Metalloproteomic approach to liver tissue of rats exposed to mercury.

The aims of this study were to identify mercury-associated protein spots in the liver tissue of rats exposed to low concentrations of mercury and to elucidate the physiological and functional aspects of the proteins identified in the protein spots. Therefore, proteomic analysis of the liver tissue of Wistar rats exposed to mercury chloride (4.60 µg kg-1 in Hg2+) was performed for thirty days (Hg-30 group) and sixty days (Hg-60 group). The proteomic profile of the liver tissue of the rats was obtained by two-dimensional electrophoresis (2D-PAGE), and the determinations of total mercury in the liver tissue, pellets and protein spots were performed by graphite furnace atomic absorption spectrometry (GFAAS). ImageMaster 2D Platinum 7.0 software was used to identify the differentially expressed mercury-associated protein spots, which were then characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The determinations by GFAAS indicated a total mercury bioaccumulation of 2812% in the Hg-30 group and 3298% in the Hg-60 group and 10 mercury-associated protein spots with a concentration range of 51 ± 1.0 to 412 ± 6.00 mg kg-1 in the 2D PAGE gels from the liver tissue of the Hg-60 group. The LC-MS/MS analyses allowed the identification of 11 metal binding proteins in mercury-associated protein spots that presented fold change with upregulation >1.5, downregulation < -1.7 or that were expressed only in the Hg-60 group. Using the FASTA sequences of the proteins identified in the mercury-associated protein spots, bioinformatics analyses were performed to elucidate the physiological and functional aspects of the metal binding proteins, allowing us to infer that enzymes such as GSTM2 presented greater mercury concentrations and downregulation < -3; Acaa2 and Bhmt, which showed expression only in the Hg-60 group, among others, may act as potential mercury exposure biomarkers.

Investigation of Protein Biomarkers and Oxidative Stress in Pinirampus pirinampu Exposed to Mercury Species from the Madeira River, Amazon-Brazil.

In recent decades, the scientific community has widely debated the contamination of fish in the Amazon region by mercury species. As the diet of riverside populations in the Amazon region is based mainly on fish, these populations are exposed to mercurial species that can cause serious and irreversible damage to their health. The risks of consuming fish exposed to mercurial species in the Amazon region have motivated toxicological investigations. However, the effect of mercurial species on protein and enzyme levels is still controversial. In this work, analytical and bioanalytical techniques Two-dimensional polyacrylamide gel electrophoresis [2D-PAGE] Graphite Furnace Atomic Absorption Spectrometry [GFAAS], and Mass Spectrometry in Sequence with Electrospray Ionization [ESI-MS/MS] were used to identify proteins associated with mercury (metal-binding protein) in muscle and liver tissues of the fish species Pinirampus pirinampu from the Madeira River, in the Brazilian Amazon. Enzymatic and lipid peroxidation analyses were also used to assess changes related to oxidative stress. Determinations of total mercury by GFAAS indicated higher concentrations in liver tissue (555 ± 19.0 µg kg-1) when compared to muscle tissue (60 ± 2.0 µg kg-1). The fractionation process of tissue proteomes by 2D-PAGE and subsequent mapping of mercury by GFAAS in the protein spots of the gels identified the presence of mercury in three spots of the liver tissue (concentrations in the range of 0.800 to 1.90 mg kg-1). The characterization of protein spots associated with mercury by ESI-MS/MS identified the enzymes triosephosphate isomerase A, adenylate kinase 2 mitochondrial, and glyceraldehyde-3-phosphate dehydrogenase as possible candidates for mercury exposure biomarkers. The muscle tissue did not show protein spots associated with mercury. Enzymatic activity decreased proportionally to the increase in mercury concentrations in the tissues.

Metalloproteomic Strategies for Identifying Proteins as Biomarkers of Mercury Exposure in Serrasalmus rhombeus from the Amazon Region.

This manuscript describes the results of a metalloproteomic study of mercury in samples of muscle and liver tissue of the species Serrasalmus rhombeus, popularly known as black piranha and characterised as the most voracious and aggressive predator in the Brazilian Amazon. The metalloproteomic study involved using two-dimensional electrophoresis (2D PAGE) to fractionate the proteome of the muscle and liver tissue samples, along with atomic absorption spectrometry in a graphite furnace (GFAAS) to identify mercury associated with protein SPOTs and mass spectrometry with electrospray ionisation (ESI-MS/MS) to characterise the mercury-binding proteins. The protein SPOTs characterised showed concentrations in the order of 156 mg kg-1, which ranks as the highest concentrations of mercury determined so far in metalloproteomic studies involving fish species in the Amazon region. Based on FASTA sequences of proteins characterised by ESI-MS/MS, bioinformatics studies were performed that allowed identifying nine proteins with characteristics of biomarkers of mercury exposure. Of those proteins, glutathione peroxidase stands out as an enzyme of great importance in the antioxidant defence of organisms subjected to oxidative stress caused by xenobiotics.