Susceptibility of cultured rat hepatocytes to oxidative stress by peroxides and iron. The extracellular matrix aects the toxicity of tert-butyl hydroperoxide K. Hagen a , C. Zhu a ,O È . Melefors b , R. Hultcrantz a, * a Department of Gastroenterology and Hepatology, Karolinska Hospital, Karolinska Institute, 171 76 Stockholm, Sweden b Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden Received 7 November 1997; accepted 22 September 1998 Abstract The aim of this study was to set up an in vitro model for studying the importance of an altered extra-cellular matrix composition and its importance for the resistance to oxidative stress, in hepatocytes from normal and iron loaded rats. Primary cultures of hepatocytes from iron loaded and normal rats were plated on a laminin rich extracellular matrix or on collagen type I, and incubated with tert-butyl hydroperoxide (TBH). Malon dialdehyde (MDA) and the activities of lactate dehydrogenase (LDH) in cell culture medium were analyzed. The protein synthesis, the concentrations of glutathione and the expression of manganese-superoxide dismutase and ferritin genes were measured. All hepatocytes contained lower concentrations of glutathione when plated on collagen than on EHS. Ferritin H and Mn-SOD gene expression showed no dierence. The rate of lipid peroxidation in iron loaded hepatocytes exposed to TBH was higher on collagen than in those plated on EHS (0.95 20.28 mM MDA vs. 1.62 20.22 mM MDA, p < 0.05). Iron loaded cells were in general more susceptible to TBH than were normal hepatocytes (MDA, LDH, protein synthesis and glutathione content). Lipid peroxidation could be prevented by adding desferrioxamine. In conclusion, we show that the combination of iron overload and collagen matrix in rat hepatocytes leads to an increased susceptibility to oxidative stress. These ®ndings may be of interest for the further studies on eects of iron overload and the altered matrix composition in liver ®brosis. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Extracellular matrix; Iron; Rat hepatocytes; Oxidative stress; Glutathione; Collagen 1. Introduction Iron overload in humans caused by a defect in iron absorption (genetic hemochromatosis) [1] leads to liver damage and cirrhosis in some of the patients ± mainly males [2±4]. Hepatic stellate cells are responsible for the deposition of The International Journal of Biochemistry & Cell Biology 31 (1999) 499±508 1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S1357-2725(98)00115-0 PERGAMON * Corresponding author. Fax: +46-8-5177-2637. Abbreviations: EDTA, ethylenediaminetetraacetic acid, EHS, Engelbreth±Holm±Swarm, GSH, glutathione, HEPES, N-[2-hydroxyethyl] piperazine-N 0 -[2-ethanesulphonic acid], HPLC, high performance liquid chromatography, LDH, lac- tate dehydrogenase, MDA, malon dialdehyde, MEM, minimal essential medium, Mn-SOD, Manganese-superoxide dismu- tase, PBS, phosphate buered saline, TBH, tert-butyl hydro- peroxide.