A comparative assessment of okadaic acid (OA) toxicity in Caco-2 and Caco-PXR intestinal in vitro models

Okadaic Acid (OA) is a potent, lipophilic phycotoxin produced by marine dinoflagellates. It is able to accumulate in shellfish and represents the primary cause of diarrhetic shellfish poisoning (DSP) in humans. The key-mechanism of toxicity is the inhibition of serine/threonine protein phosphatases (PPs), particularly PP2A. Most published studies utilized the standard Caco-2 cell line; however, the low constitutive expression of drug-metabolizing enzymes (DMEs) remains a limiting factor to fully understand OA mechanistic toxicity. In the present study, we used a comparative in vitro approach to better understand OA toxicity and address the limitations of the Caco-2 model. In addition to the standard model we employed a Caco-PXR clone transfected with the human pregnane-X (PXR) receptor, a transcription factor involved in the regulation of cytochrome P450 A3A4 (CYP3A4), a DME thought to be involved in OA metabolism. We assessed OA cytotoxicity using three complementary assays, establishing OA half maximal inhibitory concentration (IC50) across different exposure times (24 and 48h). We also evaluated potential changes in tight junction integrity and paracellular permeability caused by OA in the Caco-PXR cell line, using the lucifer yellow (LY) assay combined with measurements of transepithelial electrical resistance (TEER) Additionally, to evaluate the differences in constitutive expression of target genes between the two cell lines, we set up conditions for the execution of targeted quantitative Real Time RT-PCR (qPCR) assays. The neutral red uptake (NRU) assay revealed an OA time- and dose-dependent cytotoxicity in Caco-2 cells, with IC50 values of 490 and 330nM at 24 and 48h, respectively. However, the Caco-PXR cells proved to be more resistant to OA, with IC50 values of 600nM at 24h. The LY assay and TEER measurements were performed only in the PXR cell line and revealed a clear disturbance of the monolayer with significant reduction of TEER and increased permeability of LY at the highest concentration after 12 and 24h of OA exposure. Targeted qPCR assays are currently running in the laboratory, and results will be available and discussed during the thesis defense. Overall, this study contributes to understanding of OA toxicity and proposes a new alternative to the standard Caco-2 cell model.