List of Abstracts 2016: SESSION D - MONITORING AND TOXICITY OF NANOMATERIALS
Date: October 20, 2016
Physicochemical properties of nanomaterials differ significantly from those of corresponding bulk chemicals, mainly because of their high surface area relative to mass, which tends to increase reactivity. They are also better able to penetrate biological membranes. Therefore the hazards associated with nanomaterials cannot simply be extrapolated from those of bulk chemicals. Risk assessment presents a challenge in view of the large and increasing number of nanomaterials in production and use, and so there is an emphasis on developing high throughput methods and high content analysis for assessing nanomaterial toxicity. This approach, in addition to the savings in cost and time, allows testing at different concentrations and in diverse cell types, and reduces the effect of inter-experimental variation.
An important aspect of toxicity is damage to DNA, which is most conveniently measured with the comet assay. Experiments with four common nanomaterials, derived from ZnO, CeO2, TiO2 and Ag, have demonstrated direct strand breakage as well as oxidation damage (the latter being detected by digestion with the enzyme Fpg). Different levels of damage were seen depending on whether incubation with the nanomaterials continued for 3 or 24 h. While Ag and ZnO nanomaterials were clearly cytotoxic, CeO2 and TiO2 were not. All four induced significant levels of DNA damage at non-cytotoxic concentrations.
Recommendations for nanomaterial genotoxicity assessment should include testing over a range of concentrations, parallel testing for cytotoxicity/cell viability, and including Fpg in the comet assay. ACKNOWLEDGEMENTS: (Supported by EC FP7 NANoREG (Grant Agreement NMP4-LA-2013-310584) and by the Research Council of Norway, project NorNANoREG (239199/O70).)
We evaluated possible biological effects of inhalation of ZnO nanoparticles in adult female ICR mice. The mice were exposed to doses of 6x104 and 2x106 nanoparticles/cm3 for three days or three months to mimic acute and chronic NP exposure effects. To analyze gene expression changes, we used mRNA extracted from lung tissue. To study potential immunotoxic and immunomodulatory effects of ZnO nanoparticles, we used spleen cells from mice after three months exposure to ZnO. For gene expression analysis, we selected genes whose products are involved in oxidative stress, immune response, inflammation, apoptosis, DNA damage and repair, and cell cycle regulation. We used a novel approach based on next-generation sequencing (MiSeq, Illumina) of 298 splice junctions of 68 selected genes. Unlike standard Illumina microarray analysis, this method allowed us to evaluate gene expression changes associated with individual splice junctions thus giving us the opportunity to elucidate their role in the response of the organism to the inhalation exposure to NP.
We observed the presence of ZnO nanoparticles in the lungs after the chronic exposure to 2x106 particles/cm3, although their number in the tissue was low. After acute exposure, we mostly detected significant downregulation of gene expression, while chronic exposure was associated with increased gene expression levels. In the spleen cells, ZnO exposure caused significant decrease in the percentage of granulocytes; the production of pro-inflammatory and anti-inflammatory cytokines was not affected. ACKNOWLEDGEMENTS: Supported by the Ministry of Youth, Education and Sports of the Czech Republic (LO1508) and Czech Science Foundation (P503/12/G147).
Rat mesenchymal stem cells (rMSCs) labeled with (1) superparamagnetic iron oxide nanoparticles coated with poly-L-lysine (SPION) or (2) silica-coated cobalt-zinc-iron nanoparticles (CZF-NP) were implanted into the left brain hemisphere of rats with the aim to assess their effects on the levels of oxidative damage to biological macromolecules in brain tissue. Controls were implanted with unlabeled rMSCs. Animals were sacrificed 24 hours and 4 weeks after the treatment and the implantation site with surrounding tissue was isolated from the brains. The samples from the right untreated hemisphere were taken as a negative control. At the same intervals, parallel groups of animals were scanned in vivo by magnetic resonance imaging (MRI).The comet assay with enzymes of excision DNA repair (ENDO III and FPG) was used to analyze breaks and oxidative damage to DNA in the brain tissue. Oxidative damage to proteins and lipids was determined by measuring the levels of carbonyl groups and 15 F2t-Isoprostane (ELISA).
MRI displayed implants of labeled cells as extensive hypointense areas in the brain tissue. Both contrast labels caused similar response in the T2-weighted MR image and the signal was clearly visible within the four weeks after implantation of rMSCs. No increase of oxidative damage to DNA, lipids and proteins over the control values was detected in any sample of brain tissue from treated animals. Both types of nanoparticles thus appear as prospective and safe label for tracking of transplanted cells by magnetic resonance. ACKNOWLEDGEMENTS: Supported by projects 7F14057, RI NanoEnviCz (LM2015073), and OPPK (CZ.2.16/3.1.00/24507).
Nanoparticles of anthropogenic origin are continuously becoming more prevalent in the environment - both those purposefully produced and those originating from other human activities. Since exposition to nanoparticle pollution can be harmful to both human health and ecosystems, monitoring of their presence and concentration is of high concern, especially in the production facilities and their vicinity. Although biomonitoring of various pollutants utilizing bryophyte thalli is a rather wide-spread and well-known method of assessment of the environmental burden, it has not been applied for the monitoring of nanoparticles so far. In the experiment, moss thalli were exposed to titanium dioxide nanoparticle suspension up to eight weeks and analysed for the amount of the accumulated titanium in order to assess its suitability for nanoparticle biomonitoring. The amount of accumulated metal was in a strong linear relationship (R2 > 0.9, p < 0.05) with the time of exposition and hence the amount of the titanium dioxide the moss was exposed to. The study has thus proven the practicability of the bryophyte use in the nanoparticle pollution biomonitoring.
To model the quantitative relationship of the nanoparticle toxicity we can use theoretical molecular descriptors or physical-chemical characteristics. The former provide an auspicious interpretation of the toxicity mechanisms, however their computation may be very demanding, namely in the nanoscale. The latter are on the other hand fully observable, yet scarcely available for all the toxicity-assessed particles. Currently, there are large initiatives generating data for QNTR, including their toxicity and physical-chemical features. Resulting data are naturally very heterogeneous because of multiple subjects involved in the project. In this study, we investigate whether data generated from such large projects are sufficient to induce well-generelizing models. We used the data generated by MODENA-COST, consisting of the toxicity measurements and physical-chemical characteristics of 192 nanoparticles. We build several machine-learning based models and focused on their statistical validity. The internal evaluation of these models (i.e. protocol using the same data set, such as cross-validation) suggests quite good validity of these models. Hence, the result were not so optimistic at all. Instead, they seem valid only for a well-defined set of experimental conditions. ACKNOWLEDGEMENTS: This research is supported by the Czech Ministry of Education, Youth and Sports (Grants No. LD 14002 and LO1508).
Recent studies suggest that [C60] fullerenes and their derivatives are good antioxidants, anti-viral agents and can potentially be used as anticancer medicine and nanovectors for the drug delivery across biological barriers. But the effect of such derivatives on human cells is still controversially discussed and requires further investigation. We studied the effect of 3 [C60]fullerene derivatives with different moieties (F-243, F-827 and F-828) on breast cancer cell line MCF-7. F-828 and F-827 affect MCF-7 in a similar way: NOX4 expression increases, level of ROS elevates leading to oxidative damage of the DNA. 3 days after start NOX4 decreases as well as ROS production and reparation starts. In response to DNA breaks increases expression of ATM and ATR, Rad54 (17 - 27 %), survival-regulating STAT3 mRNA (6-fold) and protein STAT3 (50%), antiapoptotic Bcl2 (10%). After 3 days start overcoming the negative effect of F-827 and F-828. Substance F-243 is more toxic for MCF-7. The cells are trying to recover after a day of incubation with F-243, but reparation system remains inactive (BRCA1 remains on control level), ATM, ATR and STAT3 gene expression decreases as well as STAT3 protein level. NOX4 gene expression increases 4-fold and produces ROS, that should result in higher levels of DNA breaks and oxidative damage, which, however, isnt`t the case. Most likely the damaged cells are dying due to the reparation system not being activated - the level of apoptosis 3 days after start of incubation increases 15% compared to control.