Proteomic Changes in Primary Rat Cortical Cultures following Acute exposure to Triethyltin Bromide (TET)
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There is a need to develop quick and sensitive chemical screening workflows such as utilization of in vitro new approach methods (NAMs) to reduce the number of animals used and increase efficiency to make informed decisions before proceeding to in vivo testing for neurotoxicity (NT) and developmental neurotoxicity (DNT). By using data from current NAMs such as neurite outgrowth (NO) and microelectrode arrays (MEA), current effort is to identify adverse outcome pathways (AOPs) in vitro with primary rat cortical cultures and proteomic approach to establish unique molecular signatures needed for empirical modeling and screening of untested chemicals. We first used a known neurotoxicant, triethyltin bromide (TET), which causes neuronal death, axonopathy, demyelination, inhibition of glutathione-S transferase, and changes in calcium homeostasis. At 7 days in vitro (DIV) primary cortical cultures were exposed to TET (0 to 50 µM) in Locke’s buffer for 3-48hrs. By measuring the cytosolic levels of lactate dehydrogenase with the CytoTox96® Non-Radioactive Cytotoxic Assay, we obtained time-course and dose-response data. In agreement with other in vitro studies, 24hr following stimulation the cytotoxic state (EC50=1.33+/-0.367 µM) was evident with a potency of 1.563 µM (p = 6 x 10-5; n=3-9 per group). MEA data was collected at DIV 12-13 with TET concentrations like those above. There was a significant decrease in mean firing rate at 500 nM, 1 µM, and 5 µM TET in a dose-dependent manner (9.5%, 45%, and 55%, respectively), which correlates with cytotoxicity data above. We then selected two time points (3 and 24hr) and a subtoxic dose (100 nM; <10% cytotoxicity) for proteomic study. Total protein was extracted using a modified Sample Preparation by Easy Extraction and Digestion method, quantified with Pierce Quantitative Colorimetric Peptide assay, and diluted to 200-µg/µL. Nanoflow LC-MS analysis was used to acquire proteomic data. A total of 2,017 high-confidence proteins were identified and quantified using the Proteome Discoverer v.2.5 software. Comparisons between 0nM/100nM were made for each timepoint and proteins with an increase or decrease in fold-change were selected. At both timepoints TET affected proteins associated with golgi apparatus (Glg1), and stathmins 3 & 4. At 24-hr only, there was an overexpression of proteins associated with cadherins (Arvcf), spliceosomes (Clns1a), cholesterol modulation (Hmgcs1, Idi1, and Fdps), heat-shock proteins (Hsp90A, Hsp90B), oxidative stress indicators (Rsp27a) and calcium modulation (Camsap1). This data is the first step in the developing NAMs using proteomics based AOPs and also support the currently used NAMs such as NO and MEA for screening and prioritization of untested chemicals for NT and DNT. (This abstract does not necessarily reflect USEPA policy).