Dosimetria biologica delle radiazioni ionizzanti: valutazione della dose con metodologie citogenetiche tramite la costruzione di curve di calibrazione

The assessment of the risk for biological damage of an individual after overexposure to ionizing radiation due to an accident or a terroristic attack passes through the evaluation of the absorbed dose received from the person/s involved. This evaluation, corresponding also to how dangerous the overexposure is, using methodologies typical of the biological dosimetry, is done with the help of reference calibration curves and it presumes that all individuals exposed have the same individual radiosensitivity, which is true when the biological system into account are human lymphocytes of the peripheral blood (PBL). It supposes that a certain dose of radiation produces the same quantity of variation (chromosomal aberrations) in that biological system in all the exposed. The goal of this Ph.D. thesis is the application of various cytogenetic methodologies to detect in a reliable and possibly fast way chromosomal aberrations non stable (dicentrics and centromeric rings) in the lymphocytes of the human peripheral blood after gamma irradiation and the construction of calibration curves dose effect able to give rapidly a response of the absorbed dose in case of radiation accident. The time needed to give a reliable response on the absorbed dose is crucial and this is especially true in radiation emergency medicine. The objective was to standardize, develop and apply sophisticate, sensitive and reliable methodologies for the estimation of the absorbed doses after accidental gamma radiation overexposures for radiation protection purposes. Specifically, in the present study the following were performed: I. Application of the classic “golden standard” methodology of biological dosimetry which provides stimulation of the lymphocytes and Giemsa staining of the chromosomes at mitosis II. Development, consolidation and application of original methodologies in biological dosimetry using fluorescence in situ hybridization (FISH) and peptide nucleic acid (PNA) probes for painting specifically chromosome telomers and centromeres III. Application and consolidation of the method of premature chromosome condensation (PCC) where no stimulation at lymphocytes is needed IV. Development of a fast, sensitive and reliable methodology, absolutely original in the field of biological dosimetry for dose evaluation combining the PCC method with the FISH using PNA probes for centromers and telomers V. Construction for all the above methodologies, using in vitro experiments, of calibration curves dose-effect where unstable chromosomal aberrations where plotted against gamma dose given. The dose interval investigated was between 0.3 Gy and 8 Gy; more than 12.500 slides with metaphases and interphases were scored using Microscopic analysis of chromosome aberrations. Dose effect curves are linear quadratic and for their construction a minimum of 500 cells or 100 dicentrics per dose were analysed. The Poisson distribution of the chromosomal aberrations was tested for every dose and the coefficients alpha and beta of the linear quadratic equation, their errors and p value were calculated. Finally, all calibration curves obtained with the different methodologies were compared and the advantages of each methodology reported.