title: Translocation of iron from lysosomes to mitochondria during acetaminophen-induced hepatocellular injury: Protection by starch-desferal and minocycline.
abstract: Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and the mitochondrial permeability transition (MPT). Iron is a critical catalyst for ROS formation, and reactive oxygen species (ROS) play an important role in APAP-induced hepatotoxicity. Previous studies show that APAP disrupts lysosomes, which release ferrous iron (Fe(2+)) into the cytosol to trigger the MPT and cell killing. Here, our aim was to investigate whether iron released from lysosomes after APAP is then taken up into mitochondria via the mitochondrial electrogenic Ca(2+), Fe(2+) uniporter (MCFU) to cause mitochondrial dysfunction and cell death. Hepatocytes were isolated from fasted male C57BL/6 mice. Necrotic cell killing was assessed by propidium iodide fluorimetry. Mitochondrial membrane potential (ΔΨ) was visualized by confocal microscopy of rhodamine 123 (Rh123) and tetramethylrhodamine methylester (TMRM). Chelatable Fe(2+) was monitored by quenching of calcein (cytosol) and mitoferrofluor (MFF, mitochondria). ROS generation was monitored by confocal microscopy of MitoSox Red and plate reader fluorimetry of chloromethyldihydrodichlorofluorescein diacetate (cmH2DCF-DA). Administered 1h before APAP (10mM), the lysosomally targeted iron chelator, starch-desferal (1mM), and the MCFU inhibitors, Ru360 (100nM) and minocycline (4µM), decreased cell killing from 83% to 41%, 57% and 53%, respectively, after 10h. Progressive quenching of calcein and MFF began after ~4h, signifying increased cytosolic and mitochondrial chelatable Fe(2+). Mitochondria then depolarized after ~10h. Dipyridyl, a membrane-permeable iron chelator, dequenched calcein and MFF fluorescence after APAP. Starch-desferal, but not Ru360 and minocycline, suppressed cytosolic calcein quenching, whereas starch-desferal, Ru360 and minocycline all suppressed mitochondrial MFF quenching and mitochondrial depolarization. Starch-desferal, Ru360 and minocycline also each decreased ROS formation. Moreover, minocycline 1h after APAP decreased cell killing by half. In conclusion, release of Fe(2+) from lysosomes followed by uptake into mitochondria via MCFU occurs during APAP hepatotoxicity. Mitochondrial iron then catalyzes toxic hydroxyl radical formation, which triggers the MPT and cell killing. The efficacy of minocycline post-treatment shows minocycline as a possible therapeutic agent against APAP hepatotoxicity.
authors.affiliation: Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston, SC 29425, United States; Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States.
authors.forename: Jiangting
authors.initials: J
authors.lastname: Hu
authors.affiliation: Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston, SC 29425, United States; Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States.
authors.forename: Andaleb
authors.initials: A
authors.lastname: Kholmukhamedov
authors.affiliation: Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Lindsey
authors.affiliation: Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States.
authors.forename: Craig C
authors.initials: CC
authors.lastname: Beeson
authors.affiliation: Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, United States.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Jaeschke
authors.affiliation: Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston, SC 29425, United States; Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States; Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, United States; Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, United States; Institute of Theoretical & Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russian Federation. Electronic address: JJLemasters@musc.edu.
authors.forename: John J
authors.initials: JJ
authors.lastname: Lemasters
mesh_terms: D000082:Acetaminophen; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000188:drug therapy / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology*; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D008810:Mice, Inbred C57BL; D008911:Minocycline / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D017975:Ruthenium Compounds / Q000494:pharmacology; D013213:Starch / Q000494:pharmacology
chemical_list: D007502:Iron Chelating Agents; D017382:Reactive Oxygen Species; C112020:Ru 360; D017975:Ruthenium Compounds; D000082:Acetaminophen; D013213:Starch; D007501:Iron; D008911:Minocycline; D003676:Deferoxamine
keywords: APAP; Hepatocytes; Iron; Lysosome; Mitochondria