In vivo radioprotective activities of diethyldithiocarbamate (DDC)

Copyright

0360.3016184 $03.00 + .Xl 0 1984 Pergamon Press Ltd.

o Original Contribution IN

YZVO RADIOPROTECTIVE ACTIVITIES DIETHYLDITHIOCARBAMATE (DDC)

LUKA MILAS, HISAO The University

of Texas

OF

M.D., PH.D.,* NANCY HUNTER, B.S., M.Sc.,* ITO, M.D.* AND LESTER J. PETERS, M.D.7

M.D. Anderson Hospital, and Tumor Institute at Houston, Houston, TX 77030

Studies were performed to determine whether diethyldithiocarbamate (DDC) protects against radiation damage to bone marrow, jejunal crypts, testicular tubules, hair follicles, tissues in the leg responsible for leg contractures, and a fibrosarcoma (FSA) of GHf/Kam mice. In most experiments, DDC at a dose of 400 mg/kg or 1000 mg/ kg body weight was given i.p. 30 minutes before single doses of gamma radiation. DDC (1000 mg/kg) given 30 minutes before whole-body irradiation protected hematopoietic stem cells by a factor (PF) of 1.59, as assessed by the LD50,3,1 assay, and by PFs of 1.32-1.55, as assessed by the endogenous spleen colony assay. A dose of 400 mg/kg DDC was less effective. Protection was also significant against hair loss and leg contractures; PFs produced by 1000 mg/kg DDC were 1.44 and 1.38-1.51, respectively. Jejunum was protected by 400 mg/kg DDC (PF = 1.2), but not by 1000 mg/kg. The opposite was observed with testis: 1000 mg/kg was protective (PF = 1.2), but not 400 mg/kg. DDC also protected the FSa tumor, either as lung micrometastases or as a solitary tumor in the leg. Both 400 mg/kg and 1000 mg/kg DDC protected 4 day-old micrometastases by a PF of approximately 1.1. DDC at a dose of 1000 mg/kg protected 8 mm leg tumors by a PF of 1.24 at the TCDS,, level. Therefore, DDC protected both normal tissues and FSA, but the degree of protection varied greatly. A therapeutic gain was achieved in some instances. DDC, Radioprotection,

Normal tissues, Tumors, Metastases.

peutic gain of radiotherapy, a search for new radiosensitizers and radioprotectors with potential clinical applicability is warranted. Nearly 30 years ago, diethyldithiocarbamate (DDC), a potent chelating agent, was found to significantly protect mice against lethal effects of whole body irradiation (WBI).2.32More recently, on the basis of in vitro experiments using plateau phase cultures of mammalian cells, it has been suggested that this compound might be a useful clinical radioprotector, especially since it is relatively nontoxic.‘6.30 In this report we describe our studies on the in vivo radioprotective potential of DDC, using several normal tissues and a solid fibrosarcoma in mice. A considerable variation in the degree of radioprotection was observed among normal tissues. In addition, DDC protected the tumor, providing a therapeutic gain only in some situations.

INTRODUCTION

Over the past decade, chemical radiosensitizers and radioprotectors have been the subject of significant research because of their potential for increasing the therapeutic gain when combined with radio- and chemotherapy. Misonidazole has become a classic prototype of radiosensitizers and WR-2721 a classic prototype of radioprotectors. Considerable experimental evidence shows that misonidazole radiosensitizes tumors preferentially over normal tissues, and WR-272 1 radioprotects normal tissues better than it does tumors. 1*3*5.‘8,21,38 Both these drugs have undergone clinical testing, but their undesirable side effects 23,3’limit the usefulness of these compounds. Since preferential protection of normal tissues and preferential sensitization of tumors increase the thera* Department of Experimental Radiotherapy. t Division of Radiotherapy. Reprint requests to: Dr. Luka Milas, Department of Experimental Radiotherapy, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, TX 77030. Acknowledgements-We wish to thank Hank Faykus and Ilona Kiss for their technical assistance, Rozanne Goddard for her assistance in the preparation of this manuscript, and Lane Watkins and his staff for the supply and care of animals used in these studies. Animals used in this study were maintained

in facilities approved by the American Association for Accreditation of Laboratory Animal Care, and in accordance with current regulations and standards of the United States Department of Agriculture and Department of Health and Human Services, National Institutes of Health. This investigation was supported in part by grant number CA-06294 and CA-16672, awarded by the National Cancer Institute, Department of Health and Human Services. Accepted for publication 3 July 1984.

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METHODS

AND MATERIALS

Mice Inbred CsHf/Kam mice of both sexes bred and maintained in our own specific pathogen-free mouse colony were used. They were 9 to 14 weeks old at the beginning of the experiments. Within each experiment, mice of the same sex were housed 4 to 7 per cage. DDC DDC* was dissolved in 0.9% sodium chloride solution and injected i.p. in a volume equal to 0.01 ml/g body weight. The doses of DDC ranged from 200 to 1000 mg/kg body weight and were given to mice from 5 days before to 1 day after irradiation. In most experiments, however, a dose of 1000 mg/kg or 400 mg/kg of DDC was given 30 minutes before irradiation. Assays for normal tissue radiation damage Gut. The microcolony assay introduced by Withers and Elkind3’ was used to assay the survival of crypt epithelial cells in the jejunum of mice exposed to ionizing radiation. Mice were exposed to WBI with single doses of gamma rays ranging from 12-19.5 Gy delivered by a small-animal irradiator with a single’37Cs source. The dose rate was 2.23 Gy/minute, and the distance between the source of radiation and mid-mouse plane was 28 cm. At day 3.5 after irradiation, mice were killed, and the jejunum prepared for histological examination. The regenerating crypts in the jejunal crosssection were counted. In order to construct radiation survival curves, the number of regenerating crypts was converted to the number of surviving cells by applying a Poisson correction for crypts regenerating from more than one stem ce11.35y37 Lines were fitted to data points by least-squares regression analysis. Testis. The assay for determining stem cell survival of testis seminiferous tubules is described in detail by Withers et al.36 Local irradiation of testes was performed with a small-animal irradiator with two parallel-opposed 13’Cs sources at a dose rate of 8.79 Gy/minute. Each mouse (10 weeks old) was contained without anesthesia in an acrylic box placed in such a way that the testes were within the 3 cm-diameter irradiation field. Thirtyfive days after irradiation, mice were killed, and their testes were removed and processed for histological analysis. The tubules containing spermatogenic epithelium and the tubules sectioned were counted. The average number of colony-forming stem cells surviving per tubule cross-section was determined for each animal, and its geometric mean was computed for each group of 5 mice at a given dose point. Survival curves were obtained in the same manner as described for the gut assay. Hematopoietic tissue. Mouse lethality (LD50& and endogenous spleen colony formation were the assays used to test the response of hematopoietic tissue to

* Sigma Chemical

Company,

St. Louis, MO.

December 1984, Volume IO, Number 12

irradiation and its modification by DDC. In the LD50 assay, mice were exposed to single doses of gamma rays ranging from 6.2 to 13 Gy to the whole body using the same irradiator and conditions as described above for the gut. Mice mortality was checked daily for 30 days. Radiation dose-response curves for lethality were constructed and fitted by a logit analysis. In the endogenous spleen colony assay, mice were exposed to WBI with single doses ranging from 5 to 8.2 Gy. Nine days later the mice were killed, and their spleens removed and fixed in Bouin’s solution. The colonies on the surface of the spleen were counted with the naked eye. Hair loss and reduction in leg extension. Hair loss (epilation) was examined on irradiated legs of mice in the TCDso experiment (see below, TCDso assay) 43 days after irradiation. Only mice without recurrent tumors were used for the determination of radiation-induced hair loss. At each irradiation dose point, the number of mice having 100% epilation was scored. The EDso was then determined by the logit method of analysis.’ Radiation-induced leg contraction (reduction in leg extension) was also determined on mice in the TCDso assay that had no recurrent tumors present. It was measured at day 107 after local leg irradiation using the method introduced by Dr. H. Stone (oral communication, March 1974). This method has frequently been used in our laboratory. ‘4,2o For measurements, mice were placed in an acrylic jig with tails between the vertical posts of the jig. Both the nonirradiated and irradiated legs were extended over a scale measuring millimeters. Readings were made at the ankle. The leg extension reduction values were obtained by subtracting the length of the irradiated leg from that of the nonirradiated leg. The data were analyzed by linear regression analysis. Assays for tumor response to radiation FSA is a methylcholanthrene-induced tumor that has been used often in our laboratory for studies on different aspects of tumor radiobiology. Single-cell suspensions from this tumor were prepared by trypsin digestion of nonnecrotic tumor tissue.” Viability of the cells was more than 95% as assessed by trypan blue exclusion and phase-contrast microscopy. FSA micrometastases in the lung. The assay for testing radiation-response of tumor micrometastases in the lung, previously developed in our laboratory,” was used in the present study. Micrometastases were generated by i.v. injection of lo4 viable FSA cells mixed with lo6 heavily irradiated FSA cells into mice exposed to 6 Gy WBI 1 day earlier. Four days after injection of tumor cells, mice were exposed to single doses of local thoracic irradiation (LTI) that ranged from 7.5 to 13.5 Gy. Irradiation was delivered to unanesthesized mice with a double-headed 13’Cs irradiator at 8.79 Gy/minute. The

Radioprotection with DDC 0 L. MILAS et

irradiation portal was a 3 cm-diameter circle, with the inferior margin at the level of the xiphisternum. Sixteen days after LTI, mice were killed and the number of lung nodules was determined. The number of lung nodules in mice not exposed to LTI was determined 14 days after tumor cell injection. The number of surviving clonogenic tumor cells was plotted for each irradiation dose, and survival curves were fitted to the data by leastsquares regression analysis. TCDsO assay. Mice were given injections of 5 X lo5 viable FSA cells into the right thighs. The resulting tumors were exposed to single doses of 32 to 57 Gy gamma radiation when they grew to 8 mm in diameter, which occurred 8 to 11 days after tumor cell transplantation. Irradiation was delivered by a dual-source 13’Cs irradiator, as described for testes irradiation, the difference being that the mice were not constrained in an acrylic box, but instead were immobilized in a jig. During irradiation, the tumor was centered in the circular radiation field of 3 cm in diameter. The dose rate was 8.79 Gy/minute. Mice were checked for the presence of tumor at the irradiated site for up to 107 days. TCDso values were computed by the logit method of analysis.’ Tumor growth delay. Tumors were generated by 3 X lo5 viable FSA cells injected into the right thighs. When the tumors reached 5 mm in diameter, mice were treated with 25, 30, or 35 Gy single doses of local tumor irradiation. Regression and regrowth of tumors was followed 3 times a week until tumor diameter reached 16 to 18 mm. To obtain tumor growth curves, three mutually orthogonal diameters of tumors were measured with a vernier caliper, and the mean values were calculated.

RESULTS DDC toxicity Six to 11 mice per group were given i.v. injections of DDC in doses ranging from 1000 to 3000 mg/kg, and ensuing deaths were recorded. About 10% of the mice that received 1500 mg/kg died, whereas doses of 2000 mg/kg or larger were lethal for all mice. The deaths occurred within the first day after drug injection. The LD50,30value was 1660 (1560/ 1770) mg/kg (numbers in parenthesis are 95% confidence limits). ModiJication of normal tissue response to irradiation Gut. Initial studies on the radioprotective effect of DDC were performed using the jejunum microcolony assay. Doses of 200,400, or 800 mg/kg DDC were given 30 or 60 minutes before exposure of mice to 16 Gy single-dose WBI. The surviving crypts per jejunal crosssection were counted 3.5 days later, and the number of surviving crypt cells was calculated. The results showed (Figure 1) that all 3 doses of DDC radioprotected crypt cells, with 400 mg/kg being the most effective dose. There was no apparent difference in protection if the drug was given 30 or 60 minutes before radiation

2331

al.

20.

/ 11’ /

lo-

5-

/4 / /

2-

i

DOSE OF DDC (mg/kg)

Fig. 1. Effect of different doses of DDC on radiation injury to the jejunal mucosa. Mice were treated with 16 Gy alone (A) or with DDC 30 min (0) or 1 hr (0) before exposure to irradiation. Bars, S.E.

exposure, except in the case of 200mg/kg. The effect of DDC on the radiation survival curve of jejunal crypt cells is shown in Figure 2A. Single doses of radiation ranged from 12 to 19.5 Gy; 400 or 1000 mg/kg DDC was given 30 minutes before irradiation. Radioprotection was achieved with 400, but not with 1000 mg/kg DDC, the lower dose yielding protection factors (PF) of 1.12 to 1.13. Testis. The protective effect of DDC against radiation damage to the stem cells of testis seminiferous tubules was investigated in two separate experiments. The results of both experiments showed only a small protection of this organ. Figure 2B shows the results of one experiment in which testes were exposed to 9 to 15 Gy single-dose radiation 30 minutes after treatment of mice with 400 or 1000 mg/kg DDC. Testes were protected with 1000, but not 400 mg/kg DDC: PFs for 1000 mg/kg were 1.12 to 1.13. Hematopoietic tissue. Protective effects of DDC against radiation damage of hematopoietic tissue were determined by LD50,30and endogenous spleen colony assays. Figure 3 shows the radiation response curves for the LDso in mice exposed to radiation alone, or in mice that received 400 or 1000 mg/kg DDC 30 minutes before irradiation. Both doses of DDC were protective, in particular 1000 mg/kg, which resulted in a PF of 1.59 at the LDso level. In another experiment, we examined the affect of giving DDC immediately after, 1 day after, or 1 day before WBI. The LD50 values were 7.3 (7.2-7.4) Gy to mice exposed to radiation only, and 7.90 (7.6-8.2) Gy, 7.45 (7.25-7.6), and 7.55 (7.35-7.7) in mice that received 1000 mg/kg DDC immediately after, 1 day after, and 1 day before radiation, respectively. Only administration

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0.1

December 1984, Volume 10, Number 12

c

t

PF = 1.12

t

PF = 1.12

PF = 1.12

RADIATION DOSE ( G Y I

Fig. 2. Radiation dose survival curves for jejunal crypt cells (A) and testis stem cells (B) in mice untreated (0) or treated with 400 mg/kg (A) or 1000 mg/kg (0) DDC. DDC was given i.p. 30 minutes before irradiation. Bars. SE.

5

RADIATION DOSE (GY)

Fig. 3. Radiation dose response curves for lethality of mice exposed to WBI alone (0) or mice that received 400 (A) or 1000 mg/kg (0) DDC 30 minutes before irradiation. Bars, 95% confidence limits at the LDso level.

of DDC immediately after irradiation was significantly radioprotective (PF = 1.08). The effect of DDC on the radioresponse of hematopoietic cells that form endogenous spleen colonies is presented in Figure 4. DDC at a dose of 1000 mg/kg was given 30 minutes before WBI with doses ranging from 5 to 8.2 Gy. At all radiation doses the number of spleen colonies was higher in mice that received DDC and radiation than in mice exposed to radiation only. PF values were 1.32 to 1.55. Since DDC was protective when given 1 hour after irradiation in the LD5~,30assay, the possibility that DDC stimulates proliferation of hematopoietic cells and through that mechanism protects against lethality was tested by determining the number of endogenous spleen colonies in mice that received DDC 5 days, 1 day, or 30 minutes before or 30 minutes after WBI. Results presented in Table 1 show the number of endogenous colonies was greatly increased at all time points of DDC administration, the drug being most effective when given

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Radioprotection with DDC 0 L. MILAS et al.

Table 2. Effect of DDC on radiation-induced in C3Hf/Kam mice*

hair loss

Number of mice with hair loss/ total number of mice

Irradiation dose (Gy) 32 38 42 45 48 52 51 EDso 95% Confidence limit

RADIATION DOSE (GY 1

Fig. 4. Effect of DDC on formation of endogenous spleen colonies. Mice were treated with WBI alone (0) or with irradiation 30 minutes after injection of 1000 mg/kg DDC

(0). Bars, SE.

Table 1. Effect of the time of application of DDC in relation to WBI on endogenous spleen colonies

None 5 days before WBI 1 day before WBI 30 min before WBI 30 min after WBI

DDC + Irradiation

O/3

-

O/l 616 818 616 717

O/l O/4 O/2 O/4

616

39.7 (3 1.9-49.4)

016 316 -57

* Mice with g-mm FSA in the right thighs were exposed to single doses of radiation over that thigh (see Table 3) and 43 days later the presence of hair in the irradiated area was determined in mice that had no macroscopic tumor. DDC at the dose of 1000 mg/kg was given i.p. 30 min before irradiation. was 57 (55.6-58.4)

30 minutes before irradiation. This supports the hypothesis that some of the bone marrow protective effect of DDC is caused by a proliferative stimulus rather than true radioprotective effect. Hair loss. The protective effect of DDC against hair loss after irradiation was determined on the same mice that were used in the TCDso experiment (see below). Mice bearing FSA in the right thigh were irradiated with single doses of 32 to 57 Gy gamma irradiation. Approximately one-half of the mice were given 1000 mg/kg DDC 30 minutes prior to irradiation. At 43 days after irradiation, mice free of macroscopic tumor (recurrence) were checked for hair loss on the portion of the thigh exposed to radiation. Results presented in Table 2 show the proportion of mice with complete epilation at each radiation dose level, and also the EDso values. The EDso value for mice exposed to radiation only was 39.7 (3 1.949.4) Gy; for mice treated with DDC and irradiation it

Time of application of DDC*

Irradiation

Endogenous spleen colonies in mice exposed to WBI with 5.8 Gy 2.5 f 0.8t 12.4 + 1.9

6.6 Gy 1.2 + 12.0 + 20.6 + >40 3.7 +

0.6 3.6 8.6 0.8

* Mice were injected i.p. with 1000 mg/kg DDC. t Mean + SE. The number of colonies was determined days after WBI. Each group contained 5 mice.

9

(numbers in parentheses are 95% confidence limits). The PF was 1.44. Reduction in leg extension. Protection against radiation-induced leg contraction by DDC treatment was also determined in the same mice that were used in the TCDso experiment. Mice with no recurrence in the irradiated leg were examined for reduction in leg extension caused by irradiation at 107 days after radiation. This amount of time was needed for maximal expression of the radiation damage as measured by this technique.14 Results presented in Figure 5 show that higher doses of radiation caused more profound limitation of leg extension. These changes were less severe in mice that were given 1000 mg/kg DDC prior to irradiation. PF values calculated at the level of 1.5 and 2.5 mm contractures were 1.38 and 1.5 1, respectively. Modification of tumor response to irradiation Micrometastases in the lung. Four-day-old FSA micrometastases, generated by i.v. injection of lo4 tumor cells, were exposed to single doses of local tumor irradiation ranging from 7.5 to 13.5 Gy. Groups of mice were given injections of DDC at a dose of 400 or 1000 mg/kg 30 minutes prior to irradiation. The number of lung tumor nodules was determined 16 days after local tumor irradiation. Figure 6 shows the radiation doseresponse curves in DDC-treated and untreated mice. Only a slight protection (PF = 1.1) of micrometastases was observed, with no difference in the activity between the two doses of DDC. Treatment of mice with DDC alone did not affect the number of metastases. The experiment was repeated, and it provided similar results. Solitary tumors in the leg. Tumor growth delay and TCDso assay were used to determine whether DDC protected FSA growing in the leg as solitary tumors. In the former assay, 5 mm tumors, generated by injection of 3 X 10’ FSA cells into the right thighs of mice, were

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December 1984, Volume 10, Number 12

7 show that at all three radiation dose levels, tumors in mice that received DDC prior to irradiation regrew more rapidly. The regrowth curves of tumors exposed to 25 Gy without DDC or 30 Gy with DDC are superimposed, yielding a PF of 1.20. In the TCDSo assay, 8 mm FSA tumors, generated by injection of 5 X lo5 tumor cells into the right thighs, were exposed to graded single doses of radiation ranging from 32 to 57 Gy. DDC at a dose of 1000 mg/kg was given 30 minutes before local tumor irradiation. Table 3 shows the TCDso values as determined at 100 days after irradiation, as well as the proportion of tumor cures at each dose of radiation. TCDso was 37 (32.741.8) Gy in normal mice and 45.8 (41.7-50.3) Gy in DDC-treated mice, giving a PF of 1.24.

6-

I

30

.

.

.

.

40

DISCUSSION

I

50

60

RADIATKJN DOSE (GY)

Fig. 5. Extent of leg contractures at 107 days after irradiation of mice untreated (0) or treated with DDC (0). DDC (1000 mg/kg) was given i.p. 30 minutes before irradiation of legs. Bars, S.E.

exposed to 25, 30, or 35 Gy single-dose gamma rays. Approximately one-half of the mice were given an i.p. injection of DDC at a dose of 1000 mg/kg 30 minutes prior to tumor irradiation. Results presented in Figure

DDC is a potent chelating agent that possesses many different biological activities, which are briefly reviewed here. DDC has been used as a therapeutic agent for Wilson’s Disease,30 and nickel poisoning.28929The drug also alters the effect of many other agents, mainly those that damage tissues through free radical formation, such as ionizing radiation and certain chemotherapeutic agents. Interestingly, however, alterations can be manifested as either increased or decreased damage by such agents. In vitro cell cultures treated with DDC exhibit

100

C

PF = 1.12

t

PF = 1.10

C

PF = 1.07

RADlATlON DOSE (GY) Fig. 6. Radiation dose survival curves for FSA micrometastases in lungs of mice untreated (0) or treated with 400 (A) or 1000 mg/kg (0) DDC. DDC was given i.p. 30 minutes before LTI 4 days after injection of tumor cells. Bars, SE.

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u

2u

1U

YU

40

DAYS AFTER TREATMENT Fig. 7. Growth of FSA leg tumors in mice untreated (0) or treated with 25 Gy (A), 30 Gy (H), 35 Gy (O), 25 Gy plus DDC (A), 30 Gy plus DDC (O), 35 Gy plus DDC (0), or DDC alone (0). DDC (1000 mg/kg) was given i.p. 30 minutes before irradiation when tumors had grown to 5 mm in diameter.

increased susceptibility to bleomycin,16 ionizing radiation, ‘5,26*33and hyperthermia.8 Experimental animals treated with DDC show increased oxygen toxicity” and lethality caused by ozone and paraquat.” A major mechanism for these sensitizing effects of DDC is considered to be the inhibition of superoxide dismutase (SOD).13 SOD is an enzyme present in all aerobic organisms, and it functions as a scavenger of superoxide anion, which is a highly reactive free radical generated from molecular oxygen.22 Table 3. Effect of DDC on TCDSo of 8-mm FSA in the leg of C3Hf/Kam mice* Tumor cures in mice treated with

Irradiation

dose (Gy)

32 38 42 45 48 52 57 TCDSO 95% Confidence

Irradiation 3/V l/7 617 W3 617 717 ($6

limit

(32.77:1.8)

DDC + Irradiation O/6 l/7 417 2/8 417 517 616 45.8 (41.7-50.3)

* Mice were given injections of 5 X 10’ FSA cells into the right thighs. When tumors grew to 8 mm in diameter, they were exposed to single doses of gamma rays. Mice were checked for tumor cures up to 107 days. DDC in the dose of 1000 mg/kg was given i.p. to mice before local tumor irradiation. t Number of mice cured over total number of mice.

In contrast to the above sensitizing effects of DDC, several reports show that DDC can reduce cell and tissue damaging effects of a number of agents. For example, inhibition by DDC of cis-platinum nephrotoxicity and cyclophosphamide bladder toxicityI without compromising the antitumor effect of these chemotherapeutic agents, was recently reported. The suggested mechanisms for protection were competitive chelation and removal of platinum4 and adduct formation with acrolein, which is the causative factor in cyclophosphamide-induced cystitis. ‘* DDC protected against mouse lethality caused by WB12.32and against hepatic necrosis induced by halothane6 in rats, these effects being ascribed primarily to the free-radical scavenging by DDC. More recently, DDC was found to protect in vitro plateau phase cultures of mammalian cells against ionizing radiation and cis-platinum.7 This activity was ascribed to the ability of DDC to enhance the repair of potentially lethal damage (PLD) caused by radiation and cis-platinum. Therefore, while inhibition of SOD appeared to be a major mechanism for the sensitizing effects of DDC, protective effects of DDC are mediated via chelation, free-radical scavenge, and enhancement of PLD repair. Our data presented in this paper show that DDC treatment of mice resulted in increased radioresistance of all normal tissues tested. The degree of protection, however, varied considerably among tissues, and it depended on dose and time of DDC administration in relation to radiation treatment. Bone marrow, hair follicles, and connective tissues responsible for leg contractures were protected much better (PF = 1.32-1.59)

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than jejunum and testis (PF = 1.12-l. 13). The reason for this difference is not known, but the possibility that DDC accumulates differently in different tissues should first be considered. When injected into mice, 35S-labeled DDC gave rise to a high concentration of free thiol; only a small fraction (l-3%) was bound to protein.27 In general, there was no preferential accumulation of DDC in any of the tissues tested, although, relevant to our study, a somewhat higher concentration of DDC was found in bone marrow than in the gut. Thus, no clearcut correlation exists between tissue accumulation of DDC2’ and extent of tissue radioprotection by this compound (present study). Also, no correlation was found between the dose of DDC (over the range used in this study) and the degree of radioprotection. Whereas bone marrow and testis were better protected with higher doses, 400 mg/kg appeared to be optimal and 1000 mg/kg was entirely ineffective for gut protection, and the dose of DDC had no influence on the extent of radioprotection of FSA micrometastases. It is quite possible that the pharmacokinetics of the drug is different in different tissues, and that DDC might even be toxic to some cells. The latter could be one reason for the loss of radioprotection of jejunum when 1000 mg/kg instead of 400 mg/kg DDC was used. However, as discussed above, DDC is also a potent inhibitor of SOD, which is by itself a freeradical scavenger.’ 3322In fact, the radiosensitizing effects of DDC in certain in vitro conditions were ascribed to its inhibition of SOD. Since the concentration of SOD is not the same in all tissues,22 the extent of interaction between SOD and DDC will vary depending on the tissue in which the reaction takes place, which will then likely be reflected in different degrees of radioprotection. However, the role of this interaction in the in vivo radioprotective effect of DDC is not known. In most of our experiments DDC was given 30 minutes before irradiation. However, it was equally radioprotective when given 1 hour or 30 minutes before irradiation, as determined by the protection of jejunum. This suggests that the timing of application of DDC prior to irradiation in order to achieve maximal protection might not be critical. It is also important to note that DDC reduced mouse mortality from WBI even if given 1 hour after irradiation, although the protection was much smaller than that achieved by applying DDC 30 minutes before WBI. Furthermore, DDC increased the number of endogenous spleen colonies in mice exposed to WBI when administered 1 or 5 days before or 30 minutes after irradiation, although the increase in the number of hematopoietic colonies was not as great

December 1984, Volume 10, Number 12

as when DDC was given 30 minutes before irradiation. This shows that DDC does not protect bone marrow cells only by interfering with radiation-produced free radicals, but also through some other mechanisms, most likely through direct stimulation of hematopoietic cell proliferation. It has recently been reported that DDC can act as an immunopotentiating agent;24.25this activity is likely related to the ability of DDC to induce proliferation of hematopoietic cells. In addition to its ability to protect normal tissues against radiation damage, DDC protected the fibrosarcoma FSA, whether the tumor grew as micrometastases in the lung or as solitary tumors of an appreciable size in the legs of mice. The protection of micrometastases was, however, less marked. It is possible that DDC accumulates more in hypoxic regions of larger tumors or that hypoxic conditions favor radioprotection with DDC. Our observation that DDC induces relatively good radioprotection of hair follicles in the skin (see Table 2) a tissue considered to be hypoxic to some degree. 34 Recently, DDC was reported to enhance the repair of PLD in cultured cells exposed to ionizing radiation or cis-platinum.’ It is possible that this mechanism could also have played a role in the protection of FSA, especially when the tumor grew as a solitary mass, since metabolic conditions there would be more conducive to PLD repair than in the tiny cell aggregates that compose 4 day-old micrometastases. However, other factors could also account for this, including differences in SOD levels or differences in the phase of cell cycle between small and large tumors. Definite conclusions about the therapeutic benefit of DDC treatment from our data would be premature. Although protection of FSA growing in the leg was considerable (PF = 1.24), hair follicles and tissues responsible for leg contractures were better protected (PF = 1.44 and 1.5 1, respectively), which indicates that a therapeutic gain was achieved. Thus, when these normal tissues or the bone marrow limit the radiation dose, one might expect to achieve therapeutic gain. On the other hand, no benefit of combining DDC with radiotherapy is likely to result when jejunum represents a critical normal tissue. It should be noted, however, that DDC can be given in large doses to humans without apparent toxicity,‘6,30 which might be an advantage in using this drug instead of some other, even more potent radioprotective agents, whose toxicities limit their application. Further studies appear warranted to define the in vivo radioprotective potential of DDC in more detail and to explore its ability to increase the therapeutic gain when combined with radiotherapy.

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3. Biaglow, J.E., Varnes, M.E., Astor, M., Hall, E.J.: Nonprotein thiols and cellular response to drugs and radiation.

Int. J. Radiat. Oncol. Biol. Phys. 8: 719-723, 1982. 4. Borch, R.F., Pleasants, M.E.: Inhibition of cis-platinum nephrotoxicity by diethyldithiocarbamate rescue in a rat model. Proc. Natl. Acad. Sci. U.S.A. 76: 66 11-66 14, 1979.

Radioprotection with DDC 0 L. MILAS c/ al. 5. Brown, J.M.: The mechanisms mosensitization by misonidazole

of cytotoxicity and cheand other nitroimidazoles.

Int. J. Radiat. Oncol. Biol. Phys. 8: 675-682, 1982. G.H., Lucas, K., Mitchell, 6. Eade, O.E., Millward-Sadler, J., Wright, R.: Hepatic necrosis in rats following halothane administration: Protective effect of diethyldithiocarbamate. Stand. .I. Gastroenterol. 15: 859-864, 1980. 7. Evans, R.G., Engel, C., Wheatley, C., Nielsen, J.: Modification of the sensitivity and repair of potentially lethal damage by diethyldithiocarbamate during and following exposure of plateau-phase cultures of mammalian cells to radiation and cis-diamminedichloroplatinum (II). Cancer

Rex 42: 3074-3078,

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