Proteome profiling of emamectin-treated primary rat cortical cells following up to twelve days in vitro exposure
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SOT 2025 Conference abstract:
Proteome profiling of emamectin-treated primary rat cortical cells following up to twelve days in vitro exposure
W.M. Winnik, W. Padgett, D. Freeborn, D.W. Herr, P.R.S. Kodavanti
Background and Purpose: In vitro new approach methods (NAMs) have been of growing importance as they are intended to reduce the use of vertebrate test animals and afford efficiency to make informed decisions before in vivo testing for neurotoxicity (NT) and developmental neurotoxicity (DNT). This study is a step towards identifying key events in adverse outcome pathways (AOPs) in vitro, using primary rat cortical cultures and a proteomic approach to provide unique molecular signatures needed for future empirical modeling and screening of untested chemicals. Here we describe the effects of emamectin benzoate (EMA) insecticide, a semi-synthetic derivative of avermectin used for control pests on vegetables and field crops, that is a positive allosteric modulator to GABA-gated chloride channels, on the proteome in cortical cell cultures. Few studies have been done regarding the neurotoxicity potential of this chemical. The current study is a preliminary in vitro assessment of the impact of the chemical exposure on the developing nervous system.
Methods: High-resolution mass spectrometry and label-free quantitation (LFQ) was used to investigate neuron protein changes in primary rat cortical cultures from one day old pups of Long-Evans rats. One million cortical cells in four ml were exposed to EMA at 2 hours after plating. The selected concentrations of EMA are 0.625, 0.9375, and 1.25 µM (based on acute cytotoxicity studies where no significant cytotoxicity was observed). Vehicle (0.2% DMSO) controls were also examined. The media along with EMA were changed on days in vitro (DIV) 5 and DIV 9. The EMA exposures were terminated at DIVs 2, 5, 7, and 12 by scraping the cells and centrifuged at low speed (300 rpm) for 10 min. The pellet containing cells were quick-frozen on dry ice and stored at -80oC until proteomic analysis. The LFQ analytical protocol was described previously, including the protein identification, quantitation and the data statistics, and was performed using the Proteome Discoverer 3.0TM software [Winnik, et al, J. Proteome Res. 2023, 22(7) 2460]. Briefly, nano-LC and high-resolution mass spectrometry was used for peptide-based protein quantitation (OrbitrapTM) and tandem mass spectrometry for protein identification by the protein library and database MSMS spectral matching.
Results: Out of the 1,822 proteins identified with high confidence in the 96 individual samples (six biological repeats per analytical group), 143 proteins showed at least one statistically-significant (ANOVA Benjamini-Hochberg adjusted p-value ≤ 0.05; used as the data acceptance criteria) for the normalized protein abundance ratio(s): LFQ log2 (Protein Abundance_N_DIVx / Protein Abundance at 0 µM EMA treatment control DIVx), where DIVx = 2, 5, 7, 12; EMA concentration N = B (0.625 µM), C (0.9375 µM), D (1.25 µM). All the significant protein ratios were changed by at least 20%. Expectedly, the largest number of the significant protein ratios (104) were recorded for the highest treatment duration and concentration (1.25 µM at DIV12), whereby 85 proteins were downregulated. Within this protein data subset, there were 30% protein ratio change trends progressing consistently across at least a part of the range of the EMA concentrations and the investigated DIV times. The number of such consistent trends increased to 68% upon adopting the less stringent ANOVA- t-test p-value of 0.05 threshold instead of the adjusted p-value threshold for the ratios of the 104 top proteins. For instance, the top upregulated Cyp51a1 14x for 1.25 µM at DIV12, 7.3x for 0.9375 µM at DIV12 and 2.2x for the lowest concentration (0.625 µM) at DIV5. (Continued in the attachment below for this sub-product).