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2012 28: 675 originally published online 27 October 2011Toxicol Ind HealthMurthy Prakhya

Surekha Pasupuleti, Srinivas Alapati, Selvam Ganapathy, Goparaju Anumolu, Neelakanta Reddy Pully and BalakrishnaToxicity of zinc oxide nanoparticles through oral route

  

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Toxicity of zinc oxide nanoparticlesthrough oral route

Surekha Pasupuleti1, Srinivas Alapati1,Selvam Ganapathy1, Goparaju Anumolu1,Neelakanta Reddy Pully2, and Balakrishna Murthy Prakhya1

AbstractThis experiment was aimed to determine the significance of dose by comparing acute oral toxicologicalpotential of nano-sized zinc oxide (20 nm) with its micro-sized zinc oxide. Sprague Dawley rats, 8 to 9 weeksold, were administered with 5, 50, 300, 1000 and 2000 mg/kg body weight (b.w.) of nano- and micro-sized zincoxide suspended in distilled water once through oral gavage. The effects of the micro- and nano-sized zincoxide on biochemical and hematological parameters were analyzed on day 14 of administration. The organswere collected for histopathology. Interestingly, inverse dose-dependent increase was noted in aspartateaminotransferase, alanine aminotransferase serum levels of nano-size zinc oxide groups when comparedwith their micro-sized zinc oxide. Clotting time was effected in all the male groups of nano-size zinc oxide,except in 1000 mg/kg b.w. The incidences of microscopic lesions in liver, pancreas, heart and stomach werehigher in lower doses of nano-size zinc oxide compared to higher dose. However, the incidences of abovelesions were higher in rats treated with a high dose of micro-sized zinc oxide. We conclude that nano-sizezinc oxide exhibited toxicity at lower doses, thus alarming future nanotoxicology research needs to befocused on importance of dose metrics rather following the conventional methods while conducting in vivoexperiments.

KeywordsZinc oxide, nanoparticles, dose, oral, acute toxicity

Introduction

Due to the wide application of nanomaterials in indus-

try, agriculture, business, medicine and public health

nanotechnology has gained a great deal of public

interest. Nanotechnology includes the integration of

these nanoscale structures into larger material compo-

nents and systems and construction of new and

improved materials at the nanoscale (Ju-Nam and

Lead, 2008). Nanoscience involves research to dis-

cover new behavior and properties or materials with

dimension at the nanoscale (NNI, 2008). Thorough

evaluation of desirable versus adverse effects is

required for safe applications of engineered nanoma-

terials (ENMs) and major challenges lie ahead to

answer the key questions of nanotoxicology

(Oberdorster, 2010).

Since the investment in nanotechnology research

and development is growing, the value of products

utilizing these technologies may exceed to $3 trillion

by 2015 (Lux Research, 2009); hence understanding,

identifying and addressing the potential risks of these

novel materials to human health and the environment

is of great interest (Chemical Industry Vision, 2020;

ICON 2008; Luther, 2004; Maynard, 2006; Maynard

et al., 2006; Oberdorster et al., 2005; PCAST, 2010;

RCEP, 2008; SCENIHR, 2009, 2005).

1International Institute of Biotechnology and Toxicology (IIBAT),Padappai, Tamil Nadu, India2Central Leather Research Institute, Adyar, Chennai, Tamil Nadu,India

Corresponding author:Pasupuleti Surekha, Department of Toxicology, InternationalInstitute of Biotechnology and Toxicology (IIBAT), Padappai601301, Tamil Nadu, IndiaEmail: santhoshmbt@gmail.com

Toxicology and Industrial Health28(8) 675–686ª The Author(s) 2012Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0748233711420473tih.sagepub.com

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Currently, metal oxide nanoparticles have not been

comprehensively assessed in regard to the potential

effects on human health, due to exposure (accidental

or otherwise) in the workplace during the production

of nanoparticles or exposure through use in commer-

cial products (e.g. titanium dioxide [TiO2] or ZnO

sunscreens).

Zinc oxide (ZnO) nanoparticles are widely used in

various applications including cosmetics, paints, as

drug carriers and fillings in medical materials (Dufour

et al., 2006). These metal oxide nanoparticles can also

be employed in environmental remediation due to

their good absorptive and photocatalytic properties

for elimination or degradation of pollutants in water

or air (Qiang, 2001). In addition, photocatalytic stud-

ies indicated that these nanomaterials showed good

photocatalytic performance for organic pollutants in

water (Wu and Huanga, 2010).

In spite of the fact that ZnO is a widely used ingre-

dient in dermatological preparations and sunscreens

and many other products, only two investigations

have focused on the effects of these nanoparticles

through oral administration on mice and through diet-

ary exposure on snails (Croteau et al., 2011; Wang

et al., 2008).

Although many studies aimed to understand the

importance of physicochemical properties such as

size, surface area, crystal structure, agglomerating

property and surface charge in influencing the biolo-

gical activity (Hoet et al., 1999, 2001; Hoshino

et al., 2004; Magrez et al., 2006; Nel et al., 2006;

Oberdorster et al., 2005; Wick et al., 2007), the use

of appropriate dose metric needs to be carefully con-

sidered. Keeping in mind the uncertainity in dose

selection, we have made an attempt to understand the

importance of dose by comparing nano-size ZnO with

its micro-size counterpart at different dose levels.

Materials and methods

ZnO nanoparticles (Stock No. 5810HT) were purchased

from Nanostructured and Amorphous Materials, Inc.

USA. The ZnO (Product No. ZO385) was purchased

from Sigma Aldrich, USA.

Physicochemical characterization

ZnO nanoparticles were synthesized by wet chemistry

method. The manufacturer specifications for nanoma-

terial characterization were confirmed by the

following techniques. The characteristics of ZnO

nanoparticles were assessed in the as-synthesized

form prior to use in experiments or after dispersion

in the vehicle (water) for dosing. Solution (water)

characteristics were measured with dynamic light

scattering (DLS), at Malvern Aimil Ltd, Bangalore.

The size of nano-size ZnO was determined with scan-

ning electron microscopy (SEM), at Anna University,

Chennai. SEM produces images by rastering a pri-

mary electron beam across the sample surface while

detecting secondary or backscattered electrons, which

are emitted from the surface. Therefore, the images

obtained in an SEM provide a 3D quality and greater

resolution. In this study, Hitachi S-520 SEM was used

at an accelerating voltage of 10,000 V after depositing

the samples onto aluminum stubs with double-sided

carbon adhesive tape. Photon correlation spectro-

scopy or DLS is an analytical technique capable

of measuring the size of very small particles, at

low sample concentrations. Measurement of parti-

cle size of nano ZnO in solution was determined

with DLS on a Malvern Zetasizer nanoseries (Nano

ZS) with Malvern application software version

6.20. This instrument can measure particle sizes

ranging from 0.6 nm to 6 mm using noninvasive

back scatter (NIBS) technology and DLS. The Mal-

vern Zetasizer can also provide zeta potential mea-

surements in aqueous and nonaqueous dispersions

using M3-phase analysis light scattering (PALS)

technology. Zeta potential is defined as the accu-

mulation of charge around the surface of a particle

in solution and gives an indication of the stability

of the colloidal system.

Animals and housing conditions

Experimental animals were obtained from in-house

animal facility. All procedures using animals were

reviewed and approved by the institutional animal

ethics committee.

Acute oral toxicity test in rats

Acute oral toxicity—acute toxic class method was

conducted per the Organization for Economic

Co-operation and Development (OECD) 423 guide-

lines (OECD, 2001) with modifications in terms of

different dose levels, usage of sexes, animal number,

and inclusion of hematology, biochemical parameters

and histopathology evaluation. The healthy Sprague

Dawley rats of both sex, aged between 8 and 9 weeks

and body weights of 180–220 g were used. Females

were nulliparous and nonpregnant. The animals were

procured from breeding facilities of International

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Institute of Biotechnology and Toxicology (IIBAT).

Animals were housed in polypropylene cages with

stainless steel grills and gamma-irradiated corn cobs

were used as bedding. Bedding material, cages, grills

and water bottles were changed on alternate days.

Animals were housed individually sex wise and group

wise. Animals were acclimated for a minimum period

of 5 days in the controlled environment (temperature:

22 + 3�C; relative humidity: 50 + 20% and light:

12-h light/dark cycle) and ad libitum supply of reverse

osmosis water and a standard rodent pellet feed

(supplier: M/s. Tetragon Chemie Pvt. Ltd, Bangalore,

India). Feed alone was withdrawn overnight prior

to the dosing and following dosing, for a period of

3 hours.

One hundred and ten animals were distributed

randomly into different groups (Table 1).

The test materials (either micro-size ZnO or nano-

size ZnO) were suspended in distilled water and

administered through oral gavage once at dose levels

of 5, 50, 300, 1000 and 2000 mg/kg body weight

(b.w.). The test solution was prepared shortly prior

to the administration. The dose volume maintained

for all the groups was maximum (10 ml/kg b.w.).

Similarly, control group of animals (5 males and

5 females) were dosed with distilled water alone.

Animals were observed for mortality/morbidity,

clinical signs of toxicity, weekly body weight and

weekly food consumption during the experimental

period.

At the end of 14 days of administration, the animals

were killed and the blood was obtained through

ophthalmic vein. The organs such as esophagus,

stomach, small and large intestines, liver, spleen,

thymus, mandibular and mesenteric lymphnodes, kid-

ney, urinary bladder, heart, pancreas, brain, lungs ovar-

ies and testes were collected, and all the organs were

kept in 10% buffered formalin and the testes in mod-

ified Davidson fluid.

Clinical biochemistry

The serum obtained after centrifuging was analyzed

for biochemical parameters such as creatinine,

albumin, alkaline phosphatase (ALP), alanine amino-

transferase (ALT), amylase, aspartate aminotransfer-

ase (AST), blood urea nitrogen (BUN), calcium,

cholinesterase, total cholesterol, glucose, HDL choles-

terol, iron, phosphorus, total protein, triglycerides,

urea and zinc using Humastar 300 fully automated

biochemistry analyzer (Human GmbH., Germany),

and sodium, potassium and chlorides in serum were

analyzed on day 14 by means of a humalyte electrolyte

analyzer (Human GmbH., Germany).

Hematology

Blood treated with EDTA was used for analyzing

hematology parameters such as erythrocyte count (red

blood cell [RBC]), hemoglobin, hematocrit (HCT),

mean corpuscular volume (MCV), mean corpuscular

hemoglobin (MCH), mean corpuscular hemoglobin

concentration (MCHC), platelet (PLT) count, total

leucocyte count (white blood cell [WBC]) and differ-

ential count (five parameters namely neutrophils,

eosinophils, basophils, lymphocytes and monocytes)

were determined on day 14 using Bayer ADVIA

120, fully automated hematology analyzer (Bayer,

Table 1. Groups and dose levels of micro- and nano-sizezinc oxide

Group Dose No. of animals

Micro-size zinc oxideG1 Vehicle control 5 Males and 5 femalesG2 (M-5) 5 mg/kg b.w. 5 Males and 5 femalesG3 (M-50) 50 mg/kg b.w. 5 Males and 5 femalesG4 (M-300) 300 mg/kg b.w. 5 Males and 5 femalesG5 (M-1000) 1000 mg/kg b.w. 5 Males and 5 femalesG6 (M-2000) 2000 mg/kg b.w. 5 Males and 5 females

Nano-size zinc oxideG7 (N-5) 5 mg/kg b.w. 5 Males and 5 femalesG8 (N-50) 50 mg/kg b.w. 5 Males and 5 femalesG9 (N-300) 300 mg/kg b.w. 5 Males and 5 femalesG10 (N-1000) 1000 mg/kg b.w. 5 Males and 5 femalesG11 (N-2000) 2000 mg/kg b.w. 5 Males and 5 females Figure 1. Scanningelectronmicroscopic (SEM) imageof nano

zinc oxide.

Pasupuleti et al. 677

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Germany). Clotting time of blood was determined by

capillary tube method.

Necropsy

Gross pathology was performed at the end of experi-

mental period (day 14).

Histopathology

Histopathology of organs (liver, spleen, kidneys,

heart, adrenals, lungs, pancreas, stomach, esophagus,

small and large intestine) were evaluated. Tissues

were collected and preserved in 10% buffered forma-

lin. All tissues required for histopathology evaluation

were subjected to dehydration procedure and processed

in tissue processor, embedded in paraffin wax and

prepared sections of 5–8 mm thickness and stained

with hematoxylin-eosin stain.

Statistical analysis

The data was expressed as mean þ/� standard devia-

tion for statistical analysis. A comparison of treated

rats with control groups was done using Newman–

Table 2. Nano zinc oxide characterization

Average sizea Size using SEM Size in distilled waterb Polydispersity index Surface areac (m2/g) Zeta potentiald

20 nm 63 nm 224.7 nm 0.305 50 �30.9

SEM: scanning electron microscopy.aAccording to the manufacturer.bUsing dynamic light scattering (DLS).cUsing BET (Brunauer, Emmett, Teller) analysis.dUsing zeta sizer.

Table 3. Clinical biochemistry parameters (males and females)

Group ALT AST Ca

MalesG1 65.0 + 14.1 141 + 16.6 8.0 + 0.6G2 (M-5) 83.2 + 12.9 129.2 + 9.0 8.0 + 0.6G3 (M-50) 71.0 + 13.6 135.6 + 6.3 8.4 + 0.7G4 (M-300) 72.8 + 19.8 139.8 + 11.6 9.1 + 0.3G5 (M-1000) 80.0 + 14.8 135.6 + 11.0 8.8 + 0.6G6 (M-2000) 73.0 + 14.0 151.8 + 6.9 8.3 + 0.5G7 (N-5) 747.0 + 120.6a,b 687.4 + 83.1a,b 10.1 + 0.1a,b

G8 (N-50) 514 + 88.0a,b 553.6 + 55.5a,b 10.3 + 0.2a,b

G9 (N-300) 428.6 + 111.5a,b 509.6 + 41.5a,b 10.6 + 0.4a,b

G10 (N-1000) 395.2 + 57.8a,b 411.8 + 66.6a,b 10.2 + 0.2a,b

G11 (N-2000) 298.4 + 119.1a,b 357.4 + 69.3a,b 10.1 + 0.1a,b

FemalesG1 68.6 + 12.2 150.6 + 11.5 9.2 + 0.6G2 (M-5) 78.6 + 8.4 152.8 + 10.7 9.6 + 0.8G3 (M-50) 71.8 + 8.8 152.2 + 12.0 9.2 + 0.8G4 (M-300) 79.2 + 6.4 157.4 + 9.1 9.4 + 0.8G5 (M-1000) 79.2 + 6.4 157.4 + 9.1 9.4 + 0.8G6 (M-2000) 76.0 + 15.0 140.8 + 12.4 9.7 + 0.6G7 (N-5) 712.2 + 169.5a,b 501.6 + 49.3a,b 10.0 + 0.3G8 (N-50) 521.4 + 39.8a,b 404.8 + 86.7a,b 9.6 + 0.8G9 (N-300) 464.2 + 61.2a,b 345.8 + 50.1 9.6 + 0.7G10 (N-1000) 355.6 + 106.6a,b 276.6 + 47.6a,b 10.2 + 0.8G11 (N-2000) 248.6 + 126.4a,b 226.8 + 18.8a,b 10.0 + 0.4

ALP: alkaline phosphatase, ALT: alanine transaminase, AST: aspartate transaminase, Ca: calcium.aStatistically different from control; p < 0.05 (n ¼ 5); mean þ/� standard deviation.bStatistically different from micro-size dose group; p < 0.05 (n ¼ 5); mean þ/� standard deviation.

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Keuls multiple comparison test. The data found to be

heterogeneous were subjected to nonparametric-

Kruskal–Wallis multiple comparison Z value test. The

comparison of nano- and micro-size ZnO dose groups

were done by Student’s t test and the heterogenous

data subjected to Mann–Whitney U test. The alpha

level at which all tests were conducted is 0.05, and the

NCSS 2007 software was used for analysis.

Results

Physicochemical characterization

The average size of the nano ZnO is 63 nm in SEM

analysis (Figure 1). The properties of the solution of

nanomaterials in distilled water were examined for

changes in size due to agglomeration using DLS

(Dufour et al., 2006). Average size was calculated

Male

Dose

AL

T (

U/L

)

2000 1000 300 50 50

200

400

600

800

1000

Figure 2. Alanine aminotransferase (ALT) activity in malestreated with nano zinc oxide at different dose levels (mg/kgbody weight [b.w.]).

Female

Dose

AL

T (

U/L

)

2000 1000 300 50 50

200

400

600

800

1000

Figure 3. Alanine aminotransferase (ALT) activity in femalestreated with nano zinc oxide at different dose levels (mg/kgbody weight [b.w.]).

Male

Dose

AST

(U

/L)

2000 1000 300 50 50

200

400

600

800

1000

Figure 4. Aspartate aminotransferase (AST) activity inmalestreated with nano zinc oxide at different dose levels (mg/kgbody weight [b.w.]).

Female

Dose

AST

(U

/L)

2000 1000 300 50 50

200

400

600

Figure 5. Aspartate aminotransferase (AST) activity infemales treated with nano zinc oxide at different doselevels (mg/kg body weight [b.w.]).

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by the software from the intensity, volume and num-

ber distributions measured (Table 2). The DLS results

illustrate that depending on the material, the nanoma-

terials in solution do not necessarily retain their nano

size (Dufour et al., 2006). The average size of nano

ZnO (in solution) was 224.7 nm. The polydispersity

index was 0.305. The zeta potential of nano-size ZnO

was �30.9 mV (Table 2).

Clinical biochemistry

The effects of micro-size and nano-size ZnO on rats

are presented in Table 3. The results indicated that the

plasma ALT and AST of nano-ZnO-treated rats were

significantly higher than the controls and with the

micro-size counterparts in both the sexes. There is

an inverse dose-dependent increase in the AST and

ALT serum levels in animals treated with nano-size

ZnO compared with their micro-size counterparts

(Figures 2–5). Calcium levels in the serum were also

significantly high from control as well as with the

micro-size ZnO-treated groups in males. Females

have no difference in the calcium levels. However,

there are no significant differences in rest of the para-

meters analyzed (Table 3; Figure 6).

Hematology

There were no statistically significant changes in the

hematologic parameters when compared to control.

Although few parameters such as PLT, HCT and MCV

are statistically different from their micro-size counter-

parts, the values are within the historical range of the

institute. A statistically significant increase in clotting

time was observed in all the treatment groups of nano-

size ZnO with that of micro-size ZnO dose groups except

group 10 ([G10] 1000 mg/kg b.w.; Table 4; Figure 7).

Necropsy

No gross pathological lesions were observed in any of

the treatment groups.

Histopathology

Animals treated with nano- and micro-size ZnO

showed lesions in liver and pancreas. In addition to

Male

Dose

Cal

cium

(m

g/dl

)

2000 1000 300 50 50

5

10

15

Figure 6. Calcium levels in males treated with nano zincoxide at different dose levels (mg/kg body weight [b.w.]).

Table 4. Hematology parameters—males

Group Clotting time (sec)

G1 (control) 83.20 + 9.71G2 (M-5) 107.20 + 9.98G3 (M-50) 111.40 + 12.82G4 (M-300) 105.40 + 26.47G5 (M-1000) 129.60 + 4.16G6 (M-2000) 97.00 + 35.19G7 (N-5) 127.60 + 8.26a

G8 (N-50) 144.80 + 20.91a,b

G9 (N-300) 115.20 + 14.02a,b

G10 (N-1000) 81.60 + 31.33G11 (N-2000) 124.80 + 22.93a,b

a Statistically different from control p < 0.05 (n ¼ 5); + standarddeviation.b Statistically different from micro-size dose group p < 0.05 (n¼ 5);+ standard deviation.

Male

Dose

CT

(se

c)

2000 1000 300 50 50

50

100

150

200

Figure 7. Clotting time inmales treatedwithnanozinc oxideat different dose levels (mg/kg body weight [b.w.]).

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this, animals treated with nano-size ZnO at 2000,

1000, 300, 50 and 5 mg/kg b.w. showed lesions in

stomach and heart as well (Figures 8–11). No lesions

were observed in the similar treatment groups of

micro-size ZnO (Tables 5 and 6).

Discussion

The current study investigated the toxicity response

of nano-size ZnO at different dose levels in Spra-

gue Dawley rats in comparison with their micro-

size ZnO counterparts. Although Wang et al.

(2008) has conducted studies on oral toxicity of

nano-size ZnO (20 and 120 nm) and zinc powder

in mice, they concluded that future research needs

to focus on the toxicity induced by exposure to low

oral dose of small-sized (20-nm) ZnO. We have

conducted the study with lowest dose ranging from

5 mg/kg b.w. to the highest dose of 2000 mg/kg

b.w.

A significant increase in the AST and ALT lev-

els was recorded in all the dose groups treated with

of nano-size ZnO (5–2000 mg/kg b.w. [N-5–N-

2000]). There was an inverse dose-dependent

response suggesting that the damage to liver is high

in the low-dose (5 mg/kg b.w.) group when com-

pared to the high-dose group (2000 mg/kg b.w.).

AST and ALT are the two liver enzymes whose

serum level gets elevated during necrosis, degen-

eration, hepatitis and inflammatory condition (Ellis

et al., 1978; Kellerman, 1995). The incidence of

histopathology lesions of liver correlated with the

elevated liver enzyme levels. Surekha et al. (in

press) have reported the inverse dose response of

nano-size ZnO through dermal route. There is no

change in AST and ALT levels in any of the

groups treated with micro ZnO (5–2000 mg/kg

b.w. [M-5–M-2000]). This may be due to their

large size. The nano-size ZnO at all the dose levels

has raised the above enzyme levels depicting

severe liver damage at the lowest dose levels, thus

suggesting the importance of particle number con-

centration rather than conventional mass concentra-

tion dose metrics. In contradictory to our findings,

previous studies (Maynard and Kuempel, 2005;

Oberdorster, 2000) through inhalation route have

(d) (e)(((((e(e((e(e(e(e(ee(ee(e(e((((e(((e(e(e(e(eeee(ee(e(e(e(e((e(eee(e(e((e(((e(e(e(((e(e(e(((ee(((e(e(e((e(e(e((e(e(e((e(e((((e(((((e((((((((((((((((((e(((ee(((((((((e((e(e(((((ee(((((((e(((((e(((((e((((((((((((((((((((((((((((((((((((((((((((( )))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))((d(d(d(d(d(d(d(d(ddd(d(dd(d(d(d(dddd(d((d(d(d(d(d(dd(d(dd(ddd((d(d(d(d(d(d(d((d(ddd(d(d(d(d(d((d(d(ddd(dddd(dd(d(d(dd((dd(dd((ddd(((dd(d(d((((dd(d((d(d(d(d(d((dddd(((((d(((((d((((d((((d((dddd(((dd(((dd(((((((((((((((((((((((((((((((((((((((((((( )))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))

(a) (b) (c)

Figure 8. (a) Normal structure of liver of control group. Degeneration of liver treated (b) with micro-size zincoxide at dose of 2000 mg/kg body weight (b.w.) and (c) with nano zinc oxide at dose of 2000 mg/kg b.w. Focalnecrosis of liver treated with (d) micro zinc oxide at a dose of 2000 mg/kg b.w. and (e) nano zinc oxide at a dose of2000 mg/kg b.w. (�20).

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suggested particle surface area as relevant metric

for small insoluble particles instead of mass concen-

tration which may not necessarily be applicable to the

oral route.

Wang et al. (2008) reported inflammatory cell

infiltration in pancreas at higher dose level (5 g/

kg b.w.) with 120 nm size ZnO powder in CD-

ICR mice. Although similar pancreatic lesion was

observed in the high dose (6 of 10), the incidence

of the lesions was higher in the lower dose (7 of

10) level (5 mg/kg b.w.). Histopathology evalua-

tion of heart and stomach revealed inflammatory

cell infiltration in higher incidence at lower dose

(5 mg/kg b.w.) group when compared to the higher

dose (2000 mg/kg b.w.). The inflammation may be

due to the generation of free radicals by the metal

oxide nanoparticles which leads to oxidative stress

(Kreyling et al., 2006).

Although there is increase in clotting time in males

of all the groups treated with nano-size ZnO except in

G10 (N-1000) the increase is not dose- and sex-

dependent. The increase in clotting time may be due

to decreased synthesis of proteins involved in the

coagulation cascade in the liver (Bloom and Brandt,

2001).

It is important to note that the doses used in pre-

viously published studies were higher than those to

which most people are likely to be exposed.

(a) (b)

(c)

Figure 9. (a) Normal architecture of pancreas of control group. Inflammatory cell infiltration treated (b) with micro-size zinc oxide at a dose of 2000 mg/kg body weight (b.w.) and (c) with nano-size zinc oxide at a dose of 2000 mg/kgb.w. (�20).

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Oberdorster (2010) recommended that, the results

of toxicological studies using extraordinarily high

experimental doses should be interpreted with cau-

tion. The high toxicity at low doses which were

evident in the present study may be due to the less

number of nanoparticles in that mass, which may

result in less agglomeration, making them to pene-

trate into the cells. In support to our hypothesis

Zook et al. (2011) reported that large agglomerates

of silver nanoparticles cause significantly less

hemolytic toxicity than small agglomerates.

Information about toxicity of nano-enabled prod-

ucts combined with the knowledge of unintentional

human exposure or intentional delivery for medic-

inal purposes must be necessary to determine real

or perceived risks of nanomaterials. Hence, our

results suggest that significance of dose—one of

the key concepts of nanotoxicology—needs to be

(a)

(b)

Figure 10. (a) Normal structure of stomach in controlgroup. (b) Inflammatory cell infiltration, polymorphstreated with nano-size zinc oxide at a dose of 5 mg/kgbody weight [b.w.] �20).

(a)

(b)

Figure 11. (a) Normal structure of heart in control group.(b) Inflammatory cell of heart at a dose of 5 mg/kg bodyweight ([b.w.] nano-sized zinc oxide; �20).

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addressed while evaluating the toxicity of engineered

nanomaterials.

Conflict of interest

The authors declared no conflicts of interest.

Funding

The research received no specific grant from any funding

agency in the public, commercial, or not-for-profit sectors.

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