Translocation of iron from lysosomes into mitochondria is a ... : Hepatology (original) (raw)

Liver Biology/Pathobiology

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

Uchiyama, Akira1,2; Kim, Jae-Sung3; Kon, Kazuyoshi2; Jaeschke, Hartmut4; Ikejima, Kenichi2; Watanabe, Sumio2; Lemasters, John J.1,5*

1_Departments of Pharmaceutical & Biomedical Sciences, Medical University of South Carolina, Charleston, SC_

2_Department of Gastroenterology, Juntendo University School of Medicine, Tokyo, Japan_

3_Department of Surgery, University of Florida, Gainesville, FL_

4_Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS_

5_Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC_

* Address reprint requests to: Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology, Medical University of South Carolina, 280 Calhoun Street, MSC 140, Charleston, SC 29425

Email:[email protected]

Received 18 November 2007; Accepted 18 June 2008

Published online in Wiley InterScience (www.interscience.wiley.com).

Supported by National Institutes of Health; Grant Numbers: DK070195 DK073336 DK37034 C06 RR015455.

Potential conflict of interest: Nothing to report.

fax: 843-792-1617.

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 starch-deferoxamine (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 μM 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 μM), 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.)

Abbreviations: cmDCF, chloromethyldichlorofluorescein; cmH2DCF-DA, chloromethyldihydrodichlorofluorescein diacetate; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; MPT, mitochondrial permeability transition; O2•−, superoxide; OH•, hydroxyl radical; PI, propidium iodide; ROS, reactive oxygen species; t-BuOOH, tert-butylhydroperoxide; TMRM, tetramethylrhodamine methylester.