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Published on: February 7, 2018
Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically
Christine L Merrill1, Hong Ni, Lawrence W Yoon
1Department of Microbiology, Pathology and Parasitology, North Carolina State University, Raleigh 27606, USA. clm70753@gsk.com
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.
Insights
Etomoxir, a carnitine palmitoyltransferase-1 inhibitor, causes oxidative stress and impairs mitochondrial energy metabolism. This study reveals etomoxir-induced changes in redox and cell cycle gene expression, confirming its toxic effects.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Carnitine palmitoyltransferase-1 (CPT-1) inhibitors like etomoxir (ET) are effective antidiabetic agents.
- Limited data exists on the toxic effects of CPT-1 inhibitors.
Purpose of the Study:
- To investigate the toxic effects of etomoxir (ET) on HepG2 cells.
- To analyze etomoxir-induced changes in gene expression and cellular metabolism.
Main Methods:
- HepG2 cells were treated with 1 mM etomoxir for 6 hours.
- Microarray analysis and real-time quantitative PCR (RT-PCR) were used to measure mRNA expression.
- Levels of reduced glutathione (GSH), oxidized glutathione (GSSG), mitochondrial membrane potential (MMP), and ATP were assessed.
Main Results:
- Etomoxir modulated the expression of redox and cell cycle-related mRNAs, including upregulation of HO1, OGG1, GSR, CDKN1, and SOD2, and downregulation of CYP1A1 and HSPA1A.
- RT-PCR confirmed changes in CYP1A1, HO1, GSR, CDKN1, GCLM, TXNRD1, and TOP2A.
- Etomoxir induced oxidative stress, evidenced by decreased GSH, GSH/GSSG ratio, MMP, and ATP levels, and increased GSSG and superoxide generation.
Conclusions:
- Etomoxir treatment leads to significant alterations in gene expression related to redox balance and cell cycle control.
- The study provides the first evidence of etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism.
