Cell viability and proteomic analysis in cultured neurons exposed to methylmercury

Abstract

<jats:p> 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 &amp; Experimental Toxicology (2007) 26, 263-272 </jats:p>

Influence of prenatal exposure to environmental pollutants on human cord blood levels of glutamate

“…Specific attention should be paid to its organometallic compound, methylmercury, because: i) it has recognized properties as a human neurodevelopmental toxicant (Grandjean and Landrigan, 2006); ii) poisoning incidents with methylmercury have demonstrated the potential of this pollutant as neurotoxicant and its serious health consequences (Castoldi et al, 2008;Ekino et al, 2007;Grandjean et al, 2010;Nakagawa et al, 2002); iii) millions of people are nowadays chronically exposed to this contaminant and there is epidemiological (Marsh et al, 1987; Pinheiro et al, 2007;Yokoo et al, 2003) and experimental (Castoldi et al, 2008;Farina et al, 2009;Vendrell et al, 2007Vendrell et al, , 2010 evidence of the toxic effects of chronic exposure to this metal especially for the nervous system; and iv) previous results from the INMA birth cohort of Valencia (Spain) showed elevated levels of this pollutant in umbilical cord blood, where 70% of newborns had mercury levels above US EPA recommended level, i.e. 5.8 μg/L of MeHg (Ramon et al, 2008).…”

In Vitro Models for Methylmercury Neurotoxicity: Effects on Glutamatergic Cerebellar Granule Neurons

“…MeHg-induced cell death was concentration-and time-dependent, with LC 50 values of 508 ± 199 , 345 ± 47 , and 243 ± 45 nM after exposure for 6, 11, and 16 days in vitro, respectively ( p = 0.05, one-way ANOVA) (Vendrell et al 2010 ) . Contrarily to the effects observed after short term and high concentration (>1 m M) exposure to MeHg, exposure to 100 nM MeHg for 10 days in vitro or to 300 nM MeHg for 5 days in vitro, which did not induce cell death, did not result in inhibition of the glutamate transport nor in changes of intracellular glutamate levels (Vendrell et al 2007(Vendrell et al , 2010Farina et al 2009 ) . All together these results suggest that impairment of neuronal glutamate homeostasis is a key event in the acute-high level toxicity of MeHg, whereas other mechanisms underlie the cellular toxic effects of long-term and low-level exposure to MeHg.…”

“…Prolonged exposure of CGCs to submicromolar concentrations of MeHg also resulted in oxidative stress inducing lipid peroxidation (Fig. 14.2a ) and increasing cell vulnerability against peroxides such as hydrogen peroxide and t-butyl hydroxy peroxide (Vendrell et al 2007 ;Farina et al 2009 ) . The analysis of endogenous regulators of red-ox homeostasis revealed that reduction of GPx activity was the fi rst event induced by prolonged exposure to low MeHg concentration before changes in glutathione (GSH), catalase and glutathione reductase (GR) could be observed (Farina et al 2009 ) .…”

“…Cell death induced by low MeHg concentrations is through an apoptotic pathway that is accompanied by neurite beading and microtubule fragmentation long before nuclear condensation occurred (Castoldi et al 2000 ;Vendrell et al 2007 ) . A proteomic study of CGCs exposed to low concentrations of MeHg ( £ 1 m M) for several days revealed both a decrease in the phosphorylation of the cytosolic protein cofi lin and the translocation of cofi lin from the cytosol to the mitochondria (Fig.…”

Effects of organic and inorganic mercury(II) on gene expression via DNA conformational changes

“…Although recent transcriptomic and proteomic analyses revealed a comprehensive set of cellular factors responding to Hg exposure (Hwang et al, 2011;Toyama et al, 2011;Vendrell et al, 2007), it is noteworthy that previous studies have reported the effects of Hg on mRNA and protein syntheses using radiolabeled substrates with brain tissue from regions including the cerebellum, a specific target of Hg (Sarafian and Verity, 1985;Syversen, 1982;Yoshino et al, 1966). These previous reports suggested the hypothesis, which has not been studied recently, that Hg might exert toxicity via modulation of gene expression itself, in addition to altering the profile of gene expression that occurs as a cellular response to Hg.…”