Chronic co-exposure to BPA and alcohol synergizes hepatotoxicity via dysregulation of glycerolipid metabolism and ceramide/HtrA2-driven apoptosis in HepG2 cells.

Bisphenol A (BPA) and alcohol (ALC) are recognized hepatotoxicants to which humans are chronically exposed through environmental sources, dietary habits, and preferences for alcoholic beverages. However, there is currently a lack of conclusive evidence concerning the synergistic hepatotoxic effects associated with co-exposure. This study provides mechanistic insights into BPA and ALC co-exposure-induced subcellular changes mimicking hepatotoxicity using acute and chronic 2D models and 3D HepG2/LX-2 spheroids. Results demonstrated that co-exposure amplified oxidative stress, DNA damage, and apoptosis, while promoting lipid droplet accumulation, thereby manifesting synergistic hepatic lipotoxicity. These findings were corroborated in a physiologically relevant 3D HepG2/LX-2 co-culture spheroid model, where co-exposure induced significantly greater cytotoxicity than single-agent treatments. Mechanistically, chronic co-exposure specifically dysregulated LIPIN-mediated glycerolipid homeostasis by synergistically inducing the expression of LIPIN-2 and LIPIN-3. Furthermore, integrated transcriptomic and lipidomic analyses revealed that the ceramide metabolic pathway is uniquely involved in triggering high-temperature requirement A2 (HtrA2)-mediated apoptosis and oncogenic signaling under conditions of chronic co-exposure to BPA and ALC. Crucially, in vitro oncogenic transformation assays identified the co-exposure as a tumor promoter rather than an initiator, significantly enhancing colony formation following 3-MCA initiation. TIMER database analysis further associated aberrant HTRA2 expression with hepatocellular carcinoma (HCC) development and poor prognosis, which are potentially mediated through downregulation of the detoxification enzyme UDP-glucuronosyltransferase family 2 member B10 (UGT2B10), a factor critical for normal liver cell function. Overall, this study establishes glycerolipid metabolism and HtrA2 as early biomarkers of synergistic hepatic injury and further hepatocarcinogenesis, highlighting critical implications for food safety and risk assessment of chemical-behavioral co-exposures.