Reactive nitrogen species-driven oxidative and nitrative damage underlying cadmium/mercury co-exposure: A multimodal fluorescent visualization study.

Heavy metal contamination severely threatens ecological environment and human health. Few studies have established an integrated mechanistic framework linking heavy metal co-exposure, reactive nitrogen species (RNS) burst, and progressive cellular injury, especially for multi-metal combined pollution that better reflects real environmental scenarios. As downstream substance of nitric oxide (NO) and representative RNS, peroxynitrite (ONOO-) possess stronger oxidation/nitration capacity compared with hydrogen peroxide (H2O2). The oxidative/nitrifying stress ignited by RNS outburst under cadmium ion (Cd2+) and mercury ion (Hg2+) has not been seemingly clarified yet. Here, we rationally designed a ONOO- fluorescent probe and establish single/co-exposure models to visualize ONOO- dynamics, with the goal of defining a novel RNS-mediated mechanism for Cd2+/Hg2+ synergistic toxicity. Upon addition of ONOO-, the quenching of fluorophore by the recognition group was disrupted, and intramolecular charge transfer (ICT) mechanism restored, showing enhanced fluorescence. The probe has high recognition sensitivity for ONOO- (LOD = 36.4 nM) and good selectivity. Bcy-dpp was employed to observe exogenous and endogenous ONOO- level fluctuation in RAW264.7, HepG2 cells, zebrafish and cockscomb flowers. Our imaging results directly establish a causal link between heavy metal co-exposure, RNS burst, and cellular damage, providing a unified conceptual basis for understanding heavy metal combined toxicology. It is adequately envisioned that our proposed strategy could offer new insights for exploring the toxicology of heavy metal stress.