Engineered nanomaterial-relevant AOPs; network creation and identification of key nodes for adverse outcomes

Background, Motivation and Objective
Adverse Outcome Pathways (AOPs) are a useful framework for organizing and presenting scientific data related to toxicological processes. As a step in the progress towards developing New Approach Methodologies (NAM) in the EU project HARMLESS, partners identified, compiled and organized existing and tentative AOPs relevant for engineered nanomaterial (ENM) induced adverse outcomes (AOs) to human health and ecology.

Statement of Contribution/Methods
ENM-relevant AOPs were identified from previous EU projects, the AOPwiki and through other approaches. The AOPs were then submitted to selection criteria aligned with the overall aims of HARMLESS, e.g. related to exposure routes, and AOPs not meeting these were excluded from the AOP list. Networks related to major ENM-induced human adverse outcomes were created for visualizing relationships and nodes, and to identify central key events (KEs) for further NAM development.

Results/Discussion
Following a thorough search based on the listed search criteria, 25 AOPs relevant for human toxicology and 19 AOPs relevant for ecotoxicology were identified. Based on exclusion criteria, the list of AOPs relevant for human toxicology was shortened to 21 AOPs. For ecotoxicology, the list of 19 relevant AOPs was shortened to 7 AOPs relevant to HARMLESS following a ranking and selection process. Of these, 5 were overlapping with AOPs identified as relevant for human toxicology. Short descriptions of each AOP were provided, including their development status.
Four networks for human toxicology were created based on AOs: lung fibrosis, lung cancer/mesothelioma, decreased lung function and liver. Each identified KE node was assessed for its use as a potentially predictive marker for the AO. For lung fibrosis, KE nodes could be divided into early and late KE nodes. The early KE nodes were related to inflammation and later KE nodes were related to extracellular matrix, in particular collagen, deposition. The lung cancer/mesothelioma network was highly intertwined, with several major KE nodes. Inflammation and oxidative stress were identified as the central initial KEs, leading to the node KEs related to oxidative DNA damage and mutations, and subsequently to increased cell proliferation. In contrast, no KE nodes were identified for decreased lung function. The AOPs in the liver network were also relatively detached, but common node KEs were identified for liver fibrosis-related events, such as inflammation, stellate cell activation and collagen deposition. Ecotoxicology AOPs could not be merged into meaningful groups and networks were therefore not created for these. However, homologies were identified in KEs of AOPs applicable to humans and fish related to endocytic lysosomal uptake, oxidative stress responses and glutathione homeostasis perturbation leading to hepatotoxicty, liver fibrosis and mortality.
Collectively, AOP network visualization and node identification highlighted important key events for ENM-induced progression towards AOs. This information is useful as targeted guidance for NAM development and constructing assays for hazard screening of new nanomaterials.

Funding information
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 953183 (HARMLESS).