Approved Abstracts

Cerebellar proteomic profile underling biochemical, morphological and functional impairments triggered by methylmercury long-term exposure in rats



Author(s): Bittencourt LO; Matta PP; Aragão WAB; Nascimento PC; Eiró LG; Silva MCF; Fernandes LMP; Dionizio A; Buzalaf MAR; Maia CSF; Crespo-López ME; Lima RR;
Presenter: Leonardo Oliveira Bittencourt

Methylmercury (MeHg) is a hazardous toxicant and its presence in the environment may occur by natural or anthropogenic actions, mainly due to the industrial and artisanal gold mining activities. Due to its chemical characteristics and its biogeochemical cycle, the MeHg is able to bioaccumulate and biomagnify in aquatic trophic chain. In this way, several populations are exposed to MeHg for long periods and low doses by the consumption of contaminated fish and seafood. This environmental contamination can provide risks of intoxication to riverside populations that consume local water and fish, featuring an exposure profile to low doses for a long period. The literature presents several evidences regarding MeHg neurotoxicity, however the investigation of the global proteomic profile that underlies others neurotoxic mechanisms, are scarce, especially when considering a representative exposure model. In this way, this study aimed to investigate molecular, morphological, and functional damages in the cerebellum of rats exposed to MeHg. For this, forty adult Wistar rats were divided into an exposed group, which received 0.04 mg/kg/day of MeHg, through orogastric gavage for 60 days, and a control group, which received only vehicle. After the exposure period, the motor functions were analyzed by open field test, to assess spontaneous vertical and horizontal locomotion and rotarod test, to assess balance and forced locomotion. Then, the cerebellums were collected to determinate the total mercury (Hg) content, to analyze oxidative biochemistry through the levels of malondialdehyde (MDA), nitrites and antioxidant capacity against peroxyl radicals (ACAP), and to assess the global proteomic profile by mass spectrometry (UPLC/MS), with further bioinformatic approaches using gene ontology annotations with Cytoscape software and over-representation (ORA) analysis with R programing language. In addition, the neural cell populations were evaluated by counting Punkinje cells, and by immunohistochemistry using antibodies for mature neurons, astrocytes and microglia, besides the evaluation of myelin sheath integrity, by anti- myelin basic protein (MBP) labeling and synaptic vesicles, by anti-synaptophysin (SYP). Statistical analyses were performed by Student's t-test, considering p <0.05. The proteomic statistical analysis was performed by ProteinLynx GlobalSERVER software. The results showed that long-term exposure to low doses of MeHg increase total Hg levels in cerebellar parenchyma and triggered oxidative stress by decreasing ACAP and increasing MDA and nitrite levels. Moreover, it was observed a significant decrease on Purkinje cells, mature neurons and astrocytes, while the density of microglia increased. In addition, it was observed a decrease on MBP labeling and an increase on SYP labeling. The proteomic profile showed a total of 597 proteins with different status of regulation, being 279 exclusives in the exposed or control group and 318 with status up or down-regulated. According to the ORA analysis, the proteins are involved mainly in cytoskeleton regulation, oxidative balance, synaptic signaling and energy metabolism. The behavioral assessment showed a decrease on the horizontal and vertical exploration in the exposed group compared to control, as well an increase on the number of falls. Therefore, we verified that MeHg administered at low dose for a prolonged period is associated with a neurodegenerative pattern and astrocytes density decrease, while there is an immunological response evidenced by microglial density increase. Corroborating with the functional impairments, it was observed that MeHg causes impacts on myelin sheath and increase on synaptic vesicle marker, suggesting a compromise on neuronal signaling, which drove to a poor motor performance. This study was financed in part by the Brazilian National Council for Scientific and Technological Development (CNPq).

Keywords: Methylmercury; Myelin sheath; Synaptophysin

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