Chemische Charakterisierung und Spurenanalytik
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Eingeladener Vortrag
- nein (83)
Mykotoxine werden als sekundäre Metabolite von Schimmelpilzen gebildet und haben diverse schädliche Effekte auf Menschen, Tiere und Pflanzen. Laut FAO sind weltweit bis zu 25 % aller Lebens- und Futtermittel mit Mykotoxinen kontaminiert, wodurch jährlich immense ökonomische Verluste entstehen.
Neben den eigentlichen Mykotoxinen rücken zunehmend deren Transformationsprodukte (TPs) in den Fokus. Diese können als natürliche Stoffwechselprodukte toxischer sein als das eigentliche Mykotoxin und stellen eine potenzielle zusätzliche Gefahr für den Verbraucher dar. TPs werden überwiegend mit in vitro und in vivo Techniken untersucht, welche sowohl zeit- als auch kostenintensiv sind. Ziel war es nun, schnelle und zudem verlässliche Techniken zur Simulation von TPs diverser Mykotoxine zu testen. Die Kopplung von Elektrochemie und Massenspektrometrie, kurz EC/MS, als rein instrumenteller Ansatz ist seit vielen Jahren in der pharmazeutischen Forschung etabliert. Hierbei wird mithilfe einer elektrochemischen Durchflusszelle der oxidative Metabolismus einer Substanz simuliert und zugleich analysiert.
Unter Verwendung von EC/MS, EC/LC/MS und HPLC-MS Techniken (MS/MS und FT-ICR) wurden nun die Mykotoxine Zearalenon, Citrinin und Dihydroergocristin (als Modellsubstanz für die Ergotalkaloide) elektrochemisch bei Potentialen bis zu 2500 mV vs. Pd/H2 oxidiert und deren TPs mit mikrosomalen in vitro Ansätzen verglichen. Hierbei konnten mehrere Übereinstimmungen hydroxylierter Spezies, sowohl elektrochemisch als auch biochemisch generiert, festgestellt werden. Die EC/MS ist somit auch in der Lebensmittelanalytik als nützliches und vielseitiges Werkzeug verwendbar.
Quantifizierung der Schädigung von DNA in wässriger Lösung unter direkter Elektronenbestrahlung
(2018)
To cure cancer radiation therapy is used to kill tumor cells.
It is based on radiation induced damage to biomolecules.
Especially DNA damage is of key interest due to its central role in apoptosis and mutation.
Because of the high amount of water in biological tissue, most of the damage is caused by the secondary particles produced by the inelastic scattering of ionizing radiation and water.
A detailed understanding of the underlying molecular processes under physiological conditions is the prerequisite to develop more efficient therapies.
Goal of this work is to quantify the DNA damage caused by ionizing radiation in dependence of the inelastic scattering events and the energy deposit within the microscopic target volume of biological relevance.
The irradiations have to be performed in liquid, under consideration of the chemical environment.
Therefore, a new combination of experiment and Monte-Carlo simulations was developed and tested.
To make it possible to irradiate liquids with electrons within scanning electron microscopes a new sample holder was constructed incorporating an electron transparent nanomembrane.
It makes it possible to irradiate DNA, proteins or cells at different pH, salinity and in the presence of cosolutes.
%The most important results of this work are as follows:
The median lethal dose for a model system of plasmid DNA and water was determined by the combination of experimental data, particle scattering simulations (Geant4-DNA) and diffusion calculations as D0.5=(1.7+-0.3) Gy.
From the convolution of plasmid positions and the spatially resolved energy deposit, as determined by electron scattering simulations, the histogram of the energy deposit within the target volume of the plasmids and the microscopic median lethal energy deposit was calculated as E0.5=6+-4eV.
It could be deduced that on average less than two ionization events are sufficient to cause a single-strand-break.
The relation of single-strand-breaks (SSB) to double-strand-breaks (DSB), which is of importance for microdosimetric modeling, was determined as SSB:DSB = 12:1.
The presented method for the determination of microscopic dose-damage relations was further extended to be applicable for general irradiation experiments.
It becomes independent of the type of primary radiation used, the experimental geometry, and the diffusional properties of the molecules under investigation.
This way different experimental systems with varying, inhomogeneous energy deposit characteristics become comparable with each other, which is not possible when only macroscopic averaged values are taken into account.
In addition, the radiation protection properties of the compatible solute ectoine, as well as its influence on the water properties and biomolecules were investigated.
%In addition, the influence of the compatible solute ectoine on water, biomolecules and its radiation protection properties were investigated.
Raman spectroscopy revealed a concentration dependent increase of the collective water modes in the OH-stretching region, which was found to be independent of the sodium chloride concentration.
Molecular dynamic simulations showed that the zwitterionic properties of ectoine lead to its half-chair conformation.
The hydrogen bonds in the first hydration shell are more stable and have an increased lifetime compared to the bulk water.
Irradiation experiments with DNA in the presence of 1M ectoine revealed an increase of the survival rate by a factor of 1.41 as compared to the absence of ectoine.
The protective properties of ectoine result from the increase of the inelastic scattering probabilities of low energy electrons at the acoustic vibrational modes of water and its properties as OH-radical scavenger.
This was shown by Raman spectroscopy and electron paramagnetic resonance measurements (EPR).
The overall interest in nanotoxicity, triggered by the increasing use of nanomaterials in the material and life sciences, and the synthesis of an ever increasing number of new functional nanoparticles calls for standardized test procedures1,2 and for efficient approaches to screen the potential genotoxicity of these materials. Aiming at the development of fast and easy to use, automated microscopic methods for the determination of the genotoxicity of different types of nanoparticles, we assess the potential of the fluorometric γH2AX assay for this purpose. This assay, which can be run on an automated microscopic detection system, relies on the detection of DNA double strand breaks as a sign for genotoxicity3. Here, we provide first results obtained with broadly used nanomaterials like CdSe/CdS and InP/ZnS quantum dots as well as iron oxide, gold, and polymer particles of different surface chemistry with previously tested colloidal stability and different cell lines like Hep-2 and 8E11 cells, which reveal a dependence of the genotoxicity on the chemical composition as well as the surface chemistry of these nanomaterials. These studies will be also used to establish nanomaterials as positive and negative genotoxicity controls or standards for assay performance validation for users of this fluorometric genotoxicity assay. In the future, after proper validation, this microscopic platform technology will be expanded to other typical toxicity assays.