Comparative cytotoxicity of 3,5-dichloro-2-hydroxybenzoic acid and 3,5-dichloro-4-hydroxybenzoic acid and underlying mechanisms: Isomer-specific cytotoxicity of phenolic disinfection byproducts

Phenolic halogenated DBPs are an emerging class of highly toxic DBPs in drinking water. However, the underlying mechanisms for isomer-specific cytotoxicity of phenolic disinfection byproducts are still largely veiled. In this work, comparative cytotoxicity of 3,5-dichloro-2-hydroxybenzoic acid (3,5-DC-2-HBA) and 3,5-dichloro-4-hydroxybenzoic acid (3,5-DC-4-HBA) in diverse mammalian cell lines was investigated, SOD activity in Chinese hamster ovary cells treated by 3,5-DC-2-HBA and 3,5-DC-4-HBA was compared, and their binding interactions with Cu/Zn-superoxide dismutase (Cu/Zn-SOD) were analyzed. Results indicated that 3,5-DC-2-HBA commonly presented significantly higher cytotoxicity compared to 3,5-DC-4-HBA. Exposure to 3,5-DC-2-HBA and 3,5-DC-4-HBA caused a dose-dependent increase in intracellular SOD activity, with the trend paralleling the cytotoxicity of the two isomeric compounds. In addition, this rank order was consistently observed in the conformational changes, secondary structure alterations, as well as enzymatic activity modulation of Cu/Zn-SOD induced by these isomeric compounds. Molecular docking simulations further elucidated the binding mechanisms, demonstrating that van der Waals interactions and hydrogen bonding energies predominantly drive complex formation between these two halogenated phenolic DBPs and Cu/Zn-SOD. Notably, the interaction with Cu/Zn-SOD may be generally applied to explain the isomeric structure-derived cytotoxic pattern of various groups of phenolic DBPs. This systematic investigation advances our mechanistic understanding of isomer-specific cytotoxicity in phenolic DBPs, providing valuable insights for risk assessment and mitigation strategies in drinking water treatment processes.