Variation in U.V. primary fluorescence-intensity of vital cells depending on 60Co gamma-radiation dose.

Using impulse-cytofluorophotometry in the ultra-violet spectral region the author has shown on vital, unstained Ehrlich ascites tumour cells that the primary fluorescence intensity of this tumour is on day 11 after transplantation 20 per cent higher than on day 8. Storage of the vital cells for 25 min at 20 degrees C has no effect on this result. When the cells are exposed to 60Co to gamma-radiation on day 6, a new stable fluorescence level is established afted 20 hours. Measurements of the primary fluorescence intensity depending on dose have shown a significant rise starting from 75 rad at 48 hours after irradiation. The fluorescence intensity rises by 42.5 per cent of the control value at 3000 rad, but only by 31.5 per cent on exposure to 4000 rad.     

Fluorescence study of thymus lymphocytes of X-irradiated mice and their progeny.

The mechanisms of changes in the ultra-violet fluorescence (U.V.F.) intensity of mouse thymus lymphocytes 24 hours and 30 days after whole-body X-irradiation have been studied. The thymus lymphocytes of the first generation offspring (F1) from X-irradiated males and unirradiated females were also investigated. At 24 hours after irradiation the U.V.F. intensity decreased for small doses (50 and 65 rad) and increased for doses of more than 100 rad. The changes in U.V.F. intensity were related to a size-independent mechanism. It was found that the U.V.F. increase for doses of 100-700 rad was not connected with the appearance of non-viable (eosin test) cells. The changes in U.V.F. intensity and cellular composition of the thymus were the same 30 days after irradiation and for F1 mice. The increase in U.V.F. intensity was about 14% and did not depend on dose between 50 and 500 rad. About one-half of this increase was connected with an increase in the proportion of medium and large lymphocytes in the thymus. The rest of the effect was related to a size-independent mechanism.     

Mechanism of action of small doses of radiation

Opposite changes occur in the intensity of UV-fluorescence (UVF) in irradiated (0.1 Gy and 5.0 Gy) HeLa cells. The radiometric study has demonstrated that there is a correlation between the number of tryptophan-containing proteins and UVF intensity in nonirradiated and irradiated (5.0 Gy) cells during culture growth. Such a correlation was absent in cells exposed to 0.1 Gy radiation. Low radiation doses (0.1 Gy) have maximum action on cytoplasm membrane fluorescence. Low-level radiation changes the intensity of the ANS probe fluorescence connected with cell membranes, and the intensity of the cell protein UVF. High radiation doses increase and low doses decrease the probe fluorescence.     

Effect of gamma-radiation on ultraviolet fluorescence of rat lymphocytes

Changes in the level of the proper UV-fluorescence ( UVF ) of rat blood lymphocytes are considered as a function of radiation dose and time after gamma-irradiation of animals. 24 h following irradiation with a dose of 0.5 Gy the UVF intensity decreased; with doses of 1-8 Gy, the level of cytofluorescence exceeded the control values. The effect of fluorescence at this time was not related to changes in a cell diameter whereas at later times following irradiation part of the effect was related to the growth of cell dimensions.     

Changes in the ultraviolet fluorescence and the size of Ehrlich ascites carcinoma cells in the 1st hours after X-ray irradiation

Using the Ehrlich ascite carcinoma cells, a decrease in ultraviolet fluorescence (UVF) was demonstrated one hour following a 10 Gy X-irradiation, and an increase in UVF 5 hours following the same irradiation. The same changes were demonstrated for cell turbidity. Association between the optical changes and those in cell radius and membrane protein state is discussed.     

Effects of gamma irradiation on histomorphology of some endocrine glands of the rain quail, Coturnix coromandelica (Gmelin)

Effects of single, whole-body 60Co-gamma irradiation in different doses (250 rad to 15 k rad) on histology of thyroid, adrenal and pancreatic islets of the rain quail were studied. A low dose of 250 rad failed to evoke any change in histology of the glands studied. Doses of 500 rad and 1 k rad resulted in hypoactivity of thyroid but could not affect adrenal and pancreatic islets. Exposure to 1.5 k rad and higher doses caused hyperplasia and hypertrophy of thyroid and hypertrophy of adrenal gland. Thyroid was injured by heavy irradiation. Doses up to 3 k rad did not bring about any change in islet cells, however, higher doses resulted in degenerative changes in islet cells. alpha-islets were affected by 7 and 15 k rad but necrotic changes in beta-islets were observed only after exposure to 15 k rad. The results indicate that thyroid is the most sensitive and pancreatic islet, highly resistant to gamma radiation.     

He—Ne激光照射离体鼠巨噬细胞对线粒体跨膜电势的影响

目的：研究Ｈｅ－Ｎｅ激光照射鼠巨噬细胞对线粒体跨膜电势的影响,及其与激光剂量的关系。方法：用亲脂性阳离子荧光染料Ｒｈｏｄａｍｉｎｅ１２３对鼠巨噬细胞线粒体作荧光标记,以不同的激光剂量照射,采用图像分析系统（ＩＡＳ）和荧光显微镜观察线粒体跨膜电势荧光强度的变化。结果：低功率Ｈｅ－Ｎｅ激光照射５,１０,１５ｍｉｎ,激光剂量分别为０．６４９,１．３８８和２．０８２Ｊ／ｃｍ＾２,巨噬细胞线粒体跨膜电势荧光     

Cell cycle arrest determines the intensity of the global transcriptional response of Saccharomyces cerevisiae to ionizing radiation

Whole-genome analysis was performed using DNA microarrays to define the changes in the gene expression patterns occurring in Saccharomyces cerevisiae cells exposed to ionizing radiation. The effects of sublethal dose on wild-type, rad53 (enhanced sensitivity to radiation and impaired in a cell cycle damage checkpoint), and rad6 (enhanced sensitivity to radiation and functional cell cycle block by radiation) mutant backgrounds and of a higher dose on the wild-type and G(2)-phase-arrested cells were analyzed. Several gene pathways were identified as being implicated in the response to radiation. In particular, the cell cycle blockage that occurred in the wild-type strain after a high radiation dose and in the rad6 mutant after a lower dose entailed modifications of defined gene expression patterns, which are described here and are compared with the gene modulation patterns observed in the rad53 strain in the absence of efficient blockage. Loss of the RAD53 function caused a major increase in the number of genes modulated by radiation. Given that Rad53-Sad1p, the protein encoded by RAD53, has functions other than those directly connected to cell cycle arrest, we determined the gene patterns that were modulated upon irradiation of rad53 cells that had been forced to arrest in G(2) phase by nocodazole treatment. These differential whole-genome analyses shed light on the multiplicity of functions of the pivotal Rad53-Sad1p protein. The results obtained describe how the cells respond to different irradiation conditions by modulating important gene classes, including those associated with stress defense, ribosomal proteins, histones, ergosterol and GCR1-controlled sugar metabolism.     

Growth regulation in X-irradiated mouse skin; the possible role of chalones.

Extracts of hairless mouse skin were tested for their content of epidermal G1 inhibitor and G2 inhibitor at daily intervals after X-irradiation with 4 500 or 2 250 rad. After either dose the skin extracts lacked G1 inhibitory activity on days 5 and 6 respectively after irradiation. This coincided with the time when the epidermal mitotic rate again became normal and started a period of over-shoot. The time interval of 5-6 days corresponds to the turnover time of the differentiating cells in hairless mouse back epidermis. The findings indicate that the proliferating cells in epidermis can respond to changes in local chalone concentration, even after X-irradiation at the tested doses, and that the irradiated epidermal cell population still retains some important properties inherent in a cybernetically regulated system. The local G2-inhibitory activity also varied after irradiation, but these variations could not be directly related to the corresponding mitotic rates.     

DNA replication in mammalian cells after the action of physical, chemical or biological factors. VI. The recovery of DNA synthesis in a culture of irradiated cells

The kinetics of DNA synthesis restoration in cultured HeLa cells and in L-929 mouse fibroblasts irradiated by gamma-rays of 60Co with a dose of 10 Gy was studied. Early after irradiation the rate of DNA synthesis in HeLa cells measured with 3H-thymidine incorporation was seen to decrease. Two hours later the incorporation starts to increase to reach the control level 4 hours after irradiation and then becomes even higher than this level. The distribution of cells among phases of the cell cycle measured with flow cytometry undergoes changes. 4-6 hours after irradiation part of S-phase cells increased contributing presumably to the elevating of 3H-thymidine incorporation observed at this time. The restoration of the incorporation was suppressed by inhibitors of protein and RNA synthesis--cycloheximide and actinomycin D. It is suggested that the processes of restoration of DNA synthesis in irradiated cells can be of inducible nature. In irradiated HeLa and L-929 cells the restoration of DNA synthesis is resistant to novobiocin, an inhibitor of DNA replication.     

Effects of dose rate and dose fractionation of irradiation on pulmonary alveolar macrophage colony-forming cells

We have studied the effects of dose rate and dose fractionation on murine pulmonary alveolar macrophage colony-forming cells (AL-CFC). The dose-response curve of AL-CFC to ionizing irradiation has a Dq of about 100 rad, reflecting the cells' ability to repair sublethal damage. For comparison, we investigated the effect of dose schedule on the committed bone marrow stem cells for both granulocytes and monocytes (GM-CFC) since their dose-response curve has a very small shoulder. We compared the results of dose rates of 3 and 10 rad/min to those obtained with a dose rate of 85 rad/min. We determined survival after giving 100, 300, and 500 rad either in vivo or in vitro. A significant dose rate effect was observed. To study the effect of dose fractionation, a total of 600 rad was given either as a single fraction, three fractions of 200 rad on 3 consecutive days, or six fractions of 100 rad in 3 days. The most dramatic effect was seen in the group that received six 100-rad fractions. No reduction in the number of AL-CFC was seen in this group. In sharp contrast, only a minimal dose schedule effect was observed with GM-CFC.     

RESULTS OF IRRADIATING SACCHAROMYCES WITH MONOCHROMATIC ULTRA-VIOLET LIGHT : II. THE INFLUENCE OF MODIFYING FACTORS

Possible variation in the probability that absorbed quanta of ultra-violet energy will produce observable inhibitory and lethal effects in the yeast cell, due to non-uniformity in sensitivity of the different regions of the cell, may be further modified by the reproductive stage of the cell at the time of irradiation. Tests of the survival of yeast cells of 15 day and 24 hour cultures indicate that the older resting cells are more resistant to ultra-violet irradiation effects than cells undergoing rapid cell division. The effects of temperature changes within the range of normal growth are evidently small, as judged from the temperature coefficient (1.10). Possible inhibitory effects due to the action of ultra-violet radiation on the malt agar medium and to toxic substances diffused from cells killed by irradiation were not found under the conditions of the experiments. Tests of the validity of the Bunsen-Roscoe reciprocity law for variation in the intensity of the incident ultra-violet radiation up to 30 per cent indicate that for this range the rate of absorption of quanta by the cell does not produce any marked change in the lethal effects observed.     

Dose-rate effects of 8-methoxypsoralen plus 365-NM irradiation on cell killing in Saccharomyces cerevisiae.

Tow types of dose-rate effect that alter the survival response of haploid yeast cells to 8-methoxypsoralen (8-MOP) plus treatment with irradiation at 365 nm were studied. (1) When the concentration of 8-MOP was varied between 9.2 X 10(-5) and 2.3 X 10(-8) M and the dose rate of 365-nm irradiation kept constant, the efficiency of the irradiation for killing increased relatively to that of 8-MOP whe the concentration of 8-MOP decreased. This indicated that there was no strict reciprocity between radiation dose and concentration of drug. (2) When the dose rate of radiation was varied between 0.66 X 10(3) and 108 X 10(3) J m-2 h-1 and the concentration of 8-MOP was kept constant, the survival of wild-type cells increased strikingly at low dose rates of radiation as compared with high dose rates. Cells responded more to changes at low dose rates than to equal changes a high dose rates. The high resistance of wild-type cells to 8-MOP plus radiation delivered at low dose rates absent from rad 1-3 cells defective in excision-repair. This suggests that the dose-rate effect seen in wild-type cells depended at least in part on an active excision-repair function. At low dose rates of radiation, the shoulder of the survival curve for rad1-3 cells, i.e. the ability to accumulate sub-lethal damage, was increased by a factor of about 2 when compared with that seen at a high dose rate. Thus it is likely that at low dose rates a repair function other than excision-resynthesis may operate in rad1-3 cells.     

Effect of polyamines on the cellular radiation reaction

The influence of polyamines (e.g. putrescine, spermine and spermidine) on the survival rate of HeLa cells and the mitotic index of A. cepa meristem cells, as well as a change in a radiation response of cells under the effect of polyamines have been investigated. Putrescine was shown to produce the lowest cytotoxic effect on mammalian cells, whereas the cytotoxic effect on plant cells was either insignificant or absent at all. One-hour incubation of HeLa cells with putrescine of 5 x 10(-4)-5 x 10(-5) M prior to or after irradiation with a dose of 6 Gy increased the survived cell fraction. Spermine of 10(-3) M increased considerably the mitotic index of the exposed meristem as compared to irradiated meristem untreated with spermine. The role of polyamines in the formation of radiation damage to a cell is discussed.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase

Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.     

Mechanisms of increasing the intensity of ultraviolet fluorescence of irradiated cells

As shown on the in vitro irradiated Ehrlich ascites tumor cells the increase in the intensity of ultraviolet fluorescence (UVF) of the exposed cells occurs only in such incubation conditions when an increase in the tryptophan content of irradiated cells becomes possible. The increase in the UVF intensity and in the tryptophan content are quantitatively identical. It is concluded that the increase in the content of the fluorescing substance, tryptophan, in the irradiated cells is the cause of the increase in the UVF intensity.     

The enhancement of haemopoietic stem cell recovery in irradiated mice by prior treatment with cyclophosphamide.

Studies are reported of the enhancement of stem cell recovery following whole body irradiation as a result of prior administration of cyclophosphamide. It is shown that the much larger enhancement of regeneration observed for the hosts own surviving stem cells, compared to the regeneration of injected bone marrow stem cells, is due to the different numbers of stem cells initiating the regeneration in conjunction with the time course of stem cell regeneration. The results show that the environmental changes produced by cyclophosphmide greatly enhance haemopoietic recovery even though at the dose used this agent is relatively toxic to stem cells. Furthermore it has been shown that the level of stem cell regeneration is nearly independent of the gamma-ray dose in the range 3-8 gray (300-800 rad). If human bone marrow should respond similarly it follows that regeneration produced by cytotoxic drugs administered prior to radiation embodies a considerable safety factor as far as recovery of the haemopoietic system is concerned.     

THE ENHANCEMENT OF HAEMOPOIETIC STEM CELL RECOVERY IN IRRADIATED MICE BY PRIOR TREATMENT WITH CYCLOPHOSPHAMIDE

Studies are reported of the enhancement of stem cell recovery following whole body irradiation as a result of prior administration of cyclophosphamide. It is shown that the much larger enhancement of regeneration observed for the hosts own surviving stem cells, compared to the regeneration of injected bone marrow stem cells, is due to the different numbers of stem cells initiating the regeneration in conjunction with the time course of stem cell regeneration. The results show that the environmental changes produced by cyclophosphamide greatly enhance haemopoietic recovery even though at the dose used this agent is relatively toxic to stem cells. Furthermore it has been shown that the level of stem cell regeneration is nearly independent of the γ-ray dose in the range 3–8 gray (300–800 rad). If human bone marrow should respond similarly it follows that regeneration produced by cytotoxic drugs administered prior to radiation embodies a considerable safety factor as far as recovery of the haemopoietic system is concerned.     

Radiosensitivity of vascular tissue. II. Differential radiosensitivity of aortic cells in vitro

The cellular outgrowths from three layers of rabbit and monkey aorta were used as primary cultures. Irradiation of the tissue fragments at the time of explanation resulted in a reduction in outgrowth of 50% with a dose of 200 rad, and in a reduction of over 90% with doses of 300 rad and above. When comparable cultures were irradiated after 2 months in vitro as a mature actively metabolizing but slowly proliferating cell population, radioresistance was increased. Subcultures of medial smooth muscle cells irradiated during their logarithmic growth phase showed a linear dose response in the cell number parameter up to 150 rad. A dose of 250 rad resulted in complete flattening of the growth curve, with a reduction in labeling index, after a 3-hr terminal [3H]TdR pulse. On the other hand, the labeling index indicated some recovery 3 days after irradiation in cultures receiving less than 250 rad. Under the same experimental conditions, cells derived from the intima of the same aorta showed no recovery when increase in cell numbers over time, or the number of labeled cells per area, were used as parameters. Cells derived from adventitia showed a relative increase in the number of labeled cells per area 4 and 7 days after irradiation following an initial decrease on Day 1.