Citation:

Pitzer, E., T. Beasley, G. Jung, K. McDaniel, W. Padgett, W. Winnik, AND D. Herr. Pathway analysis of developmental rat cerebellar and cortical proteomes following perinatal agonism of GABA-gated chloride channels. Society of Toxicology (SOT) 63rd annual meeting, Salt Lake City, UT, March 10 - 14, 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, as well as measure functional changes that can then be related to these protein level changes. These 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. Data presented herein relay proteomic profile pathway analyses following developmental exposure to prototypical neurotoxicants to apply to an AOP framework. This is a proposed poster abstract for the 2024 SOT conference. 

Description:

The period of neurodevelopment is vulnerable to various insults, including those produced by environmental compounds. We have previously observed behavioral alterations (uncoordinated hindlimb gait, decreased startle response, altered motor activity ontogeny) following perinatal exposure to emamectin benzoate (EB), a positive allosteric modulator to GABA-gated chloride channels in rat offspring. Here we used a proteomic approach to examine protein changes following perinatal EB exposure, to understand biological processes that may be altered that could account for the observed behavioral changes. Pregnant Long Evans rats were gavaged with EB (3.78 mg/kg in 5mL deionized water) or vehicle (control) from gestational day 6 to postnatal day (PND) 21. Region specific brain tissues were collected from offspring at either PND 2, 8, 15, or 22 for proteomic analysis (N= 10 rat pups/treatment/sex/timepoint). Offspring were perfused with phosphate buffered saline, and multiple brain regions were dissected on ice and stored at -80°C. Cerebellar and cortex samples (6/group) 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). A total of 2502 and 2587 proteins were identified for the cerebellum and cortex, respectively, when examining results across the PND timepoints. Few significant treatment-related proteomic changes were found when examining treated compared with control at individual PND timepoints. Proteomic data was then examined across the PND timepoints for each treatment. Examining changes between PND 2 and 22 (for both treatment by sex), we found the top canonical pathways among all groups were EIF2 signaling, Mitochondrial Dysfunction, Synaptogenesis signaling, Huntington’s disease signaling and Sirtuin signaling. Although similar pathways were identified for all groups for PND 2 vs 22, we predicted proteins within these pathways to differ, potentially shifting a path to a disease state in treated animals due to observed behavioral changes. Protein expression changes in male offspring between PND 15 and 22 revealed greater z scores for treated than controls for both Mitochondrial Dysfunction (treated: 3.81 (cortex), and 3.5 (cerebellum); control: 2.777 (cortex), and 2.433 (cerebellum)) and Sirtuin signaling (treated: 3.272 (cortex), and 1.414 (cerebellum); control: 2.837 (cortex), and 1.095 (cerebellum)). Greater z scores were found for Sirtuin signaling in EB treated females (1.3) than controls (0.378), in the cerebellum. This could indicate a potential shift towards an increased activation of these pathways for EB treated rats compared with controls, as well as sex specific differences in pathways implicated. When examining top diseases and functional annotations identified in IPA comparing PND 15 to 22 “Motor dysfunction/movement disorder”, “Familiar encephalopathy”, and “Familiar central nervous system disease” were identified among the top 5 for treated rats (both regions and sexes), with more molecules identified in these functional groups for treated rats compared with controls. When examining treated offspring across timepoints there appear to be different proteomic profiles from the control offspring, as well as potential differences between males and females.  Pathway analysis indicates a predicted shift to activation of neurodegenerative pathways, which could account for some of the previously observed behavioral alterations, although this requires further assessment. Additional measures are being performed, following up with differential expression analyses of proteins across the timepoints for each treatment to examine a potential shift in expression profiles. This is an abstract of a proposed presentation and does not necessarily reflect US EPA policy.

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