Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury

Abstract

Iron overload exacerbates various liver diseases. In hepatocytes, a portion of non-heme iron is sequestered in lysosomes and endosomes. The precise mechanisms by which lysosomal iron participates in hepatocellular injury remain uncertain. Here, our aim was to determine the role of intracellular movement of chelatable iron in oxidative stress-induced killing to cultured hepatocytes from C3Heb mice and Sprague-Dawley rats. Mitochondrial polarization and chelatable iron were visualized by confocal microscopy of tetramethylrhodamine methylester (TMRM) and quenching of calcein, respectively. Cell viability and hydroperoxide formation (a measure of lipid peroxidation) were measured fluorometrically using propidium iodide and chloromethyl dihydrodichlorofluorescein, respectively. After collapse of lysosomal/endosomal acidic pH gradients with bafilomycin (50 nM), an inhibitor of the vacuolar proton-pumping adenosine triphosphatase, cytosolic calcein fluorescence became quenched. Deferoxamine mesylate and starchdeferoxamine (1 mM) prevented bafilomycin-induced calcein quenching, indicating that bafilomycin induced release of chelatable iron from lysosomes/endosomes. Bafilomycin also quenched calcein fluorescence in mitochondria, which was blocked by 20 M Ru360, an inhibitor of the mitochondrial calcium uniporter, consistent with mitochondrial iron uptake by the uniporter. Bafilomycin alone was not sufficient to induce mitochondrial depolarization and cell killing, but in the presence of low-dose tert-butylhydroperoxide (25 M), bafilomycin enhanced hydroperoxide generation, leading to mitochondrial depolarization and subsequent cell death. Conclusion: Taken together, the results are consistent with the conclusion that bafilomycin induces release of chelatable iron from lysosomes/endosomes, which is taken up by mitochondria. Oxidative stress and chelatable iron thus act as two "hits" synergistically promoting toxic radical formation, mitochondrial dysfunction, and cell death. This pathway of intracellular iron translocation is a potential therapeutic target against oxidative stress-mediated hepatotoxicity. (HEPATOLOGY 2008;48:1644-1654 C helatable iron and other transition metals such as copper catalyze formation of highly reactive hydroxyl radical (OH • ) from H 2 O 2 and superoxide (O 2 •Ϫ ), which damages DNA, proteins, and membranes. 1 Cytoprotection by deferoxamine in various models of oxidative stress and hypoxia/ischemia suggests a role for iron in the pathogenesis of injury. [2][3][4][5][6] In hepatocytes, generation of reactive oxygen species (ROS), including

Inhibitory effects of Schisandra chinensis on acetaminophen-induced hepatotoxicity

“…However, Schisandra chinensis may be able to treat acute liver injury though protection of the mitochondria. Disruption of lysosomes in hepatocytes following acetaminophen treatment has been observed in previous studies (13,15). Notably, Schisandra chinensis was also found to protect lysosomes in the present study.…”

“…Studies have indicated that lysosomal disruption occurs subsequent to numerous types of cellular stresses in hepatocytes and other cell types (11)(12)(13). A breakdown of lysosomes may result in cell death by necrosis, which is associated with an increase in cytosolic acidification (14).…”

Transcription factor NRF2 protects mice against dietary iron-induced liver injury by preventing hepatocytic cell death

“…A striking feature of necrotic hepatocytes from Nrf2 -/-mice fed iron-rich diet was the extreme swelling of mitochondria. In hepatocytes, non-heme iron that is initially sequestered in lysosomes may be released and taken up into mitochondria, where it catalyzes toxic ROS-mediated reactions [31]. This may open nonspecific permeability transition pores in the mitochondrial inner membrane that cause mitochondrial membrane depolarization, uncoupling of oxidative phosphorylation, and large scale mitochondrial swelling, leading to hepatocyte necrosis from Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 adenosine triphosphate depletion.…”

Ethanol Suppresses Ureagenesis in Rat Hepatocytes

“…Hepatocyte Isolation and Culture-Hepatocytes were isolated from 200-to 250-g overnight-fasted male Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) by collagenase perfusion as described previously (33,34). Hepatocytes were resuspended in Waymouth's MB-752/1 growth medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 nM insulin (Squibb-Novo, Princeton, NJ), 100 nM dexamethasone (LyphoMed, Rosemont, IL), 100 units/ml penicillin, and 100 g/ml streptomycin.…”