Metabolic Signature of Ethanol-Induced Hepatotoxicity in HepaRG Cells by Liquid Chromatography–Mass Spectrometry-Based Untargeted Metabolomics

Alcoholic
liver disease is highly prevalent but poorly identified
and characterized, leading to knowledge gaps, which impairs early
diagnosis. Excessive alcohol consumption is known to alter lipid metabolism,
followed by progressive intracellular lipid accumulation, resulting
in alcoholic fatty liver disease. In this study, HepaRG cells were
exposed to ethanol at IC10 and 1/10 IC10 for
24 and 48 h. Metabolic alterations were investigated intra- and extracellularly
with liquid chromatography–high-resolution mass spectrometry.
Ion mobility was added as an extra separation dimension for untargeted
lipidomics to improve annotation confidence. Distinctive patterns
between exposed and control cells were consistently observed, with
intracellular upregulation of di- and triglycerides, downregulation
of phosphatidylcholines and phosphatidylethanolamines, sphingomyelins,
and S-adenosylmethionine, among others. Several intracellular metabolic
patterns could be related to changes in the extracellular environment,
such as increased intracellular hydrolysis of sphingomyelins, leading
to increased phosphorylcholine secretion. Carnitines showed alterations
depending on the size of their carbon chain, which highlights the
interplay between β-oxidation in mitochondria and peroxisomes.
Potential new biomarkers of ethanol-induced hepatotoxicity have been
observed, such as ceramides with a sphingadienine backbone, octanoylcarnitine,
creatine, acetylcholine, and ethoxylated phosphorylcholine. The combination
of the metabolic fingerprint and footprint enabled a comprehensive
investigation of the pathophysiology behind ethanol-induced hepatotoxicity.