Effect of treatment time and dose of fluoride chronically administered on protein expression in the liver of rats

Abstract

In a previous study conducted by our group it was observed that fluoride can cause alterations in the expression of many hepatic proteins. In addition, it has been reported that these alterations are dose- and time-dependent. Thus, the objective of this study is to analyze the alterations in liver of rats submitted to chronic intoxication with fluoride for different treatment times and doses, using proteomic and histological analyses as tools. Thirty-six 21-day-old male Wistar rats will be included. They will be divided into 2 groups (n=18 animals per group), according with duration of the treatment (20 days, group A, or 60 days, group B). Each group will be split into 3 subgroups (n=6 animals for subgroup), according with the dose of fluoride to be administrated through drinking water, as follows: 0 mg/L (control- subgroups A1 and B1), 15 mg/L (subgroups A2 and B2) ou 50 mg/L (subgroups A3 and B3). All animals will receive, during the treatment, AIN-93 diet (low fluoride concentration). After the experimental period, the animals will be anaesthetized using 0.5 mL/Kg b.w. of Anasedan + 1.5 mL/Kg b.w of quetamin. Liver and blood will be collected. Fluoride analyses in plasma hepatic tissue will be done with the ion specific electrode, after hexamethyldisiloxane-facilitated difusion. Part of the liver will be fixed for histological analysis. After the extraction of the hepatic proteins, they will be submitted to 2-D electrophoresis. Gels from control and treated groups will be scanned and analyzed using the software ImageMaster Platinum. Spots of interest will then be excised and analyzed by LC-MS/MS for the identification of the proteins differentially expressed. After assumption of normal distribution and homogeneity of standard deviations, data will be analyzed by appropriate statistical tests (p<0.05). (AU)