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Protection by phenytoin and calcium channel blocking agents against the toxicity of diisopropylfluorophosphate
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
83,
584-589
(1986)
SHORT COMMUNICATIONS Protection by Phenytoin and Calcium Channel Blocking Agents against the Toxicity of Diisopropylfluorophosphate’ Protection by Phenytoin and Calcium Channel Blocking Agents against the Toxicity of Diisopropylfluorophosphate. DRETCHEN, K. L., BOWLES, A. M., AND RAINES, A. (1986). Tmicol. Appl. Pharmacol. 83, 584-589. Pretreatment of male Swiss-Webster mice with phenytoin, 25 mg/kg, verapamil. 25 to 3.0 mg/kg. nifedipine, 0.05 to 0. I mg/kg, nitrendipine, 0. I mg/kg. and nimodipme, 1 to 2.5 mg/kg, elevated the LD50 of diisopropylfluorophosphate (DFP) to a significant degree. In addition, these agents enhanced the protection that can be obtained from atropine and 2-pralidoxime. The protective effects of phenytoin cannot be attributed to an anticonvulsant action, per se, since carbamazepine. phenobarbital. and diphenylbarbituric acid in anticonvulsant doses did not influence DFP lethality. The mechanism of action of phenytoin and the other effective calcium channel blockers in providing protection over and above that achieved with atropine and 2-pralidoxime appears to be due to a protective action of the former agents on central respiratory centers and peripheral nicotinic sites and may involve the movement of calcium into excitable membranes. C 1986 Academic Press. Inc
Organophosphates are important insecticides which account for thousands of intoxications annually in the United States alone. Furthermore, stock piling of these agents as potential weapons represents an ongoing societal concern. The toxic and lethal effects of organophosphates are mediated largely through hyperactivity of central and peripheral cholinergic synapses. The observed constellation of effects includes miosis, salivation, bronchosecretion and constriction. fasciculations, gastrointestinal hypermotility and hypersecretions, cardiovascular depression, convulsions, and respiratory failure-often with a fatal outcome. The mechanisms of action of these agents appear to involve at least two processes: (a) the classical essentially irreversible phosphorylation of cholinesterase leading to acetylcholine cumulation at central and peripheral cholinergic neuroeffector junctions (Holmstedt, 1959) and (b) the induction of presynaptic repetitive discharges leading to augmented postjunctional responses (Standaert and Riker, 1967). Both of these effects lead to an inten-
sification and prolongation of the effects of acetylcholine. The cholinergic crisis produced by anticholinesterase drugs may be attenuated by pretreatment or treatment with atropine and/or 2-pralidoxime (‘-PAM). The use of both of these agents represents optimum treatment by blocking muscarinic receptor sites in the central nervous system (CNS) and peripherally. If administered promptly, 2-PAM may regenerate active cholinesterase (Wilson, 1959). Thus a combination of atropine and 2-PAM provides maximum protection from cholinesterase inhibition and represents optimum contemporary therapy. Whereas anticholinesterases induce nerve terminal repetitive discharges, phenytoin (DPH) and verapamil have been shown to suppress such discharges (Raines and Standaert, 1966: Dretchen et al.. 1977) and the accompanying fasciculations. Since these agents share a common capacity to impair the intracellular movement of Ca2+ into motor nerve terminal structures (Dretchen et al., 1977; Pincus, 1977; Yaari et al.. 1977, 1979) and that Ca’+ movements are associated with neurotransmitter release, it appeared fruitful to evaluate the influence of several calcium
’ This work was supported by a research grant from the office of Naval Research, NO00 14-83K-0047. 0041-008x/86 Copyright
$3.00
D 1986 by Academic
AU rights of reprcduct~on I” any
Press. Inc form reserved.
584
SHORT
585
COMMUNICATIONS
channel blocking agents on organophosphate toxicity. Our data indicate that some calcium blocking agents are effective antagonists to diisopropylfluorophosphate (DFP), a prototype organophosphate anticholinesterase, and they substantially enhance protection afforded by atropine and 2-PAM.
Drugs were obtained from the following sources: diisopropylfluorophosphate (Sigma); atropine sulfate, injection vial of 0.4 mg/ml (Elkins-Sinn Inc.): pralidoxime chloride (Ayerst Laboratories, Inc.): verapamil, ampule 5 mg/2 ml (Knoll Pharmaceutical Co.); phenytoin (Parke-Davis); nitrendipine and nimodipine (Miles Laboratories): nifedipine (Pfizer Laboratories): phenobarbital (Sigma); carbamazepine (Ciba-Geigy): carbowax PEG 400 (Fisher Scientific Co.): diphenylbarbiturate was prepared in our laboratory (Raines et al., 1973).
METHODS Adult male Swiss-Webster mice obtained from Flow laboratories (Dublin, Va.) were used in all experiments. They were allowed free access to feed and water except during experimental trials. At the time of experimentation. the mice weighed from 20 to 2.5 g. Solutions were freshly prepared as follows: Atropine and verapamil were diluted with saline from commercially available (see below) parenteral solutions. Pralidoxime and sodium phenobarbital were dissolved in saline. Diphenylbarbituric acid (DPB) was dissolved in saline by the addition of a minimal amount of sodium hydroxide; carbamazepine was dissolved in a 50% solution of PEG 400 and saline. Phenytoin sodium powder (Parke-Davis, Detroit, Mich.) was dissolved in a minimal volume of saline. adjusted to a pH of 10.5 to 11.5 with sodium hydroxide. and then diluted to final volume with saline. DFP solutions were prepared in saline and used within 2 hr. All solutions were prepared in concentrations so that 0.1 ml contained the amount of material to be given per 10 g body weight. All solutions were administered by ip injection. The lethal dose (LD50) of DFP in the absence or presence of potentially protective treatments was determined with four dose amounts of DFP; ten mice per dose were employed: lethality was assessed within the first hour after DFP: LD50 values were calculated by the method of probits (Finney. I97 1). The data were generated over a 6-month period and DFP LD50 values were repeatedly obtained. Variations of the DFP LD50 over this period, involving hundreds of mice, were minimal. Group comparisons were made by an analysis of variance with orthoginal contrasts of the means determined with Duncan’s new multiple range test (Steel and Torrie. 1960). In all cases a p < 0.05 was considered to achieve statistical significance. We attempted to elevate the lethal dose of DFP in the mouse by pretreatment with atropine and 2-PAM. Optimal doses for these agents were determined in preliminary experiments and utilized as described below. Times to peak effect for verapamil(30 min), phenytoin (2 hr), phenobarbital (30 min), carbamazepine (45 min), and DPB (3 hr) were derived from other studies demonstrating these to be the times ofpeak activity in the mouse (Raines et al.. 1979; Knoll Pharmaceuticals product literature).
RESULTS Diisopropylfluorophosphate administered ip to mice, produced an LD50 of 6.0 mg/kg (kO.23 SE). Early signs of toxicity included tremors, respiratory distress, urination, and/ or defecation. Subsequently, convulsions often occurred prior to death. Death, when it occurred, always ensued in 10 to 15 min. Animals that survived over 15 min were left with no residual drug effects. In preliminary experiments we determined that doses of atropine in excess of 1 mg/kg afforded no greater protection than 1 mg/kg. Similarly, doses of 2-PAM in excess of 10 mg/ kg were no more effective than 10 mg/kg. The administration of atropine or 2-PAM produced protection (cf. Table 1). The combination of the optimal doses of atropine and 2-PAM ( 1.Oand 10.0 mg/kg, respectively) was designated the standard combined pretreat-
TABLE PROTECTIVE
EFFECTS
AGAINST
Pretreatment None Atropine SO, 2-PAM Atropine plus 2-PAM (SCP)
DOSe (w/kg)
1
OF ATROPINE
2-PAM
Pretreatment time (min) -
1.0 10.0 1.0 10.0
AND
DFP TOXICITY
20 10 20 10
DFP LD50 6.0 7.3 8.1 8.7
t i + +
0.23 (SE) 0.09” 0.24” 0.25”.b
Note. SCP, standard combined pretreatment. L1Significantly greater than LDSO DF’P alone; p < 0.05. b Significantly greater than LD50 DFP plus atropine or 2-PAM; p < 0.05.
SHORT COMMUNICATIONS
586
rnent (SCP). The SCP raised the LD50 to 8.7 mg/kg, a value in excessof that achieved with either protective drug alone. The capacity of a variety of agents, which are either calcium antagonists, anticonvulsants, or both, was examined for protection against DFP lethality. The resultsofthe former appear in Table 2. As can be seen,verapamil was protective in dosesbetween 2.5 and 6.0 mg/kg; larger doses were not protective; at doses of 7.8 mg/kg and greater, verapamil lowered the LD50 of DFP. With this dosethe LD50 of DFP was 4.7 mg/kg; at 12.5 mg/kg
TABLE 2 PROTECTWE
EFFECTS OF CALCIUM ANTAGONISTS AGAINST DFP TOXICITY
Pretreatment
-
Dose hdkg)
Pretreatment time (min)
None (DFP alone)
DFP LD50 6.0 2 0.23
SCP (see Table I)
-
Verapamil Verapamil + SCP Verapamil Verapamil + SCP Verapamil Verapamil
2.5 2.5 3.0 3.0 6.0 7.8
30 30 30 30 30 30
6.8 9.3 7.5 8.8 6.7 4.7
Nifedtpine Nlfedlpine Nifedipine Nifedipine Nifedipine Nifedlpine
0.05 0.10 0.10 0.50 1.0 2.0
35 35 35 35 35 35
7.x i 7.x i 13.6~ 5.1 k 6.2 + 4.5 iv
O.lOQ 0.29” 1.7’ 0.34 0.83 0.77
0.05 0.10 0.10 1.0
30 30 30 30
6.4 6.9 8.7 6.4
2 2 ? f
0.30 1.0” 0.84 0.18
0.5 1.0 2.5 2.5
30 30 30 30
6.8 1.6 7.5 7.2
+ + + +
0.230 0.46” 0.39’ 0.29”
2.0 2.0 10.0
30 30 30
5.8 k 0.65 7.6 + 0.14 6.1 IL 0.58
Nitrendipine Nitrendipme Nitrendipine Nitrendipine Nimodipine Nimodlpine Nimodipine Nimodipine Dilriazem Diltiazem Diltiazem
+ SCP
+ SCP
+ SCP + SCP
8.1 f 0.25 k0.17” T. 0.2bb kO.10” + 0.05 ? 0.26” ? 0.60
kre. SCP. standard combined pretreatment (atropine. I mg/ kg, + 2.PAM, 10 mg/kg,ip). ’ Significantly greater than LD50 DFP alone: p < 0.05. ’ SIgnilicantly greater protection than SCP alone: p < 0.05.
the LD50 of DFP was 3.5 mg/kg and at 25 mg/kg it was lessthan 2.0 mg/kg. At a dose of 2.5 mg/kg verapamil enhanced the protective effect of the SCP. Nifedipine was also effective in protecting against DFP toxicity. At dosesof 0.05 and 0.1 mg/kg, nifedipine produced a significant protective effect. As with verapamil, a biphasic dose-response relationship obtains; higher dosesof nifedipine were lesseffective and, in fact, reduced the LD50 of DFP. Nifedipine (0.1 mg/kg) significantly and substantially enhanced the efficacy of the SCP (Table 2). Nimodipine and nitrendipine exhibited a modest but statistically significant protective effect. However, they did not appear to enhance the effectiveness of the SCP. Diltiazem, in doses of 2 to 10 mg/kg, did not alter the LD50 of DFP nor did diltiazem increasethe efficacy of the SCP. Dosesof 30 mg/kg reduced the LD50 of DFP to 3.9 mg/kg. Larger doseswere not evaluated. The effects of the four anticonvulsant agents appear in Table 3. Of these agents only phenytoin exhibited protective effects. The drug was maximally protective at a doseof 25 mg/ kg. As with verapamil and nifedipine, the administration of phenytoin produced a significant elevation in the LD50 of DFP. when given with the SCP. Carbamazepine, phenobarbital. and diphenylbarbiturate were without protective actions given alone or as adjuncts to the SCP. DISCUSSION Given the ubiquitous distribution and varied functions of acetylcholine asa neurotransmitter, death by DFP and other organophosphate anticholinesterasesis the result of a disordering of several organ systems. In the periphery, skeletal neuromuscular failure, bronchiolar secretions, and smooth muscle spasm are prominent features. In the CNS. convulsions and respiratory failure appear to be most important. The present experiments demonstrating a protective effect of atropine and Z-PAM
587
SHORT COMMUNICATIONS TABLE 3 PROTECTIVE
EFFECTS OF ANTICONWLSANTS
AGAINST
DFY TOXICITY
Dose (m/W
Pretreatment time (mm)
DFP LD50
None (DFP alone)
-
-
6.0 f 0.23
SCP (see Table 1)
-
-
8.7 f 0.25
Phenytoin Phenytoin + SCP Phenytoin Phenytoin + SCP Phenytoin Phenytoin + SCP
15.0 15.0 25.0 25.0 50.0 50.0
120 120 120 120 120 120
Phenobarbital Phenobarbital + SCP
30.0 30.0
30 30
6.0 -t 0.08 8.5 k 0.05
Carbamazepine Carbamazepine + SCP Carbamazepine Carbamazepine + SCP
12.5 12.5 25.0 25.0
45 45 45 45
6.2 7.4 5.5 8.3
f 0.20 AI 0.33 f 0.40 + 0.45
+ SCP
50.0 50.0 100.0
180 180 100
f 0.34 * 0.45 + 0.20
+ SCP
100.0
100
6.0 7.3 5.6 7.5
Pretreatment
Diphenylbarbiturate Diphenylbarbiturate Diphenylbarbiturate Diphenylbarbiturate
6.8 f 7.8 f 6.5 f 10.6 f 6.2 f 8.8 f
0.06” 0.34 0.20 0.12* 0.30 0.20
t- 0.0 I
Note. SCP, standard combined pretreatment (atropine, 1 mg/kg, + 2-PAM. IO mg/kg, ip). ’ Significantly greater than LD50 DFP alone; p < 0.05. b Significantly greater protection than SCP alone; p < 0.05.
against DFP poisoning confirm the work of others (Horton et al., 1946; Ring and Poulsen, 1958: O’Leary et al., 196 1). Thus, atropine elevated the LD50 of DFP by 20% and the SCP increased the LD50 by 45%. On the other hand, when verapamil, phenytoin, or nifedipine was added to the SCP, protection against DFP of 60, 80, and 120% above control was observed. All of these values were statistically significantly greater than the SCP. The protection observed, although statistically significant, was not spectacular. The mouse appears to be a rather insensitive animal for the performance of studies such as those here performed. Thus O’Leary et al. (1961) demonstrated substantially greater protection in the rabbit than we report in the mouse. The actions of anticholinesterases appear to be mediated by at least two seemingly sepa-
rable actions at cholinergic neuroeffector junctions. First, the better known is the cumulation of acetylcholine at these sites, due to enzyme inhibition which produces excessive activation and subsequent failure. Second, these agents exert a multiplier effect by converting a single action potential in the nerve terminal into a train of repetitive action potentials which are transmitted to postjunctional elements (Standaert and Riker, 1967; Riker and Okamoto, 1969). The conversion of a single action potential to a train of potentials would be expected to be accompanied by a substantial increase in acetylcholine liberation. With regard to DFP toxicity, clearly these mechanisms are complementary and perhaps synergistic. The seemingly limited capacity of atropine to exert appreciable protection in our experiments is probably due to the limited
588
SHORT COMMUNICATIONS
specificity of the drug. Atropine only produces a partial block of the actions of acetylcholine at parasympathetic neuroeffector junctions and exerts no significant actions at nicotinic sites. Nicotinic sites, however, appear from our experiments to be influenced profoundly by phenytoin and other agents. The present series of experiments are phenomenologic and addressed only the exploration of the capacity of a variety of drugs to protect against death from DFP exposure. That is, it was our intent presently to identify agents that are protective. We did not in this series of investigations explore the underlying mechanisms of action which result in elevations in the dose of DFP necessary to prove lethal. However, some speculation regarding probable mechanisms is appropriate. The substantial enhancement of protection by phenytoin may be due to an effect on presynaptic nerve terminal structures. The drug antagonizes physostigmine-induced presynaptitally generated repetitive discharges and muscle fasciculations (Raines and Standaert, 1966). The stabilization of motor nerve terminals by phenytoin has been attributed to a blockade of the intracellular movement of calcium into motor nerve endings (Yaari et al., 1977, 1979) and synaptosomes (Sohn and Ferrendelli, 1976). In this regard, verapamil and phenytoin are quite similar in their actions on the cat neuromuscular junction in that both suppress neural repetitive discharges induced by tetanic stimulation and drugs (Dretchen ef al., 1977). This presynaptic stabilization of motor nerve endings prevents the postjunctional augmented responses. It therefore seems reasonable to presume that the actions of phenytoin and other calcium antagonists prevent a calcium-mediated depolarization of motor nerve terminals and subsequent excessive neurotransmitter release. It is also probable that at least some of these agents relax bronchiolar smooth muscle (Patel, 1981; Pate1 et al., 1983; Corris et al., 1983) and hence ameliorate one of the contributing factors to DFP-induced fatality. Additional crucial sites for both DFP action
and protection
against DFP are within the CNS. One possible means of exerting protection in the CNS would be to prevent convulsions which are a prominent feature of DFP poisoning. The data, however, indicate that an anticonvulsant action, per se, is not protective. The central respiratory centers are vulnerable to DFP and are depressed with lesser doses than required for neuromuscular impairment (Marx et al., 1984). In addition, we have recently observed that phenytoin pretreatment confers protection against DFP-induced respiratory depression in the cat (Marx et al., 1984).
Thus, it appears that phenytoin and several calcium antagonists have the capacity to enter the brain and exert an action to protect vital central functions. It is unclear at this time why diltiazem, a nondihydropyridine calcium antagonist, failed to exert a protective effect. Perhaps the variant chemical structure of diltiazem does not penetrate the blood-brain barrier in quantities sufficient to influence DFP poisoning. The present experiments demonstrate that a new class of agents, namely, drugs which impair calcium movements, exert a protective action against DFP poisoning and substantially and significantly enhance protection afforded by optimum contemporary pretreatments. REFERENCES CORRIS.P. A.. NARIMAN, S.. AND GIBSON, G. J. (1983). Nifedipine in the prevention of asthma induced by exercise and histamine. Amer. Rev. Respir. Dis. 128,99 I 992.
DRETCHEN, K. L.. STANDAERT, F. G., AND RAINES, A. (1977). Effects of phenytoin on the cyclic nucleotide system in the motor nerve terminal. Epilepsia 18. 337348.
FINNEY, D. J. (197 I j. Staiistical Methods m Biological .4ssay Griffin, London. HOLMSTEDT, B. (I 959). Pharmacology of organophosphorus cholinesterase inhibitors. Pharmacol. Rer. 11, 567-688.
HORTON, R. G., KOELL, G. B., MCNAMMARA, B. P.. AND PRATT, H. (1946). The acute toxicity of diisopropylfluorophosphate. J. Pharmacol. Exp. Ther. 87.4 14-420.
589
SHORT COMMUNICATIONS KING, T.. AND POULSEN, E. (1958). The action of an aldoxime (2-pyridine aldoxime methiodide) on acute alkylphosphate poisoning in mice. Arch. Int. Pharmacodyn. Ther. 114, 118-121.
MARX, K. A., ANASTASI, N-C., HERNANDEZ, Y. M., FIVOZINSKY, K. B., RAINES, A., AND DRETCHEN, K. L. ( 1984). Protection by phenytoin against the toxic effects of organophosphates on central respiratory centers. Sot. Neurosci.
10, 709.
O’LEARY, J. F., KUNKEL, A. M., AND JONES,A. H. (196 I). Efficacy and limitations of oxime-atropine treatment of organophosphorus anticholinesterase poisoning. J. Pharmacol.
E.xp. Ther. 132, 50-57.
PATEL, K. R. (198 I). Calcium antagonists in exercise-induced asthma. Brit. Med. J. 282, 932-933. PATEL, K. R.. AL-SHAMA, M. R., AND KERR, J. W. ( 1983). The effect of inhaled verapamil on allergen-induced bronchoconstriction. C/in. Allergy 13, 119-122. PINCUS, J. H. (1977). Anticonvulsant actions at a neuromuscular synapse. Neurology 27, 374-375. RAINES.A., BLAKE, G. J., RICHARDSON,B.. AND GILBERT, M. B. (1979). Differential selectivity of several barbiturates on experimental seizures and neurotoxicity in the mouse. Epilepsia 20, 105-I 13. RAINES, A., NINER. J. M., AND PACE, D. G. (1973). A comparison of the anticonvulsant, neurotoxic and lethal effects of diphenylbarbituric acid, phenobarbital and diphenylhydantoin in the mouse. J. Pharmacok Exp. Ther. 186,3
15-322.
RAINES,A., AND STANDAERT, F. G. (1966). Pre- and postjunctional effectsofdiphenylhydantoin at the cat soleus
neuromuscular junction. J. Pharmacol.
Exp. Ther. 153,
36 1-366.
RIKER, W. F., AND OKAMOTO, M. (1969). Pharmacology of motor nerve terminals. Annu. Rev. Pharmacol. 9. 173-208.
SOHN, R. S., AND FERRENDELLI, J. A. (1976). Anticonvulsant drug mechanisms. Arch. Neurol. 33, 626-629. STANDAERT, F. G., AND RIKER, W. F. (1967). The consequences of cholinergic drug actions on motor nerve terminals. Ann. N. Y. Acad. Sci. 144, 5 17-533. STEEL, R., AND TORRIE, J. H. (1960). Principles and Procedures of Sfatisks. McGraw-Hill, New York. WILSON, I. B. (1959). Molecular complementarity and antidotes for alkylphosphate poisoning. Fed. Proc. 18, 752-758.
YAARI, Y.. PINCUS, J. H., AND ARGOV, 2. (1977). Depression of synaptic transmission by diphenylhydantoin. Ann. Neural. 1, 334-338. YAARI, Y., PINCUS,J. H., AND ARGOV. 2. (1979). Phenytoin and transmitter release at the neuromuscular junction of the frog. Brain Res. 160, 497. KENNETH L. DRETCHEN ALYCE M. BOWLES ARTHUR RAINES Department of Pharmacology Georgetown University Schools qf Medicine and Dentistry 3900 Reservoir Road, N. W. Washington. DC 20007 Received Ju1.v 1, 1985
AND
APPLIED
PHARMACOLOGY
83,
584-589
(1986)
SHORT COMMUNICATIONS Protection by Phenytoin and Calcium Channel Blocking Agents against the Toxicity of Diisopropylfluorophosphate’ Protection by Phenytoin and Calcium Channel Blocking Agents against the Toxicity of Diisopropylfluorophosphate. DRETCHEN, K. L., BOWLES, A. M., AND RAINES, A. (1986). Tmicol. Appl. Pharmacol. 83, 584-589. Pretreatment of male Swiss-Webster mice with phenytoin, 25 mg/kg, verapamil. 25 to 3.0 mg/kg. nifedipine, 0.05 to 0. I mg/kg, nitrendipine, 0. I mg/kg. and nimodipme, 1 to 2.5 mg/kg, elevated the LD50 of diisopropylfluorophosphate (DFP) to a significant degree. In addition, these agents enhanced the protection that can be obtained from atropine and 2-pralidoxime. The protective effects of phenytoin cannot be attributed to an anticonvulsant action, per se, since carbamazepine. phenobarbital. and diphenylbarbituric acid in anticonvulsant doses did not influence DFP lethality. The mechanism of action of phenytoin and the other effective calcium channel blockers in providing protection over and above that achieved with atropine and 2-pralidoxime appears to be due to a protective action of the former agents on central respiratory centers and peripheral nicotinic sites and may involve the movement of calcium into excitable membranes. C 1986 Academic Press. Inc
Organophosphates are important insecticides which account for thousands of intoxications annually in the United States alone. Furthermore, stock piling of these agents as potential weapons represents an ongoing societal concern. The toxic and lethal effects of organophosphates are mediated largely through hyperactivity of central and peripheral cholinergic synapses. The observed constellation of effects includes miosis, salivation, bronchosecretion and constriction. fasciculations, gastrointestinal hypermotility and hypersecretions, cardiovascular depression, convulsions, and respiratory failure-often with a fatal outcome. The mechanisms of action of these agents appear to involve at least two processes: (a) the classical essentially irreversible phosphorylation of cholinesterase leading to acetylcholine cumulation at central and peripheral cholinergic neuroeffector junctions (Holmstedt, 1959) and (b) the induction of presynaptic repetitive discharges leading to augmented postjunctional responses (Standaert and Riker, 1967). Both of these effects lead to an inten-
sification and prolongation of the effects of acetylcholine. The cholinergic crisis produced by anticholinesterase drugs may be attenuated by pretreatment or treatment with atropine and/or 2-pralidoxime (‘-PAM). The use of both of these agents represents optimum treatment by blocking muscarinic receptor sites in the central nervous system (CNS) and peripherally. If administered promptly, 2-PAM may regenerate active cholinesterase (Wilson, 1959). Thus a combination of atropine and 2-PAM provides maximum protection from cholinesterase inhibition and represents optimum contemporary therapy. Whereas anticholinesterases induce nerve terminal repetitive discharges, phenytoin (DPH) and verapamil have been shown to suppress such discharges (Raines and Standaert, 1966: Dretchen et al.. 1977) and the accompanying fasciculations. Since these agents share a common capacity to impair the intracellular movement of Ca2+ into motor nerve terminal structures (Dretchen et al., 1977; Pincus, 1977; Yaari et al.. 1977, 1979) and that Ca’+ movements are associated with neurotransmitter release, it appeared fruitful to evaluate the influence of several calcium
’ This work was supported by a research grant from the office of Naval Research, NO00 14-83K-0047. 0041-008x/86 Copyright
$3.00
D 1986 by Academic
AU rights of reprcduct~on I” any
Press. Inc form reserved.
584
SHORT
585
COMMUNICATIONS
channel blocking agents on organophosphate toxicity. Our data indicate that some calcium blocking agents are effective antagonists to diisopropylfluorophosphate (DFP), a prototype organophosphate anticholinesterase, and they substantially enhance protection afforded by atropine and 2-PAM.
Drugs were obtained from the following sources: diisopropylfluorophosphate (Sigma); atropine sulfate, injection vial of 0.4 mg/ml (Elkins-Sinn Inc.): pralidoxime chloride (Ayerst Laboratories, Inc.): verapamil, ampule 5 mg/2 ml (Knoll Pharmaceutical Co.); phenytoin (Parke-Davis); nitrendipine and nimodipine (Miles Laboratories): nifedipine (Pfizer Laboratories): phenobarbital (Sigma); carbamazepine (Ciba-Geigy): carbowax PEG 400 (Fisher Scientific Co.): diphenylbarbiturate was prepared in our laboratory (Raines et al., 1973).
METHODS Adult male Swiss-Webster mice obtained from Flow laboratories (Dublin, Va.) were used in all experiments. They were allowed free access to feed and water except during experimental trials. At the time of experimentation. the mice weighed from 20 to 2.5 g. Solutions were freshly prepared as follows: Atropine and verapamil were diluted with saline from commercially available (see below) parenteral solutions. Pralidoxime and sodium phenobarbital were dissolved in saline. Diphenylbarbituric acid (DPB) was dissolved in saline by the addition of a minimal amount of sodium hydroxide; carbamazepine was dissolved in a 50% solution of PEG 400 and saline. Phenytoin sodium powder (Parke-Davis, Detroit, Mich.) was dissolved in a minimal volume of saline. adjusted to a pH of 10.5 to 11.5 with sodium hydroxide. and then diluted to final volume with saline. DFP solutions were prepared in saline and used within 2 hr. All solutions were prepared in concentrations so that 0.1 ml contained the amount of material to be given per 10 g body weight. All solutions were administered by ip injection. The lethal dose (LD50) of DFP in the absence or presence of potentially protective treatments was determined with four dose amounts of DFP; ten mice per dose were employed: lethality was assessed within the first hour after DFP: LD50 values were calculated by the method of probits (Finney. I97 1). The data were generated over a 6-month period and DFP LD50 values were repeatedly obtained. Variations of the DFP LD50 over this period, involving hundreds of mice, were minimal. Group comparisons were made by an analysis of variance with orthoginal contrasts of the means determined with Duncan’s new multiple range test (Steel and Torrie. 1960). In all cases a p < 0.05 was considered to achieve statistical significance. We attempted to elevate the lethal dose of DFP in the mouse by pretreatment with atropine and 2-PAM. Optimal doses for these agents were determined in preliminary experiments and utilized as described below. Times to peak effect for verapamil(30 min), phenytoin (2 hr), phenobarbital (30 min), carbamazepine (45 min), and DPB (3 hr) were derived from other studies demonstrating these to be the times ofpeak activity in the mouse (Raines et al.. 1979; Knoll Pharmaceuticals product literature).
RESULTS Diisopropylfluorophosphate administered ip to mice, produced an LD50 of 6.0 mg/kg (kO.23 SE). Early signs of toxicity included tremors, respiratory distress, urination, and/ or defecation. Subsequently, convulsions often occurred prior to death. Death, when it occurred, always ensued in 10 to 15 min. Animals that survived over 15 min were left with no residual drug effects. In preliminary experiments we determined that doses of atropine in excess of 1 mg/kg afforded no greater protection than 1 mg/kg. Similarly, doses of 2-PAM in excess of 10 mg/ kg were no more effective than 10 mg/kg. The administration of atropine or 2-PAM produced protection (cf. Table 1). The combination of the optimal doses of atropine and 2-PAM ( 1.Oand 10.0 mg/kg, respectively) was designated the standard combined pretreat-
TABLE PROTECTIVE
EFFECTS
AGAINST
Pretreatment None Atropine SO, 2-PAM Atropine plus 2-PAM (SCP)
DOSe (w/kg)
1
OF ATROPINE
2-PAM
Pretreatment time (min) -
1.0 10.0 1.0 10.0
AND
DFP TOXICITY
20 10 20 10
DFP LD50 6.0 7.3 8.1 8.7
t i + +
0.23 (SE) 0.09” 0.24” 0.25”.b
Note. SCP, standard combined pretreatment. L1Significantly greater than LDSO DF’P alone; p < 0.05. b Significantly greater than LD50 DFP plus atropine or 2-PAM; p < 0.05.
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586
rnent (SCP). The SCP raised the LD50 to 8.7 mg/kg, a value in excessof that achieved with either protective drug alone. The capacity of a variety of agents, which are either calcium antagonists, anticonvulsants, or both, was examined for protection against DFP lethality. The resultsofthe former appear in Table 2. As can be seen,verapamil was protective in dosesbetween 2.5 and 6.0 mg/kg; larger doses were not protective; at doses of 7.8 mg/kg and greater, verapamil lowered the LD50 of DFP. With this dosethe LD50 of DFP was 4.7 mg/kg; at 12.5 mg/kg
TABLE 2 PROTECTWE
EFFECTS OF CALCIUM ANTAGONISTS AGAINST DFP TOXICITY
Pretreatment
-
Dose hdkg)
Pretreatment time (min)
None (DFP alone)
DFP LD50 6.0 2 0.23
SCP (see Table I)
-
Verapamil Verapamil + SCP Verapamil Verapamil + SCP Verapamil Verapamil
2.5 2.5 3.0 3.0 6.0 7.8
30 30 30 30 30 30
6.8 9.3 7.5 8.8 6.7 4.7
Nifedtpine Nlfedlpine Nifedipine Nifedipine Nifedipine Nifedlpine
0.05 0.10 0.10 0.50 1.0 2.0
35 35 35 35 35 35
7.x i 7.x i 13.6~ 5.1 k 6.2 + 4.5 iv
O.lOQ 0.29” 1.7’ 0.34 0.83 0.77
0.05 0.10 0.10 1.0
30 30 30 30
6.4 6.9 8.7 6.4
2 2 ? f
0.30 1.0” 0.84 0.18
0.5 1.0 2.5 2.5
30 30 30 30
6.8 1.6 7.5 7.2
+ + + +
0.230 0.46” 0.39’ 0.29”
2.0 2.0 10.0
30 30 30
5.8 k 0.65 7.6 + 0.14 6.1 IL 0.58
Nitrendipine Nitrendipme Nitrendipine Nitrendipine Nimodipine Nimodlpine Nimodipine Nimodipine Dilriazem Diltiazem Diltiazem
+ SCP
+ SCP
+ SCP + SCP
8.1 f 0.25 k0.17” T. 0.2bb kO.10” + 0.05 ? 0.26” ? 0.60
kre. SCP. standard combined pretreatment (atropine. I mg/ kg, + 2.PAM, 10 mg/kg,ip). ’ Significantly greater than LD50 DFP alone: p < 0.05. ’ SIgnilicantly greater protection than SCP alone: p < 0.05.
the LD50 of DFP was 3.5 mg/kg and at 25 mg/kg it was lessthan 2.0 mg/kg. At a dose of 2.5 mg/kg verapamil enhanced the protective effect of the SCP. Nifedipine was also effective in protecting against DFP toxicity. At dosesof 0.05 and 0.1 mg/kg, nifedipine produced a significant protective effect. As with verapamil, a biphasic dose-response relationship obtains; higher dosesof nifedipine were lesseffective and, in fact, reduced the LD50 of DFP. Nifedipine (0.1 mg/kg) significantly and substantially enhanced the efficacy of the SCP (Table 2). Nimodipine and nitrendipine exhibited a modest but statistically significant protective effect. However, they did not appear to enhance the effectiveness of the SCP. Diltiazem, in doses of 2 to 10 mg/kg, did not alter the LD50 of DFP nor did diltiazem increasethe efficacy of the SCP. Dosesof 30 mg/kg reduced the LD50 of DFP to 3.9 mg/kg. Larger doseswere not evaluated. The effects of the four anticonvulsant agents appear in Table 3. Of these agents only phenytoin exhibited protective effects. The drug was maximally protective at a doseof 25 mg/ kg. As with verapamil and nifedipine, the administration of phenytoin produced a significant elevation in the LD50 of DFP. when given with the SCP. Carbamazepine, phenobarbital. and diphenylbarbiturate were without protective actions given alone or as adjuncts to the SCP. DISCUSSION Given the ubiquitous distribution and varied functions of acetylcholine asa neurotransmitter, death by DFP and other organophosphate anticholinesterasesis the result of a disordering of several organ systems. In the periphery, skeletal neuromuscular failure, bronchiolar secretions, and smooth muscle spasm are prominent features. In the CNS. convulsions and respiratory failure appear to be most important. The present experiments demonstrating a protective effect of atropine and Z-PAM
587
SHORT COMMUNICATIONS TABLE 3 PROTECTIVE
EFFECTS OF ANTICONWLSANTS
AGAINST
DFY TOXICITY
Dose (m/W
Pretreatment time (mm)
DFP LD50
None (DFP alone)
-
-
6.0 f 0.23
SCP (see Table 1)
-
-
8.7 f 0.25
Phenytoin Phenytoin + SCP Phenytoin Phenytoin + SCP Phenytoin Phenytoin + SCP
15.0 15.0 25.0 25.0 50.0 50.0
120 120 120 120 120 120
Phenobarbital Phenobarbital + SCP
30.0 30.0
30 30
6.0 -t 0.08 8.5 k 0.05
Carbamazepine Carbamazepine + SCP Carbamazepine Carbamazepine + SCP
12.5 12.5 25.0 25.0
45 45 45 45
6.2 7.4 5.5 8.3
f 0.20 AI 0.33 f 0.40 + 0.45
+ SCP
50.0 50.0 100.0
180 180 100
f 0.34 * 0.45 + 0.20
+ SCP
100.0
100
6.0 7.3 5.6 7.5
Pretreatment
Diphenylbarbiturate Diphenylbarbiturate Diphenylbarbiturate Diphenylbarbiturate
6.8 f 7.8 f 6.5 f 10.6 f 6.2 f 8.8 f
0.06” 0.34 0.20 0.12* 0.30 0.20
t- 0.0 I
Note. SCP, standard combined pretreatment (atropine, 1 mg/kg, + 2-PAM. IO mg/kg, ip). ’ Significantly greater than LD50 DFP alone; p < 0.05. b Significantly greater protection than SCP alone; p < 0.05.
against DFP poisoning confirm the work of others (Horton et al., 1946; Ring and Poulsen, 1958: O’Leary et al., 196 1). Thus, atropine elevated the LD50 of DFP by 20% and the SCP increased the LD50 by 45%. On the other hand, when verapamil, phenytoin, or nifedipine was added to the SCP, protection against DFP of 60, 80, and 120% above control was observed. All of these values were statistically significantly greater than the SCP. The protection observed, although statistically significant, was not spectacular. The mouse appears to be a rather insensitive animal for the performance of studies such as those here performed. Thus O’Leary et al. (1961) demonstrated substantially greater protection in the rabbit than we report in the mouse. The actions of anticholinesterases appear to be mediated by at least two seemingly sepa-
rable actions at cholinergic neuroeffector junctions. First, the better known is the cumulation of acetylcholine at these sites, due to enzyme inhibition which produces excessive activation and subsequent failure. Second, these agents exert a multiplier effect by converting a single action potential in the nerve terminal into a train of repetitive action potentials which are transmitted to postjunctional elements (Standaert and Riker, 1967; Riker and Okamoto, 1969). The conversion of a single action potential to a train of potentials would be expected to be accompanied by a substantial increase in acetylcholine liberation. With regard to DFP toxicity, clearly these mechanisms are complementary and perhaps synergistic. The seemingly limited capacity of atropine to exert appreciable protection in our experiments is probably due to the limited
588
SHORT COMMUNICATIONS
specificity of the drug. Atropine only produces a partial block of the actions of acetylcholine at parasympathetic neuroeffector junctions and exerts no significant actions at nicotinic sites. Nicotinic sites, however, appear from our experiments to be influenced profoundly by phenytoin and other agents. The present series of experiments are phenomenologic and addressed only the exploration of the capacity of a variety of drugs to protect against death from DFP exposure. That is, it was our intent presently to identify agents that are protective. We did not in this series of investigations explore the underlying mechanisms of action which result in elevations in the dose of DFP necessary to prove lethal. However, some speculation regarding probable mechanisms is appropriate. The substantial enhancement of protection by phenytoin may be due to an effect on presynaptic nerve terminal structures. The drug antagonizes physostigmine-induced presynaptitally generated repetitive discharges and muscle fasciculations (Raines and Standaert, 1966). The stabilization of motor nerve terminals by phenytoin has been attributed to a blockade of the intracellular movement of calcium into motor nerve endings (Yaari et al., 1977, 1979) and synaptosomes (Sohn and Ferrendelli, 1976). In this regard, verapamil and phenytoin are quite similar in their actions on the cat neuromuscular junction in that both suppress neural repetitive discharges induced by tetanic stimulation and drugs (Dretchen ef al., 1977). This presynaptic stabilization of motor nerve endings prevents the postjunctional augmented responses. It therefore seems reasonable to presume that the actions of phenytoin and other calcium antagonists prevent a calcium-mediated depolarization of motor nerve terminals and subsequent excessive neurotransmitter release. It is also probable that at least some of these agents relax bronchiolar smooth muscle (Patel, 1981; Pate1 et al., 1983; Corris et al., 1983) and hence ameliorate one of the contributing factors to DFP-induced fatality. Additional crucial sites for both DFP action
and protection
against DFP are within the CNS. One possible means of exerting protection in the CNS would be to prevent convulsions which are a prominent feature of DFP poisoning. The data, however, indicate that an anticonvulsant action, per se, is not protective. The central respiratory centers are vulnerable to DFP and are depressed with lesser doses than required for neuromuscular impairment (Marx et al., 1984). In addition, we have recently observed that phenytoin pretreatment confers protection against DFP-induced respiratory depression in the cat (Marx et al., 1984).
Thus, it appears that phenytoin and several calcium antagonists have the capacity to enter the brain and exert an action to protect vital central functions. It is unclear at this time why diltiazem, a nondihydropyridine calcium antagonist, failed to exert a protective effect. Perhaps the variant chemical structure of diltiazem does not penetrate the blood-brain barrier in quantities sufficient to influence DFP poisoning. The present experiments demonstrate that a new class of agents, namely, drugs which impair calcium movements, exert a protective action against DFP poisoning and substantially and significantly enhance protection afforded by optimum contemporary pretreatments. REFERENCES CORRIS.P. A.. NARIMAN, S.. AND GIBSON, G. J. (1983). Nifedipine in the prevention of asthma induced by exercise and histamine. Amer. Rev. Respir. Dis. 128,99 I 992.
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HORTON, R. G., KOELL, G. B., MCNAMMARA, B. P.. AND PRATT, H. (1946). The acute toxicity of diisopropylfluorophosphate. J. Pharmacol. Exp. Ther. 87.4 14-420.
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SHORT COMMUNICATIONS KING, T.. AND POULSEN, E. (1958). The action of an aldoxime (2-pyridine aldoxime methiodide) on acute alkylphosphate poisoning in mice. Arch. Int. Pharmacodyn. Ther. 114, 118-121.
MARX, K. A., ANASTASI, N-C., HERNANDEZ, Y. M., FIVOZINSKY, K. B., RAINES, A., AND DRETCHEN, K. L. ( 1984). Protection by phenytoin against the toxic effects of organophosphates on central respiratory centers. Sot. Neurosci.
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SOHN, R. S., AND FERRENDELLI, J. A. (1976). Anticonvulsant drug mechanisms. Arch. Neurol. 33, 626-629. STANDAERT, F. G., AND RIKER, W. F. (1967). The consequences of cholinergic drug actions on motor nerve terminals. Ann. N. Y. Acad. Sci. 144, 5 17-533. STEEL, R., AND TORRIE, J. H. (1960). Principles and Procedures of Sfatisks. McGraw-Hill, New York. WILSON, I. B. (1959). Molecular complementarity and antidotes for alkylphosphate poisoning. Fed. Proc. 18, 752-758.
YAARI, Y.. PINCUS, J. H., AND ARGOV, 2. (1977). Depression of synaptic transmission by diphenylhydantoin. Ann. Neural. 1, 334-338. YAARI, Y., PINCUS,J. H., AND ARGOV. 2. (1979). Phenytoin and transmitter release at the neuromuscular junction of the frog. Brain Res. 160, 497. KENNETH L. DRETCHEN ALYCE M. BOWLES ARTHUR RAINES Department of Pharmacology Georgetown University Schools qf Medicine and Dentistry 3900 Reservoir Road, N. W. Washington. DC 20007 Received Ju1.v 1, 1985