Halogen-Induced Chemical Injury to the Mammalian Cardiopulmonary Systems

Abstract

The halogens chlorine (Cl₂) and bromine (Br₂) are highly reactive oxidizing elements with widespread industrial applications and a history of development and use as chemical weapons. When inhaled, depending on the dose and duration of exposure, they cause acute and chronic injury to both the lungs and systemic organs that may result in the development of chronic changes (such as fibrosis) and death from cardiopulmonary failure. A number of conditions, such as viral infections, coexposure to other toxic gases, and pregnancy increase susceptibility to halogens significantly. Herein we review their danger to public health, their mechanisms of action, and the development of pharmacological agents that when administered post-exposure decrease morbidity and mortality.

Introduction

The halogens chlorine (Cl₂) and bromine (Br₂) are highly reactive, oxidizing elements with widespread industrial applications and a history of development and use as chemical weapons. Both are encountered in gaseous form at room temperature and both are only slightly soluble in water with solubilities of 7.25 and 42 g/L at 20°C, respectively. Despite their low physical solubility, both Cl₂ and Br₂ produce high concentrations of their hydrolysis products due to their high reactivity with water. In the reactions

$$C{l}_2+H_{2}O\rightleftarrows HOCl+HCl\rightleftarrows H^{+}+OC{l}^{-}+H^{+}+C{l}^{-}$$

and

$$B{r}_2+H_{2}O\rightleftarrows HOBr+HBr\rightleftarrows H^{+}+OB{r}^{-}+H^{+}+B{r}^{-}$$

they readily produce hypohalous (HOCl and HOBr) and hydrohalic acids (HCl and HBr), also known as hydrogen halydes. While hypohalous acids dissociate relatively poorly and do not reduce pH substantially, they are highly reactive. Hydrohalic acids are not reactive but dissociate readily to protons and halide ions, producing acidic pH. The high reactivity of hypohalous acids with organic molecules is considered to be the main cause of halogen toxicity to all living things.

There are numerous instances of both intentional and unintentional human exposure to halogen gasses resulting in morbidity and in some cases mortality. Depending on the concentration and duration of exposure, symptoms range from mild mucosal membrane irritation to severe lung and cardiac injury, in some cases culminating in death (1–3). Survivors may develop chronic ailments including pulmonary and cardiac fibrosis (3, 4). Exposure of pregnant animals to halogens results in significant mortality and cardiovascular injury to both the dams and their fetuses (5).

There has been significant interest in developing suitable animal and cell models to investigate the mechanisms by which Cl₂ and Br₂ damage cardiomyocytes as well as lung epithelial, smooth muscle, and endothelial cells along with developing countermeasures which may prevent and reverse this injury. Herein, we briefly review the epidemiology of halogen gas exposure, summarize what is known concerning the generation of secondary reactive intermediates and how these species contribute to pulmonary and systemic injury, and review some of the most promising countermeasures.

Halogens and Public Health

Cl₂ and Br₂ have widespread industrial applications and are used to sterilize drinking water, to treat swimming pools and in numerous industrial applications including the production of paper, plastic, dyes, textiles, and medicines (2, 6, 7). Most human exposures occur accidentally during manufacturing, transportation, or storage of halogens for industrial purposes. Exposures to Cl₂ and Br₂ at water treatment facilities and swimming pools are well documented. The toxic properties of halogens and their derivatives were inadvertently exhibited near the start of the 1900s during administration of chloroform as an anesthetic where the open flames used to illuminate the operating rooms of the time caused its decomposition into Cl₂ and phosgene (COCl₂) leading to respiratory tract symptoms and in some cases the death of patients and physicians (8). Simultaneously, efforts to harness this toxic potential for use on the battlefields of World War I were ongoing. Br₂-based tear gas agents, along with Cl₂, COCl₂, diphosgene, C4H8Cl2S (mustard gas), and various combinations of each, were all refined and deployed at various conflicts (6). Unfortunately, the use of halogen-based chemical weapons has continued into the modern era with Cl₂ attacks documented in Iraq and during the ongoing Syrian civil war (1, 9) and with Br₂ storage tanks in Israel serving as the apparent target of a failed terrorist attack in 2004 (10). The US Department of Homeland Security identified industrial sites dedicated to Cl₂ as possible targets of terrorist activity, with some projections estimating that a successful attack could result in thousands of fatalities and 100,000 hospitalization (The Homeland Security Council. Planning Scenarios: Executive Summaries. 004; 8-1).

Lessons Learned from Accidental Halogen Exposures

As mentioned, industrial and manufacturing mishaps represent the most likely interface between humans and halogen gas. Through exploring the results of these accidents, one can begin to piece together a picture of both the acute and long-term sequelae resulting from various exposure intensities. Thirty large-scale Cl₂-related accidents have occurred in urban centers over the past two decades with hundreds of documented smaller scale incidents. In one prominent example, a train was accidentally diverted causing a collision with a parked train car carrying Cl₂. The tanker ruptured, spilling 54,422 kg (120,000 lb) of Cl₂ and engulfing the city of Graniteville in a toxic cloud of gas. Eight victims died before arrival to a hospital. Seventy-one patients were hospitalized due to acute Cl₂ exposure. Of these, there was 1 in-hospital death and 25 (35%) individuals who required intensive care (11). Patients who survive acute lung injury frequently develop chronic lung disease, with airflow obstruction, fibrosis, airway hyperreactivity, and impaired gas exchange (3). These patients are more likely to require hospital admission (1, 12) and more susceptible to bacterial pneumonia (13). People that smoke and those with respiratory infections and asthma are much more sensitive to the toxic effects of Cl₂ and may experience airway hyperresponsiveness at concentrations that cause no symptoms in people with no respiratory disease (7, 14–17).

Airway hyperresponsiveness, termed reactive airways dysfunction syndrome, is also reported in individuals exposed to Cl_2, and asthma-like symptoms can persist for long periods (11). In the year following the Graniteville train accident, a 4% decrease in mean forced expiratory volume at 1 s (FEV₁) was observed in patients when compared with test results the year before the incident. During the subsequent year, a partial recovery in the forced vital capacity was noted, but the predicted average FEV₁ continued to decrease over time. Severe annual FEV₁ decline was most prevalent in the year of the accident and independent of smoking status (1, 3, 4, 18). Intriguingly, hospitalizations for hypertension doubled from 110 per 10,000 residents in 2005 to 220 hospitalizations per 10,000 residents in 2012 in the larger Graniteville population. This increase was found in all age groups, races, and both sexes. Cardiomegaly was observed during autopsy in eight of the nine immediate victims (11).

In other accidents, exposure to high levels of Cl₂ gas led to vascular injury and depressed of cardiac function (19). Risk of stroke over time may also be impacted due to oxidation of low-density lipoproteins (LDL) by hypochlorous acid and the generated oxidized LDL contributing to the vessel wall inflammation and thus promoting atherosclerosis (20, 21).

Chemical accidents due to inadvertent release of Br₂ have occurred in Netherlands, Germany, Belgium, Israel, United States, Russia, and many other countries (22). A major train accident involving Br₂ occurred in Chelyabinsk, Russia (population 1.1 million) leading to 42 hospitalizations and 200 patients requiring medical attention. In another train accident, a freight train collided with three rail cars in Dimona Israel, one of which was carrying liquid bromine. There are numerous reports of incidents worldwide, where trucks carrying Br₂ overturned, spilling their contents in the atmosphere.

Presently, treatments following halogen gas exposure are mainly supportive, including humidified oxygen administration for correction of hypoxemia, β₂-agonists for reversal of for bronchospasm, and antibiotics for potential infections (23). In severe cases of acute respiratory distress syndrome (ARDS), positive pressure ventilation with or without intubation may be necessary. Corticosteroids and sodium bicarbonate can alleviate ARDS following Cl₂ exposure in animal studies, but similar efficacy in humans has yet to be demonstrated (24, 25).

Animal Models of Cl₂ Toxicity

Mice, rats, rabbits, pigs, sheep, dogs, and monkeys have been used to study the cardiopulmonary sequelae of exposure to Cl₂ and the response to various treatments (reviewed in Refs. 1, 2). Rodents are either placed in environmental chambers (whole body exposures) or breathe Cl₂ via a head-only exposure system. The advantages and disadvantages of each system have been previously described in detail (26). One important consideration is that Cl₂ injury to the skin induces the endoplasmic reticulum stress and unfolded protein response which may contribute to lung injury (27). Notably, animals exposed through nose-only exposure require restraint which may impose an additional stress. Rodents can be exposed to halogens either while sedated or when fully conscious (and treated with an analgesic rather than anxiolytic). On the other hand, large animals need to be anesthetized, intubated, and ventilated. Thus halogens are delivered through the endotracheal tube.

Experiments in rodents show that the degree of lung damage after Cl₂ exposure depends on total dose (i.e., concentration × time of exposure) as well as the specifics of exposure concentration and time (28). Acute exposure to doses of 1 ppm may cause irritation (29), and chronic exposure to even lower doses (0.1–0.4 ppm) may lead to ocular irritation and degeneration of the airway epithelium (30, 31). Irritant effects are primarily mediated through transient receptor potential ankyrin 1 (TRPA1) ion channels and their interplay with neurons within the airways that mediates the noxious reflex to inhaled irritant exposure, including the cough reflex (32, 33). These channels, which are found in trigeminal nerve fibers (upper airway innervation) as well as in sensory fibers of the vagus nerve (larynx and lower airways), mediate protective mechanisms including the cough reflex, sneezing, decreased respiratory rate, sensations of irritation and pain, and glandular secretions (33, 34). TRPA1 channels act through increasing intracellular calcium and other cations, thus promoting neuronal excitation (35). Pharmacologic inhibition of TRPA1 channels before exposure to Cl₂ prevents the decrease in respiratory rate. The consequent increase in respiratory rate improves ventilation but, as an undesirable consequence, may result in higher levels of lung injury because of the higher levels of inhaled oxidant gases due to the increase in minute ventilation (32, 36). On the other hand, inhibition of TRPA1 channels decreases lung injury and inflammation post-acrolein exposure, an unsaturated aldehyde generated during incomplete combustion as in tobacco smoke and indoor fire (37, 38). Mice exposed to Cl₂ develop decreased locomotion, which was alleviated by the administration of buprenorphine, a common analgesic agent, indicating that this effect is probably due Cl₂-induced pain, because of activation of TRPA1 channels (39). The effects of TRPA1 inhibitors in halogen-induced toxicity are currently under investigation.

To draw valid conclusions from animal studies that will prove relevant to humans, it is important to use exposure models that approximate conditions found during release of Cl₂ in the atmosphere. With the use of validated plume models (40) in the Graniteville accident, it was determined that Cl₂ levels during a 30-min exposure period were 4,428, 550, and 161 ppm at 0.2, 0.5 and 1 km downwind from the epicenter of the accident. Persons exposed to 550 ppm Cl₂ for 30 min required hospitalization, and a number of them develop ARDS. Similarly, the large majority of rodents exposed to 500–600 ppm Cl₂ for 30 min survive the exposure although they develop bradypnea and signs of respiratory distress (41, 42). At various times in the first 48 h post-exposure, rodents develop airway epithelial sloughing, interstitial and alveolar edema secondary to injury of the microvascular and alveolar epithelial cells, massive infiltration of inflammatory cells (mainly neutrophils), hypoxemia and respiratory acidosis, impaired surfactant function, decreased active Na⁺ transport across alveolar epithelial cells, airway hyperreactivity, alveolar hypercoagulation, and systemic hypocoagulation (2, 41, 43–53). In addition, significant cardiac injury as well as injury to placentas and developing fetuses of pregnant mice were present as well (FIGURE 1).

FIGURE 1.

Halogen (Cl₂ and Br₂)-induced acute lung and systemic injury A: schema showing interaction of halogens with various components of the epithelial lining fluid and lung epithelial cells. At higher concentrations (>50 ppm), inhaled halogens reach the distal lung regions (from Ref. 48, with permission from the publisher). ASC, ascorbate; GSH, reduced glutathione. B: extensive denudation of airway epithelium in a rat exposed to Cl₂ (400 ppm for 30 min) and returned to room air for 24 h. Lung sections were stained with hematoxylin-eosin. C: lungs of a mouse exposed to Cl₂ (600 ppm and 45 min) and returned to room air for 24 h. There was significant hemorrhage and alveolar spaces were filled with protein-rich edema. D: Cl₂ decreases endothelial nitric oxide synthase (eNOS) protein expression. I and II: representative immunofluorescence images for eNOS staining from aorta collected 24 h after exposure to air (I) or Cl₂ (400 ppm, 30 min) (II). Red: eNOS; blue: Hoechst staining for nuclei (modified from Ref. 46, with permission from the publisher). E: bromine (Br₂) inhalation causes disruption of the cardiac cytoskeleton and loss of the normal highly organized linear mitochondrial sarcomere integrity. Representative transmission electron microscopy (×3,200) images demonstrating a normal control heart (top left). The remaining 3 images demonstrate myofibrillar loss, contraction band necrosis (red arrow), loss of I bands, and disruption of z-disks (yellow arrowheads) in the left ventricle 3 h after Br₂ exposure in addition to mitochondrial swelling (yellow arrows) and cristae lysis (red asterisk) [nucleus (N)] (modified from Ref. 54, with permission from the publisher). F: pregnant [embryonic day 14.5 (E14.5)] mice were exposed to air or 600 ppm Br₂ for 30 min, returned to room. Representative hematoxylin-eosin-stained placenta sections at E18.5 with the junctional zone demarcated with yellow highlighting (left) as well as periodic acid-Schiff staining (right) of Br₂-exposed pregnant mice revealed a reduced junctional zone (black bars) at E18.5 (modified from Ref. 55, with permission from the publisher). M, male; F, female; A, air; B, bromide. G: pregnant mice were exposed to air or Br₂ (400 or 600 ppm for 30 min) at E14.5 and were returned to room air. Fetuses were delivered by C-section on E18.5. Notice marked fetal growth restriction that was dependent on Br₂ concentration (modified from Ref. 55).

More than 50% of mice or rats exposed to 600 ppm Cl₂ for 30–45 min die from respiratory failure within 5 days post-exposure. Animals that survive the initial Cl₂ insult exhibit resolution of many of the initial symptoms observed in the acute phase of injury post-exposure but often develop recurring or progressively worsening elevated airway resistance in response to methacholine challenge, mucous cell metaplasia, abnormal epithelial repair, airway fibrosis, alveolar enlargement, increased respiratory system compliance, bronchiolitis obliterans, and demonstrate changes in gene expression (3, 7, 49, 56–68) (FIGURE 2). Furthermore, Cl₂ and HOCl react with amino acids in carbohydrate recognition domains of surfactant protein (SP)-A, hindering the capability to bind and eliminate pathogens (69). HOCl disrupts disulfide crosslinking of SP-D and inhibits pathogen aggregating activity after an infectious challenge (122, 123). Exposure of mice to Cl₂ increases their susceptibility infection by the mold Aspergillus fumigatus by 500-fold (70). The diminished antifungal defense is potentially explained by the inhibitory effect of Cl₂ exposure on superoxide generation and production of IL-17A and IL-22 by myeloid cells in the lung (70).

FIGURE 2.

Halogen (Cl₂ and Br₂) induced chronic lung injury A: loss of epithelial integrity results in inflammatory cell infiltration, mesenchyme infiltration, and collagen deposition. I: hematoxylin-eosin staining of tracheal epithelium, 2 days after exposure to Cl₂ (350 ppm for 30 min). II: immunofluorescent staining for CD11b (red) in a similar day 2 tissue section. In I and II, scale bars = 25 μm. Immunofluorescent staining of either CD11b (red) or collagen I (green) in a control tracheal section and sections harvested on days 5, 7, and 9 after high-dose Cl₂ and CD11b (III, VII, XI, and XV, respectively) and collagen I (IV, VIII, XII, and XVI, respectively; scale bar, = 200 μm). In VII, arrows denote collagen 1⁺ cells infiltrating the lumen. Hematoxylin-and-eosin staining of tracheal sections harvested on days 5, 7, and 9 after high-dose Cl₂ (V, IX, and XII, respectively; scale bar, = 200 μm); magnified images are presented VI, X, and XIV, respectively (scale bar, = 25 μm) In VI, arrowheads denote macrophages, whereas arrows denote neutrophils. All tissue sections are representative of 5–6 tracheas at each time. Asterisks denote the lumen (from Ref. 66, with permission from the publisher). B: histopathological effects of Cl₂ inhalation in proximal airways, 7 days after exposure. Rats were exposed to air or 400 ppm Cl₂ for 30 min. Tissue was collected 7 days after exposure, embedded in paraffin, and stained with Alcian blue and period acid-Schiff stain. I and II: representative light micrographs are of proximal airways from air-exposed (I) or 400 ppm Cl₂ for 30 min (II). Arrows, mucous cells; arrowheads, basal lamina. Magnification bar = 50 μm (modified from Ref. 59, with permission from the publisher). C and D: male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 min) and returned to room air: peripheral lung tissue with Masson’s trichrome (C) or hematoxylin-eosin stain (H&E; D); notice increased accumulation collagen deposition (blue stain) primarily around airways on days 14 and 21 after Br₂ (C) and marked enlargement of alveolar spaces (modified from Ref. 68, with permission from the publisher). E and F: quasistatic inflation and deflation pressure volume lung relationships (measured by flexiVent) (E) and lung compliance (F), measured as the slope of the deflation pressure-volume curve of mice exposed to Br₂ gas (400 ppm, 30 min) and returned to room air at the indicated time points (modified from Ref. 68).

Hemodynamic responses of pigs to lung injury resemble those of humans (71). Thus experiments in larger animals allow a more complete assessment of cardiopulmonary and systemic changes. When anesthetized and ventilated pigs were exposed to 400 ppm Cl₂ for 15 min and then returned to room air, they developed arterial hypoxemia and pulmonary arterial hypertension within 30 min post-exposure. Although they demonstrated improvement over the next 5–7 h, both variables remained abnormal up to 23 h post-exposure (72). In another study, Gunnarsson et al. (73) reported that exposure of young pigs to 140 ppm Cl₂ for 10 min led to severe hypoxemia, increased pulmonary pressure, and decreased lung compliance starting at 1 h post-exposure. Microscopic examination of lung tissue showed sloughing of the bronchial epithelium and early infiltration with leukocytes, but the alveolar structure remained generally intact. Interstitial edema and increased number of inflammatory cells in the lung spaces appeared at the later stages of exposure.

It is important to note that these exposures were conducted in the context of general anesthesia, endotracheal intubation, and mechanical positive pressure ventilation. Thus these findings may not be generalizable to an awake, spontaneously ventilating human exposed to Cl₂ in the industrial or chemical warfare scenario. For instance, Cl₂ delivery to the distal airways is likely greater when introduced via an endotracheal tube. In addition, airway reflexes are inherently ablated in the process of induction of general anesthesia to facilitate endotracheal intubation further compounding the limitations of extrapolating these data to human exposures. Despite these limitations, taken as a whole, these studies indicate that exposures of both small and large animals to Cl₂ in concentrations likely to be encountered in the vicinity of industrial accidents compromise gas exchange through dose-dependent damage to the respiratory and alveolar epithelia. Exposures cause significant atelectasis and ventilation-perfusion (VA/Q) mismatch leading to severe hypoxemia and decreased lung compliance most likely due to injury to pulmonary surfactant (48, 49). Injury to the alveolar and pulmonary vasculature results over time in the accumulation of protein-rich (noncardiogenenic) edema fluid in both the interstitial and eventually in the alveolar space. Alveolar edema is exacerbated by injury to epithelial Na⁺ channels during and following exposure of rodents to Cl₂ (47, 74), decreasing the ability of alveolar cells to clear edema fluid. Most importantly, these effects persist long after the exposure and result in either death from respiratory failure or the development of chronic lung injury such as fibrosis and bronchiolitis obliterans.

Animal Models of Br₂ Toxicity

Humans exposed to 0.9 ppm Br₂ for 5 min developed cough, eye, and airwar irritation leading bronchospasm (22, 75). At higher doses, Br₂ exposure leads to acute lung injury and adult respiratory distress syndrome. Survivors may develop reactive airway disease and pulmonary fibrosis (76). Additional information of the toxic effects of Br₂ inhalation comes from animal studies. Bitron and Aharonson (77) found that the median lethal time (Lt50) at a concentration of 750 ppm was 9 min, and the Lt50 at a concentration of 240 ppm was 100 min. No deaths occurred during a 5-min exposure at 750 ppm or during a 20-min exposure at 240 ppm. Schlagbauer and Henschler (78) reported a 30-min lethal concentration (LC₅₀) for mice exposed to Br₂ was 174 ppm. In other studies, mice exposed to 600 ppm of Br₂ gas for 30 min manifest significant lung injury within 24 h post-exposure, as demonstrated by increased protein extravasation, total cell infiltration into the bronchoalveolar lavage fluid, disruption of the airway parenchyma, and increased lung cellularity (22, 79). In these mice, Br₂ gas inhalation resulted in lung protein oxidation, increased airway resistance following methacholine challenge, elevated lung wet-to-dry weight ratio, and correlates with the observed hypoxemia and uncompensated respiratory acidosis. There is also significant inhibition of lung alveolar Na⁺ transport which worsens pulmonary edema (74). In mice exposed to Br₂ at 600 ppm for 30 min, there was a gradual increase in mortality, reaching 50% by 5 days post-exposure.

Recently Aggarwal et al. (68) reported that mice that survived exposure to Br₂ at 400 ppm for 30 min developed significant emphysematous-type changes 14–21 days later characterized by significant alveolar enlargement, increased pulmonary compliance, and increased total lung capacity along with increased levels of lung elastase. Peribronchial fibrosis was also noted at that time but the overall phenotype was predominantly consistent with changes seen in pulmonary emphysema (FIGURE 2). The mechanisms involved are discussed in detail in the following section.

Cardiovascular Effects of Halogen Toxicity

Cl₂ exposure has also been shown to injure systemic organs and impair vascular function (FIGURE 1). Rats exposed to Cl₂ (250–400 ppm) for 30 min had significantly decreased endothelial nitric oxide synthase (eNOS) protein expression and displayed marked attenuation in acetylcholine-induced eNOS-dependent vasodilation at 24 to 48 h post-exposure, suggesting that nitric oxide (NO) dysregulation may underlie the pathophysiology of Cl₂-inhalation induced systemic endothelial dysfunction (46). However, during the initial stages of Cl₂ induced injury inflammatory cell derived inducible NO synthase (iNOS) may compensate for the loss of eNOS and prevent the increase in the blood pressure. However, when iNOS was inhibited by administration of 1,400 W, significantly higher systemic blood pressures were recorded (46). This observation led some investigators to consider administration of nitrite, which is converted to NO in hypoxic and acidotic tissue, as a therapeutic agent. Post-Cl₂ administration of nitrite in rats and mice improved hypoxemia, injury to the alveolar and airway epithelia, lung inflammation pulmonary edema, and survival within 24 h post-exposure (42, 45, 80, 81) (FIGURE 3).

FIGURE 3.

Countermeasures against halogen toxicity. Post-Cl₂ administration of antioxidants increase survival Mice were exposed to 600 ppm of Cl₂ for 45 min and returned to room air. A: they received intramuscular injections of ascorbate (Asc; 2 mg) and deferoxamine (Desf; 0.3 mg) in saline starting at 1 h after exposure and every 12 h thereafter up to 60 h after exposure. They also received aerosols of ascorbate (150 mg/mL) and deferoxamine (0.3577 mg/mL) at 1.5, 24, and 48 h after exposure in sterile water. Control mice received vehicle (saline for intramuscular injections; sterile water for aerosols) instead of antioxidants using identical protocols. Data points were fitted with Kaplan-Meier survival curves and compared with the log-rank test (P = 0.0007) (from Ref. 41). B: post-Br₂ exposure administration of hemopexin increases survival. C57BL/6 mice were exposed to Br₂ (400 ppm for 30 min) and returned to room air. At either 1 h or 5 days post-exposure they were given a single intraperitoneal injection of purified human hemopexin (Hx) (4 µg/g body wt) or vehicle. Survival was assessed in the next 14 days. The Kaplan-Meier curve demonstrated that Hx reduced mortality after Br₂ exposure, even when was given 5 days later. *P < 0.05 vs. Br₂ + saline by one-way ANOVA followed by Tukey’s post hoc test (68). C: post-Br₂ administration of tadalafil (Cialis; TDF or TAD) decreases mortality of pregnant (P) mice. Nonpregnant (NP) and P gestational day 14.5 (E14.5) mice were exposed to air or to Br₂ at 600 ppm for 30 min and returned to room air; they received tadalafil (TAD; 2 mg/kg body weight in 0.1 mL of sterile saline) or vehicle via oral gavage at 1 h post-exposure and every 24 h thereafter. Data show Kaplan-Meyer curves of P and NP mice with tadalafil or vehicle, post-Br₂ exposure. NP mice exposed to Br₂ and returned to room air lived longer than similarly exposed P mice (*P < 0.05) (from Ref. 55). D: post-Cl₂ administration of nitrite improves survival. Male or female mice were exposed to Cl₂ at 600 ppm, 45 min and then brought back to room air, and nitrite was administered by intramuscular injection 30 min post-exposure. Data show Kaplan-Meier survival curves. P < 0.03 between male and female Cl₂-alone groups; P < 0.02 for nitrite therapy in males and P = 0.09 for nitrite therapy in females.

Cardiotoxicity leads to severe short-term sequelae after Cl₂-exposure. In rats, exposure to 500 ppm Cl₂ for 30 min increased the concentration of lactate in the coronary sinus, indicative of a myocardial oxygen supply/demand mismatch and consequent anaerobic metabolism. In these experiments, Cl₂ also attenuated myocardial contractile force, decreased systemic blood pressure, and led to biventricular failure and substantial mortality (19, 82). An increased left ventricular ejection fraction was also observed. The authors surmised that this effect was related to a decrease in systemic afterload in concert with a hyperadrenergic state and a consequent increase in inotropy (82).

Similarly, significant cardiac damage was found after exposure of rats to Br₂, characterized by increased troponin I, heart-type fatty acid-binding protein, and NH₂-terminal pro-brain natriuretic peptide. In these experiments, left ventricular (LV) systolic and diastolic dysfunction was observed at 7 days post-exposure (54). Lambert et al. (55) also reported that exposure of pregnant mice to Br₂ led to significant cardiac injury (to be reviewed in more detail in a subsequent section). Cumulatively these studies indicate that cardiovascular derangements caused by acute exposure to either Cl₂ or Br₂ may lead to cardiac dysfunction, hypotension, and even biventricular failure.

Halogens and the Proteome

Addis et al. (84) used a combination of proteomics analysis of lung tissues along with a systems biology analysis to identify the lung proteome of mice exposed to Br₂ and returned to room for 24 h. They reported alterations in proteins coinciding with regulation of three processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability (FIGURE 4). Similarly, confluent monolayers of lung cells in primary culture exhibited zona occludens-1 dissociated from cell wall localization, increased phosphorylation, internalization of E-cadherin, and increased actin stress fiber formation 24 h post-exposure to Br₂ exposure, consistent with increased permeability.

FIGURE 4.

Global lung protein changes 24 h post-Br₂ exposure. Adult male C57BL/6 mice, 8–10 wk old, were exposed to Br₂ gas (600 ppm for 30 min) or air in environmental chambers and returned to room air Twenty-four hours later, their lungs were removed and proteins were processed for global proteomics analysis. A: Venn diagram demonstrating the total number of proteins identified across both groups in addition to those proteins found to be significantly changed following exposure to Br₂ (increased vs. the other group). B: volcano plot of the log₁₀(P) value vs. log₂ fold change (Br₂/air) demonstrating the distribution of the entire data set of proteins with upper limits (above the line) indicating statistically significant changes and outer limits (to the right and left of each line) indicating significant fold changes as outlined in materials and methods under statistics. Note that although fold change is visualized as log2, the cutoff value of ±1.5 was applied to the fold change before logging, thereby yielding the indicated ±0.6 limits. Various proteins that play a role in vascular permeability are identified by the arrows (from Ref. 84).

Mechanisms of Halogen Toxicity

Inhaled halogens exert their toxicity by 1) direct interaction of Cl₂ and Br₂ with important cellular targets in the epithelial lining fluid or on the surface of lung airway and epithelial cells; 2) by generating long-acting toxic intermediates capable of reaching sites (such as the heart and placenta) which are beyond their mean free paths; and 3) by increasing levels of reactive oxygen and nitrogen species which promote an exuberant inflammatory response.

Direct Interactions of Halogens with Biological Targets

The toxicities of halogens have been mainly attributed to the reactivities of HOCl and HOBr, generated during the reaction of Cl₂ and Br₂ with water as detailed in the introduction. HOCl and HOBr, like Cl₂ and Br₂, are strong oxidants and electrophiles. HOCl is a stronger oxidant and a weaker electrophile, while Br₂ is a stronger electrophile and a weaker oxidant of the two, which is possibly behind differences in their toxic effects (85). HCl and HBr are neutralized by bicarbonate in the epithelial lining fluid (ELF) (86, 87) and probably do not play a major role in halogen toxicity. Prior studies illustrate that Cl₂ gas is more toxic to the lung than aerosolized HCl (65). However, aspiration of the stomach contents or instillation of large quantities of HCl at pH <2 are known to damage the blood gas barrier and result in acute and chronic lung injury via neutrophil dependent mechanisms (24, 88, 89).

Based on the reaction rate constants and the large concentrations of ascorbate and reduced glutathione in the ELF, Cl₂ and Br₂ will react with ascorbate and reduced glutathione, which exist in mM concentrations in the ELF, before being converted to HOCl (48, 90). Exposure of rats to Cl₂ decreases ascorbate in bronchoalveolar lavage fluid and lung tissues (49, 91); furthermore, aerosolized and intramuscular injections of ascorbate and deferoxamine, an iron chelator, in rats and mice post-Cl₂ exposure replenished lung ascorbate stores and decreased lipid peroxidation, airway hyperresponsiveness inflammation, the accumulation of plasma proteins in the alveolar space, and airway thickening and hyperplasia, and mortality (41, 49, 59) (FIGURE 3). Other antioxidants such as dimethylthiourea (92) and N-acetylcysteine (NAC) (93) have also shown some effectiveness in reducing various indices of lung injury (but not mortality) when administered in mice either pre or post-Cl₂ exposure. NAC also potentiated the effects of corticosteroids (94) as well as ascorbate and deferoxamine (49) in decreasing Cl₂ toxicity. In addition, AEOL 10150, a compound that scavenges peroxynitrite, inhibits lipid peroxidation, and has superoxide dismutase and catalase-like activities, diminished airway hyperresponsiveness and lung injury in Cl₂-exposed mice (63). The results of various countermeasures in mitigating the extent of halogen injury to adult and newborn animals are shown in Table 1 and FIGURE 3.

Table 1.

Mitigation of Cl₂ and Br₂ injury in animals by various classes of compounds

Target Pathway(s)/Agents	Reference No.	Species	Halogen	Variables Affected
Antioxidants
N-acetylcysteine	(93)	Isolated perfused lungs; rats	Cl2	Cytokines, airway contraction. permeability
Dimethylthiourea	(92)	Mice	Cl2	Permeability. lipid peroxidation
AEOL 10150	(63)	Mice	Cl2	Airway hyperreactivity. Permeability. inflammatory cells
Ascorbic acid plus deferoxamine	(41, 59)	Mice	Cl2	Survival, permeability. airway hyperplasia. inflammatory cells
Anti-inflammatory
Corticosteroids	(95)	Pigs	Cl2	Airway pressures. arterial blood gases. lung wet/dry weights
cAMP modulation
Forskolin	(96)	Mice	Cl2	Na+ channel activation
Rolipram	(50)	Mice	Cl2	Pulmonary edema, airway hyperreactivity
β2-Agonists	(57)	Mice	Cl2	Airway hyperreactivity, inflammation, cytokines
NO modulation
Nitrite	(42, 80, 97)	Rats, mice, rabbits	Cl2	Permeability, inflammation, survival, inflammatory cell accumulation, airway reactivity, vessel dilation
Heme and coagulation
Aerosolized heparin	(44)	Mice	Cl2	Coagulation, permeability, inflammatory cells
Hemopexin	(68, 74)	Mice	Cl2 and Br2	Permeability, inflammatory cells, airway hyperreactivity, survival, Na+ channels, wet/dry weights, pulmonary emphysema, fibrosis
TRP ion channels
TRPV4 inhibitors		Mice	Cl2, HCl	Pulmonary edema, oxygen saturation, inflammatory cells, cytokines, permeability
SERCA activators
Ranolazine	(98)	Rats, cardiomyocytes	Cl2	Prevented cardiomyocyte death in rats, prevented mitochondrial injury in cells
Hyaluronan
Yabro	(58, 99)	Mice, human cells	Cl2 and Br2	Airway hyperreactivity, permeability, inflammatory cells, membrane potential, RhoA, calcium sensor
Type 5-phosphodiesterase inhibitors
Tadalafil	(5, 55)	Pregnant mice	Br2	Increased survival, fetal growth, systemic blood pressure, inflammation, decreased placental injury
VEGF
VEGF-121	(83)	Pregnant mice	Br2	Lung permeability, lung wet/dry weights, survival, decreased placental injury, fetal growth development

NO, nitric oxide; TRP, transient receptor potential; SERCA, sarcoendoplasmic Ca²⁺ ATPase.

Important targets of Cl₂, HOCl, and OCl⁻ include proteins and amino acids like cysteine (100, 101), methionine, side-chain amino groups in amino acids, terminal amino groups (creating corresponding chloramines) (102), and aromatic amino acids like tyrosine (creating 3-chlorotyrosine) (103–105). Chloramines are intrinsically toxic and cause injury directly as well as synergizing to exacerbate the Cl₂/HOCl/OCl⁻ damage (47, 98, 106–109). HOCl activates the mitogen-activated protein kinase pathway, and the cell-permeable glycine and taurine chloramines regulate NF-κB activity through oxidation of IκBa (110, 111). The consequent release of chemokines and cytokines by inflammatory cells stimulates the secondary release of millimolar concentrations of HOCl, generated through catalytic activity of eosinophil and neutrophil-derived peroxidases with Cl⁻ and H₂O₂ functioning as substrates (112). HOCl also damages DNA and impairs poly-ADP-ribose polymerase, a DNA-repair enzyme (113, 114), thus impairing the repair of damaged DNA.

Contributing to the overall pro-oxidant environment, exposure of mice to Cl₂ gas upregulates the inducible form of nitric oxide synthase (iNOS) in alveolar epithelial cells and macrophages (65). However, as mentioned previously, NO derived from iNOS may be essential in maintaining blood pressure following Cl₂ induced injury to eNOS. In addition, one study reported that exposure of iNOS^(−/−) mice to Cl₂ does not blunt the hyperresponsiveness of airway resistance to methacholine as compared with wild type mice (57).

Pulmonary surfactant, consisting of lipids and four specific protein (SP-A, B, C, and D), is a critical Cl₂ target. Higher minimum surface tensions of concentrated bronchoalveolar lavage fluid were measured post-Cl₂ exposure in rats (49), although levels of surfactant lipids were normal. These findings are consistent with injury to the lipid soluble surfactant apoproteins SP-B and SP-C, which are responsible for interacting with surfactant lipids to generate a minimum surface tension while surface area decreases, thus preventing alveolar collapse. Damage to SP-B and SP-C has been reported following exposure of rabbits to hyperoxia (115, 116) and other oxidants (116) as well as exposure of surfactant mixtures containing surfactant lipids and SP-B and SP-C to peroxynitrite (117), HOCl, and Fenton reagents (118). Increased levels of plasma proteins in the ELF due to increased permeability of the blood gas barrier following exposure to halogens (2, 48, 49, 68, 79) may further interfere with the ability of pulmonary surfactant to reach a minimum surface tension (53, 119). Damage to the pulmonary surfactant system will result in hypoxemia and may contribute to the development of ARDS (116). Exposure of surfactant protein A (SP-A) to HOCl or peroxynitrite led to oxidation, nitration, and chlorination of amino acids found within the carbohydrate recognition domain, impairing mannose residue binding, a step required for sequestration and elimination of pathogens (69, 120, 121). Surfactant protein D (SP-D), like SP-A, is a lectin with a variety of important roles in the innate immune response and maintenance of physiology respiratory homeostasis. Exposing SP-D to HOCl prevented pathogen aggregation and led to generation of abnormal disulfide cross-linked oligomers (122, 123). Therefore, it is reasonable to conclude that after Cl₂ exposure, mammals are at increased risk of bacterial pneumonia due to a compromised innate immune response.

Active transport of Na⁺ ions down the electrochemical gradient generated by the Na/K/ATPase from the epithelial lining fluid to the interstitium is an important function of a normal functioning alveolar epithelium (124–126). Sodium (Na⁺) ions enter epithelial cells through amiloride-sensitive (ENaC) or cation channels located in their apical membranes and are extruded into the interstitium by the basolateral Na/K/ATPase. To maintain electroneutrality, Cl⁻ ions passively move across paracellular junctions of alveolar epithelial cells or transcellularly. Vectorial ionic movement of Na⁺ and Cl⁻ ions is followed passively by fluid from the airspace to the interstitium [the physiological concept of airspace fluid clearance (AFC)]. This is necessary to allow the reabsorption of pulmonary edema fluid (reviewed in Refs. 126, 127). Patients with acute lung injury with intact AFC have lower mortality and lower hospital stays as compared with those with compromised AFC (128, 129).

A variety of studies have shown that halogens damage the activity of amiloride-sensitive Na⁺ channels either directly or by upregulation of signal transduction pathways. Direct recordings of single Na⁺ channels activity in alveolar type I and type II cells in the lungs of mice at 1 and 24 h post-exposure to either Cl₂ or Br₂ in their native environment (lung slices) showed significant injury to ENaC (74, 96), as evidenced by decreased single channel activity. Exposure of alveolar type II (ATII) cell monolayers to Cl₂ increased levels of reactive intermediates, leading to ERK1/2 phosphorylation and decreased apical ENaC and transepithelial Na⁺ transport; these changes were prevented and reversed by inhibitors of ERK1/2 and of the proteasomes and lysosome systems (96). Chloramines formed by the reactions of HOCl with taurine inhibited ENaC activity across Xenopus oocytes injected with the three ENaC subunits (47). These data indicate that halogens inhibit ENaC and may contribute to the development of pulmonary edema seen posthalogen exposure. Agents that upregulate cAMP levels, such as rolipram (50), β-agonists (57), forskolin, and cAMP (47, 96), increase Na⁺ transport, decreased pulmonary edema in Cl₂-exposed mice by increasing ENaC levels and activity. Agents that increase cAMP also improve airway hyperresponsiveness by relaxing airway smooth muscles. These finding explain the beneficial role of β-agonists in persons exposed to halogens.

Reactive Species: Mitochondrial Bioenergetic Dysfunction

Mitochondria are key regulators of cell survival in response to stress, and this has stimulated the development of mitochondrially targeted therapies (130–132). The exposure of cells to reactive oxygen species (ROS) generated during inflammation decrease mitochondrial quality, as evidenced by damage to the respiratory chain, damage to mitochondrial DNA, and increased generation of ROS (133–138). The continual generation of reactive oxygen species by the respiratory chain may promote proinflammatory signaling and thus prevent cellular recovery (134, 139, 140) along with activating a number of danger signals, which play a critical pathogenic role by activating downstream inflammatory cascades. Progressive injury to mitochondria results in the opening of the mitochondrial permeability transition pore, release of cytochrome c, and necrotic or apoptotic cell death (141–143). Maintaining mitochondrial quality control through selective removal of damaged, ROS-generating mitochondria, is an important property of macroautophagy known as mitophagy (130, 144). Proteins or organelles modified by reactive species are targeted for removal by the lysosomal-autophagy system (130, 145, 146).

In vitro studies showed that exposure of human club cell-like lung epithelial cells (H441) to Cl₂ gas decreased their maximal mitochondrial oxygen consumption rate (OCR) (43) and their bioenergetic reserve capacity, an index of cellular energy for repair and resistance to oxidative damage (138). Cl₂ also increased nonmitochondrial OCR, which represents an increased capacity to generate reactive oxygen species (43). Along these lines, Cl₂ increased superoxide production in the mitochondria of H441 cells (43) (FIGURE 5) and in primary cultures of alveolar type II cells (96) for up to 12 h post-exposure. Treatment of Cl₂-exposed H441 cells with MitoQ, an antioxidant targeted to the mitochondria), can attenuate the Cl₂-dependent reduction in maximal OCR (43). Cl₂ also reduced the mitochondrial membrane potential, and this effect was similarly attenuated following MitoQ pretreatment (43). Cl₂ significantly decreased the maximum extracellular acidification rate, which suggests a Cl₂-dependant impairment in glycolysis (44). Cl₂ specifically inhibits complex I and II of the mitochondrial electron transport chain (43). Increased reactive oxygen species were detected in lungs of Cl₂- and Br₂-exposed mice as shown by the presence of products of malondialdehyde adducts and free heme (22, 68, 74). In addition, F2a-isoprostanes (prostaglandin F2-like compounds derived from the nonenzymatic oxidation of arachidonic acid by chloramines or HOCl) were detected in lung tissue of rats exposed to Cl₂ (48).

FIGURE 5.

Production of secondary mediators A: exposure to Cl₂ increases mitochondrial superoxide/hydrogen peroxide. I–VI: H441 cells immersed in 50 µl of artificial epithelial lining fluid were exposed to 95% air-5% CO₂ as control (I–III) or Cl₂ (100 ppm for 15 min) (IV–VI) and returned to 95% air-5% CO₂ for 1 h. Before imaging with confocal laser microscopy, the cells were incubated with MitoSOX (red) for 15 min and MitoTracker (green). II and V: most of the Cl₂-exposed H441 cells showed higher levels of red fluorescence compared with controls. VI: MitoSOX fluorescence localized with MitoTracker (yellow), consistent with the presence of reactive species in mitochondria. Nuclei were counterstained with DAPI (blue). Quantitative analysis showed increased production of reactive intermediates at 6 h post-exposure (modified from Ref. 43, with permission from the publisher). B: plasmalogen-derived Cl₂ and Br₂ oxidation products. The vinyl ether bond of plasmalogens is targeted by Cl₂, Br₂, HOCl, and HOBr resulting in 2-chlorofatty(bromo)aldehyde production including 2-Cl(Br)-Pald and 2-Cl(Br)-Sald. The 2-chloro(bromo)fatty aldehydes are either oxidized to the 2-chloro(bromo)fatty acids and 2-Cl(Br)-PA and 2-Cl(Br)l-SA or reduced to the 2-chloro(bromo)fatty alcohols, 2-chloro(bromo)palmitoyl alcohol, and 2-chloro(bromo)stearoyl alcohol. Alternatively, nucleophilic attack of 2-chloro(bromo)fatty aldehydes by GSH results in either palmitaldehyde or stearaldehyde GSH adduct formation. R1 = C14H29 or C16H33 (modified from Ref. 147). C: effects of Cl-lipids on lung permeability and inflammation. C57bl/6 male mice were exposed to saline, ethanol, palmitate (PA), palmitic aldehyde (Pald), 2-Cl-PA, or 2-Cl-Pald by intranasal administration and lung injury was assessed by measuring total protein in the bronchoalveolar lavage levels of protein. Data are means ± SE (n = 4–6). *P < 0.05 relative to corresponding native fatty acid (from Ref. 147). BAL, bronchoalveolar lavage. D: plasma cell-free heme (CFH) and chlorinated lipids are elevated in humans and mice exposed to Cl₂ gas. Blood was collected from patients (pts) exposed to Cl₂ gas in the emergency room of the University of Alabama at Birmingham, 3–4 h post-exposure, stored for 72 h at 4°C at which time it was analyzed. Blood from human volunteers as controls was treated in the same fashion. I: plasma CFH levels in persons exposed to Cl₂ were higher than the age- and sex-matched human controls. II and III: Cl₂-exposed individuals also had elevated levels of 16ClFA (n = 4–5) (II) and 18ClFA (III). Similarly, adult male C57BL/6 mice exposed to Cl2 gas (400 ppm, 30 min) had increased levels of heme in plasma 24 h post-exposure. Individual values and means ± SE. *P < 0.05 vs. unexposed humans or air exposed mice; by unpaired t test (from Ref. 74). E: cardiac dysfunction and cardiomyocyte death with 2-bromohexadecanal (Br-HDA). I: left ventricular (LV) diastolic dysfunction was reflected by the decrease in mitral valve (MV) early and late wave velocities 4 h after injection of Br-HDA into the LV cavity. II: Br-HDA caused an increase in LV end-diastolic dimension and end-systolic dimension, resulting in a decrease in fractional shortening. Image demonstrates M-mode echocardiography of the LV before and 4 h after intracardiac injection of Br-HDA. III: Br-HDA caused extensive disruption of the cardiac cytoskeleton and loss of the normal highly organized linear mitochondrial sarcomere integrity. Transmission electron microscopy (×3,200 at top and middle and ×8,000 at bottom) demonstrated contraction band necrosis (red arrows), loss of I bands, and disruption of z-disk (yellow arrowheads) in the LV of rats administered Br-HDA as well as mitochondrial swelling (yellow arrows) and cristae lysis (red asterisk) (from Ref. 54). F: Br₂-exposed pregnant mice exhibit diminished cardiac function at gestational day 18.5 (E18.5). Nonpregnant and pregnant (E14.5) mice were exposed to air or 600 ppm Br₂ for 30 mi and returned to room air. Representative echosonography left ventricular (LV) and right ventricular (RV) traces at E18.5 of pregnant mice exposed to air (left) or Br₂ with demarcation of ventricular sizes (ED, end diastole; ES, end systole) (from Ref. 55).

Providing further evidence for mitochondrial injury of H441 cells by exposure to Cl₂, upregulation of autophagy by pretreatment with trehalose (which activates autophagy) improved bioenergetics function. Conversely, the administration of the autophagy inhibitor 3-methyladenine exacerbated the deterioration of bioenergetics after Cl₂ exposure. Critically, treatment of mice with trehalose decreased some manifestations of Cl₂-induced lung injury (43).

Injured epithelial cells produce mediators including reactive oxygen species, cytokines, and platelet-activating factor leading to recruitment of inflammatory cells (mainly neutrophils). Significant increases in a number of cytokines (such as TNFa, IL6, and KC) were observed in the plasma and lung tissues post-Cl₂ and -Br₂ exposures (57, 79). These cytokines stimulate release of arachidonic acid and result in eicosanoid production, further stimulating the secretion of mucus and exacerbating inflammation.

Mitochondrial injury may also mediate Cl₂-induced cardiotoxicity. As briefly mentioned previously, rats exposed to Cl₂ showed a decrease in ATP content in primary cardiomyocytes along with an increase in lactate within the coronary sinus indicative of increased myocardial anaerobic metabolism (98). Sedlic et al. (148) attributed this impaired mitochondrial function to Cl₂ reactants in the circulation as well as chlorination causing inactivation of cardiac sarcoendoplasmic Ca²⁺ ATPase (SERCA) with a corresponding cytosolic Ca^2 + overload. Increased cytosolic Ca²⁺ leads to a concurrent increase in the mitochondrial production of reactive oxygen species. Additionally, reactive oxygen species in turn cause disturbances in cytosolic Ca²⁺ homeostasis and may contribute to myocardial dysfunction by bromine (149). SERCA regulates Ca²⁺ homeostasis in the heart through transport of cytosolic Ca²⁺ into the sarcoendoplasmic reticulum (lowering intracellular Ca²⁺). SERCA activity is impaired by HOCL (98, 150). Notably, ranolazine (a SERCA stabilizer) and istaroxime (a SERCA activator) preserve ATP levels, preserve the membrane potential across the mitochondria, and, importantly, can prevent Cl₂-induced cardiomyocyte death after Cl₂ exposure (98). Similar results were reported following exposure of rats to Br₂ (FIGURE 1) (54).

Formation of Secondary Mediators: Halogenated Lipids

Plasmalogens are important components of plasma membrane phospholipids in mammals (151). They facilitate solvation of transmembrane ion channels and transport proteins and storage of arachidonic acid (152). A number of studies have shown that Cl₂ and Br₂ (along with the hydration products HOCl and HOBr) react with lung plasmalogens to form halogenated lipid aldehydes (147, 153). Major molecular species of the reactions include 2-(chloro)bromopalmitaldehyde (2-XPALD) and 2-(chloro)bromostearaldehyde (2-XrSALD), which are either reduced to alcohol or oxidized to 2-(chloro)bromomopalmitic acid (2-XPA) and 2-chloro(bromo)stearic acid (2-XSA), respectively (FIGURE 5). The fatty acids exist either in the esterified or free forms (154). 2-BrPA is a potent inhibitor of mitochondrial fatty acid oxidation (155) and of protein palmitoylation (156). Endogenously generated, myeloperoxidase-derived α-ClFALD has also been implicated in eNOS inhibition (157), neutrophil chemotaxis (158), and myocardial contractile dysfunction (159). Thus α-ClFALD may be responsible for the vascular eNOS inhibition reported in mice post-Cl₂ (45, 46) exposure. Decreased NO bioavailability was also reported in pregnant mice exposed to Br₂, which contributed to the subsequent development of a preeclamptic-like syndrome and higher incidence of fetal demise (5, 55, 83). Furthermore, incubation of lung cells with brominated lipids (fatty acids and aldehydes) resulted in activation of RhoA and its downstream kinase Rho-associated kinase 2 (ROCK-2), coinciding with significant increases in permeability as detected by measuring significant decreases of electrical resistance. The decreased barrier function was accompanied by formation of actin stress fibers, phosphorylation and internalization of VE-cadherin as well as disruption of ZO-1 localization (84). Hartman et al. (160) also reported that a synthetic analogue of chlorofatty acids localized in the Weibel-Palade bodies of human endothelial cells resulting in the release of P-selectin, von Willebrand factor, and angiopoietin-2 from endothelial cells . Chlorinated fatty acids also lead to platelet aggregation due to neutrophil adherence and contribute to an increase in endothelial permeability, a phenomenon related to the release of angiopoietin-2 (160).

Chlorinated and brominated fatty acids and aldehydes were detected in large quantities in the plasma, lung tissues, and bronchoalveolar lavage of mice and rats up to 24–48 h post-Cl₂ and -Br₂ exposure (79, 161) in the left ventricular tissues of rats exposed to Br₂ (54), as well as in the plasma of humans at 6 h post-exposure to Cl₂ (74) (FIGURE 5). Intranasal administration of 2-Cl-PA or 2-Cl-PALD at doses like those formed in the lung after Cl₂ gas exposure led to increased permeability of lung epithelial cells to plasma proteins, neutrophil influx, and systemic endothelial dysfunction characterized by loss of eNOS-dependent vasodilation (FIGURE 5) (147). Furthermore, injection of brominated aldehydes into the left ventricular cavity of an air breathing rats caused acute left ventricular enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage (54). These findings suggest that the halogen-induced mitochondrial injury may be mediated at least in part by halogenated lipids (FIGURE 5).

Chlorinated lipids have also been detected in the lung of patients with ARDS secondary to sepsis (162) and their levels correlated with the severity of clinical symptoms. In this case, HOCl originated from the action of neutrophil myeloperoxidase on H₂O₂ and chloride ions. However, the concentrations measured in persons exposed to Cl₂ (74) were a least 100-fold higher than what has been reported in patients with sepsis (162). Overall, these findings indicate that halogenated fatty acids may be both biomarkers of and contributing to the pathogenesis of halogen cardiopulmonary injury.

In addition to lung and cardiac cells, halogenated fatty acids are known to damage red blood cells, causing the release of free heme in the plasma. Heme is an essential functional group of many intracellular proteins. However, nonencapsulated cell-free heme (CFH), a breakdown component of proteins such as hemoglobin, myoglobin, cytochromes, is a significant source of reactive species, impairs cellular integrity (163), and is implicated in the pathogenesis of several disorders (164–170). CFH is an abundant source of redox-active iron capable of damaging lipids, proteins, and DNA (171) and causing cell necrosis (172). Under normal conditions, circulating CFH is maintained at low levels by serum albumin, haptoglobin, and most importantly hemopexin (173–175). Significant concentrations of nonencapsulated free heme are present in the plasma, lung tissue, and bronchoalveolar lavage of mice post-Cl₂ and -Br₂ exposure (68, 74, 79) (FIGURE 5). In addition, free heme was detected in the plasma of humans exposed to Cl₂ gas in a recent incident in Birmingham, AL at 6 h post-exposure (74) (FIGURE 5).

As mentioned above, it is highly unlikely that inhaled Cl₂ or Br₂ will be present in the plasma. This has led investigators to question whether chlorinated lipids are responsible for red blood cell (RBC) injury. Ex vivo incubation of RBC with chlorinated and brominated lipids followed by mechanical agitation resulted in the release of free heme (74). There was significant carbonylation of RBC membranes following incubation with halogenated lipids, as shown by oxyblot analysis (68, 74, 84). Studies of RBCs isolated from mice 24 h postbromine exposure identified six putative carbonylation sites in lysines within the spectrin α-chain and one site in the β-spectrin, while Cl₂ caused carbonylation only in the α-spectrin chain (74). Carbonylation of spectrin may form aggregates, which may destabilize the RBC membrane rendering RBCs more susceptible to mechanical stress as they cross capillaries (176). In a positive feedback loop, free heme can destabilize the RBC membrane by altering the conformation of cytoskeletal proteins, such as spectrin, protein 4.1 (177), and other cytoskeletal proteins (178, 179).

Two lines of evidence point to the potential importance of heme in the pathophysiology of halogen toxicity. First, mice overexpressing heme oxygenase-1 (HO-1), which catalyzes the first and rate-limiting step in heme degradation into equimolar amounts of iron, carbon monoxide (CO), and biliverdin, exhibit decreased mortality and lung injury when exposed to Br₂, while mice lacking HO-1 are highly susceptible to exposures to both Cl₂ and Br₂ (79). Second, injection of hemopexin, an endogenous plasma protein with a high affinity for heme, decreased lung heme levels and lung oxidative stress, ameliorating the associated tissue damage and respiratory dysfunction after both Br₂ and Cl₂ inhalation as well as the development of pulmonary fibrosis and emphysema, and prolonged survival posthalogen exposure (68, 74) (FIGURE 3).

Low-Molecular Weight Hyaluronan

Increased airway resistance and airway hyperresponsiveness (AHR) are important pathological events following exposure to Cl₂ and Br_2, and may result in persistent asthma-like symptoms and exacerbation of allergic airway inflammation (56, 57, 65), which may progress to lung fibrosis (60, 61, 68). Below we discuss some findings indicating that low-molecular-weight hyaluronan (LMW-HA), generated by the action of halogens, on high-molecular-weight hyaluronan (HMW-HA) may be an important mediator of halogen toxicity.

HMW-HA, a major structural component of the extracellular matrix, promotes cell survival and has antiangiogenic properties and anti-inflammatory effects on immune cells mediated by binding to its membrane receptors, CD44 TLR2, and TLR4 (180–183). On the contrary, the binding of LMW-HA (L-HA ∼300 kDa) fragments to the same receptors act as endogenous innate immune ligands and promote inflammatory responses, angiogenesis, and epithelial to mesenchymal transition (180, 181, 184, 185). LMW-HA fragments stimulate cytokine production and activate the innate immune response via binding to CD44 and Toll-like receptor (TLR) signaling in an MyD-88- and NF-κB-dependent fashion, whereas HMW-HA inhibits TLR-2 signaling in vitro and in vivo (185). Binding of either HMW-HA or LMW-HA to CD44 is enhanced by the inter-α-inhibitor (IαI), a serum protease inhibitor consisting of three polypeptides (180, 186–188). LMW-HA is both necessary and sufficient for the development of AHR after exposure to ozone (180, 189) and ischemia-reperfusion (190) among other injury patterns. LMW-HA leads to an increase in permeability through activation of RhoA and ROCK, triggering cytoskeletal reorganization, and through inhibition of cell-cell junctional integrity (191). Conversely, the administration of HMW-HA is protective in injury models including ozone exposure (189), bleomycin administration (192), smoke inhalation, and sepsis (193, 194).

In a series of experiments, Lazrak et al. (99) showed the presence of LMW-HA in the bronchoalveolar lavage fluid and the peribronchial spaces of C57BL/6 mice exposed to Cl₂ for up to 24 h post-exposure. The increase in LMW-HA was accompanied by an increase in hyalruonan synthases expression and presence of hyaluronidases. LMW-HA was also increased in bronchoalveolar lavage fluid of mice exposed to Br₂ (58). Instilling HMW-HA in the nares posthalogen-exposure decreased AHR (58, 99). Cl₂-induced AHR was mitigated by instillation of an antibody against the inter-α-trypsin inhibitor (IαI), which inhibited HA signaling (99). Mice infected with respiratory syncytial virus (RSV) develop an increased sensitivity to Cl₂, and this sensitivity was attenuated by high molecular weight hyaluronan (15).

In vitro studies demonstrated that exposure of human airway smooth muscle to Cl₂, Br₂, or to LMW-HA increased intracellular Ca²⁺ levels and RhoA activity_. This resulted in activation of the RhoA downstream kinase ROCK2 and activation of TMEM16, a Ca²⁺-activated Cl⁻ channel, resulting in membrane depolarization (58). Post-exposure administration of HMW-HA reversed these sequelae. Activation of RhoA leads to AHR, alters airway smooth muscle contractility, and leads to an increase in pulmonary vascular permeability (195–197). In subsequent experiments, it was shown that LMW-HA increased the expression of calcium-sensing receptor (Ca-SR) 24 h post-Cl₂ or -Br₂ (58). The effect of LMW-HA on Ca-SR was reversed when cells were treated with HMW-HA, added after the incubation with LMW-HA. Instillation of Ca-SR inhibitor (calcilytic) NPS2143, in the external nares of mice at 1 h and 23 h post-Br₂ exposure, resulted in normal airway responsiveness to methacholine (58). HMW-HA reversed the LMW-HA Ca-SR overexpression by either inhibiting downstream signaling cascades or by displacing LMW-HA from its receptors. The mechanisms involved are currently under investigation.

HMW-HA may be fragmented to LMW-HA by a variety of mechanisms. Reactive oxygen species (ROS), including superoxide, hydrogen peroxide, nitric oxide and peroxynitrite, hypochlorous, and hypobromous acids, are known to degrade HMW-HA (88, 99, 198–200). Inhaled ozone and Cl₂ can fragment HMW-HA, while neutralization of ROS, through superoxide dismutase or its mimetics, decreases HA degradation and resulting inflammation (201, 202). Whether halogenated lipids or nonencapsulated heme contributes to the fragmentation of hyaluronan has not been determined.

While generally the impact of the halogens is remarkably similar, it is interesting to note that although Cl₂ and Br₂ are both halogens, there are important differences in their physical and chemical properties and reactivity with biomolecules. To name just a few: 1) Br₂ is almost 10 times more soluble than Cl₂ and thus likely to react more readily on biological targets in upper airways; 2) chlorination of the plasmalogen vinyl ether bond occurs at acidic pH (203), while bromination occurs at neutral pH (204); 3) 2-bromofatty acids have a reduced charge in their carboxyl group compared with 2-chlorofatty acid due to the disparate electron withdrawing properties of these two halogens; and 4) while Br₂ exposure increases LMW-HA along with AHR through similar mechanisms as Cl₂ (119), the AHR observed after Cl₂ exposure is due to an increase in Newtonian resistance (large airway resistance) whereas the AHR seen after Br₂ exposure is related to tissue viscoelasticity and resistance (possibly related to small airway resistance) (58).

Toxic Effects on the Parturient

A detailed review of the impact of halogen gas exposure on the parturient was recently published (5). Vulnerable populations may demonstrate unique and specific sequelae after exposure to halogen gas. Up to 5% of women of reproductive age in the United States are pregnant or within 6 wk postpartum (205). Physiologic maternal adaptations begin early in pregnancy to prime the cardiopulmonary system for the demands of carrying a fetus to term, laboring, and the immediate postpartum period. These changes may create a milieu more susceptible to certain cardiopulmonary toxins. Fortunately, halogen exposure during pregnancy is rare; thus animal models are relied on to explore and understand these differential risks and sequelae unique to the parturient. Exposure of pregnant mice to Br₂ leads to a preeclamptic-like syndrome characterized by increased systemic and pulmonary pressures, endothelial dysfunction, a decrease in cardiac output, placental disruption, an increase in inflammatory cytokines, and fetal growth restriction (5, 55, 83) (FIGURES 1 AND 5). Exposed pregnant mice demonstrate an increase in the level of soluble fms-like tyrosine kinase 1 (sFlt-1) over time that corresponds to worsening pulmonary edema. Notably, pregnant halogen-exposed mice respond favorably to treatment with exogenous vascular endothelial growth factor (VEGF) and tadalafil (a phosphodiesterase-5 specific inhibitor) with increased maternal survival (FIGURE 3) and attenuation of pulmonary and cardiac injury (5, 55, 83). Neither exogenous VEGF nor tadalafil is efficacious in reducing injury in nonpregnant mice indicating that this is a unique phenomenon to pregnancy. The precise mechanisms by which halogen inhalation leads to placental hypoperfusion and injury, causes alterations in the ratio of sFlt-1:VEGFR1, and culminates in fetal demise have not been identified. Halogenated lipids, nonencapsulated heme, as well as the inflammatory response likely all contribute to this injury.

Halogens Impair Newborn Lung Development

Damage to the developing lung can lead to impaired development and permanent adverse pulmonary remodeling. In some cases, this could lead to early mortality or a lifelong increased risk of respiratory disease(s). In the single study in the literature, Jilling et al. (206) reported that exposure of newborn mice to bromine for 30 min at postnatal day 3 had impaired alveolar development characterized by alveolar simplification, decreased lung compliance, hypoxemia, and increased mRNAs of inflammatory cytokines in lung tissues. In addition, gene expression analysis of lung tissues revealed persistent abnormalities in gene expression profiles in genes involved in pulmonary development. Clearly, additional studies are needed to understand the mechanisms involved.

Conclusions

Exposure of humans and animals to the halogens Cl₂ and Br₂ damages the cardiopulmonary system and may, in severe cases, cause death from cardiopulmonary failure. Survivors may develop significant sequalae including pulmonary and cardiac fibrosis, increased compliance and alveolar simplification, and airway hyperresponsiveness. Pregnant animals are particularly vulnerable to halogens and develop preeclampsia even after a short exposure to halogens. Fetuses are born stillborn or with severe growth restrictions while newborns exposed to halogens develop alveolar simplification and bronchopulmonary dysplasia type symptoms. The toxic effects of halogens are exerted by oxidizing and carbonylating targets in the epithelial lining fluid and lung epithelial cells or by generating reactive intermediates and reactive species capable of reaching distant targets (such as the vascular system, heart, and placenta). Although several countermeasures demonstrate significant promise in reversing cardiopulmonary injury in animals, none of them have been approved for human treatment. Since clinical trials cannot be conducted, additional work is needed to satisfy Food and Drug Administration requirements under the Animal Rule.