Differential Gene Expression Analysis of Okadaic Acid in the Porcine Intestinal Epithelial Cell Line IPEC-J2: A Bulk RNA-Seq Study at Food-Safety Relevant Concentrations

Okadaic acid (OA) is a lipophilic polyether phycotoxin derived from marine sources and produced by the dinoflagellate taxa Dinophysis and Prorocentrum which are the main cause of diarrhetic shellfish poisoning (DSP), through the consumption of toxin-tainted bivalve shellfish and mollusks. OA main mechanism of action is the strong inhibition of serine/threonine protein phosphatases (PP), especially protein phosphatase 2A (PP2A). It induces a series of biochemical reactions resulting in cellular hyper-phosphorylation and disruption of multiple signaling cascades. Although OA toxicity has been studied in several intestinal cell lines, very little information is available of the effect of OA on gene expression at sub-lethal concentrations that would be relevant for food safety. The aim of this thesis is to examine the effect of OA exposure on the porcine intestinal epithelial cell line IPEC-J2 at exposure to OA at a non-toxic reference concentration (5 nM), a sub-cytotoxic concentration corresponding to the EU regulatory limit for a 160µg OA/kg and 100-gram serving of shellfish (20 nM), and one cytotoxic concentration (80nM, corresponding with the IC50) corresponding with the EU regulatory limits of a 400-gram serving of shellfish using bulk RNA sequencing (RNA-seq). The IPEC-J2 porcine intestinal epithelial cell line model, a non-tumorigenic cell model with an apparent similarity to the human enterocyte cell line in terms of morphology and function, was used in this study for the assessment of the molecular effect of OA-induced intestinal toxicity. Bulk RNA sequencing was carried out on four biological replicates per sample were sequenced using the paired-end approach with the Illumina NextSeq 500 platform (150 bp) to obtain around 28–31 million reads per sample (>96% Q30 base quality). The raw data from sequencing were analyzed using the nf-core/rnaseq pipeline (v3.21.0), where the transcriptional level was quantified using SALMON, using the Sus scrofa reference genome. Differential gene expression (DEG) was determined with DESeq2 in RStudio (significance thresholds: absolute log₂ fold-change ≥ 0.58, corresponding to an approximately 1.5-fold change, and adjusted p-value < 0.05), followed by Gene Ontology (GO) over-representation analysis and Gene Set Enrichment Analysis (GSEA). The results identified that rather than a simple binary cytotoxic response, OA produced a dose-dependent transcriptomic progression. Even at the lowest tested concentration of 5 nM, a hidden program of proliferation and metabolism was activated as evidenced by the enrichment of signature pathways involving E2F, MYC, G2/M checkpoint, glycolysis, mTORC1 and hypoxia. At the physiologically relevant dose of 20 nM, stress and inflammatory pathways were enriched, including the NF-κB and p53 pathways. At the cytotoxic dose of 80 nM, inflammation and genotoxic stress characterized by loss of epithelial identity and acquisition of characteristics typical for the process of EMT were observed. Notably, while GO was performed successfully exclusively at 80 nM, GSEA identified pathways at each concentration tested. This implies that sub-cytotoxic concentrations lead to unique molecular alterations, which cannot be detected by single gene-based analysis alone. In summary, this study reveals molecular pathways associated with intestinal toxicity mediated by OA and includes regulation of cell cycle, metabolic adaptations, tight junction proteins and actin filaments, and inflammatory responses. Moreover, the transcriptome alteration occurs at concentrations, which may be reached due to dietary consumption of contaminated shellfish.