Redox Proteomics and Immunohistology to Study Molecular Events During Ischemia-Reperfusion in Human Liver

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doi:10.1016/j.transproceed.2007.05.082 Copyright © 2007 Elsevier Inc. All rights reserved.

Ischemia reperfusion injury

Redox Proteomics and Immunohistology to Study Molecular Events During Ischemia-Reperfusion in Human Liver

C. Avellinia, U. Baccaranib, , G. Trevisana, L. Cesarattoc, C. Vascottoc, F. D’Aurizioa, M. Pandolfia, G.L. Adanib and G. Tellc aDepartment of Medical and Morphological Sciences, University of Udine, Udine, ItalybDepartment of Surgery & Transplantation, University of Udine, Udine, ItalycDepartment of Biomedical Sciences and Technologies, University of Udine, Udine, Italy. Available online 8 August 2007. Abstract

Oxidative stress is a condition occurring in liver disorders and causing liver damage due to ischemia-reperfusion (I/R) during liver transplantation. Several markers of chronic oxidative stress are well known; however, early protein targets of oxidative injury are not well defined. To identify them, we used a differential proteomics approach to HepG2 human liver cells that has been treated for 10 minutes with 500 μmol/L H2O2. By differential proteomic analysis, using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins sensitive to H2O2 treatment that underwent posttranslational modification of native polypeptides. Three of the proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, PrxI, PrxII, and Prx VI, that showed changes in their pI as result of hyperoxidation. Mass mapping experiments demonstrated specific modification of the peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining an increased negative charge. The oxidation kinetics of all peroxiredoxins were extremely rapid and sensitive, occurring at H2O2 doses unable to affect common markers of cellular oxidative stress. A differential proteomics approach was also applied to liver needle biopsies after cold (T1) and warm (T2) ischemia. Proteomic analysis of this material was related to histological changes and immunophenotypic expression of APE1/Ref-1. Hyperoxidation of PrxI occurring during I/R upon liver transplantation is dependent on the time of warm ischemia. Histological changes and APE1/Ref-1 expression parallel Peroxiredoxin changes. Our present data may be relevant to better graft preservation and evaluation for transplantation.

Address reprint requests to Dr Umberto Baccarani, Dipartimento di Scienze Chirurgiche, Università of Udine, P.le S. della Misericordia, 33100 Udine, Italia.

Transplantation Proceedings Volume 39, Issue 6, July-August 2007, Pages 1755-1760