- Email: [email protected]
Anticonvulsive effects of 2,4-dichlorophenoxyacetic acid in mice susceptible to audiogenic seizures
EXPERIMENTAL
NEUROLOGY
6,
30-35 (1962)
Anticonvulsive Effects of 2,4-Dichlorophenoxyacetic Acid in Mice Susceptible to Audiogenic Seizures B. ESSMAN,
FREDERICK N. SUDAK, WALTER Departments
of Physiology
amd Amto~y,
Albert
AND MAX Einstein
College
HAMBURGH~ of Medicine,
New York, New York Received
February
27, 1962
The anticonvulsive effects of 2,4-dichlorophenoxyacetic acid (2,4-D) were lnvestigated in a strain of mice genetically predisposed to audiogenic seizure. Animals injected intraperitoneally with 100, 125, and 150mg per kg of 2,4-D failed to respond within 60sec to a 90-db noise with a convulsive seizure. The ED,, of this drug was found to be 75 mg per kg. An increase in latency to seizure occurred in mice treated with 50 and 7.5mg per kg. The onset of action of 100mg per kg, as indicated by an increase in latency to seizure, was found 5 min after treatment, and complete protection against convulsive attack occurred 1.5min following injection. The protective action of this compound (1OOmg per kg) remained effective for 24 hours, but was no longer active in seizure-susceptible mice 48 hours after treatment. Introduction
Sound-induced seizures have been studied in rodents in relation to the protective effect offered by various drugs. In general, phenothiazine ataractics (6), sympathomimetic agents, and a number of compounds that reduce metabolic rate, etc. (l), have been found to be effective in reducing the incidence of audiogenic seizure in seizure-susceptible animals. Drawbacks in making use of such compounds are the interactive physiological side-effects that these drugs initiate. One is faced with the problem of separating the primary causes from the secondary effects of such compounds on audiogenic seizure. With anesthetics and barbiturates, especially (2)) the effects upon motor and sensory functions may obviate any direct effects that such agents exert upon the seizure mechanism. The purpose of the present investigation was to study the effect on 1 This study was supported in part by Grants M-5140 National Institutes of Health. 30
and B-1716 from the
EFFECTS
OF
31
2,4-D
audiogenic seizure of 2,4-dichlorophenoxyacetic acid (2,4-D), a compound which is neither an anesthetic nor behaviorally depressing. It has been shown previously to act as a thermolytic agent in rodents (3,7), although it does not interfere with performance or cognitive functions (3). Initially, we proposed to utilize this drug to facilitate reduction of body temperature in mice to study temperature reduction as an independent variable in audiogenic seizure. We noted that even in the absence of reduced body temperature the compound had a protective effect against convulsive seizure when auditory stimulation was presented. Consequently, our purpose became one of specifically examining the parameters of 2,4-D action in relation to audiogenic seizures. Methods One hundred and fourteen Swiss albino mice, weighing approximately 15 gm, inbred for susceptibility to audiogenic seizure,2 were used in three experiments designed to study the protective effects of 2,4-D on audiogenic seizure. The apparatus used to induce seizure consisted of a wire-enclosed cage 30.5 cm in diameter placed 12.7 cm below a bell capable of generating a 90-db noise. Mice were tested for seizure-susceptibility 24 to 48 hours before they were used in experiments and latency to seizure recorded. The time was manually recorded by means of stop watch from the beginning of bell ringing to the end of running and the onset of clonus. The auditory stimulus was stopped at the onset of clonus. The auditory stimulus was stopped at the onset of the convulsive attack, or at 60sec if no seizure was induced. Positive-pressure artificial respiration was administered to convulsing mice until spontaneous respiration was re-established. . Experimental animals received intraperitoneal injections of 2,4-D, while control animals were given intraperitoneal injections of an equivalent volume of physiological saline solution. Experiment 1. To determine the dose-response relationship, fifty-six animals received 2,4-D as follows: nine, 25 mg per kg; ten, 50 mg per kg; ten, 75 mg per kg; seven, 100 mg per kg; eleven, 125 mg per kg; and nine, 150 mg per kg. The eleven controls received physiological saline solution (0.9% NaCl), 2.5 ml per kg. They were tested for susceptibility to audiogenic seizure 30 min after injection. 2 The mice were York, New York.
obtained
from
a commercial
supplier,
Mrs.
Flora
O’Grady,
New
32
SUDAK, ESSMAN, AND HAMBURGH
Experiment 2. The duration of protective action of 2,4-D was fnvestigated after having established the EDloo at 100 mg per kg. Mice tested for susceptibility to audiogenic seizure 24 to 48 hours prior to the experiment were randomly assigned to four groups of seven, eleven, nine, and eleven mice. The drug was injected as follows: group 1, 100 mg per kg; group 2, 125 mg per kg; and group 3, 150 mg per kg. Group 4 received 2.5 ml per kg of saline solution. Animals in each group were tested at 24, 48, and 72 hours following injection. Experiment 3. The time of onset of the protective effect of 100 mg per kg of 2,4-D following intraperitoneal injection was determined in the following manner. Twenty seizure-susceptible mice were randomly assigned to three groups. Eight mice were tested for seizure at 5 min; six at 10 min; and six at 15 min following injection of 2,4-D. Results
Experiment 1. Table 1 contains a summary of the results of the relationship between various doses of 2,4-D, and the protective effect upon RELATIONSHIP
OF DOSE OF
TABLE 1 2.4-D TO EFFECT ON AUDIOCENIC
SEIZURE
Mean response latency to seizure Dose 2,4-D Control 25w/kg 50 mdkg 75 w/kg 100 mg/kg 125 mg/kg lSOmg/kg
N 11 9 10 10
7 11 9
Seizure
Initiala
Testb
N
%
-FY 7.5 7.9 8.1 10.2 7.3 7.2
set 8.0 9.6 38.3 36.0 -
11 9 6 5 0 0 0
100 100 60 50 0 0 0
0 Latency to seizure 24 to 28 hours prior to experiment. b Latency to seizure after injection.
audiogenic seizure. Doses of 100 mg per kg (EDloo) and above provided complete protection in the mice tested. Some animals in this group responded to the auditory stimulus by running, but stopped shortly after and failed to convulse within 60 set of stimulation. Other animals failed to run at all during the test period. The ED5,, was established at 75 mg per kg. Lower doses, while they did not afford complete protection against audiogenic seizure, increased the threshold to such seizures.
100
12.5
150 Control
to seizure to seizure
2
3 4
0 Latency b Latency
(w/W
2,4-D
1
Grp.
Dose 7
N
prior
7.2 7.7
7.3
6.4
a.5
13.9
27.5
10.8
24
TABLE OF VARIOUS
Mean lat.” (set)
to experiment.
Before Mean lat.a (set)
OF PRCYTECTIVE ACTION
24 to 48 hours after injection.
9 11
11
DURATION
78
100
73 12.4 8.6
9.3
7.5
a5
48
injection
Seiz. (%)
after
89 100
92
loo
7.1 8.3
7.1
7.5
Mean lat. (set)
SEIZURES
Seiz. (%>
ON AUDIOGENE
Mean lat. (set)
Hours
2 DOSES OF 2,4-D
72
100 100
82
loo
Seiz. ($3)
g Iu P u
z 2 2
34
SUDAK,
ESSMAN,
AND
HAMBURGH
The mean latencies to seizure of animals treated with 50 and 75 mg per kg increased from 7.9 to 38.3 set and from 8.1 to 36 set, respectively. All animals treated with 25 mg per kg of 2,4-D convulsed when tested, as did control mice injected with physiological saline solution. The mean TABLE ONSET OF PROTECTIVE ACTION Grp.
N
1 2 3
8 6 6
0 Latency b Latency
Mean initial laba (set)
3 OF 100~~
Time after inj. (min)
7.3 7.0 7.3
to seizure 24 to 48 hours to seizure after injection.
5 10 15 prior
PER KG 2.4-D Mean test lat.b (set) 21.1 58.8 -
Seiz. (%) 100 33 0
to experiment.
latencies to seizure of both of these latter groups fell within control limits. Effective doses of 100 and 125 mg per kg of 2,4-D, while conferring complete protection against audiogenic seizure, did not appear to affect motor activity appreciably or produce confusion in these mice. Mild myotonia became apparent only in the group treated with 150 mg per kg of 2,4-D. None of the other doses used was toxic to the animals. Experiment 2. Duration of the protective action of the various doses of 2,4-D in mice susceptible to audiogenic seizure is summarized in Table 2. No trace of the protective effect of 100 mg per kg, as measured by either an increase in response latency or by the percentage of animals showing seizures, was present by 48 hours following injection. Larger doses (150 mg per kg) were completely ineffective after 72 hours. Experiment 3. The latency to seizure had increased approximately three-fold by 5 min after injection of 100 mg per kg of 2,4-D. Complete protection to sound-induced seizure was established for all mice tested 15 min after administration of the drug (Table 3). Discussion
These experiments demonstrate that 2,4-D in doses of 100 to 150 mg per kg completely protected mice susceptible to audiogenic seizure against convulsive attack. Doses of 50 and 75 mg per kg offered some protection, in that the latency to seizure was increased. The observation that neither the EDS0 nor the E.Dloo doses were toxic to audiogenic mice corroborates the work by Hill and Carlisle (S), who have reported the LDSO of 2,4-D to be 37.5 mg per kg in their strain of mice. Based on these findings, the “therapeutic index” of 2,4-D for protecting mice against
EFFECTS OF 2,4-D
35
sound-induced seizure was quite large and indicated a wide margin of safety in its use. Our experiments suggest that 2,4-D raises the threshold to seizure. Although dosesof 150 mg per kg produced myotonia in some animals, the implication that myotonia serves to elevate seizure-threshold seems unlikely because no gross manifestations of myotonia were evident in those animals completely protected from audiogenic seizure with 100 mg per kg of 2,4-D. The observation (Expt. 2) that 125 mg per kg was more effective in providing complete protection against audiogenic seizure at 72 hours than the higher dose of 150 mg per kg may be accounted for by factors other than those related to dosage, and may reside in the heterogeneity of the population used. The suggestion advanced by Fink and Swinyard (4), that any drug capable of interfering with running behavior protects the animal against seizure, is not borne out in the present investigation. Doses of 2,4-D affording complete protection against audiogenic seizure did not suppress preconvulsive running behavior in all animals. Conditions prerequisite for seizure probably involve intact auditory and motor pathways. Effective dosesof 2,4-D, while protecting against seizure, did not completely inhibit preconvulsive motor activity or suppress the startle response typically elicited in mice in response to sound. This demonstrates the intactness of both motor and auditory pathways in the presence of this compound. It may be, therefore, that 2,4-D does not exert its effect on a peripheral mechanism, but upon central nervous system mechanismsinvolved in audiogenic seizure. References
1. BEVAN, W. 1955. Sound-precipitated convulsions. Psychol. Bull. 62: 473-504. 2. CAIN, J., and J. MERCIER. 1948. Influence de certain medicaments anticonvulsants sur la crisi audiogene du rat albinos. Compt. rend. sot. biol. 142: 688-691. 3. ESSMAN, W. B., and F. N. &DAK. 1962. Effect of body temperature reduction on response acquisition in mice. J. Appl. Phyriol. 17: 113-116. 4. FINK, G. B., and E. A. SWINYARD. 1959. Modification of maximal audiogenic and electroshock seizures in mice by psychopharmacologic drugs. J. Pharmacol. Exptl. Therap. 127: 318-324. 5. HILL, E. V., and H. CARLISLE. 1947. Toxicity of 2,4-Dichlorophenoxyacetic acid for experimental animals. /. Znd. Hyg. Tmicol. 29: 85-95. 6. PLOTNIKOFF, N. 1961. Drug resistance due to inbreeding. Science 134: 1881-1882. 7. SUDAK, F. N., and C. L. CLAN. 1960. Effect of 2,4-Dichlorophenoxyacetic acid on thermoregulation in rats. Federation Proc. 19: 43.
NEUROLOGY
6,
30-35 (1962)
Anticonvulsive Effects of 2,4-Dichlorophenoxyacetic Acid in Mice Susceptible to Audiogenic Seizures B. ESSMAN,
FREDERICK N. SUDAK, WALTER Departments
of Physiology
amd Amto~y,
Albert
AND MAX Einstein
College
HAMBURGH~ of Medicine,
New York, New York Received
February
27, 1962
The anticonvulsive effects of 2,4-dichlorophenoxyacetic acid (2,4-D) were lnvestigated in a strain of mice genetically predisposed to audiogenic seizure. Animals injected intraperitoneally with 100, 125, and 150mg per kg of 2,4-D failed to respond within 60sec to a 90-db noise with a convulsive seizure. The ED,, of this drug was found to be 75 mg per kg. An increase in latency to seizure occurred in mice treated with 50 and 7.5mg per kg. The onset of action of 100mg per kg, as indicated by an increase in latency to seizure, was found 5 min after treatment, and complete protection against convulsive attack occurred 1.5min following injection. The protective action of this compound (1OOmg per kg) remained effective for 24 hours, but was no longer active in seizure-susceptible mice 48 hours after treatment. Introduction
Sound-induced seizures have been studied in rodents in relation to the protective effect offered by various drugs. In general, phenothiazine ataractics (6), sympathomimetic agents, and a number of compounds that reduce metabolic rate, etc. (l), have been found to be effective in reducing the incidence of audiogenic seizure in seizure-susceptible animals. Drawbacks in making use of such compounds are the interactive physiological side-effects that these drugs initiate. One is faced with the problem of separating the primary causes from the secondary effects of such compounds on audiogenic seizure. With anesthetics and barbiturates, especially (2)) the effects upon motor and sensory functions may obviate any direct effects that such agents exert upon the seizure mechanism. The purpose of the present investigation was to study the effect on 1 This study was supported in part by Grants M-5140 National Institutes of Health. 30
and B-1716 from the
EFFECTS
OF
31
2,4-D
audiogenic seizure of 2,4-dichlorophenoxyacetic acid (2,4-D), a compound which is neither an anesthetic nor behaviorally depressing. It has been shown previously to act as a thermolytic agent in rodents (3,7), although it does not interfere with performance or cognitive functions (3). Initially, we proposed to utilize this drug to facilitate reduction of body temperature in mice to study temperature reduction as an independent variable in audiogenic seizure. We noted that even in the absence of reduced body temperature the compound had a protective effect against convulsive seizure when auditory stimulation was presented. Consequently, our purpose became one of specifically examining the parameters of 2,4-D action in relation to audiogenic seizures. Methods One hundred and fourteen Swiss albino mice, weighing approximately 15 gm, inbred for susceptibility to audiogenic seizure,2 were used in three experiments designed to study the protective effects of 2,4-D on audiogenic seizure. The apparatus used to induce seizure consisted of a wire-enclosed cage 30.5 cm in diameter placed 12.7 cm below a bell capable of generating a 90-db noise. Mice were tested for seizure-susceptibility 24 to 48 hours before they were used in experiments and latency to seizure recorded. The time was manually recorded by means of stop watch from the beginning of bell ringing to the end of running and the onset of clonus. The auditory stimulus was stopped at the onset of clonus. The auditory stimulus was stopped at the onset of the convulsive attack, or at 60sec if no seizure was induced. Positive-pressure artificial respiration was administered to convulsing mice until spontaneous respiration was re-established. . Experimental animals received intraperitoneal injections of 2,4-D, while control animals were given intraperitoneal injections of an equivalent volume of physiological saline solution. Experiment 1. To determine the dose-response relationship, fifty-six animals received 2,4-D as follows: nine, 25 mg per kg; ten, 50 mg per kg; ten, 75 mg per kg; seven, 100 mg per kg; eleven, 125 mg per kg; and nine, 150 mg per kg. The eleven controls received physiological saline solution (0.9% NaCl), 2.5 ml per kg. They were tested for susceptibility to audiogenic seizure 30 min after injection. 2 The mice were York, New York.
obtained
from
a commercial
supplier,
Mrs.
Flora
O’Grady,
New
32
SUDAK, ESSMAN, AND HAMBURGH
Experiment 2. The duration of protective action of 2,4-D was fnvestigated after having established the EDloo at 100 mg per kg. Mice tested for susceptibility to audiogenic seizure 24 to 48 hours prior to the experiment were randomly assigned to four groups of seven, eleven, nine, and eleven mice. The drug was injected as follows: group 1, 100 mg per kg; group 2, 125 mg per kg; and group 3, 150 mg per kg. Group 4 received 2.5 ml per kg of saline solution. Animals in each group were tested at 24, 48, and 72 hours following injection. Experiment 3. The time of onset of the protective effect of 100 mg per kg of 2,4-D following intraperitoneal injection was determined in the following manner. Twenty seizure-susceptible mice were randomly assigned to three groups. Eight mice were tested for seizure at 5 min; six at 10 min; and six at 15 min following injection of 2,4-D. Results
Experiment 1. Table 1 contains a summary of the results of the relationship between various doses of 2,4-D, and the protective effect upon RELATIONSHIP
OF DOSE OF
TABLE 1 2.4-D TO EFFECT ON AUDIOCENIC
SEIZURE
Mean response latency to seizure Dose 2,4-D Control 25w/kg 50 mdkg 75 w/kg 100 mg/kg 125 mg/kg lSOmg/kg
N 11 9 10 10
7 11 9
Seizure
Initiala
Testb
N
%
-FY 7.5 7.9 8.1 10.2 7.3 7.2
set 8.0 9.6 38.3 36.0 -
11 9 6 5 0 0 0
100 100 60 50 0 0 0
0 Latency to seizure 24 to 28 hours prior to experiment. b Latency to seizure after injection.
audiogenic seizure. Doses of 100 mg per kg (EDloo) and above provided complete protection in the mice tested. Some animals in this group responded to the auditory stimulus by running, but stopped shortly after and failed to convulse within 60 set of stimulation. Other animals failed to run at all during the test period. The ED5,, was established at 75 mg per kg. Lower doses, while they did not afford complete protection against audiogenic seizure, increased the threshold to such seizures.
100
12.5
150 Control
to seizure to seizure
2
3 4
0 Latency b Latency
(w/W
2,4-D
1
Grp.
Dose 7
N
prior
7.2 7.7
7.3
6.4
a.5
13.9
27.5
10.8
24
TABLE OF VARIOUS
Mean lat.” (set)
to experiment.
Before Mean lat.a (set)
OF PRCYTECTIVE ACTION
24 to 48 hours after injection.
9 11
11
DURATION
78
100
73 12.4 8.6
9.3
7.5
a5
48
injection
Seiz. (%)
after
89 100
92
loo
7.1 8.3
7.1
7.5
Mean lat. (set)
SEIZURES
Seiz. (%>
ON AUDIOGENE
Mean lat. (set)
Hours
2 DOSES OF 2,4-D
72
100 100
82
loo
Seiz. ($3)
g Iu P u
z 2 2
34
SUDAK,
ESSMAN,
AND
HAMBURGH
The mean latencies to seizure of animals treated with 50 and 75 mg per kg increased from 7.9 to 38.3 set and from 8.1 to 36 set, respectively. All animals treated with 25 mg per kg of 2,4-D convulsed when tested, as did control mice injected with physiological saline solution. The mean TABLE ONSET OF PROTECTIVE ACTION Grp.
N
1 2 3
8 6 6
0 Latency b Latency
Mean initial laba (set)
3 OF 100~~
Time after inj. (min)
7.3 7.0 7.3
to seizure 24 to 48 hours to seizure after injection.
5 10 15 prior
PER KG 2.4-D Mean test lat.b (set) 21.1 58.8 -
Seiz. (%) 100 33 0
to experiment.
latencies to seizure of both of these latter groups fell within control limits. Effective doses of 100 and 125 mg per kg of 2,4-D, while conferring complete protection against audiogenic seizure, did not appear to affect motor activity appreciably or produce confusion in these mice. Mild myotonia became apparent only in the group treated with 150 mg per kg of 2,4-D. None of the other doses used was toxic to the animals. Experiment 2. Duration of the protective action of the various doses of 2,4-D in mice susceptible to audiogenic seizure is summarized in Table 2. No trace of the protective effect of 100 mg per kg, as measured by either an increase in response latency or by the percentage of animals showing seizures, was present by 48 hours following injection. Larger doses (150 mg per kg) were completely ineffective after 72 hours. Experiment 3. The latency to seizure had increased approximately three-fold by 5 min after injection of 100 mg per kg of 2,4-D. Complete protection to sound-induced seizure was established for all mice tested 15 min after administration of the drug (Table 3). Discussion
These experiments demonstrate that 2,4-D in doses of 100 to 150 mg per kg completely protected mice susceptible to audiogenic seizure against convulsive attack. Doses of 50 and 75 mg per kg offered some protection, in that the latency to seizure was increased. The observation that neither the EDS0 nor the E.Dloo doses were toxic to audiogenic mice corroborates the work by Hill and Carlisle (S), who have reported the LDSO of 2,4-D to be 37.5 mg per kg in their strain of mice. Based on these findings, the “therapeutic index” of 2,4-D for protecting mice against
EFFECTS OF 2,4-D
35
sound-induced seizure was quite large and indicated a wide margin of safety in its use. Our experiments suggest that 2,4-D raises the threshold to seizure. Although dosesof 150 mg per kg produced myotonia in some animals, the implication that myotonia serves to elevate seizure-threshold seems unlikely because no gross manifestations of myotonia were evident in those animals completely protected from audiogenic seizure with 100 mg per kg of 2,4-D. The observation (Expt. 2) that 125 mg per kg was more effective in providing complete protection against audiogenic seizure at 72 hours than the higher dose of 150 mg per kg may be accounted for by factors other than those related to dosage, and may reside in the heterogeneity of the population used. The suggestion advanced by Fink and Swinyard (4), that any drug capable of interfering with running behavior protects the animal against seizure, is not borne out in the present investigation. Doses of 2,4-D affording complete protection against audiogenic seizure did not suppress preconvulsive running behavior in all animals. Conditions prerequisite for seizure probably involve intact auditory and motor pathways. Effective dosesof 2,4-D, while protecting against seizure, did not completely inhibit preconvulsive motor activity or suppress the startle response typically elicited in mice in response to sound. This demonstrates the intactness of both motor and auditory pathways in the presence of this compound. It may be, therefore, that 2,4-D does not exert its effect on a peripheral mechanism, but upon central nervous system mechanismsinvolved in audiogenic seizure. References
1. BEVAN, W. 1955. Sound-precipitated convulsions. Psychol. Bull. 62: 473-504. 2. CAIN, J., and J. MERCIER. 1948. Influence de certain medicaments anticonvulsants sur la crisi audiogene du rat albinos. Compt. rend. sot. biol. 142: 688-691. 3. ESSMAN, W. B., and F. N. &DAK. 1962. Effect of body temperature reduction on response acquisition in mice. J. Appl. Phyriol. 17: 113-116. 4. FINK, G. B., and E. A. SWINYARD. 1959. Modification of maximal audiogenic and electroshock seizures in mice by psychopharmacologic drugs. J. Pharmacol. Exptl. Therap. 127: 318-324. 5. HILL, E. V., and H. CARLISLE. 1947. Toxicity of 2,4-Dichlorophenoxyacetic acid for experimental animals. /. Znd. Hyg. Tmicol. 29: 85-95. 6. PLOTNIKOFF, N. 1961. Drug resistance due to inbreeding. Science 134: 1881-1882. 7. SUDAK, F. N., and C. L. CLAN. 1960. Effect of 2,4-Dichlorophenoxyacetic acid on thermoregulation in rats. Federation Proc. 19: 43.