Synthesis, characterization and biological evaluation of octyltrimethylammonium tetrathiotungstate

Article abstract of DOI:10.1007/s10534-020-00267-9

Abstract: Octyltrimethylammonium tetrathiotungstate salt (ATT-C8) was synthesized and its ability to chelate copper was evaluated. The biological and toxic aspects were evaluated by in vitro and in vivo assays, using bovine aorta endothelial cells (BAEC) and zebrafish (Danio rerio) embryos. The obtained results suggest that ATT-C8 has better biocompatibility, showing a significantly lower lethal concentration 50 (LC₅₀) value in comparison to ammonium tetrathiotungstate (ATT). Zebrafish embryos assay results indicate that both tetrathiotungstate salts at the studied concentrations increase the hatching time. Even more, an in vivo assay showed that synthesized materials behave as copper antagonists and have the ability to inhibit its toxicological effects. Also, both materials were found to be active for the in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The characterization of the materials was carried out using the following spectroscopic techniques: Ultraviolet–Visible (UV–Vis), Fourier Transform Infrared (FTIR) and proton nuclear magnetic resonance (¹H-NRM). Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10534-020-00267-9

Biometals
https://doi.org/10.1007/s10534-020-00267-9(0123456789(). -, volV
(
)
0
1
2
3
4
5
8
6
9
7
(
)
-
,
v
o
l
V
)
Synthesis, characterization and biological evaluation
of octyltrimethylammonium tetrathiotungstate
.
.
.
.
Karla Vega-Granados Juan Cruz-Reyes Jos e´ F. Horta-Marr o´ n
.
.
Manuel Mar ´ı -Beffa Laura D ´ı az-Rubio Iv a´ n C o´ rdova-Guerrero
.
.
.
Daniel Ch a´ vez-Velasco M. Carmen Oca n˜ a Miguel A. Medina
Lilian B. Romero-S a´ nchez
Received: 28 February 2020 / Accepted: 30 October 2020
Ó Springer Nature B.V. 2020
Abstract Octyltrimethylammonium
tetrathio-
embryos assay results indicate that both tetrathio-
tungstate salts at the studied concentrations increase
the hatching time. Even more, an in vivo assay showed
that synthesized materials behave as copper antago-
nists and have the ability to inhibit its toxicological
effects. Also, both materials were found to be active
for the in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay. The characterization of the materials was
carried out using the following spectroscopic tech-
niques: Ultraviolet–Visible (UV–Vis), Fourier Trans-
form Infrared (FTIR) and proton nuclear magnetic
tungstate salt (ATT-C8) was synthesized and its
ability to chelate copper was evaluated. The biological
and toxic aspects were evaluated by in vitro and
in vivo assays, using bovine aorta endothelial cells
(
BAEC) and zebrafish (Danio rerio) embryos. The
obtained results suggest that ATT-C8 has better
biocompatibility, showing a significantly lower lethal
concentration 50 (LC ) value in comparison to
5
0
ammonium tetrathiotungstate (ATT). Zebrafish
1
resonance ( H-NRM).
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10534-020-00267-9) con-
tains supplementary material, which is available to authorized
users.
K. Vega-Granados Á J. Cruz-Reyes Á
M. C. Oca n˜ a Á M. A. Medina
J. F. Horta-Marr o´ n Á L. D ´ı az-Rubio Á
Department of Molecular Biology and Biochemistry,
Faculty of Science, University of Malaga, 29071 Malaga,
Spain
I. C o´ rdova-Guerrero Á L. B. Romero-S a´ nchez (&)
Faculty of Chemical Sciences and Engineering,
Autonomous University of Baja California (UABC),
Universidad 14418, Parque Internacional Industrial
Tijuana, 22390 Tijuana, BC, Mexico
e-mail: lilian.beatriz.romero.sanchez@uabc.edu.mx
M. Mar ´ı -Beffa
Department of Cell Biology, Genetics and Physiology,
University of Malaga, 29071 Malaga, Spain
D. Ch a´ vez-Velasco
Center for Graduates and Research in Chemistry, National
Technological Institute of Mexico/ Technological Institute
of Tijuana, 22510 Tijuana, BC, Mexico
123

Biometals
Graphic abstract
precursors, (NR ) MS , (where R = alkyl radical and
4
2
4
M = Mo, W). There was a notable advance in the
synthesis of these materials when Alonso et al.
prepared the (NR ) MS precursors (where R = H,
4
2
4
CH , C H and M = Mo, W) in aqueous media, which
4 9
3
afterwards were decomposed into MoS and WS
2
2
(Alonso et al. 1998, 2000). In this sense, it would be of
interest to evaluate the influence of superficial carbons
dosed by alkyl chains in the chelation properties of
salts derived from these compounds when adminis-
tered to animal models.
Keywords Tetrathiotungstate salts Á Zebrafish
embryo Á Antioxidant activity Á Anticopper therapy Á
Toxicity
Zebrafish embryo is becoming a new model of
choice for preclinical studies. Several interesting tests
that use these embryos have been proposed as rapid,
high-throughput, and cost-effective drug and chemical
screens (Dooley and Zon 2000; Garc ´ı a-Caballero et al.
2018; Tobia et al. 2011; Tran et al. 2007; Wilkinson
Introduction
and Van Eeden 2014). A number of different biolog-
ical aspects such as toxicity, inflammation, organ
regeneration or angiogenesis are studied in these
screens to test drugs and biomaterials as chemical
modulators. Recent studies with zebrafish embryos
have evaluated the pro-angiogenic modulation of
copper either directly administrated to water or
lixiviated from biomaterials (Romero-S a´ nchez et al.
2018).
Diverse studies suggest that, as ammonium
tetrathiomolybdate (ATM), ammonium tetrathio-
tungstate (ATT) presents excellent chelating proper-
ties (McQuaid et al. 1994; Young et al. 1982). ATMꢀs
chelating properties have been thoroughly studied in
the treatment of Wilson’s disease (WD) (Brewer 2009;
Brewer et al. 1994, 1991). ATM forms complexes with
copper and albumin that decrease the bioavailability of
copper in the blood. Nowadays, ATM is in phase II
and phase III of multinational clinical trials as an
anticopper therapy for the treatment of neurological
symptoms in patients with WD (Aggarwal and Bhatt
The objective of the present work is to explore the
biological aspects and the copper chelating ability of a
new tetrathiotungstate salt. The study includes the
antioxidant activity and toxic effects of a modification
of ATT with an alkyl group (octyltrimethylammonium
tetrathiotungstate, ATT-C8). With this objective, the
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scav-
enging assay, in vitro endothelial cell viability and
migration assays, and an in vivo toxicity zebrafish
embryo assay were used.
2
018; Brewer et al. 1996). More recently, it was found
that ATM has excellent efficacy in the treatment of
animal models of fibrotic, inflammatory, and autoim-
mune diseases, as well cancer (Brewer
2
003, 2005, 2016; Brewer et al. 2000). Nevertheless,
research of the potential of ATT treatments against
degenerative diseases is scarce, compared to ATM.
The catalytic activity in hydrotreatment processes
of the sulfides obtained from the decomposition of
Methods and materials
these thiometallates, MoS2 and WS , shows an
2
Spectroscopy characterization and molecular
modeling
increase when presenting superficial carbon available
from the synthesis method, generating the MoS2-XCX
and WS2-XC species (Berhault et al. 2001; Chianelli
X
The chemical structures of ATT and ATT-C8 com-
pounds were characterized using the spectroscopic
techniques of Ultraviolet–Visible (UV–Vis), Fourier
Transform Infrared (FTIR) and proton nuclear mag-
and Pecoraro 1981; Kelty et al. 2007; Pecoraro and
Chianelli 1985) With this knowledge, new synthesis
.
routes have been developed, where the carbon is dosed
during the process, in the form of alkyl chains,
generating the tetraalkylammonium thiometallate
1
netic resonance ( H-NRM). UV–Vis data was
obtained using
a
HACH DR-6000 UV–Vis
1
23

Biometals
-
1
1
Spectrometer in the k = 200–700 nm range. FTIR
spectra were acquired in transmission configuration
with a Perkin-Elmer Spectrum GX over the range of
FTIR (cm ): 460 (m Mo-S); 3128, 1395 (m N–H). H-
NMR (CDCl ): d 7.14 (s, 8H, (NH ) ).
3
4 2
-
1 1
4
000–400 cm . H-NMR spectra were recorded on a
Synthesis of ATT-C8
Bruker Avance III spectrometer 400 MHz. The chem-
ical shifts (d) are presented with tetramethylsilane
ATT-C8 was prepared by the reaction between ATT (1
equivalent) and octyltrimethylammonium bromide (2
equivalent) in aqueous solution following the method
reported by Alonso et al. (1998, 2000). Freshly
prepared (NH ) WS ATT (2.0 g, 5.7 mmol) was
(
TMS) (d: 0.00) as the internal standard. The 2D and
D-structure molecular modeling of the synthesized
compound was plotted using ChemDraw Professional
6. The UV–Vis, FT-IR and RMN spectra with their
3
1
4
2
4
corresponding data, as well as the 3-D molecular
structure of the obtained tungsten salts are available in
the supplementary information section.
dissolved in 25.0 mL of distilled water. While stirring,
25.0 mL of a second aqueous solution was added,
containing octyltrimethylammonium bromide (CH3
(CH ) N (Br) (CH ) ; Sigma-Aldrich) (2.9 g,
2 7 3 3
Synthesis of materials
11.5 mmol). The reaction mixture is kept at rest for
2 h and the resulting precipitate is recovered by
1
Synthesis of ATT
sedimentation using a centrifuge at 2000 rpm. Finally,
the yellow product ATT-C8 ( (CH3 (CH ) N
2 7
ATT ((NH ) WS ) was prepared according to the
4
(CH ) ) WS ) was dried in an oven at 30 °C and
4
2
3 3 2
4
method described by Ramanathan and Weller (1985).
Ammonium metatungstate ((NH ) H W O
18H O; Sigma-Aldrich) (15.0 g, 5.1 mmol) is
stored, being named as ATT-C8. The molecular
structure of ATT-C8 is show in Fig. 1b. UV–Vis
1
4
6
2
12 40-
-
9
kmax (nm): 216, 276, 393. FTIR (cm ): 460 (m Mo-S);
3128, 1395 (m N–H); 3005, 2922, 2852 (m C-H); 1468
2
dissolved in 75.0 mL of distilled water containing
2.5 mL of an ammonium hydroxide solution (NH4-
1
5
(d C-H). H-NMR (CDCl ): d 3.34 (m, 11H, CH -N),
3
2
OH (20%); Sigma-Aldrich). The resulting ammonia
3.08 (s, 18H, CH -N), 1.68 (m, 4H, CH -CH -N), 1.30
3 2 2
solution was heated in a temperature bath to 60 °C for
(m, 20H, (CH ) -CH -CH -N), 0.87 (t, 6H,
2 5 2 2
6
h, while hydrogen sulfide was bubbled (H S (g)) into
CH terminal).
3
2
the solution. The reaction mixture was then cooled in
an ice bath and allowed to stand for 12 h, yielding
yellow crystals. The product was vacuum-filtered,
washed with isopropyl alcohol (C H O; Sigma-
Antioxidant activity
The antioxidant capacity of the synthetized materials
was evaluated using the DPPH radical scavenging
method proposed by Blois (1958). In the method, the
DPPH (C H N O ; Sigma-Aldrich) free radical is
3
8
Aldrich) stored under N atmosphere at 15 °C and it
2
was named as ATT. The molecular structure of ATT is
shown in Fig. 1a. UV–Vis kmax (nm): 216, 276, 393.
18
12 5 6
Fig. 1 Structure of the synthesized compounds a ATT and b ATT-C8
123

Biometals
dissolved showing a purple coloration, that upon
reduction by an antioxidant or a radical species,
changes to yellow due to an electron or proton transfer.
The change in absorbance is spectrophotometrically
monitored at a wavelength of 517 nm. The DPPH
radical scavenging effect was calculated according the
following equation:
manufacturers. Half-maximal inhibitory concentra-
tion (IC ) values were calculated as the concentra-
5
0
tions of compound yielding 50% cell survival, taking
the values obtained for control as 100%.
Cell migration assay
Migration of BAEC was assessed using the so-called
‘‘wound-healing’’ assay. Cells were grown, and once
they reached confluence, different concentrations of
ATT and ATT-C8 were added with fresh medium and
a cross-shaped scratch was done using a pipette tip.
Wounded areas were observed under a microscope
after 4, 7 and 24 h of incubation, photographs were
taken from the same areas as those recorded at zero
time. Images were analyzed with NIH Image J 1.6
software. The regrowth of BAEC into the cell-free
area was expressed as the percentage of the initial
wounded area (time 0) recovered by cells migration to
different incubation times.
DPPH-scavenging effect ð%Þ
ꢂ
The half maximal inhibitory concentration (IC )
ꢀ
ꢁꢃ
Absorbance sample
Absorbance control
¼
1 À
 100
5
0
value was interpreted as the concentration of antiox-
idant material necessary to scavenge 50% of the initial
DPPH radicals. The IC50 values were calculated by
linear regression resulting from plotting the % of
DPPH-scavenging activity versus the concentration of
the test samples. Analyses were performed in three
independent assays.
Cell assays
Zebrafish embryo assays
Cell culture
Zebrafish collection
Bovine aortic endothelial cells (BAEC) were isolated
from bovine aortic arches as previously described
C a´ rdenas et al. 2006) and maintained in Dulbecco’s
Zebrafish (Danio rerio) embryos were obtained from
fish facilities at the University of Malaga following
standard procedures (Kimmel et al. 1995; Truong et al.
2011).
(
modified Eagle’s medium (DMEM) containing glu-
cose (1 g/L) and supplemented with glutamine
(
2 mM), penicillin (50 U/mL), streptomycin (50
U/mL) and 10% FBS. Cells were maintained at 37
C under a humidified 5% CO atmosphere.
A breeding stock of adult zebrafish was kept on a
recirculating system at 27 ± 1 °C. Fishes were main-
tained with a standard 12:12-h light–dark photoperiod.
Embryos were produced by one-to-one or one-to-two
female-male mating in fish hatch box. Fertilized eggs
are collected and disinfected using a 0.5% bleach
solution during 1 min and washed several times with
E3 embryo media (5.00 mM NaCl, 0.17 mM KCl,
8
2
Cell viability assay
3
.5 9 10 cells were seeded in 96-well microplates
2
using a total volume of 100 ll of culture medium
containing different concentrations of the ATT and
ATT-C8 materials. Cells were incubated for 72 h (37
0.33 mM CaCl
, 0.33 mM MgSO , 0.00003% C16-
4
2
H
18ClN S; Sigma-Aldrich). Clean eggs were incu-
3
8
C, 5% CO in a humid atmosphere). Then, 0.5 mg/
2
bated in E3 medium in a Petri dish at 28 ± 0.1 °C.
Fishes were handled under National Laws (Law
9/2003, April 25, and Law 31/1995, November 8) by
notification A/ES/12/I-22 and activity A/ES/12/24.
The experiments were carried out under the proce-
dures established and approved by the UMA Bioethics
Commission under the BIO2014-56092-R grant.
mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) were added to the wells and
incubated an additional 4 h in the dark. HCl iso-
propanol was used for resuspension of formazan
crystals and asbsorbance data (550 nm) was collected
using an Eon Microplate Spectrophotometer from Bio-
Tek Instruments (Winooski, VT, USA). Data was
collected by Gen5 software from the same
1
23

Biometals
Zebrafish embryo exposure assay
evident at all the tested concentrations of ATT
1.25–50 lg/mL) and ATT-C8 (0.63–30 lg/mL). It
(
At 24 h after fertilization (hpf) 15 zebrafish embryos
were transferred to wells of a 6-well plate. The
embryos were incubated in 3 mL of culture medium
with different concentrations of the ATT and ATT-C8
materials (0.1, 10, 100, 250, 750, 1000, 10,000 lg/ml)
for 96 h at 28 °C. All solutions with different material
concentrations were prepared in E3 medium. The
survival rate of the embryos from each treatment
group was determined. Zebrafish were observed
directly on the 6-well plate using a Nikon AZ100
multizoom microscope. The hatching rate, the changes
in morphology throughout development and the
tetrathiotungstates effect in the copper induced
embryo toxicity were also evaluated. Zebrafish
embryos were exposed to control (10 lg/mL CuCl2)
was found that the DPPH scavenging effect of
tetrathiotungstates was dose dependent.
Figure 2b displays the DPPH IC50 values of ATT
and ATT-C8 calculated by the regression equation of
the calibration curve. The lower the IC50 value means
higher DPPH radical scavenging of tetrathio-
tungstates, implying a higher antioxidant activity.
ATT-C8 (p \ 0.001) was found to have better free
radical scavenging ability in comparison with ATT,
having an IC50 value of 15.04 ± 0.01 lg/mL and
46.64 ± 0.36 lg/mL, respectively. The capacity of
materials to scavenging free radicals could be
attributed to the electron donor nature of the sub-
stituents -NH and -CH present in the samples, since
3
they are known to be good hydrogen donors (Schubert
et al. 1962).
and two experimental (10 lg/mL CuCl , 250 lg/mL
2
ATT; and 10 lg/mL CuCl , 250 lg/mL ATT-C8)
Antioxidants are molecules with the ability to
eliminate or neutralize the free radicals responsible for
the oxidative stress associated with cancer, arterioscle-
rosis, inflammatory processes and degenerative dis-
eases (Goodman et al. 2011). In fact, bioactive
materials studies use antioxidant capacity as an
indicator of biological activity. However, in the
knowledge of the authors, there are not existent
reports of the antioxidant properties of this type of
coordination compounds. Thus, although the results
cannot be compared with other bioinorganic materials,
the IC50 values for ATT and ATT-C8 are important
compared to some DPPH assay reference antioxidants,
such as the butyrated hydroxyl toluene (BHT)
(19.4–86.6 lg/mL), vitamin C (5.8–110.7 lg/mL)
and tocopherol (27.1–96.0 lg/mL) (Mishra et al.
2012).
2
conditions and their hatching rate studied. All assays
were repeated three times and at least 60 embryos were
used per concentration. Mortality curves were gener-
ated using MS Excel 2016 and the lethal concentration
5
0 (LC ) was calculated applying a nonlinear
50
regression test (sigmoidal dose–response curve).
LC50 indicates the concentration that kills half of
tested population in zebrafish embryo model.
Statistical analysis
Data are shown as mean ± standard deviation (SD;
9
9% confidence interval) resulting from triplicate
evaluations. Statistical significance was calculated
using a Student t-test of control-experimental group
pairs using the Statistical Package for the Social
Sciences (SPSS) version 25 software (IBM, SPSS).
Differences were considered significant when p-val-
ues are p \ 0.05 (*), p \ 0.01 (**), and p \ 0.001
Cell assays
(
***).
In this study, BAEC was incubated in the presence of
ATT and ATT-C8 materials to determine their influ-
ence on cell growth and cell migration capacity.
Figure 3 displays the effects in cellular growth
measured up to 72 h-culture, in the presence of ATT
and ATT-C8 solutions with concentrations of
Results and discussion
Antioxidant activity
0.1–200 lg/mL. The IC50 values of 1.02 ± 0.24 lg/
The DPPH scavenging activity percentage of
tetrathiotungstates at varying concentrations was
measured and the results are displayed in Fig. 2a.
Significant DPPH radical scavenging activity was
mL and 4.27 ± 0.95 lg/mL were obtained for ATT
and ATT-C8 respectively, showing that both materials
have cytotoxic effects over BAEC cells; with ATT
123

Biometals
Fig. 2 DPPH free radical scavenging assay results: a Concen-
tration–response curve of antioxidant activity and b Bar graph
with IC50 (lg/mL) values of synthesized materials. Values
represent mean ± SD of three replicates. The figure includes the
statistical significance of the ATT-C8 group measured with
respect to the control group (ATT); ***p \ 0.001 by Student’s t
test
the control group. After 4 h, a slight promotion in
cellular migration was observed for the ATT-C8
culture at low concentration (2 lg/mL), in comparison
to the bare medium control culture. However, there
were no significant differences between the experi-
mental BAEC cultures, suggesting that the studied
materials have no angiogenic influence at the studied
concentrations (Fig. 4).
Zebrafish embryo assays
Developmental toxicity and biological responses of
zebrafish embryos exposed to an ATT and ATT-C8
dissolution were evaluated (Fig. 5). Figure 6 shows
the survival rate of zebrafish embryos treated with
different concentrations of tetrathiotungstates for
several days. The control group, without the addition
of tetrathiotungstate salts, presented a normal behavior
with an overall mortality rate \ 1%. The tetrathio-
tungstate salts induced lower than 5% mortality
zebrafish embryos incubated at 0.1–100 lg/mL
concentrations.
Fig. 3 In vitro BAEC cell growth assay obtained with the MTT
assay after exposure to ATT and ATT-C8 materials at different
concentrations for a 72 h incubation period. The plots represent
the mean ± SD of three independent experiments. Dose–
response curves were calculated using a nonlinear regression
test
presenting more acute toxic effects with respect to
ATT-C8.
At 72 hpf, embryos under these concentrations were
anatomically indistinguishable from control (Fig. 6a,
b). The absence of sublethal effects, such as the lack of
instant motility, depigmentation and development of
edemas or clots, indicate that at the studied concen-
trations (1–100 lg/mL), ATT and ATT C-8 do not
have any toxic or teratogenic effects in zebrafish
The wound healing assay with BAEC was used to
evaluate the inhibitory or stimulating effects on
angiogenesis. At short periods (4 and 7 h), cell wound
filling area is due exclusively to its migration and not
to its proliferation. Figure 4 shows cell migration after
4
, 7 and 24 h of the wound healing assay compared to
1
23

Biometals
Fig. 4 Wound assay was used to assess in vitro cell migration of BAEC. The graphics present frequency of wound recovery in a ATT
and b ATT-C8 culture cells. The values are presented as the mean ± SD of three independent experiments
Fig. 5 Representative images of zebrafish larvae (72 hpf) after
8 h exposure to a bare medium; b 100 lg/mL ATT; c 250 lg/
mL ATT; d 250 lg/mL ATT-C8; e 500 lg/mL ATT; f 500 lg/
mL ATT-C8; g 750 lg/mL ATT; h 750 lg/mL ATT-C8;
i 1000 lg/mL ATT and j 1000 lg/mL ATT-C8. Scale bar:
10 lm
4
embryos. A significantly higher mortality rate than the
one measured in the control group was initially
observed at a concentration of 250 lg/mL of tetrathio-
tungstate salts (Fig. 6), and there was a 0% survival
rate when the embryos were exposed to concentrations
above 1000 lg/mL of ATT for more than 48 h and
above 10,000 lg/mL ATT-C8 for more than 24 h.
123

Biometals
Fig. 6 Survival rate of zebrafish embryos exposed to different concentrations of a ATT and b ATT-C8 during 24, 48 and 72 h. All
results are representative of three independent experiments
Fig. 7 Mortality curves of zebrafish embryos exposed to a ATT and b ATT-C8 materials at different concentrations during 24, 48 and
7
2 h exposure. All the experiments were done in triplicate. Dose–response curves were calculated using a nonlinear regression test
Figure 7 shows the mortality rate plotted for
embryos exposed to ATT and ATT-C8 materials for
4, 48 and 72 h. The LC50 values of ATT and ATT-C8
Table 1) were estimated based on these mortality
dependent toxic profile with an LC50 of 1211 ± 84,
542 ± 57 and 316 ± 68 lg/mL at 24, 48 and 72 h of
exposure, respectively. Consistent with the obtained
IC50 values in the cell viability assay, zebrafish is more
sensitive to the ATT sample at 72 h of exposure. In the
case of ATT-C8, constant IC50 values were observed
at around 700 lg/mL (p \ 0.001) at 24, 48 and 72 h of
exposure. In this regard, the results suggest that the
incorporation of carbon into the structure improves
ATT biocompatibility.
2
(
curves. ATT shows a concentration and time
Table 1 Results of LC50 at to 24, 48 and 72 h
Material
LC50 (lg/mL)
4 h
2
48 h
72 h
Figure 8 displays the hatching rate of zebrafish
embryos incubated for 72 h to the 250 lg/mL solu-
tions of tetrathiotungstate salts. The results show an
increase in the hatching rate of zebrafish embryos of
the groups exposed to tetrathiotungstate salts, com-
pared to the control group. All of the embryos exposed
ATT
1211 ± 84
735 ± 25
542 ± 57
722 ± 31
316 ± 68
689 ± 29
ATT-C8
Values represent the mean ± SD of three independent
experiments
1
23

Biometals
Fig. 8 Hatching rate of zebrafish embryos exposed to 250 lg/
mL ATT and ATT-C8 materials during 72 h. All the experi-
ments were done in triplicate
Fig. 9 Hatching rate of zebrafish embryos exposed to 10 lg/
mL CuCl solution (negative control), a mixture of 10 lg/mL
CuCl and 250 lg/mL ATT and a mixture of 10 lg/mL CuCl
2
2
2
and 250 lg/mL ATT-C8. All results are representative of three
independent experiments
to ATT and ATT-C8 hatched at 72 hpf. It is known that
the activity of chorionic hatching enzymes (ZHE) and
embryo movements affect their hatching time
the mixture of copper and tetrathiotungstate salts hatch
at 48 hpf. All these results suggest that tetrathio-
tungstate salts could be effective protectors against
copper toxicity. As reported for ATM and ATT, ATT-
C8 also acts as a copper antagonist, becoming an
alternative chelating agent in anticopper therapies.
(
Nechaev and Pavlov 2004). In this case, the embryo
membranes were very deteriorated just before hatch-
ing, suggesting that the studied compounds do not
inactivate the involved enzymes or embryo motility.
Other reports mention that the hatching of zebrafish
embryos is plastic in response to potential stress
changes or changes in the salinity of the environment
(
Ord 2019; Warkentin 2011). In this way, it could be
Conclusions
inferred that an increase in the concentration of
external ions causes moderate hypoxia that accelerates
the hatching time. Overall, tetrathiotungstate salts
were found to accelerate the hatching rate of zebrafish
embryos at low concentrations and cause acute death
or chronic lethal toxicity at high concentrations.
In order to evaluate whether tetrathiotungstate salts
revert the copper-mediated embryo toxicity, embryos
The copper chelating abilities of ATM and its use in
clinical assays have opened new research opportuni-
ties for treatments against diseases where an increase
of copper levels is involved. In this work, we have
studied the bioactive potentialities of a new tetrathio-
tungstate salt, ATT-C8 in different in vitro and in vivo
assays.
were treated with 10 lg/mL of CuCl and a mixture of
Our results show that ATT and ATT-C8 behave as
copper antagonists. This copper chelating ability of the
tested tetrathiotungstate salts was evaluated in a
zebrafish embryo hatching assay, validating their
potential use in anticopper therapies. Toxicity results
suggest that the materials have toxic effects in BAEC
cells and zebrafish embryos over a determined
concentration, which increase at higher doses and
exposure time. This effect ameliorates (72 h data) with
the addition of the eight carbon residues, cell migra-
tion increases and zebrafish hatching time decreases.
Further studies of these promising compounds are
2
1
0 lg/mL CuCl and 250 lg/mL of ATT or ATT-C8
2
for 72 h. It is well documented that copper interferes
with the enzymatic activity of the ZHE, delays
hatching and induces abnormal morphology and
mortality (Lin et al. 2013; Romero-S a´ nchez et al.
2
018). The obtained results show that copper signif-
icantly delays hatching up to 96 hpf and 120 hpf, in
good agreement with reported results. Tetrathio-
tungstate salts thus significantly inhibit the delaying
effect of copper in the hatching process (p \ 0.001).
Figure 9 shows that 90% of embryos incubated into
123

Biometals
needed to strictly define these dose-dependent effects
and to analyze the stability of compounds. It is
interesting to note that the results of the in vitro assays
suggest that the tetrathiotungstate salts could neutral-
ize free radicals (antioxidant activity), a property that
is useful in the treatment of oxidative stress-induced
degenerative diseases.
hydrotreating catalysts. J Catal 198(1):9–19. https://doi.
org/10.1006/jcat.2000.3124
Blois MS (1958) Antoxidant determinations by the use of a
stable free radical. Nature 181:1199–1200. https://doi.org/
10.2307/3280933
Brewer GJ (2003) Copper-lowering therapy with
tetrathiomolybdate for cancer and diseases of fibrosis and
inflammation. J Trace Elem Exp Med 16(4):191–199.
https://doi.org/10.1002/jtra.10045
Brewer GJ (2005) Copper lowering therapy with
tetrathiomolybdate as an antiangiogenic strategy in cancer.
Curr Cancer Drug Targets 5(3):195–202. https://doi.org/
10.2174/1568009053765807
Brewer GJ (2009) Zinc and tetrathiomolybdate for the treatment
of Wilson’s disease and the potential efficacy of anticopper
therapy in a wide variety of diseases. Metallomics
1(3):199–206. https://doi.org/10.1039/b901614g
Brewer GJ (2016) Copper in Wilson’s and Alzheimer’s dis-
eases, copper-lowering therapy in cancer and other dis-
eases, and copper deficiency. In: Molecular, genetic, and
nutritional aspects of major and trace minerals. Elsevier
Inc. https://doi.org/10.1016/B978-0-12-802168-2.00010-5
Brewer GJ, Dick RD, Yuzbasiyan-Gurkin V, Tankanow R,
Young AB, Kluin KJ (1991) Initial therapy of patients with
Wilson’s disease with tetrathiomolybdate. Arch Neurol
Acknowledgements The authors gratefully acknowledge the
financial support provided by the UABC-UNAM SENER-
CONACYT 117373 project. Authors thank David Navas
Fernandez for excellent technical support in these
experiments, as well as Dr. Ra u´ l Romero Rivera for his
valuable comments. The scientific work developed by MAM’s
group is supported by grants PID2019-105010RB-I00 (Spanish
Ministry of Science, Innovation and Universities), and UMA18-
FEDERJA-220 (Andalusian Government and FEDER) and
funds from group BIO 267 (Andalusian Government), as well as
funds from ‘‘Plan Propio de Investigaci o´ n y Transferencia’’
(
U. M a´ laga).
Author contributions KV-G: Investigation, Writing-
Reviewing and Editing. JC-R: Conceptualization, Funding
acquisition. JFH-M: Methodology, Data curation. MM-B:
Conceptualization, Visualization. LJD-R: Formal analysis. IC-
G: Resources. DC-V: Validation. MCO: Investigation. MAM:
Supervision. LBR-S: Writing-Original draft preparation, Project
administration.
48:42–47.
00530130050019
https://doi.org/10.1001/archneur.1991.
Brewer GJ, Dick RD, Johnson V, Wang Y, Yuzbasiyan/Gurkan
V, Kluin K, Fink JK, Aisen A (1994) Treatment of Wil-
son’s disease with ammonium tetrathiomolybdate. Arch
Neurol 51:545–554. https://doi.org/10.1001/archneur.
1
994.00540180023009
Brewer GJ, Johnson V, Fink JK, Brunberg A, Dick RD, Kluin KJ
1996) Treatment of Wilson disease with ammonium
Compliance with ethical standards
(
Conflict of interest The authors declare that they have no
known competing financial interests or personal relationships
that could have appeared to influence the work reported in this
paper.
tetrathiomolybdate. Arch Neurol 53:1017–1025. https://
doi.org/10.1001/archneur.1996.00550100103019
Brewer GJ, Dick RD, Grover DK, LeClaire V, Tseng M, Wicha
M, Pienta K, Redman BG, Jahan T, Sondak VK, Straw-
derman M, LeCarpentier G, Merajver SD (2000) Treatment
of metastatic cancer with tetrathiomolybdate, an anticop-
per, antiangiogenic agent: phase I study. Clin Cancer Res
6
(1):1–10
References
C a´ rdenas C, Quesada AR, Medina MA (2006) Evaluation of the
anti-angiogenic effect of aloe-emodin. Cell Mol Life Sci
Aggarwal A, Bhatt M (2018) Advances in treatment of Wilson
disease. Tremor Hyperkinetic Mov 8:1–13. https://doi.org/
6
6
3(24):3083–3089. https://doi.org/10.1007/s00018-006-
399-6
1
0.7916/D841881D
Chianelli RR, Pecoraro TA (1981) United States Patent
,288,422. Method of preparing chalcogenides of group
Alonso G, Aguirre G, Rivero IA, Fuentes S (1998) Synthesis and
characterization of tetraalkylammonium thiomolybdates
and thiotungstates in aqueous solution. Inorg Chim Acta
4
VIII by low temperature precipitation from monaqueous
solution, the products produced by daid method and their
use as catalysts. https://doi.org/10.1016/0375-6505
2
1
74(1):108–110.
693(97)05901-X
https://doi.org/10.1016/S0020-
(
85)90011-2
Alonso G, Del Valle M, Cruz-Reyes J, Licea-Claverie A,
Fuentes S (2000) Preparation of WS2 catalysts by in situ
decomposition of tetraalkylammonium thiotungstates.
Appl Catal A 197(1):87–97. https://doi.org/10.1016/
S0926-860X(99)00536-0
Berhault G, Mehta A, Pavel AC, Yang J, Rendon L, Y a´ caman
MJ, Araiza LC, Moller AD, Chianelli RR (2001) The role
of structural carbon in transition metal sulfides
Dooley K, Zon LI (2000) Zebrafish: a model system for the
study of human disease. Curr Opin Genet Dev
1
4
0(3):252–256.
37X(00)00074-5
https://doi.org/10.1016/S0959-
Garc ´ı a-Caballero M, Quesada AR, Medina MA, Mar ´ı -Beffa M
2018) Fishing anti (lymph)angiogenic drugs with zebra-
(
fish. Drug Discovery Today 23(2):366–374. https://doi.
org/10.1016/j.drudis.2017.10.018
1
23

Biometals
Goodman M, Bostick RM, Kucuk O, Jones DP (2011) Clinical
trials of antioxidants as cancer prevention agents: past,
present, and future. Free Radical Biol Med
Ramanathan K, Weller SW (1985) Characterization of tungsten
sulfide catalysts. J Catal 95:249–259. https://doi.org/10.
1016/0021-9517(85)90025-9
´
51(5):1068–1084.
freeradbiomed.2011.05.018
https://doi.org/10.1016/j.
Romero-S a´ nchez LB, Mar ´ı -Beffa M, Carrillo P, Medina MA,
D ´ı az-Cuenca A (2018) Copper-containing mesoporous
bioactive glass promotes angiogenesis in an in vivo zeb-
rafish model. Acta Biomater 68:272–285. https://doi.org/
10.1016/j.actbio.2017.12.032
Kelty SP, Berhault G, Chianelli RR (2007) The role of carbon in
catalytically stabilized transition metal sulfides. Appl Catal
A 322(suppl):9–15. https://doi.org/10.1016/j.apcata.2007.
0
1.017
Schubert WM, Murphy RB, Robins J (1962) Electron donor and
acceptor properties of alkyl substituents. Tetrahedron
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling
TF (1995) Stages of embryonic development of the zeb-
rafish. Dev Dyn 203(3):253–310. https://doi.org/10.1002/
aja.1002030302
Lin S, Zhao Y, Ji Z, Ear J, Chang CH, Zhang H, Low-Kam C,
Yamada K, Meng H, Wang X, Liu R, Pokhrel S, Madler L,
Damoiseaux R, Xia T, Godwin HA, Lin S, Nel AE (2013)
Zebrafish high throughput screening to study the impact of
dissolving metal oxide nanoparticles on the hatching
enzyme, ZHE1. NIH Public Access 9:1776–1785. https://
doi.org/10.1038/jid.2014.371
17(3–4):199–214.
4020(01)99020-9
https://doi.org/10.1016/S0040-
Tobia C, de Sena G, Presta M (2011) Zebrafish embryo, a tool to
study tumor angiogenesis. Int J Dev Biol 55(4–5):505–509.
https://doi.org/10.1387/ijdb.103238ct
Tran TC, Sneed B, Haider J, Blavo D, White A, Aiyejorun T,
Baranowski TC, Rubinstein AL, Doan TN, Dingledine R,
Sandberg EM (2007) Automated, quantitative screening
assay for antiangiogenic compounds using transgenic
zebrafish. Can Res 67(23):11386–11392. https://doi.org/
10.1158/0008-5472.CAN-07-3126
McQuaid A, Lamand M, Mason J (1994) Thiotungstate-copper
interactions II. The effects of tetrathiotungstate on systemic
copper metabolism in normal and copper-treated rats.
J Inorg Biochem 53(3):205–218. https://doi.org/10.1016/
Truong L, Harper SL, Tanguay RL (2011) Evaluation of
embryotoxicity using the zebrafish model. Methods Mol
Biol 691:271–279.
9780470464151
https://doi.org/10.1002/
0
162-0134(94)80005-7
Mishra K, Ojha H, Chaudhury NK (2012) Estimation of anti-
radical properties of antioxidants using DPPH- assay: a
critical review and results. Food Chem 130(4):1036–1043.
https://doi.org/10.1016/j.foodchem.2011.07.127
Nechaev IV, Pavlov ADS (2004) The species specificity of
hatching enzyme and its effect on the duration of
embryogenesis in the Fish Cichlasoma nigrofasciatum
(Cichlidae) 394(6):845–848. https://doi.org/10.1023/b:
dobs.0000017136.04664.66
Ord J (2019) Ionic stress prompts premature hatching of Zeb-
rafish ( Danio rerio ) Embryos. Fishes. https://doi.org/10.
Warkentin KM (2011) Plasticity of hatching in amphibians:
evolution, trade-offs, cues and mechanisms. Integr Comp
Biol 51(1):111–127. https://doi.org/10.1093/icb/icr046
Wilkinson RN, Van Eeden FJ M (2014) The zebrafish as a model
of vascular development and disease. In: Progress in
molecular biology and translational science, vol 124, 1st
ed. Elsevier Inc. https://doi.org/10.1016/B978-0-12-
386930-2.00005-7
Young BW, Bremmer I, Mills CF (1982) Effects of iron on
copper metabolism and copper on iron metabolism in rats.
J Inorg Biochem 15:121–134. https://doi.org/10.1152/
ajplegacy.1973.224.3.514
3
390/fishes4010020
Pecoraro TA, Chianelli RR (1985) United States Patent
,528,089. Hydrogenation proccess using carbon-contain-
ing molybdanum and tungsten sulfide catalysts
4
Publisher’s Note Springer Nature remains neutral with
regard to jurisdictional claims in published maps and
institutional affiliations.
123