Marine Environmental Research xxx (2014) 1e6

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Effects of in vitro exposure to titanium dioxide on DNA integrity of bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes Giada Frenzilli a,1, Margherita Bernardeschi a,1, Patrizia Guidi a, Vittoria Scarcelli a, Paolo Lucchesi a, Letizia Marsili b, Maria Cristina Fossi b, Andrea Brunelli c, Giulio Pojana d, Antonio Marcomini c, Marco Nigro a, * a

Dipartimento di Medicina Clinica e Sperimentale, Università di Pisa, sezione di Biologia applicata e genetica, Via A. Volta, 4-56126 Pisa, Italy Dipartimento di Scienze Ambientali, Università di Siena, Via Mattioli, 4-53100 Siena, Italy DAIS-Dipartimento di Scienze Ambientali, Informatiche e Statistiche, Università Ca’ Foscari, Calle Larga S. Marta 2137, 30123 Venice, Italy d Dipartimento di Filosofia e Beni Culturali, Università Ca’ Foscari, Dorsoduro 3484/D, 30123 Venice, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 November 2013 Received in revised form 7 January 2014 Accepted 9 January 2014

In the present study, the genotoxic potential of nanosized TiO2 anatase and micro-sized rutile on bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes was investigated. Human and mouse cells were also studied in order to compare susceptibility to TiO2 in different mammalian species. Cell lines were exposed for 4, 24, and 48 h to different concentrations of TiO2 (20, 50, 100, 150 mg/ml) and DNA damage was investigated by single cell gel electrophoresis (Comet assay). Both anatase and rutile induced increased DNA damage, even though statistically significant effects were scattered according to species and cell lines. Bottlenose dolphin leukocytes and murine fibroblasts exhibited increased DNA damage after rutile exposure at some doses/times, while human fibroblasts showed a significant doseeresponse effect after a 4 h exposure to anatase. Human leukocytes were tolerant to both anatase and rutile. Ultrastructural investigation showed that TiO2 particles entered the cell and were compartmentalized within membrane-bound vesicles. Ó 2014 Published by Elsevier Ltd.

Keywords: Titanium dioxide Nanomaterials DNA damage Bottlenose dolphin Comet assay

1. Introduction Among particulate metal oxides, titanium dioxide (TiO2) is one of the most widely used in the composition of such consumer products as cosmetics, toothpastes, sunscreens, and pharmaceuticals; moreover, it is also used for many industrial products and processes, such as paints, plastics, building materials, paper, and waste water photo-catalytic treatment (Aitken et al., 2006; Robichaud et al., 2009). Due to its large-scale and ever-increasing production, TiO2 has the potential to become an “emerging contaminant” of marine ecosystems in the near future. Although the data on TiO2 toxicity are still controversial, many papers report that TiO2 (mainly nanosized) is responsible for oxidative damage, inflammation, fibrosis and lung tumor in mammalian systems (Bermudez et al., 2004; Warheit et al., 2007). Based on this evidence, the International Agency for Research on Cancer classified TiO2 as “possibly carcinogenic to humans” (Group 2B) (IARC, 2010).

* Corresponding author. Tel.: þ39 050 2219113; fax: þ39 050 2219101. E-mail address: [email protected] (M. Nigro). 1 These authors contributed equally to the present work.

For this reason numerous studies have dealt with the genotoxic potential of TiO2 (Singh et al., 2009). However, such studies often report conflicting results, some depicting TiO2 as fundamentally non-genotoxic (Falck et al., 2009; Hackenberg et al., 2010, 2011; Wang et al., 2011), while others report significant DNA and/or chromosomal damage after exposure to TiO2, either alone or in combination with UV radiation (Gurr et al., 2005; Wang et al., 2007; Falck et al., 2009; Shukla et al., 2011; Jugan et al., 2012; Guichard et al., 2012). However, published genotoxicological data for nanomaterials are difficult to compare even for the same NP, due to a range of factors including level of characterization, experimental conditions, sample purity and stability, dispersion protocols, and other physicalechemical properties (Magdenolova et al., 2013). Only during the last decade has the potential impact of TiO2 on aquatic ecosystems become a topic of interest for ecotoxicologists (Moore, 2006; Menard et al., 2011). This is related to the fact that up to 7.5 million tons of particulate TiO2 are expected to enter the marine environment in the near future (Owen and Depledge, 2005; Farrokhpay et al., 2010). Although the environmental fate of TiO2 is hardly predictable, it has been noted that filter and/or deposit feeders are capable of accumulating TiO2 and could potentially

0141-1136/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.marenvres.2014.01.002

Please cite this article in press as: Frenzilli, G., et al., Effects of in vitro exposure to titanium dioxide on DNA integrity of bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.01.002

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transfer it through the marine food web (Holbrook et al., 2008; Galloway et al., 2010; Montes et al., 2012; Barmo et al., 2013). At present, data relating to the aquatic ecotoxicology of TiO2 is limited to a few freshwater and marine invertebrates (Lovern and Klaper, 2006; Galloway et al., 2010; Barmo et al., 2013; D’Agata et al., 2014) and in vitro and in vivo fish studies (Federici et al., 2007; Vevers and Jha, 2008; Boyle et al., 2013). To date limited attention has been paid to the potential impact on marine top predators, which might be exposed through the food web. To the best of our knowledge, the only published toxicological data on the subject are our preliminary results on Tursiops truncatus leukocytes (Bernardeschi et al., 2010). In light of this paucity of information, the main purpose of the present investigation is to improve the data set on bottlenose dolphin leukocytes from captive specimens, to extend the study to fibroblasts sampled by skin biopsy from freeranging animals, and to observe the susceptibility to TiO2 of other mammalian models besides dolphins, namely human and mouse cells. Fibroblasts and leukocytes cannot be considered target cells of alimentary exposure to TiO2; however, these cell types can be sampled through non-destructive methods and can be easily cultured for in vitro studies. The use of skin biopsy has been validated as a non-destructive method for studying the susceptibility of free-ranging cetaceans to different pollutants and to assess their health status (Fossi et al., 2000, 2003, 2007; Panti et al., 2011). Similarly, primary cultures of circulating leukocytes have been used for ecotoxicological investigations on bottlenose dolphins reared in captivity (Betti and Nigro, 1996; Taddei et al., 2001) as well as on wild specimens (Lee et al., 2013). Nanometric TiO2 anatase and micrometric rutile particles were selected on the basis of literature data which reported nano-sized anatase and sub-micrometric rutile as genotoxic TiO2 forms, in the absence of photo-activation (Gurr et al., 2005; Karlsson et al., 2009; Guichard et al., 2012). Alkaline single-cell gel electrophoresis (Comet assay) was used to quantify DNA integrity loss. 2. Material and methods 2.1. Chemicals Anatase (batch number 59496MJ) and Rutile (batch number 03008JS) particles, Methyl Methane Sulfonate (MMS), Low Melting Agarose (LMA), Normal Melting Agarose (NMA), Ethidium Bromide (EtBr), Triton X-100, Ethylenediaminetetraacetic acid (EDTA), Trizma Base, Dimethyl Sulfoxide (DMSO), NaOH, TriseHCl, and Trypan blue were purchased from SigmaeAldrich (Germany). Chemicals and cell culture media for storing and processing T. truncatus skin biopsies were purchased from GibcoÒ, specifically: MEM Eagle Earle’s salts w/L-glutamine and sodium bicarbonate supplemented with 10% gamma irradiated fetal calf serum with 1% MEM non-essential aminoacids solution 100, 1% Penicillin/ Streptomycin 100, 0.1% Amphotericin B 100, Earle’s Balanced Salt Solution (EBSS), Trypsin-EDTA 1. Chemicals and cell culture media used with human skin (HuDE) and mouse embryo fibroblasts (3T3) were obtained from EuroClone SpA (Italy), specifically: Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), antibiotic and antimycotic solution 1% Pen/Strep, 1% L-Glut Trypsin 2.5%; RPMI. All other chemicals were obtained locally and were of analytical reagent grade. Cell culture wares were obtained from EuroClone SpA (Italy). 2.2. Characterization of TiO2 particles According to the Supplier, the TiO2 powders had the following characteristics: anatase: 99.7% purity (metals basis); nominal

size < 25 nm; specific gravity/density, 4 g/cm3; rutile: 99.9% purity (metals basis); nominal size 13) and electrophoresed for 200 at 25 V and 300 mA. After electrophoresis, the slides were neutralized with TriseHCl (0.4 M, pH 7.5), stained with ethidium bromide and observed under a fluorescence microscope (400). The percentage of DNA that had migrated toward the anode (tail DNA) was quantified by an image analyzer (Kinetic Imaging Ltd., Komet, Version 5). At least 25 nuclei per slide and two slides per sample were scored and the mean calculated (Tice et al., 2000). At least two independent experiments were carried out for each treatment. Before and at the end of the experiments, an aliquot of both exposed and control cells was used for the assessment of cell viability by the Trypan blue dye exclusion technique, mixing 0.4% Trypan blue solution with the cell pellet, smearing the mixture on a Bürker chamber, and scoring white (live) and blue (dead) cells.

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Table 1 Primary physicalechemical properties of TiO2 particles. Crystal structure

Particle shape

Size

Surface area m2/g

Purity (%)

Surface coating

Density g/cm3

Anatase Rutile

Elongated Irregular

20e50 nm(2) 1e5 mm(3)

101.0(4) 4.0(4)

99.7(1) 99.9(1)

No No

4.00(1) 4.23(1)

(1) Data declared by the supplier; (2) by TEM; (3) by SEM; (4) by BET.

powder was highly homogeneous, being composed of elongated crystals (20e25  45e50 nm), while the rutile powder was heterogeneous, containing 1.7% particles less than 1 mm in size, 83.6% in the range between 1 and 3 mm, 6% in the range between 3 and 4 mm, and 8.6% measuring >4 mm. The surface area e measured by BET e was 101 m2/g and 4 m2/g for anatase and rutile respectively. Dynamic Light scattering analysis showed that both TiO2 forms agglomerated in the media used for exposure. Initially, anatase size frequency distribution was unimodal in RPMI (prevailing size 810  130 nm) and bi-modal in DeMEM (peaks at 27  4 and 1780  385 nm); after 24 h, a large amount of particles was visible on the bottom of the vial and a multimodal size distribution e from 70 to over 6000 in RPMI and from 30 to 1500 nm in DMEM e was detected. After 48 h, the maximum diameter of suspended anatase particles was reduced to 800 and 400 nm in RPMI and DMEM respectively, likely as a consequence of the sedimentation of larger agglomerates. The early size frequency distribution of rutile was bi-modal in RPMI (peaks at 853  148 and 8681  1319 nm) and multimodal in DMEM (from 99 to over 5300 nm). After 24 h, the maximum diameter of suspended rutile particles was reduced to 1500 and 2000 nm in RPMI and DMEM respectively, likely due to the sedimentation of larger agglomerates. The maximum diameter of suspended rutile particles was 6000 and 4000 nm in RPMI and DMEM respectively after a 48 h exposure, when large amounts of particles were found at the bottom of the vial. These data are consistent with further particle agglomeration and sedimentation.

2.6. Statistical analysis The effect of exposure dose, time, cell type, experiment, culture, and replicate were evaluated by the multifactor analysis of variance (MANOVA). A multiple range test (MRT) was performed in order to detect differences (p < 0.05) among means from different experimental groups. 2.7. Uptake and intracellular compartmentation of TiO2 The cellular uptake of TiO2 particles was assessed by TEM. The pellets of treated and control cells were fixed with Karnovsky solution for 5 h at 37  C, washed overnight with 0.1 M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide for 2 h at room temperature, and dehydrated in ethanol. Samples were preembedded in Epon Araldite-propilene oxide 1:1 mixture and embedded in EponAraldite at 60  C for 48 h. Ultra-thin sections (70e90 nm) were cut using an Reichert-Jung Ultracut E ultramicrotome, collected on a 150-mesh formvar carbon-coated copper grid and stained with uranyl acetate and lead citrate. The sections were then observed with a JEOL JEM 100 SX transmission electron microscope (JEOL, Tokyo, Japan) at 80 KV. 3. Results 3.1. Particle characterization The main characteristics of the TiO2 used for the exposures are reported in Table 1. Electron microscopy revealed that the anatase

3.2. Genotoxicity Fibroblasts of the three investigated species exhibited different amounts of DNA damage after exposure to nanometric anatase and micrometric rutile, depending on cell type, dose, and exposure time. Bottlenose dolphin fibroblasts were found generally tolerant to TiO2: indeed, only a few scattered statistically significant increases of DNA strand breaks were observed with respect to controls. These occurred after a 24 h exposure to 20 mg/ml anatase and after a 4 and 48 h treatment with 50 and 150 mg/ml rutile, respectively (Table 2). Anatase did not induce genotoxic effects in mouse fibroblasts (3T3) at any dose/time investigated. 3T3 were susceptible to rutile, however: indeed, almost all the doses were responsible for a statistically significant induction of DNA strand breaks after a 4 h exposure, and so were the two highest ones after a 24 h treatment (Table 2). DNA damage increased in a dosee response manner in human fibroblasts (HuDE) after a 4 h anatase exposure, followed by a complete recovery 24 and 48 h later (Table 2); however, HuDE DNA integrity was not impacted by rutile (Table 2). Bottlenose dolphin leukocytes were tolerant to anatase, but showed a statistically significant increase of DNA damage after 24 and 48 h rutile exposure at the highest doses (Table 3). Finally, human leukocytes were completely resistant to TiO2: no evidence of genotoxicity was detected by the Comet assay. Exposure to the known genotoxicant MMS (0.5 mM), used as a positive control, resulted in a significant increase of DNA damage (% tail DNA) ranging from 28.4  4.3 to 31.0  6.3 in HuDE; from

Please cite this article in press as: Frenzilli, G., et al., Effects of in vitro exposure to titanium dioxide on DNA integrity of bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.01.002

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Table 2 DNA damage in bottlenose dolphin (BDF), mouse (3T3) and human (HuDE) fibroblasts after exposure to TiO2 anatase and rutile particles. Cell type

Dose

Anatase 4h % Tail DNA

BDF

3T3

HuDE

a

C 20 50 100 150 C 20 50 100 150 C 20 50 100 150

22.6 34.6 31.1 34.8 21.2 17.2 24.4 21.8 13.8 18.8 12.1 16.5 18.6 23.4 25.0

              

6.5 10.5 8.0 7.2 9.6 4.2 3.1 4.3 2.7 2.0 1.8 1.9 3.3a 4.7a 2.6a

Rutile 24 h % Tail DNA 17.6 38.4 25.6 21.9 25.0 14.5 21.4 26.0 14.5 15.9 13.7 14.0 16.3 17.3 16.4

              

2.1 2.5a 5.1 1.9 0.1 2.7 14.9 9.1a 4.8 1.8 2.3 3.7 5.7 2.9 8.6

48 h % Tail DNA 13.5 32.7 27.3 25.2 25.9 22.1 18.3 28.3 21.0 26.3 20.3 20.3 20.9 21.0 20.8

              

5.2 14.8 9.3 2.4 7.6 5.3 5.1 10.1 3.9 4.9 6.6 5.3 1.7 4.1 6.7

4h % Tail DNA 22.6 34.3 39.2 28.7 15.6 17.6 33.7 29.3 28.3 34.0 16.2 19.5 18.0 20.7 20.8

              

6.5 12.4 8.8a 5.8 4.2 4.4 14.2a 12.4a 10.9 11.5a 4.5 6.5 3.6 3.5 7.7

24 h % Tail DNA 17.6 19.1 25.1 24.7 22.1 19.5 21.4 26.0 29.6 34.8 18.5 17.1 17.1 18.4 19.2

              

2.1 0.9 5.4 1.4 1.9 8.5 4.9 10.0 12.4a 1.9a 7.4 2.1 2.3 6.8 2.0

48 h % Tail DNA 13.5 30.8 29.8 30.8 46.4 22.1 30.7 28.4 28.8 21.8 20.6 22.5 24.3 18.2 22.3

              

5.2 4.0 5.5 3.2 17.9a 7.8 14.0 3.9 11.4 6.9 7.0 6.3 7.0 2.4 3.9

¼ significant differences with control (p < 0.05).

31.5  3.6 to 37.0  4.7 in 3T3; and from 30.9  12.6 to 43.9  17.9 in bottlenose dolphin fibroblasts. 3.3. Cell viability The trypan blue exclusion test showed that cell viability was always 80%, even after exposure to the highest doses of particulate TiO2 at all experimental conditions. In particular, the range of viable cells was between 93 and 100% in HuDE and 3T3, between 80 and 95% in bottlenose dolphin fibroblasts and leukocytes, and between 97 and 100% in human leukocytes. 3.4. Intracellular compartmentation of TiO2 Transmission electron microscopy revealed that TiO2 anatase entered the cells being observed inside membrane-bound vesicles mainly located in the peripheral cytoplasm (Fig. 1). Rutile was also observed within the exposed cells, both by light and electron microscopy; however, the presence of large rutile crystals/aggregates did not make it possible to obtain sufficiently intact sections from epoxy resin (data not shown). Titanium dioxide particles were never seen inside the nucleus. 4. Discussion The need to acquire information on the susceptibility of toothed cetaceans to emerging pollutants led us to investigate the genotoxic potential of titanium dioxide on bottlenose dolphin cell lines, obtained from skin biopsies collected from free-ranging specimens

and from blood samples of reared animals. T. truncatus was selected as the study species because it inhabits coastal and estuarine environments (Leatherwood and Reeves, 1983), and may thus potentially be exposed to pollutants deriving from rivers and continental run off. In this respect, Wise et al. (2011) reported variable levels of titanium in the skin biopsies of sperm whales around the world, showing that the Mediterranean specimens had the highest Ti levels. Because these samples were collected prior to extensive use of TiO2 nanotechnology in consumer products, these data provide a valuable global baseline for interpreting the expected impact of this new class of chemicals. Our data showed that cell viability was generally high for all exposed cell types confirming that TiO2 is only slightly cytotoxic (Bhattacharya et al., 2009; Shukla et al., 2011). On the other hand, the genotoxic response to TiO2 exposure varied depending on the crystalline form and/or dimension of the particles, cell type, species and exposure time/dose. However, the absence of micro anatase and nano rutile from the study did not allow us to determine whether the observed differences were due to the crystallographic form of the material or to particle size. Dolphin leukocytes were more susceptible to micro-rutile than nano-anatase: they were affected by the highest doses of rutile after 24 and 48 h and by a single dose of anatase after 24 h. This result essentially confirms our previous observation of a limited genotoxic effect of TiO2 on bottlenose dolphin leukocytes, based on a preliminary data set (Bernardeschi et al., 2010). Murine fibroblasts were affected by TiO2 rutile mostly after 4 and 24 h exposure, although there was not a doseeresponse trend. These findings are in agreement with data by Guichard et al. (2012),

Table 3 DNA damage in bottlenose dolphin (BDL) and human (HL) leukocytes after exposure to TiO2 anatase and rutile particles. Cell type

Dose

Anatase 4h % Tail DNA

BDL

HL

a

C 20 50 100 C 20 50 100

25.5 33.8 27.8 35.3 8.3 10.6 12.3 13.2

       

10.6 15.1 7.8 15.9 2.3 4.5 4.4 4.8

Rutile 24 h % Tail DNA 35.2 44.5 50.4 47.5 11.8 14.6 11.2 13.1

       

19.5 22.6 19.4a 16.2 3.2 5.9 2.5 2.5

48 h % Tail DNA 36.1 29.5 44.9 43.9 10.0 14.7 14.4 12.6

       

14.3 9.7 18.8 12.1 2.1 3.2 7.8 5.1

4h % Tail DNA 29.9 35.1 32.8 34.9 12.8 14.3 14.4 18.2

       

14.3 19.6 15.4 12.4 3.4 6.3 6.4 8.0

24 h % Tail DNA 35.2 46.4 45.9 55.5 10.8 12.6 13.8 12.5

       

19.5 23.3 20.7 22.7a 2.7 4.4 5.8 5.8

48 h % Tail DNA 34.6 40.2 50.7 48.5 10.9 12.5 12.3 15.8

       

13.6 8.3 16.0a 12.8a 2.6 3.4 3.2 7.6

¼ significant differences with control (p < 0.05).

Please cite this article in press as: Frenzilli, G., et al., Effects of in vitro exposure to titanium dioxide on DNA integrity of bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.01.002

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fibroblasts by Comet assay. Even if in vitro results cannot be directly used to predict the effect on the whole organism, our data suggest a limited impact of TiO2 on the DNA integrity of bottlenose dolphin cells, at least with respect to the dose range and experimental conditions we used. These results do not mean that TiO2 is to be considered environmentally safe, as our data are limited to one aspect of the potential interaction between TiO2 and biota. Moreover, further studies are needed to see whether TiO2 particles can interact with other inorganic and organic pollutants, thus possibly leading to Trojan horse effects, as recently reported in the case of marine mussels (Canesi et al. in press). Finally, the present paper also confirms the use of skin biopsy from free-ranging cetaceans as a profitable, non-destructive method for developing laboratory investigations on endangered species.

Acknowledgments

Fig. 1. Transmission electron micrograph of the peripheral portion of exposed (50 mg/ ml) fibroblast (3T3) cell line showing clustered TiO2 anatase particles within membrane bound vacuoles. bar ¼ 1m.

according to which micrometric rutile particles, but not nanometric ones, are genotoxic for Syrian hamster embryo cells. Only human fibroblasts were proved to be susceptible to TiO2 anatase nanoparticles, even if exclusively after a short exposure (4 h); indeed, DNA integrity appeared to be completely unaffected 24 and 48 h later. Similar results were observed in human lung carcinoma cells (A549) exposed to 25 nm TiO2 anatase, which exhibited a recovery at 48 h (Jugan et al., 2012). Furthermore, the accumulation of p53 and activation of DNA damage checkpoint kinases was proved for nano- TiO2 treated lymphocytes by Kang et al (2008). Contrary to what was observed for T. truncatus leukocytes, human leukocytes were completely tolerant to TiO2. Such a remarkable resistance of human cell lines to TiO2 exposure was previously reported for leukocytes (Hackenberg et al., 2011; Kang et al., 2008), nasal mucosa (Hackenberg et al., 2010) and lung cells (Bhattacharya et al., 2009). Apart from DNA repair, differences in terms of antioxidant defense efficiency might also be responsible for the different responses to TiO2 exhibited by various cell types and species. Indeed, the induction of oxidative stress has been often reported as the pivotal mechanism accounting for the genotoxic effect of TiO2 (Gurr et al., 2005; Trouiller et al., 2009; Barillet et al., 2010; Di Virgilio et al., 2010). However, TiO2 uptake is not always accompanied by a strong intracellular elevation of ROS, indicating that oxidative stress is only one of the possible mechanisms mediating TiO2 genotoxicity (Toyooka et al., 2012). Transmission electron microscopy showed that TiO2 particles were incorporated into the exposed cells, confirming data from the literature (Di Virgilio et al., 2010; Barillet et al., 2010; Toyooka et al., 2012). In the vast majority of cases, nanosized TiO2 particles were found within membrane-bound cytoplasmic vacuoles deriving from endocytosis (Barillet et al., 2010). The cytoplasm compartmentation of nanosized TiO2 supports the hypothesis that there is an indirect mechanism mediating DNA damage. Although methods for the assessment of the potential genotoxic hazard of NPs still need to be standardized and validated (Magdenolova et al., 2013), the present work is the first attempt to assess the genotoxic potential of TiO2 on bottlenose dolphin

This work is part of the PhD thesis of M.B. The Authors are grateful to Claudio Ghezzani for his technical assistance with image analysis and to Laura Carletti for preparing the bottlenose dolphin cell cultures. The Authors wish to thank the staff of the dolphinarium “Oltremare” (Riccione) for providing dolphin blood. The Authors gratefully acknowledge R. Cossi (Qi srl, Italy) for his technical support with DLS measurements.

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Please cite this article in press as: Frenzilli, G., et al., Effects of in vitro exposure to titanium dioxide on DNA integrity of bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.01.002