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Chlorine (Cl2)-induced lung injury is a serious public health threat that may result from industrial and household accidents. Post-Cl2 administration of aerosolized ascorbate in rodents decreased lung injury and mortality. However, the extent to which aerosolized ascorbate augments depleted ascorbate stores in distal lung compartments has not been assessed.
We exposed rats to Cl2 (300 ppm for 30min) and returned them to room air. Within 15–30min postexposure, rats breathed aerosolized ascorbate and desferal or vehicle (mean particle size 3.3μm) through a nose-only exposure system for 60min and were euthanized. We measured the concentrations of reduced ascorbate in the bronchoalveolar lavage (BAL), plasma, and lung tissues with high-pressure liquid chromatography, protein plasma concentration in the BAL, and the volume of the epithelia lining fluid (ELF).
Cl2-exposed rats that breathed aerosolized vehicle had lower values of ascorbate in their BAL, ELF, and lung tissues compared to air-breathing rats. Delivery of aerosolized ascorbate increased reduced ascorbate in BAL, ELF, lung tissues, and plasma of both Cl2 and air-exposed rats without causing lung injury. Based on mean diameter of aerosolized particles and airway sizes we calculated that approximately 5% and 1% of inhaled ascorbate was deposited in distal lung regions of air and Cl2-exposed rats, respectively. Significantly higher ascorbate levels were present in the BAL of Cl2-exposed rats when aerosol delivery was initiated 1h post-Cl2.
Aerosol administration is an effective, safe, and noninvasive method for the delivery of low molecular weight antioxidants to the lungs of Cl2-exposed individuals for the purpose of decreasing morbidity and mortality. Delivery is most effective when initiated 1h postexposure when the effects of Cl2 on minute ventilation subside.
Exposure to chlorine (Cl2) represents a serious and underestimated threat to public health. The accidental mixing of bleach (sodium hypochlorite) with acidic solutions in household settings results in the generation of Cl2, which may cause severe bronchoconstriction especially in people with preexisting lung diseases.(1,2) Children and adults inadvertently exposed to Cl2 in swimming pool accidents (such as the recent malfunction of Cl2 delivery systems to a water park near Sacramento, CA, http://sanfrancisco.cbslocal.com/2011/08/15/20-hospitalized-from-chlorine-leak-at-sacramento-water-park/) experienced bronchoconstriction and lung function impairment lasting for several months.(3) Furthermore, the accidental release of large amounts of Cl2 in 30 large cities world-wide during the last 20 years (see, e.g., ref (4)) and the deliberate release of Cl2 during acts of terrorism by insurgents in the Iraq conflict,(5) caused significant mortality and morbidity to humans and animals. In addition to these public incidents, there were about 9000 calls for Cl2 related injuries to U.S. poison control centers each year from 2000–2005.(6)
The extent of Cl2-induced injury in humans and animals depends on the concentration and duration of exposure. Our theoretical analysis shows that Cl2 first reacts with antioxidants in the lung epithelial lining fluid (ELF).(1) When antioxidants are depleted, Cl2 hydrolyzes to form hypochlorous acid (HOCl) and its conjugate base (OCl−), which react with proteins, components of the extracellular matrix, and unsaturated fatty acids. Products of these reactions (chloramines, lipid hydroperoxides, and low molecular weight hyaluronic acid fragments) are considerably toxic. In previous studies we assessed the onset and development of lung injury in rodents exposed to Cl2 concentrations likely to be encountered in the vicinity of industrial accidents. A prominent finding, in agreement with our theoretical analysis, was the significant and sustained decrease of lung ascorbate, a key antioxidant in both rodents and humans.(2,7) This was followed by injury to respiratory and alveolar epithelial cells resulting in compromised ion transport, increased airway resistance and hyperreactivity, surfactant dysfunction, increased levels of protein in the alveolar space, pulmonary edema, and even death from respiratory failure.(2,5,7–11) In addition to pulmonary toxicity, Cl2 caused systemic injury and endothelial dysfunction, resulting from the inactivation of endothelial nitric oxide synthase in pulmonary arteries.(12)
These studies led to the exciting and novel concept that replenishment of lung ascorbate post Cl2 exposure decreases the extent of lung injury. In order for antioxidants to work efficiently, they must be delivered in such a fashion as to replete antioxidants stores in the lung epithelial lining fluid and tissues rapidly. Aerosols are an effective and noninvasive means of delivering antioxidants to large airways and distal lung spaces. However, bronchoconstriction as well as local edema may limit the effectiveness of aerosolized antioxidants. Herein, we exposed rats to sublethal concentrations of Cl2, returned them to room air, and assessed the efficiency of aerosolized ascorbate to deplete depleted ascorbate levels in lung epithelial lining fluid, lung tissues, and plasma if administered shortly after return to room air. Furthermore, we measured levels of H2O2 in the bronchoalveolar lavage (BAL) of rats following administration of ascorbate to test the possibility that ascorbate may act as a pro-oxidant by generating hydrogen peroxide (H2O2).(13–16) Our results demonstrate that inhalation of aerosolized ascorbate via nose-only inhalation is an effective means of delivering ascorbate to distal lung regions of Cl2- exposed rats without causing significant damage to the airway and alveolar epithelia.
Ascorbic acid for injection USP 500mg/mL without preservative (American Regent Inc., Shirlez, NY) and deferoxamine mesylate for injection USP 500mg/vial lyophilized (Hospira Inc., Lake Forest, IL) were used in the preparation of the solutions to be administered. All solutions were prepared in a Class II biosafety cabinet (Labonco Inc., Kansas City, MO). Only pyrogen free consumables were used. All glassware was heat treated accordingly. Dilutions of the ascorbic acid stock solution and the lyophilized deferoxamine were done with sterile and pyrogen free saline 0.9% 308 mOsmol/liter (Hospira Inc.). Solutions to be aerosolized were prepared with sterile deionized water (Hospira Inc.).
Male Sprague-Dawley rats (Harlan Inc., Indianapolis, IN), with a body weight between 200 and 250g, were used for all experiments. All rats were housed in the University of Alabama at Birmingham Animal Facility under standard conditions. Food (Purina rodent chow, Dyets Inc., Bethlehem, PA) and autoclaved water were provided ad libitum. All animal experiments were approved by the UAB Institutional Animal Care and Use Committee.
Rats were exposed to a mixture of 300 ppm Cl2 in air for 30min in environmental chambers and returned to room air as previously described.(7,8) Within 15–30min from the end of exposure, rats breathed a mixture of aerosolized ascorbic acid and deferoxamine (desferal) mesylate by nose-only inhalation for 1h. The nebulizer concentrations were 150mg/mL for ascorbate and 0.357mg/mL for deferoxamine. We opted to use a combination of ascorbate and desferal because at high concentrations ascorbate may act as a pro-oxidant by reducing transition metals, thus initiating a variety of radical reactions, including the formation of hydroxyl radicals through Fenton chemistry.(13) Deferoxamine decreases the pro-oxidant activity of ascorbate by chelating ferric ions and at the same time maintaining ascorbate in the reduced state and thereby ensuring that ascorbate is not oxidized during the aerosol administration. In another set of experiments, air- or Cl2-exposed rats were connected to the aerosol delivery system and breathed vehicle (water) under identical conditions. The aerosol delivery system has been described previously,(8) and is shown in Figure 1. Peripheral oxygen saturations were measured with a MouseOx Small Animal Oxymeter (STARR Life Sciences, Allison Park, PA) connected to a computer equipped with MouseOx software.(11) The sensor was placed on the rat tail.
Shortly (within 5min) after the completion of aerosol delivery, rats were anesthetized via intraperitoneal (i.p.) injection of 0.4mL diazepam (5mg/mL) (Hospira Inc.) and 0.4mL ketamine HCl (5mg/mL) (IVX Animal Health Inc., St. Joseph, MO). Depth of anesthesia was verified by lack of toe pinch reflex. The chest was opened and a 1.5mL blood sample was drawn from the left ventricle in a heparinized syringe. The lungs were then perfused with ice-cold saline until clear of blood, and lavaged with 8mL ice-cold saline, which was instilled and withdrawn twice. The BAL fluid was centrifuged at 200×g to pellet cells and debris and stored in ice. The lungs were then removed and large airways as well as nonpulmonary tissue were trimmed. Plasma, BAL and lung tissue were then processed for measurements of reduced ascorbate with high-pressure liquid chromatography using electrochemical detection, as described previously.(7,8) Most of the ascorbate is in the reduced form. The concentrations of total proteins in the BAL were measured with the Bradford method using the Bio Rad Quick Start™ protein assay kit. Standard curves were generated using bovine serum albumin (Bio Rad Laboratories, Hercules, CA) for each experiment.
We calculated the volume of the ELF by measuring the concentrations of urea in both the plasma and recovered pooled BAL fluid as previously described.(2,17) Briefly, ELF=(BAL)×[urea]BAL/([urea]Plasma−[urea]BAL), where ELF and BAL are the volumes of the ELF and of the BAL used for lavage (8mL) and [urea]BAL and ([urea]Plasma are the concentrations of urea in the BAL and plasma, respectively. We then calculated the concentrations of ascorbate and total protein in the ELF as follows:
where [Z]ELF is the concentration of ascorbate or protein in the ELF, BAL, and ELF are the volumes of BAL and EKL and [Z]BAL is the concentration of ascorbate or protein in the ELF.(2)
The concentration of hydrogen peroxide in BAL fluid was determined by using an Amplex Red hydrogen peroxide assay kit (Invitrogen Corp., Carlsbad, CA). The amount of hydrogen peroxide is quantified by the production of resorufin, which has a fluorescence emission maxium at 571nm. Quantification was done with at 540nm excitation and 590nm emission wavelength with a fluorescence plate reader (BMG Labrechnologies, Inc., Durham, NC 27703).
Statistical significance of multiple group means was assessed by ANOVA followed by the Bonferonni modification of the t-test. Group differences were considered to be statistically significant when p≤0.05. Data are represented as mean±standard error (SE) of the mean. The presence of normal distribution was tested using the Kolmogorov-Smirnov method and equal standard deviation between groups was tested using Bartlett's test. In cases where there was a significant difference of standard deviations between the tested groups, data was normalized by calculating their log10 values. Statistical differences based on calculations with normalized data are labeled. Significant changes in arterial oxygen saturation of aerosol administrated animals were determined with two-tailed paired t-test.
All rats survived the exposure to Cl2. Exposure to Cl2 resulted in significant decreases of peripheral oxygen saturations (Fig. 2), which returned toward baseline in rats receiving either antioxidants (AA) or vehicle. Aerosol administration caused a small but nonsignificant decrease of oxygen saturations in air-breathing rats.
Typical particle size distribution data for aerosolized AA are shown in Figure 3. The mean mass median aerodynamic diameter (MMAD), and mean geometric standard deviation (GSD) were 3.3±0.1μm and 1.9±0.2, respectively, consistent with our previous studies employing aerosol administration.(8)
In all studies, we measured reduced ascorbate only. In Cl2-exposed rats, a fraction of ascorbate in the BAL, ELF, lung tissue, and plasma may have been oxidized by Cl2, HOCl, and their reactive intermediates to dehydroascorbate. It would be difficult to measure dehydroascorbate because its half-life in the ELF is less than 10min and its steady-state concentration is insignificant compared to ascorbate. The ascorbate that is oxidized to dehydroascorbate decomposes to various other products that by definition do not contribute to “total ascorbate” (ascorbate+dehydroascorbate). Ascorbate may also be transported from the surface of the lung to various tissues. Thus, measurements of ascorbate in Cl2-exposed rats may underestimate the total ascorbate delivered.
As shown in Figure 4, air-breathing rats not receiving aerosols had mean ELF volumes of 112±22μL; ELF values of air-breathing rats receiving aerosolized vehicle (water) or AA for 60min and sacrificed shortly after were not statistically different from this value. Similarly, aerosolized AA did not increase the concentrations of proteins in the ELF (Fig. 5); rats exposed to Cl2 had significantly higher ELF volume and protein values, which were unchanged by aerosolized delivery of AA (Figs. 4 and and5).5). These findings indicate that aerosolized AA did not cause significant damage to the airway and alveolar epithelial of normal and Cl2-exposed rats.
As shown in Figures 6, ,7,7, and and8,8, aerosol delivery of AA in air breathing rats resulted in significant augmentation of ascorbate levels in BAL, ELF, and lung tissue. Aerosol delivery 15–30min postexposure also increased the corresponding variables in Cl2-exposed rat lungs, albeit at much lower levels, most likely due to depressed ventilation and bronchoconstriction. However, aerosolized AA restored depleted values of ascorbate to their normal controls. In addition, aerosol administration of AA increased ascorbate in the plasma (Fig. 9).
The inhaled ascorbate dose (ID) was calculated by the following equation as described in the online supplement of ref. (8): ID=CT×VE×T, where CT=aerosol concentration in the plenum, that is, inhaled concentration (mg/L), VE=minute ventilation (L/min), and T=aerosol delivery time (min). The concentration of inhaled ascorbate in the plenum was calculated by the weight gain of a filter placed in an empty plenum port during aerosol delivery (corrected for the weight of other solutes (NaCl and preservatives) in the solution(18)), averaged 2.8mg/L. Measured values for VE for normal, conscious, spontaneously breathing rats vary from 60 to 45mL/mim/100g body weight,(19,20) which agree well with the predicted values for VE based on body weight (VE=2.1×BW,g)0.75. (21) Thus, assuming a mean body weight of 220g and a mean VE of 0.130 L/min, we calculated an average ID of 22mg, which corresponds to approximately 124,000nmol of inhaled ascorbate (based on a MW of 176). The amounts of ascorbate present in the ELF and lung tissues of air breathing rats (i.e., the difference values among air breathing rats receiving AA and vehicle) shortly after the completion of aerosol deliveries were 5136 and 1065nmol, respectively. Thus, the total amount of ascorbate delivered in distal lung tissues (6201nmol) was about 5% of the inhaled dose. This value is an underestimate, because plasma ascorbate increased by 1000nmol (based on a rat plasma volume of about 10mL) as well, due to the diffusion of ascorbate from the lungs into plasma and eventually into systemic tissues (Fig. 9). Based on the mean MMAD of our aerosolized particles (3.3μm)(8) and existing information in the literature,(22) we calculated that approximately 8% of the inhaled ascorbate should be deposited in the distal lung regions of rats, which agrees well with our experimental findings.
In Cl2-exposed rats, assuming a 50% drop in VE based on measurements of respiratory frequency shortly after return to room air,(18) the corresponding values are: ID=11mg (62,000nmol); ELF=200 nmol; lung tissue=400 nmol; % delivered in the distal lung=1%. This was sufficient to normalize depleted ascorbate levels in the ELF and lung tissues. The remaining inhaled ascorbate was most likely deposited in the external nares and upper airways. As mentioned above, this measurement is an underestimate, because plasma ascorbate increased by 1000nmol as well, reflecting diffusion of ascorbate from the lungs into plasma and eventually into systemic tissues. In addition, in Cl2-exposed rats, a fraction of ascorbate was probably oxidized to dehydroascorbate and was not detected by our method. In the next series of experiments we tested the hypothesis that delivery to the distal lungs can be improved by delaying the onset of aerosol delivery to 1h postexposure. These rats also breathed aerosolized ascorbate and desferal through a nose only exposure for 60min, euthanized shortly thereafter and their lungs lavaged in the same fashion. As shown in Figure 10, the ascorbate concentration the BAL was 10-fold higher when compared to the corresponding value when aerosol administration was initiated within 15–30min postexposure. This was most likely due to increased ventilation, decreased bronchoconstriction, and partial resolution of nasal congestion as well as less oxidation of ascorbate.
The concentrations of H2O2 in Cl2-exposed rats that breathed vehicle or ascorbate starting at 15–30min postexposure for 60min were 33±19 and 122±20 pM (mean±1 SE; n=6 and 4, respectively; p<0.05). Only background levels of H2O2 (<10 pM) were detected in the BAL of rats breathing vehicle.
The severity of Cl2-induced lung injury varies with the level and duration of exposure.(1,2,23) People and animals that inhale Cl2 at concentrations less than 50 ppm developed reversible increased mucous production and airway resistance and a decreased respiratory rate most likely due to the activation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channels, located in a subset of airway sensory neurons.(24,25) At higher concentrations Cl2 penetrates into distal lung regions(26) and interacts with critical components of the pulmonary surfactant system, as well as with alveolar epithelial cells resulting in severe hypoxemia, pulmonary edema, and even death from respiratory failure.(5,7,9,10,27–29)
Precise measurements of Cl2 concentrations at the accident scene are not possible. In the Graniteville, SC, accidents,(4) average Cl2 concentrations during a 30-min exposure period were estimated to be 4428, 550, and 161 ppm at 0.2, 0.5, and 1km downwind from the epicenter of the Cl2 release (Dr. Eric Svendsen, Tulane University; personal communication). Most people and animals exposed within 0.5km of the epicenter developed acute lung injury but eventually recovered. Thus, to mimic sublethal injury, in our previous work we exposed rats and mice to Cl2 concentrations of 187–400 ppm for 30min and returned them to room air. Rats and mice developed hypoxemia, increased alveolar permeability to plasma proteins, loss of surface and tissue ascorbate, decreased ability to clear fluid, functional surfactant deficiency, pulmonary edema, and reactive airway disease syndrome starting at 1h postexposure.(7,11,30,31) More than 90% were alive at 7 days post-Cl2 exposure and exhibited normal behavior; however, physiological and quantitative morphological measurements revealed the presence of severe airway hyperreactivity,(30) mucous metaplasia and airway thickening(18) up to 7 days postexposure.
On the other hand, mice exposed to 600 ppm Cl2 for 45min developed symptoms consistent with the presence of adult respiratory distress syndrome starting at 2h post-Cl2 exposure. About 75% of the mice died within 36h postexposure. Surviving mice had considerable injury to their alveolar epithelium.(8) Humans exposed to equivalent Cl2 concentrations in Graniteville, Iraq, and in other accidents developed similar symptoms with considerably mortality.(4,5,32) A recent study also pointed out that the physiological sequelae of Cl2 exposure deviate from the Haber's law postulate, necessitating that that evaluation of countermeasures against chlorine-induced lung injury should be performed in multiple types of exposure scenarios.(33)
Currently, there is no treatment specific for Cl2-exposed persons. Supplemental oxygen (to alleviate hypoxemia)(23) and administration of β2 agonists and agents that increase cAMP (to decrease bronchoconstriction and inflammation and enhance alveolar fluid clearance)(30,31,34) have been used to treat both animals and humans exposed to Cl2. In addition, agents that inhibit TRPA1 channels prevent the Cl2-induced decrease of respiration in mice after low concentration exposures.(35)
Exposure of rats to Cl2 resulted in significant depletion of ascorbate in their epithelial lining fluid and lung tissues(8,18) and increased levels of F2-isoprostanes in their lung, a reliable index of lipid peroxidation,(11) indicating the presence of reactive intermediates after the cessation of Cl2 breathing. Similarly, increased levels of reactive intermediates were detected in alveolar epithelial type II cells exposed to Cl2 in vitro (as evidenced by Electron Paramagnetic Resonance and the oxidation of redox-sensitive dyes).(36) Thus, postexposure administration of ascorbate, an effective hydrophilic antioxidant that also reduces Vitamin E radicals (generated during reduction of lipid hydroperoxides or peroxyl radicals (1,37)) and decreased the generation of reactive intermediates by inhibiting endothelial cell NADPH oxidase,(38) and prevented tumor necrosis factor-alpha (TNF-α) challenge-related apoptosis of endothelial cells in vitro,(39) seems a viable strategy for decreasing Cl2 induced morbidity and mortality. Furthermore, because the primary sites of Cl2 injury are the airway and alveolar epithelia, inhalation of aerosolized antioxidants appeared an effective and noninvasive means of delivering ascorbate to the Cl2-exposed lungs. Although a large fraction of inhaled ascorbate was probably trapped in the external nares, sufficient levels reached the upper airways and distal lung regions to restore depleted levels of ascorbate in both the epithelial lining fluid and lung tissue. Furthermore, our data clearly demonstrate that significantly higher levels of ascorbate may be delivered to distal lungs if aerosol administration is delayed to 1h postexposure, a likely scenario in mass casualty situations.
Ascorbate may also act as a pro-oxidant.(13) Our data clearly demonstrate the presence of small amounts of H2O2 (<100 pM) in Cl2-exposed rats following ascorbate administration. H2O2 becomes a cosubstrate for specific peroxidases and thereby generates reactive centers that in turn react with critical protein thiols forming disulfide linkages in key signaling elements. Fe+3, likely to be present in the epithelial lining due to Cl2-induced cell injury, in conjunction with excess H2O2, may result in the formation of hydroxyl radicals, which can cause extensive tissue injury.(40) Hydrogen peroxide may also trigger various signaling pathways.(40) Thus, we opted to coadminister desferal with ascorbate, which will scavenge trivalent ions, and minimize any pro-oxidant effects of ascorbate while at the same time prevent hydroxyl radical production via Fenton reactions.
Based on measurements of ELF volume and protein content, we concluded that aerosolized antioxidants did not damage the distal lung epithelium of normal and Cl2- exposed rats at 1h postexposure, attesting to the safety of the procedure. In previous studies(18) using quantitative morphological techniques, we found that administration of aerosolized ascorbate and desferal to air breathing rats did not alter the number of ciliated cells or epithelial thickness in large airways up to 7 days postexposure. Intravenous injection of up to 110g of ascorbate a day in patients with severe burns decreased the extent of lung injury and caused no adverse effect. Others have reported that doses of 5g/kg body weight of ascorbate are safe and well tolerated by patients with severe burns.(41) However, the safety of combined aerosolized administration of ascorbate and desferal in humans has yet to be demonstrated. Although each agent is FDA approved for human use, the combination of the two via aerosols is not. Previously, we have shown that aerosolized ascorbate did not increase airway resistance in response to methacholine challenge up to 7 days postexposure.(18) Additional studies are needed to show safety in humans.
In conclusion, data presented here show that aerosolized administration of ascorbate in rats post-Cl2 exposure increases ascorbate levels in the ELF, lung tissue, and plasma without damaging airway and alveolar epithelia. Based on the results of our studies, we concluded that administration of aerosolized ascorbate is most effective when initiated at least 1h postexposure. In humans, because of the larger diameter of their airways, about 60% of inhaled ascorbate will reach the distal lung spaces.(22) Because of the higher deposition fraction of inhaled ascorbate and the larger minute ventilation of humans (about 6 L/min), considerable shorter inhalation periods (10–20min) are need to deliver sufficient levels to replete depleted ascorbate levels in distal lung spaces of human following exposure to Cl2 regiments likely to be encountered within 0.5–1 mile from the epicenters of industrial accidents (200–600 ppm for 30min). Thus, aerosolized antioxidant delivery, especially coupled with β2 agonists to decrease airway resistance,(30) may be a viable strategy for the decrease of lung injury in persons exposed to Cl2.
This research was supported by the CounterACT Program, National Institutes of Health, Office of the Director, and the National Institute of Environmental Health Sciences, Grant Number U54ES017218 and 5U01ES015676-05.
The authors declare that no conflicting financial interests exist.