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Tabun-inhibited rat tissue and blood cholinesterases and their reactivation with the combination of trimedoxime and HI-6 in vivo
Chemico-Biological Interactions 187 (2010) 287–290
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Tabun-inhibited rat tissue and blood cholinesterases and their reactivation with the combination of trimedoxime and HI-6 in vivo Jiri Bajgar a,∗ , Jana Zdarova Karasova a , Jiri Kassa a , Jiri Cabal a , Josef Fusek a , Vaclav Blaha b , Sandra Tesarova c a b c
Department of Toxicology, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic Department of Multidisciplinary Studies, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic 7th Field Hospital, Buzulucka 897, 500 02 Hradec Kralove, Czech Republic
a r t i c l e
i n f o
Article history: Available online 16 February 2010 Keywords: Tabun Acetylcholinesterase Rat Brain parts Diaphragm Blood Reactivators
a b s t r a c t 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. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The treatment of OP/nerve agent poisoning comprises administration of anticholinergics, cholinesterase reactivators and anticonvulsants. While the use of atropine as anticholinergic drug and diazepam as anticonvulsant is usually accepted, there are contraversies in the use of reactivators [1–5]. Some of them are effective against nerve agents, some of them are more effective against OP insecticides and up to now, a universal reactivator against all these agents is not available. There were (and are) some attempts to synthesize a universal reactivator able to reactivate cholinesterases inhibited by all types of nerve agents/OP but without practical results [6–8]. Therefore another approach was described using a combination of two reactivators differently reactivating enzyme inhibited by different inhibitors. This approach was tested in vitro with obidoxime and HI-6 on acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by tabun; it was demonstrated that reactivating effect is due to the effective oxime (i.e. obidoxime in the case of tabun-inhibited AChE) and combination of obidoxime with HI-6 copied the effect of obidoxime alone [9]. This com-
∗ Corresponding author. Tel.: +420 973 251 507; fax: +420 495 518 094. E-mail address: [email protected] (J. Bajgar). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.02.009
bination was tested in experiments with therapeutic efficacy [10,11]. We tried to know if this effect will be observed in vivo. For better distinguishing of different oxime’s action, we used trimedoxime. Trimedoxime is a very effective reactivator of tabun-inhibited AChE in vitro and when combined with atropine, it is one of the best therapies against tabun poisoning [12–14]. The percentages of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats showed trimedoxime to be the most efficacious reactivator [13,14]. Testing the extent of reactivation by trimedoxime and HI-6 in vivo of tabun-inhibited cholinesterases, especially in the different parts of the central nervous system was the aim of this study. 2. Materials and methods 2.1. Animals Female Wistar rats (VELAZ Prague), weighing 200–220 g, were used in this study. The animals were divided into groups of 6 animals each. Housing of rats was realized in the Central Vivarium of the Faculty of Military Health Sciences under veterinary control. All experiments were performed under permission and supervision of the Ethics Committee of the Faculty of Military Health Sciences, Hradec Kralove (permission no. 153/06) according to §17 of the Czech law no. 207/2004.
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J. Bajgar et al. / Chemico-Biological Interactions 187 (2010) 287–290
Table 1 AChE activity in biological samples following treated and untreated tabun poisoning. Group
Blood
Diaphragm
Frontal cortex
Basal ganglia
Pontomedullar AREA
Control
30.1 + 4.2 nc 6.13 + 0.3 nc 9.53 + 0.4 14.2 6.78 + 0.7 2.7 24.55 + 4.0 76.8 17.37 + 4.9 46.9 11.20 + 1.3 31.2 21.54 + 1.2 64.3
70.4 + 7.2 nc 1.52 + 0.4 nc 8.96 + 1.5 10.5 5.17 + 2.2 5.3 23.28 + 2.4 31.6 19.68 + 4.2 26.4 5.82 + 1.0 6.3 18.8 + 4.4 25.2
247.4 + 15.4 nc 3.29 + 0.24 nc 11.1 + 0.8 3.2 3.93 + 0.8 0.3 51.64 + 2.4 19.8 31.53 + 19.4 11.6 10.27 + 1.8 2.9 53.52 + 19.2 20.6
1103 + 132 nc 824 + 91 nc 934 + 93 nc 764 + 121 nc 1141 + 94 nc 1090 + 89 nc 1123 + 98 nc 1060 + 102 nc
380.1 + 20.3 nc 5.93 + 0.9 nc 14.18 + 1.2 2.2 9.78 + 4.1 1.0 98.75 + 4.0 24.8 69.28 + 32.4 16.9 12.13 + 1.6 1.7 99.24 + 23.2 24.9
Tabun 5TR 5H 5H + 5TR 5H + 2.5TR 2.5H + 2.5TR 2.5H + 5TR
Doses of oximes. 5TR: 5% of the lethal dose of trimedoxime, i.e. 3.75 mg/kg; 5H: 5% of the lethal dose of HI-6, i.e. 19.5 mg/kg; the numbers (5 and 2.5) in other groups indicated the dose ratio of oximes. The first line: AChE activity in kat/l (blood) or kat/kg wet weight tissue (mean ± SED); the second line: % of reactivation; nc: not calculated. In bold – statistically significant difference (p < 0.05) from control group – control (for AChE activity). In bold italics – statistically significant difference (p < 0.05) from group with tabun treated with atropine only – Tabun. Reactivation was not statistically evaluated.
2.2. Chemicals Tabun was obtained from Military Technical Institute of Protection (Brno, Czech Republic). It was of minimally 95% purity and stored in glass ampullas (1 ml). The solutions of the agents for experiments were prepared before use. The oximes (trimedoxime dibromide, m.w. 443.98 and HI-6 dichloride monohydrate, m.w. 377.22) were synthesized at the Department of Toxicology of the Faculty of Military Health Sciences (Hradec Kralove, Czech Republic). Their purities were analyzed using HPLC technique. All other chemicals of analytical purity were obtained commercially and used without further purification. All substances were administered i.m. at a volume of 1 ml/kg body weight. 2.3. Intoxication and treatment Control group: The animals were injected with saline i.m. and 1 min later, they were injected once again with saline i.m. (0.1 ml/100 g). The decapitation and sampling was realized 30 min after the last saline injection. Tabun group: The animals were injected with tabun (i.m.) in a dose of 1.5 × LD50 , i.e. 300 g/kg; 1 min later, the animals were injected with atropine (i.m., 21 mg/kg). 31 min after the intoxication, the animals were decapitated and the blood and tissues were collected for biochemical examinations. Treated groups: The animals received tabun (i.m.) at a dose of 1.5 × LD50 , i.e. 300 g/kg, followed by 21 mg/kg atropine sulfate (i.m.) without or with an oxime (i.m.), 1 min later. Animals were decapitated and tissues were immediately collected 31 min after tabun. Oxime doses are given in Table 1. Six animals were used for each group. 2.4. Biochemical determination of AChE The blood was obtained by bleeding from the carotid artery. The brain and diaphragm were prepared. The tissues were frozen and the brain parts (frontal cortex—FC; pontomedullar area—PM, and basal ganglia—BG) were prepared. After thawing, tissues were homogenized (1:10, distilled water, Ultra Turrax homogenizer) and homogenates were used for enzymatic analysis. Concentration of the wet weight tissue was 2 mg per cuvette (2 ml). AChE activ-
ity was determined according the method of Ellman et al. [15] as described elsewhere [16]. Acetylthiocholine iodide (0.5 mM) was used as substrate (Tris–HCl buffer pH 7.6) and 5,5 -dithiobis-2-nitrobenzoic acid (0.5 mM) as chromogen. UVIKON 752 spectrophotometer was used for the determination of absorbancy at 412 nm. The activity was expressed as kat/kg wet weight tissue or as % of control values. The blood was haemolysed with distilled water and in haemolysates, activity of AChE was determined. The activity was expressed as kat/l or as % of control values. 2.5. Statistical evaluation Enzyme activities determined by biochemical method were expressed as a mean ± SD or % of control values and statistical differences were tested by t-test. The reactivation (%) was determined using the AChE activity values:
1−
ao − ar ao − ai
× 100
where ao is the activity in control group (with administration of saline), ar is the activity in tabun-intoxicated group treated with atropine and reactivator, ai is the activity in tabun-intoxicated group treated with atropine only. 3. Results Normal AChE activity varied from high (BG) to low (FC, blood and diaphragm) values. The AChE activity in the brain areas, blood and diaphragm were inhibited following tabun untreated intoxication and treated with atropine only (Table 1). Percentual inhibition was highest in PM, FC and diaphragm, containing about 2% of the control activity. Residual activity of about 20% was observed in the blood, and AChE activity in BG was relatively resistant preserving 75% of control values. Following tabun intoxication, in groups treated with atropine and different doses of reactivators, AChE reactivation was the lowest in group treated with atropine and HI-6, varying from 0.3 to 5.3%. Treatment with atropine and trimedoxime had higher effect—reactivation from 2 to 14%. Combination of trimedoxime and HI-6 in highest doses caused the highest percentage of reactivation. Relative resistance towards reactivation of tabun-inhibited brain AChE is apparent. HI-6 augments the reactivation brought by
J. Bajgar et al. / Chemico-Biological Interactions 187 (2010) 287–290
trimedoxime, particularly in peripheral tissues. Comparing of AChE reactivation showed that trimedoxime is the better reactivator in vivo. When we compare the reactivation caused by a combination of the oximes used, higher reactivation was demonstrated not corresponding to simple summation of the single oximes.
4. Discussion The enzyme activity determined in our experiments is represented by two cholinesterases present in examined tissues: AChE and butyrylcholinesterase (BuChE, EC 3.1.1.8). Their ratio in the biological material is very variable. The activity determined in the whole rat blood corresponds to about 29% of BuChE and 71% of AChE [1,17]; in the brain, the ratio is different representing about 10% of BuChE in the brain structures [18]. For present experiments, the differentiation between these enzymes was not made; however, for further reactivation studies it will be useful to determine AChE and BuChE separately. From the point of its particular inhibitory effects on blood and central cholinesterases, tabun, compared to the other nerve agents such as soman and sarin, seems to be the optimal candidate as a model for the action of two reactivators. There are oximes without reactivation efficacy to tabun-inhibited AChE (e.g. HI-6) and others reactivating AChE inhibited by this agents (trimedoxime and obidoxime). In preventing the lethality of tabun, atropine and trimedoxime were effective compared with other reactivators (obidoxime was comparatively effective); however, HI-6 was practically ineffective [12–14,19,20]. It is of interest that in cholinergic system in the brain, the main cholinergic pathways are represented inter alia by septal nuclei, thalamus, cortex and hippocampus [18]. Some of these structures were studied in our paper and their susceptibility towards tabun was different. The non-uniform AChE inhibition in the different brain structures following untreated intoxication with nerve agents in rats and guinea pigs [21–23] was demonstrated. These results suggest that trimedoxime is an important drug for the treatment of tabun poisoning. Its generally known effect – reactivation of AChE after its inhibition by tabun – is not limited to AChE but it is focused differently to various structures of the brain depending on the particular brain area. AChE activity/inhibition in the frontal cortex and pontomedullar area could be of special importance as it was described for survival/death of rats intoxicated with sarin, soman and VX [24]. These changes were observed for other nerve agents such as sarin including inhalation exposure, too [25]. It appears from our results that areas containing different AChE activities are differentially affected (inhibition/reactivation). Interestingly, tabun inhibited similar amount of AChE in the brain areas tested, i.e. about 300 kat/kg. Possibly, the amount of tabun exposing BG AChE was too low to completely block the abundant enzyme in this region. Surprisingly, the effect of oxime combination does not respond to a simple summation. It would be possible that the oximes blocked the AChE from phosphonylation by tabun or its reactivation by trimedoxime is enhanced allosterically by HI-6 but further explanation could be easily solved by in vitro experiments. Another explanation could be done by interaction of AChE with tabun (slow) and so that 1 min after administered oxime have a real chance to protect part of the enzyme. The affinity of HI-6 and trimedoxime to intact AChE supports this idea [20]. Moreover, AChE activity for different four therapeutic approaches shows that groups with equal doses of HI-6 and trimedoxime differ between these two groups: lower dose of oxime results in lower protection. Simultaneously, toxicity of trimedoxime is higher than that of HI-6 [12,19,20]. Thus, the concentration of HI-6 is higher than trimedoxime and it cannot be excluded that reactivation by trimedoxime
289
and protection by HI-6 are combined. Different pharmacokinetics of oximes was demonstrated for HI-6 and obidoxime [26]. It would be not a main reason of potentiation because the maximum concentration of oximes was achieved before the interval measured, i.e. 10–20 min after the oxime administration [26]. It is difficult to interpret higher reactivation following administration of two oximes. Studies in vitro did not demonstrate any potentiation (tabuninhibited human AChE and reactivation with HI-6 and obidoxime). AChE inhibited by tabun showed that reactivating effect was caused by the effective oxime (obidoxime) while HI-6 in combination used was ineffective [9]. On the other hand, one study [19] in vivo described also higher AChE reactivation in the rat blood following tabun intoxication and treatment with three oximes (HI-6, K 203 and obidoxime). Combinations of oximes in vivo was higher than the reactivating efficacy of the most effective individual oxime. However, the results cannot be directly compared due to marked species differences and different oximes (human and rat AChE, obidoxime–trimedoxime). Therefore, the explanation of the effect in vivo needs further studies. 5. Conclusions - AChE reactivation in different tissues of the rat following treated tabun intoxication is not of the same quantity. - Trimedoxime is a better reactivator than HI-6 for AChE inhibited by tabun. - AChE reactivation by combination of oximes (trimedoxime and HI-6) in vivo does not respond to simple summation. Conflict of interest None. Acknowledgements The authors are indebted to Mrs J. Uhlirova for skilful technical assistance. Financial support of the Ministry of Defence (grant no. FVZ 0000501) is gratefully acknowledged. References [1] J. Bajgar, Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis and treatment, Adv. Clin. Chem. 38 (2004) 151–216. [2] T.C. Marrs, Organophosphate anticholinesterase poisoning, Toxic Subst. Mech. 15 (1996) 357–388. [3] S.W. Wiener, R.S. Hoffman, Nerve agents: a comprehensive review, J. Int. Care Med. 19 (2004) 22–37. [4] J.S. Rotenberg, J. Newmark, Nerve attacks on children: diagnosis and management, Pediatrics 112 (2003) 648–658. [5] P. Eyer, The role of oximes in the management of organophosphorus pesticides poisoning, Toxicol. Rev. 22 (2003) 165–190. [6] J. Bajgar, J. Fusek, K. Kuca, L. Bartosova, D. Jun, Treatment of organophosphate intoxication using cholinesterase reactivators: facts and fiction, Mini Rev. Med. Chem. 7 (2007) 461–466. [7] J. Bajgar, K. Kuca, D. Jun, L. Bartosova, J. Fusek, Cholinesterase reactivators: the fate and effects in the organism poisoned with organophosphates/nerve agents, Curr. Drug Metab. 8 (2007) 803–809. [8] K. Kuca, K. Musilek, D. Jun, J. Bajgar, J. Kassa, Novel oximes, in: R.C. Gupta (Ed.), Handbook of Toxicology of Chemical Warfare Agents, Elsevier, 2009, pp. 997–1021 (Chapter 66). [9] F. Worek, N. Aurbek, H. Thiermann, Reactivation of organophosphate-inhibited human AChE by combinations of obidoxime and HI-6 in vitro, J. Appl. Toxicol. 27 (2007) 582–588. [10] M. Maksimovic, D. Pantelic, V. Kovacevic, Z. Binenfeld, Protective effect of HI-6 and Toxogonin combinations in soman- and tabun-poisoned rats, Acta Pharm. Yugoslav 37 (1987) 227–229. [11] J.G. Clement, J.D. Shiloff, C. Gennings, Efficacy of a combination of acetylcholinesterase reactivators, HI-6 and obidoxime, against tabun and soman poisoning of mice, Arch. Toxicol. 61 (1987) 70–75. [12] J. Kassa, K. Kuca, J. Cabal, Comparison of the efficacy of currently available oximes against tabun in rats, Biologia 60 (Suppl. 17) (2005) 77–79.
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[13] J. Kassa, J. Karasova, J. Bajgar, K. Kuca, K. Musilek, I. Popelikova, A comparison of the reactivating and therapeutic efficacy of newly developed bispyridinium oximes (K250, K251) with commonly used oximes against tabun in rats and mice, J. Enzym. Inhib. Med. Chem. 24 (2009) 1041–1044. [14] J. Kassa, V. Humlicek, A comparison of the potency of newly developed oximes (K074, K075) and currently available oximes (obidoxime, trimedoxime, HI-6) to counteract acute toxic effects of tabun and cyclosarin in mice, Drug Chem. Toxicol. 31 (2008) 127–135. [15] G.L. Ellman, D.K. Courtney, V. Andres, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [16] J. Bajgar, Biological monitoring of exposure to nerve agents, Brit. J. Ind. Med. 49 (1992) 648–653. [17] J. Bajgar, Time course of acetylcholinesterase inhibition in the medulla oblongata of the rat by O-ethyl S-(2-dimethylaminoethyl) methylphosphonothioate in vivo, Brit. J. Pharmacol. 45 (1972) 368–371. [18] G. Giacobini, Cholinesterases in human brain: the effect of cholinesterase inhibitors on Alzheimer’s disease related disorders, in: E. Giacobini, G. Pepeu (Eds.), The Brain Cholinergic System in Health and Disease, Informa Healthcare, Abingdon, UK, 2006, pp. 235–264. [19] J. Kassa, J. Karasova-Zdarova, R. Pavlikova, J. Misik, F. Caisberger, J. Bajgar, The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of tabun poisoning in rats and mice, J. Appl. Toxicol. 30 (2010) 120–124.
[20] S. Berend, A. Lucic Vrdoljak, B. Radic, K. Kuca, New bispyridinium oximes: in vitro and in vivo evaluation of their biological efficiency in soman and tabun poisoning, Chem. Biol. Interact. 175 (2008) 413–416. [21] R.C. Gupta, G.T. Patterson, W.-D. Dettbarn, Biochemical and histochemical alterations following acute soman intoxication in the rat, Toxicol. Appl. Pharmacol. 87 (1987) 393–402. [22] T.-M. Shih, R.K. Kan, J.H. McDonough, In vivo cholinesterase inhibitory specificity of organophosphorus nerve agents, Chem. Biol. Interact. 157–158 (2005) 293–303. [23] J. Bajgar, P. Hajek, D. Slizova, O. Krs, J. Fusek, K. Kuca, D. Jun, L. Bartosova, V. Blaha, Changes of acetylcholinesterase activity in different brain areas following intoxication with nerve agents: biochemical and histochemical study, Chem. Biol. Interact. 165 (2007) 14–21. [24] J. Bajgar, J. Fusek, J. Kassa, D. Jun, K. Kuca, P. Hajek, An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents, Chem. Biol. Interact. 175 (2008) 281–285. [25] J. Bajgar, L. Sevelova, G. Krejcova, J. Fusek, J. Vachek, J. Kassa, L. de Jong, H. Benschop, Biochemical and behavioral effects of soman vapors in low concentrations, Inhal. Toxicol. 16 (2004) 497–507. [26] J. Karasova-Zdarova, L. Novotny, K. Antos, H. Zivna, K. Kuca, Time-dependent changes in concentration of two clinically used acetylcholinesterase reactivators (HI-6 and obidoxime) in rat plasma determined by HPLC techniques after in vivo administration, Anal. Sci. 26 (2010) 63–67.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Tabun-inhibited rat tissue and blood cholinesterases and their reactivation with the combination of trimedoxime and HI-6 in vivo Jiri Bajgar a,∗ , Jana Zdarova Karasova a , Jiri Kassa a , Jiri Cabal a , Josef Fusek a , Vaclav Blaha b , Sandra Tesarova c a b c
Department of Toxicology, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic Department of Multidisciplinary Studies, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic 7th Field Hospital, Buzulucka 897, 500 02 Hradec Kralove, Czech Republic
a r t i c l e
i n f o
Article history: Available online 16 February 2010 Keywords: Tabun Acetylcholinesterase Rat Brain parts Diaphragm Blood Reactivators
a b s t r a c t 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. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The treatment of OP/nerve agent poisoning comprises administration of anticholinergics, cholinesterase reactivators and anticonvulsants. While the use of atropine as anticholinergic drug and diazepam as anticonvulsant is usually accepted, there are contraversies in the use of reactivators [1–5]. Some of them are effective against nerve agents, some of them are more effective against OP insecticides and up to now, a universal reactivator against all these agents is not available. There were (and are) some attempts to synthesize a universal reactivator able to reactivate cholinesterases inhibited by all types of nerve agents/OP but without practical results [6–8]. Therefore another approach was described using a combination of two reactivators differently reactivating enzyme inhibited by different inhibitors. This approach was tested in vitro with obidoxime and HI-6 on acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by tabun; it was demonstrated that reactivating effect is due to the effective oxime (i.e. obidoxime in the case of tabun-inhibited AChE) and combination of obidoxime with HI-6 copied the effect of obidoxime alone [9]. This com-
∗ Corresponding author. Tel.: +420 973 251 507; fax: +420 495 518 094. E-mail address: [email protected] (J. Bajgar). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.02.009
bination was tested in experiments with therapeutic efficacy [10,11]. We tried to know if this effect will be observed in vivo. For better distinguishing of different oxime’s action, we used trimedoxime. Trimedoxime is a very effective reactivator of tabun-inhibited AChE in vitro and when combined with atropine, it is one of the best therapies against tabun poisoning [12–14]. The percentages of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats showed trimedoxime to be the most efficacious reactivator [13,14]. Testing the extent of reactivation by trimedoxime and HI-6 in vivo of tabun-inhibited cholinesterases, especially in the different parts of the central nervous system was the aim of this study. 2. Materials and methods 2.1. Animals Female Wistar rats (VELAZ Prague), weighing 200–220 g, were used in this study. The animals were divided into groups of 6 animals each. Housing of rats was realized in the Central Vivarium of the Faculty of Military Health Sciences under veterinary control. All experiments were performed under permission and supervision of the Ethics Committee of the Faculty of Military Health Sciences, Hradec Kralove (permission no. 153/06) according to §17 of the Czech law no. 207/2004.
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Table 1 AChE activity in biological samples following treated and untreated tabun poisoning. Group
Blood
Diaphragm
Frontal cortex
Basal ganglia
Pontomedullar AREA
Control
30.1 + 4.2 nc 6.13 + 0.3 nc 9.53 + 0.4 14.2 6.78 + 0.7 2.7 24.55 + 4.0 76.8 17.37 + 4.9 46.9 11.20 + 1.3 31.2 21.54 + 1.2 64.3
70.4 + 7.2 nc 1.52 + 0.4 nc 8.96 + 1.5 10.5 5.17 + 2.2 5.3 23.28 + 2.4 31.6 19.68 + 4.2 26.4 5.82 + 1.0 6.3 18.8 + 4.4 25.2
247.4 + 15.4 nc 3.29 + 0.24 nc 11.1 + 0.8 3.2 3.93 + 0.8 0.3 51.64 + 2.4 19.8 31.53 + 19.4 11.6 10.27 + 1.8 2.9 53.52 + 19.2 20.6
1103 + 132 nc 824 + 91 nc 934 + 93 nc 764 + 121 nc 1141 + 94 nc 1090 + 89 nc 1123 + 98 nc 1060 + 102 nc
380.1 + 20.3 nc 5.93 + 0.9 nc 14.18 + 1.2 2.2 9.78 + 4.1 1.0 98.75 + 4.0 24.8 69.28 + 32.4 16.9 12.13 + 1.6 1.7 99.24 + 23.2 24.9
Tabun 5TR 5H 5H + 5TR 5H + 2.5TR 2.5H + 2.5TR 2.5H + 5TR
Doses of oximes. 5TR: 5% of the lethal dose of trimedoxime, i.e. 3.75 mg/kg; 5H: 5% of the lethal dose of HI-6, i.e. 19.5 mg/kg; the numbers (5 and 2.5) in other groups indicated the dose ratio of oximes. The first line: AChE activity in kat/l (blood) or kat/kg wet weight tissue (mean ± SED); the second line: % of reactivation; nc: not calculated. In bold – statistically significant difference (p < 0.05) from control group – control (for AChE activity). In bold italics – statistically significant difference (p < 0.05) from group with tabun treated with atropine only – Tabun. Reactivation was not statistically evaluated.
2.2. Chemicals Tabun was obtained from Military Technical Institute of Protection (Brno, Czech Republic). It was of minimally 95% purity and stored in glass ampullas (1 ml). The solutions of the agents for experiments were prepared before use. The oximes (trimedoxime dibromide, m.w. 443.98 and HI-6 dichloride monohydrate, m.w. 377.22) were synthesized at the Department of Toxicology of the Faculty of Military Health Sciences (Hradec Kralove, Czech Republic). Their purities were analyzed using HPLC technique. All other chemicals of analytical purity were obtained commercially and used without further purification. All substances were administered i.m. at a volume of 1 ml/kg body weight. 2.3. Intoxication and treatment Control group: The animals were injected with saline i.m. and 1 min later, they were injected once again with saline i.m. (0.1 ml/100 g). The decapitation and sampling was realized 30 min after the last saline injection. Tabun group: The animals were injected with tabun (i.m.) in a dose of 1.5 × LD50 , i.e. 300 g/kg; 1 min later, the animals were injected with atropine (i.m., 21 mg/kg). 31 min after the intoxication, the animals were decapitated and the blood and tissues were collected for biochemical examinations. Treated groups: The animals received tabun (i.m.) at a dose of 1.5 × LD50 , i.e. 300 g/kg, followed by 21 mg/kg atropine sulfate (i.m.) without or with an oxime (i.m.), 1 min later. Animals were decapitated and tissues were immediately collected 31 min after tabun. Oxime doses are given in Table 1. Six animals were used for each group. 2.4. Biochemical determination of AChE The blood was obtained by bleeding from the carotid artery. The brain and diaphragm were prepared. The tissues were frozen and the brain parts (frontal cortex—FC; pontomedullar area—PM, and basal ganglia—BG) were prepared. After thawing, tissues were homogenized (1:10, distilled water, Ultra Turrax homogenizer) and homogenates were used for enzymatic analysis. Concentration of the wet weight tissue was 2 mg per cuvette (2 ml). AChE activ-
ity was determined according the method of Ellman et al. [15] as described elsewhere [16]. Acetylthiocholine iodide (0.5 mM) was used as substrate (Tris–HCl buffer pH 7.6) and 5,5 -dithiobis-2-nitrobenzoic acid (0.5 mM) as chromogen. UVIKON 752 spectrophotometer was used for the determination of absorbancy at 412 nm. The activity was expressed as kat/kg wet weight tissue or as % of control values. The blood was haemolysed with distilled water and in haemolysates, activity of AChE was determined. The activity was expressed as kat/l or as % of control values. 2.5. Statistical evaluation Enzyme activities determined by biochemical method were expressed as a mean ± SD or % of control values and statistical differences were tested by t-test. The reactivation (%) was determined using the AChE activity values:
1−
ao − ar ao − ai
× 100
where ao is the activity in control group (with administration of saline), ar is the activity in tabun-intoxicated group treated with atropine and reactivator, ai is the activity in tabun-intoxicated group treated with atropine only. 3. Results Normal AChE activity varied from high (BG) to low (FC, blood and diaphragm) values. The AChE activity in the brain areas, blood and diaphragm were inhibited following tabun untreated intoxication and treated with atropine only (Table 1). Percentual inhibition was highest in PM, FC and diaphragm, containing about 2% of the control activity. Residual activity of about 20% was observed in the blood, and AChE activity in BG was relatively resistant preserving 75% of control values. Following tabun intoxication, in groups treated with atropine and different doses of reactivators, AChE reactivation was the lowest in group treated with atropine and HI-6, varying from 0.3 to 5.3%. Treatment with atropine and trimedoxime had higher effect—reactivation from 2 to 14%. Combination of trimedoxime and HI-6 in highest doses caused the highest percentage of reactivation. Relative resistance towards reactivation of tabun-inhibited brain AChE is apparent. HI-6 augments the reactivation brought by
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trimedoxime, particularly in peripheral tissues. Comparing of AChE reactivation showed that trimedoxime is the better reactivator in vivo. When we compare the reactivation caused by a combination of the oximes used, higher reactivation was demonstrated not corresponding to simple summation of the single oximes.
4. Discussion The enzyme activity determined in our experiments is represented by two cholinesterases present in examined tissues: AChE and butyrylcholinesterase (BuChE, EC 3.1.1.8). Their ratio in the biological material is very variable. The activity determined in the whole rat blood corresponds to about 29% of BuChE and 71% of AChE [1,17]; in the brain, the ratio is different representing about 10% of BuChE in the brain structures [18]. For present experiments, the differentiation between these enzymes was not made; however, for further reactivation studies it will be useful to determine AChE and BuChE separately. From the point of its particular inhibitory effects on blood and central cholinesterases, tabun, compared to the other nerve agents such as soman and sarin, seems to be the optimal candidate as a model for the action of two reactivators. There are oximes without reactivation efficacy to tabun-inhibited AChE (e.g. HI-6) and others reactivating AChE inhibited by this agents (trimedoxime and obidoxime). In preventing the lethality of tabun, atropine and trimedoxime were effective compared with other reactivators (obidoxime was comparatively effective); however, HI-6 was practically ineffective [12–14,19,20]. It is of interest that in cholinergic system in the brain, the main cholinergic pathways are represented inter alia by septal nuclei, thalamus, cortex and hippocampus [18]. Some of these structures were studied in our paper and their susceptibility towards tabun was different. The non-uniform AChE inhibition in the different brain structures following untreated intoxication with nerve agents in rats and guinea pigs [21–23] was demonstrated. These results suggest that trimedoxime is an important drug for the treatment of tabun poisoning. Its generally known effect – reactivation of AChE after its inhibition by tabun – is not limited to AChE but it is focused differently to various structures of the brain depending on the particular brain area. AChE activity/inhibition in the frontal cortex and pontomedullar area could be of special importance as it was described for survival/death of rats intoxicated with sarin, soman and VX [24]. These changes were observed for other nerve agents such as sarin including inhalation exposure, too [25]. It appears from our results that areas containing different AChE activities are differentially affected (inhibition/reactivation). Interestingly, tabun inhibited similar amount of AChE in the brain areas tested, i.e. about 300 kat/kg. Possibly, the amount of tabun exposing BG AChE was too low to completely block the abundant enzyme in this region. Surprisingly, the effect of oxime combination does not respond to a simple summation. It would be possible that the oximes blocked the AChE from phosphonylation by tabun or its reactivation by trimedoxime is enhanced allosterically by HI-6 but further explanation could be easily solved by in vitro experiments. Another explanation could be done by interaction of AChE with tabun (slow) and so that 1 min after administered oxime have a real chance to protect part of the enzyme. The affinity of HI-6 and trimedoxime to intact AChE supports this idea [20]. Moreover, AChE activity for different four therapeutic approaches shows that groups with equal doses of HI-6 and trimedoxime differ between these two groups: lower dose of oxime results in lower protection. Simultaneously, toxicity of trimedoxime is higher than that of HI-6 [12,19,20]. Thus, the concentration of HI-6 is higher than trimedoxime and it cannot be excluded that reactivation by trimedoxime
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and protection by HI-6 are combined. Different pharmacokinetics of oximes was demonstrated for HI-6 and obidoxime [26]. It would be not a main reason of potentiation because the maximum concentration of oximes was achieved before the interval measured, i.e. 10–20 min after the oxime administration [26]. It is difficult to interpret higher reactivation following administration of two oximes. Studies in vitro did not demonstrate any potentiation (tabuninhibited human AChE and reactivation with HI-6 and obidoxime). AChE inhibited by tabun showed that reactivating effect was caused by the effective oxime (obidoxime) while HI-6 in combination used was ineffective [9]. On the other hand, one study [19] in vivo described also higher AChE reactivation in the rat blood following tabun intoxication and treatment with three oximes (HI-6, K 203 and obidoxime). Combinations of oximes in vivo was higher than the reactivating efficacy of the most effective individual oxime. However, the results cannot be directly compared due to marked species differences and different oximes (human and rat AChE, obidoxime–trimedoxime). Therefore, the explanation of the effect in vivo needs further studies. 5. Conclusions - AChE reactivation in different tissues of the rat following treated tabun intoxication is not of the same quantity. - Trimedoxime is a better reactivator than HI-6 for AChE inhibited by tabun. - AChE reactivation by combination of oximes (trimedoxime and HI-6) in vivo does not respond to simple summation. Conflict of interest None. Acknowledgements The authors are indebted to Mrs J. Uhlirova for skilful technical assistance. Financial support of the Ministry of Defence (grant no. FVZ 0000501) is gratefully acknowledged. References [1] J. Bajgar, Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis and treatment, Adv. Clin. Chem. 38 (2004) 151–216. [2] T.C. Marrs, Organophosphate anticholinesterase poisoning, Toxic Subst. Mech. 15 (1996) 357–388. [3] S.W. Wiener, R.S. Hoffman, Nerve agents: a comprehensive review, J. Int. Care Med. 19 (2004) 22–37. [4] J.S. Rotenberg, J. Newmark, Nerve attacks on children: diagnosis and management, Pediatrics 112 (2003) 648–658. [5] P. Eyer, The role of oximes in the management of organophosphorus pesticides poisoning, Toxicol. Rev. 22 (2003) 165–190. [6] J. Bajgar, J. Fusek, K. Kuca, L. Bartosova, D. Jun, Treatment of organophosphate intoxication using cholinesterase reactivators: facts and fiction, Mini Rev. Med. Chem. 7 (2007) 461–466. [7] J. Bajgar, K. Kuca, D. Jun, L. Bartosova, J. Fusek, Cholinesterase reactivators: the fate and effects in the organism poisoned with organophosphates/nerve agents, Curr. Drug Metab. 8 (2007) 803–809. [8] K. Kuca, K. Musilek, D. Jun, J. Bajgar, J. Kassa, Novel oximes, in: R.C. Gupta (Ed.), Handbook of Toxicology of Chemical Warfare Agents, Elsevier, 2009, pp. 997–1021 (Chapter 66). [9] F. Worek, N. Aurbek, H. Thiermann, Reactivation of organophosphate-inhibited human AChE by combinations of obidoxime and HI-6 in vitro, J. Appl. Toxicol. 27 (2007) 582–588. [10] M. Maksimovic, D. Pantelic, V. Kovacevic, Z. Binenfeld, Protective effect of HI-6 and Toxogonin combinations in soman- and tabun-poisoned rats, Acta Pharm. Yugoslav 37 (1987) 227–229. [11] J.G. Clement, J.D. Shiloff, C. Gennings, Efficacy of a combination of acetylcholinesterase reactivators, HI-6 and obidoxime, against tabun and soman poisoning of mice, Arch. Toxicol. 61 (1987) 70–75. [12] J. Kassa, K. Kuca, J. Cabal, Comparison of the efficacy of currently available oximes against tabun in rats, Biologia 60 (Suppl. 17) (2005) 77–79.
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