Okadaic acid influences xenobiotic metabolism in HepaRG cells

Abstract

Okadaic acid (OA) is an algae-produced lipophilic marine biotoxin that accumulates in the fatty tissue of filter-feeding shellfish. Ingestion of contaminated shellfish leads to the diarrheic shellfish poisoning syndrome. Furthermore, several other effects of OA like genotoxicity, liver toxicity and tumor-promoting properties have been observed, probably linked to the phosphatase-inhibiting properties of the toxin. It has been shown that at high doses OA can disrupt the physical barrier of the intestinal epithelium. As the intestine and the liver do not only constitute a physical, but also a metabolic barrier against xenobiotic exposure, we here investigated the impact of OA on the expression of cytochrome P450 (CYP) enzymes and transporter proteins in human HepaRG cells liver cells in vitro at non-cytotoxic concentrations. The interplay of OA with known CYP inducers was also studied. Data show that the expression of various xenobiotic-metabolizing CYPs was downregulated after exposure to OA. Moreover, OA was able to counteract the activation of CYPs by their inducers. A number of transporters were also mainly downregulated. Overall, we demonstrate that OA has a significant effect on xenobiotic metabolism barrier in liver cells, highlighting the possibility for interactions of OA exposure with the metabolism of drugs and xenobiotics.

Keywords: CYP enzymes; HepaRG cells; okadaic acid.

Figure 1. Effect of OA on cytochrome P450 (CYP) mRNA levels. Differentiated HepaRG cells were treated with 33 nM OA, alone or in combination with 5 µM CITCO, 5 µM BkF, 20 µM SR12813, or with the respective solvent. Analysis of mRNA levels was performed by qPCR. The heatmap shows the log₂ values of the resulting fold changes as mean of three independent replicates, relative to the solvent control. Statistical analysis (n=3) was performed using one-way ANOVA followed by Dunnett's post-hoc test (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 2. Effect of OA on CYP protein expression. HepaRG cells were treated as outlined in the legend to Figure 1. Cells were lysed and protein amounts were determined using the BCA assay. Proteins were proteolyzed using trypsin and analyzed using LC-MS. Raw data were processed using Skyline software and TraceFinder 4.1. Peptide amounts were calculated by forming the ratios of the integrated peaks of the endogenous peptides and the isotope-labeled standards. Samples were then normalized to the solvent-treated control. Statistical analysis was performed by one-way ANOVA followed by Dunnett's posthoc test (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3. Activity of selected CYP enzymes. HepaRG cells were treated as outlined in the legend to Figure 1 and then incubated with CYP substrates for 3 h. Metabolite formation equal to the enzymatic activity of each CYP was determined using a mass-spectrometric approach. The results were normalized to the solvent-treated control. Statistical analysis (n=3) was performed by one-way ANOVA followed by Dunnett's post-hoc test (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4. Effect of OA on transporter mRNA (A) and protein (B) levels. HepaRG cells were treated as outlined in the legend to Figure 1, and mRNA levels were determined by qPCR and normalized to the housekeeping genes GUSB and GAPDH, while protein levels were assessed using LC-MS and calculated relative to isotope-labeled standards. The heatmaps shows the resulting fold changes of three independent replicates. Marking with “+” indicates a fold change value outside of the color scheme (i.e. value >10). Statistical analysis was performed using one-way ANOVA followed by Dunnett's posthoc test (*p < 0.05; **p < 0.01; ***p < 0.001).