RUNX3 Methylation: An Epigenetic Biomarker for Early Liver Damage Induced by Co-Exposure to Aflatoxin B1 and Hepatitis B Virus

Aflatoxin B1 (AFB1), a well-established hepatic carcinogen, has limited research on early-stage epigenetic biomarkers for aflatoxin-induced liver damage. In this study, we investigated 168 unpackaged peanut oil (UPP) consumers to evaluate associations among AFB1 exposure, HBV infection, <i>RUNX3</i> methylation, and liver function. Our findings indicated an average daily AFB1 intake of 3.14 ng/kg·bw/day from UPP oil consumption. The high AFB1 exposure group exhibited significantly elevated gamma-glutamyl transferase (GGT) levels compared with the low AFB1 exposure group (<i>p</i> = 0.030). AFB1 exposure was negatively correlated with methylation status at the 2nd, 8th, and 9th CpG sites of <i>RUNX3</i> (r_s = -0.196, -0.192, -0.181, <i>p</i> = 0.021, 0.024, 0.036). Furthermore, methylation at the 8th and 9th CpG sites positively correlated with GGT (r_s = 0.206, 0.203, <i>p</i> = 0.019, 0.024). HBV infection significantly influenced <i>RUNX3</i> methylation, with the HBsAg⁺ group exhibiting 16.25% lower methylation (<i>p</i> < 0.05). Stratified analysis by HBV and AFB1 revealed that in the low AFB1 exposure subgroup, <i>RUNX3</i> methylation in the HBsAg⁺ group exhibited a significant 26.38% reduction compared with the HBsAg^- group. These results indicated that AFB1 and HBV independently and synergistically promote site-specific <i>RUNX3</i> hypomethylation. Our results implicated <i>RUNX3</i> methylation as a critical mediator in HBV-AFB1 co-exposure hepatotoxicity, potentially serving as a novel epigenetic biomarker for early liver damage detection.