Brain Research, 518 (1990) 115-119 Elsevier
115
BRES 15522
Transport into brain of buthionine sulfoximine, an inhibitor of glutathione synthesis, is facilitated by esterification and administration of dimethylsulfoxide Reuben Steinherz, Johannes Mhrtensson, Daniel Wellner and Alton Meister Department of Biochemistry, Cornell University Medical College, New York, N Y 10021 (U.S.A.) (Accepted 14 November 1989)
Key words: Glutathione; Buthionine sulfoximine; Dimethyisulfoxide; Esterification; Therapy Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, is poorly transported into the brain of adult mice, and only a slight decrease (~10%) in the level of brain glutathione is found 30-60 min after intraperitoneal administration of BSO. When BSO is given as the ethyl ester, the brain level of BSO increases substantially after 5-15 rain, and the glutathione level decreases by about 25% after 30-60 min. When BSO or its ester is given in 15% dimethylsulfoxide solution the brain levels of BSO are increased significantly and the brain glutathione levels are decreased by 20-35%. These observations suggest procedures that may be useful in decreasing the glutathione levels of the brains of adult animals. The finding that administration of BSO ethyl ester led to about a 25% decrease in the brain level of glutathione within 15 min suggests that a fraction of brain glutathione turns over very rapidly and may therefore be of special physiological significance. INTRODUCTION T r e a t m e n t of mice and rats with buthionine sulfoximine ( B S O ) , an irreversible inhibitor of y-glutamylcysteine synthetase, leads to a decrease of glutathione levels in a n u m b e r of tissues 16'17'2s. The glutathione levels of cells with relatively high overall turnover rates of glutathione (e.g. kidney, liver, lung, leukocytes) are most readily affected by B S O because substantial cellular e x p o r t of glutathione continues whereas there is little or no synthesis of glutathione. B S O has served as a useful agent for a variety of experimental studies on the t r a n s p o r t and m e t a b o l i s m of glutathione 27. B S O has also b e e n used for the p u r p o s e of decreasing glutathione levels of tumors, thus sensitizing them to chemotherapeutic agents and to radiation 26'2s'29'31'44. B S O is currently being tested in clinical trials. The effectiveness of B S O in studies of glutathione m e t a b o l i s m in the brain and for t r e a t m e n t of brain tumors is limited by the fact that B S O is p o o r l y t r a n s p o r t e d into the brain in adult animals. B S O is t r a n s p o r t e d into brain to a greater extent in preweanling mice than in adults 38, and B S O has been a d m i n i s t e r e d intracerebroventricularly to rats 32. The toxicity of B S O to the brain has not been extensively studied, but a p p e a r s to be chiefly related to its effect on glutathione synthesis 21. In the present work we have e x p l o r e d the effects of esterification o f B S O on its u p t a k e by brain and also the effect of dimethylsulfoxide ( D M S O ) administration on
the uptake of BSO. Esterification of certain c o m p o u n d s changes the lipid solubility to a form that is m o r e favorable for transport, and esterification of certain c o m p o u n d s m a y facilitate their transport into cells2' 7,11-14,24,26,33,35,40-42,46 In this a p p r o a c h , the ester is t r a n s p o r t e d m o r e rapidly than the original c o m p o u n d , and after transport the ester is h y d r o l y z e d intracellularly. In another approach, D M S O has been r e p o r t e d to increase the transport of several c o m p o u n d s into brain 36,19,20. The findings that D M S O facilitates transport into brain of c o m p o u n d s that differ m a r k e d l y in structure, e.g., pemoline 3 and horseradish peroxidase 5 are consistent with the view that D M S O might reversibly open the blood brain barrier; however, d a t a that are not in accord with this conclusion have been published 8,1°'15'22'23'3°'36'45. MATERIALS AND METHODS 5,5'-Dithiobis(2-nitrobenzoic acid), glutathione disulfide reductase, and NADPH, were obtained from Sigma. DMSO was obtained from Mailinckrodt. Swiss Webster mice (male, 28-30 g) were obtained from Taconic Farms, Germantown, NY. The ethyl ester of L-buthionine-SR-sulfoximine (BSO) 16,zs was prepared by the general procedures given by Anderson2 and Steinherz et al. 4°. Thin layer chromatography on silica gel plates (MKGF, Analtech) at 25 °C (30 min) with a solvent consisting of n-propanol, acetic acid, and water (10:5:1 (v/v)) was used to follow esterification. The plates were sprayed with 0.5% ninhydfin in acetone; BSO gave a red-brown color whereas BSO ethyl ester gave a brown-yellow color. The Rf values for BSO and its ethyl ester were, respectively, 0.5 and 0.7. The stability of BSO ester was examined in mixtures containing the amino acid ester (2 mM), mouse serum (50%; v/v), and DMSO (7.5%; v/v) at pH 6.8. For analysis, the mixtures were acidified by adding
Correspondence: A. Meister, Department of Biochemistry, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
116 5-sulfosalicylic acid (final concentration, 4.3%) and (after centrifugation of the mixtures containing serum) portions were analyzed on a Durrum model 500 amino acid analyzer using sodium buffers. The elution time for BSO was 62.1 min. Under these conditions, no hydrolysis of BSO ester was detected in freshly prepared solutions. Analysis of solutions of BSO ester that had been allowed to stand at 25-27 °C for 24 h indicated 72% hydrolysis. The presence of 7.5% DMSO stabilized the ester considerably; thus, only 18% hydrolysis was found after 24 h. In the mixtures containing serum, 26% of the initial BSO ester present was hydrolyzed per min at 37 °C; in the presence of 7.5% DMSO, the corresponding value for hydrolysis was 8.5%. Mice were injected intraperitoneally with freshly prepared solutions of BSO or BSO ethyl ester. The compounds were dissolved in water or in aqueous 15% (v/v) DMSO at a final concentration of 300 mOsm (pH 6.7-6.8). At the indicated intervals after injection, samples were taken from mice that were anesthetized by intraperitoneal injection of a mixture of xylazine (5 mg/kg) and ketamine (100 mg/kg) 3 rain before the samples were taken. The right ventricle was sectioned to bleed the animal. Cold saline (10 ml) was perfused through the left ventricle for 1 rain. Samples of the cortical area of the brain were excised, blotted, weighed, and homogenized in 5 volumes of 14.4% 5-sulfosalicylic acid. The homogenates were centrifuged and the supernatant solutions were extracted with 0.3 volume of chloroform for 30 min. After centrifugation, 80% of the aqueous layer was removed by aspiration and neutralized with sodium hydroxide. The sample was then evaporated to dryness in a Speed Vac apparatus and the residue was taken up in 2.5% sulfosalicylic acid such that the volume was one-third of the original volume of the homogenate sample. A portion (20/A) of this solution was used for analysis for BSO on the amino acid analyzer. The supernatant solutions obtained from the homogenates were analyzed for total glutathione as describedI'43 Blood plasma was obtained from samples (100/A) withdrawn from the right ventricle; the samples were treated with 1 /4 of 250 mM Na2EDTA and centrifuged at 10,000 g at 4 °C for 1 min. Portions (36 H1) of the supernatant solution were treated with 4 HI of 43% sulfosalicylic acid and centrifuged for 5 min as stated above. The supernatant solutions were analyzed for BSO on the amino acid analyzer. RESULTS
equimolar amount of BSO ethyl ester (in water) led to a significantly higher level of BSO (Fig. IA, Curve 2). The BSO level decreased rapidly within 30-60 rain to about the same value found after BSO administration. Addition of 15% DMSO to the BSO injection solution increased the brain cortex BSO level by about 100% after 30 and 60 min (compare Curve 3 with Curve 1, Fig. 1); DMSO did not significantly affect the 5 min value. The result obtained after injection of BSO ethyl ester in DMSO (Curve 4) reflects both the effect of DMSO and that of esterification. Thus, a significantly higher BSO level was found 5 and 15 rain after giving BSO ester in DMSO than found after giving BSO in DMSO. The data given in Fig. 2 indicate that the increased brain uptake of BSO after administration of BSO ester, and of BSO and its ester in DMSO solution, is reflected by a greater decrease in glutathione levels than found after administration of BSO in water. Administration of BSO in water led to about a 10% decline of the glutathione level. Administration of BSO ester led to a decline of about 25% after 15 min. Administration of BSO or of BSO ester in 15% DMSO led to greater decreases in glutathione levels than found after giving these compounds in water. Data on the total BSO levels in the blood plasma under these conditions are given in Fig. 3. The values found after giving BSO ester in water were similar to those found after giving BSO in water, except for the 5 min value, which was somewhat higher after giving BSO ester than after giving BSO. In general, the values for total plasma BSO after administration of BSO and BSO ester
BSO appeared in the brain cortex 5 min after intraperitoneal injection of an aqueous solution of BSO to adult mice (Fig. 1A, Curve 1). After 60 min, the BSO level had decreased by about 50%. Administration of an
0.3
A [H203
0.3
'
I B[DMSO]
BSO ESTER --o- BSO
~
o.2
o.2
0.1
.I
'
I
'
'
I
'
'
'
I
:
II
I
°
\ ~ - BSO(DMSO)
BSO ESTER BSO o
~'~.-"';'
I
Ld > .J O CO 123
I BSOESTER \
I 1 30
I 60
I 90
I 120 0
l 30
i 60
1 90
(°MS°' \
/
/
I 120
MINUTES
Fig. 1. Effect of esterification of BSO and of treatment with DMSO on BSO uptake by brain. Mice were injected with BSO or BSO ethyl ester (4 mmol/kg). These compounds were dissolved in water or in 15% (v/v) aqueous DMSO. The experimental details are given in the text. The data are given as means + S.D., n = 3.
0
30
60 MINUTES
90
120
Fig. 2. Effect of administration of BSO and BSO ethyl ester dissolved in water or in 15% (v/v) aqueous DMSO on brain glutathione levels. Data are given as means; S.D. _+ 5% n = 3-4.
117 5.0
4.0
:~
A [H20]
B [DMSO]
. - o - BSO BSO ESTER
3.0
4.0 ]I
.-a,-- BSO ESTER
3.0
~f4 m
2.0
0
2.0
30
60
90
120 0
30
60
90
120
MINUTES
Fig. 3. Effect of administration of BSO and BSO ethyl ester dissolved in water or 15% (v/v) aqueous DMSO on plasma BSO levels. Data are given as means _+ S.D.; n = 3-5. in DMSO were somewhat higher than those found after giving the compounds in water (except for the 15 min values). It seems of interest that the values for BSO and BSO ester (given in DMSO) at 60 min were clearly greater than the corresponding values obtained after giving these compounds in water indicating that administration of DMSO decreases the plasma clearance of BSO. DISCUSSION The data indicate that administration of BSO ethyl ester leads to a higher level of brain BSO than found after giving BSO. This effect is seen 5 and 15 rain after administration (Fig. 1A), and is associated with an appreciable decline of the glutathione level which persists for at least 120 min (Fig. 2). The finding of a significant ( ~ 2 5 % ) decrease in the level of glutathione in the brain 15 min after administration of BSO ethyl ester is of particular interest because it suggests that a fraction of brain glutathione turns over very rapidly. Previous studies led to the conclusion that the overall rate of turnover of brain glutathione is relatively slow compared to that of kidney and liver 9"17'32. The overall rate of glutathione turnover in rat brain was estimated to be about 70 h 9, whereas that in mouse kidney was found to be 29 min 37. The present observations suggest that further work on the nature and sites of this apparently rapidly turning over fraction of brain glutathione might be rewarding. Recent work indicating that glutathione is
present in high concentrations in cultured astrocytes but not in cultured neurons 34 may be of interest in this connection. That the brain level of BSO after giving BSO ester declines rapidly (Fig. 1A, Curve 2) is consistent with the finding that BSO ethyl ester is rapidly hydrolyzed, and suggests that BSO is effectively exported from brain. Administration of BSO or of its ester in DMSO leads to higher and more sustained brain levels of BSO (Fig. 1B) and to more effective depletion of brain glutathione (Fig. 2). The studies on plasma levels of BSO (Fig. 3) indicate that administration of DMSO leads to more sustained plasma levels of BSO after either BSO or its ester is given. Although DMSO may increase transport by acting on the blood-brain barrier as suggested 3-6A9'2°, its apparent effect on plasma clearance of BSO may also be related to an increase in uptake of BSO (and its ester) from the peritoneal cavity, or to a decrease in renal clearance; such effects would lead to sustained plasma levels of BSO and would presumably promote uptake by the brain. The mechanism or mechanisms by which DMSO affects plasma clearance of BSO need to be studied. The effect of BSO ester and DMSO on brain glutathione levels may be ascribed to an effect on entry of the ester into the brain or to high blood levels of BSO or its ester, or to both. We have also carried out studies in which adult rats (250-300 g) were treated with BSO and DMSO as described here, and in which similar results were observed. Administration of DMSO alone did not affect brain cortex glutathione levels in rats or mice. The dose of DMSO given in the studies reported here (about 2.2 g/kg b. wt.) is substantially lower than the reported 39 value for the LDs0 for DMSO given intraperitoneally to mice (14.7-17.7 g/kg). The present findings suggest that esterification of BSO facilitates its uptake by brain, and that administration of DMSO also facilitates uptake of this amino acid and its ester into brain. It is notable that the synthesis of glutathione in brain is more effectively inhibited after administration of BSO in DMSO than by giving BSO in water. Although DMSO is apparently not a panacea for facilitating transport of compounds into the brain s, 10,15,22,23,30,36,45, it seems possible that DMSO may be useful in this regard for certain compounds such as amino acids and their esters. Although these studies suggest that esterified amino acids may be more readily transported into brain than the parent amino acid, the usefulness of amino acid a-esters is likely to be affected by the rates of their hydrolysis in the blood plasma*. DMSO does not
* Preliminary studies on the uptake into brain of L-phenylalanine and its methyl ester indicate that phenylalanine methyl ester is more readily taken up than is phenylalanine, and that administration in DMSO solution, as in the case of BSO, leads to increased uptake.
118 stabilize BSO ethyl ester in the plasma to a great extent, but it might stabilize other esters more effectively. Although BSO has been of value in studies on glutathione metabolism as an in vivo inhibitor of 7-glutamylcy-
present findings suggest useful approaches that may overcome this limitation.
steine synthetase, and may therefore be a useful therapeutic agent, its value in studies on the brain and its
Acknowledgments. This research was supported in part by a grant to A.M. from the American Cancer Society. J.M. acknowledges stipendary support from the Swedish Medical Research Council (05644-06B), the Throne-Hoist Foundation, and the Killough Trust Foundation.
potential for treatment of brain tumors has been limited by the slow rate of its penetration into this organ. The REFERENCES 1 Anderson, M.E., Determination of glutathione and Glutathione disulfide in biological samples, Methods Enzymol., 113 (1985) 548-555. 2 Anderson, M.E., Powrie, E, Puri, R.N. and Meister, A., Glutathione monoethyl ester; preparation, uptake by tissues, and conversion to glutathione, Arch. Biochem., 239 (1985) 538-548. 3 Brink, J.J. and Stein, D.G., Pemoline levels in brain: enhancement by dimethyl sulfoxide, Science, 158 (1967) 1479-1480. 4 Brink, J.J. and Stein, D.G., Dimethyl sulfoxide: breakdown of blood-brain barrier?, Science, 160 (1968) 1472-1473. 5 Broadwell, R.D., Salcman, M. and Kaplan, R.S., Morphologic effect of dimethyl sulfoxide on the blood-brain barrier, Science, 217 (1982) 164-166. 6 De La Torre, J.C., Relative penetration of L-Dopa and 5-HTP through the brain barrier using dimethyl sulfoxide, Experientia, 26 (1970) 1117-1118. 7 Dingman, W. and Sporn, M.B., The penetration of proline and proline derivatives into brain, J. Neurochem., 4 (1959) 148-153, 8 Dixon, R.L., Adamson, R.H., Ben, M. and Rail, D.P., Apparent lack of interaction between dimethyl sulfoxide (DMSO) and a variety of drugs, Proc. Soc. Exp. Biol. Med., 118 (1965) 756-759. 9 Douglas, G.W. and Mortensen, R.A., The rate of metabolism of brain and liver glutathione in the rat studied with C14-glycine,J. Biol. Chem., 222 (1956) 581-585. 10 Egorin, M.J., Kaplan, R.S., Salcman, M., Aisner, J., Colvin, M., Wiernik, P.H. and Bachur, N.R., Cyclophosphamide plasma and cerebrospinal fluid kinetics with and without dimethyl sulfoxide, Clin. PharmacoL Ther., 32 (1982) 122-128. 11 Gahl, W.A., Tietze, F., Bashan, N., Steinherz, R. and Schulman, J.D., Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes, J. Biol. Chem., 257 (1982) 9570-9575. 12 Goldman, R,, Dipeptide hydrolysis within intact lysosomes in vitro, FEBS Lett., 33 (1973) 208-212. 13 Goldman, R, and Kaplan, A., Rupture of rat liver lysosomes mediated by e-amino acid esters, Biochim. Biophys. Acta, 318 (1973) 205-216. 14 Goldman, R. and Naider, F., Permeation and stereospecificity of hydrolysis of peptide esters within intact lysosomes in vitro, Biochim. Biophys. Acta, 338 (1974) 224-233. 15 Greig, N.H., Sweeney, D.J. and Rapoport, S.I., Inability of dimethyl sulfoxide to increase brain uptake of water-soluble compounds: implications to chemotherapy for brain tumors, Cancer Treat. Rep., 68 (1985) 305-312. 16 Griffith, O.W. and Meister, A., Potent and Specific Inhibition of glutathione synthesis by BSO (S-n-butyl homocysteine sulfoximine), J. Biol. Chem,, 254 (1979) 7558-7560. 17 Griffith, O.W. and Meister, A., Glutathione: interorgan translocation, turnover and metabolism, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5606-5610. 18 Griffith, O.W., Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis, J. Biol. Chem., 257 (1982) 13,704-13,712.
19 Hanig, J.P., Morris, J.M., Jr. and Krop, S., Increase of blood-brain barrier permeability to catecholamines by dimethyl sulphoxide in the neonate chick, J. Pharm. Pharmacol., 23 (1971) 386-387. 20 Iwen, P.C. and Miller, N.G., Enhancement of ketoconazole penetration across the blood-brain barrier of mice by dimethyl sulfoxide, Antimicrob. Agents Chemother., 30 (1986) 617-618. 21 Jain, A., M~rtensson, J. and Meister, A., unpublished data. 22 Keane, D.M., Gray, I. and Panuska, J.A., Ineffectiveness of dimethyl sulfoxide in altering the permeability of the blood-brain barrier, Cryobiology, 14 (1977) 592-597. 23 Kocsis, J.J., Harkaway, S. and Vogel, W.H., Dimethyl sulfoxide: breakdown of the blood-brain barrier?, Science, 160 (1968) 1472-1473. 24 Long, W.M., Chua, B.H.L., Lautensack, N. and Morgan, H.E., Effects of amino acid methyl esters on cardiac lysosomes and protein degradation, Am. J. Physiol., (1983) C101-112. 25 Mhrtensson, J., Jain, A., Frayer, W. and Meister, A,, Glutathione metabolism in the lung: inhibition of its synthesis leads to lamellar body and mitochondrial defects, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5296-5300. 26 Meister, A., Novel drugs that affect glutathione metabolism. In Mechanisms of Drug Resistance in Neoplastic Cells: Part H. Enzymatic Basis of Drug Resistance, Chapter 7, Academic Press, New York, 1988, pp. 99-126. 27 Meister, A., Metabolism and function of glutathione. In D. Dolphin, R. Poulson and O. Avramovic (Eds.), Glutathione: Chemical, Biochemical and Medical Aspects, Chapter 11, Wiley, New York, 1989, pp. 367-474. 28 Meister, A. and Griffith, O.W., Effects of methionine sulfoximine analogs on the synthesis of glutamine and glutathione; possible chemotherapeutic implications, Cancer Treat. Rep., 63 (1978) 1115-1121. 29 Meister, A., Selective modification of glutathione metabolism, Science, 220 (1983) 471-477. 30 Neuwelt, E.A., Barnett, P., Barranger, J., McCormick, C., Pagel, M. and Frenkel, E., Inability of dimethyl sulfoxide and 5-fluorouracil to open the blood-brain barrier, Neurosurgery, 12 (1983) 29-34. 31 Ozols, R.F., Hamilton, T.C., Louie, K.G., Behrens, B.C. and Young, R.C., Glutathione depletion with buthionine sulfoximine: potential clinical applications. In F.A. Valeriote and L.H. Baker (Eds.), Biochemical Modulation of Anticancer Agents: Experimental and Clinical Approaches, Boston, MA, 1986, pp. 277-293. 32 Pileblad, E. and Magnusson, T., Marked reduction of rat brain glutathione following intracerebroventricular administration of the glutathione synthesis inhibitor e-buthionine sulfoximine, Neurosci. Lett., 95 (1988) 302-306. 33 Puri, R.N. and Meister, A., Transport of glutathione as y-glutamylcysteinylglycylester, into liver and kidney, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 5258-5260. 34 Raps, S.P., Lai, J.C.K., Hertz, L. and Cooper, A.J.L., Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons, Brain Research, 493 (1989) 398-401. 35 Reeves, J.P., Accumulation of amino acids by lysosomes incubated with amino acid methyl esters, J. Biol. Chem., 254
119 (1979) 8914-8921. 36 Rubinstein, E. and Lev-E1, A., The effect of dimethyl sulfoxide on tissue distribution of gentamicin, Experientia, 36 (1980) 92-93. 37 Sekura, R. and Meister, A., Glutathione turnover in the kidney; considerations relating to the ),-glutamyl cycle and the transport of amino acids, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 2969-2972. 38 Slivka, A., Spina, M.B., Calvin, H.I. and Cohen, G., Depletion of brain glutathione in preweanling mice by L-buthionine sulfoximine, J. Neurochem., 50 (1988) 1391-1393. 39 Smith, E.R., Hadidian, Z. and Mason, M.M., The single-and repeated-dose toxicity of dimethyl sulfoxide, Ann. N.Y. Acad. Sci., 141 (1967) 97-109. 40 Steinherz, R., Tietze, F., Raiford, D., Gahl, W.A. and Schuiman, J.D., Patterns of amino acid efflux from isolated normal and cystinotic human leucocyte lysosomes, J. Biol. Chem., 257 (1982) 6041-6049. 41 Steinherz, R., Tietze, E, Gahl, W.A., Triche, T.J., Chiang, H., Modesti, A. and Schulman, J.D., Cystine accumulation and
42 43 44 45 46
clearance by normal and cystinotic leukocytes exposed to cystine dimethyl ester, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 4446-4450. Suzuki, E, Hayashi, H., Ito, S. and Hayaishi, O., Methyl ester of prostaglandin D2 as a delivery system of Prostaglandin D2 into brain, Biochim. Biophys. Acta, 917 (1987) 224-230. Tietze, F., Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione, Anal. Biochem., 27 (1969) 502-522. Vistica, D., In Y. Sakamoto et al. (Eds.), Glutathione Centennial: Molecular Perspectives and Clinical Implications, Academic Press, New York, 1989. Waiters, A., Jackson-Lewis, V. and Fahn, S., Little effect of dimethyl sulfoxide on blood-brain barrier to dopamine, Experientia, 40 (1984) 859-861. Wellner, V.P., Anderson, M.E., Puri, R.N., Jensen, G.L., and Meister, A., Radioprotection by glutathione ester; transport of glutathione ester into human lymphoid cells and fibroblasts, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 4732-4735.
115
BRES 15522
Transport into brain of buthionine sulfoximine, an inhibitor of glutathione synthesis, is facilitated by esterification and administration of dimethylsulfoxide Reuben Steinherz, Johannes Mhrtensson, Daniel Wellner and Alton Meister Department of Biochemistry, Cornell University Medical College, New York, N Y 10021 (U.S.A.) (Accepted 14 November 1989)
Key words: Glutathione; Buthionine sulfoximine; Dimethyisulfoxide; Esterification; Therapy Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, is poorly transported into the brain of adult mice, and only a slight decrease (~10%) in the level of brain glutathione is found 30-60 min after intraperitoneal administration of BSO. When BSO is given as the ethyl ester, the brain level of BSO increases substantially after 5-15 rain, and the glutathione level decreases by about 25% after 30-60 min. When BSO or its ester is given in 15% dimethylsulfoxide solution the brain levels of BSO are increased significantly and the brain glutathione levels are decreased by 20-35%. These observations suggest procedures that may be useful in decreasing the glutathione levels of the brains of adult animals. The finding that administration of BSO ethyl ester led to about a 25% decrease in the brain level of glutathione within 15 min suggests that a fraction of brain glutathione turns over very rapidly and may therefore be of special physiological significance. INTRODUCTION T r e a t m e n t of mice and rats with buthionine sulfoximine ( B S O ) , an irreversible inhibitor of y-glutamylcysteine synthetase, leads to a decrease of glutathione levels in a n u m b e r of tissues 16'17'2s. The glutathione levels of cells with relatively high overall turnover rates of glutathione (e.g. kidney, liver, lung, leukocytes) are most readily affected by B S O because substantial cellular e x p o r t of glutathione continues whereas there is little or no synthesis of glutathione. B S O has served as a useful agent for a variety of experimental studies on the t r a n s p o r t and m e t a b o l i s m of glutathione 27. B S O has also b e e n used for the p u r p o s e of decreasing glutathione levels of tumors, thus sensitizing them to chemotherapeutic agents and to radiation 26'2s'29'31'44. B S O is currently being tested in clinical trials. The effectiveness of B S O in studies of glutathione m e t a b o l i s m in the brain and for t r e a t m e n t of brain tumors is limited by the fact that B S O is p o o r l y t r a n s p o r t e d into the brain in adult animals. B S O is t r a n s p o r t e d into brain to a greater extent in preweanling mice than in adults 38, and B S O has been a d m i n i s t e r e d intracerebroventricularly to rats 32. The toxicity of B S O to the brain has not been extensively studied, but a p p e a r s to be chiefly related to its effect on glutathione synthesis 21. In the present work we have e x p l o r e d the effects of esterification o f B S O on its u p t a k e by brain and also the effect of dimethylsulfoxide ( D M S O ) administration on
the uptake of BSO. Esterification of certain c o m p o u n d s changes the lipid solubility to a form that is m o r e favorable for transport, and esterification of certain c o m p o u n d s m a y facilitate their transport into cells2' 7,11-14,24,26,33,35,40-42,46 In this a p p r o a c h , the ester is t r a n s p o r t e d m o r e rapidly than the original c o m p o u n d , and after transport the ester is h y d r o l y z e d intracellularly. In another approach, D M S O has been r e p o r t e d to increase the transport of several c o m p o u n d s into brain 36,19,20. The findings that D M S O facilitates transport into brain of c o m p o u n d s that differ m a r k e d l y in structure, e.g., pemoline 3 and horseradish peroxidase 5 are consistent with the view that D M S O might reversibly open the blood brain barrier; however, d a t a that are not in accord with this conclusion have been published 8,1°'15'22'23'3°'36'45. MATERIALS AND METHODS 5,5'-Dithiobis(2-nitrobenzoic acid), glutathione disulfide reductase, and NADPH, were obtained from Sigma. DMSO was obtained from Mailinckrodt. Swiss Webster mice (male, 28-30 g) were obtained from Taconic Farms, Germantown, NY. The ethyl ester of L-buthionine-SR-sulfoximine (BSO) 16,zs was prepared by the general procedures given by Anderson2 and Steinherz et al. 4°. Thin layer chromatography on silica gel plates (MKGF, Analtech) at 25 °C (30 min) with a solvent consisting of n-propanol, acetic acid, and water (10:5:1 (v/v)) was used to follow esterification. The plates were sprayed with 0.5% ninhydfin in acetone; BSO gave a red-brown color whereas BSO ethyl ester gave a brown-yellow color. The Rf values for BSO and its ethyl ester were, respectively, 0.5 and 0.7. The stability of BSO ester was examined in mixtures containing the amino acid ester (2 mM), mouse serum (50%; v/v), and DMSO (7.5%; v/v) at pH 6.8. For analysis, the mixtures were acidified by adding
Correspondence: A. Meister, Department of Biochemistry, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
116 5-sulfosalicylic acid (final concentration, 4.3%) and (after centrifugation of the mixtures containing serum) portions were analyzed on a Durrum model 500 amino acid analyzer using sodium buffers. The elution time for BSO was 62.1 min. Under these conditions, no hydrolysis of BSO ester was detected in freshly prepared solutions. Analysis of solutions of BSO ester that had been allowed to stand at 25-27 °C for 24 h indicated 72% hydrolysis. The presence of 7.5% DMSO stabilized the ester considerably; thus, only 18% hydrolysis was found after 24 h. In the mixtures containing serum, 26% of the initial BSO ester present was hydrolyzed per min at 37 °C; in the presence of 7.5% DMSO, the corresponding value for hydrolysis was 8.5%. Mice were injected intraperitoneally with freshly prepared solutions of BSO or BSO ethyl ester. The compounds were dissolved in water or in aqueous 15% (v/v) DMSO at a final concentration of 300 mOsm (pH 6.7-6.8). At the indicated intervals after injection, samples were taken from mice that were anesthetized by intraperitoneal injection of a mixture of xylazine (5 mg/kg) and ketamine (100 mg/kg) 3 rain before the samples were taken. The right ventricle was sectioned to bleed the animal. Cold saline (10 ml) was perfused through the left ventricle for 1 rain. Samples of the cortical area of the brain were excised, blotted, weighed, and homogenized in 5 volumes of 14.4% 5-sulfosalicylic acid. The homogenates were centrifuged and the supernatant solutions were extracted with 0.3 volume of chloroform for 30 min. After centrifugation, 80% of the aqueous layer was removed by aspiration and neutralized with sodium hydroxide. The sample was then evaporated to dryness in a Speed Vac apparatus and the residue was taken up in 2.5% sulfosalicylic acid such that the volume was one-third of the original volume of the homogenate sample. A portion (20/A) of this solution was used for analysis for BSO on the amino acid analyzer. The supernatant solutions obtained from the homogenates were analyzed for total glutathione as describedI'43 Blood plasma was obtained from samples (100/A) withdrawn from the right ventricle; the samples were treated with 1 /4 of 250 mM Na2EDTA and centrifuged at 10,000 g at 4 °C for 1 min. Portions (36 H1) of the supernatant solution were treated with 4 HI of 43% sulfosalicylic acid and centrifuged for 5 min as stated above. The supernatant solutions were analyzed for BSO on the amino acid analyzer. RESULTS
equimolar amount of BSO ethyl ester (in water) led to a significantly higher level of BSO (Fig. IA, Curve 2). The BSO level decreased rapidly within 30-60 rain to about the same value found after BSO administration. Addition of 15% DMSO to the BSO injection solution increased the brain cortex BSO level by about 100% after 30 and 60 min (compare Curve 3 with Curve 1, Fig. 1); DMSO did not significantly affect the 5 min value. The result obtained after injection of BSO ethyl ester in DMSO (Curve 4) reflects both the effect of DMSO and that of esterification. Thus, a significantly higher BSO level was found 5 and 15 rain after giving BSO ester in DMSO than found after giving BSO in DMSO. The data given in Fig. 2 indicate that the increased brain uptake of BSO after administration of BSO ester, and of BSO and its ester in DMSO solution, is reflected by a greater decrease in glutathione levels than found after administration of BSO in water. Administration of BSO in water led to about a 10% decline of the glutathione level. Administration of BSO ester led to a decline of about 25% after 15 min. Administration of BSO or of BSO ester in 15% DMSO led to greater decreases in glutathione levels than found after giving these compounds in water. Data on the total BSO levels in the blood plasma under these conditions are given in Fig. 3. The values found after giving BSO ester in water were similar to those found after giving BSO in water, except for the 5 min value, which was somewhat higher after giving BSO ester than after giving BSO. In general, the values for total plasma BSO after administration of BSO and BSO ester
BSO appeared in the brain cortex 5 min after intraperitoneal injection of an aqueous solution of BSO to adult mice (Fig. 1A, Curve 1). After 60 min, the BSO level had decreased by about 50%. Administration of an
0.3
A [H203
0.3
'
I B[DMSO]
BSO ESTER --o- BSO
~
o.2
o.2
0.1
.I
'
I
'
'
I
'
'
'
I
:
II
I
°
\ ~ - BSO(DMSO)
BSO ESTER BSO o
~'~.-"';'
I
Ld > .J O CO 123
I BSOESTER \
I 1 30
I 60
I 90
I 120 0
l 30
i 60
1 90
(°MS°' \
/
/
I 120
MINUTES
Fig. 1. Effect of esterification of BSO and of treatment with DMSO on BSO uptake by brain. Mice were injected with BSO or BSO ethyl ester (4 mmol/kg). These compounds were dissolved in water or in 15% (v/v) aqueous DMSO. The experimental details are given in the text. The data are given as means + S.D., n = 3.
0
30
60 MINUTES
90
120
Fig. 2. Effect of administration of BSO and BSO ethyl ester dissolved in water or in 15% (v/v) aqueous DMSO on brain glutathione levels. Data are given as means; S.D. _+ 5% n = 3-4.
117 5.0
4.0
:~
A [H20]
B [DMSO]
. - o - BSO BSO ESTER
3.0
4.0 ]I
.-a,-- BSO ESTER
3.0
~f4 m
2.0
0
2.0
30
60
90
120 0
30
60
90
120
MINUTES
Fig. 3. Effect of administration of BSO and BSO ethyl ester dissolved in water or 15% (v/v) aqueous DMSO on plasma BSO levels. Data are given as means _+ S.D.; n = 3-5. in DMSO were somewhat higher than those found after giving the compounds in water (except for the 15 min values). It seems of interest that the values for BSO and BSO ester (given in DMSO) at 60 min were clearly greater than the corresponding values obtained after giving these compounds in water indicating that administration of DMSO decreases the plasma clearance of BSO. DISCUSSION The data indicate that administration of BSO ethyl ester leads to a higher level of brain BSO than found after giving BSO. This effect is seen 5 and 15 rain after administration (Fig. 1A), and is associated with an appreciable decline of the glutathione level which persists for at least 120 min (Fig. 2). The finding of a significant ( ~ 2 5 % ) decrease in the level of glutathione in the brain 15 min after administration of BSO ethyl ester is of particular interest because it suggests that a fraction of brain glutathione turns over very rapidly. Previous studies led to the conclusion that the overall rate of turnover of brain glutathione is relatively slow compared to that of kidney and liver 9"17'32. The overall rate of glutathione turnover in rat brain was estimated to be about 70 h 9, whereas that in mouse kidney was found to be 29 min 37. The present observations suggest that further work on the nature and sites of this apparently rapidly turning over fraction of brain glutathione might be rewarding. Recent work indicating that glutathione is
present in high concentrations in cultured astrocytes but not in cultured neurons 34 may be of interest in this connection. That the brain level of BSO after giving BSO ester declines rapidly (Fig. 1A, Curve 2) is consistent with the finding that BSO ethyl ester is rapidly hydrolyzed, and suggests that BSO is effectively exported from brain. Administration of BSO or of its ester in DMSO leads to higher and more sustained brain levels of BSO (Fig. 1B) and to more effective depletion of brain glutathione (Fig. 2). The studies on plasma levels of BSO (Fig. 3) indicate that administration of DMSO leads to more sustained plasma levels of BSO after either BSO or its ester is given. Although DMSO may increase transport by acting on the blood-brain barrier as suggested 3-6A9'2°, its apparent effect on plasma clearance of BSO may also be related to an increase in uptake of BSO (and its ester) from the peritoneal cavity, or to a decrease in renal clearance; such effects would lead to sustained plasma levels of BSO and would presumably promote uptake by the brain. The mechanism or mechanisms by which DMSO affects plasma clearance of BSO need to be studied. The effect of BSO ester and DMSO on brain glutathione levels may be ascribed to an effect on entry of the ester into the brain or to high blood levels of BSO or its ester, or to both. We have also carried out studies in which adult rats (250-300 g) were treated with BSO and DMSO as described here, and in which similar results were observed. Administration of DMSO alone did not affect brain cortex glutathione levels in rats or mice. The dose of DMSO given in the studies reported here (about 2.2 g/kg b. wt.) is substantially lower than the reported 39 value for the LDs0 for DMSO given intraperitoneally to mice (14.7-17.7 g/kg). The present findings suggest that esterification of BSO facilitates its uptake by brain, and that administration of DMSO also facilitates uptake of this amino acid and its ester into brain. It is notable that the synthesis of glutathione in brain is more effectively inhibited after administration of BSO in DMSO than by giving BSO in water. Although DMSO is apparently not a panacea for facilitating transport of compounds into the brain s, 10,15,22,23,30,36,45, it seems possible that DMSO may be useful in this regard for certain compounds such as amino acids and their esters. Although these studies suggest that esterified amino acids may be more readily transported into brain than the parent amino acid, the usefulness of amino acid a-esters is likely to be affected by the rates of their hydrolysis in the blood plasma*. DMSO does not
* Preliminary studies on the uptake into brain of L-phenylalanine and its methyl ester indicate that phenylalanine methyl ester is more readily taken up than is phenylalanine, and that administration in DMSO solution, as in the case of BSO, leads to increased uptake.
118 stabilize BSO ethyl ester in the plasma to a great extent, but it might stabilize other esters more effectively. Although BSO has been of value in studies on glutathione metabolism as an in vivo inhibitor of 7-glutamylcy-
present findings suggest useful approaches that may overcome this limitation.
steine synthetase, and may therefore be a useful therapeutic agent, its value in studies on the brain and its
Acknowledgments. This research was supported in part by a grant to A.M. from the American Cancer Society. J.M. acknowledges stipendary support from the Swedish Medical Research Council (05644-06B), the Throne-Hoist Foundation, and the Killough Trust Foundation.
potential for treatment of brain tumors has been limited by the slow rate of its penetration into this organ. The REFERENCES 1 Anderson, M.E., Determination of glutathione and Glutathione disulfide in biological samples, Methods Enzymol., 113 (1985) 548-555. 2 Anderson, M.E., Powrie, E, Puri, R.N. and Meister, A., Glutathione monoethyl ester; preparation, uptake by tissues, and conversion to glutathione, Arch. Biochem., 239 (1985) 538-548. 3 Brink, J.J. and Stein, D.G., Pemoline levels in brain: enhancement by dimethyl sulfoxide, Science, 158 (1967) 1479-1480. 4 Brink, J.J. and Stein, D.G., Dimethyl sulfoxide: breakdown of blood-brain barrier?, Science, 160 (1968) 1472-1473. 5 Broadwell, R.D., Salcman, M. and Kaplan, R.S., Morphologic effect of dimethyl sulfoxide on the blood-brain barrier, Science, 217 (1982) 164-166. 6 De La Torre, J.C., Relative penetration of L-Dopa and 5-HTP through the brain barrier using dimethyl sulfoxide, Experientia, 26 (1970) 1117-1118. 7 Dingman, W. and Sporn, M.B., The penetration of proline and proline derivatives into brain, J. Neurochem., 4 (1959) 148-153, 8 Dixon, R.L., Adamson, R.H., Ben, M. and Rail, D.P., Apparent lack of interaction between dimethyl sulfoxide (DMSO) and a variety of drugs, Proc. Soc. Exp. Biol. Med., 118 (1965) 756-759. 9 Douglas, G.W. and Mortensen, R.A., The rate of metabolism of brain and liver glutathione in the rat studied with C14-glycine,J. Biol. Chem., 222 (1956) 581-585. 10 Egorin, M.J., Kaplan, R.S., Salcman, M., Aisner, J., Colvin, M., Wiernik, P.H. and Bachur, N.R., Cyclophosphamide plasma and cerebrospinal fluid kinetics with and without dimethyl sulfoxide, Clin. PharmacoL Ther., 32 (1982) 122-128. 11 Gahl, W.A., Tietze, F., Bashan, N., Steinherz, R. and Schulman, J.D., Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes, J. Biol. Chem., 257 (1982) 9570-9575. 12 Goldman, R,, Dipeptide hydrolysis within intact lysosomes in vitro, FEBS Lett., 33 (1973) 208-212. 13 Goldman, R, and Kaplan, A., Rupture of rat liver lysosomes mediated by e-amino acid esters, Biochim. Biophys. Acta, 318 (1973) 205-216. 14 Goldman, R. and Naider, F., Permeation and stereospecificity of hydrolysis of peptide esters within intact lysosomes in vitro, Biochim. Biophys. Acta, 338 (1974) 224-233. 15 Greig, N.H., Sweeney, D.J. and Rapoport, S.I., Inability of dimethyl sulfoxide to increase brain uptake of water-soluble compounds: implications to chemotherapy for brain tumors, Cancer Treat. Rep., 68 (1985) 305-312. 16 Griffith, O.W. and Meister, A., Potent and Specific Inhibition of glutathione synthesis by BSO (S-n-butyl homocysteine sulfoximine), J. Biol. Chem,, 254 (1979) 7558-7560. 17 Griffith, O.W. and Meister, A., Glutathione: interorgan translocation, turnover and metabolism, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5606-5610. 18 Griffith, O.W., Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis, J. Biol. Chem., 257 (1982) 13,704-13,712.
19 Hanig, J.P., Morris, J.M., Jr. and Krop, S., Increase of blood-brain barrier permeability to catecholamines by dimethyl sulphoxide in the neonate chick, J. Pharm. Pharmacol., 23 (1971) 386-387. 20 Iwen, P.C. and Miller, N.G., Enhancement of ketoconazole penetration across the blood-brain barrier of mice by dimethyl sulfoxide, Antimicrob. Agents Chemother., 30 (1986) 617-618. 21 Jain, A., M~rtensson, J. and Meister, A., unpublished data. 22 Keane, D.M., Gray, I. and Panuska, J.A., Ineffectiveness of dimethyl sulfoxide in altering the permeability of the blood-brain barrier, Cryobiology, 14 (1977) 592-597. 23 Kocsis, J.J., Harkaway, S. and Vogel, W.H., Dimethyl sulfoxide: breakdown of the blood-brain barrier?, Science, 160 (1968) 1472-1473. 24 Long, W.M., Chua, B.H.L., Lautensack, N. and Morgan, H.E., Effects of amino acid methyl esters on cardiac lysosomes and protein degradation, Am. J. Physiol., (1983) C101-112. 25 Mhrtensson, J., Jain, A., Frayer, W. and Meister, A,, Glutathione metabolism in the lung: inhibition of its synthesis leads to lamellar body and mitochondrial defects, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5296-5300. 26 Meister, A., Novel drugs that affect glutathione metabolism. In Mechanisms of Drug Resistance in Neoplastic Cells: Part H. Enzymatic Basis of Drug Resistance, Chapter 7, Academic Press, New York, 1988, pp. 99-126. 27 Meister, A., Metabolism and function of glutathione. In D. Dolphin, R. Poulson and O. Avramovic (Eds.), Glutathione: Chemical, Biochemical and Medical Aspects, Chapter 11, Wiley, New York, 1989, pp. 367-474. 28 Meister, A. and Griffith, O.W., Effects of methionine sulfoximine analogs on the synthesis of glutamine and glutathione; possible chemotherapeutic implications, Cancer Treat. Rep., 63 (1978) 1115-1121. 29 Meister, A., Selective modification of glutathione metabolism, Science, 220 (1983) 471-477. 30 Neuwelt, E.A., Barnett, P., Barranger, J., McCormick, C., Pagel, M. and Frenkel, E., Inability of dimethyl sulfoxide and 5-fluorouracil to open the blood-brain barrier, Neurosurgery, 12 (1983) 29-34. 31 Ozols, R.F., Hamilton, T.C., Louie, K.G., Behrens, B.C. and Young, R.C., Glutathione depletion with buthionine sulfoximine: potential clinical applications. In F.A. Valeriote and L.H. Baker (Eds.), Biochemical Modulation of Anticancer Agents: Experimental and Clinical Approaches, Boston, MA, 1986, pp. 277-293. 32 Pileblad, E. and Magnusson, T., Marked reduction of rat brain glutathione following intracerebroventricular administration of the glutathione synthesis inhibitor e-buthionine sulfoximine, Neurosci. Lett., 95 (1988) 302-306. 33 Puri, R.N. and Meister, A., Transport of glutathione as y-glutamylcysteinylglycylester, into liver and kidney, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 5258-5260. 34 Raps, S.P., Lai, J.C.K., Hertz, L. and Cooper, A.J.L., Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons, Brain Research, 493 (1989) 398-401. 35 Reeves, J.P., Accumulation of amino acids by lysosomes incubated with amino acid methyl esters, J. Biol. Chem., 254
119 (1979) 8914-8921. 36 Rubinstein, E. and Lev-E1, A., The effect of dimethyl sulfoxide on tissue distribution of gentamicin, Experientia, 36 (1980) 92-93. 37 Sekura, R. and Meister, A., Glutathione turnover in the kidney; considerations relating to the ),-glutamyl cycle and the transport of amino acids, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 2969-2972. 38 Slivka, A., Spina, M.B., Calvin, H.I. and Cohen, G., Depletion of brain glutathione in preweanling mice by L-buthionine sulfoximine, J. Neurochem., 50 (1988) 1391-1393. 39 Smith, E.R., Hadidian, Z. and Mason, M.M., The single-and repeated-dose toxicity of dimethyl sulfoxide, Ann. N.Y. Acad. Sci., 141 (1967) 97-109. 40 Steinherz, R., Tietze, F., Raiford, D., Gahl, W.A. and Schuiman, J.D., Patterns of amino acid efflux from isolated normal and cystinotic human leucocyte lysosomes, J. Biol. Chem., 257 (1982) 6041-6049. 41 Steinherz, R., Tietze, E, Gahl, W.A., Triche, T.J., Chiang, H., Modesti, A. and Schulman, J.D., Cystine accumulation and
42 43 44 45 46
clearance by normal and cystinotic leukocytes exposed to cystine dimethyl ester, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 4446-4450. Suzuki, E, Hayashi, H., Ito, S. and Hayaishi, O., Methyl ester of prostaglandin D2 as a delivery system of Prostaglandin D2 into brain, Biochim. Biophys. Acta, 917 (1987) 224-230. Tietze, F., Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione, Anal. Biochem., 27 (1969) 502-522. Vistica, D., In Y. Sakamoto et al. (Eds.), Glutathione Centennial: Molecular Perspectives and Clinical Implications, Academic Press, New York, 1989. Waiters, A., Jackson-Lewis, V. and Fahn, S., Little effect of dimethyl sulfoxide on blood-brain barrier to dopamine, Experientia, 40 (1984) 859-861. Wellner, V.P., Anderson, M.E., Puri, R.N., Jensen, G.L., and Meister, A., Radioprotection by glutathione ester; transport of glutathione ester into human lymphoid cells and fibroblasts, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 4732-4735.
