Rationally Optimized Fluorescent Probe for Imaging Mitochondrial SO2 in HeLa Cells and Zebrafish

Article abstract of DOI:10.1021/acs.analchem.8b01505

Organisms have built up immunological systems, where mitochondrial SO₂ plays conflicting roles in regulating cell apoptosis. However, no exploration on the influence and regulating principle of mitochondrial SO₂ to the specific apoptosis type can be found, which brings about a challenge to fluorescent probes. Herein, we optimize the fluorophore and develop a new fluorescent probe FHMI ((E)-4-(3-formyl-4-hydroxystyryl)-1-methylpyridin-1-iumiodide) by equipping an ICT (intramolecular charge transfer) fluorophore HMII ((E)-4-(4-hydroxystyryl)-1-methylpyridin-1-ium iodide) with an aldehyde group that serves as both fluorescence quencher and reporting group. After the optimization, although the nonconjugated electron donor is formed when sensing SO₂, the preset ICT fluorophore HMII is permitted to release the fluorescence at the enlarged wavelength. Compared with the traditional design, the probe FHMI exhibits obvious enhanced fluorescence with large red shift. FHMI is successfully applied to the mechanistic exploration of the dichotomous effects of mitochondrial SO₂ to cells apoptosis, showing that mitochondrial SO₂ regulates the early apoptosis of HeLa cells via the reduction of mitochondrial membrane potential. FHMI is applied to explore the dichotomous bioinfluence of mitochondrial SO₂ to HeLa cells under oxidative stress, visualizing the regulative role of mitochondrial SO₂ in the apoptotic process. For the first time, the mitochondrial SO₂ is visually found to be closely associated with the early apoptosis of HeLa cells. Moreover, FHMI proves to be readily applicable to monitoring endogenous SO₂ in zebrafish. This probe can act as an effective optical tool for exploring SO₂ in biospecimen.

Full text of DOI:10.1021/acs.analchem.8b01505

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Article
A rationally optimized fluorescent probe for imaging
mitochondrial SO2 in HeLa cells and zebrafish
Guang Chen, Wei Zhou, Chenyang Zhao, Yuxia Liu, Tao Chen, Yulin Li, and Bo Tang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01505 • Publication Date (Web): 09 Oct 2018
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Analytical Chemistry
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A rationally optimized fluorescent probe for imaging mitochondrial
SO2 in HeLa cells and zebrafish
Guang Chena, b, Wei Zhoua, Chenyang Zhaoa, Yuxia Liu*a,b, Tao Chenc, Yulin Lic and Bo Tang*b
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a. The Key Laboratory of LifeꢀOrganic Analysis; Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural
Medicine, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China.
b. College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized
Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Educ
ation, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China.
*Eꢀmail: tangb@sdnu.edu.cn; Tel.: +86 0531ꢀ86180010. Fax: +86 0531ꢀ86180017; *Eꢀmail: liuyuxia2008@163.com.
c. Key Laboratory of Tibetan Medicine Research & Qinghai Key Laboratory of QinghaiꢀTibet Plateau Biological Resources,
Northwest Institute of Plateau Biology, Chinese Academy of Science, Xining 810001, Qinghai, PR China.
ABSTRACT: Organism has built up immunologic system, where mitochondrial SO2 plays the conflicting roles in regulating cell
apoptosis. However, no exploration on the influence and regulating principle of mitochondrial SO2 to the specific apoptosis type
can be found, which brings about challenge to fluorescent probe. Herein, we optimize the fluorophore and develop a new fluoresꢀ
cent probe FHMI ((E)ꢀ4ꢀ(3ꢀformylꢀ4ꢀhydroxystyryl)ꢀ1ꢀmethylpyridinꢀ1ꢀiumiodide) by equipping an ICT (intramolecular charge
transfer) fluorophore HMII ((E)ꢀ4ꢀ(4ꢀhydroxystyryl)ꢀ1ꢀmethylpyridinꢀ1ꢀium iodide) with an aldehyde group that serves as both
fluorescence quencher and reporting group. After the optimization, although the nonꢀconjugated electron donor is formed when
sensing SO2, the preset ICT fluorophore HMII is permitted to release the fluorescence at the enlarged wavelength. Comparing with
the traditional design, the probe FHMI exhibit obvious enhanced fluorescence with large red shift. FHMI is successfully applied to
the mechanistic exploration of the dichotomous effects of mitochondrial SO2 to cells apoptosis, showing that mitochondrial SO2
regulates the early apoptosis of HeLa cells via the reduction of mitochondrial membrane potential. FHMI is applied to explore the
dichotomous bioꢀinfluence of mitochondrial SO2 to HeLa cells under oxidative stress, visualizing the regulative role of mitochonꢀ
drial SO2 in the apoptotic process. For the first time, the mitochondrial SO2 is visually found to be closely associated with the early
apoptosis of HeLa cells. Moreover, FHMI proves to be readily applicable to monitoring endogenous SO2 in zebrafish. This probe
can act as an effective optical tool for exploring SO2 in bioꢀspecimen.
Mammalian cells are always kept in the oxidative siege
where their survival requires an appropriate balance beꢀ
tween oxidants and antioxidants. Overproduction of reacꢀ
tive oxygen species (ROS) or the failure in antioxidant deꢀ
fenses against ROS will cause pathogenesis, such as metaꢀ
bolic syndrome1, DNA damage2 and even carcinogenesis3.
Evidence shows that an altered redox status accompanied
by the elevated ROS has nearly always been observed in
cancer cells, where the redox signaling regulates various
transcription factors4. As the reflection of oxidative stress,
the transcription factors in turn control the expression of
antiꢀapoptosis, proliferation and angiogenesis5,6. By virtue
of natural evolution, organism has built up immunologꢀ
ic mechanism, through which the antioxidant agents can
counter the rising of ROS by suppressing the pathway of
oxidative stress7. Therefore, exploration of reciprocal relaꢀ
tionship between oxidativeꢀstress with the antioxidant caꢀ
pacity is of great significance to organism8,9.
containing amino acids or the decomposition of sulfinylpyꢀ
ruvate10. Increasing studies suggest that SO2, participates
actively in regulating the redox status as an antioxidant
against ROS11ꢀ14. However, SO2 also possesses the potenꢀ
tial oxidative ability15,16, thus in the complicated bioꢀ
environment, SO2 may act as both the signaling molecule
and the oxidative stressꢀinducer. Therefore, exploring the
crossꢀtalk influence of SO2 with ROS to cells is very signifꢀ
icant for further understanding the dichotomous role of SO2
inside the cell17. Mitochondria plays the key roles in metaꢀ
bolic regulation of the innate and adaptive immune reꢀ
sponses to cellular damage and stress18. With the glutaꢀ
mate, mitochondria can localize the aspartate aminotransꢀ
ferase, catalyzing the reactions of Lꢀcysteine to generate
SO219,20. Given the antiꢀoxidative role of SO2, whatever the
absolute amount of mitochondrial SO2, it is likely that the
mitochondrial SO2 may suppress the oxidative stress and
retard the progression of cell apoptosis under oxidative
stress. Conversely, given the oxidative effect of SO2, the
damaged mitochondria may set in motion a vicious cycle of
ꢀ
Sulfur dioxide (SO2), existing with the form of HSO3 in
organism, can be produced via the oxidation of sulphurꢀ
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cell apoptosis, leading to the increased ROS production
which in turn leads to more cell apoptosis21ꢀ23. Taking into
account the conflicting properties of SO2 together, we reaꢀ
son that inꢀdepth exploring the dichotomous role of mitoꢀ
chondrial SO2 will provide invaluable information for celꢀ
lular regulatory mechanisms, promising to promote the
development of chemotherapeutic intervention for cancer
cells. Unfortunately, no report yet describing the conflictꢀ
ing effects of mitochondrial SO2 on the apoptotic regulation
of cancer cells was found by us. Furthermore, as far as the
apoptosis types are concerned, different apoptosis, such as
early or late apoptosis, reflect their respective regulating
principles. For example, oxidative stress causes the DNA
fragmentation that is closely associated with late
apoptosis24,25. However, no exploration on the influence
and regulating principle of mitochondrial SO2 to the specifꢀ
ic apoptosis type can be found. Therefore, the growing inꢀ
terest in mitochondrial SO2 drives us to focus on these
studies.
diformylphenol (215 mg, 45% yield) as yellow powder.
Characterizations: H NMR (500 M, d6ꢀDMSO): 11.55 (s,
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1H), 10.00 (s, 1H), 9.94 (s, 1H), 8.14 (d, J = 2.1 Hz, 1H),
8.07 (dd, J = 8.7, 2.1 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H). m/z
calcd for [+HꢀC8H6O3]: 151.06, found: 151.0131. A soluꢀ
tion of 4ꢀmethylpeidine (4 mL, 2 mmol) in diethyl ether (12
mL) was treated with methyl iodide (1.5 mL, 2.5 mmol).
After reaction, the crystalline 1,4ꢀdimethylpyridinꢀ1ꢀium
1
iodide was isolated. Characterizations: H NMR (500 M,
d6ꢀDMSO): δ 9.13 (d, J =6.5, Hz, 1H), 7.87 (d, J =6.0, Hz,
2H), 4.61 (s, 3H), 2.67 (s, 3H). m/z calcd for [C7H10N+]:
108.08, found: 108.0804. A mixture of 2,4ꢀdiformylphenol
(10 mg, 0.067 mmol), piperidine (100 ꢁL, 0.133 mmol) and
4ꢀmethylpeidine iodoide (31.2 mg, 0.133 mmol) was disꢀ
solved in EtOH (10 mL). The mixture was stirred for 30
min at 80 oC under an N2 atmosphere to give the compound
FHMI as a yellow solid (20 mg, 80% yield). Characterizaꢀ
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tions for FHMI: H NMR (500 MHz, d6ꢀDMSO): δ= 9.39
(s, 1H), 8.79 (d, J =7.0 Hz, 2H), 8.01 (s, 1H), 7.97 (s, 2H),
7.91 (d, J =6.5 Hz, 1H), 7.80 (d, J =2.0 Hz, 1H), 7.67 (s,
1H), 7.64 (s, 1H), 7.39 (dd, J =2.5, 2.5 Hz, 1H), 6.32(d, J
=9.0 Hz, 1H), δ 4.10 (s, 3H). 13C NMR (125.8 MHz, d6ꢀ
DMSO): δ= 187.38, 179.58, 155.65, 144.25, 143.01,
Though many excellent fluorescent probes for SO2 have
been developed26ꢀ32, no report yet describing the conflicting
effects of mitochondrial SO2 on apoptotic regulation can be
found. Moreover, many probes suffer the blue shift due to
the nonꢀcoplanar addition reaction of SO2, which was not
satisfactory enough for biological imaging. Herein, we
conceived a smart ICT probe FHMI by optimize the fluorꢀ
ophore. As illustrated in Scheme 1, first, we designed an
ICT fluorophore HMII which exhibited the strong fluoresꢀ
cence at 525 nm. Then, we equipped the aldehyde to the
HMII as both the quenching and the weak electronꢀ
withdrawing effect, and achieved the probe FHMI. When
reacted with SO2, FHMI exhibited the strong fluorescence
at 560 nm. Comparing with the traditional design as shown
in FMPI/HTMI, HHMI showed the enhanced fluorescence
as well as the 100 nm of read shift. FHMI was used to inꢀ
depth explore the bioꢀeffect and the regulating principle of
mitochondrial SO2 on the cells apoptosis. For the first time,
the mitochondrial SO2 was found to be closely related with
the early apoptosis of HeLa cells via the reduction of mitoꢀ
chondrial membrane potential with the aid of fluorescence
imaging. Under the oxidative stress, the dichotomous influꢀ
ence of mitochondrial SO2 to HeLa cells was explored. In
addition, the developed probe FHMI was applied to the
fluorescence imaging of SO2 in zebrafish, showing its roꢀ
bust applicability.
+
121.88, 119.49, 117.37, 46.39. m/z calcd for [C15H14NO2 ]:
240.10, found: 240.2533. The compounds HMII and FMPI
were also prepared (Supporting Information).
Visualizing mitochondrial SO2 on cells apoptosis
under oxidative stress. To investigate the dichotomous
roles of mitochondrial SO2, the crossꢀtalk experiments were
performed. First, HeLa cells were pretreated with the
DMEM (Dulbecco′s modified eagle media) medium conꢀ
taining FHMI (10 ꢁM) for 30 min and the medium containꢀ
ing MTR (MitoꢀTracker®Red, 0.08 ꢁM) for 15 min, reꢀ
spectively.
Then
BTSA
(Nꢀbenzylꢀ2,4ꢀdiniꢀ
trophenylsulfonamide, 200 ꢁM) was added to endogenousꢀ
ly generate the SO2 in absence or presence of NaClO (100
ꢁM). The resultant cells were incubated for 3 h. As the conꢀ
trol experiment, HeLa cells pretreated with probe FHMI
(10 ꢁM) and MTR (0.08 ꢁM) were directly stimulated by
the media containing NaClO (100 ꢁM) for 3 h. The whole
process was followed by confocal laser scanning microꢀ
scope (Japan Olympus Co., Ltd) with an objective lens
(×10) and the flow cytometry (ACEA NovoCyte, Hangꢀ
zhou, China) to report the homeostasis of mitochondrial
SO2 and the cellular apoptosis rate. Annexin VꢀFITC (fluoꢀ
rescein isothiocyanate) and PI (propidium iodide) was used
to indicate the different population of cells in the apoptosis
analysis. To follow the change of mitochondrial membrane
potential, HeLa cells was incubated with JCꢀ1 stainꢀ
ing working solution (50 ꢁL JCꢀ1 in 8000 ꢁL H2O and
2000 ꢁL staining buffer) in absence or presence of BTSA
(200 ꢁM) for 3 h. The resultant cells were analyzed with
the confocal laser scanning microscope with excitation
wavelengths set as 515 and 488 nm and collection waveꢀ
lengths set as 600−700 nm and 500ꢀ600, respectively.
EXPERIMENTAL SECTION
Synthesis of probe. To the solution of phenol (300 mg,
3.19 mmol) in toluene (20 mL), acetic acid (25 mL) and
hexamethylenetetramine (983 mg, 7.03 mmol) were sucꢀ
cessively added. The orange solution was refluxed until all
the starting material was consumed (18 hrs). The mixture
was then cooled to room temperature and poured into 6M
HCl (30 mL). The product was extracted with ethyl acetate
(3 × 20 mL). The combined organic extracts were washed
with saturated brine (40 mL) and then dried over Na2SO4.
The crude product was purified by column chromatography
(silica gel, ethyl acetate: hexane = 1:10) to obtain 2,4ꢀ
Fluorescence imaging in zebrafish. Zebrafishes were
provided by HuanTe biological corporation (Hangzhou,
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China). Zebrafishes were incubated in E3 media (15 mM
could be constructed, the probe would be in the fluoresꢀ
cence off state. To put this conception into action, we perꢀ
formed Scheme 1A (c) and obtained probe FHMI. As a
result, FHMI exhibited weak fluorescence (Ø=0.02), indiꢀ
cating that aldehyde effectively quenched the fluorescence.
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NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM
KH2PO4, 0.05 mM Na2HPO4 and 0.7 mM NaHCO3; pH
7.5) containing 10 ꢁM FHMI probe at 28 °C for 1 h, and
then, the residual probe was washed by the E3 media. To
ꢀ
ꢀ
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image the HSO3 , zebrafish larvae (threeꢀdays) were put
Upon the addition reaction by HSO3 , FHMI was transꢀ
into a 50 mm Petri dish filled with E3 media containing
BTSA for 1~5 h. Fluorescence imaging was subsequently
carried out with a confocal laser scanning microscope (Jaꢀ
pan Olympus Co., Ltd) with an objective lens (×10). The
excitation wavelengths were 515 nm and 488 nm, and colꢀ
lection windows were at 600−700 nm and 500ꢀ600 nm,
respectively. All animal experiments were performed in
full compliance with international ethical guidelines.
formed to the product HHMI (Ø=0.38) which exhibited the
obviously enhanced fluorescence. Moreover, comparing
with FMPI, probe FHMI exhibited large red shift (100 nm)
in emission, indicating that aldehyde served as not only the
fluorescence quencher but also the weak electronꢀ
withdrawing group. To confirm this point, we performed
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series of investigation. HNMR titration of FHMI (Figure
S2) indicated, upon addition of NaHSO3, the peak of aldeꢀ
hyde H at 9.39 ppm disappeared and a new peak at 6.51
ppm rose up, demonstrating the transformation of aldeꢀ
hydeꢀH to quaternary carbonꢀH. Meanwhile, the appearꢀ
ance of peaks at 3.63 ppm indicated the formation of hyꢀ
droxyl H in this quaternary carbon. Moreover, the appearꢀ
ance of peaks at 10.15 ppm displayed the sulfonic H in this
quaternary carbon. These results confirmed that probe unꢀ
derwent a nucleophilic addition reaction with analyte. To
validate the nonꢀcoplanar structure and the electronic beꢀ
havior, we calculated the molecular structure with density
functional theory (DFT)33ꢀ36. As displayed in Scheme 2B,
the molecule FHMI had a large planar conjugated structure
in which the hydroxyl oxygen (donor), the ketonic oxygen
(acceptorꢀ2) as well as the positive nitrogen (acceptorꢀ1)
RESULTS AND DISCUSSION
Optimizing the design for probe FHMI. Our inspiraꢀ
tion came from the optical properties of FMPI (Scheme 1A
(b)). We found that the two acceptors coꢀplanar in FMPI
led to the weak response at 450 nm. This phenomena indiꢀ
cated that, without a donor, the ICT suffered fluorescence
quenching when SO2 had a nonꢀcoplanar addition reaction
with probe. Thus, we thought about to improve the fluoresꢀ
cence intensity by equipping an electron donor to the Π
system. Accordingly, we synthesized HMII following
Scheme 1A (a) and investigated the contribution of a donor
to fluorescence enhancement. As expected, HMII exhibited
both the strong fluorescence intensity (Ø=0.66) and the red
shift of emission (75 nm) relative to FMPI (Figure 1A).
Optical comparison of HMII and FMPI provided us the
enlightenment that if the electronꢀwithdrawing quencher
ꢀ
were included. When sensing HSO3 , as illustrated in
Scheme 2B, FHMI was transformed to the product HHMI,
forming a saturated hydroxyl oxygen. Clearly, this oxygen
Scheme 1. A) Optimization of probe design; B) Theoretic calculation for the optimized structures of FHMI and HHMI; C): Dissection of
the HOMO molecular orbital for illustrating the switch of electron pushꢀpull. Green and red shapes are corresponding to the different
phases of the molecular wave functions for HOMO orbitals.
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was no more coꢀplanar with the large conjugated system
Page 4 of 9
ons, metal ions and RSS, resulted in insignificant interferꢀ
ence. Amino acids were investigated to evaluate whether
the amino group would cause interferences through the
reaction with aldehyde group of this probe. Results in Figꢀ
ure S12 showed that amino acids caused insignificant interꢀ
ference. Thus, probe FHMI could selectively report SO2
without being interfered by biologically relevant comꢀ
pounds. In addition, we established linear response of
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and therefore was no more competitive with the positive
nitrogen (acceptorꢀ1) in electronꢀwithdrawing. To further
confirm this phenomena, we dissected the molecular orbitꢀ
als for both FHMI and HHMI. As shown in Scheme 2C,
molecular orbital of ketonic group in FHMI displayed the
continuously conjugated Π orbital with hydroxyl oxygen
(electron donor) and positive nitrogen (electron acceptor).
ꢀ
ꢀ
In contrast, the molecular orbitals of HSO3 ꢀadded ketonic
FHMI to HSO3 (10ꢀ100 ꢁM) (Figure S11) and obtained
group in HHMI was fragmented and no more included in
the Π orbital. Consequently, above optical properties, exꢀ
perimental results and theoretical calculation demonstrated
that the probe FHMI was rationally constructed after an
optimization of designing.
detection limit of 13.1 nM, which was almost the lowest
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among the reported SO2 probes26ꢀ31,39,40. Therefore, the deꢀ
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ꢀ
signed probe FHMI was competent in detecting HSO3 in
biological samples.
Intracellular properties of FHMI. Dynamic fluoresꢀ
cence imaging (Figure S9) indicated that FHMI could rapꢀ
Optical properties of probe FHMI. Timeꢀdependent
fluorescence (Figure S6) of FHMI versus HSO3 showed
ꢀ
ꢀ
idly respond to intracellular HSO3 . Fluorescence intensity
that maximal response could be achieved within 60 s. This
rapid response could meet the requirement of inꢀsitu moniꢀ
toring. As the time progressed, the fluorescence exhibited a
steady signal, indicating the satisfactory photostability. The
remained almost unchanged for 300 min, indicating the
satisfactory photostability. Before cells analysis, cytotoxiciꢀ
ty of FHMI was evaluated by MTT assay (Supporting Inꢀ
formation). Figure S13 showed that more than 94% cells
remained alive when 10 ꢁM FHMI probe was added for 24
h, demonstrating the low toxicity of FHMI. Since, probe
FHMI was designed with a positive group, we speculated
that this probe would accumulate in the mitochondria. Thus
we carried out the coꢀstaining experiments in HeLa cells
ꢀ
pHꢀdependent fluorescent response of FHMI to HSO3 was
investigated. As shown in Figure S7, the probe FHMI itself
did not respond to the tested pH values (4.80~10.34). In
presence of NaHSO3, the pKa was estimated as 7.10.
Figure 1. A) Fluorescence properties for the synthesized comꢀ
Figure 2. Colocalization experiments of probe FHMI (10 µM)
with MitoTracker Red (MTR) (0.08 µM) in HeLa cells. A): MTR;
B): FHMI in presence of NaHCO3; C): Merged images of A) and
B); D) Colocalizationꢀcorrelation between red and yellow chanꢀ
nels in C) (P is the Pearson’s coꢀlocalization coefficient); E) 3D
perspective observation of C). F) Pixel correlation across line in
C). Scale bar: 20 ꢁm.
pounds (FHMI (10 ꢁM), HMII (10 ꢁM), FMPI (10 ꢁM),
ꢀ
HHMI(FHMI (10 ꢁM)+ HSO3 (300 ꢁM)) and HTMI (FMPI (10
ꢀ
ꢁM)+ HSO3 (300 ꢁM))) (FHMI and HHMI: λex=460 nm; HMII:
λex=400 nm; FMPI and HTMI: λex=360 nm); B) Fluorescence
ꢀ
response of probe FHMI (10 ꢁM) to HSO3 (0ꢀ100 ꢁM) All experꢀ
iments were performed in DMSO/PBS buffer solution (9/1, v/v,
pH = 7.4) at 37 °C.
Though there were the response change during pH of
6.84~7.40, this change was insignificant. Moreover, the
mitochondrial pH value was in the range 7.5~8.537,38. Thus,
the heterogeneous pH in mitochondria would not influence
the working of probe FHMI. Temperatureꢀdependent fluoꢀ
rescence was tested (Figure S8), showing that the highest
using FHMI with MitoTracker Red (MTR). After being
incubated with 10 ꢁM FHMI for 30 min, the HeLa cells
were stained with 0.08 ꢁM MTR for 15 min at 37 °C. Figꢀ
ure 2 showed the fluorescence image of HeLa cells with
good morphology after coꢀstaining. As illustrated in Figure
2a~b, the mitochondria profiles for MTR and probe FHMI
were obtained in two channels. The correlation was estiꢀ
mated with the dependency of fluorescence pixels (Figure
2c, 2f), showing the good correlation between the two
group pixels. Pearson's coꢀlocalization coefficient was deꢀ
termined to be 0.90, indicating that FHMI predominantly
accumulated in mitochondria of living cells. Therefore,
ꢀ
response of FHMI to HSO3 could be achieved at the physꢀ
o
iological temperature (37 C). Therefore, FHMI exhibited
the potential applicability for biological sample. Before the
ꢀ
intracellular application, the selectivity of FHMI to HSO3
against other possible interference was tested (Figure S12).
Relative fluorescence intensity showed that excessive aniꢀ
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probe FHMI could localize in mitochondria, the main orga
the early apoptosis caused by mitochondrial SO2 was exꢀ
plored with fluorescence imaging. To perform the further
investigation of this phenomena, we imposed artificial inꢀ
tervention of late apoptosis to cells. NaClO could induce
the late apoptosis49, and thus was added before the subseꢀ
quent addition of BTSA. Figure 4 Cꢀ3 showed the remarkꢀ
able late apoptosis, demonstrating that ClOꢀ specifically
caused the elevated level of late apoptotic cells rather than
early apoptotic cells49. As a result, the rate of early apoptoꢀ
sis was significantly reduced in contrast to group 2 and
correspondingly, the rather weak emission in yellow chanꢀ
nel (Figure 4 Aꢀ3) was observed. Clearly, the upꢀregulated
late apoptosis and the downꢀregulated early apoptosis as
well as the decreased mitochondrial SO2 demonstrated the
association of early apoptosis with mitochondrial SO2.
Thus, with the aid of FHMI, the injurious effect of mitoꢀ
chondrial SO2 to cells was visually demonstrated.
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nelle for endogenously generating SO2 in vivo. To investiꢀ
ꢀ
gate the response of this probe toward intracellular HSO3 ,
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we incubated HeLa cells with FHMI (10 ꢁM) and MTR
(0.08 ꢁM) in presence of different levels of HSO3 (Figure
ꢀ
3), in which, MTR served as both the reference and the
control for recording the fluorescence from the site where
FHMI and MTR coꢀlocalized. As can be seen from Figure
3, the yellow channel showed enhanced fluorescence with
ꢀ
the increase of HSO3 . The pseudocolored ratio images visꢀ
ualized the increased ratio of Iprobe/IMTR (0.55ꢀ9.21). These
results indicated that the developed probe FHMI was suitaꢀ
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ꢀ
ble for monitoring the mitochondrial HSO3 .
Exploring bio-influence of mitochondrial SO2 to
cells apoptosis. SO2 can be endogenously generated from
the sulphurꢀcontaining amino acids. Generally, SO2 exerts
the reducing property, serving as an antioxidant to elimiꢀ
nate the excessive ROS41,42 or as a therapeutic prodrug43.
While, SO2 also holds the oxidative potential to cause the
injury to cell and tissue44,45. Therefore, the reciprocal relaꢀ
tionship between oxidativeꢀstress with the antiꢀoxidative
stress is of great significance to organism. In the following
experiments, we monitored the mitochondrial SO2 during
cell apoptosis, so that to explore the apoptotic and/or antiꢀ
apoptotic effect of mitochondrial SO2 on cancer cells. HeLa
cells were first pretreated by FHMI (10 ꢁM) and MTR
(0.08 ꢁM) as the control group. The control cells were then
treated with the SO2 donor BTSA (100 ꢁM) for 3 h in abꢀ
sence or presence of NaClO (100 ꢁM). Besides, the control
cells were incubated directly with NaClO (100 ꢁM) for 3 h.
As shown in Figure 4 Aꢀ1, cells showed mainly the fluoꢀ
rescence in red channel but the negligible fluorescence in
yellow channel, indicating the quite low level of mitochonꢀ
drial SO2 in the normal cells. Under this condition, we
found 97.70% of cells were viable, 1.97% of cells were
necrotic, and the apoptotic cells were negligible (Figure 4
Bꢀ1; Cꢀ1). Stimulation by BTSA would trigger the burst of
mitochondrial SO2. As expected, the obvious yellow emisꢀ
sion and the increased ratio of Iprobe/IMTR were found in
Figure 4 Aꢀ2, indicating the elevated level of mitochondrial
SO2. Correspondingly, the apoptosis rate was obviously
increased (10.04(early)+13.93% (late)), implying the injuꢀ
rious influence of SO2 to cells.
Since the early apoptosis was always accompanied by
the disruption of inner mitochondrial membrane potential46,
we hypothesized that the excessive mitochondrial SO2 cont
ributed to the variation of inner mitochondrial membrane
potential (MMP) thereby causing the upregulated early
apoptosis47,48. To confirm this point, we investigated the
MMP by incubating the group 1 and 2 with JCꢀ1 (50 ꢁL
JCꢀ1 in 8000 ꢁL H2O and 2000 ꢁL staining buffer) in abꢀ
sence (left) or presence (right) of BTSA (100 ꢁM) for 3 h
(Figure S14). We observed the green fluorescence in group
2 whereas the red fluorescence in group 1. This result conꢀ
firmed that MMP was reduced in presence of SO2, which,
together with the above findings, justified that the upreguꢀ
lated early apoptosis was associated with the mitochondrial
SO2. To the best of our knowledge, this is the first time that
Figure 3. Confocal fluorescence images for HeLa cells preꢀ
treated with probe FHMI (10 ꢁM) with addition of different
ꢀ
levels of HSO3 (1: 80 ꢁM, 2: 60 ꢁM, 3: 40 ꢁM, 4: 20 ꢁM, 5: 0
ꢁM) for 30 min. a): the red channel for MitoTracker Red (0.08
ꢁM); b): the bright field; c) the yellow channel for FHMI; d):
the pseudo color images for fluorescence ratio of FHMI with
MTR. The color band represented the ratios (Iprobe /IMTR: 1)
9.21; 2) 7.10; 3) 4.82; 4) 1.79; 5) 0.55.). Scale bar: 20 ꢁm.
Since, SO2 might play the dichotomous roles in the
transduction of the apoptotic and antiꢀapoptotic signals
with its oxidizing and antiꢀoxidizing properties50, we were
inspired to explore the other effect of SO2 on regulating the
apoptotic system. Apoptotic analysis induced directly by
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Figure 4. Confocal microscopy images (A), apoptosis analyꢀ
sis (B) and apoptotic population (C). HeLa cells were incuꢀ
bated respectively with probe FHMI (10 ꢁM) for 30 min and
then MTR (0.08 ꢁM) for 15 min, to obtain the control group
(Aꢀ1), and then incubated with BTSA (100 ꢁM) for 3 h in
absence (Aꢀ2) or presence (Aꢀ3) of NaClO (100 ꢁM). To
perform comparison, HeLa cells in control group were incuꢀ
bated with the media consisting of NaClO (100 ꢁM) for 3h
(Aꢀ4). The confocal fluorescence images were collected, inꢀ
cluding a: the red channel (for IMTR), b: the yellow channel
(for Iprobe), c: bright field, d: the pseudo colour images for the
fluorescence ratio of Iprobe/IMTR. The colour band represented
the ratios (Iprobe/IMTR: 1) 0.79; 2) 5.12; 3) 2.17; 4) 0.69.). The
apoptotic population were obtained with the average means
from replicated experiments (n=15) and the representative
apoptosis distribution was illustrated in C, where 1~4 referred
to the four groups of cells. The statistical analysis for apoptoꢀ
sis experiments were performed with Student’s tꢀtest: *P
<0.05. Scale bar: 20 ꢁm.
Figure 5. Fluorescence images for zebrafish larvae. 1: The
zebrafish was fed with probe FHMI (10 ꢁM) in E3 media for 1 h
and was then washed by E3 media; 2: larva preꢀtreated with proꢀ
cedure 1 was incubated with NaHSO3 (100 ꢁM) for 1 h and was
then washed by E3 media; 3: larva preꢀtreated with procedure 1
was incubated with BTSA (100 ꢁM) for 3 h; 4: larva was incubatꢀ
ed with 1 mM Nꢀethylmaleimide (NEM) and probe FHMI (10
ꢁM) for respective 1.5 h and 1 h, and then was incubated with
BTSA for 3 h. Fluorescence images from a to c represented: a):
the yellow channel; b): brightꢀfield; c): merged images of a) and
b). Scale bar: 20 ꢁm.
stress was verified. In fact, based on the above experiꢀ
ments, it could be expected that either the SO2 (induce earꢀ
ly apop tosis) or the ClOꢀ (induce late apoptosis) would
cause lower cells viability. As expected, group 3 showed
the higher viable rate than both group 2 and group 4, imꢀ
plying that mitochondrial SO2 might participate the posiꢀ
tive modulation of antiꢀoxidant system to the cells viability
under the oxidative stress42,48,51,52. Overall, these studies
demonstrated that, FHMI was an effective tool to explore
the biological role of mitochondrial SO2.
Imaging endogenous SO2 in zebrafish. With the satꢀ
isfactory properties of FHMI, we then tested its applicabilꢀ
ity in vivo. Since most genes of zebrafish were homoloꢀ
gous with those of human, zebrafish was selected as the
model for in vivo experiment 53,54. First, we had the
zebrafish incubated in E3 media (15 mM NaCl, 0.5 mM
KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05
mM Na2HPO4 and 0.7 mM NaHCO3; pH 7.5) containing
10 ꢁM FHMI for 1 h. As shown in group 1 of Figure 5,
almost no fluorescent emission was detected in the yellow
NaClO was carried out. As shown in group 4 of Figure 4,
almost no fluorescence emission in yellow channel was
observed, indicating that NaClO effectively consumed the
intracellular SO2. Meanwhile, as seen from the Figure 4 Bꢀ
4 and Figure 4 Cꢀ4, the late apoptosis rate, was obviously
increased comparing with those in group 3. This finding
indicated that without the endogenous SO2, ClOꢀ lead to
more remarkable late apoptosis. To further confirm this
result, the late apoptosis was validated by the detection of
DNA fragmentation (Figure S15), showing that the inꢀ
creased ClOꢀ (simultaneously decreased SO2) caused upꢀ
regulated late apoptosis. In return, this result confirmed that
in group 3, it was the mitochondrial SO2 that contributed to
the downꢀregulated late apoptosis. Therefore, the antiꢀ
oxidative effect of mitochondrial SO2 under the oxidative
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Analytical Chemistry
Markers
(KLDTTM2015ꢀ6;
KLDTTM2015ꢀ9),
and
channel, suggesting that the spontaneously generated SO2
in zebrafish was quite low. Then, the NaHSO3 (100 ꢁM)
was added to the medium of group 1 and the incubation
was kept for 1 h. As shown in group 2, the fluorescence
exhibited obvious enhancement. This result indicated that
the Natural Science Foundation of Shandong Province
(ZR201709240033).
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REFERENCES
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the elevated HSO3 inside the zebrafish triggered the fluoꢀ
rescence of FHMI. To confirm this point, we incubated the
zebrafish of group 1 with BTSA (100 ꢁM) for 3 h. Obviꢀ
ously, the enhanced fluorescence intensity was observed in
the yellow channel (Figure 5ꢀ3), indicating that the amount
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before the incubation with BTSA. As a result, the Figure 5ꢀ
4 displayed the rather weak fluorescence intensity. This
result indicated that even in presence of the BTSA, the
amount of SO2 in zebrafish was decreased under the inhibꢀ
itive effect of NEM. Therefore, these experiments demonꢀ
strated that the applicability of probe FHMI was robust for
various bioꢀspecimen, thus providing the opportunity for
the new investigations of wholeꢀorganism analysis.
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In summary, we rationally constructed a new mitochondrial
SO2 probe by optimizing the optical properties of the
quencher/donor influenced fluorophore. Such a control
avoided the potential defects including the fluorescence
quenching and emission shortening when sensing SO2 via
the nonꢀplanar addition reaction. Comparing with the tradiꢀ
tional design, the present probe FHMI exhibit obvious enꢀ
hanced fluorescence with large red shift. With FHMI, miꢀ
tochondrial SO2 was found to be related with the early
apoptosis of HeLa cells through reducing the mitochondrial
membrane potential. Under the oxidative stress, mitochonꢀ
drial SO2 played the regulative role in apoptosis process.
Besides, FHMI proved to be applicable in zebrafish for
visualizing the endogenously generated SO2. We therefore
envision that this probe can find the extensive application
in monitoring mitochondrial SO2 in biological systems.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge
on the ACS Publications website.
Additional detailed information, NMR spectra, mass
spectrum, and biological experiments.
AUTHOR INFORMATION
Corresponding Author
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organs of mice: sulfur dioxide is a systemic oxidative damage
agent. Inhal Toxicol 2003, 15, 181.
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Zhao, X.; Xia, L.; Chen, L.; Wang, H.; You, J. A novel dualꢀ
ratiometricꢀresponse fluorescent probe for SO2/ClO− detection in
* tangb@sdnu.edu.cn; liuyuxia2008@163.com
ACKNOWLEDGMENT
This work was supported by National Natural Science Founꢀ
dation of China (Nos: 21535004, 91753111, 21390411,
21475074, 21403123), the China Postdoctoral Science Foundation
(2017M622254; 2016M600531), the Open Funds of the Shandong
Province Key Laboratory of Detection Technology for Tumor
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