Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.
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.
Merrill
Christine L
CL
Department of Microbiology, Pathology and Parasitology, North Carolina State University, Raleigh 27606, USA. clm70753@gsk.com
Ni
Hong
H
Yoon
Lawrence W
LW
Tirmenstein
Mark A
MA
Narayanan
Padma
P
Benavides
Gina R
GR
Easton
M J
MJ
Creech
Donald R
DR
Hu
Catherine X
CX
McFarland
David C
DC
Hahn
Laura M
LM
Thomas
Heath C
HC
Morgan
Kevin T
KT
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
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