Impact of E-Cigarette Liquid Flavoring Agents on Activity of Microsomal Recombinant CYP2A6, the Primary Nicotine-Metabolizing Enzyme.

Nicotine is the primary psychoactive chemical in both traditional and electronic cigarettes (e-cigarettes). Nicotine levels in both traditional cigarettes and e-cigarettes are an important concern for public health. Nicotine exposure due to e-cigarette use is of importance primarily due to the addictive potential of nicotine, but there is also concern for nicotine poisoning in e-cigarette users. Nicotine concentrations in e-liquids vary widely. Additionally, there is significant genetic variability in the rate of metabolism of nicotine due to polymorphisms of CYP2A6, the enzyme responsible for the metabolism of approximately 80% of nicotine. Recent studies have shown CYP2A6 activity is also reduced by aromatic aldehydes such as those added to e-liquids as flavoring agents, which may increase nicotine serum concentrations. However, the impacts of flavored e-liquids on CYP2A6 activity are unknown. In this study, we investigated the impact of three flavored e-liquids on microsomal recombinant CYP2A6. Microsomal recombinant CYP2A6 was challenged at e-liquid concentrations ranging up to 0.125% (v/v) and monitored for metabolic activity using a probe molecule approach. Two e-liquids exhibited dose-dependent inhibition of CYP2A6 activity. Mass spectrometry was conducted to identify flavoring agents in flavored e-liquids that inhibited CYP2A6. Microsomal recombinant CYP2A6 was subsequently exposed to flavoring agents at concentrations ranging from 0.03 µM to 500 µM. Cinnamaldehyde and benzaldehyde were found to be the most potent inhibitors of microsomal CYP2A6 of the flavoring agents tested, with identified IC50 values of 1.1 µM and 3.0 µM, respectively. These data indicate certain aromatic aldehyde flavoring agents are potent inhibitors of CYP2A6, which may reduce nicotine metabolism in vivo. These findings indicate an urgent need to evaluate the effects of flavoring agents in e-cigarette liquids on the pharmacokinetics of nicotine in vivo.

A Fulvic Acid-like Substance Participates in the Pro-inflammatory Effects of Cigarette Smoke and Wood Smoke Particles.

We tested the postulates that (1) a fulvic acid (FA)-like substance is included in cigarette smoke and wood smoke particles (WSP) and (2) cell exposure to this substance results in a disruption of iron homeostasis, associated with a deficiency of the metal and an inflammatory response. The fluorescence excitation-emission matrix spectra of the water-soluble components of cigarette smoke condensate and WSP (Cig-WS and Wood-WS) approximated those for the standard reference materials, Suwanee River and Nordic fulvic acids (SRFA and NFA). Fourier transform infrared spectra for the FA fraction of cigarette smoke and WSP (Cig-FA and Wood-FA), SRFA, and NFA also revealed significant similarities (O-H bond in alcohols, phenols, and carboxylates, C═O in ketones, aldehydes, and carboxylates, and a significant carboxylate content). After exposure to Cig-WS and Wood-WS and the FA standards, iron was imported by respiratory epithelial cells, reflecting a functional iron deficiency. The release of pro-inflammatory mediators interleukin (IL)-8 and IL-6 by respiratory epithelial cells also increased following exposures to Cig-WS, Wood-WS, SRFA, and NFA. Co-exposure of the respiratory epithelial cells with iron decreased supernatant concentrations of the ILs relative to exposures to Cig-WS, Wood-WS, SRFA, and NFA alone. It is concluded that (1) a FA-like substance is included in cigarette smoke and WSP and (2) respiratory epithelial cell exposure to this substance results in a disruption of iron homeostasis associated with both a cell deficiency of the metal and an inflammatory response.

Ozone Reacts With Carbon Black to Produce a Fulvic Acid-Like Substance and Increase an Inflammatory Effect.

Exposure to ambient ozone has been associated with increased human mortality. Ozone exposure can introduce oxygen-containing functional groups in particulate matter (PM) effecting a greater capacity of the particle for metal complexation and inflammatory effect. We tested the postulate that (1) a fulvic acid-like substance can be produced through a reaction of a carbonaceous particle with high concentrations of ozone and (2) such a fulvic acid-like substance included in the PM can initiate inflammatory effects following exposure of respiratory epithelial (BEAS-2B) cells and an animal model (male Wistar Kyoto rats). Carbon black (CB) was exposed for 72 hours to either filtered air (CB-Air) or approximately 100 ppm ozone (CB-O3). Carbon black exposure to high levels of ozone produced water-soluble, fluorescent organic material. Iron import by BEAS-2B cells at 4 and 24 hours was not induced by incubations with CB-Air but was increased following coexposures of CB-O3 with ferric ammonium citrate. In contrast to CB-Air, exposure of BEAS-2B cells and rats to CB-O3 for 24 hours increased expression of pro-inflammatory cytokines and lung injury, respectively. It is concluded that inflammatory effects of carbonaceous particles on cells can potentially result from (1) an inclusion of a fulvic acid-like substance after reaction with ozone and (2) changes in iron homeostasis following such exposure.

Oleic acid and derivatives affect human endothelial cell mitochondrial function and vasoactive mediator production.

BACKGROUND: Inhalation of common air pollutants such as diesel and biodiesel combustion products can induce vascular changes in humans which may contribute to increased mortality and morbidity associated with fine particulate matter exposures. Diesel, biodiesel, and other combustion byproducts contain fatty acid components capable of entering the body through particulate matter inhalation. Fatty acids can also be endogenously released into circulation following a systemic stress response to some inhaled pollutants such as ozone. When in the circulation, bioactive fatty acids may interact with cells lining the blood vessels, potentially inducing endothelial dysfunction. To examine whether fatty acids could potentially be involved in human vascular responses to air pollutants, we determined the effects of fatty acids and derivatives on important vascular cell functions. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to oleic acid (OA) or OA metabolites for 4-48 h. Cytotoxicity, vasodilator production (by ELISA measurement), mitochondrial function (using Sea Horse assays), and iron metabolism (inferred by ICP-OES measurements) were examined, with standard statistical testing (ANOVA, t-tests) employed. RESULTS: Dose-dependent cytotoxicity was noted at 24 h, with 12-hydroxy OA more potent than OA. Mitochondrial stress testing showed that 12-hydroxy OA and OA induce mitochondrial dysfunction. Analysis of soluble mediator release from HUVEC showed a dose-dependent increase in prostaglandin F2α, a lipid involved in control of vascular tone, at 24 h (85% above controls) after OA-BSA exposure. RT-PCR analysis revealed OA did not induce changes in gene expression at noncytotoxic concentrations in exposed HUVEC, but 12-OH OA did alter ICAM and COX2 gene expression. CONCLUSIONS: Together, these data demonstrate that FA may be capable of inducing cytotoxic effects and altering expression of mediators of vascular function following inhalation exposure, and may be implicated in air pollutant-induced deaths and hospitalizations. (267 of max 350 words).

Quartz Disrupts Iron Homeostasis in Alveolar Macrophages To Impact a Pro-Inflammatory Effect.

The biological response of bronchial epithelial cells to particles is associated with a sequestration of cell metal by the particle surface and a subsequent disruption in host iron homeostasis. The macrophage is the cell type resident in the respiratory tract that is most likely to make initial contact with inhaled particles. We tested the postulates that (1) silica, a prototypical particle, disrupts iron homeostasis in alveolar macrophages (AMs); and (2) the altered iron homeostasis results in both an oxidative stress and pro-inflammatory effects. Human AMs (1.0 × 106/mL) demonstrated an increased import of iron following particle exposure with nonheme iron concentrations of 0.57 ± 0.03, 1.72 ± 0.09, 0.88 ± 0.09, and 3.21 ± 0.11 ppm in cells exposed for 4 h to media, 500 µM ferric ammonium citrate (FAC), 100 µg/mL silica, and both silica and FAC, respectively. Intracellular ferritin concentrations and iron release were similarly increased after AM exposure to FAC and silica. Silica increased oxidant generation by AMs measured using both dichlorofluorescein diacetate fluorescence and reduction of nitroblue tetrazolium salt. Concentrations of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α in macrophage supernatant increased following 100 µg/mL silica exposure for 24 h. Treatment of AMs with 500 µM FAC decreased both oxidant generation and cytokine release associated with silica exposure, supporting a dependence of these effects on sequestration of cell metal by the particle surface. We conclude that (1) silica exposure disrupts iron homeostasis resulting in increased import, accumulation, and release of the metal; and (2) the altered iron homeostasis following silica exposure impacts oxidant generation and pro-inflammatory effects.

12-hydroxy oleic acid impairs endothelium-dependent vasorelaxation.

Diesel and biodiesel emissions exposures reduce vascular responsiveness in vivo, but the components of PM responsible for this effect are poorly understood. Fatty acids (FAs) represent a significant fraction of the compounds that make up organic combustion by-products, and may be involved in vascular responses following inhalation. It was hypothesized that vascular tissue exposed to a model FA might impair responses to vasoactive agonists ex vivo. Rat aortic rings were exposed to oleic acid or 12-hydroxy oleic acid and responses determined by myography. 12-Hydroxy oleic acid was found to significantly reduce endothelium-dependent vasodilation at sub-cytotoxic concentrations. This approach demonstrates the potential for FAs, especially oxidized forms, to play a role in the vascular responses observed following air pollution exposure.

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.

The toxicology of air pollution predicts its epidemiology.

The epidemiologic investigation has successively delineated associations of air pollution exposure with non-malignant and malignant lung disease, cardiovascular disease, cerebrovascular disease, pregnancy outcomes, perinatal effects and other extra-pulmonary disease including diabetes. Defining these relationships between air pollution exposure and human health closely parallels results of an earlier epidemiologic investigation into cigarette smoking and environmental tobacco smoke (ETS), two other particle-related exposures. Humic-like substances (HULIS) have been identified as a chemical component common to cigarette smoke and air pollution particles. Toxicology studies provide evidence that a disruption of iron homeostasis with sequestration of host metal by HULIS is a fundamental mechanistic pathway through which biological effects are initiated by cigarette smoke and air pollution particles. As a result of a common chemical component and a shared mechanistic pathway, it should be possible to extrapolate from the epidemiology of cigarette smoking and ETS to predict associations of air pollution exposure with human disease, which are currently unrecognized. Accordingly, it is anticipated that the forthcoming epidemiologic investigation will demonstrate relationships of air pollution with COPD causation, peripheral vascular disease, hypertension, renal disease, digestive disease, loss of bone mass/risk of fractures, dental disease, eye disease, fertility problems, and extrapulmonary malignancies.

Human lung injury following exposure to humic substances and humic-like substances.

Among the myriad particles the human respiratory tract is exposed to, a significant number are distinctive in that they include humic substances (HS) and humic-like substances (HULIS) as organic components. HS are heterogeneous, amorphous, organic materials which are ubiquitous occurring in all terrestrial and aqueous environments. HULIS are a complex class of organic, macromolecular compounds initially extracted from atmospheric aerosol particles which share some features with HS including an aromatic, polyacidic nature. As a result of having a variety of oxygen-containing functional groups, both HS and HULIS complex metal cations, especially iron. Following particle uptake by cells resident in the lung, host iron will be sequestered by HS- and HULIS-containing particles initiating pathways of inflammation and subsequent fibrosis. It is proposed that (1) human exposures to HS and HULIS of respirable size (<10 µm diameter) are associated with inflammatory and fibrotic lung disease and (2) following retention of particles which include HS and HULIS, the mechanism of cell and tissue injury involves complexation of host iron. Human inflammatory and fibrotic lung injuries following HS and HULIS exposures may include coal workers' pneumoconiosis, sarcoidosis, and idiopathic pulmonary fibrosis as well as diseases associated with cigarette smoking and exposures to emission and ambient air pollution particles.

A paler shade of green? The toxicology of biodiesel emissions: Recent findings from studies with this alternative fuel.

BACKGROUND: Biodiesel produced primarily from plants and algal feedstocks is believed to have advantages for production and use compared to petroleum and to some other fuel sources. There is some speculation that exposure to biodiesel combustion emissions may not induce biological responses or health effects or at a minimum reduce the effects relative to other fuels. In evaluating the overall environmental and health effects of biodiesel production to end use scenario, empirical data or modeling data based on such data are needed. SCOPE OF REVIEW: This manuscript examines the available toxicology reports examining combustion derived biodiesel emissions since approximately 2007, when our last review of the topic occurred. Toxicity derived from other end uses of biodiesel - e.g., spills, dermal absorption, etc. - are not examined. Findings from biodiesel emissions are roughly divided into three areas: whole non-human animal model exposures; in vitro exposures of mammalian and bacterial cells (used for mutation studies primarily); and human exposures in controlled or other exposure fashions. MAJOR CONCLUSIONS: Overall, these more current studies clearly demonstrate that biodiesel combustion emission exposure- to either 100% biodiesel or a blend in petroleum diesel- can induce biological effects. There are reports that show biodiesel exposure generally induces more effects or a greater magnitude of effect than petroleum diesel, however there are also a similar number of reports showing the opposite trend. It is unclear whether effects induced by exposure to a blend are greater than exposure to 100% biodiesel. Taken together, the evidence suggest biodiesel emissions can have some similar effects as diesel emissions on inflammatory, vascular, mutagenic, and other responses. GENERAL SIGNIFICANCE: While acute biodiesel exposures can show toxicity with a variety of endpoints, the potential effects on human health need further validation. Additionally there are few or no findings to date on whether biodiesel emissions can induce effects or even a weaker response that petroleum diesel with repeated exposure scenarios such as in an occupational setting. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

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.

Ozone Exposure Increases Circulating Stress Hormones and Lipid Metabolites in Humans.

RATIONALE: Air pollution has been associated with increased prevalence of type 2 diabetes; however, the mechanisms remain unknown. We have shown that acute ozone exposure in rats induces release of stress hormones, hyperglycemia, leptinemia, and glucose intolerance that are associated with global changes in peripheral glucose, lipid, and amino acid metabolism. OBJECTIVES: To examine ozone-induced metabolic derangement in humans using serum metabolomic assessment, establish human-to-rodent coherence, and identify novel nonprotein biomarkers. METHODS: Serum samples were obtained from a crossover clinical study that included two clinic visits (n = 24 each) where each subject was blindly exposed in the morning to either filtered air or 0.3 parts per million ozone for 2 hours during 15-minute on-off exercise. Serum samples collected within 1 hour after exposure were assessed for changes in metabolites using a metabolomic approach. MEASUREMENTS AND MAIN RESULTS: Metabolomic analysis revealed that ozone exposure markedly increased serum cortisol and corticosterone together with increases in monoacylglycerol, glycerol, and medium- and long-chain free fatty acids, reflective of lipid mobilization and catabolism. Additionally, ozone exposure increased serum lysolipids, potentially originating from membrane lipid breakdown. Ozone exposure also increased circulating mitochondrial ß-oxidation-derived metabolites, such as acylcarnitines, together with increases in the ketone body 3-hydroxybutyrate. These changes suggested saturation of ß-oxidation by ozone in exercising humans. CONCLUSIONS: As in rodents, acute ozone exposure increased stress hormones and globally altered peripheral lipid metabolism in humans, likely through activation of a neurohormonally mediated stress response pathway. The metabolomic assessment revealed new biomarkers and allowed for establishment of rodent-to-human coherence. Clinical trial registered with www.clinicaltrials.gov (NCT 01492517).

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.

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.

Comparative toxicity and mutagenicity of soy-biodiesel and petroleum-diesel emissions: overview of studies from the U.S. EPA, Research Triangle Park, NC.

Biodiesel use as a fuel is increasing globally as an alternate to petroleum sources. To comprehensively assess the effects of the use of biodiesel as an energy source, end stage uses of biodiesel such as the effects of inhalation of combusted products on human health must be incorporated. To date, few reports concerning the toxicological effects of the emissions of combusted biodiesel or blends of biodiesel on surrogates of health effects have been published. The relative toxicity of the combusted biodiesel emissions compared to petroleum diesel emissions with short term exposures is also not well known. To address the paucity of findings on the toxicity of combusted biodiesel emissions, studies were undertaken at the U.S. Environmental Protection Agency laboratories in Research Triangle Park, North Carolina. The studies used a variety of approaches with nonhuman animal models to examine biological responses of the lung and cardiovascular systems induced by acute and repeated exposures to pure biodiesel and biodiesel blended with petroleum diesel. Effects of the emissions on induction of mutations in bacterial test strains and mammalian DNA adducts were also characterized and normalized to engine work load. The emissions were characterized as to the physicochemical composition in order to determine the magnitude of the differences among the emissions utilized in the studies. This article summarizes the major finding of these studies which are contained within this special issue of Inhalation Toxicology. The findings provided in these articles provide information about the toxicity of biodiesel emissions relative to petroleum diesel emissions and which can be utilized in a life cycle analyses of the effects of increased biodiesel usage.

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.

Diesel exhaust modulates ozone-induced lung function decrements in healthy human volunteers.

The potential effects of combinations of dilute whole diesel exhaust (DE) and ozone (O3), each a common component of ambient airborne pollutant mixtures, on lung function were examined. Healthy young human volunteers were exposed for 2 hr to pollutants while exercising (~50 L/min) intermittently on two consecutive days. Day 1 exposures were either to filtered air, DE (300 µg/m³), O3 (0.300 ppm), or the combination of both pollutants. On Day 2 all exposures were to O3 (0.300 ppm), and Day 3 served as a followup observation day. Lung function was assessed by spirometry just prior to, immediately after, and up to 4 hr post-exposure on each exposure day. Functional pulmonary responses to the pollutants were also characterized based on stratification by glutathione S-transferase mu 1 (GSTM1) genotype. On Day 1, exposure to air or DE did not change FEV1 or FVC in the subject population (n = 15). The co-exposure to O3 and DE decreased FEV1 (17.6%) to a greater extent than O3 alone (9.9%). To test for synergistic exposure effects, i.e., in a greater than additive fashion, FEV1 changes post individual O3 and DE exposures were summed together and compared to the combined DE and O3 exposure; the p value was 0.057. On Day 2, subjects who received DE exposure on Day 1 had a larger FEV1 decrement (14.7%) immediately after the O3 exposure than the individuals' matched response following a Day 1 air exposure (10.9%). GSTM1 genotype did not affect the magnitude of lung function changes in a significant fashion. These data suggest that altered respiratory responses to the combination of O3 and DE exposure can be observed showing a greater than additive manner. In addition, O3-induced lung function decrements are greater with a prior exposure to DE compared to a prior exposure to filtered air. Based on the joint occurrence of these pollutants in the ambient environment, the potential exists for interactions in more than an additive fashion affecting lung physiological processes.

E-Cigarette Liquids and Aldehyde Flavoring Agents Inhibit CYP2A6 Activity in Lung Epithelial Cells.

Certain e-liquids and aromatic aldehyde flavoring agents were previously identified as inhibitors of microsomal recombinant CYP2A6, the primary nicotine-metabolizing enzyme. However, due to their reactive nature, aldehydes may react with cellular components before reaching CYP2A6 in the endoplasmic reticulum. To determine whether e-liquid flavoring agents inhibited CYP2A6 in a cellular system, we investigated their effects on CYP2A6 using BEAS-2B cells transduced to overexpress CYP2A6. We demonstrated that two e-liquids and three aldehyde flavoring agents (cinnamaldehyde, benzaldehyde, and ethyl vanillin) exhibited dose-dependent inhibition of cellular CYP2A6.