European Review for Medical and Pharmacological Sciences 2014; 18: 1813-1828

Prophylactic role of α-lipoic acid and vitamin E

against zinc oxide nanoparticles induced
metabolic and immune disorders in rat’s liver
N.M. AL-RASHEED1,2, N.M. AL-RASHEED1, N.A. ABDEL BAKY1, L.M. FADDAH1,
A.J. FATANI1, I.H. HASAN1, R.A. MOHAMAD3
1
Pharmacology Department, Faculty of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia
2
Pharmacology Department, College of Pharmacy, Princess Nora Bint Abdul Rahman University,
Kingdom of Saudi Arabia
3
Department of Anatomy, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia

Abstract. – OBJECTIVES: Potential health Introduction

hazard is associated with the wide use of
nanoparticles. The prophylactic role of either α-
α-lip) or vitamin E (vit E) against the
lipoic acid (α
toxic effect of zinc oxide nano-particles (ZnO-
NPs) induced metabolic disorder, inflammation
and DNA damage in rat livers was studied.
MATERIALS AND METHODS: ZnO-NPs were
administered orally using two doses (600 mg
and 1 g/kg body weight/day for 5 conscutive
days). Some biomarkers of tissue damage,
metabolic disorder, and DNA damage were in-
vestigated to explore the protective mecha-
nisms of α-lip or vit E against ZnO-NPs induced
hepatotoxicity.
RESULTS: Co-administration of either α -lip
(200 mg/kg body weight) or vit E (100 mg/kg
body weight) daily for three weeks to ZnO-NPs
intoxicated rats, significantly down-modulated
the marked increase in serum ALT (marker of liv-
er damage) and also serum glucose level (mark-
er of metabolic disorder) as well as the pro-in-
flammatory biomarkers including nitric oxide
α (TNF-α α), inter-
(NO), tumor necrosis factor-α
leukin-6 (IL-6), C-reactive protein (CRP), and im-
munoglobin G (IGg). Reduced glutathione level
was decreased while caspase3 level was elevat-
ed in liver tissues of ZnO-NPs treated group
compared with intoxicated one . Moreover
histopathological examination of liver tissue
supported the previous biochemical markers.
Furthermore, ZnO-NPs induced hepatic oxidative
DNA damage.
CONCLUSIONS: Either α-lip or vit E proved
to be hepatoprotective agents against ZnO-
NPs toxicity because they ameliorated meta-
bolic and immune disorders related to liver
damage and modulated the previous measured
parameters.
Key words:
α-lipoic acid, Vitamin E, Zinc oxide nano-particles, De-
oxyribonucleic acid, Tumor necrosis factor-α.
In the last decade, several epidemiological
studies have found high associations of ambient
ultrafine particles (d < 100 nm) with respiratory
and cardiovascular diseases of humans1,2. It has
demonstrated that nanoparticles, may cause more
inflammation than larger particles of the same
materials at a same mass dose delivery3-5.
Nanoparticles are conceived to stick to cell
membrane, get inside specific cells in the body,
and pass through cells. The surfaces of nanoparti-
cles are sometimes also modified to avoid being
recognized and eliminated by the immune
system6. Due to their small size, nanoparticles can
translocate from these entry portals into the circu-
latory and lymphatic systems, and ultimately to
body tissues and organs. Some nanoparticles, de-
pending on their composition and size, can pro-
duce irreversible damage to cells by oxidative
stress and/or organelle injury7,8. Translocation to
other organs, such as liver, spleen, brain, heart and
kidney may lead to disfunction of these organs.
Exposure to some nanoparticles is associated to
the occurrence of autoimmune diseases9. It also
has been proposed that the size of nanoparticles
surface area greatly increases their ability to pro-
duce reactive oxygen species (ROS)10,11.
Inflammation is a complex, concerted group of
responses that, although defensive against infec-
tion, is harmful when induced chronically by en-
vironmental stimuli such as inhaled particles12,14.
Due to their unique properties and diverse nanos-
tructures, zinc oxide nano-particles (ZnO-NPs)
are widely applied in optoelectronics, cosmetics,
catalysts, ceramics, pigments, etc.15,16. However,
toxicological studies indicated that ZnO-NPs had
adverse impacts on human health and environ-

Corresponding Author: Iman H. Hasan, MD; e-mail: ihasan@ksu.edu.sa 1813

N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad
mental species. The bio-safety of ZnO-NPs is
still a controversial issue.
Recently, in vivo cell experiments showed that
exposure to ZnO-NPs resulted in oxidative dam-
age and inflammation response in vascular/lung
endothelial cells17,18. Animal experiments indicat-
ed that liver, spleen, heart, pancreas, and bone
were target organs of oral exposure to 20- and
120-nm ZnO19. Compared with the conventional
toxicology, the dose of ZnO-NPs is no longer a
sole factor in evaluating the toxicity of nanoparti-
cles, but the physicochemical properties of its
nanoparticles, such as size, shape, chemical com-
position, aggregation, high specific surface area
and its solubility may play a more important role
in its toxicity20-22.
A central feature of the pathophysiology of in-
flammation is the production of multiple proin-
flammatory mediators such as the proinflamma-
tory transcription nuclear factor-kappa B (NF-
kB). Specifically, reactive oxygen species (ROS),
reactive nitrogen species (RNS), cytokines and
chemokine’s23. Although these proinflammatory
mediators are required for an adequate host-de-
fense response, dysregulation of their production
can lead to cardiovascular hyporeactivity, in-
travascular coagulation, multiple organ failure,
and death24. The possible role of naturally occur-
ring antioxidants in protection against inflamma-
tory response and oxidative damage have been
describe25,26.
α-lipoic acid (α-lip) is a natural dithiol com-
pound which is known to be a co-factor in α-ke-
toacid dehydrogenase mitochondrial complex and
for its potent antioxidant properties. The antioxi-
dant effects of α-lip are attributed to direct radical
scavenging and metal chelation25. α-lip is an ef-
fective antidote against heavy metal toxicity27. α-
lip has high redox potential and can regenerate
antioxidants (such as vitamin E, vitamin C and
GSH) from their radical or inactive form. A sub-
stantial inhibition of hexavalent chromium, Cr
(6+), induced DNA damage by α-lip has been re-
ported 28. α-lip prevents vascular endothelial
growth factor (VEGF) upregulation29. α-lip was
also, shown to inhibit linoleic acid-induced apop-
tosis in human aortic endothelial cells, and data
obtained in rats suggest that its beneficial effects
on vascular dysfunction in the aorta and retina
could relate to its antiapoptotic activity30. It also
found to be effective against oxidative tissue in-
jury26, including its possible role as a chemopre-
ventive agent in inflammation-associated tumori-
genesis and colitis26,31, and hepatic disorder32.
1814
Based on its lipophilicity, vit E is considered to
be the major chain-breaking antioxidant prevent-
ing the propagation of oxidative stress, especially
in biological membranes33. Vit E specifically α-
tocopherol has been reported to be able to modu-
late signal transduction and gene expression via
its antioxidant and non-antioxidant properties34.
Also, previous study demonstrates the anti-in-
flammatory and anti-carcinogenic activities of vit
E in the lung and colon suggesting these activities
is associated with the reduction of oxidative dam-
age, trapping of reactive nitrogen species35. All to-
copherols are effective antioxidants, known to
trap reactive nitrogen species and is beneficial as
anti-inflammatory agents36. Additional studies are
shown its immuno-regulatory functions during In-
flammation37, reducing damaged DNA and sup-
port normal cell division38.
The aim of this work is to investigate the potential
hepatoprotective role of α-lip and vit E against the
metabolic disorder, inflammation and DNA damage
induced by toxicity of ZnO-NPs in rat liver.
Materials and methods
Chemicals
All chemicals used were of high analytical
grade, product of Sigma and Merck companies.
Kits used for the quantitative determination of
different parameters were purchased from Sigma
Aldrich Biogamma, Stanbio, Germany. 50-nm
ZnO powders were purchased from Sigma Co.,
St. Louis, MO, USA.
Animals and treatments
Fifty Wistar albino rats weighing 180-200 g were
used. The rats were obtained from Experimental
Animal Care Center, College of Pharmacy, King
Saud University. Animals have been kept in special
cages, and maintained on a constant 12-h light/12-h
dark cycle with air conditioning and temperature
ranging 20-22 °C and humidity (60%). Rats were
fed with standard rat pellet chow with free access to
tap water ad libitum for one week before the experi-
ment for acclimatization. Animal utilization proto-
cols were performed in accordance with the guide-
lines provided by the Experimental Animal Labora-
tory and approved by the Animal Care and Use
Committee of the King Saud University, College of
Pharmacy. After one week acclimation, the rats
were kept fasting over night before treatment and
randomly divided into two classes according to the
dose of ZnO-NPs administered to rats.
 Prophylactic role of α-lipoic acid and vitamin E
Class I, consists of five groups (each of ten rats):
G1: Normal healthy animals
G2-G4 groups of animals administered orally
600 mg/kg body weight/day ZnO-NPs for 5
constitutive days39 and divided as follow
G2: ZnO-NPs-intoxicated animals with oral low
dose (600 mg/kg/day) daily for 5 constitutive
days.
G3: ZnO-NPs-intoxicated animals co-adminis-
tered α-lip (200 mg/kg) daily40
G4: ZnO-NPs-intoxicated animals co-adminis-
tered vit E (100 mg/kg) daily40
Class II consists of 4 groups (G5-G7), each of
ten rats, administered orally 1 g/kg body
weight/day ZnO-NPs for 5 constitutive days39
and divided as follow
G5: ZnO-NPs-treated animals with oral high
dose (1 g/kg/day) daily for 5 days
G6: ZnO-NPs-intoxicated animals co-adminis-
tered α lip (200 mg/kg) daily
G7: ZnO-NPs-intoxicated animals co-adminis-
tered vit E (100 mg/kg) daily
α-lip and vit E were orally administered daily
for three constitutive weeks from the beginning
of the experiment. Three weeks later, and after
24 hour of the last dose administration, rats were
fasted overnight then sacrificed and the blood
was collected. Serum was separated by centrifu-
gation at 3000 r.p.m. for 10 minutes and kept at –
80 °C for different biochemical estimations. Liv-
er were harvested rinsed in cold isotonic saline,
homogenized, and frozen at −80°C for COMET
assay. Three livers were put in 4% formalin for
histopathological examination.
Biochemical serum assay
ALT was estimated using Stanbio kit produced
by Stanbio Labs (Boerne, TX, USA). TNF-α
was quantified using a commercial ELISA kit
(Endogen, Woburn, MA, USA).
Fasting blood glucose was measured according
to method adopted previously by41 using a glu-
cose kit (enzymatic method) (Wako Diagnos-
tics, Mountain View, CA, USA).
TNF-α in serum was determined using commer-
cially available ELISA assays following the in-
structions supplied by the manufacturer (DuoSet
kits, R&D Systems; Minneapolis, MN, USA).
The results are shown as pg of cytokine per mL.
IL-6 was measured by ultra-sensitive ELISA
(Quantikine HS Human IL-6 Immunoassay;
R&D Systems, Minneapolis, MN, USA) with
an analytical CV of 6.3% and a detection level
of 0.04 pg/mL
CRP was measured with latex-enhanced im-
munonephelometry on a Behring BN II Neph-
elometer (Dade Behring). In this assay, poly-
styrene beads coated with rat monoclonal an-
tibodies bind CRP present in the serum sam-
ple and form aggregates. The intensity of the
scattered light is proportional to the size of
the aggregates and thus reflects concentration
of CRP present in the sample. The intra-assay
and interassay coefficients of variation for
CRP were 3.3% and 3.2%, respectively. The
lower detection limit of the assay was 0.15
mg/L42.
IgG level was measured in serum using (ELISA).
1 µg/ml of goat anti-rat IgG (Kirkegaard & Per-
ry Laboratories, Inc., Gaithersberg, MD, USA)
was used as capture antibodies. Standards were
prepared from rat IgG (Sigma Chemical Co., St.
Louis, MO, USA) Goat anti-rat IgG peroxidase
conjugates diluted 1:250 in PBS/BSA (from
Kirkegaard & Perry Laboratories, Inc.,
Gaithersberg, MD, USA) were used as detect-
ing antibodies.
Biochemical assay of liver tissue
Determination of hepatic reduced
glutathione (GSH) level
Liver tissue levels of acid-soluble thiols and re-
duced GSH were determined colorimetrically at
412 nm43. Homogenates were precipitated with
trichloroacetic acid, and after centrifugation, su-
pernatants were used for the estimation of protein
thiols (Protein-SH) expressed as µmol/g tissue.
Determination of hepatic caspase 3 level
Caspase 3-like protease was assayed according
to the method described by Vaculova and Zhivo-
tovsky, 200844.
Comet analysis
The alkaline comet assay was performed ac-
cording to45 the parameters measured to analyze
the electrophoretic patterns were: tail length
measured from the middle of the head to the end
of the tail, relative DNA content in the tail.
Histopathological examinate
Samples of liver tissues were collected, and
were fixed in a 4% formaldehyde solution for 24
h. They were dehydrated in ascending grades of
ethyl alcohol, cleared in xylene, and embedded in
paraffin. Paraffin blocks were cut at 4-µm thick-
1815
N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad

Table I. Protective effect of α-lip and vit E on serum ALT of intoxicated rats with either low or high doses of ZnO-NPs.

Groups High dose ZnO-NPs Low dose ZnO-NPss

Normal 126.06 ±1.05a 126.06 ± 1.05a

ZnO- NPs 138.04 ± 0.8 139.8 ± 1.2
α-lip 126.2 ±1.3a,n,c 133.1 ± 0.7a,*,b
vit E 129.9 ± 0.6a,b 127.4 ± 0.9a,n

Data are presented as mean ± SE of 6 rats, ap ≤ 0.001, compared with ZnO-NPs intoxicated group, nnon significance compared with
normal group, *p ≤ 0.01, compared with normal group, bp ≤ 0.05, compared with vit E group, cp ≤ 0.001, compared with vit E group.

ness, then fixed on glass slides. Sections were hematoxylin was added to stain the nuclei, and af-
stained with hematoxylin and eosin (H&E) by the terwards eosin was added to the cytoplasm. An-
following method: they were hydrated using other set of unstained slides were stained with
washes with descending grades of alcohol, then Masson’s trichrome after dewaxing and rehydra-

Figure 1. Effect of α-lip or vit E treatments on

serum glucose (A), TNF-α, (B), IL-6 (C), CRP (D)
and IgG (E) levels in intoxicated rats with low dose
of ZnO-NPs. Values are expressed as mean ± SE. ap
< 0.001, bp < 0.01, cp < 0.05 compared to normal
control group, *p < 0.001,**p < 0.01 compared to
ZnO-NPs intoxicated group, ππp < 0.001 compared
with vit E group using ANOVA followed by Bonfer-
roni as a post-ANOVA test.

B C

D E

1816
 Prophylactic role of α-lipoic acid and vitamin E

tion of unstained slides. Sections were immersed Results

in Weigert’s working hematoxylin for 10 min then
washed with water for 5 min, and was rinsed in
distilled water. They were then stained with
Biebrich scarlet for 5 min followed by rinsing in
distilled water. Slides were immersed in phospho-
tungstic/phosphomolybdic acid for 10 min. The
solution was discarded. After that sections were
transferred directly into Light Green for 5 min fol-
lowed by rinsing in distilled water. The sections
were immersed in 10.1% acetic acid solution for 1
min to visualize deposition of collagen fibers. This
usually makes the sample turn green46. The histo-
logical changes were estimated semi-quantitative-
ly, as being mild, moderate, or marked by micro-
scopic examination of histological sections.
The level of serum ALT was significantly in-
creased in mice administered with either low or
high repeated doses of ZnO-NPs for 5 consecu-
tive days (Table I). Co-administration of either α-
lip or vit E significantly attenuated ZnO-NPs in-
duced elevation of serum ALT.
The protective effect of either α-lip or vit E on
levels of serum glucose (marker of metabolic dis-
order) and some immunological pro-inflammato-
ry biomarkers in serum including, NO TNF-α,
IL-6, CRP, and IGg in different groups of ZnO-
NPs intoxicated rats with either the low or the
high repeated doses are illustrated in (Figures 1
and 2) respectively. It can be observed that these

Figure 2. Effect of α-lip or vit E treatments on

serum glucose (A), TNF-α, (B), IL-6 (C), CRP (D)
and IgG (E) levels in intoxicated rats with high dose
of ZnO-NPs particles. Values are expressed as mean
± SE. ap < 0.001, bp < 0.01 compared to normal con-
trol group, *p < 0.001, **p < 0.01 compared to ZnO-
NPs intoxicated group, πp ≤ 0.05, ππp < 0.001 com-
pared with vit E group, respectively, using ANOVA
A followed by Bonferroni as a post-ANOVA test.

B C

D E

1817
N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad

biomarkers were elevated dramatically in rat sera
intoxicated with either two repeated doses of
ZnO-NPs in relation to normal group. The intake
of α-lip or vit E immediately along with ZnO-
NPs ingestion markedly reduced the elevated
glucose and inhibited the induced inflammatory
mediators versus intoxicated animals. Treatment
by either the antioxidant agents increased GSH
level which was decreased in ZnO-NPs group,
while caspase 3 level was decreased by the treat-
ment of these agents (Figure 3).
The effect of either α-lip or vit E against the
toxicity of either two doses of ZnO-NPs on he-
patic DNA of rats is shown in (Figures 4, 5 and
6). The result revealed a significant increase in
the tail length and DNA % (tail DNA contents) in
livers of rats intoxicated with either two doses of
ZnO-NPs. This effect was severe in rat livers in-
gested the high dose of the NPs. Co-administra-
tion of the current agents, α-lip or vit E, to ZnO-
NPs intoxicated rats with either the studied two
doses effectively protected their livers from DNA
damage indicated by a decrease in tail length and
DNA % compared with intoxicated rats.
The results of the biochemical markers were
supported by the histopathological examination
of liver tissues stained with HE. This examina-
tion revealed that the animals which received a
low dose of ZnO-NPs showed many hepatocytes
with karyolysis compared with control group
(Figure 7A). Vit E treatment showed minimal
protective effects on liver tissues of intoxicated
rats as examined liver sections showed moderate
number of hepatocytes with karyolysis (Figure 7
D). However, ZnO-NPs intoxicated animals
treated with α-lip showed a major improvement
in the histopathological injury observed in liver
tissues as shown by miniml hepatocytes with
karyolysis (Figure 7 C). On the other hand, the
livers of rats treated with a high dose of ZnO-
NPs showed numerous hepatocytes with karyoly-
sis and pyknotic nuclei in addition to inflamma-

A B

C D

Figure 3. Effect of α-lip or vit E treatment on hepatic GSH (A, B) and caspase-3 activity (C, D) in intoxicated rats with low
and high dose of ZnO-NPs. Values are expressed as mean ± SE. ap < 0.001, bp < 0.01, cp < 0.05, compared to normal control
group, *p < 0.001, **p < 0.01, ***p < 0.05 compared to ZnO-NPs intoxicated group, πp < 0.001 compared to vit E group, re-
spectively, using ANOVA followed by Bonferroni as a post-ANOVA test.

1818
 Prophylactic role of α-lipoic acid and vitamin E

A B

C D

Figure 4. Tail length and DNA percentage in tail of comets obtained from liver tissue of intoxicated rats with low (A, B respective-
ly) and high dose (C, D respectively) of ZnO-NPs particles, and the effect of α-lip or vit E treatment on reduction of DNA damage.
Values are means ±S.D (n=10). ap < 0.001, bp < 0.01, cp < 0.05 compared to normal control group, ***p < 0.001 compared to ZnO-NPs
intoxicated group, $p ≤ 0.05 compared with vit E treated group, using ANOVA followed by Bonferroni as post ANOVA test.

tory cellular infiltration (Figure 8B). However,
the animals which received ZnO-NPs and vit E
showed moderate histopathological changes in
the liver tissue as compared to liver samples of
control group. Where, moderate number of hepa-
tocytes with karyolysis were observed upon ex-
amining liver tissues of vit E treated rats (Figure
8D). Meanwhile, the animals that received ZnO-
NPs and α-lip showed few hepatocytes with py-
knotic nuclei and karyolysis (Figure 8C).
Liver tissues of animals intoxicated with ZnO-
NPs and stained with Masson’s trichrome
showed moderate with low and high dose of
ZnO-NPs and showed increase of collagen fiber
deposits in the portal area (Figures 9B and 10B)
compared with the normal control group (Figures
9A and 10A). However, either intoxicated ani-
mals with low and high dose of ZnO-NPs that re-
ceived α-lip or vit E showed minimum collagen
deposition similar to the control group (Figures
9C and D, 10C and D respectively).
Discussion
Several studies have found high toxicity of ul-
trafine particles (d < 100 nm) was associated
with respiratory, cardiovascular and liver
diseases47-50. It was demonstrated that NPs, in-
cluding ZnO, may cause more inflammatory tis-
sue damage than larger particles of the same ma-
terials at a same mass dose delivery50-52.
The current study revealed either two doses
of ZnO-NPs (600 mg/kg/day and 1 g/kg/day for
5 days) induced liver damage documented by
the elevation of serum ALT compared to control
group, implying cellular leakage and loss of the
functional integrity of cell membranes in liver19.
This finding reflect that liver is one of the target
organs of such NPs toxicity. It has been reported
that liver damage could be induced by excess
oral zinc salt and zinc powder administration53-55
reported that high dietary zinc caused liver toxi-
city of mice and resulted in inhibiting the activi-
ty of GOT in liver homogenate of mice.
Co-administration of either α-lip or vit E to
ZnO-NPs intoxicated rats with either low or high
doses, significantly reduced serum ALT level
compared with intoxicated rats. This may indi-
cate that the used agents act as effective hepato-
protective against liver dysfunction caused by
nano materials toxicity. The hepato-protective ef-
fect of either α-lip or vit E against hepatic tissue
injury was documented56,57.

1819
N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad
A B
C D
Some reports have demonstrated that patho-
genic mechanisms initiated by NPs are dominat-
ed by inflammation-driven effects, including, ox-
idative stress, apoptosis and DNA damage50,58,59.
In the present study, it was found that a marked
increase in serum glucose level as well as im-
munological pro-inflammatory biomarkers in-
cluding TNF-α, IL-6, CRP, and IGg in rat sera
intoxicated with either two doses of ZnO-NPs in
relation to normal group implying metabolic and
immune disorder.
TNF-α is one of the most commonly inflam-
matory injurous chemokine immunological
markers increased in response to different metal
oxide NPs-toxicity including ZnO60,61. The up-
regulation of this cytokine triggers the production
A B
C D
1820
Figure 5. DNA damage in the liver tis-
sue in intoxicated rats with low dose of
ZnO-NPs particles, and the effect of α-lip
or vit E treatment on the level of DNA
damage. COMET assay showing degree
of DNA damage in liver tissue of (A) nor-
mal control group, (B) group intoxicated
with small dose of ZnO-NPs particles, (C)
intoxicated group treated with α-lip, and
(D) intoxicated group treated with vit E.
of other cytokines and IL-6, the chief stimulator
of the production of CRP inducing inflammatory
liver injury62. IL-6 triggers the activation of tran-
scription factors that bind to DNA elements and
stimulate increased transcription of CRP, result-
ing in a rise in its serum level62,63.
CRP is a member of the pentaxin protein
family involved in pattern recognition and in-
nate immunity; it is synthesized primarily by
the liver in response to inflammation62. It was
reported that up-regulation of CRP is closely as-
sociated with metabolic disturbances including,
insulin resistance and related complications
such as fatty liver disease and hyperglycemia63.
It also has a principle role in the activation of
proinflammatory pathways in various cell types64,65.
Figure 6. DNA damage in the liver tis-
sue in intoxicated rats with high dose of
ZnO-NPs particles, and the effect of α-lip
or vit E treatment on the level of DNA
damage. COMET assay showing degree
of DNA damage in liver tissue of (A) nor-
mal control group, (B) group intoxicated
with high dose of ZnO-NPs particles, (C)
intoxicated group treated with α-lip and
(D) intoxicated group treated with vit E.
 Prophylactic role of α-lipoic acid and vitamin E

Figure 7. Photomicrographs of liver sections, stained with H&E, from rats received low dose of ZnO. Scale bars = 50 µm. (A)
Liver section from control rat showing normal architecture of hepatocytes (arrow), separated by well-organized blood sinu-
soids (curved arrow). (B) Liver section from rat received low dose of ZnO showing many hepatocytes with karyolysis (hollow
arrow). (C) Liver section from rat received low dose of ZnO and lipoic acid showing few hepatocytes with karyolysis (hollow
arrow). (D) Liver section of rat received low dose of ZnO and vitamin E, showing moderate number of hepatocytes with kary-
olysis (hollow arrow).

Figure 8. Photomicrographs of liver sections, stained with H&E, from rats received high dose of ZnO. Scale bars = 50 µm.
(A) Liver section from control rat showing normal architecture of hepatocytes (arrow), separated by blood sinusoids (curved
arrows). (B) Liver section from rat received high dose of ZnO showing pyknotic nuclei (arrow) or karyolysis (hollow arrow).
A mononuclear inflammatory cellular infiltrate was observed (arrowhead). (C) Liver section from rat received high dose of
ZnO and lipoic acid showing few hepatocytes with pyknotic nuclei (arrow) or karyolysis (hollow arrow). The portal area
shows mild mononuclear inflammatory infiltrate (arrowhead). (D) Liver section of rat received high dose of Zn O and vitamin
E, showing moderate number of hepatocytes with karyolysis (hollow arrow).

1821
N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad
So, the increase in serum glucose level in re-
sponse to ZnO-NPs toxicity may be related to
CRP up-regulation. The marked increase in the
ciculating IgG in rat sera intoxicated with either
doses of ZnO-NPs is another response to im-
mune disorder induced by this NPs toxicity. It
was suggested that the increase in the circulating
antibody production is the result of production of
different inflammatory cytokines including TNF-
α with potential impact on immunoglobulin pro-
1822
Figure 9. Photomicrographs
of liver sections, stained with
Masson’s trichrome, from
rats received low dose of
ZnO. Scale bars = 50 µm. (A)
Liver section from control rat
showing few collagen fibers
in the portal area (arrow). (B)
Liver section from rat re-
ceived low dose of ZnO
showing few collagen fibers
distribution in the portal area
(arrow). (C) Liver section
from rat received low dose of
ZnO and lipoic acid, showing
also few deposition of colla-
gen fibers in the portal area
(arrow). (D) Liver section of
rat received low dose of ZnO
and vitamin E, showing few
collagen fibers deposition
(arrow).
duction during inflammation66. These result may
indicate that ZnO-NPs induced inflammatory liv-
er injury through production of the inflaamatory
maediators. Thus, a protective strategy attenuates
production of inflammatory mediators could pre-
dispose to both liver injury and remote organ
dysfunction
The intake of α-lip or vit E immediately with
ZnO-NPs ingestion presented in this study was
beneficial in the prevention of this NPs induced
Figure 10. Photomicro-
graphs of liver sections,
stained with Masson’s
trichrome, from rats received
high dose of ZnO. Scale bars
= 50 µm. (A) Liver section
from control rat showing few
collagen fibers in the portal
area (arrow). (B) Liver sec-
tion from rat received high
dose of ZnO showing few
collagen fibers distribution in
the portal area (arrow). (C)
Liver section from rat re-
ceived high dose of ZnO and
lipoic acid, showing also few
deposition of collagen fibers
in the portal area (arrow). (D)
Liver section of rat received
high dose of ZnO and vitamin
E, showing few collagen
fibers deposition (arrow).
 Prophylactic role of α-lipoic acid and vitamin E
serum glucos elevation as well as inflammatory
liver injury. The reduced in serum glucose by
treatment with either α-lip or vit E may attrib-
uted to both agents attenuate the metabolic dis-
turbance induced by ZnO-NPs. Previous pub-
lished data revealed the benficial effect of α-lip
in improving of insulin sensitivity in patients
with the metabolic syndrome67. Also some au-
thors revealed that vit E supplementation could
improve glycemic control and reduce blood sugar
in diabetic patients68.
The protective actions of these antioxidant
agents against ZnO-NPs toxicity induced inflam-
matory mediators in rats may attributed to their
abilities in the inhibition of these mediators ex-
pression. Previous studies showed that α-lip ex-
hibits strong anti-inflammatory activities in vitro
and in vivo by reducing inflammatory mediators
such as proinflammatory cytokine production
and CRP24,67,69.
The anti-inflammatory role of vit E is associ-
ated with the amelioration of oxidative damage,
trapping of reactive nitrogen species35,37, as well
as down-regulating of other inflammatory mark-
ers as IL-6, CRP70,71. Furthermore, a decrease in
oxidative stress by either α-lip and vit E may al-
so be involved in their anti-inflammatory ef-
fects33.
Hepatic GSH levels were significantly reduced
after ZnO-NPs treatment, while treatment with
either α-lip or vit E increased GSH content in
liver cells when compared with ZnO-NPs intoxi-
cated rats. This was attributed to the antioxidant
properties of both agents. Previous investigations
reported that vit E supplementation protects
against oxidative damage caused by different
pathological conditions through inhibition of
ROS production72,73. Additionally, α-lip has the
ability to correct thiol deficiency within cells74
increase the de novo synthesis of glutathione75,
and enhance glutathione synthesis in normal ani-
mals76. Moreover, α-lip can maintain high levels
of vitamin C and participate in vitamin E recy-
cling, thus complementing some of the functions
of glutathione77. These data augment the current
results in which either of the used antioxidants
modalities significantly decreased liver damage
due to ZnO-NPs by synergistically increasing
cellular levels of GSH (Figure 3).
Apoptosis represents a key event after oxidative
DNA damage 78. The data of the current work
showed a marked increased in activity of the apop-
totic biomarker, caspase 3, in liver tissue of rats in-
toxicated with either low or high dose of ZnO-NPs,
suggesting that apoptosis might contribute to this
NPs induced DNA damage. Administration of ei-
ther α-lip or vit E to ZnO-NPs intoxicated rats ben-
eficially down regulate the increase in liver caspase
3 activity. This can be attributed to the strong anti-
apoptotic effect of these compound79,80.
Penetration of NPs into the nucleus has been
shown in a number of studies81. Some authors82
have shown that 1.4 nm gold (Au55) particle
clusters interact with DNA in a way which may
be the reason for the strong toxicity of these tiny
particles towards human cancer, since it is gener-
ally known that DNA double-strand breaks may
cause cancer.
The comet assay is considered to be a rapid,
sensitive and a relative simple assay for decting
DNA damage at the level of individual cells45. In-
creased DNA migration results from the induc-
tion of DNA single-strand breaks, alkalilabile
sites, and incomplete excision repair sites at the
time of lysis. Increased DNA migration also ac-
companies the DNA fragmentation associated
with the cell death arising from a non-DNA-
mediated process or apoptosis83. With an increas-
ing number of breaks, DNA pieces migrate freely
into the tail of the comet, and in extreme cases
(the apoptofic cell) the head and the tail are well
separated. Tail length, percentage of total DNA
in the tail, reflect DNA damage, though the per-
centage of tail DNA generally seems to be the
most useful, as it is directly related to the fre-
quency of breaks over a wide range of damage84.
Use of the very sensitive single-cell elec-
trophoresis assay to detect DNA damage indicated
that ZnO-NPs induced liver DNA damage docu-
mented by a significant increase in the tail length
and DNA % in the tail in livers of rats intoxicated
with either two doses of ZnO-NPs. This effect was
severe in rat livers ingested the high dose of the
NPs. DNA damaging potential of ZnO-NPs was
ensured by experimental studies85,86.
Also, the present data is supported by a previ-
ous study conducted by Dufour et al87 who re-
ported a concentration increase in chromosome
aberrations upon ZnO-NPs exposure (< 100 nm)
in CHO cells although at a very high concentra-
tion (≥ 105 µg/ml). As there is a well document-
ed link between NPs and oxidative stress, one of
the possible modes that can be suggested for
ZnO-NPs induced DNA damage may be lipid
peroxidation and oxidative stress 50. ROS are
known to react with DNAmolecule causing dam-
age to both purine and pyrimidine bases as well
as DNA backbone88. Another important outcome
1823
N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad
of ROS production is lipid peroxidation which
generates a variety of products reactive towards
cellular macromolecules including DNA. One of
the major products of lipid peroxidation malondi-
aldehyde, is a proven mutagen and carcinogenic
compound which reacts with DNA to form
adducts to deoxyguanosine, deoxyadenosine and
deoxycytidine89,90. DNAdamage resulting from
any of these probable mechanisms may trigger
signal transduction pathways leading to apoptosis
or cause interferences with normal cellular
processes thereby causing cell death. Co-admin-
istration of the current antioxidant agents, α-lip
and vit E to ZnO-NPs intoxicated rats with either
the studied two doses effectively protected their
livers from DNA damage.
The possible role of naturally occurring antioxi-
dants in protection against oxidative DNA damage
has been studied, vit E inhibited the increase in
liver DNA damage induced by the hepatocarcino-
gen 2-nitropropane in rats91. Also, in vitro studies
documented the ability of vit E to reduce the ox-
idative DNA damage38. The capacity of vit E to
decrease some target organ/cell specific induction
of oxidative DNA damage would explain on the
basis that vit E easily intercept the increases in
ROS generated further away (e.g. by metabolism
of xenobiotics in the cytoplasm) before it can dif-
fuse to the nucleus and damage the DNA92. On the
other hand, previous work demonstrate that vit E
deficiency also increases oxidative damage to
DNA and that basal levels can be subsequently re-
stored by reintroduction of vit E to the diet. Such
damage to DNA may produce mutations that
cause permanent alterations in the genetic mes-
sage when the cell replicates its DNA and, thus,
increases risk of carcinogenesis. These results,
therefore, support epidemiological studies impli-
cating low intakes or blood levels of vit E with the
subsequent risk of cancers at various sites93. α-lip
significantly reduced the metal toxicity-induce ox-
idative DNA in a dose dependent manner by re-
ducing the formation of 8-hydroxy-2-
deoxyguanosine (8-OH-dG) in DNA28,94. The pro-
tective effect of α-lip against DNA oxidative dam-
age related to its ability to decreases oxidative
stress and restores the normal levels of other an-
tioxidants in vivo under various physiological and
pathophysiological conditions95.
Our results were supported by the histopatho-
logical examination of liver tissues, which clari-
fied that animals received either low or high dos-
es of ZnO-NPs showed moderate to massive
numbers of hepatocytes with karyolysis and py-
1824
knotic nuclei in addition to inflammatory cellular
infiltration. These hazardous effects were dose
dependent. Treatment of ZnO-NPs intoxicated
rats with either vit E or α-lip, significantly im-
proved most of the deviated histopathological al-
teration and nearly prevented the damaging effect
due to ZnO-NPs intoxication that was manifested
microscopically by extensive improvements in
the histological features of liver cells, as well as
marked reduction in the deposition of collagen
fibers in the portal area.
Conclusions
These findings suggest that vit E and α-lip
acid supplementation as prophylactic agents may
beneficial against inflammatory responses in-
duced liver injury and oxidative DNA damage
caused by toxic effect of metal oxide nano-parti-
cles.
––––––––––––––––––––
Acknowledgements
The authors gratefully acknowledge the Strategic Tech-
nique Program of the National Plane for Science, Technolo-
gy, and Innovation (NPST) in Riyadh, Kingdom of Saudi
Arabia for the financial support of this work in the form of
Research Fellowship.
–––––––––––––––––-––––
Conflict of Interest
The authors declare that they have no conflict of interest.
References
1) PEKKANEN J, PETERS A, HOEK G, TIITTANEN P, BRUNEKREEF
B, DE HARTOG J, HEINRICH J, IBALD-MULLI A, KREYLING
WG, LANKI T, TIMONEN KL, VANNINEN E. Particulate air
pollution and risk of ST-segment depression during
repeated submaximal exercise tests among sub-
jects with coronary heart disease: the exposure
and risk assessment for fine and ultrafine particles
in ambient air ultra study. Circulation 2002; 106:
933-938.
2) PENTTINEN P, TIMONEN KL, TIITTANEN P, MIRME A, RU-
USKANEN J, PEKKANEN J. Ultrafine particles in urban
air and respiratory health among adult asthmat-
ics. Eur Resp J 2001; 17: 428-435.
3) RAHMAN Q, LOHANI M, DOPP E, PEMSEL H, JONAS L,
WEISS DG, SCHIFFMAN D. Evidence that ultrafine ti-
tanium dioxide induces micronuclei and apoptosis
in syrian hamster embryo fibroblasts. Environ
Health Perspect 2002; 110: 797-800.
 Prophylactic role of α-lipoic acid and vitamin E
4) OBERDÖRSTER G, FINKELSTEIN JN, JOHNSTON C. Acute
pulmonary effects of ultrafine particles in rats and
mice. Res Rep Health Eff Inst 2000; 96: 5-74.
5) LAM CW, JAMES JT, MCCLUSKEY R, HUNTER RL. Pul-
monary toxicity of single-wall carbon nanotubes in
mice 7 and 90 days after intratracheal instillation.
Toxicol Sci 2004; 77: 126-134.
6) SALATA OV. Application of nanoparticles in biology
and medicine. J Nanobiotechnol 2004; 2: 3.
7) NAM J-M, THAXTON CS, MIRKIN CA. Nanoparticle-
based bio-bar codes for the ultrasensitive detec-
tion of proteins. Science 2003; 301: 1884-1886.
8) LANZA GM, WICKLINE SA. Targeted ultrasonic con-
trast agents for molecular imaging and therapy.
Curr Probl Cardiol 2003; 28: 625-653.
9) BUZEA C, PACHECO II, ROBBIE K. Nanomaterials and
nanoparticles: sources and toxicity. Biointerphas-
es 2007; 2: MR17-71.
10) LI N, SIOUTAS C, CHO A, MISRA C, SEMPF J, WANG M,
OBERLEY T, FROINES J, NEL A. Ultrafine particulate
pollutants induce oxidative stress and mitochron-
drial damage. Environ Health Perspect 2003; 111:
455-460.
11) MØLLER P, JACOBSEN NR, FOLKMANN JK, DANIELSEN PH,
MIKKELSEN L, HEMMINGSEN JG, VESTERDAL LK, FORCH-
HAMMER L, WALLIN H, LOFT S. Role of oxidative dam-
age in toxicity of particulates. Free Radic Res
2010; 44: 1-46.
12) DONALDSON K, AITKEN R, TRAN L, STONE V, DUFFIN R,
FORREST G. Carbon nanotubes: a review of their
properties in relation to pulmonary toxicology and
workplace safety. Toxicol Sci 2006; 92: 5-22.
13) MROZ RM, SCHINS RP, LI H, JIMENEZ LA, DROST EM,
HOLOWNIA A. Nanoparticle-driven DNA damage
mimics irradiation-related carcinogenesis path-
ways. Eur Respir J 2008; 31: 241-251.
14) O BERDORSTER G, O BERDORSTER E, O BERDORSTER J.
Nano-toxicology: an emerging discipline evolving
from studies of ultrafine particles. Environ Health
Perspect 2005; 113: 823-839.
15) ENVIRONMENTAL PROTECTION AGENCY. Science Policy
Council, Nanotechnology Workgroup Nanotech-
nology White Paper, 2007.
16) WANG XL, RAINWATER DL, MAHANEY MC, STOCKER R.
Cosupplementation with vitamin E and coenzyme
Q10 reduces circulating markers of inflammation
in baboons. Am J Clin Nutr 2004; 80: 649-655.
17) GOJOVA A, GUO B, KOTA RS, RUTLEDGE JC, KENNEDY
IM, BARAKAT AI. Induction of inflammation in vascu-
lar endothelial cells by metal oxide nanoparticles:
effect of particle composition. Environ Health Per-
spect 2007; 115: 403-409.
18) LIN WS, XU Y, HUANG CC, MA Y, SHANNON KB, CHEN
DR, HUANG YW. Toxicity of nano- and micro-sized
ZnO particles in human lung epithelial cells. J
Nanopart Res 2009; 11: 25-29.
19) WANG B, FENG WY, WANG M, WANG TC, GU YQ, ZHU
MT, OUYANG H, SHI JW, ZHANG F, ZHAO YL, CHAI ZF,
WANG HF, WANG J. Acute toxicological impact of nano-
and submicro-scaled zinc oxide powder on healthy
adult mice. J Nanopart Res 2008; 10: 263-276.
20) LOVERN SB, K LAPER R. Daphnia magna mortality
when exposed to titanium dioxide and fullerene
(C60) nanoparticles. Environ Toxicol Chem 2006;
25: 1132-1137.
21) FRANKLIN NM, ROGERS NJ, APTE SC, BATLEY GE, GADD
GE, CASEY PS. Comparative toxicity of nanoparticu-
late ZnO, bulk ZnO, and ZnCl2 to a freshwater mi-
croalga (Pseudokirchneriella subcapitata): the im-
portance of particle solubility. Environ Sci Technol
2007; 4124: 8484-8490.
22) WANG Z, ZHOU J, FAN J, QIU SJ, YU Y, HUANG XW,
TANG ZY. Effect of rapamycin alone and in combi-
nation with sorafenib in an orthotopic model of
human hepatocellular carcinoma. Clin Cancer
Res 2008; 14: 5124-5130.
23) CHEN LC, PACE JL, RUSSELL SW, MORRISON DC. Al-
tered regulation of inducible nitric oxide synthase
expression in macrophages from senescent mice.
Infect Immun 1996; 64: 4288-4298.
24) ZHANG WJ, WEI H, HAGEN T, FREI B. Lipoic acid at-
tenuates LPS-induced inflammatory responses by
activating the phosphoinositide 3-kinase/Akt sig-
naling pathway. PNAS 2007; 104: 4077-4082.
25) BILSKA A, WLODEK L. Lipoic acid–the drug of the fu-
ture? Pharmacol Rep 2005; 57: 570-577.
26) SEHIRLI AO, TATLÝDEDE, E, YÜKSEL M, ÇETINEL S, ERZIK
C, YEÐEN B, SENER G. Protective effects of alpha-
lipoic acid against oxidative injury in TNBS-in-
duced colitis. Erciyes Med J 2009; 31: 15-26.
27) BUDHWAR R, KUMAR S. Substantial inhibition of chro-
maten induced DNA-protein crosslink formation in
vivo by alpha-lipoic acid. Environ Toxicol Pharma-
col 2005; 20: 246-249.
28) KUMAR S, BUDHWAR R, NIGAM A, PRIYA S. Cytoprotec-
tion against Cr61-induced DNA damage by alpha-
lipoic acid: implications in reducing occupational
cancer risk. Mutagenesis 2009; 24: 495-500.
29) OBROSOVA IG, MINCHENKO AG, MARINESCU V, FATHALLAH
L, KENNEDY A, STOCKERT CM, FRANK RN, STEVENS MJ.
Antioxidants attenuate early up regulation of retinal
vascular endothelial growth factor in streptozotocin
diabetic rats. Diabetologia 2001; 44: 1102-1110.
30) LEE WJ, LEE IK, KIM HS, KIM YM, KOH EH, WON JC,
HAN SM, KIM MS, JO I, OH GT, PARK IS, YOUN JH,
PARK SW, LEE KU, PARK JY. Lipoic acid prevents en-
dothelial dysfunction in obese rats via activation
of AMP-activated protein kinase. Arterioscler
Thromb Vasc Biol 2005; 25: 2488-2494.
31) HO YS, LAI CS, LIU HI. Dihydrolipoic acid inhibits
skin tumor promotion through anti-inflammation
and anti-oxidation. Biochem Pharmacol 2007; 73:
1786-1795.
32) PACKER L, WITT EH, TRITSCHLER HJ. Alpha-lipoic as bi-
ological antioxidant a review. Free Rad Biol Med
1995; 19: 227-250.
33) SCHAFFER S, MULLER WE, ECKERT GP. Tocotrienols:
constitutional effects in aging and disease. J Nutr
2005; 135: 151-154.
34) ZINGG JM. Modulation of signal transduction by vit-
amin E. Mol Aspect Med 2007; 28: 481-506.
35) QURESHI AA, REIS JC, QURESHI N, PAPASIAN CJ, MORRI-
SON DC, SCHAEFER DM. δ-Tocotrienol and quercetin
reduce serum levels of nitric oxide and lipid para-
meters in female chickens. Lipid Health Dis 2011;
10: 39-58.
1825
N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad
36) YANG CS, LU G, JU J, LI GX. Inhibition of inflam-
mation and carcinogenesis in the lung and
colon by tocopherols. Ann N Y Acad Sci 2010;
1203: 29-34.
37) TAHAN G, AYTAC E, AYTEKIN H, GUNDUZ F, DOGUSOY G,
AYDIN S, TAHAN V, UZUN H. Vitamin E has a dual ef-
fect of anti-inflammatory and antioxidant activities
in acetic acid-induced ulcerative colitis in rats.
Can J Surg 2011; 54: 333-338.
38) MAKPOL S, DURANI LW, CHUA KH, YUSOF YAM, NGAH
WZW. Tocotrienol-rich fraction prevents cell cycle
arrest and elongates telomere length in senes-
cent human diploid fibroblasts. J Biomed Biotech-
nol 2011; 2011: 506171.
39) WANG B, W Y, FENG ET AL. Acute toxicological im-
pact of nano-and submicro-scaled zinc oxide
powder on healthy adult mice. J Nano Particl Res
2008; 10: 263-276.
40) SHARMA M, GUPTA YK. Effect of alpha lipoic acid on
intracerebroventricular streptozotocin model of
cognitive impairment in rats. Eur Neuropsy-
chopharmacol 2003; 13: 241-247.
41) MIWA I, OKUDA J, MAEDA K, OKUDA G. Mutarotase
effect on colorimetric determination of blood glu-
cose with D-glucose oxidase. Clin Chem Acta
1972; 37: 538-540.
42) KIM N, KIM YJ, CHO DK. Gold nanoparticle-based
signal augmentation of quartz crystal microbal-
ance immunosensor measuring C-reactive pro-
tein. Curr Appl Phys 2010; 10: 1227-1230.
43) MORON MS, DEPIERRE JW, MANNERVIK B. Levels of
glutathione, glutathione reductase and glu-
tathione S-transferase activities in rat lung and liv-
er. Biochim Biophys Acta 1979; 582: 67-78.
44) VACULOVA A, ZHIVOTOVSKY B. Caspases: determina-
tion of their activities in apoptotic cells. Methods
Enzymol 2008; 442: 157-181.
45) SINGH NP, MCCOY MT, TICE RR, SCHNEIDEREL. A sim-
ple technique foe quantitation of low levels of
DNA damage in individaul cells. Exp Cell Res
1988; 175: 184-191.
46) SMITH A, BRUTON J. A colour Atlas of histological
staining techniques. J Clin Pathol 1978; 31: 298.
47) P E K K A N E N J, P E T E R S A, H O E K G, T I I T TA N E N P,
BRUNEKREEF B, DE HARTOG J, HEINRICH J, IBALD-MULLI
A, KREYLING WG, LANKI T, TIMONEN KL, VANNINEN E.
Particulate air pollution and risk of ST-segment
depression during repeated submaximal exercise
tests among subjects with coronary heart dis-
ease: the exposure and risk assessment for fine
and ultrafine particles in ambient air ultra)
study. Circulation 2002; 106: 933-938.
48) PENTTINEN P, TIMONEN KL, TIITTANEN P, MIRME A, RU-
USKANEN J, PEKKANEN J. Ultrafine particles in urban
air and respiratory health among adult asthmat-
ics. Eur Resp J 2001; 17: 428-435.
49) VON KLOT S, WOLKE G, TUCH T, HEINRICH J, DOCKERY
DW, SCHWARTZ J, KREYLING WG, WICHMANN HE, PE-
TERS A. Increased asthma medication use in asso-
ciation with ambient fine and ultrafine particles.
Eur Respir J 2002; 20: 691-702.
50) XIONG D, FANG T, YU L, SIMA X, ZHU W. Effects of
nano-scale TiO2, ZnO and their bulk counterparts
1826
on zebrafish: acute toxicity, oxidative stress and
oxidative damage. Sci Total Environ 2011; 409:
1444-1452.
51) OBERDÖRSTER G, FINKELSTEIN JN, JOHNSTON C. Acute
pulmonary effects of ultrafine particles in rats and
mice. Res Rep Health Eff Inst 2000; 96: 5-74.
52) RAHMAN Q, LOHANI M, DOPP E, PEMSEL H, JONAS L,
WEISS DG, SCHIFFMAN D. Evidence that ultrafine ti-
tanium dioxide induces micronuclei and apoptosis
in Syrian hamster embryo fibroblasts. Environ
Health Perspect 2002; 110: 797-800.
53) DING H, PENG R, CHEN J. Effects of high dietary
zinc on liver function, hepatic drug metabolism
enzymes and membrane fluidity in mice. Wei
Sheng Yan Jiu 1998; 27: 180-182.
54) CHEN RH, QIN R, WANG FD, WANG JP, LU TX. The
effects of oral excess zinc on the zinc level and
morphology of tissues. Zhonghua Yixue Zazhi
1992; 72: 391-393.
55) WANG B, FENG WY, WANG TC, JIA G, WANG M, SHI
JW, ZHANG F, ZHAO YL, CHAI ZF. Acute toxicity of
nano- and micro-scale zinc powder in healthy
adult mice. Toxicol Lett 2006; 161: 115-123.
56) ASUKU O, ATAWODI SE, ONYIKE E. Antioxidant, he-
patoprotective, and ameliorative effects of
methanolic extract of leaves of grewia mollis juss.
on carbon tetrachloride-treated albino rats. J Med
Food 2012; 15: 83-88.
57) MORSY MA, ABDALLA AM, MAHMOUD AM, ABDELWAHAB
SA, MAHMOUD ME. Protective effects of curcumin,
α-lipoic acid, and N-acetylcysteine against carbon
tetrachloride-induced liver fibrosis in rats. J Physi-
ol Biochem 2012; 68: 29-35.
58) BORM PJ, ROBBINS D, HAUBOLD S, KUHLBUSCH T, FISSAN
H, DONALDSON K, SCHINS R, STONE V, KREYLING W, LADE-
MANN J, KRUTMANN J, WARHEIT D, OBERDORSTER E. The
potential risks of nanomaterials: a review carried
out for ECETOC. Part Fibre Toxicol 2006; 3: 11.
59) LU S, DUFFIN R, POLAND C, DALY P, MURPHY F, DROST
E. Efficacy of simple short-term in vitro assays for
predicting the potential of metal oxide nanoparti-
cles to cause pulmonary inflammation. Environ
Health Perspect 2009; 117: 241-247.
60) SAYES CM, REED KL, WARHEIT DB. Assessing toxicity
of fine and nanoparticles: comparing in vitro mea-
surements to in vivo pulmonary toxicity profiles,
Toxicol Sci 2007; 97: 163-180.
61) VERANTH JM, KASER EG, VERANTH MM, KOCH M, YOST
GS. Cytokine responses of human lung cells
(BEAS-2B) treated with micron-sized and
nanoparticles of metal oxides compared to soil
dusts. Particle and Fibre Toxicology 2007; 4: 2-19.
62) KERNER A, AVIZOHAR O, SELLA R, BARTHA P, ZINDER O,
MARKIEWICZ W, YISHAI L, BROOK G J, ARONSON D. As-
sociation between elevated liver enzymes and C-
reactive protein possible hepatic contribution to
systemic inflammation in the metabolic syndrome.
Arterioscler Thromb Vasc Biol 2005; 25: 93-197.
63) PATEL DN, KING CA, BAILEY SR. Interleukin-17 stimu-
lates C-reactive protein expression in hepatocytes
and smooth muscle cells via p38 MAPK and
ERK1/2-dependent NF-kappaB and C/EBPbeta
activation. J Biol Chem 2007; 282: 27229-27238.
 Prophylactic role of α-lipoic acid and vitamin E
62) PEPYS MB, BALTZ ML. Acute phase proteins with
special reference to C-reactive protein and relat-
ed proteins (pentaxins) and serum amyloid A pro-
tein. Adv Immunol 1983; 34: 141-212.
63) XI L, XIAO C, BANDSMA RHJ, NAPLES M, ADELI K, LEWIS
GF. C-reactive protein impairs hepatic insulin sen-
sitivity and insulin signaling in rats: role of mito-
gen-activated protein kinases. Hepatology 2011;
53: 127-135.
64) HATTORI Y, MATSUMURA M, KASAI K. Vascular smooth
muscle cell activation by C-reactive protein. Car-
diovasc Res 2003; 58: 186-195.
65) D’ALESSANDRIS C, LAURO R, PRESTA I, SESTI G. C-reac-
tive protein induces phosphorylation of insulin re-
ceptor substrate-1 on Ser307 and Ser 612 in L6
myocytes, thereby impairing the insulin signaling
pathway that promotes glucose transport. Dia-
betologia 2007; 50: 840-849.
66) DAVIS GS, PFEIFFER LM, HEMENWAY DR. Persistent
overexpression of interleukin-1 beta and tumor
necrosis factor-alpha in murine silicosis. J Environ
Pathol Toxicol Oncol 1998; 17: 99-114.
67) MCMACKIN CJ, WIDLANSKY ME, HAMBURG NM, ALEX L.
HUANG AL, WELLER S, HOLBROOK M, GOKCE N, HAGEN
TM, KEANEY JF, VITA JA. Effect of combined treat-
ment with α-lipoic acid and acetyl-L-carnitine on
vascular function and blood pressure in patients
with coronary artery disease. J Clin Hypertens
2007; 9: 249-255.
68) KHABAZ M, RASHIDI M, KASEB F, ARDEKANI MA. Effect
of vitamin E on blood glucose, lipid profile and
blood pressure in Type 2 diabetic patients. Iranian
J Diabetes Obesity 2009; 1: 11-15.
69) ALLEVA R, TOMASETTI M, SARTINI D, EMANUELLI M, NA-
SOLE E, DI DONATO F, BORGHI B, SANTARELLI L, NEUZIL
J. α-lipoic acid modulates extracellular matrix and
angiogenesis gene expression in non-healing
wounds treated with hyperbaric oxygen therapy.
Mol Med 2008; 14: 175-183.
70) WANG XL, RAINWATER DL, MAHANEY MC, STOCKER R.
Cosupplementation with vitamin E and coenzyme
Q10 reduces circulating markers of inflammation
in baboons. Am J Clin Nutr 2004; 80: 649-655.
71) LEE Y, KIM M, CHOI K, KIM J, BAE W, KIM S, SOHN C.
Relationship between inflammation biomarkers,
antioxidant vitamins, and bone mineral density in
patients with metabolic syndrome. Nutr Res Pract
2011; 5: 150-156.
72) QURESHI AA, REIS JC, QURESHI N, PAPASIAN CJ, MORRI-
SON DC, SCHAEFER DM. δ-Tocotrienol and quercetin
reduce serum levels of nitric oxide and lipid para-
meters in female chickens. Lipids Health Dis
2011; 10: 39.
73) TAHAN G, AYTAC E, AYTEKIN H, GUNDUZ F, DOGUSOY G,
AYDIN S, TAHAN V, UZUN H. Vitamin E has a dual ef-
fect of anti-inflammatory and antioxidant activities
in acetic acid-induced ulcerative colitis in rats.
Can J Surg 2011; 54: 333-338.
74) SEN CK, ROY S, HAN D, PACKER L. Regulation of cel-
lular thiols in human lymphocytes by alphalipoic
acid: a flow cytometric analysis. Free Radic Biol
Med 1997; 22: 1241-1257.
75) HAN D, HANDELMAN G, MARCOCCI L, SEN CK, ROY S,
KOBUCHI H, TRITSCHLER HJ, FLOHE L, PACKER L. Lipoic
acid increases de novo synthesis of cellular glu-
tathione by improving cystine utilization. Biofac-
tors 1997; 6: 321-338.
76) KHANNA P, WANG L, PEREZ-POLO RJ, ANSARI NH. Ox-
idative defense enzyme activity and mRNA levels
in lenses of diabetic rats. J Toxicol Environ Health
1997; 51: 541-555.
77) M AITRA I, S ERBINOVA E, T RISCHLER H, PACKER L. α-
Lipoic acid prevents buthionine sulfoximinein-
duced cataract formation in newborn rats. Free
Radic Biol Med 1995; 18: 823-829.
78) SHARMA V, SHUKLA R K, SAXENA N, PARMAR D, DAS M,
DHAWAN A. DNA damaging potential of zinc oxide
nanoparticles in human epidermal cells. Toxicol
Lett 2009; 185: 211-218.
79) LEE WJ, LEE IK, KIM HS, KIM YM, KOH EH, WON JC,
HAN SM, KIM MS, JO I, OH GT, PARK IS, YOUN JH,
PARK SW, LEE KU, PARK JY. α-lipoic acid prevents en-
dothelial dysfunction in obese rats via activation
of AMP-activated protein kinase. Arterioscler
Thromb Vasc Biol 2005; 25: 2488-2494.
80) ABOUL-SOUD MAM, OTHMAN AM, EL-DESOKY GE, AL-
OTHMAN ZA, YUSUF K, AHMAD J, AL-KHEDHAIRY AA.
Hepatoprotective eff ect of vitamin E/selenium
against malathion induced injuries on nthe antiox-
idant status and apoptosis realted gene expres-
sion in rats. J Toxicol Sci 2011; 36: 285-296.
81) GURR JR, WANG AS, CHEN CH, JAN KY. Ultrafine tita-
nium dioxide particles in the absence of phopacti-
vation can induce oxidative DNA damage to hu-
man bronchial epithelial cells. Toxicol 2005; 15:
66-73.
82) AGARWAL S, TAFEL AA, KANAAR R. DNA double-strand
break repair and chromosome translocations.
DNA Repair (Amst) 2006; 5: 1075-1081.
83) TICE RR, AND STRAUSS GHS. The single cell gel eec-
trophoresis comet assay—a potential tool for de-
tecting radiation-induced DNA damage in hu-
mans. Stem Cells 1995; 13(Suppl 1): 207-214.
84) COLLINS AR, DUSINSKA M, GEDIK CM, AND STETINA R.
Oxidative damage to DNA: do we have a reliable
biomarker? Environ Health Perspect 1996; 104:
465-469.
85) HUANG TH, LEE H, KAO CT. Evaluation of the geno-
toxicity of zinc oxide eugenol-based, calcium hy-
droxide-based, and epoxy resin-based root canal
sealers by comet assay. J Endod 2001; 27: 744-
748.
86) SHARMA V, SHUKLA RK, SAXENA N, PARMAR D, DAS M,
DHAWAN A. DNA damaging potential of zinc oxide
nanoparticles in human epidermal cells. Toxicol
Lett 2009; 185: 211-218.
87) DUFOUR EK, KUMARAVEL T, NOHYNEK GJ, KIRKLAND D,
TOUTAIN H. Clastogenicity, photo-clatogenicity or
pseudo-photo-clastogenicity: genotoxic effect of
zinc oxide in the dark, in pre-irradiatd or simulta-
neously irradiated Chinese hamster ovary cells.
Mutat Res 2006; 607: 215-224.
88) MARTINEZ GR, LOUREIRO AP, MARQUES SA, MIYAMOTO
S, YAMAGUCHI LF, ONUK J, ALMEIDA EA, GARCIA CC,
BARBOSA LF, MEDEIROS MH, DI MASCIO P. Oxidative
and alkylating damage in DNA. Mutat Res 2003;
544: 115-127.
1827
N.M. Al-Rasheed, N.M. Al-Rasheed, N.A. Abdel Baky, L.M. Faddah, A.J. Fatani, I.H. Hasan, R.A. Mohamad
89) M ARNETT LJ. Oxy radicals, lipid peroxidation
and DNA damage. Toxicology 2002; 182: 219-
222.
90) N IEDERNHOFER LJ, D ANIELS JS, R OUZER CA, G REENE
RE, M ARNETT LJ. Malondialdehyde, a product of
lipid peroxidation, is mutagenic in human cells.
J Biol Chem 2003; 278: 31426-31433.
91) T A K A G I A , S A I K , U M E M U R A T, H A S E G A W A R ,
K UROKAWA Y. Inhibitory effects of vitamin E and
ellagic acid on 8-hydroxy-deoxyguanosine for-
mation in liver nuclear DNA of rats treated with
2-nitropropane. Cancer Lett 1995; 91: 139-
144.
92) CADENAS S, BARJA1 G, POULSEN HE, LOFT S. Oxida-
tive DNA damage estimated by oxo8dG in the liv-
er of guinea-pigs supplemented with graded di-
1828
etary doses of ascorbic acid and a-tocopherol.
Carcinogenesis 1997; 18: 2373-2377.
93) LEVI F, PASCHE C, LUCCHINI F, LA VECCHIA C. Dietary
intake of selected micronutrients and breast-can-
cer risk. Int J Cancer 2001; 91: 260-263.
94) BURILLO SL, TAN DX, MAYO JC, SAINZ RS, MANCHES-
TER LC, R EITER RJ. Melatonin, xanthurenic acid,
resveratrol, EGCG, vitamin C and α-lipoic acid
differentially reduce oxidative DNA damage in-
duced by Fenton reagents: a study of their indi-
vidual and synergistic actions. J Pineal Res
2003; 34: 269-277.
95) R YBAK LP, HUSSAIN K, WHITEWORTH C, SOMANI SM.
Dose dependent protection by lipoic acid against
cisplatin-induced ototoxicity in rats. Antioxidant
defense system. Toxicol Sci 1999; 47: 195-202.