Crotalus atrox Induced Cellular Injury Triggers an Oxidative Stress Gene Response in HEK‐293T Cells

Wound and regeneration involves a series of complex processes that can induce oxidative stress by elevating intracellular reactive oxygen species (ROS) levels. In this study, we used RNA-seq and qPCR to examine the molecular signature of Human Embryonic Kidney cells (HEK 293T) stimulated with Crotalus atrox venom. NGS data was assembled and annotated using BLASTx and UNIPROT. Results showed 243 genes were differentially expressed (106 upregulated, 137 downregulated) between Crotalus atrox stimulated and non-stimulated cells (p<0.05). HEK 293T stimulated with Crotalus atrox venom for 4 hours showed an increase in the expression of oxidative stress response genes (e.g., TOMM6, EGR (1,2,3), HSP70 (1A, 1B), UPK3BL (1,2)), and protein synthesis (ETV5, EMP1) (fc>2.0). Significantly downregulated (log2(fc)<-1.20) genes were associated with protein coding (e.g., ABCG1, ABCA1, PPARA, HMGA2, TLCD5). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 8 significant KEGG pathways, which included an amphetamine addiction pathway (p<0.03). Genes identified in this pathway such as FosB proto-oncogene (FosB) and activity regulated cytoskeleton protein (ARC) are connected to oxidative stress responses. Quantitative PCR (qPCR) was used to evaluate several genes identified from RNA-seq data. Cells stimulated with Crotalus atrox venom for 4 hours induced an up-regulation of HSPA1A (2.49±0.49), HSPA1B (2.04±0.12), and cytochrome P450 1A1 (CYP1A1) (3.24±0.33), matching NGS data trends. Pre-incubation with the zinc chelator tris(2-pyridylmethyl) amine (TPA) prior to venom stimulation induced a significant up-regulation in HSPA1B (13.2±0.04). Extending Crotalus atrox venom stimulation to 8 hours increased the gene expression of metallothionein 1X (MT1X, 4.68±0.26) and peroxiredoxin-4 (PRDX4, 3.34±0.11) compared to non-stimulated cells. Interestingly, pre-incubating cells with polyethylene glycol (PEG) catalase then stimulating cells with Crotalus atrox venom for 8 hours produced PRDX4 and MT1X gene expression values that were not significantly different from the non-stimulated control. In contrast, glutathione peroxidase-1 (GPx-1) was significantly downregulated (0.27±0.16) after an 8 hour stimulation with Crotalus atrox venom, compared to non-stimulated cells. Our data suggests Crotalus atrox induced cellular injury involves several gene families connected to oxidative stress responses.