Studying the Toxicity of Trolox on Hepatocytes and Drug‐Induced Liver Injury

ABSTRACT The Vitamin E analogue Trolox (Tx) is a well‐known reference compound utilized in various In Vitro antioxidant assays. In Vivo experimentation of the scavenging and protective potential of Tx is increasing, however, its impact on healthy and stressed models has not been thoroughly examined. The current study evaluates Tx under both In Vitro and In Vivo settings, employing the classical acetaminophen (APAP)‐induced hepatotoxicity model to assess its ability to manage and prevent liver injury. Primary mouse hepatocytes and C57/B6 mice were treated with Tx either alone or before exposure to APAP. Cell modality and viability were evaluated In Vitro, along with In Vivo hepatic functions and histological changes. The mRNA levels of stress response and cell‐death associated genes were analyzed in liver homogenates, along with the levels of NRF2 proteins, a crucial intracellular antioxidant regulator. The conducted experiments revealed that Tx (1 mM) reduced cell viability, triggered apoptosis, and led to DNA leakage in cells, while exacerbating injury when administered as a pretreatment before APAP exposure in mice. This damage was correlated with dose‐dependent hemorrhagic necrosis observed in liver tissue sections and a dose‐dependent increase in serum LDH. Tx affected the mRNA expression of CYP metabolism enzymes, as well as Sult1a1 expression and genes related to NRF2/ARE pathway. Furthermore, NRF2 activity was diminished following pretreatment with Tx doses. Despite its recognized antioxidant properties, Tx induced liver injury in a concentration‐dependent manner in both normal and stressed liver models. Consequently, the use of Tx may pose injury that is evident and requires further investigation across various pathological models.