title: Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.
abstract: Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.
authors.affiliation: Department of Microbiology, Pathology and Parasitology, North Carolina State University, Raleigh 27606, USA. clm70753@gsk.com
authors.forename: Christine L
authors.initials: CL
authors.lastname: Merrill
authors.forename: Hong
authors.initials: H
authors.lastname: Ni
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Yoon
authors.forename: Mark A
authors.initials: MA
authors.lastname: Tirmenstein
authors.forename: Padma
authors.initials: P
authors.lastname: Narayanan
authors.forename: Gina R
authors.initials: GR
authors.lastname: Benavides
authors.forename: M J
authors.initials: MJ
authors.lastname: Easton
authors.forename: Donald R
authors.initials: DR
authors.lastname: Creech
authors.forename: Catherine X
authors.initials: CX
authors.lastname: Hu
authors.forename: David C
authors.initials: DC
authors.lastname: McFarland
authors.forename: Laura M
authors.initials: LM
authors.lastname: Hahn
authors.forename: Heath C
authors.initials: HC
authors.lastname: Thomas
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Morgan
mesh_terms: D006528:Carcinoma, Hepatocellular; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004798:Enzymes / Q000235:genetics / Q000378:metabolism; D004852:Epoxy Compounds / Q000633:toxicity*; D005786:Gene Expression Regulation; D005978:Glutathione / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007004:Hypoglycemic Agents / Q000633:toxicity*; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D004798:Enzymes; D004852:Epoxy Compounds; D007004:Hypoglycemic Agents; D012333:RNA, Messenger; C085214:gamma-glutamyl-cysteinyl-glutamic acid; D004247:DNA; C516124:TXNRD1 protein, human; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione; C054207:etomoxir