62, 315–320 (2001) Copyright © 2001 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Effect of 4-Vinylcyclohexene Diepoxide Dosing in Rats on GSH Levels in Liver and Ovaries Patrick J. Devine,* I. Glenn Sipes,† ,‡ and Patricia B. Hoyer* ,‡ ,1 Departments of *Physiology and †Pharmacology and Toxicology, The University of Arizona, 1501 N. Campbell Ave., P.O. Box 245051, Tucson, Arizona 85724 –5051; and ‡Southwest Environmental Health Sciences Center, The University of Arizona, Tucson, Arizona 85724 –5051 Received February 2, 2001; accepted May 1, 2001

The reproductive success of mammalian females is dependent upon the pool of primordial ovarian follicles that is established during the latter part of fetal development and in the early neonatal period (Hirshfield, 1991). Oocytes contained in those follicles are arrested in meiosis and therefore, once destroyed, cannot be regenerated (Hirshfield, 1991). As a result, exposure to chemicals that cause depletion of the primor1

To whom correspondence should be addressed. Fax: (520) 626-2382. E-mail: [email protected].

dial follicle pool can jeopardize the reproductive capacity of females and, in women, may cause early menopause (Hoyer and Sipes, 1996). Of the initial cohort of primordial follicles, only a few progress successfully through each stage of follicular development to ovulation. Instead, the vast majority are lost through a degradative process called atresia (Hirshfield, 1991). The atretic process is believed to occur via physiological cell death, apoptosis (Hughes and Gorospe, 1991; Tilly et al., 1991). Loss of follicles can also occur in response to exposure to toxic chemicals (Hoyer and Sipes, 1996), though this may or may not be through physiological processes. 4-Vinylcyclohexene (VCH) and its monoepoxide (VCME) and diepoxide (VCD) metabolites belong to a group of occupational chemicals that has been shown to have selective toxicity in small ovarian (primordial and primary) follicles (Smith et al., 1990). VCH is a product of pesticide, flame retardants, rubber, and plastics production (IARC, 1982; Rappaport and Fraser, 1977), whereas VCD is used industrially as a diluent for other epoxides (IARC, 1976). Female rats administered the ovotoxic form, VCD (0.57 mmol/kg, 80 mg/kg), daily for 15 days demonstrate a 50% loss of primordial and primary follicles (Kao et al., 1999; Springer et al., 1996a). The exact mechanism causing this loss is unknown, but it has been proposed to involve acceleration of the natural process of atresia (Borman et al., 1999). Because apoptosis has been associated with cellular increases in oxidative stress in other tissues, such a mechanism may also be involved in VCDinduced destruction of ovarian preantral follicles. GSH, a ubiquitous intracellular antioxidant, is a tripeptide (␥-glu-cys-gly) present in millimolar concentrations in most cells. It is important for maintaining the intracellular environment in a reduced state, for protecting against both oxidative stress and electrophilic compounds. It also participates in protein folding and proper formation of disulfide bonds (DeLeve and Kaplowitz, 1991). Liver is the most active tissue in production and use of GSH, but all cells synthesize and utilize it to some degree (DeLeve and Kaplowitz, 1991; Meister, 1995). The rate-limiting enzyme for GSH synthesis is ␥-glutamylcysteinyl synthetase (GCS), and its activity can be irreversibly inhibited by the widely used chemical, buthionine sulfoximine

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Repeated daily dosing of rats with the occupational chemical 4vinylcyclohexene or its diepoxide metabolite (VCD) for 15 days destroys the smallest ovarian follicles. VCD acutely reduced hepatic levels of the antioxidant, glutathione (GSH); therefore, these studies were designed to evaluate whether GSH concentrations mediate VCD-induced ovotoxicity. Immature female Fischer 344 rats were dosed once or daily for 15 days with VCD (0.57 mmol/ kg, ip) or the GSH synthesis inhibitor buthionine sulfoximine (BSO, 2 mmol/kg, ip). Animals were euthanized 2, 6, or 26 h following a single dose, and 2 or 26 h following 15 days of daily dosing. Reduced (p < 0.05) hepatic GSH was seen within 2 h of a single dose of either VCD (51 ⴞ 5% of control) or BSO (42 ⴞ 9%), but only BSO reduced ovarian GSH (71 ⴞ 5% at 6 h, p ⴝ 0.05) as measured by HPLC. Within 26 h, GSH levels had returned to control levels with either treatment. Hepatic GSH levels were reduced (p < 0.05) 2 h after 15 daily doses with BSO (42 ⴞ 5%) or VCD (70 ⴞ 4%), but only BSO decreased ovarian GSH (64 ⴞ 3%). GSH levels in 15-day tissues were similar to controls 26 h after the final dose. Neither BSO nor VCD increased hepatic or ovarian concentrations of the oxidized dimer of GSH (GSSG) or thiobarbituric acid-reactive substances (TBARS), indicators of oxidative stress. These results suggest these treatments did not cause an oxidative stress. Histological counts of ovarian small follicle numbers were reduced (p < 0.05) in 15-day VCD-treated rats, whereas BSO did not affect follicle numbers, even though BSO reduced ovarian GSH content. These results support the conclusion that alterations in ovarian GSH levels are not involved in VCD-induced ovotoxicity. Key Words: 4-vinylcyclohexene diepoxide; ovarian follicle; glutathione; buthionine sulfoximine; ovary.

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MATERIALS AND METHODS Materials. D,L-Buthionine sulfoximine and HPLC-grade methanol were purchased from Fischer Scientific (Pittsburgh, PA). Perchloric acid and bathophenanthralinedisulfonic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Animals, treatments, and tissue collection. Female Fischer 344 rats (21 days of age) were purchased from Harlan (Indianapolis, IN). Animals were housed in plastic cages, given food and water ad libitum, and maintained on a 12-h light/dark cycle. Animals were allowed to acclimate for 1 week before experiments began. Experiments were approved by the University’s Institutional Animal Care and Use Committee. VCD (0.57 mmol/kg) or BSO (2 mmol/kg) was administered to rats (28 days of age) in sesame oil or in 0.9% saline, respectively, through ip injection either once or once daily between 0700 and 0900 h for 15 days as described

previously (Kao et al., 1999; Springer et al., 1996b). The VCD dosing regime was chosen because it has been shown to cause 50% primordial and primary follicle depletion after 15 days of daily dosing (Springer et al., 1996a). The dose of BSO was selected from a published report (Kang and Uthus, 1996) and from preliminary experiments that demonstrated this dose to be maximally effective (data not shown). Any variability in GSH concentrations due to circadian rhythm, as reported by Farooqui and Ahmed et al. (1984), was minimized with the use of matched controls sacrificed at the same time of day as treated animals. Animals treated for multiple days were killed between 1000 and 1100 h either the day of or the day after the final injection. All rats were subjected to euthanasia by CO 2 inhalation, and tissues (liver and one ovary) were excised and snap frozen in liquid nitrogen. The other ovary from each animal was processed for histological evaluation. Tissues were weighed immediately before sample preparation for GSH or lipid oxidation (TBARS) measurements. Ovarian follicle counts. Ovaries from treated and control animals were excised and put into Bouin’s fixative for 4 h. Tissues were dehydrated and embedded in paraffin. Blocks were sectioned at 4 ␮m and the numbers of follicles at each developmental stage were counted in every 40th section, classifying primordial follicles as those with a single layer of squamous granulosa cells, primary follicles as those with a single layer of cuboidal granulosa cells, and secondary follicles as those with 2–3 layers of granulosa cells but no antrum, as previously described (Smith et al., 1990). GSH measurement. The measurement of GSH in ovarian and liver tissue was performed as described by Reed et al. (1980). Tissues were homogenized using a Tissue Tearor (Bio Spec, Inc., Bartlesville, OK) homogenizer in 1 ml of 10% perchloric acid with 1 mM bathophenanthralinedisulfonic acid. Precipitated protein was removed by centrifugation at 16,000 x g for 5 min, and GSH and GSSG were measured in the supernatant after protection of free sulfhydryls with iodoacetic acid and derivitization with fluorodinitrobenzene. Reverse-phase HPLC was performed on these samples using a Beckman System Gold HPLC system with a 25 cm ⫻ 0.4 cm aminopropyl silica column (5 ␮m particle size, Varian) and a methanol/sodium acetate gradient system to separate sample components. Analytes were detected at 365 nm on a UV detector. All peak areas were normalized to an internal standard (␥-glutamateglutamate) that had been added to each sample before derivitization. Measurement of thiobarbituric acid-reactive substances (TBARS). Lipid peroxidation products, which have been reported to be mainly malondialdehyde, were measured as TBARS as previously described (Draper and Hadley, 1990; Esterbauer and Cheeseman, 1990). Briefly, tissues were homogenized in 1% KCl with 0.05% butylated hydroxytoluene, protein was precipitated by addition of 10% trichloroacetic acid, and the supernatant was reacted with 0.67% 2-thiobarbituric acid in boiling water for 30 min. After cooling, samples were extracted with 2ml n-butanol. Fluorescent products were measured in the butanol using a Hitachi fluorescence spectrofluorometer (exc. 515, em. 553). 1,1,3,3-Tetraethoxypropane was hydrolyzed in 1% sulfuric acid (v/v) for 2 h at room temperature and used as a malondialdehyde standard (7.5–250 nM). Statistics. Data were analyzed by one-way ANOVA, and where appropriate, by Fischer PLSD and Scheffe F-test post-hoc tests. Significance was assigned at p ⬍ 0.05. All comparisons were made between treatment and its respective time-matched vehicle control. That is, BSO-treated animals were compared with saline-treated animals, and VCD-treated animals were compared with sesame oil-treated animals. Results represent data from at least 7 animals in each treatment group.

RESULTS

Single Exposure Liver and ovaries in female rats treated with either vehicle (saline-treated or sesame oil-treated) contained similar amounts of GSH or GSSG (p ⬎ 0.05, Figs. 1 and 2). GSH

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(BSO). The result of inhibition of GCS, both in vivo and in vitro, is eventual depletion of GSH as it is exported from the cell or utilized without regeneration. GSH has two potential mechanisms for protecting the cell against electrophilic compounds (DeLeve and Kaplowitz, 1991). First, through the activity of GSH transferases, GSH can become conjugated to electrophilic compounds, detoxifying them and allowing for more rapid excretion. Diethyl maleate is an example of a chemical shown to deplete intracellular GSH through conjugation and excretion (Boyland and Chausseaud, 1968). Second, within a cell, electrophilic compounds that generate reactive oxygen species, such as H 2O 2 and superoxide radicals, can cause oxidative damage to lipids, proteins, and DNA. However, these reactive oxygen intermediates can be directly reduced and neutralized by GSH in a coupled reaction involving GSH peroxidase in which oxidation of the reduced sulfhydryl group forms the oxidized dimer (GSSG) of GSH. Subsequently, the enzyme, GSH reductase, restores GSSG to the reduced form (GSH) with the consumption of NADPH (DeLeve and Kaplowitz, 1991). Menadione is an example of a chemical that depletes intracellular GSH as a result of the production of reactive oxygen species (Doroshow et al., 1990). VCH, VCME, and VCD administered to mice (500 mg/kg, ip) have also been reported to deplete hepatic levels of GSH (Giannarini et al., 1981). Additionally, polar metabolites, which may be products of GSH conjugation with VCD, were found in urine from female B6C3F1 mice and Fischer 344 rats that had been given a single dose of VCD (100 mg/kg, Salyers and Sipes, unpublished). Therefore, there is evidence to support that the effect of VCH and its epoxide metabolites may impact hepatic GSH by both of the possible mechanisms. Thus, the present study was conducted to examine the potential role of GSH depletion in VCD-induced ovotoxicity. The experimental approach was designed to investigate whether VCD dosing causes an initial and/or protracted depletion of GSH concentrations in liver or ovaries following a single dose or repeated daily dosing. Further, it was important to evaluate a direct association of ovarian GSH with VCD-induced follicle loss.

VCD EFFECT ON GSH IN LIVER AND OVARIES

content was reduced in liver to 48% of control levels 2 h after a single dose of VCD (p ⬍ 0.05). However, by 6 h after VCD treatment, the GSH content had returned to control levels (Fig. 1). BSO also significantly reduced hepatic GSH concentrations to 42% within 2 h and 35% at 6 h following a single dose (p ⬍ 0.05, Fig. 1). Ovarian GSH content was not significantly affected by VCD at any time point measured (Fig. 2). Con-

FIG. 3. Effects of repeated dosing with VCD or BSO on hepatic GSH concentrations. Female Fischer 344 rats were dosed daily for 15 days with VCD (0.57 mmol /kg, ip, in sesame oil), BSO (2 mmol/kg, ip, in saline), or vehicle control (sesame oil or saline), and GSH was measured in tissue homogenates by HPLC as described in Methods in samples collected 2 or 26 h after the final dose. Values represent means ⫾ standard error; *p ⬍ 0.05, different from control, n ⫽ 8 –12 animals per group.

versely, ovarian GSH content was decreased to 71% of control values 6 h following a single dose of BSO (p ⬍ 0.05, Fig. 2) but these levels were not different from control by 26 h. No increase in GSSG concentrations was detected in response to VCD or BSO treatment (p ⬎ 0.05, data not shown). Neither ovarian nor hepatic TBARS concentrations were significantly different between control and VCD-treated animals (p ⬎ 0.05, data not shown). Repeated Exposures

FIG. 2. Alterations of GSH concentrations in ovarian tissue following a single dose of VCD or BSO. GSH concentrations were measured as described in Methods in tissue homogenates by HPLC 2, 6, or 26 h after a single dose of VCD (0.57 mmol/kg, ip, in sesame oil), BSO (2 mmol/kg, ip, in saline), or vehicle control (sesame oil or saline) administered to female Fischer 344 rats. Values represent means ⫾ standard error; *p ⬍ 0.05, different from control, n ⫽ 8 –12 animals per group.

The effect of repeated VCD or BSO dosing for 15 days on overall GSH stores was determined in samples taken 2 or 26 h after the final dose. The effect of dosing for 15 days on hepatic and ovarian GSH content was similar to that seen following a single dose. Two h following the last dose, hepatic GSH content was reduced by VCD, as well as BSO (p ⬍ 0.05, Fig. 3). By 26 h following the last dose, hepatic GSH had recovered to control levels in VCD-treated rats (p ⬎ 0.05, Fig. 3), but remained significantly reduced in BSO-treated rats (p ⬍ 0.05, Fig. 3). The reduction in hepatic GSH concentrations was greater (p ⬍ 0.05) 2 h following a single dose as compared with 2 h following 15 daily doses. Ovarian concentrations of GSH were unaffected by VCD treatment, whereas BSO reduced those concentrations to 64% of control at this time point (p ⬍ 0.05, Fig. 4). Other time points were also examined during repeated dosing with VCD, but neither hepatic nor ovarian levels were reduced 26 h following 5 or 10 daily doses (p ⬎ 0.05, Fig. 5). Hepatic GSH content in control sesame oil-treated animals differed between the different days of sampling (Fig. 5). Furthermore, on Day 10, GSH levels in VCD-treated rats were

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FIG. 1. Alterations of GSH concentrations in liver following a single dose of VCD or BSO. GSH levels were measured as described in Methods in tissue homogenates by HPLC 2, 6, or 26 h after a single dose of VCD (0.57 mmol/kg, ip, in sesame oil), BSO (2 mmol/kg, ip, in saline), or vehicle control (sesame oil or saline) administered to female Fischer 344 rats. Values represent means ⫾ standard error; *p ⬍ 0.05, different from control, n ⫽ 8 –12 animals per group.

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greater than in matched controls. However, there was no effect of VCD on other days. The effect of repeated dosing with BSO was not examined earlier than Day 15. GSSG remained low in liver and ovaries at all times, and there were no significant differences among treatments (data not shown). After repeated dosing with VCD, follicle counts were selec-

FIG. 6. The effect of VCD or BSO on small ovarian follicle numbers. Female Fischer 344 rats were dosed daily for 15 days with VCD (0.57 mmol/kg, ip, in sesame oil), BSO (2 mmol/kg, ip, in saline), or vehicle control (sesame oil or saline). Ovaries were collected and fixed 26 h following the last dose. Ovarian follicles were classified and counted in every 40th section (4 ␮m sections) as previously described (Flaws et al., 1994). Values represent means ⫾ standard error; *p ⬍ 0.05, different from control, n ⫽ 8 –12 animals per group.

tively reduced relative to sesame oil-treated controls for both primordial and primary follicles (p ⬍ 0.05, Fig. 6). Numbers of secondary follicles were unaffected (data not shown). BSO treatment for 15 days did not cause reductions in any follicle population when compared to saline-treated controls (Fig. 6), even though total ovarian GSH concentrations had been decreased by this dosing regime. DISCUSSION

FIG. 5. Effects of repeated dosing with VCD at several time points on ovarian and hepatic GSH concentrations. Female Fischer 344 rats were dosed daily for 5, 10, or 15 days with VCD (0.57 mmol/kg, ip, in sesame oil) or vehicle control (sesame oil), and GSH concentrations were measured in tissue homogenates by HPLC as described in Methods in samples collected 26 h following the final dose. Values represent means ⫾ standard error; *p ⬍ 0.05, different from control, n ⫽ 8 –12 animals per group.

These experiments investigated whether dosing of rats with the ovotoxicant, VCD, causes acute (single dose) or protracted (repeated dosing) GSH depletion in liver and ovaries. GSH concentrations decreased rapidly in liver following a single dose of VCD but were restored to control levels within 6 h. Furthermore, there was no persistent depletion of GSH in liver following repeated daily dosing with VCD for 15 days. Whereas temporary reductions in intracellular GSH did occur, VCD did not induce a detectable accumulation of GSSG. Therefore, these results are consistent with utilization of GSH in the liver to form GSH-VCD conjugates for detoxification and excretion of the xenobiotic rather than GSH concentrations being reduced through a direct induction of oxidative stress. Additionally, no reduction in GSH content of whole ovarian tissue by VCD was seen at any time. This appears to rule out a direct role for GSH in mediating ovarian toxicity induced by VCD. This possibility is underscored by the ability of BSO to reduce ovarian GSH concentrations without altering preantral follicle numbers. On the other hand, it must be considered that localized GSH depletion within the target population of small

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FIG. 4. Effects of repeated dosing with VCD or BSO on ovarian GSH concentrations. Female Fischer 344 rats were dosed daily for 15 days with VCD (0.57 mmol /kg, ip, in sesame oil), BSO (2 mmol/kg, ip, in saline), or vehicle control (sesame oil or saline), and GSH levels were measured in tissue homogenates by HPLC as described in Methods in samples collected 2 or 26 h after the final dose. Values represent means ⫾ standard error; *p ⬍ 0.05, different from control, n ⫽ 8 –12 animals per group.

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ACKNOWLEDGMENTS This work was supported by NIH grant ES08979 and Center Grant ES06694. We would like to thank Husam Younis (The University of Arizona) for his technical assistance.

REFERENCES Borman, S. M., VanDePol, B. J., Kao, S. W., Thompson, K. E., Sipes, I. G., and Hoyer, P. B. (1999). A single dose of the ovotoxicant 4-vinylcyclohexene diepoxide is protective in rat primary ovarian follicles. Toxicol. Appl. Pharmacol. 158, 244 –252. Boyland, E., and Chasseaud, L. F. (1968). Enzymes catalyzing conjugations of glutathione with ␣␤-unsaturated carbonyl compounds. Biochem. J. 109, 651– 661. DeLeve, L. D., and Kaplowitz, N. (1991). Glutathione metabolism and its role in hepatotoxicity. Pharmacol. Ther. 52, 287–305. Doroshow, J. H., Akman, S., Chu, F. F., and Esworthy, S. (1990). Role of the glutathione-glutathione peroxidase cycle in the cytotoxicity of the anticancer quinones. Pharmacol. Ther. 47, 359 –370. Draper, H. H., and Hadley, M. (1990). Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186, 421– 431. Esterbauer, H., and Cheeseman, K. H. (1990). Determination of aldehydic lipid peroxidatoin products: Malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186, 407– 421. Farooqui, M. Y., and Ahmed, A. E. (1984). Circadian periodicity of tissue glutathione and its relationship with lipid peroxidation in rats. Life Sci. 34, 2413–2418. Flaws, J. A., Doerr, J. K., Sipes, I. G., and Hoyer, P. B. (1994). Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide. Reprod. Toxicol. 8, 509 –514. Giannarini, C., Citti, L., Gervasi, P. G., and Turchi, G. (1981). Effects of 4-vinylcyclohexene and its main oxirane metabolite on mouse hepatic microsomal enzymes and glutathione levels. Toxicol. Lett. 8, 115–121. Griffith, O. W. (1982). Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J. Biol. Chem. 257, 13704 –13712. Hirshfield, A. N. (1991). Development of follicles in the mammalian ovary. Int. Rev. Cytol. 124, 43–101. Hoyer, P. B., and Sipes, I. G. (1996). Assessment of follicle destruction in chemical-induced ovarian toxicity. Annu. Rev. Pharmacol. Toxicol. 36, 307–331. Hughes, F. M., and Gorospe, W. C. (1991). Biochemical identification of apoptosis (programmed cell death) in granulosa cells: Evidence for a potential mechanism underlying follicular atresia. Endocrinology 129, 2415– 2422. IARC (1976). Cadmium, nickel, some epoxides, miscellaneous industrial chemicals and general considerations on volatile anaesthetics. In IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, Vol. 11, pp. 141–145. International Agency for Research on Cancer, Lyon, France. IARC (1982). 4-Vinylcyclohexene. In IARC Monographs on the Evaluation of Carcinogenic Risks of Chemicals to Humans: Some Industrial Chemicals, Vol. 60, pp. 347–359. International Agency for Research on Cancer, Lyon, France. Jarrell, J. F., Sevcik, M., and Stuart, G. (1992). Regulation of total ovarian glutathione content in the rat. Reprod. Toxicol. 6, 133–135. Kang, Y. J., and Uthus, E. O. (1996). Suppression of plasma estradiol and progesterone concentrations by buthionine sulfoximine in female rats. Biochem. Pharmacol. 51, 567–570.

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ovarian follicles may have been induced by VCD dosing. Because of the heterogeneity of ovarian structures, and the fact that the smallest compartments (preantral follicles) are those selectively targeted by VCD, if such alterations had occurred, they may have been masked by unaltered GSH levels in larger, nonresponsive, ovarian compartments. The kinetics of GSH depletion by either VCD or BSO were similar to experiments performed in mice (dosed once, ip) by Giannarini et al. (1981) and Griffith (1982), respectively. Maximal effects of VCD were expected in the liver after approximately 2 h (500 mg/kg, Giannarini et al., 1981). Approximately 70% of a single dose of VCD is excreted within 6 h of administration in rats (100 mg/kg, Salyers and Sipes, unpublished). Thus, changes in GSH levels would be expected to occur rapidly after dosing. This was confirmed by the reduction in hepatic GSH within 2 h of VCD dosing followed by partial recovery after 6 h. BSO has also been reported to be excreted rapidly, with greater than 85% of an administered dose found in the urine after 24 h (Griffith, 1982). Jarrell et al. (1992) demonstrated maximal depletion (50%) of ovarian GSH 8 h after treatment with 4.5 mmol/kg BSO, while Griffith (1982) reported that 2 mmol/kg caused depletion in liver and kidney 2–10 h after administration. Whereas multiple doses of VCD are required to cause VCDinduced follicle loss (Springer et al., 1996a), cumulative effects on ovarian GSH were not observed with repeated dosing. Thus, the effect of VCD appears to be primarily on hepatic content of GSH. In comparing the effect of a single dose versus repeated dosing, there was less of an impact in liver on Day 15. Relative to the respective controls, GSH concentrations were significantly lower on Days 1 and 15. Furthermore, the decrease was greater (p ⬍ 0.05) on Day 1 (50% of control) than on Day 15 (69% of control) when analyzed either as absolute values or normalized to controls. This finding further points out a lack of association between VCD-induced ovotoxicity and alterations in GSH content. This also suggests that enzymes that synthesize GSH may be induced in the livers of VCDtreated animals after repeated dosing. In summary, the results presented here support the hypothesis that GSH conjugation with VCD in liver provides a pathway for excretion (detoxification) of the ovotoxicant. Additionally, no support for an involvement of GSH in mediating a response to oxidative stress in the liver or ovary was provided. Therefore, it is likely that GSH is not involved in mediating VCD-induced ovotoxicity beyond its role in hepatic metabolism, detoxification, and excretion of the ovotoxic form. Future studies will be aimed at identifying urinary metabolites of GSH-VCD as well as at determining whether GSH plays a compartmentalized, direct role in ovarian toxicity caused by VCD. Such an understanding will increase our awareness of generalized mechanisms of action utilized by chemicals that act as ovarian toxicants.

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Kao, S. W., Sipes, I. G., and Hoyer, P. B. (1999). Early effects of ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats and mice. Reprod. Toxicol. 13, 67–75. Meister, A. (1995). Glutathione metabolism. Methods Enzymol. 251, 3–7. Rappaport, S. M., and Fraser, D. A. (1977). Air sampling and analysis in a rubber vulcanization area. Am. Ind. Hyg. Assoc. J. 38, 205–210. Reed, D. J., Babson, J. R., Beatty, P. W., Brodie, A. E., Ellis, W. W., and Potter, D. W. (1980). High performance liquid chromatography analysis of nanomolar levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106, 55– 62. Smith, B. J., Mattison, D. R., and Sipes, I. G. (1990). The role of epoxidation

in 4-vinylcyclohexene-induced ovarian toxicity. Toxicol. Appl. Pharmacol. 105, 372–381. Springer, L. N., McAsey, M. E., Flaws, J. A., Tilly, J. L., Sipes, I. G., and Hoyer, P. B. (1996a). Involvement of apoptosis in 4-vinylcyclohexene diepoxideinduced ovotoxicity in rats. Toxicol. Appl. Pharmacol. 139, 394 – 401. Springer, L. N., Tilly, J. L., Sipes, I. G., and Hoyer, P. B. (1996b). Enhanced expression of bax in small preantral follicles during 4-vinylcyclohexene diepoxide-induced ovotoxicity in the rat. Toxicol. Appl. Pharmacol. 139, 402– 410. Tilly, J. L., Kowalski, K. I., Johnson, A. L., and Hsueh, A. J. W. (1991). Involvement of apoptosis in ovarian follicular atresia and post-ovulatory regression. Endocrinology 129, 2799 –2801.

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