Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.
Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity.
PEDEGO Research Center and Medical Research Center Oulu, P.O. Box 5000, FIN-90014, University of Oulu, Oulu, Finland; Department of Children and Adolescents, Division of Pediatric Neurology, Oulu University Hospital, P.O. Box 23, FI-90029 OYS, Oulu, Finland. Electronic address: mikakomu@student.oulu.fi.
Tuomas
T
Komulainen
Nuffield Department of Obstetrics and Gynaecology, Women's Centre, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX6 9DU, United Kingdom.
Tiffany
T
Lodge
PEDEGO Research Center and Medical Research Center Oulu, P.O. Box 5000, FIN-90014, University of Oulu, Oulu, Finland; Department of Children and Adolescents, Division of Pediatric Neurology, Oulu University Hospital, P.O. Box 23, FI-90029 OYS, Oulu, Finland.
Reetta
R
Hinttala
PEDEGO Research Center and Medical Research Center Oulu, P.O. Box 5000, FIN-90014, University of Oulu, Oulu, Finland; Department of Children and Adolescents, Division of Pediatric Neurology, Oulu University Hospital, P.O. Box 23, FI-90029 OYS, Oulu, Finland.
Maija
M
Bolszak
Faculty of Medicine, Institute of Biomedicine, Department of Anatomy and Cell Biology, P.O. Box 5000, FI-90014, University of Oulu, Oulu, Finland.
Mika
M
Pietilä
Biocenter Oulu, Faculty of Biochemisty and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, P.O. Box 5400, FI-90014, University of Oulu, Finland.
Peppi
P
Koivunen
Faculty of Medicine, Institute of Biomedicine, Department of Pharmacology and Toxicology, P.O. Box 5000, FI-90014, University of Oulu, Oulu, Finland; Medical Research Center Oulu, P.O. Box 5000, FIN-90014, University of Oulu, Oulu University Hospital, Oulu, Finland.
Jukka
J
Hakkola
Nuffield Department of Obstetrics and Gynaecology, Women's Centre, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX6 9DU, United Kingdom.
Joanna
J
Poulton
Nuffield Department of Obstetrics and Gynaecology, Women's Centre, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX6 9DU, United Kingdom.
Karl J
KJ
Morten
PEDEGO Research Center and Medical Research Center Oulu, P.O. Box 5000, FIN-90014, University of Oulu, Oulu, Finland; Department of Children and Adolescents, Division of Pediatric Neurology, Oulu University Hospital, P.O. Box 23, FI-90029 OYS, Oulu, Finland.
Johanna
J
Uusimaa
D000255:Adenosine Triphosphate / Q000378:metabolism; D000927:Anticonvulsants / Q000633:toxicity*; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D019069:Cell Respiration / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D045222:Electron Transport Chain Complex Proteins / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D028361:Mitochondrial Diseases / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D018384:Oxidative Stress / Q000187:drug effects; D019289:Pyruvic Acid / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014635:Valproic Acid / Q000633:toxicity*; D000099156:Superoxide Dismutase 2
D000255:Adenosine Triphosphate; D000927:Anticonvulsants; D045222:Electron Transport Chain Complex Proteins; D005690:Galactose; D005947:Glucose; D019289:Pyruvic Acid; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D014635:Valproic Acid; D000099156:Superoxide Dismutase 2
HepG2; Hepatotoxicity; Mitochondrial respiration; Mitochondrial toxicity; Reactive oxygen species; Sodium valproate