Increase in vulnerability to oxidative damage in cholesterol-modified erythrocytes exposed to t -BuOOH Abel Lo ´ pez-Revuelta, Jose ´ I. Sa ´nchez-Gallego, Angel Herna ´ndez-Herna ´ndez, Jesu ´s Sa ´nchez-Yagqe, Marcial Llanillo T Departamento de Bioquı ´mica y Biologı ´a Molecular, Universidad de Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain Received 23 December 2004; received in revised form 15 February 2005; accepted 15 February 2005 Available online 5 March 2005 Abstract During the course of radical oxidation, cholesterol may exert seemingly contradictory effects. In order to gain a better understanding of the relationship between cholesterol levels and membrane susceptibility to oxidative damage induced by reactive oxygen species (ROS), here we analyze the integrity and structural stability of cholesterol-modified (enriched or depleted) and unmodified (control) erythrocytes exposed to tert -butyl hydroperoxide. The oxidant significantly increased ROS production, with almost complete oxidation of hemoglobin and a reduction in GSH content in the different erythrocyte groups at 2 mM concentration. These changes were accompanied by losses of cholesterol and total phospholipids, the main decreases being in phosphatidylethanolamine and phosphatidylcholine. The highest lipid loss was found in the cholesterol-depleted group. Fatty acid analyses revealed changes only in peroxidized cholesterol-modified erythrocytes, with decreases in linoleic and arachidonic acids. Fluorescence anisotropy studies showed an increase in the fluidity of the negatively charged surface of peroxidized control erythrocytes. Increased hemolysis and a positive correlation between cellular osmotic fragility and malondialdehyde contents were found in all peroxidized groups. These findings provide evidence that the modification of cholesterol levels in the erythrocyte membrane has provoking effects on peroxidation, with corresponding increases in oxidative damage in the treated cell, possibly as a consequence of lipid bilayer destabilization. D 2005 Elsevier B.V. All rights reserved. Keywords: Cholesterol; Oxidative damage; Lipid oxidation; tert -Butyl hydroperoxide; Erythrocyte 1. Introduction A major target of free radicals is the cell membrane, which is involved in the control of cell function through integral maintenance of the spatial and intermolecular arrangements of its components. Because of their suscept- ibility to oxidation, erythrocytes have been used as a model to investigate oxidative damage in biomembranes. These cells are considered prime targets for free radical attack owing to the presence of both high membrane concen- trations of polyunsaturated fatty acids (PUFA) and the oxygen transport associated with redox active hemoglobin molecules, which are potent promoters of reactive oxygen species (ROS) [1]. To counteract the potential deleterious effects of ROS, erythrocytes have a well-integrated network of antioxidant mechanisms to combat this oxidative stress, whose specific purpose is to protect the functional and structural integrity of biologically essential macromolecules [2]. Unbalanced ROS production and antioxidant cell defences have been reported to occur in the pathophysiology of many human diseases, including several forms of non- 1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2005.02.004 Abbreviations: BCD, h-cylodextrin; Ch, cholesterol; DCF, 2V ,7V - dichlorofluorescein; DPH, 1,6-diphenyl-1,3,5-hexatriene; Hb, hemoglobin; MBCD, methyl-h-cyclodextrin; MDA, malonyldialdehyde; MetHb, meth- emoglobin; PL, phospholipids; PC, phosphatidylcholine; PE, phosphatidy- lethanolamine; PS+PI, phosphatidylserine+phospatidylinositol; PUFA, polyunsaturated fatty acids; PVP, polyvinylpyrrolidone; ROS, reactive oxygen species; SM, sphingomyelin; t -BuOOH, tert -butylhydroperoxide; TBARS, thiobarbituric acid-reactive substances; TMA-DPH, 1[4V -(trime- thylammonium)phenyl]-6-phenyl-1,3,5-hexatriene T Corresponding author. Tel.: +34 923 294 465; fax: +34 923 294 579. E-mail address: llanillo@usal.es (M. Llanillo). Biochimica et Biophysica Acta 1734 (2005) 74 – 85 http://www.elsevier.com/locate/bba