Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure.

Bromine (Br2) is an organohalide found in nature and is integral to many manufacturing processes. Br2 is toxic to living organisms, and high concentrations can prove fatal. To meet industrial demand, large amounts of purified Br2 are produced, transported, and stored worldwide, providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br2 exposure, we have surveyed the lung proteomes of C57BL/6 male mice and human lung-derived microvascular endothelial cells (HMECs) at 24 h following exposure to Br2 in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 h after Br2 exposure. These experiments revealed significant increases in the filtration coefficient (Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br2 and Br-lipid-treated HMECs exhibited differential levels of zona occludens-1 that were found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadherin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br2 toxicity.

Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats.

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.

Respiratory syncytial virus infection increases chlorine-induced airway hyperresponsiveness.

Exposure to chlorine (Cl2) damages airway and alveolar epithelia resulting in acute lung injury and reactive airway hyperresponsiveness (AHR) to methacholine. However, little is known about the effect of preexisting respiratory disease on Cl2-induced lung injury. By using a murine respiratory syncytial virus (RSV) infection model, we found that preexisting RSV infection increases Cl2 (187 ppm for 30 min)-induced lung inflammation and airway AHR at 24 h after exposure (5 days after infection). RSV infection and Cl2 exposure synergistically induced oxygen desaturation and neutrophil infiltration and increased MCP-1, MIP-1ß, IL-10, IFN-γ, and RANTES concentrations in the bronchoalveolar lavage fluid (BALF). In contrast, levels of type 2 cytokines (i.e., IL-4, IL-5, IL-9, and IL-13) were not significantly affected by either RSV infection or Cl2 exposure. Cl2 exposure, but not RSV infection, induced AHR to methacholine challenge as measured by flexiVent. Moreover, preexisting RSV infection amplified BALF levels of hyaluronan (HA) and AHR. The Cl2-induced AHR was mitigated by treatment with inter-α-trypsin inhibitor antibody, which inhibits HA signaling, suggesting a mechanism of HA-mediated AHR from exacerbated oxidative injury. Our results show for the first time that preexisting RSV infection predisposes the lung to Cl2-induced injury. These data emphasize the necessity for further research on the effects of Cl2 in vulnerable populations and the development of appropriate treatments.

Upregulation of autophagy decreases chlorine-induced mitochondrial injury and lung inflammation.

The mechanisms of toxicity during exposure of the airways to chlorinated biomolecules generated during the course of inflammation and to chlorine (Cl2) gas are poorly understood. We hypothesized that lung epithelial cell mitochondria are damaged by Cl2 exposure and activation of autophagy mitigates this injury. To address this, NCI-H441 (human lung adenocarcinoma epithelial) cells were exposed to Cl2 (100 ppm/15 min) and bioenergetics were assessed. One hour after Cl2, cellular bioenergetic function and mitochondrial membrane potential were decreased. These changes were associated with increased MitoSOX signal, and treatment with the mitochondrial redox modulator MitoQ attenuated these bioenergetic defects. At 6h postexposure, there was significant increase in autophagy, which was associated with an improvement of mitochondrial function. Pretreatment of H441 cells with trehalose (an autophagy activator) improved bioenergetic function, whereas 3-methyladenine (an autophagy inhibitor) resulted in increased bioenergetic dysfunction 1h after Cl2 exposure. These data indicate that Cl2 induces bioenergetic dysfunction, and autophagy plays a protective role in vitro. Addition of trehalose (2 vol%) to the drinking water of C57BL/6 mice for 6 weeks, but not 1 week, before Cl2 (400 ppm/30 min) decreased white blood cells in the bronchoalveolar lavage fluid at 6h after Cl2 by 70%. Acute administration of trehalose delivered through inhalation 24 and 1h before the exposure decreased alveolar permeability but not cell infiltration. These data indicate that Cl2 induces bioenergetic dysfunction associated with lung inflammation and suggests that autophagy plays a protective role.

Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury.

Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca(2+), and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca(2+), blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca(2+) channels of airway smooth muscle cells, increasing their contractility and thus causing AHR.

TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury.

The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.

Chlorine induces the unfolded protein response in murine lungs and skin.

Chlorine (Cl2) is an important industrial chemical. Accidental full body exposure to Cl2 poses an environmental, occupational, and public health hazard characterized mainly by injury to the lung, skin, and ocular epithelia. The cellular mechanisms underlying its acute toxicity are incompletely understood. This study examined whether whole body exposure of BALB/c mice to Cl2 in environmental chambers leads to the up-regulation of the unfolded protein response (UPR) in their lungs and skin. Shaved BALB/c mice were exposed to a sublethal concentration of Cl2 (400 ppm for 30 min) and returned to room air for 1 or 6 hours and killed. IL-6 and TNF-α were increased significantly at 1 and 6 hours after Cl2 exposure in the lungs and at 6 hours in the skin. These changes were accompanied by increased UPR signaling (i.e., activation of protein kinase RNA-like endoplasmic reticulum kinase, inositol-requiring enzyme 1 α, and activating transcription factor 6α) at these time points. The expression of hepcidin, which regulates tissue accumulation and mobilization of iron, was increased in the skin and lungs of Cl2-exposed mice. The data shown herein indicate for the first time the up-regulation of UPR signaling and hepcidin in the skin and lungs of Cl2-exposed mice, which persisted when the mice were returned to room air for 6 hours.

Chlorine gas exposure increases susceptibility to invasive lung fungal infection.

Chlorine (Cl2) is a highly irritating and reactive gas with potential occupational and environmental hazards. Acute exposure to Cl2 induces severe epithelial damage, airway hyperreactivity, impaired alveolar fluid clearance, and pulmonary edema in the presence of heightened inflammation and significant neutrophil accumulation in the lungs. Herein, we investigated whether Cl2 exposure affected the lung antimicrobial immune response leading to increased susceptibility to opportunistic infections. Mice exposed to Cl2 and challenged intratracheally 24 h thereafter with the opportunistic mold Aspergillus fumigatus demonstrated an >500-fold increase in A. fumigatus lung burden 72 h postchallenge compared with A. fumigatus mice exposed to room air. Cl2-exposed A. fumigatus challenged mice also demonstrated significantly higher lung resistance following methacholine challenge and increased levels of plasma proteins (albumin and IgG) in the bronchoalveolar lavage fluid. Despite enhanced recruitment of inflammatory cells to the lungs of Cl2-exposed A. fumigatus challenged mice, these cells (>60% of which were neutrophils) demonstrated a profound impairment in generating superoxide. Significantly higher A. fumigatus burden in the lungs of Cl2 exposed mice correlated with enhanced production of IL-6, TNF-α, CXCL1, CCL2, and CCL3. Surprisingly, however, Cl2-exposed A. fumigatus challenged mice had a specific impairment in the production of IL-17A and IL-22 in the lungs compared with mice exposed to room air and challenged with A. fumigatus. In summary, our results indicate that Cl2 exposure markedly impairs the antimicrobial activity and inflammatory reactivity of myeloid cells in the lung leading to increased susceptibility to opportunistic pathogens.

Targeted aerosolized delivery of ascorbate in the lungs of chlorine-exposed rats.

BACKGROUND: Chlorine (Cl(2))-induced lung injury is a serious public health threat that may result from industrial and household accidents. Post-Cl(2) administration of aerosolized ascorbate in rodents decreased lung injury and mortality. However, the extent to which aerosolized ascorbate augments depleted ascorbate stores in distal lung compartments has not been assessed. METHODS: We exposed rats to Cl(2) (300 ppm for 30 min) and returned them to room air. Within 15-30 min postexposure, rats breathed aerosolized ascorbate and desferal or vehicle (mean particle size 3.3 µm) through a nose-only exposure system for 60 min and were euthanized. We measured the concentrations of reduced ascorbate in the bronchoalveolar lavage (BAL), plasma, and lung tissues with high-pressure liquid chromatography, protein plasma concentration in the BAL, and the volume of the epithelia lining fluid (ELF). RESULTS: Cl(2)-exposed rats that breathed aerosolized vehicle had lower values of ascorbate in their BAL, ELF, and lung tissues compared to air-breathing rats. Delivery of aerosolized ascorbate increased reduced ascorbate in BAL, ELF, lung tissues, and plasma of both Cl(2) and air-exposed rats without causing lung injury. Based on mean diameter of aerosolized particles and airway sizes we calculated that approximately 5% and 1% of inhaled ascorbate was deposited in distal lung regions of air and Cl(2)-exposed rats, respectively. Significantly higher ascorbate levels were present in the BAL of Cl(2)-exposed rats when aerosol delivery was initiated 1 h post-Cl(2). CONCLUSIONS: Aerosol administration is an effective, safe, and noninvasive method for the delivery of low molecular weight antioxidants to the lungs of Cl(2)-exposed individuals for the purpose of decreasing morbidity and mortality. Delivery is most effective when initiated 1 h postexposure when the effects of Cl(2) on minute ventilation subside.

Post-exposure antioxidant treatment in rats decreases airway hyperplasia and hyperreactivity due to chlorine inhalation.

We assessed the safety and efficacy of combined intravenous and aerosolized antioxidant administration to attenuate chlorine gas-induced airway alterations when administered after exposure. Adult male Sprague-Dawley rats were exposed to air or 400 parts per million (ppm) chlorine (a concentration likely to be encountered in the vicinity of industrial accidents) in environmental chambers for 30 minutes, and returned to room air, and they then received a single intravenous injection of ascorbic acid and deferoxamine or saline. At 1 hour and 15 hours after chlorine exposure, the rats were treated with aerosolized ascorbate and deferoxamine or vehicle. Lung antioxidant profiles, plasma ascorbate concentrations, airway morphology, and airway reactivity were evaluated at 24 hours and 7 days after chlorine exposure. At 24 hours after exposure, chlorine-exposed rats had significantly lower pulmonary ascorbate and reduced glutathione concentrations. Treatment with antioxidants restored depleted ascorbate in lungs and plasma. At 7 days after exposure, in chlorine-exposed, vehicle-treated rats, the thickness of the proximal airways was 60% greater than in control rats, with twice the amount of mucosubstances. Airway resistance in response to methacholine challenge was also significantly elevated. Combined treatment with intravenous and aerosolized antioxidants restored airway morphology, the amount of airway mucosubstances, and airway reactivity to control levels by 7 days after chlorine exposure. Our results demonstrate for the first time, to the best of our knowledge, that severe injury to major airways in rats exposed to chlorine, as characterized by epithelial hyperplasia, mucus accumulation, and airway hyperreactivity, can be reversed in a safe and efficacious manner by the post-exposure administration of ascorbate and deferoxamine.

Regulation of alveolar epithelial Na+ channels by ERK1/2 in chlorine-breathing mice.

The mechanisms by which the exposure of mice to Cl(2) decreases vectorial Na(+) transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na(+) channels (ENaCs) after exposure to Cl(2), and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (I(amil)) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na(+) channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl(2), the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl(2) increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased I(amil) and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl(2) decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in I(amil) and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl(2) activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.

Mitigation of chlorine gas lung injury in rats by postexposure administration of sodium nitrite.

Nitrite (NO(2)(-)) has been shown to limit injury to the heart, liver, and kidneys in various models of ischemia-reperfusion injury. Potential protective effects of systemic NO(2)(-) in limiting lung injury or enhancing repair have not been documented. We assessed the efficacy and mechanisms by which postexposure intraperitoneal injections of NO(2)(-) mitigate chlorine (Cl(2))-induced lung injury in rats. Rats were exposed to Cl(2) (400 ppm) for 30 min and returned to room air. NO(2)(-) (1 mg/kg) or saline was administered intraperitoneally at 10 min and 2, 4, and 6 h after exposure. Rats were killed at 6 or 24 h. Injury to airway and alveolar epithelia was assessed by quantitative morphology, protein concentrations, number of cells in bronchoalveolar lavage (BAL), and wet-to-dry lung weight ratio. Lipid peroxidation was assessed by measurement of lung F(2)-isoprostanes. Rats developed severe, but transient, hypoxemia. A significant increase of protein concentration, neutrophil numbers, airway epithelia in the BAL, and lung wet-to-dry weight ratio was evident at 6 h after Cl(2) exposure. Quantitative morphology revealed extensive lung injury in the upper airways. Airway epithelial cells stained positive for terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL), but not caspase-3. Administration of NO(2)(-) resulted in lower BAL protein levels, significant reduction in the intensity of the TUNEL-positive cells, and normal lung wet-to-dry weight ratios. F(2)-isoprostane levels increased at 6 and 24 h after Cl(2) exposure in NO(2)(-)- and saline-injected rats. This is the first demonstration that systemic NO(2)(-) administration mitigates airway and epithelial injury.

Ascorbate and deferoxamine administration after chlorine exposure decrease mortality and lung injury in mice.

Chlorine (Cl(2)) gas exposure poses an environmental and occupational hazard that frequently results in acute lung injury. There is no effective treatment. We assessed the efficacy of antioxidants, administered after exposure, in decreasing mortality and lung injury in C57BL/6 mice exposed to 600 ppm of Cl(2) for 45 minutes and returned to room air. Ascorbate and deferoxamine were administered intramuscularly every 12 hours and by nose-only inhalation every 24 hours for 3 days starting after 1 hour after exposure. Control mice were exposed to Cl(2) and treated with vehicle (saline or water). Mortality was reduced fourfold in the treatment group compared with the control group (22 versus 78%; P = 0.007). Surviving animals in the treatment group had significantly lower protein concentrations, cell counts, and epithelial cells in their bronchoalveolar lavage (BAL). Lung tissue ascorbate correlated inversely with BAL protein as well as with the number of neutrophils and epithelial cells. In addition, lipid peroxidation was reduced threefold in the BAL of mice treated with ascorbate and deferoxamine when compared with the control group. Administration of ascorbate and deferoxamine reduces mortality and decreases lung injury through reduction of alveolar-capillary permeability, inflammation, and epithelial sloughing and lipid peroxidation.

Mechanisms and modification of chlorine-induced lung injury in animals.

Chlorine (Cl(2)) is a reactive oxidant gas used extensively in industrial processes. Exposure of both humans and animals to high concentrations of Cl(2) results in acute lung injury, which may resolve spontaneously or progress to acute respiratory failure. Injury to airway and alveolar epithelium may result from chemical reactions of Cl(2), from HOCl (the hydrolysis product of Cl(2)), and/or from the various reaction products, such as chloramines, that are formed from the reactions of these chlorinating species with biological molecules. Subsequent reactions may initiate self-propagating reactions and induce the production of inflammatory mediators compounding injury to pulmonary surfactant, ion channels, and components of lung epithelial and airway cells. Low-molecular-weight antioxidants, such as ascorbate, glutathione, and urate, present in the lung epithelial lining fluid and tissue, remove Cl(2) and HOCl and thus decrease injury to critical target biological targets. However, levels of lung antioxidants of animals exposed to Cl(2) in concentrations likely to be encountered in the vicinity of industrial accidents decrease rapidly and irreversibly. Our measurements show that prophylactic administration of a mixture containing ascorbate and desferal N-acetyl-cysteine, a precursor of reduced glutathione, prevents Cl(2)-induced injury to the alveolar epithelium of rats exposed to Cl(2). The clinical challenge is to deliver sufficient quantities of antioxidants noninvasively, after Cl(2) exposure, to decrease morbidity and mortality.