title: ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes.
abstract: OBJECTIVE
  Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress.
  
  
  METHODS
  Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH).
  
  
  RESULTS
  ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity.
  
  
  CONCLUSIONS
  We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.
authors.affiliation: Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Xin-ming
authors.initials: XM
authors.lastname: Qi
authors.forename: Ling-ling
authors.initials: LL
authors.lastname: Miao
authors.forename: Yan
authors.initials: Y
authors.lastname: Cai
authors.forename: Li-kun
authors.initials: LK
authors.lastname: Gong
authors.forename: Jin
authors.initials: J
authors.lastname: Ren
mesh_terms: D000818:Animals; D044182:Benzylisoquinolines / Q000494:pharmacology*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis*; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D044182:Benzylisoquinolines; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; C009438:tetrandrine; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1
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