P 32 ISSRNS 2012: Abstracts / Synchrotron Radiation in Natural Science Vol. 11, No 1 – 2 (2012)
FIRST APPROACH OF THE FTIR MICROSPECTROSCOPY FOR STUDYING THE EFFECT OF IONISING RADIATION
IN SINGLE CELLS
E. Lipiec1∗, G. Birarda2, J. Lekki1, L. Vaccari2, A. Wieche´c1, and W.M. Kwiatek1
1The Henryk Niewodniczanski Institute of Nuclear Physics, PAN, Krak´ow, Poland
2ELETTRA Synchrotron Light Laboratory, Area Science Park, 34012 Basovizza, Trieste, Italy Keywords: FTIR microspectroscopy, DNA damage, single cell irradiation, DU-145
∗e-mail : Ewelina.Lipiec@ifj.edu.pl
The application of microprobe [1] made a very important development in studies of the response of biological systems to radiation exposure. Research on the well localized radiation dose-dependent bio- logical effects (such as DNA double strand breaks
— the most important DNA damage produced by ionizing radiation) enhances the understanding of the mechanisms leading to cell death [2]. Among many experimental techniques applied in this field, optimization of FTIR microspectroscopy to study radiation damage in single cells allows fast detec- tion of damage in lipids, proteins and nucleic acids on molecular level at the same time. Therefore, the aim of this study was the application of SR-FTIR microspectroscopy to investigate the DNA damage in single cells exposed to proton microbeam.
The DNA damage in single cells was induced by 1 MeV protons from the focused horizontal mi- crobeam generated by the Van de Graaff accelerator at the Institute of Nuclear Physics PAN in Krak´ow, Poland. The prostate cancer cells DU-145 were ir- radiated by the specific number (50, 200, 400, 2000, 4000) of protons per cell. Cell FTIR spectra were obtained using both: a) the synchrotron radiation source at SISSI beamline at ELETTRA with the Mercury-Cadmium-Telluride (MCT) detector and b) globar source with the Focal Plane Array (FPA) also at ELETTRA Laboratory, Trieste, Italy. In the presented study, experimental FTIR data obtained from both measurements were analyzed separately to investigate the changes in DNA backbone spec- tral range (950 cm−1– 1250 cm−1) of irradiated and control (untreated by ionizing radiation) cells. The results were then compared in order to evaluate the experimental approach for this study.
To show the structure in the relationships between the data and to detect the bands, which are different for the each group of spectra, the Principal Component Analysis (PCA) of the spectral region 950 cm−1 – 1250 cm−1 was performed using Statis- tica 8.0 software. The results showed distinct clus- ters for all groups of cells spectra, even for those
irradiated by the smallest dose of protons. The cellular spectral bands (about 20 for every FTIR spectrum, derived from proteins, nucleic acids and lipids [3, 4]) were fitted in spectral range 950 cm−1– 1250 cm−1 with Gaussian-Lorentzian curves after the Mie scattering effect correction [5]. In both cases (cell spectra collected using MCT and FPA detector) the fitting analysis produced comparable results. The dose-depended changes in the rela- tive intensities of DNA peaks: 960 cm−1 (ribose- phosphate skeletal motions), and 1095 cm−1 (sym- metric and stretching of O-P-O band), as well as shape and intensity modification of the 1105 cm−1 peak (symmetric stretching of P-O-C band) were observed.
Acknowledgments: This work was supported by European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 20110367.
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