Investigation of metallothionein in fish from the Jirau region - Madeira River basin - Rondônia

Author(s)

José Cavalcante Souza Vieira

• Document type: Doctoral Thesis
• Press: Botucatu. 2017-05-24.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2017-03-24
• Advisor: Pedro de Magalhães Padilha

Abstract

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. (AU)

• FAPESP's process: 14/02668-1 - Utilization of metalloproteomic strategies for metallothionein evaluation in liver tissue samples of fishes collected in the reservoir of Ahe Jirau - Madeira River Basin, RO, Brazil
• Grantee: José Cavalcante Souza Vieira
• Support Opportunities: Scholarships in Brazil - Doctorate