Protocols of two-dimensional difference gel electrophoresis to investigate mechanisms of toxicity.

In recent years, several global omics technologies have been increasingly used to better understand the molecular mechanisms of drug toxicity. Two-dimensional difference gel electrophoresis (2D-DIGE) is a large-scale proteomics high-resolution gel-based quantitative method widely used to detect protein expression alterations after drug treatment. The 2D-DIGE technology is based on the labeling of proteins with different fluorescent dyes, allowing the separation of different samples on the same gel with the use of an internal standard, thus reducing the complexity of spot pattern comparison and providing a reliable method applied to toxicology studies for the detection of modulated proteins in targeted organs.

Optimization of SELDI for biomarker detection in plasma.

The surface-enhanced laser desorption ionization (SELDI) technology is a promising approach not only for the research of biomarkers in the blood of patients in clinical applications but also in preclinical studies to assess the drug-induced toxicities. The optimization of the SELDI platform is a crucial step before running plasma samples from preclinical toxicity studies. First, mass spectrometer parameters such as the laser energy and ion focus mass values should be assessed in order to obtain the highest quality of spectra. Second, the coefficient of variation of the intensity, resolution, and signal-to-noise ratio of the peaks detected in reference samples should be evaluated and used as quality control criteria. Last, a systematic evaluation of technical bias such as the spot and chip position and the bioprocessor sequence number may be achieved using the appropriate multivariate statistical analyses.

Proteomic characterization of the effects of clofibrate on protein expression in rat liver.

Clofibrate is a peroxisome proliferator known to induce liver tumours in rats. A proteomics study was conducted to provide new insights into the molecular mechanisms of clofibrate-induced non-genotoxic hepatocarcinogenesis. Rats were treated with 250 mg/kg day clofibrate orally and sacrificed after 7 days. Proteins extracted from the liver were analysed by 2-DE using DIGE technology. The protein identification performed by MS showed that clofibrate induced up-regulation of 77 proteins and down-regulation of 27 proteins. The highest expression ratios corresponded to proteins involved in a series of biochemical pathways such as lipid metabolism, fatty acid metabolism, amino acid metabolism, protein metabolism, citric acid cycle, xenobiotic detoxification and oxidative stress. Proteins implicated in cell proliferation and apoptosis, such as prohibitin, 10-formyl tetrahydrofolate dehydrogenase, senescence marker protein-30, pyridoxine 5'-phosphate oxidase and vimentin, were also identified as being regulated. These results provide leads for further investigations into the molecular mechanisms of liver tumours induced by clofibrate. In addition, MS results showed that a series of regulated proteins were detected as several spots corresponding to different pI and/or M(r). Differential effects on those variants could result from specific PTM and could be a specific molecular signature of the clofibrate-induced protein expression modulation in rat liver.