Some short-term cell kinetic effects of ionizing radiation on mouse bladder urothelium

Early morphological changes and the pattern of reactive proliferation of the hairless mouse urinary bladder urothelium after irradiation are reported. Groups of female hairless mice were anaesthetized with sodium pentobarbital and irradiated over the bladder region with 0, 10, 20 and 30 Gy. Control groups were sham-treated. Short-term cell kinetic changes were monitored using incorporation of tritiated thymidine and flow cytometry. Only minor radiation-induced alterations in the cell kinetic pattern were recorded, and no significant histomorphological changes were seen. However, a marked increase in the thymidine incorporation was seen in the control animals on the first day after anaesthesia. Radiation proctitis induced early deaths in the 30 Gy irradiated animals. The present results are in accordance with commonly accepted radiobiological theories, but not in agreement with results previously published by others.     

Some cell kinetic effects of combined injury with ionizing radiation and cyclophosphamide on mouse bladder urothelium

Cyclophosphamide was given intraperitoneally to groups of eight female mice 48 h after local electron irradiation to the bladder with 0, 10 and 20 Gy respectively. The reactions in the urothelium were monitored by histology, incorporation of tritiated thymidine and flow cytometry. A wave of increased thymidine incorporation combined with an increase in the proportion of diploid S-phase cells was seen in the unirradiated bladders 24 h after the drug treatment, followed by normalization after 1 week. This response was significantly less pronounced in the irradiated animals. In the unirradiated animals a similar wave characterized by an increased proportion of octaploid cells was also seen, but this wave occurred later in the irradiated animals. Severe injury was observed in the rectum of the 20 Gy-irradiated animals. Irradiation prior to drug treatment led to only small effects, but a decreased ability for regenerative DNA synthesis after drug injury seems to persist. This affects both proliferation and the building up of polyploidy.     

Radiotoxicity of incorporated [3H]thymidine Consequences for the interpretation of FLM data of irradiated cell populations

Abstract. Problems in the interpretation of FLM data of externally irradiated cell populations are mainly due to the interference of radiation effects with radiotoxic effects originating from incorporated [³H]thymidine. These problems were investigated using L-929 cells flash labelled in vitro with [³H]thymidine (30 min, 0.3 μCi/ml, 40 Ci/mM) and irradiated with 2 Gy of 200 kV X-rays; the fractions of labelled mitoses and the index of labelled and of unlabelled mitoses were determined. The results showed that the FLM is not an adequate parameter to quantify the early cell kinetic changes in irradiated cell populations.     

Cell kinetics of mouse urinary bladder epithelium

A dose of 0.2 ml propylene glycol (1,2 propanediol) was injected subcutaneously into 12 hairless mice three times a week for three months. Four animals were killed at 1, 2 and 3 months and micro-flow fluorometric histograms of the bladder epithelial cells were made. The proportion of cells in diploid S phase was not much altered, but the proportion of tetraploid S-phase cells was significantly reduced and at three months DNA synthesis in tetraploid cells completely disappeared. The proportion of diploid cells increased, the proportion of tetraploids was slightly reduced and almost all octoploid cells disappeared. The changes are qualitatively similar to those seen after the bladder carcinogen dibutylnitrosamine, and after repeated injections of cyclophosphamide, but quantitatively much less pronounced. They can be explained as a result of cell toxicity whereby propylene glycol kills some bladder epithelial cells and disturbs the mechanism of repeated DNA synthesis. Propylene glycol is thus not a completely harmless solvent and when the kinetic effects of bladder carcinogens dissolved in propylene glycol are studied, the effect of the solvent alone must be accounted for.     

Local regulation of haemopoietic stem cell proliferation in mice following irradiation

Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.     

Local regulation of haemopoietic stem cell proliferation in mice following irradiation

Abstract Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.     

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.     

Cell cycle changes and cytotoxicity in irradiated cultures of bovine aortic endothelial cells

The purpose of this experiment was to determine the effect of ionizing radiation on cell number, lactate dehydrogenase (LDH) release, cell cycle distribution, [3H]thymidine incorporation, and autoradiographic labeling index in bovine aortic endothelial cells in vitro. Confluent endothelial monolayers were exposed to single doses of 0.5-10 Gy of 60Co gamma rays and were analyzed from 2 to 24 h postirradiation. Irradiated monolayers exhibited a time- and dose-dependent decrease in cell number, increase in LDH release, and redistribution of cells in the cell cycle. Cell cycle redistribution included an increase in the proportion of cells in S phase at 4 h after irradiation and a decrease in S phase at 24 h. The cells also exhibited a decrease in [3H]thymidine incorporation as early as 2 h after 5 Gy. This represented the most rapid radiation response observed in the present study. These data demonstrate that radiation cytotoxicity in confluent, plateau-phase endothelial monolayers is accompanied by changes in the cell cycle distribution of adherent cells, and that reduced [3H]thymidine incorporation is an early marker of radiation injury in this clinically important cell type.     

Some long-term cell kinetic effects of ionizing radiation on mouse bladder urothelium

The cell kinetics of the mouse bladder urothelium were followed with tritiated thymidine pulse labelling and flow cytometry for one year after irradiation with electrons. No perturbations were registered after 10 Gy. Three to four weeks after 20 Gy an elevation of the labelling index with a subsequent return to normal was seen. Flow cytometry revealed some increase in the proportion of octaploid nuclei at the same time. From about six months after irradiation the normal polyploidization decreased. The urothelium turned into a mainly diploid cell population. The proportion of diploid S phase cells also increased. The data give some support to the model hypothesis of reactive proliferation in a 'flexible' tissue, according to Wheldon et al. (1982).     

Some Long-Term Cell Kinetic Effects of Ionizing Radiation On Mouse Bladder Urothelium

Abstract. The cell kinetics of the mouse bladder urothelium were followed with tritiated thymidine pulse labelling and flow cytometry for one year after irradiation with electrons. No perturbations were registered after 10 Gy. Three to four weeks after 20 Gy an elevation of the labelling index with a subsequent return to normal was seen. Flow cytometry revealed some increase in the proportion of octaploid nuclei at the same time. From about six months after irradiation the normal polyploidization decreased. the urothelium turned into a mainly diploid cell population. the proportion of diploid S phase cells also increased. the data give some support to the model hypothesis of reactive proliferation in a 'flexible' tissue, according to Wheldon et al. (1982).     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

The effect of microwave radiation on the cell genome

Cultured V79 Chinese hamster cells were exposed to continuous radiation, frequency 7.7 GHz, power density 30 mW/cm2 for 15, 30, and 60 min. The parameters investigated were the incorporation of [3H]thymidine and the frequency of chromosome aberrations. Data obtained by 2 methods (the incorporation of [3H]thymidine into DNA and autoradiography) showed that the inhibition of [3H]thymidine incorporation took place by complete prevention of DNA from entering into the S phase. The normal rate of incorporation of [3H]thymidine was recovered within 1 generation cycle of V79 cells. Mutagenic tests performed concurrently showed that even DNA macromolecules were involved in the process. In comparison with the control samples there was a higher frequency of specific chromosome lesions in cells that had been irradiated. Results discussed in this study suggest that microwave radiation causes changes in the synthesis as well as in the structure of DNA molecules.     

Continuous induction of unscheduled DNA synthesis by gamma irradiation

The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA. Usually, in an in vivo-in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process--with centrifugation in alkaline sucrose a half-life of some minutes is found--an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation.     

Effect of metronidazole on the free thymidine content in the blood serum of irradiated mice

The influence of a radiosensitizer, metronidazole, on the free thymidine content of blood serum of irradiated mice was studied in aerobic and hypoxic conditions. A heated metronidazole solution (1 mg/g) was administered intraperitoneally 30 min before irradiation of animals with a dose of 3 Gy. Thymidine concentration in blood serum was determined by the radioimmunological technique. The influence of metronidazole on the level of thymidinemia was only noted in the animals exposed under hypoxic conditions.     

Computerized video time-lapse (CVTL) analysis of cell death kinetics in human bladder carcinoma cells (EJ30) X-irradiated in different phases of the cell cycle

The purpose of this study was to quantify the modes and kinetics of cell death for EJ30 human bladder carcinoma cells irradiated in different phases of the cell cycle. Asynchronous human bladder carcinoma cells were observed in multiple fields by computerized video time-lapse (CVTL) microscopy for one to two cell divisions before irradiation (6 Gy) and for 6-11 days afterward. By analyzing time-lapse movies collected from these fields, pedigrees were constructed showing the behaviors of 231 cells irradiated in different phases of the cell cycle (i.e. at different times after mitosis). A total of 219 irradiated cells were determined to be non-colony-forming over the time spans of the experiments. In these nonclonogenic pedigrees, cells died primarily by necrosis either without entering mitosis or over 1 to 10 postirradiation generations. A total of 105 giant cells developed from the irradiated cells or their progeny, and 30% (31/105) divided successfully. Most nonclonogenic cells irradiated in mid-S phase (9-12 h after mitosis) died by the second generation, while those irradiated either before or after this short period in mid-S phase had cell deaths occurring over one to nine postirradiation generations. The nonclonogenic cells irradiated in mid-S phase also experienced the longest average delay before their first division. Clonogenic cells (11/12 cells) divided sooner after irradiation than the average nonclonogenic cells derived from the same phase of the cell cycle. The early death and long division delay observed for nonclonogenic cells irradiated in mid-S phase could possibly result from an increase in damage induced during the transition from the replication of euchromatin to the replication of heterochromatin.     

Interleukin 4 promotes survival of lethally irradiated mice in the absence of hematopoietic efficacy

The therapeutic potential of Il4 in lethally irradiated mice was evaluated in C57BL6/J mice subjected to 7 to 10 Gy total-body irradiation (TBI) from a (60)Co gamma-ray source. Il4 was administered 2 h after TBI either in a single injection or for 5 consecutive days. Il4 treatment increased 30-day survival of mice irradiated with doses as high as 8.5 Gy, which caused 100% mortality in placebo-treated animals. By convention, hematopoietic failure would induce death over a period of up to 30 days. However, in our study, the Il4-enhanced survival of mice within this period could not be attributed to significantly accelerated hematopoietic reconstitution as shown by blood cell counts and progenitor cell contents in the bone marrow and spleen. Our data strongly suggest that aplasia is not the only cause of death of animals irradiated with doses around the LD(50) and that Il4-treated animals can survive in spite of a very poor hematopoietic activity.     

Adaptive response and split-dose effect of radiation on the survival of mice

Although the importance of radiation-induced adaptive response has been recognized in human health, risk assessment and clinical application, the phenomenon has not been understood well in terms of survival of animals. To examine this aspect Swiss albino mice were irradiated with different doses (2–10 Gy) at 0015 Gy/s dose rate and observed on a regular basis for 30 days. Since almost 50% lethality was seen with 8 Gy, it was selected as the challenging dose for further studies. Irradiation of mice with conditioning doses (0.25 or 0.5 Gy) and subsequent exposure to 8 Gy caused significant increase in the survival of mice compared to irradiated control. The splitting of challenging dose did not influence the efficiency of conditioning doses (0.25 Gy and 0.5 Gy) to induce an adaptive response. However conditioning doses given in fractions (0.25 Gy + 0.25 Gy) or (0.5 Gy + 0.5 Gy) were able to modulate the response of challenging dose of 8 Gy. These results clearly showed the occurrence of adaptive response in terms of survival of animals. The conditioning dose given in small fractions seemed to be more effective. The findings have been discussed from a mechanistic point of view. The possible biological implications, potential medical benefits, uncertainties and controversies related to adaptive response have also been addressed     

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.     

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity

Abstract. The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1–24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5¢-amino-5¢-deoxythymidine (5¢-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.