Radioresistance and radiosensitivity: a biophysical approach on bacterial cells robustness.

The study of radiosensitivity and radioresistance of organisms exposed to ionizing radiation has acquired additional relevance since a new bio-concept, coined as The primacy of Proteome over Genome, was proposed and demonstrated elsewhere a few years ago. According to that finding, genome integrity would require an actively functioning Proteome. However, when exposure to radiation takes place, Reactive Oxygen Species (ROS) from water radiolysis induce protein carbonylation (PC), an irreversible oxidative Proteome damage. The bio-models used in that study were the radiosensitive Escherichia coli and the extraordinarily robust Deinococcus radiodurans. The production of ROS induces protective reactions rendering them non-reactive forms. Protective entities present in the cytosol, moieties smaller than 3 kDa, shield the Proteome against ROS, yielding protection against carbonylation. Shown in the present study is the fact that the fate of proteins functionality is determined by the magnitude of the Protein Carbonylation Yield (YPC), a quantity here analytically defined using published YPC numerical results. Analytical YPC expressions for E. coli and D. radiodurans were the input for a phenomenological approach, where the radiobiological magnitudes PP and PN, the probabilities for production of protein damage and ROS neutralization, respectively, were also analytically deduced. These highly relevant magnitudes, associated with key radiosensitivity and radioresistance issues, are addressed and discussed in this study. Among the plethora of information and conclusions derived from the present study, those endowed with higher conceptual degree, vis-à-vis the "Primacy of Proteome over Genome" concept, are as follows: (1) the ROS neutralization process in D. radiodurans reaches a maximum at a dose interval corresponding to the repairing shoulder. Therefore, it is a signature of the higher efficiency of the PC neutralization process. (2) ROS neutralization in D. radiodurans is nearly one order of magnitude higher than in E. coli, thus accounting for its extraordinary radioresistance. (3) Both physical (ROS-induced carbonyl radicals) and biological (protein modifications) processes are imbedded in the Protein Carbonylation Yield. The amalgamation of these two processes was accomplished by means of a statistical formalism.

Exposure of Deinococcus radiodurans to both static magnetic fields and gamma radiation: observation of cell recuperation effects.

The extremophilic bacterium Deinococcus radiodurans displays an extraordinary ability to withstand lethal radiation effects, due to its complex mechanisms for both proteome radiation protection and DNA repair. Published results obtained recently at this laboratory show that D. radiodurans submitted to ionizing radiation results in its DNA being shattered into small fragments which, when exposed to a "static electric field' (SEF), greatly decreases cell viability. These findings motivated the performing of D. radiodurans exposed to gamma radiation, yet exposed to a different exogenous physical agent, "static magnetic fields" (SMF). Cells of D. radiodurans [strain D.r. GY 9613 (R1)] in the exponential phase were submitted to 60Co gamma radiation from a gamma cell. Samples were exposed to doses in the interval 0.5-12.5 kGy, while the control samples were kept next to the irradiation setup. Exposures to SMF were carried out with intensities of 0.08 T and 0.8 T delivered by two settings: (a) a device built up at this laboratory with niobium magnets, delivering 0.08 T, and (b) an electromagnet (Walker Scientific) generating static magnetic fields with intensities from 0.1 to 0.8 T. All samples were placed in a bacteriological incubator at 30 °C for 48 h, and after incubation, a counting of colony forming units was performed. Two sets of cell surviving data were measured, each in triplicate, obtained in independent experiments. A remarkable similarity between the two data sets is revealed, underscoring reproducibility within the 5% range. Appraisal of raw data shows that exposure of irradiated cells to SMF substantially increases their viability. Data interpretation strongly suggests that the increase of D. radiodurans cell viability is a sole magnetic physical effect, driven by a stochastic process, improving the efficiency of the rejoining of DNA fragments, thus increasing cell viability. A type of cut-off dose is identified at 10 kGy, above which the irradiated cellular system loses recovery and the cell survival mechanism collapses.

DNA fragmentation by gamma radiation and electron beams using atomic force microscopy.

Double-stranded pBS plasmid DNA was irradiated with gamma rays at doses ranging from 1 to 12 kGy and electron beams from 1 to 10 kGy. Fragment-size distributions were determined by direct visualization, using atomic force microscopy with nanometer-resolution operating in non-tapping mode, combined with an improved methodology. The fragment distributions from irradiation with gamma rays revealed discrete-like patterns at all doses, suggesting that these patterns are modulated by the base pair composition of the plasmid. Irradiation with electron beams, at very high dose rates, generated continuous distributions of highly shattered DNA fragments, similar to results at much lower dose rates found in the literature. Altogether, these results indicate that AFM could supplement traditional methods for high-resolution measurements of radiation damage to DNA, while providing new and relevant information.