Research Article

Transcriptional Profiling Identifies PhysicochemicalProperties of Nanomaterials That Are Determinants of

the In Vivo Pulmonary Response

Sabina Halappanavar,1* Anne Thoustrup Saber,2 Nathalie Decan,1

Keld Alstrup Jensen,2 Dongmei Wu,1 Nicklas Raun Jacobsen,2Charles Guo,1

Jacob Rogowski,1 Ismo K. Koponen,2 Marcus Levin,2 AnneMette Madsen,2

Rambabu Atluri,3Valentinas Snitka,4 Renie K. Birkedal,2 David Rickerby,5

Andrew Williams,1 H�kanWallin,2,6Carole L.Yauk,1 and Ulla Vogel2,7

1Environmental Health Science and Research Bureau, Health Canada, Ottawa,Canada

2National Research Centre for the Working Environment, Copenhagen,Denmark

3Nanologica, Stockholm, Sweden currently at National Research Center for theWorking Environment, Copenhagen, Denmark

4The Research Centre for Microsystems and Nanotechnology, KTU, Kaunas,Lithuania

5European Commission Joint Research Centre Institute for Environment and Sus-tainability, I-21027 Ispra, VA, Italy

6Institute of Public Health, University of Copenhagen, Denmark7Department of Micro- and Nanotechnology, Technical University of Denmark,

Lyngby, Denmark

We applied transcriptional profiling to elucidatethe mechanisms associated with pulmonaryresponses to titanium dioxide (TiO2) nanopar-ticles (NPs) of different sizes and surface coat-ings, and to determine if these responses aremodified by NP size, surface area, surfacemodification, and embedding in paint matrices.Adult C57BL/6 mice were exposed via singleintratracheal instillations to free forms ofTiO2NPs (10, 20.6, or 38 nm in diameter)with different surface coatings, or TiO2NPsembedded in paint matrices. Controls wereexposed to dispersion medium devoid of NPs.TiO2NPs were characterized for size, surfacearea, chemical impurities, and agglomerationstate in the exposure medium. Pulmonary tran-

scriptional profiles were generated using micro-arrays from tissues collected one and 28 dpostexposure. Property-specific pathway effectswere identified. Pulmonary protein levels of spe-cific inflammatory cytokines and chemokineswere confirmed by ELISA. The data were col-lapsed to 659 differentially expressed genes (P� 0.05; fold change � 1.5). Unsupervised hier-archical clustering of these genes revealed thatTiO2NPs clustered mainly by postexposure time-point followed by particle type. A pathway-based meta-analysis showed that the combina-tion of smaller size, large deposited surfacearea, and surface amidation contributes toTiO2NP gene expression response. Embeddingof TiO2NP in paint dampens the overall tran-

Grant sponsor: Health Canada’s Chemicals Management Plan and

Genomics Research and Development Initiative.

Grant sponsor: Danish Centre for Nanosafety; Grant number:

20110092173/3.

Grant sponsor: Nanokem; Grant number: 20060068816.

Grant sponsor: Danish Working Environment Research Foundation.

Grant sponsor: European Community’s Seventh Framework Programme

(FP7/2007-2013); Grant number: 247989.

*Correspondence to: Sabina Halappanavar, Mechanistic Studies Divi-

sion, Environmental Health Science and Research Bureau, Healthy Envi-

ronments and Consumer Safety Branch, Health Canada, 50 Colombine

Driveway, Ottawa, Ontario K1A 0K9, Canada.

E-mail: sabina.halappanavar@hc-sc.gc.caAdditional Supporting Information may be found in the online versionof this article.

Received 7 September 2014; provisionally accepted 21 November 2014;

and in final form 00 Month 2014

DOI 10.1002/em.21936

Published online 00 Month 2014 in

Wiley Online Library (wileyonlinelibrary.com).

VC 2014Wiley Periodicals, Inc.

Environmental andMolecular Mutagenesis 00:00^00 (2014)

scriptional effects. The magnitude of the expres-sion changes associated with pulmonary inflam-mation differed across all particles; however,the underlying pathway perturbations leading toinflammation were similar, suggesting a general-ized mechanism-of-action for all TiO2NPs. Thus,

transcriptional profiling is an effective tool todetermine the property-specific biological/toxic-ity responses induced by nanomaterials. Envi-ron. Mol. Mutagen. 00:000–000, 2014. VC

2014 Wiley Periodicals, Inc.

Key words: titanium dioxide nanoparticles; toxicogenomics; paint dusts; matrix-embedded nanomate-rials; lung inflammation; hyperspectral microscopy

INTRODUCTION

Nanoparticles (NPs) of titanium dioxide (TiO2) are

among the most widely manufactured NPs globally. Com-

mercial applications of TiO2NPs continue to grow in elec-

tronics, optics and pharmaceutical fields. A number of

consumer products containing TiO2NPs (sunscreens, cos-

metics, personal care products and paints) are becoming

available on the market. Enhanced demand for manufac-

tured raw TiO2NPs and their extensive use has resulted in

increased potential for their release into the environment

and heightened risk for human exposure. Although

inhaled bulk TiO2 is considered biologically and toxico-

logically inert under non-overload conditions in both

experimental animals and humans [ILSI Risk Science

Institute Workshop Participants, 2000], a wide variety of

characteristics associated with nano (<100 nm in size),

forms of TiO2 render them biologically active and toxic

under certain circumstances (reviewed in [Shi et al.,

2013]).

A decade of nanotoxicology research has demon-

strated that nanomaterials including TiO2NPs have

increased ability to interact with cellular membranes,

access deeper regions of tissues, interfere with cellular

signalling by binding proteins, act as carriers for other

toxicants, and are toxicologically more active than large

particles of similar chemical composition. Their small

size and high surface-to-volume ratio are suggested to

be the primary determinants of their toxic potential. For

example, increased inflammation is observed in rat

lungs following pulmonary deposition of TiO2NPs com-

pared with the same airborne mass of fine TiO2 (a

standard white pigment in paints and plastics exhibiting

a primary particle size of 0.1–2.5 mm that is regularly

used as a control dust in toxicology studies) [Fabian

et al., 2008; van Ravenzwaay et al., 2009]. Increased

pulmonary retention, deeper penetration into interstitial

regions of alveoli, inflammation, and alveolar dysfunc-

tion occur following acute exposure to 5–20 nm

TiO2NPs relative to larger TiO2NPs or bulk TiO2 (50–

250 nm), suggesting that the smaller particle size and

larger surface area of the poorly soluble TiO2NP can

lead to delayed clearance and longer biopersistence in

the target organ, ultimately resulting in augmented

response [Ferin, 1994; Oberdorster et al., 1994; Ober-

dorster, 2001; Sager et al., 2008].

In addition to their nanosize, TiO2NPs may vary signif-

icantly in their physicochemical properties (e.g., agglom-

eration state in the biological medium, shape, crystalline

structure, surface properties, and charge), which are also

suggested to profoundly impact TiO2NP-induced pulmo-

nary toxicity (reviewed in [Shi et al. 2013]). Noel et al.

[2012] examined aerosolized TiO2NPs exhibiting two dif-

ferent agglomeration states (<100 and >100 nm) and

demonstrated that pulmonary response to inhaled NPs is

influenced by the dimension and concentrations of the

agglomerated NPs [Noel et al., 2012]. In another study,

intratracheal instillation of mixed (80/20 anatase/rutile)

TiO2NP (exhibiting particle size distribution of 130 nm in

water) induced 40 times more pulmonary inflammation

and lung damage than an equal mass dose of 100% rutile

forms of TiO2NP (exhibiting 136 or 149 nm size in

water), suggesting that the crystallite forms of TiO2NP is

another important property that contributes to toxicity

[Sager et al., 2008]. Other studies have shown enhanced

pulmonary inflammation following exposure to function-

alized TiO2NPs or TiO2NPs coated with zirconium, sili-

con, aluminium and polyalcohol [Husain et al., 2013].

Apart from these primary characteristics, incorporation of

TiO2NPs in matrices such as paint, plastic or emulsions

are also suggested to exert a large influence on the toxi-

cological outcome of exposure to TiO2NPs [Wohlleben

et al., 2011; Saber et al., 2012c)]. However, it has yet to

be determined, which of these particle characteristics and

exposure contexts are more important to toxicological

outcome.

In addition to a lack of clear understanding of the role

of particle properties in toxicity, it is also unclear if the

biological responses associated with specific NP proper-

ties represent differences in: (1) the primary mechanisms

affecting diverse biological functions/processes leading to

a toxicological outcome or (2) the severity of the toxico-

logical response. A better understanding and characteriza-

tion of the tissue response and comprehensive knowledge

of underlying mechanisms-of-action of TiO2NPs are

essential to determining the unique toxicities that are

associated with diverse particle features. This task is not

simple and cannot be achieved employing conventional,

single endpoint based toxicological methods; thus, alter-

native tools and strategies are urgently needed in this

field. Genomics tools provide a unique means to globally

Environmental and Molecular Mutagenesis. DOI 10.1002/em

2 Halappanavar et al.

analyse all of the biological pathways perturbed in

response to toxicant exposure in a tissue, enabling the

identification of potential hazards, mechanisms associated

with toxicological responses, dose-response relationships,

and sensitive markers that can ultimately be used for

human biomonitoring. Gene expression profiles have been

developed that can identify specific types of toxicities

associated with chemical structure [Hamadeh et al.,

2002a,b], to categorize chemical mechanisms-of-action

(reviewed in [Waters and Fostel, 2004]), to identify

tissue-specific responses [Labib et al., 2013] and to differ-

entiate between carcinogenic and noncarcinogenic chemi-

cals [Bercu et al., 2010].

Numerous studies have applied genomics to study the

in vivo effects of NPs. For example, Oberd€orster et al.

[2006] employed genomics in conjunction with EPA-

approved traditional ecotoxicity assays to evaluate the

toxicity of reactive nano-iron particles in fathead min-

nows. The authors concluded that genomics was more

sensitive in detecting subtle changes early following

exposure to nano-iron particles, enabling faster and

cheaper ecotoxicity testing than traditional methods

[Oberd€orster et al., 2006]. In another study [Yang et al.,

2010] genomics was employed to investigate the underly-

ing mechanisms of nano-copper-induced hepatotoxicity in

rats. Observed changes in gene expression were examined

against conventional toxicological parameters. The results

demonstrate that gene expression profiles are useful in

detecting responses to low, subtoxic doses of nano-copper

and enable the identification of mechanism-of-action-

based markers of overt toxicity at higher doses [Yang

et al., 2010]. By assessing gene expression profiles, Henry

et al. [2007] were able to attribute the effects of aqueous

C60 nano-aggregates to decomposition products of the

vehicle that was used to administer C60 particles in larval

zebrafish [Henry et al., 2007]. Using a combination of

gene and protein expression profiling and bioinformatic

analyses, we have previously: (1) elucidated the mecha-

nisms by which inhaled surface-coated TiO2NPs induce

pulmonary toxicity at occupationally relevant doses

[Halappanavar et al., 2011]; (2) characterized the reper-

cussions of local inflammation in the lungs on secondary

tissues (e.g., liver) following exposure to NPs [Bourdon

et al., 2012]; and (3) validated the relevance of in vitro

data to predict in vivo responses of exposure to NPs

[Poulsen et al., 2013]. Collectively, these genomics stud-

ies and others have demonstrated that the approach can

be used to predict toxicity before a phenotype is mani-

fested, enhance the existing mechanistic knowledgebase

of how toxicants exert their effects, facilitate the develop-

ment of biomarkers of exposure and effect, and provide

new and complementary information for risk assessment.

The main objective of this study is to employ genomics

and bioinformatics to investigate the gene and protein

expression changes induced by TiO2NPs with different

properties, provide insight into mechanisms-of-action and

identify distinct property-specific toxicogenomic

responses (or signatures of toxicity). TiO2NPs of different

sizes, surface coatings, and TiO2NPs embedded in paint

matrices were investigated. Mice were exposed to various

doses of free form TiO2NPs with diameters of 10, 20.6,

or 38 nm in parallel with specific TiO2NPs embedded in

paint. In addition, responses to pristine 10 nm TiO2NPs

(uncharged surface) were compared to those with ami-

nated surfaces (i.e., positively charged surfaces). Pulmo-

nary gene expression was profiled one and 28 days

postexposure using DNA microarrays. A meta-analysis of

the gene expression changes was conducted to explore

the pathway perturbations associated with particle features

and provide insight into mechanisms-of-action. This

meta-analysis also investigated the particle properties that

are responsible for eliciting the observed responses in

gene expression.

MATERIALS ANDMETHODS

TiO2NP Types and Their Properties

The key physico-chemical characteristics of the types of TiO2NP

powders and sanding dusts of paints used in this study are summarized

in Table I. In brief, five rutile TiO2NPs were tested. TiO2NP10.5 and

TiO2NP10 were obtained from NanoAmor (Nanostructured and Amor-

phous Materials, Houston). They represent different batches of the same

TiO2NP and exhibit an average crystalline size of 10 nm [Kermanizadeh

et al., 2013]. TiO2NP38 was obtained from NaBond (NaBond Technolo-

gies, Limited, Shenzhen, China). TiO2NP101 is the surface functional-

ized form of TiO2NP10 (contains positively charged amino groups on the

surface). Surface modification of TiO2NP101 has been described in [Ker-

manizadeh et al., 2013]. UV-Titan L181 (TiO2NP20.6, Kemira, Pori, Fin-

land) is a 20.6 nm TiO2NP surface-coated with traces of Al, Si, Zr, and

polyalcohol.

Wooden boards that were painted with one of two types of alcyde

paints containing different amounts of TiO2NPs as fillers (paint layers

were a few mm thick) were provided by Fl€ugger A/S; these are known

as SD-TiO2NP10.5138 (12 wt % of TiO2NP10.5and 24 wt % of TiO2NP38)

and SD-TiO2NP38 (36 wt % of TiO2NP38) [Gomez et al., 2014]. The

Danish paint and lacquer industry provided SD-TiO2NP20.6 acrylic paint

consisting of 10 wt % of TiO2NP20.6, or SD paint with no NPs [Saber

et al., 2012a]. The results of exposure to SD-TiO2NP20.6 were compared

with previously published results of pulmonary mRNA responses to free

forms of UV-Titan L181 [Saber et al., 2012a; Husain et al., 2013].

Generation and Sampling of Paint Dust

Details of dust generation were described previously [Koponen et al.,

2011; Saber et al., 2012b; Gomez et al., 2014]. Sanding of wooden

boards painted with SD-TiO2NP38 and SD-TiO2NP10.5138 was performed

using a commercially available hand-held orbital sander (Metabo Model

FSR 200 Intec, Nurtingen, Germany) placed onto a rotating painted

wooden board. The aerosol was characterized using an Electrical Low

Pressure Impactor (ELPI1, Dekati, Finland). For boards painted with

SD and SD-TiO2NP20.6, the orbital sander was moved manually onto the

painted wooden surface. A Fast Mobility Particle Sizer (FMPS 3091,

TSI, Shoreview, MN) and an Aerodynamic Particle Sizer (APS 3321,

TSI, Shoreview, MN) were used to characterize the aerosol. In both

cases, particles were collected using a commercial electrostatic

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genomic Responses of Titanium Dioxide Nanoparticles 3

precipitator, previously described by [Sharma et al., 2007]. The collected

dust was stored at 220�C until the analysis.

Physicochemical Characterization of Test Materials

The details of characterization of TiO2NP10.5 and TiO2NP38 are provided

below and in Table I. The characterization data for TiO2NP10, TiO2NP101,

TiO2NP20.6, SD-TiO2NP10.5138, SD-TiO2NP38, SD-TiO2NP20.6, and SD

have been published before and are summarized in Table I.

In brief, the X-ray diffraction (XRD) analysis was performed on

TiO2NP10.5 and TiO2NP38 using a Phillips X’PERT X-ray diffractometer

(Philips Analytical B.V., 7602 EA Almelo, The Netherlands) using

monochromated 0.15406 nm Cu Ka1 radiation (40 mA, 35 kV) with a

scan speed of 1�/min and a step-size of 0, 1�, and 2�. The TiO2NP sam-

ples were prepared by compressing the powder into a glass substrate (76

3 26 3 1 mm; Hirschmann Laborgerate glass). Average crystallite sizes

were calculated from the powder XRD data using the MAUD software

(http://www.ing.unitn.it/~maud/) that is based on the Rietveld refinement

method.

Atomic Force Microscopy (AFM, NT-MDT Co. Moscow 124482,

Russia) was conducted to assess the primary particle size-distributions of

TiO2NP10.5 and TiO2NP38. The AFM analyses were conducted in the tap-

ping mode (NT-MDT Co. Moscow 124482, Russia) using commercial sil-

icon cantilevers NSG11 with a force constant of 5 nm21. The TiO2NPs

suspended in water (1 mg/ml) were filtered using a 200 nm polyethersul-

fone pore membrane (Chromafil PES-20/25, Macherey-Nagel). Size-

distributions were obtained using NOVA software for image acquisition

(NT-MDT, http://nt-mdt.com/software) and the data were processed using

ImageAnalysis (NT-MDT, http://nt-mdt.com/software).

The specific surface area was evaluated by the Brunauer-Emmett-

Teller (BET) nitrogen adsorption, adsorption/desorption method using a

Micromeritics TriStar II volumetric adsorption analyzer (Micromeritics

Instrument Corporation, GA). The BET equation was used to calculate

the surface area from the adsorption data obtained at the relative pres-

sure (p/p0) range of 0.05–0.3.

Thermogravimetric analysis (TGA) was conducted to determine the

amount of evaporable and combustible compounds including water, and

nonspecific ingredients associated with the particles. The TiO2NP10.5 and

TiO2NP38 were measured using a Perkin Elmer Pyrus TGA [Ytkemiska

Institutet (YKI), Stockholm, Sweden]. The TiO2NP10 and TiO2NP101

were measured using a Mettler Toledo TGA/SDTA 851e (Mettler-Tol-

edo International A/S, Glostrup, Denmark) using an oxygen atmosphere

and a heating rate of 10 K/min. The weight-loss was determined at the

temperature range 25–1000�C. The sample crucibles were made of alu-

mina for both instruments.

The SEM analyses were performed by using a Zeiss NVision 40

Cross-Beam Focused Ion Beam machine, equipped with a high resolu-

tion Gemini Field Emission Gun (FEG) scanning electron microscope

column. The instrument was also equipped with an Oxford INCA 350

energy dispersive X-ray spectrometer (EDS) incorporating an X-act sili-

con drift detector with an energy resolution of 129 eV at the Mn ka

line.

In Vivo Exposure

Female C57BL/6 mice that were 5–7 weeks old were obtained from

Taconic (Ry, Denmark) and were allowed to acclimatise for 1–3 weeks

before the exposure. All mice were given food (Altromin no.1324,

Christian Petersen, Din vivoenmark) and water ad libitum. Mice were

grouped in polypropylene cages with sawdust bedding at controlled tem-

perature (21 6 1�C) and humidity (50 6 10%) in a 12 h-light: dark

cycle. The experiments were approved by the Danish “Animal Experi-

ments Inspectorate” and carried out following their guidelines for ethical

conduct and care when using animals in research.

TABLE I. TiO2NP Types and Their Physicochemical Properties

TiO2NP types

Referred to in the

text as

Size

(nm)

Wt % of TiO2NPs

in paint

Crystalline

phase

Surface

area

(m2/g)

Elemental

impurities References

Free TiO2NPs

NanoAmor

(NRCWE-030)

TiO2NP10.5 10.5 Rutile 139.1 NAa [Gomez et al.,

2014]

Nabond

(NRCWE-025)

TiO2NP38 38 Rutile 28.2 NAa [Gomez et al.,

2014]

NRCWE-001 (No

charge)

TiO2NP10 10 Rutile 99 NAa [Kermanizadeh

et al., 2013]

NRCWE-002

(Positively

charged)

TiO2NP101 10 Rutile 84 NAa [Kermanizadeh

et al., 2013]

UV-Titan L181 TiO2NP20.6 20.6 Rutile 107.7 Si, Al, Zr, and

polyalcohol

[Saber et al.,

2012a]

Sanding dusts

Sanding dust -

NRCWE-033

SD-TiO2NP10.5138 TiO2NP10.5 - 12% 1.20 Not measured

TiO2NP38 - 24%

Sanding dust

NRCWE-032

SD-TiO2NP38 TiO2NP38 - 36 % 0.82 Not measured

Sanding dust-

Indoor-

nanoTiO2

SD-TiO2NP20.6 TiO2NP20.6 - 10

%

NAb Not measured

Sanding dust-

Indoor-R

SD No NPs NAb Not measured

aTotal amount of elemental impurities is below 1%.bBET analysis was not possible for the paint dust samples due to insufficient amount of material.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

4 Halappanavar et al.

Animal exposures and sample collection for SD-TiO2NP10.5138, SD-

TiO2NP38, TiO2NP10, TiO2NP101, TiO2NP20.6, SD-TiO2NP20.6, and SD

were conducted as part of previous studies [Saber et al., 2012b; Kobler

et al., 2014]. All of the gene and protein expression data produced in

this study, with the exception of TiO2NP20.6 [Husain et al., 2013], have

not been published elsewhere. Exposures were conducted in four sepa-

rate studies: (1) TiO2NP10.5 or TiO2NP38; (2) SD-TiO2NP10.5138 or SD-

TiO2NP38; (3) TiO2NP10 or TiO2NP101; and (4) TiO2NP20.6, SD-

TiO2NP20.6, or SD. Each experiment included a separate vehicle-treated

control mice and data from exposed mice were compared with their

respective controls within the group.

For free TiO2NPs, each mouse received 18, 54, or 162 mg of

TiO2NPs or only the vehicle via single intratracheal instillation. For

TiO2NPs embedded in paints, mice were exposed to 54, 162, or 486 mg

of paint dust. Table II provides details of the exposure doses of paint

dust and the exact particle load in mgs in each dose. Mice were anaes-

thetized with 3% Isoflurane and intratracheally instilled with particle

suspensions as described previously. Each control and treatment group

consisted of five animals. At the time of sampling, mice were anesthe-

tized by subcutaneous injection of 0.2 ml of HypnormVR and DormicumVR

and killed by cardiac puncture [Jackson et al., 2011]. Whole lung tissues

were collected 24 hr and 28 d postexposure, flash frozen in liquid nitro-

gen and stored at 280�C until analysis.

Preparation of Exposure Stocks

TiO2NPs were dispersed in 2% serum in water for groups 1, 2, and 3

or in MilliQ-filtered water with 10% bronchoalveolar lavage fluid

(BAL), 0.9% NaCl for group 4 as described in [Kobler et al., 2014;

Saber et al., 2012b]. Free TiO2NPs (3.24 mg/ml for particles in groups 1

and 2 or 4.05 mg/ml for TiO2NP20.6) and the dust suspensions (9.72 mg/

ml for SD-TiO2NP10.5138 and SD-TiO2NP38, and 12.15 mg/ml for SD-

TiO2NP20.6 and SD) were dispersed by sonication (in Scott-Durham

Scintillation vials using 300 Watt and 20kHz Branson Sonifier S-450D

equipped with a 13 mm Titania disruptor horn, model number 101-147-

037) as described previously [Saber et al., 2012b; Kobler et al., 2014].

The exposure medium for control animals was similarly prepared with-

out any TiO2NPs or sanding dust. The stock suspensions were further

diluted as required to obtain the right exposure doses. Suspensions were

mixed by pipetting between the dilutions.

Particle size distributions in the exposure medium were analysed

using Dynamic Light Scattering (DLS, Malvern zetasizer Nano ZS

equipped with a 633 nm He-Ne laser, Malvern, UK) as detailed in

[Roursgaard et al., 2010]. In brief, the results were calculated using Mal-

vern DTS software version 6.11 and 7.11. The intensity-derived average

hydrodynamic diameters, Zave (zeta potential), and polydispersivity indi-

ces (PDI) of each of the dispersions used for toxicological testing were

derived. Sanding dust particles in the exposure medium were also ana-

lyzed by Scanning Electron Microscopy (SEM; Quanta 200 FEG MK11

SEM). Samples for microscopy were prepared and the analysis was con-

ducted as described in [Saber et al., 2012a].

Dose Selection

The 18, 54, and 162 mg of free TiO2NP doses represent 1.5, 5, and

15 eight-hr working days at the maximum permitted Danish occupa-

tional exposure level for TiO2, which is 6.0 or 10 mg TiO2/m3. This cal-

culation is based on the assumption that �9% of the inhaled mass is

deposited in the pulmonary region [Hougaard et al., 2010] at a volume

ventilation rate of 1.8 L per hour. The estimated TiO2NP doses (Table

II]) from the sanding dusts of paints SD-TiO2NP10.5138 and SD-

TiO2NP38 (54, 162, and 486 mg by intratracheal instillation) approxi-

mately correspond to 19, 58, and 175 mg doses of free TiO2NPs, respec-

tively. The amounts of individual TiO2NP10.5 or TiO2NP38 in paint dusts

are provided in Table II. For SD-TiO2NP20.6, the lowest SD-TiO2NP20.6

(54 mg) contained 5 mg of free TiO2NP20.6, the 162 and 486 mg doses

correspond to 16 and 48 mg of free TiO2NP20.6. The total instilled sur-

face area was calculated based on the BET surface area times the dose.

Tissue RNA Extraction and Purification

Total RNA was isolated from a random section of the lung tissue as

previously described [Halappanavar et al., 2011; Husain et al., 2013].

Briefly, a small frozen section of lung tissue (n 5 5/treatment group)

was homogenized in Trizol (Invitrogen, Carlsbad, CA) using the Retsch

Mixer MM 400. RNA was isolated using phenol:chloroform and purified

using RNeasy Mini Kits (Qiagen, Mississauga, ON, Canada). All RNA

samples showed high integrity with an A260/280 ratio between 2.0 and

2.2 and RNA integrity number above 7.0.

Microarray Hybridization

Agilent Linear Amplification kits (Agilent Technologies, Mississauga,

ON, Canada) were used to synthesize cDNA and labeled cRNA from

TABLE II. Total Amount (mg) of NPs in Each of the Exposure Doses of Free and Paint Embedded TiO2NPs

Free TiO2NPs

Content of TiO2NP in mgs

Low Medium High

TiO2NP38 18 54 162

TiO2NP10.5 18 54 162

TiO2NP10 18 54 162

TiO2NP101 18 54 162

TiO2NP20.6 18 54 162

Paint dusts

Content of TiO2NP (mgs)

Low (54 mg) Medium (162 mg) High (486 mg)

TiO2NP10.5138 TiO2NP38 - 13 mg TiO2NP38 - 39 mg TiO2NP38 - 117 mg

TiO2NP10.5 - 6 mg TiO2NP10.5 - 19 mg TiO2NP10.5 - 58 mg

SD-TiO2NP38 TiO2NP38 - 19 mg TiO2NP38 - 58 mg TiO2NP38 - 175 mg

SD-TiO2NP20.6 SD-TiO2NP20.6 – 5 mg SD-TiO2NP20.6 - 16 mg SD-TiO2NP20.6 - 48 mg

SD No NPs No NPs No NPs

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genomic Responses of Titanium Dioxide Nanoparticles 5

200 ng of total RNA derived from each individual mouse lung or com-

mercially available Universal Mouse Reference RNA (UMRR, Strata-

gene, Mississauga, ON, Canada). Cyanine-labelled cRNA was in vitro

transcribed using T7 RNA polymerase and purified using RNeasy Mini

Kits (Qiagen, Mississauga, ON, Canada). Experimental samples were

labelled with Cyanine-5 and the UMRR was labelled with Cyanine-3.

Equal amounts (300 ng) of labelled cRNA from each experimental sam-

ple were hybridized to Whole Mouse Genome GE 4x44K microarrays

(Agilent Technologies, Mississauga, ON, Canada). The arrays were

washed and scanned on an Agilent G2505B scanner. Feature extraction

10.7.3.1 (Agilent Technologies, Mississauga, ON, Canada) was used to

extract the data.

Microarray Data Normalization

Microarray data were analysed as previously described in [Husain

et al., 2013]. Briefly, a randomized block design [Kerr, 2003; Kerr and

Churchill, 2007] was used to analyse the data and the data were normal-

ized using the locally weighted scatterplot smoothing regression model-

ing method. The ratio intensity plots and heat maps were constructed for

the raw and normalized data to identify outliers. The microarray experi-

ments were repeated for the outliers. The microarray analysis of variance

(MAANOVA) [Wu et al., 2003] in R statistical software (http://www.r-

project.org) was used to determine the statistical significance of the dif-

ferentially expressed genes (DEG). The Fs statistic [Cui et al., 2005]

with residual shuffling was used to test the treatment effects and to esti-

mate P-values, respectively. The false discovery rate (FDR) multiple

testing correction [Benjamini and Hochberg, 1995] was applied to

reduce false positives. Fold change calculations were based on the least-

square means. A probe/gene was considered to be expressed if the probe

signal intensity was above background in 4 out of 5 samples in at least

one experimental condition. Genes showing expression changes of at

least 1.5 fold in either direction compared to their matched controls with

FDR P � 0.05 were considered significantly differentially expressed and

were used in the downstream analysis. We refer to these as DEGs in the

rest of the text. The final dataset was assembled using results from 350

microarrays. All microarray data have been deposited in the NCBI gene

expression omnibus database and can be accessed via the accession

number GSE60801 through http://www.ncbi.nlm.nih.gov/geo/query/acc.

cgi?token?avohseiehpwznwv&acc=GSE60801.

Cluster Analysis

Each experimental condition across the four studies was collapsed to

a group average using the median signal intensity. These values were

then normalized to the time matched controls. The data were further

reduced by only considering genes that showed FDR P � 0.05 and fold

change �1.5 in any of the pairwise comparisons, yielding 659 genes.

Hierarchical cluster analysis was then conducted using the one minus

correlation (Spearman) dissimilarity metric with average linkage.

Microarray Data Analysis

Various bioinformatics and pathway analysis tools were used to iden-

tify the altered biological functions or processes in the lungs in response

to exposure to different types of TiO2NPs. The biological and molecular

functions associated with DEGs were explored in Ingenuity Pathway

Analysis (IPA, Ingenuity Systems, Redwood City, CA) and MetaCore

(Thomson Reuters Scientific, Philadelphia, PA. http://www.genego.com/

metacore.php). The significance of the association between the DEGs

and the canonical pathways or functions was measured using the Fish-

er’s exact test in IPA. Upstream regulator analysis was used to identify

the upstream transcriptional regulators that may be involved in the

observed gene expression changes in lungs. Upstream regulators with a

Z-score above 2.0 were considered for interpretation of the data.

Expression Analysis of Inflammatory Proteins

Total protein was extracted from the frozen lung tissues (n 5 3–4

per condition) from experimental and control mice using Bioplex cell

lysis kits (BioRad Laboratories, Mississauga, ON, Canada), and quanti-

fied using Bradford protein assay kits (BioRad Laboratories, Missis-

sauga, ON, Canada). Protein expression of pro-inflammatory cytokines

was assessed using a custom-made 12-plex assay kit (BioRad Laborato-

ries, Mississauga, ON, Canada) as previously described [Husain et al.,

2013]. Twelve cytokines and chemokines prioritized for investigation

were selected based on: (1) they are components of published immune

and inflammation response pathways; (2) the mRNA for these proteins

was altered in this study; and (3) previous studies have shown that the

expression of these proteins is altered during inflammation following NP

exposure.

Detection of TiO2NP in the Lung Tissues

Frozen lung tissue samples (n 5 2) from the high dose group sampled

on day 1 and day 28, and matched controls, were sliced into 5 lm thick

sections for staining with hematoxylin and eosin (H-E). Two lung sections

per sample were analyzed by hyperspectral imaging using a Cytoviva

Darkfield Hyperspectral Imaging system (Cytoviva, Auburn, AL), which

combines a concentric imaging visible and near-infrared (VNIR) spectro-

photometer (400–1000 nm) with an integrated CCD camera. Image acqui-

sition (1003 magnification) was carried out using a Dage Excel Color

Cooled-M camera attached to an Olympus BX 43 optical microscope (as

described in [Husain et al., 2013]) and images were analyzed by the Envi-

ronment for Visualization (ENVI 4.8, Cytoviva, Auburn, AL) software.

Reference spectral libraries were built for individual TiO2NPs, paint dusts

and for lung tissues derived from control mice prior to the analysis of sam-

ples. Spectra from TiO2NPs or paint dusts exposed lung tissues were com-

pared with the established reference libraries by Spectral Angle Mapping,

an automated spectral classification system in ENVI that uses an n-D angle

to match pixels from the treated samples to reference spectra. The algo-

rithm establishes the spectral similarity between two spectra by calculating

the angle between them and converting them to vectors in a space with

dimensionality equal to the number of bands. Smaller angles represent

closer matches to the reference spectrum. The maximum angle (radians)

threshold for spectral similarity was set to 0.1. Pixels further away than the

specified maximum angle threshold in radians were not classified.

RESULTS

Characterization of Free TiO2NPs

Several material characterization techniques were used

to determine the size, surface area, crystalline phase,

chemical impurities, and size distributions of TiO2NPs in

the exposure media. The results are summarized in Tables

I and III. In brief, TiO2NP10.5, TiO2NP38, and TiO2NP10

were identified as pure rutile with average particle sizes

of 10.5, 38.1, and 10 nm, respectively. Reasonable com-

parability was observed between the Rietveld refinement

data on the about 10 nm average crystallite sizes for

TiO2NP10.5 and TiO2NP10. However, the specific surface

areas and amount of evaporable and combustible com-

pounds were notably different with about 139.1 m2/g and

9 wt % mass-loss and about 99 m2/g and 4 wt % mass-

loss for TiO2NP10.5 and TiO2NP10, respectively.

TiO2NP10.5 and TiO2NP10 are the same TiO2NP, except

that they are derived from different batches. Surface

Environmental and Molecular Mutagenesis. DOI 10.1002/em

6 Halappanavar et al.

modification of TiO2NP10 with a positively charged

amino group resulted in a 15 m2/g reduction in the spe-

cific area of TiO2NP101 and increased the mass-loss. The

results of TGA varied across TiO2NPs with <1

(TiO2NP38) to 9–9.9 wt % (TiO2NP10.5 and TiO2NP101).

The average crystallite sizes and specific surface areas of

TiO2NP38 and TiO2NP20.6 were 38.1 nm and 28.2 m2/g,

and 20.6 nm and 107.7 m2/g, respectively. The

TiO2NP20.6was the only one that exhibited significant ele-

mental impurities (16.7 wt % elements plus oxygen) and

polyol (6.1 wt %) as organic coating [Hougaard et al.,

2010]. Total elemental impurities as assessed by induc-

tively coupled plasma mass spectrometry revealed <1%

impurities for all other particle types.

Dust Characterization

The sanding dusts SD-TiO2NP10.5138, SD-TiO2NP38,

SD-TiO2NP20.6, and SD, were extensively characterized

and published in [Saber et al., 2012b, 2012c; Gomez et al.,

2014]. Briefly, SEM of SD-TiO2NP10.5138 and SD-

TiO2NP38 dust samples showed that TiO2NP particles

mostly remained attached to the matrix and consisted of

aggregates of sub-mm-size particles. Energy Dispersive

X-ray analysis of 25 particles collected from SD-

TiO2NP10.5138 and SD-TiO2NP38 revealed Ti signals for

80% of the 25 particles. Similarly, the collective size-

ranges for SD-TiO2NP20.6 varied from about 10 nm to

about 1.7 mm. Particles were contained within the paint

matrix and no free particles devoid of paint matrix were

found in the collected dust. SEM analysis of aggregate

structures consisting of particles revealed that the size

ranges of particles in the aggregates were compatible with

the corresponding free TiO2NPs (for SEM images please

refer to Fig. 1, and [Saber et al., 2012a, 2012b, 2012c]).

Measurements of airborne particles produced during

the sanding process of SD-TiO2NP10.5138 and SD-

TiO2NP38 showed a bi-modal distribution with a narrow

peak at 15 nm, which originates from the engine of the

sanding apparatus [Koponen et al., 2011]. The sanding of

SD-TiO2NP20.6 and SD gave a multimodal distribution

with a similar peak at 15 nm from the sanding apparatus,

and additional peaks at 180 nm and 1 mm for SD-

TiO2NP20.6 and 160 nm and 1 mm for SD.

Endotoxin content in the supernatants of particle suspen-

sions was assessed as described in [Saber et al., 2012b].

The amount of endotoxin found in the 162 mg dose for all

of the tested particle types and dusts except one was below

0.10 EU, which is approximately equivalent to 6 pg of

endotoxin or 0.45 ng endotoxin/kg body weight. Six pg of

endotoxin is the established safe (no increases in cytokine

levels) level of endotoxin that should be administered to a

20-g mouse via intravenous injection, which equals 0.1 EU

or 6 pg of endotoxin administered over 1-hr period. The

SD-TiO2NP38 samples showed 8 pg of endotoxin in the

162 mg dose of sanding dust. The endotoxin contamination

may have been introduced during the process of sanding.

Characterization of Dispersion of TiO2NPs and SandingDusts in the ExposureMedia

Table III summarizes the DLS results of the average

intensity-derived hydrodynamic sizes of the free TiO2NPs

and sanding dusts dispersed in different exposure media.

TABLE III. Particle Size Distributions in the Exposure Medium as Analyzed Using DLS

Types of Materials Zave (PDI)

CommentsFree TiO2NPs 18 mg 54 mg 162 mg

TiO2NP10.5 133 6 2 (0.136) 130 6 3 (0.159) 126 6 2 (0.157) Minor amounts of ca. 3–6 mm-size particles detected

in the intensity size-distribution. Stable dispersion.

TiO2NP38 219 6 5 (0.152) 178 6 86 (0.122) 214 6 6 (0.172) Minor amounts of ca. 3–6 mm-size particles detected

in the intensity size-distribution. Stable dispersion.

TiO2NP10 109 6 1 (0.150) 108 6 1 (0.144) 108 6 1 (0.145) Finely dispersed and no large agglomerates detected.

Stable dispersion.

TiO2NP101 1898 6 117 (0.161) 1978 6 153 (0.325) 1719 6 166 (0.362) Highly agglomerated particles. Unstable dispersion.

TiO2NP20.6 – 5224 6 832 (0.571) – Some particles out of DLS range. Unstable dispersion.

5 mm filtering show 485 6 102 nm-size particles

[Saber et al., 2010].

Sanding Dusts

SD-TiO2NP10.5138 276 6 19 (0.195) 280 6 16 (0.204) 274 6 16 (0.157) Minor amounts of ca. 3–6 mm-size particles detected

in the intensity size-distribution. Slightly unstable

dispersion.

SD-TiO2NP38 372 6 49 (0.188) 517 6 60 (0.345) 494 6 48 (0.272) Minor amounts of ca. 3–6 mm-size particles detected

in the intensity size-distribution. Unstable dispersion.

SD-TiO2NP20.6 – – – Coarse particles out of DLS range. 0.2 mm filtering

revealed presence of 158 6 6 nm-size particles [Saber

et al., 2010].

SD – 386 6 14 (0.214) – Data only available for a 54 mg dose exposure test

[Saber et al., 2010].

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genomic Responses of Titanium Dioxide Nanoparticles 7

The results show that TiO2NP101 and TiO2NP20.6 were

extensively agglomerated to form mm-size particles. The

finest dispersion was observed for TiO2NP10 and

TiO2NP10.5 (Table III). In comparison, sanding dust aero-

sols showed sub-micron sized particles [Koponen et al.,

2011; Gomez et al., 2014]. The SD-TiO2NP20.6 showed

extensive agglomeration. The other sanding dust samples

appeared to be relatively well-dispersed and dominated

by sub-mm particles and the presence of a small fraction

of mm-size particles by intensity. Although minor differ-

ences in the average hydrodynamic size were observed

between the different batch preparations of the same

material, overall the results were reproducible. The great-

est batch-to-batch variation was observed for SD-

TiO2NP38, suggesting unstable dispersion for this

material.

Overview of Pulmonary mRNA Responses FollowingExposure to a Variety of TiO2NPs

The following comparisons were made between gene

expression profiles: (1) size-related effects were obtained

by comparing TiO2NP10.5 to TiO2NP38; (2) surface

property-related effects were obtained by comparing

TiO2NP10 (pristine surface) to TiO2NP101 (positively

charged aminated surface); and (3) toxicity-related to free

particle exposure versus exposure to matrix embedded

particles was obtained by comparing TiO2NP10.5 and

TiO2NP38 to SD-TiO2NP10.5138 and SD-TiO2NP38, or by

comparing the results of exposure to free TiO2NP20.6 par-

ticles to SD-TiO2NP20.6.

Meta-Analysis of Pulmonary Responses

A meta-analysis was conducted on all of the datasets

derived from 350 microarrays. A total of 659 genes were

differentially expressed in at least one condition. Support-

ing Information Table Ia provides a list of all DEGs

across particle types, dose, and post-exposure timepoints.

Hierarchical cluster analysis of the DEGs revealed that

the expression patterns were similar for individual mice

within exposure groups. Cutting the tree at level three

(indicated by the horizontal dotted line in Fig. 2) revealed

clear separation of samples in three distinct clusters

Fig. 1. Scanning electron microscope images showing TiO2NP or TiO2NP containing sanding dusts in instillation vehi-

cle. A: TiO2NP38 (162 mg dose), B: TiO2NP10.5 (162 mg dose), C: SD-TiO2NP10.5 1 38 (486 mg dose), and D: SD-

TiO2NP38 (486 mg dose).

Environmental and Molecular Mutagenesis. DOI 10.1002/em

8 Halappanavar et al.

(indicated by the different colors in Fig. 2). Cluster-1

consists of all doses of TiO2NP10.5, TiO2NP38, and corre-

sponding paint dust samples (SD-TiO2NP10.5138 and SD-

TiO2NP38) for the 28 d postexposure timepoint. Cluster-2

and -3 are on a separate branch. Cluster-2 includes 24 hr

and 28 d samples of TiO2NP10 and TiO2NP101, and 24 hr

samples of TiO2NP10.5, TiO2NP38 and corresponding 24

hr paint dust samples. Cluster-2 also contains a few

TiO2NP20.6 samples. Cluster-3 is mainly driven by the

samples of SD-TiO2NP20.6, SD paint dust representing

both 24 hr and 28 d post-exposure timepoints along with

28 d samples from mice exposed to lower doses of free

TiO2NP20.6.

The results of the cluster analysis showed that in gen-

eral samples from the same experiment clustered more

closely than samples from two separate experiments. The

lung tissue samples exposed to free TiO2NPs collected 24

hr post-exposure exhibiting the largest transcriptional

response were all clustered together. The matrix embed-

ded TiO2NP-exposed samples exhibiting subtle responses

subclustered separately from free NPs. A detailed analysis

of DEGs in each cluster was conducted to derive common

gene expression patterns (data not shown). The genes that

were present within a specific cluster and that showed

fold change values �1.5 in the same direction for all con-

ditions within that specific cluster (i.e., eliminating genes

that were unchanging in the samples in that subcluster, or

were not consistently changed in the same direction) were

analysed further by DAVID to determine the biological

drivers of these clusters. The results revealed that the

Cluster-1 and Cluster-3 were enriched with functional

annotations such as inflammation and wound healing;

however, none were significant. Cluster-2 showed highly

significant functional groups that included annotations

such as cytokines and chemokines, in addition to inflam-

mation and wound healing (data not shown). Thus, the

overall global pulmonary gene expression patterns were

very similar between the particle types and were predomi-

nantly associated with the activation of innate immune

response and inflammation pathways (Figs. 3a and 3b,

described in detail below).

Each individual data set was analyzed separately to

identify the specific molecular changes and magnitude of

the response (detailed descriptions are provided below).

Table IV summarizes the up- and down-regulated genes

for each particle type. Supporting Information Figures SI–

SIV show the results of Venn analysis. Since mice

exposed to SD-TiO2NP20.6 or SD exhibited very few

DEGs (Table IV), and since the results of TiO2NP20.6

were previously published [Husain et al., 2013], these

data sets were not included in the downstream analysis

presented below.

Size-Related Pulmonary Responses

Size-related effects were assessed by comparing

responses to TiO2NP10.5 and - TiO2NP38. Supporting

Information Table SIb lists DEGs following exposure to

TiO2NP10.5 or TiO2NP38. Supporting Information Figures

SIa and SIb shows commonly altered DEGs between the

doses for each time point following exposure to

TiO2NP10.5 or TiO2NP38, respectively. In general, the

extent of response varied between the two particle sizes

studied, TiO2NP10.5 and TiO2NP38. A total of 344 DEGs

were found in the lungs in mice exposed to TiO2NP10.5

sampled 24 hr postexposure: 5 (2 down-regulated and 3

up-regulated), 60 (20 down-regulated and 40 up-regu-

lated), and 281 genes (72 down-regulated and 209 up-

regulated) in the 18, 54, and 162 mg dose groups, respec-

tively. Mice exposed to TiO2NP38 and sampled 24 hr

postexposure exhibited far fewer DEGs (23 in total): one

gene in each of the 18 and 54 mg dose groups, and 21

Fig. 2. Gene expression relationships among the TiO2NP varieties tested.

Hierarchical clustering was conducted using 659 genes with expression

changes of at least 1.5 fold compared with matched controls and with

FDR P � 0.05 in any of the conditions. The dotted line indicates the

level at which the tree was cut. Colors show individual clusters. [Color

figure can be viewed in the online issue, which is available at wileyonli-

nelibrary.com.]

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genomic Responses of Titanium Dioxide Nanoparticles 9

DEGs (5 down-regulated and 16 up-regulated) in the 162

mg dose group. Eighteen of these 23 genes overlapped

with TiO2NP10.5, suggesting a high degree of overlap in

the DEGs induced by the less responsive TiO2NP38 with

TiO2NP10.5 (Supporting Information Fig. SIc). DEGs

were mainly associated with immune and inflammatory

response pathways, reflecting leukocytes and phagocytic

movement, and proliferation.

The response at the 28 d timepoint decreased to a large

extent for TiO2NP10.5, but remained relatively unchanged

for TiO2NP38. A total of 49 genes (23 down-regulated

and 26 up-regulated) for TiO2NP10.5 and 18 genes (14

down-regulated and 4 up-regulated) for TiO2NP38 were

differentially expressed 28 days postexposure. Only 6

genes were in common to both particle types (Supporting

Information Fig. SId). Several genes belonging to the

heat shock protein family were down-regulated at this

late timepoint.

Surface Property-Related Effects

The pulmonary responses to TiO2NP10 and TiO2NP101

were compared with assess the influence of surface func-

tionalization. A total of 263 genes were significantly differ-

entially expressed following exposure to TiO2NP10 (10 nm,

pristine surface) at the 24 hr postexposure timepoint; 4

genes (3 down-regulated and 1 up-regulated), 21 genes (4

down-regulated and 18 up-regulated), and 238 genes (75

down-regulated and 163 up-regulated) were differentially

expressed in the 18, 54, and 162 mg dose groups, respec-

tively. Addition of an amino group to the surface resulting

in positive surface charge (TiO2NP101) resulted in an over-

all reduction in response, with 80 genes (25 down-regulated

and 55 up-regulated) in the 54 mg dose group and 112 genes

(24 down-regulated and 88 up-regulated) in the 162 mg dose

group at the 24 hr postexposure timepoint. The 18 mg dose

group did not show any response (Supporting Information

Table SIc). A greater response was observed in the medium

dose group of TiO2NP101 than TiO2NP10. Although fewer

genes were altered in response to TiO2NP101, the magni-

tude of the response (fold changes associated with altered

genes) was much larger for TiO2NP101. Supporting Infor-

mation Figure SIIa (TiO2NP10) and IIb (TiO2NP101) show

the overlapping DEGs between the doses for each timepoint.

A high degree of overlap was found between TiO2NP10 and

TiO2NP101, with 98 genes in common at the 24 hr time-

point (Supporting Information Fig. SIIc).

Fig. 3. Ingenuity pathway analysis to identify significantly altered canonical pathways, biological processes, and upstream

regulators. A: pathways (top panel), biological processes (middle panel), or predicted activation of upstream regulators

(bottom panel) associated with DEGs at the 24 hr and (B) pathways (top panel), biological processes (middle panel), or

predicted activation of upstream regulators (bottom panel) associated with DEGs at the 28 d postexposure timepoints.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

10 Halappanavar et al.

At the 28 d postexposure timepoint the overall response

was diminished. Only 4 DEGs in response to TiO2NP101

(162 mg) and 44 DEGs (42 genes in the 18 mg and 2

genes in the 54 mg dose groups) in response to TiO2NP10

were noted with only one gene common to both particles

(Supporting Information Fig. SIId). The DEGs were

mainly heat shock chaperones and associated with andro-

gen signaling pathways.

Responses to Particles Embedded in Paint Matrix^SD-TiO2NP

10.5138 and SD-TiO2NP38

We assessed the effects of embedding TiO2NPs in

complex matrices such as paint. As described in Figure 2,

pulmonary effects of exposure to sanding dusts of paints

containing TiO2NPs were very subtle across all doses

and timepoints tested compared to free particles

Fig. 3. (Continued).

TABLE IV. Summary of the number of up- and down-regulated DEGs following pulmonary exposure to TiO2NPs and sandingdusts containing TiO2NPs

Postexposure (24 h)

Dose TiO2NP10.5 TiO2NP38 TiO2NP10 TiO2NP101 TiO2NP20.6 SD-TiO2NP10.5 1 38 SD-TiO2NP38 SD-TiO2NP20.6 SD

Low Up 2 1 1 0 1 0 0 0 0

Down 3 0 3 0 0 0 0 2 0

Medium Up 40 1 18 55 7 2 6 0 0

Down 20 0 4 25 0 0 0 0 0

High Up 209 16 163 88 197 50 4 7 1

Down 72 5 75 24 40 22 0 3 0

Postexposure (28 d)

Low Up 19 2 38 0 7 3 1 1 0

Down 11 6 4 0 0 3 1 9 8

Medium Up 2 1 2 0 0 2 2 0 0

Down 6 3 0 0 0 6 1 1 5

High Up 5 1 0 0 33 4 1 0 0

Down 6 5 0 4 6 10 5 7 2

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genomic Responses of Titanium Dioxide Nanoparticles 11

(Table IV and Supporting Information Table SId). SD-

TiO2NP10.5138, consisting of 12 wt % of TiO2NP10.5, and

24 wt % of TiO2NP38, had the maximum number of

DEGs of the paint dusts at both the 24 hr and 28 d post-

exposure timepoints. A total of 74 DEGs were found; 2

in the 162 mg dose and 72 in the 486 mg dose group at 24

hr. There were 28 DEGs (6, 8, and 14 genes in the 54,

162, and 486 mg dose groups, respectively) at the 28 d

timepoint. In contrast, the overall response to SD-

TiO2NP38 (36 wt % of TiO2NP38) included 21 genes,

with 10 and 11 DEGs at 24 hr and 28 d timepoints,

respectively (Table IV and Supporting Information Table

SId). There were not many genes in common between the

doses (Supporting Information Fig. SIIIa and SIIIb).

To determine the similarity in responses between paint

dusts and corresponding free particles, a Venn analysis

was performed and overlapping genes were identified.

There were no commonalities between SD-TiO2NP38 and

free TiO2NPs (Supporting Information Fig. SIIIc). Com-

parison between SD-TiO2NP10.5138 and TiO2NP10.5

revealed 28 DEGs in common. These DEGs represent

�50% of the total DEGs (all doses combined) from SD-

TiO2NP10.5138; these included 28 genes in common with

the 162 mg dose, and 9 genes in common with the 54 mg

dose of TiO2NP10.5 at 24 hr. About five genes overlapped

within the 28 d groups (Supporting Information Fig.

SIIId). Similar comparisons of SD-TiO2NP10.5138 with

TiO2NP38 revealed six genes in common (Supporting

Information Fig. SIIIc and SIIId). Similarly, comparison

of responses between TiO2NP20.6, SD-TiO2NP20.6, and

SD showed one overlapping gene (Supporting Information

Fig. SIIIe).

Comparison of Responses to TiO2NP10.5 and TiO2NP

10

In addition to the comparisons described above, tran-

scriptional responses to intratracheal instillation of

TiO2NP10.5 and TiO2NP10 were compared. These two

TiO2NPs represent different batches of the same mate-

rial, and thus are expected to behave in a similar manner

biologically. Table IV shows that the total number of

DEGs altered in response to TiO2NP10.5 was greater

than TiO2NP10. In addition, only 46 DEGs were com-

mon to both of these TiO2NP types (Supporting Infor-

mation Fig. SIV). Moreover, TiO2NP10.5 induced larger

fold changes in the common DEGs, suggesting that the

overall lung transcriptional response was different

between the two particle types. Although there were

uniquely altered genes (different genes) in response to

TiO2NP10.5 and TiO2NP10, they were associated with

similar pathways and biological functions. Thus, despite

minor differences in specific genes, the biological func-

tions and pathways that were altered were not distinct to

the particle types.

Biological Pathways and Functions Altered

The DEGs were analyzed to identify specific biological

functions, processes, or pathways altered. This analysis

aimed to identify similarities as well as particle-specific

or property-specific responses or toxicological mecha-

nisms. In general, pronounced pulmonary responses were

mainly observed at the highest dose following exposure

to TiO2NPs (explained in detail in the following para-

graphs). Two particle types, TiO2NP10.5 and TiO2NP10,

showed some response at the lowest dose (18 mg) 28 d

postexposure. However, analyses of the DEGs altered at

this timepoint showed five or fewer genes involved in any

single canonical pathway. The aldosterone signaling and

protein ubiquitination pathways were altered in response

to TiO2NP10.5. The perturbed genes associated with these

pathways were identical (Dnaja1, Dnajb1, Hspa8,Hspa1a, and Hsph1) and were downregulated. The canon-

ical pathways that were altered in response to the low

dose of TiO2NP10 included circadian rhythm (Arntl,Bhlhe40, Creb5, and Per2), protein ubiquitination

(Dnaja1, Dnajb1, Hspa8, Hspa1a, and Hsph1), glucocor-

ticoid receptor signaling (Fkbp5, Hspa8, Hspa1a, Slp1,

and Tsc22d3) and aldosterone signaling (Dnaja1, Hspa8,Hspa1a, and Hsph1). Given the small proportion of genes

affected in these pathways, it is difficult to determine if

these changes are relevant to persistent effects of expo-

sure to TiO2NP.

Since the low dose response was subtle across the time

points, the DEGs from only the highest dose for all parti-

cle types and timepoints were considered in further analy-

sis. A high degree of overlap in the canonical pathways

across all particle types 24 hr and 28 d post-exposure was

found (Figs. 3a and 3b, respectively). The average linkage

and Euclidean distance metric was used to generate a

heatmap of canonical pathways (Figs. 3a and 3b top pan-

els). Pathways that exhibited a –log(P-value) score >5.0

[the –log(P-value) from the Fisher’s exact test] in any

one of the observation are shown in Figure 3a. Among

the major pathways perturbed 24 hr postexposure, granu-

locyte adhesion and diapedesis, agranulocyte adhesion,

and diapedesis, IL-17F in allergic inflammatory airway

diseases, LXR/RXR activation, and acute phase signaling

had the highest scores. The first three of these pathways

were also the highest scoring at the 28 d postexposure

timepoint following exposure to free particles; however,

these pathways did not meet the statistical cut-off at 28 d

(Fig. 3b, top panel). Nevertheless, this suggests that some

of the processes induced within 24 hr of exposure to

TiO2NPs persist until 28 d (Fig. 3b, top panel) after the

exposure. The other canonical pathway that was altered

one and 28 d postexposure was pattern recognition recep-

tors involved in recognizing bacteria and viruses. Aldoste-

rone signaling in epithelial cells was specific to the 28 d

postexposure timepoint (Fig. 3b top panel). In agreement

Environmental and Molecular Mutagenesis. DOI 10.1002/em

12 Halappanavar et al.

with the results of canonical pathways, a detailed analysis

of biological functions associated with DEGs mainly

pointed to altered proliferation and movement of different

types of inflammatory cells (Figs. 3a and 3b middle

panel) at 24 hr.

IPA’s upstream regulator analysis was used to deter-

mine the upstream transcription factors and/or membrane

receptors responsible for up- or down-regulation of DEGs

associated with specific pathways for all particle types.

Direction of expression changes in DEGs that are targets

of specific upstream regulators are compared to the

expected direction of change to predict activation or inhi-

bition of the upstream regulator. This analysis revealed

altered activity of several upstream regulators related to

significantly altered canonical pathways including inflam-

mation, cytokine production, and pattern recognition

receptor pathways. Predicted activation of NFjB was

observed only at the 24 hr timepoint, whereas TNF,

IFNG, and TLRs were among the many transcription fac-

tors that were predicted to be activated at both the 24 hr

and 28 d timepoints (Figs. 3a and 3b bottom panel). In

general, compared with the response observed in samples

exposed to free particles, the responses to paint dusts

were subtle at 24 hr. No significant pathways, functions,

or upstream regulators were altered 28 d following expo-

sure to paint dusts. In addition, despite a huge overlap in

the altered pathways and functions between free and

paint-embedded particles across all sample sets, the mag-

nitude of the response (number of DEGs in each of the

perturbed pathway and associated fold changes) varied

across the TiO2NP types.

ELISA

Changes in the mRNA expression levels of several of

the inflammatory cytokines and chemokines were con-

firmed at the protein level in lung tissue by ELISA for all

the particle types for the highest dose. Due to the limited

availability of tissue samples, a custom-ordered multiplex

ELISA containing 12 cytokines and chemokines was used

to investigate IL-1b, IL-4, IL-5, IL-6, IL-13, IL-17, G-

CSF, GM-CSF, CXCL-1, CCL-2, CCL-3, and CCL-4. In

alignment with the microarray results, changes in pulmo-

nary protein levels were observed at the 24 hr post-

exposure timepoint (Table V), with all TiO2NPs exhibit-

ing somewhat similar responses. The protein analyses

provide confirmation of an inflammatory phenotype.

There were no significant changes in proteins observed 28

d postexposure (data not shown).

Detection of Particles in Lungs

To determine the postexposure status of matrix-

embedded NPs within the lungs, lung tissue sections from

the high dose group 24 hr and 28 d post-exposure were

analyzed by hyperspectral imaging. Figure 4 shows the

representative spectral libraries constructed for various

different types of materials on the left and corresponding

hyperspectral images and spectral angle mapping results

on the right. Figures 4a–4c represent the reference libra-

ries constructed for vehicle-dispersed TiO2NPs, paint dust

consisting of TiO2NPs and control lung tissues exposed

to vehicle, respectively. Endogenously fluorescing non-

specific objects that did not map to the reference libraries

were observed in all samples, including controls, and

thus, were filtered out. Figures 4d and 4e show the unique

spectrum and hyperspectral mapping of one of the free

TiO2NPs in lung tissue from the high dose group for both

the timepoints. Figures 4f and 4g show the spectral pro-

files of paint-embedded TiO2NPs and of the paint matrix.

Significant retention of free and paint-embedded TiO2NPs

was observed for all particle types at both timepoints.

Since the material available for the analysis was small,

the exact amount of TiO2NPs retained in the lung tissue

was not quantified. Similarly, it was not possible to assess

whether pulmonary retention of TiO2NPs was influenced

by their specific properties. In all samples exposed to

paint-embedded TiO2NPs, both paint matrix (red dots)

and TiO2NPs (white dots) were observed (Figs. 4f and

4g). The spectra of TiO2NPs and paint dust were similar

at the 24 hr and 28 d timepoints. The results suggest that

matrix dissociation or in vivo transformation of TiO2NPs

did not occur.

DISCUSSION

In general, a larger effect on gene expression was

observed following exposure to free forms of smaller

TiO2NPs; TiO2NP10.5 exhibited the largest number of

DEGs of all the particles examined. The response to 38

nm TiO2NP38 was much lower than the other 10 nm

TiO2NPs. However, two other 10 nm TiO2NPs (TiO2NP10

and TiO2NP101) altered expression of fewer DEGs than

TiO2NP10.5. As described above, TiO2NP10.5 and

TiO2NP10 are the same TiO2NPs and differ only in their

batch numbers. TiO2NP101 is the surface functionalized

form of TiO2NP10. However, the surface areas of both

TiO2NP10 and TiO2NP101 are smaller than TiO2NP10.5,

suggesting that the smaller surface area may have contrib-

uted to the observed reductions in the mRNA responses

to TiO2NP10 and TiO2NP101. These results are consistent

with previously published reports suggesting that particle

size and the total deposited surface area are main contrib-

utors to TiO2NP-induced biological response [Fabian

et al., 2008; van Ravenzwaay et al., 2009]. However,

since these materials were tested in separate experiments,

further investigation is required to rule out the possibility

that the subtle differences could be the results of

experiment-to-experiment variations. Thus, pulmonary

transcriptional responses to the four TiO2NPs could be

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genomic Responses of Titanium Dioxide Nanoparticles 13

ordered as follows based on the number of DEGs:

TiO2NP10.5 (139.1 m2/g) > TiO2NP10 (99 m2/g) >TiO2NP101 (84 m2/g)> TiO2NP38 (28.2 m2/g). Regardless

of the number of DEGs, all four TiO2NPs altered the

same biological pathways associated with innate immune

response and inflammation (Figs. 3a and 3b). Although

the aminated TiO2NP101 NPs induced fewer DEGs than

the other two 10 nm TiO2NPs, the magnitude of the

response (fold change associated with individual DEGs)

was comparable to that of TiO2NP10.5. In addition, more

DEGs were found in mice treated with 54 mg of

TiO2NP101 than 54 mg of TiO2NP10.5 or TiO2NP10 (Table

IV). Although subtle, these results suggest that a combi-

nation of small size and aminated surface resulting in a

positive surface charge may be more effective in inducing

gene expression changes at lower doses than small sized

TiO2NPs with pristine surfaces.

Interestingly, we did not find a correlation between the

agglomerated state of particle types and the pulmonary

response. This is consistent with other study results sug-

gesting that the primary particle size and total surface

area of the instilled particles correlate better with the pul-

monary response [Duffin et al., 2007; Saber et al., 2012b,

2013, 2014]. It is known that the agglomeration state of

TiO2NPs and CB NPs are driven by the dispersion vehi-

cle. Thus, although the agglomeration states of TiO2NPs

govern pulmonary deposition and distribution patterns,

they do not appear to influence the biological response.

Pulmonary Acute Phase Response

One of the genes commonly up-regulated in response

to all of the different types of TiO2NPs tested in the pres-

ent study is Saa3, an acute phase response gene whose

expression levels in lungs increase in response to various

stimulants that cause pulmonary inflammation. Upregu-

lated Saa3 levels are specifically used as a marker of pul-

monary acute phase response. Previously, we have shown

large increases in Saa3 expression following inhalation

and intratracheal exposure to TiO2NP and other types of

particles [Halappanavar et al., 2011; Bourdon et al., 2012;

Jackson et al., 2012; Poulsen et al., 2013; Saber et al.,

2013]. Acute phase response is a risk factor for cardiovas-

cular disease [Saber et al., 2014]. Blood levels of SAA

are predictive of cardiovascular disease in prospective

epidemiological studies [Ridker et al., 2000]. Interest-

ingly, a positive correlation was observed between pulmo-

nary Saa3 mRNA levels and the total instilled surface

area of carbon black Printex90 and TiO2NP20.6 [Saber

et al., 2014], and between Saa3 mRNA levels and pulmo-

nary neutrophil influx [Saber et al., 2013, 2014]. Further-

more, it was shown that increased pulmonary Saa3expression was accompanied by increased serum levels of

SAA [Saber et al., 2013]. These results link the total

deposited surface area of particles with pulmonary acute

phase response and, in turn with risk of cardiovascular

disease. Pulmonary Saa3 expression levels may therefore

be used as a marker of particle-induced inflammation,

acute phase response, and risk of cardiovascular disease.

Thus, we assessed the correlation between Saa3 mRNA

levels and the total instilled BET surface area of

TiO2NP10.5, TiO2NP38, TiO2NP20.6, TiO2NP10, and

TiO2NP101. The results (Fig. 5) show that primary parti-

cle size is a strong predictor of the pulmonary acute

phase response. These results also suggest that

TiO2NP101 is likely to be more potent than the other free

forms of TiO2NPs. However, studies involving a library

of TiO2NPs of the same size or surface functionalization

derived from different sources are required to confirm

these results.

Responses to Paint-Embedded TiO2NPs

Similar to the small, free forms of TiO2NPs, response

to paint-embedded TiO2NPs appears to be influenced by

the primary size of the embedded particles. The SD-

TiO2NP38 paint dust that contained only TiO2NP38 did

not induce significant mRNA changes across doses and

timepoints, and only the highest dose of SD-

TiO2NP10.5138 caused any substantive changes in gene

TABLE V. The Results of the Multiplex ELISA Assay Measuring Cytokine Levels (Fold Increase Compared With MatchedVehicle-Treated Controls) in Lung Tissues 24 hr Post Exposure

Protein Name TiO2NP10.5 TiO2NP38 TiO2NP10 TiO2NP101 SD-TiO2NP38110.5 SD-TiO2NP38

IL-1b 1.47a 1.47a 1.99 1.71a 1.46a

IL-4 1.40a 1.74 1.46

IL-5 1.55a 1.86a 1.67

IL-6 2.39a 2.37a 2.11a 1.51 1.95a 2.28a

G-CSF 6.24a 4.03a 6.56a 3.08a 5.10a 2.74a

CXCL-1 6.58a 5.15a 4.99a 3.16a 6.75a 7.19a

CCL-2 2.19a 2.31 2.46a 2.31 1.98 2.26a

CCL-3 8.19a 6.98a 3.42a 2.73 7.92a 5.13a

CCL-4 1.76a 1.87a 1.56a

aIndicates statistically significant (P < 0.05).

Environmental and Molecular Mutagenesis. DOI 10.1002/em

14 Halappanavar et al.

Fig. 4. Hyperspectral imaging and Spectral Angle Mapping to detect

free TiO2NPs and matrix-embedded TiO2NPs in lungs. This figure shows

the results of VNIR hyperspectral imaging analysis of Free TiO2NPs

(TiO2NP10.5), paint-embedded TiO2NPs (TiO2NP10.5138) suspended in dis-

persion vehicle, lung tissue samples from mice exposed to 162 mg of free

or paint embedded TiO2NPs harvested 24 hr and 28 d post-exposure

along with lung tissues from matched controls. In (A–C), representative

spectra from reference libraries are shown on the left. For each spectrum,

a hyperspectral image and corresponding spectral angle mapping (SAM)

is shown on the right. White/gray refers to TiO2NP, red refers to paint,

and blue refers to lung tissue. TiO2NP in exposed lung tissues 24 hr and

28 d postexposure are shown in (D and E). Hyperspectral mapping of

TiO2NP and paint matrix in lung tissue 1 and 28 days postexposure is

shown in (F and G).

expression (72 DEGs at 24 hr). For comparison, the high-

est dose (486 mg) of SD-TiO2NP10.5138 contains 58 mg of

the 10 nm TiO2NP10.5 (comparable to 54 mg dose of

TiO2NP10.5) and 117 mg of 38 nm TiO2NP38. The total

number of DEGs altered in the 486 mg dose of SD-

TiO2NP10.5138 corresponds to the total number of DEGs

in the 54 mg dose of TiO2NP38. Since the large TiO2NP38-

caused only a subtle response in its free form and since

the highest dose of SD-TiO2NP38 (consisting of �162 mg

of large TiO2NP38) did not yield any response, response

to SD-TiO2NP10.5138 may thus be due to the presence of

TiO2NP10.5 with the rest of the paint matrix contributing

little to the response. Overall, our results indicate that

responses to matrix-embedded TiO2NPs are influenced by

the primary size of the particles. SD, which did not con-

tain any TiO2NPs induced little changes in gene expres-

sion with 1 differentially regulated gene after 24 hr and

15 after 28 days suggesting that the paint matrix itself

induces little response.

Saber et al. [2012a, 2012b, 2012c] previously investi-

gated inflammatory and genotoxic responses in the mice

used in this study following intratracheal instillation of

the free TiO2NP20.6, SD-TiO2NP20.6 (consisting of 10%

TiO2NP20.6) or SD devoid of NPs. Inflammatory

responses to paint-embedded TiO2NP20.6 were lower per

mass unit than effects induced by free TiO2NP20.6, and

there was no difference between the SD-TiO2NP20.6 and

SD, suggesting that the paint matrix masked the responses

of free TiO2NPs. No genotoxicity was observed [Saber

et al. 2012a, 2012b, 2012c]. Wohlleben et al. [2011]

reported no additional toxicity in rats following exposure

to sanding dusts of thermoplastics and concrete contain-

ing carbon nanotubes compared to the toxicity induced by

the reference test materials without nanomaterials [Wohl-

leben et al., 2011]. The authors of these studies proposed

that free forms of particles are more toxic than matrix-

bound and suggested that the matrices in which the par-

ticles are embedded contribute to the toxicity rather than

the particles themselves.

NPs have been suggested to undergo transformation

when embedded in matrices or dissociate from the matrix

[Kaegi et al., 2008, 2010] during the process of sample

preparation or after the exposure, all of which may alter

their toxic potential. Hyperspectral analysis of lung sec-

tions exposed to free or matrix embedded particles did

not show any alterations between the spectral profiles col-

lected from these samples (Fig. 4). Moreover, particles

were observed to be matrix bound at all times (Figs. 4f

and 4g), which was also implied by [Saber et al., 2012c]

in their study. Thus, the observed effects following expo-

sure to SD-TiO2NP10.5138 were not the result of trans-

formed particles.

A Model for TiO2NP Induced Pulmonary Inflammation

The results of our study show that the major acute

inflammatory mediators induced were the same across the

TiO2NPs studied, suggesting that similar mechanisms

operate across the particle types; however, the magnitude

of the change in their expression varied from NP to NP.

DEGs from the top five significant pathways (granulo-

cyte/agranulocyte adhesion and diapedesis, role of IL-17F

in allergic inflammatory airway diseases, LXR/RXR acti-

vation, and acute phase signaling) and upstream regula-

tors were used along with published knowledge to

propose a working model to explain how TiO2NPs may

Fig. 5. Correlation between the gene expression changes of Saa3 and instilled TiO2NP surface areas. Fold change of

pulmonary Saa3 mRNA expression levels relative to the corresponding vehicle controls are depicted against the total

instilled surface area of TiO2NP10.5, TiO2NP38, TiO2NP10, TiO2NP101, and TiO2NP20.6. [Color figure can be viewed in

the online issue, which is available at wileyonlinelibrary.com.]

Environmental and Molecular Mutagenesis. DOI 10.1002/em

16 Halappanavar et al.

induce inflammation. DEGs from the highest dose were

used to construct a model of TiO2NPs induced pulmonary

inflammation. As schematically shown in Figure 6, imme-

diately after exposure to TiO2NPs (inducers of inflamma-

tion), resident tissue macrophages in lungs are activated,

releasing proinflammatory mediators such as Il-1 and Il-6

into the circulation. Il-6 and Il-1 are proposed to engage

the tissue neuroendocrine axis during the acute phase

response via tissue acute phase signaling, subsequently

leading to glucocorticoid release from the adrenal glands,

which will activate production of cytokine receptors

including Il-6 and Il-1, and acute phase reactants such as

the Saa family of genes [Gruys et al., 2005]. This facili-

tates cytokine receptor-mediated signaling involving

inflammatory mediators and sensors such as Il-1, Il-6,

IFNg, etc. An activated cytokine-mediated signaling cas-

cade will trigger leukocyte migration to the tissue sites of

exposure or injury via the granulocyte/agranulocyte adhe-

sion and diapedesis pathway, resulting in additional secre-

tion and activation of various adhesion molecules,

chemokines, cytokines, and their receptors. During the

process, numerous proinflammatory transcription factors

are activated including NFjB and D site of albumin pro-

moter binding protein, which will further contribute to the

synthesis and release of acute phase reactants, inflamma-

tory cytokines, and molecules involved in extracellular

matrix recognition and digestion. Excessive synthesis and

activation of a multitude of cytokines, chemokines, and

acute phase proteins will eventually lead to infiltration of

inflammatory cells (macrophages, eosinophils, neutro-

phils/granulocytes, and others) into the lung space and a

cascade of inflammation is initiated [Medzhitov, 2008].

An increased neutrophil count is routinely reported in

response to several types of NPs including TiO2NPs in

this study [Saber et al. unpublished]. Thus, acute

responses to TiO2NP exposure follow a similar mecha-

nism-of-action.

A similar analysis at 28 d postexposure revealed that

the transcriptional response at a later timepoint was char-

acterized primarily by reduced NFkB-mediated

Fig. 6. Proposed mechanism of action for TiO2NP-induced pulmonary

inflammation. Individual DEGs from each dataset associated with the top

five significantly altered canonical pathways were used in the construction

of the pathway. Fold changes are colored to show the magnitude of the

response to each of the TiO2NPs. Gradients of green indicate down-

regulation and gradients of yellow show up-regulation. Dark red and

brown colors indicate fold changes >30. Each column in the heat map

indicates specific TiO2NP. From left to right: TiO2NP10.5, TiO2NP38,

TiO2NP10, TiO2NP101, SD - TiO2NP10.5138, and SD-TiO2NP38. Mole-

cules in purple refer to predicted activation of membrane receptors and

upstream regulators. Black arrows refer to experimental observations from

this study, and blue arrows refer to the observations from the literature.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Genomic Responses of Titanium Dioxide Nanoparticles 17

proinflammatory signaling resulting in decreased expres-

sion of cytokines, chemokines, matrix proteinases, and

acute phase reactants. In contrast, genes associated with

the pattern recognition receptors (PRR) pathway were

increased (2-5-oligoadenylate synthatase 1 and C-type

lectin domain family 1 genes), which can be activated in

response to pathogen stimulation (reviewed in [Lee and

Kim, 2007]). In addition, a prominent inducer of IFN

responsive signaling, transcription factor IRF-7 was up-

regulated. IRF-7-mediated type-1 interferon signaling is

known to contribute to innate immune response induced

by PRR [Honda et al., 2005]. Upstream regulator analysis

predicted activation of several TLRs involved in PRR-

mediated activation of IRF-7 and thus IFN signaling.

Decreased expression of SAA and MMPs, and increased

expression of PRR genes, is consistent with type-1 inter-

feron signaling [Lee and Kim, 2007]. Increased expres-

sion of IRF-7 and PRR genes has been observed

following pulmonary exposure to silica particles or gas

metal arc stainless steel welding fume [Erdely et al.,

2012]. However, IRF-7 activation was not observed in

lung tissues exposed to carbon nanotubes, suggesting that

IRF-7 mediated IFN-gamma signaling could be metal-

specific [Erdely et al., 2012]. Although IRF-7-mediated

interferon signaling is involved in promoting inflamma-

tion, it is also implicated in immunosuppression. There-

fore, it can be hypothesized that at 28 d post-exposure

IRF-7 is negatively regulating inflammatory processes to

aid in restoring homeostasis, which is supported by the

down-regulation of several inflammatory molecules. In

contrast, IRF-7 could be induced as a compensatory

mechanism in response to reduced NF-KB mediated

inflammatory signaling. This hypothesis agrees with sig-

nificant particle retention in lungs 28 d postexposure (Fig.

4 also reported by [Husain et al., 2013]) with no evidence

of inflammation. IRF-7-induced IFN signaling could be a

cellular effort to reinitiate inflammatory cascades to

remove retained particles. Further studies involving

chronic exposures and sampling at later postexposure

time points are required to confirm the implications of

activating IRF-7 mediated IFN signaling.

Overall, our pathway analysis suggests that TiO2NPs

with different properties induce pulmonary inflammation

via common mechanisms. Based on our observations,

gene expression changes recorded at 24 hr postexposure

accurately reflect the high neutrophil influx and inflamma-

tory phenotype described for TiO2NPs. While the long

term implications of activation of this process and the

consequences of retention of TiO2NP in lungs is not

clear, we propose that candidate markers of inflammation

identified in this study can serve to discern the inflamma-

tory TiO2NPs from the inert TiO2NPs without a need for

extensive confirmation of inflammatory phenotype involv-

ing tissue histology or differential cellular counts in the

BAL fluid.

CONCLUSION

In this study, we show that although different TiO2NPs

types caused differences in the magnitude of gene and

protein expression changes, the pathway perturbations

were similar across the particles suggesting that similar

mechanisms are operating in producing pulmonary

inflammatory phenotypes. We also demonstrate that pri-

mary size and the total deposited surface area of the par-

ticles contribute significantly to the overall magnitude of

the gene expression changes induced by TiO2NPs.

Changes in the surface characteristics of small sized par-

ticles, such as the addition of positively charged amino

groups, can further enhance their potential to induce

changes in inflammatory pathways, whereas, embedding

of TiO2NPs in paint dampens the overall inflammatory

pathway response.

ACKNOWLEDGMENTS

The authors thank the Danish Coatings and Adhesives

Association, and especially the following Danish paint

companies who kindly supplied the painted boards and

UV-Titan L181: Fl€ugger A/S, Boesens Fabrikker ApS,

and Beck&Jørgensen A/S.

AUTHORCONTRIBUTIONS

ATS, UV, HW, and NRJ planned animal experiments,

conducted animal exposures, collected samples. KAJ,

ML, RA, VS, RKB, AMM, and HW characterized the

materials, analyzed, and interpreted the characterization

data. CG, JR, and DW were responsible for the microar-

ray experiments. ND conducted microscopic analysis and

assisted in revising the manuscript. AW conducted the

statistical analysis. SH acquired funds for the study,

designed all microarray experiments, analyzed, and inter-

preted the microarray data, wrote the manuscript. CY

contributed to the design and analysis of microarray

experiments, and preparation of the manuscript.

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