CHAPTER 5

GENOTOXICITY SCREENING: CHROMOSOME

ABERRATION AND KARYOTYPING

5.1 Introduction

Genotoxicity evaluation is an ideal way to assess the molecular level

toxicity of nano sized materials and nano materials-based medical devices. There

are many methods to evaluate the genotoxicity of chemicals, bio materials and

nano sized materials, both in vivo and in vitro. The in vivo methods include

mammalian bone marrow chromosome aberration, mammalian bone marrow

micronucleus test, terratogenicity study, dominant lethal evaluation etc. The in

vitro methods include Ames test, in vitro mammalian chromosome aberration, in

vitro micronucleus, commet assay, sister chromatid exchange etc. Among these

the most suitable test for demonstrating chromosome aberrations is the in vitro

chromosome aberration in the metaphase chromosome using human peripheral

blood lymphocytes (ISO10993-3, 2003).

The chromosomes are most condensed and visualized during the

metaphase stage of the cell division. During this period the chromosomes will

arrange along the metaphase plate without separating the chromatids and

connected to the spindle fibres, which pulls both the chromatids to the poles in the

next phase of cell division. To visualise the metaphase chromosomes a metaphase

arrester/spindle poison can be used in the culture at appropriate time point.

Generally cochicine/colcemide is used as spindle poison.

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Chromosome aberrations and related events can cause many human

genetic diseases. Alterations in oncogenes and tumour suppressor genes in

somatic cells by chromosomal aberrations can induce cancer in humans and

experimental animals. In vitro chromosome aberrations study can check whether a

substance is capable of inducing structural chromosome aberration in cultured

mammalian cells (Evans, 1976, Ishidate and Sofuni, 1985 , Galloway et al.,

1987). Structural aberrations are of two types: Chromosome and chromatid

aberrations. Majority of chemicals induce chromatid type aberrations. As per

Organisation for Economic Co-operation and Development (OECD473, 1997), an

international standard, in the context of in vitro chromosome aberration test, the

cell culture can be exposed to the test substance with and without an exogenous

metabolic activator. This can be done with cell lines or primary culture. The

cytotoxicity or cell proliferation is another parameter to be evaluated along with

chromosome aberration. In the case of cell lines this can be evaluated by the

methods like confluency, cell number, cell integrity, viable cell counts, apoptosis,

necrosis, metaphase counting or population doubling time. In the lymphocyte

culture this can be done by mitotic index (MI). Human peripheral blood

lymphocyte culture for chromosome aberration is suggested to evaluate genotoxic

nature of MHA and NHA if any. The chromosome aberration has to be evaluated

at least with three concentrations of the test compound in presence and absence of

a metabolic activator for obtaining a valid result (OECD473, 1997). In the present

study five concentrations of MHA and NHA were analysed with and without

metabolic activation.

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Evaluation of number and appearance of chromosomes in the nucleus of a

eukaryotic cell or the complete set of chromosomes in a species, or an individual

organism is known as karyotyping. Karyotype is the arrangement of metaphase

chromosomes according to their size, shape and structure. G banding with trypsin

gives a distinct banding pattern to each chromosome which allows identifying

chromosomal imbalance, both structural and numerical. The arrangement of

chromosomes in a standard format according to the size, position of centromere

and banding pattern is called as karyogram (Stebbins, 1950). The major

applications of karyotyping are the detection of chromosome aberrations like

deletions, duplication, translocation and ploidy. This is a very valuable tool in

diagnosing reproductive disorders, oncohematology and genetic abnormalities.

This has many applications in nanotechnology also (Durnev, 2008, Yixin and

Max, 2013).

The production and applications of nano sized particles are enormously

increasing, subsequently human exposure to these materials also increased. This

may cause adverse health effects, so that toxicity evaluation of these particles is

necessary to confirm for its safety (Lakshmanan et al., 2013). The complete

physicochemical properties of the nano sized materials are essential for assessing

the toxicity of these particles. The main physicochemical properties include size

distribution, shape and other morphological features, chemistry of the material,

solubility, surface area, state of dispersion, surface chemistry etc (Powers et al.,

2006, Kunikazu et al., 2006). Many researchers reported several adverse effects of

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different nano particles. It was reported that the metal nano particles can cause

chromosomal aberrations, DNA strand breaks, oxidative DNA damage, and

mutations (Xie, 2011). The genotoxic effects of nano sized particles not only

depend on particle size, surface modification, and exposure route, but also on

exposure time (Klien, 2012). Metal ions can produce reactive oxygen species,

leading to metal nano particle induced genotoxicity (Liming et al., 2012).

The ceramic micro and nano particles (MHA and NHA) are indented to be

used for drug delivery or many biomedical applications. As this is designed for

biological application, there is interaction of these materials with the cells. Hence

the genotoxicity of these materials are to be evaluated to confirm the safety of

their uses. The objective of the present study is to evaluate the genotoxic effects

of different concentrations of MHA and NHA on human peripheral blood

lymphocytes by in vitro chromosome aberration test and by karyotyping. The

method followed is OECD 473 and G banding technique for chromosome

aberration and karyotyping of metaphase chromosomes.

5.2 Materials and methods

5.2.1 Chemicals
Human peripheral blood karyotyping medium (Invitrogen, India), RPMI

1640 medium, Foetal calf serum, colcemide and cyclophosphamide (Sigma

Aldrich), S9 fraction (Krishgen bio systems, India), Magnesium chloride, Sodium

phosphate, Heparin, Potassium chloride (KCl), DPX mountant, Methanol and

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Glacial Acetic acid (Merk, India), Glucose-6-phosphate (G-6-P) and NADP

(Himedia, India) were procured with AR grade.

5.2.2 Equipments
Autoclave (NatSteel, India), Laminar Bench (Mark air particulars, India),

Carbon dioxide incubator (Sanyo, Japan), Centrifuge (Remi, India), Microscope

(Leica, India), Automated microscope with metafer 4 software (Carl Zeiss, India),

Culture tubes (Tarson, India), microscopic slides and Cover clips (Blue Star,

India).

5.2.3 Chromosome aberration

The in vitro genotoxic potential of MHA and NHA was assessed by

chromosomal aberration study as per International standard: OECD 473: 1997 in

cultured human peripheral blood lymphocytes. Human peripheral blood was

collected from healthy voluntary donors in a sterile screw capped tube with

heparin at a dose of 40 units/mL, under aseptic conditions. Five hundred micro

litre blood was added to 4.5 mL of peripheral blood karyotyping medium, mixed

well and incubated at 37º C with 5% CO2 in a carbon dioxide incubator for 72 h,

mixed the culture in between. The culture was done in presence and absence of S9

fraction to check the genotoxic effect of MHA and NHA in presence and absence

of metabolic activator. S9 fraction is a post mitochondrial fraction of liver

homogenate of rodents, treated with enzyme-inducing agents such as Aroclor

1254, a rich source of metabolizing enzymes like P-450 (Thomas, 1992). After 48

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h of incubation, the cultures without S9 (S9-) fraction were treated with different

concentrations of MHA and NHA (10, 25, 50, 100, 250 µg/mL). The culture with

S9 (S9+) was centrifuged and transferred the cells to a low serum medium (RPMI

1640 with 2% foetal calf serum). S9 mix was prepared by mixing the S9 fraction

with 6M potassium chloride, 0.25 mM magnesium chloride, 0.2 M glucose-6-

phosphate, 0.04 M NADP, 0.2 M Sodium phosphate at 4º C and filter sterilized.

S9 mix (250 µL) and different concentrations of MHA and NHA were added to

each culture tube. After 3 h of incubation, the cultures were centrifuged and

removed the supernatant. The cell pellet was washed thrice with RPMI 1640

without serum. Resuspended the culture in the karyotyping medium and

incubated. At 70th h, 100 µL of colcemide was added to each culture tubes (S9+

and S9-) to arrest the metaphase and at 72 h harvested the cells. The harvested

cells were processed as follows:

 Hypotonic treatment: The cells were treated with 0.56% KCl at 37º C for

15 minutes.

 Fixation: The cells were treated with freshly prepared methanol acetic

acid mixture in 3:1ratio. This was repeated thrice.

 Dropping: Resuspended the cells with 0.5 mL of fresh fixative and

dropped the swelled and fixed cells to a pre chilled microscopic slide.

Dried the slides by flaming.

 Staining: The slides were stained with 10% Geimsa.

 Mounting: After drying, the stained slides were mounted with DPX

mountant.

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The stained and mounted slides were evaluated for chromosome aberration under

light microscope. The culture was done in duplicates and was repeated.

Physiological saline was used as negative control and cyclophosphamide (100

µg/mL) was used as positive control in S9- and S9+ cultures. Hundred well spread

metaphases were evaluated for chromosome aberrations. Each metaphase was

observed for chromosomal and chromatid aberrations like, chromosome break,

chromosome gap and chromatid break, chromatid gap, dicentric chromosomes

and chromosome or chromatid fragments. Each type of aberration was converted

to percentage aberration by the formula:

 Percentage aberration = Total number of aberrations in 100

metaphase/4600* X 100
(* 46 chromosomes in one metaphase plate x 100 metaphases)

The cell proliferation was evaluated by mitotic index.

Mitotic index was done by counting 1000 cells/culture and calculated by the

formula:

 Mitotic index = Number of cells in metaphase/1000 X 100 (expressed in

percentage).

The chromosome aberrations were expressed by photomicrographs and

numerically by means (% aberration) ± standard error.

5.2.4 Karyotying

Karyotyping was done by G banding technique of Seabright (Seabright,

1971). Cultures were set as same as for the chromosome aberration study.

Physiological saline was used as negative control and cyclophosphamide as

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positive control in S9- and S9+ cultures. At 69.5 h of culture, colcemide (100 µL)

was added and at 70 h, the cells were harvested, processed, metaphase spreads

were prepared as for chromosome aberration. The chromosome spreads prepared

were stained for karyotyping as follows. The slides were treated with 0.025%

Trypsin EDTA for 30 seconds for digestion and band formation. The banded

chromosome spreads were stained with 4% Geimsa. After drying, the slides were

mounted in DPX. The well spread metaphase chromosomes were analysed for

abnormalities in an automated microscope with Metafer 4 software. The results

were expressed as karyograms.

5.2.5 Statistical analysis

Statistical analysis was done for chromosome aberration study by One-way

Analysis of Variance, followed by Tukey-Kramer Multiple Comparisons Test

using GraphPad InStat software version 3.10 and ‘p’ value less than 0.05 is

considered as significant. All the experiments were done in triplicates and were

repeated twice.

5.3 Results
5.3.1 Chromosome aberration

MHA and NHA induced chromosome aberration was evaluated by in vitro

culture of human peripheral blood lymphocytes following OECD 473 guidelines.

The aberrations counted were converted to total aberrations by adding up all the

calculated percentage breaks (chromosomal and chromatid), gaps (chromosomal

and chromatid) and other aberrations (fragments, dicentric chromosomes etc) for

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both MHA and NHA in S9+ and S9-. The results are depicted quantitatively in

Tables 5.1 & 5.2 and Figures 5.1 & 5.2. The chromosomal aberrations induced by

the NHA and MHA were comparable with the negative control in the experiments

in S9+ and S9- (p > 0.05). Positive control exhibited significant increase in the

chromosomal aberrations than the negative control as well as the NHA and MHA

(p < 0.05). More over the positive control in S9+ showed increased number of

aberrations than positive control in S9-. Positive control in S9- showed aberrations

like chromatid gap and dicentric chromosome (Figure5.4 a, b) and in S9+ showed

chromatid break and dicentric chromosomes (Figures 5.4c, d). The chromosome

aberrations at same concentrations of MHA and NHA did not show any

significant difference both in S9- and S9+. The results are depicted qualitatively

in Figures 5.3 a, b, 54 a-d, 5.5 a-j and 5.6 a-j. In S9+ both NHA and MHA

induced aberrations comparable with the negative control. These showed a dose

dependant increase in aberrations but were not statistically significant. In S9-

NHA and MHA the aberrations were comparable to each other as well as with the

negative control. NHA and MHA in S9+ and S9- did not induce any chromosome

aberration at the concentrations tested in this study.

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Table 5.1: Total chromosome aberration induced by NHA and MHA in

S9+.Values are mean ± SE.

Concentration of NHA S9+ MHAS9+

Materials (µg/mL) Total chromosome aberration (%)
Negative control S9+ 0.1521±0.0069 0.1521±0.0069
Positive control S9+ 2.9456±0.0689* 2.9456±0.0689*
10 0.1358±0.0128 0.1355±0.01284
25 0.1576±0.0081 0.1458±0.0080
50 0.1695±0.0049 0.1467±0.0081
100 0.1730±0.0071 0.1521±0.0121
250 0.1705±0.0051 0.1724±0.0073
* indicates significance

Figure 5.1: Graph showing total chromosome aberration induced by NHA and MHA in
S9+. Values are mean±SE.

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Table 5.2: Total chromosome aberration induced by NHA and MHA in

S9-.Values are mean ± SE.

Concentration of NHA S9- MHAS9-

Materials (µg/mL) Total chromosome aberration (%)
Negative control
S9- 0.1358±0.0081 0.1358±0.0081
Positive control S9- 1.4510±0.0529* 1.4510±0.0529*
10 0.1252±0.0081 0.1630±0.0110
25 0.1565±0.0053 0.1545±0.0043
50 0.1533±0.0028 0.1502±0.0040
100 0.1537±0.0035 0.1559±0.0076
250 0.1574±0.0066 0.1497±0.0050
* indicates significance

Figure 5.2: Graph showing total chromosome aberration induced by NHA and MHA in
S9-. Values are mean±SE.

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Figure 5.3 a, b: Photomicrographs of metaphase chromosomes of

negative control in S9- (a) and S9+ (b)

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Figure 5.4 a, b: Photomicrographs of metaphase chromosomes of

positive control in S9- (a & b)

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Figure 5.4 c, d: Photomicrographs of metaphase chromosomes of

positive control in S9+ (c & d)

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Figure 5.5 a, b: Photomicrographs of metaphase chromosomes of NHA,

10µg/mL in S9- (a) and S9+ (b)

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Figure 5.5 c, d: Photomicrographs of metaphase chromosomes of NHA,

25µg/mL in S9- (c) and S9+ (d)

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Figure 5.5 e, f: Photomicrographs of metaphase chromosomes of NHA,

50µg/mL in S9- (e) and S9+ (f)

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Figure 5.5 g, h: Photomicrographs of metaphase chromosomes of NHA,

100µg/mL in S9- (g) and S9+ (h)

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Figure 5.5 i, j: Photomicrographs of metaphase chromosomes of NHA,

250µg/mL in S9- (i) and S9+ (j)

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Figure 5.6 a, b: Photomicrographs of metaphase chromosomes of MHA

10µg/mL in S9- (a) and S9+ (b)

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Figure 5.6 c, d: Photomicrographs of metaphase chromosomes of MHA

25µg/mL in S9- (c) and S9+ (d)

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Figure 5.6 e, f: Photomicrographs of metaphase chromosomes of MHA

50µg/mL in S9- (e) and S9+ (f)

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Figure 5.6 g, h: Photomicrographs of metaphase chromosomes of MHA

100µg/mL in S9- (g) and S9+ (h)

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Figure 5.6 i, j: Photomicrographs of metaphase chromosomes of MHA

250µg/mL in S9- (i) and S9+ (j)

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Table 5.3: Mitotic Index of NHA and MHA in S9+ and S9-. Values are mean±SD

Mitotic Index (%)

Controls S9+ S9-
Negative control 4.7 ± 0.10 4.6 ± 0.06
Positive control 2.6 ± 0.08* 3.0 ± 0.04*
Concentration of
NHA S9+ MHA S9+ NHA S9- MHA S9-
materials (µg/mL)
10 4.3 ± 0.11 4.4 ± 0.07 4.3 ± 0.07 4.4 ± 0.04
25 4.2 ± 0.10 4.2 ± 0.06 4.1 ± 0.08 4.1 ± 0.08
50 4.3 ± 0.11 4.4 ± 0.08 4.4 ± 0.06 4.4 ± 0.04
100 4.2 ± 0.10 4.2 ± 0.10 4.6 ± 0.07 4.5 ± 0.06
250 4.4 ± 0.04 4.4 ± 0.02 4.2 ± 0.08 4.3 ± 0.07
* Indicates significance

Mitotic index was also counted and calculated. The results of mitotic index

are given in Table 5.3. The MI of NHA and MHA did not show any significant

difference in S9+ and S9- in comparison with the negative control. The MI of

positive control revealed significant decrease both in S9+ and S9-. Positive

control with S9+ showed lesser MI than the S9-.

5.3.2 Karyotyping

The karyotyping was done by G banding technique and the results are

represented in Figures 5.7 a, b; 5.8 a, b; 5.9 a-j and 5.10 a-j. The pictoral

demonstration of chromosomes according to the size, position of centromere and

the banding pattern is known as karyogram. The chromosomes are arranged in 23

pairs according to the size, position of centromere and the banding pattern with

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the help of Metafer 4 software. The arranged chromosomes were analysed for any

abnormalities. The karyograms of different concentrations of MHA and NHA was

normal as negative control. The positive control induced abnormalities like

chromatid break, chromosome break and dicentric chromosomes (Figure 5.8a, b).

Positive control in S9+ showed Chromatid breaks in chromosome number 1 and

2. The chromosome number 16 of the same showed chromosome break and

chromosome 1 also showed dicentricity. Positive control in S9- showed

dicentricity in chromosomes 1, 4, 6, 12 and 14. The negative control and different

concentrations of NHA and MHA exhibited normal karyograms.

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Figure 5.7 a, b: Karyograms of negative control in S9- (a)and S9+ (b)

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Figure 5.8 a, b: Karyograms of positive control in S9- (a) and S9+ (b)

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Figure 5.9 a, b: Karyograms of NHA, 10µg/mL in S9- (a) and S9+ (b)

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Figure 5.9 c, d: Karyograms of NHA, 25µg/mL in S9- (c) and S9+ (d)

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Figure 5.9 e, f: Karyograms of NHA, 50µg/mL in S9- (e) and S9+ (f)

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Figure 5.9 g, h: Karyograms of NHA, 100µg/mL in S9- (g) and S9+ (h)

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Figure 5.9 i, j: Karyograms of NHA, 250µg/mL in S9- (i) and S9+ (j)

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Figure 5.10 a, b: Karyograms of MHA, 10µg/mL in S9- (a) and S9+ (b)

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Figure 5.10 c, d: Karyograms of MHA, 25µg/mL in S9- (c) and S9+ (d)

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Figure 5.10 e, f: Karyograms of MHA, 50µg/mL in S9- (e) and S9+ (f)

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Figure 5.10 g, h: Karyograms of MHA, 100µg/mL in S9- (g) and S9+ (h)

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Figure 5.10 i, j: Karyograms of MHA, 250µg/mL in S9- (i) and S9+(j)

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5.4 Discussion
The genotoxicity of MHA and NHA was evaluated by in vitro

chromosomal aberration study and karyotyping in human peripheral blood

lymphocytes. The present study used these two standard tests for genotoxicity

evaluation of nano particles. Zuzana and others reported that standard

genotoxicity tests can be used to evaluate DNA damage and chromosomal

aberrations induced by nano particles (Zuzana et al., 2012).

The results of in vitro chromosomal aberration revealed that NHA and

MHA did not induce any significant level of chromosome aberration. Different

concentrations of MHA and NHA were evaluated for chromosomal aberrations

and compared with negative as well as positive controls. It was found that the

aberrations induced by MHA and NHA were comparable with the negative

control. Positive control showed a significant increase in the chromosome

aberrations than MHA and NHA treatment as well as with the negative control.

Many researchers reported that silver nano particles can induce

genotoxicity in several types of cells (Asharani et al., 2009a, Asharani et al.,

2009b, Kumari et al., 2009, Kawata et al., 2009, Kim et al., 2011). There are

reports revealing that metal nanoparticles are capable of inducing genotoxicity by

the production of reactive oxygen species (Kunikazu et al., 2006). The ROS will

act as a signal molecule and interfere with cell cycle and induce oxidative DNA

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damage (Boonstra and Post, 2004, Hussain et al., 2005, Carlson et al., 2008, Xia

et al., 2006).

Nano particles of size < 100 nm can enter the cells, < 40 nm can enter the

nucleus and < 35 nm can cross blood brain barrier (Mytych and Wnuk, 2013). It

was also reported that the nano particles like metallic, metal oxide, semiconductor

nanoparticles, polymeric nano particles and carbon based nano particles can cause

cytotoxic effects, which is dose, cell type and treatment time dependent

(Dechsakulthorn et al., 2007, Arora et al., 2012). Silver nano particles of size 15

nm, molybdenum and aluminium nano particles of 30 nm were able to cause

adverse effects on mouse spermatogonia (Braydich-Stolle et al., 2005). Gold nano

particle’s toxicity not related to the ligand formation but with its size (Pan et al.,

2007). In the present study the ceramic particles (MHA and NHA) of different

sizes did not induce chromosomal aberration or genontoxicity in human peripheral

blood lymphocytes exposed to different concentrations.

The present study evaluated different exposure times. In the experiment

with S9+ the cells are exposed to ceramic particles both MHA and NHA for

nearly 24 h and in S9- the cells are exposed for 3 h. In both the experiments the

MHA and NHA did not induce chromosomal aberrations. This is in agreement

with the earlier studies in the titanium dioxide and zinc oxide nanoparticles. It was

also reported that zinc oxide and titanium dioxide nano particles on exposure for

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24 h induced tremendous toxic effects on human skin fibroblasts but 4 h exposure

caused only mild toxicity (Dechsakulthorn et al., 2007).

Higher concentrations of titanium dioxide nano particles could not induce

mutation but C-60 nano particles induced a dose dependant mutation in mouse

embryonic fibroblast (MEF) (Wise et al., 2010). In the present study the human

peripheral blood lymphocytes were exposed to different concentrations of MHA

and NHA. The results revealed that there are no significant observations in the

chromosome aberrations when the cells are exposed to different concentrations of

ceramic materials, MHA and NHA. It was reported that titanium dioxide nano

particles induced DNA and chromosome damage in in vitro condition than in in

vivo conditions (Tao et al., 2014).

Titanium dioxide nano particles can induce DNA single-strand breaks,

double-strand breaks, oxidative DNA or chromosomal damage in bone marrow

cells and can cause DNA deletions in offspring when it is exposed maternally

during pregnancy (Trouiller et al., 2009). Dobrzyńska and co-workers reported

that the single exposure of silver and titanium dioxide nanoparticles in rats

induced genotoxicity by micronucleus formation (Dobrzyńska et al., 2014). The

gold nano particles are capable of causing DNA double strand breaks and induce

cytokinesis arrest in cancer cells which in turn result in abnormal cell division and

cell death (Kang et al., 2010). Fe3O4 nanoparticles were found to cause cell cycle

arrest in G2/M phase of rat pheochromocytoma cells (Wu and Sun, 2011).

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The internalization and interaction of nano particles can lead to

cytotoxicity and genotoxicity, which depends on its size. Larger particles will

bind to the cell membrane proteins and induce permanent damage, but the smaller

particles will pass through the membrane and damage the organelles (Suh et al.,

2009). It was reported that human white blood cells do not have phagocytic

potential (Colognato et al., 2008). Hence internalization of nano particles is

happened in vitro by passive membrane diffusion process after a protein layer

formation over the nano particles from the serum present in the culture medium

(Oberdörster et al., 2005b, Oberdörster et al., 2005a). Even though the MHA and

NHA are interacting with the cells it is not capable of inducing chromosomal

aberrations.

It was reported that surface-modified silica particles did not express any

cytotoxic or genotoxic effects, as determined by various methods like, cell

viability, apoptosis/necrosis, oxidative DNA damage, chromosome aberrations

(Anna et al., 2013). From the results of the present investigation it can be

concluded that, irrespective of size, concentration and exposure time, MHA and

NHA did not cause genotoxicity in human peripheral blood lymphocytes up to a

concentration of 250 µg/mL in presence and absence of metabolic activator.

These findings are also in agreement with the results obtained in DNA adduct

formation (8-OHdG assay) study.

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5.5 References

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Krystyna, R.-W., Maria, W. & Stanislaw, S. 2013. Effect of surface
modification of silica nanoparticles on toxicity and cellular uptake by
human peripheral blood lymphocytes in vitro. Nanotoxicology, 7, 235-250.
Arora, S., Rajwade, J. M. & Paknikar, K. M. 2012. Nanotoxi- cology and in Vitro
Studies: The Need of the Hour. Toxicology and Applied Pharmacology,
258, 151-165.
Asharani, P. V., Hande, M. P. & Valiyaveettil, S. 2009a. Anti-proliferative
activity of silver nanoparticles. BMC Cell Biology, 10, 65.
Asharani, P. V., Low Kah Mun, G., Hande, M. P. & Valiyaveettil, S. 2009b.
Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS
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Boonstra, J. & Post, J. A. 2004. Molecular events associated with reactive oxygen
species and cell cycle progression in mammalian cells. Gene, 337, 1-13.
Braydich-Stolle, L., Hussain, S., Schlager, J. J. & Hofmann, M. C. 2005. In Vitro
Cytotoxicity of Nanoparticles in Mammalian Germline Stem Cells.
Toxicological Sciences, 88, 412-419.
Carlson, C., Hussain, S. M., Schrand, A. M., Braydich-Stolle, L. K., Hess, K. L.,
Jones, R. L. & Schlager, J. J. 2008. Unique cellular interaction of silver
nanoparticles: size-dependent generation of reactive oxygen species. The
Journal of Physical Chemistry B, 112, 13608-13619.
Colognato, R., Bonelli, A., Ponti, J., Farina, M., Bergamaschi, E., Sabbioni, E. &
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