Toxicological Effects of Titanium Dioxide Nanoparticles: a Review of in Vitro Mammalian Studies

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European Review for Medical and Pharmacological Sciences 2011; 15: 481-508 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies

I. IAVICOLI, V. LESO, L. FONTANA, A. BERGAMASCHI

Institute of Occupational Medicine, School of Medicine, Catholic University of the Sacred Heart, Rome (Italy)

Abstract. – Background and Objective: Introduction Recent rapid advances in nanotechnology raise concerns about development, production route, Nanotechnology is a broad interdisciplinary and diffusion in industrial and consumer prod- branch of science, grouping physical, chemical, ucts of titanium dioxide nanoparticles (TiO2-NPs). In fact, compared to recent increase in applica- biological, and engineering expertise, that has the tions of this nanomaterial, the health effects of ability to manipulate matter at the level of single human exposure have not been systematically in- atoms and small groups of atoms to produce new vestigated. The aim of this review was to provide structures, materials and devices with unique a comprehensive overview on the current knowl- physical and chemical properties1. Research in edge regarding the effects of TiO -NPs on mam- 2 nanoscale technologies is growing rapidly world- malian cells. 2 Evidence and Information Sources: This re- wide. The U.S. National Science Foundation es- view is based on an analysis of the current liter- timates that, by 2015, nanotechnology will have ature on this topic. a $1 trillion impact on the global economy and

State of the Art: Fine TiO2 particles have will employ 2 million workers. The industrial ap- been considered as safe and to pose little risk to plications of nanomaterials are very wide includ- humans, suggesting that exposure to this materi- ing those that may lead to more efficient water al is relatively harmless. However, available data purification, stronger and lighter building materi- in the literature showed that TiO2-NPs can cause several adverse effects on mammalian cells such als, increased computing power and speed, im- as increase of reactive oxygen species (ROS) pro- proved generation and conservation of energy, duction and cytokines levels, reduction of cell vi- and new tools for the diagnosis and treatment of ability and proliferation, induction of apoptosis diseases3. To achieve these multiple objectives and genotoxicity. nanotechnology, in the manufacturing of differ- Perspectives and Conclusions: Additional re- ent consumer and industrial products, uses sever- search is needed to obtain up-to-date knowledge al types of nanoparticles (NPs) such as carbon on health effects of TiO2-NPs and to avoid any potential risk correlated to their exposure. Conse- nanotubes, silica and metal NPs. quently, future studies need to: (1) use an homoge- Among the manufactured NPs, titanium diox- neous and rigorous exposure classification to clari- ide NPs (TiO2-NPs) are the earliest industrially 4 fy how the physicochemical properties of TiO2-NPs produced nanomaterials and, according to the correlate with their toxicological effects; (2) assess U.S. National Nanotechnology Initiative, they are the potential adverse effects of low level expo- one of the most highly manufactured in the sures to TiO2-NPs, as most of the information cur- 5 rently available originates from studies in which world . In fact, they are widely used in paints, exposure levels were excessively and unrealisti- printing ink, rubber, paper, cosmetics, pharma- cally high; (3) identify the possible roles of TiO2- ceuticals, sunscreens, car materials, cleaning air NPs in genotoxicity and carcinogenicity (4) carry products, bio-medical ceramic and implanted out epidemiologic studies of exposed workers to biomaterials, sterilization, industrial photocat- provide an assessment of possible risks correlated alytic processes and decomposing organic mat- to the occupational exposure to TiO -NPs. 2 ters in wastewater6-8. Key Words: Titanium dioxide is a natural, highly insoluble, thermally stable and non-flammable, non-silicate Titanium dioxide, Nanoparticles, In vitro studies, Ef- mineral oxide found primarily in the form of the fects, Genotoxicity. minerals rutile, anatase, brookite and as the iron-

Corresponding Author: Ivo Iavicoli MD, PhD; e-mail: [email protected]. 481 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi

9-13 containing mineral ilmenite . The major natural part, it is reasonable to assume that TiO2-NPs source of TiO2 is ilmenite, whereas rutile and have also a quite different toxicological profile. anatase polymorphs are mainly manufactured In the literature there are several in vitro and in commercially13. Though both rutile and anatase vivo studies that have investigated the toxicologi- belong to the tetragonal crystal system, rutile has cal effects of TiO2-NPs, even if the former stud- a denser arrangement of atoms. The luster and ies are much more numerous and extremely use- hardness of anatase and rutile are also similar, ful means to investigate underlying mechanistic but their cleavages differ. Common impurities in processes and to decide on the dose levels for the rutile include iron, tantalum, niobium, chromi- latter studies. Nevertheless, there is a lack of an um, vanadium and tin, whereas those in anatase overall evaluation on these effects to define an include iron, tin, vanadium and niobium14. Due adequate risk assessment, in particular to protect to its excellent physicochemical properties of exposed workers and general population sub- good fatigue strength, resistance to corrosion, jects. Therefore, in this review we will evaluate machineability, biocompatibility, whitening and current knowledge regarding the in vitro toxic ef- photocatalysis, as well as its excellent optical fects of TiO2-NPs to identify research areas performance and electrical properties, TiO2 has a where further studies should be carried out in or- 15,16 wide range of applications . In fact, it is used der to reach a fuller understanding of TiO2-NPs mainly in paints, varnishes, lacquer, paper, plas- toxicity. tics, ceramics, printing ink, welding rod coatings, floor coverings, catalysts, coated fabrics and tex- tiles, cosmetics, food colorants, glassware, phar- In vitro studies maceuticals, roofing granules, rubber tire manufacturing and in the production of electronic Lung Cells components and dental impressions14,17-19. Studies conducted on lung cells (Table I)

The potential adverse health effects of TiO2 demonstrate that several parameters seem to be have been investigated in different experimental correlated with the toxicity of the TiO2-NPs, prin- 20-23 investigations and in some epidemiological cipally the form of the TiO2, but also the size, studies24-27. On the basis of results and data collect- shape, surface chemistry and surface area of NPs. ed by researchers TiO2 was considered to exhibit Concerning the form of TiO2, different studies relatively low toxicity28 and inhaled fine particles have reported several toxic effects due to anatase of this material are generally regarded, at least un- TiO2-NPs relative to other forms. In fact, anatase 29,30 der nonoverload conditions , biologically inac- TiO2-NPs have been found to produce greater re- tive and physiologically inert21,29,31,32. The Interna- sponses, particularly a reduction of cell viabili- tional Agency for Research on Cancer (IARC) has ty46-51, an increase of inflammatory indices (e.g., 33 recently reclassified TiO2 (IARC, 2006) as “pos- lactate dehydrogenase (LDH) and interleukin sibly carcinogenic to humans” (group 2B) because (IL)-8)46 and an increase in radical oxygen high concentrations of pigment-grade (< 2.5 µm) species (ROS) generation46,49,51-54, that in some 49,51 and ultrafine (< 100 µm) TiO2 dust can cause respi- studies was shown to be dose-dependent . Fi- ratory tract cancer in exposed rats34,35. However, nally, anatase NPs are also able to induce cell the epidemiological studies of workers exposed to death by an intrinsic apoptosis pathway51. This pigment-grade TiO2 have not be able to detect an higher toxicity of anatase TiO2-NPs may be cor- association between occupational exposure and an related to their photocatalytic activity, as suggest- increased risk for lung cancer36,37. ed by Sayes et al46. With the advent of nanotechnology, some of Furthermore, though the aforementioned stud- the assumptions concerning the safety of TiO2 ies were performed with all due diligence, it is would be challenged. In fact, the chemical, opti- important to note that is difficult to extrapolate cal, magnetic, biological and structural properties the toxic effects observed in vitro studies with of NPs may differ considerably from those of monocellular cultures to the real in vivo situation larger particles composed of the same materi- of the lung, and consequently, these findings als38,39. Since available data in the literature show should be considered with caution. Indeed, this that the physico-chemical characteristics of NPs point of view is confirmed by the fact that in a are closely associated with their biological ef- triple culture model that simulates the real cellu- fects40-45, and considering that they are deeply un- lar conformation of the lung, exposed to anatase like in TiO2-NPs, respect to their bulk counter- TiO2-NPs, no statistically significant increases in

482 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies ). 2 ters ). Continued 2 ); 2 > nano TiO 2 ); 2 ). 2 . 2 located in cytosol as aggregates. located in cytosol 2 in vacuoles (early) or lamellar bodies; in vacuoles 2 externally associated with plasma membrane. externally on GSH had no effect to produce ROS, failed phagosomes. up into lose-fitting taken and lamellar bodies; incorporated in vacuoles 2 2 2 2 • 14-17% of apoptotic cells (micro and nano TiO • TiO production (ultrafine ROS Increased • Micro and nano TiO • fragmentation (micro and nano TiO 7-8% DNA ~ • GSH depletion with both TiO Human alveolar Human alveolar macrophage cells 264.7, THB-1 and A549, respectively. RAW epithelial cells • TiO THP-1 cells;A549 levels; mRNA cxcl-5 • (THP-1 cells); MCP-1(CCL2) protein level Increased Chinese hamster • Reduction of cell viability; Lung cells 2 2 -NPs on lung cells. 2 50 0.25, 5, 40 µg/ml A549 human lung • TiO Phagocytized 200 5-402-60 5 µg/ml 264.7; RAW • ~10, ~5, ~2 µg/ml for Anatase EC50 (cell death) was (THB-1); A549. > 200 (BEAS-2B) 30-50 1-100 µg/cm 10; 20 10 µg/ml Human bronchial • stress; of oxidative Induction 40-300 3, 16, 80, A549 • TiO 180-250 0.2 µg/ml parame above-mentioned the on effect slight a only or No 30; 1 µm 5- 200 µg/ml A549 • of granularity cells (micro TiO Increase 10.1 ± 1.0 3.2 ± 0.34 3 µg/ml-3 mg/ml A549 • High LDH release; • IL-8 production. Enhanced 2 : 20-30 10 µg/ml Murine alveolar • TiO 2 2 2 2 2 2 2 2 -NPs N.A. 15- 250 µg/ml A549 • and protein levels; of IL-8 mRNA Increase 2 2 2 ) treated (V79) 5 : 80% anatase; TiO 20-80 • TiO (ultrafine IL-8 levels Increased 2 2 2 2 2 2 O 2 Crystal phase Particle Doses of 99.5% pure rutile 21 0.1, 0.2, 0.5 µg/ml Human monocytes • and increased and protein levels PIGF mRNA Increased TiO Rutile TiO 100% rutile TiO rutile mixture TiO anatase TiO Rutile TiO 20% rutile 264.7) (RAW • TiO ≥ - 20% rutile TiO (V studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results – (47) – (57) 80% anatase - – (58) P25 Degussa macrophage cells depletion and on Heme Oxygenase-1 expression; 400 µg/cm – (68) Xia et al, 2006 TiO P25 Degussa Singh et al, 2007 Pure anatase TiO Stearns et al, 2001 TiO – (46) 60% anatase - 40% 5.2 ± 0.34 for 48 hr • Decreased mithocondrial activity; Sayes et al, 2006 100% anatase TiO – (52) 200 epithelial cells • of cell growth. Inhibition Gurr et al, 2005 Anatase TiO et al, 2007Park TiO Chen et al, 2006 – (61) TiO 2007 – (62) Soto et al, 2007) Anatase TiO Monteiller et al, TiO – (67) Bhattacharya et al, Anatase TiO 2008 – (53) pentoxide Vanadium 30-50 lung fibroblasts • of ROS. Induction Table I. Table

483 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi values α and CXCL2; α release. release. -NPs in all cell lines and culture types; α α 2 -NPs were clustered in cytoplasm vacuole like vacuole -NPs were clustered in cytoplasm -NPs located in the cytoplasm, isolated or in vacuoles. -NPs located in the cytoplasm, 2 2 ternalization of TiO were lower than expected values. than expected were lower • No increase in TNF- • production; Dose and time dependent increase of ROS • Decreased intracellular reduced GSH; • stress related genes; of oxidative Induction • of IL-1, IL-6, IL-8, TNF- levels Increased • of apoptosis. Induction Human monocyte dendritic derivated cells (MDDC) derivated macrophagesderivated (MDM); all cell types; in • No increase in TNF- MDDC. • production; Increased ROS Chinese hamster • Reduction of cell viability; BEAS-2B; • TiO bronchial fibroblasts (IMR 90) • • only in IMR90; observed Cytotoxic effects production. Dose-dependent intracellular ROS MDDC. • IL-8 and TNF- In the triple co-culture TAC, Lung cells 2 2 -NPs on lung cells. 2 21 5-40 µg/ml BEAS-2B • Dose and time dependent reduction of cell viability; < 100 2-50 µg/cm 30-50 1-100 µg/cm 20-30 2.5 µg/ml A549; • In 0.02-0.03 µm 5 µg/ml0.02-0.03 µm A549 5 µg/ml • Membrane-bound aggregates; A549; • Single particles unsurrounded by membranes and 2 2 , 25 ± 7 0.25-100 µg/ml A549 • Cell membrane damage; , 9 ± 3 2 2 2 2 2 2 2 , 142 ± 36 2 ) treated (V79) 5 , 95% anatase 12 ± 3 • TiO 2 O 2 Crystal phase Particle Doses of Sigma TiO anatase TiO 100% rutile 100% anatase Sigma rutile TiO (V TiO studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results – (59) Rothen-Rutishauser 99.9% anatase TiO Bhattacharya et al, Anatase TiO Muller et al, Anatase TiO et al, 2007 – (55)et al, 2008 – (54) Human monocyte • free in cytoplasm Single particles and small aggregates MDM; in membrane-bound agglomerates; Rothen-Rutishauser 99.9% anatase TiO Bhattacharya et al, Anatase TiO 2008 – (53) pentoxide Vanadium 30-50 lung fibroblasts • of ROS. Induction et al, 2008 – (60) 75% anatase CEA • Reduction of cell viability; 2009 – (49) et al, 2008Park TiO P25 Degussa Human diploid structures or lysosomes; Simon-Deckers Simon-Deckers TiO P25 Degussa 2010 – (56) MDM; • in MDDCs); (except of ROS Induction Table I. Table

484 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies 2 , in- β α mRNA; α forms; -NPs. 2 δ 2+ . nanospheres uptake. α by TiO 2 α protein-1; N.A., not applicable; PIGF, protein (RAW 264.7) and TNF- protein (RAW α , chemokine (C-X-C motif) ligand 5; CX- and IL-18 levels. , tumor necrosis factor- β hydroethidine; IL-1, interleukin- 1; IL-1 α particles increased GM CSF mRNA and particles increased GM CSF mRNA ong nanobelt were “free-floating” in cytoplasm; 2 were more toxic than Na(x)TiO receptor was involved in TiO involved receptor was δ 2+ nanospheres and short nanobelt were taken up in nanospheres and short nanobelt were taken 2 ong nanobelt were cytotoxic and increased Cathepsin B ong nanobelt were cytotoxic GM-CSF release. activated macrophage supernatants elicited CXCL-1 and activated CXCL-8 mRNA. and cell morphology was altered. and cell morphology was • of apoptosis; Induction • Dose dependent increase of ROS. • structures were inside cells in needle-like Nanofilaments • IL-6, TNF- Dose dependent increase of GM-CSF, • contents; intracellular cytokine Increased • Absorption of GM-CSF and TNF- Human bronchial (16HBE14o-) • inside endosomes or free in the cytoplasm; Aggregates 16HBE14o- • 15 nm TiO BEAS 2B • Dose dependent citotoxic effects; • by followed the highest cytoxicity, Fine rutile showed -NPs, titanium dioxide nanoparticles; TNF- 2 Lung cells 2 2 2 g/ml Human lung tumor • impaired cell proliferation; Nanofilaments g/ml BEAS-2B • Dose dependent reduction of cell viability; μ μ -NPs on lung cells. 2 m loaded AM (581-591) • IL-1 Increased μ m 0-500 µg/ml 264.7; MDM; RAW • of TNF- Induction 40 1-160 µg/cm μ 40 nm, by silica coated NPs; (MDM) × 15 0-160 µg/cm 25 (MRC-9) fibroblasts • expression of CXCL-1 and CXCL-8 mRNA Induction × < 25 30-40 Human pulmonary and protein (MDM) by silica coated NPs; mRNA width: macrophages (AM);• MARCO width: macrophages 60-200 0-250 µg/ml C57BL/6 primary • TiO < 5 µm < 5 10 needle-like • with silica coated TiO Stimulation of MRC-9 fibroblasts length 15-30 AM; levels; ≈ width: 60-300; C11-BODIPY • and lipid peroxidation; levels ROS Increased 75 nm nanowires cells (H596) • HyTiO ; 15µg/cm 5-160 2 2 2 2 2 2 or 12 nm nanotubes 0.02-2 2 2 2 2 δ 2+ 2 2 δ 2 + coated with the least cytotoxic anatase, while nanosized rutile was (< 5%) 2 2 2 Crystal phase Particle Doses of rutile TiO Anatase TiO 99.7% anatase TiO Anatase TiO Silica coated Anatase TiO long nanobeltsshort nanobelts 60-300; length 0.8-4 MARCO null mice • L SiO 99.9% rutile TiO 35% rutile TiO studies that investigated the adverse effects of TiO effects the adverse studies that investigated ; IL-6, interleukin-6; IL-8, interleukin-8; IL-18, interleukin-18; LDH, lactate dehydrogenase; MCP-1, monocytes chemoattractant ; IL-6, interleukin-6; IL-8, interleukin-8; IL-18, interleukin-18; LDH, lactate dehydrogenase; MCP-1, monocytes β In vitro Reference composition size (Nm) exposure Cell lines Results – (51) TiO – (50) Val et al, 2009Val 99.9% anatase TiO 2009 – (65) Hy TiO – (66) Rutile/anatase TiO Hussain et al, 99.9% anatase TiO Magrez et al, Na (x) TiO Falck et al, Falck 99.5% pure rutile 10 Rossi et al, 2010 Rutile TiO Shi et al, 2010 99.7% anatase < 25 0-100 Hamilton et al, Anatase TiO 2009 – (63) 65% anatase - 2009 – (48) 25-75 TiO epithelial cells • cells; production in HE positive ROS Increased 2009 – (64) nanospheres murine alveolar l lysosomes, CL-8, chemokine (C-X-C motif) ligand 8; GM-CSF, granulocyte-macrophage colony-stimulating factor; GSH, reduced glutathione; HE, factor; colony-stimulating granulocyte-macrophage CL-8, chemokine (C-X-C motif) ligand 8; GM-CSF, CCL2, chemokine (C-C motif) ligand 2; CXCL-1, (C-X-C 1; CXCL2, cxcl-5 terleukin-1 placenta growth factor; ROS, reactive oxygen species; TAC, total antioxidant capacity; TiO oxygen species; TAC, reactive ROS, factor; placenta growth Table I. Table

485 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi

ROS production and inflammatory parameters dependent. However, internalization is neither in- α 54-56 58,60,68 (i.e., IL-8 and TNF- ) were observed . Sur- fluenced by the form of the TiO2-NPs nor prisingly, however, these parameters were actual- by their shape64,65. Particles were detected in the ly lower than the expected values experimentally cytosol of cells, particularly in the peri-region of calculated from the monocultures used by the the nucleus, and trapped in vacuoles, lamellar Authors. bodies and/or lysosomes49,58,59 even though free Other investigations have reported conflicting nanoparticles60 and needle-like nanofilament 65 data on the effects of another form of TiO2-NPs: structures were observed at early treatment P25 Degussa. In fact, it was observed that P25 times. This phenomenon has been observed in all Degussa NPs are unable to induce ROS produc- types of lung cells examined to date and in the tion57, and subsequently, other research groups triple cell co-culture model55,56. The internaliza- have shown that these NPs induce ROS generation was also correlated with the oxidative stress tion58,59, IL-8 release58, decrease of glutathione and inflammatory responses63. (GSH) levels59 and a time- and dose-dependent reduction of cell viability59. Interestingly, increased ROS generation appeared to stimulate Nervous Cells cytotoxicity by an apoptotic process, even if only Different in vitro researches have evaluated the 59 in BEAS-2B cells . toxic effects of TiO2-NPs and their internaliza- Concerning the rutile form, several studies46- tion in nervous cells (Table II). The findings sug- 48,60 reported the minimal toxicity of this type of gest that an important parameter determining NP 61 NPs even if in a single study toxic effects were toxicity is the form of TiO2, in particular, P25 observed. In particular it was shown a significant Degussa69-72 and anatase NPs73. Furthermore, a induction of placenta growth factor expression in recent study74 correlated the toxicity of these NPs the monocytes and A549 cells at the mRNA, pro- with their surface chemistry investigating the ef- tein and secreted protein levels. fects of rutile TiO2-NPs coated with SiO2 on the Another study60 analyzed the effects of expo- immortalized murine neural stem cell line. After sure to NPs composed of different forms of TiO2 24 h of incubation with these NPs, a dose-depen- in A549 cells and found that the size of the NPs dent decline in proliferation of cells and a differ- has an important role in determining toxic ef- entiation tendency towards neurons from ex- fects. In fact, the Authors observed that the posed neural stem cells were observed. Indeed, smallest anatase and P25 TiO2-NPs exerted the the Authors correlated these findings with the greatest cytotoxic effects. ability of TiO2-NPs to induce neuronal differenti- Conversely, other Authors have highlighted ation. To gain insight into the possible molecular the importance of the surface area of TiO2-NPs mechanism(s) of this neuronal differentiation, as being correlated to a stronger pro-inflammato- Liu et al74 mapped target proteins in lysates of 62,63 ry effect and oxidative stress . stem cells treated and untreated with TiO2-NPs, Concerning the shape, as parameter of and nine protein levels significantly changed.

TiO2-NP toxicity recenty it has been observed Subsequent mass spectrometric assay demon- that nanobelt- or nanofilament-shaped TiO2- strated that these altered proteins were signaling NPs, as opposed to other shapes of the same molecules, molecular chaperones, cytoskeletal nanomaterial, induce cytotoxicity64,65, likely components, and nucleoproteins. Furthermore, via apoptosis64. the Authors conducted also a gene expression

The toxicity of TiO2-NPs seems to be also analysis. They concluded by suggesting that the correlated to the surface chemistry of nanoma- regulation of protein kinase C epsilon gene ex- terials65, which affects inflammatory respons- pression is indirectly correlated with the neuronal 66 es . In fact, different from other anatase and differentiation of this stem cells exposed to TiO2- rutile NPs, only exposure of murine and human NPs. macrophages to silica-coated NPs resulted in sig- The aforementioned researches also report the α nificant induction of TNF- and neutrophil-at- internalization of TiO2-NPs by different types of tracting chemokines. neural cells, in particular, microglia cells69,70, Finally in several in vitro researches it was dopaminergic neurons70 and neural stem cells74. evaluated also the internalization of NPs in lung Particles were detected in the cytosol of cells as cells, another important aspect of TiO2-NP toxic- aggregates in membrane-bound vacuoles or in ity, which was shown to be dose-67,68 and time-67 endosomal vesicles69,70,74.

486 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies - 2 ; α ; – 2 and O 2 O (BV2 cells); 2 – 2 and of O particles caused a reduction of cell 2 2 O 2 increased the LPS induced TNF- in LPS augmented NF-kB activity 2 2 , superoxide; ROS, reactive oxygen species; TiO reactive , superoxide; ROS, – 2 mall clusters phagocytised and internalized into the cells mall clusters phagocytised pyrophosphatase (inorganic)1; Rho GDP dissociation (inorganic)1; pyrophosphatase inhibitor (GDI) alpha; 1, peroxiredoxin-6 nuclear mitotic 1- like pyrophosphoryle apparatus protein isoform CRA_b, tropomyosin 4,vimentin. change, energy production and hypoxia pathways, Nrf2- production and hypoxia pathways, change, energy stress. mediated oxidative • mithocondrial lying. Disrupted and swollen • protein 1, proteins: T-complex Down-regulated • of genes associated with adaptive Down-regulation • in N27. ATP Increased U87 cells;(HFF1) viability in U87 and HFF1 cells cells (NSCs) line C17.2 • Dose-dependent inhibition of cellular proliferation; • proteins: tumor reaction antigen gp96; Up-regulated cells • levels. Dose-dependent increase of ROS neurons;Primary cultures of embryonic rat striatum • with apoptosis, death of genes involved Up-regulation • Induction of apoptosis in embryonic striatum cells calcium signaling, inflammation, cell receptor families, stress; and oxidative cycling Nervous cells g/ml Human astrocytoma • Micro and nano TiO g/ml Mouse neural stem • (endosome vesicles); into cytoplasm Internalisation g/ml PC12 rat • Reduction of cell viability; μ μ μ . -NPs on nervous cells. -NPs on nervous 2 α m 0.1-100 μ B, nucleic factor-KB; Nrf2, Nuclear Factor-E2-related factor 2; O factor Nrf2, Nuclear Factor-E2-related B, nucleic factor-KB; κ 21 1-100 80-100 0-250 1 µm (fine) 25-200 µg/ml BV2 stimulated • TiO Ultrafine : ~ 30: 300-350; 2.5-120 mg/kg Immortalized mous 2.5-120 mg/kg • Rapid and sustained release of H BV2; • Rapid release of H 2 2 , tumor necrosis factor- 2 α 2 2 2 2 2 (80% anatase - 21 nm or not with LPS • TiO Ultrafine 2 Crystal phase Particle Doses of 99.7% pure anatase TiO < 2520% rutile) Human fibroblasts (ultrafine) stimulated cells. 30% rutile size) (aggregate mesencephalic • of caspase 3/7 in BV2 and N27 cells; activity Increased 30% rutile (BV2) cytoplasm; studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results , hydrogen peroxide; LPS, Lypopolysaccaride; NF- , hydrogen peroxide; LPS, Lypopolysaccaride; 2 O – (72) anatase) (largely pheochromocytoma • Increase of percentage apoptotic cells; – (73) rutile TiO – (70) 70% anatase - 800-1900 Rat N27 • Internalization in BV2 and N27 cytoplasm; – (69) 70% anatase - e brain microglia • S – (74) coated by SiO Long et al, 2006 P25 TiO Degussa Liu et al, 2010 P25 TiO Liu et al, 2010 Rutile TiO – (71) TiO Long et al, 2007 P25 TiO Degussa Shin et al, 2010 100% rutile TiO Lai et al, 2008 > 99% pure beta- 1-1.3 2 H NPs, titanium dioxide nanoparticles; TNF- Table II. Table

487 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi

Dermal and Mucosal Cells cells. On the basis of these results, the Authors The form of TiO2 is also an important parame- concluded that TiO2-NPs directly cause cardiac ter of TiO2-NP toxicity in dermal cells (Table cell damage, affecting the function of the cells. III). In various studies46,75-78, it has been reported However, further researches are needed to verify that anatase TiO2 is correlated with toxicity in if these effects are also caused by TiO2-NPs with human and murine dermal cells, though a recent different characteristics and using other cardiac study79 failed to report these effects on primary cell lines. human epithelial cells. The photocatalytic activi- Few studies have investigated the effects of ty of the anatase form may explain this greater TiO2-NPs on vascular cells (Table IV). Some toxicity. studies86-88 examined the effects of NPs on hu- Some of the aforementioned studies have also man and murine endothelial cells but did not find reported NP internalization by dermal cells. In par- significant effects, though the experimental mod- ticular, it was reported that anatase TiO2-NPs are els are limited. In particular, the Authors did not 75,78 internalized in the cytoplasm and, surprisingly, indicate the form of TiO2 used, which, as we have as aggregates in the nucleus of a small percentage previously seen, is an important parameter corre- of cells79. Kiss et al75 also reported that NP inter- lated with the toxicity of NPs. The lack of signif- nalization is correlated to the type of cells. icant effects following TiO2-NP exposure was al- Pan et al77 showed that NP internalization in so observed by Courtois et al89,90 using rat arter- human dermal fibroblasts is independent of the ies that simulate the in vivo situation. NP forms; only anatase NPs induced vesiculated In conclusion, further research is needed, tak- nuclei. Furthermore, these Authors underscored ing in consideration the characterization of the the importance of the NP surface chemistry, NPs and using experimental models that simulate which, if changed, altered adhesion to and pene- the in vivo situation, to clarify the effects of tration of the cell membrane. TiO2-NPs on vascular cells. Unfortunately, the scientific value of these internalization investigations is limited because Hepatic and Intestinal cells other in vitro studies have demonstrated that Few Authors have investigated the effects of

TiO2-NPs do not penetrate intact human or pig TiO2-NPs on hepatic and intestinal cells, and skin. In fact, they were found only in the superfi- there are not in vitro investigations on gastric cial layer of the stratum corneum but not in the cells (Table V). Indeed, there are only four stud- 91-94 epidermis and dermis. This phenomenon was ob- ies in the literature on the toxicity of TiO2- served with NPs of various forms, sizes, surface NPs on hepatic cells, and it seems that these NPs chemistries, and shapes, as well as with different do not exert direct effects, with the exception of applied formulations80-84. Finally, the lack of increase production of malonaldehyde (MDA)

TiO2-NP penetration in the skin was also ob- and ROS. On the contrary, after TiO2-NP expo- served in hair-removed skin, a model of damaged sure, significant alterations were observed with skin84. contemporary exposure of cells to visible light or to dichlorodiphenyltrichloroethane (p,p’-DDT). Cardiovascular Cells To date, there are only two articles that investi- Currently, there is only one in vitro study that gated the effects of TiO2-NPs on intestinal cells. 95 investigated the effects of TiO2-NPs on cardiac In the first , human colon carcinoma cells were 85 cells (Table IV). To this aim, Helfenstein et al exposed to TiO2-NPs with or without UVA irra- exposed patterned growth strands of cultured diation. The Authors observed that TiO2-NPs neonatal rat ventricular cardiomyocytes to alone were not cytotoxic. However, when the anatase TiO2-NPs. After 24 h it was observed a cells were exposed to UVA irradiation and TiO2- dose-dependent increase in impulse conduction NPs, they died at a much faster rate that was cor- velocity, maximal action potential upstroke ve- related to the NP concentration and duration of locity and ROS production. Moreover, laser scan- irradiation. The second study by Koeneman et ning microscopy revealed that the myofibrillar al96 investigated the pathways by which a mixture structure in cells treated with a 0.25 µg/ml TiO2 of anatase and rutile TiO2-NPs crossed a model preparation was less organized than in unexposed of the intestinal epithelium layer composed of cells. After a 24-h incubation with 2.5 µg/ml human Caco-2 cells. The Authors reported that

TiO2-NPs, TEM analysis demonstrated that Z the NPs were able to cross the intestinal epitheli- lines were thicker and not as regular as in control um layer by transcytosis without damaging its in-

488 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies Continued ] in fibroblasts ] in fibroblasts 2+ dose increased, 2 decreased involucrin, decreased involucrin, 2 and UVA in the presence of and UVA tical determination limit; 2 (> 94.2%) was recovered in the recovered (> 94.2%) was 2 TiO receptor fluid. 2 ount found in tape strips and skin preparations in the receptor fluid; 2 g/cm was found in the first 10 tape strips. found in the first was decreased cells stiffness; detected in the perinuclear area of HDFs and was μ 2 2 2 -carotene prevented photo-oxidative stiffness alterations. stiffness photo-oxidative -carotene prevented SOD levels. β • Increased lysosomes, and damaged organelles; • Dose-dependent reduction of cell viability; • and decreased GSH and LDH levels ROS Increased • Anatase decreased mithocondrial activity; • compared to rutile. of ROS Anatase induced greater levels skin fibroblasts TiO fibroblasts (L929)fibroblasts condensated fragmented chromatin and necrosed cells; HaCaT keratinocytecell line; HDF; cytoplasm; melanocyte • in [Ca elevation and reversible Slow Primary humanmelanocytes. on keratinocytes desmoglein-1 and P-Cadherin levels Human immortalizedsebaceous gland and melanocytes. cell line (SZ95); • Dose and time dependent decrease of cellular growth; • TiO markers: Differentiation Full thickness intact • were 98-100% and recoveries mean total Ti The Abdominal human • 50.7 Human immortalized • TiO CCL-110 (ATCC) • 2 2 2 g/ cm g/ml Mouse subcutaneous • Round and shrank cells as TiO μ μ g/ml Normal human • UVC alone or with TiO g/ml- Human dermal • Dose and time-dependent decrease of cell viability; μ μ Dermal and mucosal cells -NPs on dermal and mucosal cells. 2 10 4 mg/cm × 54 54 9 0.15-15 5 3-600 10.1 ± 1.0 3 3.2 ± 0.34 2 2 2 2 2 2 2 particles coatedparticles • by washing; removed applied was All Ti compartment (88.8% recovery); 2 2 Crystal phase Particle Doses of 40% rutile mixture TiO coated with • TiO trimethyloctylsilane particles coated with methicone (3.5-5.5%) TiO with silica (2-5wt %)(4.5-6.5%)methicone and SF emulsion withT-Lite 10% needle-likeTiO 100% rutile TiO hydrophobically •am Ti was in The the order of analy • No Ti in the • No TiO TiO studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results – (134) – (46) 60% anatase - 5.2 ± 0.34 3 mg/ml (HDF). fibroblast • Anatase increased LDH release; – (75) – (81) 3% T805 Degussa skin explants stratum corneum, 5.6% in epidermis, < 0.1% dermal – (80) with 10% needle-like porcine skin S, respectively; SF-S and T-Lite 86-93% in the T-Lite Gamer et al, 2006 SF-S emulsion T-Lite 30-60 Mavon et al, 2007Mavon UV emulsion with 20 2 mg/cm – (78) Vileno et al, 2007Vileno Anatase TiO Sayes et al, 2006 100% anatase TiO Jin et al, 2008 100% anatase TiO Kiss et al, 2008 Anatase TiO Table III. Table

489 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi -NPs); 2 ). 2 -NPs); 2 fects (higher for TiO ; 2 ARP, poly (ADP-ribose) polymerase; ARP, (11% cells) and nucleus invasion (11% cells) and nucleus invasion 2 altered expression levels of MMP-9 and levels altered expression 2 tration was higher in hair-removed skin treated higher in hair-removed tration was was not detected in epidermis and dermis. was 2 ecrease in the cells area (rutile), with actin fibers thinner ecrease in the cells area (rutile), with actin fibers less than 80% control decrease in contract matrix collagen. • Both particles types were internalised in cytoplasm; • Reduction of cell viability; • traction forces, ~ 59% reduction in migration and Weak • not into hair follicles but into vacant observed was Ti • No reduction of cell viability. Dermatomedhuman skin • not detectable as assessed by a Skin absorption was cell system. diffusion Bronaugh-type flow-trough JB6 cells • micropigsof Yucatan of apoptosis (TiO Induction with coated 35 nm TiO 2 2 2 cm tape strips (uppermost layers of the stratum corneum); 2 g/ml Epithelial cells of • of TiO Uptake g/ml HaCaT cells • in inflammation of genes involved Over-representation × μ -NPs, titanium dioxide nanoparticles; UVA, ultraviolet A; UVC, ultraviolet C. A; UVC, ultraviolet ultraviolet -NPs, titanium dioxide nanoparticles; UVA, μ 2 g/cm l of 10% Intact, stripped and • No penetration in intact or stripped skin; suspension skin hair removed •concen Ti μ Dermal and mucosal cells μ 2 -NPs on dermal and mucosal cells. 2 m citochrome c and decrease of Bcl – 2. cleavage, 100 dermis or viable epidermis. μ 21 0.1-100 Mouse epidermal •25 ef cytotoxic Dose-dependent 10-100 × 10 ± 1 solution applied porcin skin • 5 detected in the first was Only a trace amount of Ti < 5 90 ± 10. 60 ± 10; area • TiO 200 ± 13 0.1-0.5 mg/ml (rutile and anatase TiO and less extended 15.0 ± 3.5 0.4, 0.8 mg/ml HDF • D 35 and 250 2 2 2 2 2 2 2 2 2 2 2 (ST-01)(ST-21)(ST-41) 60.2 • 7 nm TiO MUC-4. 2 2 2 2 Crystal phase Particle Doses of Bulk 100% anataseTiO 20 cell adhesion. and 99.5% pure rutilehydrophilic TiO 25 ± 5; on 2 cm rutile TiO silicon coatedMixture of alumina 10 applied on 1 cm 20% rutile)Rutile TiO • and caspase 3, PARP of caspase 8, Bid, BAX Activation Bulk 100% anataseTiO 200 ITGB-6, and mucin FN-1, integrin MMP-10, fibronectin coated and silicon coated rutile TiO studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results – (77) Anatase TiO Pan et al, 2009Pan Rutile TiO 2009 – (135)Senzui et al, (80% anatase - Uncoated rutile TiO Wu et al, 2009Wu – (82) 99.5% pure anatase 5 ± 1; hydrophobic TiO 5% (w/w) ear Isolated • detected in receptor fluid; was No Ti Van der Merwe Van Nanocrystalline < 1 50 mg/cm 2010 – (84) Alumina/silica/ 35 TiO Hackenberg et al, Hackenberg Anatase TiO Zhao et al, TiO P25 Degussa 2010 – (79) human nasal mucosa (4% cells). et al, 2009 – (83)Fujita et al, TiO 2009 – (76) Bulk 100% anatase TiO 7 47.0, 58.8, Human keratinocyte • Reduction of cell viability; Table III. Table GSH, reduced glutathione; LDH, lactate dehydrogenase; MMP-9, matrix metalloproteinase-9; MMP-10, metalloproteinase-10; P ROS, reactive oxygen species; SOD, superoxide dismutase; Ti, titanium; TiO oxygen species; SOD, superoxide dismutase; Ti, reactive ROS,

490 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies , prostaglandin α and relaxant α particles did not impair Ach-induced 2 nogen activator inhibitor-1, PGF2 inhibitor-1, nogen activator was internalised into autophagic vacuoles; was 2 response to Ach were not modified. lines thicker and not regular compared to control cells. and not regular lines thicker rat arteries • Contractile response to KCl or PGF2 mice vascular mice vascular endothelial cells(MPMVEC) • No increase of DCF fluorescence and p38 phosphorylation; • expression. No time response increase of PAI-1 endothelial cells(HDMEC) proliferation; • IL-8 release. Increased (HAECs) cardiomyocytes(NRVM) • levels; Dose-dependent increase of ROS • with Z structure resulted less organized, Myofibrillar arteries and/or endothelial bovine cells Cardiovascular cells Cardiovascular -NPs on cardiovascular cells. -NPs on cardiovascular 2 15 200 µg/ml28 of rat Segments • Both TiO 0-10 µg/ml Pulmonary C57BL/6J • No reduction of cell viability; 20-30 0.0025-2.5 Neonatal rat • and maximal impulse conduction velocity Increased 70 (range 0.5-50 Human dermal • TiO -NPs, titanium dioxide nanoparticles. 2 2 nanotubes 30 N.A. Human aortic • endothelial cell proliferation and motility; Enhanced 2 2 2 2 2 Crystal phase Particle Doses of studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results ; ROS, reactive oxygen species; TiO reactive ; ROS, α Yu et al, 2010 Yu TiO – (87) Peng et al, TiO 2004 – (86)Courtois et al, TiO 20-160) µg/ml microvascular • on cell No reduction of cell viability and no effect Helfenstein et al, Anatase TiO 2010 – (88) 100 endothelial cells • Decreased inflammatory and coagulation molecules. 2008 – (85)Peters et al, TiO µg/ml ventricular action potential upstroke; 2008 – (89)Courtois et al, 2010 – (90) P25 Degussa TiO 15 0.14 µm 0-200 µg/ml Intralobar pulmonary • tone; No alterations of vascular intrapulmonary relaxation. Ach; acetylcholine; DCF, dichlorofluorescein; IL-8, interleukin-8; KCl, potassium chloride; N.A., not applicable; PAI-1, plasmi dichlorofluorescein; IL-8, interleukin-8; KCl, potassium chloride; N.A., not applicable; PAI-1, Ach; acetylcholine; DCF, 2F Table IV. Table

491 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi 2 -NPs, titanium dioxide 2 after 10 day treatment; alone increased ROS alone increased ROS 2 2 was toxic to cells with or was alone increased MDA 2 2 - DDT alone. I - DDT had higher ROS and MDA and MDA - DDT had higher ROS I and p,p 2 and p,p ; 2 2 catenin could be seen in epithelial sheet at 1-100 observed. levels; γ compared to TiO levels, while only 1 µg/ml TiO levels, intracellular free calcium. • TiO Mixed • reduction of cell viability; Weak • and increased organization Changes in microvillar lines (Ls-174-t) • Condensed cellular shape ad fragmented nuclei were cells (RLE) without UV irradiation. Hepatoma cell line • Induction of apoptosis with visible light illumination for intestinal cell line,Caco-2 • Human fetus not at 1000 µg/ml TiO µg/ml, but • were observed No dose and time response differences 2 3 2 mol/l L-02. apoptosis; μ -DDT • concentration of TiO Every I g/ml TiO Liver and intestinal cells μ combined with hepatic cell line treatment groups for cell viability and between any -NPs on liver and intestinal cells. -NPs on liver 2 40 0-250 µg/ml Immortalized rat • observed was effects No cytotoxic 21.2 200-1000 Human colon • irradiation. TiO Reduction of cell viability after UVA coated\ 2 2 (CDT) 2 2 2 2 2 Crystal phase Particle Doses of -DDT, dichlorodiphenyltrichloroethane; ROS, reactive oxygen species; TEER, transepithelial electrical resistance; TiO reactive ROS, dichlorodiphenyltrichloroethane; -DDT, I TiO rutile TiO UV - TITAN M160 UV - TITAN rutile TiO 20% rutile) p,p (80% anatase - 0-0.1 with alumina and\ stearic acid TiO studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results – (93) doped anatase – (94) TiO (Bel 7402) 10 min. Hussain et al, TiO 2005 – (92) et al, 2007Wang Ce element (IV) 21 10 µg/cm cells (BRL 3A) liver Linnainmaa et al, P25 Degussa 20 0-200 epithelial Rat liver • TiO Neither of the ultrafine 2004 – (95) µg/ml carcinoma cell alone resulted non cytotoxic; Zhang and Sun, TiO Koeneman et al, Koeneman 2010 – (96) Mixture of anatase and < 40 1-1000 Human brush µg/ml • to 1000 TEER dropped 6 days after chronic exposure border expressing µg/ml TiO 1997 – (91) uncoated anatase µg/cm Shi et al, 2010 P25 Degussa 25 0-1 Table V. Table MDA, malonaldehyde; p,p MDA, nanoparticles; UV, ultraviolet; UVA, ultraviolet A. ultraviolet UVA, ultraviolet; nanoparticles; UV,

492 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies tegrity and only causing subtle effects. The tran- NP dose induced a time-dependent viability re- scytosis of the epithelial cells, even at low levels, duction. TiO2-NPs inhibited population growth demonstrates the ability of NPs to be internalized according to a dose- and time-dependent rela- by cells and supports the role of the gastrointesti- tionship. Apoptosis was observed too. Unfortu- nal tract as a possible route of TiO2-NP entry into nately, details on the form of TiO2 used are miss- organisms. The alterations reported in individual ing. cells, although non-lethal, could impair the in- Similar effects were also observed in testinal mucosal homeostasis, as well as nutrient macrophage cell lines, particularly a decrease in absorption, with potentially health-impacting cell viability and an increase in apoptosis101. consequences, particularly in cases of chronic ex- However, no description of the TiO2 form used is posure. Moreover, these results suggest the need available. These findings also provide informa- to discover markers of early tissue damages to tion on the functional impairment of white blood detect toxic effects of TiO2-NPs. cells induced by TiO2-NPs. Indeed, impairment Finally, further researches are needed to verify of the phagocytic ability, phagosomal transport the effects of TiO2-NPs on hepatic and gastroin- and cytoskeletal stiffness has been reported in testinal cells, comparing to their characterization murine macrophages. Previously, similar results and NP internalization in these cells. Indeed, the were reported by Renwick et al102, though in this limited number of the studies did not allow us to study, phagocytosis was more strongly inhibited correlate any NP parameters (e.g., the form, size, by ultrafine TiO2 than by fine TiO2. Unfortunate- surface area, etc. of the TiO2-NPs) with adverse ly, the form of the TiO2 used is not described. effects. The pro-inflammatory action of TiO2-NPs has also been described16,103,104. In particular, Schanen Hematopoietic Cells et al103 investigated the immunogenicity of Data regarding the effects of TiO2-NPs on anatase TiO2-NPs, rutile TiO2-NPs and TiO2 nan- hematopoietic cells are heterogeneous due to the otubes on a human immunologic system (MIM- extremely different characteristics of these cells, IC) composed of blood vein endothelial cells and particularly regarding their functional properties monocyte-derived dendritic cells (DCs). In par- and the various endpoints evaluated by the pub- ticular it was observed an increase of pro-inflam- lished studies (Table VI). matory cytokines, such as IL-1 α, IL-1β, IL-6, IL-8, INF-γ and TNF-α, and the maturation of White Blood Cells DCs and the expression of co-stimulatory mole- Several Authors have investigated the cytotox- cules on their surfaces. Finally, TiO2-NP-pulsed ic effects of TiO2-NPs on white blood cells. In DCs were able to activate naive CD4(+) T cells particular, the cytotoxic effects of P25 Degussa and promote their proliferation. 104 TiO2-NPs were studied on human peripheral In a recent study , human neutrophils ex- 19 blood lymphocytes by Kang et al that reported posed to anatase TiO2-NPs manifested morpho- a dose- and time-dependent reduction in cell via- logical changes, such as irregular shape, in a bility, a dose-dependent increase of ROS in TiO2 dose-dependent manner. The exposure to NPs treated cells which was inhibited by the addition caused also a rapid tyrosine phosphorylation of of N-acetylcysteine. In a subsequent study97, the multiple proteins, including P38 and the Erk-1/2 same researchers found that P25 Degussa TiO2- protein kinases. NPs caused apoptosis of human peripheral blood Conversely, Morishige et al16 investigated the lymphocytes. Data regarding mixtures of anatase role of TiO2 form and NP size/shape in the in- and rutile are conflicting. Both necrotic and creased production of IL-1β detected in THP-1 apoptotic cells were detected when human lym- cells treated with TiO2 particles with or without phoma cells were treated with a suspension of LPS stimulation. Considering the TiO2 form, ru- mixed anatase and rutile NPs98. Conversely, Wan tile and spicular particles caused greater IL-1β et al99 failed to observe cytotoxic effects of simi- production than anatase NPs at all concentra- lar TiO2NPs on the same cell line. tions. Considering particle size, the smallest (10- μ The cytotoxic effects of TiO2-NPs on human nm) anatase particles and the largest (<5- m) ru- lymphoblastoid cells were observed by Wang et tile particles induced higher IL-1β levels than 100 al . other particles of the same TiO2 form, respec- In this investigation cell viability, decreased in tively. Moreover, the spicular rutile TiO2 parti- a dose-dependent manner, but only the highest cles caused greater IL-1 β production than

493 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi α Continued , and TNF- γ , IL-6, IL-8 INF- β , IL-1 α particles compared to 220 nm. 2 types reduced and increased percentage of 2 creased IL-1 in pro- MMP2 and MMP9 activities. • levels; No increase of ROS • nor changes neither in mRNA, expression No significant immunological • In (BD-AM)mouse macrophages(J744A.1) • Reduction of cell viability and enhanced apoptosis by • Reduction of cell proliferation in J744A.1; 20 nm TiO line (WIL2-NS) tumor monocytic-macrophages cellline (J774.2) J774.2 cells capable and unable of phagocytosing indicator beads, respectively. (U937) 2 g/ml Human B-cell • Dose and time dependent decrease of cell viability; g/ml U937 • on cell viability; observed No effect μ Hematopoietic cells M (MIMIC): construct levels; μ g/ml blood lymphocytes • cleavage; of caspase 9, 3, 8 - Bid and PARP Activation g/ml blood lymphocytes • levels. Dose dependent increase of ROS μ g/mm μ μ mg/ml lymphoma cells • aspects of apoptosis and necrosis. TEM images revealed μ -NPs on hematopoietic cells. 2 2920 0.0975-0.78 Mouse (BALB/C) 10-320 µg/ml • Both TiO Beagle dog alveolar • retard relaxation in both cell types; Increased 2525 0-100 20-100 Human peripheral • Dose and time dependent decrease of cell viability; Human peripheral • sub-G1 cells percentage; Increased 220 BALB/c/NIH • capacity in J744A.1 cells; Impaired phagocytic 7-10 0.05-100 Human N.A. 0-130 15-20 length: dendritic derived 70-150 cells (PBMC) diameter: endothelial (HUVEC) • of maturation (CD83, CCR7) and Increased 2 2 2 2 2 2 2 2 2 2 Crystal phase Particle Doses of 30-15% rutile) 30-15% rutile) • Mithocondrial depolarisation. TiO nanotubes 10-15; and monocytes- (CD86) molecules on dendritic cells. costimulatory studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results Kang et al, et al,Vamanu 2008 – (98) TiO P25 Degussa Anatase and rutile 99% pure TiO < 1 00 0.005-4 Human histiocytic • of apoptosis; Induction Renwick., et al, TiO 2002 – (101)2008 – (19)2009 – (97) (70-85% anatase, (70-85% anatase, macrophages; • in both cell types; stiffness cytoskeleton Increased 2007 – (100)Kang et al, TiO P25 Degussa lymphoblastoid cell • Dose dependent increase of apoptotic cells. Wang et al, Wang 99% pure TiO 2001 – (102)Möller et al, TiO 250 2009 – (103) Rutile TiO Wan et al, Wan 90% anatase and 20 0-20 2008 – (99)Schanen et al, 10% rutile TiO Anatase TiO Table VI. Table

494 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies ); 2 ); than 2 β ). ; 2 2 , with LPS; β m were attached to the g/ml) causing 50% μ , without LPS, while anatase μ β than the other anatase and rutile β -ribose) polymerase; ROS, reactive oxy- reactive -ribose) polymerase; ROS, aggregates were mainly attached along aggregates 2 estimated dose ( . 2 α particles and small aggregates were found particles and small aggregates 2 tile induced higher IL-1 respectively; than amorphous, rutile, and nano-anatase, respectively. complexes. membrane, not within cells. the membrane; particles, respectively; • of P38 and Erk-1/2 MAPK; Activation • Dose-dependent decrease of apoptosis; • of IL-8 levels. Increase • than 0.2 larger Aggregates • Dose-dependent increase of hemolysis; • Nano TiO • (nano TiO Dose-dependent increase of MDA , tumor necrosis factor- α Hematopoietic cells -NPs on hematopoietic cells. 2 ; IL-6, interleukin-6; IL-8, interleukin-8; LPS, Lypopolysaccaride; MAPK, mitogen-activated protein kinase; MAPK, mitogen-activated ; IL-6, interleukin-6; IL-8, interleukin-8; LPS, Lypopolysaccaride; β 40 0.1-0.2 mg/ml Human platelets • not changed by TiO CD62P (P selectin) was 40 induced higher IL-1 -NPs, titanium dioxide nanoparticles; TNF- 2 × × µm. blood cells inside cells; 4-6 0-100 Human neutrophils • shape; Dose-dependent induction of irregular 200 • agglutination (nano TiO of erythrocytes Induction < 25< 25< 50 0-20 mg/ml Human erythrocytes • TiO The < 5000 nm, anatase 25 rutile and amourphous, • 73, 11 and 1.3 times more potent Micro-anatase was 10 nm presence of LPS leukaemia cell • Considering the form, rutile induced higher IL-1 < 5000 444, 564, 4960, 32414 for anatase hemolysis was 10 < 5 µm; line (THP-1) anatase; ~10 < 25 nm; absence or in monocytic and spicular rutile induced higher IL-1 30-40 nm • Considering the size, 10 nm anatase and < 5 µm rutile , interleukin-1 β 2 2 ; IL-1 2 2 α 2 2 nanorods 4-6 0.4-10 µg/ml Rat whole blood • Dose-dependent increase of platelet aggregation. 2 2 2 2 2 Crystal phase Particle Doses of , interleukin- 1 Amorphous TiO Rutile spicular TiO Rutile spherical TiO α ; IL-1 studies that investigated the adverse effects of TiO effects the adverse studies that investigated γ In vitro Reference composition size (Nm) exposure Cell lines Results , interferon- γ – (106) TiO 2010 – (104)Rothen-Rutishauser 99.9% anatase 0.02-0.03 5 µg/ml Human redBihari et al, µg/ml • TiO 99.5% rutile TiO • of tyrosine phosphorylation; Induction Morishige et al, Anatase spherical < 50 µm; 20-500 µg/ml Human acute • Ru 2009 – (109)Nemmar et al, Rutile TiO for 10 min • on platelet granulocyte observed was No effect 2010 – (16) TiO et al, 2006 – (105) TiO 2008 – (108) Li et al, 2008 99.8% anatase 20 0-800 µg/ml Rabbit erythrocytes • sedimentation rate (nano TiO erythrocyte Increased 2009 – (107) Rutile TiO Aisaka et al, Anatase TiO Gonçalves et al, Gonçalves Anatase TiO INF- MDA, malonaldehyde; MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; N.A., not applicable; PARP, poly (ADP malonaldehyde; MMP-2, matrix metalloproteinase-2; MMP-9, metalloproteinase-9; N.A., not applicable; PARP, MDA, TiO gen species; TEM, transmission electron microscopy; Table VI. Table

495 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi

spherical rutile particles, which only differed ferent shape or size of these TiO2 nanomaterials from the spicules in shape. In addition, TiO2 in- or if NPs with other characteristics cause similar duced IL-1β production by caspase-1, ROS and or different effects. cathepsin B. In conclusion, these investigations suggest that Reproductive Cells the form, size and shape of NPs influence the ef- There are only three studies that investigated fects of TiO2-NPs, in particular, on the pro-in- the effects of TiO2-NPs on reproductive cells flammatory action of white blood cells. However, (Table VII). In the first study, Komatsu et al110 further studies are needed to clarify the role of studied the direct effect of TiO2-NPs on the these characteristics in the TiO2-NP toxicity on mouse testis Leydig cell line. However, the form white blood cells. of TiO2 used was not detailed. The findings showed that NPs were internalized by the cells Red Blood Cells and were found as agglomerates in the cytoplasm There are few papers in the literature that inves- but not inside the nucleus. Furthermore it was al- tigated the response of red blood cells to TiO2-NP so detected a dose-dependent reduction in cell vi- 105 exposure. Rothen-Rutishauser et al incubated ability. A concentration of 100 µg/ml TiO2-NPs human red blood cells with anatase TiO2-NPs for 4 for 24 h reduced proliferation, which, however, to 24 h. TiO2-NPs or < 0.2-µm small aggregates was restored after this time point. Additionally, were detected inside cells, while larger aggregates no oxidative stress was determined by TiO2-NP were observed only outside the cell, though at- exposure. tached to the membrane. These results are very im- The other two studies were conducted on a portant considering of the fact that erythrocytes cell line of the female reproductive system, sup- have no specific phagocytic properties. porting a potential role of ROS production in in- Furthermore, on the basis of their experiments, ducing reproductive cellular alterations111,112. Li et al106 suggested that the membrane attach- In conclusion, additional studies are needed to ment of similar NPs could change the native sur- verify these findings and the presence of other face properties of the red blood cells, leading to effects, particularly those implicated in mutagen- hemoagglutination, while TiO2-NP trans-mem- esis. brane insertion breaks the erythrocytes causing hemolysis106. Hemolysis was also observed in Renal Cells human erythrocytes in a subsequent study107. The In the literature, there is only one study113 that

Authors exposed these erythrocytes to anatase investigated the in vitro effects of TiO2-NPs on re- TiO2-NPs, anatase TiO2 microparticles, rutile nal cells (Table VII). This report indicates that TiO2 microparticles and amorphous TiO2-NPs for TiO2-NPs are cytotoxic for tubular but not mesan- 1 h. Hemolysis was then assessed by the percent- gial cells. Further, the size of NPs seems to be im- age of hemoglobin released from the cells. plicated in the onset of this damage. Finally other

Anatase TiO2 microparticles resulted in approxi- in vitro studies are needed to verify these results mately 73, 11, 1.3 times more hemolysis than and the observed internalization of TiO2-NPs. amorphous TiO2, rutile TiO2 and nano-sized anatase TiO2 particles, respectively. These find- Bone Cells ings suggest that hemolysis may also be correlat- There are two papers in the literature concern- 114,115 ed with the form of TiO2. ing the effects of TiO2-NPs on bone cells (Table VII). The Authors demonstrated that in Platelets MG-63 cells cultured on anatase-coated disks, Data regarding platelet aggregation are con- several genes were significantly up- or down-reg- flicting. Nemmar et al108 demonstrated dose-de- ulated. These genes are implicated in signal pendent platelet aggregation caused by rutile transduction, immunity, cell cycle regulation, 109 TiO2 nanorods. However, Bihari et al showed lysosome composition and vesicular transport, that incubation of human whole blood with rutile cell adhesion, proliferation, apoptosis, and as cy-

TiO2-NPs for 10 min did not affect platelet P-se- toskeletal and extracellular matrix components. lectin expression, nor the formation of platelet- Unfortunately the release of TiO2-NPs into the granulocyte complexes. cell culture medium was not quantified. Thus, al- Thus, further researches are needed to verify if tered gene regulation cannot be correlated to spe- these contrasting results are correlated to the dif- cific doses of NPs released.

496 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies (100 µg/ml) 2 - radicals after UVA 2 cell lines; agglomerates in the cytoplasm but agglomerates in the cytoplasm 2 were incorporated into cytoplasmic vesicles. were incorporated into cytoplasmic 2 ction of cell proliferation; ction of inflammatory and coagulation molecules. oduction of OH- and CO transduction, immunity, cell cycle regulation, lysosomes regulation, cell cycle transduction, immunity, proliferation and apoptosis. induced cytotoxicity without UVA irradiation; without UVA induced cytotoxicity • Dose-dependent reduction of cell viability; • Reduction of cell proliferation. ters ovary- • Pr (UMR 106)cells (HASM) treated cells. isoprotrenol relaxation. K1 cells (CHO-K1) irradiation. cell line (IP15);proximal epithelial tubularcell line (LLC-PK1) observed in both • of F-actin Vesicles •TiO nm 15 coated line (MG63) 2 2 Other mammalian cells disks transport, cell adhesion, and vesicular composition µg/cm -NPs, titanium dioxide nanoparticles; UVA, ultraviolet A. ultraviolet -NPs, titanium dioxide nanoparticles; UVA, 2 -NPs on other mammalian cells (reproductive, renal, bone and muscular cells). -NPs on other mammalian cells (reproductive, 2 5 10-500 µg/mlhams Chinese 17 1525 0-300 µg/ml Rat osteosarcoma 0-200 µg/ml Human airway • effects; of cytotoxic Induction • of cell viability; Increase 2-70 0 -1000 µg/ml Mouse testis Leydig • Dispersed TiO , 30 • viability. Rutile did not affect 2 2 2 . N.A. Cells cultured on Osteoblast cell • in signal of genes involved Up- or down-regulation 2 2 2 2 2 , 99% anatase, , > 1% anatase, 37 , 91% anatase,, 75% anatase, 15, 78% anatase, 0-100 µg/ml 90 Chinese hamsters • Dose-dependent reduction of cell viability (anatase • High concentration of anatase form TiO 2 2 2 2 2 nanotubes 30 N.A. smooth muscle Vascular • Redu 2 2 Crystal phase Particle Doses of St-3 TiO 1% rutile Amorphous TiO < 99% rutile F1-R 3% anatase, TiO2, 97% rutile 93 65% anatase/rutile TiO 25-75 Normal pig kidney • detachment were Abnormal cell size and surface 81% anatase, 19% rutile F-1 TiO Anatase form TiO F-4 TiO 25% rutile 22% rutile St-2 TiO studies that investigated the adverse effects of TiO effects the adverse studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results , carboxyl radical; N.A., not applicable; OH-, hydroxyl TiO – 2 – (112) – (113) rutile TiO Komatsu et al, Komatsu TiO Sollazzo et al, Anatase TiO 2010 – (115)Berntsen et al, 2010 – (116)Peng et al, TiO Degussa TiO cell line -derived smooth muscle • in materials were observed with phagocyted Vesicles • histamine contraction and Altered cell stiffness, Uchino et al, F-6 TiO 2008 – (110) cell line TM3 not in nucleus; L’Azou et al, 2008L’Azou 98% anatase/ 15 et al,Di Virgilio 100% anatase TiO 0.625-160 Human mesangial • effects; of cytotoxic Induction 2008 – (114) anatase TiO Dodd et al, 2009 Anatase TiO 2010 – (88) cells (VSMCs) • Redu 2002 – (111) 9% rutile cells (CHO) ovary irradiation; form) after UVA CO Table VII. Table

497 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi

Further, Di Virgilio et al115 studied the cytotox- length. An increased mutation frequency was al- 119 ic effects of 5 to 300 µg/ml anatase TiO2-NPs ap- so recently reported by Xu et al . The other ef- plied for 24 h to rat osteosarcoma-derived cells. fect (i.e., an increase in DNA tail length) report- A significant increase in adsorbance was deter- ed by Wang et al100 was also previously shown by mined at ≥150 µg/ml and ≥25 µg/ml NPs, in both Nakagawa et al120 who observed an increase in neutral red uptake and MTT assays, respectively. the frequency of aberrant chromosomes. The trypan blue method revealed that cell prolif- Finally, recentely, Bhattacharya et al49 reported eration also decreased with exposure to NP con- an increase in DNA adduct formation and the ab- centrations of up to 100 µg/ml for 24 to 96 h. sence of DNA damage. TEM was also used to observe intracellular vesi- Similar to the results above, other researches cles with phagocytosed material in cells treated did not confirm the genotoxic effects (e.g., DNA 79 121 with 50 or 100 µg/ml TiO2-NPs for 24 h. damage , chromosome aberration and MN The findings of these studies, though limited, generation with or without UV irradiation91) of suggest toxic effects of TiO2-NPs on bone cells, TiO2-NPs. which should be further tested by in vivo studies. All of these conflicting data do not reveal a However, to our knowledge, such studies are clear correlation between genotoxicity and the lacking. characteristics of NPs, likely due to the different

cell lines and TiO2-NPs used, the various end- Muscular Cells points investigated, and the assays utilized. In There are two recent articles that evaluated the any event, the studies suggest that care should be effects of TiO2-NPs on smooth muscle cells taken when considering parameters such as UV (Table VII). In the first study116, human airway irradiation120, co-exposure with other sub- smooth muscle cells were exposed to Degussa stances94 and NP surface chemistry (which may

TiO2-NPs. The researchers measured cell viabili- play a role or have synergistic effects in inducing ty and cell stiffness and agonist-induced contrac- genotoxic alterations53) for a correct interpreta- tility, respectively. TiO2 dose-dependently in- tion of these results. Ultimately, further investi- creased the nominal viability by up to 20% at gations are needed to clarify the genotoxic role

200 µg/ml. Further, doses of up to 50 µg/ml TiO2 of TiO2-NPs. altered cell stiffness, the contraction induced by histamine and relaxation induced by isopro- terenol by no more than 50%; these alterations exhibited no clear dose dependence. In the other Discussion study88, gene expression analysis of vascular smooth muscle cells exposed to TiO2 nanotubes The recent rapid growth of nanotechnology for 24 h revealed a reduction in proliferation and and the specific attractive properties of nanoma- in the expression of molecules involved in in- terials have led to the widespread application of flammation and coagulation. TiO2-NPs in the field of life sciences, as well as The findings of these studies suggest that in the biotechnology, pharmaceutical, cosmetics

TiO2-NPs affect smooth muscle cells indepen- and textile industries. Several concerns regarding dent of the form of TiO2 and the shape of the the potentially greater biological activity of TiO2 NPs. Regardless, further research is required to in the nanoparticulate form, related to the smaller clarify and evaluate other effects. size and corresponding larger surface area122, and

the increasing worldwide TiO2-NP distribution, Genotoxicity with the consequent augmented likelihood of hu- Data regarding the genotoxicity induced by man exposure, have emerged. Moreover, the yet-

TiO2-NPs are conflicting and heterogeneous (Table limited knowledge of TiO2-NP toxicological VIII). Different studies have principally reported properties do not allow one to extrapolate toxico- 11,19,48,52,53,93,94,100,117 micronuclei (MN) formation or logical conclusions to TiO2-NPs. the presence of multinucleated cells100,118. Some Based on the studies that we reviewed, though of these investigations reported also other geno- they differ in the cell lines, the intensity and du- toxic effects, such as inter-nucleosomal fragmen- ration of exposure, endpoint parameters, and tation11 and DNA damage19,48,52,94. In particular, measurement techniques, interesting and critical Wang et al100 also observed an increased frequen- points emerge that we argue will be the object of cy of mutations and an increase in DNA tail future research.

498 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies Continued ); 2 treated 5 O 2 anatase). 5 O 2 was toxic to cells with or was 2 increased MN; 2 samples increased MN with or without UV 2 showed a dose-dependent increase in showed 2 either of the ultrafine TiO either of the ultrafine without UV irradiation; nuclear periphery; • block proliferation index; Decrease of cytokinesis • tail moment (Comet assay); 5-fold increase in olive • (HPRT). 2.5-fold increase in mutation frequency • of chromatin at the Compaction and marginalization • fragmentation of the DNA. Internucleosomal embryo cells (SHE) • TiO (ultrafine effects Induction of cytotoxic Hepatoma cell • with visible light Detection of MN after 4 hr exposure Chinese hamster • were induced by V Genotoxic effects 2 3 Genotoxicity 2 2 µg/cm -NPs on mammalian cells. 62.5-250; cells ovary that of controls at any not increased above groups was 2 750-2500 µg/ml concentration. 20 0.5-10 Syrian hamster • TiO Ultrafine 200 200 • 10 and 200 nm sized anatase increased MN. ≤ N.A. 0-130 µg/ml WIL2-NS • Increased micronucleated binucleated cells; 30-50 1-100 µg/cm 0.42 µm cell line CHL/IU with irradiation. chromosome aberration frequency 0.021 µm0.255 µm0.255 µm 0-3200 µg/ml Mouse lymphoma • Dose-dependent increase in mean tail length (p-25, WA cell line (L5178Y); Chinese hamster and TP-3); • P25 TiO 10 and 20 10 µg/ml BEAS-2B • damage by 10, 20 nm DNA of oxidative Induction ated 20 0-200 RLE • N 2 2 2 2 2 with 2 2 2 2 2 2 coated irradiation. 2 2 ) treated (V79) • increase of MN (V Threefold (CDT) 5 2 2 O 2 Crystal phase Particle Doses of anatase TiO UV - TITAN M160UV - TITAN • None TiO TP-3 rutile TiO WR rutile TiO USP purity Rutile TiO (V with alumina and stearic acid Pigmentary uncoatedanatase TiO 170 rutile TiO TiO studies that investigated the genotoxic effects of TiO the genotoxic effects studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results Bhattacharya Anatase TiO Nakagawa et al,Nakagawa P25 anatase TiO Wang et al, Wang Ce element (IV) 21 10 µg/cm Rahman et al, TiO 1997 – (120)Linnainmaa et al, anatase TiO WA unco Degussa P25 2005 – (52)2007 – (100) > 200 anatase and 200 nm rutile; Warheit et al,Warheit 2007 – (121)et al, 2008 – (53) 79% rutile-21% anatase TiO pentoxide Vanadium 140 ± 44 25-100; Chinese hamster • percentage of cells with aberration in the treated The lung fibroblasts anatase, not by untreated anatase; 2002 – (11) > 200 µg/cm Gurr et al, Anatase TiO Wang et al, Wang 99% pure TiO 1997 – (91) anatase TiO 2007 – (93) doped anatase line (Bel 7402) illumination. Table VIII. Table

499 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi ; 2 ); 2 2 – DDT as regards – DDT as regards I and p,p 2 nthine-guanine phosphoribosyltrans- ction of cell viability (40 nm TiO led to interstices around nuclear 2 time-dependent activation of ERK1/2 cascade; time-dependent activation citotoxicity in hydrophobic than hydrophilic TiO telophase, higher subG1 phase population at long term exposure. checkpoint kinases. • of micronucleated BEAS 2B cells frequency Increased -NPs, titanium dioxide nanoparticles; UV, ultraviolet. -NPs, titanium dioxide nanoparticles; UV, 2 IMR 90 • of 8-OHdG. Induction hepatic cell breaks and MN; DNA Genotoxicity 2 2 2 -NPs on mammalian cells. 3 days up • of MN in long term exposure; Increase to 12 wks • of progression at anaphase and affection G2/M delay, 2 10 µg/ml every 10 µg/ml every fibroblasts • of BNMN cells; Increase 40 1-100 BEAS 2B • rutile and anatase); damage (fine of DNA Induction 5 0.1-100 µg/ml Gpt delta transgenic •redu Dose-dependent × 4015 for 24 hr; 0-50 µg/ml mouse primary NIH 3T3; • Increase of mutation frequencies at red/gam gene loci; • production; of ROS Increase 25 0-1 µg/ml Human fetus • action of TiO Synergistic 25; 0-100 µg/ml Human peripheral • Dose-dependent increase of MN; < 25 < 25 10-100 Epithelial cells of • by comet assay. observed damage was No DNA < 100 2-50 BEAS-2B; • No induction of DNA-breakage; < 5 µm 325 mesh 0.1-30 µg/ml embryo fibroblasts • stress. of oxidative Induction major axis types co-cultures; in diameter for 3 days (MEF) 2 2 2 2 2 2 2 3 3 2 2 coated with µg/cm (84-92%) minor axis;(77-86%) cells • Higher membrane and multinucleated cells. (< 5%) (anatase). 2 2 2 2 2 Crystal phase Particle Doses of 99% anatase TiO -DDT, dichlorodiphenyltrichloroethane; ROS, reactive oxygen species; TiO reactive ROS, dichlorodiphenyltrichloroethane; -DDT, treated with ZrO2Al(OH) I 20% rutile) line L-02 • Increase of 8-OHdG formation. 99.7% anatase TiO ≥ SiO ZrO2Al(OH) and steric acid 30-15% rutile) XRDtreated with • damage of DNA Accumulation of p53 and activation 200-300 • in both TiO observed was conversion Tumorigenic Hydrophobic rutileTiO • Hydrophobic TiO 99.9% rutile TiO studies that investigated the genotoxic effects of TiO the genotoxic effects studies that investigated In vitro Reference composition size (Nm) exposure Cell lines Results – (19) (70-85% anatase, ~30 nm in blood lymphocytes • breakage; Dose and time dependent increased DNA Hackenberg et al, Hackenberg Anatase TiO Kang et al, 2008 TiO P25 Degussa Bhattacharyaet al, 2009 – (49) Anatase TiO µg/cm 2010 – (79) µg/ml human nasal mucosa – (119) 99.9% anatase TiO – (94) (80% anatase- TiO Huang et al, 2009– (117) TiO Onuma et al,2009 – (118) Hydrophilic rutile TiO 40-70 312 µg/ml QR2 fibrosarcoma for 24-72 hr; • levels; of ROS Increase Human HFW • and Dose Falck et al, 2009Falck – (48) 99.5% pure rutile 10 TiO Shi et al, 2010 TiO P25 Degussa Xu et al, 2009 99.7% anatase TiO Table VIII. Table 8-OHdG, 8-hydroxyl-2-deoxyguanosine; BNMN, binucleated micronucleated; ERK 1/2, extracellular-receptor kinase 1/2; HPRT, hypoxa kinase 1/2; HPRT, 8-OHdG, 8-hydroxyl-2-deoxyguanosine; BNMN, binucleated micronucleated; ERK 1/2, extracellular-receptor ferase, MN, micronulei; p,p

500 Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies

Firstly, it should be noted that most of the ef- and susceptibility between cell types129,131,132, im- fects observed in in vitro studies were obtained plying that more studies are required to deter- using excessively and unrealistically high doses mine the conditions in which TiO2-NP genotoxi- of NPs15,123. Thus, the direct extrapolation of city occurs. However, for these engineered nano- these results to humans under realistic, lower ex- materials, there is simply no adequate epidemio- posure scenarios must be questioned124. However, logical information to conclude whether or not though the results of such studies have to be in- there is an association between exposure and terpreted with caution and the relevance of these lung cancer risk to date133. In particular, it will be data is dubious, they could lead to focused future interesting to elucidate if TiO2-NPs act by induc- works on the detection of early signs of more se- ing modifications directly on DNA or regulating vere damages induced by higher dose exposures. gene expression, namely through mutagenic or From our review, it is evident that additional epigenetic mechanisms, respectively. Moreover, research is needed to more thoroughly investigate because carcinogenesis is a multistep process, the toxic effects derived from NP exposure, par- the role of TiO2-NP action in inducing the initia- ticularly in relation to the particle characterization event or in the subsequent processes of pro- tion, which, in our opinion, is the first step to motion or progression should be clarified. Tests reach a comprehensive and appropriate identifi- evaluating mutagenicity should be widely per- cation of the TiO2-NP hazards. Various physico- formed in both in vitro and in vivo studies to con- chemical features (e.g., size, shape, composition, firm the alterations induced by TiO2-NPs and in charge, crystallinity, solubility, added functional relation to the particle characteristics to under- groups and impurities) can be combined in any stand the mechanisms of action and to establish particular type of NP, leading to different toxic threshold values. potentials125-127. Unfortunately, the lack of and the The use of nanotechnological products will heterogeneity in the TiO2-NP parameters exam- likely increase sharply over the next decade. Be- ined in different studies leads to a non-homoge- cause of this increase, NP-related research should neous classification of the exposures and to a bi- carefully balance the improvement of nanotech- ased assessment of the exposure-response rela- nology and human health through the optimal tionships125. To overcome this potential bias, a properties of nanomaterials with the need to iden- homogeneous exposure classification should be tify potential hazards derived from unintentional used in future studies to thoroughly clarify how or intentional exposures122. In fact, the same NP the physicochemical properties of TiO2 nanocrys- properties that may be useful for the development tals correlate with their toxicological effects. of nanomedicine and specific industrial processes Useful parameters for systematic categorization (e.g., antioxidant activity, carrier capacity for could include the TiO2 form, number concentra- therapeutics and catalytic capacity for chemical tion, surface area, mass concentration, weighted reactions) could be harmful when NPs interact size distribution, state of agglomeration, surface with cells by inducing toxicity, oxidative stress or reactivity (e.g., the ability to produce radicals cellular dysfunction122. A database composed of and zeta potential), chemical composition and the results of toxicological tests could provide a 38 morphology . comprehensive set of information useful for TiO2- Moreover, an argument that merits greater fo- NP material safety data sheets, as well as a basis cus in future research (due to the limited number for potential NP risk assessments and risk man- of studies in that regard) is the possible roles of agement122. Currently, the public awareness of

TiO2-NPs in genotoxicity and carcinogenicity. nanotechnology in everyday life is limited. How- The IARC recently classified TiO2 as possibly ever, some pressing concerns are related to the 33 carcinogenic to humans (Group 2B) . TiO2-NPs risks posed by occupational exposure, particularly had a carcinogenic effect on lungs in rats128, and during manufacturing or processing for industrial several studies have reported positive in vitro and applications. Nevertheless, evidence is not exten- in vivo genotoxicity of this nanomateri- sive or definitive, and there are no published stud- al11,19,48,52,53,93,94,100,117-119,129, though these effects ies on the risks of occupational exposure to engi- were not detected in other studies79,91,121,130. Dif- neered NPs125. Moreover, studies on long-term ferences in findings between studies may be due exposure and chronic effects have not been car- to how TiO2-NPs differ in terms of production, ried out to date because such exposure is relative- particle size, degree of aggregation, preparation ly recent and generally occurs in controlled situa- method, incubation or exposure conditions, dose

501 I. Iavicoli, V. Leso, L. Fontana, A. Bergamaschi

tions. Therefore, given the increasing use of TiO2- References NPs, epidemiologic investigations of exposed workers will be needed in the near future to pro- 1) NIOSH (NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY vide an assessment of possible risks. Epidemio- AND HEALTH). Approaches to Safe Nanotechnolo- logic investigation will form an important link in gy: managing the health and safety concerns associated with engineered nanomaterials. Centers understanding health outcomes associated with for Disease Control and Prevention, Atlanta, GA; exposure to potentially hazardous materials. Such 2009. studies will form the basis for quantitative risk es- timations to establish levels that protect human 2) NSF (NATIONAL SCIENCE FOUNDATION). Societal implications of nanoscience and nanotechnology. health. 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