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Mutagenesis Advance Access published online on May 25, 2008
Mutagenesis, doi:10.1093/mutage/gen024
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro

R. Colognato1,4, A. Bonelli1, J. Ponti2,4, M. Farina2, E. Bergamaschi3, E. Sabbioni2 and L. Migliore1,*

1Department of Human and Environmental Sciences, Faculty of Medicine, University of Pisa, Via S. Giuseppe 22, 56100 Pisa, Italy 2ECVAM Unit, European Commission, DG-Joint Research Centre, Institute of Health and Consumer Protection, Via E. Fermi 1, 21020 Ispra (VA), Italy 3Department of Medical Clinic, Nephrology and Prevention Sciences, Section of Labour Medicine, University of Parma, Viale Gramsci 14, 43100 Parma, Italy 4Present address: NMI Unit, European Commission, DG-Joint Research Centre, Institute of Health and Consumer Protection, TP 203, Via E. Fermi 1, 21020 Ispra (VA), Italy

Owing to the increasing development of nanotechnology, there is a need to assess how engineered nanomaterials can interact with living cells. The main purpose of the present study was to assess whether metal cobalt nanoparticles (CoNP 100–500 nm) are genotoxic compared to cobalt ions (Co2+). Uptake experiments were carried out by incubating peripheral blood leukocytes (PBLs) with 57Co2+ (added to stable Co2+ 10–2 M to obtain concentrations in the range of 10–5 to 10–4 M) or with 60CoNP for 24 and 48 h. Whereas intracellular Co2+ showed slight or no variations over the baseline levels, CoNP were taken up efficiently leading to intracellular CoNP concentrations of 485 ± 106.1 and 970 ± 99 fg per cell after 24 and 48 h, respectively. The genotoxicity end points considered in this study were the frequency of binucleated micronucleated (BNMN) cells and the percentage of tail DNA (% Tail DNA) fragmentation by means of the comet assay. Genotoxic effects were evaluated by incubating PBLs of three healthy donors with subtoxic concentrations (10–5 to 8 x 10–5M) of Co2+ in the form of cobalt chloride, CoNP and ‘washed’ CoNP, the latter to exclude any interference by Co2+. On a group basis, Co2+ induced a clear trend in the increase of the BNMN frequency, whereas CoNP showed only minor changes. Moreover, we observed a high variability among donors in the induction of micronuclei. The comet assay showed a statistically significant dose-related increase in % Tail DNA for CoNP (P < 0,001), whereas Co2+ did not induce significant changes over control values. These findings suggest that nanosized Co can be internalized by human leukocytes and can interact with DNA leading to the observed genotoxic effects, which are, however, modulated both by donor's characteristics and/or by Co2+ release.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Nanotechnology has been developed rapidly during the past decade and nowadays it has already a wide range of technological applications. The most important productive fields will benefit from the use of engineered nanomaterials and in particular of nanoparticles (NP) (1Go). There is still little knowledge about the potential toxic effects on mankind and on the environment, especially because NP can be taken up through different ‘ports of entry’ and can accumulate preferentially in specific subcellular structures (2Go). Manufactured nanoscale materials may behave as ‘traditional’ ultrafine particles, and thus they can be transported across cell membranes leading to a broad spectrum of adaptive or toxic effects, including genotoxic ones.

Among nanoscale materials, metal NP are already commercially available for several applications in the fields of biology, medicine and pharmacology. One of the most interesting chemical elements used as NP for biomedical applications is cobalt (Co); it can be produced as Co oxide, as an organometal compound or as a biopolymer (3Go). In spite of its physiological role as a cofactor of vitamin B12, Co cannot be regarded only as an essential element. In occupational settings, Co exposure can lead to various lung diseases, such as interstitial pneumonitis, fibrosis and asthma (4Go–6Go). Moreover, Co has been regarded as the possible aetiological factor of the severe forms of lung disease observed in the workers occupationally exposed to hard metal dusts (hard metal disease) (7Go). There is also concern for long-term outcomes following exposure to compounds containing tungsten carbide (80–95%) (WC) with matrices formed by Co (5–20%) and nickel (Ni) (0–5%) (8Go–12Go). On the basis of these considerations, Co has been classified by the International Agency for Research on Cancer (13Go) as ‘possibly carcinogenic to humans’ (group 2B) but, when associated with tungsten, it has been included recently in group 2A (i.e. probably carcinogenic to humans) (14Go). Therefore, for workers employed in the hard metal industry, where Co concentrations usually exceed the currently adopted threshold limit value, time-weighted average concentration (TLV-TWA) of 0.01 mg/m3, strict dust level control and periodic health examination are recommended (15Go). Occupational exposure to hard metal dusts is associated with an increased risk of lung cancer; De Boeck et al. (16Go) showed that WC–Co particles induce in vivo genotoxic effects. Several authors have shown that a WC–Co mixture induces higher genotoxic effects than Co or WC alone (17Go,18Go). A synergistic effect of Co and WC in triggering an enhanced production of reactive oxygen species (ROS) and in inducing DNA fragmentation was found (19Go). Co2+ is genotoxic both in vitro and in vivo, potentially involving oxidative stress and DNA repair inhibition (20Go–25Go), whereas the genotoxicity of Co alone is still a controversial issue. It is still a matter of concern whether cobalt nanoparticles (CoNP) can cause cytotoxic or genotoxic effects, in view of their possible risk for human health as nanotechnology products. The purpose of this study was to analyse the potential genotoxic effects of CoNP in vitro, compared with Co2+.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals and radiochemicals
As a source of cobalt ions (Co2+), we used cobalt chloride [CoCl2 (Cas. No. 7791-13-1)] which was supplied by Alfa (Karlsruhe, Germany). CoNP (100–500 nm), with a median value of 246 nm, were supplied by Laboratory of Biomaterials, University of Modena and Reggio Emilia (Modena, Italy). Co2+ (CoCl2) was dissolved in MilliQ water at a concentration of 10–2 M, then sterilized using a 0.2-µm filter (Millipore, Milan, Italy) and diluted in complete culture medium to get the final concentrations. CoNP were suspended in MilliQ water at the concentration of 10–2 M. The freshly prepared stock solutions were ultrasonicated for 15 min and immediately diluted to test concentrations with complete culture medium. Washed CoNP (CoNPw) were obtained by diluting CoNP in MilliQ water at the concentration of 10–2 M and centrifuging twice at 11 000x g for 15 min to remove Co2+ that arose from NP synthesis (10–15% of the initial concentration) and to exclude any interferences in the bioassays.

The compounds were analysed for the elemental impurities by high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS ELAN-DRCII, Perkin-Elmer SCIEX, Ontario, Canada). The results of the analysis (data not shown) indicate a degree of purity of 98.83% which is the value obtained by analysing the presence of 25 metal elements when the concentration of 10–2 M of CoNPs were suspended in complete culturing medium (26Go). CoNP were characterized in water and in complete culture medium for their morphology by scanning electron microscopy technique and by nanoparticles tracking analysis (NanoSight LM20 Nanoparticles Analysis System, Salisbury, UK) (Figure 1). Samples for scanning electron microscopy analysis were prepared by depositing 20 µl of a 10–2 M CoNP solution water or culture medium on a silicon substrate (0.5 x 0.5 cm). Drops were dried for 2 h under an infrared lamp and immediately analysed. CoNP size analyses were performed by injecting directly a freshly prepared stock solution (10–2 M) both in water and in complete culture medium. The nanoparticles tracking analysis was performed in culture medium with or without serum and no significant differences were observed.


Figure 1
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  		Fig. 1. CoNP size distribution by nanoparticles tracking analysis (Nanosight) of CoNP suspension (10–2 M) in water. Size distribution shows a median peak at 246 nm and provides a quantitative result from the qualitative image that shows polidispersed particles with aggregates up to 500 nm.

 
CoNP were also characterized for their sizes by dynamic light scattering and scanning electron microscopy techniques.

57Co2+ was supplied by Amersham Biosciences (Milan, Italy) and it was added to stable Co2+ 10–2 M to obtain 57Co2+ at concentrations in the range of 10–5 to 10–4 M. 60CoNP were prepared by irradiation of CoNP in a nuclear reactor (HFR, Petten, Netherland) in a thermal neutron flux of 2 x 1014 neutrons/cm2/sec1. 60CoNP were washed as described above for CoNP and added to culture medium to give Co concentrations of 10–5 and 2 x 10–5 M. Radioactivity was measured by an automatic {gamma}-counting system (Wallac 1480 3'', Wallac, Sweden) equipped with a well-type NaI (TI) detector and using the characteristic line of 800–2000 KeV (60CoNP) and 50–800 KeV (57Co) photon emission.

Blood collection and leukocyte isolation
Experiments were carried out by using human peripheral blood leukocytes (PBLs) from three healthy male volunteers. Approximately 6 ml of blood was drawn by venipuncture in Li-heparin vials (Emoplast, Vacutest, Lot No. 1734) according to a standard protocol. The donors fulfilled the following criteria: <40 years old, non-smokers, no medication for at least 3 weeks previously and not having undergone radiological examination within the previous 3 months. For the micronucleus assay, the experiments were performed using whole blood, whereas for the comet assay and Co uptake, isolated leukocytes were used. Isolation procedure was performed on FICOLL-Paque Plus® gradient (Amersham Pharmacia, Milan, Italy). Briefly, 3 ml of Ficoll-Paque Plus® were carefully stratified in a plastic centrifuge tube and 4 ml of the blood sample were dropped onto it. The resulting solution was then centrifuged at 400x g for 10 min at room temperature. The cells were then aspirated and washed twice, by centrifugation at 60–100x g for 10 min, with three volumes sterile Hanks’ balanced salt solution. The cell pellet was suspended in RPMI 1640, supplemented with 10% FBS and 1% penicillin–streptomycin, for further experiment.

Co uptake assay
PBLs were isolated as described above and placed into a rotating 15-ml sterile tube, filled with RPMI 1640, supplemented with 10% FBS and 1% penicillin–streptomycin, at a concentration of 2 x 106/ml. After a resting period, cells were treated with suspensions of 60CoNP and solutions of 57Co2+ for 24 and 48 h at the concentration of 2 x 10–5 M, corresponding to the first subtoxic concentration. Cells were then centrifuged and washed with PBS and counted. Radioactivity was directly analysed in the pellet treated with 57Co2+. The majority of NP not bound to cells were removed with two washes, while those membrane bound were removed using a Percoll® gradient (10% v/v NaCl 1.5 M, Sigma-Aldrich, Milan, Italy; 48% v/v Percoll®, Amersham Pharmacia, Milan, Italy and 42% v/v ultra-pure water; 20 000x g, 15min). After centrifugation, three bands were analysed and the cells in the first band were counted for the number of cells and 60CoNP uptake.

Cytokinesis-block micronucleus assay
The cytokinesis-block micronucleus assay was performed according to the procedure described by Migliore et al. (27Go). Whole blood was used to prepare two paired independent cultures of lymphocytes for each dose level. Co2+ and CoNP were previously screened with a wide range of concentrations (10–6 to 10–3 M) to identify a subtoxic dose using the cytokinesis-block proliferation index (CBPI), as a cytotoxicity marker (a 50% CBPI reduction was considered subtoxic). Moreover, to evaluate whether any interference of Co2+ with genotoxic end points may occur, CoNP were ‘washed’. Cell cultures were performed in RPMI 1640, added with 10% FBS, 1.5% phytohaemagglutinin and 1% penicillin–streptomycin). Twenty-four hours after the seeding, the cells were exposed to the different concentrations of all the compounds. In all, 0.51 mM mitomycin C was used as a positive control. Before the cultures were harvested after 72 h, cytochalasin B (6 µg/ml) was added at 44 h (28Go). Cells were then treated with an hypotonic solution (0.075 M KCl), prefixed in 3:5 methanol:acetic acid, washed with methanol and fixed with a 7:1 methanol:acetic acid fixative solution. Staining was performed in a 2% Giemsa solution. After the slides were coded, they were analysed using an optical microscope (final magnification of x400). Two thousands binucleated cells for each experimental point were examined blindly, following the scoring criteria adopted by the Human Micronucleus Project (29Go). We evaluated the number of binucleated micronucleated (BNMN) lymphocytes on 2000 binucleated lymphocytes containing one or more micronuclei. Moreover, 500 lymphocytes were scored to evaluate the percentage of binucleated cells and the CBPI was calculated according to Surrallès et al. (30Go).

Comet assay
The modified alkaline comet assay (31Go) was carried out on isolated leukocytes, seeded at a concentration of 1.5 x 105/ml, according to the following procedure. Incubation was performed for 2 h with Co2+ and CoNP ranging from 10–5 to 10–4 M. In order to verify the protocol procedure, H2O2 (10–2 M) was used as positive control. After treatment, the cell pellet was used for the comet assay. Briefly, the slides were spread with 1% normal melting agarose (Sigma-Aldrich) in PBS (Sigma-Aldrich) and left to solidify at 4°C. Cell pellets were suspended in 85 µl of 0.5% low-melting agarose (Agarose wide range, Sigma-Aldrich) in PBS at 37°C and two slides per cultures were prepared. The cell suspension was dropped on top of the first agarose layer, covered with a coverslip and allowed to solidify at 4°C. A final layer of 0.5% low-melting agarose was then added to the slide. The lysis procedure was performed in cold lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 10% DMSO and 1% Triton X-100, pH 10.0) for 1 h at 4°C. After, the slides were placed for 20 min in alkaline buffer (300 mM NaOH and 1 mM Na2EDTA, pH > 13.0) and electrophoresed for another 20 min at 25 V adjusted at 300 mA. The slides were neutralized in 0.4 M Tris buffer, pH 7.5, for 5 min, twice. Coded slides were scored after staining with ethidium bromide (20 µg/ml) using a fluorescence microscope (Nikon Eclipse E800) at x20 magnification. A hundred randomly selected cells, fifty for each replica, were analysed using a Comet Image Analysis System, version 5.5 (Kinetic Imaging Ltd, Nottingham, UK). Results were reported as percentage of Tail DNA (% Tail DNA).

Statistical analysis
Data were analysed using the STATGRAPHICS Plus software package for windows (SWGIN, version 5.1). Obtained values are expressed as mean and standard error of mean (SEM) of two independent experiments (both for CoNP and CoNPw); the statistical significance was calculated by Fisher's exact test. Differences were considered statistically significant with a P value of <0.05. Moreover, to evaluate the overall trend and the variability observed, data were also elaborated through the multifactorial analysis of the variance (MANOVA) including the frequency of BNMN lymphocytes as dependent variable, the dose level and the donors as factors and the experiments as covariates.


   Results
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 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Uptake experiments were carried out by incubating PBLs with 57Co2+ or with 60CoNP for 24 and 48 h, and the results are summarized in Table I. Whereas intracellular Co2+ showed slight or no variations over the baseline levels, CoNP were taken up by PBLs within a few hours leading to an average intracellular CoNP concentration of 580 fg per cell for donor 1 and of 445 fg per cell for donor 2 after 24 h. After 48 h, the CoNP intracellular average concentration was of 880 fg per cell for donor 1 and 980 fg for donor 2 (Table I).


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  		Table I. Uptake of Co by human peripheral leukocytes exposed to Co2+ and CoNP

 
To verify the concentration intervals where a genotoxic effect was observable, preliminary experiments with Co2+, CoNP and CoNPw were performed with a concentration range between 10–6 and 10–3 M. Using CBPI as cytotoxicity marker, a subtoxic interval ranging from 10–5 to 8 x 10–5 M was identified (see Table II) and subsequently two paired independent cultures for each concentration level were set up. At non-cytotoxic concentrations tested, no genotoxic effects were observed (Table II).


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  		Table II. Frequency of BNMN cells and CBPI of Co2+- and CoNP-treated lymphocytes in preliminary experiments

 
Co2+ induced a clear concentration-dependent increase in the frequency of BNMN cells, which was statistically significant starting at the concentration of 4 x 10–5 M (P < 0.05) (Figure 2), as well as a concentration-dependent decrease of CBPI (Figure 3). Data represent mean ± SEM of the BNMN frequency and the CBPI of three donors.


Figure 2
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  		Fig. 2. Frequency of BNMN cells in Co2+-, CoNP- and CoNPw-treated cultures. Graph shows the values of the frequency of BNMN lymphocytes treated with Co2+, CoNP and CoNPw. Data represent mean ± SEM obtained by pooling the BNMN frequency of three donors and for each single donor by analysing two independent experiments for CoNP and CoNPw. Asterisk, filled circle and plus represent the statistical significance for Co2+, CoNP and CoNPw and refer to P < 0.05, P < 0.01 and P < 0.001 when indicated by one, two or three symbols, respectively, calculated by Fisher's exact test.

 


Figure 3
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  		Fig. 3. CBPI of Co2+-, CoNP- and CoNPw-treated lymphocytes. Graph shows the values of the cytoxicity index CBPI of lymphocytes treated with Co2+ (filled diamonds), CoNP (filled squares) and CoNPw (filled triangles). Data represent mean ± SEM obtained by pooling the CBPI of three donors and for each single donor by analysing two independent experiments. All values starting from the concentration of 2 x 10–5 M show a statistical significance of P < 0.001 compared to control (calculated by Fisher's exact test).

 
Also both CoNP and CoNPw produced changes on the frequency of BNMN, which were statistically significant starting from the dose of 2 x 10–5 M (Figure 2), and a cytotoxic effect indicated by the decrease of CBPI (Figure 3).

In contrast, when single donors were considered, BNMN (Table III), but not CBPI, displayed a high variability of response; in fact, two subjects showed an increase in BNMN frequency, whereas the first donor's cells were apparently not influenced by any treatments (Table III). MANOVA showed statistically significant differences between donors for CoNP (P = 0.0026), unlike the concentration (P = 0.146). For CoNPw, both the donor and the concentration level significantly affected the results (P = 0.015 and P = 0.04, respectively). For both CoNP and CoNPw, introducing the experiment as covariate, no statistical significance was observed.


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  		Table III. Frequency of BNMN cells and CBPI of Co2+-, CoNP- and CoNPw-treated lymphocytes of three different donors

 
The comet assay was performed on isolated PBLs of three different donors to detect the DNA damage induced by Co2+ and CoNP at concentrations of 10–5, 5 x 10–5 and 10–4 M (Figure 4). Since Co2+ did not determine DNA damage at any of the concentrations tested and we could exclude any activity of Co2+, the subsequent analysis of the genotoxicity was limited to CoNP, avoiding to test also CoNPw (Figure 3). CoNP determined a concentration-dependent increase in the percentage of DNA in the tail (% Tail DNA) already at the concentration of 5 x 10–5 M (P < 0.05) and also at higher concentrations (P < 0.001). Differently from the BNMN frequency, this effect was reproducible for all individuals (Figure 4).


Figure 4
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  		Fig. 4. Dose dependent modulation of Co2+- and CoNP-induced DNA damage in isolated human peripheral leukocytes of three donors after 2 h of exposure. Values refer to the different donors and are expressed as mean of % tail DNA of 100 cells counted. Symbols *, ** and *** represent a statistical significance of P < 0.05, P < 0.01 and P < 0.001, respectively (calculated by Fisher's exact test).

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The chemical and physical behaviour of nanomaterials is not well known; some NP form aggregates and rapidly drop out of suspension so that constant re-suspension is necessary in order to create an homogenous solution. In our experiments, this problem occurred for the CoNP and consequently we first verified if CoCl2 and CoNP could be internalized by human leukocytes. The results from the uptake experiments showed that the CoNP are internalized at a high rate by human leukocytes; we estimated a > 80-fold increase if compared to Co2+ after a 24-h incubation period and a >140-fold increase after 48 h (Table I).

The possible mechanisms involved in such an efficient cellular uptake are intriguing, in view of the hydrophobic nature of CoNP. Nevertheless, for other types of NP, recent studies have demonstrated that the absorption can occur by various processes, such as phagocytosis, pinocytosis or receptor-mediated endocytosis (32Go–34Go). The uptake and the distribution of ‘poly(DL-lactide-co-glycolide)’ (PLGA) were investigated in human arterial smooth muscle cells (35Go) and in the HUVEC cell line (32Go).

A key factor for the internalization process seems to be the surface charge of the NP: in fact Harus-Frenkel et al. (36Go) showed that negatively charged PLGA was endocytosed at a lower rate and this process was independent from the clathrin-mediated pathway. On the other hand, positively charged PLGA was taken up more rapidly by a clathrin-mediated pathway.

Since human white blood cells do not possess any phagocytic capacity, the CoNP internalization could involve an uptake mechanism triggered by a passive membrane diffusion process possibly by activation of an unspecific receptor-mediated event. It is conceivable that the hydrophobic nature of CoNP can be modified in vitro, since the particles may absorb onto their surface proteins present in culture medium, and be more easily taken up by the cells (1Go,2Go).

Until now the genotoxicity of metallic Co has been controversial; nevertheless, there are data supporting that Co alone is able to induce DNA damage to a lower extent than WC–Co dust. This could be due to the synergistic effects with other components of the ‘hard metal’ mixture (namely, WC and/or Ni) (17Go,18Go,37Go,38Go). Studies on possible genotoxic effects of Co nanosized compounds are still lacking. In the present work, we addressed the question of the possible genotoxicity of CoNP as compared to that of Co2+. Moreover, to exclude any interference of Co2+, the CoNP preparation was washed, just to eliminate or reduce the possibility of Co2+ leaching into the culture medium. The results show that Co2+ are able to induce a marked increase in the frequency of BNMN and a decrease of CBPI in human peripheral lymphocytes, whereas CoNP seem less effective and the observed changes are limited to CBPI (Figures 2 and 3). Recently, Papargeorgiou et al. (39Go) compared the cytotoxic and genotoxic effects of NP and micron-sized particles of Co chrome alloy in cultured human fibroblasts. They observed that NP induced more DNA damage than micron-sized particles and more aneuploidy and cytotoxicity at the equivalent volumetric dose (39Go).

We found a high variability in the genotoxic effects, for both Co2+ and CoNPw, among different donors (Table III). In a previous study, we already observed a different susceptibility for two donors to some metals tested (Tl and Sb) with the same in vitro assay (40Go), both for the threshold of induction of genotoxic effects and for toxic concentrations. Interesting data have been reported by Mateuca et al. (23Go), who found that the genotoxic effects in Co-exposed workers are modulated by the presence of Ser/Cys or Cys/Cys polymorphism of the repair gene hOGG1 compared to subjects with the Ser/Ser genotype. The comet assay showed a blunted effect of Co2+ on DNA damage since the treatment with CoCl2 did not induce single-strand breaks and/or alkali-labile sites. In contrast, CoNP caused a concentration-dependent increase in % Tail DNA (Figure 4). However, the incubation time with the Co compounds for the comet assay was rather short (2 h compared to 24 or 48 h in the other experiments). Thus, the lack of DNA strand break induction by Co2+ might be due to this circumstance since metal ions are usually taken up less efficiently relative to CoNP (see Table I). Our data are in agreement with those reported by Anard et al. (41Go) who tested Co2+ both in human leukocytes and purified DNA and observed that the concentration of 10–4 M did not produce any significant DNA damage. However, De Boeck et al. (37Go) observed significant concentration (0.6–6.0 µg/ml) and time-dependent DNA damage using Co2+, by means of the alkaline comet assay.

The genotoxic activity of Co seems to be due to the production of ROS (9Go). Mao et al. (42Go) showed that the oxidative potential of Co2+ (10–3 M) could be modulated by chelator agents that interfere with the ability to produce ROS in H2O2-treated cells. Moreover, Zou et al. (43Go) demonstrated that the CoCl2 induces apoptosis in PC12 cells in a time- and concentration-dependent way (10–4 to 5 x 10–4 M). The addition of antioxidants in culture media blunted these effects, suggesting that the apoptotic process is mediated by free radical generation.

The ability of Co2+ to induce DNA damage could also imply the impairment of the repair enzymes proteins in binding DNA by competing with Mg2+ (22Go). Besides free radical generation, several authors emphasized that magnetic NP preferentially localize at the mitochondrial level, altering oxidative phosphorylation (44Go–46Go). Recent in vitro studies concerning the concurrent cytotoxicity and transforming capacity of CoNP in a Balb/3T3 cell line have revealed that such NP can gradually dissolve in culture media while producing Co2+ (47Go).

Taken together, our results suggest that CoNP are able to induce genotoxic effects in vitro. They are, however, in part modulated by the differences in their chemical properties and their physical interactions with cellular components. Nevertheless, they highlight the need to better understand the molecular mechanisms involved in the genotoxicity of CoNP for a safer and responsible utilization.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Italian Ministry of University and Scientific Research (PRIN 2006069554).


   Acknowledgments
 
Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Dipartimento di Scienze dell'Uomo e dell'Ambiente, Università di Pisa, Via S. Giuseppe 22, 56126 Pisa, Italy. Tel: +39 0502211029; Fax: +39 050551290; Email: l.migliore{at}geog.unipi.it


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on December 20, 2007; revised on April 14, 2008; accepted on April 14, 2008.


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