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Real-time imaging of oxidative and nitrosative stress in the liver of live animals for drug-toxicity testing

Abstract

Current drug-safety assays for hepatotoxicity rely on biomarkers with low predictive power. The production of radical species, specifically reactive oxygen species (ROS) and reactive nitrogen species (RNS), has been proposed as an early unifying event linking the bioactivation of drugs to hepatotoxicity and as a more direct and mechanistic indicator of hepatotoxic potential. Here we present a nanosensor for rapid, real-time in vivo imaging of drug-induced ROS and RNS for direct evaluation of acute hepatotoxicity. By combining fluorescence resonance energy transfer (FRET) and chemiluminescence resonance energy transfer (CRET), our semiconducting polymer–based nanosensor simultaneously and differentially detects RNS and ROS using two optically independent channels. We imaged drug-induced hepatotoxicity and its remediation longitudinally in mice after systemic challenge with acetaminophen or isoniazid. We detected dose-dependent ROS and RNS activity in the liver within minutes of drug challenge, which preceded histological changes, protein nitration and DNA double-strand-break induction.

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Figure 1: Design of CF-SPN for detection of ROS and RNS.
Figure 2: Spectral characterization, specificity and sensitivity of CF-SPN in vitro.
Figure 3: Real-time in vivo imaging of hepatotoxicity after APAP administration to mice.
Figure 4: Longitudinal, in vivo monitoring of the remediation of APAP-induced hepatotoxicity with enzyme inhibitors and antioxidant scavengers.
Figure 5: Real-time in vivo imaging of dose-dependent hepatotoxicity in mice after INH administration.
Figure 6: The ability of CF-SPN to differentially and simultaneously detect H2O2 and ONOO provides mechanistic insights into parent drug bioactivation.

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Acknowledgements

This work was supported by the US National Institutes of Health National Cancer Institute (NCI) grants R01CA135294, R01DK099800-06A1, R21CA138353A2, the Stanford University NCI CCNE-T grant (U54CA151459) and In Vivo Cellular and Molecular Imaging Centers grant (P50CA114747). A.J.S. acknowledges Susan G. Komen For The Cure for fellowship support. We acknowledge the use of the SCi3 Core Facility and the Neuroscience Microscopy Service Facility at Stanford University, P. Chu for her expertise in preparation of histology samples, E.J. McWalter for assistance with MatLab script, and S. Machtaler for assistance with tissue preparation and imaging by confocal microscopy.

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A.J.S., K.P. and J.R. conceived of the nanosensor design, and A.J.S., K.P., J.P.U. and J.R. designed the experiments. L.C. synthesized the galactose moiety. K.P. synthesized PS-g-PEG-Gal and CF-SPN, performed in vitro characterization of CF-SPN and data analysis. A.J.S. performed in vivo studies, analyzed in vivo data, and acquired histology images. A.J.S., K.P., J.P.U. and J.R. discussed the results and co-wrote the manuscript.

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Correspondence to Jianghong Rao.

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Stanford University has filed a provisional patent application (serial number 61/841,958) to protect part of the technology described in the study.

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Shuhendler, A., Pu, K., Cui, L. et al. Real-time imaging of oxidative and nitrosative stress in the liver of live animals for drug-toxicity testing. Nat Biotechnol 32, 373–380 (2014). https://doi.org/10.1038/nbt.2838

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