OBJECTIVE:Ammonia (NH3) inhalation is a common occupational exposure, causing injuries similar to acute lung injury (ALI). Medical management is limited to supportive care, as no specific antidotes are currently available. This study aimed to evaluate the efficacy of potential therapeutic interventions to mitigate NH3-induced damage using both in vivo (mouse) and in vitro (A549 alveolar epithelial cells) models. METHODS:BALB/c mice received 91.0 mg/kg NH3 via intratracheal instillation, followed by intraperitoneal dexamethasone (100 mg/kg) at 1, 5, and 23 h post-exposure to assess therapeutic effects. Analyses were performed on days 1 and 7. The complementary studies in A549 cells examined whether therapeutic interventions could counteract NH3-induced toxicity affecting cell viability and function. RESULTS:Dexamethasone-treatment did not counteract the lethal damage in mice or significantly reduce the severity of ALI that intensified over time, including increased lung inflammatory cell infiltration, lung hemorrhages, and coagulation abnormalities. However, treatment reduced methacholine-induced AHR, and MMP-9 and SP-D levels at 20h post-exposure. Most treatments in A549 cells failed to prevent apoptotic and necrotic cell death, oxidative stress, mitochondrial dysfunction, and membrane damage caused by NH3 exposure however, the membrane stabilizer Poloxamer 188 (P188) highlighted the importance of stabilizing the cellular membrane damage to prevent further damages. CONCLUSIONS:While standard treatment with corticosteroids offered limited protection in NH3-exposed mice, the study's complementary in vitro investigations on new medical counter measures highlighted the complexity and severity of NH3-induced lung injury. Together, the in vivo and in vitro findings emphasize the urgent need for effective medical countermeasures.

Preclinical evaluation of oximes as antidotes for organophosphorus nerve agent (OPNAs) poisoning is predominantly based on protection ratio of the antidote against lethal doses. Developing protection indexes involves considerable animal distress and, due to the limited precision, requires large animal cohorts. This study aimed to establish an in vivo model for evaluating new therapeutic substances more aligned with the 3R principles that also enables detailed quantification of specific biological effects to better understand the impact of treatment. Anesthetized Sprague-Dawley rats were tracheostomized and connected to a small animal ventilator allowing simultaneous registration of respiratory function. Rats were exposed to 1xLD50 VX or tabun, and progression of poisoning was monitored in real-time through measurements of respiratory resistance (RRS) over a 30-min period. Additional assessments included clinical symptoms and acetylcholine esterase (AChE) inhibition in blood. Pre-treatment with oxime (obidoxime, HI-6, RS194B) or atropine were used to validate the model. Exposure to OPNAs resulted in rapid increases in RRS to 250-300 % above baseline. HI-6 and obidoxime were the most effective treatments, mitigating both respiratory and enzymatic effects of OPNA poisoning, while RS194B treatment delayed onset of symptoms but did not fully reverse toxicity. Tabun-inhibited AChE was generally more resistant to reactivation with oximes than VX- inhibited enzymes. The findings indicate that maintaining AChE activity above 15-20 % is sufficient to restore respiratory function and alleviate symptoms to levels comparable to unexposed controls. This study highlights the value of high-sensitivity, real-time monitoring of physiological metrics like respiratory resistance in evaluating novel antidotes for OPNA poisoning.

Chlorine is a toxic industrial chemical that has been used as a chemical weapon in recent armed conflicts. Confirming human exposure to chlorine has proven challenging, and there is currently no established method for analyzing human biomedical samples to unambiguously verify chlorine exposure. In this study, two chlorine-specific biomarkers: palmitoyl-oleoyl phosphatidylglycerol chlorohydrin (POPG-HOCl) and the lipid derivative oleoyl ethanolamide chlorohydrin (OEA-HOCl) are shown in bronchoalveolar lavage fluid (BALF) samples from spontaneously breathing pigs after chlorine exposure. These biomarkers are formed by the chemical reaction of chlorine with unsaturated phospholipids found in the pulmonary surfactant, which is present at the gas-liquid interface within the lung alveoli. Our results strongly suggest that lipid chlorohydrins are promising candidate biomarkers in the development of a verification method for chlorine exposure. The establishment of verified methods capable of confirming the illicit use of toxic industrial chemicals is crucial for upholding the principles of the Chemical Weapons Convention (CWC) and enforcing the ban on chemical weapons. This study represents the first published dataset in BALF revealing chlorine biomarkers detected in a large animal. Furthermore, these biomarkers are distinct in that they originate from molecular chlorine rather than hypochlorous acid.

Exhaled breath (EB) contains various volatile organic compounds (VOCs) that can indicate specific biological or pathological processes in the body. Analytical techniques like gas chromatography-mass spectrometry (GC-MS) can be used to detect and measure these exhaled biomarkers. In this study, the objective was to develop a non-invasive method of EB sampling in animals that were awake, as well as to analyze EB for volatile biomarkers specific for chlorine exposure and/or diagnostic biomarkers for chlorine-induced acute lung injury (ALI). To achieve this, a custom-made sampling device was used to collect EB samples from 19 female Balb/c mice. EB was sampled both pre-exposure (serving as internal control) and 30 min after exposure to chlorine. EB was collected on thermal desorption tubes and subsequently analyzed for VOCs by GC-MS. The following day, the extent of airway injury was assessed in the animals by examining neutrophils in the bronchoalveolar lavage fluid. VOC analysis revealed alterations in the EB biomarker pattern post-chlorine exposure, with eight biomarkers displaying increased levels and six exhibiting decreased levels following exposure. Four chlorinated compounds: trichloromethane, chloroacetone, 1,1-dichloroacetone and dichloroacetonitrile, were increased in chlorine-exposed mice, suggesting their specificity as chlorine EB biomarkers. Furthermore, chlorine-exposed mice displayed a neutrophilic inflammatory response and body weight loss 24 h following exposure. In conclusion, all animals developed an airway inflammation characterized by neutrophil infiltration and a specific EB pattern that could be extracted after chlorine exposure. Monitoring EB samples can readily and non-invasively provide valuable information on biomarkers for diagnosis of chlorine-induced ALI, confirming chlorine exposures.

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.

Chlorine (Cl-2) gas is a toxic industrial chemical (TIC) that poses a hazard to human health following accidental and/or intentional (e.g. terrorist) release. By using a murine model of sub-lethal Cl-2 exposure we have examined the airway hyper responsiveness, cellular infiltrates, transcriptomic and proteomic responses of the lung. In the "crisis " phase at 2 h and 6 h there is a significant decreases in leukocytes within bronchoalveolar lavage fluid accompanied by an upregulation within the proteome of immune pathways ultimately resulting in neutrophil influx at 24 h. A flip towards "repair " in the transcriptome and proteome occurs at 24 h, neutrophil influx and an associated drop in the lung function persisting until 14 d post-exposure and subsequent "recovery " after 28 days. Collectively, this research provides new insights into the mechanisms of damage, early global responses and processes of repair induced in the lung following the inhalation of Cl-2.

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.

Abstract 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-1β, 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-1β) 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 is a toxic chemical that has been used as a chemical warfare agent in recent armed conflicts. There is an urgent need for methods to verify alleged uses of chlorine, and phospholipid chlorohydrins (PL-HOCl) derived from the pulmonary surfactant of exposed victims have previously been proposed as biomarkers of chlorine exposure. Here, we describe an improved protocol for the chemical analysis of these biomarkers and its applicability to biomedical samples from chlorine-exposed animals. By the use of a polymeric solid-phase-supported transesterification of PL-HOCl using ethanolamine, a common biomarker, oleoyl ethanolamide chlorohydrin (OEA-HOCl), was derived from all the diverse oleoyl PL-HOCl that may be formed by chlorine exposure. Compared to native lipid biomarkers, OEA-HOCl represents a larger biomarker pool and is better suited for nano-liquid chromatography tandem mass spectrometry (nLC-MS-MS analysis), generating 3 amol Limit of Detection (LOD) and a reduced sample carry-over. With the improved protocol, significantly elevated levels of OEA-HOCl were identified in bronchoalveolar lavage fluid (BALF) of chlorine-exposed rats, 2-48 hours after exposure. The difficulty of BALF sampling from humans limits the methods usefulness as a verification tool of chlorine exposure. Conversely, nasal lavage fluid (NLF) is readily collected without advanced equipment. In NLF from chlorine-exposed rats, PL-HOCl were identified and significantly elevated levels of the OEA-HOCl biomarker were detected 2-24 hours after exposure. In order to test the potential of NLF as a biomedical sample for verification of human exposure to chlorine, in-vitro chlorination of human NLF samples was performed. All human in-vitro chlorinated NLF samples exhibited elevated OEA-HOCl biomarker levels, following sample derivatization. These data indicate the potential of human NLF as a biomedical sample for the verification of chlorine exposure, but further work is required to develop and validate the method for the use on real-world samples.

Exposure to high concentrations of ammonia (NH3) can cause life-threatening lung damages. The objective of this study was to establish a translational in vitro model for NH3-induced lung injury. Precision-cut lung slices (PCLS) from rats were exposed to NH3 and toxicological responses and cell viability were quantified by analysis of LDH, WST-1, inflammatory mediators (IL-1 beta, IL-6, CINC-1, MMP-9, RAGE and IL-18), and by microscopic evaluation of bronchoconstriction induced by electric-fieldstimulation (EFS) or methacholine (MCh). Different treatment strategies were assessed to prevent or reverse the damages caused by NH3 using anti-inflammatory, anti-oxidant or neurologically active drugs. Exposure to NH3 caused a concentration-dependent increase in cytotoxicity (LDH/WST-1) and IL-1 beta release in PCLS medium. None of the treatments reduced cytotoxicity. Deposition of NH3 (24-59 mM) on untreated PCLS elicited an immediate concentration-dependent bronchoconstriction. Unlike MCh, the EFS method did not constrict the airways in PCLS at 5 h after NH3-exposure (47-59 mM). Atropine and TRP-channel antagonists blocked EFS-induced bronchoconstriction but these inhibitors could not block the immediate NH3-induced bronchoconstriction. In conclusion, NH3 exposure caused cytotoxic effects and lung damages in a concentration-dependent manner and this PCLS method offers a way to identify and test new concepts of medical treatments and biomarkers that may be of prognostic value. (c) 2021 Elsevier B.V. All rights reserved.

High-level concentrations of chlorine (Cl-2) can cause life-threatening lung injuries and the objective in this study was to understand the pathogenesis of short-term sequelae of Cl-2-induced lung injury and to evaluate whether pre-treatment with the antioxidant N-acetyl cysteine (NAC) could counteract these injuries using Cl-2-exposed precision-cut lung slices (PCLS). The lungs of Sprague-Dawley rats were filled with agarose solution and cut into 250 mu m-thick slices that were exposed to Cl-2 (20-600 ppm) and incubated for 30 min. The tissue slices were pre-treated with NAC (5 - 25 mM) before exposure to Cl-2. Toxicological responses were analyzed after 5 h by measurement of LDH, WST-1 and inflammatory mediators (IL-1 beta, IL-6 and CINC-1) in medium or lung tissue homogenate. Exposure to Cl-2 induced a concentration-dependent cytotoxicity (LDH/WST-1) and IL-1 beta 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 Cl-2 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 Cl-2-exposed slices. In conclusion, Cl-2 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 Cl-2-induced lung injuries.

Objective: To establish a rat model with respiratory and pulmonary responses caused by inhalation exposure to non-lethal concentrations of ammonia (NH3) that can be used for evaluation of new medical countermeasure strategies for NH3-induced acute lung injury (ALI). This is of great value since no specific antidotes of NH3-induced injuries exist and medical management relies on supportive and symptomatically relieving efforts. Methods: Female Sprague-Dawley rats (8-9 weeks old, 213g +/- 2g) were exposed to NH3 using two different exposure regimens; nose-only inhalation or intratracheal instillation. The experiment was terminated 5 h, 24 h, 14 and 28 days post-exposure. Results: Nose-only inhalation of NH3 (9000-15 000 ppm) resulted in increased salivation and labored breathing directly post-exposure. Exposure did not increase inflammatory cells in bronchoalveolar lavage fluid but exposure to 12 000 ppm NH3 during 15 min reduced body weight and induced coagulation abnormalities by increasing serum fibrinogen levels. All animals were relatively recovered by 24 h. Intratracheal instillation of NH3 (1%) caused early symptoms of ALI including airway hyperresponsiveness, neutrophilic lung inflammation and altered levels of coagulation factors (increased fibrinogen and PAI-1) and early biomarkers of ALI (IL-18, MMP-9, TGF beta) which was followed by increased deposition of newly produced collagen 14 days later. Histopathology analysis at 5 h revealed epithelial desquamation and that most lesions were healed after 14 days. Conclusions: This study demonstrates that intratracheal instillation can reproduce several early hallmarks of ALI. Our findings therefore support that the intratracheal instillation exposure regimen can be used for new medical countermeasure strategies for NH3-induced ALI.

Diseases in the respiratory tract rank among the leading causes of death in the world, and thus novel and optimized treatments are needed. The lungs offer a large surface for drug absorption, and the inhalation of aerosolized drugs are a well-established therapeutic modality for local treatment of lung conditions. Nanoparticle-based drug delivery platforms are gaining importance for use through the pulmonary route. By using porous carrier matrices, higher doses of especially poorly soluble drugs can be administered locally, reducing their side effects and improving their biodistribution. In this study, the feasibility of mesoporous silica particles (MSPs) as carriers for anti-inflammatory drugs in the treatment of airway inflammation was investigated. Two different sizes of particles on the micron and nanoscale (1 µm and 200 nm) were produced, and were loaded with dexamethasone (DEX) to a loading degree of 1:1 DEX:MSP. These particles were further surface-functionalized with a polyethylene glycol–polyethylene imine (PEG–PEI) copolymer for optimal aqueous dispersibility. The drug-loaded particles were administered as an aerosol, through inhalation to two different mice models of neutrophil-induced (by melphalan or lipopolysaccharide) airway inflammation. The mice received treatment with either DEX-loaded MSPs or, as controls, empty MSPs or DEX only; and were evaluated for treatment effects 24 h after exposure. The results show that the MEL-induced airway inflammation could be treated by the DEX-loaded MSPs to the same extent as free DEX. Interestingly, in the case of LPS-induced inflammation, even the empty MSPs significantly down-modulated the inflammatory response. This study highlights the potential of MSPs as drug carriers for the treatment of diseases in the airways.

Inhalation of sulfur dioxide (SO 2 ) primarily affects the lungs and exposure to high concentrations can be immediately dangerous to life. The response after inhalation of SO 2 implicates that the early response involves tissue injury, neutrophilic lung inflammation and airway hyperresponsiveness (AHR). In rats, lung fibrosis is evident 14 days post exposure and early treatment with a single dose of Dexamethasone (DEX) significantly down-modulates the acute inflammatory response in airways. However, this treatment is not sufficient for complete protection against the pulmonary toxicity. The aim was to evaluate whether repeated treatment with DEX alone or in combination with the antioxidant N-acetyl-L-cysteine(NAC) and the anti-fibrotic substance, Pirfenidone (PFD), administered 1h, 5h and 23h after SO 2 -exposure (2200ppm, 10min) could counteract the inflammatory responses, AHR and lung fibrosis in Sprague-Dawley rats. All treatment approaches significantly reduced the total leukocyte response but only combined DEX and NAC reduced the number of neutrophils in BAL. PFD reduced methacholine-induced AHR to almost control levels, in contrast to DEX and DEX/NAC which only provided partial protection against AHR. Only DEX treatment reduced the collagen formation in lung tissue. In conclusion, all treatment protocols reduced the acute SO 2 -induced inflammatory response in airways with DEX/NAC treatment slightly more efficient than the other protocols. PFD appeared to be more efficient than DEX and DEX/NAC combination in reduction of AHR. Only DEX treatment was efficient in the reduction of long-term effects monitored 14 days post exposure. In the future, studies addressing both anti-inflammatory and anti-fibrotic treatment is higly motivated.

Inhalation of chlorine (Cl2) may cause oxidative acute lung injury (ALI) characterized by pulmonary edema, pneumonitis, and hyperreactive airways. The aim of the study was to identify possible biomarkers for Cl2-induced ALI. Female BALB/c mice were exposed to Cl2 for 15min using two protocols 1) concentration-dependent response (25-200ppm) and 2) time-kinetics (2h-14days post-exposure). Exposure to 50-200ppm Cl2 caused a concentration-dependent inflammatory response with increased expression of IL-1β, IL-6 and CXCL1/KC in bronchoalveolar lavage fluid 2-6h after exposure which was followed by increased lung permeability and a neutrophilic inflammation 12-24h post-exposure. The early inflammatory cytokine response was associated with a clear but transient increase of 8-isoprostane, a biomarker for oxidative stress, with its maximum at 2h after exposure. An increase of 8-isoprostane could also be detected in serum 2h after exposure to 200ppm Cl2, which was followed by increased levels of IL-6 and CXCL1/KC and signs of increased fibrinogen and PAI-1. Melphalan, a non-oxidizing mustard gas analog, did not increase the 8-isoprostane levels, indicating that 8-isoprostane is induced in airways through direct oxidation by Cl2. We conclude that 8-isoprostane represents an early biomarker for oxidative stress in airways and in the blood circulation following Cl2-exposure.

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 TGFβ1 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.

We investigated acute and delayed respiratory changes after inhalation exposure to chlorine (Cl2) with the aim to understand the pathogenesis of the long-term sequelae of Cl2-induced lung-injury. In a rat model of nose-only exposure we analyzed changes in airway hyperresponsiveness (AHR), inflammatory responses in airways, expression of pro-inflammatory markers and development of lung fibrosis during a time-course from 5h up to 90days after a single inhalation of Cl2. A single dose of dexamethasone (10mg/kg) was administered 1h following Cl2-exposure. A 15-min inhalation of 200ppm Cl2 was non-lethal in Sprague-Dawley rats. At 24h post exposure, Cl2-exposed rats displayed elevated numbers of leukocytes with an increase of neutrophils and eosinophils in bronchoalveolar lavage (BAL) and edema was shown both in lung tissue and the heart. At 24h, the inflammasome-associated cytokines IL-1β and IL-18 were detected in BAL. Concomitant with the acute inflammation a significant AHR was detected. At the later time-points, a delayed inflammatory response was observed together with signs of lung fibrosis as indicated by increased pulmonary macrophages, elevated TGF-β expression in BAL and collagen deposition around airways. Dexamethasone reduced the numbers of neutrophils in BAL at 24h but did not influence the AHR. Inhalation of Cl2 in rats leads to acute respiratory and cardiac changes as well as pulmonary inflammation involving induction of TGF-β1. The acute inflammatory response was followed by sustained macrophage response and lack of tissue repair. It was also found that pathways apart from the acute inflammatory response contribute to the Cl2-induced respiratory dysfunction.

We investigated acute and late inflammatory changes after exposure to chlorine (Cl2) with the aim to identify biomarkers of adverse inflammatory responses. Such biomarkers would be of value to diagnose whether an exposed individual would be at risk for persistent lung injuries.This study compares changes in pulmonary function (AHR),selected endpoints in serum, and bronchoalveolar lavage fluid (BALF) following a single, 15-min nose-only exposure of rats to 200 ppm Cl2. The time-course of lung injury was locally and systematically examined up to 14 days post exposure with the objective to identify early diagnostic biomarkers suitable to guide countermeasures to accidental exposures. One exposure to Cl2 was non-lethal and all rats (female Sprague-Dawley) displayed signs of anorexia and lethargy up to 24 h post exposure. Cl2-exposed rats displayed elevated numbers of total leukocytes with a significant increase of neutrophils (p<0.001) and eosinophils (p<0.05) in BALF. Cl2 induced increased lung weight (p<0.01) but no pulmonary edema. The methacholine-induced AHR of the central airways was increased more than two-fold in Cl2-exposed rats as compared to the control group at 24 h post exposure. On day 14, there were signs of lung fibrosis as indicated by increased collagen deposition in tissue. By the use of this model,there is a potential for understanding the mechanisms by which exposure to chemical compounds, such as Cl2, causes long-term effects. These results provide a foundation for future studies aimed at identification of biomarkers for adverse inflammatory responses, as well as for evaluation of new concepts for treatment of chemical-induced lung injury.