Removal, transformation and toxicity changes of halobenzoquinones by digestive enzymes

Halobenzoquinones (HBQs) are an emerging class of unregulated disinfection byproducts with high toxicity and resistance to conventional treatment, yet their fate during digestion remains unclear. Here, we systematically investigated the removal, transformation and toxicity change of HBQs mediated by digestive enzymes (salivary amylase, pepsin, and trypsin). Simulated digestion markedly accelerated HBQs removal, with intestinal digestion achieving the highest efficiency (87.1∼96.0 %). The k_obs for HBQs removal during simulated oral, gastric, and intestinal phases were 0.1247∼0.3187 h⁻¹, 0.0890∼0.1290 h⁻¹, and 0.3843∼0.5869 h⁻¹, respectively. Among HBQs, 2,6-DCBQ exhibited the strongest binding affinity to the three enzymes, as evidenced by its higher binding constants and greater number of binding sites. QM/MM simulations revealed that enzyme-induced polarization decreased E_LUMO, increased the electrophilicity index (ω), and expanded the electrophilicity gap between unsubstituted and chlorinated carbons. Transformation product analysis revealed that enzyme-driven conversion of the C = O bond to -CH-OH occurred, and HBQs were found to form covalent adducts with digestive enzymes through reactions with -SH and -NH₂ groups. Colon organoid assays demonstrated that trypsin mediated degradation was 1.20∼2.06 times more effective than cysteine mediated degradation in reducing HBQs cytotoxicity and 1.14∼1.50 times more effective in reducing genotoxicity, by forming low oxidative stress products. However, the toxicity was not completely eliminated. These findings highlight the critical role of digestive enzymes in HBQs removal, transformation and toxicity change, providing new insight into human health risk assessment of drinking water contaminants.