Convulsive Seizure Induced by Intracerebral Injection of Semicarbazide (An Anti Vitamin B6) in the Mouse

J. Nutr. Sci. Vitaminol., 22, 1-6, 1976

CONVULSIVE SEIZURE INDUCED BY INTRACEREBRAL INJECTION OF SEMICARBAZIDE (AN ANTI VITAMIN B6) IN THE MOUSE

Junko YAMASHITA1

Department of Nutrition, Jikei University School of Medicine, Tokyo 105, Japan (Received August 4, 1975)

Summary The direct injection of semicarbazide (SC), an antivitamin B6 (anti-B6), into the lateral ventricle of the mouse brain induced convul sion and tremors at a smaller dose after a shorter latent period than that in systemic administration. The symptoms were prevented by pyri doxine, aminooxyacetic acid or acetone, while they enhanced by pyridoxal, pyridoxal phosphate, or some other anti-B6. In mice fed a vitamin B6 (B6)-deficient diet, convulsion and tremors occurred at smaller doses of SC than those in mice given control food, and were counteracted by pyridoxine. On the other hand, mice into which SC had been inject ed in the neighboring site of the lambda first showed running fits, which was followed by convulsion and tremors.

Reports have shown that numerous anti-B6 induce tonic and clonic convul sions and so-called running fits in many species of animals. Investigators have

attempted to correlate the induction of convulsions and running fits with the

decrease of ƒÁ-aminobutyrate (1) by inhibition of pyridoxal kinase (EC 2.7.1.35) and/or glutamate decarboxylase (EC 4.1.1.15). These attempts, however, have not yet given a convincing explanation of the considerably long delay of the onset of the symptoms after the administration of anti-B6. Moreover, due to this elon gated delay, the elucidation of the mode of action of anti-B6 is rather laborious. In the present study, the shortening of this 'delay' was attempted by intracerebral administration of SC, an anti-B6, and this was expected to contribute to establish ing the mechanism of the convulsive events by anti-B6.

MATERIALS AND METHODS

Male mice of ddy strain, about 4-week-old, obtained commercially were fed on normal food except in the specially mentioned case, and offered to the experi ment after a few days. 1 山下洵子

1 2 J. YAMASHITA

SC hydrochloride, thiosemicarbazide, isoniazide, pyridoxal hydrochloride,

and pyridoxamine hydrochloride were purchased from Wako Pure chemicals,

pyridoxine hydrochloride from National Institute of Hygienic Sciences, pyri doxamine phosphate monohydrochloride from Mann Research Lab. Inc., 4

- deoxypyridoxine hydrochloride and DL-penicillamine from Nakarai Chemicals, Ltd., and toxopyrimidine and castrix from Takeda chemical Ind. Amino

oxyacetic acid was kindly supplied by Dr. K. Horisaka of Showa University and semicarbazone of pyridoxal phosphate by Dr. K. Makino of Jikei University. Acidic reagents were neutralized with NaOH before using.

SC and some other reagents dissolved in 5ƒÊl of water or saline were directly

injected into the left lateral ventricle, i.e. intracerebrally (i.c.), after the method of NAKAJIMA (2) except in the specially mentioned case. Some reagents were also

administered intraperitoneally (i.p.) or orally. All the control mice were given an

equal volume of water or saline as a substitute for the reagent solution. The treated animals were individually caged to be observed for 3 hr.

RESULTS Effect of injection of SC on mice Mice exhibited tonic and clonic convulsions and tremors following a latent period of about 15min after the injection of SC (Table 1). Such violent behavior as dashing, jumping, and leaping often occurred about 15min thereafter. These manifestations resembled those induced by systemic administration of SC, but running fits, which is a characteristic behavior induced by SC and other many anti-B6 when administered systemically, was not observed.

Table 1. Convulsant effect of intracerebral injection of semicarbazide hydrochloride in mice.

a Within 3 hr after semicarbazide hydrochloride injection .

Antidotal effect of B6, acetone, or aminooxyacetic acid on SC-induced convulsion Convulsion and tremors were prevented when pyridoxine (i.p. or i.c.) was administered simultaneously with SC. The antidotal effect was not afforded by pyridoxamine or pyridoxamine phosphate. Administration of either pyridoxal or pyridoxal phosphate had an augmentative rather than preventive effect on the convulsive symptoms; incidence of convulsions increased. Pyridoxal phosphate, CONVULSION BY INTRACEREBRAL INJECTION OF SEMICARBAZIDE 3

Table 2. Anticonvulsant effect of vitamin B6 on semicarbazide-induced convulsion in mice.

a Mice were injected i .c. vitamin B6 (ƒÊg/animal). b Mice were administered i .p. 10ƒÊl/g of water as controls. Semicarbazide (SC), pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and pyridoxamine phosphate (PMP) are all hydrochloride. Five mice in each group were given SC and vitamin B6, PN, PL, pyridoxal phosphate (PLP), PM or PMP. however, did not produce convulsion in mice treated with SC at subconvulsive doses (Table 2). Acetone and aminooxyacetic acid were effective antidotes for SC toxicity (Table 3). Effect of SC on acute or chronic Bs-deficient mice Some mice were given SC, thiosemicarbazide, isoniazide, DL-penicillamine, 4-deoxypyridoxine, toxopyrimidine, or castrix at subconvulsive doses to acutely deprive them of B6. Administration of SC (i.p.), isoniazide (i.c.), DL-penicillamine (i.c.), 4-deoxypyridoxine (i.c.), or toxopyrlmldlne (i.c.) induced convulsion when followed by injection of subconvulsive dose of SC. Neither thiosemicarbazide, nor castrix enhanced SC toxicity (Table 3). Mice fed on B6-deficient diet (Clea Japan, Co.) for one or three weeks after four weeks of age showed high susceptibility to SC: convulsion and tremors were induced by SC at small doses which were ineffective in those fed on control diet. These symptoms were countered with the administration of a pyridoxine antidote (Table 4). 4 J. YAMASHITA

Table 3. Synergetic effect of another anti-B6 and acetone on semicarbazide-induced convulsion in mice. Five mice were used in each group.

a All the anti -B6 reagents were preliminarily examined not to induce convulsion at the shown doses within 2hr after administration.

Table 4. Effect of vitamin B6 deficiency on semicarbazide-induced convulsion in mice. Five mice in each group were fed on vitamin B6-deficient food(-B6) or control food

(font) for 3 weeks since 4 weeks of age, but micea for one week. Mice in each group were given semicarbazide hydrochloride (30ƒÊg/animal; i.c.) 30min after pyridoxine hydrochloride (50ƒÊg/g; i.p.) or water (10ƒÊl/g; i.p.).

Table 5. Effect of intracerebral injection of semicarbazone of pyridoxal phosphate. Five mice were used in each group. CONVULSION BY INTRACEREBRAL INJECTION OF SEMICARBAZIDE 5

Table 6. Running fits induced by semicarbazide injected into various sites of the brain.

Semicarbazide hydrochloride (20ƒÊg/animal) was injected at the volume of 2ƒÊl/animal. The other technical methods followed that of NAKAJIMA (2).

a One of three exhibited startle reflex 14min after injection .

Effect of intracerebral injection of semicarbazone of pyridoxal phosphate on mice

Semicarbazone of pyridoxal phosphate (i.c.) induced convulsion and tremors similar to those by SC at doses equimolar to, or less than, the convulsive dose of

SC (Table 5).

Effect of SC injection on various sites of the brain

SC (20ƒÊg/animal) was injected into various sites of the brain. When SC was administered in the vicinity of the lambda and 2-5mm below the skull, run ning fits were first induced (Table 6). The fits were often followed by tonic and clonic convulsions, and tremors 10-60min thereafter.

DISCUSSION The direct injection of a reagent into the lateral ventricle is considered to in flict a severe mechanical injury to the brain. The method, however, would simplify the induced phenomenon at least in comparison with systemic admin istration. The present experiment indicates that intracerebral injection of SC at considerably small doses reduces the latent period of the first symptom as com pared with the data reported on the study of systemic administration of SC and many other anti-B6 (3), and also of intracerebral administration of other anti-B6 (2). This finding might deserve attention, because research on the mode of action of massive doses of convulsants administered systemically has been complicated by diverse physiological and biochemical changes of the brain during that long latency. Since JENNEYand LEE'S results were published (4), it is well known that systemic administration of SC induces tonic and clonic convulsions as well as running fits, which are inhibited by acetone (4), pyridoxine (5), or aminooxyacetic acid. The present study revealed that intracerebral injection of SC induced 6 J. YAMASHITA similar convulsions to those by systemic administration, which were also countered by acetone, pyridoxine, or aminooxyacetic acid antidotes. Moreover, the con vulsions were enhanced by some anti-B6, besides mice fed on B6-deficient diet were more sensitive to SC than to controls. It appears likely that symptoms produced by intracerebral injection of SC are due essentially to mechanisms com mon to those by SC and other many anti-Bfl administered systemically. It is not clear why both pyridoxal and pyridoxal phosphate augmented con vulsion in mice receiving SC, while pyridoxine prevented convulsion. The hypo thetical explanation is that only pyridoxine itself has an antidotal effect on SC convulsion or that pyridoxal and pyridoxal phosphate made from pyridoxine in vivo are effective, but exogenous ones are not. And both exogenous pyridoxal and pyridoxal phosphate appear to be toxic rather than anticonvulsant substances. This assumption is consistent with the report that pyridoxal phosphate (i.c.) alone is a convulsant in mice (6). Because pyridoxal kinase is inhibited in vitro by SC and severely by semi carbazone of pyridoxal (7), it is conceivable that the combination of SC with pyridoxal and/or pyridoxal phosphate is responsible for the induction of the con vulsive phenomena. This is plausibly supported by the present experimental result that small doses of semicarbazone of pyridoxal phosphate (i.c.) induced convulsion and tremors similar to those by SC. SC injected in the vicinity of lambda, 2-5mm below the skull, produced running fits which was often followed by tonic and clonic convulsions and tremors 10-60min thereafter. Considering this result, the data obtained from the in jection of SC into the lateral ventricle, and the fact that systemic administration of SC induces convulsion, tremors, and also running fits, SC seems to act upon two (or more) sites of the brain; one related to convulsion and tremors, and the other to running fits. The former appears likely to be adjacent to the lateral ventricle or the site where SC penetrates with facility from the lateral ventricle. The latter is assumed to be the midbrain, judging from the sites of administration. And the difference between the latent periods of systemic and intracerebral ad ministration is presumed to relate to the time of penetration of SC into the target loci of the brain.

The author would like to thank Drs. M. Matsuda and Y. Hirata for many helpful discussions.

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