Science of the Total Environment 407 (2009) 5243–5246

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Science of the Total Environment

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Genotoxicity of silver nanoparticles in Allium cepa

Mamta Kumari, A. Mukherjee, N. Chandrasekaran ⁎Nanobio-Medicine Group, School of Bio Sciences & Technology, VIT University, Vellore 632014, India

⁎ Corresponding author. Environmental Biotechnolognology, Chemical and Biomedical Engineering, VIT UniTel.: +91 416 2202624/2320; fax: +91 416 2243092/04

E-mail addresses: nchandrasekaran@vit.ac.in, nchan(N. Chandrasekaran).

0048-9697/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.scitotenv.2009.06.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 April 2009Received in revised form 17 June 2009Accepted 22 June 2009Available online 17 July 2009

Keywords:Silver nanoparticlesAllium cepaCytotoxicityMitotic indexChromosomal aberrations

Potential health and environmental effects of nanoparticles need to be thoroughly assessed before theirwidespread commercialization. Though there are few studies on cytotoxicity of nanoparticles on mammalianand human cell lines, there are hardly any reports on genotoxic and cytotoxic behavior of nanoparticles inplant cells. This study aims to investigate cytotoxic and genotoxic impacts of silver nanoparticles using roottip cells of Allium cepa as an indicator organism. A. cepa root tip cells were treated with four differentconcentrations (25, 20, 75, and 100 ppm) of engineered silver nanoparticles (below 100 nm size) dispersion,to study endpoints like mitotic index, distribution of cells in mitotic phases, different types of chromosomalaberrations, disturbed metaphase, sticky chromosome, cell wall disintegration, and breaks. For eachconcentration five sets of microscopic observations were carried out. No chromosomal aberration wasobserved in the control (untreated onion root tips) and the mitotic index (MI) value was 60.3%. Withincreasing concentration of the nanoparticles decrease in the mitotic index was noticed (60.30% to 27.62%).The different cytological effects including the chromosomal aberrations were studied in detail for the treatedcells as well as control. We infer from this study that silver nanoparticles could penetrate plant system andmay impair stages of cell division causing chromatin bridge, stickiness, disturbed metaphase, multiplechromosomal breaks and cell disintegration. The findings also suggest that plants as an importantcomponent of the ecosystems need to be included when evaluating the overall toxicological impact of thenanoparticles in the environment.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Nanotechnology, a rapidly developing industry, can have sub-stantial impacts on economy, society and environment. Potentialhealth and environmental impacts of nanomaterials and nanotech-nology on human, non-human biota, and ecosystems are yet to bethoroughly assessed. Nanometer-sized particles have shown specialtoxicity and are usually more toxic than the bulkmaterial of larger size(Donaldson et al., 1999). When inhaled as single particles, particleshaving less than 50 nm diameters proved to be highly toxic(Oberdörster, 1996). Nanoparticles like fullerene, carbon nanotubes,and metal oxides pose toxicity to human cells, bacteria, and rodents(Brunner et al., 2006; Hussain et al., 2005; Jia et al., 2005; Lam et al.,2006; Soto et al., 2005). Data on potential toxicity of nanoparticles toecological terrestrial test species is still limited (USEPA, 2007). Studieshave shown that, nanoparticles exert oxidative stress and cause severelipid peroxidation in fish brain tissue (Oberdörster, 2004).

y Division, School of Biotech-versity, Vellore-632014, India.11.dra40@hotmail.com

ll rights reserved.

Few studies have reported both positive and negative effects ofnanoparticles on higher plants. Nanoscale SiO2 and TiO2 enhancednitrate reductase activity in soybean, and apparently hastened itsgermination and growth (Lu et al., 2002). Nano-TiO2 promotedphotosynthesis and nitrogen metabolism, and improved growth ofspinach (Honget al., 2005a, b;Yang andWatts, 2005; Zhenget al., 2005).

The antimicrobial properties of silver nanoparticles are beingincreasingly exploited in consumer products like deodorants, clothingmaterials, bandages, and also in cleaning solutions and sprays (Chenand Schluesener 2008; Tripathy et al., 2008). In the near future there is arisk of enhanced bioavailability of the nanoparticles in the environment.

Historically plants have been used as indicator organisms, instudies on mutagenesis in higher eukaryotes. Plant systems have avariety of well-defined genetic endpoints including alterations inploidy, chromosomal aberrations, and sister chromatid exchanges. TheAllium root chromosomal aberration assay is an established plantbioassay validated by the International Programme on ChemicalSafety (IPCS, WHO) and the United Nations Environment Programme(UNEP) as an efficient and standard test for the chemical screeningand in situ monitoring for genotoxicity of environmental substances(Cabrera and Rodriguez, 1999). Allium cepa has been used forevaluating chromosomal aberrations since 1920s (Grant, 1982). andKanaya et al., 1994).

Table 1aOccurrences of cytological effects in Allium cepa root tip cells after treatment with silver nanoparticles dispersion.

Sample number Replicates Treatments Number ofcounted cells

Normalmetaphase

Normalanaphase

Stickychromosome

Disturbedmetaphase

Break Gap Iso-chromatidexchange

Control (deionized distilled water)1 Sample 1 1000 + + − − − − −2 Sample 2 1000 + + − − − − −3 Sample 3 1000 + + − − − − −4 Sample 4 1000 + + − − − − −5 Sample 5 1000 + + − − − − −

Nanoparticle (25 ppm)6 Sample 1 1000 + − + + + + +7 Sample 2 1000 + + + + + + +8 Sample 3 1000 + − + + + + +9 Sample 4 1000 + + + + + + −10 Sample 5 1000 + + + + + + −

Nanoparticle (50 ppm)11 Sample 1 1000 + + + + − + +12 Sample 2 1000 + + + + − + −13 Sample 3 1000 + + + + − − −14 Sample 4 1000 + + + + − − −15 Sample 5 1000 + + + + − − −

Nanoparticle (75 ppm)16 Sample 1 1000 + + + + − + +17 Sample 2 1000 + + + + − − +18 Sample 3 1000 + + + + + − −19 Sample 4 1000 + + + + − − −20 Sample 5 1000 + + + + − − −

Nanoparticle (100 ppm)17 Sample 1 1000 + + + + + + +18 Sample 2 1000 + − + + + − +19 Sample 3 1000 + − + + + + +20 Sample 4 1000 − − + + + + −21 Sample 5 1000 + − + + + + −

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The reports from few previous studies have advanced our under-standing of nanotoxicology for several types of nanomaterials. Thereare still many unresolved issues and challenges concerning the

Table 1bDistribution of Allium cepa root tip cells treated with different concentrations of silver nano

Dividing cell (total) Prophase Metaphase

ControlSample 1 622 597 15Sample 2 596 581 11Sample 3 625 621 3Sample 4 536 520 12Sample 5 636 623 8

25 ppmSample 1 481 471 4Sample 2 402 394 3Sample 3 501 493 2Sample 4 403 397 2Sample 5 463 459 3

50 ppmSample 1 381 378 1Sample 2 400 398 1Sample 3 362 358 2Sample 4 365 359 2Sample 5 393 390 1

75 ppmSample 1 342 341 1Sample 2 334 330 1Sample 3 360 358 2Sample 4 332 329 1Sample 5 389 383 2

100 ppmSample 1 290 288 1Sample 2 278 277 1Sample 3 321 319 2Sample 4 252 252 0Sample 5 240 239 1

a Statistically significant based on ‘t’ test at 5% level of significance.

biological effects of nanoparticles. Therefore, the present study isdesigned to investigate cytotoxic and genotoxic impacts of silvernanoparticles on A. cepa.

particles.

Anaphase Telophase Mitotic index (%) Mean±SE (%)

7 3 62.2 60.3±4.97%3 1 59.61 0 62.54 0 53.64 1 63.6

5 1 48.1 36.96±3.665 0 40.24 2 50.13 1 40.31 0 46.3

2 0 38.1 38.2±2.12a

1 0 40.01 1 36.23 1 36.52 0 39.3

0 0 34.2 29.14±7.24a

2 1 33.40 0 36.02 1 33.23 1 38.9

1 0 29.0 27.62±2.70a

0 0 27.80 0 32.10 0 25.20 0 24.0

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2. Materials and methods

2.1. Nanoparticles

Silver nanoparticles was obtained from Sigma Aldrich, USA. Thephysical characteristics of the particles according to manufacturersdata are; size (b100 nm), purity: (99.5%) trace metal basis, surfacearea (5.0 m2/g) density (10.49 g/cc).

2.2. Test system and treatment

The silver nanoparticles were suspended directly in deionizedwater (DI-water) and dispersed by ultrasonic vibration (100 W,30 kHz) for 30 min to produce four different concentrations at25 ppm, 50 ppm, 75 ppm, and 100 ppm. Four healthy onion bulbs(20–25 g) were grown in the dark in a cylindrical glass beaker at roomtemperature (28±0.5) °C and given renewed water supply every24 h. When the roots reached 2 to 3 cm in length they were treated

Fig. 1. Silver nanoparticles induced chromosomal aberrations in root tip cells A. cepa.a) Chromatin bridge, b) stickiness, c) disturbed metaphase, d) multiple chromosomalbreaks, e) cell wall disintegration.

with different concentrations of silver nanoparticles suspension for4 h. Three replicates were made for each concentration.

2.3. Microscopic examination

Four bulbs were used for each concentration of silver nanoparticlesdispersion; eight new root tips were used for each concentration. Themicro slides were prepared for each concentration and the controlfollowing Saffranin squash technique. The root tips were kept in 1 MHCl for about 6 min followed by staining with 40–45% Saffranin.Staining was continued for about 5–6 min. The slides were analyzed at×1000 magnification for cytological changes. The mitotic index wascalculated as the number of dividing cells per number of 1000observed cells (Fiskesjo, 1997). The number of aberrant cells wasnoted per total cells scored at each concentration (Bakare et al., 2000).

3. Results

The effect of the silver nanoparticles suspension on cell divisionand chromosome behavior of A. cepa is presented in Tables 1a, and 1b.No chromosomal aberration was observed in the control (untreatedonion root tips) and the mitotic index (MI) value was 60.3%. Mitoticindex was the lowest, 27.6% at the 100 ppm concentration. Withincreasing concentration of the nanoparticles, concentration depen-dent decrease in the mitotic index was noticed. The effect of silvernanoparticles concentration on mitotic index was significantlydifferent (pb0.05) for 50, 75 and 100 ppm as compared to the control.The occurrence of different cytological effects including the chromo-somal aberrations is tabulated (Table 1b). For each concentration fivesets of microscopic observations were carried out.

For 50-ppm concentration, we observed chromatin bridge, sticki-ness, and disturbed metaphase; for 75 ppm we have observedchromosomal breaks; and at 100 ppm there was complete disintegra-tion of cell walls for most of the cells. Representative aberrations aregiven in Fig. 1(a)–(e).

4. Discussion

In this study the silver nanoparticles exhibited cytotoxicity bydecreasing the mitotic index in a dose dependent manner. This provesthat the silver nanoparticles may havemito-depressive effect on the A.cepa. Many investigations have demonstrated that reduction in cellactivity could be due to changes in duration of mitotic cycle. A fewauthors attributed the inhibition of mitosis to the increase in S phaseduration (Webster and Davidson, 1969; Mcleod, 1969).

Chromosomal aberrations are changes in chromosome structureresulting from a break or exchange of chromosomal material. In thisstudy different kinds of chromosomal aberrations were observed withdifferent concentrations of silver nanoparticle suspensions. Bothphysiological and clastogenic aberrations like stickiness, breaks,gaps, disturbed metaphase, and cell wall disintegration were notice-able in the treated cells. Chromosomal stickiness was observed inmetaphase and anaphase stages. Darlington and Mc Leish (1951)suggested that stickiness might be due to degradation or depolymer-ization of chromosomal DNA. Stickiness has also been attributed toentanglement of inter chromosomal chromatin fibers. Stickiness is acommon sign of toxic influence on the chromosomes and is probablyan irreversible effect. The disturbedmetaphase observedmight be dueto disturbance in the spindle apparatus. The cell wall disintegrationappeared in the cells treated with 100 ppm nanoparticle suspension.

Previous studies (Li et al., 2003; Chen and von Mickecz, 2005) withmammalian cell lines also demonstrated that the nanoparticles pene-trated subcellular structures such as the mitochondria and nucleuscausing uncoupling of respiration and increased oxidative stress. Chromo-somal aberrations in the root meristematic cells of agricultural plants Zeamayswhen cultivated in the presence of aqueous tetramethylammonium

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hydroxide (TMA-OH) coated magnetic nanoparticles of ferrofluid, wasobserved (Racuciu and Creanga, 2007).

Mechanism of nanotoxicity remains a largely unexplored area;however, it would be closely related to the chemical composition,chemical structure, particle size and surface area of the nanoparticles.Toxicity of nanoparticles may be attributed to two different actions(Brunner et al., 2006): (1) a chemical toxicity based on the chemicalcomposition, e.g., release of (toxic) ions; and (2) stress or stimulicaused by the surface, size and/or shape of the particles. It has beenconfirmed that solubility of oxide nanoparticles greatly affected thecell culture response (Brunner et al., 2006).

Further studies are underway to correlate cytotoxic and genotoxiceffects observed with physicochemical parameters pertaining to theparticles, transport of the particles to the cells, and the biouptake ofthese particles by the cells.

5. Conclusions

Silver nanoparticles could penetrate plant system and mayinterfere with intracellular components, causing damage to celldivision. The mitotic index decreased from the control (60.3%) tothat of the 100 ppm treated (27.62%). The cell divisionwas arrested, atmetaphase stage, showing chromatin bridge, stickiness and chromo-somal breaks. The findings suggest that plants, as an importantcomponent of the environmental and ecological systems, need to beincluded when evaluating the overall fate, transport, and exposurepathways of nanoparticles in the environment.

Acknowledgement

The authors from VIT University are grateful to Mr. SekarVishwanathan, Pro-Chancellor of the University for the FinancialGrant provided for Nanobiotechnology research.

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