Acetaldehyde impairs mitochondrial glutathione transport in HepG2 cells through endoplasmic reticulum stress.
OBJECTIVE
Ethanol impairs the mitochondrial transport of reduced glutathione (GSH), resulting in lower mitochondrial GSH (mGSH) levels. Our purpose was to evaluate the role of acetaldehyde on the regulation of mGSH in HepG2 cells.


METHODS
mGSH levels and transport, mitochondrial membrane microviscosity, and lipid composition were determined in mitochondria isolated from acetaldehyde-treated HepG2 cells.


RESULTS
The major ultrastructural changes of acetaldehyde-treated HepG2 cells included cytoplasmic lipid droplets and appearance of swollen mitochondria. Acetaldehyde depleted the mGSH pool size in a time- and dose-dependent fashion with spared cytosol GSH levels. Kinetics of GSH transport into isolated mitochondria from HepG2 cells showed 2 saturable, adenosine triphosphate-stimulated, high- and low-affinity components. Treatment with acetaldehyde increased the Michaelis constant for the high- and low-affinity components, with a greater impact on the former. These changes were due to increased mitochondrial microviscosity by enhanced cholesterol deposition because preincubation with the fluidizing agent, 2-(2-methoxyethoxy) ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, normalized the initial transport rate of GSH into isolated mitochondria. Isolated mitochondria from rat liver enriched in free cholesterol reproduced the disturbing effects of acetaldehyde on GSH transport. The acetaldehyde-stimulated mitochondrial cholesterol content was preceded by increased levels of endoplasmic reticulum (ER)-responsive gene GADD153 and transcription factor sterol regulatory element-binding protein 1 and mimicked by the ER stress-inducing agents tunicamycin and homocysteine. Finally, the mGSH depletion induced by acetaldehyde sensitized HepG2 cells to tumor necrosis factor (TNF)-alpha-induced apoptosis that was prevented by cyclosporin A, GSH ethyl ester, and lovastatin.


CONCLUSIONS
Acetaldehyde sensitizes HepG2 cells to TNF-alpha by impairing mGSH transport through an ER stress-mediated increase in cholesterol.
Lluis
Josep M
JM
Liver Unit, Institut of Malalties Digestives, Hospital Clinic i Provincial, IDIBAPS, Department of Experimental Pathology, IIBB, Consejo Superior Investigaciones Científicas, Barcelona, Spain.
Colell
Anna
A
García-Ruiz
Carmen
C
Kaplowitz
Neil
N
Fernández-Checa
José C
JC
D000079:Acetaldehyde / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004221:Disulfiram / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008560:Membrane Fluidity / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D013312:Stress, Physiological / Q000378:metabolism*; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D022762:CCAAT-Enhancer-Binding Proteins; C497249:DDIT3 protein, human; D004268:DNA-Binding Proteins; D005227:Fatty Acids; D010743:Phospholipids; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051742:Transcription Factor CHOP; D002784:Cholesterol; D005978:Glutathione; D000079:Acetaldehyde; D004221:Disulfiram