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Interaction of cobalt nanoparticles withplants: a cytogenetical aspectNitisha Srivastavaa

a Plant Genetics Laboratory, Department of Botany, University ofAllahabad, Allahabad 211002, IndiaPublished online: 24 Apr 2014.

To cite this article: Nitisha Srivastava (2014): Interaction of cobalt nanoparticles with plants: acytogenetical aspect, Journal of Experimental Nanoscience, DOI: 10.1080/17458080.2014.895061

To link to this article: http://dx.doi.org/10.1080/17458080.2014.895061

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Interaction of cobalt nanoparticles with plants: a cytogenetical aspect

Nitisha Srivastava*

Plant Genetics Laboratory, Department of Botany, University of Allahabad, Allahabad 211002, India

(Received 22 June 2013; final version received 12 February 2014)

In vivo cytogenetical assay in root meristems has been carried out to study the effectof cobalt and cobalt oxide nanos on active mitotic index and chromosomalaberrations of green manure crop, Sesbania pea. Nanoparticles are nanosizeparticles and can enter freely into the cells and can interfere in cell’s normalfunction. Studies to justify its probable after-effects on living beings are scarce. Soit is important to study its effect on living beings. For this purpose, seeds ofSesbania cannabina variety ND-1 were soaked in distilled water for 14 hours andafter soaking, the seeds were treated with three different treatments, namely, nano-cobalt, nano-cobalt oxide and ethanol for three hours. The treated root meristemsexhibited various types of chromosomal aberrations such as metaphasic platedistortion, unorientation at metaphase, breaking of chromosomes, fragmentation,spindle dysfunctioning, stickiness, scattering, precocious movement at metaphaseand bridge, unequal separation, multiple bridge, fragmentation, scattering, laggardand unorientation at anaphase, etc.

Keywords: nanoparticles; cobalt; Sesbania cannabina

1. Introduction

Nanoscience is the study of phenomena and manipulation of materials at atomic, molecu-

lar and macromolecular scales, where properties differ significantly from those at a largerscale. Nanoparticles (NPs) are atomic or molecular aggregates with at least one dimension

between 1 and 100 nm,[1] that can drastically modify their physicochemical properties

compared to the bulk material.[2] Particle size and size distribution are the most important

characteristics of NP systems and are the major factors for the determination of its in vivo

distribution, biological fate, toxicity and the targeting ability of NP systems. In addition,

they can influence the drug loading, drug release and stability of NPs. Nanomaterials are

increasingly produced over the last decades and are expected to play an increasing role in

future science, technology and medicine. Despite numerous applications and uses of nano-technology, its potential risk and effect on human being and environment are poorly

known and investigated.[1,3] The possible toxic effects of NPs have been studied in a wide

variety of organisms. However, interaction of NPs with plants and other organism such as

algae have been poorly investigated and consequences of exposure of nanoparticles to

these organisms remain unclear.

There is now an extensive debate about the risks and benefits of the many manufac-

tured nanomaterials into the environment (USEPA 2007), and in order to evaluate their

*Email: srivastava_nitisha@yahoo.com

� 2014 Taylor & Francis

Journal of Experimental Nanoscience, 2014

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potential adverse effects on the ecosystems and on human health, the scientific communityis working with increased attention on this topic. The literature on the ecotoxicity of NPs

and nanomaterials as well as the chemistry of both manufactured and natural NPs is sum-

marised in recent reports.[4,5] Cobalt oxide is an important material that finds applications

in different fields such as catalysis, various types of sensors and electrochromic, electrical

and other opto-electronic devices.

The present study was undertaken to find out the possible toxic effects of NPs on plant

cells. The testing material used in this study was a green manure crop Sesbania cannabina,

commonly known as Dhaincha, Prickly sesban, Sesbania pea, etc. S. cannabina is a quickgrowing, succulent, suffructicose, annual leguminous plant and ideally suited as a green

manure crop. In India, the species has been introduced as green manure to enhance

organic matter in rice fields. Sesbania green manuring has substantially improved grain

yield up to 72%.[6] Because of its ability to grow in heavy metal soils and to withstand

water logging and tolerate soil salinity, it is often the preferred green manure crop for rice

and wheat. It yields a strong useful fibre, durable especially under water. S. cannabina

shows promises for early fodder and fuel production.[7] During this study, germinated

seeds of S. cannabina were treated with cobalt NPs prepared in different media (double dis-tilled water [DDW] and ethanol), and its impact on mitotic index and the chromosomal

aberration frequency has been studied. The value of active mitotic index (AMI) showed

the percentage of dividing cells in every sample, while chromosomal aberration index rep-

resents the percentage of aberrant cell divisions.

2. Materials and method

2.1. Cobalt and cobalt oxide nanoparticles

The above-mentioned cobalt and cobalt oxide NPs were obtained from Laser Spectros-

copy and Nanomaterials Lab, Department of Physics, University of Allahabad, Allaha-

bad, India. Nano-cobalt and nano-cobalt oxides used in the present study were in colloidal

form (5–80 nm size). When cobalt NPs are synthesised in water, they get oxidised and

become cobalt oxide, after oxidation they are quite stable.

2.2. Procurement of seeds

Seeds of S. cannabina variety ND-1 were obtained from Sunn-Hemp Research Station,

Pratapgarh, UP, India.

2.3. Treatment and fixation of root meristems

Dry and healthy seeds of S. cannabina were soaked in distilled water for 14 hours and afterthat they were kept at room temperature for rooting. After rooting, the seeds were treated

with cobalt, cobalt oxide NPs and ethanol solution for three hours (three or five hours’

time period has been used for genotoxicity assays in many experiments). Control was

maintained separately in distilled water. Treated as well as control seeds were fixed in

carnoy’s fixative (1:3, glacial acetic acid:absolute alcohol) and changed to 90% alcohol

after 24 hours, and were stored in refrigerator and used for chromosomal aberration assay.

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2.4. Chromosomal aberration assay

Three different treatment sets (ethanol, nano-cobalt and nano-cobalt oxide) and a control

were used for chromosomal aberration assay. For cytological analysis, treated as well as

control root tips were hydrolysed in 1 N HCl at 60 �C for five minutes. After hydrolysis,

the root tips were thoroughly washed several times in water to remove HCl. The root tips

were then dried between folds of filter paper for removing moisture and were kept in 2%acetocarmine solution for staining. After staining, the dark-stained tip portion of root tips

was used for slide preparation.

2.5. Scoring of slides

Slides were observed under Nikon microscope (40�). The AMI and total abnormality per-centages were calculated for each treated and control set. The assay slides were scored for

chromosomal aberrations during metaphase and telophase and for each treated set more

than 500 cells were observed.

3. Result

3.1. Effect of cobalt nanoparticle on AMI of S. cannabina

The AMI% at different treated sets has been presented in Table 1. The highest AMI% was

observed in case of the control set (15.83), which decreased up to 11.68% for nano-cobaltoxide-treated set. While ethanol-treated set exhibited a slightly lower value than the con-

trol (14.42%). Thus, from AMI% it was clear that nano-cobalt oxide was more toxic than

nano-cobalt and caused a significant reduction in AMI.

3.2. Effect of cobalt nanoparticles on mitotic division of S. cannabina

The mitotoxic effects of cobalt nanoparticles on root meristematic cells of S. cannabina

were estimated on the basis of total abnormality percentages. Percentages of different

types of chromosomal aberrations at different treated sets have been shown in Table 2.

Control seeds showed a normal mitotic behaviour (Figure 1(a) and 1(b)). While, the root

tips treated with ethanol, nano-cobalt and nano-cobalt oxide solution showed various

types of chromosomal abnormalities (Figure 1(c)–(i)) such as metaphasic plate distortion,

unorientation at metaphase, breaking of chromosomes, fragmentation, spindle dysfunc-

tioning, stickiness, scattering, precocious movement at metaphase and bridge, unequal

Table 1. Effect of nanoparticles on active mitotic index (AMI) % of S. cannabina.

Treatment Total no. of cells observed No. of dividing cells AMI (%)

Control 600 95 15.83Ethanol 617 89 14.42Nano-cobalt 519 67 12.90Nano-cobalt oxide 522 61 11.68

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Table2.EffectofnanoparticlesonmitoticdivisionofS.cannabina.

Totalno.

ofcells

No.of

dividing

No.of

abnorm

ally

dividing

Metaphasicabnorm

ality

%Anaphasicabnorm

ality

%Total

Treatm

ent

observed

cells

cells

Pldis

Un

Bre

Fr

Spin

St

Sc

Pr

Br

UneMBr

Fr

Sc

Lg

Un

abnorm

ality

%

Control

600

95

––

––

––

––

––

––

––

––

–Ethanol

617

89

35

0.48

0.16

–0.32

1.0

–1.0

0.32

0.64

––

0.64

0.64

0.16

0.64

6.0

Nano-cobalt

519

67

47

2.0

0.38

0.38

2.0

1.15

0.57

0.38

–0.57

0.77

0.57

0.57

––

–9.0

Nano-cobalt

oxide

522

61

54

2.0

––

3–

–1.14

0.19

1.5

1.3

––

1.0

0.57

–10.0

Note:PlDis

-metaphasicplate

distortion;Un-unorientation;Bre

-breakingofchromatids;

Fr-fragmentation;Spin

-spindle

dysfunctioning;St-stickiness;

Sc-scattering;Pr-precociousmovem

ent;Br-bridge;Une-unequalseparation;MBr-multiplebridge;Lg-laggards.

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separation, multiple bridge, fragmentation, scattering, laggard and unorientation at ana-

phase. Among these, the most dominant aberrations were metaphasic plate distortion,fragmentation at metaphase, scattering at metaphase, bridges at anaphase and unequal

separation at anaphase. The total abnormality% was found to be highest (10%) in case of

Figure 1. Nano-cobalt and nano-cobalt oxide induced chromosomal aberrations in Sesbania canna-bina. (a) Normal metaphase (2n ¼ 24). (b) Normal anaphase (24:24). (c) Laggard at anaphase I.(d) Scattering at metaphase I. (e) Precocious movement at metaphase I. (f) Stickiness at anaphase I.(g) Multivalent formation at metaphase I. (h) Bridge formation and unorientation at anaphaseI. (i) Asynchronisation at anaphase I.

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nano-cobalt oxide treatment set, while it was lowest in case of ethanol-treated set (6%). It isclear from Table 2 that the highest types of chromosomal variation were observed in case

of nano-cobalt treated set, while it was lowest in case of nano-cobalt oxide treated set. The

most dominant chromosomal aberrations were fragmentation at metaphase (3%), meta-

phasic plate distortion (2%), bridges at anaphase (1.5%) and unequal separation at ana-

phase (1.3%).

4. Discussion

Nowadays nanomaterials and NPs have received considerable attention due to their physi-

cal nature and uses in modern sciences and technology. Thus, before its wide utilisation

and applications in our daily life, we must know its possible impacts on living beings and

our environment. Thus the present study was undertaken to know the possible harmful

effects of NPs on plant genetic material, for which AMI and chromosomal aberration

assay were considered.

In this study, treatment of cobalt and cobalt oxide NPs on root meristems of S. canna-

bina showed that nanoparticles have mitotoxic and genotoxic effects and it causes reduc-tion in AMI and also disturbed normal mitotic behaviour of cells by inducing various

types of chromosomal aberrations. Nano-cobalt and nano-cobalt oxide used in this study

were in colloidal form (5–80 nm), and they can directly diffuse inside cells (in the micro-

metre range). It was found in this study that nano-cobalt oxide was more chromotoxic and

mitodepressive than nano-cobalt. The reduction in AMI may be due to slower progression

of cells from S (DNA synthesis) phase to M (mitosis) phase of the cell cycle as a result of

treatment. It may also be due to disturbance in microtubule synthesis.

Similar chromotoxic effects of NPs were also reported by Kumari et al. [8] and Pandaet al. [9] Similar study on root cells of Allium cepa reported that ZnO NPs have cytotoxic

and genotoxic effects, including lipid peroxidation, mitotic index reduction and increase of

micronuclei and chromosomal aberration indexes.[8] Li et al. [10] and Chen et al. [11] while

working on mammalian cell lines, demonstrated that the NPs penetrated subcellular struc-

tures such as mitochondria and nucleus causing uncoupling of respiration and increased

oxidative stress.

The breaking of chromatids represents the DNA double strand breaks that may not

have undergone G2 repair. Any such irreversible DNA damages will lead to the chromo-somal aberrations. Precocious movement of chromosomes observed during the present

study might have occurred due to disturbed homology for chromosome pairing, disturbed

spindle mechanism or inactivation of spindle mechanism.[12] Laggards in the present

study have been attributed to delayed terminalisation and/or failures of chromosomal

movement following spindle fibre discrepancies. The fragments which appeared on the

breakage of bridges as a result of spindle fibres functioning to pull the chromosome

towards poles formed laggards.[13] Sticky chromosomes may be resulted from defective

functioning of one or two types of specific non-histone proteins involved in the chromo-some organisation, which is needed for chromatid separation and segregation. Chromo-

somal stickiness may also be due to disturbance in nucleic acid metabolism in cells.[14]

Unorientation at metaphase and scattering of chromosomes may be due to either the inhi-

bition of spindle formation or the destruction of spindle fibres formed.[15] Bridge forma-

tion may occur due to the failure of chiasmata in a bivalent to terminalise and the

chromosome stretched between the poles. Single and multiple chromosome bridges may

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be due to the occurrence of dicentric chromosomes formed as a result of breakage fusionbridge cycles.[7]

The high frequency of mitotic abnormalities such as metaphasic plate distortion and

fragmentation of chromosomes induced by nano-cobalt primarily reflects its effects on

mitotic spindles, altering the orientation of chromosomes at various stages of the cell cycle.

Impairment of mitotic spindle function is probably due to the interaction of NPs with

tubulin-SH (sulphydryl) group.[16] The induction of chromosomal breaking by nano-

cobalt indicates the clastogenic potential of the test chemical, which may lead to a loss of

genetic material and these have been regarded as an indication of mutagenicity of theinducers.[17]

Present investigation clearly concludes the genotoxic behaviour of cobalt and cobalt-

oxide NPs. Thus before its utilisation in our daily life and its industrialisation, we should

check its probable harmful effects on our environment and human beings for safety point

of view.

Acknowledgements

The author is thankful to Laser Spectroscopy and Nanomaterials Laboratory, Department of Phys-ics, University of Allahabad, Allahabad, India for providing nanoparticles and Sunn Hemp ResearchStation, Pratapgarh, India for providing seeds of S. cannabina.

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