RESUMEN
Maintaining a long-term continuous and stable reactivator blood concentration to treat organophosphorus nerve agent poisoning using acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) is very important yet difficult. Because the flexible framework of MIL-88B(Fe) nanoparticles (NPs) can swell in polar solvents, pralidoxime chloride (2-PAM) was loaded in MIL-88B(Fe) NPs (size: ca. 500 nm) by stirring and incubation in deionized water to obtain 2-PAM@MIL-88B(Fe), which had a maximum drug loading capacity of 12.6 wt %. The as-prepared composite was characterized by IR, powder X-ray diffraction (P-XRD), scanning electron microscopy (SEM), ζ-potential, Brunauer-Emmett-Teller (BET), and thermogravimetry/differential thermal analysis (TG/DTA). The results showed that under constant conditions, the maximum drug release rates of 2-PAM@MIL-88B(Fe) in absolute ethanol, phosphate-buffered saline (PBS) solution (pH = 7.4), and PBS solution (pH = 4) at 150 h were 51.7, 80.6, and 67.1%, respectively. This was because the composite showed different swelling behaviors in different solvents. In PBS solution with pH = 2, the 2-PAM@MIL-88B(Fe) framework collapsed after 53 h and released 100% of 2-PAM. For mice after intragastric poisoning with sarin (a neurotoxic agent), an atropine-assisted 2-PAM@MIL-88B(Fe) treatment experiment revealed that 2-PAM@MIL-88B(Fe) continuously released 2-PAM for more than 72 h so that poisoned AChE was continuously and steadily reactivated. The reactivation rate of AChE was 56.7% after 72 h. This composite is expected to provide a prolonged, stable therapeutic drug for the mid- and late-stage treatment of neurotoxic agent poisoning.
Asunto(s)
Estructuras Metalorgánicas/química , Agentes Nerviosos/farmacología , Compuestos de Pralidoxima/farmacología , Sarín/antagonistas & inhibidores , Acetilcolinesterasa/análisis , Acetilcolinesterasa/metabolismo , Administración Oral , Animales , Atropina/administración & dosificación , Atropina/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos , Nanopartículas/química , Agentes Nerviosos/química , Compuestos de Pralidoxima/administración & dosificación , Compuestos de Pralidoxima/química , Sarín/administración & dosificación , Sarín/toxicidadRESUMEN
Oxime-based molecules are used for the treatment of patients to reactivate acetylcholinesterase (AChE) function after organophosphate intoxication. However, their efficacy is limited by low penetration through the blood-brain barrier and fast elimination. In this work, the cucurbit[7]uril (CB[7]) carrier was used for the encapsulation of the clinical agent asoxime to enhance brain bioavailability and the treatment window. We present a pharmacokinetic study of asoxime and the asoxime-CB[7] complex in an in vivo mouse model. Ultrahigh-performance liquid chromatography with electrospray ionization-mass spectrometry detection was developed to determine asoxime and CB[7] in biological fluids and tissues after thorough optimization of chromatographic conditions. The dihydroxypropane-silica stationary phase using hydrophilic interaction liquid chromatography conditions provided the best chromatographic performance. The final method was validated and applied for the pharmacokinetic study of mouse plasma, urine, bile, liver, kidney, and brain samples at different times after administration of asoxime and the asoxime-CB[7] complex. The results showed a greater than 3-fold increase in the area under the curve (AUC) in the brain for asoxime administered as a complex with CB[7] relative to that for the administration of asoxime alone. The effectiveness of the treatment strategy was evaluated using a reactivation study and a functional observatory battery. Protection of brain AChE activity is crucial for saving human lives or reducing the consequences of poisoning. The asoxime administered as a complex increased the brain activity by approximately 30% compared to that with atropine alone. CB[7] coadministration improved the AChE activity by 11%, which agrees with the higher asoxime AUC assessed in the pharmacokinetic study.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/química , Reactivadores de la Colinesterasa/administración & dosificación , Portadores de Fármacos/química , Imidazoles/química , Intoxicación por Organofosfatos/tratamiento farmacológico , Oximas/farmacocinética , Compuestos de Piridinio/farmacocinética , Acetilcolinesterasa/metabolismo , Animales , Área Bajo la Curva , Barrera Hematoencefálica/metabolismo , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/toxicidad , Reactivadores de la Colinesterasa/farmacocinética , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Pruebas de Enzimas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , Ratones , Oximas/administración & dosificación , Compuestos de Piridinio/administración & dosificación , Sarín/administración & dosificación , Sarín/toxicidadRESUMEN
Warfare neurotoxicants such as sarin, soman or VX, are organophosphorus compounds which irreversibly inhibit cholinesterase. High-dose exposure with nerve agents (NA) is known to produce seizure activity and related brain damage, while less is known about the effects of acute sub-lethal dose exposure. The aim of this study was to characterize behavioral, brain activity and neuroinflammatory modifications at different time points after exposure to 4-nitrophenyl isopropyl methylphosphonate (NIMP), a sarin surrogate. In order to decipher the impacts of sub-lethal exposure, we chose 4 different doses of NIMP each corresponding to a fraction of the median lethal dose (LD50). First, we conducted a behavioral analysis of symptoms during the first hour following NIMP challenge and established a specific scoring scale for the intoxication severity. The intensity of intoxication signs was dose-dependent and proportional to the cholinesterase activity inhibition evaluated in mice brain. The lowest dose (0.3 LD50) did not induce significant behavioral, electrocorticographic (ECoG) nor cholinesterase activity changes. Animals exposed to one of the other doses (0.5, 0.7 and 0.9 LD50) exhibited substantial changes in behavior, significant cholinesterase activity inhibition, and a disruption of brainwave distribution that persisted in a dose-dependent manner. To evaluate long lasting changes, we conducted ECoG recording for 30 days on mice exposed to 0.5 or 0.9 LD50 of NIMP. Mice in both groups showed long-lasting impairment of theta rhythms, and a lack of restoration in hippocampal ChE activity after 1-month post-exposure. In addition, an increase in neuroinflammatory markers (IBA-1, TNF-α, NF-κB) and edema were transiently observed in mice hippocampus. Furthermore, a novel object recognition test showed an alteration of short-term memory in both groups, 1-month post-NIMP intoxication. Our findings identified both transient and long-term ECoG alterations and some long term cognitive impairments following exposure to sub-lethal doses of NIMP. These may further impact morphopathological alterations in the brain.
Asunto(s)
Ondas Encefálicas/efectos de los fármacos , Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Disfunción Cognitiva/inducido químicamente , Sarín/toxicidad , Animales , Ondas Encefálicas/fisiología , Inhibidores de la Colinesterasa/administración & dosificación , Colinesterasas/metabolismo , Disfunción Cognitiva/enzimología , Disfunción Cognitiva/fisiopatología , Electrocorticografía/efectos de los fármacos , Electrocorticografía/métodos , Masculino , Ratones , Sarín/administración & dosificaciónRESUMEN
Objective: Sarin is an irreversible organophosphate cholinesterase inhibitor and a highly toxic, volatile warfare agent. Rats and guinea pigs exposed to sarin display cholinergic excitotoxicity which includes hyper-salivation, respiratory distress, tremors, seizures, and death. Here we focused on the characterization of the airways injury induced by direct exposure of the lungs to sarin vapor and compared it to that induced by the intramuscularly route. Materials and methods: Rats were exposed to sarin either in vapor (â¼1LCT50, 34.2 ± 0.8 µg/l/min, 10 min) or by i.m. (â¼1LD50, 80 µg/kg), and lung injury was evaluated by broncho-alveolar lavage (BAL). Results and discussion: BAL analysis revealed route-dependent effects in rats: vapor exposed animals showed elevation of inflammatory cytokines, protein, and neutrophil cells. These elevations were seen at 24 h and were still significantly higher compared to control values at 1 week following vapor exposure. These elevations were not detected in rats exposed to sarin i.m. Histological evaluation of the brains revealed typical changes following sarin poisoning independent of the route of administration. The airways damage following vapor exposure in rats was also compared to that induced in guinea pigs. The latter showed increased eosinophilia and histamine levels that constitutes an anaphylactic response not seen in rats. Conclusions: These data clearly point out the importance of using the appropriate route of administration in studying the deleterious effects of volatile nerve agents, as well as the selection of the appropriate animal species. Since airways form major target organs for the development of injury following inhalation toxicity, they should be included in any comprehensive evaluation of countermeasures efficacy.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Pulmón/patología , Sarín/administración & dosificación , Sarín/toxicidad , Administración por Inhalación , Animales , Lavado Broncoalveolar , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/toxicidad , Cobayas , Inflamación , Inyecciones Intramusculares , Dosificación Letal Mediana , Pulmón/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
A direct approach for the determination of a specific hydrolysis product of organophosphorus nerve agents such as methylphosphonic acid (MPA) in urine by ion chromatography and tandem mass spectrometry (IC-MS/MS) has been developed. The first advantage of the proposed approach is a rapid and simple sample preparation, which does not require a large sample volume, complicated and laborious preconcentration and derivatization steps, and takes less than 7min per sample. The second advantage is the fast and selective IC determination of MPA carried out on a noncommercial anion exchanger based on a poly(styrene-co-divinylbenzene) (PS-DVB) substrate with a high degree of crosslinking and a covalently-bonded branched functional layer, which enables complete resolution of MPA from major urine matrix components and allows one to overcome matrix effects. Hyphenation of IC with tandem mass spectrometry results in highly sensitive and reliable MPA determination with the lowest detection limit (4ngmL-1) reported so far for HPLC determination of MPA in urine. The proposed approach is successfully applied for the analysis of urine from rats exposed to nonlethal doses of organophosphorus nerve agents such as sarin, soman, and VR in up to 13days after initial exposure, which shows the possibility to verify the nerve agent exposure after a long period of time.
Asunto(s)
Agentes Nerviosos/metabolismo , Compuestos Organofosforados/orina , Espectrometría de Masas en Tándem/métodos , Animales , Límite de Detección , Modelos Lineales , Compuestos Organofosforados/metabolismo , Compuestos Organotiofosforados/administración & dosificación , Compuestos Organotiofosforados/metabolismo , Ratas , Reproducibilidad de los Resultados , Sarín/administración & dosificación , Sarín/metabolismo , Soman/administración & dosificación , Soman/metabolismoRESUMEN
The ability of two novel bispyridinium oximes K727 and K733 and currently available oximes (HI-6, obidoxime) to reactivate sarin-inhibited acetylcholinesterase and to reduce acute toxicity of sarin was evaluated. To investigate the reactivating efficacy of the oximes, the rats were administered intramuscularly with atropine and oximes in equitoxic doses corresponding to 5% of their LD50 values at 1 min after the intramuscular administration of sarin at a dose of 24 µg/kg (LD50). The activity of acetylcholinesterase was measured at 60 min after sarin poisoning. The LD50 value of sarin in non-treated and treated mice was assessed using probit-logarithmical analysis of death occurring within 24 h after intramuscular administration of sarin at five different doses. In vivo determined percentage of reactivation of sarin-inhibited rat blood, diaphragm and brain acetylcholinesterase showed that the potency of both novel oximes K727 and K733 to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to the reactivating efficacy of obidoxime. On the other hand, the oxime HI-6 was found to be the most efficient reactivator of sarin-inhibited acetylcholinesterase. While the oxime HI-6 was able to reduce the acute toxicity of sarin >3 times, both novel oximes and obidoxime decreased the acute toxicity of sarin <2 times. Based on the results, we can conclude that the reactivating and therapeutic efficacy of both novel oximes K727 and K733 is significantly lower compared to the oxime HI-6 and, therefore, they are not suitable for the replacement of the oxime HI-6 for the antidotal treatment of acute sarin poisoning.
Asunto(s)
Antídotos/uso terapéutico , Inhibidores de la Colinesterasa/toxicidad , Reactivadores de la Colinesterasa/uso terapéutico , Síndromes de Neurotoxicidad/tratamiento farmacológico , Oximas/uso terapéutico , Compuestos de Piridinio/uso terapéutico , Sarín/toxicidad , Acetilcolinesterasa/sangre , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Animales no Consanguíneos , Atropina/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/química , Diafragma/efectos de los fármacos , Diafragma/enzimología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Masculino , Ratones , Antagonistas Muscarínicos/uso terapéutico , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/enzimología , Síndromes de Neurotoxicidad/sangre , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/metabolismo , Cloruro de Obidoxima/uso terapéutico , Ratas Wistar , Sarín/administración & dosificación , Sarín/antagonistas & inhibidoresRESUMEN
Sarin is an organophosphate nerve agent that is among the most lethal chemical toxins known to mankind. Because of its vaporization properties and ease and low cost of production, sarin is the nerve agent with a strong potential for use by terrorists and rouge nations. The primary route of sarin exposure is through inhalation and, depending on the dose, sarin leads to acute respiratory failure and death. The mechanism(s) of sarin-induced respiratory failure is poorly understood. Sarin irreversibly inhibits acetylcholine esterase, leading to excessive synaptic levels of acetylcholine and, we have previously shown that sarin causes marked ventilatory changes including weakened response to hypoxia. We now show that LD50 sarin inhalation causes severe bronchoconstriction in rats, leading to airway resistance, increased hypoxia-induced factor-1α, and severe lung epithelium injury. Transferring animals into 60% oxygen chambers after sarin exposure improved the survival from about 50% to 75% at 24h; however, many animals died within hours after removal from the oxygen chambers. On the other hand, if LD50 sarin-exposed animals were administered the bronchodilator epinephrine, >90% of the animals survived. Moreover, while both epinephrine and oxygen treatments moderated cardiorespiratory parameters, the proinflammatory cytokine surge, and elevated expression of hypoxia-induced factor-1α, only epinephrine consistently reduced the sarin-induced bronchoconstriction. These data suggest that severe bronchoconstriction is a critical factor in the mortality induced by LD50 sarin inhalation, and epinephrine may limit the ventilatory, inflammatory, and lethal effects of sarin.
Asunto(s)
Broncoconstricción/efectos de los fármacos , Sustancias para la Guerra Química/toxicidad , Epinefrina/farmacología , Enfermedades Pulmonares/tratamiento farmacológico , Oxígeno/farmacología , Sarín/toxicidad , Enfermedad Aguda , Administración por Inhalación , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Inhibidores de la Colinesterasa/toxicidad , Relación Dosis-Respuesta a Droga , Precursores Enzimáticos/metabolismo , Gelatinasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Dosificación Letal Mediana , Pulmón/efectos de los fármacos , Pulmón/patología , Enfermedades Pulmonares/inducido químicamente , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratas , Ratas Endogámicas F344 , Sarín/administración & dosificaciónRESUMEN
The protective effects of selected anesthetic regimens on sarin (GB) were investigated in domestic swine. At 30% oxygen, the toxicity of this agent in isoflurane anesthetized animals (LD(50)=10.1µg/kg) was similar to literature sited values in awake swine (LD(50)=11.8µg/kg) and slightly higher than that of both ketamine (LD(50)=15.6µg/kg) and propofol (LD(50)=15.3µg/kg) anesthetized swine. Use of 100% oxygen in ketamine anesthetized animals resulted in three-fold protective effects compared to 30% oxygen. Use of 100% oxygen in both isoflurane and propofol anesthetized animals, compared to 30% resulted in profound protection against GB poisoning (>33×). There were no differences in the severity of the poisoning or recovery time in animals treated over dose ranges of 10-350µg/kg (isoflurane) or 15-500µg/kg GB (propofol). Survivors of high GB challenges that were revived from propofol anesthetic exhibited no signs of cognitive impairment seven days later. Protective treatments did not attenuate cholinesterase (ChE) inhibition; survivors of otherwise supralethal GB concentrations exhibited very low blood ChE activities. This work indicates that propofol has protective effects against GB, and that oxygen tension may have an important role in treating nerve agent casualties. More importantly, it demonstrates that non-cholinergic protective mechanisms exist that may be exploited in the future development of medical countermeasures against organophosphorous nerve agents.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Isoflurano/farmacología , Ketamina/farmacología , Propofol/farmacología , Sarín/toxicidad , Anestésicos Disociativos/administración & dosificación , Anestésicos Disociativos/farmacología , Anestésicos por Inhalación/administración & dosificación , Anestésicos por Inhalación/farmacología , Anestésicos Intravenosos/administración & dosificación , Anestésicos Intravenosos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Ketamina/administración & dosificación , Dosificación Letal Mediana , Masculino , Oxígeno/administración & dosificación , Propofol/administración & dosificación , Sarín/administración & dosificación , Índice de Severidad de la Enfermedad , Porcinos , Factores de TiempoRESUMEN
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.
Asunto(s)
Antídotos/uso terapéutico , Sustancias para la Guerra Química/toxicidad , Antagonistas Colinérgicos/uso terapéutico , Inhibidores de la Colinesterasa/toxicidad , Exposición por Inhalación/efectos adversos , Sarín/toxicidad , Escopolamina/uso terapéutico , Aerosoles , Animales , Antídotos/administración & dosificación , Conducta Animal/efectos de los fármacos , Antagonistas Colinérgicos/administración & dosificación , Inhibidores de la Colinesterasa/administración & dosificación , Relación Dosis-Respuesta a Droga , Cobayas , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Oxígeno/sangre , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/uso terapéutico , Mucosa Respiratoria/efectos de los fármacos , Sarín/administración & dosificación , Escopolamina/administración & dosificación , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Análisis de Supervivencia , Temblor/inducido químicamente , Temblor/prevención & control , Pérdida de Peso/efectos de los fármacosRESUMEN
The main injuries among victims of the terrorist act in the Tokyo subway resulted from sub-lethal inhalation and whole body exposure to sarin vapor. In order to study the long term effects of such exposure and to simulate these conditions, freely moving rats were exposed to sarin vapor (27.2±1.7 µg/l) for 10 min. About 50% of the rats showed no overt symptoms and the rest had mild to moderate clinical symptoms that subsided within 4h following exposure. A reduction of weight was noted during the first 3 days with full recovery on the 4th day. Rat's heart was challenged with epinephrine 1 and 6 months post exposure. A significant reduction in the threshold for epinephrine-induced arrhythmia (EPIA) was noted in rats exposed to sarin. A time dependent increase in the kD and Bmax values of muscarinic auto receptors (M2) was recorded in the rat's cortex and striatum. No changes were recorded in the rats' brain trans locator protein (TSPO) levels, concomitant with no observed changes in the animals' performance in A Morris water maze test. A significant increase in open field activity was noted 6 months following exposure to sarin vapor as well as a significant decrease in prostaglandin E2 (PGE2) production in the brain. It is speculated that down regulation of the M2 auto receptor function, caused hyper reactivity of the cholinergic system which leads to the changes described above. The continuous reduction in M2 auto-receptor system through an unknown mechanism may be the cause for long lasting decline in sarin-exposed casualties' health.
Asunto(s)
Encéfalo/efectos de los fármacos , Corazón/efectos de los fármacos , Exposición por Inhalación/efectos adversos , Sarín/administración & dosificación , Sarín/toxicidad , Animales , Encéfalo/fisiopatología , Corazón/fisiopatología , Dosificación Letal Mediana , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , VolatilizaciónRESUMEN
The goal of this study was to assess acetylcholinesterase (AChE) inhibition at different regions of the gastrointestinal (GI) tract following inhalation exposure to nerve agent sarin. Seven major regions of the GI tract were removed from saline control animals (n=3) and 677.4 mg/m(3) sarin-exposed animals at 4h (n=4) and 24h (n=4) post-exposure. AChE activity was determined in blood and homogenized tissue supernatant by specific Ellman's assay using Iso-OMPA, a BChE inhibitor, and expressed as activity/optical density of hemoglobin for blood and activity/mg protein for tissues. Our data showed that the AChE activity was significantly decreased for groups both 4h and 24h post-sarin exposure. Among the seven chosen regions of the guinea pig GI tract, duodenum showed the highest AChE activity in control animals. The AChE activity was significantly decreased in the stomach (p=0.03), duodenum (p=0.029), jejunum (p=0.006), and ileum (p=0.006) 4h following sarin exposure. At 24h post-sarin exposure the AChE activity of duodenum (p=0.029) and ileum (p=0.006) was significantly inhibited. Esophagus showed no inhibition following sarin exposure at both 4h and 24h groups. These results suggest that the AChE activity is different in different regions of the GI tract and highest levels of AChE inhibition following sarin exposure were seen in regions exhibiting higher overall AChE activity and cholinergic function.
Asunto(s)
Acetilcolinesterasa/metabolismo , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/enzimología , Intubación Intratraqueal , Sarín/administración & dosificación , Sarín/toxicidad , Acetilcolinesterasa/sangre , Administración por Inhalación , Animales , Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/toxicidad , Cobayas , Instilación de Medicamentos , MasculinoRESUMEN
We evaluated the effects, in rats, of single and multiple low-level inhalation exposures to sarin. Rats were trained on a variable-interval, 56 s (VI56) schedule of food reinforcement and then exposed to sarin vapor (1.7-4.0 mg/m(3) x 60 min) or air control. The exposures did not produce clinical signs of toxicity other than miosis. Subsequently, performance on the VI56 and acquisition of a radial-arm maze spatial memory task was evaluated over approximately 11 weeks. Single exposures did not affect performance on the VI56 and had little effect on acquisition of the radial-arm maze task. Multiple exposures (4.0 mg/m(3) x 60 min/day x 3) disrupted performance on the VI56 schedule during the initial post-exposure sessions. The disruption, however, resolved after several days. Multiple exposures also produced a deficit on the radial-arm maze task in that sarin-exposed rats tended to take it longer to complete the maze and to make more errors. The deficit, however, resolved during the first three weeks of acquisition. These results demonstrate that in rats, inhalation exposure to sarin at levels below those causing overt signs of clinical toxicity can produce cognitive and performance deficits. Furthermore, the observed deficits do not appear to be persistent.
Asunto(s)
Conducta Animal/efectos de los fármacos , Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Sarín/toxicidad , Acetilcolinesterasa/sangre , Administración por Inhalación , Animales , Butirilcolinesterasa/sangre , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/sangre , Condicionamiento Operante/efectos de los fármacos , Interpretación Estadística de Datos , Alimentos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Esquema de Refuerzo , Sarín/administración & dosificación , Sarín/sangreRESUMEN
An analysis method for determining isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA), the metabolic hydrolysis products of toxic organophosphorus nerve agents isopropyl methylphosphonofluoridate (sarin, GB) and cyclohexyl methylphosphonofluoridate (cyclosarin, GF), respectively, has been developed and validated using high-performance liquid chromatography-mass spectrometry with negative ion electrospray ionization with time-of-flight detection (LC-ESI-MS-TOF). The linear range of quantitation was 5 to 125 ng/mL in plasma with a method detection limit of 2 ng/mL for each compound. This method was developed to determine the amount of metabolic hydrolysis that was formed during and after nerve agent exposure in minipigs to account for a major pathway of GB and GF elimination that had not been previously characterized in the bloodstream, particularly during low-level whole-body inhalation experiments. Metabolic hydrolysis accounted for 70% to 90% of the recoverable agent in the bloodstream during exposure, when compared to both unbound and cholinesterase bound agent recovered by fluoride ion reactivation analysis for the same samples. The estimated half-life of IMPA and CMPA in plasma was determined to be 44 and 61 min, respectively. The method utilizes the mass selectivity of LC-ESI-MS-TOF using a bench-top instrument to achieve a detection limit that is consistent with reported LC-MS-MS methods analyzing blood samples.
Asunto(s)
Compuestos Organofosforados/sangre , Compuestos Organofosforados/metabolismo , Sarín/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Administración por Inhalación , Animales , Biomarcadores/sangre , Sustancias para la Guerra Química/análisis , Sustancias para la Guerra Química/metabolismo , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/sangre , Inhibidores de la Colinesterasa/metabolismo , Cromatografía Liquida/métodos , Exposición a Riesgos Ambientales/análisis , Monitoreo del Ambiente/métodos , Semivida , Compuestos Organofosforados/administración & dosificación , Reproducibilidad de los Resultados , Sarín/administración & dosificación , Sarín/sangre , Extracción en Fase Sólida/métodos , Porcinos , Porcinos EnanosRESUMEN
Freely moving rats were exposed to sarin vapor (34.2+/-0.8 microg/l) for 10 min. Mortality at 24 h was 35% and toxic sings in the surviving rats ranged from sever (prolonged convulsions) through moderate to almost no overt signs. Some of the surviving rats developed delayed, intermittent convulsions. All rats were evaluated for long-term functional deficits in comparison to air-exposed control rats. Histological analysis revealed typical cell loss at 1 week post inhalation exposure. Neuronal inflammation was demonstrated by a 20-fold increase in prostaglandin (PGE(2)) levels 24 h following exposure that markedly decreased 6 days later. An additional, delayed increase in PGE(2) was detected at 1 month and continued to increase for up to 6 months post exposure. Glial activation following neural damage was demonstrated by an elevated level of peripheral benzodiazepine receptors (PBR) seen in the brain 4 and 6 months after exposure. At the same time muscarinic receptors were unaffected. Six weeks, four and six months post exposure behavioral evaluations were performed. In the open field, sarin-exposed rats showed a significant increase in overall activity with no habituation over days. In a working memory paradigm in the water maze, these same rats showed impaired working and reference memory processes with no recovery. Our data suggest long lasting impairment of brain functions in surviving rats following a single sarin exposure. Animals that seem to fully recover from the exposure, and even animals that initially show no toxicity signs, developed some adverse neural changes with time.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Neuronas/patología , Síndromes de Neurotoxicidad/patología , Síndromes de Neurotoxicidad/psicología , Sarín/toxicidad , Administración por Inhalación , Animales , Encéfalo/patología , Química Encefálica/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/análisis , Cognición/efectos de los fármacos , Dinoprostona/metabolismo , Gases , Dosificación Letal Mediana , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Memoria a Corto Plazo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Sarín/administración & dosificación , Sarín/análisisRESUMEN
We determined the threshold concentration of sarin vapor exposure producing miosis in African green monkeys (Chlorocebus aethiops). Monkeys (n=8) were exposed to a single concentration of sarin (0.069-0.701mg/m3) for 10min. Changes in pupil size were measured from photographs taken before and after the exposure. Sarin EC50 values for miosis were determined to be 0.166mg/m3 when miosis was defined as a 50% reduction in pupil area and 0.469mg/m3 when miosis was defined as a 50% reduction in pupil diameter. Monkeys were also evaluated for behavioral changes from sarin exposure using a serial probe recognition test and performance remained essentially unchanged for all monkeys. None of the concentrations of sarin produced specific clinical signs of toxicity other than miosis. Sarin was regenerated from blood sampled following exposure in a concentration-dependent fashion. Consistent with a predominant inhibition of acetylcholinesterase (AChE), more sarin was consistently found in RBC fractions than in plasma fractions. Further, elimination of regenerated sarin from RBC fractions was slower than from plasma fractions. Blood samples following exposure also showed concentration-dependent inhibition of AChE activity and, to a lesser extent, butyrylcholinesterase activity. At the largest exposure concentration, AChE inhibition was substantial, reducing activity to approximately 40% of baseline. The results characterize sarin exposure concentrations that produce miosis in a large primate species in the absence of other overt signs of toxicity. Further, these results extend previous studies indicating that miosis is a valid early indicator for the detection of sarin vapor exposure.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Miosis , Sarín/toxicidad , Piel/citología , Piel/efectos de los fármacos , Administración por Inhalación , Animales , Carga Corporal (Radioterapia) , Chlorocebus aethiops , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/sangre , Interpretación Estadística de Datos , Eritrocitos/metabolismo , Femenino , Gases , Memoria/efectos de los fármacos , Pupila/efectos de los fármacos , Reconocimiento en Psicología/efectos de los fármacos , Sarín/administración & dosificación , Sarín/sangreRESUMEN
The objective of this study was to determine levels of DNA fragmentation in blood leukocytes and parietal cortex from guinea pigs following repeated low-level exposure to the chemical warfare nerve agent (CWNA) sarin. Guinea pigs were injected (s.c.) once a day for 10 days with saline, or 0.1, 0.2, or 0.4 LD50 (50% mean lethal dose) sarin dissolved in sterile physiological saline. Blood and parietal cortex was collected after injection at 0, 3, and 17 days recovery and evaluated for DNA fragmentation using single-cell gel electrophoresis (Comet assay). Cells were imaged using comet analysis software and three parameters of DNA fragmentation measured: tail length, percent DNA in the tail, and tail moment arm. Repeated low-dose exposure to sarin produced a dose-dependent response in leukocytes at 0 and 3 days post-exposure. There was a significant increase in all measures of DNA fragmentation at 0.2 and 0.4 LD50, but not at 0.1 LD50. There was no significant increase in DNA fragmentation in any of the groups at 17 days post-exposure. Sarin did not produce a systematic dose-dependent response in parietal cortex at any of the time points. However, significant increases in DNA fragmentation at 0.1 and 0.4 LD50 were observed at 0 and 3 days post-exposure. All measures of DNA fragmentation in both leukocytes and neurons returned to control levels by 17 days post-exposure, indicating a small and non-persistent increase in DNA fragmentation following repeated low-level exposure to sarin.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Fragmentación del ADN/efectos de los fármacos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Sarín/toxicidad , Animales , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Cobayas , Dosificación Letal Mediana , Masculino , Sarín/administración & dosificaciónRESUMEN
To improve toxicity estimates from sublethal exposures to chemical warfare nerve agents (CWNA), it is necessary to generate mathematical models of the absorption, distribution, and elimination of nerve agents. However, current models are based on representative data sets generated with different routes of exposure and in different species and are designed to interpolate between limited laboratory data sets to predict a wide range of possible human exposure scenarios. This study was performed to integrate CWNA sublethal toxicity data in male Duncan Hartley guinea pigs. Specific goal was to compare uptake and clearance kinetics of different sublethal doses of sarin (either 0.1 x or 0.4 x LC50) in blood and tissues of guinea pigs exposed to agent by acute whole-body inhalation exposure after the 60-min LC50 was determined. Arterial catheterization allowed repeated blood sampling from the same animal at various time periods. Blood and tissue levels of acetylcholinesterase, butyrylcholinesterase, and regenerated sarin (rGB) were determined at various time points during and following sarin exposure. The following pharmacokinetic parameters were calculated from the graph of plasma or RBC rGB concentration versus time: time to reach the maximal concentration; maximal concentration; mean residence time; clearance; volume of distribution at steady state; terminal elimination-phase rate constant; and area under plasma concentration time curve extrapolated to infinity using the WinNonlin analysis program 5.0. Plasma and RBC t(1/2) for rGB was also calculated. Data will be used to develop mathematical model of absorption and distribution of sublethal sarin doses into susceptible tissues.
Asunto(s)
Exposición por Inhalación/análisis , Sarín/administración & dosificación , Sarín/farmacocinética , Animales , Cámaras de Exposición Atmosférica , Cobayas , Dosificación Letal Mediana , Masculino , Sarín/sangre , Distribución TisularRESUMEN
Inhalation of subclinical doses of sarin suppresses the antibody-forming cell (AFC) response, T-cell mitogenesis, and serum corticosterone (CORT) levels, and high doses of sarin cause lung inflammation. However, the duration of these changes is not known. In these studies, rats were exposed to a subclinical dose of sarin (0.4 mg/m3/h/day) for 1 or 5 days, and immune and inflammatory parameters were assayed up to 8 weeks before sarin exposure. Our results showed that the effects of a 5-day sarin exposure on the AFC response and T-cell receptor (TCR)-mediated Ca2+ response disappeared within 2-4 weeks after sarin exposure, whereas the CORT and adrenocorticotropin hormone (ACTH) levels remained significantly decreased. Pretreatment of rats with chlorisondamine attenuated the effects of sarin on the AFC and the TCR-mediated Ca2+ response, implicating the autonomic nervous system (ANS) in the sarin-induced changes in T-cell function. Moreover, exposure to a single or five repeated subclinical doses of sarin upregulated the mRNA expression of proinflammatory cytokines in the lung, which is associated with the activation of NFkappaB in bronchoalveolar lavage cells. These effects were lost within 2 weeks of sarin inhalation. Our results suggest that while sarin-induced changes in T cells and cytokine gene expression were short lived, suppression of CORT and ACTH levels were relatively long lived and might represent biomarkers of sarin exposure. Moreover, while the effects of sarin on T-cell function were regulated by the ANS, the decreased CORT levels by sarin might result from its effects on the hypothalamus-pituitary-adrenal axis.
Asunto(s)
Sistema Nervioso Autónomo/efectos de los fármacos , Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Inflamación/inducido químicamente , Exposición por Inhalación , Neuroinmunomodulación/efectos de los fármacos , Sistemas Neurosecretores/efectos de los fármacos , Sarín/toxicidad , Hormona Adrenocorticotrópica/sangre , Animales , Sistema Nervioso Autónomo/metabolismo , Biomarcadores/sangre , Calcio/metabolismo , Clorisondamina/farmacología , Inhibidores de la Colinesterasa/administración & dosificación , Corticosterona/sangre , Citocinas/genética , Citocinas/metabolismo , Bloqueadores Ganglionares/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Inflamación/metabolismo , Inflamación/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , FN-kappa B/metabolismo , Sistemas Neurosecretores/metabolismo , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Receptores de Antígenos de Linfocitos T/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/metabolismo , Sarín/administración & dosificación , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
The extrapolation from animal data to therapeutic effects in humans, a basic pharmacological issue, is especially critical in studies aimed to estimate the protective efficacy of drugs against nerve agent poisoning. Such efficacy can only be predicted by extrapolation of data from animal studies to humans. In pretreatment therapy against nerve agents, careful dose determination is even more crucial than in antidotal therapy, since excessive doses may lead to adverse effects or performance decrements. The common method of comparing dose per body weight, still used in some studies, may lead to erroneous extrapolation. A different approach is based on the comparison of plasma concentrations at steady state required to obtain a given pharmacodynamic endpoint. In the present study, this approach was applied to predict the prophylactic efficacy of the anticholinergic drug caramiphen in combination with pyridostigmine in man based on animal data. In two species of large animals, dogs and monkeys, similar plasma concentrations of caramiphen (in the range of 60-100 ng/ml) conferred adequate protection against exposure to a lethal-dose of sarin (1.6-1.8 LD(50)). Pharmacokinetic studies at steady state were required to achieve the correlation between caramiphen plasma concentrations and therapeutic effects. Evaluation of total plasma clearance values was instrumental in establishing desirable plasma concentrations and minimizing the number of animals used in the study. Previous data in the literature for plasma levels of caramiphen that do not lead to overt side effects in humans (70-100 ng/ml) enabled extrapolation to expected human protection. The method can be applied to other drugs and other clinical situations, in which human studies are impossible due to ethical considerations. When similar dose response curves are obtained in at least two animal models, the extrapolation to expected therapeutic effects in humans might be considered more reliable.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Intoxicación por Organofosfatos , Intoxicación/prevención & control , Animales , Sustancias para la Guerra Química/envenenamiento , Antagonistas Colinérgicos/administración & dosificación , Antagonistas Colinérgicos/farmacocinética , Antagonistas Colinérgicos/uso terapéutico , Ciclopentanos/sangre , Ciclopentanos/farmacocinética , Ciclopentanos/uso terapéutico , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Humanos , Infusiones Intravenosas , Infusiones Parenterales , Dosificación Letal Mediana , Masculino , Tasa de Depuración Metabólica , Organofosfatos/administración & dosificación , Organofosfatos/sangre , Papio anubis , Intoxicación/sangre , Bromuro de Piridostigmina/sangre , Bromuro de Piridostigmina/farmacocinética , Bromuro de Piridostigmina/uso terapéutico , Sarín/administración & dosificación , Sarín/envenenamiento , Especificidad de la Especie , Resultado del TratamientoRESUMEN
Acetylcholinesterase activity in defined brain regions was determined using biochemical and histochemical methods 30 min after treating rats with sarin, soman or VX (0.5 x LD(50)). Enzyme inhibition was high in the pontomedullar area and frontal cortex, but was low in the basal ganglia. Histochemical and biochemical results correlated well. Determination of the activity in defined brain structures was a more sensitive parameter than determination in whole brain homogenate where the activity was a "mean" of the activities in different structures. The pontomedullar area controls respiration, so that the special sensitivity of acetylcholinesterase to inhibition by nerve agents in this area is important for understanding the mechanism of death caused by nerve agents. Thus, acetylcholinesterase activity is the main parameter investigated in studies searching for target sites following nerve agent poisoning.