Toxicological Effects of Titanium Dioxide Nanoparticles: A Review of In Vivo Studies

(1)

Volume 2012, Article ID 964381,36pages doi:10.1155/2012/964381

Review Article

Toxicological Effects of Titanium Dioxide Nanoparticles:

A Review of

In Vivo

Studies

Ivo Iavicoli, Veruscka Leso, and Antonio Bergamaschi

Istituto di Medicina del Lavoro, Universit`a Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy Correspondence should be addressed to Ivo Iavicoli,[email protected]

Received 2 October 2011; Accepted 14 February 2012 Academic Editor: Paul A. Schulte

Copyright © 2012 Ivo Iavicoli et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The essence of nanotechnology is the production of nanoparticles (NPs) with unique physicochemical properties allowing worldwide application in new structures, materials, and devices. The consequently increasing human exposure to NPs has raised concerns regarding their health and safety profiles. Titanium dioxide (TiO2) has been reported to induce adverse pulmonary responses in exposed animals. However, the potential more dangerous biological activities of TiO2NPs compared to their fine-sized counterparts are not fully understood. Therefore, this work is aimed to provide a comprehensive evaluation of the toxic eﬀects induced by TiO2NPs in in vivo experiments. It is intended to deeply understand the toxicological behaviour of TiO2NPs and to predict potential human health eﬀects. Moreover, it may be an instrument to extrapolate relevant data for human risk evaluation and management and to identify those critical aspects that deserve great attention in future population and epidemiologic research.

1. Introduction

Nanotechnology is the manipulation of matter on a near-atomic scale to produce new structures, materials, and devises. It has became an important industry in the twenty-first century, and the U.S. National Science Foundation esti-mated it will grow into a trillion-dollar business, employing millions of workers worldwide, within the next decade [1–3]. The essence of nanotechnology is the synthesis of engineered nanoparticles (NPs) that exhibit characteris-tics, such as small size, large surface area to mass ratio, shape, crystallinity, surface charge, reactive surface groups, dissolution rate, state of agglomeration, or dispersal that confer them properties substantially diﬀerent from those of the bulk particles of the same composition [4–6]. These properties oﬀer great opportunities for the development of new NP industrial applications increasing their worldwide distribution and enhancing the likelihood of environmental

and human exposure [5,7].

Considerable work has been carried out to advance nan-otechnology and its applications. Nevertheless, our under-standing of the general and occupational health and safety

aspects of NPs is still in its formative phase and greater eﬀort

is needed to understand how NPs interact with the human

body [8,9]. In this regard, nanotoxicology and nano-risk

have been attracting the increasing attention of toxicologists and regulatory scientists [10] particularly in relation to the unique properties of NPs that may render them potentially more dangerous than their fine-sized counterpart and may

cause unexpected adverse health eﬀects to exposed people

[6,11].

Titanium dioxide (TiO2) is an example of a fine, white,

crystalline, odorless, low-solubility powder which was con-sidered to exhibit relatively low toxicity [12–16]. It is a natu-ral, thermally stable and nonflammable, nonsilicate min-eral oxide found primarily in the form of the minmin-erals rutile, anatase, brookite, and as the iron-containing mineral ilmenite [17–21]. It has excellent physicochemical properties, such as good fatigue strength, resistance to corrosion, machineability, biocompatibility, whitening and photocatal-ysis, as well as excellent optical performance and electrical

properties [22, 23]. With regard to its potential adverse

health eﬀects, several studies have defined TiO2, at least

under nonoverload conditions, as biologically inactive and physiologically inert in both humans and animals and thus as little risk to humans [24–27]. Pulmonary inflamma-tion, fibrosis, epithelial hyperplasia, and tumorigenesis were

(2)

particle lung burden due to suﬃciently high dose and/or

duration of exposure [13,15,28,29]. In 2006, the

Interna-tional Agency for Research on Cancer (IARC), classified and

in 2010 reassessed TiO2as “possibly carcinogenic to humans”

on the basis of the suﬃcient evidence of carcinogenicity in experimental animals and inadequate evidence in humans

(Group 2B) [28,29].

TiO2is a versatile compound that has broadly been used

in nanoparticulate form [21]. According to the National

Nanotechnology Initiative of America, nanosized TiO2

parti-cles are among those most widely manufactured on a global

scale [30]. TiO2 NPs are widely used in paints, printing

ink, rubber, paper, cosmetics, sunscreens, car materials, cleaning air products, industrial photocatalytic processes, and decomposing organic matters in wastewater due to their unique physical, chemical, and biological properties (including the inherent advantages of physical stability, anticorrosion and nanoscale-enhanced photocatalysis) [31].

However, the toxicological profile of TiO2 NPs is not

completely understood and several concerns have emerged

on the potential undesirable eﬀects of the TiO2 NP

prop-erties, in regard to the harmful interactions with biological systems and the environment [11]. The recently recognized

occupational carcinogenic potential of the inhaled TiO2NPs

have ulteriourly enhanced these scientific concerns [15]. Therefore, an appropriate assessment of the risks for the

general and occupational exposed population requiring TiO2

NP hazard identification and dose-response data seems necessary [32].

In this regard, our previous work [33] reviewed several

toxic eﬀects of NPs assessed by in vitro experiments. These

studies represent a valid instrument to investigate TiO2NP,

induced cellular changes at biochemical and molecular levels and to determine their underlying mechanistic processes. Moreover, providing interesting information about doses of exposure and endpoint parameters to evaluate, this kind of investigation constitutes a significant step in planning animal research. Unfortunately, several limitations inherent to in vitro assay/cell culture systems have appeared in simulating the complex biological eﬀects of particles administered to experimental animals. These include, but are not limited to, unrealistic particle dose, selection of cell types for simulating the diﬀerent system microenvironments (single-cell culture systems or coculture systems), particle/cell exposure

interac-tions in culture versus biological fluids, time course of eﬀects

represented by 1-, 4-, 24-, or 48-hour incubation in vitro versus acute or chronic exposure in vivo, and appropriate end points for hazard evaluation [34]. These biases do not allow to obtain data that may be representative of human

exposure/eﬀects and applicable for a correct population risk

assessment.

Therefore, in the present review, we will provide a com-prehensive evaluation of the current knowledge regarding

the toxic eﬀects induced by TiO2 NPs on organ systems

investigated in in vivo studies. This overview is intended to be a useful tool to gain a thorough insight into the

toxicological profile of TiO2 NPs and to predict potential

human health eﬀects. Moreover, it may reveal itself as a valid instrument to extrapolate relevant data for human risk

evaluation and management and to identify those critical aspects that deserve great attention in future population and epidemiologic research.

2. In Vivo

Studies

2.1. Respiratory System. Considering the relevance of the

respiratory system as a route for TiO2 NP exposure in

humans, the scientific community has focused on the toxicological eﬀects of these NPs on animal respiratory models. Such experiments provide the basis to obtain more detail data regarding the hazard of these NPs and to extrapolate evaluations for a correct human risk assessment, in particular, in relation to the characteristics of the particles. Indeed, several animal pulmonary studies were carried out

to clarify the role played by TiO2form, particle size, shape,

surface area, and chemistry in determining inflammatory responses, particle lung distribution, and carcinogenic eﬀects (Table 1).

2.1.1. Inflammatory Responses

TiO2Form. Regarding the TiO2form, diﬀerent studies have

demonstrated the pulmonary toxicity of acute exposure to anatase in terms of increased bronchoalveolar lavage (BAL) inflammatory parameters [35–38], lung tissue structural

damage, and inflammatory infiltration [22,35,36,39,40]

although it is modest in both acute and subacute exposure [41]. However, as assessed by Liu et al. [36], only a slight increase in toxicity from anatase exposure was evident

relative to rutile and P25 Degussa TiO2NP treatments.

Only four studies have evaluated the ability of rutile TiO2

to induce toxic eﬀects on lung tissues, reporting conflicting

results following acute [42–45] or subchronic exposure [46]. While the first two studies demonstrated an increase of inflammatory cells in BAL [43], alterations in gene expression [42], and pathological changes in lung tissue

[42,43], the latter two failed to reveal such changes [44–46].

When comparing the lung toxicity induced by rutile NPs with that caused by P25 Degussa NPs, the former were not able to induce alterations of the lung parameters examined

suggesting greater toxic eﬀects of the mixture compared

to rutile alone [44, 45]. Regarding the toxic eﬀects of P25

Degussa NPs on the lung, other studies have reported that these particles are able to induce increased inflammatory parameters in the BAL of acutely exposed animals [47, 48], though some interspecies diﬀerences in inflammatory responses were evident in animals subchronically treated [49]. The species diﬀerences detected between hamsters, rats, and mice may reflect the capacity of some animals to rapidly clear particles from the lung, as previously demonstrated in the same species subchronically exposed to pigmentary

fine TiO2 particles [50,51]. Interestingly, Gustafsson et al.

[48] demonstrated a dynamic inflammatory response to P25 Degussa NPs characterized by a transient innate immune activation followed by a late-phase, long-lasting recruitment of lymphocytes involved in adaptive immunity.

(3)

Ta b le 1: In vi vo studies that in vestigat ed the ad ve rse eﬀ ects of T iO 2 NP s o n respir at or y syst em. R espir at or y syst em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals Re su lt s T ak enaka et al., 1986 [ 52 ] A n atase T iO 2 (15–40) Inhalat ion: aer o sols of 8.6 m g/m 3Ti O2 for 7 hr/da y, 5 da y/wk for u p to 1 year . 24 female W istar ra ts L ung bur d en: Ti O2 was found in macr o p hages and in the p er iv ascular spac es. F er in et al., 1992 [ 53 ] Ru ti le T iO 2 (12; 230) A n atase T iO 2 (21; 250) In tr at ra ch eal inst illat ion :a ll T iO 2 : 500 μg; 21 nm T iO 2 :65–1000 μg/r at; 250 nm T iO 2 :1000 μg/r at. Inhalat ion: 23 .5 ± 3. 2m g/ m 321 nm Ti O2 ;2 3. 0 ± 4. 1m g/ m 3250 nm T iO 2 for 6 h /d ay ,5 d ay /w k fo ru pt o1 2 w k s. In st illat ion stud y :4 – 8 male Fisc her 344 ra ts Inhalat ion stud y :4m al e Fisc her 344 ra ts per gr o up . In st illat ion stud y :unla vagable lung bur d en decr eased fr om 12 nm to 250 nm par tic les. Inhalat ion stud y :g reat er 21 nm T iO 2 NP co nc ent ration in the int erstitium, epithelial ce ll s, an d alve o la r sp ace . Basic int ra tr ac heal inst illat ion: 500 μgo f 250 and 2 0 n m T iO 2 . Basic inst illat ion: 4m al e Fisc her 344 ra ts per gr o up . Basic inst illat ion: hig her lung re tained amount and gr eat er inflammat o ry re sponse in the 2 0 n m T iO 2 NP tr eat ment. O b er dorst er et al., 1992 [ 54 ] A n atase T iO 2 ( ∼ 20; ∼ 250) Ru ti le T iO 2 ( ∼ 12; ∼ 220) In tr at ra ch eal reinst illat ion: 104 ± 8 μg 20 nm fr ee T iO 2 ,104 ± 10 μg ,20 nm T iO 2 phagocitiz ed b y AMs, 100 μgs er u m co at ed par tic les, 6. 8 × 10 6AMs, 2. 2 × 10 6PMNs. R einst illat ion: 4–10 male Fisc her 344 ra ts per gr o up . R einst illat ion: gr eat er amount of fr ee par tic les retained in la vaged lung s. In tr at ra ch eal d ose -re sp on se :500, 1000 μg 250 nm T iO 2 ;65–1000 μg2 0 n m Ti O2 ;500 μg 220 nm T iO 2 ;500 μg1 2 n m Ti O2 . Dose-r esp o nse: 20 nm T iO 2 NP s caused a d o se-dependent PMN lung influx. O b er dorst er et al., 1994 [ 55 ] A n atase T iO 2 (20; 250) Inhalat ion: aer o sols of 23 .5 ± 2. 9m g/ m 3 20 nm T iO 2 and 2 2. 3 ± 4. 2m g/ m 3 250 nm T iO 2 for 6 hr/da y, 5 da y/wk for u p to 1 2 w k s. 64 male Fisc her 344 ra ts Inflammat o ry act ion: AMs, PMNs, T PC incr eased in B A L. P u lmonar y re tent ion :incr eased aft er 2 0 n m Ti O2 . Hi st o lo g y :l ung fibr o sis aft er 20 nm T iO 2 . H einr eic h et al., 1995 [ 56 ] P25 D egussa T iO 2 (15–40) Inhalat ion: 10 mg/m 3Ti O2 for 1 8 h r a da y for 24 (r ats) and 13.5 (mic e) m onths. 100 female W istar ra ts 80 NMRI mic e Rats: adenocar cinomas, squamous ce ll car cinomas, adenomas incr eased. Mi ce :no di ﬀ er enc es in tumor ra te s. Bagg s et al., 1997 [ 57 ] Pig m ent-g rade T iO 2 (250) Degussa T iO 2 (20) Inhalat ion: aer o sols of 22.3 m g/m 3of 250 nm T iO 2 and 23.5 m g/m 3of 20 nm Ti O2 for 6 hr/da y, 5 da y/wk for u p to 12 wks. 3-4 m ale F isc h er 344 ra ts pe r gr o up Hi st o lo g y :al ve olar epithelial thic kness and septal fibr o sis aft er both T iO 2 tr ea tm en ts . Zhang et al., 1998 [ 58 ]T iO 2 (28) In tr at ra ch eal inst illat ion :1 m g T iO 2 . 4–6 m ale W istar rats p er gr o u p Inflammat o ry act ion: T C C, NEU s, A Ms, TPC, and L DH incr eased in B A L. A faq et al., 1998 [ 59 ]T iO 2 (30) In tr at ra ch eal inst illat ion :2 m g T iO 2 pe r animal. 6 female A lbino ra ts per gr o u p Inflammat o ry act ion: AMs, A C P, and L DH incr eased in B A L. Oxidat iv e st re ss: LPO and antio xidant acti vi ties incr eased.

(4)

Ta b le 1: C o ntin ued. R espir at or y syst em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals Re su lt s O b er dorst er et al., 2000 [ 60 ] Ti O2 (20; 250) In tr at ra ch eal inst illat ion :n om o re de tailed Rats M ic e Inflammat o ry act ion: NEU s incr eased aft er 20 nm T iO 2 . H ¨ohr et al., 2002 [ 61 ] Na ti ve o r m et h yl at ed T iO 2 (20–30; 180) In tr at ra ch eal inst illat ion :e q u iv al en t mass (1 or 6 m g) and sur fac e doses (100, 500, 600 and 3000 cm 2)o fT iO 2 pe r animal. 5 female W istar rats p er gr o u p Inflammat o ry act ion: nati ve T iO 2 NP s incr eased T CC, PMNs, T PC, A LP ,L DH, GGT ,N A G and β gl ucur onidase in B A L. Ber m udez et al., 2004 [ 49 ] P25 D egussa T iO 2 (21) Inhalat ion: aer o sols of 0.5–10 mg/m 3 Ti O2 for 6 hr/da y, 5 da ys/w eek, for 1 3 we ek s. G roups of 25 animals for eac h species: F emale 3C3F1/C rlBR m ic e; CDF (F344)/C rlBR ra ts; L ak:L V G (S Y R )B Rh am st er s. L ung bur d ens: equi valent in ra ts and m ic e, lo w er in h amst ers. Inflammat o ry act ion: incr ease in T CC, AMs, NEU s, LYMs (r ats and m ic e); T PC and LDH in B AL (r ats); o nly N EU s in h amst ers; Hi st o lo g y :t er m inal b ro nc hiolar and al ve olar cel l replication. R en w ic k et al., 2004 [ 62 ] Ti O2 (29; 250) In tr at ra ch eal inst illat ion :125, 500 μg Ti O2 . Ma le W is ta r ra ts Inflammat o ry act ion: NEU s, GGT ,T PC, LDH incr eased aft er 2 9 n m T iO 2 in B A L. AM phagocy tot ic abilit y :d ecr eased. W ar h eit et al., 2006 [ 39 ] R u tile pig ment gr ade T iO 2 (300) A n atase T iO 2 ro ds (length: 92–233; w ide: 20–35) A n atase T iO 2 dots (5.8–6.1) In tr at ra ch eal inst illat ion :1 ,5 m g/ k g Ti O2 . 5 m ale C rl:CD(SD)IGS BR ra ts pe r gr o u p Inflammat o ry act ion: incr ease in N EU s (al l par tic les) and L DH (T iO 2 ro d s) . Hi st o lo g y :Ti O2 -c ontaining agg regat ed macr o p hages in lung (all par tic les). W ar h eit et al., 2007 [ 44 , 45 ] ∼ 98% ru tile T iO 2 ,2 % al u mina ( ∼ 100) ∼ 88 w t% rutile T iO 2 , ∼ 7w t% amor phous silica, ∼ 5w t% al umina ( ∼ 100) P25 D egussa T iO 2 ( ∼ 25) ∼ 99% ru tile T iO 2 , ∼ 1% al umina (382 in wat er) In tr at ra ch el inst illat ion: 1, 5 m g/kg Ti O2 . 4-5 m ale C rl:CD(SD)IGS BR ra ts per gr o up Inflammat o ry act ion: NEU s, L DH, and micr o p ro te in incr eased in B A L b y P 25 T iO 2 Cell pr olifer at ion: air w ay and lung par enc h ymal cells pr olifer ation incr eased aft er P 25 T iO 2 . Hi st o lo g y :AM ac cum u latio n w ith tiss ue thic ke ning aft er P 25 T iO 2 . Chen et al., 2006 [ 42 ] Ru ti le T iO 2 (21) Ti O2 (180–250) In tr at ra ch eal inst illat ion :0 .1, 0 .5 mg Ti O2 . 6m al eI C R m ic e p erg ro u p L ung mor p holog y :emph ysemat ous change and al veolar epithelial thic kness aft er 21 nm Ti O2 NP s. A p o p to sis: incr eased in AMs and PNEs. G rassian et al., 2007 [ 41 ] A n atase T iO 2 (3.5 ± 1.0) Inhalat ion: aer o sols of 0.77, 7.22 mg/m 3 Ti O2 for 4 hr (acut e ex posur e); and 8. 88 ± 1. 98 mg/m 3Ti O2 for 4 hr/da y for 10 da ys (subacut e exposur e). 6m al eC 5 7 B l/ 6m ic ep er gr o u p Ac u te ex p o su re :T C C and AMs incr eased in BA L . S u bacut e exp o sur e: AMs incr eased in B A L. Li et al., 2007 [ 35 ] A n atase T iO 2 (3) Ti O2 (20) In tr at ra ch eal inst illat ion :0 .4–40 m g/kg Ti O2 . 5 M ale K unming mic e per gr o u p B A L b ioc h emical par amet ers: TPC, ALB , ALP ,and A CP incr eased b y both T iO 2 . Hi st o lo g y :al ve olar w all thic ke ning and dest ru ction.

(5)

Ta b le 1: C o ntin ued. R espir at or y syst em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals Re su lt s Geiser et al., 2005 [ 63 ]T iO 2 (4) Inhalat ion: 1 0 ra ts were ex p o se d to 0.11 mg/m 3Ti O2 aer o sols for 1 hr . 10 male WKY/NC rl B R rats Ti O2 dist ri but ion: lu minal side o f air w ay s/al ve oli (79.3%), epithelial or end o thelial cells (4.6%), connecti ve tissue (4.8%), capil lar ies (11.3%). M ¨uhlfeld et al., 2007 [ 64 ] See G eiser et al., 2005 [ 63 ] Ti O2 dist ri but ion: co nnecti ve tissue and the capillar y lu men w er e the pr efer ential target of NP s at 1 and 2 4 h r, re sp ec ti ve ly . N emmar et al., 2008 [ 43 ] Ru ti le T iO 2 nanor o ds (4–6) In tr at ra ch eal inst illat ion :1 ,5 m g/ k g Ti O2 6-7 m ale W istar rats p er gr o u p Inflammat o ry act ion: AMs, PMNs incr eased in B A L. T issue injur y :edema in lung and h ear t. Larsen et al., 2010 [ 65 ]T iO 2 (28) In tr ap er it oneal inject ions : imm unization w ith 1 μgO V A al o n eo ri n co mbination w ith either 2, 10, 50 or 250 μgT iO 2 . 7-8 inbr ed female B ALB/cJ mic e per gr o up O V A -sp ecific ant ibo dies in ser um: Ti O2 incr eased Ig E and Ig G1 le ve ls co mpar ed to the O V A -c ont rols. Inflammat o ry act ion: EOSs, N EU s, LY Ms, IL -4, and IL -5 incr eased in B A L. H amilt on et al., 2009 [ 37 ] A n atase T iO 2 nanospher es (60 ∼ 200) A n atase T iO 2 long nanobelts (w idth: 60 ∼ 300 nm; length 1 5 ∼ 30 nm) A n atase T iO 2 shor t n anobelts (w idth: 60 ∼ 300; length 0.8–4 μm) Phar yngeal aspir at ion :3 0 μg/mouse Ti O2 . M ale C57BL/6 m ic e Inflammat o ry act ion: cathepsins, IL -1 β, IL -18 incr eased aft er long n anobelts aspir ation. K o ba ya shi et al., 2009 [ 38 ] A n atase T iO 2 (4.9) (1st and 2nd ex per iment) A n atase T iO 2 (23.4) A n atase T iO 2 (154.2) In tr at ra ch eal inst illat ion :5 m g/ k gT iO 2. 5m al eC rl :C D( S D )r at s pe r gr o up Inflammat o ry act ion: 1st: 4.9 and 23.4 n m T iO 2 NP s incr eased T CC, NEU s, L DH; 2nd: agg lomer at ed T iO 2 incr eased T CC, NEU s, L DH. Hi st o lo g y :epithelium h yper tr o ph y in all tr eat ed gr oups. V an R ave n zw aay et al ., 2009 [ 66 ] A n atase-r u tile T iO 2 mixtur e (20–30) Ru ti le T iO 2 (200) Inhalat ion :a er osols of 100 and 250 mg/m 3unc oat ed and pig mentar y Ti O2 ,r esp ec ti vely for 6 h /da y on 5 co n se cu tive d ay s. In tr av enous inject ions :5 m g/ k g. 3–6 m ale W istar rats (st ra in C rl:WI (H an) per gr o up Ti O2 dist ri but ion: lung and m ediastinal ly mph n ode aft er inhalation; m ostly li ver and spleen aft er injection. Inflammat o ry act ion: both T iO 2 incr eased T CC, PMNs, T PC, A LP ,L DH, GGT ,N A G in BA L . M a-H oc k et al., 2009 [ 67 ] R u tile-anatase T iO 2 mixtur e (25.1) Inhalat ion :a er osols of 2–50 mg/m 3Ti O2 for 6 hr/da y for 5 da ys. 5-6 m ale W istar rats (st ra in C rl: WI (H an)) per gr o up Inflammat o ry act ion: PMNs, GGT ,T PC, LDH, ALP ,N A G incr eased in B A L. Cel l re plicat ion: incr eased in br onc h i and br onchioles.

(6)

Ta b le 1: C o ntin ued. R espir at or y syst em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals Re su lt s Liu et al., 2009 [ 36 ] A n atase T iO 2 (5) P25 D egussa T iO 2 (21) Ru ti le T iO 2 (50) In tr at ra ch eal inst illat ion :0 .5–50 m g/kg . 12 male and female Spr ague D aw le y rats p er gr o u p Inflammat o ry act ion: LDH and ALP incr eased in lung b y 5 and 50 nm T iO 2 . Hi st o lo g y :inflammat o ry infilt ration, al ve olar w all thic ke ning . AM phagocy tic abilit y :alt er ed b y 5 and 50 nm T iO 2 . Sager et al., 2008 [ 12 ] P25 T iO 2 (21) Ru ti le T iO 2 (1 μm) In tr at ra ch eal inst illat ion : 0.26–1.04 mg/r at for 2 1 n m T iO 2 and 5.35–21.41 mg/r at for 1 μmT iO 2 . M ale Fisc her C DF (F344/DuC rl) ra ts Inflammat o ry act ion: both T iO 2 incr eased PMNs, L DH, A LB ,T NF α,I L -1 β,M IP 2 . Oxidat iv e st re ss: incr eased b y both T iO 2 . Sager and Cast ra no va, 2009 [ 68 ] P25 T iO 2 (21) In tr at ra ch eal inst illat ion : 0.26–1.04 mg/r at T iO 2 . 8 m ale F isc h er CDF (F344/DuC rl) ra ts p er gr o u p Inflammat o ry act ion: incr ease in P MNs, ALB ,and L DH in B A L. P ar k et al., 2009 [ 69 ]P 2 5 T iO 2 (21) In tr at ra ch eal inst illat ion :5 ,20, 50 mg/kg T iO 2 . 10–12 ICR m ic e p er gr oup Inflammat o ry act ion :I L -1 ,T N F -α ,I L -6 , IL -12, IFN-γ, IL4, IL -5, IL -10, and IgE incr eased in B A L. Hi st o lo g y :inflammat o ry pr ot eins, gr an ulomas. Gene expr ession: upr egulation of genes in vo lv ed in antigen p re sentation and imm une ce ll ch emotaxis. Chen et al., 2009 [ 22 ] A natase T iO 2 (80–110) In tr ap er it oneal inject ions : 324–2592 mg/kg T iO 2 . 10 male and female ICR mic e per gr o up Hi st o lo g y :a lv eolar septal thic ke ning and int erstitial p ne umonia. M o on et al., 2010 [ 47 ]P 2 5 T iO 2 (21) In tr ap er it oneal inject ions :4 0 m g/ k g Ti O2 alone o r 3 0 m in aft er 5 mg/kg L PS injection. 10 B A LB/c mic e per gr o up Inflammat o ry act ion: N E U s,T P C ;T N F -α , IL -1 β, MIP2 incr eased b y T iO 2 or T iO 2 + LPS. R o ssi et al., 2010 [ 70 ] Ru ti le T iO 2 (< 5 μm) R u tile-anatase T iO 2 mixtur e (30–40) A n atase T iO 2 (< 25) Si lica co at ed ru tile T iO 2 ( ∼ 10 × 40) A n atase T iO 2 /br o okit e (21) Inhalat ion: 10 ± 2m g/ m 3Ti O2 for 2 hr ; 2h r o n 4 co n se cu ti ve d ay s; 2h r o n 4 co nsecuti ve d ay s for 4 w ks. 8 female B ALB/c/Sca mic e pe r gr o up Inflammat o ry act ion: NEU s in B A L, CX CL1 lung tissue, T NF -α in AMs incr eased by si li ca T iO 2 . R o ssi et al., 2010 [ 71 ] Si lica co at ed ru tile T iO 2 ( ∼ 10 × 40) Ru ti le T iO 2 (< 5 μm) Inhalat ion :1 0 ± 2m g/ m 3Ti O2 for 2 hr a d ay ,3d ay saw k ,f o r 4w k s. 8 female B ALB/c/Sca mic e pe r gr o up Inflammat o ry act ion: EOSs, LYMs, AMs, PA S + gl o b et ce ll s, IL -1 β,T N F -α ,I L -4, -13, -10 d ecr eased b y silica T iO 2 . Ai rw ay re ac ti v it y :decr eased b y silica T iO 2 ; incr eased b y fine T iO 2 Scur i et al., 2010 [ 72 ] P 25 Degussa T iO 2 (21) Inhalat ion :1 2 m g/ m 3Ti O2 for 5 .6 hr a da y, for 3 co nsecuti ve d ay s. 29 male and female F isc h er 344 ra ts N eur ot ro phin expr ession: NGF ,BDNF and their rec ept o rs incr eased in 2 d ay and 2 wk old ra ts. A ir w ay re sistanc e: incr eased in 2 w k old mic e. Li et al., 2010 [ 73 ] A natase T iO 2 (3) In tr at ra ch eal inst illat ion: 3.3 m g/kg Ti O2 onc e a w k for 4 w ks. 13 male K unming mic e per gr o u p Inflammat o ry act ion: A C P, ALP incr eased in B A L. Hi st o lo g y :dest ro ye d al veolar w al ls.

(7)

Ta b le 1: C o ntin ued. R espir at or y syst em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals Re su lt s Liu et al., 2010 [ 74 ] Ti O2 (5) Ti O2 (200) In tr at ra ch eal inst illat ion: 0.5–50 mg/kg Ti O2 3 m ale and 3 female Spr ague-Da wle y ra ts p er gr o u p AM phagocy tic and chemotact ic abilit y : re d u ce d b y T iO 2 NP s. T ang et al., 2010 [ 75 ]T iO 2 (5) In tr at ra ch eal inst illat ion: 0.8–20 mg/kg Ti O2 . 8 m ale S pr ague Da wle y ra ts pe r gr o up Ti O2 NP ag g re g at ion: pr esent in lung at the lo w est d o ses. Hi st o lo g y :l ung gaps ex panded, h yp er emia T ang et al., 2011 [ 40 ] A natase T iO 2 (5 ± 1) In tr at ra ch eal inst illat ion: 0.8–20 mg/kg Ti O2 . 8 m ale S pr ague Da wle y ra ts pe r gr o up Hi st o lo g y :AM incr ease, lung gaps ex panded, h yp er emia, al veolar thic kness. Cho et al., 2010 [ 32 ]T iO 2 (30–40) In tr at ra ch eal inst illat ion: 50 and 150 cm 2/r at 5 female W istar rats p er gr o u p Inflammat o ry act ion in B A L and hist olog y of the lung :n oe ff ect. L eppanen et al., 2011 [ 76 ] A n atase + B rookit e T iO 2 (20 n m) Inhalat ion: 8 – 30 mg/m 3for 0 .5 hr (acut e ex p o su re ); 3 0 m g/ m 3for 1 hr a d ay ,4 da ys a w k for 4 w ks (sub-c hr onic ex posur e) 4–6 m ale C rl:OF1 mic e p er gr o u p A ir fl o w limitat ion eff ect: re duction in ex pir at or y fl o w in all the ex posur e situations. Inflammat o ry act ion: no eff ect M o ri mot o et al., 2011 [ 46 ] Ru ti le T iO 2 (35) Inhalat ion :2 .8 ± 0. 9 × 10 5/cm 36 h r a da y for 4 w k s. 30 male W istar ra ts per gr o u p Inflammat o ry act ion: no eff ect. H o ugaar d et al., 2010 [ 77 ] R u tile UV -titan L181, modified w ith Zr ,S i, Al and coat ed w ith poly alc o hols (20 .6 ± 0. 3) Inhalat ion: 42 .4 ± 2. 9T iO 2 mg/m 3for 1 h r a da y, for 1 1 gestational co nsecuti ve da ys. F emale time-mat ed C57BL/6BomT ac m ic e Inflammat o ry act ion: incr eased NEU s, and LY Ms, decr eased AMs in B AL. H alappana var et al., 2011 [ 78 ] See H ougaar d et al., 2010 [ 77 ] Inflammat o ry act ion: Ti O2 incr eased mRN A for S aa1, S aa3, se ver al C-X-C and C-C m otif ch emokines, TNF -α . N emmar et al., 2011 [ 79 ] R u tile F e-d o p ed nanor o d T iO 2 (length: 80; diamet er :7) In tr at ra ch eal inst illat ion: 1,5 m g/kg Ti O2 4 m ale W istar rats p er gr o u p Inflammat o ry act ion: NEU s, IL -6 incr eased, SOD acti vi ty decr eased in B A L. Hi st o lo g y : inflammat o ry ce ll infilt ration. H u ssain et al., 2011 [ 80 ] A n atase T iO 2 (15) Or o p har yngeal aspir at ion : ∼ 0.8 m g/kg Ti O2 5-6 m ale B ALB/c mic e Ai rw ay re ac ti v it y :incr eased b y T iO 2 in TDI sensitiz ed mic e Inflammat o ry act ion: Ti O2 incr eased NEU s and A Ms in B A L o f T DI sensitiz ed mic e. G u stafsson et al., 2011 [ 48 ] P25 D egussa T iO 2 In tr at ra ch eal inst illat ion: 1, 5, and 7 .5 mg/kg T iO 2 5–20 male Dar k A gouti ra ts pe r gr o up Inflammat o ry act ion :t ra nsient incr ease in EOSs and N EU s in B AL, fol lo w ed b y a re cr uit ment of D C s and NKs. Ele vat ed le ve ls of IL -1, IL -2, IL -6, C INC-1, and G M-CSF . A C P, acid phosphatase; ALB , albumin; ALP , alkaline phosphatase; AM, al veolar m acr o phage; B A L, br onc h oal veolar la vage; BDNF , b ra in-der iv ed n eur o tr ophic fact o r; CINC-1, cy to kine induc ed neut ro phil ch emoatt ra ctant-1; CX CL -1, chemokine (C-X-C motif ) ligand 1; D C , d endr itic ce ll; N K, natur al k iller ; EOS, eosinophil; GGT ,γ -g lu tam yl tr anspeptidase; G M-CSF , gr an ulocy te -macr o phage colon y-stim ulating fact or ; Ig , imm u nog lobulin; IL -, int erleukin; IFN-γ, int erfer o n-γ; L DH, lactat e deh ydr ogenase; LPO , lipid per o xidation; L PS, lipopolysac ch ar ide; LY M, ly mphocy te ; M IP -2, m acr o phage-inflammat o ry pr ot ein-2; N A G, N-ac et yl-g lu co saminidase; N EU , n eut rophil; NGF , ner ve gr o w th fact o r; P A S, P er iodic A cid-Sc hi ff ; P MN, p oly m or phon uclear ; PNE, p neumocy te; Saa, ser u m am yloid A -; S OD , sup er o xide dism u tase; T iO 2 NP s, titanium dio xide nanopar tic les; T CC, to tal cell count; T DI, to lu ene diisocy anat e; T NF -α ,t umor necr osis fact or -α ;T PC, total pr ot ein cont ent.

(8)

Other mixtures of anatase and rutile particles were also able to induce increases in BAL fluid inflammatory parameters and lung histopathological alterations in rats

subacutely exposed by inhalation [66,67], while mixtures

of anatase and brookite (3 : 1) failed to induce such eﬀects in mice after acute, subacute, or subchronic inhalations

[70,76].

Other studies report similar changes after acute or suba-cute exposure, such as increased levels of inflammatory

mediators in BAL [58,59,62,73], enhanced reactive oxygen

species (ROS) production [59] and pathological alterations

at the histopathology examination of the lung [57,73,75]

but lack details regarding the TiO2form. Interestingly, Baggs

et al. [57] demonstrated that chronic exposure to TiO2NPs

induces lesions that are able to regress during a 1-year period following cessation of exposure. Surprisingly, Cho et al. [32], investigating the acute eﬀects of intratracheal instillations of

TiO2NPs, failed to demonstrate any alteration among BAL

inflammatory parameters and histological lung characteris-tics. Finally, Liu et al. [74] demonstrated that intratracheal

instillation of not characterized TiO2NPs damaged alveolar

macrophage (AM) phagocytic and chemotactic ability. Size. Several studies described greater inflammatory eﬀects of NPs compared to their fine counterparts both after acute

[38,53,54,62] and chronic exposure [53,55].

In line with these results on NP size-related eﬀects, a more recent intratracheal instillation study [38] pointed out that the smaller particles lead to greater inflammation in short-term observations. In contrast, no clear relationship was found between pulmonary inflammation and treatments

with diﬀerent agglomeration states of the same primary TiO2

NPs.

Shape. The critical role of shape in TiO2 NP bioactivity

is supported by the increased markers of inflammation detected in BAL of anatase nanobelt aspiration-treated mice compared with those determined in animals treated with

TiO2nanospheres [37].

Surface Area. High mass or volume dose of poorly soluble, low-toxicity (PSLT) fine particles in the lungs has been associated with overloading, while ultrafine particles impair lung clearance at lower mass or volume doses [15]. The increased lung retention and inflammatory response of nanosized PSLT particles compared to fine PSLT particles

correlate better to the particle surface area dose [81, 82].

Indeed, the larger inflammatory response after TiO2 NP

treatment compared to larger particles may be ascribed to an increase in NP surface area. This conclusion has been questioned, and conflicting results are present in literature on this topic. Some studies support the hypothesis that surface

area may be the more appropriate dose metric for TiO2

NP pulmonary toxicity studies [12,60,61]. When the same

mass doses were acutely introduced into rats and mice via

intratracheal instillation, TiO2NPs induced a much greater

pulmonary-inflammatory response than fine TiO2particles

[12,60]. However, when the doses were normalized to the

particle-administered surface area, the response in the lung

for both nanosized and fine TiO2particles showed the same

dose-response curve. Thus, in pulmonary toxicity studies, surface area, for particles of diﬀerent sizes but of the same chemistry, proved to be a better dosemetric parameter than particle mass or number.

However, other studies are in conflict with the notion that the inflammatory response is expected to be more

severe with higher surface area NPs [39, 44, 45]. In fact,

similar BAL cell count alterations were reported in rats after intratracheal administrations of the same dose of fine

rutile TiO2particles, nanosized TiO2anatase rods and dots,

although the latter had a >6-fold increase in surface area

compared with the nanorods [39]. In a subsequent study, the

same authors [44, 45] confirmed the previous evaluations

and concluded that having a larger surface area does not necessarily indicate that NPs will produce greater pulmonary inflammation and cytotoxicity compared to larger particles of a similar composition. In line with the Warheit et al. [39,

44,45] results, 2- to 5-nm anatase NPs (i.e., with the highest

surface area and smallest particle size) were not particularly toxic in the Grassian et al. [41] subacute inhalation study. Furthermore, in Li et al. [35], 3-nm anatase NPs did not elicit more pulmonary toxicity compared to 20-nm NPs, irrespective of their smaller size and greater surface area.

However, an aspect to take in consideration for a correct interpretation of these data is the potential influence of “lung overload” [13]. In the last decade, it has become clear that a breakdown in normal AM-mediated clearance of particles is seen in overload [83]. This is thought to be a consequence

of volumetric overload of the AM [84, 85] or a response

to the greater particle surface area per mass dose, as in the case of NPs, which is associated with decreased AM clearance

and inflammation [13,86]. In this regard, the importance

of the NP surface area overlaps with the role of the dose administered to rats. Most of the studies herein described

showed greater TiO2NP eﬀects at higher exposure doses [35,

36,39,40,43–45,48,49,61,62,65,67,79]. Unfortunately,

the large dose range applied in these studies does not allow one to establish a direct relationship between the diﬀerent treatments and pulmonary eﬀects. Moreover, when a study employes unrealistically high doses of NPs, such as in the case of Chen et al. [22], its relevance is dubious at best. In this light, when analyzing the NP dose role in lung damage, caution should be applied in extrapolating data for human evaluations.

Chemistry. Warheit et al. [44, 45] found that only non-coated particles resulted in BAL alteration and lung histo-pathological changes compared to their coated counterparts, suggesting a possible influencing role of surface chemistry

in nano-TiO2-induced lung toxicity. In this regard, several

studies have investigated the influence of particle chemical surface properties, surface coatings, and functionalization on

pulmonary responses showing conflicting results [61,70,77,

78].

Methylated-hydrophobic TiO2NPs acutely decreased the

(9)

intratracheally administered rats [61], a result in line with those of Oberd¨orster [87], but in contrast with those of

Pott et al. [88] showing that hydrophobic, silanized TiO2

NPs induced acute mortality in intratracheally exposed rats. Other studies reported significant inflammatory reactions

after acute [70,79] and subacute [70,77,78] exposure to

coated NPs, particularly silica-coated rutile TiO2 NPs [70],

rutile TiO2NPs surface modified with unspecified amount of

zirconium, silicon, aluminum, and coated with polyalcohols

[77, 78] and rutile Fe-doped TiO2 NPs [79]. However, at

present, it is unclear what changes lead to diﬀerences in the

toxicity induced by surface modified TiO2NPs. In additional

experiments, P25 Degussa TiO2NPs caused more persistent

pulmonary toxicity than carbon black NPs when compared on an equivalent particle surface area basis [68].

On the other hand, several studies investigated the TiO2

NP role as an adjuvant in promoting allergic sensitization or in influencing allergic lung inflammation. In this regard, the results obtained by Larsen et al. [65] and Park et al. [69] supported the adjuvant eﬀect of not characterized and

P25 Degussa TiO2 NPs, respectively, through the

promo-tion of a T-helper (Th)-2 immune response assessed by increased levels of interleukin (IL)-4, IL-5, and IL-10 in BAL. These alterations could stimulate allergic reactions, though the underlying mechanisms are not fully understood. Interestingly, in two-day- and two-week-old rats, but not

in adult animals, subacute inhalation of P25 Degussa TiO2

NPs produced upregulation of lung neurotrophins which was associated with a greater airway hyperresponsiveness

[72]. The age-dependent response to TiO2 NPs suggests

that the risk of developing asthma after NP exposure is higher in the earlier stages of lung development. Hussain

et al. [80] demonstrated that intrapulmonary doses of TiO2

NPs can aggravate pulmonary inflammation and airway hyperreactivity in a mouse model of diisocyanate-induced

asthma. Moreover, mixture of anatase and brookite TiO2

NPs caused airflow limitation in both acute and repeated 4-week-inhalation exposures [76]. In contrasts to these results

[65,69,72,76,80], a recent study [71] demonstrated the

role of silica-coated rutile TiO2 NPs as inhibitors of most

soluble and cellular mediators of allergic asthma. In fact, in ovalbumin-sensitized mice, the number of inflammatory cells, and the airway hyperreactivity were dramatically

reduced after exposure to TiO2NPs. The conflicting results

of the previous studies could be ascribed to the diﬀerent

health status of the animals, the diﬀerent type of particle

employed, or the diﬀerent route of exposure. Rossi et al. [71] hypothesized that anti-inflammatory Th-2 response caused by allergen sensitization may be suppressed by the competing

proinflammatory response elicited by TiO2exposure.

How-ever, future studies are needed to deeply clarify the TiO2

NP role in allergic reactions and the molecular mechanisms involved.

2.1.2. Lung Distribution. Particle size was able to aﬀect the fate of NPs, particularly lung accumulation and distribution in lung compartments, after pulmonary exposure. In fact, several studies described greater NP lung amounts compared

to their fine counterparts [12,52–55,60]. Ferin et al. [53]

suggested that TiO2 particle passage in the interstitium

could be promoted by the smaller sizes of the particles and the higher concentrations. Though data regarding the accumulation of NPs in the interstitium are too limited to allow a comprehensive evaluation of its consequences, this aspect is of particular interest considering that particle retention in the lung may give rise to potential enhancement

of local eﬀects and the increased possibility of systemic

redistribution.

Translocation of NPs from pulmonary airways into other pulmonary compartments, such as epithelium/endothelium, connective tissue, the capillary lumen, or into systemic circulation, is the subject of controversial discussion in the literature. Two studies of the same group demonstrated

conflicting results in that regard [63,64]. The first showed

that NP distribution was only related to the volume fractions of the corresponding compartments [63], while the second detected a correlation with the time points considered [64]. In fact, it found that NPs were preferentially located in the connective tissue and in the capillary lumen at 1 and 24 h after exposure, respectively. The epithelium and the connective tissues were not deposition sites but only passageways for NPs to reach the capillary circulation. However, whether NPs can translocate from air-filled spaces to the systemic circulation is still not fully understood, and further investigation is needed.

2.1.3. Carcinogenic Eﬀects. Animal and human

epidemio-logical data have led TiO2 to be designated as “possibly

carcinogenic” to humans [28,29,89]. Surprisingly, however,

few studies have investigated the carcinogenicity of TiO2

NPs, and only recently, NIOSH concluded that inhaled

ultrafine TiO2 is a potential occupational carcinogen [15].

The most relevant data for assessing the health risk for workers are results from a chronic animal inhalation study performed by Heinrich et al. [56], in which rats exposed by

inhalation to P25 Degussa TiO2NPs showed increased rates

of adenocarcinomas compared to controls. Interestingly, in this study, mice exposed to the same NPs, according to the same methodology, did not show diﬀerences in tumor rates compared to controls. Aside from demonstrating that

TiO2 NPs can induce lung cancer in exposed animals, an

interesting finding of this work is the diﬀerence in carcino-genicity between rats and mice. The species diﬀerence in response to insoluble and low toxicity dust, and the controversial approach to classify such a dust as a potential human carcinogen is subject to debate. Moreover, NIOSH

has concluded that TiO2 is not a direct-acting carcinogen,

but acts through a secondary genotoxic mechanism primarily related to particle size and surface area [15], as supported by the comprehensive analysis of the data reported by Heinrich et al. [56] and those obtained by Lee et al. with

fine-sized TiO2 [90]. However, considering the pressing

concern regarding cancerogenic eﬀects of TiO2NP exposure

on general and occupational populations and the limited number of in vivo studies on this topic, additional researches seem highly necessary.

(10)

2.2. Nervous System. Regarding TiO2 NP neurotoxicity,

diﬀerent studies have demonstrated the ability of these NPs to translocate into the brain, irrespective of the route of

exposure [31, 73, 91–94], the form of the TiO2, and the

size of the NPs and to accumulate in this organ [31,91–93]

inducing numerical and structural changes in the neuronal

architecture [31,92,93] (Table 2).

Several studies have unequivocally showed that rutile

TiO2NPs, taken up by intranasal exposure, could be

translo-cated into the central nervous system (CNS) via the olfactory pathway in mice and accumulate in the entire brain, mainly

in the hippocampus regions [31,91,92]. However, to verify

the generalization of this phenomenon, further studies are needed in other animal species. Regardless, this neuronal translocation pathway should be taken into account for

health risk assessments of TiO2NPs. In particular, it could be

useful to avoid possible toxic eﬀects to the general population but principally to subjects that work with these NPs. Brain accumulation was also demonstrated in mice intratracheally

instilled with no characterized TiO2 NPs able to penetrate

alveolar-capillary barrier, enter blood circulation, and fur-ther penetrate blood-brain barrier inducing tissue damage [73].

The deposition of TiO2 NPs in brain was reported to

induce changes in the release and metabolism of neuro-transmitters, although with the same conflicting results,

maybe due to the diﬀerent route of exposure [31, 91,93,

94]. Particularly, the levels of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) increased after intranasal expo-sure [91] while decreased in response to intragastric

admin-istrations of anatase TiO2 NPs [93]. Dopamine (DA),

3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic (HVA), and 5-hydroxyindole acetic acid (5-HIAA) contents were

reduced by both types of treatment [91, 94]. Moreover,

intranasal instillation of rutile [31] and intragastric

admin-istrations of anatase TiO2 NPs [94] increased catalase

and acetylcholinesterase (AchE) activity, soluble protein carbonyl, acetylcholine (Ach), glutamic acid (Glu), and

NO content. Intraperitoneally injected anatase TiO2 NPs

increased NO, while decreased Glu content and AchE activity [92]. Interestingly, the study performed by Hu et al. [94] demonstrated also that the contents of Ca, Mg, Na, K,

Fe, and Zn in brain were significantly altered after TiO2

NP exposure. The disturbed homeostasis of trace elements, neurotransmitters, and enzymes in brain may be responsible of the spatial recognition memory impairments reported in treated mice [94]. However, as assessed by a proteomic

analysis, proteins of the mouse brain resulted diﬀerentially

expressed in response to TiO2NP treatment even if NPs were

not detected in the tissue [95].

As observed in vitro [96–98], oxidative stress-related damage with a consequent alteration in the balance between oxidative and antioxidative activities was also reported

in in vivo studies [31, 73, 92, 93, 95]. Malondiadehyde

(MDA) levels increased after intranasal instillations [31,92],

intraabdominal injections [93], and intratracheal instillation in mice [73]. Similarly, Li et al. [73], and Ma et al. [93]

demonstrated that the level of superoxide anion (O2−),

per-oxide (H2O2) [73,93] and hydroxyl radicals [73] increased

in treated animals. TiO2 NPs are able also to induce an

inflammatory eﬀect in brain of treated mice, as detected

by the increased cytokine release [92, 99]. In the latter

study, rutile and P25 Degussa TiO2 NPs intraperitoneally

injected in mice after lipopolysaccharide (LPS) increased the

mRNA levels of IL-1β and tumor necrosis factor (TNF)-α

and also those of IL-1β protein. Interestingly, LPS induction

was necessary to cause this phenomenon, suggesting the importance of a trigger element or of a possible synergistic

eﬀect in tissue responses to TiO2NPs.

Finally, several studies demonstrated the embryotoxic role of maternal intravenous injection of no characterized

TiO2NPs [100], or subcutaneous injections of anatase TiO2

NPs [101–103]. Following this latter treatment, TiO2

accu-mulation was detected in the oﬀspring cerebral cortex and olfactory bulb and numerous olfactory bulb cells resulted positive to markers of apoptosis [101]. Moreover, the same prenatal exposure induced an altered expression of genes involved in brain development, cell death, and the response to oxidative stress in the newborn pups [102]. Finally, the

influence of prenatal TiO2NP exposure on the dopaminergic

system was demonstrated by the increased DA, DOPAC, HVA and 3-methoxytyramina hydrochloride levels in the prefrontal cortex and in the neostriatum of exposed mice [103].

These data confirm that TiO2 NPs can be transferred

from mothers to the fetal brain, inducing toxic eﬀects on

fetal brain development carrying a series of nervous system disorders. In this context, a deep knowledge regarding the

influence of TiO2NPs on neuronal cells is becoming urgent.

Though additional studies are necessary to support these findings, they should be taken in consideration for the

correct risk assessment of TiO2 NP exposure, particularly

during a complex biological status like pregnancy and the early stage of life.

2.3. Dermal and Mucosal System. Studies regarding the

eﬀects of TiO2NPs on the dermal system (in particular, the

percutaneous absorption of NPs) show more homogeneous findings, demonstrating a clear absence of penetration

through the intact epidermal barrier when TiO2NP

formula-tions are applied on diﬀerent animal [104,105] and human

[106–110] skins (Table 3). Considering the widespread use

of TiO2 NPs in cosmetic sunscreens (due to their

broad-spectrum UV absorption and high esthetical acceptance) [107], this is an important finding.

Sadrieh et al. [104] analyzed the correlation between

diﬀerent forms of TiO2 and percutaneous absorption in

micropig skin, demonstrating that irrespective of the form, after a subchronic exposure, NPs were found in the stratum corneum but not in the deeper epidermal strata. Anatase

TiO2NPs subacutely exposed to hairless rat skin were only

detected in the horny layer of the interfollicular epidermis, without penetration into viable cell layers or induction of any cellular changes [111].

Neither the shape [104,105,107,108] nor the surface

chemistry [107–109] seems to influence NP penetration after acute and subacute exposure.

(11)

Ta b le 2: In vi vo studies that in vestigat ed the ad ve rse eﬀ ects of T iO 2 NP s o n n er vo us syst em. Ne rv o u s sy st em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals Re su lt s W ang et al., 2007 [ 91 ]T iO 2 (25, 80, 155) In tr anasal inst il lat ion :5 0 m g/ k g T iO 2 B W ev er y o ther da y for 20 da ys. F emale CD mic e Ti b ra in co n te n t: incr eased. N eur ot ra nsmitt ers :N E and 5-HT incr eased; D A, D O P A C, HV A, and 5 -HIAA decr eased. W ang et al., 2008 [ 31 ] Ru ti le T iO 2 (80) A n atase T iO 2 (155) In tr anasal inst il lat ion: 500 μgT iO 2 ev er y other d ay for 1 5 times. 15 female CD-1(ICR) m ic e T i br ain d ist ribut ion: mainly in olfact o ry bulb and hippocampus. Oxidat iv e st re ss: CA T ,MD A, P r. C ar b . incr eased, SOD decr eased. N eur ot ra nsmitt ers: A ch E ,G lu an dN O incr eased. W ang et al., 2008 [ 92 ] S ee W ang et al., 2008 [ 31 ] In tr anasal inst il lat ion :500 μgT iO 2 ev er y other d ay for 1 5 times. 10 female CD-1(ICR) m ic e T i br ain d ist ribut ion: mainly in hippocampus. Oxidat iv e st re ss: GSH-Px, G ST ,SOD ,G SH, and M D A incr eased. Hi st o lo g y :ir re gular n eu ro nal ar rangement, co ndensat ed chr omatin. Inflammat o ry act ion: incr eased TNF -α and IL -1 β le ve ls. Shimizu et al., 2009 [ 102 ] A n atase T iO 2 (25–70) S u bcutaneous inject ions :100 μLo fT iO 2 at 1 μg/ μL o n gestational da ys 6, 9, 12, and 15. 15 pr eg nant ICR m ic e, 21 male fetuses and pups Gene expr ession :up-r egulat ed cell d eath, apo p to sis, br ain d ev elo p ment, o xidati ve st re ss, apo p to sis, ne ur ot ra nsmitt er genes. T ak eda et al., 2009 [ 101 ] A natase T iO 2 (25–70) S u bcutaneous inject ions :100 μLo fT iO 2 at 1 m g/mL at 3, 7, 10 and 1 4 d ay s post-c o itum. 6 p re gn ant S lc:ICR mic e pe r gr o up Ti O2 oﬀ spr ing br ain d ist ribut ion: co rt ex and olfact o ry bulb . A p o p to sis: pr esenc e of mar kers and featur es in olfact o ry ce ll s. T akahashi et al., 2010 [ 103 ] A n atase T iO 2 (25–70) S u bcutaneous inject ions :100 μLo fT iO 2 at 1 m g/mL at gestational d ay s 6 ,9 ,12, 15, 18. P reg nant Slc:ICR m ic e p er gr o u p M o noamine lev els: DA ,D O P A c, H V A , 3-MT incr eased in the p re fr ontal cor te x and neost riatum. M a et al., 2010 [ 93 ] A n atase T iO 2 (5) Bu lk T iO 2 (10–15 nm.) In tr a-abd o minal inject ions: 5–150 mg/kg n ano-T iO 2 and 150 mg/kg bulk T iO 2 ev er y d ay fo r 1 4 d ay s, re sp ec ti ve ly . 10 female CD-1(ICR) m ic e pe r gr o up Ti b ra in co n te n t: hig her incr ease w ith n ano Ti O2 . Oxidat iv e st re ss :O 2 − ,H 2 O2 ,M D A ,NOS, iNOS, NO incr eased; antio xidati ve enzy mes, GL U ,A chE decr eased. Hi st o lo g y :fi lament ous-shaped ne ur ons and inflammat o ry ce lls. Shin et al., 2010 [ 99 ] Ru ti le T iO 2 (1 μm) P25 T iO 2 (21) In tr ap er it oneal inject ions: 40 mg/kg Ti O2 30 min aft er ve hic le or 5 m g/kg LPS. 3–6 m ale C 57BL/6 m ic e p er gr o u p Inflammat o ry act ion: aft er L PS, T iO 2 NP s incr eased IL -1 β,T N F -α and iNOS and induc ed micr og lial acti vation. Oxidat iv e st re ss: aft er L PS T iO 2 NP s enhanc ed R OS.

(12)

Ta b le 2: C o ntin ued. Ne rv o u s sy st em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals Re su lt s H u et al., 2010 [ 94 ] A natase T iO 2 (5) In tr ag ast ric administ rat ion :5 – 5 0 m g/ k g Ti O2 suspension ev er y d ay for 6 0 d ay s. 20 female mic e per gr o up N eur ot ra nsmitt ers: A C h, Gl u, and N O in cr ea se d ;N E ,D A ,D O P A C ,5 -H T ,a n d 5-HIAA decr eased. Enzy m e act iv it y :decr eased N a +/K +,C a 2+, Ca 2+ /Mg 2+ A T P ase; p ro mot ed A ch E, and iNOS. Li et al., 2010 [ 73 ] A natase T iO 2 (3) In tr at ra ch eal inst illat ion: 3.3 m g/kg Ti O2 onc e a w k for 4 w ks. 13 male K unming mic e per gr o u p Ti b ra in co n te n t: incr eased Oxidat iv e st re ss: O2 −,O H −,H 2 O2 ,M D A incr eased in br ain. Hi st o lo g y :e xudat es, inflammat o ry infilt ration and necr osis. Y amashita et al., 2011 [ 100 ] Ti O2 (35) In tr av enous inject ion: 0.8 m g T iO 2 for 2 co nsecuti ve gestational da ys. P reg nant mic e T i dist ri but ion: Ti O2 det ect ed in fetal b ra in Je on et al., 2011 [ 95 ]T iO 2 N.A N .A. P rot eomic analysis: al te re d p ro te in ex pr ession. Oxidat iv e st re ss: antio xidant and A ch E acti vi ties re d u ce d. 5-HIAA, 5-h ydr o xy indole ac etic acid; 5-HT , 5 -h yd ro xy tr yp tamine; 3 -MT , 3.metho xy ty ramine-h yd ro cl hor ide; A ch , ac et ylc holine; A ch E, ac et ylc h o linest er ase; CA T catalase acti vit y; NE, n or epinephr ine; D A, dopamine; D OP A C , 3 , 4 -dih yd ro xy phen ylac etic acid; Gl u, gl utamic acid; GSH, re duc ed gl u tathione; G SH-Px, gl utathione p er o xidase; G ST , gl u tathio ne-S-t ra nsfer ase; H V A , h omo vanillic acid; IL -, int erleukin; iNOS, inducible nit ri c o xide synthase; L PS, lipopolysac ch ar ide; MD A, malondialdeh yd e; NE, n or epinephr ine; N O , nit ri c o xide; N OS, nit ri c o xide sy nthase; P r. Car b ., P rot ein car bon yls; R OS, reacti ve o xygen species; SOD ,super o xide dism u tase; T iO 2 NP s, titanium dio xide nanopar tic les; TNF -α ,t umor necr osis fact or -α .

(13)

Ta b le 3: In vi vo studies that in vestigat ed the ad ve rse eff ects of T iO 2 NP s o n d er mal syst em. Der m al and m uc osal syst em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals/subjects Re su lt s T an et al., 1996 [ 112 ] Sunscr een containing 8% micr ofine T iO 2 C u taneous applicat ion: 2 times/da y for 2–6 w ks. 13 male and female vo lunt eer p atients Ti O2 absor p ti on: le ve ls of T i in der m is w er e hig her in tr eat ed patients co mpar ed to co nt rols, b ut not sig nificantly . Lademann et al., 1999 [ 113 ] W /O em ulsion UV -T itan M 160 C u taneous applicat ion: 2m g/ cm 2on the volar for ear m, 5 times on the fi rst 3 da ys, and o nc e o n the 4th. H u man vol unt eers Ti O2 absor p ti on: mostly locat ed in the upper st. co rn eu m. Pfl üc ke r et al., 2001 [ 107 ] W/O em u lsion T 805 co at ed w ith tr imeth yl o ct ylsilane T iO 2 (20) W/O em u lsion E usole x T -2000 Ti O2 co at ed w it h A l2 O3 and SiO 2 (10–15) W/O em u lsion T io ve il A Q T iO 2 co at ed w ith Al and SiO 2 (100) C u taneous applicat ion: 4m g/ cm 2on fo re ar ms k inf o r 6 h r. H u man vol unt eer Ti O2 absor p ti on: Ti O2 solely localiz ed o n the o ut er most sur fac e o f the st. cor ne um, not in the deeper subcutaneous la ye rs. Sc h u lz et al., 2002 [ 108 ] See P fl üc ke r et al., 2001 [ 107 ] Ti O2 absor p ti on: locat ed o n the out er m ost sur fac e of the st. co rn eu m and not in the deeper subcutaneous la ye rs. M enz el et al., 2004 [ 105 ] Sunscr een containing T iO 2 (length: 45–150; w idth: 17–35) C u taneous applicat ion: on the b ac k pig skin for 8 –48 h r. Pig s Ti O2 absor p ti on: Ti O2 pe netra ted thr o ug h int er ce ll ular spac es of the st. co rn eu m int o the st. gr an u losum. Ke rt ész et al., 2005 [ 106 ] H ydr o p hobic em u lsion T iO 2 NP s. C u taneous applicat ion: on human for eskin gr afts for 1 –48 h r. H u man gr afts tr ansplant ed int o S CID m ic e Ti O2 absor p ti on: co rn eo cy te la ye rs o f th e st. cor ne um. M av o n et al., 2007 [ 109 ] Em ulsion w ith 3% T805 Degussa Ti O2 co at ed w ith tr imeth yl oct ylsilane (20) C u taneous applicat ion: 2m g/ cm 2on upper ar m s. 3 h uman vo lunt eers Ti O2 absor p ti on: 93% of the T iO 2 applied w as re co ve re d in the st. cor ne um. K iss et al., 2008 [ 110 ] H ydr o p hobic em u lsion co ntaining micr oniz ed T iO 2 . C u taneous applicat ion: 2m g/ cm 2on h u man for eskin gr afts for 2 4 h r. H u man gr afts tr ansplant ed int o S CID m ic e Ti O2 absor p ti on: out er m ost la yers o f the st. cor ne um. Y anag isa wa et al., 2009 [ 114 ] Ru ti le T iO 2 (15, 50, 100) In tr ader mal inject ions: of 20 μgT iO 2 wi th o r wi th o u t m it e al le rg en o n th e ea rs . 11 male C/NgaTndC rj (NC/Nga) mic e per gr o up A to p ic der m at it is: al lergen + T iO 2 enhanc ed ear thic ke ning . Inflammat o ry act ion: al lergen + T iO 2 incr eased EOSs, IL -4, MCs and decr eased IFN-γ;T iO 2 incr eased IL -13. W u et al., 2009 [ 115 ] A n atase T iO 2 (5 ± 1, 10 ± 1) Ru ti le T iO 2 (25 ± 5, 60 ± 10) P25 D egussa T iO 2 ( ∼ 21) Ti O2 (0.3–0.5 μm) C u taneous applicat ion :2 4 m gT iO 2 for m ulations on the pig ear for 3 0 d ay s and o n the mouse int erscapular skin for 60 co nsecuti ve d ay s. 3 m ale rear ed pig s p er gr oup 6 hairless mic e (B ALB/c/n u/n u ) p er gr oup Ti O2 absor p ti on in pig s: Ti O2 de te ct ed in the st. co rn eu m, gr an ulosum, p ri ck le and b asal ce ll la ye r, not in the der m is; Ti O2 absor p ti on in mic e: incr eased MD A, re duc ed H YP co nt ent, and ex ce ssi ve ke ra tinisation in skin.

(14)

Ta b le 3: C o ntin ued. Der m al and m uc osal syst em Re fe re n ce s C rystal p hase co mposition (par tic le siz e in n m) Ty p e o f ex p o su re T ype and n umber o f animals/subjects Re su lt s Sadr ieh et al., 2010 [ 104 ] P25 D egussa T iO 2 (30–50) Ru ti le T iO 2 co at ed w it h al uminium h ydr o xide/dimethic one co poly mer (diamet er :20–30, length: 50–150) Su bmicr o n T iO 2 (300–500) C u taneous applicat ion: 2 m g cr eam/cm 2 skin for 4 times/da y, 5 d ay s/w eek, 4 w eeks of. 3 female Y ucatan minipig s pe r gr o up Ti O2 absor p ti on: Ti O2 det ect ed in the upper st. co rn eu m and follicular lu men, w ith few p ar tic les obser ved in der m al la ye rs as a contaminat ion re sults. A d ac hi et al., 2010 [ 111 ] W/O em u lsion containing anatase T iO 2 (26 .4 ± 9. 5) C u taneous applicat ion: 4m g/ cm 2Ti O2 on d o rsal skin. M ale hairless W istar Y ag i ra ts Ti O2 absor p ti on: Ti O2 det ect ed in the hor n y la yer of the int er follicular epider mis. M o on et al., 2011 [ 116 ]T iO 2 (< 25; < 100) In tr ap er it oneal inject ions: onc e a d ay for 2 8 consecut iv e da ys b efor e subcutaneous implantation w ith B16F10 melanoma ce lls. Mi ce T umor g ro w th: incr eased F u ru ka wa et al., 2011 [ 117 ] Co at ed T iO 2 (long axis: 50–100; shor t axis: 10–20) C u taneous applicat ion :5 – 2 0 m gT iO 2 in the p ostinitiation phase in a skin car cinogenesis m odel. 10–20 CD1 (ICR) female mic e per gr o up Car cinogenicit y :no incr eased de ve lopment o f skin n o d ules EOS, eosinophil; H YP ,h yd ro xy pr oline; IL -, int erleukin; IFN-γ, int erfer o n-γ; M C, mast ce ll; M D A ,m alondialdeh yd e; T iO 2 NP s, titanium dio xide nanopar tic les.

(15)

Pfl¨ucker et al. [107] demonstrated that all TiO2NP types

applied on skin areas of human forearm were only detected on the outermost layer of the stratum corneum, irrespective of their surface chemistry, NP size, and shape. The same results were confirmed by Schulz et al. [108]. Similarly, after

application of lanceolate TiO2NPs on the backs of pigs, high

concentrations of Ti were found in the stratum corneum and granulosum, but not into the stratum spinosum [105].

Mavon et al. [109] demonstrated that 93% of the TiO2

T805 NPs hydrophobically coated with trimethyloctylsilane applied on human upper arms were found in the stratum

corneum, decreasing continuously with depth. No TiO2

penetration into viable skin layers was assessed confirming the in vitro findings of the study.

The studies investigating the Ti penetration and distri-bution in biopsies obtained from human skin grafts trans-planted into severe combined immune deficiency (SCID) mice could only detect Ti in the outermost layers of the stratum corneum of the skin biopsies, irrespective of the various NP formulation applied and the time points

considered (1–48 h) [106,110].

As also confirmed by Menzel et al. [105], Lademann et al. [113] failed to demonstrate a role for hair follicles as a percutaneous absorption pathway. In fact, Ti was detected in the stratum corneum and in the follicle channels but not in the interfollicular space under the stratum corneum or into the viable layers of the forearm skin of human volunteers.

Only a few reports detail Ti penetration into human

dermis [112,115]. For instance, in a pilot study conducted

in 1996, Tan et al. [112] demonstrated percutaneous pen-etration of Ti in patients subchronically applied sunscreen

containing microfine TiO2. Although Ti levels in the dermis

of exposed subjects were higher than controls, these results were not statistically significant and were not confirmed by subsequent studies. Moreover, Wu et al. [115] reported the

ability of TiO2 NPs in diﬀerent form, anatase, rutile, P25

Degussa, and diﬀerent sizes to penetrate through the skin, reaching diﬀerent tissues (e.g., liver, spleen, and heart) where pathological lesions were induced after subchronic dermal exposure of hairless mice. Moreover, increased MDA levels were determined in the skin, which also showed excessive keratinization, thinner dermis and wrinkled epidermidis. These data could not be confirmed when reared pigs were treated with two types of the previously employed NPs for shorter periods of time.

The same interesting points of discussion emerged from this latter study. It seems that the penetrative ability of similar

TiO2 NPs into the cutaneous layers depends on the animal

species studied and on the period of exposure. Furthermore, pathological skin lesions due to NP application are likely ascribed to oxidative stress, which was also detected on human and murine dermal fibroblasts in vitro [118–120].

Intradermal injection of TiO2 NPs aggravated atopic

dermatitis (AD) symptoms related to mite allergen in mice assumed to show skin barrier dysfunction/defect through Th-2 immune inflammatory responses and histamine release [114]. These data support the importance of an intact dermal

defense system to prevent uptake of TiO2 NPs, which can

lead to skin damage, activation of the immune system, and eventually a decrease in the allergy threshold [105].

Ultimately, the importance of the human studies on

per-cutaneous TiO2 absorption is underlined by the fact that

these experiments alone are able to provide a relevant ap-proach to direct human risk assessment and are the first step in the complex process of generalizing implications regarding environmental or occupational health.

Regarding skin carcinogenicity, conflicting results were

reported [116,117]. Tumor growth was increased when mice

were intraperitoneally injected with TiO2 NPs prior to the

subcutaneous implantation of B16F10 melanoma cells [116].

The authors suggest that TiO2NPs might have the potential

to enhance tumor growth in situ through immunomod-ulation of B- and T-lymphocytes, macrophages, and NK cells as supported also by results obtained in the spleen tissue. In contrast to these findings, Furukawa et al. [117]

investigated the safety of coated and uncoated TiO2NPs in

a mouse medium-term skin carcinogenesis bioassay. Both

TiO2 NPs applied to mouse skin in the postinhitiation

phase did not increase the development of skin nodules histopathologically diagnosed as squamous cell hyperplasia, sebaceous gland hyperplasia, squamous cell papilloma, and

keratoacanthoma. According to these data, TiO2NPs do not

possess postinhitiation potential and there is no carcinogenic

risk relevant to percutaneous application of TiO2NP

prepa-rations.

2.4. Cardiovascular System. Due to the complexity of the car-diovascular system, studies regarding the potential eﬀects of

TiO2NPs on this system have focused on diﬀerent functional

aspects, such as cardiotoxicity [121,122], cardiovascular

pa-rameters, such as systolic blood pressure (SBP) and heart rate

(HR) variability [79], induction of thrombosis [22,123], and

the alteration of vasomotor responses [124–128] (Table 4).

TiO2 NPs are able to induce high LDH, creatine kinase

(CK), alpha-hydroxybutyrate dehydrogenase (HBDH), and aspartate aminotransferase (AST) activities used as markers

of myocardial lesions, irrespective of the form of TiO2,

anatase [122] or a mixture of anatase and rutile [129], or the route of exposure, intraperitoneal injections in mice [122], or oral gavage in rats [129]. Moreover, acute cardiotoxicity, in terms of higher serum LDH and alpha-HBDH enzymes,

was demonstrated after a single oral gavage of TiO2 NPs

administered to mice [121]. Unfortunately, the lack of data regarding the LDH isoform prevents a clear correlation between these increases and cardiac insults. Moreover, the study performed by Wang et al. [121] did not provide

information on TiO2 form, and consequently, it does not

allow comparisons with the two previous described works

[122,129].

When intratracheally instilled in rats, rutile Fe-doped

TiO2nanorods significantly increased SBP and HR in treated

animals, which could be the consequence of the systemic inflammation induced by the same particles [79].

Regarding the TiO2 NP prothrombotic eﬀect, the

cur-rently available data are conflicting. For instance, Chen et al. [22] observed thrombosis in the pulmonary vascular system