Haloacetonitriles Induce Structure-Related Cellular Toxicity Through Distinct Proteome Thiol Reaction Mechanisms

Haloacetonitriles (HANs) are a class of toxic drinking water disinfection byproducts (DBPs). However, the toxicity mechanisms of HANs remain unclear. We herein investigated the structure-related in vitro toxicity of 6 representative HANs by utilizing complementary bioanalytical approaches. Dibromoacetonitrile (DBAN) displayed strong cytotoxicity and Nrf2 oxidative stress responses, followed by monohalogenated HANs (monoHANs) while other polyhalogenated HANs (polyHANs) exhibited little toxicity. Activity based protein profiling (ABPP) revealed that toxic HANs adduct to human proteome thiols, supporting thiol reactivity as the primary toxicity mechanism for HANs. By using glutathione (GSH) as a thiol surrogate, monoHANs reacted with GSH via S_N2 while polyHANs reacted through ultrafast addition reactions. In contrast, DBAN generated an unexpected fully debrominated product and glutathione disulfide (GSSG). The unique reaction of DBAN with GSH was found to be mediated by radicals which was supported by electron paramagnetic resonance (EPR) spectroscopy and by radical trapping reagent reaction quenching. Shotgun proteomics further revealed that monoHANs and DBAN adducted to proteome thiols in live cells forming dehalogenated adducts. Multiple antioxidant proteins, SOD1, CSTB, and GAPDH, were adducted by toxic HANs at specific cysteine residues. This study highlights the structurally selective toxicity of HANs in human cells, which are attributed to their distinct reactions with proteome thiols.