Hiro-aki Yamamoto

Preventive effect of melatonin against cyanide-induced seizures and lipid peroxidation in mice

Subcutaneous injection of potassium cyanide (6, 8, and 9 mg/kg) caused a severe tonic seizure in a dose-dependent manner. However, the incidence of seizures induced by potassium cyanide was significantly inhibited by preadministration of melatonin (20 mg/kg, s.c.) Lipid peroxidation in homogenates from whole brain of mice was significantly increased (39%, 106% and 132%, respectively) by the exposure to potassium cyanide (0.01, 0.05, and 0.1 mM). The potassium cyanide (0.1 mM)-induced lipid peroxidation was prevented by melatonin (0.1, 0.5, 1.0, or 2 mM) in a concentration-dependent manner. These results suggest that free radicals formation and subsequent lipid peroxidation may contribute in part to the development of seizures induced by cyanide in mice.

Preventive effect of melatonin against brain mitochondria DNA damage, lipid peroxidation and seizures induced by kainic acid.

The effects of kainic acid on mitochondria DNA (mtDNA) or lipid peroxidation in mice brain and, preventive effects of melatonin against its effects were investigated in vivo. Broad-spectrum limbic and severe sustained seizures were observed in all mice when kainic acid (45 mg/kg) was injected intraperitoneally (ip) to eight mice. These seizures were completely abolished by the simultaneous administration of melatonin (20 mg/kg, ip), a potent scavenger of hydroxyl radical. However, slight limbic seizures or severe sustained seizures were observed when melatonin was injected in animals 30 min before or 15 min after the kainic acid administration. The administration of kainic acid caused damage to mtDNA in brain frontal and middle cortex. These effects were abolished when melatonin was injected in animals 0 or 30 min before, but not 15 min after the kainic acid administration. In vitro or in vivo exposure of kainic acid elicited an increase in lipid peroxidation in a concentration- or dose-dependent manner. The increased lipid peroxidation induced by kainic acid was attenuated by co-treatment with melatonin. These results indicate that there may be a positive relationship among seizures, brain mtDNA damages and increased lipid peroxidation. Hence, our present results suggest that the hydroxyl radicals produced by kainic acid cause damage on mtDNA and the increase of lipid peroxidation in brain, leading to severe seizures. These effects were completely prevented by co-treatment with melatonin, a potent scavenger of hydroxyl radicals.

Melatonin attenuates L‐cysteine‐induced seizures and lipid peroxidation in the brain of mice

Abstract: The effect of melatonin, a potent free radical scavenger, on L‐cysteine‐induced seizures and lipid peroxidation was investigated in mice. When L‐cysteine (1.25, or 5.0 μmol/animal) was injected intracerebroventricularly (i.c.v.) into mice, severe tonic seizures were observed for over 20 sec in 75% and 100% of the treated mice, respectively. However, when melatonin (20 or 100 mg/kg) was injected subcutaneously (sc) into mice 15 min before L‐cysteine injection (1.25 umol/animal, i.c.v.), the incidence of seizures was observed in only 35% and 20% of the treated mice, respectively. Furthermore, when L‐cysteine (1.25 or 5.0 (amol/animal, i.c.v.) was injected into mice, lipid peroxidation in whole brain 20 min after injection was significantly increased by 56% or 67% as compared to that of the control. However, when the seizures induced by L‐cysteine (1.25 μmol/animal) were abolished by preadminstration of melatonin, the increased lipid peroxidation induced by L‐cysteine was prevented. These results suggest that there may be a positive correlation between free radical formation and seizures induced by L‐cysteine and that melatonin affords protection against the seizures as well as against the associated lipid peroxidation.

Effects of amino acids, especially taurine and γ-aminobutyric acid (GABA), on analgesia and calcium depletion induced by morphine in mice

The analgesic effect of morphine was antagonized in mice by intracerebroventricular pretreatment with taurine, gamma-aminobutyric acid (GABA) or glycine and was potentiated by ethylene glycol tetra-acetic acid (EGTA) but not altered by L-glutamate or L-aspartate. The potentiation of morphine analgesia by EGTA was reversed by a concentration of taurine that did not alter the tail-flick response. The selective depletion of 45Ca2+ from synaptic vesicles observed with morphine administration was significantly inhibited by taurine injection (1.2 mumol/brain, i.vt.) but was not altered by the same dose of GABA. Inhibition of ATP-dependent 45Ca2+ uptake in synaptosomes by morphine was also completely reversed by taurine (10(-2)M which by itself did not alter 45Ca2+ uptake. These results suggest that antagonism of morphine analgesia by taurine may be caused by blockade of the morphine-induced inhibition of both ATP-dependent synaptosomal 45Ca2+ uptake and changes in synaptic vesicular 45Ca2+ localization, while the antagonism by GABA was not associated with synaptosomal Ca2+.

Antagonistic effect of melatonin against cyanide-induced seizures and acute lethality in mice

The effect of melatonin on potassium cyanide-induced neurotoxicity was investigated in vivo. The ED50 value of potassium cyanide, as measured by induction of tonic and clonic seizures, was significantly increased by 1.5- or 1.8-fold by s.c. preinjection of melatonin (20, 100 or 345 mg/kg) in mice. The preventive effect of melatonin against potassium cyanide-induced seizures was dose dependent. The LD50 value of potassium cyanide, based on 24-h mortality, was also significantly increased by 1.3-fold by preinjection of melatonin. Potassium cyanide (8 mg/kg, s.c.) increased lipid peroxidation in whole brain of mice, and the increased lipid peroxidation was completely abolished when cyanide-induced seizures were stopped by preadministration of melatonin. These results suggest that melatonin, a pineal hormone, may protect against cyanide-induced neurotoxicity with its free radical scavenging effects in mice.

Melatonin attenuates brain mitochondria DNA damage induced by potassium cyanide in vivo and in vitro

The effect of potassium cyanide on mitochondria DNA (mtDNA) in mouse brain was investigated in vivo and in vitro. When potassium cyanide (0, 0.1, 1.0 or 2.0 mM) was incubated with a crude mitochondria fraction prepared from mouse brain at 37 degrees C for 60 min, the damage of mtDNA was observed in a concentration-dependent manner. However, the mtDNA damage was prevented by a co-treatment with melatonin (1.5 mM), a scavenger of hydroxyl radicals (*OH). Furthermore, a subcutaneous injection of potassium cyanide (7mg/kg) caused both brain mtDNA damage and severe seizures in mouse. The damage of mtDNA and seizures induced by potassium cyanide were abolished by the pre-injection of melatonin (20 mg/kg). Hydrogen peroxide (1.5 mM) inflicted damage to brain mtDNA in the presence of Fe(2+) (3.0 microM). The damage was abolished by the co-treatment with melatonin. Furthermore, when cyanide (0, 0.1 or 1.0 mM) was incubated with the crude mitochondria fraction prepared from mouse brain, the lipid peroxidation was significantly increased in a concentration-dependent manner. The increased lipid peroxidation was completely inhibited by the co-treatment with melatonin (1.0 mM). These results suggest that reactive oxygen species including the *OH may play a cardinal role for mtDNA damage induced by potassium cyanide. Hence, the present study concluded that melatonin protects against DNA damage induced by the *OH produced by cyanide or hydrogen peroxide.

Hyperammonemia, increased brain neutral and aromatic amino acid levels, and encephalopathy induced by cyanide in mice

The correlation among hyperammonemia, brain neutral amino acid, and encephalopathy induced by cyanide was investigated in mice. Subcutaneous injection of 10 mg/kg of potassium cyanide increased the levels of blood ammonia by 2.5-fold as compared to those of the corresponding controls and caused loss of consciousness in 100% of the treated mice. alpha-Ketoglutarate (500 mg/kg, ip) completely blocked the development of loss of consciousness and hyperammonemia induced by cyanide. Furthermore, the same doses of potassium cyanide increased by 50-150% the levels of brain neutral and aromatic amino acids such as leucine, isoleucine, tyrosine, and phenylalanine, while the levels of acidic amino acid such as taurine, glutamate, and aspartate did not change. alpha-Ketoglutarate also significantly inhibited the increase of the neutral and aromatic amino acid levels in brain. These findings suggest that the hyperammonemia and the increase of neutral and aromatic amino acids may play an important role in development of loss of consciousness induced by cyanide.

Effect of α-ketoglutarate and oxaloacetate on brain mitochondrial DNA damage and seizures induced by kainic acid in mice

The effects of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA (mtDNA) damage and seizures induced by kainic acid were examined both in vivo and in vitro. An intraperitoneal (ip) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. The seizures were abolished when alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg) was injected intraperitoneally in the animals 1 min before kainic acid administration. In addition, the administration of kainic acid caused damage to mtDNA in brain frontal and middle cortex of mice. These effects were completely abolished by the ip preinjection of alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) to brain homogenate inflicted damage to mtDNA in a concentration-dependent manner. The damage of mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with alpha-ketoglutarate (2.5 or 5.0 mM) or oxaloacetate (0.75 or 1.0 mM). Furthermore, in vivo and in vitro exposure of kainic acid elicited an increase in lipid peroxidation. However, the increased lipid peroxidation was completely inhibited by cotreatment of alpha-ketoglutarate or oxaloacetate. These results suggest that alpha-keto acids such as alpha-ketoglutarate and oxaloacetate play a role in the inhibition of seizures and subsequent mtDNA damage induced by the excitotoxic/neurotoxic agent, kainic acid.

Ganglioside GT1B and melatonin inhibit brain mitochondrial DNA damage and seizures induced by kainic acid in mice

The effects of ganglioside GT1b or melatonin on damage to brain mitochondrial DNA (mtDNA) and seizures induced by kainic acid were investigated both in vivo and in vitro. An intraperitoneal (i.p.) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. These seizures were completely abolished by an intracerebroventricular (i.c.v.) injection of ganglioside GT1b (90 nmol/brain), a potent inhibitor of glutamate receptor mediated activation and translocation of protein kinase C and lipid peroxidation, or an i.p. injection of melatonin (20 mg/kg), a potent scavenger of hydroxyl radicals (*OH). The administration of kainic acid caused damage to mtDNA in brain frontal and central portion of cortex in mice. The damage to mtDNA was abolished by pre-injection of ganglioside GT1b (90 nmol/brain, i.c.v.) or melatonin (20 mg/kg, i.p.). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) inflicted damage to mtDNA in a concentration-dependent manner. The damage to mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with 60 microM ganglioside GT1b or 1.5 mM melatonin. Furthermore, kainic acid (0.5 or 1.0 mM) increased lipid peroxidation in a concentration-dependent manner when incubated with a homogenate prepared from mice brain at 37 degrees C for 20 or 60 min. However, the increased lipid peroxidation was completely abolished by the co-treatment with ganglioside GT1b (60 microM) or melatonin (1.5 mM). These results suggest that reactive oxygen species including hydroxyl radical (*OH) may play a role in the damage to brain mtDNA and seizures induced by kainic acid. We conclude that the preventive effect of melatonin or ganglioside GT1b against kainic acid-induced mtDNA damage or seizures may be due to its scavenging of reactive oxygen species including the *OH.

Protection against cyanide-induced convulsions with α-ketoglutarate

Abstract Protection against convulsions induced by cyanide was observed after treatment with α-ketoglutarate, either alone or in combination with sodium thiosulfate, a classical antagonist for cyanide intoxication. However, sodium thiosulfate alone did not protect against cyanide (30 mg/kg)-induced convulsions. γ-Aminobutyric acid (GABA) levels in brain were decreased by 31% in KCN-treated mice exhibiting convulsions. The combined administration of α-ketoglutarate and sodium thiosulfate completely abolished the decrease of GABA levels induced by cyanide. Furthermore, sodium thiosulfate alone also completely abolished the decrease of GABA levels. These results suggest that the depletion of brain GABA levels may not directly contribute to the development of convulsions induced by cyanide. On the other hand, cyanide increased calcium levels by 32% in brain crude mitochondrial fractions in mice with convulsions. The increased calcium levels were completely abolished by the combined administration of α-ketoglutarate and sodium thiosulfate, but not affected by sodium thiosulfate alone. These findings support the hypothesis proposed by Johnson et al. (Toxicol. Appl. Pharmacol., 84 (1986) 464) and Robinson et al. (Toxicology, 35 (1985) 59) that calcium may play an important role in mediating cyanide neurotoxicity.