Biol Trace Elem Res (2017) 175:360–366
DOI 10.1007/s12011-016-0770-8
Exposure to Zinc Oxide Nanoparticles Induces Neurotoxicity
and Proinflammatory Response: Amelioration by Hesperidin
Sabah Ansar 1 & Manal Abudawood 1 & Sherifa Shaker Hamed 2,3 & Mukhtar M. Aleem 4
Received: 9 May 2016 / Accepted: 30 May 2016 / Published online: 14 June 2016
# Springer Science+Business Media New York 2016
Abstract Zinc oxide nanoparticles (ZnONPs) are widely
used in food packaging and may enter the body directly if
exposed. Hereby, in this study, the oral administration was
selected as the route of exposure for rats to nanoparticles
and the effect of hesperidin (HSP, 100 mg/kg bwt) was eval-
uated on ZnONP (600 mg/kg bwt)-induced neurotoxicity in
rats. ZnONPs were characterized using transmission electron
microscopy. Neurotoxicity was observed as seen by elevation
in serum inflammatory markers including tumor necrosis fac-
tor alpha (TNF-α), interleukin 1 (IL-1β), interleukin-6 (IL-6),
C-reactive protein (CRP), and activities of catalase (CAT),
glutathione peroxidase (GPx), glutathione reductase (GR),
and glutathione (GSH) content in rat brains. Pretreatment of
rats with HSP in ZnONP-treated group elevated activities of
antioxidant enzymes. HSP also caused decrease in TNF-α, IL-
1β, IL-6, and CRP levels which was higher in the ZnONP-
treated group. The results suggest that HSP augments antiox-
idant defense with anti-inflammatory response against
ZnONP-induced neurotoxicity. The increased antioxidant en-
zymes enhance the antioxidant potential to reduce oxidative
stress.
* Sabah Ansar
sabi787@yahoo.com
1
Clinical Laboratory Sciences, Applied Medical Science, King Saud
University, Riyadh, Saudi Arabia
2
Zoology Department, College of Science, King Saud University,
Riyadh, Saudi Arabia
3
Zoology Department, Faculty of Science, University of Alexandria,
Moharram Bey, Alexandria 21511, Egypt
4
Chemistry and Biochemistry Department, University of California,
Santa Cruz, CA, USA
Keywords Zinc oxide nanoparticles (ZnONP) . Tumor
necrosis factor alpha (TNF-α) . Interleukin-6 (IL-6) .
Neurotoxicity
Introduction
Zinc is known to be an important trace element in the body
and is involved in preventing free radical formation,
protecting biological structures from damage, and correcting
the immune functions. Zinc oxide nanoparticles (ZnONPs) are
known to have antimicrobial properties and are widely utilized
in many products, such as toothpaste, beauty agents, wall
paints, building materials, sunscreens, and textiles. Their
microbial selectivity, stability and low cost make them a
possible alternative to silver nanoparticles (NPs) or antimicro-
bial peptides. ZnONPs are extensively used in the industrial
field, and may have exposure to humans via several possible
routes including inhalation or ingestion or uptake by the
gastrointestinal tract. Recently, there have been reports that
NPs can reach the brain via blood–brain barrier (BBB)
penetration and subsequently cause damage by the induction
of oxidative stress, inflammatory responses, and cytotoxicity
[1–4]. It has also been found that ZnONPs could reach the
brain after oral administration in animals or translocation
along the olfactory nerve pathway [5, 6] and may induce the
changes in the spatial learning and memory ability of rats by
altering the synaptic plasticity [7], and induce toxic effects in
the blood and brain [8]. Earlier, ZnONPs have shown toxicity
in mice brain tumor cell lines (Neuro-2a) when compared with
similar sized particles of Al2O3, TiO2, Fe3O4, and CrO3 [9]. In
recent years, an increasing number of investigations have
indicated that particulate matter (PM) and NPs in the
environment may represent important risk factors for
Protection against ZnONP-Induced Neurotoxicity
neurodegenerative diseases [10] including Parkinson’s
disease, Huntington’s disease, and Alzheimer’s disease.
Additional risks can arise while washing off cosmetics and
sunscreens into sinks and showers that flow into wastewater.
The environmental impacts of these nanoparticles include
damage to ecosystems and the ability to bioaccumulate with
the food chain. Engineered nanoparticles are detected in the
water supply due to environmental release of them within the
industrial and domestic discharge [11, 12]. They have wide-
scale applications but may induce toxicity due to their
increased surface reactivity which leads to their toxicity and
may produce reactive oxygen species [13–15]. Recent studies
showed toxicity of zinc oxide nanoparticles and related
alteration in some hemato-biochemical parameters [16–18].
A number of agents which are included in our diet afford
protection against the onset of various diseases including
cancer and counteract the increased amount of oxidants
generated by toxicants. Hesperidin (HSP, 3,5,7-trihydroxy-4-
methoxy-flavanone-7-rhamnoglucoside), largely isolated
from citrus fruits and a member of the flavanone group,
exhibits antioxidative, anti-inflammatory [19, 20],
antihypercholesterolemic [21, 22], and antihyperglycemic
[23] activities. In recent studies, beneficial effects of hesperi-
din following toxicity by some compounds have been shown
[24–28]. Also, HSP significantly protected against hepatotox-
icity induced by lipopolysaccharide [29], cadmium [30],
acetaminophen [31], and carbon tetrachloride [32] in rats.
Henceforth, the objective of this study was to investigate the
protective effect of hesperidin against ZnONP-induced
oxidative stress and neurotoxicity.
Materials and Methods
Chemicals
Hesperidin powder and ZnONP were purchased from Sigma-
Aldrich, USA. All other chemicals used in the study were of
analytical grade, and were from Sigma and Merck.
Nanoparticles Characterization Technique
In this study, the ZnO nanoparticles powder was approximate-
ly 50 nm. For transmission electron microscope (TEM), the
powder was firstly put in ethanol, and the ultrasonic dispersed
solution was dropped on a copper net.
Animals
Male wistar rats weighing about 200-220 gm were kept for a
month in animal house prior to experiments. The institutional
ethics committee approved the experimental protocols and
was carried out in compliance with the declarations of
361
National Research Council [33]. All the animals used in this
study were placed in cages in an air-conditioned room main-
tained with a 12-h light/dark cycle. Twenty-four normal
healthy rats were used in this study. The rats were divided into
four groups, each consisting of six animals as the following
groups: (1) control rats (vehicle), (2) rats that received oral
administration of 600 mg/kg of ZnONPs, (3) rats that received
oral administration of 100 mg/kg of HSP, and (4) rats that
received ZnONP 1 h after pre-administration of HSP for
7 days. Oral administration took place by gavage. The exper-
imental duration was 7 days. The dose of hesperidin and
ZnONPs used in the present study were in accordance with
previous reports, respectively [31, 34]. After 24 h of last treat-
ment, the animals were sacrificed and blood samples were
collected through heart puncture without an anticoagulant.
After 30 min at room temperature, the blood samples were
centrifuged at 2000×g for 15 min at 4 °C. The serum was
subsequently transferred to clean test tubes and stored at
−80 °C.
After the rats were euthanized, brain tissues were homog-
enized in an ice bath with physiological saline. The homoge-
nates were centrifuged at 3000 rpm for 10 min at 4 °C. The
supernatants were collected and assayed for oxidative bio-
markers. The protein contents were determined using the
Bradford method with bovine serum albumin (BSA) as the
standard substance.
Cytokines Assay
BTumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1β),
interleukin-6 (IL-6), and C-reactive protein (CRP) levels were
determined by the sandwich ELISA method using a commer-
cially available kit from Pierce-Endogen (Rockford, IL). All
appropriate controls and standards as specified by the manu-
facturer’s kit were used; the data are expressed as picograms
per milliliter plasma. In the cytokine assay, control samples
were analyzed each time to check the variation from plate to
plate on different days of analyses. Protein content of the
supernatant was determined [35] using bovine serum albumin
as standard^.
Measurement of Glutathione, Glutathione Peroxidase,
Catalase, and Glutathione Reductase
For enzymatic antioxidant status, tissue was used for the de-
termination of glutathione (GSH) by the method of [36], cat-
alase activity (CAT) [37], glutathione peroxidase activity
(GPx) [38], and glutathione reductase activity (GR) [39].
GPx activity was measured using H2O2 as substrate. The re-
action was monitored indirectly as the oxidation rate of
NADPH at 240 nm for 3 min.
362
Malondialdehyde and Superoxide Dismutase
Measurements
BThe oxidant–antioxidant status of the rat was assessed by
determining the level of malondialdehyde (MDA) and the
activity of superoxide dismutase (SOD). Lipid peroxidation
was determined by measuring the level of MDA, which is
considered to be a standard marker for oxidative lipid damage.
The activity of SOD indicates the level of antioxidation in
serum [40, 41]. MDA levels were measured by the method
of Draper and Hadley [40]. The results were expressed as
micromoles per liter. Serum SOD activity was measured by
the method of Sun et al. [41]. The results were expressed as
units per liter^.
Statistical Analysis
BResults were analyzed using SPSS software and expressed as
the mean ± standard error of the mean (SEM). One-way
ANOVA of variance was applied to test for the significance
of biochemical data of the different groups. Results were con-
sidered significant when p ≤ 0.05^.
Results
Characterization of ZnO Nanoparticles
To examine the size and morphology of oxide nanocrystals,
transmission electron microscopy was used (Fig. 1).
Transmission electron microscopic image revealed ZnO nano-
particles formation at different magnifications and confirmed
the hexagonal plane to the prepared nanoparticles.
Effect of nZnO on Total Bodyweight of Rats
Concerning the total body weight, there was no significant
difference in the total body weight between different treated
groups compared with the control group.
Fig. 1 Transmission electron microscopy (TEM) image of synthesized
ZnO nanoparticles
Ansar et al.
Effect of HSP on nZnO-Induced Neurotoxicity
by Modulating the Antioxidant Defense System
ZnONP treatment led to decrease in the glutathione (GSH)
levels as shown in Fig. 2 However, pretreatment with HSP
increased levels of GSH as compared to group II. Antioxidant
enzyme activities of CAT, GPx, and GR between the HSP
treated, ZnONP treated, and HSP + ZnONP were compared
to the control group (Table 1). There was significant decrease
in the given enzymatic activities in the ZnO-treated groups
compared to the control group. However, all enzyme activities
were significantly increased in the HSP plus ZnONP-treated
group compared to the ZnONP-treated group II (P < 0.05).
Effect of HSP on nZnO-Induced Superoxide Dismutase
and Malonialdehyde Levels
Malonialdehyde (MDA) and superoxide dismutase (SOD)
levels were measured in brain tissue. MDA levels were higher,
and SOD levels were lower in the ZnONP-treated group
(p < 0.05 for MDA and p < 0.05 for SOD) when compared
with those of the control group and recovery was more signif-
icant in the HSP-pretreated group IV (p < 0.01 for MDA and
p < 0.01 for SOD) as compared to the ZnONP-treated group
II. However, there were no significant differences between the
same parameters in the HSP-alone-treated group III and con-
trol group I (Fig. 3).
Effect of HSP on nZnO-Induced Proinflammatory
Response
ZnONPs effect on the inflammatory markers IL-1β, IL-6,
TNF-α, and CRP in the serum is shown in Fig. 4(a-d). As
compared to group 1, treatment of ZnONP to rats resulted in
elevation of these biomarkers (p<0.05). Comparing group IV
1.0
0.8
0.6
#
0.4
*
0.2
0.0
Fig. 2 Effect HSP on ZnONP-induced glutathione level. The data
represent means ± SEM. *Significant change at p < 0.05 with respect to
the control group. #Significant change at p < 0.05 with respect to the
ZnONP group
Protection against ZnONP-Induced Neurotoxicity
Table 1 Effect of HSP on nZnO-
induced antioxidant status in Parameters
brain tissue
CAT (U/mg prot)
GPx (U/mg prot)
GR (nmol/mg prot)
All values are mean ± SEM, n = 6. I vs. II (*P < 0.05). II vs. IV (**P < 0.05)
with group II, pre-treatment of HSP led to decrease in their
levels (p<0.05). HSP (group III) did not affect the levels of
these markers when compared to group I rats.
Discussion
As extensive application of ZnO nanoparticles in the industrial
field increases, nanotechnology and nanoparticles have be-
come a risk factor for neuropathological and toxicological
processes. Earlier it has been shown that release of ionic
Zn2+ is responsible for the toxicity of ZnO nanowires in mac-
rophages is due to intracellular dissolution. [42]. Because of
their special properties including small size and high specific
surface area, the biological safety of nanomaterials has re-
ceived wide attention. Furthermore, ZnONPs have been
regarded as a possible treatment for cancer and autoimmune
diseases [43]. In the past, toxicity of ZnONPs has been exten-
sively studied in different animal systems and cell types as
ZnONPs could reach various organs after systemic distribu-
tion, and induce toxicity on the blood and other organs
[44–48]. Also, the cytotoxicities have also been present in
many cultured cells such as epidermal cells, macrophages,
human lung epithelial cells, CHO cells, and vascular endothe-
lial cells [43, 49, 50].
The results of the current study indicate that the ZnONPs-
treated group decreased levels of GSH and antioxidant en-
zyme activities (Cat, GPx, and GR). However, supplementa-
tion of hesperidin (HSP) to the ZnONP-treated group in-
creased GSH levels and antioxidant enzyme activities. This
could be explained by the major role of natural antioxidants in
inhibiting toxicity and in protecting the tissues and cells.
Fig. 3 Superoxide dismutase
(SOD) and malondialdehyde
(MDA) levels in the brain. a SOD
level b MDA level. The data
represent means ± SEM.
*Significant change at p < 0.05
with respect to the control group.
#
Significant change at p < 0.05
with respect to the nZnO group
363
Experimental groups
Control (I) ZnONP (II) HSP (III) HSP + ZnONP (IV)
0.38 ± 0.023 0.25 ± 0.015* 0.37 ± 0.021 0.32 ± 0.011**
0.78 ± 0.077 0.33 ± 0.017* 0.71 ± 0.056 0.59 ± 0.063**
10.11 ± 3.21 5.34 ± 0.16 * 10.14 ± 5.36 8.31 ± 1.13 **
Earlier antioxidant and anti-inflammatory activities, antican-
cer as well as vascular protection properties of HSP, have been
reported [27, 28, 51, 52]. Bioflavonoid like HSP may inhibit
induction of antioxidant enzyme activities and may play a role
in tissue protection. Neuroprotective effect of hesperidin on
aluminum chloride induced Alzheimer’s disease in Wistar rats
has been shown earlier [53]. Protective activities of HSP
against microbes and several toxicities induced by oxidants,
chemicals, toxins, chemotherapy, and radiotherapy agents has
been shown earlier in in vitro and in vivo studies [54].
The rise in MDA and decrease in SOD in ZnONPs-treated
rats as observed in this study could be due to the increased
generation of reactive oxygen species and the excessive oxi-
dative damage generated in rats. MDA content manifests the
level of lipid peroxidation and represents the level of damage
of the cell and tissue. The elevated level of MDA could be due
to enhanced formation of free radicals. However, decrease in
MDA and increase in SOD levels was observed in the HSP-
pretreated group. This could be attributed to the antioxidant
properties of HSP against oxidative stress which is consistent
with other studies that have reported protective role of HSP
against oxidative stress [55–59]. HSP also inhibits oxidative
stress and inflammation in ultraviolet B irradiation-induced
skin damage in mice [60].
In this study, hesperidin shows desired protective activity
as the cytokine levels were reduced compared with the
ZnONP treated group with alleviation in TNF α, IL-1β, IL-
6, and CRP levels. Zinc oxide particles induce inflammatory
responses in vascular endothelial cells via NF-kappaB signal-
ing has been shown earlier [61]. Also, zinc oxide elevated
circulating levels of different cytokines at low concentrations
of [62]. This is in accordance with the present study as
364
Fig. 4 Effect of HSP on ZnONP-induced inflammatory markers in the
brain compared with the respective control groups a IL-1β level, b IL-6
level, c TNF-α level, and d CRP level. The data represent means ± SEM.
ZnONPs significantly elevated cytokines levels in rat serum.
ZnONP also decreases the activity of human SHSY5Y neu-
ronal cells and mice neural stem cells by inducing DNA dam-
age and cell apoptosis [63].
Results in this study show that HSP can ameliorate
ZnONP-induced neurotoxicity, and may be useful in the tox-
icity prevention. Similarly, recent study also suggested that
HSP pretreatment protects mice via inhibiting the production
of TNF-alpha and IL-6 [64]. Elevation of CRP level post
ZnONP administration compared with the normal control
group in the current work was in accordance with previous
studies [65]. CRP is related to the incidence of many patho-
logical conditions as coronary heart disease, hypertension, and
inflammation. However, HSP treatment reduced CRP level
compared with the ZnONP-intoxicated group. These results
suggest that HSP also has an anti-inflammatory effect as evi-
dent by significant decrease in elevated inflammatory cyto-
kine levels, including TNF α, IL-1β, IL-6, and CRP, in rat
serum.
Conclusion
ZnO nanoparticles have the ability to induce various alter-
ations in brain tissue, indicating neurotoxicity presumably
by oxidative stress. In this study, ZnONPs lead to neurotoxic-
ity has been demonstrated. This was evidenced from the ele-
vation in inflammatory cytokines and decreased glutathione
and antioxidant levels. Hesperidin proved to be beneficial in
Ansar et al.
*Significant change at p < 0.05 with respect to the control group.
#
Significant change at p < 0.05 with respect to the nZnO group
attenuating the ZnONP-induced oxidative toxicity. Results in
this study suggest that HSP can be used as a neuroprotective
agent against ZnONP-induced neurotoxicity.
Acknowledgments This research project was supported by the
BResearch Center of the Center for Female Scientific and Medical
Colleges^, Deanship of Scientific Research, King Saud University,
Riyadh, Saudi Arabia. Also, we would like to thank Ms. Jennifer
Hudson and Ms. Sarah Paulson for their help in completion of the study.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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