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There is a great interest in a better knowledge of the health effects caused by nanomaterials exposures and, in particular to those induced by titanium dioxide nanoparticles (nano-TiO2) due to its high use and increasing presence in the environment. To add new information on its potential genotoxic/carcinogenic risk, we have carried out experiments using chronic exposures (up to 4 weeks), low doses, and the BEAS-2B cell line that, as a human bronchial epithelium cells, can be considered a good cell target. Cell uptake has been assessed by transmission electron microscopy (TEM) and flow cytometry (FC); genotoxicity was evaluated using the comet and the micronucleus (MN) assays; and cell-transforming ability was evaluated using the soft-agar assay to detect anchorage-independent cell growth. Results show an important cell uptake at all the tested doses and sampling times used (except for 1 µg/mL and 24-h exposure). Nevertheless, no genotoxic effects were observed in the comet and in the MN assays. This lack of genotoxic effect agrees with the FC results showing no induction of intracellular reactive oxygen species (ROS), the data from the comet assay with formamidopyrimidine DNA glycosylase (FPG) enzyme showing no induction of oxidized bases, and the lack of induction of expression of heme-oxygenase (HO-1) gene both at the RNA and protein level. On the contrary, significant increases in the number of clones growing in an anchorage-independent way were observed. This study would indicate a potential carcinogenic risk associated to nano-TiO2 exposure, not mediated by a genotoxic mechanism. [ABSTRACT FROM AUTHOR]

A weak aspect of the in vitro studies devoted to get information on the toxic, genotoxic and carcinogenic properties of nanomaterials is that they are usually conducted under acute-exposure and high-dose conditions. This makes difficult to extrapolate the results to human beings. To overcome this point, we have evaluated the cell transforming ability of cobalt nanoparticles (CoNPs) after long-term exposures (12 weeks) to sub-toxic doses (0.05 and 0.1 µg/mL). To get further information on whether CoNPs-induced oxidative DNA damage is relevant for CoNPs carcinogenesis, the cell lines selected for the study were the wild-type mouse embryonic fibroblast (MEF Ogg1+/+) and its isogenic Ogg1 knockout partner (MEF Ogg1−/−), unable to properly eliminate the 8-OH-dG lesions from DNA. Our initial short-term exposure experiments demonstrate that low doses of CoNPs are able to induce reactive oxygen species (ROS) and that MEF Ogg1−/− cells are more sensitive to CoNPs-induced acute toxicity and oxidative DNA damage. On the other hand, long-term exposures of MEF cells to sub-toxic doses of CoNPs were able to induce cell transformation, as indicated by the observed morphological cell changes, significant increases in the secretion of metalloproteinases (MMPs) and anchorage-independent cell growth ability, all cancer-like phenotypic hallmarks. Interestingly, such changes were significantly dependent on the cell line used, the Ogg1−/− cells being particularly sensitive. Altogether, the data presented here confirms the potential carcinogenic risk of CoNPs and points out the relevance of ROS and Ogg1 genetic background on CoNPs-associated effects. [ABSTRACT FROM AUTHOR]

The presence of nanomaterials (NMs) in the environment may represent a serious risk to human health, especially in a scenario of chronic exposure. To evaluate the potential relationship between NM-induced epigenetic alterations and carcinogenesis, the present study analyzed a panel of 33 miRNAs related to the cell transformation process in BEAS-2B cells transformed by TiO2NP and long-term MWCNT exposure. Our battery revealed a large impact on miRNA expression profiling in cells exposed to both NMs. From this analysis, a small set of five miRNAs (miR-23a, miR-25, miR-96, miR-210, and miR-502) were identified as informative biomarkers of the transforming effects induced by NM exposures. The usefulness of this reduced miRNA battery was further validated in other previously generated transformed cell systems by long-term exposure to other NMs (CoNP, ZnONP, MSiNP, and CeO2NP). Interestingly, the five selected miRNAs were consistently overexpressed in all cell lines and NMs tested. These results confirm the suitability of the proposed set of mRNAs to identify the potential transforming ability of NMs. Particular attention should be paid to the epigenome and especially to miRNAs for hazard assessment of NMs, as wells as for the study of the underlying mechanisms of action. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Health risk assessment of nanomaterials is an emergent field, genotoxicity being an important endpoint to be tested. Since in vivo studies offer many advantages, such as the study of the bioavailability of nanomaterials to sensitive target cells, we propose Drosophila as a useful model for the study of the toxic and genotoxic risks associated with nanoparticle exposure. In this work we have carried out a genotoxic evaluation of silver nanoparticles in Drosophila by using the wing somatic mutation and recombination test. This test 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 larval cells, which are expressed as mutant spots on the wings of adult flies. Silver nanoparticles were supplied to third instar larvae at concentrations ranging from 0.1-10 mM. The results showed that small but significant increases in the frequency of total spots were observed, thus indicating that silver nanoparticles were able to induce genotoxic activity in the wing spot assay of D. melanogaster, mainly via the induction of somatic recombination. These positive results obtained with silver nanoparticles contrast with the negative findings obtained when silver nitrate was tested. [ABSTRACT FROM AUTHOR]

Several studies suggested that gold nanoparticles (NPs) could be genotoxic in vitro and in vivo. However, gold NPs currently produced present a wide range of sizes and functionalization, which could affect their interactions with the environment or with biological structures and, thus, modify their toxic effects. In this study, we investigated the role of surface charge in determining the genotoxic potential of gold NPs, as measured by the induction of DNA damage (comet assay) and chromosomal damage (micronucleus assay) in human bronchial epithelial BEAS-2B cells. The cellular uptake of gold NPs was assessed by hyperspectral imaging. Two core sizes (~5 nm and ~20 nm) and three functionalizations representing negative (carboxylate), positive (ammonium), and neutral (poly(ethylene glycol) (PEG)ylated) surface charges were examined. Cationic ammonium gold NPs were clearly more cytotoxic than their anionic and neutral counterparts, but genotoxicity was not simply dependent on functionalization or size, since DNA damage was induced by 20-nm ammonium and PEGylated gold NPs, while micronucleus induction was increased by 5-nm ammonium and 20-nm PEGylated gold NPs. The 5-nm carboxylated gold NPs were not genotoxic, and evidence on the genotoxicity of the 20-nm carboxylated gold NPs was restricted to a positive result at the lowest dose in the micronucleus assay. When interpreting the results, it has to be taken into account that cytotoxicity limited the doses available for the ammonium-functionalized gold NPs and that gold NPs were earlier described to interfere with the comet assay procedure, possibly resulting in a false positive result. In conclusion, our findings show that the cellular uptake and cytotoxicity of gold NPs are clearly enhanced by positive surface charge, but neither functionalization nor size can single-handedly account for the genotoxic effects of the gold NPs.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used for preparations of sunscreens, cosmetics, food and personal care products. However, the possible genotoxic risk associated with this nano-scale material exposure is not clear, especially in whole organisms. In the present study, we explored the in vivo genotoxic activity of TiO2 NPs as well as their TiO2 bulk form using two well-established genotoxic assays, the wing spot test and the comet assay in Drosophila melanogaster. To determine the extent of tissue damage induced by TiO2 NPs in Drosophila larvae, the trypan blue dye exclusion test was also applied. Both compounds were supplied to third instar larvae by ingestion at concentration ranging from 0.08 to 1.60 mg/mL. The results obtained in the present study indicate that TiO2 NPs can reach and induce cytotoxic effects on midgut and imaginal disc tissues of larvae, but they do not promote genotoxicity in the wing-spot test of Drosophila. However, when both nano- and large-size forms of TiO2 were evaluated with the comet assay in Drosophila hemocytes, a significant increase in DNA damage, with a direct dose-response pattern, was observed for TiO2 NPs. The results obtained with the comet assay suggest that the primary DNA damage associated with TiO2 NPs exposure in Drosophila could be associated with specific physico-chemical properties of nano-TiO2, since no effects were observed with the bulk form. This study remarks the usefulness of using more than one genetic end-point in the evaluation of the genotoxic potential of nanomaterials., (Copyright © 2014 Elsevier B.V. All rights reserved.)