Linkages between AOP key events with Developmental Neurotoxicity (DNT) screening assays: Utilizing proteomics to further AOP development and identify knowledge gaps

New Approach Methodologies (NAMs) are being developed to foster efficient screening for neurotoxicity (NT), and reliability in these methods for regulatory purposes is necessary, entailing in vivo assessments for corroboration. We have shown alignment of NAMs with terms in NT Adverse Outcome Pathways (AOPs) found on the Wiki site, i.e., cellular death, synaptogenesis, and network function. Here we aim to assess the unique protein signatures following exposures to known neurotoxicants in Long Evans rats, to apply to an AOP framework as biological key events, furthering work to develop reliability in NAMs. Adult male Long Evans rats were acutely exposure to kainic acid (KA; targets ionotropic glutamatergic receptors; 6 mg/mL, s.c., did not produce myoclonic seizures; or vehicle control of buffered saline (0 mg/mL)) and tissues were collected at either 3 or 24 hours after exposure. Brain tissues (hippocampus, cortex, cerebellum) were assessed via LC-MS/MS and pathway analysis was completed with varying bioinformatic tools (IPA, DAVID, Reactome). Modest protein changes were observed at this sub-seizure exposure (significantly altered proteins/total protein count: 32/945 at 3h, 25/996 at 24h). Top identified pathways in IPA following KA exposure included those involved in mitochondrial function (3h), metabolism and neuronal signaling (3 and 24h). Although not directly related to current NAM measures, this could aid in identifying gaps in areas of research.  Outside of top pathways, glutamatergic system pathways and pathways relating to synaptogenesis (24h only) and neurotransmitters (24h only) are observed. Concordance with IPA results was observed in Reactome and DAVID on pathways related to metabolism of molecules and neuronal signaling being impacted. Although, some pathways observed align with NAM measures (synaptogenesis), others do not, highlighting gaps where further development is needed to capture these biological events. This is an abstract of a proposed presentation and does not necessarily reflect US EPA policy.

Impact/Purpose

New Approach Methodologies (NAMs) are being developed to foster assessment of Neurotoxicity and Developmental Neurotoxicity (DNT). Confidence in the use of these NAMs for risk decisions under FIFRA, TSCA and other authorities is increased when the outcomes in these assays are linked in a biologically plausible context to adverse outcomes of regulatory interest through Adverse Outcome Pathways (AOPs). This project aims to assess the unique protein signatures following developmental exposure to known neurotoxicants in vivo, to link impacted pathways to in vitro NAMs. Alterations in proteomics can then be applied to the AOP framework as biological key events. Ultimately these in vivo proteomic biomarkers will be compared to those observed in in vitro counterparts. Concordance of 'omics results from in vitro cell culture to ex vivo or in vivo experiments will help verify the predictive validity of the DNT NAMs.

Citation

Pitzer, E., G. Jung, Kathy McDaniel, W. Padgett, W. Winnik, AND D. Herr. Linkages between AOP key events with Developmental Neurotoxicity (DNT) screening assays: Utilizing proteomics to further AOP development and identify knowledge gaps. 5th Edition of Global Experts Meeting on FRONTIERS IN NEUROSCIENCES, Vienna, AUSTRIA, July 09 - 10, 2025.