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Die Anschlage mit Sarin in Matsumoto und Tokio (1994, 1995) und der befurchtete Einsatz von C-Waffen im letzten Irakkrieg haben die terroristische und militarische Bedrohung durch chemische Kampfstoffe oder toxische Industriechemikalien ins Bewusstsein der Offentlichkeit gerufen. Als chemische Kampfstoffe bezeichnet man chemische Verbindungen, die fur kriegerische Zwecke vorgesehen sind und den Gegner toten, handlungsunfahig machen oder schadigen sollen. Eine ernst zu nehmende Gefahrenquelle sind auch toxische Verbindungen, die in der chemischen Industrie oder anderweitig wirtschaftlich Verwendung finden und im Sinne chemischer Kampfstoffe oder fur terroristische Anschlage genutzt werden konnen. Krankenhauser sollten sich entsprechend der Bedrohungslage und ihrer Ressourcen auf eine solche Bedrohung vorbereiten. Die vorliegende Arbeit mochte Hilfestellung zu allgemeinen Schutzmasnahmen des Krankenhauses in derartigen Szenarien geben. Daruber hinaus werden die wichtigsten Vergiftungen durch chemische Kampfstoffe in Klinik und Therapie vorgestellt.

Sulfur mustard (bis(2-chloroethyl) sulfide, HD) is an alkylating agent causing erythema and blistering with a latency of several hours after skin exposure. In the present in vitro study the influence of HD (1 microM-1 mM for 30 min or 4 h) on the viability and growth of SCL II cells was investigated. No significant differences in cytotoxicity were observed as assessed by formazan formation from XTT tetrazolium salt at 24, 48 and 72 h after exposure. Sulfur mustard concentrations of >500 microM were associated with an increasing portion of apoptotic cells without change in necrosis rate as assessed by nuclear morphology and gel electrophoresis of the DNA. The ATP levels were not affected up to 6 h after HD exposure ( 500 microM. Colony-forming ability was impaired at concentrations of

The direct reaction of seven pyridinium oximes with the nerve agents sarin, soman, and tabun was followed by a spectrophotometric method. The half-lives (t 1/2) of the oximes, the first- and second-order rate constants (k 1, k 2), and the maximal reaction velocity (\(\roundv \) max) were calculated according to changes in the absorbance of the zwitterion (betaine) peak. In all cases the reaction velocity of the nerve agents with any of the oximes was highest with tabun, followed by sarin and then soman. Comparing the reaction rates of three therapeutically used oximes with the same nerve agent, the highest rate was obtained for soman with obidoxime, for sarin with 2-PAM, and for tabun with HI 6. The maximal reaction velocities reveal that the detoxification of the nerve agents by direct reaction with oximes and the subsequent decomposition of the phosphonyl oxime in vivo do not substantially contribute to the therapeutic effect of these antidotes.

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.

The repeated misuse of highly toxic organophosphorus-type (OP) chemical warfare agents (‘nerve agents’) emphasizes the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators (‘oximes’) is considered to be ineffective with certain nerve agents due to low oxime efficacy. Therefore, pretreatment with carbamate-type compounds, e.g. pyridostigmine, was recommended to improve antidotal efficacy. Recently, the clinically used reversible AChE inhibitors metoclopramide (MCP) and ranitidine (RAN) were shown to exhibit some protective effect against the OP pesticide paraoxon in vitro and in vivo. The present study was undertaken to investigate a potential protective effect of MCP and RAN against inhibition of human AChE by the nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl)methylphosphonothioate). Hemoglobin-free human erythrocyte membranes were incubated with various, human relevant MCP (0.5–2 µm) and RAN (0.5–5 µm) concentrations starting 1 min before addition of VX (1–40 nm). Both compounds failed to increase VX IC50 values. In addition, human AChE was incubated with higher than human relevant therapeutic concentrations of MCP (1 µm–1 mm) and RAN (1 µm–2.0 mm) and inhibited by 40 nm VX. At concentrations higher than 100 µm MCP and RAN caused a concentration dependent increase of residual AChE activity 15 min after addition of VX. These data indicate that MCP and RAN may be ineffective in protecting human AChE against inhibition by the nerve agent VX at human relevant doses. Copyright © 2005 John Wiley & Sons, Ltd.

Chemical and biological warfare agents constitute a low-probability, but high-impact risk both to the military and to the civilian population. The use of hazardous materials of chemical or biological origin as weapons and for homicide has been documented since ancient times. The first use of chemicals in terms of weapons of mass destruction goes back to World War I, when on April 22, 1915 large amounts of chlorine were released by German military forces at Ypres, Belgium. Until around the 1970s of the 20th century, the awareness of the threat by chemical and biological agents had been mainly confined to the military sector. In the following time, the development of increasing range delivery systems by chemical and biological agents possessors sensitised public attention to the threat emanating from these agents. Their proliferation to the terrorists field during the 1990s with the expanding scale and globalisation of terrorist attacks suggested that these agents are becoming an increasing threat to the whole world community. The following article gives a condensed overview on the history of use and development of the more prominent chemical and biological warfare agents.

Acute organophosphorus pesticide poisoning causes tens of thousands of deaths each year across the developing world. Standard treatment involves administration of intravenous atropine and oxime to counter acetylcholinesterase inhibition at the synapse. The usefulness of oximes, such as pralidoxime and obidoxime, has been challenged over the past 20 years by physicians in many parts of the world, who have failed to see benefit in their clinical practice.To find the clinical trial evidence for oximes producing clinical benefit in acute organophosphorus pesticide-poisoned patients.We carried out a systematic search to find randomised clinical trials (RCTs) of oximes in acute organophosphorus pesticide poisoning, using MEDLINE, EMBASE and Cochrane databases. All articles with the text words 'organophosphate' or 'oxime' together with 'poisoning' or 'overdose' were examined. (Search last updated November 2003.)Articles that could possibly be randomised clinical trials were retrieved to determine if this was the case.The published methodology of the possible RCTs located is not clear. One was found in abstract form only and two other published trials also had many gaps in the published methodology. We have attempted to contact the principal authors of all three trials but have been unable to obtain further information.Two RCTs have been published, involving 182 patients treated with pralidoxime. These trials did not find benefit. However, the studies did not take into account a number of issues important for outcome and the methodology is unclear. Therefore, a generalised statement on effectiveness cannot be supported by the published results. In particular, characteristics at baseline were not evenly balanced, the dose of oxime was much lower than recommended in guidelines, there were substantial delays to treatment, and the type of organophosphate was not taken into account. The abstract of the third trial, a small possible RCT, is uninterpretable without further data.Current evidence is insufficient to indicate whether oximes are harmful or beneficial in the management of acute organophosphorus pesticide poisoning. A much larger RCT is required to compare the World Health Organization recommended pralidoxime regimen (30 mg/kg bolus followed by8 mg/kg/hr infusion) with placebo. There are many theoretical and practical reasons why oximes may not be useful to patients with overwhelming self-poisoning. Such a study will need to be designed with pre-defined sub-group analysis to allow identification of patient sub-groups that may benefit from oximes.

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.

Mitochondria have been suggested as key players in apoptotic cell death of neurons and many other tissues, since the release of proapoptotic molecules from mitochondria is implicated in caspase activation. As a potential release mechanism, the occurrence of a large pore opening in the inner membrane (mitochondrial permeability transition pore, PTP) has been proposed, but has not yet been observed directly in neurons. We investigated whether the calcein/Co 2+ -quenching technique introduced by Petronilli et al. 〚Biofactors 8 (1998) 263〛, which allows direct observation of PTP opening, can be applied to neurons. Exposure of calcein-loaded neurons to Co 2+ ions resulted in the fading of diffuse cytoplasmic calcein fluorescence, with organelle-restricted fluorescent spots remaining. These spots were colocalized with mitochondrially-entrapped tetramethylrhodamineethylester (TMRE) fluorescence and corresponded to colocalization of calcein and TMRE fluorescence in digitonin-permeabilized neurons. Importantly, extensive neuronal calcium loading, which is assumed to induce PTP opening, resulted in significant fading of mitochondrial fluorescence, suggesting the occurrence of permeability transition. This fluorescence decrease could be completely prevented by the PTP blocker cyclosporin A.

Obidoxime is an antidote approved for reactivation of inhibited acetylcholinesterase in organophosphate poisoning. HPLC methods were described for its determination in blood or aqueous solutions but not for the determination in urine. Since data for renal obidoxime excretion ranged from 2.2 to 84% of administered dose in healthy volunteers depending on the route of administration and little is known about pharmacokinetics of obidoxime in severely intoxicated patients we developed an internal standard (HI 6) reversed-phase HPLC method for determining obidoxime in urine. The mobile phase consisted of methanol, the counter ion 1-heptane sulfonic acid and tetrabutylammonium phosphate, the stationary phase involved a 5 microm reversed-phase column (125x4 mm). Obidoxime was detected spectrophotometrically at 288 nm. The limit of quantification (LOQ) was 1 microM, the limit of detection (LOD) 0.5 microM. Linear calibration curves were obtained in a concentration range from 1 to 1000 microM. Intra- and inter-day precision C.V.s were below 4%. Accuracy was 95.9% in the LOQ range. Using this method, we were able to quantify obidoxime in urine of an organophosphate poisoned patient. Based on this data we calculated that 58% of the administered dose was excreted in the urine.

Until now the efficacy of new antidotes against organophosphorus poisoning has been primarily evaluated in animal models. However, the transmission of these results to humans is hampered by species differences. Besides atropine, oximes are considered as main antidotes. The most important effect of these compounds is the reactivation of inhibited acetylcholinesterase (AChE). The reactivating potency of oximes can be investigated with human material in vitro and was evaluated in the present study with human erythrocyte AChE after inhibition by different nerve agents and pesticides.

Inhibition of tissue acetylcholinesterase (AChE) is generally thought to be the principal biochemical lesion in organophosphate (OP) poisoning. Depending on the structure of the OPs, various phosphorylated AChEs are formed that differ in the rates for spontaneous and oxime-induced reactivation and for irreversible inhibition by “aging”.

2,2-Dichlorodiethylsulfide (sulfur mustard, SM) can alkylate DNA and other cell constituents. Skin exposure to SM results in erythema and blistering.

The enzyme acetylcholinesterase (AChE; EC 3.1.1.7) is essential for the function of cholinergic synaptic transmission. Inhibited acetylcholine (ACh) elimination and its accumulation in the synaptic cleft result in malfunction of various organs and tissues, including impairment of vital functions as respiration and circulation. Major symptoms of poisoning by AChE inhibitors are shown in Tab. 1. AChE is the target of the most potent synthetic poisons known. Highly effective AChE inhibitors (organophosphorus compounds (OP) and carbamates), were synthesized as pesticides and chemical warfare agents and have become a serious threat.

The effect of atropine and of the bispyridinium oximes, HI6 and HLö 7, on the electrocardiographic pattern was investigated in acutely nerve agent-poisoned guinea-pigs. The electrocardiographic, circulatory and respiratory parameters were recorded in female urethane-anaesthetized Pirbright-white guinea-pigs. After base line measurements, the animals received pyridostigmine (0.05 mumol/kg) and, 30 min later, tabun (5xLD50), sarin (5xLD50), soman (5xLD50 or 10xLD50) or VX (10xLD50 or 20xLD50), followed by saline or atropine (10 mg/kg) or atropine plus HI 6 or or HLö 7 (30 mumol/kg) 2 minutes later. Nerve agent poisoning resulted in respiratory arrest within 2-3 minutes, followed by circulatory arrest a few minutes later in nontreated animals. Antidote treatment rapidly restored heart rate and mean arterial pressure and improved the respiratory function to various extent. The nerve agent injection caused a marked sinus bradycardia and a subsequent complete atrioventricular block within 1-2 minutes, followed by idioventricular rhythm. No ventricular tachyarrhythmias were observed in these groups just before death. Atropine and atropine plus oxime administration immediately restored sinus rhythm which persisted in animals with sufficient respiration50% of base line) throughout the observation period (60 minutes). In guinea-pigs with depressed respiratory function (50%), intermittent ST-T wave alterations and second degree atrioventricular block were observed. In some cases, especially in tabun and soman (10xLD50) poisoning, sinus rhythm converted to deleterious ventricular tachycardia within 1 minute after treatment. These results suggest that atropine-containing antidote combinations may induce lethal arrhythmias in nerve agent poisoning, which may be of clinical importance during intravenous treatment of severe inhalative intoxications.

The effect of methantheline, a quaternary ammonium compound, on the reactivation by HI 6 of soman-inhibited human erythrocyte acetylcholinesterase (AChE) was investigated in vitro using purified human erythrocyte AChE or washed human erythrocytes. Methantheline itself was found to be a mixed competitive/non-competitive inhibitor of AChE (Kii 360 +/- 70 mumol/l; Ki 240 +/- 10 mumol/l). In all experiments the enzyme was first inhibited by soman for 30 min under conditions preventing ageing (pH 10.0 degrees C) and then ageing was allowed by changing the pH to 7.3 and the temperature to 37 degrees C. Methantheline addition (0.36 or 3.6 mmol/l) at the start of ageing increased the portion of AChE which could be reactivated by HI 6 (0.32 mmol/l) added 5 min later, from 24.6 +/- 1.0% (mean +/- SEM) of the original activity to 42.1 +/- 1.8% or 45 +/- 2.9%, respectively. With HI 6 alone, the portion of AChE activity which could be reactivated 25 min after the start of ageing decreased rapidly with the delay of the oxime addition (100% of the original activity at immediate addition), the half-life being approximately 2.5 min. With methantheline alone (0.36 mmol/l) the AChE activity was lower after immediate addition (37% of the original value), but the loss of activity due to the increasing delay of methantheline addition exhibited a similar half-life as with HI 6. Finally, when methantheline (0.36 mmol/l) was added at the start of ageing and HI 6 at various intervals thereafter the half-life of AChE activity loss due to the delay of HI 6 addition at least doubled, compared to incubations without methantheline.

The acute cardiovascular and respiratory effects of poisoning by high soman doses and its therapy by atropine, as well as the underlying mechanisms, are not well known as yet. Therefore, it was the purpose of this study to analyze the circulatory and respiratory functions in soman-poisoned and atropine-treated guinea-pigs. Female Pirbright-white guinea-pigs were anaesthetized with urethane (1.8 g/kg) and the arteria carotis, vena jugularis and trachea were cannulated. After base line measurements, soman (32, 80 or 160 micrograms/kg, i.v.; i.e. 2, 5 or 10 x LD50, respectively) or saline were injected and 2 min later, atropine (10 mg/kg, i.v.) or saline were applied. Respiratory and circulatory parameters were recorded for 60 minutes or until death of the animals. Poisoning by soman resulted in a rapid respiratory arrest, followed by circulatory failure and death after a few minutes. Atropine treatment restored circulatory parameters after soman (2 x LD50) and improved respiration to about 80% of base line values. All animals survived the 60 minutes period. After soman (5 x LD50), atropine improved respiratory parameters only slightly and circulatory parameters markedly. Only 2 of the 8 guinea-pigs survived. Atropine was almost completely ineffective after soman (10 x LD50), despite of a transitory improvement of circulation, and all animals died within a few minutes. In control animals, atropine did not impair respiration or circulation. The results of this study suggest that the soman-induced respiratory depression is primarily caused at the central nervous level and that a significant peripheral neuromuscular block develops only at very high soman doses. The circulatory disturbances are mainly the result of bradycardia due to peripheral muscarinic stimulation. Atropine has a high therapeutic effect in the restoration of circulatory function and may even improve respiration at high soman doses.

Glucose treatment improved survival and symptoms in mice poisoned with As2O3. In order to get more insight into the mechanisms involved, postmortum changes in glucose and glycogen content of various organs and pathology were investigated in mice acutely poisoned with As2O3 and treated with glucose. Forty mice each received 12.9 mg As2O3/kg sc as a single injection. The first group of 10 mice had no further treatment. Fifteen minutes after the As2O3 injection and every 2 h thereafter, the second group (10 mice) received saline and the third group (10 mice) received 5% glucose ip. Groups 4 and 5 (5 mice each) received either saline or glucose only. The injection volume in all groups was 10 microliters/g mouse. Group 6 (5 mice) had no treatment whatsoever. Immediately after death brain, muscle and kidneys were prepared for the enzymatic determination of glucose and glycogen. Samples of the brain, muscle, kidneys, liver, small intestine, colon and spleen were taken for microscopic examination. Independent of the therapeutic procedure, decreases in the glycogen in livers and increases in fat in livers and muscles were observed in mice which died. All mice which died showed heavy leucocytes disruptions, increased fat, and decreased glycogen in the spleen. Intrafolliculary disruptions of leucocytes in the small intestine and colon, as well as patchy hyperemias and hemorrhagic spots in the papilla of the kidneys, were observed in non-survivor mice. Decreased glucose and glycogen was in the brains of non-survivor mice; no differences in glucose and glycogen were found in brains of the mice which survived.(ABSTRACT TRUNCATED AT 250 WORDS)

Atropine (AT) and HI6 are effective antidotes in organophosphate (OP) poisoning. However, AT may enhance oxime-toxicity (Boskovic et al 1984), and a small overdose of HI6 may impair circulation seriously (Lipp and Dola 1980).

The detoxification of soman (1,2,2-dimethyl-propyl methylphosphonofluoridate) was measured in rat livers, using hemoglobin-free, non-recirculating perfusion in situ. Since the detoxification processes may differ in perivenous and periportal zones of liver parenchyma, soman uptake, stereoselective metabolism and inhibition of esterases were compared in antegrade and retrograde perfusion experiments. At low concentrations of soman (up to about 10 mumol l-1 for 5 min) soman was taken up by the liver nearly quantitatively. About 5% recovery rate in the perfusate corresponded well to the intrahepatic shunt flow. Infusions of higher amounts yielded increasing recovery rates. The racemic infusion medium contained the four isomers of soman, C(-)P(-) to C(+)P(+), in nearly identical amounts, whereas in the effluent perfusate only P(-) isomers were found. Even small amounts of soman (5-120 nmol g-1 liver wet weight) caused significant inhibition of hepatic aliesterase activity. Doses higher than 200 nmol g-1 suppressed esterase activity by more than 90%. No essential differences in soman uptake, stereoselective metabolism or inhibition of esterases were found between antegrade and retrograde perfusion experiments.

The effects of atropine and the oxime HI 6 on running performance, brain and plasma cholinesterase activity and brain catecholamines were investigated in mice intoxicated with sublethal doses of soman (100 micrograms/kg s.c.). The running time on a rotating mash wire drum (total running time 60 min) after injection of soman was reduced to 17.2 min. Treatment with atropine (10 mg/kg i.p.) or HI 6 (55 mg/kg i.p.) improved the running performance to 48.2 and 44.8 min, respectively. Cholinesterase activity was decreased in soman poisoned mice to 47.3% in plasma and 43.5% in brain. Therapy with the oxime HI 6 resulted in a reactivation of soman-inhibited peripheral cholinesterase to 76.6%, but failed to reactivate central cholinesterase. Dopamine levels in mice brain were elevated in soman poisoning by 23.2%, whereas noradrenaline levels remained unchanged. The increase in brain dopamine levels was antagonized by atropine as well as by HI 6. The results of this study lead to the speculation that central dopaminergic mechanisms may be involved in soman toxicity as well as in the antidotal action of atropine and the mainly peripherally acting oxime HI 6.

The direct reaction of seven pyridinium oximes with the organophosphorus compounds (OPCs) crotylsarin, cyclosarin, and VX was studied by spectrophotometry. This method allows to quantify different parameters: (a) the half-life times (t (1/2)) of the oxime-OPC reactions on the basis of the changes in the absorption at the zwitterion (betaine) peak maximum, (b) the first- and second-order rate constants (k (1), k (2)), and (c) the maximum reaction velocities (v (max)). The results of the study show that the reaction velocity of the nerve agents with any of the oximes investigated decreased in the order crotylsarincyclosarinVX. The comparison of the reaction rates of the three therapeutically used oximes (2-PAM, obidoxime, HI 6) with the respective OPC gave the highest rate for crotylsarin and cyclosarin with obidoxime and to a similar degree with HI 6, while in the case of VX the most reactive oxime was HI 6. The reaction velocity of the nerve agents with the monopyridinium oxime 2-PAM was lower as compared to the bispyridinium oximes (obidoxime, HI 6). The results obtained with the two sarin analogues indicate that the direct reaction with 2-PAM, obidoxime, or HI 6 could be used for non-corrosive decontamination purposes, especially, if sensitive biological surfaces like skin, mucous membranes, or wounds are considered. However, in view of the concentrations of nerve agents and oximes, which could be expected during OPC poisoning in man, the maximum reaction velocities would not be high enough to contribute markedly to the detoxication of nerve agents in vivo.

The pharmacokinetics of labelled DMPS (sodium-1,3 14C-2,3-dimercaptopropane-1-sulphonate) have been studied in four beagle dogs following bolus intravenous injection (65.7 mumol kg-1) and oral administration (197 mumol kg-1). Following intravenous injection the main kinetic parameters were t 1/2 = 43 min, V beta = 160 ml kg-1, and plasma clearance Clp = 2.6 ml min-1 kg-1. Following oral administration 14C-DMPS is rapidly absorbed with peak concentrations (478 +/- 25 mumol l-1) measured after 30-45 min. About 60 per cent of the oral dose was absorbed. Estimates of t 1/2, V beta, and Clp after oral administration were in close agreement with the values obtained in the intravenous study. 14C-DMPS is eliminated from the body by the kidneys. About 70 per cent of 14C-DMPS in dog plasma are bound to proteins. Binding is even higher in plasma from rat and man.

Acetylcholinesterase (AChE) inhibited by the organophosphate soman (1,2,2-trimethyl-propylmethylphosphonofluoridate) rapidly becomes resistant to reactivation by oximes due to dealkylation of the soman-enzyme complex. This reaction is called aging. The effect of the four mono- and bisquaternary ammonium compounds tetramethylammonium (TMA), hexamethonium, decamethonium and suxamethonium on the reactivatability of soman-inhibited, solubilized AChE from human erythrocytes was investigated in vitro. All compounds were reversible inhibitors of AChE; the respective dissociation constants and the type of inhibition exhibited considerable differences. The affinities to both the active and the allosteric site were considerably higher for suxamethonium (Kii 81.3 microM; Ki 15.9 microM) and decamethonium (Kii 15.4 microM; Ki 4.4 microM) than for TMA (Kii 1 mM; Ki 289.6 microM) and hexamethonium (Kii 4.5 mM; Ki 331.8 microM). The reactivation experiments were performed in a four-step procedure (soman-inhibition at 0 degree and pH 10, aging at 37 degrees and pH 7.3, reactivation by the oxime HI 6 at 37 degrees and pH 7.3 followed by AChE assay). After these four steps (total duration 55 min), AChE was inhibited by soman to 95-100%. HI 6 could reactivate about 20% of the inhibited enzyme. All effectors increased the AChE reactivatability by HI 6 when added before aging was started. The maximal increase in reactivatability was higher in the presence of 1.6 mM suxamethonium (+35.8%) and 150 microM decamethonium (+40%) than of 22 mM TMA (+22.5%) and 8.3 mM hexamethonium (+19.2%). If the effectors were added after 5 min of aging they increased the activity of soman-inhibited AChE, but to a considerably smaller extent than HI 6. A good correlation of the respective Kii values and the effective concentrations of these drugs was observed, indicating that an allosteric binding site of AChE might be involved in the protective effect of these drugs.

The effects of monofluoroacetate (FAC) on suspensions of isolated rat kidney tubules were investigated. FAC inhibited gluconeogenesis from lactate, pyruvate, fructose, dihydroxyacetone, alpha-ketoglutarate, and succinate. The gluconeogenesis from pyruvate, ketoglutarate, and lactate was less sensitive to FAC than that from other substrates. FAC also caused a decrease in oxygen consumption, hydroxybutyrate to acetoacetate ratio, alpha-ketoglutarate, ATP, and total adenine nucleotide content; the citrate content was increased. Addition of alpha-ketoglutarate, 5 mmol/l, caused a reversal of gluconeogenesis inhibition, an increase in ATP content, and a delay in citrate accumulation in isolated rat kidney tubules incubated with FAC.

1) The effect of As2O3 and As2O5 on gluconeogenesis from various substrates in the liver and kidney of rats was investigated. 2) A concentration-dependent inhibition by As2O3 was found. The effect was not dependent on the amount of investigated material (hepatocytes or kidney tubules). For either hepatocytes or kidney tubules the extent of inhibition depended strongly on the substrate used. The highest degree of inhibition was observed in incubations with pyruvate. The inhibition of glucose formation was accompanied to a lesser extent by a diminution in O2 consumption and ATP content. The effect was also dependent on the substrate used. Maximum effect was found in incubations with pyruvate. 3) Oleate, 0.5 mmol/l, increased gluconeogenesis from pyruvate. The effect was not abolished by As2O3. 4) A decrease in the content of acetyl-CoA, 3-hydroxybutyrate, and reduced glutathione was found in suspensions of isolated rat kidney tubules or hepatocytes incubated with As2O3. 5) About 10 times higher concentrations of As2O5 were necessary to induce a similar extent of inhibition of gluconeogenesis, decrease in O2 consumption, and in ATP content as compared with As2O3. The extent of the As2O5 effect depended on the concentration of the toxicant and on the substrate used. Gluconeogenesis from pyruvate exhibited the highest sensitivity to As2O5. 6) All findings can be largely explained by inhibition of pyruvate dehydrogenase as the central target for arsenicals. The subsequent depletion of acetyl CoA results in impaired formation of reducing equivalents in the citric acid cycle, decrease in high energy phosphates and, acetyl CoA being a strong positive modulator of pyruvate carboxylase, in gluconeogenesis inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

The traditional pharmacotherapy of organophosphate poisoning follows three principles: a) reduction of muscarinic symptoms caused by acetylcholine accumulation with an anticholinergic drug, mostly atropine; b) prevention of seizures with an anticonvulsant drug, mostly diazepam; and c) restoration of neuromuscular transmission by removal of the organophosphate from the acetylcholinesterase with an oxime, mostly pralidoxime or obidoxime.

The effect of tris-(2-chloroethyl)-amine (HN-3) on RNA and DNA was investigated spectrophotometrically. The shift in the absorbance spectrum caused by the addition of HN-3 was used to test a variety of compounds for their ability to inhibit RNA alkylation. The effect of HN-3 on the activity of several enzymes was also investigated. The activities of ribonuclease A, desoxyribonuclease I, acetylcholinesterase, diaphorase, glutathione reductase, adenosine desaminase, glyoxalase I, 3-hydroxyacyl-CoA-dehydrogenase, xanthine oxidase, glucose-6-phosphate dehydrogenase, hexokinase and the microsomal N-oxygenation of aniline were not changed by HN-3, whereas the activity of cytochrome-c-reductase exhibited a dose dependent diminution in the presence HN-3. Of 105 compounds tested only 14, namely, sodium thiosulfate, dithioxanthine, thiosalicylic acid, 1,2,4-triazole-5-thiol, 2-thiocytosine, 2-thiohistadine, 2,3-dithiosuccinic acid, thioglycolic acid, 3-mercapto-D-valine,6-amino-2-thiouracil, thionicotine amide, dithiothreitol, sodium sulfite, and ergothioneine prevented the alkylation of RNA. All of them also reacted with HN-3 in absence of RNA. No correlation was found between the reaction constant of the reaction compound:HN-3 in the absence of RNA and the concentration of the compound which inhibited RNA alkylation by 50%. The compounds which were effective in vitro were also tested in mice for their ability to reduce HN-3 toxicity in vivo. Only sodium thiosulfate, d-penicillamine, and dithiosuccinic acid were effective. A 3.9fold increase in the LD50 of HN-3 was achieved in mice treated with sodium thiosulfate 3330 mg/kg i.p., a 1.7fold with 2125 mg dithiosuccinic acid/kg, and a 2fold increase with 2500 mg/kg d-penicillamine. The compound tested was injected i.p. 0.5 to 1 min after the s.c. injection of HN-3.

1. Addition of 4-dimethylaminophenol (DMAP) to suspensions of isolated rat kidney tubules increased extracellular lactate dehydrogenase (LDH) at concn. which did not markedly affect gluconeogenesis. ATP content was also decreased by DMAP but this did not occur until the membrane became permeable to LDH. There was no similar leakage of the mitochondrial enzyme glutamate dehydrogenase. 2. After i.v. injection of DMAP to rats in doses which did not inhibit gluconeogenesis, kidney glutathione was decreased and the urinary LDH was increased. DMAP was irreversibly bound to tissue in rat, with the highest binding in the kidney. The highest binding occurs in those tissues in which DMAP causes necrosis. 3. In isolated rat hepatocytes, DMAP caused toxic effects which were similar but less extensive than occur on addition of DMAP to kidney tubules. The formation of acid-soluble metabolites was higher in isolated rat hepatocytes (20 nmol/mg protein) than in rat kidney tubules (4 nmol/mg protein). DMAP-glucuronide and DMAP-sulphate comprised the major acid-soluble metabolites in both preparations; conjugates of DMAP with glutathione or cysteine were also found.