The Generation of ROS by Exposure to Trihalomethanes Promotes the IκBα/NF-κB/p65 Complex Dissociation in Human Lung Fibroblast

<b>Background:</b> Disinfection by-products used to obtain drinking water, including halomethanes (HMs) such as CH₂Cl₂, CHCl₃, and BrCHCl₂, induce cytotoxicity and hyperproliferation in human lung fibroblasts (MRC-5). Enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) modulate these damages through their biotransformation processes, potentially generating toxic metabolites. However, the role of the oxidative stress response in cellular hyperproliferation, modulated by nuclear factor-kappa B (NF-κB), remains unclear. <b>Methods:</b> In this study, MRC-5 cells were treated with these compounds to evaluate reactive oxygen species (ROS) production, lipid peroxidation, phospho-NF-κB/p65 (Ser536) levels, and the activities of SOD, CAT, and GPx. Additionally, the interactions between HMs and ROS with the IκBα/NF-κB/p65 complex were analyzed using molecular docking. <b>Results:</b> Correlation analysis among biomarkers revealed positive relationships between pro-oxidant damage and antioxidant responses, particularly in cells treated with CH₂Cl₂ and BrCHCl₂. Conversely, negative relationships were observed between ROS levels and NF-κB/p65 levels in cells treated with CH₂Cl₂ and CHCl₃. The estimated relative free energy of binding using thermodynamic integration with the p65 subunit of NF-κB was -3.3 kcal/mol for BrCHCl₂, -3.5 kcal/mol for both CHCl₃ and O₂^•, and -3.6 kcal/mol for H₂O₂. <b>Conclusions:</b> Chloride and bromide atoms were found in close contact with IPT domain residues, particularly in the RHD region involved in DNA binding. Ser281 is located within this domain, facilitating the phosphorylation of this protein. Similarly, both ROS interacted with the IPT domain in the RHD region, with H₂O₂ forming a side-chain oxygen interaction with Leu280 adjacent to the phosphorylation site of p65. However, the negative correlation between ROS and phospho-NF-κB/p65 suggests that steric hindrance by ROS on the C-terminal domain of NF-κB/p65 may play a role in the antioxidant response.