Granulocyte Colony-Stimulating Factor (Neupogen®; Filgrastim) Accelerates Neutrophil Recovery in a Rodent Model of Sulfur Mustard-Induced Hematologic Toxicity.

OBJECTIVE: Evidence of myelosuppression has been negatively correlated with patient outcomes following cases of high dose sulfur mustard (SM) exposure. These hematologic complications can negatively impact overall immune function and increase the risk of infection and life-threatening septicemia. Currently, there are no approved medical treatments for the myelosuppressive effects of SM exposure. METHODS: Leveraging a recently developed rodent model of SM-induced hematologic toxicity, post-exposure efficacy testing of the granulocyte colony-stimulating factor drug Neupogen® was performed in rats intravenously challenged with SM. Before efficacy testing, pharmacokinetic/pharmacodynamic analyses were performed in naïve rats to identify the apparent human equivalent dose of Neupogen® for efficacy evaluation. RESULTS: When administered 1 d after SM-exposure, daily subcutaneous Neupogen® treatment did not prevent the delayed onset of hematologic toxicity but significantly accelerated recovery from neutropenia. Compared with SM controls, Neupogen®-treated animals recovered body weight faster, resolved toxic clinical signs more rapidly, and did not display transient febrility at time points generally concurrent with marked pancytopenia. CONCLUSIONS: Collectively, this work corroborates the results of a previous pilot large animal study, validates the utility of a rodent screening model, and provides further evidence for the potential clinical utility of Neupogen® as an adjunct treatment following SM exposure.

Assessment of Beta-2 Microglobulin Gene Edited Airway Epithelial Stem Cells as a treatment for Sulfur Mustard Inhalation.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient's immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

A Rodent Model of Sulfur Mustard Hematologic Toxicity for the Efficacy Evaluation of Candidate Medical Countermeasures.

INTRODUCTION: While exposure to sulfur mustard (SM) is commonly associated with the production of vesicating dermal, ocular, and respiratory injuries, systemic damage to bone marrow and lymphatic tissue can decrease critical immune cell populations leading to higher susceptibility to life-threatening infection and septicemia. There are currently no approved medical countermeasures for SM-induced myelosuppression. An intravenous SM challenge model was developed in adult rats as a preliminary proof-of-principle platform to evaluate the efficacy of candidate immunostimulants. MATERIALS AND METHODS: Adult male and female Sprague Dawley rats were exposed to SM through tail vein injection. Toxicity progression was monitored through clinical observations, body weights, body temperatures, hematology, serum clinical chemistry, and flow cytometry of blood and bone marrow samples. RESULTS: Following SM exposure, overt toxicity progression was characterized by weight loss, changes in body temperature, and manifestation of toxic clinical signs (diarrhea, lethargy, hunched posture, rough hair coat, respiratory distress, and death). Drastic alterations in complete blood cell profiles included an early-onset lymphopenia followed by a delayed-onset neutropenia and thrombocytopenia. Only transient changes in serum clinical chemistry parameters were observed. Flow cytometry analysis of circulating blood revealed that B-cells were more predominantly affected by SM exposure than T-cells. Challenge with SM resulted in loss of hematopoietic and mesenchymal stem cell populations in the bone marrow. CONCLUSIONS: The small animal model developed in this study replicates many key aspects of human SM exposures and should serve as a relevant, rapid, and cost-effective platform to screen candidate medical countermeasures for SM-induced hematologic toxicity.

A novel sulfur mustard (HD) vapor inhalation exposure model of pulmonary toxicity for the efficacy evaluation of candidate medical countermeasures.

OBJECTIVE: To develop a novel inhalation exposure system capable of delivering a controlled inhaled HD dose through an endotracheal tube to anesthetized rats to investigate the lung pathophysiology and evaluate potential medical countermeasures. MATERIALS AND METHODS: Target HD vapor exposures were generated by a temperature-controlled vapor generator, while concentration was monitored near real-time by gas chromatography. Animal breathing parameters were monitored real-time by in-line EMKA/SciReq pulmonary analysis system. Individual exposures were halted when the target inhaled doses were achieved. Animals were observed daily for clinical observations and lethality with scheduled termination at 28 days post-exposure. Upon scheduled or unscheduled death, animals underwent a gross necropsy and lung and trachea were collected for histopathology. RESULTS: Controlled HD concentrations ranged from 60 to 320 mg/m3. Delivered inhaled doses range from 0.3 to 3.20 mg/kg with administered doses within 3% of the target. The 28-day inhaled LD50 is 0.80 mg/kg (95% CI = 0.42-1.18 mg/kg). Post exposure respiratory abnormalities were observed across all dose levels though the higher dose levels had earlier onset and higher frequency of occurrence. Histopathologic alterations were not qualitatively altered in accordance with dose but instead showed a relationship to an animals' time of death, with early deaths demonstrating acute damage and later deaths displaying signs of repair. DISCUSSION/CONCLUSION: This novel exposure system administers targeted HD inhaled doses to generate a small animal model that can be used to evaluate physiological toxicities of inhaled HD on the lungs and for evaluation of potential medical countermeasure treatments.

Development of a clinical assay to measure chlorinated tyrosine in hair and tissue samples using a mouse chlorine inhalation exposure model.

Chlorine is a toxic industrial chemical with a history of use as a chemical weapon. Chlorine is also produced, stored, and transported in bulk making it a high-priority pulmonary threat in the USA. Due to the high reactivity of chlorine, few biomarkers exist to identify exposure in clinical and environmental samples. Our laboratory evaluates acute chlorine exposure in clinical samples by measuring 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Individuals can have elevated biomarker levels due to their environment and chronic health conditions, but levels are significantly lower in individuals exposed to chlorine. Historically these biomarkers have been evaluated in serum, plasma, blood, and bronchoalveolar lavage (BAL) fluid. We report the expansion into hair and lung tissue samples using our newly developed tissue homogenization protocol which fits seamlessly with our current chlorinated tyrosine quantitative assay. Furthermore, we have updated the chlorinated tyrosine assay to improve throughput and ruggedness and reduce sample volume requirements. The improved assay was used to measure chlorinated tyrosine levels in 198 mice exposed to either chlorine gas or air. From this animal study, we compared Cl-Tyr and Cl2-Tyr levels among three matrices (i.e., lung, hair, and blood) and found that hair had the most abundant chlorine exposure biomarkers. Furthermore, we captured the first timeline of each analyte in the lung, hair, and blood samples. In mice exposed to chlorine gas, both Cl-Tyr and Cl2-Tyr were present in blood and lung samples up to 24 h and up to 30 days in hair samples.

A Novel, Modified Human Butyrylcholinesterase Catalytically Degrades the Chemical Warfare Nerve Agent, Sarin.

Chemical warfare nerve agents (CWNAs) present a global threat to both military and civilian populations. The acute toxicity of CWNAs stems from their ability to effectively inhibit acetylcholinesterase (AChE). This inhibition can lead to uncontrolled cholinergic cellular signaling, resulting in cholinergic crisis and, ultimately, death. Although the current FDA-approved standard of care is moderately effective when administered early, development of novel treatment strategies is necessary. Butyrylcholinesterase (BChE) is an enzyme which displays a high degree of structural homology to AChE. Unlike AChE, the roles of BChE are uncertain and possibilities are still being explored. However, BChE appears to primarily serve as a bioscavenger of toxic esters due to its ability to accommodate a wide variety of substrates within its active site. Like AChE, BChE is also readily inhibited by CWNAs. Due to its high affinity for binding CWNAs, and that null-BChE yields no apparent health effects, exogenous BChE has been explored as a candidate therapeutic for CWNA intoxication. Despite years of research, minimal strides have been made to develop a catalytic bioscavenger. Furthermore, BChE is only in early clinical trials as a stoichiometric bioscavenger of CWNAs, and large quantities must be administered to treat CWNA toxicity. Here, we describe previously unidentified mutations to residues within and adjacent to the acyl binding pocket (positions 282-285 were mutagenized from YGTP to NHML) of BChE that confer catalytic degradation of the CWNA, sarin. These mutations, along with corresponding future efforts, may finally lead to a novel therapeutic to combat CWNA intoxication.

Characterization of Cholinesterases From Multiple Large Animal Species for Medical Countermeasure Development Against Chemical Warfare Nerve Agents.

Organophosphorus (OP) compounds, which include insecticides and chemical warfare nerve agents (CWNAs) such as sarin (GB) and VX, continue to be a global threat to both civilian and military populations. It is widely accepted that cholinesterase inhibition is the primary mechanism for acute OP toxicity. Disruption of cholinergic function through the inhibition of acetylcholinesterase (AChE) leads to the accumulation of the neurotransmitter acetylcholine. Excess acetylcholine at the synapse results in an overstimulation of cholinergic neurons which manifests in the common signs and symptoms of OP intoxication (miosis, increased secretions, seizures, convulsions, and respiratory failure). The primary therapeutic strategy employed in the United States to treat OP intoxication includes reactivation of inhibited AChE with the oxime pralidoxime (2-PAM) along with the muscarinic acetylcholine receptor antagonist atropine and the benzodiazepine, diazepam. CWNAs are also known to inhibit butyrylcholinesterase (BChE) without any apparent toxic effects. Therefore, BChE may be viewed as a "bioscavenger" that stoichiometrically binds CWNAs and removes them from circulation. The degree of inhibition of AChE and BChE and the effectiveness of 2-PAM are known to vary among species. Animal models are imperative for evaluating the efficacy of CWNA medical countermeasures, and a thorough characterization of available animal models is important for translating results to humans. Thus, the objective of this study was to compare the circulating levels of each of the cholinesterases as well as multiple kinetic properties (inhibition, reactivation, and aging rates) of both AChE and BChE derived from humans to AChE and BChE derived from commonly used large animal models.

Efficacy and pharmacokinetic/pharmacodynamic study of 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate in guinea pigs and rhesus macaques exposed to cyclosarin.

Male Hartley guinea pigs and male rhesus macaques were used to determine an efficacious dose of 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate (MMB4 DMS) that would result in 80% survival, 24 hours following a single exposure to cyclosarin (GF). The pharmacokinetic/pharmacodynamic relationship between acetylcholinesterase activity and MMB4 plasma concentrations relative to survival was evaluated. Guinea pigs and non-human primates (NHPs) were concurrently administered MMB4 DMS (guinea pigs: 0, 10, 30, or 40 mg/kg, intramuscular [IM] and NHPs: 0.1, 1, 5, 10, or 20 mg/kg, IM), atropine, and diazepam following a 3 × median lethal dose (LD50) GF challenge. Clinical observations were evaluated using a quality-of-life (QOL) scoring system. All GF-exposed animals exhibited typical signs of nerve agent poisoning immediately following challenge. In guinea pigs, 24-hour survival was 0%, 50%, 90%, and 90% for 0, 10, 30, and 40 mg/kg MMB4 DMS groups, respectively. In addition, nearly all animals surviving to 24 hours were clinically normal, with many in the 30 and 40 mg/kg MMB4 DMS dose group observed as normal by 4 hours post-challenge. In NHPs, survival was 100% for all treatment groups, with all animals noted as clinically normal by 48 hours. Following treatment with atropine/MMB4 DMS/diazepam, NHPs exhibited dose- and temporal-related decreases in incidence and duration of the clinical signs of toxicity. The QOL scores improved with increasing MMB4 DMS dose in both species. The estimated ED80s were 25.5 mg/kg MMB4 DMS (human equivalent dose [HED] of 5.5 mg/kg) and ≤ 0.1 mg/kg (HED of 0.03 mg/kg) in guinea pigs and NHPs, respectively.

In vivo acetylcholinesterase reactivation in male guinea pigs and rhesus macaques following cyclosarin exposure and treatment with 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate.

Acetylcholinesterase (AChE) reactivation studies were conducted in guinea pigs (GPs) and nonhuman primates (NHPs) to determine the 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate (MMB4 DMS) dose that reactivated at least 20% of blood AChE within 15 minutes following cyclosarin (GF) dosing (used as the criterion for efficacy). Male GPs and male rhesus macaques (NHPs) were pretreated with atropine 15 minutes prior to GF administration (1 × median lethal dose [LD50]) and MMB4 DMS 15 minutes following GF administration. The GP survival was 5 of 8, 8 of 8, 8 of 8, and 6 of 8 for the 0.75, 3.0, 6.0, or 12.0 mg/kg MMB4 DMS treatment groups, respectively. In NHPs, survival was 6 of 6 at 0.5, 1.2, 3.0, or 9.3 mg/kg MMB4 DMS, respectively, 24 hours post-challenge, with the majority of animals noted as clinically normal by 24 hours. Pharmacokinetic/pharmacodynamic modeling revealed that 1.8 mg/kg in GPs or 0.013 mg/kg in NHPs would result in an average 20% reactivation; human equivalent doses were calculated as 0.39 mg/kg (based on GP data) and 0.004 mg/kg (based on NHP data). The model suggested that MMB4 plasma concentrations of 1000 ng/mL and AChE reactivation of 80% would be most effective. Although a 0.5 mg/kg MMB4 DMS dose in NHPs resulted in 100% survival and an average of 78% AChE reactivation, adverse effects associated with GF administration were still observed 24 hours post-challenge (tremors, mydriasis, and weakness were observed in 3 of 6 animals). In comparison, 6 of 6 animals treated with 1.2 mg/kg MMB4 DMS were observed as clinically normal 24 hours post-challenge.

Indicators of oxidative stress and apoptosis in mouse whole lung and Clara cells following exposure to styrene and its metabolites.

In mice, styrene is hepatotoxic, pneumotoxic, and causes lung tumors. One explanation for the mechanism of toxicity is oxidative stress/damage. Previous studies have shown decreased glutathione levels, linked to increased apoptosis, in lung homogenates and isolated Clara cells 3 h following styrene or styrene oxide (SO) administration or in vitro exposure. The objective of the current studies was to determine what effects styrene and its active metabolites, primarily styrene oxide, had on indicators of oxidative stress and attendant apoptosis in order to understand better the mechanism of styrene-induced toxicity. Three hours following in vitro exposure of Clara cells to styrene or SO there were increases in reactive oxygen species (ROS). Following administration of styrene or styrene oxide ip, increases in ROS, superoxide dismutase (SOD), and 8-hydroxydeoxyguanosine (8-OHdG) formation were observed. Since increases in ROS have been linked to increases in apoptosis ratios of bax/bcl-2, mRNA and protein expression were determined 3-240 h following the administration of styrene and R-styrene oxide (RSO). The bax/bcl-2 mRNA ratio increased 12 and 24 h following R-SO and 120 h following styrene administration. However, the bax/bcl-2 protein ratio was not increased until 240 h following R-SO, and 24 and 240 h following styrene administration. However, only a slight increase in caspase 3 was observed. These results indicated that oxidative stress occurred 3h following styrene or styrene oxide as evidenced by increased ROS and SOD. This increased ROS may be responsible for the increased 8-OHdG formation. Our findings of limited apoptosis in Clara cells following acute exposure to styrene or SO are in agreement with others and may reflect the minimal extent to which apoptosis plays a role in acute styrene toxicity. It is clear, however, that oxidative stress and oxidative effects on DNA are increased following exposure to styrene or styrene oxide, and these may play a role in the lung tumorigenesis in mice.

Effect of multiple doses of styrene and R-styrene oxide on CC10, bax, and bcl-2 expression in isolated Clara cells of CD-1 mice.

Styrene exposure is highest among workers in the reinforced plastics industry with exposure seen for 5 consecutive days during the work week. Styrene is both hepatotoxic and pneumotoxic in mice, in addition to causing lung tumors. Human epidemiological studies are inconclusive as to the carcinogenicity of styrene so it is important to understand the mechanism responsible for styrene tumors in mice. Previous studies showed significant decreases in CC10 protein for 5 days following a single dose of the active metabolite R-styrene oxide (R-SO), yet little change in the bax/bcl-2 protein ratio was seen until 10 days following styrene or R-SO administration. Styrene or R-SO was given to CD-1 mice for 5 consecutive days. Mice were euthanized 24h, 10 days or 30 days following the last dose, and CC10, bax and bcl-2 mRNA and protein levels were determined in isolated Clara cells. CC10 mRNA levels were decreased at 24h for both styrene and R-SO. R-SO decreased CC10 protein levels up to 10 days following the last dose. Increases in the bax/bcl-2 mRNA and protein ratio were seen 24h following R-SO administration. Styrene did not significantly increase the bax/bcl-2 mRNA ratio until 10 days after treatment, with the bax/bcl-2 protein ratio increased at both 10 days and 30 days. It is likely that oxidative stress is involved in the toxicity caused by styrene and that minimal apoptosis may be involved. Chronically decreased CC10 levels may lead to increases in oxidative stress in Clara cells, the main target for styrene toxicity in the lung, and may be an early indicator for lung carcinogenesis in mice.

CC10 mRNA and protein expression in Clara cells of CD-1 mice following exposure to styrene or its metabolites styrene oxide or 4-vinylphenol.

Styrene, widely used in manufacturing, has both acute and chronic effects in humans. In mice, styrene is both hepato- and pneumo-toxic and causes lung tumors. The primary site for styrene metabolism and its effects in mouse lung is the Clara cell, which secretes Clara cell 10kDa protein (CC10) and surfactant protein A (SPA). Both play important roles in host defenses and inflammation prevention. The mode of action for styrene-induced lung tumor formation has yet to be elicited, yet one possibility relates to oxidative stress and decreased CC10 levels. CC10 mRNA and protein expression were measured in isolated Clara cells 3, 12, and 24h following in vivo administration of styrene (600mg/kg i.p.) or its metabolites [R-, S-, racemic styrene oxide (SO) (300mg/kg i.p.), 4-vinylphenol (100mg/kg i.p.)]. The largest decreases in CC10 mRNA expression were seen with R-SO and racemic SO at 24h. To determine if rebound effects would be seen, CC10 mRNA and protein expression were determined 48, 120, and 240h following styrene and R-SO administration. The CC10 protein level did not reach its lowest point to correlate with mRNA expression until 120h after R-SO administration. Styrene exposure caused a significant decrease in CC10 protein after 24h, rebounding through 240h. SPA protein expression showed little change from control levels, indicating a more specific effect on CC10 in the Clara cell by styrene and its metabolites. These studies demonstrate that acute changes in lung CC10 protein and mRNA expression do occur following in vivo treatment with styrene and its metabolites. These changes may be early indicators for a potential mechanism for lung tumor formation in mice as it relates to oxidative stress and the possibility deserves further study.

Comparison of styrene and its metabolites styrene oxide and 4-vinylphenol on cytotoxicity and glutathione depletion in Clara cells of mice and rats.

Styrene is a widely used compound in the manufacturing industry. In mice and rats, it is both hepatotoxic and pneumotoxic. It causes lung tumors in mice, but not in rats. The Clara cell is the main target for the toxicity of styrene and its metabolites, and it also has the greatest activity for styrene metabolism. Therefore, Clara cells isolated from CD-1 mice and Sprague-Dawley rats were used to compare the cytotoxicities induced by styrene and its metabolites. The cytotoxicity of styrene was greater in vitro than that of its metabolites styrene oxide (racemic, R- and S-) and 4-vinylphenol in contrast with what has been observed in vivo in previous studies on hepatotoxicity and pneumotoxicity. Susceptibility of rats to styrene and its metabolites are 4-fold less than that observed with mice. Glutathione levels were also measured in mice following addition of the chemicals in vitro and treatment of the CD-1 mice in vivo. Decreases in glutathione concentrations were seen even at doses which did not cause the death of mouse Clara cells. Significant decreases in glutathione were observed 3h after treatment with racemic SO and R-SO. At 12h, rebound effects were seen for all compounds, with all but R-SO rebounding above controls. These studies suggest that in vitro cytotoxicity of styrene and its metabolites does not strictly follow in vivo effects and that decreases in mouse glutathione levels may be related to oxidative stress.