Chemosphere 83 (2011) 1124–1132

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Cellular uptake and mutagenic potential of metal oxide nanoparticlesin bacterial cells

Ashutosh 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 Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O. Box 80,Lucknow 226 001, Uttar Pradesh, Indiab Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, Andhra Pradesh, India

a r t i c l e i n f o

Article history:Received 28 July 2010Received in revised form 24 November 2010Accepted 11 January 2011Available online 9 February 2011

Keywords:Nanoparticle uptakeBacteriaS. typhimuriumFlow cytometryZnO and TiO2 nanoparticlesAmes mutagenicity test

0045-6535/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2011.01.025

⇑ Corresponding authors. Tel.: +91 522 2230749, faxE-mail addresses: rishi@iitr.res.in (R. Shank

(A. Dhawan).

a b s t r a c t

Extensive production and consumption of nanomaterials such as ZnO and TiO2 has increased their releaseand disposal into the environment. The accumulation of nanoparticles (NPs) in ecosystem is likely to posethreat to non-specific targets such as bacteria. The present study explored the effect of ZnO and TiO2 NPsin a model bacterium, Salmonella typhimurium. The uptake of ZnO and TiO2 bare NPs in nano range with-out agglomeration was observed in S. typhimurium. TEM analysis demonstrated the internalization anduniform distribution of NPs inside the cells. Flow cytometry data also demonstrates that both ZnO andTiO2 NPs were significantly internalized in the S. typhimurium cells in a concentration dependent manner.A significant increase in uptake was observed in the S. typhimurium treated even with 8 and 80 ng mL�1 ofZnO and TiO2 NPs with S9 after 60 min, possibly the formation of micelles or protein coat facilitated entryof NPs. These NPs exhibited weak mutagenic potential in S. typhimurium strains TA98, TA1537 and Esch-erichia coli (WP2uvrA) of Ames test underscoring the possible carcinogenic potential similar to certainmutagenic chemicals. Our study reiterates the need for re-evaluating environmental toxicity of ZnOand TiO2 NPs presumably considered safe in environment.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Rapid advancement in the synthesis of nanoparticles (NPs) hasenabled the production of new materials for industrial, medicaland consumer applications. The use of NPs in electronics, tyres, fuelcells, and filters among other applications is leading to their inad-vertent release in surface and subsurface environment throughlandfills and other waste disposal methods (Oberdorster et al.,2005). Personal-care products such as cosmetics and sunscreensare a significant component of over 1000 nanotech based con-sumer products in market (Bradford et al., 2009). NPs releasedfrom various products through washing or disposal may reachthe environment leading to adverse effects in organisms therebyaffecting the eco-system health (Lemos et al., 2009). The distinctlack of information on human health and environmental impactof engineered nanomaterials has drawn increasing attention overthe last few years (RSRAE, 2004). Recently, the pulmonary damagein industrial workers in China has fueled the debate over the safetyprecautions to be taken in the work environment (Gilbert, 2009;Song et al., 2009).

ll rights reserved.

: +91 522 2628227/2611547.er), alokdhawan@iitr.res.in

Metal and metal oxide NPs (e.g. nanoiron magnetite, titaniumoxide) 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 (Esterkinet al., 2005). Zinc oxide (ZnO) and titanium (IV) dioxide (TiO2)NPs are the most common metal oxide NPs being used in consumerproducts such as cosmetics, sunscreen, etc. due to their uniqueoptical properties (Kiss et al., 2008). Their widespread use couldexpose biological systems through inhalation, dermal contact,ingestion or absorption through the digestive tract and ultimatelyaffect 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 isstill scanty, toxicity and mutagenicity of these compounds cannotbe predicted reliably on the physical and chemical behavior of thebulk materials and solutes that are used to make the NPs. Studieshave shown that size, shape, chemistry, crystallinity, surfaceproperty and agglomeration state appears to be a criticalparameter for toxicity (Pan et al., 2007; Jiang et al., 2008).

It is well known that algae and higher plants are primaryproducers while bacteria act as decomposers and play animportant role in maintaining the ecosystem. However, very fewstudies have been done to assess the mutagenic potential of theZnO NPs (Sawai et al., 1998; Yoshida et al., 2009) and TiO2 NPs

A. Kumar et al. / Chemosphere 83 (2011) 1124–1132 1125

(Wang et al., 2007; Kang et al., 2008; Karlsson et al., 2008) in bac-teria. Our earlier study with human epidermal cells has shown thatZnO nanoparticles possess DNA damaging potential (Sharma et al.,2009). In the present study, we have attempted to elucidate the up-take 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 preparationZinc oxide nanopowder (ZnO; CAS No. 1314-13-2, purity >99%),

titanium (IV) dioxide nanopowder (TiO2; CAS No. 1317-70-0, pur-ity 99.7%, anatase) were purchased from Sigma Chemical Co. Ltd.(St. Louis, MO, USA). Nanoparticle stock suspension (80 lg mL�1)was prepared by suspending 0.8 mg of ZnO or TiO2 nanoparticlesin 10 mL of 0.22 lm filtered Milli-Q water. The stock suspensionwas sonicated (Sonics Vibra cell, Sonics & Material Inc., New Town,CT, USA) for 10 min at 30 W.

2.1.2. CharacterizationDynamic light scattering (DLS): Size (hydrodynamic diameter)

and zeta potential of the nanoparticles stock suspension weredetermined using dynamic light scattering and phase analysis lightscattering respectively in a Zetasizer Nano-ZS (Model ZEN3600;Malvern Instrument Ltd., UK) facilitated with 4.0 mW, 633 nmlaser.

2.1.3. Transmission electron microscopy (TEM)For transmission electron microscopy, the samples were pre-

pared by drop-coating the NPs suspension (8 lg mL�1) on carbon-coated copper TEM grids and scanned at 120 kV (JEM-2100, JEOLLtd., 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 li-ver S9 fraction was prepared according to the method described byMaron and Ames (1983).

2.3. Detection of nanoparticles uptake in bacteria

2.3.1. Transmission electron microscopy (TEM)S. typhimurium cell suspension (109 CFU mL�1) was treated with

ZnO or TiO2 NPs at a concentration of 8 lg mL�1 for 30 min at37 �C. Treated bacterial cell suspension was fixed with 2.5% glutar-aldehyde and pelleted. After washing with 0.1% phosphate bufferthe pellet was post fixed in 1% osmium tetraoxide. Fixed pelletwas then washed and dehydrated through grades (30–100%) ofacetone. Sample was infiltrated with araldite resin overnight atroom temperature and finally embedded in pure resin. The blockswere cured at 60 �C for 72 h. After incubation ultrathin sections(60 nm) were prepared using Reichert–Jung ultra microtome. Thesections were stained with uranyl acetate and Reynold’s lead cit-rate. The grids were examined under a TEM (JEM-2100, JEOL Ltd.,Tokyo, Japan) operated at an accelerating voltage of 100 kV usinga 20l aperture.

2.3.2. Flow cytometry (FCM)S. typhimurium cell suspension (109 CFU ml�1) was treated

with different concentrations of ZnO or TiO2 nanoparticles(0.008–8 lg mL�1) for 1 h at 37 �C with and without S9 fraction.To assess whether the NPs are retained in the cell, the uptakewas evaluated for several generations. A 100 lL aliquot of treated

S. typhimurium culture was re-inoculated in 10 ml fresh LB mediaand incubated at 37 �C in an environmental shaker–incubator at200 rpm. The cells (107 CFU) treated for 1 h were re-inoculated infresh media and used to assess size and granularity after 30, 60and 90 min. The uptake of NPs in the cells was analyzed using aflow cytometer (FCM; BD FACSCanto II, BD Biosciences, San Jose,CA, USA). The forward scatter and side scatter intensities indicatingthe size and intracellular density of cells respectively were re-corded. Analysis of the data was performed using BD FACSDiva6.1.2 software.

2.4. Bacterial mutagenicity test (Ames test)

The pre-incubation assay was performed according to the meth-od of Ames et al. (1975) as described below. The tester strains usedin this study – S. typhimurium TA98, TA100, TA1535 TA1537 andEscherichia. coli (WP2 uvrA) were purchased from Xenomatrix AG(Allschwil, Switzerland). The tester strains were checked for theirgenetic integrity for histidine dependence, biotin dependence, his-tidine/biotin dependence, rfa marker (crystal violet) and thepresence of plasmid pKM101 (ampicillin resistance) beforeexperiments.

Six experimental groups were taken as follows:

� Group 1: Vehicle control.� Group 2: Positive controls.

(a) In absence of S9: 2-nitroflourene (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 uvrA).

(b) In presence of S9: 2-amino anthracene (2AA; 5 lg/plate)was used for all the strains (TA98, TA100, TA1535 TA1537and E. coli).

� Group 3: Treatment with 0.008 lg mL�1 – ZnO/TiO2 NPs.� Group 4: Treatment with 0.08 lg mL�1 – ZnO/TiO2 NPs.� Group 5: Treatment with 0. 8 lg mL�1 – ZnO/TiO2 NPs.� Group 6: Treatment with 8 lg mL�1 – ZnO/TiO2 NPs.

The tester strains were freshly prepared by pre-culturing theminto the nutrient broth No. 2 for 12–17 h at 37 �C in an environ-mental shaker at 250 rpm in presence of 50 lg mL�1 ampicillin.Cofactor mix was prepared by adding 0.04 M KCl, 0.01 MMgCl2�6H2O, 0.142 M NaH2PO4, 0.007 M glucose-6-phosphate and0.005 M NADP in a final volume of 3.5 mL. A 30% S9 mix was pre-pared by adding 1.5 mL of S9 fraction to 3.5 mL cofactor mix. Thetop agar was prepared by dissolving 0.6 g of Bacto agar and 0.5 gNaCl in 100 ml distilled water. The agar was sterilized by autoclav-ing for 15 min at 15 lbs inch�1 and 121 �C. A mixed aqueous solu-tion of L-histidine (0.5 mM L�1) and D-biotin (0.5 mM L�1) for S.typhimurium and tryptophan (0.5 mM L�1) for E. coli (WP2 uvrA),was added to top agar medium immediately before use. Hundredmicrolitre of overnight grown culture (109 CFU mL�1) of Salmonellatester strains were incubated at 37 �C for 30 min at 180 rpm in asterile glass tube containing 500 lL of metabolic activation mix(+S9) or sodium phosphate buffer (0.1 mM, pH 7.4; for�S9 system)along with the desired concentrations of the nanoparticles. Afterincubation, 2 mL of top agar maintained at 45 �C was added tothe mixture and then poured on a plate of minimal glucose agarmedia. The plates were left to solidify the top agar. After solidifyingthe top agar, plates were incubated for 48 h at 37 �C and the revert-ant colonies were counted. Plates were used in triplicate for eachconcentration along with the respective negative and positive con-trol and mean value was calculated. Each experiment was repeatedthree times and average revertants from these three atypicalexperiments were reported. The results of the Ames assay were

1126 A. Kumar et al. / Chemosphere 83 (2011) 1124–1132

interpreted using the twofold rule (Mortelmans and Zeiger, 2000;Eick et al., 2002; Katzer et al., 2003; Ali et al., 2008).

3. Results

3.1. Particle characterization

The mean hydrodynamic diameter of the ZnO and TiO2 NPs inMilli-Q as measured by dynamic light scattering (DLS) was165 nm and 124 nm whereas zeta potential was �26 mV and�17.6 mV respectively (Table 1). The average size of ZnO andTiO2 observed by transmission electron microscopy (TEM) were30 nm and 50 nm respectively (Table 1). The NPs suspensions werestable, mono-dispersed and have a lower poly dispersity index.

3.2. Detection of nanoparticles uptake through TEM

S. typhimurium treated with ZnO and TiO2 NPs showed a signif-icant cellular uptake of the nanoparticle when compared to con-trols as evident from the TEM microphotographs (Fig. 2B and D).NPs of smaller size were found to be uniformly distributed inside

Table 1Size and zeta potential of ZnO and TiO2 NPs.

ZnO TiO2

DLS 165 nm 124 nmTEM 30 nm 50 nmBET 30 nm 5–10 nmZeta potential (f) �26 mV �17.6 mV

DLS – dynamic light scattering, TEM – transmission electron microscopy, BET –Brunauer–Emmett–Teller.

Table 2Effect of ZnO NPs on Salmonella and E. coli (WP2 uvrA) strains using Ames test.

TA98 TA100

�S9 +S9 �S9 +S9

Control 16 ± 2 18 ± 2 80 ± 3 95 ± 4ZnO NPs 0.008 lg/plate 26 ± 3 45 ± 4 110 ± 6 109 ± 5ZnO NPs 0.08 lg/plate 28 ± 3 36 ± 4 103 ± 4 115 ± 4ZnO NPs 0.8 lg/plate 32 ± 4 36 ± 3 94 ± 2 120 ± 2ZnO NPs 8 lg/plate 22 ± 3 33 ± 3 92 ± 2 104 ± 5Positive control 400 ± 40 450 ± 48 650 ± 38 700 ± 50

Data represent the mean revertants ± SD of data from three independent experiments, eThe values in bold are twofold or more and are significant when compared to controls.The positive control used during �S9 mix was 2 nitroflourene (2NF) for TA98 at 5 lg/platefor TA1535, 9-aminoacridine at 50 lg/plate for TA1537 strain and methyl methane sulfonamino anthracene (2AA) at a concentration of5 lg/plate for all strains.

Table 3Effect of TiO2 NPs on Salmonella and E. coli (WP2 uvrA) strains using Ames test.

TA98 TA100

�S9 +S9 �S9 +S9

Control 16 ± 2 18 ± 2 85 ± 3 88 ± 3TiO2 NPs 0.008 lg/plate 37 ± 3 50 ± 3 132 ± 6 143 ± 5TiO2 NPs 0.08 lg/plate 38 ± 4 45 ± 4 138 ± 5 149 ± 5TiO2 NPs 0.8 lg/plate 32 ± 3 42 ± 4 145 ± 8 130 ± 4TiO2 NPs 8 lg/plate 22 ± 2 30 ± 3 120 ± 4 90 ± 4Positive control 400 ± 40 450 ± 48 650 ± 38 700 ± 50

Data represent the mean revertants ± SD of data from three independent experiments, eThe values in bold are twofold or more and are significant when compared to controls.The positive control used during �S9 mix was 2 nitroflourene (2NF) for TA98 at 5 lg/platefor TA1535, 9-aminoacridine at 50 lg/plate for TA1537 strain and methyl methane sulfonamino anthracene (2AA) at a concentration of5 lg/plate for all strains.

the cells whereas agglomerated particles having bigger size ad-hered on the membrane. NPs were also observed in dividing cells(Fig. 2E).

3.3. Detection of nanoparticles uptake through flow cytometry (FCM)

A significant uptake of NPs was observed in S. typhimurium cellstreated with different concentrations of ZnO and TiO2 NPs (0.008–8 lg mL�1) in various experimental conditions (with and withoutS9), as evident by an increase in intensity of side scatter (SSC;Fig. 4,) as compared to the control (Fig. 4A). Gated population P1shows percent cells having increase in granularity but no increasein the size.

S. typhimurium cells after 60 min treatment exhibited a pro-nounced concentration dependent increase in the uptake of ZnONPs as indicated by an increase in the intensity of SSC (indicatinggranularity) in the presence of S9 (24%, 45%, 47%, 104% increaseat 0.008, 0.08, 0.8 and 8 lg mL�1 concentration respectively) thanin the absence (10%, 12%, 15.5%, 33% increase at 0.008, 0.08, 0.8and 8 lg mL�1 concentration respectively) when compared to thecontrol. Cells treated with ZnO NPs also exhibited a minor increasein size as evident by FSC in the presence and absence of S9.

A statistically significant concentration dependent increase inthe uptake was also observed in the SSC of S. typhimurium treatedwith different concentrations of TiO2 NPs after 60 min in the ab-sence of S9 (12%, 20%, 82.4%, 108.5% increase at 0.008, 0.08, 0.8and 8 lg mL�1 concentration respectively). Whereas the presenceof S9 enhanced the uptake (34.7%, 52.3% increase at 0.008,0.08 lg mL�1 concentration) at lower concentrations, however athigher concentrations the difference of induction in SSC was notmarked. There was no significant increase in size (FSC) after TiO2

NPs exposure.

TA1535 TA1537 E. coli (WP2 uvrA)

�S9 +S9 �S9 +S9 �S9 +S9

12 ± 2 15 ± 3 9 ± 2 12 ± 2 44 ± 3 50 ± 415 ± 2 18 ± 4 15 ± 3 28 ± 3 70 ± 2 108 ± 515 ± 3 20 ± 3 16 ± 2 26 ± 3 80 ± 4 109 ± 314 ± 3 19 ± 3 22 ± 2 25 ± 2 88 ± 2 90 ± 114 ± 2 17 ± 2 15 ± 3 22 ± 3 92 ± 3 85 ± 3

412 ± 36 170 ± 21 132 ± 20 221 ± 18 367 ± 14 316 ± 17

ach having three replicates (n = 9).

, 4nitroquinoline 1 oxide (4NQO) at 5 lg/plate for TA100, sodium azide at 5 lg/plateate at 500 lg/plate was used for E. coli. The positive control used with S9 mix was 2

TA1535 TA1537 E. coli (WP2 uvrA)

�S9 +S9 �S9 +S9 �S9 +S9

12 ± 2 15 ± 2 9 ± 2 12 ± 2 44 ± 3 50 ± 418 ± 3 23 ± 3 19 ± 2 38 ± 4 105 ± 14 116 ± 419 ± 3 25 ± 3 21 ± 2 34 ± 3 102 ± 5 111 ± 322 ± 3 24 ± 3 18 ± 2 30 ± 3 93 ± 3 106 ± 218 ± 2 19 ± 2 15 ± 1 21 ± 3 85 ± 4 98 ± 3

412 ± 36 170 ± 21 132 ± 20 221 ± 18 367 ± 14 316 ± 17

ach having three replicates (n = 9).

, 4nitroquinoline 1 oxide (4NQO) at 5 lg/plate for TA100, sodium azide at 5 lg/plateate at 500 lg/plate was used for E. coli. The positive control used with S9 mix was 2

A. Kumar et al. / Chemosphere 83 (2011) 1124–1132 1127

In the multi-generation study of ZnO NPs treated culture, a sharpdecline in the intensity of SSC (granularity) was observed withoutS9 when incubated for 30–90 min in NP free medium. However inpresence of S9 there was a gradual decrease in the intensity ofSSC, indicating lesser cell granularity. A concomitant decrease inthe intensity of FSC, indicating size of the cells, was also observed.

In cells treated with TiO2 NPs without S9, a gradual decrease inthe intensity of SSC (granularity) was observed with no change inthe intensity of FSC (cell size). However, the decrease in SSC (gran-ularity) was also accompanied with a concomitant decrease in theintensity of FSC (size) in cells treated with TiO2 NPs + S9.

3.4. Bacterial mutagenicity test

The concentration of ZnO and TiO2 NPs used in this study didnot show any cytotoxic effect in any of the tester strains as deter-mined by CFU count and flow cytometry (data not shown).

ZnO NPs showed a twofold increase in revertant colonies com-pared to the negative control in TA98, TA1537 and E. coli (WP2uvrA) strains with S9 (Table 2) while other strains TA100 andTA1535 did not show a significant change. The maximum muta-genic response was observed at concentration 0.008 lg/plate inboth TA98 and TA1537 strains.

TiO2 NPs also showed a statistically significant increase in thenumber of revertant colonies in TA98, TA1537 and E. coli (WP2 uvrA)strains with and without S9 at concentrations of 0.008–0.8 lg/platewhen compared to the negative control (Table 3). The mutagenic re-sponse was slightly higher in the presence of S9 fraction. The max-imum mutagenic response was observed at a concentration of0.008 lg/plate in both TA98 and TA1537 strains with S9 fraction.The positive controls showed several fold increase in number ofrevertant colonies as compared to negative control (Tables 2 and 3).

4. Discussion

The present study demonstrates for the first time, the uptake ofZnO and TiO2 NPs in S. typhimurium (TA98 and TA1537) strains aswell as their weak mutagenic potential leading to frameshift muta-tions. TiO2 NPs showed mutagenicity, both in presence and ab-sence of metabolic activation. However, ZnO NPs exhibitedmutagenic potential in the presence of metabolic activation (S9)only. This could be correlated to an increase in the uptake ofTiO2 NPs compared to that of ZnO NPs as evident from the TEMand flow cytometry data. This observation is of significance aschemical compounds exhibiting a positive response in the Amestest are likely to be carcinogenic in mammals and hence, may af-fect human somatic and germ cells leading to infertility and cancer(Mortelmans and Zeiger, 2000). Sharma et al. (2009) have shownthat ZnO NPs induce DNA damage in human epidermal cells. Ourobservations substantiate the findings of Sharma et al. (2009)and also provide possible explanation for genotoxic effects of these

200 nm

CharacterizationDLS, TEM, BET

Cellular UTEM, Flow c

Fig. 1. Schematic showin

NPs. Earlier studies have shown the negative mutagenic responseof the ZnO NPs. In these studies, Yoshida et al. (2009) used synthe-sized water soluble ZnO NPs capped with tetramethylammoniumhydroxide (TMAOH) whereas Sawai et al. (1998) carried out muta-genicity evaluations of bare 2.6 lm NPs. Possibly, the capping andsize of the bare particles tested was responsible for no mutageniceffect. We therefore reasoned to demonstrate uptake of NPs, itwould be necessary to ensure availability of bare nanoparticles inthe nano range without agglomeration. A schematic representationof the study design is depicted in Fig. 1. Nanoparticles were sys-tematically characterized to provide a clear picture of size andother physical properties such as poly dispersity index (PDI) andzeta potential. These parameters allow better interpretation ofthe differences in the activity of two different types of particlesor particles of the same material having different sizes. In the pres-ent study, characterization of NPs was carried out through DLS,TEM and Brunauer Emmett Teller (BET) methods. In these samplesno polydispersity was observed as evident by a sharp peak for thehydrodynamic diameter of the ZnO (165 nm) and TiO2 NPs(124 nm) through DLS as well as the lower PDI values 0.187 forZnO and 0.226 for TiO2 NPs. The zeta potential was �26 mV and�17.6 mV for ZnO and TiO2 NPs, respectively. The average size ofZnO and TiO2 NPs observed by TEM were 30 and 50 nm whilethe BET technique indicates 30 and 5–10 nm, concurrent withthe reported values in the datasheet of commercial supplier. Thisvariation in size of a nanoparticle by different methods is due todifferent principles of DLS, TEM and BET (Lin et al., 2009; Sharmaet al., 2009). The size obtained from DLS was more than the sizemeasured by TEM or BET because (1) DLS measures Brownian mo-tion and subsequent size distribution of an ensemble collection ofparticles in solution to give mean hydrodynamic diameter, usuallylarger than other technique as it includes a few solvent layers (2)there is a tendency of particles to aggregate in aqueous state there-by giving the average size of clustered particles and individual par-ticles during DLS measurement (3) DLS reports an intensityweighted average hydrodynamic diameter of a collection of parti-cles so any polydispersity of the sample will skew the averagediameter towards larger particle sizes.

The hypothesis behind the uptake experiment by flow cytome-try is based on the differential forward and side scattering propertyof blood cells (Shapiro, 2001). Different populations of blood cellsare identified in flow cytometry according to their intra cellularscattering property (granularity). Flow cytometry studies revealedthat nanoparticles are taken up by the bacteria, while few adhereto the outer membrane. This was further confirmed by the findingsof TEM of NPs treated cells, which showed that at non bactericidaldoses a large number of NPs were taken up. NPs of up to 70 nmwere uniformly distributed inside the cells while some agglomer-ated particles adhered outside the membrane as evident by TEM(Fig. 2C and D). This demonstrates that smaller NPs can enter thebacterial cells and may interact with the cellular macromolecules

ptakeytometry

MutagenicityAmes Test

g the study design.

Fig. 2. TEM microphotographs of Salmonella typhimurium TA98 showing: (A) control cell (B and C) internalization of ZnO nanoparticles (D) internalization of TiO2

nanoparticles (E) retention of nanoparticles in dividing cell.

1128 A. Kumar et al. / Chemosphere 83 (2011) 1124–1132

causing adverse effect including cell death. The NPs were alsofound in the dividing cells indicating transmission to the newlyformed cells as constituent of the cytoplasm. Non-specific diffu-sion, non-specific membrane damage and specific uptake (silCBAgene transportation system, through porins) are the possiblemechanism through which the NPs could pass through the bacte-rial cell wall and membranes but the precise mechanism is still un-known. Cellular uptake of the ZnO NPs in E. coli cells have also beenshown in some previous studies using TEM (Brayner et al., 2006;Huang et al., 2008; Tam et al., 2008). However, in the present studywe could also demonstrate the uptake of NPs in S. typhimuriumusing flow cytometry. Suzuki et al. (2007) and Xu et al. (2009) haveshown the increase in granularity (SSC) upon cellular uptake of theNPs in human cells using flow cytometry. A similar observationwas made in this study where the granularity of S. typhimuriumTA98 cells treated with NPs was found to increase with a concom-

itant induction in the cellular uptake of NPs as evident by the TEMstudies.

Cells treated with ZnO NPs in the presence and absence of S9showed a significant increase in granularity while the increase insize was not momentous. However, the increase in the intensityof SSC (granularity) was more prominent with liver S9 fraction.This could either be due to the formation of micelles of NPs in pres-ence of the liver fraction (Sereemaspun et al., 2008) or the proteinpresent in the S9 fraction may coat NPs and thus facilitate entryinto the cells (Romberg et al., 2008). Scanning electron microscopycoupled with energy dispersive X-ray spectroscopy (SEM-EDS)confirmed the protein coating over NPs. The EDS spectrum of NPslocated in SEM revealed a significant increase in the concentrationof nitrogen and carbon 13.9% and 46.2%, in case of ZnO + S9 and3.6% and 39.3% in case of TiO2 + S9 when compared to NPs alone(ZnO, N: 0.92%, C: 5.05%; TiO2, N: 0.02%, C: 11.32%). These observa-

Fig. 3. SEM microphotographs (A–D) of the ZnO, ZnO + S9, TiO2 and TiO2 + S9 nanoparticles with their corresponding EDX profife (A1, B1, C1, D1) recorded on marked portionof microphotograph.

A. Kumar et al. / Chemosphere 83 (2011) 1124–1132 1129

tions further confirmed that the NPs are coated with of proteinsfrom the S9 fraction (Fig. 3). However, S. typhimurium cells treatedwith TiO2 NPs with and without S9 showed a concentration depen-dent increase in granularity but not in size, which shows that themajority of NPs were taken up by the cells. This could be possiblydue to the smaller size of the TiO2 NPs as well as the difference insurface charge and consequential arrangement of the NPs on inter-action with native biomolecules.

Granularity of the S. typhimurium cells was also found to de-crease with time as evident in the multi-generation study. Thiscould be attributed to the fact that division of cells in a NPs freemedium reduces the concentration of NPs inside the cell and inturn the granularity. Our results demonstrate that ZnO NPs treatedS. typhimurium cells in absence of S9 when re-inoculated showed adecrease in granularity and size after 30 min incubation reachingvalues comparable with unexposed cells. The rapid decrease in sizecould be due to the removal of NPs from the cell wall. Whereas thegranularity of treated cells with ZnO + S9, TiO2 and TiO2 + S9showed a gradual decrease up to 90 min of incubation.

The Scientific Committee on Emerging and Newly-IdentifiedHealth Risks (SCENIHR, 2007) reiterates that any mutagenicity orgenotoxicity shown by nanoparticles may be detected using cur-rently available protocols, in spite of the uncertainties involvedin the testing procedures. Therefore, the mutagenicity of NPs wasdetermined using the Salmonella microsome test (OECD, 1997).

Ames test detects mutation in single gene (point mutations) inprokaryotic cells with and without S9 fraction. Liver S9 fraction isused to supplement metabolic enzymes such as cytochrome P450sas metabolic oxidation system. Different genotypic strains are rec-ommended by OECD Guideline (1997) for testing of compounds fordifferent mutation-mechanisms. In TA 98 hisG gene: hisD3052 hasone frameshift deletion, which is reverted to wild-type by variousframe shift mutagens, where as hisG46 marker in TA 100 resultsfrom the substitution of a leucine (GAG/CTC) by a proline (GGG/CCC). E. coli WP2uvrA used to detect oxidizing mutagens, such asfree radical generators. This strain carries a tryptophan-depen-dence due to a substitution in allele trpE65 by mechanisms of mis-replication or misrepair at AT sequence; mutagens which induce

Fig. 4. NPs uptake assessed by flow cytometry in Salmonella typhimurium cells. (A) Control (B and C) exposed to different concentrations of ZnO NPs, (D and E) exposed todifferent concentrations of TiO2 NPs (F) Control cell with S9 fraction (G and H) exposed to different concentrations of ZnO NPs with S9 (I and J) exposed to differentconcentrations of TiO2 NPs with S9 (0.008 and 8 lg mL�1 respectively) for 60 min.

1130 A. Kumar et al. / Chemosphere 83 (2011) 1124–1132

base-pair substitution can revert these mutations. Ames testerstrains were made for assessing chemical mutagenesis, howeverwith nanoparticles, there is a limitation that the strains do not ac-count for the physical interactions of the particles with DNA whichcould lead to mutation. This is in contrast to the mechanism ofchemical induced mutations.

Our study demonstrates ZnO nanoparticles were weakly muta-genic in TA98, TA1537 and E. coli (WP2 uvrA) strains in presence of

S9 (Table 2). However, no significant difference was observed inTA100 and TA1535 strains in presence or absence of S9. This indi-cates that ZnO nanoparticles have the potential to induce frame-shift mutation in presence of S9 fraction. This could partly bedue to an increase in cellular uptake of NPs in presence of S9 frac-tion as well as the tendency of particles to induce oxidative stressin the bacteria. Higher vertebrates possess an arsenal of xenobiot-ics metabolizing enzymes in liver that modify the chemical

Fig. 4 (continued)

A. Kumar et al. / Chemosphere 83 (2011) 1124–1132 1131

structure. It is hypothesized that ZnO nanoparticles can activatevarious stress pathways (including ROS generation), ultimatelyleading to frameshift mutation in metabolically active organism.

TiO2 NPs induced frameshift mutations in TA98, TA1537 andoxidative mutagenic response to E. coli (WP2 uvrA) strains bothin presence and absence of S9 fraction (Table 3), while no signif-icant change were observed in the number of revertant coloniesin TA100 and TA1535 strains. This data shows that TiO2 NPs havethe potential to elicit a mutagenic response in metabolicallymore competent eukaryotes, as well as in the bacteria, whichdo not possess the metabolic activation system. Our observationsare consistent with recent reports that have shown the muta-genic potential of TiO2 NPs using HPRT gene mutation assayand cytokinesis blocked micronucleus assay in human lympho-blastoid cells (Wang et al., 2007; Kang et al., 2008; Karlssonet al., 2008).

In a recent study the mild mutagenic potential of the metaloxide NPs (Including bare ZnO and TiO2, size <100 nm) has beenattributed to reduced particle size (Pan et al., 2010). However,the issues related to comprehensive characterization, low dose re-sponse, uptake of NPs have not been addressed. In contrast, ourstudy design and observations are not only based on smaller sizeNPs but also the doses that are approximately thousand fold lowerthan the lowest dose used by Pan et al. (2010). Our observationsusing DLS, TEM, flow cytometry and Ames test conclusively dem-onstrate that ZnO and TiO2 used in this study was in nano rangein different experimental conditions and taken up by the bacterialcells resulting weak mutagenic response. Auffan et al. (2009) havepointed out that as the particle size gets reduced to the nano range,the surface/volume (S/V) ratio increases and more atoms get ex-posed to the surface (35–40% for <10 nm and 20–25% for >30 nmparticle size). Further, it is known that the TiO2 NPs behave as anexcellent catalyst for the oxygen reduction and CO oxidation reac-tion (Auffan et al., 2009). It can be reasoned that these characteris-tics might be responsible for the mutagenicity of the bare NPs ascompared to the capped NPs.

In conclusion, the study demonstrates that ZnO and TiO2

nanoparticles are taken up by the S. typhimurium cells and inducemutagenicity in TA98 and TA1537 strains. ZnO nanoparticles causeframe shift mutation in the presence of S9 fraction where asmutagenic potential of TiO2 nanoparticles is independent of meta-bolic activation system. These observations once again reiteratethe concern about the safety of ZnO and TiO2 NPs in consumerproducts.

Conflict of interest

The authors do not have any conflicts of interest.

Acknowledgement

The authors wish to thank the Dr. K.C. Gupta, Director, IndianInstitute of Toxicology Research, Lucknow for his keen interest inthe study. This study was financially supported by the Departmentof Science and Technology, New Delhi, India [SR/S5/NM-01/2007].The authors also gratefully acknowledge the funding from CSIR,New Delhi under its network project [NWP34, NWP35], Supra-institutional Project [SIP-08], the Department of Science and Tech-nology-UK India Education and Research Initiative [UKIERI] Re-search Award [DST/INT/UKIERI/SA/P-10/2008].

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