Studies On Damage Of DNA And Cell Membrane Induced By X-ray And ¹²C⁶⁺ Beam Irradiation In Saccharomyces Cerevisiae Cell

Purpose:Growth regularity and biomass accumulation process of Saccharomyces cerevisiae M2014483 were analyzed. The dose-effect relationship and function models of DNA damage, DNA repair process, and the changes of cell membrane permeability and integrity induced by X-ray and 12C6+ irradiation was investigated in S. cerevisiae cells.Materials and methods:The piecewise regression equations of OD600 (Optical density at 600 nm) values were established in S. cerevisiae M2014483 suspension with different dilution ratio. OD600 values were substituted into the corresponding regression equations to draw and compare the growth curves that were made by OD600 regression values and plate count. The relations among S. cerevisiae cells numbers, dilution ratio and OD600 values were researched. The survival curves of S. cerevisiae after X-ray and 12C6+beam irradiation were tested. By means of comet assay optimized was performed to elucidate and quantify the DNA damage and repair. The dose-effect relationship of DNA damage, the time-effect relationship and dynamic function model of DNA repair were then studied. We measured the protein elution and nucleotide diffusion by ELIASA after X-ray and 12C6+treatment. FCM (Flow cytometry) of FSC (Forward scatter light) versus SSC (Side scatter light), and double-staining with FDA (Fluorescein diacetate)-PI (Propidium iodide) were used to assess the changes of cell morphology, cell membrane integrity and cellular enzyme activity.Results:1. The growth curves of M2014483 were obtained by OD600 regression values and plate count. At the same time, the corresponding relation among OD600 values, cells numbers and dilution ratio was gained in credible range.2. The optimal parameters of comet assay fitting to S. cerevisiae cell for lysis time, unwinding time, and electrophoresis time were 10,20, and 8 min, respectively, and the appropriate voltage was 0.5 V/cm. OTM(Olive tail moment) was an only indicator to DNA damage induced by X-ray and 12C6+ treatment. Quadratic function fitted the dose-effect relationship and cubic function could describe the dose-effect curves for various repair times (3,6,12 and 24 h). A kinetics model was also used to evaluate the process of DNA repair.3. After X-ray and 12C6+exposure, S. cerevisiae cells showed protein elution and nucleotide diffusion. The cell morphology, size and granularity were markedly altered. The increasing of permeabilized cells and decreasing of esterase activity depended on the levels and types of radiation, and affected non-viable cell numbers.Conclusions:1. OD600 regression values and S. cerevisiae cells numbers display excellent linearity in log phase, but OD600 regression values do not estimate the cell density and growth process after stable phase. OD600 values and dilution ratio was power function rather than multiple relationship.2. The comet assay optimized can be applied to the quantitative detection of DNA lesions in S. cerevisiae. The dose-effect relation of DNA damage exposed to X-ray and 12C6+ and repair process are fitted for function models and kinetics models.3. S. cerevisiae cell permeabilization and loss of metabolic activity as a result of X-ray and 12C6+ irradiation may be an irreversible phenomenon. Besides DNA damage, cell membrane lesions also play pivotal roles in the cell death induced by radiation. The patterns of cell inactivity due to X-ray and 12C6+ irradiation may be similar in terms of cell membrane damage.