Two-photon fluorescent probe for revealing drug-induced hepatotoxicity: Via mapping fluctuation of peroxynitrite

Article abstract of DOI:10.1039/c7sc00303j

Drug-induced injury has attracted increasing attention in public health issues. Among them, hepatotoxicity has been regarded as the leading clinical problem caused by drug toxicity. However, owing to the complexity of the involved pathophysiological mechanisms and the lack of noninvasive, straightforward, and real-time tools, drug-induced hepatotoxicity has rarely been predicted satisfactorily. In this paper, by utilizing the reactive species peroxynitrite (ONOO^-) as a biomarker, we present a two-photon fluorescent probe, TP-KA, holding rapid response, high specificity and sensitivity towards ONOO^-, to investigate drug (acetaminophen and tolcapone)-related liver injury and the remediate effect of N-acetyl cysteine (NAC). With the support of TP-KA, we obtained direct and visual evidence of the upregulation of ONOO^- during drug challenge both in live cells and mice, which was accompanied by liver tissue injury and tyrosine nitration. These findings demonstrate that ONOO^- is a good and appropriate biomarker of hepatotoxicity, and nitrosative stress may be necessary for acetaminophen and tolcapone to exert their toxicity. Moreover, TP-KA can be employed as a powerful tool to pre-detect drug-induced organism injury and study the effect of antidotes.

Full text of DOI:10.1039/c7sc00303j

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Two-photon fluorescent probe for revealing drug-induced
hepatotoxicity via mapping fluctuation of peroxynitrite
Yong Li†, Xilei Xie†, Xiu’e Yang, Mengmeng Li, Xiaoyun Jiao, Yuhui Sun, Xu Wang* and Bo Tang*
Received 00th January 20xx,
Accepted 00th January 20xx
Drug-induced injury has attracted increasing attention in public health issue. Among them, hepatotoxicity has been
regarded as the leading clinical problem caused by drug toxicity. However, owing to the complexity of the involved
pathophysiological mechanisms and lack of noninvasive, straightforward, and real-time tools, drug-induced hepatotoxicity
has rarely been predicted satisfactorily. Herein, in this paper, utilizing reactive specie peroxynitrite (ONOO−) as a
biomarker, we presented a two-photon fluorescent probe, TP-KA, holding rapid response, high specificity and sensitivity
towards ONOO−, to investigate drug (acetaminophen and tolcapone)-related liver injury and the remediate effect of N-
acetyl cysteine (NAC). With support of TP-KA, we obtained a direct and visual evidence of upregulation of ONOO− during
drug challenge both in live cells and mice, which was accompanied by liver tissue injury and tyrosine nitration. These
findings demonstrate that ONOO− is a good and appropriate biomarker of hepatotoxicity, and nitrosative stress may be
necessary for acetaminophen and tolcapone to exert their toxicity. Moreover, TP-KA can be employed as a powerful tool
DOI: 10.1039/x0xx00000x
www.rsc.org/
to
pre-detect
drug-induced
organism
injury
and
study
the
effect
of
antidote.
Peroxynitrite (ONOO−), a kind of RNS, which is generated by
●−
the diffusion-limited reaction of superoxide anion radical (O2 )
Introduction
with nitric oxide (NO), could be excellent alternative because
of its high oxidative and nitrosative activity towards
biomolecules.15,16 Hence, efficient tracking of ONOO− during
drug challenge will add benefits to the in-depth understanding
of hepatotoxicity mechanism. However, the elusive nature of
ONOO− under physiological condition,17 such as extremely
Drug-induced injury, as a long-term concerning, has attracted
increasing attention in public health issue. Moreover, it cites as
the most common consideration for an approved drug to be
withdrawn from the market, or labelled with a black box
warning.1,2 Attributed to the central role of transforming and
clearing of exogenous and endogenous xenobiotics in human
body, the liver is the most frequently susceptible to the
damage of drugs, thus ultimately resulting in hepatotoxicity
being regarded as the leading clinical problem.3−5 As such,
preclinical interrogation of drug-induced hepatotoxicity is
profound to improve patient safety and therapeutic promoting.
Unfortunately, owing to the complexity of the involved
pathophysiological mechanisms, drug-induced hepatotoxicity
has rarely been predicted satisfactorily.6−8 Generally, it is
reported that the reactive metabolite’s formation of drugs is
necessary for most of hepatotoxicity,9−11 which is usually
accompanied by generation of reactive small molecules,
including reactive oxygen species (ROS) and reactive nitrogen
species (RNS).12,13 Therefore, ROS and RNS might be employed
as the biomarkers to predict drug-related hepatotoxicity.8,14
short half-life (< 10 ms), nanomolar homeostasis concentration,
and high chemical reactivity, precludes its tracking directly in
situ. Recently, fluorescence imaging has gained much attention
for its outstanding properties in terms of direct visualization
and satisfied temporal and spatial resolution,18−25 and a
number of fluorescent probes have been developed for
ONOO− detection in biological systems.26−35 Nonetheless, few
probes have been utilized to investigate the roles ONOO− plays
in the drug-
induced hepatotoxicity,8 especially probes with
two-photon excitability since two-photon probes shows
advantages of deep tissue penetration, less photodamage, and
good resolution.36−39
In this work, a new two-photon fluorescent probe, TP-KA
,
was designed and synthesized for ONOO− detection with rapid
response, high specificity, and good sensitivity. Capitalizing on
its outstanding features, TP-KA was successfully employed in
exploring hepatotoxicity events caused by two drugs, namely
the analgesic and antipyretic acetaminophen (APAP), and anti-
Parkinson's disease drug tolcapone. Encouragingly, with aid of
TP-KA, we verified that the hepatotoxicity mechanism of these
two drugs was involved in ONOO− upregulation, which was
accompanied by liver tissue injury and tyrosine nitration.
Moreover, the remediation of drug-induced damage was
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achieved by using N-acetyl cysteine (NAC), which might serve the reactivity of TP-KA toward H2O2 and ONOO– was evaluated
as a superior alternative antidote through enhancing nontoxic (Fig. S2). Encouragingly, even treated with 200 equivalents
metabolism of these drugs. These results demonstrated that H2O2, TP-KA displayed no obvious fluorescence enhancement
through efficient ONOO− evaluation, TP-KA may be a powerful at 560 nm. Conversely, when incubated with only 10
tool to pre-detect clinical drug-induced liver damage and study equivalents ONOO–, TP-KA exhibited a high turn-on ratio.
the effect of antidote.
Therefore, TP-KA was capable of recognizing ONOO– without
the interference of H2O2. The selectivity was probably due to
that 1,8-naphthalimide decreased the electrophilicity of the α-
ketoamide moiety and thus TP-KA could be only attacked by
the stronger nucleophile ONOO–.
Results and discussion
Design, synthesis, spectral properties, and reactivity.
Kinetic investigation of TP-KA. The kinetic characters of
Previously, we fabricated a hydrogen peroxide (H2O2) probe by
TP-KA were investigated (Fig. S3). In the absence of ONOO−,
the constant and weak fluorescence intensity indicated that
TP-KA was low background and stable towards light irradiation.
In the presence of ONOO−, a rapid and drastic fluorescence
enhancement was observed within 40 s, and then leveled off,
hinting that TP-KA was able of efficiently capturing ONOO− in
spite of its short lifetime and high activity.
installing
α-ketoamide
moiety
onto
hemicyanine
fluorophore.40 The α-ketoamide could be cleaved by H2O2, and
finally release the hemicyanine dye. ONOO– could also initiate
the similar reaction process, and simultaneously destroy the
conjugated skeleton of hemicyanine dye,33 thus it failed to
trigger on the reported H2O2 probe. Actually, ONOO– is
supposed to possess much stronger nucleophilicity than H2O2
under physiological condition.41 Therefore, in this work, we
attempted to construct a new probe (TP-KA) specific for
ONOO– by installing α-ketoamide onto much more stable
fluorophore, 1,8-naphthalimide.42,43 The tert-butyl ester group
was introduced to increase lipophilicity and thus enabled TP-
KA to penetrate through cell membrane smoothly. We
anticipated that TP-KA presented weak and blue-shifted
fluorescence emission because the intramolecular charge
transfer (ICT) effect was weakened by the electron-
withdrawing α-ketoamide group. Once reaction with ONOO−,
the fluorophore 1,8-naphthalimide would be released and
emit striking fluorescence attributing to the restored ICT
process (Scheme 1).
The specificity of TP-KA toward ONOO−. The specificity of
TP-KA toward ONOO− was performed. Gratifyingly, as
illustrated in Fig. 1, no obvious fluorescence intensity change
was trigged after incubation of TP-KA with a panel of highly
•−
oxidizing ROS or RNS, including H2O2, ClO−, •OH, TBHP, O2
,
1O2, and NO. Meanwhile, fluorescence response of TP-KA
towards reductive reactive species in cells, such as glutathione
(GSH), cysteine (Cys), homocysteine (Hcy), hydrogen sulfide
(H2S), and ascorbic acid (Vc), were also evaluated. Satisfactorily,
no significance fluorescence increment was observed. In
addition, negligible fluorescence augmentation was induced
after incubation of TP-KA with various cations (Na+, K+, Mg2+,
Ca2+, Fe2+, Co2+, Cu2+, Zn2+, and Fe3+), anions (NO2−, NO3
,
−
HCO3 , CH3COO−, CO3 −, S2O3 −, SO3 −, SO42−, and PO4 −), and
amino acids (Ser, Pro, Thr, Gly, and Val), which commonly
existed in biological systems. Collectively, these results
demonstrated the high specificity of TP-KA toward ONOO−
over other bioanalytes.
−
2
2
2
3
Scheme
mechanism.
1 Chemical structure of TP-KA and the corresponding response
Fig.
1 Fluorescence response of TP-KA (1.0 μM) with various bioanalytes,
including 10.0 μM ONOO−, 200 μM H2O2, 10.0 μM other ROS and RNS, 5.0 mM
GSH and Cys, and 100 mM other interfering substances. The data was recorded
after incubation of TP-KA with various bioanalytes for 10 min at 37 °C in PBS
buffer (50 mM, 7.4) with 1% DMSO. Values are the mean ± s.d. for n = 3. λex/λem
= 430/560 nm.
The synthetic procedure was displayed in Scheme S1. With
TP-KA and compound in hand, their spectral properties were
1
firstly assessed in 50 mM PBS buffer (pH 7.4, 1% DMSO) at 37
˚C. As can be seen in Fig. S1, TP-KA itself displayed maximal
absorbance/emission at 375 nm/454 nm. Due to the
weakened ICT effect caused by α-ketoamide, TP-KA showed a
Fluorescence response of TP-KA toward ONOO−. On the
basis of the excellent specificity of TP-KA to ONOO−, the
fluorescence spectra variation of TP-KA in the presence of
ONOO− was then investigated to profile the response behavior
in detail. As can be seen from Fig. 2, upon incubated with
different concentrations (0−10 μM) of ONOO−, gradually dose-
dependent fluorescence enhancement was obtained. The
fluorescence intensity of TP-KA exhibited an excellent linear
spectral blue shift compared to its reaction product
1 (430
nm/560 nm). In addition, TP-KA manifested negligible
fluorescence signal when excited at 430 nm. Therefore, to
obtain a high signal-to-noise ratio, the following in vitro
detection systems were all excited at 430 nm. Subsequently,
2 | J. Name., 2012, 00, 1−5
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response (R2 = 0.995) to ONOO− with a regression equation of
F = −9086.50 + 11698.06 [ONOO−] (μM). The limit of detection
(LOD) was calculated to be as low as 25 nM based on 3 σ/k (σ
is the standard deviation of 11 blank samples, k is the slope of
the linear equation), which enabled TP-KA sensitive enough for
tracing ONOO− in biosystems. Furthermore, the effect of pH
was investigated (Fig. S4). In the absence of ONOO−, the Fig. 3 TPM images of exogenous ONOO− in HepG2 cells. (a) Cells were stained
with TP-KA (5.0 µM, 20 min). (b) Cells were exposed to SIN-1 (1.0 mM, 30 min)
and then incubated with TP-KA (5.0 µM, 20 min). (c) Cells were exposed to SIN-1
(1.0 mM, 30 min) and then treated with uric acid (100 μM, 3 h), followed by
incubated with TP-KA (5.0 µM, 20 min). (d) Relative fluorescence intensity of (a),
(b), and (c). The images were obtained with 800 nm excitation and 500−600 nm
collection. Values are the mean ± s.d. for n = 3, ***p < 0.001. Scale bar = 15 µm.
fluorescence intensity of TP-KA itself remained unchanged in a
wide pH range of 5.0−9.0, indicating that TP-KA was hardly
influenced by pH. Meanwhile, upon addition of ONOO−,
unchanged and strong fluorescence signals were detected in a
pH range of 5.0−9.0, which well covers the physiological
environment pH range. Therefore, combined with the results
of outstanding response behavior, TP-KA exhibited robust
analytical potential in the biological application.
Visualizing exogenous ONOO− in live cells. Encouraged by
the distinguished response properties of TP-KA, its feasibility
of mapping ONOO− level in biological system was assessed
with two-photon excitation microscopy system (TPM). Prior to
cell imaging, MTT assay was performed to identify the
cytotoxicity of TP-KA, and the results (Fig. S6) implied TP-KA
presented excellent biocompatibility (IC50 = 63 μM). Then,
utilizing SIN-1 as ONOO− donor, the exogenous ONOO− imaging
was performed in HepG2 cells. As displayed in Fig. 3, in the
control group, negligible fluorescence signal was observed in
cells loaded with TP-KA
.
Contrastingly, significant
enhancement of intracellular fluorescence intensity was
obtained upon SIN-1 exposure. And this increment was
efficiently blunted by uric acid, an ONOO− scavenger.
Furthermore, the bright field images displayed intact cells,
implying good viability of the cells throughout the experiment.
Consequently, the data confirmed that TP-KA was applicable
and promising to act as a mapping tool to explore ONOO−
fluctuation under biological conditions.
Fig. 2 (a) Fluorescent response of TP-KA (1 μM) to ONOO− (0−10 μM) in PBS
buffer (50 mM, pH 7.4) with 1% DMSO. (b) The linear curve derived from
fluorescent intensity at 560 nm and ONOO− concentration. The spectra were
recorded after incubation of TP-KA with ONOO− for 10 min at 37 °C. Values are
the mean ± s.d. for n = 3. λex/λem = 430/560 nm.
The proposed mechanism for sensing of ONOO−. The
reaction mechanism of TP-KA with ONOO− was further
confirmed by high resolution mass spectrometer analysis (Fig.
S5). Upon incubation of TP-KA with ONOO− for 10 min, the
reaction mixture was found mass peaks at m/z 341.1672 and
Imaging drug-induced hepatotoxicity and remediation
effect of NAC. Typically, hepatotoxicity hints chemical-driven
liver damage.44 It is well-known that overdose of
acetaminophen (APAP), a common household medication used
to treat pain and fever, could induce hepatotoxicity relating to
overproduction of ROS and RNS.45 Therefore, guiding with
imaging biomarker ONOO−, APAP-induced damage was
visualized with the aid of TP-KA in cells. As presented in Fig.
4a−4e, after treatment of APAP, apparently stronger
fluorescence signal in HepG2 cells was detected than that in
normal condition. Therefore, the results revealed that
intracellular ONOO− was upregulated under administration of
APAP, thus contributing to oxidative and nitrosative stress
injury. Furthermore, as can be seen, the strong fluorescence
intensity could be effectively attenuated by N-acetyl cysteine
(NAC), which remediated cell injury via depleting ROS and RNS
levels through increasing GSH level and binding with the toxic
products of APAP.46,47 Therefore, the results indicated that TP-
KA exhibited excellent properties of visualizing cell damage
induced by APAP and NAC displayed good antidote effect.
m/z 166.0194, corresponding to compound
1 and the
byproduct 4-nitrobenzoic acid, respectively. Both the spectral
variations and the mass analysis results definitely validated the
proposed sensing mechanism displayed in Scheme S2.
Two-photon fluorescent properties of TP-KA. The two-
photon absorption cross-section (δ) of TP-KA and its sensing
product
two-photon imaging probe. The δmax value of TP-KA and
compound
were acquired at 800 nm as 95 GM (1 GM = 10−50
m4 s photon−1) and 193 GM, respectively. The higher two-
photon absorption cross-section of compound over the TP-
1 were investigated, as δ is a critical parameter for a
1
1
KA could be attributed to its electron donating moiety, 4-
amino group. Moreover, the high δmax value enabled TP-KA to
be applied in two-photon fluorescence microscopy imaging.
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before drug administration, negligible fluorescence signal
change was observed in both APAP and tolcapone group,
confirming the protective effect of NAC in the liver. These
results verified that both APAP and tolcapone could induce
hepatotoxicity via upregulation of ONOO−.
Fig. 4 TPM images of drug-induced injury with HepG2 cells. (a) and (f) Cells were
stained with TP-KA (5.0 µM, 20 min). (b) and (c) Cells were exposed to 250 and
500 µM APAP for 1 h, respectively, and then treated with TP-KA (5.0 µM, 20 min).
(d) Cells were pretreated with NAC (1.0 mM, 1 h), and then exposed to 500 µM
APAP, followed by treated with TP-KA (5.0 µM, 20 min). (g) and (h) Cells were
exposed to 250 and 500 µM tolcapone for 1 h, respectively, and then treated
with TP-KA (5.0 µM, 20 min). (i) Cells were pretreated with NAC (1.0 mM, 1 h),
and then exposed to 500 µM tolcapone, followed by treated with TP-KA (5.0 µM,
20 min). (e) Relative fluorescence intensity of (a), (b), (c), and (d). (j) Relative
fluorescence intensity of (f), (g), (h), and (i). The images were obtained with 800
nm excitation and 500−600 nm collection. Values are the mean ± s.d. for n = 3,
**p<0.01, ***p < 0.001. Scale bar = 15 µm.
Fig. 5 TPM images of liver slices of mice. The mice were injected intraperitoneally
with various substances and then injected TP-KA (50 µM, 100 µL) via tail vein. (a)
and (f) PBS buffer only as control group; (b) and (c) APAP (100 mg/kg and 300
mg/kg, respectively); (d) NAC (300 mg/kg) pretreated and then APAP (300
mg/kg); (g) and (h) tolcapone (100 mg/kg and 300 mg/kg, respectively); (i) NAC
(300 mg/kg) pretreated and then tolcapone (300 mg/kg); (e) relative
fluorescence intensity of (a), (b), (c), and (d); (j) relative fluorescence intensity of
(f), (g), (h), and (i). The images were obtained with 800 nm excitation and
500−600 nm collection. Values are the mean ± s.d. for n = 3, *p<0.05, ***p <
0.001. Scale bar = 20 µm.
Then, the cell injury of another drug, tolcapone, was
explored. Tolcapone was used initially for the treatment of
Parkinson’s disease, and was withdrawn from market in the EU
and labelled with black box warning in the US because of its
severe hepatotoxicity.48,49 No clear mechanism has been
proposed regarding the tolcapone-induced liver toxicity yet.
But it has been hypothesized that it is associated with
dysfunctional mitochondrial respiration due to the uncoupling
of oxidative phosphorylation.50 Accordingly, with the support
of TP-KA, the injury of tolcapone in the cell level was mapped
to explore the possible injury mechanism (Fig. 4f−4j). As can be
seen, HepG2 cells exposed to tolcapone demonstrated dose-
dependently brighter fluorescence as compared to the control
group, suggesting upregulation of ONOO− level. This means in
common with APAP, tolcapone could also induce HepG2 cells
damage via overproduction of ONOO− through induction of
oxidative and nitrosative stress. Meanwhile, the apparently
decreased fluorescence intensity of cells treated with NAC
before tolcapone treatment implied a remedial role of NAC in
tolcapone-induced cell damage.
Next, the histological and immunohistochemical analysis
on liver tissues were carried out to identify histological
changes during drugs administration. Nitrotyrosine was
employed as the immunohistochemical biomarker to confirm
the nitrosative stress of ONOO− to protein tyrosine residues.51
Briefly, Kunming mice liver sections were prepared from three
groups processed with various conditions, including PBS buffer
only, drug administration, and NAC pretreatment. As can be
seen in Fig. 6 and Fig. S7, both histological changes (i.e.,
cellular shrinkage or blebbing, rupture of cell membrane, and
condensation of chromatin) and nitrotyrosine formation could
be obviously observed after two drugs administration,
implying liver injury happened because of drug metabolism.
However, the mice in NAC pretreatment group displayed no
distinct changes in histological and immunohistochemical
analysis, indicating that the liver injury could be effectively
suppressed by NAC. Taking together, these results were in
good agreement with the observation of in vitro fluorescent
imaging using ONOO− as a biomarker (Fig. 4 and Fig. 5).
Subsequently, the hepatotoxicity of APAP and tolcapone
was visualized in tissue level to deeply verify the toxicity
mechanism. Generally, Kunming mice were divided into three
groups. As the control group, the mice were processed with an
intraperitoneal injection of PBS buffer before a tail-vein
injection of TP-KA
. The second group was given an
intraperitoneal injection of PBS buffer solution containing
various concentrations of APAP or tolcapone, and then a tail-
vein injection of TP-KA 30 min later. The mice in the last group
were pretreated with an intraperitoneal injection of NAC 1 h
before drug administration. Then all mice were anesthetized
and dissected to isolate the livers, which were cut into slices
for TPM fluorescence imaging. As shown in Fig. 5, weak
fluorescence intensity was obtained in mice liver tissue in
control group, implying low ONOO− level under normal
condition. By contrast, after administration of either APAP or
tolcapone, the mice liver in second group emitted obvious
fluorescence, indicating up-regulated ONOO− after drug
treatment. Meanwhile, for the mice pretreated with NAC
Fig.6 Representative H&E staining images of the liver of mice treated with
various conditions. (a) and (e) PBS buffer only as control group; (b) and (c) APAP
(100 mg/kg and 300 mg/kg, respectively); (d) NAC (300 mg/kg) pretreated and
then APAP (300 mg/kg); (f) and (g) tolcapone (100 mg/kg and 300 mg/kg,
respectively); (h) NAC (300 mg/kg) and then tolcapone (300 mg/kg). Scar bar = 50
µM.
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(20g) were purchased from School of Medicine at Shandong
University. All animal experiments were in agreement with the
guidelines of the Institutional Animal Care and Use Committee.
Finally, tolcapone-induced hepatotoxicity and NAC
remediation were monitored in vivo through a real-time way.
As can be seen from Fig. 7, after tolcapone administration, the
fluorescent intensity of mice liver displayed gradual increment
in a time-dependent manner. In especial, the observable
fluorescence intensity augmentation at 15 min indicated that
ONOO−-related stress began to show up after drug
administration in such a short time. In addition, the clearance
assay was also performed using uric acid to confirm the
overproduction of ONOO− after tolcapone administration.
Moreover, with the passage of time, no obvious intensity
augment was observed in NAC pretreatment group, indicating
effective NAC remediation after drug treatment. It should be
noted that for the first time, the early warning potential of
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APAP
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4
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TP-KA may be employed as a powerful tool to pre-detect drug-
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This work was supported by 973 Program (2013CB933800) and
National Natural Science Foundation of China (21390411,
21535004, 21375080, and 21505088). Male Kunming mice
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Table of contents entry
A peroxynitrite-specific two-photon fluorescent probe was developed for revealing
drug-induced hepatotoxicity by using peroxynitrite as a biomarker