https://doi.org/10.52973/rcfcv-e34481
Received: 04/07/2024 Accepted: 28/08/2024 Published: 05/12/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34481
ABSTRACT
As a result of the increasing use of quantum dots (QDs) and increased
exposure of human beings to quantum dots, the study of the toxicity
of the particles has become an important issue. In this study, the
protective activity of silymarin and mitoquinone (MitoQ), which are
known to have antioxidant properties, on the histopathological and
biochemical changes observed in the liver of mice treated with CdTe
QDs was investigated. 26 male Swiss mice were randomly divided
into four groups: Control (G1), CdTe QDs (G2), silymarin + CdTe QDs
(G3), mitoquinone + CdTe QDs (G4) application groups. Animals were
sacriced 24 hours (h) after injections and hyperspectral microscopy
images were obtained. According to the ICP–MS results, the CdTe
QDs injected through the tail vein accumulated in the liver at the
end of 24 h and caused tissue damage according to the hematoxylin
& eosin examination, and better preservation was observed with
the antioxidant pre–treatment. The immunouorescence results
showed increased inammation and apoptosis in the QDs group. It
was observed that silymarin and mitoquinone decreased anti–MMP–9,
anti–IL–10, anti–IL–1b, anti–TNF–α, and anti–caspase–9, TUNEL–
positive cell ratio, liver MDA levels. There was no signicant difference
in serum TAS (P=0.509), TOS (P=0.588) levels, but antioxidants also
increased tissue SOD and CAT levels. Antioxidants had no signicant
effect on anti–MT–MMP2 and anti–caspase–8 levels (P<0.001). In
conclusion, it was shown that pretreatment of CdTe QD–administered
mice with silymarin and mitoquinone can reduce oxidative stress in
liver tissue and may have a protective effect through reduction of
apoptosis and inammation.
Key words: Antioxidant; quantum dot; mitoquinone; oxidative
stress; silymarin
RESUMEN
Como consecuencia del creciente uso de puntos cuánticos (QD) y de
la mayor exposición de los seres humanos a los mismos, el estudio
de la toxicidad de las partículas se ha convertido en una cuestión
importante. En este estudio se investigó la actividad protectora de
la silimarina y la mitoquinona (MitoQ), conocidas por sus propiedades
antioxidantes, sobre los cambios histopatológicos y bioquímicos
observados en el hígado de ratones tratados con CdTe QDs. Se
dividieron aleatoriamente 26 ratones suizos macho en cuatro grupos:
Control (G1), CdTe QDs (G2), silimarina + CdTe QDs (G3), mitoquinona +
CdTe QDs (G4) grupos de aplicación. Los animales fueron sacricados
24 horas (h) después de las inyecciones y se obtuvieron imágenes
de microscopía hiperespectral. Según los resultados de ICP–MS, los
CdTe QDs inyectados a través de la vena de la cola se acumularon en
el hígado al cabo de 24 h y causaron daños tisulares según el examen
de hematoxilina y eosina, y se observó una mejor conservación con el
pretratamiento antioxidante. Los resultados de la inmunouorescencia
mostraron un aumento de la inamación y la apoptosis en el grupo
de QDs. Se observó que la silimarina y la mitoquinona disminuyeron
los niveles de anti–MMP–9, anti–IL–10, anti–IL–1b, anti–TNF–α y anti–
caspasa–9, la proporción de células TUNEL positivas y los niveles
de MDA hepáticos. No hubo diferencias signicativas en los niveles
séricos de TAS (P=0.509), TOS (P=0.588), pero los antioxidantes también
aumentaron los niveles tisulares de SOD y CAT. Los antioxidantes no
tuvieron un efecto signicativo en los niveles de anti–MT–MMP2 y anti–
caspasa–8 (P<0.001). En conclusión, se demostró que el pretratamiento
de ratones tratados con CdTe QD con silimarina y mitoquinona, que
tienen fuertes propiedades antioxidantes, puede reducir el estrés
oxidativo en el tejido hepático y puede tener un efecto protector gracias
a la reducción de la apoptosis y la inamación.
Palabras clave: Antioxidante; punto cuántico; mitoquinona; estrés
oxidativo; silimarina
Protective Effect of Silymarin and Mitoquinone (MitoQ) Aganist
Hepatotoxicity of Cadmium Telluride Quantum Dot (CdTe QDs)
Nanoparticles in Mice
Efecto protector de la silimarina y la mitoquinona (MitoQ) contra la hepatotoxicidad de las
nanopartículas de puntos cuánticos de telururo de cadmio (CdTe QDs) en ratones
Seda Şimşek
1
* , Merve Solmaz
1
, İsmail Hakkı Nur
2
, Muslu Kazım Körez
3
, Nejat Ünlükal
1
, Ender Erdoğan
1
1
Selcuk University, School of Medicine, Department of Histology and Embryology. Konya, Türkiye.
2
Erciyes University, School of Veterinary, Department of Anatomy. Kayseri, Türkiye.
3
Selcuk University, School of Medicine, Department of Biostatistics. Konya, Türkiye.
*Corresponding Author: sedaatay89@gmail.com
FIGURE 1. Creation of experimental groups. Group 1: Control group. Only 100 µL/animal physiological saline was administered. Group 2: CdTe QDs 10 mg·kg
–1
dose
injected into the tail vein of 100 µL/animal is the toxicity group. Group 3: 100 µL /animal at a dose of 100 mg·kg
–1
silymarin was administered ip. After 2 hours, CdTe
QDs (10 mg·kg
–1
dose) was injected into the tail vein of 100 µL/animal. Group 4: 100 µL /animal at a dose of 5 mg·kg
–1
mitoquinone was administered ip. After 2 hours,
CdTe QDs (10 mg·kg
–1
dose) was injected into the tail vein of 100 µL /animal. After 24 hours, the animals were sacriced
Protective role of Silymarin and MitoQ against CdTe QD-induced hepatotoxicity / Şimşek et al.____________________________________
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INTRODUCTION
Since the discovery of semiconductor engineered nanoparticles
(NPs) various types of QDs (CdTe, CdSe, ZnS etc.) have been
synthesized. These luminescent nanomaterials have shown great
promise for a variety of applications, in particular in optoelectronics
and biological labelling in recent years [1]. Thanks to its small
dimensions, large surface areas, and unique optical, electronic, and
chemical properties, it has increasing usage areas and so long–term
potential damage to the environment and public health has therefore
become a issue in recent years.
QDs have been shown to enter the bloodstream and ultimately
accumulate in the liver and the mechanisms of potential hepatotoxicity
are complex. QDs can be cytotoxic and cause cell stress. In particular,
the toxicity of cadmium–based QDs is thought to be related to the
leaching of free Cd
2+
from the QDs core [1]. Some general mechanisms
have been studied, such as cellular stress, mitochondrial dysfunction,
immune responses, inhibition and biotransformation of various
biomolecules, and most importantly, the formation of reactive
metabolites that trigger this process [2]. Studies have shown that
QDs also have the ability to activate macrophages and increase the
expression of inammatory factors [3]. However, a number of other
studies have shown that the inhibition of the production of ROS is the
only way to protect cells from the oxidative stress or DNA damage
caused by QDs [4].
Silymarin, the seed extract of milk thistle, has long been used as
a broad–spectrum herbal extract to protect the liver from various
toxic substances and to treat liver damage, hepatitis and cirrhosis
[5]. Studies to date have shown that silymarin and its avonolignans
have signicant antioxidant, anti–inammatory, and pro–apoptotic
properties. It has very diverse biological and pharmacological
activities with different biomolecular mechanisms [6, 7].
MitoQ is one of the most widely used antioxidants that targets
the mitochondria. MitoQ is formed by the covalent attachment
of ubiquinone or coenzyme Q, an endogenous antioxidant and
component of the mitochondrial electron transport chain (ETC),
to triphenylphosphonium (TPP
+
) ions. TPP
+
is a lipophilic cation
that pushes the ubiquinone moiety to the inner mitochondrial
membrane with a negative electrochemical potential [8]. Mito Q
has in vitro properties that prevent lipid peroxidation, reduce protein
carbonylation and ROS levels and prevent apoptosis [9].
CdTe QDs are still widely used and reducing the adverse effects by using
some antioxidant compounds would be clinically important. Although
there are some in vitro and in vivo studies on using antioxidants to
counteract nanoparticle toxicity [10, 11, 12], but there is no study on
the hepatoprotective effect of silymarin and mitoquinone antioxidants
against liver toxicity of CdTe QDs. Therefore, in this study, we aimed to
nd the pharmacological way to reduce the hepatoxicity of CdTe QDs by
investigating the effects of the antioxidants silymarin and mitoquinone.
MATERIALS AND METHODS
Experimental design
The study used 26 male Swiss albino mice (Mus musculus), 8 weeks
old. All animals were maintained according to the animal care guidelines
of SÜDAM Ethics Committee at Selçuk University Experimental
Medicine Application and Research Centre during the experiment.
26 male Swiss albino mice, were randomly divided into groups;
Group1 (G1): Control group {100 μL/animal physiological saline}:
(n=5), Group2 (G2): CdTe QDs {100 μL/animal, 10 mg·kg
–1
CdTe
core type (Sigma–Aldrich, USA) – COOH (carboxyl) functionalized,
intravenously (iv)} group: (n=7), Group 3 (G3): Silymarin {(100 mg·kg
–1
silymarin (Sigma–Aldrich, USA), intraperitoneally (ip), 2 hours (h) ago)
+ CdTe QDs (100μL/animal, 10 mg·kg
–1
)} group: (n=7), Group 4 (G4):
Mitoquinone {(5 mg·kg
–1
MitoQ (Thermo Fisher, USA), ip, 2 h ago) +
CdTe QDs (100 μL/animal, 10 mg·kg
–1
)}: (n=7). The experimental groups
summarised in detail in FIG. 1.
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Following a 24–hour period, the animals were anaesthetised with
60 mg·kg
–1
ketamine hydrochloride (Ketalar®, Parke–Davis, Pzer,
Istanbul, Turkey) and 5 mg/ An ip injection of 0.6 mg·kg
–1
xylazine
hydrochloride (Rompun®, Bayer AG, Leverkusen, Germany) was
administered, and in vivo hyperspectral uorescence microscope
images were obtained to visualize the overall distribution of CdTe
QDs in the body following injection. Subsequently, the animals were
sacriced, and blood was collected via intracardiac puncture for the
purpose of measuring total oxidant and antioxidant levels.
The liver tissues were removed without causing trauma, weighed
on a precision balance, and a portion of the tissue was utilized for
histological examinations, including general histopathological
evaluations with H&E, immunohistochemical analysis, and anti–MT–2A
for metallothionein binding to Cd+ released in the tissue . Levels of Mt–
MMP2 and MMP9 antibodies and related IL10, IL–1beta and TNF alpha
antibodies were measured as markers of acute inammation. Anti–
Caspases 3 and 8 levels were also measured as apoptosis markers.
An ICP–MS (inductively coupled plasma–mass spectrometry) analysis
was conducted on a portion of the tissue to quantify the accumulation of
Cd in the tissue. Additionally, an ELISA (enzyme–linked immunosorbent
assay) analysis was performed on the remaining portion of the tissue
to assess the levels of oxidative stress markers, including MDA
(malondialdehyde), SOD (superoxide dismutase), and CAT (catalase).
In vivo hyperspectral uorescent imaging
An in vivo imaging system (Syngene GBOX–XRQ, Cambridge, UK) was
used to acquire digital images of animals in the CdTe QDs injected
group under anaesthesia.
Whole body images and images of epidididymis, testis, stomach,
spleen, brain, lung, heart, kidney and liver organs obtained after
sacrication were taken. Imaging was performed using an epimid
wave, 302 nm UV excitation and 710 nm emission lter with a scan
time between 720 and 900 ms. The instrument’s software program
was used to process the images.
Confocal microscopy
The liver tissues of G1 and G2 xed in 4% paraformaldehyde and
embedded in cryomatrix and sections were cut at 50 μm slides using
a cryostat. Confocal microscope (NIKON /Nikon A1R1, NY, USA) images
of sections covered medium with DAPI (nuclear marker) (Sigma–
Aldrich, MO, USA) and obtained at 10× and 40× objective magnication.
Inductively coupled plasma mass spectrometry (ICP–MS)
ICP–MS (Agilent 7500A, Tokyo, Japan) was used to analyze the
quantitative measurement of the accumulation of CdTe QDs in
liver, kidney, spleen, brain, heart and testis tissues after 24 h. It was
performed according to the method of the Nordic Committee for
Food Analysis (NMKL,186) [13]. Tissue samples were homogenised
with 2 mL of 10.3 M HNO
3
at 95°C for 60 min. Samples were made
up to a nal volume of 5 mL with MilliQ® water. Samples were then
analysed. Cumulative value was obtained by combining the organs
of all animals in the G1 and G2.
Histological studies
Hematoxylin & Eosin (H&E) staining
For HE staining, tissue preparations were kept in 99.9% absolute
alcohol for a few seconds and then treated with water and then kept
in Harris Hematoxylin ( HHS32, Sigma–Aldrich,MO, USA ) [14]. After
passing through water and absolute alcohol respectively, they were
kept in eosin stain (HT110116, Sigma–Aldrich,MO, USA) for 1 minute. It
was passed through alcohol series with increasing concentration and
covered with a coverslip and entellan (1.07960, Sigma–Aldrich,MO, USA).
Immunouorescence staining
To determine the changes in the amount of metallotheonine
bound to free Cd
+2
in the tissue, 4 μm thick liver frozen sections were
labeled with polyclonal anti–MT2A primary antibody (DF6755 Anity
Biosciences, Japan). To evaluate the inammation sections were
labelled with anti–MT–MMP2 (sc–80213, Santa Cruz Biotechnology,
USA), anti–MMP9 (sc–10737, Santa Cruz Biotechnology, USA), anti–
TNF alpha (ABIN343428, antibodies.com, UK), conjugated anti–
IL–10 (A–2) (Alexa Fluor 594, sc–365858, Santa Cruz Biotechnology,
USA) and, conjugated anti IL–1 beta (Alexa Fluor 594, sc–32294,
Santa Cruz Biotechnology, USA) antibodies. For the evaluation of
apoptotic pathways, liver sections were labeled with anti–caspase–8
(ab4052, Abcam, United Kingdom) and anti–caspase–9 antibody
(ab4053, Abcam, United Kingdom) as primary antibodies. In this
immunohistochemical analysis, each section obtained from the tissue
was treated individually with all these antibodies and these antibodies
were marked with fluorescently labeled secondary antibodies,
making the presence of these markers selected for inammation
and apoptosis visible, and at the same time, the level was determined
with the appropriate program.
For this purpose, the sections were incubated with PBS containing
5% BSA and 0.2% Triton X–100 for 30 min. After incubation with
protein block solution, tissues were treated with primer antibodies
(1/200 to 1/500 dilutions). Tissues were stored at 4°C overnight and
then treated with uorescently labelled secondary antibody (donkey
anti–rabbit IgG–FITC (sc–2090, Santa Cruz Biotechnology, USA) and
covered with DAPI–containing uorescent cover medium.
TUNEL was performed with the Andy FluorTM 488 Apoptosis
Detection Kit (ABP Biosciences, MD, USA). The internucleosomal
cleavage of DNA is one of the hallmarks of apoptosis. Using terminal
deoxynucleotidyl transferase (TdT)–mediated dUTP nick–end labeling
(TUNEL), DNA cleavage in apoptotic cells can be detected in situ
in xed cells or tissue sections. TUNEL is highly selective for the
detection of apoptotic cells. It does not detect necrotic cells or
cells with DNA strand breaks resulting from irradiation or drug
treatment. In the TUNEL assay, the TdT enzyme is used to catalyze
the addition of tagged dUTP to the 3’ ends of cleaved DNA fragments.
uorescent dye–conjugated dUTP can be used for direct detection
of fragmented DNA by uorescence microscopy. The TUNEL Andy
Fluor™ 488 Apoptosis Detection Kit contains dUTP conjugated to
biotin and streptavidin conjugated to bright and photostable Andy
Fluor™ 488 green uorescent dye for bright uorescent TUNEL
staining. TUNEL (+) marked cells and DAPI (+) marked cells were
counted using the Image J (National Institutes of Health, Bethesda,
MD, USA) program. The apoptotic index (AI) was calculated using the
formula “total apoptotic cells/total cells × 100”.
TABLE I
Total weight of animals before sacrication and liver and kidney weights after sacrication
Control G2 G3 G4 P–value
Total weight (g) 30.40 ± 0.75 29.43 ± 0.84 31.43 ± 0.72 30.57 ± 0.37 0.288
Liver weight (mg) 1891.40 ± 104.791960.29 ± 46.99
b
1605 ± 101.53
a
1736.86 ± 59.21 0.028
a,b
:Dierent small superscript letters indicate that statistically signicant dirence after multiple comparison
Protective role of Silymarin and MitoQ against CdTe QD-induced hepatotoxicity / Şimşek et al.____________________________________
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All immunohistochemical procedures were performed in a dark
and humid environment. Examined under a uorescence microscope
(Olympus BX51 Trinocular fluorescence microscope, Hamburg,
Germany). Labelled area percentages in the recorded images were
calculated using the Image J program.
TAS (Total Antıoxıdant Stress), TOS (Total Oxidative Stress), OSI
(Oxidative Stress Index)
TAS and TOS levels were measured using the commercially available
TAS (RL0017) and TOS (RL0024) Rel Assay Diagnostics Kit (Relassay,
Turkey). The automated method is based on the photometric method
using the characteristic bleaching of a more stable ABTS 2,2′–azino–
bis (3–ethylbenzothiazoline–6–sulphonic acid). Results are expressed
as mmol Trolox equivalent·L
–1
[15].
The oxidising agents present in the sample oxidise the ferrous
ion–o–dianisidine complex to the ferric ion. A automated colorimetric
method was used to measure total oxidant status [16]. The test has
been calibrated with hydrogen peroxide and results are expressed
in micromolar hydrogen peroxide equivalents per litre (μmol
H
2
O
2
equivalent·L
–1
).
It is calculated according to the formula OSI = [TOS (μmol H
2
O
2
·L
-1
) /TAS
(μmol Trolox equivalent·L
–1
)] × 100. It indicates the oxidative stress load.
Liver SOD1 (Superoxide dismutase), CAT (Catalase), and MDA
(Malondialdehyde) Levels
Superoxide dismutase (Mouse SOD1 ELISA KIT, Fine Test EM0419–,
China), and catalase (Mouse CAT ELISA KIT, Fine Test EM0357–, China),
which are antioxidant enzymes, and malondialdehyde, which is a
quantitative indicator of fatty acid oxidation (Mouse MDA ELISA KIT),
Fine Test EM1723–, China) were measured using ELISA kits. These kits
are based on Double antibody–Sandwich ELISA detection method.
The microplate provided in these kit have been precoated with anti
SOD1, CAT and MDA antibodies.
The tissue homogenates were prepared for the measurements.
Residual blood was removed by washing the tissue with pre–cooled
PBS buffer (0.01 M, pH=7.4). The tissue was weighed and fragmented
using a homogeniser. The homogenates were centrifuged (Sorvall
ST1R Plus Thermo Scientic™, MA, USA) at 5,000 G to obtain the
supernatant. The total protein concentration was determined
using the BCA kit ( BCA Assay Kit, Thermo Scientic™, MA , USA).
Appropriate dilutions were made using sample dilution buffer. The
results were read in the ELISA microplate reader (Multiskan™ FC,
Thermo Scientic™, MA , USA) and expressed in units appropriate
for the kit.
Statistical analysis
All statistical analyses were performed using the R statistical
software language, version 4.2.1 (www.r–project.org). Before analyses,
normality of data was checked using Shapiro–Wilk’s normality test
and Q–Q plots, and homogeneity of group variances was checked
using Levene’s test. Results are presented as mean ± standard error.
The liver and organ weights which were weighed on an electronic
digital scale (Bovoisin, U.S.A.) , MDA, SOD, CAT levels in liver,
biochemical parameters and immunouorescence staining results
of the animals according to the study groups were analysed by using
One–Way Analysis of Variance if the data were normal distributed and
the group variances were homogeneous; Welch F (Robust ANOVA)
test if the data were normal distributed but the group variances were
not homogeneous; Kruskal Wallis test if the data were not normal
distributed. Tukey HSD, Games–Howell test and Dunn test with FDR
(false discovery rate) correction were used for multiple comparisons
for the parameters that were found to be different between the groups
as a result of these tests, respectively.
All results were also presented in FIGUREs using box plots.
Differences between groups are indicated by ‘*’. The signicance
level was set at 5%. For FDR correction, the signicance level was
corrected to 0.0083.
RESULTS AND DISCUSSION
A comparison of the total weights of 26 animals before sacrice
and liver weights after sacrice by study groups is shown in TABLE I.
According to the results obtained, the live weights of the animals
were similar between all groups (P=0.288). After sacrice, the liver
weights of animals in the G2 group were signicantly higher than
those in the silymarin treatment group (G3) (adjusted P=0.039). In
accordance with previous studies, it is thought that there is an
increase in liver weight due to increased cell inltration as an acute
toxicity response [17, 18].
In Vivo Hyperspectral and Confocal Microscope Images of CdTe QDs
The distribution of CdTe QDs in the body after injection can be
seen where there is an intense glow in the hyperspectral images.
In the non–sacriced animals, the distribution in the organs is not
clearly visible due to their thick skin and fur, while the injection site
is clearly visible from the tail vein (FIG. 2A). According to the graph of
uorescence intensity between organs, the liver, kidney and spleen
accumulated more than other organs (FIGS. 2B, 2C, 2D).
Confocal microscopy was used to conrm the presence of CdTe–
QDs injected through the tail vein in the liver. In the images obtained,
CdTe QDs were seen in red colour with Rhodamine lter (exiemission
range 690 nm–730 nm), whereas nuclei were seen in blue colour with
DAPI lter (emission range 425 nm–475 nm) and the merged images
of the two are shown in FIG. 2. CdTe QDs accumulated in liver cells
and sinusoids.
A B C D
E F G
Liver Kidney Spleen Heart
0
2
4
6
8
5.567
2.660
6.224
1.535
0000
ICP-MS RESULTS
Cd
2+
mg·kg
-1
accumulatedintissues
Control CdTe
FIGURE 2. (A) Hyperspectral microscope images of two animals. Images were
taken under anaesthesia. Imaging was performed using an epimid wave,
302nm UV excitation and 710 nm emission lter with a scan time between 720
and 900 ms. (B) Spleen, heart, kidney and liver organs from left to right in two
dierent animals after sacrication. (C) Top view of CdTe QDs density plot in
organs. (D) Side view of the density plot of CdTe QDs deposited in the organs
in the middle image. Colours used in the density plot; the density decreases
from yellow to blue. At the end of 24 hours, images of QDs (red)(E) and nuclear
dye DAPI (blue)(F) and merged images of QDs in liver tissue (G) obtained by
confocal microscopy. Red coloured images were obtained with 640 nm excitation
and Rhodamine emission lter (emission range 690 nm–730 nm), blue coloured
nucleus images were obtained with 405 nm excitation and DAPI emission lter
(emission range 425 nm–475 nm). Merged images were obtained by overlapping
the two lters. Images were obtained at 40× objective magnication (scale
20μm). (H) Results of ICP–MS measurement of Cd
2+
accumulated in liver, kidney,
spleen, heart. The results were obtained only from the animals in the CdTe QDs
treated group and a cumulative evaluation was made by combining the organs
from each animal. The results were given as mg Cd
2+
per kg organ
H
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Detection of Cadmium (Cd
+2
) in tissues using ICP–MS
According to cumulative evaluation of ICP–MS results, Cd
2+
levels
were highest in the spleen at the end of 24 hours, while liver and
kidney reached signicantly higher Cd
2+
levels than the other tissues
(FIGS. 2E, 2F, 2G). According to ICP–MS results, liver, spleen and
kidney showed the highest cadmium levels, indicating that these
tissues are preferential sites of CdTe–QDs accumulation [19, 20, 21,
22, 23].
10× 40× 40×
Group 1Group 2Group 3Group 4
FIGURE 3. H&E images of the liver at 10× and 40× objective magnications. Images were obtained by light microscopy. Group 1: Hepatocyte cords arranged along the
central vessel (black circle), hepatic sinusoids (black arrow), hepatocytes with uniform nuclei (black arrowheads). Group 2: Loss of lobular structure (yellow circle),
congestion (thick yellow arrow), inammatory cell inltration (yellow arrowheads), dilated sinusoids (blue arrow), hepatocyte necrosis (green arrows), dilated central
vein (yellow arrow). Group 3 and Group 4: Dilated sinusoids (blue arrow), activated Kuppfer cells (ne–tipped black arrow) (Scale Bar: 10×=100 μm, 40×=20 μm)
Protective role of Silymarin and MitoQ against CdTe QD-induced hepatotoxicity / Şimşek et al.____________________________________
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Effects of CdTe–QDs on the morphology of the tissue
Histological sections from the liver tissues of control mice showed
a normal structure. Hepatocytes were organised as cords of cells with
large, globular centric nuclei, showing a smooth radial architecture
around the central vein. There was no inammatory cell inltration
or necrosis. (FIG. 3).
In G2, where CdTe QDs were applied, dilated central veins and
sinusoids, congestion and loss of lobular structure, hepatocyte
necrosis and occasional inammatory cell inltration were observed.
In G3 and G4, where silymarin and mitoquinone antioxidants were
applied, dilated sinusoids, binuclear hepatocytes and activated
Kuppfer cells were observed. In particular, the dilated areas were
less in the silymarin group than in the mitoquinone group. (FIG. 3).
As mentioned earlier, congestion and inammatory cell inltration,
which are thought to be the cause of liver weight gain, were reduced
with silymarin. We believe that this result is due to the antioxidant
and immunomodulatory effects of silymarin.
TABLE II
Fluorescence labelled percentages of Liver anti–MT2A,
MT–MMP2, MMP9, IL–10, IL–1 Beta, TNF alpha
Liver Control G2 G3 G4 P–value
Metallothionein 0.68 ± 0.22
a
8.20 ± 1.19
b
8.93 ± 1.21
b
9.25 ± 0.69
b
<0.001
MT–MMP2 3.09 ± 0.47
a
15.95 ± 0.61
b
14.60 ± 0.83
b
14.08 ± 0.52
b
<0.001
MMP 9 0.50 ± 0.16
a
15.95 ± 0.61
b
12.81 ± 0.43
c
14.22 ± 0.69
bc
<0.001
IL–10 3.92 ± 0.68
a
8.60 ± 1.19
b
6.91 ± 0.66
b
7.03 ± 0.61
b
0.012
IL–1β 0.94 ± 0.25
a
11.23 ± 0.75
b
6.59 ±1.02
c
5.99 ± 0.45
c
<0.001
TNF α 2.12 ± 0.98
a
16.65 ± 1.58
b
7.49 ± 0.42
c
3.66 ± 0.57
a
<0.001
P–value optained using one–way ANOVA.
a,b
: Dierent small superscript letters indicate
that statistically signicant dierence after multiple comparison
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Immunouorescence staining results
It was shown by means of hyperspectral and confocal microscopy
that CdTe QDs injected intravenously into mice accumulate in liver
tissue after 24 h. CdTe QD application caused an increase in the
percentage of anti–metallothionein. MT can serve as a very sensitive
biomarker when the living body is exposed to Cd
2+
stress [24]
Furthermore, Lin et al. reported that only free Cd
2+
dissociated from
QDs, but not the QDs themselves, can induce MT production in tissues
[25]. Therefore, a good biological index of QDs degradation in vivo may
be the expression of an increase in MT. CdTe QD application caused
an increase in the percentage of anti–metallothionein. However,
metallothionein levels in the groups pre–treated with silymarin and
MitoQ prior to CdTe QD application were found to be at the same level
as in the group treated with CdTe QDs alone. According to this result,
neither silymarin nor MitoQ had any effect on the release of Cd
2+
from
CdTe QDs. It is known that this situation is not within the mechanism of
action of the selected antioxidants and that the antioxidant properties
are exerted by very different mechanisms [26, 27]. The results are
summarised in TABLE II.
An increase in the levels of all inammatory response–related
markers was observed in the CdTe QDs (G2) treated group compared
to the control group. The increased level of anti–matrix–type
metalloproteinase 2 (anti–MT–MMP2) after CdTe QD application was
not affected by silymarin or mitoquinone pretreatment and was higher
than in the control group. The level of anti–MMP9 was increased after
CdTe QD application compared to the control group, while it was
lower in the silymarin pre–treated group. The anti–MMP9 level in the
mitoquinone pre–treated group (G4) was similar to that in the G2 and
G3 groups (TABLE II). The percentage of uorescent area labelled with
anti–IL–10 antibody increased in the CdTe QD–treated group compared
to the control group, and silymarin and mitoquinone pretreatment
showed increased IL–10 levels compared to the control group, while
it was observed at a similar level in the CdTe QD group (TABLE II). A
signicant increase in IL–1 beta levels was observed in the CdTe QD
group compared to the control group. IL–1 beta levels in the silymarin
pretreatment group and in the mitoquinone pretreatment group were
higher than in the control group, but lower than in the CdTe QD group
(TABLE II). The percentage of TNF–alpha labelled area increased with
CdTe QD application and was lower in the silymarin pre–treatment
group, whereas it was at the same level in the mitoquinone pre–
treatment control group (TABLE II).
According to immunolabelling results of this study, the anti–
MT–MMP 2, anti–MMP 9, anti–IL–1 beta, anti–TNF–a and anti–IL–10
antibodies imaged under fluorescence microscope that CdTe
QDs increased inammation. MMPs have regulatory functions in
inammation and immunity [28]. In recent years, there has been
great interest in elucidating the roles of MMPs in acute liver injury.
MMP–9 is an inducible gelatinase expressed by leukocytes in acutely
damaged livers [29]. In a study, Si QDs administration resulted in
a two – to three–fold increase in gene expression levels of matrix
metalloproteinases (MMP 2 and MMP–9) [28]. According to the
data obtained from studies that QDs increase the production of
proinammatory and anti–inammatory cytokines such as IL–1β, 2,
4, 6, 8, and 18, and INF–γ, TGF–β, CRP, MIF, TNF–α, NF–kB, and CYP1A1
[21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33].
According to the anti–metallothionine immunolabelling results,
silymarin had no protective effect on the degradation of CdTe QDs.
Silymarin has been shown to signicantly reduce inammation and
associated MMP–9 levels through suppression of IL–1beta and TNF–a
levels. In antioxidant therapy studies, silymarin was found to reduce
the expression of MMPs [6, 34, 35, 36].
It was observed that MitoQ cannot prevent the degradation of CdTe
QDs. It can reduce anti–TNF α from inammatory responses to control
level in the liver and reduce anti–IL–1 beta and anti–IL–10 levels.
Confocal microscopy images of antibody labelling associated with
the inammatory response in liver tissue are shown in FIG 4.
Evaluation of Apoptosis
CdTe QDs accumulated in the liver increased the anti–caspase
8 and 9 levels. Pretreatment of silymarin and mitoquinone before
the CdTe QDs could not change these levels (FIG 5A, 5B). FIGURE 5
shows a uorescence microscopic image of liver tissue stained with
anti–anti–caspase 8 and 9.
CdTe QD application signicantly increased the percentage of
TUNEL positive cells in the liver. Both silymarin and mitoquinone
pretreatments decreased the percantage of TUNEL positive cells.
A uorescence microscope image of the TUNEL–positive cells in
the liver is shown in FIGURE 5.
It has also been found to CdTe QDs increase anti–caspase 8, which
is part of the extrinsic apoptosis pathway and anti–caspase 9, which
is part of the intrinsic apoptosis pathway, and apoptotic cell death
(TUNEL positive cells).
Silymarin was shown to reduce apoptotic cell death (TUNEL), but not
caspase–8 activation. Apoptosis has complex pathways, and it appears
that silymarin could not protect the increasing extrinsic pathways of
cell death markers, but it could clearly protect against DNA damage.
It has been observed that MitoQ does not signicantly alter the
levels of anti–caspases. However, it decreased the number of TUNEL–
positive cells. During the design of the study, it was thought that the
protective effect of MitoQ could be distinguished from silymarin by
the change in caspase 9 levels, as it directly targets the mitochondria.
This is because the main protective effect of MitoQ was to reduce the
generation of oxidative stress by protecting mitochondrial integrity.
However, according to the results, MitoQ did not produce a signicant
difference in anti–caspase 8 and 9 levels. The anti–apoptotic effect
was thought to prevent DNA damage by reducing free radicals in
the area.
GRP1 GRP2 GRP3 GRP4
MT–MMP2MMP9IL–10IL–1βTNF-α
FIGURE 4. Confocal microscope images of liver MT–MMP2, MMP 9, IL–10, IL–1β, TNFα. MT–MMP2 and MMP 9 are seen in the green colour under the FITC lter (emission
range 500 nm – 550 nm).TRITC lter (emission range 570nm–620nm) was used for IL–10 and coloured red in Group 1 and Group 2 and yellow in Group 3 and Group 4. For
IL–1β, TRITC lter (emission range 570nm–620nm) was used and IL–1β, was coloured red and QDs were coloured yellow in Groups 1, 2 and 4 (colouring was done to avoid
colour mixing due to the close emission range of conjugated antibodies and QDs for IL–10 and IL–1β,). Nuclei are seen in blue with DAPI lter (emission range 425 nm –
475 nm) and CdTe QDs with 710 nm excitation are seen in red with Rhodamine lter (emission range 650 nm – 720 nm). Sections were taken at a thickness of 50 µm. All
images were obtained by superimposing these three lters. They are shown at 10× (left column) and 40× (right column) magnication (Scale 10×= 100 µm, X40= 20 µm)
Protective role of Silymarin and MitoQ against CdTe QD-induced hepatotoxicity / Şimşek et al.____________________________________
8 of 12
TABLE IV
TAS, TOS and OSI Results
Control G2 G3 G4 P–value
TAS 1.27 ± 0.26 1.33 ± 0.24 1.49 ± 0.60 1.60 ± 0.43 0.509
TOS 2.74 ± 1.25 4.29 ± 2.28 3.77 ± 2.30 4.06 ± 1.70 0.588
OSI 0.21 ± 0.07 0.32 ± 0.17 0.24 ± 0.10 0.26 ± 0.12 0.491
P–value optained using one–way ANOVA
GRP1 GRP2 GRP3 GRP4
Caspase 8Caspase 9TUNEL
FIGURE 5. Liver Caspase 8, 9 and TUNEL uorescence microscope images and percentage of Caspase 8, 9 and TUNEL positive cells levels. Caspase 8, 9 and the nuclei of
TUNEL positive cells are seen green with FITC lter (emission range (500 nm – 550 nm) and nuclei are seen blue with DAPI lter (emission range (425 nm – 475 nm). The
merged image was obtained by overlapping the two lters. Sections were taken at a thickness of 4 μm. All images were obtained at 40× magnication (Scale; 40×= 20μm).
(A) Percentage of Caspase 8 antibody level and statistical dierences between groups (B) Percentage of Caspase 9 antibody level and statistical dierences between
groups. (C) Percentage of TUNEL positive cells level and statistical dierences between groups.
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Effect of CdTe QDs on oxidation and antioxidation levels in serum
No signicant change was observed in the oxidant and antioxidant
stress parameters of CdTe application and silymarin and mitoquinone
pretreatment in serum. Therefore, no signicant change was observed
in the oxidative stress index (OSI), which is the percentage ratio of
TAS and TOS values showing the oxidative stress load (TABLE IV).
FIGURE 6. (A) Results of liver MDA and statistical dierences between the groups (B) Results of liver CAT and statistical dierences between the groups (C) Results of
liver SOD1[Cu–Zn] and statistical dierences between the groups
Protective role of Silymarin and MitoQ against CdTe QD-induced hepatotoxicity / Şimşek et al.____________________________________
10 of 12
Organ oxidative stress levels
CdTe QDs signicantly increased the MDA level and the MDA level
in the G4 was at the same level with the control group. In the G3, an
increase in MDA level was observed compared to the control group
(FIG. 6A). The SOD1 level of G3 and G4 was found to be higher than
that of G2. The application of CdTe QDs caused an increase in SOD1
levels compared to the control group (FIG. 6B). According to the data
obtained, CdTe QDs caused an increase in catalase level compared to
the control group. In the G3 catalase level reached the highest value
compared to the other groups. Mitoquinone pretreatment also caused
an increase in catalase level compared to the control group (FIG. 6C).
It was shown that TAS, TOS and OSI levels analysed in serum as
markers of oxidative stress were not signicantly changed. However,
MDA, SOD and CAT levels analysed in liver tissue were signicantly
increased. According to results, although there was no change in
blood oxidant and antioxidant levels, the increase in tissue oxidative
stress caused by CdTe QDs was demonstrated in agreement with
other studies [17, 37].
While a decrease in MDA levels was observed, tissue SOD and CAT
levels increased. In their study, Negahdary et al. [38] observed an
increase in GPX, SOD and CAT activities and a decrease in MDA levels
in the group treated with silymarin and MgO NPs. In this study, an
increase in malondialdehyde (MDA), superoxide dismutase (SOD)
and catalase (CAT) levels was observed in the group treated with
cadmium telluride (CdTe) quantum dots (QDs) nanoparticles (NPs).
However, in their study, Negahdary et al. [38] observed a decrease
in GPX, SOD and CAT activities and an increase in MDA levels in the
group treated with magnesium oxide (MgO) NPs. It is hypothesised
that the principal factor contributing to this discrepancy in SOD and
CAT levels is associated with the variation in the dose, size, load or
structure of the administered nanoparticle, in addition to the time
interval between administration and analysis. It can be postulated
that these toxic particles initially stimulate the activity of natural
antioxidant enzymes, resulting in an increase in their concentration
after 24 h. However, in prolonged applications and analyses, these
particles deplete the antioxidant reserves, leading to a decline in
enzyme activity [37]. Since nanoparticles have harmful effects on
the body by generating ROS, this study showed that silymarin can
use its antioxidant property to reduce free radicals generated by
MgO NPs, as previously observed in our study [38].
In the group pre–treated with MitoQ, SOD and CAT levels increased,
MitoQ prevented lipid peroxidation caused by oxidative damage in
mitochondria and reduced MDA levels to control levels. It has been
reported that MitoQ exerts its antioxidant effect by increasing the
activation of the transcription factor Nrf2. Nrf2 upregulates the
expression of antioxidant enzymes, including SOD and CAT [39].
Studies investigating the effect of mitoquinone on oxidative damage–
induced SOD and CAT levels showed that MitoQ treatment signicantly
increased SOD and CAT mRNA levels in rats that had undergone
traumatic brain injury [40]. In another study, mitoquinone treatment
normalised impaired SOD and CAT expression in Sprague–Dawley rats
in a common bile duct ligation (CBDL)–induced cirrhosis model [41]. It
is thought that the antioxidants silymarin and mitoquinone, which we
used, lowered the MDA level to a greater extent while increasing the
levels of SOD and CAT, and in this way provided cellular protection.
CONCLUSION
The results of this study show that CdTe QDs accumulate in liver
tissue and adversely affect it, so pretreatment with silymarin and
mitoquinone reduces tissue oxidative damage, especially regulating
the inammatory response. In view of the fact that both antioxidants
have effects via different pathways, it is normal that they do not have
the same effect on the parameters. However, there was no apparent
difference between the two of them in terms of their usefulness.
Different results in CdTe QDs toxicity studies may be dependent on
various parameters such as animal species, age, sex, physical and
chemical properties of the QDs used, and the route and dose of QD
administration. However, in vivo studies of QDs toxicity, which are
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11 of 12
scarce in the literature, are very valuable, and in particular studies with
antioxidants will shed light on both the toxicity and the mechanism
of action of antioxidants in future.
ACKNOWLEDGMENTS
The authors would like to thank SÜDAM experimental animals unit
for their contribution to his study.
Financial support
This article was supported by Selcuk University Faculty Member
Training Programme (ÖYP) Coordinatorship with the project
number 2018–ÖYP–012.
Conict of Interest Statement
The authors declare no conict of interest.
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