ReproductiveToxicology,VoL 6, pp. 533-539, 1992
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THE EFFECT OF ESTROUS CYCLE AND BUTHIONINE SULFOXIMINE ON GLUTATHIONE RELEASE FROM THE IN VITRO PERFUSED RAT OVARY NICHOLAS CLAGUE,* MARGARET SEVCIK,* GAVIN STUART,* MATS BRANNSTROM,~" PER OLOF JANSON,'~ a n d JOHN F. JARRELL~ *Department of Obstetrics & Gynaecology,University of Calgary, Calgary, Alberta, Canada; tDepartment of Obstetrics and Gynaecology, University of G6teborg, Grteborg, Sweden;,Clara Christie Professorand Head, Department of Obstetrics & Gynaecology,University of Calgary/FoothillsHospital, Calgary, Alberta, Canada Abstract - - There is little known regarding the intracellular mechanisms of modification of damage in the ovary. Ovarian perfusion of en block dissections of the rat right ovary with aorta and vena cava were done to determine (a) if glutathione (GSH) is released by the ovary, (b) if the release is cycle dependent, and (c) if GSH released is the product of de novo ovarian synthesis. All perfused ovaries released GSH and the release was maximal at estrus and least at metestrus. Perfnsion with buthionine sulfoximine, a specific inhibitor of ~/-glutamylcysteine synthetase, resulted in a dose-dependent reduction in GSH released, indicating inhibition of de novo synthesis during perfusion. Key Words." ovary; perfusion; glutathione; buthionine sulfoximine.
in association with anovulation, has been reported to be a radiosensitizing agent in pulmonary (12), endometrial (13,14), and even ovarian tissues (15). Despite the above, there remains little known about endogenous intracellular modifiers of cell damage. There is, however, growing interest in glutathione and other intracellular nonprotein sulphydryls. Glutathione protects against irradiation and oxidative stress and is also important in the detoxification of endogenous and exogenous toxicants (16-18). Depletion ofintracellular glutathione by buthionine sulfoximine (BSO) enhances cell damage induced by chemotherapeutic agents (19,20) and radiation (21-
INTRODUCTION In recent years, there has been increasing interest in the effects of cytotoxic drugs and ionizing radiation on the human reproductive tract (1-7). In the female, these agents induce premature ovarian failure associated with sterility and severe estrogen deficiency ( 15). In the male, seminiferous tubule failure and azoospermia have been reported (2,6,7). Advances in the treatment of a number of malignancies, particularly those malignancies affecting young people, make considerations of the effects of such treatments on reproductive and endocrine function extremely important. Attempts to prevent ovarian damage during cancer therapy include the administration of oral contraceptives (8). Female rats given gonadotropin releasing hormone analogs have been shown to be partially protected against the effects ofcytotoxic drugs (9) and ovarian irradiation (10), although attempts to protect fecundity with analogs in response to radiation have not been successful (11). In addition, medroxyprogesterone acetate, frequently used during chemotherapy and radiotherapy in young women with cancer to control heavy menstrual bleeding of panycytopenia
23). Because glutathione has been shown to be present in ovarian tissue (24), the object of the first part of this study was to investigate variations in glutathione content during different stages of the estrous cycle of the rat and to evaluate its release from the in vitro perfused rat ovary. It was postulated that there are variations in ovarian glutathione content during the estrous cycle that may account for the reported differing sensitivity of the ovary to damage by anticancer treatments using hormonal manipulations. The object of the second part of this study was to determine whether the rat ovary has the ability to synthesize glutathione. De novo synthesis ofglutathione has been reported in a number of different tissues (16). Extensive interorgan translocation of glutathione is known to occur (16,25), and this provides a
Address correspondence to Dr. J. F. Jarrell, D e p a r t m e n t o f Obstetrics & Gynaecology, Foothills Hospital, 1403 29 Street N W , C a l o r y , Alberta, C a n a d a T 2 N 2T9. Received I l July 1991; Revision received 24 January 1992; Accepted I February 1992. 533
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rapid method of delivery to sites of utilization. It was therefore possible that any glutathione detected in the perfusate could have been originally derived from another source. In order to address this question, BSO, a specific inhibitor of ~,-glutamylcysteine synthetase, a key enzyme in glutathione biosynthesis, was added to the perfusion medium. Inhibition of 3,-glutamylcysteine synthetase by BSO is rapid and irreversible (26). If BSO were to cause a reduction in the rate of production of glutathione, this would suggest that the rat ovary synthesizes glutathione de novo and that this synthesis may be inhibited in the presence of BSO. In order to ensure the viability of the ovarian preparations throughout the study period, lactate and pyruvate determinations were performed hourly on perfusion medium samples. Measurements of lactate and pyruvate in the perfusion medium were used as checks for both oxygenation and metabolic activity. Increased lactate production by tissues sometimes indicates inadequate oxygen supply. This is not the case if an increase in lactate concentration is also matched by an increase in pyruvate concentration (27). M A T E R I A L S AND M E T H O D S
Animals Batches of mature female Sprague-Dawley rats were purchased from Charles River at 36 days of age. They were housed six rats per cage, placed on a 14 h light/10 h dark cycle and were given food and water ad libitum. Daily vaginal smears were performed on all animals between 0800 and 0900 h. Animals with 4-day estrous cycles were used and animals cycled 4 to 5 times prior to use, in order to characterize the cycle of each animal. Experimental animals were controlled for cycle and age.
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were ligated, and the two major vessels themselves were then ligated, superiorly to the ovarian vessels and inferiorly behind the insertion of the cannulae. Specimens was pre-perfused by hand with warm (37 °C) 0.9% NaC1. Only specimens in which there was no visible leakage and where the ovary readily blanched were included. Specimens were pre-perfused until the effluent was clear before attaching them to the in vitro perfusion system (29,30). This generally required up to 4 mL of 0.9% NaC1 to be passed through them. The left ovary was excised, dried, weighed and the glutathione content determined.
Perfusion procedure Perfusions were performed for 8 h in a recirculating perfusion system. This system is ideally suited to enable measurement of the small quantities ofglutathione produced in the nmol range. The apparatus has been described in detail by Br/innstr6m and colleagues (30) and Janson and colleagues (31). Briefly, the apparatus consisted of a perfusion chamber, oxygenator reservoir connected to a gas humidifier, roller pump, bubble trap, and mercury manometer. All glassware was siliconized, and the connecting tubing was made of Teflon® or Viton ®, except for Tygon® in the roller pump. The system contained 80 mL medium M199 with Earle's salts, to which gentamicin (50 ug/mL), heparin sulfate (0.2 IU/mL), insulin (0.02 IU/mL), and 4% bovine serum albumin had been added. The temperature of the perfusion medium was maintained between 37 and 38 *C, and the pH was kept at 7.4 by continuously gassing with 95% 02 and 5% CO2. The perfusion pressure was maintained between 60 and 80 mmHg throughout all experiments. The average flow through the ovary was approximately 0.9 mL/min and 99% of the medium bypassed the ovary (30).
Surgical techniques All surgeries were performed in the morning in order to control for the endogenous gonadotropin surge in the afternoon (28). Rats were anesthetized with 50 mg/kg pentobarbital i.p. and given heparin sulfate 300 IU i.p. The right ovary, with its arterial supply and venous drainage, was then surgically isolated and removed according to the procedure described in detail by Koos and Br~innstr6m and colleagues (29,30). Briefly, this consisted of cannulating the aorta and the vena cava in a cephalic direction close to their bifurcations with two 20-gauge Teflon ® cannulae. The two cannulae were advanced within 3 to 5 mm of the right ovarian artery and vein, and then secured in place using 4-0 silk suture. All other branches of the aorta and vena cava
Glutathione, lactate, and pyruvate measurements Samples were assayed for glutathione using the spectrophotometric method described by Brehe and Burch (32). This measures total glutathione, that is, both oxidized and reduced forms (GSSG and GSH). For the purposes of these studies, the term glutathione refers to total glutathione. Samples were also assayed for lactate and pyruvate using commercial kits supplied by the Sigma Chemical Co. (St. Louis, MO).
Statistics Experimental results are expressed as mean + SEM. Differences between groups were analyzed using multiple analysis of variance, followed by Dun-
Perfusedovarianglutathionerelease• N. CLAGUEETAL.
Perfusions were run for 8 h. Samples of 1 mL medium were taken hourly for total glutathione, lactate, and pyruvate measurements, and replaced with an equivalent volume of fresh medium on each occasion.
can's test for multiple comparisons or the Student t test where applicable (33). Statistical significance was accepted as P < 0.05.
Experiment 1 protocol An evaluation of glutathione release from the in vitro perfused ovary during the estrous cycle of the rat.
RESULTS
Experiment 1
There were six animals in each group with the exception of the estrus group where there were seven. The mean age at the time of surgery was 57 _+ 2 days. Immediately prior to commencing a perfusion, 1 mL of medium was removed from the apparatus for glutathione assay. Samples of 1 mL were taken hourly for 8 h and on each occasion replaced with an equivalent volume of fresh medium. Samples once taken were labelled and immediately frozen at - 2 0 °C.
Preliminary findings demonstrated no difference in total ovarian glutathione between right and left ovaries. Perfusions of the aorta and vena cava did not demonstrate glutathione release. Figure 1 shows glutathione concentrations in the medium at different stages of the estrous cycle throughout the 8 h perfusion period. Glutathione was detected in the perfusate of all ovaries. There was an initial rapid increase in the concentration ofglutathione detected in the medium in all groups in the first hour, and thereafter a more gradual rise. Because blood contains large quantities of glutathione (16), the concentration of glutathione detected in the medium was corrected to zero at T = 1 h. Subsequent concentrations detected in the medium were adjusted with respect to the concentration detected at T= 1 h. The capacity of the ovary to release glutathione into the medium was estimated by comparing the concentration of glutathione in the medium in each group at T = 8 h. At this time, the concentration of glutathione was greatest in the estrus group of animals being 0.44 + 0.20 nmol/mL. The concentration of glutathione was least in the metestrus group 0.20
Experiment 2 protocol The effect of BSO on glutathione releasefrom the in vitro perfused rat ovary. There were four experimental groups ofestrous animals in the study with six animals in each group. Group 1 was the control group in which medium alone was perfused. In groups 2 and 3, BSO was present in the medium from the commencement of the perfusion in concentrations of 1.25 nmol/mL and 12.5 nmol/mL, respectively. In group 4, 1 mmol BSO was added to the perfusion medium in the oxygenator reservoir at T = 2 h, to make a final concentration in the medium of 12.5 nmol/ mE.
0.8"
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Fig. 1. Concentration of total glutathione in the rccirculated pcrfusion media (mean + SEM). Concentrations are expressed in nmol/mL. Six animals in each group v~th the exception ofestms, where there were seven.
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ReproductiveToxicology
+ 0.07 n m o l / m L . Concentrations in the proestrus and diestrus groups were 0.39 + 0.12 n m o l / m L and 0.35 + 0.14 n m o l / m L , respectively. Significant differences in the concentration ofglutathione detected in the m e d i u m o f the proestrus and estrus groups were observed at T = 6 and T = 7 h. Significant differences between proestrus and metestrus groups were observed at T = 3 h. Significant differences between estrus and metestrus groups were observed from T = 4 until T = 8 h, and significant differences between estrus and diestrus groups were observed at T=7h. When glutathione release per milligram ovary was examined, a similar relationship was seen as for whole organ release rates although differences between groups were less marked. T h r o u g h o u t the 8-h perfusion period, the concentrations of glutathione detected in the m e d i u m were greatest in the estrus group and least in the metestrus group. On completion o f the perfusion study at T = 8 h, concentrations o f glutathione in proestrus, estrus, metestrus, and diestrus groups were 0.014 + 0.005 n m o l / m L / m g , 0.012 + 0.005 n m o l / m L / m g , 0.007 + 0.003 n m o l / m L / m g , and 0.010 + 0.004 n m o l / m L / m g , respectively. Differences between estrus and metestrus groups were significant at T = 6 and T = 7 h. Differences between other groups failed to achieve statistical significance. The mean rates of glutathione efllux from the in vitro perfused rat ovary were 2.25 + 0.72 × 10 -3, 2.81 + 1.25 × 10 3, 1.35 + 0.69 × 10 -3, and 1.75 + 0.64 × 10 -3 in proestrus, estrus, metestrus, and di-
7 o >
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Volume6, Number 6, 1992 estrus, respectively. Results are expressed in nmol/ m i n / m g ovarian tissue and indicate average glutathione efflux over the 7-h perfusion period from T = 1 until T = 8 h. The glutathione content o f the left (unperfused) ovary was determined for each animal (Figure 2). Glutathione concentrations were 3.40 _+ 0.42 nmol/ mg, 5.35 _+ 0.39 nmol/mg, 3.78 _+ 0.36 nmol/mg, and 4.14 _+ 0.43 n m o l / m g in proestrus, estrus, metestrus, and diestrus, respectively. The m a x i m u m concentration was in the estrus group. Differences between estrus and other groups achieved statistical significance with the exception of estrus and diestrus which just failed to reach statistical significance (P = 0.053). Differences between other groups were not statistically significant.
Experiment 2 Release of glutathione. Figure 3 illustrates the concentration of glutathione detected in the perfusion m e d i u m for each experimental group through the 8-h perfusion period. Concentrations are expressed in n m o l / m L . In keeping with Figure 1, concentrations o f glutathione are corrected to zero at T =lh. The capacity for the ovary to release glutathione into the m e d i u m was estimated by comparing the concentration in the perfusion m e d i u m at T = 8 h. This was maximal in the control estrous group o f animals being 0.55 +_ 0.12 n m o l / m L . In group 2, the concentration ofglutathione detected in the m e d i u m
I---I proestrus estrus , / J metestrus =7- J d i e s t r u s
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Fig. 2. Concentration of total glutathione in unperfused left ovaries (mean +_ SEM). Concentrations are expressed in nmol/ mg ovary. Six animals in each group with the exception of estrus where there were seven.
Perfused ovarian glutathione release • N. CLAGUE ET AL.
1.0. 0.8.
O m O control • • BSO 1.25nmol/.ml A A BS0 12.5nmol/.ml • • BSO 12.Snmol/ml added at T=2hrs.
537
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Fig. 3. Concentration of total glutathione in the recirculated perfusion media (mean + SEM). Concentrations are expressed in nmol/mL. Six animals in each group.
at T = 8 h was 0.52 + 0.11 nmol/mL. In group 3, the concentration of glutathione detected in the medium at T = 8 h was 0.01 + 0.01 nmol/mL. In group 4, the concentration ofglutathione detected in the medium at T = 8 h was 0.28 + 0.05 nmol/mL. Significant differences between groups 1 and 2 were not observed. Significant differences between groups 1 and 3 were observed from T = 3 until T = 8 h, and significant differences between groups 1 and 4 were observed from T = 4 until T = 8 h, when all perfusion experiments were terminated. The mean rates of glutathione efflux from the in vitro perfused rat ovary are illustrated in Table 1 for each group. Results are expressed in nmol/min/mg ovarian tissue. In group 4, the mean rate of glutathi-
one eflqux from the ovary is expressed both before and after the addition of BSO to the perfusion medium. Groups 1 and 2 were not significantly different. In group 3, the mean rate of glutathione eftlux was significantly reduced with respect to group 1 (P = 0.013). In group 4, the addition of BSO in the perfusion medium to a final concentration of 12.5 nmol/ mL at T = 2 h resulted in an apparent reduction in the rate of subsequent glutathione efflux with respect to the control group, although this just failed to achieve statistical significance (P = 0.074).
General metabolic response. Concentrations of lactate and pyruvate in the perfusion medium were found to increase with the duration ofperfusion in all groups. Significant differences between groups were not observed.
Table 1. Mean rate ofglutathione efflux from the in vitro perfused rat ovary Group
BSO Dose
1 2 3 4
Controlestrus 1.25 n m o l / m L B S O 12.5 n m o l / m L BSO 12.5 n m o l / m L BSO (added at T = 2 h)
G S H Eflux nmol/min/mg/ovary 3.93 4.17 0.07 3.57 1.38
+ 1.08 _+ 1.10 + 0.03 +__ 1.70 + 0.09
X X X X X
10 -3
10 -3 10 -3 10 -3
10 -3
Results are expressed in n m o l / m i n / m g ovavian tissue, a n d in the case o f groups l to 3 d e m o n s t r a t e the average glutathione effiux over the 7 h perfusion period from T = l to T = 8 h. In the case o f group 4, the upper figure refers to the average glutathione efflux from T = l to T = 2 h. T h e lower figure refers to the average glutathione etilux from the preparation after the addition o f BSO at T -- 2 h until completion o f the perfusion at T = 8 h.
DISCUSSION There has been growing interest in the modifying role that glutathione has in many tissues exposed to radiation and chemotherapy (15-20). Glutathione may have a role in the evolution and expression of tumors (34) as well as the modification of response to anti-cancer therapy, although the concentration may differ regionally within the same tumors (35). This study was intended to determine the characteristics of glutathione release from the normal ovary with respect to the estrous cycle and to determine whether the ovary synthesizes glutathione de
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Reproductive Toxicology
novo. The in vitro ovarian perfusion system used in this study has been previously characterized with respect to steroid release and ovulation induction suggesting that the system is capable of the maintenance of normal physiologic function during perfusion (2931). Under these in vitro circumstances, all ovaries were found to release glutathione into the perfusate, in keeping with previous reports of its presence in normal ovarian tissue (24). This suggests that the ovary is, like the liver, capable of the production and release of glutathione into the systemic circulation. Significantly the release of glutathione from the isolated rat ovary is within an order of magnitude of that produced by the isolated rat liver when release is examined on a milligram tissue basis. The isolated liver is reported to secrete glutathione at a rate of 13.8 _+ 0.4 X 10 3 nmol/min/mg (36). Because the liver has the highest capacity in the body to produce glutathione (38), this suggests that ovarian production is considerable. It remains important to determine the oxidative state ofglutathione released into the perfusate however. In order to determine whether the glutathione detected in the perfusate was from de novo synthesis, BSO, a specific and potent inhibitor of 3,-glutamylcysteine synthetase (26,37), was added to the perfusion medium. At a concentration of 12.5 nmol/mL, this caused a rapid and marked decline in subsequent glutathione production. This suggests that the ovary does synthesize glutathione de novo and that turnover is normally rapid. Lactate and pyruvate production, used as other parameters of ovarian metabolic activity, were unaffected. The rate of glutathione production and ovarian glutathione content were found to vary according to the stage of the estrous cycle with maximal production in the proestrus and estrus groups. This finding is consistent with the hypothesis that in the rat ovary, glutathione homeostasis is somehow influenced by the HPO axis. Possibly pituitary gonadotropins regulate ~,-glutamylcysteine synthetase, since the timing of maximum glutathione production coincides with the timing of the gonadotropin surges (28). Glutathione production was least in the metestrus group. During this time progesterone levels are elevated due to the functional activity of the corpora lutea. Consequently gonadotropin levels are low (28). The observation of maximal glutathione production during proestrus and estrus also suggests a possible relationship with follicle development and the process of ovulation. Because glutathione is important in normal cellular function (16) it would seem appropriate that the activity of the 3,-glutamyl cycle
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should increase during this time of increased mitotic and synthetic activity. The role ofglutathione during the ovulatory process in unknown. A number of inflammatory mediators have been implicated however (39,40). It seems likely therefore that free radicals are involved, and glutathione may have an important modulatory role here. Glutathione may therefore play an important physiologic role in normal ovarian function. - - This work has been supported by the NCIC and the Nat Christie Foundation. The secretarial assistance of G. Brittain is appreciated.
Acknowledgments
REFERENCES 1. Warne GL, Fairley KF, Hobbs JB, Martin FIR. Cyclophosphamide induced ovarian failure. N Engl J Med. 1973;289:1159-1162. 2. D'Angio GJ. Complications of treatment encountered in lymphoma-leukemia long-term survivors. Cancer (Phila). 1978;42:1015-1025. 3. Chapman RM, Sutcliffe SB, Malpas JS. Cytotoxic-induced ovarian failure in women with Hodgkin's disease. 1. Hormone function. J Am Med Assoc. 1979;242:1877-1881. 4. Homing SJ, Hoppe RT, Kaplan HS, Rosenberg SA. Female reproductive potential after treatment for Hodgkin's disease. N Engl J Med. 1981 ;304:1377-1382. 5. Whitehead E, Shalet SM, Blackledge G, Todd I, Crowther D, Beardwell CG. The effect of combination chemotherapy on ovarian function in women treated for Hodgkin's disease. Cancer (Phila). 1983;52:988-993. 6. Fairley KF, Barry JU, Johnson W. Sterility and testicular atropy related to cyclophosphamide therapy. Lancet. 1972;1:568-569. 7. Chapman RM, Sutcliffe SB, Rees LR, Edwards CRW, Malpas JS. Cyclical combination chemotherapy and gonadal function: retrospective study in males. Lancet. 1979;1:285-289. 8. Chapman R, Sutcliffe J. Protection of ovarian function by oral contraceptives in women receiving chemotherapy for Hodgkin's disease. Blood. 1981;58:849-851. 9. Ataya KM, McKanna JA, Weintraub AM, Clark MR, LeMaire WJA. A luteinizing hormone agonist for the prevention of chemotherapy-induced ovarian follicular loss in rats. Cancer Res. 1986;45:3651-3655. 10. Jarrell JF, YoungLai EV, McMahon A, Barr R, O'Connell G, Belbeck L. Effects of ionizing radiation and pre-treatment with (d-leu6,des-Glyl0) luteinizing hormone-releasing hormone ethylamide on developing rat ovarian follicles. Cancer Res. 1987;47:5005-5008. 11. Jarrell J, McMahon A, Barr R, YoungLai EV. The agonist (dleu-6, des-gly-10)-LHRH-ethylamide does not protect the fecundity of rats exposed to high dose unilateral ovarian irradiation. Reprod Toxicol. 1991 ;5:385-388. 12. De Greve J, Warson F, Deleu D, Storme G. Fatal pulmonary toxicity by the association of radiotherapy and medroxyprogesterone acetate. Cancer. 1985;56:2434-2436. 13. Bonte J, Decoster JM, lde P. Radiosensitization of endometrial adenocarcinoma of the uterus by means of medroxyprogesterone. Cancer. 1970;25:907-910. 14. Huber H, Husslein P, Michalica W, Wagenbilcher P. Radiosensitizing effect of medroxyprogesterone acetate on endometrial cancer cells in vitro. Cancer. 1984;54:999-1001. 15. Jarrell J, YoungLai EV, McMahon A, Barr R, O'Connell GD, Belbeck L. The effect of medroxyprogesterone acetate (Provera) on ovarian radiosensitivity. Am J Obstet Gynecol. 1989; 160:990-994. 16. Meister A, Anderson ME. Glutathione. Ann Rev Biochem. 1983;52:711-760.
Perfused ovarian glutathione release • N. CLAGUEET AL. 17. Arrick BA, Nathan CF. Glutathione metabolism as a determinant of therapeutic efficacy. Cancer Res. 1984;44:42244232. 18. Edwards PG. Evidence that glutathione may determine the differential cell-cycle phase toxicity of a platinum (IV) antitumour agent. J Natl Cancer Inst. 1988;80:734-738. 19. Green JA, Vistica DT, Young RC, Hamilton TC et al. Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion. Cancer Res. 1984;44:54275431. 20. Ono K, Komuro C, Tsutsui K, Shibamoto Y, Takahashi M, Abe M, Shrieve DC. Combined effect of buthionine sulfoximine and cyclophosphamide upon murine turnouts and bone marrow. BrJ Cancer. 1986;54:749-754. 2 I. Dethmers JK, Meister A. Glutathione export by human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation. Proc Natl Acad Sci USA. 1981;78:7492-7496. 22. Biagiow JE, Varnes ME, Clark EP, Epp ER. The role ofthiols in cellular response to radiation and drugs. Radiat Res. 1983;95:437-455. 23. Bertsche U, Schorn H. Glutathione depletion by DL-Buthionine-SR-Sulfoximine (BSO) potentiate x-ray induced chromosome lesions after liquid holding recovery. Radiat Res. 1986;105:351-369. 24. Mattison DR, Shiromizu K, Pendergrass JA, Thorgeirsson SS. Ontogeny of ovarian glutathione and sensitivity to primordial oocyte destruction by cyclophosphamide. Paediatr Pharmacol. 1983;3:49-55. 25. Griffith OW, Meister A. Glutathione: interorgan translocation, turnover and metabolism. Proc Nati Acad Sci USA. 1979;76:5606-5610. 26. Griffith OW. Mechanism of action, metabolism and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors ofglutathione synthesis. J Biol Chem. 1982;257:1370413712. 27. Huckabee WE. Relationship of pyruvate and lactate during anaerobic metabolism, i. Effects of infusion of pyruvate or glucose and of hyperventilation. J Clin Invest. 1958;37:244254. 28. Butcher FL, Collins WE, Fugo NW. Plasma concentrations of LH, FSH, prolactin, progesterone and estradiol-17/5 through-
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out the 4 day estrous cycle of the rat. Endocrinology. 1974;94:1704-1708. Koos RD, Jaccarino FJ, Magaril RA, LeMaire WJ. Perfusion of the rat ovary in vitro: Methodology induction of ovulation and pattern of steroidogenesis. Biol Reprod. 1984;30:1135ll41. Br~innstrrm M, Johansson BM, Sogn J, Janson PO. Characterization of an in vitro perfused rat ovary model: ovulation rate, oocyte maturation, steroidogenesis and influence of P.M.S.G. priming. Acta Physiol Scand. 1987;130:107-114. Janson PO, LeMaire W J, K~illfelt B, Holmes PV, et al. The study of ovulation in the isolated perfused rabbit ovary. I. Methodology and pattern of steroidogenesis. Biol Reprod. 1982;26:456--465. Brehe JE, Burch HB. Enzymatic assay for glutathione. Anal Biochem. 1976;74:189-197. Godfrey K. Statistics in practice: comparing the means of several groups. N Engl J Med. 1985;313:1450-1456. Novi AM. Regression of Alphatoxin-bl-induced hepatocellular carcinomas by reduced glutathione. Science. 1981;212:541-542. Lee FY, Vessey A, Rofstad E, Siemann DW, Sutherland RM. Heterogenicity ofglutathione content in human ovarian cancer. Cancer Res. 1989;49:5244-5248. Sies H, Bartoli GM, Burk RF, Waydhas C. Glutathione efllux from perfused rat liver after phenobarbital treatment, during drug oxidations, and selenium deficiency. Eur J Biochem. 1978;89: 113-118. Gritfith OW, Meister A. A potent and specific inhibition ofglutathione synthesis by buthionine sulfoxmine (s-n-butyl homocysteine sulfoximine). J Biol Chem. 1979;254:7558-7560. Lauterberg BH, Adams JD, Mitchell JR. Hepatic glutathione homeostatis in the rat. Ettlux accounts for glutathione turnover. Hepatology. 1984;4:585-590. Br~innstrrm M, Koos RD, LeMaire WJ, Janson PO. Cyclic adenosine 3',5'-monophosphate-induced ovulation in the perfused rat ovary and its mediation by prostaglandins. Biol Reprod. 1987;37:1047-1053. Br~innstrrm M, Woessner JF Jr, Koos RD, Sear CH, LeMaire WJ. Inhibitors of mammalian tissue collagenase and metalloproteinases suppress ovulation in the in vitro perfused rat ovary. Endocrinology. 1988; 122:1715-172 I.
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Printed in the U.S.A.All fightsreserved.
THE EFFECT OF ESTROUS CYCLE AND BUTHIONINE SULFOXIMINE ON GLUTATHIONE RELEASE FROM THE IN VITRO PERFUSED RAT OVARY NICHOLAS CLAGUE,* MARGARET SEVCIK,* GAVIN STUART,* MATS BRANNSTROM,~" PER OLOF JANSON,'~ a n d JOHN F. JARRELL~ *Department of Obstetrics & Gynaecology,University of Calgary, Calgary, Alberta, Canada; tDepartment of Obstetrics and Gynaecology, University of G6teborg, Grteborg, Sweden;,Clara Christie Professorand Head, Department of Obstetrics & Gynaecology,University of Calgary/FoothillsHospital, Calgary, Alberta, Canada Abstract - - There is little known regarding the intracellular mechanisms of modification of damage in the ovary. Ovarian perfusion of en block dissections of the rat right ovary with aorta and vena cava were done to determine (a) if glutathione (GSH) is released by the ovary, (b) if the release is cycle dependent, and (c) if GSH released is the product of de novo ovarian synthesis. All perfused ovaries released GSH and the release was maximal at estrus and least at metestrus. Perfnsion with buthionine sulfoximine, a specific inhibitor of ~/-glutamylcysteine synthetase, resulted in a dose-dependent reduction in GSH released, indicating inhibition of de novo synthesis during perfusion. Key Words." ovary; perfusion; glutathione; buthionine sulfoximine.
in association with anovulation, has been reported to be a radiosensitizing agent in pulmonary (12), endometrial (13,14), and even ovarian tissues (15). Despite the above, there remains little known about endogenous intracellular modifiers of cell damage. There is, however, growing interest in glutathione and other intracellular nonprotein sulphydryls. Glutathione protects against irradiation and oxidative stress and is also important in the detoxification of endogenous and exogenous toxicants (16-18). Depletion ofintracellular glutathione by buthionine sulfoximine (BSO) enhances cell damage induced by chemotherapeutic agents (19,20) and radiation (21-
INTRODUCTION In recent years, there has been increasing interest in the effects of cytotoxic drugs and ionizing radiation on the human reproductive tract (1-7). In the female, these agents induce premature ovarian failure associated with sterility and severe estrogen deficiency ( 15). In the male, seminiferous tubule failure and azoospermia have been reported (2,6,7). Advances in the treatment of a number of malignancies, particularly those malignancies affecting young people, make considerations of the effects of such treatments on reproductive and endocrine function extremely important. Attempts to prevent ovarian damage during cancer therapy include the administration of oral contraceptives (8). Female rats given gonadotropin releasing hormone analogs have been shown to be partially protected against the effects ofcytotoxic drugs (9) and ovarian irradiation (10), although attempts to protect fecundity with analogs in response to radiation have not been successful (11). In addition, medroxyprogesterone acetate, frequently used during chemotherapy and radiotherapy in young women with cancer to control heavy menstrual bleeding of panycytopenia
23). Because glutathione has been shown to be present in ovarian tissue (24), the object of the first part of this study was to investigate variations in glutathione content during different stages of the estrous cycle of the rat and to evaluate its release from the in vitro perfused rat ovary. It was postulated that there are variations in ovarian glutathione content during the estrous cycle that may account for the reported differing sensitivity of the ovary to damage by anticancer treatments using hormonal manipulations. The object of the second part of this study was to determine whether the rat ovary has the ability to synthesize glutathione. De novo synthesis ofglutathione has been reported in a number of different tissues (16). Extensive interorgan translocation of glutathione is known to occur (16,25), and this provides a
Address correspondence to Dr. J. F. Jarrell, D e p a r t m e n t o f Obstetrics & Gynaecology, Foothills Hospital, 1403 29 Street N W , C a l o r y , Alberta, C a n a d a T 2 N 2T9. Received I l July 1991; Revision received 24 January 1992; Accepted I February 1992. 533
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rapid method of delivery to sites of utilization. It was therefore possible that any glutathione detected in the perfusate could have been originally derived from another source. In order to address this question, BSO, a specific inhibitor of ~,-glutamylcysteine synthetase, a key enzyme in glutathione biosynthesis, was added to the perfusion medium. Inhibition of 3,-glutamylcysteine synthetase by BSO is rapid and irreversible (26). If BSO were to cause a reduction in the rate of production of glutathione, this would suggest that the rat ovary synthesizes glutathione de novo and that this synthesis may be inhibited in the presence of BSO. In order to ensure the viability of the ovarian preparations throughout the study period, lactate and pyruvate determinations were performed hourly on perfusion medium samples. Measurements of lactate and pyruvate in the perfusion medium were used as checks for both oxygenation and metabolic activity. Increased lactate production by tissues sometimes indicates inadequate oxygen supply. This is not the case if an increase in lactate concentration is also matched by an increase in pyruvate concentration (27). M A T E R I A L S AND M E T H O D S
Animals Batches of mature female Sprague-Dawley rats were purchased from Charles River at 36 days of age. They were housed six rats per cage, placed on a 14 h light/10 h dark cycle and were given food and water ad libitum. Daily vaginal smears were performed on all animals between 0800 and 0900 h. Animals with 4-day estrous cycles were used and animals cycled 4 to 5 times prior to use, in order to characterize the cycle of each animal. Experimental animals were controlled for cycle and age.
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were ligated, and the two major vessels themselves were then ligated, superiorly to the ovarian vessels and inferiorly behind the insertion of the cannulae. Specimens was pre-perfused by hand with warm (37 °C) 0.9% NaC1. Only specimens in which there was no visible leakage and where the ovary readily blanched were included. Specimens were pre-perfused until the effluent was clear before attaching them to the in vitro perfusion system (29,30). This generally required up to 4 mL of 0.9% NaC1 to be passed through them. The left ovary was excised, dried, weighed and the glutathione content determined.
Perfusion procedure Perfusions were performed for 8 h in a recirculating perfusion system. This system is ideally suited to enable measurement of the small quantities ofglutathione produced in the nmol range. The apparatus has been described in detail by Br/innstr6m and colleagues (30) and Janson and colleagues (31). Briefly, the apparatus consisted of a perfusion chamber, oxygenator reservoir connected to a gas humidifier, roller pump, bubble trap, and mercury manometer. All glassware was siliconized, and the connecting tubing was made of Teflon® or Viton ®, except for Tygon® in the roller pump. The system contained 80 mL medium M199 with Earle's salts, to which gentamicin (50 ug/mL), heparin sulfate (0.2 IU/mL), insulin (0.02 IU/mL), and 4% bovine serum albumin had been added. The temperature of the perfusion medium was maintained between 37 and 38 *C, and the pH was kept at 7.4 by continuously gassing with 95% 02 and 5% CO2. The perfusion pressure was maintained between 60 and 80 mmHg throughout all experiments. The average flow through the ovary was approximately 0.9 mL/min and 99% of the medium bypassed the ovary (30).
Surgical techniques All surgeries were performed in the morning in order to control for the endogenous gonadotropin surge in the afternoon (28). Rats were anesthetized with 50 mg/kg pentobarbital i.p. and given heparin sulfate 300 IU i.p. The right ovary, with its arterial supply and venous drainage, was then surgically isolated and removed according to the procedure described in detail by Koos and Br~innstr6m and colleagues (29,30). Briefly, this consisted of cannulating the aorta and the vena cava in a cephalic direction close to their bifurcations with two 20-gauge Teflon ® cannulae. The two cannulae were advanced within 3 to 5 mm of the right ovarian artery and vein, and then secured in place using 4-0 silk suture. All other branches of the aorta and vena cava
Glutathione, lactate, and pyruvate measurements Samples were assayed for glutathione using the spectrophotometric method described by Brehe and Burch (32). This measures total glutathione, that is, both oxidized and reduced forms (GSSG and GSH). For the purposes of these studies, the term glutathione refers to total glutathione. Samples were also assayed for lactate and pyruvate using commercial kits supplied by the Sigma Chemical Co. (St. Louis, MO).
Statistics Experimental results are expressed as mean + SEM. Differences between groups were analyzed using multiple analysis of variance, followed by Dun-
Perfusedovarianglutathionerelease• N. CLAGUEETAL.
Perfusions were run for 8 h. Samples of 1 mL medium were taken hourly for total glutathione, lactate, and pyruvate measurements, and replaced with an equivalent volume of fresh medium on each occasion.
can's test for multiple comparisons or the Student t test where applicable (33). Statistical significance was accepted as P < 0.05.
Experiment 1 protocol An evaluation of glutathione release from the in vitro perfused ovary during the estrous cycle of the rat.
RESULTS
Experiment 1
There were six animals in each group with the exception of the estrus group where there were seven. The mean age at the time of surgery was 57 _+ 2 days. Immediately prior to commencing a perfusion, 1 mL of medium was removed from the apparatus for glutathione assay. Samples of 1 mL were taken hourly for 8 h and on each occasion replaced with an equivalent volume of fresh medium. Samples once taken were labelled and immediately frozen at - 2 0 °C.
Preliminary findings demonstrated no difference in total ovarian glutathione between right and left ovaries. Perfusions of the aorta and vena cava did not demonstrate glutathione release. Figure 1 shows glutathione concentrations in the medium at different stages of the estrous cycle throughout the 8 h perfusion period. Glutathione was detected in the perfusate of all ovaries. There was an initial rapid increase in the concentration ofglutathione detected in the medium in all groups in the first hour, and thereafter a more gradual rise. Because blood contains large quantities of glutathione (16), the concentration of glutathione detected in the medium was corrected to zero at T = 1 h. Subsequent concentrations detected in the medium were adjusted with respect to the concentration detected at T= 1 h. The capacity of the ovary to release glutathione into the medium was estimated by comparing the concentration of glutathione in the medium in each group at T = 8 h. At this time, the concentration of glutathione was greatest in the estrus group of animals being 0.44 + 0.20 nmol/mL. The concentration of glutathione was least in the metestrus group 0.20
Experiment 2 protocol The effect of BSO on glutathione releasefrom the in vitro perfused rat ovary. There were four experimental groups ofestrous animals in the study with six animals in each group. Group 1 was the control group in which medium alone was perfused. In groups 2 and 3, BSO was present in the medium from the commencement of the perfusion in concentrations of 1.25 nmol/mL and 12.5 nmol/mL, respectively. In group 4, 1 mmol BSO was added to the perfusion medium in the oxygenator reservoir at T = 2 h, to make a final concentration in the medium of 12.5 nmol/ mE.
0.8"
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Fig. 1. Concentration of total glutathione in the rccirculated pcrfusion media (mean + SEM). Concentrations are expressed in nmol/mL. Six animals in each group v~th the exception ofestms, where there were seven.
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ReproductiveToxicology
+ 0.07 n m o l / m L . Concentrations in the proestrus and diestrus groups were 0.39 + 0.12 n m o l / m L and 0.35 + 0.14 n m o l / m L , respectively. Significant differences in the concentration ofglutathione detected in the m e d i u m o f the proestrus and estrus groups were observed at T = 6 and T = 7 h. Significant differences between proestrus and metestrus groups were observed at T = 3 h. Significant differences between estrus and metestrus groups were observed from T = 4 until T = 8 h, and significant differences between estrus and diestrus groups were observed at T=7h. When glutathione release per milligram ovary was examined, a similar relationship was seen as for whole organ release rates although differences between groups were less marked. T h r o u g h o u t the 8-h perfusion period, the concentrations of glutathione detected in the m e d i u m were greatest in the estrus group and least in the metestrus group. On completion o f the perfusion study at T = 8 h, concentrations o f glutathione in proestrus, estrus, metestrus, and diestrus groups were 0.014 + 0.005 n m o l / m L / m g , 0.012 + 0.005 n m o l / m L / m g , 0.007 + 0.003 n m o l / m L / m g , and 0.010 + 0.004 n m o l / m L / m g , respectively. Differences between estrus and metestrus groups were significant at T = 6 and T = 7 h. Differences between other groups failed to achieve statistical significance. The mean rates of glutathione efllux from the in vitro perfused rat ovary were 2.25 + 0.72 × 10 -3, 2.81 + 1.25 × 10 3, 1.35 + 0.69 × 10 -3, and 1.75 + 0.64 × 10 -3 in proestrus, estrus, metestrus, and di-
7 o >
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Volume6, Number 6, 1992 estrus, respectively. Results are expressed in nmol/ m i n / m g ovarian tissue and indicate average glutathione efflux over the 7-h perfusion period from T = 1 until T = 8 h. The glutathione content o f the left (unperfused) ovary was determined for each animal (Figure 2). Glutathione concentrations were 3.40 _+ 0.42 nmol/ mg, 5.35 _+ 0.39 nmol/mg, 3.78 _+ 0.36 nmol/mg, and 4.14 _+ 0.43 n m o l / m g in proestrus, estrus, metestrus, and diestrus, respectively. The m a x i m u m concentration was in the estrus group. Differences between estrus and other groups achieved statistical significance with the exception of estrus and diestrus which just failed to reach statistical significance (P = 0.053). Differences between other groups were not statistically significant.
Experiment 2 Release of glutathione. Figure 3 illustrates the concentration of glutathione detected in the perfusion m e d i u m for each experimental group through the 8-h perfusion period. Concentrations are expressed in n m o l / m L . In keeping with Figure 1, concentrations o f glutathione are corrected to zero at T =lh. The capacity for the ovary to release glutathione into the m e d i u m was estimated by comparing the concentration in the perfusion m e d i u m at T = 8 h. This was maximal in the control estrous group o f animals being 0.55 +_ 0.12 n m o l / m L . In group 2, the concentration ofglutathione detected in the m e d i u m
I---I proestrus estrus , / J metestrus =7- J d i e s t r u s
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Fig. 2. Concentration of total glutathione in unperfused left ovaries (mean +_ SEM). Concentrations are expressed in nmol/ mg ovary. Six animals in each group with the exception of estrus where there were seven.
Perfused ovarian glutathione release • N. CLAGUE ET AL.
1.0. 0.8.
O m O control • • BSO 1.25nmol/.ml A A BS0 12.5nmol/.ml • • BSO 12.Snmol/ml added at T=2hrs.
537
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Fig. 3. Concentration of total glutathione in the recirculated perfusion media (mean + SEM). Concentrations are expressed in nmol/mL. Six animals in each group.
at T = 8 h was 0.52 + 0.11 nmol/mL. In group 3, the concentration of glutathione detected in the medium at T = 8 h was 0.01 + 0.01 nmol/mL. In group 4, the concentration ofglutathione detected in the medium at T = 8 h was 0.28 + 0.05 nmol/mL. Significant differences between groups 1 and 2 were not observed. Significant differences between groups 1 and 3 were observed from T = 3 until T = 8 h, and significant differences between groups 1 and 4 were observed from T = 4 until T = 8 h, when all perfusion experiments were terminated. The mean rates of glutathione efflux from the in vitro perfused rat ovary are illustrated in Table 1 for each group. Results are expressed in nmol/min/mg ovarian tissue. In group 4, the mean rate of glutathi-
one eflqux from the ovary is expressed both before and after the addition of BSO to the perfusion medium. Groups 1 and 2 were not significantly different. In group 3, the mean rate of glutathione eftlux was significantly reduced with respect to group 1 (P = 0.013). In group 4, the addition of BSO in the perfusion medium to a final concentration of 12.5 nmol/ mL at T = 2 h resulted in an apparent reduction in the rate of subsequent glutathione efflux with respect to the control group, although this just failed to achieve statistical significance (P = 0.074).
General metabolic response. Concentrations of lactate and pyruvate in the perfusion medium were found to increase with the duration ofperfusion in all groups. Significant differences between groups were not observed.
Table 1. Mean rate ofglutathione efflux from the in vitro perfused rat ovary Group
BSO Dose
1 2 3 4
Controlestrus 1.25 n m o l / m L B S O 12.5 n m o l / m L BSO 12.5 n m o l / m L BSO (added at T = 2 h)
G S H Eflux nmol/min/mg/ovary 3.93 4.17 0.07 3.57 1.38
+ 1.08 _+ 1.10 + 0.03 +__ 1.70 + 0.09
X X X X X
10 -3
10 -3 10 -3 10 -3
10 -3
Results are expressed in n m o l / m i n / m g ovavian tissue, a n d in the case o f groups l to 3 d e m o n s t r a t e the average glutathione effiux over the 7 h perfusion period from T = l to T = 8 h. In the case o f group 4, the upper figure refers to the average glutathione efflux from T = l to T = 2 h. T h e lower figure refers to the average glutathione etilux from the preparation after the addition o f BSO at T -- 2 h until completion o f the perfusion at T = 8 h.
DISCUSSION There has been growing interest in the modifying role that glutathione has in many tissues exposed to radiation and chemotherapy (15-20). Glutathione may have a role in the evolution and expression of tumors (34) as well as the modification of response to anti-cancer therapy, although the concentration may differ regionally within the same tumors (35). This study was intended to determine the characteristics of glutathione release from the normal ovary with respect to the estrous cycle and to determine whether the ovary synthesizes glutathione de
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Reproductive Toxicology
novo. The in vitro ovarian perfusion system used in this study has been previously characterized with respect to steroid release and ovulation induction suggesting that the system is capable of the maintenance of normal physiologic function during perfusion (2931). Under these in vitro circumstances, all ovaries were found to release glutathione into the perfusate, in keeping with previous reports of its presence in normal ovarian tissue (24). This suggests that the ovary is, like the liver, capable of the production and release of glutathione into the systemic circulation. Significantly the release of glutathione from the isolated rat ovary is within an order of magnitude of that produced by the isolated rat liver when release is examined on a milligram tissue basis. The isolated liver is reported to secrete glutathione at a rate of 13.8 _+ 0.4 X 10 3 nmol/min/mg (36). Because the liver has the highest capacity in the body to produce glutathione (38), this suggests that ovarian production is considerable. It remains important to determine the oxidative state ofglutathione released into the perfusate however. In order to determine whether the glutathione detected in the perfusate was from de novo synthesis, BSO, a specific and potent inhibitor of 3,-glutamylcysteine synthetase (26,37), was added to the perfusion medium. At a concentration of 12.5 nmol/mL, this caused a rapid and marked decline in subsequent glutathione production. This suggests that the ovary does synthesize glutathione de novo and that turnover is normally rapid. Lactate and pyruvate production, used as other parameters of ovarian metabolic activity, were unaffected. The rate of glutathione production and ovarian glutathione content were found to vary according to the stage of the estrous cycle with maximal production in the proestrus and estrus groups. This finding is consistent with the hypothesis that in the rat ovary, glutathione homeostasis is somehow influenced by the HPO axis. Possibly pituitary gonadotropins regulate ~,-glutamylcysteine synthetase, since the timing of maximum glutathione production coincides with the timing of the gonadotropin surges (28). Glutathione production was least in the metestrus group. During this time progesterone levels are elevated due to the functional activity of the corpora lutea. Consequently gonadotropin levels are low (28). The observation of maximal glutathione production during proestrus and estrus also suggests a possible relationship with follicle development and the process of ovulation. Because glutathione is important in normal cellular function (16) it would seem appropriate that the activity of the 3,-glutamyl cycle
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should increase during this time of increased mitotic and synthetic activity. The role ofglutathione during the ovulatory process in unknown. A number of inflammatory mediators have been implicated however (39,40). It seems likely therefore that free radicals are involved, and glutathione may have an important modulatory role here. Glutathione may therefore play an important physiologic role in normal ovarian function. - - This work has been supported by the NCIC and the Nat Christie Foundation. The secretarial assistance of G. Brittain is appreciated.
Acknowledgments
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Perfused ovarian glutathione release • N. CLAGUEET AL. 17. Arrick BA, Nathan CF. Glutathione metabolism as a determinant of therapeutic efficacy. Cancer Res. 1984;44:42244232. 18. Edwards PG. Evidence that glutathione may determine the differential cell-cycle phase toxicity of a platinum (IV) antitumour agent. J Natl Cancer Inst. 1988;80:734-738. 19. Green JA, Vistica DT, Young RC, Hamilton TC et al. Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion. Cancer Res. 1984;44:54275431. 20. Ono K, Komuro C, Tsutsui K, Shibamoto Y, Takahashi M, Abe M, Shrieve DC. Combined effect of buthionine sulfoximine and cyclophosphamide upon murine turnouts and bone marrow. BrJ Cancer. 1986;54:749-754. 2 I. Dethmers JK, Meister A. Glutathione export by human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation. Proc Natl Acad Sci USA. 1981;78:7492-7496. 22. Biagiow JE, Varnes ME, Clark EP, Epp ER. The role ofthiols in cellular response to radiation and drugs. Radiat Res. 1983;95:437-455. 23. Bertsche U, Schorn H. Glutathione depletion by DL-Buthionine-SR-Sulfoximine (BSO) potentiate x-ray induced chromosome lesions after liquid holding recovery. Radiat Res. 1986;105:351-369. 24. Mattison DR, Shiromizu K, Pendergrass JA, Thorgeirsson SS. Ontogeny of ovarian glutathione and sensitivity to primordial oocyte destruction by cyclophosphamide. Paediatr Pharmacol. 1983;3:49-55. 25. Griffith OW, Meister A. Glutathione: interorgan translocation, turnover and metabolism. Proc Nati Acad Sci USA. 1979;76:5606-5610. 26. Griffith OW. Mechanism of action, metabolism and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors ofglutathione synthesis. J Biol Chem. 1982;257:1370413712. 27. Huckabee WE. Relationship of pyruvate and lactate during anaerobic metabolism, i. Effects of infusion of pyruvate or glucose and of hyperventilation. J Clin Invest. 1958;37:244254. 28. Butcher FL, Collins WE, Fugo NW. Plasma concentrations of LH, FSH, prolactin, progesterone and estradiol-17/5 through-
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