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Acute respiratory toxicity following inhalation exposure to soman in guinea pigs
Toxicology and Applied Pharmacology 245 (2010) 171–178
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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Acute respiratory toxicity following inhalation exposure to soman in guinea pigs Michael W. Perkins a, Zdenka Pierre a, Peter Rezk a, Praveena Sabnekar a, Kareem Kabra b, Soma Chanda b, Samuel Oguntayo b, Alfred M. Sciuto a, Bhupendra P. Doctor b, Madhusoodana P. Nambiar b,c,⁎ a b c
Medical/Analytical Toxicology, US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010, USA Brain Dysfunction and Blast Injury Division, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
a r t i c l e
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Article history: Received 9 November 2009 Revised 18 February 2010 Accepted 19 February 2010 Available online 4 March 2010 Keywords: Organophosphates Chemical warfare Respiratory toxicity Cholinesterases Neuroprotection Inhalation exposure Central nervous system Nerve agents
a b s t r a c t Respiratory toxicity and lung injury following inhalation exposure to chemical warfare nerve agent soman was examined in guinea pigs without therapeutics to improve survival. A microinstillation inhalation exposure technique that aerosolizes the agent in the trachea was used to administer soman to anesthetized age and weight matched male guinea pigs. Animals were exposed to 280, 561, 841, and 1121 mg/m3 concentrations of soman for 4 min. Survival data showed that all saline controls and animals exposed to 280 and 561 mg/m3 soman survived, while animals exposed to 841, and 1121 mg/m3 resulted in 38% and 13% survival, respectively. The microinstillation inhalation exposure LCt50 for soman determined by probit analysis was 827.2 mg/m3. A majority of the animals that died at 1121 mg/m3 developed seizures and died within 15–30 min post-exposure. There was a dose-dependent decrease in pulse rate and blood oxygen saturation of animals exposed to soman at 5–6.5 min post-exposure. Body weight loss increased with the dose of soman exposure. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase and butyrylcholinesterase activity was inhibited dose-dependently in soman treated groups at 24 h. BAL cells showed a dose-dependent increase in cell death and total cell counts following soman exposure. Edema by wet/dry weight ratio of the accessory lung lobe and trachea was increased slightly in soman exposed animals. An increase in total bronchoalveolar lavage fluid protein was observed in soman exposed animals at all doses. Differential cell counts of BAL and blood showed an increase in total lymphocyte counts and percentage of neutrophils. These results indicate that microinstillation inhalation exposure to soman causes respiratory toxicity and acute lung injury in guinea pigs. Published by Elsevier Inc.
Introduction Mass casualties due to chemical warfare nerve agent (CWNA) exposure on the battlefield and civilian sector has increased worldwide security and health concerns. CWNA such as tabun, VX, soman, sarin, and cyclosarin are potent irreversible inhibitors of acetylcholinesterase (AChE), resulting in the accumulation of neurotransmitter acetylcholine. AChE inhibition results in a variety of cholinergic toxic effects including hypersecretions, bronchoconstriction, respiratory depression,
Abbreviations: AChE, Acetylcholinesterase; BChE, Butyrylcholinesterase; OP, Organophosphate; CWNA, Chemical warfare nerve agent; BAL, Bronchoalveolar lavage; BALC, Bronchoalveolar lavage cells; BALF, Bronchoalveolar lavage fluid; DTNB, Dithiobis 2-nitrobenzoic acid; i.m, Intramuscular; VX, O-ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothiolate; i.p., Intraperitoneal; s.c., Subcutaneous; Iso-OMPA, tetraisopropyl pyrophosphoramide; OP, Organophosphate; GD, pinacolyl methylphosphonoflouridate, Soman; ATCH, Acetylthiocholine iodide; BTCH, Butyrylthiocholine iodide; PBS, Phosphate buffered saline. ⁎ Corresponding author. Brain Dysfunction and Blast Injury Division, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring MD, 20910-7500, USA. E-mail address: [email protected] (M.P. Nambiar). 0041-008X/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.taap.2010.02.016
muscular tremors, severe seizures, and death due to respiratory failure (Bajgar, 2004). The major route of exposure to CWNAs during a terrorist attack or war is predicted to be through inhalation (Niven and Roop, 2004; Bogucki and Weir, 2002; Greenfield et al., 2002; Leikin et al., 2002; Munro, 1994; Martin and Lobert, 2003; Sidell, 1996; Volans and Karalliedde, 2002). Studies on the effects of inhalation exposure to CWNA on respiratory toxicity and lung injury in animals not treated with drugs to increase survival, are limited. Soman (GD) is considered to be one of the most toxic CWNAs, because it permanently inactivates AChE complexes within minutes after exposure and is resistant to reactivation by oximes (Worek et al., 2004). The process of “aging” which consists of the spontaneous dealkylation of the AChE organophosphate complex intensifies nerve agent induced toxicity (Shafferman et al., 1996). The short half-life of aging makes it difficult to successfully treat soman exposure with available combination treatments of antimuscarinic antidotes, AChE reactivators, or oximes, at later time points after exposure (Harris et al., 1984; Worek et al., 2005; Volans and Karalliedde, 2002). Immediate therapeutic treatment is a critical factor for medical countermeasures against soman exposure (Talbot et al., 1988; Shafferman et al., 1997).
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Respiratory disturbances play a central role in CWNA induced toxicity and respiratory failure is the most common cause of death (Kadivar and Adams, 1991; Okumura et al., 1996; Okumura et al., 2005; Vale, 2005; Vale, 2005; Masuda et al., 1995; Bide and Risk, 2004; DeLorenzo, 2001; Inoue, 2003; Niven and Roop, 2004; Karalliedde et al., 1991; Martin and Lobert, 2003). Although a combination of respiratory failure and neuropathology are the mechanism of toxicity due to CWNAs, the extent of contribution of the two mechanisms leading to mortality is not known (Taysse et al., 2006). It has been reported that inhalation exposure to organophosphates and CWNAs causes acute respiratory toxicity and lung injuries (Segura et al., 1999). Inhalation exposure to GD in baboons induces cardiac arrhythmias, apnea, hypoxia, and respiratory disturbances (Anzueto et al., 1990). GD has been shown to induce extremely severe spasms of the smooth muscle of bronchi (Chary et al., 1957). The guinea pig model is considered to be one of the best nonhuman primate models for determining the efficacy of therapeutics for organophosphate poisoning (Inns and Leadbeater, 1983). Differences in the levels of organophosphorus metabolizing carboxylesterases in various animal models contribute to their sensitivity to CWNAs. Rats and mice have comparatively higher levels of carboxylesterases, while guinea pigs have lower levels similar to humans (Maxwell and Brecht, 1991; Ray et al., 1991; Cohan et al., 1976; Shih et al., 1999). Guinea pigs have sensitive airways and undergo bronchoconstriction unlike mice and rats (Bice et al., 2000). We have developed a microinstillation methodology of inhalation exposure for the assessment of lung injury, respiratory toxicity and evaluation of therapeutics following inhalation of CWNAs (Che et al., 2008; Graham et al., 2006; Nambiar et al., 2006; Wright et al., 2006). This method bypasses the detoxification of the CWNAs by high levels of carboxylesterases and mucous present in the upper airways of rodents (Caranto et al., 1994; Jimmerson et al., 1989b; Jimmerson et al., 1989a; Maxwell and Brecht, 1991). Systemic absorption of CWNAs through the skin, pelt, and eye can be minimized by microinstillation inhalation exposure. The technique requires smaller amounts of CWNA to conduct the experiments in comparison to noseonly and whole-body exposure systems. We investigated the effect of GD without any treatments, on respiratory toxicity and lung injury following microinstillation inhalation exposure in guinea pigs. In the past, we have reported the effects of GD on the respiratory dynamics following microinstillation inhalation exposure in guinea pigs treated with nasal atropine (Katos et al., 2009). Nasal atropine blocks airway secretions induced by GD and increases survival of animals. We utilized survival, pulse rate, blood oxygen saturation, edema, body weight loss and biochemical analysis of blood, bronchoalveolar lavage cells and fluid to demonstrate the respiratory toxicity of GD in guinea pigs. Materials and methods Animals. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals. It adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, published by the National Academy Press, 1996, and the Animal Welfare Act of 1966, as amended. Adult male Hartley guinea pigs (250– 300 grams, Charles River Laboratories, Wilmington, MA) served as subjects. Animals were housed individually under standard conditions with 12 h light/dark cycle and food and water available ad libitum. Guinea pigs were quarantined for 1 week prior to microinstillation inhalation exposure. The study protocol was approved by the Institution Animal Care and Use Committee, USAMRICD, Edgewood, MD. A total of 36 guinea pigs were used for this study. Materials. Soman (pinacolyl methylphosphonofluoridate or GD) was obtained from Edgewood Chemical and Biological Center,
Edgewood, MD and diluted in saline. Telazol was purchased from Wyeth Pharmaceuticals (Madison, NJ). Meditomidine was obtained from Pfizer Pharmaceuticals (New York, NY). Saline solution was purchased from Pierce Chemical Co. (Rockford, IL). Heparin, acetylthiocholine iodide (ATCH), tetraisopropyl pyrophosphoramide (Iso-OMPA), butyrylthiocholine iodide (BTCH) and Huperzine A were obtained from Sigma-Aldrich (St. Louis, MO). Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce Chemical Company (Rockford, IL). Microinstillation inhalation exposure to soman. Microinstillation exposure to soman was performed as described earlier by our group (Nambiar et al., 2006). Microinstillation equipment was obtained from Trudell Medical, (Ontario, Canada). All animals (GD and saline exposed) were anesthetized using a combination of Telazol (40 mg/kg, im) and Medetomidine (0.125 mg/kg, sc) 15 min prior to the intubation procedure (Buchanan et al., 1998). The animals were intubated with a 10-cm piece of translucent polystyrene tube with a stylet and placed 2.5 cm above the bifurcation of the trachea (Nambiar et al., 2007). The microinstillation catheter was passed through the intubation tube and placed 2 cm above the bifurcation of the trachea. Successful intubation was verified by condensation of vapor at the end of the translucent intubation tube. The animals were exposed to different concentrations of soman, 40 pulses per minute for 4 min. Each pulse aerosolized 1.4 μL of GD in a volume of 1.1 ml air. After exposure, the intubation tube and microcatheter were removed and the animals were allowed to recover at room temperature for 15 min before taking them out from the exposure hood. Blood oxygen saturation and pulse rate. The blood oxygen saturation (SpO2) and pulse rate were recorded every 30 s for saline and soman (280, 561, 841, and 1121 mg/m3) exposed animals. Readings were recorded pre-intubation, post-intubation and during exposure at 30 s intervals for a total of 15 min using a pulse oximeter (Noni Medical IN., Minneapolis, MN). Bronchoalveolar lavage (BAL). Animals that survived 24 h after soman exposure were anesthetized and euthanized by exsanguination. BAL was obtained by lavaging the lungs three times with 7 ml of oxygen-free saline. The average volume of BAL recovered was 5.0 ml ±10%. BAL cells (BALC) were separated from BAL fluid (BALF) by centrifugation at 2000 rpm for 10 min using a Sorvall table top centrifuge. Blood and BALF acetylcholinesterase and butyrylcholinesterase activity assay. Approximately 100 µL of guinea pig ear blood were collected by capillary action into heparinized tubes at 5, 20, 40, and 60 min after removing animals from the exposure hood. Cardiac blood and BALF was collected 24 h post-exposure. Ear blood and cardiac blood was diluted 50-fold using de-ionized water. AChE and BChE activity of the blood and BALF were determined using Ellman's assay (Ellman et al., 1961) with acetylthiocholine iodide (ATCh) and butyrylthiocholine iodide (BTCh), respectively, as the substrates. Samples were incubated for 30 min at room temperature with 400 nM final concentration of freshly prepared Huperzine A, an AChE inhibitor, for the determination of BChE activity using BTCh substrate. Similarly, AChE was determined after incubating the samples for 30 min at room temperature with 4 μM final concentration of iso-OMPA, a BChE inhibitor, and using ATCh as the substrate. AChE and BChE activities are normalized as Vmax/optical density (OD) of hemoglobin measured at 415 nm for blood and protein levels at 562 nm for BALF. All spectrophotometric readings were determined on a microtiter plate reader by Spectra Plus 384 from Molecular Devices (Sunnyvale, CA). Protein analysis. Protein levels in the BALF samples were determined by a BCA protein assay (Pierce Chemical Company, Rockford, IL). Twenty microliters of BALF was directly used to assay for protein concentrations
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read using the Spectramax Plus microplate reader and Softmax Plus 4.3 LS software. Body weight loss. Animals were weighed before administration of anesthesia and prior to euthanasia, 24 h post-exposure, to calculate the percent body weight loss. Evaluation of trachea and lung edema by wet/dry weight ratio. The accessory lung lobe was tied off using surgical string before lavage, removed, and weighed using an analytical balance. A section of the trachea, anterior of the lavage collection site, was removed and weighed using an analytical balance. Tissue was placed in 100 °C dry heat for 7 days. The wet/dry weight (W/D) ratio was determined to evaluate the degree of lung edema. Cell death and differential cells in blood and BALC. BAL cells were diluted 20-fold with trypan blue dye and cells were counted on a Nebauer cell counting slide at 10× magnification using a phasecontrast microscope (Olympus IX51, Melville, NY). The total number of viable and dead cells was counted. All cell counts were done in triplicates. The percentage of neutrophils and total number of leukocytes in blood and BALC were counted using Analyzer Analyst III from Hemagen Diagnostics (Columbia, MD).
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Figs. 1A and B. SpO2 decreased in all GD-exposed animals between 3.5 to 7.5 min post-exposure with a significant difference observed at 561 mg/m3. SpO2 decreased about 55% at 5.0 min in comparison to saline controls in 561 mg/m3 GD-exposed animals. Animals exposed to 841 mg/m3 showed a decrease in SpO2 that didn't recover to values comparable to all other experimental groups after 6.5 min. The time for peak reduction of SpO2 seems to decrease with an increase in the dose of GD. Pulse rate in all GD-exposed animals in comparison to saline control is shown in Fig. 1B. Saline controls showed stable pulse rate throughout the observation period of 15 min. There was a decrease in pulse rate at 3.5 to 7.5 min in all GD-exposed animals in comparison to saline treated animals. Animals exposed to 841 mg/m3 of GD experienced a significant (p = 0.001) drop in pulse rate from 6.5 min to 7.5 min compared to other doses (Fig. 1B). Blood AChE and BChE inhibition A time course of ear blood AChE activity inhibition is shown in Fig. 2A. Ear blood for AChE activity was collected at 5, 20, 40, and
Data analysis. Statistical analyses were performed using the Graph Pad Prism V4.03 software (Graph Pad Software Inc. San Diego, CA). The Mann–Whitney test was used to calculate the p values. A p-value less than 0.05 was considered significant. Results Soman microinstillation inhalation exposure and guinea pig survival The survival of guinea pigs following exposure to 280, 561, 841, and 1121 mg/m3 of GD by microinstillation inhalation is described in Table 1. All saline controls (n = 8) and animals exposed to 280 and 561 mg/m3 of GD survived (n = 6, for each group). A total of 3 animals survived out the total of 8, which resulted in a survival percentage of 38% for animals exposed to 841 mg/m3. The highest mortality was observed in animals exposed to 1121 mg/m3, in which seven out of eight died resulting in a survival rate of 13% (Table 1). A majority of the animals that died at 841, and 1121 mg/m3 showed respiratory failure within 5–20 min post-exposure. Increases in saliva and lacrimal secretion were evident in all GD-exposed animals within 5–10 min post GD exposure. Only animals exposed to 841 and 1121 mg/m3 developed seizures/convulsions in the forelimb, hind limbs or both, with a majority of the of animals experiencing forelimb seizures. All of the animals that experienced seizures/ convulsions symptoms died. SpO2 and pulse rate The SpO2 and pulse rate were recorded continuously every 30 s for saline and GD-exposed animals for a total of 15 min as shown in
Table 1 Survival (24 h) of guinea pigs following inhalation exposure to soman. Treatment
Survival (survived/total)
Percentage (%)
Saline Soman (280 mg/m3) Soman (561 mg/m3) Soman (841 mg/m3) Soman (1121 mg/m3)
8/8 6/6 6/6 3/8 1/8
100 100 100 38 13
Fig. 1. Blood oxygen saturation and pulse rate. The blood oxygen saturation (SpO2) and pulse rate (A; B) were recorded continuously every 30 s for saline and soman (280.3, 561 and 841 mg/m3) exposed animals for a total of 15 min. A. Blood O2 saturation was plotted against time. B. Pulse rate (bpm) was plotted against time; saline (n = 8), 280 mg/m3 (n = 6), 561 mg/m3 (n = 6), and 841 mg/m3 (n = 3).
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decrease (p = 0.004 and 0.024) in AChE activity was observed at 561 and 841 mg/m3 GD. Significant inhibition of cardiac blood BChE activity was observed in 280, 561 and 841 mg/m3 GD-exposed animals (p = 0.009, 0.004 and 0.024) in comparison to saline controls as illustrated in Fig. 2C. AChE and BChE followed similar patterns of inhibition after 24 h following GD exposure. Effects of soman exposure on bronchoalveolar lavage fluid and blood BAL cells were diluted 20-fold with trypan blue dye and cells were counted using Nebauer cell counting slide at 10× magnification with a phase-contrast microscope. The percent error of the mean for all GD and saline cell counts were 3.6–5.7%, respectively. A dose-dependent increase in cell death was observed in GD-exposed animals with significant increases (p = 0.005 and 0.018) observed at 561 and 841 mg/m3 respectively (Fig. 3A). There was an increase in the total number of cells (viable and dead), with a significant increase (p = 0.008 and 0.048) in animals exposed to 561 and 841 mg/m3 GD in comparison to saline control as shown in Fig. 3B. Body weight loss and edema of trachea and lung There was an increase in the body weight loss of 6–7% in all GD (280, 561, 841, and 1121 mg/m3) exposed animals, while a loss of 3.5% was observed in saline controls (Fig. 4A). A significant loss in the
Fig. 2. Blood AChE and BChE enzyme activity after soman microinstillation inhalation exposure. A. Ear blood AChE activity at 5, 20, 40, and 60 min after taking out the animal from the exposure hood (280, 561 and 841 mg/m3) (n ≥ 2–6 for each soman doses at each time point. B. Cardiac blood AChE activity was determined 24 h post soman exposure. The data shows a significant (p = 0.004 and 0.024) decrease in AChE activity at 560 and 841 mg/m3 in comparison to saline controls (n = 3–8). C. Blood BChE activity. BChE activity in 280, 561 and 841 mg/m3 soman exposed animals and saline controls (n = 3–8). P-value is 0.009, 0.004 and 0.024 for 280, 561 and 841 mg/m3 soman exposure respectively. AChE and BChE activity was normalized as Vmax per optical density (OD) hemoglobin measured at 415 nm. Asterisk indicates statistical significance.
60 min after removing animals from the exposure hood (280, 561 and 841 mg/m3 GD). There was a significant decrease (p = 0.008, 0.016 and 0.008) in AChE activity at 280, 561 and 841 mg/m3 GD, respectively in comparison to saline treated controls. Saline controls showed no significant change in AChE activity. Blood time course analysis for all soman exposed samples showed a dose-dependent decrease in AChE activity. Dose-dependent inhibition of AChE activity was also seen in cardiac blood collected at 24 h post-exposure (Fig. 2B). A significant
Fig. 3. Effects of soman exposure on BALC cell death and total cell count. A. Bronchoalveolar lavage (BAL) cells death count. A significant (p = 0.005, 0.018) increase in cell death was observed at 561 and 841 mg/m3 soman exposures respectively (n = 3–8). B. Total cell count, The data showed a significant (p = 0.008 and 0.048) increase in total cell count a in animals exposed to 561 and 841 mg/m3 of GD respectively in comparison to saline control (n = 3–8). Asterisk indicates statistical significance.
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Biochemical analysis of bronchoalveolar lavage fluid There was an increase in BALF total protein in all GD-exposed animals in comparison to saline controls, with a significant increase (p = 0.003, 0.024 and 0.017) at 280, 561 and 841 mg/m3 respectively (Fig. 5A). The AChE activity was inhibited in all of the GD-exposed
Fig. 4. Percent body weight loss, lung and trachea edema. A. Percent body loss, There was an increase in the percent body weight loss in all GD (280, 561, 841, and 1121 mg/m3) exposed animals. The data showed a significant increase in the percent body weight loss for animals exposed to 561 and 841 mg/m3 (p =0.008 and 0.048 respectively) in comparison to saline controls. B. Accessory lung lobe edema was increased minimally in 280 and 841 mg/m3 soman exposed animals (n =3–8). C. There was a minimal increase in trachea edema at 560 and 841 mg/m3, but not significant (n= 3–8). Asterisk indicates statistical significance.
percent body weight loss was observed in animals exposed to 561 and 841 mg/m3 GD (p = 0.008 and 0.048) in comparison to saline controls. The accessory lobe and trachea edema calculated as a ratio of wet/dry weight is shown in Figs. 4B and C. Marginal increases in accessory lung lobe edema were observed in all GD-exposed animals in comparison to saline controls. Similarly, a small increase in tracheal edema was observed in GD-exposed animals at higher doses compared to saline controls.
Fig. 5. Bronchoalveolar lavage fluid total protein, AChE and BChE activity following inhalation exposure to soman. A. BALF total protein. Significant increase in BALF total protein was observed for all soman exposed animals with significant (p = 0.003, 0.024 and 0.017) increases at 280, 561 and 841 mg/m3 respectively in comparison to saline controls (n = 3–8). B. BALF AChE activity. There was a decrease in the AChE activity in soman exposed animals, with a significant (p = 0.008, 0.032 and 0.036) decrease observed at 280, 561 and 841 mg/m3 respectively in comparison to saline controls (n = 3–8). C. BALF BChE activity. The data shows significant inhibition of BChE activity in 280, 561 and 841 mg/m3 soman exposed animals (p = 0.008 and 0.008 and 0.035 respectively) in comparison to saline controls (n = 3–8). Asterisk indicates statistical significance.
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animals, with a significant inhibition at 280, 561, and 841 mg/m3 (p = 0.008, 0.032 and 0.036, respectively) in comparison to saline controls (Fig. 5B). Significant inhibition of BChE activity was also observed in all GD-exposed animals (p = 0.008 and 0.008 and 0.036, respectively) as shown in Fig. 5C. GD exposure increases blood and BAL lymphocyte and neutrophils counts Increases in white blood cell counts in BALC and blood samples in GD-exposed animals are shown in Figs. 6A and C. Significant increases (p = 0.029, 0.006 and 0.048) were observed in BALC total white blood count in 280, 561 and 841 mg/m3 GD-exposed animals, while blood samples resulted in a significant increase (p = 0.045) in animals exposed to 280 mg/m3. An increase in total neutrophil number in BALC and blood is shown in Figs. 6B and D. There was a significant increase (p = 0.002 and 0.012) in the total BALC neutrophil number at 280 and 841 mg/m3 GD. Fig. 6D shows an increase in the total neutrophil percentage in the blood with a significant increase (p = 0.041) in animals exposed to 561 mg/m3 GD in comparison to saline controls. Discussion In the present study, we have shown the toxic effects of GD exposure by microinstillation inhalation without additional treatments to provide an accurate assessment of respiratory toxicity
and acute lung injury in guinea pigs. In the past, we have reported the effects of GD exposure by microinstillation inhalation on respiratory dynamics in the presence of nasal atropine treatment. Administration of nasal atropine 30 s post-exposure to 841 mg/m3 GD by microinstillation inhalation resulted in 70% survival, in contrast to 38% observed in untreated animals in the current study (Katos et al., 2009). The LCt50 of GD by microinstillation inhalation exposure to be 827.2 mg/m3 by probit analysis. Langenberg et al. (1998) obtained an LCt50 dose for GD by nose-only exposure of 480 mg for 1 min with an 8 min exposure time in atropinized guinea pigs. The saline controls showed no sign of abnormalities indicating that the procedure of handling and microinstillation inhalation exposure caused minimal changes in the animals. Exposure to GD resulted in a dose-dependent increase in mortality. There was 100% survival with 280 and 561 mg/ m3 GD after 24 h. However, these animals did demonstrate some of the acute toxic signs of CWNA exposure, such as increased salivation, lacrimation, defecation, urination and mild muscular fasciculation. Animal anesthesia may potentially interact with soman and could affect the toxicity. It has been shown that anesthetics such as ketamine protect against soman induced neuropathology under sub anesthetic doses (Dorandeu et al., 2005). On the other hand, anesthetic doses of barbiturate increases the toxicity of soman (Clement, 1984). Guinea pigs that survived exposure to 841, and 1121 mg/m3 of GD took on an average of 15–30 min longer to fully recover from anesthesia and GD exposure, than those exposed to 280 and 561 mg/m3 and saline controls. There was an obvious increase in the manifestation of toxicity with increased soman concentration of 841, and 1121 mg/m3. Seizures
Fig. 6. Effects of soman exposure on Blood and BAL lymphocyte and neutrophils counts: Broncholaveolar lavage (BAL) cells were counted using an automatic cell counter. A. Total lymphocytes count. There was a significant (p = 0.029, 0.006 and 0.048) increase in the number of total lymphocytes in 280, 561 and 841 mg/m3 soman exposed animals respectively in comparison to saline controls (n = 3–8). B. Total number of neutrophils. There was a significant increase in the number of neutrophils at 280 and 841 mg/m3 GD (p = 0.002 and 0.012 respectively) in comparison to saline controls (n = 3–8). C. Total white blood count. There was an increase in the total number of white blood cell count in soman exposed animals with a significant (p = 0.045) increase at 280 mg/m3 (n = 3–8). D. Blood neutrophils percentage. There was an increase in neutrophils percentage in soman exposed groups in comparison saline groups, with a significant (p = 0.041) increase at 561 mg/m3. Asterisk indicates statistical significance.
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were only observed in the animals exposed to 841and 1121 mg/m3. None of the animals that produced seizures survived. All animals that died from seizures died within 3–10 min post GD exposure suggesting a rapid systemic uptake of the agent after microinstillation exposure. Development of seizures due to nerve agent exposure also produces irreversible brain damage and increased neuropathology (Shih et al., 1999; McDonough et al., 1995). This supports the fact that simultaneous protection against neuropathology and respiratory toxicity/lung injury is needed for the treatment of CWNA exposure. A decrease in the oxygen saturation and pulse rate in all GD-exposed animals is consistent with our previous studies with sarin and VX exposed guinea pigs (Che et al., 2009; Katos et al., 2007). The minimal decrease in oxygen saturation and pulse rate in saline treated animals in the current study was also seen previously in saline treated animals (Che et al., 2009). All saline control animals in the previous and present study survived and showed no indication of toxicity. It is possible, that saline aerosol may have produced minor alterations in the oxygen saturation at the end of the 4 min exposure. The data also showed a higher decrease in SpO2 with an increase in the dose of GD. Also the time for peak reduction in SpO2 decreased with an increase in the dose of soman indicating that a higher dose of soman leads to more rapid respiratory toxicity. At higher doses the decrease in SpO2 does not return to normal levels after exposure indicating persistent toxicity. The moderate to severe salivation that was present after GD exposure may be restricting the airway and affecting the normal breathing patterns. A significant decrease in the mean blood pressure (MBP) and arterial oxygen pressure has been observed following GD inhalation in baboons (Anzueto et al., 1986, 1990). The reduction in breathing and oxygen saturation induced by GD exposure may be alleviated with administration of therapeutics, which prevent copious secretions. Similar to SpO2, results of pulse rate showed a significant decrease in the pulse rate, directly after GD exposure, which was previously reported with sarin exposed guinea pigs (Che et al., 2008). The decreased pulse rate is drastic only at higher concentrations of soman and it remained below the saline control levels. It is not clear whether the decrease in SpO2 leads to a decrease in pulse rate or a decrease in pulse rate leads to a decrease in SpO2. It is becoming more evident that if there is a reduction in SpO2 and the pulse rate is near normal, then the SpO2 comes back to normal after exposure. On the other hand if the pulse rate is decreased, SpO2 does not come back to normal after GD exposure. Increased weight loss, a characteristic of acute toxicity, was observed in all GD-exposed animals. The percentage of body weight loss was higher in animals exposed to a higher dose of soman indicating a dose-dependent toxicity following microinstillation inhalation exposure. Minimal edema in the trachea and lung was observed following GD exposure at all concentrations. The lung edema was not dose-dependent and such an effect was reported earlier with VX exposure (Wright et al., 2006). In previous studies with other nerve agents, we also reported that higher doses of a nerve agent caused less edema compared to lower doses (Che et al., 2008; Rezk et al., 2007). This is likely due to tightening of the smooth muscle that possibly prevents edema formation following nerve agent exposure (Wright et al., 2006). Protein concentrations in the BALF increased following exposure at different concentrations of GD. As expected, BALF AChE and BChE activity was inhibited strongly at 24 h post GD exposure. In the past, we have observed an increase in BALF BChE in sub-lethal concentrations of VX exposed animals probably due to lung injury and plasma influx (Graham et al., 2006). An increase in BALF protein, which may be due to leakage of blood and plasma following nerve agent exposure, has also been seen in guinea pigs exposed to VX and sarin (Che et al., 2008; Wright et al., 2006). Similar to BALF, inhibition of cardiac blood AChE and BChE activity was dose-dependent 24 h post GD exposure. It has been reported that intravenous administration of GD to mice resulted in specific GD binding depots in the skin and lungs that may further induce toxicity and lung injury (Kadar et al., 1985). Similar levels of inhibition of AChE and BChE levels in the cardiac blood and BAL suggest that there may not be any depots of GD in the lungs following
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microinstillation inhalation exposure. AChE and BChE inhibition of different organs and tissues are being determined to investigate differential distribution of soman following microinstillation inhalation exposure. Unpublished data on the alterations in the respiratory dynamics and function at 24 h following GD exposure are consistent with the onset of respiratory toxicity observed in the present study (Perkins et al., unpublished data). A dose-dependent increase in cell death in the BALC and a total BALC cell indicates that microinstillation inhalation exposure to soman leads to acute lung injury. Differential cell counts in the BALF resulted in increases in lymphocytes and neutrophils indicating neutrophil infiltration following soman exposure. Inhalation studies with soman in baboons have been shown to cause persistent lung injury characterized by an increase in the total white cell population and neutrophils (Anzueto et al., 1990). Damaged or necrotic lung tissue was observed during necropsy and tissue collection in animals exposed to 841 and 1121 mg/m3 of GD, which indicates lung injury. Previous studies with VX and sarin microinstillation inhalation exposure all showed CWNA induced respiratory toxicity and acute lung injury as observed in the current study with soman (Che et al., 2008; Wright et al., 2006). The severity and the onset of seizures/convulsions were more prevalent in GD-exposed guinea pigs in comparison to sarin exposed animals, and minimal seizures were not observed in VX exposed animals. The cholinergic effect to CWNA was present in VX, sarin and soman microinstillation inhalation exposed groups. Hypersecretion seen in GD-treated animals was similar to sarin exposure, but VX exposed guinea pigs showed more pronounced viscous mucus secretions from the nostrils. Dose-dependent increases in the BAL protein in the present study, were also consistent in VX and sarin exposed animals (Wright et al., 2006). Significant body weight loss was observed in guinea pigs exposed to GD in a dose-dependent manner, while body weight loss was lower in animals exposed to higher doses of VX (Wright et al., 2006). A gross observation of lung tissue during tissue collection showed more damage to the lung lobes in GD and sarin exposed animals in comparison to VX exposed animals. In summary, we have shown that microinstillation inhalation exposure to GD induces respiratory toxicity and lung injury that is different in comparison to nerve agents such as VX and GB (Che et al., 2008). The study will assist in the evaluation of therapeutics to counteract the respiratory toxicity and lung injury as a result of inhalation exposure to GD. Due to the quick aging of GD inhibited AChE, immediate therapeutics are required to protect against the respiratory toxicity and lung injury following GD exposure, unlike sarin and VX. In addition to the immediate effects, respiratory toxicity and lung injury may remain for an extended period of time in GD-exposed animals. The development of therapeutics against CWNA induced respiratory toxicity will be more challenging in GD-exposed animals compared to other nerve agents. The present study underscores the importance of the development and evaluation of effective therapeutic countermeasures against GD induced respiratory toxicity and lung injury to provide protection to the military and civilian sector. Acknowledgments The project described was supported by the Defense Threat Reduction Agency. Its contents, opinions and assertions contained herein are private views of the authors are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. References Anzueto, A., Berdine, G.G., Moore, G.T., Gleiser, C., Johnson, D., White, C.D., Johanson Jr., W.G., 1986. Pathophysiology of soman intoxication in primates. Toxicol. Appl. Pharmacol. 86, 56–68. Anzueto, A., deLemos, R.A., Seidenfeld, J., Moore, G., Hamil, H., Johnson, D., Jenkinson, S.G., 1990. Acute inhalation toxicity of soman and sarin in baboons. Fundam. Appl. Toxicol. 14, 676–687.
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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Acute respiratory toxicity following inhalation exposure to soman in guinea pigs Michael W. Perkins a, Zdenka Pierre a, Peter Rezk a, Praveena Sabnekar a, Kareem Kabra b, Soma Chanda b, Samuel Oguntayo b, Alfred M. Sciuto a, Bhupendra P. Doctor b, Madhusoodana P. Nambiar b,c,⁎ a b c
Medical/Analytical Toxicology, US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010, USA Brain Dysfunction and Blast Injury Division, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
a r t i c l e
i n f o
Article history: Received 9 November 2009 Revised 18 February 2010 Accepted 19 February 2010 Available online 4 March 2010 Keywords: Organophosphates Chemical warfare Respiratory toxicity Cholinesterases Neuroprotection Inhalation exposure Central nervous system Nerve agents
a b s t r a c t Respiratory toxicity and lung injury following inhalation exposure to chemical warfare nerve agent soman was examined in guinea pigs without therapeutics to improve survival. A microinstillation inhalation exposure technique that aerosolizes the agent in the trachea was used to administer soman to anesthetized age and weight matched male guinea pigs. Animals were exposed to 280, 561, 841, and 1121 mg/m3 concentrations of soman for 4 min. Survival data showed that all saline controls and animals exposed to 280 and 561 mg/m3 soman survived, while animals exposed to 841, and 1121 mg/m3 resulted in 38% and 13% survival, respectively. The microinstillation inhalation exposure LCt50 for soman determined by probit analysis was 827.2 mg/m3. A majority of the animals that died at 1121 mg/m3 developed seizures and died within 15–30 min post-exposure. There was a dose-dependent decrease in pulse rate and blood oxygen saturation of animals exposed to soman at 5–6.5 min post-exposure. Body weight loss increased with the dose of soman exposure. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase and butyrylcholinesterase activity was inhibited dose-dependently in soman treated groups at 24 h. BAL cells showed a dose-dependent increase in cell death and total cell counts following soman exposure. Edema by wet/dry weight ratio of the accessory lung lobe and trachea was increased slightly in soman exposed animals. An increase in total bronchoalveolar lavage fluid protein was observed in soman exposed animals at all doses. Differential cell counts of BAL and blood showed an increase in total lymphocyte counts and percentage of neutrophils. These results indicate that microinstillation inhalation exposure to soman causes respiratory toxicity and acute lung injury in guinea pigs. Published by Elsevier Inc.
Introduction Mass casualties due to chemical warfare nerve agent (CWNA) exposure on the battlefield and civilian sector has increased worldwide security and health concerns. CWNA such as tabun, VX, soman, sarin, and cyclosarin are potent irreversible inhibitors of acetylcholinesterase (AChE), resulting in the accumulation of neurotransmitter acetylcholine. AChE inhibition results in a variety of cholinergic toxic effects including hypersecretions, bronchoconstriction, respiratory depression,
Abbreviations: AChE, Acetylcholinesterase; BChE, Butyrylcholinesterase; OP, Organophosphate; CWNA, Chemical warfare nerve agent; BAL, Bronchoalveolar lavage; BALC, Bronchoalveolar lavage cells; BALF, Bronchoalveolar lavage fluid; DTNB, Dithiobis 2-nitrobenzoic acid; i.m, Intramuscular; VX, O-ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothiolate; i.p., Intraperitoneal; s.c., Subcutaneous; Iso-OMPA, tetraisopropyl pyrophosphoramide; OP, Organophosphate; GD, pinacolyl methylphosphonoflouridate, Soman; ATCH, Acetylthiocholine iodide; BTCH, Butyrylthiocholine iodide; PBS, Phosphate buffered saline. ⁎ Corresponding author. Brain Dysfunction and Blast Injury Division, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring MD, 20910-7500, USA. E-mail address: [email protected] (M.P. Nambiar). 0041-008X/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.taap.2010.02.016
muscular tremors, severe seizures, and death due to respiratory failure (Bajgar, 2004). The major route of exposure to CWNAs during a terrorist attack or war is predicted to be through inhalation (Niven and Roop, 2004; Bogucki and Weir, 2002; Greenfield et al., 2002; Leikin et al., 2002; Munro, 1994; Martin and Lobert, 2003; Sidell, 1996; Volans and Karalliedde, 2002). Studies on the effects of inhalation exposure to CWNA on respiratory toxicity and lung injury in animals not treated with drugs to increase survival, are limited. Soman (GD) is considered to be one of the most toxic CWNAs, because it permanently inactivates AChE complexes within minutes after exposure and is resistant to reactivation by oximes (Worek et al., 2004). The process of “aging” which consists of the spontaneous dealkylation of the AChE organophosphate complex intensifies nerve agent induced toxicity (Shafferman et al., 1996). The short half-life of aging makes it difficult to successfully treat soman exposure with available combination treatments of antimuscarinic antidotes, AChE reactivators, or oximes, at later time points after exposure (Harris et al., 1984; Worek et al., 2005; Volans and Karalliedde, 2002). Immediate therapeutic treatment is a critical factor for medical countermeasures against soman exposure (Talbot et al., 1988; Shafferman et al., 1997).
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Respiratory disturbances play a central role in CWNA induced toxicity and respiratory failure is the most common cause of death (Kadivar and Adams, 1991; Okumura et al., 1996; Okumura et al., 2005; Vale, 2005; Vale, 2005; Masuda et al., 1995; Bide and Risk, 2004; DeLorenzo, 2001; Inoue, 2003; Niven and Roop, 2004; Karalliedde et al., 1991; Martin and Lobert, 2003). Although a combination of respiratory failure and neuropathology are the mechanism of toxicity due to CWNAs, the extent of contribution of the two mechanisms leading to mortality is not known (Taysse et al., 2006). It has been reported that inhalation exposure to organophosphates and CWNAs causes acute respiratory toxicity and lung injuries (Segura et al., 1999). Inhalation exposure to GD in baboons induces cardiac arrhythmias, apnea, hypoxia, and respiratory disturbances (Anzueto et al., 1990). GD has been shown to induce extremely severe spasms of the smooth muscle of bronchi (Chary et al., 1957). The guinea pig model is considered to be one of the best nonhuman primate models for determining the efficacy of therapeutics for organophosphate poisoning (Inns and Leadbeater, 1983). Differences in the levels of organophosphorus metabolizing carboxylesterases in various animal models contribute to their sensitivity to CWNAs. Rats and mice have comparatively higher levels of carboxylesterases, while guinea pigs have lower levels similar to humans (Maxwell and Brecht, 1991; Ray et al., 1991; Cohan et al., 1976; Shih et al., 1999). Guinea pigs have sensitive airways and undergo bronchoconstriction unlike mice and rats (Bice et al., 2000). We have developed a microinstillation methodology of inhalation exposure for the assessment of lung injury, respiratory toxicity and evaluation of therapeutics following inhalation of CWNAs (Che et al., 2008; Graham et al., 2006; Nambiar et al., 2006; Wright et al., 2006). This method bypasses the detoxification of the CWNAs by high levels of carboxylesterases and mucous present in the upper airways of rodents (Caranto et al., 1994; Jimmerson et al., 1989b; Jimmerson et al., 1989a; Maxwell and Brecht, 1991). Systemic absorption of CWNAs through the skin, pelt, and eye can be minimized by microinstillation inhalation exposure. The technique requires smaller amounts of CWNA to conduct the experiments in comparison to noseonly and whole-body exposure systems. We investigated the effect of GD without any treatments, on respiratory toxicity and lung injury following microinstillation inhalation exposure in guinea pigs. In the past, we have reported the effects of GD on the respiratory dynamics following microinstillation inhalation exposure in guinea pigs treated with nasal atropine (Katos et al., 2009). Nasal atropine blocks airway secretions induced by GD and increases survival of animals. We utilized survival, pulse rate, blood oxygen saturation, edema, body weight loss and biochemical analysis of blood, bronchoalveolar lavage cells and fluid to demonstrate the respiratory toxicity of GD in guinea pigs. Materials and methods Animals. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals. It adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, published by the National Academy Press, 1996, and the Animal Welfare Act of 1966, as amended. Adult male Hartley guinea pigs (250– 300 grams, Charles River Laboratories, Wilmington, MA) served as subjects. Animals were housed individually under standard conditions with 12 h light/dark cycle and food and water available ad libitum. Guinea pigs were quarantined for 1 week prior to microinstillation inhalation exposure. The study protocol was approved by the Institution Animal Care and Use Committee, USAMRICD, Edgewood, MD. A total of 36 guinea pigs were used for this study. Materials. Soman (pinacolyl methylphosphonofluoridate or GD) was obtained from Edgewood Chemical and Biological Center,
Edgewood, MD and diluted in saline. Telazol was purchased from Wyeth Pharmaceuticals (Madison, NJ). Meditomidine was obtained from Pfizer Pharmaceuticals (New York, NY). Saline solution was purchased from Pierce Chemical Co. (Rockford, IL). Heparin, acetylthiocholine iodide (ATCH), tetraisopropyl pyrophosphoramide (Iso-OMPA), butyrylthiocholine iodide (BTCH) and Huperzine A were obtained from Sigma-Aldrich (St. Louis, MO). Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce Chemical Company (Rockford, IL). Microinstillation inhalation exposure to soman. Microinstillation exposure to soman was performed as described earlier by our group (Nambiar et al., 2006). Microinstillation equipment was obtained from Trudell Medical, (Ontario, Canada). All animals (GD and saline exposed) were anesthetized using a combination of Telazol (40 mg/kg, im) and Medetomidine (0.125 mg/kg, sc) 15 min prior to the intubation procedure (Buchanan et al., 1998). The animals were intubated with a 10-cm piece of translucent polystyrene tube with a stylet and placed 2.5 cm above the bifurcation of the trachea (Nambiar et al., 2007). The microinstillation catheter was passed through the intubation tube and placed 2 cm above the bifurcation of the trachea. Successful intubation was verified by condensation of vapor at the end of the translucent intubation tube. The animals were exposed to different concentrations of soman, 40 pulses per minute for 4 min. Each pulse aerosolized 1.4 μL of GD in a volume of 1.1 ml air. After exposure, the intubation tube and microcatheter were removed and the animals were allowed to recover at room temperature for 15 min before taking them out from the exposure hood. Blood oxygen saturation and pulse rate. The blood oxygen saturation (SpO2) and pulse rate were recorded every 30 s for saline and soman (280, 561, 841, and 1121 mg/m3) exposed animals. Readings were recorded pre-intubation, post-intubation and during exposure at 30 s intervals for a total of 15 min using a pulse oximeter (Noni Medical IN., Minneapolis, MN). Bronchoalveolar lavage (BAL). Animals that survived 24 h after soman exposure were anesthetized and euthanized by exsanguination. BAL was obtained by lavaging the lungs three times with 7 ml of oxygen-free saline. The average volume of BAL recovered was 5.0 ml ±10%. BAL cells (BALC) were separated from BAL fluid (BALF) by centrifugation at 2000 rpm for 10 min using a Sorvall table top centrifuge. Blood and BALF acetylcholinesterase and butyrylcholinesterase activity assay. Approximately 100 µL of guinea pig ear blood were collected by capillary action into heparinized tubes at 5, 20, 40, and 60 min after removing animals from the exposure hood. Cardiac blood and BALF was collected 24 h post-exposure. Ear blood and cardiac blood was diluted 50-fold using de-ionized water. AChE and BChE activity of the blood and BALF were determined using Ellman's assay (Ellman et al., 1961) with acetylthiocholine iodide (ATCh) and butyrylthiocholine iodide (BTCh), respectively, as the substrates. Samples were incubated for 30 min at room temperature with 400 nM final concentration of freshly prepared Huperzine A, an AChE inhibitor, for the determination of BChE activity using BTCh substrate. Similarly, AChE was determined after incubating the samples for 30 min at room temperature with 4 μM final concentration of iso-OMPA, a BChE inhibitor, and using ATCh as the substrate. AChE and BChE activities are normalized as Vmax/optical density (OD) of hemoglobin measured at 415 nm for blood and protein levels at 562 nm for BALF. All spectrophotometric readings were determined on a microtiter plate reader by Spectra Plus 384 from Molecular Devices (Sunnyvale, CA). Protein analysis. Protein levels in the BALF samples were determined by a BCA protein assay (Pierce Chemical Company, Rockford, IL). Twenty microliters of BALF was directly used to assay for protein concentrations
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read using the Spectramax Plus microplate reader and Softmax Plus 4.3 LS software. Body weight loss. Animals were weighed before administration of anesthesia and prior to euthanasia, 24 h post-exposure, to calculate the percent body weight loss. Evaluation of trachea and lung edema by wet/dry weight ratio. The accessory lung lobe was tied off using surgical string before lavage, removed, and weighed using an analytical balance. A section of the trachea, anterior of the lavage collection site, was removed and weighed using an analytical balance. Tissue was placed in 100 °C dry heat for 7 days. The wet/dry weight (W/D) ratio was determined to evaluate the degree of lung edema. Cell death and differential cells in blood and BALC. BAL cells were diluted 20-fold with trypan blue dye and cells were counted on a Nebauer cell counting slide at 10× magnification using a phasecontrast microscope (Olympus IX51, Melville, NY). The total number of viable and dead cells was counted. All cell counts were done in triplicates. The percentage of neutrophils and total number of leukocytes in blood and BALC were counted using Analyzer Analyst III from Hemagen Diagnostics (Columbia, MD).
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Figs. 1A and B. SpO2 decreased in all GD-exposed animals between 3.5 to 7.5 min post-exposure with a significant difference observed at 561 mg/m3. SpO2 decreased about 55% at 5.0 min in comparison to saline controls in 561 mg/m3 GD-exposed animals. Animals exposed to 841 mg/m3 showed a decrease in SpO2 that didn't recover to values comparable to all other experimental groups after 6.5 min. The time for peak reduction of SpO2 seems to decrease with an increase in the dose of GD. Pulse rate in all GD-exposed animals in comparison to saline control is shown in Fig. 1B. Saline controls showed stable pulse rate throughout the observation period of 15 min. There was a decrease in pulse rate at 3.5 to 7.5 min in all GD-exposed animals in comparison to saline treated animals. Animals exposed to 841 mg/m3 of GD experienced a significant (p = 0.001) drop in pulse rate from 6.5 min to 7.5 min compared to other doses (Fig. 1B). Blood AChE and BChE inhibition A time course of ear blood AChE activity inhibition is shown in Fig. 2A. Ear blood for AChE activity was collected at 5, 20, 40, and
Data analysis. Statistical analyses were performed using the Graph Pad Prism V4.03 software (Graph Pad Software Inc. San Diego, CA). The Mann–Whitney test was used to calculate the p values. A p-value less than 0.05 was considered significant. Results Soman microinstillation inhalation exposure and guinea pig survival The survival of guinea pigs following exposure to 280, 561, 841, and 1121 mg/m3 of GD by microinstillation inhalation is described in Table 1. All saline controls (n = 8) and animals exposed to 280 and 561 mg/m3 of GD survived (n = 6, for each group). A total of 3 animals survived out the total of 8, which resulted in a survival percentage of 38% for animals exposed to 841 mg/m3. The highest mortality was observed in animals exposed to 1121 mg/m3, in which seven out of eight died resulting in a survival rate of 13% (Table 1). A majority of the animals that died at 841, and 1121 mg/m3 showed respiratory failure within 5–20 min post-exposure. Increases in saliva and lacrimal secretion were evident in all GD-exposed animals within 5–10 min post GD exposure. Only animals exposed to 841 and 1121 mg/m3 developed seizures/convulsions in the forelimb, hind limbs or both, with a majority of the of animals experiencing forelimb seizures. All of the animals that experienced seizures/ convulsions symptoms died. SpO2 and pulse rate The SpO2 and pulse rate were recorded continuously every 30 s for saline and GD-exposed animals for a total of 15 min as shown in
Table 1 Survival (24 h) of guinea pigs following inhalation exposure to soman. Treatment
Survival (survived/total)
Percentage (%)
Saline Soman (280 mg/m3) Soman (561 mg/m3) Soman (841 mg/m3) Soman (1121 mg/m3)
8/8 6/6 6/6 3/8 1/8
100 100 100 38 13
Fig. 1. Blood oxygen saturation and pulse rate. The blood oxygen saturation (SpO2) and pulse rate (A; B) were recorded continuously every 30 s for saline and soman (280.3, 561 and 841 mg/m3) exposed animals for a total of 15 min. A. Blood O2 saturation was plotted against time. B. Pulse rate (bpm) was plotted against time; saline (n = 8), 280 mg/m3 (n = 6), 561 mg/m3 (n = 6), and 841 mg/m3 (n = 3).
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decrease (p = 0.004 and 0.024) in AChE activity was observed at 561 and 841 mg/m3 GD. Significant inhibition of cardiac blood BChE activity was observed in 280, 561 and 841 mg/m3 GD-exposed animals (p = 0.009, 0.004 and 0.024) in comparison to saline controls as illustrated in Fig. 2C. AChE and BChE followed similar patterns of inhibition after 24 h following GD exposure. Effects of soman exposure on bronchoalveolar lavage fluid and blood BAL cells were diluted 20-fold with trypan blue dye and cells were counted using Nebauer cell counting slide at 10× magnification with a phase-contrast microscope. The percent error of the mean for all GD and saline cell counts were 3.6–5.7%, respectively. A dose-dependent increase in cell death was observed in GD-exposed animals with significant increases (p = 0.005 and 0.018) observed at 561 and 841 mg/m3 respectively (Fig. 3A). There was an increase in the total number of cells (viable and dead), with a significant increase (p = 0.008 and 0.048) in animals exposed to 561 and 841 mg/m3 GD in comparison to saline control as shown in Fig. 3B. Body weight loss and edema of trachea and lung There was an increase in the body weight loss of 6–7% in all GD (280, 561, 841, and 1121 mg/m3) exposed animals, while a loss of 3.5% was observed in saline controls (Fig. 4A). A significant loss in the
Fig. 2. Blood AChE and BChE enzyme activity after soman microinstillation inhalation exposure. A. Ear blood AChE activity at 5, 20, 40, and 60 min after taking out the animal from the exposure hood (280, 561 and 841 mg/m3) (n ≥ 2–6 for each soman doses at each time point. B. Cardiac blood AChE activity was determined 24 h post soman exposure. The data shows a significant (p = 0.004 and 0.024) decrease in AChE activity at 560 and 841 mg/m3 in comparison to saline controls (n = 3–8). C. Blood BChE activity. BChE activity in 280, 561 and 841 mg/m3 soman exposed animals and saline controls (n = 3–8). P-value is 0.009, 0.004 and 0.024 for 280, 561 and 841 mg/m3 soman exposure respectively. AChE and BChE activity was normalized as Vmax per optical density (OD) hemoglobin measured at 415 nm. Asterisk indicates statistical significance.
60 min after removing animals from the exposure hood (280, 561 and 841 mg/m3 GD). There was a significant decrease (p = 0.008, 0.016 and 0.008) in AChE activity at 280, 561 and 841 mg/m3 GD, respectively in comparison to saline treated controls. Saline controls showed no significant change in AChE activity. Blood time course analysis for all soman exposed samples showed a dose-dependent decrease in AChE activity. Dose-dependent inhibition of AChE activity was also seen in cardiac blood collected at 24 h post-exposure (Fig. 2B). A significant
Fig. 3. Effects of soman exposure on BALC cell death and total cell count. A. Bronchoalveolar lavage (BAL) cells death count. A significant (p = 0.005, 0.018) increase in cell death was observed at 561 and 841 mg/m3 soman exposures respectively (n = 3–8). B. Total cell count, The data showed a significant (p = 0.008 and 0.048) increase in total cell count a in animals exposed to 561 and 841 mg/m3 of GD respectively in comparison to saline control (n = 3–8). Asterisk indicates statistical significance.
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Biochemical analysis of bronchoalveolar lavage fluid There was an increase in BALF total protein in all GD-exposed animals in comparison to saline controls, with a significant increase (p = 0.003, 0.024 and 0.017) at 280, 561 and 841 mg/m3 respectively (Fig. 5A). The AChE activity was inhibited in all of the GD-exposed
Fig. 4. Percent body weight loss, lung and trachea edema. A. Percent body loss, There was an increase in the percent body weight loss in all GD (280, 561, 841, and 1121 mg/m3) exposed animals. The data showed a significant increase in the percent body weight loss for animals exposed to 561 and 841 mg/m3 (p =0.008 and 0.048 respectively) in comparison to saline controls. B. Accessory lung lobe edema was increased minimally in 280 and 841 mg/m3 soman exposed animals (n =3–8). C. There was a minimal increase in trachea edema at 560 and 841 mg/m3, but not significant (n= 3–8). Asterisk indicates statistical significance.
percent body weight loss was observed in animals exposed to 561 and 841 mg/m3 GD (p = 0.008 and 0.048) in comparison to saline controls. The accessory lobe and trachea edema calculated as a ratio of wet/dry weight is shown in Figs. 4B and C. Marginal increases in accessory lung lobe edema were observed in all GD-exposed animals in comparison to saline controls. Similarly, a small increase in tracheal edema was observed in GD-exposed animals at higher doses compared to saline controls.
Fig. 5. Bronchoalveolar lavage fluid total protein, AChE and BChE activity following inhalation exposure to soman. A. BALF total protein. Significant increase in BALF total protein was observed for all soman exposed animals with significant (p = 0.003, 0.024 and 0.017) increases at 280, 561 and 841 mg/m3 respectively in comparison to saline controls (n = 3–8). B. BALF AChE activity. There was a decrease in the AChE activity in soman exposed animals, with a significant (p = 0.008, 0.032 and 0.036) decrease observed at 280, 561 and 841 mg/m3 respectively in comparison to saline controls (n = 3–8). C. BALF BChE activity. The data shows significant inhibition of BChE activity in 280, 561 and 841 mg/m3 soman exposed animals (p = 0.008 and 0.008 and 0.035 respectively) in comparison to saline controls (n = 3–8). Asterisk indicates statistical significance.
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animals, with a significant inhibition at 280, 561, and 841 mg/m3 (p = 0.008, 0.032 and 0.036, respectively) in comparison to saline controls (Fig. 5B). Significant inhibition of BChE activity was also observed in all GD-exposed animals (p = 0.008 and 0.008 and 0.036, respectively) as shown in Fig. 5C. GD exposure increases blood and BAL lymphocyte and neutrophils counts Increases in white blood cell counts in BALC and blood samples in GD-exposed animals are shown in Figs. 6A and C. Significant increases (p = 0.029, 0.006 and 0.048) were observed in BALC total white blood count in 280, 561 and 841 mg/m3 GD-exposed animals, while blood samples resulted in a significant increase (p = 0.045) in animals exposed to 280 mg/m3. An increase in total neutrophil number in BALC and blood is shown in Figs. 6B and D. There was a significant increase (p = 0.002 and 0.012) in the total BALC neutrophil number at 280 and 841 mg/m3 GD. Fig. 6D shows an increase in the total neutrophil percentage in the blood with a significant increase (p = 0.041) in animals exposed to 561 mg/m3 GD in comparison to saline controls. Discussion In the present study, we have shown the toxic effects of GD exposure by microinstillation inhalation without additional treatments to provide an accurate assessment of respiratory toxicity
and acute lung injury in guinea pigs. In the past, we have reported the effects of GD exposure by microinstillation inhalation on respiratory dynamics in the presence of nasal atropine treatment. Administration of nasal atropine 30 s post-exposure to 841 mg/m3 GD by microinstillation inhalation resulted in 70% survival, in contrast to 38% observed in untreated animals in the current study (Katos et al., 2009). The LCt50 of GD by microinstillation inhalation exposure to be 827.2 mg/m3 by probit analysis. Langenberg et al. (1998) obtained an LCt50 dose for GD by nose-only exposure of 480 mg for 1 min with an 8 min exposure time in atropinized guinea pigs. The saline controls showed no sign of abnormalities indicating that the procedure of handling and microinstillation inhalation exposure caused minimal changes in the animals. Exposure to GD resulted in a dose-dependent increase in mortality. There was 100% survival with 280 and 561 mg/ m3 GD after 24 h. However, these animals did demonstrate some of the acute toxic signs of CWNA exposure, such as increased salivation, lacrimation, defecation, urination and mild muscular fasciculation. Animal anesthesia may potentially interact with soman and could affect the toxicity. It has been shown that anesthetics such as ketamine protect against soman induced neuropathology under sub anesthetic doses (Dorandeu et al., 2005). On the other hand, anesthetic doses of barbiturate increases the toxicity of soman (Clement, 1984). Guinea pigs that survived exposure to 841, and 1121 mg/m3 of GD took on an average of 15–30 min longer to fully recover from anesthesia and GD exposure, than those exposed to 280 and 561 mg/m3 and saline controls. There was an obvious increase in the manifestation of toxicity with increased soman concentration of 841, and 1121 mg/m3. Seizures
Fig. 6. Effects of soman exposure on Blood and BAL lymphocyte and neutrophils counts: Broncholaveolar lavage (BAL) cells were counted using an automatic cell counter. A. Total lymphocytes count. There was a significant (p = 0.029, 0.006 and 0.048) increase in the number of total lymphocytes in 280, 561 and 841 mg/m3 soman exposed animals respectively in comparison to saline controls (n = 3–8). B. Total number of neutrophils. There was a significant increase in the number of neutrophils at 280 and 841 mg/m3 GD (p = 0.002 and 0.012 respectively) in comparison to saline controls (n = 3–8). C. Total white blood count. There was an increase in the total number of white blood cell count in soman exposed animals with a significant (p = 0.045) increase at 280 mg/m3 (n = 3–8). D. Blood neutrophils percentage. There was an increase in neutrophils percentage in soman exposed groups in comparison saline groups, with a significant (p = 0.041) increase at 561 mg/m3. Asterisk indicates statistical significance.
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were only observed in the animals exposed to 841and 1121 mg/m3. None of the animals that produced seizures survived. All animals that died from seizures died within 3–10 min post GD exposure suggesting a rapid systemic uptake of the agent after microinstillation exposure. Development of seizures due to nerve agent exposure also produces irreversible brain damage and increased neuropathology (Shih et al., 1999; McDonough et al., 1995). This supports the fact that simultaneous protection against neuropathology and respiratory toxicity/lung injury is needed for the treatment of CWNA exposure. A decrease in the oxygen saturation and pulse rate in all GD-exposed animals is consistent with our previous studies with sarin and VX exposed guinea pigs (Che et al., 2009; Katos et al., 2007). The minimal decrease in oxygen saturation and pulse rate in saline treated animals in the current study was also seen previously in saline treated animals (Che et al., 2009). All saline control animals in the previous and present study survived and showed no indication of toxicity. It is possible, that saline aerosol may have produced minor alterations in the oxygen saturation at the end of the 4 min exposure. The data also showed a higher decrease in SpO2 with an increase in the dose of GD. Also the time for peak reduction in SpO2 decreased with an increase in the dose of soman indicating that a higher dose of soman leads to more rapid respiratory toxicity. At higher doses the decrease in SpO2 does not return to normal levels after exposure indicating persistent toxicity. The moderate to severe salivation that was present after GD exposure may be restricting the airway and affecting the normal breathing patterns. A significant decrease in the mean blood pressure (MBP) and arterial oxygen pressure has been observed following GD inhalation in baboons (Anzueto et al., 1986, 1990). The reduction in breathing and oxygen saturation induced by GD exposure may be alleviated with administration of therapeutics, which prevent copious secretions. Similar to SpO2, results of pulse rate showed a significant decrease in the pulse rate, directly after GD exposure, which was previously reported with sarin exposed guinea pigs (Che et al., 2008). The decreased pulse rate is drastic only at higher concentrations of soman and it remained below the saline control levels. It is not clear whether the decrease in SpO2 leads to a decrease in pulse rate or a decrease in pulse rate leads to a decrease in SpO2. It is becoming more evident that if there is a reduction in SpO2 and the pulse rate is near normal, then the SpO2 comes back to normal after exposure. On the other hand if the pulse rate is decreased, SpO2 does not come back to normal after GD exposure. Increased weight loss, a characteristic of acute toxicity, was observed in all GD-exposed animals. The percentage of body weight loss was higher in animals exposed to a higher dose of soman indicating a dose-dependent toxicity following microinstillation inhalation exposure. Minimal edema in the trachea and lung was observed following GD exposure at all concentrations. The lung edema was not dose-dependent and such an effect was reported earlier with VX exposure (Wright et al., 2006). In previous studies with other nerve agents, we also reported that higher doses of a nerve agent caused less edema compared to lower doses (Che et al., 2008; Rezk et al., 2007). This is likely due to tightening of the smooth muscle that possibly prevents edema formation following nerve agent exposure (Wright et al., 2006). Protein concentrations in the BALF increased following exposure at different concentrations of GD. As expected, BALF AChE and BChE activity was inhibited strongly at 24 h post GD exposure. In the past, we have observed an increase in BALF BChE in sub-lethal concentrations of VX exposed animals probably due to lung injury and plasma influx (Graham et al., 2006). An increase in BALF protein, which may be due to leakage of blood and plasma following nerve agent exposure, has also been seen in guinea pigs exposed to VX and sarin (Che et al., 2008; Wright et al., 2006). Similar to BALF, inhibition of cardiac blood AChE and BChE activity was dose-dependent 24 h post GD exposure. It has been reported that intravenous administration of GD to mice resulted in specific GD binding depots in the skin and lungs that may further induce toxicity and lung injury (Kadar et al., 1985). Similar levels of inhibition of AChE and BChE levels in the cardiac blood and BAL suggest that there may not be any depots of GD in the lungs following
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microinstillation inhalation exposure. AChE and BChE inhibition of different organs and tissues are being determined to investigate differential distribution of soman following microinstillation inhalation exposure. Unpublished data on the alterations in the respiratory dynamics and function at 24 h following GD exposure are consistent with the onset of respiratory toxicity observed in the present study (Perkins et al., unpublished data). A dose-dependent increase in cell death in the BALC and a total BALC cell indicates that microinstillation inhalation exposure to soman leads to acute lung injury. Differential cell counts in the BALF resulted in increases in lymphocytes and neutrophils indicating neutrophil infiltration following soman exposure. Inhalation studies with soman in baboons have been shown to cause persistent lung injury characterized by an increase in the total white cell population and neutrophils (Anzueto et al., 1990). Damaged or necrotic lung tissue was observed during necropsy and tissue collection in animals exposed to 841 and 1121 mg/m3 of GD, which indicates lung injury. Previous studies with VX and sarin microinstillation inhalation exposure all showed CWNA induced respiratory toxicity and acute lung injury as observed in the current study with soman (Che et al., 2008; Wright et al., 2006). The severity and the onset of seizures/convulsions were more prevalent in GD-exposed guinea pigs in comparison to sarin exposed animals, and minimal seizures were not observed in VX exposed animals. The cholinergic effect to CWNA was present in VX, sarin and soman microinstillation inhalation exposed groups. Hypersecretion seen in GD-treated animals was similar to sarin exposure, but VX exposed guinea pigs showed more pronounced viscous mucus secretions from the nostrils. Dose-dependent increases in the BAL protein in the present study, were also consistent in VX and sarin exposed animals (Wright et al., 2006). Significant body weight loss was observed in guinea pigs exposed to GD in a dose-dependent manner, while body weight loss was lower in animals exposed to higher doses of VX (Wright et al., 2006). A gross observation of lung tissue during tissue collection showed more damage to the lung lobes in GD and sarin exposed animals in comparison to VX exposed animals. In summary, we have shown that microinstillation inhalation exposure to GD induces respiratory toxicity and lung injury that is different in comparison to nerve agents such as VX and GB (Che et al., 2008). The study will assist in the evaluation of therapeutics to counteract the respiratory toxicity and lung injury as a result of inhalation exposure to GD. Due to the quick aging of GD inhibited AChE, immediate therapeutics are required to protect against the respiratory toxicity and lung injury following GD exposure, unlike sarin and VX. In addition to the immediate effects, respiratory toxicity and lung injury may remain for an extended period of time in GD-exposed animals. The development of therapeutics against CWNA induced respiratory toxicity will be more challenging in GD-exposed animals compared to other nerve agents. The present study underscores the importance of the development and evaluation of effective therapeutic countermeasures against GD induced respiratory toxicity and lung injury to provide protection to the military and civilian sector. Acknowledgments The project described was supported by the Defense Threat Reduction Agency. Its contents, opinions and assertions contained herein are private views of the authors are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. References Anzueto, A., Berdine, G.G., Moore, G.T., Gleiser, C., Johnson, D., White, C.D., Johanson Jr., W.G., 1986. Pathophysiology of soman intoxication in primates. Toxicol. Appl. Pharmacol. 86, 56–68. Anzueto, A., deLemos, R.A., Seidenfeld, J., Moore, G., Hamil, H., Johnson, D., Jenkinson, S.G., 1990. Acute inhalation toxicity of soman and sarin in baboons. Fundam. Appl. Toxicol. 14, 676–687.
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