Depletion of intracellular GSH and NPSH by buthionine sulfoximine and diethyl maleate: Factors that influence enhancement of aerobic radiation response

??Session I

DEPLETION OF INTRACELLULAR GSH AND NPSH BY BUTHIONINE SULFOXIMINE AND DIETHYL MALEATE: FAmORS THAT INFLUENCE ENHANCEMENT OF AEROBIC RADIATION RESPONSE MARIE E. VARNES, PH.D., JOHN. E. BIAGLOW, PH.D., LAURIE ROIZIN-TOWLE, PH.D. AND ERIC J. HALL, D. SC. Department

of Radiology. Case Western Reserve University. Cleveland, OH 44106 and Department College of Physicians and Surgeons of Columbia University, New York. NY 10032

of Radiolog>.

Manyinvestigators have

observed aerobic sensitization of V79, CHO and AS49 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently we discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiois be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of CSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH because this agent irreversibly inhibits y-giutamyl synthetase. For A549 monolayer cultures, there is approximately 50% regeneration 6 hr after removal of 0.01 mM BSO, 20% 6 hr after 0.1 mM BSO, and only SW 6 hr after 0.5 mM BSO. We found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of AS49 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH. After removal of DEM, AS49 cells immediately begin GSH resynthesis, and return to control levels occurs within 2 hr. Exogenous 5 mM CSH increases the rate of resynthesis of GSH in DEM-treated cells, but not in BSO-treated cells. Glutathione, Nonprotein thiols, L-Buthionine radiosensitivity, A549 cells.

sulfoximine,

INTRODUCTION

Diethyl

maleate,

WR-2721,

N-acetylcysteine.

Aerobic

Many investigators have shown that depletion of intracellular GSH by DEM or BSO increases the sensitivity ’ However, in contraof hypoxic cells to radiation. 2.6.7.8.’

A mechanism for radioprotection by endogenous cellular thiols was proposed by Alper several years ago to account for observations of reduced OER in GSH-deficient cell lines, and enhanced hypoxic radiosensitivity in normal cells treated with N-ethylmaleimide or diamide.‘.3 More recently, reagents of relatively high specificity for GSH have made possible the beginnings of detailed mechanistic studies of the role of thiols in cellular radiation response. DEM. which depletes GSH via a GSH-transferase catalyzed covalent binding and BSO, which inhibits GSH synthesis so that depletion occurs via natural catabolic pathways. have been found useful for studies on cellular radiosensitivity.2.3.6.”

diction to the Alper model, Biaglow et u/..‘.~ Mitchell et al., ’’ Epp et al.’ and Hall et ~l..~ have observed aerobic

sensitization upon depletion of GSH from V79. CHO or A549 cells using BSO. In the study of Mitchell ef al.. aerobic effects of DEM were also found. The phenomenon of aerobic sensitization by thiol depleting agents is quite disturbing from a clinical standpoint, since initially many investigators felt that thiol depletors might be used. either alone or in combination with other radiosensitizers. to enhance the differential killing of hypoxic tumor cells. Recently Epp el al.’ as well as ourselves. have found that aerobic sensitization of cells by BSO pretreatment

Manuscript submitted to the Conference on Chemical Modifiers of Cancer Treatment. Banff. Canada. Nov. 27-Dec. 1. 1983. Repnnr requests to: Marie E. Barnes. Ph.D. .~c,kno,tIrdge,)lmrs-We wish to thank Mr. Gregg Bell and Mr. Keith Freeman for their technical assistance. and Ms. Helen

Kurz for typmg this manuscript. This work was supported bb research Grants No. CA 13747. Ca 12536. and CA 18596 awarded to Case Western Reserve Universit) and to the College of Physicians and Surgeons of Columbia Umverslt, b) the National Cancer Institute. DHHS. Accepted for publication 22 March 1984. 1229

I230

Radiation Oncology 0 Biology ??Physics

August 1984, Volume NPSH

0

” 10-7

10-6 8SO

/ mg Protein

D CHO

30.2

. v79 0 A549

23.0 108.0

10-5

10-4

IO. Number 8

10-3

( Molar 1

\

/ i

0.001

1

‘-1 / Srav

Fig. I. Upper panel: NPSH depletion after 24 hour treatment of monolayer cultures with various concentrations of L-BSO. Lower panel: The effect of BSO on the aerobic radiation response of A549 and CHO cells. Monolayer cultures were treated with 5 X IO-* M BSO for 24 hours before being harvested and irradiated as described in

Methods and Materials. Each data point represents the average of triplicate counts for an experiment, variation between flasks of 10% or less. P.E. for AS49 cells was 0.45 and for CHO cells 0.70. can be reversed if radioprotectors, such as WR-2721 or N-acetylcysteine (NAC), are added prior to irradiation. This study concerns regeneration of NPSH in cells after BSO or DEM treatment, and the factors that influence the rate of regeneration. We have examined the effect of exogenous thiols on NPSH levels of A549 cells depleted with BSO, in order to determine if the reversal of the aerobic radiation response could be correlated to either uptake of the exogenous thiols or enhancement of the rate of GSH resynthesis.

METHODS

AND

with a

MATERIALS

A549 human lung carcinoma cells were obtained from the American Type Culture Collection. and were grown as monolayers in McCoy’s 5A medium supplemented with 5% fetal calf serum and 10% calf serum. The medium was buffered at pH 7.2 by addition of 0.02 M HEPES. CHO cells were obtained from Dr. Ed Clark’ and grown in the same way. For irradiation experiments cells were harvested with trypsin-EDTA and resuspended in Cat+-, Mg++- and bi-

Depletion of GSH by BSO and DEM 0 M. E.

carbonate-free Hank’s balanced salt solution, buffered with HEPES at pH 7.2. Cells at 106/ml were suspended in T-25 cm2 flasks and irradiated at 37” using a 280 kV x ray unit.* The flasks were rotated on a shake table to insure adequate oxygenation during irradiation. Immediately after irradiation cells were diluted and plated in the medium used for growth. Cells were refed after 7 days, and scored for colony survival after 14 days2 BSO, when used, was added to the monolayers and removed before irradiation by washing the cells three times with cysteinefree McCoy’s medium. NAC and WR-2721 were added to trypsinized cells just prior to and during irradiation. NPSH were determined with a modified Ellman’s procedure, as previously described.‘* In the present studies acid extraction was performed by adding 10% sulfosalicylic acid directly to the monolayers. Cells were not washed after BSO or DEM treatment, but were rinsed three times with Hank’s balanced salt solution in cases where radioprotectors had been added to the flasks. GSH/ GSSG was measured using the Tietze assay.’ L-BSO was obtained from Chemi1og.t DEM, GSH and NAC from Sigma, and WR-2721 was kindly supplied by Dr. John Yuhas, University of Pennsylvania. RESULTS The upper panel of Figure 1 shows the effect of various BSO concentrations on NPSH depletion in three cell lines. Treatments were for 24 hr, and the monolayer cultures were fed with fresh medium at the time of BSO addition. With all three lines, maximal thiol depletion was found with a BSO concentration of 5 X lO-4 M. This concentration was sufficient to deplete GSH to 0, and NPSH to between 0 and 5% of control values. The data shown are representative for each cell line; variations of up to 10% have been found with BSO treatments of different cell preparations. The lower panel of Figure 1 shows the effect of BSO pretreatment of A549 or CHO cells on the aerobic radiation response. In these representative experiments (see Biaglow ef al., 3.4the dose reduction factor, measured at 10 Gy, was 1.3 for A549 and 1.27 for CHO cells. Interestingly, BSO pretreatment removed the shoulder of the CHO, but not the A549, survival curve. With A549 cells we have found maximal aerobic radiosensitization when the cells are suspended in Ca++-, Mg++- and bicarbonate-free Hank’s balanced salts. However, an aerobic effect, with a DRF of about 70% of that above. is also observed when the cells are irradiated in complete growth medium (data not shown). Figure 2 shows the effect of initial BSO concentration on regeneration of NPSH after BSO treatment. In the A549 cell line GSH constitutes 90-95% of the NPSH. We have performed regeneration studies using the Tietze

* Picker Vanguard.

1231

VARNES CI u/

120 i

1

too ”

z $ 0

Untreated

Cells

80-

I

2

Time

After

3

4

8SO Removal

5

6

(hrs)

Fig. 2. Regeneration of NPSH in A549 cells after 24 hour treatment with various concentrations of BSO. Comparisons using Ellman’s and Tietze assays showed GSH to he 90 to 95% of the NPSH in this cell line. All values are relative to the NPSH value of untreated cells at time 0, that is, just prior to refeeding.

assay and have obtained results similar to those of Figure 2. However, in the present work we measured NPSH because we were interested in studying the effects of low molecular weight thiols other than GSH (see below), and the Tietze assay would not measure possible cellular uptake of these agents. To insure that the low rates of NPSH regeneration seen in Figure 2 are not due to the presence of residual extracellular BSO after refeeding, we washed the monolayer cultures three times before addition of fresh medium, with careful aspiration of excess fluid between washings. The increase in NPSH levels of untreated cells after refeeding, as seen in Figure 2, was a generally observed phenomenon. Reversal of the BSO-induced aerobic radiosensitization of A549 cells by thiol radioprotecting agents is shown in Table 1. We have found that the exogenous thiols must be present during irradiation in order to reverse the BSO effect. If they are removed after 30 min incubation but before irradiation, aerobic sensitization remains. Even though the thiols listed above were chosen because of their low rates of auto-oxidation (data not shown), catalase was added to the medium to further insure against formation of toxic oxygen species as well as to inhibit oxygen consumption. Recently, we have observed that ascorbate can also reverse the BSO effect.

t Chemical Dynamics Corp., South Plainfield. NJ.

I232

Oncology ??Biology 0 Physics

Radiation

Table

I. Reversal of BSO-induced aerobic sensitization A549 cells bv addition of exoPenous thiols Addition

of

S.F. at IO

irradiation

Gray

None

None

0.005

0.5 mhl BSO

None

0.0005

None

5 mM

WR-2721

0.0065

None

5 mM

NAC

0.003

0.5 mM

BSO

5 mM

WR-2721

0.007

0.5 mM

BSO

5 mM

NAC

0.007

Note: BSO pretreatments and Materials”. penvons

Additions

of IO” cells/ml

trypsmization.

were for 24 hrs. as described In “Methods of WR-2721

in Hank’s

and NAC

balanced

P.E.‘s for control cells were 0.44 +/0.03.

were made to sus-

salt solution,

following

0.02. and for BSO

pretreated cells were 0.44

+/-

of triplicate plates. which

showed a variation of approximately IO%in

Data represents the mean survival

each case.

In Figure 3 (A and B). regeneration of NPSH after BSO treatment is compared with regeneration after treatment with I mM DEM for two hours. DEM. unlike BSO,

c A.

140

Regeneration After Treatment

t

5

IO. Number

8

B.

BSO

Regeneration After Treatment

EM

C+ NAC-. C + G9-i~~

0 No Additions CI + GSH a + NAC

t

5

1984. Volume

has no effect on the enzymes of GSH synthesis.“.’ and regeneration begins in A549 cells as soon as the DEM is washed away. The rate of GSH synthesis in the A549 line is approximately 20 nmoles/hr/mg protein.J Since exogenous thiols reverse BSO-induced aerobic sensitization, we were interested in determining whether addition of GSH, NAC or WR-272 1 to BSO treated cells would result in regeneration of the intracellular thiols. In addition to the data shown above, we have found that WR-272 1 has no significant effect on NPSH regeneration after BSO treatment. We have also examined the ability of precursors of GSH synthesis to increase the rate of GSH regeneration, and have found no effect of cysteine. glutamate, or glutamine (an important energy source for tumor cells) when added to the medium used for refeeding. These chemicals are, however, already present in McCoy’s 5A in low concentrations. 2-Deoxyglucose inhibited the resynthesis of GSH because of the energy requirement for resynthesis from precursors.“’ In control cultures, GSH, NAC and WR-2721 have some ability to increase intracellular NPSH levels. This varies with the age of the culture and NPSH level at the time of refeeding. However,

immediately

prior to and during Pretreatment

August

60 :

0 /-, 0

t 30

L

I

I

120

60 Time

Fig. 3. Panel A: The effect of 5 mM with 5 X 10e4 M BSO for 24 hours. and refed with McCoy’s 5A complete to inhibit auto-oxidation of GSH or The effect of 5 mM GSH on NPSH 2 hr.

After

1

1

180 BSO

1

,

0 or DEM

I

30

60

Removal

L

120

I

I80

(hrs)

GSH or 5 mM NAC on NPSH regeneration after treatment of A549 cells Pretreated cells were washed three times with Hank’s balanced salt solution medium (see Methods and Materials) to which IO pLg/rnlcatalase was added NAC. pH was maintained at 7.4 for all regeneration experiments. Panel B: regeneration in cells depleted of thiols by treatment with I mM DEM for

Depletion of

GSH by BSOand

even in those cultures where control NPSH levels are most affected by addition of exogenous thiols, there is no increase in intracellular NPSH levels of the BSOtreated cells. Addition of WR-2721 or NAC to control cultures does not result in aerobic radioprotection with the A549 cell line.

DISCUSSION The results presented show that there is a considerable difference between the effects of BSO and DEM with respect to cellular ability to regenerate NPSH after depletion. Initially we thought that the rate of NPSH regeneration might be related to aerobic sensitization, since DEM. which does not inhibit the enzymes of GSH synthesis, has only a marginal effect on aerobic response of A549 cells, compared to BSO which has a considerable effect.2 However, the failure of exogenous thiols, which reverse BSO-induced aerobic sensitization, to increase intracellular thiol levels either by affecting GSH synthesis or by merely being taken up into the cells. argues against such a hypothesis. In addition. the observation that exogenous thiols do not reverse the BSO effect, if removed just prior to irradiation. is further evidence that the cellular environment at the time of radiation is the major determinant of aerobic sensitivity of GSH-depleted cells. We are currently investigating whether DEM or BSO pretreatment of cells affects the intracellular redox state, as indicated by ability to reduce peroxides, hydroperoxides, and drugs that require NAD(P)H for their metabolism. Another possibility is that the exogenous thiols protect GSH-depleted cells by reducing oxidized groups on the external cell membrane. An objective in testing the effect of exogenous GSH on GSH regeneration in A549 cells after DEM-induced depletion was to determine if replenishment would be as

DEM 0 M. E. VARNES el al.

1233

as reported for CHO cells by Bump et u/.~ and by Koch.’ We found that exogenous GSH could increase intracellular thiol levels in DEM-pretreated, though not in BSO-pretreated cells. However, regeneration of GSH to control levels after DEM treatment took about 2 hours as compared to the 15 min reported with the CHO line. The data of Figure 3 indicate that replenishment of GSH after its removal occurs only via resynthesis. Intact cells are not permeable to exogenous GSH. Thus BSO-treated cells, which can not resynthesize GSH because of irreversible inhibition of y-glutamyl synthetase, are not affected by the thiol content of the medium. DEM-treated cells, on the other hand. are not inhibited in their synthesizing capacity and begin to replenish their GSH as soon as the excess reagent is removed. We believe exogenous GSH increases the rate of resynthesis by removing DEM trapped in the cell membrane. This is likely since DEM is quite hydrophobic and would be difficult to wash out completely. and since the rate of resynthesis in the presence of exogenous GSH is closer to the rate of GSH synthesis in untreated cells, as determined by Biagiow PI ~1.~However. we cannot exclude the possibility that DEM has caused some membrane damage. and thus the apparent higher rate of GSH.resynthesis in the presence of added GSH is due in part to uptake from the medium. Even though intracellular GSH replenishment does not seem to be a factor in the aerobic radioresponse of GSHdeficient cells, the significant differences between DEM and BSO with respect to their effect on GSH resynthesis could have important consequences for combined studies. such as those using nitroimidazole sensitizers and thiol depletors. GSH is known to be involved in the detoxification of nitro compounds,3 ’ and effects of prolonged GSH depletion, as seen following BSO treatment. are still largely unknown.

rapid

REFERENCES I. Alper. T.: The modification of damage caused by primary ionization of biological targets. Radiat. Res. 5: 573-:586. 1956. 2. Biaglow. J.E.. Clark. E.. Epp. E.. Morse-Gaudio. M.. Vames. M.. Mitchell. J.: Nonprotein thiols and the radiation response of A549 human lung carcinoma cells. lnr J. Radial

Biol 44: 489-495.

1983.

3. Biaglow. J.E.. Varnes. M.E.. Clark. E.P.. Epp. E.R.: The role of thiols in cellular response to radiation and drugs.

Rudm

4.

Ret 95: 437-455.

1983.

Biaglow. J.E., Varnes. M.E.. Epp. E.R.. Clark. E.P.. Astor. M.: Factors involved in the depletion of giutathione from A_549human lung carcinoma cells: Implications for radiotherapy. Int J Rudm! Onwl. Riot Phj~. IO: 0000-0000. 1984. 5. Bump. E.A.. Taylor. Y.C.. Brown. J.M.: Role ofglutathione in the hypoxic cell qtotoxicity of misonidazole. Cunccr Rc~.c43: 997-100’. 1983. 6. Bump. E.A.. 1’~. N.Y.. Brown. J.M.: The use of drugs which deplete intracellular glutathione in hypoxic cell radlosensitizatlon. In/ J Radial Oncoi. Biol. F’h,~,q8: 439442. 1981. 7. Epp. F.. Clark. E.. Morse-Gaudio. M.. Biaglou. J.: Glu-

tathione depletion and cell radiosensitization in three cell lines irradiated in air: Importance of exogenous thiols. Inr J Radiut. Oncol. Biol. Ph?*.s.10 (In press) 1984. 8. Hall. E.J.. Astor. M.B.. Biaglow. J.E.. Hanog. B.: The radiation response of hypoxic V79 cells pretreated with BSO alone or in combination with misonidazole. Ir?/ J Rudm Oncol Bwl. PhJ,s IO (In press) 1984.

9.

Koch. C.J.: Competition between radiation protectors and radiation sensitizers in mammalian cells. In RNdif,~n,rc~c,ror.c und.4nrl~arcino)~en.s. O.F. Nygaard and M.G. Simic (Eds.). N.Y.. Academic Press. 1983. pp. 275-295. IO. Meister. A.: Glutathione metabolism and transport. In Ra dioprorcc~m and .-lnrit,arc‘ino,~cn(.O.F. Nygaard and M.G. Simic (Eds.). NY. .Academic Press 1983. pp. 12 l-l 52. I 1. Mitchell. J.B.. Russo, A.. Biaglon. J.E.. McPherson. S.J.: Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine and its effects on the oxygen enhancement ratio. Radiur Rcs 94: 613. 1983: RN~WI Rr\ f In press) 1984. 12. Vames. M.E.. Biaglou. J.E.. Hall. E.J.. Koch. C.J.: Depletion of non-protein thiols of hypoxic cells by misonidazole and metronidazole. In Rudratwn .Smtr/rxr\. I- M’ Brad! (Ed.). U.)‘.. Masson. 1980. pp. 121-126