Phosphine (PH) is a toxidrome-spanning chemical that is widely used as an insecticide and rodenticide. Exposure to PH 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 PH. An integrated plethysmography system was used to collect respiratory parameters in real-time before, during and after PH 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 LCt, determined by probit analysis, was 23,270 ppm × min (32,345 mg × min/m). PH exposure affects both pulmonary and cardiac function. Unlike typical pulmonary toxicants, PH 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, PH 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.

Extracellular vesicles (EVs) have emerged as key regulators of cell-cell communication during inflammatory responses to lung injury induced by diverse pulmonary toxicants including cigarette smoke, air pollutants, hyperoxia, acids and endotoxin. Many lung cell types, including epithelial cells and endothelial cells, as well as infiltrating macrophages generate EVs. EVs appear to function by transporting cargo to recipient cells that, in most instances, promotes their inflammatory activity. Biologically active cargo transported by EVs include miRNAs, cytokines/ chemokines, damage-associated molecular patterns (DAMPs), tissue factor (TF)s and caspases. Findings that EVs are taken up by target cells such as macrophages, and that this leads to increased proinflammatory functioning provide support for their role in the development of pathologies associated with toxicant exposure. Understanding the nature of EVs responding to toxic exposures and their cargo may lead to the development of novel therapeutic approaches to mitigating lung injury.

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.

The Mycobacterium tuberculosis H37Rv genome has been sequenced and annotated over 20 years ago, yet roughly half of the protein-coding genes still lack a predicted function. We characterized two genes of unknown function, rv3679 and rv3680, for which inconsistent findings regarding their importance for virulence in mice have been reported. We confirmed that a rv3679-80 deletion mutant (Δrv3679-80) was virulent in mice and discovered that Δrv3679-80 suffered from a glycerol-dependent recovery defect on agar plates following mouse infection. Glycerol also exacerbated killing of Δrv3679-80 by nitric oxide. Rv3679-Rv3680 have previously been shown to form a complex with ATPase activity and we demonstrate that the ability of M. tuberculosis to cope with elevated levels of glycerol and nitric oxide requires intact ATP-binding motifs in both Rv3679 and Rv3680. Inactivation of glycerol kinase or Rv2370c, a protein of unknown function, suppressed glycerol mediated toxicity in Δrv3679-80. Glycerol catabolism led to increased intracellular methylglyoxal pools and Δrv3679-80 was hypersusceptible to extracellular methylglyoxal suggesting that glycerol toxicity in Δrv3679-80 is caused by methylglyoxal. Rv3679 and Rv3680 interacted with Rv1509, and Rv3679 had numerous additional interactors including proteins of the type II fatty acid synthase (FASII) pathway and mycolic acid modifying enzymes linking Rv3679 to fatty acid or lipid synthesis. This work provides experimentally determined roles for Rv3679 and Rv3680 and stimulates future research on these and other proteins of unknown function. Importance A better understanding of the pathogenesis of tuberculosis requires a better understanding of gene function in M. tuberculosis. This work provides the first functional insight into the Rv3679/Rv3680 ATPase complex. We demonstrate that M. tuberculosis requires this complex and specifically its ATPase activity to resist glycerol and nitric oxide toxicity. We provide evidence that the glycerol-derived metabolite methylglyoxal causes toxicity in the absence of Rv3679/Rv3680. We further show that glycerol-dependent toxicity is reversed when glycerol kinase (GlpK) is inactivated. Our work uncovered other genes of unknown function that interact with Rv3679 and/or Rv3680 genetically or physically, underscoring the importance of understanding uncharacterized genes.

Nitrogen mustard (NM) causes acute lung injury, which progresses to fibrosis. This is associated with a macrophagedominant inflammatory response and the production of proinflammatory/profibrotic mediators, including tumor necrosis factor alpha (TNF-α). Herein, we refined magnetic resonance imaging (MRI) and computed tomography (CT) imaging methodologies to track the progression of NM-induced lung injury in rodents and assess the efficacy of anti-TNF-α antibody in mitigating toxicity. Anti-TNF-α antibody was administered to rats (15 mg/kg, every 8 days, intravenously) beginning 30 min after treatment with phosphate-buffered saline control or NM (0.125 mg/kg, intratracheally). Animals were imaged by MRI and CT prior to exposure and 1-28 days postexposure. Using MRI, we characterized acute lung injury and fibrosis by quantifying high-signal lung volume, which represents edema, inflammation, and tissue consolidation; these pathologies were found to persist for 28 days following NM exposure. CT scans were used to assess structural components of the lung and to register changes in tissue radiodensities. CT scans showed that in control animals, total lung volume increased with time. Treatment of rats with NM caused loss of lung volume; anti-TNF-α antibody mitigated this decrease. These studies demonstrate that MRI and CT can be used to monitor lung disease and the impact of therapeutic intervention.