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Repair and repopulation in mouse skin during fractionated neutron and X-irradiation
Europ. J. Cancer Vol. 10, pp. 241-247. Pergamon Press 1974. Printed in Great Britain
Repair and Repopulation in Mouse Skin During Fractionated Neutron and X-Irradiation j. DENEKAMP and S. B. FIELD Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex, England; and M R C Cyclotron Unit, Hammersmith Hospital, Ducane Road, London W.11, England Abstract--The relative importance of repair and repopulation after single large doses and after multiple small doses has been investigated both for X-rays and neutrons, using a new experimental design. A considerable dose increment is necessary within 24 hr after 1000 rad of X-rays or 450 rad of neutrons; no dose increment is necessary in thefollowing 8 days, but by 15 days 400 rad are necessary in both cases to counteract repopulation. During multifraction irradiation (with 4, 9 or 14fractions of 100 rad of neutrons or 300 rad of X-rays) considerable dose sparing is observed. However, 24 hr after the last fraction of X-rays, a large dose increment was necessary, but after neutrons the skin was more sensitive (i.e. a negative increment); this is presumed to be a result of synchrony. The repopulation rate increased with increasing number of fractions with X-rays. After 4 and 9fractions of neutrons a similar result was obtained. But after 14fractions of neutrons it appeared that the repopulation in 8 days was much smaller than after X-rays. R B E values of 2"2 at I000 rad of X-rays single dose and 3"0 at 300 rad of X-rays per fraction were confirmed.
1. The repair of sublethal injury which for X-rays is complete within 8-24 hr [3]. 2. Reassortment of ceils throughout the cell cycle because of differences in the sensitivity of cells of different ages, and also because of the accumulation after a dose of radiation in particular parts of the cell cycle; for example accumulation in G2 because of mitotic delay [1]. 3. Repopulation of the survivors in response to the reduction in population size [1, 4]. We are interested in the relative contributions of these three processes in organised tissue during fractionated radiotherapy, both with X-rays and with neutrons. It is known that less sublethal injury can be accumulated after neutron irradiation than after X-irradiation, i.e. the extrapolation number is lower for neutron than for X-ray survival curves. Similarly
INTRODUCTION GROSS SKIN reactions after irradiation probably reflect the survival of the basal layer cells of the epidermis as the LD 50tad reflects the survival of intestinal crypt cells. Experiments in which the survival of epidermal clones in vivo and gross skin reactions on the feet of mice have been compared give very similar responses in terms of the dose increments required to counteract repair and repopulation, and in terms of the RBE of fast neutrons relative to X-rays [i, 2]. Thus the dose response curves which have been measured by using skin reactions will be interpreted in terms of cell survival. Some processes which are likely to occur in normal tissues between fractions are: Accepted 22nd February 1974 241
242
J. Denekamp and S. B. Field
the value of ( D 2 - D 1 ) 2 4 h r measured in a number of normal tissues is smaller after neutrons than after X-rays [e.g. 2, 5, 6]. It has been found that ( D 2 - D t)2*hr is large, after a single priming dose of X-radiation for skin, for gut, and for several other normal tissues when measured in vivo [1, 5-7]. If this dose increment is interpreted in terms of D,, then it is considerably larger than most values found for Dq when measured for cells in vitro. Similar large values for D, have however been measured for tumour cells grown as spheroids [8], but D~ was reduced if the cells were irradiated as a single cell suspension. Thus some intimate cell contact may be necessary, as generally occurs in organised tissue in order to achieve the large amount of recovery of sublethal injury associated with a large D~. The variation in sensitivity through the cell cycle and the consequent synchrony that is induced by a priming dose of irradiation can be considerable in skin after X-irradiation [1]. However, this variation in sensitivity may be smaller after high L E T radiation than after a low L E T radiation such as X-rays [9,10]. In this case less effects of synchrony would be expected with neutrons both in vitro and in tissues measured in vivo. The contribution of repopulation to the dose increment required between fractionated doses is not well understood in any tissue. The timing of the response probably depends upon the time at which the death of cells which have been damaged by the irradiation is recognised by the organism. This will be dependent on both dose, the cell cycle time, (which will differ in different tissues) and possibly the turnover time of the differentiated component of the tissues, (which m a y differ in the same tissue measured in different parts of the body, e.g. as a function of skin thickness). It is possible that the time at which damage to the basal layer is perceived by the surviving ceils may depend on the time over which the superficial layers are removed. There is no a priori reason for differences in the rates of reaction after irradiation with X-rays or neutrons, if equivalent biological damage is being caused and is being registered in the same cells in the same overall time. Early attempts to investigate this were based on studies of the pattern of healing of the skin reaction after equivalent peak reactions, caused with X-rays or neutrons [11]. The rate of development of the reaction does not appear to be dose dependent but the peak and healing rate are, and are believed to depend on the level of depletion of the basal layer and the
rate of repopulation [11]. For equivalent peak reactions the healing rates matched well after single or fractionated treatments [12]. The present paper will describe a new design of experiment aimed at measuring the relative contribution of repair and repopulation after fractionated irradiation performed with X-rays or neutrons. The extent of repair of sublethal injury will be measured by the dose increment required in the 24-hr interval after a pretreatment course. This will of course be interfered with by any synchrony induced in the population which might be expected to be greater after X-rays than after neutrons [9, 10]. The repopulation component will be estimated from the additional dose required if an interval greater than 24 hr, for example 8 days, is allowed between the pretreatment and the time of testing. In this case the dose increment between one and eight days will be interpreted as dose necessary to sterilise new cells born through the process of repopulation. The skin reaction of the left hind foot of albino mice has been measured in all the experiments which will be described below. Two strains of mice have been used, SAS/TO non-inbred mice and W H T inbred albino mice. The foot is irradiated with some form of pretreatment, X-rays or neutrons, with various fractionation schedules followed by graded test doses of X-rays to give a dose response curve at various times later (Table 1). Table 1
Pretreatment
1000 rad X-rays 4x 300 rad X-rays 9 x 300 rad X-rays 1 4 x 300 rad X-rays
450 rad neutrons 4x 100 rad neutrons 9 x 100 rad neutrons 14 x 100 rad neutrons
Tested with a range of X-ray doses to obtain a measurable response at
0, 1, . . . . . 15 days 0, 1, - . . . . 15 days 0, 1, - . . . . 15 days 0, 1, . . . . .
15 days
.... 0, 1, - . . . . 0, 1, - . . . . 0, 1, - . . . .
15 days 8 days 8 days 8 days
0, 1, -
After irradiation the skin reactions on the feet of the animals have been scored on a scale of erythema through dry desquamation to ulceration, from shortly after irradiation either 3 or 5 times per week for at least 30 days. The skin scoring system has been described in detail in m a n y previous publications [e.g. 3, 4]. Dose-response curves have been constructed by taking the average skin reaction over a period of 8-30 days after single doses or over an equivalent period for the fractionated doses,
Repair and Repopulation in Mouse Skin During Fractionated Neutron and X-Irradiation
determined by matching the leading edges of the reaction vs time curves for a fairly high reaction level.
increment which we interpret as being necessary to counteract repair after the last of the nine fractions, repair after the first eight being assumed to be complete. This dose is considerable, and is in excess of 300 tad after the last 300 rad dose. The separation of the 1 day curve, and the curves obtained when a greater time is allowed to elapse before giving the test doses, gives us an indication of the dose increment necessary to counteract repopulation after the repair of sublethal injury following 9 daily fractions of 300 tad. In this particular experiment the curves are well separated showing that additional dose is necessary to counteract repopulation in a situation where the cells of the epidermis have been induced to proliferate more rapidly because of the damage caused by the nine fractions of X-rays. This has been discussed in detail elsewhere [4]. Figure 2 shows a similar experiment in which the test doses of X-rays have been given after 9 fractions of 100 tad of neutrons. Again the average dose required to counteract repair of sublethal injury can be derived from the relative positions of the single dose and the "0 day" curves. The curve obtained 24 hr after the last of the nine fractions is displaced to the left of the curve obtained if the test doses are given at day 0. This indicates that there is a negative dose increment necessary 24 hr after the last fraction, i.e. that the population is more sensitive 24 hr after the last fraction than immediately after the last fraction. The curve for animals tested at 8 days after the last of the 9 neutron fractions is displaced well to the right of the 0 and 1 day curves, indicating that this additional dose is necessary
RESULTS
The dose-response curves obtained using X-rays only are shown in Fig. 1. The average 2'~
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reaction is shown as a point for each group of animals and curves have been constructed through those points for animals which were given the same fractionation schedule. A curve is also shown for animals which have been given a single dose of X-rays, i.e. no pre-treatment. The first curve from the left is for animals which have been given 9 daily fractions of X-rays followed by graded test doses immediately after the last 300 tad fraction. This is labelled "0 days". The curve labelled "1 d a y " is for animals which were given their graded test doses of X-rays 24 hr after the last of the 9 small fractions. Similarly curves are shown for animals which were given their range of test doses at 4, 8 or 15 days after the last of the nine 300 rad X-ray fractions. It can be derived e.g. that for a skin reaction of 1.5 a total dose of 3800 rad is required (i.e. 9 x 300+ 1100) if the test dose is given immediately after the ninth fraction, versus a dose of 2500 rad for a single dose of X-rays. This is probably because of the processes of repair, synchrony, and repopulation occurring between each of the 9 fractions, leading to additional dose being necessary. By comparing the single dose curve with the 0 day curve an average dose increment for each of the 8 intervals can be calculated and this is equivalent to approximately 200 rad per fraction interval. If we then compare the 0 day curve with the 1 day curve we obtain the dose
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244
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priming dose of 450 rad of neutrons the dose increment necessary in the first 24 hr is approximately 275 rad of X-rays; between day 1 and day 8 no dose increment is necessary (negative), suggesting that synchrony is obscuring any effect due to repopulation. However, the dose increment necessary between day 1 and day 15 is approximately 450 rad of X-rays. As after the nine X-ray fractions this is equivalent to approximately 30 rad per day necessary to counteract repopulation over this whole period, but the effect is not apparent for the first 8 days. Figure 3 (b) shows the effect after 4 fractions of X-rays or neutrons. After 4 fractions of
to counteract repopulation after treatment with 9 fractions of neutrons.
DISCUSSION
One w a y of comparing the results which have been obtained with neutrons and with X-rays is shown in Fig. 3. Cuts have been taken across the curves in Fig. 1 and Fig. 2 at a reaction level of 1-5. In Fig. 3 the additional dose o f X-rays which is necessary to produce this reaction level after a single, large priming dose, or after 4, 9 or 14 small fractions is shown plotted against the time interval after 3
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Fig. 3. The additional X-ray dose necessary to produce an average reaction level of 1"5 against the interval between pretreatment and the final test X-ray doses. (a) After 1,000 rad of X-rays or 450 rad of neutrons; (b) after 4 x 300 rad of X-rays or 4 x 100 tad of neutrons; (c) after 9 x 300 rad of X-rays or 9 x 100 rad of neutrons; (d) after 14 x 300 rad of X-rays or 14 x 100 tad of neutrons. For (b), (c) and (d) : different lines refer to results of different experiments.
the last fraction at which the range of test X-ray doses was given. Thus, in Fig. 3(a) the additional X-ray dose is plotted against days after giving a single dose of 1000 rad of X-rays or 450 t a d neutron (chosen using an RBE value from Field and Hornsey [13]). Points are shown at 0 days, i.e. immediately after the test dose, at 1 day, 8 days and 15 days. From these curves the amount of dose necessary to counteract repair of sublethal injury, together with the induced synchrony, and the dose necessary to counteract repopulation can be seen. After 1000 rad of X-rays approximately 550 rad are necessary to counteract repair in the first 24 hr. Thereafter approximately 30 t a d per day are necessary to counteract repopulation over the next two weeks. After a
X-rays additional dose is necessary in the first 24 hr to counteract the repair of sublethal injury; in the 8 or 15 days following this however no dose is necessary to counteract repopulation. After 4 fractions of neutrons no additional dose is necessary in the first 24 hr. The cells appear to be more sensitive 24 hr after the 4th fraction than immediately after the 4th fraction, by about 300 rad. Between 1 and 8 days a very small additional dose is necessary which however is still not as great as the dose needed immediately after the fourth fraction. In Fig. 3(c) the results of the test radiation after 9 fractions of X-rays or neutrons are shown. After 9 fractions of X-rays approximately 300 rad were necessary in the following
Repair and Repopulation in Mouse Skin During Fractionated Neutron and X-Irradiation
24 hr to counteract repair. Between 1 and 8 days approximately 60 rad per day are necessary to counteract repopulation, but beyond 8 days a lower value of about 20 rad per day is required. After 9 fractions of neutrons, however, a negative increment appeared to be necessary to counteract repair in the first 24 hr, equal to about 150 rad of X-rays. Beyond 24 hr a positive dose increment is necessary again between 1 and 8 days in order to counteract repopulation and this amounts to almost 80 rad of X-rays per day. This is very similar to the value observed after 9 fractions of X-rays, as shown by the similarity of the slopes in Fig. 3(b). In Fig. 3(d) the response after 14 fractions of X-rays or neutrons are shown. After 14 fractions of X-rays approximately 200 rad are required in the next 24 hr to counteract repair, whereas the dose required to counteract repopulation in the following week has increased enormously to between 100 and 150 rad/per day. This is equivalent to a cell doubling time of less than one day. After 14 fractions of neutrons however a negative increment was again observed for the first 24 hr of about 150 rad. Beyond 24 hr, between the 1st and 8th day, a very small additional dose is necessary to counteract repopulation being only approximately 30 rad per day, i.e. quite different from the measurements obtained after 14 fractions of X-rays. These results are summarised in Fig. 4(a) and (b). Fig. 4(a) shows the "repair" occurring in 2 4 h r (measured as the dose increment
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Table 2. X-ray dose increments necessary to obtain an average skin reaction of 1"5 after pretreatment.
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necessary in the first 2 4 h r after the last fraction in order to produce the standard reaction level of 1.5), against the number of fractions used as pretreatment. The dose increment is positive for all the multifraction X-ray treatments, but negative after neutrons. In Fig. 4(b) the increment necessary to counteract repopulation between day 1 and day 8 is shown against the number of fractions. The response after 4 fractions and after 9 fractions is very similar, but not after 14 fractions. This data is also shown in Table 2 where the dose increments necessary after the various pretreatments are shown.
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Fig. 4. The dose increments necessary to counteract "repair of sublethal injury" and "repopulation" as a function of the number of daily fractions (of 100 rad of neutrons or 300 rad of X-rays). (a) The dose increment in the first 24 hr following the last daily fraction: This is positive after X-rays but negative after neutrons; (b) The dose increment needed between 1 and 8 days after irradiation. This is similar after X-rays and neutrons except for the 14 fraction experiments.
1. The RBE predicted from Field's curve [13] of RBE as a function of dose per fraction has fairly accurately predicted the neutron dose that was equivalent to 1000 rad of X-rays (450 rad of neutrons, RBE = 2"2) and the dose equivalent to 300 rad fractions of X-rays (100 rad of neutrons, R B E - 3 . 0 ) . Figures 3(b), (c) and (d) show that slightly more X-ray dose was necessary immediately after 4, 9 or 14 fractions of neutrons than after the equivalent X-ray scheme. However, if the doses administered
246
J. Denekamp and S. B. Field
one day later are considered, when repair from the last fraction has been allowed, somewhat smaller additional doses are necessary after neutrons than after X-rays. Thus an average of about 3 is obtained, being a little larger or smaller depending on which criterion is used. 2. There was a considerable dose increment necessary in the 24 hr following 4, 9 or 14 fractions of X-rays; in each case with a similar neutron schedule a negative increment was required, i.e. the skin was more sensitive 24 hr after than immediately after the last fraction. This result is not understood. It seems likely that it is attributable to synchrony of the surviving cells, which are in a sensitive phase 2 4 h r after the fourth, ninth or fourteenth fraction of neutrons, but in a more resistant phase after an equivalent schedule of X-rays. We did not anticipate such an effect of synchrony after neutron irradiation. 3. The average dose increment required to counteract the processes occurring between the 4, 9 or 14 fractions can be estimated by comparing the single dose required to give a 1.5
reaction level (2080 rad X-rays) with the additional dose required 0 days after the last fraction (Table 2). Only 50 rad less of X-rays is necessary after 4 fractions of neutrons, 690 less after 9 fractions and 950 less after 14 fractions. Thus a large part of each neutron fraction is "repaired" when the average values are considered. 4. The dose necessary to counteract repopulation in the 8 days following the last of the fractionated treatments is very similar after 4 or 9 fractions of neutrons or X-rays; the results are probably within the experimental errors of the system. However, after 14 fractions of neutrons, the very large increase in the rate of repopulation which was observed after 14 fractions of X-rays is no longer evident. The reason for this is not understood. It is the first time that a suggestion of reduced proliferation rates after neutrons has been obtained experimentally and does not agree with the similar rates of healing observed after fifteen equal fractions in other experiments. Further experiments are clearly needed to clarify the mechanisms involved.
RE~'TA~_,NCES J. DENEKAMP,M. M. BALL and J. F. FOWLER,Recovery and repopulation in mouse skin as a function of time after X-irradiation. Radiat. Res. 37,36 1 (1969). 2. J. DEI~mKAMP,E. W. EMERY and S. B. FIELD, Response of mouse epidermal cells to single and divided doses of fast neutrons. Radiat. Res. 45, 80 (1971). 3. J . F . FOWLER,K. KRAOT, R. E. ELLIS, P. J. LINDOP and R. J. BERRY,The effect of divided doses of 15 MeV electrons on the skin response of mice. Int. d. Radiat. Biol. 9, 241 (1965). 4. J. DEm~KAMP,Changes in the rate ofrepopulation during multifraction irradiation of mouse skin. Brit. d. Radiol. 46, 381 (1973). 5. D . K . BEWLEY,J. F. FOWLER,R. L. MORGAN,J. A. SILVESTER,B. A. TURNER and R. H. THOMLINSON,Pre-therapeutic experiments with the fast neutron beam from the M R C Cyclotron. VII. Experiments on the skin of pigs with fast neutrons and 8 MV X-rays including some effects of dose fractionation. Brit. d. Radiol. 36, 107 (1963). 6. S. HORNSEY,The effectiveness of fast neutrons compared with low LET radiation on cell survival measured in the mouse jejunum. Radiat. Res. 55, 58 (1973). 7. T. L. PHILLIPS, Discussion in Proc. Conf. on Time and Dose Relationships in Radiation Biology as Applied to Radiotherapy, Carme, Calif., 1969. BNL Report 50203 (C-57), p. 194 (1970). 8. R . E . DURAI~ and R. M. SUTHERLAND,Effects of intercellular contact on repair of radiation damage Exp. Cell Res. 71, 75 (1972). 9. E . J . HALL, W. GROSS, R. F. DVORAK, A. M. I~LLEmm and H. H. RossI, Survival curves and age response functions for Chinese hamster cells exposed to X-rays or high LET alpha particles. Radiat. Res. 52, 88 (1972). 10. R. BIRD and J. BURKI, Inactivation of mammalian ceils at different stages of the cell cycle as a function of radiation linear energy transfer. In Biophysical Aspects of Radiation Quality, IAEA, Vienna, p. 241 (1971). 11. J. DEI~mKA~P, J. F. FOWLER, K. KRAOT, C. J. PARNELL and S. B. FIELD, Recovery and repopulation in mouse skin after irradiation with cyclotron neutrons as compared with 250 kV X-rays or 15 MeV electrons. Radiat. Res. 29, 71 (1966). 1.
Repair and Repopulation in Mouse Skin During Fraetionated Neutron and X-Irradiation 12.
13.
S . B . FIELD, T. JONES and R. H. TItOMLINSON,T h e relative effects of fast neutrons and X rays on tumour and normal tissue in the rat. II. Fractionation, recovery and reoxygenation. Brit. J. Radiol. 41~ 597 (1968). S . B . FIELD and S. HORNSEY, RBE values for cyclotron neutrons for effects on normal tissues and tumours as a function of dose and dose fractionation. Europ. J. Cancer7~ 161 (1967).
247
Repair and Repopulation in Mouse Skin During Fractionated Neutron and X-Irradiation j. DENEKAMP and S. B. FIELD Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex, England; and M R C Cyclotron Unit, Hammersmith Hospital, Ducane Road, London W.11, England Abstract--The relative importance of repair and repopulation after single large doses and after multiple small doses has been investigated both for X-rays and neutrons, using a new experimental design. A considerable dose increment is necessary within 24 hr after 1000 rad of X-rays or 450 rad of neutrons; no dose increment is necessary in thefollowing 8 days, but by 15 days 400 rad are necessary in both cases to counteract repopulation. During multifraction irradiation (with 4, 9 or 14fractions of 100 rad of neutrons or 300 rad of X-rays) considerable dose sparing is observed. However, 24 hr after the last fraction of X-rays, a large dose increment was necessary, but after neutrons the skin was more sensitive (i.e. a negative increment); this is presumed to be a result of synchrony. The repopulation rate increased with increasing number of fractions with X-rays. After 4 and 9fractions of neutrons a similar result was obtained. But after 14fractions of neutrons it appeared that the repopulation in 8 days was much smaller than after X-rays. R B E values of 2"2 at I000 rad of X-rays single dose and 3"0 at 300 rad of X-rays per fraction were confirmed.
1. The repair of sublethal injury which for X-rays is complete within 8-24 hr [3]. 2. Reassortment of ceils throughout the cell cycle because of differences in the sensitivity of cells of different ages, and also because of the accumulation after a dose of radiation in particular parts of the cell cycle; for example accumulation in G2 because of mitotic delay [1]. 3. Repopulation of the survivors in response to the reduction in population size [1, 4]. We are interested in the relative contributions of these three processes in organised tissue during fractionated radiotherapy, both with X-rays and with neutrons. It is known that less sublethal injury can be accumulated after neutron irradiation than after X-irradiation, i.e. the extrapolation number is lower for neutron than for X-ray survival curves. Similarly
INTRODUCTION GROSS SKIN reactions after irradiation probably reflect the survival of the basal layer cells of the epidermis as the LD 50tad reflects the survival of intestinal crypt cells. Experiments in which the survival of epidermal clones in vivo and gross skin reactions on the feet of mice have been compared give very similar responses in terms of the dose increments required to counteract repair and repopulation, and in terms of the RBE of fast neutrons relative to X-rays [i, 2]. Thus the dose response curves which have been measured by using skin reactions will be interpreted in terms of cell survival. Some processes which are likely to occur in normal tissues between fractions are: Accepted 22nd February 1974 241
242
J. Denekamp and S. B. Field
the value of ( D 2 - D 1 ) 2 4 h r measured in a number of normal tissues is smaller after neutrons than after X-rays [e.g. 2, 5, 6]. It has been found that ( D 2 - D t)2*hr is large, after a single priming dose of X-radiation for skin, for gut, and for several other normal tissues when measured in vivo [1, 5-7]. If this dose increment is interpreted in terms of D,, then it is considerably larger than most values found for Dq when measured for cells in vitro. Similar large values for D, have however been measured for tumour cells grown as spheroids [8], but D~ was reduced if the cells were irradiated as a single cell suspension. Thus some intimate cell contact may be necessary, as generally occurs in organised tissue in order to achieve the large amount of recovery of sublethal injury associated with a large D~. The variation in sensitivity through the cell cycle and the consequent synchrony that is induced by a priming dose of irradiation can be considerable in skin after X-irradiation [1]. However, this variation in sensitivity may be smaller after high L E T radiation than after a low L E T radiation such as X-rays [9,10]. In this case less effects of synchrony would be expected with neutrons both in vitro and in tissues measured in vivo. The contribution of repopulation to the dose increment required between fractionated doses is not well understood in any tissue. The timing of the response probably depends upon the time at which the death of cells which have been damaged by the irradiation is recognised by the organism. This will be dependent on both dose, the cell cycle time, (which will differ in different tissues) and possibly the turnover time of the differentiated component of the tissues, (which m a y differ in the same tissue measured in different parts of the body, e.g. as a function of skin thickness). It is possible that the time at which damage to the basal layer is perceived by the surviving ceils may depend on the time over which the superficial layers are removed. There is no a priori reason for differences in the rates of reaction after irradiation with X-rays or neutrons, if equivalent biological damage is being caused and is being registered in the same cells in the same overall time. Early attempts to investigate this were based on studies of the pattern of healing of the skin reaction after equivalent peak reactions, caused with X-rays or neutrons [11]. The rate of development of the reaction does not appear to be dose dependent but the peak and healing rate are, and are believed to depend on the level of depletion of the basal layer and the
rate of repopulation [11]. For equivalent peak reactions the healing rates matched well after single or fractionated treatments [12]. The present paper will describe a new design of experiment aimed at measuring the relative contribution of repair and repopulation after fractionated irradiation performed with X-rays or neutrons. The extent of repair of sublethal injury will be measured by the dose increment required in the 24-hr interval after a pretreatment course. This will of course be interfered with by any synchrony induced in the population which might be expected to be greater after X-rays than after neutrons [9, 10]. The repopulation component will be estimated from the additional dose required if an interval greater than 24 hr, for example 8 days, is allowed between the pretreatment and the time of testing. In this case the dose increment between one and eight days will be interpreted as dose necessary to sterilise new cells born through the process of repopulation. The skin reaction of the left hind foot of albino mice has been measured in all the experiments which will be described below. Two strains of mice have been used, SAS/TO non-inbred mice and W H T inbred albino mice. The foot is irradiated with some form of pretreatment, X-rays or neutrons, with various fractionation schedules followed by graded test doses of X-rays to give a dose response curve at various times later (Table 1). Table 1
Pretreatment
1000 rad X-rays 4x 300 rad X-rays 9 x 300 rad X-rays 1 4 x 300 rad X-rays
450 rad neutrons 4x 100 rad neutrons 9 x 100 rad neutrons 14 x 100 rad neutrons
Tested with a range of X-ray doses to obtain a measurable response at
0, 1, . . . . . 15 days 0, 1, - . . . . 15 days 0, 1, - . . . . 15 days 0, 1, . . . . .
15 days
.... 0, 1, - . . . . 0, 1, - . . . . 0, 1, - . . . .
15 days 8 days 8 days 8 days
0, 1, -
After irradiation the skin reactions on the feet of the animals have been scored on a scale of erythema through dry desquamation to ulceration, from shortly after irradiation either 3 or 5 times per week for at least 30 days. The skin scoring system has been described in detail in m a n y previous publications [e.g. 3, 4]. Dose-response curves have been constructed by taking the average skin reaction over a period of 8-30 days after single doses or over an equivalent period for the fractionated doses,
Repair and Repopulation in Mouse Skin During Fractionated Neutron and X-Irradiation
determined by matching the leading edges of the reaction vs time curves for a fairly high reaction level.
increment which we interpret as being necessary to counteract repair after the last of the nine fractions, repair after the first eight being assumed to be complete. This dose is considerable, and is in excess of 300 tad after the last 300 rad dose. The separation of the 1 day curve, and the curves obtained when a greater time is allowed to elapse before giving the test doses, gives us an indication of the dose increment necessary to counteract repopulation after the repair of sublethal injury following 9 daily fractions of 300 tad. In this particular experiment the curves are well separated showing that additional dose is necessary to counteract repopulation in a situation where the cells of the epidermis have been induced to proliferate more rapidly because of the damage caused by the nine fractions of X-rays. This has been discussed in detail elsewhere [4]. Figure 2 shows a similar experiment in which the test doses of X-rays have been given after 9 fractions of 100 tad of neutrons. Again the average dose required to counteract repair of sublethal injury can be derived from the relative positions of the single dose and the "0 day" curves. The curve obtained 24 hr after the last of the nine fractions is displaced to the left of the curve obtained if the test doses are given at day 0. This indicates that there is a negative dose increment necessary 24 hr after the last fraction, i.e. that the population is more sensitive 24 hr after the last fraction than immediately after the last fraction. The curve for animals tested at 8 days after the last of the 9 neutron fractions is displaced well to the right of the 0 and 1 day curves, indicating that this additional dose is necessary
RESULTS
The dose-response curves obtained using X-rays only are shown in Fig. 1. The average 2'~
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reaction is shown as a point for each group of animals and curves have been constructed through those points for animals which were given the same fractionation schedule. A curve is also shown for animals which have been given a single dose of X-rays, i.e. no pre-treatment. The first curve from the left is for animals which have been given 9 daily fractions of X-rays followed by graded test doses immediately after the last 300 tad fraction. This is labelled "0 days". The curve labelled "1 d a y " is for animals which were given their graded test doses of X-rays 24 hr after the last of the 9 small fractions. Similarly curves are shown for animals which were given their range of test doses at 4, 8 or 15 days after the last of the nine 300 rad X-ray fractions. It can be derived e.g. that for a skin reaction of 1.5 a total dose of 3800 rad is required (i.e. 9 x 300+ 1100) if the test dose is given immediately after the ninth fraction, versus a dose of 2500 rad for a single dose of X-rays. This is probably because of the processes of repair, synchrony, and repopulation occurring between each of the 9 fractions, leading to additional dose being necessary. By comparing the single dose curve with the 0 day curve an average dose increment for each of the 8 intervals can be calculated and this is equivalent to approximately 200 rad per fraction interval. If we then compare the 0 day curve with the 1 day curve we obtain the dose
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244
J . Denekamp and S. B. Field
priming dose of 450 rad of neutrons the dose increment necessary in the first 24 hr is approximately 275 rad of X-rays; between day 1 and day 8 no dose increment is necessary (negative), suggesting that synchrony is obscuring any effect due to repopulation. However, the dose increment necessary between day 1 and day 15 is approximately 450 rad of X-rays. As after the nine X-ray fractions this is equivalent to approximately 30 rad per day necessary to counteract repopulation over this whole period, but the effect is not apparent for the first 8 days. Figure 3 (b) shows the effect after 4 fractions of X-rays or neutrons. After 4 fractions of
to counteract repopulation after treatment with 9 fractions of neutrons.
DISCUSSION
One w a y of comparing the results which have been obtained with neutrons and with X-rays is shown in Fig. 3. Cuts have been taken across the curves in Fig. 1 and Fig. 2 at a reaction level of 1-5. In Fig. 3 the additional dose o f X-rays which is necessary to produce this reaction level after a single, large priming dose, or after 4, 9 or 14 small fractions is shown plotted against the time interval after 3
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Fig. 3. The additional X-ray dose necessary to produce an average reaction level of 1"5 against the interval between pretreatment and the final test X-ray doses. (a) After 1,000 rad of X-rays or 450 rad of neutrons; (b) after 4 x 300 rad of X-rays or 4 x 100 tad of neutrons; (c) after 9 x 300 rad of X-rays or 9 x 100 rad of neutrons; (d) after 14 x 300 rad of X-rays or 14 x 100 tad of neutrons. For (b), (c) and (d) : different lines refer to results of different experiments.
the last fraction at which the range of test X-ray doses was given. Thus, in Fig. 3(a) the additional X-ray dose is plotted against days after giving a single dose of 1000 rad of X-rays or 450 t a d neutron (chosen using an RBE value from Field and Hornsey [13]). Points are shown at 0 days, i.e. immediately after the test dose, at 1 day, 8 days and 15 days. From these curves the amount of dose necessary to counteract repair of sublethal injury, together with the induced synchrony, and the dose necessary to counteract repopulation can be seen. After 1000 rad of X-rays approximately 550 rad are necessary to counteract repair in the first 24 hr. Thereafter approximately 30 t a d per day are necessary to counteract repopulation over the next two weeks. After a
X-rays additional dose is necessary in the first 24 hr to counteract the repair of sublethal injury; in the 8 or 15 days following this however no dose is necessary to counteract repopulation. After 4 fractions of neutrons no additional dose is necessary in the first 24 hr. The cells appear to be more sensitive 24 hr after the 4th fraction than immediately after the 4th fraction, by about 300 rad. Between 1 and 8 days a very small additional dose is necessary which however is still not as great as the dose needed immediately after the fourth fraction. In Fig. 3(c) the results of the test radiation after 9 fractions of X-rays or neutrons are shown. After 9 fractions of X-rays approximately 300 rad were necessary in the following
Repair and Repopulation in Mouse Skin During Fractionated Neutron and X-Irradiation
24 hr to counteract repair. Between 1 and 8 days approximately 60 rad per day are necessary to counteract repopulation, but beyond 8 days a lower value of about 20 rad per day is required. After 9 fractions of neutrons, however, a negative increment appeared to be necessary to counteract repair in the first 24 hr, equal to about 150 rad of X-rays. Beyond 24 hr a positive dose increment is necessary again between 1 and 8 days in order to counteract repopulation and this amounts to almost 80 rad of X-rays per day. This is very similar to the value observed after 9 fractions of X-rays, as shown by the similarity of the slopes in Fig. 3(b). In Fig. 3(d) the response after 14 fractions of X-rays or neutrons are shown. After 14 fractions of X-rays approximately 200 rad are required in the next 24 hr to counteract repair, whereas the dose required to counteract repopulation in the following week has increased enormously to between 100 and 150 rad/per day. This is equivalent to a cell doubling time of less than one day. After 14 fractions of neutrons however a negative increment was again observed for the first 24 hr of about 150 rad. Beyond 24 hr, between the 1st and 8th day, a very small additional dose is necessary to counteract repopulation being only approximately 30 rad per day, i.e. quite different from the measurements obtained after 14 fractions of X-rays. These results are summarised in Fig. 4(a) and (b). Fig. 4(a) shows the "repair" occurring in 2 4 h r (measured as the dose increment
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245
Table 2. X-ray dose increments necessary to obtain an average skin reaction of 1"5 after pretreatment.
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Day 15-day 1 "repopulation": x 400 N 400
necessary in the first 2 4 h r after the last fraction in order to produce the standard reaction level of 1.5), against the number of fractions used as pretreatment. The dose increment is positive for all the multifraction X-ray treatments, but negative after neutrons. In Fig. 4(b) the increment necessary to counteract repopulation between day 1 and day 8 is shown against the number of fractions. The response after 4 fractions and after 9 fractions is very similar, but not after 14 fractions. This data is also shown in Table 2 where the dose increments necessary after the various pretreatments are shown.
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CONCLUSIONS
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Fig. 4. The dose increments necessary to counteract "repair of sublethal injury" and "repopulation" as a function of the number of daily fractions (of 100 rad of neutrons or 300 rad of X-rays). (a) The dose increment in the first 24 hr following the last daily fraction: This is positive after X-rays but negative after neutrons; (b) The dose increment needed between 1 and 8 days after irradiation. This is similar after X-rays and neutrons except for the 14 fraction experiments.
1. The RBE predicted from Field's curve [13] of RBE as a function of dose per fraction has fairly accurately predicted the neutron dose that was equivalent to 1000 rad of X-rays (450 rad of neutrons, RBE = 2"2) and the dose equivalent to 300 rad fractions of X-rays (100 rad of neutrons, R B E - 3 . 0 ) . Figures 3(b), (c) and (d) show that slightly more X-ray dose was necessary immediately after 4, 9 or 14 fractions of neutrons than after the equivalent X-ray scheme. However, if the doses administered
246
J. Denekamp and S. B. Field
one day later are considered, when repair from the last fraction has been allowed, somewhat smaller additional doses are necessary after neutrons than after X-rays. Thus an average of about 3 is obtained, being a little larger or smaller depending on which criterion is used. 2. There was a considerable dose increment necessary in the 24 hr following 4, 9 or 14 fractions of X-rays; in each case with a similar neutron schedule a negative increment was required, i.e. the skin was more sensitive 24 hr after than immediately after the last fraction. This result is not understood. It seems likely that it is attributable to synchrony of the surviving cells, which are in a sensitive phase 2 4 h r after the fourth, ninth or fourteenth fraction of neutrons, but in a more resistant phase after an equivalent schedule of X-rays. We did not anticipate such an effect of synchrony after neutron irradiation. 3. The average dose increment required to counteract the processes occurring between the 4, 9 or 14 fractions can be estimated by comparing the single dose required to give a 1.5
reaction level (2080 rad X-rays) with the additional dose required 0 days after the last fraction (Table 2). Only 50 rad less of X-rays is necessary after 4 fractions of neutrons, 690 less after 9 fractions and 950 less after 14 fractions. Thus a large part of each neutron fraction is "repaired" when the average values are considered. 4. The dose necessary to counteract repopulation in the 8 days following the last of the fractionated treatments is very similar after 4 or 9 fractions of neutrons or X-rays; the results are probably within the experimental errors of the system. However, after 14 fractions of neutrons, the very large increase in the rate of repopulation which was observed after 14 fractions of X-rays is no longer evident. The reason for this is not understood. It is the first time that a suggestion of reduced proliferation rates after neutrons has been obtained experimentally and does not agree with the similar rates of healing observed after fifteen equal fractions in other experiments. Further experiments are clearly needed to clarify the mechanisms involved.
RE~'TA~_,NCES J. DENEKAMP,M. M. BALL and J. F. FOWLER,Recovery and repopulation in mouse skin as a function of time after X-irradiation. Radiat. Res. 37,36 1 (1969). 2. J. DEI~mKAMP,E. W. EMERY and S. B. FIELD, Response of mouse epidermal cells to single and divided doses of fast neutrons. Radiat. Res. 45, 80 (1971). 3. J . F . FOWLER,K. KRAOT, R. E. ELLIS, P. J. LINDOP and R. J. BERRY,The effect of divided doses of 15 MeV electrons on the skin response of mice. Int. d. Radiat. Biol. 9, 241 (1965). 4. J. DEm~KAMP,Changes in the rate ofrepopulation during multifraction irradiation of mouse skin. Brit. d. Radiol. 46, 381 (1973). 5. D . K . BEWLEY,J. F. FOWLER,R. L. MORGAN,J. A. SILVESTER,B. A. TURNER and R. H. THOMLINSON,Pre-therapeutic experiments with the fast neutron beam from the M R C Cyclotron. VII. Experiments on the skin of pigs with fast neutrons and 8 MV X-rays including some effects of dose fractionation. Brit. d. Radiol. 36, 107 (1963). 6. S. HORNSEY,The effectiveness of fast neutrons compared with low LET radiation on cell survival measured in the mouse jejunum. Radiat. Res. 55, 58 (1973). 7. T. L. PHILLIPS, Discussion in Proc. Conf. on Time and Dose Relationships in Radiation Biology as Applied to Radiotherapy, Carme, Calif., 1969. BNL Report 50203 (C-57), p. 194 (1970). 8. R . E . DURAI~ and R. M. SUTHERLAND,Effects of intercellular contact on repair of radiation damage Exp. Cell Res. 71, 75 (1972). 9. E . J . HALL, W. GROSS, R. F. DVORAK, A. M. I~LLEmm and H. H. RossI, Survival curves and age response functions for Chinese hamster cells exposed to X-rays or high LET alpha particles. Radiat. Res. 52, 88 (1972). 10. R. BIRD and J. BURKI, Inactivation of mammalian ceils at different stages of the cell cycle as a function of radiation linear energy transfer. In Biophysical Aspects of Radiation Quality, IAEA, Vienna, p. 241 (1971). 11. J. DEI~mKA~P, J. F. FOWLER, K. KRAOT, C. J. PARNELL and S. B. FIELD, Recovery and repopulation in mouse skin after irradiation with cyclotron neutrons as compared with 250 kV X-rays or 15 MeV electrons. Radiat. Res. 29, 71 (1966). 1.
Repair and Repopulation in Mouse Skin During Fraetionated Neutron and X-Irradiation 12.
13.
S . B . FIELD, T. JONES and R. H. TItOMLINSON,T h e relative effects of fast neutrons and X rays on tumour and normal tissue in the rat. II. Fractionation, recovery and reoxygenation. Brit. J. Radiol. 41~ 597 (1968). S . B . FIELD and S. HORNSEY, RBE values for cyclotron neutrons for effects on normal tissues and tumours as a function of dose and dose fractionation. Europ. J. Cancer7~ 161 (1967).
247