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Smart Tools for Gauging Nano Hazards

Periodic Reporting for period 2 - SmartNanoTox (Smart Tools for Gauging Nano Hazards)

Reporting period: 2017-03-01 to 2018-08-31

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 second period of the project was focused on data generation for quantification of toxicity and properties of selected NMs. We aimed to process the omics data using systems biology methods to identify MIEs and KEs for each selected AOP, and develop an optimum strategy for the assessment of the NM's ability to induce those events.

Main results of the project to date:
In WP1 we
• Generated aerosols and suspensions and performed animal exposure experiments (inhalation and instillation) with carbonaceous, metal oxide and quartz NMs
• Prepared suspensions and exposed cell lines to metal oxides and carbonaceous materials using submerged or murine dose-controlled ALI setups
• Performed toxicology and histology analyses of in vivo samples, analyzed and scored in vivo pathological outcomes
• Demonstrated correlation between the total deposited BET and level of neutrophil influx in lung after instillation
• Demonstrated correlation of acute pulmonary inflammation between intratracheal instillation and inhalation (based on lung deposited surface area)
In WP2 we
• Performed in vitro NM tracking and characterisation in lung epithelial cells and macrophages
• Developed advanced in vitro lung barrier model using a realistic surfactant layer
• Evaluated the suitability and relevance of surfactant from different sources in a novel in vitro setup using the Constrained Drop Surfactometer
• Optimized advanced super-resolved imaging techniques 2PE, STED and 2PE STED for more reliable tracking and identification of NP under in vitro and in vivo condition
• Identified new candidates for MIE and KE such as lipid wrapping around NP and associating the phenomena to known MIE/KE and AOPs
• Performed proteomics and lipidomics studies on protein/lipid corona/wraps of NM to identify molecular cascades after first contact between lung barrier and NM
• Assessed selected molecular markers to identify and colocalize molecular events accompanying the interaction of NP within the lung epithelial/macrophage cell layer
• Developed management and sharing protocols for experimental data on bionano interactions
In WP3 we
• Developed and submitted two respiratory AOPs to OECD, 5 more are in progress
• Analyzed transcriptomic data for rodents exposed by inhalation to CNTs
• Analyzed transcriptomes on the rat alveolar macrophage model
• Performed proteomics analysis of BALF and NM corona for CNT in rat samples from animals exposed by inhalation and instillation
• Developed a statistical framework for inferring transcription regulatory programs for the identification of crucial transcription factors
In WP4 we
• Computed adsorption free energies of biomolecules to selected NMs
• Developed methodology of systematic coarse-graining of protein models
• Computed descriptors of NMs such as band gap for metal oxide NPs or hydration enthalpy
In WP5 we
• Identified several KEs for fibrosis AOP, acute phase response, and CNT-induced cancer
• Developed in silico prediction methods for NM-lipid membrane interaction and prediction of lysosomal damage
In WP6 we
• Proposed structures for 7 selected AOPs based on information from literature and from WP3 and WP5
• Identified a suite of in vitro/in silico/in vivo assays matching the AOP-specific MIEs/KEs
• Performed statistical analysis of the in vivo toxicology data and in silico data
In WP7 we
• Launched project website
• Published 28 papers in refereed journals
• Made 68 presentations at conferences
• Co-organised 4 international conferences
• Created project brochure and outreach video
We envision that the following project results to make maximum impact:

Research impacts
• Gene expression profiles for in vivo respiratory exposure
• Novel analysis protocols for inference of GRNs from transcriptomics data, identification of Core Regulatory Genes
• Novel NM labelling techniques
• Novel protocols for corona analysis
• Novel algorithms for image analysis / colocalization
• Protein corona-based NM fingerprints
• NM tracking techniques, post-uptake characterisation data
• Atomistic, coarse-grained force fields for common NMs
• Multiscale simulation tools for bionano-interface
• Novel advanced NM and protein descriptors
• Data in an accessible and searchable database

Industry/Regulation impacts:
• Development and validation a novel testing strategy that can be used for risk assessment of new NMs
• Description of respiratory AOPs, KE/MIE
• Novel ALI systems imitating realistic exposure conditions
• Demonstration of mapping inhalation-instillation
• Demonstration of equivalence between rat/mouse/human models
• Novel toxicity endpoints bound to in vivo AOPs
• Novel in vitro assays targeting MIE/KE based on reporter gene
• Elucidation of toxicity mechanisms for oxides, carbonaceous materials
• Creation of basis for grouping NMs by their ability to induce specific AOPs
• Creation of basis for read-across and safety by design through identification of NM properties of concern
• Mechanism-aware QSARs relating NM properties to biological activity