Marie Carrière

Titanium dioxide nanoparticles exhibit genotoxicity and impair DNA repair activity in A549 cells

Abstract Titanium dioxide nanoparticles (TiO2-NPs) are produced in large quantities, raising concerns about their impact for human health. The aim of this study was to deeply characterize TiO2-NPs genotoxic potential to lung cells, and to link genotoxicity to physicochemical characteristics, e.g., size, specific surface area, crystalline phase. A549 cells were exposed to a panel of TiO2-NPs with diameters ranging from 12 to 140 nm, either anatase or rutile. A set of complementary techniques (comet and micronucleus assays, gamma-H2AX immunostaining, 8-oxoGuanine analysis, H2-DCFDA, glutathione content, antioxidant enzymes activities) allowed us to demonstrate that small and spherical TiO2-NPs, both anatase and rutile, induce single-strand breaks and oxidative lesions to DNA, together with a general oxidative stress. Additionally we show that these NPs impair cell ability to repair DNA, by inactivation of both NER and BER pathways. This study thus confirms the genotoxic potential of TiO2-NPs, which may preclude their mutagenicity and carcinogenicity.

Size-, Composition- and Shape-Dependent Toxicological Impact of Metal Oxide Nanoparticles and Carbon Nanotubes toward Bacteria

Ecotoxicological effects of nanoparticles (NP) are still poorly documented while their commercialization for industrial and household applications increases. The aim of this study was to evaluate the influence of physicochemical characteristics on metal oxide NP and carbon nanotubes toxicological effects toward bacteria. Two strains of bacteria, Cupriavidus metallidurans CH34 and Escherichia coli MG1655 were exposed to TiO(2) or Al(2)O(3) NP or to multiwalled-carbon nanotubes (MWCNT). Particular attention was paid on optimizing NP dispersion to obtain nonagglomerated suspensions. Our results show that NP toxicity depends on their chemical composition, size, surface charge, and shape but not on their crystalline phase. MWCNT toxicity does not depend on their purity. Toxicity also depends on the bacterial strain: E. coli MG1655 is sensitive to NP, whereas C. metallidurans CH34 is not. Interestingly, NP are accumulated in both bacterial strains, and association between NP and bacteria is necessary for bacterial death to occur. NP may then represent a danger for the environment, causing the disappearance of some sensitive bacterial strains such as E. coli MG1655, but also being mobilized by nonsensitive strains such as C. metallidurans CH34 and transported through the whole ecosystem.

In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes

If released in the environment, nanomaterials might be inhaled by populations and cause damage to the deepest regions of the respiratory tract, i.e., the alveolar compartment. To model this situation, we studied the response of A549 human pneumocytes after exposure to aluminium oxide or titanium oxide nanoparticles, and to multi-walled carbon nanotubes. The influence of size, crystalline structure and chemical composition was investigated. After a detailed identification of nanomaterial physico-chemical characteristics, cells were exposed in vitro and viability and intracellular accumulation were assessed. In our conditions, carbon nanotubes were more toxic than metal oxide nanoparticles. Our results confirmed that both nanotubes and nanoparticles are able to rapidly enter into cells, and distribute in the cytoplasm and intracellular vesicles. Among nanoparticles, we demonstrate significant difference in biological response as a function of size, crystalline phase and chemical composition. Their toxicity was globally lower than nanotubes toxicity. Among nanotubes, the length did not influence cytotoxicity, neither the presence of metal catalyst impurities.

Accumulation, translocation and impact of TiO2 nanoparticles in wheat (Triticum aestivum spp.): Influence of diameter and crystal phase

Intensive production of TiO(2) nanoparticles (TiO(2)-NPs) would lead to their release in the environment. Their ecotoxicological impact is still poorly documented, while their use in commercial goods is constantly increasing. In this study we compare root accumulation and root-to-shoot translocation in wheat of anatase and rutile TiO(2)-NPs with diameters ranging from 14 nm to 655 nm, prepared in water. NP distribution in plant tissues was mapped by synchrotron-radiation micro-X-ray fluorescence, observed by transmission electron microscopy and quantified in the different compartments of plant roots by micro-particle-induced X-ray emission. Our results provide evidence that the smallest TiO(2)-NPs accumulate in roots and distribute through whole plant tissues without dissolution or crystal phase modification. We suggest a threshold diameter, 140 nm, above which NPs are no longer accumulated in wheat roots, as well as a threshold diameter, 36 nm, above which NPs are accumulated in wheat root parenchyma but do not reach the stele and consequently do not translocate to the shoot. This accumulation does not impact wheat seed germination, biomass and transpiration. It does not induce any modification of photosynthesis nor induce oxidative stress. However exposure of wheat plantlets to the smallest NPs during the first stages of development causes an increase of root elongation. Collectively, these data suggest that only the smallest TiO(2)-NPs may be accumulated in wheat plants, although in limited amounts and that their impact is moderate.

NLS bioconjugates for targeting therapeutic genes to the nucleus

One of the major steps limiting non-viral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of transfected cells. Trafficking of nuclear proteins from the cytoplasm into the nucleus through nuclear pore complexes is mediated by the presence of nuclear localization sequences (NLS) on proteins. Viral DNA and RNA also require interaction with cellular machinery for efficient nuclear import. In this article, we review the various strategies used to provide plasmid DNA with nuclear localization sequences, and discuss the possibility of developing efficient gene delivery systems based on these strategies.

Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery

Nickel is a cofactor for various microbial enzymes, yet as a trace element, its scavenging is challenging. In the case of the pathogen Helicobacter pylori, nickel is essential for the survival in the human stomach, because it is the cofactor of the important virulence factor urease. While nickel transport across the cytoplasmic membrane is accomplished by the nickel permease NixA, the mechanism by which nickel traverses the outer membrane (OM) of this Gram‐negative bacterium is unknown. Import of iron‐siderophores and cobalamin through the bacterial OM is carried out by specific receptors energized by the TonB/ExbB/ExbD machinery. In this study, we show for the first time that H. pylori utilizes TonB/ExbB/ExbD for nickel uptake in addition to iron acquisition. We have identified the nickel‐regulated protein FrpB4, homologous to TonB‐dependent proteins, as an OM receptor involved in nickel uptake. We demonstrate that ExbB/ExbD/TonB and FrpB4 deficient bacteria are unable to efficiently scavenge nickel at low pH. This condition mimics those encountered by H. pylori during stomach colonization, under which nickel supply and full urease activity are essential to combat acidity. We anticipate that this nickel scavenging system is not restricted to H. pylori, but will be represented more largely among Gram‐negative bacteria.

Foliar exposure of the crop Lactuca sativa to silver nanoparticles: Evidence for internalization and changes in Ag speciation

The impact of engineered nanomaterials on plants, which act as a major point of entry of contaminants into trophic chains, is little documented. The foliar pathway is even less known than the soil-root pathway. However, significant inputs of nanoparticles (NPs) on plant foliage may be expected due to deposition of atmospheric particles or application of NP-containing pesticides. The uptake of Ag-NPs in the crop species Lactuca sativa after foliar exposure and their possible biotransformation and phytotoxic effects were studied. In addition to chemical analyses and ecotoxicological tests, micro X-ray fluorescence, micro X-ray absorption spectroscopy, time of flight secondary ion mass spectrometry and electron microscopy were used to localize and determine the speciation of Ag at sub-micrometer resolution. Although no sign of phytotoxicity was observed, Ag was effectively trapped on lettuce leaves and a thorough washing did not decrease Ag content significantly. We provide first evidence for the entrapment of Ag-NPs by the cuticle and penetration in the leaf tissue through stomata, for the diffusion of Ag in leaf tissues, and oxidation of Ag-NPs and complexation of Ag(+) by thiol-containing molecules. Such type of information is crucial for better assessing the risk associated to Ag-NP containing products.

Titanium dioxide nanoparticle impact and translocation through ex vivo, in vivo and in vitro gut epithelia

BackgroundTiO2 particles are commonly used as dietary supplements and may contain up to 36% of nano-sized particles (TiO2-NPs). Still impact and translocation of NPs through the gut epithelium is poorly documented.ResultsWe show that, in vivo and ex vivo, agglomerates of TiO2-NPs cross both the regular ileum epithelium and the follicle-associated epithelium (FAE) and alter the paracellular permeability of the ileum and colon epithelia. In vitro, they accumulate in M-cells and mucus-secreting cells, much less in enterocytes. They do not cause overt cytotoxicity or apoptosis. They translocate through a model of FAE only, but induce tight junctions remodeling in the regular ileum epithelium, which is a sign of integrity alteration and suggests paracellular passage of NPs. Finally we prove that TiO2-NPs do not dissolve when sequestered up to 24 h in gut cells.ConclusionsTaken together these data prove that TiO2-NPs would possibly translocate through both the regular epithelium lining the ileum and through Peyer’s patches, would induce epithelium impairment, and would persist in gut cells where they would possibly induce chronic damage.

In vitro evaluation of SiC nanoparticles impact on A549 pulmonary cells: Cyto-, genotoxicity and oxidative stress

Silicon carbide (SiC) is considered a highly biocompatible material, consequently SiC nanoparticles (NPs) have been proposed for potential applications in diverse areas of technology. Since no toxicological data are available for these NPs, the aim of this study was to draw their global toxicological profile on A549 lung epithelial cells, using a battery of classical in vitro assays. Five SiC-NPs, with varying diameters and Si/C ratios were used, and we show that these SiC-NPs are internalized in cells where they cause a significant, though limited, cytotoxic effect. Cell redox status is deeply disturbed: SiC-NP exposure cause reactive oxygen species production, glutathione depletion and inactivation of some antioxidant enzymes: glutathione reductase, superoxide dismutase, but not catalase. Finally, the alkaline comet assay shows that SiC-NPs are genotoxic. Taken together, these data prove that SiC-NPs biocompatibility should be revisited.

Quantitative evaluation of multi-walled carbon nanotube uptake in wheat and rapeseed

Environmental contamination with carbon nanotubes would lead to plant exposure and particularly exposure of agricultural crops. The only quantitative exposure data available to date which can be used for risk assessment comes from computer modeling. The aim of this study was to provide quantitative data relative to multi-walled carbon nanotube (MWCNT) uptake and distribution in agricultural crops, and to correlate accumulation data with impact on plant development and physiology. Roots of wheat and rapeseed were exposed in hydroponics to uniformly (14)C-radiolabeled MWCNTs. Radioimaging, transmission electron microscopy and raman spectroscopy were used to identify CNT distribution. Radioactivity counting made it possible absolute quantification of CNT accumulation in plant leaves. Impact of CNTs on seed germination, root elongation, plant biomass, evapotranspiration, chlorophyll, thiobarbituric acid reactive species and H(2)O(2) contents was evaluated. We demonstrate that less than 0.005‰ of the applied MWCNT dose is taken up by plant roots and translocated to the leaves. This accumulation does not impact plant development and physiology. In addition, it does not induce any modifications in photosynthetic activity nor cause oxidative stress in plant leaves. Our results suggest that if environmental contamination occurs and MWCNTs are in the same physico-chemical state than the ones used in the present article, MWCNT transfer to the food chain via food crops would be very low.