Fluorescent probe for the imaging of superoxide and peroxynitrite during drug-induced liver injury

Abstract

Drug-induced liver injury (DILI) is an important cause of potentially fatal liver disease. Herein, we report the development of a molecular probe (<b>LW-OTf</b>) for the detection and imaging of two biomarkers involved in DILI. Initially, primary reactive oxygen species (ROS) superoxide (O₂˙^-) selectively activates a near-infrared fluorescence (NIRF) output by generating fluorophore <b>LW-OH</b>. The C[double bond, length as m-dash]C linker of this hemicyanine fluorophore is subsequently oxidized by reactive nitrogen species (RNS) peroxynitrite (ONOO^-), resulting in cleavage to release xanthene derivative <b>LW-XTD</b>, detected using two-photon excitation fluorescence (TPEF). An alternative fluorescence pathway can occur through cleavage of <b>LW-OTf</b> by ONOO^- to non-fluorescent <b>LW-XTD-OTf</b>, which can react further with the second analyte O₂˙^- to produce the same <b>LW-XTD</b> fluorescent species. By combining NIRF and TPEF, <b>LW-OTf</b> is capable of differential and simultaneous detection of ROS and RNS in DILI using two optically orthogonal channels. Probe <b>LW-OTf</b> could be used to detect O₂˙^- or O₂˙^- and ONOO^- in lysosomes stimulated by 2-methoxyestradiol (2-ME) or 2-ME and SIN-1 respectively. In addition, we were able to monitor the chemoprotective effects of <i>tert</i>-butylhydroxyanisole (BHA) against acetaminophen (APAP) toxicity in living HL-7702 cells. More importantly, TPEF and NIRF imaging confirmed an increase in levels of both O₂˙^- and ONOO^- in mouse livers during APAP-induced DILI (confirmed by hematoxylin and eosin (H&E) staining).