samples., Significance: We present an inventory and critical review of available assays for measuring environmental exposure biomarkers in DBS and NDBS samples to help facilitate this sampling medium as an emerging tool for public health (e.g., screening programs, temporal biomonitoring) and environmental epidemiology (e.g., field-based studies)., (© 2022. The Author(s).)

Tunable laser spectroscopy (TLS) near 2683 nm was used to measure carbon dioxide and water vapor inside a pilot mask during jet fighter flights. Measurement frequency was 100 Hz in order to capture breathing profiles and other gas flow dynamics. Analysis of the full inhalation and exhalation breathing cycle allowed precise monitoring of breathing performance and interaction of the pilot with the life-support system. Measurements revealed dynamic phenomena pertaining to mechanical gas flow and pilot respiration that may be used to understand gas delivery stresses imposed upon the pilot and pilot physiology during flight. Typically, such measurements are made with non-dispersive infrared instrumentation for only carbon dioxide with intrinsic challenges regarding time and optical resolution. The TLS approach is a major advance because the sensor is placed directly into the mask improving its time response and enabling use of water vapor measurements that are less impacted from memory effects. This article presents the implementation of TLS and shows highly time-resolved pilot breathing data for high-performance aircraft tests., (Creative Commons Attribution license.)

The U.S. EPA CompTox Chemicals Dashboard is a freely available web-based application providing access to chemistry, toxicity, and exposure data for ∼900 000 chemicals. Data, search functionality, and prediction models within the Dashboard can help identify chemicals found in environmental analyses and human biomonitoring. It was designed to deliver data generated to support computational toxicology to reduce chemical testing on animals and provide access to new approach methodologies including prediction models. The inclusion of mass and formula-based searches, together with relevant ranking approaches, allows for the identification and prioritization of exogenous (environmental) chemicals from high resolution mass spectrometry in need of further evaluation. The Dashboard includes chemicals that can be detected by liquid chromatography, gas chromatography-mass spectrometry (GC-MS) and direct-MS analyses, and chemical lists have been added that highlight breath-borne volatile and semi-volatile organic compounds. The Dashboard can be searched using various chemical identifiers (e.g. chemical synonyms, CASRN and InChIKeys), chemical formula, MS-ready formulae monoisotopic mass, consumer product categories and assays/genes associated with high-throughput screening data. An integrated search at a chemical level performs searches against PubMed to identify relevant published literature. This article describes specific procedures using the Dashboard as a first-stop tool for exploring both targeted and non-targeted results from GC-MS analyses of chemicals found in breath, exhaled breath condensate, and associated aerosols.

Pneumonia is a significant risk for critically ill, mechanically ventilated (CIMV) patients. Diagnosis of pneumonia generally requires a combination of clinician-guided diagnoses and clinical scoring systems. Exhaled breath condensate (EBC) can be safely collected non-invasively from CIMV patients. Hundreds of biomarkers in EBC are associated with acute disease states, including pneumonia. We evaluated cytokines in EBC from CIMV patients and hypothesized that these biomarkers would correlate with disease severity in pneumonia, sepsis, and death. EBC IL-2 levels were associated with chest radiograph severity scores (odds ratio = 1.68; 95% confidence interval = 1.09-2.60; P = 0.02). EBC TNF-α levels were also associated with pneumonia (odds ratio = 3.20; 95% confidence interval = 1.19-8.65; P = 0.02). The techniques and results from this study may be useful for all mechanically ventilated patients.

In contrast to blood and urine samples, breath is invisible and ubiquitous in the environment. Different precautions are now necessary beyond the usual 'Universal Precautions'. In the era of COVID-19, breath (especially the aerosol fraction) can no longer be considered as harmless in the clinic or laboratory. As Journal of Breath Research is a primary resource for breath-related research, we (the editors) are presently developing safety guidance applicable to all breath research , not just for those projects that involve known COVID-19 infected subjects. We are starting this process by implementing requirements on reporting safety precautions in research papers and notes. This editorial announces that authors of all new submissions to JBR henceforth must state clearly the procedures undertaken for assuring laboratory and clinical safety, much like the existing requirements for disclosing Ethics Committee or Institutional Review Board protocols for studies on human subjects. In the following, we additionally make some recommendations based on best practices drawn from our experience and input from the JBR Editorial Board.

To better understand the absorption of combustion byproducts during firefighting, we performed biological monitoring (breath and urine) on firefighters who responded to controlled residential fires and examined the results by job assignment and fire attack tactic. Urine was analyzed for metabolites of polycyclic aromatic hydrocarbons (PAHs) and breath was analyzed for volatile organic compounds (VOCs) including benzene. Median concentrations of PAH metabolites in urine increased from pre-firefighting to 3-h post firefighting for all job assignments. This change was greatest for firefighters assigned to attack and search with 2.3, 5.6, 3.9, and 1.4-fold median increases in pyrene, phenanthrene, naphthalene, and fluorene metabolites. Median exhaled breath concentrations of benzene increased 2-fold for attack and search firefighters (p < 0.01) and 1.4-fold for outside vent firefighters (p = 0.02). Compared to interior attack, transitional attack resulted in 50% less uptake of pyrene (p = 0.09), 36% less uptake phenanthrene (p = 0.052), and 20% less uptake of fluorene (p < 0.01). Dermal absorption likely contributed to firefighters' exposures in this study. Firefighters' exposures will vary by job assignment and can be reduced by employing a transitional fire attack when feasible.

This dataset contains raw area counts and percent recoveries of polycyclic aromatic hydrocarbon (PAH) standards desorbed from selected sorbent tubes and analyzed using thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS). The results of this study were published in the article "Recovery and reactivity of polycyclic aromatic hydrocarbons collected on selected sorbent tubes and analyzed by thermal desorption-gas chromatography/mass spectrometry" in Journal of Chromatography A [1]. The sorbent tubes studied include stainless steel Carbograph 2TD/1TD, glass quartz wool-Carbograph 2TD, inert-coated stainless steel Carbograph 2TD, glass and stainless steel Tenax TA, PAH (chemical weapons), and glass and stainless steel XRO-440 sorbent tubes. Tables listing the experimental conditions, TD methods, and types of sorbent tubes are included in the manuscript. Data for experiments, including the investigation of incomplete desorption of PAHs from Carbograph 2TD/1TD and XRO-440 sorbent tubes, the comparison of PAH recoveries from three different TD methods, the analysis of PAH breakthrough from sorbent tubes, the investigation of the effect of heat on PAH percent recovery from sorbent tubes, and the formation of reaction products during PAH loading and desorption are included in Appendix A. These data can be used to guide sorbent tube selection for PAH analyses in future studies.

Early identification of disease onset is regarded as an important factor for successful medical intervention. However, cancer and other long-term latency diseases are rare and may take years to manifest clinically. As such, there are no gold standards with which to immediately validate proposed preclinical screening methodologies. There is evidence that dogs can sort samples reproducibly into yes/no categories based on case-control training, but the basis of their decisions is unknown. Because dogs are sniffing air, the distinguishing chemicals must be either in the gas-phase or attached to aerosols and/or airborne particles. Recent biomonitoring research has shown how to extract and analyze semi- and non-volatile compounds from human breath in exhaled condensates and aerosols. Further research has shown that exhaled aerosols can be directly collected on standard hospital-style olefin polypropylene masks and that these masks can be used as a simple sampling scheme for canine screening. In this article, detailed liquid chromatography-high resolution mass spectrometry (LC-HR-MS) with Orbitrap instrumentation and gas chromatography-mass spectrometry (GC-MS) analyses were performed on two sets of masks sorted by consensus of a four-dog cohort as either cancer or control. Specifically, after sorting by the dogs, sample masks were cut into multiple sections and extracted for LC-MS and GC-MS non-targeted analyses. Extracts were also analyzed for human cytokines, confirming the presence of human aerosol content above levels in blank masks. In preliminary evaluations, 345 and 44 high quality chemical features were detected by LC-MS and GC-MS analyses, respectively. These features were used to develop provisional orthogonal projection to latent structures-discriminant analysis (OPLS-DA) models to determine if the samples classified as cancer (case) or non-cancer (control) by the dogs could be separated into the same groups using analytical instrumentation. While the OPLS-DA model for the LC-HR-MS data was able to separate the two groups with statistical significance, although weak explanatory power, the GC-MS model was not found to be significant. These results suggest that the dogs may rely on the less volatile compounds from breath aerosol that were analyzed by LC-HR-MS than the more volatile compounds observed by GC-MS to sort mask samples into groups. These results provide justification for more expansive studies in the future that aim to characterize specific chemical features, and the role(s) of these features in maintaining homeostatic biological processes.

Exhaled breath aerosol (EBA) is an important non-invasive biological medium for detecting exogenous environmental contaminants and endogenous metabolites present in the pulmonary tract. Currently, EBA is typically captured as a constituent of the mainstream clinical tool referred to as exhaled breath condensate (EBC). This article describes a simpler, completely non-invasive method for collecting EBA directly from different forms of hard-surface plastic respirator masks and disposable hospital paper breathing masks without first collecting EBC. The new EBA methodology bypasses the complex EBC procedures that require specialized collection gear, dry ice or other coolant, in-field sample processing, and refrigerated transport to the laboratory. Herein, mask samples collected from different types of plastic respirators and paper hospital masks worn by volunteers in the laboratory were analyzed using high resolution-liquid chromatography-mass spectrometry (HR-LC-MS) and immunochemistry. The results of immunochemistry analysis revealed that cytokines were collected above background on both plastic respirator surfaces and paper hospital masks, confirming the presence of human biological constituents. Non-targeted HR-LC-MS analyses demonstrated that larger exogenous molecules such as plasticizers, pesticides, and consumer product chemicals as well as endogenous biochemicals, including cytokines and fatty acids were also detected on mask surfaces. These results suggest that mask sampling is a viable technique for EBA collection to assess potential inhalation exposures and endogenous indicators of health state., Competing Interests: CONFLICT OF INTEREST The authors declare no conflicts of interest.

This article describes the optimization of methodology for extending the measurement of volatile organic compounds (VOCs) to increasingly heavier polycyclic aromatic hydrocarbons (PAHs) with a detailed focus on recent sorbent tube technology. Although PAHs have lower volatility than compounds such as benzene, toluene, ethylbenzene and xylenes, these semi-volatile compounds can be detected in air and breath samples. For this work, PAHs were captured on sorbent tubes and subsequently analyzed using automated thermal desorption gas chromatography - mass spectrometry (ATD-GC/MS). While many different sorbent tubes are commercially available, optimization for airborne PAH sampling using sorbent tubes has not been previously considered. Herein, several commercially available sorbent tubes, including Carbograph 2 TD/1TD, Tenax TA, XRO-440, and inert-coated PAH tubes are compared to determine the relative recovery for eight PAHs commonly found in the environment. Certain types of sorbent materials were found to be better suited for PAH recovery during thermal desorption, and PAH reaction products were observed on several types of sorbent tubes, including graphitized carbon black sorbents with stainless steel tube materials. As such, selection of sorbent tube media should be carefully considered prior to embarking on a PAH study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Training fires may constitute a major portion of some firefighters' occupational exposures to smoke. However, the magnitude and composition of those exposures are not well understood and may vary by the type of training scenario and fuels., Objectives: To understand how structure fire training contributes to firefighters' and instructors' select chemical exposures, we conducted biological monitoring during exercises involving combustion of pallet and straw and oriented strand board (OSB) or the use of simulated smoke., Methods: Urine was analyzed for metabolites of polycyclic aromatic hydrocarbons (PAHs) and breath was analyzed for volatile organic compounds (VOCs) including benzene., Results: Median concentrations of nearly all PAH metabolites in urine increased from pre-to 3-hr post-training for each scenario and were highest for OSB, followed by pallet and straw, and then simulated smoke. For instructors who supervised three trainings per day, median concentrations increased at each collection. A single day of OSB exercises led to a 30-fold increase in 1-hydroxypyrene for instructors, culminating in a median end-of-shift concentration 3.5-fold greater than median levels measured from firefighters in a previous controlled-residential fire study. Breath concentrations of benzene increased 2 to 7-fold immediately after the training exercises (with the exception of simulated smoke training). Exposures were highest for the OSB scenario and instructors accumulated PAHs with repeated daily exercises., Conclusions: Dermal absorption likely contributed to the biological levels as the respiratory route was well protected. Training academies should consider exposure risks as well as instructional objectives when selecting training exercises., (Published by Elsevier GmbH.)

This report discusses advances in instrumentation based on soft chemical ionization followed by high-resolution real-time mass spectrometry (HR-MS), specifically in relation to developments in proton transfer reaction mass spectrometry (PTR-MS) technology. It is part of a Journal of Breath Research series that describes recent technical developments in breath related research relevant to human health and analytical chemistry from scientific conferences. Herein we discuss the current state of PTR-MS as presented at the 8th International Conference on Proton Transfer Reaction - Mass Spectrometry held in Innsbruck, Austria, February 2-8, 2019, attended by the authors.

Biomarker measurements can provide unambiguous evidence of environmental exposures as well as the resultant biological responses. Firefighters have a high rate of occupational cancer incidence, which has been proposed to be linked in part to their increased environmental exposure to byproducts of combustion and contaminants produced during fire responses. In this article, the uptake and elimination of targeted volatile organic compounds were investigated by collecting the exhaled breath of firefighters on sorbent tubes before and after controlled structure burns and analyzing samples using automated thermal desorption-gas chromatography (ATD-GC/MS). Volatile organic compounds exposure was assessed by grouping the data according to firefighting job positions as well as visualizing the data at the level of the individual firefighter to determine which individuals had expected exposure responses. When data were assessed at the group level, benzene concentrations were found to be elevated post-exposure in both fire attack, victim search, and outside ventilation firefighting positions. However, the results of the data analysis at the individual level indicate that certain firefighters may be more susceptible to post-exposure volatile organic compounds increases than others, and this should be considered when assessing the effectiveness of firefighting protective gear. Although this work focuses on firefighting activity, the results can be translated to potential human health and ecological effects from building and forest fires.

Background: The human exposome, defined as ' …everything that is not the genome ', comprises all chemicals in the body interacting with life processes. The exposome drives genes x environment (GxE) interactions that can cause long-term latency and chronic diseases. The exposome constantly changes in response to external exposures and internal metabolism. Different types of compounds are found in different biological media. Objective: Measure polar volatile organic compounds (PVOCs) excreted in urine to document endogenous metabolites and exogenous compounds from environmental exposures. Methods: Use headspace collection and sorbent tube thermal desorption coupled with bench-top gas chromatography-mass spectrometry (GC-MS) for targeted and non-targeted approaches. Identify and categorize PVOCs that may distinguish among healthy and affected individuals. Results: Method is successfully demonstrated to tabulate a series of 28 PVOCs detected in human urine across 120 samples from 28 human subjects. Median concentrations range from below detect to 165 ng/mL. Certain PVOCs have potential health implications. Conclusions: Headspace collection with sorbent tubes is an effective method for documenting PVOCs in urine that are otherwise difficult to measure. This methodology can provide probative information regarding biochemical processes and adverse outcome pathways (AOPs) for toxicity testing.

A non-targeted analysis workflow was applied to analyze exhaled breath samples collected from firefighters pre- and post-structural fire suppression. Breath samples from firefighters functioning in attack and search positions were examined for target and non-target compounds in automated thermal desorption-GC/MS (ATD-GC/MS) selected ion monitoring (SIM)/scan mode and reviewed for prominent chemicals. Targeted chemicals included products of combustion such as benzene, toluene, xylenes, and polycyclic aromatic hydrocarbons (PAH) that serve as a standard assessment of exposure. Sixty unique chemical features representative of exogenous chemicals and endogenous compounds, including single-ring aromatics, polynuclear aromatic hydrocarbons, volatile sulfur-containing compounds, aldehydes, alkanes, and alkenes were identified using the non-targeted analysis workflow. Fifty-seven out of 60 non-targeted features changed by at least 50% from pre- to post-fire suppression activity in at least one subject, and 7 non-targeted features were found to exhibit significantly increased or decreased concentrations for all subjects as a group. This study is important for (1) alerting the firefighter community to potential new exposures, (2) expanding the current targeted list of toxicants, and (3) finding biomarkers of response to firefighting activity as reflected by changes in endogenous compounds. Data demonstrate that there are non-targeted compounds in firefighters' breath that are indicative of environmental exposure despite the use of protective gear, and this information may be further utilized to improve the effectiveness of personal protective equipment.

High-resolution mass spectrometry (HR-MS) is an important tool for performing non-targeted analysis for investigating complex organic mixtures in human or environmental media. This perspective demonstrates HR-MS compound identification strategies using atom counting, isotope ratios, and fragmentation pattern analysis based on 'exact' or 'accurate' mass, which allows analytical distinction among mass fragments with the same integer mass, but with different atomic constituents of the original molecules. Herein, HR-MS technology is shown to narrow down the identity of unknown compounds for specific examples, and ultimately inform future analyses when these compounds reoccur. Although HR-MS is important for all biological media, this is particularly critical for new methods and instrumentation invoking exhaled breath condensate, particles, and aerosols. In contrast to standard breath gas-phase analyses where 1 mass unit (Da) resolution is generally sufficient, the condensed phase breath media are particularly vulnerable to errors in compound identification because the larger organic non-volatile molecules can form identical integer mass fragments from different atomic constituents which then require high-resolution mass analyses to tell them apart.

Human breath, along with urine and blood, has long been one of the three major biological media for assessing human health and environmental exposure. In fact, the detection of odor on human breath, as described by Hippocrates in 400 BC, is considered the first analytical health assessment tool. Although less common in comparison to contemporary bio-fluids analyses, breath has become an attractive diagnostic medium as sampling is non-invasive, unlimited in timing and volume, and does not require clinical personnel. Exhaled breath, exhaled breath condensate (EBC), and exhaled breath aerosol (EBA) are different types of breath matrices used to assess human health and disease state. Over the past 20 years, breath research has made many advances in assessing health state, overcoming many of its initial challenges related to sampling and analysis. The wide variety of sampling techniques and collection devices that have been developed for these media are discussed herein. The different types of sensors and mass spectrometry instruments currently available for breath analysis are evaluated as well as emerging breath research topics, such as cytokines, security and airport surveillance, cellular respiration, and canine olfaction., (Published by Elsevier B.V.)

Due to the ∼86,000 chemicals registered under the Toxic Substances Control Act and increasing ethical concerns regarding animal testing, it is not economically or technically feasible to screen every registered chemical for toxicity using animal-based toxicity assays. To address this challenge, regulatory agencies are investigating high-throughput screening in vitro methods to increase speed of toxicity testing, while reducing the overall cost. One approach for rapid toxicity testing currently being investigated is monitoring of volatile emissions produced by cell lines in culture. Such a metabolomics approach would measure gaseous emissions from a cell line and determine if such gaseous metabolites are altered upon exposure to a xenobiotic. Herein, we describe the history and rationale of monitoring endogenously produced volatiles for identification of pathologic conditions, as well as emerging applications in toxicity testing for such an approach., Competing Interests: The authors declare that no competing financial interests exist.

Currently in the United States there are no regulatory standards for ambient concentrations of polycyclic aromatic hydrocarbons (PAHs), a class of organic compounds with known carcinogenic species. As such, monitoring data are not routinely collected resulting in limited exposure mapping and epidemiologic studies. This work develops the log-mass fraction (LMF) Bayesian maximum entropy (BME) geostatistical prediction method used to predict the concentration of nine particle-bound PAHs across the US state of North Carolina. The LMF method develops a relationship between a relatively small number of collocated PAH and fine Particulate Matter (PM2.5) samples collected in 2005 and applies that relationship to a larger number of locations where PM2.5 is routinely monitored to more broadly estimate PAH concentrations across the state. Cross validation and mapping results indicate that by incorporating both PAH and PM2.5 data, the LMF BME method reduces mean squared error by 28.4% and produces more realistic spatial gradients compared to the traditional kriging approach based solely on observed PAH data. The LMF BME method efficiently creates PAH predictions in a PAH data sparse and PM2.5 data rich setting, opening the door for more expansive epidemiologic exposure assessments of ambient PAH.

The data contained in this article are PubMed search strings and search string builders used to curate breath research manuscripts published from 1995-2016 and the respective number of articles found that satisfied the search requirements for selected categories. Breath sampling represents a non-invasive technique that has gained usefulness for public health, clinical, diagnostic, and environmental exposure assessment applications over the years. This data article includes search strings that were utilized to retrieve publications through the PubMed database for different breath research-related topics that were related to the analysis of exhaled breath, exhaled breath condensate (EBC), and exhaled breath aerosol (EBA) as well as the analysis of cellular headspace. Manuscripts were curated for topics including EBC, EBA, Direct MS, GC-MS, LC-MS, alcohol, and sensors. A summary of the number of papers published per year for the data retrieved using each of the search strings is also included. These data can be utilized to discern trends in the number of breath research publications in each of the different topics over time. A supplementary Appendix A containing the titles, author lists, journal names, publication dates, PMID numbers, and EntrezUID numbers for each of the journal articles curated using the finalized search strings for the seven breath research-related topics can also be found within this article. The selected manuscripts can be used to explore the impact that breath research has had on expanding the scientific knowledge in each of the investigated topics.

Human biomonitoring is the foundation of environmental toxicology, community public health evaluation, preclinical health effects assessments, pharmacological drug development and testing, and medical diagnostics. Within this framework, the intra-class correlation coefficient (ICC) serves as an important tool for gaining insight into human variability and responses and for developing risk-based assessments in the face of sparse or highly complex measurement data. The analytical procedures that provide data for clinical and public health efforts are continually evolving to expand our knowledge base of the many thousands of environmental and biomarker chemicals that define human systems biology. These chemicals range from the smallest molecules from energy metabolism (i.e., the metabolome), through larger molecules including enzymes, proteins, RNA, DNA, and adducts. In additiona, the human body contains exogenous environmental chemicals and contributions from the microbiome from gastrointestinal, pulmonary, urogenital, naso-pharyngeal, and skin sources. This complex mixture of biomarker chemicals from environmental, human, and microbiotic sources comprise the human exposome and generally accessed through sampling of blood, breath, and urine. One of the most difficult problems in biomarker assessment is assigning probative value to any given set of measurements as there are generally insufficient data to distinguish among sources of chemicals such as environmental, microbiotic, or human metabolism and also deciding which measurements are remarkable from those that are within normal human variability. The implementation of longitudinal (repeat) measurement strategies has provided new statistical approaches for interpreting such complexities, and use of descriptive statistics based upon intra-class correlation coefficients (ICC) has become a powerful tool in these efforts. This review has two parts; the first focuses on the history of repeat measures of human biomarkers starting with occupational toxicology of the early 1950s through modern applications in interpretation of the human exposome and metabolic adverse outcome pathways (AOPs). The second part reviews different methods for calculating the ICC and explores the strategies and applications in light of different data structures.

Exhaled breath condensate (EBC) and associated exhaled breath aerosols (EBA) are valuable non-invasive biological media used for the quantification of biomarkers. EBC contains exhaled water vapor, soluble gas-phase (polar) organic compounds, ionic species, plus other species including semi- and non-volatile organic compounds, proteins, cell fragments, DNA, dissolved inorganic compounds, ions, and microbiota (bacteria and viruses) dissolved in the co-collected EBA. EBC is collected from subjects who breathe 'normally' through a chilled tube assembly for approximately 10 min and is then harvested into small vials for analysis. Aerosol filters without the chilled tube assembly are also used to separately collect EBA. Unlike typical gas-phase breath samples used for environmental and clinical applications, the constituents of EBC and EBA are not easily characterized by total volume or carbon dioxide (CO 2 ) concentration, because the gas-phase is vented. Furthermore, EBC and associated EBA are greatly affected by breathing protocol, more specifically, depth of inhalation and expelled breath velocity. We have tested a new instrument developed by Loccioni Gruppa Humancare (Ancona, Italy) for implementation of EBC collection from human subjects to assess EBC collection parameters. The instrument is the first EBC collection device that provides instantaneous visual feedback to the subjects to control breathing patterns. In this report we describe the operation of the instrument, and present an overview of performance and analytical applications.

Traditionally, gas chromatography-mass spectrometry (GC/MS) analysis has used a targeted approach called selected ion monitoring (SIM) to quantify specific compounds that may have adverse health effects. Due to method limitations and the constraints of preparing duplicate samples, the information that could be obtained from separately collecting the full scan chromatogram of the sample has often been sacrificed. However, the hybrid technique called synchronous SIM/scan mode alternates between the two acquisition modes, maintaining the accuracy and sensitivity of SIM for targeted analysis while also providing the full scan chromatogram for discovery of non-target compounds. This technology was assessed using calibration data and real-world breath samples from a joint EPA/NIOSH collaboration that investigated the safety of firefighters' protective gear during controlled structure burns. Collecting field samples is costly and must be performed strategically to ensure that time points and replicates are accurate and representative of the intended population. This is difficult to accomplish with firefighters who are working under volatile conditions. The synchronous SIM/scan method decreases the number of field samples that need to be collected by half and reduces error in trying to recreate time points since a breath sample from a single sorbent tube can be used to collect both the SIM and scan data simultaneously. As a practical demonstration of the method, we investigate thirty-six firefighter breath samples, document organic compounds of interest, and identify additional non-target compounds., (Published by Elsevier B.V.)

Human biomonitoring is an indispensable tool for evaluating the systemic effects derived from external stressors including environmental pollutants, chemicals from consumer products, and pharmaceuticals. The aim of this study was to explore consequences of environmental exposures to diesel exhaust (DE) and ozone (O 3 ) and ultimately to interpret these parameters from the perspective of in vitro to in vivo extrapolation. In particular, the objective was to use cytokine expression at the cellular level as a biomarker for physiological systemic responses such as blood pressure and lung function at the systemic level. The values obtained could ultimately link in vivo behavior to simpler in vitro experiments where cytokines are a measured parameter. Human exposures to combinations of DE and O 3 and the response correlations between forced exhaled volume in 1 second (FEV 1 ), forced vital capacity (FVC), systolic and diastolic blood pressure (SBP and DBP, respectively), and 10 inflammatory cytokines in blood (interleukins 1β, 2, 4, 5, 8, 10, 12p70 and 13, IFN-γ, and TNF-α) were determined in 15 healthy human volunteers. Results across all exposures revealed that certain individuals displayed greater inflammatory responses compared to the group and, generally, there was more between-person variation in the responses. Evidence indicates that individuals are more stable within themselves and are more likely to exhibit responses independent of one another. Data suggest that in vitro findings may ultimately be implemented to elucidate underlying adverse outcome pathways (AOP) for linking high-throughput toxicity tests to physiological in vivo responses. Further, this investigation supports assessing subjects based upon individual responses as a complement to standard longitudinal (pre vs. post) intervention grouping strategies. Ultimately, it may become possible to predict a physiological (systemic) response based upon cellular-level (in vitro) observations.

Exhaled breath has joined blood and urine as a valuable resource for sampling and analyzing biomarkers in human media for assessing exposure, uptake metabolism, and elimination of toxic chemicals. This article focuses current use of exhaled gas, aerosols, and vapor in human breath, the methods for collection, and ultimately the use of the resulting data. Some advantages of breath are the noninvasive and self-administered nature of collection, the essentially inexhaustible supply, and that breath sampling does not produce potentially infectious waste such as needles, wipes, bandages, and glassware. In contrast to blood and urine, breath samples can be collected on demand in rapid succession and so allow toxicokinetic observations of uptake and elimination in any time frame. Furthermore, new technologies now allow capturing condensed breath vapor directly, or just the aerosol fraction alone, to gain access to inorganic species, lung pH, proteins and protein fragments, cellular DNA, and whole microorganisms from the pulmonary microbiome. Future applications are discussed, especially the use of isotopically labeled probes, non-targeted (discovery) analysis, cellular level toxicity testing, and ultimately assessing "crowd breath" of groups of people and the relation to dose of airborne and other environmental chemicals at the population level.

Biomarker datasets often include entries 'below the level of quantitation' (LoQ) wherein the instrumentation is no longer able to provide values that meet required analytical quality standards, a phenomenon referred to as 'left-censored' data. Generally, some form of imputation for missing values is required to allow calculating distributions and summary statistics for comparing datasets to each other. This article discusses the available options for imputing (modeling) left-censored data, presents the methods for implementing different procedures, and provides examples and guidelines using realistic data to assess relative performance. Ultimately, multiple ordered value imputation is identified as the best method; therein, the overall distribution of actual measures is used in conjunction with an estimate of relative order of the missing values to provide the most likely estimates below the LoQ.

One of the main uses of biomarker measurements is to compare different populations to each other and to assess risk in comparison to established parameters. This is most often done using summary statistics such as central tendency, variance components, confidence intervals, exceedance levels and percentiles. Such comparisons are only valid if the underlying assumptions of distribution are correct. This article discusses methodology for interpreting and evaluating data distributions using quartile-quartile plots (QQ-plots) and making decisions as to how to treat outliers, interpreting effects of mixed distributions, and identifying left-censored data. The QQ-plot graph is shown to be a simple and elegant tool for visual inspection of complex data and deciding if summary statistics should be performed after log-transformation.

Epidemiological observations of urban inhalation exposures to diesel exhaust (DE) and ozone (O3) have shown pre-clinical cardiopulmonary responses in humans. Identifying the key biological mechanisms that initiate these health bioindicators is difficult due to variability in environmental exposure in time and from person to person. Previously, environmentally controlled human exposure chambers have been used to study DE and O3 dose-response patterns separately, but investigation of co-exposures has not been performed under controlled conditions. Because a mixture is a more realistic exposure scenario for the general public, in this study we investigate the relationships of urban levels of urban-level DE exposure (300 μg/m3), O3 (0.3 ppm), DE + O3 co-exposure, and innate immune system responses. Fifteen healthy human volunteers were studied for changes in ten inflammatory cytokines (interleukins 1β, 2, 4, 5, 8, 10, 12p70 and 13, IFN-γ, and TNF-α) and counts of three white blood cell types (lymphocytes, monocytes, and neutrophils) following controlled exposures to DE, O3, and DE+O3. The results show subtle cytokines responses to the diesel-only and ozone-only exposures, and that a more complex (possibly synergistic) relationship exists in the combination of these two exposures with suppression of IL-5, IL-12p70, IFN-γ, and TNF-α that persists up to 22-hours for IFN-γ and TNF-α. The white blood cell differential counts showed significant monocyte and lymphocyte decreases and neutrophil increases following the DE + O3 exposure; lymphocytes and neutrophils changes also persist for at least 22-hours. Because human studies must be conducted under strict safety protocols at environmental levels, these effects are subtle and are generally only seen with detailed statistical analysis. This study indicates that the observed associations between environmental exposures and cardiopulmonary effects are possibly mediated by inflammatory response mechanisms.

Cytokines, low-molecular-weight messenger proteins that act as intercellular immunomodulatory signals, have become a mainstream preclinical marker for assessing the systemic inflammatory response to external stressors. The challenge is to quantitate from healthy subjects cytokine levels that are below or at baseline and relate those dynamic and complex cytokine signatures of exposures with the inflammatory and repair pathways. Thus, highly sensitive, specific, and precise analytical and statistical methods are critically important. Investigators at the U.S. Environmental Protection Agency (EPA) have implemented advanced technologies and developed statistics for evaluating panels of inflammatory cytokines in human blood, exhaled breath condensate, urine samples, and murine biological media. Advanced multiplex, bead-based, and automated analytical platforms provided sufficient sensitivity, precision, and accuracy over the traditional enzyme-linked immunosorbent assay (ELISA). Thus, baseline cytokine levels can be quantified from healthy human subjects and animals and compared to an in vivo exposure response from an environmental chemical. Specifically, patterns of cytokine responses in humans exposed to environmental levels of ozone and diesel exhaust, and in rodents exposed to selected pesticides (such as fipronil and carbaryl), were used as case studies to generally assess the taxonomic applicability of cytokine responses. The findings in this study may aid in the application of measureable cytokine markers in future adverse outcome pathway (AOP)-based toxicity testing. Data from human and animal studies were coalesced and the possibility of using cytokines as key events (KE) to bridge species responses to external stressors in an AOP-based framework was explored.

Exposure-based risk assessment employs large cross-sectional data sets of environmental and biomarker measurements to predict population statistics for adverse health outcomes. The underlying assumption is that long-term (many years) latency health problems including cancer, autoimmune and cardiovascular disease, diabetes, and asthma are triggered by lifetime exposures to environmental stressors that interact with the genome. The aim of this study was to develop a specific predictive method that provides the statistical parameters for chronic exposure at the individual level based upon a single spot measurement and knowledge of global summary statistics as derived from large data sets. This is a profound shift in exposure and health statistics in that it begins to answer the question "How large is my personal risk?" rather than just providing an overall population-based estimate. This approach also holds value for interpreting exposure-based risks for small groups of individuals within a community in comparison to random individuals from the general population.

Environmental health science aims to link environmental pollution sources to adverse health outcomes to develop effective exposure intervention strategies that reduce long-term disease risks. Over the past few decades, the public health community recognized that health risk is driven by interaction between the human genome and external environment. Now that the human genetic code has been sequenced, establishing this "G × E" (gene-environment) interaction requires a similar effort to decode the human exposome, which is the accumulation of an individual's environmental exposures and metabolic responses throughout the person's lifetime. The exposome is composed of endogenous and exogenous chemicals, many of which are measurable as biomarkers in blood, breath, and urine. Exposure to pollutants is assessed by analyzing biofluids for the pollutant itself or its metabolic products. New methods are being developed to use a subset of biomarkers, termed bioindicators, to demonstrate biological changes indicative of future adverse health effects. Typically, environmental biomarkers are assessed using noninvasive (excreted) media, such as breath and urine. Blood is often avoided for biomonitoring due to practical reasons such as medical personnel, infectious waste, or clinical setting, despite the fact that blood represents the central compartment that interacts with every living cell and is the most relevant biofluid for certain applications and analyses. The aims of this study were to (1) review the current use of blood samples in environmental health research, (2) briefly contrast blood with other biological media, and (3) propose additional applications for blood analysis in human exposure research.

This commentary is the second of a series outlining one specific concept in interpreting biomarkers data. In the first, an observational method was presented for assessing the distribution of measurements before making parametric calculations. Here, the discussion revolves around the next step, the choice of using standard error of the mean or the calculated standard deviation to compare or predict measurement results.

Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe application to exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer was evaluated for analytical performance for inflammatory cytokines (IL-6, TNF-α, IL-1β and IL-8). The system was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r(2)  >  0.99). Sensitivities varied by analyte, but were robust from ~0.006 (IL-6) to ~0.01 (TNF-α) pg ml(-1). All analytes demonstrated response suppression when diluted with deionized water and so assay buffer diluent was found to be a better choice. Analytical runs required ~45 min setup time for loading samples, reagents, calibrants, etc., after which the instrument performs without further intervention for up to 288 separate samples. Currently, available kits are limited to single-plex analyses and so sample volumes require adjustments. Sample dilutions should be made with assay diluent to avoid response suppression. Automation performs seamlessly and data are automatically analyzed and reported in spreadsheet format. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (<1.3 pg ml(-1)). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels.

Background: Each year, the U.S. NHANES measures hundreds of chemical biomarkers in samples from thousands of study participants. These biomarker measurements are used to establish population reference ranges, track exposure trends, identify population subsets with elevated exposures, and prioritize research needs. There is now interest in further utilizing the NHANES data to inform chemical risk assessments., Objectives: This article highlights a) the extent to which U.S. NHANES chemical biomarker data have been evaluated, b) groups of chemicals that have been studied, c) data analysis approaches and challenges, and d) opportunities for using these data to inform risk assessments., Methods: A literature search (1999-2013) was performed to identify publications in which U.S. NHANES data were reported. Manual curation identified only the subset of publications that clearly utilized chemical biomarker data. This subset was evaluated for chemical groupings, data analysis approaches, and overall trends., Results: A small percentage of the sampled NHANES-related publications reported on chemical biomarkers (8% yearly average). Of 11 chemical groups, metals/metalloids were most frequently evaluated (49%), followed by pesticides (9%) and environmental phenols (7%). Studies of multiple chemical groups were also common (8%). Publications linking chemical biomarkers to health metrics have increased dramatically in recent years. New studies are addressing challenges related to NHANES data interpretation in health risk contexts., Conclusions: This article demonstrates growing use of NHANES chemical biomarker data in studies that can impact risk assessments. Best practices for analysis and interpretation must be defined and adopted to allow the full potential of NHANES to be realized.