Eduard Rodríguez-Farré

Neurotoxicity of Organomercurial Compounds

Mercury is a ubiquitous contaminant, and a range of chemical species is generated by human activity and natural environmental change. Elemental mercury and its inorganic and organic compounds have different toxic properties, but all them are considered hazardous in human exposure. In an equimolecular exposure basis organomercurials with a short aliphatic chain are the most harmful compounds and they may cause irreversible damage to the nervous system. Methylmercury (CH3Hg+) is the most studied following the neurotoxic outbreaks identified as Minamata disease and the Iraq poisoning. The first description of the CNS pathology dates from 1954. Since then, the clinical neurology, the neuropathology and the mechanisms of neurotoxicity of organomercurials have been widely studied. The high thiol reactivity of CH3Hg+, as well as all mercury compounds, has been suggested to be the basis of their harmful biological effects. However, there is clear selectivity of CH3Hg+ for specific cell types and brain structures, which is not yet fully understood. The main mechanisms involved are inhibition of protein synthesis, microtubule disruption, increase of intracellular Ca2+ with disturbance of neurotransmitter function, oxidative stress and triggering of excitotoxicity mechanisms. The effects are more damaging during CNS development, leading to alterations of the structure and functionality of the nervous system. The major source of CH3Hg+ exposure is the consumption of fish and, therefore, its intake is practically unavoidable. The present concern is on the study of the effects of low level exposure to CH3Hg+ on human neurodevelopment, with a view to establishing a safe daily intake. Recommendations are 0.4 μg/kg body weight/day by the WHO and US FDA and, recently, 0.1 μg/kg body weight/day by the US EPA. Unfortunately, these levels are easily attained with few meals of fish per week, depending on the source of the fish and its position in the food chain.

Antioxidant compounds and Ca2+ pathway blockers differentially protect against methylmercury and mercuric chloride neurotoxicity

The effects of the environmental contaminants methylmercury (MeHg) and inorganic mercury (HgCl2) on cell viability, intracellular calcium concentration ([Ca2+]i), and reactive oxygen species (ROS) generation were studied in rat cerebellar granule neuron cultures using fluorescent methods. MeHg exhibited an LC50 (2.47 μM) tenfold lower than that of HgCl2 (26.40 μM). To study the involvement of oxidative stress and Ca2+ homeostasis disruption in mercury‐induced cytotoxicity, we tested the neuroprotective effects of several agents that selectively interfere with these mechanisms. After a 24 hr exposure, the cytotoxic effect of both mercury compounds was reduced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+‐ATPase; the Ca2+ channel blocker flunarizine; and the Na+/Ca2+ exchanger blocker benzamil. All these compounds decreased the mercury‐mediated [Ca2+]i rise. These results indicate that Ca2+ influx through Ca2+ channels and the Na+/Ca2+ exchanger and Ca2+ mobilization from the endoplasmic reticulum are involved in mercury‐mediated cytotoxicity. The antioxidants probucol and propyl gallate reduced the HgCl2 toxicity. Probucol and vitamin E partially inhibited the MeHg toxicity after a 24 hr period, whereas propyl gallate completely prevented this effect. Probucol slightly reduced ROS generation in methylmercury‐exposed cultures and decreased mercury‐mediated rise of [Ca2+]i. Propyl gallate abolished ROS generation and partially inhibited the increase of [Ca2+]i induced by both mercury compounds. Propyl gallate also protected human cerebral cortical neuron cultures from the MeHg effect even after 72 hr of MeHg exposure, thus showing a long‐lasting effect. Our data suggest that disruption of redox equilibrium and Ca2+ homeostasis contribute equally to HgCl2‐mediated toxicity, whereas oxidative stress is the main cause of MeHg neurotoxicity. J. Neurosci. Res. 66:135–145, 2001.

Evaluation of fluorescent dyes for measuring intracellular glutathione content in primary cultures of human neurons and neuroblastoma SH-SY5Y

Reduced glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions. To study GSH content in human neuronal cell cultures, thiol‐sensitive fluorescent techniques requiring a small number of cells may be of great value, but their GSH specificity has not been established in these cells.

Astrocytes aged in vitro show a decreased neuroprotective capacity

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100β increased at 90 days when cells were positive for the senescence β‐galactosidase marker. In long‐term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn‐superoxide dismutase (SOD‐1) and haeme oxygenase‐1 (HO‐1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90‐DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H2O2 exposure. Enhanced glutamate uptake was probably because of up‐regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age‐related neurodegenerative processes.

Differential Estrogenic Effects of the Persistent Organochlorine Pesticides Dieldrin, Endosulfan, and Lindane in Primary Neuronal Cultures

The organochlorine chemicals endosulfan, dieldrin, and γ-hexachlorocyclohexane (lindane) are persistent pesticides to which people are exposed mainly via diet. Their antagonism of the γ-aminobutyric acid-A (GABA(A)) receptor makes them convulsants. They are also endocrine disruptors because of their interaction with the estrogen receptor (ER). Here, we study the effects of dieldrin, endosulfan, and lindane on ERs in primary cultures of cortical neurons (CN) and cerebellar granule cells (CGC). All the compounds tested inhibited the binding of [(3)H]-estradiol to the ER in both CN and CGC, with dieldrin in CGC showing the highest affinity. We also determined the effects of the pesticides on protein kinase B (Akt) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Dieldrin and endosulfan increased Akt phosphorylation in CN, which was inhibited by the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol. Instead, Akt and ERK1/2 phosphorylation induced by dieldrin in CGC was mediated by multiple activation of ERα, ERβ, and G protein-coupled receptor 30. Lindane did not activate these pathways, but it inhibited estradiol-mediated Akt and ERK1/2 activation. In CN, all the chemicals activated ERK1/2 through a mechanism involving GABA(A) and glutamate receptors. Long-term exposure to these pesticides reduced the levels of ERα, but not of ERβ. Moreover, extracts of CN treated with endosulfan, dieldrin, or lindane induced cell proliferation in MCF-7 human breast cancer-derived cells, whereas only extracts of CGC treated with dieldrin induced MCF-7 cell proliferation. Overall, the observed alterations on ER-mediated signaling and ER levels in neurons might contribute to the neurotoxicity of these organochlorine pesticides.

Induction of cyclooxygenase-2 in the rat brain after a mild episode of focal ischemia without tissue inflammation or neural cell damage

Cyclooxygenase-2, a key enzyme in prostanoid synthesis, is induced by inflammatory stimuli and it is associated with cell death after cerebral ischemia. Here we evaluated whether cyclooxygenase-2 was induced after a short (10-min) episode of focal ischemia, mild enough not to cause inflammation or cell death. One-hour ischemia leading to brain infarct was studied for comparative purposes. Induction of cyclooxygenase-2 mRNA and protein was detected after both 10-min and 1-h ischemia. However, signs of edema were only apparent after 1-h, but not 10-min ischemia, and only rats subjected to 1-h ischemia had developed brain infarct at 4 days. Therefore, cyclooxygenase-2, not linked with neural cell death or inflammation, is induced after focal ischemia.

Cell viability and proteomic analysis in cultured neurons exposed to methylmercury

Methylmercury is an environmental contaminant with special selectivity for cerebellar granule cells. The aim of this study was to determine the effect of long-term methylmercury exposure on cell viability and cellular proteome in cultured cerebellar granule cells. Primary cultures of mice cerebellar granule cells were treated with 0-300 nM methylmercury at 2 days in vitro (div) and afterwards the cells were harvested at 12 div. 100 nM methylmercury produced loss of cell viability, reduced intracellular glutamate content and increased lipid peroxidation. Glutamate transport was not modified by methylmercury treatment. Cell death induced by 300 nM methylmercury at 8 div was apoptotic without producing activation of caspase 3. Extracts of total protein were separated by 2D electrophoresis. Around 800 protein spots were visualized by silver staining in SDS-polyacrylamide gels. Gel images were digitized and protein patterns were analysed by image analysis. Several spots were identified through a combination of peptide mass fingerprinting and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The mitochondrial protein 3-ketoacid-coenzyme A transferase I was decreased up to 39% of controls at concentrations of methylmercury that did not produce cytotoxic effects, whereas the cytoplasmic proteins lactate dehydrogenase chain B and actin did not change. Human & Experimental Toxicology (2007) 26, 263-272

Early 72-kDa heat shock protein induction in microglial cells following focal ischemia in the rat brain

Focal cerebral ischemia in the adult rat produces induction of 72-kDa heat shock protein (HSP-72) in neurons, glia and endothelial cells. Double antigen immunocytochemistry was carried out to find out whether microglial cells express HSP-72 following 1-h middle cerebral artery (MCA) occlusion. A monoclonal antibody against the CR3 complement receptor (OX-42) specific for microglia was used followed by a monoclonal antibody against HSP-72. Co-localization of these antibodies was seen in cells of the ipsilateral corpus callosum and striatum at 3 h following 1-h MCA occlusion, and in the ipsilateral striatal penumbra, corpus callosum and cortex at 8 h. Results demonstrate that stellate microglial cells show an early response to 1-h MCA occlusion by expressing inducible HSP-72, thus suggesting that microglial cells are sensitive to the ischemic insult.

Lindane inhibition of [35S]TBPS binding to the GABAA receptor in rat brain

The inhibition of [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) binding to the GABAA receptor by the insecticide gamma-hexachlorocyclohexane, lindane, was studied in several brain regions and using different membrane preparation methods, both in vitro and after dosing the animals with the chemical. In the latter studies, the amount of lindane remaining in the membrane suspensions used for binding assays was determined. In vitro data showed values of IC50 from 150 to 1675 nM, varying in function of the membrane preparation method used. This may account for the discrepancies in IC50 values found in the literature. IC50 values within the range of 150-250 nM were determined using extensively washed membranes from several brain regions, so no evidence arose for brain regional differences in the affinity of lindane for the TBPS binding site. After different schedules of acute treatment with lindane, we found a manifest relationship between the extent of the observable inhibition of [35S]TBPS binding and the lindane amount remaining in the membrane suspensions used for binding assays. This relationship was in good agreement with the in vitro data, so no support for an in vivo acute regulation of the binding site was obtained.

Expression of c-fos and inducible hsp-70 mRNA following a transient episode of focal ischemia that had non-lethal effects on the rat brain.

Expression of c-fos and inducible hsp-70 mRNA was studied with in situ hybridization techniques at different times following an episode of middle cerebral artery (MCA) occlusion not resulting in any apparent lethal effect on the rat brain. hsp-70 and c-fos mRNA were found in the ipsilateral striatum and adjacent cortex. In the striatum, levels of hsp-70 mRNA increased from 1 to 2 and 4 h of reperfusion, whereas levels of c-fos mRNA decreased from 1 to 4 h of reperfusion. These results demonstrate that following non-lethal focal ischemia the brain areas within the MCA territory show high c-fos and hsp-70 mRNA expression response, illustrating the concomitant induction of these mRNAs in cells that survive the ischemic insult.