Untersuchungen zu zellulären Effekten von Allicin, einem physiologisch aktiven Vertreter Reaktiver Schwefelspezies, aus Knoblauch (Allium sativum L.), in verschiedenen biologischen Systemen

  • Investigation of the physiological active Reactive Sulfur Species Allicin from garlic (Allium sativum L.) in different biological systems

Gruhlke, Martin Clemens Horst; Slusarenko, Alan (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2014)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2014

Abstract

Allicin is the quantitatively most important sulfur-containing volatile compound produced in damaged garlic tissue. Since allicin is a thiosulfinate, it acts as a ‘Reactive Sulfur Species’ (RSS) which oxidizes thiols under physiological conditions and thus has redox-modulating activity. The central hypothesis reported in this thesis is that the redox-modulatory properties are important for the biological activity of allicin. An important prerequisite of this project was to establish an efficient synthesis-protocol for allicin. Allicin’s effects were studied in Saccharomyces cerevisiae (baker’s yeast, a model fungus), Arabidopsis thaliana (model plant) and various mammalian cell lines. Changes in the absolute concentrations of reduced (GSH) and oxidized glutathione (GSSG) after allicin treatment were determined using the glutathione reductase recycling assay. Mutants and cell-biological methods were used to study the induction of apoptosis and the role of signalling components that contribute to the resistance or susceptibility of yeast cells to allicin. Also in Arabidopsis a mutant-based approach was used to identify putative factors that contribute to the response of plants to allicin. In mammalian cell cultures, changes in redox status, cell proliferation and induction of (apoptotic) cell death were followed upon treatment with either synthetic allicin or garlic juice using standard staining assays. Allicin decreased the total concentration of the GSH in the glutathione pool and increased the proportion of GSSG in yeast cells. Furthermore, mutants in glutathione metabolism were hypersensitive to allicin. Actin was shown to be a target for allicin und the subsequent RAS/cAMP/Protein Kinase A (PKA) signalling route is important for allicin-induced cell death as well as inhibition of cell proliferation in yeast. The redox-sensitive transcription factor YAP1, which coordinates the oxidative stress response in yeast, was activated by sub-lethal doses of allicin shown both by localization studies and expression of a YAP1-target gene and is necessary for full resistance. Various phytotoxic effects of allicin were observed, e.g. light-dependent bleaching which is typical for oxidative stress, inhibition of seed germination und root development. Furthermore, GSH is important in resistance as illustrated by the enhanced sensitivity of the pad2 mutant (20% of wt GSH). In mammalian cell lines, treatment with synthetic allicin or garlic juice, lead to an increase in oxidation of the glutathione pool. Effects of chemically synthesized allicin or garlic juice on cell proliferation, viability and apoptosis induction and correlation to the redox-status will be discussed in detail. The data presented in this thesis show that the effects of allicin on cellular redox-conditions are important for its toxicity und correlate with the induction of apoptosis in yeast. The identification of the RAS/cAMP/PKA pathway that previously was shown for allicin-induced apoptosis in human cancer cells, validates the yeast model for mechanistic studies of the molecular basis of allicin’s toxicity. Phytotoxic effects of allicin were investigated for the first time und show that allicin is suitable model substance to study the impact of Reactive Sulfur Species (RSS) in plants. The redox-correlated effects on different mammalian cell-cultures provide evidences for a medical application potential.

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