N-acetyl cysteine mitigates lung damage and inflammation after chlorine exposure in vivo and ex vivo.

The objective of this study was to explore the effects of antioxidant treatments, specifically N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA), in a mouse model of chlorine (Cl2)-induced lung injury. Additionally, the study aimed to investigate the utility of pig precision-cut lung slices (PCLS) as an ex vivo alternative for studying the short-term effects of Cl2 exposure and evaluating antioxidant treatments. The toxicological responses were analyzed in Cl2-exposed mice (inflammation, airway hyperresponsiveness (AHR)) and PCLS (viability, cytotoxicity, inflammatory mediators). Airways contractions were assessed using a small ventilator for mice and electric-field stimulation (EFS) for PCLS. Antioxidant treatments were administered to evaluate their effects. In Cl2-exposed mice, NAC treatment did not alleviate AHR, but it did reduce the number of neutrophils in bronchoalveolar lavage fluid and inflammatory mediators in lung tissue. In PCLS, exposure to Cl2 resulted in concentration-dependent toxicity, impairing the lung tissue's ability to respond to EFS-stimulation. NAC treatment increased viability, mitigated the toxic responses caused by Cl2 exposure, and maintained contractility comparable to unexposed controls. Interestingly, NACA did not provide any additional treatment effect beyond NAC in both models. In conclusion, the establishment of a pig model for Cl2-induced lung damage supports further investigation of NAC as a potential treatment. However, the lack of protective effects on AHR after NAC treatment in mice suggests that NAC alone may not be sufficient as a complete treatment for Cl2 injuries. Optimization of existing medications with a polypharmacy approach may be more successful in addressing the complex sequelae of Cl2-induced lung injury.

Ammonia exposure by intratracheal instillation causes severe and deteriorating lung injury and vascular effects in mice.

OBJECTIVE: Ammonia (NH3) is a corrosive alkaline gas that can cause life-threatening injuries by inhalation. The aim was to establish a disease model for NH3-induced injuries similar to acute lung injury (ALI) described in exposed humans and investigate the progression of lung damage, respiratory dysfunction and evaluate biomarkers for ALI and inflammation over time. METHODS: Female BALB/c mice were exposed to an NH3 dose of 91.0 mg/kg·bw using intratracheal instillation and the pathological changes were followed for up to 7 days. RESULTS: NH3 instillation resulted in the loss of body weight along with a significant increase in pro-inflammatory mediators in both bronchoalveolar lavage fluid (e.g. IL-1ß, IL-6, KC, MMP-9, SP-D) and blood (e.g. IL-6, Fibrinogen, PAI-1, PF4/CXCL4, SP-D), neutrophilic lung inflammation, alveolar damage, increased peripheral airway resistance and methacholine-induced airway hyperresponsiveness compared to controls at 20 h. On day 7 after exposure, deteriorating pathological changes such as increased macrophage lung infiltration, heart weights, lung hemorrhages and coagulation abnormalities (elevated plasma levels of PAI-1, fibrinogen, endothelin and thrombomodulin) were observed but no increase in lung collagen. Some of the analyzed blood biomarkers (e.g. RAGE, IL-1ß) were unaffected despite severe ALI and may not be significant for NH3-induced damages. CONCLUSIONS: NH3 induces severe acute lung injuries that deteriorate over time and biomarkers in lungs and blood that are similar to those found in humans. Therefore, this model has potential use for developing diagnostic tools for NH3-induced ALI and for finding new therapeutic treatments, since no specific antidote has been identified yet.

Chlorine exposure induces Caspase-3 independent cell death in human lung epithelial cells.

Chlorine (Cl2) is a common toxic industrial gas and human inhalation exposure causes tissue damage with symptoms ranging from wheezing to more severe symptoms such as lung injury or even death. Because the mechanism behind Cl2-induced cell death is not clearly understood, the present study aimed to study the cellular effects in vitro after Cl2 exposure of human A549 lung epithelial cells. In addition, the possible treatment effects of the anti-inflammatory antioxidant N-acetyl cysteine (NAC) were evaluated. Exposure of A549 cells to Cl2 (100-1000 ppm) in the cell medium induced cell damage and toxicity within 1 h in a dose-dependent manner. The results showed that 250 ppm Cl2 increased cell death and formation of apoptotic-like bodies, while 500 ppm Cl2 exposure resulted in predominantly necrotic death. Pre-treatment with NAC was efficient to prevent cell damage at lower Cl2 concentrations in part by averting the formation of apoptotic-like bodies and increasing the expression of the anti-apoptotic proteins clusterin and phosphorylated tumour protein p53(S46). Analysis showed that Cl2 induced cell death by a possibly caspase-independent mechanism, since no cleavage of caspase-3 could be detected after exposure to 250 ppm. Currently, these results justifies further research into new treatment strategies for Cl2-induced lung injury.

N-acetyl cysteine protects against chlorine-induced tissue damage in an ex vivo model.

High-level concentrations of chlorine (Cl2) can cause life-threatening lung injuries and the objective in this study was to understand the pathogenesis of short-term sequelae of Cl2-induced lung injury and to evaluate whether pre-treatment with the antioxidant N-acetyl cysteine (NAC) could counteract these injuries using Cl2-exposed precision-cut lung slices (PCLS). The lungs of Sprague-Dawley rats were filled with agarose solution and cut into 250 µm-thick slices that were exposed to Cl2 (20-600 ppm) and incubated for 30 min. The tissue slices were pre-treated with NAC (5-25 mM) before exposure to Cl2. Toxicological responses were analyzed after 5 h by measurement of LDH, WST-1 and inflammatory mediators (IL-1ß, IL-6 and CINC-1) in medium or lung tissue homogenate. Exposure to Cl2 induced a concentration-dependent cytotoxicity (LDH/WST-1) and IL-1ß release in medium. Similar cytokine response was detected in tissue homogenate. Contraction of larger airways was measured using electric-field-stimulation method, 200 ppm and control slices had similar contraction level (39 ± 5%) but in the 400 ppm Cl2 group, the evoked contraction was smaller (7 ± 3%) possibly due to tissue damage. NAC-treatment improved cell viability and reduced tissue damage and the contraction was similar to control levels (50 ± 11%) in the NAC treated Cl2-exposed slices. In conclusion, Cl2 induced a concentration-dependent lung tissue damage that was effectively prevented with pre-treatment with NAC. There is a great need to improve the medical treatment of acute lung injury and this PCLS method offers a way to identify and to test new concepts of treatment of Cl2-induced lung injuries.

Inhaled sulfur dioxide causes pulmonary and systemic inflammation leading to fibrotic respiratory disease in a rat model of chemical-induced lung injury.

Inhalation of high concentrations of sulfur dioxide (SO2) affects the lungs and can be immediately dangerous to life. We examined the development of acute and long-term effects after exposure of SO2 in Sprague-Dawley rats, in particular inflammatory responses, airway hyperresponsiveness (AHR) and lung fibrosis. Animals were subjected to a single exposure of 2200ppm SO2 during 10min and treated with a single dose of the anti-inflammatory corticosteroid dexamethasone 1h following exposure. Exposed rats showed labored breathing, decreased body-weight and an acute inflammation with neutrophil and macrophage airway infiltrates 5h post exposure. The acute effects were characterized by bronchial damage restricted to the larger bronchi with widespread injured mucosal epithelial lining. Rats displayed hyperreactive airways 24h after exposure as indicated by increased methacholine-induced respiratory resistance. The inflammatory infiltrates remained in lung tissue for at least 14 days but at the late time-point the dominating granulocyte types had changed from neutrophils to eosinophils. Analysis of immunoregulatory and pro-inflammatory cytokines in serum and airways implicated mixed macrophage phenotypes (M1/M2) and T helper cell activation of both TH1 and TH2 subtypes. Increased expression of the pro-fibrotic cytokine TGFß1 was detected in airways 24h post exposure and remained increased at the late time-points (14 and 28 days). The histopathology analysis confirmed a significant collagen deposition 14 days post exposure. Treatment with dexamethasone significantly counteracted the acute inflammatory response but was insufficient for complete protection against SO2-induced adverse effects, i.e. treatment only provided partial protection against AHR and the long-term fibrosis.

N-acetyl cysteine improves the effects of corticosteroids in a mouse model of chlorine-induced acute lung injury.

Chlorine (Cl2) causes tissue damage and a neutrophilic inflammatory response in the airways manifested by pronounced airway hyperreactivity (AHR). The importance of early anti-inflammatory treatment has previously been addressed. In the previous study, both high-dose and low-dose of dexamethasone (DEX) decreased the risk of developing delayed effects, such as persistent lung injuries, while only high-dose treatment could significantly counteract acute-phase effects. One aim of this study was to evaluate whether a low-dose of DEX in combination with the antioxidant N-acetyl cysteine (NAC) and if different treatments (Triptolide, Reparixin and Rolipram) administered 1h after Cl2-exposure could improve protection against acute lung injury in Cl2-exposed mice. BALB/c mice were exposed to 300 ppm Cl2 during 15 min. Assessment of AHR and inflammatory cells in bronchoalveolar lavage was analyzed 24h post exposure. Neither of DEX nor NAC reduced the AHR and displayed only minor effects on inflammatory cell influx when given as separate treatments. When given in combination, a protective effect on AHR and a significant reduction in inflammatory cells (neutrophils) was observed. Neither of triptolide, Reparixin nor Rolipram had an effect on AHR but Triptolide had major effect on the inflammatory cell influx. Treatments did not reduce the concentration of either fibrinogen or plasminogen activator inhibitor-1 in serum, thereby supporting the theory that the inflammatory response is not solely limited to the lung. These results provide a foundation for future studies aimed at identifying new concepts for treatment of chemical-induced lung injury. Studies addressing combination of anti-inflammatory and antioxidant treatment are highly motivated.

Inhalation of chlorine causes long-standing lung inflammation and airway hyperresponsiveness in a murine model of chemical-induced lung injury.

Chlorine is highly irritating when inhaled, and is a common toxic industrial gas causing tissue damage in the airways followed by an acute inflammatory response. In this study, we investigated mechanisms by which chlorine exposure may cause reactive airways dysfunction syndrome (RADS) and we examined the dose-dependency of the development of symptoms. Mice were exposed to 50 or 200 ppm Cl(2) during a single 15 min exposure in a nose-only container. The experiment terminated 2, 6, 12, 24, 48, 72 h and 7, 14, 28 and 90 days post exposure. Inflammatory cell counts in bronchoalveolar lavage (BAL), secretion of inflammatory mediators in BAL, occurrence of lung edema and histopathological changes in lung tissue was analyzed at each time-point. Airway hyperresponsiveness (AHR) was studied after 24 and 48 h and 7, 14, 28 and 90 days. The results showed a marked acute response at 6h (50 ppm) and 12h (200 ppm) post exposure as indicated by induced lung edema, increased airway reactivity in both central and peripheral airways, and an airway inflammation dominated by macrophages and neutrophils. The inflammatory response declined rapidly in airways, being normalized after 48 h, but inflammatory cells were sustained in lung tissue for at least seven days. In addition, a sustained AHR was observed for at least 28 days. In summary, this mouse model of chlorine exposure shows delayed symptoms of hyperreactive airways similar to human RADS. We conclude that the model can be used for studies aimed at improved understanding of adverse long-term responses following inhalation of chlorine.

Corticosteroid treatment inhibits airway hyperresponsiveness and lung injury in a murine model of chemical-induced airway inflammation.

CONTEXT: Exposure to toxic alkylating mustard agents causes both acute and long-term effects to the lungs as indicated by increased number of inflammatory cells in airways, lung edema and lung tissue fibrosis. We have previously demonstrated that treatment with the corticosteroid dexamethasone 1 h after lung exposure to the nitrogen mustard analog melphalan protects mice from acute and sub-acute inflammatory responses, as well as from lung tissue fibrosis. OBJECTIVE: In order to address the importance of early anti-inflammatory treatment, we investigated the therapeutic effect of dexamethasone administered 1, 2 or 6 h following exposure to melphalan. METHODS: C57BL/6 mice were exposed to melphalan and treated with dexamethasone 1, 2 or 6 h after exposure. Twenty hours or 14 days post exposure mice were subjected to analysis of respiratory mechanics where the effects of incremental doses of methacholine on central and peripheral lung components were measured. We also determined the amount of inflammatory cells in the bronchoalveolar lavage fluid and measured the amount of collagen content in the lungs. RESULTS: Melphalan exposure increased airway hyperresponsiveness in both central and peripheral airways and induced an airway inflammation dominated by infiltration of macrophages and neutrophils. Dexamethasone given 1 h after exposure to melphalan provided better protection against airway inflammation than administration 2 or 6 h after exposure. Collagen deposition 14 days after exposure was decreased due to dexamethasone treatment. CONCLUSION: Early treatment with dexamethasone is important in order to reduce the airway hyperresponsiveness and inflammation caused by toxic alkylating mustards such as melphalan.