SiSiO 2quantum dots cause cytotoxicity in lung cells through redox [618700]

Si/SiO 2quantum dots cause cytotoxicity in lung cells through redox
homeostasis imbalance
Miruna S. Stana,1, Indira Memeta,1, Cornelia Simab, Traian Popescuc, Valentin S. Teodorescuc,
Anca Hermeneand,e, Anca Dinischiotua,⇑
aDepartment of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, 050095 Bucharest, Ro mania
bNational Institute for Laser, Plasma and Radiation Physics, 409 Atomistilor, 077125 Bucharest-Magurele, Romania
cNational Institute of Materials Physics, 105 bis Atomistilor, 077125 Bucharest-Magurele, Romania
dDepartment of Histology, Faculty of Medicine, Pharmacy and Dentistry, Vasile Goldis Western University of Arad, 86 Rebreanu, 310414 Arad, Romania
eInstitute of Life Sciences, Vasile Goldis Western University of Arad, 86 Rebreanu, 310414 Arad, Romania
article info
Article history:
Received 20 February 2014
Received in revised form 5 June 2014
Accepted 19 June 2014Available online 30 June 2014
Keywords:Quantum dotsOxidative stressReduced glutathioneProtein oxidationS-glutathionylationabstract
Si/SiO 2quantum dots (QDs) are novel particles with unique physicochemical properties that promote
them as potential candidat: [anonimizat], oxidative stress appears to be the main factor involved in the cytotoxicity
of these nanoparticles. In this study, we show for the first time the influence of Si/SiO
2QDs on cellular
redox homeostasis and glutathione distribution in human lung fibroblasts. The nanoparticles morphol-ogy, composition and structure have been investigated using high resolution transmission electron
microscopy (HRTEM), selected area electron diffraction (SAED), energy-dispersive X-ray spectroscopy
(EDX) and X-ray diffraction (XRD) analysis. MRC-5 cells (human lung fibroblasts) were incubated withvarious concentrations of Si/SiO
2QDs ranging between 25 and 200 lg/mL for up to 72 h. The results of
the MTT and sulforhodamine B assays showed that exposure to QDs led to a time-dependent decrease
in cell viability and biomass. The increase in reactive oxygen species (ROS) and malondialdehyde(MDA) levels together with the lower glutathione content suggested that the cellular redox homeostasiswas altered. Regarding GSH distribution, the first two days of treatment resulted in a localization of GSH
mainly in the cytoplasm, while at longer incubation time the nuclear/cytoplasmic ratio indicated a
nuclear localization. These modifications of cell redox state also affected the redox status of proteins,which was demonstrated by the accumulation of oxidized proteins and actin S-glutathionylation. In addi-
tion, the externalization of phosphatidylserine provided evidence that apoptosis might be responsible for
cell death, but necrosis was also revealed. Our results suggest that Si/SiO
2quantum dots exerted cytotox-
icity on MRC-5 cells by disturbing cellular homeostasis which had an effect upon protein redox status.
/C2112014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Quantum dots (QDs) are crystalline semiconductor nanoparti-
cles with extremely small sizes (typically between 2 and 10 nm).
This peculiarity confers QDs specific electronic properties, leading
to size-dependent optical and spectroscopic characteristics such
as including broad absorption spectrum, narrow and tunable emis-
sion, high photostability and long luminescence lifetime [1]. These
remarkable characteristics have led to the use of QDs in many bio-
logical applications including cancer therapy, cellular imaging and
delivery of various molecules (drugs, peptides, nucleic acids) into
cells [2].
A major concern about using QDs in vivo is their potential
toxicity. In many cases, QDs consist of a core-shell structure, the
core typically involving toxic heavy metals such as Cd or Pb [3].
An increased biocompatibility of such QDs can be achieved by
http://dx.doi.org/10.1016/j.cbi.2014.06.020
0009-2797/ /C2112014 Elsevier Ireland Ltd. All rights reserved.Abbreviations: QDs, quantum dots; GSH, reduced glutathione; GSSG, glutathione
disulfide; ROS, reactive oxygen species; XRD, X-ray diffraction; TEM, transmissionelectron microscopy; HRTEM, high resolution transmission electron microscopy;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TCA, trichlo-roacetic acid; DCFDA, 2
0,70-dichlorofluorescein diacetate; DCF, 20,70-dichlorofluo-
rescein; PBS, phosphate-buffered saline; CMFDA, 5-chloromethylfluoresceindiacetate; FBS, fetal bovine serum; EDTA, ethylene diamine tetraacetic acid; DTNB,5,5
0-dithiobis-2-nitrobenzoic acid; TNB, 5-thio-2-nitrobenzoic acid; MDA, malondi-
aldehyde; EGTA, ethylene glycol tetraacetic acid; SDS, sodium dodecyl sulfate;
PMSF, phenylmethanesulfonyl fluoride; DNPH, 2,4-dinitrophenylhydrazine; PVDF,polyvinylidene difluoride; HRP, horseradish peroxidase; DAB, 3,3
0-diaminobenzi-
dine tetrahydrochloride; FITC, fluorescein isothiocyanate; PI, propidium iodide;SAED, selected area electron diffraction; EDX, energy-dispersive X-rayspectroscopy.
⇑Corresponding author. Tel./fax: +40 21 318 1575.
E-mail addresses: ancadinischiotu@yahoo.com , adin@bio.unibuc.ro
(A. Dinischiotu).
1These authors contributed equally to this work.Chemico-Biological Interactions 220 (2014) 102–115
Contents lists available at ScienceDirect
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substituting the toxic core-forming metals with biocompatible
substances such as Si or encapsulating the nanoparticles in a lipid,
PEG or SiO 2coating [4–8] . Silicon quantum dots were found to
have a lower toxicity compared to those that contain heavy metals
[9]. However, it has been proposed that high concentrations of Si
QDs may generate oxygen radicals that could affect the structural
integrity of membranes, inducing cytotoxicity [10].
Furthermore, several in vitro studies reported that various types
of nanoparticles could alter glutathione level, significantly dimin-
ishing cellular GSH content [11–13] . A connection between glutathi-
one depletion and an increased level of reactive oxygen species
(ROS) was observed, which led to the idea that the lack of GSH
may compromise the cellular antioxidant defense system and an
accumulation of ROS can occur [14]. Nevertheless, an important
attribute of glutathione redox homeostasis is the rapid response tooxidative stress by increasing the intracellular content of GSH [15].
In addition to its key role in maintaining an optimal cell redox
state, glutathione regulates the oxidation state of protein thiols
and protects cysteine residues from irreversible oxidation during
oxidative stress [16–18] . The most common marker of protein oxi-
dation is the formation of carbonyl groups, whose accumulation
induces cellular dysfunction and toxicity [19]. A different type of
protein redox modification is S-glutathionylation, which is
involved in redox signaling, enabling cells to respond to the
changes in cell redox status and serving as a protective mechanism
against oxidative stress [20]. Recent evidence also suggests a role
for GSH and S-glutathionylation in the modulation of apoptosis
[21,22] . The important influence of glutathione on cellular prolifer-
ation is supported by the findings that cellular redox state and
nuclear glutathione play essential roles in cell cycle progression
[23,24] .
As far as we know, our study is the first to analyze the impact of
exposure to Si/SiO 2QDs on the cell redox status and intracellular
distribution of glutathione. At present, there is no information
regarding the modifications in GSH compartmentalization caused
by QDs and the correlation between this outcome and other types
of cellular damage. Thus, the purpose of this study is to investigate
the effects of Si/SiO 2QDs synthesized by pulsed laser ablation on
cell redox homeostasis and their mechanisms of toxicity, in terms
of structural and functional damage to human lung cells.
2. Materials and methods
2.1. Synthesis of quantum dots
The Si/SiO 2QDs used in this study were produced by pulsed
laser ablation at the Laser Department from the National Institute
of Lasers, Plasma and Radiation Physics, Bucharest-Magurele.
Detailed descriptions of the used synthesis method were previ-
ously published by Grigoriu et al. [25,26] and Petrache et al. [14].
2.2. Structural, morphological and compositional characterization
X-ray diffraction measurements (XRD) have been performed
using Cu jaradiation (CuK a1 1.540598 Å and CuK a2 1.544426 Å)
at room temperature on a D8 Advance Powder X-ray Diffractome-
ter from Bruker AXS. Due to the small amount of material available
for the XRD measurements, the nanoparticles were placed on an
amorphous glass substrate (instead of the standard sample holders
from Brucker). The sample prepared as described above was
scanned with an angular step of 0.02 degrees and a scan speed of
0.4 deg /C1min/C01. To account for the glass contribution, the substrate
was separately scanned under identical conditions.
The transmission electron microscopy (TEM) and high resolu-
tion TEM (HRTEM) studies were performed using a JEOL ARM200 F electron microscope. The TEM specimens were prepared by
dispersions in ethyl alcohol and deposited on carbon holey copper
grids. The solvent was afterwards evaporated under ambient
conditions.
2.3. Cell culture
The human fetal lung fibroblast cell line (MRC-5) was selected
in the present study as an in vitro model to assess Si/SiO 2QDs cyto-
toxicity, providing an attractive alternative to animals and also,
rapid and effective information on how nanoparticles interact with
healthy lung cells. This cell line has been shown to be a relevant
cell culture model for nanotoxicological studies to humans [27],
being previously used in order to evaluate the nanoparticles toxic-
ity[28–30] . MRC-5 cells (ATCC CCL-171) were cultured in Mini-
mum Essential Medium (Gibco, USA) supplemented with 10%
fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin and
100lg/mL streptomycin, in a humidified atmosphere containing
5% CO 2at 37 /C176C. Culture medium was changed every 2 days until
cells reached confluence. The cells were afterwards detached with
0.25% trypsin – 0.03% ethylene diamine tetraacetic acid (EDTA)
solution (Sigma–Aldrich, USA).
2.4. Culture treatment protocol
A Si/SiO 2QDs stock suspension prepared in 0.9% NaCl was ster-
ilized by autoclaving at 120 /C176C for 20 min. After that, the
suspension was sonicated using an ultrasonic processor Hielscher
UP50H (Hielscher Ultrasonics GmbH, Germany) for 3 intervals of
5 min each before adding the suspension to the medium in
order to achieve a homogenous suspension and to avoid the parti-
cles’ tendency to settle. The cells were seeded at a density of
2/C2104cells/cm2into 96-well plates (for 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT), sulforhodamine B,
intracellular ROS and annexin V assays), on coverslips placed in
8.8 cm2petri dishes (for GSH fluorescence distribution) or in
75 cm2flasks (for glutathione level, lipid peroxidation, protein oxi-
dation and S-glutathionylation analysis), and were exposed to 25,
50, 100, 150 or 200 lg/mL Si/SiO 2QDs for 24, 48 or 72 h. Controls
were performed for each analysis.
2.5. MTT assay
Cell viability was measured by the MTT assay [31]. Following
each treatment interval, the cells were observed by phase contrast
microscopy on an inverted Olympus IX71 microscope to establish
the morphological changes and were subsequently incubated with
1 mg/mL MTT (Sigma–Aldrich, USA) for 3 h at 37 /C176C. The medium
was then removed and 100 lL of isopropyl alcohol were added to
each well to dissolve the produced formazan crystals. After thor-
oughly mixing, the optical density, which is directly correlated
with the number of viable cells, was read at 595 nm (TECAN GEN-
ios plate reader). Survival rate was expressed as a percentage of
control. In parallel, non-cellular experiments with the same QDs
concentration were performed in order to establish if a MTT-QDs
reaction occurs. The unchanged absorbance at 595 nm revealed
no interaction. The obtained cell viability data were plotted against
incubation time and the LC50 values were determined by
non-linear regression, using a first order exponential decay
function generated with Microsoft Excel 2010.
2.6. Sulforhodamine B assay
The cytotoxic potential of QDs was assessed using the In Vitro
Toxicology Assay Kit Sulforhodamine B based (Sigma–Aldrich,
USA) and following the manufacturer’s protocol. After each treat-M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115 103

ment interval, the cells were fixed by adding cold 50% trichloroace-
tic acid (TCA) on top of growth media in a 1/4 ratio (v/v), incubated
for 1 h at 4 /C176C and stained for 20 min with 0.4% sulforhodamine B.
Next, the cells were rinsed quickly with 1% acetic acid and the
incorporated dye was solubilized with 10 mM Tris solution. The
absorbance at 550 nm was measured using the Tecan GENios
microplate reader and the results were expressed relative to
control. In parallel, non-cellular experiments with the same QDs
concentration were performed in order to establish if a sulforhoda-
mine B-QDs reaction occurs. The unchanged absorbance at 550 nm
revealed no interaction.
2.7. Intracellular ROS level
The level of ROS was measured by labeling the cells with 10 lM
20,70-dichlorofluorescein diacetate (DCFH-DA, Sigma–Aldrich, USA).
The cells were incubated with the dye added to the culture media
for 30 min at 37 /C176C. The excess dye was afterwards removed, and
the cells were collected and resuspended in PBS before measuring
their fluorescence at JASCO FP-6300 spectrofluorometer
kex/em = 488/515 nm). The results were expressed as fold increase
of control after dividing the fluorescence intensity to the number of
viable cells.
2.8. Preparation of cell lysates
MRC-5 cells were harvested from culture flasks, washed with
PBS and lysed by sonication (3 /C2for 30 s) on ice with an ultrasonic
processor (Hielscher UP50H, Germany). The cell lysates were cen-
trifuged at 3,000 gfor 10 min at 4 /C176C and the supernatants were col-
lected for glutathione and lipid peroxidation assays. The protein
concentration was determined using the Bradford Reagent
(Sigma–Aldrich, USA) and bovine serum albumin as the standard
protein.
2.9. Cellular glutathione content
The cell lysates were deproteinized with 5% sulfosalicylic acid
(Sigma–Aldrich, USA) and used to measure the total glutathione
content (reduced glutathione and glutathione disulfide) according
to the manufacturer’s instructions of the commercial Glutathione
Assay kit (Sigma–Aldrich, USA). In addition, the reduced glutathi-
one (GSH) level was established spectrophotometrically based on
the reduction of 5,50-dithiobis-2-nitrobenzoic acid (DTNB) from
the above mentioned kit into 5-thio-2-nitrobenzoic acid (TNB)
without adding glutathione reductase and NADPH, preventing
the reduction of glutathione disulfide (GSSG). The absorbance
was recorded at 405 nm using a microtiter plate reader (GENios
Tecan) and GSSG levels were calculated as the difference between
total glutathione and GSH. The concentration calculated in nmoles/
mg protein was presented as a percentage of control.
2.10. Lipid peroxidation
Malondialdehyde level was assessed as a marker of lipid perox-
idation according to the fluorimetric method described by Dinisch-
iotu et al. [32]. Briefly, 200 lL of cell lysate was incubated with
0.1 N HCl (700 lL) for 20 min at room temperature. After that,
900lL of 0.025 M thiobarbituric acid was added, and the mixture
was incubated for 65 min at 37 /C176C. Relative fluorescence units
(RFU) recorded at Spectrofluorometer FP-6300 JASCO ( kex= 520
nm; kem= 549 nm) were converted to nmoles malondialdehyde
(MDA) using 1,1,3,3-tetramethoxypropane as standard, calculated
in nmoles/mg protein and presented as fold increase of control.2.11. GSH fluorescent detection and quantification
After 24, 48 and 72 h of treatment, the medium was removed,
and the cells were first stained to detect GSH with 5 lM CellTrac-
ker Green 5-chloromethylfluorescein diacetate (CMFDA; Molecular
Probes from Invitrogen, USA) in culture medium without FBS for
30 min at 37 /C176C and 5% CO 2. Subsequently, the cells were incubated
for 30 min at 37 /C176C and 5% CO 2in fresh media to allow the hydro-
lysis of CMFDA to the fluorescent 5-chloromethylfluorescein (CMF)
by intracellular esterases and the conjugation with GSH or the dif-
fusion of the unconjugated dye. The nuclei were counterstained
with 1 lg/mL Hoechst 33342 (Invitrogen, USA). The labeled cells
were observed using an Olympus IX71 fluorescence microscope
equipped with proper filter cubes for CMF and Hoechst signal
detection.
The area around the nucleus and the entire cellular area were
drawn based on the Hoechst staining of the nucleus and the phase
contrast image. The GSH-CMF complex’s fluorescence intensity in
the marked areas was quantified and the results were expressed
as nuclear and cytoplasmic GSH variation in treated cells relative
to control. In addition, the nuclear/cytoplasmic ratio for GSH was
presented as fold of control. The area drawing and the fluorescence
intensity quantification were performed for 200 cells in three inde-
pendent experiments using Image J 1.48.
2.12. Subcellular fractionation
For the cellular fractionation protocol, MRC-5 cells were
detached with 0.25% trypsin – 0.03% EDTA, washed with PBS,
resuspended in hypotonic buffer [20 mM Tris–HCl pH 7.4; 10 mM
NaCl; 3 mM MgCl 2] and incubated for 15 min on ice. Afterwards,
Tergitol type NP-40 (Sigma–Aldrich, USA) was added to a final con-centration of 0.5% and the samples were vortexed at maximum
speed for 10 s. After centrifugation at 700 gand 4 /C176C for 10 min,
the supernatant representing the cytoplasmic fraction was col-
lected. The nuclear pellet was resuspended in cell extraction buffer
[100 mM Tris–HCl pH 7.4; 2 mM Na
3VO4; 100 mM NaCl; 1% Triton
X-100; 1 mM EDTA; 10% glycerol; 1 mM ethylene glycol tetraacetic
acid (EGTA); 0.1% sodium dodecyl sulfate (SDS); 0.5% sodium
deoxycholate; 1 mM phenylmethanesulfonyl fluoride (PMSF) and
protease inhibitor cocktail P8340 (1:2000, Sigma–Aldrich, USA)]
and incubated on ice for 30 min with vortexing every 10 min.
The samples were centrifuged at 14,000 gand 4 /C176C for 30 min and
the supernatant that represented the nuclear fraction was retained.
To validate the nuclear and cytoplasmic extraction, the immunode-
tection of DNA topoisomerase I (Topo I), localized exclusively in
the nucleus, and of heat shock protein 60 (Hsp60), found only in
the mitochondria and cytoplasm, was performed using mouse
monoclonal antibodies anti-Topo I and anti-Hsp60 (dilution
1:200; Santa Cruz Biotechnology, USA) (data not shown).
2.13. Protein oxidation
The detection of carbonyl groups introduced into proteins by
oxidative reactions was accomplished with the OxyBlot Protein
Oxidation Detection Kit (Millipore, USA). Equal amounts (10 lg)
of protein from cytoplasmic and nuclear fractions obtained as
described above were derivatized with 2,4-dinitrophenylhydrazine
(DNPH) following manufacturer’s instructions and separated on a
10% SDS-polyacrylamide gel. The samples were transferred on a
0.4lm polyvinylidene difluoride (PVDF) membrane and the oxi-
dized proteins were identified using a rabbit anti-DNP primary
antibody diluted 1:150. The bands that resulted after chromogenic
detection (using WesternBreeze Chromogenic Kit Anti-Rabbit,
Invitrogen, USA) were imaged at a G:Box Chemi XR5 system104 M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115

(Syngene) using the GeneSys software and quantified with
GelQuant.NET software.
2.14. Protein S-glutathionylation
At each treatment interval, the culture medium was removed.
The cells were fixed with 4% paraformaldehyde in PBS for 10 min
at room temperature and processed as described in the S-glutath-
ionylated Protein Detection Kit (Cayman Chemical, USA). Briefly,
the permeabilized whole cells were incubated with 100 lL PSSG
Blocking Reagent for 30 min to block protein free thiol (P-SH)
groups, followed by the enzymatic cleavage of protein-S-glutathi-
one (PSSG) adducts with 100 lL PSSG Reduction Reagent for
15 min at 37 /C176C. The newly-formed protein free thiols were labeled
with a thiol-specific biotinimide (100 lL PSSG Labeling Reagent for
1 h). The lysis of cell samples was performed by incubating for
30 min on ice with 50 lL PSSG Lysis Reagent. Equal amounts
(5lg) of protein from each resulting sample were separated on a
10% SDS-polyacrylamide gel in non-reducing conditions and blot-
ted on a 0.4 lm PVDF membrane. The free sites on the membrane
were blocked with 0.1% bovine serum albumin in PBS for 1 h. The
detection of S-glutathionylated proteins was achieved using avidin
coupled to horseradish peroxidase (HRP) (dil. 1:150). The bands
were revealed with a 3,30-diaminobenzidine based peroxidase sub-
strate (0.05% DAB and 0.01% H 2O2) and quantified with Gel-
Quant.NET based on the image captured with the GeneSyssoftware at G:Box Chemi XR5 system (Syngene). Actin identifica-
tion was done based on the fact that it is the most S-glutathiony-
lated protein in human cells [33] and on its molecular weight
(using a SeeBlue Plus2 Pre-Stained Standard from Invitrogen, USA).
2.15. Annexin V/propidium iodide (PI) double staining assay
The cells harvested after QDs’ exposure were subjected to apop-
tosis analysis using the FITC Annexin V/Dead Cell Apoptosis Kit
(Invitrogen, USA). The fibroblasts were washed twice with cold
PBS and resuspended in 1X annexin-binding buffer at a final con-
centration of 1 /C2106cells/mL. The staining with fluorescein isothi-
ocyanate (FITC)-labeled Annexin V and PI was done according tomanufacturer’s protocol and was observed using an Olympus
IX71 microscope. Annexin V
+/PI/C0staining was defined as early
apoptosis, Annexin V+/PI+staining was defined as late apoptosis
and Annexin V/C0/PI+was defined as necrosis. The apoptosis and
necrosis rate among the total cell population was calculated based
on the fluorescence images, counting 104cells for each concentra-
tion and time point with Cell Counter plugin from ImageJ 1.48 soft-
ware. Despite the fact that the flow cytometry method is
recommended, a manual counting was performed due to theself-fluorescence of the QDs which interfered with the measure-
ments run on a cytometer.
2.16. Statistical evaluation
All results were calculated as mean values ± standard deviation
(SD) of at least three independent experiments and expressed rel-
ative to control. The data were analyzed for statistical difference by
Student’s t-test. A value p<0.05 was considered significant.
3. Results
3.1. QDs characteristics
The XRD pattern of the tested sample is illustrated in Fig. 1 A.
The diffraction peaks show the presence of crystalline silicon.
The amorphous contribution which is more prominent at small
angles belongs to the glass substrate ( Fig. 1 B). No other phases
were detected based on the XRD analysis.
TEM investigations show that most of the studied material con-
sists of irregularly shaped nanoparticle aggregates made of parti-
cles with spherical morphology and diameters between 6 and
8n m( Fig. 2 a). The observed particles are closely packed. The SAED
image of such an aggregate shows the characteristic reflections of
cubic silicon and an amorphous component ( Fig. 2 b).
The amorphous substance forms some of the particles in the
aggregate or forms shells around crystalline Si cores as observed
by HRTEM at the edge of the aggregate ( Fig. 3 ). The thickness of
the observed amorphous shells lies between 1 and 2 nm.
These nanometric aggregates form larger size porous agglomer-
ates on the TEM grids ( Fig. 2 a and Fig. 6 a). These agglomerates are
easily disrupted and dispersed by sonication. The effect of low
power sonication (ultrasonic bath) on the observed agglomerates
is shown in Fig. 4 .
For the preparation of the biological samples, the nanoparticle
suspensions were carefully sonicated and dispersed using an ultra-
sonic processor Hielscher UP50H (Hielscher Ultrasonics GmbH,
Germany), which ensures a much better dispersion than the ultra-
sonic bath.
The energy-dispersive X-ray spectroscopy (EDX) analysis
showed the presence of silicon, oxygen, copper and carbon in the
tested samples ( Fig. 4 ). The determined copper and carbon con-
tents arise from the carbon holey copper grids for TEM used as sub-
strates for the studied powders. Silicon and oxygen were found in a
proportion of Si:O = 1:2, corresponding to a pure SiO 2composition
(Fig. 5 ).
Besides QDs aggregates, TEM determinations also reveal the
presence of larger silicon monocrystals with sizes of the order of
1 micron ( Fig. 6 ). These silicon drops give the main contribution
Fig. 1. XRD pattern of the studied nanomaterial: (A) indexed crystalline silicon lines; (B) amorphous contribution of the glass substrate.M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115 105

in the XRD signal, leading to the narrow silicon peaks shown in
Fig. 1 .
3.2. Si/SiO 2QDs affect cell viability and morphology
The morphological changes of MRC-5 cells resulted after expo-
sure to Si/SiO 2QDs were analyzed by phase contrast microscopy(Fig. 7 A). In comparison to control, a progressive decrease in the
number of cells was observed, as well as an accumulation of
vesicles in the cytoplasm around the nuclei of treated cells
(Fig. 7 A – long arrows). The images revealed an increase in the
number and size of these structures during the incubation. After
longer incubation periods, the QDs that were not taken up by cells
formed agglomerates that interacted with the cellular membranes
of fibroblasts ( Fig. 7 A – short arrows). Therefore, cells did not inter-
nalize all nanoparticles from the culture medium, some of these
associating with cell membranes as it was previously observed
by Hussain et al. [34]. Another structural modification induced
by QDs was the decrease in cell volume. The fibroblasts became
lengthier and thinner during the incubation with nanoparticles
compared to control. They displayed very long cytoplasmic exten-
sions after 72 h of treatment.
The toxic effects of QDs exposure were first studied by the MTT
assay ( Fig. 7 B). A time- and dose-dependent decrease of cell viabil-
ity was observed, the cytotoxicity of QDs being prominent for expo-
sure times of more than 24 h in particular. In addition, low levels of
viability were registered for concentrations over 100 lg/mL QDs
after 48 and 72 h. To better describe the cytotoxicity of QDs, the
LC50 values representing the concentration of nanoparticles which
have been lethal to 50% of cells, were calculated based on the results
obtained after the MTT assay. The LC50 values determined for 24,
48 and 72 h are shown in Fig. 7 C. It must be noticed that for the
shortest exposure interval the LD50 value was not in the range of
concentrations tested, being over 200 lg/mL QDs. Furthermore,
the sulforhodamine B assay was used in order to evaluate the cell
biomass modification as a direct effect of QDs toxicity ( Fig. 7 D).
The QDs exposure for 24 h resulted in a dose-dependent decrease
of cell biomass. After 48 and 72 h, the total cell mass was signifi-
cantly ( p< 0.5) diminished, but only in a dose-dependent manner,
the values for the same QDs concentration being very similar
irrespective of the treatment interval.
Fig. 2. Representative transmission electron microscopy (TEM) and selected area electron diffraction (SAED) images of the studied nanomaterial: (a) TEM i mage of a typical
QDs aggregate; (b) SAED image revealing the characteristic reflections of cubic silicon and an amorphous component.
Fig. 3. HRTEM image showing: (a) the crystalline silicon core surrounded by a thin amorphous SiO 2layer; (b) detailed view of a nanoparticle aggregate.
Fig. 4. Disruption of agglomerates into nanometric aggregates by low power
sonication.106 M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115

3.3. Si/SiO 2QDs induce oxidative stress in lung cells
In order to assess whether the toxicity of QDs is determined by
oxidative stress, the level of ROS was quantified using the fluores-
cence intensity of dichlorofluorescein (DCF). Our results show that
exposing the cells to QDs led to an increase of ROS level, their gen-
eration taking place rapidly, even in the first 24 h of the interaction
between cells and nanoparticles ( Fig. 8 A). After 24 h of incubation
with nanoparticles, the level of ROS was 2.2 and 2.6-fold higher
compared to control for 25 lg/mL and 50 lg/mL treated group,
respectively, and 3.4-fold higher for the 200 lg/mL dose
(p< 0.01). The fluorescence intensity of DCF continued to elevate
during the QDs incubation in a dose- and time-dependent manner.
At longer exposure intervals, the increase in ROS generation was
more pronounced, 5.5 times (200 lg/mL, 48 h) and 7.0 times
(200lg/mL, 72 h) higher in comparison to control cells.
To evaluate the antioxidant defense system involved in the cel-
lular protection from oxidative damage, the total glutathione, GSH
and GSGG levels were measured. Fig. 8 B shows a slight increase of
total glutathione content with 27.1%, 16.6% and 10.0% after 24 h in
25, 50 and 200 lg/mL treated cells, respectively, compared tocontrol. Although the total glutathione amount was near the con-
trol or decreased only with 12.7% in the cells treated up to 48 h
with 50 and 200 lg/mL QDs, after 72 h of exposure the level was
diminished by 46.7% and 55.7%, respectively, for these concentra-
tions of QDs compared to control. Further, an important difference
between GSH and GSSG levels after 24 h can be observed in Fig. 8 C
and D, respectively. While the amount of GSH decreased with
13.3%, the GSSG content raised up to 173% of control for the higher
concentration of QDs. Also, a significant loss in GSH and GSSG con-
tent of cells incubated with 50 and 200 lg/mL QDs for 72 h was
noticed. The absolute values for 100% GSH were 18.67 ± 0.70,
17.29 ± 0.12 and 14.0 ± 0.75 nmoles/mg protein after 24, 48 and
72 h, respectively, and for 100% GSSG were 3.42 ± 0.37,
3.01 ± 0.34 and 2.36 ± 0.45 nmoles/mg protein after 24, 48 and
72 h, respectively.
3.4. Intracellular GSH distribution during Si/SiO 2QDs exposure
In the view of the results obtained after ROS measurement, we
have studied whether QDs exposure might have an impact on GSH
compartmentalization due to redox homeostasis imbalance. The
Fig. 5. EDX spectrum showing a composition of 66% O and 33% Si, corresponding to a pure SiO 2composition of the investigated sample.
Fig. 6. Particle morphology. (a) Micron-sized spherical silicon monocrystals (arrowed). (b) A larger (600 nm) and a smaller (60 nm) silicon spheres surroun ded by aggregates
of Si/SiO 2nanoparticles.M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115 107

analysis of intracellular GSH distribution as a result of the observa-
tion of the complex GSH-CMF fluorescence intensity revealed that
QDs treatment up to 72 h produced a decrease in the level of both
nuclear and cytoplasmic GSH compared to control ( Fig. 9 ). In addi-
tion, a significant decrease in cell volume could be noticed after
72 h of QDs exposure. As expected, the nuclear green fluorescence
reached the maximum intensity after 24 of culture in the control
cells and clearly diminished after 72 h due to the fact that cell cul-
ture reached confluence and significant differences between
nucleus and cytoplasm could not be seen anymore. This aspect
was in accordance with previous findings regarding GSH distribu-
tion in fibroblasts [35].
Quantification of GSH-CMF fluorescence intensity showed a
progressive decrease in nuclear GSH during the 72 h of cells treat-
ment with 50 and 200 lg/mL QDs (with almost 0.4-fold lower for
both concentrations compared to control after 72 h) ( Fig. 10 A).
Regarding the cytoplasmic compartment, values up to 1.3-fold
after 24 h and close to control were observed after 24 and 48 h,
respectively, followed by a reduction to half of GSH content after
72 h ( Fig. 10 B). To better highlight the variation in GSH distribution
during treatment with QDs, the ratio between GSH content from
nucleus and cytoplasm was calculated and plotted as a time-course
variation in Fig. 9 C. This analysis revealed that the ratio was less
than control during the first 48 h, and became higher after 72 h
of QDs exposure.
3.5. Effect of Si/SiO 2QDs on the protein redox state and lipid
peroxidation
We studied the level of protein oxidation and S-glutathionyla-
tion in order to reveal the possible consequences of the increased
ROS generation after QDs exposure on the redox status of proteins.
The protein oxidation revealed by carbonyl groups introduced intothe side chains of nuclear and cytoplasmic proteins is shown inFig. 11 A and B, respectively. The protein profiles obtained suggest
an increase of protein oxidation that is manifested at both Si/SiO
2
QDs concentrations and all treatment intervals in comparison withcontrol ( Fig. 11 C and D). A time-dependent increase of protein oxi-
dation in the cytoplasm can be observed. Also, in this cellular com-
partment, the 25
lg/mL QDs concentration produced a higher level
of oxidized proteins compared to the 50 lg/ml dosage. This was in
contrast to the nucleus, where the content of protein carbonyl
groups was about the same for both QDs concentrations.
Fig. 11 E shows the glutathionylated actin which is the most eas-
ily detected and S-glutathionylated protein (at Cys374)[36,37] .
Exposure to QDs induced an increase in actin S-glutathionylation
in MRC-5 cells for both concentrations tested ( Fig. 11 F). The level
of this protein modification increased in a dose-dependent man-
ner, and not progressively during the treatment. Therefore, the
peak of glutathionylation was reached after 48 h (3.5 and 9 times
higher for 25 and 50 lg/mL QDs compared to control), but the
increase of S-glutathionylation at 72 h was less pronounced than
in the preceding interval.
To further examine the level of QDs-induced oxidative damage
to lipids, the MDA content was measured ( Fig. 12 ). The data
showed a time-dependent increase in lipid peroxidation afterQDs exposure proving a strong positive correlation with the pro-
tein oxidation levels.
3.6. Evaluation of Si/SiO 2QDs-induced apoptosis
In order to assess the cell death type induced by Si/SiO 2QDs,
and its extent, Annexin V/PI double-staining assay was performed.
Fig. 13 A shows cells labeled with both annexin V and PI during the
72 h of exposure indicating that these QDs caused both apoptosis
and necrosis. The apoptotic and necrosis rate increased in a
Fig. 7. Si/SiO 2QDs reduce the viability of human lung cells. (A) Representative phase contrast images showing cellular morphology of MRC-5 cells untreated or expos ed to 50
and 200 lg/mL QDs for 24, 48 and 72 h. Scale bar = 50 lm. Note accumulation of vesicles in cytoplasm around the nuclei of treated cells ( long arrows ). Note QDs agglomerates
that interacted with the cellular membranes of fibroblasts ( short arrows ). (B) Cell viability was detected by MTT assay and (C) cellular biomass was measured by absorbance of
protein-bound sulforhodamine B at 550 nm for cells treated with 0-200 lg/mL QDs for three time periods (24, 48 and 72 h). The results were normalized to control cells and
the data shown as mean ± SD ( n= 3) were analyzed by Student’s t-test.⁄p< 0.05,⁄⁄p< 0.01 and⁄⁄⁄p< 0.001 compared to control. (D) Calculated LC50 values of nanoparticles
based on MTT results. LC50 values represent the concentration of QDs that decreased the cell viability by 50%. Values were determined by fitting a curve using non-linear
regression.108 M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115

Fig. 8. Time-dependent biochemical analysis of Si/SiO 2QDs-induced oxidative stress in lung fibroblasts. MRC-5 cells were exposed to 25, 50 and 200 lg/mL QDs for 24, 48
and 72 h. (A) DCF fluorescence intensity was measured in order to reflect ROS levels. Total glutathione content (B), GSH (C) and GSSG (D) levels were estab lished as described
in ‘‘Materials and methods’’ section. The results were normalized to control cells (the absolute values for 100% GSH were 18.67 ± 0.70, 17.29 ± 0.12 and 14.0 ± 0.75 nmoles/mg
protein after 24, 48 and 72 h, respectively, and for 100% GSSG were 3.42 ± 0.37, 3.01 ± 0.34 and 2.36 ± 0.45 nmoles/mg protein after 24, 48 and 72 h, respecti vely) and the data
shown as mean ± SD ( n= 3) were analyzed by Student’s t-test.⁄p< 0.05 and⁄⁄p< 0.01 compared to control.
Fig. 9. Cellular distribution of GSH is altered by Si/SiO 2QDs exposure. Cells were incubated in the presence of 50 and 200 lg/mL QDs for 24, 48 and 72 h and were labeled
with CMFDA to mark GSH. Representative fluorescence images of GSH staining are shown. Scale bar = 50 lm.M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115 109

time-dependent manner, but there was no dependence on nano-
particles concentration ( Fig. 13 B and C). In addition, the apoptosis
rate was much higher than the necrosis rate induced by Si/SiO 2
QDs.
4. Discussion
The progress of nanotechnology has brought innovative nano-
particles as well as new biotechnological and medical applications
for them, but inevitably, has led to toxicological and environmental
concerns [38]. Presently, silicon quantum dots are a class of prom-
ising semiconductor nanocrystals in the field of biology and med-
icine, but there are very few studies to date that investigated
directly or indirectly their potential toxicity and the effects oncellular behavior. Thus, the current study provides for the first time
information regarding the changes induced by Si/SiO 2QDs on the
cellular distribution of the antioxidant GSH and protein redox sta-
tus in an in vitro model, i.e., human lung fibroblast MRC-5 cell line.
The treatment intervals chosen allowed us to establish the poten-
tial of QDs to induce acute toxicity on MRC-5 cells, and to highlight
the mechanisms involved in regulating cellular functions in
response to the physiological stress caused by both low and high
concentrations of quantum dots.
The studied aggregates consisted of two types of nanoparticles:
core-shell structures with a crystalline silicon core and an amor-
phous SiO 2shell and nanoparticles composed of amorphous SiO 2
only. The facile dispersion of the agglomerates formed by the nano-sized aggregates on the TEM grids supports the highly dispersed
character of the particle suspensions used for cell treatment. In cul-ture media, the agglomeration processes are diminished by the
presence of FBS, known to reduce agglomeration and promote
the stability of nanomaterial suspensions (including SiO
2)
[39,40] . The eventually remained nanosized agglomerates would
have high porosity (as revealed by TEM determinations) and thus,
would not largely decrease the specific surface area and reactivity
of the tested nanomaterial.
The EDX analysis indicated a pure SiO 2composition of the stud-
ied sample, in spite of the crystalline silicon content identified by
both XRD and SAED methods. The excess of oxygen resulted from
the EDX determinations revealed the overstoichiometric oxygena-
tion of the silica surface. This surface oxygen may either bind
organic molecules or participate in the formation of hydroxyl spe-
cies on the surface of the amorphous SiO 2phase. Based on the syn-
thesis method which did not involve any organic components and
the aging of the synthesized nanomaterial under ambient atmo-
sphere conditions [25,26] , the hydroxylation of the SiO 2surface
[41] is straightforwardly expected. The implications of surface
hydroxylation and degree of crystallinity upon the toxicity of
SiO 2nanoparticles have been previously addressed in a series of
studies [42]. Some of these reported that surface hydroxyl (silanol)
groups ( „Si–OH) induced membranolysis [43] and toxicity in alve-
olar cells [44], the distribution and abundance of silanols being
generally considered to play cell toxicity modulating roles [45].
Our results of phase contrast microscopy showed an altered
morphology of fibroblasts characterized by an accumulation of ves-
icles during the QDs exposure. This is probably due to endosomes
formation because endocytosis represents the major pathway for
nanoparticle uptake in cultured cells [46,47] , although secondary
pathways of internalization could also exist depending on cell type
and nanoparticles-intrinsic properties (e.g., passive diffusion or
phagocytosis). The cell size reduction noticed in our case was also
previously highlighted for other types of nanoparticles [34,48,49]
that correlated the cellular shrinkage to the induction of apoptosis.
The decrease of cell volume in the initial stages of apoptosis is
known as AVD-apoptotic volume decrease and it was shown that
it plays an active role in regulating the activity of apoptotic nucleas-
es and the activation of caspases [50]. Being a hallmark of apoptosis,
this morphological change could be an indication of the mecha-
nisms responsible for the cell death determined by QDs [51].
The morphological changes induced by QDs were reflected very
well by the dose-dependent decrease of cell viability and biomass.
The significant cytotoxicity obtained after the longest interval of
incubation (72 h) could be explained, in part, by the nanoparticles
tendency to form agglomerates which modify their physico-chem-
ical properties, precipitating on top of the cells and leading to
much more toxic effects compared to a well dispersed suspension
as it was observed for other nanoparticles [52,53] . On the other
hand, LC50 value was over 200 lg/mL at 24 h indicating that this
type of QDs did not affect the cell viability after short time
exposure, even in great concentrations.
Fig. 10. Time-course variation of nuclear (A) and cytoplasmic (B) GSH content after
50 and 200 lg/mL Si/SiO 2QDs exposure. The fluorescence images of cells stained
with CMFDA (as presented in Fig. 8 ) were analyzed as described in ‘‘Materials and
methods’’ section. The variation of the nuclear/cytoplasmic GSH ratio during
exposure is presented in panel C. The results were normalized to control cells andthe data shown as mean ± SD ( n= 3) were analyzed by Student’s t-test.
⁄p< 0.05 and
⁄⁄⁄p< 0.001 statistical significance for 50 lg/mL QDs-treated cells compared to
control.#p< 0.05,##p< 0.01 and###p< 0.001 statistical significance for 200 lg/mL
QDs-treated cells compared to control.110 M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115

Cell redox homeostasis is defined as the balance between pro-
oxidants and antioxidants, its disturbance being associated with
several pathological conditions [54]. Generally, one importantprocess involved in nanotoxicity is considered to be oxidative
stress [55]. To further analyze this aspect after exposure of sili-
con-based QDs on MRC-5 cells, ROS and cellular glutathione levelswere measured. Our data revealed a dose- and time-dependent
increase of ROS production suggesting the potential of this type
of QDs to induce the generation of ROS. This can be closely linked
to one of their physico-chemical features, i.e., the amorphous silica
coating that covers the crystalline silicon core could be an impor-
tant determinant for ROS accumulation [56].
Being an important antioxidant, GSH is a key molecule in regu-
lating redox homeostasis and its function is carried out either
directly or indirectly through GSH-dependent enzymes. The
reduced and oxidized form of glutathione act together with other
redox active compounds to maintain the cellular redox status
[57]. The fine changes in total glutathione concentration compared
to control during the first 48 h of treatment proved that Si/SiO 2
QDs were less toxic than other types of QDs previously studied[58,59] . The slight increased GSH level after exposure to 25
lg/mL QDs compared to control was probably due to up-regulated
expression of glutamate cysteine ligase as it was previously
demonstrated by McConnachie et al. [60] for amphiphilic
Fig. 11. Si/SiO 2QDs-mediated protein redox state modifications. MRC-5 cells were exposed to 25 and 50 lg/mL QDs for 24, 48 and 72 h. Oxidized proteins from nucleus (A)
and from cytoplasm (B), and actin S-glutathionylation (E) were detected by Western blot analysis. The densitometry results for the level of oxidatio n of nuclear (C) and
cytoplasmic (D) proteins, and S-glutathionylation (F) were normalized to control cells. The data are presented as mean ± SD ( n= 3) and were analyzed by Student’s t-test.
⁄p< 0.05,⁄⁄p< 0.01 and⁄⁄⁄p< 0.001 compared to control.
Fig. 12. Si/SiO 2QDs–induced lipid peroxidation. MRC-5 cells were exposed to 25
and 50 lg/mL QDs for 24, 48 and 72 h. The MDA content was assessed as a measure
of lipid peroxidation and the results were normalized to control cells. The data are
presented as mean ± SD ( n= 3) and were analyzed by Student’s t-test.⁄⁄p< 0.01 and
⁄⁄⁄p< 0.001 compared to control.M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115 111

polymer-coated CdSe/ZnS QDs. In addition, the high GSSG content
observed in the first 24 h for all concentrations accumulated in
response to oxidant insult, an elevated GSSG/GSH ratio being a bio-
chemical measure of oxidative stress. Further, the GSH level was
halved after 72 h of exposure to 50 and 200 lg/mL QDs. This could
be due to the loss in cell membrane integrity and to the fact that
glutathione can be reversibly bound to protein cysteine residues
by S-glutathionylation [18]. These findings revealed that silicon-
based QDs induced oxidative stress that was manifested by an
increased production of reactive oxygen species and a significant
decline in the glutathione levels. The ability of this type of QDs
to induce oxidative stress was also shown in carp kidney [14].
The absence of a significant change in glutathione content when
the viability decreased up to 50% after 48 h can be due to the fact
that the glutathione concentration (nmoles/mL) was normalized to
the protein concentration (mg/mL) of each sample. The progressive
decrease in total biomass of the cells exposed to QDs relative to
that of untreated cells determined a lower protein concentration
in these cells which influenced the value of glutathione content
(nmoles/mg of total protein).
Despite being synthesized in the cytosol, GSH is distributed in
different intracellular organelles where it fulfils many physiologi-
cal functions specific for each cell compartment, in relation to itsrole in the regulation of redox status [61]. To gain insights into
the silicon QDs effects on GSH distribution, CMFDA staining in lung
fibroblasts was performed. The nuclear GSH decreased during the
72 h of QDs exposure, but the cytoplasmic levels elevated after
the first 24 h followed by a significant reduction at 72 h. The profile
of cytoplasmic GSH variation correlated very well with the total
glutathione level determined by the spectrophotometric method.
Similar results regarding the lack of dose dependence in the
case of GSH depletion induced by SiO 2nanoparticles were previ-
ously reported by Berg et al. [62]. In our case, this characteristic
may arise as a consequence of the dose dependent agglomeration
of the tested nanoparticles, under the used in vitro conditions. Such
agglomeration processes reduce the specific surface areas and
reactivity of the tested nanomaterials and alter their internaliza-
tion by cells. The obtained data indicate a GSH depletion pathway
mediated by smaller (less agglomerated) particles.
The nuclear/cytoplasmic GSH ratio showed that the first two days
of treatment resulted in a localization of GSH mainly in the cyto-
plasm. This suggests a translocation of GSH from nucleus to cytosol
or a decrease of nuclear GSH correlated with de novo synthesis of
GSH in cytoplasm in order to protect from oxidative damage induced
by QDs internalized into fibroblasts cytoplasm. The nuclear GSH
localization after three days of QDs exposure indicated that nano-
Fig. 13. Induction of apoptosis by Si/SiO 2QDs. MRC-5 cells were exposed to 25 and 50 lg/mL QDs for 24, 48 and 72 h. (A) Representative fluorescence images of Annexin V/PI
double-staining assay (green: stained with annexin V for detection of exposed phosphatidylserine; red: stained with propidium iodide for detectio n of damaged cells) are
shown. (B) Apoptosis and (C) necrosis rates were assessed based on the Annexin V/PI double-staining assay. The data are presented as mean ± SD ( n= 3) and were analyzed by
Student’s t-test.⁄p< 0.05,⁄⁄p< 0.01 and⁄⁄⁄p< 0.001 compared to control. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)112 M.S. Stan et al. / Chemico-Biological Interactions 220 (2014) 102–115

particles had a certain influence on cellular redox homeostasis. This
GSH accumulation in nucleus could be required for maintaining the
redox status of thiol groups from proteins involved in DNA synthesis
and repair, functions essential for cell survival [63], and also for tel-
omerase activity [64]. Probably, the fibroblasts responded in this
manner to achieve a reduced medium that can contribute to geno-
mic DNA protection against oxidative stress generated by nanopar-
ticles and thus, to increase the cellular resistance to apoptosis. Being
involved in modulation of the redox intracellular environment, GSH
plays a key role in regulation of cellular signaling events and its
depletion can lead to significant cell/tissue damage [65] and altered
physiological condition.
The main targets of ROS include proteins, membrane lipids and
nitrogenous bases of DNA which can undergo oxidation [66].I n
this study, Si/SiO 2QDs exposure induced protein oxidation and
lipid peroxidation in MRC-5 cells probably as a consequence of oxi-
dative stress. The carbonyl groups formation in the structure of
proteins increased in both nucleus and cytoplasm of fibroblasts
treated. The high level of nuclear carbonylated proteins could be
used to predict cell death during oxidative stress [67]. The protein
oxidation could be caused directly through ROS generated in high
amount during QDs exposure or by reactions with secondary prod-
ucts formed as a result of oxidative stress. MDA, an end-product of
lipid peroxidation can bind covalently to Lys, His and Cys residues
and lead to protein carbonylation [68]. Taking into account that
lipid peroxidation pathway involves a free radical-mediated mech-
anism and MDA is a very reactive aldehyde, probably the genera-
tion of MDA-protein adducts occurred from the first 24 h of
exposure despite the fact that the loss in GSH level was noticed
only after 72 h. In addition, the increase in GSSG content during
the first 24 h for these QDs concentrations might explain the initi-
ation of oxidative damage. Altogether, these could explain why the
protein oxidation preceded the glutathione decrease. Protein car-
bonyl derivatives can further react with a-amino groups of Lys res-
idues generating intra- or inter-molecular cross-links that can lead
to the formation of high molecular weight aggregates which are
highly resistant to proteolysis and may inhibit both lysosomal
and proteasomal degradation pathways. As a result, the protein
aggregates can be potentially harmful, their accumulation altering
cellular functions and leading to necrosis and/or apoptosis [19].
Furthermore, S-glutathionylation of many proteins has been
reported during oxidative stress [69]. In our study, the elevated
level of glutathionylation in treated cells, with at least one order
of magnitude higher than that in control, highlights the oxidative
stress induction as it was previously shown by Lind et al. [70]. This
post-translational modification might be responsible for a decrease
in actin polymerization into filaments and cell adhesion, but also
for membrane ruffling [71–73] . Taken together, the accumulation
of ROS during QDs-induced oxidative stress could have an impor-
tant influence on cell structure and fate by disturbing protein func-
tion, cell signaling pathways and cytoskeleton organization.
Various studies have shown that apoptosis has been implicated
as a major mechanism of cell death induced by different types of
nanoparticles [74,75] . The progressive increase in apoptotic cells
during QDs exposure, as the Annexin/PI staining showed, corre-
lated very well with the cellular shrinkage noticed by phase con-
trast microscopy. The low level of GSH loss in the first 48 h could
be responsible for the moderate rate of apoptosis. Based on the ele-
vated ROS level, the QDs-induced apoptosis could be ROS-medi-
ated by oxidative stress [76]. Also, the increase in intracellular
GSSG content observed after 24 h can trigger apoptosis indepen-
dent of ROS and GSH levels, as indicated by other studies [77,78] .
Moreover, the Annexin V+/PI+and Annexin V/C0/PI+staining after
this treatment validated the induction of these two types of cell
death, late apoptosis and necrosis. This aspect could involve a
necrosis-apoptosis convergence after death has taken place [79].5. Conclusion
In conclusion, the results obtained from the present analysis of
cell redox status after Si/SiO 2QDs exposure showed that the
increased level of ROS, the decrease in GSH content and the accu-
mulation of oxidized proteins contributed to the formation of a
highly oxidative environment. Our study demonstrated that high
concentration of Si/SiO 2QDs exhibited toxic effects on lung fibro-
blasts by disturbing cellular redox homeostasis. Further studies
are now needed to clarify the molecular mechanisms involved in
QDs endocytosis together as well as the main type of cell death.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
TheTransparency document associated with this article can be
found in the online version.
Acknowledgements
M.S. Stan gratefully acknowledges the support of the European
Social Fund through the contract POSDRU/159/1.5/S/133391. T.
Popescu acknowledges support under the Sectorial Operational
Programme Human Resources Development (SOP HRD), contract
number SOP HRD/107/1.5/S/82514.
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