title: Proteomics investigations of drug-induced hepatotoxicity in HepG2 cells.
abstract: Unexpected hepatotoxicity is one of the major reasons of drugs failing in clinical trials. This emphasizes the need for new screening methods that address toxicological hazards early in the drug discovery process. Here, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxic compounds. Drug-induced hepatotoxicity is mainly divided in hepatic steatosis, cholestasis, or necrosis. For each class, a compound was selected, respectively amiodarone, cyclosporin A, and acetaminophen. The changes in protein expressions in HepG2, after exposure to these test compounds, were studied using quantitative two-dimensional differential gel electrophoresis. Identification of differentially expressed proteins was performed by Maldi-TOF/TOF MS and liquid chromatography-tandem mass spectrometry. In this study, 254 differentially expressed protein spots were detected in a two-dimensional proteome map from which 86 were identified, showing that the proteome of HepG2 cells is responsive to hepatotoxic compounds. cyclosporin A treatment was responsible for most differentially expressed proteins and could be discriminated in the hierarchical clustering analysis. The identified differential proteins show that cyclosporin A may induce endoplasmic reticulum (ER) stress and disturbs the ER-Golgi transport, with an altered vesicle-mediated transport and protein secretion as result. Moreover, the differential protein pattern seen after cyclosporin A treatment can be related to cholestatic mechanisms. Therefore, our findings indicate that the HepG2 in vitro cell system has distinctive characteristics enabling the assessment of cholestatic properties of novel compounds at an early stage of drug discovery.
authors.affiliation: Department of Human Biology, Maastricht University, 6200 MD Maastricht, The Netherlands. a.vansummeren@maastrichtuniversity.nl
authors.forename: Anke
authors.initials: A
authors.lastname: Van Summeren
authors.forename: Johan
authors.initials: J
authors.lastname: Renes
authors.forename: Freek G
authors.initials: FG
authors.lastname: Bouwman
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Noben
authors.forename: Joost H M
authors.initials: JH
authors.lastname: van Delft
authors.forename: Jos C S
authors.initials: JC
authors.lastname: Kleinjans
authors.forename: Edwin C M
authors.initials: EC
authors.lastname: Mariman
mesh_terms: D000082:Acetaminophen / Q000008:administration & dosage / Q000009:adverse effects; D000638:Amiodarone / Q000008:administration & dosage / Q000009:adverse effects; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology* / Q000235:genetics / Q000378:metabolism; D016000:Cluster Analysis; D016572:Cyclosporine / Q000008:administration & dosage / Q000009:adverse effects; D064420:Drug-Related Side Effects and Adverse Reactions; D015180:Electrophoresis, Gel, Two-Dimensional; D056945:Hep G2 Cells; D006801:Humans; D004364:Pharmaceutical Preparations / Q000008:administration & dosage; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D004364:Pharmaceutical Preparations; D011506:Proteins; D000082:Acetaminophen; D016572:Cyclosporine; D000638:Amiodarone