The effect of altered fractionation schedule on the spinal cord response: radiobiological considerations

Increased fractionation spares late reacting normal tissues more than acute reacting normal tissues. A linear quadratic model is valid from large dose per fraction down to dose per fraction of 2 Gy. Experimental studies on animals and clinical studies on the spinal cord tolerance have shown incidences of myelopathy at doses lower than 50 Gy. The α/β value of the linear quadratic model have been lower for low doses per fraction, indicating a sparing effect of altered fractionation for spinal cord myelitis. Animal data, clinical and radiobiological explanations suggest limitation of the radiobiological models. Further data suggest that one must not assume the spinal cord to have a greater tolerance at doses per fraction below the conventional dose per fraction of 2 Gy.     

Transformation of C3H 10T1/2 cells with 4.3 MeV alpha particles at low doses: effects of single and fractionated doses.

Oncogenic transformation of C3H 10T1/2 cells was determined after exposure to graded doses of 4.3-MeV alpha particles LET = 101 keV/microns. The source of alpha particles was 244Cm and the irradiation was done in an irradiation chamber built for the purpose. Graded doses in the range of 0.2 to 300 cGy were studied with special emphasis on the low-dose region, with as many as seven points in the interval up to 10 cGy. The dose-effect relationship was a complex function. Transformation frequency increased with dose up to 2 cGy; it seemed to flatten at doses between 2 and 20 cGy but increased again at higher doses. A total of 21 cGy was delivered in a single dose or in 3 or 10 equal fractions at an interval of 1.5 h. An inverse dose-protraction effect of 1.4 was found with both fractionation schemes. Measurements of the mitotic index of the population immediately before the various fractions revealed a strong effect on the rate of cell division even after very low doses of radiation. Mitotic yield decreased markedly with the total dose delivered, and it was as low as 50% of the control value after 4.2 cGy and 20% after 14 cGy with both fractionation schemes.     

Dose fractionation effects in plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts

A comparison of gamma-ray dose fractionation effects was made using plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts in an attempt to simulate basically similar populations of cells that differ primarily in their turnover rates. The status of cell populations with respect to their turnover rates may be an important factor influencing dose fractionation effects in early- and late-responding tissues. In this cell culture system, the rate of cell turnover was approximately three times higher for the plateau-phase transformed cultures. While the single acute dose survival curves for log-phase cells were indistinguishable, there were significant differences between the survival curves for plateau-phase cultures of the two cell types. These differences were qualitatively similar to the differences recently postulated for the survival of target cells governing early and late tissue responses. Both cell lines had a similar capacity for repair of sublethal damage, but untransformed cells had a much greater capacity to repair potentially lethal damage in plateau phase. Further, untransformed plateau-phase cultures were much more sensitive to a radiation-induced G1 (or G0 to G1) delay than transformed cultures. Multifraction survival curves were determined for both cell lines for doses per fraction ranging from 9.0 to 0.8 Gy, and from these isoeffect curves of log total dose versus dose per fraction were derived. The isoeffect curve for the slowly cycling, untransformed cells was found to be appreciably steeper than that for the more rapidly cycling transformed cells, a finding consistent with previously reported differences in dose fractionation isoeffect curves for early- and late-responding tissues in vivo.     

An 'incomplete-repair' model for survival after fractionated and continuous irradiations

An incomplete-repair (IR) model of survival after fractionated or continuous irradiation is derived from the concept of 'dose-equivalent' of incomplete repair. The model gives reasonably good predictions of the effect of interfraction interval, dose per fraction, and dose rate on cell survival in vivo and on tissue responses. This model is compared to the 'lethal, potentially lethal' (LPL) model after the latter has been generalized to an arbitrary number of fractions and to low dose-rate, continuous exposures. It is shown that the two models are equivalent, given certain constraints on the size of dose per fraction and dose rate. For example, in a particular cell line the equivalence of fractionation models breaks down if dose per fraction is well in excess of 4 Gy (the IR model employs the linear-quadratic survival model). The equivalence of low dose rate models breaks down for dose rates well in excess of 20 cGy/min. The assumptions on which the generalized LPL model is based are used to give a radiobiological interpretation to the incomplete-repair model. The larger beta/alpha ratio characteristic of late-responding normal tissues is interpreted in terms of the relatively faster fixation of potentially reparable lesions in the target cells of acutely responding tissues, on account of progression in the cell cycle. According to this interpretation the beta/alpha ratios estimated from isoeffective fractionation regimens are directly related to the parameters of clonogenic cell killing.     

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.     

Role of radioadaptation on radiation-induced thymic lymphoma in mice

Thymic lymphoma (TL) was observed in different stages of development in 46% of male mice (23/50) following exposure to an acute challenge dose of 2 Gy ⁶⁰Co γ-rays. With an adapting dose of 1 cGy 24 h prior to the challenge dose of 2 Gy, similar growth of TL was seen in 42.5% of mice (17/40). TL was not found in unirradiated control mice (0/50) or in the group treated with 1 cGy (0/50). Multiple adapting doses for 5 or 10 consecutive days induced TL in 8/50 and 9/50 mice, respectively (17% in average). When multiple adapting doses were followed by the challenge dose, the yield of TL was much lower, 16% (8/50) and 30% (15/30), respectively. By 15, 30, 60, 90, and 120 days after exposure with 3 Gy of ⁶⁰Co γ-rays, TL developed in 30, 70, 70, 80 and 85% of the female mice, respectively. When mice were conditioned with an adapting dose of 1 cGy 24 h prior to the challenge dose, TL was not found 15 days post-irradiation, while about a 25% reduction in the occurrence of TL was noticed at all other intervals. The results suggested that an adapting dose could play a role in bringing about a change in terms of delay and inhibition of the acute effects of radiation, i.e., the onset of TL in mice.     

The kinetics of repair in mouse lung after fractionated irradiation

The kinetics of repair of sublethal damage in mouse lung was studied after fractionated doses of 137Cs gamma-rays. A wide range of doses per fraction (1.7-12 Gy) was given with interfraction intervals ranging from 0.5 to 24 h. The data were analysed by a direct method of analysis using the incomplete repair model. The half-time of repair (T1/2) was 0.76 h for the pneumonitis phase of damage (up to 8 months) and 0.65 h for the later phase of damage up to 12 months. The rate of repair was dependent on fraction size for both phases of lung damage and was faster after large dose fractions than after small fractions. The T1/2 was 0.6 h (95 per cent c.1. 0.53, 0.69) for doses per fraction greater than 5 Gy and 0.83 h (95 per cent c.1 0.76, 0.92) for doses per fraction of 2 Gy. Repair was nearly complete by 6 h, at least for the pneumonitis phase of damage. To the extent that extrapolation of these data to humans may be valid, these results imply that treatments with multiple fractions per day that involve the lung will not be limited by the necessity for interfraction intervals much longer than 6 h.     

Effect of verification imaging on in vivo dosimetry results using Gafchromic EBT3 film

In this work, the apparent treatment dose that kV planar or CBCT imaging contributes to Gafchromic EBT3 film used for in vivo dosimetry, was investigated. Gafchromic EBT3 film pieces were attached to a variety of phantoms and irradiated using the linear accelerator’s built-in kV imaging system, in both kV planar mode and CBCT mode. To evaluate the sensitivity of the film in the clinical scenario where dose contributions are received from both imaging and treatment, additional pieces of film were irradiated using base doses of 50 cGy and then irradiated using selected kV planar and CBCT techniques. For kV planar imaging, apparent treatment doses of up to 3.4 cGy per image pair were seen. For CBCT, apparent treatment doses ranged from 0.22 cGy to 3.78 cGy. These apparent doses were reproducible with and without the inclusion of the 50 cGy base dose. The contribution of apparent treatment dose from both planar kV as well as CBCT imaging can be detected, even in conjunction with an actual treatment dose. The magnitude of the apparent dose was found to be dependent on patient geometry, scanning protocol, and measurement location. It was found that the apparent treatment dose from the imaging could add up to 8% of additional uncertainty to the in vivo dosimetry result, if not taken into account. It is possible for this apparent treatment dose to be accounted for by subtraction of the experimentally determined apparent doses from in vivo measurements, as demonstrated in this work.     

Life shortening in mice exposed to fission neutrons and gamma rays. VIII. Exposures to continuous gamma radiation

Data are presented on the mean aftersurvival of male B6CF1 mice exposed for 22 h per day, 5 days per week, to 60Co gamma radiation at dose rates of 1.36 to 12.64 x 10(-3) cGy/min for 23 weeks or 1.36 to 6.32 x 10(-3) cGy/min for 59 weeks. For deaths from all causes, linear dose-response curves were obtained with slopes (days of life lost/cGy) of 0.158 +/- 0.016 and 0.077 +/- 0.002 for 23- and 59-week exposures, respectively. These values were not significantly altered when the analysis was restricted to those mice dying with tumors (92% of the total) or to those presumably dying from tumors (82% of the total). Analysis of mortality rates showed that about 90% of the radiation-specific excess mortality was tumor related. The 59-week exposure series induced only a small increase in the number of days of life lost/cGy/weekly fraction over that induced by 23 weeks of irradiation, 4.53 +/- 0.15 compared to 3.64 +/- 0.36 days lost/cGy/weekly fraction. This lower than expected value for 59 weeks of exposure may signal the approach to the final linear, additive, injury term postulated from earlier studies at this laboratory with low-dose-rate, daily, duration-of-life 60Co gamma irradiation.     

Peculiarities of the effect of low-dose-rate radiation simulating high-altitude flight conditions on mice in vivo

In the present work, the effect of a low-dose rate of high-LET radiation in polychromatic erythrocytes of mice bone marrow was investigated in vivo. The spectral and component composition of the radiation field used was similar to that present in the atmosphere at an altitude of about 10 km. The dose dependence, adaptive response, and genetic instability in the F₁ generation born from males irradiated under these conditions were examined using the micronucleus test. Irradiation of the mice was performed for 24 h per day in the radiation field behind the concrete shield of the Serpukhov accelerator. Protons of 70 GeV were used over a period of 15–31 days, to accumulate doses of 11.5–31.5 cGy. The experiment demonstrated that irradiation of mice in vivo in this dose range leads to an increase in cytogenetic damage to bone marrow cells, but does not induce any adaptive response. In mice pre-irradiated with a dose of 11.5 cGy, an increase in sensitivity was observed after an additional irradiation with a dose of 1.5 Gy. The absence of an adaptive response suggests existence of genetic instability.     

Life shortening in mice exposed to fission neutrons and gamma rays. VII. Effects of 60 once-weekly exposures

A total of 6316 B6CF1 mice were exposed to 60 equal once-weekly doses of 0.85-MeV fission neutrons (0.033 to 0.67 cGy per weekly fraction) or 60Co gamma rays (1.67 to 10 cGy per weekly fraction) and were observed until they died. The mean aftersurvival times showed that the dose-response curves for both neutron and gamma-ray exposures were indistinguishable from linear over all doses except the highest neutron dose. The relative biological effectiveness (RBE) for neutrons, calculated as the ratio of the initial slopes of the dose-response curves, was about 20 for both males and females. Essentially the same value was obtained by a number of other analyses of the data. Virtually all of the radiation-specific excess mortality could be attributed to tumors; after decrementation of the population for nontumor deaths, the value of the RBE was not significantly changed.     

Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages

The induction of congenital malformations among the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages has been studied in two experiments. Firstly, animals were exposed to varying doses (108–504 cGy) of X-rays and mated at various time intervals (1–7, 8–14, 15–21 and 64–80 days post-irradiation), so as to sample spermatozoa, spermatids and spermatogonial stem cells. In the second experiment, only treated spermatogonial stem cells were sampled. One group of males was given a single 500-cGy dose, a second group a fractionated dose (500 + 500 cGy, 24 h apart) and a third group was left unexposed.In the first experiment, induced post-implantation dominant lethality increased with dose, and was highest in week 3, in line with the known greater radiosensitivity of the early spermatid stage. Preimplantation loss also increased with dose and was highest in week 3. There was no clear induction of either pre-implantation or post-implantation loss at spermatogonial stem cell stages.There was a clear induction of congenital malformations at post-meiotic stages, the overall incidence being 2.0 ± 0.32% in the irradiated series and 0.24 ± 0.17% among the controls. The induction was statistically significant at each dose. At the two highest doses the early spermatids (15–21 days) appeared more sensitive than spermatozoa, and at this stage the incidence of malformations increased with dose. The data from Expt. 1 on the induction of malformations by irradiation of spermatogonial stages were equivocal. In contrast, Expt. 2 showed a statistically significant induction of malformations at both dose levels (2.2 ± 0.46% after 500 cGy and 3.1 ± 0.57% after 500 + 500 cGy). The relative sensitivities of male stem cells, post-neiotic stages and mature oocytes to the induction of congenital malformations were reasonably similar to their sensitivities for specific-locus mutations, except that the expected enhancing effect of the fractionation regime used was not seen.Dwarfism and exencephaly were the two most commonly observed malformations in all series.     

Effets de la radiothérapie sur la fonction testiculaire de l'adulte

Radiation therapy plays a major role in the curative management of numerous neoplasms, such as Hodgkin's disease or testicular cancer. However, the adverse effects of low-dose radiation scattered to radiosensitive normal tissues adjacent to the radiation fields, such as the testes, have been recognized. Experimental studies performed on healthy volunteers showed that no lesion was detectable on sperm counts or testicular biopsies after single doses of less than 10 cGy. Oligospermia has been reported after 15 cGy and 100 cGy result in a 90% incidence of azoospermia. In the radiotherapy of cancer, fractionated regimens are used to increase the differential effect between normal and tumoral tissues. For the same dose, a fractionated radiation regimen results in a higher incidence and a longer period of azoospermia than a single dose irradiation. Fractionated doses of >50 cGy result in a 100% incidence of azoospermia. For doses up to 200 cGy, recovery occurs but normal sperm production remains uncertain. Although the recovery time can be very long (more than 10 years), there is a risk of definitive azoospermia after doses of >200 cGy. Spermatogonia are the most radio-sensitive cell type and their depletion after small irradiation doses explain the effect of radiotherapy on fertility. Clinical hypogonadism is very unfrequent in usual practice, what seems to prove a relative radio-resistance of the Leydig cells. However, functionals studies show that there is a rise in serum LH with increasing dose to the testes. A decrease in testosterone levels has been reported after high testicular doses.     

A test of equal effect per fraction in the kidney of the mouse.

Measurements of renal damage in the mouse were made to determine if there was an equal effect per fraction during a course of repeated 240-kVp X-ray doses. An X-ray dose of 2 Gy was given 2, 8, 14, or 20 times with interfraction intervals of 12 h. Some animals were also irradiated with twenty 2-Gy doses using a 5-h interfraction interval. The underlying effect per fraction (-logeSF of the notional target cell population) was determined from the additional top-up dose of d(4)-Be neutrons needed to produce measurable renal impairment assessed by decreased clearance from the plasma of [51Cr]EDTA and by a reduction in the hematocrit at 25, 29, 33, and 39 weeks after treatment. There was no significant influence of the time of assay on the values of underlying effect measured. A mean value of underlying effect was therefore calculated for the two different assays of each mouse, from the measurements at the four times. This gave approximately 40 estimates (one for each animal assessed) with each assay of the effectiveness of 2-Gy fractions in each of the four fractionation schedules, a total of 321 determinations in the study with 12-h intervals. Regression analysis showed that there was no significant trend in underlying effect per fraction with number of fractions, i.e., the damage per fraction was constant regardless of the number of fractions used. With underlying effect normalized to 1 unit of damage for a single 2-Gy dose, the slope of this plot was -0.0013 per fraction2 +/- 0.0097 (95% CL). The assumption of equal effect per fraction was therefore not invalidated in the kidney of the mouse. With a 5- instead of a 12-h interfraction interval, the 20-fraction schedule was 7% more effective as measured by the two assays analyzed together; this was significant at P = 0.0001. This shows that 5 h is not sufficient time between fractions for full repair to occur in the kidney, and underlines the need for intervals of at least 6 h between the doses in clinical radiotherapy using more than one fraction per day. The data are consistent with an alpha/beta ratio approximately 1.6 Gy, with a repair half-time approximately 1.3 h. However, these experiments were not designed to determine these parameters and their values should be regarded only as rough estimates.     

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.     

Effects of exposure to low-dose-rate (60)co gamma rays on human tumor cells in vitro

Cells of three asynchronously growing human tumor cell lines, PC3 (human prostate carcinoma), T98G and A7 (human glioblastomas), which have been shown previously to demonstrate low-dose hyper-radiosensitivity to low acute single doses, were irradiated with (60)Co gamma rays at low dose rates (2 cGy-1 Gy h(-1)). Instead of a dose-rate sparing response, these cell lines demonstrated an inverse dose-rate effect on cell survival at dose rates below 1 Gy h(-1), whereby a decrease in dose rate resulted in an increase in cell killing per unit dose. A hyper-radiosensitivity-negative cell line, U373MG, did not demonstrate an inverse dose-rate effect. Analysis of the cell cycle indicated that this inverse dose-rate effect was not due to accumulation of cells in G(2)/M phase or to other cell cycle perturbations. T98G cells in reversible G(1)-phase arrest also showed an inverse dose-rate effect at dose rates below 30 cGy h(-1) but a sparing effect as the dose rate was reduced from 60 to 30 cGy h(-1). We conclude that this inverse dose-rate effect in continuous exposures reflects the hyper-radiosensitivity seen in the same cell lines in response to very small acute single doses.     

Radiation-induced renal damage: the effects of hyperfractionation

The response of mouse kidneys to multifraction irradiation was assessed using three nondestructive functional end points. A series of schedules was investigated giving 1, 2, 4, 8, 16, 32, or 64 equal X-ray doses, using doses per fraction in the range of 0.9 to 16 Gy. The overall treatment time was kept constant at 3 weeks. Kidney function was assessed from 19 to 48 weeks after irradiation by measuring changes in isotope clearance, urine output, and hematocrit. The degree of anemia (assessed from the hematocrit measurements) is a newly developed assay which is an early indicator of the extent of renal damage after irradiation. All three assays yielded steep dose-effect curves from which the repair capacity of kidney could be estimated by comparing the isoeffective doses in different schedules. There was a marked influence of fractionation, with increasing dose being required to achieve the same level of damage for increasing fraction number, even between 32 and 64 fractions. The data are well fitted by a linear quadratic dose-response equation, and analysis of the data in this way yields low values (approximately 3.0 Gy) for the ratio alpha/beta. This would suggest that hyperfractionation , using extremely small X-ray doses per fraction, would spare kidneys relative to tumors and acutely responding tissues.     

Low-dose irradiation of human fibroblasts leads to delayed acceleration of proliferation of their progeny

We have demonstrated for the first time that a single exposure to γ-radiation at a dose of 3 cGy on HELF-104 human embryonic lung fibroblasts in early passages leads to the delayed stimulation of proliferation of the progeny of irradiated cells by 30–37 days. Moreover, the general changes in dynamics of proliferation after irradiation with low doses (3 and 5 cGy) are more pronounced than after high-dose irradiation (2 Gy). We suggest that this effect may play an important role in the formation of the specific effects of low doses of ionizing radiation, as detected by integral endpoints at higher levels of organization of living matter.     

Melatonin and protection from whole-body irradiation: survival studies in mice

The radioprotective ability of melatonin was investigated in mice exposed to an acute whole-body gamma radiation dose of 815 cGy (estimated LD50/30 dose). The animals were observed for mortality over a period of 30 days following irradiation. The results indicated 100% survival for unirradiated and untreated control mice, and for mice treated with melatonin or solvent alone. Forty-five percent of mice exposed to 815 cGy radiation alone, and 50% of mice pretreated with solvent and irradiated with 815 cGy were alive at the end of 30 days. Irradiated mice which were pretreated with 125 mg/kg melatonin exhibited a slight increase in their survival (60%) (p=0.3421). In contrast, 85% of irradiated mice which were pretreated with 250 mg/kg melatonin were alive at the end of 30 days (p=0.0080). These results indicate that melatonin (at a dose as high as 250 mg/kg) is non-toxic, and that high doses of melatonin are effective in protecting mice from lethal effects of acute whole-body irradiation.     

Skin response to X-irradiation in the guinea-pig.

Skin reaction to X-irradiation has been studied in the albino guinea-pig; early response in limited-field irradiations of the flank is comparable to that commonly seen in rodents, swine and man, and is dose-dependent with a dynamic range from mild erythema to moist desquamation. The peak early skin reaction is seen between 14 and 21 days after irradiation, and declines before 30 days except at the highest doses used. Fractionation of the X-ray dose at 24 hours results in a 'sparing' of about 340 rad. Permanent partial epilation is detectable at doses in excess of 1400 rad, and complete epilation at 1 year occurs in 50 per cent of irradiated fields at 1740 rad. Twenty-four hour two-dose fractionation results in a 'sparing' of about 500 rad for epilation. Palpable dermal 'fibrosis' is detectable at 3 months after irradiation in fields given more than 2070 rad, and at 1 year after irradiation in fields given more than 1800 rad; 50 per cent of fields showed palpable 'fibrosis' at 1 year at 1930 rad. Unlike domestic swine and man, skin fields in the guinea-pig showed no dimensional contraction after X-ray doses which produced gross early skin damage.