Souhrn Na samcích myší kmene NMRI byl testován vliv perorálně podaného profylaktického prostředku PANPAL na akutní toxické účinky tabunu a srovnávána jeho účinnost s běžně používaným pyridostigminem cestou stanovení indexu účinnosti. Stejným způsobem byl srovnáván účinek antidotní terapie akutní otravy tabunem v závislosti na výběru anticholinergika či přidání látky s antikonvulzivním účinkem. Dále byla na samcích laboratorního potkana kmene Wistar testována reaktivační účinnost vybraných oximů cestou stanovení procenta reaktivace tabunem inhibované acetylcholinesterázy v krvi, bránici a mozku otrávených potkanů 30 minut po otravě a následné terapii. Na potkanech byl též testován vliv farmakologické profylaxe a výběru vhodného reaktivátoru na tabunem vyvolané příznaky neurotoxicity pomocí Funkční pozorovací baterie. Získané výsledky potvrdily, že ze současných reaktivátorů acetylcholinesterázy se jako nejvhodnější jeví obidoxim, zatímco oxim HI-6, tak účinný proti somanu, je vůči tabunu téměř neúčinný. Jako perspektivní reaktivátor acetylcholinesterázy se pro terapii akutních otrav tabunem nabízí též trimedoxim. Pro dosažení dostatečné úrovně antidotní terapie akutní otravy tabunem je navíc vhodné nahradit běžně používané anticholinergikum atropin za cholinolytikum s prohloubenou centrální účinností (biperiden, benaktyzin či skopolamin). Pokud je k antidotní terapii akutní otravy tabunem použit atropin, je žádoucí přidat k takovéto terapii látku s antikonvulzivním účinkem (např. diazepam). Použijeme-li v případě hrozby expozice živé síly tabunem český originální profylaktický prostředek PANPAL, dosáhneme nejen zvýšení odolnosti exponovaného organismu vůči letálním účinkům tabunu, ale také zvýšíme účinnost následné antidotní terapie otravy. Na rozdíl od PANPALu je efekt samotného pyridostigminu vůči akutní toxicitě tabunu jen minimální.

The aim of this study was to determine optimal conditions for in vitro skin decontamination using water and detergents as decontamination agents and to test the cleansing efficiency of selected detergents. Experiments were performed using a peristaltic pump for showering of pig skin in modified static diffusion cells. Several conditions were tested including different flow rates (from 5 to 33mls(-1)), quantity of rinsing fluid (from 40 to 400ml) and concentration of detergents (2; 5; 10%). Further, several types of detergents/commercial decontamination agents were evaluated under the selected conditions to find the most effective means of decontamination. The amount of paraoxon removed from the skin surface following wet-type decontamination was detected in the rinsing fluid spectrophotometrically after hydrolysis of paraoxon - a model contaminant. The efficacy of rinsing by water/Spolapon AES 253 increased with flow rate up to 25mls(-1) and a rinsing volume of 200ml. Lutensol AT 25 achieved maximum efficacy at the lowest tested concentration (2%). A flow rate of 16mls(-1), rinsing volume of 100ml (values from the middle part of the sigmoid curve) and 5% concentration of decontaminant solution were used for further evaluation of detergents as cleansing agents under the selected conditions. Cetylpyridinium bromide (cationic surfactant), carbethopendecinii bromidum (cationic surfactant) and polyoxyethylene-10-tridecyl ether (non-ionic surfactant), SDS (anionic surfactant), althosan MB (cationic surfactant), sodium dodecylbenzene sulphonate (anionic surfactant), neodekont (mixture), tergitol NPX (non-ionic surfactant), Korynt P (non-ionic surfactant) were found to be the most effective. These decontaminants were able to wash away more than 92% of paraoxon from the contaminated skin.

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.

Objectives: Highly toxic organophosphorus compounds (V- and G-nerve agents) were originally synthesized for warfare or as agricultural pesticides. Data on their acute toxicity are rare and patchy. Therefore, there is a need for integrated summary comparing acute toxicity of organophosphates using different administration routes in the same animal model with the same methodology. Based on original data, a summary of in vivo acute toxicity of selected V- and G-nerve agents (tabun, sarin, soman, VX, Russian VX) and organophosphates paraoxon (POX) and diisopropyl fluorophosphate (DFP) in rats has been investigated. Materials and methods: Male Wistar rats were exposed to organophosphates in several administration routes (i.m., i.p., p.o, s.c., p.c.). The acute toxicity was evaluated by the assessment of median lethal dose (LD50, mg kg(-1)) 2, 4, and 24 hours post exposure. Results: V-agents were the most toxic presented with LD50 ranged from 0.0082 mg kg(-1) (VX, i.m.) to 1.402 mg kg(-1) (Russian VX, p.o.), followed by G-agents (LD50 = 0.069 mg kg(-1)/soman, i.m./ - 117.9 mg kg(-1)/sarin, p.c./), organophosphate POX and DFP (LD50 = 0.321 mg kg(-1)/POX, i.m./ - 420 mg kg(-1)/DFP, p.c./). Generally, i.m. administration was the most toxic throughout all tested agents and ways of administration (LD50 = 0.0082 mg kg(-1)/VX/ - 1.399 mg kg(-1)/DFP/) whereas p.c. way was responsible for lowest acute toxicity (LD50 = 0.085 mg kg(-1)/VX/ - 420 mg kg(-1)/DFP/). Conclusion: The acute toxicity of selected organophosphorus compounds is summarized throughout this study. Although the data assessed in rats are rather illustrative prediction for human, it presents a valuable contribution, indicating the toxic potential and harmfulness of organophosphates.

Highly toxic organophosphorus compounds (OPs) were originally developed for warfare or as agricultural pesticides. Today, OPs represent a serious threat to military personnel and civilians. This study investigates the in vivo decontamination of male Wistar rats percutaneously exposed to paraoxon and two potent nerve agents - soman (GD) and VX. Four commercial detergents were tested as decontaminants - NeodekontTM, ArgosTM, DermogelTM, and FloraFreeTM. Decontamination performed 2 min after exposure resulted in a higher survival rate in comparison with non-decontaminated controls. The decontamination effectiveness was expressed as protective ratio (PR, median lethal dose of agent in decontaminated animals divided by the median lethal dose of agent in untreated animals). The highest decontamination effectiveness was consistently achieved with ArgosTM (PR=2.3 to 64.8), followed by DermogelTM (PR=2.4 to 46.1). NeodekontTM and FloraFreeTM provided the lowest decontamination effectiveness, equivalent to distilled water (PR=1.0 to 43.2).

Objective: The genotoxic vesicant sulphur mustard [bis-2-(chloroethyl)sulphide] is a chemical warfare agent which is easily available due to its relatively simple synthesis. Thus, sulphur mustard is a potential agent for mass contamination. In this study, we focused on sulphur mustard toxicity and decontamination in a rat model using commercially available detergent mixtures for dermal decontamination. Methods: Male Wistar rats were percutaneously treated with sulphur mustard and subjected to wet decontamination 2 min postexposure. Commercially produced detergents Neodekont™, Argos™, Dermogel™ and FloraFree™ were tested for their decontamination efficacy against an exposed group and their protective ratios determined. Results and conclusion: The results showed that all tested detergent solutions produced an increase in the median lethal dose [LD50 = 9.83 (5.87–13.63) mg·kg−1] in comparison to controls, which led to increased survival of experimental animals. In general, all tested detergents provided modest decontamination efficacy (PR = 2.0–5.7). The highest protective ratio (5.7) was consistently achieved with Argos™. Accordingly, Argos™ should be considered in further investigation of mass casualty decontamination.

Equipment for fast and accurate detection of organophosphate nerve agents is developed and tested. The method is based on the spectrophotometric monitoring of the enzyme activity of butyrylcholinesterase after its contact with air in a special absorption unit (a "scrubber") developed for the purpose. The scrubber was made from a glass tube filled with glass beads (diam. 3 mm) and filled with approx. 5 ml of butyrylcholinesterase in a phosphate buffer of pH 7.4. The air sample was bubbled through this solution for 20 s at a flow rate of 80 l hour(-1). Thereafter 8 mu l of the enzyme solution were aspirated into the micro-SIA-LOV analyzer and the activity of the enzymes were evaluated by using Ellman's reagent, i.e. 2.5 mmol l(-1) butyrylthiocholine iodide and 0.25 mmol 5,5'-dithiobis (2-nitrobenzoic acid). The absorbance of the coloured reaction product was measured at 412 nm after the reaction time of 60 s. The residue of the absorption liquid was washed away from the absorber and the system was washed with the enzyme solution prior to next analysis. The contaminated air caused partial inhibition of the enzyme activity of the absorption liquid. The activity of the contaminated sample was compared with the activity of the unaffected enzyme (blank measurement). The analysis was controlled by two PCs. The effect of the concentration of analyte in the absorption liquid on the enzyme activity was tested for 10(-5)-10(-9) mol l(-1) sarin. A single analysis (including the absorption step) took <130 s.

Cationic tensides are a widespread group of surface active agents.These compounds have lots of applications in various branches of industry and research.Quaternary isoquinolinium salts differing in alkyl chain length are members of a cationic surfactant group with quaternary nitrogen in its structure.The members of this group can be used as micellar catalysts or disinfectants.Decontamination (chemical warfare agents) or disinfection (bacteria or fungi) for very similar compounds was described several times.In this work, the preparation of isoquinoline-derived cationic surfactants differing in the length of the side alkylating chain from C8 to C20 is described.An HPLC method used for distinction of all prepared long-chain isoquinolinium analogues has been successfully developed.

Quaternary quinolinium salts differing in alkyl chain length are members of a widespread group of cationic surfactants. These compounds have numerous applications in various branches of industry and research. In this work, the preparation of quinoline-derived cationic surface active agents differing in the length of the side alkyl chains (from C-8 to C-20) is described. An HPLC method was successfully developed for distinction of all members of the series of prepared long-chain quinolinium derivatives. In conclusion, some possibilities of intended tests or usage have been summarized. In vitro testing using a microdilution broth method showed good activity of a substance with a C12 chain length against Gram-positive cocci and Candida species.

The purpose of this in vivo study was to assess a new, putatively optimised method for mass casualty decontamination ("ORCHIDS protocol") for effectiveness in removing the chemical warfare agent VX from the skin of anaesthetised, domestic white pigs. ORCHIDS protocol consists of a 1.5-minute shower with a mild detergent (Argos(TM)) supplemented by physical removal. A standard method of wet decontamination was used for comparison. Experimental animals were divided into four groups (A-D). Two groups were exposed to a supra-lethal percutaneous dose (5 X LD50; 300 mu g kg(-1)) of VX for 1 h prior to decontamination with either the ORCHIDS (C) or standard protocol (D). A third (B, positive control) group was exposed but not subject to decontamination. Blank controls (A) received anaesthesia and the corresponding dose of normal saline instead of VX. Observations of the clinical signs of intoxication were supplemented by measurements of whole blood cholinesterase (ChE) performed on samples of arterial blood acquired at 30-minute intervals for the duration of the study (up to 6 h). Untreated (B) animals displayed typical cholinergic signs consistent with VX intoxication (local fasciculation, mastication, salivation, pilo-erection and motor convulsions) and died 165-240 min post exposure. All animals in both decontamination treatment groups (C, D) survived the duration of the study and exhibited less severe signs of cholinergic poisoning. Thus, both the standard and ORCHIDS protocol were demonstrably effective against exposure to the potent nerve agent VX, even after a delay of 1 h. A critical advantage of the ORCHIDS protocol is the relatively short shower duration (11/2 min compared to 3 min). In practice, this could substantially improve the rate at which individuals could be decontaminated by emergency responders following exposure to toxic materials such as chemical warfare agents.

Detergent-based mixtures and pure surfactants used as decontaminants in mass casualty scenarios were investigated for their acute 24 h toxicity.Commercial detergents Neodekont TM , Argos TM , Dermogel TM , and FloraFree TM were tested in male Wistar rats after percutaneous, peroral and intramuscular administration.Pure surfactants althosan MB, sodium alkylbenzene sulphonate, triton X, benzyl dimethyl dodecyl ammonium chloride, and N-dodecyl pyridinium bromide were investigated in male ICR mice.Estimated LD 50 of surfactants varied from < 100 mg.kg -1 (i.m.) to more than 2000 mg.kg -1 (p.o, p.c.) in mice.All detergents tested in rats were rather less toxic (LD 50 > 6 ml.kg -1 ), thus could be considered as safe if used as external skin decontaminant or if low volume is accidentally ingested.

An integral part of the research and development of new antidotes against nerve agents (herein referred to as NAs) is the verification of their efficacy by means of in vitro tests.The purpose of these tests is to verify the ability of an antidote to protect cholinesterases from inhibition or to reactivate inhibited cholinesterases.For this purpose cholinesterases of different species and with a different degree of purification are traditionally used.Using immobilized cholinesterases proves to be new and advantageous for a number of reasons.The authors of the presented work verified the usability of Detehit detector (Oritest Praha Ltd) containing the pig's brain acetylcholinesterase for this purpose.In this article we see the results of tests focused on verifying the effect of different reaction conditions on the activity of acetylcholinesterase, especially with regard to reproducibility of these measurements.

Misuse of various chemicals, such as chemical warfare agents, industrial chemicals or pesticides during warfare or terrorists attacks requires adequate protection. Thus, development and evaluation of novel decontamination dispositives and techniques are needed. In this study, in vitro permeation and decontamination of a potentially hazardous compound paraoxon, an active metabolite of organophosphorus pesticide parathion, was investigated. Skin permeation and decontamination experiments were carried out in modified Franz diffusion cells. Pig skin was used as a human skin model. Commercially produced detergent-based washing solutions FloraFree (TM) and Argos (TM) were used as decontamination means. The experiments were done under "warm", "cold", "dry" and "wet" skin conditions in order to determine an effect of various physical conditions on skin permeation of paraoxon and on a subsequent decontamination process. There was no significant difference in skin permeation of paraoxon under warm, cold and dry conditions, whereas wet conditions provided significantly higher permeation rates. In the selected conditions, decontamination treatments performed 1 h after a skin exposure did not decrease the agent volume that permeated through the skin. An exception were wet skin conditions with non-significant decontamination efficacy 18 and 28% for the FloraFree (TM) and Argos (TM) treatment, respectively. In contrast, the skin permeation of paraoxon under warm, cold and dry conditions increased up to 60-290% following decontamination compared to non-decontaminated controls. This has previously been described as a skin wash-in effect.

The purpose of this study was to assess the ability of selected detergents (althosan MB 50%, triton X) and one commercial decontamination mean (Argos™) to permeate the pig skin, which could indicate a potential toxic side effect of mass casualty decontaminants.A method of static diffusion cells and UV spectrometry was used to evaluate the skin permeation.All tested agents did not permeate the skin in detectable amounts as 5% water solutions.In concentrated form, triton X and Argos™ did not permeate the skin whereas althosan MB 50% shown specific permeation rate of 6.134 µg.cm -1 in 24 hours.

Usage and misusage of pesticides represent a health risk to military and civilian, especially to agricultural workers; also a possible terrorist threat is considered.The major route of low-volatility pesticides intoxication is percutaneous.Hence, skin permeation characteristics of pesticides are intensively investigated.In this study, in vitro measurement of skin permeation is presented on the example of pesticide paraoxon.Permeation experiments were performed in Franz-type of static diffusion cells using a pig skin.Paraoxon which permeated through the skin was determined enzymatically by modified Ellman's method.During 8 hours experiment, approx.0.1 % of applied paraoxon has permeated through the skin.It was shown that pre-treatment by water simulating wet or sweated skin enhanced the paraoxon permeation.

The present study is aimed at construction of biosensors consisting of a screen printed sensor with a platinum working electrode and immobilized acetylcholinesterase (AChE) as a biorecognition element. Several immobilization protocols such as precipitation with glutaraldehyde, immobilization onto spherical graphite microparticles, sorption, interception into gelatin, and sol-gel were used in order to construct biosensors suitable for practical performance. The activity of AChE was evaluated by means of voltammetric oxidation of thiocholine originating from acetylthiocholine. Square wave and cyclic voltammetry were used throughout the experiments. Suitability of the constructed biosensors was evaluated using the toxic organophosphate diisopropylfluorophosphate.

A dipstick for fast assay of nerve agents and organophosphate pesticides was developed. Indicator pH papers were used as detectors. The principle of the assay is based on enzymatic hydrolysis of acetylcholine into acetic acid and choline by acetylcholinesterase. Acidification of the reaction medium due to accumulation of acetic acid was visible. The colour changed from dark red to yellow as the pH indicator recognized pH shift. Presence of an organophosphate pesticide or a nerve agent results in irreversible inhibition of acetylcholinesterase intercepted on the dipstick. The inhibition stops the enzymatic reaction. The inhibition appears as no change of the medium pH. Three compounds were assayed: paraoxon-ethyl as representative organophosphate pesticides and nerve agents sarin and VX. The achieved limit of detection was 5×10−8M for paraoxon-ethyl and 5×10−9M for sarin and VX. Dipsticks were found stable for at least one month. Suitability of these dipsticks for routine assay is discussed.

Up to now, intensive attempts to synthesize a universal reactivator able to reactivate cholinesterases inhibited by all types of nerve agents/organophosphates were not successful. Therefore, another approach using a combination of two reactivators differently reactivating enzyme was used: in rats poisoned with tabun and treated with combination of atropine (fixed dose) and different doses of trimedoxime and HI-6, changes of acetylcholinesterase activities (blood, diaphragm and different parts of the brain) were studied. An increase of AChE activity was observed following trimedoxime treatment depending on its dose; HI-6 had very low effect. Combination of both oximes showed potentiation of their reactivation efficacy; this potentiation was expressed for peripheral AChE (blood, diaphragm) and some parts of the brain (pontomedullar area, frontal cortex); AChE in the basal ganglia was relatively resistant. These observations suggest that the action of combination of oximes in vivo is different from that observed in vitro.

Biosenzory jsou analyticka zařizeni založena na těsnem spojeni tzv. biorekognicniho elementu a fyzikalně chemickeho převodniku. V teto studii byl použit sitotiskový senzor s platinovou pracovni elektrodou jako převodnik a acetylcholinesterasou jako biorekognicnim elementem. Acetylcholinesterasa byla imobilizovana rozdilným způsobem, vcetně sol-gel technologie, imobilizace na grafitova nanovlakna, precipitace glutaraldehydem a zachyceni do sferických grafitových mikrocastic. Pro jednotlive imobilizacni postupy byla stanovena ucinnost a diskutuje se o praktickem dopadu konstrukce biosenzoru.

Chemical warfare agents are toxic compounds showing negative effects on living organisms. This review focuses on detection of nerve and blister agents, which are two most important classes of chemical warfare agents. Main attention is paid to the construction and use of biosensors. Acetylcholinesterase, butyrylcholinesterase and phosphotriesterase are presented as convenient biorecognition components of biosensors for detection of nerve agents, while dehalogenases are useful for detection of mustard agents.