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RBE values of 15 MeV neutrons for effects on normal tissues
Europ. 07. CancerVol. 7, pp. 17i-177. Pergamon Press 1971. Printed in Great Britain
RBE Values of 15 MeV Neutrons for Effects on Normal Tissues j. j. BROERSE, G. W. BARENDSEN, G. FRERIKS and L. M. VAN PUTTEN Radiobiological Institute TWO, 151 Lange Kleiweg, R~iswijk Z.H., The Netherlands A b s t r a c t - For the application of fast neutrons in radiotherapy, it is important to evaluate the responses of various types of normal tissues in comparison with responses of experimental tumours after fast neutron- and X-irradiation. R B E values of 15 Me V neutrons are reported for effects on normal rodent tissues, namely mouse bone marrow, mouse intestine and rat skin. In general it has beenfound that these values are lower than the R B E values for effects on animal tumours. However, more data have to be obtained for the responses of normal tissues after fractionated irradiation with 15 Me V neutrons and X-rays before a definitive evaluation with respect to a possible therapeutic advantage of 15 M e V neutrons can be given.
INTRODUCTION FOR AN assessment of the potential advantage of fast neutrons for radiotherapy, effects of neutron irradiations on tumours have to be compared with effects on normal tissues. Various responses of a rhabdomyosarcoma in the rat and an osteosarcoma in the mouse have been studied for exposures to 15 MeV neutrons and 300 kV X-rays [1-3]. In this paper results will be presented on the effects of 15 MeV neutrons on normal tissues in experimental animals, namely rat skin, mouse bone marrow and mouse intestine. Application of fast neutrons in radiotherapy will only provide an advantage if the RBE values for the tumour response are larger than the RBE values for effects on normal tissues. A comparison of the relevant RBE values will be given.
Van de Graaff generator (400 kV, 250 BA of deuterons). In order to ensure an accurate positioning in the different exposure arrangements, rats were irradiated in nylon cylinders. These cylinders with the rats were turned 180 ° halfway the neutron irradiation in order to achieve a homogeneous dose distribution. Mice were irradiated in perforated Lusteroid centrifuge tubes. The mice in centrifuge tubes were mounted on a Styrofoam cylinder, which was rotated in front of the target during irradiations. This permitted compensation for any inhomogeneities in the neutron flux density distribution while the rotation of the mice caused multilateral exposure. The neutron dosimetry was carried out with a tissue equivalent ionization chamber and sulphur activation detectors [4, 5].
MATERIAL AND METHODS Irradiation techniques and dosimetry X-ray exposures were carried out with a General Electric Maxitron Generator (250 kVp, 30mA, HVL 2"1 mm Cu) and a PhilipsMiiller X-ray generator (300 kV, 10 mA, HVL 3 . 0 r a m Cu). 15 MeV neutrons were produced through the D - T reaction using a
Rat skin In order to evaluate differences between effects of various types of ionizing radiation on skin, without interference o f unirradiated cells migrating from outside the irradiated area and of damage to blood vessels and connective tissue, a transplantation technique 171
172
ft. 07. Broerse, G. IV. Barendsen, G. Freriks and L. M. van Putten
was employed to study radiation-induced damage to the skin of rats. Circular pieces of skin, about 20 mm in dia., were cut from the backs of white WAG/Rij rats after whole body irradiation with different doses of 300 kV Xrays and 15 MeV neutrons. These pieces of skin were transplanted onto the backs of brown (WAG/Rij x BN/Bi)F1 hybrid rats in which a graft bed was prepared by excision of similar pieces of skin. Due to the difference in colour the grafted skin can easily be distinguished from the host skin. Immunological complications are avoided by this choice of graft and host. The area of grafted skin remaining was measured over a three-months period after transplantation and the ratio of the treated and untreated areas has been used as a parameter for skin damage.
Mouse bone marrow and mouse intestine Acute bone marrow death and acute intestinal death have been studied in (CBA/Rij X C57BL/Rij)F1 hybrid mice after irradiation with X-rays and 15 MeV neutrons. LD50/30 and LD50/5 d values have been determined for different exposure periods [6]. Survival of the haemopoietic stem cells in these hybrid mice was investigated with the spleen colony technique [7] after single and fractionated exposures. The donor cells were irradiated either in vitro or in vivo. For each in vitro experiment one single bone marrow suspension was irradiated in a culture dish. The in vitro irradiations were carried out under hypoxic and oxygenated conditions, by flushing the dish with nitrogen or air, 10 min prior to the irradiation and during exposure. At different radiation doses part of this cell suspension was removed and appropriate dilutions were made. In the in vivo experiments the mice were killed immediately after the irradiation. The bone marrow suspensions were obtained by washing the marrow from the femoral shafts with Tyrode's solution and passing the resulting suspension through fine mesh nylon gauze. Suitable dilutions of the suspensions were injected intravenously in the recipient animals irradiated with 750 rads of X-rays. The recipients were sacrificed on the ninth day after irradiation. Subsequently the spleens were fixed in Telleyesniczky's solution and the number of superficial colonies was counted. The effect of dose fractionation in Vivo was studied by dividing the total dose in five equal fractions administered at 1-day intervals. The mice were sacrificed immediately after the last dose. The surviving fraction of colony-
forming units (CFU) was calculated relatively to the number of spleen colonies found per femur in a group of simultaneously studied unirradiated donors. The survival of intestinal crypt cells has been investigated with the microcolony method of Withers and Elkind [8]. About 3.5 days after total body irradiation of the F1 hybrid mice the animals were killed. Segments of the jejunum were fixed in Telleyesniczky's fluid. After histological preparation, transverse sections of the jejunum were stained with H.E. (haematoxylin and eosin). The number of regenerating crypts per circumference was counted and the number of surviving crypt stem cells was calculated on the basis of Poisson statistics. In unirradiated control mice, an average number of 90 crypts was found per circumference.
RESULTS Rat skin During the first 14 days after transplantation of the skin graft, little difference between irradiated and non-irradiated skin is observed macroscopically and microscopically with respect to the healing of the graft. After about 14 days, however, the irradiated skin grafts start to decrease in area. The ratio of the remaining areas of the irradiated skin graft relative to the area of unirradiated grafts approaches a constant value after about 8 weeks. This ratio depends on the dose and can be used as a measure of the skin damage produced by the irradiation. In Fig. 1 relations between the area of remaining skin and the radiation dose have been presented for single doses and two daily fractions of 15MeV neutrons and 300 kV X-rays. The X-ray as well as the neutron curve show large shoulders and consequently at low doses of up to 600 rads an RBE value cannot be estimated. At single doses in excess of about 1200 rads of 15 MeV neutrons and in excess of 1600 rads of 300 kV X-rays, very small areas of grafted skin remain, which cannot be measured accurately. An estimate of the RBE of 15 MeV neutrons can only be made with sufficient accuracy for the dose region where a reduction of the skin area to a fraction between 60% and 20% is obtained. For this region the RBE of 15 MeV neutrons after single irradiations can be calculated to be equal to 1.454-0-2. For two daily fractions, an RBE of 1-654-0.2 has been obtained whereas preliminary results for five daily fractions indicate an RBE in excess of 2" 0.
173
R.BE Values of 15 M e V Neutrons for Effects on Normal Tissues 1.:
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Fig. 1. Dose-effect relationsfor reduction of the area of skin grafts from irradiated WAG/R~j rats, transplanted on the backs of unirradiated (WAG[R~j× BW[Bi)Fx hybrid rats, measured 4-6 months after transplantation for single doses and two daily fractions of 15 MeV neutrons (curves 1) and 300 kV X-rays (curves 2).
Mouse bone marrow The relative mortality after X- and neutron irradiation has been investigated at different time intervals after irradiation as a function of the dose. For X-ray exposures in the lowest dose interval a distinct mortality peak is found on the eleventh day after irradiation. A similar peak is not present for the neutron exposures, but in the lowest lethal dose range a considerable proportion of animals survive more than 7 days after irradiation. O n the basis of the LDso/so d values for 2-hr exposures an RBE of 1.124-0.02 was obtained for the haemo-
Table 1.
poietic syndrome after 15 M e V neutron irradiation [6]. The effects on intact animals have been compared with the effects at the cellular level. Survival data of the haemopoietic stem cells have been obtained after neutron- and Xirradiations. The survival curve parameters are presented in Table 1. The survival curves for in vitro irradiation with X-rays and neutrons under oxygenated conditions show a similar slope, but a significant difference in extrapolation number. This results in a decrease of the RBE with decreasing surviving fractions. For a C F U survival of 0-1%, an R.BE of 1.10 has been obtained. The results for single dose irradiations of bone marrow stem cells in viva indicate a slightly higher Do for both radiations as compared with in vitro irradiation whereas the extrapolation numbers for neutrons and X-rays again differ significantly from each other. The R B E of the 15 M e V neutrons appears to be slightly lower as compared with in vitro irradiation, being 1.03 at a surviving fraction of 0 . 1 % . For a valid comparison of the effects of neutron- and X-irradiation on the intact animal and the effects on the haemopoietic stem cells comparable survival levels should be considered. Doses in the LDs0/30 d range correspond to surviving fractions between 10-s and 10 -4 . It should be emphasized that the RBE values for C F U survival at these levels are based on extrapolation estimates which have a low accuracy. Taking this into account it can be concluded that the RBE for the LDso/3o d is in good agreement with the RBE values for the C F U survival at a surviving fraction of 0 . 1 % . If the mice are subjected to five daily doses
Survival curve parameters for haemopoietic stem cells irradiated with neutrons or X-rays CFU X-rays Do n (rads)
RBE for cell survival of: neutrons Do n (rads)
10-1
10-3
10-s
1.25 1.21
1.39 1-90
1-18 1 "68
1.10 1.60
1.19 1.19 1-67
1.22 0.89 0.82
1.08 0"90 0.91
1.03 0.90 0.96
In vitro Air
62.2
4.03
65" 6
177.9
2" 68
123.6
Single dose
72.8
2" 53
78.0
Single dose delayed Five daily doses
69.2 92.8
1- 16 0.94
76.8 90.1
Nitrogen
In viva
174
07. 07. Broerse, G. W. Barendsen,
of radiation and the number of CFU per femur is assayed immediately after the last dose, the dose-effect curve for fast neutrons is not significantly different from that for X-rays, as illustrated in Fig. 2. However, in this case the X-rays seem slightly more effective especially at low dose levels. This result is the more surprising if one considers the small shoulder in the survival curve for fast neutrons and the smaller repair which is usually observed in split dose neutron irradiations in comparison with X-rays. A partial explanation for this finding is obtained from another series of experiments on the effects of single doses of X-rays and fast neutrons in vivo, in which a delay of 24 hours was allowed between irradiation and sacrifice of the donors for assay. The delayed assay causes a marked reduction of the recovery of colony forming cells from the bone marrow of X-irradiated mice. For the neutron exposures however, this loss of CFU has not been observed. Due to these phenomena, the RBE values for immediate assay are higher than the RBE for delayed assay and the RBE for fractionated exposures. As indicated in Table 1 for the assay after a 24-hr interval, an RBE of 0" 9 has been obtained independent of the dose. The loss of CFU after X-irradiation has been described by a number of investigators and presently it seems most likely that it is caused by differentiation of colony-forming cells to a cell type which has lost the capacity for proliferation to the size of a visible spleen
GFreriks . and L. M. van Putten colony [9]. However, at present there is no explanation for the absence of this phenomenon after neutron irradiation. In addition to the in vitro irradiation of the haemopoietic stem cells under oxygenated conditions, the response of the CFU was studied under anoxic conditions. At a 0' 1% survival level, OER values of 2"7 and 1.9 have been obtained for X- and 15 MeV neutron irradiations, respectively.
Mouse intestine A mean survival time of 4.5 days was found for death due to the intestinal syndrome. On the basis of the LD50/50 d values for 2-hr exposures an RBE of 1.424-0.02 can be calculated for the intestinal syndrome for 15 MeV neutrons with respect to X-rays [6]. The survival curves for jejunal-crypt stem cells in mice after irradiation with 15 MeV neutrons and X-rays are presented in Fig. 3. Do values of about 140 rads are obtained for both survival curves. An RBE value of 1.4 can be calculated at radiation doses in the lethal range for these mice. This value is in good agreement with the RBE for the LDs0/s d.
DISCUSSION A good agreement has been found between the RBE values observed with fast neutrons for the haemopoietic and intestinal syndromes and the RBE values for the effects on bone marrow stem cells and intestinal crypt cells respectively.
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Fig. 2. Survival of haemopoieti¢ stem cells in thefemora of mice after in vivo irradiation with five equal daily doses of 300 kV X-rays (curve 1) and 15 MeV neutrons (curve 2). The animals were satrif~ed for spleen colony assay immediately aider the last dose.
R B E Values of 15 M e V aVeutronsfor Effects on Normal Tissues
at Manchester on the CFU survival in mouse bone marrow after in vivo irradiation with D - T neutrons, 300 kV X-rays and e°Co and 137Cs gamma-rays [14, 15]. At a surviving fraction of 0" 1% the Manchester group obtained an RBE of 1.25 for CFU survival. As discussed in detail elsewhere [15] the discrepancy with our results is partly explained by a 8% difference in neutron dosimetry and partly by differences in the neutron spectra used. The Manchester experiments were performed inside a steel collimator. It has been demonstrated that in their experimental conditions a proportion of 10-20% of the neutron dose is delivered by scattered neutrons [16]. The contribution of these low energy neutrons will tend to increase the RBE, since it has been shown in other studies [17] that the RBE increases with decreasing neutron energy. The RBE of 1.4 for the LDs0t5 d derived from our data is very close to the RBE values of 1.5 to 1.6 obtained by Yamamoto [18] for the intestinal syndrome in mice. Withers et al. [19] studied the response of jejunal crypt cells in mice after single dose and split dose irradiation with 14 MeV neutrons. At a dose of about 900 rads of fast neutrons, at which mortality due to the intestinal syndrome will occur, an RBE of 1.4 can be calculated for a single dose irradiation. This value is in close agreement with the RBE values which we have found for the LDs0t5 ~ and the crypt cell survival. For the irradiation with two doses separated by a 24-hr interval an RBE of 1.5 can be calculated from their survival curves. For the survival of cultured cells of human kidney origin it has already been shown that the RBE values increase with decreasing neutron energy [17]. A similar dependence has been found for the RBE for the bone marrow syndrome and the intestinal syndrome in dependence on the neutron energy as shown in Table 2. The RBE values for the two radiation syndromes show a distinct difference for the three different neutron energies. A few years ago we postulated that there are intrinsic differences in radiosensitivity between
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Survival ofjejunal-crypt stem cells in mice after single dose irradiation with 15 MeV neutrons (curve 1) and 300 kV X-rays (curve 2). The standard deviations pertain to the variation within each separate experiment,
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This agreement is gratifying from the point of view of interpreting such very complex reactions in intact animals on the basis of responses of a single type of cells. The RBE for the LD50/30 d is in agreement with the value of 1.14 found by Sawada and Yoshinaga [10]. In accordance with this low RBE value, Davis et al. [11] found no consistent qualitative difference in lymphocyte and granulocyte counts after the administration of equal absorbed doses of 14 MeV neutrons and X-rays. Recently largely different values have been reported for the LD~0180 d in mice exposed to 14MeV neutrons. Strike [12] found RBE values of 1.6 and 1.5 for C57BL and C3H mice and Graul et al. [13] obtained an RBE of 1.9 for N M R I mice. With regard to the latter results it can be concluded, however, that death due to the intestinal syndrome interfered with death due to the bone marrow syndrome. Additional evidence for the low RBE value reported in this paper was obtained from collaborative experiments with Duncan et al. Table 2.
175
R_BE values for bone marrow ~yndrome and gastro-intestinal syndrome in mice aj2er fast neutron irradiation 15 M e V [6]
7 MeV neutrons [20, 21]
Fission neutrons [22]
Bone marrow syndrome
I" 1
1.5
1 "9
Gastro-intestinal syndrome
1.4
2.3
3.0
neutrons
1 76
07. 07. Broerse, G. W. Barendsen, G. Freriks and L. M. van Putten
bone marrow cells and intestinal cells, which are related to differences in the shapes of the survival curves for intestinal cells and bone marrow cells after neutron- and X-irradiation [23]. The recent experimental results on the dose survival characteristics of the stem cells of the bone marrow and the jejunal crypt cells support this hypothesis. In RBE studies of cyclotron produced neutrons for mammalian tissues a similar distinction has been made between the response of haemopoietie tissues and the response of a large number of other normal tissues [24]. A summary of the RBE values for effects on normal tissues and tumours has been given in Table 3. The tumour results will be discussed
Table 3.
in detail elsewhere [1-3]. As far as the effects on mouse intestine and rat skin are concerned very limited data are available at present. The results of the fractionated irradiation of these normal tissues indicate an increase in RBE with decreasing dose per fraction. It has to b e concluded, however, that more data have to be obtained for the responses of normal tissues after fractionated irradiation with 1 5 M e V neutrons and X-rays. At present no definitive evaluation can be given with respect to a possible therapeutic advantage of 1 5 M e V neutrons. Acknowledgements---The
authors are indebted to
Miss H. Roelse, Miss H. Sissingh, Mr. A. C. Engels and Mr. P. Lelieveld for skilful technical assistance.
R B E values of 15 Me V neutrons relative to 300 k V X-rays for single andfractionated irradiations of animal tumours and normal tissues
ml
Biological system
Irradiation conditions
RBE
Rhabdomyosarcoma in the rat (cloning technique*)
in vitro, single dose in vivo, single dose in vivo, 10 daily fractions in vivo, 15 daily fractions
1.6 2"9 3.0 2.9
Osteosarcoma in the mouse (end-point dilution assay*)
in vitro, single dose in vivo, single dose in vivo, 5 daily fractions
1.6 1.8 2" 5
Haemopoietic tissue in the mouse (spleen colony assay*)
in vitro, single dose in vivo, single dose in vivo, 5 daily fractions
1" I0 1.03 0-96
(Haemopoietic syndrome)
single dose
1.12
Gastro-intestinal tract in the mouse (microcolony technique)
in vivo, single dose
I" 4
(G.I. syndrome)
single dose
1.42
Skin of the rat (20-60% reduction in surface of graft)
single dose 2 daily fractions 5 daily fractions
1"45 1-65 >2" 0 ill
i
i
*RBE values for cell survival are calculated at surviving fractions of 0"001.
~ C , E $ 1. G. W. BAR~.m~SENand J. J. BROERS~., Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. I. Effects of single exposures. Europ. 07. Cancer 5, 373 (1969). 2. G . W . BAR~NDSENand J. j . BgoEgs~., Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. II. Effects of fractionated treatments, applied five times a week for several weeks. Europ. 07. Cancer 6~ 89 (1970). 3. L. M. VAN PUTTEN, P. LELX~V~ and J. J. BRom~sE, Response of a poorly reoxygenating mouse osteosarcoma to X-rays and fast neutrons. Europ. 07. Cancer 7, 153 (1971).
R B E Values of 15 M e V Neutrons for Effects on Normal Tissues 4. J . J . BROERSE, Dosimetry for fast-neutron irradiations of cultured cells and intact animals. 2. Comparison between activation and ionization methods. Int. 07. Radiat. Biol. 1O, 429 (1966). 5. J . J . BROERSEand H. VAN AMMERS,Dosimetry for fast-neutron irradiations of cultured cells and intact animals. 1. Characteristics of tissue-equivalent ionization chambers. Int. 07. Radiat. Biol. 1O, 417 (1966). 6. J . J . BROERSE,Dose-mortality studies for mice irradiated with X-rays, gammarays and 15 MeV neutrons. Int. 07. Radiat. Biol. 159 115 (1969). 7. J . E . TILL and E. A. McCuLLOCH,Direct measurement of radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213 (1961). 8. H. R. WITHERS and M. M. ELKIND, Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. 07. Radiat. Biol. 17, 261 (1970). 9. S.K. LAmm and L. M. VANPUTTEn, Modification of growth kinetics of colonyforming units in vivo. In Radiation-Induced Cancer, p. 109. IAEA, Vienna (1969). I0. S. SAWADAand H. YOSHINAOA,The relative biological effectiveness of X-ray, Co 60 gamma-ray, and 14.1 MeV fast neutron for acute death in mice. Nippon Acta Radiol. 23, 1080 (1963). 11. M . L . DAvis, E. B. DAVa~ENand G. E. COSGROVE,Early hematologic effects of whole-body 14 MeV neutron irradiation in mice. Acta Radiol. 3, 87 (1965). 12. T . A . STroKE and R. H. CRUTCHF.R,Acute mortality of mice and rats exposed to 14 MeV neutrons. In Armed Forces Radiobiology Research Institute Annual Research Report ARR-3, p. 13 (1969). 13. E . H . GRAUL,W. ROTHER and H. KROOER, Untersuchungen fiber die relative biologische Wirksamkeit yon 14-MeV-Neutronen an Laboratoriumstieren und HeLa-Zellen. Strahlentherapie 138, 699 (1969). 14. W. DUNCAN, D. GREENE, A. HOWARDand J. B. MASSE'Z,The RBE of 14 MeV neutrons. Observations on colony-forming units in mouse bone marrow. Int. J. Radiat. Biol. 15, 397 (1969). 15. J . J . BROERSE, W. DUNCAN, A. C. ENGELS, C. W. GILBERT,D. GRE~.NE,J. H. HENRY, A. HOWARD, P. LELIEWLD,J. B. MASSE',"and L. M. VAN PUTa~N, The survival of colony-forming units in mouse bone marrow after in vivo irradiation with D-T neutrons, X- and gamma-radiation. To be published in Int. 07. Radiat. Biol. 16. D. GREENE and R. L. THOMAS,An experimental unit for fast neutron radiotherapy. Brit. 07. Radiol. 41, 455 (1968). 17. J . J . BROERSE,G. W. BARENDSENand G. R. VANKERSEN,Survival of cultured human cells after irradiation with fast neutrons of different energies in hypoxic and oxygenated conditions. Int. 07. Radiat. Biol. 13, 559 (1968). 18. 0. YAMAMOTO,On the variation of LD-50 in time after 14.1 MeV fast neutronsand 180 kVp X-rays-irradiation for young and adult mice. Nippon Acta Radiol. 26, 1361 (1967). 19. H . R . WITHERS,J. T. BRENNANand M. IV[. ELKIND,The response of stem cells of intestinal mucosa to irradiation with 14 MeV neutrons. Brit. 07. Radiol. 43, 796 (1970). 20. S. HORNSEY,Private communication (I 970). 21. S. HORNSEY,S. VATISTAS,D. K. BEWLEY and C. J. PARNELL,The effect of fractionation on four-day survival of mice after whole-body neutron irradiation. Brit. 07. Radiol. 38, 878 (1965). 22. J . A . G . DAVIDS,Relative biological effectiveness of fission neutrons for production of the bone marrow syndrome in mice. Int. 07. Radiat. Biol. 10, 299 (1966). 23. J . J . BROERSE, Effects of energy dissipation by monoenergetic neutrons in mammalian cells and tissues. Thesis, Amsterdam (1966). 24. S.B. FIELD,The relative biological effectiveness of fast neutrons for mammalian tissues. Radiology 93, 915 (1969).
177
RBE Values of 15 MeV Neutrons for Effects on Normal Tissues j. j. BROERSE, G. W. BARENDSEN, G. FRERIKS and L. M. VAN PUTTEN Radiobiological Institute TWO, 151 Lange Kleiweg, R~iswijk Z.H., The Netherlands A b s t r a c t - For the application of fast neutrons in radiotherapy, it is important to evaluate the responses of various types of normal tissues in comparison with responses of experimental tumours after fast neutron- and X-irradiation. R B E values of 15 Me V neutrons are reported for effects on normal rodent tissues, namely mouse bone marrow, mouse intestine and rat skin. In general it has beenfound that these values are lower than the R B E values for effects on animal tumours. However, more data have to be obtained for the responses of normal tissues after fractionated irradiation with 15 Me V neutrons and X-rays before a definitive evaluation with respect to a possible therapeutic advantage of 15 M e V neutrons can be given.
INTRODUCTION FOR AN assessment of the potential advantage of fast neutrons for radiotherapy, effects of neutron irradiations on tumours have to be compared with effects on normal tissues. Various responses of a rhabdomyosarcoma in the rat and an osteosarcoma in the mouse have been studied for exposures to 15 MeV neutrons and 300 kV X-rays [1-3]. In this paper results will be presented on the effects of 15 MeV neutrons on normal tissues in experimental animals, namely rat skin, mouse bone marrow and mouse intestine. Application of fast neutrons in radiotherapy will only provide an advantage if the RBE values for the tumour response are larger than the RBE values for effects on normal tissues. A comparison of the relevant RBE values will be given.
Van de Graaff generator (400 kV, 250 BA of deuterons). In order to ensure an accurate positioning in the different exposure arrangements, rats were irradiated in nylon cylinders. These cylinders with the rats were turned 180 ° halfway the neutron irradiation in order to achieve a homogeneous dose distribution. Mice were irradiated in perforated Lusteroid centrifuge tubes. The mice in centrifuge tubes were mounted on a Styrofoam cylinder, which was rotated in front of the target during irradiations. This permitted compensation for any inhomogeneities in the neutron flux density distribution while the rotation of the mice caused multilateral exposure. The neutron dosimetry was carried out with a tissue equivalent ionization chamber and sulphur activation detectors [4, 5].
MATERIAL AND METHODS Irradiation techniques and dosimetry X-ray exposures were carried out with a General Electric Maxitron Generator (250 kVp, 30mA, HVL 2"1 mm Cu) and a PhilipsMiiller X-ray generator (300 kV, 10 mA, HVL 3 . 0 r a m Cu). 15 MeV neutrons were produced through the D - T reaction using a
Rat skin In order to evaluate differences between effects of various types of ionizing radiation on skin, without interference o f unirradiated cells migrating from outside the irradiated area and of damage to blood vessels and connective tissue, a transplantation technique 171
172
ft. 07. Broerse, G. IV. Barendsen, G. Freriks and L. M. van Putten
was employed to study radiation-induced damage to the skin of rats. Circular pieces of skin, about 20 mm in dia., were cut from the backs of white WAG/Rij rats after whole body irradiation with different doses of 300 kV Xrays and 15 MeV neutrons. These pieces of skin were transplanted onto the backs of brown (WAG/Rij x BN/Bi)F1 hybrid rats in which a graft bed was prepared by excision of similar pieces of skin. Due to the difference in colour the grafted skin can easily be distinguished from the host skin. Immunological complications are avoided by this choice of graft and host. The area of grafted skin remaining was measured over a three-months period after transplantation and the ratio of the treated and untreated areas has been used as a parameter for skin damage.
Mouse bone marrow and mouse intestine Acute bone marrow death and acute intestinal death have been studied in (CBA/Rij X C57BL/Rij)F1 hybrid mice after irradiation with X-rays and 15 MeV neutrons. LD50/30 and LD50/5 d values have been determined for different exposure periods [6]. Survival of the haemopoietic stem cells in these hybrid mice was investigated with the spleen colony technique [7] after single and fractionated exposures. The donor cells were irradiated either in vitro or in vivo. For each in vitro experiment one single bone marrow suspension was irradiated in a culture dish. The in vitro irradiations were carried out under hypoxic and oxygenated conditions, by flushing the dish with nitrogen or air, 10 min prior to the irradiation and during exposure. At different radiation doses part of this cell suspension was removed and appropriate dilutions were made. In the in vivo experiments the mice were killed immediately after the irradiation. The bone marrow suspensions were obtained by washing the marrow from the femoral shafts with Tyrode's solution and passing the resulting suspension through fine mesh nylon gauze. Suitable dilutions of the suspensions were injected intravenously in the recipient animals irradiated with 750 rads of X-rays. The recipients were sacrificed on the ninth day after irradiation. Subsequently the spleens were fixed in Telleyesniczky's solution and the number of superficial colonies was counted. The effect of dose fractionation in Vivo was studied by dividing the total dose in five equal fractions administered at 1-day intervals. The mice were sacrificed immediately after the last dose. The surviving fraction of colony-
forming units (CFU) was calculated relatively to the number of spleen colonies found per femur in a group of simultaneously studied unirradiated donors. The survival of intestinal crypt cells has been investigated with the microcolony method of Withers and Elkind [8]. About 3.5 days after total body irradiation of the F1 hybrid mice the animals were killed. Segments of the jejunum were fixed in Telleyesniczky's fluid. After histological preparation, transverse sections of the jejunum were stained with H.E. (haematoxylin and eosin). The number of regenerating crypts per circumference was counted and the number of surviving crypt stem cells was calculated on the basis of Poisson statistics. In unirradiated control mice, an average number of 90 crypts was found per circumference.
RESULTS Rat skin During the first 14 days after transplantation of the skin graft, little difference between irradiated and non-irradiated skin is observed macroscopically and microscopically with respect to the healing of the graft. After about 14 days, however, the irradiated skin grafts start to decrease in area. The ratio of the remaining areas of the irradiated skin graft relative to the area of unirradiated grafts approaches a constant value after about 8 weeks. This ratio depends on the dose and can be used as a measure of the skin damage produced by the irradiation. In Fig. 1 relations between the area of remaining skin and the radiation dose have been presented for single doses and two daily fractions of 15MeV neutrons and 300 kV X-rays. The X-ray as well as the neutron curve show large shoulders and consequently at low doses of up to 600 rads an RBE value cannot be estimated. At single doses in excess of about 1200 rads of 15 MeV neutrons and in excess of 1600 rads of 300 kV X-rays, very small areas of grafted skin remain, which cannot be measured accurately. An estimate of the RBE of 15 MeV neutrons can only be made with sufficient accuracy for the dose region where a reduction of the skin area to a fraction between 60% and 20% is obtained. For this region the RBE of 15 MeV neutrons after single irradiations can be calculated to be equal to 1.454-0-2. For two daily fractions, an RBE of 1-654-0.2 has been obtained whereas preliminary results for five daily fractions indicate an RBE in excess of 2" 0.
173
R.BE Values of 15 M e V Neutrons for Effects on Normal Tissues 1.:
8"~o°~..~,
i..-on s gte
0.1-
o ._c
.~ 0.01 two dailyfractions
1= E
01,
001
~
.
.
.
.
.
.
.
5
R
.
.
.
.
~
.
.
.
10
.
~
.
.
.
.
%
.
15
.
.
.
2
.
. . . . . . .
20 25 30 dosein rods x 100
Fig. 1. Dose-effect relationsfor reduction of the area of skin grafts from irradiated WAG/R~j rats, transplanted on the backs of unirradiated (WAG[R~j× BW[Bi)Fx hybrid rats, measured 4-6 months after transplantation for single doses and two daily fractions of 15 MeV neutrons (curves 1) and 300 kV X-rays (curves 2).
Mouse bone marrow The relative mortality after X- and neutron irradiation has been investigated at different time intervals after irradiation as a function of the dose. For X-ray exposures in the lowest dose interval a distinct mortality peak is found on the eleventh day after irradiation. A similar peak is not present for the neutron exposures, but in the lowest lethal dose range a considerable proportion of animals survive more than 7 days after irradiation. O n the basis of the LDso/so d values for 2-hr exposures an RBE of 1.124-0.02 was obtained for the haemo-
Table 1.
poietic syndrome after 15 M e V neutron irradiation [6]. The effects on intact animals have been compared with the effects at the cellular level. Survival data of the haemopoietic stem cells have been obtained after neutron- and Xirradiations. The survival curve parameters are presented in Table 1. The survival curves for in vitro irradiation with X-rays and neutrons under oxygenated conditions show a similar slope, but a significant difference in extrapolation number. This results in a decrease of the RBE with decreasing surviving fractions. For a C F U survival of 0-1%, an R.BE of 1.10 has been obtained. The results for single dose irradiations of bone marrow stem cells in viva indicate a slightly higher Do for both radiations as compared with in vitro irradiation whereas the extrapolation numbers for neutrons and X-rays again differ significantly from each other. The R B E of the 15 M e V neutrons appears to be slightly lower as compared with in vitro irradiation, being 1.03 at a surviving fraction of 0 . 1 % . For a valid comparison of the effects of neutron- and X-irradiation on the intact animal and the effects on the haemopoietic stem cells comparable survival levels should be considered. Doses in the LDs0/30 d range correspond to surviving fractions between 10-s and 10 -4 . It should be emphasized that the RBE values for C F U survival at these levels are based on extrapolation estimates which have a low accuracy. Taking this into account it can be concluded that the RBE for the LDso/3o d is in good agreement with the RBE values for the C F U survival at a surviving fraction of 0 . 1 % . If the mice are subjected to five daily doses
Survival curve parameters for haemopoietic stem cells irradiated with neutrons or X-rays CFU X-rays Do n (rads)
RBE for cell survival of: neutrons Do n (rads)
10-1
10-3
10-s
1.25 1.21
1.39 1-90
1-18 1 "68
1.10 1.60
1.19 1.19 1-67
1.22 0.89 0.82
1.08 0"90 0.91
1.03 0.90 0.96
In vitro Air
62.2
4.03
65" 6
177.9
2" 68
123.6
Single dose
72.8
2" 53
78.0
Single dose delayed Five daily doses
69.2 92.8
1- 16 0.94
76.8 90.1
Nitrogen
In viva
174
07. 07. Broerse, G. W. Barendsen,
of radiation and the number of CFU per femur is assayed immediately after the last dose, the dose-effect curve for fast neutrons is not significantly different from that for X-rays, as illustrated in Fig. 2. However, in this case the X-rays seem slightly more effective especially at low dose levels. This result is the more surprising if one considers the small shoulder in the survival curve for fast neutrons and the smaller repair which is usually observed in split dose neutron irradiations in comparison with X-rays. A partial explanation for this finding is obtained from another series of experiments on the effects of single doses of X-rays and fast neutrons in vivo, in which a delay of 24 hours was allowed between irradiation and sacrifice of the donors for assay. The delayed assay causes a marked reduction of the recovery of colony forming cells from the bone marrow of X-irradiated mice. For the neutron exposures however, this loss of CFU has not been observed. Due to these phenomena, the RBE values for immediate assay are higher than the RBE for delayed assay and the RBE for fractionated exposures. As indicated in Table 1 for the assay after a 24-hr interval, an RBE of 0" 9 has been obtained independent of the dose. The loss of CFU after X-irradiation has been described by a number of investigators and presently it seems most likely that it is caused by differentiation of colony-forming cells to a cell type which has lost the capacity for proliferation to the size of a visible spleen
GFreriks . and L. M. van Putten colony [9]. However, at present there is no explanation for the absence of this phenomenon after neutron irradiation. In addition to the in vitro irradiation of the haemopoietic stem cells under oxygenated conditions, the response of the CFU was studied under anoxic conditions. At a 0' 1% survival level, OER values of 2"7 and 1.9 have been obtained for X- and 15 MeV neutron irradiations, respectively.
Mouse intestine A mean survival time of 4.5 days was found for death due to the intestinal syndrome. On the basis of the LD50/50 d values for 2-hr exposures an RBE of 1.424-0.02 can be calculated for the intestinal syndrome for 15 MeV neutrons with respect to X-rays [6]. The survival curves for jejunal-crypt stem cells in mice after irradiation with 15 MeV neutrons and X-rays are presented in Fig. 3. Do values of about 140 rads are obtained for both survival curves. An RBE value of 1.4 can be calculated at radiation doses in the lethal range for these mice. This value is in good agreement with the RBE for the LDs0/s d.
DISCUSSION A good agreement has been found between the RBE values observed with fast neutrons for the haemopoietic and intestinal syndromes and the RBE values for the effects on bone marrow stem cells and intestinal crypt cells respectively.
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•
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2
~ .
1o- 3.
0 ••% •%•% 10"
I;0
~0
3;0
4;0
5;0
6;0
7;0
SO0
9~
101]0
dose in rods
Fig. 2. Survival of haemopoieti¢ stem cells in thefemora of mice after in vivo irradiation with five equal daily doses of 300 kV X-rays (curve 1) and 15 MeV neutrons (curve 2). The animals were satrif~ed for spleen colony assay immediately aider the last dose.
R B E Values of 15 M e V aVeutronsfor Effects on Normal Tissues
at Manchester on the CFU survival in mouse bone marrow after in vivo irradiation with D - T neutrons, 300 kV X-rays and e°Co and 137Cs gamma-rays [14, 15]. At a surviving fraction of 0" 1% the Manchester group obtained an RBE of 1.25 for CFU survival. As discussed in detail elsewhere [15] the discrepancy with our results is partly explained by a 8% difference in neutron dosimetry and partly by differences in the neutron spectra used. The Manchester experiments were performed inside a steel collimator. It has been demonstrated that in their experimental conditions a proportion of 10-20% of the neutron dose is delivered by scattered neutrons [16]. The contribution of these low energy neutrons will tend to increase the RBE, since it has been shown in other studies [17] that the RBE increases with decreasing neutron energy. The RBE of 1.4 for the LDs0t5 d derived from our data is very close to the RBE values of 1.5 to 1.6 obtained by Yamamoto [18] for the intestinal syndrome in mice. Withers et al. [19] studied the response of jejunal crypt cells in mice after single dose and split dose irradiation with 14 MeV neutrons. At a dose of about 900 rads of fast neutrons, at which mortality due to the intestinal syndrome will occur, an RBE of 1.4 can be calculated for a single dose irradiation. This value is in close agreement with the RBE values which we have found for the LDs0t5 ~ and the crypt cell survival. For the irradiation with two doses separated by a 24-hr interval an RBE of 1.5 can be calculated from their survival curves. For the survival of cultured cells of human kidney origin it has already been shown that the RBE values increase with decreasing neutron energy [17]. A similar dependence has been found for the RBE for the bone marrow syndrome and the intestinal syndrome in dependence on the neutron energy as shown in Table 2. The RBE values for the two radiation syndromes show a distinct difference for the three different neutron energies. A few years ago we postulated that there are intrinsic differences in radiosensitivity between
l0 2.
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N K
>
E
0
.
.
400 .
.
.
800
l i O0 16'o0 ' dose in rods
Survival ofjejunal-crypt stem cells in mice after single dose irradiation with 15 MeV neutrons (curve 1) and 300 kV X-rays (curve 2). The standard deviations pertain to the variation within each separate experiment,
F / g . 3.
This agreement is gratifying from the point of view of interpreting such very complex reactions in intact animals on the basis of responses of a single type of cells. The RBE for the LD50/30 d is in agreement with the value of 1.14 found by Sawada and Yoshinaga [10]. In accordance with this low RBE value, Davis et al. [11] found no consistent qualitative difference in lymphocyte and granulocyte counts after the administration of equal absorbed doses of 14 MeV neutrons and X-rays. Recently largely different values have been reported for the LD~0180 d in mice exposed to 14MeV neutrons. Strike [12] found RBE values of 1.6 and 1.5 for C57BL and C3H mice and Graul et al. [13] obtained an RBE of 1.9 for N M R I mice. With regard to the latter results it can be concluded, however, that death due to the intestinal syndrome interfered with death due to the bone marrow syndrome. Additional evidence for the low RBE value reported in this paper was obtained from collaborative experiments with Duncan et al. Table 2.
175
R_BE values for bone marrow ~yndrome and gastro-intestinal syndrome in mice aj2er fast neutron irradiation 15 M e V [6]
7 MeV neutrons [20, 21]
Fission neutrons [22]
Bone marrow syndrome
I" 1
1.5
1 "9
Gastro-intestinal syndrome
1.4
2.3
3.0
neutrons
1 76
07. 07. Broerse, G. W. Barendsen, G. Freriks and L. M. van Putten
bone marrow cells and intestinal cells, which are related to differences in the shapes of the survival curves for intestinal cells and bone marrow cells after neutron- and X-irradiation [23]. The recent experimental results on the dose survival characteristics of the stem cells of the bone marrow and the jejunal crypt cells support this hypothesis. In RBE studies of cyclotron produced neutrons for mammalian tissues a similar distinction has been made between the response of haemopoietie tissues and the response of a large number of other normal tissues [24]. A summary of the RBE values for effects on normal tissues and tumours has been given in Table 3. The tumour results will be discussed
Table 3.
in detail elsewhere [1-3]. As far as the effects on mouse intestine and rat skin are concerned very limited data are available at present. The results of the fractionated irradiation of these normal tissues indicate an increase in RBE with decreasing dose per fraction. It has to b e concluded, however, that more data have to be obtained for the responses of normal tissues after fractionated irradiation with 1 5 M e V neutrons and X-rays. At present no definitive evaluation can be given with respect to a possible therapeutic advantage of 1 5 M e V neutrons. Acknowledgements---The
authors are indebted to
Miss H. Roelse, Miss H. Sissingh, Mr. A. C. Engels and Mr. P. Lelieveld for skilful technical assistance.
R B E values of 15 Me V neutrons relative to 300 k V X-rays for single andfractionated irradiations of animal tumours and normal tissues
ml
Biological system
Irradiation conditions
RBE
Rhabdomyosarcoma in the rat (cloning technique*)
in vitro, single dose in vivo, single dose in vivo, 10 daily fractions in vivo, 15 daily fractions
1.6 2"9 3.0 2.9
Osteosarcoma in the mouse (end-point dilution assay*)
in vitro, single dose in vivo, single dose in vivo, 5 daily fractions
1.6 1.8 2" 5
Haemopoietic tissue in the mouse (spleen colony assay*)
in vitro, single dose in vivo, single dose in vivo, 5 daily fractions
1" I0 1.03 0-96
(Haemopoietic syndrome)
single dose
1.12
Gastro-intestinal tract in the mouse (microcolony technique)
in vivo, single dose
I" 4
(G.I. syndrome)
single dose
1.42
Skin of the rat (20-60% reduction in surface of graft)
single dose 2 daily fractions 5 daily fractions
1"45 1-65 >2" 0 ill
i
i
*RBE values for cell survival are calculated at surviving fractions of 0"001.
~ C , E $ 1. G. W. BAR~.m~SENand J. J. BROERS~., Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. I. Effects of single exposures. Europ. 07. Cancer 5, 373 (1969). 2. G . W . BAR~NDSENand J. j . BgoEgs~., Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. II. Effects of fractionated treatments, applied five times a week for several weeks. Europ. 07. Cancer 6~ 89 (1970). 3. L. M. VAN PUTTEN, P. LELX~V~ and J. J. BRom~sE, Response of a poorly reoxygenating mouse osteosarcoma to X-rays and fast neutrons. Europ. 07. Cancer 7, 153 (1971).
R B E Values of 15 M e V Neutrons for Effects on Normal Tissues 4. J . J . BROERSE, Dosimetry for fast-neutron irradiations of cultured cells and intact animals. 2. Comparison between activation and ionization methods. Int. 07. Radiat. Biol. 1O, 429 (1966). 5. J . J . BROERSEand H. VAN AMMERS,Dosimetry for fast-neutron irradiations of cultured cells and intact animals. 1. Characteristics of tissue-equivalent ionization chambers. Int. 07. Radiat. Biol. 1O, 417 (1966). 6. J . J . BROERSE,Dose-mortality studies for mice irradiated with X-rays, gammarays and 15 MeV neutrons. Int. 07. Radiat. Biol. 159 115 (1969). 7. J . E . TILL and E. A. McCuLLOCH,Direct measurement of radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213 (1961). 8. H. R. WITHERS and M. M. ELKIND, Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. 07. Radiat. Biol. 17, 261 (1970). 9. S.K. LAmm and L. M. VANPUTTEn, Modification of growth kinetics of colonyforming units in vivo. In Radiation-Induced Cancer, p. 109. IAEA, Vienna (1969). I0. S. SAWADAand H. YOSHINAOA,The relative biological effectiveness of X-ray, Co 60 gamma-ray, and 14.1 MeV fast neutron for acute death in mice. Nippon Acta Radiol. 23, 1080 (1963). 11. M . L . DAvis, E. B. DAVa~ENand G. E. COSGROVE,Early hematologic effects of whole-body 14 MeV neutron irradiation in mice. Acta Radiol. 3, 87 (1965). 12. T . A . STroKE and R. H. CRUTCHF.R,Acute mortality of mice and rats exposed to 14 MeV neutrons. In Armed Forces Radiobiology Research Institute Annual Research Report ARR-3, p. 13 (1969). 13. E . H . GRAUL,W. ROTHER and H. KROOER, Untersuchungen fiber die relative biologische Wirksamkeit yon 14-MeV-Neutronen an Laboratoriumstieren und HeLa-Zellen. Strahlentherapie 138, 699 (1969). 14. W. DUNCAN, D. GREENE, A. HOWARDand J. B. MASSE'Z,The RBE of 14 MeV neutrons. Observations on colony-forming units in mouse bone marrow. Int. J. Radiat. Biol. 15, 397 (1969). 15. J . J . BROERSE, W. DUNCAN, A. C. ENGELS, C. W. GILBERT,D. GRE~.NE,J. H. HENRY, A. HOWARD, P. LELIEWLD,J. B. MASSE',"and L. M. VAN PUTa~N, The survival of colony-forming units in mouse bone marrow after in vivo irradiation with D-T neutrons, X- and gamma-radiation. To be published in Int. 07. Radiat. Biol. 16. D. GREENE and R. L. THOMAS,An experimental unit for fast neutron radiotherapy. Brit. 07. Radiol. 41, 455 (1968). 17. J . J . BROERSE,G. W. BARENDSENand G. R. VANKERSEN,Survival of cultured human cells after irradiation with fast neutrons of different energies in hypoxic and oxygenated conditions. Int. 07. Radiat. Biol. 13, 559 (1968). 18. 0. YAMAMOTO,On the variation of LD-50 in time after 14.1 MeV fast neutronsand 180 kVp X-rays-irradiation for young and adult mice. Nippon Acta Radiol. 26, 1361 (1967). 19. H . R . WITHERS,J. T. BRENNANand M. IV[. ELKIND,The response of stem cells of intestinal mucosa to irradiation with 14 MeV neutrons. Brit. 07. Radiol. 43, 796 (1970). 20. S. HORNSEY,Private communication (I 970). 21. S. HORNSEY,S. VATISTAS,D. K. BEWLEY and C. J. PARNELL,The effect of fractionation on four-day survival of mice after whole-body neutron irradiation. Brit. 07. Radiol. 38, 878 (1965). 22. J . A . G . DAVIDS,Relative biological effectiveness of fission neutrons for production of the bone marrow syndrome in mice. Int. 07. Radiat. Biol. 10, 299 (1966). 23. J . J . BROERSE, Effects of energy dissipation by monoenergetic neutrons in mammalian cells and tissues. Thesis, Amsterdam (1966). 24. S.B. FIELD,The relative biological effectiveness of fast neutrons for mammalian tissues. Radiology 93, 915 (1969).
177