Comparative in Vitro Cytotoxicity Study of Silver Nanoparticle on Two Mammalian Cell Lines

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1 Dublin Institute of Tehnology Artiles Shool of Physis 28 Comparative in Vitro Cytotoxiity Study of Silver Nanopartile on Two Mammalian Cell Lines Gordon Chambers Dublin Institute of Tehnology, Sanhali Mukherjee Dublin Institute of Tehnology Alan Casey Dublin Institute of Tehnology, Niall Ó Claonadh Dublin Institute of Tehnology Follow this and additional works at: Part of the Biohemistry, Biophysis, and Strutural Biology Commons Reommended Citation Mukherjee, SG., O'Claonadh, N., Casey, A., and Chambers G. Comparative in vitro ytotoxiity study of silver nanopartile on two mammalian ell lines. Toxiol In Vitro. Mar; 26(2):238-51(212) doi:1.116/j.tiv This Artile is brought to you for free and open aess by the Shool of Physis at It has been aepted for inlusion in Artiles by an authorized administrator of For more information, please ontat This work is liensed under a Creative Commons Attribution- Nonommerial-Share Alike 3. Liense

2 Toxiology in Vitro 26 (212) Contents lists available at SiVerse SieneDiret Toxiology in Vitro journal homepage: Comparative in vitro ytotoxiity study of silver nanopartile on two mammalian ell lines Sanhali Gupta Mukherjee a,b,, Niall O Claonadh a, Alan Casey a, Gordon Chambers a,b a Nanolab Researh Centre, Foas Institute, Dublin Institute of Tehnology, Kevin Street, Dublin 8, Ireland b Shool of Physis, Dublin Institute of Tehnology, Kevin Street, Dublin 8, Ireland artile info abstrat Artile history: Reeived 3 June 211 Aepted 6 Deember 211 Available online 14 Deember 211 Keywords: Physial haraterization Cytotoxiity assessment Mitohondrial metabolism Alamar Blue assay Coomassie Blue assay Lysosomal ativity Cellular proliferative apaity LD5 ROS Glutathione Adenylate kinase Apoptosis Comparative ytotoxiity In this study the ytotoxi effet of ommerially available silver (Ag) nanopartile was evaluated using human dermal and ervial aner ell lines. Prior to the ellular studies a full partile size haraterisation was arried out using Dynami Light Sattering (DLS), Transmission Eletron Mirosopy and Sanning Eletron Mirosopy in distilled water and ell ulture media. The Zeta Potential (ZP) assoiated with the Ag nanopartile was also determined in order to assess its stability in the solutions and its possible interation with the media. The DLS and ZP study have suggested interation of Ag nanopartiles with the media, whih an lead to seondary toxiity. The toxi effets of Ag nanopartiles were then evaluated using different ytotoxi endpoints namely the lysosomal ativity, mitohondrial metabolism, basi ellular metabolism, ellular protein ontent and ellular proliferative apaity. The ytotoxi effet of Ag nanopartile was dependant on dose, exposure time and on the ell line tested. Further investigation was arried out on HeLa and HaCaT ell lines to eluidate the mehanism of its ytotoxiity. The Ag nanopartile was noted to indue elevated levels of oxidative stress, glutathione depletion and damage to the ell membrane as found from the adenylate kinase assay and that leads to the apoptosis. Overall, signifiant differenes were observed between the sensitivity of the two ell lines whih an be understood in terms of their natural antioxidant levels. Ó 211 Elsevier Ltd. All rights reserved. 1. Introdution Corresponding author at: Nanolab Researh Centre, Foas Institute, Dublin Institute of Tehnology, Kevin Street, Dublin 8, Ireland. Tel.: ; fax: address: sanhali.guptamukherjee@student.dit.ie (S.G. Mukherjee). Silver (Ag) nanopartiles offer remarkably different physiohemial and biologial harateristis from that of bulk silver, suh as inreased optial, eletromagneti, atalyti properties and antimirobial ativity (Choi et al., 29). Ag has traditionally been used as an antimirobial agent for many years. Ag displays an oligodynami effet whih is an inherent antimirobial property assoiated with mainly heavy metals (Shuval et al., 1995). The exat mehanism by whih this ours is still largely unknown however, some literature proposes that Ag interats with the thiol groups on proteins neessary for mirobial respiration (Cho et al., 25). Ag ions are seen to atively seek out the thiol groups of proteins and enzymes thus making them unsuitable for mirobial development (Liau et al., 1997). Ag has also been shown to diretly interat with the ell membranes of bateria ausing breaks in the membrane and the ell to essentially burst (Perival et al., 25). It is this innate property that has lead to an array of potential appliations of Ag nanopartiles in both the medial arena and onsumer produts. In terms of medial appliations Ag nanopartiles has found appliations in wound dressings, ontraeptive devies, surgial instruments and bone prostheses whih an be oated or embedded with the Ag nanopartiles (Cheng et al., 24; Chen et al., 26; Muangman et al., 26; Cohen et al., 27; Lansdown, 26; Zhang and Sun, 27). In addition, onsumer produts ontaining Ag nanopartiles inlude antibaterial room spays, laundry detergents, water purifiants and wall paint (Cheng et al., 24; Zhang and Sun, 27). Ag nanopartiles are also inorporated into textiles for manufature of lothing, undergarments and soks (Lee et al., 27; Vigneshwaran et al., 27). As a result of these appliations exposure of Ag nanopartiles to us and our environment are beoming inreasingly widespread. Consequently, silver in the form of nanopartiles has gained an inreasing aess to tissues, ells and biologial moleules within the human body. One reahing nanosale, ertain materials do exhibit signifiant toxiity to mammalian ells even if they are biohemially inert and bioompatible in bulk size, e.g. arbon (Magrez et al., 26; Shvedova et al., 25). Several studies exist in literature showing that the release of Ag ions inhibit the normal respiratory funtions of the ells and ause ell death. A study /$ - see front matter Ó 211 Elsevier Ltd. All rights reserved. doi:1.116/j.tiv

3 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) arried out by Hussain et al. (25) have shown that Ag nanopartiles was ytotoxi to rodent liver ells ausing them to hange morphologially and also interfering with mitohondrial funtion. It has also been shown that Ag nanopartiles are highly toxi to the osteoblast ell lines (Moaddab et al., 211). One study has shown a very high toxi response of Ag nanopartile of 5 1 nm diameter on hepatoma ell line, HepG2, by ausing oxidative stress and DNA damage (Kim et al., 29). The human body has several semi-open interfaes for diret substane exhange with the environment, i.e. the respiratory trat, gastrointestinal trat (GIT) and skin. In view of Ag nanopartile s diverse forms of existene, they are also the priniple routes of exposure. The irreversible pigmentation of skin and/or eye, i.e. argyria or argyrosis, due to the silver deposition, may develop after prolonged exposure to silver or silver ompounds (Van devoorde et al., 25; Eturska and Obreshkova, 1979; Spener et al., 198). Female genital trat is also an entryway of potential importane sine Ag nanopartiles has been inorporated into maternal hygiene produts (Cheng et al., 24; Zhang and Sun, 27). At these sites, nanopartiles an undergo a series of proesses like binding and reating with proteins, phagoytosis, deposition, learane and transloation. On the other hand nanopartiles an eliit a spetrum of tissue responses suh as ell ativation, generation of Reative Oxygen Speies (ROS), inflammation and ell death (Chen et al., 26; Xia et al., 26; Ahn et al., 25). Therefore, the wide range of appliations of Ag nanopartiles ould potentially result in multiple sites of exposure to Ag nanopartiles and studies into their biologial effets are of great importane. The aim of this study is to examine the effets of Ag nanopartiles on ell models of two possible routes of entry and exposure for this two human ell lines were employed a dermal ell line and a ervial anerous ell line. The effet has been evaluated using variety of end-points to postulate a mehanism of ation of Ag nanopartiles. A battery of standard ytotoxiity assays, viz., Alamar Blue (AB) assay, Neutral Red (NR) assay, MTT assay, Coomassie Blue (CB) assay, were employed to study the effet of Ag nanopartiles on both the ell lines. AB, NR, CB and MTT assays show the effet of Ag nanopartile on different aspets of ellular ativity. Therefore the interpretation of the dose response from these assays is very important to understand the ellular response upon Ag nanopartile exposure. The interpretation of the ation of the AB assay is somewhat varied. One report has suggested that it may indiate the early inhibition of ytoplasmi metabolism and ell growth (O Brien et al., 2) while another suggests that it indiates the inhibition of mitohondrial metabolism (De Fries and Mitsuhashi, 1995). But in general it is suggested that it shows basi ellular metabolism in terms of it oxido-redutase enzyme ativity (Mukherjee et al., 21b). NR is a olorimetri assay for the quantifiation of the membrane permeability and lysosomal ativity of ells in response to pharmaeutial, hemial and environmental ompounds, and nutrients (Mukherjee et al., 21a,b). The CB assay quantifies the amount of protein present in the ells (Clare and Cormak, 1996). The MTT olorimetri assay determines the ability of viable ells to redue the soluble, yellow tetrazolium salt [3-(4,5-dimehtylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) into an insoluble, purple formazan. Tetrazolium salts aept eletrons from oxidized substrates or enzymes, suh as NADH and NADPH. Redution of MTT takes plae at the ubiquinone and ytohrome b and sites of the mitohondrial eletron transport hain due to suinate dehydrogenase ativity (Mosmann, 1983). The redution of MTT to formazan within ells helps in the estimation of the mitohondrial metabolism and hene the number of viable ells after exposure to nanopartiles. Apart from aessing the effet of Ag nanopartile on different ellular organelles and metabolism, we have also aessed its effet on the ellular proliferative apaity by lonogeni assay. Intraellular reative oxygen speies (ROS) generation, glutathione (GSH) depletion, adenylate kinase (AK) release from the ells and apoptosis indution upon Ag nanopartile exposure was also studied. Adenylate kinase (also known as ADK or myokinase) is a phosphotransferase enzyme that atalyzes the nuleotide phosphoryl exhange reation 2ADP M AT- P + AMP, ritial in ell life. They are found in ytosol and mitohondria of the ells. Adenylate kinase will only leak from ells whose ell membrane integrity has been ompromised. Apoptosis is an energy driven proess; ATP levels are maintained in metabolially ative ells, but upon ell death residual ATP is degraded (Bradbury et al., 2; Crouh et al., 1993). With the onset of apoptosis, ells show an inrease in the levels of ellular ADP (adenosine diphosphate) but without an appreiable drop in ATP. As ells pass into seondary nerosis the ADP levels ontinue to rise as hydrolysis of ATP starts to our. In primary nerosis ATP levels fall rapidly as a result of exposure to high onentrations of toxi agents with a orresponding inrease in the ellular ADP (Bradbury et al., 2). Finally the differenes between the responses from the two ell lines are ompared and the possible reason behind the differene in the responses is eluidated. 2. Materials and methods 2.1. Test materials and reagents Silver (Ag) nanopowder of < nm was purhased from Sigma Aldrih Ltd. (Dublin, Ireland). 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), Neutral Red (NR), Commassie Blue (CB) and 2,7 -dihlorofluoresin diaetate (DCF) as well as ell ulture media, supplements, the trypsinisation solution were all purhased from were purhased from Sigma Aldrih Ltd. (Ireland). Alamar Blue (AB) was purhased from Biosienes (Dublin, Ireland). Carbol fuhsin was purhased from BDH (UK). ToxiLight and ApoGlow Ò kit were purhased from Lonza Ltd. (Wokingham, UK) Cell ulture HaCaT ells, an immortal non-anerous human keratinoyte ell line (kindly provided by Prof. Dr. Boukamp, Heidelberg), HeLa ells (ATCC, CCL-2 ) an epithelial adenoarinoma ell line of the ervix were employed in this study. HaCaT ells were ultured in Dulbeo s Modified Eagle s Medium Nutrient Mixture F-12 HAM (DMEM F-12 HAM) with 2 mm L-glutamine supplemented with 1% fetal bovine serum (FBS), 45 IU/ml peniillin and 45 IU/ml streptomyin (HaCaT media) at 37 C in 5% CO 2. HeLa ells were ultured in RPMI-164 medium supplemented with 1% FBS, 45 IU/ml peniillin and 45 IU/ml streptomyin (HeLa media) at 37 C in 5% CO Charaterization of Ag nanopartiles Prior to the ytotoxiity testing Ag nanopartiles were subjeted to an extensive haraterisation proess, where possible measurements were performed on Ag nanopartile as purhased and on test suspensions of them. The suspensions of Ag nanopartiles were prepared in distilled water (dh 2 O), HaCaT media and in HeLa media using bath-soniator (Degussa-Ney ULTRAsonik 57X, 5/ 6 Hz, California, USA) prior to size and zeta potential measurements. Dynami light sattering sizing measurements and zeta potential measurements were performed with the aid of a Malvern Zeta Sizer Nano ZS (Malvern Instruments, Worestershire, UK) operating with version 5.3 of the systems Dispersion Tehnology Software (DTS Nano). For size measurement, DTS12 disposable

4 24 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) sizing uvettes were used. The refrative index (RI) of Ag nanopartiles, dh 2 O, HaCaT media and HeLa media were 1.6, 1.33, 1.332, 1.3, respetively. Prior to analysis, visosity measurements were performed on all media and dh 2 O with the aid of a Visometer SV- 1 (A&D Instruments Ltd., UK) at 25 C and the reorded values used in all DLS size estimations. The visosity of dh 2 O, HaCaT media and HeLa media at 25 C were.887p,.82p,.55p, respetively. The samples were equilibrated at 25 C for 3 min before eah measurement. For zeta potential measurements DTS16C lear disposable zeta ells were used and the measurements were performed at 5 V. Smoluhowski equation was used in the DTS software to alulate the zeta potential of the Ag nanopartiles. For all zeta potential measurements analysis was performed on the solutions with the automati model setting, using an applied voltage of 5 V, to minimize artefats and harring of media proteins during analysis. The speifi surfae area of the partiles was estimated with a Mirometris GEMINI BET. BET sample holders were filled with a known mass of the powdered Ag nanopartiles and measured. The sample was degassed for 2 h at room temperature with nitrogen gas prior to analysis, nitrogen gas was used as the absorptive gas, a multipoint method was used in the estimation of speifi surfae area and the sample was analysed without porosity analysis. transmission eletron mirosopy (TEM) was also used to estimate the size of the Ag nanopartiles from its images. The instrument used was a JEOL CX model TEM. Samples were prepared by dispersing Ag nanopartiles in ethanol to a onentration of 1 mg/l by soniation 75 watts Ultrasoni Proessor tip (Branson Ultrasonis, Ultra soni proessor VCX- 75 W) soniator at 4% amplitude for a total operating time of 45 s. Samples were then drop ast onto 3 mm FORMVAR oated Cu grids (AGAR Sientifi). One the solvent had evaporated partile size was measured at a range of magnifiations up to 8,. The aelerating voltage and probe urrent used for TEM analysis were 25 kv and 18 la, respetively. SEM was also performed to analyse partile size. Hitahi SU 66 FESEM instrument was used. The SEM was alibrated with Au on Carbon standard provided by Agar Sientifi (Essex, UK). Sample preparation was idential to that of the TEM preparation. Solutions were drop ast onto a pure silion wafer whih had been thoroughly leaned by soniation in aetone for a period of 3 min followed by boiling in propanol for 3 min. The silion wafers were then left to air dry in a dust free environment and the nanopartile solution was drop asted onto them 24 h prior to measurement Cytotoxiity evaluation The AB, NR, CB, MTT and the lonogeni assay were performed for assessment of the ytotoxiity of Ag nanopartiles to the two ell lines. A preliminary or range finding test was onduted to determine the range of onentrations of interest for the definitive test. The definitive test used a onentration range (at least eight onentrations) in whih effets was likely to our, thereby permitting the alulation of the respetive Lethal Dose (LD 5 ), no observed effet onentration (NOEC) and lowest observed effet onentration (LOEC). In all ases, results were ompared to an unexposed ontrol, eliminating any dependene of ell line exposures on volumes, well type, seeding effiieny and numbers, exposure times, et. A stok solution of Ag nanopartile was prepared aseptially, from whih different onentrations of Ag nanopartiles were prepared in the respetive HaCaT media and HeLa ell ulture media, followed by bath soniation (Degussa-Ney ULTR- Asonik 57X, California, USA) for 1 min at room temperature before exposure. As a positive ontrol, 1% dimethyl sulfoxide (DMSO) prepared in the respetive media was used parallel to the Ag nanopartile exposures Alamar Blue, Neutral Red and Coomassie Blue assays For the AB and NR assays, ells were seeded in 96 well miroplates (Nun, Denmark) at a density of ells/ml in ll of respetive media ontaining 1% FBS. At least three independent experiments were onduted and six repliate wells were employed per onentration per plate in eah independent experiment. After 24 h of ell attahment, plates were washed with ll/well PBS and the ells were treated with inreasing onentrations of Ag nanopartiles, prepared in 1% FBS ontaining media for 24, 48, 72 and 96 h. All inubations were performed at 37 C in a 5% CO 2 humidified inubator. The AB, NR and CB assays were onduted onseutively on the same set of plates. The AB assay was performed first followed by the NR assay and then lastly the CB assay. The assays were arried out aording to manufaturer s instrution. Briefly, ontrol media or test exposures were removed; the ells were rinsed with PBS and ll of AB/NR medium (5% [v/v] solution of AB and 1.25% [v/v] of NR dye) prepared in fresh media (without FBS or supplements) were added to eah well. After 3 h inubation AB fluoresene was measured at the respetive exitation and emission wavelength of 531 nm and 595 nm in a VICTOR 3 V 142 Multilabel Counter (Perkin Elmer, USA). Wells having only AB and media were used as blanks. After measurement, the wells were then washed with PBS and ll of NR fixative (5% ethanol, 49% dh 2 O and 1% glaial aeti aid) was added to eah well and the plates were shaken at 24 rpm for 1 min. The NR fluoresene was then measured at the exitation and emission wavelength of 531 and 642 nm, respetively in the same instrument. Protein determinations were performed on the same plates immediately by CB assay following NR uptake study. Exess NR dye was removed from the ells by washing with ll de-staining solution. COMMASSIE dye was added to eah well and the plate agitated for 1 min. The dye was removed and the plate washed with an aeti aid ethanol solution. The wash solution was disarded and the dye extrated with measuring solution (1 M potassium aetate). The plate was shaken at 24 rpm for 1 min and the absorbane of the extrated dye was read at 57 nm (referene filter 34 nm) using the miroplate reader. For these three assays, mean fluoresene/absorbane units for the six repliate ultures were alulated for eah exposure treatment MTT assay A parallel set of at least 3 repliate plates was set up for the MTT assay and seeded and exposed in an idential manner as desribed in Setion After 24, 48 and 72 h of Ag nanopartile (AgNP) exposure, the medium for the ontrol or test exposures was removed, the ells were washed with PBS and ll of freshly prepared MTT in media (5 mg/ml of MTT in media [without FBS or supplements]) were added to eah well. After 3 h inubation, the medium was disarded and the ells were rinsed with PBS and ll of MTT fixative solution (isopropanol with.4 N HCl) were added to eah well and the plates were shaken at 24 rpm for 1 min. The absorbane was then measured at 595 nm in a TECAN GENios (Grodig, Austria) plate reader Clonogeni assay The lonogeni assay was developed by Puk and Marus (1956) and further standardized by Franken et al. (26). The lonogeni assay was performed in 6 well plates (Nun, Denmark) and eah well was seeded with 2 ml of respetive 1% FBS ontaining media at a ell density of 25 ells/ml. For both ell lines, the ells were allowed to attah for 12 h, whih is shorter than their reported doubling time of 23 h for HaCaT ells (Boukamp et al., 1988) and 22 h for Hela ells (York and Majerus, 1994). Therefore it is assumed that predominantly single ells were present at the time of exposure. After 12 h of ell attahment, plates were washed

5 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) with 2 ml/well PBS and the ells were treated with inreasing onentrations of AgNP prepared in 1% FBS ontaining respetive media. The plates were then inubated for 8 days. Two repliate wells were used for eah ontrol and test onentrations per 6-well plate. Following exposure, the test solution was removed; the ells were washed with PBS and finally fixed and stained with 2% arbol in formalin solution (BDH, Poole, UK). The olonies were then manually ounted. The minimum size of a olony was onsidered to be 5 ells/olony. In all these assays 1% DMSO in 1% FBS supplemented media was used as a positive ontrol parallel to the Ag nanopartile test solutions Optial mirosopi study Interation of Ag nanopartiles with HaCaT and HeLa ells lines was studied by optial mirosopy. Both the ell lines were seeded at a density of ells/ml in petri dishes (Nun, Denmark) and the Petri dishes were kept in a 37 C, 5% CO 2 inubator. After 24 h of ell attahment, the ells were washed twie with PBS and were exposed to 5 mg/l, mg/l and 2 mg/l Ag nanopartile onentrations in the respetive media and the Petri dishes were kept in a 37 C, 5% CO 2 inubator for 24 h. The Ag nanopartile test solutions were prepared in the same way as stated in Setion 2.4. Following exposure, the test solutions were removed; the ells were washed twie with PBS and were imaged in 37 C PBS using a Nikon Elipse E6 mirosope. All images were reorded using a Spot RT digital amera using Spot software (version 3.2.4, Diagnosti Instruments, In., USA) Reative oxygen speies (ROS) study Intraellular oxidative stress was quantified with the aid of 2,7 -dihlorofluoresin diaetate (DCFH-DA), whih was reported to detet intraellular hydroperoxides (Cathart et al., 1983) and also a probe for wide range of reative oxygen speies (Invitrogen, Ireland, website us/en/home/referenes/moleular-probes-the-handbook/tables/ Reative-oxygen-speies.html). Briefly, healthy onfluent ells were harvested and seeded ( ells/well) into blak bottomed 96 well plates (Nun, Denmark) and allowed to attah for a period of 24 h prior to exposure. For ROS quantifiation three independent experiments were performed for eah independent experiment eight repliate wells were used for ontrol, eight repliate wells were employed for the positive ontrol and eight repliate wells were used for eah test onentration per miroplate. A working stok of 2 lm DCFH-DA in PBS was prepared and all test onentrations, unexposed negative ontrols and positive ontrols were prepared and exposed to the ells in this working stok. The negative ontrol onsisted of the working stok solely namely a 2 lm DCFH-DA solution in PBS, the positive ontrol onsisted of a 1 lm Hydrogen Peroxide (H 2 O 2 ) prepared in the 2 lm DCFH-DA/PBS working stok solution and finally the test onentrations onsisted of a onentration range of Ag nanopartiles (for HaCaT 7.81 to 125 mg/l and for HeLa mg/l) prepared in the working stok a 2 lm DCFH- DA/PBS solution. After the initial 24 h attahment period the media was removed, the ells were subsequently washed with ll of PBS and treated with ll of the negative ontrol, positive ontrol and the test onentrations of AgNP and inubated for 3, 6, 12, 18, 24, 3 min. The rate of intraellular oxidative stress was then monitored by monitoring the emission at 529 nm (by 54 nm exitation) of the DCFH-DA dye at various time intervals of 3 3 min (the exposure plates were re-inubated for the remaining time after eah measurement had been reorded) Intraellular redued glutathione level measurement A ommerial kit ThiolTraker Violet was used to estimate the levels of intraellular redued glutathione (GSH) in HaCaT ells upon AgNP exposure. The fluoresent dye an be used for intraellular GSH detetion in flow ytometry or by fluoresent imaging in living ells ( T96?ICID=searh-produt). In this study, a fluoresene miroplate reader was employed to quantify the fluoresene intensity of the dye and therefore the intraellular GSH levels in ontrol and exposed HaCaT ultures. The study was performed in 96-well miroplate format, wherein the ells were seeded at a density of ells/ml in ll of respetive media ontaining 1% FBS. After 24 h of ell attahment, the ells were washed twie with ll/well PBS and were exposed to 25 2 mg/l AgNP in 1% FBS supplemented media for 24 h. 1% DMSO in the respetive media were also used as positive ontrols. Following exposure, the ells were washed with ll/well PBS and exposed to the Thiol- Traker Violet dye, prepared in PBS at a onentration of 2 lm in ll/well and the plates were kept in a 37 C, 5% CO 2 inubator for 3 min. Following inubation, the dye solution was removed, the ells were washed thrie with ll/well PBS and the fluoresene was measured at exitation and emission wavelengths of 45 and 535 nm, respetively, in a VICTOR3 V 142 Multilabel Counter plate reader (Perkin Elmer, UK) Adenylate kinase (AK) release assay The bioluminesent ToxiLight kit (Lonza Ltd., Wokingham, UK) was employed to measure the onentration of AK present in the supernatants of test solutions after different time point exposures. The kit is based on the bioluminesent measurement of AK whih is present in all ells. This kit quantitatively measures the release of AK from damaged ells. A loss of ell integrity, through damage to the plasma membrane, results in the leakage of a number of fators from ells ultured in vitro into the surrounding medium. The measurement of the release of AK from the ells allows the aurate and sensitive determination of ytotoxiity and ytolysis. The reation involves two steps. The first involves the addition of ADP as a substrate for AK. In the presene of the enzyme, AK, the ADP is onverted to ATP for assay by bioluminesene: Mg þþ Adenylate ADP þ ADP $ Mg þþ Kinase ATP þ AMP The bioluminesent method utilizes an enzyme luiferase, whih atalyses the formation of light from ATP and luiferin aording to the following reation: Luiferase ATP þ Luiferin þ O 2 ƒƒƒƒƒƒ ƒ! Oxyluiferin þ AMP þ PPi þ CO 2 þ Light Mg þþ (Lonza Rokland, In., ombining the two reations, the emitted light intensity is linearly related to the AK onentration. To quantify AK release, the exposed test solutions from the AB, NR and CB assays were olleted after their respetive time point exposures and were stored at 8 C. Upon thawing, 2 ll of eah sample (whih ontained the pooled supernatants from six repliate treatments per plate) were removed and transferred to a 96-well white luminesene plate (Falon, VWR Ireland) for AK measurement. Media was assayed in tripliate for AK release. AK Detetion Reagent (AKDR) was then reonstituted in Assay Buffer supplied with a kit. Leave for 15 min at room temperature to ensure omplete rehydration. One-hundred mirolitres of AK detetion reagent was then added to eah well and the plate

6 242 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) inubated at room temperature (RT) for 5 min and a 1 s integrated luminesene reading was reorded Apoptosis study Ag nanopartiulate indue apoptosis was studied using Apo- Glow Ò kit of Lonza Ltd. (Wokingham, UK). In brief, ll the ells were seeded in 96-well white luminesene plates (Falon, VWR Ireland) at a density of ells/ml in ll of respetive media. Following 24 h ell attahment, the plates were washed with ll/well PBS and the ells were treated with inreasing onentrations of Ag nanopartiles, prepared in 1% FBS ontaining media for 24 h. All inubations were performed at 37 C in a 5% CO 2 humidified inubator. Six repliate wells were used for eah ontrol and test onentrations per miroplate. After the exposure the apoptosis assay was performed following the manufaturers manual. In brief, the miroplates were removed from the inubator following 24 h Ag nanopartile exposure and were allowed to ool to room temperature. The luminometer was programmed to take a 1 s integrated reading. Then ll/well of nuleotide releasing reagent (NRR) was added and kept for at least 5 min. After that, 2 ll/well of NMR was added and a 1 s integrated reading (Reading A) was taken in luminometeri plate reader. The plates were then kept for 1 min and 1 s integrated reading (Reading B) was taken. Then finally 2 ll/well of ADP-CR was added and again held for 5 min and finally 1 s integrated reading (Reading C) was taken. Finally the ADP:ATP ratio was alulated from the readings using the following formula (Reading C Reading B)/Reading A. This assay is based upon the bioluminesent measurement of ATP that is present in all metabolially ative ells. The bioluminesent method uses luiferase, whih generates light from ATP and luiferin aording to the following reation: Luiferase ATP þ Luiferin ƒƒƒƒƒƒƒ! Oxyluiferin þ AMP þ PPi þ CO 2 Mg þþ (Lonza Rokland, In., The emitted light intensity is linearly related to the ATP onentration and is measured using a luminometer. The assay is onduted at ambient temperature (18 22 C), the optimal temperature for luiferase. This kit measures the intraellular AD- P:ATP ratio whih an be used to distinguish between the different modes of ell death, whether the ell dies via apoptosis or nerosis Statistial analysis At least three independent experiments were onduted for eah toxiity endpoint. Test results for eah assay were expressed as perentage of the unexposed ontrol ± standard deviation (SD). Control values were set as %. Differenes between samples and the ontrol were evaluated using the statistial analysis pakage SPSS 14.. Statistially signifiant differenes were set at p <.1. Normality of data was onfirmed with Q Q perentile plots and Kolmogorov Smirnov tests. Equality of varianes was evaluated using Levène tests. One-way analysis of varianes (ANO- VA) followed by Dunnett s multiple omparison tests were arried out for normally distributed samples with homogeneous varianes. Cytotoxiity data was fitted to a sigmoidal urve and a four parameter non-linear logisti model used to alulate the Lethal Dose of nanomaterial that aused a 5% inhibition in omparison to untreated ontrols (LD 5 ). All LD 5 values were alulated using the average ytotoxiity data of the three independent experimental results and their assoiated errors. LD 5 values are reported ±95% onfidene intervals (±95% CI). Lethal dose 5 (LD 5 ) values were estimated using Xlfit3, a urve fitting add-on for Mirosoft Ò Exel (ID Business Solutions, UK). 3. Results 3.1. Partile haraterization The partile size of the Ag nanopartiles was evaluated using a variety of methods namely DLS, TEM and SEM. DLS study have shown average number size-distribution peak-sizes of mg/l Ag nanopartiles in dh 2 O, HeLa media and in HaCaT media as 28.41, and nm, respetively (Fig. 1a). Size distribution plots of Ag nanopartiles in dh 2 O and HaCaT media are also shown as an example in Fig. 1b and Fig. 1, respetively. The sizes of the Ag nanopartiles were alulated from the highest peak value of size/number in DTS Nano software. The variation in size an be attributed to hanges in the hydrodynami radius of the partile in the different media due to partile and media interations, where steri stabilization in the media was observed for the Ag nanopartiles of nm size-range and the larger partiles of around 5 nm size-range, as observed in Fig. 1, were may be due to partile agglomeration upon dispersion in the media. In fat DLS measures the hydrodynami radius and not the physial partile size. The average diameter yielded from the TEM study was nm (Fig. 1d) and SEM analysis was <5 nm (Fig. 1e). Both of whih reflet more aurately the physial partile diameter. These ombined studies onfirmed the test partiles had an average diameter in dh 2 O of approximately 27 nm. The DLS size measurements of HaCaT and HeLa media was also performed as ontrol and were found to be 8.14 and 9.24 nm, respetively (Fig. 1a). BET analysis was performed to estimate the surfae area of the Ag nanopartiles and was found to be 2.3 ±.1 m 2 /g. The average zeta potential measured of mg/l Ag nanopartiles in dh 2 O, HeLa media and in HaCaT media were 33.33, 2.67 and mv (Fig. 1f). Zeta potential values of this nature would suggest that upon dispersion with the bath soniator the partiles form an unstable dispersion and would preipitate out with time. The average zeta potential of HaCaT and HeLa media without Ag nanopartile were found to be 6.61 and 6.22 mv (Fig. 1d) Standard ytotoxiity assays For all assays, i.e., AB, NR, Coomassie Blue and MTT, a dose dependent response was observed and an LD 5 values were alulated. Cytotoxiity data for the Ag nanopartiles and the HaCaT and HeLa ells are presented in Table 1 and Figs The LD 5 values presented in table suggest that MTT assay was the most sensitive assay among the four assays in both the ell lines. Coomassie Blue assay was found to be least sensitive in Ha- CaT ells, while in HeLa ells Neutral Red assay was found to be least sensitive. Viability was found to be dereasing with the inreasing exposure time in AB, NR, CB, MTT assay in both the ell lines. LD 5 values of HeLa ells are muh lower than HaCaT ells at all exposure time points in Coomassie Blue and Neutral Red assay. In Alamar Blue and MTT assay the LD 5 values of HeLa ells was slightly higher as ompared to HaCaT ells after 24 h of exposure, but then again get lowered after 48 and 72 h exposure time. The lonogeni assays showed dereasing olony numbers with the inreasing onentration of Ag nanopartiles (Fig. 6). In the lonogeni assay the average plating effiienies for the HaCaT and HeLa ells were 37% and 48%, respetively. The LD 5 values alulated from the lonogeni assay were 7.74 mg/l (onfidene interval ±2.97) for the HaCaT ell line and 1.16 mg/l (onfidene interval ±1.9) for that of the HeLa ells. At 2.5 and 5 mg/l Ag nanopartile onentrations the perentage of olonies formed in HeLa ells were 13.96% and %, respetively. While in HaCaT ells at 2.5 mg/l and 5 mg/l Ag nanopartile onentration there was 83.16% and 62.5% respetive olonies were observed, the olonies

7 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) a b 6 Diameter (nm) HaCaT media HeLa media mg/l Ag NP in dh2 mg/l Ag NP in HeLa media mg/l Ag NP in HaCaT media d e f Zeta Potential (mv) HaCaT media HeLa media mg/l Ag NP in HaCaT media mg/l Ag NP in HeLa media mg/l Ag NP in dh2 Fig. 1. Size measurement of mg/l Ag nanopartiles: (a) DLS in dh 2 O, HeLa media and HaCaT media, (b) size distribution plot of Ag nanopartiles in dh 2 O, () size distribution plot of Ag nanopartiles in HaCaT media, (d) TEM image of 1 mg/l Ag nanopartile, (e) SEM image of 1 mg/l Ag nanopartile, (f) Zeta potential in dh 2 O, HeLa media and HaCaT media. formed in HaCaT ells are muh higher in perent than HeLa ells at same onentration. This shows that the proliferative apaity to form olonies of HaCaT ells in omparison to HeLa ells on exposure of Ag nanopartiles is muh higher Optial mirosopi study Optial mirosopi study have shown, with the inreasing onentration of Ag nanopartiles the HaCaT (Fig. 7) and HeLa (Fig. 8) ells were looking unhealthy and the ells were dying out of the population. The population of HeLa ells were found to me more redued at 2 mg/l Ag nanopartile exposure for 24 h in omparison to HaCaT ells Reative oxygen speies study The ROS study has shown that in HaCaT ells with the inreasing time of Ag nanopartiles exposure more ROS was produed at their respetive onentration (Fig. 9a). In HeLa ells, similarly the amount of ROS was found to be inreasing with the inreasing time of exposure at their respetive onentrations (Fig. 9b). In HeLa ells the maximum amount of ROS was produed at 2 mg/ l onentration after 5 h exposure of Ag nanopartiles, while in Ha- CaT ells maximum ROS prodution was observed at mg/l onentration after 5 h exposure (Fig. 9). This study also shows that there was more ROS prodution in HeLa ells as ompared to HaCaT ells.

8 244 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) Table 1 Calulated LD 5 (mg/l) values resulting to exposure to Ag nanopartiles for the AB, NR, CB and MTT assays in the HaCaT and HeLa ell lines. Cell lines CB assay LD 5 (mg/l) (onfidene interval) HaCaT 181 (±3.92) HeLa 88.6 (±24.6) NR assay LD 5 (mg/l) (onfidene interval) AB assay LD 5 (mg/l) (onfidene interval) MTT assay LD 5 (mg/l) (onfidene interval) 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 14.5 (±26.37) 16.4 (±11.56) (±58.55) 1.7 (±3.87) 2.1 (±.91) 18.6 (±28.12) 116 (±32.99) 18.4 (±8.62) (±19.34) (±22.13) 12.7 (±6.4) 8.9 (±29.54) 35.2 (±5.37) 14.3 (±1.14) 29 (±2.99) 6.9 (±2.76) 51.8 (±19.14) 56.4 (±13.93) 35.6 (±6.79).9 (±.15) 3.4 (±8.64).4 (±.18) a b %DMSO 1%DMSO Intraellular GSH study The intraellular GSH measurement study have shown derease in the intraellular GSH level with the inreasing onentration of Ag nanopartile in both HaCaT and HeLa ell lines after 24 h exposure (Fig. 1). The rate of intraellular GSH level depletion is muh higher in HeLa ells in ompare to HaCaT ells. mg/l Ag nanopartile exposures to HaCaT and HeLa ells for 24 h have shown 5.3% and 59.57% respetive GSH depletion (Fig. 1) Adenylate kinase release study The adenylate kinase release study have shown that with the inreasing onentration of Ag nanopartiles, the perentage of adenylate kinase release in HaCaT (Fig. 11a) and HeLa (Fig. 11b) h 48h 72h Fig. 2. Cytotoxiity of Ag nanopartiles after 24, 48, and 72 h exposures determined by the Alamar Blue assay (a) in HaCaT ells, (b) in HeLa ells. Data are expressed as perent of ontrol mean ± SD of three independent experiments. Data are expressed as perent of ontrol mean ± SD of three independent experiments. denotes a statistially signifiant (p <.1) differene from the unexposed ontrol. 24h 48h 72h 25 a b %DMSO 25 5 ells were inreased as ompare to the ontrols. With the inreasing hours of exposure, the adenylate kinase release from both the ell lines further inreases (Fig. 11a and b). Upon mg/l Ag nanopartile exposure for 24, 48 and 72 h the perentage of AK release as ompare to the ontrols from the HaCaT ells were %, % and %, respetively, while in HeLa ells the perentage of AK release at same onentration and exposure time points was found to be 156.5%, % and %, respetively. Therefore, more AK was released from HeLa ells as ompared to HaCaT ell. The huge differene in AK ativity was observed at the highest onentration in both the ell lines, in HeLa ells the perentage of AK ativity was % at 15 mg/l while in HaCaT ells it was % at 8 mg/l after 72 h of Ag nanopartile exposure. This shows that there was muh more loss of ell integrity, through %DMSO Fig. 3. Cytotoxiity of Ag nanopartiles after 24, 48, and 72 h exposures determined by the Neutral Red assay (a) in HaCaT ells, (b) in HeLa ells. Data are expressed as perent of ontrol mean ± SD of three independent experiments. denotes a statistially signifiant (p <.1) differene from the unexposed ontrol. 2 24h 48h 72h 25 24h 48h 72h

9 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) a b the damage of plasma memberane was ourred of HeLa ells as ompared to HaCaT ells upon Ag nanopartile exposure Apoptosis study The apoptosis study was shown an inrease in the ADP:ATP ratios over ontrol ratios, whih shows ells has entered apoptoti pathway. In HaCaT and HeLa ell lines with inreasing onentration of Ag nanopartiles there was a signifiant inrease in apoptoti ell population (Fig. 12). When both the ell lines were ompared it was found HeLa ells are undergoing more apoptoti ell death than HaCaT ells at same onentration and exposure time. Upon mg/l of Ag nanopartile exposure for 24 h, ATP:ADP ratio in HeLa was.31 while in HaCaT it was.22. This shows a muh lower level of ATP prodution in HeLa ells than HaCaT ells when ompared to their respetive ontrols upon exposure to the same onentration of Ag nanopartile. The assay also displayed an inrease in the intraellular ADP level in HeLa ells in omparison to the HaCaT ells, this resulted in an inrease in the ADP:ATP ratios ompared to their assoiated ontrols. 4. Disussion 1%DMSO 25 5 Exposure of Ag nanopartiles to the body is beoming inreasingly widespread and intimate. Consequently, there is an inreased h 48h 72h 1%DMSO h 48 h 72 h Fig. 4. Cytotoxiity of Ag nanopartiles after 24, 48, and 72 h exposures determined by the Coomassie Blue assay (a) in HaCaT ells, (b) in HeLa ells. Data are expressed as perent of ontrol mean ± SD of three independent experiments. Data are expressed as perent of ontrol mean ± SD of three independent experiments. denotes a statistially signifiant (p <.1) differene from the unexposed ontrol. a b %DMSO 25 5 potential for diret ontat between Ag nanopartiles, biomoleules and ells in speifi outer and inner organs. This study examines the effet of Ag nanopartiles on human dermal nonanerous (HaCaT) and ervial aner (HeLa) ell lines. The toxiity of Ag nanopartile is well known (AshaRani et al., 29; Moaddab et al., 211; Kim et al., 29) but how ells of diverse origin reat with the same Ag nanopartile in different manner and the possible reason behind this differene has been illustrated in this paper. The physio-hemial harateristis of nanopartiles play a signifiant role in their effet on biologial systems (Oberdörster et al., 2; Donaldson et al., 24). The prinipal parameters of nanopartiles are their shape (inluding aspet ratios where appropriate), size, and the morphologial sub-struture of the substane. The hemial omposition and the intrinsi toxiologial properties of the hemial are also of importane for toxiity of partiles (Renwik et al., 24). The zeta potential of the partile has been reported to play a signifiant role in its interation with different biomoleules (Vila et al., 22) and the hange in the zeta potential in the exposure medium has been shown to orrelate well with toxi response (Naha et al., 29; Mukherjee et al., 21a). The size measurement of AgNP by DLS tehnique shows its diameter in dh2o was nm, but after dispersing it in the ell ulture media supplemented with 1% FBS the diameter inreases to nm in HeLa media and nm in HaCaT media (Fig. 1a, b and ). That indiates the possible interation of AgNP with the %DMSO h 48h 72h 24 h 48 h 72 h Fig. 5. Cytotoxiity of Ag nanopartiles after 24, 48, and 72 h exposures determined by the MTT assay (a) in HaCaT ells, (b) in HeLa ells. Data are expressed as perent of ontrol mean ± SD of three independent experiments. Data are expressed as perent of ontrol mean ± SD of three independent experiments. denotes a statistially signifiant (p <.1) differene from the unexposed ontrol. 25

10 246 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) (number of olonies) HaCaT HeLa Fig. 6. Cytotoxiity of Ag nanopartiles after 8 days exposures determined by the Clonogeni assay in HaCaT ells and HeLa ells. ell ulture media omponents, whih have been widely reported with different nanopartiles that leads to the formation of protein orona (Lynh and Dawson, 28; Lundqvist et al., 28). The zeta potential study have also shown the derease in the negative zeta potential of the AgNP upon its dispersion in the 1% FBS supplemented ell ulture media, where in dh 2 O it was mv and in HeLa and HaCaT media it was 2.67 and mv, respetively (Fig. 1f). The zeta potential study further onfirms the interation of AgNP with the ell ulture media omponents. Suh an interation of the nanopartiles with the omponents of the ell ulture medium has been demonstrated to eliit a seondary or indiret toxi response in the ase of single walled arbon nanotubes (Casey et al., 27, 28) and it is probable that there are similar ontributions to the toxi response observed here. Therefore, in an independent experiment to investigate the ytotoxiity of Ag nanopartile interated media was performed. Where different onentrations of Ag nanopartile were first prepared in the respetive ell ulture media and kept in 37 C inubator for 24 h and then following inubation Ag nanopartiles were removed from the media by entrifuging it at 14, rpm for 2 min. The supernatants were then olleted and filtered using ellulose aetate.2 lm syringe filters. Both the tested ell lines were then exposed to this supernatant for h and ellular viability assessed using the AB assay. The toxiity found from supernatant media was not omparable in magnitude to the ytotoxiity observed from diret nanopartile exposure and was only observed at the 72 h exposure time-point. Upon exposure of mg/l Ag nanopartile supernatant in HeLa ells after 72 h exposure to the Fig. 7. Optial mirograph of HaCaT ells after 24 h Ag nanopartile exposure at (a) mg/l, (b) 5 mg/l, () mg/l and (d) 2 mg/l.

11 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) Fig. 8. Optial mirograph of HeLa ells after 24 h Ag nanopartile exposure at (a) mg/l, (b) 5 mg/l, () mg/l and (d) 2 mg/l. supernatant, the ell viability observed was 86.74%, while no ytotoxiity was observed in HaCaT ells (data not shown). This data suggests that there is indeed a ontribution of seondary ytotoxiity from the Ag nanopartile and ell ulture media interation however the observed effets were only noted at longer exposure time-points. Following physiohemial haraterization a series of toxiologial studies were performed using a battery of assays to assess the in vitro ytotoxi effets of Ag nanopartile on these two ell lines of diverse tissues and to understand the differene in the response by the two ell lines. In Table 1 dose response from AB assay shows that in HaCaT and HeLa ell lines the ytotoxi response was inreasing with the inreasing onentration and time points of exposure. This indiates partiles have a toxi effet on the basi ellular metabolism and proliferation of the ells as there was a derease in the redution of the Alamar Blue dye from Resazurin to Resorufin as observed from this assay. This Redox reation ould not happen as ytosoli and mitohondrial oxidoredutase enzyme ativities were dereased with the inrease in Ag nanopartile onentration and exposure time (Fig. 2). Upon mg/l AgNP exposure for 72 h, the derease in the AB dye redution than the ontrol in HaCaT and HeLa ells were 16.75% and 1.11%, respetively. This further indiates the higher sensitivity of HeLa ells than HaCaT ells upon Ag nanopartile exposure. The dose response of Ag nanopartiles to HaCaT and HeLa ells in NR assay shows that with the inreasing onentration and time of exposure of AgNPs, less NR dye was uptaken by the ellular lysosomes, whih indiated the ellular viability was dereasing with the inreasing onentration and time of exposure of AgNPs. In dead ells, NR an no longer retain in the ytoplasmi vauoles and the plasma membrane does not at as a barrier to retain the NR within the ells. Fig. 3 has shown the derease in NR dye uptake in HaCaT and HeLa ells were 25.38% and 27.42%, respetively upon mg/l Ag nanopartile exposure for 72 h. CB assay shows that with the inreasing onentration and exposure time the CB dye binding was dereased in both HaCaT and HeLa ells (Fig. 4). That suggests the degradation of ellular protein or redution of protein synthesis upon AgNP exposure (Clare and Cormak, 1996). The perentage of CB dye binding to the ells than ontrol upon mg/l AgNP exposure for 72 h in Ha- CaT and HeLa ells were 51.34% and 15.75%, respetively. In this assay again the HeLa ells were found to be more sensitive than HaCaT ells. The MTT assay determines the ability of viable ell s mitohondria to redue the soluble, yellow MTT into an insoluble, purple formazan. The redution of MTT to formazan indiates the derease in mitohondrial metabolism of the ells. Therefore, the absorbane of formazan formed, diretly orrelates to the number of ells whose mitohondrial metabolism is intat even after the

12 248 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) a ROS indution (fold inrease) b ROS indution (fold inrease) ontrol 3 mins 6 mins 12 mins 18 mins 24 mins 3 mins mins 6 mins 12 mins 18 mins 24 mins 3 mins exposure of Ag nanopartiles. Dose response of AgNP in HaCaT and HeLa ells shows the derease in the redution of MTT to formazan with the inreasing onentration and time of exposure. Upon mg/l AgNP exposure for 72 h in HaCaT and HeLa ells, the perentage of MTT onversion to formazan dereases to 17.71% and 3.7%, respetively (Fig. 5). Therefore MTT assay also shows the higher sensitivity of HeLa ells to the Ag nanopartiles than HaCaT ells. Clonogeni assay indiates the proliferative apaity of a single ell upon nanopartile exposure for longer time period, whih in this study was 8 days. Dose response from this assay shows the derease in the olony number with the inreasing onentration of AgNP in both the ell lines (Fig. 6). However, it also shows that the olony forming apaity in HeLa ells were muh lesser than HaCaT ells upon AgNP exposure. Exposure of 2.5 mg/l AgNP to Ha- CaT ells an form 83% olonies ompare to the ontrol, while HeLa ells an only form 14% olonies. This is also refleted in the LD 5 values obtained from this study. The LD 5 in HaCaT ells was 7.74 mg/l, while that in HeLa ells was 1.16 mg/l. Clonogeni assay therefore also suggest the higher sensitivity of HeLa ells when ompare to HaCaT ells. The responses from all the standard ytotoxiologial assays (AB, NR, CB, MTT and Clonogeni assays) employed to test the Ag nanopartile toxiity on the HaCaT and HeLa ells were found to Fig. 9. ROS generation after different time points of Ag nanopartile exposure in (a) HaCaT ells, (b) HeLa ells. % of GSH depletion HaCaT HeLa 1% DMSO onentration of Ag nanopartiles in mg/l Fig. 1. Perentage of intraellular GSH depletion upon different onentrations of Ag nanopartile exposures for 24 h to HaCaT and HeLa ells. a % of AK release b % of AK release h 48 h 72 h 1% DMSO 24 h 48 h 72 h 1% DMSO Conentration of Ag nanopartiles in mg/l Fig. 11. Adenylate kinase release study upon Ag nanopartile exposure in (a) HaCaT ells, (b) HeLa ells. Negative ontrol is set to zero. be both dose and time dependant. In both ell lines the LD 5 values were seen to derease with inreasing exposure time. In all ases the ytotoxi effet was noted to be greater in HeLa ells than in HaCaT ells. Among all the olorimetri based short term exposure (up to 72 h) aute toxiity assays (i.e., AB, NR, CB and MTT assays) in both the ell lines, the MTT assay was noted to be the most sensitive as indiated by the lower LD 5 values (Table 1) evaluated from this assay. The AB assay showed less sensitivity than MTT

13 S.G. Mukherjee et al. / Toxiology in Vitro 26 (212) ATP:ADP ratio haat hela onentration of Ag nanopartiles in mg/l Fig. 12. Apoptosis indution in terms of inrease in ADP:ATP ratio upon different onentrations of AgNP exposure in HaCaT and HeLa ells. assay in both the ell lines. Whereas in the 72 h exposure study in HaCaT ells, NR assay was found to be less sensitive than AB assay, and CB assay was found to be least sensitive among these four assays. The 72 h exposure study in HeLa ells showed CB assay as less sensitive than AB assay and NR assay as the least sensitive among these four assays. The lonogeni assay yielded signifiantly lower LD 5 values than that of the olorimetri endpoints, with the exeption of the MTT assay in HeLa ell line. The partile exposure time in lonogeni assay is longer than other assays, i.e., 8 days, indiating hroni effets are more severe than aute effets. This assay verifies the findings of the olorimetri assays and also gives an indiation that the presene of the Ag nanopartile has an effet on the proliferative apaity of a single ell of the tested ell lines by the redution in the number of olonies formed. The dose responses from these assays suggest that upon Ag nanopartile exposure the mitohondrial metabolism was most affeted followed by the basi ellular metabolism and then the lysosomal ativity and finally beause of the toxi effet of these nanopartiles on the ells the ellular protein ontents were also dereased. Among both the ell lines, HeLa ells were found to be more sensitive than HaCaT ells. One signifiant differene between the two ell lines is their natural antioxidant levels. The natural glutathione (GSH) level of HaCaT ell is 73 nmol/mg protein (Snow et al., 25) and HeLa ell is 22 nmol/mg protein (Tipnis et al., 1999). This huge differene in their natural GSH level ould be an important reason for higher sensitivity of HeLa ells over Ha- CaT ells. Following the standard ytotoxiity assays intraellular ROS generation was also monitored. In both ell lines a signifiant inrease in the levels of ROS (Fig. 9) was observed in omparison to the unexposed ontrols. It was noted that the onset of oxidative stress in all the tested onentrations started typially within 3 min in both the ell lines. In HaCaT ell line a signifiant inrease in ROS was noted in exposure onentrations well below the alulated LD 5 values. A response of this nature would that at onentrations even were there was no observed ytotoxiity from the above standard ytotoxiity assays the presene of Ag nanopartiles an indue high levels of ellular oxidative stress. The amount of ROS generated upon AgNP exposure was higher in HeLa ells than in HaCaT ells (Fig. 9). The fold inrease of ROS than ontrol upon 1 2 mg/l AgNP exposure in HeLa ells were , whereas upon 15 mg/l AgNP exposure that in HaCaT ells was This ould be due to the muh lower natural GSH level in HeLa ells (Tipnis et al., 1999) than HaCaT ells (Snow et al., 25) as GSH is an important antioxidant and ROS savenger of the ell. Therefore an independent GSH study has shown that the rate of depletion of intraellular GSH level was muh higher in HeLa ells when ompared to that of HaCaT ells (Fig. 1). Suh differenes would aount for the higher amount of ROS prodution observed in HeLa ells in omparison to HaCaT ells. ROS plays an important role in triggering many ellular pathways whih an lead to ellular death, inluding ytokine ativation (Brown et al., 24) and aspase ativation (Kim et al., 29, Stennike et al., 1998). They an also ause damage to the nulear DNA by altering the hemial struture of the nuleotide bases and the deoxyribosyl bakbone (Cooke et al., 23, Valko et al., 24). Therefore, intraellular ROS generation upon AgNP exposure is an important fator to its ytotoxiity. ToxiLight Ò Assay assay quantifies the release of adenylate kinase from the ells whih is an indiation of the damage of the ell membrane. One the plasma membrane has been damaged by ytotoxi ation the AK will be available for detetion in the surrounding medium. With the inreasing Ag nanopartile onentration in both the ell lines, an inreasing release of adenylate kinase from the ells was observed whih also inreases with the exposure time (Fig. 11). This assay therefore suggests that with the inreasing onentration and exposure time the Ag nanopartile auses more damage to the ell membrane. Upon AgNP exposure more AK was found to be released from HeLa ells than HaCaT ells at lower dose (Fig. 11). This damage in the ell membrane also indiates that more ell population is possibly dying by either neroti or apoptoti pathway. This was onfirmed when apoptosis study was performed by ApoGlow Ò Kit of Lonza Ltd (Wokingham, UK). ApoGlow Ò Kit helps in the differentiation of normal healthy proliferative ells, apoptoti ells and neroti ells from the ellular ADP:ATP ratio. If a ell undergoes apoptosis, the test gives lower levels of ATP ompare to ontrols, but show an inrease in ADP, this eventually gives an inrease in the ADP:ATP ratios over ontrol ratios. In normal healthy ells the ADP:ATP ratio was observed below.11, whereas in apoptoti ells the ratio was in between.11 and 1. and in ells undergoing nerosis by heat shok treatment the AD- P:ATP ratio was observed in exess of 15. (Bradbury et al., 2). The apoptosis study shows the ADP:ATP ratio in HaCaT and HeLa ells upon AgNP exposure were higher than that of the ontrol and was within the range of apoptoti ell population (Fig. 12). The ADP:ATP ratio in HaCaT and HeLa ells upon 24 h mg/l Ag nanopartile exposure were.22 and.31, respetively. This indiates upon Ag nanopartile exposure the ells were entering apoptosis and HeLa ells had more apoptoti ell population than HaCaT ells. This further indiates the higher sensitivity of HeLa ells than HaCaT ells. Overall, the standard ytotoxiity assays shows more sensitivity of MTT assay upon short term exposure over other assays followed by AB assay, NR and CB assays were the least sensitive ones. This further indiates upon Ag nanopartile exposure mitohondria was getting affeted first followed by basi ellular metabolism and lysosomal ativity was dereasing later on. Optial mirosopi study had also shown the ytotoxi effet of Ag nanopartiles on HaCaT (Fig. 7) and HeLa (Fig. 8) ells with their inreasing onentration. Ag nanopartiles generate reative oxygen speies upon entering the ells and that reates oxidative stress to the ells. ROS plays an important role in the mitohondrial damage, therefore its generation upon AgNP exposure ausing the higher mitohondrial toxiity. Ag nanopartiles also damage the ell membrane and therefore more AK release from the ells was observed upon inreasing onentration and time of exposure of Ag nanopartiles. All these effets further ativate the apoptoti pathways and therefore signifiant apoptoti ell populations were observed upon 24 h Ag nanopartile exposure and eventually ell dies. The interation of AgNP with the media omponents, as indi-