Increase of tyrosinase activity at the wound site in zebrafish imaged by a new fluorescent probe

Article abstract of DOI:10.1039/d0cc08134e

Tyrosinase plays a pivotal role in the hyperpigmentation of wounds. Here, we develop a new fluorescent probe and with it, we reveal an increase of tyrosinase activity at the wound site in zebrafish.

Full text of DOI:10.1039/d0cc08134e

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Increase of tyrosinase activity at the wound site in
zebrafish imaged by a new fluorescent probe†
Cite this: Chem. Commun., 2021,
57, 2764
ab
a
ab
Ziyin Chai,ab Jizhen Shang,a Wen Shi,
*
Xiaohua Li
and Huimin Ma
Received 15th December 2020,
Accepted 2nd February 2021
DOI: 10.1039/d0cc08134e
rsc.li/chemcomm
Tyrosinase plays a pivotal role in the hyperpigmentation of wounds. inflammation;9 however, the tyrosinase variation in the wound
Here, we develop a new fluorescent probe and with it, we reveal an has not been studied to our knowledge.
increase of tyrosinase activity at the wound site in zebrafish.
In this work, we were interested in developing a long
wavelength fluorescent probe for in vivo imaging of zebrafish,
There is a broad spectrum of skin color within the human race, and studying the change of tyrosinase in their wounds. In
born from years of evolution and migration. The pigments in particular, we have synthesized CF-TYR (Scheme 1) as a new
the skin play a beneficial role in protecting the inner layer of probe with red emission. The probe exhibited high selectivity
tissue from harmful solar ultraviolet (UV) radiation.1 However, and sensitivity toward tyrosinase, and has been used to image
due to cultural and social factors, approximately 15% of the the tyrosinase variation in the wounds of zebrafish after fin
world population invests in skin whitening products.2 Other amputation, revealing an increase of tyrosinase at the wound
demands for skin color modulation come from treating site. In addition, some drugs to inhibit hyperpigmentation have
the hyperpigmentation in wounds, such as acne scars.3 An also been tested with the probe, demonstrating its potential for
important molecular mechanism of lightening skin is to reduce drug screening in zebrafish.
the melanin, the main determinant of the skin color, which is
As shown in Scheme 1, the probe consists of a spectroscopic
derived from the melanogenic pathway.4 To this end, tyrosinase moiety of dicyanomethylenedihydrofuran (CF–NH2) and a
inhibitors have been regarded as an effective therapy because recognition moiety of 3-hydroxyphenyl. CF–NH2, bearing a
tyrosinase initiates the oxidation of L-tyrosine to dopaquinone, typical electron push–pull structure from amine to cyano
which is the speed-limit step of melanogenesis.5 In this respect, groups, was chosen as the fluorophore (quantum yield = 1.1%
a number of methods for analyzing tyrosinase activity and in water) because its long emission wavelength (612 nm) is
screening its inhibitors have been developed.
suitable for zebrafish imaging.10 Modification of the amine
Because of the convenience and imaging ability, fluorescent group with carbamate blocks the electron-donating ability, and
probes for tyrosinase have aroused much interest.6 Our group thus quenches the fluorescence of CF–NH2. Reaction of the
has recently proposed 3-hydroxyphenyl as a recognition moiety recognition moiety with tyrosinase leads to a domino decom-
to construct highly selective tyrosinase probes without the position, which releases the fluorophore and recovers the
interference from reactive oxygen species (ROS).7 Moreover, strong fluorescence.
some probes have been used to explore the relationship
The absorption spectra of CF-TYR before and after reaction
between skin pigmentation and UV exposure by detecting with tyrosinase were studied first. CF-TYR itself has a maximum
tyrosinase.8 Nevertheless, little effort has been made towards
tyrosinase regulation in hyperpigmentation in wounds. It has
been reported that the fin wounds of zebrafish recruit melano-
cytes to the damaged site to cause hyperpigmentation through
a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical
Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, China. E-mail: shiwen@iccas.ac.cn
b University of Chinese Academy of Sciences, Beijing 100049, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
Scheme 1 The sensing mechanism of CF-TYR towards tyrosinase.
d0cc08134e
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inorganic salts (KCl, CaCl2, CuCl2, ZnCl2, and MgCl2) and bio-
logical molecules (cysteine, glucose, glutathionÀe, and vitamin C).
1
In addition, ROS (ꢀOH, H2O2, TBHP, O2, OCl , TBOꢀ, ONOOÀ,
and O2À) and some enzymes (biotinidase, nitroreductase, and
ꢀ
quinone oxidoreductase) were tested as well. These substances
showed negligible influence on the fluorescence of the probe
(Fig. S6, ESI†), indicating that the probe has prominent selectivity
toward tyrosinase.
To confirm the increased fluorescence arising from the
enzymatic reaction, the reaction mixture was subjected to
HPLC and mass spectral analysis. As shown in Fig. S7 (ESI†),
the distinct peak of CF–NH2 in the reaction solution can be
detected in both ways, confirming the release of the fluoro-
phore. Furthermore, kojic acid, an inhibitor of tyrosinase,11 was
added to the reaction system, which suppressed significantly the
fluorescence increase (Fig. S8, ESI†). In particular, 100 mM and
200 mM kojic acid can inhibit 82% and 91% of the tyrosinase
activity in the system, respectively. Meanwhile, kojic acid itself
showed negligible effect on the fluorescence of both CF-TYR and
CF–NH2 (Fig. S9, ESI†). These results fully proved the enzymatic
mechanism of fluorescence response. In addition, the half
Fig. 1 (A) Absorption and (B) fluorescence spectra of CF-TYR (10 mM)
before (curves a) and after (curves b) reaction with tyrosinase. (C) Kinetic
curves of CF-TYR (10 mM) with tyrosinase at varied concentrations.
(D) Linear curve toward the concentration of tyrosinase from 20 to
200 U mLÀ1. lex/em = 560/612 nm.
absorption band at about 440 nm, while the reaction solution of maximal inhibitory concentrations (IC50) of three inhibitors,
CF-TYR with tyrosinase shows a maximum absorption at 510 nm kojic acid, hydroquinone and vitamin C were determined to be
(Fig. 1A). To achieve low background fluorescence analysis, 19.2 Æ 1.5 mM, 22.1 Æ 8.9 mM, and 22.3 Æ 8.4 mM with CF-TYR,
560 nm as the excitation wavelength of the system was used to respectively (Fig. S10, ESI†).
avoid the effect of CF-TYR. In this way, probe CF-TYR displays
Furthermore, we investigated whether the probe can be used
little fluorescent background (quantum yield o0.1%), but its to image tyrosinase activity in cells. Before imaging, MTT assay
reaction product with tyrosinase shows a large fluorescence was performed to test the cytotoxicity of CF-TYR to B16 and
enhancement at 612 nm (Fig. 1B). This fluorescence off–on HeLa cells, disclosing that the probe exhibited significant
response makes CF-TYR feasible for sensitive detection of cytotoxicity when its concentration was above 10 mM (Fig. S11,
tyrosinase activity.
ESI†). Besides, B16 cells were more vulnerable to the probe than
Then, the reaction conditions of the probe with tyrosinase HeLa cells. This might be because a higher level of tyrosinase in
including pH and temperature were optimized. As shown in B16 cells12 leads to more benzoquinone, which may cause more
Fig. S4A (ESI†), when pH varies from 5 to 9, CF-TYR itself severe cell damage. Consequently, we used 5 mM of the probe in
displays unaltered weak fluorescent background; after the the following cell imaging experiments with B16 and HeLa cells.
reaction, the maximal fluorescence enhancement appears at
physiological pH 7.4. Meanwhile, the temperature changing
from 25 1C to 40 1C has almost no influence on the fluorescence
signal of the system (Fig. S4B, ESI†). Thus, the probe is quite
compatible with physiological conditions and biological
applications.
Under the optimized conditions, the kinetics of the reaction
were investigated. The reaction showed a nearly linear fluorescence
increase in the initial 3 h, and the increase rates were dependent
on the dose of tyrosinase (Fig. 1C). By evaluating the initial rate
within 0.5 h, we obtained the Michaelis constant (Km) and the
initial reaction rate (Vmax) to be 77.5 mM and 0.37 mM minÀ1
,
respectively, showing a high affinity and rapid reaction of
the probe (Fig. S5, ESI†). Furthermore, a good linear equation
of DF/Dt (minÀ1) = 0.015 Â C (U mLÀ1) À 0.13 (R = 0.998) in the
range of 20–200 U mLÀ1 can be gained through initial rate
assay (Fig. 1D), where DF/Dt is the fluorescence increase rate
in the initial 1.5 h. The detection limit was determined to be
2.26 U mLÀ1 (k = 3).
Fig. 2 Fluorescence and differentiation interference contrast (DIC)
images of B16 and HeLa cells. The 1st, 2nd, 3rd and 4th columns: cells
were incubated with CF-TYR for 0, 1, 2 and 3 h, respectively. The 5th
column: the cells were pretreated with kojic acid for 2 h, and then
incubated with CF-TYR for 3 h. Scale bar, 50 mm.
To study the selectivity toward tyrosinase, CF-TYR was
examined with different relevant bio-substances, including
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Fig. 3 (A) Fluorescence and DIC images of 3 day-old zebrafish tails: (a) tail-cutting zebrafish without CF-TYR (control); (b) intact zebrafish (without
wound) incubated with the probe (another control; 5 mM); (c) tail-cutting zebrafish incubated with the probe (5 mM); (d) tail-cutting zebrafish pretreated
with kojic acid (200 mM), and then incubated with the probe (5 mM); (e) tail-cutting zebrafish pretreated with hydroquinone (200 mM), and then incubated
with the probe (5 mM); (f) tail-cutting zebrafish pretreated with vitamin C (200 mM), and then incubated with the probe (5 mM). Scale bar = 100 mm. (B) The
ratio of mean intensity of ROI 1 (green line 1 on the intact or wound tail edge) against ROI 2 (green line 2 on the nearby background tail) in different groups
(n = 5 fishes in each group). ***p o 0.001, two-sided Student’s t-test.
As shown in Fig. 2, both B16 and HeLa cells treated with demonstrates that our probe may be used as a convenient tool
the probe manifest a distinct fluorescence increment with to screen the potential drugs for hyperpigmentation therapies.
time, and the pre-treatment with kojic acid (200 mM, which is
In summary, we reported CF-TYR as a new tyrosinase probe,
non-cytotoxic as shown in Fig. S12, ESI†) can notably inhibit which shows fluorescence response toward tyrosinase activity
the fluorescence, verifying that the tyrosinase activity leads with long wavelength as well as high selectivity and sensitivity.
to fluorescence enhancement (see also Fig. S13, ESI†). In This permits the probe to be capable of imaging the tyrosinase
contrast, HeLa cells exhibited no obvious fluorescence activity in both cells and zebrafish. Notably, the higher tyrosi-
response after being incubated with the probe for 3 h. Thus, nase activity at the wound site in zebrafish was first uncovered
CF-TYR can effectively detect the tyrosinase activity in by our probe, which might be implicated with the hyperpig-
different cells.
mentation of wounds. In addition, the probe may serve as a
To study the tyrosinase function in the wound, the fin amputa- convenient tool to screen the potential inhibitors of tyrosinase
tion model of zebrafish was adopted because the transparent for treating hyperpigmentation.
fin facilitates the fluorescence observation. The background
This work is supported by grants from the NSF of China (No.
tyrosinase in zebrafish larvae was imaged first as a control, which 21922412, 21820102007, 21775152, and 22074151).
displayed ubiquitous distribution of fluorescence except in the
zebrafish yolk sac (Fig. S14, ESI†). This is consistent with our
Conflicts of interest
previous finding.7a Nevertheless, when the zebrafish with
amputated fins were treated with the probe for 3 h, the wound
site displayed much brighter fluorescence than the other region
(Fig. 3), suggesting an increase of the tyrosinase activity. The
ratio of the mean intensity of the wound site (ROI 1) against the
nearby background tail (ROI 2) shows a significant difference
(compare columns b and c in Fig. 3B). In contrast, adding kojic
acid in the feeding media can inhibit fluorescence significantly,
proving that tyrosinase is indeed responsible for the fluores-
cence of the wound. The postulated reason for the elevated
tyrosinase at the wound site is the protective effect of melanin
synthesis through highly oxidative and toxic byproducts such as
quinone.13 Furthermore, we examined the efficacy of hydro-
quinone and vitamin C, which are used in treating hyperpig-
mentation, to inhibit the tyrosinase at the wound site. Hydro-
quinone, the first-line therapy for hyperpigmentation, and
vitamin C, a safe complementary drug, block the synthesis of
melanin in different pathways.14 As shown in Fig. 3A (images e
and f), both the compounds display significant suppression of
fluorescence at the wound site. These phenomena can also
be quantitatively described by the ratio of fluorescence at
the wound site versus unhurt tail regions (Fig. 3B), which
There are no conflicts to declare.
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