Exploring the comprehensive toxicity of PPD pollutants: Perspective of combining spectral experiments with molecular modeling and network toxicology.

Para-phenylenediamine (PPD) compounds, widely used in the automotive industry, are significant environmental pollutants posing human exposure risks. However, their environmental behavior, specific toxicological profiles, and molecular mechanisms of toxicity involving interference with key physiological proteins remain largely unclear. This study integrates multispectral analysis, molecular simulations, and network toxicology to explore the molecular interactions and potential toxicity mechanisms of three representative PPDs: N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), N-isopropyl-N'-phenyl-p-phenylene diamine (IPPD), and N, N'-diphenyl-p-phenylenediamine (DPPD). Specifically, we investigated their binding with human serum albumin (HSA) and pepsin (PEP) to assess transport and gastric stability, while employing network toxicology to predict potential hepatic toxicity pathways. Results showed that PPDs formed stable non-covalent complexes with PEP/HSA via a single-site, two-state binding model. Fluorescence quenching revealed static quenching for the HSA-6PPD, HSA-IPPD, and PEP-6PPD complexes, whereas the HSA-DPPD and PEP-IPPD/DPPD complexes exhibited mixed quenching. Binding free energy calculations identified van der Waals interactions as the dominant stabilizing force, significantly outweighing electrostatic contributions. Network toxicology predicted PPDs bind hub proteins (e.g., EGFR, SRC) to dysregulate liver function via PI3K-Akt, MAPK, and Ras pathways. Distinct risks emerged from N-substituents: 6PPD is predicted to target targets metabolic signaling via PIK3R1, IPPD poses endocrine risks via ESR1, and DPPD impairs liver regeneration via MET. This work provides structural insights into PPD-protein interactions and generates data-driven hypotheses for evaluating the health impacts of environmental pollutants.