Analyzing the effects of single and mixtures of potato glycoalkaloids on gene expression in intestinal epithelial cells
The main aims of this dissertation were first, to determine the molecular mechanism(s) underlying glycoalkaloid-induced effects, in particular their membrane disruptive effect in intestinal epithelial cells. Second, to evaluate the usefulness of DNA microarrays in discriminating individual glycoalkaloids and glycoalkaloid mixtures of varying alpha-chaconine/alpha-solanine ratios based on their differences in effect severity and potential toxicities. By exploring the application/sensitivity of transcriptomic techniques in identifying early indicators of toxicity and screening between similar class/effect compounds, these studies clarify the potential application of this technique to 'whole foods' safety assessments incorporating different models of impaired nutritional states. The transcriptional effects of individual and mixtures of glycoalkaloids were studied in the intestinal epithelial Caco-2 cell line. The principal finding was the induction of cholesterol biosynthesis genes by non-cytotoxic glycoalkaloid concentrations, which to some extent is in line with their well documented mechanism of membrane disruption. Various genes involved in the MAPK, PI3K/AKT, chemokine and growth signaling, cell cycle and apoptosis pathways also were affected. Confirmatory apoptosis and cell cycle analyses revealed that glycoalkaloids induced necrotic/apoptotic death and disproportionate accumulation of cells in the G2M phase. The DNA microarray data were in line with the results from previous studies demonstrating that potato glycoalkaloids have similar mechanisms of action but differ mainly in their adverse effect potency. In addition, this technology could discriminate among the different glycoalkaloid treatments with respect to effect severity, which correlated well with their effects on lactate dehydrogenase membrane leakage. DNA microarrays are recognized to be useful tools for generating hypotheses and elucidating mechanism of action. Therefore, the effect of alpha-chaconine on SREBP-2 protein levels and the importance of MAPK and PI3K/AKT pathways in glycoalkaloid-induced transcription of cholesterol biosynthesis genes were determined. Alpha-Chaconine induced proteolytic cleavage of SREBP-2 and phosphorylation of ERK, JNK and AKT kinases. However, the MAPK and PI3K/AKT pathways were not crucial for glycoalkaloid-induced expression of cholesterol biosynthesis genes. In this dissertation, the usefulness of DNA microarrays in identifying hitherto unknown mechanisms of action, identifying potential toxicity biomarkers, and assessing the effects of simple mixtures of compounds was demonstrated. The studies presented will contribute towards the elucidation of the toxic and potential beneficial effects of potato glycoalkaloids, which may subsequently enhance current efforts to develop transgenic potatoes with altered glycoalkaloid levels/ratios, and the utility of these tools for assessing the safety of whole foods.
European Union (NOFORISK FOOD-CT-2003-506387) and the Dutch Ministry of Agriculture, Nature, and Food Quality.
glycoalkaloids; DNA microarrays; potatoes; food safety; alpha-chaconine; alpha-solanine; intestinal epithelial cells
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