Research article

Received: 18 September 2016, Accepted: 14 October 2016 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.3415

Cytotoxicity, oxidative stress and inflammation

induced by ZnO nanoparticles in endothelial
cells: interaction with palmitate or
lipopolysaccharide
Yu Gong, Yuejia Ji, Fang Liu, Juan Li* and Yi Cao*
ABSTRACT: Recent studies showed that ZnO nanoparticles (NPs) might induce the toxicity to human endothelial cells. However,
little is known about the interaction between ZnO NPs and circulatory components, which is likely to occur when NPs enter the
blood. In this study, we evaluated ZnO NP-induced cytotoxicity, oxidative stress and inflammation in human umbilical vein en-
dothelial cells (HUVECs), with the emphasis on the interaction with palmitate (PA) or lipopolysaccharide (LPS), because PA and
LPS are normal components in human blood that increase in metabolic diseases. Overall, ZnO NPs induced cytotoxicity and in-
tracellular reactive oxygen species (ROS) at a concentration of 32 μg ml 1, but did not significantly affect the release of inflamma-
tory cytokines or adhesion of THP-1 monocytes to HUVECs. In addition, exposure to ZnO NPs dose-dependently promoted
intracellular Zn ions in HUVECs. PA and LPS have different effects. Two hundred μM PA significantly induced cytotoxicity and
THP-1 monocyte adhesion, but did not affect ROS or release of inflammatory cytokines. In contrast, 1 μg ml 1 LPS significantly
induced ROS, release of inflammatory cytokines and THP-1 monocyte adhesion, but not cytotoxicity. The presence of ZnO NPs
did not significantly affect the toxicity induced by PA or LPS. In addition, the accumulation of Zn ions after ZnO NP exposure
was not significantly affected by the presence of PA or LPS. We concluded that there was no interaction between ZnO NPs and
PA or LPS on toxicity to HUVECs in vitro. Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: ZnO nanoparticles (ZnO NPs); Palmitate (PA); Lipopolysaccharide (LPS); Human umbilical vein endothelial cells (HUVECs);
interaction

Introduction human blood, and the elevation of circulatory PA is implicated in

With the rapid development of nanotechnology, nanoparticles
(NPs) are increasingly produced and used in a number of commer-
cially available products, but the potential health effects of NPs are
not fully understood. According to a recent survey, metal and
metal oxide NPs are among the most popular NPs used in
commercially available products (they account for 37% of 1814
products evaluated by the project), which can lead to particle
exposure to human beings in daily life mainly through dermal,
inhalational and oral exposure (Vance et al., 2015). Therefore, there
is an urgent need to assess the potential adverse health effects of
metal and metal oxide NPs to ensure the safe use of them. In
recent years, some studies have shown that metal and metal oxide
NPs may induce adverse vascular effects due to the direct translo-
cation of NPs into human blood or the indirect effects induced by
oxidative stress and/or inflammatory mediators (Møller et al., 2011,
2016). In addition, considering the potential applications of metal
and metal oxide NPs in biomedicine, the direct contact of human
blood vessels with metal and metal oxide NPs is expected to
increase. Therefore, the evaluation of the toxicity of these NPs to
relevant cell lines lining blood vessels is necessary (Tomaszewski
et al., 2015). However, little is known about the interactions of
metal and metal oxide NPs and the components in human blood,
which is likely to occur when NPs enter the blood.
The interactions between NPs and palmitate (PA) or lipopolysac-
charide (LPS) on the toxicity to relevant cells of blood vessels may
need further studies. PA is one of the basic fatty acids present in
the development of metabolic diseases (Gregor & Hotamisligil,
2011; Mancini et al., 2015). In addition, high levels of PA have been
shown to induce cytotoxicity, oxidative stress and inflammatory re-
sponses to human endothelial cells in vitro (Lu et al., 2015;
Sathanoori et al., 2015; Shikama et al., 2015). LPS is a normal com-
ponent of Gram-negative bacteria, and the translocation rate of
LPS from gut into blood is increased to mediate a prolonged in-
flammatory response in metabolic diseases (Gnauck et al., 2016;
Hersoug et al., 2016). To the best of knowledge, only a recent study
investigated the interactions between PA and NPs on endothelial
cells, and results showed that the presence of PA promoted
multiwalled carbon nanotube-induced monocyte adhesion to hu-
man endothelial cells (Cao et al., 2016b). No previous study inves-
tigated the interactions between LPS and NPs on relevant cells of
human blood vessels, but a recent study indicated the combined
effects of LPS and multiwalled carbon nanotubes on lung epithelial
cells to promote fibrogenesis (Pacurari et al., 2016).
*Correspondence to: Juan Li or Yi Cao, Key Laboratory of Environment-Friendly
Chemistry and Application of Ministry of Education, Laboratory of Biochemistry,
College of Chemistry, Xiangtan University, Xiangtan 411105, People’s Republic of
China.
E-mail: juanli@xtu.edu.cn; caoyi39@xtu.edu.cn
Key Laboratory of Environment-Friendly Chemistry and Application of Ministry of
Education, Laboratory of Biochemistry, College of Chemistry, Xiangtan University,
Xiangtan 411105, People’s Republic of China

J. Appl. Toxicol. 2016 Copyright © 2016 John Wiley & Sons, Ltd.

In this study, we investigated the cytotoxicity, oxidative stress
and inflammatory responses in human umbilical vein endothelial
cells (HUVECs) induced by ZnO NPs, with the emphasis on the in-
teractions with PA or LPS. HUVECs were used as an in vitro model
because endothelial cells are the crucial cells lining the blood ves-
sels that can regulate the function of them (Gimbrone &
Garcia-Cardena, 2016; Moore et al., 2013). ZnO NPs were used
because they are one of the most popular metal and metal oxides
NPs (Vance et al., 2015). In addition, ZnO NPs may also have impor-
tant biomedical applications for their anticancer (Soenen et al.,
2015) or antidiabetes activities (Nazarizadeh & Asri-Rezaie, 2016;
Umrani & Paknikar, 2014). HUVECs were exposed to various
concentrations of ZnO NPs with or without the presence of PA or
LPS, and cytotoxicity, intracellular reactive oxygen species (ROS),
release of inflammatory mediators and THP-1 monocyte adhesion
to HUVECs were measured.
Materials and methods
Cell cultures
HUVECs (passage 1), basal endothelial cell medium (ECM) and re-
lated cell culture reagents were purchased from ScienCell Research
Laboratories (Carlsbad, CA, USA). The ECM was supplemented with
5% (v/v) fetal bovine serum (FBS), 1% (v/v) endothelial cell growth
supplement and 1% (v/v) penicillin/streptomycin solution to make
the full endothelial medium. HUVECs were initially expanded and
then used between passages 3 and 6, as previously described ( Ji
et al., 2016). For each experiment, 1 × 104 per well (in 96-well
plates) or 4 × 104 per well (in 24-well plates) HUVECs were seeded
and grown for 2 days before exposure.
THP-1 monocytes (ATCC, Manassas, VA, USA) were cultured in
RPMI 1640 medium (Thermo-Fisher, USA) supplemented with
10% FBS (Gibco, South Africa), 1% penicillin/streptomycin solution
(Beyotime, Nantong, China) and 1× sodium pyruvate (Thermo-
Fisher; RPMI 1640 medium with the supplement was denoted as
full THP-1 medium) in a CO2 incubator at 37 °C. The cells were used
within 3 months to keep their best characteristics.
Particles and exposure
ZnO NPs (code NM110; BASF Z-Cote; uncoated, 100 nm), originally
received from the European Commission Joint Research Centre
Nanomaterials Repository, were kindly provided by Prof. Peter
Møller (Department of Public Health, University of Copenhagen).
NM110 has been well characterized before by using transmission
electron microscopy, X-ray diffractogram, Brunauer–Emmett–
Teller technology, dynamic light scattering and NP tracking analy-
sis (Cao et al., 2015b; Kermanizadeh et al., 2013). The X-ray
diffractogram size is 70 to >100 nm. The transmission electron mi-
croscopy size is 20–250/50–350 nm. The Brunauer–Emmett–Teller
surface area is 14 m2 g 1. The dynamic light scattering size in
medium is 306 nm (Kermanizadeh et al., 2013). The NP tracking
analysis size in water is about 155  3 nm, which was increased
by the presence of proteins in water (Cao et al., 2015b).
To characterize further the NM110 in this study, a scanning elec-
tron microscopy (SEM; Zeiss EVO18, Germany) was used to investi-
gate the morphology of the particle. The NM110 sample was
imaged with a magnification of 100 000 times and an acceleration
potential of 20 keV. The hydrodynamic size distribution and zeta
potential were measured using 16 μg ml 1 NM110 suspended in
a different suspension medium (MilliQ water, 200 μM PA or
wileyonlinelibrary.com/journal/jat Copyright © 2016 John Wiley & Sons, Ltd.
Y. Gong et al.
1 mg ml 1 LPS) by Zetasizer Nano ZS90 (Malvern, UK). The size
and zeta potential were measured three times, and mean  SD
was calculated.
To make the suspension of NM110, 2.56 mg ml 1 particles in
MilliQ water containing 2% FBS were sonicated continuously for
8 min with continuous cooling on ice using an ultrasonic processor
FS-250 N (20% amplitude; Shanghai Shengxi, Shanghai, China) and
then diluted in full endothelial medium to expose the HUVECs. It
has been shown before that NM110 significantly induced cytotox-
icity in HUVECs at concentrations ≥32 μg ml 1 (Danielsen et al.,
2015); therefore, in the present study we used NM110 at concen-
trations from 2 to 32 μg ml 1. To make the suspension of PA, a
stock solution of 20 mM PA (Sigma-Aldrich, Saint Louis, MO, USA)
in MilliQ water was heated to 70 °C in a water bath until dissolved,
and this stock solution of PA was diluted to 200 μM for the expo-
sure. For LPS, a stock solution of LPS (Sigma-Aldrich) was made
at 200 μg ml 1 in full endothelial medium and then diluted to
1 μg ml 1 to expose HUVECs. For all the experiments, HUVECs
were exposed to various concentrations of NM110, with or without
the presence of 200 μM PA or 1 μg ml 1 LPS.
Cytotoxicity assay
The cytotoxicity was measured by using neutral red uptake, water-
soluble tetrazolium-1 (WST-1) and lactate dehydrogenase (LDH)
kits according to the manufacturer’s instructions (Beyotime). Neu-
tral red is a dye that 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 to 24-well plates were
incubated with 400 μl full endothelial medium containing various
concentrations of NM110, with or without the presence of
200 μM PA or 1 μg ml 1 LPS. After 24 h exposure, the cells were
rinsed once with Hanks’ solution, and then incubated with 10%
neutral red for 2 h. After being 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 enzyme-linked immunosorbent assay
(ELISA) reader (Synergy HT; BioTek, Tacoma, Washington, USA).
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, HUVECs seeded in to 24-well plates were exposed to
various concentrations of NM110, with or without the presence of
PA or LPS for 24 h, as indicated above, rinsed and 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).
LDH is a cytoplasmic enzyme that could be released into extra-
cellular fluid when the membrane is damaged. The LDH assay was
done in parallel with the WST-1 assay. Briefly, after exposure, the
supernatant was collected and 60 μl supernatant from each sam-
ple was mixed with 120 μl LDH reaction buffer provided by the
kit for 30 min. The absorbance was read at 490 nm with 690 nm
as reference. To induce 100% of LDH release, the positive control
cells were incubated with 10% lysis buffer (provided by the kit)
1 h before the assay and then the LDH activity was measured as in-
dicated above.
Reactive oxygen species
The intracellular ROS was estimated by using
2′,7′-dichlorofluorescein diacetate (DCFH-DA) as previously de-
scribed (Cao et al., 2015a). DCFH-DA is a cell-permeable probe,
J. Appl. Toxicol. 2016
Toxicity of ZnO NPs to endothelial cells
which becomes fluorescent when it is oxidized by the reaction
with a variety of ROS inside the cells. A stock solution of
DCFH-DA was made at 10 mM in methanol and stored at 20 °C
before use. HUVECs on a black 96-well plate were incubated with
200 μl full endothelial medium containing various concentrations
of NM110 with or without the presence of PA or LPS for 3 h, rinsed
once with Hanks’ solution and then incubated with 10 μM
DCFH-DA diluted in 100 μl ECM for 30 min. After being rinsed once
again, the fluorescence was read at excitation (ex) 485  20 nm
and emission (em) 528  20 nm by an ELISA reader. Here the cells
were exposed for only 3 h because 24 h exposure to PA signifi-
cantly induced cytotoxicity to HUVECs (see Results below).
Previous work showed that a 3 h exposure of HUVECs to a variety
of solid particles could rapidly induce ROS, which may mediate
the toxic effects of the particles (Cao et al., 2014a,b, 2015a;
Danielsen et al., 2015).
Intracellular Zn ions
The accumulation of Zn ions inside the HUVECs after 3 h expo-
sure to various concentrations of NM110 was measured using a
cell-permeable Zn ion fluorescent probe Zinquin ethyl ester
(Sigma-Aldrich). A stock solution of Zinquin ethyl ester was made
as 1 mg ml 1 (2.4 mM) in dimethyl sulfoxide and stored at 20 °C
before use. After exposure, the HUVECs were rinsed once by
using Hanks’ solution and then incubated with ECM containing
24 μM Zinquin ethyl ester for 30 min. After another wash, the fluo-
rescence was read at ex 360  44 nm and em 460  40 nm by an
ELISA reader.
Enzyme-linked immunosorbent assay
Supernatant from the exposed HUVECs was collected and stored
at 80 °C before the ELISA analysis. Inflammatory mediators inter-
leukin (IL)-6, soluble monocyte chemotactic protein-1 (sMCP-1)
and soluble vascular cell adhesion molecule-1 (sVCAM-1) was mea-
sured by ELISA kits according to the manufacturer’s instructions
(Neobioscience Technology Co., Ltd., Guangzhou, China). Some
of the samples were diluted for the measurement.
THP-1 adhesion
The adhesion of THP-1 monocytes to HUVECs was done as
previously described (Cao et al., 2016b). Briefly, HUVECs on a
black 96-well plate were exposed to 200 μl full endothelial
medium containing various concentrations of NM110 with or
without the presence of 200 μM PA or 1 μg ml 1 LPS for 24 h.
THP-1 monocytes were labeled with 10 μM CellTrackerTM Green
CMFDA (5-chloromethylfluorescein diacetate; Invitrogen,
Carlsbad, CA, USA) for 30 min in RPMI 1640 medium. The free
probe was removed by centrifuge, and 5 × 104 per well labeled
THP-1 cells were incubated with the exposed HUVECs for 1 h
for adhesion. Here, a short time (1 h) for the adhesion assay
was used 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.
J. Appl. Toxicol. 2016 Copyright © 2016 John Wiley & Sons, Ltd.
Statistics
All the data were expressed as mean  SEM of three independent
experiments carried out on three independent days (n = 3 for each
independent experiment). Two-way ANOVA (concentrations and
treatment as categorical factors) followed by Tukey honest signifi-
cant difference test using R 3.2.2; P < 0.05 was considered statisti-
cally significant.
Results
The size, zeta potential and morphology of NM110
Figure 1(A,B) represents the size distribution and zeta potential of
NM110 in different suspensions. The size of NM110 in water was
206.7  7.9 nm, which did not seem to be affected by the presence
of LPS but was apparently increased by the presence of PA. The
zeta potential of NM110 in water was 19.8  1.3 mV, which was
slightly decreased by the presence of LPS and largely decreased
by the presence of PA (Table 1). The SEM morphology of NM110
is shown in Fig. 1(C), which indicated aggregation and/or agglom-
eration of particles. The SEM morphology of NM110 after sonica-
tion has been previously reported, which showed that NM110
agglomerated in the dispersion medium after sonication and over
time (Cao et al., 2015b).
Cytotoxicity
The cytotoxicity of NM110 to HUVECs under different conditions is
shown in Fig. 2. The exposure of HUVECs to NM110 was associated
with significantly increased cytotoxicity at concentrations
≥32 μg ml 1, as assessed by neutral red uptake (P < 0.01; Fig. 2A),
WST-1 (P < 0.01; Fig. 2B) but not LDH assay (P > 0.05; Fig. 2C). The
presence of 200 μM PA (P < 0.01), but not 1 mg ml 1 LPS (P > 0.05),
significantly promoted cytotoxicity as assessed by the three inde-
pendent assays used. ANOVA analysis indicated an interaction be-
tween concentrations of NM110 and the presence of PA as
assessed by neutral red uptake (P < 0.01) and WST-1 (P < 0.01),
but not LDH assay (P > 0.05).
Reactive oxygen species
As shown in Fig. 3, the exposure of HUVECs to NM110 signifi-
cantly induced intracellular ROS at 32 μg ml 1 (P < 0.05). The
presence of PA did not significantly affect ROS (P > 0.05),
whereas LPS exposure was associated with significantly in-
creased ROS (P < 0.01). ANOVA analysis indicated no interaction
between concentrations of NM110 and the presence of PA or
LPS on ROS (P > 0.05).
Intracellular Zn ions
The accumulation of intracellular Zn ions in NM110-exposed
HUVECs under different conditions is shown in Fig. 4. There was
single factor effect of NM110 (P < 0.01), but no effect of PA or
LPS or interaction between NM110 and PA or LPS (P > 0.05). The
exposure to NM110 was associated with significantly increased in-
tracellular Zn ions at the concentrations of 8 μg ml 1 (P < 0.05),
16 μg ml 1 (P < 0.01) and 32 μg ml 1 (P < 0.01).
wileyonlinelibrary.com/journal/jat
 Y. Gong et al.

Figure 2. Cytotoxicity of NM110 to HUVECs under different conditions.

Figure 1. Representative figures showing the hydrodynamic size distribu-
tion (A) and zeta potential (B) in different suspensions as well as the
scanning electron microscopy morphology of NM110 (C). LPS, lipopolysac-
charide; PA, palmitate.
HUVECs were exposed to various concentrations of NM110 with or without
1
the presence of 200 μM PA or 1 μg ml LPS for 24 h, followed by neutral
red uptake (A), WST-1 (B) and LDH (C) assay to indicate the cytotoxicity.
*P < 0.01, compared with control, ANOVA. HUVECs, human umbilical vein
endothelial cells; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; PA,
palmitate; WST-1, water-soluble tetrazolium-1.

Table 1. Size and zeta potential of NM110 in different

suspensions Inflammatory mediators
The release of inflammatory mediators is shown in Fig. 5. The expo-
Size (nm) Zeta potential (mV)
sure to various concentrations of NM110 did not significantly affect
NM110 206.7  7.9 –19.8  1.3 the release of IL-6, sMCP-1 and sVCAM-1 (P > 0.05). The presence
NM110 + PA 375.3  32.8 –38.6  0.8 of PA did not affect the release of these inflammatory mediators ei-
NM110 + LPS 196.2  26.3 –23.0  0.8 ther, whereas LPS significantly promoted the release of sMCP-1
and sVCAM-1 (P < 0.01), but not the release of IL-6 (P > 0.05). There
LPS, lipopolysaccharide; PA, palmitate.
was no interaction between NM110 and the presence of PA or LPS
Data represent means  SD of three measurement.
(P > 0.05).

wileyonlinelibrary.com/journal/jat Copyright © 2016 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2016
Toxicity of ZnO NPs to endothelial cells

Figure 3. Intracellular ROS in HUVECs under different conditions. HUVECs

were exposed to various concentrations of NM110 with or without the pres-
1
ence of 200 μM PA or 1 μg ml LPS for 3 h, and intracellular ROS was mea-
sured by using the fluorescent probe 2′,7′-dichlorofluorescein. *P < 0.05,
compared with control, ANOVA. HUVECs, human umbilical vein endothelial
cells; LPS, lipopolysaccharide; PA, palmitate; ROS, reactive oxygen species.

Figure 4. Intracellular Zn ions in HUVECs under different conditions.

HUVECs were exposed to various concentrations of NM110 with or without
1
the presence of 200 μM PA or 1 μg ml LPS for 3 h, and intracellular Zn ions
were measured by using the fluorescent probe Zinquin ethyl ester.
*P < 0.05, compared with control, ANOVA. HUVECs, human umbilical vein
endothelial cells; LPS, lipopolysaccharide; PA, palmitate.
THP-1 adhesion
The adhesion of THP-1 monocytes to HUVECs is shown in Fig. 6. Ex-
posure to PA (P < 0.01) or LPS (P < 0.01), but not NM110 (P > 0.05),
significantly promoted the adhesion of THP-1 monocytes to
HUVECs. There was no interaction between NM110 and the pres-
ence of PA or LPS on THP-1 adhesion (P > 0.05).
Discussion
In this study, we investigated the toxicity of NM110 to HUVECs,
with emphasis on the interactions between NM110 and PA or
LPS. The endothelial viability, oxidative stress and inflammation
were used as the main endpoints as they are key events in the
early development of atherosclerosis (Gimbrone & Garcia-Cardena,
2016; Moore et al., 2013). Recently it has been shown that exposure
to ZnO NPs may induce toxicity to human endothelial cells
Figure 5. Release of IL-6 (A), sMCP-1 (B) and sVCAM-1 (C). Human umbil-
ical vein endothelial cells were exposed to various concentrations of NM110
1
with or without the presence of 200 μM PA or 1 μg ml LPS for 24 h, and
the release of the indicated inflammatory mediators was determined by
enzyme-linked immunosorbent assay. *P < 0.01, compared with control,
ANOVA. IL-6, interleukin-6; LPS, lipopolysaccharide; sMCP-1, soluble mono-
cyte chemotactic protein-1; PA, palmitate; sVCAM-1, soluble vascular cell
adhesion molecule.
(Chuang et al., 2016; Danielsen et al., 2015; Liang et al., 2016; Suzuki
et al., 2014). However, to the best of knowledge, we are the first to
investigate the interactions between ZnO NPs and the circulatory
components PA or LPS.
Our data showed that NM110 induced cytotoxicity to HUVECs at
32 μg ml 1 (Fig. 2), which is consistent with a recent report by
using the same NPs (Danielsen et al., 2015). Intracellular ROS was
only significantly induced by NM110 at 32 μg ml 1, but not at
lower concentrations (2–16 μg ml 1) that are non-cytotoxic (Fig. 3).
Recently, Liang et al. (2016), showed that the antioxidant α-lipoic
acid protected human endothelial cells from ZnO NP-induced

J. Appl. Toxicol. 2016 Copyright © 2016 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

Figure 6. THP-1 adhesion to HUVECs. HUVECs were exposed to various
concentrations of NM110 with or without the presence of 200 μM PA or
1
1 μg ml LPS for 24 h, and the adhesion of THP-1 monocytes to HUVECs
was determined. *P < 0.01, compared with control, ANOVA. HUVECs, hu-
man umbilical vein endothelial cells; LPS, lipopolysaccharide; PA, palmitate.
apoptosis. Thus, it is possible that high concentrations of ZnO
NP-induced cytotoxicity to HUVECs through the induction of
intracellular ROS. There was different impact of PA and LPS on
cytotoxicity and intracellular ROS. PA significantly induced cyto-
toxicity but not intracellular ROS, whereas LPS significantly in-
duced intracellular ROS without obvious effect on cytotoxicity
(Figs 2 and 3). There was no interaction between NM110 and
the PA or LPS on LDH or intracellular ROS (P > 0.05), although
ANOVA analysis indicated an interaction between NM110 and
PA on neutral red uptake and WST-1 assay (P < 0.01). However,
we noticed that PA did not obviously affect the trend of dose-
dependent curve for NM110. For example, there was a marked
decrease of neutral red uptake and WST-1 viability from the con-
centration 16 to 32 μg ml 1 after NM110 exposure, and this trend
was not obviously changed by the presence of PA. A previous
study found an interaction between vitamin C and ZnO NPs on
cytotoxicity by showing that vitamin C changed ZnO NPs at
non-cytotoxic concentrations to be cytotoxic through the
augmentation of NP uptake (Wang et al., 2014). Therefore, we
proposed that the interaction between NM110 and PA is indeed
from a combined toxicity of NM110 and PA, rather than a
synergistic effect of PA on the cytotoxicity of NM110.
The results from the present study showed that NM110 did not
induce the release of IL-6, sMCP-1 or sVCAM-1 (Fig. 5) or the adhe-
sion of THP-1 monocytes to HUVECs, which indicated a minimal ef-
fect of NM110 on inflammatory responses. Results from the ENPRA
project showed that NM110 only induced inflammatory responses
in pulmonary and renal cells, but not cardiovascular and hepatic
cells, which indicated that the inflammatory responses induced
by NM110 exposure is dependent on the types of cells (Danielsen
et al., 2015; Kermanizadeh et al., 2013, 2016). LPS, but not PA, sig-
nificantly promoted the release of sMCP-1 and sVCAM-1 (Fig. 5),
whereas both PA and LPS significantly induced THP-1 monocyte
adhesion to HUVECs (Fig. 6). The inflammatory potential of PA
and LPS to human endothelial cells is generally in agreement with
previous reports (Cao et al., 2016b; Lu et al., 2015; Ming et al., 2015;
Wu et al., 2015). With the presence of PA or LPS, the release of in-
flammatory mediators or THP-1 monocyte adhesion was not en-
hanced further by the exposure to NM110. This is in contrast to a
recent study showing that the presence of PA promoted
wileyonlinelibrary.com/journal/jat Copyright © 2016 John Wiley & Sons, Ltd.
Y. Gong et al.
multiwalled carbon nanotube exposure induced THP-1 monocyte
adhesion to HUVECs, associated with a modest increase of IL-6 re-
lease but not intracellular ROS. Therefore, we concluded that there
is no interaction between NM110 and PA or LPS on inflammatory
responses to HUVECs.
ZnO NPs may induce the toxicity to cells through the disruption
of Zn ion homeostasis due to the dissolution of the particles inside
and/or outside the cells (Kao et al., 2012; Mihai et al., 2015; Mu et al.,
2014). It has been shown before that NM110 is partially soluble
such that 50–60% of NM110 can dissolute into Zn ions in cell me-
dium after 24 h (Kermanizadeh et al., 2013). To address this issue,
we determined the accumulation of intracellular Zn ions by using
a fluorescent probe. As expected, NM110 dose-dependently pro-
moted the accumulation of intracellular Zn ions (Fig. 4). Compared
with the exposure to 16 μg ml 1 NM110, there was a decrease of
Zn ion accumulation after exposure to 32 μg ml 1 NM110, which
could be explained by the increased efflux activities in response
to toxic NP exposure (Dorier et al., 2015).
It has been suggested that the presence of nutrients and biolog-
ical molecules might affect the physicochemical properties of NPs
and consequently the uptake of NPs into cells (Cao et al., 2016a;
Docter et al., 2015). In this study, we also found that PA, but not
LPS, affected the size and zeta potential of NM110 (Fig. 1A,B),
which indicated a coating effect. The increase of NM110 size by
the presence of PA is in agreement with a recent study (Cao
et al., 2015b), although that study used a much higher concentra-
tion of PA. However, the accumulation of intracellular Zn ions in-
duced by NM110 exposure was not significantly affected by the
presence of PA or LPS (Fig. 4). A recent study showed that ZnO
NPs with a positive zeta potential resulted in higher cytotoxicity
due to higher NP uptake in comparison with the counterparts with
negative zeta potential (Abdelmonem et al., 2015). In our study,
the NM110 had negative zeta potential in all suspensions, which
may account for the unaltered uptake of NM110 into HUVECs with
or without the presence of PA or LPS. This may also explain the
minimal interaction between NM110 and PA or LPS on the toxicity
to HUVECs.
Collectively, we showed in this study that NM110 (ZnO NPs) in-
duced cytotoxicity and intracellular ROS at high concentrations,
but did not affect the release of inflammatory cytokines or adhe-
sion of THP-1 monocytes to HUVECs. PA induced cytotoxicity and
THP-1 monocyte adhesion, but did not affect ROS or release of in-
flammatory cytokines, whereas LPS induced ROS, release of inflam-
matory cytokines and THP-1 monocyte adhesion without an effect
on cytotoxicity. ANOVA analysis indicated no interaction between
NM110 and the presence of PA or LPS on almost all the endpoints
except cytotoxicity as assessed by neutral red uptake and WST-1
assay. However, the interaction appears to be a combined effect
of PA and NM110 on cytotoxicity. The uptake of NM110 was not
significantly affected by the presence of PA or LPS, which may
explain the minimal interaction. In perspective, we are developing
co-culture models to study the possible interactions between NPs
and circulatory components in vitro in a more complex system.
Acknowledgments
We appreciate Prof. Peter Møller (University of Copenhagen) to
kindly provide the ZnO NPs (NM110) to us. This work was
financially supported by the Scientific Research Fund of Hunan
Provincial Education Department (15C1331), Xiangtan University
start-up grant (15QDZ14) and Xiangtan University grant
(15XZX18).
J. Appl. Toxicol. 2016
Toxicity of ZnO NPs to endothelial cells
Conflict of interest
The authors did not report any conflict of interest.
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