Enantioselective in-vitro elimination kinetics of nerve agents in blood monitored by derivatization and LC-MS/MS analysis.

We present a simple method for chiral separation and analysis of organophosphorus nerve agents and apply it to monitor the enantioselective blood elimination kinetics of sarin in-vitro. The method is implemented in standard reverse phase LC-MS operating conditions, relieving the user of the dedicated operating conditions frequently demanded in chiral LC-MS analysis. The method consists of formation of diastereomers by a rapid derivatization with (R)-2-(1 aminoethyl) phenol, followed by LC-MS/MS analysis. Derivatization enantioselectivity was studied by comparing the reaction of optically pure sarin and racemic sarin, proving no substantial enantiomeric preference in the reaction and demonstrating the enantiomeric discrimination abilities of the technique. Enantioselective sarin elimination pathways were probed in-vitro by following the fast elimination kinetics of the two sarin enantiomers as well as its hydrolysis metabolite (isopropyl methyl-phosphonic acid, IMPA) in whole blood and plasma compared to water. Sarin enantiomers showed the known marked differences in elimination kinetics with rapid elimination of the (+) enantiomer and slower elimination of the (-) enantiomer in whole blood and plasma as well as dose-dependent kinetics (faster elimination at lower concentrations). We found that small amounts of acetonitrile in plasma prevent the rapid elimination of the (+) enantiomer, resulting in similar, slower elimination kinetics for both enantiomers.

Retrospective determination of regenerated nerve agent sarin in human blood by liquid chromatography-mass spectrometry and in vivo implementation in rabbit.

The highly toxic nerve agent sarin (o-isopropyl methyl-phosphonofluoridate, GB) has been used in several armed conflicts and terror attacks in recent decades. Due to its inherent high sensitivity, liquid chromatography-mass spectrometry (LC-MS/MS) has the potential to detect ultratrace levels of fluoride-regenerated G and V agents after appropriate chemical derivatization. A new method for the retrospective determination of exposure to sarin was developed. The method is based on sarin regeneration from blood using the fluoride-induced technique followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP) and LC-ESI-MS/MS (MRM) analysis. The validated method presents good linear response in the concentration range of 5-1000 pg/mL with a limit of quantitation (LOQ) of 5 pg/mL, 13.8% accuracy, 16.7% precision and a total recovery of 62% ± 9%. This new analytical approach has several advantages over existing GC/GC-MS-based methods in terms of sensitivity, specificity and simplicity, in addition to a short LC-MS cycle time of 12 min. The method was successfully applied in an in vivo experiment for retrospective determination of sarin in a rabbit exposed to 0.1 LD50 sarin (1.5 µg/kg, i.v.). GB-2-DMAMP was easily determined in samples drawn up to 11 days after exposure. The high S/N ratio (500) observed for the GB-2-DMAMP signal in the 11day sample poses the potential for an extended time frame of months for analysis with this new method for the retrospective detection of sarin exposure. To the best of our knowledge, this is the first report on LC-MS/MS trace analysis of regenerated GB from biological matrices.

Natural Detoxification Capacity to Inactivate Nerve Agents Sarin and VX in the Rat Blood.

BACKGROUND: The method of continual determination of the rat blood cholinesterase activity was developed to study the changes of the blood cholinesterases following different intervetions. AIMS: The aim of this study is registration of cholinesterase activity in the rat blood and its changes to demonstrate detoxification capacity of rats to inactivate sarin or VX in vivo. METHODS: The groups of female rats were premedicated (ketamine and xylazine) and cannulated to a. femoralis. Continual blood sampling (0.02 ml/min) and monitoring of the circulating blood cholinesterase activity were performed. Normal activity was monitored 1-2 min and then the nerve agent was administered i.m. (2×LD50). Using different time intervals of the leg compression and relaxation following the agent injection, cholinesterase activity was monitored and according to the inhibition obtained, detoxification capacity was assessed. RESULTS: Administration of sarin to the leg, then 1 and 5 min compression and 20 min later relaxation showed that further inhibition in the blood was not observed. On the other hand, VX was able to inhibit blood cholinesterases after this intervention. CONCLUSIONS: The results demonstrated that sarin can be naturally detoxified on the contrary to VX. Described method can be used as model for other studies dealing with changes of cholinesterases in the blood following different factors.

In vitro kinetics of nerve agent degradation by fresh frozen plasma (FFP).

Great efforts have been undertaken in the last decades to develop new oximes to reactivate acetylcholinesterase inhibited by organophosphorus compounds (OP). So far, a broad-spectrum oxime effective against structurally diverse OP is still missing, and alternative approaches, e.g. stoichiometric and catalytic scavengers, are under investigation. Fresh frozen plasma (FFP) has been used in human OP pesticide poisoning which prompted us to investigate the in vitro kinetics of OP nerve agent degradation by FFP. Degradation was rapid and calcium-dependent with the G-type nerve agents tabun, sarin, soman and cyclosarin with half-lives from 5 to 28 min. Substantially longer and calcium-independent degradation half-lives of 23-33 h were determined with the V-type nerve agents CVX, VR and VX. However, at all the tested conditions, the degradation of V-type nerve agents was several-fold faster than spontaneous hydrolysis. Albumin did not accelerate the degradation of nerve agents. In conclusion, the fast degradation of G-type nerve agents by FFP might be a promising tool, but would require transfusion shortly after poisoning. FFP does not seem to be suitable for detoxifying relevant agent concentrations in case of human poisoning by V-type nerve agents.

Differential mRNA expression of acetylcholinesterase in the central nervous system of rats with acute and chronic exposure of sarin & physostigmine.

A time-course study was carried out to measure the acetylcholinesterase (AChE) gene expression in the brain of female rats exposed to different doses of sarin and physostigmine. Short-term effects were studied with an acute single subcutaneous dose (s.c.) of 80 microg kg(-1) (0.5 x LD(50)) sarin. Cortex and cerebellum showed a significant decline in AChE mRNA expression at 2.5, 24 and 72 h. Biochemical studies showed that plasma butrylcholinesterase (BChE) and brain AChE activities were significantly decreased at 2.5 h, which came back to near control values by 24 h in both cases. For long-term chronic studies, three groups of female rats received daily doses of physostigmine (0.1 mg kg(-1) day(-1)) intramuscularly (i.m.), sarin (15 microg kg(-1) day(-1)) s.c. independently and a combined dose of physostigmine (i.m.) (0.1 mg kg(-1) day(-1)) followed by sarin (s.c.) (15 microg kg(-1) day(-1)) continuously for 30 days. Differential AChE mRNA levels in cortex and cerebellum of rat brain were observed after 30 days and after a lag period of another 30 days with no further administration. Plasma (BChE) and brain (AChE) showed irregular inhibition profile in biochemical studies at 30 days and returned to control levels after 60 days. The acute single subcutaneous administration of sarin for short-term as well as chronic long-term studies showed that AChE inhibition alone does not lead to observed changes in mRNA expression of AChE gene. These observations further suggest that route of administration as well as dose exposure regimen also contributes to the regulation of AChE mRNA expression.

Evaluation of cognitive and biochemical effects of low-level exposure to sarin in rhesus and African green monkeys.

We investigated the potential of low-level exposures to the chemical warfare nerve agent, sarin, to produce adverse effects. Rhesus (Macaca mulatta) and African green monkeys (Chlorocebus acthiops) were trained on a serial probe recognition (SPR) task before IM administration of a low-level concentration (5.87 microg/kg or 2.93 microg/kg) of sarin. Blood was sampled before agent administration and at various times following administration. Sarin administration did not disrupt performance on the SPR task in either species. Major dependent measures characterizing performance (accuracy, number of completed trials per session, average choice response time) were largely unaffected on the day sarin was administered as well as on subsequent testing sessions occurring over several weeks following administration. Analyses of red blood cell (RBC) and plasma samples revealed that sarin administration produced a substantial degree of inhibition of circulating acetylcholinesterase (AChE) in RBC fractions and butyrylcholinesterase (BChE) in plasma fractions, which only slowly recovered. In this regard, AChE activity was inhibited to a greater extent than BChE activity. Blood samples were also evaluated for regenerated sarin, which was found in RBC and plasma fractions in both species and showed orderly elimination functions. More sarin was regenerated from RBC fractions than from plasma fractions. Elimination of regenerated sarin was much slower in RBC than plasma and exceeded the expected time of AChE aging, suggesting the presence of additional sarin binding sites. In general, effects were similar in both species. Taken together, our results show that while the concentrations of sarin administered were clearly biochemically active, they were below those that are required to produce a disruption of behavioral performance.

Kinetics of sarin (GB) following a single sublethal inhalation exposure in the guinea pig.

To improve toxicity estimates from sublethal exposures to chemical warfare nerve agents (CWNA), it is necessary to generate mathematical models of the absorption, distribution, and elimination of nerve agents. However, current models are based on representative data sets generated with different routes of exposure and in different species and are designed to interpolate between limited laboratory data sets to predict a wide range of possible human exposure scenarios. This study was performed to integrate CWNA sublethal toxicity data in male Duncan Hartley guinea pigs. Specific goal was to compare uptake and clearance kinetics of different sublethal doses of sarin (either 0.1 x or 0.4 x LC50) in blood and tissues of guinea pigs exposed to agent by acute whole-body inhalation exposure after the 60-min LC50 was determined. Arterial catheterization allowed repeated blood sampling from the same animal at various time periods. Blood and tissue levels of acetylcholinesterase, butyrylcholinesterase, and regenerated sarin (rGB) were determined at various time points during and following sarin exposure. The following pharmacokinetic parameters were calculated from the graph of plasma or RBC rGB concentration versus time: time to reach the maximal concentration; maximal concentration; mean residence time; clearance; volume of distribution at steady state; terminal elimination-phase rate constant; and area under plasma concentration time curve extrapolated to infinity using the WinNonlin analysis program 5.0. Plasma and RBC t(1/2) for rGB was also calculated. Data will be used to develop mathematical model of absorption and distribution of sublethal sarin doses into susceptible tissues.

Equine butyrylcholinesterase protects rats against inhalation exposure to sublethal sarin concentrations.

Protection experiments were conducted using different doses of equine serum butyrylcholinesterase (Eq BuChE) as pretreatment in rats. Cholinesterase activities were determined in blood [whole blood, red blood cells (RBC) acetylcholinesterase (AChE), and plasma BuChE] before and after sarin inhalation exposure in untreated rats and those pretreated with Eq BuChE. Brain AChE activity was also determined in the frontal cortex, basal ganglia and pontomedullar areas following exposure. Dose-dependent increases in plasma BuChE activity and no changes in the RBC and brain AChE activities were demonstrated following i.p. injection of different amounts of Eq BuChE. Decreases in plasma BuChE activity and RBC and brain AChE activities were observed in control rats following sarin inhalation exposure. In rats pretreated with Eq BuChE this inhibition was lower than in control animals. These results demonstrate protective effects of Eq BuChE pretreatment in rats intoxicated with sublethal concentrations of sarin by inhalation.

A systems dynamics approach to the efficacy of oxime therapy for mild exposure to sarin gas.

The use of nerve agents such as sarin is as much a threat today as any other time in our history. The events in Syria in 2013 are proof of this. "The Obama administration asserted Sunday for the first time that the Syrian government used the nerve gas sarin to kill more than 1,400 people (August 21, 2013) in the world's gravest chemical weapons attack in 25 years." With these recent events clear in our mind, we must focus on the horrific nature of these chemical agents to devise a strategy that will enable first responders to counteract these insidious chemicals. This paper presents research on a physiologically based pharmacokinetic model to determine whether the current treatment protocol prescribed by the Center for Disease Control (CDC) and the US Army is effective in treating victims suffering from acute exposure symptoms. The model was used to determine what treatment should be used for victims suffering from mild exposure symptoms. The results indicate that the current CDC and US Army treatment is effective, but treatment with oxime therapy was not effective in alleviating symptoms of mild exposure. By applying these results, an effective treatment protocol was developed.

Alteromonas prolidase for organophosphorus G-agent decontamination.

Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

Sarin: health effects, metabolism, and methods of analysis.

Sarin (O-isopropylmethylphosphonofluoridate) is a highly toxic nerve agent produced for chemical warfare. Sarin is an extremely potent acetylcholinesterase (AchE) inhibitor with high specificity and affinity for the enzyme. Death by sarin is due to anoxia resulting from airway obstruction, weakness of the muscles of respiration, convulsions and respiratory failure. The main clinical symptoms of acute toxicity of sarin are seizures, tremors and hypothermia. Exposure to sarin during incidents in Japan in 1994, 1995 and 1998, and possible exposure to low levels of sarin during the Gulf War, resulted in the deaths and injury of many people in Japan and caused possible long-term health effects on Gulf War veterans. Symptoms related to sarin poisoning in Japan still exist 1-3 years after the incident and include fatigue, asthenia, shoulder stiffness and blurred vision. Sarin produced seizures in rats and pigs. Recent studies showed that long-term exposure to low levels of sarin caused neurophysiological and behavioral alterations. Toxicity from sarin significantly increased following concurrent exposure to other chemicals such as pyridostigmine bromide. Further research to examine effects of sarin on the cellular and the molecular levels, gene transcription, endocrine system as well as its long-term impact is needed.

Subchronic effects following a single sarin exposure on blood-brain and blood-testes barrier permeability, acetylcholinesterase, and acetylcholine receptors in the central nervous system of rat: a dose-response study.

Subchronic neurotoxic effects of sarin (O-isopropyl methylphosphonofluoridate) treatment at various doses in male Sprague Dawley rats were studied. The animals were treated with a single intramuscular (im) injection of 0.01, 0.1, 0.5, or 1 x LD50 (100 microg/kg). The animals were maintained for 90 d thereafter. [3H]Hexamethonium iodide was used to monitor the changes in blood-brain barrier (BBB) permeability in cortex, brainstem, midbrain, and cerebellum. Brainstem exhibited a significant decrease (approximately 58% of control) in uptake of [3H]hexamethonium iodide at 1 x LD50 dose. No significant changes were observed in BBB permeability in cortex, midbrain, and cerebellum at any dose. Plasma butyrylcholinesterase (BChE) activity remained unchanged, reflecting recovery of the enzyme activity from the initial inhibition following single exposure of 1 x LD50 sarin. Acetylcholinesterase (AChE) activity in the cortex remained inhibited (approximately 29%), whereas in the brainstem there was an increase (approximately 20%) at 1 x LD50 dose of sarin. The m2-selective muscarinic acetylcholine receptor (m2-mAChR) ligand binding was inhibited significantly at 1 x LD50 in the cortex, whereas brainstem showed significantly increased (approximately 45%) ligand binding at 1 x LD50 dose. Nicotinic acetylcholine receptor (nAChR), on the other hand, showed a biphasic response in ligand binding in the cortex with a decrease (approximately 30%) at 0.01 x LD50 but an increase (approximately 40%) at 1 x LD5O. Brainstem did not show any significant change in nAChR ligand binding. These results suggest that single exposure of sarin could lead to changes that may play an important role in neuropathological abnormalities in the central nervous system.

Quantitation of fluoride ion released sarin in red blood cell samples by gas chromatography-chemical ionization mass spectrometry using isotope dilution and large-volume injection.

A new method for measuring fluoride ion released isopropyl methylphosphonofluoridate (sarin, GB) in the red blood cell fraction was developed that utilizes an autoinjector, a large-volume injector port (LVI), positive ion ammonia chemical ionization detection in the SIM mode, and a deuterated stable isotope internal standard. This method was applied to red blood cell (RBC) and plasma ethyl acetate extracts from spiked human and animal whole blood samples and from whole blood of minipigs, guinea pigs, and rats exposed by whole-body sarin inhalation. Evidence of nerve agent exposure was detected in plasma and red blood cells at low levels of exposure. The linear method range of quantitation was 10-1000 pg on-column with a detection limit of approximately 2-pg on-column. In the course of method development, several conditions were optimized for the LVI, including type of injector insert, injection volume, initial temperature, pressure, and flow rate. RBC fractions had advantages over the plasma with respect to assessing nerve agent exposure using the fluoride ion method especially in samples with low serum butyrylcholinesterase activity.

Intravenous and inhalation toxicokinetics of sarin stereoisomers in atropinized guinea pigs.

We report the first toxicokinetic studies of (+/-)-sarin. The toxicokinetics of the stereoisomers of this nerve agent were studied in anesthetized, atropinized, and restrained guinea pigs after intravenous bolus administration of a dose corresponding to 0.8 LD50 and after nose-only exposure to vapor concentrations yielding 0.4 and 0.8 LCt50 in an 8-min exposure time. During exposure the respiratory minute volume and frequency were monitored. Blood samples were taken for gas chromatographic analysis of the nerve agent stereoisomers and for measurement of the activity of blood acetylcholinesterase (AChE). In all experiments, the concentration of (+)-sarin was below the detection limit (<5 pg/ml). The concentration-time profile of the toxic isomer, i.e., (-)-sarin, after an intravenous bolus was adequately described with a two-exponential equation. (-)-Sarin is distributed ca. 10-fold faster than C(-)P(-)-soman, whereas its elimination proceeds almost 10-fold slower. During nose-only exposure to 0.4 and 0.8 LCt50 of (+/-)-sarin in 8 min, (-)-sarin appeared to be rapidly absorbed. The blood AChE activity decreased during the exposure period to ca. 15 and 70% of control activity, respectively. There were no effects on the respiratory parameters. A significant nonlinearity of the toxicokinetics with dose was observed for the respiratory experiments.

Metabolite pharmacokinetics of soman, sarin and GF in rats and biological monitoring of exposure to toxic organophosphorus agents.

This study reports on the pharmacokinetics of the elimination of the metabolites of three toxic organophosphorus compounds (soman, sarin and GF). Urine, blood and lung tissue were collected from rats dosed subcutaneously at 75 micrograms kg-1. Urinary excretion of the metabolite was the major elimination route for these three compounds. The major differences among them were primarily the extent and rate of excretion. The hydrolyzed form, alkylmethylphosphonic acid, was the single major metabolite formed and excreted in urine by a non-saturable mechanism. Nearly total recoveries of the given doses for sarin and GF in metabolite form were obtained from the urine. The terminal elimination half-lives in urine were 3.7 +/- 0.1 and 9.9 +/- 0.8 h for sarin and GF, respectively. Soman metabolite showed a biphasic elimination curve with terminal half-lives of 18.5 +/- 2.7 and 3.6 +/- 2.2 h. Soman was excreted at a slower rate with a recovery of only 62%. Lung was the major organ of accumulation for soman. In blood the toxic agents were concentrated more in red blood cells than in plasma. The acid metabolites can serve as a better chemical marker for monitoring organophosphorus exposure in humans via their higher concentration and longer half-life in urine than the parent compounds.

Distribution of [3H]diisopropylfluorophosphate, [3H]soman, [3H]sarin, and their metabolites in mouse brain.

The brain area distribution of [3H]diisopropylfluorophosphate, [3H]soman, and [3H]sarin and their metabolites in mice was studied after iv administration of sublethal doses. At the appropriate time after the injection of the radiolabeled organophosphate, the mice were decapitated and their brains were dissected into seven areas. There was a relatively even distribution of the parent compounds and their metabolites in all brain areas except the hypothalamus, which contained concentrations of parent compounds the metabolites that were 2-5 times greater than those in other brain areas. Concentrations of the parent compounds and free metabolites declined steadily throughout the time course, whereas concentrations of the bound metabolites remained relatively constant between 6 and 24 hr. There was no correlation between the disposition of soman, and its metabolites, and cholinesterase inhibition in brain areas, which implicates other central mechanisms in the production of organophosphate effects. However, the higher concentrations of organophosphates and their metabolites in the hypothalamus suggest that this area might be important with respect to the pharmacological effects or the toxicity of these compounds.

The Arg192 isoform of paraoxonase with low sarin-hydrolyzing activity is dominant in the Japanese.

The high-density-lipoprotein-associated enzyme paraoxonase, which has a role in the detoxification of organophosphorus compounds, is known to be polymorphic in humans. The Arg192 isoform of paraoxonase hydrolyzes paraoxon more rapidly than the Gln192 isoform. However, with respect to the hydrolysis of toxic nerve agents, such as diazoxon, soman, and sarin, the Arg192 isoform displays a lower activity than the other isoform. To evaluate the possibility that the genetic polymorphism was involved in the aggravated extent of human injury in the sarin gas poisoning incident in the Tokyo subway in March 1995, we investigated the prevalance of this polymorphism in the Japanese population. We found that the Arg192 allele is more common in the Japanese (allele frequency: 0.66) than in people of other races (ranging 0.24-0.31). In the Japanese, 135 out of the 326 subjects (41.4%) investigated were homozygous for the Arg192 allele, which shows a very low hydrolysis activity for sarin. Thus, there seems to be a racial difference in vulnerability to toxic nerve agents, such as sarin. The dominance of the Arg192 allele in the Japanese population probably worsened the tragedy of March 1995 in the Tokyo subway.

The application of the fluoride reactivation process to the detection of sarin and soman nerve agent exposures in biological samples.

The fluoride reactivation process was evaluated for measuring the level of sarin or soman nerve agents reactivated from substrates in plasma and tissue from in vivo exposed guinea pigs (Cava porcellus), in blood from in vivo exposed rhesus monkeys (Macaca mulatta), and in spiked human plasma and purified human albumin. Guinea pig exposures ranged from 0.05 to 44 LD50, and reactivated nerve agent levels ranged from 1.0 ng/mL in plasma obtained from 0.05 LD50 sarin-exposed guinea pigs to an average of 147 ng/g in kidney tissue obtained from two 2.0 LD50 soman-exposed guinea pigs. Positive dose-response relationships were observed in all low-level, 0.05 to 0.4 LD50, exposure studies. An average value of 2.4 ng/mL for reactivated soman was determined in plasma obtained from two rhesus monkeys three days after a 2 LD50 exposure. Of the five types of guinea pig tissue studied, plasma, heart, liver, kidney and lung, the lung and kidney tissue yielded the highest amounts of reactivated agent. In similar tissue and with similar exposure procedures, reactivated soman levels were greater than reactivated sarin levels. Levels of reactivated agents decreased rapidly with time while the guinea pig was alive, but decreased much more slowly after death. This latter chemical stability should facilitate forensic retrospective identification. The high level of reactivated agents in guinea pig samples led to the hypothesis that the principal source of reactivated agent came from the agent-carboxylesterase adduct. However, there could be contributions from adducts of the cholinesterases, albumin and fibrous tissue, as well. Quantitative analysis was performed with a GC-MS system using selected ion monitoring of the 99 and 125 ions for sarin and the 99 and 126 ions for soman. Detection levels were as low as 0.5 ng/mL. The assay was precise and easy to perform, and has potential for exposure analysis from organophosphate nerve agents and pesticides in other animal species.

Interaction of organophosphorus compounds with carboxylesterases in the rat.

Carboxylesterases (CarbE) are involved in detoxication of organophosphorus compounds (OPC) through two mechanisms: hydrolysis of ester bonds in OPC which contain them and binding of OPC at the active site of CarbE which reduces the amount of OPC available for acetylcholinesterase inhibition. This study of the interaction of rat plasma and liver CarbE with dichlorvos, soman and sarin in vitro and in vivo was undertaken in order to contribute to better understanding of the role of CarbE in detoxication of OPC. The results obtained have shown that inhibitory potency (I50) of dichlorvos, sarin and soman towards rat liver CarbE was 0.2 microM, 0.5 microM and 4.5 microM, respectively, for 20-min incubation at 25 degrees C. Second-order rate constants (k(a)) for liver CarbE inhibition were 2.3 x 10(5) M-1 min-1, 6.9 x 10(4) M-1 min-1 and 1.1 x 10(4) M-1 min-1 for dichlorvos, sarin and soman, respectively. The corresponding values for plasma CarbE could not be calculated because of dominant spontaneous reactivation of inhibited CarbE. CarbE inhibited with these OPC in vitro spontaneously reactivate with half-times of 18, 143 and 497 min for sarin, dichlorvos and soman in plasma and 111, 163 and 297 min for sarin, soman and dichlorvos in liver, respectively. These results were also confirmed in experiments in vivo in which rats were subcutaneously treated with 0.5 LD50 of these agents. The half-times of spontaneous reactivation of rat plasma CarbE in vivo were 1.2, 2.0 and 2.7 h for dichlorvos, sarin and soman, respectively. These findings have changed current understanding of the mechanism of interaction of CarbE with OPC and involvement of the enzymes in detoxication of OPC, suggesting an active and important role of the enzymes in metabolic conversions of OPC to their less toxic metabolites.