Organophosphate Nerve Agents Research Articles

Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates

Methyl parathion hydrolase (MPH) has been displayed on the surface of microorganisms for the first time using only N- and C-terminal domains of the ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. A shuttle vector pINCM coding for INPNC-MPH was constructed and used to target MPH onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, overcoming the potential substrate uptake limitation. Over 90% of the MPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of the INPNC-MPH fusion was further verified by Western blot analysis and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without growth inhibition. Compared to organophosphorus hydrolase-displaying systems reported, changes in substrate specificity highlight an important potential use of the engineered strain for the clean-up of specific organophosphate nerve agents.

Detection of Toxic Organophosphate Nerve Agents Using DBR Porous Silicon Chip

The efficient detection method based on nanostructured photonic DBR PSi has been developed for DMMP, which is a simulant for G-type nerve agents. The manufactured DBR PSi chip exhibits a sharp photonic band gap at 520 nm. The detection method involves the shift of DBR peak in reflectivity spectra under the exposure of vapors of analyte. Rapid detection has been achieved in few seconds, in situ, and observed by the red-shift of DBR peak resulted from the increase of refractive indices in DBR PSi. When DBR PSi chip is exposed to DMMP, TEP, and DEEP-saturated air, DBR peak in reflectivity is red shifted by 10 nm, 25 nm, and 10 nm, respectively. Real-time detection for the nerve gases indicates that the measurement is reversible. Detection limit of DMMP (1.5 ppm) using DBR PSi is 8.8 mg/m3 for 1 min.

Catalytic hydrolysis of p-nitrophenyl acetate by electrospun polyacrylamidoxime nanofibers

Modification of polyacrylonitrile (PAN) by hydroxylamine resulted in polyacrylamidoxime (PANOx), the oxime groups of which are nucleophilic and capable of hydrolyzing esters. PANOx fiber mats with submicrometer fiber diameters ranging from tens to 300 nm were produced by electrospinning a suspension of PANOx blended with PAN (1:1 by weight) in a mixture of N, N-dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO) (85:15 by weight). Catalytic properties of the PANOx nanofibers were tested by the hydrolysis of p-nitrophenyl acetate (PNPA), which mimics toxic organophosphate nerve agents and insecticides. The presence of PANOx fibers significantly accelerated the hydrolysis of PNPA compared to its spontaneous hydrolysis. The rate constants for the hydrolysis ( k 1) and the deacetylation ( k 2) reactions for the fibers were obtained using a proposed kinetic model. The effect of the fiber size on reaction rate indicated that intra-fiber diffusional resistances might limit the accessibility of the oxime catalytic sites in the fibers and affect their catalytic activity.

Detection of organophosphorous nerve agents using liquid crystals supported on chemically functionalized surfaces

Abstract We report use of thin films of the nematic liquid crystal E7 supported on chemically functionalized surfaces to indicate the presence of vapors of the organophosphorous nerve agents sarin (GB), soman (GD), tabun (GA) and VX. The surfaces were prepared by the deposition of metal perchlorate salts onto carboxylic acid-terminated self-assembled monolayers. When using surfaces prepared from aluminum perchlorate salts, the nematic film of E7 underwent a transition from a perpendicular orientation to a tilted or planar orientation upon exposure to vapors of GB, GD, GA or VX generated from a small drop of agent placed on a piece of filter paper. The orientational transition of the liquid crystal was readily apparent as an optical signal that was visible to the naked eye. The agent VX, which has the lowest vapor pressure of these organophosphorous nerve agents (∼140 ppb), was reported by the liquid crystal within 60 s. The agents GB, GD, and GA, which have higher vapor pressures, triggered responses in the liquid crystal that were evident within 15 s, 15 s, and 60 s, respectively. By preparing surfaces from perchlorate salts of aluminum(III), zinc(II) and iron(III) it was possible to distinguish between GB, VX and either GD or GA, thus demonstrating the feasibility of designing surfaces patterned with metal salts to detect and positively identify chemical warfare agents.

Quantitative magnetic resonance brain imaging in US army veterans of the 1991 Gulf War potentially exposed to sarin and cyclosarin

Background In March 1991, a munitions storage complex at Khamisiyah, Iraq was destroyed, potentially exposing more than 100,000 US troops to low levels of the organophosphate nerve agents sarin and cyclosarin. Little is known about the neurophysiological effects of low-dose exposure to sarin/cyclosarin in humans, although some research has indicated subtle but persistent neurobehavioral and neurochemical changes in individuals exposed to sarin/cyclosarin at levels insufficient to produce obvious clinical symptoms. However, the neuroanatomical correlates of these changes are unclear. The current study examined the association between modeled estimates of sarin/cyclosarin exposure levels and volumetric measurements of gross neuroanatomical structures in 1991 Gulf War veterans with varying degrees of possible low-level sarin/cyclosarin exposure. Methods Twenty-six GW-deployed veterans recruited from the Devens Cohort Study participated. Magnetic resonance images of the brain were acquired and analyzed using morphometric techniques, producing volumetric measurements of white matter, gray matter, right and left lateral ventricles, and cerebrospinal fluid. Volumetric data were analyzed using exposure estimates obtained from refined models of the 1991 Khamisiyah presumed exposure hazard area. Results Binary comparisons of sarin/cyclosarin ‘exposed’ ( N = 13) and ‘unexposed’ ( N = 13) veterans revealed no differences in volumetric measurements of discrete brain tissues. However, linear trend analyses showed a significant association between higher levels of estimated sarin/cyclosarin exposure and both reduced white matter (adjusted parameter estimate = −4.64, p < 0.0001) and increased right lateral ventricle (adjusted parameter estimate = .11, p = 0.0288) and left lateral ventricle (adjusted parameter estimate = .13, p < 0.0001) volumes. Conclusions These findings suggest subtle but persistent central nervous system pathology in Gulf War veterans potentially exposed to low levels of sarin/cyclosarin and argue for further investigation of the long-term effects of low-dose sarin/cyclosarin exposures in humans.

Activation of the Binuclear Metal Center through Formation of Phosphotriesterase−Inhibitor Complexes

Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate.

Transient and reversible nephrotoxicity of sarin in rats

Organophosphate (OP) poisoning, which inhibits cholinesterase activity, leads to severe cholinergic symptoms. Effective and quick management of these symptoms is considered critical to the clinical outcome. Acute renal damage following exposure to OP insecticides has been reported. Similar complications might occur following exposure to OP nerve agents, however, this subject has been studied only sporadically. In the present study, the effect of the nerve agent sarin on renal function was examined in rats. A single dose of sarin ( approximately 0.9 LD(50)) led to a significant reduction (of 45%) in renal function during the first 2 days post exposure, as exhibited by evaluation of the glomerular filtration rate, through measuring the clearance of ( 99m)Tc-DTPA. The urine volume was reduced by 50%, the urine specific gravity increased to 104% of the control value and massive hematuria and glucosuria were recorded 24-48 h post exposure. In addition, around 60% decrease in urine electrolytes was monitored during the first 2 days following exposure, with a recovery after 8 days. Post mortem gross inspection of the bladder, 24 h post exposure, revealed severe edema and hemorrhage. Treatment with the muscarinic antagonist atropine and the oxime TMB-4, at excessive doses administered 1 min post exposure, did not prevent most renal impairments. It has been concluded that sarin caused an acute renal dysfunction, possibly accompanied by bladder damage. These impairments were reversible, recovered spontaneously within 3-8 days, and were probably related to the state of shock and hypovolemia caused by the poisoning. However, if renal impairments are left unattended, they might contribute to the overall toxic manifestation and as a result aggravate the clinical state of intoxicated casualties.

Acetylcholinesterase: Converting a vulnerable target to a template for antidotes and detection of inhibitor exposure

Applications of recombinant DNA technology, chemical synthesis on biological templates and fluorescence detection of organophosphorylation provide unexplored avenues for development of antidotes and approaches for remote detection of organophosphate nerve agents and pesticides. We discuss here how acetylcholinesterase (AChE), through appropriate mutations, becomes more susceptible to oxime reactivation. Since the reaction between organophosphate and the mutated enzyme remains rapid, regeneration of active enzyme by oxime becomes the rate-limiting step in the process to complete a catalytic cycle for generation of active enzyme. Accordingly, “Oxime-assisted Catalysis” by AChE provides a potential means for catalyzing the hydrolysis of organophosphates in plasma prior to their reaching the cellular target site. In turn, AChE, when conjugated with organophosphate, is employed as a template for ‘click-chemistry, freeze-frame’ synthesis of new nucleophilic reactivating agents that could potentially prove useful in AChE reactivation at the target site as well as in catalytic scavenging of organophosphates in plasma. Finally, substituted AChE molecules can be conjugated to fluorophores giving rise to shifts in emission spectra for detection of dispersed organophosphates. Since external reagents do not have to be added to detect the fluorescence change, the modified enzyme would serve as a remote sensor.

Development of Miotic Cross-Tolerance Between Pyridostigmine and Sarin Vapor

The organophosphorous nerve agent sarin (GB) and the carbamate pyridostigmine bromide (PB) both inhibit acetylcholinesterase (AChE), leading to overstimulation of muscarinic receptors. Both GB and PB produce miosis through stimulation of ocular muscarinic receptors. This study investigated 2 hypotheses: (1) that the miotic response to PB would decrease following repeated injections; and (2) that repeated administration of PB would result in tolerance to the miotic effect of GB vapor. Rats were injected intramuscularly with saline, 0.04 mg/kg, 0.5 mg/kg, or 1.4 mg/kg of PB twice daily for 8 consecutive days. After day 3, animals injected with 1.4 mg/kg PB developed miotic tolerance. Twenty-four (24) h following the final PB injection, the rats were exposed to GB vapor (4.0 mg/m(3)). A similar magnitude of miosis was observed in all groups after GB exposure. However, the rate of recovery of pupil size in animals pretreated with 0.5 and 1.4 mg/kg PB was significantly increased. Twenty (20) h following exposure to GB vapor, the pupils of animals pretreated with 1.4 mg/kg PB had recovered to 77% +/- 4% of their pre-exposure baseline, whereas the saline-injected controls had recovered to only 52% +/- 2% of their pre-exposure baseline. The increased rate of recovery does not appear to be a result of protection of pupillary muscarinic receptors by the higher doses of PB, as there was no longer PB present in the animal at the time of GB exposure. These results demonstrate the development of tolerance to the miotic effect of PB following repeated exposures, and also suggest that cross-tolerance between PB and GB occurs. However, because the magnitude of the response was not reduced, the PB pretreatment and its associated miotic cross-tolerance does not appear to diminish the effectiveness of miosis as a biomarker of acute exposure to nerve agent vapor.

Sarin experiences in Japan: Acute toxicity and long-term effects

Two terrorist attacks with the nerve agent Sarin affected citizens in Matsumoto and Tokyo, Japan in 1994 and 1995, killing 19 and injuring more the 6000. Sarin, a very potent organophosphate nerve agent, inhibits acetylcholinesterase (AchE) activity within the central, peripheral, and autonomic nervous systems. Acute and long-term Sarin effects upon humans were well documented in these two events. Sarin gas inhalation caused instantaneous death by respiratory arrest in 4 victims in Matsumoto. In Tokyo, two died in station yards and another ten victims died in hospitals within a few hours to 3 months after poisoning. Six victims with serum ChE below 20% of the lowest normal were resuscitated from cardiopulmonary arrest (CPA) or coma with generalized convulsion. Five recovered completely and one remained in vegetative state due to anoxic brain damage. EEG abnormalities persisted for up to 5 years. Miosis and copious secretions from the respiratory and GI tracts (muscarinic effects) were common in severely to slightly affected victims. Weakness and twitches of muscles (nicotinic effects) appeared in severely affected victims. Neuropathy and ataxia were observed in small number (less than 10%) of victims, which findings disappeared between 3 days and 3 months. Leukocytosis and high serum CK levels were common. Hyperglycemia, ketonuria, low serum triglyceride, hypopotassemia were observed in severely affected victims, which abnormalities were attributed to damage of the adrenal medulla. Oximes, atropine sulphate, diazepam and ample intravenous infusion were effective treatments. Pralidoxime iodide IV reversed cholinesterase and symptoms quickly even if administered 6 h after exposure. Post Traumatic Stress Disorder (PTSD) was less than 8% after 5 years. However, psychological symptoms continue in victims of both incidents. In summary, both potent toxicity and quick recovery from critical ill conditions were prominent features. Conventional therapies proved effective in Sarin incidents in Japan.

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 x 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 x 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 x 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.

Currents: Chip-based DNA analysis | Subsurface imaging of nanostructures | Complex, static microfluidic gradients | Low-abundance proteins in blood | FTIR-SPR in live cells | Detecting organophosphate nerve agents

Currents: Chip-based DNA analysis | Subsurface imaging of nanostructures | Complex, static microfluidic gradients | Low-abundance proteins in blood | FTIR-SPR in live cells | Detecting organophosphate nerve agents

Charge Transfer from Metallic Single-Walled Carbon Nanotube Sensor Arrays

This paper explores the possibility of using arrays of metallic carbon nanotubes as sensors. Unlike their semiconducting counterparts, single-walled carbon nanotube arrays or networks that are dominated by metallic conduction pathways have not been investigated for their environmental sensitivity. In this work, we demonstrate transduction of molecular adsorption via charge transfer through predominantly metallic single-walled carbon nanotubes. Raman spectroscopy and electric field dependent transport confirm that signal transduction takes place through primarily large diameter metallic nanotubes. This unique signal transduction mechanism might have implications for novel sensors. The scaling of the signal with array impedance is well described using an irreversible binding model developed previously. The arrays have several advantages including a simple, two-electrode fabrication, rapid regeneration, and a responsivity that scales predictably and linearly with the number of adsorption sites. An array-assisted hydrolysis of reactive analytes is found to regenerate the nanotube surface from hydrolyzable species which include important organophosphate nerve agents.

Disposable Electrochemical Sensor for Determination of Nitroaromatic Compounds by a Single-Run Approach

Environmental and security applications have generated major demands for effective field-deployable tools for detecting nitroaromatic compounds, such as chloramphenicol (an antibiotic), parathion (an organophosphate nerve agent), and TNT (2,4,6-trinitrotoluene, an explosive) in a fast, simple, sensitive, reliable, and cost-effective manner. We report here a single-run approach for such a purpose. The reduction potential of different nitroaromatic compounds was found to systematically shift with the substituent group at an electrochemically preanodized screen-printed carbon electrode. The preanodization treatment makes the peak sharp and hence provides a precise way to identify the substituent effect on nitroaromatic compounds. By using potential shifts as analytical characteristics of nitroaromatic compounds, a suitable internal standard can be chosen based on the criteria of well-separated peak potential and rarely found in the real sample of interest. Simply by measuring the ratio of peak currents between analytes of interest and internal standard, the analysis can be done in a single-run measurement. Both the matrix effect and the variation of electrode during the preparation process can be canceled out in this approach and thus allows for a high-precision analysis. Just by placing a 20-microL drop on a single-use amperometric sensor strip incorporating a three-electrode configuration is enough for rapid and sensitive detection of nitroaromatic compounds by square-wave voltammetry. For example, the linear detection range can be up to 100 microM with a detection limit of 0.42 microM (S/N = 3) in the detection of chloramphenicol. This approach was successfully demonstrated in real sample analysis to verify the applicability of the method. The promising performances open new possibilities for rapid determination of nitroaromatic compounds in environmental and biological samples.

LC–UV and LC–MS evaluation of stress degradation behaviour of avizafone

It has been known for many years that benzodiazepine compounds effectively antagonize seizures induced by organophosphorous nerve agents. In the event of poisoning, a combination of three drugs is commonly used: an anticholinergic drug (e.g. atropine), an oxime used as cholinesterase reactivator (e.g. pralidoxime or HI-6) and an anticonvulsant (i.e. benzodiazepine). Most of anticholinergics and oximes are freely soluble in water, whereas many benzodiazepines are not. However, a water-soluble prodrug form of diazepam, avizafone, has been adopted by French armed forces for the immediate treatment of nerve agent seizure. The degradation behaviour of this new drug was investigated under different stress degradation conditions (hydrolytic, oxidative, photolytic and thermal) as recommended by International Conference on Harmonization. Successful separation of the active pharmaceutical ingredient from decomposition products formed under stress conditions was achieved using liquid chromatography. The method was validated with respect to specificity, linearity, precision and accuracy.

Cholinesterases in the Milk of Domestic Animals

Human serum butyrylcholinesterase (Hu BChE) is currently under development as a prophylaxis that can protect soldiers from organophosphate (OP) nerve agents. Due to the high cost of production of this enzyme, recombinant Hu BChE is being produced in the milk of transgenic goats. The objective of this study was to determine the presence of endogenous cholinesterases (ChEs) in the milk of cow, goat, sheep, and pig to determine the suitability of goats for the production of recombinant Hu BChE. ChE activity was measured using acetyl-, butyryl- and propionylthiocholine as substrates using the Ellman assay. Detectable levels of ChEs (2-4 U/ml) were present in pig milk only. ChEs in milk from other animals were < 0.1 U/ml and could be detected following centrifugation and enrichment on a procainamide column only. ChE activity in milk varied with the number of pregnancy. ChE-specific inhibitors were used to calculate the proportions of acetylcholinesterase (AChE) and BChE activities. Milk from all four species contained predominantly BChE activity, varying from 100 % in pig to 55 % in sheep. Monoclonal antibodies (MAbs), produced against fetal bovine serum AChE that inhibit AChEs from ruminants only, were also used to confirm the identity of AChEs. Studies with MAbs showed that BChE activity in milk varied from 100 % in pig to 40 % in sheep. Taken together, these results support the choice of goat as the transgenic animal for the expression of recombinant Hu BChE.

Effects of VX and soman on vascular contractility in isolated rat and guinea pig aorta

The mechanism of organophosphorous nerve agents is inhibition of cholinesterase. The effect of in vivo nerve agent exposure on isolated vascular contractility is unknown. Since disruption of the cholinergic signaling cascade by nerve agent exposure could have detrimental effects on vascular function, we hypothesized that exposure to nerve agents would alter isometric force development. Integrity of vascular smooth muscle voltage and receptor-mediated contractile responses was evaluated in adult male rats and guinea pigs. Aortic rings were isolated from the animals one hour after a sublethal exposure (0.4, 0.6, or 0.8 LD50) to VX or soman (GD). Response to potassium chloride (KCl) and phenylephrine (PE) was measured in an organ bath apparatus. Force development to KCl or PE was depressed in aorta from VX-exposed rats, but increased after GD exposure. Guinea pig aorta exhibited a slight depression in KCl and PE induced at the highest VX dose. Conversely, the lower dose of GD caused an increase in guinea pig aortic force development, while the higher doses remained at, or near, control levels. These data suggest a nerve agent-induced change in vascular force regulation that is species-specific and distinct between VX and GD. The results will provide an indication of the functional consequences following in vivo exposure to nerve agents and assist in developing medical countermeasures against chemical warfare agents.

Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides

Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.

Microbial biosensor for direct determination of nitrophenyl-substituted organophosphate nerve agents using genetically engineered Moraxella sp.

A microbial biosensor consisting of a dissolved oxygen electrode modified with the genetically engineered PNP-degrader Moraxella sp. displaying organophosphorus hydrolase (OPH) on the cell surface for sensitive, selective, rapid and direct determination of p-nitrophenyl (PNP)-substituted organophosphates (OPs) is reported. Surface-expressed OPH works in tandem with the PNP oxidation machinery of the Moraxella sp. to degrade PNP-substituted OPs and PNP simultaneously while consuming oxygen, that is proportional to the analyte concentration. The optimum performance was obtained by electrodes constructed using 0.35 mg dry weight of cell and operating at pH 7.5. Operating at optimum conditions the biosensor was able to measure as low as 0.1 μM (27.5 ppb) of paraoxon and had excellent selectivity against triazines, carbamates and OPs without PNP substitutent. The biosensor was stable for a week when stored at 4 °C. The applicability of the biosensor to measure OPs in lake water was demonstrated.

Recent Advances In Treatment of Acute Organophosphorous Nerve Agents Poisoning

Organophosphorous (OP) chemical warfare nerve agents mainly sarin and tabun were used during the Iran-Iraq war with high mortalities. In addition to atropine and oximes, the followings have recently been used successfully for the treatment of OP poisoning. 1. Sodium Bicarbonate: Infusion of high doses of sodium bicarbonate (5 mEq/kg in 60 min. followed by 5-6 mEq/kg/day to obtain arterial blood pH of 7.45 to 7.55) revealed positive effects in patients with acute OP poisoning in Mashhad. 2. Magnesium Sulfate: Intravenous magnesium sulfate in a dose of 4 g only on the first day after admission was also effective in acute human OP poisoning. 3. Antioxidants: The toxicity of OP compounds is mediated by generation of nitric oxide and other free radicals. These toxic molecules can be counteracted by antioxidants such as vitamins C and E, spin traps, melatonin and low molecule weight thiols. The latter compounds can also increase the synthesis of glutathione, which can both ameliorate the OP-induced oxidative stress and enhance OP detoxification. It is concluded Sodium bicarbonate, Magnesium sulfate and the antioxidants should be added to the standard treatment of OP poisonings.