Abstract:
Copper is a micronutrient that serves important structural and catalytic roles in many proteins and enzymes. However, because of its redox potential, uncontrolled levels may be deleterious to the cell. Ceruloplasmin (Cp) belongs to the multi copper oxidase family of proteins that play a significant role in iron homeostasis particularly in Fe loading into serum transferrin. Defective copper loading into apoceruloplasmin may result from mutations in the ATP7B gene characterizing Wilson Disease (WD). Patients with WD are treated with copper chelators. Since copper mediates Cp ferroxidase activity, we postulate that in WD patients copper chelators will decrease level of copper incorporation into Cp, which would affect its ferroxidase activity and consequently disturb iron homeostasis. Therefore, our study aimed at investigating the in vitro effects of copper chelators, Penicillamine (PA) and Bathocuproine Disulfonate (BCS), on: viability of HepG2 cells (MTT assay); Cp ferroxidase activity (P-Phenylenediamine [pPD] oxidase assay); mitochondrial depolarization (Mito-JC1); ROS generation (NBT reduction); Electron transport chain (ETC) enzyme activities; and expression of Cp, ferritin, and transferrin (Western Blotting). We further examined serum iron level, Cp ferroxidase activity and expression of iron binding proteins in WD patients treated with PA.Copper reduced the viability of HepG2 cells in a dose but not time dependent manner (EC50 = 50µM). Cu induced mitochondrial membrane depolarization and reduced ETC enzymes (I and II) activities without generating ROS. HepG2 cells co-treated with Cu and BCS (50 µM: 250 µM) showed restoration of cell viability while maintaining mitochondrial membrane depolarization. However, co-treatment of HepG2 cells with Cu and PA (50 µM: 250 µM) altered cell morphology, increased cytotoxicity resulting in further decrease in viability, increased ROS generation, depolarized mitochondrial membrane, inhibited ETC enzyme activities, and induced cell cycle arrest
Description:
Thesis. M.Sc. American University of Beirut. Department of Biochemistry and Molecular Genetics 2014. W 4 M233e 2014
Advisor: Dr. Julnar Usta, Professor, Department of Biochemistry and Molecular Genetics; Co-Advisor: Dr. Kassem Barada, Professor, Internal Medicine, AUBMC ; Committee member: Dr. George Nemer, Associate Professor, Department of Biochemistry and Molecular Genetics.
Includes bibliographical references (leaves 71-80)