The aim of this study is tocharacterise two TiO

2 NPs and

one ZnO NP to assess theireffects on neuronal cells

Industrial Hygiene

CYTOTOXICITY CAUSED BY METAL OXIDE NANOPARTICLES IN HUMAN

NEURONAL CELLS

Metal oxide nanoparticles (NPs) like those of titanium dioxide (TiO2) and

zinc dioxide (ZnO) are used in a great variety of industrial and medical

applications, including hi-tech materials, plastics, paint, artificial

orthopaedic implants, paper derivatives, cosmetics and sun cream. Studies

of the effects of these NPs on the nervous system are very few and far

between. The objective of this article is to characterise three metal oxide

NPs (one of ZnO and two of TiO2) and assess their effects on SHSY5Y human

neuroblastoma cells, treated at different concentration levels and over

various exposure times. Results show that the behaviour of the two types of

TiO2 NPs is comparable, despite their different crystalline composition. This

study enhances the existing stock of knowledge on the impact of metal

oxide NPs on human health in general and the nervous system in particular.

By B. LAFFON LAGE. Doctor in Pharmacy. Tenure-holding psychobiology professor of

Universidad de A Coruña (Spain) (Unidad de Toxicología, Universidad de A Coruña, (blaffon@udc.es)

V. VALDIGLESIAS GARCÍA. Doctor in Biology. Research fellow of Instituto IRCCS San Raffaele Pisana, Rome (Italy).

C.S. TRINIDADE DA COSTA. Doctor in Biomedical Sciences. Research fellow of Instituto Nacional de Saúde Dr. Ricardo Jorge, Oporto

(Portugal).

G. KILIÇ. MSc in Molecular, Cellular and Genetic Biology. PhD student at Universidad de A Coruña (Spain).

S.C. BASTOS DA COSTA. MSc in Environmental Health. PhD student at Instituto Nacional de Saúde Dr. Ricardo Jorge, Oporto (Portugal).

J.P. FERNANDES TEIXEIRA. Doctor in Biomedical Sciences. Director of the Departamento de Saúde Ambiental of the Instituto Nacional de

Saúde Dr. Ricardo Jorge, Oporto (Portugal).

E. PÁSARO MÉNDEZ. Doctor in Biology. Chair-holding professor of Psychobiology at Universidad de A Coruña (Spain).

Nanoparticles (NPs) are widely used in today’s nanotechnology, with a huge industrial and commercial takeup. Their main

advantage is that they can be made from practically all solid substances handled on a daily basis in labs, producing a huge

range of nanoobjects with different properties from the starting material. These properties can be modified by controlling

their shape and size in a range of 5-100 nm[1].

More than a thousand NP-containing products of daily use are currently known, including sun cream, paint, medical

prostheses, make-up and various medicaments[2]. It is for this very reason that any possible NP-exposure health risk has

become a subject of increasing concern to the scientific community. This is largely due to the fact that the possible toxic

effects of these NPs have not yet been properly determined and might differ notably from the properties of their

component material on a larger scale [3,4].

Metal oxide NPs, like titanium dioxide (TiO2) and zinc oxide (ZnO) have by now

become important nanomaterials habitually used in a vast range of industrial and

medical applications, including hi-tech materials, plastics, paint, orthopaedic

implants, paper derivatives, cosmetics and sun cream[5]. Due to such a

widespread use the toxicity of these NPs has been widely studied in various cell

An analysis was made of cell NPcapture and the capacity of

inducing cytotoxicity,genotoxicity by diverse

mechanisms, DNA oxidativedamage and apoptosis

lines, mainly keratinocytes and lung cells, proving that exposure thereto causes genotoxicity, cytotoxicity and oxidative

damage[5-8].

Nonetheless, studies of any possible neurotoxicity and the effects of metal oxide NPs on the nervous system have been very

thin on the ground to date. There is now a pressing need to study and determine the particular effects of NP exposure on

neuronal tissue, especially after a recent article has given irrefutable proof that certain NPs of industrial use (e.g., gold NP

of 6 nm and 18 nm), entering the body intravenously or through the lungs, are capable of crossing the blood brain barrier

and reaching the brain of small animals[9].

To test for metal-oxide NP neurotoxicity on human cells, it is the objective of this study to characterise three of the metal

oxide NPs of most widespread daily use (one of zinc, ZnO and two of titanium, TiO2) as well as assessing their effects on

human neuroblastoma SHSY5Y neuronal cells, treated at different concentration levels and over varying exposure times.

Materials and methods

Preparation and characterisation of the NP suspensionsTo carry out this study three of the metal oxide NPs most widely in the manufacture of daily products were collected: zinc

oxide NP – ZnO – and titanium dioxide NP –TiO2– , taken from two different commercial houses (Sigma, TiO

2-S and Degussa,

TiO2-D) and with a different crystalline composition (100% anatase for TiO

2-S and 80% anatase and 20% rutile for TiO

2-D).

The NPs were suspended in deionised water or complete culture medium at a final concentration of 150 μg/ml for TiO2 NPs

and 80 μg/ml for the ZnO NP; they were the subjected to 30W ultrasonication for 5 minutes. The main hydrodynamic size,

size distribution and zeta potential were all determined by Dynamic Light Scattering (DLS).

Cell CultureThe human neuroblastoma SHSY5Y neuronal cell line was used to test for any neurotoxic effects of these NPs as the most

widely used neuronal model in many neurochemical, neurobiological and neurotoxicological studies. The cell line was taken

from the European Collection of Cell Cultures (ECCC) and kept in a medium composed of EMEM/F12 (1:1) with 1%

non-essential amino acids, 1% antibiotic and antimycotic, and 10% fetal bovine serum in an atmosphere with 5% CO2 at 37º

C. Twenty four hours before conducting the experiments the cells were transferred to new wells at a concentration of

about 2.5x105 cells per well. Treatment was carried out at 1% of final volume. Complete medium was used as negative

control in all cases; camptothecin (10 μM), mitomycin C (1.5 μM) or bleomycin (1 μg/ml) were used as positive controls for

tests of apoptosis, genotoxicity and oxidative damage, respectively.

Morphological alterations and cell viabilitySeven concentrations were tested (0-150 μg/ml for TiO

2 NPs and 0-80 μg/ml for

the ZnO NP) and three exposure times (3, 6 and 24 hours). The cell morphology

changes induced by NP treatment were observed in an inverted-field phase-

contrast microscope. Cell viability was analysed by means of the MTT test

[3-(4.5-dimetiltiazol-2-ilo)-2.5-difeniltetrazol] and the neutral red uptake (NRU)

test, following the test protocols described by Mossman (1983)[10] and

Borenfreund and Puerner (1985)[11], respectively. From the morphological-

analysis and cell-viability results a selection was then made of three

concentrations (80, 120 and 150 μg/ml for TiO2 NP and 20, 30 and 40 μg/ml for ZnO NPs) and two exposure times (3 and 6

hours) for conducting the subsequent tests.

Cell CaptureThe NP’s capacity of penetrating neuronal cells was analysed by means of a flow cytometry technique, based on

determining the cell size and granularity according to the protocol of Suzuki et al (2007)[12].

GenotoxicityMicronuclei Test

After NP treatment the cells were incubated over an additional 24-hour period. Micronuclei (MN) were assessed by

flow cytometry following the protocol described by Valdiglesias et al. (2011)[13].

Comet Assay

Primary DNA damage was determined by means of the single cell gel electrophoresis test or comet assay according to

the description given by Laffon et al. (2002)[14]. The chosen DNA damage parameter was the percentage of DNA in the

comet tail (% tDNA).

γH2AX Assay

Phosphorylation of histone H2AX ( H2AX) was analysed by means of the flow cytometry method described by

Valdiglesias et al. (2011)[13].

Oxidative DNA damageOxidative DNA damage was assessed by a modification of the comet assay, incorporating an incubation stage with the repair

enzyme OGG1, following the protocol put forward by Smith et al. (2006)[15].

ApoptosisThe cell apoptosis rate was determined by means of the annexin V-propidium iodide double staining regime using flow

cytometry and a commercial kit (BD Pharmingen™ Annexin V–FITC Apoptosis Detection Kit I).

The mitochondrial membrane potential (MMP) was also analysed by flow cytometry after cell staining with the colorant JC-1

at 10 μmol/L for 30 minutes at 37° C.

Statistical AnalysisAt least three independent experiments were conducted for each experimental condition tested. Data were expressed as

mean ± standard deviation. The distribution of response variables did not tally with the normal findings of the Kolmogorov-

Smirnov test so the non-parametric tests of Kruskal-Wallis and Mann Whitney were used to evaluate any differences

between the groups. Any P-value below 0.05 was deemed to be significant.

Results and Discussion

NP CharacterisationBefore going ahead with the toxicological assessment experiments, the physico-chemical properties of the tested NPs were

determined by DLS (Table 1). The mean hydrodynamic diameter of the NPs suspended in water was found to be 447.9 nm

for TiO2-S, 160.5 nm for TiO

2-D and 243.7 nm for ZnO. In all cases the NPs showed a slight increase in their hydrodynamic

size when characterised in complete culture medium. This could be due to a slight increase in coagulation. Nonetheless,

the size dispersion graphs obtained during the analysis show a good dispersion of the NPs tested.

Table 1. Characterisation of the NPs used

TiO2-S TiO

2-D ZnO

Particle sizea (nm) 25 (TEM) 25 (TEM) 100 (BET)

Specific surface areaa (m2/g)a (m2/g) 200-220 35-45 15-25

Hydrodynamic diameter (nm) (DLS)

Water 447.9 160.5 243.7

Medium 504.5 228.3 273.4

Zeta Potential (mV) (DLS)

Water -9.96 -27.8 -8.23

Medium -10.7 -10.7 -11.7

a Manufacturer’s specification

TEM: Transmission Electron Microscope, BET: Brunauer-Emmett-Teller; DLS: Dynamic Light Scattering.

Morphological AlterationsAfter the NP treatment of the neuronal cells for 3, 6 and 24 hours a microscopic evaluation was made of their morphology

and general look. Figure 1 shows the images taken at the highest tested concentrations for each NP. At the end of the

treatment there was no visible sign of any change in the cell cultures treated with TiO" NPs though NP aggregates were

observed 24 hours after the start of the treatment, bearing out the instability of the suspension under these conditions.

Conversely, morphological alterations were observed in the cells treated with ZnO NP at the highest concentrations and

even at low concentrations during the longest exposure times. These alterations included formation of cell aggregates, loss

of transparency, cell rounding and detachment from the culture flask.

Figure 1. SHSY5Y cells treated with TiO2 and ZnO NPs for different exposure times

Cell ViabilityThe cell-viability effects of the various NPs were assessed by MTT and NRU. Both tests are complementary since MTT

furnishes information on mitochondrial cell activity while NRU shows the lisosomal activity. Seven different concentrations

of each NP were tested for three exposure times (3, 6 and 24 hours). The findings for both assays are shown in figure 2.

None of the treatments with TiO2 NP had any effect on neuronal cell viability, with very similar results in both assays.

These findings tally with those of Petkovic et al. (2011)[16], who exposed hepatic cells to TiO2 NP (anatase and rutile) in

concentrations and conditions very similar to those used in this study.

The behaviour of the two typesof TiO

2 NPs is comparable,

despite their differentcrystalline composition

Figure 2. MTT and NRU results in neuronal cells treated with NPs of TiO2-S (a and b), TiO

2-D (c and d) and ZnO (e and f).

Conversely, exposure to ZnO NP does reduce cell viability regardless of the dose

in the three exposure times tested, although the drop is sharper at 24 hours.

These results tally with findings reported in earlier studies with other cell

types[17,18] .

Cell captureTiO

2 NPs showed a high, dose-dependent cell capture rate (figure 3); it was also slightly higher in cells treated for 6 hours

than in cells treated for 3 hours. The capture percentages were similar in TiO2-D and TiO

2-S NPs.

Conversely, and surprisingly, no cell capture was observed by the ZnO NP under any of the test conditions. Lesniak et al.

(2010)[19] found that the NPs suspended in supplemented complete medium can re-cover themselves in proteins from the

medium, making them less prone to cell capture. It has also been reported that the NP internalisation rate in cells might be

affected by parameters other than NP size and shape [20]. It would therefore seem that the conditions used in our study,

especially in terms of the medium supplemented with fetal bovine serum, somehow limit ZnO NP penetration in the cells.

GenotoxicityNP-induced genotoxicity was assessed by means of the micronuclei (MN) assay, the comet assay and analysis of the

phosphorylation of histone H2AX. MNs are small cytoplasmic nuclei containing complete chromosomes or chromosome

fragments that lagged behind in the anafase cell division stage. They thus give information on both clastogenesis and

aneugenesis processes. The phosphorylation level of histone H2AX (γH2AX) was determined as DNA double strand breakage

markers. One of the first cell responses to any breakage of the double strand in genetic material is phosphorylation of the

histone H2AX tails near the breakage zone. The DNA repair mechanism recognises these phosphorylated forms and then

starts to repair the broken strands. This test therefore gives information on clastogenicity-related events. For its part the

comet assay assesses primary DNA damage: single and double strand breakages, alkali labile sites and incomplete excision

repair zones. The findings of these three tests are shown in figures 4 to 6.

Figure 5. Comet assay results on neuronal cells treated with NPs of TiO2-S (a), TiO

2-D (b) and ZnO (c). *P<0.05, **P<0.01 significant

difference from the negative control. PC: positive control

Figure 6. γH2AX assay results on neuronal cells treated with NPs of TiO2-S (a), TiO

2-D (b) and ZnO (c). *P<0.05, **P<0.01 significant

difference from the negative control. PC: positive control.

TiO2 NPs penetrate the cells but

do not cause cytotoxicity. Theyinduce genotoxicity, not relatedto double strand breakage of the

DNA or oxidative damage, andalso intrinsic-pathway apoptosis

Although ZnO NPs do notpenetrate the cells, they do

induce cytotoxicity, genotoxicity(including oxidative DNA

damage) and non-mitochondrialapoptosis

Results for both TiO2 NPs were similar. The MN test showed significant increase

in damage only after 6 hours of treatment. No effect was observed on

phosphorylation of histone H2AX, while the comet assay showed up positive

effects after 3 and 6 hours of exposure. Comet assay results and H2AX

phosphorylation results are usually similar, since both detect DNA double strand

breakages. Nonetheless, as already mentioned, the comet assay also detects

other types of damage not necessarily bound up with phosphorylation of histone

H2AX. It would therefore seem that the TiO2 NPs induce DNA damage other than

double strand breakages.

The ZnO NP produced no genotoxic effects after 3 hours of exposure. There were, however, signs of a significant,

dose-dependent induction of MN after 6 hours, accompanied by an increase in phosphorylation of histone H2AX and in

primary DNA damage. These results chime in with the few past studies that have been published on genotoxic effects of

ZnO NPs on nerve cells, reporting DNA damage in rat primary neuronal cells[21] and induced MN formation in glioma cells[22],

at concentrations very similar to those used in this study.

Oxidative DNA DamageOxidative DNA damage was assessed by a variant of the comet assay, including an incubation stage with the DNA glycosylase

enzyme OGG1, which detects oxidative damage by recognition of the 8-oxo-7.8-dihidroguanine adduct. This highly

mutagenic adduct is one of the commonest DNA lesions [23], and has by now become well established as a suitable

biomarker of oxidative stress [24].

No oxidative damage was observed after TiO2 NP treatment at any concentration

or exposure time (Figure 7). Although other studies report involvement of

oxidative stress in TiO2 NP-induced cell damage[16,25], oxidative damage is not

always observed after treatment with titanium NP and other mechanisms have

been proposed for the toxicity of these NPs, such as interaction with different

cell components like microtubules[26].

Nonetheless ZnO NPs did induce oxidative DNA damage after incubation of 3 and

6 hours. These results are similar to those obtained in previous studies on

different cell types [27,28] and experimentation animals[18].

Figure 7. Results of the comet assay with the OGG1 enzyme on neuronal cells treated with NPs of TiO2-S (a), TiO

2-D (b) and ZnO (c).

*P<0.05, **P<0.01 significant difference from the buffer. PC: positive control

ApoptosisCell death by apoptosis in NP-treated neuronal cells was assessed by two different methods. Firstly by annexin V-propidium

iodide double staining, which shows up cells in early apoptosis states, and secondly the mitochondrial membrane potential

to ascertain the potential participation of this organule in the apoptosis cell death process, since alteration of this

potential is one of the first steps in intrinsic pathway apoptosis.

After both TiO2 NP exposure times, dose-dependent increases were observed in the percentage of apoptotic cells together

with significant falls in the mitochondrial membrane potential (Figure 8). This suggests involvement of this organule in

intrinsic pathway apoptosis. Our findings coincide with earlier studies that reported an increase in intrinsic-pathway

apoptosis in various types of cells treated with TiO2 NP[29,30].

The findings of this studyenhance our knowledge of theimpact of metal oxide NPs on

human health in general and thenervous system in particular

Figure 8. Results of apoptosis analysis with annexin V-propidium iodide double staining and analysis of the mitochondrial membranepotential (MMP) on neuronal cells treated with NPs of TiO

2-S (a and b), TiO

2-D (c and d) and ZnO (e and f). *P<0.05, **P<0.01 significant

difference from the control. PC: positive control

In the case of ZnO NP, significant increases were also observed in the percentage of apoptotic cells, although in this case

there was no sign of any significant alterations in the mitochondrial membrane potential. Our findings therefore suggest

that ZnO NPs induce non-mitochondrial-related cell death. In keeping with our results, ZnO NP-induced apoptosis was

observed by other authors in several cell types, including neuronal cells[22,28,31].

Conclusions

Behaviour of the two types of TiO2 NPs is comparable, despite their different crystalline composition. They do not alter cell

viability but are effectively internalised by SHSY5Y cells. They induce dose-dependent genotoxicity unrelated with the

production of DNA double strand breakage or oxidative damage, and intrinsic-pathway apoptosis. While the genotoxic

effects of both types of TiO2 NPs were very similar, the TiO

2-S NPs were more effective at cytotoxicity induction, in

keeping with their higher cell capture rate.

Although ZnO NPs do not penetrate inside the neuronal cells used in this study,

exposure to these NPs reduces cell viability and induces morphological

alterations, genotoxicity, (including oxidative DNA damage) and

non-mitochondrial apoptosis; most of these effects are proportional to the NP

dose and exposure time.

The findings of this study enhance our knowledge of the impact of metal oxide

NPs on human health in general and the nervous system in particular. Further

research, however, is still needed to arrive at a full understanding of the molecular mechanisms underlying the effects

observed in this study and also to develop safety measures and exposure limits for people working with materials containing

these types of NPs and those who might be exposed thereto in commercial, industrial and medical applications.

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