Peter Rezk

Protective efficacy of catalytic bioscavenger, paraoxonase 1 against sarin and soman exposure in guinea pigs §

Human paraoxonase 1 (PON1) has been portrayed as a catalytic bioscavenger which can hydrolyze large amounts of chemical warfare nerve agents (CWNAs) and organophosphate (OP) pesticides compared to the stoichiometric bioscavengers such as butyrylcholinesterase. We evaluated the protective efficacy of purified human and rabbit serum PON1 against nerve agents sarin and soman in guinea pigs. Catalytically active PON1 purified from human and rabbit serum was intravenously injected to guinea pigs, which were 30 min later exposed to 1.2 × LCt₅₀ sarin or soman using a microinstillation inhalation exposure technology. Pre-treatment with 5 units of purified human and rabbit serum PON1 showed mild to moderate increase in the activity of blood PON1, but significantly increased the survival rate with reduced symptoms of CWNA exposure. Although PON1 is expected to be catalytic, sarin and soman exposure resulted in a significant reduction in blood PON1 activity. However, the blood levels of PON1 in pre-treated animals after exposure to nerve agent were higher than that of untreated control animals. The activity of blood acetylcholinesterase and butyrylcholinesterase and brain acetylcholinesterase was significantly higher in PON1 pre-treated animals and were highly correlated with the survival rate. Blood O₂ saturation, pulse rate and respiratory dynamics were normalized in animals treated with PON1 compared to controls. These results demonstrate that purified human and rabbit serum PON1 significantly protect against sarin and soman exposure in guinea pigs and support the development of PON1 as a catalytic bioscavenger for protection against lethal exposure to CWNAs.

Development of a rat pilocarpine model of seizure/status epilepticus that mimics chemical warfare nerve agent exposure

We developed a rat pilocarpine seizure/status epilepticus (SE) model, which closely resembles 1.6-2.0/LD50 soman exposure, to analyse the molecular mechanism of neuronal damage and to screen effective neuroprotectants against cholinergic agonist and chemical warfare nerve agent (CWNA) exposure. Rats implanted with radiotelemetry probes capable of recording electroencephalogram (EEG), electrocardiogram (ECG), temperature, and physical activity were treated with lithium chloride (5 mEq/kg, im), followed 24 h later by (ip) doses of pilocarpine hydrochloride. Based on radiotelemetry analysis, a dose of 240 mg/kg (ip) pilocarpine generated seizure/SE analogous to 1.6-2.0/LD50 of soman. The model was refined by reducing the peripheral convulsions without affecting the central nervous system (CNS) by administering methylscopolamine bromide (1 mg/kg, ip), an anti-cholinergic that does not cross the blood brain barrier. However, when methylscopolamine bromide was administered, a higher dose of pilocarpine (320 mg/kg, ip) was required to generate the equivalent seizure/SE. Histopathology data indicated that pilocarpine induces significant damage to the hippocampal region of the brain, with similar neuropathology to that of 1.6-2.0/LD50 soman exposure. There was a reduction in body temperature after the administration of pilocarpine, as observed in organophosphate (OP) nerve agents exposure. The heart-rate of pilocarpine-treated animals increased compared to the normal range. The pilocarpine seizure/SE model was also reproducible in the absence of lithium chloride. These results support that pilocarpine seizure/SE model is useful in studying the molecular mechanisms of neuropathology and screening neuroprotectants following cholinergic agonist and CWNA exposure.

Acute lung injury following inhalation exposure to nerve agent VX in guinea pigs.

A microinstillation technique of inhalation exposure was utilized to assess lung injury following chemical warfare nerve agent VX [methylphosphonothioic acid S-(2-[bis(1-methylethyl)amino]ethyl) O-ethyl ester] exposure in guinea pigs. Animals were anesthetized using Telazol-meditomidine, gently intubated, and VX was aerosolized using a microcatheter placed 2 cm above the bifurcation of the trachea. Different doses (50.4 μg/m3, 70.4 μ g/mm3, 90.4 μg/mm3) of VX were administered at 40 pulses/min for 5 min. Dosing of VX was calculated by the volume of aerosol produced per 200 pulses and diluting the agent accordingly. Although the survival rate of animals exposed to different doses of VX was similar to the controls, nearly a 20% weight reduction was observed in exposed animals. After 24 h of recovery, the animals were euthanized and bronchoalveolar lavage (BAL) was performed with oxygen free saline. BAL was centrifuged and separated into BAL fluid (BALF) and BAL cells (BALC) and analyzed for indication of lung injury. The edema by dry/wet weight ratio of the accessory lobe increased 11% in VX-treated animals. BAL cell number was increased in VX-treated animals compared to controls, independent of dosage. Trypan blue viability assay indicated an increase in BAL cell death in 70.4 μg/mm3 and 90.4 μg/mm3 VX-exposed animals. Differential cell counting of BALC indicated a decrease in macrophage/monocytes in VX-exposed animals. The total amount of BAL protein increased gradually with the exposed dose of VX and was highest in animals exposed to 90.4 μg/mm3, indicating that this dose of VX caused lung injury that persisted at 24 h. In addition, histopathology results also suggest that inhalation exposure to VX induces acute lung injury.

Development of a Microinstillation Model of Inhalation Exposure to Assess Lung Injury Following Exposure to Toxic Chemicals and Nerve Agents in Guinea Pigs

Respiratory disturbances due to chemical warfare nerve agents (CWNAs) are the starting point of mass casualty and the primary cause of death by these weapons of terror and mass destruction. However, very few studies have been implemented to assess respiratory toxicity and exacerbation induced by CWNAs, especially methylphosphonothioic acid S-(2-(bis(1-methylethyl)amino)ethyl)O-ethyl ester (VX). In this study, we developed a microinstillation technique of inhalation exposure to assess lung injury following exposure to CWNAs and toxic chemicals. Guinea pigs were gently intubated by placing a microcatheter into the trachea 1.5 to 2.0 cm centrally above the bifurcation. This location is crucial to deliver aerosolized agents uniformly to the lungs lobes. The placement of the tube is calculated by measuring the distance from the upper front teeth to the tracheal bifurcation, which is typically 8.5 cm for guinea pigs of equivalent size and a weight range of 250 g to 300 g. The catheter is capable of withstanding 100 psi pressure; the terminus has five peripheral holes to pump air that aerosolizes the nerve agent that is delivered in the central hole. The microcatheter is regulated by a central control system to deliver the aerosolized agent in a volume lower than the tidal volume of the guinea pigs. The average particle size of the nerve agent delivered was 1.48 ± 0.07 micrometer. The microinstillation technology has been validated by exposing the animals to Coomassie brilliant blue, which showed a uniform distribution of the dye in different lung lobes. In addition, the concentration of the dye in the lungs correlated with the dose/time of exposure. Furthermore, histopathological analysis confirmed the absence of barotraumas following micoinstillation. This novel technique delivers the agent safely, requires less amount of agent, avoids exposure to skin, pelt, and eye, and circumvents the concern of deposition of the particles in the nasal and palette due to the switching of breathing from nasal to oronasal in whole-body dynamic chamber or nose only exposure. Currently, we are using this inhalation exposure technique to investigate lung injuries and respiratory disturbances following direct exposure to VX.

Recombinant paraoxonase 1 protects against sarin and soman toxicity following microinstillation inhalation exposure in guinea pigs

To explore the efficacy of paraoxonase 1 (PON1) as a catalytic bioscavenger, we evaluated human recombinant PON1 (rePON1) expressed in Trichoplusia ni larvae against sarin and soman toxicity using microinstillation inhalation exposure in guinea pigs. Animals were pretreated intravenously with catalytically active rePON1, followed by exposure to 1.2 X LCt₅₀ sarin or soman. Administration of 5 units of rePON1 showed mild increase in the blood activity of the enzyme after 30 min, but protected the animals with a significant increase in survival rate along with minimal signs of nerve agent toxicity. Recombinant PON1 pretreated animals exposed to sarin or soman prevented the reduction of blood O₂ saturation and pulse rate observed after nerve agent exposure. In addition, rePON1 pretreated animals showed significantly higher blood PON1, acetylcholinesterase (AChE), and butyrylcholinesterase activity after nerve agent exposure compared to the respective controls without treatments. AChE activity in different brain regions of rePON1 pretreated animals exposed to sarin or soman were also significantly higher than respective controls. The remaining activity of blood PON1, cholinesterases and brain AChE in PON1 pretreated animals after nerve agent exposure correlated with the survival rate. In summary, these data suggest that human rePON1 protects against sarin and soman exposure in guinea pigs.

Acute respiratory toxicity following inhalation exposure to soman in guinea pigs

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/m(3) concentrations of soman for 4 min. Survival data showed that all saline controls and animals exposed to 280 and 561 mg/m(3) soman survived, while animals exposed to 841, and 1121 mg/m(3) resulted in 38% and 13% survival, respectively. The microinstillation inhalation exposure LCt(50) for soman determined by probit analysis was 827.2mg/m(3). A majority of the animals that died at 1121 mg/m(3) 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 24h. 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.

Acute toxic effects of nerve agent VX on respiratory dynamics and functions following microinsillation inhalation exposure in guinea pigs.

Exposure to a chemical warfare nerve agent (CWNA) leads to severe respiratory distress, respiratory failure, or death if not treated. We investigated the toxic effects of nerve agent VX on the respiratory dynamics of guinea pigs following exposure to 90.4 μg/m3 of VX or saline by microinstillation inhalation technology for 10 min. Respiratory parameters were monitored by whole-body barometric plethysmography at 4, 24, and 48 h, 7 d, 18 d, and 4 wk after VX exposure. VX-exposed animals showed a significant decrease in the respiratory frequency (RF) at 24 and 48 h of recovery (p value.0329 and.0142, respectively) compared to the saline control. The tidal volume (TV) slightly increased in VX exposed animals at 24 and significantly at 48 h (p = .02) postexposure. Minute ventilation (MV) increased slightly at 4 h but was reduced at 24 h and remained unchanged at 48 h. Animals exposed to VX also showed an increase in expiratory (Te) and relaxation time (RT) at 24 and 48 h and a small reduction in inspiratory time (Ti) at 24 h. A significant increase in end expiratory pause (EEP) was observed at 48 h after VX exposure (p = .049). The pseudo lung resistance (Penh) was significantly increased at 4 h after VX exposure and remained slightly high even at 48 h. Time-course studies reveal that most of the altered respiratory dynamics returned to normal at 7 d after VX exposure except for EEP, which was high at 7 d and returned to normal at 18 d postexposure. After 1 mo, all the monitored respiratory parameters were within normal ranges. Bronchoalveolar lavage (BAL) 1 mo after exposure showed virtually no difference in protein levels, cholinesterase levels, cell number, and cell death in the exposed and control animals. These results indicate that sublethal concentrations of VX induce changes in respiratory dynamics and functions that over time return to normal levels.

Butyrylcholinesterase in guinea pig lung lavage: a novel biomarker to assess lung injury following inhalation exposure to nerve agent VX.

Respiratory disturbances play a central role in chemical warfare nerve agent (CWNA) induced toxicity; they are the starting point of mass casualty and the major cause of death. We developed a microinstillation technique of inhalation exposure to nerve agent VX and assessed lung injury by biochemical analysis of the bronchoalveolar lavage fluid (BALF). Here we demonstrate that normal guinea pig BALF has a significant amount of cholinesterase activity. Treatment with Huperzine A, a specific inhibitor of acetylcholinesterase (AChE), showed that a minor fraction of BALF cholinesterase is AChE. Furthermore, treatment with tetraisopropyl pyrophosphoramide (iso-OMPA), a specific inhibitor of butyrylcholinesterase (BChE), inhibited more than 90% of BChE activity, indicating the predominance of BChE in BALF. A predominance of BChE expression in the lung lavage was seen in both genders. Substrate specific inhibition indicated that nearly 30% of the cholinesterase in lung tissue homogenate is AChE. BALF and lung tissue AChE and BChE activities were strongly inhibited in guinea pigs exposed for 5 min to 70.4 and 90.4 μ g/m3 VX and allowed to recover for 15 min. In contrast, BALF AChE activity was increased 63% and 128% and BChE activity was increased 77% and 88% after 24 h of recovery following 5 min inhalation exposure to 70.4 μ g/m3 and 90.4 μg/m3, respectively. The increase in BALF AChE and BChE activity was dose dependent. Since BChE is synthesized in the liver and present in the plasma, an increase in BALF indicates endothelial barrier injury and leakage of plasma into lung interstitium. Therefore, a measure of increased levels of AChE and BChE in the lung lavage can be used to determine the chronology of barrier damage as well as the extent of lung injury following exposure to chemical warfare nerve agents.

Post-exposure treatment with nasal atropine methyl bromide protects against microinstillation inhalation exposure to sarin in guinea pigs.

We evaluated the protective efficacy of nasal atropine methyl bromide (AMB) which does not cross the blood-brain barrier against sarin inhalation exposure. Age and weight matched male guinea pigs were exposed to 846.5 mg/m(3) sarin using a microinstillation inhalation exposure technique for 4 min. The survival rate at this dose was 20%. Post-exposure treatment with nasal AMB (2.5 mg/kg, 1 min) completely protected against sarin induced toxicity (100% survival). Development of muscular tremors was decreased in animals treated with nasal AMB. Post-exposure treatment with nasal AMB also normalized acute decrease in blood oxygen saturation and heart rate following sarin exposure. Inhibition of blood AChE and BChE activities following sarin exposure was reduced in animals treated with nasal AMB, indicating that survival increases the metabolism of sarin or expression of AChE. The body weight loss of animals exposed to sarin and treated with nasal AMB was similar to saline controls. No differences were observed in lung accessory lobe or tracheal edema following exposure to sarin and subsequent treatment with nasal AMB. Total bronchoalveolar lavage fluid (BALF) protein, a biomarker of lung injury, showed trends similar to saline controls. Surfactant levels post-exposure treatment with nasal AMB returned to normal, similar to saline controls. Alkaline phosphatase levels post-exposure treatment with nasal AMB were decreased. Taken together, these data suggest that nasal AMB blocks the copious airway secretion and peripheral cholinergic effects and protects against lethal inhalation exposure to sarin thus increasing survival.

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

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