In!. J. Radialron Oncology Rio/. Phys.. Vol. 12, pp. 119 I-I I93 0360-3016/86 $3.00 + .OO Printed in the U.S.A. All rights reserved. Copyright 0 1986 Pergamon Journals Lfd

??Session II

INHIBITION OF THE PROTECTIVE EFFECT OF CYCLOPHOSPHAMIDE BY PRE-TREATMENT WITH BUTHIONINE SULFOXIMINE

JAMES CARMICHAEL, M.B.CH.B.,* NORMAN FRIEDMAN, M.S.,2 ZELIG TOCHNER, M.D.,2

DAVID J. ADAMS, PH.D.,~ C. ROLAND WOLF, PH.D.,~ JAMES B. MITCHELL, PH.D.~

AND ANGELO Russo, M.D., PH.D.~

‘NCI-Navy Medical Oncology Branch, Division of Cancer Treatment, ‘Radiation Oncology Branch. Division of Cancer Treatment, National Cancer Institute and Naval Hospital, Bethesda, MD 208 14 USA; 31mperial Cancer Research Fund,

Laboratory of Molecular Pharmacology, George Square, Edinburgh, Scotland

Low dose cyclophosphamide (CTX) is protective against a subsequent challenge with a lethal dose of the same drug administered 5 days later. At the time of maximal protection, elevation of glutathione (GSH) and glutathione transferase (GST) levels are detectable in the bone marrow of pre-treated animals. Elevation of GSH levels in the bone marrow was inhibited with the use of D,L-buthionine-S,R-sulfoximine (BSO), and this resulted in loss of the protective effect of CTX pre-treatment. In contrast, the overshoot in GST levels observed in these animals was not affected by BSO therapy. Bone marrow GSH levels in animals treated with BSO alone were minimally depleted (68% of control); whereas, animals pre-treated with CTX followed by BSO exhibited a greater reduction in GSH levels (47% of control). These results suggest that GSH is important in the protective effect afforded by low dose CTX pre-treatment and that the elevation of GSH levels observed is the result of a rebound synthetic process. In CTX pre-treated animals, BSO treatment resulted in greater than predicted depletion in GSH levels, and, therefore, caution is recommended with the potential use of combinations of BSO and cytotoxic drugs in the presence of a regenerating bone marrow.

Buthionine sulfoximine, Cyclophosphamide, Glutathione, Glutathione transferases, Bone marrow.

INTRODUCTION

It has previously been reported that if a high, apparently lethal, dose of CTX* is preceded by a ‘priming’ dose, then the bone marrow toxicity and lethality of the high dose are reduced.’ This protective effect was confirmed in a recent report,’ where CTX (75 mg/kg) was shown to pro- tect against the lethality from a CTX dose of 350 mg/kg administered 5-7 days later. At the time of maximal pro- tection, in this study, increased levels of GSH and GST were detected in bone marrow samples, and fluorescence activated cell sorting showed these increases to be re- stricted to the granulocyte fraction.2 The question re- mained whether the elevated GSH and GST levels ob- served in the granulocyte fraction related to enhanced synthesis following an initial depletion in levels, or merely reflected the preferential survival of cells with higher GSH and GST values. To investigate this question, BSO,t a

compound that depletes intracellular GSH levels by com- petitive inhibition of the enzyme y-glutamyl cysteine synthetase,3 was used.

METHODS AND MATERIALS

C3H mice, (16-23 g), 8-12 weeks of age were used in this study. All drugs were administered intraperitoneally.

Lethality studies Lethality was assessed in the six treatment groups, de-

scribed later, using 8 mice per group, with survival assessed on day 14 following the high dose CTX. Two separate doses of CTX were used for the high dose; 350 mg/kg, which was used in previous studies using CBA mice,‘,2 and 500 mg/kg, as the lower dose was not lethal in control C3H mice. A ‘priming’ dose of CTX 75 mg/kg was ad-

Reprint requests to: Dr. James Carmichael, NCI-Navy Med- ical Oncology Branch, Building 8, Room 6 109, Division of Can- cer Treatment, National Cancer Institute and Naval Hospital, Bethesda, Maryland 208 14 USA.

Presented at the Chemical Modifiers of Cancer Treatment Conference, Clearwater, Florida, 20-24 October 1985.

The opinions or assertions contained herein are the private

views of the authors and are not to be construed as official or as reflecting the views of the Department of the Navy or the Department of Defense.

Accepted for publication 11 February 1986. * Sigma Chemicals Ltd. P.O. Box 14508 St. Louis, MO. t Chemical Dynamics Corporation, South Plainfield, NJ.

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1192 I. J. Radiation Oncology 0 Biology 0 Physics July 1986, Volume 12, Number 7

ministered 5 days before the high dose in appropriate groups. BSO, 26.5 mg/mouse, was given either 6 hr before, or twice, 6 and 12 hr prior to high dose CTX.

6. CTX 75 mg/kg + BSO X 2-prior to high dose CTX or death.

RESULTS Bone marrow studies

All six treatment groups were studied, using 3 additional mice per group. At the time of high dose CTX adminis- tration to mice for the lethality studies, animals were killed by cervical dislocation and femurs were excised rapidly. Pooled bone marrow samples were obtained by pushing 2 ml of ice cold phosphate buffered saline through the femurs of 3 mice, and cell counts were determined using a particle counter. Specimens were sonicated at 4°C pro- tein precipitated using 0.6% sulphosalicylic acid and, GSH assays performed immediately using the method described by Tietze,’ GST assays were carried out using l-chloro- 2,4_dinitrobenzene as a substrate, as previously described.6 GSH values are expressed as pg/106 cells and, also, as a percentage of control values. GST values are likewise ex- pressed as specific activity/lo6 cells and as a percentage of control.

The results of the lethality experiments are summarized in Figures 1 a and 1 b. Fifty percent survival was observed in animals pre-treated with CTX, 75 mg/kg, 5 days prior to a lethal dose of 500 mg/kg. The protective effect of a priming dose of CTX on survival is lost in the animals subsequently treated with BSO. In fact, enhanced toxicity is observed in BSO-treated animals receiving 350 mg/kg CTX, whether or not they had received a priming dose of CTX. A dose of 350 mg/kg CTX was not lethal in control or CTX primed animals not receiving BSO.

The six treatment groups are listed below.

Treatment groups 1.

2.

3. 4.

5.

Control-saline 5 days prior to high dose CTX or death.

GSH and GST levels are shown in Table 1. Elevations are observed for both measurements following low dose CTX. BSO treatment resulted in lowering of the bone marrow GSH level in untreated mice to 68% of control, but in CTX ‘primed mice, BSO treatment resulted in bone marrow levels falling to 47% of control. GST values were elevated in animals pre-treated with cyclophospha- mide, irrespective of subsequent therapy, with BSO having no effect on these levels. These experiments were repeated on three occasions and quantitatively similar results ob- tained.

CTX 75 mg/kg-5 days prior to high dose CTX or death.

DISCUSSION

BSO X l-6 hr prior to high dose CTX or death. BSO X 2-6 and 12 hr prior to high dose CTX or death. CTX 75 mg/kg + BSO X l-prior to high dose CTX

This study confirms previous reports of the protective effect of low dose alkylating agents when administered several days prior to challenge with a normally lethal dose of the same or similar compound.‘,2,6,7 The protection afforded by a priming dose of CTX was inhibited by sub- or death.

A

CTX 350 mg/kg

100

m 80

> .- > 5

60

CO

8 40

20

0 1 2 3 4 5 6

B

CTX 500 mg/kg

I__ 123456

Group Number

Fig. 1. Fourteen day lethality in groups of 8 mice to 2 doses of CTX 350 mg/kg and 500 mg/kg. Treatment groups l-6 are described in the Methods and Materials Section.

Pre-treatment with buthionine sulfoximine 0 J. CARMICHAEL el al.

Table 1. GSH and GST levels in murine bone marrow

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Group GSH* Control % GSTt Control %

1. Control 0.247 * .033 100.0 6.87 f 2.24 100.0 2. CTX 75 mg/kg 0.353 f .03 143.0 13.38 t- 1.18 194.8 3. BSO X I 0.166 f .038 67.2 6.55 f 2.89 95.3 4. BSO x 2 0.172 f ,024 69.6 7.55 t 1.9 109.8 5. CTX 75 mg/kg + BSO X 1 0.123 f .036 49.8 13.36 -t 3.14 194.5 6. CTX 75 mg/kg + BSO X 2 0.110 ?I ,031 44.8 15.70 + 1.96 228.5

* Glutathione content expressed in pg per IO6 cells + S.D. t Glutathione-S-transferase Specific Activity (X 1 Om4) per 1 O6 cells + S.D.

sequent BSO treatment with, in fact, enhanced toxicity in mice treated with both drugs prior to the high dose CTX. The cause of death in these animals was not deter- mined: however, it is possible that BSO related reduction of glutathione levels in other tissues such as bladder, lung, or heart caused the observed enhancement of toxicity. Although alterations in glutathione homeostasis in the bone marrow were observed by these manipulations, it is not proven that these changes directly resulted in loss of protection. The protection afforded by low dose alkylating agents has previously been shown to be associated with an accelerated recovery of bone marrow elements,’ in- cluding granulocytes. Pre-treatment with CTX has been shown to increase GSH and GST levels in murine bone marrow,’ and this study confirms these findings. The in- crease in bone marrow GSH and GST levels has been shown to predominantly affect the granulocytic fraction.* The elevation of GSH normally associated with CTX ‘priming’ was totally inhibited by treatment with BSO. In fact, GSH levels were lower in mice initially given low dose CTX followed by BSO treatment than in animals treated with BSO alone. This suggests that an active syn- thetic process is being interrupted by BSO therapy, re- sulting in the lowering of GSH levels, below that normally achievable in bone marrow. These observations suggest that the elevation in GSH and GST levels observed in

CTX treated animals, is more likely to be caused by a compensatory synthetic process following an initial de- pletion in values, than merely the survival of cells with higher initial levels of GSH and GST. Minimal depletion in GSH levels was observed following BSO therapy alone; this is in agreement with previous studies showing min- imal potentiation of melphalan bone marrow toxicity with BSO.*

BSO therapy had no effect on GST levels and did not inhibit the overshoot normally observed following a CTX ‘priming’ dose. This indicates that in the presence of a low GSH level, elevation of GST levels to 200% of control had no protective effect on survival from a toxic dose of CTX, although glutathione depletion may have affected the baseline sensitivity of these mice to CTX. BSO therapy has minimal effect on normal bone marrow, glutathione levels 68% of control, and does not enhance hematological toxicity when used in combination with melphalan.’ However, because of the increased glutathione depletion observed in mice treated with BSO following low-dose cyclophosphamide, caution is suggested with the use of BSO in the presence of a regenerating bone marrow, re- gardless of the cause.

The protective effect of ‘priming’ doses of alkylating agents is an interesting phenomenon, and these results offer further insight into the etiology of this protection.

REFERENCES

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2.

3.

4.

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Adams, D.J., Carmichael, J., Wolf, C.R.: Altered tissue glu- tathione and glutathione transferase levels in response to cytotoxins: Studies with mouse bone marrow. Cancer Rex 45: 1669-1673, 1985. Carmichael, J., Adams, D.J., Wolf, C.R.: Glutathione and glutathione transferase levels in mouse granulocytes follow- ing cyclophosphamide administration. Cancer Res. 46: 735- 739, 1986. Griffith, O.W., Meister, A.: Potent and specific inhibition ofglutathione synthesis by buthionine sulfoximine (S-n-bu- tyl homocysteine sulfoximine). J. Biol. Chem. 254: 7558- 7560, 1979. Habig, W.H., Pabst, M.J., Jakoby, W.B.: Glutathione-S- transferases. The first enzymatic step in mercaptic acid for- mation. J. Biol. Chem. 249: 7130-7139, 1974. Hedley, D.W., McElwain, E.J., Miuar, J.L., Gordon, M.Y.: Acceleration of bone marrow recovery by pre-treatment with

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