Longer reported amiodarone use is associated with FLI-defined hepatic steatosis and a nutlin-3a-sensitive lipid phenotype in HepG2 cells

Amiodarone can cause hepatic steatosis, but the relationship between treatment duration and steatosis burden and the cellular pathways involved remain uncertain. We examined duration-related associations among current amiodarone users and explored candidate hepatic lipid-response pathways. We conducted a cross-sectional, survey-weighted analysis of ten National Health and Nutrition Examination Survey cycles from 1999 to 2018. The primary sample comprised 116 current amiodarone users with complete Fatty Liver Index (FLI) data. Age- and sex-adjusted models evaluated associations of reported treatment duration with FLI ≥ 60 and continuous FLI. Network toxicology and enrichment analyses were used to prioritize candidate pathways, and molecular docking with co-crystal ligand redocking was used to generate structural hypotheses. HepG2 cells received 20 μM amiodarone for 24 h. In the combined-treatment groups, β-naphthoflavone or nutlin-3a was added 2 h before amiodarone and remained present during the subsequent 24-h co-incubation. Each additional year of reported amiodarone use was associated with higher odds of FLI ≥ 60 (odds ratio, 1.130; 95% confidence interval, 1.022–1.250; p = 0.0209) and a 1.122-point increase in continuous FLI (95% confidence interval, 0.267–1.977; p = 0.0144). Use for ≥2,190 days versus <365 days was associated with higher odds of FLI ≥ 60 (odds ratio, 5.350; 95% confidence interval, 1.527–18.747; p = 0.0138). Restricted cubic spline analysis identified an overall association without evidence of nonlinearity. The primary association remained significant in multiple sensitivity analyses but was not significant in the smaller fasting-reference FLI and USFLI subsamples. Network analysis identified 220 candidate targets and 16 core targets enriched in xenobiotic- and lipid-metabolism pathways. Docking favored CYP2C19, CYP2C8, and CYP2C9 over CYP1A2 and TP53 and did not support classification of TP53 as a direct amiodarone target. In HepG2 cells, amiodarone increased Oil Red O staining and the p-AKT(Thr308)/AKT ratio but did not significantly alter basal CYP1A2 or TP53 abundance. nutlin-3a co-treatment was associated with lower lipid staining and higher TP53 and CYP1A2 abundance than amiodarone alone, whereas β-naphthoflavone did not significantly reduce lipid staining. Longer reported amiodarone use was associated with higher FLI among current users, while acute exposure produced lipid accumulation and AKT-related signaling in HepG2 cells. The nutlin-3a-sensitive phenotype supports a TP53-responsive component linked to CYP1A2 abundance but does not establish direct binding or causal mediation. Prospective clinical studies and targeted genetic and enzymatic experiments are required.