Comparative Analysis of Genotoxic Modes of Action and Underlying Molecular Mechanisms of Four Classes of Unregulated DBPs in Human Uroepithelial (SV-HUC-1) Cells

Abstract Increasing evidence suggests that the contribution of unregulated disinfection by-products (UR-DBPs) to the overall toxicity of disinfected water is underestimated. However, the molecular mechanisms by which they promote bladder carcinogenesis remain largely unexplored. Using the nontumorigenic human uroepithelial cell line SV-HUC-1, we sought to elucidate the potential genotoxic mechanisms underlying the bladder cancer risk associated with four classes of typical UR-DBPs: haloacetonitriles (HANs), haloacetamides (HAMs), halobenzoquinones (HBQs), and halobenzoquinone imines (HQCs). Genotoxicity was evaluated by the SOS/umu assay and the micronucleus assay. Only three of the eight UR-DBPs gave positive SOS/umu responses. However, all tested UR-DBPs showed clear cytotoxicity and micronucleus effects, with a consistent toxicity order: HANs > HAMs > HBQs and HQCs. Principal component analysis (PCA) of transcriptomic data indicated that the eight UR-DBPs may act through two distinct modes of action (MOAs). Gene set variation analysis (GSVA) further revealed that HANs and HAMs activated DNA repair pathways, including base excision repair (BER) and nonhomologous end-joining for double-strand break repair (DSBR-NHEJ), while HBQs and HQCs suppressed DNA damage repair pathways or the regulatory pathways involved in nucleotide excision repair (NER), mismatch repair (MMR), and double-strand break repair (DSBR). Western blotting confirmed that HBQs and HQCs significantly downregulated proteins critical for DNA damage repair regulation. This is possibly attributable to their stronger protein-binding capacities relative to other UR-DBPs. Collectively, these findings argue that the impairment of DNA repair regulation constitutes a distinct genotoxic mechanism that should be integrated into DBP risk evaluation alongside direct DNA damage.