Identification of Halophenol-Deoxyguanosine Adducts in SV-HUC-1 Cells Exposed to Halobenzoquinones via the Neutral Loss and Information-Dependent Acquisition Strategy

Halobenzoquinones (HBQs), emerging disinfection byproducts (DBPs) in drinking water, were prioritized as potential bladder carcinogens. This study aimed to elucidate the interaction between HBQs and DNA, which may represent a critical initial molecular event in the adverse outcome pathway toward carcinogenesis. By combining high- and low-resolution mass spectrometry (MS), we developed a highly selective and sensitive analytical strategy integrating pentose neutral loss and information-dependent acquisition to characterize trace-level HBQ-DNA adducts in complex biological matrices. Deoxyguanosine (dG) was identified as the primary target of HBQs, and in vitro reaction revealed 16 mono-, di-, and trihalogenated 1:1 and 2:1 (mol:mol) adducts, including HBQ-dG, HHQ-dG, HPH-dG, and HBQ-HPH-dG (HHQ and HPH denoted halohydroquinone and halophenol, respectively). The stepwise formation involved HBQ-HHQ interconversion, cyclization of OH or halogen in HBQ with N1 and N2 in dG, and ether bond formation between HBQ and HPH. Four adducts were identified in reaction with calf thymus DNA, and in particular, 1-Cl-3-OH-PH-6,5-N1,N2-dG was identified in human urothelial cells SV-HUC-1. Molecular docking and spectroscopic analyses pinpointed intercalation and groove binding as the dominant noncovalent interactions between HBQs and DNA. This study provided the first evidence of intracellular HBQ-DNA adduct formation, linking the genotoxicity and carcinogenic relevance of HBQ DBPs.