Titanium dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Data source

Reference

Reference Type:

publication

Title:

DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells.

Author:

Petković, J. et al.

Year:

2011

Bibliographic source:

Nanotoxicology 5(3): 341-353.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study human hepatoma HepG2 cells were exposed to TiO2 NPs at the concentrations 0, 1, 10, 100, 250 µg/mL in a comet asssay.

GLP compliance:

not specified

Remarks:

publication

Type of assay:

comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Titanium dioxide

EC Number:

236-675-5

EC Name:

Titanium dioxide

Cas Number:

13463-67-7

Molecular formula:

O2Ti

IUPAC Name:

dioxotitanium

Test material form:

solid: nanoform

Details on test material:

PARTICLE CHARACTERIZATION (by authors)
The size and morphology of TiO2 NPs were observed by field-emission-gun scanning electron microscopy. The crystal phase of the powders was identified by X-ray diffraction. UV/Vis (diffuse reflectance) spectroscopic characterization of the samples was recorded. The specific surface area was determined by gas adsorption using the BET method (Gemini 2370, Micromeritics).

- Name of test material (as cited in study): TiO2-Ru
- Source: purchased from Sigma-Aldrich
- Crystalline phase: rutile
- Size: ca. 10x40 nm
- Specific surface area (BET): 116.7 m²/g
- FEG-SEM (supplied material) Particle size within the agglomerates/aggregates: <100 nm
- Shape: elongated crystallites
- XRD: Rutile type: tetragonal crystallographic system; P42/mnm space group (according to JCPDS: 34–0180); characteristic reflections (110) at 27.3; (101) at 36.0; (211) at 54.1, 2θ.
- UV-Vis spectroscopy: rutile: absorption peaks at lmax = 305 nm and λmax = 205 nm

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

mammalian cell line, other: human hepatoma HepG2 cells

Details on mammalian cell type (if applicable):

HepG2 cells were obtained from European Collection of Cell Cultures (ECACC). Cells were grown in EMEM containing 10% fetal bovine serum, 1% non-essential amino acid solution, 2 mM L-glutamine and 100 U/mL penicillin plus 100 µg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO2.

Metabolic activation:

not specified

Test concentrations with justification for top dose:

1, 10, 100, and 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffered saline (PBS)
Powdered TiO2 NPs were suspended in PBS at a concentration 1 mg/mL and sonicated for 30 minutes in an ultrasonic bath (Sonorex, Bandelin electronic, Germany) at a frequency of 60 kHz, voltage of 220 V and an electric current of 0.5 A to ensure uniform suspension. This stock solution was subsequently diluted in the complete cell growth medium to yield concentrations. These samples were then sonicated for 30 minutes to produce a stable, less-agglomerated nanocrystalline suspension before exposure of cells in culture.

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

cell growth medium containing 10% PBS

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: tert-butyl hydroperoxide

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

cell growth medium containing 10% PBS

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

PARTICLE SIZE DISTRIBUTION IN TREATMENT MEDIUM
Particle size distribution of TiO2 NPs in the treatment medium was measured by laser scattering, using a particle size distribution analyzer Horiba LA-920 (Japan). The particles were dispersed in Eagle Minimal Essential Medium (EMEM) medium at 30 µg/mL and sonicated using the same conditions as for preparation of particle suspensions for the cell treatments.

CYTOTOXICITY ASSAY
Cytotoxicity was determined with the MTT assay according to Mossman (1983)* with minor modifications (Zegura et al. 2003)*. The HepG2 cells were seeded onto 96-well microplates (Nunc, Naperville, IL, USA) at a density of 40,000 cells/mL and incubated for 20 hours at 37°C to attach. The medium was then replaced by fresh complete medium containing 0, 1, 10, 100 and 250 µg/mL of TiO2 NPs, and incubated for 4, 24 and 48 hours. In each experiment a vehicle control (cell growth medium containing 10% PBS) was included. MTT (final concentration 0.5 mg/mL) was then added, incubated for an additional 3 hours, the medium with MTT was then removed and the formed formazan crystals dissolved in DMSO. The optical density (OD) was measured at 570 nm (reference filter 690 nm) using a microplate reading spectrofluorometer (Tecan GENios, Austria). Viability was determined by comparing the OD of the wells containing the NPs treated cells with those of the vehicle (cell growth medium containing 10% PBS) treated cells. Five replicates per concentration point and three independent experiments were performed.

COMET ASSAY
HepG2 cells were seeded at a density of ~ 60,000 cells/mL into 12-well microtiter plates (Corning Costar Corporation, Corning, NY, USA). After incubating the cells at 37°C in 5% CO2 for 20 hours to attach to the plates, the growth medium was replaced with fresh medium containing 0, 1, 10, 100 and 250 µg/mL TiO2 NPs and incubated for 2, 4 and 24 hours. In each experiment positive controls (0.5 mM t-BOOH and 50 mM BaP) and a vehicle control (cell growth medium containing 10% PBS) were included.
At the end of the exposure the cells were harvested and the DNA damage determined by the protocol of Singh et al. (1988)* with minor modifications (Žegura and Filipic 2004)*. Images of 50 randomly selected nuclei per experimental point were analyzed with image analysis software Comet Assay IV (Perceptive Instruments, UK). Three independent experiments were performed for each of the treatment conditions. The percent of tail DNA was used to measure the level of DNA damage.
The level of oxidized purines/pyrimidines was determined with the modified comet assay as described by Collins et al. (1996)*. After the cell lysis the slides were washed three times for 5 minutes with endonuclease buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL bovine serum albumin, pH 8.0). Fifty microlitre aliquots of Fpg/Endo III solution or enzyme buffer without Fpg/Endo III were added, covered with a cover glass and incubated at 37°C for 30/45minutes. The slides were then processed as described above.

*References:
- Mossman T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63.
- Zegura B, Sedmak B, Filipic M. 2003. Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 41:41–48.
- Singh NP, McCoy MT, Tice RR, Schneider EL. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191.
- Zegura B, Lah TT, Filipic M. 2004. The role of reactive oxygen species in microcystin-LR-induced DNA damage. Toxicology
200:59–68.
- Collins AR, Dusinska M, Gedik CM, Stetina R. 1996. Oxidative damage to DNA: Do we have a reliable biomarker? Environ Health Perspect 104(3):465–469.

Evaluation criteria:

no data

Statistics:

One-way analysis of variance (ANOVA, Kruskal-Wallis) was used to analyze the differences between treatments within each experiment. Dunnet’s test was used for comparing median values of percentage tail DNA; P < 0.05 was considered as statistically significant.

Results and discussion

Test results

Species / strain:

mammalian cell line, other: human hepatoma HepG2 cells

Metabolic activation:

not specified

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

PARTICLE SIZE DISTRIBUTION IN TREATMENT MEDIUM
The particle size distribution determined by laser scattering particle size distribution analysis showed that in the medium both types of TiO2 NPs are highly aggregated and agglomerated with an average size of aggregates and agglomerate size at the micron level (TiO2-Ru: 1542 ± 760 nm). The portion of submicron-sized particles is much higher TiO2-Ru NPs compared to TiO2-An NPs. Micron-sized aggregates and agglomerates were confirmed also by FEG-SEM.

CYTOTOXICITY ASSAY
The viability of HepG2 cells exposed to 0, 1, 10, 100 and 250 µg/mL of TiO2-Ru NPs for 4, 24 and 48 h was not significantly affected.

COMET ASSAY
- Exposure to TiO2-Ru NPs induced significantly more strand breaks only in cells exposed for 4 hours to 100 µg/mL TiO2-Ru NPs.
- In cells exposed to TiO2-Ru NPs, a significant increase of Fpg-sensitive sites was detected only after 24 hours exposure to 10 and 100 µg/mL NPs.
- Induction of Endo III-sensitive sites by TiO2 NPs was much lower than that of Fpg-sensitive sites and was not dose-dependent. In cells exposed to TiO2-Ru, a significant increase of Endo III-sensitive sites was observed at the highest concentration (250 µg/mL) after 2 hours and after 24 hours exposure.

Applicant's summary and conclusion

Conclusions:

Petković, J. et al. (2011) examined the DNA damaging potential of TiO2-Ru NPs in Hep-G2 cells using the conventional as well as modified comet assays. The colorimetric MTT assay was exploited in order to investigate on NP’s cytotoxicity.

The authors conclude that TiO2-Ru NPs was practically ineffective to induce DNA damage.

According to the evaluator of the publication, TiO2-Ru did not show an increase of DNA damage.