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Tumor radiosensitization with concomitant vone marrow radioprotection: A study in mice using diethyldithiocarbamate (DDC) under oxygenated and hypoxic conditions
In! J Radiation Oncology Biol Phys.. Vol. Pnnted I” the U.S.A. All rights reserved.
0360-3016/85 $03.00 + .X7 Copyright 8 ,985 Pergamon Press Ltd.
I I, pp. 1163-l 169
??Original Contribution
TUMOR RADIOSENSITIZATION WITH CONCOMITANT BONE MARROW RADIOPROTECTION: A STUDY IN MICE USING DIETHYLDITHIOCARBAMATE (DDC) UNDER OXYGENATED AND HYPOXIC CONDITIONS RICHARD G. EVANS, PHD., Fricke Radiobiology
Research
Laboratories,
M.D.
Mayo Clinic & Mayo Foundation,
Rochester,
MN 55905
We have established, both in vitro and in viva, that Diethyldithiocarbamate(DDC) protects mammaliancells from radiation. The in viva protection, when non-toxic concentrations of DDC are present one-half hour before irradiation, is reflected by a dose modification factor (DMF) of 1.9 based on LDs,po and 1.5 using survival of CFU, as an endpoint. Further experiments in viva have extended our knowledge to the differential radioprotective effects of DDC on the bone marrow of animals breathing room air compared to a 5.5% oxygen in nitrogen mixture. The DMF (LDsopo) for DDC in air breathing animals, previously established as 1.9, can be contrasted with a DMF, obtained in the present study, of 1.2 for animals irradiated in the hypoxic state. Moreover the DMF (CFU, survival) previously established at 1.5 for air breathing animals, can be compared to a value of 1.3, obtained in the present study, for mice irradiated under hypoxic conditions. Modification of the dose response by DDC, for both bone marrow and tumor, was also examined in animals bearing a RIF sarcoma. Although protection of the bone marrow was confirmed (DMF = 2.1), the striking finding was that ILL tumor cells Were sensirized, in both air breathing and nitrogen breathing animals, by the addition of DDC one-half hour before the radiation exposure. Moreover, the tumor radiosensitization, a factor greater than 2, in air breathing animals, appeared to be independent of dose (4 = 200 rad, with or without DDC). The tumor radiosensitization was even more marked in the nitrogen breathing mice, in which a factor of 10 difference in survival was noted, together with a tendency towards greater sensitization at radiation doses in the clinical range. The results, demonstrating bone marrow radioprotection by DDC (aerobic > hypoxic) with concomitant tumor radiosensitization (hypoxic > aerobic) strongly suggest a large therapeutic gain factor (TGF) which could be further explored in a clinical setting. Radiosensitization,
Radioprotection,
DDC.
INTRODUCTION
of the “hypoxic tumor cell” remains questionable due not only to the phenomenon of reoxygenation but to the evolving evidence that slight natural hypoxia may exist in some critical normal tissues. Recently, the role of tissue oxygen concentration has been underlined in experiments designed to demonstrate the radioprotective properties of various thiols.6,20 In the study of Denekamp et al.,’ using an epidermal clone assay and WR-2721, the skin protection was maximum in air and diminished at lower and higher concentrations of oxygen. Parkins et a1.,22 using breathing rate in mice as an end point following lung irradiation, found that the radioprotection afforded by WR-2721 decreased as the oxygen tension was lowered from 100% to 7%. Similar observations have been noted in spheroids (using
It has now been over a quarter of a century since Gray and his colleaguesr4 suggested that severely hypoxic cells within tumors might survive a normally sterilizing dose of radiation and following treatment become reoxygenated, recover their clonogenic potential and cause the tumor to regrow. As Kaplan has pointed out,r7 the thesis was so seductively plausible as to have become an article of faith by many investigators. To this day it has still not been well established that a sufficient number of hypoxic cells remain, following a fractionated course of radiotherapy, and that the survivors have the clonogenic potential to cause tumor regrowth. Moreover, as Hendry has noted,16 the magnitude of the therapeutic importance
Supported in part by the Mayo Clinic Comprehensive Cancer Center (CA 15083) and the Mayo Foundation. Reprint requests to: R. G. Evans, Ph.D., M.D. Acknowledgements-I am indebted to Christine Wheatley, Carol Engel and James Nielsen for excellent technical assistance and to Bonnie Hanson for her patience in the many revisions of this manuscript.
This work was supported in part by the Mayo Clinic Comprehensive Cancer Center (CA 15083) and the Mayo Foundation. Accepted for publication 7 January 1985.
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Radiation Oncology 0 Biology 0 Physics
WR-2721) when the best radioprotection was noted at intermediate levels of oxygen and the weakest protection for well oxygenated or anoxic cells.7 Our laboratory, working with DDC and plateau-phase cultures of mammalian cells,’ has shown protection factors of 1.2 for intermediate levels of oxygenation (4 ml of medium overlaying the monolayers)’ but have noted essentially no protection under well oxygenated (2 ml) or hypoxic conditions (9 ml) (Evans, R. G., unpublished data, Nov. 1983). The radioprotection afforded by DDC, and other thiols, is likely to depend on many factors, including degree of tissue oxygenation (tumor size), level of protector in the particular tissue or tumor, and size of the radiation fraction. We have established that DDC protects the bone marrow of mice from radiation as judged by modifications in the LD50,30 and survival of CFU,.9 We report here our observations on the protection afforded by DDC, as a function of the oxygen tension, within the bone marrow of Fl mice, at the time of irradiation. In addition, we present data showing tumor radiosensitization by DDC (under both oxygenated and hypoxic conditions) with concomitant bone marrow protection in the same tumor-bearing animals. MATERIALS
AND METHODS
Experimental animals and tumor The mice used were Fl hybrid (C57 Bl X BALB/c) and C3H/Km, which were bred and maintained in a specific pathogen-free colony. They were handled aseptically in laminar flow hoods for all experimental manipulations. The RIF-1 in C3H/Km mice was used throughout as the tumor model. Tumor cells were passed in animals and in culture as described by Twentyman et a1.,29 Tumors were obtained by injecting lo5 cells intradermally into the flank of 16 to 18 week-old male (C3H/Km mice. The size of the tumors, at the time of irradiation and DDC exposure was restricted to the range 150-200 mm3 (7-8 mm diameter). Irradiation procedure Mice were restrained within individual compartments in an acrylic jig and irradiated in groups of 20 using a 300 KVP x-ray machine* (2 mm copper filtration, HVL 1.4 mm copper) delivering 47 rad/minute, at the jig (114 rad/min in animals irradiated under hypoxic conditions). After irradiation the mice were housed five to an acrylic cage in light and temperature controlled rooms and allowed water and mouse chow ad libitum. The mortality was noted daily. The LD50,30 values with 95% confidence intervals were determined by the method of Litchfield and Wilcoxan.”
* General
Electric
Maxitron.
June 1985, Volume 11,Number 6
An in vivo irradiation and in vitro cell survival assay was used to determine the radiation response of RIF tumors to different radiation fraction sizes as described by Brown et al.’ Total body irradiation (dose rate 47 rad/min) was used to irradiate animals bearing the RIF tumor in their flank. In experiments designed to test the radiation response, with or without DDC, of bone marrow and RIF tumor in animals under hypoxic conditions, the animals were bathed in an atmosphere of 5.5% oxygen in nitrogen mixture for five minutes prior to and during radiation exposure.25 The cell survival assay in the tumor-bearing animals was initiated immediately after irradiation (animals had died an hypoxic death). The tumor was cut into several pieces and these made into a fine brei which was then mixed with 30 ml of complete Waymouth’s medium containing the following concentrations of enzyme: 0.02% DNase, 0.05% pronase and 0.02% collagenase as described by Brown et al4 All cells that excluded dye were scored as viable tumor cells. Following appropriate dilution procedures of the single cell suspension, cells were plated into 100 mm polystyrene petri dishes in 5 ml of medium and incubated for 8 to 10 days. Colonies were fixed and stained with crystal violet and those containing 50 or more cells scored using a magnification method. Plating efficiencies were in the range 20-30%. Diethyldithiocarbamate (DDC)? The sodium salt of DDC was made up in saline before each experiment and injected i.p. (0.1 ml per 10 grams body weight) one-half hour before initiation of irradiation. LD50,30experiments were performed with at least 10 animals per dose point and appropriate controls were used. The same dose of DDC (1000 mg/kg) was used throughout, i.e., in CFU, and tumor studies. Spleen colony assay for mouse bone marrow cells The methods of Till and McCulloch28 and Ainsworth and Larsen’ were used to obtain bone marrow survival curves, with and without the presence of DDC at the time of total body irradiation (TBI). Nine hundred and fifty rad of TBI were used to prepare the recipient mice, and no endogenous colonies were formed in the spleens of either the Fl hybrid or the C3H/Km mice at this dose. Surviving fractions were based on injecting bone marrow cells, from mice irradiated in vivo, into 10 recipient mice and determining the number of spleen colonies eight days later. In experiments using DDC, the drug was injected i.p., at a concentration of 1000 mg/kg, 30 minutes before the TBI. The survival of CFU, with and without DDC, in mice breathing air or a 5.5% oxygen-nitrogen mixture, was determined in non tumorbearing Fl hybrid mice. Additional data on CFU, survival were obtained, with and without DDC, from bone
t Sigma Chemical
Corporation,
St. Louis, MO.
Radiosensitization-protection:
marrow in air-breathing C3H/Km RIF tumor in their flank.
animals bearing a
RESULTS
Modification of the LD501j0 of Fl hybrid mice by DDC under oxygenated and hypoxic conditions The increase in LD50,30from 780 to 1490 rad, attributable to the presence of DDC at the time of irradiation, in air-breathing animals has been reported previously.g A dose modification factor (DMF,i, = LD50,30wDx i LD50,30w,0DDc)of 1.9 was obtained, as shown in Table 1. In animals breathing a 5.5% oxygen in nitrogen mixture, the LD50,30w,ooDC was raised to 1660 rad indicating that an hypoxic state had been reached at the time of total body irradiation (TBI). When animals, breathing the same mixture, received TBI in the presence of DDC an LD,o,jo of 2000 rad was obtained giving a DMFhypoxia of 1.2, as noted in Table 1. It would appear therefore, at least based on LD50,30as an endpoint, that DDC confers a greater radioprotection to well oxygenated bone marrow (DMF,i, = 1.9) than it does to hypoxic bone marrow (DMFhypoxia= 1.2). CFU, survival,for Fl bone marrow cells, with and without DDC, in air-breathing compared to 5.5% oxygen in nitrogen-breathing animals The two lower survival curves in figure 1, previously reported,g obtained in air-breathing animals allowed us to calculate a DMFai, of 1.5. When similar experiments were performed, in Fl mice breathing a 5.5% oxygen in nitrogen mixture the upper two curves in Figure 1 were obtained. The Do of 180 rad for the CFU, radiation response, with no DDC present at the time of irradiation, resulted in an oxygen enhancement ratio (OER) of 2.3 (180 f 80) indicating that an hypoxic state existed in the bone marrow of the animals breathing the oxygen in nitrogen mixture. The D,‘s of the upper two curves resulted in a dose modification factor (DFMhypoxia) of 1.3 (240 + 180). These data, based on survival of CFU,, are in concert with the findings noted in Table 1, using L50,30 as an endpoint, that DDC confers somewhat greater protection of the bone marrow stem cells in well oxygenated bone marrow (DMFai, = 1.5) compared to hypoxic bone marrow (DMFhypoxia= 1.3).
Table
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DDC in viva 0 R. G. EVANS
CFU, survival for bone marrow cells in RIF-bearing C3H/Km mice with and without DDC The data in Figure 2 were obtained at a single TBI dose of 400 rad in air-breathing animals, with and without the addition of DDC one-half hour before the irradiation. The Do of 140 rad for bone marrow stem cells in C3H mice obtained in the absence of DDC is somewhat greater than the Do of 80 rad obtained for the Fl hybrid mice, also in the absence of DDC. However, this radiation response of bone marrow stem cells in the C3H mice is in keeping with that noted by others.28 When the radiation was given one-half hour after the i.p. injection of DDC, a Do of 300 rad was obtained for the radiation response of the CFU,. The dose modification factor (DMF,,,), as noted in Figure 2, has a value of 2.1. This is somewhat larger than the value of 1.5 obtained for the radioprotection of CFU, in the FI hybrid mice breathing air. EJect of DDC on the dose response of RIF tumor in C3H/Km mice breathing air The data in Figure 3 were obtained from mice irradiated when their flank tumors were in the range 150200 mm3 (7-8 mm diameter). The Do of 200 rad for animals irradiated in the absence of DDC, together with the broad shoulder (D4 = 400 rad) is a similar response to that observed by others following irradiation in airbreathing animals.4 Of particular note is that although the Do for the survival curve obtained in the presence of DDC, is similar to that obtained in animals in the absence of DDC, the D, has been reduced from 400 to 175 rad. This “reduction in shoulder” implies a modification by DDC of repair of sub-lethal damage. Modijication of radiation response qf RIF tumor by DDC in N2-breathing mice When the animals were allowed to breath nitrogen for five minutes prior to and during the irradiation the data shown in Figure 4 were obtained. In the absence of DDC an extremely large “shoulder” with a D, of the order of 725 rad was obtained similar to that noted by others with the same tumor model.4 However, the striking finding was that when these tumor bearing animals were irradiated one-half hour following the i.p. injection of DDC, surviving fractions were markedly
1. LDSo,Xo as a function of presence or absence of DDC (1000 mg/m2, 30 min prior to TBI) in Fl hybrid mice breathing room air or 5.5% O2 in N2 mixture
Conditions
LDso,30 rad (95% confidence intervals)
Room Air, without DDC Room Air, with DDC
780 1490
(710-820) (1430-1550)
5.5% 02 in N2, without DDC 5.5% 02 in N2, with DDC
1660 2000
(1600-1725) (1900-2100)
Dose modification
factor (DMF)
1490 DMFai,: = 1.9 780 2000 DMF,,ypox~a: ~ = 1.2 1600
Radiation Oncology 0 Biology0 Physics
June1985, Volume II, Number6
1.0
RIF tumor (C3HIKm) Air-breathing P E. = 0.25
.-g z ?! ; s ‘.-5 2:
01
0.0 1
0
1000
mice
mg/kg
0.5 hr before
DOC irradiation
01
z
0001
0
100
200
300
400
Dose
500
600
0.01 0
250
500
750
1000
Dose in rad
in rad
Fig. 1. Survival of CFU, under oxygenated (lower two curves) and hypoxic conditions (upper two curves) in the absence of DDC (circles) and the presence of DDC (triangles). Open symbols represent data obtained from air-breathing animals; closed symbols from animals breathing a 5.5% oxygen in nitrogen mixture. The i.p. dose of DDC was 1000 mg/kg added 0.5 hr before the i~diation. Ten recipient mice were used to determine the survival at each dose point. One to six animals were used as bone marrow donors at each point, depending upon the surviving fraction. The oxygen enhancement ratio (OER) was obtained by the ratio of the Do’s of the survival curves obtained, without the presence of DDC, under hypoxic and oxygenated conditions.
Fig. 3. Radiation response of the RIF tumor in air breathing mice with and without the presence of DDC. Each data point represents the mean of four experiments & one standard error (SE). The plating efficiency (P.E.) had a mean value of -25. The size of the tumors at the time of irradiation were in the range 150-200 mm3 (7-8 mm) but the majority were in the range 170- 180 mm3. The surviving fractions, with and without DDC, were compared at three dose points using a two sample t test for different means: 250 rad @ = 0.002), 500 rad @ = 0.007) and 750 (p = 0.008).
lowered. The reduction was particularly significant at a 250 rad dose in that the surviving fraction in the presence of DDC was an order of magnitude lower than in its absence.
Recent work from our laboratory has demonstrated different tissue dist~butions of DDC (kidney > bone marrow > lung > tumor),9 radioprotection of bone mar-
\
LXX
0
\
DISCUSSION
\ \ 01
\
‘\O
\oD”=i40rad 0
5
07
DMF
0.01
3
0
D =-z-z0 Wwl DOC 8Ttr 0 ouio DDC
No DDC
o,o, i 300 140
0 100
200
300 Oose
400 in
500
[yzE+ilyi:,
2.1
600
rad
Fig. 2. Survival of CFW, with and without the presence of DDC (1000 mg/kg 0.5 hr before irradiation). Ten recipient mice were used to determine survival at each dose point and two to four animals served as donors depending upon the surviving fraction. The dose modification factor for air breathing animals (DMF,,) = 2.1.
250
500
750 Dose
yyi”
10001250
1500
in rad
Fig, 4. Radiation response of RIF tumor in animals breathing nitrogen, with and without the presence of DDC. Data points represent the mean from two to three experiments * one standard error (SE). The plating efficiency (P.E.) had a mean value of .20. The size of the tumors at the time of irradiation were in the range 150 to 200 mm3 (7-8 mm), but the majority were in the range 170- 180 mm’.
Radiosensitization-protection:
row in mice by DDC (LD50,30, survival of CFU,),9 the ability of DDC either to radiosensitize or radioprotect plateau-phase cultures of mammalian cells depending upon the irradiation conditions,* protection of bone marrow in mice exposed to high dose cis-platinum,‘2,24 enhancement of the therapeutic effect of cis-platinum in lymphoma-bearing mice by DDC” and the ability of DDC to enhance the heat sensitivity of mammalian cells in tissue culture. lo It appears, therefore, that under some conditions DDC can act as a sensitizer and in others as a protector. Lin et al.,‘* using exponentially growing cells, showed a sensitization of X ray killing by 0.1 mM DDC (enhancement ratio = 1.7) and Westman and Marklund,3’ using a 3 mM concentration of DDC, showed an enhancement ratio of 1.23 for X rays, also using exponentially growing cells. Allalunis-Turner and Chapman have recently reported* radioprotection of bone marrow in mice by single doses of DDC (DMFs in the range 1.4 to 2.9). They note that part of this “apparent” protection is due to stimulation of the bone marrow by DDC alone resulting in “real” DMFs ranging from 0.9 to 1.6. We have been unable to demonstrate, using a similar exogeneous spleen colony technique, any stimulation of the bone marrow by DDC alone and moreover, would argue that it is the end result that is important and not how much of the protection is “real”. Milas et al.*’ have demonstrated a small protective effect by DDC in a mouse fibrosarcoma, either as lung micrometastases or as a solitary leg tumor (protection factors 1. l-l .2). Penhaligon has noted that the degree of protection conferred by WR-272 1 to a RIF- 1 fibrosarcoma is dependent upon the irradiation procedure.23 If RIF-1 bearing mice are restrained in lead jigs, during local tumor irradiation, little or no protection is noted by prior treatment with WR-272 1, whereas if the mice are confined in large perspex jigs and given whole body irradiation then the tumor demonstrates marked protection by WR-2721. The experimental evidence suggests that the modification of radiation response by thiols, such as DDC and WR-272 1, is multifaceted. Factors likely to be important in the radiation modification conferred by DDC include selectivity for and the proliferative state of different normal tissues and tumor (DDC accessibility and loss), the size of the tumor at the time of irradiation (growth fraction, hypoxic fraction), the state of oxygenation of the tissues at the time of exposure (free radicals, oxygen enhancement), the radiation fraction size (both single and fractionated), the endpoint chosen (LD50,30, CFUs survival, tumor survival), and the irradiation conditions. Although we can provide no data to support active absorption of DDC by normal tissues versus passive absorption by tumor as noted with WR-272 1,33it appears that organs, relevant from a clinical standpoint regarding radioprotection (bone marrow, kidney and lung) appear to have a greater uptake than tumor cells (lymphoma’; RIF sarcoma, Evans, R. G., unpublished data, July
DDC in viva 0 R. G. EVANS
1167
1983). However, uptake per se, of thiols, has not been well correlated with degree of protection. We are presently exploring the effect of tumor size on the radiation modification conferred by DDC, using both single and fractionated doses. It is entirely possible that the radiation response of tumors as a function of size will reflect the different oxygenated/hypoxic compartments within the tumor at the time of irradiation and also DDC accessibility to these compartments. In view of the clinical and radiobiological relevance of the “hypoxic cell,” we examined whether DDC conferred different radioprotection to oxygenated versus hypoxic tumor and bone marrow (a key normal tissue). There is extensive evidence that thiols, such as DDC, protect by competing with oxygen for the fixation of radiation induced damage or by scavenging radiation induced radicals (not necessarily mutually exclusive mechanisms and does not preclude protection by hypoxia and protection by thiols operating through independent mechanisms*$ see recent review by Ward3’). As we noted in the Introduction, the radiation protection conferred by thiols appears to be intimately related to the level of oxygenation at the time of the radiations~6~20~22 and that the greatest radioprotection may be observed at “intermediate levels” of oxygenation. Our method in the present study, of obtaining an hypoxic state, in both the bone marrow and the tumors of the mice, is artificial by nature and results in a state of acute hypoxia rather than the chronic hypoxic situation that might arise naturally in an air-breathing animal or patient. However, the data from these experiments can at least serve as a guide to future experimentation. The dose modification factor (DMF, mxia) of 1.2 conferred on animals breathing 5.5% oxygen in nitrogen using LD50,30 as an endpoint is significantly lower than the DMFai, of 1.9, noted previously in air-breathing animals (Table 1). When the effect of DDC on the bone marrow of the Fl hybrid mice was examined in a somewhat different fashion, i.e., fraction of CFU, surviving, then a DMFhypoxiaof 1.3 was obtained, which is less than the 1.5 noted previously for DMF,i,. There are two lines of evidence, therefore, suggesting that DDC protects oxygenated bone marrow to a greater degree than it does hypoxic marrow. The radiation response of a RIF tumor in an air-breathing animal, determined in vitro following in vivo irradiation, was in concert with that noted previously.4 However, the finding of radiosensitization, as judged by the shoulder reduction (Figure 3) when DDC was added one-half hour before the irradiation, was unexpected. The important finding was that we had observed tumor radiosensitization in the low dose range, the significance of which will be discussed later. The effect of the induction of the hypoxic state in RIF-bearing animals, presumably in all tissues of the animal including the tumor, is reflected in a significant change in the slope (OER = 1.6) as well as the shoulder region of the radiation survival curve of the tumor cells (Figure 4). We again note that
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Radiation Oncology 0 Biology0 Physics
the presence of DDC one-half hour before the irradiation, causes a marked change in the survival of the tumor cells in the low dose range; a sensitization factor greater than 10 at 250 rad. (The terminal slopes of the survival curves with and without DDC are very similar, D,, = 320 rad.) It would appear therefore that the hypoxic tumor cells, at least at a 250 rad fraction size, are sensitized more by the DDC than the tumor cells in an air-breathing animal (a factor of 13 compared to 2.6). Split dose experiments exploring repair of sub-lethal damage in tumors, with and without DDC, are in progress. The significant therapeutic gain factor, i.e., the tumor radiosensitizing effect of DDC, with concomitant radio-
June 1985, Volume 11, Number 6 of the bone marrow, we have demonstrated in the RIF tumor model is most thought provoking and has direct application to the clinic. The ability of nontoxic concentrations of DDC to protect normal tissues such as bone marrow while concomitantly causing sensitization of a sarcoma warrants consideration for the use of this drug in clinical trials. There is ample evidence in the clinical literature of the minimal toxicity of DDC (treatment of nickel-carbonyl poisoning2’ and alcoholics) in contrast to the toxicity observed with WR-2721, a protective agent that has been explored in the clinic.3*‘3,32 The expertise garnered from the clinical trials with WR2721 could be most advantageously applied to trials of DDC with radiation therapy.
protection
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M.J., Chapman, J.D.: Evaluation of diethyldithiocarbamate as a radioprotector of bone marrow. Int. J. Radiat. Oncol. Biol. Phys. 10: 1569-1573, 1984. Blumberg, A.L., Nelson, D.F., Gramkowski, N., Glover, D., Glick, J.H., Yuhas, J.M., Kligerman, M.M.: Clinical trials of WR-2721 with radiation therapy. Znt. J. Radiat. Oncol. Biol. Phys. 8: 561-563, 1981. Brown, J.M., Twentyman, P.R., Zamvil, S.S.: X-radiation (cell survival, regrowth delay, and tumor control), chemotherapeutic agents and activated macrophages. J. NCZ 64: 805-811, 1980. Denekamp, J., Michael, B.D., Rojas, A., Stewart, F.A.: Radioprotection of mouse skin by WR-2721: The critical influence of oxygen tension. Int. J. Radiat. Oncol. Biol. Phys. 8: 531-534, 1981. Denekamp, J., Michael, B.D., Rojas, A., Stewart, F.A.: Thiol radioprotection in vivo: The critical role of tissue oxygen concentration. Brit. J. Radiol. 54: 1112-l 113, 1981. Durand, R.E.: Radioprotection by WR-2721 in vitro at low oxygen tensions: Implications for its mechanism of action. Brit. J. Cancer 47: 387-392, 1983. Evans, R.G., Engel, C., Wheatley, C., Nielsen, J.: Modification of the sensitivity and repair of potentially lethal damage by diethyldithiocarbamate (DDC) during and following exposure of plateau-phase cultures of mammalian cells to radiation and cis-Diamminedichloroplatinum (II). Cancer Res. 42: 3074-3078,
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10. Evans, R.G., Nielsen, J., Engel, C., Wheatley, C.: Enhancement of heat sensitivity and modification of repair of potentially lethal damage in plateau-phase cultures of mammalian cells by diethyldithiocarbamate. Radiat. Res. 93: 319-325,
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11. Evans, R.G., Wheatley, C.R., Engel, C.R., Nielsen, J.R.: Enhancement of the therapeutic effect of cis-platinum by DDC: A study in lymphoma-bearing mice. Cancer Chemother. Pharmacol. (In press) 1985. 12. Evans, R.G., Wheatley, C., Engel, C., Nielsen, J., Ciborowski, L.J.: Modification of the bone marrow toxicity of cis-Diamminedichloroplatinum (II) in mice by diethyldi-
thiocarbamate (DDC). Cancer Res. 44: 3686-3690, 1984. 13. Glick, J.H., Glover, D.J., Weiler, C., Blumberg, A.L., Nelson, D.F., Yuhas, J.M., Kligerman, M.M.: Phase I clinical trials of WR-2721 with alkylating agent chemotherapy. Int. Radiat. Oncol. Biol. Phys. 8: 575-590, 1981. 14. Gray, L.H., Conger, A.E., Ebert, N., Hornsey, S., Scott, O.C.A.: The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Brit. J. Radiol. 26: 638-645,
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17. Kaplan, H.S.: On the relative importance of hypoxic cells for the radiotherapy of human tumors. Europ. J. Cancer 10: 275-280, 1974. 18. Lin, P.S., Kwock, L., Butterfield, C.E.: Diethyldithiocarbamate enhancement of radiation and hyperthermic effects on Chinese hamster cells in vitro. Radiat. Res. 77: 501511, 1979.
19. Litchfield, J.T., Wilcoxan, F.: A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96: 99-103,
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20. Lunec, J.: A cautionary note on the use of thiol compounds to protect normal tissues in radiotherapy. Brit. J. Radiol. 54: 428-429, 1981. 21. Milas, L., Ito, H., Hunter, N., Peters, L.J.: Zn vivo radioprotection of normal tissues and a tumor by diethyldithiocarbamate (DDC). Int. J. Radiat. 0~01. Biol. Phys. 10: 1806, 1984. 22. Parkins, C., Fowler, J.F., Denekamp, J.A.: Low radioprotection by thiol in lung: The role of local tissue oxygenation. Europ. J. Cancer Clin. Oncol. 19: 1169-1172, 1983. 23. Penhaligon, M.: Radioprotection of mouse skin vasculature and the RIF-1 fibrosarcoma by WR-272 1. Int. J. Radiat. Oncol. Biol. Phys. 10: 1541-1544, 1984. 24. Peskin, A.V., Koen, Y.M., Zbarsky, LB., Konstantinov, A.A.: Superoxide dismutase and glutathione peroxidase activities in tumors. FEBS LETT 78: 41-45, 1977. 25. Phillips, T.L.: Qualitative alteration in radiation injury under hypoxic conditions. Radiol. 91: 529-536, 1968. 26. Shewell, J.: Combined chemical and hypoxic protection
Radiosensitization-protection: DDC in vivo 0 R. G. EVANS against whole body X irradiation Radiat. Rex 36: 508-520, 1968.
in the nestling
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27. Sunderman, F.W.: The treatment of acute nickel-carbonyl poisoning with sodium Diethyldithiocarbamate. Ann. Clin. Rex 3: 182-185, 1971. 28. Till, J.E., McCulloch, E.A.: A direct measurement of the radiosensitivity of normal mouse bone marrow cells. Rad. Rex 14: 213-222, 1961. 29. Twentyman, P.R., Brown, J.M., Gray, J.W., Franko, A.J., Stoles, M.A., Kallman, R.F.: A new mouse tumor model system RIF-1 for comparison of end-point studies. J. NCZ 64: 595-603, 1980. 30. Ward, J.F.: Chemical aspects of DNA radioprotection. In
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Radioprotectors and Anticarcinogens, O.F. Nygaard and M.G. Simic (Eds.). Orlando, FL, Academic Press. 1983, pp. 73-85. S.: Diethyldithiocarbamate 31. Westman, G., Marklund, (DDC), a superoxide dismutase inhibitor, decreases the radioresistance of Chinese hamster cells. Radiat. Res. 83: 303-311, 1980. 32. Woolley, P.V., Ayoob, M.J., Smith, F.P., Dritschilo, A.: Clinical trial of the effect of S-2-(3-Aminopropylamino)ethylphosphorothioic Acid (WR-2721) on the toxicity of cyclophosphamide. J. Clin. Oncol. 1: 198-203, 1983. 33. Yuhas, J.M.: Active versus passive absorption kinetics as the basis for selective protection of normal tissues by WR2721. Cancer Res. 40: 1519-1524, 1980.
0360-3016/85 $03.00 + .X7 Copyright 8 ,985 Pergamon Press Ltd.
I I, pp. 1163-l 169
??Original Contribution
TUMOR RADIOSENSITIZATION WITH CONCOMITANT BONE MARROW RADIOPROTECTION: A STUDY IN MICE USING DIETHYLDITHIOCARBAMATE (DDC) UNDER OXYGENATED AND HYPOXIC CONDITIONS RICHARD G. EVANS, PHD., Fricke Radiobiology
Research
Laboratories,
M.D.
Mayo Clinic & Mayo Foundation,
Rochester,
MN 55905
We have established, both in vitro and in viva, that Diethyldithiocarbamate(DDC) protects mammaliancells from radiation. The in viva protection, when non-toxic concentrations of DDC are present one-half hour before irradiation, is reflected by a dose modification factor (DMF) of 1.9 based on LDs,po and 1.5 using survival of CFU, as an endpoint. Further experiments in viva have extended our knowledge to the differential radioprotective effects of DDC on the bone marrow of animals breathing room air compared to a 5.5% oxygen in nitrogen mixture. The DMF (LDsopo) for DDC in air breathing animals, previously established as 1.9, can be contrasted with a DMF, obtained in the present study, of 1.2 for animals irradiated in the hypoxic state. Moreover the DMF (CFU, survival) previously established at 1.5 for air breathing animals, can be compared to a value of 1.3, obtained in the present study, for mice irradiated under hypoxic conditions. Modification of the dose response by DDC, for both bone marrow and tumor, was also examined in animals bearing a RIF sarcoma. Although protection of the bone marrow was confirmed (DMF = 2.1), the striking finding was that ILL tumor cells Were sensirized, in both air breathing and nitrogen breathing animals, by the addition of DDC one-half hour before the radiation exposure. Moreover, the tumor radiosensitization, a factor greater than 2, in air breathing animals, appeared to be independent of dose (4 = 200 rad, with or without DDC). The tumor radiosensitization was even more marked in the nitrogen breathing mice, in which a factor of 10 difference in survival was noted, together with a tendency towards greater sensitization at radiation doses in the clinical range. The results, demonstrating bone marrow radioprotection by DDC (aerobic > hypoxic) with concomitant tumor radiosensitization (hypoxic > aerobic) strongly suggest a large therapeutic gain factor (TGF) which could be further explored in a clinical setting. Radiosensitization,
Radioprotection,
DDC.
INTRODUCTION
of the “hypoxic tumor cell” remains questionable due not only to the phenomenon of reoxygenation but to the evolving evidence that slight natural hypoxia may exist in some critical normal tissues. Recently, the role of tissue oxygen concentration has been underlined in experiments designed to demonstrate the radioprotective properties of various thiols.6,20 In the study of Denekamp et al.,’ using an epidermal clone assay and WR-2721, the skin protection was maximum in air and diminished at lower and higher concentrations of oxygen. Parkins et a1.,22 using breathing rate in mice as an end point following lung irradiation, found that the radioprotection afforded by WR-2721 decreased as the oxygen tension was lowered from 100% to 7%. Similar observations have been noted in spheroids (using
It has now been over a quarter of a century since Gray and his colleaguesr4 suggested that severely hypoxic cells within tumors might survive a normally sterilizing dose of radiation and following treatment become reoxygenated, recover their clonogenic potential and cause the tumor to regrow. As Kaplan has pointed out,r7 the thesis was so seductively plausible as to have become an article of faith by many investigators. To this day it has still not been well established that a sufficient number of hypoxic cells remain, following a fractionated course of radiotherapy, and that the survivors have the clonogenic potential to cause tumor regrowth. Moreover, as Hendry has noted,16 the magnitude of the therapeutic importance
Supported in part by the Mayo Clinic Comprehensive Cancer Center (CA 15083) and the Mayo Foundation. Reprint requests to: R. G. Evans, Ph.D., M.D. Acknowledgements-I am indebted to Christine Wheatley, Carol Engel and James Nielsen for excellent technical assistance and to Bonnie Hanson for her patience in the many revisions of this manuscript.
This work was supported in part by the Mayo Clinic Comprehensive Cancer Center (CA 15083) and the Mayo Foundation. Accepted for publication 7 January 1985.
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Radiation Oncology 0 Biology 0 Physics
WR-2721) when the best radioprotection was noted at intermediate levels of oxygen and the weakest protection for well oxygenated or anoxic cells.7 Our laboratory, working with DDC and plateau-phase cultures of mammalian cells,’ has shown protection factors of 1.2 for intermediate levels of oxygenation (4 ml of medium overlaying the monolayers)’ but have noted essentially no protection under well oxygenated (2 ml) or hypoxic conditions (9 ml) (Evans, R. G., unpublished data, Nov. 1983). The radioprotection afforded by DDC, and other thiols, is likely to depend on many factors, including degree of tissue oxygenation (tumor size), level of protector in the particular tissue or tumor, and size of the radiation fraction. We have established that DDC protects the bone marrow of mice from radiation as judged by modifications in the LD50,30 and survival of CFU,.9 We report here our observations on the protection afforded by DDC, as a function of the oxygen tension, within the bone marrow of Fl mice, at the time of irradiation. In addition, we present data showing tumor radiosensitization by DDC (under both oxygenated and hypoxic conditions) with concomitant bone marrow protection in the same tumor-bearing animals. MATERIALS
AND METHODS
Experimental animals and tumor The mice used were Fl hybrid (C57 Bl X BALB/c) and C3H/Km, which were bred and maintained in a specific pathogen-free colony. They were handled aseptically in laminar flow hoods for all experimental manipulations. The RIF-1 in C3H/Km mice was used throughout as the tumor model. Tumor cells were passed in animals and in culture as described by Twentyman et a1.,29 Tumors were obtained by injecting lo5 cells intradermally into the flank of 16 to 18 week-old male (C3H/Km mice. The size of the tumors, at the time of irradiation and DDC exposure was restricted to the range 150-200 mm3 (7-8 mm diameter). Irradiation procedure Mice were restrained within individual compartments in an acrylic jig and irradiated in groups of 20 using a 300 KVP x-ray machine* (2 mm copper filtration, HVL 1.4 mm copper) delivering 47 rad/minute, at the jig (114 rad/min in animals irradiated under hypoxic conditions). After irradiation the mice were housed five to an acrylic cage in light and temperature controlled rooms and allowed water and mouse chow ad libitum. The mortality was noted daily. The LD50,30 values with 95% confidence intervals were determined by the method of Litchfield and Wilcoxan.”
* General
Electric
Maxitron.
June 1985, Volume 11,Number 6
An in vivo irradiation and in vitro cell survival assay was used to determine the radiation response of RIF tumors to different radiation fraction sizes as described by Brown et al.’ Total body irradiation (dose rate 47 rad/min) was used to irradiate animals bearing the RIF tumor in their flank. In experiments designed to test the radiation response, with or without DDC, of bone marrow and RIF tumor in animals under hypoxic conditions, the animals were bathed in an atmosphere of 5.5% oxygen in nitrogen mixture for five minutes prior to and during radiation exposure.25 The cell survival assay in the tumor-bearing animals was initiated immediately after irradiation (animals had died an hypoxic death). The tumor was cut into several pieces and these made into a fine brei which was then mixed with 30 ml of complete Waymouth’s medium containing the following concentrations of enzyme: 0.02% DNase, 0.05% pronase and 0.02% collagenase as described by Brown et al4 All cells that excluded dye were scored as viable tumor cells. Following appropriate dilution procedures of the single cell suspension, cells were plated into 100 mm polystyrene petri dishes in 5 ml of medium and incubated for 8 to 10 days. Colonies were fixed and stained with crystal violet and those containing 50 or more cells scored using a magnification method. Plating efficiencies were in the range 20-30%. Diethyldithiocarbamate (DDC)? The sodium salt of DDC was made up in saline before each experiment and injected i.p. (0.1 ml per 10 grams body weight) one-half hour before initiation of irradiation. LD50,30experiments were performed with at least 10 animals per dose point and appropriate controls were used. The same dose of DDC (1000 mg/kg) was used throughout, i.e., in CFU, and tumor studies. Spleen colony assay for mouse bone marrow cells The methods of Till and McCulloch28 and Ainsworth and Larsen’ were used to obtain bone marrow survival curves, with and without the presence of DDC at the time of total body irradiation (TBI). Nine hundred and fifty rad of TBI were used to prepare the recipient mice, and no endogenous colonies were formed in the spleens of either the Fl hybrid or the C3H/Km mice at this dose. Surviving fractions were based on injecting bone marrow cells, from mice irradiated in vivo, into 10 recipient mice and determining the number of spleen colonies eight days later. In experiments using DDC, the drug was injected i.p., at a concentration of 1000 mg/kg, 30 minutes before the TBI. The survival of CFU, with and without DDC, in mice breathing air or a 5.5% oxygen-nitrogen mixture, was determined in non tumorbearing Fl hybrid mice. Additional data on CFU, survival were obtained, with and without DDC, from bone
t Sigma Chemical
Corporation,
St. Louis, MO.
Radiosensitization-protection:
marrow in air-breathing C3H/Km RIF tumor in their flank.
animals bearing a
RESULTS
Modification of the LD501j0 of Fl hybrid mice by DDC under oxygenated and hypoxic conditions The increase in LD50,30from 780 to 1490 rad, attributable to the presence of DDC at the time of irradiation, in air-breathing animals has been reported previously.g A dose modification factor (DMF,i, = LD50,30wDx i LD50,30w,0DDc)of 1.9 was obtained, as shown in Table 1. In animals breathing a 5.5% oxygen in nitrogen mixture, the LD50,30w,ooDC was raised to 1660 rad indicating that an hypoxic state had been reached at the time of total body irradiation (TBI). When animals, breathing the same mixture, received TBI in the presence of DDC an LD,o,jo of 2000 rad was obtained giving a DMFhypoxia of 1.2, as noted in Table 1. It would appear therefore, at least based on LD50,30as an endpoint, that DDC confers a greater radioprotection to well oxygenated bone marrow (DMF,i, = 1.9) than it does to hypoxic bone marrow (DMFhypoxia= 1.2). CFU, survival,for Fl bone marrow cells, with and without DDC, in air-breathing compared to 5.5% oxygen in nitrogen-breathing animals The two lower survival curves in figure 1, previously reported,g obtained in air-breathing animals allowed us to calculate a DMFai, of 1.5. When similar experiments were performed, in Fl mice breathing a 5.5% oxygen in nitrogen mixture the upper two curves in Figure 1 were obtained. The Do of 180 rad for the CFU, radiation response, with no DDC present at the time of irradiation, resulted in an oxygen enhancement ratio (OER) of 2.3 (180 f 80) indicating that an hypoxic state existed in the bone marrow of the animals breathing the oxygen in nitrogen mixture. The D,‘s of the upper two curves resulted in a dose modification factor (DFMhypoxia) of 1.3 (240 + 180). These data, based on survival of CFU,, are in concert with the findings noted in Table 1, using L50,30 as an endpoint, that DDC confers somewhat greater protection of the bone marrow stem cells in well oxygenated bone marrow (DMFai, = 1.5) compared to hypoxic bone marrow (DMFhypoxia= 1.3).
Table
1165
DDC in viva 0 R. G. EVANS
CFU, survival for bone marrow cells in RIF-bearing C3H/Km mice with and without DDC The data in Figure 2 were obtained at a single TBI dose of 400 rad in air-breathing animals, with and without the addition of DDC one-half hour before the irradiation. The Do of 140 rad for bone marrow stem cells in C3H mice obtained in the absence of DDC is somewhat greater than the Do of 80 rad obtained for the Fl hybrid mice, also in the absence of DDC. However, this radiation response of bone marrow stem cells in the C3H mice is in keeping with that noted by others.28 When the radiation was given one-half hour after the i.p. injection of DDC, a Do of 300 rad was obtained for the radiation response of the CFU,. The dose modification factor (DMF,,,), as noted in Figure 2, has a value of 2.1. This is somewhat larger than the value of 1.5 obtained for the radioprotection of CFU, in the FI hybrid mice breathing air. EJect of DDC on the dose response of RIF tumor in C3H/Km mice breathing air The data in Figure 3 were obtained from mice irradiated when their flank tumors were in the range 150200 mm3 (7-8 mm diameter). The Do of 200 rad for animals irradiated in the absence of DDC, together with the broad shoulder (D4 = 400 rad) is a similar response to that observed by others following irradiation in airbreathing animals.4 Of particular note is that although the Do for the survival curve obtained in the presence of DDC, is similar to that obtained in animals in the absence of DDC, the D, has been reduced from 400 to 175 rad. This “reduction in shoulder” implies a modification by DDC of repair of sub-lethal damage. Modijication of radiation response qf RIF tumor by DDC in N2-breathing mice When the animals were allowed to breath nitrogen for five minutes prior to and during the irradiation the data shown in Figure 4 were obtained. In the absence of DDC an extremely large “shoulder” with a D, of the order of 725 rad was obtained similar to that noted by others with the same tumor model.4 However, the striking finding was that when these tumor bearing animals were irradiated one-half hour following the i.p. injection of DDC, surviving fractions were markedly
1. LDSo,Xo as a function of presence or absence of DDC (1000 mg/m2, 30 min prior to TBI) in Fl hybrid mice breathing room air or 5.5% O2 in N2 mixture
Conditions
LDso,30 rad (95% confidence intervals)
Room Air, without DDC Room Air, with DDC
780 1490
(710-820) (1430-1550)
5.5% 02 in N2, without DDC 5.5% 02 in N2, with DDC
1660 2000
(1600-1725) (1900-2100)
Dose modification
factor (DMF)
1490 DMFai,: = 1.9 780 2000 DMF,,ypox~a: ~ = 1.2 1600
Radiation Oncology 0 Biology0 Physics
June1985, Volume II, Number6
1.0
RIF tumor (C3HIKm) Air-breathing P E. = 0.25
.-g z ?! ; s ‘.-5 2:
01
0.0 1
0
1000
mice
mg/kg
0.5 hr before
DOC irradiation
01
z
0001
0
100
200
300
400
Dose
500
600
0.01 0
250
500
750
1000
Dose in rad
in rad
Fig. 1. Survival of CFU, under oxygenated (lower two curves) and hypoxic conditions (upper two curves) in the absence of DDC (circles) and the presence of DDC (triangles). Open symbols represent data obtained from air-breathing animals; closed symbols from animals breathing a 5.5% oxygen in nitrogen mixture. The i.p. dose of DDC was 1000 mg/kg added 0.5 hr before the i~diation. Ten recipient mice were used to determine the survival at each dose point. One to six animals were used as bone marrow donors at each point, depending upon the surviving fraction. The oxygen enhancement ratio (OER) was obtained by the ratio of the Do’s of the survival curves obtained, without the presence of DDC, under hypoxic and oxygenated conditions.
Fig. 3. Radiation response of the RIF tumor in air breathing mice with and without the presence of DDC. Each data point represents the mean of four experiments & one standard error (SE). The plating efficiency (P.E.) had a mean value of -25. The size of the tumors at the time of irradiation were in the range 150-200 mm3 (7-8 mm) but the majority were in the range 170- 180 mm3. The surviving fractions, with and without DDC, were compared at three dose points using a two sample t test for different means: 250 rad @ = 0.002), 500 rad @ = 0.007) and 750 (p = 0.008).
lowered. The reduction was particularly significant at a 250 rad dose in that the surviving fraction in the presence of DDC was an order of magnitude lower than in its absence.
Recent work from our laboratory has demonstrated different tissue dist~butions of DDC (kidney > bone marrow > lung > tumor),9 radioprotection of bone mar-
\
LXX
0
\
DISCUSSION
\ \ 01
\
‘\O
\oD”=i40rad 0
5
07
DMF
0.01
3
0
D =-z-z0 Wwl DOC 8Ttr 0 ouio DDC
No DDC
o,o, i 300 140
0 100
200
300 Oose
400 in
500
[yzE+ilyi:,
2.1
600
rad
Fig. 2. Survival of CFW, with and without the presence of DDC (1000 mg/kg 0.5 hr before irradiation). Ten recipient mice were used to determine survival at each dose point and two to four animals served as donors depending upon the surviving fraction. The dose modification factor for air breathing animals (DMF,,) = 2.1.
250
500
750 Dose
yyi”
10001250
1500
in rad
Fig, 4. Radiation response of RIF tumor in animals breathing nitrogen, with and without the presence of DDC. Data points represent the mean from two to three experiments * one standard error (SE). The plating efficiency (P.E.) had a mean value of .20. The size of the tumors at the time of irradiation were in the range 150 to 200 mm3 (7-8 mm), but the majority were in the range 170- 180 mm’.
Radiosensitization-protection:
row in mice by DDC (LD50,30, survival of CFU,),9 the ability of DDC either to radiosensitize or radioprotect plateau-phase cultures of mammalian cells depending upon the irradiation conditions,* protection of bone marrow in mice exposed to high dose cis-platinum,‘2,24 enhancement of the therapeutic effect of cis-platinum in lymphoma-bearing mice by DDC” and the ability of DDC to enhance the heat sensitivity of mammalian cells in tissue culture. lo It appears, therefore, that under some conditions DDC can act as a sensitizer and in others as a protector. Lin et al.,‘* using exponentially growing cells, showed a sensitization of X ray killing by 0.1 mM DDC (enhancement ratio = 1.7) and Westman and Marklund,3’ using a 3 mM concentration of DDC, showed an enhancement ratio of 1.23 for X rays, also using exponentially growing cells. Allalunis-Turner and Chapman have recently reported* radioprotection of bone marrow in mice by single doses of DDC (DMFs in the range 1.4 to 2.9). They note that part of this “apparent” protection is due to stimulation of the bone marrow by DDC alone resulting in “real” DMFs ranging from 0.9 to 1.6. We have been unable to demonstrate, using a similar exogeneous spleen colony technique, any stimulation of the bone marrow by DDC alone and moreover, would argue that it is the end result that is important and not how much of the protection is “real”. Milas et al.*’ have demonstrated a small protective effect by DDC in a mouse fibrosarcoma, either as lung micrometastases or as a solitary leg tumor (protection factors 1. l-l .2). Penhaligon has noted that the degree of protection conferred by WR-272 1 to a RIF- 1 fibrosarcoma is dependent upon the irradiation procedure.23 If RIF-1 bearing mice are restrained in lead jigs, during local tumor irradiation, little or no protection is noted by prior treatment with WR-272 1, whereas if the mice are confined in large perspex jigs and given whole body irradiation then the tumor demonstrates marked protection by WR-2721. The experimental evidence suggests that the modification of radiation response by thiols, such as DDC and WR-272 1, is multifaceted. Factors likely to be important in the radiation modification conferred by DDC include selectivity for and the proliferative state of different normal tissues and tumor (DDC accessibility and loss), the size of the tumor at the time of irradiation (growth fraction, hypoxic fraction), the state of oxygenation of the tissues at the time of exposure (free radicals, oxygen enhancement), the radiation fraction size (both single and fractionated), the endpoint chosen (LD50,30, CFUs survival, tumor survival), and the irradiation conditions. Although we can provide no data to support active absorption of DDC by normal tissues versus passive absorption by tumor as noted with WR-272 1,33it appears that organs, relevant from a clinical standpoint regarding radioprotection (bone marrow, kidney and lung) appear to have a greater uptake than tumor cells (lymphoma’; RIF sarcoma, Evans, R. G., unpublished data, July
DDC in viva 0 R. G. EVANS
1167
1983). However, uptake per se, of thiols, has not been well correlated with degree of protection. We are presently exploring the effect of tumor size on the radiation modification conferred by DDC, using both single and fractionated doses. It is entirely possible that the radiation response of tumors as a function of size will reflect the different oxygenated/hypoxic compartments within the tumor at the time of irradiation and also DDC accessibility to these compartments. In view of the clinical and radiobiological relevance of the “hypoxic cell,” we examined whether DDC conferred different radioprotection to oxygenated versus hypoxic tumor and bone marrow (a key normal tissue). There is extensive evidence that thiols, such as DDC, protect by competing with oxygen for the fixation of radiation induced damage or by scavenging radiation induced radicals (not necessarily mutually exclusive mechanisms and does not preclude protection by hypoxia and protection by thiols operating through independent mechanisms*$ see recent review by Ward3’). As we noted in the Introduction, the radiation protection conferred by thiols appears to be intimately related to the level of oxygenation at the time of the radiations~6~20~22 and that the greatest radioprotection may be observed at “intermediate levels” of oxygenation. Our method in the present study, of obtaining an hypoxic state, in both the bone marrow and the tumors of the mice, is artificial by nature and results in a state of acute hypoxia rather than the chronic hypoxic situation that might arise naturally in an air-breathing animal or patient. However, the data from these experiments can at least serve as a guide to future experimentation. The dose modification factor (DMF, mxia) of 1.2 conferred on animals breathing 5.5% oxygen in nitrogen using LD50,30 as an endpoint is significantly lower than the DMFai, of 1.9, noted previously in air-breathing animals (Table 1). When the effect of DDC on the bone marrow of the Fl hybrid mice was examined in a somewhat different fashion, i.e., fraction of CFU, surviving, then a DMFhypoxiaof 1.3 was obtained, which is less than the 1.5 noted previously for DMF,i,. There are two lines of evidence, therefore, suggesting that DDC protects oxygenated bone marrow to a greater degree than it does hypoxic marrow. The radiation response of a RIF tumor in an air-breathing animal, determined in vitro following in vivo irradiation, was in concert with that noted previously.4 However, the finding of radiosensitization, as judged by the shoulder reduction (Figure 3) when DDC was added one-half hour before the irradiation, was unexpected. The important finding was that we had observed tumor radiosensitization in the low dose range, the significance of which will be discussed later. The effect of the induction of the hypoxic state in RIF-bearing animals, presumably in all tissues of the animal including the tumor, is reflected in a significant change in the slope (OER = 1.6) as well as the shoulder region of the radiation survival curve of the tumor cells (Figure 4). We again note that
1168
Radiation Oncology 0 Biology0 Physics
the presence of DDC one-half hour before the irradiation, causes a marked change in the survival of the tumor cells in the low dose range; a sensitization factor greater than 10 at 250 rad. (The terminal slopes of the survival curves with and without DDC are very similar, D,, = 320 rad.) It would appear therefore that the hypoxic tumor cells, at least at a 250 rad fraction size, are sensitized more by the DDC than the tumor cells in an air-breathing animal (a factor of 13 compared to 2.6). Split dose experiments exploring repair of sub-lethal damage in tumors, with and without DDC, are in progress. The significant therapeutic gain factor, i.e., the tumor radiosensitizing effect of DDC, with concomitant radio-
June 1985, Volume 11, Number 6 of the bone marrow, we have demonstrated in the RIF tumor model is most thought provoking and has direct application to the clinic. The ability of nontoxic concentrations of DDC to protect normal tissues such as bone marrow while concomitantly causing sensitization of a sarcoma warrants consideration for the use of this drug in clinical trials. There is ample evidence in the clinical literature of the minimal toxicity of DDC (treatment of nickel-carbonyl poisoning2’ and alcoholics) in contrast to the toxicity observed with WR-2721, a protective agent that has been explored in the clinic.3*‘3,32 The expertise garnered from the clinical trials with WR2721 could be most advantageously applied to trials of DDC with radiation therapy.
protection
REFERENCES 1. Ainsworth, E.J., Larsen, R.N.: Colony forming units and survival of irradiated mice treated with AET or endotoxin. Radiat. Rex 40: 149-176,
1969.
2. Allalunis-Turner, 3.
4.
5.
6.
7.
8.
M.J., Chapman, J.D.: Evaluation of diethyldithiocarbamate as a radioprotector of bone marrow. Int. J. Radiat. Oncol. Biol. Phys. 10: 1569-1573, 1984. Blumberg, A.L., Nelson, D.F., Gramkowski, N., Glover, D., Glick, J.H., Yuhas, J.M., Kligerman, M.M.: Clinical trials of WR-2721 with radiation therapy. Znt. J. Radiat. Oncol. Biol. Phys. 8: 561-563, 1981. Brown, J.M., Twentyman, P.R., Zamvil, S.S.: X-radiation (cell survival, regrowth delay, and tumor control), chemotherapeutic agents and activated macrophages. J. NCZ 64: 805-811, 1980. Denekamp, J., Michael, B.D., Rojas, A., Stewart, F.A.: Radioprotection of mouse skin by WR-2721: The critical influence of oxygen tension. Int. J. Radiat. Oncol. Biol. Phys. 8: 531-534, 1981. Denekamp, J., Michael, B.D., Rojas, A., Stewart, F.A.: Thiol radioprotection in vivo: The critical role of tissue oxygen concentration. Brit. J. Radiol. 54: 1112-l 113, 1981. Durand, R.E.: Radioprotection by WR-2721 in vitro at low oxygen tensions: Implications for its mechanism of action. Brit. J. Cancer 47: 387-392, 1983. Evans, R.G., Engel, C., Wheatley, C., Nielsen, J.: Modification of the sensitivity and repair of potentially lethal damage by diethyldithiocarbamate (DDC) during and following exposure of plateau-phase cultures of mammalian cells to radiation and cis-Diamminedichloroplatinum (II). Cancer Res. 42: 3074-3078,
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9. Evans, R.G., Engel, C.R., Wheatley, C.L., Nielsen, J.R., Ciborowski, L.J.: An in vivo study of the radioprotective effect of diethyldithiocarbamate (DDC). Znt. J. Radiat. Oncol. Biol. Phys. 9: 1635-1640,
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10. Evans, R.G., Nielsen, J., Engel, C., Wheatley, C.: Enhancement of heat sensitivity and modification of repair of potentially lethal damage in plateau-phase cultures of mammalian cells by diethyldithiocarbamate. Radiat. Res. 93: 319-325,
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11. Evans, R.G., Wheatley, C.R., Engel, C.R., Nielsen, J.R.: Enhancement of the therapeutic effect of cis-platinum by DDC: A study in lymphoma-bearing mice. Cancer Chemother. Pharmacol. (In press) 1985. 12. Evans, R.G., Wheatley, C., Engel, C., Nielsen, J., Ciborowski, L.J.: Modification of the bone marrow toxicity of cis-Diamminedichloroplatinum (II) in mice by diethyldi-
thiocarbamate (DDC). Cancer Res. 44: 3686-3690, 1984. 13. Glick, J.H., Glover, D.J., Weiler, C., Blumberg, A.L., Nelson, D.F., Yuhas, J.M., Kligerman, M.M.: Phase I clinical trials of WR-2721 with alkylating agent chemotherapy. Int. Radiat. Oncol. Biol. Phys. 8: 575-590, 1981. 14. Gray, L.H., Conger, A.E., Ebert, N., Hornsey, S., Scott, O.C.A.: The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Brit. J. Radiol. 26: 638-645,
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15. Hahn, G.M., Bagshaw, M.A., Evans, R.G., Gordon, L.F.: Repair of potentially lethal lesions in X-irradiated, denselyinhibited Chinese hamster cells: Metabolic effects and hypoxia. Radiat. Res. 55: 280-290, 1973. 16. Hendry, J.H.: Quantitation of the radiotherapeutic importance of naturally-hypoxic normal tissues from collated experiments with rodents using single doses. Znt. J. Radiat. Oncol. Biol. Phys. 5: 971-976,
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17. Kaplan, H.S.: On the relative importance of hypoxic cells for the radiotherapy of human tumors. Europ. J. Cancer 10: 275-280, 1974. 18. Lin, P.S., Kwock, L., Butterfield, C.E.: Diethyldithiocarbamate enhancement of radiation and hyperthermic effects on Chinese hamster cells in vitro. Radiat. Res. 77: 501511, 1979.
19. Litchfield, J.T., Wilcoxan, F.: A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96: 99-103,
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20. Lunec, J.: A cautionary note on the use of thiol compounds to protect normal tissues in radiotherapy. Brit. J. Radiol. 54: 428-429, 1981. 21. Milas, L., Ito, H., Hunter, N., Peters, L.J.: Zn vivo radioprotection of normal tissues and a tumor by diethyldithiocarbamate (DDC). Int. J. Radiat. 0~01. Biol. Phys. 10: 1806, 1984. 22. Parkins, C., Fowler, J.F., Denekamp, J.A.: Low radioprotection by thiol in lung: The role of local tissue oxygenation. Europ. J. Cancer Clin. Oncol. 19: 1169-1172, 1983. 23. Penhaligon, M.: Radioprotection of mouse skin vasculature and the RIF-1 fibrosarcoma by WR-272 1. Int. J. Radiat. Oncol. Biol. Phys. 10: 1541-1544, 1984. 24. Peskin, A.V., Koen, Y.M., Zbarsky, LB., Konstantinov, A.A.: Superoxide dismutase and glutathione peroxidase activities in tumors. FEBS LETT 78: 41-45, 1977. 25. Phillips, T.L.: Qualitative alteration in radiation injury under hypoxic conditions. Radiol. 91: 529-536, 1968. 26. Shewell, J.: Combined chemical and hypoxic protection
Radiosensitization-protection: DDC in vivo 0 R. G. EVANS against whole body X irradiation Radiat. Rex 36: 508-520, 1968.
in the nestling
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