Visualization of mercury(ii) accumulation: In vivo using bioluminescence imaging with a highly selective probe

Article abstract of DOI:10.1039/c8ob00398j

Mercury is a highly toxic environmental pollutant that negatively affects human health. Thus, an in vivo method for noninvasive imaging of mercury(ii) and visualization of its accumulation within living systems would be advantageous. Herein, we describe a reaction-based bioluminescent probe for detection of mercury(ii) in vitro and accumulation in vivo. The application of this probe would help to shed light on the intricate contributions of mercury(ii) to various physiological and pathological processes.

Full text of DOI:10.1039/c8ob00398j

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Takeharu Haino et al.
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Visualization of mercury (II) accumulation in vivo using
bioluminescence imaging with a highly selective probe
Received 00th January 20xx,
Accepted 00th January 20xx
Bowen Ke,‡ Hui Chen,‡,a,b Lin Ma, Sarah Zingales, Deying Gong, Die Hu, Lupei Du, and Minyong
,a
b
c
a
a
b
b,*
Li
DOI: 10.1039/x0xx00000x
www.rsc.org/
Mercury is a highly toxic environmental pollutant that negatively
Given the clear correlation between mercury accumulation and
affects human health. Thus, an in vivo method for noninvasive human health, the establishment of a rapid and reliable strategy for
imaging of mercury (II) and visualization of its accumulation within detecting mercury species in diverse environmental settings and
living systems would be advantageous. Herein, we describe a complicated biological systems could greatly advance
reaction‐based bioluminescent probe for detection of mercury (II) understanding of the consequences of mercury pollution and help
in vitro and accumulation in vivo. The application of this probe find an appropriate remedy. To this end, a number of fluorescent
would help to shed light on the intricate contributions of mercury probes have been fashioned for the detection and quantification of
(
II) to various physiological and pathological processes.
mercury (II), which is a common inorganic mercury species in the
1
0‐18
environment.
Probe‐based detection methods have advantages
over instrumental technologies such as atomic absorption/emission
As Mercury is abundant in the environment and is derived from
various natural processes, such as volcanoes, thermal springs,
oceanic emissions, and weathering of rocks, it was not considered
as an environmental concern of primary importance until the
1
9
spectroscopy (AAS/AES) and atomic fluorescence spectroscopy
20
(
AFS), because probe‐based methods do not require sophisticated
procedures and expensive instrumentation, affording a versatile
method for recognizing analytes in diverse matrices. More
importantly, this approach is particularly suitable for on‐site
monitoring which provides an essential insight into complex
systems, especially biological system. However, the inherent
limitations of fluorescence, such as photobleaching and
autofluorescence, limit its further biological applications. Thus,
developing a strategy to visualize dynamic changes and biological
processes of mercury (II) in a living system is still a challenging
endeavour.
1
mercury poisoning of Minamata Bay in the 1950s. Anthropogenic
activities over centuries, such as mining and waste disposal,
aggravate the already serious mercury contamination and pose a
2
substantial threat to ecosystems and human health. The facile
binding of mercurous and mercuric ions to nucleophilic functional
groups of biomolecules results in perturbation of function, including
DNA damage3 and neuronal migration disruption.4 A variety of
diseases are closely associated with the adverse effects of mercury,
such as immunotoxicity, genotoxicity, carcinogenicity, and
5
teratogenicity. Dietary mercury intake and environmental
To establish a suitable approach for matching the stringent
criteria of in vivo studies, we sought to utilize bioluminescence as
an imaging tool. Bioluminescence is a natural phenomenon widely
prevalent among both marine and terrestrial species, wherein
chemical energy is converted into light. Basically, an enzyme
exposure to mercury leads to arrhythmias, stomatitis, gastritis,
6
‐9
colitis, renal tubule degeneration, and haemorrhagic gastritis.
Mercury accumulation is also correlated with cognitive and motor
disorders. Thus, the World Health Organization (WHO) has
established a maximum limit of 0.001 mg/L for total mercury in
drinking water.
catalyzes the oxidation of a substrate to yield photons through
21‐24
chemical reactions in the presence of co‐factors.
This natural
process does not require light absorption, thus avoiding auto
2
5
fluorescence.
Recently, we developed the first bioluminescent
probe based on the deprotection of dithioacetal to decanal for the
a
Laboratory of Anaesthesiology & Critical Care Medicine, West China Brain Research
2
6
detection of mercury (II) in living cells and animals. However, that
bioluminescent probe is not suitable for detection of mercury (II) at
high levels because of its limited detection range (2.5‐10 µM). In
addition, that probe requires local injection of an E. coli culture for
in situ enzyme production, which precludes simultaneous
monitoring of mercury (II) in multiple regions of the animal or in the
whole animal.
Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
b
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE),
School of Pharmacy, Shandong University, Jinan, Shandong 250012, China. Tel./fax:
c
+
86‐531‐8838‐2076; E‐mail: mli@sdu.edu.cn
Department of Chemistry and Physics, Armstrong State University, Savannah, GA
1419, USA
3
†
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
‡
These authors contributed equally.
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through acylation of D‐aminoluciferin with a self‐immolative
benzylic linker; and probe 3 was prepared in two steps from 2‐
Cyano‐6‐hydroxybenzothiazole through introduction of a butynyl
group (Scheme S1). The detailed synthetic procedure and structural
characterizations of probes are provided in the Supporting
Information.
Scheme 1. The detection of mercury (II) by using new generation
bioluminescent probes based on the luciferin/luciferase system.
Due to the inherent limitations of our first‐generation probe, we
chose the luciferin/luciferase system as a scaffold for constructing a
new reporter system to detect mercury (II) and monitor its
accumulation in biological systems. Highly sensitive bioluminescent
probes based on the luciferin/luciferase system have been
Figure 2. Chemical structures of the bioluminescent mercury probes
2
7
developed for a variety of biomolecules, such as caspase, β‐
1‐3.
2
8
29, 30
31, 32
lactamase, aminopeptidase N,
nitroreductase,
hydrogen
In addition,
peroxide,3
3,
34
fluoride and hydrogen sulfide.36,
35
37
2
+
With these probes in hand, Hg ‐mediated bioluminescent
responses were initially evaluated in Tris‐HCl buffer (50 mM at pH
.4, 10 mM MgCl ). Probes 1‐3 were each incubated with 50 µM
HgCl at 25°C for 60 minutes, followed by addition of luciferase.
luciferin/luciferase bioluminescence was found to be a promising
tool for in vivo applications due to its efficient photon generation
7
2
3
8
and high signal‐to‐noise ratio. More importantly, this system
could offer a convenient and feasible means for whole‐animal
imaging. The caging approach is a well‐established strategy to
design turn‐on bioluminescent probes for selective detection of
target analytes.21 By taking advantage of the high specificity of
enzyme‐substrate interaction, the appropriate chemical
modification of firefly luciferin could suppress the response of
substrate to luciferase and effectually prevent the luminescence
production. In the presence of an analyte, probes could be
triggered to release the free luciferin for the catalytic enzyme‐
substrate reaction, in which luciferin is oxidized by oxygen to
provide energy for light production. Our strategy was to create a
highly selective probe utilizing the oxymercuration‐reduction
reaction that converts an alkene/alkyne into the corresponding
alcohol/ketone. In this reaction mercury would promote the
cleavage of the specific bioluminescence‐masking group and yield
the release of luciferin (Scheme 1). The details of the sensing
mechanism are shown in Figure 1. We expected that the
introduction of vinyl or 1‐butynyl ether could offer these probes
sufficient specificity toward mercury.
2
High bioluminescent productions from both probes 1 and 2 were
monitored with a living optical imaging system. However, unlike
probe 1 and 2, probe 3 did not exhibit any luminescent signal in the
assay. A subsequent experiment determined that high temperature
2
+
(
60°C) is required for 3 to react with Hg . Obviously, this
experimental condition is not suitable for biological applications;
thus probe 3 was not further examined. Next, we evaluated the
2
+
ability of probes 1 and 2 to detect Hg in a concentration‐
dependent manner. Incubation of probes with various Hg2 levels in
the Tris‐HCl buffer at 25°C for 70 minutes was followed by addition
of the enzyme and subsequently detection of light production.
Probe 1 exhibited a clear positive linear relationship between
+
2
+
2
luminescence and Hg concentration (R = 0.993 in the range of
2
+
2
.5‐100 µM Hg ). Probe 2 was not linearly proportional with
respect to luminescence and Hg concentration: light production
first increased, reached a maximum at 10 µM of Hg , and then
decreased with increasing concentrations of analyte (Figure S1).
Thus, probe 2 was not further examined.
2
+
2
+
2
+
Having confirmed the excellent responsiveness of probe 1 to Hg ,
we then turned our attention to confirming its selectivity by
treating a 25 µM solution of probe 1 with a variety of relevant
metal ions (100 µM) at 25°C. After a 60‐minute incubation, ATP and
luciferase were added and the relative total photon flux was
measured to calculate the amount of luciferin release from probe in
each condition. As depicted in Figure 3, consistent with our
expectations, little to no bioluminescence was observed by the
ArOH
HgCl
2
Ar
OH
O
2
ClHgCH CHO
+
HCl
Ar
HgCl
2
H O
O
OH
+
(
2
CH2CHO) Hg
Ar
HgR
HCl
O
ArOH
2
+
2+
2+
Figure 1. The sensing mechanism of probe with Hg2+ in water.
presence of other transition metal ions like Cu , Pd , and Fe or
alkali/alkaline earth metal cations such as K or Ca . In contrast, the
addition of Hg significantly induced a ca. 130‐fold increase in
emission intensity, even at low concentration (100 µM). The
excellent selectivity of probe 1 toward Hg over other species
under physiological conditions suggests that probe 1 is a promising
+
2+
2+
To validate this proof of concept, three bioluminescent probes
were designed and synthesized (Figure 2). Briefly, probe 1 was
prepared in three steps (substitution, elimination, and cyclization)
from 2‐Cyano‐6‐hydroxybenzothiazole; probe 2 was prepared
2
+
2
| J. Name., 2012, 00, 1‐3
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chemical tool for the specific detection of Hg2+ in the complicated of bioluminescence imaging of probe 1 with varying concentrations
biological environment. of HgCl solution in the cell. All assays were performed in triplicate
2
and represented as the mean ± SEM.
After establishing that probe 1 had the capability of reliably
monitoring mercury (II) in aqueous solution and in cell culture, our
next step was to determine whether this probe would be able to
detect mercury in vivo and keep its advantages, including high
sensitivity, low background, excellent biocompatibility, and deep
2
+
tissue penetration. The in vivo imaging of Hg by probe 1 was
performed with transgenic FVB‐luc+ mice, which ubiquitously
express firefly luciferase. Mice were given a sub‐lethal dose of HgCl
0.02 μmol) or the vehicle control (normal saline, NS, 0.9% NaCl))
2
(
by intravenous tail vein injection, and then an intraperitoneal
injection of 20 μL probe 1 (100 μM) 3 hours later. The
bioluminescence signals generated from the animal model were
Figure 3. The relative bioluminescence intensity of probe 1 with real‐time detected using a CCD immediately following injection of
2
+
+
+
2+
3+
2+
2+
3+
2+
2+
2
various anions (Hg , Li , Na , Ca , Cr , Mn , Fe , Fe , Co , Ni , probe 1. As demonstrated in Figure 5, treatment with HgCl and
2
+
2+
3+
+
2+
2+
Cu , Zn , Au , Ag , Pd , and Cd ) after adding luciferase and ATP. probe 1 resulted in robust bioluminescence. The emission signal
reached its maximum value within 18 minutes; a ca. 260% increase
Encouraged by the results of ex vivo experiments, we further in bioluminescence relative to control animal was clearly measured.
examined probe 1 in biological systems. ES‐2‐Fluc cells, which Treatment of FVB‐luc+ mice with NS lead to a weak, but measurable
express firefly luciferase, were utilized as a model system to assess bioluminescence background, suggesting that probe 1 may be
2
+
sensitive enough to respond to basal levels of Hg2+ obtained from
the ability of probe 1 to respond to changes in Hg levels in vitro.
First, the cytotoxicity of 1 was assessed using a standard MTT the diet.
assay.39 Probe 1 exhibited low cytotoxicity (IC50 = 697.8 µM) toward
the cultured cells, demonstrating its safety at the concentrations
used in our assays (Figure S2). Next, we examined the ability of
2
+
probe 1 to respond to Hg . ES‐2‐Fluc cells were pre‐treated with 25
µM probe 1 at 37°C for 30 minutes. After removal of the medium,
the cells were then supplemented with different concentrations of
2
HgCl ranging from 0 to 100 μM and incubated for an additional 5
minutes to increase intracellular levels of mercury. A gradual and
remarkable increase in bioluminescence was observed in
concentration‐dependent manner with increased exogenous Hg
levels. As shown in Figure 4, there was a good linear correlation (R
a
2
+
2
2
+
=
0.993) between Hg concentrations and total bioluminescence,
2
+
indicating that probe 1 is capable of monitoring labile Hg in vitro.
Figure 5. Representative bioluminescence imaging of FVB‐luc+ mice
2
+
injected with probe 1 ± Hg .
Since the accumulation of mercury in organisms through the food
chain is a major concern, to better understand this issue and its
relation to health effects, we next applied probe 1 and in vivo
imaging to determine mercury accumulation over time in a living
5
5
4
1
1 .. 00
8..00
6..00

x 101 0
2
animal. Mice were dosed with 0.02 μmol HgCl or the vehicle
control (NS) by intravenous tail vein injection, and then dosed by
4
8

x 10 10
st
intraperitoneal injection of 20 μL probe 1 (100 μM) again on the 1 ,
4
4
rd
th
2+
6

x 10 10
3 , and 7 day after Hg treatment. As illustrated in Figure 6, the
2
+
Hg ‐treated mice injected with probe 1 generated significantly
greater bioluminescence compared to non‐Hg treated mice. On
the 1 and 3 days, relatively significant luminescence signals were
recorded, and no sign of decreasing of luminescence signal was
observed, suggesting that considerable levels of Hg persist for 72
4
4
4
y=714.6x+14758
R =0.9928
4.0x10

4.0 10
2
2+
4
st
rd
2
.0

10
2
.0x10
0
0
2
+
0
50
100
2
+
HgCl concentration
（
μM)
hours after an exogenous Hg addition. The bioluminescence signal
from Hg ‐treated mice was weaker on the 7 day, but its intensity
2
2
+
th
2
+
Figure 4. Probe 1 responds to Hg and measured dynamic changes was clearly still stronger than that from mice in the control group.
in Hg2 levels ranging from 0 to 100 μM in living cells. The total flux This demonstrates the ability of probe 1 to examine the long‐term
+
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bioaccumulation of mercury. Notably, stronger luminescence
signals from the kidney regions were observed, likely due to the
renal handling of mercury.
Figure 6. (A) The bioluminescence imaging of probe 1 in FVB‐luc+ mice; (B) Quantification of the total flux (photon/s) from the whole‐body
2
+
st
rd
th
area except tail in the presence or absence of Hg on the 1 day, 3 day, and 7 day. (C) Bioluminescent intensities measured at various
time points (3, 6, 9, 12, 15, 18 min) on the 1 day, (D) 3 day, and (E) 7th day. All assays were performed in duplicate and represented as
st
rd
the mean ± SEM. **: P < 0.05, ***: P < 0.01
In conclusion, we have developed a next‐generation mercury
bioluminescent probe and evaluated its optical properties and
biological applications. Probe 1 exhibited high sensitivity, good
selectivity, and a robust “turn‐on” response toward mercury (II).
Compared to other standard methods, our strategy can easily be
used to perform real‐time detection of mercury (II) in complex
biological systems. Probe 1 was successfully applied to in vitro
detection, as well as to in vivo imaging of organ accumulation, toxic
potency, and the biological effects of mercury.
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This work was supported by the National Natural Science
Foundation of China (Nos. 81773685 and 81673393), the 1.3.5
Project for Disciplines of Excellence, West China Hospital, Sichuan
University (Nos. ZY2016101 and KY2016203), the Taishan Scholar
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Visualization of mercury (II) accumulation in vivo using bioluminescence imaging
with a highly selective probe
‡,a
‡,a,b
b
c
a
a
b
Bowen Ke, Hui Chen,
Lin Ma, Sarah Zingales, Deying Gong, Die Hu, Lupei Du, and Minyong
b,*
Li