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Radiosensitization by diamide analogs and arsenicals
hf. J. Radiation Onm/ogy Biol. Phys.. Vol. 12, pp. 1533-1535 Printed in the U.S.A. AU rights reserved.
Copyright
0
036J.M016/86 1986 Pergamon
$3.00 + .CKl Journals Ltd.
??Session VIII
RADIOSENSITIZATION BY DIAMIDE ANALOGS AND ABSENICALS EDWARD A. BUMP, PH.D.,’ GEOFFREY P. JACOBS,PH.D.,2 WILLIAM W. LEE, PH.D.~ AND J. MARTIN BROWN, D.PHIL.~ ‘Joint Center for Radiation Therapy, Harvard Medical School, 50 Binney St., Boston, MA 02 115; ‘Department of Pharmacy, School of Pharmacy, Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 9 1120, Israel; 3SRI International, Menlo Park, CA 94025; 4Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 Several analogs of the glutathlone (GSH) oxidll
reagent dhunlde [diinedicarboxyllc acid bllN,N’dlethylamlde)] were tested as radiosensitlzers of aerobic cells. In general, radiosensitization correlates with the rate of reaction with cellular reducing agents and occurs only when the reductive capacity of the cell is exceeded. SR-lO77, [dhuenedicarboxylic acid bis(N,N-piperidide], is particularly suitable for mechanistic studies, because it is less cytotoxic than diamide, but is equally reactive toward cellular GSH. SR-4077 sensitizes CHO cells to X rays under aerobic conditions, even when the drug is added after irradiation. Radiosensitization is expressed both as a change in the exponential slope of the radiation cell survival curve and as a decrease in the shoulder of the survival curve. Phenylarsine oxide, a dithiol-binding reagent, sensitizes aerobic CHO cells to X rays by modllcatlon of the shoulder of the survival curve. The results are consistent with the hypothesis that the shoulder-modifying effect of GSH oxidants is caused by the loss of protein thiols, which might be involved in the repair of X ray-induced DNA damage. Glutathione, Diamide, Radiosensitization.
INTRODUCTION
CHO (HA- 1) cells were grown as monolayers in Eagle’s minimum essential medium (MEM) containing 15% fetal
bovine serum and antibiotics. For cell survival studies, plates were seeded (2 X lo5 cells/60 mm plate) 48 hrprior to experiment (finaldensity:2-3 X lo6 cells/plate). Drugs were added in fresh medium at room temperature 1 hr before irradiation. Drugs were removed 1 hr after irradiation and cells were replated for clonogenic assay of survival. For determination of cellular reaction rates with diamide analogs, cells were harvested by trypsinization and resuspended in Dulbecco’s phosphate-buffered saline containing 1 mgjml glucose at lo6 cells/ml. Reaction rates were based upon loss of absorbance of the diazene moiety. CHO-To cells were grown in suspension culture in Alpha MEM containing 10% fetal bovine serum and antibiotics. This cell line was used for the studies on SR-4077, as indicated. Cells were harvested by centrifugation and resuspended in fresh medium. In these studies, cells were incubated in suspension at 37” under 5% CO2 in air, except during irradiation, when the flasks were immersed in ice. Diamide analogs were dissolved in medium or PBS, as indicated. Phenylarsine oxide was dissolved in DMSO and
Presented at the Chemical Modifiers of Cancer Treatment Conference, Clearwater, Florida, 20-24 October 1985. This investigation was supported in part by Grant Number CA 1520 1- 12 and Contract Number NO 1-CM-476 11 from the
National Cancer Institute, Department of Health and Human Services. Reprint requests to: Edward A. Bump. Accepted for publication 25 February 1986.
Diamide was introduced by Kosower et al. ‘* as a reagent for oxidation of intracellular glutathione (GSH). Several studies have shown that diamide sensitizes cells to ionizing radiation under aerobic or hypoxic conditions.3T4,6 The mechanism of radiosensitization by diamide has not been established and probably is a composite of several mechanisms, since diamide affects cellular metabolism in many ways.’ Several reports indicate that diamide inhibits the repair of X ray-induced DNA single-strand breaks.2,13,‘5 Harris et al8 have reported that the diamide analog, DIP, which reacts more slowly than diamide with cellular GSH, also sensitizes cells to radiation. Their findings predominantly indicate a shoulder-modifying effect and a preferential sensitization of hypoxic cells. They also noted preliminary indications that radiosensitization could be achieved by adding DIP after irradiation. METHODS
AND
MATERIALS
1533
1534
I.
J. Radiation Oncology 0 Biology 0 Physics
diluted at least 1: 10,000 into medium. Irradiation with a 250 kVP X ray machine.*
was
RESULTS A series of diamide analogs (disubstituted diazenes) were tested as radiosensitizers of CHO (HA- 1) cells under aerobic conditions. In general, no cytotoxicity or radiosensitization was seen until drug doses were sufficient to exceed the reductive capacity of the cell (data not shown). The maximum reductive capacity of HA-l monolayers at 37” was 5.8 nanomoles per 1O6cells per min, based on the loss of diazene absorbance in the medium. SR-4222 (4-methoxyphenyldiazenecarboxylic acid thioamide) was reduced by HA-l cells at one-half the rate that diamide was reduced; and twice the dose of SR-4222 was required to achieve the same cytotoxicity and radiosensitization as diamide. Addition of dehydroisoandrosterone (DHEA), an inhibitor of glucose 6-P dehydrogenase, decreased the maximum reductive capacity of the cell and resulted in cytotoxicity and radiosensitization at lower doses of SR-
August 1986, Volume 12, Number 8
4222. These results suggest that the effects observed were caused by oxidative perturbation and not other interactions between the drug and the cell. A brief report of these results has been presented.’ SR-4077 was the only diamide analog less cytotoxic than diamide at an equal level of oxidative perturbation. While radiosensitization with diamide or other analogs was always associated with cytotoxicity (Surviving Fraction after drug alone: 0.5-0.75), very little cytotoxicity was seen with SR-4077. Radiosensitization of CHO-To cells was observed when the drug was added either before or after irradiation. Figure 1 shows the radiosensitization of CHO-To cells by SR-4077 at 37” when the drug was added after irradiation for 15 or 30 min. The surviving fraction of cells treated with drug alone was 0.88 after 15 min and 0.74 after 30 min. Data are corrected for drug cytotoxicity. Radiosensitization is manifested by both a decrease in the shoulder of the survival curve and an increase in the exponential rate of cell-killing.
r
1
I
I
lo*0 12 3 RADlAibN
DO& (Gy)
Fig. 1. Aerobic radiosensitization of CHO cells by SR-4077. CHO-To cells (lo6 cells/ml) were incubated in suspension at 37’ under 5% CO1 in air, except during irradiation, when the flasks were immersed in ice. SR-4077 (300 PM) was added after irradiation, for 15 (A) or 30 min (0). Solid circles are controls. Survival was determined clonogenically. Survival values are corrected for cytotoxicity. Results are averages of 2-3 experiments f S.E.
* Drugs were obtained from SRI International, Menlo Park, CA, as part of a drug development program supported by Contract No. NO 1-CM-476 11. Tissue culture reagents were obtained
----T-3 RADlA;lON DO;E
(Gy)
Fig. 2. Aerobic radiosensitization of CHO cells by phenylarsine oxide. CHO (HA- 1) cells were plated 48 hr before the experiment at 2 X lo5 cells/60 mm plate. Phenylarsine oxide, 0.5 FM (A) and 1.O PM (0) was added 10 min before irradiation and removed 60 min alter irradiation. Cells were at 37’, except during irradiation, which was carried out at room temperature. Cells were harvested with trypsin and replated for clonogenic survival assay. Data are corrected for cytotoxicity (S.F. for 0.5 PM: 0.92; S.F. for 1.OPM: 0.43). Results are averages horn two experiments + S.E. Controls are indicated with closed circles.
from GIBCO Laboratories, New York. All other reagents were obtained from Sigma Chemical Co., St. Louis, MO.
1535
Radiosensitization by diamide analogs 0 E. A. BUMP et al.
Figure 2 shows the effect of the dithiol-binding reagent, phenylarsine oxide, on the aerobic radiosensitivity of CHO (HA-l) cells. The effect appears to be a reduction of the shoulder of the survival curve. In these experiments, cells were treated as monolayers and the drug added 10 min before irradiation and removed 60 min after irradiation. Results are corrected for cytotoxicity. Preliminary experiments indicate that the magnitude of radiosensitization depends on the length of exposure to the drug and not on the timing between drug addition and irradiation.
DISCUSSION The redox state of GSH is an important parameter of cell function.“,’ ’ Thiol-disulfide exchange enzymes catalyze the oxidation of protein thiols by oxidized glutathione (GSSG);’ and numerous enzymes are known to require reduced thiols.’ Consequently, perturbation of the GSH redox state can affect virtually every major metabolic pathway in the cell. Since GSH oxidation leads to widespread disruption of cellular metabolism and treatment with agents such as diamide can upset the redox state of
non-thiol redox couples,’ it is difficult to assign mechanistic importance to any one of these perturbations in regard to radiosensitization. Our finding that radiosensitization by SR-4077 can be achieved by adding the reagent after irradiation rules out a mechanism involving rapid radical reactions, since these occur within milliseconds of irradiation. In addition, results obtained with GSH depleting agents appear to eliminate the importance of the loss of reduced GSH, since these cells are not sensitized to radiation by thiol depletion under aerobic conditions. Since GSH oxidation leads to oxidation of protein thiols and vicinal dithiols are particularly prone to oxidation, it is probable that SR-4077 inactivates some dithiol proteins. Ribonucleotide reductase is an example of an enzyme which requires a dithiol for activity.14 It is interesting in this regard that phenylarsine oxide, at a concentration which would favor specific binding to dithiols in the cell, also sensitizes CHO cells to radiation under aerobic conditions. However, it should be noted that dithiols are required for a variety of cellular processes. Arsenicals are highly toxic compounds, and the precise mechanism of interaction with radiation will require further study.
REFERENCES 1. Bump, E.A., Brown, J.M., Lee, W.W.: Sensitization of CHO cells to X rays by perturbation of intracellular thiols (Abstr.).
DIP and DIP+2 as glutathione oxidants and radiation sensitizers in cultured Chinese hamster cells. Znt. J. Radiat.
Fed. Proc. 44: 853, 1985. 2. Bump, E.A., Yu, N.Y., Brown, J.M.: The use ofdrugs which
Biol. 28: 439-445, 1975. 9. Jocelyn, P.C.: Biochemistry of the SH Group. London, Ac-
deplete intracellular glutathione in hypoxic cell radiosensitization. Int. J. Radiat. Oncol. Biol. Phys. 8: 439-442, 1982. 3. Chapman, J.D., Reuvers, A.P., Borsa, J., Greenstock, C.L.: Chemical radioprotection and radiosensitization of mammalian cells growing in vitro. Radiat. Res. 56: 291-306,
ademic Press, 1972. 10. Kosower, N.S., Kosower, E.M.: The glutathione status of cells. Int. Rev. Cytol. 54: 109- 160, 1978. 11. Kosower, N.S., Kosower, E.M.: Glutathione and cell membrane thiol status. In Functions ofglutathione: biochemical, physiological, Toxicological and clinical aspects, A. Iarsson, (Ed.). NY, Raven Press, 1983. 12. Kosower, N.S., Kosower, E.M., Wertheim, B.: Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem. Biophys. Res. Comm. 37: 593-
1973. 4. Clark, E.P., Michaels, H.B., Peterson, E.C., Epp, E.R.: Ir-
radiation of mammalian cells in the presence of diamide and low concentrations of oxygen at conventional and at ultrahigh dose rates. Radiat. Rex 93: 479-49 1, 1983. 5. Eriksson, S., Askelof, P., Axelsson, K., Carlberg, I., Gunthenberg, C., Mannervik, B.: Resolution of glutathionelinked enzymes in rat liver and evaluation of their contribution to disulfide reduction via Thiol-Disulfide interchange. Acta Chemica Scandinavica B 28: 922-930, 1974. 6. Harris, J.W.: Mammalian cell studies with diamide. Pharmat. Ther. 7: 375-391, 1979. 7. Harris, J.W., Biaglow, J.E.: Non-specific reactions of the
glutathione
oxidant
‘diamide’ with
mammalian
Biochem. Biophys. Res. Comm. 46: 1743-1749,
cells.
1972.
8. Harris, J.W., Power, J.A., Kosower, N.S., Kosower, E.M.:
596, 1969. 13. Meyn, R.E., Jenkins, W.T., Jenkins, S.F.: Post-irradiation
treatment of CHO cells with diamide inhibits DNA strand break rejoining (Abst.). Radiat. Res. 94: 614, 1983. 14. Thelander, L., Gmslund, A., Thelander, M.: Continual presence of oxygen and iron required for mammalian ribonucleotide reduction: possible regulation mechanism. Biochem. Biophys. Res. Comm. 110: 859-865, 1983. 15. Ward, J.F., Joner, E.I., Blakely, W.F.: Effect of inhibitors of DNA strand break repair on HeLa cell radiosensitivity. Cancer Res. 44: 59-63,
1984.
Copyright
0
036J.M016/86 1986 Pergamon
$3.00 + .CKl Journals Ltd.
??Session VIII
RADIOSENSITIZATION BY DIAMIDE ANALOGS AND ABSENICALS EDWARD A. BUMP, PH.D.,’ GEOFFREY P. JACOBS,PH.D.,2 WILLIAM W. LEE, PH.D.~ AND J. MARTIN BROWN, D.PHIL.~ ‘Joint Center for Radiation Therapy, Harvard Medical School, 50 Binney St., Boston, MA 02 115; ‘Department of Pharmacy, School of Pharmacy, Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 9 1120, Israel; 3SRI International, Menlo Park, CA 94025; 4Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 Several analogs of the glutathlone (GSH) oxidll
reagent dhunlde [diinedicarboxyllc acid bllN,N’dlethylamlde)] were tested as radiosensitlzers of aerobic cells. In general, radiosensitization correlates with the rate of reaction with cellular reducing agents and occurs only when the reductive capacity of the cell is exceeded. SR-lO77, [dhuenedicarboxylic acid bis(N,N-piperidide], is particularly suitable for mechanistic studies, because it is less cytotoxic than diamide, but is equally reactive toward cellular GSH. SR-4077 sensitizes CHO cells to X rays under aerobic conditions, even when the drug is added after irradiation. Radiosensitization is expressed both as a change in the exponential slope of the radiation cell survival curve and as a decrease in the shoulder of the survival curve. Phenylarsine oxide, a dithiol-binding reagent, sensitizes aerobic CHO cells to X rays by modllcatlon of the shoulder of the survival curve. The results are consistent with the hypothesis that the shoulder-modifying effect of GSH oxidants is caused by the loss of protein thiols, which might be involved in the repair of X ray-induced DNA damage. Glutathione, Diamide, Radiosensitization.
INTRODUCTION
CHO (HA- 1) cells were grown as monolayers in Eagle’s minimum essential medium (MEM) containing 15% fetal
bovine serum and antibiotics. For cell survival studies, plates were seeded (2 X lo5 cells/60 mm plate) 48 hrprior to experiment (finaldensity:2-3 X lo6 cells/plate). Drugs were added in fresh medium at room temperature 1 hr before irradiation. Drugs were removed 1 hr after irradiation and cells were replated for clonogenic assay of survival. For determination of cellular reaction rates with diamide analogs, cells were harvested by trypsinization and resuspended in Dulbecco’s phosphate-buffered saline containing 1 mgjml glucose at lo6 cells/ml. Reaction rates were based upon loss of absorbance of the diazene moiety. CHO-To cells were grown in suspension culture in Alpha MEM containing 10% fetal bovine serum and antibiotics. This cell line was used for the studies on SR-4077, as indicated. Cells were harvested by centrifugation and resuspended in fresh medium. In these studies, cells were incubated in suspension at 37” under 5% CO2 in air, except during irradiation, when the flasks were immersed in ice. Diamide analogs were dissolved in medium or PBS, as indicated. Phenylarsine oxide was dissolved in DMSO and
Presented at the Chemical Modifiers of Cancer Treatment Conference, Clearwater, Florida, 20-24 October 1985. This investigation was supported in part by Grant Number CA 1520 1- 12 and Contract Number NO 1-CM-476 11 from the
National Cancer Institute, Department of Health and Human Services. Reprint requests to: Edward A. Bump. Accepted for publication 25 February 1986.
Diamide was introduced by Kosower et al. ‘* as a reagent for oxidation of intracellular glutathione (GSH). Several studies have shown that diamide sensitizes cells to ionizing radiation under aerobic or hypoxic conditions.3T4,6 The mechanism of radiosensitization by diamide has not been established and probably is a composite of several mechanisms, since diamide affects cellular metabolism in many ways.’ Several reports indicate that diamide inhibits the repair of X ray-induced DNA single-strand breaks.2,13,‘5 Harris et al8 have reported that the diamide analog, DIP, which reacts more slowly than diamide with cellular GSH, also sensitizes cells to radiation. Their findings predominantly indicate a shoulder-modifying effect and a preferential sensitization of hypoxic cells. They also noted preliminary indications that radiosensitization could be achieved by adding DIP after irradiation. METHODS
AND
MATERIALS
1533
1534
I.
J. Radiation Oncology 0 Biology 0 Physics
diluted at least 1: 10,000 into medium. Irradiation with a 250 kVP X ray machine.*
was
RESULTS A series of diamide analogs (disubstituted diazenes) were tested as radiosensitizers of CHO (HA- 1) cells under aerobic conditions. In general, no cytotoxicity or radiosensitization was seen until drug doses were sufficient to exceed the reductive capacity of the cell (data not shown). The maximum reductive capacity of HA-l monolayers at 37” was 5.8 nanomoles per 1O6cells per min, based on the loss of diazene absorbance in the medium. SR-4222 (4-methoxyphenyldiazenecarboxylic acid thioamide) was reduced by HA-l cells at one-half the rate that diamide was reduced; and twice the dose of SR-4222 was required to achieve the same cytotoxicity and radiosensitization as diamide. Addition of dehydroisoandrosterone (DHEA), an inhibitor of glucose 6-P dehydrogenase, decreased the maximum reductive capacity of the cell and resulted in cytotoxicity and radiosensitization at lower doses of SR-
August 1986, Volume 12, Number 8
4222. These results suggest that the effects observed were caused by oxidative perturbation and not other interactions between the drug and the cell. A brief report of these results has been presented.’ SR-4077 was the only diamide analog less cytotoxic than diamide at an equal level of oxidative perturbation. While radiosensitization with diamide or other analogs was always associated with cytotoxicity (Surviving Fraction after drug alone: 0.5-0.75), very little cytotoxicity was seen with SR-4077. Radiosensitization of CHO-To cells was observed when the drug was added either before or after irradiation. Figure 1 shows the radiosensitization of CHO-To cells by SR-4077 at 37” when the drug was added after irradiation for 15 or 30 min. The surviving fraction of cells treated with drug alone was 0.88 after 15 min and 0.74 after 30 min. Data are corrected for drug cytotoxicity. Radiosensitization is manifested by both a decrease in the shoulder of the survival curve and an increase in the exponential rate of cell-killing.
r
1
I
I
lo*0 12 3 RADlAibN
DO& (Gy)
Fig. 1. Aerobic radiosensitization of CHO cells by SR-4077. CHO-To cells (lo6 cells/ml) were incubated in suspension at 37’ under 5% CO1 in air, except during irradiation, when the flasks were immersed in ice. SR-4077 (300 PM) was added after irradiation, for 15 (A) or 30 min (0). Solid circles are controls. Survival was determined clonogenically. Survival values are corrected for cytotoxicity. Results are averages of 2-3 experiments f S.E.
* Drugs were obtained from SRI International, Menlo Park, CA, as part of a drug development program supported by Contract No. NO 1-CM-476 11. Tissue culture reagents were obtained
----T-3 RADlA;lON DO;E
(Gy)
Fig. 2. Aerobic radiosensitization of CHO cells by phenylarsine oxide. CHO (HA- 1) cells were plated 48 hr before the experiment at 2 X lo5 cells/60 mm plate. Phenylarsine oxide, 0.5 FM (A) and 1.O PM (0) was added 10 min before irradiation and removed 60 min alter irradiation. Cells were at 37’, except during irradiation, which was carried out at room temperature. Cells were harvested with trypsin and replated for clonogenic survival assay. Data are corrected for cytotoxicity (S.F. for 0.5 PM: 0.92; S.F. for 1.OPM: 0.43). Results are averages horn two experiments + S.E. Controls are indicated with closed circles.
from GIBCO Laboratories, New York. All other reagents were obtained from Sigma Chemical Co., St. Louis, MO.
1535
Radiosensitization by diamide analogs 0 E. A. BUMP et al.
Figure 2 shows the effect of the dithiol-binding reagent, phenylarsine oxide, on the aerobic radiosensitivity of CHO (HA-l) cells. The effect appears to be a reduction of the shoulder of the survival curve. In these experiments, cells were treated as monolayers and the drug added 10 min before irradiation and removed 60 min after irradiation. Results are corrected for cytotoxicity. Preliminary experiments indicate that the magnitude of radiosensitization depends on the length of exposure to the drug and not on the timing between drug addition and irradiation.
DISCUSSION The redox state of GSH is an important parameter of cell function.“,’ ’ Thiol-disulfide exchange enzymes catalyze the oxidation of protein thiols by oxidized glutathione (GSSG);’ and numerous enzymes are known to require reduced thiols.’ Consequently, perturbation of the GSH redox state can affect virtually every major metabolic pathway in the cell. Since GSH oxidation leads to widespread disruption of cellular metabolism and treatment with agents such as diamide can upset the redox state of
non-thiol redox couples,’ it is difficult to assign mechanistic importance to any one of these perturbations in regard to radiosensitization. Our finding that radiosensitization by SR-4077 can be achieved by adding the reagent after irradiation rules out a mechanism involving rapid radical reactions, since these occur within milliseconds of irradiation. In addition, results obtained with GSH depleting agents appear to eliminate the importance of the loss of reduced GSH, since these cells are not sensitized to radiation by thiol depletion under aerobic conditions. Since GSH oxidation leads to oxidation of protein thiols and vicinal dithiols are particularly prone to oxidation, it is probable that SR-4077 inactivates some dithiol proteins. Ribonucleotide reductase is an example of an enzyme which requires a dithiol for activity.14 It is interesting in this regard that phenylarsine oxide, at a concentration which would favor specific binding to dithiols in the cell, also sensitizes CHO cells to radiation under aerobic conditions. However, it should be noted that dithiols are required for a variety of cellular processes. Arsenicals are highly toxic compounds, and the precise mechanism of interaction with radiation will require further study.
REFERENCES 1. Bump, E.A., Brown, J.M., Lee, W.W.: Sensitization of CHO cells to X rays by perturbation of intracellular thiols (Abstr.).
DIP and DIP+2 as glutathione oxidants and radiation sensitizers in cultured Chinese hamster cells. Znt. J. Radiat.
Fed. Proc. 44: 853, 1985. 2. Bump, E.A., Yu, N.Y., Brown, J.M.: The use ofdrugs which
Biol. 28: 439-445, 1975. 9. Jocelyn, P.C.: Biochemistry of the SH Group. London, Ac-
deplete intracellular glutathione in hypoxic cell radiosensitization. Int. J. Radiat. Oncol. Biol. Phys. 8: 439-442, 1982. 3. Chapman, J.D., Reuvers, A.P., Borsa, J., Greenstock, C.L.: Chemical radioprotection and radiosensitization of mammalian cells growing in vitro. Radiat. Res. 56: 291-306,
ademic Press, 1972. 10. Kosower, N.S., Kosower, E.M.: The glutathione status of cells. Int. Rev. Cytol. 54: 109- 160, 1978. 11. Kosower, N.S., Kosower, E.M.: Glutathione and cell membrane thiol status. In Functions ofglutathione: biochemical, physiological, Toxicological and clinical aspects, A. Iarsson, (Ed.). NY, Raven Press, 1983. 12. Kosower, N.S., Kosower, E.M., Wertheim, B.: Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem. Biophys. Res. Comm. 37: 593-
1973. 4. Clark, E.P., Michaels, H.B., Peterson, E.C., Epp, E.R.: Ir-
radiation of mammalian cells in the presence of diamide and low concentrations of oxygen at conventional and at ultrahigh dose rates. Radiat. Rex 93: 479-49 1, 1983. 5. Eriksson, S., Askelof, P., Axelsson, K., Carlberg, I., Gunthenberg, C., Mannervik, B.: Resolution of glutathionelinked enzymes in rat liver and evaluation of their contribution to disulfide reduction via Thiol-Disulfide interchange. Acta Chemica Scandinavica B 28: 922-930, 1974. 6. Harris, J.W.: Mammalian cell studies with diamide. Pharmat. Ther. 7: 375-391, 1979. 7. Harris, J.W., Biaglow, J.E.: Non-specific reactions of the
glutathione
oxidant
‘diamide’ with
mammalian
Biochem. Biophys. Res. Comm. 46: 1743-1749,
cells.
1972.
8. Harris, J.W., Power, J.A., Kosower, N.S., Kosower, E.M.:
596, 1969. 13. Meyn, R.E., Jenkins, W.T., Jenkins, S.F.: Post-irradiation
treatment of CHO cells with diamide inhibits DNA strand break rejoining (Abst.). Radiat. Res. 94: 614, 1983. 14. Thelander, L., Gmslund, A., Thelander, M.: Continual presence of oxygen and iron required for mammalian ribonucleotide reduction: possible regulation mechanism. Biochem. Biophys. Res. Comm. 110: 859-865, 1983. 15. Ward, J.F., Joner, E.I., Blakely, W.F.: Effect of inhibitors of DNA strand break repair on HeLa cell radiosensitivity. Cancer Res. 44: 59-63,
1984.