Observation of the generation of peroxynitrite in mouse liver after acetaminophen overdose with a boronate-based ratiometric fluorescence probe

Article abstract of DOI:10.1039/c8ra10053e

A ratiometric fluorescent probe, BTPB, for the selective monitoring of hepatic peroxynitrite (ONOO^-) in situ after acetaminophen (APAP) overdose has been developed. Our study provided direct evidence for supporting the generation of ONOO^- in APAP-induced liver injury. This new probe will be a useful tool for studying the roles of ONOO^-in vivo.

Full text of DOI:10.1039/c8ra10053e

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Observation of the generation of peroxynitrite in
mouse liver after acetaminophen overdose with
a boronate-based ratiometric fluorescence probe†
a
Cite this: RSC Adv., 2019, 9, 6510
Ying Ma,a Haigang Tian,a Zhengyu Jin,a Xiaoyong Lib and Yiping Li
*
Received 6th December 2018
Accepted 15th February 2019
A ratiometric fluorescent probe, BTPB, for the selective monitoring of hepatic peroxynitrite (ONOOꢀ) in situ
after acetaminophen (APAP) overdose has been developed. Our study provided direct evidence for
supporting the generation of ONOOꢀ in APAP-induced liver injury. This new probe will be a useful tool
for studying the roles of ONOOꢀ in vivo.
DOI: 10.1039/c8ra10053e
rsc.li/rsc-advances
Acetaminophen (APAP) is a safe and effective antipyretic/
During resulting in MPT, it was recognized that the strong
analgesic drug when used at recommended doses, and is one oxidant and nitrating peroxynitrite (ONOOꢀ), formed from
of the most commonly used drugs in the US and throughout the superoxide (O2cꢀ) and nitric oxide (NO), is being generated in
West.1 However, APAP overdose can cause severe acute liver hepatocytes and a critical mediator in the injury mechanism.
injury with the potential to progress to liver failure. It is The generation of ONOOꢀ was only demonstrated by immu-
responsible for almost 80 000 emergency department visits in nohistochemical staining for 3-nitrotyrosine protein adducts.7,8
the US each year,2 as well as 26 000 hospitalizations and nearly The well-known nitration mechanism involves the initial one-
500 deaths.3 In fact, APAP hepatotoxicity is one of the most electron oxidation of tyrosine to yield tyrosyl radical followed
common etiologies of acute liver failure.4
by a diffusion-controlled reaction with nitrogen dioxide (cNO2)
Despite substantial progress in understanding the mecha- to yield 3-nitrotyrosine.9,10 However, in biological systems, cNO2
nism of APAP-induced liver injury during the last four decades, arises from a variety of sources, except most notably the
many details are still unknown. At therapeutic levels, about 50– decomposition of ONOOꢀ, but the hemeperoxidase-dependent
70% of the administered APAP is glucuronidated, and 25–35% oxidation of nitrite,10,11 which is a ONOOꢀ-independent mech-
undergoes sulfation, while only 5–10% is converted to the anism. Actually, protein 3-nitrotyrosine, which was initially
reactive electrophilic intermediate N-acetyl-p-benzoquinone conceived as a specic footprint of ONOOꢀ, typically represents
imine (NAPQI) by cytochrome P450 enzymes.5 NAPQI can be a fairly stable end product that is generated by the action of NO-
detoxied by reaction with the cysteine sulydryl group on derived oxidants.12 It seems insufficient for the generation of
glutathione (GSH). Once the administration of APAP overdose, ONOOꢀ aer the administration of APAP overdose only
NAPQI can deplete GSH stores, and bind to protein sulydryl according to the observation of the formation of protein 3-
groups, especially mitochondrial proteins, which triggers nitrotyrosine. Therefore, there is an urgent need to provide the
mitochondrial oxidant stress. The amplication of mitochon- direct evidence for supporting the generation of ONOOꢀ aer
drial oxidative stress further leads to the opening of the mito- APAP overdose.
chondrial membrane permeability transition (MPT) pore, then
The lifetime of ONOOꢀ in biological systems is limited to
the rupture of the outer mitochondrial membrane. As a result, only a few milliseconds, which makes it impossible to quanti-
there is extensive release of proteins from the intermembrane tate ONOOꢀ in processed cell or tissue samples directly.13
space, including endonuclease
G and apoptosis-inducing Fluorescence based techniques are advantageous for detection
factor, both of which can translocate to the nucleus because of ONOOꢀ. In accordance with the multiplicity of ONOOꢀ
of their nuclear localization sequences. This then results in biochemistry,14 the triuorocarbonyl (HKGreen 1-3)15–17 and
DNA fragmentation and, nally, oncotic necrosis.6
phenyl boronic esters (CBA)18 based uorescent probes, which
can directly detect OONOꢀ, have been reported, mainly for in
vitro applications. However, these probes are not suitable for
biological studies in vivo because of their high molecular
weight, bearing easily metabolized lactone group, such as
HKGreen 1-3, or a low response signal, such as CBA. Chem-
iluminescence (CL) probes19,20 can distinguish ONOOꢀ from
a variety of other ROS owing to the radicals from the
aSchool of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an 710061,
Shaanxi, China. E-mail: yipingli@mail.xjtu.edu.cn
bSchool of Science, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China. E-mail:
lixy6658@mail.xjtu.edu.cn
† Electronic supplementary information (ESI) available: Experimental procedures,
supplemental spectra, and the 1H-, 13C-NMR, and MS spectra. See DOI:
10.1039/c8ra10053e
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decomposition of ONOOꢀ, such as ONOOH, cOH and O2cꢀ, not probe sensitive. More importantly, benzothiazol group is
directly to the reaction with ONOOꢀ. Additionally, CL probes metabolically stable and a usual drug fragments,26,27 and this
were not used to detect OONOꢀ in vivo.
probe has a low molecular weight and appropriate water solu-
In this regard, to provide direct and strong evidence for the bility, which makes this probe suitable to use in vivo. The design
formation of OONOꢀ in liver aer APAP overdose, we developed rationale is depicted in Fig. 1, and illustrated as follows.
a novel and low background phenyl boronic acid-containing
Probe BTPB was prepared using the one-step reaction shown
uorogenic probe for sensitive and selective detecting of in ESI.†28 2-(Benzothiazol-2-yl) phenol, BTP, the expected
OONOꢀ. This probe was introduced into the mice adminis- product resulting from reaction of BTPB with ONOOꢀ, was also
trated APAP overdose by heart perfusion to monitor in situ the prepared utilizing a previously described method.29 The struc-
formation of OONOꢀ in liver.
tures of BTPB and BTP were conrmed by H NMR, 13C NMR,
1
The new uorescent probe was designed by the direct and HRMS spectra (Fig. S1–S6†), which match those of
conjugation of an boronic acid reaction group with a (phenyl-2- literatures.28,29
yl)benzothiazol scaffold, serving as a uorophore. Phenyl
Firstly, the spectroscopic response of BTPB to ONOOꢀ was
boronic acid group was selected because the group can react investigated in an abiotic system. We examined the spectroscopic
with OONOꢀ directly, rapidly and stoichiometrically,21 which is properties of BTPB (1.0 mM) towards ONOOꢀ (1 equiv.) in
more suitable to detect OONOꢀ than other groups that react 100.0 mM PBS buffer with 1.0 mM cetyltrimethylammonium
with those secondary radicals derived from OONOꢀ, including bromide (CTAB) at pH 7.4 at 25.0 ꢁ 0.1 ꢂC. 50 mL of BTPB
HONOO, ONOOCO2ꢀ, or metal–OONOꢀ complexes. Moreover, (1.0 mM) was added to ONOOꢀ (1.0 equiv.), then vigorously
phenyl boronic acid group was recently reported to be highly stirred for 30 seconds. Aer dilution to 1.0 mM with 100.0 mM
reactive to OONOꢀ nearly a million times faster than H2O2 and PBS buffer 1.0 mM CTAB, the mixture was vigorously stirred for
two hundred times faster than ClOꢀ.22,23 The (phenyl-2-yl) 1 min before measurement. The absorption and uorescence
benzothiazol scaffold was selected because the probe spectra of BTPB before and aer reaction with ONOOꢀ are shown
designed reacts with OONOꢀ to yield corresponding hydroxyl in Fig. 2. As is seen, BTPB shows a major UV-absorption centered
derivatives 2-(benzothiazol-2-yl)phenol, which possesses the at 296 nm, but its reaction with ONOOꢀ produces two absorption
excellent photophysical properties, such as high photo- peak at 290 nm and 338 nm. Moreover, BTPB itself displays a low
stabilities and high uorescence quantum yields, of members uorescence with the maximum emission peak at 372 nm,
of this family.24,25 The boronic acid group is converted to the whereas the reaction with ONOOꢀ leads to a uorescence
hydroxyl by the reaction with OONOꢀ, with the conversions enhancement with the maximum emission peak at 462 nm.
offering ratiometric responses because 2-(benzothiazol-2-yl)
The uorescence response of BTPB to varied concentrations
phenylboronic acid (BTPB) and 2-(benzothiazol-2-yl) phenol of ONOOꢀ (0–1.0 mM) was examined. When the emission
(BTP) uoresce at different wavelengths, which makes this wavelength was set to 372 nm, upon addition of ONOOꢀ, the
Fig. 1 Structure of BTPB and detection of peroxynitrite by oxidation of BTPB.
Fig. 2 Absorption spectra (a) and fluorescence spectra (b) of BTPB (1.0 mm) before and after reaction with ONOOꢀ (1.0 mm). Spectra were
obtained in 100.0 mm phosphate buffer (pH 7.4) with 1.0 mm cetyltrimethylammonium bromide (CTAB) under vigorous stirring for 1 min after
incubation of the probe with ONOOꢀ for 30 seconds at 25.0 ꢂC, lex ¼ 296 nm.
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main excitation peak of BTPB at 296 nm decreases with introduced into the aqueous phase. Thus BTPB molecules could
a concurrent increase in excitation peaks at 296 nm, 332 nm be encapsulated in micelles, which makes more intense emis-
and 386 nm when the emission wavelength was 462 nm (Fig. 3a sion of BTPB in CTAB-added PBS than that in PBS without CTAB
and b). Thus, the ratiometric feature in the spectra was obtained (Fig. S7†). The formation of micelles was also conrmed by
at an excitation wavelength of 296 nm.
means of scanning electron microscopy (Fig. S8†).
As expected, in the absence of ONOOꢀ, BTPB (1.0 mM) in
The selectivity of BTPB (1.0 mM) toward ONOOꢀ was deter-
100.0 mM PBS buffer with 1.0 mM CTAB at pH 7.4 showed a very mined by measuring uorescence changes that take place in the
poor uorescence at 372 nm (fF ¼ 0.05); upon addition of presence of various ROS and RNS including superoxide (O2cꢀ),
ONOOꢀ (1.0 mM), a dramatic uorescence enhancement was hypochlorite (ClOꢀ), hydroxyl radicals (cOH), hydrogen peroxide
observed immediately at 462 nm (fF ¼ 0.33). The ratio of (H2O2), single oxygen (1O2), tert-butoxyl radical (cOtBu), nitrite
emission intensities (F462/F372) upon excitation at 296 nm varies (NO2ꢀ) and nitric oxide (NO) in phosphate buffer (100 mM, pH
ꢀ
from 0.41 in the absence of ONOOꢀ to 26.45 aer reaction with 7.4, 1.0 mM CTAB) at 25 C. As shown in Fig. 4, only ONOO
ꢂ
ONOOꢀ, a ca. 65-fold emission ratio change (Fig. 3c), which is promotes a dramatic enhancement in uorescence intensity at
more than the response signal of CBA.18 As shown in Fig. 3d, 462 nm (F462/F372 ¼ 26.5). In contrast, no changes in emission
The ratio of emission intensities (F462/F372) exhibits a good intensities occur when the probe was incubated with O2cꢀ,
ꢀ
linear relationship in the concentration range of 0–1.0 mM H2O2, cOH, 1O2, cOtBu, NO2 and NO. ClOꢀ induces a small
ONOOꢀ with an equation of F ¼ 27.27 ꢃ [ONOOꢀ] (mM) + 0.41 uorescence response (F462/F372 ¼ 1.2). Taken together, these
(R2 ¼ 0.988), where F represents the ratio of uorescence results established that BTPB is sensitive enough and also
intensity of the reaction solution (F462/F372). The detection limit considerably selective toward ONOOꢀ over other ROS or RNS
was calculated to be 12.3 nM ONOOꢀ. These results revealed under the conditions of low concentration (1.0 mM) and short
that BTPB could potentially test ONOOꢀ in abiotic systems, reaction time (1.0 min). The observations also corroborate the
which is similar with physiological conditions, both qualita- previous nding that boronate-containing small mꢀolecules
tively and quantitatively.
react with ONOOꢀ much faster than with H2O2 or ClO .22,23
It is known that CTAB is a cationic surfactant and forms
Our approach to develop a ONOOꢀ specic probe relies on
spherical micelles within the concentration range 0.9–100 mM the use of oxidation of phenyl boronic acid group of BTPB to
in water. Both BTPB and BTP are low soluble in water; to apply hydroxyl group of BTP. Subsequently, to conrm this reaction,
them efficiently in an aqueous environment, CTAB was the process taking place between BTPB and ONOOꢀ was
Fig. 3 (a) Fluorescence excitation spectra of BTPB (1.0 mm) to OONOꢀ (0–1 equiv.), lem ¼ 372 nm. (b) Fluorescence excitation spectra of BTPB
(1.0 mm) to OONOꢀ (0–1 equiv.), lem ¼ 462 nm. (c) Fluorescence emission spectra of BTPB (1.0 mm) to OONOꢀ (0–1 equiv.), lex ¼ 296 nm. (d)
Linear relationship of emission intensity ratio (F462/F372) to OONOꢀ (0–1.0 mm). All spectra were obtained in 100.0 mm phosphate buffer (pH 7.4)
with 1.0 mm cetyltrimethylammonium bromide (CTAB) under vigorous stirring for 1 min after incubation of the probe with ONOOꢀ for 30
seconds at 25.0 ꢂC.
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Fig. 4 (a) Fluorescence emission spectra of BTPB (1.0 mm) to various ROS and RNS (1 equiv. ONOOꢀ and 10 equiv. for other ROS and RNS). (1)
Blank; (2) ONOOꢀ; (3) cOH; (4) ClOꢀ; (5) H2O2; (6) NO2ꢀ; (7) NO; (8) O2cꢀ; (9) 1O2; (10) cOtBu. (b) Emission intensity ratio (F462/F372) of various ROS
and RNS.
monitored by using HPLC. HPLC analysis of the mixture transfer of BTPB molecule and results in uorescent emission
generated from the reaction of ONOOꢀ with BTPB showed that enhancement. The red shis in emission maxima could be
in the presence of various concentrations of ONOOꢀ, the attributed to the lower-energy emission of the phenolate anion
intensity of the peak of BTPB (eluted at 2.0 min) gradually of BTP in PBS at pH 7.4, which matches the peak emission of the
decreased, in the meanwhile that of the peak of oxidation deprotonated phenolate anion of benzothiazol derivative in
products BTP eluted at 5.5 min emerged and gradually aqueous solution.30,31
increased (Fig. S9†). The results conrmed that the uorescent
MTT assays (Fig. S10†) revealed that BTPB of concentrations
product was indeed BTP, resulting from the ONOOꢀ induced below 40 mM does not exhibit obvious cytotoxicity living HepG2
oxidation reaction of BTPB. The ratiometric uorescence cells. Finally, encouraged by the above promising results,
mechanism is concerned with internal charge transfer (ICT). especially fast response rate (within 30 seconds), excellent
The boron atom in boronic acid group is sp2 hybridized and sensitivity (detection limit ¼ 12.3 nM) and high selectivity,
possesses ercely electron-withdrawing character due to the BTPB was employed for confocal imaging of ONOOꢀ in the liver
availability of its vacant p orbitals, forming the ICT state with tissue sample. ONOOꢀ formation has long been implicated in
benzothiazol moity as the electron-donating group. Aer the APAP-induced liver injury on the basis of indirect detection, i.e.,
reaction with ONOOꢀ, the boronic acid group is converted to immunostaining of protein nitration, which, however, cannot
the electron-donating hydroxyl, which interferes internal charge be used as unique evidence for ONOOꢀ formation. To address
Fig. 5 Hematoxylin and eosin staining of liver sections for hepatic necrosis (a–d) and confocal microscopic fluorescence images of liver sections
for detecting of ONOOꢀ generation via BTPB (e–h) in controls and 6 h after 400 mg kgꢀ1 APAP. (a) Control, ꢃ100. (b) Control, ꢃ200. (c) 6 h after
400 mg kgꢀ1 APAP, ꢃ100. (d) 6 h after 400 mg kgꢀ1 APAP, ꢃ200. (e) Control, ꢃ100. (f) 6 h after 400 mg kgꢀ1 APAP, ꢃ100. (g) 6 h after 400 mg
kgꢀ1 APAP, ꢃ200. (h) 6 h after 400 mg kgꢀ1 APAP, ꢃ400.
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this question, the mice administrated APAP overdose
(400 mg kgꢀ1) were employed to monitor ONOOꢀ formation in
situ. Six hours aer administrated APAP overdose, living heart
tissues were perfused with BTPB (1.0 mM) in saline for 3 min
under anesthesia. Mouse livers were then excised and cryosec-
tioned at 10 mm intervals for confocal imaging and hematoxylin
and eosin (HE) staining. The mice without APAP administration
were perfused with BTPB as the control.
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In summary, we present herein a ratiometric uorescent probe
for the highly sensitive and selective detection and hepatic
imaging of ONOOꢀ aer APAP overdose. This probe was
designed based on a ONOOꢀ specic reaction with the boronic
acid group. Our study provided the direct evidence for sup-
porting the generation of ONOOꢀ in APAP-induced liver injury.
We believe that the probe with excellent spectroscopic proper-
ties will nd its applications in understanding the actions of
ONOOꢀ in vivo.
Conflicts of interest
The authors declare no conicts of interest.
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Acknowledgements
We acknowledge the National Natural Science Foundation of
China (Grant No. 81272448).
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6514 | RSC Adv., 2019, 9, 6510–6514
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