Toxicology Letters 190 (2009) Contents lists available at ScienceDirect. Toxicology Letters. journal homepage:

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1 Toxicology Letters 190 (2009) Contents lists available at ScienceDirect Toxicology Letters journal homepage: Mini-review Medical treatment of acute poisoning with organophosphorus and carbamate pesticides Milan Jokanović a,b, a Faculty of Medicine, University of Nish, Nish, Serbia b Academy of Sciences and Arts of Republic Srpska, Banja Luka, Republic Srpska, Bosnia and Herzegovina article info abstract Article history: Received 13 July 2009 Accepted 27 July 2009 Available online 3 August 2009 Keywords: Organophosphorus compounds Pyridinium oximes Antidotes Pralidoxime Obidoxime Trimedoxime HI-6 Atropine Diazepam Warfare nerve agents Acetylcholinesterase Pesticides Carbamates Medical treatment Organophosphorus compounds (OPs) are used as pesticides and developed as warfare nerve agents such as tabun, soman, sarin, VX and others. Exposure to even small amounts of an OP can be fatal and death is usually caused by respiratory failure. The mechanism of OP poisoning involves inhibition of acetylcholinesterase (AChE) leading to inactivation of the enzyme which has an important role in neurotransmission. AChE inhibition results in the accumulation of acetylcholine at cholinergic receptor sites, producing continuous stimulation of cholinergic fibers throughout the nervous systems. During more than five decades, pyridinium oximes have been developed as therapeutic agents used in the medical treatment of poisoning with OP. They act by reactivation of AChE inhibited by OP. However, they differ in their activity in poisoning with pesticides and warfare nerve agents and there is still no universal broad-spectrum oxime capable of protecting against all known OP. In spite of enormous efforts devoted to development of new pyridinium oximes as potential antidotes against poisoning with OP only four compounds so far have found its application in human medicine. Presently, a combination of an antimuscarinic agent, e.g. atropine, AChE reactivator such as one of the recommended pyridinium oximes (pralidoxime, trimedoxime, obidoxime and HI-6) and diazepam are used for the treatment of OP poisoning in humans. In this article the available data related to medical treatment of poisoning with OP pesticides are reviewed and the current recommendations are presented Elsevier Ireland Ltd. All rights reserved. Contents 1. Introduction Interaction of cholinesterases with organophosphorus and carbamate compounds Clinical presentation of acute poisoning with organophosphorus compounds Antidotes in the treatment of human poisoning with organophosphorus pesticides Atropine Diazepam Pyridinium oximes Medical treatment of acute poisoning with organophosphorus pesticides General measures Specific therapy Atropine Pyridinium oximes Clinical experience with pyridinium oximes Clinical aspects of medical treatment of poisoning with carbamate pesticides Conclusions Conflict of interest Acknowledgements References Correspondence address: Experta Consulting, Nehruova 57, Belgrade, Serbia. Tel.: addresses: milan.jokanovic@gmail.com, mikatox@yahoo.com /$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi: /j.toxlet

2 108 M. Jokanović / Toxicology Letters 190 (2009) Introduction Organophosphorus compounds (OPs) have been used as pesticides and developed as warfare nerve agents such as soman, sarin, tabun, VX and others. OP pesticide self-poisoning is an important clinical problem in rural regions of the developing world that kills an estimated 200,000 people every year. Unintentional poisoning kills far fewer people but is an apparent problem in places where highly toxic OP pesticides are available. Medical management is difficult, with case fatality generally more than 15% (Eddleston et al., 2008). Carbamates (CBs) are used as pesticides and some of them (e.g. physostigmine, pyridostigmine) have been registered as human drugs. Exposure to even small amounts of an OP compound can be fatal; death is usually caused by respiratory failure resulting from paralysis of the diaphragm and intercostal muscles, depression of the brain respiratory center, bronchospasm, and excessive bronchial secretions. The mechanism of OP poisoning involves phosphorylation of the serine hydroxyl group in the active site of acetylcholinesterase (AChE) leading to the inactivation of this essential enzyme which has an important role in neurotransmission. The CB also act by carbamylating the same site on AChE which reversibly inhibits the enzyme activity. AChE inhibition results in the accumulation of acetylcholine at cholinergic receptor sites, producing continuous stimulation of cholinergic fibers throughout the central and peripheral nervous systems. Presently, a combination of an antimuscarinic agent, e.g. atropine, AChE reactivator such as one of the recommended pyridinium oximes (pralidoxime, trimedoxime, obidoxime and HI-6) and diazepam are used for the treatment of OP poisoning in humans. Both atropine and oximes were introduced into clinical practice in the 1950s without any clinical trials. As a consequence, we still do not know the ideal regimens for either therapy. This article reviews the mechanisms of action of OP, CB, and antidotes used in current medical treatment of human poisonings with OP and CB pesticides. 2. Interaction of cholinesterases with organophosphorus and carbamate compounds There are different types of cholinesterases in the human body, which differ in their location in tissues, substrate affinity, and physiological function. The principal ones are acetylcholinesterase (EC , AChE), found in the nervous system and also present in the outer membrane of erythrocytes, and plasma cholinesterases (EC , ChE), which are a group of enzymes present in plasma, liver, cerebrospinal fluid and glial cells. Under normal physiological conditions, AChE performs the breakdown of acetylcholine (ACh), which is the chemical mediator responsible for conduction of nerve impulses at the sites of cholinergic transmission. However, its physiological role in blood is not understood. On the other hand, ChE is a circulating plasma glycoprotein synthesised in the liver, which does not serve any known physiological function. It was proposed that ChE may have roles in cholinergic neurotransmission and involved in other nervous system functions (cellular proliferation and neurite growth during the development of the nervous system) and in neurodegenerative disorders (Darvesh et al., 2003). The function of AChE is termination of action of ACh at the junctions of the various cholinergic nerve endings with their effector organs or post-synaptic sites. OP and CB are the most important AChE inhibitors and often called anticholinesterases. In the presence of inhibitors, AChE becomes progressively inhibited and is not further capable of hydrolyzing ACh to choline and acetic acid (Jokanović and Maksimović, 1997). Consequently, ACh accumulates at cholinergic receptor sites and produces effects equivalent to excessive stimulation of cholinergic receptors throughout the central and peripheral nervous system. Both substrate and inhibitors react covalently with the esterases in essentially the same manner, because acetylation of the serine residue at AChE catalytic site is analogous to phosphorylation or carbamylation. In contrast to the acetylated enzyme which rapidly separates acetic acid and restores the catalytic site, the phosphorylated enzyme is stable (Fig. 1). Inhibited enzyme can be spontaneously reactivated at different rates depending on the inhibitor for CB it occurs very rapidly with half-time of an hour or less, while for OP having branched alkyl groups it may occur at a very slow rate. In addition, for OP pesticides containing dimethyl phosphate groups the half-time of spontaneous reactivation of phosphorylated AChE in vitro is h, in vivo 2.1 h and the half-time of aging is 3.7 h. For diethyl phosphates the rate of spontaneous reactivation is slower (31 57 h in vitro) as well as the rate of aging (31 h) (Reiner and Pleština, 1979; Worek and Diepold, 1999; Mason et al., 2000; Eyer, 2003). As the result, CB and dimethyl phosphates are recognized as reversible AChE inhibitors, while other OP having branched alkyl groups are practically irreversible AChE inhibitors. The variations in the acute toxicity of OP are the result of their different chemical structures and rates of spontaneous reactivation and aging. Aged form of phosphorylated AChE is resistant to both spontaneous and oxime-induced reactivation. The aging reaction, although appearing with many phosphorylated AChE complexes, has the only major clinical importance and an imperative problem particularly in the treatment of soman poisoning. Aging with soman occurs so fast that no clinically relevant spontaneous reactivation of AChE can occur before aging has taken place. Hence, recovery of function depends on relatively slow resynthesis of AChE. It is important to immediately administer atropine and oximes so that some extent of AChE reactivation occurs before all AChE has aged. Even though aging occurs slowly and reactivation relatively rapidly in the case of OP insecticides and other nerve agents, early oxime administration is clinically important particularly in patients poisoned with these agents. 3. Clinical presentation of acute poisoning with organophosphorus compounds Signs and symptoms of acute poisoning with anticholinesterase agents are predictable from their biochemical mechanism of action and are directly related to the levels of AChE activity. In cases of human intoxication, general acute symptoms of peripheral nicotinic and muscarinic intoxication are clearly apparent (World Health Organization, 1986). These symptoms include miosis (unreactive to light); sweating, rhinorrhea, lacrimation, and salivation; abdominal cramps and other gastrointestinal symptoms; respiratory difficulties and cough; dyspnea, constriction sensation in the chest, wheezing; twitching of facial muscles and tongue, tremors, and fasciculations; bradycardia and ECG changes, pallor, and cyanosis; anorexia, nausea, vomiting, diarrhea, and involuntary urination and defecation. These signs and symptoms are accompanied by central effects such as dizziness, tremulousness, and confusion; ataxia; headache, fatigability, and paresthesia. Finally, seizures, convulsions, twitching, coma, and respiratory failure may occur. If the subject survives past the day of poisoning, there are personality changes, mood swings, aggressive events and psychotic episodes including schizoid reactions, paranoid delusions, and exacerbations of preexisting psychiatric problems. Sleep is poor from nightmares and hallucinations; disturbances or deficits in memory and attention, and additional delayed effects also occur (Karchmar, 2007; World Health Organization, 1986; IPCS, 1989; Marrs and Vale, 2006). The first 4 6 h are the most critical in acute poisoning with OP pesticides. If there is improvement in symptoms after initial treatment then the patient is very likely to survive if adequate treatment

3 M. Jokanović / Toxicology Letters 190 (2009) Fig. 1. Physiological role (a) and interaction of acetylcholinesterase (AChE) and other esterases (E) with organophosphorus (b) and carbamate (c) compounds. Reaction 1 shows interaction of organophosphate molecule with the serine hydroxyl group at the active site of AChE via formation of an intermediate Michaelis Menten complex leading to phosphorylated enzyme (Reaction 2). Reaction 3 is a spontaneous reactivation of inhibited AChE which occurs very slowly for most OP and very rapidly for carbamates. Reaction 4, called aging, represents non-enzymatic time-dependent loss of one alkyl group (R) bound to the phosphorus. The aging reaction depends on the chemical structure of the inhibitor and leads to a stable non-reactivatable form of phosphorylated AChE. is continued (IPCS, 1989). The duration of effects is determined mainly by the properties of the compound: its liposolubility, the stability of the OP AChE complex and whether it is reactivatable after the use of cholinesterase reactivators (such as oximes). It is important to note that only OP containing P O bond (known as direct inhibitors) are potent AChE inhibitors; those having a P S group (indirect inhibitors) must be metabolically activated to P O group (Jokanović, 2001). The signs and symptoms of poisoning with direct inhibitors appear quickly during or after exposure, while those with indirect inhibitors appear slowly and last longer, even up to several days after cessation of exposure. Clinical diagnosis is relatively simple and is based on medical history, circumstances of exposure, clinical presentation, and laboratory tests. Confirmation of diagnosis can be made by measurement of erythrocyte AChE or plasma ChE. Activities of these enzymes are accepted as biomarkers of exposure and/or toxicity of OP and CB. Erythrocyte AChE is identical to the enzyme present in the target synapses and its levels are assumed to reflect the effects in target organs. Thus, erythrocyte AChE is regarded as biomarker of toxicity of these compounds. However, it must be kept in mind that the above assumption is only correct when the inhibitor has equal access to blood and synapses. It is difficult to know, due to pharmacokinetic reasons, how closely AChE inhibition in erythrocytes reflects that in the nervous system since access to blood is always easier than access to brain. Thus, the inhibition of AChE in erythrocytes may be overestimated relative to that in brain (Jokanović and Maksimović, 1997). In addition, AChE in brain is restored by de novo synthesis more rapidly than in erythrocytes where AChE activity is recovered via erythropoesis. The level of activity of ChE should be carefully interpreted since the normal range in healthy subjects is relatively wide, rendering interpretation in individual patients difficult unless the results of previous estimations in the patient are available. Inhibition of ChE does not provide accurate information related to clinical severity of the poisoning. Many OP insecticides (e.g. chlorpyrifos, demethon and malathion) appear to be more potent inhibitors of ChE than they are of erythrocyte AChE and, as the consequence, ChE inhibition might occur to a greater extent than AChE inhibition. The rate of spontaneous reactivation (Fig. 1, Reaction 3) can be accelerated by pyridinium oximes that have a chemical structure which fits the structure of the inhibited AChE. The oximes can only be of benefit as long as inhibited AChE is not completely converted to the aged form. 4. Antidotes in the treatment of human poisoning with organophosphorus pesticides 4.1. Atropine Atropine acts by blocking the effects of excess concentrations of acetylcholine at muscarinic cholinergic synapses following OP inhibition of AChE. Atropine is the initial drug of choice in acute OP poisoning. Atropine sulphate in combination with an oxime has been used in traditional therapy for OP intoxications including insecticides. Atropine can relieve the following symptoms of OP poisoning: sweating, salivation, rhinorrhoea, lacrimation, nausea, vomiting and diarrhea, and can help control of bradycardia and circulatory depressions, dilating the bronchi and abolishing bronchorrhoea. Atropine does not bind to nicotinic receptors and cannot relieve nicotinic effects in OP poisoning. It has been shown that atropine may have anticonvulsant effects and prevent development of convulsions and brain damage induced by certain OP (McDonough et al., 1987). Other authors have stated that atropine can only partly block convulsions after exposure to these agents since other transmitter systems (GABA, glutamate) become involved in cholinergic overstimulation in brain (Zilker, 2005; Antonijević and Stojiljković, 2007). The effects of atropine in OP poisoning are far more complex than muscarinic blokade. In a study in rats it was found that atropine treatment reduced local use of cerebral glucose and brain damage during seizures induced with soman (Pazdernik et al., 1986). Although the clinical efficacy of atropine in OP poisoning is well established, no controlled studies have been published. Atropine dosing is discussed later in this text Diazepam Benzodiazepines are CNS depressants, anxiolytics and muscle relaxants. Their main site of action is at the gamma-aminobutyric acid (GABA) receptor. The GABA A receptor is a ligand gated chloride ion channel and part of a superfamily of receptors which also includes the nicotinic acetylcholine receptor and the glycine receptor. GABA is the major inhibitory neurotransmitter in the mammalian central nervous system. Benzodiazepines, including diazepam, alter GABA binding at the GABA A receptor in an allosteric fashion, but do not directly activate the receptors (Sellström, 1992; Marrs, 2004). Currently, the most important anticonvulsant is diazepam. The combination of atropine and diazepam is more effective in reducing mortality than atropine or oxime alone. It was also shown that diazepam enhanced the efficacy of low doses of atropine. In the cholinergic nervous system, diazepam appears to decrease the synaptic release of ACh. The main consequence of the action of benzodiazepines in CNS is hyperpolarization of neurons making them significantly less susceptible to cholinergically induced depolarization. The ultimate result is cessation of propagation of convulsions (Sellström, 1992; Marrs, 2004; Antonijević and Stojiljković, 2007). In patients poisoned with OP, benzodiazepines have a beneficial effect in reducing anxiety and restlessness, reducing muscle fasciculation, arresting seizures, convulsions, controlling apprehension and agitation and possibly reducing morbidity and mortality when

4 110 M. Jokanović / Toxicology Letters 190 (2009) used in conjunction with atropine and an oxime. Diazepam should be given to patients poisoned with OP whenever convulsions or pronounced muscle fasciculation are present. In severe poisoning, diazepam administration should be considered even before these complications develop. The recommended dose of diazepam in cases of OP poisoning is 5 10 mg intravenously in the absence of convulsions and mg intravenously in cases with convulsions, which may be repeated as required (Johnson and Vale, 1992; Antonijević and Stojiljković, 2007). WHO recommends the dose of diazepam of 5 10 mg intravenously slowly over 3 min which may be repeated every min (maximum 30 mg) in adults and mg/kg intravenously slowly over 3 min in children (maximum 5 mg in children up to 5 years old, and 10 mg in children older than 5 years) (IPCS, 1989) Pyridinium oximes Extensive studies over the past decades have investigated the mechanism of action of pyridinium oximes. There is convincing evidence that the antidotal potency of pyridinium oximes is primarily attributed to their ability to reactivate the phosphorylated cholinesterases. Oximes reactivate phosphorylated cholinesterases by displacing the phosphoryl moiety from the enzyme by virtue of their high affinity for the enzyme and their powerful nucleophilicity. Reactivation proceeds as a two-step reaction via formation of an intermediate Michaelis Menten complex leading to formation of more stable phosphoryl residue bound to the hydroxyl group of serine at the active site of AChE. The rate of reactivation depends on the structure of the phosphoryl moiety bound to the enzyme, the source of the enzyme, the structure and concentration of oxime which is present at the active site, and the rate of postinhibitory dealkylation known as aging. Phosphorylated oximes are formed during reactivation reaction and some of them appear to be potent inhibitors of AChE (Luo et al., 1999; Ashani et al., 2003; Worek et al., 2007). The structure activity relationship for pyridinium oximes developed as AChE reactivators was recently discussed by Jokanović and Prostran (2009). In addition to performing AChE reactivation in OP poisoning, pyridinium oximes also show direct pharmacological effects that are discussed in detail in other publications (Jokanović and Stojiljković, 2006; Jokanović and Prostran, 2009). Pyridinium oximes are effective against OP-inhibited AChE in the peripheral nervous system, but have a limited penetration across the blood brain barrier due to their pharmacokinetic profile and the presence of quaternary nitrogen atom(s) in their structure. However, it appears that oxime penetration through blood brain barrier is underestimated since soman can cause seizure-related opening of the blood brain barrier (Carpentier et al., 1990; Grange-Messent et al., 1999) and enable passage of higher oxime concentrations into the brain. Two other processes may contribute to better penetration of oximes through the blood brain barrier: the induction of local inflamatory processes and increase of brain blood flow (Shrot et al., 2009). It was shown that LD50 of sarin caused a dose-dependent increase in permeability of blood brain barrier in midbrain, brainstem, cerebrum and cerebellum in rats 24 h after poisoning (Abdel-Rahman et al., 2002). Sakurada et al. (2003) have determined the amount of PAM-2 passing across the blood brain barrier at approximately 10% of the given dose which may be effective in reactivation of OP-inhibited AChE in brain. Additional data indicate that in OP poisoning, when given with atropine, PAM-2 can pass blood brain barrier at higher concentrations. Some clinicians from Asia have reported that pralidoxime is not sufficiently effective in treatment of OP pesticide poisoning and their opinion is based on poorly designed studies (suboptimal dose, short duration of treatment, long delay before pralidoxime is given, studies did not follow WHO recommendations, the chemical strucutre of OP pesticide not taken into account, etc.) (De Silva et al., 1992; Singh et al., 1995; Johnson et al., 1996; Cherian et al., 2005). Possible reasons why oximes may not be effective in OP poisoning were discussed by Johnson et al. (2000), Eyer (2003) and Milatović and Jokanović (2009). Among those reasons the most important are the following: a. The oxime dose may be inadequate to produce the optimal concentration required to achieve the desired reactivation and it may not be present at target sites when mostly needed (i.e. when AChE inhibition reaches its maximum and when OP is present in blood at high concentrations). Proposed minimumeffective plasma levels for oximes of 4 mg/l were also supported by other scientists (Bokonjić et al., 1987; Shiloff and Clement, 1987; Kušić et al., 1991). In severe cases of OP pesticide poisoning, higher oxime concentrations may be necessary, especially in the case of pralidoxime. However, Eyer (2003) disagrees with the 4 mg/l concept suggesting necessity of a higher oxime concentration. He proposed that for the most frequently used OP, pralidoxime plasma concentrations of around 80 mol/l (13.8 mg/l pralidoxime chloride) or 10 mol/l of obidoxime (3.6 mg/l obidoxime chloride) should be adequate and maintained for as long as the OP is present in the body. Oxime treatment may be required for up to 10 days. b. In addition to an inadequate initial dose, subsequent treatment with oxime may not be sufficiently persistent. Oximes are rapidly cleared from the body and although some reactivation may be achieved, another cycle of inhibition and eventually aging of inhibited AChE, due to continuing presence of OP in blood, may follow. This is particularly possible in the case of massive overdose where residual OP pesticide may persist in the body for several days. In such cases only persistent administration of oximes, by means of continuous infusion or repeated oxime administration, can be expected to result in permanent clinical improvement. Eyer (2003) suggests that the most appropriate dosing regimen consists of a bolus short infusion followed by a maintenance dosage. For pralidoxime chloride a 1 g bolus over 30 min followed by an infusion of 0.5 g/h appears appropriate to maintain the concentration of 13 mg/l. For obidoxime chloride the proposed dosing regimen is a 0.25 g bolus followed by an infusion of 0.75 g/24 h. It is important that the concentrations were well tolerated and effective in keeping the active levels of AChE. c. Treatment with oxime may be started too late or terminated too soon. In all cases of OP poisoning it would be ideal to start antidotal treatment as soon as possible and to maintain the treatment as long as needed. In cases of poisoning with persistent OP pesticides it is appropriate to start oxime therapy at adequate dose levels up to 10 days after exposure or even later. Eddleston et al. (2006) suggest that oximes may be effective if given within about 120 h for diethyl OP poisoning and 12 h for dimethyl OP poisoning. The treatment should be continued until obviously no longer needed and the decision about this can be made on the basis of clinical status of the patient, relatively high AChE activity in erythrocytes when compared to control values and the absence of OP and/or OP metabolites in urine. Observational studies of pralidoxime and obidoxime suggest that the ability to reverse AChE inhibition with oximes varies with the pesticide ingested. AChE inhibited by diethyl OP pesticides, such as parathion and quinalphos, seems to be effectively reactivated by oximes, but AChE inhibited by dimethyl OP, such as dimethoate, monocrotophos and oxydemeton-methyl, apparently responds poorly. AChE inhibited by S-alkyl-linked OP, such as profenofos, is not reactivated by oximes at all. This difference is

5 M. Jokanović / Toxicology Letters 190 (2009) probably caused by variations in the rate of aging on inhibited AChE induced by different OP pesticides. 5. Medical treatment of acute poisoning with organophosphorus pesticides 5.1. General measures Treatment of OP pesticide poisoning should begin with decontamination and resuscitation if needed. Decontamination is vital in reducing the dose of the pesticide absorbed, but care must be taken not to contaminate others, such as medical and paramedical workers. In the case of ingestion, lavage can be performed, and activated charcoal administered. The patient should be observed carefully during the early stages of treatment because respiratory arrest may occur. Solvent vehicles and other components of the formulated OP pesticides may complicate the clinical picture and should be taken into consideration (IPCS, 1989). Supportive measures should be directed towards the cardiorespiratory system with particular emphasis on maintenance of ventilation, cardiac rhythm and blood pressure; the removal by suction of respiratory and oral secretions which may cause respiratory distress; and the oxygenation of the patient. Severely poisoned patients disconnected from the ventilator when the general condition improves, must be carefully watched for rapid deterioration and development of the intermediate syndrome during the following few days in the Intensive Care Unit (IPCS, 1989). In addition, the patients should be warned to report to hospital if signs of organophosphate-induced delayed polyneuropathy appear 2 3 weeks after exposure. Ingested organophosphates should be removed by early gastric aspiration and then lavage, with protection of the airway because they are mostly dissolved in aromatic hydrocarbons; this may be the best remedy in unconscious patients. Gastric lavage is most effective within 30 min of ingestion, but might be still effective up to 4 h post-ingestion, as organophosphates are rapidly absorbed from the gastrointestinal tract (World Health Organization, 1986). Administration of oral activated charcoal, in conventional doses, may be considered for reducing further absorption of some OP pesticides (World Health Organization, 1986). This recommendation was supported by Peng et al. (2004) who conducted a randomized controlled clinical trial involving 108 patients aimed to assess the efficacy of hemoperfusion with charcoal in treatment of acute severe dichlorvos poisoning. The authors concluded that the rapid fall in blood dichlorvos level and the dramatic clinical response suggest that hemoperfusion with charcoal is effective in the treatment of acute severe dichlorvos poisoning. Two recent clinical trials designed to evaluate the effectiveness of activated charcoal in OP poisoned patients failed to confirm these results. A randomized controlled trial of single and multiple doses of activated charcoal in Sri Lanka failed to find a significant benefit of either regimen over placebo in more than 1000 patients poisoned with pesticides (Eddleston et al., 2005a). In addition, Eddleston et al. (2008) conducted an open-label, parallel group, randomized, controlled trial in three Sri Lankan hospitals aimed to assess whether routine treatment with multiple-dose activated charcoal offers benefit compared with no charcoal. Among 2338 patients who ingested pesticides (1310 cases of poisoning with OP and carbamate pesticides) there were no differences in mortality between patients treated with or no charcoal. The authors concluded that they cannot recommend the routine use of multiple dose activated charcoal in poisonings with OP and carbamate pesticides and suggest that further studies of early charcoal administration might be useful. Fig. 2. Chemical structure of pyridinium oximes used in medical treatment of human OP poisoning. X stands for an anion (chloride or methylsulphate) Specific therapy Atropine According to IPCS (1989) an initial trial dose of atropine, 1 2 mg (0.05 mg/kg) intravenously, should be given slowly over 3 min, and then repeated every 5 10 min if there is no observable adverse effect. In symptomatic children, intravenous dose of mg/kg atropine should be administered every 15 min as needed. Atropine may then be repeated or increased in increments at min intervals until bronchosecretion is cleared and the patient is atropinized (dilated pupils, dry skin, and skin flushing) which should be maintained during further treatment. Repeated evaluations of the quantity of the secretions through regular auscultation of the lungs is the only adequate measure of atropinization in the severely poisoned patient. The dose may be increased as required. Patients poisoned with OP appear to be resistant to toxic effects of atropine and may require relatively large doses of atropine administered during prolonged periods. In severe OP poisoning total dose of atropine given during 5 weeks of treatment can be as high as 30,000 mg (IPCS, 2002) Pyridinium oximes Among the many classes of oximes investigated so far, those with clinical application can be divided in two groups the monopyridinium and bispyridinium oximes. Currently, the only used monopyridinium oxime is pralidoxime (PAM-2), while the most significant bispyridinium oximes comprise: trimedoxime (TMB-4), obidoxime (LüH-6, Toxogonin) and asoxime (HI-6), and their chemical structure is presented in Fig. 2. There is still no international consensus on the choice of most effective oxime and on dosing regimen Pralidoxime. Pralidoxime administered to human volunteers at a dose of 10 mg/kg by intramuscular route, produced a plasma concentration of >4 mg/l within 5 10 min and maintained levels above this threshold for an hour (Sidell and Groff, 1971). Adverse effects of PAM-2 iodide in volunteers include dizziness, blurred vision, occasional diplopia, impaired accomodation, nausea and headache (Jagger and Stagg, 1958; Sidell and Groff, 1971). The clinical experience with the use of PAM-2 iodide, given with atropine and diazepam, in the treatment of the victims of Tokyo sarin attack victims in 1995 was extremely favourable (Stojiljković and Jokanović, 2005). However, PAM-2 should not be recommended as the drug of choice in poisoning with warfare nerve

6 112 M. Jokanović / Toxicology Letters 190 (2009) agents due to its lack of efficacy against tabun and soman (Kassa, 2005). In poisoning with OP pesticides pralidoxime chloride should be administered to adults in a dose of 500 mg/h, continuously maintained until clinical improvement is obtained, or 30 mg/kg body weight bolus intravenously over 4 6 h or 8 10 mg/kg/h intravenously until full recovery occurs. In children, pralidoxime chloride should be administered in a dose of 25 mg/kg intravenously for min, followed by a continuous infusion of mg/kg/h. The therapy can continue for 18 h or longer, depending on the clinical status (IPCS, 1989). It is essential to adjust the appropriate plasma concentration, i.e. for pralidoxime mg/l and for obidoxime about 4 mg/l. This concentration is usually attained by a daily dose of g PAM- 2 Cl and g obidoxime, respectively, either given divided in 4 6 single bolus doses or, preferably, by continuous intravenous infusion, following the first loading dose (2 g pralidoxime and 0.25 g obidoxime, respectively) (IPCS, 1989) Obidoxime. When administered to human volunteers by intramuscular route obidoxime 5 mg/kg produced a plasma concentration >4 mg/l, from 5 min after injection to 3 h (Sidell and Groff, 1970). Adverse effects of obidoxime in male volunteers were described as pallor, nausea, burning sensation, headache, generalized weakness, sore throat, and paresthesia of the face (Simon and Pickering, 1976; Eyer, 2003; Marrs and Vale, 2006). Following high doses of obidoxime (several grams per day) in severely OPpoisoned patients, hepatotoxic effects were occasionally observed including increased serum transaminases, jaundice and cholestasis (Eyer, 2003). Obidoxime should be administered in adults at dose of 250 mg given by slow intravenous injection followed by continuous infusion of 750 mg/24 h (0.4 mg/kg/h) to reach plasma concentrations of mol/l. Intramuscular dosing is possible when the intravenous route is inaccessible. In children, the dose of obidoxime is 3 6 mg/kg slowly administered intravenously over at least 5 min (IPCS, 1989) Asoxime (HI-6). Clinical studies showed that HI-6 dosed at either 250 mg or 500 mg by intramuscular route reached plasma concentrations >4 mg/l in 4 6 min. This concentration was maintained for 125 min following the lower dose (250 mg) and 200 min following the higher dose (500 mg) (Kušić et al., 1985, 1991). These authors have administered HI-6 four times a day as a single intramuscular injection of 500 mg with atropine and diazepam treatment. Oxime therapy was started on admission and continued for 2 7 days. A clinical study performed on 22 healthy human volunteers did not reveal any adverse effects when HI-6 was given in doses up to 500 mg by oral route (Jovanović et al., 1990). HI-6 is considered to be a very promising bispyridinium oxime in medical treatment following exposure to most nerve agents. A disadvantage of HI-6 compared to other available oximes is its lack of stability in aqueous solutions. HI-6 was considered to be an effective antidote (in combination with atropine and diazepam) in treatment of patients poisoned with OP insecticides (Kušić et al., 1991). It is important to note that oximes are not effective for improvement of outcomes if the patient develops severe complications such as aspiration pneumonia or hypoxic brain injury before treatment. Such complications take place with fast-acting pesticides such as parathion and dichlorvos (Eddleston et al., 2008) Clinical experience with pyridinium oximes A particular problem in interpreting the beneficial role and efficacy of oximes in clinical practice is a deficiency of published data, especially those evaluated in controlled clinical trials. Studies related to the efficacy of oximes in clinical setting showed the heterogeneity of therapeutic approaches (i.e. dose regimen, oxime choice and final outcome of the treatment). In most reports cited in this section chemical structure of OP pesticides was identified in blood/urine and there were adequate data on therapeutic measures taken. Eddleston et al. (2005b) reported the results of a prospective study on 802 patients self-poisoned with chlorpyrifos, dimethoate, or fenthion. Compared with chlorpyrifos (8.0%), the proportion dying was significantly higher with dimethoate (23.1%) or fenthion (16.2%) as was the proportion requiring endotracheal intubation (chlorpyrifos, 15.0%; dimethoate, 35.2%; fenthion, 31.3%). Patients poisoned by diethyl OP pesticide (chlorpyrifos) responded well to pralidoxime, whereas those poisoned by two dimethyl OP pesticides (dimethoate, fenthion) responded poorly. Poor efficacy of pralidoxime in treatment of human dimethoate and fenthion poisonings was in agreement with experimental studies conducted by Jokanović and Maksimović (1995) who found that antidotal efficacy of obidoxime, trimedoxime, pralidoxime and HI-6 (given with atropine and diazepam) in rats dosed with 2 LD50 of the dimethoate, was low. However, there was a discrepancy between fenthion-poisoned patients and animals in that pralidoxime was ineffective as an antidote in patients, while the four oximes showed considerable efficacy in rats. Kušić et al. (1991) have tested the oxime HI-6 in OP pesticide poisoning in 60 patients. HI-6 was administered four times a day as a single intramuscular injection of 500 mg with atropine and diazepam treatment. Oxime therapy was started on admission and continued for 2 7 days. Most patients were treated with HI-6 and nine patients severely poisoned with quinalphos were treated with PAM-2 chloride (1000 mg four times per day). HI-6 rapidly reactivated human erythrocyte AChE inhibited by diethoxy OPs (phorate, pyridaphenthion, quinalphos) as well as that inhibited by dichlorvos (a dimethoxy OP) with reactivation half-lives ranging from 0.5 h to 3.5 h. AChE inhibited with other dimethoxy OPs (dimethoate, phosphamidon) was reported to be resistant to HI-6 treatment, whereas reactivation with malathion was slow (reactivation halftime 10 h). Both HI-6 and PAM-2 successfully reactivated AChE in quinalphos-poisoned patients, with HI-6 acting as a faster AChE reactivator than PAM-2. No adverse effects were seen in patients treated with the oximes. Nine patients intoxicated with OP pesticides were treated with PAM-2 methylsulphate (Contrathion) using a dose of 4.42 mg/kg as a bolus injection followed by continuous infusion 2.14 mg/kg/h. In patients with ethylparathion and methylparathion poisonings, enzyme reactivation could be obtained in some patients at oxime concentrations as low as 2.88 mg/l. In others, however, oxime concentration as high as 14.6 mg/l were ineffective. The therapeutic effect of the oxime seemed to depend on the plasma concentrations of ethyl parathion and methyl parathion. Due to AChE reinhibition, reactivation was absent as long as these concentrations remained above 30 g/l (Aragao et al., 1996). Willems et al. (1993) reported that ethyl parathion and methyl parathion could be effectively treated with PAM-2 methylsulphate (plasma concentrations 4 mg/l) and atropine when pesticide concentrations in plasma were relatively low. In severe poisoning with pesticide levels in plasma above 30 g/l, high PAM-2 concentrations in plasma (14.6 mg/l) did not provide any improvement. In addition, PAM-2 at concentrations of 6.3 mg/l was not effective in AChE reactivation in dimethoate poisoning where AChE was inhibited with its active metabolite omethoate. It was reported that in cases of life-threatening parathion poisoning obidoxime (Toxogonin) (250 mg administered intravenously as a bolus followed by infusion of 750 mg per day) was effective (Thiermann et al., 1997, 1999). However, AChE reactivation did not occur until the concentration of paraoxon in plasma

7 M. Jokanović / Toxicology Letters 190 (2009) was low. Oxydemeton methyl poisoning responded to obidoxime therapy only when the oxime was instituted shortly after poisoning. In cases when obidoxime treatment started too late there was no reactivation of erythrocyte AChE and one out of six treated patients died. In a clinical study of 63 patients poisoned with OP pesticides, patients were divided into three groups: one was treated with atropine only, while the other two received atropine and either PAM-2 or obidoxime. Initial and maintenance intravenous doses for PAM-2 were 30 mg/kg and 8 mg/kg/h, respectively, and 8 mg/kg and 2 mg/kg/h, respectively, for obidoxime. The major clinical findings or AChE activities at the time of admission did not show statistically significant differences among the groups. Although the severity of intoxications (based on respiratory complications and duration of hospitalization) was higher in the atropine plus oxime groups, 12% and 50% of patients in the atropine and atropine plus obidoxime groups died, respectively. No mortality was found in the PAM-2 plus atropine group. Incidence of recurrent twitching and convulsions, repeated respiratory arrest, required mechanical respiration, required intensive care unit therapy and duration of hospitalization were lower in the atropine plus obidoxime group than in the atropine plus PAM-2 group. Three of the patients who received the obidoxime combination therapy developed hepatitis and two of them died due to hepatic failure, which may indicate overdosage of obidoxime (Balali-Mood and Shariat, 1998). AChE inhibited by several OP pesticides, including dimethoate, demethon, triamiphos, ethoprophos, profenofos, fenamiphos and pyridafenthion, resists any attempt of reactivation with any oxime, probably due to variations in phosphoryl moiety and distribution of electronic charge (Jokanović and Maksimović, 1995; Bismuth et al., 1992). In a randomized controlled trial, Pawar et al. (2006) studied the effect of very-high dose pralidoxime iodide (2 g loading dose, then 1 g either every hour or every 4 h for 48 h, then 1 g every 4 h until recovery) in 200 patients with moderate OP poisoning (excluding severely ill patients). Among OP pesticides involved there were chlorpyrifos (diethyl OP) and dimethoate (dimethyl OP). The highdose regimen was associated with reduced case fatality, fewer cases of pneumonia, and reduced time on mechanical ventilation. This study suggests that large doses of pralidoxime could have benefit if patients are treated early and have good supportive care. 6. Clinical aspects of medical treatment of poisoning with carbamate pesticides Cases of accidental overexposure to or suicide attempts with various CB pesticides have followed similar clinical courses characteristic of cholinergic poisoning like that in poisoning with OP. Differences in severity, duration, and outcome have corresponded to differences in effective doses and in promptness and appropriateness of treatment. Spontaneous recovery without medical treatment has occurred generally within 4 h of exposures producing symptoms of headache, dizziness, weakness, excessive salivation, nausea, or vomiting. More severe symptoms have generally prompted medical treatment. Following treatment with sufficient atropine, individuals have recovered from poisoning that produced such symptoms as visual disturbances, profuse sweating, abdominal pain, incoordination, fasciculations, breathing difficulties, or changes in pulse rate. Recovery has been complete in some cases within 2 h and in all cases within 1 day. CB poorly penetrate the blood brain barrier and effects on central nervous system seen in OP poisoning are absent or minimal. Deaths have resulted in severe cases where treatment was delayed and/or insufficient atropine was administered. It is important to note, however, that treatment with atropine combined with general supportive treatment, such as artificial respiration and administration of fluids, has resulted in recovery even in cases where symptoms progressed to pulmonary edema or coma (Baron, 1991; Rotenberg et al., 1995). Carbamylation of AChE is apparently a short-lived phenomenon, as CB are reversible AChE inhibitors that spontaneously reactivate with a half-life in the order of an hour or less. Although the immediate clinical picture of CB poisoning is similar to that of OP, reversible inhibition with spontaneous hydrolysis of the carbamylated AChE moiety results in less severe and less prolonged toxicity. Dimethyl compounds are a special case: they produce a carbamylated AChE, which may be reactivated with oximes. However, oximes are harmful when employed in animals poisoned with monomethyl carbamate, and a man who ingested carbaryl died 6 h after a PAM-2 treatment (Karchmar, 2007). The use of oximes in the case of CB poisoning is controversial and considered contraindicated by some authors. Lieske et al. (1992) have found that pyridinium oximes (obidoxime, trimedoxime, pralidoxime, and HI-6) enhance inhibition of both eel AChE and human serum ChE induced by carbaryl. The authors have proposed that oximes act as allosteric effectors of cholinesterases in carbaryl poisoning resulting in enhanced inhibition rates and potentiation of carbaryl toxicity. In spite of this, some authors have reported beneficial effects of pralidoxime in aldicarb poisoning in humans (Garber, 1987; Burgess et al., 1994). In experimental studies, oximes have been shown to be beneficial, alone and/or with atropine, in countering the toxicity of the carbamates isolan, thimetilan, pyramat, dimetilan, aldicarb, neostigmine, physostigmine, pyridostigmine and others (Bošković et al., 1976; Sterri et al., 1979; Dawson, 1995). It appears that the only CB whose toxicity was increased by an oxime was carbaryl (Dawson, 1995). 7. Conclusions Medical management of acute OP pesticide poisoning in humans includes general (decontamination and supportive measures) and specific treatment with atropine, oximes (pralidoxime, trimedoxime, obidoxime, and HI-6) and diazepam. About a half of the century has passed since the introduction of the antidotes to medical treatment of patients poisoned with OP compounds and there is still no agreement on how these substances should best be given. While the use of atropine and diazepam in human OP poisoning has been widely accepted throughout the world, there are apparently conflicting results related to the importance of pralidoxime treatment. When given with atropine and diazepam, pyridinium oximes were successful in the treatment of most cases of OP poisoning in European toxicology clinics where the recommendations proposed by World Health Organization were followed. On the other hand, reports from developing countries indicated that pralidoxime treatment was not sufficiently beneficial in their patients, but their studies were not designed according to the recommendations. These problems of effectiveness of oxime treatment may be solved in randomized clinical trial(s) comparing the WHO-recommended regimen with a placebo to assess the value of pralidoxime, and other oximes (obidoxime, trimedoxime, and HI-6) as well, in acute OP poisoning. The trial(s) will have to be carefully designed and take into account many factors that may have impact on their results. Conflict of interest None declared. Acknowledgements The research of M.J. was supported by grants from the Serbian Ministry of Science (Projects and ).

8 114 M. Jokanović / Toxicology Letters 190 (2009) References Abdel-Rahman, A., Shetty, A.K., Abou-Donia, M., Acute exposure to sarin increases blood brain barrier permeability and induces neuropathological changes in the rat brain: dose response relationships. Neuroscience 113, Antonijević, B., Stojiljković, M.P., Unequal efficacy of pyridinium oximes in acute organophosphorus poisoning. Clin. Med. Res. 5, Aragao, I., Soares, M.E., Bastos, M.L., Lopes, M., Continuous infusion of obidoxime and the importance of plasma levels monitoring in organophosphate poisoning. Intensive Care Med. 22 (Suppl. 3), S388, 512. Ashani, Y., Bhattacharjee, A.K., Leader, H., Saxena, A., Doctor, B.P., Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators. Biochem. Pharmacol. 66, Balali-Mood, M., Shariat, M., Treatment of organophosphate poisoning. Experience of nerve agents and acute pesticide poisoning on the effects of oximes. J. Physiol. Paris 92, Baron, R.L., Carbamate insecticides. In: Hayes, W.J., Laws Jr., E.R. (Eds.), Handbook of Pesticide Toxicology, vol. 3. Academic Press, Inc., San Diego, p Bismuth, C., Inns, R.H., Marrs, T.C., Efficacy, toxicity and clinical use of oximes in anticholinesterase poisoning. In: Ballantyne, B., Marrs, T.C. (Eds.), Clinical & Experimental Toxicology of Organophosphates & Carbamates. Butterworth- Heinemann, Oxford, pp Bokonjić, D., Jovanović, D., Jokanović, M., Maksimović, M., Protective effects of oximes HI-6 and PAM-2 applied by osmotic minipumps in quinalphos-poisoned rats. Arch. Int. Pharmacodyn. 288, Bošković, B., Vojvodić, V., Maksimović, M., Granov, A., Besarović-Lazarev, S., Binenfeld, Z., Effect of mono and bis-quaternary pyridinium oximes on the serum cholinesterase inhibitory activity of dioxacarb, carbaryl and carbofuran. Arh. Hig. Rada 27, Burgess, J.L., Bernstein, J.N., Hurlbut, K., Aldicarb poisoning. A case report with prolonged cholinesterase inhibition and improvement after pralidoxime therapy. Arch. Int. Med. 154, Carpentier, P., Delamanch, I., Le Bert, M., Blanchet, G., Bouchard, C., Seizurerelated opening of the blood brain barrier induced by soman: possible correlation with the acute neuropathology observed in poisoned rats. Neurotoxicology 11, Cherian, M.A., Roshini, C., Peter, J.V., Cherian, A.M., Oximes in organophosphorus poisoning. Indian J. Crit. Care Med. 9, Darvesh, S., Hopkins, D.A., Geula, C., Neurobiology of butyrylcholinesterase. Nat. Rev. Neurosci. 4, Dawson, R.M., Oxime effects on the rate constants of carbamylation and decarbamylation of acetylcholinesterase for pyridine, physostigmine and insecticidal carbamates. Neurochem. Int. 26, De Silva, H.J., Wijewickrema, R., Senanayake, N., Does pralidoxime affect outcome of management in acute organophosphate poisoning? Lancet 339, Eddleston, M., Juszczak, E., Buckley, N.A., 2005a. Randomised controlled trial of routine single or multiple dose superactivated charcoal for self-poisoning in a region with high mortality. Clin. Toxicol. 43, Eddleston, M., Eyer, P., Worek, F., Mohamed, F., Senarathna, L., von Meyer, L., Juszczak, E., Hittarage, A., Azhar, S., Dissanayake, W., Sheriff, M.H.R., Szinicz, L., Dawson, A.H., Buckley, N.A., 2005b. Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study. Lancet 366, Eddleston, M., Singh, S., Buckley, N., Organophosphorus poisoning (acute). Clin. Evid. 15, Eddleston, M., Juszczak, E., Buckley, N.A., Senarathna, L., Mohamed, F., Dissanayake, W., Hittarage, A., Azher, S., Jeganathan, K., Jayamanne, S., Sheriff, M.H.R., Warrell, D.A., Multiple-dose activated charcoal in acute self-poisoning: a randomised controlled trial. Lancet 371, Eyer, P., The role of oximes in the management of organophosphorus pesticide poisoning. Toxicol. Rev. 22, Garber, M., Carbamate poisoning: the other insecticide. Pediatrics 79, Grange-Messent, V., Bouchaud, C., Jamme, M., Lallement, G., Foguin, A., Carpentier, P., Seizure-related opening of the blood brain barrier produced by the anticholinesterase compound, soman: new ultrastructural observations. Cell Mol. Biol. 45, IPCS, Organophosphorus pesticides. In: International Programme on Chemical Safety Poisons Information Monograph G001. World Health Organization, Geneva, Updated Available online at: documents/pims/chemical/pimg001.htm. IPCS, Antidotes for poisoning by organophosphorus pesticides. In: Monograph on Atropine. International Programme on Chemical Safety Evaluation, World Health Organization, Geneva. Jagger, B.V., Stagg, G.N., Toxicity of diacetylmonoxime and of pyridine-2- aldoxime methiodide in man mg/kg 2-PAM: no effects on EEG in a healthy man. Bull. Johns Hopkins Hosp. 102, Johnson, M.K., Vale, J.A., Clinical management of acute organophosphate poisoning: an overview. In: Ballantyne, B., Marrs, T.C. (Eds.), Clinical & Experimental Toxicology of Organophosphates & Carbamates. Butterworth- Heinemann, Oxford, pp Johnson, M.K., Jacobsen, D., Meredith, T.J., Eyer, P., Heath, A.J., Ligtenstein, D.A., Marrs, T.C., Szinicz, L., Vale, A.J., Haines, J.A., Evaluation of antidotes for poisoning by organophosphorus pesticides. Emerg. Med. 12, Johnson, S., Peter, J.V., Thomas, K., Jeyaseelan, L., Cherian, A.M., Evaluation of two treatment regimens of pralidoxime (1 gm single bolus dose vs 12 gm infusion) in the management of organophosphorus poisoning. J. Assoc. Physicians India 44, Jokanović, M., Maksimović, M., A comparison of trimedoxime, obidoxime, pralidoxime and HI-6 in treatment of oral organophosphorus insecticide poisoning. Arch. Toxicol. 70, Jokanović, M., Maksimović, M., Abnormal cholinesterase activity: understanding and interpretation. Eur. J. Clin. Chem. Clin. Biochem. 35, Jokanović, M., Biotransformation of organophosphorus compounds. Toxicology 166, Jokanović, M., Stojiljković, M.P., Current understanding of the application of pyridinium oximes as cholinesterase reactivators in treatment of organophosphate poisoning. Eur. J. Pharmacol. 553, Jokanović, M., Prostran, M., Pyridinium oximes as cholinesterase reactivators. Structure activity relationship and efficacy in the treatment of poisoning with organophosphorus compounds. Curr. Med. Chem. 16, Jovanović, D., Maksimović, M., Joksović, D., Kovačević, V., Oral forms of the oxime HI-6: a study of pharmacokinetics and tolerance after administration to healthy volunteers. Vet. Hum. Toxicol. 32, Karchmar, A.G., Anticholinesterases and war gases. In: Karchmar, A.G. (Ed.), Exploring the Vertebrate Central Cholinergic Nervous System. Springer, pp Kassa, J., The role of oximes in the antidotal treatment of chemical casualties exposed to nerve agents. In: Monov, A., Dishovsky, C. (Eds.), Medical Aspects of Chemical and Biological Terrorism: Chemical Terrorism and Traumatism. Publishing House of the Union of Scientists in Bulgaria, Sofia, Bulgaria, pp (Available at Kušić, R., Bošković, B., Vojvodić, V., Jovanović, D., HI-6 in man: blood levels, urinary excretion, and tolerance after intramuscular administration of the oxime to healthy volunteers. Fundam. Appl. Toxicol. 5, S89 S97. Kušić, R., Jovanović, D., Ran delović, S., Joksović, D., Todorović, V., Bošković, B., Jokanović, M., Vojvodić, V., HI-6 in man: efficacy of the oxime in poisoning by organophosphorus insecticides. Hum. Exp. Toxicol. 10, Lieske, C.N., Clark, J.H., Maxwell, D.M., Zoeffel, L.D., Sultan, W.E., Studies of the amplification of carbaryl toxicity by various oximes. Toxicol. Lett. 62, Luo, C., Saxena, A., Smith, M., Garcia, G., Radić, Z., Taylor, P., Doctor, B.P., Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium. Biochemistry 38, Marrs, T.C., Diazepam. Antidotes for Poisoning by Organophosphorus Pesticides. International Programme on Chemical Safety, World Health Organization, Geneva. Marrs, T.C., Vale, J.A., Management of organophosphorus pesticide poisoning. In: Gupta, R.C. (Ed.), Toxicology of Organophosphorus and Carbamate Compounds. Elsevier Academic Press, Amsterdam, pp Mason, H.J., Sams, C., Stevenson, A.J., Rawborne, R., Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides. Hum. Exp. Toxicol. 19, McDonough Jr., J.H., McLeod Jr., C.G., Nipwoda, M.D., Direct micro-injection of soman or VX into the amygdala produces repetitive limbic convulsions and neuropathology. Brain Res. 435, Milatović, D., Jokanović, M., Pyridinium oximes as cholinesterase reactivators in the treatment of poisoning with organophosphorus compounds. In: Gupta, R.C. (Ed.), Handbook of Toxicology of Chemical Warfare Agents. Elsevier, pp Pazdernik, T.L., Nelson, S.R., Cross, R., Churchill, L., Giesler, M., Samson, F.E., Effects of antidotes on soman-induced brain damage. Arch. Toxicol. 9, Pawar, K.S., Bhoite, R.R., Pillay, C.P., Chavan, S.C., Malshikare, D.S., Garad, S.G., Continuous pralidoxime infusion versus repeated bolus injection to treat organophosphorus pesticide poisoning: a randomised controlled trial. Lancet 368, Peng, A., Meng, F.-Q., Sun, L.-F., Ji, Z.-S., Li, Y.-H., Therapeutic efficacy of charcoal hemoperfusion in patients with acute severe dichlorvos poisoning. Acta Pharmacol. Sin. 25, Reiner, E., Pleština, R., Regeneration of cholinesterase activities in humans and rats after inhibition by O,O-dimethyl-O-dimethyl-2,2-dichlorvinyl phosphate. Toxicol. Appl. Pharmacol. 49, Rotenberg, M., Shefi, M., Dany, S., Dore, I., Tirosh, M., Almog, S., Differentiation between organophosphate and carbamate poisoning. Clin. Chim. Acta 234, Sakurada, K., Matsubara, K., Shimizu, K., Shiono, H., Seto, Y., Tsuge, K., Yoshino, M., Sakai, I., Mukoyama, H., Takatori, T., Pralidoxime iodide (2-PAM) penetrates accross the blood brain barrier. Neurochem. Res. 28, Sellström, A., Anticonvulsants in anticholinesterase poisoning. In: Ballantyne, B., Marrs, T.C. (Eds.), Clinical & Experimental Toxicology of Organophosphates & Carbamates. Butterworth-Heinemann, Oxford, pp Shiloff, J.D., Clement, J.G., Comparison of serum concentration of the acetylcholinesterase oxime reactivatiors HI-6, obidoxime and PAM to efficacy against sarin (isopropyl methylphosphonofluoridate) poisoning in rats. Toxicol. Appl. Pharmacol. 89, Shrot, S., Markel, G., Dushnitsky, T., Krivoy, A., The possible use of oximes as antidotal therapy in organophosphate-induced brain damage. Neurotoxicology 30,