Protective effect of 2-hydroxyestrone and 2-hydroxyestradiol against chemically induced hepatotoxicity in vitro and in vivo.

Ferroptosis is a form of regulated cell death closely associated with glutathione depletion and accumulation of reactive lipid peroxides. In this study, we seek to determine whether 2-hydroxyestrone (2-OH-E₁) and 2-hydroxyestradiol (2-OH-E₂), 2 major metabolites of endogenous estrone (E₁) and 17β-estradiol (E₂) formed by cytochrome P450 in the liver, can protect against erastin- and RSL3-induced ferroptosis in hepatoma cells (H-4-II-E and HuH-7) in vitro and acetaminophen-induced mouse liver injury in vivo. We find that 2-OH-E₁ and 2-OH-E₂ can protect, in a dose-dependent manner, H-4-II-E hepatoma cells against erastin/RSL3-induced ferroptosis. A similar protective effect of 2-OH-E₁ and 2-OH-E₂ against erastin- and RSL3-induced ferroptosis is also observed in HuH-7 hepatoma cells. These 2 estrogen metabolites can strongly abrogate erastin- and RSL3-induced accumulation of cellular NO, reactive oxygen species (ROS), and lipid-ROS. Mechanistically, 2-OH-E₁ and 2-OH-E₂ protect cells against chemically induced ferroptosis by binding to cellular protein disulfide isomerase and then inhibiting its catalytic activity and reducing protein disulfide isomerase-mediated activation (dimerization) of inducible nitric oxide synthase, abrogating cellular NO, ROS, and lipid-ROS accumulation. Animal studies show that 2-OH-E₁ and 2-OH-E₂ also exhibit strong protection against acetaminophen-induced liver injury in mice. Interestingly, although E₁ and E₂ have a very weak protective effect in cultured hepatoma cells, they exhibit a similarly strong protective effect as 2-OH-E₁ and 2-OH-E₂ in vivo, suggesting that the metabolic conversion of E₁ and E₂ to 2-OH-E₁ and 2-OH-E₂ contributes importantly to their hepatoprotective effect. This study reveals that 2-OH-E₁ and 2-OH-E₂ are important endogenous factors for protection against chemically induced liver injury in vivo. SIGNIFICANCE STATEMENT: Ferroptosis is an iron-dependent and lipid reactive oxygen species-dependent form of regulated cell death. Recent evidence has shown that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for ferroptosis protection. This study shows that 2-hydroxyestrone and 2-hydroxyestradiol are 2 inhibitors of PDI that can strongly protect against chemically induced ferroptotic hepatocyte death in vitro and in vivo. This work supports a PDI-mediated, estrogen receptor-independent mechanism of hepatocyte protection by 2-hydroxyestrone and 2-hydroxyestradiol.