Development of a model for nerve agent inhalation in conscious rats

Abstract

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.     

Short-term inhalation study of graphene oxide nanoplates

Graphene oxides possess unique physicochemical properties with important potential applications in electronics, pharmaceuticals, and medicine. However, the toxicity following inhalation exposure to graphene oxide has not yet been clarified. Therefore, this study conducted a short-term graphene oxide inhalation toxicity analysis using a nose-only inhalation exposure system and male Sprague–Dawley rats. A total of four groups (15 rats per group) were exposed: (1) control (fresh air), (2) low concentration (0.76?±?0.16?mg/m³), (3) moderate concentration (2.60?±?0.19?mg/m³), and (4) high concentration (9.78?±?0.29?mg/m³). The rats were exposed to graphene oxide for 6?h/day for 5 days, followed by recovery for 1, 3, and 21 days. No significant body or organ weight changes were noted after the short-term exposure or during the recovery period. Similarly, no significant systemic effects of toxicological importance were noted in the hematological assays, bronchoalveolar lavage fluid (BAL) inflammatory markers, BAL fluid cytokines, or blood biochemical assays following the graphene oxide exposure or during the post-exposure observation period. Moreover, no significant differences were observed in the BAL cell differentials, such as lymphocytes, macrophages, or polymorphonuclear cells. Graphene oxide-ingested alveolar macrophages as a spontaneous clearance reaction were observed in the lungs of all the concentration groups from post 1?day to post 21 days. Histopathological examination of the liver and kidneys did not reveal any significant test-article-relevant histopathological lesions. Importantly, similar to previously reported graphene inhalation data, this short-term nose-only inhalation study found only minimal or unnoticeable graphene oxide toxicity in the lungs and other organs.     

The in vitro exposure to cypermethrin does not inhibit the proliferative response of peripheral blood mononuclear cells

This study evaluated the effect of in vitro exposure to cypermethrin on peripheral blood mononuclear cells proliferative response, considering reduced peripheral blood mononuclear cells proliferative response observed in individuals occupationally exposed to pyrethroids. Peripheral blood mononuclear cells were obtained from 21 healthy subjects (28.0?±?9.0 years old). The effect of cypermethrin (at 0.5, 1.0 and 5.0?mg/ml) on cell viability was evaluated by flow cytometry using an apoptosis detection kit. Cell proliferation (PI) was evaluated by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence decay using flow cytometry. Cells labeled with CFSE were exposed, in vitro, to cypermethrin (0.5, 1.0, 2.0, 2.5 and 4?μg/ml) and stimulated with phytohemagglutinin (PHA 1.0 or 5.0?μg/ml) for 5?d (37?°C, 5% CO₂). The in vitro treatment of peripheral blood mononuclear cells with cypermethrin did not induce apoptosis or necrosis after 5?d in culture. Stimulation by PHA induced cell proliferation (PI?=?1.29?±?1.09 and 2.01?±?0.62, PHA at 1.0 and 5.0?μg/ml, respectively, mean?±?SD) and in vitro exposure to cypermethrin did not alter cellular proliferative response to PHA (PI?=?1.80?±?0.50, 2.60?±?0.05 and 2.10?±?1.20 for cypermethrin at 1.0, 2.0 and 4.0?μg/ml, respectively, and PHA at 5.0?μg/ml). In vitro treatment of peripheral blood mononuclear cells with cypermethrin, at the doses tested, does not affect cell viability or proliferation. These findings suggest that the reduction of proliferation observed on lymphocytes derived from individuals occupationally exposed to pesticides may be related to other mechanisms than direct action of cypermethrin on lymphocytes.     

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.     

Lung damage following whole body,but not intramuscular,exposure to median lethality dose of sarin: findings in rats and guinea pigs

Objective: Sarin is an irreversible organophosphate cholinesterase inhibitor and a highly toxic, volatile warfare agent. Rats and guinea pigs exposed to sarin display cholinergic excitotoxicity which includes hyper-salivation, respiratory distress, tremors, seizures, and death. Here we focused on the characterization of the airways injury induced by direct exposure of the lungs to sarin vapor and compared it to that induced by the intramuscularly route.

Materials and methods: Rats were exposed to sarin either in vapor (～1LCT50, 34.2?±?0.8?µg/l/min, 10?min) or by i.m. (～1LD50, 80?µg/kg), and lung injury was evaluated by broncho-alveolar lavage (BAL).

Results and discussion: BAL analysis revealed route-dependent effects in rats: vapor exposed animals showed elevation of inflammatory cytokines, protein, and neutrophil cells. These elevations were seen at 24?h and were still significantly higher compared to control values at 1?week following vapor exposure. These elevations were not detected in rats exposed to sarin i.m. Histological evaluation of the brains revealed typical changes following sarin poisoning independent of the route of administration. The airways damage following vapor exposure in rats was also compared to that induced in guinea pigs. The latter showed increased eosinophilia and histamine levels that constitutes an anaphylactic response not seen in rats.

Conclusions: These data clearly point out the importance of using the appropriate route of administration in studying the deleterious effects of volatile nerve agents, as well as the selection of the appropriate animal species. Since airways form major target organs for the development of injury following inhalation toxicity, they should be included in any comprehensive evaluation of countermeasures efficacy.     

Subacute inhalation toxicity study of synthetic amorphous silica nanoparticles in Sprague-Dawley rats

Synthetic amorphous silica nanoparticles (SiNPs) are one of the most applied nanomaterials and are widely used in a broad variety of industrial and biomedical fields. However, no recent long-term inhalation studies evaluating the toxicity of SiNPs are available and results of acute studies are limited. Thus, we conducted a subacute inhalation toxicity study of SiNPs in Sprague-Dawley rats using a nose-only inhalation system. Rats were separated into four groups and target concentrations selected in this study were as follows: control (fresh air), low- (0.407?±?0.066?mg/m³), middle- (1.439?±?0.177?mg/m³) and high-concentration group (5.386?±?0.729?mg/m³), respectively. The rats were exposed to SiNPs for four consecutive weeks (6 hr/day, 5 days/week) except for control group of rats which received filtered fresh air. After 28-days of inhalation exposure to SiNPs, rats were sacrificed after recovery periods of one, seven and 28 days. Although there were minimal toxic changes such as temporary decrease of body weight after exposure, increased levels of red blood cells (RBCs) and hemoglobin (Hb) concentration, the lung histopathological findings and inflammatory markers in bronchoalveolar lavage (BAL) fluid including polymorphonuclear (PMN) leukocyte, lactate dehydrogenase (LDH), albumin and protein did not show significant changes at any recovery period. The results of this study suggest that the subacute inhalation of SiNPs had no toxic effects on the lung of rats at the concentrations and selected time points used in this study.     

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/m³ 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.     

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.     

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/m³) 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.     

Acute secretory cell toxicity and epithelial exfoliation after smoke inhalation injury in sheep: An electron and light microscopic study

Smoke inhalation injury promotes exfoliation of the upper airway columnar epithelium. Tracheal tissues from sheep 30?min after smoke exposure show intact epithelial areas, areas of epithelial disruption with loss of columnar cells and areas denuded of columnar cells. In intact areas detaching ciliated cells can be seen raised above the apical surface. This study aims to assess cell-specific toxicity by examining intact epithelium after inhalation injury. The junctional adhesion integrity between columnar and basal cells and the type of cells initially being displaced were also studied using light (LM) and transmission electron microscopy (TEM). TEM assessment of intact areas of sheep tracheal tissue (n?=?3) 30?min after exposure showed secretory cell toxicity including extrusion of cytoplasmic contents. In cells with severe secretory cell cytoplasmic disruption, loss of desmosomal junctions between the secretory and adjacent ciliated cells was evident. The number of desmosomes visible between columnar cells and basal cells was reduced (2.8 ± 1.8) in smoke-exposed animals compared to those in uninjured animals (5.0 ± 2.7), p?=?0.008. Serial sections of intact regions found 52 cells being displaced from the epithelium. All detaching cells were identified as ciliated cells. These studies show that the acute effects of inhalation injury include selective secretory cell toxicity which is associated with loss of junctional adhesion mechanisms and displacement of ciliated cells. Improved understanding of acute hypersecretory responses and epithelial integrity after exposure to toxic agents may improve understanding of epithelial fragility in airway disease.     

Methyl isocyanate inhalation induces tissue factor-dependent activation of coagulation in rats

Methyl isocyanate (MIC) is a highly toxic industrial chemical causing acute lethality after inhalation. The objective of this study was to determine whether alterations in hemostasis also occur in the immediate hours after exposure. Male rats were exposed to MIC (125–500?ppm) by nose-only vapor inhalation for 30?min. Arterial O₂ saturation was monitored prior to exposure, and hourly thereafter. Rats were euthanized at 1, 2, 4, and 8?hr and plasma analyzed for recalcification clotting time, tissue factor (TF) activity, and protein levels. Hypoxemia, as assessed by pulse oximetry, was an early feature of MIC inhalation. In contrast to sham or low (125?ppm) concentrations, 250 and 500?ppm MIC caused significant declines in blood oxygen saturation (% SpO₂) at 1?hr, which remained at deficit during the postexposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1?hr after MIC inhalation (sham treatment: 955?±?62.8?s; 125?ppm MIC: 790?±?62?s; 250?ppm: 676?±?28.0?s; 500?ppm: 581?±?175?s). This procoagulant effect was transient, with no difference observed between sham and all MIC groups by 8?hr. Similarly, elevated TF activity and protein were detected in plasma 1?hr after MIC inhalation, each of which showed a progressive decline back to control levels at later timepoints. This study demonstrates that MIC inhalation resulted in hypoxemia and transient hypercoagulability of blood. Accelerated clotting occurred rapidly and was likely due to intravascular TF, which initiates the extrinsic coagulation pathway.     

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

Abstract

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/m³ 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/m³. Exposure to soman resulted in increases in respiratory frequency (RF) in animals exposed to 560 and 600?mg?×?min/m³ with significant increases following exposure to 560?mg?×?min/m³ 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/m³. Peak inspiratory (PIF) and expiratory flow (PEF) followed similar patterns and increased 24?h post-exposure to 600?mg?×?min/m³ of soman. Results demonstrate that inhalational exposure to 600?mg?×?min/m³ 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.     

The effect of pentobarbital on the carbohydrate metabolism of glial cells in culture

Hamster astroblast glial cells (clone NN) and dissociated glial cells from newborn rat brains in primary culture were exposed to from 5 × 10^?5 to 1 × 10^?3 M pentobarbital-Na and 1 × 10^?4M or 2.5 × 10^?4 M morphine hydrochloride. After various periods of time the following enzymatic activities were measured in the cell culture: fructose-diphosphate-aldolase (ALD), pyruvate kinase (PK.), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH) and NADH cytochrome c reductase (CCR). The long-term exposure to pentobarbital resulted in increases in LDH, MDH and GDH activities and a consistent decrease in PK activity. Continuous exposure to morphine resulted in minor, uniformly depressive changes. When neuroblastoma cells (clone M1) or rat fibroblasts in primary culture were exposed to pentobarbital no specific changes could be detected. The results are interpreted to suggest that barbiturates affect the carbohydrate metabolism of glial cells in a drug- and cell-dependent fashion.     

5-Day repeated inhalation and 28-day post-exposure study of graphene

Abstract

Graphene has recently been attracting increasing attention due to its unique electronic and chemical properties and many potential applications in such fields as semiconductors, energy storage, flexible electronics, biosensors and medical imaging. However, the toxicity of graphene in the case of human exposure has not yet been clarified. Thus, a 5-day repeated inhalation toxicity study of graphene was conducted using a nose-only inhalation system for male Sprague-Dawley rats. A total of three groups (20 rats per group) were compared: (1) control (ambient air), (2) low concentration (0.68?±?0.14?mg/m³ graphene) and (3) high concentration (3.86?±?0.94?mg/m³ graphene). The rats were exposed to graphene for 6?h/day for 5 days, followed by recovery for 1, 3, 7 or 28 days. The bioaccumulation and macrophage ingestion of the graphene were evaluated in the rat lungs. The exposure to graphene did not change the body weights or organ weights of the rats after the 5-day exposure and during the recovery period. No statistically significant difference was observed in the levels of lactate dehydrogenase, protein and albumin between the exposed and control groups. However, graphene ingestion by alveolar macrophages was observed in the exposed groups. Therefore, these results suggest that the 5-day repeated exposure to graphene only had a minimal toxic effect at the concentrations and time points used in this study.     

An effort to test the embryotoxicity of benzene,toluene, xylene,and formaldehyde to murine embryonic stem cells using airborne exposure technique

Benzene, toluene, xylene, and formaldehyde are well-known indoor air pollutants, especially after house decoration. They are also common pollutants in the working places of the plastic industry, chemical industry, and leather industry. It has been reported that these pollutants cause people to be irritated, sick, experience a headache, and be dizzy. They also have the potential to induce asthma, aplastic anemia, and leukemia, even cause abortion or fetus malformation in humans. In this study, the airborne toxicity of benzene, toluene, xylene, and formaldehyde to murine embryonic stem cells (mES cells) were tested using airborne exposure technique to evaluate the mES cell airborne exposure model on embryotoxicity prediction. Briefly, mES cells were cultured on Transwell inserts and were exposed to an airborne surrounding of test chemicals in a chamber for 1?h at 37°C. Cytotoxicity was determined using the MTT assay after further culture for 18?h at 37°C in normal medium. The airborne IC₅₀ (50% inhibition concentration) of benzene, toluene, xylene, and formaldehyde derived from the fitted dose-response curves were 17,400?±?1290, 16,000?±?250, 4680?±?500, and 620?±?310 ppm, respectively. Formaldehyde was found to be the compound most toxic to mES cells compared to benzene homologues. The toxicity data had good correlation with the in vivo data. The results showed that the mES airborne exposure model may be used to predict embryotoxicity of volatile organic compounds.     

Tiotropium bromide suppresses smoke inhalation and burn injury-induced ERK 1/2 and SMAD 2/3 signaling in sheep bronchial submucosal glands

The effects of tiotropium bromide on ERK 1/2, SMAD 2/3 and NFκB signaling in bronchial submucosal gland (SMG) cells of sheep after smoke inhalation and burn injury (S?+?B) were studied. We hypothesized that tiotropium would modify intracellular signaling processes within SMG cells after injury. Bronchial tissues were obtained from uninjured (sham, n?=?6), S?+?B injured sheep 48?h after injury (n?=?6), and injured sheep nebulized with tiotropium (n?=?6). The percentage (mean?±?SD) of cells showing nuclear localization of phosphorylated ERK 1/2, pSMAD 2/3, and NFκB (p65) was determined by immunohistochemistry. Nuclear pERK 1/2 staining was increased in injured animals as compared to sham, (66?±?20 versus 14?±?9), p?=?0.0022, as was nuclear pSMAD, 84?±?10 versus 20?±?10, p?=?0.0022. There was a significant decrease in pERK 1/2 labeling in the tiotropium group compared to the injured group (31?±?20 versus 66?±?20, p?=?0.013), and also a decrease in pSMAD labeling, 62?±?17 versus 84?±?10, p?=?0.04. A significant increase for NFκB (p65) was noted in injured animals as compared to sham (73?±?16 versus 7?±?6, p?=?0.0022). Tiotropium-treated animals showed decreased p65 labeling as compared to injured (35?±?17 versus 74?±?16, p?=?0.02). The decrease in nuclear expression of pERK, pSMAD and NFκB molecules in SMG cells with tiotropium treatment is suggestive that their activation after injury is mediated in part through muscarinic receptors.     

Melatonin mitigates neomycin-induced hair cell injury in zebrafish

Context: Ototoxicity due to medications, such as aminoglycosides, is irreversible, and free radicals in the inner ear are assumed to play a major role. Because melatonin has an antioxidant property, we hypothesize that it might mitigate hair cell injury by aminoglycosides.

Objective: The objective of this study was to evaluate whether melatonin has an alleviative effect on neomycin-induced hair cell injury in zebrafish (Danio rerio).

Methods: Various concentrations of melatonin were administered to 5-day post-fertilization zebrafish treated with 125?μM neomycin for 1?h. Surviving hair cells within four neuromasts were compared with that of a control group. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The changes of ultrastructure were confirmed using a scanning electron microscope.

Results: Melatonin alleviated neomycin-induced hair cell injury in neuromasts (neomycin?+?melatonin 100?μM: 13.88?±?0.91 cells, neomycin only: 7.85?±?0.90 cells; n?=?10, p?Conclusion: Melatonin is effective in alleviating aminoglycoside-induced hair cell injury in zebrafish. The results of this study demonstrated that melatonin has the potential to reduce apoptosis induced by aminoglycosides in zebrafish.     

Aerosolized scopolamine protects against microinstillation inhalation toxicity to sarin in guinea pigs

Sarin is a volatile nerve agent that has been used in the Tokyo subway attack. Inhalation is predicted to be the major route of exposure if sarin is used in war or terrorism. Currently available treatments are limited for effective postexposure protection against sarin under mass casualty scenario. Nasal drug delivery is a potential treatment option for mass casualty under field conditions. We evaluated the efficacy of endotracheal administration of muscarinic antagonist scopolamine, a secretion blocker which effectively crosses the blood-brain barrier for protection against sarin inhalation toxicity. Age and weight matched male Hartley guinea pigs were exposed to 677.4?mg/m³ or 846.5?mg/?m³ (1.2?×?LCt₅₀) sarin by microinstillation inhalation exposure for 4?min. One minute later, the animals exposed to 846.5?mg/?m³ sarin were treated with endotracheally aerosolized scopolamine (0.25?mg/kg) and allowed to recover for 24?h for efficacy evaluation. The results showed that treatment with scopolamine increased the survival rate from 20% to 100% observed in untreated sarin-exposed animals. Behavioral symptoms of nerve agent toxicity including, convulsions and muscular tremors were reduced in sarin-exposed animals treated with scopolamine. Sarin-induced body weight loss, decreased blood O₂ saturation and pulse rate were returned to basal levels in scopolamine-treated animals. Increased bronchoalveolar lavage (BAL) cell death due to sarin exposure was returned to normal levels after treatment with scopolamine. Taken together, these data indicate that postexposure treatment with aerosolized scopolamine prevents respiratory toxicity and protects against lethal inhalation exposure to sarin in guinea pigs.     

Impact of acute and subchronic inhalation exposure to PbO nanoparticles on mice

Lead nanoparticles (NPs) are released into air from metal processing, road transport or combustion processes. Inhalation exposure is therefore very likely to occur. However, even though the effects of bulk lead are well known, there is limited knowledge regarding impact of Pb NPs inhalation. This study focused on acute and subchronic exposures to lead oxide nanoparticles (PbO NPs). Mice were exposed to PbO NPs in whole body inhalation chambers for 4–72?h in acute experiment (4.05?×?10⁶ PbO NPs/cm³), and for 1–11 weeks in subchronic experiment (3.83?×?10⁵ particles/cm³ in lower and 1.93?×?10⁶ particles/cm³ in higher exposure group). Presence of NPs was confirmed in all studied organs, including brain, which is very important considering lead neurotoxicity. Lead concentration gradually increased in all tissues depending on the exposure concentration and duration. The most burdened organs were lung and kidney, however liver and brain also showed significant increase of lead concentration during exposure. Histological analysis documented numerous morphological alterations and tissue damage, mainly in lung, but also in liver. Mild pathological changes were observed also in kidney and brain. Levels of glutathione (reduced and oxidized) were modulated mainly in lung in both, acute and subchronic exposures. Increase of lipid peroxidation was observed in kidney after acute exposure. This study characterized impacts of short to longer-term inhalation exposure, proved transport of PbO NPs to secondary organs, documented time and concentration dependent gradual increase of Pb concentration and histopathological damage in tissues.     

Attempts to counteract phosgene-induced acute lung injury by instant high-dose aerosol exposure to hexamethylenetetramine,cysteine or glutathione

Phosgene is an important high-production-volume intermediate with widespread industrial use. Consistent with other lung irritants causing ALI (acute lung injury), mode-of-action-based countermeasures remain rudimentary. This study was conducted to analyze whether extremely short high-level exposure to phosgene gas could be mitigated using three different inhaled nucleophiles administered by inhalation instantly after exposure to phosgene. Groups of young adult male Wistar rats were acutely exposed to carbonyl chloride (phosgene) using a directed-flow nose-only mode of exposure of 600?mg/m³ for 1.5?min (225?ppm?×?min). Immediately after exposure to phosgene gas the rats were similarly exposed to three strong nucleophiles with and without antioxidant properties for 5 or 15?min. The following nucleophiles were used: hexamethylenetetramine (HMT), l-cysteine (Cys), and l-glutathione (GSH). The concentration of the aerosol (mass median aerodynamic diameter 1.7–2?µm) was targeted to be in the range of 1?mg/L. Cys and GSH have antioxidant properties in addition. The calculated alveolar molar dosage of phosgene was 9 µmol/kg. At 15-min exposure duration, the respective inhaled dose of HMT, Csy, and GSH were 111, 103, and 46 µmol/kg, respectively. The alveolar dose of drugs was ~10-times lower. The efficacy of treatment was judged by protein concentrations in bronchoalveolar lavage fluid (BALF) collected 1 day post-exposure. In spite of using optimized aerosolization techniques, none of the nucleophiles chosen had any mitigating effect on BALF-protein extravasation. This finding appear to suggest that inhaled phosgene gas acylates instantly nucleophilic moieties at the site of initial deposition and that the resultant reaction products can not be reactivated even following instant inhalation treatment with competing nucleophilic agents. In spite of using maximal technically attainable concentrations, it appears to be experimentally challenging to deliver such nucleophiles to the lower respiratory tract at high dosages.