Mechanistic insight into Metabolic Activation Mechanism of Aniline Disinfection Byproducts catalyzed by P450 Enzymes from Distinct Species: N-hydroxylation Mechanism.

Disinfection by-products (DBPs) are emerging environmental pollutants generated from reactions between disinfectants and precursor compounds in the water treatment, which pose a serious toxicity effect to the ecological environment and human health. Metabolism, especially by P450 enzymes, is responsible for both the toxification and detoxification of xenobiotics in humans and other organisms. Multidisciplinary theoretical approaches utilizing density functional theory (DFT), quantitative structure-activity relationship (QSAR), and quantum mechanics/molecular mechanics (QM/MM) are used to investigate the metabolic mechanism of aniline DBPs (AN-DBPs) catalyzed by P450 enzymes. The high-throughput DFT calculations confirm that the N-hydroxylation represents a feasible metabolic pathway for AN-DBPs, with the OH rebound reaction serving as the rate-determining step. A machine learning driven QSAR model is developed to predict the energy barriers of the rate-determining step involved in N-hydroxylation for AN-DBPs through employing four molecular descriptors derived from Mordred platform. The QSAR model shows relatively high predictive accuracy, with R2 values of approximately 0.8. The QM/MM calculations characterize the reaction energy barriers and structural details for the H-abstraction and OH rebound pathways of 2,6-dichloroaniline catalyzed by P450 enzymes from four different species, revealing that zebrafish P450 enzyme is the most readily activated metabolically upon exposure to AN-DBPs. Furthermore, the potential toxicity effects of AN-DBPs and their metabolites are predicted by in silico methods, and the results demonstrate that the metabolites generated from P450-mediated biotransformation process exhibit higher predicted toxicity than the parent AN-DBPs. These unveiled metabolic profiles offer valuable insights for the risk evaluation of AN-DBPs, which can guide future research and regulatory actions.