RESUMO
Methylmercury (MeHg) and Ethylmercury (EtHg) are toxic to the central nervous system. Human exposure to MeHg and EtHg results mainly from the consumption of contaminated fish and thimerosal-containing vaccines, respectively. The mechanisms underlying the toxicity of MeHg and EtHg are still elusive. Here, we compared the toxic effects of MeHg and EtHg in Saccharomyces cerevisiae (S. cerevisiae) emphasizing the involvement of oxidative stress and the identification of molecular targets from antioxidant pathways. Wild type and mutant strains with deleted genes for antioxidant defenses, namely: γ-glutamylcysteine synthetase, glutathione peroxidase, catalase, superoxide dismutase, mitochondrial peroxiredoxin, cytoplasmic thioredoxin, and redox transcription factor Yap1 were used to identify potential pathways and proteins from cell redox system targeted by MeHg and EtHg. MeHg and EtHg inhibited cell growth, decreased membrane integrity, and increased the granularity and production of reactive species (RS) in wild type yeast. The mutants were predominantly less tolerant of mercurial than wild type yeast. But, as the wild strain, mutants exhibited higher tolerance to MeHg than EtHg. Our results indicate the involvement of oxidative stress in the cytotoxicity of MeHg and EtHg and reinforce S. cerevisiae as a suitable model to explore the mechanisms of action of electrophilic toxicants.
Assuntos
Antioxidantes/farmacologia , Compostos de Etilmercúrio/farmacologia , Compostos de Metilmercúrio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismoRESUMO
Despite the vast distribution among tissues, the central nervous system (CNS) represents the main target of methylmercury (MeHg) toxicity. However, few studies have evaluated the effects of MeHg exposure on the CNS at equivalent doses to human environmental exposure. In our study, we evaluated the motor cortex, an important area of motor control, in adult rats chronically exposed to MeHg in a concentration equivalent to those found in fish-eating populations exposed to mercury (Hg). The parameters evaluated were total Hg accumulation, oxidative stress, tissue damage, and behavioral assessment in functional actions that involved this cortical region. Our results show in exposed animals a significantly greater level of Hg in the motor cortex; increase of nitrite levels and lipid peroxidation, associated with decreased antioxidant capacity against peroxyl radicals; reduction of neuronal and astrocyte density; and poor coordination and motor learning impairment. Our data showed that chronic exposure at low doses to MeHg is capable of promoting damages to the motor cortex of adult animals, with changes in oxidative biochemistry misbalance, neurodegeneration, and motor function impairment.
Assuntos
Compostos de Metilmercúrio/farmacologia , Córtex Motor/efeitos dos fármacos , Córtex Motor/fisiopatologia , Destreza Motora/efeitos dos fármacos , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Masculino , Compostos de Metilmercúrio/administração & dosagem , Córtex Motor/patologia , Ratos , Ratos WistarRESUMO
Methylmercury (MeHg) is an environmental neurotoxicant that inhibits neuronal migration. This process requires several cyclic steps involving the formation of membrane protrusions (lamellipodia and filopodia) and focal adhesion turnover. FAK and Src are critical proteins that regulate both processes. The FAK-Src complex promotes the activation of Rac1 and Cdc42, two GTPases involved in the remodeling of the actin cytoskeletal network. Here, we studied the effect of MeHg (1, 10, 100, 500 and 1000nM) on cell migration, the formation of cell protrusions, focal adhesion location and the activation of FAK, Src, Rac1 and Cdc42 using the SH-SY5Y neuroblastoma cell line stimulated with PDGF-BB (PDGF). The data show that MeHg (1-500nM) inhibited PDGF-stimulated cell migration. In PDGF-stimulated cells, MeHg (100-1000nM) decreased protrusions and increased the size of the p-FAKY397 clusters. MeHg also inhibited PDGF-induced FAK and Src activation and, at 100nM, MeHg inhibited the activation of Rac1 and Cdc42. Altogether, the findings show that low concentrations of MeHg inhibit SH-SY5Y cell migration by disrupting the activation and disassembly of FAK. This negatively affects the activation of Src, Rac1 and Cdc42, all of which are critical proteins for the regulation of cell movement. These effects could be related to the MeHg-mediated inhibition of PDGF-induced formation of lamellipodia and filopodia, focal adhesion disassembly and PDGF-induced movement.
Assuntos
Movimento Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Compostos de Metilmercúrio/farmacologia , Neuroblastoma/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
In the present work, we focused on mechanisms of methylmercury (MeHg) toxicity in primary astrocytes and neurons of rats. Cortical astrocytes and neurons exposed to 0.5-5 µM MeHg present a link among morphological alterations, glutathione (GSH) depletion, glutamate dyshomeostasis, and cell death. Disrupted neuronal cytoskeleton was assessed by decreased neurite length and neurite/neuron ratio. Astrocytes presented reorganization of actin and glial fibrillary acidic protein (GFAP) networks and reduced cytoplasmic area. Glutamate uptake and Na+K+ATPase activity in MeHg-treated astrocytes were preserved; however, downregulated EAAC1-mediated glutamate uptake was associated with impaired Na+K+ATPase activity in neurons. Oxidative imbalance was found in astrocytes and neurons through increased 2'7'-dichlorofluorescein (DCF) production and misregulated superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GPX) activities. Glutathione (GSH) levels were downregulated in both astrocytes and neurons. MeHg reduced neuronal viability and induced caspase 3-dependent apoptosis together with downregulated PI3K/Akt pathway. In astrocytes, necrotic death was associated with increased TNF-α and JNK/MAPK activities. Cytoskeletal remodeling and cell death were fully prevented in astrocytes and neurons by GSH, but not melatonin or Trolox supplementation. These findings support a role for depleted GSH in the cytotoxicity of MeHg leading to disruption of the cytoskeleton and cell death. Moreover, in neurons, glutamate antagonists also prevented cytoskeletal disruption and neuronal death. We propose that cytoskeleton is an end point in MeHg cytotoxicity. Oxidative imbalance and glutamate mechanisms mediate MeHg cytoskeletal disruption and apoptosis in neurons. Otherwise, redox imbalance and glutamate-independent mechanisms disrupted the cytoskeleton and induced necrosis in MeHg-exposed astrocyte.
Assuntos
Astrócitos/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Compostos de Metilmercúrio/farmacologia , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Feminino , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Organoseleno-compounds have been investigated for its beneficial effects against methylmercury toxicity. In this way, diphenyl diselenide (PhSe)2 was demonstrated to decrease Hg accumulation in mice, protect against MeHg-induced mitochondrial dysfunction, and protect against the overall toxicity of this metal. In the present study we aimed to investigate if co-treatment with (PhSe)2 and MeHg could decrease accumulation of Hg in liver slices of rats. Rat liver slices were co-treated with (PhSe)2 (0.5; 5 µM) and/or MeHg (25 µM) for 30 min at 37 °C and Se and Hg levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) in the slices homogenate, P1 fraction, mitochondria and incubation medium. Co-treatment with (PhSe)2 and MeHg did not significantly alter Se levels in any of the samples when compared with compounds alone. In addition, co-treatment with (PhSe)2 and MeHg did not decrease Hg levels in any of the samples tested, although, co-incubation significantly increased Hg levels in homogenate. We suggest here that (PhSe)2 could exert its previously demonstrated protective effects not by reducing MeHg levels, but forming a complex with MeHg avoiding it to bind to critical molecules in cell.
Assuntos
Derivados de Benzeno/farmacologia , Fígado/química , Fígado/efeitos dos fármacos , Mercúrio/análise , Compostos de Metilmercúrio/farmacologia , Compostos Organosselênicos/farmacologia , Selênio/análise , Animais , Derivados de Benzeno/administração & dosagem , Masculino , Espectrometria de Massas , Compostos de Metilmercúrio/administração & dosagem , Mitocôndrias Hepáticas/química , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Compostos Organosselênicos/administração & dosagem , Ratos , Ratos WistarRESUMO
Environmental contamination by methylmercury (MeHg) is an enormous public health problem in world regions such as Amazonia. MeHg toxic effects seem to be influenced by environmental and genetic factors. However, few studies have evaluated the genetic influences of MeHg toxicity in humans. Therefore, the aim of this study was to evaluate the genetic influence of Ala16Val manganese superoxide dismutase gene polymorphism (Ala16Val-MnSOD) on the cytotoxic effects of in vitro human leukocytes exposed to MeHg. Subjects were selected from 100 individuals aged 26.4 ± 7.3 years genotyped to Ala16Val-MnSOD polymorphism (AA = 6, VV = 6, and AV = 12) to perform in vitro testing using white blood cells (WBCs). Reactive oxygen species production was measured using 2',7'-dichlorofluorescein diacetate fluorimetric assay, and cell viability was measured using MTT assay on WBC samples from the same subjects that were both exposed and not exposed to MeHg (2.5 µM for 6 h). The results showed that AA- and VV-WBCs exposed to MeHg did not display increased reactive oxygen species levels compared to those in cells that were not exposed. However, AV-leukocytes exposed to MeHg displayed increased ROS levels. Cellular viability comparison among genotypes exposed to MeHg showed that the viability of AA-WBCs was lower than that of VV-WBC, with mean values of 3.46 ± 0.13 and 3.08 ± 0.77 (standard error), respectively (P = 0.033), whereas heterozygous cells (AV) displayed intermediate values. This difference was likely due to the higher basal H2O2 production of AA-WBCs compared to that of other genotypes. These results suggest that the Ala16Val-MnSOD polymorphism has toxicogenetic effects in human cells exposed to MeHg.
Assuntos
Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Compostos de Metilmercúrio/farmacologia , Polimorfismo Genético , Superóxido Dismutase/genética , Alelos , Substituição de Aminoácidos , Sobrevivência Celular/efeitos dos fármacos , Frequência do Gene , Genótipo , Humanos , Espécies Reativas de OxigênioRESUMO
Interest in organoselenide chemistry and biochemistry has increased in the past three decades, mainly due to their chemical and biological activities. Here, we investigated the protective effect of the organic selenium compound diphenyl diselenide (PhSe)2 (5 µmol/kg), in a mouse model of methylmercury (MeHg)-induced brain toxicity. Our group has previously demonstrated that the oral and repeated administration (21 days) of MeHg (40 mg/L) induced MeHg brain accumulation at toxic concentrations, and a pattern of severe cortical and cerebellar biochemical and behavioral. In order to assess neurotoxicity, the neurochemical parameters, namely, mitochondrial complexes I, II, II-III and IV, glutathione peroxidase (GPx) and glutathione reductase (GR) activities, the content of thiobarbituric acid-reactive substances (TBA-RS), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and brain-derived neurotrophic factor (BDNF), as well as, metal deposition were investigated in mouse cerebral cortex. Cortical neurotoxicity induced by brain MeHg deposition was characterized by the reduction of complexes I, II, and IV activities, reduction of GPx and increased GR activities, increased TBA-RS and 8-OHdG content, and reduced BDNF levels. The daily treatment with (PhSe)2 was able to counteract the inhibitory effect of MeHg on mitochondrial activities, the increased oxidative stress parameters, TBA-RS and 8-OHdG levels, and the reduction of BDNF content. The observed protective (PhSe)2 effect could be linked to its antioxidant properties and/or its ability to reduce MeHg deposition in brain, which was here histochemically corroborated. Altogether, these data indicate that (PhSe)2 could be consider as a neuroprotectant compound to be tested under neurotoxicity.
Assuntos
Antineoplásicos/farmacologia , Derivados de Benzeno/farmacologia , Córtex Cerebral/efeitos dos fármacos , Modelos Animais de Doenças , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Animais , Antineoplásicos/química , Derivados de Benzeno/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Masculino , Compostos de Metilmercúrio/química , Compostos de Metilmercúrio/farmacologia , Camundongos , Fármacos Neuroprotetores/química , Compostos Organosselênicos/química , Relação Estrutura-AtividadeRESUMO
Mercury is a toxic pollutant and spreads to several compartments in the environment. Previous in-vitro studies showed that roots of aquatic macrophytes are sites of methylmercury formation, performed mainly by sulfate-reducing bacteria (SRB). The objective of this study was to observe MMHg formation and distribution among filtered water (0.2µm), suspended and settled particles and macrophyte roots during seventeen days, in (203)Hg- spiked mesocosms with and without live Eichhornia crassipes whole plants and a SRB inhibitor. Root samples were also incubated in-vitro for comparison of MM(203)Hg formation under in-vitro and in-vivo conditions. To evaluate the effect of SRB inhibition by sodium molybdate on total heterotrophic activity, the latter was measured by (3)H-leucine uptake. Inhibition of Hg methylation by sodium molybdate decreased with time in mesocosms. MMHg averaged 10, 12.4 and 0.23 percent of total (203)Hg present in filtered water, suspended particles and roots respectively. In vitro MMHg formation in roots averaged 5.54 percent of total added (203)Hg, with a clearer SRB inhibition effect than in mesocosms. Though significant, MMHg formation in roots from in-vivo mesocosms was one order of magnitude lower than previously found in in-vitro incubations of roots alone.
Assuntos
Eichhornia/metabolismo , Mercúrio/metabolismo , Bactérias/metabolismo , Eichhornia/química , Eichhornia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Mercúrio/análise , Metilação/efeitos dos fármacos , Compostos de Metilmercúrio/farmacologia , Molibdênio/farmacologia , Raízes de Plantas/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismoRESUMO
The thioredoxin (Trx) system, involving redox active Trxs and thioredoxin reductases (TrxRs), sustain a number of important Trx-dependent pathways. These redox active proteins support several processes crucial for cell function, cell proliferation, antioxidant defense, and redox-regulated signaling cascades. Methylmercury (MeHg) is an important environmental toxicant that has a high affinity for thiol groups and can cause oxidative stress. The Trx system is the major system responsible for maintaining the redox state of cells and this function involves thiol reduction mediated by selenol groups in TrxRs. MeHg has a great affinity to thiols and selenols, thus the potential toxic effects of MeHg on TrxR inhibition were determined in the current study. A single administration of MeHg (1, 5, and 10 mg/Kg) caused a marked inhibition of kidney TrxR activity, while significant inhibition was observed in the liver after exposure to 5 and 10 mg/Kg of MeHg. TrxR activity was determined 24 h after MeHg. In the brain, MeHg did not inhibit TrxR activity. In vitro exposure to MeHg indicated that MeHg inhibits cerebral (IC(50), 0.158 µM), hepatic (IC(50), 0.071 µM), and renal TrxR activity (IC(50), 0.078 µM). The results presented herein demonstrated for the first time that renal and hepatic TrxRs can serve as an in vivo target for MeHg. This study suggests that MeHg can bind to selenocysteine residues present in the catalytic site of TrxR, in turn causing enzyme inhibition that can compromise the redox state of cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Compostos de Metilmercúrio/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Inibidores Enzimáticos/metabolismo , Técnicas In Vitro , Concentração Inibidora 50 , Rim/efeitos dos fármacos , Rim/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Compostos de Metilmercúrio/metabolismo , Camundongos , Selenocisteína/metabolismoRESUMO
Methylmercury (MeHg), a potent neurotoxicant, easily passes through the blood-brain barrier and accumulates in brain causing severe irreversible damage. However, the underlying neurotoxic mechanisms elicited by MeHg are still not completed defined. In this study, we aimed to investigate the in vitro toxic effects elicited by crescent concentrations (0-1500 microM) of MeHg on creatine kinase (CK) activity, thiol content (NPSH) and protein carbonyl content (PCC) in mouse brain preparations. In addition, CK activity, MTT reduction and DCFH-DA oxidation (reactive oxygen species (ROS) formation) were also measured in C6 glioma cell linage. CK activity was severely reduced by MeHg treatment in mouse brain preparations. This inhibitory effect was positively correlated to the MeHg-induced reduction of NPSH levels and increment in PCC. Moreover, the positive correlation between brain CK activity and NPSH levels was observed at either 15 or 60 min of MeHg pre-incubation. In addition, MeHg-treated C6 cells showed also a significant inhibition of CK activity at MeHg concentrations, as low as, 50 microM in parallel to reduced mitochondrial function and increased ROS production. Taking together, these data demonstrate that MeHg severely affects CK activity, an essential enzyme for brain energy buffering to maintain cellular energy homeostasis. This effect appears to be mediated by oxidation of thiol groups that might cause subsequent oxidative stress.