effects of glutathione depletion by buthionine sulphoximine on radiosensitization by oxygen and...

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1798 Radiation Oncology 0 Biology 0 Physics September 1984. Volume 10, Number 9 In these studies the GSH-, total thiol (TSH)- and nonprotein thiol (NPSH)-contents of Ehrlich ascites tumour cells (EATC) and HeLa cells after BSO application were determined. After X-irradiation the colony forming ability and micronucleus induction were used as endpoints of the radiation reaction. The depletion of GSH after incubation with BSO for time intervals up to 72 hours was more pronounced and less toxic than comparable treatments with other known thiol depleting agents. Treatment of EATC and HeLA cells with BSO resulted in a decrease of GSH to 6% resp. I% of controls. The NPSH content was not reduced to the same degree, suggesting that cysteine cannot substitute for GSH with regard to some modification of the radiobiological response. Hypoxic radiosensitization was found to depend on the applied BSO concentration with a maximum at about 250 uM. At this concentration no shoulder in the survival curve could be observed and the survival was below that of the oxic control. A biphasic concentration dependence was found in survival as well as in micronucleus measurements with a reduction of the sensi- tization at values above 400 uM. Thus. a possible relationship between GSH and the oxygen effect must be restricted to lower BSO concen- trations. Studies using BSO for inhibition of GSH biosynthesis may help to understand the specific role of glutathione in radiobiological effects and therefore should stimulate the possible application of buthionine sulfox- imine in the tumor treatment by ionizing radiations. (A pan of this investigation was supported by EURATOM contract No. 20576-I-BIO-D.) EFFECTS OF GLUTATHIONE DEPLETION BY BUTHIONINE SULPHOXIMINE ON RADIOSENSITIZATION BY OXYGEN AND MISONIDAZOLE IN VITRO DENNIS C. SHRIEVE, JULIANA DENEKAMP AND ANDREW I. MINCHIN~ON Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood. Middlesex HA6 2RN. England The radiosensitizing action of oxygen and of misonidazole (MISO) can be modified by the addition or depletion of intmoellular thiols. Buthionine sulphoximine (BSO) has been used to deplete glutathione (GSH) in V79- 379 cells in vitro, and the effect on the efficiency of oxygen and MIS0 as radiosensitizers has been determined. Untreated V79-379 cells contained 5-8 fmol/cell(5-8 mM) of GSH. Cells treated with 50 or 500 PM BSO showed a rapid decline in GSH content, falling to less than 5% after IO h exposure to these doses of BSO (T,,r = I.6 h). Cells washed free of the BSO showed rapid regen- eration of GSH after 50 hM BSO. but no regeneration was observed over the subsequent 10 h period after 500 PM. Treatment with these levels of BSO for up to 14 h did not affect cell growth or viability. GSH was measured as its monobromobimane derivative by high performance liquid chromatography. Cells irradiated in monolayer on glass had an oxygen enhancement ratio (OER) of 3. I, with a K value of0.7% OZ. After IO-14 h pretreatment with 50 PM BSO, washed cells showed sensitization in all oxygen tensions tested. The OER was reduced to 2.6, due to greater sensitization of hypoxic cells by GSH depletion. The maximum effect (ER = 1.53) was observed near the K value. The GSH depletion had the effect of shifting the K curve to lower oxygen tensions. making oxygen appear more efficient by a factor of approximately 2. Similar experiments were performed with MISO. IO-14 h pretreatment with 50 pM BSO increased the MIS0 radiosensitizing efficiency by a factor of 25. An enhancement ratio of 2.0 could be achieved with 0.2 mM MIS0 in BSO pretreated cells. instead of with 5 mM MIS0 in untreated cells. Although the effect of GSH depletion was qualitatively similar for oxygen and for MISO. it was quantitatively different. The apparent increased efficiency of MIS0 was more than IO times greater than for oxygen. Both effects can be largely explained on the basis of sensitization of hypoxic cells by GSH depletion alone (ER = 1.3). Further sensitization by oxygen or MIS0 appeared to be nearly additive with less sensitizer being required to achieve a specific degree of sensitization relative to control cells. (Supported by The Cancer Research Campaign.) EFFECT OF GLUTATHIONE DEPLETION ON RADIOSENS1TIVt-R IN VI VO A. ROJAS, J. P. SORANSON, K. A. SMITH, A. 1. MINCHINTON, R. W. MIDDLETON AND J. DENEKAMP Gray Laboratory of the Cancer Research Campaign. Mount Vernon Hospital, Northwood. Middlesex HA6 2RN. England The influence of glutathione (GSH) depletion on skin and tumor ra- diosensitivity has been studied using X rays alone, with misonidazole and oxygen being supplied at various concentrations. GSH depletion has been achieved in mice by repeated i.p. administration of BSO for periods up to 9.5 hours. Tumor regrowth delay, skin reactions, epidermal clones and pharmacology results will be presented. BSO alone is apparently non toxic and depletes GSH in tumors to 50% (i.e. less depletion than in liver, kidney and muscle). It sensitizes skin slightly with prolonged exposure (9.5 hours) but no sensitization has been seen in CA MT tumors (up to 8 hours). Prolonged BSO exposure combined with an acute dose of miso (given once or on 5 successive days) does not result in further GSH depletion in tumors. However, the combination increases the effectiveness of miso as a radiosensitizer of hypoxic cells in tumors (by a factor of -5 in CA MT and -3 in SA FA). A similar enhancement was seen in artificially hypoxic mouse skin but none was seen in well oxygenated skin. The effectiveness of oxygen as a radiosensitizer is also increased, with the “K value” for mouse skin clones being decreased by a factor of about 2. The enhancement of miso radiosensitization in tumon by BSO was not observed in a 5 fraction schedule, presumably because of reoxy- genation. The use of BSO in viva to enhance the effectiveness of misonidazole will be constrained by the limited GSH depletion in tumors, increased miso toxicity, some skin sensitization and because its effect is confined to hypoxic cells. RADIOSENSITIZATION OF HYPOXIC CELLS BY GLUTATHIONE DEPLETION WITH BSO H. SCHORN,’ U. BERTSCHE’ AND 0. Vos’ ‘Euratom DG. XII-GSF; 2 GSF. Abt. Biophys. Strahlenforschg., Frankfurt-Germany; ‘Medical Biologial Laboratory. TNO, Rijswijk and Erasmus University, Rotterdam. The Netherlands Buthionine Sulfoximine (BSO) is a selective inhibitor of y-glutamyl- cysteine synthetase. Therefore BSO may serve to clarify the influence of endogeneous glutathione on radiosensitivity. In these studies the glu- tathione. total thiol (TSH) and nonprotein thiol (NPSH) content of EAT and Hela cells after BSO application were determined. After X ray irradiation, colony forming ability and micronuclei formation were used as endpoints. The glutathione depletion after BSO was more pronounced and less toxic than treatment with other commonly used thioldepleting agents. Incubation of EAT and Hela cells with BSO resulted in a decrease in cellular glutathione to 6% and 1% of the respective controls. The NPSH concentration was not reduced to the same degree, suggesting that cysteine cannot substitute for glutathione with regard to modification of radio- biological response. Hypoxic radiosensitization was found to be dependent on the con- centration of BSO applied, with a maximum at C = 250 mM. At this concentration no shoulder could be observed and the sensitization was below the oxic control value, where a shoulder is usually found. BSO was found to act in a biphasic manner. At concentrations above 400 PM the effect was smaller than at 250 PM. The reduction of the oxygen enhancement ratio (OER) of both cell lines after treatment with BSO suggests a relationship between cellular glutathione and the oxygen effect. Studies with the inhibitor of glutathione biosynthesis, BSO, may help us to understand the specific role of glutathione in radiobiology and could stimulate its possible application in radiotherapy of tumors. (A part of the investigations were supported by the EURATOM con- tract No-20576- I-B10 D.)

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1798 Radiation Oncology 0 Biology 0 Physics September 1984. Volume 10, Number 9

In these studies the GSH-, total thiol (TSH)- and nonprotein thiol (NPSH)-contents of Ehrlich ascites tumour cells (EATC) and HeLa cells after BSO application were determined. After X-irradiation the colony forming ability and micronucleus induction were used as endpoints of the radiation reaction.

The depletion of GSH after incubation with BSO for time intervals up to 72 hours was more pronounced and less toxic than comparable treatments with other known thiol depleting agents. Treatment of EATC and HeLA cells with BSO resulted in a decrease of GSH to 6% resp. I% of controls. The NPSH content was not reduced to the same degree, suggesting that cysteine cannot substitute for GSH with regard to some modification of the radiobiological response. Hypoxic radiosensitization was found to depend on the applied BSO concentration with a maximum at about 250 uM. At this concentration no shoulder in the survival curve could be observed and the survival was below that of the oxic control. A biphasic concentration dependence was found in survival as well as in micronucleus measurements with a reduction of the sensi- tization at values above 400 uM. Thus. a possible relationship between GSH and the oxygen effect must be restricted to lower BSO concen- trations.

Studies using BSO for inhibition of GSH biosynthesis may help to understand the specific role of glutathione in radiobiological effects and therefore should stimulate the possible application of buthionine sulfox- imine in the tumor treatment by ionizing radiations.

(A pan of this investigation was supported by EURATOM contract No. 20576-I-BIO-D.)

EFFECTS OF GLUTATHIONE DEPLETION BY BUTHIONINE SULPHOXIMINE ON RADIOSENSITIZATION BY OXYGEN

AND MISONIDAZOLE IN VITRO

DENNIS C. SHRIEVE, JULIANA DENEKAMP AND ANDREW I. MINCHIN~ON

Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood. Middlesex HA6 2RN. England

The radiosensitizing action of oxygen and of misonidazole (MISO) can be modified by the addition or depletion of intmoellular thiols. Buthionine sulphoximine (BSO) has been used to deplete glutathione (GSH) in V79- 379 cells in vitro, and the effect on the efficiency of oxygen and MIS0 as radiosensitizers has been determined.

Untreated V79-379 cells contained 5-8 fmol/cell(5-8 mM) of GSH. Cells treated with 50 or 500 PM BSO showed a rapid decline in GSH content, falling to less than 5% after IO h exposure to these doses of BSO (T,,r = I.6 h). Cells washed free of the BSO showed rapid regen- eration of GSH after 50 hM BSO. but no regeneration was observed over the subsequent 10 h period after 500 PM. Treatment with these levels of BSO for up to 14 h did not affect cell growth or viability. GSH was measured as its monobromobimane derivative by high performance liquid chromatography.

Cells irradiated in monolayer on glass had an oxygen enhancement ratio (OER) of 3. I, with a K value of0.7% OZ. After IO-14 h pretreatment with 50 PM BSO, washed cells showed sensitization in all oxygen tensions tested. The OER was reduced to 2.6, due to greater sensitization of hypoxic cells by GSH depletion. The maximum effect (ER = 1.53) was observed near the K value. The GSH depletion had the effect of shifting the K curve to lower oxygen tensions. making oxygen appear more efficient by a factor of approximately 2.

Similar experiments were performed with MISO. IO-14 h pretreatment with 50 pM BSO increased the MIS0 radiosensitizing efficiency by a factor of 25. An enhancement ratio of 2.0 could be achieved with 0.2 mM MIS0 in BSO pretreated cells. instead of with 5 mM MIS0 in untreated cells.

Although the effect of GSH depletion was qualitatively similar for oxygen and for MISO. it was quantitatively different. The apparent increased efficiency of MIS0 was more than IO times greater than for oxygen. Both effects can be largely explained on the basis of sensitization of hypoxic cells by GSH depletion alone (ER = 1.3). Further sensitization by oxygen or MIS0 appeared to be nearly additive with less sensitizer being required to achieve a specific degree of sensitization relative to control cells.

(Supported by The Cancer Research Campaign.)

EFFECT OF GLUTATHIONE DEPLETION ON RADIOSENS1TIVt-R IN VI VO

A. ROJAS, J. P. SORANSON, K. A. SMITH, A. 1. MINCHINTON, R. W. MIDDLETON AND J. DENEKAMP

Gray Laboratory of the Cancer Research Campaign. Mount Vernon Hospital, Northwood. Middlesex HA6 2RN. England

The influence of glutathione (GSH) depletion on skin and tumor ra- diosensitivity has been studied using X rays alone, with misonidazole and oxygen being supplied at various concentrations. GSH depletion has been achieved in mice by repeated i.p. administration of BSO for periods up to 9.5 hours. Tumor regrowth delay, skin reactions, epidermal clones and pharmacology results will be presented.

BSO alone is apparently non toxic and depletes GSH in tumors to 50% (i.e. less depletion than in liver, kidney and muscle). It sensitizes skin slightly with prolonged exposure (9.5 hours) but no sensitization has been seen in CA MT tumors (up to 8 hours).

Prolonged BSO exposure combined with an acute dose of miso (given once or on 5 successive days) does not result in further GSH depletion in tumors. However, the combination increases the effectiveness of miso as a radiosensitizer of hypoxic cells in tumors (by a factor of -5 in CA MT and -3 in SA FA). A similar enhancement was seen in artificially hypoxic mouse skin but none was seen in well oxygenated skin. The effectiveness of oxygen as a radiosensitizer is also increased, with the “K value” for mouse skin clones being decreased by a factor of about 2.

The enhancement of miso radiosensitization in tumon by BSO was not observed in a 5 fraction schedule, presumably because of reoxy- genation.

The use of BSO in viva to enhance the effectiveness of misonidazole will be constrained by the limited GSH depletion in tumors, increased miso toxicity, some skin sensitization and because its effect is confined to hypoxic cells.

RADIOSENSITIZATION OF HYPOXIC CELLS BY GLUTATHIONE DEPLETION WITH BSO

H. SCHORN,’ U. BERTSCHE’ AND 0. Vos’ ‘Euratom DG. XII-GSF; 2 GSF. Abt. Biophys. Strahlenforschg.,

Frankfurt-Germany; ‘Medical Biologial Laboratory. TNO, Rijswijk and Erasmus University, Rotterdam. The Netherlands

Buthionine Sulfoximine (BSO) is a selective inhibitor of y-glutamyl- cysteine synthetase. Therefore BSO may serve to clarify the influence of endogeneous glutathione on radiosensitivity. In these studies the glu- tathione. total thiol (TSH) and nonprotein thiol (NPSH) content of EAT and Hela cells after BSO application were determined. After X ray irradiation, colony forming ability and micronuclei formation were used as endpoints.

The glutathione depletion after BSO was more pronounced and less toxic than treatment with other commonly used thioldepleting agents. Incubation of EAT and Hela cells with BSO resulted in a decrease in cellular glutathione to 6% and 1% of the respective controls. The NPSH concentration was not reduced to the same degree, suggesting that cysteine cannot substitute for glutathione with regard to modification of radio- biological response.

Hypoxic radiosensitization was found to be dependent on the con- centration of BSO applied, with a maximum at C = 250 mM. At this concentration no shoulder could be observed and the sensitization was below the oxic control value, where a shoulder is usually found. BSO was found to act in a biphasic manner. At concentrations above 400 PM the effect was smaller than at 250 PM. The reduction of the oxygen enhancement ratio (OER) of both cell lines after treatment with BSO suggests a relationship between cellular glutathione and the oxygen effect.

Studies with the inhibitor of glutathione biosynthesis, BSO, may help us to understand the specific role of glutathione in radiobiology and could stimulate its possible application in radiotherapy of tumors.

(A part of the investigations were supported by the EURATOM con- tract No-20576- I-B10 D.)