RESUMO
Fish is an important source of protein, vitamins, and minerals. However, this food is also a major source of human exposure to toxic contaminants such as mercury. Thus, this paper aimed to evaluate mercury-binding proteins for possible application as biomarkers of mercury contamination in hepatic and renal tissues of Plagioscion squamosissimus (carnivorous fish) and Colossoma macropomum (omnivorous fish) from the Amazon region using metalloproteomic approach. The proteome of hepatic and renal tissues of fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the mercury concentrations in protein spots were determined by graphite furnace atomic absorption spectrometry (GFAAS). Finally, the protein spots associated to mercury were characterized by electrospray ionization mass spectrometry (ESI-MS/MS). The activity of antioxidant enzymes (SOD, CAT, GPx, and GST) and lipid peroxidation (LPO) were also determined. The results showed that the highest concentrations of mercury were found in the carnivorous species (P. squamosissimus) and that the accumulation pattern of this metal was higher in hepatic tissues than in renal tissues for both species. A tendency was observed for greater enzymatic activity in the hepatic and renal tissues of P. squamosissimus, the species with the highest concentration of mercury. Only GPx activity in the kidney and GST in the liver were lower for the P. squamosissimus species, and this finding can be explained by the interaction of mercury with these enzymes. The data obtained by ESI-MS/MS allowed for the characterization of the protein spots associated to mercury, revealing proteins involved in energy metabolism, biomolecules transport, protein synthesis and degradation, cell differentiation, gene regulation, and the antioxidant system. The results obtained in the present study can contribute to understanding the physiological processes underlying mercury toxicity and have provided new perspectives on possible candidates for mercury contamination biomarkers in fish.
Assuntos
Fígado , Animais , Biomarcadores , Proteínas de Transporte , Humanos , Mercúrio , Proteômica , Espectrometria de Massas em Tandem , Poluentes Químicos da ÁguaRESUMO
High concentrations of mercury found in soils, sediments, fish, and humans of the Amazon region have gained prominence in scientific studies during the last decade. However, studies related to the elucidation of mercury toxicity mechanisms in ichthyofauna at the molecular and metallomic levels that seek to elucidate physiological and functional aspects, as well as the search for biomarkers of mercury exposure, are still sparse. In the search for these answers, the present study analyzed the hepatic tissue proteome of the Arapaima gigas (pirarucu) fish species collected in the Jirau hydroelectric power plant reservoir in the state of Rondônia state, Brazil, in order to identify mercury-related metal-binding proteins and to elucidate their physiological and functional aspects. The proteomic profile of the hepatic tissue of Arapaima gigas was obtained by two-dimensional electrophoresis (2D-PAGE) and the presence of mercury was mapped in the protein SPOTS by graphite furnace atomic absorption spectrometry(GFAAS). Mercury was detected in 18 protein SPOTS with concentrations ranging from 0.13⯱â¯0.003 to 131.00⯱â¯3â¯mgâ¯kg-1. The characterization of the protein SPOTS associated with mercury was performed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), and 10 proteins were identified. Bioinformatics analyses showed that most of the proteins found linked to mercury were involved in cellular component processes and biological processes. For the most part, protein sequences have cellular functions comprising catalytic, binding, sense of localization, and metabolic processes.
Assuntos
Proteínas de Transporte/química , Mercúrio/química , Proteômica/métodos , Animais , Brasil , Peixes , HumanosRESUMO
This paper presents a slurry sampling method for total mercury determination by graphite furnace atomic absorption spectrometry (GFAAS) in tissue of fish from the Amazon. The tissue samples were lyophilized and macerated, and then the slurry samples were prepared by putting 20 mg of tissue, added to a solution containing Triton X-100, Suprapur HNO3, and zirconium nitrate directly in sampling vials of a spectrometer. Mercury standard solutions were prepared under the same conditions as the slurry samples. The slurry samples and the mercury standard solutions were sonicated for 20 s. Twenty microliters of slurry samples were injected into the graphite tube, which contained an internal wall lined with tungsten carbide. Under these conditions, it was possible to thermally stabilize the mercury up to an atomization temperature of 1700 °C. The method was validated by mercury determination in reference materials DORM-4 and DOLT-4. The LOD and LOQ were 0.014 and 0.045 mg kg-1, respectively, and recovery percentages in relation to the concentration values were certified in the order of 98%.
Assuntos
Peixes , Fígado/química , Mercúrio/análise , Músculos/química , Animais , Brasil , Grafite/química , Temperatura Alta , Mercúrio/normas , Padrões de Referência , Reprodutibilidade dos Testes , Espectrofotometria Atômica/métodosRESUMO
This study presents data on the extraction and characterization of proteins associated with mercury in the muscle and liver tissues of jaraqui (Semaprochilodus spp.) from the Madeira River in the Brazilian Amazon. Protein fractionation was carried out by two-dimensional electrophoresis (2D-PAGE). Mercury determination in tissues, pellets, and protein spots was performed by graphite furnace atomic absorption spectrometry (GFAAS). Proteins in the spots that showed mercury were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The highest mercury concentrations were found in liver tissues and pellets (426 ± 6 and 277 ± 4 µg kg-1), followed by muscle tissues and pellets (132 ± 4 and 86 ± 1 µg kg-1, respectively). Mercury quantification in the protein spots allowed us to propose stoichiometric ratios in the range of 1-4 mercury atoms per molecule of protein in the protein spots. The proteins characterized in the analysis by ESI-MS/MS were keratin, type II cytoskeletal 8, parvalbumin beta, parvalbumin-2, ubiquitin-40S ribosomal S27a, 39S ribosomal protein L36 mitochondrial, hemoglobin subunit beta, and hemoglobin subunit beta-A/B. The results suggest that proteins such as ubiquitin-40S ribosomal protein S27a, which have specific domains, possibly zinc finger, can be used as biomarkers of mercury, whereas mercury and zinc present characteristics of soft acids.
Assuntos
Caraciformes/metabolismo , Proteínas de Peixes/metabolismo , Fígado/metabolismo , Mercúrio/toxicidade , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismoRESUMO
In the present study, a simple, rapid and sensitive method was developed for the determination of mercury concentrations in the muscle tissue of fish from the Brazilian Amazon using graphite furnace atomic absorption spectrometry (GFAAS) following acid mineralization of the samples in an ultrasonic cold water bath. Using copper nitrate as a chemical modifier in solution and sodium tungstate as permanent modifier, we were able to attain thermal stabilization of the mercury up to the atomisation temperature of 1600 °C in the GFAAS assay. The calculated limits of detection (LOD) and quantification (LOQ) were 0.014 and 0.047 mg kg(-1), respectively.
Assuntos
Mercúrio/análise , Músculo Esquelético/química , Alimentos Marinhos/análise , Espectrofotometria Atômica/métodos , Animais , Brasil , Peixes , Contaminação de Alimentos/análise , Limite de DetecçãoRESUMO
Descreve-se o método para determinação de residuos de mercúrio em peixes. Baseia-se na destruição da matéria orgânica com ácido sulfurico, ácido nítrico e catalizador á base de dióxido de titânio; extração do mercúrio em meio fortemente ácido com solução de ditizona em tetracloreto de carbono de carbono, e deternminação da absorbância em espectrofotômetro, a 490nm. Foi determinado o mercúrio residual em 10 amostras de peixes de água doce e 109 de água salgada, sendo que continham mercúrio 4 amostras no primeiro caso e no segundo, 27 amostras. A totalidade das amostras positivas e 50% da amostras negativas foram controladas por espectrofotometria de absorção atômica, havendo plena concordância nos resultados(AU).