Lysosomal iron mobilization and induction of the mitochondrial permeability transition in acetaminophen-induced toxicity to mouse hepatocytes.
Acetaminophen induces the mitochondrial permeability transition (MPT) in hepatocytes. Reactive oxygen species (ROS) trigger the MPT and play an important role in AAP-induced hepatocellular injury. Because iron is a catalyst for ROS formation, our aim was to investigate the role of chelatable iron in MPT-dependent acetaminophen toxicity to mouse hepatocytes. Hepatocytes were isolated from fasted male C3Heb/FeJ mice. Necrotic cell killing was determined by propidium iodide fluorometry. Mitochondrial membrane potential was visualized by confocal microscopy of tetramethylrhodamine methylester. Chelatable ferrous ion was monitored by calcein quenching, and 70 kDa rhodamine-dextran was used to visualize lysosomes. Cell killing after acetaminophen (10mM) was delayed and decreased by more than half after 6 h by 1mM desferal or 1mM starch-desferal. In a cell-free system, ferrous but not ferric iron quenched calcein fluorescence, an effect reversed by dipyridyl, a membrane-permeable iron chelator. In hepatocytes loaded with calcein, intracellular calcein fluorescence decreased progressively beginning about 4 h after acetaminophen. Mitochondria then depolarized after about 6 h. Dipyridyl (20mM) dequenched calcein fluorescence. Desferal and starch-desferal conjugate prevented acetaminophen-induced calcein quenching and mitochondrial depolarization. As calcein fluorescence became quenched, lysosomes disappeared, consistent with release of iron from ruptured lysosomes. In conclusion, an increase of cytosolic chelatable ferrous iron occurs during acetaminophen hepatotoxicity, which triggers the MPT and cell killing. Disrupted lysosomes are the likely source of iron, and chelation of this iron decreases acetaminophen toxicity to hepatocytes.
Kon
Kazuyoshi
K
Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Kim
Jae-Sung
JS
Uchiyama
Akira
A
Jaeschke
Hartmut
H
Lemasters
John J
JJ
D000082:Acetaminophen / Q000633:toxicity*; D000818:Animals; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D018613:Microscopy, Confocal; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects*; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species / Q000378:metabolism
D005452:Fluoresceins; D007502:Iron Chelating Agents; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D000082:Acetaminophen; D007501:Iron; C007740:fluorexon