ABSTRACT

Nanogenotoxicology is an emergent area of

estimating the potential

research , relevant for

carcinogenic risk of nanomaterials. The

present study is to analyse about the toxicological risk of nano copper

particles. The toxicological effect of nano particles was studied on
Drosophila

melanogaster

.The

nanoparticles

formed

characterized by UV-VIS spectroscopy ,FTIR and Scanning

where
Electron

Microscopy .Their genotoxicity analyses were studied on Drosophila

melanogaster.
1 INTRODUCTION
Nanotoxicology is a multi-disciplinary area of applied science and
engineering , It deals with design and manufacture of extremely small
components in nanosizes .
The

nanoparticles

behave

according

to

the

law

of

quantum

mechanism. One of the benefits of nanoparticles is that their property

differ from that of bulk material , the nanoparticles can be easily varied
by changing their size ,shape and chemical environment.

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

metal nanoparticles are used in various field due to high surface area
and antimicrobial activity . copper is a nontoxic , safe inorganic,
antibacterial agent .
Copper has an excellent electrical conductivity. Due to relatively low costs, this
metal plays a significant role in modern electronic circuit . Because of its
excellent electrical conductivity, catalytic behaviour, good compatibility and
surface enhanced Raman scattering activity, Cu nanoparticles have drawn the
attention of scientists to be used as essential component in the future nanodevices ( Sulekha Chandra et al ,Avdesh Kumar et al, [raveen kumar
Tomar et al 2011)
1.1

COPPER NANOPARTICLES

Copper is a Block D, Period 4 element. It is a ductile metal with very high thermal
and electrical conductivity. The morphology of copper nanoparticles is round, and
they appear as a brown to black powder.Copper Nanoparticles are a class of
materials with properties which differ from their characteristics and find use in
different areas such as electronic, magnetic, pharmaceutical, cosmetic energy,
catalytic and materials applications.

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

1.2 GENOTOXICITY OF COPPER NANOPARTICLES

copper nanotoxicity
Toxicological studies have shown increased toxicity of nanoparticles (<100nm)
compared to micrometer particles of the same composition, which has raised
concern about the impact on human health from nanoparticles. (Hanna L et al,
Karlson 1 et al,

johanna gustafssun et al,

Printus cornholm et al ,

Lennart molle et al 2009)

it was reported that the toxicity of copper particles increases with the
decrease of the particle size on a mass basis. The results suggest that
when the sizes of particles become small and down to a nanoscale,
copper becomes extremely reactive in a simulative intracorporeal
environment. The nanosized copper particlesUltrahigh reactivity and
grave nanotoxicity of copper nanoparticles
consume the hydrogen ions in stomach more quickly than micron
ones. These processes further convert the copper nanoparticles into
cupric ions whose toxicity is very high in vivo. (Huan Menget al,Zhen
Chen et al Gengmei Xing et al ,Hui Yuan et al 2007)

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

Nanotechnology is concerned with materials and systems whose structures and

components exhibit novel and significantly improved physical, chemical and
biological properties, phenomena and processes due to their nano scale size.
Recently, studies on the biological effects of nanomaterials show signs that some
of the manufactured nanoparticles exhibit unexpected toxicity to living organisms.
It has previously been reported that the copper particles possess size-depended
toxicity (Huan Menga et al ,Zhen Chena et al , Gengmei xiinga et al, Hui
yuana et al 2007)
Kidney, liver and spleen are found to be target organs of nano-copper particles.
Nanoparticles induce gravely toxicological effects and heavy injuries on kidney,
liver and spleen (Zhen chen et al ,Gengmei xing et al ,Chunying Chen et
al, 2006)
Copper is highly toxic to aquatic organisms and may cau d reproduction .
Although copper is considered an essential element, its high concentrations in
water are toxic to freshwater organisms.(Tao wang et al, xiaohua long et al,
yongzuou cheng et al, zhaopu liu et al, shaohua yan et al 2014)

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

As a result of the growing nanotechnology applications, many nano-metals are

discharged into the aquatic habitats that affect its biota
The genotoxic effect of

copper nanoparticles were studied on

drosophila melanogaster.
1.2.1 Drosophila melanogaster
Drosophila melanogaster, is commonly called the fruit fly.It is the most
common model organism for the study of eukaryotic biology .it is the
common model organism for research as its behavioral pattern are
parallel

to human beings with homology upto 70% .It gives a wide

range of genetic information

1.2.2 morphology
The body of Drosophila is divided into head ,thorax ,abdomen . The
head carries pair of antennae , red coloured compound eyes and three
simple dorsal eyes.the abdomen consist of seven to eight visible
segments in female and five to six segments in male , The thorax is
composed of three segments

namely prothorax , mesothorax and

metathorax which are fused. There are three pairs of jointed legs

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

.There are two pairs of wings present , the first pair is attached to
mesothorax region and second pair is small and is known an halters.
The body is yellowish brown in colourand has transverse black rings
across the abdomen . The male is easily distinguishable from the
female
1.2.3 Life cycle
EGGS : A single female lays 50 to 75 eggs in a say and maximum of
500 eggs are laid in 10 days
LARVA : The egg hatches into a worm like white larva in about 20-30
hours .They feed on the medium with the help of black chitinous
jaws.there are three larval stages and these are the first , second and
third instars.The larva grows upto a length of about 4.5mm
PUPA : The mature larva creeps on the sides of culture vial or bottle
then it pupates within its larval skin ,the cuticle of the third instar
becomes hardened and darkened in colour,thus orming pre-pupa .It
undergoes metamorphism during which the adult structure and
internal organs develop

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

IMAGO : Oncethe metamorphism is complete ,young adults emerge out

of the pupal case .the fly is fragile light in colour with long abdomen
and unexpanded wings, after a few hours the body colour darkens ,
abdomen becomes rounded and the wings extend

Life cycle in Days from the time of laying eggs

Day 0 : Egg is laid
Day 1 : Egg hatches
Day 2: First instar
Day 3: second instar
Day 5:Third instar
Day7:Pupa formation occurs
Day 11-12 Eclosion (adults emerge from pupa case) females become
sexually mature

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

1.2.4 Advantages of using Drosophila melanogaster

1. Small and easy to grow in laboratories
2. Has high frequency rate
3. Has short lifecycle
4.Exhibits sexual dimorphism
5.Giant salivary gland chromosome
6 Demonstartes genetic variability
7Phenotypes are easily distinguishable

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

SYNTHESIS AND CHARACTERISATION

BY CHEMICAL REDUCTION METHOD

CHEMICALS USED:
Copper sulphate - 0.319g in 20ml of water
PEG 6000

- 2.4g in 20ml of water

Ascorbic acid

-0.070 in 20ml of water

Sodium hydroxide -0.0799 in 20ml of water

Sodium boro hydrate -0.07566 g in 20ml water

1)copper sulphate

- 0.319g of Copper sulphate

was dissolved in 20ml of

distilled water and stored in a brown bottle.

2) PEG 6000

-2.4 g of poly ethylene glycol was dissolved in 20ml of distilled

water
3)ASCORBIC ACID

-0.070g of ascorbic acid was dissolved in

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

20 ml of distilled water
4)SODIUM HYDROXIDE-0.0799 Of NaoH was dissolved in 20ml of distilled
water
5)SODIUM BOROHYDRATE -0.07566g of sodium borohydrate was dissolved in
20ml of distilled water
METHOD:
Copper sulphate was first dissolved in 20ml of water,shows the presence of blue colour
,and then to it PEG 6000 dissoled in 20 ml was and mixed well, white solution appears, to
this ascorbic acid and sodium hydroxide each dissolved in 20ml of water was added and
mixed well ,yellow color appears ,finally sodium boro hydrate was added and mixed well
, the final colour is black colour this confirms the presence of copper nanoparticles since
the confirmatory step is the appearance of black color

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

2.3

CHARACTERISATION

OF

THE

SYNTHESIZED

COPPER

NANOPARTICLES
2.3.1 VISUAL INSPECTION

The reduction of metal ions was monitored by visual inspection of the color
change in the solution . The conversion of the colourless reaction mixture to a black color
clearly indicated the formation of copper nanoparticles.
RESULT

The original colour of the reaction mixture after the addition of coppersulphate to
water gives blue colour .addition of coppersulphate to PEG 6000 gives white colour
solution .when mixed with ascorbic acid and sodium hydroxide yellow colour solution is
formed.when sodium borohydrate was added he solution turns black. The colour changes
observed is shown below.

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

Figure : Visual observation after the addition of sodiumborohydrate

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

2.3 UV-VIS spectroscopy

UV-VIS spectroscopy is the measurement of the wavelength and
intensity of absorption of near-ultraviolet and visible light by sample.
Ultraviolet and visible lights are energetic enough to promote outer electrons to higher
energy levels. UV-VIS spectroscopy is usually applied to molecules and inorganic ions or
complexes in solution. The UV-VIS spectra have broad features that are very useful for
quantitative measurements. The reduction of metal ions was monitored by measuring the
UV-VIS spectroscopy of the solution according to the method of Mie (1908), by the
sampling of aliquots (3ml) of the aqueous component

RESULT

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

2.4 SCANNING ELECTRON MICROSCOPY (SEM)

SEM investigations provide valuable information about morphology ,
surface and composition of sample .they are mostly preffered because
they require little preparation of specimen and the image can be
obtained rapidly
RESULT

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

2.5 FOURIER TRANSFORMATION INFRARED SPECTROSCOPY (FTIR)

Fourier transform infrared spectroscopy(FTIR)is a technique which is used to

obtain Infrared sprectruof absorption or emission of a solid, liquid or gas. An FTIR
spectrometer simultaneously collects high spectral resolution data over a wide
spectral range. This confers a significant advantage over a dispersive
spectrometer which measures intensity over a narrow range of wavelengths at a
time.
RESULT

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

2.6

X RAY DIFFRACTION (XRD)

X-ray crystallography is a tool used for identifying the atomic and

molecular structure of a crystal, in which the crystalline beam of
incident X-rayto diffract into many specific directions. By measuring
the angles and intensities of these diffracted beams, a crystallographer
can produce a three-dimensional picture of the density of electrons
within the crystal. From this electron density, the mean positions of the
atoms in the crystal can be determined, as well as their chemical
bonds, their disorder and various other information.
Result:

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

MATERIALS AND METHOD

INVIVO STUDIES
3.1

MAINTANENCE OF DROSOPHILA CULTURE

MATERIALS:
1

soap solution

0.1M hydrochloric acid

Distilled water

4 Autoclave

PROCEDURE :
1 The glasswares were soaked in soap water and were washed and rinsed
well
2 Materials were soaked in warm water containing dilute HCL overnight

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

3Then they were washed again in distilled water

4 Micropipette tips,centrifuge tubes , eppendrof tube were dried in microvave
oven
5 The materials were then dried completely using hot air oven and
autoclaved at 1210 C for 15 minutes at 15lbs per inch square
6 Autoclaved materials were dried completely in hot air oven and stored in
clean dry place
3.2

DNA ISOLATION

MATERIALS

micropipette and tips

vials

Aproximately 30 flies

Dry ice

Mortar and pestel

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

Solution A (Tris Hcl- 100mM,EDTA- 100mM , Nacl -100mM ,SDS-0.5%)

Buffer saturated phenol -30ml

Isopropyl alcohol -50ml

70% ethanol-50ml

Tris Acetate EDTA buffer (TAE buffer) -20ml

PROCEDURE
The flies were transferred to fresh vials with corresponding lables and these
were placed in glass beaker containin ice in refrigerator for 30 minutea
after 30 minutes flies were transferred and mortor and pestel and crushed
using 1 ml of solution A
the crushed flies were transferred to sterile

eppendorfs with appropiate

labelling and placed in ice for 15minutes

after this the eppendorfs were incubated in waterbath at 700C for 30minutes
the samples were centifuged at 13000RPM for 15minutes at 40C

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

the supernatant was transferred to fresh eppendorfs and equal volume of

buffer saturated phenol was added
centrifugation was done at 13000RPM for 5minutes at 40C
the supernatant was transferred to fresh eppendorfs and phosphate saline
wash was repeated
to the supernatan 150ml isopropyl alcohol and mixedwell
it was spun at 13000rpm for 5minutes at 40C
to the supernatant 70% ethanol was added and spun at 13000rpm for
minutes at 4
the pellet was air dried and resuspended in 100ml TE buffer ( Tris EDTA
buffer) and stored at 20OC

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

3.3

AGAROSE GEL ELECTROPHORESIS

MATERIALS :
1. power pack
2 .hot plate
3 .Transilluminator
4. Conical flask
5. Polythene sheet
6. Marker pen
CHEMICALS:
10X TAE BUFFER :
dissolve 48.4 g of Tris base,10.9 ml of Glacial acetic acid and 2.92g of EDTA IN
800ML of distilled water. Adjust pH to 8.0 and makeup the volume to 1ltre.
1X TAE BUFFER :Take 10 ml 10X TAE buffer and make upto 1 liter

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

Ethidium Bromide (10mg/ml) : Dissolve 1gm ethidium bromide in 100ml of

distilled water and store at 4 degrees centigrade .it is just diluted before use of a
concentrated 10mg/L ethidium bromide.
Glycerol and bromophenol blue Agarose Gel Loading

Buffer (6X

concentrate):
Dissolve 3ml of glycerol (30%) and 25mg of bromophenol blue (0.25%)in 10ml of
distilled water. Store in small aliquots at 40c
PROCEDURE :
the electrophoretic unit and gel boats were washed with sterile water before use.
The two edges of the gel boats were covered with adhesive tape and placed on
level led table .The combs were placed over the gel boat .2000mg of agarose
was dissolved In 100ml of 0.5X TAE buffer ny heating on an electrical heater
.molten agarose was cooled to about 60oc and poured into gel plates without
trapping bubbles .After gelling ,the comb was carefully lifted up yo obtained
undisturbed wells

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

The gel boats were transferred on

to the platform of submarine gel

electrophoresis unit until after removing the adhevsive tape.the TAE buffer
(1X)was poured into buffer tank in such a way that the buffer was about 2mm
above the gel and without trapping air bubbles In the gel.The DNA samples were
carefully loaded in to the wells using micropipette .the unit was connected to a
power pack and current passed initially at 80 and later increaded to 100
tvolts.when the marker dye reaches almost the other end of the gel,the power
bank and supply to the unit was disconnected

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

DNA FRAGMENTATION ASSAY

The DNA after being checked for its quality was subjected to fragmentation
assay using 2% agarose gel,to study the DNA damage .The results obtained for
different concentrations were as follows

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

Well number

sample

DNA ladder

PC - EMS

NC

0.2 mg/ml

0.3mg/ml

0.4mg/ml

0.5mg/ml

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

REFERENCE
1)The potential toxicity of copper nanoparticles and copper sulphate on juvenile
Epinephelus coioides
Tao Wang, , Yongzhou Cheng, Zhaopu Liu, Shaohua Yan
2)Acute toxicological effects of copper nanoparticles in vivo
Toxicology Letters Volume 163, Issue 2, 25 May 2006, Pages 109120
Zhen Chen, Gengmei Xinga, Chunying Chena, Yuliang Zhaoa, Guang
Jiab, Tiancheng hui,Chang Yea, Feng Zhaoa, Zhifang Chaia,Chuanfeng Zhu,
Lijun Wanc
3)Size-dependent toxicity of metal oxide particlesA comparison between nanoand micrometer size
Toxicology Letters Volume 188, Issue 2, 24 July 2009, Pages 112118
Hanna L. Karlsson1, Johanna Gustafsson1, Pontus Cronholm, Lennart
Mlle

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster

4)Ultrahigh reactivity provokes nanotoxicity: Explanation of oral toxicity of nanocopper particles

Toxicology Letters

Volume 175, Issues 13, 10 December 2007, Pages 102

110
Huan Menga, Zhen Chena, Gengmei Xinga, Hui Yuana, Chunying
Chena,Feng Zhaoa,Chengcheng Zhanga, Yuliang Zhaoa,
5)Ultrahigh reactivity and grave nanotoxicity of copper nanoparticles
Journal of Radioanalytical and Nuclear Chemistry
June 2007,Volume 272,i ssue 3, pp 595-598
Huan Meng, ZhenChen, Gengmei Xing ,Hui Yuan,Chunying Chen,Feng
Zhao, Chengcheng Zhang, yun Wang, Yuliang Zhao
6)Synthesis and characterization of copper nanoparticles by reducing agent
Sulekh Chandra, Avdhesh Kumar Praveen Kumar Tomar
Department of Chemistry, Zakir Husain College (University of Delhi), J.L.N.
Marg, New Delhi 110 002, India
Received 25 March 2011, Accepted 14 June 2011, Available online 30 June 2011

Synthesis ,Characterization and Genotoxic evaluation of Copper nanoparticles on Drosophila

melanogaster