Genomic changes in primary cortical neurons in culture following in vitro exposure to Emamectin
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Background and Purpose: Humans are exposed to thousands of chemicals throughout their lifetime, yet more than 100,000 of these compounds remain untested for their potential neurotoxic effects. The National Academy of Science has suggested utilizing advances in molecular biology to identify adverse outcome pathways (AOPs) to develop new approach methods (NAMs) to prioritize chemicals that lack hazard data for neurotoxicity (NT) and developmental neurotoxicity (DNT). We hypothesize that there is an overlap in the AOPs of neurotoxic chemicals and that by identifying commonalities between chemical signatures (proteomics/transcriptomics), an empirical model based on a set of common genes/proteins can be created for screening untested chemicals for their NT/DNT. An in vitro model using rat primary neuronal cultures was developed to conduct broad range proteomics and transcriptomics, aimed at generating proteomic and genomic signatures for each test chemical. We describe the effects of emamectin benzoate (EMA) insecticide on the transcriptome in cortical cell cultures. This is one of the selected test chemicals among several others. EMA has been used globally in pest control on vegetable and field crops.
Methods: Primary cortical cultures were prepared on postnatal day 0 to1 old pups from pregnant Long Evans rats and maintained in the incubator at a density of 1 million cortical cells in 4 mL of media in 6-well plates. Following 2 hours of plating, the cells were exposed to EMA at concentrations of 0.625, and 1.25 µM (concentrations based on acute cytotoxicity studies where no significant cytotoxicity was observed). Vehicle (0.2% DMSO) controls were also maintained. 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 adding Trizol solution and scraping the cells. The cell suspension in Trizol was quick-frozen on dry ice and stored at -80oC until genomic analysis by BioSpyder Technologies Inc.
Results: Differential gene expression was assessed using the DESeq2 package in R, and read counts were assessed for treatment-related effects. All differential expression analysis used significance cutoffs of FDR < .05 and absolute fold change > 20%. Sample sizes for all included analyses were 3-4/group. EMA exposure resulted in 6 differentially expressed genes (DEG) at 0.625 ppm on DIV2 with 2 downregulated and 4 upregulated genes, with those minimal changes absent at DIV7 and DIV12. At 1.25 ppm, EMA exposure resulted in 423 DEG as early as DIV2 with 93 down- and 330 upregulated, with 416 DEG persisting until DIV7 with 94 down- and 323 upregulated, and 100 DEG at the final DIV12 timepoint with 46 down- and 54 upregulated. There are 23 DEG induced by EMA shared by more than one DIV, which include Mobp, a gene that encodes a structural constituent of the myelin sheath, Tubb4a, a gene that encodes beta-tubulin, and Gabrb1, a gene that encodes GABA A receptor beta 1 subunit. Hallmark and Reactome pathway analysis were performed on the in vitro developmental EMA data. The pathways identified as significantly altered due to exposure primarily involve caspase-mediated cell death, NF-kB regulation, cholesterol homeostasis, translation and protein synthesis, pathways involved in mitochondrial bioenergetics, and inflammatory pathways driving oxidative stress.
Conclusions: These transcriptomic results indicate that developmental EMA exposure disrupts multiple transcriptomic processes involved in mitochondrial permeabilization, cell migration, oxidative stress, and cell metabolism in rat cortical cell cultures. These changes could lead to EMA-induced adverse effect in vitro such as cytotoxicity. (This abstract does not necessarily reflect US EPA policy).