Research Article Safety Evaluation of Engineered Nanomaterials...

Hindawi Publishing CorporationISRN ToxicologyVolume 2013 Article ID 825427 13 pageshttpdxdoiorg1011552013825427

Research ArticleSafety Evaluation of Engineered Nanomaterials for HealthRisk Assessment An Experimental Tiered Testing ApproachUsing Pristine and Functionalized Carbon Nanotubes

Teresa Coccini Luigi Manzo and Elisa Roda

Laboratory of Clinical Toxicology IRCCS Maugeri Foundation Medical Institute of Pavia and University of Pavia Via Maugeri1027100 Pavia Italy

Correspondence should be addressed to Teresa Coccini teresacoccinifsmit

Received 8 February 2013 Accepted 20 March 2013

Academic Editors A Botta G C Jagetia M Pacheco and G T Ramesh

Copyright copy 2013 Teresa Coccini et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Increasing application of engineered nanomaterials within occupational environmental and consumer settings has raised the levelsof public concern regarding possible adverse effects on human health We applied a tiered testing strategy including (i) a first invitro stage to investigate general toxicity endpoints followed by (ii) a focused in vivo experiment Cytotoxicity of laboratory-madefunctionalized multiwalled carbon nanotubes (CNTs) (ie MW-COOH and MW-NH2) compared to pristine MWCNTs carbonblack and silica has been assessed in human A549 pneumocytes by MTT assay and calceinpropidium iodide (PI) staining Purityand physicochemical properties of the test nanomaterials were also determined Subsequently pulmonary toxic effectswere assessedin rats 16 days afterMWCNTs it administration (1mgkg bw) investigating lung histopathology andmonitoring several markersof lung toxicity inflammation and fibrosis In vitro data calceinPI test indicated no cell viability loss after all CNTs treatmentMTTassay showed false positive cytotoxic response occurring not dose dependently at exceedingly low CNT concentrations (1 120583gmL)In vivo results demonstrated a general pulmonary toxicity coupled with inflammatory response without overt signs of fibrosisand granuloma formation irrespective of nanotube functionalizationThis multitiered approach contributed to clarifying the CNTtoxicity mechanisms improving the overall understanding of the possible adverse outcomes resulting from CNT exposure

1 Introduction

Nanotechnology is one of the fastest emerging fields involvingdevelopment and manipulation of materials le100 nm in sizeThere are numerous potential perspectives for the applica-tions arising from nanotechnology which include their usein a wide range of fields for example medicine environ-ment occupational setting chemistry energy productioninformation and communication heavy industry and con-sumer goods Human exposure to engineered nanomaterials(ENMs) can occur at different stages of their life cycle(manufacture use and disposal) and several concerns havebeen expressed about their potential to cause unanticipatedadverse effects in humans

The remarkable diversity of engineered nanomateri-als (ENMs) together with their unique properties andbehaviour complicates their risk assessment there are cur-rently about 50000 different types of carbon nanotubes

obtained by different rawmaterials and production processesSimilar diversity applies to other types of ENMs rendering adhoc risk assessment of all of these materials an immense task[1 2]

The REACH Regulation (Registration EvaluationAuthorization and Restriction of Chemicals) is the currentregulatory framework for chemical risk assessment andmanagement in European Union Although REACH shouldbe applied to ENMs the Technical Guidance Documents[3] for preparing a risk assessment currently include verylittle reference to substances in particulate form thus lackingin addressing specific characteristics of ENMs In 2007 and2009 the European Union (EU) Scientific Committee onEmerging and Newly Identified Health Risks (SCENIHR)concluded the applicability of the current risk assessmentapproachesmethodologies to identify the ENM-associatedhazard though pointing to main limitations thus stressingthe need for several adjustments [4ndash6]

2 ISRN Toxicology

Nevertheless though particular ENMs properties andbehaviour (ie translocation and capability to cross biologi-cal barriers) have been described in several studies (ie modeof action toxicity targets dose-response relationships andthe potential to react with constituents of cells at the portalof entry and beyond [2]) several aspects concerning NP-associated risks are still unknown and critical steps in the riskassessment of ENMs remain so far the same as those used forconventional chemicals

Furthermore there are major challenges in assessingexposure (both airborneadministered and internal dose ieparticle deposition in lung) ENMs are produced from manysubstances in many forms and sizes and with a variety ofsurface coatings The health risk assessment for such diversematerials requires validated analytical methods permittingtheir characterization in bulk samples and their detection andmeasurement in workplace air [7] Efforts are also neededfor the identification of properties that trigger ENMs toxicity(eg contaminants impurities and defects)

Today there are not enough studies to validate the poten-tial hazards posed by these novel materials and hence thedefinitive conclusions and tools technologies systems andmethods to obviate the risks Several tools for the assessmentof risks are still in the conceptual stage and further thereare considerable uncertainties on how to assess nanoparticles(NPs) exposure (ie which metrics to adopt systematically)and what methods to use to assess toxicity

Thus being unable to perform a quantitative risk assess-ment for ENMs due to the lack of sufficient data on exposurebiokinetics and organ toxicity it should be made mandatoryto prevent exposure by appropriate precautionary measuresand practicing best industrial hygiene to avoid future shockscenarios from environmental or occupational exposures [8]

In the safety assessment area two basic questions stillneed to be addressed (i) do nanomaterial properties neces-sitate a new toxicological science and (ii) what are the bio-logical and biokinetic properties to be considered for toxicitytesting in particular can biology and certain mechanismsof effects of ENMs (eg proinflammatory action oxidativestress mitochondrial perturbation generation of neoanti-gens and protein complexes in the body enhanced proteindegradation at the large surface area of NPs complexes etc)be used as a basis for studying NPs toxicity and risk assess-ment Addressing these questions is of crucial importanceto define adequate strategies and establish whether ENM-tailored testing methods should be added to conventionaltoxicity testing protocols to comply with regulatory demandand properly characterize the ENMs potential hazard

According to the major institutions [7 9 10] and interna-tional consensusmeetings [11] the proposedmultitiered test-ing protocols could be used to address toxicological researchand health risk assessment for NPs Toxicity testing shouldfirstly include a careful physicochemical characterization (egparticle size particle size distribution reactivity surface areaparticle mass impurity and aggregation tendency) assistedby using reference materials as well as the use of acellularsystems to explore the reactivity of the materials in acellularenvironments Subsequent steps include the use of validatedcellular (in vitro) models to support evidence-based testing

process followed by a limited series of in vivo studies guidedby information generated from in vitro studies especially thetoxicological data relevant to ranking of ENMs designingappropriate exposure concentrations and defining the criticalhealth endpoints to be monitored

Biosafety should be evaluated by tests examining generaltoxicity target organ toxicity and biocompatibility in linewith regulatory requirements limiting the use of lab animalsin toxicological research [12 13] to identify molecular end-points and multiple toxicity pathways

The present work represents an example of a tieredapproach for toxicity testing employing CNTs as a practicalmodel suitable to validate a two-level strategy combining invitro and in vivo experiments Such investigation is relatedto our recent studies on the pulmonary toxicity of a series offunctionalized multiwalled carbon nanotubes (MWCNTs)

Human A549 pneumocytes selected as potential targetcells during respiratory exposure associated with occupa-tional setting and environment [1 7 14ndash18] have beenused to assess in vitro the cytotoxicity of MWCNTs withdifferent degrees of functionalization using MTT assay andcalceinpropidium iodide (PI) staining

Subsequently the pulmonary toxic effects were assessedin rats 16 days after it administration of the previouslyreported type of CNTs (1mgkg bw) Major endpointstested included (i) histopathology of lung tissue (haema-toxylineosin staining) (ii) apoptoticproliferating featuresexamined by TUNEL and PCNA immunostaining and (iii)presencedistribution of (1) transforming growth factor-beta1(TGF1205731) (2) interleukin-6 (IL-6) and (3) collagen (TypeI) investigated by immunochemical methods as markers oflung toxicity inflammation and fibrosis respectively

11 CNTs State of the Art Among the several types ofengineered nanoparticles CNTs are emerging as one of themost promising and revolutionary nanomaterials widelyemployed within commercial environmental and energeticsectors due to their unusual one-dimensional hollow nanos-tructure and unique physicochemical properties [19 20]CNTs have been also proposed in medicine as nanovectorsfor vaccine and drug delivery nanodevices or as substratesfor tissue engineering [21 22] Consequently this expandingusage paralleled by occupational exposure at all phases of thematerial life cycle may lead to widespread human exposurevia skin contact inhalation and via intravenous injection inmedical applications

Moreover in view of some similar aspects to fibers suchas structural characteristics extreme aspect ratio low specificdensity and low solubility CNTs might exhibit toxicitysimilar to those observed with other fibrous particles suchas asbestos [1] Further their small size accompanied by highsurface area defines the chemical reactivity of CNTs induc-ing changes in permeability and conductivity of biologicalmembranes in fact a typical behaviour has been reportedregarding translocation and distribution of certain ENMs inthe body and their capability to cross internal barriers [2]Moreover chemical functionalization not only extends theapplications of CNTs conferring them new functions that

ISRN Toxicology 3

Table 1 Physicochemical properties of nanoparticulate materials

MWCNTs MW-COOH MW-NH2 CB SiO2

Primary particle size range (nm) 500ndash2000 100ndash300 100ndash300 10ndash20 7Aggregate size range (nm) le2000lowast 300ndash1000lowast 300ndash1000lowast 100ndash1000 150ndash200Specific surface area BET (m2g) 60 sim100 sim100 sim240 sim200Zeta potential (mV) in deionised H2O minus44 minus50 minus48 minus22 minus30

lowastDetermined by NC-AFM on samples deposited on high resolution mica sheets

cannot otherwise be acquired by pristine nanomaterials butalso impacts on biological response to CNTs modifying theirtoxicological profile [22 23]

Thus the growing use together with the suspensewhetherCNTs have negative impact on human health and environ-ment evokes concern by worldwide public and regulatoryinstitutions

Nowadays a number of in vivo and in vitro studieshave also been performed on CNT toxic effects evaluatingdifferent mechanistic endpoints [14 24ndash30] However theexisting data are controversial and findings have been difficultto be interpreted in some cases Toxicity and reactivity ofCNTs were shown to vary depending on characteristics ofthe specific type of material tested such as fiber length fiberdiameter surface area tendency to agglomerate dispersibil-ity in media and the method used for synthesis which canproduce impurities and leave metal catalyst residues [31ndash38]

Some scientists have suggested that metal traces associ-ated with the commercial nanotubes are responsible for thecytotoxicity and that CNTs show no signs of acute toxicity[29] It has also to be taken into consideration that pristineCNTs are insoluble in almost all solvents and effects of CNTscan also be modified by chemical functionalization [39]

In certain studies a high degree of functionalization wasshown to mitigate toxic effects [39 40] while contrarilyin other studies functionalization increased lung toxicity ofCNTs in mice exposed by inhalation [41] Further dissimilarresults from in vivo studies have been ascribed to differencesin CNT size and surface area rodent species used route ofexposure and differences in observation period [42ndash47]

2 Materials and Methods

21 RawMaterial Commercial MWCNTs (95 purity) wereobtained from Cheap Tubes Inc (Brattleboro USA) and CBfrom Aldrich-Fluka (Milan Italy) SiO

2particles were kindly

provided by Degussa AG (Germany) (Table 1)

22 Preparation of Lab-Made Functionalized MWCNTsMWCNTs with different functionalization were preparedas described by Fagnoni et al 2009 [48] The lab-madematerials produced included carboxyl (COOH) functional-ized MW-COOH and amine-containing nanotubes with lowamino groups content (MW-NH

2)

221 Physicochemical Characterization of CNTs Before thein vitro and in vivo exposure exhaustive physicochemi-cal characterization of differently functionalized CNTs was

performed by IR spectroscopy thermogravimetric analysis(TGA) and by noncontact atomic force microscopy (NC-AFM)

It has been estimated that one functional chain isanchored each sim16 carbon atoms of the CNTs surface for theMW-COOHand eachsim100 carbon atoms of theCNTs surfacefor the MW-NH

2

All the three types of CNTs had an outer diameter of20ndash30 nm and the wall thickness was 1-2 nm The particlelength was 100ndash300 nm for both functionalized CNTs (egMW-COOH and MW-NH

2) and 500ndash2000 nm for the pris-

tine MWCNTs Other physicochemical characteristics aredetailed in Table 1

The content of iron (Fe) cobalt (Co) and nickel (Ni) inboth pristine and functionalized MWCNTs was determinedby inductively coupled plasma optical emission spectrometry(ICP-OES) analysis Results indicated the presence of Feand Ni impurities at concentrations of 025 and 15respectively in pristineMWCNTs Considerably lower metalconcentrations were found in the functionalized nanotubesIn these materials the content of Fe and Ni was 005ndash015and 03ndash05 respectively

222 Nanotubes Dispersion Previous to the experiments astock of 1mgmL and a suspension of 1mgkg bw of eachtest material (ie MWCNT MW-COOH MW-NH

2 SiO2

and CB) were prepared in DMEM plain or in NaCl 09for the in vitro expo pure and in vivo exposure respectivelyJust before use these suspensions were sonicated for 15minwith an ultrasonic Sonopuls (Bandelin Electronics BerlinGermany) in a short break every two minutes also vortexingthe suspension on ice to further force the CNT dispersionavoiding the agglomeration and the formation of bundle-like structures No surfactants or solvents were used Thesuspensions of the test materials were immediately used forthe treatment

23 In Vitro Tests

231 A549 Human Cells All cell culture reagents wereobtained from Sigma Aldrich (Milan Italy) A549 cellsfrom a human Caucasian lung adenocarcinoma with thealveolar type II phenotype were obtained from ECACC(Sigma Aldrich Milan Italy) The cells were cultured in Dul-beccorsquosmodified Eaglemedium (DMEM) supplementedwith10 fetal calf serum (FCS) 2mM L-glutamine 50 IUmLpenicillin and 50 120583gmL streptomycin in a humidifiedatmosphere containing 5 CO

2at 37∘C and grown to 80

4 ISRN Toxicology

confluence Exposures to the different nanomaterials weredone on subconfluent cells

At the end of the incubation period cell cultures wereexamined by phase contrast microscopy using a ZeissAxiovert 25 light microscope

232 Exposure Conditions Cells were seeded in 96-wellplates at a density of 1 times 104cm2 in complete medium After24 h of cell attachment plates were washed with 100120583Lwellof phosphate-buffered saline (PBS) Cells were then exposedto suspensions (10 120583L) of the test materials (pristine MW-CNTs MW-COOH MW-NH

2 CB or SiO

2) at concentra-

tions as 1 10 and 100120583gmL for 24 or 48 h No fetal bovineserum was used in these preparations as it was proven tointeract with nanotubes [49]

The doses tested (1ndash100 120583gmL) and the time points ofdeterminations (24 and 48 hr) have been derived from previ-ous studies showing different adverse effects (eg oxidativestress inflammatory response cell death loss of cellularmorphology and gene expression levels changes) caused byvarious carbon nanotubes

Six replicate wells were used on each 96-well platefor all the treatments and each experiment was repeatedindependently three times Identical treated cultures weretaken as replicate measure for statistical tests Significanteffects (119875 lt 005) were determined by one-way analyses ofvariance (ANOVA) (software package SPSS Inc 1999)

In graphfigures data are presented asmeanplusmn SDover themean experimental values of each of the three independentexperiments

233 Cytotoxicity Assays Two dye-based methods namelythe Live (calcein-green fluorescence)-Dead (propidiumiodide-red fluorescence) staining (calceinPI) and the MTTassay were used to assess cytotoxicity

The livedead viability test detects cell membraneintegrity and measures the number of damaged cellsFluorescence microscopy labels both live (green colour) anddead (red colour) cells simultaneously thereby permittingvisualization and enumeration of all cells by a single countingprocedure Viability was expressed as a percent of the totalnumber of cells counted

The MTT assay uses tetrazolium salts to assess mito-chondrial dehydrogenase activity Only active mitochon-dria contain dehydrogenases enzymes able to cleave thetetrazolium ring and reduce MTT to insoluble dark-blueformazan crystals and therefore the reaction only occurs inviable cells

Absorbance directly proportional to cell viability wasdetermined at 550 nm in a Biorad microplate reader Theabsorbance values were normalized by the controls andexpressed as percent viability

After the incubation period all cultures were also exam-ined and photographed in phase contrast using a ZeissAxiovert 25 light microscope combined with a digital camera(Canon Powershot G8)

24 In Vivo Tests

241 Animals and Treatment with Carbon Nanotubes byIntratracheal Instillation (it) All experimental proceduresinvolving animals were performed in compliance with theEuropean Council Directive 86609EEC on the care and useof laboratory animals

Adult Sprague-Dawley rats (25 males 12 weeks old) werepurchased from Charles River Italia (Calco Italy) at least2 weeks before treatment and allowed to acclimatize for 3weeks Throughout the experiment animals were kept in anartificial 12 h light 12 h dark cycle with humidity at 50plusmn10Animals were provided with rat chow (4RF21 diet) and tapwater ad libitum

For the treatment groups of 6 rats were anesthetized withpentobarbital sodium for veterinary use and were intratra-cheally instilled with MWCNTs pristine or functionalizeddispersed at a dose of 1mgkg bw in NaCl 09 (seeSection 222)

Sixteen days after the it treated and control rats weredeeply anesthetized with an overdose ip injection of 35chloral hydrate (100 120583L100 g bw) lung preparation formicroscopic evaluations was done by vascular perfusion offixative Briefly the trachea was cannulated and laparotomywas performed The pulmonary artery was cannulated viathe ventricle and an outflow cannula was inserted into theleft atrium In quick succession the tracheal cannula wasconnected to about 7 cm H

2O pressure source to inflate the

lungs with air and clearing solution (saline with 100UmLheparin 350 mosM sucrose) was perfused via the pulmonaryartery After blood was cleared from the lungs the perfusatewas switched to fixative consisting of 4 paraformaldehydein 01M phosphate buffer (pH 74) After fixation the lungswere carefully removed washed in NaCl 09 and postfixedby immersion for 7 h in 4 paraformaldehyde in 01Mphosphate buffer (pH 74) then the tissues were dehydratedthrough a graded series of ethanol and finally embedded inParaplast Eight 120583M thick sections of the samples were cut inthe transversal plane and collected on silane-coated slides

242 Histology Immunocytochemistry and TUNEL StainingTo avoid possible staining differences due to small changes inthe procedure all the reactions were carried out simultane-ously on slides of control and treated animals at all stages

Lung sections were stained with haematoxylineosin(HampE) for histological examination

Immunohistochemistry was performed using commer-cial antibodies on rat lung specimens to assess the presenceand distribution of (i) transforming growth factor-beta1(TGF1205731) (ii) interleukin-6 (IL-6) (iii) collagen (type I)and (iv) proliferating cell nuclear antigen (PCNA-PC10)as typical markers of general lung toxicity inflammationfibrosis and cell proliferation respectively

Lung sections were incubated overnight at room temper-ature with (i) a primary rabbit polyclonal antibody againstTGF1205731 (Santa Cruz Biotechnology Santa Cruz CA USA)diluted 1 100 (ii) a primary rabbit polyclonal antibodyagainst collagen (type I) (Chemicon Temecula CA USA)diluted 1 400 (iii) a primary goat polyclonal antibody

ISRN Toxicology 5

against IL-6 (Santa Cruz Biotechnology) diluted 1 100 (iv) aprimary mouse monoclonal antibody against PCNA (Amer-ican Biotechnology Plantation USA) diluted 1 5 in PBSBiotinylated anti-rabbit anti-goat and anti-mouse secondaryantibodies and an avidin biotinylated horseradish peroxidasecomplex (Vector Laboratories Burlingame CA USA) wereused to reveal the sites of antigenantibody interaction331015840-Diaminobenzidine tetrahydrochloride (DAB Sigma StLouis MO USA) was used as the chromogen followed by alight counterstaining with haematoxylin Then the sectionswere dehydrated in ethanol cleared in xylene and finallymounted in Eukitt (Kindler Freiburg Germany)

In the case of negative controls some sections wereincubated with phosphate-buffered saline instead of theprimary antibodies no immunoreactivity was observed inthese conditions

In addition to morphological criteria the apoptotic celldeath was assayed by in situ detection of DNA fragmentationusing the terminal deoxynucleotidyl-transferase (TUNEL)assay (OncogeneRes Prod BostonMAUSA)The lung sec-tions were incubated for 5min with 20120583gmLminus1 proteinase-K solution at room temperature followed by treatmentwith 3 H

2O2to quench endogenous peroxidase activity

After incubation with the TUNEL solution (90min withTdTbiotinylated dNTP and 30min with HRP-conjugatestreptavidin) in a humidified chamber at 37∘C the reactionwas developed using 005 3-amino-9-ethylcarbazole (AEC)in 01M TRIS buffer (pH 76) with 02 H

2O2 in some

specimens the reaction was developed using a 01 DABsolution

As a negative control the TdT incubationwas omitted nostaining was observed in these conditions

243 Cytochemical Assessment (a) Scoring different spec-imens the immunostaining for IL-6 TGF1205731 and col-lagen (type I) was evaluated in conventional brightfieldmicroscopy by recording the localization and intensityof labelling according to a semiquantitative scale fromabsentundetectable (minus) to maximal (++++) Then to assessthe significance of the immunohistochemical results aKruskal-Wallis nonparametric analysis of the semiquantita-tive data was performed A 119875 value of lt005 was consideredsignificant

(b)The evaluation of PCNA- andTUNEL-cytochemicallypositive cells (PCNA LI TUNEL LI) was calculated as thepercentage (Labelling Index) of a total number (about 500)of bronchiolar alveolar and macrophagic cells for each ani-mal and experimental condition in different representativemicroscopic fields Statistical analyses among the differentbiological situations were performed by the Studentrsquos t-testand differences between medians were considered significantat lowast119875 lt 005

The slides were observed and scored with a brightfieldZeiss Axioscop Plus Microscope The images were recordedwith an Olympus Camedia C-2000 Z digital camera andstored on a PC running Olympus software

3 Results and Discussion

Laboratory-made functionalized multiwalled CNTs (MW-COOH and MW-NH

2) were tested in comparison with

pristine MWCNTs in cultures of A549 pneumocytes Car-bon black (CB) and silica (SiO

2) were also investigated as

reference nanoparticles [50 51] Purity and physicochemicalproperties of the tested nanomaterials are reported in Table 1

In vitro cytotoxicity was assayed in parallel by twoclassical dye-based cell viability assays for example MTTmetabolism and livedead-calceinPI staining

In preparations treated with all nanotube types (pristineand functionalized) and CB the calceinPI test indicatedno loss of cell viability (Figure 2) whereas MTT data ofpristine MWCNTs MW-COOH and MW-NH

2apparently

showed cytotoxic response occurring not dose-dependentlyat exceedingly low CNT concentrations (1 120583gmL) with 50viability loss at 10 120583gmL and no further cell death increaseat 100120583gmL (Figure 1) Similar cytotoxicity pattern wasobserved byMTT in preparations exposed to CB Loss of cellviability was apparentlymore pronouncedwith CB comparedto SiO

2(Figure 1)

Notably as shown in Figure 2 the data obtained by cal-ceinPI staining considerably differed from the MTT resultsin that (i) cell viability was unaffected after treatment withpristine MWCNTs functionalized MWCNTs or CB even atthe higher concentrations and for longer time of exposure (ie48 h) (ii) loss of cell viability was noted in cells treated withSiO2with approximately 40 cell death occurring after 24 h

exposure to 100 120583gmL SiO2(Figure 2)

Pristine MWCNTs MW-COOH and MW-NH2were

either insoluble or little soluble in polar solvents and dif-ficult to evenly disperse in an aqueous matrix They weresonicated to force their dispersion in the medium beforeapplication to cell cultures In cells incubated with thesematerials significant agglomeration occurred CNTs wereshown to adhere to each other forming dense micron-sizedassemblies completely covering the cell surface (Figure 3)Agglomeration increased with increasing doses at bothincubation time points (24 and 48 h) The dosimetry ofthese nanoparticles tested in vitro is a fundamental questionincluding not only amount and time but also primary particlecharacteristics physical properties (eg size shape andagglomeration state) core particle and surface chemistryThe present data on (i) dose metric characteristics (Table 1)(ii) agglomeration tendency (Figure 3) and (iii) in vitrocytotoxicity suggest some considerations Specifically overtin vitro toxic effects were observed after exposure to SiO

2

(viability results from both MTT and calceinPI) whilecontrasting results were obtained from MTT and calceinPIafter pristine and functionalized MWCNTs As far as physic-ochemical properties concern although the functionalizednanomaterials possessed lower aggregate size and particlesize ranges (Table 1) than those displayed by the pristineones these characteristics seemed not to markedly modifytheir cytotoxic potential when compared to that of pristineMWCNTs

Further according to the literature reporting interactionsbetween CNTs and colorimetric dyes commonly used in

6 ISRN Toxicology

0

20

40

60

80

100

MW MW-COOH

CB

MTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

24 hr-exposure

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

1 120583gmL10 120583gmL100 120583gmL

(a)

0

20

40

60

80

100

MW MW-COOH

CBMTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

48 hr-exposure

1 120583gmL10 120583gmL100 120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowast lowastlowast

lowastlowast lowastlowast

lowast

(b)

Figure 1 Histograms showing cell viability measured by MTTassay in A549 cells after 24 h and 48 h exposure to pristine mul-tiwalled CNTs MWCNT laboratory-made functionalized nan-otubes MW-COOH (carboxyl functionalized) and MW-NH

2

(amino-functionalized) carbon black CB silica SiO2 Data repre-

sent the mean plusmn SD over the mean experimental values of each ofthe three independent experiments lowast119875 lt 005 compared to control(100 cell viability)

classical dye-based cytotoxicity assays such as MTT andneutral red assays [28 51ndash54] the present MTT data seemedmore likely to reflect a false positive cytotoxicity signalpossibly due to nonspecific CNT interaction with cell culturecomponents or MTT formazan salt

Subsequently the second focused in vivo step addressedthe pulmonary effects of the three different previously men-tionedMWCNTs (pristine versus two diverse lab-made func-tionalized MWCNTs ie MW-COOH and MW-NH

2) on

some cell kinetic (ie TUNEL and PCNA) and cytochemicalparameters (ie IL-6 TGF1205731 and collagen) investigated inrats after 16 days from a single it exposure (1mgkg bw)

After it exposure to all the three different CNTs lungmorphology micrographs clearly showed a marked uptake ofthe CNTs into the macrophages (Figure 4) Noticeably thewidespread presence of dark particulate-laden macrophageswas evident as a consequence of the CNTs engulfing atalveolar stromal and also bronchiolar levels in accordance

Table 2 Localization and expression of immunolabeling for IL-6TGF-1205731 and Collagen (type I) on a semiquantitative evaluation

IL-6 TGF-1205731 Collagen-IControl

Bronchiolar cells plusmn minus +Alveolar cells plusmn minus plusmn

Stromal cells plusmn plusmn +PristineMW

Bronchiolar cells ++plusmn +plusmn +plusmnAlveolar cells +plusmn + +Stromal cells +plusmn +++ +plusmn

MW-NH2

Bronchiolar cells ++++ ++ +plusmnAlveolar cells ++ + +plusmnStromal cells ++plusmn ++++ ++

MW-COOHBronchiolar cells ++plusmn ++ +plusmnAlveolar cells +plusmn + +plusmnStromal cells +plusmn +++plusmn ++119875 value

Bronchiolar cells lt005 lt005 nsAlveolar cells lt005 lt005 nsStromal cells lt005 lt005 ns

Degree of staining intensity from undetectable (minus) to strong (++++)119875 values calculated by Kruskal-Wallis testns not statistically significant

with the poor solubility of the MWCNTs (pristine and lab-made MW-COOH and MW-NH

2) already evidenced in the

first in vitro step as described previously and irrespectively ofthe nature of functionalization

Histologically alteration of lung architecture was alsoobserved in several areas showing collapsed thick-walledalveoli and presence of microhaemorrhagic foci (Figure 4)not accompanied by evident signs of fibrotic reactionOccasionally inflammatory shedding of leucocyte clusterscharacterized the parenchyma even though the occurrenceof true granulomas was never detected

TUNEL and PCNA staining employed as typicalmarkersof apoptosis and cell proliferation respectively showed a sig-nificant increase of reactivity in different cell populations (iebronchiolar alveolar cells andmacrophages) as expression of(i) diverse sensitivity of different cell categories to the insultand (ii) an improved cellular turnover aimed at replacingdamaged elements and finalizing a repair process (Figures 5and 6)

In agreement with previous literature data reporting toxicpulmonary effects including inflammation (characterized byan increase in alveolar cell number and in cytokines egTNF-alpha and IL-1) and oxidative stress [24 25 44 45 55]our experimental results showed an extensive spreading inthe bronchiolar alveolar and stromal cells of pulmonary IL-6 and TGF1205731 evidencing the cellular inflammatory reactionto the CNTs instillation (Table 2 and Figure 7) while differ-ently evident changes for collagen were not detected

ISRN Toxicology 7

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

SiO2

(d)

CB

(e)

MW-NH2

(f)

Figure 2 A549 cells examined by calceinPI staining (a) Untreated cells Representative micrographs showing cell preparations after 24 hexposure to 100 120583gmL (b) pristineMWCNT (c) MW-COOH laboratory-made carboxyl functionalized nanotubes (d) SiO

2 silica (e) CB

carbon black (f)MW-NH2 laboratory-made amino-containing functionalized nanotubes Viable (green) and damaged (red) cells are shown

Objective Magnification 32x (times4)

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

MW-NH2

(d)

CB

(e)

SiO2

(f)

Figure 3 Phase contrast micrographs showing A549 cell cultures (a) Untreated cells (bndashf) Bundle-like agglomerates covering cell surfaceafter 24 h exposure to 100 120583gmL (b) MWCNTs (c) MW-COOH (d) MW-NH

2 or (e) CB (f) absence of agglomeration after exposure to

SiO2(100120583gmL) Objective magnification 32x (times4)

8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)

Figure 4 Representative HampE-stained lung parenchyma specimens (a b) Normal lung architecture in control animals (cndashf) Structuralalterations detected in treated rats it exposed to 1mgkg bw MW-COOH (c e) or MW-NH

2(d f) Dark particulate-laden macrophages

evident as a consequence of the carbon nanotubes engulfing at alveolar (c d) bronchiolar (e) and stromal (f) levels (d) Thickened-walledcollapsed alveoli accompanied by the presence of widespread microhaemorrhagic foci Objective magnification 40x (andashd) 60x (e f)

The persistence of inflammation still observable 16 daysafter instillation could be explained as the consequence offailure to eliminate the inciting agent (ie different MWC-NTs) as demonstrated by the durability of fibrocarbonaceousnanomaterial internalized by macrophages Differently thelack of a marked collagen accumulation could be attributableto a physiological tissue repair in which the tissue remod-elling occurred moderately

These research studies on CNTs clearly indicated thatcertain negative properties that are typical of classic CNTssuch as cytotoxicity poor blood compatibility inflammo-genic effects and target-organ toxicity are maintained tosome extent in functionalized CNTs supporting the view

that overcoming the safety problems remains a considerablechallenge for these nanomaterials

4 Conclusions

In summary our first in vitro step investigation reportedconflicting results on the cytotoxic effects of pristine anddifferently functionalized MWCNTs specifically all thetested CNTs exhibited mild to moderate cytotoxicity whentested using MTT assay with changes occurring not dose-dependently already at very low CNT concentrations Bycontrast the calceinPI test showed that cell viability was

ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL

Figure 5 (andashc) Cell proliferation and (dndashg) apoptosis detected by PCNA immunolabelling and TUNEL staining respectively after in vivoexposure to 1mgkg bw (c d e) MW-COOH and (a b f g) MW-NH

2 PCNA-positive epithelial cells at (a) bronchiolar and (b c) alveolar

levels TUNEL-positive CNT-laden macrophages in both (d) normal and (e) inflammatory stromal areas as well as (f g) at alveolar levelsObjective magnification 60x (andashe) 100x (f g)

unaffected by all tested CNTs even at the higher con-centrations and for longer exposure time These divergingresults highlighted some limitations intrinsical to classical invitro cytotoxicity tests applied to the study of the ENMsdue to the peculiar physicochemical characteristics of thesenew nanomaterials fully supporting the notion [56 57]that a number of issues remain to be resolved before theexclusive use of in vitro studies can be accepted in the safetyevaluation of nanomaterials calling for research approachescomplementary to the in vitro studies aimed at understandingeffects at all physiological levels and predicting human healthhazards

Strategies for the safety assessment of ENMs and agglom-erates are under discussion and development in severalnational and international projects It has been suggested[58] to develop a tiered strategy of tests starting with anassessment of cell viability providing a first-stage attemptat screening particles allowing their benchmarking beforechoosing specific particles for further testing perhaps in vivo

Since investigations in laboratory animals may giveessential insight the in vivo study being the subsequentsecond essential stage has provided fundamental informa-tion contributing to clarifying the CNT toxicity mechanismsand to understanding the different CNT toxicity targets at

10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling

MW-COOH Pristine MWControl

75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2

(b)Figure 6 Histograms showing changes in percentage of (a) PCNA and (b) TUNEL Labelling Index of bronchiolar alveolar andmacrophagiccells after it exposure to different CNTs (pristine MWCNTs versus lab-made functionalized MW-COOH and MW-NH

2) A significant

increase (Studentrsquos 119905-test) of the above-mentioned cell types was clearly observed in lungs from all CNT-treated rats Data are expressed asmean plusmn SD ( lowast119875 lt 005)

Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH

(f)Figure 7 (andashd) IL-6 and (e f) TGF-1205731 immunostaining patterns in controls (a b) and differently treated rats (c d) MW-NH

2or (e f) MW-

COOH (a) IL-6 and (b) TGF-1205731 low labelling detected in control rats at all lung districts (eg bronchiolar stromal and alveolar levels) (ce) Strongly IL-6-immunoreactive bronchiolar areas and collapsed alveoli showing several markedly immunopositive cells (arrows) togetherwith the presence of widespread black particulate (d f) Strong TGF-1205731 immunoreactivity at stromal level (arrows) of some collapsed alveolarzones with evident immunopositive cells (arrows) Objective magnification 40x

ISRN Toxicology 11

different pulmonary cytochemical levels in order to improvethe overall understanding of the possible adverse outcomesresulting from CNT exposure

In particular our in vivo data demonstrated that intratra-cheal instilled MWCNTs can induce lung toxicity associatedwith inflammation even in absence of fibrosis irrespective ofnanotubes functionalizationThis latter finding suggests cau-tion before considering chemical functionalization as a broadway to improve biocompatibility and safety characteristics ofCNTs being evenmore important forCNTs that are proposedfor diagnostic or therapeutic applications

These overall experimental results further support theuse of a multitiered strategy for the safety assessment ofthe human health impact of novel nanotechnology-basedproducts

Conflict of Interests

The authors declare no conflict of interestsThe authors aloneare responsible for the content and writing of the paper

Acknowledgments

This work was supported by the Italian Ministries of HealthResearch and Education (PRIN 2007) and Cariplo Founda-tion (Grant no 2009-2440)

References

[1] A D Maynard P A Baron M Foley A A Shvedova ER Kisin and V Castranova ldquoExposure to carbon nanotubematerial aerosol release during the handling of unrefinedsingle-walled carbon nanotube materialrdquo Journal of Toxicologyand Environmental Health Part A vol 67 no 1 pp 87ndash107 2004

[2] K Savolainen H Alenius H Norppa L Pylkkanen T Tuomiand G Kasper ldquoRisk assessment of engineered nanomaterialsand nanotechnologiesmdasha reviewrdquo Toxicology vol 269 no 2-3pp 92ndash104 2010

[3] ECHA (European Chemicals Agency) ldquoREACH Guidance onInformation Requirements and Chemicals Safety AssessmentrdquoEuropean Chemicals Agency 2008 httpguidanceechaeuro-paeuguidance enhtm

[4] SCENIHR ldquoOpinion on the appropriateness of the risk assess-ment methodology in accordance with the technical guidancedocuments for new and existing substances for assessingthe risk of nanomaterialsrdquo Scientific Committee on Emerg-ing and Newly Identified Health Risks Opinion adoptedJune 2007 httpeceuropaeuhealthph riskcommittees04scenihrdocsscenihr o 004cpdf

[5] SCENIHR ldquoRiskAssessment of Products ofNanotechnologiesrdquoScientific Committee on Emerging and Newly IdentifiedHealth Risks Opinion adopted January 2009 httpeceuro-paeuhealthph riskcommittees04 scenihrdocsscenihr o023pdf

[6] K Aschberger H J Johnston V Stone et al ldquoReview of carbonnanotubes toxicity and exposure-appraisal of human healthrisk assessment based on open literaturerdquo Critical Reviews inToxicology vol 40 no 9 pp 759ndash790 2010

[7] NIOSH ldquoCurrent Intelligence Bulletin Occupational Expo-sure to Carbon Nanotubes and Nanofibersrdquo National Insti-tute for Occupational Safety and Health 2010 httpwwwcdcgovnioshdocketreviewdocket161A

[8] G Oberdorster ldquoSafety assessment for nanotechnology andnanomedicine concepts of nanotoxicologyrdquo Journal of InternalMedicine vol 267 no 1 pp 89ndash105 2010

[9] EPA ldquoDraft nanomaterial research strategy (NRS)rdquo EPA600S-08002 January 2008

[10] NCI National Cancer Institute httpnclcancergovworkingassay-cascadeasp

[11] D B Warheit P J A Borm C Hennes and J LademannldquoTesting strategies to establish the safety of nanomaterialsconclusions of an ECETOC workshoprdquo Inhalation Toxicologyvol 19 no 8 pp 631ndash643 2007

[12] REACH ldquoRegulation (EC) No 19072006 of the EuropeanParliament and of the Council of 18 December 2006 concerningthe Registration Evaluation Authorisation and Restrictionof Chemicals (REACH) establishing a European ChemicalsAgency amending Directive 199945EC and repealing CouncilRegulation (EEC) No 79393 and Commission Regulation (EC)No 148894 as well as Council Directive 76769EEC andCommissionDirectives 91155EEC 9367EEC 93105EC and200021ECrdquo

[13] S Creton I C Dewhurst L K Earl et al ldquoAcute toxicity testingof chemicalsmdashopportunities to avoid redundant testing and usealternative approachesrdquo Critical Reviews in Toxicology vol 40no 1 pp 50ndash83 2010

[14] A A Shvedova V Castranova E R Kisin et al ldquoExposure tocarbon nanotube material assessment of nanotube cytotoxicityusing human keratinocyte cellsrdquo Journal of Toxicology andEnvironmental Health Part A vol 66 no 20 pp 1909ndash19262003

[15] S K Smart A I Cassady G Q Lu and D J Martin ldquoThebiocompatibility of carbon nanotubesrdquo Carbon vol 44 no 6pp 1034ndash1047 2006

[16] J H Han E J Lee J H Lee et al ldquoMonitoring multiwalled car-bon nanotube exposure in carbon nanotube research facilityrdquoInhalation Toxicology vol 20 no 8 pp 741ndash749 2008

[17] D Bello A J Hart K Ahn et al ldquoParticle exposure levelsduring CVD growth and subsequent handling of vertically-aligned carbon nanotube filmsrdquo Carbon vol 46 no 6 pp 974ndash977 2008

[18] D Bello B L Wardle N Yamamoto et al ldquoExposure tonanoscale particles and fibers during machining of hybridadvanced composites containing carbon nanotubesrdquo Journal ofNanoparticle Research vol 11 no 1 pp 231ndash249 2009

[19] M Pacurari V Castranova and V Vallyathan ldquoSingle- andmulti-wall carbon nanotubes versus asbestos are the carbonnanotubes a new health risk to humansrdquo Journal of Toxicologyand Environmental Health Part A vol 73 no 5-6 pp 378ndash3952010

[20] H J Johnston G R Hutchison F M Christensen et al ldquoAcritical review of the biological mechanisms underlying the invivo and in vitro toxicity of carbon nanotubes the contributionof physico-chemical characteristicsrdquo Nanotoxicology vol 4 no2 pp 207ndash246 2010

[21] X Hu S Cook P Wang H M Hwang X Liu and Q LWilliams ldquoIn vitro evaluation of cytotoxicity of engineeredcarbon nanotubes in selected human cell linesrdquo Science of theTotal Environment vol 408 no 8 pp 1812ndash1817 2010

12 ISRN Toxicology

[22] I Fenoglio E Aldieri E Gazzano et al ldquoThickness of multi-walled carbon nanotubes affects their lung toxicityrdquo ChemicalResearch in Toxicology vol 25 no 1 pp 74ndash82 2012

[23] B Coto I Antia M Blanco et al ldquoMolecular dynamics studyof the influence of functionalization on the elastic propertiesof single and multiwall carbon Nanotubesrdquo ComputationalMaterials Science vol 50 no 12 pp 3417ndash3424 2011

[24] C W Lam J T James R McCluskey and R L HunterldquoPulmonary toxicity of single-wall carbon nanotubes in mice7 and 90 days after intractracheal instillationrdquo ToxicologicalSciences vol 77 no 1 pp 126ndash134 2004

[25] A A Shvedova E R Kisin R Mercer et al ldquoUnusual inflam-matory and fibrogenic pulmonary responses to single-walledcarbon nanotubes in micerdquoAmerican Journal of Physiology vol289 no 5 pp L698ndashL708 2005

[26] D Cui F Tian C S Ozkan M Wang and H Gao ldquoEffectof single wall carbon nanotubes on human HEK293 cellsrdquoToxicology Letters vol 155 no 1 pp 73ndash85 2005

[27] G Jia HWang L Yan et al ldquoCytotoxicity of carbon nanomate-rials single-wall nanotube multi-wall nanotube and fullerenerdquoEnvironmental Science and Technology vol 39 no 5 pp 1378ndash1383 2005

[28] J M Worle-Knirsch K Pulskamp and H F Krug ldquoOopsthey did it again Carbon nanotubes hoax scientists in viabilityassaysrdquo Nano Letters vol 6 no 6 pp 1261ndash1268 2006

[29] K Pulskamp S Diabate and H F Krug ldquoCarbon nanotubesshow no sign of acute toxicity but induce intracellular reactiveoxygen species in dependence on contaminantsrdquo ToxicologyLetters vol 168 no 1 pp 58ndash74 2007

[30] DW Porter A FHubbs R RMercer et al ldquoMouse pulmonarydose- and time course-responses induced by exposure tomulti-walled carbon nanotubesrdquo Toxicology vol 269 no 2-3 pp 136ndash147 2010

[31] K Donaldson R Aitken L Tran et al ldquoCarbon nanotubes areview of their properties in relation to pulmonary toxicologyand workplace safetyrdquo Toxicological Sciences vol 92 no 1 pp5ndash22 2006

[32] E Herzog A Casey F M Lyng G Chambers H J Byrne andM Davoren ldquoA new approach to the toxicity testing of carbon-based nanomaterials-The clonogenic assayrdquo Toxicology Lettersvol 174 no 1ndash3 pp 49ndash60 2007

[33] A Nel T Xia LMadler andN Li ldquoToxic potential of materialsat the nanolevelrdquo Science vol 311 no 5761 pp 622ndash627 2006

[34] A A Shvedova E R Kisin D Porter et al ldquoMechanismsof pulmonary toxicity and medical applications of carbonnanotubes two faces of Janusrdquo Pharmacology andTherapeuticsvol 121 no 2 pp 192ndash204 2009

[35] P P Simeonova ldquoUpdate on carbon nanotube toxicityrdquoNanomedicine vol 4 no 4 pp 373ndash375 2009

[36] XWang G Jia HWang et al ldquoDiameter effects on cytotoxicityof multi-walled carbon nanotubesrdquo Journal of Nanoscience andNanotechnology vol 9 no 5 pp 3025ndash3033 2009

[37] P Wick P Manser L K Limbach et al ldquoThe degree and kind ofagglomeration affect carbon nanotube cytotoxicityrdquo ToxicologyLetters vol 168 no 2 pp 121ndash131 2007

[38] Y-G Han J Xu Z-G Li G-G Ren and Z Yang ldquoIn vitrotoxicity of multi-walled carbon nanotubes in C6 rat gliomacellsrdquo Neurotoxicology vol 33 no 5 pp 1128ndash1134 2012

[39] CM Sayes F Liang J L Hudson et al ldquoFunctionalization den-sity dependence of single-walled carbon nanotubes cytotoxicityin vitrordquo Toxicology Letters vol 161 no 2 pp 135ndash142 2006

[40] LW Zhang L Zeng A R Barron andN AMonteiro-RiviereldquoBiological interactions of functionalized single-wall carbonnanotubes in human epidermal keratinocytesrdquo InternationalJournal of Toxicology vol 26 no 2 pp 103ndash113 2007

[41] H Tong J K McGee R K Saxena U P Kodavanti R BDevlin and M I Gilmour ldquoInfluence of acid functionaliza-tion on the cardiopulmonary toxicity of carbon nanotubesand carbon black particles in micerdquo Toxicology and AppliedPharmacology vol 239 no 3 pp 224ndash232 2009

[42] J McDonald and L Mitchell ldquoTo the editorrdquo ToxicologicalSciences vol 101 no 1 pp 181ndash182 2008

[43] K E Driscoll D L Costa G Hatch et al ldquoIntratrachealinstillation as an exposure technique for the evaluation ofrespiratory tract toxicity uses and limitationsrdquo ToxicologicalSciences vol 55 no 1 pp 24ndash35 2000

[44] D B Warheit B R Laurence K L Reed D H Roach GA M Reynolds and T R Webb ldquoComparative pulmonarytoxicity assessment of single-wall carbon nanotubes in ratsrdquoToxicological Sciences vol 77 no 1 pp 117ndash125 2004

[45] J Muller F Huaux N Moreau et al ldquoRespiratory toxicity ofmulti-wall carbon nanotubesrdquo Toxicology and Applied Pharma-cology vol 207 no 3 pp 221ndash231 2005

[46] J G Li W X Li J Y Xu et al ldquoComparative study ofpathological lesions induced by multiwalled carbon nanotubesin lungs of mice by intratracheal instillation and inhalationrdquoEnvironmental Toxicology vol 22 no 4 pp 415ndash421 2007

[47] D Elgrabli S Abella-Gallart F Robidel F Rogerieux JBoczkowski and G Lacroix ldquoInduction of apoptosis andabsence of inflammation in rat lung after intratracheal instilla-tion of multiwalled carbon nanotubesrdquo Toxicology vol 253 no1ndash3 pp 131ndash136 2008

[48] M Fagnoni A Profumo D Merli D Dondi P Mustarelliand E Quartarone ldquoWater-miscible liquid multiwalled carbonnanotubesrdquo Advanced Materials vol 21 no 17 pp 1761ndash17652009

[49] A Casey E Herzog M Davoren F M Lyng H J Byrne andG Chambers ldquoSpectroscopic analysis confirms the interactionsbetween single walled carbon nanotubes and various dyescommonly used to assess cytotoxicityrdquo Carbon vol 45 no 7pp 1425ndash1432 2007

[50] W Lin YW Huang X D Zhou and YMa ldquoIn vitro toxicity ofsilica nanoparticles in human lung cancer cellsrdquo Toxicology andApplied Pharmacology vol 217 no 3 pp 252ndash259 2006

[51] N A Monteiro-Riviere and A O Inman ldquoChallenges forassessing carbon nanomaterial toxicity to the skinrdquoCarbon vol44 no 6 pp 1070ndash1078 2006

[52] A Casey M Davoren E Herzog F M Lyng H J Byrne andG Chambers ldquoProbing the interaction of single walled carbonnanotubes within cell culture medium as a precursor to toxicitytestingrdquo Carbon vol 45 no 1 pp 34ndash40 2007

[53] N AMonteiro-Riviere A O Inman and LW Zhang ldquoLimita-tions and relative utility of screening assays to assess engineerednanoparticle toxicity in a human cell linerdquo Toxicology andApplied Pharmacology vol 234 no 2 pp 222ndash235 2009

[54] A Simon-Deckers B Gouget M Mayne-LrsquoHermite N Herlin-Boime C Reynaud and M Carriere ldquoIn vitro investigation ofoxide nanoparticle and carbon nanotube toxicity and intracel-lular accumulation in A549 human pneumocytesrdquo Toxicologyvol 253 no 1ndash3 pp 137ndash146 2008

[55] J B Mangum E A Turpin A Antao-Menezes M F Cesta EBermudez and J C Bonner ldquoSingle-walled carbon nanotube

ISRN Toxicology 13

(SWCNT)-induced interstitial fibrosis in the lungs of rats isassociated with increased levels of PDGF mRNA and theformation of unique intercellular carbon structures that bridgealveolarmacrophages In Siturdquo Particle and Fibre Toxicology vol3 article 15 pp 1ndash13 2006

[56] K Donaldson P J A Borm V Castranova and M GulumianldquoThe limits of testing particle-mediated oxidative stress invitro in predicting diverse pathologies relevance for testing ofnanoparticlesrdquo Particle and Fibre Toxicology vol 6 article 132009

[57] M V D Z Park D P K Lankveld H van Loveren and WH de Jong ldquoThe status of in vitro toxicity studies in the riskassessment of nanomaterialsrdquo Nanomedicine vol 4 no 6 pp669ndash685 2009

[58] V Stone H Johnston and R P F Schins ldquoDevelopment of invitro systems for nanotoxicology methodological considera-tionsrdquo Critical Reviews in Toxicology vol 39 no 7 pp 613ndash6262009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of

2 ISRN Toxicology

Nevertheless though particular ENMs properties andbehaviour (ie translocation and capability to cross biologi-cal barriers) have been described in several studies (ie modeof action toxicity targets dose-response relationships andthe potential to react with constituents of cells at the portalof entry and beyond [2]) several aspects concerning NP-associated risks are still unknown and critical steps in the riskassessment of ENMs remain so far the same as those used forconventional chemicals

Furthermore there are major challenges in assessingexposure (both airborneadministered and internal dose ieparticle deposition in lung) ENMs are produced from manysubstances in many forms and sizes and with a variety ofsurface coatings The health risk assessment for such diversematerials requires validated analytical methods permittingtheir characterization in bulk samples and their detection andmeasurement in workplace air [7] Efforts are also neededfor the identification of properties that trigger ENMs toxicity(eg contaminants impurities and defects)

Today there are not enough studies to validate the poten-tial hazards posed by these novel materials and hence thedefinitive conclusions and tools technologies systems andmethods to obviate the risks Several tools for the assessmentof risks are still in the conceptual stage and further thereare considerable uncertainties on how to assess nanoparticles(NPs) exposure (ie which metrics to adopt systematically)and what methods to use to assess toxicity

Thus being unable to perform a quantitative risk assess-ment for ENMs due to the lack of sufficient data on exposurebiokinetics and organ toxicity it should be made mandatoryto prevent exposure by appropriate precautionary measuresand practicing best industrial hygiene to avoid future shockscenarios from environmental or occupational exposures [8]

In the safety assessment area two basic questions stillneed to be addressed (i) do nanomaterial properties neces-sitate a new toxicological science and (ii) what are the bio-logical and biokinetic properties to be considered for toxicitytesting in particular can biology and certain mechanismsof effects of ENMs (eg proinflammatory action oxidativestress mitochondrial perturbation generation of neoanti-gens and protein complexes in the body enhanced proteindegradation at the large surface area of NPs complexes etc)be used as a basis for studying NPs toxicity and risk assess-ment Addressing these questions is of crucial importanceto define adequate strategies and establish whether ENM-tailored testing methods should be added to conventionaltoxicity testing protocols to comply with regulatory demandand properly characterize the ENMs potential hazard

According to the major institutions [7 9 10] and interna-tional consensusmeetings [11] the proposedmultitiered test-ing protocols could be used to address toxicological researchand health risk assessment for NPs Toxicity testing shouldfirstly include a careful physicochemical characterization (egparticle size particle size distribution reactivity surface areaparticle mass impurity and aggregation tendency) assistedby using reference materials as well as the use of acellularsystems to explore the reactivity of the materials in acellularenvironments Subsequent steps include the use of validatedcellular (in vitro) models to support evidence-based testing

process followed by a limited series of in vivo studies guidedby information generated from in vitro studies especially thetoxicological data relevant to ranking of ENMs designingappropriate exposure concentrations and defining the criticalhealth endpoints to be monitored

Biosafety should be evaluated by tests examining generaltoxicity target organ toxicity and biocompatibility in linewith regulatory requirements limiting the use of lab animalsin toxicological research [12 13] to identify molecular end-points and multiple toxicity pathways

The present work represents an example of a tieredapproach for toxicity testing employing CNTs as a practicalmodel suitable to validate a two-level strategy combining invitro and in vivo experiments Such investigation is relatedto our recent studies on the pulmonary toxicity of a series offunctionalized multiwalled carbon nanotubes (MWCNTs)

Human A549 pneumocytes selected as potential targetcells during respiratory exposure associated with occupa-tional setting and environment [1 7 14ndash18] have beenused to assess in vitro the cytotoxicity of MWCNTs withdifferent degrees of functionalization using MTT assay andcalceinpropidium iodide (PI) staining

Subsequently the pulmonary toxic effects were assessedin rats 16 days after it administration of the previouslyreported type of CNTs (1mgkg bw) Major endpointstested included (i) histopathology of lung tissue (haema-toxylineosin staining) (ii) apoptoticproliferating featuresexamined by TUNEL and PCNA immunostaining and (iii)presencedistribution of (1) transforming growth factor-beta1(TGF1205731) (2) interleukin-6 (IL-6) and (3) collagen (TypeI) investigated by immunochemical methods as markers oflung toxicity inflammation and fibrosis respectively

11 CNTs State of the Art Among the several types ofengineered nanoparticles CNTs are emerging as one of themost promising and revolutionary nanomaterials widelyemployed within commercial environmental and energeticsectors due to their unusual one-dimensional hollow nanos-tructure and unique physicochemical properties [19 20]CNTs have been also proposed in medicine as nanovectorsfor vaccine and drug delivery nanodevices or as substratesfor tissue engineering [21 22] Consequently this expandingusage paralleled by occupational exposure at all phases of thematerial life cycle may lead to widespread human exposurevia skin contact inhalation and via intravenous injection inmedical applications

Moreover in view of some similar aspects to fibers suchas structural characteristics extreme aspect ratio low specificdensity and low solubility CNTs might exhibit toxicitysimilar to those observed with other fibrous particles suchas asbestos [1] Further their small size accompanied by highsurface area defines the chemical reactivity of CNTs induc-ing changes in permeability and conductivity of biologicalmembranes in fact a typical behaviour has been reportedregarding translocation and distribution of certain ENMs inthe body and their capability to cross internal barriers [2]Moreover chemical functionalization not only extends theapplications of CNTs conferring them new functions that

ISRN Toxicology 3















2)




2






2 SiO2


23 In Vitro Tests



4 ISRN Toxicology




2 CB or SiO

2) at concentra-










24 In Vivo Tests











ISRN Toxicology 5






2O2 in some














2apparently


2(Figure 1)





2



6 ISRN Toxicology

0

20

40

60

80

100

MW MW-COOH

CB

MTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

24 hr-exposure

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

1 120583gmL10 120583gmL100 120583gmL

(a)

0

20

40

60

80

100

MW MW-COOH

CBMTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

48 hr-exposure

1 120583gmL10 120583gmL100 120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowast lowastlowast


lowast

(b)


2





2) on








MW-NH2











ISRN Toxicology 7

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

SiO2

(d)

CB

(e)

MW-NH2

(f)


2 silica (e) CB



Control

(a)

MWCNTs

(b)

MW-COOH

(c)

MW-NH2

(d)

CB

(e)

SiO2

(f)




8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)







4 Conclusions


ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 3















2)




2






2 SiO2


23 In Vitro Tests



4 ISRN Toxicology




2 CB or SiO

2) at concentra-










24 In Vivo Tests











ISRN Toxicology 5






2O2 in some














2apparently


2(Figure 1)





2



6 ISRN Toxicology

0

20

40

60

80

100

MW MW-COOH

CB

MTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

24 hr-exposure

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

1 120583gmL10 120583gmL100 120583gmL

(a)

0

20

40

60

80

100

MW MW-COOH

CBMTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

48 hr-exposure

1 120583gmL10 120583gmL100 120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowast lowastlowast


lowast

(b)


2





2) on








MW-NH2











ISRN Toxicology 7

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

SiO2

(d)

CB

(e)

MW-NH2

(f)


2 silica (e) CB



Control

(a)

MWCNTs

(b)

MW-COOH

(c)

MW-NH2

(d)

CB

(e)

SiO2

(f)




8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)







4 Conclusions


ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

4 ISRN Toxicology




2 CB or SiO

2) at concentra-










24 In Vivo Tests











ISRN Toxicology 5






2O2 in some














2apparently


2(Figure 1)





2



6 ISRN Toxicology

0

20

40

60

80

100

MW MW-COOH

CB

MTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

24 hr-exposure

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

1 120583gmL10 120583gmL100 120583gmL

(a)

0

20

40

60

80

100

MW MW-COOH

CBMTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

48 hr-exposure

1 120583gmL10 120583gmL100 120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowast lowastlowast


lowast

(b)


2





2) on








MW-NH2











ISRN Toxicology 7

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

SiO2

(d)

CB

(e)

MW-NH2

(f)


2 silica (e) CB



Control

(a)

MWCNTs

(b)

MW-COOH

(c)

MW-NH2

(d)

CB

(e)

SiO2

(f)




8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)







4 Conclusions


ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 5






2O2 in some














2apparently


2(Figure 1)





2



6 ISRN Toxicology

0

20

40

60

80

100

MW MW-COOH

CB

MTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

24 hr-exposure

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

1 120583gmL10 120583gmL100 120583gmL

(a)

0

20

40

60

80

100

MW MW-COOH

CBMTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

48 hr-exposure

1 120583gmL10 120583gmL100 120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowast lowastlowast


lowast

(b)


2





2) on








MW-NH2











ISRN Toxicology 7

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

SiO2

(d)

CB

(e)

MW-NH2

(f)


2 silica (e) CB



Control

(a)

MWCNTs

(b)

MW-COOH

(c)

MW-NH2

(d)

CB

(e)

SiO2

(f)




8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)







4 Conclusions


ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

6 ISRN Toxicology

0

20

40

60

80

100

MW MW-COOH

CB

MTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

24 hr-exposure

lowastlowastlowast

lowastlowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

1 120583gmL10 120583gmL100 120583gmL

(a)

0

20

40

60

80

100

MW MW-COOH

CBMTT

met

abol

ism (

cont

rol)

SiO2MW-NH2

48 hr-exposure

1 120583gmL10 120583gmL100 120583gmL

lowastlowast lowast

lowastlowast

lowastlowast

lowast lowastlowast


lowast

(b)


2





2) on








MW-NH2











ISRN Toxicology 7

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

SiO2

(d)

CB

(e)

MW-NH2

(f)


2 silica (e) CB



Control

(a)

MWCNTs

(b)

MW-COOH

(c)

MW-NH2

(d)

CB

(e)

SiO2

(f)




8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)







4 Conclusions


ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 7

Control

(a)

MWCNTs

(b)

MW-COOH

(c)

SiO2

(d)

CB

(e)

MW-NH2

(f)


2 silica (e) CB



Control

(a)

MWCNTs

(b)

MW-COOH

(c)

MW-NH2

(d)

CB

(e)

SiO2

(f)




8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)







4 Conclusions


ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

8 ISRN Toxicology

Control

(a)

Control

(b)

MW-COOH

(c)

MW-NH2

(d)

MW-COOH

(e)

MW-NH2

(f)







4 Conclusions


ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 9

MW

-NH2

MW

-NH2

MW

-CO

OH

MW

-CO

OH

MW

-CO

OH

MW

-NH2

PCNA

(a)

(b) (e)

(d)

(c) (f) (g)

TUNEL







10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

10 ISRN Toxicology

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()

PCNA labelling


75

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

MW-NH2

(a)

0 15 3 45 6

Bronchiolar cells

Alveolar cells

Macrophages

Frequency ()


TUNEL labelling

75

lowastlowast

lowast

lowastlowastlowast

lowastlowast

lowast

MW-NH2


2) A significant


Con

trol

IL-6

(a)

TGF-1205731

Con

trol

(b)

MW

-NH2

(c)

MW

-NH2

(d)

MW

-CO

OH

(e)

MW

-CO

OH


2or (e f) MW-


ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 11






Acknowledgments


References






















12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

12 ISRN Toxicology



































ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Toxicology 13









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Safety Evaluation of Engineered Nanomaterials...

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