Evaluation of histopathological and ultrastructural...

Indian Journal of Biotechnology

Vol. 17, January 2018, pp 9-15

Evaluation of histopathological and ultrastructural changes in the

testicular cells of Wistar rats post chronic exposure to gold nanoparticles

Himanshu Gupta1, Dipty Singh

2, Geeta Vanage

2, D S Joshi

1,3 and Mansee Thakur

3*

1Department of Genetics, MGM Institute of Health Sciences, Kamothe, Navi Mumbai, Maharashtra, India 2National Centre for Preclinical Reproductive and Genetic Toxicology (NIRRH),

National Institute of Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai, Maharashtra, India 3MGMCET & Department of Medical Biotechnology, Central Research Laboratory, MGM Medical College,

MGMIHS, Kamothe, Navi Mumbai, Maharashtra, India

Received 20 June 2016; revised 19 July 2016; accepted 28 July 2016

Gold nanoparticles (GNP) have numerous therapeutic potentials due to their ability to cross blood barriers. However, limited

data is available showing GNPs crossing the blood testicular barrier. Here we report results of chronic exposure (90 days) to

GNPs ranging in size 5 to 20 nm in male Wistar rats. Histopathological and transmission electron microscopy (TEM) analysis

show GNPs distributed and accumulated in majority of the testicular tissues. This shows the ability of GNPs of specific sizes to

cross the blood testicular barrier effectively, indicating possible insignificant toxicity to spermatogenesis process due to chronic

exposure. Thus, GNPs of smaller size can possibly be used for various therapeutic and diagnostic purposes.

Keywords: Gold nanoparticles (GNP), histopathology, blood testicular barrier, Wistar rats

Introduction

Advancements in the field of nanotechnology have

helped usage of nanoparticles in various fields of

medicine including diagnosis1, imaging

2 and therapy

3.

Nanoparticles (NPs) are easy to synthesize and their

physical properties (size and shape) can also be

altered easily. Moreover, NPs can be tagged with

specific ligands (drugs, antibodies or aptamers) for

enhanced targeting applications4-6

. Although the

potential of nanoparticles in the field of medicine is

promising, the lack of documented evidence of

toxicological effects of these nanoparticles is

concerning7,8

. It is therefore of critical interest to

study the effects of nanomaterials on animal systems,

their patterns of bioaccumulation in various organs

(including reproductive organs) and subsequent

biological consequences. Among the various classes

of nanoparticles gold based ones attract considerable

attention because of their unique properties pertaining

to therapeutic potential and drug delivery

mechanisms9. Ayurveda, the ancient Indian system

medicine has been using suvarnabhasmas, allegedly

containing Au-nanoparticles, as a potent drug in

treating many diseases10

. By altering the size and

shape of gold nanoparticles (GNPs) their plasmonic

resonance can be shifted to the near infrared

spectrum11

, at which the biological materials have low

absorption (optical therapeutic window). These GNPs

altered for absorption spectrum have high tissue

penetration and can be used in imaging and cancer

therapies12

. Decreasing the size of GNPs increases the

surface to volume ratio which manifests novel

changes in physical and chemical properties of these

NPs. Moreover, the decrease in size is expected to

affect the GNP’s interaction with the cells and tissues.

Size-dependent distributions of GNPs in various

organs have been studied in vivo12-17

. Although,

previous studies in mice based on oral administration

of GNPs have documented the inverse correlation

between size of the particles and absorption or

distribution of GNPs in various tissues and organs,

research on toxicity of GNPs are scant and more

specifically on reproductive toxicity. There are only a

few studies documenting the individual toxicity of

GNP in vivo, therefore it would be of interest to evaluate

the toxicity of gold nanoparticles18

. Currently, there are a

few data available regarding the accumulation of

nanoparticles in vivo after their repeated administration.

Here, we report the effect of chronic exposure of

GNPs in rat testicular tissue. We studied the ability of

——————

*Author for correspondence:

Mobile: 09769909212

[email protected]

INDIAN J BIOTECHNOL, JANUARY 2018

10

GNPs to cross blood testes barrier by histopathological

and ultra structural analysis transmission electron

microscopy (TEM). For the first time we present here

the GNP accumulation in testicular tissues and their

proliferation following chronic exposure.

Materials and Methods

Gold nanoparticles (GNPs) were freshly prepared and

confirmed by spectrophotometer. GNPs with average

diameter of about 15 nm were chemically synthesized in

the Department of Biotechnology, MGM Institute of

Health Sciences, Navi Mumbai, India19,20

. All the

reagents were used of analytical reagent grade. To

determine the size, shape, and aggregation state,

GNPs were analysed by TEM and UV–Visible

Spectrophotometer. The kinetics of particle development

was followed at λ = 519 nm. UV–visible spectroscopy

using 1 cm quartz cuvette on a Thermo-Scientific,

Evolution 201 series spectrophotometer. TEM was used

to characterize the particles. Sample was prepared by

placing a drop of solution containing nanoparticles on a

carbon-coated grid. Transmission electron microscopy

(Tecnai G2, 120kV TEM – FEI) at a voltage of 120 kV.

Animals and Treatment Groups

Ten to twelve weeks old adult male Wistar rats,

weighing approximately 150 ± 5 gm, were obtained

from the Haffkin Institute, Mumbai, India. The

animals were housed in humidity- and temperature-

controlled ventilated cages on a 12 h day/night cycle,

with free access to standard laboratory food and tap

water. The animals were randomly divided into 2

groups of 8 animals each. One group served as control

and received ordinary drinking water. Experimental

group was administered GNPs orally at a repeated

dosing of 20 µg/g for duration of 90 days. For oral

administration of the dose of nanoparticles gavage

was prepared by bending the tip of the metal tube and

coating it with silicone. Animal experiments were

carried out with the approval of Institutional Animal

Ethics Committee of the MGM Medical College,

Navi Mumbai. Rats were sacrificed 12–16 h after the

drug administration on the final day of drug

administration. The right and left testes were

removed, weighed and fixed in 10% formalin for

histological examination.

Histopathology

Rats were sacrificed by cervical dislocation, and

the testes were removed and fixed in 10% formalin

solution. Histopathological analysis was performed as

explained previously by Thakur et al20

.

Ultrastructural Analysis

Ultrastructural analysis of testes for presence or

absence of GNP’s was performed using TEM as

explained previously by Thakur et al20

.

Results

Synthesis and Characterization of GNPs

GNPs of average size of 15 nm were synthesized by

reduction of HAuCl4 with sodium citrate according

with the procedure described by Turkervich et al19

the

solution turn bright red after the addition of 2 ml of

sodium citrate(Fig. 1a). The GNPs synthesized showed

a defined plasmon resonance peak at 520 nm. This

gives brilliant red colour to gold nanoparticles (GNPs),

varies according to their size distribution. The colloidal

gold synthesized in experiment exhibited absorption

max at 520 nm (Fig. 1a). The extremely low

absorbance at wavelengths greater than 600 nm

indicated their well dispersed state in solution 20

. GNPs

were further analyzed by TEM analysis of the colloidal

gold solution. The size of gold nanoparticles has been

determined by measuring the diameter of whole

particles on TEM images (Fig. 1b). The average

diameter of colloidal gold was average size 15 nm.

Moreover the TEM images show that most of the gold

nano-spheres are round or spherical in shape at

different scale bar (Fig. 1c & d) showed particles of

small sizes (15.0 ± 5 nm) with regular shapes and

narrow size distribution. The size distribution of the

GNPs was determined.

The major objective of these studies was to

investigate if GNPs treatment produces toxicity due to

chronic exposure to GNPs in the rats. For this

purpose, it was necessary to determine if GNPs cross

blood testes barrier if so, their biodistribution and its

possible consequences. Our result indicates indicate

that despite the prolonged exposure to nanoparticles,

no mortality, morbidity or gross behavioural changes

were observed in rats receiving GNPs at the doses

studied. Our finding is similar to previous short

exposure studies where no mortality and gross

behavioral changes were observed21

. There was no

effect observed on the body weight. In tissue size no

evidence of atrophy, congestion, or inflammation could

be observed in the treated animals. These observations

indicate that extensive inflammation might not be

induced in the rat after administration of gold

GUPTA et al: POST CHRONIC EXPOSURE OF GOLD NANOPARTICLES IN TESTICULAR CELLS OF WISTAR RATS

11

nanoparticles, which is confirmed by the macroscopic

morphological examination.

Testes Histology

The rats from control group displayed normal

testicular architecture indicating seminiferous tubules

of various shapes and sizes. Stratified epithelium

(Fig. 2a) was visible with an orderly arrangement of

germinal cells (spermatogonia, primary spermatocytes,

secondary spermatocytes and spermatids in addition to

spermatozoa) and Sertoli cells. These tissues were

separated from one another by a delicate connective

tissue stroma (interstitial tissue) containing interstitial

Leydig cells. In contrast, sections in testes of treated rat

displayed seminiferous tubules in various shapes and sizes with mild sloughing of germ cells and detachment from the basement membrane. The degenerative tubules were lined by very few spermatogenic cells (Fig. 2b).

Fig. 1 — Characterization results of gold nanoparticle. 1a – Cherry red colour of synthesized gold nanoparticles & UV/Vis spectrum of

GNP’s (λmax – 519 nm), 1b - Histogram representing the number-based distribution of the mean core diameter of the nanoparticles, 1c-d.

transmission electron micrographs (TEM) of Au-particles prepared: c. scale bar at 50 nm, d. at scale bar of 200 nm.

Bio distribution of gold NPs in Testes

Fig. 2 — Histology section: 2a. Control- Testes histo-architecture of the rats that received normal saline (control) showed no damage to

the seminiferous tubules (ST-seminiferous tubule) and 2b. Treatment group -Test that received 20 ug/kg /bodyweight of GNP for

90 days showed less damage of the seminiferous tubules such as showing mild sloughing of germ cells (SGC) from the basement

membrane. Magnifications 40X.


12

Transmission Electron Microscopy: Effects on the Ultra

structure of Testes

Testicular Ultrastructure of Control Group

Electron micrographs of control group of animals

showed normal germ cells development starting from

spermatogonia to spermatids. Sertoli cells had

irregular large nucleus, prominent nucleolus and

cytoplasmic extension from the basal lamina of the

seminiferous tubule to the lumen supporting the germ

cells (Fig. 3A). Cytoplasm of the Sertoli cells

contained prosecretory granules, rosettes of glycogen

granules and free ribosome (Fig. 3A). Basal lamina

was surrounded by myoid cells having elongated

nucleus (Fig. 3B). Spermatogonia with a round or

oval nucleus and patchy chromatin materials were

observed near to the basal lamina (Fig. 3C). In control

group animals, a number of spermatocytes and round

spermatids were present towards luminal part of

seminiferous tubules (Fig. 3D). The spermatocytes

showed round prominent nucleus with distinct

chromatin networks and well-defined nuclear

membranes. Round spermatids had smaller nuclei

and their cytoplasm had mitochondria (Fig. 3E). The

testes of control group showed different stages of

elongating and elongated spermatids with normal

ultrastructure (Fig. 3F). Elongated spermatozoa also

presented with normal ultrastructure with the

characteristic shape of head, intact cell membranes,

acrosomes, and homogenous nuclei. The cytoplasm

organelles of different cells had no evident

morphological abnormalities.

Testicular Ultrastructure of Treated Group

Considerable bioaccumulation of GNPs was found

in the testes of treated group of animals. This indicates

that the GNPs can cross the blood testes barrier and

accumulate in the testes tissue. However, no

preferential site of accumulation was seen. Electron

micrographs of testes tissues showed abundunt gold

nanoparticles (GNPs) aggregated in interstitial spaces

of the testes (Fig. 4 A, B & C). GNPs could be clearly

seen in large aggregates near the Leydig cells. GNPs

were detected crossing the outer membrane of the

Leydig cells (Fig. 4D). Interestingly enough, GNPs did

not appear to induce any apparent toxicity to the

Leydig cell as they appeared structurally intact. Leydig

cell of treated group showed normal cytoplasm and

nucleus containing prominent rim of heterochromatin

attached to the nuclear membrane.

In experimental group, majority of the developing

germ cell were found to be structurally intact.

However, some spermatocytes and spermatids

presented with vacuolated cytoplasm (Fig. 4E) and

disrupted nuclear membrane. Membrane bound GNPs

were found to be present close to the developing

spermatids. Ectoplasmic specializations were disrupted

in those germ cells (Fig. 4F). Damage such as

chromatin clumping and fragmentation were also seen

in these germ cells (Fig. 4G). Aggregates of GNPs

were largely entrapped in vesicular structures and

distributed all over the Setorli cell cytoplasm (Fig. 4H).

Some of the spermatogenic cells showed degenerative

changes having deformed nucleus (Fig. 4I).

Fig. 3 — Transmission electron microscopy images of control group testicular cells: A. Sertoli cell nucleus (N) with nucleolus (Nu) and

cytoplasm (Cy). Scale bar = 2 µm, B. Myoid cells (My). Scale bar = 2 µm, C. Spermatogonial cell. Scale bar = 2 µm, D. Spermatocytes.

Scale bar = 2 µm, E. Round spermatid. Scale bar = 1 µm, F. Elongated spermatid. Scale bar= 0.5 µm.


13

Electron micrographs of the unstained ultrathin

sections of testes tissue from GNPs treated group

showed some very interesting results. In these

sections testes ultrastructure was faintly visible

whereas, aggregates of GNPs were clearly visible

distributed all over the interstitial spaces and Sertoli

cell epithelium. This was due to the fact that, gold

being heavy metal; GNPs can scatter electrons and

generate better contrast under electron microscope.

GNPs could be clearly located inside the testes and

exclude the probability of any kind of artefacts. We

could clearly see the GNPs inside the Leydig cell

nucleus which suggest that GNPs can cross nuclear

pore and bind to the chromatin (Fig. 5A). There were

abundant GNPs present in the Sertoli as well as in

germ cell cytoplasm entrapped in lysosomal bodies

(Fig. 5B, C, D, E, and F). The ultrastructure of the

testicular cell was not significantly hampered though

few abnormalities were noticed such as elongated

spermatid having deformed head and tail (Fig. 5G &

H). The other remarkable finding was the presence of

GNPs in the elongated sperm nucleus (Fig. 5I). In

some cases of the developing germ cells, cytoplasm

was also noticed vacuolated with presence of GNPs

(Fig. 5J).

Discussion

The ability to engineer nanoparticles with desired

characteristics has allowed nanoparticles to find

increased applications in various fields of medicine.

Studies have documented the bio distribution and

functions of nanoparticles as a function of their size

and shape22,23

in vitro24

and in vivo19

.

Among nanoparticles gold based ones are gaining

interest due to certain unique properties25

. Although,

gold nanoparticles are considered inert and

biocompatible, limited studies in mice have shown

dose dependent toxicity including erythrocyte cell

death26

and nephrotoxicity27

. In addition,

histopathological studies of testicular tissues from

mice acutely exposed to GNPs have shown them

crossing the blood testicle barrier16,28

. Our study was

aimed at exposing rats to chronic doses of GNPs and

to study their ability to cross the blood testicle barrier

and to study if there is any reproductive toxicity. This

is the first report showing the effects of chronic (90

days) exposure of gold nanoparticles on rat germ cells

in the peer-reviewed literature. Similar type of post

chronic exposure of GNP in testicular cells of Wistar

rat was studied by our team20

.

Fig. 4 — Transmission electron microscopy of treated testes

showing accumulation of GNPs near Leydig cells, inter testicular

spaces and germ cells: A. Leydig cells (Ly). Scale bar = 2 µm,

B. Accumulation of GNPs in intertubular spaces (arrow). Scale

bar = 1 µm, C. GNPs in intertubular spaces (arrow) at higher

magnification. Scale bar = 0.5 µm, D. GNPs entering to the

Leydig cell. Scale bar = 0.5 µm, E. spermatocytes presented with

vacuolated cytoplasm (arrow). Scale bar = 2 µm, F. Elongated

spermatid showing perturbed ectoplasmic specialization, membrane

bound GNPs (arrow) Scale bar = 1 µm, G. Germ cell with

vacuolated cytoplasm and deformed nucleus. Scale bar = 2 µm, H.

Sertoli cell cytoplasm showing GNPs accumulation (arrow). Scale

bar = 2 µm, and I. Spermatid having perturbed nucleus. Scale

bar = 1 µm.

Fig. 5 — Electron micrograph of treated group testes showing

presence of GNPs in nucleus and cytoplasm: A. Electron micrograph

showing GNPs (arrow) inside the Leydig cell nucleus. Scale bar =

1 µm. (B, C, D, E, F) GNPs (arrow) entrapped in lysosomal bodies,

B. Scale bar = 1 µm, C. Scale bar = 0.5 µm, D. Scale bar = 1 µm, E.

Scale bar = 0.5 µm, F. Scale bar = 1 µm. (G & H) image showing

elongated spermatid having deformed head and tail, G. Scale

bar = 0.5 µm, H. Scale bar = 2 µm and I & J. Presence of GNPs in

the elongated sperm nucleus (arrow) and vacuolated (arrowhead)

germ cell cytoplasm, I. Scale bar = 1 µm, J. Scale bar = 1 µm.


14

The structural and functional integrity of testes is

essential for normal reproductive capacity in males.

Studies shows germinal cells are extremely vulnerable

to the interference of external agents. Among them

nanoparticles pose a greater concern due to their

ability to cross the blood testicle barrier. Previous

studies28

in mice show GNPs crossing the blood

testicle barrier after acute exposure with the same.

Our TEM analysis of rat testicular tissues post 90

days exposure to GNPs shows accumulation of and

distribution of GNPs in majority of the testicular

tissues. For this study we used GNPs size range of

15 nm, and our ultrastructural analysis reveals the

presence of the same sized nanoparticles in the

testicular tissues. This indicates GNPs of the above-

mentioned size effectively crosses the blood testicle

barrier. Moreover, after crossing the blood testicle

barrier the GNPs was found present in the

seminiferous tubules and various types of cells in the

testes including spermatocytes, Leydig and Sertoli

cells20,29

. Distribution of GNPs in testicular tissues

was shown to be dependent on time and size of

exposure28

. One study, showed significantly higher

distribution of GNPs after a sixty-day exposure

compared to a week’s exposure. Similarly, the

distribution of GNPs sized between 10 and 50 nm are

effective compared to large sizes (>50 nm). Our

results with GNPs of average size of 15 nm for 90

days in rat testicles corroborate with these previous

studies. Prolonged exposure to GNPs could lead to

reproductive toxicity through degeneration of

testicular tissues. As seen by histological analysis

degeneration of testes occurs by sloughing of the

germinal epithelium from the basement membrane

and reduction in the population of the germ cells. Our

results from TEM analysis of treated rat testes

sections also show mild degenerative changes. Studies

have shown the effect on germ cells and Leydig cell

viability20,31

post exposure to TiO2, silver and Cb

based nanoparticles. Moreover, GNPs were shown to

reduce sperm motility. Cormier et al32

pointed out that

particles will affect the human reproductive system,

but what kind of nanoparticles (NPs) or (non NPs)

affect the human reproductive system were not

pointed out. However, it is demonstrated that not all

nanoparticles contribute towards reproductive

toxicity. Also supplements were shown to have a

positive effect on sperms in goats33

.

Thus it becomes impertative to investigate GNPs

induced toxicity depending on the size and time of

exposure. Even though we observed mild

degeneration of testicular tissues, our

histopathological results show presence of all the

different stages of testicular cells. They appear

healthy and they were highly similar with untreated

tissues. Moreover, the testicular tubules remained

healthy and normal and also, the relative proportions

of spermatogenic cells were not affected. Thus, our

results show the ability of the GNPs to cross the blood

testicle barrier and prove the distribution of

nanoparticles is dependent on their size and time of

exposure. The less severity in toxicity inspite of

distribution and accumulation of GNPs could possibly

be due to the size of the nanoparticles. Further studies

would be necessary to conclusively prove GNPs of

this size could be safely used for treatment and other

purposes at chronic doses. Understanding the effects

of nanotoxicology on male reproductive organ will be

beneficial in understanding the problem of male

infertility and setting up plans to solve it.

References 1 Kerman K, Morita Y, Takamura Y, Ozsoz M & Tamiya E,

Modification of Escherichia coli single-stranded DNA

binding protein with gold nanoparticles for electrochemical

detection of DNA hybridization, AnalyticaChimicaActa, 510

(2004) 169-174.

2 Agarwal A, Shao X, Rajian J, Zhang H, Chamberland D

et al, Dual-mode imaging with radiolabeled gold nanorods,

J Biomed Opt,16 (2011) 051307-1-051307-7.

3 Torchilin V P, Passive and active drug targeting: Drug

delivery to tumors as an example, Drug Delivery, 197 (2010) 3-53.

4 Shah D, Kwon S, Bale S, Banerjee A, Dordicket et al,

Regulation of stem cell signaling by nanoparticle-mediated

intracellular protein delivery, Biomaterials, 32 (2011)

3210-3219.

5 Chen B & Wu Wang, Magnetic iron oxide nanoparticles for

tumor-targeted therapy, CCDT, 11 (2011) 184-189.

6 Paulo C, Neves Ricardo P & Ferreira L, Nanoparticles for

intracellular-targeted drug delivery, Nanotechnology, 22 (2011)

494002.

7 Dziendzikowska K, Gromadzka-Ostrowska J, Lankoff A,

Oczkowski M, Krawczyska A et al, Time-dependent

biodistribution and excretion of silver nanoparticles in male

Wistar rats, J Appl Toxicol, 32 (2012) 920-928.

8 Kim Y, Song M, Park J, Song K, Ryu H et al, Subchronic oral

toxicity of silver nanoparticles, Part Fibre Toxicol, 7 (2010) 20.

9 Hu M, Chen J, Li Z, Au L, Hartland G et al, Gold

nanostructures: engineering their plasmonic properties for

biomedical applications, Chem Soc Rev, 35 (2006) 1084.

10 M R, Swarnabhasmas do contain nanoparticles? Int

J Pharma & Bio Sciences, 4 (2013) 243.

11 Galanzha E, Kokoska M, Shashkov E, Kim J, Tuchin V et al,

In vivo fiber-based multicolor photo acoustic detection and

photothermal purging of metastasis in sentinel lymph nodes

targeted by nanoparticles, J Biophoton, 2 (2009) 528-539.


15

12 Kamat P, ChemInform abstract: Photophysical,

photochemical and photocatalytic aspects of metal

nanoparticles, Chem Inform, (2002) 33(43).

13 Shipway A, Katz E & Willner I, Nanoparticle arrays on

surfaces for electronic, optical, and sensor applications,

ChemPhysChem,1 (2000) 18-52.

14 Kuwahara Y, Akiyama T & Yamada S, Facile fabrication of

photoelectrochemical assemblies consisting of gold

nanoparticles and a tris (2,2-bipyridine) ruthenium

(ii) viologen linked thiol, Langmuir, 17 (2001) 5714-5716.

15 Mirkin C, Letsinger R, Mucic R & Storhoff J, A DNA-based

method for rationally assembling nanoparticles into

macroscopic materials, Nature, 382 (1996) 607-609.

16 Lasagna-Reeves C, Gonzalez-Romero D, Barria M,

Olmedo I, Clos A et al, Bioaccumulation and toxicity of

gold nanoparticles after repeated administration in

mice, Biochem Biophys Res Commun, 393 (2010) 649-655.

17 Sonavane G, Tomoda K & Makino K, Biodistribution of

colloidal gold nanoparticles after intravenous administration:

Effect of particle size,Colloids and Surfaces B: Biointerfaces,

66 (2008) 274-280.

18 Lansdown A, Physiological and toxicological changes in the

skin resulting from the action and interaction of metal

ions, Crit Rev Toxicol, 25 (1995) 397-462.

19 Turkevich J, Stevenson P & Hillier J, A study of the

nucleation and growth processes in the synthesis of colloidal

gold, Discuss Faraday Soc, 11(1951) 55.

20 Thakur M, Gupta H, Singh D, Mohanty I, Maheswari U et al,

Histopathological and ultrastructural effects of nanoparticles

on rat testes following 90 days (chronic study) of repeated oral

administration, J Nanobiotechnol, 12 (2014) 42.

21 Frens G, Controlled nucleation for the regulation of the

particle size in monodisperse gold suspensions, Nat Phys Sci,

241 (1973) 20-22.

22 Rathore M, Mohanty I, Maheswari U, Dayal N, Suman R

et al, Comparative in vivo assessment of the subacute toxicity

of gold and silver nanoparticles, J Nanoparticle Res, 16

(2014) 12.

23 Lasagna-Reeves C, Gonzalez-Romero D, Barria M, Olmedo

I, Clos et al, Bioaccumulation and toxicity of gold

nanoparticles in vivo after repeated administration in

mice, Biochem Biophys Res Commun, 393 (2010) 649-655.

24 Zhao F, Zhao Y, Liu Y, Chang X, Chen C et al, Cellular

uptake, intracellular trafficking, and cytotoxicity of

nanomaterials, 7 (2011) 1322-37.

25 Lewinski N, Colvin V & Drezek R, Cytotoxicity of

nanoparticles, Small, 4 (2008) 26-49.

26 Schrand A, Braydich-Stolle L, Schlager J, Dai L & Hussain

S,Can silver nanoparticles be useful as potential biological

labels? Nanotechnology, 19 (2008) 235104.

27 Alkilany A & Murphy C, Toxicity and cellular uptake of

gold nanoparticles: what we have learned so far?

J Nanoparticle Res, 12 (2010) 2313-2333.

28 Gibson M, Danial M & Klok H, Sequentially modified,

polymer-stabilized gold nanoparticle libraries: convergent

synthesis and aggregation behavior, ACS Combinatorial

Science,13 (2011) 286-297.

29 Sopjani M, Foller M, Gulbins E & Lang F, Suicidal death

of erythrocytes due to selenium compounds, Cell Physiol

Biochem, 22 (2008) 387–394.

30 Balasubramanian S, Jittiwat J, Manikandan J, Ong C, Yu L et al,

Biodistribution of gold nanoparticles and gene expression

changes in the liver and spleen after intravenous

administration in rats, Biomaterials, 31 (2010) 2034-2042.

31 Ema M, Kobayashi N, Naya M, Hanai S & Nakanishi J,

Reproductive and developmental toxicity studies of manufactured

nanomaterials, Reprod Toxicol, 30 (2010) 343-352.

32 Cormier S, Lomnicki S, Backes W & Dellinger B, Origin and

health impacts of emissions of toxic by-products and fine

particles from combustion and thermal treatment of hazardous

wastes and materials, Environ Health Perspect, 114 (2006) 810-

817.

33 Shi L, Xun W, Yue W, Zhang C, Ren Y et al, Effect of sodium

selenite, Se-yeast and nano-elemental selenium on growth

performance, Se concentration and antioxidant status in growing

male goats, Small Ruminant Research, 96 (2011) 49-52.

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