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Glutathione depletion in tissues after administration of buthionine sulphoximine
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036&3016/84 $03.00 + 00 Cu I984 Pergamon Press Lid
??Session I
GLUTATHIONE
DEPLETION IN TISSUES AFTER ADMINISTRATION OF BUTHIONINE SULPHOXIMINE
ANDREW I. MINCHINTON, B.Sc., ANAMARIA ROJAS, M.D., K. ANNE SMITH, B.Sc., JULIE A. SORANSON, B.Sc., DENNIS C. SHRIEVE, PH.D., NIGEL R. JONES, B.Sc. AND JANE C. BREMNER, B.Sc. Gray Laboratory of the Cancer Research Campaign. Mount Vernon Hospital. Northwocxi,MiddlesexHA6 2RN, England
Buthioninesulphoximine(BSO)an inhibitorof glutathione(CSH)biosynthesis,wasadministered to mice and repeated doses of 0.5, 1 and 5 kidney, skeletal muscle and three levels of co. 20% of controls after but at a slower rate after a single days to obtain a similar degree of
in single mmol kg-’ (i.p.). The resultant pattern of CSH depletion was studied in liver, types of murine tumor. Liver and kidney exhibited a rapid depletion to CSH single doses of l-5 mmol kg-’ BSO. Muscle was depleted to a similar level, dose. All three tumors required repeated administration of BSO over several depletion to that shown in the other tissues.
Glutathione
sulphoximine,
depletion,
Buthionine
HPLC.
INTRODUCIION Buthionine sulphoximine (BSO) was identified by Griffith and Meiste$ as a potent inhibitor of y-glutamyl cysteine synthetase, a key enzyme involved in the biosynthesis of glutathione (GSH). Depletion of GSH has been shown by many workers to render cells more sensitive to radiation (for review see ref. 1). Using BSO to deplete GSH in tissues it may be possible to enhance the usefulness of radiosensitizers in radiotherapy. Accordingly BSO has been used in this laboratory to evaluate the therapeutic potential of glutathione depletion in vitro* and in vivo (Rojas, A., personal communication, September 1983). This paper characterizes the kinetics of glutathione depletion and its subsequent regeneration, as a function of dose and time, in liver, kidney, skeletal muscle and three transplantable tumour types in mice.
MT or fibrosarcoma SA FA and female CBA/HtGyBSVS mice bearing the fat sarcoma Sa F. Mice were killed by decapitation, and samples of liver, kidney, sartorius muscle and tumour were removed, immediately weighed and homogenized in ice cold 5-sulphosalicylic acid (20 mmol dmT3).* A modification of the thiol assay of Newton er al.’ utilizing the thiol derivatizing compound monobromobimane and subsequent separation by high performance liquid chromatography, was adopted.6 RESULTS
AND DISCUSSION
to female mice. bearing the anaplastic tumor Ca
The rate of depletion, the maximum level of depletion and the subsequent pattern of regeneration after a single dose of BSO varied for different tissues. This may be a result of a variety of factors: the size of the dose, the availability of the drug to the different tissues, the inherent rate of utilization and biosynthesis of glutathione and the transport of glutathione from one tissue to another.‘.5 The pattern of glutathione depletion in liver after various single and repeated administrations of BSO is shown in Figures l(a) and l(b) respectively. Initially there was a rapid fall in the GSH concentration. relative to the control, reaching 50% within 2 hours. After a single dose of 1 mmol kg-’ BSO, there was no further depletion and
Based upon a poster presentation at the Conference on Chemical Modifiers of Cancer Treatment Banff. Canada. 27 Nov.-l Dec. 1983. Reprint requests to: A. I. Minchinton. .-lckno\c,ledgements-This work is supported by the Cancer Research Campaign. The authors wish to express their thanks to
Dick Middleton for synthesis of the BSO and to Julie Denekamp. Mike Stratford and Peter Wardman for helpful discussion. to P. Russell and the animal house staff for care and provision of the mice and to D. Woodman. for typing the manuscript. Accepted for publication 22 March 1984. * Sigma Chemical Co.
METHODS AND MATERIALS DL-buthionine sulphoximine (BSO) was synthesised by R. W. Middleton, Brunel University, England.4It was dissolved in distilled water at concentrations from 0.125 to 0.25 mol dm-3 prior to i.p. administration
WHT/GyfBSVS
1262
Radiation Oncology 0 Biology 0 Physics
120 r
August 1984. Volume 10, Number 8
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Fig. I. Profile of GSH concentration in liver in WHT/GyfBSVS mice after 1.p. administration of BSO. Untreated liver GSH concentration was 7.4 f 1.3 mmol kg-‘. (Mean + s.e.m.). (a) (0, 0): single dose of I and 5 mmol kg-’ respectively. (b) (0, <), A, 0); repeated doses of I mmol kg-’ every 0.5, I. 4. and I2 hours respectively and All repeated administrations (+, A, a) repeated doses of 5 mmol kg-’ every I. 4 and 12 hours respectively. commence at time 0. The lines in fig (b) are those from (a) for comparison with the single-dose regimen.
the GSH levels regenerated to control values after 12 hours. In contrast, a dose of 5 mmol kg’ resulted in further depletion to about 20% of controls within 4 to 6 hours, followed by regeneration at a similar rate to that of a 1 mmol kg-’ dosage, reaching approximately control levels after 24 hours and overshooting to 115% after 48 hours. Multiple injections of 1 and 5 mmol kg-’ every
1 hour and 4 hours, respectively, could maintain even higher degrees of depletion. In kidney an even more rapid depletion was observed. Single doses (Figure 2(a)) of both 1 or 5 mmol kg-’ resulted in depletion of GSH to 20 to 25% after I to 5 hours, followed by regeneration which was dose dependent and appeared to be incomplete after 24-48 hours. Again mul-
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Fig. 2. Profile ofGSH concentration in kidney in WHT/GygBSVS mice after i.p. administration kidney GSH concentration was 3.8 + 0.1 mmol kg-‘. Key as Figure I.
25't%%o
of BSO. Untreated
Glutathione depletion. buthionine sulphoximine, HPLC 0 A. I.
tiple administration of these doses could maintain, but not significantly increase, the degree of depletion. The two tumors Ca MT and SA FA appeared to respond similarly to BSO administration (Figure 3) resulting in a slow rate of GSH depletion similar to muscle. After a single dose of 1 mmol kg-’ the lowest observed GSH concentration was 75% recovering to near control levels after 24 hours (Figure 3(a)). With a single dose of 5 mmol kg-’ the maximum depletion achieved was 55% in the Ca MT tumor after 8 hours and 37% in the SA FA tumor after 12 hours. Repeated administration of 1 and 5 mmol kg-’ every 4 hours resulted in further depletion to approximately 30% (Figure 3(b)). The relatively slow rate of GSH depletion in muscle is illustrated in Figure 4(a): 50% depletion is achieved after about 9 hours irrespective of dose: even after a single injection of 1 mmol kg-’ the GSH level was still falling at 24 hours. With a single dose of 5 mmol kg-’ the GSH level reached 15% after 48 hours and with repeated administration of 5 mmol kg-’ every 12 hours, levels of less than 5% were observed after 72 hours. In a third tumor, the Sa F grown in CBA/HtGyfBSVS female mice. repeated administrations of 5 mmol kg-’ were given at 7.5 hour intervals and the observed GSH depletion is illustrated in Figure 4(b). The rate of tumor GSH depletion was similar to that observed in the single dose studies and resulted in maximal depletion to 12% of control values after 30 hours (7.5 hours following the last of the 4 injections). However, the liver concentration of GSH was seen to recover more rapidly in this mouse
MINCHINTON
1263
CI (11
(dotted line) compared to the previous studies using the WHT mice, and this was substantiated when mice were dosed with 10 and 20 mmol kg ’(data not shown), which also indicated that these higher doses did not result in enhanced GSH depletion in the tumor. This regimen (Figure 4(b)) illustrates that despite achieving a high degree of tumor depletion, the liver GSH concentration can recover to control values between doses. Thus treatment with drugs and/or radiation can be given when the tumor GSH concentration is compromised and the liver GSH concentration is normal. From the tumor studies shown. and further studies using higher doses of IO and 20 mmol kg- ’ (data not presented) there appears to be a level of around 10%. beyond which further GSH depletion cannot be achieved. One possible explanation (A. Meister. personal communication, December 1983) is that BSO cannot enter and therefore deplete GSH within the mitochondria: the contribution that this mitochondrial pool makes to the overall measurement of GSH in tissue may account for the residual GSH seen after maximal depletion. At no time was a substantial increase in the concentration of cysteine found in any of the tissues studies. CONCLUSIONS This work has confirmed previous work by Griffith and Meister’ showing a differential response in various tissues of GSH depletion after BSO administration. Tumor tissue is relatively slowly depleted of GSH compared
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Fig. 3. Profile of GSH concentration in: SA FA (S) and Ca MT (C) tumors after i.p. administration of BSO. Untreated GSH concentrations for SA FA and Ca MT tumors were 1.15 ? 0.2 and 1.60 + 0.1 mmol kg-‘. respectively. Key as Figure 1.
Radiation Oncology 0 Biology 0 Physics
I264
August 1984. Volume IO. Number 8
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Fig. 4. (a) Profile of GSH concentration in sartorius muscle in WHT/GyfBSVS mice after i.p. administration of BSO. Untreated muscle GSH concentration was 0.78 f 0.02 mmol kg-‘. Key as Figure I. (b) Profile of GSH concentration in CBA/HtGyBSVS mice bearing the Sa F tumor after repeated administrations every 7.5 hours of 5 mmol kg-’ BSO. 0; tumour, A; liver, 0; kidney. Cl: muscle. Untreated normal tissue GSH concentrations in these mice were not significantly different from WHT mice. Untreated tumour GSH concentration was 1.69 rt 0.12 mmol kg-‘.
to liver and kidney and requires multiple carefully timed
administrations to achieve maximal depletion. This differential response is important since it might be possible to devise a dose schedule in which some normal tissues have regenerated their GSH before administration of a radiosensitizer. This might avoid, for example, the com-
plications of increased toxicity associated with depletion of hepatic GSH as seen with other drugs, e.g. acetaminophen’. This exploratory investigation illustrates the requirement of careful monitoring of tissue glutathione levels when the effects of BSO administration are being studied.
REFERENCES Biagiow, J.E., Vames, M.E.. Clark, E.P., Epp, E.R.: The role of thiols in cellular response to radiation and drugs. Radiat. Res. 95: 437-455.
1983.
Griffith, O.W., Meister, A.: Glutathione: Interorgan translocation, turnover and metabolism. Proc. Nat/. Acad. Sci. USA 76: 5606-5610,
1979.
Griffith, O.W., Meister, A.: Potent and specific inhibition of glutathione synthesis by buthionine sulphoximine (S-nButylhomocysteine Sulphoximine). J. Biol. Chem. 254: 7558-7560.
1979.
Hodgkiss, R.J., Middleton, R.W.: Enhancement of misonidazole radiosensitization by an inhibitor of glutathione biosynthesis. Int. J. Radiat. Biol. 43: 179-183, 1983.
5. Meister, A.: On cycles of ghrtathione metabolism and transport. Curr. Top. Cell. Rex 18: 2 l-58, 198 1, 6. Minchinton. AI.: Measurements of glutathione and other thiols in cells and tissues: A simplified procedure based on the HPLC separation of monobromobimane derivatives of thiols. Int. J. Radiat. Oncol. Biol. Phys. 10 (In press) 1984. 7. Mitchell, J.R., Jollow, D.J.. Potter, W.Z., Gillette, J.R.. Brodie, B.B.: Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187: 21 l-217. 1973. 8. Newton, G.L., Dorian,
R., Fahey, R.C.: Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse phase high performance liquid chromatography. Anal. Biochem. 114: 383-387, 1981.
036&3016/84 $03.00 + 00 Cu I984 Pergamon Press Lid
??Session I
GLUTATHIONE
DEPLETION IN TISSUES AFTER ADMINISTRATION OF BUTHIONINE SULPHOXIMINE
ANDREW I. MINCHINTON, B.Sc., ANAMARIA ROJAS, M.D., K. ANNE SMITH, B.Sc., JULIE A. SORANSON, B.Sc., DENNIS C. SHRIEVE, PH.D., NIGEL R. JONES, B.Sc. AND JANE C. BREMNER, B.Sc. Gray Laboratory of the Cancer Research Campaign. Mount Vernon Hospital. Northwocxi,MiddlesexHA6 2RN, England
Buthioninesulphoximine(BSO)an inhibitorof glutathione(CSH)biosynthesis,wasadministered to mice and repeated doses of 0.5, 1 and 5 kidney, skeletal muscle and three levels of co. 20% of controls after but at a slower rate after a single days to obtain a similar degree of
in single mmol kg-’ (i.p.). The resultant pattern of CSH depletion was studied in liver, types of murine tumor. Liver and kidney exhibited a rapid depletion to CSH single doses of l-5 mmol kg-’ BSO. Muscle was depleted to a similar level, dose. All three tumors required repeated administration of BSO over several depletion to that shown in the other tissues.
Glutathione
sulphoximine,
depletion,
Buthionine
HPLC.
INTRODUCIION Buthionine sulphoximine (BSO) was identified by Griffith and Meiste$ as a potent inhibitor of y-glutamyl cysteine synthetase, a key enzyme involved in the biosynthesis of glutathione (GSH). Depletion of GSH has been shown by many workers to render cells more sensitive to radiation (for review see ref. 1). Using BSO to deplete GSH in tissues it may be possible to enhance the usefulness of radiosensitizers in radiotherapy. Accordingly BSO has been used in this laboratory to evaluate the therapeutic potential of glutathione depletion in vitro* and in vivo (Rojas, A., personal communication, September 1983). This paper characterizes the kinetics of glutathione depletion and its subsequent regeneration, as a function of dose and time, in liver, kidney, skeletal muscle and three transplantable tumour types in mice.
MT or fibrosarcoma SA FA and female CBA/HtGyBSVS mice bearing the fat sarcoma Sa F. Mice were killed by decapitation, and samples of liver, kidney, sartorius muscle and tumour were removed, immediately weighed and homogenized in ice cold 5-sulphosalicylic acid (20 mmol dmT3).* A modification of the thiol assay of Newton er al.’ utilizing the thiol derivatizing compound monobromobimane and subsequent separation by high performance liquid chromatography, was adopted.6 RESULTS
AND DISCUSSION
to female mice. bearing the anaplastic tumor Ca
The rate of depletion, the maximum level of depletion and the subsequent pattern of regeneration after a single dose of BSO varied for different tissues. This may be a result of a variety of factors: the size of the dose, the availability of the drug to the different tissues, the inherent rate of utilization and biosynthesis of glutathione and the transport of glutathione from one tissue to another.‘.5 The pattern of glutathione depletion in liver after various single and repeated administrations of BSO is shown in Figures l(a) and l(b) respectively. Initially there was a rapid fall in the GSH concentration. relative to the control, reaching 50% within 2 hours. After a single dose of 1 mmol kg-’ BSO, there was no further depletion and
Based upon a poster presentation at the Conference on Chemical Modifiers of Cancer Treatment Banff. Canada. 27 Nov.-l Dec. 1983. Reprint requests to: A. I. Minchinton. .-lckno\c,ledgements-This work is supported by the Cancer Research Campaign. The authors wish to express their thanks to
Dick Middleton for synthesis of the BSO and to Julie Denekamp. Mike Stratford and Peter Wardman for helpful discussion. to P. Russell and the animal house staff for care and provision of the mice and to D. Woodman. for typing the manuscript. Accepted for publication 22 March 1984. * Sigma Chemical Co.
METHODS AND MATERIALS DL-buthionine sulphoximine (BSO) was synthesised by R. W. Middleton, Brunel University, England.4It was dissolved in distilled water at concentrations from 0.125 to 0.25 mol dm-3 prior to i.p. administration
WHT/GyfBSVS
1262
Radiation Oncology 0 Biology 0 Physics
120 r
August 1984. Volume 10, Number 8
r
.--- k--”
(b)
(a)
/* _...._..__....._..._........_...........-.............__......... o
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20 I 25 c/28 time (hrs)
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Fig. I. Profile of GSH concentration in liver in WHT/GyfBSVS mice after 1.p. administration of BSO. Untreated liver GSH concentration was 7.4 f 1.3 mmol kg-‘. (Mean + s.e.m.). (a) (0, 0): single dose of I and 5 mmol kg-’ respectively. (b) (0, <), A, 0); repeated doses of I mmol kg-’ every 0.5, I. 4. and I2 hours respectively and All repeated administrations (+, A, a) repeated doses of 5 mmol kg-’ every I. 4 and 12 hours respectively. commence at time 0. The lines in fig (b) are those from (a) for comparison with the single-dose regimen.
the GSH levels regenerated to control values after 12 hours. In contrast, a dose of 5 mmol kg’ resulted in further depletion to about 20% of controls within 4 to 6 hours, followed by regeneration at a similar rate to that of a 1 mmol kg-’ dosage, reaching approximately control levels after 24 hours and overshooting to 115% after 48 hours. Multiple injections of 1 and 5 mmol kg-’ every
1 hour and 4 hours, respectively, could maintain even higher degrees of depletion. In kidney an even more rapid depletion was observed. Single doses (Figure 2(a)) of both 1 or 5 mmol kg-’ resulted in depletion of GSH to 20 to 25% after I to 5 hours, followed by regeneration which was dose dependent and appeared to be incomplete after 24-48 hours. Again mul-
(a)
(b)
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Fig. 2. Profile ofGSH concentration in kidney in WHT/GygBSVS mice after i.p. administration kidney GSH concentration was 3.8 + 0.1 mmol kg-‘. Key as Figure I.
25't%%o
of BSO. Untreated
Glutathione depletion. buthionine sulphoximine, HPLC 0 A. I.
tiple administration of these doses could maintain, but not significantly increase, the degree of depletion. The two tumors Ca MT and SA FA appeared to respond similarly to BSO administration (Figure 3) resulting in a slow rate of GSH depletion similar to muscle. After a single dose of 1 mmol kg-’ the lowest observed GSH concentration was 75% recovering to near control levels after 24 hours (Figure 3(a)). With a single dose of 5 mmol kg-’ the maximum depletion achieved was 55% in the Ca MT tumor after 8 hours and 37% in the SA FA tumor after 12 hours. Repeated administration of 1 and 5 mmol kg-’ every 4 hours resulted in further depletion to approximately 30% (Figure 3(b)). The relatively slow rate of GSH depletion in muscle is illustrated in Figure 4(a): 50% depletion is achieved after about 9 hours irrespective of dose: even after a single injection of 1 mmol kg-’ the GSH level was still falling at 24 hours. With a single dose of 5 mmol kg-’ the GSH level reached 15% after 48 hours and with repeated administration of 5 mmol kg-’ every 12 hours, levels of less than 5% were observed after 72 hours. In a third tumor, the Sa F grown in CBA/HtGyfBSVS female mice. repeated administrations of 5 mmol kg-’ were given at 7.5 hour intervals and the observed GSH depletion is illustrated in Figure 4(b). The rate of tumor GSH depletion was similar to that observed in the single dose studies and resulted in maximal depletion to 12% of control values after 30 hours (7.5 hours following the last of the 4 injections). However, the liver concentration of GSH was seen to recover more rapidly in this mouse
MINCHINTON
1263
CI (11
(dotted line) compared to the previous studies using the WHT mice, and this was substantiated when mice were dosed with 10 and 20 mmol kg ’(data not shown), which also indicated that these higher doses did not result in enhanced GSH depletion in the tumor. This regimen (Figure 4(b)) illustrates that despite achieving a high degree of tumor depletion, the liver GSH concentration can recover to control values between doses. Thus treatment with drugs and/or radiation can be given when the tumor GSH concentration is compromised and the liver GSH concentration is normal. From the tumor studies shown. and further studies using higher doses of IO and 20 mmol kg- ’ (data not presented) there appears to be a level of around 10%. beyond which further GSH depletion cannot be achieved. One possible explanation (A. Meister. personal communication, December 1983) is that BSO cannot enter and therefore deplete GSH within the mitochondria: the contribution that this mitochondrial pool makes to the overall measurement of GSH in tissue may account for the residual GSH seen after maximal depletion. At no time was a substantial increase in the concentration of cysteine found in any of the tissues studies. CONCLUSIONS This work has confirmed previous work by Griffith and Meister’ showing a differential response in various tissues of GSH depletion after BSO administration. Tumor tissue is relatively slowly depleted of GSH compared
(a)
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Fig. 3. Profile of GSH concentration in: SA FA (S) and Ca MT (C) tumors after i.p. administration of BSO. Untreated GSH concentrations for SA FA and Ca MT tumors were 1.15 ? 0.2 and 1.60 + 0.1 mmol kg-‘. respectively. Key as Figure 1.
Radiation Oncology 0 Biology 0 Physics
I264
August 1984. Volume IO. Number 8
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Fig. 4. (a) Profile of GSH concentration in sartorius muscle in WHT/GyfBSVS mice after i.p. administration of BSO. Untreated muscle GSH concentration was 0.78 f 0.02 mmol kg-‘. Key as Figure I. (b) Profile of GSH concentration in CBA/HtGyBSVS mice bearing the Sa F tumor after repeated administrations every 7.5 hours of 5 mmol kg-’ BSO. 0; tumour, A; liver, 0; kidney. Cl: muscle. Untreated normal tissue GSH concentrations in these mice were not significantly different from WHT mice. Untreated tumour GSH concentration was 1.69 rt 0.12 mmol kg-‘.
to liver and kidney and requires multiple carefully timed
administrations to achieve maximal depletion. This differential response is important since it might be possible to devise a dose schedule in which some normal tissues have regenerated their GSH before administration of a radiosensitizer. This might avoid, for example, the com-
plications of increased toxicity associated with depletion of hepatic GSH as seen with other drugs, e.g. acetaminophen’. This exploratory investigation illustrates the requirement of careful monitoring of tissue glutathione levels when the effects of BSO administration are being studied.
REFERENCES Biagiow, J.E., Vames, M.E.. Clark, E.P., Epp, E.R.: The role of thiols in cellular response to radiation and drugs. Radiat. Res. 95: 437-455.
1983.
Griffith, O.W., Meister, A.: Glutathione: Interorgan translocation, turnover and metabolism. Proc. Nat/. Acad. Sci. USA 76: 5606-5610,
1979.
Griffith, O.W., Meister, A.: Potent and specific inhibition of glutathione synthesis by buthionine sulphoximine (S-nButylhomocysteine Sulphoximine). J. Biol. Chem. 254: 7558-7560.
1979.
Hodgkiss, R.J., Middleton, R.W.: Enhancement of misonidazole radiosensitization by an inhibitor of glutathione biosynthesis. Int. J. Radiat. Biol. 43: 179-183, 1983.
5. Meister, A.: On cycles of ghrtathione metabolism and transport. Curr. Top. Cell. Rex 18: 2 l-58, 198 1, 6. Minchinton. AI.: Measurements of glutathione and other thiols in cells and tissues: A simplified procedure based on the HPLC separation of monobromobimane derivatives of thiols. Int. J. Radiat. Oncol. Biol. Phys. 10 (In press) 1984. 7. Mitchell, J.R., Jollow, D.J.. Potter, W.Z., Gillette, J.R.. Brodie, B.B.: Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187: 21 l-217. 1973. 8. Newton, G.L., Dorian,
R., Fahey, R.C.: Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse phase high performance liquid chromatography. Anal. Biochem. 114: 383-387, 1981.