Associated Detection of Superoxide Anion and Mercury(II) under Chronic Mercury Exposure in Cells and Mice Models via a Three-Channel Fluorescent Probe

As a cytotoxic heavy metal ion, mercury(II) ion (Hg²⁺) induces severe oxidative stress and further results in physiological dysfunction. Although mercury poisoning can be treated with many drugs, such as sodium selenite, the therapeutic effect is relatively poor, and it seems that the damage to human health continues. However, the interpretation for the pathogenesis has not been clarified yet. We supposed that the reason is attributed to Hg²⁺-caused intracellular oxidative stress. To confirm our hypothesis, we strived to design a three-channel ratio fluorescent probe, HCy-SeH, for superoxide anion (O₂^•-) and Hg²⁺ combined detection. O₂^•- is a vital precursor for other reactive oxygen species (ROS), which is involved in many physiological and pathological processes. However, until now there is no efficient chemical tool for O₂^•- and Hg²⁺ combined detection in cells and in vivo. The fluorescence response of our probe is initiated by a hydrogen abstraction reaction from the hydrocyanine fluorophore moiety. Once oxidized by O₂^•-, HCy-SeH recovers its π-conjugated system back to a heptamethine cyanine derivative, Cy-SeH. Cy-SeH coexists with its conjugate base, Cy═Se. One emits red fluorescence, and the other one emits green fluorescence. The response unit, -SeH, can trap Hg²⁺ via a Se-Hg antagonism reaction to afford an orange-emitting final product, Keto-Cy. The probe offers high selectivity and sensitivity toward O₂^•- and Hg²⁺. When applied for O₂^•- and Hg²⁺ detection in HEK 293 cells, the imaging results indicate that our probe can provide a combined response for O₂^•- and Hg²⁺ in real time and in situ. Flow cytometry analysis is well-consistent with the results from fluorescence imaging. When applied to image O₂^•- and Hg²⁺ in mice models, we find that Hg²⁺ dominantly accumulates in the kidney and induces a burst of O₂^•-. We confirm that chronic mercurialism can cause severe oxidative damage and renal fibrosis. HCy-SeH further provides a new information that, even when intracellular Hg²⁺ has been antagonized, the outbreak of O₂^•- caused by mercury poisoning still lasts.