Effects of intermediate frequency magnetic fields on male fertility indicators in mice.

Human exposure to intermediate frequency (IF) fields is increasing due to new applications such as electronic article surveillance systems, wireless power transfer and induction heating cookers. However, limited data is available on effects of IF magnetic fields (MF) on male fertility function. This study was conducted to assess possible effects on fertility indicators from exposure to IF MF. Male C57BL/6J mice were exposed continuously for 5 weeks to 7.5kHz MF at 12 and 120µT. Sperm cells from cauda epididymis were analysed for motility, total sperm counts, and head abnormalities. Motile sperm cells were classified as progressive or non-progressive. Testicular spermatid heads were counted as well. The body weight development and reproductive tissue weights were not affected. No exposure-related differences were observed in sperm counts or sperm head abnormalities. Proportion of non-motile cells was significantly decreased in the 120µT group, and a corresponding increase was seen in the percentage of motile cells (significant in non-progressive motile cells). In conclusion, no adverse effects on fertility indicators were observed. Increased sperm motility is an interesting finding that needs to be confirmed in further studies.

Synergistic proinflammatory interactions of microbial toxins and structural components characteristic to moisture-damaged buildings.

Indoor exposure to microbes and their structural and metabolic compounds is notoriously complex. To study proinflammatory interactions between the multiple microbial agents, macrophages derived from human THP-1 monocytic cells were exposed to several concentrations of microbial toxins alone (emodin, enniatin B, physcion, sterigmatocystin, valinomycin) and in combination with microbial structural components (bacterial lipopolysaccharide [LPS] or fungal ß-glucan). While the expression of proinflammatory cytokines TNFα and IL-1ß to single toxins alone was modest, low-dose co-exposure with structural components increased the responses of emodin, enniatin B, and valinomycin synergistically, both at the mRNA and protein level, as measured by RT-qPCR and ELISA, respectively. Co-exposure of toxins and ß-glucan resulted in consistent synergistically increased expression of several inflammation-related genes, while some of the responses with LPS were also inhibitory. Co-exposure of toxins with either ß-glucan or LPS induced also mitochondrial damage and autophagocytosis. The results demonstrate that microbial toxins together with bacterial and fungal structural components characteristic to moisture-damaged buildings can have drastic synergistic proinflammatory interactions at low exposure levels.

Cell death and production of reactive oxygen species by murine macrophages after short term exposure to phthalates.

The effects of four phthalates, i.e., di-2-ethylhexyl phthalate (DEHP), butyl benzyl phthalate (BBP), dibutyl phthalate (DBP) and diisobutyl phthalate (DIBP) on necrotic and apoptotic cell death, and production of reactive oxygen species (ROS) were studied on mouse macrophage cell line RAW 264.7. All the phthalates caused negligible and non-dose-dependent ROS production compared to control experiment. DEHP and BBP did not cause significant necrotic nor apoptotic cell death at any of the studied doses. Both DIBP and DBP caused dose-dependent necrotic cell death at the two highest concentrations (100 microM and 1 mM). Both doses (500 microM and 1 mM) of DIBP increased apoptosis by 31- and 60-fold, respectively, whereas the increase in apoptotic cell death caused by DBP was only two and fourfold, that however, was not statistically significant. In conclusion, DIBP caused a substantially different apoptotic cell death effect on murine macrophages from the three other phthalates, and this effect was not related to ROS production. Thus, toxicological and health risks of DIBP and DBP should be assessed separately in the future.

Ornithine decarboxylase activity of L929 cells after exposure to continuous wave or 50 Hz modulated radiofrequency radiation--a replication study.

A replication study with some extensions was made to confirm enhancement of ornithine decarboxylase (ODC) activity in murine L929 fibroblasts after radiofrequency (RF) field exposure reported in earlier studies. L929 cells purchased from two cell banks were exposed for 2, 8, or 24 h to continuous wave or DAMPS (burst modulated at 50 Hz, with 33% duty cycle) signals at specific absorption rate (SAR) levels of 2.5 or 6.0 W/kg. Exposures were carried out in Crawford and waveguide chambers, at frequencies 835 and 872 MHz, respectively. The results did not confirm findings of previous studies reporting increased ODC activity in RF-exposed cells. When Crawford cell exposure system was used, ODC activity was either not affected (in the case of 8 or 24 h exposures) or decreased after 2 h exposure at the highest SAR level (6 W/kg). The decrease was most pronounced when cooling with air flow was not used, and is most likely related to increased temperature. The minor methodological differences (use of antibiotics, increased sensitivity of ODC assay) are not likely to explain the inconsistency of the findings of the present and previous studies. Different results were obtained in experiments with the waveguide system that involves more efficient temperature control. In this exposure system, ODC activity was increased after 8 h exposure at 6 W/kg. Further studies are warranted to explore whether this finding reflects a true non-thermal effect. The present study did not provide evidence for modulation-specific effects reported in earlier studies.

Ornithine decarboxylase activity is affected in primary astrocytes but not in secondary cell lines exposed to 872 MHz RF radiation.

PURPOSE: The effects of radiofrequency (RF) radiation on cellular ornithine decarboxylase (ODC) activity were studied in fibroblasts, two neural cell lines and primary astrocytes. Several exposure times and exposure levels were used, and the fields were either unmodulated or modulated according to the characteristics of the Global System for Mobile (GSM) communications. MATERIALS AND METHODS: Murine L929 fibroblasts, rat C6 glioblastoma cells, human SH-SY5Y neuroblastoma cells, and rat primary astrocytes were exposed to RF radiation at 872 MHz in a waveguide exposure chamber equipped with water cooling. Cells were exposed for 2, 8, or 24 hours to continuous wave (CW) RF radiation or to a GSM type signal pulse modulated at 217 Hz, at specific absorption rates of 1.5, 2.5, or 6.0 W/kg. Cellular ODC activities of cell samples were assayed. RESULTS: ODC activity in rat primary astrocytes was decreased statistically significantly (p values from 0.003 to <0.001) and consistently in all experiments performed at two exposure levels (1.5 and 6.0 W/kg) and using GSM modulated or CW radiation. In the secondary cell lines, ODC activity was generally not affected. CONCLUSIONS: ODC activity was affected by RF radiation in rat primary neural cells, but the secondary cells used in this study showed essentially no response to similar RF radiation. In contrast to some previous studies, no differences between the modulated and continuous wave signals were detected. Further studies with primary astrocytes are warranted to confirm the present findings and to explore the mechanisms of the effects.

Modest increase in temperature affects ODC activity in L929 cells: Low-level radiofrequency radiation does not.

The effects of low-level radiofrequency (RF) radiation and elevated temperature on ornithine decarboxylase (ODC) activity were investigated in murine L929 fibroblasts. The cells were exposed at 900 MHz either to a pulse-modulated (pulse frequency 217 Hz; GSM-type modulation) or a continuous wave signal at specific absorption rate (SAR) levels of 0.2 W kg(-1) (0.1-0.3 W kg(-1)) and 0.4 W kg(-1) (0.3-0.5 W kg(-1)) for 2, 8, or 24 h. RF radiation did not affect cellular ODC activity. However, a slight increase in temperature (0.8-0.9 degrees C) in the exposure system lead to decreased ODC activity in cell cultures. This was verified by tests in which cells were exposed to different temperatures in incubators. The results show that ODC activity is sensitive to small temperature differences in cell cultures. Hence, a precise temperature control in cellular ODC activity studies is needed.

Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb2+) in immortalized mouse hypothalamic GT1-7 neuronal cells.

We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1-7 cells, it clearly enhances Pb2+-induced cytotoxicity. It is likely that Pb2+ must enter cells to exert most of its toxic effects. Pb2+ is known to substitute for Ca2+ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb2+ into GT1-7 neuronal cells with a special focus on the role of extracellular calcium (Ca2+), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb2+ (1 microM or 10 microM), i.e. without any external stimulus, clearly increased in nominally Ca2+-free buffer and was partially abolished by 13 mM Ca2+ when compared to uptake in the presence of a physiological concentration of extracellular Ca2+ (1.3 mM). Depolarization by 25 mM K+, or antagonists of VSCCs, verapamil (10 microM) or flunarizine (10 microM) had no clear effect on basal Pb2+ uptake. Glutamate (1 mM) increased Pb2+ uptake, but only when cells were treated with 1 microM Pb2+ in the presence of 1.3 mM Ca2+. Our data suggest that Pb2+ competes for the same cellular uptake pathways with Ca2+, although not via VSCCs. In addition, enhancement of Pb2+-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb2+.

The effects of wood dusts on the redox status and cell death in mouse macrophages (RAW 264.7) and human leukocytes in vitro.

Wood dusts are classified as carcinogenic to humans and also produce other toxic, allergic, and acute effects in woodworkers. However, little is known about causative agents in wood dusts and their mechanisms of action. The effects of different tree species and particle size for biological activity were studied. The differences in the production of reactive oxygen species (ROS) and cell death (necrotic and apoptotic) between mouse macrophage (RAW 264.7) cells and human polymorphonuclear leukocytes (PMNL) for pine, birch, and beech dust exposures were investigated in vitro. The pine and birch dust exposure (1-100 microg/ml) produced concentration-dependent ROS production in both the cells, which was one order of magnitude higher with pine dust. The ROS production was faster in human PNML than murine RAW cells. The higher concentrations (500 and/or 1000 microg/ml) decreased ROS formation. With pine and birch dust exposure, this was probably due to the necrotic cell death. The pine dust concentrations of 500 and 1000 microg/ml were cytotoxic to human PMNL. The beech dust exposure activated the ROS production and decreased the cell viability only at the highest concentrations, being least potent of the three dusts. A sign of the apoptotic cell death in the murine RAW cells was observed at the pine dust concentration of 100 microg/ml. The exposure to the birch and beech dusts with a smaller particle size (<5 microm) produced greater ROS production than exposure to the corresponding dust with a wide range of particle sizes. However, changing the particle size did not affect the cell viability. The results indicate that the type of wood dust (tree species and possibly particle size) has a significant impact on the function and viability of phagocytic cells.

EC-SOD gene therapy reduces paracetamol-induced liver damage in mice.

BACKGROUND: Paracetamol overdose causes acute liver damage which leads to severe centrilobular hepatic necrosis. The hepatotoxic effect is caused by reactive metabolites and oxidative stress. Since extracellular superoxide dismutase (EC-SOD) protects tissues against the harmful effects of superoxide anion, the hypothesis that systemic adenovirus-mediated EC-SOD gene transfer could reduce liver damage was tested. METHODS: Mice were given paracetamol (600 mg/kg) enterally 2 days after adenovirus-mediated gene transfer of EC-SOD (2 x 10(9) pfu). Five days after gene transfer, plasma and tissue samples were collected for clinical chemistry analyses and tissue pathology evaluation. RESULTS: EC-SOD was expressed in a dose-dependent manner with the highest enzyme activity occurring 3 days after the gene transfer. Clinical chemistry and tissue pathology analyses showed that adenoviral EC-SOD gene transfer significantly attenuated release of liver enzymes and inhibited necrosis and apoptosis caused by paracetamol overdose. CONCLUSION: The results indicate the involvement of superoxide anion in paracetamol-mediated liver damage and suggest a possible protective role for EC-SOD gene transfer in paracetamol-induced liver damage.

Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation.

To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation. Most of the UV energy was in the UVB range (280-320 nm). The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2. We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs). In addition, the effect of coexposure on cell cycle kinetics was investigated. Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation. The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation. Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations. These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth.

False-positive apoptosis signal in mouse kidney and liver detected with TUNEL assay.

Apoptosis is a physiological, programmed process for the elimination of cells from living organisms. Currently, one of the most frequently used methods to detect apoptosis is TUNEL assay. It has provided valuable information about apoptosis in various tissues. However, the sensitivity and the specificity of TUNEL technique have also been criticized. We detected an intense false-positive apoptotic signal in nude and Balb/c mice kidney and liver. In kidney the signal was confined to the proximal, distal and collecting tubular cells, and in liver to hepatocytes. Both tissues appeared normal in light microscopy, and no DNA ladder formation or increase in caspase-3 enzyme activity was detected. BrdU labelling and Ki-67 immunostaining did not reveal increased cell proliferation in these tissues. On the other hand, false-positive signal was not detected in testis, spleen, pancreas or renal cell carcinoma from the same animals. Also, no false-positive signal was seen in human liver or kidney samples. Although factors known to produce false-positive staining related to sample harvesting, preparation and staining protocols were eliminated, the cause of the false- positive apoptotic signal remains unknown. We conclude that caution must be exercised when examining apoptosis in mouse tissues with TUNEL assay.

Glutamate-stimulated ROS production in neuronal cultures: interactions with lead and the cholinergic system.

Oxidative stress may be an important factor in several pathological brain conditions. A contributing factor in many such conditions is excessive glutamate release, and subsequent glutamatergic neuronal stimulation, that causes increased production of reactive oxygen species (ROS), oxidative stress, excitotoxicity and neuronal damage. Glutamate release is also associated with illnesses such as Alzheimer's disease, stroke, and brain injury. Glutamate may interact with an environmental toxin, lead, and this interaction may result in neuronal damage. Glutamate-induced ROS production is greatly amplified by lead in cultured neuronal cells. Alterations in protein kinase C (PKC) activity seem to be important both for glutamate-induced ROS production, and for the amplification of glutamate-induced ROS production by lead. It is possible that the neurotoxic effects of lead are amplified through glutamate-induced neuronal excitation. Cholinergic stimulation can also trigger ROS production in neuronal cells. PKC seems to play a key-role also in cholinergic-induced ROS production superoxide anion being the primary reactive oxygen species. There seems to be a close relationship between the responses of cholinergic muscarinic and glutamatergic receptors because glutamate receptor antagonists inhibit cholinergic-induced activation of human neuroblastoma cells. Glutamatergic neuronal stimulation may be a common final pathway in several brain conditions in which oxidative stress and ensuing excitotoxicity plays a role.

Modification of glutamate-induced oxidative stress by lead: the role of extracellular calcium.

The role of extracellular calcium in glutamate-induced oxidative stress, and the role of glutamatergic neuronal stimulation and oxidative stress in lead neurotoxicity were explored in mouse hypothalamic GT1-7 cells. Glutamate increased the production of reactive oxygen species (ROS) whether or not extracellular calcium was present. Glutamate-induced ROS production was amplified by lead acetate (PbAc), but only in the absence of extracellular calcium. However, PbAc on its own did not increase the production of ROS. A PKC inhibitor (Ro 31-8220) and superoxide dismutase (SOD) abolished the amplification of glutamate-induced production of ROS by PbAc, but did not inhibit ROS production induced by glutamate alone. Both glutamate and PbAc decreased the levels of intracellular glutathione (GSH), and amplified each other's effect on GSH depletion. Glutamate did not decrease cell viability, whereas the cytotoxicity of PbAc was amplified by glutamate. Extracellular calcium, a PKC inhibitor, or SOD did not modify the effects of glutamate, PbAc or their combination on the levels of GSH or cell viability. These data indicate that in GT1-7 cells extracellular calcium is not essential for glutamate-induced ROS production, which is amplified by PbAc, but only without extracellular calcium. The joint cytotoxicity of glutamate and PbAc is mainly induced by PbAc, preferentially through mechanisms other than ROS production.

Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead.

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 (ROS), 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-kappaB 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.

Cholinergic-induced production of reactive oxygen species in human neuroblastoma cells.

Stimulation of human SH-SY5Y neuroblastoma cells by a muscarinic receptor agonist, carbachol (CCh; 1 mM), elevated levels of free intracellular calcium and subsequently increased the production of reactive oxygen species (ROS). Quinuclidinylbenzilate (QNB) binding increased at 1 h after CCh, but returned back to the control level at 3 h. Production of ROS increased, however, during the 3 h time period. CCh also increased the translocation of protein kinase C (PKC) to the membrane. ROS production was completely blocked by atropine and a PKC inhibitor, Ro 31-8220. These results show that increased ROS production was a result of muscarinic receptor stimulation, and that PKC had an active role in this cellular stimulation. ROS production upon cellular stimulation by CCh was completely inhibited also by superoxide dismutase, and partially by catalase, indicating that the formation of superoxide anion dominated in cholinergic-induced generation of ROS in human neuroblastoma cells. These results also show that muscarinic stimulation causes sustained ROS production in human neuroblastoma cells. The slow increase in ROS production by CCh suggest a stepwise cascade of events leading to oxidative stress with a triggering role of cholinergic muscarinic receptors in this process.

Amplification of glutamate-induced oxidative stress.

Glutamate is a ubiquitous neurotransmitter which causes excess neuronal excitotoxicity and neurodegenerative insults such as stroke, trauma and seizures. A salient feature of the activation of glutamate receptors is the induction of oxidative burst. Moreover, glutamate stimulates Ca2+ influx and translocates protein kinase C (PKC). PKC mediates cellular processes mediated via phosphorylations which may be essential for oxidative burst in many cells. Subsequent oxidative stress may be a causal factor of neurodegenerative diseases. Increased glutamate release and oxidative burst may thus both be essential in the cascade of events leading to neuronal damage. Glutamate may also mediate neurotoxic effects of environmental toxic agents such as lead which amplify glutamate excitotoxicity. In these interactions, excessive activation of glutamate receptors and oxidative burst may converge into a common pathway leading to cell death through a cascade involving PKC or other protein important in oxidative burst in neurons.

Lead amplifies glutamate-induced oxidative stress.

Lead markedly amplified L-glutamate-induced oxidative stress, that is, increased L-glutamate-induced production of reactive oxygen species, decreased cellular glutathione, and induced cytotoxicity in human neuroblastoma cells. It was notable that oxidative burst induced by L-glutamate alone was observed only when neuronal glutathione was depleted. A role of protein kinase C (PKC) in glutamate-induced production of reactive oxygen species is likely because it was blocked by a PKC inhibitor. We suggest here that the mechanism whereby lead causes its neurotoxicity may be through the amplification of glutamate-induced oxidative stress, possibly through PKC activation.

Mineral fiber-induced leukocyte activation: the role of intra- and extracellular calcium.

The role of intra- and extracellular calcium in the activation of human polymorphonuclear leukocytes (PMNL) to produce reactive oxygen metabolites (ROM) were studied by using soluble, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), or particulate stimuli, quartz or chrysotile. A calcium channel inhibitor, verapamil, attenuated only quartz-induced elevation of free intracellular calcium ([Ca2+]i) and ROM production. Likewise, ethyleneglycol-bis (aminoethyl ether) tetraacetic acid (EGTA) attenuated quartz-, chrysotile- and fMLP-induced elevation of [Ca2+]i and ROM production. It also inhibited PMA-induced ROM production. A calcium ionophore, A23187 amplified ROM production by all of these stimuli. These results suggest that both intra- and extra-cellular calcium are required for the full activation of respiratory burst by soluble and particulate stimuli in human PMNL.

The effects of N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA) on the changes in free intracellular calcium and the production of reactive oxygen metabolites in human leukocytes.

Human leukocytes were exposed to N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA), a postulated impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981. Changes induced by 5-VTPA alone and together with a chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), a tumor promoter, phorbol myristate acetate (PMA), or a synthetic diacylglycerol, dioctanoyl-s,n-glycerol (DiC8) in free intracellular calcium levels ([Ca2+]i) and in the induction of oxidative burst were measured. 5-VTPA elevated dose-dependently [Ca2+]i and induced the production of reactive oxygen metabolites in leukocytes. 5-VTPA also amplified FMLP-induced increase in [Ca2+]i, but was without an effect on FMLP-induced oxidative burst. On the contrary, 5-VTPA amplified dose-dependently PMA- and DiC8-induced respiratory burst. The present results indicate that 5-VTPA may interfere with transmembrane signalling in human leukocytes. 5-VTPA may elevate [Ca2+]i by acting directly on the membrane, or by acting through Ca(2+)-mobilizing receptors. Moreover, 5-VTPA also clearly amplified responses produced through protein kinase C stimulation. Thus, 5-VTPA may act on human leukocytes by affecting Ca(2+)-metabolism and the activity of protein kinase C.

Phosphoinositide second messengers in cholinergic excitotoxicity.

Acetylcholine (ACh) is a powerful excitotoxic neurotransmitter in the brain. By stimulating Ca(2+)-mobilizing receptors, ACh, through G-protein(s), stimulates phospholipase C and causes the hydrolysis of a membrane phospholipid, phosphatidylinositol-4,5-bisphosphate to two second messengers, inositol-1,4,5-trisphosphate (ins-(1,4,5)-P3), and diacylglycerol. Ins-(1,4,5)-P3 is important in cholinergic neuronal stimulation, and injury. Cholinergic agonists cause tonic-clonic convulsions which may be either transient or persistent. Even short-term cholinergic convulsions may be associated with neuronal injury, especially in the basal forebrain and the hippocampus. Cholinergic-induced convulsions also elevate levels of brain Ca2+ which precede neuronal injury. Female sex and senescence increase the sensitivity of rats to cholinergic excitotoxicity. Even if cholinergic-induced brain phosphoinositide signalling is likely to trigger cholinergic excitotoxicity, several other processes may be involved in the ensuing neuronal injury. Once initiated, cholinergic convulsions cannot be stopped with cholinergic antagonists such as atropine even though they are effective when given prior to a cholinergic agonist. However, glutaminergic antagonists, and GABAergic agonists, are effective in the attenuation of ongoing cholinergic status epilepticus. Cholinergic brain stimulation may be, in fact, under a partial control of brain GABAergic tonus, but also cause the release of glutamate. Glutamate stimulates inositol lipid signalling in several neuronal cells and, therefore, underlines the significance of inositol lipid signalling in cholinergic-induced excitotoxicity. Moreover, the anatomical distribution of cholinergic brain damage correlates well with that of glutaminergic neurons. Furthermore, glutamate increases neuronal oxidative stress, i.e. it increases the levels of free intracellular calcium, the production of reactive oxygen species, and causes the depletion of neuronal glutathione. The role of excitatory amino acids as common mediators of cholinergic excitotoxicity may offer new insights into the neurotoxic consequences of cholinergic neuronal stimulation.