036C-3016/79/081313-03$02.00/O
Inr. J. Radiurio~ Oncology Biol. Phyt., Vol. 5. pp. 1313-1315 ’ Pergamon Press Ltd., 1979. Printed in the U.S.A.
0 Adriamycin
THE INTERACTION OF IRRADIATION AND ADRIAMYCIN INTESTINAL CRYPT CELLS GLENDA
Y. Ross,? LAWRENCE
THEODORE L. PHILLIPS,?+ S. GoLDsTEIN,t PH.D.
M.D.
IN
and
The intestinal crypt cell assay was used to evaluate Adriamycin (ADR) and radiation injury in LAFl mice. The MTD (LD01/60) for ADR was 8 mg/kg. Crypt cell survival was reduced to 30% of the level of survival after radiation alone when drug was given at the MTD 2 hr before or 3 hr after irradiation. Determination of crypt cell survival from -48 to +48 hr relative to 1210 rad showed little fluctuation and no evidence of a time interval yielding enhanced response. When a radiation survival curve was measured 2 hr after a dose of 8 mg/kg, it paralleled the control curve with a shift toward lower radiation doses reflecting a 70% cell kill by drug. The 3 hr split dose survival ratio of 4-6 was similar to the control values in two separate experiments. ADR appears to be toxic to intestinal crypt cells, but does not inhibit repair of radiation injury. Irradiation,
Adriamycin,
Jejunal crypt cells.
INTRODUCTION Gastrointestinal toxicity to Adriamycin (ADR) is not dose-limiting in currently used clinical schedules.3 There is evidence, however, to suggest that ADR used in conjunction with irradiation will significantly increase mortality due to gastrointestinal complications,4 since both ADR and irradiation produce similar damage including single and double strand breaks in DNA6 and chromosome fragmentation. l3 Different forms of sublethal damage may also be induced by radiation and ADR.2 In order to establish the gastrointestinal toxicity, the survival of jejunal crypt cells was measured after treating with irradiation and ADR.
Whole-body irradiation was performed in a selfcontained 2000 Ci 137Cs irradiator. The mice were placed in a rotating lucite chamber and irradiated at 235 rad min-‘. The dose rate was determined with LiF thermoluminescent dosimeters placed in paraffin phantoms. Male LAF, mice aged 10-12 weeks were caged in groups of four and given food and water ad libitum. Survival of jejunal crypt cells was determined using a procedure modified from Withers and Elkind.13 Three Y3 days after irradiation, 4 to 6 mice per group were killed by cervical dislocation. The jejuna were excised and were fixed for 24 hr in Tellyesniczky’s solution. Ten sections were cut from each jejunum and prepared for histology, providing 40-60 5 p cross sections for counting. Jejunal crypts were counted under light microscopy and the data corrected for multiplicity by Poisson statistics.
METHODS AND MATERIALS ADR was dissolved in sterile distilled water and the MTD (LD,, I,,,) was determined to be 8 mg/kg by an analysis of the survival of mice given graded doses of the drug intraperitoneally. A stock solution (0.8 mg/ml) was made immediately before each experiment and the appropriate volume injected intraperitoneally corresponding to final dosages of 3/4, l/2 and l/4 of the MTD.
RESULTS Except for simultaneous administration of ADR and radiation, the survival of jejunal crypt cells was uniformly depressed to about 30% of the value for radiation alone, whether the drug preceded or fol-
tDepartment of Radiation Oncology, University of California, San Francisco, CA 94143. *Laboratory of Radiobiology, University of California, San Francisco, CA 94143. Reprint requests to: Glenda Y. Ross. Acknowledgements-This investigation was supported by National Cancer Institute Research Grant CA-20529 and
by Contract EY-76-C-03-1012 from the U. S. Department of Energy to the Laboratory of Radiobiology, University of California, San Francisco, CA. Martha Spence provided technical assistance. Adriamycin was obtained from the Developmental Therapeutic Program, Chemotherapy Division, National Cancer Institute, Bethesda, MD. 1313
RadiationOncology 0 Biology 0 Physics ADRIAMYCIN,
August
1979, Volume
5, Number
8
ADRIAMYCIN
8mg/Kg
2 HR. BEFORE
IRRADIATION 0 ‘3’cs 0 Bmg/Kg V ISmg/Kg
“‘Cr CONTROL, ‘NNN/NN///,/7/w,Ywmm~
-46
-36
-24 TIME
1210
RAD
-12 OF
/
0 DRUG
t12 INJECTION.
t24
t36
t40
HR.
FIG. 1. Crypt cell survival as a function of time of ADR administration (8 mgikg + 1210 rad 137Cswhole-body irradiation). lowed irradiation and for all time intervals from 2 to 48 hr between treatments (Fig. 1). When radiation and ADR were given concurrently, the number of surviving crypts was the same as controls given radiation alone. This dose of ADR given alone caused no detectable change in the number of crypts. When ADR (8 mg/kg) was administered at the MTD 2 hr before irradiation, the radiation survival curve showed little change in the slope (D,=130 rad), but the curve was displaced to the left corresponding to a reduction in the shoulder of about 250 rad (Fig. 2.). The dose reduction factor (DRF=ratio of isoeffect doses for radiation alone and radiation with drug) of 1.2 most likely reflects cell killing by the drug. When ADR was given at a dose of 15 mg/kg, which corresponds to the LDSo&,,, the resulting survival curve had a smaller slope than that with radiation alone (D,=180 rad), and the DRF (measured at 10 surviving crypt cells) was 1.7. With an increase in dose of ADR given either 2 hr before or 3 hr after an exposure of 1100 rad, the cell killing also increased (Fig. 3). In this case, giving the drug after irradiation was more effective than when the drug was given prior to irradiation. DISCUSSION
The additive effect for combined treatment with ADR and radiation is consistent with observations made for cells grown in vitro. Although some forms of damage are induced by both ADR and ionizing radiation,7*1’,1* other data suggest that the damage induced by the two agents is in part different and there is the capacity for interaction.* Certainly the
V
’
/
1
600
1
I
1000
I
I
1400
DOSE,
1
1800
RAD
FIG. 2. Radiation survival curve for crypt cells in mice treated
with
15 mg/kg (V) or 8 mgikg (Cl) 2 hr before irradiation. L37Csalone (0). ADRIAMYCIN 0 INJ. 2 MR. 0
INJ.
BEFORE
3 MR. AFTER
IRRADIATION IRRADIATION
200
l3'CS CONTROL, 1100 RAD
au 0
2 .o
6.0
4.0 ADY
DOSE,
6.0
mg/Kg
FIG. 3. Crypt ceil survival for mice given whole-body irradiation (1100 rad, 137Cs) and graded drug doses. Drug given 2 hr before irradiation (0) and drug given 3 hr after irradiation (0).
agents differ must regard response of ADR-induced
in their effect on the cell cycle5 and one the data in terms of the homeostatic the tissue to radiation-induced and damage.
Interaction of Irradiation and ADR 0 Ross et (11.
Irradiation causes epithelial hyperplasia in mouse jejunal crypt cells and soon after irradiation, cells accumulate at the G,S border.‘O Although in vitro work suggest that cells in S-phase are more sensitive to the lethal action of ADR’, our data (Figs. 1 and 3) show only a slight increase in cell killing when the drug is given 3 hr after exposure as compared to 2 hr before irradiation. Epithelial hyperplasia in jejunal crypt cells after treatment with ADR is found after 48 hr.5 In vitro data suggest that cells treated in S-phase* or at the S-G,border” accumulate in G, and thus the cohort of cells irradiated within 48 hr of ADR treatment may all be in the same part of the cell cycle resulting in a rather uniform radioresponse (Fig. 1). The observation that ADR does not affect cell survival when given immediately before irradiation is consistent with data of Burholt et al.* and may indicate that some hours are needed for the drug to reach the target tissue. Since the radiosensitivity of jejunal crypt cell depends on the concentration of the drug (Figs. 2 and 3), our data suggest that simultaneous administration does not permit sufficient time for possible interaction. There is little effect on the D, of the radiation survival curve when ADR was given at the MTD, and a decrease in slope at doses approximately twice the MTD (Fig. 2). We interpret the latter as suggest-
1315
ing that irradiation has been wasted in cells which have already received a lethal dose of ADR. Since it is difficult to draw any conclusions about the possible action of ADR on repair of radiation damage from the shape of the radiation survival curve after ADR administration, we performed a split-dose experiment in which ADR (8 mg/kg) was given 2 hr before a dose of 1170 rad or before two 585 rad exposures separated by 3 hr. The survival ratio was 5.6 for controls and 3.6 in the presence of drug. Therefore, ADR does not appear to substantially depress the repair of sublethal damage induced by radiation in jejunal crypt cells. These data are consistent with those found in vitro.2.” We conclude that when ADR and irradiation are given within 48 hr of each hour, there is enhancement which depresses the number of viable jejunal crypt cells. This effect appears to be independent of the time or order of administration except when ADR and irradiation are given simultaneously at which time little enhancement is found. We could find no evidence that ADR interfered with the repair of sublethal radiation damage. These data suggest that although gastrointestinal complications are not dose-limiting when ADR is given alone, they may be more pronounced when drug and radiation are given in a combined treatment schedule.
REFERENCES 1. Barranco, S.C.: A review of the survival and cell kinetics effects of Adriamycin (NSC 123127) on mammalian cells. Cancer Chemotherapy Rpt. 6: (Part 3), 147-152, 1975. 2. Belli, J.A., Pero, A.J.: The interaction between radiation and Adriamycin damage in mammalian cells. Cancer Res. 37: 1624-1630, 1977. 3. Benjamin, R.S.: A practical approach to Adriamycin (NSC 123127) Toxicology. Cancer Chemotherapy Rpt. 6: (Part 3): 195-202, 1975. 4. Burholt, D.R., Hagemann, R.F., Cooper, J.W., Schenken, L.L., Lesher, S.: Damage and recovery assessment of the mouse jejunum to abdominal X-ray and Adriamycin treatment. Brit. J. Radiol. 48: 908-912, 1975. 5. Burholt, D.R., Hagemann, R.F., Schenken, L.L., Lefier, S.: InlIuence of Adriamycin and Adriamycinradiation combination on jejunal proliferation in the mouse. Cancer Res. 31: 22-27, 1977. 6. Byfield, J.E., Lee, Y.C., Tu, L.: Molecular interactions between Adriamycin and X-ray damage in mammalian tumor cells. Int. J. Cancer 19: 186-193, 1977. 7. Byfield, J.E., Lynch, M., Kulhanian, F., Chan,
P.Y.M.: Cellular effects of combined Adriamycin and X-irradiation in human tumor cells. Int. J. Cancer 19: 194-204, 1977. 8. Clarkson, J.M.,
Humphrey, R.M.: The effect of Adriamycin on cell cycle progression and DNA replication in Chinese hamster ovary cells. Cancer Res. 37:
2OCL205, 1977. 9. Kimler, B.F., Leeper,
D.B.: The effect of Adriamycin and radiation on G, progression. Cancer Res. 36: 3212-3215, 1976. 10. Lesher, S., Cooper, J., Hagemann, R., Lesher, J.: Proliferative patterns in the mouse jejunal epithelium after fractionated abdominal X-irradiation. Curr. Top. Radiat. Res. 10: 229-261, 1975. Il. Lett, J.T., Caldwell, I., Dean, C.J., Alexander, P.: Rejoining of X-ray-induced breaks in DNA of leukemia cells. Nature (Lond.) 214: 790-792, 1967. 12. Vig, B.K.: Chromosome aberration induced in human leukocytes by the antileukemic antibiotic Adriamycin. Cancer Res. 31: 32-38, 1971. 13. Withers, H.R., Elkind, M.M.:
Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radiat. Biol. 17: 261-267, 1970.
Inr. J. Radiurio~ Oncology Biol. Phyt., Vol. 5. pp. 1313-1315 ’ Pergamon Press Ltd., 1979. Printed in the U.S.A.
0 Adriamycin
THE INTERACTION OF IRRADIATION AND ADRIAMYCIN INTESTINAL CRYPT CELLS GLENDA
Y. Ross,? LAWRENCE
THEODORE L. PHILLIPS,?+ S. GoLDsTEIN,t PH.D.
M.D.
IN
and
The intestinal crypt cell assay was used to evaluate Adriamycin (ADR) and radiation injury in LAFl mice. The MTD (LD01/60) for ADR was 8 mg/kg. Crypt cell survival was reduced to 30% of the level of survival after radiation alone when drug was given at the MTD 2 hr before or 3 hr after irradiation. Determination of crypt cell survival from -48 to +48 hr relative to 1210 rad showed little fluctuation and no evidence of a time interval yielding enhanced response. When a radiation survival curve was measured 2 hr after a dose of 8 mg/kg, it paralleled the control curve with a shift toward lower radiation doses reflecting a 70% cell kill by drug. The 3 hr split dose survival ratio of 4-6 was similar to the control values in two separate experiments. ADR appears to be toxic to intestinal crypt cells, but does not inhibit repair of radiation injury. Irradiation,
Adriamycin,
Jejunal crypt cells.
INTRODUCTION Gastrointestinal toxicity to Adriamycin (ADR) is not dose-limiting in currently used clinical schedules.3 There is evidence, however, to suggest that ADR used in conjunction with irradiation will significantly increase mortality due to gastrointestinal complications,4 since both ADR and irradiation produce similar damage including single and double strand breaks in DNA6 and chromosome fragmentation. l3 Different forms of sublethal damage may also be induced by radiation and ADR.2 In order to establish the gastrointestinal toxicity, the survival of jejunal crypt cells was measured after treating with irradiation and ADR.
Whole-body irradiation was performed in a selfcontained 2000 Ci 137Cs irradiator. The mice were placed in a rotating lucite chamber and irradiated at 235 rad min-‘. The dose rate was determined with LiF thermoluminescent dosimeters placed in paraffin phantoms. Male LAF, mice aged 10-12 weeks were caged in groups of four and given food and water ad libitum. Survival of jejunal crypt cells was determined using a procedure modified from Withers and Elkind.13 Three Y3 days after irradiation, 4 to 6 mice per group were killed by cervical dislocation. The jejuna were excised and were fixed for 24 hr in Tellyesniczky’s solution. Ten sections were cut from each jejunum and prepared for histology, providing 40-60 5 p cross sections for counting. Jejunal crypts were counted under light microscopy and the data corrected for multiplicity by Poisson statistics.
METHODS AND MATERIALS ADR was dissolved in sterile distilled water and the MTD (LD,, I,,,) was determined to be 8 mg/kg by an analysis of the survival of mice given graded doses of the drug intraperitoneally. A stock solution (0.8 mg/ml) was made immediately before each experiment and the appropriate volume injected intraperitoneally corresponding to final dosages of 3/4, l/2 and l/4 of the MTD.
RESULTS Except for simultaneous administration of ADR and radiation, the survival of jejunal crypt cells was uniformly depressed to about 30% of the value for radiation alone, whether the drug preceded or fol-
tDepartment of Radiation Oncology, University of California, San Francisco, CA 94143. *Laboratory of Radiobiology, University of California, San Francisco, CA 94143. Reprint requests to: Glenda Y. Ross. Acknowledgements-This investigation was supported by National Cancer Institute Research Grant CA-20529 and
by Contract EY-76-C-03-1012 from the U. S. Department of Energy to the Laboratory of Radiobiology, University of California, San Francisco, CA. Martha Spence provided technical assistance. Adriamycin was obtained from the Developmental Therapeutic Program, Chemotherapy Division, National Cancer Institute, Bethesda, MD. 1313
RadiationOncology 0 Biology 0 Physics ADRIAMYCIN,
August
1979, Volume
5, Number
8
ADRIAMYCIN
8mg/Kg
2 HR. BEFORE
IRRADIATION 0 ‘3’cs 0 Bmg/Kg V ISmg/Kg
“‘Cr CONTROL, ‘NNN/NN///,/7/w,Ywmm~
-46
-36
-24 TIME
1210
RAD
-12 OF
/
0 DRUG
t12 INJECTION.
t24
t36
t40
HR.
FIG. 1. Crypt cell survival as a function of time of ADR administration (8 mgikg + 1210 rad 137Cswhole-body irradiation). lowed irradiation and for all time intervals from 2 to 48 hr between treatments (Fig. 1). When radiation and ADR were given concurrently, the number of surviving crypts was the same as controls given radiation alone. This dose of ADR given alone caused no detectable change in the number of crypts. When ADR (8 mg/kg) was administered at the MTD 2 hr before irradiation, the radiation survival curve showed little change in the slope (D,=130 rad), but the curve was displaced to the left corresponding to a reduction in the shoulder of about 250 rad (Fig. 2.). The dose reduction factor (DRF=ratio of isoeffect doses for radiation alone and radiation with drug) of 1.2 most likely reflects cell killing by the drug. When ADR was given at a dose of 15 mg/kg, which corresponds to the LDSo&,,, the resulting survival curve had a smaller slope than that with radiation alone (D,=180 rad), and the DRF (measured at 10 surviving crypt cells) was 1.7. With an increase in dose of ADR given either 2 hr before or 3 hr after an exposure of 1100 rad, the cell killing also increased (Fig. 3). In this case, giving the drug after irradiation was more effective than when the drug was given prior to irradiation. DISCUSSION
The additive effect for combined treatment with ADR and radiation is consistent with observations made for cells grown in vitro. Although some forms of damage are induced by both ADR and ionizing radiation,7*1’,1* other data suggest that the damage induced by the two agents is in part different and there is the capacity for interaction.* Certainly the
V
’
/
1
600
1
I
1000
I
I
1400
DOSE,
1
1800
RAD
FIG. 2. Radiation survival curve for crypt cells in mice treated
with
15 mg/kg (V) or 8 mgikg (Cl) 2 hr before irradiation. L37Csalone (0). ADRIAMYCIN 0 INJ. 2 MR. 0
INJ.
BEFORE
3 MR. AFTER
IRRADIATION IRRADIATION
200
l3'CS CONTROL, 1100 RAD
au 0
2 .o
6.0
4.0 ADY
DOSE,
6.0
mg/Kg
FIG. 3. Crypt ceil survival for mice given whole-body irradiation (1100 rad, 137Cs) and graded drug doses. Drug given 2 hr before irradiation (0) and drug given 3 hr after irradiation (0).
agents differ must regard response of ADR-induced
in their effect on the cell cycle5 and one the data in terms of the homeostatic the tissue to radiation-induced and damage.
Interaction of Irradiation and ADR 0 Ross et (11.
Irradiation causes epithelial hyperplasia in mouse jejunal crypt cells and soon after irradiation, cells accumulate at the G,S border.‘O Although in vitro work suggest that cells in S-phase are more sensitive to the lethal action of ADR’, our data (Figs. 1 and 3) show only a slight increase in cell killing when the drug is given 3 hr after exposure as compared to 2 hr before irradiation. Epithelial hyperplasia in jejunal crypt cells after treatment with ADR is found after 48 hr.5 In vitro data suggest that cells treated in S-phase* or at the S-G,border” accumulate in G, and thus the cohort of cells irradiated within 48 hr of ADR treatment may all be in the same part of the cell cycle resulting in a rather uniform radioresponse (Fig. 1). The observation that ADR does not affect cell survival when given immediately before irradiation is consistent with data of Burholt et al.* and may indicate that some hours are needed for the drug to reach the target tissue. Since the radiosensitivity of jejunal crypt cell depends on the concentration of the drug (Figs. 2 and 3), our data suggest that simultaneous administration does not permit sufficient time for possible interaction. There is little effect on the D, of the radiation survival curve when ADR was given at the MTD, and a decrease in slope at doses approximately twice the MTD (Fig. 2). We interpret the latter as suggest-
1315
ing that irradiation has been wasted in cells which have already received a lethal dose of ADR. Since it is difficult to draw any conclusions about the possible action of ADR on repair of radiation damage from the shape of the radiation survival curve after ADR administration, we performed a split-dose experiment in which ADR (8 mg/kg) was given 2 hr before a dose of 1170 rad or before two 585 rad exposures separated by 3 hr. The survival ratio was 5.6 for controls and 3.6 in the presence of drug. Therefore, ADR does not appear to substantially depress the repair of sublethal damage induced by radiation in jejunal crypt cells. These data are consistent with those found in vitro.2.” We conclude that when ADR and irradiation are given within 48 hr of each hour, there is enhancement which depresses the number of viable jejunal crypt cells. This effect appears to be independent of the time or order of administration except when ADR and irradiation are given simultaneously at which time little enhancement is found. We could find no evidence that ADR interfered with the repair of sublethal radiation damage. These data suggest that although gastrointestinal complications are not dose-limiting when ADR is given alone, they may be more pronounced when drug and radiation are given in a combined treatment schedule.
REFERENCES 1. Barranco, S.C.: A review of the survival and cell kinetics effects of Adriamycin (NSC 123127) on mammalian cells. Cancer Chemotherapy Rpt. 6: (Part 3), 147-152, 1975. 2. Belli, J.A., Pero, A.J.: The interaction between radiation and Adriamycin damage in mammalian cells. Cancer Res. 37: 1624-1630, 1977. 3. Benjamin, R.S.: A practical approach to Adriamycin (NSC 123127) Toxicology. Cancer Chemotherapy Rpt. 6: (Part 3): 195-202, 1975. 4. Burholt, D.R., Hagemann, R.F., Cooper, J.W., Schenken, L.L., Lesher, S.: Damage and recovery assessment of the mouse jejunum to abdominal X-ray and Adriamycin treatment. Brit. J. Radiol. 48: 908-912, 1975. 5. Burholt, D.R., Hagemann, R.F., Schenken, L.L., Lefier, S.: InlIuence of Adriamycin and Adriamycinradiation combination on jejunal proliferation in the mouse. Cancer Res. 31: 22-27, 1977. 6. Byfield, J.E., Lee, Y.C., Tu, L.: Molecular interactions between Adriamycin and X-ray damage in mammalian tumor cells. Int. J. Cancer 19: 186-193, 1977. 7. Byfield, J.E., Lynch, M., Kulhanian, F., Chan,
P.Y.M.: Cellular effects of combined Adriamycin and X-irradiation in human tumor cells. Int. J. Cancer 19: 194-204, 1977. 8. Clarkson, J.M.,
Humphrey, R.M.: The effect of Adriamycin on cell cycle progression and DNA replication in Chinese hamster ovary cells. Cancer Res. 37:
2OCL205, 1977. 9. Kimler, B.F., Leeper,
D.B.: The effect of Adriamycin and radiation on G, progression. Cancer Res. 36: 3212-3215, 1976. 10. Lesher, S., Cooper, J., Hagemann, R., Lesher, J.: Proliferative patterns in the mouse jejunal epithelium after fractionated abdominal X-irradiation. Curr. Top. Radiat. Res. 10: 229-261, 1975. Il. Lett, J.T., Caldwell, I., Dean, C.J., Alexander, P.: Rejoining of X-ray-induced breaks in DNA of leukemia cells. Nature (Lond.) 214: 790-792, 1967. 12. Vig, B.K.: Chromosome aberration induced in human leukocytes by the antileukemic antibiotic Adriamycin. Cancer Res. 31: 32-38, 1971. 13. Withers, H.R., Elkind, M.M.:
Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radiat. Biol. 17: 261-267, 1970.
