In Vivo and in Vitro Effects of Melatonin or Ganglioside GT1B on L-Cysteine–Induced Brain Mitochondrial DNA Damage in Mice

Hiro-aki Yamamoto*,1 and Parayanthala V. Mohanan{dagger}

* The University of Tsukuba, Institute of Community Medicine, Tsukuba, Ibaraki, 305-8575 Japan; and {dagger} Recipient of a Research Fellowship for Foreign Scientists on the Japan Society for the Promotion of Science

Received January 16, 2003; accepted March 17, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of L-cysteine on mitochondrial DNA (mtDNA) in mouse brain were investigated both in vivoandin vitro. An intracerebroventricular (icv) injection of L-cysteine (1.25 µmol/animal) caused mtDNA damage in brain frontal and central portions of the cortex, broad-spectrum limbic and severe sustained seizures in mice, and increased lipid peroxidation in the whole brain. The L-cysteine–mediated effects were prevented by an intraperitoneal (ip) preinjection of melatonin (20 mg/kg) or an intracerebroventricular preinjection of ganglioside GT1b (90 nmol/animal). Furthermore, in in vitroexperiments, L-cysteine (0.05, 0.5, or 1.0 mM) caused damage to brain mtDNA and increased lipid peroxidation in a concentration-dependent manner when incubated at 37°C for 20 or 60 min with a homogenate prepared from whole mouse brains. However, the mtDNA damage and the increased lipid peroxidation were completely abolished by a cotreatment with melatonin (1.5 mM), a potent scavenger of the hydroxyl radical (•OH), or ganglioside GT1b (60 µM), a potent inhibitor of glutamate-receptor-mediated activation and translocation of protein kinase C and lipid peroxidation. These results suggest that reactive oxygen species including the •OH may be involved in L-cysteine–induced brain mtDNA damage, lipid peroxidation, and development of seizures in mice. Therefore, we concluded that •OH scavengers, such as the pineal hormone melatonin and ganglioside GT1b, can protect against brain mtDNA damage, seizures, and lipid peroxidation induced by reactive oxygen species producers such as L-cysteine.

Key Words: melatonin; ganglioside GT1b; L-cysteine; mitochondrial DNA; lipid peroxidation; seizures; hydroxyl radical.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
L-cysteine is a sulfur-containing neurotoxic substance with some properties characteristic of neurotransmitters. It depolarizes neuronal membranes (Olney et al., 1990Go) and is released from rat brain slices in a calcium-dependent manner (Keller et al., 1989Go; Zängerle et al., 1992Go). L-cysteine plays a role in the development of neurological disorders including motor neuron disease, Parkinson’s, and Alzheimer’s disease (Heafield et al., 1990Go). It has also been reported that L-cysteine is markedly elevated during brain ischemia in gerbils (Slivka and Cohen, 1993Go).

A number of reports have suggested that L-cysteine, administered to perinatal mice or rats that possess an immature blood-brain barrier, produces widespread neurodegeneration and brain atrophy of the cerebral cortex, hippocampus, thalamus, and striatum (Olney and Ho, 1970Go; Olney et al., 1971Go). Furthermore, our previous reports showed that an intracerebroventricular (icv) injection of L-cysteine produces severe seizures in adult animals (Yamamoto, 1996Go; Yamamoto and Tang, 1996aGo,bGo). L-cysteine activates selectively the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, which in turn generates reactive oxygen species including the hydroxyl free radical, •OH. The L-cysteine–induced lipid peroxidation in the mouse brain and is blocked by the •OH scavenger, melatonin (Yamamoto and Tang, 1996aGo,bGo). The role that NMDA receptor plays in epilepsy, seizure-induced brain damage, and glutamate excitotoxicity is well established (Choi, 1985Go; Choi et al., 1988Go; Dingledine et al., 1990Go).

Mitochondria generate most of the free radicals in the cell, and it has been estimated that up to 2% to 4% of the inhaled oxygen is converted to free radicals (Tully et al., 2000Go). Oxidative damage to mitochondrial DNA (mtDNA) may induce specific mutations, deletions, or point mutations within the mitochondrial genome (Jazin et al., 1996Go). A major emphasis in the past has been on the interaction of chemical toxins with nuclear DNA; subsequently it was observed that damage to mtDNA is greater than that of nuclear DNA (Duara et al., 1993Go; Edland et al., 1996Go; Shigenaga et al., 1994Go).

It is well documented that DNA is susceptible to •OH damage. Reactive oxygen species such as •OH cause damage to many biomolecules, and we have found that H2O2/Fe2+-induced destruction of DNA is a result of •OH (Uz et al., 1996Go; Yamamoto and Mohanan, 2001bGo). Recently, we have also reported that melatonin inhibits DNA damage induced by kainic acid, paraquat, and cyanide in the mouse, using both in vitro and in vivo methods (Mohanan and Yamamoto, 2002Go; Yamamoto and Mohanan, 2001aGo,bGo, 2002Go, 2003Go).

Gangliosides are glycosphingolipids containing sialic acid residues. They exist in mammalian cell membrane and are particularly abundant in neuronal cell membrane (Stults et al., 1989Go; Wiegandt, 1995Go). Gangliosides attenuate excitotoxic neuronal injury (Dawson et al., 1995Go; Favaron et al., 1988Go). Several mechanisms have been suggested to underlie the neuroprotective effects of gangliosides, including inhibition of protein kinase C translocation (Vaccarino et al., 1987Go), inhibition of lipid peroxidation (Tyurina et al., 1990Go, 1993Go), inhibition of nitric oxide synthase (Dawson et al., 1995Go), and phosphorylation of the nerve growth factor receptor Trk (Ferrari et al., 1995Go; Rabin et al., 1995Go). Favaron et al. (1988)Go reported that gangliosides prevent kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex. It has been reported that gangliosides inhibit the intensification of free radicals reactions by glutamate (Avrova et al., 1998Go) and also protect polyenoic fatty acids from oxidative destruction in synaptosomes of rat brain (Tyurina et al., 1990Go, 1993Go). Rahmann (1987)Go reported that seizure-inducing agents such as tetrazolium, bemegride, and cobalt reduce the ganglioside content in brain. Our previous reports have suggested that in vivo and in vitro exposures to cyanide cause severe seizures and damage to the brain mtDNA in mice (Monahan and Yamamoto, 2002Go; Yamamoto, 1995Go), and the cyanide-induced seizures are abolished by an icv injection of gangliosides (Yamamoto, 1995Go). In this study, we evaluated the effects of L-cysteine on mtDNA and lipid peroxidation in the mouse brain and the neuroprotective effects of melatonin or ganglioside GT1b against those effects.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
L-cysteine, ganglioside GT1b, ethidium bromide, TAE, TE buffer (pH8.0), agarose, 0.06 sodium barbital solution (pH 8.6), and mtDNA extractor CT kit were purchased from WAKO Pure Chemicals Co.(Osaka, Japan). Hind II and RNase were obtained from Nippon Gene Co., Ltd. (Toyama, Japan). Melatonin was purchased from Aldrich Chemical Company Inc. (Milwaukee, WI, USA). 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 housed in groups of six mice per plastic cage, with wood shavings as bedding. They were given a diet of standard laboratory chow, supplied by Oriental Kobo Ltd. (Tokyo, Japan) and water ad libitum. All procedures were performed in accordance with approved institutional protocols, the 1996 National Research Council Guide for the Care and Use of Laboratory Animals, and the Society for Neuroscience Policy on the Use of Animals in Neuroscience Research.

Experimental design.
Thirty-six animals were divided into six experimental groups as follows: Group I, Saline only; Group II, L-cysteine (1.25 µmol/animal; 5µl/animal) plus saline (10ml/kg, injected 30 min before L-cysteine administration); Group III, L-cysteine (1.25 µmol/animal; 5 µl/animal) plus melatonin (20 mg/kg, injected 30 min before L-cysteine administration); Group IV L-cysteine (1.25 µmol/animal) plus ganglioside GT1b (90 nmol/animal, injected 1 min before L-cysteine administration); Group V, melatonin (20 mg/kg) only; Group VI, ganglioside GT1b (90 nmol/animal) only.

L-cysteine and ganglioside GT1b were injected intracerebroventrically, and melatonin was injected intraperitoneally as described by our previous report (Yamamoto, 1995Go; Yamamoto and Tang, 1996bGo). Behavior of the treated animals was observed for 30 min after administration of L-cysteine.

Brain collection and preservation.
Thirty min after dosing, L-cysteine–treated groups, were decapitated, and their brains were immediately removed under aseptic conditions. Animals of other groups were also decapitated at the same time, and brains were collected in the same manner. The brains were cut and separated into frontal and central cortical regions with weights of approximately 100 mg each. All the brains were immediately frozen at -20°C for mtDNA isolation.

Preparation of brain homogenate.
Frozen brain regions (100 mg) were immediately homogenized with ice-cold buffer (1.0 ml) supplied with the mtDNA extractor kit (mtDNA extractor CT kit, Wako, Japan), at a speed of 1000 r.p.m. using a Potter-Elvehjem’s glass homogenizer (Iuchi, Tokyo, Japan) fitted with a Teflon pestle with a clearance of 0.23 mm, to prepare a 10% (w/v) brain homogenate. The brain homogenate was kept on ice, mixed uniformly, and was used for mtDNA extraction. All the procedures were carried out under sterile conditions.

Preparation of crude mitochondria fraction from brain.
The brain homogenate was uniformly mixed and after centrifugation at 1,000 x g for 1 min, the nuclei and cell debris was removed. The supernatant solution was carefully separated and was further centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was discarded, and the crude mitochondrial pellet was directly subjected to mtDNA isolation without further treatment.

In vitro experiments.
One ml of 10% (w/v) homogenate of whole fresh brain was incubated with L-cysteine (0, 0.05, 0.5, or 1.0 mM) in the presence or absence of melatonin (1.5 mM) or ganglioside GT1b (60 µM) at 37°C for 60 min in a shaking water bath. After incubation, the homogenate was centrifuged at 1000 x g for 1 min at 4°C using a micro-centrifuge (Iuchi, Tokyo, Japan) and the supernatant solution was carefully separated and further centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was discarded and this crude mitochondrial pellet was used for mtDNA isolation.

Isolation of brain mtDNA using mtDNA extractor CT kit (Kit containing buffer, solution I, II A & B, III, sodium iodide and washing solution).
The crude mitochondria pellet was resuspended in 50 ml of solution I and vortexed thoroughly. Further, 100 ml of solution IIA and B (equal volume of Solution II A and B) was added in the suspension and vortexed again and kept on ice for 5 min. To this solution, 75 ml of Solution III was added, slowly mixed and kept on ice for 5 min again. The aliquots were centrifuged at 12,000 x g for 5 min at 4°C, and 200 ml of the supernatant solution was transferred to another tube. Subsequently, 300 ml of sodium iodide solution and 500 ml of isopropyl alcohol were added to this solution and vortexed. The aliquots were centrifuged at 12,000 x g for 10 min at room temperature. The supernatant solution was discarded, and the pellet was resuspended in washing solution and centrifuged at 12,000 x g for 5 min (two times) at room temperature. The final mtDNA pellet was dried under vacuum and resuspended in TE buffer, pH 8.0 solution; this was used for restriction endonuclease digestion.

Restriction endonuclease digestion.
The mtDNA was completely digested with restriction endonuclease as indicated below. A total volume of 26 ml digestion mixture contained 20.4 ml of mtDNA (contains 1.0 mg of mtDNA), 2 ml of Hind II enzyme (20 units), 2.6 ml of enzyme buffer, and 1 ml of RNase (10 mg/ml). The digestions were carried out at 37°C for 2 h in a shaking water bath.

Gel electrophoresis.
Agarose (0.8%) slab gels were prepared by dissolving in 0.06 M barbital sodium solution at pH 8.6. Agarose gel electrophoresis was performed in a running TAE buffer at a voltage of 5V/cm at room temperature using a Mini-Gel Electrophoresis system (Mupid-2, Advance Co. Ltd., Tokyo, Japan). Following electrophoresis, gels were stained with ethidium bromide solution (0.5 mg/ml) for 20 min and irradiated from below with a UV transilluminator box at a wavelength 254nm and photographed through an orange filter.

Lipid peroxidation assay.
Lipid peroxidation was carried out by in vitro and in vivo methods. Lipid peroxidation of brain homogenate was determined as previously described (Yamamoto and Tang, 1996aGo). For in vitro studies, aliquots (1 ml) of 10% (w/v) whole brain homogenate of mice were incubated, either in a shaking water bath for 20 min at 37°C or at 0°C on ice (time zero samples), with a concentration of L-cysteine (0.05, 0.5 or 1.0 mM) in the presence or absence of melatonin (1.0 mM) or ganglioside GT1b (60 µM). Each experiment was performed on a pool of brain extracts from eight mice; three aliquots were used from each homogenate. For in vivo studies, aliquots (1 ml) of 10% (w/v) whole brain homogenate of mouse treated with saline, melatonin (20 mg/kg) only, ganglioside GT1b (90 nmol/animal) only, L-cysteine (1.25 µmol/animal) only, L-cysteine (1.25 µmol/animal) plus melatonin (20 mg/kg), or L-cysteine (1.25 µmol/animal) plus ganglioside GT1b were incubated under the same conditions mentioned above. The samples were cooled on ice for 10 min. Thereafter, the reaction was stopped and the samples were centrifuged at 10,000 x g for 15 min in a refrigerated centrifuge (Kokusan Co., Tokyo, Japan) and immediately tested for lipid peroxidation by measuring the concentration of malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) in the tissues. Lipid peroxidation was expressed in terms of MDA + 4-HDA per mg protein. The Bioxytech LPO-586 kit was used for these measurements. Protein concentrations were determined by the method of Lowry et al.(1951)Go, with bovine serum albumin as standard. All data were statistically analyzed by an analysis of variance (ANOVA). Where a significant difference was indicated, the means were determined by Student-Newman-Keul’s test; p < 0.05 was considered as significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
When L-cysteine (1.25 µmol/animal) was injected intracerebroventrically into six mice, severe seizures were observed in five animals within 5 min after injection (see Table 1Go). However, L-cysteine (1.25 µmol/animal) induced seizures were completely abolished by the preadministration of melatonin (20 mg/kg) or ganglioside GT1b (90 nmol/animal) (Table 1Go). These data confirmed our previous results (Yamamoto, 1995Go; Yamamoto and Tang, 1996bGo). Agarose gel electrophoresis of mtDNA isolated from brain frontal cortex of mice administered with or without L-cysteine, L-cysteine + melatonin, or L-cysteine + ganglioside GT1b is shown in Figures 1Go and 2Go. The data indicate that administration of L-cysteine inflicted damage to mtDNA in the frontal cortex, and its effects were significantly inhibited by the preinjection of melatonin or ganglioside GT1b. L-cysteine also caused damage to mtDNA in the central cortical regions, and its effects were strongly inhibited by the preinjection of melatonin or ganglioside GT1b (data not shown).


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TABLE 1 Animal Behavior after Injection of L-Cysteine, L-Cysteine + Melatonin, or L-Cysteine + Ganglioside GT1b
 


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FIG. 1. Agarose gel electrophoresis of mtDNA isolated from brain frontal cortex of mice administered L-cysteine, or L-cysteine + melatonin. Lanes 1, 2, and 3: control; lanes 4, 5, and 6: L-cysteine (1.25 µmol/animal) alone; lanes 7, 8, and 9: L-cysteine (1.25 µmol/animal) + melatonin (20 mg/kg, injected 30 min before L-cysteine administration); lanes 10, 11, and 12: melatonin (20 mg/kg) alone. The experiment was replicated on a second set of three mice per group and showed consistent results.

 


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FIG. 2. Agarose gel electrophoresis of mtDNA isolated from brain frontal cortex of mice administered L-cysteine, or L-cysteine + ganglioside GT1b. Lanes 1, 2, and 3: control; lanes 4, 5, and 6: L-cysteine (1.25 µmol/animal) alone; lanes 7, 8, and 9: L-cysteine (1.25 µmol/animal) + ganglioside GT1b (90 nmol/animal, injected 1 min before L-cysteine administration); lanes 10, 11, and 12: ganglioside GT1b (90 nmol/animal) alone. The experiment was conducted on a second set of three animals per group and showed consistent results.

 
MtDNA was isolated from the homogenate of cortical tissue in mouse brain incubated with different concentrations of L-cysteine (0, 0.05, 0.5 and 1.0 mM) at 37°C for 60 min and was further digested with Hind II at 37°C for 2 h and electrophoresed using 0.8% agarose gel. Agarose gels of mtDNA treated with or without L-cysteine are shown in Figure 3Go. The data indicated that L-cysteine inflicted damage on mtDNA of mouse brain in a concentration-dependent manner. However, L-cysteine–induced damage of brain mtDNA was completely abolished by the cotreatment with melatonin (1.5 mM) (Fig. 3Go) or ganglioside GT1b (60 µM) (Fig. 4Go). Furthermore, the preventive effects of melatonin or ganglioside GT1b against L-cysteine–induced DNA damage were concentration dependent (Figs. 5Go and 6Go).



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FIG. 3. Agarose gel electrophoresis of mtDNA isolated from brain cortical tissue of mice treated in vitro with different concentration of L-cysteine. Lanes 1 and 2: control; lanes 3 and 4: L-cysteine (0.05 mM) alone; lanes 5 and 6: L-cysteine (0.5 mM) alone; lanes 7 and 8: L-cysteine (1.0 mM) alone; lanes 9 and 10: L-cysteine (1.0 mM) + melatonin (1.5 mM); lanes 11 and 12: Melatonin (1.5 mM) alone. The experiment was replicated on two additional sets of two mice per group and showed consistent results.

 


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FIG. 4. Agarose gel electrophoresis of mtDNA isolated from brain cortical tissue of mice treated in vitro with different concentration of L-cysteine. Lanes 1 and 2: control; lanes 3 and 4: L-cysteine (0.05 mM) alone; lanes 5 and 6: L-cysteine (0.5 mM) alone; lanes 7 and 8: L-cysteine (1.0 mM) alone; lanes 9 and 10: L-cysteine (1.0 mM) + ganglioside GT1b (60 µM); lanes 11 and 12: ganglioside GT1b (60 µM) alone. The experiment was replicated on two additional sets of mice and showed consistent results.

 


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FIG. 5. Agarose gel electrophoresis of mtDNA isolated from brain cortical tissue of mice treated in vitro with of L-cysteine or L-cysteine + different concentration of melatonin. Lanes 1 and 2: control; lanes 3 and 4: L-cysteine (1.0 mM) alone; lanes 5 and 6: L-cysteine (1.0 mM) + melatonin (0.1 mM); lanes 7 and 8: L-cysteine (1.0 mM) + melatonin (0.5 mM); lanes 9 and 10: L-cysteine (1.0 mM) + melatonin (1.5 mM). The experiment was replicated on two additional sets of mice and showed consistent results.

 


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FIG. 6. Agarose gel electrophoresis of mtDNA isolated from brain cortical tissue of mice treated in vitro with of L-cysteine or L-cysteine + different concentration of ganglioside GT1b. Lanes 1 and 2: control; lanes 3 and 4: L-cysteine (1.0 mM) alone; lanes 5 and 6: L-cysteine (1.0 mM) + ganglioside GT1b (0.6 µM); lanes 7 and 8: L-cysteine (1.0 mM) + ganglioside GT1b (6.0 µM); lanes 9 and 10: L-cysteine (1.0 mM) + ganglioside GT1b (60 µM). The experiment was conducted on two additional sets of mice and showed consistent results.

 
The exposure of mouse brain homogenates to L-cysteine (0.05, 0.5 or 1.0 mM) at 37°C for 20 min caused a significant increase in the concentration of MDA + 4-HDA (compared to control samples) in a concentration-dependent manner (Table 2Go). The increased lipid peroxidation induced by L-cysteine (1.0 mM) was attenuated by coincubation of the homogenate with melatonin (1.5 mM) or ganglioside GT1b (60 µM). Furthermore, lipid peroxidation of brain homogenate from mice administered L-cysteine ((1.25 µmol/animal) was increased significantly from that of mice administered saline. Increased lipid peroxidation in brain homogenate of mice administered L-cysteine was completely abolished by the coadministration of melatonin (20 mg/kg) or ganglioside GT1b (90 nmol/animal).


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TABLE 2 In Vitro and in Vivo Effects of L-Cysteine, Melatonin, and Ganglioside GT1b on Lipid Peroxidation in Mouse Brain
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present results have clearly demonstrated that in vivo and in vitro exposures of L-cysteine caused damage to brain mtDNA and increased lipid peroxidation in mouse brain, and these effects were abolished by the cotreatment with melatonin, a scavenger of •OH or ganglioside GT1b, a potent inhibitor of glutamate receptor mediated activation and translocation of protein kinase C and lipid peroxidation. When the mice were observed during the posttreatment period, the broad spectrum limbic and severe sustained seizures consistently induced by the icv injection of L-cysteine were absent in mice preadministered melatonin or ganglioside GT1b (see Table 1Go).

The L-cysteine contains a thiol group which provides a special feature that may explain its neurotoxicity, and it produces reactive oxygen species such as •OH. The autoxidation of thiols in the presence of iron generates reactive oxygen species (Misra, 1974Go; Nath and Salahudeen, 1993Go; Saez et al., 1982Go; Yamamoto and Mohanan, 2001bGo; Yamamoto and Tang, 1996aGo). A number of reports suggest that the neurotoxicity of reactive oxygen species may be based on activation of NMDA receptors by the reduction of the redox site on the NMDA receptor-ion channel complex (Goel et al., 1993Go; Lipton and Stamler, 1994Go); this may be via the modification of the NMDA receptor by oxidation of the SH group of proteins at the redox site (Aizemnman et al., 1990Go) or by the nonvesicular basal release of aspartate (Gilman et al., 1994Go), and/or by activation of non-NMDA receptors (Hall et al., 1978Go; Melchiorri et al., 1995Go; Mohanan and Yamamoto, 2002Go). It is well known that L-cysteine activates selectively the NMDA subtype of glutamate receptor (Olney et al., 1990Go) and results in a transmembrane ion imbalance, especially via induction of calcium influx. Elevation of cytosolic Ca2+ is believed to activate nitric oxide (NO) synthase, resulting in the generation of NO. Many reports suggest that NO is a mediator of cell toxicity (Beckman, 1991Go; Yamamoto, 1996Go; Youdim et al., 1993Go). NO is a free radical which reacts with superoxide (O2-) to form peroxynitrite anions (ONOO-) (Blough and Zafiriou, 1985Go). ONOO-can generate •OH (Beckman, 1991Go). This means that L-cysteine may produce •OH by both the activation of NMDA receptors and autoxidation of thiols. In support of this hypothesis, our previous reports showed that L-cysteine and NMDA-induced neurotoxicity is prevented by NG-nitro-L-arginine, NO synthase inhibitor (Yamamoto, 1996Go) or melatonin, a scavenger of •OH (Yamamoto and Tang. 1998Go). It is also suggested that •OH attacks macromolecules within neurons, resulting in membrane lipid peroxidation and DNA strand breaks. Our results suggest that L-cysteine–induced mtDNA damage and lipid peroxidation may be due to reactive oxygen species including •OH. Hence, the mtDNA damage and increased lipid peroxidation induced by L-cysteine were completely inhibited by in vivo and in vitro exposure of the reactive oxygen species scavenger, melatonin or ganglioside GT1b.

Since it is known that cyclic AMP attenuates reactive oxygen species-mediated neuron toxicity (Keller et al., 1998Go) and ganglioside GT1b inhibits cyclic AMP-dependent protein kinase (Yates et al., 1989Go), a part of preventive effect of ganglioside GT1b against L-cysteine–induced brain mtDNA damage may be due to its inhibition of cyclic AMP dependent pathway in mouse brain, however, further studies may be necessary to elucidate this hypothesis.


    ACKNOWLEDGMENTS
 
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 program on the Japan Society for the Promotion of Science.


    NOTES
 
1 To whom correspondence should be addressed at the University of Tsukuba, Institute of Community Medicine, Tsukuba, Ibaraki 305-8575, Japan. Fax: 81-29-853-3485 or 81-29-858-3443. E-mail: hiro_aki{at}d4.dion.ne.jp. Back


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