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Leg contracture in mice: an assay of normal tissue response
0360-3016/84 SO3 00 + 00 Copynghl 6 19X4 Pcrgamon Press Lid
Inr I Rudiamn Oncdogy Bml Phys. Vol. IO. pp. 1053-1061 Pnnted in the U.S.A All rights reserved
@ Original Contribution LEG CONTRACI’URE IN MICE: AN ASSAY OF NORMAL TISSUE RESPONSE HELEN Radiation
B. STONE, PH.D.
Oncology Research Laboratory, CED-200, Department of Radiation University of California. San Francisco, CA 94143
Oncology,
Leg coutracture, defined as the difference in extensibility of the control and irradiated hind legs of mice, was found to correhte with shtgk doses of radiation from about 20 to 80 Gy. The time of development of the early phase of the response coincided with that reported for the appearance of the acute skin response, and in some cases, partially reversed as this reaction healed. The contracture then progressed again at a moderate rate through 90 days, and then more slowly through one year. Skin contraction, measured by decrease in intertattoo distance, was assayed in the same mice. It followed the same time course as leg contracture, but had a different doseresponse relationship. Maximal contraction occurred following doses of Xl Gy or more, reaching this level sooner following higher doses. The early reactions in individual mice were not reliible in predicting late response for either assay. To determine the contribution of skin contraction to the overall leg contracture response, mice were sacrificed and the leg contracture measured before and after the removal of the skin of the leg. After doses of up to 30 Gy, little contracture remained from skiing the leg, indicating that skin contraction was largely responsible for leg contracture in this dose range. After doses of about 45 Gy and above, some contracture remained in the skinned legs, although less than in intact legs. This indicated that injury to the deeper tissues of the leg as well as to the skin was responsible for sontracture at these higher doses. There was little or no enhancement of either skin contraction or leg contracture by the hypoxic cell sensitizers metronidazole or misonidazole. Hypoxic cell sensitizers, Metronidazole, Misonidazole, Late normal tissue response, Leg contracture, traction.
INTRODUCTION Normal tissue response governs the total radiation dose that can be administered in a given regimen in an attempt to eradicate a tumor. New therapies designed to enhance local tumor control will not benefit the patient if they increase the rate of normal tissue complications. Many assays have been developed to evaluate the radioresponse of various normal tissues in animals. Those most commonly used fall into two categories: those involving acute responses that result from injury to rapidly proliferating epithelial cell populations such as those of the skin,s.29*30 or the intestine,3’ and those that involve late responses that result from injury to slowly proliferating cells and interactions between vascular, connective and parenchymal tissues.4*‘o*2*~32 Included in the latter category are assays for radiation myelopathy,’ nephritis,‘* and pneumonitis.” Contracture, atrophy and fibrosis can result
Acknowledgments-1 am grateful to Vi&i Woodard Zelle, Judith &eider, Nancy Hunter and Paul Hagopian for excellent technical assistance; to Donald Highfield, Paul Hagopian, and Karla Cavarra for preparing the figures; to Bailey Moore and his staff and S. L. Gillespie for making the jigs; and to Drs. John Hicks and Geoff Ibbott for dosimetry; and to Drs. W. C. Dewey and H. R. Withers for helpful discussions of the manuscript.
Skin con-
from radiotherapy administered to the limbI and are considered to be late responses in man. In this paper, we describe an assay for leg contracture in mice, showing the response to single doses of radiation, the time course of the development of contracture through one year after treatment, and the effect of hypoxic cell sensitizers. Skin contraction was also assessed in the same mice, using the tattoo separation method of Hayashi and SuitV9and compared to the leg contracture response.
METHODS
AND MATERIALS
These experiments were performed from 1975 to 1976 at the University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, and from 1976 to 1982 at Colorado State University. Table 1 summarizes the combinations of mice and radiation sources.
This investigation was supported by Grant Numbers CA 11138, CA 06294 and CA 20344, awarded by the National Cancer Institute, DHEW. Accepted for publication 21 March 1984.
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July 1984, Volume 10, Number 7
Table 1. Irradiators and mice Year
Site
Irradiator
1975-76 1976-77 1978431
UTMDAH csu csu
Cesium, dual source 300 kVp X ray Cesium, single source
Dose rate 10.7-10.5 Gy/mint 5.9 Gy/min$ 9.9-9.1 Gy/mint
Mice’ C3Hf/Bu specific pathogen-free C3H/HeJ conventional C3H/HeJ conventional
UTMDAH: The University of Texas Cancer Center, M.D. Anderson Hospital and Tumor Institute, Section of Experimental Radiotherapy, Houston, TX; CSU: Colorado State University, Department of Radiology and Radiation Biology, Fort Collins, CO. * C3Hf/Bu mice were from the specific pathogen-free colony of the Section of Experimental Radiotherapy, UTMDAH. C3H/HeJ mice were from Jackson Laboratory, Bar Harbor, ME. t Lithium fluoride dosimetry.
$ Ion chamber dosimetry. Mice Female C3Hf/Bu and C3H/HeJ mice were irradiated when 12 to 16 weeks old. There were 6 to 11 mice per assay point, as noted in the figure legends. Irradiation In the “‘Cs irradiator at M.D. Anderson Hospital,* the field was 3.0 cm in diameter.12 The mice were restrained without anesthesia in a jig with the right hind leg extended across the 3 cm opening in the jig and the heel 0 to 1 mm outside the opening.*’ The feet were not irradiated. The 13’Csirradiator at Colorado State University? had a field 3 X 18 cm, and the mice were irradiated in a jig similar to that used at M.D. Anderson, the right hind leg extended across the shorter dimension of the beam. Anesthesia was not used. Data from mice treated similarly on the two cesium irradiators were indistinguishable, and have been plotted together where appropriate. A second radiation source at Colorado State University was a 300 kVp X ray unit* with an HVL of 1.36 mm Cu. The field was 2.7 cm in diameter. The mice irradiated on this machine were anesthetized with sodium pentobarbital, 0.065 mg/g body weight, and half of the treatment was administered from the dorsal and half from the ventral side. These data were not pooled with those from animals treated with 13’Cs radiation. Animals in a given treatment group were not caged together,
and the person
making
measurements
did not
know the treatment group. Drugs Metronidazole
was obtained from Searle and Co., San Misonidazole was obtained from Roche Products, Ltd., Welwyn Garden City, Hertfordshire, England, courtesy of Dr. Carey Smithen, and from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. The drugs were dissolved fresh daily in Ringer’s solution to give concentrations of 10 and 20 mg/ml, respectively. The doses were 0.8 to 1.0 mg/g body weight
Juan,
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* Custom model, Atomic Energy of Canada, Ltd., Ottawa, Canada.
and were administered i.p. 30 min before irradiation In our experience these doses give maximum sensitization of hypoxic cells in tumors. 23*24Controls were given an equivalent volume of Ringer’s solution i.p. 30 min before irradiation. Leg contracture assay Extensibility of both the treated and control hind legs in each mouse was measured using the jig shown in Fig. 1. The tail was placed between the vertical posts and held taut while each leg was extended against a millimeter ruler embedded in the base. Both the control and irradiated legs could be extended easily to a certain point beyond which there was considerably greater resistance to further extension. Readings were made at this point, measuring to the tip of the central digit, and were reproducible to within + 1 mm when repeated by the same person or a different individual. In untreated mice, the difference between the two legs was rarely greater than 1 mm when care was taken to avoid rotating the pelvis of the mouse. From the difference in extensibility of the two legs, group means and standard deviations were calculated. The mice were not anesthetized during any of the measurements, as each measurement was made within about 2 seconds. Discomfort appeared to be minimal. Skin contraction assay This assay was performed in the same mice that were used for the leg contracture assay. The hair on the hind legs was removed with an electric clipper 6 or more days before irradiation, and tattoo marks approximately 1 cm apart were made in the skin overlying the tibia-fibula, using India ink. The distance between the centers of the tattoo marks was measured before irradiation and at various intervals afterward. The data were expressed as a percentage of the pre-irradiation distance between the marks, corrected for the change in values on the untreated contralateral legs (by multiplying by pre-treatment/posttreatment measurements). Group means and standard deviations were calculated.
t Custom model, J. L. Shepherd and Assoc., Glendale, CA. $ Maxitron 300, General Electric Co. Milwaukee, WI.
Leg contracture in mice 0 H. B. STONE
1055
was evident below 20 Gy, but an increasing response was observed with increasing doses through 80 Gy. In contrast, the skin contraction response, measured in the same mice, showed a correlation with dose between 15 to 30 Gy, but at 50 Gy and above, was not responsive to dose (Fig. 3). With both endpoints, the shapes of the dose-response relationships were similar at 23 days to those at 320 days after irradiation, but damage was less severe at 23 days.
no damage
Time courses The time course of the leg contracture response is shown in Fig. 4. Error bars have been omitted for clarity, but
Fig. I. Jig for measuring leg contracture. The tail is held so that the rump is firmly placed against the posts. Each leg is extended along the respective scale using forceps, and measurements are read at the tip of the toes. RESULTS
Dose-response relationships
were similar to those in Figs. 2 and 3 in all experiments. Leg contracture developed rapidly during the first 23 days after irradiation. At the next measurement, at 44 days, the contracture had partially reversed or plateaued. Then, for doses 30 Gy and above, it progressed at a slower rate through 90 days followed either by a very gradual increase or no further progression through one year. A similar time course was observed for skin contraction (Fig. 5), except that contraction ceased when the skin shrank to 24 to 32% of the pre-treatment length. Although contraction developed rapidly during the first 23 days, most of this appeared to take place between day 14 and
The leg contracture responses 23 and 320 days after single doses of radiation are shown in Figure 2. Little or
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Fig. 3. Dose-response relationship for skin contraction 23 and 320 days after irradiation. Open circles were measurements made 138 days after treatment in a separate experiment. Error bars were omitted from these points for clarity, but were of similar magnitude to those shown. Solid symbols: 6 mice/point; open symbols: 10 mice/point, initially.
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sontracture in mice 0 H B. STONE
Efect of weekly measurement Because the extension of the legs of the mice during measurement might have a therapeutic effect, reducing the amount of contraction, we compared both skin contraction and leg contracture in mice measured at weekly intervals with those measured only at 6 months after :-,.>I_.:-_ * &51x “Z..--_rL._.^.. ^:-_:C,,_. A:I IllilUlall”ll. ,4L II1”11WS,.I.^_^ LIICICZ wiw ^^ 11”>‘ g‘1111Ldlll uuference between the two groups with either response (Table 3). Correlation between skin contraction and leg contracture The relationship between skin contraction and leg contracture is shown in Fig. 6. The data used in these cal-
culations were from mice irradiated with doses of 23.8 to 66.6 Gy, and scored from 37 to 200 days after irradiation. These were both doses and times at which contracture was observed. The solid line and its dashed extrapolation are based on a least squares fit of the data from 0 to 0.8 cm contracture. A significant amount of skin contraction occurred in the absence of leg contracture (tattoo separation at 80% for 0 cm leg contracture), reflecting a slightly higher threshold dose for leg contracture. Above 1.O cm leg contracture, the skin contraction started to level off. At the termination of two experiments at 138 and 190 days after irradiation, respectively, leg contracture was measured, first in live animals, and then immediately after cervical dislocation, in intact animals and following removal of the skin of the hind legs. The results are shown in Fig. 7. Although the data differed slightly in the two experiments, ieg contracture was reduced in sicinned iegs at all doses tested. At the lower doses, little residual contracture was evident in skinned legs, but at higher doses there was residual contracture The extensibility of the legs was slightly greater in dead than in live mice, averaging 0.06 +- 0.01 (se.) cm greater in the treated legs, and 0.02 :C 0.01 cm in the control
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Fig. 6. Correlation between skin contraction and leg wntracture. Data were pooled from several experiments, from mice treated with 23.6 Gy to 66.6 Gy, and from 37 to 200 days after irradiation. The solid line is a least squares fit to the points from 0 to 0.8 cm contracture. The dashed line is extrapolated from the solid line.
Table 3. Effect of weekly measurement of skin and leg contracture 6 months after irradiation Dose, GY Skin wnuaction-_$ _^
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Meas. weekly of preirradiation
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Note: None of the differences were statistically significant. All groups contained 9 to 10 animals. Mice were irradiated with 300 kVp X rays, and were anesthetized barbital.
with sodium pento-
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Radiation Oncology 0 Biology 0 Physics
legs. Thus, 0.04 f 0.02 cm of the decrease in contracture in the skinned legs resulted from differences in extensi. .. . bility between iive and dead mice. Skinning resuited in an increase of 0.01 + 0.01 cm in the extensibility of the control legs. Effect of hypoxic cell sensitizers
The hypoxic cell sensitizers metronidazole and misonidazole were given i.p. to groups of mice 30 min before irradiation to determine whether the skin and leg contracture responses would be enhanced by these agents. The results are shown in Fig. 8a and b. There was no significant enhancement of either response by the sensitizers. Correlation between early and late responses in individual animals
Late leg contracture and skin contraction (at 280 to 320 days after irradiation) have been plotted in Fig. 9a and b, respectively, as functions of the early reactions (at 23 days) in individual mice. Although both plots show general correlations between early and late damage, there was considerable scatter of the data. The leg contracture data from mice treated with metronidazole or misonidazole showed more scatter than the control data (Fig. 9a), although neither sensitizer affected the scatter in the relationshinr hetwwn _____________ (Fie. \_ _5). --_..__-- P~AV ___‘, ag8 ia?e ~kjn COntractinn 9b). In both assays, however, very few individuals showed a late response that was less than or equal to the early responses (dashed lines in Fig. 9).
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DISCUSSION Th_e lep SPCQV l&R” lit~ the ~ltin mntmrtinn a mntrwtnrp _.,_~1.__._~_ UUJ, . ..w .,_.I .,“......“.I”SI assay, can be used as a measure of late response. Repeated measurements in the same animals give information on the time course of the development of contracture. The response correlates with single radiation doses from about 20 to 80 Gy, a range in which several transplantable murine tumors are locally controlled.23*24~26 It can therefore be used to estimate therapeutic gain in treatments involving combined modalities. For example, we found a TCDSO of 64.2 Gy for the mammary carcinoma MDAH-MCa-4, and in mice given 1.O mg misonidazole/ g body weight before irradiation, a TCDSO of 27.5 Gy.*’ From the dose-response curve in Fig. 8a, these doses would give, at about 120 days after irradiation, approximately 1.5 and 0.5 cm leg contracture, respectively, clearly a therapeutic gain when misonidazole was used. The skin contraction assay
The present studies have shown that the maximum level of skin contraction is about 24 to 32% of the original tattoo distance. Single doses of 50 Gy and above produced this level by 90 days, but after 30 Gy it was not reached until about 300 days. Thus, this assay has a useful range from about 15 to 30 Gy (single doses) but this may be extended to 40 to 50 Gy if measurements are made within icK1
li_ays.
The time course of the skin contraction response has been reported by Hayashi and Suit,’ and Masuda et al.,” and Pearson and Steel.16 Our data generally agree with
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Leg contracturein mice 0 H. B. STONE
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Fig. 9. Correlation between early (23 days) and late (280 to 320 days) leg contracture (a) or skin contraction (b) in individual mice. The dashed lines represent equal levels of early and late response. Metronidazole (“Metro”) and misonidazole (“Miso”) were given i.p. 30 min before irradiation. The doses were 0.8 to 1.O mg/g body weight.
of damage was observed between 90 and 365 days only in the groups exhibiting damage at 90 days. However, Masuda et sml.Is found that a group treated with 12 Gy did not start to show contraction until 320 days, but that this group and others treated with higher doses continued to contract through 540 days. The response itself or the dose-response relationship may depend on strain or species, because Berry, e6 aL2 found no dimensional changes in the skin of guinea pigs after doses of up to 26 Gy, although these animals did exhibit acute skin reactions. Differences in both the time course and dose-response relationship have been observed I.., D~a,-wv-. “,.A Ct~l16 lult..ra~... i..rr\ rt,.dnc .4 m;,..e “J 1 &daIJvli 411&h h.z?C~b,I ubCWlrc.‘I &“” aucuII* “1 Lll‘bA.. theirs. Progression
Early reactions The underlying mechanisms in the development of skin and leg contracture after irradiation are complex and not well understood. The early wave of contraction occurs at the time of the development of acute desquamation, although contraction may occur in the absence of ulceration.’ Desquamation itself is primarily a consequence of killing of stem cells in the basal epithelium,29 but the delay in its appearance after irradiation corresponds to the time required for the radioresistant cells in subsequent stages of differentiation to complete their life cycles and to be s10ughed.‘~ Masuda et al.” have stated that early skin shrinkage similarly reflects depletion of basal epitheiial ceiis. The correspondence is not direct, however, because other cellular and non-cellular tissue components
contribute to the overall response so that the magnitude of contraction is not as great as might be expected for the level of stem cell depletion: at 20 Gy, cell survival is on the order of low6 29 but skin remains at 70% of its original dimensions and leg contracture is about 0.2 cm. Possible mechanisms, late reactions The similarity of skin contraction and leg contracture suggest that the two assays measure the same phenomena. At doses of 15 to 30 Gy, this may indeed be the case: both assays correlate with dose in this range (Figs. 2 and 3), the lower levels of skin contraction correlate well with lxx+
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legs abolished most of the leg contracture (Fig. 7). At higher doses, however, leg contracture continued to increase with dose (Fig. 2), but skin contraction did not (Fig. 3). This was seen also in the correiation between the two responses (Fig. 6). Residual contracture after removal of the skin (Fig. 7) indicated that skin contraction was not the only cause of leg contracture at doses of 45 Gy or more. Leg contracture does not result from shortening of the bones of the leg when adult mice are irradiated (Kouji Masuda, personal communication, January, 1976). The extent to which early reactions are responsible for the development of late reactions is not known. They may have some, but probably not all, mechanisms in common. Although there was a general correlation between early and late damage for both skin contraction
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Radiation Oncology 0 Biology 0 Physics
and leg contracture, there were wide individual differences so that the early reactions were of little value in predicting late damage in a given animal (Fig. 9). This has been observed in other animal studies6*9,‘4*20and in hu-___ ,521 m-- ~~ . n. .I& c mans.-‘-‘-- rearson ana 31eel” round that CBA mice had more severe acute skin reactions than C57B1 mice, but had less severe late reactions. This suggests that factors other than the severity of the early skin reaction may also contribute to the development of late damage. Nevertheless, cells that die early and are not replaced contribute to the atrophy observed after the early reactions have subsided. Whether either skin contraction or leg contracture in mice respond to fractionated regimens in a manner similar to that of early or late endpoints of damage, as shown by Thames et uI.,~’is not presently known. This may depend on the time after treatment. Eflect of hypoxic
July 1984, Volume 10, Number 7
ulation might be masked by the response of a predominantly aerobic population in both the skin contraction and leg contracture assays. Foot ioss
Hopewell” has described a lesion at the flexure between the leg and foot of rats that developed with a latency of 2 or more months after irradiation. The lesion led to edema, necrosis and amputation of the foot. Foot loss in our mice may have occurred as a result of a similar lesion. Although the feet of our mice were shielded during irradiation, the ankle region received a substantial proportion of the dose to the leg, and atrophy of the tissues proximal to the ankle may have produced constriction of the vascular and lymphatic channels, leading to the observed edema, fracture and necrosis.
cell sensitizers
The failure to find significant sensitization of either skin contraction or leg contracture by metronidazole or misonidazole with our usual irradiation conditions was unexpected, in view of the observation that acute skin responses were sensitized by both these agents3*25.33and by oxygen.7.30 The data of Wondergem et al.33 have suggested that hypoxia in mouse skin is the result of stress-induced vasoconstriction, or of vascular obstruction during irradiation. However, sensitization of a small hypoxic subpop-
CONCLUSIONS The leg contracture assay is easy to use and can provide information on the time course of the development of late damage, as well as its modification by other therapeutic modalities. It is dose-responsive over a wide range of doses, including those at which local control of a variety of murine tumors occurs. Further studies are needed to understand more fully the factors that influence the development of this response and the time-dose relationships in multifraction radiotherapy.
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Arcangeii, G., Friedman, M.. Paoiuzi, R.: A quantitative study of late radiation effect on normal skin and subcutaneous tissues in human beings. Brit. J. Radiol. 47: 44-
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50, 1974. Beny, R.J., Mole, R.H., Barnes, D.W.: Skin response lo X-irradiation in the guinea pig. Int. J. Radiat. Biol. 30: 535-541, 1976.
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3. Brown, J.M.: Selective radiosensitization 4. 5. 6.
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of the hypoxic cells of mouse tumors with the nitroimidazoles metronidazole and Ro 7-0582. Radial. Res. 64: 633-647, 1975. Casarett, G.W.: Basic mechanisms. Cancer 37: 1002-1010, 1976. Chu, F., Glicksman, A.S., Nickson, J.J.: Late consequences of early skin reactions. Radiology 94: 669-672, 1970. Field, S.B., Homsey, S.: RBE values for cyclotron neutrons for eitects on normai tissues and tumours as a function of dose and dose fractionation. Europ. J. Cancer 7: I6 I- 169, 1971. Fowler, J.F., Kragt, K.. Ellis, R.E., Lindop, P.J.. Berry, R.J.: The effect of divided doses of I5 MeV electrons on the skin response of mice. Int. J. Radial. Biol. 9: 241-252, 1965. Goffinet, D.R., Marsa, G.W., Brown, J.M.: The effects of single and multifraction radiation courses on the mouse spinal cord. Radiology 119: 709-7 13, 1976. Hayashi, S., Suit, H.D.: Effect of fractionation of radiation dose on skin contraction and skin reaction of Swiss mice. Radiology 103: 43 l-437,
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IO. Hopewell, J.W.: The importance of vascular damage in the development of late radiation effects in normal tissues. In Radiation Biology Cancer Research, Raymond E. Meyn
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__ and H. Kodney Withers, (Eds.). New York, Raven Press. 1980, pp. 449-459. Hopewell, J.W.: Persistent and late occurring lesions in irradiated feet of rats: Their clinical relevance. Brit. J. Radioi. 55: 574-578. 1982. Hranitzky, E.B., Almond, P.R., Suit, H.D., Moore, E.B.: A cesium- I37 irradiator for small laboratory animals. Radiology 107: 641-644, 1973. Jentzsch, K., Binder, H., Cramer, H.. Glaubiger. D.L., Kessler, R.M., Bull, C., Pomeroy, T.C.. Gerber, N.L.: Leg function after radiotherapy for Ewing’s sarcoma. Cancer 47: 1267-1278. 1981. Masuda, K.: Relationship between early skin reaction and late skin reaction. Nippon Acta Radiol. 42: 665-667. 1982. Manuda. K _______, ___,Hunter. ______ -_. N.... Withers? H,R;: Late e!?& in mouse skin following single and multifractionated irradiation. Int. J. Radiat. Oncol. Biol. Phys. 6: 1539-l 544. 1980. Pearson, A.E.. Steel, G.G.: The relationship between ‘early’ and ‘late’ radiation-induced skin reactions as seen in two strains of mice that differ in radiosensitivity. Int. J. Radiar. Biol. 44: 353-362.
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17. Phillips, T.L.. Margolis, L.: Radiation pathology and the clinical response of lung and esophagus. Front. Radial. Ther. Oncol. 6: 254-273,
1972.
18. Phillips. T.L., Ross, G.: A quantitative technique for measuring renal damage after irradiation. Radiology 109: 457462, 1973.
19. Potten, C.S.: The cell kinetic mechanism for radiation-induced cellular depletion of epithelial tissue based on hi-
Leg contracture in rnxe 0
20.
21.
22.
23.
24. 25. 26,
27.
erarchical differences in radiosensitivity. Int. J. Radial. Biol. 40: 217-225, 1981. Probert, J.C., Brown, J.M.: A comparison of three and five times weekly fractionation on the response of normal and malignant tissues of the C3H mouse. Br. J. Radiol. 47: 755-780, 1974. Sause, W.T., Stewart, J.R., Plenk. H.P., Levitt. D.D.: Late skin changes following twice-weekly electron beam radiation to post-mastectomy chest walls. Int. J. Radiat. Oncol. Biol. Phys. 7: 1541-1544, 1981. Stone, H.B.: Radiotherapy of a mouse mammary carcinoma following treatment with 5-iodo_2’-deoxyuridine. Radiofogv 112: 719-725, 1974. Stone, H.B., Milas, 1.: Modification of radiation response of murine tumors by misonidazole (Ro 07-0582), host immune capability, and Corynebacrerium parvum. J. Null. Cancer Inst. 60: 887-893, 1978. Stone, H.B., Sinesi, M.S.: Testing of new hypoxic cell sensitizers in vivo. Radial. Res. 91: 186-198, 1982. Stone. H.B.. Withers, H.R.: Tumor and normal tissue response to metronidazole and irradiation in mice. Radiology 113: 441-444, 1974. C,lit T-l HvrrPrharir nx~m=n nf __.., .*.-..I-I -._,Y’ .-..v “,.JD _.. in . . . radinthwsmw a-...Y...v.-Y, . . fmlr ..,.-. mn,,c,= . . ...“_ tumors. Proc. Int. ConJ: Radial. Biol. and Cancer. Kyoto, Rad. Sot. Japan. 1966, pp. 39-43. Thames, H.D., Jr., Withers, H.R., Peters, L.J.. Fletcher,
H. B. STONE
28.
29.
30.
31.
32.
33.
1061
G.H.: Changes in early and late radiation responses with altered dose fractionation: Implications for dose-survival relationships. Int. J Radiat. Oncol. Biol PhJ1.s.8: 219-226. 1982. Van der Kogel, A.J.: Mechanisms of late radiation injury in the spinal cord. In Radiation Biology in Cancer Research, Raymond E. Meyn and H. Rodney Withers (Eds.). New York, Raven Press. 1980, pp. 46 l-470. Withers, H.R.: The dose-survival relationship for irradiation of epithelial cells of mouse skin. Br J. Radiol. 40: 187194, 1967. Withers, H.R.: The effect of oxygen and anaesthesia on radiosensitivity in vivo of epithelial cells of mouse skin. Br J. Radiol. 40: 335-343, 1967. Withers. H.R., Elkind. M.M.: Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radial. Biol. 17: 261-267. 1970. Withers. H.R., Peters, L.J., Kogelnik, H.D.: The pathobiology of late effects of irradiation. In Radiobiology in Cancer Research, Raymond E. Meyn and H. Rodney Withers (Eds.) New York, Raven Press. 1980, pp. 439-448. Wondergem, J., Haveman, J., Van der Schueren, E.: Infl~,pnr,= nf sm~wzth&~ ~k,mrt;n~ nf the Lao ..-v.aw_ Y. micnniAs~A=. ..L. “V.....UY.~, I....II...W.Yu.) C.Y.n.+r.n,6 “1 La._ a-6 and stress of the animal during treatment on the radiationinduced skin reaction of mouse feet. Int. J Radiat. Biol. 41: 689-695. 1982.
Inr I Rudiamn Oncdogy Bml Phys. Vol. IO. pp. 1053-1061 Pnnted in the U.S.A All rights reserved
@ Original Contribution LEG CONTRACI’URE IN MICE: AN ASSAY OF NORMAL TISSUE RESPONSE HELEN Radiation
B. STONE, PH.D.
Oncology Research Laboratory, CED-200, Department of Radiation University of California. San Francisco, CA 94143
Oncology,
Leg coutracture, defined as the difference in extensibility of the control and irradiated hind legs of mice, was found to correhte with shtgk doses of radiation from about 20 to 80 Gy. The time of development of the early phase of the response coincided with that reported for the appearance of the acute skin response, and in some cases, partially reversed as this reaction healed. The contracture then progressed again at a moderate rate through 90 days, and then more slowly through one year. Skin contraction, measured by decrease in intertattoo distance, was assayed in the same mice. It followed the same time course as leg contracture, but had a different doseresponse relationship. Maximal contraction occurred following doses of Xl Gy or more, reaching this level sooner following higher doses. The early reactions in individual mice were not reliible in predicting late response for either assay. To determine the contribution of skin contraction to the overall leg contracture response, mice were sacrificed and the leg contracture measured before and after the removal of the skin of the leg. After doses of up to 30 Gy, little contracture remained from skiing the leg, indicating that skin contraction was largely responsible for leg contracture in this dose range. After doses of about 45 Gy and above, some contracture remained in the skinned legs, although less than in intact legs. This indicated that injury to the deeper tissues of the leg as well as to the skin was responsible for sontracture at these higher doses. There was little or no enhancement of either skin contraction or leg contracture by the hypoxic cell sensitizers metronidazole or misonidazole. Hypoxic cell sensitizers, Metronidazole, Misonidazole, Late normal tissue response, Leg contracture, traction.
INTRODUCTION Normal tissue response governs the total radiation dose that can be administered in a given regimen in an attempt to eradicate a tumor. New therapies designed to enhance local tumor control will not benefit the patient if they increase the rate of normal tissue complications. Many assays have been developed to evaluate the radioresponse of various normal tissues in animals. Those most commonly used fall into two categories: those involving acute responses that result from injury to rapidly proliferating epithelial cell populations such as those of the skin,s.29*30 or the intestine,3’ and those that involve late responses that result from injury to slowly proliferating cells and interactions between vascular, connective and parenchymal tissues.4*‘o*2*~32 Included in the latter category are assays for radiation myelopathy,’ nephritis,‘* and pneumonitis.” Contracture, atrophy and fibrosis can result
Acknowledgments-1 am grateful to Vi&i Woodard Zelle, Judith &eider, Nancy Hunter and Paul Hagopian for excellent technical assistance; to Donald Highfield, Paul Hagopian, and Karla Cavarra for preparing the figures; to Bailey Moore and his staff and S. L. Gillespie for making the jigs; and to Drs. John Hicks and Geoff Ibbott for dosimetry; and to Drs. W. C. Dewey and H. R. Withers for helpful discussions of the manuscript.
Skin con-
from radiotherapy administered to the limbI and are considered to be late responses in man. In this paper, we describe an assay for leg contracture in mice, showing the response to single doses of radiation, the time course of the development of contracture through one year after treatment, and the effect of hypoxic cell sensitizers. Skin contraction was also assessed in the same mice, using the tattoo separation method of Hayashi and SuitV9and compared to the leg contracture response.
METHODS
AND MATERIALS
These experiments were performed from 1975 to 1976 at the University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, and from 1976 to 1982 at Colorado State University. Table 1 summarizes the combinations of mice and radiation sources.
This investigation was supported by Grant Numbers CA 11138, CA 06294 and CA 20344, awarded by the National Cancer Institute, DHEW. Accepted for publication 21 March 1984.
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Radiation Oncology 0 Biology 0 Physics
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July 1984, Volume 10, Number 7
Table 1. Irradiators and mice Year
Site
Irradiator
1975-76 1976-77 1978431
UTMDAH csu csu
Cesium, dual source 300 kVp X ray Cesium, single source
Dose rate 10.7-10.5 Gy/mint 5.9 Gy/min$ 9.9-9.1 Gy/mint
Mice’ C3Hf/Bu specific pathogen-free C3H/HeJ conventional C3H/HeJ conventional
UTMDAH: The University of Texas Cancer Center, M.D. Anderson Hospital and Tumor Institute, Section of Experimental Radiotherapy, Houston, TX; CSU: Colorado State University, Department of Radiology and Radiation Biology, Fort Collins, CO. * C3Hf/Bu mice were from the specific pathogen-free colony of the Section of Experimental Radiotherapy, UTMDAH. C3H/HeJ mice were from Jackson Laboratory, Bar Harbor, ME. t Lithium fluoride dosimetry.
$ Ion chamber dosimetry. Mice Female C3Hf/Bu and C3H/HeJ mice were irradiated when 12 to 16 weeks old. There were 6 to 11 mice per assay point, as noted in the figure legends. Irradiation In the “‘Cs irradiator at M.D. Anderson Hospital,* the field was 3.0 cm in diameter.12 The mice were restrained without anesthesia in a jig with the right hind leg extended across the 3 cm opening in the jig and the heel 0 to 1 mm outside the opening.*’ The feet were not irradiated. The 13’Csirradiator at Colorado State University? had a field 3 X 18 cm, and the mice were irradiated in a jig similar to that used at M.D. Anderson, the right hind leg extended across the shorter dimension of the beam. Anesthesia was not used. Data from mice treated similarly on the two cesium irradiators were indistinguishable, and have been plotted together where appropriate. A second radiation source at Colorado State University was a 300 kVp X ray unit* with an HVL of 1.36 mm Cu. The field was 2.7 cm in diameter. The mice irradiated on this machine were anesthetized with sodium pentobarbital, 0.065 mg/g body weight, and half of the treatment was administered from the dorsal and half from the ventral side. These data were not pooled with those from animals treated with 13’Cs radiation. Animals in a given treatment group were not caged together,
and the person
making
measurements
did not
know the treatment group. Drugs Metronidazole
was obtained from Searle and Co., San Misonidazole was obtained from Roche Products, Ltd., Welwyn Garden City, Hertfordshire, England, courtesy of Dr. Carey Smithen, and from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. The drugs were dissolved fresh daily in Ringer’s solution to give concentrations of 10 and 20 mg/ml, respectively. The doses were 0.8 to 1.0 mg/g body weight
Juan,
Puerto
Rico.
* Custom model, Atomic Energy of Canada, Ltd., Ottawa, Canada.
and were administered i.p. 30 min before irradiation In our experience these doses give maximum sensitization of hypoxic cells in tumors. 23*24Controls were given an equivalent volume of Ringer’s solution i.p. 30 min before irradiation. Leg contracture assay Extensibility of both the treated and control hind legs in each mouse was measured using the jig shown in Fig. 1. The tail was placed between the vertical posts and held taut while each leg was extended against a millimeter ruler embedded in the base. Both the control and irradiated legs could be extended easily to a certain point beyond which there was considerably greater resistance to further extension. Readings were made at this point, measuring to the tip of the central digit, and were reproducible to within + 1 mm when repeated by the same person or a different individual. In untreated mice, the difference between the two legs was rarely greater than 1 mm when care was taken to avoid rotating the pelvis of the mouse. From the difference in extensibility of the two legs, group means and standard deviations were calculated. The mice were not anesthetized during any of the measurements, as each measurement was made within about 2 seconds. Discomfort appeared to be minimal. Skin contraction assay This assay was performed in the same mice that were used for the leg contracture assay. The hair on the hind legs was removed with an electric clipper 6 or more days before irradiation, and tattoo marks approximately 1 cm apart were made in the skin overlying the tibia-fibula, using India ink. The distance between the centers of the tattoo marks was measured before irradiation and at various intervals afterward. The data were expressed as a percentage of the pre-irradiation distance between the marks, corrected for the change in values on the untreated contralateral legs (by multiplying by pre-treatment/posttreatment measurements). Group means and standard deviations were calculated.
t Custom model, J. L. Shepherd and Assoc., Glendale, CA. $ Maxitron 300, General Electric Co. Milwaukee, WI.
Leg contracture in mice 0 H. B. STONE
1055
was evident below 20 Gy, but an increasing response was observed with increasing doses through 80 Gy. In contrast, the skin contraction response, measured in the same mice, showed a correlation with dose between 15 to 30 Gy, but at 50 Gy and above, was not responsive to dose (Fig. 3). With both endpoints, the shapes of the dose-response relationships were similar at 23 days to those at 320 days after irradiation, but damage was less severe at 23 days.
no damage
Time courses The time course of the leg contracture response is shown in Fig. 4. Error bars have been omitted for clarity, but
Fig. I. Jig for measuring leg contracture. The tail is held so that the rump is firmly placed against the posts. Each leg is extended along the respective scale using forceps, and measurements are read at the tip of the toes. RESULTS
Dose-response relationships
were similar to those in Figs. 2 and 3 in all experiments. Leg contracture developed rapidly during the first 23 days after irradiation. At the next measurement, at 44 days, the contracture had partially reversed or plateaued. Then, for doses 30 Gy and above, it progressed at a slower rate through 90 days followed either by a very gradual increase or no further progression through one year. A similar time course was observed for skin contraction (Fig. 5), except that contraction ceased when the skin shrank to 24 to 32% of the pre-treatment length. Although contraction developed rapidly during the first 23 days, most of this appeared to take place between day 14 and
The leg contracture responses 23 and 320 days after single doses of radiation are shown in Figure 2. Little or
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Fig. 2. Dose-response relationship for leg contractme 23 and 320 days after irradiation. There were initially 6 mice/point.
Fig. 3. Dose-response relationship for skin contraction 23 and 320 days after irradiation. Open circles were measurements made 138 days after treatment in a separate experiment. Error bars were omitted from these points for clarity, but were of similar magnitude to those shown. Solid symbols: 6 mice/point; open symbols: 10 mice/point, initially.
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1057
sontracture in mice 0 H B. STONE
Efect of weekly measurement Because the extension of the legs of the mice during measurement might have a therapeutic effect, reducing the amount of contraction, we compared both skin contraction and leg contracture in mice measured at weekly intervals with those measured only at 6 months after :-,.>I_.:-_ * &51x “Z..--_rL._.^.. ^:-_:C,,_. A:I IllilUlall”ll. ,4L II1”11WS,.I.^_^ LIICICZ wiw ^^ 11”>‘ g‘1111Ldlll uuference between the two groups with either response (Table 3). Correlation between skin contraction and leg contracture The relationship between skin contraction and leg contracture is shown in Fig. 6. The data used in these cal-
culations were from mice irradiated with doses of 23.8 to 66.6 Gy, and scored from 37 to 200 days after irradiation. These were both doses and times at which contracture was observed. The solid line and its dashed extrapolation are based on a least squares fit of the data from 0 to 0.8 cm contracture. A significant amount of skin contraction occurred in the absence of leg contracture (tattoo separation at 80% for 0 cm leg contracture), reflecting a slightly higher threshold dose for leg contracture. Above 1.O cm leg contracture, the skin contraction started to level off. At the termination of two experiments at 138 and 190 days after irradiation, respectively, leg contracture was measured, first in live animals, and then immediately after cervical dislocation, in intact animals and following removal of the skin of the hind legs. The results are shown in Fig. 7. Although the data differed slightly in the two experiments, ieg contracture was reduced in sicinned iegs at all doses tested. At the lower doses, little residual contracture was evident in skinned legs, but at higher doses there was residual contracture The extensibility of the legs was slightly greater in dead than in live mice, averaging 0.06 +- 0.01 (se.) cm greater in the treated legs, and 0.02 :C 0.01 cm in the control
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Fig. 6. Correlation between skin contraction and leg wntracture. Data were pooled from several experiments, from mice treated with 23.6 Gy to 66.6 Gy, and from 37 to 200 days after irradiation. The solid line is a least squares fit to the points from 0 to 0.8 cm contracture. The dashed line is extrapolated from the solid line.
Table 3. Effect of weekly measurement of skin and leg contracture 6 months after irradiation Dose, GY Skin wnuaction-_$ _^
Meas. at 6 mos. only
Meas. weekly of preirradiation
tattoo separation + s.d.
77 -t i5
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30
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79* i3 76? 18 65 z!z23 -O’*
Leg contracture: 20 25 30
cm difference in leg extensibility 0.34 2 0.26 0.49 + 0.50 0.63 f 0.32
+- s.d.
0.29 10.15 0.50 + 0.37 0.88 ZL0.45
Note: None of the differences were statistically significant. All groups contained 9 to 10 animals. Mice were irradiated with 300 kVp X rays, and were anesthetized barbital.
with sodium pento-
IO
I 20
, 30
L 40
DOSE,
GY
1 50
1 60
Fig. 7. Dose-response relationship in intact (solid symbols) and skinned legs (open symbols). Measurements were from an experiment measured 138 days after irradiation (dashed lines) and from one measured 190 days after irradiation (solid lines). Legs were skinned immediately after euthanasia by cervical dislocation.
1058
Radiation Oncology 0 Biology 0 Physics
legs. Thus, 0.04 f 0.02 cm of the decrease in contracture in the skinned legs resulted from differences in extensi. .. . bility between iive and dead mice. Skinning resuited in an increase of 0.01 + 0.01 cm in the extensibility of the control legs. Effect of hypoxic cell sensitizers
The hypoxic cell sensitizers metronidazole and misonidazole were given i.p. to groups of mice 30 min before irradiation to determine whether the skin and leg contracture responses would be enhanced by these agents. The results are shown in Fig. 8a and b. There was no significant enhancement of either response by the sensitizers. Correlation between early and late responses in individual animals
Late leg contracture and skin contraction (at 280 to 320 days after irradiation) have been plotted in Fig. 9a and b, respectively, as functions of the early reactions (at 23 days) in individual mice. Although both plots show general correlations between early and late damage, there was considerable scatter of the data. The leg contracture data from mice treated with metronidazole or misonidazole showed more scatter than the control data (Fig. 9a), although neither sensitizer affected the scatter in the relationshinr hetwwn _____________ (Fie. \_ _5). --_..__-- P~AV ___‘, ag8 ia?e ~kjn COntractinn 9b). In both assays, however, very few individuals showed a late response that was less than or equal to the early responses (dashed lines in Fig. 9).
. a 1.8-
fi
DISCUSSION Th_e lep SPCQV l&R” lit~ the ~ltin mntmrtinn a mntrwtnrp _.,_~1.__._~_ UUJ, . ..w .,_.I .,“......“.I”SI assay, can be used as a measure of late response. Repeated measurements in the same animals give information on the time course of the development of contracture. The response correlates with single radiation doses from about 20 to 80 Gy, a range in which several transplantable murine tumors are locally controlled.23*24~26 It can therefore be used to estimate therapeutic gain in treatments involving combined modalities. For example, we found a TCDSO of 64.2 Gy for the mammary carcinoma MDAH-MCa-4, and in mice given 1.O mg misonidazole/ g body weight before irradiation, a TCDSO of 27.5 Gy.*’ From the dose-response curve in Fig. 8a, these doses would give, at about 120 days after irradiation, approximately 1.5 and 0.5 cm leg contracture, respectively, clearly a therapeutic gain when misonidazole was used. The skin contraction assay
The present studies have shown that the maximum level of skin contraction is about 24 to 32% of the original tattoo distance. Single doses of 50 Gy and above produced this level by 90 days, but after 30 Gy it was not reached until about 300 days. Thus, this assay has a useful range from about 15 to 30 Gy (single doses) but this may be extended to 40 to 50 Gy if measurements are made within icK1
li_ays.
The time course of the skin contraction response has been reported by Hayashi and Suit,’ and Masuda et al.,” and Pearson and Steel.16 Our data generally agree with
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July 1984, Volume 10, Number 7
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Leg contracturein mice 0 H. B. STONE
1.8 -
zl
A
CI
1059
CONTROL -A MCTDn
.
A
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DAYS
Fig. 9. Correlation between early (23 days) and late (280 to 320 days) leg contracture (a) or skin contraction (b) in individual mice. The dashed lines represent equal levels of early and late response. Metronidazole (“Metro”) and misonidazole (“Miso”) were given i.p. 30 min before irradiation. The doses were 0.8 to 1.O mg/g body weight.
of damage was observed between 90 and 365 days only in the groups exhibiting damage at 90 days. However, Masuda et sml.Is found that a group treated with 12 Gy did not start to show contraction until 320 days, but that this group and others treated with higher doses continued to contract through 540 days. The response itself or the dose-response relationship may depend on strain or species, because Berry, e6 aL2 found no dimensional changes in the skin of guinea pigs after doses of up to 26 Gy, although these animals did exhibit acute skin reactions. Differences in both the time course and dose-response relationship have been observed I.., D~a,-wv-. “,.A Ct~l16 lult..ra~... i..rr\ rt,.dnc .4 m;,..e “J 1 &daIJvli 411&h h.z?C~b,I ubCWlrc.‘I &“” aucuII* “1 Lll‘bA.. theirs. Progression
Early reactions The underlying mechanisms in the development of skin and leg contracture after irradiation are complex and not well understood. The early wave of contraction occurs at the time of the development of acute desquamation, although contraction may occur in the absence of ulceration.’ Desquamation itself is primarily a consequence of killing of stem cells in the basal epithelium,29 but the delay in its appearance after irradiation corresponds to the time required for the radioresistant cells in subsequent stages of differentiation to complete their life cycles and to be s10ughed.‘~ Masuda et al.” have stated that early skin shrinkage similarly reflects depletion of basal epitheiial ceiis. The correspondence is not direct, however, because other cellular and non-cellular tissue components
contribute to the overall response so that the magnitude of contraction is not as great as might be expected for the level of stem cell depletion: at 20 Gy, cell survival is on the order of low6 29 but skin remains at 70% of its original dimensions and leg contracture is about 0.2 cm. Possible mechanisms, late reactions The similarity of skin contraction and leg contracture suggest that the two assays measure the same phenomena. At doses of 15 to 30 Gy, this may indeed be the case: both assays correlate with dose in this range (Figs. 2 and 3), the lower levels of skin contraction correlate well with lxx+
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legs abolished most of the leg contracture (Fig. 7). At higher doses, however, leg contracture continued to increase with dose (Fig. 2), but skin contraction did not (Fig. 3). This was seen also in the correiation between the two responses (Fig. 6). Residual contracture after removal of the skin (Fig. 7) indicated that skin contraction was not the only cause of leg contracture at doses of 45 Gy or more. Leg contracture does not result from shortening of the bones of the leg when adult mice are irradiated (Kouji Masuda, personal communication, January, 1976). The extent to which early reactions are responsible for the development of late reactions is not known. They may have some, but probably not all, mechanisms in common. Although there was a general correlation between early and late damage for both skin contraction
1060
Radiation Oncology 0 Biology 0 Physics
and leg contracture, there were wide individual differences so that the early reactions were of little value in predicting late damage in a given animal (Fig. 9). This has been observed in other animal studies6*9,‘4*20and in hu-___ ,521 m-- ~~ . n. .I& c mans.-‘-‘-- rearson ana 31eel” round that CBA mice had more severe acute skin reactions than C57B1 mice, but had less severe late reactions. This suggests that factors other than the severity of the early skin reaction may also contribute to the development of late damage. Nevertheless, cells that die early and are not replaced contribute to the atrophy observed after the early reactions have subsided. Whether either skin contraction or leg contracture in mice respond to fractionated regimens in a manner similar to that of early or late endpoints of damage, as shown by Thames et uI.,~’is not presently known. This may depend on the time after treatment. Eflect of hypoxic
July 1984, Volume 10, Number 7
ulation might be masked by the response of a predominantly aerobic population in both the skin contraction and leg contracture assays. Foot ioss
Hopewell” has described a lesion at the flexure between the leg and foot of rats that developed with a latency of 2 or more months after irradiation. The lesion led to edema, necrosis and amputation of the foot. Foot loss in our mice may have occurred as a result of a similar lesion. Although the feet of our mice were shielded during irradiation, the ankle region received a substantial proportion of the dose to the leg, and atrophy of the tissues proximal to the ankle may have produced constriction of the vascular and lymphatic channels, leading to the observed edema, fracture and necrosis.
cell sensitizers
The failure to find significant sensitization of either skin contraction or leg contracture by metronidazole or misonidazole with our usual irradiation conditions was unexpected, in view of the observation that acute skin responses were sensitized by both these agents3*25.33and by oxygen.7.30 The data of Wondergem et al.33 have suggested that hypoxia in mouse skin is the result of stress-induced vasoconstriction, or of vascular obstruction during irradiation. However, sensitization of a small hypoxic subpop-
CONCLUSIONS The leg contracture assay is easy to use and can provide information on the time course of the development of late damage, as well as its modification by other therapeutic modalities. It is dose-responsive over a wide range of doses, including those at which local control of a variety of murine tumors occurs. Further studies are needed to understand more fully the factors that influence the development of this response and the time-dose relationships in multifraction radiotherapy.
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