Advancing Global Health Surveillance of Mycotoxin Exposures using Minimally Invasive Sampling Techniques: A State-of-the-Science Review.

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.

Detection and analysis of endogenous polar volatile organic compounds (PVOCs) in urine for human exposome research.

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.

Non-targeted GC/MS analysis of exhaled breath samples: Exploring human biomarkers of exogenous exposure and endogenous response from professional firefighting activity.

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.

Identifying organic compounds in exhaled breath aerosol: Non-invasive sampling from respirator surfaces and disposable hospital masks.

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.

Targeted GC-MS analysis of firefighters' exhaled breath: Exploring biomarker response at the individual level.

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.

Human biomarker interpretation: the importance of intra-class correlation coefficients (ICC) and their calculations based on mixed models, ANOVA, and variance estimates.

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.

Linking physiological parameters to perturbations in the human exposome: Environmental exposures modify blood pressure and lung function via inflammatory cytokine pathway.

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 (O3) 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 O3 and the response correlations between forced exhaled volume in 1 second (FEV1), forced vital capacity (FVC), systolic and diastolic blood pressure (SBP and DBP, respectively), and 10 inflammatory cytokines in blood (interleukins 1ß, 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.

Comparing biomarker measurements to a normal range: when to use standard error of the mean (SEM) or standard deviation (SD) confidence intervals tests.

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.

Blood-borne biomarkers and bioindicators for linking exposure to health effects in environmental health science.

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.

Breath biomarkers in toxicology.

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.

Estimating central tendency from a single spot measure: A closed-form solution for lognormally distributed biomarker data for risk assessment at the individual level.

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.

Taxonomic applicability of inflammatory cytokines in adverse outcome pathway (AOP) development.

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.

Probe molecule (PrM) approach in adverse outcome pathway (AOP) based high-throughput screening (HTS): in vivo discovery for developing in vitro target methods.

Efficient and accurate adverse outcome pathway (AOP) based high-throughput screening (HTS) methods use a systems biology based approach to computationally model in vitro cellular and molecular data for rapid chemical prioritization; however, not all HTS assays are grounded by relevant in vivo exposure data. The challenge is to develop HTS assays with unambiguous quantitative links between in vitro responses and corresponding in vivo effects, which is complicated by metabolically insufficient systems, in vitro to in vivo extrapolation (IVIVE), cross-species comparisons, and other inherent issues correlating IVIVE findings. This article introduces the concept of ultrasensitive gas phase probe molecules (PrMs) to help bridge the current HTS assay IVIVE gap. The PrM concept assesses metabolic pathways that have already been well-defined from intact human or mammalian models. Specifically, the idea is to introduce a gas phase probe molecule into a system, observe normal steady state, add chemicals of interest, and quantitatively measure (from headspace gas) effects on PrM metabolism that can be directly linked back to a well-defined and corresponding in vivo effect. As an example, we developed the pharmacokinetic (PK) parameters and differential equations to estimate methyl tertiary butyl ether (MTBE) metabolism to tertiary butyl alcohol (TBA) via cytochrome (CYP) 2A6 in the liver from human empirical data. Because MTBE metabolic pathways are well characterized from in vivo data, we can use it as a PrM to explore direct and indirect chemical effects on CYP pathways. The PrM concept could be easily applied to in vitro and alternative models of disease and phenotype, and even test for volatile chemicals while avoiding liquid handling robotics. Furthermore, a PrM can be designed for any chemical with known empirical human exposure data and used to assess chemicals for which no information exists. Herein, we propose an elegant gas phase probe molecule-based approach to in vitro toxicity testing.

Editorial: new analytical and statistical approaches for interpreting the relationships among environmental stressors and biomarkers.

The broad topic of biomarker research has an often-overlooked component: the documentation and interpretation of the surrounding chemical environment and other meta-data, especially from visualization, analytical and statistical perspectives. A second concern is how the environment interacts with human systems biology, what the variability is in "normal" subjects, and how such biological observations might be reconstructed to infer external stressors. In this article, we report on recent research presentations from a symposium at the 248th American Chemical Society meeting held in San Francisco, 10-14 August 2014, that focused on providing some insight into these important issues.

Analysis of inflammatory cytokines in human blood, breath condensate, and urine using a multiplex immunoassay platform.

A change in the expression of cytokines in human biological media indicates an inflammatory response to external stressors and reflects an early step along the adverse outcome pathway (AOP) for various health endpoints. To characterize and interpret this inflammatory response, methodology was developed for measuring a suite of 10 different cytokines in human blood, exhaled breath condensate (EBC), and urine using an electrochemiluminescent multiplex Th1/Th2 cytokine immunoassay platform. Measurement distributions and correlations for eight interleukins (IL) (1ß, 2, 4, 5, 8, 10, 12p70 and 13), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were evaluated using 90 blood plasma, 77 EBC, and 400 urine samples collected from nominally healthy adults subjects in North Carolina in 2008-2012. The in vivo results show that there is sufficient sensitivity for characterizing all 10 cytokines at levels of 0.05-0.10 ρg/ml with a dynamic range up to 100 ng/ml across all three of these biological media. The measured in vivo results also show that the duplicate analysis of blood, EBC and urine samples have average estimated fold ranges of 2.21, 3.49, and 2.50, respectively, which are similar to the mean estimated fold range (2.88) for the lowest concentration (0.610 ρg/ml) from a series of spiked control samples; the cytokine method can be used for all three biological media. Nine out of the 10 cytokines measured in EBC were highly correlated within one another with Spearman ρ coefficients ranging from 0.679 to 0.852, while the cytokines measured in blood had a mix of negative and positive correlations, ranging from -0.620 to 0.836. Almost all correlations between EBC and blood were positive. This work also represents the first successful within- and between-person evaluation of ultra trace-level inflammatory markers in blood, EBC, and urine.

Volatile Organic Compounds Off-gassing from Firefighters' Personal Protective Equipment Ensembles after Use.

Firefighters' personal protective equipment (PPE) ensembles will become contaminated with various compounds during firefighting. Some of these compounds will off-gas following a response, which could result in inhalation exposure. This study was conducted to determine the magnitude and composition of volatile organic compounds (VOCs) generated during controlled structure burns that subsequently off-gassed from the firefighters' PPE, and were systemically absorbed and exhaled in firefighters' breath. Three crews of five firefighters performed entry, suppression, and overhaul during a controlled burn. We used evacuated canisters to sample air inside the burn structure during active fire and overhaul. After each burn, we placed PPE from two firefighters inside clean enclosures and sampled the air using evacuated canisters over 15 min. Firefighters' exhaled breath was collected ∼1 hr before and 4-14 min after each burn. Using gas chromatography/mass spectrometry, the evacuated canister samples were analyzed for 64 VOCs and the exhaled breath samples were analyzed for benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS). Fourteen of the same VOCs were detected off-gassing from PPE in 50% or more of the samples. Compared to background levels, we measured >5 fold increases in mean off-gas concentrations of styrene, benzene, 1,4-dichlorobenzene, acetone, and cyclohexane. Several of the compounds detected off-gassing from PPE were also measured at concentrations above background during active fire and overhaul, including benzene, propene, and styrene. The overhaul and off-gas air concentrations were well below applicable short-term occupational exposure limits. Compared to pre-burn levels, we measured >2 fold increases in mean breath concentrations of benzene, toluene, and styrene after the burns. Air concentrations of BTEXS measured off-gassing from firefighters' used PPE and in firefighters' post-burn exhaled breath were significantly correlated. The firefighters may have absorbed BTEXS through both the dermal route (during firefighting) and inhalation route (from off-gassing PPE after firefighting). Firefighters should be made aware of the potential for inhalation exposure when doffing and traveling in confined vehicles with contaminated PPE and take measures to minimize this exposure pathway.

Are urinary PAHs biomarkers of controlled exposure to diesel exhaust?

Urinary polycyclic aromatic hydrocarbons (PAHs) were evaluated as possible biomarkers of exposure to diesel exhaust (DE) in two controlled-chamber studies. We report levels of 14 PAHs from 28 subjects in urine that were collected before, immediately after and the morning after exposure. Using linear mixed-effects models, we tested for effects of DE exposure and several covariates (time, age, gender and urinary creatinine) on urinary PAH levels. DE exposures did not significantly alter urinary PAH levels. We conclude that urinary PAHs are not promising biomarkers of short-term exposures to DE in the range of 106-276 µg/m(3).

Estimating common parameters of lognormally distributed environmental and biomonitoring data: harmonizing disparate statistics from publications.

The progression of science is driven by the accumulation of knowledge and builds upon published work of others. Another important feature is to place current results into the context of previous observations. The published literature, however, often does not provide sufficient direct information for the reader to interpret the results beyond the scope of that particular article. Authors tend to provide only summary statistics in various forms, such as means and standard deviations, median and range, quartiles, 95% confidence intervals, and so on, rather than providing measurement data. Second, essentially all environmental and biomonitoring measurements have an underlying lognormal distribution, so certain published statistical characterizations may be inappropriate for comparisons. The aim of this study was to review and develop direct conversions of different descriptions of data into a standard format comprised of the geometric mean (GM) and the geometric standard deviation (GSD) and then demonstrate how, under the assumption of lognormal distribution, these parameters are used to answer questions of confidence intervals, exceedance levels, and statistical differences among distributions. A wide variety of real-world measurement data sets was reviewed, and it was demonstrated that these data sets are indeed of lognormal character, thus making them amenable to these methods. Potential errors incurred from making retrospective estimates from disparate summary statistics are described. In addition to providing tools to interpret "other people's data," this review should also be seen as a cautionary tale for publishing one's own data to make it as useful as possible for other researchers.

Evaluating an alternative method for rapid urinary creatinine determination.

Creatinine (CR) is an endogenously produced chemical that is routinely assayed in urine specimens to assess kidney function and sample dilution. The industry-standard method for CR determination, known as the kinetic Jaffé (KJ) method, relies on an exponential rate of a colorimetric change, and can therefore require automated processing equipment for moderate- to high-throughput analysis (hundreds to thousands of samples per day). This study evaluates an alternative colorimetric method, the "plateau Jaffé" (PJ) method, which utilizes the chemistry of the KJ method, a commercially available kit, and a multipoint calibration curve. This method is amenable to moderate-throughput sample analysis and does not require automated processing equipment. Thirty-two spot urine samples from healthy adult volunteers were analyzed for creatinine concentration (CRc) using the KJ and PJ methods. Samples were also analyzed using a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) method, which acted as an analytical control. Replicate measurements of spot samples (natural log-transformed values) were used to estimate method precision, and linear regression models were used to evaluate method accuracy (LC-TOF/MS measurements were considered the analytical benchmark). Measurement precision was comparable across all three methods, with coefficent of variation estimates ranging from 3 to 6%. Regression models generally showed good agreement across methods with R(2) estimates ranging from .996 to .998, slope estimates ranging from .944 to .986, and y-intercept estimates ranging from 0.111 to 0.303. Minor bias (between 2 and 16%) was observed across methods at the tails of the measurement distributions. The provided regression equations can be used to adjust for this bias and to improve CR measurement comparisons across studies employing different methods. Considering these results, the PJ method is a suitable alternative to the industry standard KJ method for urinary CRc determination. It can be implemented for moderate-throughput sample analysis using modest and commonly available lab instrumentation and manual sample preparation techniques.

Systemic exposure to PAHs and benzene in firefighters suppressing controlled structure fires.

Turnout gear provides protection against dermal exposure to contaminants during firefighting; however, the level of protection is unknown. We explored the dermal contribution to the systemic dose of polycyclic aromatic hydrocarbons (PAHs) and other aromatic hydrocarbons in firefighters during suppression and overhaul of controlled structure burns. The study was organized into two rounds, three controlled burns per round, and five firefighters per burn. The firefighters wore new or laundered turnout gear tested before each burn to ensure lack of PAH contamination. To ensure that any increase in systemic PAH levels after the burn was the result of dermal rather than inhalation exposure, the firefighters did not remove their self-contained breathing apparatus until overhaul was completed and they were >30 m upwind from the burn structure. Specimens were collected before and at intervals after the burn for biomarker analysis. Urine was analyzed for phenanthrene equivalents using enzyme-linked immunosorbent assay and a benzene metabolite (s-phenylmercapturic acid) using liquid chromatography/tandem mass spectrometry; both were adjusted by creatinine. Exhaled breath collected on thermal desorption tubes was analyzed for PAHs and other aromatic hydrocarbons using gas chromatography/mass spectrometry. We collected personal air samples during the burn and skin wipe samples (corn oil medium) on several body sites before and after the burn. The air and wipe samples were analyzed for PAHs using a liquid chromatography with photodiode array detection. We explored possible changes in external exposures or biomarkers over time and the relationships between these variables using non-parametric sign tests and Spearman tests, respectively. We found significantly elevated (P < 0.05) post-exposure breath concentrations of benzene compared with pre-exposure concentrations for both rounds. We also found significantly elevated post-exposure levels of PAHs on the neck compared with pre-exposure levels for round 1. We found statistically significant positive correlations between external exposures (i.e. personal air concentrations of PAHs) and biomarkers (i.e. change in urinary PAH metabolite levels in round 1 and change in breath concentrations of benzene in round 2). The results suggest that firefighters wearing full protective ensembles absorbed combustion products into their bodies. The PAHs most likely entered firefighters' bodies through their skin, with the neck being the primary site of exposure and absorption due to the lower level of dermal protection afforded by hoods. Aromatic hydrocarbons could have been absorbed dermally during firefighting or inhaled during the doffing of gear that was off-gassing contaminants.