Elsevier

Toxicology Letters

Volume 143, Issue 2, 20 July 2003, Pages 115-122
Toxicology Letters

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

https://doi.org/10.1016/S0378-4274(03)00114-0Get rights and content

Abstract

The effects of α-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 α-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 α-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 α-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 α-ketoglutarate or oxaloacetate. These results suggest that α-keto acids such as α-ketoglutarate and oxaloacetate play a role in the inhibition of seizures and subsequent mtDNA damage induced by the excitotoxic/neurotoxic agent, kainic acid.

Introduction

Kainic acid is an excitatory and neuro-toxic substance. It stimulates a subtype of ionotropic receptor of the brain neurotransmitter glutamate and results in transmembrane ion imbalance especially causing calcium influx, which in turn generates reactive oxygen species such as H2O2, superoxide anion (radical dotO2) and the hydroxyl radical (radical dotOH). These reactive oxygen species attack macromolecules within neurons resulting in membrane lipid peroxidation structural and functional changes in proteins, and DNA strand breaks. In addition, a number of reports have suggested that kainic acid increases calcium uptake into brain mitochondria and then generates reactive oxygen species (Lesli et al., 2002)

Administration of kainic acid to experimental animals induces strong lasting convulsions, resembling status epilepticus in man (Ben-Ari, 1985). The kainic acid model constitutes one of the most established models of temporal lobe epilepsy, since the behavioural, electrophysiological, biochemical and histopathological symptoms are reminiscent of those described in patients with temporal lobe epilepsy. Kainic acid enhances hippocampal nitric oxide generator in a severity-related manner of the induced seizures and the enhanced nitric oxide generation upon kainic acid challenge appears mainly to be involved in seizure suppression (Kashihara et al., 1998). Recently, we have found that melatonin inhibits both lipid peroxidation and DNA damage induced by kainic acid, potassium cyanide or paraquat in mice brain by in vitro method (Yamamoto and Mohanan, 2001a, Yamamoto and Mohanan, 2001b, Mohanan and Yamamoto, 2002, Yamamoto and Mohanan, 2002, Yamamoto and Mohanan, 2003). Similarly, intraperitoneal (ip) administration of melatonin, a potent radical dotOH scavenger reduces excito-toxic brain damage triggered by kainic acid-induced seizures in rats (Uz et al., 1996).

Mitochondria is supposed to generate most of the free radicals in the living cell (Tully et al., 2000). Oxidative stress is an important participant in the process of excitotoxicity which is thought to play a critical role in epileptic brain damage and mitochondria seems to be an important source of reactive species produced during excitotoxicity (Coyle and Puttfarcken, 1993, Reynolds and Hastings, 1995). The mitochondrial DNA (mtDNA) molecules within a cell may differ in sequence containing mixtures of mutant and wild type alleles a condition known as heteroplasmy and the expressed defects in mtDNA frequently lead to metabolic defects cellular energy failure and ultimately diseases (Johns, 1995, Esposito et al., 1999). The major emphasis in the past has been on the interaction of chemical toxins with nuclear DNA, later it has been found that the extent of modification of mtDNA is greater than that of nuclear DNA (Duara et al., 1993, Shigenaga et al., 1994, Edland et al., 1996). Reports showed that the abnormal glucose metabolism in the brain can lead to the generation of superoxide and other free radicals, which then cause damage to the adjacent DNA, protein, and lipids (Elena et al., 1996, Cui et al., 2000). It is well documented that DNA is susceptible to the radical dotOH and is easily cleaved. The reactive oxygen species such as the radical dotOH cause damage to various biomolecules and further suggest that ascorbate/Fe3+, dithiothreitol/Fe3+, glutathione/Fe3+ or H2O2/Fe2+ mediated radical dotOH inflicts damage on DNA (Saraiva et al., 1985, Sims et al., 1987, Shoffner et al., 1993, Wallace, 1994, Yamamoto and Mohanan, 2001a).

It is suggested that α-keto acid inhibits the increased levels of reaction oxygen species and malondialdehyde induced by hydrogen peroxide treatments in human erythrocyte (Sokolowska et al., 1999) and cultured striatal neuroins (Desagher et al., 1997). These findings suggest that α-keto acid inhibits reactive oxygen species-dependent oxidative damages to macromolecules in cell. Hence, in this study we have investigated the effect of oxaloacetate or α-ketoglutarate on seizures and brain mtDNA damage induced by kainic acid in mice.

Section snippets

Chemicals

Kainic acid, oxaloacetate, α-ketoglutarate, ethidium bromide, TAE, TE buffer (pH 8.0), agarose, 0.06% barbital sodium solution (pH 8.6) and mtDNA extractor CT kit were purchased from WAKO Pure Chemicals Co., Osaka, Japan. HindII and RNase were obtained from Nippon Gene Co., Ltd, Toyama, Japan. Bioxytech LPO-586 kit was obtained from Funakoshi Co., Tokyo, Japan.

Animals

Male ddy strain mice (4-weeks-old weight 22–24 g) used in all the experiments were purchased from SLC, Inc, Shizuoka, Japan. They were

Results

The behavioural changes were closely monitored and are given in the Table 1. When kainic acid (45 mg/kg) was injected ip to 8 mice, broad-spectrum limbic and severe sustained seizures were observed in all animals within 30 min after injection. However, these seizures were completely abolished by the pre-administration of oxaloacetate (1 g/kg) or α-ketoglutarate (2 g/kg). Slight limbic seizures were observed in the animals injected with α-ketoglutarate (1 g/kg) before kainic acid administration.

Discussion

Kainic acid model constitutes one of the most established models of temporal lobe epilepsy since the behavioural, electrophysiological, biochemical and histopathological symptoms are reminiscent of those described in patients with temporal lobe epilepsy (Ben-Ari, 1985). The injection of kainic acid induces acute limbic seizures which are accompanied by seizure-induced brain damage and late spontaneous recurrent seizures in experimental animals and induces strong and long lasting convulsions,

Acknowledgements

This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture and a fellowship programme on the Japan Society for the Promotion of Science.

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1

Recipient of a Research Fellowship for Foreign Scientists on the Japan Society for the Promotion of Science.

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