Introduction Mitochondria are central to many cellular processes and are dynamic organelles that exist as a network in the form of elongated filaments and respond to the demands of the cell through cycles of fusion (binding of mitochondria) and fission (mitochondrial-fragmentation) which are driven primarily through multiple mitochondrial shaping proteins. Mitochondrial function is intimately associated with their morphology and while mitochondrial dysfunction has been previously correlated with alcohol consumption, there is a paucity of understanding regarding the impact of alcohol on the dynamic balance between fusion/fission and on mitochondrial morphology. The aim of this study was to investigate the impact of alcohol-induced liver damage on mitochondrial morphology, dynamics and to identify the precise mechanisms driving these changes.
Methods Ethanol metabolising-human hepatoma cell lines VL-17A (positive for alcohol-dehydrogenase and CYP2E1) were cultured in the presence of increasing doses of ethanol (EtOH), reflecting real-life alcohol consumption. Cells were cultured with EtOH at 10 mM (safe levels), 50 mM, 250 mM (high levels) and 500 mM (highly toxic levels). Cultures were incubated in presence/absence of EtOH for 24, 48, 72 and 96 h. Post-treatment the levels of mitochondrial shaping proteins including Mitofusin-1 (Mfn-1), Mitofusin-2 (Mfn-2) and Dynamin-related protein-1 (Drp-1) were analysed by detecting protein and mRNA levels. Dynamic changes in the morphology of mitochondria were assessed by confocal microscopy.
Results In the absence of alcohol, we observed no changes in the mitochondrial shaping proteins and no changes in the mitochondrial network over time. At 24 h, cells treated with 50 mM ethanol induced profound modifications in the mitochondrial network with a spot-like presentation which correlated with increased levels of Drp-1. At higher toxic levels of 500 mM, the cells display features of mitochondrial toxicity characterised by fragmentation reflecting the high level of cell death observed with this concentration. This toxicity was associated with reduced expression of Mfn-1 and Mfn-2.
Conclusion For the first time we show that alcohol can profoundly perturb the equilibrium between fusion and fission which directly affects the mitochondrial morphology. This study reveals a novel finding in the pathogenesis of alcohol-induced liver toxicity.
Competing interests None declared.
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