Effects of organophosphates on precision-cut kidney slices.

Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 µM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from the control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms, including inflammatory response, oxidative stress or activation of apoptosis to further understand the nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme-inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.

Optimization of long-term incubation of precision-cut kidney slices.

Precision-cut kidney slices (PCKS) provide a powerful model to close the gap between in vivo and in vitro research. Publications by various authors favor different incubation conditions, media, and antibiotics, that have not yet been compared in a standardized manner. After preparation, rat-PCKS were incubated in a total of nine combinations of incubation media and antibiotics for four days. We found that a combination of DMEM/F-12 and gentamicin showed the highest levels of viability. Utilizing both qualitative and quantitative methods, we observed stable levels of cellular viability for 10 days when incubated in the most suitable medium combination of DMEM and gentamicin. Additionally, a calcein acetoxymethyl/ethidium homodimer-1 based live/dead staining, analysis of total protein content and lactate dehydrogenase (LDH) were explored to assess both short- and long-term tissue viability. PCKS showed a significant decrease in total protein content, leveling off at around 60% over the duration of 10 days. To be able to evaluate viability irrespective of decreases in total protein detected, we chose to utilize the alamarBlue Cell Viability Assay. Quantifying both intra- and extracellular activity of LDH, while using different concentrations of ethanol as a positive control, we explored enzyme content as a parameter for cell membrane damage and cytotoxicity in PCKS. Overall, we showed that PCKS are suitable for both short- and long-term observation by optimizing incubation parameters, with numerous possibilities for other assays and methods in future studies.

A case report of cholinesterase inhibitor poisoning: cholinesterase activities and analytical methods for diagnosis and clinical decision making.

Suicidal ingestion of organophosphorus (OP) or carbamate (CM) compounds challenges health care systems worldwide, particularly in Southeast Asia. The diagnosis and treatment of OP or CM poisoning is traditionally based on the clinical appearance of the typical cholinergic toxidrome, e.g. miosis, salivation and bradycardia. Yet, clinical signs might be inconclusive or even misleading. A current case report highlights the importance of enzymatic assays to provide rapid information and support clinicians in diagnosis and rational clinical decision making. Furthermore, the differentiation between OP and CM poisoning seems important, as an oxime therapy will most probably not provide benefit in CM poisoning, but-as every pharmaceutical product-it might result in adverse effects. The early identification of the causing agent and the amount taken up in the body are helpful in planning of the therapeutic regimen including experimental strategies, e.g. the use of human blood products to facilitate scavenging of the toxic agent. Furthermore, the analysis of biotransformation products and antidote levels provides additional insights into the pathophysiology of OP or CM poisoning. In conclusion, cholinesterase activities and modern analytical methods help to provide a more effective treatment and a thorough understanding of individual cases of OP or CM poisoning.

Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro.

Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by de-carbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.

Effect of different buffers on kinetic properties of human acetylcholinesterase and the interaction with organophosphates and oximes.

Acetylcholinesterase (AChE) is the primary target of organophosphorus compounds (OP). The investigation into interactions between AChE, OP and oximes in vitro may be affected by the experimental conditions, e.g. by the buffer system. Hence, it was tempting to investigate the Michaelis-Menten kinetics and the inhibition and reactivation kinetics of paraoxon-ethyl, sarin, soman and VX in the presence of phosphate, MOPS, Tyrode and TRIS buffer with human AChE. Compared to phosphate buffer, the inhibition and reactivation kinetics of human erythrocyte AChE were markedly changed by TRIS and in part by MOPS, whereas Tyrode showed similar results to phosphate buffer. These results indicate an effect of the tested buffers on the properties of AChE, and an interaction between OP and oximes has to be considered for the design of in vitro studies and may impair the comparison of data from different laboratories. In view of the comparability of human in vitro kinetic data determined with phosphate buffer with data from human OP poisoning, it seems to be a suitable buffer for the investigation into interactions between AChE, OP and oximes.

Investigation of cardiac glycosides from oleander in a human induced pluripotent stem cells derived cardiomyocyte model.

The ingestion of Nerium oleander and Thevetia peruviana are common causes for poisoning in Southeast Asia. All parts of the oleander shrub contain cardiac glycosides of the cardenolide type. These glycosides act via inhibition of a Na+/K+-ATPase which might cause severe arrhythmia and subsequent death in oleander-poisoned patients. The current study uses human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) in a microelectrode array (MEA) system to assess the cardiac effects of neriifolin, oleandrin, digitoxigenin, peruvoside and thevetin A from the oleander plant. Digoxin was used as established reference compound. All tested compounds showed a corrected field potential duration (FPDc) shortening and was the lowest for 600 nM digitoxigenin with -36.9 ± 1.2 %. Next to the dose-dependent pro-arrhythmic potential, a complete beat arrest of the spontaneously beating hiPSC-CM was observed at a concentration of 300 nM for neriifolin, 600 nM for oleandrin and 1000 nM for digitoxigenin and peruvoside. Thevetin A did not cause arrhythmia up to a final concentration of 1000 nM. Thus, it was possible to establish a cardiac effect rank order of the tested substances: neriifolin > oleandrin > digitoxigenin = peruvoside > digoxin > thevetin A.

Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: a kinetic analysis.

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.

Interaction of pentylsarin analogues with human acetylcholinesterase: a kinetic study.

Previous kinetic studies investigating the interactions between human acetylcholinesterase (AChE), structurally different organophosphorus compounds (OP) and oximes did not reveal a conclusive structure-activity relationship of the different reactions. The only exception was for a homologous series of methylphosphonofluoridates bearing C1-C4 O-n- or O-i-alkyl residues. Hence, it was tempting to investigate the kinetic interactions between different pentylsarin analogues, human AChE and two oximes, obidoxime and HI 6, in order to increase the understanding of structure-activity relationship between highly toxic OP and human AChE. The rate constants for the inhibition of human erythrocyte AChE by four pentylsarin compounds (k(i)), for the spontaneous dealkylation (aging, k(a)) and reactivation (k(s)) of inhibited AChE as well as for the oxime-induced reactivation of inhibited AChE by obidoxime and HI 6 reflected by the dissociation constant (K(D)) and the reactivity constant (k(r)) were determined. All pentylsarin analogues had a high inhibitory potency towards AChE. Inhibited AChE was subject to spontaneous reactivation which outweighed aging substantially. Pentylsarin-inhibited AChE could be reactivated by oximes, HI 6 being more potent than obidoxime. The determination of inhibition, reactivation and aging kinetics of pentylsarin analogues with human AChE extends the database on interactions between AChE and methylphosphonofluoridate homologues with C1-C4 n- and i-alkyl residues demonstrating a structure-activity relationship depending on the chain length with certain differences regarding inhibition and post-inhibitory reactions. Unfortunately, no structure-activity relationship could be observed for the oxime-induced reactivation of inhibited AChE. In view of previous results with numerous structurally different organophosphates, organophosphonates and phosphoramidates it has to be concluded that up to now kinetic studies did not provide decisive information for the development of more effective oxime-based reactivators.

Inhibition, reactivation and aging kinetics of highly toxic organophosphorus compounds: pig versus minipig acetylcholinesterase.

Organophosphorus compound-based (OP) chemical warfare agents (nerve agents) represent a continuing threat to military forces and the civilian population. OPs act primarily by inhibiting acetylcholinesterase (AChE), the standard treatment for which includes AChE reactivators (oximes) in combination with antimuscarinic drugs. In the last decades, the efficacy of oximes has been investigated mostly in small animal models. In order to increase the predictive value of animal studies it is desirable to measure numerous physiological and biochemical parameters. This is difficult in small animals. Large animal models fulfil these requirements and swine are increasingly being used in toxicology studies. Swine breeds generally show considerable variability in different characteristics which may be minimised by the use of specially bred minipigs which have a known genetic background and health status. A comparative study was, therefore, initiated to investigate the kinetic properties of the White Landrace pig and Göttingen minipig AChE in respect of inhibition by the pesticide paraoxon; the nerve agents cyclosarin, VX and VR; the reactivation of inhibited AChE by oximes (obidoxime, pralidoxime and HI 6); and the aging and spontaneous reactivation of inhibited AChE. The determination of the respective kinetic constants found similarities between pig and minipig AChE which showed marked differences in comparison with human AChE values. This has to be considered in designing meaningful models for the investigation of oxime efficacy in pig or minipig experiments. The generated data indicate comparable kinetic properties of pig and minipig AChE and may provide a kinetic basis for extrapolation of data from pig studies to humans.

In vitro pharmacological characterization of the bispyridinium non-oxime compound MB327 and its 2- and 3-regioisomers.

The primary toxic mechanism of organophosphorus compounds, i.e. nerve agents or pesticides, is based on the irreversible inhibition of acetylcholinesterase. In consequence of the impaired hydrolysis, the neurotransmitter acetylcholine accumulates in cholinergic synapses and disturbs functional activity of nicotinic and muscarinic acetylcholine receptors by overstimulation and subsequent desensitization. The resulting cholinergic syndrome will become acute life-threatening, if not treated adequately. The current standard treatment, consisting of administration of a competitive mAChR antagonist (e.g. atropine) and an oxime (e.g. obidoxime, pralidoxime), is not sufficient in the case of soman or tabun intoxications. Consequently, alternative therapeutic options are necessary. An innovative approach comprises the use of compounds selectively targeting nAChRs, especially positive allosteric modulators, which increase the population of the conducting receptor state. MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)) is able to restore soman-blocked muscle-force in preparations of various species including human and was recently identified as "resensitizer". In contrast to the well-studied MB327, the pharmacological efficacy of the 2- and 3-tert-butylpyridinium propane regioisomers is unknown. As a first step, MB327 and its 3-regioisomer (PTM0001) and 2-regioisomer (PTM0002) were pharmacologically characterized using [3H]epibatidine binding assays, functional studies by solid supported membranes based electrophysiology, and in vitro muscle-force investigations of soman-poisoned rat hemidiaphragm preparations by indirect field stimulation technique. The results obtained from targets of different complexity (receptor, muscle tissue) showed that the pharmacological profiles of the 2- and 3-regioisomers were relatively similar to those of MB327. Furthermore, high concentrations showed inhibitory effects, which might critically influence the application as an antidote. Thus, more effective drugs have to be developed. Nevertheless, the combination of the methods presented is an effective tool for clarifying structure-activity relationships.

Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor.

The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [2H6]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI-MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [2H6]MB327 from target-bound [2H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [2H6]MB327 (Kd=15.5±0.9µmolL-1) and for MB327 (Ki=18.3±2.6µmolL-1) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [2H6]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR.

Preparation and characterization of dialkylphosphoryl-obidoxime conjugates, potent anticholinesterase derivatives that are quickly hydrolyzed by human paraoxonase (PON1192Q).

The potential of the most active pyridinium-4-aldoximes, such as obidoxime and trimedoxime, to reactivate phosphorylated acetylcholinesterase is not fully exploited because of inevitable formation of phosphoryloximes (POXs) with extremely high anticholinesterase activity. Hence, a topochemical equilibrium is expected at the active site, with the freshly reactivated enzyme being rapidly re-inhibited by POX produced during reactivation. In the present study, dimethylphosphoryl-, diethylphosphoryl-, and diisopropyl-obidoxime conjugates were generated and isolated in substance. Their inhibition rate of acetylcholinesterase from human red cell membranes was by a factor of 2250, 480 and 600 higher than that observed with paraoxon-methyl, paraoxon-ethyl, and diisopropyl phosphorofluoridate, respectively. All three POXs were hydrolyzed by human paraoxonase (PON1), with the alloenzyme PON1192Q being about 50-fold more active than PON1192R. The rate of hydrolysis, yielding obidoxime, was 1:6:0.03 for the three POXs, respectively. The rate of non-enzymic degradation, yielding obidoxime mononitrile, was similar with the three POXs and showed a high dependency on the reaction temperature (activation energy 83 kJ/mol), while enzymic hydrolysis required less energy (16 kJ/mol). To determine POX-hydrolase activity, we preferred a reaction temperature of 20 degrees C to reduce the noise of spontaneous degradation. A plot of POX-hydrolase versus salt-stimulated paraoxonase activity showed a highly discriminating power towards the PON1Q192R alloenzymes, which may be based on repulsive forces of the quaternary nitrogen atoms of the protonated arginine subtype and the bisquaternary POXs. It is concluded that the pharmacogenetic PON1Q192R polymorphism may be another contributor to the large variability of susceptible subjects seen in obidoxime-treated patients.

Evaluation of HI 6 treatment after percutaneous VR exposure by use of a kinetic-based dynamic computer model.

The availability of highly toxic OP-type chemical warfare agents (nerve agents) and the exertion of organophosphorus compounds during military conflicts and terrorist attacks against civilians in the past underlines the necessity of an effective treatment regimen of OP-poisoning. Presently, standard treatment includes administration of an antimuscarinic agent (e.g. atropine) and a reactivator of inhibited AChE (oxime), but is considered to be rather ineffective with certain nerve agents due to low oxime effectiveness of the currently available oximes, obidoxime and pralidoxime. The evaluation of new oximes as antidotes relies on the implementation of animal experiments for ethical reasons and is complicated by a limited extrapolation of animal data to humans. The development of a reliable animal model might accelerate the evaluation of new substances and their approval as antidotes, whereas, the pig as higher mammalian species seems to be promising as model animal. A dynamic in vitro model, which allows the calculation of AChE activities at different scenarios was developed to facilitate the definition of effective oxime concentrations and the optimization of oxime treatment of OP poisoning of humans and may furthermore be helpful by designing animal experiments. The model is based on a combination of enzyme kinetics (inhibition, reactivation, aging) of AChE with OP, toxicokinetics and oxime pharmacokinetics. By considering species-specific kinetic data this dynamic model was used for the calculation of AChE activities in humans and pigs after percutaneous exposure with 5x LD(50) VR (Russian VX) and treatment with HI 6, a promising new reactivator of OP-inhibited AChE. Due to a low affinity of HI 6 with VR-inhibited pig AChE the oxime dose that causes maximal reactivation of VR-inhibited pig AChE is conspicuously higher compared to humans. Therefore, the design of animal experiments in consideration of calculated data based on species-specific kinetic values may lead to a more reliable extrapolation of animal data to humans and may reduce the number of necessary animal experiments.

Enzyme-kinetic investigation of different sarin analogues reacting with human acetylcholinesterase and butyrylcholinesterase.

The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure-activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP (k(i)), for the spontaneous dealkylation (k(a)) and reactivation (k(s)) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure-activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HLö 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure-activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation.

Reevaluation of indirect field stimulation technique to demonstrate oxime effectiveness in OP-poisoning in muscles in vitro.

Organophosphorus (OP) pesticides or nerve agents cause severe intoxication by inhibition of acetylcholinesterase, finally resulting in death due to respiratory failure. The phrenic nerve diaphragm preparation is considered as the classic model to investigate the effect of OP intoxications and oxime treatment at the neuromuscular junction. However, this preparation is unsuitable for larger species or for muscle strips from biopsies where no nerve is available for stimulation. An alternative technique is the indirect field stimulation of muscles containing intramuscular nerve branches only. The proposed method by Wolthuis et al. [Wolthuis, O.L., Vanwersch, R.A.P., Van Der Wiel, H.J., 1981. The efficacy of some bis-pyridinium oximes as antidotes to soman in isolated muscles of several species including man. Eur. J. Pharmacol. 70, 355-369] was modified and experimentally reevaluated in isolated mouse diaphragms. To confirm that electrical field stimulation technique induced muscle contraction only via the neuromuscular endplate the nicotinic antagonists pancuronium or d-tubocurarine (1microM) were given. In the presence of a nicotinic antagonist hardly any contraction was blocked after indirect field stimulation technique with very short pulses (5micros, <0.6A), in contrast to direct muscle stimulation (broader pulse width, or higher amplitude >0.6A). During paraoxon circumfusion (20min, 1micromol/l) muscle force generation by indirect stimulation was almost completely blocked. Restoration of paralyzed muscle function to 80% of initial values could be achieved after paraoxon wash out (20min) and circumfusion with obidoxime (1micromol/l, 20min). This data correspond quite well to data shown earlier when using conventional nerve stimulation techniques.

Lessons to be learnt from organophosphorus pesticide poisoning for the treatment of nerve agent poisoning.

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.

Swine models in the design of more effective medical countermeasures against organophosphorus poisoning.

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.

Apoptosis in sulfur mustard treated A549 cell cultures.

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.

Respiratory failure in acute organophosphorus pesticide self-poisoning.

BACKGROUND: Acute organophosphorus (OP) pesticide poisoning is a major clinical problem in the developing world. Textbooks ascribe most deaths to respiratory failure occurring in one of two distinct clinical syndromes: acute cholinergic respiratory failure or the intermediate syndrome. Delayed failure appears to be due to respiratory muscle weakness, but its pathophysiology is unclear. AIM: To describe the clinical patterns of OP-induced respiratory failure, and to determine whether the two syndromes are clinically distinct. DESIGN: Prospective study of 376 patients with confirmed OP poisoning. METHODS: Patients were observed throughout their admission to three Sri Lankan hospitals. Exposure was confirmed by butyrylcholinesterase and blood OP assays. RESULTS: Ninety of 376 patients (24%) required intubation: 52 (58%) within 2 h of admission while unconscious with cholinergic features. Twenty-nine (32%) were well on admission but then required intubation after 24 h while conscious and without cholinergic features. These two syndromes were not clinically distinct and had much overlap. In particular, some patients who required intubation on arrival subsequently recovered consciousness but could not be extubated, requiring ventilation for up to 6 days. DISCUSSION: Respiratory failure did not occur as two discrete clinical syndromes within distinct time frames. Instead, the pattern of failure was variable and overlapped in some patients. There seemed to be two underlying mechanisms (an early acute mixed central and peripheral respiratory failure, and a late peripheral respiratory failure) rather than two distinct clinical syndromes.

Application of kinetic-based computer modelling to evaluate the efficacy of HI 6 in percutaneous VX poisoning.

The rife use of organophosphorus compounds (OP) as pesticides and the exertion of highly toxic OP-type chemical warfare agents (nerve agents) during military conflicts and terrorist attacks in the past emphasize the necessity of the development of effective therapeutic countermeasures. Presently, standard treatment of poisoning by OP includes administration of atropine as an antimuscarinic agent and of oximes, e.g. obidoxime or pralidoxime, as reactivators of OP-inhibited acetylcholinesterase (AChE), but is considered to be rather ineffective with certain nerve agents. The evaluation of new oximes as antidotes is only possible by implementation of animal experiments for ethical reasons and therefore complicated by a limited extrapolation of animal data to humans due to marked species differences. A computer simulation based on combination of AChE kinetic data (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics allows the calculation of AChE activities at different scenarios and may facilitate to define effective oxime concentrations and to optimize oxime dosage in OP poisoning. On the base of species-specific kinetic data this model was used to calculate AChE activities in humans and pigs after percutaneous exposure to 5 x LD50 VX and treatment with HI 6. Due to marked species differences between human and pig AChE the HI 6 dose that is necessary to cause a comparable reactivation of VX-inhibited pig AChE is conspicuously higher. Hence, designing animal experiments with the aid of computer modeling may reduce the number of animal experiments and allow a more reliable extrapolation of animal data to humans.