Acetaminophen-induced acute liver injury abrogates the cytosine methylation pathway.

PURPOSE: Acetaminophen overdose is a leading cause of drug-induced liver injury (DILI) and acute liver failure. Traditional markers such as AST, ALT, and ALP detect damage only after significant hepatocellular injury. This study aimed to identify early epigenetic events, focusing on microRNA (miRNA) regulation of ten-eleven translocation (TET) enzymes, and to evaluate their potential as biomarkers for early detection of DILI. METHODS: Next-generation miRNA sequencing was performed in HepG2 cells exposed to acetaminophen. Bioinformatic analysis identified target pathways and predicted miRNA-TET interactions. Validation was conducted in vitro (HepG2 cells) and in vivo (C57BL/6J male mice) using gene expression assays and 5 methyl cytosine profiling. RESULTS: Forty-four miRNAs were significantly upregulated following acetaminophen treatment. These miRNAs were enriched in pathways including Akt, fatty acid metabolism, ERK, and mTOR signaling. A subset directly targeted TET1 and TET2, resulting in their downregulation in both HepG2 cells and mouse liver tissue. This suppression correlated with increased global 5-methylcytosine (5mC) levels, indicating enhanced DNA methylation. Importantly, these epigenetic changes occurred at early stages of acetaminophen exposure, preceding overt necrosis. CONCLUSION: This study demonstrates that miRNA-mediated downregulation of TET enzymes and the resulting accumulation of 5mC represent early molecular events in acetaminophen-induced liver injury. These findings highlight the potential of miRNAs and DNA methylation changes as early biomarkers of DILI and suggest novel therapeutic targets for mitigating acetaminophen hepatotoxicity.