Exploring the oxidative mechanism of methotrexate on catalase enzyme: an in vitro study

Methotrexate (MTX) is a well-known anti-metabolite agent recognized for its oxidative effects, particularly in the liver where the enzyme catalase is abundant. This research aimed to clarify the impact of MTX on the behavior of liver catalase. The cytotoxicity of HepG2 cells was assessed across various concentrations of MTX. Following that, the examination focused on the generation of reactive oxygen species (ROS) and the activity of catalase. Furthermore, the kinetic activity of bovine liver catalase (BLC) was examined in the presence of MTX. Finally, the interaction between MTX and the enzyme's protein structure was investigated using docking and dynamic light scattering (DLS) methods. The results indicated a significant decrease in catalase activity and a significant increase in ROS production in HepG2 cells treated with MTX. Although the activity of BLC remained unaffected by MTX directly, molecular docking and DLS techniques revealed MTX binding to BLC, inhibiting its tetramerization. The oxidative effects of MTX were associated with elevated ROS levels in cellular processes, leading to excessive catalase activity and subsequent suicide inactivation. Furthermore, MTX influenced the protein structure of catalase.