Linkages between AOP key events with in vitro DNT assays: Examples of GABAergic and Glutamatergic modes of action
Citation:
Pitzer, E. Linkages between AOP key events with in vitro DNT assays: Examples of GABAergic and Glutamatergic modes of action. 5th International Confernce on Developmental Neurotoxicity Testing (DNT5), Konstanz, GERMANY, April 07 - 10, 2024.
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.
Description:
Neurodevelopment is a period vulnerable to various insults, including those produced by environmental compounds, and an understanding of these effects continues to be needed. Although in vitro New Approach Methodologies (NAMs) are being developed to foster efficient assessment of both neurotoxicity (NT) and developmental neurotoxicity (DNT), reliability in these methods for regulatory purposes is necessary, which entails in vivo assessments for corroboration and extrapolation. Currently there is indication of alignment of DNT NAMs with terms used in NT/DNT Adverse Outcome Pathways (AOPs) found on the Wiki site, as seen in key events such as “cell injury/death”, “decreased synaptogenesis”, or “decreased neuronal network function”. Here we aim to assess the unique protein signatures following developmental or acute exposures to known neurotoxicants in Long Evans rats, which can then be applied to an AOP framework as biological key events and further work to develop and show reliability in in vitro NAMs. Examples of proteomic results described were done in two studies. The first study was an acute exposure to kainic acid (KA; targets ionotropic glutamatergic receptors; 6 mg/mL, s.c., chosen not to produce myoclonic seizures or vehicle control of buffered saline (0 mg/mL)) in adult male rats with proteomic analysis 3 or 24 hours after exposure. A second study was conducted that involved developmental exposure to emamectin benzoate (EB; targets GABA gated chloride channels and glycine receptors; 3.78 mg/kg in 5mL deionized water or vehicle via gavage from gestational day 6 to postnatal day (PND) 21) with proteomic measures at PND2, 8, 15, or 22. Region-specific brain tissues (cortex, hippocampus, or cerebellum) were collected for both experiments following perfusions with phosphate buffered saline, and then stored at -80°C until use. Samples (6/group/region) have been analyzed for proteomic content using Orbitrap LC-MS/MS and the identified proteins were then further processed using Proteome Discover. Pathway analysis of proteomic results was completed using Ingenuity Pathway Analysis (IPA) software (significance cutoff: ≤ log2(0.8), ≥ log2(1.2), FDR p-value ≤ 0.05); data was examined both with cut offs applied and without). Altered pathways following KA exposure include synaptogenesis signaling, ephrin receptor signaling, semaphoring neuronal repulsive signaling, and autophagy, all of which can relate to current in vitro NAMs, such as synaptogenesis, apoptosis, and migration. Likewise, across all developmental timepoints for EB treated animals there appear to be impacted pathways relating to in vitro NAM measures (assay endpoints including synaptogenesis, proliferation, growth, migration, and apoptosis). This includes Hippo signaling, apoptosis signaling, unfolded protein response, and immunogenic cell death signaling among predicted pathways in the cortex, and synaptogenesis signaling and ephrin signaling among impacted pathways in the cerebellum. Further analysis includes ontological changes in proteomic profiles to examine if potential shifts in developmental protein expression differ between treatments. Also, additional doses and regions are being examined for both experimental exposures. 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. This is an abstract of a proposed presentation and does not necessarily reflect US EPA policy.