Article

Toxicology and Industrial Health

2017, Vol. 33(9) 687–695
Ascorbic acid prevents zinc oxide © The Author(s) 2017
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nanoparticle–induced intracellular DOI: 10.1177/0748233717707361
journals.sagepub.com/home/tih
oxidative stress and inflammatory
responses

Hiroko Fukui1, Hitoshi Iwahashi1, Keiko Nishio2, Yoshihisa Hagihara2,

Yasukazu Yoshida3 and Masanori Horie3

Abstract
Exposure to zinc oxide nanoparticles (ZnO NPs) promotes acute pulmonary toxicity through oxidative stress
and inflammation. Furthermore, dissolved zinc from ZnO NPs induces the formation of intracellular reactive
oxygen species (ROS). We previously reported that supplemental ascorbic acid (AA) inhibits ZnO NP-induced
acute pulmonary toxicity in a rat model; however, the mechanism of this action remains unclear. Therefore, we
investigated the effects of AA on ZnO NP-induced cytotoxicity in human lung carcinoma A549 cells. AA was
found to suppress intracellular production of ROS, and thus reduce the subsequent inflammation of ZnO NPs.
However, intracellular Zn2þ concentrations were higher in AA-treated cells than in AA-untreated cells. AA
was found to react with Zn2þ but not with the ZnO NPs themselves. These results suggest the possibility that
AA-chelated extracellular Zn2þ and the Zn–AA complex was readily taken up into cell. Even if the intracellular
Zn2þ level was high, cytotoxicity might be reduced because the Zn–AA complex was stable. Co-treatment of
AA to A549 inhibited ROS production and subsequent intracellular inflammatory responses. These results are
consistent with those previously reported from an in vivo model. Thus, two possibilities can be considered
about the cytotoxicity-reducing the effect of AA: antioxidant efficacy and chelating effect.

Keywords
Nanoparticles, metal toxicity, oxidative stress, inflammation, antioxidant

Received 21 July 2016; Revised 24 March 2017; Accepted 30 March 2017

Introduction level in natural environments, can cause significant

Zinc oxide nanoparticles (ZnO NPs) are widely
manufactured for applications ranging from catalysis
to cosmetics. With increasing ZnO NP production,
more workers are being exposed to ZnO NPs. There-
fore, it is necessary to understand ZnO NP-induced
toxicity and consider how to reduce the risk of
toxicity.
Widespread and expanding production and use of
ZnO NPs increases the potential for their release to
the environment. Ma et al. reported that toxic effects
of ZnO NPs in both aquatic and terrestrial species
were within a broad range of taxa, and for certain
species (such as Daphnia magna), the toxicity can
occur at concentrations as low as less than 1 mg/l.
This suggests that ZnO NPs, if reaching a sufficient
risk to the environmental biota (Ma et al., 2013).
The in vivo toxic effects of ZnO NPs were studied
using different treatment routes. Oral, inhalation, and
intratracheal administration routes have also been
used to evaluate the acute toxicity of ZnO NPs. In our
1
United Graduate School of Agricultural Science, Gifu University,
Yanagido, Gifu City, Japan
2
Health Research Institute (HRI), National Institute of Advanced
Industrial Science and Technology (AIST), Ikeda, Osaka, Japan
3
HRI, AIST, Takamatsu, Kagawa, Japan
Corresponding author:
Hiroko Fukui, United Graduate School of Agricultural Science,
Gifu University, 1-1, Yanagido, Gifu City, Gifu 501-1193, Japan.
Email: hiroko-fukui@aist.go.jp
688
previous study, intratracheal administration of ZnO
NPs induced pulmonary oxidative stress and inflam-
mation in rats, and Zn2þ released from ZnO NPs was
associated with pulmonary toxicity (Fukui et al.,
2012). Thus, it is known that the release of Zn2þ and
subsequent induction of oxidative stress are known to
be contributing factors to the mechanism of ZnO NP-
induced toxicity. However, there are no reports on
preventive measures for this toxicity.
Since oxidative stress is a known contributor to the
pulmonary toxicity induced by ZnO NPs, we investi-
gated the antioxidant L-ascorbic acid (AA) as a poten-
tial preventative agent. AA is a water-soluble radical
scavenger, known to protect against membrane oxida-
tion and injury. Moreover, AA has been shown to
chelate heavy metals (e.g. cadmium, mercury, and
zinc) to inhibit metal-derived oxidative stress
(Tajmir-Riahi, 1991). AA has also been reported to
inhibit intracellular oxidative stress induced by nano-
particles. For example, reports have shown that AA
treatment reduces intracellular lipid peroxidation
induced by talc nanoparticles in A549 cells and inhi-
bits reactive oxygen species (ROS) generation (and
subsequent activation of apoptosis) induced by silica
nanoparticles (Ahmad et al., 2012; Akhtar et al.,
2010). AA potentially prevents not only oxidative
stress but also inflammation. In addition, AA was
found to decrease the secretion of pro-inflammatory
cytokines (IL-1 and IL-6) in human keratinocytes
stimulated by UVA irradiation (Tebbe et al., 1997).
We have previously demonstrated that supplying
AA in water reduces acute pulmonary toxicity
(including oxidative stress, inflammation, and injury)
induced by ZnO NPs in rat lungs (Fukui et al., 2015).
However, the mechanism by which AA acts to pre-
vent ZnO NP-induced toxicities (i.e. ROS trapping
and/or Zn2þ chelating) is still unclear. Clarifying
AA’s prevention mechanism is important for reducing
the occupatonal health risk of ZnO NP-induced toxi-
cities. Therefore, the present study aimed to evaluate
the preventative role of AA in ZnO NP-induced toxi-
city and to determine its mechanism of action.
Materials and methods
ZnO NPs and chemicals
ZnO NPs were purchased from Ishihara Sangyo
Kaisha, Ltd (Osaka, Japan); the product name was
FZO-50 (lot No. 0010). According to the manufactur-
er’s material data sheets, the primary particle size was
21 nm, the purity was 97.4%, and the specific surface
Toxicology and Industrial Health 33(9)
area was 49.6 m2/g. Zinc chloride (ZnCl2) and AA
were purchased from Wako Pure Chemical Industries,
Ltd (Osaka, Japan).
Cell culture
Human lung carcinoma A549 cells were purchased
from the Riken BioResource Center (Tsukuba, Ibar-
aki, Japan). The cells were cultured in Dulbecco’s
modified Eagle medium (DMEM; Gibco, Thermo
Fisher Scientific, Waltham, MA, USA) and supple-
mented with 10% heat-inactivated fetal bovine serum
(FBS), penicillin (100 units/ml), streptomycin
(100 mg/ml), and amphotericin B (250 ng/ml) (Nacalai
Tesque Inc., Kyoto, Japan), abbreviated herein as
DMEM-FBS.
Cells were cultured in DMEM-FBS at 37 C, in an
atmosphere that contained 5% CO2 . For cellular
experiments, cells were seeded at 2  105 cells/ml
in a six-well multidish (Corning Inc., Corning, NY,
USA) and incubated for 24 h. The medium was then
replaced by ZnO dispersion, with or without 5 mM
AA, this concentration of AA was the no observed
adverse effect level in preliminary examination, and
incubated for another 6 h. The fresh medium was also
replaced for the control cells.
Preparation of the ZnO dispersion for in vitro
evaluations
The ZnO NP powder (10 mg/ml) was dispersed in a
bovine serum albumin (BSA) solution (10 mg/ml;
Nacalai Tesque Inc.) using sonication in an ultrasonic
bath for 30 min with shaking. The ZnO–BSA disper-
sion was serially diluted 10 times with DMEM-FBS
for use in the cellular experiments. The size distribu-
tion of ZnO NPs in the dispersion was measured by
dynamic light scattering (DLS) using a Zetasizer
Nano system (Malvern Instruments Limited,
Malvern, UK).
Measurement of intracellular ROS level
The intracellular ROS level was determined by the
2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA;
Sigma-Aldrich) method. A 5 mM DCFH-DA stock
solution in dimethyl sulfoxide (DMSO) was prepared
and stored at 20 C. When used in an experiment,
this stock solution was diluted 500-fold with serum-
free medium. After exposure of the cells to the ZnO
dispersion with or without AA for 6 h, the medium
was changed to serum-free DMEM that included
Fukui et al.
10 mM DCFH-DA and incubated for 30 min at 37 C.
The cells were then washed with phosphate-buffered
saline (PBS), trypsinized with 0.25% trypsin, washed
again with PBS, and resuspended in 500 ml of PBS.
The cell samples were excited with a 488-nm argon
ion laser in a Cytomics FC500 flow cytometry system
(Beckman Coulter Inc., Brea, CA, USA), and the
emission of 20 ,70 -dichlorofluorescein was recorded
at 525 nm. Data were collected from at least 5000
gated events.
Real-time polymerase chain reaction
The expression of target genes was determined by
real-time polymerase chain reaction (PCR). Total
RNA was isolated from cells using the RNeasy
mini kit (Qiagen GmbH, Hilden, Germany). cDNA
synthesis was carried out with a high-capacity
cDNA reverse transcription kit (Life Technologies
Corp., Carlsbad, CA, USA). Real-time PCR was
conducted with a StepOne real-time PCR system
(Life Technologies Corp.). Gene expression levels
were analyzed by the Ct method. PCR ampli-
fication of cells was analyzed using TaqMan® gene
expression assays (Life Technologies Corp.), with
689
Measurement of intracellular Zn2þ levels
Intracellular Zn2þ was detected using the fluorescent
ZnAF-2 DA reagent (Sekisui Medical Co. Ltd,
Tokyo, Japan) (Hirano et al., 2002). For use in cellular
experiments, the ZnAF-2 DA stock solution (5 mM in
DMSO) was diluted 500-fold with a serum-free
medium. After exposure to the ZnO dispersion for
6 h, with or without AA treatment, the medium was
changed to serum-free DMEM that included 10 mM
ZnAF-2 DA and incubated for 30 min at 37 C. The
cells were then washed with PBS, trypsinized with
0.25% trypsin, washed again with PBS, and resus-
pended in 500 ml of PBS. The cell samples were
excited with a 488-nm argon ion laser in a Cytomics
FC500 flow cytometry system, and the emission of
ZnAF-2 was recorded at 525 nm. Data were collected
from at least 5000 gated events.
The reactivity of AA with ZnO and Zn2þ
To evaluate the reactivity of AAs with ZnO, AA was
dissolved in PBS at a concentration of 40 mM. ZnO
NPs were then added to the AA solution at a concen-
tration of 0, 50, 100, and 1000 mM. The AA absor-
bance at 260 nm was measured using a scanning
spectrophotometer (model UV-3100PC UV–VIS–
the human -actin gene used as an endogenous
control. The gene expression assays for heme
oxygenase-1 (ho-1), interleukin-8 (il-8), and
metallothionein-2 (mt-2) were designated as
Hs01110250_m1, Hs00174103_m1, and
Hs02379661_g1, respectively.
Evaluation of AA uptake in A549 cells
The concentration of AA in A549 cells was measured
using a high-performance liquid chromatography sys-
tem (HPLC) system equipped with a UV detector
(SPD-10AV, 263 nm; Shimadzu, Japan) and NH2 col-
umn (Wakosil 5 NH2, 5 mm, 250  4.6 mm; Wako
Pure Chemical Industries). PBS (40 mM)/methanol
(1/9, v/v) was eluted at a flow rate of 1 ml/min. The
cells were treated with 1 and 5 mM AA for 6 h. After
treatment, the cells were washed with PBS, trypsi-
nized with 0.25% trypsin, washed again with PBS,
and resuspended in 100 ml of PBS. The cells were
then diluted with methanol (1/4, v/v) and mixed vig-
orously for 1 min using a vortex mixer, followed by
centrifugation (20,400  g, 10 min). An aliquot of
the resulting upper layer was injected into the HPLC
system for analysis.
NIR; Shimadzu). Changes in soluble zinc concentra-
tion in the ZnO DMEM-FBS dispersions, with or
without AA, were measured at 0 and 6 h after pre-
paration using 2-(5-bromo-2-pyridylazo)-5-[N-n-pro-
pyl-N-(3-sulfopropyl) amino] phenol disodium salt
dehydrate (5-Br-PAPS; Dojindo Laboratories, Kuma-
moto, Japan). The ZnO NPs dispersions were centri-
fuged at 16,000  g for 20 min, and the supernatant
was carefully collected. After suitable dilution of the
supernatant, the supernatant (400 ml), APS (100 ml,
5 mM), DTCS (100 ml, 1 mM), and 5-Br-PAPS (50 ml,
1 mM) were added to a reaction buffer (2.0 ml, pre-
pared by dissolving 6 g of sodium hydroxide in 500 ml
of 0.5 M HEPES, pH 7.8). The resulting solution was
mixed well, incubated for 10 min at room temperature,
and its absorbance at 555 nm was measured using a
DU530-spectrophotometer (Beckman Coulter Inc.,
Miami, FL, USA). The reactivity of AA with Zn2þ
was evaluated both directly and indirectly. For the
direct measurement, AA was dissolved in PBS at a
concentration of 40 mM. ZnCl2 was then added to the
AA solution at concentrations of 0, 10, 20, 50, 100,
200, 400, and 1000 mM. The absorbance of AA at
260 nm was then measured using the scanning spectro-
photometer. For the indirect measurement, 10 mM
690 Toxicology and Industrial Health 33(9)

ZnCl2 and 50 mM 5-Br-PAPS were dissolved in PBS.

AA was then added to yield concentrations ranging
from 0 to 40 mM. The absorbance of 5-Br-PAPS at
555 nm was then measured using the scanning
spectrophotometer.

Statistical analysis
Data are presented as mean + SD of at least three
separate experiments. Statistical evaluations were
performed via analysis of variance using the Dunnett
test for multiple comparisons. Individual calculation
methods are described in each figure legend.

Results
AA decreased the cytotoxicity caused by ZnO NPs
The secondary particle size of ZnO NPs in the ZnO
water dispersion was measured by DLS. The average
particle size was 90.2 nm (based on the number). The
intracellular ROS levels in A549 cells exposed to ZnO
NPs with or without AA treatment were measured by
the DCFH method (Figure 1). Intracellular ROS lev-
els increased remarkably in cells exposed to ZnO NPs
but not to AA. In contrast, AA treatment was found to
inhibit the ZnO NP-induced increase in intracellular
ROS levels. Further, ZnO NPs were found to induce
expressions of ho-1, a major oxidative stress response
enzyme protein (Figure 2(a)), and il-8, one of the
human chemotactic factors (Figure 2(b)) in the
A549 cells. Interestingly, elevation in these gene
expression levels was not observed in cells exposed
to both ZnO NPs and AA.
AA uptake in A549 cells
To evaluate AA uptake in A549 cells, intracellular
concentration was measured (Figure 3). AA was taken
up by cells following 6 h of treatment, in a dose-
dependent manner.
Effect of AA on intracellular Zn2þ levels
Gene expression of mt-2, which is known to be
induced by metals, in A549 cells, increased following
exposure to ZnO NPs. However, the co-treatment of
ZnO NPs and AA inhibited the increase in mt-2 gene
expression (Figure 4(a)). The Zn2þ level was mea-
sured in cells exposed to ZnO NPs, in the presence
or absence of AA treatment (Figure 4(b)). Exposure to
ZnO NPs significantly increased intracellular Zn2þ
levels, regardless of AA treatment. In particular, Zn2þ
Figure 1. Effect of AA on intracellular ROS level in A549
cells exposed to ZnO NPs. Cells were treated with the
ZnO-DMEM-FBS dispersion with or without AA medium
solution for 6 h. Then the intracellular ROS levels were
measured by the DCFH method using a flow cytometer.
The dispersion included 0.1 mg/ml ZnO NPs and 5 mM AA.
**p < 0.01 (vs. control, Dunnett, ANOVA). AA: ascorbic
acid; ROS: reactive oxygen species; ZnO NP: zinc oxide
nanoparticle; DCFH: 20 ,70 -dichlorofluorescein diacetate;
ZnO-DMEM-FBS: zinc oxide–Dulbecco’s modified Eagle
medium–fetal bovine serum; ANOVA: analysis of variance.
levels in cells treated with AA were remarkably
higher than those in cells not treated with AA.
Reactivity between AA and ZnO or Zn2þ
We examined the reasons for the increase in the intra-
cellular Zn2þ level with AA co-treatment. There are
two possibilities for this result: (1) co-treatment with
AA induced Zn2þ release from ZnO NPs and (2)
co-treatment with AA enhanced cellular uptake of
Zn. To confirm these possibilities, we examined
whether AA accelerates Zn2þ release of ZnO NPs.
We also examined whether AA induces cellular
uptake of Zn2þ. AA exhibits an absorption maximum
in the 244–265 nm range (Nováková et al., 2008). In
this study, we used a slightly broader range (200–290
nm) to measure AA absorption. The interaction
between AA and ZnO NPs was evaluated by measur-
ing AA absorbance at 260 nm (Figure 5(a)). When the
concentration of ZnO NPs was 0 and 1 mM, no sig-
nificant changes were observed in AA absorbance.
There were no remarkable changes in Zn2þ concen-
tration in ZnO DMEM-FBS dispersions with or with-
out AA added 0 and 6 h after preparation (Table 1).
Fukui et al.
Figure 2. Effect of AA on the gene expression of ho-1 and
il-8 in A549 cells exposed to ZnO NPs. Cells were treated
with the ZnO-DMEM-FBS dispersion with or without AA
medium solution for 6-h exposure. Gene expression of
ho-1 (a) and il-8 (b) was measured by real-time PCR after
6 h. The dispersion included 0.1 mg/ml ZnO NPs and 5 mM
AA. **p < 0.01 (vs. control, Dunnett, ANOVA). AA: ascor-
bic acid; ANOVA: analysis of variance; ZnO-DMEM-FBS:
zinc oxide–Dulbecco’s modified Eagle medium–fetal bovine
serum; ho-1: heme oxygenase-1; il-8: interleukin-8.
However, the AA absorbance at 260 nm (and the AA
absorbance curve) responded to changes in Zn 2þ
concentration in PBS buffer (pH 7.0; Figure 5(b) and
(c)). The absorbance of 5-Br-PAPS complexed with
zinc was measured at 555 nm (Figure 5(d)). This
highly sensitive reagent is used to determine zinc
concentration in solution; the 5-Br-PAPS–Zn com-
plex is known to be red in color, with an absorption
maximum at 554 nm (Makino et al., 1982). 5-Br-
PAPS–Zn absorbance was detected at increasing
AA concentrations.
691
Figure 3. Uptake of AA in A549 cells. Cells were treated
with either 1 mM or 5 mM AA medium solutions for 6 h.
The intracellular AA concentration was measured by
HPLC. **p < 0.01, *p < 0.05 (vs. control, Dunnett,
ANOVA). ANOVA: analysis of variance. AA: ascorbic acid;
HPLC: high-performance liquid chromatography.
Discussion
ZnO NPs are known to induce strong oxidative stress
in vivo and in vitro. The oxidative stress triggered by
intratracheal administration of ZnO NPs has been
associated with lung inflammation and injury in a rat
model (Fukui et al., 2012). ZnO NPs release Zn2þ into
the culture media and exhibit increased levels of intra-
cellular ROS levels associated with inflammation and
corresponding tissue damaging effects including cell
death (Cuzzocrea et al., 2000; Heng et al., 2011;
Huang et al., 2010; Kim et al., 2010a, 2010b; Pawlic-
zak, 2003; Sharma et al., 2012; Song et al., 2010;
Tsou et al., 2010; Xia et al., 2011). Thus, Zn2þ release
and intracellular ROS generation are important
predictors of cytotoxicity associated with ZnO NPs
exposure. In order to prevent ZnO NP-induced cyto-
toxicity, inhibition of ROS production must be
addressed.
We previously reported that AA prevents pulmon-
ary lipid peroxidation, inflammation, and injury in rat
lung resulting from intratracheal administration of
ZnO NPs (Fukui et al., 2012). In the present study,
A549 cells exposed to ZnO NPs and treated with AA
exhibited decreased intracellular ROS levels com-
pared to cells not treated with AA. Furthermore,
increases in ho-1 and il-8 gene expressions were also
inhibited by AA treatment. These in vitro results are
in agreement with those previously obtained in vivo.
There are two known AA membrane transporters,
SVCT1 and SVCT2, in human lungs cells (Rivas
et al., 2008; Savini et al., 2008; Tsukaguchi et al.,
692
Figure 4. Effect of the AA on gene expression of mt-2 and
intracellular Zn2þ levels in A549 cells exposed to ZnO
NPs. Cells were treated with the ZnO-DMEM-FBS
dispersion with or without AA medium solution for 6 h.
Gene expression of mt-2 was measured by real-time PCR
after 6-h exposure (a). Next, intracellular Zn2þ level was
measured by the ZnAF-DA using a flow cytometer (b). The
dispersion included 0.1 mg/ml ZnO NPs with or without
5 mM AA. **p < 0.01 (vs. control, Dunnett, ANOVA). AA:
ascorbic acid; mt-2: metallothionein-2; ZnO NPs: zinc
oxide nanoparticles; DMEM-FBS: Dulbecco’s modified
Eagle medium–fetal bovine serum; ANOVA: analysis of
variance.
1999). In this study, AA was readily taken up by A549
cells. Since AA is widely known to be a radical-
scavenging antioxidant and a zinc chelator (Tajmir-
Riahi, 1991), this agent is believed to be an ideal
candidate to prevent the oxidative stress triggered
by ZnO NPs exposure.
Toxicology and Industrial Health 33(9)
The expression of metallothioneins depends on
their binding to specific heavy metals (e.g. zinc, cad-
mium, copper, and mercury) (Kägi et al., 1984). For
example, mt-2 gene expression is highly inducible by
zinc (Karin and Richards, 1984). In the present
study, mt-2 gene expression levels increased in cells
exposed to ZnO NPs. This effect was markedly
inhibited by AA co-treatment. These findings sug-
gest that AA reduced intracellular levels of Zn2þ by
inhibiting the release of Zn2þ from ZnO NPs, most
likely via chelation. However, intracellular Zn2þ
levels significantly increased upon treating the cells
exposed to ZnO NPs with AA. With this result see-
mingly contradicting the above hypothesis, we next
focused on investigating the reaction of AA with
ZnO and Zn2þ.
We considered two possible explanations for the
observed increase in the intracellular Zn2þ level
resulting from AA treatment: increased intracellular
Zn2þ dissolution from ZnO NPs and facilitate intra-
cellular Zn2þ uptake. We believed that if AA directly
acts to promote Zn2þ dissolution from ZnO NPs
inside cells, it would also react with ZnO in culture
medium. Although AA is known to react with Zn2þ, it
is unclear whether AA directly reacts with ZnO.
Wang et al. reported that AA increased the endocy-
tosis processes of ZnO NPs, which resulted from the
increase in concentration of Zn2þ in human gastric
epithelial cells. Moreover, they reported that the acid-
ity of AA facilitates the dissolution rate of the ZnO
NPs in cells. Unquestionably, it becomes the predo-
minating mechanism for the increase of Zn ions,
hence aggravating cytotoxicity (Wang et al., 2014).
To address this question, we confirmed that the absor-
bance of AA at 260 nm was unaffected when ZnO (0–
1000 mM in PBS buffer, pH 7.0) was added. Further,
the concentration of Zn2þ in ZnO DMEM-FBS dis-
persions with AA was unchanged after 6 h. These data
suggest that AA does not react with ZnO NP dissolu-
tions in the culture medium. Thus, the extracellular or
cytoplasmic Zn2þ dissolution from ZnO NPs is most
likely not augmented by AA. However, AA absor-
bance at 260 nm increased in accordance with Zn2þ
concentration in PBS. Similarly, Maniyar et al. (2012)
reported that the absorbance of AA in solution con-
taining nickel sulfate at pH 7.0 and 7.4 was higher
than the absorbance of AA alone. Thus, the absor-
bance of an AA–metal chelate complex is most likely
more pronounced than that of unchelated AA at the
culture medium pH. Our results therefore suggest the
latter explanation of increased intracellular Zn2þ
Fukui et al. 693

Figure 5. The reactivity of AA with ZnO NPs and Zn2þ. The PBS contained 40 mM AA and ZnO NPs or ZnCl2. The
concentrations of ZnO were 0, 50, 100, and 1000 mM in PBS. The absorbance of AA at 260 nm in PBS was measured by a
spectrofluorophotometer (a). The concentrations of ZnCl2 were 0, 10, 20, 50, 100, 200, 400, and 1000 mM in PBS. The
absorbance of AA at 260 nm in PBS (pH 7.0) was measured by a spectrofluorophotometer (b). Change in the AA
absorbance curve against Zn2þ concentration in PBS (pH 7.0) (c). Reactivity between AA and zinc combined with
5-Br-PAPS (d). The PBS contained 10 mM Zn, and 50 mM 5-Br-PAPS and AA. The absorbance of 5-Br-PAPS at 555 nm was
measured by a spectrofluorophotometer. AA: ascorbic acid; ZnO NPs: zinc oxide nanoparticles; PBS: phosphate-buffered
saline; 5-Br-PAPS: 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl) amino] phenol disodium salt dehydrate.

concentration to be the most likely that AA chelates Table 1. Soluble zinc concentration changes in ZnO NPs
extracellular Zn2þ released from ZnO NPs and the DMEM-FBS dispersion.a
resulting Zn–AA complexes become incorporated
Concentration
into cells. Intracellular Zn2þ can also result from ZnO of Zn2þ (mg/ml)
NPs becoming incorporated into cells and their sub-
sequent release of ZnO. While intracellular Zn2þ is 0h 6h
the source of ZnO NP-induced cytotoxicity, its dele-
ZnO DMEM-FBS dispersion 17.8 14.6
terious effects decrease when AA forms Zn–AA com-
SD 0.616 0.407
plexes inside cells. However, the effect of the AA on
ZnO DMEM-FBS dispersion with 16.46 15.00
cell death caused by ZnO NPs was not able to evaluate
adding 5 mM AA
because influence of the Zn2þ was too large.
SD 0.782 0.481
Zinc has an important role in antioxidant mechan-
isms in vivo. AA has been reported to enhance the ZnO NPs: zinc oxide nanoparticles; DMEM-FBS: Dulbecco’s mod-
uptake of Zn2þ during oxidative stress and suppress ified Eagle medium–fetal bovine serum.
a
The soluble zinc concentration in the dispersions included
its uptake when Zn2þ deficiencies are detected (Agte 0.1 mg/ml of ZnO NPs with or without 5 mM AA at measured
et al., 2004). In this study, the cellular uptake of Zn2þ at 0 and 6 h after preparation. The absorbance of 5-Br-PAPS at
might be accelerated with the induction of oxidative 555 nm was measured by a spectrofluorophotometer.
694
stress caused by Zn2þ. The chelation of Zn2þ with AA
most likely inhibits oxidative stress and inflamma-
tion, regardless of the increase in intracellular uptake
of Zn2þ. Concentration of free Zinc is likely low in
these cells.
The Zn–AA complexes accumulated in the in vitro
situation, while these complexes may be eliminated
from the lung in the in vivo situation. Metal chelate
complexes are known to be mobilized into urine or
feces by chelating agents in vivo (Andersen, 1989;
Lihm et al., 2013).
Conclusion
In conclusion, the oxidative stress and inflammatory
response induced by ZnO NPs were prevented by
co-treatment with AA in human lung carcinoma
A549 cells. These results are consistent with those
previously reported from an in vivo model. Two pos-
sibilities can be considered regarding the reduced
cytotoxicity effect of AA. One possibility is that intra-
cellular ROS generated by ZnO NPs were removed by
AA. Another possibility is that Zn2þ was trapped by
AA as a metal-chelator antioxidant effect and/or the
chelating effect may be involved in the ZnO NP-
induced oxidative stress and inflammation reduction
by AA. In order to ensure the role of AA as a metal
chelator and antioxidant, it is necessary to evaluate
the protective effect of other metal chelators and anti-
oxidants. These findings contribute to our continued
efforts toward the prevention and treatment of
nanoparticle-induced toxicity.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest
with respect to the research, authorship, and/or publication
of this article.
Funding
The author(s) disclosed receipt of the following financial
support for the research, authorship, and/or publication of
this article: This study was partially supported by the Long-
range Research Initiative program organized by the Japan
Chemical Industry Association.
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