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Organophosphates (OPs) are commonly used pesticides worldwide. Humans are exposed to OPs via different routes viz., the respiratory tract, gastrointestinal tract, and dermal integuments. OPs induce neuropathy by either phosphorylating acetyl cholinesterase or neuropathy target esterase, or by binding specifically to nicotinic or muscarinic receptors of nervous system. Other than neurobehavioral effects in humans, OPs cause cholinergic crisis, intermediate syndrome, OP-induced delayed neuropathy, and Chronic organophosphate-induced neuropsychiatric disorders in time and dosage dependent manner. Biomonitoring of OP markers from body fluids minimizes or measures the severity of the impact, allowing for timely control of the exposure. The standard treatments for OPs poisoning which avoid secondary organ damage are atropine administration, acetylcholine esterase restoration therapy with oximes, and general intensive care. This review summarizes the toxic manifestation data available on humans and discusses potential therapeutic modalities, with the aim to highlight the importance of increasing awareness about its potential risk and reevaluation of exposure level.

Organophosphates (OPs) are a class of chemicals commonly used in agriculture as pesticides, that can often lead to severe toxicity in humans. Paraoxonase-1 (PON1) belongs to a family of A-esterases and hydrolyses several OPs while also serving other biological roles. Two main genetic polymorphisms have been shown to affect enzymatic ability; an A > G transition in the 192nd position (192 Q/R, rs662), and an A > T at codon 55 (55 M/L, rs854560). In this review, we searched PubMed for relevant articles published from its inception till June 2018 and included publications from 1996 to 2018. We aimed to address the distribution of the polymorphisms in various populations, the way they affect enzymatic activity and the possible use of PON1 as a biomarker. The polymorphisms present great heterogeneity between populations, with the data being clearer over 192 Q/R, and this heterogeneity is related to the phylogenetic origins of each population. Concerning enzymatic activity, the different genotypes react better or worse to different OP substrates, with studies presenting a variety of findings. Detecting the “paraoxonase status” of an individual -referring to PON1 function- seems to be important in predicting OP toxicity, as studies have shown that some specific-genotype individuals present symptoms of toxicity in higher rates than others. We are strongly convinced that in order for the scientific community to reach a consensus over which polymorphisms confer susceptibility to toxicity and whether PON1 can eventually be used as a biomarker, more studies need to be carried out, since the data thus far does not seem to reach a universal conclusion.

In this article the neurotoxic disorders appearing in patients exposed to organophosphorus pesticides and known mechanisms involved are reviewed. Organophosphorus compounds cause four main neurotoxic effects in humans: the cholinergic syndrome, the intermediate syndrome, organophosphate-induced delayed polyneuropathy and chronic organophosphate-induced neuropsychiatric disorder. Compared to the cholinergic syndrome, that causes millions of cases of poisoning with fatality of more than 15% each year, other disorders involve much smaller number of patients. Possible link of exposure to organophosphorus pesticides with neurodegenerative diseases, dementia, attention deficit hyperactivity disorder and Parkinson's disease in man is also approached. This article is focused on neurotoxic disorders appearing after acute and chronic exposure to organophosphates with emphasis on molecular mechanisms, clinical presentation, pathogenesis, and possibilities for prevention/medical treatment.

The term organophosphate (OP) refers to a diverse group of chemicals that are found in hundreds of products worldwide. As pesticides, their most common use, OPs are clearly beneficial for agricultural productivity and the control of deadly vector-borne illnesses. However, as a consequence of their widespread use, OPs are now among the most common synthetic chemicals detected in the environment as well as in animal and human tissues. This is an increasing environmental concern because many OPs are highly toxic and both accidental and intentional exposures to OPs resulting in deleterious health effects have been documented for decades. Some of these deleterious health effects include a variety of long-term neurological and psychiatric disturbances including impairments in attention, memory, and other domains of cognition. Moreover, some chronic illnesses that manifest these symptoms such as Gulf War Illness and Aerotoxic Syndrome have (at least in part) been attributed to OP exposure. In addition to acute acetylcholinesterase inhibition, OPs may affect a number of additional targets that lead to oxidative stress, axonal transport deficits, neuroinflammation, and autoimmunity. Some of these targets could be exploited for therapeutic purposes. The purpose of this review is thus to: 1) describe the important uses of organophosphate (OP)-based compounds worldwide, 2) provide an overview of the various risks and toxicology associated with OP exposure, particularly long-term neurologic and psychiatric symptoms, 3) discuss mechanisms of OP toxicity beyond cholinesterase inhibition, 4) review potential therapeutic strategies to reverse the acute toxicity and long term deleterious effects of OPs.

Standard therapeutic options in organophosphate (OP) poisoning are limited to the administration of atropine and oximes, a regimen often lacking in efficacy and applicability. Treatment alternatives are needed, preferably covering a broad spectrum of OP intoxications. Although recent research yielded several promising compounds, e.g. bioscavengers, modulators of the muscarinic acetylcholine (ACh) receptor or bispyridinium non-oximes, these substances still need further evaluation, especially regarding effects on the potentially lethal respiratory symptoms of OP poisoning. Aim of this study was the development of an applicable and easy method to test the therapeutic efficiency of such substances. For this purpose, airway responsiveness in viable precision cut lung slices (PCLS) from rats was analysed. We showed that ACh-induced airway contractions were spontaneously reversible in non-poisoned PCLS, whereas in OP poisoned PCLS, contractions were irreversible. This effect could be antagonized by addition of the standard therapeutic atropine, thereby presenting a clear indication for treatment efficiency. Now, candidate therapeutic compounds can be evaluated, based on their ability to counteract the irreversible airway contraction in OP poisoned PCLS.

The misuse of organophosphate compounds still represents a current threat worldwide. Treatment of poisoning with organophosphates (OPs) remains unsatisfactorily resolved despite the extensive investment in research in academia. There are no universal, effective and centrally-active acetylcholinesterase (AChE) reactivators to countermeasure OP intoxication. One major obstacle is to overcome the blood-brain barrier (BBB). The central compartment is readily accessible by the OPs which are lipophilic bullets that can easily cross the BBB, whereas first-line therapeutics, namely oxime-based AChE reactivators and atropine, do not cross or do so rather slowly. The limitation of oxime-based AChE reactivators can be ascribed to their chemical nature, bearing a positive charge which is essential either for their AChE affinity or their reactivating potency. The aim of this article is to review the methods for targeting the brain by oxime reactivators that have been developed so far. Approaches using prodrugs, lipophilicity enhancement, or sugar-based oximes have been rather unsuccessful. However, other strategies have been more promising, such as the use of nanoparticles or co-administration of the reactivator with efflux transporter inhibitors. Encouraging results have also been associated with intranasal delivery, but research in this field is still at the beginning. Further research of auspicious approaches is inevitable.

Two polyhydroxyfullerenes, which decrease organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition in vitro, were administered by the intraperitoneal (ip) route or applied topically at doses of 0.9–24 mg/kg to protect adult male mice from enzyme-inhibiting and behavioral effects indicative of OP toxicity resulting from exposure to 1.7 - 2 mg/kg diphosphorofluoridate (DFP) ip or 2.3 – 2.7 mg paraoxon topical. Dosing paradigms included OP-fullerene simultaneous administration by the ip route, and 20 min post-OP polyhydroxyfullerene treatment topically. Benefits of OP sequestration by the polyhydroxyfullerene were noted and were dependent on the OP compound as well as timing and route of the polyhydroxyfullerene treatment.

The ability to overcome cellular barriers in the body is crucial for efficient delivery of drugs to the target where intervention is needed. For drugs acting in the brain it is essential to overcome the blood-brain barrier (BBB). Such drugs include antidotes for the treatment of organophosphate poisoning, a current warfare and terroristic threat. Being lipophilic compounds, organophosphates readily penetrate the brain and block the enzyme acetylcholinesterase (AChE). They cause severe symptoms which may have fatal consequences. A major drawback of currently available oxime reactivators is their inability to reactivate AChE in the central nervous system (CNS) as they are unable to cross the blood-brain barrier. An important obstacle preventing many drugs from reaching their therapeutic target in the brain is the efflux transporter P-glycoprotein (P-gp), whose function is to prevent the penetration of potentially harmful substances. The aim of this study was to evaluate the effect of P-gp on the permeation of oximes into the brain. The study of this interaction was carried out on the CACO-2 cell line, stably expressing P-gp. As it turned out, P-gp has no essential influence on the central availability of clinically used oxime reactivators within this study.

Background Carbamates physostigmine and pyridostigmine have been used as a pretreatment against poisoning with nerve agents in order to reversibly inhibit and thus protect from irreversible inhibition a portion of acetylcholinesterase (AChE) in brain and respiratory muscles that is crucial for survival. Memantine, an adamantine derivative, has emerged as a promising alternative to carbamates, since it prevented the fasciculations and skeletal muscle necrosis induced by carbamates and organophosphates, including nerve agents. Aim This experimental study was undertaken in order to investigate and compare the protective and behavioural effects of memantine and standard carbamates physostigmine and pyridostigmine in rats poisoned with soman and treated with atropine, oxime HI-6 and diazepam. Another goal was to elucidate the mechanisms of the antidotal effect of memantine and its potential synergism with standard antidotes against nerve agents. Materials and methods Male Wistar rats were used throughout the experiments. In dose-finding experiments memantine was administered at dose interval 0–72 mg/kg sc 60 min before sc injection of soman. In time-finding experiments memantine was injected 18 mg/kg sc 0–1440 min before soman. Standard treatment antidotes - atropine 10 mg/kg, HI-6 50 mg/kg and diazepam 2.5 mg/kg – were administered im within 15 s post-exposure. Soman 0.75 LD50 was used to study its inhibitions of neuromuscular transmission on the phrenic nerve-diaphragm preparation in situ and of tissue AChE activity. Behavioural effects of the prophylactic antidotes were investigated by means of the rotarod test. Based on these data therapeutic index and therapeutic width was calculated for all three prophylactic agents. Results Memantine pretreatment (18 mg/kg sc) produced in rats poisoned with soman significantly better protective ratios (PRs) than the two carbamates – 1.25 when administered alone and 2.3 when combined with atropine pretreatment and 6.33 and 7.23 with atropine/HI-6 and atropine/HI-6/diazepam post-exposure therapy, respectively. The highest PR of 10.11 obtained in Atr/HI-6-treated rats was achieved after pretreatment with memantine 36 mg/kg. This additional protection lasted for 8 h. All three prophylactic regimens antagonised the soman-induced neuromuscular blockade, but the effect of memantine was fastest. Pretreatment with memantine assured higher AChE activity in brain and diaphragm than in unpretreated rats (46% vs 28% and 68% vs. 38%, respectively). All three prophylactic regimens affected the rotarod performance in rats, but the effect of memantine was relatively strongest. Memantine and pyridostigmine had lowest and highest therapeutic index and therapeutic width, respectively. Conclusions Although memantine assures better and longer-lasting protection against soman poisoning in rats than the two carbamates, its small therapeutic index and narrow therapeutic width seriously limit its potential as a pretreatment agent. Despite its behavioural effects, memantine seems to be beneficial antidote when administered after soman, along with atropine/HI-6/diazepam therapy. Mechanism of the antidotal effect of memantine against soman poisoning appears to be a combination of AChE-protecting and NMDA receptor-blocking action.

Based on human toxicity studies, by appropriate regulatory decisions, the number of organophosphates (OP) on Serbian market has reduced significantly over the last two decades, followed by a gradual decrease in the number of poisonings by organophoshates, treated at the National Poison Control Centre (NPCC). Methodology The aim of this retrospective study is to present data regarding the clinical management of poisoning with OP pesticides at the NPCC, that we collected during the 17 years period (1998–2014). Results In the period 1998–2014, about 17.250 patients were hospitalized at the NPCC, there were around 14.000 patients treated for poisoning by various toxic agents, and among them 410 cases (3%) due to poisoning with OP pesticides. In this period, 92% of OPI poisonings treated in the NPCC were suicidal by intention, while only 8% were due to accidental ingestion or inhalation. The most common clinical signs of poisoning in patients exposed to anticholinesterase pesticides, observed at Clinic of Toxicology of the NPCC were miosis (63.4%), bronchorrhoea (51.9%), vomiting and diarrhea (44.8%), hypotension (19.5%). Acute respiratory insufficiency was registered in 81 (19.7%) and acute cardiocirculatory failure in 16 (3.9%) patients. There were about 25% of most severely poisoned patients. Besides general supportive measures (decontamination, respiratory support), specific pharmacological treatment (atropine, oxime, diazepam) was applied. The highest total administered dose of atropine at NPCC was 6400 mg. However, the most patients received total doses of atropine up to 500 mg (32%). Conclusion Acute poisoning with OP pesticides is not frequent in Serbia, however, it represents important clinical feature due to severity, possible complications and their impact on duration and costs of hospitalization. Initial treatment involves prevention of further absorption and provision of supportive care, followed by administration of specific antidotes. According to its role, the National Poison Control Centre in Belgrade, in addition to treatment of acute poisonings, continuously performs toxicovigilance, i.e. the identification, investigation and evaluation of various toxic risks in the community in order to undertake adequate and timely procedures. Permanent efforts are being made in order to reduce availability and to improve control measures for pesticides marketing.

The effect of three newly developed bispyridinium non-oxime compounds (MB408, MB442, and MB444) on the therapeutic efficacy of a standard antidotal treatment (atropine in combination with the oxime HI-6 or obidoxime) of acute poisoning by two nerve agents (sarin and cyclosarin) in mice was studied. The therapeutic efficacy of atropine in combination with an oxime with or without one of the bispyridinium non-oximes was evaluated by determination of the 24 h LD50 values of the nerve agents studied and by measurement of the survival time after supralethal poisoning. Addition of all tested non-oximes increased the therapeutic efficacy of atropine in combination with an oxime against sarin poisoning; however, the differences were not significant. The non-oximes also positively influenced the number of surviving mice 6 h after supralethal poisoning with sarin. In the case of cyclosarin, they were also slightly beneficial in the treatment of acute poisoning. The higher dose of MB444 was able to significantly increase the therapeutic efficacy of standard antidotal treatment of poisoning with cyclosarin. The benefit of each bispyridinium non-oxime compound itself was obviously dose-dependent. In summary, the addition of MB compounds to the standard antidotal treatment of acute nerve agent poisoning was beneficial for the antidotal treatment of sarin or cyclosarin poisoning, although their benefit at 24 h after poisoning was not significant, with the exception of the higher dose of MB444 against cyclosarin.

Organophosphorus (OP) pesticides are a diverse class of acetylcholinesterase (AChE) inhibitors that are responsible for tremendous morbidity and mortality worldwide, killing approximately 300,000 people annually. Enzymatic hydrolysis of OPs is a potential therapy for acute poisoning. OpdA, an OP hydrolase isolated from Agrobacterium radiobacter, has been shown to decrease lethality in rodent models of OP poisoning. This study investigated the effects of OpdA on AChE activity, plasma concentrations of OP, and signs of toxicity after administration of dichlorvos to nonhuman primates. A dose of 75 mg/kg dichlorvos given orally caused apnea within 10 min with a progressive decrease in heart rate. Blood AChE activity decreased to zero within 10 min. Respirations and AChE activity did not recover. The mean dichlorvos concentration rose to a peak of 0.66 μg/ml. Treated monkeys received 1.2mg/kg OpdA iv immediately after poisoning with dichlorvos. In Opda-treated animals, heart and respiratory rates were unchanged from baseline over a 240-minute observation period. AChE activity slowly declined, but remained above 25% of baseline for the entire duration. Dichlorvos concentrations reached a mean peak of 0.19 μg/ml at 40 min after poisoning and decreased to a mean of 0.05 μg/ml at 240 min. These results show that OpdA hydrolyzes dichlorvos in an African green monkey model of lethal poisoning, delays AChE inhibition, and prevents lethality.

a b s t r a c t Paraoxonase (PON1) is an A-esterase capable of hydrolyzing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and in plasma. Human PON1 displays two polymorphisms in the coding region (Q192R and L55M) and several polymorphisms in the promoter and the 3 � -UTR regions. The Q192R polymorphism imparts differential catalytic activity toward some OP substrates, while the polymorphism at position −108 (C/T) is the major contributor of differences in the levels of PON1 expression. Both contribute to determining an individual's PON1 "status". Animal studies have shown that PON1 is an important determinant of OP toxicity. Administration of exogenous PON1 to rats or mice protects them from the toxicity of specific OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not of paraoxon. In vitro catalytic efficiencies of purified PON192 alloforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Evidence is slowly emerging that a low PON1 status may increase susceptibility to OP toxicity in humans. Low PON1 activity may also contribute to the developmental toxicity and neurotoxicity of OPs, as shown by animal and human studies. © 2012 Published by Elsevier Ireland Ltd.

Pesticides almost always occur in mixtures with other ones. The toxicological effects of low-dose pesticide mixtures on the human health are largely unknown, although there are growing concerns about their safety. The combined toxicological effects of two or more components of a pesticide mixture can take one of three forms: independent, dose addition or interaction. Not all mixtures of pesticides with similar chemical structures produce additive effects; thus, if they act on multiple sites their mixtures may produce different toxic effects. The additive approach also fails when evaluating mixtures that involve a secondary chemical that changes the toxicokinetics of the pesticide as a result of its increased activation or decreased detoxification, which is followed by an enhanced or reduced toxicity, respectively. This review addresses a number of toxicological interactions of pesticide mixtures at a molecular level. Examples of such interactions include the postulated mechanisms for the potentiation of pyrethroid, carbaryl and triazine herbicides toxicity by organophosphates; how the toxicity of some organophosphates can be potentiated by other organophosphates or by previous exposure to organochlorines; the synergism between pyrethroid and carbamate compounds and the antagonism between triazine herbicides and prochloraz. Particular interactions are also addressed, such as those of pesticides acting as endocrine disruptors, the cumulative toxicity of organophosphates and organochlorines resulting in estrogenic effects and the promotion of organophosphate-induced delayed polyneuropathy.

The immune system can be the target of many chemicals, with potentially severe adverse effects on the host's health. In Western countries pesticides, together with new and modified patterns of exposure to chemicals, have been implicated in the increasing prevalence of diseases associated with alterations of the immune response, such as hypersensitivity reactions, certain autoimmune diseases and cancers. Xenobiotics may initiate, facilitate or exacerbate pathological immune processes, resulting in immunotoxicity by induction of mutations in genes coding for immunoregulatory factors, modifying immune tolerance and activation pathways. The purpose of this article is to update the evidence of pesticide immunotoxicity. Even if experimental data as well as sporadic human studies indicate that some pesticides can affect the immune system, overall, existing epidemiological studies are inadequate to raise conclusions on the immunotoxic risk associated to pesticide exposure. The available studies on the effects of pesticides on human immune system have several limitations including poor indication on exposure levels, multiple chemical exposures, heterogeneity of the approach, and difficulty in giving a prognostic significance to the slight changes often observed. Further studies are necessary, and they should be preferably carried out through comparison of pre and post-exposure findings in the same group of subjects with a matched control group. Attempt should be made to define the prognostic significance of slight changes often observed. Animal and in vitro studies are also important and necessary to scientifically support epidemiological evidences on pesticide-induced immunotoxicity.

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.

The increasing threat of nerve agent use for terrorist purposes against civilian and military population calls for effective therapeutic preparedness. At present, administration of atropine and an oxime are recommended, although effectiveness of this treatment is not proved in clinical trials. Here, monitoring of intoxications with organophosphorus (OP) pesticides may be of help, as their actions are closely related to those of nerve agents and intoxication and therapy follow the same principles. To this end, the clinical course of poisoning and the effectiveness of antidotal therapy were investigated in patients requiring artificial ventilation being treated with atropine and obidoxime. However, poisoning with OP pesticides shows extremely heterogeneous pictures of cholinergic crisis frequently associated with clinical complications. To achieve valuable information for the therapy of nerve agent poisoning, cases resembling situations in nerve agent poisoning had to be extracted: (a) intoxication with OPs forming reactivatable OP-AChE-complexes with short persistence of the OP in the body resembling inhalational sarin intoxication; (b) intoxication with OPs resulting rapidly in an aged OP-AChE-complex resembling inhalational soman intoxication; (c) intoxications with OPs forming a reactivatable AChE-OP complex with prolonged persistence of the OP in the body resembling percutaneous VX intoxication. From these cases it was concluded that sufficient reactivation of nerve agent inhibited non-aged AChE should be possible, if the poison load was not too high and the effective oximes were administered early and with an appropriate duration. When RBC-AChE activity was higher than some 30%, neuromuscular transmission was relatively normal. Relatively low atropine doses (several milligrams) should be sufficient to cope with muscarinic symptoms during oxime therapy.

Antidotes against chemical warfare agents are "orphan drugs" given that these poisonings are rare. Therefore, they are of limited interest to the pharmaceutical industry. For this reason, and recognizing the increasing threat of terrorist or asymmetrical use of chemical warfare agents, the responsibility for research into medical countermeasures against these weapons is of primary interest to armies. Accordingly, the research activities of the Bundeswehr Institute of Pharmacology and Toxicology are dedicated to improving diagnosis, prophylaxis and therapy of individuals who are exposed to a chemical agent. Here, antidote development and testing are a high priority in the research program, particularly with respect to organophosphorus (OP) nerve agents and sulphur mustard. The Institute has been coordinating research activities undertaken in house and in collaboration with external researchers. The research program aims to develop primarily in vitro models to minimize the sacrifice of animals, using strategies, which involve human material early in antidote testing. An animal model using isolated mouse diaphragm demonstrated the correlation between AChE activity and neuromuscular function. A similar relationship was found between erythrocyte AChE and neuromuscular function in patients with acute OP pesticide poisoning. In vitro rate constants of the various reactions that are involved in enzyme inhibition and reactivation using human material were used for prediction of what would happen in vivo. This prediction could be confirmed in a patient with acute OP pesticide poisoning. Finally, computer models are being established to estimate the therapeutic effect of an antidote in various human poisoning scenarios. This approach is necessary to compensate for the lack of human clinical pharmacodynamic studies that are usually required for drug regulatory approval, given the obvious ethical issues preventing human volunteer studies with these agents.

In scenarios of mass destruction it is likely that victims are intoxicated by organophosphates and, at the same time, physically injured. Organophosphate compounds produce excessive cholinergic overstimulation in the CNS via blocking acetylcholinesterase activity. The specifics of acute care and anaesthesia in physically traumatized and intoxicated patients are largely unknown. Recent studies in animals and human subjects demonstrated that acetylcholinesterase inhibitors reverse anaesthesia. Two distinct mechanisms are potentially involved. First, acetylcholine produces an excitatory drive onto neurons, thereby counterbalancing the inhibitory actions of anaesthetics. Anaesthesia is reversed because it critically depends on a distinctive depression of several central nervous functions. Second, cholinergic stimulation may affect the mechanisms by which anaesthetics mediate their depressant actions on central neurons. In this case acetylcholine reverses anaesthesia by decreasing the potency of anaesthetic agents. In order to identify potential mechanisms involved in cholinergic reversal of anaesthesia we have investigated interactions between acetylcholine and the volatile anaesthetic sevoflurane in isolated cortical brain slices. Our results provide evidence that cholinergic stimulation counterbalances the effects of general anaesthetics by increasing neuronal excitability, and, in addition, by decreasing anaesthetic potency. These findings imply that in patients suffering from organophosphorus intoxication dose requirements for general anaesthetics are considerably increased.

In scenarios of terrorist attacks with organophosphorus compounds it appears likely that medical aid is required by victims not only suffering from the intoxication but also from physical trauma. These subjects may have to undergo surgical interventions, raising the need for anaesthesia. This prompts the question of how anaesthetic agents work in intoxicated patients. Organophosphates block acetylcholinesterase activity, thereby inducing excessive cholinergic overstimulation in the central nervous system. As the neocortex and spinal cord are important substrates for general anaesthetics, we investigated to what extent cholinergic overstimulation affects the potency and efficacy of the commonly used volatile anaesthetic isoflurane in depressing action potential activity of cortical and spinal neurons. We first quantified the effects of isoflurane in the absence of acetylcholine by performing extracellular voltage recordings in cultured tissue slices. Isoflurane induced a concentration-dependent decrease of neuronal activity in neocortical (EC 50 = 0.43 ± 0.08 MAC) and spinal slices (EC 50 = 0.41 ± 0.03 MAC). At concentrations above 1.5 MAC, the anaesthetic almost completely depressed action potential firing in both preparations. Next, we studied the effects of acetylcholine (10 μM) in the absence of isoflurane. Acetylcholine approximately doubled spontaneous activity in neocortical and spinal slices. When applying isoflurane together with acetylcholine, different interactions between these agents were observed in neocortical and spinal networks. Acetylcholine significantly reduced both the potency and efficacy of the anaesthetic in neocortical (efficacy 83%; EC 50 = 1.16 ± 0.02 MAC) but not in spinal (efficacy 100%; EC 50 = 0.41 ± 0.04 MAC) slices. Our results indicate that cholinergic overstimulation increases the requirement for anaesthetic agents in patients suffering from organophosphorus poisoning via enhancing neuronal background activity of neocortical and spinal neurons and in addition via decreasing drug potency and efficacy in the cortex. Raising anaesthetic concentrations into a high-dose range may not be an appropriate alternative to compensate the increased excitability, since high concentrations of anaesthetics may worsen cardiac abnormalities and hemodynamic instability frequently observed in these patients.

The ability of two types of pharmacological pretreatment (pyridostigmine alone or pyridostigmine in combination with two anticholinergic drugs) to increase the resistance of rats and mice against tabun and to increase the therapeutic efficacy of common antidotal treatment of tabun-poisoned rats and mice was compared. A significant decrease in the LD50 values of tabun was observed when mice as well as rats were pretreated with the prophylactic antidotal mixture consisting of pyridostigmine, benactyzine and trihexyphenidyle, designated PANPAL. Pyridostigmine-pretreated rats were also more resistant against acute lethal effects of tabun but pyridostigmine-induced resistance of rats was not so high as PANPAL-induced resistance. In addition, the pharmacological pretreatment with pyridostigmine alone was not able to protect mice against tabun-induced acute toxicity. The pharmacological pretreatment with pyridostigmine alone was able to increase the efficacy of currently used antidotal treatment (obidoxime in combination with atropine and diazepam) of tabun-induced poisoning, but PANPAL-induced increase in the efficacy of the same antidotal treatment was significantly higher than an increase induced by pyridostigmine alone. PANPAL-induced increase in the efficacy of antidotal treatment of tabun poisoning was also observed in mice. These findings confirm that PANPAL pretreatment of tabun-poisoned rats and mice seems to be much more suitable than currently used pyridostigmine alone.

Potent cholinesterase inhibitors such as sarin, induce an array of harmful effects including hypersecretion, convulsions and ultimately death. Surviving subjects demonstrate damage in specific brain regions that lead to cognitive and neurological dysfunctions. An early accumulation of acetylcholine in the synaptic clefts was suggested as the trigger of a sequence of neurochemical events such as an excessive outpour of glutamate and activation of its receptors. Indeed, alterations in NMDA and AMPA central receptors’ densities were detected in brains of poisoned animals. Attempts to improve the current cholinergic-based treatment by adding potent anticonvulsants or antiglutamatergic drugs produced unsatisfactory results. In light of recent events in Syria and the probability of various scenarios of military or terrorist attacks involving organophosphate (OP) nerve agent, research should focus on finding markedly improved countermeasures. Caramiphen, an antimuscarinic drug with antiglutamatergic and GABAergic facilitating properties, was evaluated in a wide range of animals and experimental protocols against OP poisoning. Its remarkable efficacy against OP exposure was established both in prophylactic and post-exposure therapies in both small and large animals. The present review will highlight the outstanding neuroprotective effect of caramiphen as the optimal candidate for the treatment of OP-exposed subjects.

There is a growing concern about the endocrine effects of long-term, low-level exposure to organophosphate (OP) compounds. Studies on experimental animals have found that OP pesticides have an impact on the endocrine system and a few clinical and epidemiological studies have also shown that OPs may affect the male hormone profile, although results are inconsistent. We have evaluated the effect of exposure to OP pesticides, measured through urinary levels of six dialkylphosphate (DAP) metabolites, on male hormone profile in 136 floriculture workers from the State of Mexico and Morelos during two agricultural periods with different degree of pesticide exposure. Generalized estimated equations (GEE) models were developed and adjusted for several potential confounders, including PON1 enzyme activity, as a biomarker of susceptibility, and serum levels of p,p'-DDE, a metabolite of the pesticide DDT widely used in Mexico until 1999 for control of agricultural pests and malaria. Exposure of male floriculture workers to OP pesticides was associated with increased serum levels of follicle-stimulating hormone (FSH) and prolactin and with decreased serum testosterone and inhibin B levels. Among all DAPs tested, only DETP was inversely associated with luteinizing hormone (LH). Estradiol showed a marginally significant positive trend with DEP and DETP derivatives. In conclusion, OP pesticides may have an impact on the endocrine function because of their potential to modify the male hormone profile as a function of the type of pesticide used as well as the magnitude of exposure.

In the event of mass destruction with nerve agents a number of victims can be expected to suffer from symptoms of cholinergic overstimulation due to intoxication as well as from physical trauma. Since previous studies have demonstrated that cholinesterase inhibitors may reverse general anaesthesia in humans this scenario raises the question of how these patients can be anaesthetised in order to enable surgical interventions. A likely reason for this reversal is a reduction of anaesthetic potency by acetylcholine as observed for volatile anaesthetics in vitro. In order to test whether a combination of cholinergic antagonists with general anaesthetics improves their potency, we investigated the effects of clinically relevant concentrations of atropine on sevoflurane potency in cortical and spinal slice cultures during cholinergic overstimulation. As the spinal cord and neocortex are important substrates for general anaesthetics cultured spinal and cortical tissue slices were obtained from embryonic and newborn mice, respectively. Drug effects were assessed by extracellular voltage recordings of spontaneous action potential activity. Application of acetylcholine elevated spontaneous activity in neocortical and spinal slices. Atropine (10 nM) reduced discharge rates and reversed the increase of spontaneous activity induced by acetylcholine. In the presence of acetylcholine and atropine sevoflurane caused a concentration-dependent decrease of neuronal activity in neocortical (EC(50)=0.35+/-0.33 MAC) and spinal slices (EC(50)=0.43+/-0.03 MAC). Comparing our results with previous studies which investigated the effects of acetylcholine on anaesthetic potency it is concluded that small concentrations of atropine increase sevoflurane potency in cortical networks during cholinergic overstimulation. Thus, in a clinical setting, we recommend that anaesthetic drugs should be co-applied with atropine for adequate performance of general anaesthesia.

The widespread use of organophosphorus compounds (OPs) as pesticides and the frequent misuse of OP nerve agents in military conflicts or terrorist attacks emphasize the high clinical relevance of OP poisoning. The toxic symptomatology is caused by inhibition of acetylcholinesterase (AChE). A mainstay of standard antidotal treatment is atropine for antagonizing effects mediated by over stimulation of muscarinic ACh-receptors and oxime to reactivate OP-inhibited AChE. For therapeutic monitoring of oxime treatment in OP poisoning, measurement of erythrocyte AChE is suitable because erythrocyte AChE is an easily accessible surrogate for synaptic AChE. However, measurement of erythrocyte AChE is not standard practice. In contrast, determination of plasma butyrylcholinesterase (BChE) activity is in routine use for monitoring the benefit of oxime therapy. As oxime efficacy is limited with certain OPs (e.g. dimethoate, tabun, soman) alternative therapeutic approaches, e.g. the application of scavengers (BChE) which may sequester OPs before they reach their physiological target, are under investigation. To assess the eligibility of BChE as laboratory parameter and (pseudo catalytic or stoichiometric) scavenger in OP poisoning we initiated an in vitro study under standardized experimental conditions with the objective of determination of kinetic constants for inhibition, reactivation and aging of plasma BChE. It could be shown that, due to limited efficacy of obidoxime, pralidoxime, HI 6 and MMB4 with OP-inhibited BChE, plasma BChE activity is an inappropriate parameter for therapeutic monitoring of oxime treatment in OP poisoning. Furthermore, oxime-induced reactivation is too slow to accomplish a pseudo catalytic function, so that administered BChE may be merely effective as a stoichiometric scavenger.

A method was developed to study exclusively those therapeutic effects of oximes that are not related to reactivation of organophosphate-inhibited acetylcholinesterase (AChE). The model uses the organophosphorus compound crotylsarin (CRS), which proved to be a potent, irreversible, peripherally and centrally active AChE inhibitor with a very short biological half-life. CRS-inhibited AChE appeared to age very rapidly, because in vitro addition of oximes immediately following inhibition, did not result in any AChE reactivation. Anaesthetized, atropinized and artificially ventilated rats were intoxicated with 3 x LD50 CRS and treated 5 min later with the bispyridinium oxime HI-6. Fifty percent of these animals survived more than 24 h following termination of artificial ventilation at 10 min after oxime treatment. The mean survival time of the remaining animals was 66 min, whereas all untreated animals died within 4 min. HI-6, when added in vitro to isolated intact hemidiaphragms, or to diaphragm or brain homogenates from rats which had been killed 1 min following 3 x LD50 CRS, failed to reactivate the inhibited AChE. If blood was sampled (before and) after HI-6 administration to CRS-intoxicated rats, no HI-6-induced AChE reactivation was observed. Yet, a clear improvement of the neuromuscular transmission in the hindleg muscles of these animals was found following HI-6 injection. With this model, decisive evidence is obtained that non-reactivating effects of HI-6 by themselves are therapeutically relevant.

The potency of newly developed monoxime bispyridinium compounds (K156, K203) in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determining percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of newly developed oxime K203 is comparable with obidoxime and trimedoxime in blood and higher than the reactivating potency of trimedoxime and obidoxime in diaphragm and brain, where the difference in reactivating efficacy of obidoxime, trimedoxime and K203 is significant. On the other hand, the potency of newly developed K156 to reactivate tabun-inhibited acetylcholinesterase is comparable with obidoxime or trimedoxime in diaphragm and brain. It is significantly lower than the reactivating efficacy of trimedoxime and obidoxime in blood. Moreover, both newly developed oximes were found to be relatively efficacious in the reduction of lethal toxic effects in tabun-poisoned mice. Especially, the oxime K203 is able to decrease the acute toxicity of tabun nearly two times. The therapeutic efficacy of K156 and K203 corresponds to their potency to reactivate tabun-inhibited acetylcholinesterase, especially in diaphragm and brain. In contrast to obidoxime and trimedoxime, the oxime HI-6 is not effective in reactivation of tabun-inhibited acetycholinesterase and in reducing tabun lethality. While the oxime K156 does not improve the reactivating and therapeutic effectiveness of currently available obidoxime and trimedoxime, the newly developed oxime K203 is markedly more effective in reactivation of tabun-inhibited acetylcholinesterase in rats, especially in brain, and in reducing lethal toxic effects of tabun in mice and, therefore, it is suitable for the replacement of commonly used oximes for the antidotal treatment of acute tabun poisoning.

Oximes K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide] and K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as pretreatment drugs in tabun poisoned mice followed by treatment with atropine plus K033, K048, K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], TMB-4 [1, 3-bis(4-hydroxyiminomethylpyridinium) propane dibromide] and HI-6 [(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride)]. Oxime doses of 25% or 5% of its LD50 were used for pretreatment 15 minutes before tabun-poisoning and for treatment one minute after tabun administration to mice. The best therapeutic effect was obtained when oxime K048 (25% of its LD50) was used in both pretreatment and treatment with atropine. This regiment insured survival of all tested animals after the application of 10 LD50 of tabun. In addition, since BChE is considered an endogenous bio-scavenger of anticholinesterase compounds and its interactions with oximes could be masked by AChE interactions, we evaluated kinetic parameters for interactions of tested oximes with native and tabun-inhibited human plasma butyrylcholinesterase (BChE ; EC 3.1.1.8) and compared them with results obtained previously for human erythrocyte acetylcholinesterase (AChE ; EC 3.1.1.7). Progressive inhibition of BChE by tabun was slightly faster than that of AChE. The reactivation of tabun-inhibited BChE by oximes was very slow, and BChE binding affinity for oximes was lower than AChE's. Therefore, BChE could scavenge tabun prior to AChE inhibition, but fast oxime-assisted reactivation of tabun-inhibited AChE or protection of AChE by oxime against inhibition with tabun would not be obstructed by interaction between BChE and oximes.

Although the three most commonly used large mammal species in the safety assessment of drugs remain the dog, the macaque and the marmoset, swine, especially minipigs, have also been widely used over the years in many toxicological studies. Swine present a number of interesting biological and physiological characteristics. Similarities in skin properties with humans have led to extensive in vitro and in vivo studies. There is a specific interest in cardiovascular research, as well as in anaesthesiology and critical care medicine due to common features of swine and human physiology. Although knowledge of swine brain structure and functions remains incomplete, data does exist. The multiple blood sampling that is necessary in pharmacokinetic and toxicokinetic studies are possible, as well as multiparametric monitoring and interventions with equipment used in human clinical settings. Practicality (handling), scientific (stress reduction) and ethical (invasive monitoring) reasons have led research teams to incorporate anaesthesia into their paradigms which makes the analysis of data increasingly difficult. Although not substantiated by scientific data, the swine appears to have an intermediate position in the scale of public perception between non-human primates and animals commonly referred to as pets (i.e. dogs and cats) and rodents. The benefits of the swine model justify the use of these animals in the design of more effective medical countermeasures against known chemical warfare agents (nerve agents, vesicants and lung damaging agents). Exposure to organophosphorus (OP) pesticides represents a severe health issue in developing countries, while OP intoxication with the more lethal military nerve agents is not only of military concern but also a terrorist threat. Tailoring therapeutic regimens to the reality of OP poisoning is of the utmost importance when little experimental data and sparse human clinical data are available in the decision making process. We will present some of the advantages and disadvantages of the swine model in OP countermeasures elaborating on two examples. First, we will present the issues related to the use of anaesthesia during experimental OP poisoning and second we will show how results from experiments with swine can be integrated into a kinetic-based dynamic model to evaluate oxime efficacy. A better knowledge of OP poisoning in swine (comparative toxicokinetics, pharmacokinetics and biochemistry) is definitely necessary before accepting it as a first choice non-rodent model. However, there exists a large amount of data in the model on anaesthesia and different types of shock favouring their use for evaluation of complex situations such as the anaesthesia of OP poisoned patients and combined injuries.

According to current knowledge, inhibition of acetylcholinesterase (AChE) is a very important toxic action of organphosphorus compounds (OP). Hence, it is obvious to follow the AChE activity in order to quantify the degree of inhibition and to assess possible reactivation. Red blood cell (RBC)-AChE provides an easily accessible source to follow the AChE status also in humans. There are many reports underlining the appropriateness of RBC-AChE as a surrogate parameter that mirrors the synaptic enzyme. With this tool at hand, we can study the kinetic parameters of inhibition, spontaneous and oxime-induced reactivation, as well as aging with human RBCs under physiological conditions in vitro. Moreover, we can simulate the influence of inhibitor and reactivator on enzyme activity and can calculate what happens when both components change with time. Finally, we can correlate under controlled conditions the AChE-status in intoxicated patients with the clinical signs and symptoms and determine the time-dependent changes of the oxime and OP concentration. Data of a clinical trial performed in Munich to analyze the value of obidoxime has elucidated that obidoxime worked as expected from in vitro studies. Following a 250mg bolus, obidoxime was administered by continuous infusion at 750mg/24h aimed at maintaining a plasma concentration of 10-20microM obidoxime. This oxime concentration reactivated RBC-AChE>20% of normal in most cases of OP poisoning by diethylphosphoryl compounds within a few hours. The degree of reactivation fitted theoretical calculations very well when the obidoxime and paraoxon concentrations were fed into the model. Only in a few cases reactivation was much lower than expected. The reason for this effect is probably based on the polymorphism of paraoxonase (PON1) in that the (192)arginine phenotype does hardly hydrolyze the arising diethylphosphoryl obidoxime. While this variable may complicate a proper assessment even more, we are confident that the in vitro evaluation of all relevant kinetic data enables the prediction of probable effectiveness in humans. These studies also help to understand therapeutic failures and to define scenarios where oximes are virtually ineffective. These include poisonings with rapidly aging phosphylated AChE, late start with an effective oxime and too early discontinuation of oximes in poisonings with a persistent OP. The experience gathered with the experimental and therapeutic approaches to human poisoning by OP pesticides may be helpful when oximes have to be selected against nerve agents.

The increased concern about terrorist use of nerve agents prompted us to search for new more effective oximes against tabun and soman poisoning. We investigated the interactions of five bispyridinium oximes: K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide], K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide], TMB-4 [1, 3-bis(4-hydroxyiminomethylpyridinium) propane dibromide] and HI-6 [(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride)] with human erythrocyte acetylcholinesterase (AChE ; E.C. 3.1.1.7) and their effects on tabun- and soman-poisoned mice. All the oximes reversibly inhibited AChE, and the enzyme-oxime dissociation constants were between 17 and 180  M. Tabun-inhibited AChE was completely reactivated by TMB-4, K027 and K048, with the overall reactivation rate constants of 306, 376 and 673 min-1M-1, respectively. The reactivation of tabun-inhibited AChE by K033 reached 50 % after 24 hours, while HI-6 failed to reactivate any AChE at all. Soman-inhibited AChE was resistant to reactivation by 1 mM oximes. All studied oximes protected AChE from phosphorylation with both soman and tabun. In vivo experiments showed that the studied oximes were relatively toxic to mice ; K033 was the most toxic (LD50=33.4 mg/kg), while K027 was the least toxic (LD50=672.8 mg/kg). The best antidotal efficacy was obtained with K048, K027 and TMB-4 for tabun poisoning, and HI-6 for soman poisoning. Moreover, all tested oximes showed no cytotoxic effect on several cell lines in concentrations up to 0.8 mM. The potency of the oximes K048 and K027 to protect mice from 5-fold LD50 of tabun and their low toxicity make these compounds leading in the therapy of tabun poisoning. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.

Anesthetized pigs were injected i.m. with 500 mg HI-6 dichloride (HI-6 2Cl) (1-[[[4-(aminocarbonyl)-pyridinio]methoxy]methyl]-2[(hydroxyimino)methyl]pyridinium dichloride; CAS 34433-31-3)) or the molar equivalent of HI-6 dimethanesulphonate (HI-6 DMS) 633 mg. Plasma HI-6 concentrations were measured by HPLC (1, 3, 5, 10, 15, 30, 60 min and every 30 min until 4 h or 6 h following the i.v. or i.m. dose respectively) while a variety of physiological responses were continuously examined. HI-6 (500 mg 2Cl or 633 mg DMS) resulted in an identical pharmacokinetic profile unaffected by atropine co-administration. Neither HI-6 salt resulted in clinically significant changes in cardiovascular or respiratory function. HI-6 DMS (1899 mg i.v.) resulted in plasma HI-6 concentrations about 10 times higher than measured following i.m. 500 mg 2Cl or 633 mg DMS and resulted in small transitory effect on mean arterial pressure. Atropine plus HI-6 DMS (1–9 mg/kg or 127–172 mg/kg i.m.) protected up to 100% of guinea pigs exposed to 5 × LD 50 of GF (cyclohexyl methyl phosphonoflouridate) or soman (pinacolyl methylphosphonofluoridate) (GD) respectively. The results suggest that the two HI-6 salts have a similar pharmacokinetic profile while HI-6 DMS appears extremely safe and effective against nerve agents and may be as suitable for human use.

It was shown that intoxications with GF-agent are rather resistant to convential oxime therapy; therefore, the development of new oximes in an effort to improve this unsatisfactory situation continues. Upon screening in vitro reactivation test for oximes, that were either newly synthesized at our department, or those that have never been tested for reactivation of GF-inhibited acetylcholinesterase (AChE), three oximes {(1,4-bis(4-hydroxyiminomethylpyridinium)butane dibromide) (K033); (1-(2-hydroxyiminomethylpyridinium)-3-(3-carbamoylpyridinium)-2-oxa-propane dichloride) (HS-6); and (1-(2-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)-but-2-ene dibromide) (BI-6)} with the highest reactivation potency were chosen for in vivo testing in our study. 1,3-Bis(4-hydroxyiminomethylpyridinium)-2-oxa-propane dibromide) (obidoxime); (1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxa-propane dichloride) (HI-6); and (1,1-bis(4-hydroxyiminomethylpyridinium)-methane dibromide) (methoxime) were chosen for comparison as a standard antidotal treatment. All the oximes were applied at the same proportion of their LD50 value (5%), and because of the different acute toxicity of the oximes, the molar concentrations of their solutions for intramuscular (i.m.) administration were considerably different. The highest therapeutic ratio was achieved for therapeutic regimen consisting of HI-6 and atropine. The significantly (P < 0.05) lowest effectivity in treatment of supralethal GF-agent poisoning in comparison with all the other therapeutic regimens, was surprisingly observed for methoxime. HS-6, K033 and BI-6 as well as obidoxime were comparably effective antidotes against GF-agent intoxication and their therapeutic ratios were similar.

The regional distribution of AChE inhibition by parathion in the human brain was examined in a comparative study of the brains of 2 victims of lethal parathion intoxication and 2 control brains matched for age and sex. AChE activity in discrete brain regions was studied by quantitative histochemistry of 40 μm-thick sagittal or coronal cryostat sections from the 4 brains. The inhibition of human brain AChE by parathion is regionally selective. The biggest decreases were observed in the cerebellum, some thalamic nuclei and cortex. Only a moderate decrease (10–30%) was observed in the substantia nigra and basal ganglia, while no effect at all was seen in white matter regions. Detailed knowledge of the brain regions affected by parathion poisoning may explain some of the clinical manifestations of organophosphate poisoning.