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Departments of Community and Environmental Medicine,3 Medicine,4 and Developmental and Cell Biology,5 University of California Irvine, Irvine, California 92617
ABSTRACT
The polycyclic aromatic hydrocarbon (PAH) 9,10-dimethyl-1,2-benzanthracene (DMBA) destroys primordial, primary, and secondary ovarian follicles in rodents, but its effects on antral follicles have received limited attention. PAHs are metabolized to reactive species, some of which can undergo redox cycling to generate reactive oxygen species (ROS). We previously showed that ROS initiate apoptosis in preovulatory follicles cultured without gonadotropin support and that glutathione (GSH) depletion induces apoptosis in the presence of gonadotropins. In the present study, we tested the hypothesis that DMBA induces apoptosis in preovulatory follicles, which is mediated by ROS and prevented by GSH. Preovulatory follicles were isolated from ovaries of 25-day-old rats 48 h after the injection of 10 IU of eCG and were cultured with DMBA in the presence of FSH for 2 to 48 h. DMBA induced granulosa cell (GC) and theca cell (TC) apoptosis at 48 h, as judged by TUNEL and activated caspase-3 immunostaining. DMBA treatment also increased the numbers of GCs and TCs that immunostained for the proapoptotic protein BAX. Follicular ROS levels were significantly increased in DMBA-treated follicles at 12, 24, and 48 h. GSH supplementation protected against and GSH depletion enhanced the induction of apoptosis in GCs and TCs by DMBA. These findings suggest that GSH is a critical protective mechanism against DMBA-induced apoptosis in antral follicles and that ROS generation may mediate DMBA-induced GC apoptosis.
apoptosis, atresia, dimethylbenzanthracene, follicle, glutathione, oxidative stress, polycyclic aromatic hydrocarbon, toxicology
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental toxicants that are formed during incomplete combustion and carbonization of organic materials [1]. Humans are exposed to PAHs through work in the production, refining, and use of coal, oil shale, or mineral oil [1, 2] and through ambient air pollution, tobacco smoke, and foods [3]. PAHs are carcinogens and ovarian toxicants. Studies in mice and rats have shown that the PAHs 9,10-dimethyl-1,2-benzanthracene (also called 7,12-dimethylbenz[a]anthracene; DMBA), 3-methylcholanthrene (3-MC), and benzo[a]pyrene (BaP) destroy ovarian follicles [4–7]. Evidence for ovarian toxicity of PAHs in humans comes from epidemiologic studies demonstrating that women who smoke have earlier onset of menopause [8, 9] and decreased fecundity [10, 11] compared with women who do not smoke. Most rodent studies have focused on the destruction of follicles in the earliest stages of development, the primordial and primary follicles. Destruction of primordial follicles can lead to premature ovarian failure, but more acute effects on fertility, such as the decreased per menstrual cycle probability of pregnancy observed in women who smoke [11], are more consistent with destruction of follicles at later stages of development, such as secondary and antral stages. However, the effects of PAHs on larger, growing follicles have received limited attention. Destruction of antral follicles was reported 2–4 days after treatment with DMBA in mice [6]. A dramatic decline in corpora lutea numbers within 1 wk of BaP treatment in mice is also consistent with an effect on antral follicles [12].
PAHs like DMBA undergo extensive metabolism. The parent compounds themselves are not thought to be toxic. The most well-characterized pathway begins with oxidation by cytochromes P450 (primarily CYP1A1 and 1B1) to arene oxides, which can be hydrolyzed by microsomal epoxide hydrolase to dihydrodiols, which can be further oxidized to highly reactive diol epoxides by CYP1A1 and CYP1B1 [13, 14]. The diol epoxides can form DNA adducts and were long thought to be the ultimate carcinogens [15, 16] and ovotoxicants [17]. However, other metabolic products of PAHs may also mediate some of these toxic effects. The dihydrodiols can undergo metabolic activation by didydrodiol dehydrogenases to quinones [18]. The BaP quinones increase reactive oxygen species (ROS) formation in cultured cells, which may result in oxidative DNA damage [18, 19].
The tripeptide glutathione (GSH) is present at millimolar concentrations in cells and is the most abundant intracellular thiol [20]. GSH detoxifies ROS by spontaneous or GSH peroxidase-catalyzed reduction [20]. GSH-S-transferase-catalyzed conjugation of electrophilic toxicants with GSH is an important detoxification mechanism for many exogenous toxicants [20]. Conjugation with GSH is the primary detoxification mechanism for the diol epoxides of PAHs [21–23]. The arene oxide and quinone metabolites of PAHs also undergo GSH conjugation [1, 18]. The ovary has moderately high GSH concentrations compared with other organs [24, 25], and ovulated oocytes have among the highest GSH concentrations of any cell type (9–10 mM) [26]. GSH is necessary for normal oocyte spindle function and formation of the female pronucleus and for normal male pronuclear decondensation [27–29]. We have shown that ovarian GSH synthesis is regulated by gonadotropin modulation of the expression of the catalytic and modulatory subunit of the rate-limiting enzyme in GSH synthesis, glutamate cysteine ligase, in a follicle stage-dependent manner, with high levels of mRNA expression in granulosa cells (GCs) of growing follicles during most of the estrous cycle [30–32]. GSH depletion increased antral follicle atresia in vivo and in vitro [24, 33].
The mechanisms by which PAHs destroy primordial follicles in fetal and neonatal mouse ovaries appear to involve the aryl hydrocarbon receptor (AHR), a member of the Per-Arnt-Sim gene family of transcription factors, and the proapoptotic BCL2 family protein BAX. Destruction of primordial follicles by DMBA appears to be driven by apoptosis of the oocyte, with secondary death of the somatic cells, much like endogenous atresia in primordial follicles [34]. DMBA treatment upregulated BAX protein expression in primordial oocytes, and treatment with an AHR receptor antagonist blocked this destruction [35]. Moreover, ovaries from mice that lacked a functioning Ahr gene or a functioning Bax gene were resistant to DMBA-induced destruction of primordial follicles [35]. However, no studies have investigated the mechanisms by which PAHs destroy larger, growing ovarian follicles in adult rodents. Because the natural process of apoptotic follicular degeneration, which is called atresia, occurs predominantly in antral follicles and is driven by GC apoptosis [34], we hypothesized that DMBA destroys antral follicles by inducing GC apoptosis. Second, because PAH metabolites have been shown to generate ROS [18, 19], we hypothesized that the induction of apoptosis by DMBA is mediated by oxidative stress. Finally, because GSH is important in detoxifying both reactive metabolites of DMBA and ROS, we hypothesized that GSH depletion would potentiate and that GSH supplementation would prevent the induction of apoptosis by DMBA.
All chemicals were purchased from Sigma or Fisher Scientific, unless otherwise noted. Tissue culture reagents were from Invitrogen. The eCG and ovine FSH (NIDDK-oFSH-20 in vivo biological potency of 4453 IU/mg) were obtained from Dr. A.F. Parlow, National Hormone and Peptide Program, NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases). WST-1 reagent, diaminobenzidine (DAB) substrate and buffer, and In Situ Cell Death Detection Kit POD (peroxidase) were from Roche Applied Science.
Sprague-Dawley rats, 22- to 23-day-old females (Crl:CD(SD)IGS BR) weighing 40–51 g, were obtained from Charles River Laboratories. Upon arrival, two to three animals per cage were housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-accredited facility, with free access to deionized water and standard laboratory chow, on a 14L:10D cycle. The experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of California, Irvine.
Preovulatory Follicle Isolation and Culture
The 25-day-old female rats were injected subcutaneously with 10 IU of eCG in 0.1 ml of sterile 0.9% NaCl to promote growth of a cohort of follicles. Forty-eight hours after the eCG treatment, the animals were killed by carbon dioxide asphyxiation for collection of ovaries. Ovaries were placed on ice in serum-free medium pregassed with 95% O2 and 5% CO2. Healthy large antral (preovulatory) follicles (8–12 per ovary) between 600 and 800 µm in diameter were isolated with iris forceps and 27-gauge needles under a stereomicroscope by an aseptic technique, as previously described [33, 36, 37]. To minimize spontaneous apoptosis during follicle preparation, only one ovary was processed at one time, and isolated follicles were kept at 4°C until ready to culture.
Isolated preovulatory follicles (five to six follicles per treatment group per experiment) were cultured in sterile 20-ml glass scintillation vials in 2 ml of serum-free culture medium (MEM) consisting of Eagle Minimal Essential Medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 0.1% fatty acid-free BSA. Each vial was gassed with 95% O2 and 5% CO2, sealed with Vaseline, covered with Parafilm, and then incubated in a shaking water bath at 37°C for 2 to 48 h. Each vial was regassed every 12 h until the time of collection. Depending on the experiment, follicles were cultured with subsets of the following treatments: 1) 75 ng/ml ovine FSH, 2) FSH plus various concentrations of DMBA, 3) FSH plus antioxidant, 4) FSH plus 100 µM buthionine sulfoximine (BSO), 5) FSH+DMBA+BSO, or 6) FSH+DMBA plus antioxidant. BSO is a specific inhibitor of GSH synthesis [38]. The concentration of BSO was chosen to suppress follicular GSH to undetectable levels based on our previous studies in cultured preovulatory follicles [33]. The antioxidants were 5 mM GSH ethyl ester (GEE), 0.5 mM dithiothreitol (DTT), and 0.2 mM butylated hydroxytoluene (BHT). GEE is a cell-permeable analog of GSH [39]. The concentration of GEE was based on our previous study of cultured preovulatory follicles [33]. DTT is a thiol antioxidant. BHT is a phenolic antioxidant. Concentrations of DTT and BHT were chosen on the basis of previously published studies [40–42]. DMBA was dissolved in dimethylsulfoxide (DMSO). Control groups received an equal volume of DMSO alone. The final concentration of DMSO in all groups was 1% (v/v).
At the time of collection, follicles were processed for several assays as follows. For 8- and 24-h GSH and oxidized GSH (GSSG) assays, six antral follicles were immediately homogenized on ice in 54 µl of TES-SB buffer (20 mM Tris, 1 mM EDTA, 250 mM sucrose, 2 mM L-serine, and 20 mM boric acid). A 6-µl aliquot of homogenate was removed for protein assay with a bicinchoninic acid kit (Pierce); then, 12 µl of 5% sulfosalicylic acid (SSA) was added to the remaining homogenate, which was incubated on ice for 15 min and centrifuged at 14 000 x g for 2 min at 4°C; supernatants were then stored at –70°C. Follicles were homogenized in 65 µl of 5% SSA for 48-h GSH assays. For TUNEL and activated caspase-3 and BAX immunostaining, follicles were prefixed in 4% paraformaldehyde in PBS for 30 min at 4°C, cryopreserved in 15% sucrose in PBS for 90 min at 4°C, embedded in Tissue Tek OCT (Sakura Finetek), and stored at –70°C until sectioning at 10-µm thickness with a cryostat. For oxidative stress assessment, follicles were immediately processed for confocal laser scanning microscopy.
Supernatants from homogenized follicles were analyzed for total GSH and GSSG with a microplate enzymatic recycling assay, modified from Griffith [30, 31, 43]. For measurement of GSSG, reduced GSH (rGSH) was first removed from the sample by conjugation with 2-vinylpyridine, followed by chloroform extraction. For both GSH and GSSG assays, triplicates of samples or standards were combined with 33 µl of metal-free water and incubated for 10 min at 30°C. The samples were then mixed with 140 µl of 0.3 mM NADPH, 20 µl of 6 mM DTNB (5,5'-dithiobis(2-nitrobenzoic acid)), and 2 µl of 50 U/ml GSH reductase. The rate of TNB formation from DTNB was proportional to the total GSH concentration in each sample. TNB formation was monitored by measuring the absorbance at 412 nm for 5 min every 10 sec with a Molecular Devices Versamax microplate reader. The concentrations of total GSH or GSSG in the samples were calculated from a standard curve generated from the slopes of the standards. The concentration of rGSH was calculated as [total GSH] – 2 x [GSSG]. The intraassay coefficients of variation (CV) for total GSH ranged from 4.1% to 8.1% for GSH, and the interassay CV was 4.4%. The GSSG samples were all measured in a single assay for which the intraassay CV was 1.8%.
TUNEL Assay for In Situ Detection of Apoptosis
TUNEL was performed with the In Situ Cell Death Detection Kit as described [32, 33]. Briefly, sections were blocked with 3% H2O2 in methanol, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate, blocked with 3% BSA, and incubated with terminal deoxynucleotidyl transferase (TdT) solution with fluorescein-labeled 2'-deoxyuridine 5'-triphosphate. After washing, sections were again blocked with 3% BSA, washed, and incubated with peroxidase converter solution, followed by color development with DAB. Lastly, slides were counterstained with hematoxylin and mounted with Permount. Negative controls were treated with label solution without TdT. Positive controls were pretreated with DNase 1 (1 U/ml) prior to labeling.
For quantification of apoptosis, the numbers of TUNEL-positive GCs or theca cells (TCs) were counted in a blinded manner in five 400x fields per follicle with an Olympus BX60 microscope. The averages of the five GC counts and the five TC counts were calculated for each follicle, and these values were used for statistical analyses.
Immunohistochemistry for Cleaved Caspase-3 and BAX
Immunohistochemistry was performed with a polyclonal cleaved caspase-3 antibody (1:5 dilution, Asp175, no. 9661, Cell Signaling Technology) or a polyclonal BAX antibody (1:150 dilution, 554106, BD Biosciences Pharmingen) and the Vectastain ABC Kit (Vector Laboratories), as previously described [33]. For antigen retrieval, slides were heated with a conventional microwave in 1 mM sodium citrate, and 1 mM citric acid buffer. The slides were sequentially blocked with 3% H2O2 in methanol, blocking serum, and avidin D/biotin blocking solution. The slides were then incubated with primary antibody overnight at 4°C, followed by biotinylated secondary antibody, ABC (avidin-biotin-horseradish peroxidase molecular complex) reagents, and DAB substrate. Finally, the slides were counterstained with hematoxylin and mounted.
The numbers of activated caspase-3 or BAX-positive GCs or TCs per 400x field were counted in five independent fields of the same follicle on two different slides. The averages were used for data analysis and presentation.
ROS were detected in whole cultured follicles by laser scanning confocal microscopy with two different probes as previously described [33]. Briefly, after 2–48 h of culture, follicles were washed with PBS and incubated with 100 µM 2',7'-dichlorofluorescein diacetate (DCF) or 100 µM dihydrorhodamine 123 (DHR; both from Molecular Probes) in MEM medium for 30 min and were washed again. DCF or DHR fluorescence was viewed and quantified with a Zeiss LSM 511 META laser scanning confocal microscope. The objective was set as Plan-Neofluor 10x/0.3, and the reflector was set as GFP (green filter). Excitation filter and tracking were configured to a single track of cy2/alexa488 fluorescein isothiocyanate, and emission filter was configured to BP-500–530. For scanning parameters, frame size of 512 x 512, scan speed of 9, and scan average of 4 were set. The Z Stack option was chosen to detect DCF or DHR fluorescence intensity in 15 evenly spaced, 10- to 25-µm-thick layers of each follicle. The mean DCF or DHR fluorescence intensity of each follicle was obtained with the Histo option, wherein the DCF or DHR intensities from all 15 layers were calculated, pooled, and averaged.
Data from two or three replicate experiments were pooled for statistical analyses. Homogeneity of variances was assessed by the Levene test. When variances were not homogeneous, a natural logarithm transformation was applied. The differences among groups were analyzed by ANOVA followed by Fisher Least Significant Difference (LSD) test. When variances remained nonhomogeneous after data transformation, the Kruskal-Wallis test for nonparametric data was used. If the Kruskal-Wallis test was significant at P < 0.05, the Mann-Whitney test was then used for intergroup comparisons. Statistical analyses were carried out by SPSS 11.0 for Mac OS X.
DMBA-Induced Apoptosis in Cultured Preovulatory Follicles
To investigate the mechanism by which DMBA destroys antral follicles, we utilized a whole follicle culture system. Previous studies have shown that preovulatory follicles spontaneously undergo apoptosis within 24 h when cultured in the absence of gonadotropin support [44]. FSH treatment inhibits GC apoptosis in these cultured follicles [44, 45], as does gonadotropin treatment in vivo [36]. To test whether DMBA induces apoptosis in large follicles, we cultured preovulatory follicles with 75 ng/ml FSH and 0, 0.1, 1, 10, or 100 µM DMBA for 12, 24, or 48 h. DMBA treatment significantly increased the numbers of TUNEL-positive, apoptotic GCs (Fig. 1, A and B) and TCs (Fig. 1, A and C) at 48 h but not at 12 or 24 h (P < 0.001 and P = 0.001, effect of DMBA concentration by the Kruskal-Wallis test at 48 h on GC and TC apoptosis, respectively). Activated caspase-3 immunostaining paralleled the results obtained by TUNEL (Fig. 1, D and E). DMBA treatment for 48 h statistically significantly increased the numbers of activated caspase-3-positive GCs and TCs (P = 0.002 and P = 0.001, effect of DMBA concentration by the Kruskal-Wallis test on GC and TCs, respectively). DMBA treatment also caused statistically significant increases in the numbers of BAX-immunopositive GCs (P < 0.001, effect of concentration by two-way ANOVA) and TCs (P < 0.001, effect of concentration) (Fig. 1, D, F, and G). One-way ANOVAs for each time point separately showed that BAX immunostaining in GCs was significantly increased above 0 µM control levels in all DMBA-treated groups at 24 h and in 1, 10, and 100 µM groups at 48 h. TC BAX immunostaining was significantly increased above 0 µM control levels in 1, 10, and 100 µM groups at 24 h.
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DMBA Did Not Alter Follicular GSH Concentrations
Conjugation with GSH is an important phase 2 biotransformation pathway for diol epoxide and other metabolites of DMBA [1, 21–23]. Reduced GSH also detoxifies ROS that may be produced as a result of PAH exposure; this results in oxidation of GSH. To determine whether treatment with DMBA depletes follicular GSH concentrations or alters the ratio of reduced GSH (rGSH) to GSSG, we measured follicular levels of total GSH and GSSG and calculated the concentration of rGSH after 8, 24, and 48 h of treatment. For the 48-h experiments, follicles were homogenized directly in 5% SSA, which precludes measuring protein concentrations in the samples. For the 8- and 24-h experiments, follicles were first homogenized in TES-SB buffer so that an aliquot of sample could be removed for protein assay prior to acidification. The results of the analyses did not differ appreciably whether GSH concentrations were expressed in nanomoles per follicle or per milligram of protein. Treatment with DMBA did not significantly alter concentrations of total GSH per follicle (Fig. 2A). DMBA treatment also did not significantly affect the rGSH or GSSG concentrations (Fig. 2, B and C). As we have previously reported [33], follicular GSH concentrations increased significantly with duration of treatment with FSH (P < 0.001, effect of time in culture for total GSH, rGSH, and GSSG; Fig. 2, A–C).
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DMBA Increased ROS in Cultured Preovulatory Follicles
To test whether ROS are generated upon culture of preovulatory follicles with DMBA, we utilized two probes, 2',7'-DCF and DHR 123, which are oxidized to fluorescent compounds in the presence of ROS [46, 47].
Preovulatory follicles were cultured with 0, 0.5, 1, and 10 µM DMBA in the presence of 75 ng/ml FSH. Additional groups of follicles were cultured without FSH in MEM medium alone as positive controls for the generation of ROS [33]. As expected, both DCF and DHR fluorescence increased markedly by 12 h of culture in MEM and remained elevated above 0-h levels thereafter (P < 0.001 and P = 0.004 effect of time for DCF [Fig. 3A] and DHR [Fig. 3B], respectively, by the Kruskal-Wallis test). To assess the effects of DMBA concentration and treatment duration on ROS generation, two-way ANOVAs were performed, excluding the positive control MEM group. There were statistically significant effects of both DMBA concentration (P = 0.004) and treatment duration (P < 0.001) on DCF fluorescence intensity, with greater concentrations of DMBA leading to greater DCF fluorescence (Fig. 3A). DCF fluorescence was significantly increased above 0-h control levels by 12 h in the 1 and 10 µM groups and by 24 h in the 0.5 µM group. The trends were similar for DHR fluorescence, which increased with both DMBA concentration and treatment duration (Fig. 3B). The effects of treatment duration (P < 0.001) and DMBA concentration x duration interaction (P = 0.037) were statistically significant, and the effect of DMBA concentration alone approached significance (P = 0.072). Because the DHR data were nonhomogeneous, even after transformation, the Kruskal-Wallis tests were also performed. The effect of treatment duration was statistically significant for all concentrations of DMBA (P < 0.05) but not for the 0 µM group cultured with FSH alone (P = 0.212), again consistent with increased ROS generation upon culture with DMBA. DHR fluorescence was significantly increased above 0-h levels in the 1 and 10 µM groups by 24 h (P < 0.01 by the Mann-Whitney test).
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Supplementation of GSH Protects Against DMBA-Induced Apoptosis
To test whether the increase in ROS mediates DMBA-induced apoptosis, we cotreated preovulatory follicles with 75 ng/ml FSH and 10 µM DMBA in the presence or absence of one of three antioxidants, GEE, DTT, or BHT. Follicular apoptosis was assessed by TUNEL and activated caspase-3 immunostaining.
Treatment with 10 µM DMBA for 48 h induced significant increases in GC and TC apoptosis as assessed by TUNEL (P = 0.002 and P < 0.001, effect of treatment on GC and TC apoptosis, respectively; Fig. 4A). The increased GC and TC apoptosis was completely prevented by cotreatment with GEE (P < 0.01, FSH+DMBA+GEE versus FSH+DMBA by LSD test). The effects on GCs were similar when apoptosis was assessed by activated caspase-3 immunostaining (P = 0.001, effect of treatment on GC apoptosis by Kruskal-Wallis test; Fig. 4B). Treatment with DMBA increased GC apoptosis by 2-fold compared with FSH alone, although the intergroup comparison was not statistically significant. Cotreatment with GEE completely prevented the increase in activated caspase-3 immunostaining. Cotreatment with two other antioxidants, DTT and BHT, did not protect against DMBA-induced GC and TC apoptosis. In fact, these agents induced significant GC and TC apoptosis, even in the absence of DMBA, compared with FSH alone (Fig. 4, A and B).
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GSH Depletion Potentiates the Proapoptotic Effect of DMBA in Cultured Preovulatory Follicles
Because GSH conjugation is an important detoxification mechanism for metabolites of DMBA and for ROS, we hypothesized that GSH depletion would sensitize follicles to the induction of apoptosis by DMBA. To test this hypothesis, we cultured follicles with 75 ng/ml FSH with or without BSO, a specific inhibitor of GSH synthesis, for 8 h to allow follicular GSH concentrations to fall prior to adding DMBA for an additional 40 h of culture [33]. We chose a concentration of DMBA, 0.5 µM, which was not expected to induce GC apoptosis alone based on the data presented in Figure 1.
Follicular GSH was suppressed to undetectable levels in follicles treated with FSH+BSO or with FSH+BSO+DMBA for 48 h (from 2.0 ± 0.2 nmol/follicle in FSH-treated and 1.9 ± 0.1 nmol/follicle in FSH+DMBA-treated to less than 0.04 nmol/follicle in FSH+BSO and FSH+DMBA+BSO groups). The number of TUNEL-positive, apoptotic GCs varied with treatment in a statistically significant manner (P = 0.002, Kruskal-Wallis test; Fig. 5A). The numbers of TUNEL-positive GCs were significantly greater in the FSH+BSO and FSH+BSO+DMBA groups than in the group cultured with FSH alone (P = 0.023 and P < 0.001, respectively, by the Mann-Whitney test). Combined treatment with BSO and DMBA caused greater GC apoptosis than either treatment alone (P = 0.010 and P = 0.376, FSH+BSO+DMBA versus FSH+DMBA and versus FSH+BSO, respectively). There was no effect of treatment on TC apoptosis (P = 0.391). Similar effects on GCs were observed when activated caspase-3 immunostaining was used as an indicator of apoptosis (Fig. 5B). The number of activated caspase-3-positive GCs varied with treatment in a statistically significant manner (P = 0.004 by the Kruskal-Wallis test). Combined treatment with BSO and DMBA again caused greater GC apoptosis than either treatment alone (P = 0.016 and P = 0.286, FSH+BSO+DMBA versus FSH+DMBA and versus FSH+BSO, respectively). In addition, combined treatment with BSO and DMBA significantly increased the number of caspase-3-positive TCs compared with either treatment alone (P = 0.002, overall effect of treatment; P = 0.016 and P = 0.016, FSH+BSO+DMBA versus FSH+DMBA and versus FSH+BSO, respectively).
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PAHs are ubiquitous environmental toxicants that destroy ovarian follicles in rodents [4–7] and in explanted human ovarian tissue [35]. Although most attention has focused on the destruction of primordial follicles by PAHs, there is evidence that these compounds are also capable of destroying secondary and antral follicles [4, 12]. We used a well-characterized model system in which antral follicles cultured without gonadotropin support spontaneously undergo apoptosis, and gonadotropin treatment prevents apoptosis, to study the effects of DMBA on apoptosis in large antral follicles. We showed that DMBA induces GC and TC apoptosis and that the onset of apoptosis is preceded by an increase in ROS. We further showed that DMBA-induced apoptosis is associated with increased numbers of BAX and activated caspase-3 immunopositive GCs and TCs. Treatment with the cell-permeable GSH analog GEE prevented the induction of apoptosis by DMBA. Depletion of GSH with BSO enhanced the apoptotic effect of DMBA on apoptosis in cultured follicles. These latter two findings demonstrate the importance of GSH in protecting against DMBA-induced follicular toxicity.
The mechanisms by which PAHs induce apoptosis have been investigated in several systems. Treatment of immature mice with DMBA or of cultured neonatal mouse ovaries with the dihydrodiol metabolite of DMBA led to increased expression of BAX protein in oocytes of primordial follicles via increased transcription of Bax and subsequent destruction of primordial follicles [35]. This upregulation of BAX and destruction of primordial follicles was inhibited by AHR receptor antagonists and did not occur in Ahr-deficient or Bax-deficient mice [35]. Moreover, evidence was presented that functional arylhydrocarbon response elements in the Bax promoter were required for the upregulation of Bax transcription in oocytes [35]. Bax and Ahr also appear to be required for the induction of apoptosis by PAHs in murine embryos [48]. In the present study, we also observed increasing BAX protein expression with DMBA treatment in GCs and TCs at 24 h, prior to the increase in TUNEL-positive cells (Fig. 1). Moreover, our observations that DMBA treatment causes ROS generation prior to the onset of apoptosis (Figs. 1 and 3) suggest a mechanism for BAX activation by DMBA in antral follicles. Oxidative stress has recently been shown to induce BAX translocation to the mitochondria, resulting in cytochrome c release [49]. Moreover, the PAH BaP induced BAX translocation to the mitochondria without increasing overall levels of BAX protein in Hepa1c1c7 cells [50].
The present study also supports a role for caspase-3 in mediating DMBA-induced follicular apoptosis, as numbers of activated caspase-3-positive GCs and TCs increased in concentration-dependent manner with DMBA treatment (Fig. 1). Activation of caspase-3 has been shown to be necessary for the induction of apoptosis by DMBA in bone marrow B cells [51]. Caspase-3 activation has also been implicated in the induction of apoptosis in Hepa1c1c7 cells by BaP [50]. All the apoptotic markers we assessed increased with a similar time course in GCs and TCs, suggesting that apoptosis of both cell types in concert drives the induction of antral follicle atresia by DMBA. It is also possible that apoptosis of one or the other cell type occurs first, between the 12- and 24-h time points that we assessed. Our study was not designed to investigate the effects of DMBA on the oocyte; however, we did not observe oocyte TUNEL or activated caspase-3 staining with DMBA treatment (data not shown). DMBA treatment also did not appear to increase BAX immunostaining in oocytes, with the exception of two oocytes in the 100 µM group at 48 h (data not shown). The endogenous process of antral follicle atresia is thought to be driven by GC apoptosis in vivo [34]. An early study of the effects of PAHs on follicular atresia in vivo described the destruction of somatic cells of large follicles occurring by 4 days after DMBA treatment, but no mention was made of whether GC or TC demise was more pronounced or had occurred earlier [6]. We are not aware of any other studies that examined the effects of PAHs on GCs and TCs of antral follicles.
PAHs require metabolic activation to reactive metabolites to exert their toxicity [18]. Therefore, our observation that DMBA induced apoptosis in cultured preovulatory follicles shows that these follicles are capable of metabolizing DMBA. This is consistent with previous studies, which showed that the major enzymes involved in PAH metabolism, CYP1A1, CYP1B1, microsomal epoxide hydrolase, and dihydrodiol dehydrogenase, are all expressed in rodent ovaries [52–56] and that cultured GCs obtained from preovulatory rat follicles are capable of metabolizing DMBA and BaP [57, 58]. Metabolism of PAHs begins with oxidation to arene oxides, which can be hydrolyzed to dihydrodiols. The dihydrodiols can be further oxidized to diol epoxides [13, 14]. A recent study showed that concentrations of another PAH, BaP, in culture medium decreased by about 30% within the first 30 min of being placed in polystyrene tissue culture plates due to binding to the plastic; however, a similar decline was not observed in polypropylene tubes [59]. Our follicles were cultured in glass scintillation vials, and binding of DMBA to the vials was likely low. The study by Kushman et al. [59] also showed that BaP was metabolized in the presence of cells expressing CYP1A1. From 30 min to 48 h, the amount of BaP per plate declined by 98% in plates with the CYP1A1-expressing cell line, while the concentration of the diol epoxide metabolite of BaP peaked at 24 h and then declined. In contrast, BaP declined by only 65% in the plates with the non-CYP1A1-expressing cells, and diol epoxide levels remained very low throughout the culture period. A similar decline in DMBA and increase in its metabolites likely occurred during the course of the culture period in our experiment.
The diol epoxide metabolites of PAHs are highly reactive and rapidly form DNA adducts [18]. A diol epoxide metabolite of BaP had a greater than 10-fold lower median effective dose for primordial and primary follicle destruction than BaP or than the parent dihydrodiol metabolite after intraovarian injection, and it was concluded that diol epoxides were therefore the ultimate ovotoxicants [17]. The dihydrodiol metabolites of PAHs can alternatively be oxidized by members of the aldo-keto reductase superfamily, the dihydrodiol dehydrogenases, to ketols, which tautomerize to catechols. The catechols undergo oxidation first to an o-semi-quinone anion radical and hydrogen peroxide and then to an o-quinone and superoxide anion [18]. Redox cycling can occur when the o-quinone undergoes either one or two electron reductions back to o-semiquinone or to catechol, respectively [18]. Treatment of cultured cells with BaP quinones has been shown to induce ROS formation [19], and oxidative DNA damage has been reported upon in vitro exposure of DNA to PAH quinones [18, 60]. DMBA treatment in vivo caused accumulation of lipid peroxidation products in intestinal mucosa [61] and in liver and erythrocytes [62]. Our observation of increased generation of ROS in preovulatory follicles cultured with DMBA prior to the onset of apoptosis and rescue of the follicles from DMBA-induced apoptosis by GSH supplementation with GEE suggests that ROS mediate DMBA-induced apoptosis in these follicles. The lack of a protective effect of two other antioxidants, DTT and BHT, is not necessarily inconsistent with this conclusion. BHT and DTT react with ROS and protect against induction of apoptosis in some cell systems [41, 63] but exert proapoptotic and prooxidant effects in other systems [64, 65]. Our results show that both of these agents are proapoptotic in preovulatory follicles.
In vivo administration of DMBA depressed GSH concentrations in intestinal mucosa, liver, and blood [61, 62]. Treatment of peripheral blood lymphocytes for 6 h with BaP 7,8-diol-9,10-epoxide, BaP 4,5-epoxide, and 7-OH-BaP, but not with BaP, DMBA, or BaP dihydrodiol, caused a decline in intracellular GSH concentrations [66]. We observed no decline in total GSH concentrations or any statistically significant effect on rGSH or GSSG concentrations in cultured preovulatory follicles treated with DMBA in the present study (Fig. 2), despite evidence that DMBA is metabolized in these cultured follicles. Our observations that enhancement of intracellular GSH concentrations with GEE prevented DMBA-induced GC and TC apoptosis (Fig. 4) and that depletion of GSH enhanced DMBA-induced apoptosis (Fig. 5) show that variations in intracellular GSH synthesis modify susceptibility to PAH-induced follicular toxicity. This is relevant to humans because polymorphisms have been identified in the promoters of both subunits of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis that affects basal and/or inducible GSH synthesis [67–69].
The present study shows that DMBA induces apoptosis in GCs and TCs of cultured preovulatory follicles. The data suggest that DMBA treatment results in the generation of ROS, which leads to increased levels of the proapoptotic protein BAX, caspase-3 activation, and ultimately cell death. The observations that 1) the depletion of the antioxidant GSH potentiates the proapoptotic effect of DMBA and 2) GSH supplementation abolishes the proapoptotic effect of DMBA in cultured preovulatory follicles further support the hypothesis that ROS mediate DMBA-induced apoptosis. The results also demonstrate the important role of GSH in protecting follicles from toxicant-induced apoptosis.
ACKNOWLEDGMENTS
We thank Dr. Zifu Wang, Manager of the University of California Irvine Microscopy and Image Analysis Facility, for his expert assistance with the confocal microscopy.
FOOTNOTES
1Supported by NIH grant K08 ES10963 (to U.L), the University of California Irvine Developmental Biology Center and Chao Family Comprehensive Cancer Center Optical Biology Shared Resource (NIH Cancer Center Support grant P50 CA62203), and the Center for Occupational and Environmental Health, University of California, Irvine. 
Correspondence: 2Ulrike Luderer, Center for Occupational and Environmental Health, 5201 California Ave., Suite 100, Irvine, CA 92617. FAX: 949 824 2345; e-mail: uluderer{at}uci.edu
Received: 25 January 2007.
First decision: 15 February 2007.
Accepted: 30 May 2007.
REFERENCES
-glutamylcysteinylglycyl ester into liver and kidney Proc Natl Acad Sci U S A 1983 805258–5260This article has been cited by other articles:
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, significantly different from FSH+DMBA by the LSD test at P < 0.05. N = 3–8 follicles/group. B) Mean + SEM number of activated caspase-3-positive GCs or TCs per x400 field. There was a statistically significant effect of treatment on activated caspase-3 in GCs by the Kruskal-Wallis test (P = 0.001). *, Significantly different from FSH by the Mann-Whitney test at P < 0.05. N = 3–7 follicles/group.
, significantly different from FSH+BSO by the Mann-Whitney test at P < 0.05.