Cytotoxicity and Fluorescence Visualization of Ergot Alkaloids in Human Cell Lines

Abstract

The ergot alkaloids as secondary metabolites from fungi of the genus Claviceps are the focus of many investigations because of their pharmacological and toxicological properties. The main effects of ergot alkaloids are referred to an interaction with several receptor systems in the human body. It is well-known that ergot alkaloids are able to isomerize with one isomer being biologically active and one being only weakly active, whereas the activity is restricted to receptor interactions. Latest investigations have proven that ergot alkaloids also show cytotoxic effects and induce apoptosis in human primary cells. These effects seem to correlate with accumulation properties. It was the aim of our current study to determine such effects in cancer cell lines, because ergot derivatives are also used in tumor therapy. Our results confirm the apoptotic effects in two cancer cell lines (HepG2 and HT-29) in a high range, and accumulation measurements show an interesting correlation between the alkaloid concentration in the cell lysate of the receptor-inactive isomers and cytotoxicity. In addition, the strong accumulative effects were first visualized by fluorescence microscopy by taking advantage of the natural fluorescence properties of ergot alkaloids.

Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes

“…Apoptosis was elucidated by detection of caspase-3 activation using cell lysates of LDH release assay in LUHMES and CCF-STTG1 cells as described before with slight modifications [33]. 10 L/30 L cell lysate (LUHMES/CCF-STTG1) were mixed with reaction buffer (50 mM PIPES, 10 mM EDTA, 0.5% CHAPS, 10 mM DTT, 80 M DEVD-AFC) in a black 96 well plate and incubated for 1 h/3 h at 37 • C. Subsequently, fluorescence activity of cleaved 7-amino-4-trifluoromethylcumarin (AFC) was determined (400 nm ex , 520 nm em ).…”

The Key Role of Peltate Glandular Trichomes in Symbiota Comprising Clavicipitaceous Fungi of the Genus Periglandula and Their Host Plants

“…Indeed, the entire steps in ergot alkaloid biosynthesis are likely to take place within the fungus because the complete set of ergot biosynthetic genes is present within the fungus [ 18 ]. At the early stage of leaf development, alkaloids can be visualized by their blue UV-autofluorescence within cells according to Mulac et al [ 33 ] ( Figure 2 D). On mature leaves, the UV-autofluorescence faded away from the hyphae.…”

Toxicological properties of the thiolated inorganic arsenic and arsenosugar metabolite thio-dimethylarsinic acid in human bladder cells

“…The CCK-8 ® test and the neutral red uptake test represent well accepted test systems to assess cell viability, and were performed as described before [19,21]. LDH activity was observed in both the cell lysates and the dosing media as described before [27] with minor modifications. Briefly, 40 L of culture medium or 10 L of cell lysates were mixed in a 96 well plate with reaction buffer (100 mM HEPES, 0.14 g/L NADH, 1.1 g/L sodium pyruvate, pH 7) to reach each a total volume of 200 L. Absorbance was detected kinetically at 355 nm every 1.5 min at 37 • C. LDH activity was calculated as mU/mL as percent of untreated control cells.…”

“…Besides SubG1 Peak formation, apoptosis was monitored via Caspase-3 activation as described before [27]. Briefly, after 24 or 48 h incubation with the respective arsenicals and lysis of cells, lysates were mixed in an equal amount of reaction buffer (50 mM PIPES, 10 mM EDTA, 0.5% CHAPS, 10 mM DTT, 80 M DEVD-AFC) in a black 96 well plate and incubated for 4 h at 37 • C. Subsequently, fluorescence activity of cleaved 7-amino-4-trifluoromethylcumarin (AFC) was determined by a plate reader (ex.…”