BIOMARKERS IDENTIFICATION OF IONIZING RADIATION EXPOSURE IN BALB/c 3T3 CELLS

Individuals are continuously exposed to ionizing radiation (IR) through the environment and in medical settings during diagnostic and therapeutic procedures. Therefore, the identification of biomarkers for IR response is strongly needed. Our aim was to identify new biological indicators of IR exposure in the BALB/c 3T3 model by analyzing alterations in gene expression related to specific biological endpoints.Preliminarily, BALB/c 3T3 cells were exposed to 137Cs radiation source (0.25 - 6 Gy) in order to predict the toxic effects of γ-irradiation. Cells were seeded at a concentration of 250 cells/60 mm dish 20 hrs after the exposure. A dose-related reduction in cloning efficiency was induced by 1.5-6 Gy doses while an increment in cell proliferation was the main outcome at lower doses, thus suggesting the possibility of a hormetic effect. We also evaluated the ability of 137Cs (0.75 - 3 Gy) to increase the occurrence of transformed foci in the in vitro cell transformation assay. This test has extensively being used for several years to assess the cancer potential of chemical and physical agents. After 3 Gy-irradiation, the transformation frequency of BALB/c 3T3 cells was significantly enhanced.The effect of different IR doses (0.25, 0.75, 3 Gy) on BALB/c 3T3 gene expression profile was investigated. Microarray experiments were carried out by using slides containing >20,000 probes. In details, total RNA was isolated 20 hrs after the exposure and labeled by incorporation of Cy3 and Cy5. Also, treated cells and non-exposed cells were compared by performing a dye-swap experiment.The data were analysed by MAANOVA (MicroArray ANalysis Of Variance) data analysis package of R programming environments. Then, the significant genes were evaluated in the context of Gene Ontology (GO) for biological interpretation.We found several GO categories which were involved in the response to radiation. Some of them were shared by all the analysed IR doses and showed a dose-related behaviour. Interestingly, many of the highlighted genes seemed to play a key role in cytoskeleton reorganisation (Actn1, Act), in nucleosome assembly (histones), in cell cycle regulation (p21, CDK4, CALM-1, GADD45) and in DNA metabolism (HDAC-1).In conclusion, the combination of biological and molecular approaches provided indications on molecular targets that could be involved in the effects induced by different doses of IR and that could represent potential biomarkers of IR exposure