Biological effects of diesel exhaust inhalation. III cardiovascular function.

OBJECTIVE: Inhalation of diesel exhaust (DE) has been shown to be an occupational hazard in the transportation, mining, and gas and oil industries. DE also contributes to air pollution, and therefore, is a health hazard to the general public. Because of its effects on human health, changes have been made to diesel engines to reduce both the amounts of particulate matter and volatile fumes they generate. The goal of the current study was to examine the effects of inhalation of diesel exhaust. MATERIALS AND METHODS: The study presented here specifically examines the effects of exposure to 0.2 and 1.0 mg/m3 DE or filtered air (6h/d for 4 d) on measures of peripheral and cardio-vascular function, and biomarkers of heart and kidney dysfunction in male rats. A Tier 2 engine used in oil and gas fracking operations was used to generate the diesel exhaust. RESULTS: Exposure to 0.2 mg/m3 DE resulted in an increase in blood pressure 1d following the last exposure, and increases in dobutamine-induced cardiac output and stroke volume 1 and 27d after exposure. Changes in peripheral vascular responses to norepinephrine and acetylcholine were minimal as were changes in transcript expression in the heart and kidney. Exposure to 1.0 mg/m3 DE did not result in major changes in blood pressure, measures of cardiac function, peripheral vascular function or transcript expression. DISCUSSION AND CONCLUSIONS: Based on the results of this study, we suggest that exposure to DE generated by a Tier 2 compliant diesel engine generates acute effects on biomarkers indicative of cardiovascular dysfunction. Recovery occurs quickly with most measures of vascular/cardiovascular function returning to baseline levels by 7d following exposure.

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.

Biological effects of inhaled crude oil vapor. II. Pulmonary effects.

Workers involved in oil exploration and production in the upstream petroleum industry are exposed to crude oil vapor (COV). COV levels in the proximity of workers during production tank gauging and opening of thief hatches can exceed regulatory standards, and several deaths have occurred after opening thief hatches. There is a paucity of information regarding the effects of COV inhalation in the lung. To address these knowledge gaps, the present hazard identification study was undertaken to investigate the effects of an acute, single inhalation exposure (6 h) or a 28 d sub-chronic exposure (6 h/d × 4 d/wk × 4 wks) to COV (300 ppm; Macondo well surrogate oil) on ventilatory and non-ventilatory functions of the lung in a rat model 1 and 28 d after acute exposure, and 1, 28 and 90 d following sub-chronic exposure. Basal airway resistance was increased 90 d post-sub-chronic exposure, but reactivity to methacholine (MCh) was unaffected. In the isolated, perfused trachea preparation the inhibitory effect of the airway epithelium on reactivity to MCh was increased at 90 d post-exposure. Efferent cholinergic nerve activity regulating airway smooth muscle was unaffected by COV exposure. Acute exposure did not affect basal airway epithelial ion transport, but 28 d after sub-chronic exposure alterations in active (Na+ and Cl¯) and passive ion transport occurred. COV treatment did not affect lung vascular permeability. The findings indicate that acute and sub-chronic COV inhalation does not appreciably affect ventilatory properties of the rat, but transient changes in airway epithelium occur.

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.

Biological effects of crude oil vapor. IV. Cardiovascular effects.

Workers in the oil and gas extraction industry are at risk of inhaling volatile organic compounds. Epidemiological studies suggest oil vapor inhalation may affect cardiovascular health. Thus, in this hazard identification study we investigated the effects of inhalation of crude oil vapor (COV) on cardiovascular function. Male rats were exposed to air or COV (300 ppm) for 6 h (acute), or 6 h/day × 4 d/wk. × 4 wk. (sub-chronic). The effects of COV inhalation were assessed 1, 28, and 90 d post-exposure. Acute exposure to COV resulted in reductions in mean arterial and diastolic blood pressures 1 and 28 d after exposure, changes in nitrate-nitrite and H2O2 levels, and in the expression of transcripts and proteins that regulate inflammation, vascular remodeling, and the synthesis of nitric oxide (NO) in the heart and kidneys. The sub-chronic exposure resulted in a reduced sensitivity to α1-adrenoreceptor-mediated vasoconstriction in vitro 28 d post-exposure, and a reduction in oxidative stress in the heart. Sub-chronic COV exposure led to alterations in the expression of NO synthases and anti-oxidant enzymes, which regulate inflammation and oxidative stress in the heart and kidneys. There seems to be a balance between changes in the expression of transcripts associated with the generation of reactive oxygen species (ROS) and antioxidant enzymes. The ability of antioxidant enzymes to reduce or inhibit the effects of ROS may allow the cardiovascular system to adapt to acute COV exposures. However, sub-chronic exposures may result in longer-lasting negative health consequences on the cardiovascular system.

Biological effects of inhaled crude oil vapor V. Altered biogenic amine neurotransmitters and neural protein expression.

Workers in the oil and gas industry are at risk for exposure to a number of physical and chemical hazards at the workplace. Chemical hazard risks include inhalation of crude oil or its volatile components. While several studies have investigated the neurotoxic effects of volatile hydrocarbons, in general, there is a paucity of studies assessing the neurotoxicity of crude oil vapor (COV). Consequent to the 2010 Deepwater Horizon (DWH) oil spill, there is growing concern about the short- and long-term health effects of exposure to COV. NIOSH surveys suggested that the DWH oil spill cleanup workers experienced neurological symptoms, including depression and mood disorders, but the health effects apart from oil dispersants were difficult to discern. To investigate the potential neurological risks of COV, male Sprague-Dawley rats were exposed by whole-body inhalation to COV (300 ppm; Macondo surrogate crude oil) following an acute (6 h/d × 1 d) or sub-chronic (6 h/d × 4 d/wk. × 4 wks) exposure regimen. At 1, 28 or 90 d post-exposure, norepinephrine (NE), epinephrine (EPI), dopamine (DA) and serotonin (5-HT) were evaluated as neurotransmitter imbalances are associated with psychosocial-, motor- and cognitive- disorders. Sub-chronic COV exposure caused significant reductions in NE, EPI and DA in the dopaminergic brain regions, striatum (STR) and midbrain (MB), and a large increase in 5-HT in the STR. Further, sub-chronic exposure to COV caused upregulation of synaptic and Parkinson's disease-related proteins in the STR and MB. Whether such effects will lead to neurodegenerative outcomes remain to be investigated.

Automated crude oil vapor inhalation exposure system.

Objective: Inhalation exposure systems are tools for delivering compounds (particles, vapors, and gases) under well-controlled conditions for toxicological testing. The objective of this project was to develop an automated computer-controlled system to expose small laboratory animals to precise concentrations of crude oil vapor (COV).Materials and Methods: Vapor from heated Deepwater Horizon surrogate oil was atomized into a fine mist then diluted with filtered air, then the air/droplet mixture was routed into an evaporation column with an high efficiency particulate air (HEPA) filter on its exit port. The HEPA filter was used to remove oil particles, thus ensuring only vapor would pass. The vapor was then introduced into a custom-built exposure chamber housing rats. A calibrated flame ionization detector was used to read the total volatile organic compounds (TVOC) in real time, and custom software was developed to automatically adjust the amount of oil entering the atomizer with a syringe pump. The software also controlled relative humidity and pressure inside the exposure chamber. Other exposure chamber environmental parameters, e.g. temperature and CO2 levels, were monitored. Four specific components within the COV were monitored during each exposure: benzene, toluene, ethylbenzene, and xylenes.Results: The TVOC vapor concentration control algorithm maintained median concentrations to within ±2 ppm of the target concentration (300 ppm) of TVOC during exposures lasting 6 h. The system could reach 90% of the desired target in less than 15 min, and repeat exposures were consistent and reproducible.Conclusion: This exposure system provided a highly automated tool for conducting COV inhalation toxicology studies.

Biological effects of inhaled hydraulic fracturing sand dust. I. Scope of the investigation.

Hydraulic fracturing ("fracking") is a process in which subterranean natural gas-laden rock is fractured under pressure to enhance retrieval of gas. Sand (a "proppant") is present in the fracking fluid pumped down the well bore to stabilize the fissures and facilitate gas flow. The manipulation of sand at the well site creates respirable dust (fracking sand dust, FSD) to which workers are exposed. Because workplace exposures to FSD have exceeded exposure limits set by OSHA, a physico-chemical characterization of FSD along with comprehensive investigations of the potential early adverse effects of FSDs on organ function and biomarkers has been conducted using a rat model and related in vivo and in vitro experiments involving the respiratory, cardiovascular, immune systems, kidney and brain. An undercurrent theme of the overall hazard identification study was, to what degree do the health effects of inhaled FSD resemble those previously observed after crystalline silica dust inhalation? In short-term studies, FSD was found to be less bioactive than MIN-U-SIL® 5 in the lungs. A second theme was, are the biological effects of FSD restricted to the lungs? Bioactivity of FSD was observed in all examined organ systems. Our findings indicate that, in many respects, the physical and chemical properties, and the short-term biological effects, of the FSDs share many similarities as a group but have little in common with crystalline silica dust.

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 VII. Neuroinflammation and altered synaptic protein expression.

Hydraulic fracturing (fracking) is a process used to recover oil and gas from shale rock formation during unconventional drilling. Pressurized liquids containing water and sand (proppant) are used to fracture the oil- and natural gas-laden rock. The transportation and handling of proppant at well sites generate dust aerosols; thus, there is concern of worker exposure to such fracking sand dusts (FSD) by inhalation. FSD are generally composed of respirable crystalline silica and other minerals native to the geological source of the proppant material. Field investigations by NIOSH suggest that the levels of respirable crystalline silica at well sites can exceed the permissible exposure limits. Thus, from an occupational safety perspective, it is important to evaluate the potential toxicological effects of FSD, including any neurological risks. Here, we report that acute inhalation exposure of rats to one FSD, i.e., FSD 8, elicited neuroinflammation, altered the expression of blood brain barrier-related markers, and caused glial changes in the olfactory bulb, hippocampus and cerebellum. An intriguing observation was the persistent reduction of synaptophysin 1 and synaptotagmin 1 proteins in the cerebellum, indicative of synaptic disruption and/or injury. While our initial hazard identification studies suggest a likely neural risk, more research is necessary to determine if such molecular aberrations will progressively culminate in neuropathology/neurodegeneration leading to behavioral and/or functional deficits.

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. VI. Cardiovascular effects.

Hydraulic fracturing is used to access oil and natural gas reserves. This process involves the high-pressure injection of fluid to fracture shale. Fracking fluid contains approximately 95% water, chemicals and 4.5% fracking sand. Workers may be exposed to fracking sand dust (FSD) during the manipulation of the sand, and negative health consequences could occur if FSD is inhaled. In the absence of any information about its potential toxicity, a comprehensive rat animal model study (see Fedan et al., 2020) 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. The goal of this study was to assess the effects of inhalation of one FSD, i.e., FSD 8, on factors and tissues that affect cardiovascular function. Male rats were exposed to 10 or 30 mg/m3 FSD (6 h/d for 4 d) by whole body inhalation, with measurements made 1, 7 or 27 d post-exposure. One day following exposure to 10 mg/m3 FSD the sensitivity to phenylephrine-induced vasoconstriction in tail arteries in vitro was increased. FSD exposure at both doses resulted in decreases in heart rate (HR), HR variability, and blood pressure in vivo. FSD induced changes in hydrogen peroxide concentrations and transcript levels for pro-inflammatory factors in heart tissues. In kidney, expression of proteins indicative of injury and remodeling was reduced after FSD exposure. When analyzed using regression analysis, changes in proteins involved in repair and remodeling were correlated. Thus, it appears that inhalation of FSD does have some prolonged effects on cardiovascular, and, possibly, renal function. The findings also provide information regarding potential mechanisms that may lead to these changes, and biomarkers that could be examined to monitor physiological changes that could be indicative of impending cardiovascular dysfunction.

Biological effects of inhaled hydraulic fracturing sand dust. IV. Pulmonary effects.

Hydraulic fracturing creates fissures in subterranean rock to increase the flow and retrieval of natural gas. Sand ("proppant") in fracking fluid injected into the well bore maintains fissure patency. Fracking sand dust (FSD) is generated during manipulation of sand to prepare the fracking fluid. Containing respirable crystalline silica, FSD could pose hazards similar to those found in work sites where silica inhalation induces lung disease such as silicosis. This study was performed to evaluate the possible toxic effects following inhalation of a FSD (FSD 8) in the lung and airways. Rats were exposed (6 h/d × 4 d) to 10 or 30 mg/m3 of a FSD collected at a gas well, and measurements were performed 1, 7, 27 and, in one series of experiments, 90 d post-exposure. The following ventilatory and non-ventilatory parameters were measured in vivo and/or in vitro: 1) lung mechanics (respiratory system resistance and elastance, tissue damping, tissue elastance, Newtonian resistance and hysteresivity); 2) airway reactivity to inhaled methacholine (MCh); airway epithelium integrity (isolated, perfused trachea); airway efferent motor nerve activity (electric field stimulation in vitro); airway smooth muscle contractility; ion transport in intact and cultured epithelium; airway effector and sensory nerves; tracheal particle deposition; and neurogenic inflammation/vascular permeability. FSD 8 was without large effect on most parameters, and was not pro-inflammatory, as judged histologically and in cultured epithelial cells, but increased reactivity to inhaled MCh at some post-exposure time points and affected Na+ transport in airway epithelial cells.

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.

Comparison of multi-wall carbon nanotube and nitrogen-doped multi-wall carbon nanotube effects on lung function and airway reactivity in rats.

Incorporation of multi-wall carbon nanotubes (MWCNT) into materials has raised concerns about their potential hazards to manufacturing workers. In animal models, airway inflammation and lung fibrosis follow aspiration, instillation, and inhalation exposures to MWCNT. However, the effects of MWCNT on pulmonary function, airway reactivity and airway epithelium function following inhalation exposure has not been studied. We investigated whether inhaled MWCNT affects lung resistance (RL) and dynamic compliance (Cdyn), reactivity to inhaled methacholine (MCh), epithelial regulation of airway reactivity to MCh in vitro, and airway epithelial ion transport. Male rats were exposed by whole body inhalation for 6 h to air or aerosolized MWCNT (0.5, 1 or 5 mg/m3) for one or nine days. Eighteen h after 1 d exposure to 5 mg/m3 MWCNT, basal RL was increased and basal Cdyn was decreased; changes did not persist for 7 d. Reactivity to MCh (RL) was increased and Cdyn responses were decreased at 18 h, but not 7 d after exposure to 1 and 5 mg/m3 MWCNT. The effects of i.t.-instilled MWCNT and nitrogen-doped MWCNT (N-MWCNT) on pulmonary function and reactivity to MCh at doses comparable to deposition after inhalation of 5 mg/m3 at 1 d and 0.5, 1, and 5 mg/m3 MWCNT 9 d-exposures were compared. Both nanoparticles increased airway reactivity (RL); N-MWCNT did not affect Cdyn responses. Lung function and airway reactivity are altered following a single MWCNT inhalation and generally subside over time. Given i.t., MWCNT's and N-MWCNT's effects were comparable, but N-MWCNT evoke smaller changes in Cdyn responses.

Flavorings-Related Lung Disease: A Brief Review and New Mechanistic Data.

Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile α-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon α-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate α-dicarbonyl compounds in airway injury and flavorings-related lung disease.

Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity.

Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive α-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this α-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the α-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease.

Altered ion transport in normal human bronchial epithelial cells following exposure to chemically distinct metal welding fume particles.

Welding fume inhalation causes pulmonary toxicity, including susceptibility to infection. We hypothesized that airway epithelial ion transport is a target of fume toxicity, and investigated the effects of fume particulates from manual metal arc-stainless steel (MMA-SS) and gas metal arc-mild steel (GMA-MS) on ion transport in normal human bronchial epithelium (NHBE) cultured in air-interface. MMA-SS particles, more soluble than GMA-MS particles, contain Cr, Ni, Fe and Mn; GMA-MS particles contain Fe and Mn. MMA-SS or GMA-MS particles (0.0167-166.7µg/cm2) were applied apically to NHBEs. After 18h transepithelial potential difference (Vt), resistance (Rt), and short circuit current (Isc) were measured. Particle effects on Na+ and Cl¯ channels and the Na+,K+,2Cl¯-cotransporter were evaluated using amiloride (apical), 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB, apical), and bumetanide (basolateral), respectively. MMA-SS (0.0167-16.7µg/cm2) increased basal Vt. Only 16.7µg/cm2 GMA-MS increased basal Vt significantly. MMA-SS or GMA-MS exposure potentiated Isc responses (decreases) to amiloride and bumetanide, while not affecting those to NPPB, GMA-MS to a lesser degree than MMA-SS. Variable effects on Rt were observed in response to amiloride, and bumetanide. Generally, MMA-SS was more potent in altering responses to amiloride and bumetanide than GMA-MS. Hyperpolarization occurred in the absence of LDH release, but decreases in Vt, Rt, and Isc at higher fume particulate doses accompanied LDH release, to a greater extent for MMA-SS. Thus, Na+ transport and Na+,K+,2Cl¯-cotransport are affected by fume exposure; MMA-MS is more potent than GMA-MS. Enhanced Na+ absorption and decreased airway surface liquid could compromise defenses against infection.

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.