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

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.

During resulting in MPT, it was recognized that the strong oxidant and nitrating peroxynitrite (ONOO À ), formed from superoxide (O 2 c À ) and nitric oxide (NO), is being generated in hepatocytes and a critical mediator in the injury mechanism. The generation of ONOO À was only demonstrated by immunohistochemical staining for 3-nitrotyrosine protein adducts. 7,8 The well-known nitration mechanism involves the initial oneelectron oxidation of tyrosine to yield tyrosyl radical followed by a diffusion-controlled reaction with nitrogen dioxide (cNO 2 ) to yield 3-nitrotyrosine. 9,10 However, in biological systems, cNO 2 arises from a variety of sources, except most notably the decomposition of ONOO À , but the hemeperoxidase-dependent oxidation of nitrite, 10,11 which is a ONOO À -independent mechanism. Actually, protein 3-nitrotyrosine, which was initially conceived as a specic footprint of ONOO À , typically represents a fairly stable end product that is generated by the action of NOderived oxidants. 12 It seems insufficient for the generation of ONOO À aer the administration of APAP overdose only according to the observation of the formation of protein 3nitrotyrosine. Therefore, there is an urgent need to provide the direct evidence for supporting the generation of ONOO À aer APAP overdose.
The lifetime of ONOO À in biological systems is limited to only a few milliseconds, which makes it impossible to quantitate ONOO À in processed cell or tissue samples directly. 13 Fluorescence based techniques are advantageous for detection of ONOO À . In accordance with the multiplicity of ONOO À biochemistry, 14 the triuorocarbonyl (HKGreen 1-3) 15-17 and phenyl boronic esters (CBA) 18 based uorescent probes, which can directly detect OONO À , 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. Chemiluminescence (CL) probes 19,20 can distinguish ONOO À from a variety of other ROS owing to the radicals from the decomposition of ONOO À , such as ONOOH, cOH and O 2 c À , not directly to the reaction with ONOO À . Additionally, CL probes were not used to detect OONO À in vivo.
In this regard, to provide direct and strong evidence for the formation of OONO À in liver aer APAP overdose, we developed a novel and low background phenyl boronic acid-containing uorogenic probe for sensitive and selective detecting of OONO À . This probe was introduced into the mice administrated APAP overdose by heart perfusion to monitor in situ the formation of OONO À in liver.
The new uorescent probe was designed by the direct conjugation of an boronic acid reaction group with a (phenyl-2yl)benzothiazol scaffold, serving as a uorophore. Phenyl boronic acid group was selected because the group can react with OONO À directly, rapidly and stoichiometrically, 21 which is more suitable to detect OONO À than other groups that react with those secondary radicals derived from OONO À , including HONOO, ONOOCO 2 À , or metal-OONO À complexes. Moreover, phenyl boronic acid group was recently reported to be highly reactive to OONO À nearly a million times faster than H 2 O 2 and two hundred times faster than ClO À . 22,23 The (phenyl-2-yl) benzothiazol scaffold was selected because the probe designed reacts with OONO À to yield corresponding hydroxyl derivatives 2-(benzothiazol-2-yl)phenol, which possesses the excellent photophysical properties, such as high photostabilities and high uorescence quantum yields, of members of this family. 24,25 The boronic acid group is converted to the hydroxyl by the reaction with OONO À , with the conversions offering ratiometric responses because 2-(benzothiazol-2-yl) phenylboronic acid (BTPB) and 2-(benzothiazol-2-yl) phenol (BTP) uoresce at different wavelengths, which makes this probe sensitive. More importantly, benzothiazol group is metabolically stable and a usual drug fragments, 26,27 and this probe has a low molecular weight and appropriate water solubility, which makes this probe suitable to use in vivo. The design rationale is depicted in Fig. 1, and illustrated as follows. Probe BTPB was prepared using the one-step reaction shown in ESI. † 28 2-(Benzothiazol-2-yl) phenol, BTP, the expected product resulting from reaction of BTPB with ONOO À , was also prepared utilizing a previously described method. 29 The structures of BTPB and BTP were conrmed by 1 H NMR, 13 C NMR, and HRMS spectra ( Fig. S1-S6 †), which match those of literatures. 28,29 Firstly, the spectroscopic response of BTPB to ONOO À was investigated in an abiotic system. We examined the spectroscopic properties of BTPB (1.0 mM) towards ONOO À (1 equiv.) in 100.0 mM PBS buffer with 1.0 mM cetyltrimethylammonium bromide (CTAB) at pH 7.4 at 25.0 AE 0.1 C. 50 mL of BTPB (1.0 mM) was added to ONOO À (1.0 equiv.), then vigorously stirred for 30 seconds. Aer dilution to 1.0 mM with 100.0 mM PBS buffer 1.0 mM CTAB, the mixture was vigorously stirred for 1 min before measurement. The absorption and uorescence spectra of BTPB before and aer reaction with ONOO À are shown in Fig. 2. As is seen, BTPB shows a major UV-absorption centered at 296 nm, but its reaction with ONOO À produces two absorption peak at 290 nm and 338 nm. Moreover, BTPB itself displays a low uorescence with the maximum emission peak at 372 nm, whereas the reaction with ONOO À leads to a uorescence enhancement with the maximum emission peak at 462 nm.
The uorescence response of BTPB to varied concentrations of ONOO À (0-1.0 mM) was examined. When the emission wavelength was set to 372 nm, upon addition of ONOO À , the  Paper main excitation peak of BTPB at 296 nm decreases with a concurrent increase in excitation peaks at 296 nm, 332 nm and 386 nm when the emission wavelength was 462 nm (Fig. 3a  and b). Thus, the ratiometric feature in the spectra was obtained at an excitation wavelength of 296 nm.
As expected, in the absence of ONOO À , BTPB (1.0 mM) in 100.0 mM PBS buffer with 1.0 mM CTAB at pH 7.4 showed a very poor uorescence at 372 nm (f F ¼ 0.05); upon addition of ONOO À (1.0 mM), a dramatic uorescence enhancement was observed immediately at 462 nm (f F ¼ 0.33). The ratio of emission intensities (F 462 /F 372 ) upon excitation at 296 nm varies from 0.41 in the absence of ONOO À to 26.45 aer reaction with ONOO À , a ca. 65-fold emission ratio change (Fig. 3c), which is more than the response signal of CBA. 18 As shown in Fig. 3d, The ratio of emission intensities (F 462 /F 372 ) exhibits a good linear relationship in the concentration range of 0-1.0 mM ONOO À with an equation of F ¼ 27.27 Â [ONOO À ] (mM) + 0.41 (R 2 ¼ 0.988), where F represents the ratio of uorescence intensity of the reaction solution (F 462 /F 372 ). The detection limit was calculated to be 12.3 nM ONOO À . These results revealed that BTPB could potentially test ONOO À in abiotic systems, which is similar with physiological conditions, both qualitatively and quantitatively.
It is known that CTAB is a cationic surfactant and forms spherical micelles within the concentration range 0.9-100 mM in water. Both BTPB and BTP are low soluble in water; to apply them efficiently in an aqueous environment, CTAB was introduced into the aqueous phase. Thus BTPB molecules could be encapsulated in micelles, which makes more intense emission of BTPB in CTAB-added PBS than that in PBS without CTAB (Fig. S7 †). The formation of micelles was also conrmed by means of scanning electron microscopy (Fig. S8 †).
The selectivity of BTPB (1.0 mM) toward ONOO À was determined by measuring uorescence changes that take place in the presence of various ROS and RNS including superoxide (O 2 c À ), hypochlorite (ClO À ), hydroxyl radicals (cOH), hydrogen peroxide (H 2 O 2 ), single oxygen ( 1 O 2 ), tert-butoxyl radical (cO t Bu), nitrite (NO 2 À ) and nitric oxide (NO) in phosphate buffer (100 mM, pH 7.4, 1.0 mM CTAB) at 25 C. As shown in Fig. 4 Our approach to develop a ONOO À specic probe relies on the use of oxidation of phenyl boronic acid group of BTPB to hydroxyl group of BTP. Subsequently, to conrm this reaction, the process taking place between BTPB and ONOO À was monitored by using HPLC. HPLC analysis of the mixture generated from the reaction of ONOO À with BTPB showed that in the presence of various concentrations of ONOO À , the intensity of the peak of BTPB (eluted at 2.0 min) gradually decreased, in the meanwhile that of the peak of oxidation products BTP eluted at 5.5 min emerged and gradually increased (Fig. S9 †). The results conrmed that the uorescent product was indeed BTP, resulting from the ONOO À induced oxidation reaction of BTPB. The ratiometric uorescence mechanism is concerned with internal charge transfer (ICT). The boron atom in boronic acid group is sp 2 hybridized and possesses ercely electron-withdrawing character due to the availability of its vacant p orbitals, forming the ICT state with benzothiazol moity as the electron-donating group. Aer the reaction with ONOO À , the boronic acid group is converted to the electron-donating hydroxyl, which interferes internal charge transfer of BTPB molecule and results in uorescent emission enhancement. The red shis in emission maxima could be attributed to the lower-energy emission of the phenolate anion of BTP in PBS at pH 7.4, which matches the peak emission of the deprotonated phenolate anion of benzothiazol derivative in aqueous solution. 30,31 MTT assays (Fig. S10 †) revealed that BTPB of concentrations below 40 mM does not exhibit obvious cytotoxicity living HepG2 cells. Finally, encouraged by the above promising results, especially fast response rate (within 30 seconds), excellent sensitivity (detection limit ¼ 12.3 nM) and high selectivity, BTPB was employed for confocal imaging of ONOO À in the liver tissue sample. ONOO À formation has long been implicated in APAP-induced liver injury on the basis of indirect detection, i.e., immunostaining of protein nitration, which, however, cannot be used as unique evidence for ONOO À formation. To address  this question, the mice administrated APAP overdose (400 mg kg À1 ) were employed to monitor ONOO À 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 cryosectioned 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.
As shown in Fig. 5, HE staining of liver sections indicate the substantial centrilobular necrosis 6 hours aer APAP administration, but no necrosis is observed in the control group without APAP administration, suggesting that APAP overdose induces liver injury, which is consistent with those studies. 32,33 Then liver sections were monitored using a confocal microscope over the wavelength range of 417-477 nm with an excitation wavelength at 408 nm. Without APAP administration no uorescence in the liver sections was observed, while strong blue uorescence in the centrilobular regions was imaged aer treatment with overdose APAP, which correlated well with the areas of necrosis. This result indicates the generation of ONOO À during APAP-induced liver injury, which is the rst direct evidence provided by our study. Also, these observations suggest that BTPB has satisfactory cell permeability and is wellretained during histological sample preparation and sensitive enough to visualize endogenous ONOO À in live tissues.

Conclusions
In summary, we present herein a ratiometric uorescent probe for the highly sensitive and selective detection and hepatic imaging of ONOO À aer APAP overdose. This probe was designed based on a ONOO À specic reaction with the boronic acid group. Our study provided the direct evidence for supporting the generation of ONOO À in APAP-induced liver injury. We believe that the probe with excellent spectroscopic properties will nd its applications in understanding the actions of ONOO À in vivo.

Conflicts of interest
The authors declare no conicts of interest.