Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury.
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 microM 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 microM), bafilomycin enhanced hydroperoxide generation, leading to mitochondrial depolarization and subsequent cell death.


CONCLUSIONS
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.
Department of Pharmaceutical & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA.
Akira
A
Uchiyama
Jae-Sung
JS
Kim
Kazuyoshi
K
Kon
Hartmut
H
Jaeschke
Kenichi
K
Ikejima
Sumio
S
Watanabe
John J
JJ
Lemasters
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000378:metabolism / Q000494:pharmacology*; D019190:Iron Overload / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000293:injuries* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D018613:Microscopy, Confocal; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D007502:Iron Chelating Agents; D007501:Iron