Singlet oxygen ((1)Delta(g))-mediated oxidation of cellular and subcellular components: ESR and AFM assays

László Forró, Sandor Kasas, Andrzej Kulik, Andrzej Sienkiewicz
Institute of Physics, 2005
Article de conférence

Résumé

We report a comprehensive in vitro study of the photo-oxidative stress on different biomolecular and cellular targets generated in the presence of fullerol C-60(OH)(n), a novel, fullerene-based and water-soluble sensitizer of singlet oxygen ((1)Delta(g)). The photodynamic efficiency of fullerol C-60(OH)(n), was checked by using a singlet oxygen scavenger, TMP-OH, and the electron spin resonance (ESR) technique, which was capable of detecting the resulting paramagnetic product, TEMPOL. ESR was also used to monitor the conformation changes occurring in the spin-labelled protein, T4L lysozyme, which Was exposed to the photo-oxidative stress in solutions containing fullerol C-60(OH)(n). Finally, atomic force microscopy (AFM) experiments were performed to monitor changes in the local elastic properties of living and glutaraldehyde-fixed cells (neurons) exposed to the toxic action of (1)Delta(g) generated in the presence of fullerol C-60(OH)(n). Remarkably, the Young's modulus values measured for both living and fixed neurons revealed a pronounced drop as a function of exposure to the toxic action of I Ag. Thus, our ESR and AFM results bring evidence that the multi-hydroxylated fullerene is an efficient (1)Delta(g)-generator in aqueous media and might be implemented as a photosensitizer for performing oxidations in biological systems

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https://infoscience.epfl.ch/record/147379?ln=fr

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Reactive Oxygen Species (ROS) is a family of oxygen-based active molecules, which are deeply involved in numerous crucial processes of cellular metabolism, where the redox equilibrium is very important. An imbalance between ROS and the cell ability to neutralize them with antioxidants results in an Oxidative Stress (OS). This leads to cell alterations and impaired physiological functions. OS is involved in a growing list of human pathologies, from brain disorder, like Alzheimer and Parkinson's disease, to various forms of cancer (skin melanoma) and eye pathologies (cataract, macular disease), as well as in the aging process. Sometimes, inducing OS selectively in diseased cells can also act as a powerful therapy. This is the basis of PhotoDynamic Therapy (PDT), a newly developed medical application to cure cancer. It is based on ROS generation through photo-sensitive molecules (also referred to as PhotoSensitizers, or PS) under specific illumination. The goal of this work was to develop an efficient way to tune and characterize OS and related damage to biological systems. For this purpose, we synthesized a water-soluble derivative of fullerene-based PS, since the pristine molecule is known to generate ROS under visible illumination at very high yield. The efficiency of our custom-made PS in generating ROS was characterized by Electron Spin Resonance (ESR). Parallel studies were performed on a porphyrin-based PS and on TiO2 nanoparticles. The effects of oxidative stress provoked via PS were investigated for selected proteins and for a large variety of cells. The extent of OS was finely tuned by adjusting the experimental conditions such as PS concentration in the buffer. The changes upon OS were studied by three modern experimental techniques: ESR, Synchrotron Infrared MicroSpectroscopy (SIRMS) and Atomic Force Microscopy (AFM). We have obtained a promising set of data on the consequences of OS detected by these local techniques. During the OS certain chemical bonds are interrupted and others are created which manifest in the change of the lattice vibrations detected for entire cells using SIRMS. These stresses affect, first of all, the constituents of the cells. For example, OS on a protein induces conformational changes as measured by ESR using spin labeled proteins. We have shown that larger organizations of proteins like cytoskeletal filaments must also change under OS, since the cell stiffness decreases considerably. AFM measurements on various cell types (neurons, fibroblast, bladder cell, glioblastoma…) and on healthy and cancerous cells have clearly shown the progressive decrease of the Young's modulus as OS was switched on.

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