- Email: [email protected]
Antidote effect of sodium fluoride against organophosphate poisoning in mice
Life Sciences, Vol. 32, pp. 1803-1810 Printed in the U.S.A.
Pergamon Press
ANTIDOTE EFFECT OF SODIUM FLUORIDE AGAINST ORGANOPHOSPHATE POISONING IN MICE1 J.G. Clement*2 and Marg Filbert?3 * Biomedical Section, Defence Research Establishment Suffield, Ralston, Alberta, Canada., TOJ 2N0 ? Neurophysiology Branch, U.S. Army Institute of Chemical Defence, Aberdeen Proving Ground, Maryland, U.S.A. (Received
in final form January 19, 1983)
Summary Pretreatment of mice with atropine (17.4 mg/kg) + NaF (5 or 15 mg/kg) had a significant antidotal effect over atropine alone against the lethality produced by soman and sarin. Atropine + NaF (15 mg/kg) was effective against tabun, whereas the lower dose of NaF was not. An effect of NaF on organophosphate inhibited acetylcholinesterase could not account for the antidotal action of NaF. NaF had no effect on liver somanase activity but inhibited aliesterase activity. Aliesterase activity in NaF pretreated somanpoisoned mice was significantly (p < 0.05) higher than in those receiving atropine alone. In CBDP-pretreated mice NaF did not significantly attenuate the toxicity of soman. It is hypothesized that the antidotal effect of NaF versus organophosphate poisoning is due to its antidesensitizing action at nicotinic receptors in the neuromuscular junction and/ or sympathetic ganglia in addition to the proposed increased hydrolysis of sarin and direct detoxification of tabun. Desensitization of cholinergic receptors at the endplate usually occurs following prolonged exposure to high concentrations of acetylcholine (1). Anticholinesterase drugs which increase the synaptic concentration of acetylcholine were found to accelerate significantly the time to desensitize receptors (2). In addition, these authors found that sodium fluoride (NaF) antagonized the acceleration of desensitization induced by anticholinesterase drugs. Recently Barritt et al. (3) reported that in vitro NaF specifically decreased the binding of an agonist to the muscarinic receptor. NaF had no effect on the binding of antagonists to the muscarinic receptor (3,4). NaF, when combined with atropine, was an effective prophylactic or therapeutic treatment for organophosphate poisoning (5). Previous investigators have reported that NaF was capable of reactivating acetylcholinesterase inhibited by sarin (5,6) and tabun (6) but not soman (6) or TEPP (7). The object of this study was to determine the extent and nature of the antidotal properties of NaF when combined with atropine against the lethality of soman, sarin and tabun in mice.
Suffield
Report No. 318
To whom all correspondence
and reprint requests should be sent
The opinions or assertations contained herein are the private views of the author and are not construed as reflecting views of the Department of the Army or the Department of Defence, U.S.A. 0024-3205/83/161803-08$03,00/O
1804
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
Methods Toxicology Male mice (CD-l@ , 25 - 30 g) obtained from Canadian Breeding Farm and Laboratories Ltd., St. Constant, Quebec, Canada were used in this study. The animals were acclimatized for at least one week in our animal facilities prior to use. The animals were usually fasted (but had free access to water) for 18 hr prior to the toxicological experiments. Following the injections the animals were given food and water ad libitum. The 24 hr LD50 values were calculated by probit analysis using the procedure of Miller and Tainter (8). All drugs were injected in a volume of 1% of body weight. Soman was injected subcutaneously (s.c.) in the back of the neck 5 min after an intraperitoneal (i.p.) injection of atropine or a solution of atropine + NaF. Acetylcholinesterase
Determinations
Acetylcholinesterase activity was determined according to the procedure of Siakotos et al. (9) using 14C-labelled acetylcholine iodide as the substrate. Mice were decapitated and exsanguinated. The whole brain, minus the cerebellum, was rinsed in 0.9% saline, blotted dry on filter paper and homogenized in enough 0.1 M phosphate buffer (pH 7.4) + 0.4 M sucrose to give a 1Vo (w/v) homogenate. The incubation time was 10 min. Whole blood was collected in a plastic beaker containing heparin. The blood was taken up into a syringe and stored in an ice water bath. Acetylcholinesterase activity was determined within 2 hrs of taking the samples. The time of incubation with substrate was 5 min for blood and 10 min for brain. Somanase Determinations Male CD-l mice were sacrificed by decapitation and exsanguinated. The liver was excised, placed in ice-cold saline, blotted dry on filter paper and weighed. A 10% w/v homogenate was prepared in 0.9% saline using a glass-teflon homogenizer. Somanase activity was assessed by the pH-stat method using soman (1.1 mM) as the substrate. Soman (10 mL) dissolved in 0.9% saline was placed in a vessel and the pH titrated automatically to pH 7.2 at 23°C using a Radiometer titragraph. Then 400 PL of the 10% liver homogenate was added to start the reaction and the amount of 0.01 N NaOH titrated over a 5 min period was recorded. The reaction was linear over this time period. NaF was added to some reactions prior to the addition of the homogenate. All solutions were made up using COzfree solutions. In addition, the reaction vessel was purged with nitrogen. Aliesterase
Determinations
Aliesterase activity was assessed by the pH-stat method using tributyrin as the substrate. Tributyrin (10 mL of 0.2% solution) in C02-free 0.9% saline was added to the reaction vessel and titrated to pH 7.9 with 0.01 N NaOH by a Radiometer titragraph. Either 50 I.IL of serum or 25 PL of 10% w/v liver homogenate was added to start the reaction. The homogenate was prepared using COz-free 0.9% saline. The addition of 0.01 N NaOH from the first to fifth min was used in determining the reaction rate. The reaction was linear over this time period. Materials The following drugs were used in this study: NaF (Fisher); atropine sulfate (J.T. Baker); 14C-acetylcholine iodide (New England Nuclear; S.A. = 4 mCi/mmol). Soman, sarin, tabun and 2-( o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP) were synthesized by the Organic Chemistry Group, Defence Research Establishment Suffield. All drugs were dissolved in 0.9% saline immediately before use except CBDP which was dissolved in DMSO. Statistically significant differences were determined using the Students ‘t’ test or the potency ratio (IO).
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
1805
Results In CD@ -1 mice NaF had a 24 hr LDso value of 55.6 mg/kg confidence limits) following i.p. administration in 0.9% NaCl.
(42.1 to 68.3; 95%
The results in Table I show that compared to those obtained with atropine alone NaF (5 mg/kg) plus atropine increased the LDso value significantly (p < 0.05) for soman and sarin but not tabun whereas, NaF (15 mg/kg) significantly (p < 0.05) increased the LDso value for all three organophosphate anticholinesterases. Increasing the dose of NaF to 30 mg/kg decreased the protective effect to that obtained with atropine alone. TABLE I Effect of NaF on the LD50 Value for Soman, Sarin and Tabun in Mice
Treatment
Sarin
Soman LD50
-
114 (104-
Atropine (AT)b
P.F.
LD50
P.F.
LD50
P.F.
-
169 (162 - 176)
-
290 (279 - 311)
-
123)a
1.42c
214 (199 - 231)
1.27
323 (313 - 330)
1.11
(5 mg) A+T 202 (186 - 231)d
1.77
330 (282 - 375)d
1.95
339 (305 - 361)
1.17
NaF (15 mg) LT 208 (194 - 228)d
1.82
444 (338 - 586)d
2.63
411 (393 - 437)d
1.42
NaF
162 (151 - 176)
Tabun
NaF (30 mg) 165 (131 - 203)
1.45
-
-
-
-
LT LD5o value in pg/kg with the 95% confidence
limits in parentheses.
Either atropine (17.4 mg/kg) alonepr atropine +. NaF (mg/kg; in the same solution) was administered 1.p. 5 mm before recelvmg the organophosphate antichohnesterase S.C. Protection
Factor (P.F.)
=
LDso of organophosphate LD50 of organophosphate
Significantly different atropine only.
(p < 0.05)
from
the treatment
+ treatment alone group
receiving
NaF has been reported to be a weak reactivator of organophosphate inhibited acetylcholinesterase (see introduction). The results in Table II demonstrate that in sarinpoisoned mice pretreated with NaF + atropine the blood but not brain acetylcholinesterase activity was significantly different (p < 0.001) from that obtained in mice pretreated with atropine. There was no significant difference between atropine- and NaF + atropinetreated mice brain or blood sIcetylcholinesterase in soman- or tabun-poisoned mice.
1806
NaF
and Organophosphate
Poisoning
Vol.
32, No. 16, 1983
TABLE II Effect of NaF on Blood and Brain Acetylchoiinesterase Activity Following
Organophosphate
Poisoninga
Acetylcholinesterase
Treatment Soman
P<
Sarin
Activity b P<
Tabun
P<
Brain: Atropine
1.6 + 0.2c(7)
Atropine
+ NaF
1.6 +_ 0.3 (6)
3.1 f 0.5 (8) NS
NSd 1.8 k 0.2 (8)
NS 1.7 + 0.2 (7)
2.6 h 0.4 (8)
Blood: Atropine
166 + 18 (6)
389 + 16 (8) NS
Atropine
+ NaF
151 f
187 + 23 (6) 0.001
15 (8)
590 f
16 (8)
NS 236 + 32 (8)
a Mice were administered either soman (130 pg/kg, s.c.), sarin (170 pg/kg, s.c.) or tabun (250 pg/kg, s.c.) 5 minutes after receiving either atropine (17.4 mg/kg, i.p.) or atropine + NaF (15 mg/kg, i.p.). All animals were sacrificed 3 hours after receiving the organophosphate. b Brain acetylcholinesterase activity is expressed as nmol acetylcholine hydrolysed/mg brain tissue/minute Blood acetylcholinesterase activity is expressed as nmol acetylcholine hydrolysed/mL of blood/minute Control brain and blood acetylcholinesterase activity is 1 I. 16 + 0.57 (X f SEM) (nmol ACh/mg/min) and 1046 f 8 (nmol ACh/mL/min), respectively. c X f
SEM with the number
d NS = not significantly
of observations
different
in parentheses.
from the atropine
control
group.
Soman is hydrolysed in vivo by A-esterase( s) ( 11) present in tissues such as the liver and plasma. The effect of NaF on somanase activity in the mouse liver was assessed in vitro. The results in Table III indicated that NaF (I mM and 10 mM) did not affect the in vitro hydrolysis of soman by somanase. TABLE III Effect of NaF on the in vitro Activity of Liver Somanase and Serum Aliesterase
Treatment
Somanase Activity (nmoles soman hydrolyzed/ g tissue/min)
Control NaF (0.1 mM) NaF (1 mM) NaF (10 mM) a mean
740 -t 45 (7)a 809 * 32 (7) 760 f 32 (3) f S.E.M.
(N)
Aliesterase Activity (nmoles tributyrin hydrolyzed/ mL serum/ min) 1684 f 86 (5) 1405 ? 58 (5) 859 + 45 (5) -
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
1807
Aliesterase, a B-esterase (1 l), present in mouse serum and liver also detoxifies soman (12). The results in Table III show that high concentrations of NaF inhibited aliesterase activity in vitro in contrast to the results obtained with somanase. CBDP, a metabolite of tri-o-cresyl phosphate, which is a potent and irreversible inhibitor of aliesterase activity (Clement, J.G., unpublished observations) increased the toxicity of soman in mice (Table IV; 13). However, NaF (15 mg/kg) when combined with atropine did not attenuate significantly the toxicity of soman in CBDP pretreated mice. The effect of NaF on the activity of aliesterase in soman poisoned animals was examined. The results in Table V indicate that soman inhibited serum aliesterase activity and that in soman poisoned mice NaF (15 mg/kg) + atropine pretreatment resulted in significantly (p < 0.05) higher aliesterase activity than mice receiving atropine (17.4 mg/ kg) only. TABLE IV Effect of Pretreatment with CBDP on Soman Toxicity and the Protective Effect of NaF Pretreatment -
CBDP
P.F. -
130 8.7
CBDP CBDP
95% Limits
Soman LD50 (&kg)
+ atropine
+ atropine
+ NaF
(7.5 - 9.7)
-
12.4
(10.8 -
14.4)
1.43
15.0
(13.6 -
16.9)
1.72
Mice were pretreated with CBDP (50 mg/kg) S.C. 60 min before soman. Atropine (17.4 mg/kg) alone or atropine + NaF (15 mg/kg) was Mortality was assessed at 24 hr. administered i.p. 5 min before soman. These mice were not fasted for 18 hr prior to use. TABLE V Effect of NaF on Serum Aliesterase Activity in Soman Poisoned Micea Aliesterase Activity (nmoles tributyrin hydrolyzed/ mL serum/ min)
Treatment
-
-
-
AT + NaF
Soman
AT
Soman
AT + NaF
1684 5 (5) 1740 + (4) 405 + (6) 501 + (6)
% Control
86b
100
62
103
27c
24
34c
30
a Mice were pretreated with either atropine (AT; 17.4 mg/kg, i.p.) or atropine + NaF (15 mg/kg, i.p.) 5 min before soman (130 pg/kg, s.c.) administration. The mice were sacrificed 1 hr after soman injection. b mean
+ S.E.M.
c The difference
(N) between means was significant
(p < 0.05).
1808
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
Discussion The results of this study demonstrate that in mice NaF provided significant antidotal action, when combined with atropine, against lethality produced by soman, sarin and tabun. However, at higher doses of NaF (30 mg/kg) the antidotal effect disappeared probably due to the toxic effects of NaF. Following sarin poisoning blood acetylcholinesterase activity in mice pretreated with NaF (15 mg/kg) + atropine was significantly different (p < 0.001) from mice receiving atropine only. These results agree with those of Albanus et al. (5). The effect of NaF on blood acetylcholinesterase in sarin poisoning could be due to protection of acetylcholinesterase before inhibition and/ or reactivation of the phosphorylated enzyme. The lack of effect of NaF on sarin-inhibited brain acetylcholinesterase is perhaps due to the fact that the fluoride ion penetrates poorly into the central nervous system (14). Soman- and tabun-inhibited blood and brain acetylcholinesterase were not affected by pretreatment with NaF. These results suggest that the mechanism for the protection provided by NaF is probably not due to an effect on acetylcholinesterase. Since soman is hydrolysed in viva by A-esterase( s) perhaps the protective effect of NaF Sterri and Fonnum (12) suggested that is due to an increased hydrolysis of soman. somanase in the liver is the enzyme important in the detoxification of soman in vivo. However, soman is not a chemically homogeneous compound. There are four stereoisomers of soman, two of which have high anticholinesterase activity (15). In vitro metabolic degradation of soman by blood and liver appears to be preferential for the relatively nonof “ - “-sarin to toxic isomers of soman ( 1.5). In vitro NaF catalysed the racemisation “ + “-sarin which is rapidly and preferentially hydrolysed by rat plasma (17). This result could account for the antidote effect of NaF against sarin poisoning. However, in this study, NaF had no significant effect on the hydrolysis of soman by mouse liver which, combined with the above, suggests that an increase in the activity of somanase is not responsible for the protective effect of NaF against soman poisoning. Aliesterase is important in the in vivo detoxification of soman (12) as evidenced by the potentiation of soman toxicity in CBDP-pretreated mice. NaF did not attenuate significantly the toxicity of soman in atropine and CBDP-pretreated mice although the protection factor was very similar to that obtained in mice not pretreated with CBDP (Table I). Aliesterase hydrolysis is the “dominant” factor in determining the acute toxicity of malathion (18). NaF pretreatment resulted in significantly (p < 0.05) higher aliesterase activity in soman poisoned mice. This higher aliesterase is perhaps due to the protective effect of NaF, i.e., NaF which inhibited aliesterase activity (Table III) protected aliesterase against the inhibitory effects of soman. It is well known that organophosphate and carbamate anticholinesterases produce receptor desensitization due to the accumulation of acetylcholine in the synaptic cleft. In addition, anticholinesterases appear to have a desensitizing action independently of their cummulative effect on acetylcholine (19). At nicotinic receptors in the neuromuscular junction Karczmar and his group have shown that NaF was one of the few substances that antagonized desensitization. This antidesensitizing action of NaF was not related to either its weak anticholinesterase action or its Ca ++ chelating action (2). Recently Barritt et al. (3) found that NaF specifically decreased the binding of the muscarinic agonist [3H] cis methyldioxolane to cardiac muscarinic receptors. In contrast NaF had no effect on the binding of antagonists such as quinuclidinyl benzilate (3) or propylbenzilylcholine (4) to muscarinic receptors. Based on the results of this study and those of Akasu and Karczmar (2) and Barritt et al. (3) we propose that the antidote action of NaF versus organophosphate poisoning is perhaps due to the antidesensitizing effect at the nicotinic receptors in the neuromuscular junction and/or sympathetic ganglia (20). The concentration of NaF required to reduce agonist binding at muscarinic receptors was 10 mM (3) in excess of the peak in vivo
vol. 32, No. 16, 1983
NaF and Organophosphate Poisoning
1809
concentration of 4.46 mM1 whereas, Akasu and Karczmar (2) reported that the antidesensitizing effect of NaF at the neur_omuscular junction was apparent at concentrations as low as 0.01 mM. In addition, Ullberg et al. (14) found that NaF did not penetrate into the CNS very readily therefore an effect on cholinergic receptors is doubtful. Also, the mice in this study were pretreated with atropine and since NaF does not affect the binding of antagonists it would not be expected that the antidesensitizing action of NaF would have an effect on muscarinic receptors in this situation. The lack of significant antidotal action of NaF in CBDP pretreated animals is perhaps due to the fact that besides inhibiting aliesterase irreversibly, CBDP (50 mg/kg, s.c.) also inhibits, significantly, acetylcholinesterase present in blood, brain (21) and diaphragm (J.G. Clement, unpublished observations). The animals were injected with CBDP = 1 hr before receiving the NaF therefore perhaps the muscarinic receptors were already in a desensitized state thus reducing the protective action of NaF. In summary, the antidesensitizing action of NaF at nicotinic receptors accounts for its major antidotal action versus soman, sarin and tabun. In sarin poisoning the catalysis of the racemisation of “ - ” -sarin to “ + “-sarin which is rapidly and preferentially hydrolysed by in plasma and protection and/ or reactivation of sarin-inhibited acetylcholinesterase (17) are additional mechanisms and perhaps accounts for the increased protection factors over that obtained with either soman or tabun. The direct reaction of NaF with tabun forming a less toxic anticholinesterase dimethylamido-ethoxy-phosphoryl fluoride (22) may explain the antidotal effect of NaF versus this anticholinesterase. 1 Assuming that the dose of absorbed and present only concentration in the blood body fluids and excretion concentrations in vivo.
NaF (15 mg/kg; i.p.) administered was completely in the blood (80 mL/ kg body weight) the maximum would be 4.46 mM. However, distribution to other of NaF by the kidneys would result in much lower Acknowledgements
The authors would like to thank Dr. P. Lockwood, Defence Research Establishment Suffield, for synthesizing soman, sarin, tabun and CBDP used in the toxicological experiments. Mr. B. Hand provided excellent technical assistance. Mrs. B. Adie decyphered the first draft and Mrs. L. Wall typed the final manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
B. KATZ and S. THESLEFF, J. Physiol. 138, 63-80 (1957). T. AKASU and A.G. KARCZMAR, Neuropharmacology 19, 393-404 (1980). D. BARRITT, H.I. YAMAMURA and W.R. ROESKE, Life Sci. 30,875-877 (1982). N. J.M. BIRDSALL, A.S.V. BURGEN, E.C. HULME and J.W. WELLS, Br. J. Pharmacol. 67, 371-377 (1979). L. ALBANUS, E. HEILBRONN and A. SUNDWALL, Biochem. Pharmacol. 14, 1375-1381 (1965). E. HEILBRONN, Biochem. Pharmacol. 14, 1363-1373 (1965). E. USDIN, Anticholinesterase Agents, Section 13, Vol. 1, pp. 45-354, Pergamon Press, Oxford (1970). L.C. MILLER and M.L. TAINTER, Proc. Sot. Exp. Biol. Med.57, 261-264 (1969). A.N. SIAKOTOS, M. FILBERT and R. HESTER, Biochem. Med. 3, l-12 (1969). J.T. LITCHFIELD and F. WILCOXON, J. Pharmacol. Exp. Ther. 76, 99-113 (1949). W.N. ALDRIDGE, B&hem. J. 53, 110-117 (1953). S.H. STERRI and F. FONNUM, Acta Pharmacol. Toxicol., Suppl. 1 49, 53 (1981). B. BOSKOVIC, Fundam. Appl. Toxicol. 1, 203-213 (1981).
1810
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
s. ULLBERG, L-E. APPELGREN, C-J. CLEMEDSON, Y. ERRICSSON, B. EWALDSON, B. SORB0 and R. SOREMARK, Biochem. Pharmacol. 13, 407-412 (1964). 15. H.P. BENSCHOP, F. BERENDS and L.P.A. DE JONG, Fundam. Appl. Toxicol. 1, 177-182 (1981). 16. S.H. STERRI, S. LYNGAAS and F. FONNUM, Acta Pharmacol. Toxicol. 49, 8-13 (1981). 17. P.J. CHRISTEN and J.A.C.M. VAN DEN MUYSENBERG, Biochem. Biophys. Acta 220, 217-220 (1965). 18. A.R. MAIN and P.E. BRAID, Biochem. J. 84, 255-263 (1962). 19. A.G. KARCZMAR and Y. OHTA, Fundam. Appl. Toxicol. 1, 135-142 (1981). 20. K. KOFETSU,, Int. J. Neuropharmacol. 5, 247-254 (1966). 21. B. BOSKOVIC, Arch. Toxicol. 42, 207-216 (1979). 22. E. HEILBRONN and B. TOLAGEN, Biochem. Pharmacol. 14, 73-77 (1965). 14.
Pergamon Press
ANTIDOTE EFFECT OF SODIUM FLUORIDE AGAINST ORGANOPHOSPHATE POISONING IN MICE1 J.G. Clement*2 and Marg Filbert?3 * Biomedical Section, Defence Research Establishment Suffield, Ralston, Alberta, Canada., TOJ 2N0 ? Neurophysiology Branch, U.S. Army Institute of Chemical Defence, Aberdeen Proving Ground, Maryland, U.S.A. (Received
in final form January 19, 1983)
Summary Pretreatment of mice with atropine (17.4 mg/kg) + NaF (5 or 15 mg/kg) had a significant antidotal effect over atropine alone against the lethality produced by soman and sarin. Atropine + NaF (15 mg/kg) was effective against tabun, whereas the lower dose of NaF was not. An effect of NaF on organophosphate inhibited acetylcholinesterase could not account for the antidotal action of NaF. NaF had no effect on liver somanase activity but inhibited aliesterase activity. Aliesterase activity in NaF pretreated somanpoisoned mice was significantly (p < 0.05) higher than in those receiving atropine alone. In CBDP-pretreated mice NaF did not significantly attenuate the toxicity of soman. It is hypothesized that the antidotal effect of NaF versus organophosphate poisoning is due to its antidesensitizing action at nicotinic receptors in the neuromuscular junction and/ or sympathetic ganglia in addition to the proposed increased hydrolysis of sarin and direct detoxification of tabun. Desensitization of cholinergic receptors at the endplate usually occurs following prolonged exposure to high concentrations of acetylcholine (1). Anticholinesterase drugs which increase the synaptic concentration of acetylcholine were found to accelerate significantly the time to desensitize receptors (2). In addition, these authors found that sodium fluoride (NaF) antagonized the acceleration of desensitization induced by anticholinesterase drugs. Recently Barritt et al. (3) reported that in vitro NaF specifically decreased the binding of an agonist to the muscarinic receptor. NaF had no effect on the binding of antagonists to the muscarinic receptor (3,4). NaF, when combined with atropine, was an effective prophylactic or therapeutic treatment for organophosphate poisoning (5). Previous investigators have reported that NaF was capable of reactivating acetylcholinesterase inhibited by sarin (5,6) and tabun (6) but not soman (6) or TEPP (7). The object of this study was to determine the extent and nature of the antidotal properties of NaF when combined with atropine against the lethality of soman, sarin and tabun in mice.
Suffield
Report No. 318
To whom all correspondence
and reprint requests should be sent
The opinions or assertations contained herein are the private views of the author and are not construed as reflecting views of the Department of the Army or the Department of Defence, U.S.A. 0024-3205/83/161803-08$03,00/O
1804
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
Methods Toxicology Male mice (CD-l@ , 25 - 30 g) obtained from Canadian Breeding Farm and Laboratories Ltd., St. Constant, Quebec, Canada were used in this study. The animals were acclimatized for at least one week in our animal facilities prior to use. The animals were usually fasted (but had free access to water) for 18 hr prior to the toxicological experiments. Following the injections the animals were given food and water ad libitum. The 24 hr LD50 values were calculated by probit analysis using the procedure of Miller and Tainter (8). All drugs were injected in a volume of 1% of body weight. Soman was injected subcutaneously (s.c.) in the back of the neck 5 min after an intraperitoneal (i.p.) injection of atropine or a solution of atropine + NaF. Acetylcholinesterase
Determinations
Acetylcholinesterase activity was determined according to the procedure of Siakotos et al. (9) using 14C-labelled acetylcholine iodide as the substrate. Mice were decapitated and exsanguinated. The whole brain, minus the cerebellum, was rinsed in 0.9% saline, blotted dry on filter paper and homogenized in enough 0.1 M phosphate buffer (pH 7.4) + 0.4 M sucrose to give a 1Vo (w/v) homogenate. The incubation time was 10 min. Whole blood was collected in a plastic beaker containing heparin. The blood was taken up into a syringe and stored in an ice water bath. Acetylcholinesterase activity was determined within 2 hrs of taking the samples. The time of incubation with substrate was 5 min for blood and 10 min for brain. Somanase Determinations Male CD-l mice were sacrificed by decapitation and exsanguinated. The liver was excised, placed in ice-cold saline, blotted dry on filter paper and weighed. A 10% w/v homogenate was prepared in 0.9% saline using a glass-teflon homogenizer. Somanase activity was assessed by the pH-stat method using soman (1.1 mM) as the substrate. Soman (10 mL) dissolved in 0.9% saline was placed in a vessel and the pH titrated automatically to pH 7.2 at 23°C using a Radiometer titragraph. Then 400 PL of the 10% liver homogenate was added to start the reaction and the amount of 0.01 N NaOH titrated over a 5 min period was recorded. The reaction was linear over this time period. NaF was added to some reactions prior to the addition of the homogenate. All solutions were made up using COzfree solutions. In addition, the reaction vessel was purged with nitrogen. Aliesterase
Determinations
Aliesterase activity was assessed by the pH-stat method using tributyrin as the substrate. Tributyrin (10 mL of 0.2% solution) in C02-free 0.9% saline was added to the reaction vessel and titrated to pH 7.9 with 0.01 N NaOH by a Radiometer titragraph. Either 50 I.IL of serum or 25 PL of 10% w/v liver homogenate was added to start the reaction. The homogenate was prepared using COz-free 0.9% saline. The addition of 0.01 N NaOH from the first to fifth min was used in determining the reaction rate. The reaction was linear over this time period. Materials The following drugs were used in this study: NaF (Fisher); atropine sulfate (J.T. Baker); 14C-acetylcholine iodide (New England Nuclear; S.A. = 4 mCi/mmol). Soman, sarin, tabun and 2-( o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP) were synthesized by the Organic Chemistry Group, Defence Research Establishment Suffield. All drugs were dissolved in 0.9% saline immediately before use except CBDP which was dissolved in DMSO. Statistically significant differences were determined using the Students ‘t’ test or the potency ratio (IO).
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
1805
Results In CD@ -1 mice NaF had a 24 hr LDso value of 55.6 mg/kg confidence limits) following i.p. administration in 0.9% NaCl.
(42.1 to 68.3; 95%
The results in Table I show that compared to those obtained with atropine alone NaF (5 mg/kg) plus atropine increased the LDso value significantly (p < 0.05) for soman and sarin but not tabun whereas, NaF (15 mg/kg) significantly (p < 0.05) increased the LDso value for all three organophosphate anticholinesterases. Increasing the dose of NaF to 30 mg/kg decreased the protective effect to that obtained with atropine alone. TABLE I Effect of NaF on the LD50 Value for Soman, Sarin and Tabun in Mice
Treatment
Sarin
Soman LD50
-
114 (104-
Atropine (AT)b
P.F.
LD50
P.F.
LD50
P.F.
-
169 (162 - 176)
-
290 (279 - 311)
-
123)a
1.42c
214 (199 - 231)
1.27
323 (313 - 330)
1.11
(5 mg) A+T 202 (186 - 231)d
1.77
330 (282 - 375)d
1.95
339 (305 - 361)
1.17
NaF (15 mg) LT 208 (194 - 228)d
1.82
444 (338 - 586)d
2.63
411 (393 - 437)d
1.42
NaF
162 (151 - 176)
Tabun
NaF (30 mg) 165 (131 - 203)
1.45
-
-
-
-
LT LD5o value in pg/kg with the 95% confidence
limits in parentheses.
Either atropine (17.4 mg/kg) alonepr atropine +. NaF (mg/kg; in the same solution) was administered 1.p. 5 mm before recelvmg the organophosphate antichohnesterase S.C. Protection
Factor (P.F.)
=
LDso of organophosphate LD50 of organophosphate
Significantly different atropine only.
(p < 0.05)
from
the treatment
+ treatment alone group
receiving
NaF has been reported to be a weak reactivator of organophosphate inhibited acetylcholinesterase (see introduction). The results in Table II demonstrate that in sarinpoisoned mice pretreated with NaF + atropine the blood but not brain acetylcholinesterase activity was significantly different (p < 0.001) from that obtained in mice pretreated with atropine. There was no significant difference between atropine- and NaF + atropinetreated mice brain or blood sIcetylcholinesterase in soman- or tabun-poisoned mice.
1806
NaF
and Organophosphate
Poisoning
Vol.
32, No. 16, 1983
TABLE II Effect of NaF on Blood and Brain Acetylchoiinesterase Activity Following
Organophosphate
Poisoninga
Acetylcholinesterase
Treatment Soman
P<
Sarin
Activity b P<
Tabun
P<
Brain: Atropine
1.6 + 0.2c(7)
Atropine
+ NaF
1.6 +_ 0.3 (6)
3.1 f 0.5 (8) NS
NSd 1.8 k 0.2 (8)
NS 1.7 + 0.2 (7)
2.6 h 0.4 (8)
Blood: Atropine
166 + 18 (6)
389 + 16 (8) NS
Atropine
+ NaF
151 f
187 + 23 (6) 0.001
15 (8)
590 f
16 (8)
NS 236 + 32 (8)
a Mice were administered either soman (130 pg/kg, s.c.), sarin (170 pg/kg, s.c.) or tabun (250 pg/kg, s.c.) 5 minutes after receiving either atropine (17.4 mg/kg, i.p.) or atropine + NaF (15 mg/kg, i.p.). All animals were sacrificed 3 hours after receiving the organophosphate. b Brain acetylcholinesterase activity is expressed as nmol acetylcholine hydrolysed/mg brain tissue/minute Blood acetylcholinesterase activity is expressed as nmol acetylcholine hydrolysed/mL of blood/minute Control brain and blood acetylcholinesterase activity is 1 I. 16 + 0.57 (X f SEM) (nmol ACh/mg/min) and 1046 f 8 (nmol ACh/mL/min), respectively. c X f
SEM with the number
d NS = not significantly
of observations
different
in parentheses.
from the atropine
control
group.
Soman is hydrolysed in vivo by A-esterase( s) ( 11) present in tissues such as the liver and plasma. The effect of NaF on somanase activity in the mouse liver was assessed in vitro. The results in Table III indicated that NaF (I mM and 10 mM) did not affect the in vitro hydrolysis of soman by somanase. TABLE III Effect of NaF on the in vitro Activity of Liver Somanase and Serum Aliesterase
Treatment
Somanase Activity (nmoles soman hydrolyzed/ g tissue/min)
Control NaF (0.1 mM) NaF (1 mM) NaF (10 mM) a mean
740 -t 45 (7)a 809 * 32 (7) 760 f 32 (3) f S.E.M.
(N)
Aliesterase Activity (nmoles tributyrin hydrolyzed/ mL serum/ min) 1684 f 86 (5) 1405 ? 58 (5) 859 + 45 (5) -
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
1807
Aliesterase, a B-esterase (1 l), present in mouse serum and liver also detoxifies soman (12). The results in Table III show that high concentrations of NaF inhibited aliesterase activity in vitro in contrast to the results obtained with somanase. CBDP, a metabolite of tri-o-cresyl phosphate, which is a potent and irreversible inhibitor of aliesterase activity (Clement, J.G., unpublished observations) increased the toxicity of soman in mice (Table IV; 13). However, NaF (15 mg/kg) when combined with atropine did not attenuate significantly the toxicity of soman in CBDP pretreated mice. The effect of NaF on the activity of aliesterase in soman poisoned animals was examined. The results in Table V indicate that soman inhibited serum aliesterase activity and that in soman poisoned mice NaF (15 mg/kg) + atropine pretreatment resulted in significantly (p < 0.05) higher aliesterase activity than mice receiving atropine (17.4 mg/ kg) only. TABLE IV Effect of Pretreatment with CBDP on Soman Toxicity and the Protective Effect of NaF Pretreatment -
CBDP
P.F. -
130 8.7
CBDP CBDP
95% Limits
Soman LD50 (&kg)
+ atropine
+ atropine
+ NaF
(7.5 - 9.7)
-
12.4
(10.8 -
14.4)
1.43
15.0
(13.6 -
16.9)
1.72
Mice were pretreated with CBDP (50 mg/kg) S.C. 60 min before soman. Atropine (17.4 mg/kg) alone or atropine + NaF (15 mg/kg) was Mortality was assessed at 24 hr. administered i.p. 5 min before soman. These mice were not fasted for 18 hr prior to use. TABLE V Effect of NaF on Serum Aliesterase Activity in Soman Poisoned Micea Aliesterase Activity (nmoles tributyrin hydrolyzed/ mL serum/ min)
Treatment
-
-
-
AT + NaF
Soman
AT
Soman
AT + NaF
1684 5 (5) 1740 + (4) 405 + (6) 501 + (6)
% Control
86b
100
62
103
27c
24
34c
30
a Mice were pretreated with either atropine (AT; 17.4 mg/kg, i.p.) or atropine + NaF (15 mg/kg, i.p.) 5 min before soman (130 pg/kg, s.c.) administration. The mice were sacrificed 1 hr after soman injection. b mean
+ S.E.M.
c The difference
(N) between means was significant
(p < 0.05).
1808
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
Discussion The results of this study demonstrate that in mice NaF provided significant antidotal action, when combined with atropine, against lethality produced by soman, sarin and tabun. However, at higher doses of NaF (30 mg/kg) the antidotal effect disappeared probably due to the toxic effects of NaF. Following sarin poisoning blood acetylcholinesterase activity in mice pretreated with NaF (15 mg/kg) + atropine was significantly different (p < 0.001) from mice receiving atropine only. These results agree with those of Albanus et al. (5). The effect of NaF on blood acetylcholinesterase in sarin poisoning could be due to protection of acetylcholinesterase before inhibition and/ or reactivation of the phosphorylated enzyme. The lack of effect of NaF on sarin-inhibited brain acetylcholinesterase is perhaps due to the fact that the fluoride ion penetrates poorly into the central nervous system (14). Soman- and tabun-inhibited blood and brain acetylcholinesterase were not affected by pretreatment with NaF. These results suggest that the mechanism for the protection provided by NaF is probably not due to an effect on acetylcholinesterase. Since soman is hydrolysed in viva by A-esterase( s) perhaps the protective effect of NaF Sterri and Fonnum (12) suggested that is due to an increased hydrolysis of soman. somanase in the liver is the enzyme important in the detoxification of soman in vivo. However, soman is not a chemically homogeneous compound. There are four stereoisomers of soman, two of which have high anticholinesterase activity (15). In vitro metabolic degradation of soman by blood and liver appears to be preferential for the relatively nonof “ - “-sarin to toxic isomers of soman ( 1.5). In vitro NaF catalysed the racemisation “ + “-sarin which is rapidly and preferentially hydrolysed by rat plasma (17). This result could account for the antidote effect of NaF against sarin poisoning. However, in this study, NaF had no significant effect on the hydrolysis of soman by mouse liver which, combined with the above, suggests that an increase in the activity of somanase is not responsible for the protective effect of NaF against soman poisoning. Aliesterase is important in the in vivo detoxification of soman (12) as evidenced by the potentiation of soman toxicity in CBDP-pretreated mice. NaF did not attenuate significantly the toxicity of soman in atropine and CBDP-pretreated mice although the protection factor was very similar to that obtained in mice not pretreated with CBDP (Table I). Aliesterase hydrolysis is the “dominant” factor in determining the acute toxicity of malathion (18). NaF pretreatment resulted in significantly (p < 0.05) higher aliesterase activity in soman poisoned mice. This higher aliesterase is perhaps due to the protective effect of NaF, i.e., NaF which inhibited aliesterase activity (Table III) protected aliesterase against the inhibitory effects of soman. It is well known that organophosphate and carbamate anticholinesterases produce receptor desensitization due to the accumulation of acetylcholine in the synaptic cleft. In addition, anticholinesterases appear to have a desensitizing action independently of their cummulative effect on acetylcholine (19). At nicotinic receptors in the neuromuscular junction Karczmar and his group have shown that NaF was one of the few substances that antagonized desensitization. This antidesensitizing action of NaF was not related to either its weak anticholinesterase action or its Ca ++ chelating action (2). Recently Barritt et al. (3) found that NaF specifically decreased the binding of the muscarinic agonist [3H] cis methyldioxolane to cardiac muscarinic receptors. In contrast NaF had no effect on the binding of antagonists such as quinuclidinyl benzilate (3) or propylbenzilylcholine (4) to muscarinic receptors. Based on the results of this study and those of Akasu and Karczmar (2) and Barritt et al. (3) we propose that the antidote action of NaF versus organophosphate poisoning is perhaps due to the antidesensitizing effect at the nicotinic receptors in the neuromuscular junction and/or sympathetic ganglia (20). The concentration of NaF required to reduce agonist binding at muscarinic receptors was 10 mM (3) in excess of the peak in vivo
vol. 32, No. 16, 1983
NaF and Organophosphate Poisoning
1809
concentration of 4.46 mM1 whereas, Akasu and Karczmar (2) reported that the antidesensitizing effect of NaF at the neur_omuscular junction was apparent at concentrations as low as 0.01 mM. In addition, Ullberg et al. (14) found that NaF did not penetrate into the CNS very readily therefore an effect on cholinergic receptors is doubtful. Also, the mice in this study were pretreated with atropine and since NaF does not affect the binding of antagonists it would not be expected that the antidesensitizing action of NaF would have an effect on muscarinic receptors in this situation. The lack of significant antidotal action of NaF in CBDP pretreated animals is perhaps due to the fact that besides inhibiting aliesterase irreversibly, CBDP (50 mg/kg, s.c.) also inhibits, significantly, acetylcholinesterase present in blood, brain (21) and diaphragm (J.G. Clement, unpublished observations). The animals were injected with CBDP = 1 hr before receiving the NaF therefore perhaps the muscarinic receptors were already in a desensitized state thus reducing the protective action of NaF. In summary, the antidesensitizing action of NaF at nicotinic receptors accounts for its major antidotal action versus soman, sarin and tabun. In sarin poisoning the catalysis of the racemisation of “ - ” -sarin to “ + “-sarin which is rapidly and preferentially hydrolysed by in plasma and protection and/ or reactivation of sarin-inhibited acetylcholinesterase (17) are additional mechanisms and perhaps accounts for the increased protection factors over that obtained with either soman or tabun. The direct reaction of NaF with tabun forming a less toxic anticholinesterase dimethylamido-ethoxy-phosphoryl fluoride (22) may explain the antidotal effect of NaF versus this anticholinesterase. 1 Assuming that the dose of absorbed and present only concentration in the blood body fluids and excretion concentrations in vivo.
NaF (15 mg/kg; i.p.) administered was completely in the blood (80 mL/ kg body weight) the maximum would be 4.46 mM. However, distribution to other of NaF by the kidneys would result in much lower Acknowledgements
The authors would like to thank Dr. P. Lockwood, Defence Research Establishment Suffield, for synthesizing soman, sarin, tabun and CBDP used in the toxicological experiments. Mr. B. Hand provided excellent technical assistance. Mrs. B. Adie decyphered the first draft and Mrs. L. Wall typed the final manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
B. KATZ and S. THESLEFF, J. Physiol. 138, 63-80 (1957). T. AKASU and A.G. KARCZMAR, Neuropharmacology 19, 393-404 (1980). D. BARRITT, H.I. YAMAMURA and W.R. ROESKE, Life Sci. 30,875-877 (1982). N. J.M. BIRDSALL, A.S.V. BURGEN, E.C. HULME and J.W. WELLS, Br. J. Pharmacol. 67, 371-377 (1979). L. ALBANUS, E. HEILBRONN and A. SUNDWALL, Biochem. Pharmacol. 14, 1375-1381 (1965). E. HEILBRONN, Biochem. Pharmacol. 14, 1363-1373 (1965). E. USDIN, Anticholinesterase Agents, Section 13, Vol. 1, pp. 45-354, Pergamon Press, Oxford (1970). L.C. MILLER and M.L. TAINTER, Proc. Sot. Exp. Biol. Med.57, 261-264 (1969). A.N. SIAKOTOS, M. FILBERT and R. HESTER, Biochem. Med. 3, l-12 (1969). J.T. LITCHFIELD and F. WILCOXON, J. Pharmacol. Exp. Ther. 76, 99-113 (1949). W.N. ALDRIDGE, B&hem. J. 53, 110-117 (1953). S.H. STERRI and F. FONNUM, Acta Pharmacol. Toxicol., Suppl. 1 49, 53 (1981). B. BOSKOVIC, Fundam. Appl. Toxicol. 1, 203-213 (1981).
1810
NaF and Organophosphate Poisoning
Vol. 32, No. 16, 1983
s. ULLBERG, L-E. APPELGREN, C-J. CLEMEDSON, Y. ERRICSSON, B. EWALDSON, B. SORB0 and R. SOREMARK, Biochem. Pharmacol. 13, 407-412 (1964). 15. H.P. BENSCHOP, F. BERENDS and L.P.A. DE JONG, Fundam. Appl. Toxicol. 1, 177-182 (1981). 16. S.H. STERRI, S. LYNGAAS and F. FONNUM, Acta Pharmacol. Toxicol. 49, 8-13 (1981). 17. P.J. CHRISTEN and J.A.C.M. VAN DEN MUYSENBERG, Biochem. Biophys. Acta 220, 217-220 (1965). 18. A.R. MAIN and P.E. BRAID, Biochem. J. 84, 255-263 (1962). 19. A.G. KARCZMAR and Y. OHTA, Fundam. Appl. Toxicol. 1, 135-142 (1981). 20. K. KOFETSU,, Int. J. Neuropharmacol. 5, 247-254 (1966). 21. B. BOSKOVIC, Arch. Toxicol. 42, 207-216 (1979). 22. E. HEILBRONN and B. TOLAGEN, Biochem. Pharmacol. 14, 73-77 (1965). 14.