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1.
Food Chem Toxicol ; 59: 554-63, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23838314

ABSTRACT

Methylmercury (MeHg) has been recognized as a very toxic contaminant present in certain foodstuffs that adversely affects health and impairs the normal function of different organs. Experimental studies have shown that selenocompounds play an important role as cellular detoxificant and protective agents against the harmful effects of mercury. The present study examined the potential preventive activities of organic selenocompounds, focused on selenocystine (SeCys), against MeHg-induced toxicity in human HepG2 cells. Combined treatment of SeCys and MeHg protected HepG2 cells against MeHg-induced cell damage, showing this selenocompound a more relevant effect than those of selenium methylselenocysteine and selenium methionine. Co-treatment with SeCys exerted a protective effect against MeHg by restraining ROS generation and glutathione decrease, and through the modulation of antioxidant enzymes activities. In addition, SeCys delayed MeHg-induced apoptosis and prevented extracellular regulated kinases (ERKs) deactivation, as well as p38 and c-Jun N-terminal kinase (JNK) stimulations in comparison to MeHg-treated cells. ERK, JNK and p38 involvement on the protective effect of SeCys against MeHg-induced cell damage was confirmed by using selective inhibitors. All these results indicate that SeCys protects against MeHg-induced cell damage by modulating the redox status and key proteins related to cell stress and survival/proliferation pathways.


Subject(s)
Apoptosis/drug effects , Cystine/analogs & derivatives , Environmental Pollutants/antagonists & inhibitors , Hepatocytes/drug effects , Methylmercury Compounds/antagonists & inhibitors , Organoselenium Compounds/pharmacology , Oxidative Stress/drug effects , Protective Agents/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cystine/pharmacology , Environmental Pollutants/toxicity , Glutathione/antagonists & inhibitors , Glutathione/metabolism , Hep G2 Cells , Hepatocytes/metabolism , Humans , MAP Kinase Signaling System/drug effects , Methylmercury Compounds/toxicity , Osmolar Concentration , Oxidation-Reduction , Oxidoreductases/metabolism , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Selenocysteine/analogs & derivatives , Selenocysteine/pharmacology , Selenomethionine/pharmacology
2.
Analyst ; 137(22): 5302-11, 2012 Nov 21.
Article in English | MEDLINE | ID: mdl-23014190

ABSTRACT

Mercury toxicity and its implications in development are a major concern, due to the major threat to ecosystems and human health that this compound represents. Although some of the effects of methylmercury (MeHg) exposure have been extensively studied, the molecular mechanisms of interaction between this compound and developing organisms are still not completely understood. To provide further insights into these mechanisms, we carried out a quantitative proteomic study (iTRAQ) using zebrafish larvae exposed to 5 µg L(-1) and 25 µg L(-1) MeHg as a model. In this study, a multidimensional approach combining isoelectric focusing (IEF) and strong cation exchange (SCX) followed by reversed phase liquid chromatography prior to MALDI TOF/TOF analysis was employed, which resulted in a substantial increase in proteome coverage. Among the proteins identified, 71 were found de-regulated by more than 1.5-fold, and implicated in embryonic development, protein synthesis, calcium homeostasis and energy production. Furthermore, morphological and histological analysis of exposed larvae was carried out, reflecting changes such as smaller swim bladder, remaining yolk, bent body axis and accumulation of blood in the heart, among others.


Subject(s)
Chromatography, High Pressure Liquid , Embryonic Development/drug effects , Methylmercury Compounds/toxicity , Proteome/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Animals , Calcium/metabolism , Chromatography, Ion Exchange , Chromatography, Reverse-Phase , Energy Metabolism , Isoelectric Focusing , Larva/drug effects , Larva/growth & development , Methylmercury Compounds/chemistry , Peptides/analysis , Zebrafish/growth & development
3.
Anal Bioanal Chem ; 404(2): 315-24, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22535442

ABSTRACT

Understanding the molecular mechanisms underlying MeHg toxicity and the way in which this molecule interacts with living organisms is a critical point since MeHg represents a well-known risk to ecosystems and human health. We used a quantitative proteomic approach based on stable isotopic labeling by amino acids in cell culture in combination with SDS-PAGE and nanoflow LC-ESI-LTQ for analyzing the differential protein expression of hepatic cells associated to MeHg exposure. Seventy-eight proteins were found de-regulated by more than 1.5-fold. We identified a number of proteins involved in different essential biological processes including apoptosis, mitochondrial dysfunction, cellular trafficking and energy production. Among these proteins, we found several molecules whose de-regulation has been already related to MeHg exposure, thus confirming the usefulness of our discovery approach, and new ones that helped to gain a deeper insight into the biomolecular mechanisms related to MeHg-induced toxicity. Overexpression of several HSPs and the proteasome 26S subunit itself showed the proteasome system as a molecular target of toxic MeHg. As for the interaction networks, the top ranked was the nucleic acid metabolism, where many of the identified de-regulated proteins are involved.


Subject(s)
Liver/drug effects , Methylmercury Compounds/toxicity , Proteins/metabolism , Apoptosis , Cell Line, Tumor , Electron Transport , Electrophoresis, Polyacrylamide Gel , Humans , Liver/cytology , Liver/metabolism , Spectrometry, Mass, Electrospray Ionization
4.
Talanta ; 82(2): 594-9, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20602941

ABSTRACT

A fast method for mercury extraction from biological samples based on the use of HCl leaching plus different enzymatic hydrolysis (with and without mercury complexing agents), and the use of focussed ultrasounds (2-mm microtip) is here proposed. Total mercury content in several biological samples was determined by FI-ICP-MS using a carrier solution consisting of 0.1% (v/v) HCl, 0.1% (v/v) 2-mercaptoethanol, to avoid memory effect, and 0.15% (w/v) KCl. For mercury speciation a RP18 chromatographic column coupled to ICP-MS was used. A mobile phase consisting of 0.1% (v/v) formic acid, 0.1% (v/v) HFBA, 2% (v/v) methanol, and 0.02% (w/v) mM L-cysteine at pH 2.1 was used for chromatographic separation of the mercury species in the sample extracts. Extraction procedures were validated by using 50 mg of tuna fish tissue CRM-463 (2.85+/-0.16 mg kg(-1) for methylmercury). The recoveries obtained were 99+/-3% and 93+/-1% after acid leaching (HCl 7 M) and enzymatic extraction (15 mg protease type XIV in 2.5% (v/v) 2-mercaptoethanol), respectively. The optimal sonication conditions (5 min of exposure time and 40% of ultrasound amplitude) were applied to 5 mg of CRM-463 (88+/-5%), 5 mg of mussel tissue (81+/-11%) and to 2 mg of zebra fish embryos (90+/-10%) obtaining good recoveries in all cases. Methylmecury was found to be the most abundant Hg specie in all samples. The developed method is simple and rapid (5 min sample treatment); it is suitable for very small samples and does not alter the original form of the mercury species. Thus, it is of special interest in those cases in which validation of the results may often be hampered by lack of sample availability.


Subject(s)
Chromatography, Liquid/methods , Fishes , Mass Spectrometry/methods , Mercury/analysis , Mercury/classification , Ultrasonics , Animals , Peptide Hydrolases/chemistry , Reference Standards , Time Factors
5.
Food Chem Toxicol ; 48(5): 1405-11, 2010 May.
Article in English | MEDLINE | ID: mdl-20226830

ABSTRACT

Methylmercury (MeHg) has been suggested to exert cytotoxicity through multiple mechanisms, but the precise biochemical machinery has not been fully defined. This study was aimed at investigating the time-course (0-24h) effect of 2mg/L MeHg on cell death in human HepG2 cells. MeHg decreased cell viability in a time-dependent manner, which was concomitant with increased LDH leakage, reduced GSH levels, CAT activity and altered activity of the antioxidant enzymes GPx and GR at the longest times of incubation (16 and 24h). Activity of the detoxifying enzyme GST was also early enhanced (2h). Caspase-3 activity reached a maximum value at 8h and continued increased up to 24h. This feature was preceded by an enhancement in the caspase-9 activity (2h), whereas caspase-8 activity remained unchanged. MeHg early diminished Bcl-x(L)/Bcl-x(S) ratio and increased levels of the pro-apoptotic Bax and Bad. Moreover, MeHg-induced cytotoxicity was completely inhibited by the antioxidants (GSH and NAC) and notably by the mitochondrial complex I inhibitor rotenone, but not by the NADH oxidase inhibitor DPI. In summary, MeHg induced an oxidative stress responsible for apoptosis in HepG2 cells through direct activation of the caspase cascade and altered the cellular antioxidant and detoxificant enzymatic system to later provoke necrosis at later stages.


Subject(s)
Environmental Pollutants/toxicity , Hepatocytes/drug effects , Methylmercury Compounds/toxicity , Antioxidants/pharmacology , Apoptosis/drug effects , Caspase 3/biosynthesis , Cell Survival/drug effects , Enzyme Activation/drug effects , Glutathione/metabolism , Hep G2 Cells , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Necrosis/chemically induced , Oxidative Stress/drug effects , Oxidoreductases/metabolism , Rotenone/pharmacology , Time Factors , Uncoupling Agents/pharmacology
6.
Anal Bioanal Chem ; 389(7-8): 2167-78, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17952420

ABSTRACT

Selenium methylselenocysteine (Se-MeSeCys) is a common selenocompound in the diet with a tested chemopreventive effect. This study investigated the potential protective effect of Se-MeSeCys against a chemical oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) on human hepatoma HepG2 cells. Speciation of selenium derivatives by liquid chromatography-inductively coupled plasma mass spectrometry depicts Se-MeSeCys as the only selenocompound in the cell culture. Cell viability (lactate dehydrogenase) and markers of oxidative status--concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR)--were evaluated. Pretreatment of cells with Se-MeSeCys for 20 h completely prevented the enhanced cell damage, MDA concentration and GR and GPx activity and the decreased GSH induced by t-BOOH but did not prevent increased ROS generation. The results show that treatment of HepG2 cells with concentrations of Se-MeSeCys in the nanomolar to micromolar range confers a significant protection against an oxidative insult.


Subject(s)
Carcinoma, Hepatocellular , Cysteine/analogs & derivatives , Liver Neoplasms , Organoselenium Compounds/pharmacology , Oxidative Stress , tert-Butylhydroperoxide/toxicity , Anticarcinogenic Agents/pharmacology , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Survival , Cysteine/pharmacology , Glutathione/analysis , Humans , Liver Neoplasms/metabolism , Malondialdehyde/analysis , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Selenocysteine/analogs & derivatives , Time Factors
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