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Smart Tools for Gauging Nano Hazards

Periodic Reporting for period 1 - SmartNanoTox (Smart Tools for Gauging Nano Hazards)

Reporting period: 2016-03-01 to 2017-02-28

A definitive conclusion about the dangers associated with human or animal exposure to a particular nanomaterial (NM) can currently be made upon complex and costly procedures including complete NM characterisation with consequent careful and well-controlled in vivo experiments. A significant progress on the possibility of the robust nanotoxicity prediction can be achieved using modern approaches based on the one hand on systems biology, on the other hand on statistical and other computational methods of analysis.
In this project, using a comprehensive self-consistent study, which includes in vivo, in vitro and in silico research, we address main respiratory adverse outcome pathways (AOP) for representative set of NMs, identify the mechanistic key events (KE) of the pathways, and relate them to interactions at the bionano interface via careful post-uptake NM characterisation and molecular modelling. Our goal is to formulate a novel set of toxicological mechanism-aware end-points that can be assessed by means of economic and straightforward tests. Using the exhaustive list of end-points and pathways for the selected NM and exposure routes, we attempt to relate the AOPs to the properties of the material via quantitative structure-activity relationships (QSAR). This will lead to grouping of NMs based on their ability to trigger the pathway, and will enable an identification of properties of concern for new NMs.

The SmartNanoTox predictive model for gauging the toxicological and biological impacts of NMs will be based on the mechanistic approach, which makes use of detailed understanding of the response of the organism to exposure to NMs from the initial contact to the adverse outcome (AO).

SmartNanoTox objectives:
· To identify main pulmonary AOs induced by common NMs, and identify associated MIE, KEs and toxicity pathways (TP) leading to AO.
· To establish relationships between physicochemical properties of NMs and KEs steering the TP leading to AO, and suggest descriptors for grouping of NMs according to their toxicological mode-of-action.
· To create a database of bionano interactions that will enable development of read-across and QSAR tools for the toxicity assessment of new NMs.
· To develop a smart screening approach, where predictions of toxicity of a NM can be made on the basis of purely computational or limited in vitro screening tests focused on crucial bionano interactions.
The first period of the project was focused on an analysis of the existing information and a development of an optimum strategy to identify in vivo AOPs for respiratory exposure to NM. We also developed, tested and validated methods for NM tracking inside the biological samples, post-uptake characterisation and bionano interface modelling. Our main achievements for the reporting period are as follows:
Work Package 1:
· Identified 5 respiratory AOPs that can be addressed within the project:
· Selected a set of relevant NMs suitable for aerosolisation and study of the chosen AOPs
· Performed transcriptomics analysis of lung samples after in vivo exposure to MWCNT from NANoREG
Work Package 2:
· Developed a protocol to assess fluorescent probe desorption from NM
· Applied the protocol for ensuring the quality of labelled NM at three major phases of experiments: functionalisation of NM, labelling and free probe removal
· Developed a NM labelling technique by embedding europium atoms into the TiO2 crystal lattice during TiO2 NP synthesis, which enables fluorescent imaging of NP after long-exposure in vivo experiments
· Developed a set of in vitro tests consisting of pristine NPs and model membranes to study the evolution of NP wraps and alleviate the determination of MIEs
· Refined the procedure for the analysis of NM protein corona
Work Package 3:
· Selected NMs, doses and protocols to identify the relevant TPs
· Isolated coated nanoparticles from in vivo samples from mice and identified proteins in corona by mass-spectrometry
· Performed proteomics analysis of in vivo samples after exposure to asbestos and CNTs
· Performed pathway reconstruction for several in vivo samples with MWCNTs
Work Package 4:
· Developed a multiscale method of modelling of NM-biomolecule interaction using two-layer NM model and potentials of mean force NM-aminoacid from atomistic simulations
· Calculated adsorption free energies of aminoacids to various CNTs
· Predicted 3D structures for over a 100 plasma proteins identified in NM protein corona
· Launched a project website
· Published 5 papers in refereed journals
· Presented 17 talks at conferences
· Published 5 press releases and created a twitter account to advertise the project activities
· Participated in OECD-ProSafe meeting on Nanosafety
The main beyond the state-of-art feature of our approach is the focus on mechanistic interpretation of the nanotoxicology data. We plan to achieve this via identification of the relevant pathways and extensive use of molecular modelling tools and post-uptake NM characterisation, which relate the physicochemical descriptors of the NM to the MIEs of respiratory TPs in terms of molecular interactions.
The planned project outcomes:
· Mechanism-aware AOP-oriented QSARs for toxicity prediction
· Toxicity assessment strategy based on in vivo study of the acute and chronic toxic effects, with relation to the AO and in-depth analysis of the pathologies
· Analysis of post-uptake state of NMs: biomolecular corona of the NMs, NM tracking inside the biological fluids and identification of molecules involved in bionano interactions
· Identification of generic properties responsible for pulmonary toxicity for carbonaceous materials, non-metal oxides.

We aim to directly address the desired impacts of both the overall NMPB Programme, and the specific call (Increasing capacity to perform nanosafety assessment):
• New screening tools to enhance the efficiency of end-rate at which NM hazard profiling can be performed
We will develop reliable in vitro and in silico tools based on the knowledge of the mechanisms of the pulmonary toxicity of NMs and bound to novel endpoints ans validate their predictive power by in vivo strength of evidence.
• Enable “safer by design” approaches, tailored to stakeholders’ needs (modelers, industry and regulators)
By systematically studying interactions between the NMs and all the building blocks of biomolecules, we will enable a prediction of the outcome of interaction of arbitrary key molecules with the NM and the content of NM-BM complexes for any NM with known physicochemical properties. By scanning main groups of engineered NMs, we will identify the NM properties of concern related to a particular AO, and thus should be modified or avoided. This will enable development of NMs that are safe by design.
• Data in a recognized and accessible database for use beyond the lifetime of the project
We will create a bionano interactions database to classify the NM according to the type of change of the NM state and type of event each particular property can be related to. The experimental results will be made available to the community using the eNanoMapper framework.
• Solutions to the long-term challenges of nanosafety and nanoregulation
We will contribute to NCBI GEO database with MIAME-compliant NM-induced gene expression profiles, contribute to OECD database with exhaustive description of new TPs (or AOPs).
Research concept and outline of the project activities