Effect of TRPV4 Antagonist GSK2798745 on Chlorine Gas-Induced Acute Lung Injury in a Swine Model.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Plutonium Systemic Biokinetic Model for Rats.

A baseline compartmental model (relative to modeling decorporation) of the distribution and retention of plutonium (Pu) in the rat for a systemic intake is derived. The model is derived from data obtained from a study designed to evaluate the behavior of plutonium in the first 28 days after incorporation. The model is based on a recently published model of americium (Am) in rats, which incorporated a pharmacokinetic (PK)-front-end modeling approach, which was used to specify transfer to and from the extracellular fluids (ECF) in the various tissues in terms of vascular flow and volumes of ECF. In the americium model, the approach was "cell-membrane limited," meaning that rapid diffusion of americium occurred throughout all the extracellular fluids (i.e., the blood plasma and interstitial fluids), while back-end rates representing transport into and out of the cells were determined empirically. However, this approach was inconsistent with the plutonium dataset. A good fit to the data is obtained by incorporating aspects of the Durbin et al. model structure, with plutonium in plasma separated into "free" and "bound" components. Free plutonium uses a cell-membrane-limited front end as for americium. Bound plutonium uses a capillary-wall-limited front end, where transfer rates from blood plasma into the interstitial fluids are relatively slow, and must be determined either empirically or from a priori knowledge. As in the Durbin et al. model, both free and bound plutonium are available for deposition in bone. In addition, our model has some bound plutonium associated with uptake to the gastrointestinal (GI) tract. Uncertainties in transfer rates were investigated using Markov Chain Monte Carlo (MCMC). It is anticipated that this model structure of plutonium will also be useful in interpreting comparable data from decorporation studies done in experimental animals.

Developing and comparing models of hematopoietic-acute radiation syndrome in Göttingen and Sinclair minipigs.

PURPOSE: Current animal models of hematopoietic-acute radiation syndrome (H-ARS) are resource intensive and have limited translation to humans, thereby inhibiting the development of effective medical countermeasures (MCM)s for radiation exposure. MATERIALS AND METHODS: To improve the MCM pipeline, we developed models of H-ARS in male Göttingen and Sinclair minipigs. Weight matched Göttingens and Sinclairs received total body irradiation (TBI; 1.50-2.10 Gy and 1.94-2.90 Gy, respectively), were observed for up to 45 days with blood collections for clinical pathology analysis, and were examined during gross necropsy. RESULTS: The lethal dose for 50% of the population over the course of 45 days (LD50/45) with 'field' supportive care (primarily antibiotics and hydration support) and implanted vascular access ports was 1.89 and 2.53 Gy for Göttingens and Sinclairs, respectively. Both minipig strains exhibited prototypical H-ARS characteristics, experiencing thrombocytopenia and neutropenia, and nadirs approximately 14 days following irradiation, slightly varying with dose. Both strains experienced increased bruising, petechia, and signs of internal hemorrhage in the lungs, GI, heart, and skin. All observations were noted to correlate with dose more closely in Sinclairs than in Göttingens. CONCLUSION: The results of this study provide a template for future MCM development in an alternate species, and support further development of the Göttingen and Sinclair minipig H-ARS models.

Dose and Dose-Rate Effects in a Mouse Model of Internal Exposure from 137Cs. Part 2: Integration of Gamma-H2AX and Gene Expression Biomarkers for Retrospective Radiation Biodosimetry.

Inhalation and ingestion of 137Cs and other long-lived radionuclides can occur after large-scale accidental or malicious radioactive contamination incidents, resulting in a complex temporal pattern of radiation dose/dose rate, influenced by radionuclide pharmacokinetics and chemical properties. High-throughput radiation biodosimetry techniques for such internal exposure are needed to assess potential risks of short-term toxicity and delayed effects (e.g., carcinogenesis) for exposed individuals. Previously, we used γ-H2AX to reconstruct injected 137Cs activity in experimentally-exposed mice, and converted activity values into radiation doses based on time since injection and 137Cs-elimination kinetics. In the current study, we sought to assess the feasibility and possible advantages of combining γ-H2AX with transcriptomics to improve 137Cs activity reconstructions. We selected five genes (Atf5, Hist2h2aa2, Olfr358, Psrc1, Hist2h2ac) with strong statistically-significant Spearman's correlations with injected activity and stable expression over time after 137Cs injection. The geometric mean of log-transformed signals of these five genes, combined with γ-H2AX fluorescence, were used as predictors in a nonlinear model for reconstructing injected 137Cs activity. The coefficient of determination (R2) comparing actual and reconstructed activities was 0.91 and root mean squared error (RMSE) was 0.95 MBq. These metrics remained stable when the model was fitted to a randomly-selected half of the data and tested on the other half, repeated 100 times. Model performance was significantly better when compared to our previous analysis using γ-H2AX alone, and when compared to an analysis where genes are used without γ-H2AX, suggesting that integrating γ-H2AX with gene expression provides an important advantage. Our findings show a proof of principle that integration of radiation-responsive biomarkers from different fields is promising for radiation biodosimetry of internal emitters.

Dose and Dose-Rate Effects in a Mouse Model of Internal Exposure to 137Cs. Part 1: Global Transcriptomic Responses in Blood.

Internal contamination by radionuclides may constitute a major source of exposure and biological damage after radiation accidents and potentially in a dirty bomb or improvised nuclear device scenario. We injected male C57BL/6 mice with radiolabeled cesium chloride solution (137CsCl) to evaluate the biological effects of varying cumulative doses and dose rates in a two-week study. Injection activities of 137CsCl were 5.71, 6.78, 7.67 and 9.29 MBq, calculated to achieve a target dose of 4 Gy at days 14, 7, 5 and 3, respectively. We collected whole blood samples at days 2, 3, 5, 7 and 14 so that we can publish the issue in Decemberfrom all injection groups and measured gene expression using Agilent Mouse Whole Genome microarrays. We identified both dose-rate-independent and dose-rate-dependent gene expression responses in the time series. Gene Ontology analysis indicated a rapid and persistent immune response to the chronic low-dose-rate irradiation, consistent with depletion of radiosensitive B cells. Pathways impacting platelet aggregation and TP53 signaling appeared activated, but not consistently at all times in the study. Clustering of genes by pattern and identification of dose-rate-independent and -dependent genes provided insight into possible drivers of the dynamic transcriptome response in vivo, and also indicated that TP53 signaling may be upstream of very different transcript response patterns. This characterization of the biological response of blood cells to internal radiation at varying doses and dose rates is an important step in understanding the effects of internal contamination after a nuclear event.

Serum Metabolomic Alterations Associated with Cesium-137 Internal Emitter Delivered in Various Dose Rates.

Our laboratory and others have use radiation metabolomics to assess responses in order to develop biomarkers reflecting exposure and level of injury. To expand the types of exposure and compare to previously published results, metabolomic analysis has been carried out using serum samples from mice exposed to 137Cs internal emitters. Animals were injected intraperitoneally with 137CsCl solutions of varying radioactivity, and the absorbed doses were calculated. To determine the dose rate effect, serum samples were collected at 2, 3, 5, 7, and 14 days after injection. Based on the time for each group receiving the cumulative dose of 4 Gy, the dose rate for each group was determined. The dose rates analyzed were 0.16 Gy/day (low), 0.69 Gy/day (medium), and 1.25 Gy/day (high). The results indicated that at a cumulative dose of 4 Gy, the low dose rate group had the least number of statistically significantly differential spectral features. Some identified metabolites showed common changes for different dose rates. For example, significantly altered levels of oleamide and sphingosine 1-phosphate were seen in all three groups. On the other hand, the intensity of three amino acids, Isoleucine, Phenylalanine and Arginine, significantly decreased only in the medium dose rate group. These findings have the potential to be used in assessing the exposure and the biological effects of internal emitters.

Americium Systemic Biokinetic Model for Rats.

In this work, a baseline compartmental model of the distribution and retention of americium in the rat for a systemic intake was derived. The model was derived from data obtained from a study designed to evaluate the behavior of americium in the first 28 days after incorporation. A pharmacokinetic (PK)-front-end modeling approach was used to specify transfer to and from the extracellular fluids (ECF) in the various tissues in terms of vascular flow and volumes of ECF. Back-end rates representing transport into and out of the cells were determined empirically. Uncertainties in transfer rates were investigated using Markov chain Monte Carlo (MCMC). The combination of PK-front-end model and the back-end model structure used allowed for extrapolation to the earliest times with small uncertainty. This approach clearly demonstrated the rapid transfer of material from ECF to liver and bone. This model provides a baseline for modeling the action of decorporation agents, such as DTPA.

Effect of dose and dose rate on temporal γ-H2AX kinetics in mouse blood and spleen mononuclear cells in vivo following Cesium-137 administration.

BACKGROUND: Cesium-137 (137Cs) is one of the major and most clinically relevant radionuclides of concern in a radiological dispersal device, "dirty bomb" scenario as well as in nuclear accidents and detonations. In this exposure scenario, a significant amount of soluble radionuclide(s) may be dispersed into the atmosphere as a component of fallout. The objectives of the present study were to investigate the effect of protracted 137Cs radionuclide exposures on DNA damage in mouse blood and spleen mononuclear cells (MNCs) in vivo using the γ-H2AX biomarker, and to develop a mathematical formalism for these processes. RESULTS: C57BL/6 mice were injected with a range of 137CsCl activities (5.74, 6.66, 7.65 and 9.28 MBq) to achieve total-body committed doses of ~ 4 Gy at Days 3, 5, 7, and 14. Close to 50% of 137Cs was excreted by day 5, leading to a slower rate of decay for the remaining time of the study; 137Cs excretion kinetics were independent of activity level within the tested range, and the absorbed radiation dose was determined by injected activity and time after injection. Measurements of γ-H2AX fluorescence in blood and spleen MNCs at each time point were used to develop a new biodosimetric mathematical formalism to estimate injected activity based on γ-H2AX fluorescence and time after injection. The formalism performed reasonably well on blood data at 2-5 days after injection: Pearson and Spearman's correlation coefficients between actual and predicted activity values were 0.857 (p = 0.00659) and 0.929 (p = 0.00223), respectively. CONCLUSIONS: Despite the complicated nature of the studied biological system and the time-dependent changes in radiation dose and dose rate due to radionuclide excretion and other processes, we have used the γ-H2AX repair kinetics to develop a mathematical formalism, which can relatively accurately predict injected 137Cs activity 2-5 days after initial exposure. To determine the assay's usefulness to predict retrospective absorbed dose for medical triage, further studies are required to validate the sensitivity and accuracy of the γ-H2AX response after protracted exposures.

The pharmacokinetics of ketamine following intramuscular injection to F344 rats.

Ketamine is a glutamate N-methyl-D-aspartate receptor antagonist that is a rapid-acting dissociative anesthetic. It has been proposed as an adjuvant treatment along with other drugs (atropine, midazolam, pralidoxime) used in the current standard of care (SOC) for organophosphate and nerve agent exposures. Ketamine is a pharmaceutical agent that is readily available to most clinicians in emergency departments and possesses a broad therapeutic index with well-characterized effects in humans. The objective of this study was to determine the pharmacokinetic profile of ketamine and its active metabolite, norketamine, in F344 rats following single or repeated intramuscular administrations of subanesthetic levels (7.5 mg/kg or 30 mg/kg) of ketamine with or without the SOC. Following administration, plasma and brain tissues were collected and analyzed using a liquid chromatography-mass spectrometry method to quantitate ketamine and norketamine. Following sample analysis, the pharmacokinetics were determined using non-compartmental analysis. The addition of the current SOC had a minimal impact on the pharmacokinetics of ketamine following intramuscular administration and repeated dosing at 7.5 mg/kg every 90 minutes allows for sustained plasma concentrations above 100 ng/mL. The pharmacokinetics of ketamine with and without the SOC in rats supports further investigation of the efficacy of ketamine co-administration with the SOC following nerve agent exposure in animal models.

Addition of ketamine to standard-of-care countermeasures for acute organophosphate poisoning improves neurobiological outcomes.

Rats poisoned with sarin enter into ahyper-cholinergic crisis characterized by excessive salivation, respiratory distress, tremors, seizures, and death. Through the use of rescue medications and an anticonvulsant, death can be avoided in many animals, with the long-term consequences of poisoning partly ameliorated, especially when countermeasures are made available immediately after exposure. However, when anticonvulsant measures are delayed by as little as 30 min, clinical, neurological, cognitive, and psychiatric abnormalities may persist long after the initial exposure. This study sought to determine if the addition of the NMDA receptor antagonist Ketamine to human standard-of-care countermeasures consisting of two rescue medications (2-PAM and atropine) and an anti-convulsant (Midazolam), would afford protection against persistent neurobiological compromise. Rats were exposed to sarin (105 µg/kg via subcutaneous injection), and treated 1 min later with 2-PAM and Atropine Methyl Nitrate (IM) to minimize mortality. One of four anti-convulsant protocols was then initiated at 50 min postsarin:Midazolam alone (MDZ, a single injection (IM) at 0.66 mg/kg); Ketamine alone (KET, a series of five injections (IM) of Ketamine at 7.5 mg/kg, 90 min apart); Midazolam + low dose Ketamine (MDZ + lowKET, a single injection of Midazolam (IM) at 0.66 mg/kg, plus five sequential doses of ketamine (IM) at 2.5 mg/kg, starting at the time of Midazolam dosing and then 90 min apart); Midazolam + high dose Ketamine (MDZ + highKET, a single injection of Midazolam (IM) at 0.66 mg/kg, plus five sequential injections of 7.5 mg/kg Ketamine (IM), starting at the time of Midazolam dosing and then 90 min apart). Animals were preassigned to groups culled at post-exposure Days 1, 7 or 30, for histopathology. For all surviving animals, EEG activity was monitored through skull electrodes for 24-h beginning immediately after sarin exposure. Surviving animals also underwent 24-h EEG monitoring on Days 6, 13, and/or 29, post-sarin. Memory assessment using the Morris Water Maze was performed on Days 1, 4, 7, 14 and 30. Following sarin exposure, 85% of surviving animals demonstrated status epilepticus within 20 min. Each of the anti-convulsant protocols was sufficient to stop convulsions within 1 h of anti-convulsant administration, but all of the animals still showed signs of electrographic status for an additional 2-12 h, without substantial differentiation between treatment groups. However, for post-sarin hours 13-24, the MDZ + highKET group showed significantly less severe EEG abnormalities than the MDZ and KET groups (Mood's Median Test, p < 0.005). At one month post-exposure, 90% of animals that had received Midazolam alone still showed evidence of some epileptiform activity. In contrast, 90% of animals that had received Midazolam + high dose Ketamine combination therapy had EEG profiles that were within normal limits. This difference in EEG outcomes was highly significant (Mood's Median Test, p < 0.001). Likewise, on the water maze, the majority of animals that had received Midazolam combined with either high or low dose Ketamine therapy returned to near baseline levels of mnemonic performance within 2 weeks, whereas the majority of the animals that had received midazolam alone or ketamine alone demonstrated persistent and significant memory impairments even at one month postexposure (Mood's Median Test, p < 0.005). With respect to neuronal necrosis, animals in the MDZ + highKET group showed significantly less overall damage than animals in other treatment groups (Mood's Median Test, p < 0.001). Of special note were findings in the hippocampus, where only 12% of animals in the MDZ + highKET group showed evidence of necrosis on H&E staining, whereas 100% of animals in the KET group, 70% of animals in the MDZ group, and 40% of animals in the MDZ + lowKET group showed evidence of hippocampal necrosis. Overall, the data demonstrate that Ketamine augmentation of an atropine, 2PAM, and Midazolam standard-ofcare for sarin exposure provides clinically-relevant additional protection against the negative neurobiological consequences of sarin, even when initiation of the anti-convulsant countermeasures is delayed by 50 min.

Development of a combined radiation and full thickness burn injury minipig model to study the effects of uncultured adipose-derived regenerative cell therapy in wound healing.

PURPOSE: To develop an approach that models the cutaneous healing that occurs in a patient with full thickness thermal burn injury complicated by total body radiation exposure sufficient to induce sub-lethal prodromal symptoms. An assessment of the effects of an autologous cell therapy on wound healing on thermal burn injury with concomitant radiation exposure was used to validate the utility of the model. METHODS: Göttingen minipigs were subjected to a 1.2 Gy total body irradiation by exposure to a 6 MV X-ray linear accelerator followed by ∼10 cm2 full thickness burns (pre-heated brass block with calibrated spring). Three days after injury, wounds were excised to the underlying fascia and each animal was randomized to receive treatment with autologous adipose-derived regenerative cells (ADRC) delivered by local or intravenous injection, or vehicle control. Blood counts were used to assess radiation-induced marrow suppression. All animals were followed using digital imaging to assess wound healing. Full-thickness biopsies were obtained at 7, 14, 21 and 30 days' post-treatment. RESULTS: Compared to animals receiving burn injury alone, significant transient neutropenia and thrombocytopenia were observed in irradiated subjects with average neutrophil nadir of 0.79 × 103/µl (day 15) and platelet nadir of 60 × 103/µl (day 12). Wound closure through a combination of contraction and epithelialization from the wound edges occurred over a period of approximately 28 days' post excision and treatment. Re-epithelialization was accelerated in wounds treated with ADRC (mean 3.5-fold increase at 2 weeks post-treatment relative to control). This acceleration was accompanied by an average 67% increase in blood vessel density and 30% increase in matrix (collagen) deposition. Similar results were observed when ADRC were injected either directly into the wound or by intravenous administration. CONCLUSIONS: Although preliminary, this study provides a reproducible minipig model of thermal burn injury complicated by myelosuppressive total body irradiation that utilizes standardized procedures to evaluate novel countermeasures for potential use following attack by an improvised nuclear device.

Quantitative Metabolomic Analysis of Urinary Citrulline and Calcitroic Acid in Mice after Exposure to Various Types of Ionizing Radiation.

With the safety of existing nuclear power plants being brought into question after the Fukushima disaster and the increased level of concern over terrorism-sponsored use of improvised nuclear devices, it is more crucial to develop well-defined radiation injury markers in easily accessible biofluids to help emergency-responders with injury assessment during patient triage. Here, we focused on utilizing ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify and quantitate the unique changes in the urinary excretion of two metabolite markers, calcitroic acid and citrulline, in mice induced by different forms of irradiation; external γ irradiation at a low dose rate (LDR) of 3.0 mGy/min and a high dose rate (HDR) of 1.1 Gy/min, and internal exposure to Cesium-137 ((137)Cs) and Strontium-90 ((90)Sr). The multiple reaction monitoring analysis showed that, while exposure to (137)Cs and (90)Sr induced a statistically significant and persistent decrease, similar doses of external γ beam at the HDR had the opposite effect, and the LDR had no effect on the urinary levels of these two metabolites. This suggests that the source of exposure and the dose rate strongly modulate the in vivo metabolomic injury responses, which may have utility in clinical biodosimetry assays for the assessment of exposure in an affected population. This study complements our previous investigations into the metabolomic profile of urine from mice internally exposed to (90)Sr and (137)Cs and to external γ beam radiation.

γ-H2AX Kinetic Profile in Mouse Lymphocytes Exposed to the Internal Emitters Cesium-137 and Strontium-90.

In the event of a dirty bomb scenario or an industrial nuclear accident, a significant dose of volatile radionuclides such as 137Cs and 90Sr may be dispersed into the atmosphere as a component of fallout and inhaled or ingested by hundreds and thousands of people. To study the effects of prolonged exposure to ingested radionuclides, we have performed long-term (30 day) internal-emitter mouse irradiations using soluble-injected 137CsCl and 90SrCl2 radioisotopes. The effect of ionizing radiation on the induction and repair of DNA double strand breaks (DSBs) in peripheral mouse lymphocytes in vivo was determined using the γ-H2AX biodosimetry marker. Using a serial sacrifice experimental design, whole-body radiation absorbed doses for 137Cs (0 to 10 Gy) and 90Sr (0 to 49 Gy) were delivered over 30 days following exposure to each radionuclide. The committed absorbed doses of the two internal emitters as a function of time post exposure were calculated based on their retention parameters and their derived dose coefficients for each specific sacrifice time. In order to measure the kinetic profile for γ-H2AX, peripheral blood samples were drawn at 5 specific timed dose points over the 30-day study period and the total γ-H2AX nuclear fluorescence per lymphocyte was determined using image analysis software. A key finding was that a significant γ-H2AX signal was observed in vivo several weeks after a single radionuclide exposure. A mechanistically-motivated model was used to analyze the temporal kinetics of γ-H2AX fluorescence. Exposure to either radionuclide showed two peaks of γ-H2AX: one within the first week, which may represent the death of mature, differentiated lymphocytes, and the second at approximately three weeks, which may represent the production of new lymphocytes from damaged progenitor cells. The complexity of the observed responses to internal irradiation is likely caused by the interplay between continual production and repair of DNA damage, cell cycle effects and apoptosis.

Serum Dyslipidemia Is Induced by Internal Exposure to Strontium-90 in Mice, Lipidomic Profiling Using a Data-Independent Liquid Chromatography-Mass Spectrometry Approach.

Despite considerable research into the environmental risks and biological effects of exposure to external beam γ rays, incorporation of radionuclides has largely been understudied. This dosimetry and exposure risk assessment is challenging for first responders in the field during a nuclear or radiological event. Therefore, we have developed a workflow for assessing injury responses in easily obtainable biofluids, such as urine and serum, as the result of exposure to internal emitters cesium-137 ((137)Cs) and strontium-90 ((90)Sr) in mice. Here we report on the results of the untargeted lipidomic profiling of serum from mice exposed to (90)Sr. We also compared these results to those from previously published (137)Cs exposure to determine any differences in cellular responses based on exposure type. The results of this study conclude that there is a gross increase in the serum abundance of triacylglycerides and cholesterol esters, while phostaphatidylcholines and lysophosphatidylcholines displayed decreases in their serum levels postexposure at study days 4, 7, 9, 25, and 30, with corresponding average cumulative skeleton doses ranging from 1.2 ± 0.1 to 5.2 ± 0.73 Gy. The results show significant perturbations in serum lipidome as early as 2 days postexposure persisting until the end of the study (day 30).

Effect of 90Sr internal emitter on gene expression in mouse blood.

BACKGROUND: The radioactive isotope Strontium-90 ((90)Sr) may be released as a component of fallout from nuclear accidents, or in the event of a radiological incident such as detonation of an improvised nuclear device, and if ingested poses a significant health risk to exposed individuals. In order to better understand the response to (90)Sr, using an easily attainable and standard biodosimetry sample fluid, we analyzed the global transcriptomic response of blood cells in an in vivo model system. RESULTS: We injected C57BL/6 mice with a solution of 90SrCl2 and followed them over a 30-day period. At days 4, 7, 9, 25 and 30, we collected blood and isolated RNA for microarray analyses. These days corresponded to target doses in a range from 1-5 Gy. We investigated changes in mRNA levels using microarrays, and changes in specific microRNA (miRNA) predicted to be involved in the response using qRT-PCR. We identified 8082 differentially expressed genes in the blood of mice exposed to (90)Sr compared with controls. Common biological functions were affected throughout the study, including apoptosis of B and T lymphocytes, and atrophy of lymphoid organs. Cellular functions such as RNA degradation and lipid metabolism were also affected during the study. The broad down regulation of genes observed in our study suggested a potential role for miRNA in gene regulation. We tested candidate miRNAs, mmu-miR-16, mmu-miR-124, mmu-miR-125 and mmu-mir-21; and found that all were induced at the earliest time point, day 4. CONCLUSIONS: Our study is the first to report the transcriptomic response of blood cells to the internal emitter (90)Sr in mouse and a possible role for microRNA in gene regulation after (90)Sr exposure. The most dramatic effect was observed on gene expression related to B-cell development and RNA maintenance. These functions were affected by genes that were down regulated throughout the study, suggesting severely compromised antigen response, which may be a result of the deposition of the radioisotope proximal to the hematopoietic compartment in bone.

A Comprehensive Metabolomic Investigation in Urine of Mice Exposed to Strontium-90.

Internal emitters such as Strontium-90 ((90)Sr) pose a substantial health risk during and immediately after a nuclear disaster or detonation of an improvised device. The environmental persistency and potency of (90)Sr calls for urgent development of high-throughput tests to establish levels of exposure and to help triage potentially exposed individuals who were in the immediate area of the disaster. In response to these concerns, our team focused on developing a robust metabolomic profile for (90)Sr exposure in urine using a mouse model. The sensitivity of modern time-of-flight mass spectrometry (TOFMS) combined with the separation power of ultra performance liquid chromatography (UPLC) was used to determine perturbations in the urinary metabolome of mice exposed to (90)Sr. The recently developed statistical suite, MetaboLyzer, was used to explore the mass spectrometry data. The results indicated a significant change in the urinary abundances of metabolites pertaining to butanoate metabolism, vitamin B metabolism, glutamate and fatty acid oxidation. All of these pathways are either directly or indirectly connected to the central energy production pathway, the tricarboxylic acid (TCA) cycle. To our knowledge, this is the first in vivo metabolomics to evaluate the effects of exposure to (90)Sr using the easily accessible biofluid, urine.

Orally administered DTPA di-ethyl ester for decorporation of (241)Am in dogs: Assessment of safety and efficacy in an inhalation-contamination model.

PURPOSE: Currently two injectable products of diethylenetriaminepentaacetic acid (DTPA) are U.S. Food and Drug Administration (FDA)-approved for decorporation of (241)Am; however, an oral product is considered more amenable in a mass casualty situation. The di-ethyl ester of DTPA, named C2E2, is being developed as an oral drug for treatment of internal radionuclide contamination. MATERIALS AND METHODS: Single-dose decorporation efficacy of C2E2 administered 24-h post contamination was determined in beagle dogs using a (241)Am nitrate inhalation contamination model. Single and multiple dose toxicity studies in beagle dogs were performed as part of an initial safety assessment program. In addition, the genotoxic potential of C2E2 was evaluated by the in vitro bacterial reverse mutation Ames test, mammalian cell chromosome aberration cytogenetic assay and an in vivo micronucleus test. RESULTS: Oral administration of C2E2 significantly increased (241)Am elimination over untreated controls and significantly reduced the retention of (241)Am in tissues, especially liver, kidney, lung and bone. Daily dosing of 200 mg/kg/day for 10 days was well tolerated in dogs. C2E2 was found to be neither mutagenic or clastogenic. CONCLUSIONS: The di-ethyl ester of DTPA (C2E2) was shown to effectively enhance the elimination of (241)Am after oral administration in a dog inhalation-contamination model and was well tolerated in toxicity studies.

NOS-2 Inhibition in Phosgene-Induced Acute Lung Injury.

Phosgene exposure via an industrial or warfare release produces severe acute lung injury (ALI) with high mortality, characterized by massive pulmonary edema, disruption of epithelial tight junctions, surfactant dysfunction, and oxidative stress. There are no targeted treatments for phosgene-induced ALI. Previous studies demonstrated that nitric oxide synthase 2 (NOS-2) is upregulated in the lungs after phosgene exposure; however, the role of NOS-2 in the pathogenesis of phosgene-induced ALI remains unknown. We previously demonstrated that NOS-2 expression in lung epithelium exacerbates inhaled endotoxin-induced ALI in mice, mediated partially through downregulation of surfactant protein B (SP-B) expression. Therefore, we hypothesized that a selective NOS-2 inhibitor delivered to the lung epithelium by inhalation would mitigate phosgene-induced ALI. Inhaled phosgene produced increases in bronchoalveolar lavage fluid protein, histologic lung injury, and lung NOS-2 expression at 24 h. Administration of the selective NOS-2 inhibitor 1400 W via inhalation, but not via systemic delivery, significantly attenuated phosgene-induced ALI and preserved epithelial barrier integrity. Furthermore, aerosolized 1400 W augmented expression of SP-B and prevented downregulation of tight junction protein zonula occludens 1 (ZO-1), both critical for maintenance of normal lung physiology and barrier integrity. We also demonstrate for the first time that NOS-2-derived nitric oxide downregulates the ZO-1 expression at the transcriptional level in human lung epithelial cells, providing a novel target for ameliorating vascular leak in ALI. Our data demonstrate that lung NOS-2 plays a critical role in the development of phosgene-induced ALI and suggest that aerosolized NOS-2 inhibitors offer a novel therapeutic strategy for its treatment.

Decorporation of systemically distributed americium by a novel orally administered diethylenetriaminepentaacetic acid (DTPA) formulation in beagle dogs.

Novel decorporation agents are being developed to protect against radiological accidents and terrorists attacks. Radioactive americium is a significant component of nuclear fallout. Removal of large radioactive materials, such as 241Am, from exposed persons is a subject of significant interest due to the hazards they pose. The objective of this study was to evaluate the dose-related efficacy of daily doses of NanoDTPA™ Capsules for decorporating Am administered intravenously as a soluble citrate complex to male and female beagle dogs. In addition, the efficacy of the NanoDTPA™ Capsules for decorporating 241Am was directly compared to intravenously administered saline and DTPA. Animals received a single IV administration of 241Am(III)-citrate on Day 0. One day after radionuclide administration, one of four different doses of NanoDTPA™ Capsules [1, 2, or 6 capsules d(-1) (30 mg, 60 mg, or 180 mg DTPA) or 2 capsules BID], IV Zn-DTPA (5 mg kg(-1) pentetate zinc trisodium) as a positive control, or IV saline as a placebo were administered. NanoDTPA™ Capsules, IV Zn-DTPA, or IV saline was administered on study days 1-14. Animals were euthanized on day 21. A full necropsy was conducted, and liver, spleen, kidneys, lungs and trachea, tracheobronchial lymph nodes (TBLN), muscle samples (right and left quadriceps), gastrointestinal (GI) tract (stomach plus esophagus, upper and lower intestine), gonads, two femurs, lumbar vertebrae (L1-L4), and all other soft tissue remains were collected. Urinary and fecal excretion profiles were increased approximately 10-fold compared to those for untreated animals. Tissue contents were decreased compared to untreated controls. In particular, liver content was decreased by approximately eightfold compared to untreated animals. The results from this study further demonstrate that oral NanoDTPA™ Capsules are equally efficient compared to IV Zn-DTPA in decorporation of actinides.

Metabolomic and lipidomic analysis of serum from mice exposed to an internal emitter, cesium-137, using a shotgun LC-MS(E) approach.

In this study ultra performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry in the MS(E) mode was used for rapid and comprehensive analysis of metabolites in the serum of mice exposed to internal exposure by Cesium-137 ((137)Cs). The effects of exposure to (137)Cs were studied at several time points after injection of (137)CsCl in mice. Over 1800 spectral features were detected in the serum of mice in positive and negative electrospray ionization modes combined. Detailed statistical analysis revealed that several metabolites associated with amino acid metabolism, fatty acid metabolism, and the TCA cycle were significantly perturbed in the serum of (137)Cs-exposed mice compared with that of control mice. While metabolites associated with the TCA cycle and glycolysis increased in their serum abundances, fatty acids such as linoleic acid and palmitic acid were detected at lower levels in serum after (137)Cs exposure. Furthermore, phosphatidylcholines (PCs) were among the most perturbed ions in the serum of (137)Cs-exposed mice. This is the first study on the effects of exposure by an internal emitter in serum using a UPLC-MS(E) approach. The results have put forth a panel of metabolites, which may serve as potential serum markers to (137)Cs exposure.