The interaction between X-rays and 3 MeV neutrons in the skin of the mouse foot

Mouse feet were irradiated with mixtures of 3 MeV neutrons and 140 kVp X-rays given simultaneously or within 24 hours of each other. The effects of different treatments were contrasted by comparing the doses required to give equal skin reactions. Irradiation was given as 1, 2, 4 or 8 equal fractions, in order to assess r.b.e. and the shapes of the underlying dose-response curves for mixed beams over a wide range of dose per fraction. All dose-effect curves were well fitted by a linear-quadratic (alpha, beta) model. For X-rays and neutrons given simultaneously, the linear coefficient (alpha) decreased by a factor of 4.80 while the quadratic coefficient (beta) increased by a factor of only 1.44 when the proton contamination in the beam increased from 11 to 100 per cent, with alpha/beta changing from 95.0 to 13.8. The data from simultaneous X-ray and neutron irradiation were consistent with full interaction of those effects from the two radiations which give rise to the total quadratic component of effect. When the two radiations are separated by up to 24 h, this interaction decreases but does not entirely disappear.     

The effect of sequential irradiation with X-rays and fast neutrons on the survival of V79 Chinese hamster cells

V79 Chinese hamster cells have been exposed to X-rays or fast neutrons or to the two radiations given sequentially. Cells exposed to a priming dose of X-rays and then exposed immediately to a series of neutron doses regard the X-ray dose as equivalent to a neutron dose giving the same surviving fraction (iso-effective). If the cells are exposed to a neutron dose followed by X-rays the resulting survival is higher than would be obtained if the primary dose had been an iso-effective X-ray dose. However, it is lower than would be expected if the two radiations acted independently. The results imply that there is interaction between the damage caused by X-rays and fast neutrons. If the two radiations are given 3 hours apart they act independently.     

Neutron RBEs for mouse skin at low doses per fraction

The effect of low doses of 240 kVp X rays or of 3 MeV neutrons has been investigated using skin reactions on mouse feet as the biological system. Eight or nine repeated small doses of radiation were used, followed by graded "top-up" doses to bring the reactions into a detectable range. By comparing dose-response curves, the RBE has been determined for neutron doses per fraction ranging from 0.25-1.0 Gy. The data are consistent with a limiting RBE of between 7 and 10 at very low doses. A review of other published RBE values for low doses per fraction shows a wide range of RBEs . Very few studies show a plateau value for the RBE. These findings are more consistent with dose-response data that fit a linear-quadratic model than with a multitarget single-hit model.     

Recovery of murine lens epithelial cells from single and fractionated doses of X rays and neutrons

Subpopulations of mouse lens epithelial cells, differing in proliferative status, were irradiated with either X rays or fission spectrum neutrons given singly or in four weekly fractions. After various times, epithelia were mitogenically stimulated by wounding and DNA synthesis responses were determined by incorporation of [3H]thymidine. At 1 h following both X and neutron irradiations, significant suppression of the wound response after single doses and a sparing effect of fractionation were evident in both the mitotically quiescent and the slowly proliferating subpopulations. At 1 week following single or fractionated doses of both radiations, recovery was evident in both subpopulations. By 4 weeks, the quiescent subpopulation showed significant recovery after both single and fractionated doses of X rays or neutrons. In contrast, a marked decreased ability to respond after neutron irradiation and, in addition, a significant enhancement effect of neutron fractionation were observed for the slowly proliferating subpopulation. Per gray, neutrons were about 7.5 times more effective than X rays as a single dose and 25 times more effective in four equal fractions. The shift from an initial sparing to a final enhancing effect of neutron fractionation for the slowly proliferating subpopulation has importance for understanding divergent early and late radiation responses following dose fractionation.     

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.     

Evidence for a constant repair capacity over 20 fractions of X-rays

The response of mouse skin to small X-ray doses (less than or equal to 4.5 Gy) has been studied using gross skin reactions to obtain dose response curves. In order to study such small doses without giving a very prolonged series of fractions, the 'top-up' or partial tolerance design of experiment has been used. Eight or twenty priming fractions of X-rays have been 'topped up' with graded single doses of 3 MeV neutrons to bring the sub-threshold X-ray damage into the measurable range. By this means the effect of the same dose could be studied, when given either 8 or 20 times. The data were analysed to see whether each fraction was equally effective in the long or short fractionation schedules. The effectiveness remained constant, showing no significant loss of the repair capacity as the fractionation schedule proceeded.     

Selection of genetically distinct,rapidly proliferating clones does not contribute to repopulation during fractionated irradiation in FaDu squamous cell carcinoma

Acceleration of clonogen repopulation during fractionated irradiation after about 3 weeks has been demonstrated previously in FaDu human squamous cell carcinoma in nude mice (Petersen et al., Int. J. Radiat. Oncol. Biol. Phys. 51, 483-493, 2001). Selection of genetically distinct, rapidly proliferating clones might contribute to this phenomenon. To address this question, three sublines (R1-R3) were established from FaDu tumors that recurred locally after fractionated irradiation. The tumors were retransplanted and irradiated under clamp hypoxia with single doses or with 18 x 3 Gy within 18 days or 36 days, followed by graded top-up doses. The results were compared with data obtained after the same treatment schedules in the parental tumor line. Histologies, tumor volume doubling times, and potential doubling times of FaDu sublines R1-R3 were not different from those of the parental line. The radiation dose required to control 50% of the tumors (TCD(50)) after single-dose irradiation of 37-38 Gy was the same for the FaDu sublines R1-R3 and the parental tumor. The top-up TCD(50) values for the FaDu sublines R1-R3 after 18 fractions within 36 days were 14-17 Gy higher than those after 18 fractions within 18 days, indicating significant repopulation. The magnitude of this effect was not significantly different between the sublines R1-R3 or between these sublines and the parental FaDu tumors. The results indicate that selection of genetically distinct, rapidly proliferating clones does not contribute to the acceleration of repopulation during fractionated irradiation in poorly differentiated FaDu tumors.     

Repopulation in murine skin after X-ray treatments with multiple fractions per day

The kinetics of repopulation of clonogens in skin after fractionated X-ray exposures was studied in a series of experiments using a top-up design. The feet of mice were exposed to small X-ray doses (1.5 or 2 Gy), given two or three times a day on consecutive days with a minimum interfraction interval of 8 h. A single top-up dose of d(4)-Be neutrons was then given at various intervals after the last X-ray fraction, typically on Days 1,4,8, 15, and 19. The acute skin reaction produced was scored an analyzed by both a standard 23-day averaging and a 7-day averaging procedure. Either method gave similar results and led to the same conclusions. The amount of top-up dose needed to produce a fixed skin reaction was used as a measure of the net effect of the X-ray treatments. This net effect is a result of the initial reduction in skin clonogens due to X rays, and their repopulation before the top-up dose was given. Repopulation was not detected during any of these courses of fractionated treatment, up to an overall time of at least 12 and possibly 16 days. On completion of X-ray schedules lasting 6-16 days, repopulation started 4 days later. In contrast, this delay lengthened to approximately 8 days for shorter overall treatment times of 3-4 days. Once repopulation started, it proceeded rapidly over 11 days so that by 15 days after the cessation of X rays, the skin was restored almost to its normal state with respect to radiosensitivity. The residual damage from Day 15 to Day 19 postirradiation was 3-13% of a full-effect level. The rate of repopulation can be expressed as a clonogen doubling time (Tclon), assuming that an average skin reaction of 1.5 is equivalent to a clonogen surviving fraction of 1.7 x 10(-5). Tclon varied inversely with the amount of initial damage inflicted by the X rays, with the shortest values (1-1.3 days) seen following X-ray doses that gave an initial damage level of 60-80% of full effect. These data are consistent with a hypothesis that damage is "sensed" only 10-12 days after the first X-ray fraction, which provides the stimulus for repopulation of the target cells in the basal layer, the keratinoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal damage in the mouse: the effect of d(4)-Be neutrons

A further study on the response of the mouse kidney to d(4)-Be neutrons (EN = 2.3 MeV) is described. The results confirm and augment the work published previously by Stewart et al. [Br. J. Radiol. 57, 1009-1021 (1984)]; the present paper includes the data from a "top-up" design of experiment which extends the measurements of neutron RBE (relative to 240 kVp X rays) down to X-ray doses of 0.75 Gy per fraction. The mean RBE for these neutrons increases from 5.8 to 7.3 as X-ray dose per fraction decreases from 3.0 to 1.5 Gy in the kidney. This agrees with the predictions from the linear quadratic (LQ) model, based on the renal response to X-ray doses above 4 Gy per fraction. The mean RBE estimate from a single dose group at 0.75 Gy per fraction of X rays is, however, 3.9. This is below the LQ prediction and may indicate increasing X-ray sensitivity at low doses. Data from this study and from those published previously have been used to determine more accurately the shape of the underlying response to d(4)-Be neutrons; an alpha/beta ratio of 20.5 +/- 3.7 Gy was found. The best value of alpha/beta for X rays determined from these experiments was 3.04 +/- 0.35 Gy, in agreement with previous values.     

Changes in bromodeoxyuridine labeling index during radiation treatment of an experimental tumor

Cell proliferation kinetics in a spontaneous mouse fibrosarcoma (FSaII) growing in C3H mice has been studied by in vivo pulse labeling of cells synthesizing DNA with bromodeoxyuridine (BrdUrd). A monoclonal antibody to BrdUrd and flow cytometry were used to quantify these cells. Labeling indices (LI) were measured before and after radiation. Unirradiated 10-mm tumors had a mean LI of 17.5%. After a single dose of 20 Gy there was depression of LI after 1 day followed by a rapid increase to greater than control values after 5 days. Analysis performed after five fractions showed that LI was dependent on the dose per fraction and interval between fractions. After 5 and 7 Gy/fraction LI remained similar to control values during daily fractionation but was significantly depressed after twice daily fractionation. With doses greater than 10 Gy/fraction there was marked depression of LI using both fractionation schedules. These changes in LI correlated well with changes in tumor volume after radiation. Tumors were also biopsied after 5 fractions of a 20-fraction course to see if LI would predict for tumor control. LIs of greater than or equal to 10% were associated with lack of tumor control at 90 days while all controlled tumors had a significant depression of LI. Changes in LI after radiation were a reasonable indication of the amount of repopulation occurring and might be useful in selecting patients for altered fractionation schedules.     

Proliferation kinetics of NK/Ly tumor cells after single and fractionated irradiation with fast electrons]

With single irradiation, the inhibition of tumor cell division and DNA synthesis was more pronounced than with fractionation irradiation. In fractionated schedules, the dose increase per fraction, and the interval prolongation between fractions, with decrease of the number of fractions (within the same time of irradiation), enhanced the effect of radiation. Yield of pahologic mitoses and extent of morphologic injury of cells was less expressed with fractionated irradiation and did not depend on the schedule of fractionation of the total dose.     

The use of 'top-up' experiments to investigate the effect of very small doses per fraction in mouse skin

The partial tolerance type of 'top-up' experiment has been investigated to determine the resolution of this approach for studying the damage to mouse skin from very small doses of X-rays and neutrons. The effect of 20 fractions, each as small as 0.10 Gy of X-rays or of 0.05 Gy of neutrons, can be detected if 3 MeV neutrons are used as the 'top-up' reference radiation. This capability results from the almost linear underlying dose-response curve and highly reproducible dose-effect relationship for the low energy neutrons. The data fit the linear quadratic model of dose fractionation for X-rays down to fractional doses of 0.75 Gy, but at lower doses there is a trend towards an increase in the skin radiosensitivity. Modelling shows that this might be consistent with a sub-population of the cells showing an exceptional radiosensitivity, and a replenishment of this subpopulation occurring in the 8 h between small dose fractions. More experiments are needed at very low doses in order to confirm this hypothesis for skin and for other tissues.     

Repair but not potentiation observed in mouse lung irradiated with neon ions

The lungs of mice were irradiated with 1, 4, or 7 fractions of X rays or neon ions in a 4-cm spread Bragg peak. Lung function as a function of total radiation dose was tested at 7 and 12 months after irradiation by measuring the resting breathing rate in a whole-body plethysmograph. The isoeffect doses increased sequentially with X rays for 1 through 4 to 7 fractions, demonstrating repair of sublethal radiation injury as previously reported. There was also a significant increase of isoeffect dose with neon ions between 1 and 4 fractions but no further increase at 7 fractions. Thus repair instead of potentiation of radiation injury in lung clearly occurred after neon ion irradiation. The effectiveness of neon ions appeared to be closer to that of neutrons with a mean energy of 8 meV than those with a mean energy of 2.3 meV.     

Ex vivo determination of the effect of whole-body exposure to fast neutrons on murine spleen cell viability and apoptosis

The effects of high-linear energy transfer (LET) radiations on lymphoid tissues and lymphocytes are not well understood. As a first approach to delineate these effects, the present work was conducted to assess the effects of high-LET radiations on murine spleen cells ex vivo and in vitro. BALB/c mice were irradiated whole-body with 65 MeV neutrons or 15 MV X rays at doses ranging from 0.2 to 3 Gy. Spleens were removed 1 day postirradiation and weighed, and single cell suspensions were prepared and cultured for several days. Apoptosis occurring in vitro was determined at different times by flow cytometry analysis of cells labeled with propidium iodide. It was found that irradiation with fast neutrons reduced spleen weight and cellularity to a greater extent than photons. Considering the spleen cellularity as end point, the relative biological effectiveness (RBE) of fast neutrons was 2. However, for both modes of irradiation, apoptosis of recovered spleen cells in vitro increased as a function of dose and the duration of culture. The level of apoptosis occurring at various times postirradiation was found to be identical for high- and low-LET radiations. Taken together, these results suggest that external as well as cellular factors might differentially modulate the sensitivity of lymphocytes to fast neutrons and photons.     

Relative cataractogenic effects of X rays, fission-spectrum neutrons, and 56Fe particles: a comparison with mitotic effects

The eyes of Sprague-Dawley rats were irradiated with doses of 2.5-10 Gy 250-kVp X rays, 1.25-2.25 Gy fission-spectrum neutrons (approximately 0.85 MeV), or 0.1-2.0 Gy 600-MeV/A 56Fe particles. Lens opacifications were evaluated for 51-61 weeks following X and neutron irradiations and for 87 weeks following X and 56Fe-particle irradiations. Average stage of opacification was determined relative to time after irradiation, and the time required for 50% of the irradiated lenses to achieve various stages (T50) was determined as a function of radiation dose. Data from two experiments were combined in dose-effect curves as T50 experimental values taken as percentages of the respective T50 control values (T50-% control). Simple exponential curves best describe dose responsiveness for both high-LET radiations. For X rays, a shallow dose-effect relationship (shoulder) up to 4.5 Gy was followed at higher doses by a steeper exponential dose-effect relationship. As a consequence, RBE values for the high-LET radiations are dose dependent. Dose-effect curves for cataracts were compared to those for mitotic abnormalities observed when quiescent lens epithelial cells were stimulated mechanically to proliferate at various intervals after irradiation. Neutrons were about 1.6-1.8 times more effective than 56Fe particles for inducing both cataracts and mitotic abnormalities. For stage 1 and 2 cataracts, the X-ray Dq was 10-fold greater and the D0 was similar to those for mitotic abnormalities initially expressed after irradiation.     

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.     

Comparison of recovery from potential mitotic abnormality in mitotically quiescent lens cells after X, neutron, and 56Fe irradiations

After exposure to various doses of 250 kVp X radiation, 0.85 Me V fission spectrum neutrons, or 600 MeV/A iron (Fe) particles, mitotically quiescent rat lens cells showed no visible evidence of radiation injury. However, following the mitogenic stimulus of wounding, mitotic abnormalities became evident when responding cells entered mitosis. Latent damage and recovery therefrom were monitored at 3, 7, 14, and 28 days after irradiation. Following doses of 1 to 10 Gy of X radiation, the recovery rate, indicated by a decrease in abnormalities with time, was proportional to dose, and the dose-effect slope decreased exponentially with time. Virtually no recovery occurred during the 28 days after 1.25 to 2.25 Gy of fission neutron radiation. After doses of 0.5 to 3.0 Gy of Fe particles, an increased expression of mitotic damage or recovery than recovery occurred. As a consequence of the differing patterns in time for expression of damage or recovery following X rays and the high-LET radiations, the relative biological effectiveness (RBE) increased from 3.6 to 16 for neutrons and from 2 to 10 for Fe particles over the 28-day observation period.     

Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.     

The F value for chromosome aberrations in atomic bomb survivors does not provide evidence for a primary contribution of neutrons to the dose in Hiroshima.

Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) proposed that the ratio of interchromosomal to intrachromosomal exchanges, termed the F value, can be a cytogenetic fingerprint of exposure to radiations of different linear energy transfer (LET). Using published data, they suggested that F values are over 10 for low-LET radiations and approximately 6 for high-LET radiations. Subsequently, as F values for atomic bomb survivors were reported to be around 6, Brenner suggested that the biological effects of atomic bomb radiation in Hiroshima are due primarily to neutrons. However, the F values used for the survivors were means from individuals exposed to various doses. As the F-value hypothesis predicts a radiation fingerprint at low doses, we analyzed our own data for the survivors in relation to dose. G-banding data for the survivors showed F values varying from 5 to 8 at DS86 doses of 0.2 to 5 Gy in Hiroshima and around 6 in Nagasaki with no evidence of a difference between the two cities. The results are consistent with our in vitro data that the F values are invariably around 6 for X and gamma rays at doses of 0.5 to 2 Gy as well as two types of fission-spectrum neutrons at doses of about 0.2 to 1 Gy. Thus, apart from a possible effect at even lower doses, current data do not provide evidence to support the proposition that the biological effects of atomic bomb radiation in Hiroshima are caused mainly by neutrons.