Transcriptomic Characterization of Inhalation Phosphine Toxicity in Adult Male Sprague-Dawley Rats.

Phosphine (PH3) is a highly toxic, corrosive, flammable, heavier-than-air gas that is a commonly used fumigant. When used as a fumigant, PH3 can be released from compressed gas tanks or produced from commercially available metal phosphide tablets. Although the mechanism of toxicity is unclear, PH3 is thought to be a metabolic poison. PH3 exposure induces multiorgan toxicity, and no effective antidotes or therapeutics have been identified. Current medical treatment consists largely of supportive care and maintenance of cardiovascular function. To better characterize the mechanism(s) driving PH3-induced toxicity, we have performed transcriptomic analysis on conscious adult male Sprague-Dawley rats following whole-body inhalation exposure to phosphine gas at various concentration-time products. PH3 exposure induced concentration- and time-dependent changes in gene expression across multiple tissues. These gene expression changes were mapped to pathophysiological responses using molecular pathway analysis. Toxicity pathways indicative of cardiac dysfunction, cardiac arteriopathy, and cardiac enlargement were identified. These cardiotoxic responses were linked to apelin-mediated cardiomyocyte and cardiac fibroblast signaling pathways. Evaluation of gene expression changes in blood revealed alterations in pathways associated with the uptake, transport, and utilization of iron. Altered erythropoietin signaling was also observed in the blood. Upstream regulator analysis identified several therapeutics predicted to counteract PH3-induced gene expression changes. These include antihypertensive drugs (losartan, candesartan, and prazosin) and therapeutics to reduce pathological cardiac remodeling (curcumin and TIMP3). This transcriptomics study has characterized molecular pathways involved in PH3-induced cardiotoxicity. These data will aid in elucidating a precise mechanism of toxicity for PH3 and guide the development of effective medical countermeasures for PH3-induced toxicity.

The Cardiopulmonary Effects of Sodium Fluoroacetate (1080) in Sprague-Dawley Rats.

Sodium fluoroacetate (1080) is a highly toxic metabolic poison that has the potential because of its lack of defined color, odor, and taste and its high water solubility to be intentionally or unintentionally ingested through food adulteration. Although the mechanism of action for 1080 has been known since the 1950's, no known antidote exists. In an effort to better understand the cardiopulmonary impacts of 1080, we utilized whole-body plethysmography and telemeterized Sprague-Dawley rats which allowed for the real-time measurement of respiratory and cardiac parameters following exposure using a non-invasive assisted-drinking method. Overall, the animals showed marked depression of respiratory parameters over the course of 24 hours post-exposure and the development of hemorrhage in the lung tissue. Tidal volume was reduced by 30% in males and 60% in females at 24 hours post-exposure, and respiratory frequency was significantly depressed as well. In telemeterized female rats, we observed severe cardiac abnormalities, highlighted by a 50% reduction in heart rate, 75% reduction in systolic blood pressure, and a 3.5-fold lengthening of the QRS interval over the course of 24 hours. We also observed a reduction in core body temperature of nearly 15°C. Our study was able to describe the severe and pronounced effects of sodium fluoroacetate poisoning on cardiopulmonary function, the results of which indicate that both tissue specific and systemic deficits contribute to the toxicological progression of 1080 intoxication and will need to be accounted for when developing any potential countermeasure for 1080 poisoning.

Changes in murine respiratory dynamics induced by aerosolized carfentanil inhalation: Efficacy of naloxone and naltrexone.

Carfentanil (CRF) is an extremely potent opioid capable of inducing fatal respiratory depression. Naloxone (NX) and naltrexone (NTX) are opioid antagonists for which the efficacy against CRF remains largely unexplored. In this study, the effects of aerosolized CRF on respiratory function were investigated using adult male CD-1 mice. Mice were exposed to 0.4 mg/m3 of CRF for 15 min using custom whole-body plethysmograph units. Minute volume (MV), respiratory frequency (f), duty cycle (DC), and tidal volume (TV) were monitored and compared to control animals exposed to aerosolized H2O. CRF exposure induced respiratory depression, characterized by a marked decrease in MV, which was sustained throughout 24 h post-exposure. Prophylactic and therapeutic treatment with intramuscular (i.m.) NX marginally improved MV, with slight dose-dependent effects. Analogous treatment with i.m. NTX returned MV to baseline levels, with all doses and intervention times performing similarly. Despite improvements in MV, treatment administration did not reverse changes in DC, a measure of respiratory timing. Overall, NX and NTX administration alleviated volumetric aspects of opioid-induced respiratory toxicity, while changes in respiratory timing remained unresolved throughout post-exposure observation. These sustained changes and differences in recovery between two aspects of respiratory dynamics may provide insights for further exploration into the underlying mechanism of action of opioids and opioid antagonists.

Pulmonary toxicity following inhalation exposure to VX in anesthetized rats: Possible roles for compromised immunity and oxidative stress-induced lung injury.

The nerve agent VX is one of the most deadly threat agents available in weapons stockpiles for intentional release. While mostly considered a percutaneous toxicant, it can be fatal when aerosolized. The objective of this study was to investigate toxic responses in the lung up to two weeks following a single 10-minute exposure to inhaled VX. Anesthetized rats were exposed singly and only once to VX. The nebulization rate in this system was 0.2-0.3 ml per minute with the delivery of a consistent particle size of 2.1 µm. Following exposure, all rats were removed from the ventilator and allowed to recover in the glovebox for 10-15 minutes. Results showed that inhaled VX altered several respiratory parameters and caused increased lung resistance up to 6 h post-exposure (PE). There was a trending increase in SOD and xanthine oxidoreductase (XOR) activities, both of which are indicative of oxidative stress. Based on increased lung tissue p38 signaling, MAP kinase expression was activated after VX exposure. IL-6 expression was also increased at 6 h post-inhalation for the 31.6 mg/m3 exposed group. Innate survival response mechanisms in rats may be present due to increased lung tissue mRNA AChE expression 6 h after exposure. Immunohistochemistry showed reduced staining for surfactant D and increased expression of iNOS, indicating that the activation of •NO precursor pathways. Bronchoalveloar lavage fluid (BALF) results from 1 h to 2 weeks PE show that inflammatory cells are highly active as evidenced by the increased production of cytokines and chemokines. This is the first study linking VX-induced lung injury to a possible innate survival amplification of AChE and possibly compromised immune function. These results could supplement medical treatment strategies with regard to therapeutic approaches against VX inhalational challenge.

The physiology and toxicology of acute inhalation phosphine poisoning in conscious male rats.

Phosphine (PH3) is a toxidrome-spanning chemical that is widely used as an insecticide and rodenticide. Exposure to PH3 causes a host of target organ and systemic effects, including oxidative stress, cardiopulmonary toxicity, seizure-like activity and overall metabolic disturbance. A custom dynamic inhalation gas exposure system was designed for the whole-body exposure of conscious male Sprague-Dawley rats (250-350 g) to PH3. An integrated plethysmography system was used to collect respiratory parameters in real-time before, during and after PH3 exposure. At several time points post-exposure, rats were euthanized, and various organs were removed and analyzed to assess organ and systemic effects. The 24 h post-exposure LCt50, determined by probit analysis, was 23,270 ppm × min (32,345 mg × min/m3). PH3 exposure affects both pulmonary and cardiac function. Unlike typical pulmonary toxicants, PH3 induced net increases in respiration during exposure. Gross observations of the heart and lungs of exposed rats suggested pulmonary and cardiac tissue damage, but histopathological examination showed little to no observable pathologic changes in those organs. Gene expression studies indicated alterations in inflammatory processes, metabolic function and cell signaling, with particular focus in cardiac tissue. Transmission electron microscopy examination of cardiac tissue revealed ultrastructural damage to both tissue and mitochondria. Altogether, these data reveal that in untreated, un-anesthetized rats, PH3 inhalation induces acute cardiorespiratory toxicity and injury, leading to death and that it is characterized by a steep dose-response curve. Continued use of our interdisciplinary approach will permit more effective identification of therapeutic windows and development of rational medical countermeasures and countermeasure strategies.

An Official American Thoracic Society Workshop Report: Chemical Inhalational Disasters. Biology of Lung Injury, Development of Novel Therapeutics, and Medical Preparedness.

This report is based on the proceedings from the Inhalational Lung Injury Workshop jointly sponsored by the American Thoracic Society (ATS) and the National Institutes of Health (NIH) Countermeasures Against Chemical Threats (CounterACT) program on May 21, 2013, in Philadelphia, Pennsylvania. The CounterACT program facilitates research leading to the development of new and improved medical countermeasures for chemical threat agents. The workshop was initiated by the Terrorism and Inhalational Disasters Section of the Environmental, Occupational, and Population Health Assembly of the ATS. Participants included both domestic and international experts in the field, as well as representatives from U.S. governmental funding agencies. The meeting objectives were to (1) provide a forum to review the evidence supporting current standard medical therapies, (2) present updates on our understanding of the epidemiology and underlying pathophysiology of inhalational lung injuries, (3) discuss innovative investigative approaches to further delineating mechanisms of lung injury and identifying new specific therapeutic targets, (4) present promising novel medical countermeasures, (5) facilitate collaborative research efforts, and (6) identify challenges and future directions in the ongoing development, manufacture, and distribution of effective and specific medical countermeasures. Specific inhalational toxins discussed included irritants/pulmonary toxicants (chlorine gas, bromine, and phosgene), vesicants (sulfur mustard), chemical asphyxiants (cyanide), particulates (World Trade Center dust), and respirable nerve agents.

Effects of inhaled aerosolized carfentanil on real-time physiological responses in mice: a preliminary evaluation of naloxone.

This study examined the real-time exposure-response effects of aerosolized carfentanil (CRF) on opioid-induced toxicity, respiratory dynamics and cardiac function in mice. Unrestrained, conscious male CD-1 mice (25-30 g) were exposed to 0.4 or 4.0 mg/m3 of aerosolized CRF for 15 min (Ct = 6 or 60 mg min/m3) in a whole-body plethysmograph chamber. Minute volume (MV), core body temperature (Tc), mean arterial blood pressure (MAP) and heart rate (HR) were evaluated in animals exposed to CRF or sterile H2O. Loss of consciousness and Straub tail were observed in before 1 min following initiation of exposure to 6 or 60 mg min/m3 of CRF. Clinical signs of opioid-induced toxicity were observed in a dose-dependent manner. Exposure to 6 or 60 mg min/m3 of CRF resulted in significant decrease in MV as compared to the controls. MAP, HR and Tc decreased 24 h in animals exposed to either 6 or 60 mg min/m3 of CRF as compared to the controls. Post-exposure administration of naloxone (NX, 0.05 mg/kg, i.m.) did not increase the MV of animals exposed to CRF to control levels within 24 h, but decreased clinical signs of opioid-induced toxicity and the duration of respiratory depression. This is the first study to evaluate real-time respiratory dynamics and cardiac function during exposure and up to 24 h post-exposure to CRF. The evaluation of toxicological signs and respiratory dynamics following exposure to CRF will be useful in the development of therapeutic strategies to counteract the ongoing threat of abuse and overuse of opioids and their synthetic variants.

Adverse respiratory effects in rats following inhalation exposure to ammonia: respiratory dynamics and histopathology.

Acute respiratory dynamics and histopathology of the lungs and trachea following inhaled exposure to ammonia were investigated. Respiratory dynamic parameters were collected from male Sprague-Dawley rats (300-350 g) during (20 min) and 24 h (10 min) after inhalation exposure for 20 min to 9000, 20,000, and 23,000 ppm of ammonia in a head-only exposure system. Body weight loss, analysis of blood cells, and lungs and trachea histopathology were assessed 1, 3, and 24 h following inhalation exposure to 20,000 ppm of ammonia. Prominent decreases in minute volume (MV) and tidal volume (TV) were observed during and 24 h post-exposure in all ammonia-exposed animals. Inspiratory time (IT) and expiratory time (ET) followed similar patterns and decreased significantly during the exposure and then increased at 24 h post-exposure in all ammonia-exposed animals in comparison to air-exposed controls. Peak inspiratory (PIF) and expiratory flow (PEF) significantly decreased during the exposure to all ammonia doses, while at 24 h post-exposure they remained significantly decreased following exposure to 20,000 and 23,000 ppm. Exposure to 20,000 ppm of ammonia resulted in body weight loss at 1 and 3 h post-exposure; weight loss was significant at 24 h compared to controls. Exposure to 20,000 ppm of ammonia for 20 min resulted in increases in the total blood cell counts of white blood cells, neutrophils, and platelets at 1, 3, and 24 h post-exposure. Histopathologic evaluation of the lungs and trachea tissue of animals exposed to 20,000 ppm of ammonia at 1, 3, and 24 h post-exposure revealed various morphological changes, including alveolar, bronchial, and tracheal edema, epithelial necrosis, and exudate consisting of fibrin, hemorrhage, and inflammatory cells. The various alterations in respiratory dynamics and damage to the respiratory system observed in this study further emphasize ammonia-induced respiratory toxicity and the relevance of efficacious medical countermeasure strategies.

Evaluation of an in vitro screening model to assess phosgene inhalation injury.

Therapeutic development against exposure to toxic gases is hindered by the lack of appropriate models to evaluate candidate compounds prior to animal efficacy studies. In this study, an in vitro, air-liquid interface exposure model has been tested to examine its potential application for screening treatments for phosgene (carbonyl chloride)-induced pulmonary injury. Epithelial cultures on Transwell® inserts, combined with a Vitrocell® exposure apparatus, provided a physiologically relevant exposure environment. Differentiated human bronchial epithelial (16HBE) cultures were exposed for 8 min to phosgene ranging from 0 to 64 ppm and assessed for changes in transepithelial electrical resistance (TEER, epithelial barrier integrity), cellular viability (XTT) and post-exposure (PE) cellular metabolic energy status. Exposure to phosgene concentrations ≥8 ppm caused dose-dependent and significant decreases in TEER and XTT which did not recover within 24-h PE. In addition, at 64 ppm the rate of oxidative glutamine metabolism was significantly inhibited at 6 and 24 h after exposure. Glycolytic activities (glucose utilization and lactate production) were also inhibited, but to a lesser extent. Decreased glycolytic function can translate to insufficient energy sources to counteract barrier function failure. Consistent and sensitive markers of phosgene exposure were TEER, cell viability and decreased metabolism. As such, we have assessed an appropriate in vitro model of phosgene inhalation that produced quantifiable alterations in markers of lung cell metabolism and injury in human airway epithelial cells. Data indicate the suitability of this model for testing classes of anti-edemagenic compounds such as corticosteroids or phosphodiesterase inhibitors for evaluating phosgene therapeutics.

Acute Gene Expression Profile of Lung Tissue Following Sulfur Mustard Inhalation Exposure in Large Anesthetized Swine.

Sulfur mustard (HD) is a vesicating and alkylating agent widely used on the battlefield during World War I and more recently in the Iran-Iraq War. It targets the eyes, skin, and lungs, producing skin burns, conjunctivitis, and compromised respiratory function; early acute effects lead to long-term consequences. However, it is the effects on the lungs that drive morbidity and eventual mortality. The temporal postexposure response to HD within lung tissue raises the question of whether toxicity is driven by the alkylating properties of HD on critical homeostatic pathways. We have established an anesthetized swine model of inhaled HD vapor exposure to investigate the toxic effects of HD 12 h postexposure. Large white female swine were anesthetized and instrumented prior to exposure to air, 60 (sublethal) or 100 µg·kg-1 (∼LD40) doses of HD (10 min). Physiological parameters were continuously assessed. Data indicate that exposure to 100 µg·kg-1 HD lowered arterial blood oxygenation and increased shunt fraction and lavage protein compared with those of air-exposed controls and the 60 µg·kg-1 dose of HD. Histopathology showed an increased total pathology score between the 100 µg·kg-1 HD group and air-exposed controls. Principal component analysis of differentially expressed genes demonstrated a distinct and separable response of inhaled HD between air-exposed controls and the 60 and 100 µg·kg-1 doses of HD. Canonical pathway analysis demonstrated changes in acute phase response signaling, aryl hydrocarbon receptor signaling, NRF-2 mediated oxidative stress, and zymosterol biosynthesis in the 60 and 100 µg·kg-1 HD dose group. Transcriptional changes also indicated alterations in immune response, cancer, and cell signaling and metabolism canonical pathways. The 100 µg·kg-1 dose group also showed significant changes in cholesterol biosynthesis. Taken together, exposure to inhaled HD had a significant effect on physiological responses coinciding with acute changes in gene expression and lung histopathology. In addition, transcriptomics support the observed beneficial effects of N-acetyl-l-cysteine for treatment of acute inhalation HD exposure.

Phosphine toxicity: a story of disrupted mitochondrial metabolism.

Rodenticides and pesticides pose a significant threat not only to the environment but also directly to humans by way of accidental and/or intentional exposure. Metal phosphides, such as aluminum, magnesium, and zinc phosphides, have gained popularity owing to ease of manufacture and application. These agents and their hydrolysis by-product phosphine gas (PH3 ) are more than adequate for eliminating pests, primarily in the grain storage industry. In addition to the potential for accidental exposures in the manufacture and use of these agents, intentional exposures must also be considered. As examples, ingestion of metal phosphides is a well-known suicide route, especially in Asia; and intentional release of PH3 in a populated area cannot be discounted. Metal phosphides cause a wide array of effects that include cellular poisoning, oxidative stress, cholinesterase inhibition, circulatory failure, cardiotoxicity, gastrointestinal and pulmonary toxicity, hepatic damage, neurological toxicity, electrolyte imbalance, and overall metabolic disturbances. Mortality rates often exceed 70%. There are no specific antidotes against metal phosphide poisoning. Current therapeutic intervention is limited to supportive care. The development of beneficial medical countermeasures will rely on investigative mechanistic toxicology; the ultimate goal will be to identify specific treatments and therapeutic windows for intervention.

Assessment of inhaled acute ammonia-induced lung injury in rats.

This study examined acute toxicity and lung injury following inhalation exposure to ammonia. Male Sprague-Dawley rats (300-350 g) were exposed to 9000, 20,000, 23,000, 26,000, 30,000 or 35,000 ppm of ammonia for 20 min in a custom head-out exposure system. The exposure atmosphere, which attained steady state within 3 min for all ammonia concentrations, was monitored and verified using a Fourier transform infrared spectroscopy (FTIR) gas analyzer. Animals exposed to ammonia resulted in dose-dependent increases in observed signs of intoxication, including increased chewing and licking, ocular irritation, salivation, lacrimation, oronasal secretion and labored breathing. The LCt50 of ammonia within this head-out inhalation exposure model was determined by probit analysis to be 23,672 ppm (16,489 mg/m(3)) for the 20 min exposure in male rats. Exposure to 20,000 or 23,000 ppm of ammonia resulted in significant body weight loss 24-h post-exposure. Lung edema increased in all ammonia-exposed animal groups and was significant following exposure to 9000 ppm. Bronchoalveolar fluid (BALF) protein concentrations significantly increased following exposure to 20,000 or 23,000 ppm of ammonia in comparison to controls. BAL cell (BALC) death and total cell counts increased in animals exposed to 20,000 or 23,000 ppm of ammonia in comparison to controls. Differential cell counts of white blood cells, neutrophils and platelets from blood and BALF were significantly increased following exposure to 23,000 ppm of ammonia. The following studies describe the validation of a head-out inhalation exposure model for the determination of acute ammonia-induced toxicity; this model will be used for the development and evaluation of potential therapies that provide protection against respiratory and systemic toxicological effects.

Conceptual approaches for treatment of phosgene inhalation-induced lung injury.

Toxic industrial chemicals are used throughout the world to produce everyday products such as household and commercial cleaners, disinfectants, pesticides, pharmaceuticals, plastics, paper, and fertilizers. These chemicals are produced, stored, and transported in large quantities, which poses a threat to the local civilian population in cases of accidental or intentional release. Several of these chemicals have no known medical countermeasures for their toxic effects. Phosgene is a highly toxic industrial chemical which was used as a chemical warfare agent in WWI. Exposure to phosgene causes latent, non-cardiogenic pulmonary edema which can result in respiratory failure and death. The mechanisms of phosgene-induced pulmonary injury are not fully identified, and currently there is no efficacious countermeasure. Here, we provide a proposed mechanism of phosgene-induced lung injury based on the literature and from studies conducted in our lab, as well as provide results from studies designed to evaluate survival efficacy of potential therapies following whole-body phosgene exposure in mice. Several therapies were able to significantly increase 24h survival following an LCt50-70 exposure to phosgene; however, no treatment was able to fully protect against phosgene-induced mortality. These studies provide evidence that mortality following phosgene toxicity can be mitigated by neuro- and calcium-regulators, antioxidants, phosphodiesterase and endothelin receptor antagonists, angiotensin converting enzymes, and transient receptor potential cation channel inhibitors. However, because the mechanism of phosgene toxicity is multifaceted, we conclude that a single therapeutic is unlikely to be sufficient to ameliorate the multitude of direct and secondary toxic effects caused by phosgene inhalation.

Vapor inhalation exposure to soman in conscious untreated rats: preliminary assessment of neurotoxicity.

Neurological toxicity and brain injury following vapor inhalation exposure to the chemical warfare nerve agent (CWNA) soman (GD) were examined in untreated non-anesthetized rats. In this study, male Sprague-Dawley rats (300-350 g) were exposed to 600 mg × min/m(3) of soman or vehicle in a customized head-out inhalation system for 7 min. Convulsant animals were observed for clinical signs and various regions of the brain (dorsolateral thalamus, basolateral amygdala, piriform cortex, and lateral cortex) were collected for pathological observations 24 h post-exposure. Signs of CWNA-induced cholinergic crises including salivation, lacrimation, increased urination and defecation, and tremors were observed in all soman-exposed animals. Soman-exposed animals at 24 h post-exposure lost 11% of their body weight in comparison to 2% in vehicle-exposed animals. Whole blood acetylcholinesterase (AChE) activity was significantly inhibited in all soman-exposed groups in comparison to controls. Brain injury was confirmed by the neurological assessment of hematoxylin-eosin (H&E) staining and microscopy in the piriform cortex, dorsolateral thalamus, basolateral amygdala, and lateral cortex. Severe damage including prominent lesions, edematous, congested, and/or hemorrhagic tissues was observed in the piriform cortex, dorsolateral thalamus, and lateral cortex in soman-exposed animals 24 h post-exposure, while only minimal damage was observed in the basolateral amygdala. These results indicate that inhalation exposure to soman vapor causes neurological toxicity and brain injury in untreated unanesthetized rats. This study demonstrates the ability of the described soman vapor inhalation exposure model to cause neurological damage 24 h post-exposure in rats.

Measurement of various respiratory dynamics parameters following acute inhalational exposure to soman vapor in conscious rats.

Respiratory dynamics were investigated in head-out plethysmography chambers following inhalational exposure to soman in untreated, non-anesthetized rats. A multipass saturator cell was used to generate 520, 560 and 600 mg × min/m(3) of soman vapor in a customized inhalational exposure system. Various respiratory dynamic parameters were collected from male Sprague-Dawley rats (300--350 g) during (20 min) and 24 h (10 min) after inhalational exposure. Signs of CWNA-induced cholinergic crisis were observed in all soman-exposed animals. Percentage body weight loss and lung edema were observed in all soman-exposed animals, with significant increases in both at 24 h following exposure to 600 mg × min/m(3). Exposure to soman resulted in increases in respiratory frequency (RF) in animals exposed to 560 and 600 mg × min/m(3) with significant increases following exposure to 560 mg × min/m(3) at 24 h. No significant alterations in inspiratory time (IT) or expiratory time (ET) were observed in soman-exposed animals 24 h post-exposure. Prominent increases in tidal volume (TV) and minute volume (MV) were observed at 24 h post-exposure in animals exposed to 600 mg × min/m(3). Peak inspiratory (PIF) and expiratory flow (PEF) followed similar patterns and increased 24 h post-exposure to 600 mg × min/m(3) of soman. Results demonstrate that inhalational exposure to 600 mg × min/m(3) soman produces notable alterations in various respiratory dynamic parameters at 24 h. The following multitude of physiological changes in respiratory dynamics highlights the need to develop countermeasures that protect against respiratory toxicity and lung injury.

Respiratory complications of organophosphorus nerve agent and insecticide poisoning. Implications for respiratory and critical care.

Organophosphorus (OP) compound poisoning is a major global public health problem. Acute OP insecticide self-poisoning kills over 200,000 people every year, the majority from self-harm in rural Asia. Highly toxic OP nerve agents (e.g., sarin) are a significant current terrorist threat, as shown by attacks in Damascus during 2013. These anticholinesterase compounds are classically considered to cause an acute cholinergic syndrome with decreased consciousness, respiratory failure, and, in the case of insecticides, a delayed intermediate syndrome that requires prolonged ventilation. Acute respiratory failure, by central and peripheral mechanisms, is the primary cause of death in most cases. However, preclinical and clinical research over the last two decades has indicated a more complex picture of respiratory complications after OP insecticide poisoning, including onset of delayed neuromuscular junction dysfunction during the cholinergic syndrome, aspiration causing pneumonia and acute respiratory distress syndrome, and the involvement of solvents in OP toxicity. The treatment of OP poisoning has not changed over the last 50 years. However, a better understanding of the multiple respiratory complications of OP poisoning offers additional therapeutic opportunities.

Acute pulmonary toxicity following inhalation exposure to aerosolized VX in anesthetized rats.

This study evaluated acute toxicity and pulmonary injury in rats at 3, 6 and 24 h after an inhalation exposure to aerosolized O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX). Anesthetized male Sprague-Dawley rats (250-300 g) were incubated with a glass endotracheal tube and exposed to saline or VX (171, 343 and 514 mg×min/m³ or 0.2, 0.5 and 0.8 LCt50, respectively) for 10 min. VX was delivered by a small animal ventilator at a volume of 2.5 ml × 70 breaths/minute. All VX-exposed animals experienced a significant loss in percentage body weight at 3, 6, and 24 h post-exposure. In comparison to controls, animals exposed to 514 mg×min/m³ of VX had significant increases in bronchoalveolar lavage (BAL) protein concentrations at 6 and 24 h post-exposure. Blood acetylcholinesterase (AChE) activity was inhibited dose dependently at each of the times points for all VX-exposed groups. AChE activity in lung homogenates was significantly inhibited in all VX-exposed groups at each time point. All VX-exposed animals assessed at 20 min and 3, 6 and 24 h post-exposure showed increases in lung resistance, which was prominent at 20 min and 3 h post-exposure. Histopathologic evaluation of lung tissue of the 514 mg×min/m³ VX-exposed animals at 3, 6 and 24 h indicated morphological changes, including perivascular inflammation, alveolar exudate and histiocytosis, alveolar septal inflammation and edema, alveolar epithelial necrosis, and bronchiolar inflammatory infiltrates, in comparison to controls. These results suggest that aerosolization of the highly toxic, persistent chemical warfare nerve agent VX results in acute pulmonary toxicity and lung injury in rats.

Inhalation toxicity of soman vapor in non-anesthetized rats: a preliminary assessment of inhaled bronchodilator or steroid therapy.

Respiratory toxicity, injury and treatment following vapor inhalational exposure to the chemical warfare nerve agent (CWNA) soman (GD) were examined in non-anesthetized rats. This study exposed male Sprague-Dawley rats (250-300g) to 520, 560, 600, 825 or 1410mg×min/m(3) of soman in a customized head-out inhalation system. Signs of CWNA-induced cholinergic crises were observed in all soman-exposed animals. The LCt50 of vaporized soman as determined by probit analysis was 593.1mg×min/m(3). All animals exposed to 825 and 1410mg×min/m(3) developed severe convulsions and died within 4-8min post-exposure. Edema measured by wet/dry weight ratio of the left lung lobe increased in a dose-dependent manner in all soman-exposed animals. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase (AChE) activities were inhibited dose-dependently in soman-exposed groups at 24h. A significant increase in total BAL protein was observed in soman-exposed animals at all doses. AChE activity was inhibited in lung and whole brain tissues in all soman-exposed animals. Histopathological analysis of the lungs of animals exposed to 600mg×min/m(3) of soman revealed prominent morphological changes including alveolar histiocytosis, hemorrhage and inflammation consisting of neutrophilic exudate. Exposure of animals to 600mg×min/m(3) of soman followed by treatment with two actuations for 10s of Combivent (21µg of ipratropium bromide and 120µg of albuterol sulfate) and Symbicort (80µg budesonide and 4.5µg formoterol) by inhalation into a modified metered dose inhaler (MDI) 10min post-exposure resulted in increased minute volume, but did not decrease mortality. These results indicate that inhalation exposure to soman vapor causes acute respiratory toxicity and injury in untreated, un-anesthetized rats and that inhalation treatment with Combivent or Symbicort did improve the respiratory outcomes, but did not influence lethality.

Development of a model for nerve agent inhalation in conscious rats.

This study characterizes the development of a head-out inhalation exposure system for assessing respiratory toxicity of vaporized chemical agents in untreated, non-anesthetized rats. The organophosphate diisopropyl fluorophosphate (DFP) induces classical cholinergic toxicity following inhalation exposure and was utilized to validate the effectiveness of this newly designed inhalation exposure system. A saturator cell apparatus was used to generate DFP vapor at 9750, 10,950, 12,200, 14,625 and 19,500 mg × min/m³ which was carried by filtered nitrogen into a glass mixing tube, where it combined with ambient air before being introduced to the custom-made glass exposure chamber. Male Sprague-Dawley rats (250-300 g) were restrained in individual head-out plethysmography chambers, which acquired respiratory parameters before, during and after agent exposure. All animals were acclimated to the exposure system prior to exposure to reduce novel environment-induced stress. The LCt50, as determined by probit analysis, was 12,014 mg × min/m³. Weight loss in exposed animals was dose-dependent and ranged from 8 to 28% of their body weight 24 h after exposure. Increased salivation, lacrimation, urination, defecation (SLUD) and mild muscular fasciculation were observed in all DFP-exposed animals during and immediately following exposure. In all exposed animals, DFP vapor produced significant inhibition of acetylcholinesterase (AChE) activity in cardiac blood, bronchoalveolar lavage fluid (BALF), whole brain and lung tissue as well as alterations in tidal volume and minute volume. These studies have provided valuable information leading to the initiation of studies evaluating inhalational toxicity and treatments following exposure to the more lethal and potent chemical warfare nerve agents.

Determination of LCt(50)s in anesthetized rats exposed to aerosolized nerve agents.

Nerve agents pose a threat to the respiratory tract with exposure that could result in acute compromised lung performance and death. The determination of toxicity by inhalation is important for the rational development of timely therapeutic countermeasures. This study was designed to deliver aerosolized dilute nerve agents in a dose-response manner to investigate the extent of lethality of nerve agents: soman, sarin, VX and VR. Male rats (240-270 g) were anesthetized intramuscularly with 10 mg/kg xylazine and 90 mg/kg ketamine. Following anesthesia, rats were intubated with a glass endotracheal tube (ET) and placed in a glove box. The ET was connected to a closed circuit nebulizer system (Aeroneb, Aerogen, Inc.) that delivered a particle size of < 2.0 µm and was in series between the ventilator and the ET. Nerve agents were delivered by a small animal ventilator set for a volume of 2.5 mL × 60-80 breaths/min. VX or VR were nebulized and delivered in concentrations ranging from 6.25-800 µg/kg over a 10-min exposure time period. Sarin (GB) or soman (GD), 6.5-1250 µg/kg, were delivered in a similar manner. Lethality by inhalation occurred either during the 10-min exposure period or less than 15 min after the cessation of exposure. Survivors were euthanized at 24 h postexposure. LCt(50) estimates (± 95% confidence intervals [CIs]) were obtained from the sequential stage-wise experiments using the probit analysis. Probit analysis revealed that the LD(50) for VX was 110.7 µg/kg (CI: 73.5-166.7), VR 64.2 µg/kg (CI: 42.1-97.8); soman (GD), 167 µg/kg (CI: 90-310), and sarin (GB), 154 µg/kg (CI: 98-242), respectively. Although VR is a structural isomer of VX, the compounds appear to be markedly different in terms of toxicity when delivered by aerosol. These relationships were converted to actual 10 min LCt(50) equivalents: VX = 632.2, VR = 367, GD = 954.3 and GB = 880 mg·min/m(3). Validation of exposure was verified by the determination of blood levels of acetylcholinesterase (AChE) across doses for the agent VR.