Lung toxicity, deposition, and clearance of thermal spray coating particles with different metal profiles after inhalation in rats.

Thermal spray coating is a process in which molten metal is sprayed onto a surface. Little is known about the health effects associated with these aerosols. Sprague-Dawley rats were exposed to aerosols (25 mg/m3 × 4 hr/d × 4 d) generated during thermal spray coating using different consumables [i.e. stainless-steel wire (PMET731), Ni-based wire (PMET885), Zn-based wire (PMET540)]. Control animals received air. Bronchoalveolar lavage was performed at 4 and 30 d post-exposure to assess lung toxicity. The particles were chain-like agglomerates and similar in size (310-378 nm). Inhalation of PMET885 aerosol caused a significant increase in lung injury and inflammation at both time points. Inhalation of PMET540 aerosol caused a slight but significant increase in lung toxicity at 4 but not 30 d. Exposure to PMET731 aerosol had no effect on lung toxicity. Overall, the lung responses were in the order: PMET885≫PMET540 >PMT731. Following a shorter exposure (25 mg/m3 × 4 h/d × 1d), lung burdens of metals from the different aerosols were determined by ICP-AES at 0, 1, 4 and 30 d post-exposure. Zn was cleared from the lungs at the fastest rate with complete clearance by 4 d post-exposure. Ni, Cr, and Mn had similar rates of clearance as nearly half of the deposited metal was cleared by 4 d. A small but significant percentage of each of these metals persisted in the lungs at 30 d. The pulmonary clearance of Fe was difficult to assess because of inherently high levels of Fe in control lungs.

Influence of Impurities from Manufacturing Process on the Toxicity Profile of Boron Nitride Nanotubes.

The toxicity of boron nitride nanotubes (BNNTs) has been the subject of conflicting reports, likely due to differences in the residuals and impurities that can make up to 30-60% of the material produced based on the manufacturing processes and purification employed. Four BNNTs manufactured by induction thermal plasma process with a gradient of BNNT purity levels achieved through sequential gas purification, water and solvent washing, allowed assessing the influence of these residuals/impurities on the toxicity profile of BNNTs. Extensive characterization including infrared and X-ray spectroscopy, thermogravimetric analysis, size, charge, surface area, and density captured the alteration in physicochemical properties as the material went through sequential purification. The material from each step is screened using acellular and in vitro assays for evaluating general toxicity, mechanisms of toxicity, and macrophage function. As the material increased in purity, there are more high-aspect-ratio particulates and a corresponding distinct increase in cytotoxicity, nuclear factor-κB transcription, and inflammasome activation. There is no alteration in macrophage function after BNNT exposure with all purity grades. The cytotoxicity and mechanism of screening clustered with the purity grade of BNNTs, illustrating that greater purity of BNNT corresponds to greater toxicity.

In vivo and in vitro toxicity of a stainless-steel aerosol generated during thermal spray coating.

Thermal spray coating is an industrial process in which molten metal is sprayed at high velocity onto a surface as a protective coating. An automated electric arc wire thermal spray coating aerosol generator and inhalation exposure system was developed to simulate an occupational exposure and, using this system, male Sprague-Dawley rats were exposed to stainless steel PMET720 aerosols at 25 mg/m3 × 4 h/day × 9 day. Lung injury, inflammation, and cytokine alteration were determined. Resolution was assessed by evaluating these parameters at 1, 7, 14 and 28 d after exposure. The aerosols generated were also collected and characterized. Macrophages were exposed in vitro over a wide dose range (0-200 µg/ml) to determine cytotoxicity and to screen for known mechanisms of toxicity. Welding fumes were used as comparative particulate controls. In vivo lung damage, inflammation and alteration in cytokines were observed 1 day post exposure and this response resolved by day 7. Alveolar macrophages retained the particulates even after 28 day post-exposure. In line with the pulmonary toxicity findings, in vitro cytotoxicity and membrane damage in macrophages were observed only at the higher doses. Electron paramagnetic resonance showed in an acellular environment the particulate generated free radicals and a dose-dependent increase in intracellular oxidative stress and NF-kB/AP-1 activity was observed. PMET720 particles were internalized via clathrin and caveolar mediated endocytosis as well as actin-dependent pinocytosis/phagocytosis. The results suggest that compared to stainless steel welding fumes, the PMET 720 aerosols were not as overtly toxic, and the animals recovered from the acute pulmonary injury by 7 days.

Effects of E-Cigarette Flavoring Chemicals on Human Macrophages and Bronchial Epithelial Cells.

E-cigarettes utilize a wide range of flavoring chemicals with respiratory health effects that are not well understood. In this study, we used pulmonary-associated cell lines to assess the in vitro cytotoxic effects of 30 flavoring chemicals. Human bronchial epithelial cells (BEAS-2B) and both naïve and activated macrophages (THP-1) were treated with 10, 100, and 1000 µM of flavoring chemicals and analyzed for changes in viability, cell membrane damage, reactive oxygen species (ROS) production, and inflammatory cytokine release. Viability was unaffected for all chemicals at the 10 and 100 µM concentrations. At 1000 µM, the greatest reductions in viability were seen with decanal, hexanal, nonanal, cinnamaldehyde, eugenol, vanillin, alpha-pinene, and limonene. High amounts of ROS were elicited by vanillin, ethyl maltol, and the diketones (2,3-pentanedione, 2,3-heptanedione, and 2,3-hexanedione) from both cell lines. Naïve THP-1 cells produced significantly elevated levels of IL-1ß, IL-8, and TNF-α when exposed to ethyl maltol and hexanal. Activated THP-1 cells released increased IL-1ß and TNF-α when exposed to ethyl maltol, but many flavoring chemicals had an apparent suppressive effect on inflammatory cytokines released by activated macrophages, some with varying degrees of accompanying cytotoxicity. The diketones, L-carvone, and linalool suppressed cytokine release in the absence of cytotoxicity. These findings provide insight into lung cell cytotoxicity and inflammatory cytokine release in response to flavorings commonly used in e-cigarettes.

Toxicology of flavoring- and cannabis-containing e-liquids used in electronic delivery systems.

Electronic cigarettes (e-cigarettes) were introduced in the United States in 2007 and by 2014 they were the most popular tobacco product amongst youth and had overtaken use of regular tobacco cigarettes. E-cigarettes are used to aerosolize a liquid (e-liquid) that the user inhales. Flavorings in e-liquids is a primary reason for youth to initiate use of e-cigarettes. Evidence is growing in the scientific literature that inhalation of some flavorings is not without risk of harm. In this review, 67 original articles (primarily cellular in vitro) on the toxicity of flavored e-liquids were identified in the PubMed and Scopus databases and evaluated critically. At least 65 individual flavoring ingredients in e-liquids or aerosols from e-cigarettes induced toxicity in the respiratory tract, cardiovascular and circulatory systems, skeletal system, and skin. Cinnamaldehyde was most frequently reported to be cytotoxic, followed by vanillin, menthol, ethyl maltol, ethyl vanillin, benzaldehyde and linalool. Additionally, modern e-cigarettes can be modified to aerosolize cannabis as dried plant material or a concentrated extract. The U.S. experienced an outbreak of lung injuries, termed e-cigarette, or vaping, product use-associated lung injury (EVALI) that began in 2019; among 2,022 hospitalized patients who had data on substance use (as of January 14, 2020), 82% reported using a delta-9-tetrahydrocannabinol (main psychoactive component in cannabis) containing e-cigarette, or vaping, product. Our literature search identified 33 articles related to EVALI. Vitamin E acetate, a diluent and thickening agent in cannabis-based products, was strongly linked to the EVALI outbreak in epidemiologic and laboratory studies; however, e-liquid chemistry is highly complex, and more than one mechanism of lung injury, ingredient, or thermal breakdown product may be responsible for toxicity. More research is needed, particularly with regard to e-cigarettes (generation, power settings, etc.), e-liquids (composition, bulk or vaped form), modeled systems (cell type, culture type, and dosimetry metrics), biological monitoring, secondhand exposures and contact with residues that contain nicotine and flavorings, and causative agents and mechanisms of EVALI toxicity.

Biological effects of inhaled hydraulic fracturing sand dust. III. Cytotoxicity and pro-inflammatory responses in cultured murine macrophage cells.

Cultured murine macrophages (RAW 264.7) were used to investigate the effects of fracking sand dust (FSD) for its pro-inflammatory activity, in order to gain insight into the potential toxicity to workers associated with inhalation of FSD during hydraulic fracturing. While the role of respirable crystalline silica in the development of silicosis is well documented, nothing is known about the toxicity of inhaled FSD. The FSD (FSD 8) used in these studies was from an unconventional gas well drilling site. FSD 8was prepared as a 10 mg/ml stock solution in sterile PBS, vortexed for 15 s, and allowed to sit at room temperature for 30 min before applying the suspension to RAW 264.7cells. Compared to PBS controls, cellular viability was significantly decreased after a 24 h exposure to FSD. Intracellular reactive oxygen species (ROS) production and the production of IL-6, TNFα, and endothelin-1 (ET-1) were up-regulated as a result of the exposure, whereas the hydroxyl radical (.OH) was only detected in an acellular system. Immunofluorescent staining of cells against TNFα revealed that FSD 8 caused cellular blebbing, and engulfment of FSD 8 by macrophages was observed with enhanced dark-field microscopy. The observed changes in cellular viability, cellular morphology, free radical generation and cytokine production all confirm that FSD 8 is cytotoxic to RAW 264.7 cells and warrants future studies into the specific pathways and mechanisms by which these toxicities occur.

Biological effects of inhaled hydraulic fracturing sand dust. II. Particle characterization and pulmonary effects 30 d following intratracheal instillation.

Hydraulic fracturing ("fracking") is used in unconventional gas drilling to allow for the free flow of natural gas from rock. Sand in fracking fluid is pumped into the well bore under high pressure to enter and stabilize fissures in the rock. In the process of manipulating the sand on site, respirable dust (fracking sand dust, FSD) is generated. Inhalation of FSD is a potential hazard to workers inasmuch as respirable crystalline silica causes silicosis, and levels of FSD at drilling work sites have exceeded occupational exposure limits set by OSHA. In the absence of any information about its potential toxicity, a comprehensive rat animal model was designed to investigate the bioactivities of several FSDs in comparison to MIN-U-SIL® 5, a respirable α-quartz reference dust used in previous animal models of silicosis, in several organ systems (Fedan, J.S., Toxicol Appl Pharmacol. 00, 000-000, 2020). The present report, part of the larger investigation, describes: 1) a comparison of the physico-chemical properties of nine FSDs, collected at drilling sites, and MIN-U-SIL® 5, a reference silica dust, and 2) a comparison of the pulmonary inflammatory responses to intratracheal instillation of the nine FSDs and MIN-U-SIL® 5. Our findings indicate that, in many respects, the physico-chemical characteristics, and the biological effects of the FSDs and MIN-U-SIL® 5 after intratracheal instillation, have distinct differences.

Mild steel and stainless steel welding fumes elicit pro-inflammatory and pro-oxidant effects in first trimester trophoblast cells.

PROBLEM: As more women join the skilled-trade workforce, the effects of workplace exposures on pregnancy need to be explored. This study aims to identify the effects of mild steel and stainless steel welding fume exposures on cultured placental trophoblast cells. METHOD OF STUDY: Welding fumes (mild steel and stainless steel) were generously donated by Lincoln Electric. Electron microscopy was used to characterize welding fume particle size and the ability of particles to enter extravillous trophoblast cells (HTR-8/SVneo). Cellular viability, free radical production, cytokine production, and ability of cells to maintain invasive properties were analyzed, respectively, by WST-1, electron paramagnetic resonance, DCFH-DA, V-plex MULTI-SPOT assay system, and a matrix gel invasion assay. RESULTS: For all three welding fume types, average particle size was <210 nm. HTR-8/SVneo cells internalized welding particles, and nuclear condensation was observed. Cellular viability was significantly decreased at the high dose of 100 µg/mL for all three welding fumes, and stainless steel generated the greatest production of the hydroxyl radical, and intracellular reactive oxygen species. Production of the cytokines IL-1ß and TNFα were not observed in response to welding fume exposure, but IL-6 and IL-8 were. Finally, the invasive capability of cells was decreased upon exposure to both mild steel and stainless steel welding fumes. CONCLUSION: Welding fumes are cytotoxic to extravillous trophoblasts, as is evident by the production of free radicals, pro-inflammatory cytokines, and the observed decrease in invasive capabilities.

An integrated electrolysis - electrospray - ionization antimicrobial platform using Engineered Water Nanostructures (EWNS) for food safety applications.

Engineered water nanostructures (EWNS) synthesized utilizing electrospray and ionization of water, have been, recently, shown to be an effective, green, antimicrobial platform for surface and air disinfection, where reactive oxygen species (ROS), generated and encapsulated within the particles during synthesis, were found to be the main inactivation mechanism. Herein, the antimicrobial potency of the EWNS was further enhanced by integrating electrolysis, electrospray and ionization of de-ionized water in the EWNS synthesis process. Detailed physicochemical characterization of these enhanced EWNS (eEWNS) was performed using state-of-the-art analytical methods and has shown that, while both size and charge remain similar to the EWNS (mean diameter of 13 nm and charge of 13 electrons), they possess a three times higher ROS content. The increase of the ROS content as a result of the addition of the electrolysis step before electrospray and ionization led to an increased antimicrobial ability as verified by E. coli inactivation studies using stainless steel coupons. It was shown that a 45-minute exposure to eEWNS resulted in a 4-log reduction as opposed to a 1.9-log reduction when exposed to EWNS. In addition, the eEWNS were assessed for their potency to inactivate natural microbiota (total viable and yeast and mold counts), as well as, inoculated E.coli on the surface of fresh organic blackberries. The results showed a 97% (1.5-log) inactivation of the total viable count, a 99% (2-log) reduction in the yeast and mold count and a 2.5-log reduction of the inoculated E.coli after 45 minutes of exposure, without any visual changes to the fruit. This enhanced antimicrobial activity further underpins the EWNS platform as an effective, dry and chemical free approach suitable for a variety of food safety applications and could be ideal for delicate fresh produce that cannot withstand the classical, wet disinfection treatments.

Assessment of reactive oxygen species generated by electronic cigarettes using acellular and cellular approaches.

Electronic cigarettes (e-cigs) have fast increased in popularity but the physico-chemical properties and toxicity of the generated emission remain unclear. Reactive oxygen species (ROS) are likely present in e-cig emission and can play an important role in e-cig toxicity. However, e-cig ROS generation is poorly documented. Here, we generated e-cig exposures using a recently developed versatile exposure platform and performed systematic ROS characterization on e-cig emissions using complementary acellular and cellular techniques: 1) a novel acellular Trolox-based mass spectrometry method for total ROS and hydrogen peroxide (H2O2) detection, 2) electron spin resonance (ESR) for hydroxyl radical detection in an acellular and cellular systems and 3) in vitro ROS detection in small airway epithelial cells (SAEC) using the dihydroethidium (DHE) assay. Findings confirm ROS generation in cellular and acellular systems and is highly dependent on the e-cig brand, flavor, puffing pattern and voltage. Trolox method detected a total of 1.2-8.9nmol H2O2eq./puff; H2O2 accounted for 12-68% of total ROS. SAEC cells exposed to e-cig emissions generated up to eight times more ROS compared to control. The dependency of e-cig emission profile on e-cig features and operational parameters should be taken into consideration in toxicological studies.

Comparison of the toxicity of sintered and unsintered indium-tin oxide particles in murine macrophage and epidermal cells.

Indium-tin oxide (ITO) is used to produce flat panel displays and several other technology products. Composed of 90% indium oxide (In2O3) and 10% tin oxide (SnO2) by weight, ITO is synthesized under conditions of high heat via a process known as sintering. Indium lung disease, a recently recognized occupational illness, is characterized by pulmonary alveolar proteinosis, fibrosis, and emphysema. Murine macrophage (RAW 264.7) and epidermal (JB6) cells stably transfected with AP-1 to study tumor promoting potential, were used to differentiate between the toxicological profiles of sintered ITO (SITO) and unsintered mixture (UITO). We hypothesized that sintering would play a key role in free radical generation and cytotoxicity. Exposure of cells to both UITO and SITO caused a time and dose dependent decrease of the viability of cells. Intracellular ROS generation was inversely related to the dose of both UITO and SITO, a direct reflection of the decreased number of viable RAW 264.7 and JB6/AP-1 cells observed at higher concentrations. Electron spin resonance showed significantly increased hydroxyl radical (OH) generation in cells exposed to UITO compared to SITO. This is different from LDH release, which showed that SITO caused significantly increased damage to the cell membrane compared to UITO. Lastly, the JB6/AP-1 cell line did not show activation of the AP-1 pathway. Our results highlight both the differences in the mechanisms of cytotoxicity and the consistent adverse effects associated with UITO and SITO exposure.

Role of engineered metal oxide nanoparticle agglomeration in reactive oxygen species generation and cathepsin B release in NLRP3 inflammasome activation and pulmonary toxicity.

Incomplete understanding of the contributions of dispersants and engineered nanoparticles/materials (ENM) agglomeration state to biological outcomes presents an obstacle for toxicological studies. Although reactive oxygen species (ROS) production is often regarded as the primary indicator of ENM bioactivity and toxicity, it remains unclear whether ENM produce ROS or whether ROS is an outcome of ENM-induced cell injury. Phagolysosomal disruption and cathepsin B release also promote bioactivity through inflammasome activation. Therefore, specific particle parameters, i.e. preexposure dispersion status and particle surface area, of two ENM (NiO and CeO2) were used to evaluate the role of ROS generation and cathepsin B release during ENM-induced toxicity. Male C57BL/6J mice were exposed to 0, 20, 40, or 80 µg of poorly or well-dispersed NiO-NP or CeO2-NP in four types of dispersion media. At 1- and 7-day postexposure, lung lavage fluid was collected to assess inflammation, cytotoxicity, and inflammasome activation. Results showed that preexposure dispersion status correlated with postexposure pulmonary bioactivity. The differences in bioactivity of NiO-NP and CeO2-NP are likely due to NiO-NP facilitating the release of cathepsin B and in turn inflammasome activation generating proinflammatory cytokines. Further, both metal oxides acted as free radical scavengers. Depending on the pH, CeO2-NP acted as a free radical scavenger in an acidic environment (an environment mimicking the lysosome) while the NiO-NP acted as a scavenger in a physiological pH (an environment that mimics the cytosol of the cell). Therefore, results from this study suggest that ENM-induced ROS is not likely a mechanism of inflammasome activation.

Evaluation of pulmonary and systemic toxicity following lung exposure to graphite nanoplates: a member of the graphene-based nanomaterial family.

BACKGROUND: Graphene, a monolayer of carbon, is an engineered nanomaterial (ENM) with physical and chemical properties that may offer application advantages over other carbonaceous ENMs, such as carbon nanotubes (CNT). The goal of this study was to comparatively assess pulmonary and systemic toxicity of graphite nanoplates, a member of the graphene-based nanomaterial family, with respect to nanoplate size. METHODS: Three sizes of graphite nanoplates [20 µm lateral (Gr20), 5 µm lateral (Gr5), and <2 µm lateral (Gr1)] ranging from 8-25 nm in thickness were characterized for difference in surface area, structure,, zeta potential, and agglomeration in dispersion medium, the vehicle for in vivo studies. Mice were exposed by pharyngeal aspiration to these 3 sizes of graphite nanoplates at doses of 4 or 40 µg/mouse, or to carbon black (CB) as a carbonaceous control material. At 4 h, 1 day, 7 days, 1 month, and 2 months post-exposure, bronchoalveolar lavage was performed to collect fluid and cells for analysis of lung injury and inflammation. Particle clearance, histopathology and gene expression in lung tissue were evaluated. In addition, protein levels and gene expression were measured in blood, heart, aorta and liver to assess systemic responses. RESULTS: All Gr samples were found to be similarly composed of two graphite structures and agglomerated to varying degrees in DM in proportion to the lateral dimension. Surface area for Gr1 was approximately 7-fold greater than Gr5 and Gr20, but was less reactive reactive per m(2). At the low dose, none of the Gr materials induced toxicity. At the high dose, Gr20 and Gr5 exposure increased indices of lung inflammation and injury in lavage fluid and tissue gene expression to a greater degree and duration than Gr1 and CB. Gr5 and Gr20 showed no or minimal lung epithelial hypertrophy and hyperplasia, and no development of fibrosis by 2 months post-exposure. In addition, the aorta and liver inflammatory and acute phase genes were transiently elevated in Gr5 and Gr20, relative to Gr1. CONCLUSIONS: Pulmonary and systemic toxicity of graphite nanoplates may be dependent on lateral size and/or surface reactivity, with the graphite nanoplates > 5 µm laterally inducing greater toxicity which peaked at the early time points post-exposure relative to the 1-2 µm graphite nanoplate.

Evaluation of the effect of valence state on cerium oxide nanoparticle toxicity following intratracheal instillation in rats.

Cerium (Ce) is becoming a popular metal for use in electrochemical applications. When in the form of cerium oxide (CeO2), Ce can exist in both 3 + and 4 + valence states, acting as an ideal catalyst. Previous in vitro and in vivo evidence have demonstrated that CeO2 has either anti- or pro-oxidant properties, possibly due to the ability of the nanoparticles to transition between valence states. Therefore, we chose to chemically modify the nanoparticles to shift the valence state toward 3+. During the hydrothermal synthesis process, 10 mol% gadolinium (Gd) and 20 mol% Gd, were substituted into the lattice of the CeO2 nanoparticles forming a perfect solid solution with various A-site valence states. These two Gd-doped CeO2 nanoparticles were compared to pure CeO2 nanoparticles. Preliminary characteristics indicated that doping results in minimal size and zeta potential changes but alters valence state. Following characterization, male Sprague-Dawley rats were exposed to 0.5 or 1.0 mg/kg nanoparticles via a single intratracheal instillation. Animals were sacrificed and bronchoalveolar lavage fluid and various tissues were collected to determine the effect of valence state and oxygen vacancies on toxicity 1-, 7-, or 84-day post-exposure. Results indicate that damage, as measured by elevations in lactate dehydrogenase, occurred within 1-day post-exposure and was sustained 7-day post-exposure, but subsided to control levels 84-day post-exposure. Furthermore, no inflammatory signaling or lipid peroxidation occurred following exposure with any of the nanoparticles. Our results implicate that valence state has a minimal effect on CeO2 nanoparticle toxicity in vivo.

Optimization of a nanotechnology based antimicrobial platform for food safety applications using Engineered Water Nanostructures (EWNS).

A chemical free, nanotechnology-based, antimicrobial platform using Engineered Water Nanostructures (EWNS) was recently developed. EWNS have high surface charge, are loaded with reactive oxygen species (ROS), and can interact-with, and inactivate an array of microorganisms, including foodborne pathogens. Here, it was demonstrated that their properties during synthesis can be fine tuned and optimized to further enhance their antimicrobial potential. A lab based EWNS platform was developed to enable fine-tuning of EWNS properties by modifying synthesis parameters. Characterization of EWNS properties (charge, size and ROS content) was performed using state-of-the art analytical methods. Further their microbial inactivation potential was evaluated with food related microorganisms such as Escherichia coli, Salmonella enterica, Listeria innocua, Mycobacterium parafortuitum, and Saccharomyces cerevisiae inoculated onto the surface of organic grape tomatoes. The results presented here indicate that EWNS properties can be fine-tuned during synthesis resulting in a multifold increase of the inactivation efficacy. More specifically, the surface charge quadrupled and the ROS content increased. Microbial removal rates were microorganism dependent and ranged between 1.0 to 3.8 logs after 45 mins of exposure to an EWNS aerosol dose of 40,000 #/cm(3).

Pulmonary toxicity of indium-tin oxide production facility particles in rats.

Indium-tin oxide (ITO) is used to make transparent conductive coatings for touch-screen and liquid crystal display electronics. Occupational exposures to potentially toxic particles generated during ITO production have increased in recent years as the demand for consumer electronics continues to rise. Previous studies have demonstrated cytotoxicity in vitro and animal models have shown pulmonary inflammation and injury in response to various indium-containing particles. In humans, pulmonary alveolar proteinosis (PAP) and fibrotic interstitial lung disease have been observed in ITO facility workers. However, which indium materials or specific processes in the workplace may be the most toxic to workers is unknown. Here we examined the pulmonary toxicity of three different particle samples that represent real-life worker exposures, as they were collected at various production stages throughout an ITO facility. Indium oxide (In2O3), sintered ITO (SITO) and ventilation dust (VD) particles each caused pulmonary inflammation and damage in rats over a time course (1, 7 and 90 days post-intratracheal instillation), but SITO and VD appeared to induce greater toxicity in rat lungs than In2O3 at a dose of 1 mg per rat. Downstream pathological changes such as PAP and fibrosis were observed in response to all three particles 90 days after treatment, with a trend towards greatest severity in animals exposed to VD when comparing animals that received the same dose. These findings may inform workplace exposure reduction efforts and provide a better understanding of the pathogenesis of an emerging occupational health issue.

Temporal changes in rat liver gene expression after acute cadmium and chromium exposure.

U.S. Service Members and civilians are at risk of exposure to a variety of environmental health hazards throughout their normal duty activities and in industrial occupations. Metals are widely used in large quantities in a number of industrial processes and are a common environmental toxicant, which increases the possibility of being exposed at toxic levels. While metal toxicity has been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify candidate biomarkers, rats were exposed via a single intraperitoneal injection to three concentrations of CdCl2 and Na(2)Cr(2)O(7), with livers harvested at 1, 3, or 7 days after exposure. Cd and Cr accumulated in the liver at 1 day post exposure. Cd levels remained elevated over the length of the experiment, while Cr levels declined. Metal exposures induced ROS, including hydroxyl radical (•OH), resulting in DNA strand breaks and lipid peroxidation. Interestingly, ROS and cellular damage appeared to increase with time post-exposure in both metals, despite declines in Cr levels. Differentially expressed genes were identified via microarray analysis. Both metals perturbed gene expression in pathways related to oxidative stress, metabolism, DNA damage, cell cycle, and inflammatory response. This work provides insight into the temporal effects and mechanistic pathways involved in acute metal intoxication, leading to the identification of candidate biomarkers.

Sintered indium-tin oxide particles induce pro-inflammatory responses in vitro, in part through inflammasome activation.

Indium-tin oxide (ITO) is used to make transparent conductive coatings for touch-screen and liquid crystal display electronics. As the demand for consumer electronics continues to increase, so does the concern for occupational exposures to particles containing these potentially toxic metal oxides. Indium-containing particles have been shown to be cytotoxic in cultured cells and pro-inflammatory in pulmonary animal models. In humans, pulmonary alveolar proteinosis and fibrotic interstitial lung disease have been observed in ITO facility workers. However, which ITO production materials may be the most toxic to workers and how they initiate pulmonary inflammation remain poorly understood. Here we examined four different particle samples collected from an ITO production facility for their ability to induce pro-inflammatory responses in vitro. Tin oxide, sintered ITO (SITO), and ventilation dust particles activated nuclear factor kappa B (NFκB) within 3 h of treatment. However, only SITO induced robust cytokine production (IL-1ß, IL-6, TNFα, and IL-8) within 24 h in both RAW 264.7 mouse macrophages and BEAS-2B human bronchial epithelial cells. Our lab and others have previously demonstrated SITO-induced cytotoxicity as well. These findings suggest that SITO particles activate the NLRP3 inflammasome, which has been implicated in several immune-mediated diseases via its ability to induce IL-1ß release and cause subsequent cell death. Inflammasome activation by SITO was confirmed, but it required the presence of endotoxin. Further, a phagocytosis assay revealed that pre-uptake of SITO or ventilation dust impaired proper macrophage phagocytosis of E. coli. Our results suggest that adverse inflammatory responses to SITO particles by both macrophage and epithelial cells may initiate and propagate indium lung disease. These findings will provide a better understanding of the molecular mechanisms behind an emerging occupational health issue.

The Effect of Cerium Oxide Nanoparticle Valence State on Reactive Oxygen Species and Toxicity.

Cerium oxide (CeO2) nanoparticles, which are used in a variety of products including solar cells, gas sensors, and catalysts, are expected to increase in industrial use. This will subsequently lead to additional occupational exposures, making toxicology screenings crucial. Previous toxicology studies have presented conflicting results as to the extent of CeO2 toxicity, which is hypothesized to be due to the ability of Ce to exist in both a +3 and +4 valence state. Thus, to study whether valence state and oxygen vacancy concentration are important in CeO2 toxicity, CeO2 nanoparticles were doped with gadolinium to adjust the cation (Ce, Gd) and anion (O) defect states. The hypothesis that doping would increase toxicity and decrease antioxidant abilities as a result of increased oxygen vacancies and inhibition of +3 to +4 transition was tested. Differences in toxicity and reactivity based on valence state were determined in RLE-6TN rat alveolar epithelial and NR8383 rat alveolar macrophage cells using enhanced dark field microscopy, electron paramagnetic resonance (EPR), and annexin V/propidium iodide cell viability stain. Results from EPR indicated that as doping increased, antioxidant potential decreased. Alternatively, doping had no effect on toxicity at 24 h. The present results imply that as doping increases, thus subsequently increasing the Ce(3+)/Ce(4+) ratio, antioxidant potential decreases, suggesting that differences in reactivity of CeO2 are due to the ability of Ce to transition between the two valence states and the presence of increased oxygen vacancies, rather than dependent on a specific valence state.

Intravenous and gastric cerium dioxide nanoparticle exposure disrupts microvascular smooth muscle signaling.

Cerium dioxide nanoparticles (CeO2 NP) hold great therapeutic potential, but the in vivo effects of non-pulmonary exposure routes are unclear. The first aim was to determine whether microvascular function is impaired after intravenous and gastric CeO2 NP exposure. The second aim was to investigate the mechanism(s) of action underlying microvascular dysfunction following CeO2 NP exposure. Rats were exposed to CeO2 NP (primary diameter: 4 ± 1 nm, surface area: 81.36 m(2)/g) by intratracheal instillation, intravenous injection, or gastric gavage. Mesenteric arterioles were harvested 24 h post-exposure and vascular function was assessed using an isolated arteriole preparation. Endothelium-dependent and independent function and vascular smooth muscle (VSM) signaling (soluble guanylyl cyclase [sGC] and cyclic guanosine monophosphate [cGMP]) were assessed. Reactive oxygen species (ROS) generation and nitric oxide (NO) production were analyzed. Compared with controls, endothelium-dependent and independent dilation were impaired following intravenous injection (by 61% and 45%) and gastric gavage (by 63% and 49%). However, intravenous injection resulted in greater microvascular impairment (16% and 35%) compared with gastric gavage at an identical dose (100 µg). Furthermore, sGC activation and cGMP responsiveness were impaired following pulmonary, intravenous, and gastric CeO2 NP treatment. Finally, nanoparticle exposure resulted in route-dependent, increased ROS generation and decreased NO production. These results indicate that CeO2 NP exposure route differentially impairs microvascular function, which may be mechanistically linked to decreased NO production and subsequent VSM signaling. Fully understanding the mechanisms behind CeO2 NP in vivo effects is a critical step in the continued therapeutic development of this nanoparticle.