Estudo de biomarcadores de mercúrio em peixes da amazônia por meio da metalômica e análise do estresse oxidativo
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Data
2017-07-07
Autores
Bittarello, Alis Correia [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O mercúrio é um metal tóxico, de distribuição ubíqua, com capacidade para bioacumulação e biomagnificação, que provoca alterações em biomoléculas importantes no metabolismo e contribui para o estabelecimento do estresse oxidativo em organismos aquáticos. Logo, o presente estudo teve por objetivo identificar e avaliar possíveis biomarcadores proteicos e/ou enzimáticos da toxicidade do mercúrio em peixes da região amazônica, por meio do estudo metaloproteômico e avaliação do estresse oxidativo. Foram utilizadas metodologias de fracionamento e identificação de proteínas por eletroforese bidimensional (2D PAGE) associada à espectrometria de massas (MS), mapeamento do mercúrio, em spots proteicos, por espectrometria de absorção atômica em forno de grafite (GFAAS) e avaliação de marcadores de estresse oxidativo. As espécies utilizadas foram o Plagioscion squamosissimus (corvina) e Colossoma macropomum (tambaqui), coletados na área da Usina Hidrelétrica de Jirau (rio Madeira-RO), que foram selecionadas em função da abundância populacional, interesse para a pesca e posição diferente na cadeia trófica (carnívoro e onívoro, respectivamente). Os tecidos amostrados foram o hepático, renal e muscular. Os resultados obtidos demonstraram maior concentração de mercúrio total no P. squamosissimus, espécie carnívora, e padrão de distribuição deste elemento igual para ambas as espécies (fígado>rim>músculo). Há tendência para maior atividade enzimática nos tecidos hepático e renal da espécie com maior concentração de mercúrio. Somente a atividade da glutationa peroxidase no rim e da glutationa-S-transferase no fígado do P. squamosissimus foi inferior, fato que pode ser explicado pela interação do mercúrio com essas enzimas, interferindo em sua conformação e função. Os dados obtidos por MS possibilitaram a identificação dos spots proteicos associados ao mercúrio, revelando proteínas envolvidas no metabolismo energético (gliceraldeído-3-fosfato, malato e Llactato desidrogenase, enolase, creatina quinase e fosfoglicerato mutase), transporte (hemoglobinas α e β, proteínas de ligação a ácidos graxos e GTPases), síntese e degradação de proteínas (ubiquitina e ribossomal S27a e ribosomal 40S S24) diferenciação celular (nucleosídeo difosfato quinase), regulação gênica (pterina-4-α-carbinolamina desidratase e histona H4), defesa imunitária (fator de aprimoramento de células natural killer), metabolismo do cálcio (parvalbumina β), adipogênese (UPF0366), metabolismo de xenobióticos e esteroidogênese (citocromo P450scc) e sistema antioxidante (glutationa peroxidase, glutationa-S-transferase, superóxido dismutase, catalase e aldeído desidrogenase). Contribuindo assim para a compreensão dos mecanismos moleculares subjacentes à toxicidade ao mercúrio e fornecendo novas perspectivas sobre possíveis candidatos a biomarcadores de monitoramento ambiental.
Mercury is a toxic metal, of ubiquitous distribution, with capacity for bioaccumulation and biomagnification, that causes changes in important biomolecules in the metabolism and contributes to the establishment of oxidative stress in aquatic organisms. Therefore, the present study aimed to identify and evaluate possible proteic and/or enzymatic biomarkers of the mercury toxicity in fish from the Amazon region, through a metalloproteomic study and evaluation of oxidative stress. Were used protein fractionation and identification methodologies by two-dimensional electrophoresis (2D PAGE) associated with mass spectrometry (MS), mapping of mercury in protein spots by atomic absorption spectrometry in graphite furnace (GFAAS) and evaluation of oxidative stress markers. The species used were Plagioscion squamosissimus (corvina) and Colossoma macropomum (tambaqui), collected in the area of the Jirau Hydroelectric Power Plant (Madeira-RO), which were selected due to the population abundance, interest for fishing and different position in the food chain (carnivorous and omnivorous, respectively). The tissues sampled were hepatic, renal and muscular. The results showed higher concentration of total mercury in P. squamosissimus, carnivorous species, and distribution pattern of this element equal to both species (liver> kidney> muscle). There is a tendency for greater enzymatic activity in the hepatic and renal tissues of the specie with the highest concentration of mercury. Only the activity of glutathione peroxidase in the kidney and glutathione-S-transferase in the liver of P. squamosissimus was lower, a fact that can be explained by the interaction of mercury with these enzymes, interfering in their conformation and function. The data obtained by MS allowed the identification of the protein spots associated with mercury, revealing proteins involved in energy metabolism (glyceraldehyde-3-phosphate, malate and Llactate dehydrogenase, enolase, creatine kinase and phosphoglycerate mutase), transport (α and β hemoglobins, fatty acid binding proteins and GTPases), synthesis and degradation of proteins (ubiquitin and ribosomal S27a and ribosomal 40S S24), cell differentiation (nucleoside diphosphate kinase), gene regulation (pterin-4-α-carbinolamine dehydratase and histone H4), immune defense (natural killer cell enhancement factor), calcium metabolism (β-parvalbumin), adipogenesis (UPF0366), xenobiotic metabolism and steroidogenesis (cytochrome P450scc) and antioxidant system (Glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, catalase and aldehyde dehydrogenase). Thus contributing to the understanding of the molecular mechanisms underlying mercury toxicity and providing new perspectives on potential candidates for environmental monitoring biomarkers.
Mercury is a toxic metal, of ubiquitous distribution, with capacity for bioaccumulation and biomagnification, that causes changes in important biomolecules in the metabolism and contributes to the establishment of oxidative stress in aquatic organisms. Therefore, the present study aimed to identify and evaluate possible proteic and/or enzymatic biomarkers of the mercury toxicity in fish from the Amazon region, through a metalloproteomic study and evaluation of oxidative stress. Were used protein fractionation and identification methodologies by two-dimensional electrophoresis (2D PAGE) associated with mass spectrometry (MS), mapping of mercury in protein spots by atomic absorption spectrometry in graphite furnace (GFAAS) and evaluation of oxidative stress markers. The species used were Plagioscion squamosissimus (corvina) and Colossoma macropomum (tambaqui), collected in the area of the Jirau Hydroelectric Power Plant (Madeira-RO), which were selected due to the population abundance, interest for fishing and different position in the food chain (carnivorous and omnivorous, respectively). The tissues sampled were hepatic, renal and muscular. The results showed higher concentration of total mercury in P. squamosissimus, carnivorous species, and distribution pattern of this element equal to both species (liver> kidney> muscle). There is a tendency for greater enzymatic activity in the hepatic and renal tissues of the specie with the highest concentration of mercury. Only the activity of glutathione peroxidase in the kidney and glutathione-S-transferase in the liver of P. squamosissimus was lower, a fact that can be explained by the interaction of mercury with these enzymes, interfering in their conformation and function. The data obtained by MS allowed the identification of the protein spots associated with mercury, revealing proteins involved in energy metabolism (glyceraldehyde-3-phosphate, malate and Llactate dehydrogenase, enolase, creatine kinase and phosphoglycerate mutase), transport (α and β hemoglobins, fatty acid binding proteins and GTPases), synthesis and degradation of proteins (ubiquitin and ribosomal S27a and ribosomal 40S S24), cell differentiation (nucleoside diphosphate kinase), gene regulation (pterin-4-α-carbinolamine dehydratase and histone H4), immune defense (natural killer cell enhancement factor), calcium metabolism (β-parvalbumin), adipogenesis (UPF0366), xenobiotic metabolism and steroidogenesis (cytochrome P450scc) and antioxidant system (Glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, catalase and aldehyde dehydrogenase). Thus contributing to the understanding of the molecular mechanisms underlying mercury toxicity and providing new perspectives on potential candidates for environmental monitoring biomarkers.
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Palavras-chave
Espécies mercuriais, Estresse oxidativo, 2D PAGE, LC-MS/MS, GFAAS, Mercury species, Oxidative stress