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Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead
Toxicology Letters 102]103 Ž1998. 363]367
Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead a Kai M. Savolainena,b,U , Jarkko Loikkanen a , Simo Eerikainen , Jonne Naaralaa,c ¨ a Department of Pharmacology and Toxicology, Uni¨ ersity of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland Department of En¨ ironmental Medicine, National Public Health Institute, P.O. Box 95, FIN-70701 Kuopio, Finland c Institute of Surgical Research, Laboratory of Neurobiology, Ludwig-Maximilians Uni¨ ersity Munich, Marchioninistrasse 15, D-81366 Munich, Germany b
Abstract Increased glutamate release is associated with serious neurological disorders such as epilepsy, stroke, Alzheimer’s disease and other brain injuries. Excessive glutamate release and subsequent glutamatergic neuronal stimulation increase the production of reactive oxygen species ŽROS., which in turn induce oxidative stress, excitotoxicity and neuronal damage. A number of studies have shown that co-exposure of neuronal cells to glutamate, and an environmental toxin, lead, can greatly amplify glutamate excitotoxicity and cell death through apoptosis or necrosis. Even though the mechanisms of excitotoxicity or those of glutamate]lead interactions have not been exhaustively delineated, there is ample evidence to suggest that increased production of ROS may play an important role in both events. Subsequently, increased DNA binding of redox-regulated transcription factors, NF-k B and AP-1, seems to be associated with these events. Induction of an immediate early gene, c-fos, is seen in neuronal cells exposed to glutamate or lead. Immediate early genes are important in regulating the expression of other neuronal genes. Elevated expressions of the genes encoding Hsp70 or cyclo-oxygenase-2 seem to be involved in the apoptosis or necrosis induced by glutamate, and may be associated with induction of several of the genes in cells exposed to lead, or to the glutamate]lead combination. Further studies are required to clarify the mechanisms of glutamate]lead neurotoxicity. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glutamate; Lead; Excitotoxicity; Oxidative stress; Apoptotic cell death; Necrotic cell death
U
Corresponding author. Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland. Tel.: q358 9 47471; fax: q358 9 4747208; e-mail: [email protected] 0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00233-1
364
K.M. Sa¨ olainen et al. r Toxicology Letters 102]103 (1998) 363]367
1. Introduction Activation of NMDA Ž N-methyl-D-aspartate., AMPA Žalpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid., kainate and metabotrobic receptor subtypes by glutamate, the most ubiquitous cerebral neurotransmitter, leads to an increase in the levels of free intracellular calcium ŽCoyle and Puttfarcken, 1993.. Glutamate release is closely associated with serious neurological disorders such as epilepsy, stroke, hypoxia, glucose deprivation and brain trauma ŽCoyle and Puttfarcken, 1993; Kalda et al., 1998.. In addition to its vital role as a neurotransmitter, glutamate at high levels is excitotoxic to neurons. The excitotoxicity is associated with marked increases in free intracellular calcium levels ŽCoyle and Puttfarcken, 1993; Whetsell, 1996.. Glutamate-induced excitotoxicity induces cytoskeletal alterations, excitatory amino acid ŽEAA. release, impaired EAA uptake, and the production of ROS ŽNaarala et al., 1995; Nieminen et al., 1996; Whetsell, 1996.. Glutamate also increases DNA binding of the redox-regulated transcription factors, NF-k B and AP-1, in human neuroblastoma cells ŽNaarala et al., unpublished., and increases the expression of the immediate early gene, c-fos, in murine neuronal cells ŽGriffiths et al., 1997.. These events occur before glutamate-induced apoptosis or necrosis in several neuronal cell types ŽBondy and Lee, 1993; Coyle and Puttfarcken, 1993; Lafon-Cazal et al., 1993; Reynolds and Hastings, 1995.. Lead, an environmental toxin, provokes central nervous system ŽCNS. symptoms such as headache, hyperactivity, learning disorders, ataxia, convulsions, and coma ŽNeedleman et al., 1990.. Lead neurotoxicity is poorly understood, but some effects of lead may be due to the interference of this metal with many calcium-dependent cellular processes due to similarities between Ca2q and Pb 2q in aqueous solutions ŽSimons, 1986, 1993.. Lead also induces the expression of c-fos in PC-12 cells ŽKim et al., 1997. suggesting that lead may alter the expression of other genes. The co-exposure of human neuroblastoma cells to lead and glutamate greatly amplifies the glutamate-induced oxidative stress and excitotoxicity ŽNaarala et al., 1995.. Thus, lead may amplify the gluta-
mate-induced production of ROS that precedes alterations in gene expression, and this may well be associated with glutamate]lead neurotoxicity. This paper discusses the interaction of glutamate and lead, especially in ROS production, oxidative stress, excitotoxicity and lead neurotoxicity. Furthermore, the role of gene expression in these events will be reviewed. 2. Interactions of glutamate with lead 2.1. Effects of glutamate and lead on ROS production and oxidati¨ e stress Several cell types have been used to explore glutamate]lead neurotoxicity. Naarala et al. Ž1995. have used human SH-SY5Y neuroblastoma cells after having demonstrated that they express a variety of glutamate receptor subtypes wNaarala et al. Ž1993.; see also Nair et al. Ž1996.x. Murine hypothalamic GT1-7 cells have also often been used for the same purpose ŽLoikkanen et al., 1998a.. PC-12 cells have been used to explore the effects of lead on gene expression ŽKim et al., 1997., primary cultures of rat granule cells were used in studies of the mechanisms by which glutamate increases ROS production in vitro ŽBondy and Lee, 1993; Bondy and LeBel, 1993., and primary cultures of murine neuronal cells have been used to examine the effects of glutamate on alterations in gene expression ŽGriffiths et al., 1997.. Bondy and Lee Ž1993. and Bondy and LeBel Ž1993. have shown in rat cerebellar granule cells that 1 m M glutamate increases the production of ROS in these cells. Naarala et al. Ž1995., in turn, demonstrated that glutamate could increase ROS production in human SH-SY5Y neuroblastoma cells subsequent to depletion of intracellular glutathione ŽGSH. levels with diethylmaleate ŽDEM.. Apparently, antioxidative defence mechanisms are more effective in human neuroblastoma cells than in rat cerebellar granule cells. Alternatively, rat cerebellar granule cells may express more glutamate receptors than the neuroblastoma cells. Glutamate-induced ROS production was completely inhibited with a putative protein kinase C ŽPKC. inhibitor, Ro 31-7549, emphasising the key
K.M. Sa¨ olainen et al. r Toxicology Letters 102]103 (1998) 363]367
role of PKC in this receptor-mediated production of ROS in human SH-SY5Y neuroblastoma cells ŽNaarala et al., 1995.. When human neuroblastoma cells were co-exposed to glutamate and lead, ROS production was greatly amplified, and intracellular GSH levels were reduced without the addition of DEM, indicating that lead provokes a marked potentiation of glutamate-induced ROS production. Amplification of glutamate-induced neuronal effects by lead could, in fact, be an important mechanism for lead neurotoxicity ŽNaarala et al., 1995.. Glutamate increased ROS production and decreased intracellular GSH levels in murine hypothalamic GT1-7 neurones both in the presence and absence of extracellular calcium. Lead, in turn, decreased GSH levels without increasing ROS production. Glutamate also amplified leadinduced decreases in intracellular GSH. Glutamate-induced ROS production did not depend on PKC because it could not be prevented by a novel putative PKC inhibitor, Ro 31-8220, in GT1-7 cells ŽLoikkanen et al., 1998a.. Lead, however, amplified glutamate-induced production of ROS in these cells. In agreement with the findings of Naarala et al. Ž1995., amplification of glutamateevoked ROS production by lead occurred only in the absence of extracellular calcium. A PKC inhibitor partially blocked this ROS production pointing to the potential role of PKC in this glutamate]lead interaction ŽLoikkanen et al., 1998a.. 2.2. Effects of glutamate and lead on transcription factors and gene expression Gahring et al. Ž1996. showed that the excitotoxicity of kainic acid was associated with increased expression of the immediate early gene, c-fos, in fibroblasts transfected with the glutamate receptor subunit, GluR1. Furthermore, several reports indicate that exposure to glutamate receptor analogues can induce the expression of a number of genes. Chen et al. Ž1995. showed that exposure to kainate can increase the expression of cyclooxygenase-2 ŽCOX-2. in rat brain; this effect could be prevented by pre-treatment of the rats with MK-801 or NBQX indicative of the involvement
365
of glutamate receptor subtypes. Increased COX-2 expression was associated with neuronal necrosis. This observation was confirmed by Nogawa et al. Ž1997. and Miettinen et al. Ž1997.. Furthermore, Kondo et al. Ž1997. have shown that the neuronal death after treatment with kainic acid in transgenic mice over-expressing human CuZn-superoxide dismutase is associated with prolonged expression of c-fos and c-jun. Lead induced the expression of c-fos in PC-12 cells ŽKim et al., 1997. and it also activated another transcription factor, NF-k B, in human lymphocytes ŽPyatt et al., 1996.. Naarala et al. Žunpublished. also demonstrated increased DNA binding of NF-k B that was not amplified by coexposure to glutamate. Since lead can increase expression of different transcription factors, it may also change the expression of other genes. Naarala et al. Žunpublished. explored the effects of glutamate, lead, or their combination on the binding of redox-regulated transcription factors, NF-k B and AP-1, to DNA in human SHSY5Y neuroblastoma cells. Glutamate andror lead increased the binding of NF-k B between 6 and 72 h after the beginning of the exposure. Glutamate increased the DNA binding of AP-1 to DNA at 3 h, the glutamate]lead combination at 12 h, but lead on its own was without effect. All these findings indicate that these compounds and their combination were able to induce the binding of these factors and, therefore, capable of causing alterations in the expression of a number of genes. These alterations in the binding of transcription factors were most likely due to increased production of ROS upon stimulation of the cells with the test compounds. 2.3. Effects of glutamate and lead on neuronal death Naarala et al. Ž1995. showed that neither glutamate or lead alone induced death of human SHSY5Y neuroblastoma cells, but when the cells were co-exposed to these compounds, cytotoxicity was apparent. The cytotoxicity seemed to correlate with ROS production and GSH depletion. Loikkanen et al. Ž1998a. demonstrated in murine hypothalamic GT1-7 cells that glutamate alone did not affect cell viability, whereas glutamate
366
K.M. Sa¨ olainen et al. r Toxicology Letters 102]103 (1998) 363]367
markedly increased lead-induced cytotoxicity. Loikkanen also concluded that the joint cytotoxicity of glutamate and lead was mainly due to lead toxicity and preferentially mediated through mechanisms other than ROS production ŽLoikkanen et al., 1998a.. These cytotoxicity data are in agreement with findings presented by other groups ŽBondy and Lee, 1993; Bondy and LeBel, 1993.. The mechanism through which glutamate-induced cell death proceeds has remained an open question until recently when several investigators have provided evidence that apoptosis may be important in this regard, and that alterations of gene expression may be critical in these events. Oberto et al. Ž1996. provided evidence that lead at micromolar concentrations induces neuronal death through apoptosis in rat cerebellar granule cells; this effect could be prevented by a Ca2q channel antagonist suggesting that entry of Pb 2q into the cells, or alternatively influx of calcium, may be important for this effect. Naarala et al. Žunpublished. have shown that lead alone and together with glutamate induces DNA fragmentation in human SH-SY5Y neuroblastoma cells at 48 and 72 h after the beginning of the exposure. Glutamate was without any effect in these cells. Moreover, Loikkanen et al. Ž1998b. found that glutamate did not induce DNA fragmentation in GT1-7 cells. In these cells, lead alone, and lead combined with glutamate, induced DNA fragmentation which was seen at 24, 48 and 72 h after the beginning of the exposure. Thus, lead-induced neuronal cell death may, at least partially, be mediated through apoptosis. 3. Conclusion There is a plethora of data that both glutamate and lead, as well as their combination, provokes the production of ROS, and induces oxidative stress in different neuronal cell lines. These events seem to be associated with increased neuronal lethality, through both apoptotic and necrotic mechanisms. The cytotoxicity, whether apoptotic or necrotic, seems to depend on the dose. Oxidative stress seems to induce increased binding of redox-regulated transcription factors to DNA, leading to regulation of the expression of a num-
ber of different genes. It is most likely that both transcription factors and alterations in gene expression are ultimately involved in apoptotic and necrotic neuronal death induced by glutamate, lead, or their combination. Acknowledgements The authors wish to thank Dr Ewen MacDonald for reading the manuscript. This study was supported by The Academy of Finland, Jenny and Antti Wihuri Foundation and Emil Aaltonen Foundation. References Bondy, S.C., LeBel, C.P., 1993. The relationship between excitotoxicity and oxidative stress in the central nervous system. Free Radic. Biol. Med. 14, 633]642. Bondy, S.C., Lee, D.K., 1993. Oxidative stress induced by glutamate receptor agonists. Brain Res. 610, 229]233. Chen, J., Marsh, T., Zhang, J.S., Graham, S.H., 1995. Expression of cyclo-oxygenase 2 in rat brain following kainate treatment. NeuroReport 6, 245]248. Coyle, J.T., Puttfarcken, P., 1993. Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689]694. Gahring, L.C., Cauley, K., Rogers, S.W., 1996. Kainic acid induced excitotoxicity and c-fos expression in fibroblasts transfected with glutamate receptor subunit, GluR1. J. Neurobiol. 31, 56]66. Griffiths, R., Malcolm, C., Ritchie, L., et al., 1997. Association of c-fos mRNA expression and excitotoxicity in primary cultures of mouse neocortical and cerebellar neurones. J. Neurosci. Res. 48, 533]542. Kalda, A., Eriste, E., Vassiljev, V., Zharkovsky, A., 1998. Medium transitory oxygen-glucose deprivation induced both apoptosis and necrosis in cerebellar granule cells. Neurosci. Lett. 240, 21]24. Kim, K., Annadata, M., Goldstein, G.W., Bressler, J.P., 1997. Induction of c-fos mRNA by lead in PC-12 cells. Int. J. Dev. Neurosci. 15, 175]182. Kondo, T., Sharp, F.R., Honkaniemi, J., Mikawa, S., Epstein, C.J., Chan, P.H., 1997. DNA fragmentation and prolonged expression of c-fos, c-jun, and Hsp70 in kainic acid-induced neuronal cell death in transgenic mice overexpressing human CuZn-superoxide dismutase. J. Cerebral Blood Flow Metab. 17, 241]256. Lafon-Cazal, M., Pietri, S., Culcasi, M., Bockaert, J., 1993. NMDA-dependent superoxide production and neurotoxicity. Nature 364, 535]537. Loikkanen, J.J., Naarala, J., Savolainen, K.M., 1998a. Modification of glutamate-induced oxidative stress by lead: the role of extracellular calcium. Free Radic. Biol. Med. 24, 377]384.
K.M. Sa¨ olainen et al. r Toxicology Letters 102]103 (1998) 363]367 Loikkanen, J., Naarala, J., Savolainen, K., 1998b. The role of glutamatergic neuronal stimulation and oxidative stress in lead neurotoxicology. International Congress of Toxicology VIII, Paris, France. Toxicol. Lett. 95, S129. Naarala, J., Nykvist, P., Tuomala, M., Savolainen, K., 1993. Excitatory amino acid-induced slow biphasic responses of free intracellular calcium in human neuroblastoma cells. FEBS Lett. 330, 222]226. Naarala, J., Loikkanen, J.J., Ruotsalainen, M.H., Savolainen, K.M., 1995. Lead amplifies glutamate-induced oxidative stress. Free Radic. Biol. Med. 19, 689]693. Nair, V.D., Niznik, H.B., Mishra, R.K., 1996. Interaction of NMDA and dopamine D2L receptors in human neuroblastoma SH-SY5Y cells. J. Neurochem. 66, 2390]2393. Needleman, H.L., Schell, A., Bellinger, D., Leviton, A., Allred, E.N., 1990. The long-term effects of exposure to low doses of lead in childhood. N. Engl. J. Med. 322, 83]88. Nieminen, A.-L., Petrie, T.G., Lemasters, J.J., Selman, W.R., 1996. Cyclosporin A delays mitochondrial depolarization induced by N-methyl-D-aspartate in cortical neurones: evidence of the mitochondrial permeability transition. Neuroscience 75, 993]997. Nogawa, S., Zhang, F., Ross, M.E., Iadecola, C., 1997. Cyclooxygenase-2 gene expression in neurones contributes to ischemic brain damage. J. Neurosci. 17, 2746]2755.
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Miettinen, S., Fusco, F.R., Yrjanheikki, J., et al., 1997. Spread¨ ing depression and focal brain ischemia induce cyclooxygenase-2 in cortical neurones through N-methyl-D-aspartic acid-receptors and phospholipase A 2 . Proc. Natl. Acad. Sci. U.S.A. 94, 6500]6505. Oberto, A., Marks, N., Evans, H.L., Guidotti, A., 1996. Lead ŽPb 2q . promotes apoptosis in newborn rat cerebellar neurons: pathological implications. J. Pharmacol. Exp. Ther. 279, 435]442. Pyatt, D.W., Zheng, J.-H., Stillman, W.S., Irons, R.D., 1996. Inorganic lead activates NF-k B in primary human CD4q T lymphocytes. Biochem. Biophys. Res. Commun. 227, 380]385. Reynolds, I.J., Hastings, T.G., 1995. Glutamate induces the production of reactive oxygen species in cultured forebrain neurones following NMDA receptor activation. J. Neurosci. 15, 3318]3327. Simons, T.J.B., 1986. Cellular interactions between lead and calcium. Br. Med. Bull. 42, 431]434. Simons, T.J.B., 1993. Lead]calcium interactions in cellular lead toxicity. Neurotoxicology 14, 77]86. Whetsell, W.O., Jr., 1996. Current concepts of excitotoxicity. J. Neuropathol. Exp. Neurol. 55, 1]13.
Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead a Kai M. Savolainena,b,U , Jarkko Loikkanen a , Simo Eerikainen , Jonne Naaralaa,c ¨ a Department of Pharmacology and Toxicology, Uni¨ ersity of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland Department of En¨ ironmental Medicine, National Public Health Institute, P.O. Box 95, FIN-70701 Kuopio, Finland c Institute of Surgical Research, Laboratory of Neurobiology, Ludwig-Maximilians Uni¨ ersity Munich, Marchioninistrasse 15, D-81366 Munich, Germany b
Abstract Increased glutamate release is associated with serious neurological disorders such as epilepsy, stroke, Alzheimer’s disease and other brain injuries. Excessive glutamate release and subsequent glutamatergic neuronal stimulation increase the production of reactive oxygen species ŽROS., which in turn induce oxidative stress, excitotoxicity and neuronal damage. A number of studies have shown that co-exposure of neuronal cells to glutamate, and an environmental toxin, lead, can greatly amplify glutamate excitotoxicity and cell death through apoptosis or necrosis. Even though the mechanisms of excitotoxicity or those of glutamate]lead interactions have not been exhaustively delineated, there is ample evidence to suggest that increased production of ROS may play an important role in both events. Subsequently, increased DNA binding of redox-regulated transcription factors, NF-k B and AP-1, seems to be associated with these events. Induction of an immediate early gene, c-fos, is seen in neuronal cells exposed to glutamate or lead. Immediate early genes are important in regulating the expression of other neuronal genes. Elevated expressions of the genes encoding Hsp70 or cyclo-oxygenase-2 seem to be involved in the apoptosis or necrosis induced by glutamate, and may be associated with induction of several of the genes in cells exposed to lead, or to the glutamate]lead combination. Further studies are required to clarify the mechanisms of glutamate]lead neurotoxicity. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glutamate; Lead; Excitotoxicity; Oxidative stress; Apoptotic cell death; Necrotic cell death
U
Corresponding author. Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland. Tel.: q358 9 47471; fax: q358 9 4747208; e-mail: [email protected] 0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00233-1
364
K.M. Sa¨ olainen et al. r Toxicology Letters 102]103 (1998) 363]367
1. Introduction Activation of NMDA Ž N-methyl-D-aspartate., AMPA Žalpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid., kainate and metabotrobic receptor subtypes by glutamate, the most ubiquitous cerebral neurotransmitter, leads to an increase in the levels of free intracellular calcium ŽCoyle and Puttfarcken, 1993.. Glutamate release is closely associated with serious neurological disorders such as epilepsy, stroke, hypoxia, glucose deprivation and brain trauma ŽCoyle and Puttfarcken, 1993; Kalda et al., 1998.. In addition to its vital role as a neurotransmitter, glutamate at high levels is excitotoxic to neurons. The excitotoxicity is associated with marked increases in free intracellular calcium levels ŽCoyle and Puttfarcken, 1993; Whetsell, 1996.. Glutamate-induced excitotoxicity induces cytoskeletal alterations, excitatory amino acid ŽEAA. release, impaired EAA uptake, and the production of ROS ŽNaarala et al., 1995; Nieminen et al., 1996; Whetsell, 1996.. Glutamate also increases DNA binding of the redox-regulated transcription factors, NF-k B and AP-1, in human neuroblastoma cells ŽNaarala et al., unpublished., and increases the expression of the immediate early gene, c-fos, in murine neuronal cells ŽGriffiths et al., 1997.. These events occur before glutamate-induced apoptosis or necrosis in several neuronal cell types ŽBondy and Lee, 1993; Coyle and Puttfarcken, 1993; Lafon-Cazal et al., 1993; Reynolds and Hastings, 1995.. Lead, an environmental toxin, provokes central nervous system ŽCNS. symptoms such as headache, hyperactivity, learning disorders, ataxia, convulsions, and coma ŽNeedleman et al., 1990.. Lead neurotoxicity is poorly understood, but some effects of lead may be due to the interference of this metal with many calcium-dependent cellular processes due to similarities between Ca2q and Pb 2q in aqueous solutions ŽSimons, 1986, 1993.. Lead also induces the expression of c-fos in PC-12 cells ŽKim et al., 1997. suggesting that lead may alter the expression of other genes. The co-exposure of human neuroblastoma cells to lead and glutamate greatly amplifies the glutamate-induced oxidative stress and excitotoxicity ŽNaarala et al., 1995.. Thus, lead may amplify the gluta-
mate-induced production of ROS that precedes alterations in gene expression, and this may well be associated with glutamate]lead neurotoxicity. This paper discusses the interaction of glutamate and lead, especially in ROS production, oxidative stress, excitotoxicity and lead neurotoxicity. Furthermore, the role of gene expression in these events will be reviewed. 2. Interactions of glutamate with lead 2.1. Effects of glutamate and lead on ROS production and oxidati¨ e stress Several cell types have been used to explore glutamate]lead neurotoxicity. Naarala et al. Ž1995. have used human SH-SY5Y neuroblastoma cells after having demonstrated that they express a variety of glutamate receptor subtypes wNaarala et al. Ž1993.; see also Nair et al. Ž1996.x. Murine hypothalamic GT1-7 cells have also often been used for the same purpose ŽLoikkanen et al., 1998a.. PC-12 cells have been used to explore the effects of lead on gene expression ŽKim et al., 1997., primary cultures of rat granule cells were used in studies of the mechanisms by which glutamate increases ROS production in vitro ŽBondy and Lee, 1993; Bondy and LeBel, 1993., and primary cultures of murine neuronal cells have been used to examine the effects of glutamate on alterations in gene expression ŽGriffiths et al., 1997.. Bondy and Lee Ž1993. and Bondy and LeBel Ž1993. have shown in rat cerebellar granule cells that 1 m M glutamate increases the production of ROS in these cells. Naarala et al. Ž1995., in turn, demonstrated that glutamate could increase ROS production in human SH-SY5Y neuroblastoma cells subsequent to depletion of intracellular glutathione ŽGSH. levels with diethylmaleate ŽDEM.. Apparently, antioxidative defence mechanisms are more effective in human neuroblastoma cells than in rat cerebellar granule cells. Alternatively, rat cerebellar granule cells may express more glutamate receptors than the neuroblastoma cells. Glutamate-induced ROS production was completely inhibited with a putative protein kinase C ŽPKC. inhibitor, Ro 31-7549, emphasising the key
K.M. Sa¨ olainen et al. r Toxicology Letters 102]103 (1998) 363]367
role of PKC in this receptor-mediated production of ROS in human SH-SY5Y neuroblastoma cells ŽNaarala et al., 1995.. When human neuroblastoma cells were co-exposed to glutamate and lead, ROS production was greatly amplified, and intracellular GSH levels were reduced without the addition of DEM, indicating that lead provokes a marked potentiation of glutamate-induced ROS production. Amplification of glutamate-induced neuronal effects by lead could, in fact, be an important mechanism for lead neurotoxicity ŽNaarala et al., 1995.. Glutamate increased ROS production and decreased intracellular GSH levels in murine hypothalamic GT1-7 neurones both in the presence and absence of extracellular calcium. Lead, in turn, decreased GSH levels without increasing ROS production. Glutamate also amplified leadinduced decreases in intracellular GSH. Glutamate-induced ROS production did not depend on PKC because it could not be prevented by a novel putative PKC inhibitor, Ro 31-8220, in GT1-7 cells ŽLoikkanen et al., 1998a.. Lead, however, amplified glutamate-induced production of ROS in these cells. In agreement with the findings of Naarala et al. Ž1995., amplification of glutamateevoked ROS production by lead occurred only in the absence of extracellular calcium. A PKC inhibitor partially blocked this ROS production pointing to the potential role of PKC in this glutamate]lead interaction ŽLoikkanen et al., 1998a.. 2.2. Effects of glutamate and lead on transcription factors and gene expression Gahring et al. Ž1996. showed that the excitotoxicity of kainic acid was associated with increased expression of the immediate early gene, c-fos, in fibroblasts transfected with the glutamate receptor subunit, GluR1. Furthermore, several reports indicate that exposure to glutamate receptor analogues can induce the expression of a number of genes. Chen et al. Ž1995. showed that exposure to kainate can increase the expression of cyclooxygenase-2 ŽCOX-2. in rat brain; this effect could be prevented by pre-treatment of the rats with MK-801 or NBQX indicative of the involvement
365
of glutamate receptor subtypes. Increased COX-2 expression was associated with neuronal necrosis. This observation was confirmed by Nogawa et al. Ž1997. and Miettinen et al. Ž1997.. Furthermore, Kondo et al. Ž1997. have shown that the neuronal death after treatment with kainic acid in transgenic mice over-expressing human CuZn-superoxide dismutase is associated with prolonged expression of c-fos and c-jun. Lead induced the expression of c-fos in PC-12 cells ŽKim et al., 1997. and it also activated another transcription factor, NF-k B, in human lymphocytes ŽPyatt et al., 1996.. Naarala et al. Žunpublished. also demonstrated increased DNA binding of NF-k B that was not amplified by coexposure to glutamate. Since lead can increase expression of different transcription factors, it may also change the expression of other genes. Naarala et al. Žunpublished. explored the effects of glutamate, lead, or their combination on the binding of redox-regulated transcription factors, NF-k B and AP-1, to DNA in human SHSY5Y neuroblastoma cells. Glutamate andror lead increased the binding of NF-k B between 6 and 72 h after the beginning of the exposure. Glutamate increased the DNA binding of AP-1 to DNA at 3 h, the glutamate]lead combination at 12 h, but lead on its own was without effect. All these findings indicate that these compounds and their combination were able to induce the binding of these factors and, therefore, capable of causing alterations in the expression of a number of genes. These alterations in the binding of transcription factors were most likely due to increased production of ROS upon stimulation of the cells with the test compounds. 2.3. Effects of glutamate and lead on neuronal death Naarala et al. Ž1995. showed that neither glutamate or lead alone induced death of human SHSY5Y neuroblastoma cells, but when the cells were co-exposed to these compounds, cytotoxicity was apparent. The cytotoxicity seemed to correlate with ROS production and GSH depletion. Loikkanen et al. Ž1998a. demonstrated in murine hypothalamic GT1-7 cells that glutamate alone did not affect cell viability, whereas glutamate
366
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markedly increased lead-induced cytotoxicity. Loikkanen also concluded that the joint cytotoxicity of glutamate and lead was mainly due to lead toxicity and preferentially mediated through mechanisms other than ROS production ŽLoikkanen et al., 1998a.. These cytotoxicity data are in agreement with findings presented by other groups ŽBondy and Lee, 1993; Bondy and LeBel, 1993.. The mechanism through which glutamate-induced cell death proceeds has remained an open question until recently when several investigators have provided evidence that apoptosis may be important in this regard, and that alterations of gene expression may be critical in these events. Oberto et al. Ž1996. provided evidence that lead at micromolar concentrations induces neuronal death through apoptosis in rat cerebellar granule cells; this effect could be prevented by a Ca2q channel antagonist suggesting that entry of Pb 2q into the cells, or alternatively influx of calcium, may be important for this effect. Naarala et al. Žunpublished. have shown that lead alone and together with glutamate induces DNA fragmentation in human SH-SY5Y neuroblastoma cells at 48 and 72 h after the beginning of the exposure. Glutamate was without any effect in these cells. Moreover, Loikkanen et al. Ž1998b. found that glutamate did not induce DNA fragmentation in GT1-7 cells. In these cells, lead alone, and lead combined with glutamate, induced DNA fragmentation which was seen at 24, 48 and 72 h after the beginning of the exposure. Thus, lead-induced neuronal cell death may, at least partially, be mediated through apoptosis. 3. Conclusion There is a plethora of data that both glutamate and lead, as well as their combination, provokes the production of ROS, and induces oxidative stress in different neuronal cell lines. These events seem to be associated with increased neuronal lethality, through both apoptotic and necrotic mechanisms. The cytotoxicity, whether apoptotic or necrotic, seems to depend on the dose. Oxidative stress seems to induce increased binding of redox-regulated transcription factors to DNA, leading to regulation of the expression of a num-
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