Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 364-369

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal weblink: http://www.jett.dormaj.com

The Effects of ZnO Nanoparticles and

ZnO/Chitosan NCs on Liver Histology and

Serum Parameters in Rats

Hajinezhad MR

Associate Professor of physiology, Basic Science Department, Faculty of Veterinary Medicine, University of Zabol, Zabol,

Iran.

Received: 20/06/2019

Accepted: 02/08/2019

Published: 01/12/2019

Abstract

There are conflicting and confusing reports about prooxidant/ activities of Zinc oxide nanoparticles and Zinc oxide NCs.

This study aimed to resolve these discrepanciesby examiningthe effects of these compounds on liver histopathology in healthy

rats. Materials and Methods: 42 adult male Wistar rats were divided into 7 groups. Rats in the treatment group received

intraperitoneal injections of ZnO nanoparticles 10, 20 and 40 mmol/ml) and ZnO nanocomposites (NCs) (10, 20 and 40

mmol/ml) for 28 days. Control rats received distilled water. At the end of the study, the following parameters were assessed:

serum liver enzymes (ALT and AST), the activity of serum catalase (CAT) and superoxide dismutase (SOD), serum BUN and

serum creatinine and liver histology. Intraperitoneal injection of ZnO nanoparticles at a concentration of 10 mmol/ml/day had

no significant effect on serum liver enzymes but at 20 and 40 mmol/ml/day significantly decreased serum catalase and SOD

activity compared with the control group (P<0.05). ZnO NCs at the concentrations of 20 and 40mmol/ml/day decreased serum

catalase activity and SOD activities and significantly elevates serum liver enzymes. Furthermore, both ZnO nanoparticles and

ZnO NCs had no significant effect on serumBUN and creatininelevels. Both nanoparticlesinduced severe histological changes

at the two higher doses (20 and 40 mmol/ml). The results suggest that proper concentrations of ZnO nanoparticles and ZnO

NCs have no toxic effects on the liver while the higher doses can induce severe histological changes.

Keywords: ZnO nanoparticles, ZnO NCs, serum biochemical parameters, Rat

unique optical, chemical, mechanical and electrical

Introduction

properties, have attracted scientific attention (26). Based

on previous reports, ZnO NPs can increase or decrease

reactive oxygen species (ROS) generation and

subsequentlyoxidative stress in different parts of the body

Zinc is an essential component of many cytoplasmic

enzymes that participates in the metabolism of

carbohydrates, cytoplasmic proteins, lipids, and nucleic

acids. This element induces many biological,

physiological, and behavioral effects (1). Zinc is a

necessary element for the growth of the body and zinc

deficiency is associated with impaired immune function,

irritability and stunting of growth. Specific age groups

includinginfants, children, and adults are more susceptible

to Zinc deficiency (2). Zinc is stable in the environment

but is seldom abundant in food chains. Natural sources of

zinc include meats, fruits, legume, and seeds. Major

anthropogenic sources of zinc in the world are usually

arising from municipal, industrial and agricultural

activities (3). The brain, hematopoietic, hepatic and renal

systems are the most important organs affected by Zinc

deficiency (4). Nowadays there is an increasing trend

toward the use of Zn in nanotechnology in the form of Zinc

nanoparticles. Nanotechnology is a field of science which

is about the preparation of nanoscale materials (5-7).

Nanoparticles exhibit new and improved characteristics

such as morphology and size distribution respectinglarger

particles from which they are made (8-25). Zinc

nanoparticles are widely used in agriculture, cosmetic

industry and medicine. ZnO nanoparticles, due to their

(

27). Reactive oxygen species are short-lived organic

compounds with uneven number of electrons that stabilize

themselves by oxidizing biological molecules (28).

Normally, the reactive oxygen-containing molecules and

other free radicals can be quickly removed by natural

defense mechanisms such as Glutathione peroxidase,

superoxidedismutase,and catalase (29). Nanochamposites

are usually synthesized by using ionic gelation of

pentasodium tripolyphosphate and chitosan. Applications

of these compounds have also become more widespread

regarding their chemical properties (30). Adding chitosan

to Zinc structureaffects itsbiological and physicochemical

properties (31) So in the present study, we decided to

examine whether adding chitosan to zinc can decrease or

increase the and pro-oxidant activities of Zinc.

2 Material and Methods

2.1 Animals

Wistar rats (201- 234 g) obtained from Laboratory

Animal Center University of Zabol were used in the

current study. Animals were maintained in well-ventilated

Corresponding author: Hajinezhad MR. Associate Professor of physiology, Basic Science Department, Faculty of Veterinary

Medicine, University of Zabol, Zabol, Iran.

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 364-369

rooms at a constant temperature of 20–23 ◦C and 12 (h)

light/dark cycles with free to standard rodent food

embedding and block making of tissue samples, serial

sections were prepared by using the hematoxylin-eosin

method and were examined under a light microscope

(Olympus, Tokyo, Japan).

(

Javaneh-Khorasan, Iran) and tap water. they were handled

in accordance with the Animal Ethics Committee of the

University of Zabol, Zabol- Iran, and Guide for the Care

and Use of Laboratory Animals (National Institutes of

Health (NIH) publication 86–23; revised 1985. 42 adult

male Wistar rats were divided into 8 groups (7 rats in each

group: one control group and six treatment group. Rats in

treatmentgroup received intraperitonealinjections of ZnO

nanoparticles (10, 20 and 40 mmol/ml/day) and ZnO NCs

2.2 Statistical analysis

The collected data were analyzed with SPSS software

(version 20.0) expressed as mean ± SD. All Multiple

comparisons were performed by using one-way analysis of

variance (ANOVA) followed by posthoc Tukey's test.

Statistical signiﬁcance was set at P< 0.05.

(

10, 20 and 40 mmol/ml/day) for 28 days. Control rats

Results

received 1 ml of distilled water intraperitoneally for 28

days. At the end of our study, blood samples were

collected via retro-orbital puncture and centrifuged (3000

rpm for 10 minutes) for serum separation. The serum

samples were immediately frozen at −80◦ C. Following

blood collection, rats were sacrificed by cervical

dislocation and whole-brain tissues were isolated. The

fresh braintissueswere immediatelywashed with 0.9%Na

Cl and stored at −20°C for further determination of lipid

peroxidation in MDA form.

Intraperitonealinjectionof ZnO NPs at a concentration

of 10 mmol/ml had no significant effect on serum

biochemical parameters (P> 0.05). Also, liver histology

was not affected by this dose (fig.2). However, ZnO NPs

at 20 and 40 mmol/ml significantly decreased serum

catalase and superoxide dismutase activity compared with

the control group (P< 0.05) (fig.1). As seen in fig.2, serum

liver enzymes ALT and AST were increased by ZnO NPs

administration (P>0.05). Furthermore, intraperitoneal

injection of two low concentrations of ZnO NPs did not

affect serum creatinine and BUN fig.3. As seen in fig 3,

ZnO NPs intraperitonealinjection at a concentration of 40

mmol/ml caused a non-significant difference in kidney

function tests (BUN and serum creatinine) fig.3 (P>0.05).

ZnO NCs at a concentration of 40 mmol/ml decreased

serum catalaseand SOD activity (P< 0.05). Intraperitoneal

injection of ZnO NCs also increased serum liver enzymes

.1 Serum biochemical parameters

Analyses of serum ALT, AST and ALP levels were

performed by using the Selectra pro, M auto analyzer,

(

Vital Scientific, SpanNeren, Netherlands) with Pars

Azmoon reagents kit (Pars Azmoon. Co., Tehran, Iran).

Serum creatinine and BUN were measured using

commercial kits (Pars Azmoon Lab, Iran), according to

manufacturer’s instructions. Serum antioxidant enzymes

were measured usingcommerciall kits (ZellBio Germany)

and according to the company instructions.

(

fig 2), while serum BUN and creatinine levels were not

affected by ZnO NCs administration fig.3. As expected,

ZnO nanoparticles and ZnO NCs had no significant effect

on serum BUN and creatinine fig.5. Liver histological

investigation of rats received a low dose of ZnO NCs

showed normal architecture. However, the rats received

the 20 mmol/ml and 40 mmol/ml of NCs showed signs of

necrosis and hemorrhage (fig.5).

.2 Histopathological examination

After animals were euthanized by diethyl ether, liver

specimens were separated and washed in water. The liver

specimens were then sliced and preservedin a 10% neutral

buffered formalin solution (NBF). After parafﬁn

Fig. 1: Serum CAT and SOD activities in rats treated with ZnO NPs (X ±SD, n=10). *P < 0.05, compared to control group.

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 364-369

Fig. 2: Serum ALT and AST activities in rats treated with ZnO NPs (X ±SD, n=10). *P < 0.05 compared to control group

Fig. 3: Serum BUN and creatinine levels in rats treated with ZnO NPs (X ±SD, n=10). *P < 0.05compared to control group.

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 364-369

Fig. 4: Liver histology in control rats (a): Normal liver architecture with intact hepatocytes and sinusoids, rats received ZnO nanoparticles 10

mmol (b): normal hepatocytes, rats received ZnO nanoparticles 20 mmol (c): necrosis (arrow), rats received ZnO nanoparticles 30 mmol (d):

necrosis (arrow) and fatty changes. . H&E staining (× 40)

Fig. 5: Liver histology in control rats (a): healthy hepatocytes and well-arranged sinusoids, rats received ZnO nanoparticles 10 mmol (b):

normal liver histological pattern, rats received ZnO nanoparticles 20 mmol (c): necrosis (arrow), rats received ZnO nanoparticles 30 mmol (d):

necrosis (arrow) and bleeding. . H&E staining (× 40).

catalase and superoxide dismutase activity is an indicator

Discussion

of pro-oxidant activities of ZnO nanoparticles (36).

Superoxide radical anion , peroxyl radicals which are the

major reactive oxygen species generated duringoxidative

stress, may induce liver fibrosis and fatty changes by

activating type I procollagen synthesis enzyme. This

process may led to the generation of lipid peroxidation

end-products. Our eukaryotic cells have a broad types

of oxidative defense mechanisms, like enzymatic

molecules such as CAT, SOD, glutathione peroxidase and

molecular scavengers such as vitamin A, reduced

The effects of ZnO NPs on serum oxidative stress

biomarkers have not been fully elaborated yet. Some

studies had reported the properties of ZnO nanoparticles

(

32) while other studies indicated oxidative damage

caused by ZnO nanoparticles (33). As seen in this study,

administration of ZnO nanoparticles decreased serum

enzyme activities. These results were in line with previous

studies about the biological effects of ZnO nanoparticles

(

34, 35) It can be concluded that the decreases in serum

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 364-369

glutathione (GSH), ascorbic acid, vitamin E (alpha

6. Alaee S, Ilani M. Effect of titanium dioxide nanoparticles on

male and female reproductive systems. Journal of Advanced

Medical Sciences and Applied Technologies. 2017;3(1):3-8.

tocopherol)

and

N-acetyl-5-

methoxytryptamine (melatonin) (37). In this investigation,

decrease in the activities of SOD and CAT might be due

to increased generation of reactive oxygen species or

increased availability of NADPH that is required to

maintain oxidative defenses mechanisms. These results

suggest that the ZnO nanoparticles have free radical

producing effect and can reduce the activity of the

Ravanshad R, Karimi Zadeh A, Amani AM, Mousavi SM,

Hashemi SA, Savar Dashtaki A, et al. Application of

nanoparticles in cancer detection by Raman scattering based

techniques.

Nano

reviews

experiments.

2018;9(1):1373551.

8. Amani A. Synthesis and biological activity of piperazine

derivatives of phenothiazine. Drug Res. 2015;65(01):5-8.

Beheshtkhoo N, Kouhbanani MAJ, Savardashtaki A, Amani

AM, Taghizadeh S. Green synthesis of iron oxide

nanoparticles by aqueous leaf extract of Daphne mezereum

endogenous enzymes. In this study,

significant

increase in serum AST and ALT levels was observed in

rats treated with ZnO nanoparticles. These results were in

line with other studies indicating hepatic damage caused

by ZnO nanoparticles or with studies indicating

hepatoprotective potential of these nanoparticles (38, 39).

As previously noted, damage to liver tissues releases the

AST, ALP and ALT into the serum, and, hence, elevation

of serum activities of these enzymes is considered a

valuable marker of liver damage (40). Serum creatinine

and blood urea nitrogen are used as indicators of

glomerular filtration rate and renal function (41). ZnO

nanoparticles or ZnO nanochitosans had no significant

effect on serum kidney function markers. These findings

were in contrast with previous studies that reported

significant increase in serum kidney markers following

treatment with ZnO nanoparticles (8-23, 42). This result

might be due to the low doses of ZnO nanoparticles or

ZnO nanochitosans used in this study. This observation led

us to conclude thatlow concentrations of thesecompounds

have potential without any toxicity in vivo.

as a novel dye removing material. Appl Phys A.

018;124(5):363.

0. Hashemi SA, Mousavi SM, Faghihi R, Arjmand M, Sina S,

Amani AM. Lead oxide-decorated graphene oxide/epoxy

composite towards X-Ray radiation shielding. Radiation

Physics and Chemistry. 2018;146:77-85.

1. Kouhbanani MAJ, Beheshtkhoo N, Amani AM, Taghizadeh

S, Beigi V, Bazmandeh AZ, et al. Green synthesis of iron

oxide nanoparticles using Artemisia vulgaris leaf extract and

their application as a heterogeneous Fenton-like catalyst for

the degradation of methyl orange. Materials Research

Express. 2018;5(11):115013.

12. Kouhbanani MAJ, Beheshtkhoo N, Fotoohiardakani G,

Hosseini-Nave H, Taghizadeh S, Amani AM. Green

Synthesis and Characterization of Spherical Structure Silver

Nanoparticles Using Wheatgrass Extract. Journal of

Environmental Treatment Techniques. 2019;7(1):142-9.

3. Kouhbanani MAJ, Beheshtkhoo N, Taghizadeh S, Amani

AM, Alimardani V. One-step green synthesis and

characterization of iron oxide nanoparticles using aqueous

leaf extract of Teucrium polium and their catalytic

application in dye degradation. Advances in Natural

Sciences:

Nanoscience

and

Nanotechnology.

Conclusion

019;10(1):015007.

Our results indicate that ZnO nanoparticles have

4. Lohrasbi S, Kouhbanani MAJ, Beheshtkhoo N, Ghasemi Y,

Amani AM, Taghizadeh S. Green Synthesis of Iron

Nanoparticles Using Plantago major Leaf Extract and Their

Application as a Catalyst for the Decolorization of Azo Dye.

BioNanoScience. 2019;9(2):317-22.

similar effects compared to ZnO NCs. Further studies are

required to elucidate the effects of these compounds in

laboratory animals.

5. Mahdavinia GH, Rostamizadeh S, Amani AM, Sepehrian H.

Fast and efficient method for the synthesis of 2-

arylbenzimidazoles using MCM-41-SO3H. Heterocycl

Commun. 2012;18(1):33-7.

6. Mousavi SM, Hashemi SA, Arjmand M, Amani AM, Sharif

F, Jahandideh S. Octadecyl amine functionalized Graphene

oxide towards hydrophobic chemical resistant epoxy

Nanocomposites. ChemistrySelect. 2018;3(25):7200-7.

7. Rostamizadeh S, Amani AM, Aryan R, Ghaieni HR, Norouzi

L. Very fast and efficient synthesis of some novel substituted

Acknowledgment

This study was based on the grant on University of

Zabol Grant number: 9618-15. We are grateful to

University of Zabol for financial support.

Conflicts of interest

There are no conflicts of interest.

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