NATURAL DETOXIFICATION CAPACITY TO INACTIVATE NERVE AGENTS SARIN AND VX IN THE RAT BLOOD

Biomedical Research Center, University Hospital Hradec Králové, Hradec Králové, Czech Republic1; Center for Basic and Applied Research, Faculty of Informatics and Management, University of Hradec Králové, Hradec Králové, Czech Republic2; Department of Toxicology and Military Pharmacy3 and Department of Teaching Support, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic4


Introduction
The toxicodynamics of nerve agents is based on irreversible acetylcholinesterase (AChE, EC 3.1.1.7)inhibition at the cholinergic synapses (4).The resulting accumulation of neuromediator acetylcholine at the cholinergic synapses overstimulates the cholinergic pathways and subsequently desensitizes the cholinergic receptor sites.Before AChE inhibition in the central and peripheral nervous system, the enzyme is inhibited in the transport system, in the blood according to the principle "first come, first serve" (6).Two enzymes in the blood are present, AChE in the erythrocytes and butyrylcholinesterase (BuChE, EC 3.1.1.8)in the plasma/ serum.However, in the blood, the binding of the agent to cholinesterases is leading to the decrease of its concentration and toxic effect.Other detoxification reactions have occured, too.These characteristics are important for diagnosis, mechanism of action of nerve agents, and, especially, for prophylaxis.
Enzymes capable of nerve agents decomposition (catalytic scavengers) are known many years.They are extensively studied with the aim to develop new ways of decontamination or to prevent nerve agent toxicity (2,10,12,14,19).
Among enzymes participating in metabolism of nerve agents, A-esterases, serum cholinesterase and carboxylesterases are involved.Their role and mechanism of action in detoxification process is different and was extensively discussed by Jokanovic (10).
Detoxification capacity was studied in rats with the aim to elucidate the action of nerve agents and, to improve prophylaxis against nerve agents and organophosphorus compounds.We developed the technique for continual monitoring of the blood cholinesterase activity (7).It allows to study the changes in the activity in the real time.The method was originally developed for the study of the blood cholinesterase changes following acute exposure with nerve agents.However, the technique can be used for modelling of effects of different factors influencing the enzyme and, in the present study, it was evaluated for the demonstration of detoxification of two nerve agents, sarin and VX.

Methodical approach
Animals: Female Wistar rats (VELAZ Prague), weighing 250-270 g, were used in this study.The animals were divid-ed into groups of 3-7 animals in each.Housing of rats was realized in the Central Vivarium of the Faculty of Military Health Sciences under veterinary control.All the experiments were performed under permission and supervision of the Ethics Committee of the Faculty of Military Health Sciences, Hradec Králové.
Chemicals: Nerve agents (sarin, VX) were obtained from Military Technical Institute of Protection (Brno, Czech Republic).They were of minimally 99% of purity and stored in glass ampullas (1 ml).The solutions in saline for experiments were prepared before use.
Continual monitoring of the rat blood cholinesterase activity: The method described by Cabal et al. (7) based on Ellman's method (8) with acetylthiocholine as substrate was used.Generally, the groups of female Wistar rats weighing 250-270 g (n = 3-7) were premedicated (ketamine and xylazine) and cannulated to a. femoralis.Continual blood sampling (0.02 ml/min) and monitoring of the circulating blood cho-linesterase activity were performed (the activity represents all enzymes hydrolyzing acetylthiocholine).Normal activity was monitored 1-2 min and then the agent (sarin or VX) was administered i.m. into one leg in dose of 2× LD 50 .Using different combinations of the leg compression and relaxation (different time intervals before/following agent injection), cholinesterase activity was monitored and according to the inhibition obtained, detoxification capacity was derived.

Results
Intoxication with sarin and VX caused strong decrease of the blood cholinesterase activity.Copies of original records of the blood cholinesterase activity is shown in Fig. 1.When administration of the agent was performed and the leg was compressed immediately (1 min) after the intoxication, the activity was decreased to 40-60% of normal activity.After relaxation 20 min later, two different changes were observed: following sarin intoxication, the inhibition was remained on the same level while following VX administration, the inhibition was continuing after relaxation (Fig. 2).When the compression was performed 5 min after the intoxication (and relaxation 20 min later), the inhibition was more expressed and continued after relaxation following VX administration while following sarin intoxication, further inhibition was not demonstrated (Fig. 3).If the compression was done later (10 min after the intoxication, then relaxation 20 min), the inhibitions by sarin and VX were not very different after relaxation in both cases (following sarin and VX administration).

Discussion
The results suggested that resorption of the agent (both sarin and VX) is very fast and 1 min after the intoxication, approximately 50% of the blood cholinesterases are inhibited.The role of compression is not significant for haemodynamic changes at the time intervals studied as it ] and with compression 5 min after the intoxication and relaxation 20 min later.The number in brackettes indicate the number of animals in group.The results are means only, SD were not higher than ±10%.was described earlier: there were observed haemodynamic changes following femoral vein ligation but the changes were demonstrated after relatively long time (6 hours) after the ligation (20).
Comparison of these results is difficult because a lack of similar data in literature.The half-lives of inhibition of the blood ChE following intoxication with VX and sarin (1) can be compared with our results and the half-times of inhibition are similar.The blood cholinesterase activity on swine was monitored following percutaneous intoxication with VX (17); the inhibition was lower in comparison with our results.Toxicokinetic data with direct determination of the agents published by Schans et al. (21) show similar results as demonstrated in our experiments with inhibition efficacy in the blood.
Quite different cholinesterase changes obtained following sarin and VX intoxication (after compression and relaxation of the leg) suggested that the loss of inhibition capacity can not be scavenger effect (in that case, similar results for both agents sarin and VX would be expected).The loss of inhibition capacity is probably caused by detoxification indicating that sarin is able to be detoxified.Similar results (detoxification of nerve agents including sarin except VX) were demonstrated by Ohmori et al. (18) in decontamination study.Degradation of G-type of nerve agents was also described (15); it was caused by carboxylesterases: their amount in mice and rats was higher than in rabbits or guinea pigs leading to higher LD 50 .
Since VX does not react with carboxylesterases, VX can not be detoxified by these enzymes (16).It could be also a reason for different cholinesterase inhibition by G-and V-nerve agents in the brain structures as demonstrated earlier (9,3).Similar problem was solved by Wills et al. (22) using another approach: fresh frozen plasma (FFP) was used to demonstrate detoxification of G-and V-agents in vitro; the authors concluded suitability of FFP for the treatment of intoxication with G-agents but not for V-agents.Using the method of continual determination of the blood cholinesterase activity, it is possible to monitor cholinesterase activity not only for irreversible but also for reversible inhibitors used for prophylaxis against nerve agent intoxication, e.g.physostigmine, tacrine, huperzine etc. (4,13).

Conclusions
Described methodical approach allows in real time and real conditions in vivo to monitor the changes of the blood cholinesterases following administration of different experimental interventions, e.g.agents administration, and the effect of detoxification.

Fig. 2 :Fig. 3 :
Fig. 2:Changes in the rat blood cholinesterase activity following intoxication with sarin and VX without compression and with compression 1 min after the intoxication and relaxation 20 min later [sarin(6), VX(7)].The number in brackettes indicate the number of animals in group.The results are means only, SD were not higher than ±10%.

Fig. 1 :
Fig. 1: Copies of original records of monitoring the rat blood cholinesterase activity.C -compression of the leg, R -relaxation of the leg.