Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=itxm20

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.

Submit your article to this journal

View related articles

View Crossmark data

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 (caoyi39@xtu.edu.cn)

JUST A

CCEPTED

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

JUST A

CCEPTED

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

JUST A

CCEPTED

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.

JUST A

CCEPTED

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

JUST A

CCEPTED

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

JUST A

CCEPTED

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).

JUST A

CCEPTED

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,

JUST A

CCEPTED

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.

JUST A

CCEPTED

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).

JUST A

CCEPTED

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).

JUST A

CCEPTED

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

JUST A

CCEPTED

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.

JUST A

CCEPTED

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

JUST A

CCEPTED

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.

JUST A

CCEPTED

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.

References

Back M, Hansson GK. (2015). Anti-inflammatory therapies for atherosclerosis. Nat Rev

Cardiol 12:199-211

Cao Y, Jacobsen NR, Danielsen PH, et al. (2014a). Vascular effects of multiwalled

carbon nanotubes in dyslipidemic ApoE-/- mice and cultured endothelial cells.

Toxicol Sci 138:104-16

Cao Y, Jantzen K, Gouveia AC, et al. (2015). Automobile diesel exhaust particles

induce lipid droplet formation in macrophages in vitro. Environ Toxicol

Pharmacol 40:164-71

Cao Y, Long J, Ji Y, Chen G, et al. (2016a). Foam cell formation by particulate matter

(PM) exposure: a review. Inhal Toxicol 28:583-90

Cao Y, Roursgaard M, Danielsen PH, et al. (2014b). Carbon black nanoparticles

promote endothelial activation and lipid accumulation in macrophages

independently of intracellular ROS production. PLoS One 9:e106711

Cao Y, Roursgaard M, Jacobsen NR, et al. (2016b). Monocyte adhesion induced by

multi-walled carbon nanotubes and palmitic acid in endothelial cells and

alveolar-endothelial co-cultures. Nanotoxicology235-244

Chen R, Huo L, Shi X, et al. (2014). Endoplasmic reticulum stress induced by zinc

oxide nanoparticles is an earlier biomarker for nanotoxicological evaluation.

ACS Nano 8:2562-74

Danielsen PH, Cao Y, Roursgaard M, et al. (2015). Endothelial cell activation,

oxidative stress and inflammation induced by a panel of metal-based

nanomaterials. Nanotoxicology 9:813-24

Eelen G, de ZP, Simons M, Carmeliet P. (2015). Endothelial cell metabolism in normal

and diseased vasculature. Circ Res 116:1231-1244

JUST A

CCEPTED

Gong Y, Ji Y, Liu F, et al. (2016). Cytotoxicity, oxidative stress and inflammation

induced by ZnO nanoparticles in endothelial cells: interaction with palmitate or

lipopolysaccharide. J Appl Toxicol in press DOI:10.1002/jat.3415

Ji Y, Zhu M, Gong Y, et al. (2016). Thermoresponsive Polymers with Lower Critical

Solution Temperature- or Upper Critical Solution Temperature-Type Phase

Behaviour Do Not Induce Toxicity to Human Endothelial Cells. Basic Clin

Pharmacol Toxicol in press DOI:10.1111/bcpt.12643

Jiang Q, Li X, Cheng S, et al. (2016). Combined effects of low levels of palmitate on

toxicity of ZnO nanoparticles to THP-1 macrophages. Environ Toxicol

Pharmacol 48:103-9

Kermanizadeh A, Gosens I, MacCalman L, et al. (2016). A Multilaboratory

Toxicological Assessment of a Panel of 10 Engineered Nanomaterials to Human

Health--ENPRA Project--The Highlights, Limitations, and Current and Future

Challenges. J Toxicol Environ Health B Crit Rev 19:1-28

Kermanizadeh A, Pojana G, Gaiser BK, et al. (2013). In vitro assessment of engineered

nanomaterials using a hepatocyte cell line: cytotoxicity, pro-inflammatory

cytokines and functional markers. Nanotoxicology 7:301-313

Lee GH, Kim DS, Kim HT, et al. (2011). Enhanced lysosomal activity is involved in

Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress

response and cell death against ER stress: involvement of vacuolar H+-ATPase

(V-ATPase). J Biol Chem 286:24743-53

Liu S, Sarkar C, Dinizo M, et al. (2015). Disrupted autophagy after spinal cord injury is

associated with ER stress and neuronal cell death. Cell Death Dis 6:e1582

Moller P, Christophersen DV, Jacobsen NR, et al. (2016). Atherosclerosis and

vasomotor dysfunction in arteries of animals after exposure to combustion-

derived particulate matter or nanomaterials. Crit Rev Toxicol 46:437-76

Moreau KL, Deane KD, Meditz AL, et al. (2013). Tumor necrosis factor-alpha

inhibition improves endothelial function and decreases arterial stiffness in

estrogen-deficient postmenopausal women. Atherosclerosis 230:390-6

Ozcan L, Tabas I. (2012). Role of endoplasmic reticulum stress in metabolic disease

and other disorders. Annu Rev Med 63:317-28

Sasaki K, Yoshida H. (2015). Organelle autoregulation-stress responses in the ER,

Golgi, mitochondria and lysosome. J Biochem 157:185-95

JUST A

CCEPTED

Setyawati MI, Tay CY, Docter D, et al. (2015). Understanding and exploiting

nanoparticles' intimacy with the blood vessel and blood. Chem Soc Rev

44:8174-99

Tomaszewski KA, Radomski MW, Santos-Martinez MJ. (2015). Nanodiagnostics,

nanopharmacology and nanotoxicology of platelet-vessel wall interactions.

Nanomedicine (Lond) 10:1451-75

Vance ME, Kuiken T, Vejerano EP, et al. (2015). Nanotechnology in the real world:

Redeveloping the nanomaterial consumer products inventory. Beilstein J

Nanotechnol 6:1769-80

Ying R, Wang XQ, Yang Y, et al. (2016). Hydrogen sulfide suppresses endoplasmic

reticulum stress-induced endothelial-to-mesenchymal transition through Src

pathway. Life Sci 144:208-17

Zhang Y, Yang X, Bian F, et al. (2014). TNF-alpha promotes early atherosclerosis by

increasing transcytosis of LDL across endothelial cells: crosstalk between NF-

kappaB and PPAR-gamma. J Mol Cell Cardiol 72:85-94

Zhou AX, Tabas I. (2013). The UPR in atherosclerosis. Semin Immunopathol 35:321-

32

JUST A

CCEPTED

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)

JUST A

CCEPTED

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.

JUST A

CCEPTED

JUST A

CCEPTED

JUST A

CCEPTED

JUST A

CCEPTED

JUST A

CCEPTED

JUST A

CCEPTED