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Analyzing the effects of single and mixtures of potato glycoalkaloids on gene expression in intestinal epithelial cells

Author
Mandimika, Tafadzwa
Abstract
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.
Sponsorship
European Union (NOFORISK FOOD-CT-2003-506387) and the Dutch Ministry of
Agriculture, Nature, and Food Quality.
Date Issued
2008-02-29Subject
glycoalkaloids; DNA microarrays; potatoes; food safety; alpha-chaconine; alpha-solanine; intestinal epithelial cells
Has other format(s)
bibid: 6397084
Type
dissertation or thesis