title: Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway.
abstract: BACKGROUND
  Ketamine is widely used as an i.v. anaesthetic agent and as a drug of abuse. Hepatocytes contribute to the metabolism of endogenous and exogenous substances. This study evaluated the toxic effects of S-(+)-ketamine and possible mechanisms using human hepatoma HepG2 cells as the experimental model.
  
  
  METHODS
  HepG2 cells were exposed to S-(+)-ketamine. Cell viability and the release of lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GPT) were measured to determine the toxicity of S-(+)-ketamine to HepG2 cells. Cell morphology, DNA fragmentation, and apoptotic cells were analysed to evaluate the mechanism of S-(+)-ketamine-induced cell death. Amounts of Bax, an apoptotic protein, and cytochrome c in the cytoplasm or mitochondria were quantified by immunoblotting. Cellular adenosine triphosphate levels were analysed using a bioluminescence assay. Caspases-3, -9, and -6 were measured fluorometrically.
  
  
  RESULTS
  Exposure of HepG2 cells to S-(+)-ketamine increased the release of LDH and GPT, but decreased cell viability (all P<0.01). S-(+)-Ketamine time-dependently caused shrinkage of HepG2 cells. Exposure to S-(+)-ketamine led to significant DNA fragmentation and cell apoptosis (P=0.003 and 0.002). S-(+)-Ketamine increased translocation of Bax from the cytoplasm to mitochondria, but decreased the mitochondrial membrane potential and cellular adenosine triphosphate levels (all P<0.01). Sequentially, cytosolic cytochrome c levels and activities of caspases-9, -3, and -6 were augmented after S-(+)-ketamine administration (all P<0.001). Z-VEID-FMK, an inhibitor of caspase-6, alleviated the S-(+)-ketamine-induced augmentation of caspase-6 activity, DNA fragmentation, and cell apoptosis (all P<0.001).
  
  
  CONCLUSIONS
  This study shows that S-(+)-ketamine can induce apoptotic insults to human HepG2 cells via a Bax-mitochondria-caspase protease pathway. Thus, we suggest that S-(+)-ketamine at a clinically relevant or an abused concentration may induce liver dysfunction possibly due to its toxicity to hepatocytes.
authors.affiliation: Department of Pediatrics, Cathay General Hospital, Taipei, Taiwan, Republic of China.
authors.forename: S-T
authors.initials: ST
authors.lastname: Lee
authors.forename: T-T
authors.initials: TT
authors.lastname: Wu
authors.forename: P-Y
authors.initials: PY
authors.lastname: Yu
authors.forename: R-M
authors.initials: RM
authors.lastname: Chen
mesh_terms: D000778:Anesthetics, Dissociative / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007649:Ketamine / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000778:Anesthetics, Dissociative; C494826:BAX protein, human; D051028:bcl-2-Associated X Protein; D007649:Ketamine; D010447:Peptide Hydrolases; D020169:Caspases