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Download by: [FU Berlin] Date: 21 December 2016, At: 22:24
Toxicology Mechanisms and Methods
ISSN: 1537-6516 (Print) 1537-6524 (Online) Journal homepage: http://www.tandfonline.com/loi/itxm20
The effects of endoplasmic reticulum stressinducer thapsigargin on the toxicity of ZnO or TiO2nanoparticles to human endothelial cells
Yuxiu Gu, Shanshan Cheng, Gui Chen, Yuexin Shen, Xiyue Li, Qin Jiang, JuanLi & Yi Cao
To cite this article: Yuxiu Gu, Shanshan Cheng, Gui Chen, Yuexin Shen, Xiyue Li, Qin Jiang, JuanLi & Yi Cao (2016): The effects of endoplasmic reticulum stress inducer thapsigargin on thetoxicity of ZnO or TiO2 nanoparticles to human endothelial cells, Toxicology Mechanisms andMethods, DOI: 10.1080/15376516.2016.1273429
To link to this article: http://dx.doi.org/10.1080/15376516.2016.1273429
Accepted author version posted online: 20Dec 2016.
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The effects of endoplasmic reticulum stress inducer thapsigargin on
the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells
Yuxiu Gu#, Shanshan Cheng#, Gui Chen, Yuexin Shen, Xiyue Li, Qin
Jiang, Juan Li, Yi Cao*
Key Laboratory of Environment-Friendly Chemistry and Application of Ministry of
Education, Laboratory of Biochemistry, College of Chemistry, Xiangtan University,
Xiangtan 411105, P.R. China
#: The first two authors contributed equally to this work
*: Send correspondence to Yi Cao ([email protected])
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The effects of endoplasmic reticulum stress inducer thapsigargin on
the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells
Abstract: It was recently shown that ZnO nanoparticles (NPs) could induce
endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells
(HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of
ER stress inducer thapsigargin (TG) should alter the response of HUVECs to
ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity,
oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with
or without the presence of TG. Moreover, TiO2 NPs were used to compare the
effects. Exposure to 32 μg/mL ZnO NPs (p<0.05), but not TiO2 NPs (p>0.05),
significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake
assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the
accumulation of intracellular Zn ions, and ZnSO4 induced similar cytotoxic
effects as ZnO NPs, which indicated a role of Zn ions. The release of
inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or
the adhesion of THP-1 monocytes to HUVECs was not significantly affected by
ZnO or TiO2 NP exposure (p>0.05). The presence of 250 nM TG significantly
induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p<0.01), but
did not significantly affect intracellular ROS or release of TNFα (p>0.05).
ANOVA analysis indicated no interaction between exposure to ZnO NPs and the
presence of TG on almost all the endpoints (p>0.05) except neutral red uptake
assay (p<0.01). We concluded ER stress is probably not associated with ZnO NP
exposure induced oxidative stress and inflammatory responses in HUVECs.
Keywords: ZnO nanoparticles; TiO2 nanoparticles; Human umbilical vein
endothelial cells (HUVECs); Thapsigargin (TG); Endoplasmic reticulum stress
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Introduction
Metal and metal oxide nanoparticles (NPs) are among the most produced and used NPs
in commercially available products according to a recent survey (Vance et al., 2015).
However, their potential adverse health effects are not fully understood and require
further extensive studies. In recent years, some studies showed that metal and metal
oxide NPs may impose potential cardiovascular health effects both in vivo and in vitro
(Tomaszewski et al., 2015;Moller et al., 2016;Cao et al., 2016a). But the molecular
mechanisms are not fully elucidated.
Endoplasmic reticulum (ER) is a crucial organelle involved in cell homeostasis
and survival, and perturbations in the normal function of ER will lead to a condition
termed as ER stress, which has been implicated in the development of many diseases,
including atherosclerosis (Zhou and Tabas, 2013;Ozcan and Tabas, 2012). Interestingly,
a recent study showed that ZnO NP, a popular metal oxide NP, could activate ER stress
pathway in human umbilical vein endothelial cells (HUVECs), and ER stress response
may be served as an earlier and sensitive endpoint for toxicological assessment of ZnO
NPs, as suggested by the authors (Chen et al., 2014). Given the crucial role of ER stress
in the development of atherosclerosis (Zhou and Tabas, 2013;Ozcan and Tabas, 2012),
it could be possible that ZnO NP activated ER stress response is associated with the
cardiovascular health effects of ZnO NPs. However, the association between ER stress
and ZnO NP exposure induced toxicity to endothelial cells remains unknown. To
address this issue, we investigated if the presence of ER stress inducer thapsigargin
(TG) could affect the cytotoxicity, oxidative stress and inflammatory responses induced
by ZnO NPs in this study. ZnO NPs were used because they are among one of the most
popular metal and metal oxide NPs produced and used (Vance et al., 2015). In addition,
ZnO NPs may also be used in medicine, and therefore the potential adverse effects to
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human blood vessels should be carefully assessed to ensure the safe use (Tomaszewski
et al., 2015). We also compared the different responses of HUVECs after exposure to
ZnO and TiO2 NPs, because we and others have shown that ZnO NPs could dissolute in
HUVECs and release Zn ions as the most significant factors associated with
cytotoxicity (Chen et al., 2014;Gong et al., 2016), whereas TiO2 NPs almost do not
dissolute (Kermanizadeh et al., 2013). The cytotoxicity was investigated by WST-1
(water soluble tetrazolium), neutral red uptake and direct observation under a light
microscope. Oxidative stress was estimated by the measurement of intracellular reactive
oxygen species (ROS), and inflammatory response was measured by determining the
release of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) and the adhesion of
THP-1 monocytes to HUVECs. The dissolution of NM110 inside HUVECs was
estimated by the quantification of intracellular accumulation of Zn ions using a
fluorescence probe.
Materials and methods
Cell cultures
HUVECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA)
and cultured in basal endothelial cell medium (ECM) supplemented with 5 % (v/v)
foetal bovine serum (FBS), 1 % (v/v) endothelial cell growth supplement (ECGS) and 1
% (v/v) penicillin/streptomycin solution (PS) as our previously described (Ji et al.,
2016). THP-1 monocytes (ATCC) were cultured in RPMI 1640 medium (Thermo-
Fisher, USA) supplemented with 10% FBS (Gibco, South Africa), 1% P/S solution
(Beyotime, China) and 1╳ sodium pyruvate (Thermo-Fisher, USA) in a CO2 incubator
at 37 °C. The cells were used within 3 month to keep their best characteristics.
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Particles and exposure
ZnO (Code NM110; BASF Z-Cote; uncoated, 100 nm) and TiO2 (Code NM101;
Hombikat UV100, rutile with minor anatase; 7 nm) NPs, originally received from the
European Commission Joint Research Centre Nanomaterials Repository, were kindly
provided by Prof. Peter Moller (Department of Public Health, University of
Copenhagen). NM110 and NM101 have been well characterized before by ENPRA
project, using transmission electron microscopy (TEM), X-ray diffractogram (XRD),
Brunauer Emmett Teller (BET) technology, dynamic light scattering (DLS) and
nanoparticle tracking analysis (NTA) (Danielsen et al., 2015;Kermanizadeh et al., 2013).
The main physicochemical properties of NM110 and NM101 were summarized in Table
1.
In this study, we also used a scanning electron microscopy (SEM; ZEISS
EVO18, Germany) to investigate the morphology of NM110 and NM101. The samples
of NM110 and NM101 were imaged with magnification of 100000 times and an
acceleration potential of 20 keV. The hydrodynamic size distribution and Zeta potential
were measured using 16 μg/mL NM110 or NM101 suspended in MilliQ water by
Zetasizer Nano ZS90 (Malvern, UK). Size and Zeta potential was measured for three
times, and mean±S.D. (standard deviation) was calculated. The size and Zeta potential
of NM110 and NM101 in full cell culture medium were not further measured in this
study, as the components in medium may interfere with the measurement.
To make the suspension of NM110 and NM101, 2.56 mg/mL particle in MilliQ
water containing 2 % FBS was sonicated continuously for 8 minutes with continuously
cooling on ice using an ultrasonic processor FS-250N (20 % amplitude; Shanghai
Shengxi, China) and then diluted in cell culture medium to 2 μg/mL, 4 μg/mL, 8 μ
g/mL, 16 μg/mL and 32 μg/mL. We used NPs up to 32 μg/mL because previous
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study showed that NM110 significantly induced cytotoxicity in HUVECs at
concentrations ≥ 32 μg/mL (Danielsen et al., 2015). The stock solution of 200 μM TG
(Sigma-Aldrich, USA) was prepared in DMSO and then diluted to 250 nM in full
endothelial medium for the exposure. For all the experiments, HUVECs were exposed
to NM110 or NM101 from 0 μg/mL (for control) up to 32 μg/mL, with or without the
presence of 250 nM TG.
Cytotoxicity assay
The cytotoxicity was measured by using WST-1 and neutral red uptake kits according to
manufacturer’s instruction (Beyotime, China). WST-1 reagent can indicate the
mitochondrial activity as it could be converted to a yellow formazan by mitochondria in
living cells. For the assay, 4╳104/well HUVECs were seeded in 24-well plates and
grown for 2 days before exposure. Then, the cells were exposed to various
concentrations of NM110 or NM101, with or without the presence of 250 nM TG for 24
h. After rinsed once by Hanks solution, the cells were incubated with 10 % WST-1
reagent for 2 h. The yellow product was read at 450 nm with 690 nm as reference by an
ELISA reader (Synergy HT, BioTek, USA). To indicate the possible role Zn ions in
cytotoxicity, HUVECs were also exposed to 25 to 400 μM ZnSO4 for 24 h (25 to 400
μM ZnSO4 equals to the same amount of Zn in ZnO NPs from 2 μg/mL to 32 μg/mL),
followed by WST-1 assay as indicated above.
Neutral red is a dye which could be incorporated into intact lysosomes of living
cells, therefore it could be used to indicate the integrity of lysosomes. For the assay,
HUVECs seeded in 24-well plates were incubated with various concentrations of
NM110 or NM101, with or without the presence of 250 nM TG. After 24 h exposure,
the cells were rinsed once with Hank’s solution, and then incubated with 10 % neutral
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red for 2 h. After rinsed once again, the neutral red incorporated into lysosomes was
dissolved in the lysis solution provided by the kit, and the absorbance was read at 540
nm with 690 nm as reference by an ELISA reader (Synergy HT, BioTek, USA).
We also used a light microscope (Olympus, Japan) to observe the morphology of
the cells after exposure and neutral red staining. The cells exposed to 0 μg/mL (for
control), 16 μg/mL or 32 μg/mL NM110 or NM101 with or without the presence of 250
nM TG were imaged.
ROS
The intracellular ROS was estimated by using 2’,7’-dichlorofluorescein diacetate
(DCFH-DA) as previously described (Cao et al., 2015). DCFH-DA is a general
indicator for oxidative stress as it could be oxidized by the reaction with a variety of
ROS when it is inside the cells (Cao et al., 2015). A stock solution of DCFH-DA was
made at 10 mM in methanol and stored at -20 ℃ before use. 1╳104/well HUVECs
were seeded in a black 96-well plate and grown for 2 days before exposure. After that,
the cells were incubated with various concentrations of NM110 or NM101 with or
without the presence of 250 nM TG for 3 h, rinsed once with Hanks solution, and then
incubated with 10 μM DCFH-DA diluted in 100 μL serum free medium for 30 min.
After rinsed once again, the fluorescence was read at ex 485±20 nm and em 528±20 nm
by an ELISA reader. Here the cells were exposed for only 3 h because 24 h exposure
significantly induced cytotoxicity (see results below). Previous work showed that 3 h
exposure of HUVECs to a variety of solid particles could rapidly induce ROS, which
may mediate the toxic effects of the particles (Danielsen et al., 2015;Cao et al.,
2015;Cao et al., 2014a;Cao et al., 2014b).
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Intracellular Zn ion accumulation
The accumulation of intracellular Zn ions in HUVECs after 24 h exposure to up to 16
μg/mL NM110 with or without the presence of TG was determined by using a
fluorescent probe Zinquin ethyl ester (Sigma-Aldrich, USA). Here we only exposed
HUVECs to up to 16 μg/mL NM110 because 32μg/mL NM110 significantly induced
cytotoxicity after 24 h exposure (see results below). The assay was done as our
previously described (Jiang et al., 2016). Briefly, the HUVECs on black 96-well plates
after exposure were rinsed once by using Hanks solution, and then incubated with the
probe diluted in serum free medium for 30 min. After washed again, the fluorescence
was read at ex 360±44 nm and em 460±40 nm by an ELISA reader.
ELISA
The supernatant from the neutral red uptake assay was collected and stored at -80 °C
before ELISA analysis. The concentrations of inflammatory mediators TNFα (detection
limit 7.8 pg/mL) and IL-6 (detection limit 3.9 pg/mL) were measured by ELISA kits
according to manufacturer’s instruction (Neobioscience Technology Co., Ltd., China).
The concentrations of TNFα and IL-6 in all the samples were higher than the detection
limit.
THP-1 adhesion
The adhesion of THP-1 monocytes to HUVECs was done as previously described (Cao
et al., 2016b). Briefly, 1╳104/well HUVECs on a black 96-well plate were exposed to
various concentrations of NM110 or NM101 with or without the presence of 250 nM
TG for 24 h. When the exposure was finished, THP-1 monocytes were labeled with 10
μM CellTrackerTM Green CMFDA (5-Chloromethylfluorescein Diacetate, Invitrogen,
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Carlsbad, CA) for 30 min in serum free medium. The free probe was removed by
centrifuge, and 5╳104/well labeled THP-1 cells were incubated with the exposed
HUVECs for 1 h for adhesion. Here we used a short time (1 h) for the adhesion assay
because it is expected that during 1 h incubation there was minimal proliferation of
THP-1 monocytes since the doubling time for THP-1 cells was measured to be 31 h
(Cao et al., 2014a). After that, the unbound THP-1 cells were washed away, and the
fluorescence was read at ex 485±20 nm and em 528±20 nm by an ELISA reader.
Statistics
All the data were expressed as mean±S.E. (standard error) of means of three
independent experiments carried out on at least three independent days (n=3 for each
experiment). Two-way ANOVA (concentrations of NM110 or NM101, and the
presence of TG as categorical factors) followed by Tukey HSD test using R 3.2.2; p
value <0.05 was considered to be statistically significant.
Results
Particle characterization
The size distribution and Zeta potential are shown in Figure 1, and the physicochemical
properties of NM110 and NM101 are summarized in Table 1. The size of NM101 is
relatively larger than that of NM110 (Figure 1A & Table 1), but the Zeta potential of
NM110 and NM101 is similar (Figure 1B & Table 1). The SEM morphology images of
NM110 and NM101 are shown in Figure 1C and 1D, respectively. The SEM images
indicate that NM101 contains relatively larger aggregates and/or agglomerates of
particles compared with that of NM110.
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Cytotoxicity
The cytotoxicity as assessed by WST-1 and neutral red uptake assay is shown in Figure
2. Exposure to NM110 was associated with significantly decreased mitochondrial
viability at the concentration of 32 μg/mL (p<0.01); whereas exposure to NM101 did
not significantly affect mitochondrial viability up to 32 μg/mL (p>0.05; Figure 2A). 250
nM TG alone significantly decreased mitochondrial viability of HUVECs (p<0.01), but
ANOVA analysis indicated no interaction between NP exposure and the presence of TG
on mitochondrial viability (p>0.05). Similarly, neutral red uptake assay showed that 32
μg/mL NM110 (p<0.01), but not that of NM101 (p>0.05), significantly affected the
lysosomal integrity (Figure 2B). 250 nM TG alone significantly decreased neutral red
uptake into lysosomes (p<0.01), and ANOVA analysis indicated that there was
significant interaction between NM110 and the presence of TG on neutral red uptake
(p<0.01).
The morphology of HUVECs after exposure to NM110 and NM101 with or
without the presence of 250 nM TG is shown in Figure 3. Exposure to 16 μg/mL
NM110 with or without the presence of TG did not obviously affect the morphology
and neutral red staining (the red color in Figure 3) in HUVECs. However, after
exposure to 32 μg/mL NM110 with or without the presence of TG, most of the cells
were removed, and the rest of cells showed round morphology with little to no neutral
red staining. In contrast, exposure to 16 μg/mL and 32 μg/mL NM101 with or without
the presence of TG did not obviously affect the morphology and neutral red staining of
HUVECs.
As shown in Figure 4, exposure to ZnSO4 induced similar cytotoxic effects in
HUVECs as NM110, with significant decrease in mitochondrial viability at 200 μM
(200 μM ZnSO4 equals to the same amount of Zn in 32 μg/mL NM110; p<0.01).
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Accumulation of intracellular Zn ions
There was dose-dependent increase of intracellular Zn ions after 24 h exposure to
NM110 (p<0.01; Figure 5). Without the presence of TG, 4 μg/mL (p<0.05), 8 μg/mL
(p<0.01) and 16 μg/mL (p<0.01) NM110 significantly promoted the accumulation of
intracellular Zn ions. TG alone did not significantly affect intracellular Zn ions
(p>0.05), and there was no interaction between NM110 and TG (p>0.05).
Intracellular ROS
As shown in Figure 6, the exposure to 32 μg/mL NM110 significantly induced
intracellular ROS (p<0.05), whereas various concentrations of NM101 did not
significantly affect intracellular ROS (p>0.05). The exposure to 250 nM TG alone did
not significantly affect intracellular ROS (p>0.05), and there was no interaction
between NP exposure and the presence of TG on intracellular ROS (p>0.05).
Inflammation
The release of inflammatory cytokines is shown in Figure 7. Exposure to NM110 or
NM101 with or without the presence of TG did not significantly affect the release of
TNFα or IL-6 (p>0.05). However, the exposure to 250 nM TG was associated with
significantly increased IL-6 concentrations (p<0.01). There was no interaction between
NP exposure and the presence of TG on IL-6 concentrations (p>0.05) as indicated by
ANOVA analysis.
The THP-1 monocyte adhesion to HUVECs is shown in Figure 8. The adhesion
was not significantly affected by the exposure to NM110 or NM101 (p>0.05), whereas
250 nM TG alone significantly promoted the adhesion of THP-1 monocytes to
HUVECs (p<0.01). Again, there was no interaction between NP exposure and the
presence of TG on the adhesion (p>0.05).
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Discussion
A recent study showed that the soluble ZnO NPs, but not the insoluble CeO2 NPs,
induced ER stress in HUVECs, which may be used as an earlier endpoint for
toxicological studies (Chen et al., 2014). If ER stress is associated the toxicity of ZnO
NPs to HUVECs, the presence of ER stress inducer TG should alter the toxicological
response of the cells to ZnO NP exposure. In this study, we addressed this issue by
testing the effects of TG on NM110 (ZnO NPs) induced cytotoxicity, intracellular ROS
and inflammation to HUVECs, and the effects were compared with the insoluble
NM101 (TiO2 NPs). We expected that the results of our study may provide important
information to further reveal the role of ER stress in the toxicity induced by ZnO NPs.
Our data showed that NM110, but not NM101, significantly induced
cytotoxicity as assessed by WST-1 (Figure 2A), neutral red uptake assay (Figure 2B)
and direct observation under light microscope (Figure 3), associated with an increase of
intracellular ROS (Figure 6). These results were generally in agreement with ENPRA
project by using the same NPs (Danielsen et al., 2015;Kermanizadeh et al., 2016). It has
been shown before that NM110, but not NM101, is partially soluble in aqueous solution
(Kermanizadeh et al., 2013). Here in this study we found a dose-dependent increase of
Zn ion accumulation associated with NM110 exposure with or without the presence of
TG, which indicated that NM110 could be dissolved in HUVECs (Figure 5). The
increase of intracellular Zn ions following NM110 exposure is also consistent with our
recent observations by using HUVECs and THP-1 macrophages (Jiang et al.,
2016;Gong et al., 2016). Moreover, in this study we showed that ZnSO4 induced similar
cytotoxic effects as NM110 at equal concentrations, which indicated that excessive Zn
ions are toxic to cells (Figure 4). Thus, it is possible that the relatively higher
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cytotoxicity of NM110 compared with that of NM101 is at least partially attributed to
the dissolved Zn ion inside cells.
We showed that TG induced cytotoxicity as assessed by WST-1 and neutral red
uptake assay (Figure 2). A recent study found that 1 μM TG significantly induced ER
stress in HUVECs (Ying et al., 2016). However, our recent study showed that 1 μM TG
was too toxic to HUVECs (Ji et al., 2016), which may make it difficult to observe the
combined effects. Therefore, we used a relatively low concentration of TG (250 nM) to
see if it may alter the response of HUVECs to NM110 or NM101 exposure. With the
presence of TG, there seems to be a relatively higher level of cytotoxicity induced by
NM110, and ANOVA analysis showed a significant interaction between NM110 and
TG as indicated by neutral red uptake assay (p<0.01; Figure 2). It has been shown
before that the activation of ER stress could induce apoptosis both in vivo and in vitro,
associated with the dysfunction of other organelles, e.g., lysosomes, due to interplay
between them (Lee et al., 2011;Liu et al., 2015;Sasaki and Yoshida, 2015). Thus, it is
possible that the presence of TG could enhance the toxicity of NM110 especially the
toxicity to lysosomes. However, we noticed that TG did not obviously affect the trend
of dose-dependent curve of NM110. For example, there was a marked decrease of
WST-1 viability and neutral red uptake from the concentration 16 μg/mL to 32 μg/mL
after NM110 exposure, and this trend was not obviously changed by the presence of TG
(Figure 2). The interaction between NM110 and TG seems to be a combined toxicity of
NM110 and TG NM110. Given the importance of ER stress in driving apoptosis and
dysfunction of organelles (Zhou and Tabas, 2013;Sasaki and Yoshida, 2015;Ozcan and
Tabas, 2012), damages of organelles especially dysfunction of lysosmes could be a
marker for the combined effects of TG and NP exposure, but it may be necessary to
investigate more endpoints associated with dysfunction of organelles in the future.
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The results of the present study indicated that neither NM110 or NM101
induced inflammatory responses showing as unaltered release of cytokines (TNFα and
IL-6) and THP-1 monocyte adhesion (Figure 7 & 8), which is consistent with a recent
report using the same NPs (Danielsen et al., 2015). TG exposure significantly promoted
the release of IL-6 and THP-1 monocyte adhesion, but did not affect the release of
TNFα (Figure 5 & 6). In our recent study, 1 μM TG treatment did not significantly
affect the release of a number of inflammatory mediators (Ji et al., 2016). Although in
this study we found that a much lower concentration of TG promoted the release of IL-
6, the release of TNFα remained unaltered (Figure 7). TNFα has been shown to play a
crucial role in the development of cardiovascular diseases because it is closely
associated with vascular dysfunction, and anti-TNFα has been considered as a plausible
way for the treatment of cardiovascular diseases (Back and Hansson, 2015;Zhang et al.,
2014;Moreau et al., 2013). Therefore, we suggested that TG was only able to trigger a
modestly inflammatory response in HUVECs. With the presence of TG, the release of
cytokines and THP-1 adhesion was not further significantly affected by NM110 or
NM101 exposure (Figure 7 & 8), and ANOVA analysis indicated no interaction
between TG and NPs on these endpoints (P>0.05). All of these results in combination
indicated that there is no interaction between TG and NM110 or NM101 on
inflammatory responses in HUVECs.
In this study, we assessed cytotoxicity, oxidative stress and inflammatory
responses in HUVECs after combined exposure to NPs and TG. Previous studies have
evaluated these endpoints in ‘healthy’ HUVECs (Danielsen et al., 2015;Kermanizadeh
et al., 2016). However, the endothelial cells behavior differently in normal and diseased
conditions (Eelen et al., 2015), and it has been suggested that the diversity of
endothelial cells under different conditions should be considered when assessing
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endothelial-NP interactions (Setyawati et al., 2015). Here we used the ER stress TG for
co-exposure with NPs to HUVECs, which may mimic the toxicological responses of
NPs under an atherosclerosis-like conditions, since ER stress is closed associated with
the development of atherosclerosis (Zhou and Tabas, 2013;Ozcan and Tabas, 2012).
The results indicated no interaction between exposure to ZnO NPs and the presence of
TG on almost all the endpoints except neutral red uptake assay (lysosomal dysfunction).
Nevertheless, it may be still necessary to assess the toxicity of NPs to endothelial cells
under different conditions (Setyawati et al., 2015), and our model by using combined
exposure of TG and NPs may serve as a tool to predict the toxicological responses of
NPs under atherosclerosis-like conditions.
In summary, the results from the present study showed that exposure to NM110
(ZnO NPs), but not NM101 (TiO2 NPs), was associated with cytotoxicity as assessed by
WST-1 and neutral red uptake assay, as well as intracellular ROS. However, neither
NM110 nor NM101 significantly affected release of TNFα or IL-6 or the adhesion of
THP-1 monocytes to HUVECs, which suggested that these NPs were not inflammatory
to HUVECs. The presence of ER stress inducer TG significantly induced cytotoxicity,
release of IL-6 and THP-1 monocyte adhesion, but did not significantly affect
intracellular ROS or release of TNFα. ANOVA analysis indicated no interaction
between exposure to NM110 and the presence of TG on almost all the endpoints except
neutral red uptake assay. However, the interaction appears to be a combined toxicity
resulted from TG and NM110. We concluded ER stress is probably not associated with
ZnO NP exposure induced oxidative stress and inflammatory responses in HUVECs.
There appears to be an interaction between TG and NP exposure on the dysfunction of
organelles, but the role of ER stress in NP induced cytotoxicity may still need further
studies.
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Acknowledgements
We appreciate Dr. Peter Moller (University of Copenhagen) to kindly provide the ZnO
(NM110) and TiO2 NPs (NM101) to us. This work was financially supported by The Scientific
Research Fund of Hunan Provincial Education Department (16C1551), Xiangtan University
grant (15XZX19) and Xiangtan University start-up grant (15QDZ47).
Conflict of interest
The authors declare no conflict of interest.
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Table 1. The main physicochemical properties of ZnO (NM110) and TiO2 (NM101)
NPs.
Names of NPs ZnO (code NM110;
uncoated NPs)
TiO2 (code NM101; phase
Anatase)
Average size (a) 100 nm 7 nm
TEM size (b) 20-250/50-350 nm 4-8/50-100 nm
XRD size (b) 70 to >100 nm 9 nm
BET surface area (b) 14 m2/g 322 m
2/g
NTA size in water (c) Mean 162 nm, Mode 138
nm
Mean 171 nm, Mode 145
nm
NTA size in RPMI (c) Mean 187 nm, Mode 177
nm
Mean 152 nm, Mode 111
nm
Zetasizer Nano size (d) 202.6±12.8 nm 279.2±11.0 nm
Zeta potential (d) -19.1±0.5 mV -19.7±0.6 mV
Note: (a) from supplier information, (b) reproduced from reference (Kermanizadeh et al.,
2013), (c) reproduced from reference (Danielsen et al., 2015), (d) measured by Zetasizer
Nano ZS90 in the present study.
Figure 1. The size distribution (1A) and Zeta potential (1B) of ZnO (NM110) or TiO2
NPs (NM101) suspended in MilliQ water. The morphology of NM110 (1C) and NM101
(1D) by scanning electron microscopy (SEM).
Figure 2. Cytotoxicity as assessed by WST-1 (2A) and neutral red uptake assays (2B).
HUVECs were exposed to 0 μg/mL (for control), 2 μg/mL, 4 μg/mL, 8 μg/mL, 16
μg/mL or 32 μg/mL ZnO (NM110) or TiO2 NPs (NM101) with or without the presence
of 250 nM thapsigargin (TG) for 24 h, followed by WST-1 and neutral red uptake
assays to indicate the toxicity. *, p<0.01, ANOVA.
Figure 3. The morphology of HUVECs after exposure and neutral red staining.
HUVECs were exposed to 0 μg/mL (for control), 16 μg/mL or 32 μg/mL ZnO (NM110)
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or TiO2 NPs (NM101) with or without the presence of 250 nM thapsigargin (TG) for 24
h, and then stained by using neutral red. The cells were imaged by a light microscope.
Figure 4. Cytotoxicity of ZnSO4 as assessed by WST-1 assay. HUVECs were exposed
to 0 μM (for control), 25 μM, 50 μM, 100 μM, 200 μM or 400 μM ZnSO4 for 24 h,
followed by WST-1 assay. *, p<0.01, ANOVA.
Figure 5. The accumulation of intracellular Zn ions. HUVECs were exposed to 0 μg/mL
(for control), 2 μg/mL, 4 μg/mL, 8 μg/mL or 16 μg/mL ZnO NPs (NM110) with or
without the presence of 250 nM thapsigargin (TG) for 24 h, and the accumulation of
intracellular Zn ions was determined by using a fluorescent probe. *, p<0.05, ANOVA.
Figure 6. The intracellular ROS as measured by DCFH-DA. HUVECs were exposed to
0 μg/mL (for control), 2 μg/mL, 4 μg/mL, 8 μg/mL, 16 μg/mL or 32 μg/mL ZnO NPs
(NM110) or TiO2 NPs (NM101) with or without the presence of 250 nM thapsigargin
(TG) for 3 h, followed by DCFH-DA assay to indicate the level of intracellular ROS. *,
p<0.05, ANOVA.
Figure 7. The release of TNFα (5A) and IL-6 (5B). HUVECs were exposed to 0 μg/mL
(for control), 4 μg/mL, or 16 μg/mL ZnO NPs (NM110) or TiO2 NPs (NM101) with or
without the presence of 250 nM thapsigargin (TG) for 24 h, followed by ELISA to
measure the concentrations of TNFα and IL-6 in the supernatants. *, p<0.01, ANOVA.
Figure 8. THP-1 adhesion to HUVECs. HUVECs were exposed to 0 μg/mL (for
control), 2 μg/mL, 4 μg/mL, 8 μg/mL, 16 μg/mL or 32 μg/mL ZnO NPs (NM110) or
TiO2 NPs (NM101) with or without the presence of 250 nM thapsigargin (TG) for 24 h,
and the adhesion of THP-1 monocytes to HUVECs was determined. *, p<0.01,
ANOVA.
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