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The effect of sodium fluoride on responses to various central nervous system agents in rats
TOXICOLOGY
AND
The
APPLIED
Effect
Various
PHARMACOLOGY
3,
of Sodium Central
31-38
(1961)
Fluoride Nervous
on
Responses
System
Agents
to in
Rats’ F. C. Lu,
IRENA
M.
AND Food
and Drug
R. S.
MAZURKIEWICZ,
Laboratories,
M. G.
GREWAL,
ALLMARK,
P. BOIVIN
Department
Ottawa, Received
of National Canada
June
Health
and
Welfare,
6, 1960
The usefulness of fluoride in the control of dental caries has long been recognized (Erhardt, 1874) and more recently has received world-wide attention (World Health Organization, 1957). In order to ascertain the safety of fluoride in general and fluoridation of drinking water in particular, the toxicity of fluoride has been studied extensively in various domestic and laboratory animals as well as in man and the data have been well documented. Certain outstanding aspects have been dealt with in a publication by Shaw (1954). Since relatively little is known about its effect on the central nervous system, the following experiments were designed in an attempt to provide some information on this point. METHODS
Two series of experiments were carried out on female rats of the Wistar strain, each weighing approximately 130 g. In the first series a group of 48 rats received sodium fluoride intraperitoneally at a dosage of 10 mg/kg/day. The same number of rats were given injections of saline and served as controls. In the second series of experiments the rats were given sodium fluoride orally. A total of 150 rats were divided into three groups. Two groups, each consisting of 50 rats, were given diets with added sodium fluoride amounting to 15 and 150 parts per million (ppm), respectively (equivalent to 7 and 70 ppm in terms of fluoride). The other 50 rats were kept on a control diet. Fluoride was added to the diet instead of to the water 1 Presented in part cology and Experimental
at the Annual Therapeutics,
Meeting April 31
of the American 14, 1959, Atlantic
Society for PharmaCity, New Jersey.
32
F. C. LU
ET
AL.
becausethe amount of diet, and therefore the amount of additional fluoride, ingested by the rats could be determined much more accurately. McClure (1939) and Machle and Largent (1932) found in the rat that NaF added to the diet, like NaF added to the drinking water, was almost completely available. The fluoride content in the control diet used in our experiments was SO-60 ppm. Since the fluoride in the control diet derived mainly from bone meal, only a small fraction of it was absorbable in the alimentary tract (Machle and Largent, 1943; Taylor and Gardner, 1959). There was therefore an appreciable difference in the amount of fluoride available in the control diet and that in the diet containing added NaF. The drinking water used by all animals contained about 0.3 ppm fluoride. These rats were allowed water and diet ad libitum. Body weight and food consumption were recorded weekly. The average food consumption was about 11 g per rat per day in the first week and about 15 g per rat per day in the fifteenth week. The initial body weight averaged 110 g: and 15 weeks later it was about 210 g. On the basis of the food consumption and body weight, the sodium fluoride ingested by the rats on the high level was about 15 mg/kg/‘day at the start; it decreased to about 11 mg/kg/day after 15 weeks’ treatment. These figures do not include the fluorides in the basic diet and the water. The amount of the additional fluoride taken by these rats was therefore comparable to that received by the treated rats of the first series. The rats on the low level of sodium fluoride received one-tenth of this amount. During the course of the experiments the sensitivity of the treated rats to a number of agents that are active on the central nervous system (CNS) was compared with that of the control rats. The following procedures were used. Pentylertetetrazol-induced convulsions. The drug was given by the intraperitoneal (i.p.) route and the percentage of rats showing convulsions on each dose was recorded. The percentages were converted to probits and the statistical analysis of the probit versus the logarithmic dose was carried out by the method of Bliss (I935a,b). In some tests, only one dose of pentylenetetrazol was given; the results were evaluated by the Chi square test. The drug was given by the intraStrychnine-induced convulsions. peritoneal route and the calculations were done as described above. ELectroskock-induced convulsions. The electroshock (60 cycle AC, 10 mA for 0.2 seconds) was applied through needle electrodes inserted
SODIUM
FLUORIDE
AND
CNS
33
EFFECT
in the ears. Tonic extensor seizure was used as the end point. The number of rats showing extensor seizures was recorded and the data were analyzed by applying the Chi square test. The anti-extensor seizure activity of diphenylhydantoin. The response of all rats to electroshock (60 cycle AC, 15 mA for 0.2 sec.) was determined on the first day. Out of each group, 16 of the rats that showed tonic extensor seizures were given diphenylhydantoin sodium (20 mg/kg, i.p.) on the following day. Their response to electroshock was tested 1.5 minutes after the injection; the number of rats showing convulsions was recorded and the data were analyzed by applying the Chi square test. Pentobarbital-induced sleeping time. Pentobarbital sodium was given by the intraperitoneal route at 1 or 2 dose levels (20 mg/kg and 25 mg/kg) . Each dose was administered to 10 rats of each of the control and the two fluoride-treated groups. These rats were kept in individual cages and the duration (in minutes) from injection to the appearance of righting reflex in each rat was recorded. Analysis of the results was done according to the method of Gaddum (19.53). RESULTS
In the first series of experiments, pentylenetetrazol was given by the intraperitoneal route to the rats after they had been on test for 4 weeks. The details are given in Table 1. The activity of pentylenetetrazol to induce convulsions in the fluoride-treated rats was 110.3 I+ 7.1 y0 in terms of that in the control rats. Therefore there was a slight but insignificant increase in the sensitivity to pentylenetetrazol after 4 weeks’ treatment with fluoride. TABLE EFFECTS
OF FLUORIDE
TREATMENT
ON THE
AND
(after Group
STRYCHNINE
NaF-treated
of rats showing
TO PENTYLENETETRAZOL
(after Dose (ma/kd
Responsea
Strychnine 5 weeks’ treatment) Responsea
72.0
9/10
2.57
t/9
60.0 50.0
S/15 4/15
2.20 1.88
5/9 2/Y
60.0 50.0
S/10 4/10
2 .oo
1.71
8/9 5/9
1.46
219
41.7
(1 Number
OF RATS
SULFATE
Pentylenetetrazol 4 weeks’ treatment)
Dose (mn/ka)
Saline-treated
1 RESPONSE
convulsions
l/10
per number
of rats
tested.
34
F. C. LU
ET
AL.
One week later, the sensitivity of these two groups of rats to strychnine was compared in a similar manner. The details are also shown in Table 1. Analysis of the data showed that the dose-response lines for the two groups were parallel and the activity of strychnine to induce convulsions in the treated rats was 131.2 A 8.8 s in terms of that in the control rats. The difference between these two groups was highly significant. Since the first test on pentylenetetrazol was inconclusive, it was repeated after the rats had been treated for 11 weeks. Pentylenetetrazol (50 mg/kg) was given intraperitoneally to the rats of both groups. The results listed in Table 2 show that the sensitivity to this CNS stimulant TABLE EFFECTS
OF FLUORIDE
TREATMENT
ON THE AND
(after
Group
2 RESPONSE
OF RATS
ro
Pentylenetetrazol 11 weeks’ treatment)
(after
Electroshock 14 weeks’treatment)
S/19 11113”
Saline-treated NaF-treated a Statistically
significant.
PENTYLENETETRAZOL
ELECTROSHOCK
2/19 u/13”
P = 0.02.
b P= 0.001.
was definitely increased in the fluoride-treated rats. These rats were also found to be more sensitive to electroshock (Table 2). This test was carried out after the rats had been treated for 14 weeks. During the course of the second series of experiments, the sensitivity to electroshock, pentylenetetrazol, and strychnine was determined in the three groups of rats after they had been on treatment for 15, 16, and 19 weeks, respectively. The results are listed in Table 3. It may be noted TABLE EFFECTS
OF FLUORIDE
Group Control Low level of NaF High level of NaF 5 43.4 mg/kg. b 1.2 mg/kg.
TREATMENT
3
ON THE SENSITIVITY STIMULATIONS
Electroshock 14/25 24/25d 17/25
OF RATS
TO SOME
Pentylenetetrazola
Strychnineb
2/10 10/13* 9/10d C Significant d Significant
CNS
4/15 l/15
lO/lV at at
P = 0.05. P = 0.01.
that the sensitivity to pentylenetetrazol was increased in both groups of fluoride-treated rats, whereas that to strychnine was raised only in the rats on the high level of fluoride. The responseto electroshock was some-
SODIUM
FLUORIDE
AND
CNS
35
EFFECT
what enigmatic since the sensitivity was enhanced in the rats receiving the low level of fluoride, but not in those on the high level. The activity of pentobarbital to induce “sleep” was also determined in these rats after they had been treated for 18 weeks. The results showed that the hypnotic activity of pentobarbital was 113.4 + 4.0 s in the rats treated with the low level of sodium fluoride and 153.9 * 3.5 5% in those on the high level, as compared to the controls. The antiextensor seizure activity of diphenylhydantoin was studied after the rats had been on test for 21 weeks. In the control group 6 rats were protected by this antiepileptic agent. In the low and high fluoride level groups, 10 and 12 were protected, respectively. The difference between the rats on the high level of fluoride-treated and the controls was significant (P = 0.02). In order to determine the reversibility of the effects of fluoride on the CNS, some of the test procedures were repeated after the fluoride-treated rats had been given the control diet for 2 weeks. The results obtained in these tests are listed in Table 4. It is evident that the effects of fluoride TABLE THE
REVERSIBILITY
OF THE
EFFECTS
Group@
Pentylenetetrazolb
Control Low level of NaF High level of NaF (1 b C d
All groups on control diet for 45 mg/kg i.p. 20mg/kg i.p. Sleeping time in minutes.
4
OF FLUORIDE
2-3
ON THE
CENTRAL
NERVOUS
Pentobarbitalcj
9/15
85.6
S/15 7/15
91.0 +- 6.6 94.6 & 5.5
SYSTEM
d
-+ 4.8
weeks.
on the activities of pentylenetetrazol and pentobarbital were reversible. It may be noted that the fluoride treatment did not appreciably affect the gain in body weight. The weights of the heart, liver, kidneys, adrenals, and thyroid were also not significantly altered. DISCUSSION
The experimental results show that the rats that had received sodium fluoride, either intraperitoneally or orally, were more sensitive to electrical as well as chemical stimulations of the central nervous system. Furthermore, they were more sensitive to CNS depressants. A simultaneous increase in sensitivity of the CNS to both stimulants and depressants is
36
F. C. LU
ET
AL.
not unique. For example, pretreatment of mice with diphenhydramine has been found to augment the effects of both pentobarbital and strychnine (Sherman, 1956). Similarly, chlorpromazine has been found to potentiate the CNS stimulant action of picrotoxin in cats (Marquardt et al., 1955) as well as to prolong the sleeping time induced by butabarbital in mice (Courvoisier et al., 1953) and increase the protective effect of diphenylhydantoin against electroshock in rats (Bertrand et al., 1955). A number of factors are known to affect the threshold of electroshock and the duration of barbiturate-induced sleep. However, the increased sensitivity in the fluoride-treated rats was unlikely a result of a deterioration in their general health or a change in their body water content. This is suggested by the normal growth rate, food consumption, and organ weights in the treated rats as compared to the controls. Fluoride is known to inhibit many enzymes (Colowick and Kaplan, 1955). In the case of cholinesterase, Nachmansohn (1939) found that fluoride, at 1 mM, causeda 38 70 inhibition of the enzyme. At 10 mM, it reduced the cholinesteraseactivity to one-third that of the control (Taylor et a,?., 1952). Since the fluoride concentration in the brain of someanimals exposed to large dosesof fluoride was as high as 5 mM (Boddie, 1949)) a significant inhibition of cholinesterasecould conceivably occur in these animals. It is of interest to note that physostigmine and other cholinesterase inhibitors potentiate the convulsant action of pentylenetetrazol (Gutierrez-Noriega, 1945; Cordova, 1948) and lower the threshold for electroshock (Gellhorn and Ballin, 1948). On the other hand, iproniazid, a monoamine oxidase inhibitor, prolongs the sleeping time induced by barbiturates (Fouts and Brodie, 1956). The effects observed in the fluoride-treated rats were therefore possibly consequential to an inhibition of these two important enzymes in the CNS. An enzyme may be inhibited by fluoride either directly (Slater and Bonner, 1952) or through a change in the concentration of such bivalent cations as Ca, Mg, Mn, etc., in the media (Colowick and Kaplan, 1955). Results concerning the effect of fluoride on the metabolism of these ions in animals are inconclusive. For example, Leone et al. (1956) found that large dosesof sodium fluoride given intravenously to dogsslightly reduced the Ca level in the blood. On the other hand, Schulz (1931) failed to observe an appreciable change in the Ca level in the plasma of rats receiving NaF. In the dogs given NaF for 5 months, Greenwood et al. (1935) found no change in the total Ca or the acid-soluble inorganic P.
SODIUM
FLUORIDE
AND
CNS
EFFECT
37
Administration of very large doses of fluoride has been shown to alter the electrical activity of the brain in the cat (Moruzzi and Bremer, 1938) and to cause convulsions and depression in various species of animals (Leone et al., 1956). The effects of smaller doses of fluoride described herein have not been reported by other investigators. The practical significance of these observations is as yet unknown. It is possible that rats are unique in this respect, and man may respond to fluoride entirely differently. Furthermore, the fact that the increased sensitivity of the CXS was reversible (Table 4) indicates that perhaps no organic damage had occurred in these rats after a S-month treatment with fluoride. SUMM.4RY Rats given sodium fluoride at 10 mg/kg daily by the intraperitoneal route were more sensitive to the convulsant action of strychnine and pentylenetetrazol and to electroshock. This increase in sensitivity to CNS stimulation w-as observed also in two other groups of rats which were given a diet with added sodium fluoride amounting to 7 and 70 parts per million in terms of fluoride. Prolonged treatment with fluoride also lengthened the sleeping time induced by pentobarbital and increased the protective effect of diphenylhydantoin against electroshock. The increase in sensitivity to some CNS-active agents in the fluoride-treated rats disappeared after these rats were given the control diet for 2 weeks, REFERENCES T., and QUIVY, D. (1935). Influence exercbe par certains ganglioplegiques et neuroplegiques sur l’effet anticonvulsivant de la diphenylhydantoine. Arch. intern, pharmacodynamie 100, 283-297. BLISS, C. I. (1935a). The calculation of the dosage-mortality curve. Ann. A#. Biol. 22, 134-167. BLISS, C. I. (1935b). The comparison of dosage-mortality data. Ann. Bppl. Biol. 22, 307-333. BODDIE, G. F. ( 1949). Effects of fluorine compounds on animals in the Fort William area. In: Industrial Fluorosis. A Study of the Hazard to Man and Animals near Fort William, Scotland. A Report to the Fluorosis Committee, Med. Research Council Mem. 1vo. 22 (by J. K. Agate et al.), H.M. Stationery Ofice, London. COLOWICK, S. P., and KAPLAN, N. 0. (eds.). (1955). Methods in En:ymoZogy, Vol. II. Academic Press, New York. CORDOVA, J. A. (1948). Efecto potenciador de 10s medicamentos anticolinoesterasicos sobre las convulsiones cardiazolicas. Rev. farmaco2. y med. ezptl. 1, 196-208. COURVOISIER, S., FOURNEL, J., DUCROT, R., KOLSKY, M., and KOETSCHET, P. (1953). Prop&& pharmacodynamiques du chlorhydrate de chloro-3-(dim&hylamino-3’propyl)-IO-phCnothiazine (4.560 R.P.) ; etude experimentale d’un nouveau corps utilise dans I’anesthbsie potentialiste et dans l’hibernation artificielle. .4rch. intern. pharmacodynamie 92, 305-361. ERHARDT, DR. (1874). II. Kali Ruoratum zur Erhaltung del Zihne. Memorabilien. Monatsh. rationelle Aerste 19, 359-360. BERTRAND,
I.,
GAYET-HALLION,
38
F. C. LU ET AL.
FoUrs, J. R., and BRODIE, B. B. (1956). On the mechanism of drug potentiation by iproniazid (Z-isopropyl-1-isonicotinyl bydrazine) . Pharmacol. Exptl. Therap. 116, 480-485. GAnnuM, J. H. (1953). Simplified mathematics for bioassays. J. Pharm. Pharmacol. 6, 343-358. GELLHORN, E., and BALLIN, H. M. (1948). Age and susceptibility to convulsions. Proc. Sot. Exptl. Biol. Med. 68, 540-543. GREENWOOD, D. A., HEWITT, E. A., and NELSON, V. E. (1935). The effects of fluorine on respiration, blood pressure, coagulation and blood calcium and phosphorus in the dog. J. Am. Vet. Med. Assoc. 86, 28-42. GUTIERREZ-NORIEGA, C. (1945). Epilepsia experimental y drogas colinergicas. Rev. neuro-psiquiatria 8, 121-134. LEONE, N. C., GEEVER, E. F., and MORAN, N. C. (1956). Acute and subacute toxicity studies of sodium fluoride in animals. Public Health Repts. (U. S.) 71, 459-467. MCCLURE, F. J. (1939). Fluorides in food and drinking water. A comparison of effects of water-ingested versus food-ingested sodium fluoride. Natl. Znsts. Health Bull. No. 172. MACHLE, W., and LARGENT, E. J. (1943). The absorption and excretion of fluoride. II. The metabolism at high levels of intake. J. 2nd. Hyg. Toxicol. 26, 112-123. MARQUARDT, P., PUPPEL, H., and SCHUMACHER, H. (1955). Die Wirkung erregender Pbarmaka nach Megapben-vorbehandlung. Klin. Wochschr. 88, 211-215. MORUZZI, G., and BREMER, F. (1938). Action du fluorure de sodium et du bleu de methylene sur I’activitk electrique, spontanee et provoqute de i’ecorce cerebrale. Compt. rend. sot. biol. 129, 884-889. NACHMANSOHN, D. (1939). Sur I’inhibition de la cholinesterase. Compt. rend. sot. biol. 130, 1065-1068. SCHULZ, J, A. (1931). Effects of the ingestion of fluorides on some of the constituents of the teeth, bones, blood and tissues of albino rats. Zowa Agr. Expt. Sta. Ann. Rept., p. 52. SHAW, J. H. (ed.). (1954). Fluoridation as a Public Health Measzrre. American Association for the Advancement of Science, Washington, D. C. Enhancement of the central nervous system effects of SHERMAN, J. F. (1956). strychnine and pentobarbital by diphenhydramine. Science 123, 1170-I 171. SLATER, E. C., and BONNER, W. D. (1952). The effect of fluoride on the succinic oxidase system. Biochem. J. 52, 185-196. TAYLOR, I. M., WELLER, J. M., and HASTINGS, A. B. (1952). Effect of cholinesterase and choline-acetylase inhibitors on the potassium concentration gradient and potassium exchange of human erythrocytes. Am. J. Physiol. 168, 658-665. TAYLOR, J. M., and GARDNER, D. E. (1959). Metabolism of water-borne fluoride in the rat. Federation Proc. 18, 450. World Health Organization (1958). First Report of the Expert Committee on Water Fluoridation. World Health Organization Tech. Rept. Ser. No. 146.
AND
The
APPLIED
Effect
Various
PHARMACOLOGY
3,
of Sodium Central
31-38
(1961)
Fluoride Nervous
on
Responses
System
Agents
to in
Rats’ F. C. Lu,
IRENA
M.
AND Food
and Drug
R. S.
MAZURKIEWICZ,
Laboratories,
M. G.
GREWAL,
ALLMARK,
P. BOIVIN
Department
Ottawa, Received
of National Canada
June
Health
and
Welfare,
6, 1960
The usefulness of fluoride in the control of dental caries has long been recognized (Erhardt, 1874) and more recently has received world-wide attention (World Health Organization, 1957). In order to ascertain the safety of fluoride in general and fluoridation of drinking water in particular, the toxicity of fluoride has been studied extensively in various domestic and laboratory animals as well as in man and the data have been well documented. Certain outstanding aspects have been dealt with in a publication by Shaw (1954). Since relatively little is known about its effect on the central nervous system, the following experiments were designed in an attempt to provide some information on this point. METHODS
Two series of experiments were carried out on female rats of the Wistar strain, each weighing approximately 130 g. In the first series a group of 48 rats received sodium fluoride intraperitoneally at a dosage of 10 mg/kg/day. The same number of rats were given injections of saline and served as controls. In the second series of experiments the rats were given sodium fluoride orally. A total of 150 rats were divided into three groups. Two groups, each consisting of 50 rats, were given diets with added sodium fluoride amounting to 15 and 150 parts per million (ppm), respectively (equivalent to 7 and 70 ppm in terms of fluoride). The other 50 rats were kept on a control diet. Fluoride was added to the diet instead of to the water 1 Presented in part cology and Experimental
at the Annual Therapeutics,
Meeting April 31
of the American 14, 1959, Atlantic
Society for PharmaCity, New Jersey.
32
F. C. LU
ET
AL.
becausethe amount of diet, and therefore the amount of additional fluoride, ingested by the rats could be determined much more accurately. McClure (1939) and Machle and Largent (1932) found in the rat that NaF added to the diet, like NaF added to the drinking water, was almost completely available. The fluoride content in the control diet used in our experiments was SO-60 ppm. Since the fluoride in the control diet derived mainly from bone meal, only a small fraction of it was absorbable in the alimentary tract (Machle and Largent, 1943; Taylor and Gardner, 1959). There was therefore an appreciable difference in the amount of fluoride available in the control diet and that in the diet containing added NaF. The drinking water used by all animals contained about 0.3 ppm fluoride. These rats were allowed water and diet ad libitum. Body weight and food consumption were recorded weekly. The average food consumption was about 11 g per rat per day in the first week and about 15 g per rat per day in the fifteenth week. The initial body weight averaged 110 g: and 15 weeks later it was about 210 g. On the basis of the food consumption and body weight, the sodium fluoride ingested by the rats on the high level was about 15 mg/kg/‘day at the start; it decreased to about 11 mg/kg/day after 15 weeks’ treatment. These figures do not include the fluorides in the basic diet and the water. The amount of the additional fluoride taken by these rats was therefore comparable to that received by the treated rats of the first series. The rats on the low level of sodium fluoride received one-tenth of this amount. During the course of the experiments the sensitivity of the treated rats to a number of agents that are active on the central nervous system (CNS) was compared with that of the control rats. The following procedures were used. Pentylertetetrazol-induced convulsions. The drug was given by the intraperitoneal (i.p.) route and the percentage of rats showing convulsions on each dose was recorded. The percentages were converted to probits and the statistical analysis of the probit versus the logarithmic dose was carried out by the method of Bliss (I935a,b). In some tests, only one dose of pentylenetetrazol was given; the results were evaluated by the Chi square test. The drug was given by the intraStrychnine-induced convulsions. peritoneal route and the calculations were done as described above. ELectroskock-induced convulsions. The electroshock (60 cycle AC, 10 mA for 0.2 seconds) was applied through needle electrodes inserted
SODIUM
FLUORIDE
AND
CNS
33
EFFECT
in the ears. Tonic extensor seizure was used as the end point. The number of rats showing extensor seizures was recorded and the data were analyzed by applying the Chi square test. The anti-extensor seizure activity of diphenylhydantoin. The response of all rats to electroshock (60 cycle AC, 15 mA for 0.2 sec.) was determined on the first day. Out of each group, 16 of the rats that showed tonic extensor seizures were given diphenylhydantoin sodium (20 mg/kg, i.p.) on the following day. Their response to electroshock was tested 1.5 minutes after the injection; the number of rats showing convulsions was recorded and the data were analyzed by applying the Chi square test. Pentobarbital-induced sleeping time. Pentobarbital sodium was given by the intraperitoneal route at 1 or 2 dose levels (20 mg/kg and 25 mg/kg) . Each dose was administered to 10 rats of each of the control and the two fluoride-treated groups. These rats were kept in individual cages and the duration (in minutes) from injection to the appearance of righting reflex in each rat was recorded. Analysis of the results was done according to the method of Gaddum (19.53). RESULTS
In the first series of experiments, pentylenetetrazol was given by the intraperitoneal route to the rats after they had been on test for 4 weeks. The details are given in Table 1. The activity of pentylenetetrazol to induce convulsions in the fluoride-treated rats was 110.3 I+ 7.1 y0 in terms of that in the control rats. Therefore there was a slight but insignificant increase in the sensitivity to pentylenetetrazol after 4 weeks’ treatment with fluoride. TABLE EFFECTS
OF FLUORIDE
TREATMENT
ON THE
AND
(after Group
STRYCHNINE
NaF-treated
of rats showing
TO PENTYLENETETRAZOL
(after Dose (ma/kd
Responsea
Strychnine 5 weeks’ treatment) Responsea
72.0
9/10
2.57
t/9
60.0 50.0
S/15 4/15
2.20 1.88
5/9 2/Y
60.0 50.0
S/10 4/10
2 .oo
1.71
8/9 5/9
1.46
219
41.7
(1 Number
OF RATS
SULFATE
Pentylenetetrazol 4 weeks’ treatment)
Dose (mn/ka)
Saline-treated
1 RESPONSE
convulsions
l/10
per number
of rats
tested.
34
F. C. LU
ET
AL.
One week later, the sensitivity of these two groups of rats to strychnine was compared in a similar manner. The details are also shown in Table 1. Analysis of the data showed that the dose-response lines for the two groups were parallel and the activity of strychnine to induce convulsions in the treated rats was 131.2 A 8.8 s in terms of that in the control rats. The difference between these two groups was highly significant. Since the first test on pentylenetetrazol was inconclusive, it was repeated after the rats had been treated for 11 weeks. Pentylenetetrazol (50 mg/kg) was given intraperitoneally to the rats of both groups. The results listed in Table 2 show that the sensitivity to this CNS stimulant TABLE EFFECTS
OF FLUORIDE
TREATMENT
ON THE AND
(after
Group
2 RESPONSE
OF RATS
ro
Pentylenetetrazol 11 weeks’ treatment)
(after
Electroshock 14 weeks’treatment)
S/19 11113”
Saline-treated NaF-treated a Statistically
significant.
PENTYLENETETRAZOL
ELECTROSHOCK
2/19 u/13”
P = 0.02.
b P= 0.001.
was definitely increased in the fluoride-treated rats. These rats were also found to be more sensitive to electroshock (Table 2). This test was carried out after the rats had been treated for 14 weeks. During the course of the second series of experiments, the sensitivity to electroshock, pentylenetetrazol, and strychnine was determined in the three groups of rats after they had been on treatment for 15, 16, and 19 weeks, respectively. The results are listed in Table 3. It may be noted TABLE EFFECTS
OF FLUORIDE
Group Control Low level of NaF High level of NaF 5 43.4 mg/kg. b 1.2 mg/kg.
TREATMENT
3
ON THE SENSITIVITY STIMULATIONS
Electroshock 14/25 24/25d 17/25
OF RATS
TO SOME
Pentylenetetrazola
Strychnineb
2/10 10/13* 9/10d C Significant d Significant
CNS
4/15 l/15
lO/lV at at
P = 0.05. P = 0.01.
that the sensitivity to pentylenetetrazol was increased in both groups of fluoride-treated rats, whereas that to strychnine was raised only in the rats on the high level of fluoride. The responseto electroshock was some-
SODIUM
FLUORIDE
AND
CNS
35
EFFECT
what enigmatic since the sensitivity was enhanced in the rats receiving the low level of fluoride, but not in those on the high level. The activity of pentobarbital to induce “sleep” was also determined in these rats after they had been treated for 18 weeks. The results showed that the hypnotic activity of pentobarbital was 113.4 + 4.0 s in the rats treated with the low level of sodium fluoride and 153.9 * 3.5 5% in those on the high level, as compared to the controls. The antiextensor seizure activity of diphenylhydantoin was studied after the rats had been on test for 21 weeks. In the control group 6 rats were protected by this antiepileptic agent. In the low and high fluoride level groups, 10 and 12 were protected, respectively. The difference between the rats on the high level of fluoride-treated and the controls was significant (P = 0.02). In order to determine the reversibility of the effects of fluoride on the CNS, some of the test procedures were repeated after the fluoride-treated rats had been given the control diet for 2 weeks. The results obtained in these tests are listed in Table 4. It is evident that the effects of fluoride TABLE THE
REVERSIBILITY
OF THE
EFFECTS
Group@
Pentylenetetrazolb
Control Low level of NaF High level of NaF (1 b C d
All groups on control diet for 45 mg/kg i.p. 20mg/kg i.p. Sleeping time in minutes.
4
OF FLUORIDE
2-3
ON THE
CENTRAL
NERVOUS
Pentobarbitalcj
9/15
85.6
S/15 7/15
91.0 +- 6.6 94.6 & 5.5
SYSTEM
d
-+ 4.8
weeks.
on the activities of pentylenetetrazol and pentobarbital were reversible. It may be noted that the fluoride treatment did not appreciably affect the gain in body weight. The weights of the heart, liver, kidneys, adrenals, and thyroid were also not significantly altered. DISCUSSION
The experimental results show that the rats that had received sodium fluoride, either intraperitoneally or orally, were more sensitive to electrical as well as chemical stimulations of the central nervous system. Furthermore, they were more sensitive to CNS depressants. A simultaneous increase in sensitivity of the CNS to both stimulants and depressants is
36
F. C. LU
ET
AL.
not unique. For example, pretreatment of mice with diphenhydramine has been found to augment the effects of both pentobarbital and strychnine (Sherman, 1956). Similarly, chlorpromazine has been found to potentiate the CNS stimulant action of picrotoxin in cats (Marquardt et al., 1955) as well as to prolong the sleeping time induced by butabarbital in mice (Courvoisier et al., 1953) and increase the protective effect of diphenylhydantoin against electroshock in rats (Bertrand et al., 1955). A number of factors are known to affect the threshold of electroshock and the duration of barbiturate-induced sleep. However, the increased sensitivity in the fluoride-treated rats was unlikely a result of a deterioration in their general health or a change in their body water content. This is suggested by the normal growth rate, food consumption, and organ weights in the treated rats as compared to the controls. Fluoride is known to inhibit many enzymes (Colowick and Kaplan, 1955). In the case of cholinesterase, Nachmansohn (1939) found that fluoride, at 1 mM, causeda 38 70 inhibition of the enzyme. At 10 mM, it reduced the cholinesteraseactivity to one-third that of the control (Taylor et a,?., 1952). Since the fluoride concentration in the brain of someanimals exposed to large dosesof fluoride was as high as 5 mM (Boddie, 1949)) a significant inhibition of cholinesterasecould conceivably occur in these animals. It is of interest to note that physostigmine and other cholinesterase inhibitors potentiate the convulsant action of pentylenetetrazol (Gutierrez-Noriega, 1945; Cordova, 1948) and lower the threshold for electroshock (Gellhorn and Ballin, 1948). On the other hand, iproniazid, a monoamine oxidase inhibitor, prolongs the sleeping time induced by barbiturates (Fouts and Brodie, 1956). The effects observed in the fluoride-treated rats were therefore possibly consequential to an inhibition of these two important enzymes in the CNS. An enzyme may be inhibited by fluoride either directly (Slater and Bonner, 1952) or through a change in the concentration of such bivalent cations as Ca, Mg, Mn, etc., in the media (Colowick and Kaplan, 1955). Results concerning the effect of fluoride on the metabolism of these ions in animals are inconclusive. For example, Leone et al. (1956) found that large dosesof sodium fluoride given intravenously to dogsslightly reduced the Ca level in the blood. On the other hand, Schulz (1931) failed to observe an appreciable change in the Ca level in the plasma of rats receiving NaF. In the dogs given NaF for 5 months, Greenwood et al. (1935) found no change in the total Ca or the acid-soluble inorganic P.
SODIUM
FLUORIDE
AND
CNS
EFFECT
37
Administration of very large doses of fluoride has been shown to alter the electrical activity of the brain in the cat (Moruzzi and Bremer, 1938) and to cause convulsions and depression in various species of animals (Leone et al., 1956). The effects of smaller doses of fluoride described herein have not been reported by other investigators. The practical significance of these observations is as yet unknown. It is possible that rats are unique in this respect, and man may respond to fluoride entirely differently. Furthermore, the fact that the increased sensitivity of the CXS was reversible (Table 4) indicates that perhaps no organic damage had occurred in these rats after a S-month treatment with fluoride. SUMM.4RY Rats given sodium fluoride at 10 mg/kg daily by the intraperitoneal route were more sensitive to the convulsant action of strychnine and pentylenetetrazol and to electroshock. This increase in sensitivity to CNS stimulation w-as observed also in two other groups of rats which were given a diet with added sodium fluoride amounting to 7 and 70 parts per million in terms of fluoride. Prolonged treatment with fluoride also lengthened the sleeping time induced by pentobarbital and increased the protective effect of diphenylhydantoin against electroshock. The increase in sensitivity to some CNS-active agents in the fluoride-treated rats disappeared after these rats were given the control diet for 2 weeks, REFERENCES T., and QUIVY, D. (1935). Influence exercbe par certains ganglioplegiques et neuroplegiques sur l’effet anticonvulsivant de la diphenylhydantoine. Arch. intern, pharmacodynamie 100, 283-297. BLISS, C. I. (1935a). The calculation of the dosage-mortality curve. Ann. A#. Biol. 22, 134-167. BLISS, C. I. (1935b). The comparison of dosage-mortality data. Ann. Bppl. Biol. 22, 307-333. BODDIE, G. F. ( 1949). Effects of fluorine compounds on animals in the Fort William area. In: Industrial Fluorosis. A Study of the Hazard to Man and Animals near Fort William, Scotland. A Report to the Fluorosis Committee, Med. Research Council Mem. 1vo. 22 (by J. K. Agate et al.), H.M. Stationery Ofice, London. COLOWICK, S. P., and KAPLAN, N. 0. (eds.). (1955). Methods in En:ymoZogy, Vol. II. Academic Press, New York. CORDOVA, J. A. (1948). Efecto potenciador de 10s medicamentos anticolinoesterasicos sobre las convulsiones cardiazolicas. Rev. farmaco2. y med. ezptl. 1, 196-208. COURVOISIER, S., FOURNEL, J., DUCROT, R., KOLSKY, M., and KOETSCHET, P. (1953). Prop&& pharmacodynamiques du chlorhydrate de chloro-3-(dim&hylamino-3’propyl)-IO-phCnothiazine (4.560 R.P.) ; etude experimentale d’un nouveau corps utilise dans I’anesthbsie potentialiste et dans l’hibernation artificielle. .4rch. intern. pharmacodynamie 92, 305-361. ERHARDT, DR. (1874). II. Kali Ruoratum zur Erhaltung del Zihne. Memorabilien. Monatsh. rationelle Aerste 19, 359-360. BERTRAND,
I.,
GAYET-HALLION,
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FoUrs, J. R., and BRODIE, B. B. (1956). On the mechanism of drug potentiation by iproniazid (Z-isopropyl-1-isonicotinyl bydrazine) . Pharmacol. Exptl. Therap. 116, 480-485. GAnnuM, J. H. (1953). Simplified mathematics for bioassays. J. Pharm. Pharmacol. 6, 343-358. GELLHORN, E., and BALLIN, H. M. (1948). Age and susceptibility to convulsions. Proc. Sot. Exptl. Biol. Med. 68, 540-543. GREENWOOD, D. A., HEWITT, E. A., and NELSON, V. E. (1935). The effects of fluorine on respiration, blood pressure, coagulation and blood calcium and phosphorus in the dog. J. Am. Vet. Med. Assoc. 86, 28-42. GUTIERREZ-NORIEGA, C. (1945). Epilepsia experimental y drogas colinergicas. Rev. neuro-psiquiatria 8, 121-134. LEONE, N. C., GEEVER, E. F., and MORAN, N. C. (1956). Acute and subacute toxicity studies of sodium fluoride in animals. Public Health Repts. (U. S.) 71, 459-467. MCCLURE, F. J. (1939). Fluorides in food and drinking water. A comparison of effects of water-ingested versus food-ingested sodium fluoride. Natl. Znsts. Health Bull. No. 172. MACHLE, W., and LARGENT, E. J. (1943). The absorption and excretion of fluoride. II. The metabolism at high levels of intake. J. 2nd. Hyg. Toxicol. 26, 112-123. MARQUARDT, P., PUPPEL, H., and SCHUMACHER, H. (1955). Die Wirkung erregender Pbarmaka nach Megapben-vorbehandlung. Klin. Wochschr. 88, 211-215. MORUZZI, G., and BREMER, F. (1938). Action du fluorure de sodium et du bleu de methylene sur I’activitk electrique, spontanee et provoqute de i’ecorce cerebrale. Compt. rend. sot. biol. 129, 884-889. NACHMANSOHN, D. (1939). Sur I’inhibition de la cholinesterase. Compt. rend. sot. biol. 130, 1065-1068. SCHULZ, J, A. (1931). Effects of the ingestion of fluorides on some of the constituents of the teeth, bones, blood and tissues of albino rats. Zowa Agr. Expt. Sta. Ann. Rept., p. 52. SHAW, J. H. (ed.). (1954). Fluoridation as a Public Health Measzrre. American Association for the Advancement of Science, Washington, D. C. Enhancement of the central nervous system effects of SHERMAN, J. F. (1956). strychnine and pentobarbital by diphenhydramine. Science 123, 1170-I 171. SLATER, E. C., and BONNER, W. D. (1952). The effect of fluoride on the succinic oxidase system. Biochem. J. 52, 185-196. TAYLOR, I. M., WELLER, J. M., and HASTINGS, A. B. (1952). Effect of cholinesterase and choline-acetylase inhibitors on the potassium concentration gradient and potassium exchange of human erythrocytes. Am. J. Physiol. 168, 658-665. TAYLOR, J. M., and GARDNER, D. E. (1959). Metabolism of water-borne fluoride in the rat. Federation Proc. 18, 450. World Health Organization (1958). First Report of the Expert Committee on Water Fluoridation. World Health Organization Tech. Rept. Ser. No. 146.