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Exposure to coarse particulate matter (PM) is associated with lung inflammation and exacerbation of respiratory symptoms in sensitive populations, but the degree to which specific emission sources contribute to these effects is unclear. We examined whether coarse PM samples enriched with diverse sources differentially exacerbate allergic airway responses. Coarse PM was collected weekly (7/2009-6/2010) from urban (G.T. Craig [GTC]) and rural (Chippewa Lake Monitor [CLM]) sites in the Cleveland, Ohio area. Source apportionment results were used to pool GTC filter PM extracts into five samples dominated by traffic, coal, steel (two samples), or road salt sources. Five CLM samples were prepared from corresponding weeks. Control non-allergic and house dust mite (HDM)-allergic Balb/cJ mice were exposed by oropharyngeal aspiration to 100 μg coarse GTC or CLM, control filter extract, or saline only, and responses were examined 2 d after PM exposures. In allergic mice, CLM traffic, CLM road salt and all GTC samples except steel-1 significantly increased airway responsiveness to methacholine (MCh) compared with control treatments. In non-allergic mice, CLM traffic, CLM steel-2 and all GTC samples except coal significantly increased bronchoalveolar lavage fluid (BALF) neutrophils, while only CLM traffic PM increased eosinophils in allergic mice. In non-allergic mice, CLM coal PM increased BALF interleukin (IL)-13 and GTC steel-1 PM increased TNF-α levels. These results demonstrate that equal masses of GTC and CLM coarse PM enriched with a variety of sources exacerbate allergic airway disease. Greater PM concentrations at the urban GTC site signify a greater potential for human health effects.

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 H 2 O 2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H 2 O 2 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., (Published by Elsevier Inc.)

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

Background: There is an urgent need to provide access to cleaner end user energy technologies for the nearly 40% of the world's population who currently depend on rudimentary cooking and heating systems. Advanced cookstoves (CS) are designed to cut emissions and solid-fuel consumption, thus reducing adverse human health and environmental impacts., Study Premise: We hypothesized that, compared to a traditional (Tier 0) three-stone (3-S) fire, acute inhalation of solid-fuel emissions from advanced natural-draft (ND; Tier 2) or forced-draft (FD; Tier 3) stoves would reduce exposure biomarkers and lessen pulmonary and innate immune system health effects in exposed mice., Results: Across two simulated cooking cycles (duration ~ 3h), emitted particulate mass concentrations were reduced 80% and 62% by FD and ND stoves, respectively, compared to the 3-S fire; with corresponding decreases in particles visible within murine alveolar macrophages. Emitted carbon monoxide was reduced ~ 90% and ~ 60%, respectively. Only 3-S-fire-exposed mice had increased carboxyhemoglobin levels. Emitted volatile organic compounds were FD ≪ 3-S-fire ≤ ND stove; increased expression of genes involved in xenobiotic metabolism (COX-2, NQO1, CYP1a1) was detected only in ND- and 3-S-fire-exposed mice. Diminished macrophage phagocytosis was observed in the ND group. Lung glutathione was significantly depleted across all CS groups, however the FD group had the most severe, ongoing oxidative stress., Conclusions: These results are consistent with reports associating exposure to solid fuel stove emissions with modulation of the innate immune system and increased susceptibility to infection. Lower respiratory infections continue to be a leading cause of death in low-income economies. Notably, 3-S-fire-exposed mice were the only group to develop acute lung injury, possibly because they inhaled the highest concentrations of hazardous air toxicants (e.g., 1,3-butadiene, toluene, benzene, acrolein) in association with the greatest number of particles, and particles with the highest % organic carbon. However, no Tier 0-3 ranked CS group was without some untoward health effect indicating that access to still cleaner, ideally renewable, energy technologies for cooking and heating is warranted., (Published by Elsevier Inc.)

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US and its impact continues to increase in women. Oxidant insults during critical periods of early life appear to increase risk of COPD through-out the life course. To better understand susceptibility to early life exposure to oxidant air pollutants we used Fisher (F344), Sprague-Dawley (SD) and Wistar (WIS) male and female neonatal rat pups to assess: (A) if strain (i.e. genetics), sex, or stage of early life development affected baseline lung antioxidant or redox enzyme levels and (B) if these same factors modulated antioxidant responsiveness to acute ozone exposure (1 ppm × 2 h) on post-natal day (PND) 14, 21, or 28. In air-exposed pups from PND14-28, some parameters were unchanged (e.g. uric acid), some decreased (e.g. superoxide dismutase), while others increased (e.g. glutathione recycling enzymes) especially post-weaning. Lung total glutathione levels decreased in F344 and SD pups, but were relatively unchanged in WIS pups. Post-ozone exposure, data suggest that: (1) the youngest (PND14) pups were the most adversely affected; (2) neonatal SD and WIS pups, especially females, were more prone to ozone effects than males of the same age and (3) F344 neonates (females and males) were less susceptible to oxidative lung insult, not unlike F344 adults. Differences in antioxidant levels and responsiveness between sexes and strains and at different periods of development may provide a basis for assessing later life health outcomes - with implications for humans with analogous genetic or dietary-based lung antioxidant deficits.

Background: The toxicity of many xenobiotic compounds is believed to involve oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically encoded probes designed for monitoring intracellular redox changes represents a methodological advance with potential applications in toxicological studies., Objective: We tested the utility of redox-sensitive green fluorescent protein (roGFP)-based redox sensors for monitoring real-time intracellular redox changes induced by xenobiotics in toxicological studies., Methods: roGFP2, a reporter of the glutathione redox potential (E(GSH)), was used to monitor EGSH in cultured human airway epithelial cells (BEAS-2B cells) undergoing exposure to 0.15-1.0 ppm ozone (O(3)). Cells were imaged in real time using a custom-built O(3) exposure system coupled to a confocal microscope., Results: O(3) exposure induced a dose- and time-dependent increase of the cytosolic EGSH. Additional experiments confirmed that roGFP2 is not directly oxidized, but properly equilibrates with the glutathione redox couple: Inhibition of endogenous glutaredoxin 1 (Grx1) disrupted roGFP2 responses to O(3), and a Grx1-roGFP2 fusion protein responded more rapidly to O(3) exposure. Selenite-induced up-regulation of GPx (glutathione peroxidase) expression-enhanced roGFP2 responsiveness to O(3), suggesting that (hydro)peroxides are intermediates linking O(3) exposure to glutathione oxidation., Conclusion: Exposure to O(3) induces a profound increase in the cytosolic E(GSH) of airway epithelial cells that is indicative of an oxidant-dependent impairment of glutathione redox homeostasis. These studies demonstrate the utility of using genetically encoded redox reporters in making reliable assessments of cells undergoing exposure to xenobiotics with strong oxidizing properties.

The estrogenic and antiestrogenic potential of perfluorooctanoic acid (PFOA) was assessed using an immature mouse uterotrophic assay and by histologic evaluation of the uterus, cervix and vagina following treatment. Female offspring of CD-1 dams were weaned at 18days old and assigned to groups of equal weight, and received 0, 0.01, 0.1, or 1mg PFOA/kg BW/d by gavage with or without 17-β estradiol (E(2), 500μg/kg/d) from PND 18-20 (n=8/treatment/block). At 24h after the third dose (PND 21), uteri were removed and weighed. Absolute and relative uterine weights were significantly increased in the 0.01mg/kg PFOA only group. Characteristic estrogenic changes were present in all E(2)-treated mice; however, they were minimally visible in the 0.01 PFOA only mice. These data suggest that at a low dose PFOA produces minimal histopathologic changes in the reproductive tract of immature female mice, and does not antagonize the histopathologic effects of E(2)., (Published by Elsevier Inc.)

Background: Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane., Results: Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependent internalization process., Conclusions: DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.

Abnormally high incidences of asbestos-related pulmonary disease have been reported in residents of Libby, Montana, because of occupational and environmental exposure to asbestos-contaminated vermiculite. The mechanism by which Libby amphibole (LA) causes pulmonary injury is not known. The purpose of this study is to compare the cellular stress responses induced in primary human airway epithelial cells (HAECs) exposed to a respirable size fraction (≤ 2.5 μm) of Libby amphibole (LA(2.5)) to a similar size fraction of a reference amphibole sample amosite (AM(2.5)). HAEC were exposed to 0, 2.64, 13.2, or 26.4 μg/cm(2) AM(2.5) or LA(2.5) or to equivalent doses of unfractionated amosite (AM) or LA for 2 or 24 h. Comparable messenger RNA transcript levels were observed for interleukin-8, cyclooxygenase-2, and heme oxygenase-1 in HAEC following a 24-h exposure to AM or LA. Conversely, exposure to AM(2.5) resulted in a 4- to 10-fold greater induction in these proinflammatory mediators compared with LA(2.5) after 24 h. Evaluation of the expression of 84 additional genes involved in cellular stress and toxicity responses confirmed a more robust response for AM(2.5) compared with LA(2.5) on an equal mass basis. Differences in total surface area (TSA) by gas adsorption, total particle number, or oxidant generation by the size-fractionated particles did not account for the observed difference in response. In summary, AM(2.5) and LA(2.5) are at least as potent in stimulating production of proinflammatory cytokines as unfractionated AM and LA. Interestingly, AM(2.5) was more potent at inducing a proinflammatory response than LA(2.5). This difference could not be explained by differences in mineral contamination between the two samples, TSA, or oxidant generation by the samples.

The synthetic surfactant, perfluorooctanoic acid (PFOA) is a proven developmental toxicant in mice, causing pregnancy loss, increased neonatal mortality, delayed eye opening, and abnormal mammary gland growth in animals exposed during fetal life. PFOA is found in the sera and tissues of wildlife and humans throughout the world, but is especially high in the sera of children compared to adults. These studies in CD-1 mice aim to determine the latent health effects of PFOA following: (1) an in utero exposure, (2) an in utero exposure followed by ovariectomy (ovx), or (3) exposure as an adult. Mice were exposed to 0, 0.01, 0.1, 0.3, 1, 3, or 5mg PFOA/kg BW for 17 days of pregnancy or as young adults. Body weight was reduced in the highest doses on postnatal day (PND) 1 and at weaning. However, the lowest exposures (0.01-0.3mg/kg) significantly increased body weight, and serum insulin and leptin (0.01-0.1mg/kg) in mid-life after developmental exposure. PFOA exposure combined with ovx caused no additional increase in mid-life body weight. At 18 months of age, the effects of in utero PFOA exposure on body weight were no longer detected. White adipose tissue and spleen weights were decreased at high doses of PFOA in intact developmentally exposed mice, and spleen weight was reduced in PFOA-exposed ovx mice. Brown adipose tissue weight was significantly increased in both ovx and intact mice at high PFOA doses. Liver weight was unaffected in late life by these exposure paradigms. Finally, there was no effect of adult exposure to PFOA on body weight. These studies demonstrate an important window of exposure for low-dose effects of PFOA on body weight gain, as well as leptin and insulin concentrations in mid-life, at a lowest observed effect level of 0.01mg PFOA/kg BW. The mode of action of these effects and its relevance to human health remain to be explored.