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.

Figure 1. Design of CF-SPN for detection of ROS and RNS

(a) The molecular components of CF-SPN are the NIR fluorescent semiconducting polymer PFODBT (dark red), a PEG-grafted poly(styrene) copolymer conjugated to galactose for hepatocyte targeting (black), the H₂O₂-specific chemiluminescent substrate CPPO (light blue) that serves as CRET energy donor, and the FRET acceptor IR775S (bright green) that degrades after oxidation by ONOO⁻ or ⁻OCl (dark green). PFODBT serves as the CRET energy acceptor and the FRET energy donor. (b) An illustration of the mechanism of simultaneous and differential detection of ONOO⁻ or ⁻OCl and H₂O₂ by CF-SPN is shown. Following drug challenge to the liver, CF-SPN report via the chemiluminescent and fluorescent channels the generation of radical metabolites at safe (left) and toxic drug doses (right). The hydrodynamic diameter distribution of CF-SPN was determined by dynamic light scattering (c). A transmission electron micrograph of CF-SPN (scale bar = 100 nm) is shown in (d).

Figure 2. Spectral characterization, specificity and sensitivity of CF-SPN in vitro

(a) The UV/Vis absorption spectrum of CF-SPN, with PFODBT maximum at 580 nm and NIR775S maximum at 775 nm. (b) Fluorescence (red) and chemiluminescence (blue) spectra of CF-SPN, indicating PFODBT and NIR775S emission maxima at 680 nm and 820 nm, respectively. Chemiluminescence was induced by the addition of 2 mM H₂O₂. (c) The fluorescence emission ratio of CF-SPN (1 μg/mL) in the presence of indicated ROS/RNS (6 μM). (d) The sensitivity and range of fluorescence ratiometric detection of ONOO⁻ with CF-SPN (1 μg/mL) following 1 μM incremental additions of NaONOO. (e) The specificity of the chemiluminescent signal of CF-SPN (5 μg/mL) was determined in the presence of ROS/RNS (6 μM). (f) The chemiluminescent response of CF-SPN to varying concentrations of H₂O₂ was assessed. Experiments were performed with CF-SPN (1 μg/mL) in 1x PBS.

Figure 3. Real-time in vivo imaging of hepatotoxicity following APAP administration to mice

(a) Representative images of mice receiving, from left to right, 300, 150, 75 mg/kg of APAP or saline i.p., followed by CF-SPN (0.8 mg) i.v. Threshold toxicity is observed for both the chemiluminescence (top row, shown at 18 min after CF-SPN administration) and fluorescence (bottom row, shown at 53 min after CF-SPN administration) channels. The emission intensities of the liver for (b) chemiluminescence and (c) normalized fluorescent percent difference (FL Index) over time are shown. The black arrows indicate the respective time points shown in (a). Values are the mean±s.d. for n=3 mice. Representative immunohistochemistry is shown for liver (d) 45 min and (e) 180 min after drug administration. Nitrotyrosine (top) and TUNEL (bottom) staining was performed, with white arrowheads representing positive cellular or nuclear staining, respectively. Scale bars represent 10 μm.

Figure 4

Longitudinal, in vivo monitoring of the remediation of APAP-induced hepatotoxicity with enzyme inhibitors and antioxidant scavengers. (a) A representation of the mechanism of APAP induced toxicity is shown, with the effects of inhibition by GSH, 1-ABT, and t-1,2-DCE; indicates inhibition. (b) Representative images of mice receiving, from left to right, saline, 300 mg/kg APAP i.p. alone, and 300 mg/kg APAP with GSH (200 mg/kg i.v.), 1-ABT (2×100 mg/kg i.p.), or t-1,2-DCE (0.2 mg/kg i.p.), followed by CF-SPN (0.8 mg) i.v. The emission intensities of the liver for the (c) chemiluminescence or (d) fluorescent ratiometric signals are shown over time. The black arrows indicate the respective time points shown in (a). Values are the mean±s.d. for n=3 mice.

Figure 5

Real-time in vivo imaging of dose-dependent hepatotoxicity in mice following INH administration. (a) Representative images of mice receiving, from left to right, 200, 100, 50 mg/kg INH, or saline i.p., followed by CF-SPN (0.8 mg i.v.). The emission intensities of the liver for the (b) chemiluminescence or (c) fluorescent ratiometric signals are shown over time. The black arrows indicate the respective time points shown in (a). Values are the mean±s.d. for n=3 mice. (d) Representative histology (H&E) of the liver of mice treated as in (a) is shown 180 min after drug administration, with corresponding image enlargements in the bottom row. Scale bars represent (top) 10 μm and (bottom enlargement) 2.5 μm, respectively.

Figure 6

The ability of CF-SPN to differentially and simultaneously detect H₂O₂ and ONOO⁻ provides mechanistic insights into parent drug bioactivation. The majority of H₂O₂ is produced following Phase I bioactivation, either through uncoupling of enzyme-mediated drug oxidation or through the initiation of redox cycling following oxidation of the parent drug. Strong chemiluminescence from CF-SPN suggests the involvement of Phase I bioactivation in the mechanism of drug toxicity. Conversely, the production of ONOO⁻ is associated with mitochondrial toxicity and uncoupling of the mitochondrial electron transport chain by reactive drug metabolites. A significant increase in the FL index indicates the production the ONOO⁻, and suggests that drug bioactivation leads to mitochondrial dysfunction.