Effects of X-ray and carbon ion beam irradiation on membrane permeability and integrity in Saccharomyces cerevisiae cells

摘要

The yeast strain used in this work was S. cerevisiae strain CICC 1308 (MATa, budding, haploid) (obtained from the Center of Industrial Culture Collection of China). The S. cerevisiae cells were inoculated into 20 ml of yeast peptone dextrose (YPD) medium (1% yeast extract, 2% peptone and 2% glucose) in 50-ml flasks incubated on a mechanical shaker (200 rpm) at 30°C. In order to obtain good strain vitality, cells were continuously cultured three times to the density of cells 2 × 107 cells/ml (OD₆₀₀ = 0.4). Saccharomyces cerevisiae cells were incubated at 30°C for 9 h (log phase) in YPD medium, centrifuged at 3500 rpm for 3 min to harvest yeast cells. The cells were resuspended in sterile water, and divided into eight groups at random. Each group was put into an individual sterilized dish of φ35 mm. These samples were irradiated with X-ray or CI beams at doses of 0, 25, 50, 75, 100, 125, 150 and 175 Gy, respectively. The irradiation experiments were conducted with the RX-650 X-ray biological irradiator (FAXITRON, USA) and the HIRFL (Heavy Ion Research Facility in Lanzhou) at IMPCAS (Institute of Modern Physics, Chinese Academy of Sciences). The energy and LET of CI beams are 100 MeV/u and 202 keV/μm, respectively. With regard to X-rays, the energy and dose rate are 100 kVp and 1.5 Gy/min, respectively. The solution was stable during irradiation and negative control was subjecting to the same handling minus irradiation. After irradiation, S. cerevisiae cells were diluted by 10-fold aliquots to the desired concentrations using sterilized water and surface-plated to solid YPD medium for colony formation in order to measure their survival rate. Three replicates were used, but when irradiation treatment resulted in low counts, more plates were used as replicates. Plates were incubated for 48 h at 30°C. Counting was used for enumeration of colonies. Survival curves were generated from experimental data by plotting doses of irradiation versus Log N/N₀ [where N is the number of colony-forming units (CFUs) at a given dose and N₀ is the negative control number of CFUs]. The non-viable cells were shown by Log N₀–Log N. After S. cerevisiae cells were irradiated with X-ray or CI beams at different doses, the samples were harvested, washed and resuspended in PBS to a final OD₆₀₀ = 0.2 (～1 × 106 cells/ml) and OD₆₀₀ = 0.4 (～1 × 107 cells/ml), while the control was incubated with YPD medium alone. In order to prevent the activity of protease, all samples were kept on ice for 2 h and harvested by centrifugation at 10 000 rpm at 4°C for 10 min. Eluting protein from S. cerevisiae cells was measured using a BCA protein assay kit (Beyotime, China) on the supernatants. The absorbances of irradiated cells, untreated control cells and BSA standards (range 0–200 μg/ml BSA) were measured at 560 nm after 2 h incubation at 37°C. The BSA standard (μg/ml) was used to draw standard curves. In addition, for calculating diffusion of intracellular nucleotides, the absorbances of cell supernatants at 260 and 280 nm were measured. The calculating equation was as follows [id="xref-ref-14-1" class="xref-bibr" href="#ref-14">14]: nucleotide (μg/ml) = (11.87 × A₂₆₀ ? 10.40 × A₂₈₀) × 100/9. Cells from the groups irradiated at 100 and 175 Gy and the control group (0 Gy) were injected into the FACSAria II flow cytometer (BD, USA) under the same voltage. Dot plots were drawn by FSC versus SSC. Moreover, double-staining with FDA (Sigma, USA) and PI (Sigma, USA) were used for FCM analysis. A blank control (non-stained and non-irradiated) was employed to determine the autofluorescence of the cells. The staining procedure was as follows: cells were incubated with 5 mg/ml (acetone) FDA at 37°C for 30 min, then cells were centrifugally washed twice by sterilized water (3000 rpm, 3 min) and suspended in 1 ml phosphate buffered saline solution (PBS buffer, pH 7.2). Afterwards, 1 mg/ml PI (dissolved in deionized water) was added to the system and incubated for 10 min at room temperature in the dark. Samples were immediately analyzed. Analysis was also performed on the FACSAria II flow cytometer (BD, USA). The flow rate was adjusted to keep the acquisition at 200 cells per second. A total of 10 000 events were registered per sample. Green fluorescence of cells stained with FDA was collected in the FL1 channel (525 ± 15 nm), and red fluorescence of cells labeled with PI was collected in the FL2 channel (620 ± 15 nm). Tests were replicated at least three times with three samples for each of the irradiation doses. The software FlowJo7.6 was used to analyze flow cytometric data. Dot plot analysis of FDA versus PI was applied to determine the fluorescence properties of the population. Qualitative images of FDA–PI double-staining were acquired with BX53 epifluorescence microsc