Investigação de metalotioneínas em peixes da região de Jirau - bacia do Rio Madeira - Rondônia
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Data
2017-03-24
Autores
Vieira, José Cavalcante Souza
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Universidade Estadual Paulista (Unesp)
Resumo
Devido a sua grande concentração de nutrientes, tais como proteínas, vitaminas e minerais, o peixe é considerado um dos alimentos mais saudáveis que se pode encontrar na natureza. No entanto, a ingestão de peixes é considerada a forma predominante de via de exposição do ser humano ao mercúrio (Hg), principalmente para as populações que vivem às margens dos rios, onde o peixe constitui a principal fonte de proteína. Na tentativa de elucidar os mecanismos de toxicidade das espécies mercuriais, o teor desse metal tem sido estudado intensamente pela comunidade científica nas últimas décadas em amostras de solo, sedimentos, humanos e peixes na Amazônia brasileira. Sabe-se que as espécies mercuriais bioacumuladas nos tecidos dos seres vivos ligam-se a metaloproteínas, e quando há uma concentração alta de metal tóxico nos organismos, esses passam a expressar proteínas de defesa, denominadas metalotioneínas (MTs) responsáveis pelo transporte e eliminação de metais tóxicos. Apesar de estudos mostrarem o aumento das metalotioneínas em animais expostos a metais potencialmente tóxicos, essas proteínas não foram caracterizadas para confirmação de sua veridicidade, são analisadas por métodos indiretos, esse fato leva a necessidade de técnicas mais precisas na identificação de metalotioneínas. Levando em consideração o exposto esse estudo teve como objetivo otimizar métodos de quantificação de mercúrio e técnica de eletroforese para identificação de possíveis metalotioneínas biomarcadoras de mercúrio em tecido muscular e hepático de peixes de interesse econômico, Tucunaré (Cichla spp.), Filhote (Brachyplatystoma filamentosum), Jaraqui (Semaprochilodus sp.) e Barba chata (Pinirampus pirinampu). Através da Espectrometria de Absorção Atômica em Forno de Grafite (GFAAS) foi demonstrado que as quantificações dos níveis de mercúrio foram eficientes, mostrando boa recuperação do metal nos pools de tecidos, pellets e spots proteicos do músculo e fígado das espécies coletadas no reservatório da Usina Hidrelétrica de Jirau (UHE Jirau) - Bacia do rio Madeira. A separação das proteínas por eletroforese bidimensional (2D-PAGE) mostrou grande eficiência, com spots proteicos bem definidos, boa repetibilidade e reprodutibilidade nas corridas. Posteriormente a técnica de espectrometria de massas com ionização por eletrospray (ESI MS/MS) foi utilizada para identificação das proteínas obtidas por 2D-PAGE. Foi encontrado mercúrio em 27 spots proteicos e destes caracterizadas 23 proteínas ligadas ao mercúrio. No entanto, não foi identificada presença de metalotioneínas nas espécies estudadas, sugerindo que as concentrações de mercúrio não foram suficientes para provocar a produção destas proteínas de defesa contra intoxicação do organismo por este metal. Já nas análises comparando a atividade de indicadoras de estresse oxidativo glutationa peroxidase (GSH-Px), catalase (CAT), superóxido dismutase (SOD) e hidroperóxido de lipídios (HP) com a concentração de mercúrio encontrada em cada espécie de peixes, foi verificado aumento do estresse oxidativo em relação ao aumento da concentração de mercúrio nos tecidos.
Due to its high concentration of nutrients, such as proteins, vitamins and minerals, fish is considered one of the healthiest foods that one can find in nature. However, fish intake is considered to be the predominant human exposure pathway to mercury (Hg), especially for populations living along riverbanks where fish are the main source of protein. In the attempt to elucidate the toxicity mechanisms of mercurial species, the content of this metal has been intensively studied by the scientific community in recent decades in soil, sediment, human and fish samples in the Brazilian Amazon. It is known that mercurial species bioaccumulated in the tissues of living beings bind to metalloproteins, and when there is a high concentration of toxic metal in organisms, they begin to express defense proteins, called metallothioneins (MTs) responsible for the transport and elimination of Toxic metals. Although studies have shown the increase of metallothioneins in animals exposed to potentially toxic metals, these proteins have not been characterized to confirm their veridicity, are analyzed by indirect methods, this fact leads to the need for more precise techniques in the identification of metallothioneins. Taking into account the above, this study aimed to optimize mercury quantification methods and electrophoresis technique for identification of possible mercury biomarkers metallothionein in muscular and hepatic tissue of fish of economic interest, Tucunaré (Cichla spp.), Filhote (Brachyplatystoma filamentosum), Jaraqui (Semaprochilodus sp.) and Barba-chata (Pinirampus pirinampu). Through Graphite Furnace Atomic Absorption Spectrometry (GFAAS) it was demonstrated that the quantification of mercury levels were efficient, showing good recovery of the metal in the pools of tissue, pellets and protein spots of the muscle and liver of the species collected in the Plant's reservoir Jirau Hydroelectric Plant (UHE Jirau) - Madeira River Basin. Separation of proteins by two-dimensional electrophoresis (2D-PAGE) showed high efficiency, with well-defined protein spots, good repeatability and reproducibility in the runs. Subsequently the mass spectroscopy technique with electrospray ionization (ESI MS/MS) was used to identify the proteins obtained by 2D-PAGE. Mercury was found in 27 protein spots and of these 23 mercury - bound proteins were characterized. However, the presence of metallothioneins in the species studied was not identified, suggesting that the concentrations of mercury were not enough to provoke the production of these defense proteins against intoxication of the organism by this metal. In the analyzes comparing the activity of oxidative stress indicators glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) and lipid hydroperoxide (HP) with the concentration of mercury found in each species of fish, Increased oxidative stress in relation to the increase in the concentration of mercury in the tissues.
Due to its high concentration of nutrients, such as proteins, vitamins and minerals, fish is considered one of the healthiest foods that one can find in nature. However, fish intake is considered to be the predominant human exposure pathway to mercury (Hg), especially for populations living along riverbanks where fish are the main source of protein. In the attempt to elucidate the toxicity mechanisms of mercurial species, the content of this metal has been intensively studied by the scientific community in recent decades in soil, sediment, human and fish samples in the Brazilian Amazon. It is known that mercurial species bioaccumulated in the tissues of living beings bind to metalloproteins, and when there is a high concentration of toxic metal in organisms, they begin to express defense proteins, called metallothioneins (MTs) responsible for the transport and elimination of Toxic metals. Although studies have shown the increase of metallothioneins in animals exposed to potentially toxic metals, these proteins have not been characterized to confirm their veridicity, are analyzed by indirect methods, this fact leads to the need for more precise techniques in the identification of metallothioneins. Taking into account the above, this study aimed to optimize mercury quantification methods and electrophoresis technique for identification of possible mercury biomarkers metallothionein in muscular and hepatic tissue of fish of economic interest, Tucunaré (Cichla spp.), Filhote (Brachyplatystoma filamentosum), Jaraqui (Semaprochilodus sp.) and Barba-chata (Pinirampus pirinampu). Through Graphite Furnace Atomic Absorption Spectrometry (GFAAS) it was demonstrated that the quantification of mercury levels were efficient, showing good recovery of the metal in the pools of tissue, pellets and protein spots of the muscle and liver of the species collected in the Plant's reservoir Jirau Hydroelectric Plant (UHE Jirau) - Madeira River Basin. Separation of proteins by two-dimensional electrophoresis (2D-PAGE) showed high efficiency, with well-defined protein spots, good repeatability and reproducibility in the runs. Subsequently the mass spectroscopy technique with electrospray ionization (ESI MS/MS) was used to identify the proteins obtained by 2D-PAGE. Mercury was found in 27 protein spots and of these 23 mercury - bound proteins were characterized. However, the presence of metallothioneins in the species studied was not identified, suggesting that the concentrations of mercury were not enough to provoke the production of these defense proteins against intoxication of the organism by this metal. In the analyzes comparing the activity of oxidative stress indicators glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) and lipid hydroperoxide (HP) with the concentration of mercury found in each species of fish, Increased oxidative stress in relation to the increase in the concentration of mercury in the tissues.
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Palavras-chave
Metalloprotein, Metallothionein, 2D-PAGE, GFAAS, ESI MS/MS