μM MeHg Research Articles

Comparison of alterations in amino acids content in cultured astrocytes or neurons exposed to methylmercury separately or in co-culture

Methylmercury (MeHg) is an environmental toxicant that induces enduring neuropsychological deficits in humans. Although the mechanisms associated with MeHg-induced neurotoxicity have not yet been fully elucidated, some lines of evidence point out to excitatory amino acids dyshomeostasis as an important outcome of MeHg exposure. The present study was designed to characterize the effects of MeHg on amino acid content in co-cultured astrocytes and neurons or in each cell type under solitary conditions. The results showed that glutamate concentrations significantly decreased in neurons, but not in astrocyte cultures exposed to 10 μM MeHg. The decrease in neurons was fully reversed when these cells were co-cultured with astrocytes. The content of other amino acids (aspartate, alanine, glycine and serine) decreased upon exposure to 10 μM MeHg in both neurons and astrocytes cultured in solitary conditions, although the effect was generally smaller in astrocytes than in neurons. However, the content of these amino acids in each of the cell types was indistinguishable from controls when co-cultures were treated with MeHg. Overall, the results indicate that astrocytes, which are more resistant to amino acid modulation by MeHg, can (i) mitigate the effects of MeHg that occur in neurons cultured in solitary conditions and (ii) become themselves more MeHg resistant in the presence of neurons. Delineating the mechanisms underlying the mutual neuroprotective effects of astrocytes and neurons in co-culture to MeHg-induced amino acid imbalance requires further investigation.

Continuous exposure to low concentrations of methylmercury impairs cerebellar granule cell migration in organotypic slice culture

Chronic, low-level perinatal exposure to methylmercury (MeHg) is associated with neurological and motor deficits that appear to result from cerebellar dysfunction. Neuropathological studies suggest that these deficits are due to impaired cerebellar granule cell (CGC) migration. Although neuronal migration in vivo and in vitro has been shown to be impaired during acute and/or high level exposure to MeHg, the cellular effects of chronic exposure to submicromolar and micromolar levels of MeHg during development are not clear. The majority of CGC migration in rats occurs between postnatal days 8 and 14 (P8 and 14); migration peaks on P10 and 11. Organotypic cultures of parasagittal slices of cerebellum from P8 rats were exposed to low levels of MeHg (0.2–5.0 μM) for 3 or 7 days, and CGC viability and migration were assessed. MeHg-induced cell death was time- and concentration-dependent. After 3 days of exposure CGC viability decreased in 3 μM MeHg and declined to 42.7% in 5 μM MeHg. Cultures treated with MeHg for 7 days showed decreased CGC viability in 1 μM MeHg, which declined to 62.8% in 3 μM MeHg. CGC migration was assessed by BrdU pulse-chase labeling. Migration into the internal granule cell layer (IGL) was impaired in cultures exposed to ≥1 μM MeHg for 3 days or ≥0.5 μM for 7 days. CGCs failed to initiate migration from the external germinal cell layer at the same level of exposure. For those cells which initiated migration, MeHg reduced the number that migrated into the IGL. This implied a slowing of migration once it had begun. These effects occurred with no overall change in cerebellar cortical structure, or loss of granule cell viability. Thus, chronic exposure to low micromolar concentrations of MeHg impairs development of the cerebellar cortex in a slice culture model.

Methyl mercury inhibits short-circuit current and Cl − influx across isolated epipodite of European lobster ( Homarus gammarus)

The effect of methyl mercuric chloride (MeHg) on short-circuit current ( I SC) was studied in the isolated perfused epipodite preparation from the branchial chamber of European lobster ( Homarus gammarus) acclimated to dilute seawater. When applied at the apical surface, 0.2, 1.0 and 3.0 μM MeHg depressed I SC by a 26%, 81% and 98%, respectively. The half-maximal inhibitory concentration (IC 50) of apically applied MeHg was 0.6 μM. Basolaterally added MeHg (3.0 μM) had no effect on I SC, whereas addition of the specific Na +,K +-ATPase inhibitor ouabain (1.5 mM) reduced I SC by ∼ 90%. Ouabain effects were reversible, and I SC fully recovered upon removal of ouabain. The MeHg-induced block of I SC was partially reversed by the reducing agent, 1,4-dithiothreitol, suggesting that the formation of S–Hg–S bridges is important in the inhibitory mechanism. A significant reduction of I SC and conductance occurred when low Na + and Cl − salines were substituted. Furthermore, in the low Na + saline, J Cl A → B fluxes were reduced by about 50%. In the highly conductive epipodite epithelium, coupling of Na + and Cl − fluxes was suggested. The effects of MeHg on I SC in the lobster epipodite are attributed to inhibition of an apical Cl − influx.

Characterization of demethylation of methylmercury in cultured astrocytes

Mercury (Hg) is a well-known neurotoxicant but its toxicity depends on the species present. A steady emergence of inorganic Hg in the brain following chronic and accidental exposure to methylmercury (MeHg) has suggested that MeHg can undergo demethylation. The objective of this study is to develop an in vitro model to study factors affecting Hg demethylation in the central nervous system. Astrocytes obtained from neonatal rat pups were cultured for 24 h with 1 μM MeHg in the presence of two pro-oxidants, buthionine sulphoximine (BSO) and rotenone. The BSO treatment produced a 21% increase in reactive oxygen species (ROS) content compared to the control (control vs. BSO; 100 ± 1.35 vs. 121 ± 1.52 relative fluorescence units (RFU) mg −1 protein, p < 0.001) but did not affect total Hg accumulation (control vs. BSO = 86.5 ± 4.14 ng mg −1 vs. 95.7 ± 9.26 ng mg −1). Rotenone increased ROS levels 107% (control vs. rotenone; 100% ± 1.35 vs. 207% ± 6.78 RFU mg −1 protein, p < 0.001) and significantly increased the accumulation of total Hg (control vs. rotenone = 86.5 ± 4.14 ng mg −1 vs. 124 ± 3.80 ng mg −1, p < 0.001). There was no detectable demethylation in the control or BSO treated group, however, the rotenone treatment significantly increased the demethylation (control vs. rotenone = −1.86 ± 5.57% vs. 16.3 ± 2.68%, p < 0.05). For the first time, we have demonstrated in an in vitro primary astrocyte culture model that MeHg can be converted to inorganic Hg and demethylation increases with oxidative stress. Our results provide a useful model to study demethylation of Hg in astrocytes and to explore potential ways to protect against Hg toxicity.

Involvement of the Lymphocytic Muscarinic Acetylcholine Receptor in Methylmercury-Induced c-Fos Expression and Apoptosis in Human Leukemic T Cells

Methylmercury (MeHg) is an environmental toxicant that is known to induce lymphocyte apoptosis; however, little is known about the molecular mechanism involved. Data showed that MOLT-3 cells were more sensitive to MeHg-induced cytotoxic effects than Jurkat clone E6-1 cells, suggesting that the lymphocytic muscarinic cholinergic system may be involved since the expressions of five subtypes (M1–M5) of muscarinic acetylcholine receptor (mAChR) in MOLT-3 cells are higher than in Jurkat cells. The role of mAChR-linked pathways in MeHg-induced apoptosis in human leukemic T cells was examined in this study. Treatment of the MOLT-3 cells with 1 μM MeHg produced induction of c-Fos expression, apoptotic cell death, and downregulation of mAChR. MeHg-induced c-Fos expression was significantly reduced by pretreatment with atropine (a nonselective mAChR antagonist), or 4-DAMP (a selective M1/M3 mAChR antagonist), whereas pirenzipine (a selective M1 mAChR antagonist) or himbazine (a selective M2/M4 mAChR antagonist) did not reduce this induction, suggesting that MeHg-induced c-Fos expression through the activation of the mAChR, at least M3 subtype, is involved. Pretreatment with 4-DAMP or SB 203580 (a specific p38 inhibitor) resulted in decreases in the level of phosphorylated p38, c-Fos expression, and apoptotic cell death induced by MeHg. Taken together, these data suggest that the mAChR-p38-dependent pathway participates in the increase of c-Fos expression, which is involved in MeHg-induced lymphocyte apoptosis. In addition, a noncytotoxic concentration of MeHg (0.1 μM) inhibited PHA/PMA-stimulated interleukin (IL)-2 production, and this inhibition was reversed by pretreatment with atropine or 4-DAMP. Overall, this study provides initial evidence that MeHg may alter the immune system by targeting the lymphocytic mAChR.

Methylmercury increases N-methyl- d-aspartate receptors on human SH-SY 5Y neuroblastoma cells leading to neurotoxicity

Methylmercury (MeHg) is a known neurotoxin, yet the mechanism for low dose chronic toxicity is still not clear. While N-methyl- d-aspartate receptors (NMDARs) were found to be induced after exposure to MeHg in a mink model, its role on neurotoxicity is not known. The aims of this study were to investigate the expression and the functional roles of NMDARs on the induction of cell death in the human SH-SY 5Y neuroblastoma cell line after exposure to MeHg. NMDARs were measured using a radiolabeled phencyclidine receptor ligand [ 3H] (MK801) and cell death was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and lactate dehydrogenase (LDH) release. We found a significant increase in NMDARs followed by increased caspase-3 activity after 4 h of exposure to MeHg (0.25–1 μM). Necrotic cell death was found after 4 and 24 h of exposure to MeHg (0.25–5 μM). The NMDAR antagonists dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d] cyclohepten-5,10-iminemaleate [(+)-MK801]) and Memantine (1-amino-3,5-dimethyl-adamantane) (10 μM) completely attenuated MeHg-mediated cell death by blocking NMDARs, thus demonstrating the importance of NMDARs in mercury neurotoxicity. Intracellular calcium chelator BAPTA-AM (1 μM) partially attenuated the neurotoxicity effect of 1 μM MeHg. These results suggest that MeHg toxicity can be mediated through the binding and increase of NMDARs.

Selenomethionine reduces visual deficits due to developmental methylmercury exposures

Developmental exposures to methylmercury (MeHg) have life-long behavioral effects. Many micronutrients, including selenium, are involved in cellular defenses against oxidative stress and may reduce the severity of MeHg-induced deficits. Zebrafish embryos (< 4 h post fertilization, hpf) were exposed to combinations of 0.0–0.30 μM MeHg and/or selenomethionine (SeMet) until 24 hpf then placed in clean medium. Fish were tested as adults under low light conditions (∼ 60 μW/m 2) for visual responses to a rotating black bar. Dose-dependent responses to MeHg exposure were evident (ANOVA, P < 0.001) as evidenced by reduced responsiveness, whereas SeMet did not induce deficits except at 0.3 μM. Ratios of SeMet:MeHg of 1:1 or 1:3 resulted in responses that were indistinguishable from controls (ANOVA, P < 0.001). No gross histopathologies were observed (H&E stain) in the retina or optic tectum at any MeHg concentration. Whole-cell, voltage-gated, depolarization-elicited outward K + currents of bipolar cells in intact retina of slices adult zebrafish were recorded and outward K + current amplitude was larger in bipolar cells of MeHg-treated fish. This was due to the intense response of cells expressing the delayed rectifying I K current; cells expressing the transient I A current displayed a slight trend for smaller amplitude among MeHg-treated fish. Developmental co-exposure to SeMet reduced but did not eliminate the increase in the MeHg-induced I K response, however, I A responses increased significantly over MeHg-treated fish to match control levels. Electrophysiological deficits parallel behavioral patterns in MeHg-treated fish, i.e., initial reactions to the rotating bar were followed by periods of inactivity and then a resumption of responses.

The effects of methylmercury on motor activity are sex- and age-dependent, and modulated by genetic deletion of adenosine receptors and caffeine administration

Adenosine and its receptors are, as part of the brain stress response, potential targets for neuroprotective drugs. We have investigated if the adenosine receptor system affects the developmental neurotoxicity caused by the fish pollutant methylmercury (MeHg). Behavioral outcomes of low dose perinatal MeHg exposure were studied in mice where the A 1 and A 2A adenosine receptors were either partially blocked by caffeine treatment or eliminated by genetic modification (A 1R and A 2AR knock-out mice). From gestational day 7 to day 7 of lactation dams were administered doses that mimic human intake via normal diet, i.e. 1 μM MeHg and/or 0.3 g/l caffeine in the drinking water. This exposure to MeHg resulted in a doubling of brain Hg levels in wild type females and males at postnatal day 21 (PND21). Open field analysis was performed at PND21 and 2 months of age. MeHg caused time-dependent behavioral alterations preferentially in male mice. A decreased response to amphetamine in 2-month-old males pointed to disturbances in dopaminergic functions. Maternal caffeine intake induced long-lasting changes in the offspring evidenced by an increased motor activity and a modified response to psychostimulants in adult age, irrespectively of sex. Similar alterations were observed in A 1R knock-out mice, suggesting that adenosine A 1 receptors are involved in the alterations triggered by caffeine exposure during development. Perinatal caffeine treatment and, to some extent, genetic elimination of adenosine A 1 receptors, attenuated the behavioral consequences of MeHg in males. Importantly, also deletion of the A 2A adenosine receptor reduced the vulnerability to MeHg, consistent with the neuroprotective effects of adenosine A 2A receptor inactivation observed in hypoxia and Parkinson's disease. Thus, the consequences of MeHg toxicity during gestation and lactation can be reduced by adenosine A 1 and A 2A receptor inactivation, either via their genetic deletion or by treatment with their antagonist caffeine.

Methylmercury causes glial IL-6 release

Methylmercury (MeHg) is an environmental toxin that causes severe neurological complications in humans and experimental animals. MeHg caused IL-6 release from the rat C6 glioma cells, the human U251HF glioma cells and the human retina pigment epithelial (ARPE-19) cells. These results plus those we reported earlier using mouse N9 microglia cells indicate that IL-6 induction may be a general property of MeHg among various glial cell types across species. MeHg caused a concentration-dependent increase of cellular oxidation with a maximal level reached by ∼10 μM MeHg, which was similar to that caused by 30 μM H 2O 2 or t-butyl hydroperoxide ( tBH). The ability of MeHg to induce IL-6 release was not affected by exogenously added H 2O 2 or t-butyl hydroperoxide. Furthermore, IL-6 release was not accompanied by other cytokine release. Given the reports by others that IL-6 could modulate neuronal survival, glia may affect MeHg neurotoxicity by their IL-6 release when exposed to this neurotoxin.

Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes

The neurotoxicity of high levels of methylmercury (MeHg) is well established both in humans and experimental animals. Astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS). Although the precise mechanisms of MeHg neurotoxicity are ill-defined, oxidative stress and altered mitochondrial and cell membrane permeability appear to be critical factors in its pathogenesis. The present study examined the effects of MeHg treatment on oxidative injury, mitochondrial inner membrane potential, glutamine uptake and expression of glutamine transporters in primary astrocyte cultures. MeHg caused a significant increase in F 2-isoprostanes (F 2-IsoPs), lipid peroxidation biomarkers of oxidative damage, in astrocyte cultures treated with 5 or 10 μM MeHg for 1 or 6 h. Consistent with this observation, MeHg induced a concentration-dependant reduction in the inner mitochondrial membrane potential (Δ Ψ m), as assessed by the potentiometric dye, tetramethylrhodamine ethyl ester (TMRE). Our results demonstrate that Δ Ψ m is a very sensitive endpoint for MeHg toxicity, since significant reductions were observed after only 1 h exposure to concentrations of MeHg as low as 1 μM. MeHg pretreatment (1, 5 and 10 μM) for 30 min also inhibited the net uptake of glutamine ( 3H-glutamine) measured at 1 min and 5 min. Expression of the mRNA coding the glutamine transporters, SNAT3/SN1 and ASCT2, was inhibited only at the highest (10 μM) MeHg concentration, suggesting that the reduction in glutamine uptake observed after 30 min treatment with lower concentrations of MeHg (1 and 5 μM) was not due to inhibition of transcription. Taken together, these studies demonstrate that MeHg exposure is associated with increased mitochondrial membrane permeability, alterations in glutamine/glutamate cycling, increased ROS formation and consequent oxidative injury. Ultimately, MeHg initiates multiple additive or synergistic disruptive mechanisms that lead to cellular dysfunction and cell death.

Diphenyl ditelluride- and methylmercury-induced hyperphosphorilation of the high molecular weight neurofilament subunit is prevented by organoselenium compounds in cerebral cortex of young rats

Organotellurides are important intermediates in organic synthesis and, consequently, the occupational exposure to them is a constant risk for laboratory workers. These compounds can elicit many neurotoxic events in the central nervous system (CNS) that are associated with several neurological symptoms. In contrast, organoselenium compounds are considered to exert neuroprotective actions on such effects. Neurofilaments (NF) are important cytoskeletal proteins and phosphorylation/dephosphorylation of NF is important to stabilize the cytoskeleton. In this work we investigated the potential protective ability of the selenium compounds ebselen and diphenyl diselenide (PhSe) 2 against the effect of diphenyl ditelluride (PhTe) 2 and methylmercury (MeHg) on the total (phosphorylated plus nonphosphorylated) and phosphorylated immunocontent of the high molecular weight neurofilament subunit (NF-H) from slices of cerebral cortex of 17-day-old rats. We observed that 1 μM MeHg induced hyperphosphorylation, increasing the total immunocontent of this subunit of the high-salt Triton insoluble NF-H. Otherwise, 15 μM (PhTe) 2 induced hyperphosphorylation of the high-salt Triton insoluble NF-H without altering the total immunocontent of this protein into the cytoskeletal fraction. Concerning the selenium compounds, 15 μM (PhSe) 2 and 5 μM ebselen did not induce alteration per se on the in vitro phosphorylation of NF-H. In addition, (PhSe) 2 and ebselen at these concentrations, presented a protective effect against the action of (PhTe) 2 and MeHg, on the immunoreactivity of NF-H. Considering that hyperphosphorylation of NF-H is associated with neuronal dysfunction it is probable that the effects of (PhTe) 2 and MeHg could be related to the remarkable neurotoxicity of these organocalcogenides. Furthermore the neuroprotective action of selenium compounds against (PhTe) 2 and MeHg effects could be a promising route to be exploited for a possible treatment of calcogenides poisoning.

Glutathione modulation influences methyl mercury induced neurotoxicity in primary cell cultures of neurons and astrocytes

Methyl mercury (MeHg) is highly neurotoxic and may lead to numerous neurodegenerative disorders. In this study, we investigated the role of glutathione (GSH) and reactive oxygen species (ROS) in MeHg-induced neurotoxicity, using primary cell cultures of cerebellar neurons and astrocytes. To evaluate the effect of GSH on MeHg-induced cytotoxicity, ROS and GSH were measured using the fluorescent indicators chloro methyl derivative of di-chloro di-hydro fluorescein diacetate (CMH 2DCFDA) and monochlorobimane (MCB). Cell-associated MeHg was measured with 14C-radiolabeled MeHg. Mitochondrial dehydrogenase activity was detected by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. MTT timeline study was also performed to evaluate the effects of both the concentration and duration of MeHg exposure. The intracellular GSH content was modified by pretreatment with N-acetyl cysteine (NAC) or di-ethyl maleate (DEM) for 12 h. Treatment with 5 μM MeHg for 30 min led to significant ( p < 0.05) increase in ROS and reduction ( p < 0.001) in GSH content. Depletion of intracellular GSH by DEM further increased the generation of MeHg-induced ROS in both cell cultures. Conversely, NAC supplementation increased intracellular GSH and provided protection against MeHg-induced oxidative stress in both cell cultures. MTT studies also confirmed the efficacy of NAC supplementation in attenuating MeHg-induced cytotoxicity. The cell-associated MeHg was significantly ( p < 0.02) increased after DEM treatment. In summary, depletion of GSH increases MeHg accumulation and enhances MeHg-induced oxidative stress, and conversely, supplementation with GSH precursor protects against MeHg exposure in vitro.

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37 degrees C for 7-8 days. Cultures were exposed to MeHg (10 microM, 100 microM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H3]-arginine to L-[H3]-citrulline. The incubation of cultured retina cells with 10 and 100 microM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 +/- 5.3 and 91.3 +/- 3.7%, respectively (data are reported as mean +/- SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Ebselen protects glutamate uptake inhibition caused by methyl mercury but does not by Hg 2+

Alterations of the neurotransmitter release systems in CNS have been reported in a variety of neuropathological processes associated with heavy metal toxicity. Neurotoxic effects of mercurials were investigated in vitro in cerebral cortex slices from young rats. The present study indicates that: (i) the environmental contaminants methylmercury (MeHg) and mercuric chloride (Hg 2+) (50 μM) inhibited the glutamate net uptake from the cerebral cortex of 17-day-old rats; (ii) ebselen (10 μM) reverted the MeHg-induced inhibition of glutamate net uptake but did not protect the inhibition caused by Hg 2+. At same time, we investigated another diorganochalcogenide, diphenyl diselenide (PhSe) 2 and it was observed that this compound did not revert the action of MeHg or Hg 2+; (iii) in addition, we observed that exposure of slices to 50 μM MeHg and Hg 2+ for 30 min followed by Trypan blue exclusion assay resulted in 58.5 and 67.5% of staining cells, respectively, indicating a decrease in cell viability. Ebselen protected slices from the deleterious effects of MeHg, but not of Hg 2+ on cell viability. Conversely, ebselen did not modify the reduction of MTT caused by MeHg and Hg 2+; (iv) the protective effect of ebselen on MeHg-induced inhibition of glutamate net uptake seems to be related to its ability in maintaining cell viability.

Methylmercury Alters Eph and Ephrin Expression During Neuronal Differentiation of P19 Embryonal Carcinoma Cells

Developmental exposure to methylmercury (MeHg) induces a spectrum of neurological impairment characterized by cognitive disturbance, sensory/motor deficit, and diffuse structural abnormalities of the brain. These alterations may arise from neural path-finding errors during brain development, resulting from disturbances in the function of morphoregulatory guidance molecules. The Eph family of tyrosine kinase receptors and their ligands, the ephrins, guide neuronal migration and neurite pathfinding mainly via repulsive intercellular interactions. The present study examined the effects of MeHg on mRNA and protein expression profiles of Ephs and ephrins in the P19 embryonal carcinoma (EC) cell line and its neuronal derivatives. Undifferentiated control P19 cells displayed low- to undetectable levels of mRNA for ephrins or Ephs, with the sole exception of EphA2 which was highly expressed. Upon differentiation into neurons, the ephrin expression increased progressively through day 10. Similarly, expression of the Ephs, including EphsA3, -A4, -A8, -B2, -B3, -B4, and -B6, increased significantly. In contrast, EphA2 expression decreased in day 2, 6 and 10 control neurons. Treatment with MeHg did not affect the expression of mRNA for ephrins or Ephs in undifferentiated P19 cells. However, treatment of differentiating neurons with MeHg for 24 h caused consistent increases in ligand mRNA expression, particularly ephrin-A5, -A6, -B1, and -B2. Similarly, MeHg induced variable increases in mRNA expression of receptors EphA2, -A3, -B3, and -B6. A trend toward a concentration–response relationship was observed for the alterations in Eph receptor mRNA expression although increases at the low and mid concentrations did not reach statistical significance. Immunoblots for ligand and receptor proteins mirrored the increases in the mRNA levels at the 0.5 and 1.5 μM MeHg concentrations but showed decreased protein levels compared to controls at the 3.0 μM concentration. Alterations in the Eph/ephrin family of repulsion molecules may represent an important mechanism in developmental MeHg neurotoxicity.

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5–5 μM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 μM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca 2+ and non-Ca 2+ divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 μM MeHg, 97.7% of Purkinje cells were viable. At 3 μM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 μM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca 2+ divalent cation released by MeHg in Purkinje neurons.

Selenoprotein W as molecular target of methylmercury in human neuronal cells is down-regulated by GSH depletion

Methylmercury (MeHg) is well known as a neurotoxic chemical. However, little is mentioned about its neurotoxic mechanism or molecular target in human neuronal cells in particular. We show in this study that exposure of human neuronal cell line, SH-SY5Y, to MeHg dose- and time-dependently impairs viability and mRNA expression of selenoprotein W (SeW) with a significant difference, unlike other selenoenzymes such as, SeP, GPX4, 5DI, and 5′DI. Using real-time RT PCR, the influence of selenium (Se) and glutathione (GSH) on SeW expression was also investigated. While Se depletion caused a weakly reduced SeW mRNA levels, additional Se caused an increase of SeW mRNA levels. Although 2 mM GSH had induced a weak shift on SeW level, the expression of SeW mRNA was down-regulated in SH-SY5Y cells treated with 25 μM BSO, an inhibitor of GSH synthesis. To understand the relationship between a decrease of SeW expression and intracellular GSH and ROS, we measured the concentration of intracellular GSH and ROS in cells treated to 1.4 μM MeHg using fluorescence based assays. A positive correlation was found between SeW mRNA level and intracellular GSH but no significant correlation was observed between intracellular ROS and SeW mRNA level or intracellular GSH contents. Therefore, we suggest that SeW is the novel molecular target of MeHg in human neuronal cells and down-regulation of this selenoenzyme by MeHg is dependent not on generation of ROS but on depletion of GSH.

Modulatory effect of glutathione status and antioxidants on methylmercury-induced free radical formation in primary cultures of cerebral astrocytes

Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-dependent neurotoxicity remains far from clear, overwhelming data give credence to a mediatory role for astrocytes, a major cell type that preferentially accumulates MeHg. To extend our recent findings of MeHg-induced increase in ROS formation (G. Shanker, J.L. Aschner, T. Syversen et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128 (2004) 48–57), the present studies were designed to assess the effect of modulating intracellular glutathione (GSH) content, on ROS generation, in the absence and presence of MeHg. Intracellular GSH was reduced by treatment with 100 μM buthionine- l-sulfoxane (BSO) for 24 h, and increased by treatment with 1 mM l-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 h. Additionally, the effects of the selective antioxidants, catalase (1000 U/ml for 1 h), an H 2O 2 scavenger, and n-propyl gallate (100 μM for 1 h), a superoxide radical ( ·O 2 −) and possibly hydroxyl radical ( ·OH) scavenger on MeHg-induced ROS formation were examined. After these treatments, astrocytes were exposed to ±10 μM MeHg for 30 min, following which the fluorescent probes, CM-H 2DCFA and CM-H 2XRos were added; 20 min later, laser scanning confocal microscopy (LSCM) images were obtained. Exposure of astrocytes for 24 h to 100 μM BSO, a GSH synthesis inhibitor, led to a significant increase in mitochondrial ROS (i.e., ·O 2 −, ·NO, and ONOO −) formation, as assessed with CM-H 2XRos mitotracker red dye. Similarly, BSO increased ROS formation in various intracellular organelles, as assessed with CM-H 2DCFDA. BSO in combination with MeHg increased fluorescence levels in astrocytes to levels above those noted with BSO or MeHg alone, but this effect was statistically indistinguishable from either of these groups (BSO or MeHg). Pretreatment of astrocytes for 24 h with 1 mM OTC abolished the MeHg-induced increase in ROS. Results similar to those obtained with OTC were observed with the free radical scavenger, n-propyl gallate (n-PG). The latter had no significant effects on astrocytic fluorescence when administered alone. This ·O 2 − and possibly ·OH radical scavenger significantly attenuated MeHg-induced ROS formation. Catalase, an H 2O 2 scavenger, was less effective in reducing MeHg-induced ROS formation. Taken together, these studies point to the important protective effect of adequate intracellular GSH content as well as antioxidants against MeHg-triggered oxidative stress in primary astrocyte cultures.

Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury

Oxidative stress has been implicated in neurotoxic damage associated with various metals, including methylmercury (MeHg). Although the mechanism(s) of MeHg-induced neurotoxicity remains unclear, evidence supports a mediatory role for astrocytes, a cell type that preferentially accumulates MeHg. Using scanning confocal microscopy (LSCM), the present study was undertaken to examine the role of astrocytes as the site of reactive oxygen species (ROS). Three redox-sensitive fluorescent probes were used for ROS analysis, (a) CM-H 2DCFDA (chloromethyl derivative of dichlorodihydrofluorescein diacetate), a probe for intracellular hydrogen peroxide (H 2O 2); (b) hydroethidine (HETH), a probe for superoxide anion (·O 2 −), and (c) CM-H 2XRos (chloromethyl derivative of dihydro X-rosamine), and a probe that is selective for mitochondrial reactive oxygen intermediates. Astrocytes were treated with 10 μM MeHg for 30 min, following which the various fluorescent probes were added; 20 min later LSCM images were collected. Astrocytes loaded with CM-H 2DCFDA and HE demonstrated a significant MeHg-induced increase in fluorescence intensity indicative of increased intracellular H 2O 2 and ·O 2 −, respectively. Similar results were obtained with the mitotracker dye, CM-H 2XRos. Additionally, exposure of astrocytes for 24 h to 100 μM buthionine- l-sulfoxane (BSO), a glutathione (GSH) synthesis inhibitor, caused a significant increase in ROS formation. Furthermore, BSO pretreatment significantly enhanced the MeHg-induced formation of ·O 2 −, indicating an important role for GSH in the maintenance of optimal cellular redox status. Time-course experiments performed in the simultaneous presence of CM-H 2XRos and CM-H 2DCFDA demonstrated that the MeHg-induced CM-H 2XRos fluorescence changes preceded those of CM-H 2DCFDA, suggesting that the mitochondria represent an early primary site for ROS formation. Taken together, these studies illustrate that MeHg induces the generation of astrocyte-derived ROS and support a role for astrocytic ROS in MeHg-associated neurotoxic damage.

Early acute necrosis and delayed apoptosis induced by methyl mercury in murine peritoneal neutrophils.

There is growing evidence that heavy metals in general, and mercurial compounds in particular, are immunotoxic. The purpose of this study was to explore the mechanism of MeHg in inducing cell death of mouse peritoneal neutrophils. In this paper we demonstrate that MeHg induces apoptosis and necrosis depending on MeHg concentration. In vitro exposure of mouse peritoneal neutrophils to MeHg resulted in a time- and concentration-dependent cell death. MeHg (15 microM) induced neutrophil necrosis in 13 min. The type of cell death was attributed to necrosis based on cells permeable to the fluorescent dye, propidium iodide and DNA appeared as a smear. With fura-2 microfluorimetric technique, we found that the entry of external Ca2+ into the cytosol played a crucial role in inducing cell necrosis by 15 microM MeHg. However, at lower concentrations, MeHg (10 microM)-induced apoptosis is confirmed by the observation of morphological features characterised by apoptotic bodies and fragmented DNA ladder. MeHg (10 microM) caused an immediate fall in pHi as revealed by the pH-sensitive fluorescent probe 2'7'-bis (carboxyethyl)-5(6)-carboxyfluorescein. We have found that MeHg induced cellular acidification prior to DNA fragmentation so as the other two apoptosis-inducing agents (ZnCl(2) and EGTA). Furthermore, acid-activated endonuclease was increased by MeHg in neutrophils, which we considered to play a possible role in chromatin digestion leading to apoptosis. Taken together, these findings indicate that MeHg induces necrosis at higher concentrations by a rapid increase of [Ca2+]i and apoptosis at lower concentrations by acid activation of endonuclease.