Monitoring redox stress in human airway epithelial cells exposed to woodsmoke at an air-liquid interface.

Wildland fires contribute significantly to the ambient air pollution burden worldwide, causing a range of adverse health effects in exposed populations. The toxicity of woodsmoke, a complex mixture of gases, volatile organic compounds, and particulate matter, is commonly studied in vitro using isolated exposures of conventionally cultured lung cells to either resuspended particulate matter or organic solvent extracts of smoke, leading to incomplete toxicity evaluations. This study aimed to improve our understanding of the effects of woodsmoke inhalation by building an advanced in vitro exposure system that emulates human exposure of the airway epithelium. We report the development and characterization of an innovative system that permits live-cell monitoring of the intracellular redox status of differentiated primary human bronchial epithelial cells cultured at an air-liquid interface (pHBEC-ALI) as they are exposed to unfractionated woodsmoke generated in a tube furnace in real time. pHBEC-ALI exposed to freshly generated woodsmoke showed oxidative changes that were dose-dependent and reversible, and not attributable to carbon monoxide exposure. These findings show the utility of this novel system for studying the molecular initiating events underlying woodsmoke-induced toxicity in a physiologically relevant in vitro model, and its potential to provide biological plausibility for risk assessment and public health measures.

Omega-3 fatty acids attenuate cardiovascular effects of short-term exposure to ambient air pollution.

BACKGROUND: Exposure to air pollution is associated with elevated cardiovascular risk. Evidence shows that omega-3 polyunsaturated fatty acids (omega-3 PUFA) may attenuate the adverse cardiovascular effects of exposure to fine particulate matter (PM2.5). However, it is unclear whether habitual dietary intake of omega-3 PUFA protects against the cardiovascular effects of short-term exposure to low-level ambient air pollution in healthy participants. In the present study, sixty-two adults with low or high dietary omega-3 PUFA intake were enrolled. Blood lipids, markers of vascular inflammation, coagulation and fibrinolysis, and heart rate variability (HRV) and repolarization were repeatedly assessed in 5 sessions separated by at least 7 days. This study was carried out in the Research Triangle area of North Carolina, USA between October 2016 and September 2019. Daily PM2.5 and maximum 8-h ozone (O3) concentrations were obtained from nearby air quality monitoring stations. Linear mixed-effects models were used to assess the associations between air pollutant concentrations and cardiovascular responses stratified by the omega-3 intake levels. RESULTS: The average concentrations of ambient PM2.5 and O3 were well below the U.S. National Ambient Air Quality Standards during the study period. Significant associations between exposure to PM2.5 and changes in total cholesterol, von Willebrand factor (vWF), tissue plasminogen activator, D-dimer, and very-low frequency HRV were observed in the low omega-3 group, but not in the high group. Similarly, O3-associated adverse changes in cardiovascular biomarkers (total cholesterol, high-density lipoprotein, serum amyloid A, soluable intracellular adhesion molecule 1, and vWF) were mainly observed in the low omega-3 group. Lag-time-dependent biphasic changes were observed for some biomarkers. CONCLUSIONS: This study demonstrates associations between short-term exposure to PM2.5 and O3, at concentrations below regulatory standard, and subclinical cardiovascular responses, and that dietary omega-3 PUFA consumption may provide protection against such cardiovascular effects in healthy adults.

Can disposable masks be worn more than once?

Disposable facemasks are a primary tool to prevent the transmission of SARS-COV-2 during the COVID-19 pandemic. However, plastic waste generated from their disposal represents a significant environmental problem that can be reduced by maximizing the service life of disposable masks. We evaluated the effect of repeated wearing on the fitted filtration efficiency (FFE) of N95, KF94, KN95, and procedure/surgical masks. The FFEs of masks were compared following extended wearing with and without washing. Results reveal that most disposable facemasks can retain a high level of their baseline FFE after extended wearing, even after 40 h of wearing. Laundering disposable masks degraded FFE in some instances. We conclude that the durability of disposable facemask performance is considerably longer than their intended single use indication, suggesting that reusing disposable masks is a safe means of reducing plastic waste in the environment.

Circulating microRNAs as putative mediators in the association between short-term exposure to ambient air pollution and cardiovascular biomarkers.

BACKGROUND: Exposure to ambient air pollution is associated with increased cardiovascular morbidity and mortality. Circulating microRNAs (miRNAs) may mediate cardiovascular effects of exposure to air pollution. This study aims to investigate whether circulating miRNAs mediate the associations between short-term human exposure to ambient air pollution and cardiovascular biomarkers. METHODS: Twenty-four healthy adults residing in the Research Triangle area of North Carolina, USA were enrolled between December 2016 and July 2019. Circulating miRNAs, protein, and lipid biomarkers were assessed repeatedly for 3 sessions separated by at least 7 days. Linear mixed-effects models were used to assess the associations between air pollutant concentrations obtained from nearby air quality monitoring stations and miRNAs controlling for covariates including omega-3 index, relative humidity, and temperature. miRNAs that were significantly altered were then matched with protein or blood lipid biomarkers using either Ingenuity Pathway Analysis or a literature search. A mediation analysis was performed to test the statistical significance of miRNA's mediating effects between exposure to air pollution and cardiovascular biomarkers. RESULTS: Short-term exposure to ambient fine particulate matter (PM2.5), ozone (O3), and nitrogen dioxide (NO2) was associated with changes in 11, 9, and 24 circulating miRNAs, respectively. Pathway analysis showed that several miRNAs including miR-125b-5p, miR-144-5p, miR-26a-5p, and miR-34a-5p may mediate the effects of air pollutant exposure on the changes of downstream protein / lipid biomarkers including serum amyloid A (SAA), C-reactive protein (CRP), soluble vascular adhesive molecules 1 (sICAM1), total cholesterol, and high-density lipoproteins (HDL). Mediation analysis showed that only miR-26a-5p significantly mediated air pollutant (PM2.5 and NO2)-induced effects on blood CRP and total cholesterol levels. For example, 34.1% of PM2.5-associated changes in CRP were significantly mediated by miR-26a-5p at lag4 [indirect effects, 0.06 (0.02, 0.10), P = 0.005]. Similarly, the proportions of indirect effects of miR-26a-5p on the association between NO2 exposure and CRP were 46.8% at lag2 [0.06 (0.02, 0.11), P = 0.003], 61.2% at lag3 [0.05 (0.00, 0.09), P = 0.04], and 30.8% at 5-day moving average [0.06 (0.02, 0.10), P = 0.01]. In addition, omega-3 index may be a significant modifying factor of the mediated effects of miRNAs. CONCLUSIONS: This study demonstrates that short-term exposure to ambient PM2.5, O3, and NO2 was associated with specific circulating miRNAs, and some of which may mediate their effects on the downstream inflammation and blood lipid markers.

The influence of dietary intake of omega-3 polyunsaturated fatty acids on the association between short-term exposure to ambient nitrogen dioxide and respiratory and cardiovascular outcomes among healthy adults.

BACKGROUND: Short-term exposure to ambient nitrogen dioxide (NO2) is associated with adverse respiratory and cardiovascular outcomes. Supplementation of omega-3 polyunsaturated fatty acids (PUFA) has shown protection against exposure to fine particulate matter. This study aims to investigate whether habitual omega-3 PUFA intake differentially modify the associations between respiratory and cardiovascular responses and short-term exposure to ambient NO2. METHODS: Sixty-two healthy participants were enrolled into low or high omega-3 groups based on their habitual omega-3 PUFA intake. Each participant was repeatedly assessed for lung function, blood lipids, markers of coagulation and fibrinolysis, vascular function, and heart rate variability (HRV) in up to five sessions, each separated by at least 7 days. This study was carried out in the Research Triangle area of North Carolina, USA between October 2016 and September 2019. Daily ambient NO2 concentrations were obtained from an area air quality monitoring station on the day of outcome assessment (Lag0), 4 days prior (Lag1-4), as well as 5-day moving average (5dMA). The associations between short-term exposure to NO2 and the measured indices were evaluated using linear mixed-effects models stratified by omega-3 levels and adjusted by covariates including relative humidity and temperature. RESULTS: The average concentration of ambient NO2 during the study periods was 5.3±3.8 ppb which was below the National Ambient Air Quality Standards (NAAQS). In the high omega-3 group, an interquartile range (IQR) increase in short-term NO2 concentrations was significantly associated with increased lung function [e.g. 1.2% (95%CI: 0.2%, 2.2%) in FVC at lag1, 2.6% (95%CI: 0.4%, 4.8%) in FEV1 at 5dMA], decreased blood lipids [e.g. -2.6% (95%CI: -4.4%, -0.9%) in total cholesterol at lag2, -3.1% (95%CI: -6.1%, 0.0%) in HDL at 5dMA, and -3.1% (95%CI: -5.5%, -0.7%) in LDL at lag2], improved vascular function [e.g. 8.9% (95%CI: 0.6%, 17.2%) increase in FMD and 43.1% (95%CI: -79.8%, -6.3%) decrease in endothelin-1 at 5dMA], and changed HRV parameters [e.g. -7.2% (95%CI: -13.6%, -0.8%) in HFn and 13.4% (95%CI: 0.2%, 28.3%) in LF/HF ratio at lag3]. In the low omega-3 group, an IQR increase in ambient NO2 was associated with elevations in coagulation markers (von Willebrand Factor, D-dimer) and a decrease in HRV (very-low frequency); however, null associations were observed between short-term NO2 exposure and changes in lung function, blood lipids, and vascular function. CONCLUSIONS: The results in this study imply that dietary omega-3 PUFA consumption may offer respiratory and vascular benefits in response to short-term exposure of healthy adults to NO2 levels below the NAAQS. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT02921048 ).

Cardiovascular health impacts of wildfire smoke exposure.

In recent years, wildland fires have occurred more frequently and with increased intensity in many fire-prone areas. In addition to the direct life and economic losses attributable to wildfires, the emitted smoke is a major contributor to ambient air pollution, leading to significant public health impacts. Wildfire smoke is a complex mixture of particulate matter (PM), gases such as carbon monoxide, nitrogen oxide, and volatile and semi-volatile organic compounds. PM from wildfire smoke has a high content of elemental carbon and organic carbon, with lesser amounts of metal compounds. Epidemiological studies have consistently found an association between exposure to wildfire smoke (typically monitored as the PM concentration) and increased respiratory morbidity and mortality. However, previous reviews of the health effects of wildfire smoke exposure have not established a conclusive link between wildfire smoke exposure and adverse cardiovascular effects. In this review, we systematically evaluate published epidemiological observations, controlled clinical exposure studies, and toxicological studies focusing on evidence of wildfire smoke exposure and cardiovascular effects, and identify knowledge gaps. Improving exposure assessment and identifying sensitive cardiovascular endpoints will serve to better understand the association between exposure to wildfire smoke and cardiovascular effects and the mechanisms involved. Similarly, filling the knowledge gaps identified in this review will better define adverse cardiovascular health effects of exposure to wildfire smoke, thus informing risk assessments and potentially leading to the development of targeted interventional strategies to mitigate the health impacts of wildfire smoke.

Supplementation with omega-3 fatty acids potentiates oxidative stress in human airway epithelial cells exposed to ozone.

BACKGROUND: Dietary intake of the omega-3 family of polyunsaturated fatty acids (ω-3 FA) is associated with anti-inflammatory effects. However, unsaturated fatty acids are susceptible to oxidation, which produces pro-inflammatory mediators. Ozone (O3) is a tropospheric pollutant that reacts rapidly with unsaturated fatty acids to produce electrophilic and oxidative mediators of inflammation. OBJECTIVE: Determine whether supplementation with ω-3 FA alters O3-induced oxidative stress in human airway epithelial cells (HAEC). METHODS: 16-HBE cells expressing a genetically encoded sensor of the reduced to oxidized glutathione ratio (GSH/GSSG, EGSH) were supplemented with saturated, monounsaturated, or ω-3 FA prior to exposure to 0, 0.08, 0.1, or 0.3 ppm O3. Lipid peroxidation was measured in cellular lipid extracts and intact cells following O3 exposure. RESULTS: Relative to cells incubated with the saturated or monounsaturated fatty acids, cells supplemented with ω-3 FA containing 5 or 6 double bonds showed a marked increase in EGSH during exposure to O3 concentrations as low as 0.08 ppm. Consistent with this finding, the concentration of lipid hydroperoxides produced following O3 exposure was significantly elevated in ω-3 FA supplemented cells. DISCUSSION: Supplementation with polyunsaturated ω-3 FA potentiates oxidative responses, as indicated by EGSH, in HAEC exposed to environmentally relevant concentrations of O3. This effect is mediated by the increased formation of lipid hydroperoxides produced by the reaction of O3 with polyunsaturated fatty acids. Given the inflammatory activity of lipid hydroperoxides, these findings have implications for the potential role of ω-3 FA in increasing human susceptibility to the adverse health effects of O3 exposure.

Macrophages from the upper and lower human respiratory tract are metabolically distinct.

The function and cell surface phenotype of lung macrophages vary within the respiratory tract. Alterations in the bioenergetic profile of macrophages may also be influenced by their location within the respiratory tract. This study sought to characterize the bioenergetic profile of macrophages sampled from different locations within the respiratory tract at baseline and in response to ex vivo xenobiotic challenge. Surface macrophages recovered from healthy volunteers by induced sputum and by bronchial and bronchoalveolar lavage were profiled using extracellular flux analyses. Oxygen consumption and extracellular acidification rates were measured at rest and after stimulation with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or 1,2-naphthoquinone (1,2-NQ). Oxygen consumption and extracellular acidification rates were highly correlated for all macrophage samples. Induced sputum macrophages had relatively higher oxygen consumption and extracellular acidification rates and were largely reliant on glycolysis. In contrast, bronchial fraction and bronchoalveolar macrophages depended more heavily on mitochondrial respiration. Bronchoalveolar macrophages showed elevated LPS-induced cytokine responses. Unlike their autologous peripheral blood monocytes, lung macrophages from any source did not display bioenergetic changes following LPS stimulation. The protein kinase C activator PMA did not affect mitochondrial respiration, whereas the air pollutant 1,2-NQ induced marked mitochondrial dysfunction in bronchoalveolar and bronchial fraction macrophages. The bioenergetic characteristics of macrophages from healthy individuals are dependent on their location within the respiratory tract. These findings establish a regional bioenergetic profile for macrophages from healthy human airways that serves as a reference for changes that occur in disease.

Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components.

Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H2O2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H2O2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air.

Inflammatory cell signaling following exposures to particulate matter and ozone.

BACKGROUND: Particulate matter (PM) and ozone (O3) are two major ambient air pollutants. Epidemiological and toxicological studies have demonstrated exposure to these pollutants is associated with a variety of adverse health effects, including cardiovascular and respiratory disease, in which inflammation is believed to be a common and essential factor. SCOPE OF REVIEW: This review mainly focuses on major inflammatory cell signaling pathways triggered by exposure to PM and O3. The receptors covered in this review include the EGF receptor, toll like receptor, and NOD-like receptor. Intracellular signaling protein kinases depicted in this review are phosphatidylinositol 3-kinase and mitogen-activated protein kinases. Activation of antioxidant and inflammatory transcription factors such as NrF2 and NFκB induced by PM and O3 is also discussed. MAJOR CONCLUSIONS: Exposure to PM or O3 can activate cellular signaling networks including membrane receptors, intracellular kinases and phosphatases, and transcription factors that regulate inflammatory responses. While PM-induced cell signaling is associated with resultant ROS, O3-induced cell signaling implicates phosphates. Notably, the cellular signaling induced by PM and O3 exposure varies with cell type and physiochemical properties of these pollutants. GENERAL SIGNIFICANCE: Cellular signaling plays a critical role in the regulation of inflammatory pathogenesis. Elucidation of cellular signaling pathways initiated by PM or O3 cannot only help to uncover the mechanisms of air pollutant toxicity but also provide clues for development of interventional measures against air pollution-induced disorders. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

Live-cell imaging approaches for the investigation of xenobiotic-induced oxidant stress.

BACKGROUND: Oxidant stress is arguably a universal feature in toxicology. Research studies on the role of oxidant stress induced by xenobiotic exposures have typically relied on the identification of damaged biomolecules using a variety of conventional biochemical and molecular techniques. However, there is increasing evidence that low-level exposure to a variety of toxicants dysregulates cellular physiology by interfering with redox-dependent processes. SCOPE OF REVIEW: The study of events involved in redox toxicology requires methodology capable of detecting transient modifications at relatively low signal strength. This article reviews the advantages of live-cell imaging for redox toxicology studies. MAJOR CONCLUSIONS: Toxicological studies with xenobiotics of supra-physiological reactivity require careful consideration when using fluorogenic sensors in order to avoid potential artifacts and false negatives. Fortunately, experiments conducted for the purpose of validating the use of these sensors in toxicological applications often yield unexpected insights into the mechanisms through which xenobiotic exposure induces oxidant stress. GENERAL SIGNIFICANCE: Live-cell imaging using a new generation of small molecule and genetically encoded fluorophores with excellent sensitivity and specificity affords unprecedented spatiotemporal resolution that is optimal for redox toxicology studies. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

Regulation of ozone-induced lung inflammation by the epidermal growth factor receptor in mice.

Human exposure to the highly reactive oxidant gas Ozone (O3 ) is associated with inflammatory responses in the airway epithelium. The mechanisms responsible have not been fully elucidated. Epidermal growth factor receptor (EGFR) has previously been shown to play a critical role in the pathogenesis of lung inflammation. To define the role of EGFR in O3 -induced lung inflammation in mice. 40 BALB/c mice were exposed to filtered air (FA) or (0.25, 0.5, 1.00 ppm) O3 for 3 h per day for 7 consecutive days. Levels of reactive oxygen species (ROS), EGF, and transforming growth factor α (TGF-α) in the bronchoalveolar lavage fluid (BALF) of mice were measured using ELISA. BALB/c mice were intratracheally instilled with the EGFR kinase inhibitor PD153035 2 h prior to O3 exposure and every other day thereafter. Phosphorylation of EGFR (Y1068) in lung sections was determined using immunohistochemical staining and western blot 24 h after exposure. Inhalation of O3 induced pronounced lung inflammation in a dose-dependent manner. Levels of ROS, TGF-α, and total proteins and cells in the BALF of mice exposed to 0.5 ppm or 1.0 ppm of O3 were markedly elevated relative to those in the BALF of the mice exposed to FA. In addition, exposure to O3 induced EGFR(Y1068) phosphorylation in the airway epithelium. Administration of PD153035 resulted in a significantly reduced lung inflammation as well as EGFR phosphorylation induced by O3 exposure. Inhalation of O3 leads to inflammatory responses that are dependent on the activation the EGFR in the airway epithelium. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2016-2027, 2016.

Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress.

Oxidative stress is a commonly cited mechanism of toxicity of environmental agents. Ubiquitous environmental chemicals such as the diesel exhaust component 1,2-naphthoquinone (1,2-NQ) induce oxidative stress by redox cycling, which generates hydrogen peroxide (H2O2). Cysteinyl thiolate residues on regulatory proteins are subjected to oxidative modification by H2O2 in physiological contexts and are also toxicological targets of oxidant stress induced by environmental contaminants. We investigated whether exposure to environmentally relevant concentrations of 1,2-NQ can induce H2O2-dependent oxidation of cysteinyl thiols in regulatory proteins as a readout of oxidant stress in human airway epithelial cells. BEAS-2B cells were exposed to 0-1000 µM 1,2-NQ for 0-30 min, and levels of H2O2 were measured by ratiometric spectrofluorometry of HyPer. H2O2-dependent protein sulfenylation was measured using immunohistochemistry, immunoblotting, and isotopic mass spectrometry. Catalase overexpression was used to investigate the relationship between H2O2 generation and protein sulfenylation in cells exposed to 1,2-NQ. Multiple experimental approaches showed that exposure to 1,2-NQ at concentrations as low as 3 µM induces H2O2-dependent protein sulfenylation in BEAS-2B cells. Moreover, the time of onset and duration of 1,2-NQ-induced sulfenylation of the regulatory proteins GAPDH and PTP1B showed significant differences. Oxidative modification of regulatory cysteinyl thiols in human lung cells exposed to relevant concentrations of an ambient air contaminant represents a novel marker of oxidative environmental stress.

Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy.

There is increasing interest in using live cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of fluorescent signals from multiple probes is that emission spectra of many fluorophores overlap, often with maxima that are only a few nanometers apart. Spectral acquisition of mixed fluorescence signals captured within a dedicated scanning range can be used to quantitatively separate signals into component spectra. We report here the development of a novel live cell application of spectral unmixing for the simultaneous monitoring of intracellular events reported by closely-emitting fluorophores responding dynamically to external stimuli. We validate the performance of dynamic spectral unmixing microscopy (DynSUM) using genetically encoded sensors to simultaneously monitor changes in glutathione redox potential (Egsh) and H2O2 production in living cells exposed to oxidizing and reducing agents. We further demonstrate the utility of the DynSUM approach to observe the relationship between the increases in Egsh and H2O2 generation induced in airway epithelial cells exposed to an environmental electrophile.

Ex vivo chemical cytometric analysis of protein tyrosine phosphatase activity in single human airway epithelial cells.

We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min(-1) mg(-1)) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min(-1) mg(-1) and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min(-1) mg(-1) resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min(-1) mg(-1) (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues.

Cardiovascular effects caused by increasing concentrations of diesel exhaust in middle-aged healthy GSTM1 null human volunteers.

CONTEXT: Epidemiological studies have shown an association between the incidence of adverse cardiovascular effects and exposure to ambient particulate matter (PM). Diesel exhaust (DE) is a major contributor to ambient PM and gaseous emissions in urban areas. OBJECTIVE: This was a pilot study designed to evaluate concentration-dependent effects of short-term exposure to whole DE on the cardiovascular system in order to identify a threshold concentration that can elicit biological responses in healthy human volunteers. MATERIALS AND METHODS: Six healthy middle-aged participants with glutathione-S-transferase-Mu 1 (GSTM1) null genotype underwent sequential exposures to 100 µg/m(3), 200 µg/m(3), and 300 µg/m(3) whole DE generated in real time using an idling diesel truck engine. Exposures were separated by 14 d and each was 2 h in duration. RESULTS: We report concentration-dependent effects of exposure to DE, with 100 µg/m(3) concentration causing minimal cardiovascular effects, while exposure to 300 µg/m(3) DE for 2 h resulted in a borderline significant reduction of baseline brachial artery diameter (3.34 ± 0.27 mm pre- versus 3.23 ± 0.25 mm post-exposure; p = 0.08). Exposure to the highest concentration of DE also resulted in increases of 5 mmHg in diastolic blood pressure as well as a decrease in indices of the frequency domain of heart rate variability (HRV). DISCUSSION AND CONCLUSIONS: These findings demonstrate that acute exposure to relatively high concentrations of DE produces cardiovascular changes in middle-aged GSTM1 null individuals. This study therefore suggests that arterial vasoconstriction and changes in HRV are responses through which traffic-related air pollution increases the risk of adverse cardiovascular outcomes.

Variation in the fitted filtration efficiency of disposable face masks by sex.

BACKGROUND & OBJECTIVE: Disposable face masks are a primary protective measure against the adverse health effects of exposure to infectious and toxic aerosols such as airborne viruses and particulate air pollutants. While the fit of high efficiency respirators is regulated in occupational settings, relatively little is known about the fitted filtration efficiencies of ear loop style face masks worn by the public. METHODS: We measured the variation in fitted filtration efficiency (FFE) of four commonly worn disposable face masks, in a cohort of healthy adult participants (N = 100, 50% female, 50% male, average age = 32.3 ± 9.2 years, average BMI = 25.5 ± 3.4) using the U.S. Occupational Safety and Health Administration Quantitative Fit Test, for an N95 (respirator), KN95, surgical, and KF94 masks. The latter three ear loop style masks were additionally tested in a clip-modified condition, tightened using a plastic clip to centrally fasten loops in the back of the head. RESULTS: The findings show that sex is a major determinant of the FFE of KN95, surgical, and KF94 masks. On average, males had an 11% higher FFE relative to females, at baseline testing. We show that a simple modification using an ear loop clip, results in improvements in the average FFE for females but provides comparatively minor changes for males. On average, females had a 20% increased FFE when a clip was worn behind the head, relative to a 6% increase for males. IMPACT: The efficacy of a disposable face mask as protection against air contaminants depends on the efficiency of the mask materials and how well it fits the wearer. We report that the sex of the wearer is a major determinant of the baseline fitted filtration efficiency (FFE) of commonly available ear loop style face masks. In addition, we show that a simple fit modifier, an ear loop clip fastened behind the head, substantially improves baseline FFE for females but produces only minor changes for males. These findings have significant public health implications for the use of face masks as a protective intervention against inhalational exposure to airborne contaminants.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Sequestration of mitochondrial iron by silica particle initiates a biological effect.

Inhalation of particulate matter has presented a challenge to human health for thousands of years. The underlying mechanism for biological effect following particle exposure is incompletely understood. We tested the postulate that particle sequestration of cell and mitochondrial iron is a pivotal event mediating oxidant generation and biological effect. In vitro exposure of human bronchial epithelial cells to silica reduced intracellular iron, which resulted in increases in both the importer divalent metal transporter 1 expression and metal uptake. Diminished mitochondrial (57)Fe concentrations following silica exposure confirmed particle sequestration of cell iron. Preincubation of cells with excess ferric ammonium citrate increased cell, nuclear, and mitochondrial metal concentrations and prevented significant iron loss from mitochondria following silica exposure. Cell and mitochondrial oxidant generation increased after silica incubation, but pretreatment with iron diminished this generation of reactive oxygen species. Silica exposure activated MAP kinases (ERK and p38) and altered the expression of transcription factors (nF-κB and NF-E2-related factor 2), proinflammatory cytokines (interleukin-8 and -6), and apoptotic proteins. All of these changes in indexes of biological effect were either diminished or inhibited by cell pretreatment with iron. Finally, percentage of neutrophils and total protein concentrations in an animal model instilled with silica were decreased by concurrent exposure to iron. We conclude that an initiating event in the response to particulate matter is a sequestration of cell and mitochondrial iron by endocytosed particle. The resultant oxidative stress and biological response after particle exposure are either diminished or inhibited by increasing the cell iron concentration.

Toxicological disruption of signaling homeostasis: tyrosine phosphatases as targets.

The protein tyrosine phosphatases (PTPs) consist of a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTPs have critical roles in maintaining signaling quiescence in resting cells and in restoring homeostasis by effecting signal termination. Interest in these enzymes has increased in recent years following the discovery that the activity of PTPs is modulated through redox mechanisms during signaling. The molecular features that enable redox regulation of PTPs during physiological signaling also render them highly susceptible to oxidative and electrophilic inactivation by a broad spectrum of structurally disparate xenobiotic compounds. The loss of PTP activity results in a profound disregulation of protein phosphotyrosine metabolism, leading to widespread and persistent activation of signaling cascades in the cell.