Biological effects of inhaled crude oil vapor. III. Pulmonary inflammation, cytotoxicity, and gene expression profile.

OBJECTIVE: Workers may be exposed to vapors emitted from crude oil in upstream operations in the oil and gas industry. Although the toxicity of crude oil constituents has been studied, there are very few in vivo investigations designed to mimic crude oil vapor (COV) exposures that occur in these operations. The goal of the current investigation was to examine lung injury, inflammation, oxidant generation, and effects on the lung global gene expression profile following a whole-body acute or sub-chronic inhalation exposure to COV. MATERIALS AND METHODS: To conduct this investigation, rats were subjected to either a whole-body acute (6 hr) or a sub-chronic (28 d) inhalation exposure (6 hr/d × 4 d/wk × 4 wk) to COV (300 ppm; Macondo well surrogate oil). Control rats were exposed to filtered air. One and 28 d after acute exposure, and 1, 28, and 90 d following sub-chronic exposure, bronchoalveolar lavage was performed on the left lung to collect cells and fluid for analyses, the apical right lobe was preserved for histopathology, and the right cardiac and diaphragmatic lobes were processed for gene expression analyses. RESULTS: No exposure-related changes were identified in histopathology, cytotoxicity, or lavage cell profiles. Changes in lavage fluid cytokines indicative of inflammation, immune function, and endothelial function after sub-chronic exposure were limited and varied over time. Minimal gene expression changes were detected only at the 28 d post-exposure time interval in both the exposure groups. CONCLUSION: Taken together, the results from this exposure paradigm, including concentration, duration, and exposure chamber parameters, did not indicate significant and toxicologically relevant changes in markers of injury, oxidant generation, inflammation, and gene expression profile in the lung.

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.

Biological effects of inhaled crude oil. VI. Immunotoxicity.

Crude oil is an unrefined petroleum product that is a mixture of hydrocarbons and other organic material. Studies on the individual components of crude oil and crude oil exposure itself suggest it has immunomodulatory potential. As investigations of the immunotoxicity of crude oil focus mainly on ingestion and dermal exposure, the effects of whole-body inhalation of 300 ppm crude oil vapor [COV; acute inhalation exposure: (6 h × 1 d); or a 28 d sub-chronic exposure (6 h/d × 4 d/wk. × 4 wks)] was investigated 1, 28, and 90 d post-exposure in Sprague-Dawley rats. Acute exposure increased bronchoalveolar lavage (BAL) fluid cellularity, CD4+ and CD8+ cells, and absolute and percent CDllb+ cells only at 1 d post-exposure; additionally, NK cell activity was suppressed. Sub-chronic exposure resulted in a decreased frequency of CD4+ T-cells at 1 d post-exposure and an increased number and frequency of B-cells at 28 d post-exposure in the lung-associated lymph nodes. A significant increase in the number and frequency of B-cells was observed in the spleen at 1 d post-exposure; however, NK cell activity was suppressed at this time point. No effect on cellularity was identified in the BALF. No change in the IgM response to sheep red blood cells was observed. The findings indicate that crude oil inhalation exposure resulted in alterations in cellularity of phenotypic subsets that may impair immune function in rats.

Understanding toxicity associated with boron nitride nanotubes: Review of toxicity studies, exposure assessment at manufacturing facilities, and read-across.

Boron nitride nanotubes (BNNT) are produced by many different methods leading to variances in physicochemical characteristics and impurities in the final product. These differences can alter the toxicity profile. The importance of understanding the potential pathological implications of this high aspect ratio nanomaterial is increasing as new approaches to synthesize and purify in large scale are being developed. In this review, we discuss the various factors of BNNT production that can influence its toxicity followed by summarizing the toxicity findings from in vitro and in vivo studies conducted to date, including a review of particle clearance observed with various exposure routes. To understand the risk to workers and interpret relevance of toxicological findings, exposure assessment at manufacturing facilities was discussed. Workplace exposure assessment of BNNT from two manufacturing facilities measured boron concentrations in personal breathing zones from non-detectable to 0.95 µg/m3 and TEM structure counts of 0.0123 ± 0.0094 structures/cm3, concentrations well below what was found with other engineered high aspect ratio nanomaterials like carbon nanotubes and nanofibers. Finally, using a purified BNNT, a "read-across" toxicity assessment was performed to demonstrate how known hazard data and physicochemical characteristics can be utilized to evaluate potential inhalation toxicity concerns.

Pulmonary toxicity and gene expression changes in response to whole-body inhalation exposure to multi-walled carbon nanotubes in rats.

Purpose: To investigate the molecular mechanisms underlying the pulmonary toxicity induced by exposure to one form of multi-walled carbon nanotubes (MWCNT-7).Materials and methods: Rats were exposed, by whole-body inhalation, to air or an aerosol containing MWCNT-7 particles at target cumulative doses (concentration x time) ranging from 22.5 to 180 (mg/m3)h over a three-day (6 hours/day) period and toxicity and global gene expression profiles were determined in the lungs.Results: MWCNT-7 particles, associated with alveolar macrophages (AMs), were detected in rat lungs following the exposure. Mild to moderate lung pathological changes consisting of increased cellularity, thickening of the alveolar wall, alveolitis, fibrosis, and granuloma formation were detected. Bronchoalveolar lavage (BAL) toxicity parameters such as lactate dehydrogenase activity, number of AMs and polymorphonuclear leukocytes (PMNs), intracellular oxidant generation by phagocytes, and levels of cytokines were significantly (p < 0.05) increased in response to exposure to MWCNT-7. Global gene expression profiling identified several significantly differentially expressed genes (fold change >1.5 and FDR p value <0.05) in all the MWCNT-7 exposed rats. Bioinformatic analysis of the gene expression data identified significant enrichment of several diseases/biological function categories (for example, cancer, leukocyte migration, inflammatory response, mitosis, and movement of phagocytes) and canonical pathways (for example, kinetochore metaphase signaling pathway, granulocyte and agranulocyte adhesion and diapedesis, acute phase response, and LXR/RXR activation). The alterations in the lung toxicity parameters and gene expression changes exhibited a dose-response to the MWCNT exposure.Conclusions: Taken together, the data provided insights into the molecular mechanisms underlying the pulmonary toxicity induced by inhalation exposure of rats to MWCNT-7.

Lung toxicity and gene expression changes in response to whole-body inhalation exposure to cellulose nanocrystal in rats.

OBJECTIVE: Human exposure to cellulose nanocrystal (CNC) is possible during the production and/or use of products containing CNC. The objectives of the current study were to determine the lung toxicity of CNC and the underlying molecular mechanisms of the toxicity. METHODS: Rats were exposed to air or CNC (20 mg/m3, six hours/day, 14 d) by whole-body inhalation and lung toxicity and global gene expression profile were determined. RESULTS: Significant increases in lactate dehydrogenase activity, pro-inflammatory cytokine levels, phagocyte oxidant production, and macrophage and neutrophil counts were detected in the bronchoalveolar lavage cells or fluid from the CNC exposed rats. Mild lung histological changes, such as the accumulation of macrophages and neutrophils, were detected in the CNC exposed rats. Gene expression profiling by next generation sequencing identified 531 genes whose expressions were significantly different in the lungs of the CNC exposed rats, compared with the controls. Bioinformatic analysis of the lung gene expression data identified significant enrichment in several biological functions and canonical pathways including those related to inflammation (cellular movement, immune cell trafficking, inflammatory diseases and response, respiratory disease, complement system, acute phase response, leukocyte extravasation signaling, granulocyte and agranulocyte adhesion and diapedesis, IL-10 signaling, and phagosome formation and maturation) and oxidative stress (NRF2-mediated oxidative stress response, production of nitric oxide and reactive oxygen species in macrophages, and free radical scavenging). CONCLUSION: Our data demonstrated that inhalation exposure of rats to CNC resulted in lung toxicity mediated mainly through the induction of inflammation and oxidative stress.

Biological effects of inhaled hydraulic fracturing sand dust. VIII. Immunotoxicity.

Hydraulic fracturing ("fracking") is a process used to enhance retrieval of gas from subterranean natural gas-laden rock by fracturing it under pressure. Sand used to stabilize fissures and facilitate gas flow creates a potential occupational hazard from respirable fracking sand dust (FSD). As studies of the immunotoxicity of FSD are lacking, the effects of whole-body inhalation (6 h/d for 4 d) of a FSD, i.e., FSD 8, was investigated at 1, 7, and 27 d post-exposure in rats. Exposure to 10 mg/m3 FSD 8 resulted in decreased lung-associated lymph node (LLN) cellularity, total B-cells, CD4+ T-cells, CD8+ T-cells and total natural killer (NK) cells at 7-d post exposure. The frequency of CD4+ T-cells decreased while the frequency of B-cells increased (7 and 27 d) in the LLN. In contrast, increases in LLN cellularity and increases in total CD4+ and CD8+ T-cells were observed in rats following 30 mg/m3 FSD 8 at 1 d post-exposure. Increases in the frequency and number of CD4+ T-cells and NK cells were observed in bronchial alveolar lavage fluid at 7-d post-exposure (10 mg/m3) along with an increase in total CD4+ T-cells, CD11b + cells, and NK cells at 1-day post-exposure (30 mg/m3). Increases in the numbers of B-cells and CD8+ T-cells were observed in the spleen at 1-day post 30 mg/m3 FSD 8 exposure. In addition, NK cell activity was suppressed at 1 d (30 mg/m3) and 27 d post-exposure (10 mg/m3). No change in the IgM response to sheep red blood cells was observed. The findings indicate that FSD 8 caused alterations in cellularity, phenotypic subsets, and impairment of immune function.

Biological effects of inhaled hydraulic fracturing sand dust. V. Pulmonary inflammatory, cytotoxic and oxidant effects.

The pulmonary inflammatory response to inhalation exposure to a fracking sand dust (FSD 8) was investigated in a rat model. Adult male Sprague-Dawley rats were exposed by whole-body inhalation to air or an aerosol of a FSD, i.e., FSD 8, at concentrations of 10 or 30 mg/m3, 6 h/d for 4 d. The control and FSD 8-exposed rats were euthanized at post-exposure time intervals of 1, 7 or 27 d and pulmonary inflammatory, cytotoxic and oxidant responses were determined. Deposition of FSD 8 particles was detected in the lungs of all the FSD 8-exposed rats. Analysis of bronchoalveolar lavage parameters of toxicity, oxidant generation, and inflammation did not reveal any significant persistent pulmonary toxicity in the FSD 8-exposed rats. Similarly, the lung histology of the FSD 8-exposed rats showed only minimal changes in influx of macrophages following the exposure. Determination of global gene expression profiles detected statistically significant differential expressions of only six and five genes in the 10 mg/m3, 1-d post-exposure, and the 30 mg/m3, 7-d post-exposure FSD 8 groups, respectively. Taken together, data obtained from the present study demonstrated that FSD 8 inhalation exposure resulted in no statistically significant toxicity or gene expression changes in the lungs of the rats. In the absence of any information about its potential toxicity, a comprehensive rat animal model study (see Fedan, J.S., Toxicol Appl Pharmacol. 000, 000-000, 2020) has been 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.

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.

A possible relationship between telomere length and markers of neurodegeneration in rat brain after welding fume inhalation exposure.

Inhalation of welding fume (WF) can result in the deposition of toxic metals, such as manganese (Mn), in the brain and may cause neurological changes in exposed workers. Alterations in telomere length are indicative of cellular aging and, possibly, neurodegeneration. Here, we investigated the effect of WF inhalation on telomere length and markers of neurodegeneration in whole brain tissue in rats. Male Fischer-344 (F-344) rats were exposed by inhalation to stainless steel WF (20 mg/m3 x 3 h/d x 4 d/wk x 5 wk) or filtered air (control). Telomere length, DNA-methylation, gene expression of Trf1, Trf2, ATM, and APP, protein expression of p-Tau, α-synuclein, and presenilin 1 and 2 were assessed in whole brain tissue at 12 wk after WF exposure ended. Results suggest that WF inhalation increased telomere length without affecting telomerase in whole brain. Moreover, we observed that components of the shelterin complex, Trf1 and Trf2, play an important role in telomere end protection, and their regulation may be responsible for the increase in telomere length. In addition, expression of different neurodegeneration markers, such as p-Tau, presenilin 1-2 and α-synuclein proteins, were increased in brain tissue from the WF-exposed rats as compared to control. These findings suggest a possible correlation between epigenetic modifications, telomere length alteration, and neurodegeneration because of the presence of factors in serum after WF exposure that may cause extra-pulmonary effects as well as the translocation of potentially neurotoxic metals associated with WF to the central nervous system (CNS). Further studies are needed to investigate the brain region specificity and temporal response of these effects.

Surface area- and mass-based comparison of fine and ultrafine nickel oxide lung toxicity and augmentation of allergic response in an ovalbumin asthma model.

Background: The correlation of physico-chemical properties with mechanisms of toxicity has been proposed as an approach to predict the toxic potential of the vast number of emerging nanomaterials. Although relationships have been established between properties and the acute pulmonary inflammation induced by nanomaterials, properties' effects on other responses, such as exacerbation of respiratory allergy, have been less frequently explored.Methods: In this study, the role of nickel oxide (NiO) physico-chemical properties in the modulation of ovalbumin (OVA) allergy was examined in a murine model. Results: 181 nm fine (NiO-F) and 42 nm ultrafine (NiO-UF) particles were characterized and incorporated into a time course study where measured markers of pulmonary injury and inflammation were associated with NiO particle surface area. In the OVA model, exposure to NiO, irrespective of any metric was associated with elevated circulating total IgE levels. Serum and lung cytokine levels were similar with respect to NiO surface area. The lower surface area was associated with an enhanced Th2 profile, whereas the higher surface area was associated with a Th1-dominant profile. Surface area-normalized groups also exhibited similar alterations in OVA-specific IgE levels and lung neutrophil number. However, lung eosinophil number and allergen challenge-induced alterations in lung function related more to particle size, wherein NiO-F was associated with an increased enhanced pause response and NiO-UF was associated with increased lung eosinophil burden.Conclusions: Collectively, these findings suggest that although NiO surface area correlates best with acute pulmonary injury and inflammation following respiratory exposure, other physico-chemical properties may contribute to the modulation of immune responses in the lung.

Sparse Supervised Classification Methods Predict and Characterize Nanomaterial Exposures: Independent Markers of MWCNT Exposures.

Recent experimental evidence indicates significant pulmonary toxicity of multiwalled carbon nanotubes (MWCNTs), such as inflammation, interstitial fibrosis, granuloma formation, and carcinogenicity. Although numerous studies explored the adverse potential of various CNTs, their comparability is often limited. This is due to differences in administered dose, physicochemical characteristics, exposure methods, and end points monitored. Here, we addressed the problem through sparse classification method, a supervised machine learning approach that can reduce the noise contained in redundant variables for discriminating among MWCNT-exposed and MWCNT-unexposed groups. A panel of proteins measured from bronchoalveolar lavage fluid (BAL) samples was used to predict exposure to various MWCNT and determine markers that are attributable to MWCNT exposure and toxicity in mice. Using sparse support vector machine-based classification technique, we identified a small subset of proteins clearly distinguishing each exposure. Macrophage-derived chemokine (MDC/CCL22), in particular, was associated with various MWCNT exposures and was independent of exposure method employed, that is, oropharyngeal aspiration versus inhalation exposure. Sustained expression of some of the selected protein markers identified also suggests their potential role in MWCNT-induced toxicity and proposes hypotheses for future mechanistic studies. Such approaches can be used more broadly for nanomaterial risk profiling studies to evaluate decisions related to dose/time-response relationships that could delineate experimental variables from exposure markers.

The Fate of Inhaled Nanoparticles: Detection and Measurement by Enhanced Dark-field Microscopy.

Assessing the potential health risks for newly developed nanoparticles poses a significant challenge. Nanometer-sized particles are not generally detectable with the light microscope. Electron microscopy typically requires high-level doses, above the physiologic range, for particle examination in tissues. Enhanced dark-field microscopy (EDM) is an adaption of the light microscope that images scattered light. Nanoparticles scatter light with high efficiency while normal tissues do not. EDM has the potential to identify the critical target sites for nanoparticle deposition and injury in the lungs and other organs. This study describes the methods for EDM imaging of nanoparticles and applications. Examples of EDM application include measurement of deposition and clearance patterns. Imaging of a wide variety of nanoparticles demonstrated frequent situations where nanoparticles detected by EDM were not visible by light microscopy. EDM examination of colloidal gold nanospheres (10-100 nm diameter) demonstrated a detection size limit of approximately 15 nm in tissue sections. EDM determined nanoparticle volume density was directly proportional to total lung burden of exposed animals. The results confirm that EDM can determine nanoparticle distribution, clearance, transport to lymph nodes, and accumulation in extrapulmonary organs. Thus, EDM substantially improves the qualitative and quantitative microscopic evaluation of inhaled nanoparticles.

Pulmonary toxicity and global gene expression changes in response to sub-chronic inhalation exposure to crystalline silica in rats.

Exposure to crystalline silica results in serious adverse health effects, most notably, silicosis. An understanding of the mechanism(s) underlying silica-induced pulmonary toxicity is critical for the intervention and/or prevention of its adverse health effects. Rats were exposed by inhalation to crystalline silica at a concentration of 15 mg/m3, 6 hr/day, 5 days/week for 3, 6 or 12 weeks. Pulmonary toxicity and global gene expression profiles were determined in lungs at the end of each exposure period. Crystalline silica was visible in lungs of rats especially in the 12-week group. Pulmonary toxicity, as evidenced by an increase in lactate dehydrogenase (LDH) activity and albumin content and accumulation of macrophages and neutrophils in the bronchoalveolar lavage (BAL), was seen in animals depending upon silica exposure duration. The most severe histological changes, noted in the 12-week exposure group, consisted of chronic active inflammation, type II pneumocyte hyperplasia, and fibrosis. Microarray analysis of lung gene expression profiles detected significant differential expression of 38, 77, and 99 genes in rats exposed to silica for 3-, 6-, or 12-weeks, respectively, compared to time-matched controls. Among the significantly differentially expressed genes (SDEG), 32 genes were common in all exposure groups. Bioinformatics analysis of the SDEG identified enrichment of functions, networks and canonical pathways related to inflammation, cancer, oxidative stress, fibrosis, and tissue remodeling in response to silica exposure. Collectively, these results provided insights into the molecular mechanisms underlying pulmonary toxicity following sub-chronic inhalation exposure to crystalline silica in rats.

Molecular mechanisms of pulmonary response progression in crystalline silica exposed rats.

An understanding of the mechanisms underlying diseases is critical for their prevention. Excessive exposure to crystalline silica is a risk factor for silicosis, a potentially fatal pulmonary disease. Male Fischer 344 rats were exposed by inhalation to crystalline silica (15 mg/m3, six hours/day, five days) and pulmonary response was determined at 44 weeks following termination of silica exposure. Additionally, global gene expression profiling in lungs and BAL cells and bioinformatic analysis of the gene expression data were done to understand the molecular mechanisms underlying the progression of pulmonary response to silica. A significant increase in lactate dehydrogenase activity and albumin content in BAL fluid (BALF) suggested silica-induced pulmonary toxicity in the rats. A significant increase in the number of alveolar macrophages and infiltrating neutrophils in the lungs and elevation in monocyte chemoattractant protein-1 (MCP-1) in BALF suggested the induction of pulmonary inflammation in the silica exposed rats. Histological changes in the lungs included granuloma formation, type II pneumocyte hyperplasia, thickening of alveolar septa and positive response to Masson's trichrome stain. Microarray analysis of global gene expression detected 94 and 225 significantly differentially expressed genes in the lungs and BAL cells, respectively. Bioinformatic analysis of the gene expression data identified significant enrichment of several disease and biological function categories and canonical pathways related to pulmonary toxicity, especially inflammation. Taken together, these data suggested the involvement of chronic inflammation as a mechanism underlying the progression of pulmonary response to exposure of rats to crystalline silica at 44 weeks following termination of exposure.

Pulmonary toxicity following acute coexposures to diesel particulate matter and α-quartz crystalline silica in the Sprague-Dawley rat.

The effects of acute pulmonary coexposures to silica and diesel particulate matter (DPM), which may occur in various mining operations, were investigated in vivo. Rats were exposed by intratracheal instillation (IT) to silica (50 or 233 µg), DPM (7.89 or 50 µg) or silica and DPM combined in phosphate-buffered saline (PBS) or to PBS alone (control). At one day, one week, one month, two months and three months postexposure bronchoalveolar lavage and histopathology were performed to assess lung injury, inflammation and immune response. While higher doses of silica caused inflammation and injury at all time points, DPM exposure alone did not. DPM (50 µg) combined with silica (233 µg) increased inflammation at one week and one-month postexposure and caused an increase in the incidence of fibrosis at one month compared with exposure to silica alone. To assess susceptibility to lung infection following coexposure, rats were exposed by IT to 233 µg silica, 50 µg DPM, a combination of the two or PBS control one week before intratracheal inoculation with 5 × 105 Listeria monocytogenes. At 1, 3, 5, 7 and 14 days following infection, pulmonary immune response and bacterial clearance from the lung were evaluated. Coexposure to DPM and silica did not alter bacterial clearance from the lung compared to control. Although DPM and silica coexposure did not alter pulmonary susceptibility to infection in this model, the study showed that noninflammatory doses of DPM had the capacity to increase silica-induced lung injury, inflammation and onset/incidence of fibrosis.

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.

Cardiovascular effects in rats after intratracheal instillation of metal welding particles.

Studies have indicated that pulmonary exposure to welding fumes can induce a series of adverse effects in the respiratory system, including infection, bronchitis, siderosis and decreased pulmonary function. Recent clinical and epidemiological studies have found that pulmonary exposure to welding fumes is also associated with a higher incidence of cardiovascular events. However, there is insufficient evidence to confirm a direct effect of welding fumes on the cardiovascular system. The present study investigated the effects of pulmonary exposure to welding fumes on the heart and the vascular system in rats. Two chemically distinct welding fumes generated from manual metal arc-hard surfacing (MMA-HS) and gas metal arc-mild steel (GMA-MS) welding were tested. Three groups of rats were instilled intratracheally with MMA-HS (2 mg/rat), GMA-MS (2 mg/rat) or saline as control once a week for seven weeks. On days 1 and 7 after the last treatment, basal cardiovascular function and the cardiovascular response to increasing doses of adrenoreceptor agonists were assessed. MMA-HS treatment reduced the basal levels of left ventricle end-systolic pressure and dP/dt(max) at 1 day post-treatment, and decreased dP/dt(min) in response to isoproterenol (ISO) at 7 days post-treatment. Unlike MMA-HS, GMA-MS only affected left ventricular end-diastolic pressure in response to ISO at 7 days post-treatment. Treatment with MMA-HS or GMA-MS did not alter heart rate and blood pressure. Our findings suggest that exposure to different welding fumes can induce different adverse effects on the cardiovascular system, and that cardiac contractility may be a sensitive indicator of cardiovascular dysfunction.

Oxidative stress and reduced responsiveness of challenged circulating leukocytes following pulmonary instillation of metal-rich particulate matter in rats.

Welding fume is an exposure that consists of a mixture of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection in animal models, the question arose whether the dysfunction persisted systemically. Our aim was to evaluate the circulating leukocyte population in terms of cellular activation, presence of oxidative stress, and functionality after a secondary challenge, following welding fume exposure. Rats were intratracheally instilled (ITI) with PBS or 2 mg of welding fume collected from a stainless steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with LPS utilizing TruCulture technology. In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Welding fume exposure had rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. These findings support previous studies of an inadequate pulmonary immune response following a metal-rich exposure and extend those findings showing leukocyte dysfunction occurs systemically.

Effects of inhaled tier-2 diesel engine exhaust on immunotoxicity in a rat model: A hazard identification study. Part II. Immunotoxicology.

Diesel exhaust (DE) is an air pollutant containing gaseous compounds and particulate matter. Diesel engines are common on gas extraction and oil sites, leading to complex DE exposure to a broad range of compounds through occupational settings. The US EPA concluded that short-term exposure to DE leads to allergic inflammatory disorders of the airways. To further evaluate the immunotoxicity of DE, the effects of whole-body inhalation of 0.2 and 1 mg/m3 DE (total carbon; 6 h/d for 4 days) were investigated 1-, 7-, and 27-days post exposure in Sprague-Dawley rats using an occupationally relevant exposure system. DE exposure of 1 mg/m3 increased total cellularity, number of CD4+ and CD8+ T-cells, and B-cells at 1 d post-exposure in the lung lymph nodes. At 7 d post-exposure to 1 mg/m3, cellularity and the number of CD4+ and CD8+ T-cells decreased in the LLNs. In the bronchoalveolar lavage, B-cell number and frequency increased at 1 d post-exposure, Natural Killer cell number and frequency decreased at 7 d post-exposure, and at 27 d post-exposure CD8+ T-cell and CD11b+ cell number and frequency decreased with 0.2 mg/m3 exposure. In the spleen, 0.2 mg/m3 increased CD4+ T-cell frequency at 1 and 7 d post-exposure and at 27 d post-exposure increased CD4+ and CD8+ T-cell number and CD8+ T-cell frequency. B-cells were the only immune cell subset altered in the three tissues (spleen, LLNs, and BALF), suggesting the induction of the adaptive immune response. The increase in lymphocytes in several different organ types also suggests an induction of a systemic inflammatory response occurring following DE exposure. These results show that DE exposure induced modifications of cellularity of phenotypic subsets that may impair immune function and contribute to airway inflammation induced by DE exposure in rats.