Objective Aromatic amine-releasing dyes (AADs) are a class of synthetic dyes that can decompose into aromatic amines (AAs) under specific conditions, such as microbial action in the environment, and are mainly used for the dyeing of leather, textiles, and other materials. Therein, some AAs are confirmed with strong carcinogenic and teratogenic properties, posing potential threats to human health. Although AADs have been completely banned, the related exposure risk to the human body persists. To address the gaps in current research on the health risks and toxic mechanisms of AADs, this study explored the toxic mechanisms of their metabolites, i.e., AAs, in the human body.

Methods Four typical AAs, namely benzidine (BZD), 2,4-dimethylaniline (DMA), 4,4′-diaminodiphenylmethane (MDA), and o-tolidine (OT), were selected as representatives. Multiple spectroscopic techniques, including molecular fluorescence, time-resolved fluorescence, three-dimensional fluorescence, and circular dichroism spectroscopy, combined with molecular docking simulation, were used to systematically investigate the interaction mechanism between AAs and human serum albumin (HSA) in the temperature range from room temperature to human physiological temperature (298-310 K). The quenching mechanism, binding sites, binding constants, and thermodynamic constants were analyzed by fitting the data using the Stern-Volmer, double logarithmic, and Van’t Hoff equations, and the effects of AAs on the conformational structure of HSA were clarified.

Results The results demonstrate that all four AAs could bind to HSA. Specifically, the binding mechanism between BZD and HSA was static quenching, while DMA, MDA, and OT all bound to HSA through a combination of static and dynamic quenching. The number of the binding site between HSA and AAs is one, i.e., AAs bind to HSA in an equimolar ratio. The binding constant K_a of BZD to HSA was 1.78×10⁶ L·mol⁻¹, while the K_a values of DMA, MDA, and OT to HSA were in the range of 1.53×10²-17.53×10² L·mol⁻¹, showing an overall moderate-to-low binding strength at 310 K. The bindings of HSA/AAs systems proceeded spontaneously (Gibbs free energy variation ΔG＜0). Besides, the binding interaction of the HSA/BZD system was dominated by the hydrophobic interactions, while the bindings of DMA, MDA, and OT to HSA were mainly driven by the hydrogen bonds and van der Waals forces. In addition, AAs were found to prefer to achieve specific binding interaction through multiple forces near the hydrophobic amino acid residues of HSA. Furthermore, the introduction of AAs caused the α-helical structure of HSA to unfold and the peptide chain to stretch, resulting in decreased hydrophobicity of its internal environment and increased molecular diameter, without altering the main structure of HSA.

Conclusions This study revealed the binding interaction characteristics between AAs and HSA as well as the conformational changes of HSA at the molecular level, confirming the diffusion risk of AAs in the human body, and these interactions may interfere with the normal physiological functions of HSA. Among these AAs, BZD may remain in the blood for a long time due to its strong affinity to HSA, thus posing a more extensive diffusion risk to the human body; DMA, MDA, and OT are more likely to exhibit local accumulation hazards in organ tissues. Clarifying the in vivo toxic mechanism of AAs, the metabolite of AADs, provides an important theoretical basis for the health risk assessment and targeted prevention and control of the entire AADs exposure path and the entire AAs application chain in leather and other related industries.