Inl. J Radiolion Oncology Biol. Phys.. Vol. 18. pp. 1387-1392 Printed in the U.S.A. All rights reserved.
Copyright
0360-3016/90 53.00 + .oO 0 1990 Pergamon Press plc
??Original Contribution
16,16-DIMETHYL PROSTAGLANDIN E2 AND/OR SYNGENEIC BONE MARROW TRANSPLANTATION INCREASE MOUSE SURVIVAL AFTER SUPRA-LETHAL TOTAL BODY IRRADIATION L. B. BERK, Department
PH.D.,
M.D.,
K. D. PATRENE,
M.S.
AND
S. S. BOGGS, PH.D.
of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA I526 1
We evaluated the effects of 16,16-dimethyl prostaglandin E2 (dm-PGE2), with and without syngeneic bone marrow transplantation (BMT) on the survival and hematopoietic recovery of mice given 14-20 Gy total body irradiation (TBI). Survival of mice given combined dm-PGE2 and BMT was improved significantly over that of mice given either treatment alone. The 30-day survival after 14, 15, 16 or 18 Gy TBI for combined treatment was 97,90, 20 or 10 percent, respectively. The corresponding 30-day survival rates for mice given BMT alone were 69, 60, 7 or 0 percent, respectively. For dm-PGE2 alone, 30-day survival was 63, 20, 10 or 0 percent, respectively. Deaths in both dm-PGE2 treated groups generally occurred after day 10 whereas deaths in the BMT group occurred before day 10. All irradiated controls were dead on or before day 10; after larger doses, deaths clustered around day 5. After 20 Gy TBI, all mice in all groups were dead by day 7. Studies of white blood cell recovery l-9 days after 14 Gy TBI showed improvement with BMT, whereas dm-PGE2 did not enhance recovery. Nucleated cells per humerus, spleen weight, and spleen iron uptake (erythropoiesis) were also improved by BMT but not dm-PGEZ. Prostaglandins,
Total body irradiation, Bone marrow Transplantation,
INTRODUCTION
Hematopoiesis.
and animal survival (24) is increased when dm-PGE2 is injected prior to TBI. The present studies were undertaken to evaluate the efficacy of combining dm-PGE2 treatment with BMT to improve overall survival after doses of TBI in excess of those that can safely be given with either treatment alone. Based on previous studies ( 13, 16), we chose to use 20 ug, given 20 minutes before irradiation and to delay marrow injection until 2-3 hr after irradiation. The combination of 20 ug dm-PGE2 and BMT was found to be much more effective than either dm-PGE2 or BMT in protecting mice from supra-lethal TBI.
The
use of bone marrow transplantation (BMT) to overmarrow toxicity has made it possible to increase the doses of cytotoxic agents including total body irradiation (TBI) which can be used against cancer. Further increases in these doses is limited by gastrointestinal toxicity. Increasing interest in the role of biological response modifiers in the protection of normal tissues against such toxicities prompted us to evaluate 16,16-dimethyl prostaglandin E2 (dm-PGE2) with and without BMT in modifying the response of mice to supra-lethal doses of TBI. Prostaglandins mediate an ever increasing array of biological functions (7). There has been particular interest in their ability to protect gut mucosa against injury by noxious agents such as boiling water, hydrochloric acid, sodium hydroxide (4), and ionizing radiation (6, 15, 19). Studies of crypt microcolonies and microscopic morphology of gut ( 12-14) have demonstrated a dramatic radioprotective effect of exogenous dm-PGE2. It has also been reported that hematopoietic cells are protected ( 12) come
METHODS
AND
MATERIALS
Mice The mice were C57BL/6J X DBA/U (B6D2FI) (Jackson Laboratories, Bar Harbor, Maine) females housed 56 per cage and given food and pH 2.4 acidified water ad libitum. For survival studies mature mice 12 to 26 weeks
Reprint requests to: Sallie S. Boggs, Ph.D., RC5 14 Scaife Hall, Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA 1526 1. Winner of the Robert D. Mosley Jr. Award in Radiation Protection in Medicine of the National Council on Radiation Protection and Measurements for 1990.
Acknowledgements-Source
of Financial Support: This investigation was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant #5ROl-DK14352-19 of the U.S. Public Health Service, Department of Health and Human Services. Accepted for publication 20 December 1989.
1387
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I. J. Radiation Oncology 0 Biology 0 Physics
old weighing 23-25 g were used. Groups to be compared consisted of lo-20 mice of comparable age and weight. 16,16-dimethyl prostaglandin E2 The dm-PGE2* was dissolved in methyl acetate ( 10 mg/ml). Just before use, the methyl acetate was removed under flowing nitrogen and the dry or near-dry prostaglandin was resuspended in absolute ethanol as a 4 mg/ ml solution. This was then diluted with normal saline to 0.2 mg/ml dm-PGE2 in 5% ethanol in normal saline. Dm-PGE2 was injected subcutaneously at the nape of the neck as a solution of 20 ug in 0.1 ml. The injections were completed within 10 min and, 10 min later, irradiation was initiated. Since irradiation lasted about 15 min, the time from injection to mid irradiation was 17-27 min. These doses and times were chosen on the basis of published data ( 13,24) to balance the beneficial effects of dmPGE2 on the gut against the possible deleterious effects on the injected marrow cells (Variable effects of PGE2 on bone marrow cell proliferation have been reported (4, 5, 9, 10, 11, 16, 17, 22). Injection of dm-PGE2 caused immediate effects on the mice. They developed diarrhea, ruffled hair and lethargy. In one case when the dm-PGE2 had been prepared by evaporating the methyl acetate under vacuum rather than under flowing NZ, the evaporation was not as complete and the injection failed to cause the acute effects and also failed to protect the mice from irradiation. Data are not shown for these mice. Irradiation Mice were exposed to 13’Cs gamma irradiation+ at a rate of 0.97 f 0.05 Gy/min in groups of 20 in a rotating lucite container. Bone marrow transplantation Two to 3 hr after irradiation, mice received tail vein injections of 0.2 ml of Hanks’ solution with or without lo7 marrow cells per ml (2 X lo6 cells/mouse). The marrow cells were harvested from the long bones of syngeneic mice of the same age. After treatment, mice were kept in laminar flow racks. Mice were observed at least twice a day for the first 7 days and at least once per day thereafter up to 30 days. From those mice dying within the first week, blood samples were obtained by sterile heart punctures and cultured on sheep blood agar plates, primarily to detect Pseudomonas septicemia. No Pseudomonas infection was found in any of the tested animals.
* Upjohn. +J. L. Shepherd and Associates, Inc., Mark I, Model 68 Irradiator, San Fernando, CA.
June 1990, Volume 18, Number 6
Spleen weights and spleen iron uptake Seventeen hours after intraperitoneal injection of 0.1 uCi 59Fe*in 0.2 ml (ferric chloride in 0.1 HCl diluted in 0.003 M citric acid) mice were killed by cervical dislocation and spleens harvested, stripped of extraneous tissue, weighed and placed in Bouin’s solution. Cell counts Peripheral blood was obtained from the retroorbital sinus in one microhematocrit tube (about 75 microliter), on two occasions either days 1 and 4, 2 and 7, or 3 and 9. Samples were diluted in Isoton II, lysed with Zapoglobin II and counted.$ Quantitative counts of nucleated marrow cells per humerus were obtained after flushing the marrow with Hanks’ solution as previously described (2). Statistics Survival groups were compared by the generalized Wilcoxon method and other values were compared by Student t test.
RESULTS Survival Figure 1 compares the survival of mice given BMT, dm-PGE2, or combined treatment, with that of irradiated controls for TBI doses of 14, 15, 16, or 18 Gy. After 20 Gy TBI all the mice died between days 5 and 7 and these data are not shown. After 18 Gy TBI, 1 of 10 mice survived with combined treatment; none of the mice given dm-PGE2 alone, BMT alone, or no treatment survived. The time to death with combined treatment was marginally extended as compared to controls. After 16 Gy there was some improvement in survival of the mice given combined treatment (20%) and either dm-PGE2 (10%) or BMT (7%) alone. Time to death was delayed somewhat in all groups. All of the control mice died between days 4 and 8, whereas 50% of the mice given the combined treatment were alive at day 8. After 15 Gy TBI, combined treatment with both dmPGE2 and BMT yielded 90% survival with only one death occurring at day 5. The mice given BMT had a 60% survival, all four deaths occurring within 10 days. The mice treated with dm-PGE2 alone had 50% survival rate at day 10 but, unlike the BMT treated mice, they continued to die until day 19. After 14 Gy TBI, times of death for BMT and dm-PGE2 treated mice were markedly different but final survival rates were similar. The mice given BMT
t Amersham, England. 5ZBI Coulter Counter.
PGE2 plus BMT protects from supra-lethal irradiation 0 L. B. BERK etal.
00
1389
18 GY
80
60 40 20 0 DAY Fig. 1. Survival of mice after irradiation with 14, IS, 16 and 18 Gy TBI if treated with dm-PGE2 (closed diamond), bone marrow (closed square), both (open square) or neither (open diamond). The numbers of mice per group were 35 for 14 Gy, 10 for 15 Gy, 20 for 16 Gy groups given dm-PGEZ, 30 for 16 Gy groups given no treatment or bone marrow, and 10 for the 18 Gy groups. Mice were given irradiation alone or 20 ug of dm-PGE2 subcutaneously lo-20 min before TBI with or without 2 X lo6 bone marrow cells 3 hr after TBI.
had a 69% survival rate, and all deaths occurred before day 10. The dm-PGE2 treated mice had a comparable survival rate of 63% but deaths occurred at or after day 10. Mice receiving combined treatment had a low mortality rate (l/35) (97% alive) and the one mouse that died did so within the first 10 days. The protection from early (< 10 day) death afforded by dm-PGE2 is reflected in the statistical analysis of the sur-
viva1 curves (Table 1). Since the generalized Wilcoxon test used considers the number of mice surviving at each time point, prolongation of life is weighted as heavily as eventual survival. With this in mind, one can see that the combined treatment was significantly more effective than no treatment for all radiation doses, more effective than dm-PGE2 alone for 15 and 18 Gy, and more effective than BMT alone for 16 and 18 Gy. Dm-PGE2 alone sig-
Table 1. Generalized Wilcoxon p values for comparisons of curves in Figure 1 Dose (GY)
PG* + BMT+ vs control
PG vs control
BMT vs control
PG + BMT vs PG
PG + BMT vs BMT
14 15 16 18
Copyright
0360-3016/90 53.00 + .oO 0 1990 Pergamon Press plc
??Original Contribution
16,16-DIMETHYL PROSTAGLANDIN E2 AND/OR SYNGENEIC BONE MARROW TRANSPLANTATION INCREASE MOUSE SURVIVAL AFTER SUPRA-LETHAL TOTAL BODY IRRADIATION L. B. BERK, Department
PH.D.,
M.D.,
K. D. PATRENE,
M.S.
AND
S. S. BOGGS, PH.D.
of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA I526 1
We evaluated the effects of 16,16-dimethyl prostaglandin E2 (dm-PGE2), with and without syngeneic bone marrow transplantation (BMT) on the survival and hematopoietic recovery of mice given 14-20 Gy total body irradiation (TBI). Survival of mice given combined dm-PGE2 and BMT was improved significantly over that of mice given either treatment alone. The 30-day survival after 14, 15, 16 or 18 Gy TBI for combined treatment was 97,90, 20 or 10 percent, respectively. The corresponding 30-day survival rates for mice given BMT alone were 69, 60, 7 or 0 percent, respectively. For dm-PGE2 alone, 30-day survival was 63, 20, 10 or 0 percent, respectively. Deaths in both dm-PGE2 treated groups generally occurred after day 10 whereas deaths in the BMT group occurred before day 10. All irradiated controls were dead on or before day 10; after larger doses, deaths clustered around day 5. After 20 Gy TBI, all mice in all groups were dead by day 7. Studies of white blood cell recovery l-9 days after 14 Gy TBI showed improvement with BMT, whereas dm-PGE2 did not enhance recovery. Nucleated cells per humerus, spleen weight, and spleen iron uptake (erythropoiesis) were also improved by BMT but not dm-PGEZ. Prostaglandins,
Total body irradiation, Bone marrow Transplantation,
INTRODUCTION
Hematopoiesis.
and animal survival (24) is increased when dm-PGE2 is injected prior to TBI. The present studies were undertaken to evaluate the efficacy of combining dm-PGE2 treatment with BMT to improve overall survival after doses of TBI in excess of those that can safely be given with either treatment alone. Based on previous studies ( 13, 16), we chose to use 20 ug, given 20 minutes before irradiation and to delay marrow injection until 2-3 hr after irradiation. The combination of 20 ug dm-PGE2 and BMT was found to be much more effective than either dm-PGE2 or BMT in protecting mice from supra-lethal TBI.
The
use of bone marrow transplantation (BMT) to overmarrow toxicity has made it possible to increase the doses of cytotoxic agents including total body irradiation (TBI) which can be used against cancer. Further increases in these doses is limited by gastrointestinal toxicity. Increasing interest in the role of biological response modifiers in the protection of normal tissues against such toxicities prompted us to evaluate 16,16-dimethyl prostaglandin E2 (dm-PGE2) with and without BMT in modifying the response of mice to supra-lethal doses of TBI. Prostaglandins mediate an ever increasing array of biological functions (7). There has been particular interest in their ability to protect gut mucosa against injury by noxious agents such as boiling water, hydrochloric acid, sodium hydroxide (4), and ionizing radiation (6, 15, 19). Studies of crypt microcolonies and microscopic morphology of gut ( 12-14) have demonstrated a dramatic radioprotective effect of exogenous dm-PGE2. It has also been reported that hematopoietic cells are protected ( 12) come
METHODS
AND
MATERIALS
Mice The mice were C57BL/6J X DBA/U (B6D2FI) (Jackson Laboratories, Bar Harbor, Maine) females housed 56 per cage and given food and pH 2.4 acidified water ad libitum. For survival studies mature mice 12 to 26 weeks
Reprint requests to: Sallie S. Boggs, Ph.D., RC5 14 Scaife Hall, Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA 1526 1. Winner of the Robert D. Mosley Jr. Award in Radiation Protection in Medicine of the National Council on Radiation Protection and Measurements for 1990.
Acknowledgements-Source
of Financial Support: This investigation was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant #5ROl-DK14352-19 of the U.S. Public Health Service, Department of Health and Human Services. Accepted for publication 20 December 1989.
1387
1388
I. J. Radiation Oncology 0 Biology 0 Physics
old weighing 23-25 g were used. Groups to be compared consisted of lo-20 mice of comparable age and weight. 16,16-dimethyl prostaglandin E2 The dm-PGE2* was dissolved in methyl acetate ( 10 mg/ml). Just before use, the methyl acetate was removed under flowing nitrogen and the dry or near-dry prostaglandin was resuspended in absolute ethanol as a 4 mg/ ml solution. This was then diluted with normal saline to 0.2 mg/ml dm-PGE2 in 5% ethanol in normal saline. Dm-PGE2 was injected subcutaneously at the nape of the neck as a solution of 20 ug in 0.1 ml. The injections were completed within 10 min and, 10 min later, irradiation was initiated. Since irradiation lasted about 15 min, the time from injection to mid irradiation was 17-27 min. These doses and times were chosen on the basis of published data ( 13,24) to balance the beneficial effects of dmPGE2 on the gut against the possible deleterious effects on the injected marrow cells (Variable effects of PGE2 on bone marrow cell proliferation have been reported (4, 5, 9, 10, 11, 16, 17, 22). Injection of dm-PGE2 caused immediate effects on the mice. They developed diarrhea, ruffled hair and lethargy. In one case when the dm-PGE2 had been prepared by evaporating the methyl acetate under vacuum rather than under flowing NZ, the evaporation was not as complete and the injection failed to cause the acute effects and also failed to protect the mice from irradiation. Data are not shown for these mice. Irradiation Mice were exposed to 13’Cs gamma irradiation+ at a rate of 0.97 f 0.05 Gy/min in groups of 20 in a rotating lucite container. Bone marrow transplantation Two to 3 hr after irradiation, mice received tail vein injections of 0.2 ml of Hanks’ solution with or without lo7 marrow cells per ml (2 X lo6 cells/mouse). The marrow cells were harvested from the long bones of syngeneic mice of the same age. After treatment, mice were kept in laminar flow racks. Mice were observed at least twice a day for the first 7 days and at least once per day thereafter up to 30 days. From those mice dying within the first week, blood samples were obtained by sterile heart punctures and cultured on sheep blood agar plates, primarily to detect Pseudomonas septicemia. No Pseudomonas infection was found in any of the tested animals.
* Upjohn. +J. L. Shepherd and Associates, Inc., Mark I, Model 68 Irradiator, San Fernando, CA.
June 1990, Volume 18, Number 6
Spleen weights and spleen iron uptake Seventeen hours after intraperitoneal injection of 0.1 uCi 59Fe*in 0.2 ml (ferric chloride in 0.1 HCl diluted in 0.003 M citric acid) mice were killed by cervical dislocation and spleens harvested, stripped of extraneous tissue, weighed and placed in Bouin’s solution. Cell counts Peripheral blood was obtained from the retroorbital sinus in one microhematocrit tube (about 75 microliter), on two occasions either days 1 and 4, 2 and 7, or 3 and 9. Samples were diluted in Isoton II, lysed with Zapoglobin II and counted.$ Quantitative counts of nucleated marrow cells per humerus were obtained after flushing the marrow with Hanks’ solution as previously described (2). Statistics Survival groups were compared by the generalized Wilcoxon method and other values were compared by Student t test.
RESULTS Survival Figure 1 compares the survival of mice given BMT, dm-PGE2, or combined treatment, with that of irradiated controls for TBI doses of 14, 15, 16, or 18 Gy. After 20 Gy TBI all the mice died between days 5 and 7 and these data are not shown. After 18 Gy TBI, 1 of 10 mice survived with combined treatment; none of the mice given dm-PGE2 alone, BMT alone, or no treatment survived. The time to death with combined treatment was marginally extended as compared to controls. After 16 Gy there was some improvement in survival of the mice given combined treatment (20%) and either dm-PGE2 (10%) or BMT (7%) alone. Time to death was delayed somewhat in all groups. All of the control mice died between days 4 and 8, whereas 50% of the mice given the combined treatment were alive at day 8. After 15 Gy TBI, combined treatment with both dmPGE2 and BMT yielded 90% survival with only one death occurring at day 5. The mice given BMT had a 60% survival, all four deaths occurring within 10 days. The mice treated with dm-PGE2 alone had 50% survival rate at day 10 but, unlike the BMT treated mice, they continued to die until day 19. After 14 Gy TBI, times of death for BMT and dm-PGE2 treated mice were markedly different but final survival rates were similar. The mice given BMT
t Amersham, England. 5ZBI Coulter Counter.
PGE2 plus BMT protects from supra-lethal irradiation 0 L. B. BERK etal.
00
1389
18 GY
80
60 40 20 0 DAY Fig. 1. Survival of mice after irradiation with 14, IS, 16 and 18 Gy TBI if treated with dm-PGE2 (closed diamond), bone marrow (closed square), both (open square) or neither (open diamond). The numbers of mice per group were 35 for 14 Gy, 10 for 15 Gy, 20 for 16 Gy groups given dm-PGEZ, 30 for 16 Gy groups given no treatment or bone marrow, and 10 for the 18 Gy groups. Mice were given irradiation alone or 20 ug of dm-PGE2 subcutaneously lo-20 min before TBI with or without 2 X lo6 bone marrow cells 3 hr after TBI.
had a 69% survival rate, and all deaths occurred before day 10. The dm-PGE2 treated mice had a comparable survival rate of 63% but deaths occurred at or after day 10. Mice receiving combined treatment had a low mortality rate (l/35) (97% alive) and the one mouse that died did so within the first 10 days. The protection from early (< 10 day) death afforded by dm-PGE2 is reflected in the statistical analysis of the sur-
viva1 curves (Table 1). Since the generalized Wilcoxon test used considers the number of mice surviving at each time point, prolongation of life is weighted as heavily as eventual survival. With this in mind, one can see that the combined treatment was significantly more effective than no treatment for all radiation doses, more effective than dm-PGE2 alone for 15 and 18 Gy, and more effective than BMT alone for 16 and 18 Gy. Dm-PGE2 alone sig-
Table 1. Generalized Wilcoxon p values for comparisons of curves in Figure 1 Dose (GY)
PG* + BMT+ vs control
PG vs control
BMT vs control
PG + BMT vs PG
PG + BMT vs BMT
14 15 16 18
