A longitudinal characterization of cardiopulmonary and systemic injury following acute chlorine inhalation in rats

Abstract

Chlorine is a highly reactive, toxic industrial gas whose accidental or intentional release poses a significant public health threat, highlighting the need for effective medical countermeasures. Well-characterized animal models are essential for understanding the mechanisms of chlorine-induced injury and evaluating potential therapeutics. In this study, we established a longitudinal preclinical rat model of acute chlorine inhalation using two near lethal (LD₅₀) exposures (500 or 600 ppm for 45 min) in unanesthetized adult Sprague Dawley rats. Pulmonary and systemic injury were evaluated at 3, 6 and 24h postexposure using clinical, biochemical, hematological, physiological and histopathological endpoints. Both exposure concentrations produced approximately 50% mortality with most deaths occurring shortly after exposure, while survivors exhibited severe respiratory distress, increased clinical scores, weight loss, bradycardia and hypoxemia. Arterial blood gas analysis demonstrated impaired gas exchange characterized by hypercapnia and compensated respiratory acidosis. Chlorine exposure induced robust pulmonary inflammation and injury, evidenced by increased BALF protein, LDH and the damage associated molecular pattern (DAMP) HMGB1, pulmonary edema, airway epithelial sloughing, alveolar damage and elevated lung injury scores. Systemic inflammation was characterized by neutrophilia with concurrent lymphopenia and monocytopenia. Vascular injury was indicated by increased syndecan-1 concentrations in BALF and plasma while cardiac involvement was demonstrated by increased heart-to-body weight ratios, elevated circulating cardiac injury biomarkers and ultrastructural myocardial damage. Collectively, this well-characterized model recapitulates the major manifestations of acute chlorine toxicity and provides a robust platform for investigating disease mechanisms and evaluating candidate medical countermeasures.

Introduction

Chlorine is a widely used toxic industrial chemical that poses significant risks to human health and the environment. Accidental releases during production and transport are common and hazardous causing fatalities and long-term health effects^1,2. Chlorine was among the first chemical weapons developed and remains a threat agent. There is no cure for chlorine-induced injuries due in part to the rarity and heterogeneity of human cases and limitations in relevant animal models, which hinder understanding of disease mechanisms.

Chlorine gas is a potent irritant that damages the eyes, nose and throat causing coughing, wheezing, dyspnea and chest tightness. Exposed individuals may develop rhinitis, tracheobronchitis, bronchospasms, airway hyperresponsiveness, bronchiolitis and reactive airway dysfunction syndrome and severe cases may progress to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)^3–6. High-concentration exposure can result in pulmonary and systemic injury leading to death^7–9. Survivors may experience persistent airway hyperresponsiveness influenced by factors such as preexisting asthma or smoking history¹⁰. Chronic occupational exposure can cause chronic bronchitis and recurrent wheezing. Although chest X-rays may show abnormalities, no specific biomarkers for chlorine exposure are currently available¹¹. Studies including our own have identified chlorinated tyrosine adducts, chloro-fatty acids and chloro-fatty-aldehyde as potential biomarkers, though detection methods remain complex^12–14. Improved preclinical models are needed to support translational studies linking therapeutic effects with clinical outcomes and biomarker profiles.

Insights from chemical warfare and industrial exposure cases are limited and most knowledge derives from preclinical models that replicate key features of human injury and underlying mechanisms^15–22. However, model limitations and the push for new approach methodologies (NAMs) have prompted the development of in vitro systems, which still face challenges for therapeutic testing^23,24. Various animal models of chlorine exposure have been reported including mice, rats, rabbits, pigs, sheep and primates^10,25. Except for few ovine studies, most of the larger animal models need to be anesthetized and intubated which may not truly recapitulate the actual exposure situations limiting real world relevance^23,26. In contrast, whole-body exposures of unanesthetized rodents offer a more representative approach^9,13,27–30. Reproducible animal models that faithfully recapitulate the route of exposure and pathophysiological responses observed in humans following chlorine inhalation are critical for elucidating the mechanisms of injury and for the preclinical evaluation of potential therapeutic interventions. Here we describe a preclinical rat model of whole-body chlorine exposure using two near-lethal dose (LD₅₀) concentrations to assess the pulmonary and systemic effects and enable longitudinal therapeutic assessment. High concentration (>200 ppm) exposure affects both sexes similarly and our prior extensive bromine studies and data with chlorine (included in the Supplement here) show comparable susceptibility in female rats^26,31,32. By characterizing the pulmonary injury, biomarker profiles, respiratory distress parameters, and systemic physiological effects associated with chlorine exposure, this model builds on our prior studies and offers a robust framework for evaluating the efficacy of candidate medical countermeasures against chlorine toxicity.

Methods:

Animals:

Male and Female Sprague Dawley rats (250–300g) were obtained (Inotiv, Lafayette IN), housed, and cared for, and used according to approved protocols and University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC) guidelines. Rats (2/cage) were housed under a 12h dim light/12h dark cycle in standard polycarbonate cages with wood chip bedding and nesting enrichment (EnviroPak, Earth City, MO). Standard diet and tap water were provided ad libitum and the room temperature was maintained at 70°F and humidity maintained between 40–60%. A priori power analysis was performed using ANOVA F-test to compute sample size. Assuming the group sizes with a power of 95% and α-error probability of 5% or less, expected mortality, and an effect size of 0.5–0.7, the number of animals required for proposed studies were calculated. In the supporting Supplementary data, weight matched female Sprague Dawley rats (250–300g) (Inotiv, Lafayette, IN) were utilized.

In vivo chlorine exposure, monitoring and sample collection.

Unanesthetized rats were exposed (whole body) to 500 or 600 ppm chlorine (Airgas, Birmingham Al) customized gas cylinders with gas concentrations in balance air certified within 2% by the manufacturer) for 45 minutes as previously described^33–35. Rats were then returned to room air (RA) and monitored continuously for 3, 6 or 24 h after exposure until the end point of evaluation was reached. The animals were assigned clinical scores based on activity and respiratory quality as outlined before³³. Oxygen saturations and heart rates were also measured in unanesthetized rats using a large collar clip sensor of MouseOx small animal oximeter (Starr Life Sciences, Oakmont, PA)³². Rats were euthanized at the end of the experiment in accordance with 2020 American Veterinary Medical Association Panel on Euthanasia Guidelines to allow for specimen collection. For blood and tissue collection general anesthesia was induced by 5% isoflurane (Piramal Critical Care, Telangana, India) and maintained by 2% isoflurane. After ensuring the depth of anesthesia, incisions were made to open the abdominal cavity, bowels were gently displaced and the abdominal descending aorta was exposed. Blood was then collected using a 25G needle and heparinized syringe and analyzed for arterial blood gas (ABG) using a EPOC blood gas and electrolyte analyzer (Heska, Loveland, CO). For complete blood cell count (CBC), blood was collected in EDTA treated tubes and analyzed with the Hemavet 950 FS hematology analyzer (Drew Scientific Inc, Plantation, FL)³⁶. Exsanguination was performed, the chest cavity was opened, and the bronchoalveolar lavage fluid (BALF) was collected, and the right upper lobe of the lung was isolated and removed for wet to dry weight ratio evaluation as described before^33,37. Tissues like heart and spleen were also collected and weighed using a precision balance.

Lung injury biomarker assessment

Lung injury was evaluated by BALF protein content measurement as previously described^33,36. We also evaluated BALF lactate dehydrogenase (LDH) content using a commercial kit (Promega, Madison, WI) to confirm airway cell death and injury as demonstrated in other lung injury models^38,39. High mobility group box-1 (HMGB1) is a damage associate molecular pattern (DAMP) protein which is released extracellularly following injury⁴⁰. We also evaluated the BALF HMGB1 content using Western blots to assess its release following chlorine exposure.

Cardiac injury and biomarker evaluation

Markers of cardiovascular injury were also evaluated. LDH was determined in plasma to evaluate vascular cell death as described above. Vascular injury markers like Syndecan-1 (Lifeome Biolabs, San Diego, CA) and cardiac injury markers CKMB (Abcam, Cambridge, UK), NT-proBNP (Novus Biologicals, Centennial, CO) and hFABP (Abcam, Cambridge, UK) were also evaluated in the plasma using commercial rat ELISA kits.

Histology and Electron Microscopy

For histology, the lungs of separate animals were filled with alcoholic formalin (10%) through intratracheal instillation⁴¹. The fixed lungs were embedded in paraffin and cut into 5 μm sections. The sections were then stained with hematoxylin and eosin (H&E), and microscopic evaluation was performed for lung injury scoring as described previously^42,43. Myocardial tissue was obtained and processed for transmission electron microscopy as described by us before³³.

Statistical Analysis

All statistical analyses were performed using Graphpad prism software version 10.1.0. Means were compared by two-tailed t-test for comparison between two groups and one-way analysis of variance (ANOVA) for multiple comparisons. The p-values of ≤0.05 were considered significant and are represented as * ≤ 0.05, ** ≤ 0.01, # ≤ 0.001 and $ ≤ 0.0001. Survival was assessed using Kaplan-Meier method and compared using a log rank test (Mantel-Cox test).

Results:

Accidental or intentional exposure to high concentrations of chlorine gas, including its use as a chemical warfare agent, can cause death and severe organ damage. The severity of injury depends on factors such as the concentration of chlorine, duration of exposure, proximity to the source and individual susceptibility of those exposed¹⁹. Previous rat studies, including our own, have demonstrated that high concentration halogen exposures (400–600 ppm for 30–60 min) results in approximately 50% mortality (LD50) and causes severe cardiopulmonary injury in adult rats^30,33,44. In these studies, we aimed to provide a comprehensive characterization of chlorine-induced injuries to improve understanding of the mechanisms of damage and to establish a longitudinal animal model for evaluating the efficacy of potential therapeutic drugs.

Acute chlorine exposure induces fatalities and respiratory distress in survivors

Exposure to 600 or 500 ppm chlorine for 45 minutes (Figure 1A, AUC values of 168 or 202 mg/m³) caused about 50% mortality in adult male rats and was consistently similar in both concentrations of gas with most mortalities occurring soon after exposure (Figure 1B & C). In our limited supplemental study weight matched female rats exposed to 600 ppm chlorine demonstrated a similar mortality rate; however, a longitudinal survival was not assessed in this group. Among the animals that did not survive, a few were euthanized after reaching humane endpoint criteria, which included respiratory distress, cumulative clinical scores exceeding 7 and oxygen saturation levels below 70%. The chlorine exposed animals stop eating and drinking that could be a contributing factor to about 10% of body weight loss observed by 24h after exposure (Figure 1D & Supplementary Figure 1A). There was extreme respiratory distress and loss of activity observed by increased clinical scores which improved with time in survivors with exposure to both concentrations of chlorine (Figure 1E, Supplementary Figure 1B). Oxygen saturations and heart rates decreased immediately after exposure but improved by 24h in the survivors of both exposure concentrations (Figure 1F, G and Supplementary Figure 1C & D).

Figure 1.

Effect of chlorine exposure on survival and non-invasive physiological parameters. (A) Schematic illustration of exposure of Sprague-Dawley rats to chlorine (500 or 600 ppm) for 45 minutes and evaluation of endpoints. (B) Demonstrates the area under the curve (AUC) showing amount of gas exposed to and (C) illustrates the Kaplan-Meier survival curve of rats in 24 hours at the two chlorine concentrations where * demonstrates significant difference (p>0.05) as compared to unexposed room air controls. (D) Demonstrates body weight change in chlorine exposed rats at different durations. (E) Demonstrates the clinical scores and (F) and (G) demonstrate tissue oxygenation (pulse ox) and heart rates respectively of rats exposed to chlorine at various time durations. Values shown are mean±SEM n=8–12/group and a p value for ≤ 0.0001 (as compared to unexposed controls) is shown by $.

Acute chlorine exposure causes pulmonary edema, organ damage and lung injury

Twenty-four hours after exposure (or at the otherwise indicated time point) to either 500 or 600 ppm of chlorine, rats were euthanized, the right upper lung lobe was dissected out and weighed, and wet-to-dry weight ratios were calculated as described in the Methods. Chlorine exposure consistently caused edema of the lungs that started early following exposure (observed at 3h) and remained in the survivors even at 24h after exposure (Figure 2A and Supplementary figure 1E for females exposed to 600 ppm chlorine and observed at 24h post exposure). There was an increase in cardiac weight but a very interesting decline of spleen weight, which was most apparent in the rats exposed to higher concentrations of chlorine at earlier time points (Figure 2B & C and Supplementary figure 1F for females where only a trend was observed for cardiac weight however spleen weights (Supplementary figure 1G) were consistently decreased). There was increased lung injury as evidenced by the bronchoalveolar lavage fluid (BALF) protein content in rats exposed to either 500 or 600 ppm chlorine (Figure 2D and Supplementary figure 1H). We also utilized BALF and plasma lactate dehydrogenase (LDH) as an index of airway or vascular cell death after chlorine exposure. LDH content was increased prominently in both the BALF and plasma of rats exposed to 500 ppm chlorine, while rats exposed to 600 ppm chlorine showed an increase in BALF content of LDH that was lower than that of the 500 ppm ones (Figure 2E, F and Supplementary figure 1IJ). We also evaluated BALF cell counts to follow the inflammatory response in the lungs and found that chlorine exposure caused a consistent increase in the BALF cell population, demonstrating initiation of an acute inflammatory response in both sexes (Figure 2G and Supplementary figure 1K). The BALF cell pellets of chlorine exposed animals also included airway epithelial sloughings (could be identified by Western blots of cell pellet lysates, as cytokeratin 5 (CK5) and club cell secretory protein (CCSP) positive) and alveolar epithelial cells (Podoplanin positive) (not shown).

Figure 2.

Chlorine exposure causes lung injury and distant organ damage. Rats were exposed to chlorine (500 or 600 ppm) for 45 minutes and transferred to room air. The rats were euthanized at various timepoints, and lung wet/dry weight ratios were measured to indicate pulmonary edema (A), heart to body weight ratio (B), and spleen weights (C) were also measured. Bronchoalveolar lavage fluid (BALF) was collected and evaluated for protein (D), lactate dehydrogenase (LDH) (E & F for plasma LDH), and cell counts (G) as described in the Methods section. Values shown are mean±SEM, n=8–12/group. The p-values demonstrating statistical significance as represented by signs are shown (* ≤ 0.05, ** ≤ 0.01, # ≤ 0.001 and $ ≤ 0.0001).

Chlorine exposure increases biomarkers of pulmonary inflammation and cardiovascular damage in the biofluids

Extracellular high mobility box protein 1 (HMGB1) is a damage associated molecular pattern (DAMP) that can signal to inflammatory regulators and modulate an inflammatory response. Our studies have demonstrated increased levels of HMGB1 in the BALF of animals challenged with lung injury caused by toxic chemicals including halogens^9,36,45–48. Both concentrations of chlorine caused increased HMGB1 in the BALF of exposed animals, identified as the expected 25–30 kD band, and an additional more prominent band at around 50–60 kD (Figure 3A–C and the whole gel images in the Supplementary Figure 2). Chlorine exposure similarly increased the DAMP HMGB1 expression in the BALF of female animals (Supplementary Figure 3A–D). We also utilized an ELISA to evaluate if HMGB1 could be detected in the plasma and indicate whether vascular injury may contribute to HMGB1 in the BALF. Plasma HMGB1 content in chlorine exposed rats was also increased significantly (not shown). Our studies have described an extensive cardiovascular impact of halogen inhalation⁹. Since 600 ppm gave a consistent increase in HMGB-1 (Figure 3BC), we assessed levels of CK-MB to evaluate chlorine-induced general muscle injury and the specific vascular injury marker Syndecan-1 in the plasma of 600 ppm chlorine-exposed rats. CK-MB levels were consistently increased in the plasma at all three time points (3, 6 and 24h) evaluated after chlorine exposure (Figure 3D). Vascular injury marker Syndecan-1 was increased significantly but peaked at 6h after chlorine exposure in the BALF (not shown) and plasma (Figure 3E). We also evaluated cardiac injury specific markers such as heart fatty acid binding protein (hFABP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) with both 500 and 600 ppm chlorine exposure in the plasma of exposed rats. Both hFABP and NT-proBNP were significantly elevated at 3h and 6h after exposure, however, at 24h after exposure, the levels of both markers decreased but were still elevated from baseline (Figure 3F, G). Significantly increased hFABP levels were also observed in female rats exposed to chlorine (600 ppm for 45 min) and observed 24h after exposure (Supplementary Figure 1L). We also assessed the ultrastructure of the right and left ventricle following chlorine exposure (Figure 3H–O). Hearts of chlorine exposed rats demonstrated consistent biventricular myofibrillar loss, contraction band necrosis, mitochondrial damage and cristae lysis (Figure 3H–O).

Figure 3.

Chlorine exposure increases damage associated molecular pattern molecules (DAMPs/HMGB1) and cardiovascular injury markers. Rats were exposed to chlorine (500 or 600 ppm) for 45 minutes and transferred to the room air. Necropsy was performed as described in the Methods section, BALF was collected to assess the presence of HMGB1 (A), levels at 3, 6 and 24 h where the quantification of 60kDa complex bands are shown in (B), and the 30 kDa HMGB1 band is shown in (C). Arterial blood was also collected and separated for plasma or serum. The serum was analyzed for CK-MB (D) and Syndecan 1 (E) levels and plasma samples were analyzed for hFABP (F) and NT-proBNP (G) levels using commercial ELISA kits as described in the Methods section. Representative transmission electron microscopy (TEM) images (x3,400 and x13,500) of naïve rats (H through K) and chlorine exposed rats (L through O). Images of chlorine exposed rat right ventricle (RV, L and M) and left ventricle (LV, N and O) showing myofibrillar loss and contraction band necrosis (yellow arrow), mitochondrial cristae lysis (red asterisk), and disruption of z-discs (yellow arrowhead) are displayed in lower panel. Values shown are mean±SEM n=8–12/group. The p-values demonstrating statistical significance as represented by the signs are shown (* ≤ 0.05, ** ≤ 0.01, # ≤ 0.001 and $ ≤ 0.0001).

Chlorine exposure impairs gas exchange and alters electrolyte balance

Chlorine exposure damages the respiratory tract and may affect the respiratory gas exchange process. We performed an arterial blood gas (ABG) analysis to measure various gas exchange parameters in the arterial blood of chlorine exposed animals at 3, 6 and 24h after exposure (Figure 4). The blood pH was significantly reduced at earlier time points (3 & 6h) after exposure to 500 or 600 ppm of chlorine, suggesting an increase in H⁺ ions. However, 24h after exposure, the pH values returned to baseline (Figure 4A and Supplementary figure 4A in female rats observed at 24h after exposure to 600 ppm chlorine). As expected, the CO₂ levels were significantly elevated at all time points measured post chlorine exposure (Figure 4BC and Supplementary figure 4BC). Similarly, the blood bicarbonate content was significantly increased following chlorine exposure (Figure 4D and Supplementary figure 4D). The partial pressure of oxygen and oxygen saturation was significantly decreased at 3h post chlorine exposure, but it improved in the survivors at later time points (Figure 4E, F). The oxygen saturation reached ~100% in survivors of 500 ppm chlorine exposure at 24h but remained decreased in 600 ppm chlorine exposed animals (Figure 4F and Supplementary figure 4A). These results corroborated a case of compensated respiratory acidosis due to chlorine exposure. This was further confirmed by the increased BE (b) and BE (ecf) values post chlorine exposure (Figure 4GH and Supplementary figure 4GH). The early anion gap changes could be either a result of dehydration or caused by respiratory distress after exposure to chlorine (Figure 4I and Supplementary figure 4I).

Figure 4.

Effect of chlorine exposure on blood gas parameters and acid-base balance. Rats were exposed to chlorine (500 or 600 ppm) for 45 minutes and transferred to room air and euthanized at different time points at which blood was collected from the descending aorta for arterial blood gas (ABG) measurement. Bar graphs are shown for pH (A), pCO₂ (B), TCO₂ (C), cHCO3- (D), pO₂ (E), cSO₂ (F), BE(b) (G), BE (ecf) (H) and AGapK (I). Values shown are mean±SEM n=8–12/group. The p-values demonstrating statistical significance as represented by signs are shown (* ≤ 0.05, ** ≤ 0.01, # ≤ 0.001 and $ ≤ 0.0001).

ABG analysis also revealed the electrolyte content in the blood of chlorine exposed animals (Figure 5A–D and Supplementary figure 5 A–D). Sodium content was significantly increased at 6 and 24h after higher concentration (600 ppm) chlorine exposure (Figure 5A). Potassium content seemed to decrease in animals exposed to both chlorine concentrations at later (6 and 24h) time points after exposure (Figure 5B). Blood calcium content increased significantly at all time points following higher concentration (600 ppm) chlorine exposure (Figure 5C), whereas chloride content decreased at 6h post chlorine exposure for both chlorine concentrations. The electrolyte content was not significantly altered in the female rats after chlorine exposure (Supplementary figure 4A–D). We also measured lactate and glucose content of blood which tend to increase under stress. The lactate levels were significantly elevated at all time points, however blood glucose seemed elevated at earlier (3h) time points but normalized in survivors at later time points (6 and 24h) (Figure 5E, F). The blood lactate and glucose remained unaltered from baseline which was already higher in female rats (and Supplementary figure 4EF). Kidney injury markers, blood urea nitrogen (BUN) and creatinine were significantly increased at all time points in the blood of the animals after exposure to both 500 and 600 ppm chlorine (Figure 5GH). Although BUN was significantly increased in female rats after chlorine exposure creatinine levels remained unaltered (Supplementary figure 4GH).

Fig. 5.

Impact of chlorine exposure on blood chemistry and electrolytes. Rats were exposed to chlorine, transferred to the room air and euthanized as detailed in the legend to Figure 4. Blood was collected from the descending aorta for arterial blood gas (ABG) measurement. (A) illustrates sodium levels at 3, 6 and 24 h after chlorine exposure. (B) demonstrates potassium content, (C) and (D) show calcium and chloride levels respectively. (E) demonstrates lactate, (F) illustrates glucose, (G) shows BUN and (H) illustrates creatinine levels after chlorine exposure. Values shown are mean±SEM n=8–12/group. The p-values as represented by signs are shown (* ≤ 0.05, ** ≤ 0.01, # ≤ 0.001 and $ ≤ 0.0001).

Chlorine exposure alters blood cell counts and hemoglobin content

Chlorine induced respiratory distress may affect blood chemistry, therefore, we evaluated the blood cell counts and hemoglobin content of the arterial blood of chlorine exposed animals with a hematology analyzer. The white blood cell (WBC) counts decreased consistently with time following exposure to both chlorine concentrations (Figure 6A and Supplementary figure 6A). However, the red blood cell (RBC) counts increased significantly 24h after exposure to 600 ppm chlorine only (Figure 6B). Platelet counts decreased in a time-dependent manner following exposure to 500 ppm chlorine (Figure 6C). Similarly, exposure to 600 ppm chlorine resulted in a significant decrease in platelet counts as early as 3h post exposure and this decrease persisted for the duration of the study (Figure 6J). The RBC count and platelet numbers were not significantly altered at 24h post exposure to chlorine (and Supplementary figure 6BC) Hemoglobin levels increased with time after chlorine exposure as assessed by ABG analysis or CBC hematology analysis (Figure 6D & E and Supplementary figure 6D). These machines may not be very sensitive in estimating the hemoglobin content, but an increased trend was consistently observed in the two methods⁴⁹. Like the RBC content, the hematocrit values were also increased upon chlorine exposure (Figure 6F). The mean corpuscular volume (MCV), which reflects the RBC size, was increased upon exposure to 600 ppm chlorine at 24h after exposure (Figure 6G). The high variability across samples in the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values made it difficult to determine statistical significance between the naïve and exposed groups, although the trends suggest they were both increased upon chlorine exposure (Figure 6H&I). HCT, MCV, MCH and MCHC remained near baseline levels 24h after exposure in females (Supplementary figure 6E–H). The red cell distribution width (RDW) remained unchanged after chlorine exposure in male rats (not shown) but increased significantly in female rats (Supplementary figure 6J).

Fig. 6.

Effect of chlorine exposure on the complete blood counts (CBC) and hemoglobin. Rats were exposed to chlorine and transferred to room air as described above. Blood collection and complete blood count (CBC) was carried out as described in the Methods section using a HEMAVET (Drew Scientific) 5-part differential blood count analyzer. A and B illustrate the WBC and RBC levels, respectively at 3, 6 and 24 h. C through J demonstrate platelets (PLT), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). Values shown are mean±SEM n=8–12/group. The p-values as represented by signs are shown (* ≤ 0.05, ** ≤ 0.01, # ≤ 0.001 and $ ≤ 0.0001).

Chlorine exposure alters blood immune cell content

The absolute neutrophil content and percentage of neutrophils were both increased soon after chlorine exposure at both 500 and 600 ppm concentrations (Figure 7A & F and Supplementary figure 7A & F). Lymphocytes were significantly decreased shortly after exposure and remained decreased at 24h after exposure (Figure 7B & G and Supplementary figure 7B & G). The neutrophil to lymphocyte ratio was calculated and found to be significantly increased after chlorine exposure when compared to the naïve animals (inset in Figure 7B top panel and Supplementary figure inset in 7B). The blood monocyte content decreased consistently with time following chlorine exposure (Figure 7C & H). The eosinophils and basophils were increased after chlorine exposure, which was most consistent in the lower chlorine (500 ppm) exposure (Figure 7D & E top and lower panel I &J). Although trends of alterations in the monocyte, eosinophils and basophils were apparent they were not significantly altered in female rats after chlorine exposure (and Supplementary figure 7C–E and H–J).

Fig. 7.

Effect of chlorine exposure on immune cell counts. Blood collection and CBC analysis was carried out as described in the Methods section using a HEMAVET (Drew Scientific) 5-part differential blood count analyzer to determine the levels of neutrophils (A), lymphocytes (B, with inset demonstrating the neutrophil (NE) to lymphocyte (LY) ratios), monocytes (MO) (C), eosinophils (EO) (D), and basophils (BA) (E) levels at 3, 6 and 24 h. Lower panels (F-J) demonstrate the respective cell percent values. Values shown are mean±SEM n=8–12/group. The p-values as represented by signs are shown (* ≤ 0.05, ** ≤ 0.01, # ≤ 0.001 and $ ≤ 0.0001).

Chlorine exposure causes acute airway damage and lung injury

Chlorine is highly corrosive and its inhalation causes severe damage to the exposed epithelium. We evaluated the hematoxylin and eosin (H&E)-stained sections of trachea of the 600 ppm chlorine exposed rats to observe the pathological changes (Figure 8A–C). Chlorine exposed tracheas had extensive and significant airway epithelial sloughing which could be quantified (Figure 8C). The residual tracheal epithelium showed extensive deciliation with multifocal epithelial denudation revealing the underlying basement membrane. The tracheal/airway lumen was filled with casts and cellular debris. The capillaries and blood vessels appeared congested with occasional neutrophil infiltration in the mucosa and the submucosa.

Fig. 8.

Effect of chlorine exposure on airway epithelium. Tracheas obtained from naïve and chlorine exposed rats (24h after exposure) were fixed, embedded in paraffin, sectioned and the slides were stained with H&E and observed under a light microscope. Sections of naïve (A) and chlorine exposed rats (B, with adjacent magnified image to demonstrate sloughed and denuded epithelium) were evaluated to assess epithelial sloughing in the tracheas (C). Upto 6 images per animal and sections of tracheas from 6 animals per group were evaluated. A p value for ≤ 0.01 (as compared to unexposed controls) is shown by **. (D-I) Effect of chlorine exposure on the lung. The lungs of chlorine exposed rats were fixed at 24 h after exposure, paraffin embedded, sectioned, and stained with H&E and histology examination was performed for naïve (D & G), and chlorine exposed rats (E and H, with adjacent magnified images to demonstrate hyaline membranes and proteinaceous debris in E, and abnormal epithelial growth in the airways shown in H) to determine the injury score (F & I) using the following parameters; neutrophils in the alveolar and interstitial space (green arrow), hyaline membranes and proteinaceous debris in the airspace (black arrowhead and red arrow) and alveolar wall thickening (open arrow) of left (top) and right upper (bottom) lung lobes. Abnormal epithelial growth inside the airways of chlorine exposed animals is shown by the red arrowhead (H). Upto 6 images per lung lobe and sections of lungs from 6 animals per group were evaluated including the representative images shown here. A p value for ≤ 0.0001 (as compared to unexposed controls) is shown by $.

To evaluate the effect of chlorine exposure on the lung pathology we determined the lung injury scoring that accounts for parameters such as neutrophils in the alveolar space and interstitial space, hyaline membranes, proteinaceous debris in the airspace and alveolar wall thickening^42,43. Although all lung lobes were evaluated histologically, lung injury and injury scores were quantified only using images obtained from the left lobe and the right upper lobe at 24h after exposure (Figure 8D–I). Chlorine exposure caused pulmonary edema characterized by exudation of fluid from the vessels, interstitial and intra-alveolar edema, neutrophil infiltration, hyaline membrane formation, and the accumulation of proteinaceous debris. Alveolar damage and septal thickening were also observed, accompanied by inflammatory cell infiltration (Figure 8D–I). Occasionally, abnormal epithelial structures were seen inside the airways of chlorine-exposed animals suggesting abnormal epithelial repair and growth (red arrowhead, Figure 8H).

Discussion:

Experimental chlorine exposure in rodents, particularly mice and rats, has been invaluable for elucidating the mechanisms of lung injury, airway hyperresponsiveness and subsequent repair. These preclinical models closely mimic several physiological and histopathological features of chlorine-induced respiratory injury in humans, making them useful tools for mechanistic and therapeutic studies. These models can vary by chlorine concentration and exposure duration. Reported exposure durations vary between minutes to hours, and low concentration exposures cause only airway hyperreactivity, irritation and inflammation whereas high concentration exposures cause severe lung injury, organ damage and may result in death. Although some studies do report LD₅₀ values for chlorine exposure, they are limited in number and generally lack detailed and consistent characterization of gas exchange impairment, physiological status or systemic consequences of exposure. In the present study, and in our published abstract, using a rat model exposed to two near lethal dose (LD₅₀) chlorine concentrations we demonstrate that chlorine-induced pulmonary injury results in impaired gas exchange and subsequent systemic physiological dysfunction⁵⁰.

Previous rat studies, including our own, have shown that high concentration (400–600 ppm for 45–60 min) halogen inhalation produces approximately 50% mortality (LD₅₀) in~24h after exposure and is associated with severe cardiopulmonary injury in adult rats^9,30,33,44. Mortality occurred predominantly during the early post-exposure period and therefore, only surviving animals were available for assessment at subsequent time points. Another study using the same species of rats reported an even higher mortality rate within 6h after exposure to similar chlorine concentrations²⁹. This difference could be attributed to several factors, including the higher altitude of the exposure site, modifications in the exposure equipment, and exposure conditions such as gas flow rates, differences in the rat body weight, and variability of the procurement source of the rats. Similar early mortality has been reported in humans following accidental chlorine gas exposure. For example during the train derailment incident in Graniteville, SC, 9 fatalities were reported on the same day⁵¹. Although the toxicity may vary with sex, age, health, exposure extent and individual susceptibility, the lowest lethal concentration reported to occur in humans is 430 ppm following a 30 min exposure⁵². The median lethal time (LT₅₀) of 7h for 50% mortality was reported at 250 ppm chlorine for both rats and mice^52,53. For a 1000 ppm chlorine exposure, the LT₅₀ was 28 min for rat and 53 min for mice⁵². It is important to note that different mouse strains have varying susceptibility to chlorine exposure which can influence the extent of injury and the resulting outcomes^54–57. Toxicity data such as LD₅₀/LC₅₀ values of an agent are crucial for risk assessment and provide a reference point for testing antidotes. In an occupational setting, they enable the establishment of exposure limits and emergency response guidelines.

Human victims of acute accidental chlorine exposure demonstrate burning and lacrimation in the eyes, coughing, and severe shortness of breath, rapid breathing, severe respiratory distress, low blood and tissue oxygenation levels, respiratory failure, and chest pain. Pulmonary edema, tracheobronchitis, and pneumonitis may also develop. Consistent with the clinical manifestations of chlorine exposure in humans, rodents develop acute respiratory distress characterized by labored breathing, gasping, stridor and reduced activity. The severity of these symptoms can be quantified using a qualitative clinical scoring system that evaluates the respiratory quality and stridor^58,59. As reported in this study, high-dose chlorine causes significant respiratory distress soon after exposure which is characterized by decreased respiratory rate, gasping, stridor and severe abdominal retractions²⁹. Oxygen supplementation is the standard of care for acute respiratory compromise following chlorine exposure. However, studies in chlorine exposed rats have shown that supplemental oxygen alone fails to rescue the loss of respiratory quality, suggesting that factors beyond hypoxemia contribute to chlorine-induced respiratory dysfunction^8,29. The animals have been observed to avoid eating and drinking briefly, which may contribute to the weight loss and dehydration reported in this study and this behavior has also been observed in previous halogen toxicity studies^9,33.

Exposure to high concentrations of chlorine can damage the respiratory tract and lungs resulting in acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) depending on the severity of the injury. Reaction of chlorine on moist airway tissue surfaces forms hydrochloric and hypochlorous acids, which damage the airway epithelium and the alveoli and increases the permeability of the alveolar capillary membrane, leading to pulmonary edema and impaired gas exchange. Acute pulmonary edema and severe hypoxemia requiring tracheal intubation and mechanical ventilation was reported in a case of accidental chlorine exposure⁶⁰. A dose-dependent increase in pulmonary edema and progressive deterioration of lung function was reported in a swine model of chlorine exposure⁶¹. Exposure to even non-lethal concentrations of chlorine caused edema in the lungs and hearts of rats⁶². Our studies have also shown acute halogen exposure-induced pulmonary and cardiac edema³³. Chlorine exposure-induced decrease in spleen weight has recently been reported in a sheep model of chlorine exposure⁶³. This could be attributed to the mobilization of red blood cells from the splenic reservoir to increase blood gas storage capacity⁶⁴. The damage to the alveolar capillary barrier allows a plasma protein leak from the bloodstream into the airspaces of the lungs which can be measured as increased protein content in the bronchoalveolar lavage fluid (BALF). Increased lactate dehydrogenase (LDH) in the BALF and plasma is an effective marker of lung damage and inflammation⁶⁵. Similarly, increased LDH or its isoenzymes in plasma may also indicate acute myocardial infarction⁶⁶. Consistent with our findings, chlorine exposure of precision-cut rat lung slices caused an increase in LDH levels indicating tissue damage⁶⁷. The lower levels of LDH associated with the higher chlorine concentration exposure in this study could be because of chlorination preventing or interfering in the activity detection⁶⁸. Aside from the increased BALF protein content and LDH levels we have also evaluated the presence of HMGB1 and IgM to characterize the extent of cell death and disruption of the capillary endothelial barrier upon lung injury caused by chemical threat agents, including sulfur mustard, arsenicals and halogens^36,44,47,48. In lung tissue sections of nitrogen mustard-exposed mice, HMGB1 staining predominantly localized to the macrophages, suggesting that HMGB1 may play a role in proinflammatory processes that contribute to lung injury progression⁶⁹. HMGB1 release in BALF has been implicated in the pathogenesis of ALI/ARDS, further indicating that our findings are consistent with an ARDS-like proinflammatory phenotype^50,70. Although increased heparanase activity, that can increase syndecan shedding from cells, was reported after chlorine exposure in mice, actual increase in syndecans has not been reported in lung tissue or plasma following chlorine inhalation⁷¹. A creatinine kinase increase occurs with myocardial damage and is known to quickly return to baseline as reported in our findings after chlorine exposure⁷². This is congruent with the increased ultrastructural damage demonstrated in the heart of chlorine exposed rats in this study. Our studies have provided extensive evidence that halogen exposure induces myocardial damage and dysfunction, which are frequently accompanied by multiorgan injury^9,30,33.

Arterial blood gas (ABG) analysis provides key information about three major physiologic functions: blood oxygenation, ventilation, and acid-base balance. Arterial blood gas testing in patients with acute chlorine exposure is crucial for monitoring respiratory distress and systemic toxicity. For victims of the 2005 Graniteville SC train derailment chlorine disaster, ABG measurements provided an early indication of outcome severity⁷³. People with acid-base disorders or a lower PO₂/FiO₂ ratio developed ALI and ARDS and had a longer hospitalization⁷³. However, survivors of chlorine exposure with normal ABG values had non-lethal outcomes. Therefore, abnormal blood gas levels may contribute to early fatalities after chlorine exposure. Pulmonary edema and dysfunctional lung mechanics post-chlorine exposure can lead to decreased oxygen saturation and partial pressure of oxygen and increased carbon dioxide¹⁶. Immediate hypoxia and hypoxemia have been reported for ventilated swine models of chlorine exposure^12,74. Respiratory acidosis, hypoxemia, and hypercapnia, followed by an increase in lactate levels in blood were reported in rats that survived exposure to chlorine^27,29. In the study where oxygen supplementation improved hypoxemia but worsened hypercapnia, the loss of alveolar ventilation and increased dead space may have caused a ventilation perfusion mismatch²⁹. Increased PaCO₂ and total CO₂ was accompanied by increased HCO3⁻ levels at earlier time points. The reduction in these values at 24h indicate onset of compensatory hyperventilation. Overall, ABG analysis demonstrated hypoxemia and hypercapnia with respiratory and metabolic acidosis (or compensated respiratory acidosis) at all time points of assessment in rats exposed to the two chlorine concentrations. In an anesthetized and ventilated pig model of chlorine exposure a combined initial respiratory and metabolic acidosis was followed by later respiratory alkalosis and metabolic acidosis¹². Blood electrolyte changes following chlorine exposure may be attributed in part to systemic effects such as dehydration resulting from reduced intake, acid-base disturbances and respiratory injury rather than a direct effect of chlorine on electrolyte homeostasis. The BUN and creatinine values are consistent with our previously reported findings of kidney damage associated with cardiopulmonary dysfunction⁹.

In addition to physiological changes chlorine-induced lung injury is characterized by cellular infiltration in the lung and increased inflammation. A 15 min nose only inhalation of 200 ppm chlorine significantly increased the leukocytes, eosinophils and neutrophils in the BALF of rats⁷⁵. A similar nose-only exposure of mice to chlorine (100 ppm, 5 min) increased BALF cell content of macrophages, neutrophils and eosinophils⁷⁶. Whole body non-lethal chlorine (300 ppm, 25 min) exposure of mice resulted lung injury and oxidative stress followed by an increase in total BALF cells, including proinflammatory macrophages⁷⁷. Interestingly, this study also demonstrated increase in soluble receptor for advanced end glycation product (sRAGE) in the BALF of chlorine-exposed mice. RAGE is a key mediator of pulmonary proinflammatory responses and an important contributor of acute lung injury. HMGB1 is a RAGE ligand and the binding of HMGB1 to RAGE initiates signaling events that cause sustained activation of NF-kB and transcription of proinflammatory genes like iNOS, MMP-9 and RAGE itself^78,79. HMGB1 along with other DAMPs, was increased in the sputum of elite swimmer athletes that demonstrated airway epithelial damage due to chlorine exposure in pools⁸⁰. Increased HMGB1 after chlorine exposure in this study is consistent with lung injury and edema due to disruption of alveolar epithelial function demonstrated by our other studies in rodent models of halogen, sulfur mustard and arsenicals^9,47,48.

Chlorine inhalation-induced significant hematological alterations indicate a systemic response to pulmonary injury. The decline in white blood cell (WBC) counts following exposure to chlorine concentration suggests an acute inflammatory response characterized by migration of leukocytes from whole blood to the injured lung. Such blood leukocyte decreases and sequestration is consistent with previous reports of chlorine-induced recruitment of neutrophils and other immune cells to the site of tissue damage^12,57. These results contrast with other studies in which the blood leukocyte counts were increased after chlorine exposure, however the chlorine exposure concentration in those studies was different⁸¹. Similarly, blood WBC counts were also increased in a ventilated pig model of chlorine exposure⁷⁴. Marked changes also occurred in the leukocyte subsets of blood after chlorine exposure Such as the rapid onset increases in neutrophils observed for both chlorine concentrations indicates an acute mobilization of innate immune cells in response to lung injury. Neutrophils may contribute to tissue damage through the release of reactive oxygen species (ROS) and proteolytic enzymes. The lymphocyte counts were significantly reduced and remained suppressed up to 24h after chlorine exposure, resulting in an elevated neutrophil to lymphocyte ratio (NLR). This shift reflects a proinflammatory state and suggests that NLR may serve as a useful and sensitive prognostic biomarker of chlorine-induced lung injury. A decline in monocyte counts may reflect recruitment to injured lung tissue to participate in inflammatory clearance and early repair processes. The increased granulocytes may suggest a secondary involvement in inflammatory response, but their relative reduction at higher concentration chlorine exposure may indicate the severity of injury and broader immune dysregulation. The significant increases in RBC counts, hemoglobin concentration, and hematocrit after exposure to 600 ppm chlorine are likely due to hemoconcentration resulting from fluid loss secondary to pulmonary edema and dehydration. Additionally, enhanced erythrocyte mobilization from the splenic reserve on account of hypoxemia caused by chlorine exposure may add to this effect⁸². De novo erythropoiesis is a less likely factor, as it may need a longer duration to increase the RBC values. The RBC turnover might also be related to the chlorine-induced membrane fragility and lysis, promoting release of cell-free hemoglobin which may further potentiate lung injury^82–84. The increase in mean corpuscular volume (MCV) following 600 ppm chlorine exposure could be a result of larger immature erythrocyte release in response to systemic stress. A distinct reduction in platelet counts suggests platelet activation and consumption in response to endothelial injury and systemic inflammation. This chlorine-induced thrombocytopenia is consistent with chlorine-induced platelet depletion reported before in a ventilated swine model¹².

The increased inflammatory response following chlorine exposure is likely to contribute to lung injury and may also play a role in the development of subsequent extrapulmonary organ damage. Histopathological assessment and lung injury scoring are key techniques to evaluate the extent and severity of lung damage. Our findings demonstrate that chlorine inhalation causes severe injury to both the conducting airways and the lung parenchyma. Airway epithelial cell apoptosis, epithelial sloughing and increased airway hyperresponsiveness was reported in mice exposed to a single dose of high concentration chlorine⁸⁵. Chlorine exposure-induced airway epithelial sloughing followed by aberrant epithelial repair resulted in persistent airway remodelling⁸⁶. Necrotic bronchiolar epithelium was observed in ventilated swine models of chlorine exposure⁷⁴. Similarly, epithelial necrosis and inflammatory cell infiltrates were observed in the airways of human victims of chlorine exposure^17,87. These findings are consistent with our histopathological observations of extensive epithelial denudation, deciliation, and airway debris formation post-chlorine exposure. The histopathological changes that were observed in the lung in this study included edema, neutrophil infiltration, hyaline membrane formation, proteinaceous exudates, and alveolar damage. These are characteristic features of ALI and ARDS. Hyaline membrane formation and alveolar damage contribute to hypoxemia and respiratory failure, which has been reported in human victims of accidental chlorine exposure⁸⁸. Acute lung injury consistent with hyaline membrane deposition and neutrophilic alveolitis was reported in a swine model of chlorine exposure¹².

This study demonstrates that chlorine inhalation produces severe acute lung injury accompanied by profound disturbances in pulmonary gas exchange, systemic physiology and inflammatory response in rats. Chlorine exposure resulted in marked respiratory distress, pulmonary edema, disruption of the alveolar capillary barrier, impaired arterial blood gases, vascular injury, and histopathological features consistent with ALI/ARDS. These pulmonary changes were associated with systemic consequences, including hematological alterations, acid base imbalance, evidence of cardiac and renal injury and early multiorgan dysfunction. The observed increases in BALF protein content, LDH levels, presence of extracellular HMGB1, and syndecans, along with inflammatory cell infiltration and elevated NLR ratios, further support the involvement of epithelial and endothelial injury, innate immune activation, and dysregulated inflammatory response in chlorine-induced toxicity. Thus, we provide a comprehensive assessment of physiological, biochemical, hematological and histopathological changes following two near lethal chlorine exposures. We also demonstrate that mortality following high dose chlorine inhalation is closely associated with severe impairment of gas exchange and systemic physiological dysfunction rather than isolated pulmonary injury alone. This well characterized rat model closely reproduces many human clinical manifestations of acute chlorine exposure. These results also improve our understanding of acute chlorine toxicity and provide a strong foundation for the development of therapeutic strategies aimed at reducing morbidity and mortality following accidental or intentional chlorine exposure.

Highlights.

• This study provides a comprehensive physiological characterization of a rodent model following high dose chlorine inhalation demonstrating that mortality is driven by the combined effects of acute respiratory failure and systemic physiological dysfunction rather than lung injury alone.

• By demonstrating that impaired gas exchange and multi-system dysfunction jointly determine the extent of injury and survival, these findings advance the understanding of acute chlorine toxicity and establish a more integrated framework for investigating halogen gas-induced toxicity and evaluating potential therapeutic interventions.