Dibutyl phthalate exposure induced hepatotoxicity via VDAC2 mediated mitochondria-dependent ferroptosis.

Widely used plasticiser, dibutyl phthalate (DBP), poses a serious threat to ecosystems and human health. Although evidence of the toxic effects of DBP is growing, the specific mechanisms underlying its hepatotoxicity is still unclear. Previous studies have confirmed that CH-223191 (AhR inhibitor) could not completely alleviate DBP-induced ferroptosis and the consequent hepatotoxicity, suggesting the existence of additional pathways. Thus, this study aims to comprehensively investigate the mechanisms underlying DBP-induced hepatotoxicity through computational modelling, in vivo assays in zebrafish, and in vitro assays in human hepatocytes and Kupffer cells. Firstly, network toxicology, molecular docking and western blotting have preliminarily confirmed the activating effect of DBP on the voltage-dependent anion channel 2 (VDAC2). Both in vitro and in vivo assays, treatment with the VDAC2 inhibitor WEHI-9625 or vdac2 siRNA attenuated the accumulation of mitochondrial superoxide and lipid peroxides, the opening of the mPTP, and iron overload, indicating that VDAC2 acts as a crucial switch for mitochondria-dependent ferroptosis. Moreover, combined treatment with CH223191 largely reversed these abnormalities, as well as high-mobility group box 1 (HMGB1) release, macrophage activation and the upregulated protein expression of toll-like receptor 4 (TLR4) and NF-κB. These results reveal that DBP directly targets VDAC2 and AhR, synergistically inducing ferroptosis and inflammation, ultimately leading to hepatotoxicity. In summary, this study provides an important theoretical foundation for enhancing the toxicological assessment of phthalate compounds.