Inter- and intra-chromosomal distribution of chromatid breaks induced by X-rays during G2 in human lymphocytes

Cultures of blood from healthy adults were irradiated 48 h after stimulation with 240 R of X-rays and fixed after various time intervals (0–2 h, 2–4 h, 4–6 h). ³HTdR was added to several cultures after irradiation. Mitotic and labelling indices were used to distinguish between two cell samples inside the irradiated G₂ population: D − cells reaching mitosis without mitotic delay and a high frequency of chromatic breaks and D + cells with mitotic delay and which, during the delay, repair most of the damage produced. After R banding 450 chromatid deletions were located in each of the two cell samples. The D + cells showed a higher frequency of breaks than the D − cells with decreasing chromosome size, in the telomeric and centromeric region and in the junction between the R + and R − bands. These results can be interpreted as indicative of a non-random distribution of repair processes both between and within chromosomes.     

Quantitative regularities and peculiarities of the development of the cerebral form of radiation sickness at fractionated irradiation

Several peculiarities in manifestations of cerebral form of radiation sickness have been revealed at a fractionated double irradiation with equal and unequal doses per fraction and different intervals between the fractions. A reliable increase in average lifespan of rats irradiated with (100 + 100 Gy) equal doses at 10 and 60 min intervals between two fractions compared to the single radiation exposure to 200 Gy has been obtained. Lifespan of rats irradiated with a total dose greater than 200 Gy in most cases of double exposures with 10 min interval was reliably less than that for animals after a single exposure. The influence of the first dose on the reduction of animal average lifespan increased with fraction dose increasing from 150 to 300 Gy and was most pronounced at the total exposure dose of 400 Gy. Reaction of rats on the repeated irradiation was significantly weakened in comparison with the reaction on the first exposure. At a study of capacitation the interval of 30 min appeared to be more favorable compared to 10 min interval. Importance of a dose value in the first fraction has been demonstrated: the higher this value the worse the capacity of the rats 3 hours after the repeated exposure.     

Translocation yield from the mouse spermatogonial stem cell following fractionated X-ray treatments: influence of unequal fraction size and of increasing fractionation interval

(1) The genetic response of the mouse spermatogonial stem cell to a high dose of X-rays given in two unequal fractions 24 h apart can be dependent upon the order in which the two fractions are given. When 1000 R was administered as 100 R followed by 900 R the recovered translocation yield (22%) was similar to that which can be obtained by extrapolation from lower doses and also to that of a 500 + 500 R 24 h fractionation. By contrast, when the 900 R preceded the 100 R the response was much lower (7.4%), yet still greater than that produced by a single 1000 R treatment (4.5%). The same order of effectiveness was observed for length of sterile period. (2) The sub-additive translocation yields previously obtained with 800 R treatments given in fractions of 500 R and 300 R at intervals of 3-12 days were found to be maintained with intervals up to at least 15 days but additivity was regained by the end of the third week. Sterile period data indicated that with these intervals the germinal epithelium had recovered sufficiently from the first fraction for spermatogenesis to restart before the second fraction was given. (3) It is concluded from the two experiments that (a) 24 h after a radiation exposure the surviving stem cells are more sensitive than formerly both to killing and genetic damage, (b) at this time they are no longer heterogeneous in their radiosensitivities, so that increasing yields of genetic damage may be obtained with increasing dose i.e. there is no fall in yield at higher doses, (c) the change in sensitivity could be a consequence of a synchronization to a sensitive stage in a cell cycle, or to a transitional phase preparatory to entering a different cell cycle. (d) to achieve rapid repopulation of the germinal epithelium the surviving stem cells are stimulated to enter a shorter cell cycle and this is the cause of the sub-additive translocation yields with fractionation intervals of 3-15 days, (e) the recommencement of spermatogenesis is associated with the reestablishment of the heterogeneity in radiosensitivity among the stem cells. At this time additive translocation yields can again be recovered.     

Differential spermatogonial stem-cell survival and mutation frequency

One group of adult C3H×101 hybrid male mice was given 3 injections of 12.5 μCi of [³H]thymidine at 9-h intervals and irradiated 24 h after the last injection with X-ray doses of 100, 300, 500, 600, 1000 R or the first fraction of a split 1000-R dose given as two 500-R exposures 24 h apart. Mice were killed 207 and 414 h after irradiation. A second group of mice was given a single injection of 12.5 μCi of [³H]thymidine 1 h before irradiation with single exposures of 300, 500, 600, 1000 R, or the first fraction of a 1000-R exposure given as two 500-R fractions 24 h apart. Mice were killed 120 and 207 h after irradiation. In both experiments, parallel groups of mice were given X-ray only as a control for the effect of [³H]thymidine. Two sets of slides were prepared for each mouse receiving [³H]thymidine: one set was not autoradiographed and was used for scoring cell survival; the second set was coated with emulsion and used for scoring percentage of labeled cells. The dose-response curves for survival at 120 and 207 h were curvilinear, with no evidence of discontinuity over the 100–1000-R range. After multiple injections of [³H]thymidine and irradiation 24 h later, percentage of labeled cells at 207 h was comparable for controls, 100, 300, and 600 R; significantly lower than controls for 1000 R; and significantly above controls after 500 + 500 R. Thus the surviving stem-cell population was qualitatively the same for that portion of the dose-response curve giving a linear increase in mutation rate but was different for both 1000-R and 500 + 500-R exposures, and the single and fractionated 1000-R exposures differed from each other. This parallelism between survival of labeled cells and mutation frequency in spermatogonial stem cells suggests that a stage in the cell cycle 24–42 h after DNA synthesis is resistant to cell killing but sensitive to mutation induction. The mutation rate after a single 1000-R exposure is low because labeled, mutation-sensitive cells have been selectively killed. Mutation frequency after the 500 + 500-R dose is increased because of synchronization induced by the first dose combined with selective killing of unlabeled cells by the second fraction. Irradiation 1 h after labeling with [³H]-thymidine demonstrated that the S phase of the spermatogonial stem-cell cycle is sensitive to radiation-induced cell killing.     

Bystander and delayed effects after fractionated radiation exposure

Human immortalized keratinocytes were exposed to a range of single or fractionated doses of gamma rays from (60)Co, to medium harvested from donor cells exposed to these protocols, or to a combination of radiation and irradiated cell conditioned medium (ICCM). The surviving fractions after direct irradiation or exposure to ICCM were determined using a clonogenic assay. The results show that medium harvested from cultures receiving fractionated irradiation gave lower "recovery factors" than direct fractionated irradiation, where normal split-dose recovery occurred. The recovery factor is defined here as the surviving fraction of the cells receiving two doses (direct or ICCM) separated by an interval of 2 h divided by the surviving fraction of cells receiving the same dose in one exposure. After treatment with ICCM, the recovery factors were less than 1 over a range of total doses from 5 mGy-5 Gy. Varying the time between doses from 10 min to 180 min did not alter the effect of ICCM, suggesting that two exposures to ICCM are more toxic than one irrespective of the dose used to generate the response. In certain protocols using mixtures of direct irradiation and ICCM, it was possible to eliminate the bystander effect. If bystander factors are produced in vivo, then they may reduce the sparing effect of the dose fractionation.     

Split-dose exposure to N-methyl-N'-nitro-N-nitrosoguanidine in BALB/3T3 C1 a31-1-1 cells: evidence of DNA repair by alkaline elution without changes in cell survival, mutation and transformation rates

Dose fractionation of a direct-acting chemical carcinogen, the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was studied for its concurrent effects on survival, DNA damage and repair, ouabain resistance (Ouar) mutations and neoplastic transformation, in the mouse embryo cell line BALB/3T3 C1A31-1-1. MNNG doses of 0.5, 1 and 2 micrograms/ml were added to the cells either as a single exposure or in two equal fractions separated by 1, 3 or 5 h intervals. No significant difference in cytotoxicity was found when single and split-dose treatments were compared. No recovery from sublethal damage was therefore found in this cell line by split-dose administration of MNNG, although such an effect was found when the same cell line was treated with single and split doses of X-rays. Repair of DNA damage as measured by alkaline elution was studied up to 24 h after a single MNNG exposure (0.5 micrograms/ml). DNA repair was rapid during the first 5 h after treatment and slow thereafter. DNA damage detected after split doses of MNNG at 1 and 5 h intervals was significantly lower than after a corresponding single dose. With both single and split doses, rejoining of single-strand breaks (ssb) was nearly complete after 24 h of repair time. Ouar mutation and neoplastic transformation frequencies were determined for single and split doses of MNNG with the second treatment being given during (1 h) or after (5 h) the period of rapid DNA repair. No significant differences in either effect were detected for dose splitting at any tested dose.     

Repair capacity and kinetics in spheroids from a lung metastasis of a human soft tissue sarcoma: a growth delay study.

Spheroids grown from the human cell line EF8 of a lung metastasis of a human malignant fibrous histiocytoma were given fractionated irradiation with 60Co gamma rays at passages 31 and 32. The mean diameter of the spheroids at the time of treatment was 250 microns. Growth delay was used as the end point in these studies. Two experiments were carried out to determine the capacity and kinetics of repair of sublethal damage. In the first experiment, one, two, and five fractions were given at three or four dose levels with fixed intervals of 360 min. In the second experiment, schedules with two and four dose fractions and intervals of 0, 20, 60, 120, and 360 min were used, each at two dose levels. Data analysis was performed by a direct method based on the alpha/beta model and first-order repair kinetics of radiation damage. In both experiments, the alpha/beta value of EF8 spheroids was estimated to be about 8 (6-10) Gy. The rate constant of repair, mu, and its 95% confidence interval were estimated to be 0.62 (0.40-0.84) 10(-2) min-1, equivalent to a half-time of repair (T1/2) of 112 (83-172) min. A more detailed analysis of the data of the second experiment revealed a significant dependence of the rate constant of repair, mu, on the total radiation effect induced by the fractionated radiation treatments with short overall times. With increasing level of effect, mu decreased. These data indicate that the half-time of recovery of a human tumor can be longer than that of the surrounding normal tissue, in this case lung, at least for a limited range of doses and for some fractionation schedules.     

Direct analysis of quantal radiation response data

A direct analysis is proposed for quantal (all-or-nothing) responses to fractionated radiation and endpoint-dilution assays of cell survival. As opposed to two-step methods such as the reciprocal-dose technique, in which ED50 values are first estimated for different fractionation schemes and then fit (as reciprocals) against dose per fraction, all raw data are included in a single maximum-likelihood treatment. The method accommodates variations such as short-interval fractionation regimens designed to determine tissue repair kinetics, tissue response to continuous exposures, and data obtained using endpoint-dilution assays of cell survival after fractionated doses. Monte-Carlo techniques were used to compare the direct and reciprocal-dose methods for analysis of small-scale and large-scale studies of response to fractionated doses. Both methods tended toward biased estimates in the analysis of the small-scale (3 fraction numbers) studies. The alpha/beta ratios showed less scatter when estimated by the direct method. Most important, the 95 per cent confidence intervals determined by the direct method were more appropriate than those determined by reciprocal-dose analysis, for which 18 per cent (small-scale study) or 8 per cent (large-scale study) of the confidence intervals did not include the 'true' value of alpha/beta.     

Exposure fractionation effects for X-ray-induced dominant lethals in immature (stage-7) oocytes of Drosophila melanogaster: A re-analysis

Young (0—4-h-old) Drosophila melanogaster females were X-irradiated with single or fractioned exposured over a range up to 6000 R and the induction of dominant lethals in immatuer (stage-7) oocytes was studied. The results show that (i) the frequencies of dominant lethals are higher after single than after fractionated exposures; (ii) at any given exposure level, the higher the number of fractions, the lower is the frequency of dominant lethals; (iii) conserquently, the reduction in dominant lethality relative to single exposures increases with increasing number of fractions; and (iv) this relative reduction in dominant lethality approaches a maximum value when the magnitude of the single X-ray exposure approaches zero (i.e., when tha egg survival after single X-ray exposure approaches 100%); the maximum, however, are different for the different fractionation regimes, being higher with increasing number of fractions.These findings are consistent with the assumed kinetics of X-ray induction of dominant lethality in stage-7 oocytes. It is shown that it is possible to predict the expected relative reduction in dominant lethality after fractionation, from appropriate dominant lethal data from single unfractioned exposures.     

Multifraction radiation response of mouse lung

The response of mouse lung to repeated doses of 60Co gamma-rays of as low as 115 cGy per fraction was measured using death from pneumonitis between 80 and 120 days after irradiation as the endpoint. A fractionation interval of 3 h was maintained for most regimens but in the longer experiments some 12 h intervals were introduced for logistic reasons. The longest overall duration (for a 43 fraction regimen) was 8 days. The total doses required to produce 50 per cent mortality increased continuously as dose/fraction was decreased, even from 160 to 115 cGy per fraction. Of clinical relevance, the steepness of the isoeffect curve over the dose range 115-500 cGy indicates that the lung shows greater sparing from dose fractionation than is characteristic of more rapidly-responding normal tissues, resembling, in this respect, other more slowly-responding tissues such as spinal cord. The plot of the reciprocal of the LD50 values as a function of dose per fraction was non-linear, suggesting that a linear quadratic dose response model may not be appropriate or that repair of cellular injury in lung is not complete in 3 h, or both.     

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.     

Translocations in mouse spermatogonia after exposure to unequally fractionated doses of x-rays

The influence of various X-ray fractionation regimes on translocation induction in mouse spermatogonia was studied. Splitting a single exposure of 600 R into two fractions of 300 R separated by 24 h did not influence the translocation yield (7.58% vs. 8.35%). When the dose was given in the unequal fractions,100 R+500 R and 500 R+100 R, with the same 24 h between the fractions, the translocation frequency was significantly altered (10,19 and 4.94%, respectively). As a possible explanation of these findings it is assumed that the relatively resistant cells surviving the first fraction of the dose are sensitized towards translocation induction when receiving the second fraction of the dose.     

Reparative processes of damaged chromosomes in non-stimulated human peripheral blood lymphocytes by the fractionated irradiation method and temperature variation]

The fractionation experiments were carried out on resting lymphocytes. Non-stimulated lymphocytes were X-irradiated at the total dose of 200 r separated into two equal parts by either 5-hour or 20-hour intervals. The whole blood samples were kept during the intervals between the exposure at the temperature of 20degrees C or 37degrees C. All the cultures were made after the last radiation exposure at 37degrees C. Dicentrics and centric rings were scored. It is shown that a fractionation effect takes place in resting lymlf-dose at 37degrees C and is absent at the temperature of 20degrees C. It is suggested that there is the repair in lymphocytes at the stage Go, at least, from the dose of 100 r.     

Simulation modelling of the postradiation restoration of the crypt-villus system

Basic principles have been developed for a discrete stochastic simulation model of an elementary proliferative unit of the intestinal epithelium, a "crypt-villus" system. The analysis of the results obtained after a single exposure of the animal's abdomen to 3 and 6 Gy radiation has demonstrated that the dynamics of the number of cells that synthesize DNA in a small intestine crypt of exposed mice depends on the rate of radiation damage repair (50 to 100 h following irradiation). The rate of repair after 6 Gy irradiation is 1.5 times lower that after 3 Gy. The changes in the shape of the labeled mitoses curve, followed up during the postirradiation recovery of the intestinal epithelium, may occur with the time parameters of the cell mitotic cycle being invariable.     

Time dependence of Elkind-type recovery in class B oocytes of Drosophila melanogaster.

Recovery from X-ray-induced damage in class B oocytes of Drosophila melanogaster was studied by the dose-fractionation technique. A total dose of 500 R was delivered either as a single exposure or as two fractions of 2000 R and 3000 R separated by increasing time intervals. The use of attached-X females made it possible to study simultaneously the induction of dominant lethals and of chromosome aberrations (detachments of the attached-X chromosome). The same repair kinetics were observed for sublethal damage and for the lesions leading to detachments. The time-response curves are of similar shape: a plateau is reached within 20 to 30 min and half of the repairable damage disappears in 5 to 7 min. It is concluded that the same type of X-ray-induced primary lesion in chromosomes is responsible for the induction of detachments and for dominant lethals. As primary lesions actual chromosome breaks or lesions leading to breaks and chromosome rearrangements are assumed.     

Influence of X-ray dose fractionation on the frequency of somatic mutations induced in tradescantia stamen hairs

X-rays were used to investigate the influence of dose fractionation on the induction of pink and colorless somatic mutations in stamen hair cells of Tradescantia clone 02. Inflorescences were exposed to a single acute dose of 60 rad, two acute doses of 30 rad, or three acute doses of 20 rad. The dose rate in all cases was 30 rad/min. Intervals between dose fractions were varied from 35 sec to 48 h and the mutation frequency was compared with that resulting after the single dose of 60 rad. The data show a reduction in mutation frequency for fractionation intervals longer than 15 and 6 min for pink and colorless mutations, respectively, but not for shorter intervals.One interpretation of the data predicts that pink mutation frequencies are reduced by 11% for fraction intervals of from 30 min to 6 h, and that colorless mutation frequencies are reduced by 24% for intervals of from 15 min to 6 h. The corresponding sparing effect of dose fractionation is equal to 6 rad for pink mutations and 9 rad at the colorless mutation endpoint. A calculation has been made which indicates that the percentages of the total repairable (presumably two-hit) damage that is repaired during fraction intervals up to 6 h, are 16 and 35% for pink and colorless mutations respectively.     

Ultraviolet Light-Induced Division Delay in Synchronized Chinese Hamster Cells

The age-dependent, ultraviolet light (UVL) (254 nm)-induced division delay of surviving and nonsurviving Chinese hamster cells was studied. The response was examined after UVL exposures adjusted to yield approximately the same survival levels at different stages of the cell cycle, 60% or 30% survival. Cells irradiated in the middle of S suffered the longest division delay, and cells exposed in mitosis or in G₁ had about the same smaller delay in division. Cells irradiated in G₂, however, were not delayed at either survival level. It was further established, after exposures that yielded about 30% survivors at various stages of the cycle, that surviving cells had shorter delays than nonsurvivors. This difference was not observed for cells in G₂ at the time of exposure; i.e., neither surviving nor nonsurviving G₂ cells were delayed in division. The examination of mitotic index vs. time revealed that most cells reach mitosis, but all of the increase in the number of cells in the population can be accounted for by the increase of the viable cell fraction. These observations suggest strongly that nonsurviving cells, although present during most of the experiment, are stopped at mitosis and do not divide. Cells in mitosis at the time of irradiation complete their division, and in the same length of time as unirradiated controls. Division and mitotic delays after UVL are relatively much larger than after X-ray doses that reduce survival to about the same level.     

Cell kinetics and radiation biology

The cell cycle, the growth fraction and cell loss influence the response of cells to radiation in many ways. The variation in radiosensitivity around the cell cycle, and the extent of radiation-induced delay in cell cycle progression have both been clearly demonstrated in vitro. This translates into a variable time of expression of radiation injury in different normal tissues, ranging from a few days in intestine to weeks, months or even years in slowly proliferating tissues like lung, kidney, bladder and spinal cord. The radiosensitivity of tumours, to single doses, is dominated by hypoxic cells which arise from the imbalance between tumour cell production and the proliferation and branching of the blood vessels needed to bring oxygen and other nutrients to each cell. The response to fractionated radiation schedules is also influenced by the cell kinetic parameters of the cells comprising each tissue or tumour. This is described in terms of repair, redistribution, reoxygenation and repopulation. Slowly cycling cells show much more curved underlying cell survival curves, leading to more dramatic changes with fractionation, dose rate or l.e.t. Rapidly cycling cells redistribute around the cell cycle when the cells in sensitive phases have been killed, and experience less mitotic delay than slowly proliferating cells. Reoxygenation seems more effective in tumours with rapidly cycling cells and high natural cell loss rates. Compensatory repopulation within a treatment schedule may spare skin and mucosa but does not spare slowly proliferating tissues. Furthermore, tumour cell proliferation during fractionated radiotherapy may be an important factor limiting the overall success of treatment.     

No elevated cell surface charge at mitosis in X-ray-irradiated mammalian cells

Rounded mitotic cells showed 30% enhanced electrophoretic mobility (EPM) when compared to spindle-formed interphase cells. This increase in EPM that was not present in interphase cells that had been rounded chemically by EDTA is considered to reflect a structural change in the cell membrane during mitosis. X-ray irradiation induced a dose-dependent EPM decrease in both interphase and mitotic cells during a 4-hour period. During the next 20 h of incubation, EPM recovery took place in cells irradiated with 250R, but not in cells exposed to 1000R. EPM was enhanced during mitosis in cells irradiated with low doses, but was absent in cells irradiated with 1000R. The ratio of colony-forming cells and of electrophoretically recovered mitotic cells after 24 h of exposure showed a good statistical correlation. These results indicate that unrepaired membrane damage contributes to mitotic cell death after irradiation.