title: Targeting mitochondria with methylene blue protects mice against acetaminophen-induced liver injury.
abstract: Acetaminophen (APAP) overdose is a frequent cause of drug-induced liver injury and the most frequent cause of acute liver failure in the Western world. Previous studies with mouse models have revealed that impairment of mitochondrial respiration is an early event in the pathogenesis, but the exact mechanisms have remained unclear, and therapeutic approaches to specifically target mitochondria have been insufficiently explored. Here, we found that the reactive oxidative metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI), caused the selective inhibition of mitochondrial complex II activity by >90% in both mouse hepatic mitochondria and yeast-derived complexes reconstituted into nanoscale model membranes, as well as the decrease of succinate-driven adenosine triphosphate (ATP) biosynthesis rates. Based on these findings, we hypothesized that methylene blue (MB), a mitochondria-permeant redox-active compound that can act as an alternative electron carrier, protects against APAP-induced hepatocyte injury. We found that MB (<3 µM) readily accepted electrons from NAPQI-altered, succinate-energized complex II and transferred them to cytochrome c, restoring ATP biosynthesis rates. In cultured mouse hepatocytes, MB prevented the mitochondrial permeability transition and loss of intracellular ATP without interfering with APAP bioactivation. In male C57BL/6J mice treated with APAP (450 mg/kg, intraperitoneally [IP]), MB (10 mg/kg, IP, administered 90 minutes post-APAP) protected against hepatotoxicity, whereas mice treated with APAP alone developed massive centrilobular necrosis and increased serum alanine aminotransferase activity. APAP treatment inhibited complex II activity ex vivo, but did not alter the protein expression levels of subunits SdhA or SdhC after 4 hours.
  
  
  CONCLUSIONS
  MB can effectively protect mice against APAP-induced liver injury by bypassing the NAPQI-altered mitochondrial complex II, thus alleviating the cellular energy crisis. Because MB is a clinically used drug, its potential application after APAP overdose in patients should be further explored.
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT.
authors.forename: Kang Kwang
authors.initials: KK
authors.lastname: Lee
authors.forename: Naoki
authors.initials: N
authors.lastname: Imaizumi
authors.forename: Sally R
authors.initials: SR
authors.lastname: Chamberland
authors.forename: Nathan N
authors.initials: NN
authors.lastname: Alder
authors.forename: Urs A
authors.initials: UA
authors.lastname: Boelsterli
mesh_terms: D000082:Acetaminophen / Q000009:adverse effects; D018712:Analgesics, Non-Narcotic / Q000009:adverse effects; D000818:Animals; D016227:Benzoquinones / Q000009:adverse effects; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D004353:Drug Evaluation, Preclinical; D042963:Electron Transport Complex II / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D022781:Hepatocytes / Q000187:drug effects*; D007097:Imines / Q000009:adverse effects; D008099:Liver / Q000187:drug effects / Q000473:pathology; D008297:Male; D008751:Methylene Blue / Q000494:pharmacology / Q000627:therapeutic use*; D008810:Mice, Inbred C57BL; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D009336:Necrosis / Q000517:prevention & control; D030421:Peroxynitrous Acid / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism
chemical_list: D018712:Analgesics, Non-Narcotic; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D007097:Imines; D030421:Peroxynitrous Acid; D000082:Acetaminophen; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; C028473:N-acetyl-4-benzoquinoneimine; D008751:Methylene Blue