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Chemosphere 83 (2011) 11241132

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Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cellsAshutosh Kumar a, Alok K. Pandey a, Shashi S. Singh b, Rishi Shanker a,, Alok Dhawan a,a Nanomaterial Toxicology Group, Indian Institute of Toxicology Research, Council of Scientic and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O. Box 80, Lucknow 226 001, Uttar Pradesh, India b Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, Andhra Pradesh, India

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a b s t r a c tExtensive production and consumption of nanomaterials such as ZnO and TiO2 has increased their release and disposal into the environment. The accumulation of nanoparticles (NPs) in ecosystem is likely to pose threat to non-specic targets such as bacteria. The present study explored the effect of ZnO and TiO2 NPs in a model bacterium, Salmonella typhimurium. The uptake of ZnO and TiO2 bare NPs in nano range without agglomeration was observed in S. typhimurium. TEM analysis demonstrated the internalization and uniform distribution of NPs inside the cells. Flow cytometry data also demonstrates that both ZnO and TiO2 NPs were signicantly internalized in the S. typhimurium cells in a concentration dependent manner. A signicant increase in uptake was observed in the S. typhimurium treated even with 8 and 80 ng mL1 of ZnO and TiO2 NPs with S9 after 60 min, possibly the formation of micelles or protein coat facilitated entry of NPs. These NPs exhibited weak mutagenic potential in S. typhimurium strains TA98, TA1537 and Escherichia coli (WP2uvrA) of Ames test underscoring the possible carcinogenic potential similar to certain mutagenic chemicals. Our study reiterates the need for re-evaluating environmental toxicity of ZnO and TiO2 NPs presumably considered safe in environment. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 28 July 2010 Received in revised form 24 November 2010 Accepted 11 January 2011 Available online 9 February 2011 Keywords: Nanoparticle uptake Bacteria S. typhimurium Flow cytometry ZnO and TiO2 nanoparticles Ames mutagenicity test

1. Introduction Rapid advancement in the synthesis of nanoparticles (NPs) has enabled the production of new materials for industrial, medical and consumer applications. The use of NPs in electronics, tyres, fuel cells, and lters among other applications is leading to their inadvertent release in surface and subsurface environment through landlls and other waste disposal methods (Oberdorster et al., 2005). Personal-care products such as cosmetics and sunscreens are a signicant component of over 1000 nanotech based consumer products in market (Bradford et al., 2009). NPs released from various products through washing or disposal may reach the environment leading to adverse effects in organisms thereby affecting the eco-system health (Lemos et al., 2009). The distinct lack of information on human health and environmental impact of engineered nanomaterials has drawn increasing attention over the last few years (RSRAE, 2004). Recently, the pulmonary damage in industrial workers in China has fueled the debate over the safety precautions to be taken in the work environment (Gilbert, 2009; Song et al., 2009).

Corresponding authors. Tel.: +91 522 2230749, fax: +91 522 2628227/2611547.E-mail addresses: (A. Dhawan). (R. Shanker),

Metal and metal oxide NPs (e.g. nanoiron magnetite, titanium oxide) have been proposed for groundwater remediation (McCormick and Adriaens, 2004; Liu et al., 2005; Mattigod et al., 2005), water treatment (Ferguson et al., 2005; Lee et al., 2005) and removal of toxic contaminants from air streams (Esterkin et al., 2005). Zinc oxide (ZnO) and titanium (IV) dioxide (TiO2) NPs are the most common metal oxide NPs being used in consumer products such as cosmetics, sunscreen, etc. due to their unique optical properties (Kiss et al., 2008). Their widespread use could expose biological systems through inhalation, dermal contact, ingestion or absorption through the digestive tract and ultimately affect the human population directly or indirectly (Gurr et al., 2005; Warheit and Frame, 2006; Warheit et al., 2007; Pan et al., 2009). While information about the safety/toxicity of these NPs is still scanty, toxicity and mutagenicity of these compounds cannot be predicted reliably on the physical and chemical behavior of the bulk materials and solutes that are used to make the NPs. Studies have shown that size, shape, chemistry, crystallinity, surface property and agglomeration state appears to be a critical parameter for toxicity (Pan et al., 2007; Jiang et al., 2008). It is well known that algae and higher plants are primary producers while bacteria act as decomposers and play an important role in maintaining the ecosystem. However, very few studies have been done to assess the mutagenic potential of the ZnO NPs (Sawai et al., 1998; Yoshida et al., 2009) and TiO2 NPs

0045-6535/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.01.025

A. Kumar et al. / Chemosphere 83 (2011) 11241132


(Wang et al., 2007; Kang et al., 2008; Karlsson et al., 2008) in bacteria. Our earlier study with human epidermal cells has shown that ZnO nanoparticles possess DNA damaging potential (Sharma et al., 2009). In the present study, we have attempted to elucidate the uptake and mutagenic potential of ZnO and TiO2 NPs in bacteria. 2. Materials and methods 2.1. Preparation and characterization of nanoparticles 2.1.1. Particle preparation Zinc oxide nanopowder (ZnO; CAS No. 1314-13-2, purity >99%), titanium (IV) dioxide nanopowder (TiO2; CAS No. 1317-70-0, purity 99.7%, anatase) were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). Nanoparticle stock suspension (80 lg mL1) was prepared by suspending 0.8 mg of ZnO or TiO2 nanoparticles in 10 mL of 0.22 lm ltered Milli-Q water. The stock suspension was sonicated (Sonics Vibra cell, Sonics & Material Inc., New Town, CT, USA) for 10 min at 30 W. 2.1.2. Characterization Dynamic light scattering (DLS): Size (hydrodynamic diameter) and zeta potential of the nanoparticles stock suspension were determined using dynamic light scattering and phase analysis light scattering respectively in a Zetasizer Nano-ZS (Model ZEN3600; Malvern Instrument Ltd., UK) facilitated with 4.0 mW, 633 nm laser. 2.1.3. Transmission electron microscopy (TEM) For transmission electron microscopy, the samples were prepared by drop-coating the NPs suspension (8 lg mL1) on carboncoated copper TEM grids and scanned at 120 kV (JEM-2100, JEOL Ltd., Tokyo, Japan). The size of particles was measured manually. 2.2. Preparation of mammalian liver S9 fraction To prepare the liver S9 fraction, polychlorinated biphenyl (PCB) mixture (Aroclor 1254) was administered in Wistar rats and the liver S9 fraction was prepared according to the method described by Maron and Ames (1983). 2.3. Detection of nanoparticles uptake in bacteria 2.3.1. Transmission electron microscopy (TEM) S. typhimurium cell suspension (109 CFU mL1) was treated with ZnO or TiO2 NPs at a concentration of 8 lg mL1 for 30 min at 37 C. Treated bacterial cell suspension was xed with 2.5% glutaraldehyde and pelleted. After washing with 0.1% phosphate buffer the pellet was post xed in 1% osmium tetraoxide. Fixed pellet was then washed and dehydrated through grades (30100%) of acetone. Sample was inltrated with araldite resin overnight at room temperature and nally embedded in pure resin. The blocks were cured at 60 C for 72 h. After incubation ultrathin sections (60 nm) were prepared using ReichertJung ultra microtome. The sections were stained with uranyl acetate and Reynolds lead citrate. The grids were examined under a TEM (JEM-2100, JEOL Ltd., Tokyo, Japan) operated at an accelerating voltage of 100 kV using a 20l aperture. 2.3.2. Flow cytometry (FCM) S. typhimurium cell suspension (109 CFU ml1) was treated with different concentrations of ZnO or TiO2 nanoparticles (0.0088 lg mL1) for 1 h at 37 C with and without S9 fraction. To assess whether the NPs are retained in the cell, the uptake was evaluated for several generations. A 100 lL aliquot of treated

S. typhimurium culture was re-inoculated in 10 ml fresh LB media and incubated at 37 C in an environmental shakerincubator at 200 rpm. The cells (107 CFU) treated for 1 h were re-inoculated in fresh media and used to assess size and granularity after 30, 60 and 90 min. The uptake of NPs in the cells was analyzed using a ow cytometer (FCM; BD FACSCanto II, BD Biosciences, San Jose, CA, USA). The forward scatter and side scatter intensities indicating the size and intracellular density of cells respectively were recorded. Analysis of the data was performed using BD FACSDiva 6.1.2 software. 2.4. Bacterial mutagenicity test (Ames test) The pre-incubation assay was performed according to the method of Ames et al. (1975) as described below. The tester strains used in this study S. typhimurium TA98, TA100, TA1535 TA1537 and Escherichia. coli (WP2 uvrA) were purchased from Xenomatrix AG (Allschwil, Switzerland). The tester strains were checked for their genetic integrity for histidine dependence, biotin dependence, histidine/biotin dependence, rfa marker (crystal violet) and the presence of plasmid pKM101 (ampicillin resistance) before experiments. Six experimental groups were taken as follows: Group 1: Vehicle control. Group 2: Positive controls. (a) In absence of S9: 2-nitroourene (2NF; 5 lg/plate) for TA98; 4, nitroquinoline-1-oxide (4NQO; 5 lg/plate) for TA100, sodium azide (5 lg/plate) for TA1535, 9-aminoacridine (50 lg/plate) for TA1537 and methyl methane sulfonate (MMS; 500 lg/plate) for E. coli (WP2 uvr