* University of Arizona, Department of Pharmacology and Toxicology, Tucson, Arizona 85721;
Northern Arizona University, Department of Biological Sciences, Flagstaff, Arizona 86011; and
University of Arizona, Department of Physiology, Tucson, Arizona 85724
Received December 3, 2001; accepted February 28, 2002
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ABSTRACT |
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Key Words: microsomal epoxide hydrolase; 4-vinylcyclohexene; ovary; ovarian follicles; mouse; confocal microscopy.
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INTRODUCTION |
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Many xenobiotic compounds that are or can form reactive epoxide metabolites are detoxified by mEH, including the epoxides of 4-vinylcyclohexene (Fig. 1). 4-Vinylcyclohexene (VCH), an industrial chemical used in the manufacture of flame retardants, insecticides, and plasticizers, causes ovarian toxicity in mice. Bioactivation of VCH by the cytochrome P450 enzymes to the diepoxide metabolite (VCD) is required to produce the ovary-specific toxic effects, which involves targeting and destruction of immature, small preantral follicles (Doerr et al., 1995
; Smith et al., 1990b
). The resulting ovotoxicity is relevant because the ovary contains a finite number of follicles which, once depleted, cannot be regenerated. Thus, widespread destruction of preantral ovarian follicles can lead to premature ovarian failure. Additionally, ovarian failure is associated with an increased incidence of ovarian neoplasms (Flaws et al., 1994a
; NTP, 1989
).
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Currently, it is not known whether ovarian mEH is involved in VCD metabolism in the mouse, although it has been proposed as a detoxification pathway in rat in vitro studies (Flaws et al., 1994b). Additionally, it is not known if mEH is compartmentalized within different sizes of ovarian follicles representing different stages of development. Because VCD specifically destroys small preantral (primordial and primary) follicles, ovarian distribution of mEH may influence either protection or destruction of specific follicle populations. Thus, the purposes of our studies were to (1) determine expression of mRNA encoding for mEH in follicles isolated from mouse ovaries, (2) determine catalytic activity of mEH in follicles isolated from mouse ovaries, (3) identify ovarian distribution of mEH protein, and (4) determine the effects of in vivo dosing with VCH/VCD on mEH expression, activity, and protein distribution.
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MATERIALS AND METHODS |
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Animals.
Day 21 (d 21) female B6C3F1 mice from Harlan Laboratories (Indianapolis, IN) were maintained in the University of Arizona Animal Care Facility for 1 week prior to study. Animals were housed in plastic cages, five animals per cage, and maintained on 12-h light/12-h dark cycles in a controlled temperature of 22 ± 2°C. The animals were provided a standard diet with ad libitum access to food and water. All animal experiments were approved by the University of Arizona's Institutional Animal Care and Use Committee.
Animal dosing.
Day 28 (d 28) female mice (810 animals/treatment group) were weighed and administered (ip) 1 single dose or 15 daily, consecutive doses (2.5 µl/g of body weight; 3050 µl/dose) of either sesame oil (vehicle) or sesame oil containing VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day). The equitoxic doses, routes of administration, and dosing time courses were based on previous studies performed in our laboratory (Borman et al., 1999; Smith et al., 1990b
). Animals were killed by CO2 inhalation 4 h following the final dose.
Follicle isolation.
Ovaries were removed and the oviduct and excess fat were trimmed away. Ovaries were minced and gently dissociated (40°C, 20 min) in Medium 199 (containing Hank's salts, L-glutamine, 25 mM HEPES) with collagenase (7.5 mg/ml), DNase (0.267 mg/ml), and BSA (40 mg/ml). The resulting suspension was filtered through a 250 µm screen via vacuum suction to exclude antral follicles (> 250 µm; F3). The filtered follicles containing small (25100 µm; fraction 1; F1) and large (100250 µm; fraction 2; F2) preantral follicles and interstitial cells (Int) were further hand sorted using calibrated Pasteur pipettes into distinct populations. Tissue was stored (80°C) until further use. Because F1 and F2 follicles are hand sorted during the procedure, these follicular fractions are relatively pure. The F3 follicles and Int cells are less pure since these fractions may be contaminated by clumps of tissue that are not completely dissociated (F3) or by follicles that are extensively dissociated resulting in the loss of individual granulosa and theca cells (Int). In both instances, the percent contamination is small and should not account for statistically significant differences between ovarian fractions.
RNA isolation.
Total RNA was extracted from isolated ovarian fractions utilizing the RNAqueous® kit protocol. Briefly, the samples were lysed and homogenized. The resulting mixture was applied to a filter cartridge, allowing the RNA to bind to the filter. After centrifugation, RNA was eluted from the filter and its concentration was determined via UV spectrometry ( = 260/280 nm; Beckman DU-64).
First strand cDNA synthesis.
Total RNA (0.75 µg) was reverse transcribed into cDNA utilizing the Reverse Transcription System®. Following reverse transcription utilizing random primers, the resulting cDNA was precipitated (ethanol, 80°C overnight). The excess supernatant was removed and the pellet was resuspended in PCR-grade water (100 µl).
Real-time polymerase chain reaction.
cDNA (1 µl) from various ovarian fractions was used to perform relative, semiquantitative PCR utilizing a LightCycler® (Idaho Technology) capable of real-time PCR. The LightCycler® quantifies the amount of PCR product generated by measuring the dye (SYBR green), which fluoresces when bound to double stranded DNA. A standard curve was generated from 1:5 serial dilutions of purified PCR product (mEH or 18S rRNA). Custom-designed primers for mEH were utilized (forward primer: 5` GGG TCA AAG CCA TCA GCC A 3`; reverse primer: 5` CCT CCA GAA GGA CAC CAC TTT; NCBI, 1997).
Amplification conditions were 95°C/0 s (denaturing), 65°C/0 s (annealing), and 72°C/6 s (extending) for 50 cycles.
Arbitrary numbers were assigned to each standard. Values were calculated for the experimental samples from the standard curve. 18S rRNA was measured in each sample as an internal standard. Final values for mEH expression were calculated as a ratio of mEH:18S rRNA.
Protein determination.
Total protein was extracted from isolated ovarian fractions. Briefly, the tissue was lysed (buffer containing Triton-X, HEPES, NaCl, glycerol, SDS, EDTA, NaF, PMSF, leupeptin, and aprotinin) and homogenized. Samples were incubated on ice for 30 min and centrifuged (16,000 x g; 10 min). The resulting supernatant was collected and the protein concentration was determined utilizing the BCA Protein Assay® kit ( = 570 nm).
mEH activity assay.
Protein (25 µg) from isolated ovarian fractions (F1, F2, F3, Int) was incubated (37°C, shaking water bath) in Tris buffer (100 mM, pH 9.0) containing 1 µl [3H]-cis-stilbene oxide (CSO) for 2 h. A 5 mM stock solution of substrate ([3H]-CSO) in ethanol was utilized. The [3H]-CSO was over 99% pure (purified by preparative TLC) and the specific activity was 1 mCi/mmol. The [3H]-CSO added to the reaction was prediluted (1:2) in nonradiolabeled CSO (5 mM in ethanol) to yield approximately 15,000 cpm/1 µl. The final substrate concentration was 12.5 µM. The final volume of the in vitro reaction was 200 µl. Ethanol made up 0.5% of the total reaction volume. Following incubation, the reaction was stopped with the addition of 200 µl 2,2,4-trimethylpentane. The samples were vortexed and centrifuged, resulting in a phase separation. By this approach, unreacted CSO partitions into the organic phase, while reacted CSO product (diol) partitions into the aqueous phase. An aliquot (150 µl) of the aqueous phase was removed and the reaction product (diol) was counted by standard scintillation counting. Additionally, experiments were performed to ensure linearity of the reaction in terms of incubation time (30 min3 h) and protein concentration (1050 µg). Optimal conditions for CSO metabolism in ovarian fractions utilized 25 µg of protein incubated for 2 h. After the incubation, the average percent of the substrate consumed was 4%.
Confocal microscopy.
Four h following the standard dosing regimen, animals (two per group) were euthanized, ovaries removed, and oviduct and excess fat trimmed away. Ovaries were fixed for 4 h in 10% buffered formalin, dehydrated, and embedded in paraffin. Every seventh section (5 µm thick) throughout the ovary was prepared and deparaffinized (approximately 24 sections/group). Sections were incubated with primary antibody against mEH (goat antirabbit; 1:50 dilution) at 4°C overnight. Secondary biotinylated antibody (horse antigoat; 1:75 dilution) was applied for 1 h, followed by Cy-5-streptavidin (1 h; 1:50 dilution). Sections were treated with Ribonuclease A (100 µg/ml) for 1 h, followed by staining with YOYO-1 (10 min; 5 nM). Slides were repeatedly rinsed with phosphate buffered saline (PBS), coverslipped, and stored in the dark (4°C) until visualization. Immunofluorescence was visualized on a Leica confocal microscope with a xenon light source and the intensity was determined via an argon-krypton laser projected through the tissue into a photomultiplier tube at = 488 and 647 nm for YOYO-1 (green) and Cy-5 (red), respectively. Relative densitometric analysis using Scion Image Software (National Institutes of Health, Bethesda, MD) was utilized to compare relative intensities of staining. Background staining intensity was subtracted from each field. Multiple readings were taken throughout the sections. Analysis was performed at controlled settings on the confocal microscope in which 3035 primordial follicles, 1020 small primary follicles, 510 large primary follicles, 25 antral follicles, and 812 interstitial compartments were evaluated per group. Because the intensity of staining was very high for the interstitial cells at the settings in which the follicles were evaluated, these settings were changed to allow more accurate assessment of staining intensity in this ovarian fraction. However, all of the analyses for the interstitial cells were also compared at the same settings. Samples were normalized to control so multiple experiments could be compared.
Statistical analyses.
Comparisons were made using one-way ANOVA. When significant differences were detected, individual groups were compared with the Fisher protected least significant difference (PLSD) multiple range test. The assigned level of significance for all tests was p < 0.05.
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RESULTS |
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DISCUSSION |
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Repeated in vivo dosing with VCH or VCD modulated expression of ovarian mEH. A significant increase in expression of mRNA encoding mEH was observed in targeted F1 follicles, as well as nontargeted F2 follicles after VCH or VCD treatment compared to vehicle-treated animals. Furthermore, the effects of VCH treatment caused a significantly greater increase in mRNA encoding mEH compared to VCD in these same follicle populations. These differences in response to VCH/VCD dosing may be explained by (1) the higher dose of VCH (7.4 mmol/kg) compared to VCD (0.57 mmol/kg), resulting in a higher concentration of metabolites being formed and/or (2) the presence of the VCH monoepoxides (vinylcyclohexene 1,2-monoepoxide or vinylcyclohexene 7,8-monoepoxide), which are also substrates for mEH and can be metabolized to nontoxic diols (vinylcyclohexene 1,2-diol or vinylcyclohexene 7,8-diol).
mEH activity in F1 follicles was also induced following VCH/VCD dosing, thus correlating with the mRNA data. A correlation between mEH mRNA levels and protein has been previously noted (Cho and Kim, 1997, 2000
; Kim et al., 1998
). The correlation between mRNA and specific activity was not seen in F2 follicles. However, induction in F2 follicles may be unimportant because of the already high levels of enzyme activity in this follicle population, relative to the other ovarian fractions. Regardless of mechanism, it appears that ovarian preantral follicles have acquired a protective response via mEH induction. However, after 12 days of repeated daily dosing, extensive follicle loss is evident in mice (Kao et al., 1999
). Therefore, the detoxification system likely becomes overwhelmed, and vulnerability of this follicle population (F1) compared to the larger, more mature follicles may in part be the reason for their greater susceptibility to the ovotoxic effects of VCD.
After 15 days of dosing, only primordial and primary follicles (contained in F1) are impacted by VCH/VCD dosing (d1-d15; Kao et al., 1999; Springer et al., 1996
). Dosing with VCH/VCD does not directly affect the population of follicles contained in F2 (large growing, preantral). However, following 30 days of dosing, there is a reduction in the number of large growing follicles (Flaws et al., 1994a
). This has been interpreted to reflect the effect of a reduced population of primordial and primary follicles from which to recruit growing follicles. Likewise, ultimately F3 follicles will also be impacted as ovarian failure occurs (Hooser et al., 1994
; Mayer et al., unpublished data). Although not directly targeted by VCH/VCD, other sizes of ovarian follicles, as well as the interstitial compartment, may be involved in the detoxification of VCH-monoepoxides and/or VCD, thus protecting the vulnerable F1 follicle population from exposure to and destruction by VCD. For instance, F2 follicles from vehicle-treated animals have a significantly greater amount of mEH activity compared to other fractions. Additionally, there was a nonsignificant trend for mEH activity to be induced in F3 follicles and interstitial cells following repeated exposure to VCH/VCD. Although, as mentioned previously, any defense provided by these nontarget populations is not sufficient to protect the ovary and prevent follicle destruction, as follicle loss was first seen in mice on day 12 of dosing (Kao et al., 1999
).
Interestingly, expression of mEH protein was upregulated in theca cells of growing, preantral follicles (F2), as observed and quantified by confocal microscopy. Theca cells are highly vascularized, surround the follicle, and may serve as a protective cell barrier between blood born toxic metabolites and the vulnerable oocyte. In contrast, primordial follicles (F1) do not have a developed theca cell layer. Thus, this extra barrier of protection is not in place, and may be another factor contributing to the specificity of targeting in this population of follicles. It is thought that a subpopulation of interstitial cells is derived from theca cells of follicles undergoing atresia, a natural form of cell death within the ovary (Erickson et al., 1985). Thus, retaining detoxification capabilities as theca cells become interstitial cells would serve as a constant protective mechanism for the ovary. The apparently large metabolic potential of the interstitial cells suggests a possible physiologic and toxicologic role of this ovarian cell type.
Although the liver is the primary organ of xenobiotic metabolism, extrahepatic tissues, including the ovary, may also be important tissue specific sites of bioactivation and detoxification. For example, Shiromizu and Mattison (1984) injected benzo(a)pyrene, B(a)P, directly into the ovary to evaluate the role of ovarian metabolism on B(a)P-induced ovotoxicity in mice. As a result, primordial follicles were destroyed, suggesting that the ovary has the capacity to metabolize B(a)P to the toxic diol-epoxide metabolite (7,8-dihydrodiol-9,10-epoxide; Mattison et al., 1983). Studies by Bengtsson et al. (1992, 1987, 1983) demonstrated ovarian metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) and 3-methylcholanthrene (3-MC) in rats. Since metabolites of these compounds are known to be ovotoxic, ovarian metabolism likely plays a role in activating polycyclic aromatic hydrocarbons. Studies by Flaws et al. (1994b) demonstrated a role for ovarian detoxification of epoxide metabolites, because follicles isolated from rat ovaries converted VCD to the nontoxic tetrol, presumably by mEH. The studies presented here further suggest a role for mEH in ovarian metabolism of epoxides by showing the capability for ovarian detoxification of both the mono- and diepoxide metabolites of VCH in mice. These findings are unique in that they have evaluated ovarian metabolic enzymes, notably mEH, in isolated ovarian compartments, whereas to date, previous studies have been performed in microsomes prepared from whole ovaries. Our approach allows investigation of differential responses within targeted and nontargeted subpopulations of ovarian tissue.
Recently, studies by Coller et al. (2001) and Hattori et al. (2000) evaluated mEH in the human ovary. By immunohistochemical analysis, Coller et al. showed that mEH is highly expressed throughout the ovary. Additionally, mEH was found to be highly expressed in ovarian tumors, including theca fibromas. Studies by Hattori et al. (2000) also evaluated mEH distribution throughout the ovary via immunohistochemistry. mEH was detected in granulosa cells and theca cells. In vitro studies utilizing the mEH inhibitor, 1,2-epoxy-3,3,3-trichloropropane, caused a dose-dependent decrease in the production of 17ß-estradiol. Thus, mEH may also be involved in different ovarian steroidogenic pathways. Most importantly, however, these reported findings support that mEH is likely to play a role in human ovarian function.
Taken together, mEH is expressed and is functional in follicles isolated from mouse ovaries. Furthermore, in vivo dosing with VCH or VCD affects both mEH mRNA levels and specific activity. Specifically, F1 follicles (those targeted by VCD) demonstrated increased expression of mRNA and greater enzyme activity, compared to other ovarian fractions following repeated dosing with VCH/VCD. Thus, after repeated exposure, F1 follicles may acquire a greater ability to participate in detoxification of VCD as a protective measure against follicle destruction. Future studies will investigate in vitro metabolism utilizing the VCH-monoepoxides and VCD as substrates to directly evaluate the role of the ovary in both bioactivation and detoxification.
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ACKNOWLEDGMENTS |
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NOTES |
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