Methadone and morphine are two widely prescribed opioids for pain management and opioid substitution therapy. While both are pharmacologically effective, their long-term use may induce hepatotoxic effects. This study aimed to evaluate the hepatotoxic potential of methadone and morphine through an integrative approach combining experimental and bioinformatics analyses. Male Norway rats were administered methadone and morphine at doses of 3 and 7 mg/kg for 30 days. Liver tissues were analyzed using qRT-PCR to assess the expression of key metabolic genes (Cyp2b3, Cyp1a2, Cyp3a2, Cyp7a1, and Ugt2b), and serum liver enzyme levels (SGOT and SGPT) were measured as markers of liver injury. To further elucidate the molecular mechanisms underlying drug-induced liver injury, RNA sequencing (RNA-seq) data were analyzed to identify differentially expressed genes and enriched biological pathways. The bioinformatics data were sourced from the Gene Expression Omnibus (GEO) database with the accession number GSE250058. Bioinformatics analysis using DESeq2 revealed significant alterations in gene expression profiles, especially within immune regulation, oxidative stress, and metabolic detoxification pathways. KEGG and GO enrichment analyses highlighted the involvement of apoptosis, autophagy, and inflammation-related processes. These findings were supported by observed elevations in liver weight and liver enzymes, particularly in the high-dose methadone group. Overall, our results demonstrate that high-dose methadone induces more severe hepatic metabolic stress and gene dys regulation compared to morphine, indicating its greater hepatotoxic potential. This integrative study underscores the importance of combining experimental data with transcriptomic analyses to enhance our understanding of opioid-induced liver injury and inform future therapeutic strategies.
