CHOLINESTERASE REACTIVATORS:
MAPPING OF THEIR EFFECT IN THE RAT BRAIN
(TOXICOLOGICAL, BIOCHEMICAL AND HISTOCHEMICAL STUDY)

Jiri Bajgar¹, Petr Hajek², Josef Fusek¹, Daniel Jun^1,3, Jiri Kassa¹, Jana Karasova¹, Otakar Krs², Kamil Kuca^1,3, Kamil Musilek^1,3, and Dasa Slizova²

¹ Department of Toxicology, Faculty of Military Health Sciences, University of Defence,
Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
² Department of Anatomy, Medical Faculty, Charles University,
Simkova 870, 500 38 Hradec Kralove, Czech Republic
³ Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defence,
Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
Phone: +420 973 251 507; Fax: +420 495 518 094; E-mail: bajgar@pmfhk.cz

ABSTRACT

INTRODUCTION

          Inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in the central and peripheral nervous system is the basic and trigger mechanism of action of organophosphate (OP) nerve agents. The current standard treatment of poisoning with these agents comprises administration of anticholinergics and cholinesterase reactivators (oximes), and in some cases anticonvulsants (Bajgar, 2004). The beneficial therapeutic effect of reactivators against OP poisoning has been described in numerous papers (e.g., Bajgar, 2004; Kassa et al., 2007; Kuca et al., 2007). The in vitro reactivation efficacy of oximes has also been described (Kuca et al., 2003a,b; 2007a,b; Puu et al. 1986; Worek et al. 1998). There is no doubt of the reactivation of AChE by oximes in vivo in the peripheral nervous system (Bajgar et al., 2007a,b). However, oxime´s role in the reactivation of the brain AChE for an organism intoxicated with nerve agents has not been examined in vivo. The central reactivation effects of oximes are not so clear; how well do oximes penetrate through the blood-brain-barrier and what is the effective concentration for AChE reactivation in the brain (Bajgar et al., 2007a)?

          We examine here the reactivation of AChE in the brain and underline its importance for the survival or death of the organism poisoned with OP/nerve agents. Tabun (O-ethyl-N,N-dimethyl phosphoroamidocyanidate)  belongs to the most toxic nerve agents (Cabal and Bajgar, 1999). Treatment of intoxication with tabun is based on administration of acetylcholinesterase reactivators, parasympatholytics and anticonvulsants. Reactivation of AChE at peripheral and central nervous system locations is very dependent on the type of reactivator. Reactivation of AChE is determined mostly via biochemical assays, without specification of its reactivtion in different brain structures (Kassa and Cabal, 1999a,b). Histochemical determination permits a fine search for different structures but without quantitative evaluation. In this contribution, the reactivation/inhibition of AChE in the central nervous system detected by both biochemical and histochemical methods is compared.

METHODS

          The study was performed on rats intoxicated with different nerve agents and treated/untreated with atropine with obidoxime, HI-6 or K048. AChE activities in different brain areas were detected using biochemical and histochemical methods.

Chemicals:

          Tabun of minimally 95% purity was obtained from Military Technical Institute of Protection (Brno, Czech Republic). The oximes (obidoxime, HI-6 and K048) were synthesized at the Department of Toxicology of the Faculty of Military Health Sciences (Hradec Kralove, Czech Republic) All substances were administered intramuscularly, i.m., with a volume of 1 mL/kg body weight.

Animals

          Female Wistar rats, weighing from 200 to 220 g, were purchased from BioTest (Konarovice, Czech Republic). The animals were maintained in an air-conditioned room (22° ± 2° C and 50 ±  10%  relative humidity, RH, with light from 7 AM to 7 PM), and were allowed free access to standard chow and tap water. All the experiments were performed under permission and supervision of the Ethic Committee of the Medical Faculty of Charles University, and Faculty of Military Health Sciences, Hradec Kralove. 

Toxicological experiments

          The therapeutic efficacy of oximes was evaluated by the assessment of the LD₅₀ values and their 95% confidence limits using probit-logarithmical analysis of death occurring 24 hours after i.m. administration of tabun at 5 different doses (8 animals per dose). The oximes were administered i.m. at equimolar doses (10 µmol/kg) in combination with atropine (21 mg/kg) 1 min after tabun administration. The effectiveness of antidotal treatment was expressed as protective index (PI), i.e. LD₅₀ value of treated rats/LD₅₀ value of untreated rats) (Kassa et al. 2006). 

Intoxication

          A single dose of 1.0xLD₅₀ of tabun was injected i.m. Control rats were treated with saline. Treated rats were intoxicated with tabun and 1 min later, they were treated with atropine (21 mg/kg, i.m.) with obidoxime, HI-6 or K048 (i.m., 100 µmol/kg). 30 min after treatment, they were decapitated and the brains were removed. For biochemical experiments, 6 animals per group, and for histochemical examination, 3 animals per group were used.

Histochemical determination of AChE  

          Removed brains were rapidly frozen and cut into series of 20 μm sections in a cryostat. Qualitative and quantitative histochemistry was performed as described elsewhere (Bajgar et al., 2007c). The activity was compared in relative percent values. We selected these sections and groups of nuclei to compare quantitative histochemistry with biochemical determination of AChE: ncleus ruber (NR); ponto-medullar area (PM), and frontal cortex (FC), respectively.

Biochemical determination of AChE

          The brains were frozen and the different sections (NR, FC, PM) were prepared. After thawing, tissue was homogenized (1:10, distilled water, Ultra Turrax homogenizer) and homogenates were used for enzymatic analysis of AChE (Ellman et al., 1961) as described earlier (Bajgar et al., 2007d) The activity was expressed as µkat/kg wet weight tissue (microkatal/kilogram, measure of catalytical activity per body weight) or as a percentage of control values.

RESULTS

Toxicological

          Protective indexes, expressed as ratios of LD₅₀s, for combinations of atropine with the oxime reactivators showed that K048 seems to be very good antidote against tabun intoxication followed by obidoxime and then HI-6 (Table 1). However, the differences between the oximes were not statistically significant.

Biochemical

          In the brain parts studied, based on the AChE activity detected after tabun administration, showed the highest sensitivities were in the FC and PM areas; the NR appeared to be relatively resistant to inhibition. Following antidotal treatment, AChE activity was practically unchanged in the NR area, but in both the FC and PM areas an increase was observed, and was more marked for obidoxime and K048 treatments. The increase in AChE activity was the lowest for HI-6 (Table 2) for all brain areas.

Histochemical

          The changes of AChE activity in the frontal cortex (FC) are shown in figure 1 as quantitative histochemical evaluation. The numbers of stained pixels were noted for treated and untreated rats. The greatest AChE reactivation was observed in FC and PM areas. AChE activity is presented in Table 2. The AChE activity in the NR was relatively resistant and agreed with the resistance noted in the biochemical analysis. The correlation between results obtained by for tabun intoxication by histochemical and biochemical methods is presented in figure 2.

DISCUSSION

          In previous work, we demonstrated that the correlation between AChE activity in the brain areas detected by biochemical and histochemical methods was good, except for basal ganglia (Bajgar et al., 2007d). A similar lack of correlation was observed in present experiments for in the NR. Both structures (basal ganglia and NR) have relatively high AChE activities (Bajgar et al., 2007c; Gupta, 2002).  A possible reason for this lack of correlation may be that because the levels of the enzyme activity are so high, not all the AChE is able to interact with inhibitor.

          The results in this paper demonstrated a good correlation between histochemical and biochemical methods of AChE activity determined after antidotal treatment. When these results are expanded to include to our earlier observations with other nerve agents (Bajgar et al., 2007c,d,e), a very good relationship between histochemical  and biochemical results was obtained; this correlation is shown in figure 3.  How does the AChE activity compare with the protective indices achieved by the oxime treatment? Comparisons of results obtained by biochemical, histochemical and toxicological methods are shown in figure 4. Thus for all nerve agents, either the histochemical pr biochemical methods are valid measures of AChE reactivation

          As noted in Table 2, following treatment with reactivators, a small but significant AChE reactivation was observed in some brain structures. The AChE activity after reactivation correlated with toxicological data (survival/death of experimental animals). The FC and PM areas were the most sensitive structures. The PM area controls respiration (Sungur and Guven, 2001; Goswany et al., 1994; Bird et al., 2003) so that the special sensitivity of AChE in this structure is important for understanding the mechanism of death/survival following nerve agent poisoning.

          The oximes differed in their ability to reactivate AChE inhibited by tabun: HI-6 was not as effective in comparison with obidoxime and K048. Research to find a universal reactivator, applicable to all nerve agent treatments, needs to be continued and the activity in the PM and FC areas of the brain may be the more sensitive measure of effectiveness.

ACKNOWLEDGEMENTS

          Support of the Grant INTOX OPUOFVZ No 200603, MoD, Czech Republic, is gratefully acknowledged.

FIGURES

Fig. 1. Top: Microphotography of 20µm sections of the rat frontal cortex following tabun intoxication and its treatment. 1 - control, 2 - tabun, 3 – HI-6, 4 – obidoxime, 5 – K048.
Bottom: Quantitative evaluation of histochemical data. Density curves of microphotographs.

Fig. 2. : Comparison of results obtained by histochemical and biochemical methods for the intoxication with tabun. 

Fig. 3.: Summary of results obtained by histochemical and biochemical methods presented in this paper and in following experiments: intoxication with sarin, soman and VX (according to Bajgar et al., 2007d); intoxication with soman pretreated with huperzine A (according to Bajgar et al., 2007c); intoxication with VX and its derivatives (according to Bajgar et al., 2007e).

Fig. 4. Summary of results obtained by toxicological (protective index, PI), biochemical and histochemical methods.

TABLES

Table 1. Protective indexes for antidotal treatment following tabun intoxication

Table 2.: AChE activities expressed as percentage of control in different brain areas following tabun intoxication and its treatment determined using histochemical/biochemical methods in rats

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