The effects of ebselen on cisplatin and diethyldithiocarbamate (DDC) cytotoxicity in rat hippocampal astrocytes

Toxicology Letters 131 (2002) 215– 226 www.elsevier.com/locate/toxlet

The effects of ebselen on cisplatin and diethyldithiocarbamate (DDC) cytotoxicity in rat hippocampal astrocytes D. Hardej, L.D. Trombetta * College of Pharmacy and Allied Health Professions, St. John’s Uni6ersity, Toxicology Program, 8000 Utopia Parkway, Jamaica, NY 11439, USA Received 15 October 2001; received in revised form 15 February 2002; accepted 18 February 2002

Abstract Ebselen is a seleno-organic compound with documented cytoprotective properties. Little work has been done, however, demonstrating ebselen’s cytoprotective properties in neural cell lines. In order to examine the effects of this compound and its mechanism of action, astrocytes were exposed to two known neurotoxicants, cisplatin and diethyldithiocarbamate (DDC). Cells were pretreated with 30 mM ebselen and subsequently treated with either 150 mM DDC for 1 h or 250 and 500 mM cisplatin for 24 h. Results indicate significant increases in viability in cells pretreated with ebselen and exposed to cisplatin. Ebselen pretreatment did not significantly increase viability in cells exposed to DDC. Light and scanning electron microscopy studies confirm the viability studies. Gross morphological damage was seen in cells treated with cisplatin, however, cells pretreated with ebselen and then exposed to cisplatin, appeared similar to controls. No differences were noted in cells pretreated with ebselen and then exposed to DDC or cells treated with DDC alone. In order to examine the mechanism of protection of this compound, glutathione status was examined. Results show that ebselen does not significantly increase reduced or oxidized glutathione (GSH, GSSG). All cell groups treated with cisplatin showed an increase in GSH levels. Ebselen showed protection in glutathione depleted cells at the 250 mM cisplatin dose. DDC treatment showed no significant increase in either reduced or oxidized glutathione. We conclude that ebselen significantly protects against cisplatin, but not DDC toxicity. We further conclude that this protection is not related to changes in glutathione status in the rat hippocampal cell line as has been reported in other cell types. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ebselen; Cisplatin; Diethyldithiocarbamate; Astrocytes; Glutathione

1. Introduction

* Corresponding author. Tel.: +1-718-990-6025; fax: + 1718-990-6439. E-mail address: [email protected] (L.D. Trombetta).

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)one) is a seleno-organic compound that has been demonstrated to have cytoprotective properties. The precise mechanism of action of this com-

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pound remains unknown. Studies have shown that it may act as a glutathione peroxidase mimic (Mu¨ ller et al., 1984; Wendel et al., 1984) and that it also has potent anti-inflammatory (Tanaka and Yamada, 1989; Schewe, 1995; Hattori et al., 1994) and antioxidant properties (Wendel et al., 1984; Narayanaswami and Sies, 1990; Lass et al., 1996). Increases in both reduced glutathione (GSH) and oxidized glutathione (GSSG) have been demonstrated with ebselen treatment (Hoshida et al., 1997). Glutathione plays an important role in the cellular oxidative defense system. This critical tripeptide acts as a substrate for the enzyme glutathione peroxidase in a reaction that converts hydroperoxides to water and alcohol. Cells maintain a normal ratio of GSH to GSSG in favor of the former. Perturbation of the ratio between GSH and GSSG may be indicative of oxidative stress (Peuchen et al., 1997). Increases in total cellular glutathione have been noted in the astroglial cell line in situations of cellular insult, such as exposure to metals (Cookson and Pentreath, 1996; Delmaestro and Trombetta, 1995). Toxicity of heavy metals, such as methylmercury, has been shown to be enhanced under conditions of decreased GSH levels and attenuated by increased GSH levels (Aschner et al., 1994). GSH has also been shown to be important in maintaining cellular sulfhydryl status (Kromidas et al., 1990). Glutathione maintains the thiol redox potential in cells keeping sulfhydryl groups of proteins in the reduced form (Cotgreave and Gerdes, 1998). Astrocytes are vital for the growth, maturation and survival of central nervous system neurons (Drukarch et al., 1997). Although once thought to play a passive role in the central nervous system, many studies have now demonstrated that astrocytes serve a protective role (Bronstein et al., 1995). Trombetta and Adachi (1985) demonstrated that cells treated with disulfiram showed degenerative changes in astrocytes prior to alterations seen in neurons, suggesting that astrocytes may be the initial target for some types of toxic insult. Deficiency in glutathione status has been shown to result in neuronal injury (Drukarch et al., 1997). Neuronal homeostasis has been shown to be dependent on astrocytes for the maintenance

of their glutathione levels. Measurements of reduced glutathione and other antioxidants in purified cultures of astrocytes or neurons have shown that the former have higher concentrations per cell (Makar et al., 1994). Several studies have shown that although exposure of astrocytes to metals may initially deplete glutathione, overall increases above control levels are seen over time (Lagarre et al., 1993; Verity and Sarafian, 1991). Cisplatin is a widely used antitumor agent used clinically for the treatment of cancers of the head and neck, certain lymphomas and for testicular and ovarian tumors (Abrams and Murrer, 1993). Therapeutic action of this agent is attributed to its ability to inhibit DNA synthesis (Lippert, 1992). The use of cisplatin is limited by its toxicity. Nephrotoxicity with this compound has been well documented, however severe neurotoxic effects have also been noted (Higa et al., 1995; Minami et al., 1996). The mechanism of cisplatin toxicity has not yet been elucidated, but research suggests that binding of platinum to critical protein sulfhydryl groups may be involved (Levi et al., 1980). Mitochondrial injury due to cisplatin is associated with loss of mitochondrial protein sulfhydryl groups subsequent to decreases in mitochondrial glutathione concentration (Zhang and Lindup, 1993). Ebselen has previously been demonstrated to protect against cisplatin toxicity in the renal cell line LLK-PK1 (Baldrew et al., 1992). Diethyldithiocarbamate (DDC) is a compound found commonly in fungicides. The toxicity of this compound has been well documented in this laboratory and has been found to exert its toxic effect by disruption of cellular thiol status and by causing alterations in intracellular metal concentration, especially copper (Delmaestro and Trombetta, 1995). In addition, this compromised sulfhydryl status has been shown to be involved in the cytoskeletal disruption (McManus and Trombetta, 1995). Disulfiram (DS) is the parent compound of DDC and is considered to be metabolically interchangeable. Stromme (1963) demonstrated that DS has the ability to bind thiol groups by the formation of mixed disulfides. Irreversible inactivation of several enzymes has also been attributed to disulfiram’s thiol binding ability (Hassinen, 1966; Schurr et al., 1978).

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The purpose of this study was to investigate the cytoprotective properties of ebselen against cisplatin and DDC. These compounds were chosen based on the fact that they both have been shown to have neurotoxic effects (Trombetta and Adachi, 1985; Higa et al., 1995; Gregg et al., 1992) and both appear to affect cellular sulfhydryl status (Stromme, 1963; Levi et al., 1980). Glutathione status was examined to determine if ebselen caused changes in GSH and GSSG levels in the rat hippocampal cell line and if these changes could be correlated to ebselen’s ability to protect against cytotoxicity. Glutathione depleted cells were also studied to determine ebselen’s cytoprotection.

2. Materials and methods

2.1. Cell cultures A continuous astrocyte cell culture line was established according to the method of McManus and Trombetta (1995). Briefly, rat hippocampal astrocytes from Sprague– Dawley rat pups were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing glutamine and 4.5 mg glucose/l, supplemented with 10% heat-inactivated fetal bovine serum (FBS), and 0.1 mg/ml gentamycin. All cultures were incubated at 37 °C in a mixture of 8% CO2 and 92% air and fed with complete medium twice weekly. Astroglial lineage of these cells was confirmed by the presence of glial fibrillary acidic protein (GFAP) determined using a peroxidase– antiperoxidase labeling technique (Ludwin et al., 1976). Astrocyte cultures were harvested just before reaching confluency by trypsinizing for 10– 15 min at 37 °C with 0.25% trypsin in Dulbecco’s phosphate buffered saline (PBS), pH 7.4.

2.2. Cell treatment and 6iability Astrocytes were grown on 96-well plates (Costar, Corning Inc., Corning, NY) at an initial seeding density of 2× 103 cells/well in a volume of 100 ml of DMEM: Nutrient Mixture F-12 (HAM) (1:1), containing glutamine, supplemented with

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10% heat-inactivated FBS and 0.1 mg/ml gentamycin, and allowed to grow to subconfluency. On day 4, all media was removed from the plates and the wells replaced with either complete media (control) or complete media containing 30 mM ebselen for 24 h. For cells to be treated with cisplatin, all media was removed, wells rinsed three times with PBS and the wells replaced with either complete media or complete media containing either 250 or 500 mM cisplatin and allowed to incubate at 37 °C for 24 h. After incubation, the MTT cell viability assay, based on the method of Mossman (1983) was performed (Sigma, St. Louis, MO). This assay utilizes (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT, which is converted by mitochondrial dehydrogenase in viable cells yielding a purple formazan product. The crystalline product is dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically at 570 nM and cell number extrapolated from the standard curve. Standard curves were established by counting cells using Trypan Blue exclusion and serial dilutions. For cells to be treated with DDC, media was removed, wells were washed three times with PBS, and replaced with complete media (control) or media containing 150 mM DDC for a period of 1 h. After incubation all media was again removed, wells rinsed three times with PBS and replaced with complete media. Plates were incubated at 37 °C for 24 h and the MTT assay was preformed. The paradigm for this treatment replicates our previous studies on the pathogenic and cytotoxicologic characteristics of DDC (Trombetta et al., 1988; McManus and Trombetta, 1995). Results obtained with MTT assay were confirmed using a Neutral Red assay (data not shown). The Neutral Red assay was preformed according to the method outlined by Dacasto et al. (2001) adapted from the method of Huso *y et al. (1993). The Neutral Red assay was set up according to the treatment procedure outlined for the MTT assay. The Neutral Red assay is a procedure used for the determination of cell viability by the uptake of dye into lysosomes. The dye is solubilized and the resulting red color is read spectrophometrically.

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2.3. Light microscopy Astrocytes were seeded at an initial density of 2× 104 cells/slide and grown to approximately 80% subconfluence in Lab-Tek Chamber Slides® (Nalge Nunc International, Naperville, IL). Cell treatment was the same as described above. Cells were examined using an inverted phase contrast microscope and photographed using a Nikon camera.

phoric acid was added in an equal volume to sample and allowed to sit 10 min at room temperature to precipitate protein. Samples were centrifuged at 3000× g for 5 min and supernatant collected for assay. All samples were frozen at − 80 °C. Reduced and oxidized glutathione were determined using the method of Baker et al. (1990). A kit was used from Cayman Chemical (Ann Arbor, MI).

2.6. Glutathione depletion 2.4. Scanning electron microscopy Cells were treated as described above, except that the astrocytes were grown on glass coverslips. After incubation, all media was removed, cover slips were rinsed three times in PBS and fixed in phosphate buffered (pH 7.4) 1.5% glutaraldehyde for 1 h at 0 – 4 °C. Dehydration was achieved through a series of water to acetone steps. Cells were critically point dried (CPD) in a Polaron E 3000 using bone dry CO2 as a transition fluid. The samples were sputter-coated with 15 nm platinum using a Polaron E 5100 series II coater for 90 s set at 2.5 kV. Samples were viewed on a Hitachi S-530 scanning electron microscope at 25 kV with an eucentric stage.

2.5. Glutathione assay Astrocytes were plated at an initial density of 8× 104 cells/flask and grown to approximately 80% confluence in T75 culture flasks. Cell treatment was the same as described above. After the 24 h incubation, cells were harvested using a cell scraper, poured into conical tubes and centrifuged at 1000× g to pellet cells. Supernatant was removed and cells were resuspended in PBS, centrifuged and supernatant removed. Cells were resuspended in 1 ml of PBS and transferred to Eppendorf tubes. Cells were subjected to five freeze-thaw cycles to lyse cells, and centrifuged at 4 °C for 20 min at 10,000× g. Supernatant was then divided into two tubes; one for protein determination and the other for glutathione assay. For the glutathione assay, 10% metaphos-

The method of Allen et al. (2001) was used for glutathione depletion. Astrocytes were grown on 96-well plates according to the methods outlined in cell treatment and viability except that on day 5 all control wells were treated with complete media plus 50 mM buthionine sulfoximine (BSO). Ebselen treated wells were treated with 30 mM ebselen plus 50 mM BSO to deplete glutathione. On day 6, all media was removed, wells rinsed three times with PBS and media replaced with complete media or complete media containing either 250 or 500 mM cisplatin and allowed to incubate at 37 °C for 24 h. The MTT assay was performed as outlined above.

2.7. Protein assay Samples for protein assay were collected as outlined above and frozen at −80 °C prior to assay. Total protein was determined according the method of Schaffner and Weissman (1973).

2.8. Statistics Statistics were preformed using the ANOVA with post-hoc analysis using the Tukey method. For measurement of glutathione values, N=3 and all absorbances were measured in duplicate. The entire experiment was performed in triplicate. For MTT results, N= 8 and the experiment was performed in triplicate. In the glutathione depletion experiment using BSO, cell viability was measured using the MTT assay, N=8 and the experiment was run in triplicate. PB 0.05 was considered significant.

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tween DDC treated cells and cells pretreated with ebselen prior to DDC treatment.

3.2. Light microscopy

Fig. 1. The effects of ebselen pretreatment on the viability of rat hippocampal astrocytes treated with either cisplatin or DDC. N = 8 *significantly different from its matched pair (PB 0.001). SEM not greater than 0.03 for any group. All groups are significantly different from control except the control+ ebselen group. Experiment performed in triplicate.

3. Results

3.1. Viability of astrocytes treated with cisplatin and DDC

Light microscopy of control cells and cells pretreated with 30 mM ebselen for 24 h appear similar (Fig. 2a and b). These cells were typically polymorphic showing both epithelioid and fusiform shape. Some cells possessed long nonbranching processes. Cells treated with 250 mM cisplatin appear severely damaged (Fig. 2c). They appeared highly vacuolated and many appeared shrunken and rounded. Cells pretreated with 30 mM ebselen for 24 h and treated with 250 mM cisplatin showed somewhat normal cells. These cells displayed normal cellular shape and processes, however, some dead cells were noted (Fig. 2d). Cells treated with DDC (Fig. 2e) appeared damaged regardless of ebselen pretreatment prior to DDC treatment (Fig. 2f). They appeared highly vacuolated, irregularly shaped and contained fragmented processes.

3.3. Electron microscopy Fig. 1 shows the effects of 250 and 500 mM cisplatin and 150 mM DDC on the viability of rat hippocampal astrocytes. Each concentration was paired with cells pretreated with 30 mM ebselen for 24 h prior to cisplatin and DDC treatment. All treatment groups were significantly different from the control group except for the control plus ebselen group. Treatment with 250 and 500 mM cisplatin was significantly different from its ebselen pretreated matched pair. DDC and DDC plus ebselen were significantly different from control although, there was no significant difference be-

Fig. 3a and b show control cells and cells pretreated with 30 mM ebselen. Both groups had a similar appearance and appeared similar to cells previously described by McManus and Trombetta (1995). Cells treated with 250 mM cisplatin alone (Fig. 3c) showed damage evidenced by rounded appearance, blebbing, loss of retraction fibers and/or irregular plasmalemma. Cells pretreated with 30 mM ebselen prior to 250 mM cisplatin (Fig. 3d), appeared similar to control cells, although many contained blebs and fragmented

Fig. 2. Light Micrographs. (a) Phase contrast micrograph of control rat hippocampal astrocytes showing both fusiform (arrows) and epithelioid shaped cells (arrow heads). All cells appear normal. ×200. (b) Phase contrast micrograph of control rat hippocampal astrocytes treated with 30 mM ebselen for 24 h. All cells appear normal. ×200. (c) Phase contrast micrograph of control rat hippocampal astrocytes treated with 250 mM cisplatin for 24 h, showing damaged cells and cellular debris. × 200. (d) Phase contrast micrograph of control rat hippocampal astrocytes pretreated with 30 mM ebselen for 24 h and then treated with 250 mM cisplatin for 24 h. Although floating and damaged cells were observed, the majority of cells showed normal appearance. × 200. (e) Phase contrast micrograph of control rat hippocampal astrocytes treated with 150 mM DDC for 1 h and allowed to recover for 24 h. Damaged cells were observed (arrows). ×200. (f) Phase contrast micrograph of control rat hippocampal astrocytes pretreated with 30 mM ebselen for 24 h and then treated with 150 mM DDC for 1 h and allowed to recover for 24 h. Damaged and floating cells were observed and numerous fragmented processes were seen. × 200.

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Fig. 2.

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Fig. 3.

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processes. Cellular debris was also noted. Cells treated with DDC (Fig. 3e and f) showed severe changes in the cell surface including an irregular and distorted plasmalemma and numerous small blebs. Ebselen pretreatment did not prevent the effects of DDC.

3.4. Glutathione status Glutathione status was measured to determine whether changes in reduced and oxidized glutathione could be correlated to ebselen’s ability to protect against toxicity. Table 1 shows the glutathione status of cells treated with cisplatin and DDC with and without 30 mM ebselen pretreatment. GSH and GSSG levels are not significantly different demonstrating no increases attributed to ebselen treatment alone. Significant increases were seen, however, in astrocytes treated with cisplatin regardless of ebselen pretreatment. No significant differences were noted in cells treated with DDC or in cells pretreated with ebselen prior to DDC treatment. In all treatment groups, the ratio of GSH to GSSG showed no change from the control group.

3.5. Depletion of glutathione BSO had no significant effect on the viability of control or ebselen pretreated astrocytes. Cells pretreated with BSO plus ebselen and treated with 500 mM cisplatin did not show a significant difference in viability from cells pretreated with BSO alone and treated with 500 mM cisplatin. However, cells pretreated with ebselen and BSO and treated with 250 mM cisplatin showed significant changes (PB0.0001) in viability from cells pre-

treated with BSO alone and treated with 250 mM cisplatin (Table 2).

4. Discussion Ebselen has been shown to protect against cytotoxicity of various compounds in a number of cell types and animal models (Schewe, 1995) although the precise mechanism for this protection remains unknown. Nephrotoxicity is a major factor that limits the use of cisplatin as a chemotherapeutic agent, although neurotoxicty and ototoxicity have been documented as well (Gregg et al., 1992; Leibbrandt et al., 1995). Baldrew et al. (1992), demonstrated that this compound was effective as a cytoprotective agent against cisplatin toxicity in LLC-PK1 kidney cell line. It was previously believed that central neurotoxicity with cisplatin use was not a major concern due to the poor penetration of this compound through the blood brain barrier (Vandiver et al., 1976; Ginos et al., 1987). Evidence is emerging, however, that penetration may occur to a greater extent than was previously thought, especially under conditions of hypoxia (Minami et al., 1996) or under conditions where lipopolysaccharide content may be increased (Minami et al., 1998). Higa et al. (1995) also showed severe neurotoxic effects with cisplatin use. DDC is an agent used commercially as a component of fungicides and industrially in the vulcanization of rubber. The parent compound of DDC, disulfiram, is used therapeutically in alcohol aversion therapy under the tradename Antabuse®, DDC and disulfiram are considered to be metabolically interchangeable (Stromme, 1963). Neurotoxic effects have been noted with

Fig. 3. Scanning electron micrographs. (a) Scanning electron micrograph of control rat hippocampal astrocyte showing processes (arrow) and refraction fibers (open arrow) Nuclear region (Nu). × 3000. (b) Scanning electron micrograph of control rat hippocampal astrocyte treated with 30 mM ebselen for 24 h. These cells appear similar to control cells. ×3000. (c) Scanning electron micrograph of control rat hippocampal astrocyte treated with 250 mM cisplatin for 24 h. Cells appear rounded and evident blebbing (arrows). × 3000. (d) Scanning electron micrograph of control rat hippocampal astrocyte pretreated with 30 mM ebselen for 24 h and then treated with 250 mM cisplatin for 24 h. Although cellular debris was noted (arrows), many cells appeared somewhat normal but cellular blebbing was noted in some cells (open arrow). × 3000. (e) Scanning electron micrograph of control rat hippocampal astrocyte treated with 150 mM DDC for 1 h and then allowed to recover for 24 h. Cells appear distorted showing an irregular plasmalemma and numerous retraction fibers (arrowheads). × 3000. (f) Scanning electron micrograph of control rat hippocampal astrocyte pretreated with 30 mM ebselen for 24 h and then treated with 150 mM DDC for 1 h and allowed to recover for 24 h. Cells appear similar to cells treated with DDC alone. × 3000.

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Table 1 Glutathione status Treatment group

GSSG (nmol/mg protein)

GSH (nmol/mg protein)

Ratio GSSG/GSH

Control Ebselen Cisplatin Cisplatin+ebselen DDC DDC+ebselen

6.629 0.80 6.649 0.96 15.21*9 0.79 15.80* 9 1.32 8.13 9 0.30 8.07 9 1.57

48.56 93.30 44.50 97.19 108.78* 9 15.90 135.12* 919.55 60.68 9 0.55 63.59 913.87

1:7.3 1:6.7 1:7.1 1:8.5 1:7.4 1:7.9

Effect of Cisplatin and DDC on GSH and GSSG in rat hippocampal astrocytes. N =3 *indicates significant difference from control group PB0.05. Experiment performed in triplicate.

disulfiram (Simonian et al., 1992; Delmaestro and Trombetta, 1995). Although the precise mechanism of toxicity is not known, it has been shown that disulfiram’s ability to act as a potent metal chelator may result in binding of extracellular copper and result in transport of excess copper into the cell (Eneanya et al., 1981; Trombetta et al., 1988). This compound has also been shown to bind sulfhydryl groups (Stromme, 1963; Hassinen, 1966; Schurr et al., 1978). Affects in sulfhydryl status cause disruption of cytoskeletal architecture, and compromised membrane integrity (McManus and Trombetta, 1995). In this present study, we demonstrated that ebselen protected rat hippocampal astrocytes against cisplatin but not DDC cytotoxicity. Cell viability studies showed that a significant increase in cell viability occurred with 250 and 500 mM concentrations of cisplatin when cells were pretreated with 30 mM ebselen. No protection was noted however with ebselen pretreatment against DDC toxicity (Fig. 1). Light and scanning electron microscopy studies confirm viability studies, showing that ebselen pretreatment against cisplatin toxicity resulted in cells that appeared similar to control cells, whereas pretreatment with ebselen against DDC treatment had no such protective effects (Figs. 2 and 3). Other studies have demonstrated that ebselen may cause changes in cellular glutathione status presumably resulting in better cellular defense against oxidative stress (Hoshida et al., 1997). Since ebselen is known to be a glutathione peroxidase mimic, subsequent increases in GSSG suggest oxidative processes may be involved. With

this in mind, we examined the effect of ebselen on the glutathione status in an astrocytic cell line to determine if this could be related to its cytoprotective properties. We found no significant changes in cells pretreated with ebselen. We did, however, find a significant increase in both reduced and oxidized glutathione in cells treated with cisplatin, regardless of pretreatment with ebselen (Table 1). These results were not surprising considering the protective nature of the astroglia in providing anti-oxidant protection for neurons. Increases in glutathione in astroglia have been noted in the presence of other metals such as cadmium and mercury (Cookson and Pentreath, 1996). It is interesting to note, however, that this increase in GSH in cisplatin treated cells did not help their viability. Table 2 Glutathione depletion Treatment group

Number of cells×104

Control 54.59 9 1.5 30 mM ebselen 52.29 9 1.0 30 mM ebselen+50 mM BSO 48.92 91.74 BSO (50 mM)+500 mM cisplatin 0 30 mM ebselen+50 mM BSO+500 mM 0 cisplatin 50 mM BSO+250 mM cisplatin 0 30 mM ebselen+50 mM BSO+250 mM 8.581 91.2* cisplatin The effects of glutathione depletion by 50 mM BSO on cell viability determined by MTT assay. N =8 *indicates significant difference between 250 mM cisplatin+BSO and astrocytes pretreated with BSO, ebselen and 250 mM cisplatin. PB 0.0001. Experiment was performed in triplicate.

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Although, no increases in either GSH or GSSG were noted in cells pretreated with ebselen, depletion of glutathione in these cells does appear to have some effect on ebselen’s cytoprotective properties (Table 2). Other studies have demonstrated that conjugation of ebselen with GSH is required for the bioactivation of ebselen (Haenen et al., 1989; Vermeulen et al., 1998) and is not surprising that depletion of GSH results in a decrease in cytoprotection by ebselen. In fact, the 500 mM cisplatin dose after pretreatment with BSO and ebselen resulted in diminished cytoprotection. However, cells pretreated with BSO plus ebselen and treated with 250 mM cisplatin dose still showed significant cytoprotection when compared to cells that were not pretreated with ebselen. Cytoprotection from cisplatin toxicity afforded by ebselen after glutathione depletion, is not as dramatic as cytoprotection noted when glutathione is present, suggesting that another mechanism is contributing to this protective effect. Since it was established at the onset of this study that ebselen afforded no obvious protection against DDC toxicity, the depletion of glutathione was not examined for its effect on DDC toxicity. From the work that has been presented here, several conclusions can be made. Ebselen is cytoprotective against cisplatin toxicity in the rat astroglial cell line. We further conclude that ebselen afforded no such protection against DDC toxicity under the conditions outlined here. Secondly, we conclude that the mechanism of cytoprotection by ebselen is not related to changes in glutathione status in this cell line. No increases in reduced or oxidized glutathione were seen in cells pretreated with ebselen. Depletion of glutathione does result in a decrease in cytoprotection by ebselen, although protection is not completely diminished. This would suggest that another mechanism is also involved. From the work presented here, it is not possible to determine why ebselen protects against cisplatin, but not DDC toxicity. Since ebselen has been known to have a number of intermediates, the possibility exists that cytoprotection favoring one toxin over another may be related to the structure of these ebselen intermediates or induction of other cellular defenses. Mechanism for ebselen protection may be more

complicated than previously thought and may involve more complex mechanisms than enhancing cellular antioxidant activity through glutathione and glutathione peroxidase activity.

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