Expression and Activity of Microsomal Epoxide Hydrolase in Follicles Isolated from Mouse Ovaries

Ellen A. Cannady*, Cheryl A. Dyer{dagger}, Patricia J. Christian{ddagger}, I. Glenn Sipes* and Patricia B. Hoyer{ddagger},1

* University of Arizona, Department of Pharmacology and Toxicology, Tucson, Arizona 85721; {dagger} Northern Arizona University, Department of Biological Sciences, Flagstaff, Arizona 86011; and {ddagger} University of Arizona, Department of Physiology, Tucson, Arizona 85724

Received December 3, 2001; accepted February 28, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microsomal epoxide hydrolase (mEH) is involved in the detoxification of xenobiotics that are or can form epoxide metabolites, including the ovotoxicant, 4-vinylcyclohexene (VCH). This industrial chemical is bioactivated by hepatic CYP450 to the diepoxide metabolite, VCD, which destroys mouse small preantral follicles (F1). Since ovarian mEH may play a role in VCD detoxification, these studies investigated the expression and activity of mEH in isolated ovarian fractions. Mice were given 1 or 15 daily doses (ip) of VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day); 4 h following the final dose, ovaries were removed, distinct populations of intact follicles (F1, 25–100 µm; F2, 100–250 µm; F3, > 250 µm) and interstitial cells (Int) were isolated, and total RNA and protein were extracted. Real-time polymerase chain reaction and the substrate cis-stilbene oxide (CSO; 12.5 µM) were used to evaluate expression and specific activity of mEH, respectively. Confocal microscopy evaluated ovarian distribution of mEH protein. Expression of mRNA encoding mEH was increased in F1 (410 ± 5% VCH; 292 ± 5% VCD) and F2 (1379 ± 4% VCH; 381 ± 11% VCD) follicles following repeated dosing with VCH or VCD. Catalytic activity of mEH increased in F1 follicles following repeated dosing with VCH/VCD (381 ± 11% VCH; 384 ± 27% VCD). Visualized by confocal microscopy, mEH protein was distributed throughout the ovary with the greatest staining intensity in the interstitial cells and staining in the theca cells that was increased by dosing (56 ± 0.8% VCH; 29 ± 0.9% VCD). We conclude that mEH is expressed and is functional in mouse ovarian follicles. Additionally,in vivo dosing with VCH and VCD affects these parameters.

Key Words: microsomal epoxide hydrolase; 4-vinylcyclohexene; ovary; ovarian follicles; mouse; confocal microscopy.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epoxide hydrolase is an important metabolic enzyme that catalyzes the addition of water to alkene epoxides and arene oxides. The five characterized classes of mammalian epoxide hydrolase include three cytosolic forms (hepoxilin A3, leukotriene A4, soluble) and two microsomal forms (cholesterol 5,6-oxide, microsomal). Both the soluble and microsomal forms of epoxide hydrolase are involved in xenobiotic metabolism. Cytosolic epoxide hydrolase preferentially metabolizes trans-substituted epoxides, while microsomal epoxide hydrolase (mEH) metabolizes cis-substituted epoxides. Microsomal epoxide hydrolase has a wide substrate specificity and has the capacity to both bioactivate and detoxify xenobiotics (Omiecinski et al., 2000Go). Some xenobiotics metabolized by mEH include the epoxide metabolites of polycyclic aromatic hydrocarbons, 1,3-butadiene, benzene, aflatoxin B1, and the anticonvulsant drugs phenytoin and carbamazepine (Fanucchi et al., 2000Go; Fretland and Omiecinski, 2000Go). Microsomal epoxide hydrolase is most abundant in the liver, but has been found in all other tissues examined to date, including the ovary (Dannan and Guengerich, 1982Go; Mukhtar et al., 1978Go; Oesch et al., 1977Go).

Many xenobiotic compounds that are or can form reactive epoxide metabolites are detoxified by mEH, including the epoxides of 4-vinylcyclohexene (Fig. 1Go). 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., 1995Go; Smith et al., 1990bGo). 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., 1994aGo; NTP, 1989Go).



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FIG. 1. Proposed scheme for hepatic metabolism of VCH. The parent compound, VCH, is bioactivated via cytochromes P450 to form the mono- and diepoxide metabolites. Detoxification via hydrolysis of epoxides is via microsomal epoxide hydrolase (Keller et al., 1997Go).

 
The exact mechanism of VCH-induced ovotoxicity is not known. However, metabolism (bioactivation and detoxification) is a key component, and is thought to be at least partially responsible for a species-specific response in which VCH is ovotoxic in the mouse, but not the rat. The VCH-monoepoxides and VCD are ovotoxic in both species (Smith et al., 1990aGo,bGo). Specifically, it is thought that the mouse may be more sensitive to the effects of VCH compared to the rat because of a greater ability to bioactivate VCH (via cytochrome P450). Previous studies have suggested that hepatic cytochrome P450 isoforms 2A, 2B, and 2E1 may be involved in the bioactivation of VCH to VCD in mice (Doerr-Stevens et al., 1999Go; Smith et al., 1990cGo). Species comparison studies showed that VCH treatment caused increased hepatic protein levels and activities of CYP2A and CYP2B in mice, but not rats. Although studies in human hepatic "supersomes" showed that human CYP2E1 and human CYP2B6 were capable of catalyzing VCH epoxidation, CYP 2E1 was not induced in mice or rats following VCH treatment. Furthermore, the same amounts of VCH 1,2-monoepoxide and VCH 7,8-monoepoxide were formed from VCH in hepatic microsomal incubations from CYP2E1 knockout and wild type mice, suggesting that CYP2E1 is not involved in VCH epoxidation in the liver (Fontaine et al., 2001aGo,bGo). Additionally, relative to the rat, the mouse has a lesser ability to detoxify VCD (via mEH) to the nontoxic tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). Studies by Salyers et al. (1993) showed that 6 h after receiving a single dose of [14C]-VCD, rats excreted 70% of the dose in the urine, whereas mice only excreted 30% of the dose in the urine. Likewise, it has been reported that mice have lower levels of mEH activity compared to humans or rats (Kitteringham et al., 1996Go; Krause et al., 1997Go). This information collectively supports the greater susceptibility of mice, compared with rats, to these chemicals.

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., 1994bGo). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Medium 199 (M199) and custom-designed primers were purchased from Gibco, Inc. (Grand Island, NY). VCH (racemic mixture; purity 95–99%) was purchased from Aldrich Chemical Co. (Milwaukee, WI). VCD (mixture of isomers, composition unknown; purity > 99%), collagenase (Clostridium histolytium type I), deoxyribonuclease type I (DNase; from bovine pancreas), bovine serum albumin (BSA), sesame oil, N-(2-hydroxyethyl)piperazine-N`-(2-ethanesulfonic acid) (HEPES), NaCl, ethylenediaminetetraacetic acid (EDTA), Tris, triton X, glycerol, sodium dodecyl sulfate (SDS), NaF, phenylmethanesulfonyl fluoride (PMSF), leupeptin, aprotinin, 2,2,4-trimethylpentane, and ribonuclease A were purchased from Sigma Chemical Co. (St. Louis, MO). 18S ribosomal RNA (18S rRNA) primers and RNAqueous kit were from Ambion Inc. (Austin, TX). Reverse transcription system was from Promega (Madison, WI). Deoxynucleotidetriphosphate (dNTP) mix, MgCl2, and enzyme diluent were purchased from Idaho Technologies (Salt Lake City, UT). Advantaq Plus taq polymerase was from Clontech Laboratories, Inc. (Palo Alto, CA), and SYBR Green dye and YOYO-1 were purchased from Molecular Probes (Eugene, OR). The bicinchoninic acid (BCA) protein assay kit was from Pierce (Rockford, IL). CytoScint and nonradioactive cis-stilbene oxide were from ICN Radiochemicals (Costa Mesa, CA). The [3H] cis-stilbene oxide was a generous gift from Dr. Bruce Hammock (UC Davis, CA). The mEH antibody (goat antirabbit) was purchased from Detroit R and D (Detroit, MI); the secondary antibody (horse antigoat) and Cy-5-streptavidin were purchased from Vector (Burlingame, CA).

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 (8–10 animals/treatment group) were weighed and administered (ip) 1 single dose or 15 daily, consecutive doses (2.5 µl/g of body weight; 30–50 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., 1999Go; Smith et al., 1990bGo). 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 (25–100 µm; fraction 1; F1) and large (100–250 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 ({lambda} = 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 ({lambda} = 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 min–3 h) and protein concentration (10–50 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 {lambda} = 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 30–35 primordial follicles, 10–20 small primary follicles, 5–10 large primary follicles, 2–5 antral follicles, and 8–12 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Real-Time Polymerase Chain Reaction
mRNA encoding mEH was evaluated in follicles isolated from mouse ovaries following in vivo pretreatment (1 single dose or 15 daily doses) with vehicle, VCH, or VCD. It was detected in tissue obtained from all treatment groups. mEH mRNA was expressed in all follicle populations that were studied (F1, small preantral, 25–100 µm; F2, large preantral, 100–250 µm; F3, antral, > 250 µm) in the ovary, as well as in the Int cells. Expression levels for mRNA encoding mEH were compared in different ovarian fractions from vehicle-treated animals, in which mature F3 follicles (13.52 ± 5.9 mEH mRNA:18S rRNA) expressed significantly more mRNA encoding mEH compared to the other fractions (F1, 4.01 ± 1.4 mEH mRNA:18S rRNA; F2, 3.06 ± 1.1 mEH mRNA:18S rRNA; Int, 2.38 ± 1.3 mEH mRNA:18S rRNA; p < 0.05). There were no significant effects on gene expression following a single dose of VCH/VCD (data not shown). In contrast, repeated daily dosing (15 days) with VCH increased (p < 0.05) expression of mRNA encoding mEH by 410 ± 5% in F1 and 1379 ± 4% in F2, compared to control (Fig. 2Go). Conversely, VCH decreased (p < 0.05) mEH expression by 60 ± 27% in Int cells compared to control. Repeated dosing with VCD increased (p < 0.05) mEH expression by 292 ± 5% in F1, 381 ± 11% in F2, and 153 ± 7% in F3, whereas VCD did not affect mEH expression in Int cells (Fig. 2Go).



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FIG. 2. Relative semiquantitative RT-PCR for mEH. Female B6C3F1 mice (d 28) were given repeated daily doses (15 days; ip) of sesame oil (vehicle), VCH (7.4 mmol/kg/day), or VCD (0.57 mmol/kg/day); n = 4–6. Four h following the final dose, ovaries were collected and follicle fractions were prepared. Total RNA was isolated from ovarian follicle fractions and reverse transcribed into cDNA using random primers. Real-time RT-PCR was utilized for semiquantitation of mRNA encoding mEH. A standard curve was generated from 1:5 serial dilutions of purified mEH PCR product. An aliquot of each dilution was used as PCR template to generate a standard curve. Arbitrary numbers were assigned to each standard and experimental samples were calculated from the standard curve. The housekeeping gene, 18S rRNA, was also determined for all samples as described for mEH. Final values for mEH expression were calculated as a ratio of mEH mRNA:18S rRNA (treatment/control; Tx/Con). *Significantly different from control; "a" and "b" significantly different from one another; p < 0.05.

 
mEH Activity Assay
Microsomal epoxide hydrolase activity was analyzed in isolated mouse ovarian follicles following in vivo dosing (1 single dose or 15 daily doses) with vehicle, VCH, or VCD. Activity was detected in all groups examined. Levels of mEH activity in d 28 vehicle-treated animals were 56.7 ± 14.5 nmol/min/mg of protein in F1 follicles, 128.7 ± 65.6 nmol/min/mg of protein in F2 follicles, 61.7 ± 30.6 nmol/min/mg of protein in F3 follicles, and 85.8 ± 26.4 nmol/min/mg of protein in Int cells. In d 42 vehicle-treated mice, mEH activity was 34.2 ± 15.5 nmol/min/mg, 147.7 ± 42.1 nmol/min/mg, 19.3 ± 9 nmol/min/mg, and 16.3 ± 6.8 nmol/min/mg of protein in F1, F2, F3, and Int cells, respectively. Interestingly, the VCH/VCD nontarget population of F2 follicles displayed significantly higher activity (p < 0.05) compared to other fractions in this group (Fig. 3Go). There were no significant effects in enzyme activity following a single dose of VCH or VCD (data not shown). However, repeated dosing with VCH or VCD did alter mEH activity (Fig. 4Go). In F1 follicles (those targeted by VCH and VCD) there was an increase (p < 0.05) in mEH activity (381 ± 11% VCH, 384 ± 27% VCD) above control. There was no effect of dosing in F2 follicles, while nonsignificant trends for increased mEH activity were seen in large antral (F3) follicles and Int cells following VCH or VCD dosing (344 ± 15% VCH, F3; 304 ± 69% VCD, F3; 482 ± 40% VCH, Int; 285 ± 28% VCD, Int) compared to control.



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FIG. 3. Unstimulated mEH activity in follicles and interstitial cells isolated from mouse ovaries. Ovaries were collected and follicle fractions prepared from vehicle-treated mice (d 42). Total protein was isolated from ovarian follicle fractions. Protein (25 µg) was incubated with [3H]-cis stilbene oxide for 2 h. 2,2,4-Trimethylpentane was added to stop the reaction and resulted in a phase separation. The unreacted cis-stilbene oxide partitions into the organic phase while the reaction product (diol) partitions into the aqueous phase. An aliquot of the aqueous phase was removed and the radioactivity counted by standard scintillation counting. Values are expressed as nmol/min/mg of protein; n = 4. *Significantly different from control, p < 0.05.

 


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FIG. 4. mEH activity in follicles and interstitial cells isolated from mouse ovaries. Female B6C3F1 mice (d 28) were given repeated daily doses (15 day; ip) of sesame oil (vehicle), VCH (7.4 mmol/kg/day), or VCD (0.57 mmol/kg/day); n = 4. Four h following the final dose, ovaries were collected, follicle fractions were prepared, and protein (25 µg) was incubated with [3H]-cis stilbene oxide for 2 h. Samples were processed as presented in Figure 3Go. Values are expressed as a ratio of treatment/control (Tx/Con). *Significantly different from control, p < 0.05.

 
Confocal Microscopy
Total mEH protein was visualized in the mouse ovary. Protein was present in oocytes, granulosa cells, and theca cells, as well as the surrounding Int cells. Following repeated dosing, VCH and VCD increased mEH protein expression in theca cells of growing preantral follicles (F2), as measured by densitometry (56 ± 0.8% VCH; 29 ± 0.9% VCD, p < 0.05) compared to all other ovarian follicle components and Int cells in vehicle-treated mice (Fig. 5Go). No autofluorescence was seen in unstained, coverslipped ovarian sections at {lambda} = 647 nm.



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FIG. 5. mEH protein distribution in ovarian follicles and interstitial cells by confocal microscopy. B6C3F1 mice (d 28) were dosed daily (15 days; ip) with sesame oil (vehicle), VCH (7.4 mmol/kg/day), or VCD (0.57 mmol/kg/day); n = 2. Ovaries were removed 4 h after the final dose, fixed in 4% formalin, sectioned (5 µm thick, every seventh section), and incubated overnight with a goat antirabbit mEH polyclonal antibody. Immunofluorescence for mEH protein 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 {lambda} = 488 nm for YOYO-1 (green, DNA stain) and {lambda} = 647 nm for Cy-5 (red; mEH). All images were captured at 40X magnification. Relative changes in staining intensity were made by densitometry using Scion Image Software. Background staining intensity was subtracted from each field. Multiple readings were taken throughout the sections. Analysis was performed on different sizes of ovarian follicles and interstitial cells at controlled settings on the confocal microscope. mEH protein was visualized in (A) F1, F2, and Int from vehicle-treated mice, (B) F1, F2, and Int from VCH-treated mice, in which the staining intensity in theca cells of growing follicles (F2) was significantly increased compared to control (p < 0.05), and (C) F1, F2, and Int from VCD-treated mice, in which the staining intensity in theca cells of growing follicles (F2) was significantly increased compared to control (p < 0.05). (D) Ovarian section stained with YOYO-1 and Cy-5, with no primary antibody added. All samples were normalized to control so multiple experiments could be compared.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ovary is a heterogeneous organ, containing follicles in various stages of maturity, from the smallest, primordial, to the most mature, large antral follicles. Our studies have demonstrated that mEH, an important metabolic enzyme, is expressed and functional within these specific follicle types, as well as in interstitial cells. Additionally, confocal studies revealed distribution of mEH throughout the ovary, with protein being expressed in the oocytes, granulosa cells, and theca cells, as well as interstitial cells, where the highest staining intensity was observed. In ovaries from vehicle-treated animals, mRNA expression of mEH was greatest in antral follicles (F3), while the greatest functional activity was measured in growing, preantral follicles (F2).

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, 1997Go, 2000Go; Kim et al., 1998Go). 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., 1999Go). 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., 1999Go; Springer et al., 1996Go). 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., 1994aGo). 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., 1994Go; 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., 1999Go).

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., 1985Go). 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., 1983Go). 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.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. Bruce Hammock at U.C. Davis for the generous gift of the [3H]-cis-stilbene oxide. This work was supported by NIH grant ES08979 (P.B.H.), NIEHS Center grant ES06694, and NIEHS training grant ES07019.


    NOTES
 
1 To whom correspondence should be addressed at University of Arizona, Department of Physiology, 1501 N. Campbell Ave., #4122, Tucson, AZ 85724. Fax: (520) 626-2382. E-mail: hoyer{at}u.arizona.edu. Back


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