* 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 November 21, 2002; accepted February 27, 2003
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ABSTRACT |
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Key Words: cytochrome P450; 4-vinylcyclohexene; ovary; ovarian follicles; mouse; confocal microscopy.
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INTRODUCTION |
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The exact mechanism of VCH induced-ovotoxicity is not known. However, it likely involves metabolism of the parent compound, VCH, to the ultimate ovotoxicant, VCD. Whether this occurs in the ovary is not known. Previous studies by Smith et al.(1990a) evaluated circulating levels of VCH and VCH-1,2-monoepoxide in blood following a single dose of VCH. Further studies by Doerr-Stevens et al.(1999)
evaluated circulating levels of VCD after repeated dosing. These studies indicated that the liver could be the major site of VCH bioactivation. Hepatic CYP450 isoforms thought to be involved in the conversion of VCH to VCD in mice include CYP2A, CYP2B, and CYP2E1 (Doerr-Stevens et al., 1999
; Smith et al., 1990c
). Studies by Fontaine et al. (2001a
,b
) showed that repeated daily dosing with VCH induced hepatic protein levels and activities of CYP2A and CYP2B in mice, but not rats. Hepatic CYP2E1 was not enhanced in mice or rats following in vivo dosing with VCH. In hepatic microsomal incubations from CYP2E1-deficient and wild-type mice, VCH could be metabolized to similar amounts of VCH 1,2-monoepoxide and VCH 7,8-monoepoxide, suggesting that CYP2E1 is not required for epoxidation of VCH in the liver.
As regards to metabolism in extrahepatic tissues, the ovary may be an important tissue-specific site of bioactivation or detoxification. For example, Mattison et al. (1979) directly evaluated the role of ovarian metabolism of benzo(a)pyrene (B(a)P) in mice. 3H-B(a)P was incubated in vitro with the S9 fraction isolated from ovaries collected from mice dosed in vivo with vehicle control or 3-methylcholanthrene (3-MC). Numerous B(a)P metabolites were detected and greater amounts of product were formed in the tissue from 3-MC-treated animals. Studies by Shiromizu and Mattison (1984)
evaluated the effect of unilateral intraovarian (i.o.) injection of B(a)P on the number of primordial oocytes in mice. B(a)P, which must be bioactivated to the toxic metabolite, was only ovotoxic in those ovaries injected with B(a)P, as evidenced by oocyte destruction. Additionally, oocyte loss was not as great when the potent CYP450 inhibitor,
-naphthoflavone, was administered (i.p.) concurrently with B(a)P (i.o.). Additional studies by Bengtsson et al. (1983
, 1987
, 1992
) have also demonstrated ovarian metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) and 3-MC in rats by granulosa cells. These studies support the hypothesis that the ovary contains CYP450-dependent monooxygenases that can metabolize xenobiotics.
Although circulating levels of VCH and its metabolites can reach the ovary via the circulation, it is not known whether bioactivation of VCH or the monoepoxides to VCD occurs in the ovary. Studies by Keller et al.(1997) showed that ovarian microsomes obtained from mice do not convert VCH to detectable levels of the epoxide metabolites in vitro, as analyzed by gas chromatography. However, previous in vivo exposure of mice to VCH may induce such a capacity. Furthermore, the possible compartmentalization of CYP450 enzymes/isoforms within different sizes of ovarian follicles has not been reported. Studies in our laboratory have demonstrated that the ovary has the capacity to be involved in detoxification reactions in both mice and rats. In mice, VCH or VCD dosing altered expression of mRNA and distribution of total protein, as well as functional activity for mEH in different sizes of ovarian follicles (Cannady et al., 2002
). In rats, Flaws et al.(1994b)
showed that isolated preantral follicles can detoxify VCD to the nontoxic tetrol metabolite [4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane]. Thus, a number of studies have provided evidence to support the idea that extrahepatic metabolism within the ovary may directly amplify or attenuate the extent of ovarian toxicity caused by xenobiotic chemicals.
Because VCD specifically destroys small preantral (primordial and primary) follicles, ovarian distribution of CYP450 may impact susceptibility to exposure within specific follicle populations. Therefore, this study was designed to investigate ovarian CYP450 isoforms (CYP2E1, CYP2A, and CYP2B) by (1) assessing expression of mRNA in follicles isolated from mouse ovaries, (2) identifying ovarian distribution of protein, (3) measuring catalytic activity in whole ovaries, and (4) determining the effects of in vivo dosing with VCH and VCD on these CYP450 isoforms.
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MATERIALS AND METHODS |
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Animals.
Female B6C3F1 mice (day 21, d21) were purchased from Harlan Laboratories (Indianapolis, IN) and maintained in the University of Arizona Animal Care Facility. Animals (acclimated for 1 week prior to study) were housed in plastic cages, 5 animals per cage, and maintained in a controlled environment (22 ± 2°C; 12 h light/12 h dark cycles). The animals were provided a standard diet with ad libitum access to food and water. All animal experiments were approved by the University of Arizonas Institutional Animal Care and Use Committee.
Animal dosing.
Female B6C3F1 mice (day 28, d 28; 10 animals/treatment group) were weighed and administered (intraperitoneal; ip) 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 (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 pooled (20 ovaries/treatment group), minced, and gently dissociated (40°C, 20 min) in Medium 199 (containing Hanks salts, L-glutamine, 25 mM HEPES) with collagenase (7.5 mg/ml), DNase (0.267 mg/ml), and BSA (40 mg/ml). The dissociated mixture was filtered through a 250-mm 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 into distinct populations, using calibrated Pasteur pipettes (Flaws et al., 1994a). Ovarian fractions were 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). However, 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 (20 pooled ovaries/treatment group) 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).
Realtime polymerase chain reaction.
cDNA (1 µl) from various ovarian fractions was used to perform relative, semi-quantitative PCR utilizing a LightCycler® (Idaho Technology) capable of realtime PCR. The LightCycler® quantifies the amount of PCR product generated by measuring the dye (SYBR green), which fluoresces when bound to double stranded DNA. Custom designed primers were utilized (CYP2E1; forward primer: 5' GTC TTT AAC CAA GTT GGC AA 3'; reverse primer: 5' CCA ATC AGA AAG GTA GGG TC 3' [Freeman et al., 1992]; CYP2A; forward primer: 5' ATT GAC CCC ACC TTC TAC CT 3'; reverse primer: 5' CAG TAT TGG GGT TCT TCT TCT CC 3' [Squires and Negishi, 1988
]; CYP2B; forward primer: 5' CTC TTC CAG TGC ATC AC 3'; reverse primer: 5' GGA ACT CCT CGA CTA CAT TG 3' [Marc et al., 1999
]). Amplification conditions for CYP2E1 were 95°C/0 s (denaturing), 57°C/0 s (annealing), and 72°C/10 s (extending) for 50 cycles. Amplification conditions for CYP2A were 95°C/0 s (denaturing), 68°C/0 s (annealing), and 72°C/12 s (extending) for 50 cycles. Amplification conditions for CYP2B were 95°C/0 s (denaturing), 64°C/0 s (annealing), and 72°C/16 s (extending) for 50 cycles. A standard curve was generated from 1:5 serial dilutions of purified PCR product (CYP2E1, CYP2A, CYP2B, or 18S rRNA). 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 extrapolated from the standard curve. 18S rRNA was measured in each sample as an internal standard. Final values were expressed as a ratio of CYP450 : 18S rRNA.
Confocal microscopy.
Four h following the standard dosing regimen, animals (2/group) were euthanized, ovaries removed, and oviduct and excess fat trimmed away. Ovaries were fixed for histology (10% buffered formalin; 4 h), 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 antibodies against either CYP2E1 (goat anti-rat; 1:50 dilution; 37°C, 60 min), CYP2A (rabbit anti-human; 1:25 dilution; 4°C, overnight), or CYP2B (goat anti-rat; 1:50 dilution; 4°C, overnight). Specificity for these primary antibodies was determined in our previous studies using Western blotting (Fontaine et al., 2001a). Secondary biotinylated antibody (rabbit anti-goat, 1:75 dilution, CYP2E1 and CYP2B; goat anti-rabbit, 1:75 dilution, CYP2A) 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), cover-slipped, 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. All images were captured at 40 x magnification. No autofluorescence was seen in unstained, coverslipped ovarian sections at
= 647 nm. Relative densitometric analysis using Scion Image Software (National Institutes of Health, Bethesda, MD) was utilized to compare relative intensities of staining, in which 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 approximately 35 primordial follicles, 20 small primary follicles, 10 large primary follicles, 5 antral follicles, and 12 interstitial compartments were evaluated per group. The confocal settings were changed to allow more accurate assessment of staining intensity in the interstitial cells, because the staining intensity was very high for this ovarian compartment. However, all of the analyses for the interstitial cells were also compared at the same settings. All groups were normalized to control, so multiple experiments could be compared.
Protein determination.
Total protein was extracted from whole ovaries (two ovaries/animal/sample). Briefly, ovaries were removed, and the oviduct and excess fat were trimmed away. Both ovaries from each animal were pooled, and 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.
Liver microsome preparation.
Following euthanization, livers were excised from B6C3F1 mice. Microsomes were prepared according to Guengerich (1989). Briefly, livers were homogenized in buffer (pH 7.4) containing 1 M TrisHCl, 1 M KCl, 100 mM EDTA, and 20 mM BHT. Microsomes were isolated by differential ultracentrifugation. The microsomal fraction was collected and the protein concentration was determined utilizing the BCA Protein Assay® kit.
CYP450 functional assays.
Specific activities for CYP2E1, CYP2A, and CYP2B were evaluated in whole ovarian homogenates utilizing model substrates. CYP2E1 catalyzes the hydroxylation of p-nitrophenol to p-nitrocatechol (Patten et al., 1992), while CYP2A catalyzes the hydroxylation of coumarin to 7-hydroxycoumarin (Waxman et al., 1991
), and at low concentrations of substrate (~5 µM), CYP2B specifically catalyzes the metabolism (7-O-deethylation) of 7-ethoxy-4-trifluoromethyl coumarin (EFC) to 7-hydroxy-4-trifluoromethyl coumarin (HFC; Code et al., 1997
). Protein (200 µg) from whole ovarian homogenates was incubated (37°C, shaking water bath) in assay buffer containing substrate and cofactor according to conditions for specific assays listed in Table 1
. Following incubation, the CYP2E1 reaction was stopped by adding 20% trichloroacetic acid to samples, and samples were placed on ice. The samples were concentrated (~50 µl), and the supernatant was transferred to a 96-well plate. Immediately prior to reading, 2-M NaOH was added to each sample or standard. In the CYP2A activity assay, 2-N hydrochloric acid was added to stop the reaction, and samples were placed on ice. The samples were extracted with dichloromethane and concentrated (~50 µl), back extracted with sodium borate (30 mM), and the aqueous layer was transferred to a 96-well plate. In the CYP2B activity assay, 2-N hydrochloric acid was added to stop the reaction and samples were placed on ice. The samples were concentrated (~50 µl), and the supernatant was transferred to a 96-well plate. Immediately prior to reading, 0.1-M Tris (pH 9.0) was added to each sample or standard. In the CYP2E1 assay, absorbance was measured at
= 520 nm on a 96-well plate reader (Molecular Devices) equipped with Soft Max Pro Software. Fluorescence for the CYP2A and CYP2B activity assays (excitation
= 410 nm; emission
= 510 nm) was measured on a fluorescent plate reader (Spectra Max Gemini XR) equipped with Soft Max Pro Software. In all CYP450 functional assays, the amount of product formed (expressed as nmol product formed/min/mg protein) in each unknown sample was determined by linear regression analysis. Additionally, experiments were performed to ensure linearity of the reaction in terms of incubation time (15 min2 h) and protein concentration (501000 µg). To verify assay measurements, the experiments were also performed in liver microsomes (data not shown). Optimal conditions for p-nitrophenol hydroxylation (CYP2E1) and EFC metabolism (CYP2B) in whole ovaries utilized 200 µg of protein incubated for 1 h.
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RESULTS |
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DISCUSSION |
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Although the present study did not directly evaluate the ovarian metabolism of VCH, the results provide indirect support for an involvement of the ovary in VCH-induced ovotoxicity. For instance, repeated in vivo dosing with VCH or VCD altered CYP450 expression. A significant increase in expression of mRNA encoding CYP2E1 was observed in F1 follicles, those follicles specifically targeted by VCD, as well as nontargeted F3 follicles after VCH or VCD dosing. Although total protein for CYP2E1 was slightly decreased in Int cells following VCH-treatment, whole ovarian CYP2E1 enzyme activity was increased. Due to low levels of enzyme activity, the measurements were performed in whole ovarian homogenates, thus making it impossible to identify the specific ovarian compartment that is functionally responsible for metabolism of the model substrate. Based on the immunohistochemical data, any size of follicle could potentially convert the model substrate, p-nitrophenol. However, due to the abundant CYP2E1 protein distribution in the interstitium, this ovarian compartment would likely contribute most significantly to this metabolism.
A significant increase in expression of mRNA encoding CYP2A was observed in the targeted F1 follicles following VCH or VCD dosing. VCH dosing also increased expression of CYP2A in the Int cells. Staining intensity for CYP2A protein was significantly increased in the Int cells. Although this correlates with the mRNA data, the changes in mRNA levels and total protein do not correlate with functional CYP2A activity towards the model substrate, coumarin, which was not detectable in any treatment group. Specific activity for CYP2A towards other model substrates was not evaluated in the study design. Thus, it is not known whether a lack of detection of CYP2A activity was due to reduced sensitivity of the enzyme towards coumarin, or whether it was because CYP2A protein is expressed in the ovary but does not possess functional activity. mRNA encoding CYP2B was increased in F1 follicles following repeated dosing with VCH. This was consistent with the VCH-induced increase in CYP2B protein in granulosa cells of small primary (F1) follicles. Furthermore, VCH and VCD dosing increased protein in F3 follicles. Although CYP2B activity was measured in whole ovarian homogenates, it was not significantly affected by VCH or VCD dosing. However, if there was a specific effect of dosing in F1 follicles, it might have been masked by unchanged activities in larger ovarian compartments.
Our studies evaluated mRNA expression, total protein, and functional activity following 15 days of repeated daily dosing with VCH or VCD. The results suggest that CYP2E1 is a likely contributor to ovarian metabolism, since baseline activity toward a model substrate was significantly higher than that of CYP2A or CYP2B. This is even more likely because activity for CYP2E1 was significantly increased by repeated dosing with VCH for 15 days. However, the effect of dosing on CYP2E1, CYP2A, and CYP2B expression profiles in the ovary are not known at earlier or later time points. Interestingly, in previous studies CYP2E1 did not appear to be involved in the bioactivation of VCH in liver (Fontaine et al., 2001a,b
). Rather, those studies showed induction of total and functional hepatic protein for CYP2A and CYP2B following 10 days of dosing with VCH in the mouse. Thus, different isoform profiles may predominate in the ovary as compared to the liver. Additionally, different dosing regimens may impact isoform expression patterns.
Our studies showed that mRNA encoding CYP2E1, CYP2A, and CYP2B is increased in the vulnerable population of F1 follicles following repeated dosing with VCH or VCD. However, other sizes of ovarian follicles and Int cells also express the metabolic enzymes and may contribute to providing the target population of follicles with the bioactive metabolite. Interestingly, there was a high level of staining intensity for all of the CYP450 isoform proteins localized in the Int compartment. Thus, this ovarian compartment may be involved in either mono-epoxidation of VCH and/or subsequent epoxidation of the monoepoxides to VCD. This hypothesis is plausible when one considers that the Int cells are in the highly vascularized region of the ovary (Erickson et.al., 1985). Such cells would likely be exposed to toxicants traveling through the blood stream. On a broader scale, this metabolic potential may serve as a generalized function for the interstitium, whose physiological function has remained largely unclear. Because enzyme induction was observed in ovarian compartments not targeted by VCH or VCD, an interesting point to consider is the protection against ovotoxicity granted to the nontargeted populations, despite the presence and activity of bioactivating enzymes. These compartments also contain detoxifying enzymes, such as mEH. The balance between bioactivation and detoxification enzymes may favor detoxification in these compartments. For example, Flaws et al. (1994b)
showed in rats that large preantral follicles (F2) converted more VCD to the inactive tetrol metabolite compared to small preantral follicles (F1). Cannady et al. (2002)
showed in mice that baseline mEH activity was greater in F2 follicles compared to other sizes of follicles and Int cells. Additionally, the susceptibility of the F1 follicles compared to other compartments may be due to inherent differences in morphology (e.g., lack of theca cell layer) as well as physical properties of the chemical, thus governing the specificity of the response.
Although the relative contribution of ovarian metabolism, compared to hepatic metabolism, in VCH-induced ovotoxicity is not known, it has been demonstrated here that the ovary has the potential (mRNA and functional protein) for bioactivation of VCH and/or the monoepoxides. Based collectively on the basal and VCH-induced levels of CYP450 activity, CYP2E1 is the most likely isoform that would be involved in ovarian metabolism. Additionally, we conclude that the interstitial (Int) compartment is potentially the most important ovarian compartment involved in bioactivation of VCH, due to its high level of expression of all three of the CYP450 isoforms. Future studies will be aimed at determining more precisely the extent to which the ovary contributes to VCH-induced ovotoxicity in mice. This information can serve to better elucidate the metabolic role of the ovary in responding to xenobiotic exposures, which may impact ovarian function in women.
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ACKNOWLEDGMENTS |
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NOTES |
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