PROTEOMIC ANALYSES IN KIDNEYS OF METHYLMERCURY EXPOSED MICE: A PRELIMINARY STUDY.

MARCO, K. C.1, SOUZA, V. C. O.1, BARBOSA Jr, F.1 1 Faculty of Pharmaceutical Sciences of Ribeirao Preto University of Sao Paulo Brazil. kdemarco@fcfrp.usp.br Introduction Mercury is a nonessential metal and one of the most harmful environment pollutants. Several toxic effects in humans after mercury exposure are very well known. Methylmercury (MeHg) is the most important form in terms of toxicity and is predominated found in fish and seafood samples. The intracellular activity of metal ions is largely controlled by several families of proteins, which act as detoxificants, or simply protective proteins involved in cell cycle, proliferation and apoptosis. Thus, the structure and amount of protein species reflect the biological activity of the cell. So a proteomic study may lead to identification of new candidate biomarkers that may better reflect the early effects after the exposure and also the mechanism associated with. Methods Mice were daily exposed to MeHg (100g) by gavage during 5 days. Animals were euthanized and kidney samples were collected and homogenized in a lysis buffer and protease inhibitor cocktail. The lysates were centrifuged and the supernatant was precipitated with TCA and acetone in an ice bath for 30 minutes. Electrophoresis buffer was added following five cycles of ultrasound bath for proteins solubilization. The protein quantification was performed by the Bradford method (1 mg of protein was applied to the gel). For the first dimension of electrophoresis were used strips with immobilized pH gradient (3-11). During this process, the IPG strips are incubated for 12 hours at 20 C. The process of electrophoretic separation was performed at 20C, using 75A/strip IPG. For the second dimension the proteins were subjected to reduction and alkylation. The SDS-PAGE was performed in vertical electrophoresis system using gels containing 12.5% acrylamide. After gels staining, the images were digitized and processed by comparative analysis of twodimensional maps using the program ImageMaster Platinum version 5.0 (GE Healthcare). Results Figure 1 Two-dimensional Gel (Kidney) after MeHg exposure. The comparative analysis of maps was obtained by using the program ImageMaster Platinum. Numbers identify the spots of the expressed proteins. These results, although preliminary, show that MeHg causes a change in expression of at least 42 proteins, as highlighted in Figure 1. It is important to emphasize that this effect was observed after a short period of exposure (5 days). Based on these findings, it is expected that longer exposures cause changed expression of a larger number of proteins and hence increase the chance of identifying a strong candidate marker for early effects of mercury in addition elucidating possible mechanism toxicity this metal. Conclusion MeHg caused a change in several kidney proteins expression and after the identification of proteins, it can be used to elucidate mechanisms of MeHg toxicity. Acknowledgments Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP).