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NAnomics in vitro DEvelopmental TOXcology

Final Report Summary - NADETOX (NAnomics in vitro DEvelopmental TOXcology)

The EU funded research project NADETOX (NAnomics in vitro DEvelopmental TOXicology) was focused on the eco-toxicological potential of metallic nanoparticles (mNPs) widely applied in medical health care and consumer products. NADETOX is based on FETAX (Frog Embryo Teratogenesis Assay-Xenopus), a whole-embryo developmental toxicity test that utilizes embryos of the South African clawed frog Xenopus laevis. FETAX can detect, by its three endpoints (embryotoxicity, teratogenicity and growth retardation), xenobiotics affecting embryonic development. Moreover, FETAX represents one of the most extensively used developmental toxicity alternative test system of chemicals and it has been applied in ecological risk assessment and in metal toxicology research, showing X. laevis very sensitive to increased concentrations of metals. Furthermore, FETAX can be integrated with peculiar spectrochemical, radiochemical, physicochemical, biochemical and molecular biology techniques that considerably increase the capability of the test allowing an interdisciplinary approach of the problem studied.

Summary of project objectives
1. Morphological and chemical characterization of Co-, Au- and Ag-NPs by microscopy, radiochemical and spectro-chemical techniques.
2. Characterization of Co-, Au- and Ag-NPs behaviour in FETAX medium and study of the potential quantitative release of ions in the growth medium.
3. Study of the potential impact of Co-, Au- and Ag-NPs on embryonic growth of Xenopus.
4. Study the toxicokinetics of Co-, Au- and Ag-NPs in Xenopus embryos/larvae.

DESCRIPTION OF THE WORK

WP 1. We measured elemental impurities present in cobalt and gold nanoparticles (Co-NPs and Au-NPs) by NAA and ICP-MS. Elemental impurities could cause toxic effects erroneously ascribable to mNPs and this misinterpretation of the collected data would completely undermine the scientific validity of the work carried out.

198Au-NPs and 195Au3+ was prepared at the Hoger Onderwijs Reactor of TU Delft and at Isochronous cyclotron U-120M of the Nuclear Physics Institut, Řež, Czech Republic, respectively. Due to several technical and experimental problems this task is, unfortunately, could not be completed.

WP 2. INAA in combination with TEM analysis was applied to assess surface modifications or even rupture of Au-NPs after neutron bombardment.

WP 3 and 4. INAA, RNAA and ICP-MS were used to measure the body elemental composition of Xenopus larvae for the understanding of the metabolic changes with their progressive development and as reference data in the context of FETAX. Moreover, the uptake of Co2+and Co-NPs in Xenopus embryos/larvae according the FETAX procedure was studied to obtain a picture of the metabolic pattern of Co2+and Co-NPs by using 57Co2+ and 60Co-NPs. Furthermore, eventual gene expression alteration resulting from exposure to Co2+and Co-NPs was studied by differential display, a molecular biology technique that allows the comparison of two or more messenger ribonucleic acid (mRNA) pools. We commenced these investigations by testing the expression level of heat shock protein 70 (HSP70) in response to Co2+ and Co-NPs exposure.

MAIN RESULTS ACHIEVED SO FAR

WP 1. Elemental impurities in Co-NPs, manufactured by American Elements Co, are at very low level and they should not cause any toxicological response in our biological system. On the contrary, the level of elemental impurities in Au-NPs, synthetized and provided by Prof. Andreas Schmidt-Ottt (Group of Nanostructure Materials, Department of Chemical Engineering, TU Delft), was too high and their application in toxicity tests was impossible. It was necessary to refine the protocol of Au-NPs synthesis in order to reduce the amount of impurities to an acceptable level. This task took a long time delaying the research work.

WP 2. Preliminary results of TEM analysis suggest that neutron irradiation do not change surface and size of mNPs.

WP 3 and 4. Macroelements (> 100 mg Kg-1), trace elements (range ng g-1) and ultratrace elements (< ng g-1) were measured in few hundreds of milligrams Xenopus larvae.

Co, in both the forms of Co tested(Co2+/Co-NPs), is taken up by embryos. Moreover, the isolation of Co–DNA adducts confirm the ability of Co to bind DNA.

HSP70 appeared to be induced in our system by both Co forms (Co2+/Co-NPs).

EXPECTED FINAL RESULTS, POTENTIAL IMPACT AND USE

WP 1. The results of this task suggested that Co-NPs can be used in toxicity tests. mNPs can be neutron activated, detected and quantified, apparently without destruction of the sample applied.

WP 2. NAA can be used to label mNPs in a non-destructive manner without modifying their characteristics.

WP 3 and 4. The results about body elemental composition of 120 h Xenopus larvae have been summarized in a paper titled “Nuclear and spectrochemical techniques in developmental metal toxicology research. Whole-body elemental composition of Xenopus laevis larvae” that was accepted by the Journal of Radioanalytical and Nuclear Chemistry. The paper is in press.

The results of the Co-uptake indicates that cobalt mNPs could cause unregulated DNA structures that could affect gene expression, preventing transcription of certain genes or changing the topology of the double helix.

Now, we are searching for other genes whose expression could be modified by Co2+ and Co-NPs exposure. This task is still in progress and carried out in collaboration with Prof. Jacob Souopgui, head at Laboratory of Developmental Genetics DBM-IBMM (Université Libre de Bruxelles, Gosselies, Belgium).