TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
112, 104- 109 ( 1992)
Central Activity of Acetylcholinesterase
Oxime Reactivators
JOHNG.CLEMENT' Biomedical Defence Section, Defence Research Establishment St&e/d, Ralston, Alberta. Canada Received March 22, 199 1; accepted September 20, 199 1
sees them rapidly eliminated from the blood stream), access Central Activity of Acetylcholinesterase Oxime Reactivators. to the central compartment is severely restricted by the CLEMENT, J. G. (1992). Toxicol. Appl. Pharmacol. 112, blood-brain barrier (Hobbiger and Vojvodic, 1967; Firemark 104-109. et al., 1964; Ligtenstein and Kossen, 1983; Ligtenstein et al., The ability of various oximes to antagonize the win-induced 1988; Lundy et al., 1990). Thus, oximes generally reactivate hypothermia and reactivate phosphorylated acetylcholmesterase phosphorylated acetylcholinesterase in the peripheral but not was used as an indicator of the central activity of oximes. HI-6, in the central compartment. but neither toxogonin nor PAM Cl, antagonized satin-induced An oxime reactivator, HI-6 [ l-(((4-(aminocarbonyl)hypothermia and reactivated brain acetylcholinesterase, in par- pyridinio)methoxy)methyl)-2-((hydroxyimino)methyl)-pyticular hypothalamic acetylcholinesterase. The satin-induced tidinium dichloride], has emerged as an effective therapy, in hypothermia appears to be a muscarinic cholinergic action since combination with atropine, in the treatment of poisoning atropine was also an effective antagonist of satin-induced hyby the highly toxic nerve agents such as soman (pinacolyl pothermia. Neither HI-6 nor toxogonin antagonized oxotremethylphosphonofluoridate), sarin (isopropyl methylphosmorine-induced hypothermia, indicating that these oximes do phonofluoridate), tabun (ethyl N-dimethylphosphoramidonot possess central cholinolytic activity. The results demonstrated that HI-6 penetrated the blood-brain barrier in a suffi- cyanidate), and VX (O-ethyl 9[2-(diisopropylamino)cient concentration to produce a biochemical and physiological ethyllmethylphosphonothioate) in a variety of species action against satin poisoning. (Boskovic et al., 1984; Clement, 198 1, 1983; Hamilton and Lundy, 1989; Maksimovic et al., 1980). Similar to other oximes, HI-6 reactivated phosphorylated acetylcholinesterase primarily in the peripheral compartment (Clement, 1982; The primary locus of action of organophosphorus comLundy and Shih, 1983) and the blood-brain barrier severely pounds is the phosphorylation of the active site of the enzyme restricted the access of HI-6 to the central compartment acetylcholinesterase. This leads to an increase in the synaptic (Ligtenstein and Kossen, 1983; Ligtenstein et al., 1988; concentration of the neurotransmitter acetylcholine which Lundy et al., 1990). results in the overstimulation of cholinergic receptors and A consequence of organophosphate poisoning in rodents symptoms such as salivation, lacrimation, tremors, miosis, is a profound transient hypothermia, of central origin diarrhea, and, if the dose is great enough, respiratory diffi(Clement et al., 1989; Meeter et al., 1971). The purpose of culties and death due to anoxia. Organophosphate poisoning is treated using a combination of a cholinolytic, such as atro- this investigation was to determine the central activity of oximes in mice by investigating the oxime antagonism of pine, to overcome the excessive muscarinic receptor stimulation and an acetylcholinesterase oxime reactivator, such sarin-induced hypothermia and the relationship to reactivation of phosphorylated brain acetylcholinesterase. as either pyridine 2-aldoxime (PAM) or toxogonin, to repair the biochemical lesion by dephosphorylating the enzyme and METHODS returning the function of acetylcholinesterase to normal. Atropine (a lipophilic tertiary amine) is effective in counAnimds. Male CD-1 mice (25-30 g) obtained from Charles River Canada teracting the muscarinic receptor overstimulation in the Ltd. (St. Constant, Quebec) were used in this study. The animals were kept central and peripheral compartments. Due to the chemical in the vivarium at De.fence Research Establishment Suffield for at least 1 nature of the oximes (usually hydrophilic mono- or bispyrweek, following their arrival, prior to experimentation. The animals were idinium oximes) and their pharmacokinetic profile (which allowed access to food and water ad libitum. The room temperature was ’ Address for reprint requests: Defence Research Establishment Suffield, Box 4000, Medicine Hat, Alberta, Canada, TlB 8K6.
2 I-22’C with a 12-hr light-dark cycle with the lights on at 0700 hr. Recording of core temperature. Core temperature was monitored using a telemetry system obtained from Data Sciences Inc. (Roseville, MN) as
104
CENTRAL
ACTIVITY
OF OXIMES
105
Toxogontn
26340 f 465
PAM
26448
10
30
s 0
10
Atropine
26770
f
540
10
HI-6
B Toxogonin V PAM a Atropine 25l
627 *
0 colltrol 0
f
‘.
”
F(4,43)=8.59 p c 0.01
ANOVA
“’ 180
”
‘I’
” 360
Time
( min)
*
’
I” 540
“‘I
720
gboxisum.apg
FIG. 1. Effect of acetylcholinesterase reactivators or atropine on satin-induced hypothermia in mice. Sarin (130 &kg&) was administered (as indicated by the first arrow) followed by saline, HI-6 (132 pmol/kg,ip), PAM (132 nmol/kg,ip), toxogonin (132 rmol/kg,ip), or atropine (17.4 mg/kg,ip) 90 min later (as indicated by the second arrow). Each point represents the mean value. The standard error bars in this and the following figure were omitted for reasons of clarity. The asterisk indicates that the value is significantly different from the control group, p < 0.0 1.
described in Clement ef al. (1989). The mice were anesthetized with sodium pentobarbital (75 m&kg, ip). An abdominal incision was made and the telemetry transmitter was implanted in the peritoneal cavity. The mice were then allowed to recover for at least 1 week prior to use in an experimental situation. The telemetry transmitter was activated by bringing a magnet close to the abdomen which turned on the battery power. The weight of the telemetry transmitter was tared prior to recording the body weight of the mouse so that the animal was injected with the proper dose of the drug based on tissue weight of the animal. Mice were placed in individual cages and the core temperature was monitored by telemetry. Typically, the first three data points established a control baseline. The drug was administered immediately after the acquisition of the third data point. The data were acquired for a total of 720 min following satin administration at 30-min intervals and 280 min following oxotremorine at IO-min intervals. The entire time period, including the control interval, was then used in the calculation of the mean minimum temperature and area under the curve (AUC). Data analysis. The data were analyzed by one way analysis of variance (ANOVA) and where a significant overall effect was found, the group means were compared using the Scheffetest, which is a rigorous multiple comparison test. A p < 0.05 was considered significant. Tissue preparation. Mice were terminated by decapitation and exsanguination. The tissues (hypothalamus, cortex, and striatum) were removed, rinsed in 0.9% saline, blotted dry on filter paper, and weighed. The tissue was homogenized (lo-20 strokes in a glass-Teflon homogenizer) in a buffer (4’C) containing 1 M NaCI, 0.05 M MgClr, 0.01 M Tris, and 1% Triton X-
100, pH 7.4. The tissue concentration of the final homogenate was 10 mg wet wt of tissue/ml buffer. The homogenates were then centrifuged at 20,OOOg for 20 min at 4°C. Fresh, unfrozen tissue was used in all experiments. Acetylcholinesterase enzyme assay. Acetylcholinesterase activity was determined, at room temperature, in a microplate assayusing the method of Ellman et al. (1961). Each fraction contained ISO-OMPA (10 PM) to selectively inhibit pseudocholinesterase activity.
MATERIALS Sarin, PAM, toxogonin. and HI-6, prepared at Defence Research Establishment Suffield, were in excessof 99% pure. The 24-hr LDSO of sarin was in the range 170-l 80 &kg (SC)(Clement, J. G., unpublished observations). The following drugs were obtained from various commercial sources: acetylthiocholine (Sigma), oxotremorine (Aldrich), and ISO-OMPA (ICN Pharmaceuticals). All drugs were dissolved in physiological saline immediately prior to injection. The volume of injection was 1% of body weight in all cases.
RESULTS Administration of sarin (130 pg/kg,sc) produced a profound hypothermia in SOme mice (Fig. 1). It was previously reported (Clement, 199 1) that the hypothermic response following sarin ( 130 pg/kg) administration was variable. There
106
JOHN G. CLEMENT
TABLE 1 Effect of Oximes on Brain AcetylcholinesteraseActivity following Administration of Sarin” Acetylcholinesterase activity (nmol/ml/min) Hypothalamus % Control
Treatment group Untreated control Satin + saline Satin + HI-6 Satin + toxogonin Sarin + PAM ANOVA
124.2 + 22.lC 34.7 + 8.1 74.7 f 14.6d 42.7 +_ 4.0 41.8 + 13.8 F(4, 28) = 36.90 p < 0.01
100 28 60 34 34
Hippocampus
90
%
Reac b 45 9 8
Control 108.6 ?z 27.7 19.7 + 6.1 40.6 f 13.5 21.0 lr 2.0 20.0 + 11.6
100 18 37 19 18
F(4,27) = 33.57 p < 0.01
Cortex
%
%
Reac
Control
24 0 0
84.6 rt 6.2 + 19.1 + 8.0 + ll.O+
11.2 3.7 6.3’ 4.0 5.2
N
16 2 6
9 5 11 3 5f
F(4,28) = 148.48 p < 0.01
a Mice were injected with satin (130 &kg, sc) and separated into responders (based on the presence of hypothermia) 90 min after the injection of satin the mice were injected with saline, HI-6 (132 pmol/kg, ip), or toxogonin (132 rmol/kg, or saline administration the mice were terminated and acetylcholinesterase activity was determined. b % Reac = % reactivation as determined from the acetylcholinesterase activity as follows: (oxime) - (saline)/(control) ’ Mean + SD. All of the treatment groups were significantly different from the untreated control group. The following the satin + saline group. dp < 0.01. cp < 0.05. IN = 5 except for the hippocampus where N = 4.
was always a certain percentage of nonresponders, i.e., mice in which hypothermia did not occur. A similar observation was reported by Fernando et al. (1985) following satin administration to rats. Thus, in this study only mice which displayed a hypothermic response within 75-90 min following sarin administration were included in the study. Sarin hypothermia was rapid in onset, reaching a maximum between 90 and 120 min following administration. Core temperature gradually recovered to the control level over the next 460 min. The mice displayed various signs of poisoning such as tremors, salivation, piloerection, and diarrhea following administration of satin (130 &kg,sc). By 24 hr, the mice appeared to be totally recovered from the satin injection (a subjective observation). Administration of HI-6 ( 132 pmol/kg,ip) 90 min following satin administration resulted in a significant reversal of the satin-induced hypothermia (Fig. l), whereas an equimolar dose of other oximes, toxogonin and PAM, was ineffective in reversing the sarininduced hypothermia. Administration of the cholinolytic atropine ( 17.4 mg/kg,ip) also produced a significant reversal of the satin-induced hypothermia (p < 0.0 1). The antagonism of the sarin hypothermia was reflected more in the AUC data than in the minimum temperature data. Since the antagonists were administered at a time close to the maximum hypothermia, as determined from control experiments, the minimum temperatures would not be expected to be changed significantly and they were not. Sarin ( 130 pg/kg,sc) significantly reduced acetylcholinesterase activity in all of the brain areas examined; however, each area was not affected to the same degree (Table 1). The
100 7 23 9 13
% Reac
approximately 75 min later. At ip). Ninety minutes after oxime - (saline) X 100. were significantly different from
oxime-induced reactivation of sarin-inhibited brain (hypothalamus, cortex, and hippocampus) acetylcholinesterase was investigated. HI-6 reactivated (Table 1) &n-inhibited acetylcholinesterase in the hypothalamus and cortex but not in the hippocampus; the greatest reactivation occurred in the hypothalamus. Neither toxogonin nor PAM reactivated sarin-inhibited acetylcholinesterase in any of the brain regions examined. The in viva central cholinolytic activity of various oximes was investigated. Oxotremorine hypothermia is a central event presumably due to stimulation of muscarinic receptors in the preoptic anterior hypothalamus (Lomax and Jenden, 1966). Atropine is an effective inhibitor of oxotremorine hypothermia (Lomax and Jenden, 1966; Clement, J. G., unpublished observations). The cholinolytic activity of HI-6 and toxogonin was evaluated against the hypothermia induced by the muscarinic agonist oxotremorine. The dose of oxotremorine used in this experiment, 156 &kg, produced approximately 70% of the maximum hypothermia (as derived from the oxotremorine dose-response curve (Clement et al., 1989)). The results in Fig. 2 indicate that neither HI-6 ( 132 pmol/kg) nor toxogonin ( 132 pmol/kg) possesses cholinolytic activity versus oxotremorine-induced hypothermia. DISCUSSION
Numerous studies have indicated that acetylcholinesterase oxime reactivators either do (Bajgar et al., 1972; Hobbiger and Vojvodic, 1966, 1967; Clement, 1982; Aarseth and Barstad, 1968; Lundy and Shih, 1983; Pantelic and Maksimovic,
CENTRAL
0 0
ACTIVITY
107
OF OXIMES
0x0 Control HI-6 Toxogonin
V
I
Toxo
gee3 2665 9972
Et 313 i 303 f 345*
28.70 28.78 30.12
f f f
1.45 1.36 1.1%
7 6 8
_
Atrop 10614 f 116 35.22 i 1.66 6 ANOVA F(3.27)= 19.24 36.49 P< 0.01 0.01
Time
(min)
FIG. 2. Cholinolytic activity of either HI-6 or toxogonin vs oxotremorine-induced hypothermia. Oxotremorine (156 &kg,ip) was administered (at the first arrow) followed by saline, HI-6 (132 pmol/kg,ip), or toxogonin (132 pmol/kg,ip) 50 min later (as indicated by the second arrow). Atropine (2.2 mg/kg,ip), included for comparison purposes, was administered 5 min before oxotremorine. The asterisk indicates that the value is significantiy different from the control and the oxime treatment groups.
1982) or do not have a central action (Brown, 1960; Kewitz and Nachmansohn, 1957; Hobbiger, 1957; Heffron and Hobbiger, 1980; Loomis, 1963; Sterri et al., 1979; Boskovic et al., 1980; Mayer and Michalek, 197 1; Lundy and Shih, 1983). One of the signs of organophosphate poisoning in rodents is a profound hypothermia which lasts for a period of hours depending upon the dose. Cholinergic-induced hypothermia in rodents is thought to be the result of muscarinic receptor stimulation in the preoptic anterior hypothalamus (Lomax and Jenden, 1966). Organophosphate-induced hypothermia is the result of a lowering of the set point for heat release and decreased metabolism (Meeter et al., 1971). In the present study, HI-6 antagonism of the sarin-induced hypothermia may result from a lowering of the set point for heat release and decreased metabolism (Meeter et al., 197 1). In the present study, HI-6 antagonism of the sarin-induced hypothermia was the result of HI-6 reactivation of phosphorylated acetylcholinesterase, most likely in the hypothalamus. These results indicate that HI-6 has a central action; i.e., it is able to cross the blood brain-barrier in large enough concentrations to repair a biochemical lesion and evoke a physiological change. Previous investigators have found that HI-6 was effective in reactivating sarin- (Clement,
1982; Lundy and Shih, 1983) and VX-inhibited brain acetylcholinesterase. The results of the present study confirm those of Ligtenstein and Kossen (1983) who reported that small amounts of HI-6 did pass the blood-brain barrier. Neither toxogonin nor PAM antagonized &n-induced hypothermia or reactivated sarin-inhibited acetylcholinesterase in the central compartment. These results indicate that neither toxogonin nor PAM enter the central compartment in biochemically or physiologically significant quantities. The degree of reactivation of acetylcholinesterase produced by HI-6 was different in the various brain regions. Similar results were reported for toxogonin (Hobbiger and Vojvodic, 1966) and PAM (Hobbiger and Vojvodic, 1967) against paraoxon poisoning. Thus, if one is measuring total brain acetylcholinesterase activity to assess oxime-induced reactivation, a significant degree of reactivation may occur in a specific area of the brain; however, this may be masked by the lower degree of reactivation in other, larger regions of the brain. The blood-brain barrier is not complete, i.e., there are certain areas in the brain (choroid plexus, area postrema, neurohypophysis, fornix) which are more accessible to quaternary ammonium compounds than other areas. The results of this study suggest that there are regional differences in the
108
JOHN G. CLEMENT
penetration of oximes into various regions of the brain. The measurement of the degree of sarin inhibition of acetylcholinesterase activity in the various brain regions may be a more sensitive indicator of the penetration of the oxime into the CNS than the measurement of the compound itself in the tissue since one is not sure if the oxime is inside or outside the blood-brain barrier. An interesting result from this study was the speed at which the antagonists reversed the sarin-induced hypothermia. Neither HI-6 nor atropine reversed the sarin-induced hypothermia quickly; i.e., it took between 30 and 60 min for the antagonism to be expressed. This result suggests that it is not simply a matter that the overexcitation be stopped, e.g., by the occupation of the receptor or by the repair of the biochemical lesion, to produce the antagonism. The lag time may be a reflection of the time it takes for the intracellular changes, which the hypothermia produced, to be reversed. Also, the intracellular changes probably occur more slowly due to the state of hypothermia. The ability of the oximes to antagonize organophosphateinduced hypothermia was used by Meeter and associates as a test to determine the central activity of oximes (Meeter et al., 197 1). However, they did not correlate the action of the various oximes in antagonizing organophosphate-induced hypothermia directly to the ability of the oximes to reactivate acetylcholinesterase in the CNS. The present study clearly shows that there is a direct relationship between the ability of the compound to reactivate the acetylcholinesterase in the hypothalamus and the antagonism of organophosphate-induced hypothermia. On the basis of the known pharmacology of the organophosphate anticholinesterases, sarin inhibits the enzyme acetylcholinesterase, which produces an increase in the synaptic concentration of acetylcholine resulting in overstimulation of choline& receptors. The results of this study indicate that sarin-induced hypothermia is the result of the muscarinic receptor overstimulation, probably in the hypothalamus, the thermoregulatory area important in causing cholinergic-induced hypothermia (Lomax and Jenden, 1966). ACKNOWLEDGMENT The author thanks Ms. N. Erhardt for performing the acetylcholinesterase measurements.
REFERENCES Aarseth, P., and Barstad, J. A. B. (1968). Blood brain barrier permeability in various parts of the central nervous system. Arch. Int. Pharmacodyn. Ther. 176,434-442. Bajgar, J., Jakl, A., and Hrdina, V. (1972). The influence of obidoxime on acetylcholinesterase activity in different parts of the mouse brain following
isopropylmethyl phosphonofluoridate intoxication. Eur. J. Pharmacol. 19, 199-202. Boskovic, B., Kovacevic, V., and Jovanovic, D. (1984). PAM-2 Cl, HI-6 and HGG-I 2 in soman and tabun poisoning. Fundam. Appl. Toxicol. 4, s106-~115. Boskovic, B., Tadic, V., and Kusic, R. (1980). Reactivating and protective effects of pro-2-PAM in mice poisoned with paraoxon. Toxicol. Appl. Pharmacol. 55,32-36. Brown, R. V. (1960). The effects of intracistemal sarin and pyridine-2-a]doxime methyl methanesulphonate in anaesthetized dogs. Br. J. Pharmacol. 15, 170-174. Clement, J. G. (198 I). Toxicology and pharmacology of bispyridinium oximes-Insight into the mechanism of action vs soman poisoning in vivo. Fundam. Appl. Toxicol. 1, 193-202. Clement, J. G. (1982). HI-6: Reactivation of central and peripheral acetylcholinesterase following inhibition by soman, sarin and tabun in vivo in the rat. Biochem. Pharmacol. 31, 1283-1287. Clement, J. G. (1983). Efficacy of mono- and bis-pyridinium oximes versus soman, sarin, and tabun poisoning in mice. Fundam. Appl. Toxicol. 3, 533-535. Clement, J. G. (199 I). Variability of sarin-induced hypothermia in mice: Investigation into the incidence and mechanism. Biochem. Pharmacol. 42, 1316-1318. Clement, J. G., Mills, P., and Brockway, B. (1989). Use of telemetry to record body temperature and activity in mice. J. Pharmacol. Methods 21, 129-140. Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (196 1). A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. I, 88-95. Fernando, J. C. R., Lim, D. K., Hoskins, B., and Ho, I. K. (1985). Variability of neurotoxicity of and lack of tolerance to the anticholinesterase soman and sarin in the rat. Res. Commun. Chem. Pathol. Pharmacol. 4!3,415430. Firemark, H., Barlow, C. F., and Roth, L. J. (1964). The penetration of 2PAM-Cl4 into brain and the effect of cholinesterase inhibitors on its transport. J. Pharmacoi. Exp. Ther. 145,252-265. Hamilton, M. G., and Lundy, P. M. (1989). HI-6 therapy of soman and tabun poisoning in primates and rodents. Arch. Toxicol. 63, 144- 149. Heffron, P. F., and Hobbiger, F. (1980). Does reactivation of phosphorylated acetylcholinesterase (ache) in the brain enhance the antidotal actions of pyridinium aldoximes? Br. J. Pharmacol. 69,3 13-3 14. Hobbiger, F. (1957). Protection against the lethal effectsof organophosphates by pytidine-2-aldoxime methiodide. Br. J. Pharmacol. 12,438-446. Hobbiger, F., and Vojvodic, V. (1966). The reactivating and antidotal actions of N,N’-trimethylenebis(pyridinium-4-aldoxime) (TMB-4) and N,N’-oxydimethylenebis(pyridinium-4-aldoxime) (toxogonin), with particular reference to their effecton phosphorylated acetylcholinesterase in the brain. Biochem. Pharmacol. 15, 1677-1690. Hobbiger, F., and Vojvodic, V. (1967). The reactivation by pyridinium aldoximes of phosphorylated acetylcholinesterase in the central nervous system. Biochem. Pharmacol. 16,455-462. Kewitz. H., and Nachmansohn, D. (1957). A specific antidote against lethal alkyl phosphate intoxication. IV. Effects in the brain. Arch. Biochem. Biophys. 66, 27 l-283. Ligtenstein, D. A., and Kossen, S. P. (1983). Kinetic profile in blood and brain of the cholinesterase reactivating oxime HI-6 after intravenous administration to the rat. Toxicol. Appl. Pharmacol. 71, 177-183. Ligtenstein, D. A., Moes, G. W. H., and Kossen, S. P. (1988). In vivo distribution of organophosphate antidotes: Autoradiography of [14C]HI-6 in the rat. Toxicol. Appl. Pharmacol. 92, 324-329.
CENTRAL
ACTIVITY
Lomax, P., and Jenden, D. J. (1966). Hypothermia following systematic and intracerebral injection of oxotremorine in the rat. Int. J. Neuropharmacol. 5,353-359. Loomis, T. A. (1963). Distribution and excretion of pyridine-2-aldoxime methiodide (PAM); atropine and PAM in sarin poisoning. Toxicol. Appl. Pharmacol. 5,489-499. Lundy, P. M., Hand, B. T.. Broxup, B. R., Yipchuck, G., and Hamilton, M. G. (1990). Distribution ofthe bispyridinium oxime [i4C]HI-6 in male and female rats. Arch. Toxicol. 64, 377-382. Lundy, P. M., and Shih, T. M. (1983). Examination of the role of central cholinergic mechanisms in the therapeutic effects of HI-6 in organophosphate poisoning. J. Neurochem. 40, 1321-1328. Maksimovic, M., Boskovic, B., Radovic, L., Tadic, V., Deljac, V., and Binenfeld. Z. ( 1980). Antidotal effectsof bis-pyridinium-2-monooxime car-
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bony1 derivatives in intoxications with highly toxic organophosphorus compounds. Acta Pharm. Jugosl. 30, 15 l-160. Mayer, O., and Michalek, H. (197 1). Effects of DFP and obidoxime on brain acetylcholine levels and on brain and peripheral cholinesterases. B&hem. Pharmacol. 20,3029-3037. Meeter, E., Wolthuis, 0. L., and Vanbenthem, R. M. J. (1971). The anticholinesterase hypothermia in the rat: Its practical application in the study of the central effectivenessof oximes. Bull. WHO 44, 25 l-257. Pantelic, D.. and Maksimovic, M. (1982). Effect of HI-6 on rat brain acetylcholinesterase inhibited by soman and VX in vivo. Acta Pharm. Jugosl. 32, 119-123. Sterri, S. H., Rognerud, B., Fiskum, S. E., and Lyngaas, S. (I 979). Effect of toxogonin and P2S on the toxicity of carbamates and organophosphorus compounds. Acta Pharmacol. Toxicol. 45, 9-15.
AND
APPLIED
PHARMACOLOGY
112, 104- 109 ( 1992)
Central Activity of Acetylcholinesterase
Oxime Reactivators
JOHNG.CLEMENT' Biomedical Defence Section, Defence Research Establishment St&e/d, Ralston, Alberta. Canada Received March 22, 199 1; accepted September 20, 199 1
sees them rapidly eliminated from the blood stream), access Central Activity of Acetylcholinesterase Oxime Reactivators. to the central compartment is severely restricted by the CLEMENT, J. G. (1992). Toxicol. Appl. Pharmacol. 112, blood-brain barrier (Hobbiger and Vojvodic, 1967; Firemark 104-109. et al., 1964; Ligtenstein and Kossen, 1983; Ligtenstein et al., The ability of various oximes to antagonize the win-induced 1988; Lundy et al., 1990). Thus, oximes generally reactivate hypothermia and reactivate phosphorylated acetylcholmesterase phosphorylated acetylcholinesterase in the peripheral but not was used as an indicator of the central activity of oximes. HI-6, in the central compartment. but neither toxogonin nor PAM Cl, antagonized satin-induced An oxime reactivator, HI-6 [ l-(((4-(aminocarbonyl)hypothermia and reactivated brain acetylcholinesterase, in par- pyridinio)methoxy)methyl)-2-((hydroxyimino)methyl)-pyticular hypothalamic acetylcholinesterase. The satin-induced tidinium dichloride], has emerged as an effective therapy, in hypothermia appears to be a muscarinic cholinergic action since combination with atropine, in the treatment of poisoning atropine was also an effective antagonist of satin-induced hyby the highly toxic nerve agents such as soman (pinacolyl pothermia. Neither HI-6 nor toxogonin antagonized oxotremethylphosphonofluoridate), sarin (isopropyl methylphosmorine-induced hypothermia, indicating that these oximes do phonofluoridate), tabun (ethyl N-dimethylphosphoramidonot possess central cholinolytic activity. The results demonstrated that HI-6 penetrated the blood-brain barrier in a suffi- cyanidate), and VX (O-ethyl 9[2-(diisopropylamino)cient concentration to produce a biochemical and physiological ethyllmethylphosphonothioate) in a variety of species action against satin poisoning. (Boskovic et al., 1984; Clement, 198 1, 1983; Hamilton and Lundy, 1989; Maksimovic et al., 1980). Similar to other oximes, HI-6 reactivated phosphorylated acetylcholinesterase primarily in the peripheral compartment (Clement, 1982; The primary locus of action of organophosphorus comLundy and Shih, 1983) and the blood-brain barrier severely pounds is the phosphorylation of the active site of the enzyme restricted the access of HI-6 to the central compartment acetylcholinesterase. This leads to an increase in the synaptic (Ligtenstein and Kossen, 1983; Ligtenstein et al., 1988; concentration of the neurotransmitter acetylcholine which Lundy et al., 1990). results in the overstimulation of cholinergic receptors and A consequence of organophosphate poisoning in rodents symptoms such as salivation, lacrimation, tremors, miosis, is a profound transient hypothermia, of central origin diarrhea, and, if the dose is great enough, respiratory diffi(Clement et al., 1989; Meeter et al., 1971). The purpose of culties and death due to anoxia. Organophosphate poisoning is treated using a combination of a cholinolytic, such as atro- this investigation was to determine the central activity of oximes in mice by investigating the oxime antagonism of pine, to overcome the excessive muscarinic receptor stimulation and an acetylcholinesterase oxime reactivator, such sarin-induced hypothermia and the relationship to reactivation of phosphorylated brain acetylcholinesterase. as either pyridine 2-aldoxime (PAM) or toxogonin, to repair the biochemical lesion by dephosphorylating the enzyme and METHODS returning the function of acetylcholinesterase to normal. Atropine (a lipophilic tertiary amine) is effective in counAnimds. Male CD-1 mice (25-30 g) obtained from Charles River Canada teracting the muscarinic receptor overstimulation in the Ltd. (St. Constant, Quebec) were used in this study. The animals were kept central and peripheral compartments. Due to the chemical in the vivarium at De.fence Research Establishment Suffield for at least 1 nature of the oximes (usually hydrophilic mono- or bispyrweek, following their arrival, prior to experimentation. The animals were idinium oximes) and their pharmacokinetic profile (which allowed access to food and water ad libitum. The room temperature was ’ Address for reprint requests: Defence Research Establishment Suffield, Box 4000, Medicine Hat, Alberta, Canada, TlB 8K6.
2 I-22’C with a 12-hr light-dark cycle with the lights on at 0700 hr. Recording of core temperature. Core temperature was monitored using a telemetry system obtained from Data Sciences Inc. (Roseville, MN) as
104
CENTRAL
ACTIVITY
OF OXIMES
105
Toxogontn
26340 f 465
PAM
26448
10
30
s 0
10
Atropine
26770
f
540
10
HI-6
B Toxogonin V PAM a Atropine 25l
627 *
0 colltrol 0
f
‘.
”
F(4,43)=8.59 p c 0.01
ANOVA
“’ 180
”
‘I’
” 360
Time
( min)
*
’
I” 540
“‘I
720
gboxisum.apg
FIG. 1. Effect of acetylcholinesterase reactivators or atropine on satin-induced hypothermia in mice. Sarin (130 &kg&) was administered (as indicated by the first arrow) followed by saline, HI-6 (132 pmol/kg,ip), PAM (132 nmol/kg,ip), toxogonin (132 rmol/kg,ip), or atropine (17.4 mg/kg,ip) 90 min later (as indicated by the second arrow). Each point represents the mean value. The standard error bars in this and the following figure were omitted for reasons of clarity. The asterisk indicates that the value is significantly different from the control group, p < 0.0 1.
described in Clement ef al. (1989). The mice were anesthetized with sodium pentobarbital (75 m&kg, ip). An abdominal incision was made and the telemetry transmitter was implanted in the peritoneal cavity. The mice were then allowed to recover for at least 1 week prior to use in an experimental situation. The telemetry transmitter was activated by bringing a magnet close to the abdomen which turned on the battery power. The weight of the telemetry transmitter was tared prior to recording the body weight of the mouse so that the animal was injected with the proper dose of the drug based on tissue weight of the animal. Mice were placed in individual cages and the core temperature was monitored by telemetry. Typically, the first three data points established a control baseline. The drug was administered immediately after the acquisition of the third data point. The data were acquired for a total of 720 min following satin administration at 30-min intervals and 280 min following oxotremorine at IO-min intervals. The entire time period, including the control interval, was then used in the calculation of the mean minimum temperature and area under the curve (AUC). Data analysis. The data were analyzed by one way analysis of variance (ANOVA) and where a significant overall effect was found, the group means were compared using the Scheffetest, which is a rigorous multiple comparison test. A p < 0.05 was considered significant. Tissue preparation. Mice were terminated by decapitation and exsanguination. The tissues (hypothalamus, cortex, and striatum) were removed, rinsed in 0.9% saline, blotted dry on filter paper, and weighed. The tissue was homogenized (lo-20 strokes in a glass-Teflon homogenizer) in a buffer (4’C) containing 1 M NaCI, 0.05 M MgClr, 0.01 M Tris, and 1% Triton X-
100, pH 7.4. The tissue concentration of the final homogenate was 10 mg wet wt of tissue/ml buffer. The homogenates were then centrifuged at 20,OOOg for 20 min at 4°C. Fresh, unfrozen tissue was used in all experiments. Acetylcholinesterase enzyme assay. Acetylcholinesterase activity was determined, at room temperature, in a microplate assayusing the method of Ellman et al. (1961). Each fraction contained ISO-OMPA (10 PM) to selectively inhibit pseudocholinesterase activity.
MATERIALS Sarin, PAM, toxogonin. and HI-6, prepared at Defence Research Establishment Suffield, were in excessof 99% pure. The 24-hr LDSO of sarin was in the range 170-l 80 &kg (SC)(Clement, J. G., unpublished observations). The following drugs were obtained from various commercial sources: acetylthiocholine (Sigma), oxotremorine (Aldrich), and ISO-OMPA (ICN Pharmaceuticals). All drugs were dissolved in physiological saline immediately prior to injection. The volume of injection was 1% of body weight in all cases.
RESULTS Administration of sarin (130 pg/kg,sc) produced a profound hypothermia in SOme mice (Fig. 1). It was previously reported (Clement, 199 1) that the hypothermic response following sarin ( 130 pg/kg) administration was variable. There
106
JOHN G. CLEMENT
TABLE 1 Effect of Oximes on Brain AcetylcholinesteraseActivity following Administration of Sarin” Acetylcholinesterase activity (nmol/ml/min) Hypothalamus % Control
Treatment group Untreated control Satin + saline Satin + HI-6 Satin + toxogonin Sarin + PAM ANOVA
124.2 + 22.lC 34.7 + 8.1 74.7 f 14.6d 42.7 +_ 4.0 41.8 + 13.8 F(4, 28) = 36.90 p < 0.01
100 28 60 34 34
Hippocampus
90
%
Reac b 45 9 8
Control 108.6 ?z 27.7 19.7 + 6.1 40.6 f 13.5 21.0 lr 2.0 20.0 + 11.6
100 18 37 19 18
F(4,27) = 33.57 p < 0.01
Cortex
%
%
Reac
Control
24 0 0
84.6 rt 6.2 + 19.1 + 8.0 + ll.O+
11.2 3.7 6.3’ 4.0 5.2
N
16 2 6
9 5 11 3 5f
F(4,28) = 148.48 p < 0.01
a Mice were injected with satin (130 &kg, sc) and separated into responders (based on the presence of hypothermia) 90 min after the injection of satin the mice were injected with saline, HI-6 (132 pmol/kg, ip), or toxogonin (132 rmol/kg, or saline administration the mice were terminated and acetylcholinesterase activity was determined. b % Reac = % reactivation as determined from the acetylcholinesterase activity as follows: (oxime) - (saline)/(control) ’ Mean + SD. All of the treatment groups were significantly different from the untreated control group. The following the satin + saline group. dp < 0.01. cp < 0.05. IN = 5 except for the hippocampus where N = 4.
was always a certain percentage of nonresponders, i.e., mice in which hypothermia did not occur. A similar observation was reported by Fernando et al. (1985) following satin administration to rats. Thus, in this study only mice which displayed a hypothermic response within 75-90 min following sarin administration were included in the study. Sarin hypothermia was rapid in onset, reaching a maximum between 90 and 120 min following administration. Core temperature gradually recovered to the control level over the next 460 min. The mice displayed various signs of poisoning such as tremors, salivation, piloerection, and diarrhea following administration of satin (130 &kg,sc). By 24 hr, the mice appeared to be totally recovered from the satin injection (a subjective observation). Administration of HI-6 ( 132 pmol/kg,ip) 90 min following satin administration resulted in a significant reversal of the satin-induced hypothermia (Fig. l), whereas an equimolar dose of other oximes, toxogonin and PAM, was ineffective in reversing the sarininduced hypothermia. Administration of the cholinolytic atropine ( 17.4 mg/kg,ip) also produced a significant reversal of the satin-induced hypothermia (p < 0.0 1). The antagonism of the sarin hypothermia was reflected more in the AUC data than in the minimum temperature data. Since the antagonists were administered at a time close to the maximum hypothermia, as determined from control experiments, the minimum temperatures would not be expected to be changed significantly and they were not. Sarin ( 130 pg/kg,sc) significantly reduced acetylcholinesterase activity in all of the brain areas examined; however, each area was not affected to the same degree (Table 1). The
100 7 23 9 13
% Reac
approximately 75 min later. At ip). Ninety minutes after oxime - (saline) X 100. were significantly different from
oxime-induced reactivation of sarin-inhibited brain (hypothalamus, cortex, and hippocampus) acetylcholinesterase was investigated. HI-6 reactivated (Table 1) &n-inhibited acetylcholinesterase in the hypothalamus and cortex but not in the hippocampus; the greatest reactivation occurred in the hypothalamus. Neither toxogonin nor PAM reactivated sarin-inhibited acetylcholinesterase in any of the brain regions examined. The in viva central cholinolytic activity of various oximes was investigated. Oxotremorine hypothermia is a central event presumably due to stimulation of muscarinic receptors in the preoptic anterior hypothalamus (Lomax and Jenden, 1966). Atropine is an effective inhibitor of oxotremorine hypothermia (Lomax and Jenden, 1966; Clement, J. G., unpublished observations). The cholinolytic activity of HI-6 and toxogonin was evaluated against the hypothermia induced by the muscarinic agonist oxotremorine. The dose of oxotremorine used in this experiment, 156 &kg, produced approximately 70% of the maximum hypothermia (as derived from the oxotremorine dose-response curve (Clement et al., 1989)). The results in Fig. 2 indicate that neither HI-6 ( 132 pmol/kg) nor toxogonin ( 132 pmol/kg) possesses cholinolytic activity versus oxotremorine-induced hypothermia. DISCUSSION
Numerous studies have indicated that acetylcholinesterase oxime reactivators either do (Bajgar et al., 1972; Hobbiger and Vojvodic, 1966, 1967; Clement, 1982; Aarseth and Barstad, 1968; Lundy and Shih, 1983; Pantelic and Maksimovic,
CENTRAL
0 0
ACTIVITY
107
OF OXIMES
0x0 Control HI-6 Toxogonin
V
I
Toxo
gee3 2665 9972
Et 313 i 303 f 345*
28.70 28.78 30.12
f f f
1.45 1.36 1.1%
7 6 8
_
Atrop 10614 f 116 35.22 i 1.66 6 ANOVA F(3.27)= 19.24 36.49 P< 0.01 0.01
Time
(min)
FIG. 2. Cholinolytic activity of either HI-6 or toxogonin vs oxotremorine-induced hypothermia. Oxotremorine (156 &kg,ip) was administered (at the first arrow) followed by saline, HI-6 (132 pmol/kg,ip), or toxogonin (132 pmol/kg,ip) 50 min later (as indicated by the second arrow). Atropine (2.2 mg/kg,ip), included for comparison purposes, was administered 5 min before oxotremorine. The asterisk indicates that the value is significantiy different from the control and the oxime treatment groups.
1982) or do not have a central action (Brown, 1960; Kewitz and Nachmansohn, 1957; Hobbiger, 1957; Heffron and Hobbiger, 1980; Loomis, 1963; Sterri et al., 1979; Boskovic et al., 1980; Mayer and Michalek, 197 1; Lundy and Shih, 1983). One of the signs of organophosphate poisoning in rodents is a profound hypothermia which lasts for a period of hours depending upon the dose. Cholinergic-induced hypothermia in rodents is thought to be the result of muscarinic receptor stimulation in the preoptic anterior hypothalamus (Lomax and Jenden, 1966). Organophosphate-induced hypothermia is the result of a lowering of the set point for heat release and decreased metabolism (Meeter et al., 1971). In the present study, HI-6 antagonism of the sarin-induced hypothermia may result from a lowering of the set point for heat release and decreased metabolism (Meeter et al., 197 1). In the present study, HI-6 antagonism of the sarin-induced hypothermia was the result of HI-6 reactivation of phosphorylated acetylcholinesterase, most likely in the hypothalamus. These results indicate that HI-6 has a central action; i.e., it is able to cross the blood brain-barrier in large enough concentrations to repair a biochemical lesion and evoke a physiological change. Previous investigators have found that HI-6 was effective in reactivating sarin- (Clement,
1982; Lundy and Shih, 1983) and VX-inhibited brain acetylcholinesterase. The results of the present study confirm those of Ligtenstein and Kossen (1983) who reported that small amounts of HI-6 did pass the blood-brain barrier. Neither toxogonin nor PAM antagonized &n-induced hypothermia or reactivated sarin-inhibited acetylcholinesterase in the central compartment. These results indicate that neither toxogonin nor PAM enter the central compartment in biochemically or physiologically significant quantities. The degree of reactivation of acetylcholinesterase produced by HI-6 was different in the various brain regions. Similar results were reported for toxogonin (Hobbiger and Vojvodic, 1966) and PAM (Hobbiger and Vojvodic, 1967) against paraoxon poisoning. Thus, if one is measuring total brain acetylcholinesterase activity to assess oxime-induced reactivation, a significant degree of reactivation may occur in a specific area of the brain; however, this may be masked by the lower degree of reactivation in other, larger regions of the brain. The blood-brain barrier is not complete, i.e., there are certain areas in the brain (choroid plexus, area postrema, neurohypophysis, fornix) which are more accessible to quaternary ammonium compounds than other areas. The results of this study suggest that there are regional differences in the
108
JOHN G. CLEMENT
penetration of oximes into various regions of the brain. The measurement of the degree of sarin inhibition of acetylcholinesterase activity in the various brain regions may be a more sensitive indicator of the penetration of the oxime into the CNS than the measurement of the compound itself in the tissue since one is not sure if the oxime is inside or outside the blood-brain barrier. An interesting result from this study was the speed at which the antagonists reversed the sarin-induced hypothermia. Neither HI-6 nor atropine reversed the sarin-induced hypothermia quickly; i.e., it took between 30 and 60 min for the antagonism to be expressed. This result suggests that it is not simply a matter that the overexcitation be stopped, e.g., by the occupation of the receptor or by the repair of the biochemical lesion, to produce the antagonism. The lag time may be a reflection of the time it takes for the intracellular changes, which the hypothermia produced, to be reversed. Also, the intracellular changes probably occur more slowly due to the state of hypothermia. The ability of the oximes to antagonize organophosphateinduced hypothermia was used by Meeter and associates as a test to determine the central activity of oximes (Meeter et al., 197 1). However, they did not correlate the action of the various oximes in antagonizing organophosphate-induced hypothermia directly to the ability of the oximes to reactivate acetylcholinesterase in the CNS. The present study clearly shows that there is a direct relationship between the ability of the compound to reactivate the acetylcholinesterase in the hypothalamus and the antagonism of organophosphate-induced hypothermia. On the basis of the known pharmacology of the organophosphate anticholinesterases, sarin inhibits the enzyme acetylcholinesterase, which produces an increase in the synaptic concentration of acetylcholine resulting in overstimulation of choline& receptors. The results of this study indicate that sarin-induced hypothermia is the result of the muscarinic receptor overstimulation, probably in the hypothalamus, the thermoregulatory area important in causing cholinergic-induced hypothermia (Lomax and Jenden, 1966). ACKNOWLEDGMENT The author thanks Ms. N. Erhardt for performing the acetylcholinesterase measurements.
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CENTRAL
ACTIVITY
Lomax, P., and Jenden, D. J. (1966). Hypothermia following systematic and intracerebral injection of oxotremorine in the rat. Int. J. Neuropharmacol. 5,353-359. Loomis, T. A. (1963). Distribution and excretion of pyridine-2-aldoxime methiodide (PAM); atropine and PAM in sarin poisoning. Toxicol. Appl. Pharmacol. 5,489-499. Lundy, P. M., Hand, B. T.. Broxup, B. R., Yipchuck, G., and Hamilton, M. G. (1990). Distribution ofthe bispyridinium oxime [i4C]HI-6 in male and female rats. Arch. Toxicol. 64, 377-382. Lundy, P. M., and Shih, T. M. (1983). Examination of the role of central cholinergic mechanisms in the therapeutic effects of HI-6 in organophosphate poisoning. J. Neurochem. 40, 1321-1328. Maksimovic, M., Boskovic, B., Radovic, L., Tadic, V., Deljac, V., and Binenfeld. Z. ( 1980). Antidotal effectsof bis-pyridinium-2-monooxime car-
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bony1 derivatives in intoxications with highly toxic organophosphorus compounds. Acta Pharm. Jugosl. 30, 15 l-160. Mayer, O., and Michalek, H. (197 1). Effects of DFP and obidoxime on brain acetylcholine levels and on brain and peripheral cholinesterases. B&hem. Pharmacol. 20,3029-3037. Meeter, E., Wolthuis, 0. L., and Vanbenthem, R. M. J. (1971). The anticholinesterase hypothermia in the rat: Its practical application in the study of the central effectivenessof oximes. Bull. WHO 44, 25 l-257. Pantelic, D.. and Maksimovic, M. (1982). Effect of HI-6 on rat brain acetylcholinesterase inhibited by soman and VX in vivo. Acta Pharm. Jugosl. 32, 119-123. Sterri, S. H., Rognerud, B., Fiskum, S. E., and Lyngaas, S. (I 979). Effect of toxogonin and P2S on the toxicity of carbamates and organophosphorus compounds. Acta Pharmacol. Toxicol. 45, 9-15.
