Tumor· and Normal Tissue Response to irradiation In Vivo: Variation with Decreasing Dose Rates 1
Radiation Biology
Karen K. Fu, M.D., Theodore L. Phillips, M.D., Lawrence J. Kane, B. S., and Vernon Smith, M.S. Continuous irradiation in vivo at dose rates ranging from 0.54 to 274 rads per minute was performed in mice using the EMT6 tumor, the gut clone system, and the bone-marrow CFU system. A progressive increase in 0 0 and decrease in were seen with decreasing dose rates in the EMT6 tumor and the gut clone system. The 00 showed little change in the bone-marrow CFU system. These findings are related to the O2 - 0 1 values obtained trom split-dose experiments at conventional dose rates. The results do not fit the various mathematical models proposed for correlating the effects of continuous low-dose-rate irradiation and acute exposure.
n
Neoplasms, experimental. Radiobiology, bone-marrow studies. Radiobiology, cell and tissue studies • Radiobiology,time-dose studies
INDEX TERMS:
Radiology 114:709-716, March 1975
HE EFFECT of the dose rate is an important factor in the radiation response of normal and tumor tissues and is of interest to radiobiologists as well as radiotherapists. Inactivation of cellular reproductive ability varies considerably at a dose rate of between 1 and 100 rads per minute; the number of cells killed decreases as the dose rate is lowered, due partly to repair of sublethal damage during the course of protracted irradiation (13). Since the width of the shoulder (D q ) of the cell survival curve following an acute exposure is a measure of such repair, one should be able to predict the effectiveness of changes in the dose rate of continuous irradiation from the shapes of the survival curves following acute exposures: if the shoulder is wide (large Dq ), the radiation response would vary considerably with the dose rate, while conversely a narrow shoulder (small Dq ) would indicate a comparatively stable radiation effect. Previous reports regarding the effects of continuous irradiation at low dose rates on normal and tumor tissues show conflicting results. In vitro irradiation of HeLa and Chinese hamster cells over a wide range of dose rates showed significant change of Do with dose rate (1, 15). Continuous irradiation in vivo at low dose rates resulted in an increase in Do and a decrease in the extrapolation number in rat rhabdomyosarcoma (21) and P388 leukemic cells (2, 3), whereas data on KHT sarcoma (19) and NCTC 2472 fibrosarcoma and bonemarrow colony-forming units (CFU) (8) showed no significant difference in response between acute and protracted irradiation. Using whole-body irradiation, Sacher and Grahn (12, 37) investigated the effects of fractionation and various dose rates on the lethality and late ef-
T
fects of irradiation in mice, and Sacher (36) has done extensive mathematical analysis of the data. There have been several attempts to predict or evaluate the biological effects of continuous irradiation at low dose rates using different theoretical and mathematical analyses. Based on the data of Elkind and Sutton (6), Lajtha and Oliver (23) described a mathematical model for estimating the cumulative effective dose of a course of continuous irradiation at a constant dose rate. Liversage (28) proposed a general formula for equating protracted and acute regimens which is independent of the size and shape of the shoulder of the cell survival curve and thus applicable to all tissues. Kirk et al. (22) developed a method for assessing and comparing the biological effects of continuous irradiation with constant dose rate based on the isoeffect curves described by Paterson (32) and Ellis (7). We performed a number of experiments with 8- to 12-week-old BALB/c female mice to study the varying responses of normal and tumor cells to acute and continuous irradiation in vivo at decreasing dose rates using the gut clone system, the bone-marrow CFU system, and the EMT6 mouse mammary tumor and to test the validity of the mathematical models formulated by various authors for correlating the effects of acute and continuous irradiation. MA TERIALS AND METHODS
Biological Assays EMT6 tumor: The characteristics of the EMT6 tumor and its tissue-culture derivatives have been described by Rockwell et al. (35). It grows in BALB/c female mice and is readily transplantable. It clones as discrete colo-
1 From the Section of Radiation Oncology (K.K.F., T.L.P., V.S.) and the Laboratory of Radiobiology (K.K.F., T.L.P., L.J.K.), University of California School of Medicine, San Francisco, Calif. Presented at the Fifty-ninth Scientific Assembly and Annual Meeting of the Radiological Society of North America, Chicago, 111., Nov. 25-30, 1973. This work was performed under the auspices of the U. S. Atomic Energy Commission and was supported in part by NIH training grant CA05177 and Clinical Cancer Center grant CA-11067. sjh
709
710
KAREN K .
lr--_-+-_ 137C.
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Fu AND OTHERS
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March 1975
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Fig. 1 Cross section of the 13 7CS irradiator, showing the positions of the radiation source, lead attenuators, and mice in rotating Lucite boxes.
nies in tissue culture and can be used for in vitro assay after treatment in situ. In vitro assay of the EMT6 tumor was carried out immediately after total-body irradiation, using an assay technique described previously (10). Briefly , irradiated mice bearing a 500- to 1000-mm3 transplanted EMT6 tumor in the flank area were sacrificed. The tumor was excised, trypsinized, and suspended in Ca++-free Eagle's Minimal Essential Medium. A known number of viable cells was plated in plastic tissue-culture flasks and incubated at 37 0 C in an atmosphere of 5% CO2 and 95% room air. Nine days later the clones were fixed, stained, and counted. Cell survival was calculated from the ratios of the plating efficiencies of the irradiated tumors and unirradiated controls. The plating efficiency for unirradiated control tumors was 35 ± 10%. Bone-marrow CFU: The technique of bone-marrow spleen colony assay has been described by McCulloch and Till (30). CFUs were irradiated in vivo and injected into recipient mice that had received 700 rads of totalbody radiation with 230-kVp x rays. Seven days later the number of colonies on the spleen surface was scored. Surviving fractions were based on the yield of CFUs per 105 nucleated cells from the femora of unirradiated animals. Gut clone system: Survival of jejunum crypt cells was determined by the microcolony assay technique described by Withers and Elkind (41). Three and onehalf days after total-body irradiation, mice were sacrificed and the jejunum was removed for histological examination. When irradiation was prolonged up to 86 hours, the mice often died earlier than three and onehalf days after the cessation of irradiation; hence it was necessary to shorten the interval between irradiation and sacrifice to two or three days when using a dose rate of 0.92 or 0.54 rads per minute. Mice were housed 5 per cage before whole-body irradiation and 4 per cage afterward. The average number of crypts per circumference in the jejunum of unirradiated control BALB/ c female mice
(1)
where x is the number of regenerating crypts in each transverse section (41). Method Of Irradiation
Pairs of mice were irradiated in rotating Lucite boxes inside a self-contained cesium irradiator at dose rates of 274, 36, 4.5, 0.92, or 0.54 rads per minute. Low dose rates were achieved with various thicknesses of lead attenuators (Fig. 1) and dosimetry was checked with thermoluminescent dosimeters. The effect of attenuators on radiation quality was determined with a combination ionization chamber/silicon p-n diode assembly calibrated with beams with known half-value layers. Since the ionization chamber had a relatively flat energy response while the diode had an increased response at lower energies, the ratio of the two readings served as an indication of quality. During protracted irradiation, mice were given food in the Lucite boxes and taken out for 15 to 30 minutes twice a day to drink water ad libitum. To determine the peak of recovery following an acute exposure, split-dose experiments were carried out at 274 rads per minute, using two doses of 500 rads each for EMT6 tumors, two doses of 150 rads each for bone-marrow CFUs, and a first dose of 700 rads.and a second dose of 500 rads for jejunum crypt cells. The second dose was delivered at 0, 2, 4, 6, 8, 10, 12: or 24 hours after the first dose. Subsequent split-dose experiments were performed with fixed first doses and graded second doses given at the peak of recovery from the first dose. In order to determine whether prolonged confinement had any effect on plating efficiency, spleen colony formation, or jejunal crypt regenerating ability, control animals were subjected to the same conditions of restraint as the irradiated animals, while a separate group of controls was given a test dose of 274 rads per minute at the end of various periods of restraint to see whether the radiation response was affected. RESULTS
Continuous Irradiation
Survival curves were calculated using least-squares regression analysis. Those represent ing EMT6 tumors subjected to continuous irradiation at dose rates ranging from 0.54 to 36 rads per minute showed a marked change in shape from the biphasic acute exposure curve expected for tumor cells irradiated at the conventional dose rate of 274 rads per minute (Fig. 2); rather, the Do increased and decreased with decreasing dose rates (TABLE I). The dose response curves of jejunum crypt cells irra-
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Vol. 114
TUMOR AND NORMAL TISSUE RESPONSE TO IRRADIATION
711
In Vivo
Radiation Biology
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Jejunal crypt cells
Bone-marrow CFUs
Variation of Do with Dose Rate Dose Rate (rads/rnin.)
Do (rads)
274 36 4.5 0.92 0.54
160/360 366 460 662 712
274 36 4.5 0.92 0.54
65 153 180 272 .740
274 36 4.5 0.92 0.54
80 113 87 76/95 76/95
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diated continuously at low dose rates also showed a significant increase in Do with decreasing dose rates (Fig. 3), and the intercept of the dose decreased as well. The ratio of Do for continuous irradiation at low dose rates (Dod to Do for acute exposures (Dos) showed similar changes in magnitude with variation of dose rate for the gut clone system and the EMT6 tumor in the range of 0.92 to 274 rads per minute (TABLE I). With a further decrease to 0.54 rads per minute, the Do increased elevenfold over that of the acute exposures, probably due in part to cell proliferation and decreased cell cycle time during protracted irradiation at this dose rate. The response of bone-marrow CFUs to continuous irradiation with decreasing dose rates differed markedly from those of the EMT6 tumor and jejunum crypt cells (Fig. 4). When the dose rate was decreased to 36 rads per minute, the Do increased to 110 rads while remained unchanged. With continuous irradiation at 4.5 87 rads per minute, the dose response curve (Do
n
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Fig. 3. Response of mouse jejunal crypt cells to 137CS irradiation with decreasing dose rates. Symbols represent mean surviving cells per circumference of at least 8 mice assayed separately; bars represent standard errors of the mean.
rads) differed very little from that for irradiation with 80 rads): but when the dose acute exposures (Do rate was reduced to less than 1 rad per minute, the dose response curves unexpectedly became biphasic. Since there seemed to be little if any difference in response whether the dose rate was 0.92 or 0.54 rads per minute, the points at these lower dose rates were fitted to a common dose response curve, which exhibited increased sensitivity in the low-dose region (Do =
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KAREN K. Fu AND OTHERS
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March 1975
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Fig. 4. Response of mouse bone-marrow CFUs to i37CS irradiation with decreasing dose rates. Symbols represent the mean survival of bone-marrow CFUs of 2-16 mice assayed separately; bars represent standard errors of the mean.
Fig. 5. Response of EMT6 tumor in vivo to a single dose and second doses given four hours atter an initial dose of 500 rads at a rate of 274 rads per minute. Symbols represent the mean survival of 2-4 tumors; bars represent standard errors of the mean.
76 rads) and decreased sensitivity in the high-dose region (Do = 95 rads). With continuous prolonged irradiation at these low dose rates, bone-marrow cellularity fell to about 80% after 9 hours and about 50% after 15.5 hours. When the survival fraction was calculated based on the ratio of the number of spleen colonies yielded per irradiated and control femora, a similar biphasic change in the dose response curve was observed, indicating that the reduction in cellularity did not. introduce an error into the estimation of cell survival.
second doses (Fig. 7); however, this was not surprising in view of the small 02 - D1 value expected for bonemarrow CFUs.
Split-Dose Irradiation
Split-dose experiments indicated that the first peak of recovery occurred four hours after the first dose for EMT6 tumors and bone-marrow CFUs and six hours for jejunum crypt cells. Subsequent split-dose experiments with graded second doses given four to six hours after the first dose showed a O2 - 0 1 value2 of 330 rads for the EMT6 tumor, estimated at the survival level of 0.1 (Fig. 5), and 195 rads for jejunum crypt cells at the level of 10 surviving cells per circumference (Fig. 6). Although split-dose experiments with two equal doses showed a survival ratio of 1.5 for bone-marrow CFUs at four hours, which would suggest a Dq or O2 - 0 1 value of approximately 32 rads (Oq = Do In n), this was not substantiated by split-dose experiments with graded 2 D, is the single dose and O2 the total of two fractionated doses separated by a sufficient time for repair of sublethal damage to produce the same level of damage. O2 - 0, has been found to approximate the value of Oq (14).
Determinations of Radiation Beam Quality
Assessment of the quality of the radiation beam indicated that the addition of lead attenuators resulted in increased scatter and hence softening of the beam. The energy of the beam ranged from 600 to 280 keV depending on the thickness of the attenuators. Although the radiobiological effectiveness (RBE) changes along with the beam energy, the degree of change is small in this energy range: it increases only 10% with x rays having a mean photonenerqy of 100 keV, based on the results of our in vitro irradiation of EMT6 cells with 230kVp x rays at 131 rads per minute and 137CS at 274 rads per minute. The results obtained were not corrected for the possible small change in RBE. Res~amtofCon~oIAn;ma~
Restraint of controls under the same conditions as irradiated animals for various periods of time up to 96 hours caused no significant change in the plating efficiency of EMT6 tumors, the number of jejunal crypts per circumference, or the spleen colony-forming ability of bone-marrow CFUs compared with unrestrained controls, nor was there any significant change in radiation response following prolonged restraint (TABLE II).
TUMOR AND NORMAL TISSUE RESPONSE TO IRRADIATION
Vol. 114
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DOSE (RADS) Fig. 6. Response of mouse jejunal crypt cells to a single acute dose and second doses given six hours after an initial dose of 700 rads at a rate of 274 rads per minute. Symbols represent the mean surviving cells per circumference of 4-8 mice; bars represent standard errors of the mean.
Fig. 7. Response of bone-marrow CFUs to a single dose and second doses given four hours after an initial dose of 150 rads at a rate of 274 rads per minute. Symbols represent the mean survival of bone-marrow CFUs of 2-8 mice; bars represent standard errors of the mean.
Table II: Plating Efficiency of EMT6 Tumor and Bone-Marrow CFUs, Number of Crypts per Circumference, and Response to Acute Radiation Exposure in Restrained Mice
ous irradiation at low dose rates: dose rate, capacity for repair of sublethal damage, cell cycle time, variation of radiosensitivity during the cell cycle, density distribution of radiosensitive and radioresistant cells, redistribution of cells over the different phases of the cell cycle, proliferation of clonogenic cells, cell loss, oxygenation, and .reoxygenation during continuous irradiation. Cell reproductive deaths due to irradiation may result from a single lethal event (single-hit phenomenon), an accumulation of several sublethal events (multihit phenomenon), or, most likely, a combination of both. Although single lethal events are independent of the dose rate, repair of the sublethal component of damage during protracted irradiation would lead to fewer cell deaths with decreasing dose rates up to a limiting dose rate below which little change would occur. With the exception of bonemarrow CFUs, most mammalian tumor and normal tissue cells studied showed an increase in Do with a decrease in dose rate (Fig. 8). It has been suggested that the total dose received by a cell during the cell cycle is an important factor in determining its response to prolonged exposure at low dose rates (5, 17), with the radiation effect being greater for cells with long cell cycle times. This is complicated further by possible changes in cell cycle time during continuous irradiation (4, 9, 26, 27, 29); for example, the generation cycle of mouse duodenal crypt cells decreased when they were exposed to chronic gamma ir-
EMT6 Tumor
Duration of Restraint (hr.) 7 48 96
Plating Efficiency 0.30 0.25 0.38
Surival
p 1,000 rads 0.067 0.07 0.053
Jejunal Crypts
Duration of Restraint (hr.) 8 48
72
Number of Crypts per Circumference 106.4 104.4 103.0
Survival ± S. E. p 1,100 rads 10.6 ± 1.1 7.4±1.0 8.4 ± 1.9
Bone-Marrow CFUs
Duration of Restraint (hr.) 2 5 15
Plating Efficiency 3.9 X 10- 4 5.2 X 10- 4 3.8 X 10- 4
Survival ± S. E. p 150 rads 0.247± 0.04 0.187± 0.03 0.194± 0.03
DISCUSSION
The effect of the dose rate on radiation response varies with the tissue. Continuous irradiation at 0.54 to 36 rads per minute produced marked variation of response in the EMT6 mouse mammary tumor, mouse jejunum crypt cells, and bone-marrow CFUs. There are many complicating factors that might influence the response of different normal and tumor tissues to continu-
714
KAREN K.
Fu AND OTHERS
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Fig. 8. Variation of Do with dose rate. • = EMT6 mouse mammary tumor; • = mouse jejunal crypt cells; A = mouse bone-marKHT sarcoma row CFUs; 0 = rat rhabdomyosarcoma (21); A P388 leukemia cells; 0 HeLa cells in vitro (15); • = Chi(19); 0 nese hamster cells in vitro (1).
=
=
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radiation at a rate of 0.91-1.09 rads per hour (9, 26, 27). It has been shown that radiosensitivity depends on the position of the cell in the cell cycle (11, 31, 38); thus the distribution of cells with different radiosensitivities in the tumor or normal tissue at the beginning of continuous low-dose-rate irradiation and subsequent redistribution of surviving cells over the different phases of the cell cycle (1, 8) might influence the variation of response to continuous irradiation. With progressively decreasing dose rate and increasing exposure time, clonogenic cells may regenerate and proliferate during irradiation (1, 4, 24, 25, 39) at a rate which depends on the cell cycle time and cell loss factor, which in turn may change during continuous low-dose-rate irradiation. Reoxygenation might take place during protracted tumor irradiation (19, 21), which would also influence the radiation response. All of these factors may be interdependent, and the relative importance of a given factor in determining the ultimate response to continuous low-dose-rate irradiation may vary in different tissues. Our studies demonstrated a marked difference in response between EMT6 tumors, jejunum crypt cells, and bone-marrow CFUs. Further wide variation in response, though not beyond the ranges we have shown, is demonstrated by the results of other experiments summarized in Figure 8. Both the EMT6 tumor and jejunum crypt cells showed relatively large O2 - 0 1 values, suggesting wide shoulders on the survival curves and relatively large capacities for repair of sublethal damage. It is not surprising that with decreasing dose rates, cell killing is less effec-
March 1975
tive and the Do increases in these cells. In contrast to the survival curve following acute exposures, that of EMT6 tumors given continuous low-dose-rate irradiation did not exhibit a biphasic effect. This type of change in the shape of survival curves following protracted irradiation has been demonstrated in rat rhabdomyosarcoma (21), KHT sarcoma (19), and NCTC fibrosarcoma (8). Two possibilities may account for this: (a) hypoxic cells may show less variation in response with dose rate, i.e., they do not repair sublethal damage, and/or (b) reoxygenation may occur during protracted irradiation, as suggested by data on KHT sarcoma (19) and rat rhabdomyosarcoma (21). The bone-marrow CFU results were both unexpected and puzzling. Our results differ from those obtained by Frindel et at. (8), who observed no significant difference between the effects of acute and continuous irradiation. Since bone-marrow CFUs showed little capacity for repair of sublethal damage, a significant change in response with decreaslnq dose rates would not be expected. Although there was a slight increase in Do when the dose rate was lowered from 274 to 36 rads per minute, a further decrease resulted in increased sensitivity in the low-dose region and slightly decreased sensitivity in the high-dose region with prolonged exposure time. Several factors might account for these changes, among them variation of sensitivity due to position in the cell cycle (18), redistribution of cells in the cell cycle (8), possible cell loss, and changes in the proportion of cycling stem cells before and during irradiation; however, further studies are necessary to elucidate the true underlying mechanisms. Although the survival curves of the EMT6 tumor and jejunum crypt cells are qualitatively consistent with the prediction of Lajtha and Oliver (23), they do not fit quantitatively with our estimates calculated using their proposed formula for correlating the effects of continuous irradiation with single acute exposures, assuming that the half-life of the radiation effect is 1.5 hours. Their analysis yields an effective dose of DE due to previous irradiation over time t in hours at a dose rate of d rads per hour defined as I
(2)
=
where I.L 0.693/half-life of damage in hours and DE is the equivalent dose for the same effect given at conventional dose rates (100-300 rads per minute). This assumes that any additional or subsequent dose undergoes exponential fading or repair. We have modifiedthis analysis to take into account the usual situation, in which part of the dose yields permanent damage while the other part yields damage that can be repaired (Appendix). If k is the fraction of the dose that is not repaired, (1-k) is the fraction that can be repaired, and ON is the nominal dose, the effective dose DE is given by
DE = kDN
+
d(! - k) [1 _ e-lJ.t] fJ.
(3)
Radiation Vol. 114
TUMOR AND NORMAL TISSUE RESPONSE TO IRRADIATION In Vivo
or, since
Table III:
(4) then
(5)
=
Biology
Effective Dose for EMT6 Mouse Mammary Tumor*
Sutviving Fraction
DN
d
DE
(rads)
(rads/rnin.)
(rads)
0.01 0.01 0.01 0.01
1,450 1,830 2,180 3,020
274 36 4.5 0.92
1,436 1,616 1,336 1,497
* Half-life (Tl/2) = 1.5 hours; k Table IV:
This formula was tested using experirtlental survival data for the EMT6 tumor and mouse jejunum crypt cells. The doses required to give a particular level of survival for the different dose rates, as read from the survival curves, were taken as values of ON and used in formula 5, and a value of DE was calculated for each value of ON. If the formula is valid, DE should be constant irrespective of the dose rate for a given cell system and level of survival. TABLE '" gives the test results for the EMT6 tumor, taking a survival value of 0.01 and assuming a half-life of 1.5 hours and k 0.475. The values for DE agree within ± 10%. TA~LE IV gives test results for the mouse jejunum crypt cells, taking 1 surviving cell per circumference as the survival level and assuming a half-life of 45 minutes and k = 0.42. The overall agreement is still about ± 10%. Our calculations suggest that the two major factors in cell survival following irradiation are the half-life of reparable damage and the fraction of damage that is reparable. These values appear to differ for EMT6 tumors and jejunal crypt cells, with both the half-life and the irreparable damage being less in the latter case. This is in agreement with Withers's results for jejunal crypt cells (40) and Homsey's finding of very rapid repair in the gut (20). Much additional information is needed in order to determine reasonable values for k and the halflife in different tissues and to relate these values to the nand Oq of the survival curves and ON/01 and O2 - 0 1 values for repair of tissues following fractionated irradiation (33). Our results also differed from what might be predicted from the method suggested by Kirk et al. (22), whose method was based on clinical impressions of connective-tissue tolerance; it is not surprising, therefore, that its application to other types of tissue and cell systems is limited. Liversage's formula (28) for correlating fractionated high-dose-rate and continuous low-dose-rate irradiation did not take into consideration the different survival curve shoulder widths of various types of cells. Our results suggest that response to continuous irradiation is at least partly influenced by the capacity for repair of sublethal damage, as reflected by the shoulder width (02 - 0 1 ) , and is reflected by the degree to which Do increases as the dose rate decreases. Thus it is evident that no single available formula can be used to adequately calculate equivalent doses for similar effects over a wide range of dose rates or be applicable to all types of cells. In clinical radiotherapy, continuous low-dose-rate irradiation with interstitial implants, intracavitary insertions,
715
= 0.475
Effective Dose for Mouse Jejunum Crypt Cells*
Surviving Cells per Circu mference
ON
d
(rads)
(rads/min.)
1 1 1
1,220 i,800
1
2,700
274 36 4.5 0.92
2,380
DE (rads)
1,218 1,484 1,169 1,127
* Half-life (TI/l) = 45 minutes; k = 0.42
or surface molds, either alone or in combination with external radiation therapy, has been used successfully in the treatment of many malignant tumors. Physically, the dose distribution favors both a higher total dose and a higher dose rate to the tumor; in addition, the biological advantage of decreased cell mortality with a lower dose rate in the adjacent normal tissue might be of advantage in the therapeutic ratio. Other factors such as the decreased oxygen enhancement ratio with lowdose-rate irradiation (16) and possible reoxygenation during protracted irradiation may also be advantages of interstitial and intracavitary radiotherapy. Our results with the EMT6 tumor support these potential advantages. External radiotherapy at low dose rates has recently been initiated (34), and early results are encouraging. The differences in response and the change in cellular kinetics between different normal and tumor tissues subjected to protracted low-dose-rate irradiation might be advantageous in the treatment of certain tumors. Although hemopoietic tissue, lymphomas, and leukemic bone marrow show no significant alteration in effect when exposed to low-dose-rate irradiation, other normal tissues such as the gastrointestinal tract may suffer less damage with this modality. However, any advantage gained with low-dose-rate external radiotherapy must be balanced against the practicality of the prolonged treatment time required for such therapy. ACKNOWLEDGMENTS: We are grateful to Mrs. Glenda Ross for her expert technical assistance and to Mimi Zeiger and Myra Goldring for their editorial and secretarial help.
Section of Radiation Oncology Universityof California School of Medicine San Francisco. Calif. 94143 REFERENCES 1. Bedford JS. Mitchell JB: Dose~rate effects in synchronous mammalian cells in culture. Radiat Res 54:316-327, May 1973 2. Berry RJ: Hypoxic protection and recovery in tumour cells irradiated at low dose-rates and assessed in vivo. Br J Radiol 41: 921-926, Dec 1968
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KAREN K.
Fu
3. Berry RJ, Cohen AB: Some observations on the reproductive capacity of mammalian tumour cells exposed in vivo to gamma radiation at low dose-rates. Br J Radiol 35:489-491, Jul 1962 4. Cairnie AB: Cell proliferation studies in the intestinal epithelium of the rat: response to continuous irradiation. Radiat Res 32: 240-264,Oct1967 5. Courtenay VC: Unpublished data; cited by LF Lamerton and VC Courtenay in The steady state under continuous irradiation. [In] Brown DG, Cragle RG, Noonan TR, ed: Proceedings of Symposium on Dose Rate in Mammalian Radiation Biology, April 29-May 1, 1968, Oak Ridge, Tenn. CONF-680410. Springfield, Va., US Dept Commerce, 1968, pp 3.1-3.12 6. Elkind MM, Sutton H: Radiation response of mammalian cells grown in culture. I. Repair of x-ray damage in surviving Chinese hamster cells. Radiat Res 13: 556-593, Oct 1960 7. Ellis F: The relationship of biological effects to dose-timefractionation factors in radiotherapy. [In] Ebert M, Howard A, ed: Current Topics in Radiation Research. Amsterdam, North-Holland, 1968, Vol 4, pp 357-397 8. Frindel E, Hahn G, Robaglia D, et al: Responses of bone marrow and tumor cells to acute and protracted irradiation. Cancer Res 32:2096-2103, Oct 1972 9. Fry RJM, Lesher S, Sallese A, et al: The generation cycle of duodenal crypt cells of mice exposed to 220 roentgens of cobalt60 gamma irradiation per day. Radiat Res 19:628-635, Aug 1963 10. Fu KK, Phillips TL, Rowe JR: The RBE of neutrons in vivo. Cancer 34:48-53, Jul1974 11. Gillette EL, Withers HR, Tannock IF: The age sensitivity of epithelial cells of mouse small intestine. Radiology 96:639-643, Sep 1970 12. Grahn 0, Sacher GA: Fractionation and protraction factors and the late effects of radiation in small mammals. [In] Brown DG, Cragle RG, Noonan TR, ed: Proceedings of Symposium on Dose Rate in Mammalian Radiation Biology, April 29-May 1, 1968, Oak Ridge, Tenn. CONF-680410. Springfield, Va., US Dept Commerce, 1968, pp 2.1-2.27 13. Hall EJ: Radiation dose-rate: a factor of importance in radiobiology and radiotherapy. Br J RadioI45:81-97, Feb 1972 14. Hall EJ: Radiobiology for the Radiologist. Hagerstown, Harper & Row, 1973, pp 108-109 15. Hall EJ, Bedford JS: Dose rate: its effect on the survival of HeLa cells irradiated with gamma rays. Radiat Res 22:305-3'15, Jun 1964 16. Hall EJ, Bedford JS, Oliver R: Extreme hypoxia; its effect on the survival of mammalian cells irradiated at high and low doserates. Br J RadioI39:302-307, Apr 1966 17. Hall EJ, Oliver R, Shepstone BJ, et al: On the population kinetics of the root meristem of Vicia faba exposed to continuous irradiation. Radiat Res 27:597-603, Apr 1966 18. Hellman S: X-irradiation of the hematopoietic stem cell compartment. [In] Radiation Effect and Tolerance, Normal Tissue. Basic Concepts in Radiation Pathology. Vol 6 of Frontiers of Radiation Therapy and Oncology, ed by JM Vaeth. Baltimore, Univ Park Press, 1972, pp 415-527 19. Hill RP, Bush RS: The effect of continuous or fractionated irradiation on a murine sarcoma. Br J RadioI46:167-174, Mar 1973 20. Hornsey S: Differences in survival of jejunal crypt cells after radiation delivered at different dose-rates. Br J Radiol 43:802806, Nov 1970 21. Kal HB, Barendsen GW: Effects of continuous irradiation at low dose-rates on a rat rhabdomyosarcoma. Br J Radiol 45:279283, Apr 1972 22. Kirk J, Gray WM, Watson ER: Cumulative radiation effect. Part II: Continuous radiation therapy-long-lived sources. Clin Radiol 23:93-105, Jan 1972 23. Lajtha LG, Oliver R: Some radiobiological considerations in radiotherapy. Br J RadioI34:252-257, Apr 1961 24. Lamerton LF: Cell proliferation under continuous irradiation. Radiat Res 27: 119-138, Jan 1966 25. Lamerton LF, Courtenay VC: The steady state under continuous irradiation. [In] Brown DG, Cragle RG, Noonan TR, ed: Proceedings of Symposium on Dose Rate in Mammalian Radiation BioI-
AND OTHERS
March 1975
ogy, April 29-May 1, 1968, Oak Ridge, Tenn. CONF-680410. Springfield, Va., US Dept Commerce, 1968, pp 3.1-3.12 26. Lesher S, Lamerton LF, Sacher GA, et al: Effect of continuous gamma irradiation of the generation cycle of the duodenal crypt cells of the mouse and rat. Radiat Res 29:57-70, Sep 1966 27. Lesher S, Fry RJM, Sacher GA: Effects of chronic gamma irradiation on the generation cycle of the mouse duodenum. Exp Cell Res 25:398-404, Nov 1961 28. Liversage WE: A general formula for equating protracted and acute regimes of radiation. Br J RadioI42:432-440, Jun 1969 29. Lord BI: Cellular proliferation in normal and continuously irradiated rat bone marrow studied by repeated labelling with tritiated thymidine. Br J HematoI11:130-143, Mar 1965 30. McCulloch EA, Till JE: The sensitivity of cells from normal mouse bone marrow to gamma radiation in vitro and in vivo. Radiat Res 16:822-832, Jun 1962 31. Mauro F, Madoc-Jones H: Age response to x-radiation of murine lymphoma cells synchronized in vivo. Proc Natl Acad Sci US 63:686-691, Jul 1969 32. Paterson R: The Treatment of Malignant Disease by Radiotherapy. Baltimore, Williams & Wilkins, 2d Ed, 1963, P 210 33. Phillips TL: Split-dose recovery in euoxic and hypoxic normal and tumor cells. Radiology 105:127-134, Oct 1972 34. Pierquin B: L'effet differential de I'irradiation continue (ou faible debit des carcinomes epidermoides (note semi-continue) preliminaire). J Radiol Electrol Med NucI51:533-536, Aug-Sep 1970 35. Rockwell SC, Kallman RF, Fajardo LF: Characteristics of a serially transplanted mouse mammary tumor and its tissue-cultureadapted derivative. J Nat Cancer Inst 49:735-749, Sep 1972 36. Sacher GA: Lethal effects of whole-body irradiation in mice: the dose-time relation for terminated and time-dependent exposure. Radiol Clin N Amer 3:227-241, Aug 1965 37. Sacher GA, Grahn 0: Survival of mice under duration-oflife exposure to gamma rays. I. The dosage-survival relation and the lethality function. J Nat Cancer Inst 32:277-321, Feb 1964 38. Sinclair WK: Dependence of radiosensitivity upon cell age. [In] Conference on Time and Dose Relationships in Radiation Biology as Applied to Radiotherapy, ed by VP Bond, HD Suit, and V Marcial. Brookhaven Nat Lab Rep BNL 50203 (C-57). Springfield, Va., Clghse Fed Sci Tech Inf, 1970, pp 97-116 39. Withers HR: Cell renewal system concepts and the radiation response. [In] Radiation Effect and Tolerance, Normal Tissue. Basic Concepts in Radiation Pathology. Vol 6 of Frontiers of Radiation Therapy and Oncology, ed by JM Vaeth. Baltimore, Univ Park Press, 1972, pp 93-107 40. Withers HR, Elkind MM: Radiosensitivity and fractionation response of crypt cells of mouse jejunum. Radiat Res 38:598-613, Jun 1969 41. Withers HR, Elkind MM: Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int J Radiat Bioi 17:261-267, Mar 1970
a
APPENDIX A
The differential form of the equation for the rate of increase of effective dose DEcan be written as
d(D E )
= d dt
- Il(D E
-
kdt)dt
The first term on the right-hand side relates to the continuous accumulation of nominal dose, while the second term relates to the part that fades. This second term is proportional to the fraction of the effective accumulated dose that can undergo fading. Upon integration, we have
d(l - k)e-lJ.t = d(l -
k) - J1(D E
-
kdt)
or, substituting for t and rearranging,
DE
= kDN +
d(l J1- k) [1 - e-lJ-DN /d]
Radiation Biology
Karen K. Fu, M.D., Theodore L. Phillips, M.D., Lawrence J. Kane, B. S., and Vernon Smith, M.S. Continuous irradiation in vivo at dose rates ranging from 0.54 to 274 rads per minute was performed in mice using the EMT6 tumor, the gut clone system, and the bone-marrow CFU system. A progressive increase in 0 0 and decrease in were seen with decreasing dose rates in the EMT6 tumor and the gut clone system. The 00 showed little change in the bone-marrow CFU system. These findings are related to the O2 - 0 1 values obtained trom split-dose experiments at conventional dose rates. The results do not fit the various mathematical models proposed for correlating the effects of continuous low-dose-rate irradiation and acute exposure.
n
Neoplasms, experimental. Radiobiology, bone-marrow studies. Radiobiology, cell and tissue studies • Radiobiology,time-dose studies
INDEX TERMS:
Radiology 114:709-716, March 1975
HE EFFECT of the dose rate is an important factor in the radiation response of normal and tumor tissues and is of interest to radiobiologists as well as radiotherapists. Inactivation of cellular reproductive ability varies considerably at a dose rate of between 1 and 100 rads per minute; the number of cells killed decreases as the dose rate is lowered, due partly to repair of sublethal damage during the course of protracted irradiation (13). Since the width of the shoulder (D q ) of the cell survival curve following an acute exposure is a measure of such repair, one should be able to predict the effectiveness of changes in the dose rate of continuous irradiation from the shapes of the survival curves following acute exposures: if the shoulder is wide (large Dq ), the radiation response would vary considerably with the dose rate, while conversely a narrow shoulder (small Dq ) would indicate a comparatively stable radiation effect. Previous reports regarding the effects of continuous irradiation at low dose rates on normal and tumor tissues show conflicting results. In vitro irradiation of HeLa and Chinese hamster cells over a wide range of dose rates showed significant change of Do with dose rate (1, 15). Continuous irradiation in vivo at low dose rates resulted in an increase in Do and a decrease in the extrapolation number in rat rhabdomyosarcoma (21) and P388 leukemic cells (2, 3), whereas data on KHT sarcoma (19) and NCTC 2472 fibrosarcoma and bonemarrow colony-forming units (CFU) (8) showed no significant difference in response between acute and protracted irradiation. Using whole-body irradiation, Sacher and Grahn (12, 37) investigated the effects of fractionation and various dose rates on the lethality and late ef-
T
fects of irradiation in mice, and Sacher (36) has done extensive mathematical analysis of the data. There have been several attempts to predict or evaluate the biological effects of continuous irradiation at low dose rates using different theoretical and mathematical analyses. Based on the data of Elkind and Sutton (6), Lajtha and Oliver (23) described a mathematical model for estimating the cumulative effective dose of a course of continuous irradiation at a constant dose rate. Liversage (28) proposed a general formula for equating protracted and acute regimens which is independent of the size and shape of the shoulder of the cell survival curve and thus applicable to all tissues. Kirk et al. (22) developed a method for assessing and comparing the biological effects of continuous irradiation with constant dose rate based on the isoeffect curves described by Paterson (32) and Ellis (7). We performed a number of experiments with 8- to 12-week-old BALB/c female mice to study the varying responses of normal and tumor cells to acute and continuous irradiation in vivo at decreasing dose rates using the gut clone system, the bone-marrow CFU system, and the EMT6 mouse mammary tumor and to test the validity of the mathematical models formulated by various authors for correlating the effects of acute and continuous irradiation. MA TERIALS AND METHODS
Biological Assays EMT6 tumor: The characteristics of the EMT6 tumor and its tissue-culture derivatives have been described by Rockwell et al. (35). It grows in BALB/c female mice and is readily transplantable. It clones as discrete colo-
1 From the Section of Radiation Oncology (K.K.F., T.L.P., V.S.) and the Laboratory of Radiobiology (K.K.F., T.L.P., L.J.K.), University of California School of Medicine, San Francisco, Calif. Presented at the Fifty-ninth Scientific Assembly and Annual Meeting of the Radiological Society of North America, Chicago, 111., Nov. 25-30, 1973. This work was performed under the auspices of the U. S. Atomic Energy Commission and was supported in part by NIH training grant CA05177 and Clinical Cancer Center grant CA-11067. sjh
709
710
KAREN K .
lr--_-+-_ 137C.
. !
! 2
Fu AND OTHERS
SOURCE
~~~'- LEAO ATTE NUATORS
--
was 113. The formula for estimating the number of surviving cells per circumference was therefore modified to
113-X )J [ (113
113 loge ......
(l\/( ~ ~l, .......
March 1975
-
)+--1------1--
ROTA TING LUCIlE lOX WITHHOLESIN THE WAll LEAD WAll
- - - - I - - LEAO DOOR
Fig. 1 Cross section of the 13 7CS irradiator, showing the positions of the radiation source, lead attenuators, and mice in rotating Lucite boxes.
nies in tissue culture and can be used for in vitro assay after treatment in situ. In vitro assay of the EMT6 tumor was carried out immediately after total-body irradiation, using an assay technique described previously (10). Briefly , irradiated mice bearing a 500- to 1000-mm3 transplanted EMT6 tumor in the flank area were sacrificed. The tumor was excised, trypsinized, and suspended in Ca++-free Eagle's Minimal Essential Medium. A known number of viable cells was plated in plastic tissue-culture flasks and incubated at 37 0 C in an atmosphere of 5% CO2 and 95% room air. Nine days later the clones were fixed, stained, and counted. Cell survival was calculated from the ratios of the plating efficiencies of the irradiated tumors and unirradiated controls. The plating efficiency for unirradiated control tumors was 35 ± 10%. Bone-marrow CFU: The technique of bone-marrow spleen colony assay has been described by McCulloch and Till (30). CFUs were irradiated in vivo and injected into recipient mice that had received 700 rads of totalbody radiation with 230-kVp x rays. Seven days later the number of colonies on the spleen surface was scored. Surviving fractions were based on the yield of CFUs per 105 nucleated cells from the femora of unirradiated animals. Gut clone system: Survival of jejunum crypt cells was determined by the microcolony assay technique described by Withers and Elkind (41). Three and onehalf days after total-body irradiation, mice were sacrificed and the jejunum was removed for histological examination. When irradiation was prolonged up to 86 hours, the mice often died earlier than three and onehalf days after the cessation of irradiation; hence it was necessary to shorten the interval between irradiation and sacrifice to two or three days when using a dose rate of 0.92 or 0.54 rads per minute. Mice were housed 5 per cage before whole-body irradiation and 4 per cage afterward. The average number of crypts per circumference in the jejunum of unirradiated control BALB/ c female mice
(1)
where x is the number of regenerating crypts in each transverse section (41). Method Of Irradiation
Pairs of mice were irradiated in rotating Lucite boxes inside a self-contained cesium irradiator at dose rates of 274, 36, 4.5, 0.92, or 0.54 rads per minute. Low dose rates were achieved with various thicknesses of lead attenuators (Fig. 1) and dosimetry was checked with thermoluminescent dosimeters. The effect of attenuators on radiation quality was determined with a combination ionization chamber/silicon p-n diode assembly calibrated with beams with known half-value layers. Since the ionization chamber had a relatively flat energy response while the diode had an increased response at lower energies, the ratio of the two readings served as an indication of quality. During protracted irradiation, mice were given food in the Lucite boxes and taken out for 15 to 30 minutes twice a day to drink water ad libitum. To determine the peak of recovery following an acute exposure, split-dose experiments were carried out at 274 rads per minute, using two doses of 500 rads each for EMT6 tumors, two doses of 150 rads each for bone-marrow CFUs, and a first dose of 700 rads.and a second dose of 500 rads for jejunum crypt cells. The second dose was delivered at 0, 2, 4, 6, 8, 10, 12: or 24 hours after the first dose. Subsequent split-dose experiments were performed with fixed first doses and graded second doses given at the peak of recovery from the first dose. In order to determine whether prolonged confinement had any effect on plating efficiency, spleen colony formation, or jejunal crypt regenerating ability, control animals were subjected to the same conditions of restraint as the irradiated animals, while a separate group of controls was given a test dose of 274 rads per minute at the end of various periods of restraint to see whether the radiation response was affected. RESULTS
Continuous Irradiation
Survival curves were calculated using least-squares regression analysis. Those represent ing EMT6 tumors subjected to continuous irradiation at dose rates ranging from 0.54 to 36 rads per minute showed a marked change in shape from the biphasic acute exposure curve expected for tumor cells irradiated at the conventional dose rate of 274 rads per minute (Fig. 2); rather, the Do increased and decreased with decreasing dose rates (TABLE I). The dose response curves of jejunum crypt cells irra-
n
Vol. 114
TUMOR AND NORMAL TISSUE RESPONSE TO IRRADIATION
711
In Vivo
Radiation Biology
274 036 •• 4.5 RADS/MIN
z
•.92 0.54
o
1
~
u
« 0:: LI..
o z
s s
0::
:::>
V')
DOSE (RADS) Fig. 2 Response of EMT6 tumor to 137CS irradiation in vivo with decreasing dose rates. Symbols represent the mean survival of 2-4 tumors assayed separately; bars represent standard errors of the mean. Table I: Tissue EMT6 tumor
Jejunal crypt cells
Bone-marrow CFUs
Variation of Do with Dose Rate Dose Rate (rads/rnin.)
Do (rads)
274 36 4.5 0.92 0.54
160/360 366 460 662 712
274 36 4.5 0.92 0.54
65 153 180 272 .740
274 36 4.5 0.92 0.54
80 113 87 76/95 76/95
1.0 _ Doc * DO a t
2.28 2.88 4.13 4.45
• SINGLE DOSE o SPLIT DOSE
.1
z
Q I-
2.35 2.77 4.18
U
« c.:::
LI..
11
o
z
1.41 1.08 0.96/1.18 0.96/1.18
* Doc = Do of survival curve for continuous irradiation t DOa = Do of survival curve for acute irradiation
.01
s
s
c.::: ::::> v»
.001
diated continuously at low dose rates also showed a significant increase in Do with decreasing dose rates (Fig. 3), and the intercept of the dose decreased as well. The ratio of Do for continuous irradiation at low dose rates (Dod to Do for acute exposures (Dos) showed similar changes in magnitude with variation of dose rate for the gut clone system and the EMT6 tumor in the range of 0.92 to 274 rads per minute (TABLE I). With a further decrease to 0.54 rads per minute, the Do increased elevenfold over that of the acute exposures, probably due in part to cell proliferation and decreased cell cycle time during protracted irradiation at this dose rate. The response of bone-marrow CFUs to continuous irradiation with decreasing dose rates differed markedly from those of the EMT6 tumor and jejunum crypt cells (Fig. 4). When the dose rate was decreased to 36 rads per minute, the Do increased to 110 rads while remained unchanged. With continuous irradiation at 4.5 87 rads per minute, the dose response curve (Do
n
=
500
Fig. 3. Response of mouse jejunal crypt cells to 137CS irradiation with decreasing dose rates. Symbols represent mean surviving cells per circumference of at least 8 mice assayed separately; bars represent standard errors of the mean.
rads) differed very little from that for irradiation with 80 rads): but when the dose acute exposures (Do rate was reduced to less than 1 rad per minute, the dose response curves unexpectedly became biphasic. Since there seemed to be little if any difference in response whether the dose rate was 0.92 or 0.54 rads per minute, the points at these lower dose rates were fitted to a common dose response curve, which exhibited increased sensitivity in the low-dose region (Do =
=
KAREN K. Fu AND OTHERS
712
100
'.
March 1975
1.0
UJ
U
Z UJ c:::
o
10
SPLIT DOSE
• SINGLE DOSE o SPLIT DOSE
.1
UJ
u... ~
+.
~
Z 0 ~ u
c:::
c::: u,
a...
e
::::>
u u
-c
UJ
.01
Z
~ LU
s sc:::
U
o
~
V)
Z
s sc:::
.001
::::>
V)
.01
1000
110.9
1200
1300
1400
1500
1600
DOSE (RADS)
DOSE (RADS)
Fig. 4. Response of mouse bone-marrow CFUs to i37CS irradiation with decreasing dose rates. Symbols represent the mean survival of bone-marrow CFUs of 2-16 mice assayed separately; bars represent standard errors of the mean.
Fig. 5. Response of EMT6 tumor in vivo to a single dose and second doses given four hours atter an initial dose of 500 rads at a rate of 274 rads per minute. Symbols represent the mean survival of 2-4 tumors; bars represent standard errors of the mean.
76 rads) and decreased sensitivity in the high-dose region (Do = 95 rads). With continuous prolonged irradiation at these low dose rates, bone-marrow cellularity fell to about 80% after 9 hours and about 50% after 15.5 hours. When the survival fraction was calculated based on the ratio of the number of spleen colonies yielded per irradiated and control femora, a similar biphasic change in the dose response curve was observed, indicating that the reduction in cellularity did not. introduce an error into the estimation of cell survival.
second doses (Fig. 7); however, this was not surprising in view of the small 02 - D1 value expected for bonemarrow CFUs.
Split-Dose Irradiation
Split-dose experiments indicated that the first peak of recovery occurred four hours after the first dose for EMT6 tumors and bone-marrow CFUs and six hours for jejunum crypt cells. Subsequent split-dose experiments with graded second doses given four to six hours after the first dose showed a O2 - 0 1 value2 of 330 rads for the EMT6 tumor, estimated at the survival level of 0.1 (Fig. 5), and 195 rads for jejunum crypt cells at the level of 10 surviving cells per circumference (Fig. 6). Although split-dose experiments with two equal doses showed a survival ratio of 1.5 for bone-marrow CFUs at four hours, which would suggest a Dq or O2 - 0 1 value of approximately 32 rads (Oq = Do In n), this was not substantiated by split-dose experiments with graded 2 D, is the single dose and O2 the total of two fractionated doses separated by a sufficient time for repair of sublethal damage to produce the same level of damage. O2 - 0, has been found to approximate the value of Oq (14).
Determinations of Radiation Beam Quality
Assessment of the quality of the radiation beam indicated that the addition of lead attenuators resulted in increased scatter and hence softening of the beam. The energy of the beam ranged from 600 to 280 keV depending on the thickness of the attenuators. Although the radiobiological effectiveness (RBE) changes along with the beam energy, the degree of change is small in this energy range: it increases only 10% with x rays having a mean photonenerqy of 100 keV, based on the results of our in vitro irradiation of EMT6 cells with 230kVp x rays at 131 rads per minute and 137CS at 274 rads per minute. The results obtained were not corrected for the possible small change in RBE. Res~amtofCon~oIAn;ma~
Restraint of controls under the same conditions as irradiated animals for various periods of time up to 96 hours caused no significant change in the plating efficiency of EMT6 tumors, the number of jejunal crypts per circumference, or the spleen colony-forming ability of bone-marrow CFUs compared with unrestrained controls, nor was there any significant change in radiation response following prolonged restraint (TABLE II).
TUMOR AND NORMAL TISSUE RESPONSE TO IRRADIATION
Vol. 114
.1
z
T~~
0 i= u
u
~
U
0::: W
~
\\~
·~ 274] l.~
\:
,
~:~~
RADS/ MIN
Q.. V) .....J .....J
0:::
W
::::>
U
V)
o
.001
.0001
100
200
300
400
*~"
500
z
s s
.1
0:::
::::>
V)
\ •
\
600
01 1000
1400
DOSE (RADS) Fig. 6. Response of mouse jejunal crypt cells to a single acute dose and second doses given six hours after an initial dose of 700 rads at a rate of 274 rads per minute. Symbols represent the mean surviving cells per circumference of 4-8 mice; bars represent standard errors of the mean.
Fig. 7. Response of bone-marrow CFUs to a single dose and second doses given four hours after an initial dose of 150 rads at a rate of 274 rads per minute. Symbols represent the mean survival of bone-marrow CFUs of 2-8 mice; bars represent standard errors of the mean.
Table II: Plating Efficiency of EMT6 Tumor and Bone-Marrow CFUs, Number of Crypts per Circumference, and Response to Acute Radiation Exposure in Restrained Mice
ous irradiation at low dose rates: dose rate, capacity for repair of sublethal damage, cell cycle time, variation of radiosensitivity during the cell cycle, density distribution of radiosensitive and radioresistant cells, redistribution of cells over the different phases of the cell cycle, proliferation of clonogenic cells, cell loss, oxygenation, and .reoxygenation during continuous irradiation. Cell reproductive deaths due to irradiation may result from a single lethal event (single-hit phenomenon), an accumulation of several sublethal events (multihit phenomenon), or, most likely, a combination of both. Although single lethal events are independent of the dose rate, repair of the sublethal component of damage during protracted irradiation would lead to fewer cell deaths with decreasing dose rates up to a limiting dose rate below which little change would occur. With the exception of bonemarrow CFUs, most mammalian tumor and normal tissue cells studied showed an increase in Do with a decrease in dose rate (Fig. 8). It has been suggested that the total dose received by a cell during the cell cycle is an important factor in determining its response to prolonged exposure at low dose rates (5, 17), with the radiation effect being greater for cells with long cell cycle times. This is complicated further by possible changes in cell cycle time during continuous irradiation (4, 9, 26, 27, 29); for example, the generation cycle of mouse duodenal crypt cells decreased when they were exposed to chronic gamma ir-
EMT6 Tumor
Duration of Restraint (hr.) 7 48 96
Plating Efficiency 0.30 0.25 0.38
Surival
p 1,000 rads 0.067 0.07 0.053
Jejunal Crypts
Duration of Restraint (hr.) 8 48
72
Number of Crypts per Circumference 106.4 104.4 103.0
Survival ± S. E. p 1,100 rads 10.6 ± 1.1 7.4±1.0 8.4 ± 1.9
Bone-Marrow CFUs
Duration of Restraint (hr.) 2 5 15
Plating Efficiency 3.9 X 10- 4 5.2 X 10- 4 3.8 X 10- 4
Survival ± S. E. p 150 rads 0.247± 0.04 0.187± 0.03 0.194± 0.03
DISCUSSION
The effect of the dose rate on radiation response varies with the tissue. Continuous irradiation at 0.54 to 36 rads per minute produced marked variation of response in the EMT6 mouse mammary tumor, mouse jejunum crypt cells, and bone-marrow CFUs. There are many complicating factors that might influence the response of different normal and tumor tissues to continu-
714
KAREN K.
Fu AND OTHERS
DOSE RATE (RADS/HR)
....-----1 700
600
60
\. I
600 I
6000 I
10
100
60000 I
e\ o
00
Do
.1
1.0
DOSE RATE (RADS/MIN)
Fig. 8. Variation of Do with dose rate. • = EMT6 mouse mammary tumor; • = mouse jejunal crypt cells; A = mouse bone-marKHT sarcoma row CFUs; 0 = rat rhabdomyosarcoma (21); A P388 leukemia cells; 0 HeLa cells in vitro (15); • = Chi(19); 0 nese hamster cells in vitro (1).
=
=
=
radiation at a rate of 0.91-1.09 rads per hour (9, 26, 27). It has been shown that radiosensitivity depends on the position of the cell in the cell cycle (11, 31, 38); thus the distribution of cells with different radiosensitivities in the tumor or normal tissue at the beginning of continuous low-dose-rate irradiation and subsequent redistribution of surviving cells over the different phases of the cell cycle (1, 8) might influence the variation of response to continuous irradiation. With progressively decreasing dose rate and increasing exposure time, clonogenic cells may regenerate and proliferate during irradiation (1, 4, 24, 25, 39) at a rate which depends on the cell cycle time and cell loss factor, which in turn may change during continuous low-dose-rate irradiation. Reoxygenation might take place during protracted tumor irradiation (19, 21), which would also influence the radiation response. All of these factors may be interdependent, and the relative importance of a given factor in determining the ultimate response to continuous low-dose-rate irradiation may vary in different tissues. Our studies demonstrated a marked difference in response between EMT6 tumors, jejunum crypt cells, and bone-marrow CFUs. Further wide variation in response, though not beyond the ranges we have shown, is demonstrated by the results of other experiments summarized in Figure 8. Both the EMT6 tumor and jejunum crypt cells showed relatively large O2 - 0 1 values, suggesting wide shoulders on the survival curves and relatively large capacities for repair of sublethal damage. It is not surprising that with decreasing dose rates, cell killing is less effec-
March 1975
tive and the Do increases in these cells. In contrast to the survival curve following acute exposures, that of EMT6 tumors given continuous low-dose-rate irradiation did not exhibit a biphasic effect. This type of change in the shape of survival curves following protracted irradiation has been demonstrated in rat rhabdomyosarcoma (21), KHT sarcoma (19), and NCTC fibrosarcoma (8). Two possibilities may account for this: (a) hypoxic cells may show less variation in response with dose rate, i.e., they do not repair sublethal damage, and/or (b) reoxygenation may occur during protracted irradiation, as suggested by data on KHT sarcoma (19) and rat rhabdomyosarcoma (21). The bone-marrow CFU results were both unexpected and puzzling. Our results differ from those obtained by Frindel et at. (8), who observed no significant difference between the effects of acute and continuous irradiation. Since bone-marrow CFUs showed little capacity for repair of sublethal damage, a significant change in response with decreaslnq dose rates would not be expected. Although there was a slight increase in Do when the dose rate was lowered from 274 to 36 rads per minute, a further decrease resulted in increased sensitivity in the low-dose region and slightly decreased sensitivity in the high-dose region with prolonged exposure time. Several factors might account for these changes, among them variation of sensitivity due to position in the cell cycle (18), redistribution of cells in the cell cycle (8), possible cell loss, and changes in the proportion of cycling stem cells before and during irradiation; however, further studies are necessary to elucidate the true underlying mechanisms. Although the survival curves of the EMT6 tumor and jejunum crypt cells are qualitatively consistent with the prediction of Lajtha and Oliver (23), they do not fit quantitatively with our estimates calculated using their proposed formula for correlating the effects of continuous irradiation with single acute exposures, assuming that the half-life of the radiation effect is 1.5 hours. Their analysis yields an effective dose of DE due to previous irradiation over time t in hours at a dose rate of d rads per hour defined as I
(2)
=
where I.L 0.693/half-life of damage in hours and DE is the equivalent dose for the same effect given at conventional dose rates (100-300 rads per minute). This assumes that any additional or subsequent dose undergoes exponential fading or repair. We have modifiedthis analysis to take into account the usual situation, in which part of the dose yields permanent damage while the other part yields damage that can be repaired (Appendix). If k is the fraction of the dose that is not repaired, (1-k) is the fraction that can be repaired, and ON is the nominal dose, the effective dose DE is given by
DE = kDN
+
d(! - k) [1 _ e-lJ.t] fJ.
(3)
Radiation Vol. 114
TUMOR AND NORMAL TISSUE RESPONSE TO IRRADIATION In Vivo
or, since
Table III:
(4) then
(5)
=
Biology
Effective Dose for EMT6 Mouse Mammary Tumor*
Sutviving Fraction
DN
d
DE
(rads)
(rads/rnin.)
(rads)
0.01 0.01 0.01 0.01
1,450 1,830 2,180 3,020
274 36 4.5 0.92
1,436 1,616 1,336 1,497
* Half-life (Tl/2) = 1.5 hours; k Table IV:
This formula was tested using experirtlental survival data for the EMT6 tumor and mouse jejunum crypt cells. The doses required to give a particular level of survival for the different dose rates, as read from the survival curves, were taken as values of ON and used in formula 5, and a value of DE was calculated for each value of ON. If the formula is valid, DE should be constant irrespective of the dose rate for a given cell system and level of survival. TABLE '" gives the test results for the EMT6 tumor, taking a survival value of 0.01 and assuming a half-life of 1.5 hours and k 0.475. The values for DE agree within ± 10%. TA~LE IV gives test results for the mouse jejunum crypt cells, taking 1 surviving cell per circumference as the survival level and assuming a half-life of 45 minutes and k = 0.42. The overall agreement is still about ± 10%. Our calculations suggest that the two major factors in cell survival following irradiation are the half-life of reparable damage and the fraction of damage that is reparable. These values appear to differ for EMT6 tumors and jejunal crypt cells, with both the half-life and the irreparable damage being less in the latter case. This is in agreement with Withers's results for jejunal crypt cells (40) and Homsey's finding of very rapid repair in the gut (20). Much additional information is needed in order to determine reasonable values for k and the halflife in different tissues and to relate these values to the nand Oq of the survival curves and ON/01 and O2 - 0 1 values for repair of tissues following fractionated irradiation (33). Our results also differed from what might be predicted from the method suggested by Kirk et al. (22), whose method was based on clinical impressions of connective-tissue tolerance; it is not surprising, therefore, that its application to other types of tissue and cell systems is limited. Liversage's formula (28) for correlating fractionated high-dose-rate and continuous low-dose-rate irradiation did not take into consideration the different survival curve shoulder widths of various types of cells. Our results suggest that response to continuous irradiation is at least partly influenced by the capacity for repair of sublethal damage, as reflected by the shoulder width (02 - 0 1 ) , and is reflected by the degree to which Do increases as the dose rate decreases. Thus it is evident that no single available formula can be used to adequately calculate equivalent doses for similar effects over a wide range of dose rates or be applicable to all types of cells. In clinical radiotherapy, continuous low-dose-rate irradiation with interstitial implants, intracavitary insertions,
715
= 0.475
Effective Dose for Mouse Jejunum Crypt Cells*
Surviving Cells per Circu mference
ON
d
(rads)
(rads/min.)
1 1 1
1,220 i,800
1
2,700
274 36 4.5 0.92
2,380
DE (rads)
1,218 1,484 1,169 1,127
* Half-life (TI/l) = 45 minutes; k = 0.42
or surface molds, either alone or in combination with external radiation therapy, has been used successfully in the treatment of many malignant tumors. Physically, the dose distribution favors both a higher total dose and a higher dose rate to the tumor; in addition, the biological advantage of decreased cell mortality with a lower dose rate in the adjacent normal tissue might be of advantage in the therapeutic ratio. Other factors such as the decreased oxygen enhancement ratio with lowdose-rate irradiation (16) and possible reoxygenation during protracted irradiation may also be advantages of interstitial and intracavitary radiotherapy. Our results with the EMT6 tumor support these potential advantages. External radiotherapy at low dose rates has recently been initiated (34), and early results are encouraging. The differences in response and the change in cellular kinetics between different normal and tumor tissues subjected to protracted low-dose-rate irradiation might be advantageous in the treatment of certain tumors. Although hemopoietic tissue, lymphomas, and leukemic bone marrow show no significant alteration in effect when exposed to low-dose-rate irradiation, other normal tissues such as the gastrointestinal tract may suffer less damage with this modality. However, any advantage gained with low-dose-rate external radiotherapy must be balanced against the practicality of the prolonged treatment time required for such therapy. ACKNOWLEDGMENTS: We are grateful to Mrs. Glenda Ross for her expert technical assistance and to Mimi Zeiger and Myra Goldring for their editorial and secretarial help.
Section of Radiation Oncology Universityof California School of Medicine San Francisco. Calif. 94143 REFERENCES 1. Bedford JS. Mitchell JB: Dose~rate effects in synchronous mammalian cells in culture. Radiat Res 54:316-327, May 1973 2. Berry RJ: Hypoxic protection and recovery in tumour cells irradiated at low dose-rates and assessed in vivo. Br J Radiol 41: 921-926, Dec 1968
716
KAREN K.
Fu
3. Berry RJ, Cohen AB: Some observations on the reproductive capacity of mammalian tumour cells exposed in vivo to gamma radiation at low dose-rates. Br J Radiol 35:489-491, Jul 1962 4. Cairnie AB: Cell proliferation studies in the intestinal epithelium of the rat: response to continuous irradiation. Radiat Res 32: 240-264,Oct1967 5. Courtenay VC: Unpublished data; cited by LF Lamerton and VC Courtenay in The steady state under continuous irradiation. [In] Brown DG, Cragle RG, Noonan TR, ed: Proceedings of Symposium on Dose Rate in Mammalian Radiation Biology, April 29-May 1, 1968, Oak Ridge, Tenn. CONF-680410. Springfield, Va., US Dept Commerce, 1968, pp 3.1-3.12 6. Elkind MM, Sutton H: Radiation response of mammalian cells grown in culture. I. Repair of x-ray damage in surviving Chinese hamster cells. Radiat Res 13: 556-593, Oct 1960 7. Ellis F: The relationship of biological effects to dose-timefractionation factors in radiotherapy. [In] Ebert M, Howard A, ed: Current Topics in Radiation Research. Amsterdam, North-Holland, 1968, Vol 4, pp 357-397 8. Frindel E, Hahn G, Robaglia D, et al: Responses of bone marrow and tumor cells to acute and protracted irradiation. Cancer Res 32:2096-2103, Oct 1972 9. Fry RJM, Lesher S, Sallese A, et al: The generation cycle of duodenal crypt cells of mice exposed to 220 roentgens of cobalt60 gamma irradiation per day. Radiat Res 19:628-635, Aug 1963 10. Fu KK, Phillips TL, Rowe JR: The RBE of neutrons in vivo. Cancer 34:48-53, Jul1974 11. Gillette EL, Withers HR, Tannock IF: The age sensitivity of epithelial cells of mouse small intestine. Radiology 96:639-643, Sep 1970 12. Grahn 0, Sacher GA: Fractionation and protraction factors and the late effects of radiation in small mammals. [In] Brown DG, Cragle RG, Noonan TR, ed: Proceedings of Symposium on Dose Rate in Mammalian Radiation Biology, April 29-May 1, 1968, Oak Ridge, Tenn. CONF-680410. Springfield, Va., US Dept Commerce, 1968, pp 2.1-2.27 13. Hall EJ: Radiation dose-rate: a factor of importance in radiobiology and radiotherapy. Br J RadioI45:81-97, Feb 1972 14. Hall EJ: Radiobiology for the Radiologist. Hagerstown, Harper & Row, 1973, pp 108-109 15. Hall EJ, Bedford JS: Dose rate: its effect on the survival of HeLa cells irradiated with gamma rays. Radiat Res 22:305-3'15, Jun 1964 16. Hall EJ, Bedford JS, Oliver R: Extreme hypoxia; its effect on the survival of mammalian cells irradiated at high and low doserates. Br J RadioI39:302-307, Apr 1966 17. Hall EJ, Oliver R, Shepstone BJ, et al: On the population kinetics of the root meristem of Vicia faba exposed to continuous irradiation. Radiat Res 27:597-603, Apr 1966 18. Hellman S: X-irradiation of the hematopoietic stem cell compartment. [In] Radiation Effect and Tolerance, Normal Tissue. Basic Concepts in Radiation Pathology. Vol 6 of Frontiers of Radiation Therapy and Oncology, ed by JM Vaeth. Baltimore, Univ Park Press, 1972, pp 415-527 19. Hill RP, Bush RS: The effect of continuous or fractionated irradiation on a murine sarcoma. Br J RadioI46:167-174, Mar 1973 20. Hornsey S: Differences in survival of jejunal crypt cells after radiation delivered at different dose-rates. Br J Radiol 43:802806, Nov 1970 21. Kal HB, Barendsen GW: Effects of continuous irradiation at low dose-rates on a rat rhabdomyosarcoma. Br J Radiol 45:279283, Apr 1972 22. Kirk J, Gray WM, Watson ER: Cumulative radiation effect. Part II: Continuous radiation therapy-long-lived sources. Clin Radiol 23:93-105, Jan 1972 23. Lajtha LG, Oliver R: Some radiobiological considerations in radiotherapy. Br J RadioI34:252-257, Apr 1961 24. Lamerton LF: Cell proliferation under continuous irradiation. Radiat Res 27: 119-138, Jan 1966 25. Lamerton LF, Courtenay VC: The steady state under continuous irradiation. [In] Brown DG, Cragle RG, Noonan TR, ed: Proceedings of Symposium on Dose Rate in Mammalian Radiation BioI-
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a
APPENDIX A
The differential form of the equation for the rate of increase of effective dose DEcan be written as
d(D E )
= d dt
- Il(D E
-
kdt)dt
The first term on the right-hand side relates to the continuous accumulation of nominal dose, while the second term relates to the part that fades. This second term is proportional to the fraction of the effective accumulated dose that can undergo fading. Upon integration, we have
d(l - k)e-lJ.t = d(l -
k) - J1(D E
-
kdt)
or, substituting for t and rearranging,
DE
= kDN +
d(l J1- k) [1 - e-lJ-DN /d]
