Eşref Demir

Zinc oxide nanoparticles: Genotoxicity, interactions with UV-light and cell-transforming potential

The in vitro genotoxic and the soft agar anchorage independent cell transformation ability of zinc oxide nanoparticles (NPs) and its bulky forms have been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells, either alone or in combination with UVB-light. The comet assay, with and without the use of FPG and Endo III enzymes, the micronucleus assay and the soft-agar colony assay were used. For the comet assay a statistically significant induction of DNA damage, with and without the enzymes, were observed up of 100μg/mL. ZnO NPs were able to increase significantly the frequency of micronuclei, and similar results were observed in the cell transformation assay where such NPs were able to induce cell-anchorage independent growth. These effects were observed at doses up 100μg/mL. Although UVB-light was able to induce genotoxic damage and cell-anchorage growth, a significant antagonist interaction effect was observed in combination with ZnO NPs. These in vitro results, obtained with the selected cell lines, contribute to increase our genotoxicity database on the ZnO NPs effects as well as to open the discussion about their risk in photo-protection sun screens.

Genotoxicity of cobalt nanoparticles and ions in Drosophila

Abstract Nanogenotoxicology is an emergent area of research, relevant for estimating the potential carcinogenic risk of nanomaterials. Since most of the approaches use in vitro studies, and neglecting the whole organism limits the accuracy of the obtained results, we have used Drosophila melanogaster to study the possible genotoxic potential of cobalt nanoparticles (Co NPs). The wing somatic mutation and recombination test has been the test of choice. This test is based on the principle that the loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3 can lead to the formation of mutant clone cells in growing up larvae, which are expressed as mutant spots on the wings of adult flies. Co NPs, as well as the ionic form cobalt chloride, were given to third instar larvae through the food, at concentrations ranging from 0.1 to 10 mM. The results obtained indicate that both cobalt forms are able to induce significant increases in the frequency of mutant clones. Although at low concentrations only Co NPs were genotoxic, the level of genetic damage obtained at the highest dose tested of cobalt chloride (10 mM) showed a significant higher increase in the frequency of total spots than those observed after the treatment with cobalt nanoparticles. As conclusion, our results indicate that Co NPs were able to induce genotoxic activity in the wing-spot assay of D. melanogaster, mainly via the induction of somatic recombination. The differences observed in the behaviour of the two selected cobalt forms may result from differences in the uptake.

Determination of TiO2, ZrO2, and Al2O3 Nanoparticles on Genotoxic Responses in Human Peripheral Blood Lymphocytes and Cultured Embyronic Kidney Cells

In this study a genotoxic evaluation of titanium dioxide (TiO2, 2.3 nm), zirconium oxide (ZrO2, 6 nm), aluminum oxide (Al2O3, 16.7 nm) nanoparticles (NP) and their ionic forms was conducted using human peripheral blood lymphocytes and cultured human embryonic kidney (HEK293) cells by means of a modified alkaline comet assay with/without the formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) enzymes. Modifications to the comet assay by using lesion-specific endonucleases, such as Endo III and Fpg, detect DNA bases with oxidative damage. Both human peripheral blood lymphocytes and cultured embryonic kidney cells were incubated with TiO2, ZrO2, or Al2O3 NP at concentrations of 1, 10, or 100 μg/ml. Our results showed no significant induction in DNA damage by the comet assay with/without the Endo III and Fpg enzymes at all concentrations of ZrO2 and Al2O3. In the case of TiO2 NP only the highest concentration of 100 μg/ml significantly induced a genotoxic response. Data thus indicate that both ZrO2 and Al2O3 NP were not genotoxic in our system and in the case of TiO2 the lowest-observed-adverse-effect level (LOAEL) for genotoxicity was 100 μg/ml. Evidence indicates that these metallic NP are considered safe in light of the fact that no genotoxicity was noted with ZrO2 and Al2O3 and that the highest TiO2 concentration is not environmentally relevant.

In vivo genotoxicity assessment of titanium, zirconium and aluminium nanoparticles, and their microparticulated forms, in Drosophila

As in vivo system, we propose Drosophila melanogaster as a useful model for study the genotoxic risks associated with nanoparticle exposure. In this study we have carried out a genotoxic evaluation of titanium dioxide (TiO2), zirconium oxide (ZrO2) and aluminium oxide (Al2O3) nanoparticles and their microparticulated forms in D. melanogaster by using the wing somatic mutation and recombination assay. This assay is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs and flare-3, can lead to the formation of mutant clones in treated larvae, which are expressed as mutant spots on the wings of adult flies. Third instar larvae were feed with TiO2, ZrO2 and Al2O3 nanoparticles, and their microparticulated forms, at concentrations ranging from 0.1 to 10mM. Although a certain level of aggregation/agglomeration was observed in solution, it must be noted than the constant digging activity of larvae ensures that treated medium pass constantly through the digestive tract ensuring exposure. The results showed that no significant increases in the frequency of all spots (e.g. small single, large single, twin, total mwh and total spots) were observed, indicating that these nanoparticles were not able to induce genotoxic activity in the wing spot assay of D. melanogaster. Negative data were also obtained with the microparticulated forms. This indicates that the nanoparticulated form of the selected nanomaterials does not modify the potential genotoxicity of their microparticulated versions. These in vivo results contribute to increase the genotoxicity database on the TiO2, ZrO2 and Al2O3 nanoparticles.

Assessment of genotoxic effects of benzyl derivatives by the comet assay.

In this study, different concentrations of four benzyl derivatives (benzyl alcohol, benzyl acetate, benzoic acid and benzaldehyde) used as flavour ingredients were investigated for genotoxicity in in vitro. By taking blood from two healthy people comet assay was carried on to investigate the potential health damages of benzyl derivatives. For the evaluation of genotoxic effects, the tail moment and % tail DNA in the treated chemicals were compared to the solvent control, which is distilled water. The alkaline comet assay showed significantly increased tail moment and % tail DNA at 25 and 50 mM concentrations of benzyl alcohol. Benzyl acetate increased both % tail DNA and tail moment at 50 mM concentrations. While % tail DNA was statistically increased at 10 mM and higher concentrations, tail moment has significant difference at 10 and 25 mM concentrations of benzaldehyde. Benzoic acid has apoptotic effects at the concentrations higher than 5 mM, for this reason we tested concentrations less than 5mM (0.05, 0.1, 0.5, 1 and 5 mM). Only the highest concentration of benzoic acid increased both tail moment and % tail DNA.

Antioxidant and antigenotoxic properties of CeO2 NPs and cerium sulphate: Studies with Drosophila melanogaster as a promising in vivo model.

Abstract Although in vitro approaches are the most used for testing the potential harmful effects of nanomaterials, in vivo studies produce relevant information complementing in vitro data. In this context, we promote the use of Drosophila melanogaster as a suitable in vivo model to characterise the potential risks associated to nanomaterials exposure. The main aim of this study was to evaluate different biological effects associated to cerium oxide nanoparticles (Ce-NPs) and cerium (IV) sulphate exposure. The end-points evaluated were egg-to-adult viability, particles uptake through the intestinal barrier, gene expression and intracellular reactive oxygen species (ROS) production by haemocytes, genotoxicity and antigenotoxicity. Transmission electron microscopy images showed internalisation of Ce-NPs by the intestinal barrier and haemocytes, and significant expression of Hsp genes was detected. In spite of these findings, neither toxicity nor genotoxicity related to both forms of cerium were observed. Interestingly, Ce-NPs significantly reduced the genotoxic effect of potassium dichromate and the intracellular ROS production. No morphological malformations were detected after larvae treatment. This study highlights the importance of D. melanogaster as animal model in the study of the different biological effects caused by nanoparticulated materials, at the time that shows its usefulness to study the role of the intestinal barrier in the transposition of nanomaterials entering via ingestion.

Genotoxic and cell-transforming effects of titanium dioxide nanoparticles

The in vitro genotoxic and the soft-agar anchorage independent cell transformation ability of titanium dioxide nanoparticles (nano-TiO2) and its microparticulated form has been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells. Nano-TiO2 of two different sizes (21 and 50 nm) were used in this study. The comet assay, with and without the use of FPG enzyme, the micronucleus assay and the soft-agar colony assay were used. For both the comet assay and the frequency of micronuclei a statistically significant induction of DNA damage, was observed at the highest dose tested (1000 µg/mL). No oxidative DNA damage induction was observed when the comet assay was complemented with the use of FPG enzyme. Furthermore, long-term exposure to nano-TiO2 has also proved to induce cell-transformation promoting cell-anchorage independent growth in soft-agar. Results were similar for the two nano-TiO2 sizes. Negative results were obtained when the microparticulated form of TiO2 was tested, indicating the existence of important differences between the microparticulated and nanoparticulated forms. As a conclusion it should be indicated that the observed genotoxic/tranforming effects were only detected at the higher dose tested (1000 µg/mL) what play down the real risk of environmental exposures to this nanomaterial.

Genotoxicity and DNA repair processes of zinc oxide nanoparticles.

Two different sizes of zinc oxide nanoparticles (ZnO NP, ≤35 nm and 50–80 nm) were tested in the human lymphoblastoid cell line TK6 to increase our knowledge on their genotoxic potential. The comet assay was the system used, and the results obtained showed that the highest concentration tested (100 μg/ml) for the two selected compounds was genotoxic. The percent DNA in tail obtained after treatment with ZnO NP (≤35 nm) was significantly higher than that of ZnO NP (50–80 nm) at all concentrations tested. To investigate the nature of the induced genotoxic damage, specific enzymes recognizing oxidized DNA bases were used. Treatments with endonuclease III (Endo III) and formamidopyrimidine DNA glycosylase (FPG) demonstrated that only ZnO NP (50–80 nm) were able to induce significant levels of net oxidative DNA damage. Further DNA repair kinetics studies revealed that DNA damage initially induced was removed in approximately 5 h. DNA damage induced by ZnO NP was repaired more slowly than damage following microparticulated ZnO exposure. No marked differences in repair kinetics of both forms of ZnO NP were observed. Evidence indicates that a high proportion of DNA damage induced by ZnO NP (50–80 nm) correlated with induction of oxidative damage, and that both forms of ZnO NP interfere with mechanisms involved in DNA damage repair.

Genotoxic analysis of four lipid-peroxidation products in the mouse lymphoma assay

Lipid-peroxidation products are formed by the thermal treatment of foodstuffs, as well as by endogenous processes. In addition, they are also common environmental pollutants originating from many different sources. Since conflicting data exist on their possible risk for humans, we have selected four lipid-peroxidation products namely acrolein, crotonaldehyde, 4-hydroxy-hexenal (4-HHE) and 4-oxo-2-nonenal (4-ONE) to determine their ability to induce mutagenicity in mammalian cells. There is an important lack of mutagenicity data on mammalian cells for such products, which presents an important gap for any risk-assessment estimation. We have used the mouse lymphoma assay (MLA) to determine the mutagenic potential of these four compounds. This assay detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The results obtained indicate that the four selected compounds are mutagenic in the MLA assay, showing a direct dose-effect relationship. The relative mutagenic potencies according to the induced mutant frequency (IMF) are as follows: crotonaldehyde (IMF=758.5×10(-6)), 4-ONE (IMF=700.5×10(-6)), acrolein (IMF=660.5×10(-6)) and 4-HHE (IMF=572×10(-6)). Although the differences between the induced mutant frequencies for these compounds are not very large, the α,β-unsaturated aldehyde 4-oxo-2-nonenal turned out to be the agent most mutagenic. This is because its induced mutant frequency was reached after treatment with 10μM, while 50μM of the other compounds was needed to reach the reported frequencies.

In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays

Currently, antimicrobial additives derived from essential oils (Eos) extracted from plants or spices, such as Origanum vulgare, are used in food packaging. Thymol and carvacrol, the major EO compounds of O. vulgare, have demonstrated their potential use as active additives. These new applications use high concentrations, thereby increasing the concern regarding their toxicological profile and especially their genotoxic risk. The aim of this work was to investigate the potential in vitro genotoxicity of thymol (0-250 μM) and carvacrol (0-2500 μM) at equivalent doses to those used in food packaging. The micronucleus (MN) test and the mouse lymphoma (MLA) assay on L5178Y/Tk(±) mouse lymphoma cells were used. The negative results for thymol with the MN with and without the S9 fraction and also with the MLA assay reinforce the view that this compound is not genotoxic in mammalian cells. However, carvacrol presented slight genotoxic effects, but only in the MN test at the highest concentration assayed (700 μM) and in the absence of metabolic activation. The lack of genotoxic response in the MLA assay after 4 and 24h of exposure indicates a low genotoxic potential for carvacrol. Alternatively, the general negative findings observed in both assays suggest that the MN results of carvacrol are marginal data without biological relevance. These results can be useful to identify the appropriate concentrations of these substances to be used as additives in food packaging.