μM MeHg Research Articles

Methylmercury Stimulates Arachidonic Acid Release and Cytosolic Phospholipase A 2 Expression in Primary Neuronal Cultures

Cytosolic phospholipase A 2 (cPLA 2) plays an important role in the stimulus-dependent hydrolysis of sn-2 ester bond from membrane phospholipids, releasing arachidonic acid (AA), which along with its metabolites is involved in a number of regulatory functions. The present study examined the effect of methylmercury (MeHg; 0, 2.5, 5.0 μM) on cPLA 2 activation in primary hippocampal neurons by assessing the release of 3 H -AA. A significant increase in AA release was observed in cultures treated with 5 μM MeHg (10, 30, 60 and 120 min). This effect was due to neuronal cPLA 2 activation, since it was completely abolished by arachidonyl trifluoromethyl ketone (AACOCF 3), a specific inhibitor of cPLA 2. Additional studies confirmed, by means of western blot analysis, that MeHg (5.0 and 10 μM; 16 h) potently increases neuronal cPLA 2 protein expression. These results suggest that cPLA 2-stimulated hydrolysis and release of AA are potential mediators of MeHg-induced neurotoxicity.

Effects of successive intrastriatal methylmercury administrations on dopaminergic system

The present study was carried out in order to determine the effects of intrastriatal administration of different doses (40 μM, 400 μM, and 4 mM) of methylmercury (MeHg) on dopaminergic system of rat striatum. Experiments were performed in conscious and freely moving rats using brain microdialysis coupled with liquid chromatography. Intrastriatal administration of MeHg produced significant increases in dopamine (DA) striatal levels (907±7%, 1870±319%, and 7971±534% for the doses of 40, 400 μM, and 4 mM, with respect to basal). The increase in DA levels was associated with significant decreases in extracellular levels of its main metabolites dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) (65.0±3.0% and 52.2±1.3%, respectively) using the dose of 4 mM MeHg, whereas nonsignificant changes in metabolite levels were observed with the doses of 40 and 400 μM MeHg. A second infusion of 4 mM MeHg 24 h after first infusion also produced a rise of DA levels, but this increase was very small as compared with that produced by first infusion (7971±534% versus 985±186%). This second infusion of 4 mM MeHg also decreased DOPAC and HVA levels, but this decrease was not significant as compared with that observed after first infusion (65.0±3.0% and 52.2±1.3% versus 62.4±5.2% and 63.4±7.4%, respectively). We discuss these effects based on a stimulated DA release and/or a decreased DA intraneuronal degradation.

Methylmercury enhances arachidonic acid release and cytosolic phospholipase A 2 expression in primary cultures of neonatal astrocytes

Cytosolic phospholipase A 2 (cPLA 2) stimulates the hydrolysis of sn-2 ester bond in membrane phospholipids releasing arachidonic acid (AA) and lysophospholipids. The present study examined the effect of methylmercury (MeHg) on cPLA 2 activation and AA release in primary cultures of neonatal rat cerebral astrocytes. Astrocytes were preloaded overnight at 37 °C with 3H-AA to metabolically label phospholipids. The effect of MeHg on the activation of cPLA 2 was measured by the release of 3H-AA from astrocytes over 120 min. MeHg (5 μM) caused a significant increase in AA release at 10, 30, 60, and 120 min, whereas 2.5 μM MeHg significantly increased AA release only at 120 min. MeHg-induced increase in 3H-AA release was due to cPLA 2 activation, since arachidonyl trifluoromethyl ketone (AACOCF 3), a selective inhibitor of cPLA 2, completely abolished MeHg’s effect. Consistent with these observations, MeHg (5.0 and 10.0 μM) increased cPLA 2 mRNA (6 h) and cPLA 2 protein expression (5.0 and 10.0 μM; 24 h). The time-course of these effects suggests an immediate direct or indirect effect of MeHg on cPLA 2 activation and 3H-AA release as well as a long-term effect involving the induction of cPLA 2. Thin layer chromatographic analysis of 3H-AA-labeled phospholipids showed that MeHg-stimulated astrocyte 3H-AA release was not due to increased incorporation of 3H-AA into the putative substrates of cPLA 2. These results invoke cPLA 2 as a putative target for MeHg toxicity, and support the notion that cPLA 2-stimulated hydrolysis and release of AA play a critical role in MeHg-induced neurotoxicity.

Methylmercury increases glutamate extracellular levels in frontal cortex of awake rats

A current hypothesis about methylmercury (MeHg) neurotoxicity proposes that neuronal damage is due to excitotoxicity following glutamate uptake alterations in the astrocyte. By sampling from a microdialysis probe implanted in the frontal cortex of adult Wistar rats, we measured the effects of acute exposure to either 10 or 100 μM MeHg through the microdialysis probe, on glutamate extracellular levels in 15 awake animals. After baseline measurements, the perfusion of MeHg during 90 min induced immediate and significant elevations in extracellular glutamate at 10 μM (9.8-fold, P<.001) and at 100 μM (2.4-fold, P=.001). This in vivo demonstration of increments of extracellular glutamate supports the hypothesis that dysfunction of glutamate neurotransmission plays a key role in MeHg-induced neural damage.

Methylmercury inhibits nitric oxide production mediated by Ca 2+ overload and protein kinase A activation

The importance of cytosolic free calcium level intracellular Ca 2+, [Ca 2+]i, in neutrophil activation prompted us to investigate changes in [Ca 2+]i of neutrophils caused by methylmercury (MeHg), which has been shown to have immunomodulatory properties. We have shown in this paper that MeHg increased [Ca 2+]i in the mouse peritoneal neutrophil. The L-type calcium channel blocker verapamil can decrease the elevated [Ca 2+]i caused by 10 μM MeHg, suggesting that Ca 2+-influx through L-type Ca 2+ channel mediates the effect of MeHg. Moreover, MeHg potently decreased nitric oxide (NO) production but also the protein and mRNA level of NO synthase induced by lipopolysaccharide. Both verapamil (1 μM) and H-89 (10 μM) can antagonize the inhibitory effect of MeHg (10 μM) on NO production. These findings lead us to conclude that MeHg inhibits NO production mediated at least in part by Ca 2+-activated adenylate cyclase–cAMP-protein kinase A pathway.

Protection of methylmercury effects on the in vivo dopamine release by NMDA receptor antagonists and nitric oxide synthase inhibitors

The possible protective effects of NMDA receptor antagonists dizocilpine (MK-801) and d(−)-2-amino-5-phosphonopentanoic acid (AP5), and nitric oxide synthase (NOS) inhibitors l-nitro-arginine methyl ester ( l-NAME) and 7-nitro-indazol (7-NI) on the methylmercury (MeHg)-induced dopamine (DA) release from rat striatum were investigated using in vivo microdialysis. Intrastriatal infusion of 400 μM or 4 mM MeHg increased the extracellular DA levels to 1941±199 and 7971±534% with respect to basal levels. Infusion of 400 μM or 4 mM MeHg in 400 μM MK-801 pretreated animals, increased striatal DA levels to 677±126 and 2926±254%, with respect to basal levels, these increases being 65 and 63% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 μM or 4 mM MeHg in 400 μM AP5 pretreated animals, increased striatal DA levels to 950±234 and 2251±254% with respect to basal levels, these increases being 51 and 72% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 μM MeHg in 100 μM l-NAME or 7-NI pretreated animals, increased the extracellular DA levels to 1159±90 and 981±292%, with respect to basal levels, these increases being 40 and 50% smaller than those induced by MeHg in non-pretreated animals. In summary, MeHg acts, at last in part, through an overstimulation of NMDA receptors with possible NO production to induce DA release, and administration of NMDA receptor antagonists and NOS inhibitors protects against MeHg-induced DA release from rat striatum.

Mechanism of action of methylmercury on in vivo striatal dopamine release: Possible involvement of dopamine transporter

Methylmercury (MeHg) produces significant increases in the spontaneous output of dopamine (DA) from rat striatal tissue. The mechanism through MeHg produces such increase in the extracellular DA levels could be due to increased DA release or decreased DA uptake into DA terminals. One of the aims of this study was to investigate the role of DA transporter (DAT) in the MeHg-induced DA release. Coinfusion of 400 μM MeHg and nomifensine (50 μM) or amphetamine (50 μM) produced increases in the release of DA similar to those produced by nomifensine and amphetamine alone. In the same way, MeHg-induced DA release was not attenuated under Ca 2+-free conditions or after pretreatment with reserpine (10 mg/kg i.p.) or tetrodotoxin (TTX), suggesting that the DA release was independent of calcium and vesicular stores, as well as it was not affected by the blockade of voltage sensitive sodium channels. Thus, to investigate whether depolarization of dopaminergic terminal was able to affect MeHg-induced DA release, we infused 75 mM KCl through the dyalisis membrane. Our results clearly showed a decrease induced by MeHg in the KCl-evoked DA release. Taken together, these results suggest that MeHg induces release of DA via transporter-dependent, calcium- and vesicular-independent mechanism and it decreases the KCl-evoked DA release.

Acute Exposure to Methylmercury Opens the Mitochondrial Permeability Transition Pore in Rat Cerebellar Granule Cells

Cerebellar granule cells are preferentially targeted during methylmercury (MeHg) poisoning. Following acute MeHg exposure, granule cells in culture undergo an increase in intracellular Ca2+ concentration ([Ca2+]i) that apparently contributes to cell death. This effect consists of several temporally and kinetically distinct phases. The initial elevation arises from release of Ca2+i stores; the second phase results from entry of Ca2+e. In these experiments, we tested the hypothesis that release of mitochondrial Ca2+ through the mitochondrial permeability transition pore (MTP) contributes to the initial release of Ca2+i. Neonatal rat cerebellar granule cells in culture and single cell microfluorimetry were used to examine MeHg-induced changes in [Ca2+]i and mitochondrial membrane potential (Ψm). Pretreatment with the MTP inhibitor cyclosporin A (CsA, 5 μM) delayed the initial phase of increased [Ca2+]i induced by 0.2 and 0.5 μM MeHg, but not 1.0 μM MeHg. CsA (5 μM) also delayed the irreversible loss of Ψm induced by 0.5 μM MeHg. Ca2+e was not required for either effect, because the effect of CsA on the first phase increase in [Ca2+]i and loss of Ψm was not altered in nominally Ca2+-free buffer. Increasing concentrations of MeHg (0.2–2.0 μM) caused increasing incidence of cell death at 24 h postexposure. Treatment with CsA provided protection against cytotoxicity at lower MeHg concentrations (0.2–0.5 μM), but not at higher MeHg concentrations (1.0–2.0 μM). Thus, the MTP appears to play a significant role in the cellular effects following acute exposure of cerebellar granule neurons to MeHg.

P21WAF1/CIP1 Inhibits Cell Cycle Progression but Not G2/M-Phase Transition Following Methylmercury Exposure

Methylmercury (MeHg) is an environmentally prevalent organometal that is particularly toxic to the developing central nervous system (CNS). Prenatal MeHg exposure is associated with reduced brain size and weight and a reduced number of neurons, which have been associated with impaired cell proliferation. We evaluate the role of p21, a cell cycle protein involved in the G1- and G2-phase checkpoint control, in the cell cycle inhibition induced by MeHg. Primary mouse embryonic fibroblasts (MEFs) of different p21 genotypes (wild-type, heterozygous, and null) were isolated at day 14 of gestation and treated at passages 4–6 with either 0, 2, 4, or 6 μM MeHg or 50 nM colchicine for 24 h. Changes in cell cycle distribution after continuous toxicant treatment were analyzed by DNA content-based flow cytometry using DAPI. MeHg induced an increase in the proportion of cells in G2/M at 2 and 4 μM MeHg (p ≤ 0.05) irrespective of p21 genotype. Effects of MeHg on cell cycle progression were subsequently evaluated using BrdU–Hoechst flow cytometric analysis. Inhibition of cell cycle progression was observed in all p21 genotypes after continuous exposure to MeHg for 24 and 48 h. p21 null (−/−) cells reached the second-round G1 at a higher fraction compared to the wild type (+/+) and heterozygous (+/−) cells (p ≤ 0.05). These data support previous observations that MeHg inhibits cell cycle progression through delayed G2/M transition. Whereas the G2/M accumulation induced by MeHg was independent of p21 status, a greater proportion of p21(−/−) cells were able to complete one round of cell division in the presence of MeHg compared to p21(+/−) or p21(+/+) cells. These data suggest a role for p21 in retarding cell cycle progression, but not mitotic inhibition, following exposure to MeHg.

Inhibition of Axonal Morphogenesis by Nonlethal, Submicromolar Concentrations of Methylmercury

We investigated the effects of sublethal concentrations of the neurotoxicant methylmercury (MeHg) on the developmental progression of cultured neurons to the stage of axonal morphogenesis. Chick (E8) forebrain neurons in vitro develop axons by a stereotyped developmental sequence nearly identical to that of widely used rat hippocampal neurons, but at much less cost and difficulty. In this chick forebrain system, 40% of neurons develop long axons after 2 days in culture, and 80% have axons after 4 days. A single, 2-h exposure to 0.5 or 0.25 μM MeHg reduced the number of neurons developing axons to approximately half that of controls without causing significant cell death for at least 2 days after treatment. Although MeHg caused an immediate depolymerization of neuronal microtubules, after 1 day of recovery the microtubule array of MeHg-treated neurons was indistinguishable by immunofluorescent assay from that of untreated cells at equivalent development stages. Thus, the inhibition of axonal development by submicromolar concentrations of MeHg did not appear to be the direct effect of microtubule disassembly. Chelation of Ca2+ during MeHg exposure appeared to exert a small immediate protective effect, as previously reported, but was itself toxic within 1 day after chelation. We suggest that this inhibition of axonal morphogenesis by acute, sublethal concentrations of MeHg may play a role in the developmental syndrome caused by environmental exposure to MeHg.

Toxicity of methylmercury conjugated with l-cysteine on rat thymocytes and human leukemia K562 cells in comparison with that of methylmercury chloride

In order to reveal the implication of use of methylmercury chloride (MeHgCl) in in vitro study, the effects of 10 μM MeHgCl on rat thymocytes and human leukemia K562 cells were compared with those of methylmercury conjugated with l-cysteine (10 μM MeHg–Cys) using a flow cytometer and fluorescent probes to monitor cellular physiological and pathological parameters. MeHgCl hyperpolarized membranes of thymocytes, followed by depolarization within a few minutes after the application, while MeHg–Cys persistently hyperpolarized them. MeHgCl increased intracellular concentration of Ca 2+, decreased cellular content of glutathione and increased generation of superoxide anion in the cells. The effects of MeHg–Cys were much less than those of MeHgCl. MeHgCl greatly increased both numbers of the cells undergoing apoptosis and dead cells in cell suspension containing thymocytes, while this was not the case for MeHg–Cys. MeHgCl reduced the cell viability of human leukemia K562 cells and completely inhibited the cell growth. The effects of MeHg–Cys on K562 cells were less than those of MeHgCl. It can be concluded that the effects of MeHgCl on rat thymocytes and K562 cells are different from those of MeHg–Cys. The results obtained from the in vitro studies using MeHgCl may be less implicit to elucidate the mechanism of MeHg intoxication in humans and experimental animals because MeHg are present in forms of MeHg–Cys and/or MeHg–S conjugate under the in vivo conditions.

Effects of Sequential Exposure to Multiple Concentrations of Methylmercury in the Rat Hippocampal Slice

Two experiments explored the effects of sequential exposure to multiple concentrations of methylmercury (MeHg) on rat hippocampal slice synaptic transmission and excitability in area CA1. When hippocampal slices were exposed to 0.1, 1, 10, and 100 μM MeHg chloride in successive 30-min exposures, MeHg produced an increase in excitability over baseline levels throughout the 1 μM exposure and the first 5 min of the 10 μM exposure, followed by profound suppression of excitability at the 100 μM level. When hippocampal slices were exposed to 10, 25, 50, 75, and 100 μM concentrations, MeHg produced an increase in excitability throughout most of the 10 and 25 μM exposures, followed by profound suppression of excitability at the 50 μM level of exposure. In both series of concentrations, MeHg suppressed local inhibitory systems prior to suppressing excitatory systems. In a third experiment, a single exposure of 50 μM MeHg suppressed both presynaptic and postsynaptic responses recorded in stratum radiatum with the same time course, suggesting that the observed suppressive effects of MeHg were not primarily synaptic.

Methylmercury Affects Multiple Subtypes of Calcium Channels in Rat Cerebellar Granule Cells

We tested the ability of methylmercury (MeHg) to block calcium channel current in cultures of neonatal cerebellar granule cells using whole-cell patch clamp techniques and Ba2+ as charge carrier. Low micromolar concentrations of MeHg (0.25–1 μM) reduced the amplitude of whole cell Ba2+ current in a concentration- and time-dependent fashion; however, this effect was not voltage-dependent and the current–voltage relationship was not altered. Increasing the stimulation frequency hastened the onset and increased the magnitude of block at both 0.25 and 0.5 μM MeHg but not at 1 μM. In the absence of stimulation, all concentrations of MeHg were able to decrease current amplitude. The ability of several Ca2+ channel antagonists (ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA, calcicludine, and nimodipine) to alter the MeHg-induced effect was tested in an effort to determine if MeHg targets a specific subtype of Ca2+ channel. Each of the antagonists tested was able to decrease a portion of whole cell Ba2+ current under control conditions. However, none were able to attenuate the MeHg-induced block of whole cell Ba2+ current, suggesting either that the mechanism of MeHg-induced block involves sites other than those influenced specifically by Ca2+ channel antagonists or that MeHg was able to “outcompete” these toxins for their binding sites. These results show that acute exposure to submicromolar concentrations of MeHg can block Ba2+ currents carried through multiple Ca2+ channel subtypes in primary cultures of cerebellar granule cells. However, it is unlikely that the presence of a specific Ca2+ channel subtype is able to render granule cells more susceptible to the neurotoxicologic actions of MeHg.

Developmental methylmercury administration alters cerebellar PSA–NCAM expression and Golgi sialyltransferase activity

Brain dysmorphogenesis and persistent psychomotor disturbances are hallmarks of developmental methylmercury (MeHg) exposure, but the molecular mechanisms underlying these effects are poorly understood. Targets of developmental MeHg exposure include neural cell adhesion molecules (NCAMs), sialoglycoconjugate molecules whose proper temporal and spatial expression is important at all stages of neurodevelopment and especially during synaptic structuring. To investigate the effects of MeHg on the temporal expression of NCAM during development, rat pups were dosed with 7.0 mg/kg MeHgCl (s.c.) on alternate days from postnatal days (PNDs) 3–13 and killed on PNDs 15, 30 and 60. Brain MeHg concentrations were determined in a subset of litters injected with CH 3 203 Hg . Expression of NCAM180 protein and of NCAM180 polysialylation was examined in whole cerebellum homogenates, cerebellar synaptosomes and isolated cerebellar growth cones by Western blotting and immunocytochemical staining. NCAM sialyltransferase activity was assayed in preparations of purified Golgi apparatus from the cerebelli of rats treated in vivo, or following in vitro incubation with 0, 1, 2.5, or 7.5 μM MeHg for 2 h. At PND15, no change in NCAM180 protein expression was observed in any cerebellar preparations, but decreased polysialylation of NCAM180 was observed in cerebellar whole homogenates, synaptosomes and isolated growth cones. At PND30, both NCAM180 protein expression and NCAM180 polysialylation were elevated in whole homogenate preparations but not in synaptosomes. NCAM180 expression in MeHg-treated rats was similar to controls at PND60, 47 days after the last methylmercury administration. In vivo studies of cerebellar Golgi sialyltransferase activity revealed significant reductions in PND15 MeHg-treated rats as compared to controls, but no changes in sialyltransferase activity in PND30 and PND60 animals. In vitro experiments revealed decreasing sensitivity of cerebellar sialyltransferases to MeHg as the developmental age of the rat increased. Toxic perturbation of the developmentally-regulated expression of polysialylated NCAM during brain formation may disturb the stereotypic formation of neuronal contacts and could contribute to the behavioral and morphological disturbances observed following MeHg poisoning.

W2/L3 - Developmental aspects in neurobehavioural toxicology: Contribution to risk assessment for environmental neurotoxicants

In these experiments we examined whether the elevations in intracellular Ca2+concentration ([Ca2+]i) induced by methylmercury (MeHg) (described in our previous study) might contribute to cerebellar granule cell mortality following exposure to MeHgin vitro.Cells were exposed to 0.5 μM MeHg for 45 min or 1 μM MeHg for 38 min, conditions previously shown to induce elevations in [Ca2+]iin these cells. Control cells were exposed to buffer alone for 60 min. Viability was assessed using the Live/Dead viability/cytotoxicity kit. At 30 min post-MeHg exposure, there was no immediate increase in cell mortality; however, by 3.5 h after the onset of MeHg exposure, cell viability decreased to 74 and 54% of control values for 0.5 and 1.0 μM MeHg, respectively. At 24.5 h after MeHg exposure, cell viability declined to approximately 27%. Losses in cell viability at 3.5 h were prevented by pretreating the granule cells for 65 min with the Ca2+chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA; 10 μM), then exposing the cells to MeHg in the continued presence of BAPTA; however, at 24.5 h, BAPTA no longer prevented MeHg-induced cell death. Exposure to the Ca2+channel blockers ω-conotoxin MVIIC (1 μM) or nifedipine (1 μM), previously shown to delay elevations in [Ca2+]iwith MeHg exposurein vitro,protected granule cells from MeHg-induced mortality at 3.5 h postexposure. These data suggest that at early time points, MeHg-induced increases in [Ca2+]imay contribute to granule cell mortality; however, the role of Ca2+at later time points is unclear.

Elevations of Intracellular Ca2+as a Probable Contributor to Decreased Viability in Cerebellar Granule Cells Following Acute Exposure to Methylmercury

In these experiments we examined whether the elevations in intracellular Ca2+concentration ([Ca2+]i) induced by methylmercury (MeHg) (described in our previous study) might contribute to cerebellar granule cell mortality following exposure to MeHgin vitro.Cells were exposed to 0.5 μM MeHg for 45 min or 1 μM MeHg for 38 min, conditions previously shown to induce elevations in [Ca2+]iin these cells. Control cells were exposed to buffer alone for 60 min. Viability was assessed using the Live/Dead viability/cytotoxicity kit. At 30 min post-MeHg exposure, there was no immediate increase in cell mortality; however, by 3.5 h after the onset of MeHg exposure, cell viability decreased to 74 and 54% of control values for 0.5 and 1.0 μM MeHg, respectively. At 24.5 h after MeHg exposure, cell viability declined to approximately 27%. Losses in cell viability at 3.5 h were prevented by pretreating the granule cells for 65 min with the Ca2+chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA; 10 μM), then exposing the cells to MeHg in the continued presence of BAPTA; however, at 24.5 h, BAPTA no longer prevented MeHg-induced cell death. Exposure to the Ca2+channel blockers ω-conotoxin MVIIC (1 μM) or nifedipine (1 μM), previously shown to delay elevations in [Ca2+]iwith MeHg exposurein vitro,protected granule cells from MeHg-induced mortality at 3.5 h postexposure. These data suggest that at early time points, MeHg-induced increases in [Ca2+]imay contribute to granule cell mortality; however, the role of Ca2+at later time points is unclear.

The effects of methylmercury on endogenous dopamine efflux from mouse striatal slices

The present study investigated the effects of in vitro methylmercury (MeHg) exposure on endogenous dopamine (DA) efflux from mouse striatal slices. MeHg produced a concentration-dependent increase in the spontaneous efflux of DA which was independent of the availability of Ca 2+ in the superfusion medium. The Ca 2+-dependent K +-evoked release of DA was significantly enhanced by 50 and 100 μM MeHg. This increase could not be solely accounted for by the MeHg-induced increase in spontaneous DA efflux. The K +-stimulated efflux of DA was enhanced by MeHg m both the presence and absence of Ca 2+in the superfusion medium, suggesting that under depolarizing conditions, DA efflux induced by MeHg has a Ca 2+-independent component. The alterations in DA efflux occurred at concentrations of MeHg previously found in the CNS of animals exhibiting symptoms of MeHg intoxication suggesting that alterations in DA neurotransmission in the striatum may contribute to the symptoms of MeHg toxicity.

Inhibition of regulatory volume decrease in swollen rat primary astrocyte cultures by methylmercury is due to increased amiloride-sensitive Na + uptake

Primary astrocyte cultures from neonatal rats were swollen by exposure to hypotonic buffer with and without 10 μM methylmercury (MeHg). We investigated the effects of MeHg on K + (using 86Rb), taurine, d-aspartate (a non metabolizable analogue of glutamate) and Na + fluxes during regulatory volume decrease (RVD), with an electrical impedance method for determination of cell volume, coupled with on-line measurements of efflux of radioactive ions and amino acids. Addition of 10 μM MeHg completely inhibited RVD in swollen astrocytes, increased the uptake of 22Na +, increased 86Rb release, and decreased 3H-taurine release. There was no effect on the rate of release of 3H- d-aspartate from swollen astrocytes. 0.5 mM amiloride completely inhibited MeHg-induced increased Na + influx during RVD, while 1 mM furosemide had no effect. When Na + in the hypotonic buffer was replaced with N-methyl- d-glucamine (NMDG), RVD in the presence of MeHg was indistinguishable from controls. These results indicate that MeHg increases cellular permeability to ions such as Na + and K +, and that an increase in Na + permeability via Na +/H + exchange, offsetting K + loss, is the primary mechanism in its inhibition of RVD in swollen astrocytes.

In vitro toxicity of methyl mercury: Effects on nerve growth factor (NGF)-responsive neurons and on NGF synthesis in fibroblasts

The effect of methyl mercury chloride (MeHgCl) on the chick sympathetic and sensory dorsal root ganglia was studied in a biological in vitro assay. These cultures were not affected by the addition of MeHg up to a concentration of 2 μM. However, after an addition of 4–5 μM MeHg the capability of the neurons to respond to added nerve growth factor (NGF) was completely inhibited. The effect of MeHg was also examined in a fibroblast cell line, mouse 3T3 cells. After the addition of mercury to the culture medium at concentrations as low as 0.1 μM, an elevated production of the NGF protein was observed. However, NGF mRNA measured in the individual fibroblast cells by in situ hybridization was found to be reduced to about 80% of the control in the low level at day 2 of exposure. These results suggest that the release of NGF is actively enhanced from the 3T3 cells by addition of low levels of mercury. The results thus show that MeHg at low to moderate concentrations has adverse effects on NGF responses in cultured neurons and moreover alter levels of NGF production in cells, suggestive of mechanisms for mercury toxicity in the developing nervous system.

Bcl-2 expression decreases methyl mercury-induced free-radical generation and cell killing in a neural cell line

Methyl mercury neurotoxicity is associated with a broad range of neuropathologic and biochemical disturbances which include induction of oxidative injury. Treatment of the hypothalamic neural cell line GT1-7 with 10 μM methyl mercury (MeHg) for 3 h resulted in increased formation of reactive oxygen species (ROS), and decreased levels of reduced glutathione (GSH), associated with 20% cell death. Cells transfected with an expression construct for the anti-apoptotic proto-oncogene, bcl-2, displayed attenuated ROS induction and negligible cell death. Twenty-four-h exposure to 5 μM MeHg killed 56% of control cells, but only 19% of bcl-2-transfected cells. By using diethyl maleate to deplete cells of GSH, we demonstrate that the differential sensitivity to MeHg was not due solely to intrinsically different GSH levels. The data suggest that MeHg-mediated cell killing correlates more closely with ROS generation than with GSH levels and that bcl-2 protects MeHg-treated cells by suppressing ROS generation.