* Department of Biotechnical and Clinical Laboratory Science,
Department of Pharmacology and Toxicology, and
Department of Gynecology-Obstetrics, State University of New York at Buffalo, Buffalo, New York 14214;
W. Harry Feinstone Center for Genomic Research, University of Memphis, Memphis, Tennessee 38152; and
¶ Women's Reproductive Health Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Received January 18, 2001; accepted March 30, 2001
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ABSTRACT |
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Key Words: endometrium; endometriosis; dioxin; 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; CYP1A1; CYP1B1; cytochrome P450 enzymes; cytochrome P450 1A1; cytochrome P450 1B1..
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INTRODUCTION |
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The pathogenesis of endometriosis is not well understood. Several theories that have been developed over the past century to explain its histogenesis have recently been reviewed (Oral and Arici, 1997; Witz, 1999
) and can be divided into endometrial, in situ, and combination theories. Endometrial theories propose that endometriosis results from the implantation of uterine endometrium at sites outside of the uterus after dissemination from the uterus by retrograde menstruation, vascular or lymphatic metastasis, direct invasion or extension through the uterine musculature, or by iatrogenic deposition. In situ theories suggest that endometriosis arises spontaneously from coelomic metaplasia of the peritoneum or germinal epithelium of the ovary, or from the differentiation of embryonic cell rests. A combination theory, called the induction theory, proposes that metaplasia is induced by endogenous factors secreted by disseminated uterine endometrium at a distant site. Uterotubal, menstrual (Oral and Arici, 1997
), genetic (Kennedy, 1999
; Oral and Arici, 1997
; Witz, 1999
), and immune factors also appear to play a role (Oral and Arici, 1997
; Witz, 1999
). In addition, the progression of the disease can be modulated by hormonal factors, notably stimulation of ectopic endometrial implants by estrogens and regression of implants in menopause or by hormonal therapies that suppress estrogen production (Oral and Arici, 1997
; Witz, 1999
).
More recently, environmental factors have also been implicated in the pathogenesis of endometriosis. In rhesus monkeys, endometriosis has been associated with exposure to radiation (Fanton and Golden, 1991; McClure et al., 1971
; Splitter et al., 1972
; Wood et al., 1983
) and dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) (Rier et al., 1993
). In particular, Rier et al. (1993) demonstrated that chronic dietary exposure to TCDD resulted in an increased incidence in the development of endometriosis, in a colony of rhesus monkeys, 10 years after the termination of treatment, and that the severity of the disease increased with the level of exposure (0, 5, or 25 ppt/day for 4 years). More recently, Rier et al. (2001) presented data at 13 years posttreatment showing that a high prevalence of endometriosis in these animals was associated with elevated serum levels of 3,3',4,4'-tetrachlorobiphenyl and 3,3',4,4',5-pentachlorobiphenyl and increased total serum toxic equivalents (TEQs), rather than with serum TCDD levels. This observation may be due to the unique, multichemical exposure history of the monkeys and the relative times following exposure when blood was sampled for analysis. The recent observation still supports a common potential mechanism of action, since TCDD and coplanar PCBs have similar dioxin-like activities. TCDD and other polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) cause a diverse spectrum of species-, strain-, sex-, age-, and concentration-specific responses (Birnbaum and DeVito, 1995
; Poland and Knutson, 1982
; Zeyneloglu et al., 1997
), including reproductive toxicity (Umbreit et al., 1987
; Zeyneloglu et al., 1997
). In addition, they have endocrine-disrupting properties acting as either estrogens or antiestrogens in a tissue- and species-specific manner (Peterson et al., 1993
).
Although no direct evidence exists for a relationship between environmental toxin exposure and the development of endometriosis in human populations, evidence is beginning to accumulate. In Belgium, Koninckx et al. (1994) reported that the incidence of endometriosis (6080%) in women with infertility and pain was among the highest in the world. They noted the parallel, but not direct, association of breast milk dioxin concentrations in this country also being among the highest in the world (Slorach and Vaz, 1985). In Israel, Mayani et al. (1997) showed that 8/44 infertile women with endometriosis had elevated dioxin levels compared to only 1/35 women with tubal infertility (p = 0.04). In a mostly German population, Gerhard and Runnebaum (1992) reported elevated serum concentrations of PCBs in women with endometriosis, and more specifically (Gerhard et al., 1999
), significantly higher serum concentrations of the abundant and persistent PCB congeners 138, 153, and 180 (2,2',3,4,4',5'-, 2,2',4,4',5,5'-, and 2,2',3,4,4',5,5'-PCB, respectively). The results of the Seveso Women`s Health Study should be available soon (Eskenazi et al., 2000
). This comprehensive study on the reproductive health of a human population exposed to TCDD in a major industrial accident should provide a more definitive picture of the relationship between dioxin exposure and the development of endometriosis in humans.
Recently, 5 studies utilizing animal models have investigated the effects of TCDD exposure on experimental endometriosis. These models are based on the widely accepted endometrial theory that endometriosis arises from the implantation of disseminated uterine endometrium.
In the most recent study, Yang et al. (2000) studied the subchronic exposure of cynomolgus monkeys to TCDD. Intact nulliparous monkeys were surgically implanted with autologous endometrium and dosed 5 days a week for a year. TCDD exposure resulted in a higher survival rate of the implants at doses of 5 and 25 ng/kg body weight (bw), and exhibited a bimodal effect on implant size, which decreased in the lowest exposure group (1 ng/kg bw) and increased in the highest exposure group (25 ng/kg bw) compared to control animals.
In the other 4 animal model studies (Cummings et al., 1996, 1999
; Johnson et al., 1997
; Yang and Foster, 1997
), female B6C3F1 mice were used in a model patterned after the rat model developed by Vernon and Wilson (1985). In this model, explants of full-thickness uterus (endometrium plus myometrium) were implanted in the peritoneal cavity. TCDD exposure promoted endometriosis in 3 of these studies (Cummings et al., 1996
, 1999
; Johnson et al., 1997
) in which intact adult mice were treated orally with TCDD (3 or 10 µg/kg bw) 3 weeks prior to the induction of endometriosis and 3, 6, 9, and 12 weeks after the induction. In one of these studies (Cummings et al., 1999
), perinatal exposure on gestation day 8, in addition to adult exposure, was found to promote endometriosis. The fourth study (Yang and Foster, 1997
) used a different experimental protocol in which endometriosis was induced in estrogen-supplemented ovariectomized mice 1 week prior to chronic subcutaneous TCDD exposure (10, 50, or 100 ng/kg bw for 28 consecutive days). In this study, TCDD exposure caused the regression of endometriosis. The conflicting results of Yang and Foster (1997), compared to the other 3 studies, could be explained by the shorter duration of TCDD exposure, the lower cumulative dose of TCDD, the route of administration of TCDD, the timing of initial TCDD exposure relative to the induction of endometriosis, and/or the timing or amount of exposure to estrogen.
Bruner-Tran et al. (1999) used a human model of endometriosis (Bruner et al., 1997) in which proliferative-phase human endometrial explants were cultured in vitro for 24 h with or without TCDD and hormones (Osteen et al., 1994
). The explants were then injected intraperitoneally into ovariectomized athymic (nude) mice treated 24 h before with subcutaneous estradiol-releasing pellets. In this model, treatment of cultured human endometrial explants with estradiol (E2) maintained both stromal- and epithelial-specific matrix metalloproteinase (MMP) secretion in vitro and spontaneously promoted the establishment of ectopic peritoneal lesions in vivo when the explants were injected into recipient animals. In contrast, treatment with progesterone (P4) in conjunction with E2 suppressed both in vitro metalloproteinase secretion and in vivo lesion formation (Bruner et al., 1997
, 1999
; Bruner-Tran et al., 1999
). This suppression was coordinately regulated by both P4 and TGF-ß (Bruner et al., 1999
). Treatment with TCDD along with E2 increased both the number and size of the lesions compared to treatment with E2 alone, and treatment with TCDD in the presence of E2 and P4 disrupted the ability of P4 to block lesion formation and metalloproteinase expression (Bruner-Tran et al., 1999
). These results provide mechanistic evidence linking the steroidal regulation of matrix metalloproteinase (MMP) secretion in human endometrial tissue with the establishment of an endometriosis-like disease in the nude mouse (Bruner et al., 1997
, 1999
) and demonstrate the ability of TCDD to disrupt this regulation (Bruner-Tran et al., 1999
).
The many effects of TCDD have been shown to be mediated through high-affinity binding to the arylhydrocarbon receptor (AhR), which forms an activated heterodimer complex with the structurally related AhR nuclear translocator protein (ARNT) upon binding to TCDD (Schrenk, 1998; Whitlock et al., 1997
). This activated complex binds to specific DNA enhancer sequences known as dioxin response elements (DREs) to induce the expression of dioxin-responsive genes (Schrenk, 1998
; Whitlock et al., 1997
). The most studied, and perhaps the most responsive member of the AhR gene battery, is cytochrome P450 enzyme 1A1 (CYP1A1) (Nerbert et al., 2000
; Whitlock et al., 1997
). A more recently discovered member is cytochrome P450 1B1 (CYP1B1; Alexander et al., 1997; Shimada et al., 1996; Sutter et al., 1994).
Both CYP1A1 and CYP1B1 are expressed in extrahepatic tissues (Hakkola et al., 1997; Safe, 1995
; Sesardic et al., 1990
; Shimada et al., 1996
; Sutter et al., 1994
) and are sensitive biomarkers of exposure to TCDD and related compounds. These enzymes are also involved in the metabolism of estradiol (Hayes et al., 1996
; Liehr and Ricci, 1996
; Spink et al., 1990
, 1992
). Since endometriosis is an estrogen-dependent disease (Oral and Arici, 1997
; Witz, 1999
), altered metabolism of estradiol by TCDD or other dioxin-like halogenated aromatic hydrocarbons (HAHs) may be involved in the pathogenesis of endometriosis. However, it should be noted that HAHs such as PCBs can exhibit a complex and diverse range of effects on estradiol metabolism. For example, PCB 169 (3,3',4,4',5,5'-hexachlorobiphenyl) was recently found to induce the expression of CYP1A1 and CYP1B1 mRNA in human MCF-7 breast cancer cells without causing elevated rates of estradiol metabolism. In fact, PCB169 directly inhibited the production of 4-methoxyestradiol without affecting the production of 2-methoxyestradiol, reflecting decreased CYP1B1 activity without induction of CYP1A1 activity (Pang et al., 1999
). Thus, it is important to characterize complex, real-world exposures to HAHs, along with the potential of a given exposure, to modulate estradiol metabolism.
The human endometrial explant culture model (Osteen et al., 1994) used by Bruner-Tran et al. (1999) in the nude mouse model of endometriosis should be an excellent model for studying the direct effects of TCDD and related compounds on human endometrium. Neither the expression of CYP1A1 and CYP1B1 nor the expressions of AhR and ARNT have been investigated in this model. The purpose of this study was to characterize this model for studying the direct effects of TCDD exposure by investigating the expressions of CYP1A1 and CYP1B1 mRNA, protein, and activity in human endometrial explants cultured with and without TCDD. The expression of AhR and ARNT mRNA in these explants was evaluated using quantitative RT-PCR with an internal standard, and is reported in a parallel companion paper (Pitt et al., 2001
).
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MATERIALS AND METHODS |
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Surgical specimens.
A specimen of endometrial tissue was collected by pipelle or curet at the beginning of the surgical procedure. The specimen was divided into 2 portions. One-half of the specimen was placed in sterile, phenol-red free, D-MEM/F-12 medium (a 50:50 mix of Dulbecco's Modified Eagle's Medium and Ham's F-12 with 15 mM HEPES buffer, L-glutamine, and pyridoxine HCl; Life Technologies, Grand Island, NY). This portion was transported to the laboratory on ice for culture preparation. The second half of the specimen was sent to the pathology laboratory in the hospital for routine histological dating, as described by Mazur and Kerman (1995). The histological phase of the endometrium was recorded as proliferative or secretory.
Tissue culture media.
D-MEM/F-12 medium (phenol red-free with 15 mM HEPES buffer, L-glutamine, and pyridoxine HCl; Life Technologies, Grand Island, NY) was used in all stages of tissue preparation and explant culture as described by Osteen, et al. (1994). An initial culture medium (3% FBS-D-MEM/F-12) was used to establish the cultures prior to the start of the experiments. This medium consisted of D-MEM/F-12 with 3% charcoal-stripped fetal bovine serum (FBS; Life Technologies), 1% antibiotic/antimycotic solution (penicillin/streptomycin/amphotericin; Life Technologies, Grand Island, NY), and either E2 (10 nM) or E2:P4 (1 nM:500 nM) with DMSO (hormone vehicle) at a final concentration of 0.08%. Charcoal-stripped FBS was prepared by mixing and stirring FBS with washed and dried charcoal (30 g charcoal/1 L FBS) at 4°C overnight, then removing the charcoal with 1.22, 0.45, and 0.22 µm filters and performing heat inactivation at 56°C for 30 min. After the cultures were established, the experiments were performed in a serum-free D-MEM/F-12 medium containing 1% ITS+ PremixTM (insulin, transferrin, selenious acid, bovine serum albumin, and linoleic acid; Becton Dickinson Labware, Bedford, MA), 0.1% Excyte III (Bovine Lipoprotein Solution; Bayer Corp., Kankakee, IL), and 1% antibiotic/antimycotic solution. The cultures were treated with E2 (10 nM) or E2:P4 (1 nM:500 nM) with either TCDD or an equivalent volume of DMSO (TCDD vehicle), such that the final concentration of DMSO was 0.08%.
The final concentration of TCDD in the medium was 10 nM for all studies except the dose-response study in which the final TCDD concentrations were 0.001, 0.01, 0.1, 1.0, or 10 nM.
Explant culture.
The surgical specimens were prepared and cultured under sterile conditions similar to the method of Osteen et al. (1994). Each specimen was washed 23 times with D-MEM/F-12 medium and blood clots were dissected away as much as possible. The endometrial sample was cut into 1 x 2-mm uniform explants with a sterile scalpel blade, and the pieces were washed once more with D-MEM/F-12 and transferred to 3% FBS-D-MEM/F-12 medium. The 3% FBS-D-MEM/F-12 medium contained either E2 (specimens from women at cycle days 113, presumed to be in the proliferative phase) or E2:P4 (specimens from women at cycle days 14 or higher, presumed to be in the secretory phase); the phase of the endometrial samples could not be confirmed until the pathology report was available 2 weeks later. If the explants contained excessive blood, the pieces were incubated in the 3% FBS-D-MEM/F-12 medium for 3 h at 37°C until the blood had dissipated. Otherwise, the pieces were immediately transferred to 0.4 µm culture plate inserts (Millicell-CM, Fisher Scientific Co., Springfield, NJ) at a concentration of 810 pieces per insert in a 24-well plate (Costar Corporation, Cambridge, MA) with 1 ml 3% FBS-D-MEM/F-12 medium containing the appropriate hormone(s). The inserts were modified prior to use, by punching 3 holes around the wall of the insert, 3 mm above the membrane surface, to allow equilibration of the medium across the membrane. The explants were incubated for 2030 h at 37°C in a humidified 5% CO2-air environment to establish the cultures. At the start of an experiment (Time 0), the 3% FBS-D-MEM/F-12 medium was replaced with serum-free D-MEM/F-12 medium containing the appropriate hormone(s) and either TCDD or the vehicle control (DMSO). After 24-h incubation, the TCDD and control media were removed, and the explants were either harvested or incubated in media containing hormones, but no TCDD, for another 2496 h (Time 48120 h), changing the medium every 24 h. At the end of the incubation, the explants were either fixed and embedded in agar for morphology and immunohistochemistry or harvested for total RNA isolation or whole-lysate preparation.
Morphology and immunohistochemistry.
At the end of the culture incubation period, the medium was removed from each explant well and insert and replaced with 10% buffered formalin for overnight fixation. The formalin was then removed and 1% agar (55°C) was placed in each insert. After the agar solidified, the bottom of the insert membrane was scored with a razor blade and gently removed, and the agar plug containing the explants was carefully pushed out of the insert and soaked in 10% formalin for at least 4 h. The explant-agar plug was then paraffin-embedded and sections (5 µm thick) were placed on Superfrost "Plus" slides (Fisher Scientific Co., Pittsburgh, PA). Slides were stained with hematoxylin and eosin for characterization of morphology or by immunohistochemistry for the localization of CYP1B1 or CYP1A1 protein.
CYP1B1 localization was performed using a modification of the procedure of Walker et al. (1998), using standard deparaffinization and rehydration protocols and a CYP1B1 primary antibody (rabbit anti His6-CYP1B1 IgG provided by T. R. Sutter, University of Memphis, Memphis, TN), which is suitable for immunohistochemical detection. For antigen retrieval, pairs of slides, facing together and standing in a mini-icecube tray filled with sodium citrate buffer (10 mM), were steamed at 80°C for 12 min in a pre-equilibrated steamer (Black & Decker, Inc., Shelton, CT) containing 10 mM sodium citrate buffer in its lower chamber. The slides were cooled for 10 min, washed twice with PBS (5 min each), washed once with 1% BSA in PBS (5 min), and treated with normal goat serum to block nonspecific binding (5 min). The slides were incubated for 23 h at room temperature, then overnight at 4°C with either primary antibody or a negative control antibody (mouse myeloma protein IgG 2A; Organon Teknik, Durham, NC) that also reacts with the secondary antibody. The slides were washed with PBS, then incubated with biotinylated goat antirabbit IgG (secondary antibody) for 30 min. The PBS wash was repeated and the slides were incubated with streptavidin-alkaline phosphatase for 30 min. The PBS wash was repeated again, and the slides were incubated twice (30 min each) with fast-red color developer, followed by 2 PBS washes and counterstaining with Mayer`s hematoxylin. The normal serum, biotinylated anti-IgG, streptavidin-alkaline phosphatase, and fast-red color developer were obtained from Signet Laboratories, Inc. (Dedham, MA).
CYP1A1 protein was localized by a modification of the above CYP1B1 procedure and the procedure described by Drahushuk et al. (1999). The antigen retrieval step and preceding PBS wash step were eliminated, primary antibody was replaced with Mab 1123 (Drahushuk et al., 1999), and negative-control antibody was replaced with mouse myeloma protein IgG 2A (Organo Teknik, Durham, NC). Primary and control antibody incubation was for 180 min. Mab 1123 (Park et al., 1986
), which was prepared against a marine fish scup CYP1A1, was generously provided by J. Stegeman (Woods Hole Institute, Woods Hole, MA) and was previously shown to react with human CYP1A1 (Drahushuk et al., 1998
).
Primer design and synthesis.
Sense and antisense oligonucleotide primers for human cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) mRNAs were designed from the mRNA sequences using GeneJocky (Biosoft, Ferguson, MO). The sequences were obtained from Entrez (National Center for Biotechnology Information, NIH). The sense primer chosen for CYP1A1 (5'-CAA GCG GAA GTG TAT CGG-3') corresponded to bp 14451462, and the antisense primer (5'-CTT CCA GAG AGT TCT TCA GAG C-3') was the complementary sequence to bp 19311952. The sense primer chosen for CYP1B1 (5'-AGT TCT ACA GTG TCC TAA GTG C-3') corresponded to bp 36633684, and the antisense primer (5'-GGA CCT GGT TGA CAT AAT GAG G-3') was the complementary sequence to bp 42494270. GIBCO BRL Custom Primers (Life Technologies, Grand Island, NY) synthesized the primers.
Isolation of total RNA.
Total RNA was isolated from human endometrial explants by the single-step acid guanidinium thiocyanate-phenol-chloroform (Reagent D) extraction method (Chomczynski and Sacchi, 1987). Explants were pooled from wells cultured under the same conditions (3 wells per data point) and collected by centrifugation (1500 x g for 5 min).
Preparation of labeled cDNA probes.
Labeled cDNA probes were prepared for CYP1A1 and CYP1B1 mRNA, and 28S ribosomal RNA (28S rRNA). The CYP1A1 and CYP1B1 cDNAs were each reverse-transcribed from 5 µg of total RNA extracted from an endometrial explant preparation cultured in the presence of TCDD. Total RNA from a TCDD-treated specimen was chosen because these specimens were more likely to contain substantial quantities of CYP1A1 and CYP1B1 mRNA. The RT reactions were performed using Superscript II Reverse Transcriptase (Life Technologies). PCR reactions contained the following: 10 µl of 10 x PCR buffer (200 mM TrisHCl, pH 8.4, 500 mM KCl ), 0.5 µl Taq polymerase (5 U/µl Life Technologies), 3 µl of 100 ng/µl antisense primer (either CYP1A1 or CYP1B1), 3 µl of 100 ng/µl of the corresponding sense primer, 7.5 µl RT mixture, 8 µl or 5 µl of 25 mM MgCl2 for the CYP1A1 or CYP1B1 PCR reaction mixture, respectively, and DEPC-treated water to give a total reaction mixture volume of 100 µl. The contents of each PCR tube was gently mixed and centrifuged and 50 µl of mineral oil was added to prevent evaporation. PCR was performed in a thermal cycler (PowerblockTM System, Ericomp Inc., San Diego, CA) using the following parameters: denaturation at 95°C for 30 s, primer annealing at 55°C for 1 min, and strand extension at 72°C for 1 min for a total of 30 cycles. The products resulting from the RT-PCR reactions were a 507-bp CYP1A1 cDNA fragment and a 609-bp CYP1B1 fragment. These fragments were cloned into pCR 2.1 as described by the manufacturer (Invitrogen Corp., Carlsbad, CA). Plasmid DNA containing the CYP1A1 and CYP1B1 clones were sequenced at CAMBI (Center for Advanced Molecular Biology and Immunology, SUNY at Buffalo), and the resulting sequences were compared with CYP1A1 and CYP1B1 sequences in Genbank using GeneJockey (Biosoft, Ferguson, MO). CYP1A1 or CYP1B1 cDNA fragments were excised from the pCR 2.1 plasmid using EcoRI, and the excised cDNA fragments were resolved on agarose gels and purified by electroelution (S&S Elutrap System, Schleicher & Schuell, Inc., Keene, NH). A cDNA probe for 28S rRNA was prepared from the Phagemid clone of human 28S rRNA in E.coli (ATCC 77237, American Type Culture Collection, Manassas, VA). The cDNA probes for CYP1A1 mRNA, CYP1B1 mRNA, and 28S rRNA were random primer labeled according to manufacturers instructions using a Multiprime DNA Labeling System (Amersham International PLC, Amersham UK) and 50 µCi of 32P-dCTP (3000 Ci/mmol, Du Pont, NEN Research Products, Boston, MA).
Northern blot analyses.
Electrophoresis of total RNA from human endometrial explants was performed in 6.0% formaldehyde/1.0% agarose gels. Each sample lane contained 15 µg of total explant RNA. Ethidium bromide (10 µg) was added to each sample immediately before electrophoresis. Gels were photographed and blotted onto nylon membranes after the electrophoresis was completed. Prehybridization and hybridization were performed in a solution containing 50% formamide, 6X SSC (Sambrook et al., 1989), 50X Denhardt's solution (0.01% Ficoll, 0.01% polyviylpyrrolidone, 0.01% BSA) (Sambrook et al., 1989
), and 0.5% sodium dodecyl sulfate. Prehybridization was performed for 23 h at 42°C. Ten million cpm of probe was added to NTE buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, pH8.0; Sambrook et al., 1989) containing 200 µg of sonicated herring sperm DNA and denatured by boiling. The probe/herring sperm mixture was added directly into the prehybridization solution, and the hybridization was allowed to proceed overnight at 42°C. The blots were washed twice with 50 ml Wash I solution (100 ml 20 x SSC and 50 ml SDS diluted to 1 liter with deionized H2O) at 42°C for 20 min, then washed with 50 ml Wash Solution II (5 ml 20 x SSC and 10 ml SDS diluted to 1 liter), 3 times at 53°C for 20 min. The washed blots were then exposed to X-ray film, and the autoradiographic signals were quantitated using a densitometer (Model GS-700 Imaging Densitometer, Bio-Rad Laboratories, Hercules, CA). Densitometric measurements of CYP1A1 or CYP1B1 mRNA were normalized to the amount of 28S rRNA in the same sample lane.
Whole-tissue lysate preparation.
Whole-tissue lysates were prepared from cultured human endometrial explants that were pooled from wells cultured under the same conditions (2 wells per data point) and collected by centrifugation (1500 x g, 5 min). The explants were suspended in PBS (a 1:10 dilution of ice-cold 10X Dulbecco's phosphate-buffered saline from Life Technologies) in a ratio of 100 mg tissue/ml PBS, then homogenized on ice by sonication (Heat Systems-Ultrasonics Model W-380 sonicator, Farmingdale, NY; 510% power, using intervals of five 0.5-s pulses with 1 min resting periods between intervals) until lysed. The homogenates were centrifuged at 200 x g for 5 min, and the supernatants were saved for protein and 7-ethoxyresorufin-O-deethylase (EROD) assays, and for Western blotting to measure CYP1B1 protein.
Protein and 7-ethoxyresorufin-O-deethylase (EROD) assays.
EROD activity was measured by a fluorometric method previously described by Prough et al. (1978) that was modified for use in a 48-well plate (Costar, Cambridge, MA), and protein concentration was measured in the same wells using fluorescamine. Each well contained whole tissue-lysate supernatant (50 µl) and 890 µl of PBS or 1 ml of bovine serum albumin (BSA) as standards for a protein calibration curve. Ethoxyresorufin (Eastman Kodak, Rochester, NY; 50 µl of 10 µM) was added to all wells containing the tissue lysates, followed by the addition of 10 µl of 10 mM NADPH. After 5 min at room temperature, the fluorescence of each well was measured using a Cytofluor II (PerSeptive Biosystems, Framingham, MA) with an excitation filter of 530 nm and an emission filter of 590 nm to obtain a baseline reading. The plate was then incubated at 37°C for 45 min and the fluorescence was measured again before and after the addition of an internal calibration standard of resorufin (Eastman Kodak, Rochester, NY; 10 µl containing 50 pmol) to all wells, including the wells for the protein calibration curve. The excitation filter was then changed to 360 nm and the emission filter to 460 nm for the protein assay. Following a baseline reading, fluorescamine (Molecular Probe, Eugene, Oregon; 100 µl of a 0.6 mg/ml solution in acetonitrile) was added to each well and the plate was incubated at room temperature, in the dark, for 10 min. The fluorescence was measured and the relative protein concentration (mg/ml) of each sample was determined from the protein calibration curve. The EROD activity was then calculated as pmol resorufin produced/min/mg lysate protein.
SDSPAGE and Western immunoblotting.
Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) was performed on whole lysates of human endometrium, essentially according to the method of Laemmli (1970). Western immunoblotting of CYP1A1 protein was performed as described by Drahushuk et al. (1998), utilizing secondary antibodies conjugated with alkaline phosphatase and the CDP-Star (Tropix, Bedford, MA) chemiluminescent substrate. This method used the Mab 1123 antibody prepared against a marine fish scup CYP1A1 (Park et al., 1986) which was previously shown to detect human liver CYP1A1 (Drahushuk et al., 1998
). Mab 1123 and marine fish scup CYP1A1 standard were generously provided by J. Stegeman (Woods Hole Oceanographic Institute, Woods Hole, MA).
CYP1B1 protein in whole lysates of human endometrium was quantitated by an adaptation of the CYP1A1 protein method. The CYP1A1 standard and the CYP1A1 primary antibody were replaced with a recombinant mouse CYP1B1 protein standard and a rabbit antimouse CYP1B1 primary antibody prepared against the protein standard (Pottenger et al., 1991). The CYP1B1 protein standard and primary antibody were gifts from C. R. Jefcoate (University of Wisconsin Medical School, Madison, WI). This antibody was found to be suitable for detection of human CYP1B1 protein by Western immunoblotting.
Statistical analysis.
Statistical analysis was performed using Systat 8.0 (SPSS Inc., Chicago, IL). The effect of TCDD exposure (with vs. without TCDD exposure) was analyzed for 4 data sets (CYP1A1 mRNA, CYP1B1 mRNA, EROD activity, and CYP1B1 protein) using the paired t-test, to take into account variations in inducibility between specimens. The CYP1A1 and CYP1B1 mRNA data were obtained at 24 h after initial TCDD exposure, and EROD activity and CYP1B1 protein data were obtained at 72 h after initial TCDD exposure. The effect of several covariates (donor age group 30 or >30, endometriosis status, smoking history, and birth control pill use; cycle phase of tissue; and hormonal treatment of tissue in culture) was analyzed on 3 different subsets of each of these 4 data sets. The subsets were as follows: (1) data obtained without TCDD exposure (constitutive expression), (2) data obtained with TCDD exposure (induced expression), and (3) the difference between the values obtained with and without TCDD exposure (a measure of the amount of induction). Each covariate was examined individually for each subset of each data set, using a 2-group (unpaired) t-test rather than a multiple regression model. The age groups of
30 and >30 were chosen based on scatter plots of induced EROD activity vs. age, which showed these 2 distinct age-group populations, unlike other data sets and subsets. The correlation between each pair of experimental parameters (CYP1A1 mRNA vs. CYP1B1 mRNA; EROD activity vs. CYP1B1 protein; CYP1A1 mRNA vs. EROD activity; CYP1B1 mRNA vs. CYP1B1 protein; and CYP1A1 mRNA vs. CYP1B1 protein) was determined by linear regression analysis. For this analysis, the data included the above 4 data sets along with data from uncultured biopsies and data used to examine the effect of TCDD dose and culture time. All results were considered significant only if p
0.05.
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RESULTS |
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Distribution of EROD Activities by Donor Age, Cycle Phase, and Type of Hormonal Treatment in Culture
Figure 6A shows the age distribution of the TCDD-induced EROD activities (+TCDD data set from Fig. 4C
plus 4 additional specimens). The results show that the highest TCDD-induced EROD activities were associated with specimens from donors of age 30 and under. The majority of these specimens (8/11) had TCDD-induced EROD activities of greater than 9 pmol resorufin formed/min/mg lysate protein. If other covariates are ignored, the difference in the TCDD-induced EROD activities between the
30 (n = 11) and >30 (n = 9) age groups is statistically significant (p = 0.01).
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None of the other covariates studied (smoking history; current birth control pill use; current endometriosis; or endometriosis, including both current and past history cases) were related to TCDD-induced EROD activity or any of the other parameters measured. Also, age and cycle phases were not related to CYP1B1 protein, CYP1A1 mRNA, or CYP1B1 mRNA. Further studies are needed to discriminate between the effects of age and cycle phase on TCDD-induced EROD activity.
Immunohistochemical Localization of CYP1B1 Protein
CYP1B1 protein was easily detected in the biopsy and explant culture specimens of human endometrium (Fig. 7). In the biopsy (Fig. 7A
), red staining was predominantly seen in the cytoplasm of the epithelial glands compared with the control (Fig. 7B
). The surface epithelial membrane was unstained, except for occasional patches of membrane. In the explant cultures before (Fig. 7C
) and after (Figs. 7D and 7E
) TCDD exposure, CYP1B1 protein was most prominent in the new surface epithelial membrane and in the glands closest to the surface epithelium compared to the 120-h control (Fig. 7F
). These results suggest that CYP1B1 protein expression is associated with the tissue remodeling process.
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DISCUSSION |
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The explant culture model developed by Osteen et al. (1994) uses fresh human endometrial biopsy tissue that contains both epithelial and stromal cells in normal relationship. This model is better for initial studies of CYP1A1 and CYP1B1 expression than models that use isolated stromal or epithelial cells (Sharpe-Timms, 1997). Osteen et al. (1994) and Bruner et al. (1995) previously demonstrated the importance of communication between epithelial and stromal cells when the 2 cell types were cocultured in separate connected chambers and evaluated for cooperation in metalloproteinase expression. In the model of Osteen et al. (1994), intact biopsies are cultured on Millicell-CM culture plate inserts (Fisher Scientific Co., Springfield, NJ) which are readily available and allow easy recovery of the tissue for biochemical analysis. Thus, the explant culture model of Osteen et al. (1994) is an ideal in vitro model for assessing the effects of environmental agents on human endometrial function.
In the current report, the viability of human endometrial explants in culture was confirmed by the re-epithelialization of the biopsies and by the maintenance of the basic tissue architecture. Secretory phase explants established in culture for 2030 h in the presence of 1 nM E2:500 nM P4 reorganized and formed a new surface epithelial membrane (Fig. 1B). The basic tissue morphology of epithelial glands and stroma was maintained for up to 120 h after initial TCDD exposure (Figs. 1C and 1D
). Similar results were seen in preliminary studies using 10 nM E2, with either proliferative or secretory phase endometrium, for up to 48 h in culture, with or without initial 24-h TCDD exposure (data not shown).
Another advantage of the human endometrial explant culture model of Osteen et al. (1994) is that viable human cultures treated with environmental agents can be subsequently implanted into athymic (nude) mice for the evaluation of the effects of tissue treatment on the development of endometriotic lesions (Bruner et al., 1997, 1999
; Bruner-Tran et al., 1999
). In this model, the explants are injected intraperitoneally into ovariectomized athymic (nude) mice treated 24 h earlier with subcutaneous estradiol-releasing pellets. This human model of endometriosis in the nude mouse offers several advantages over typical mouse models (Cummings et al., 1996
, 1999
; Johnson et al., 1997
; Yang and Foster, 1997
) and a primate model (Yang et al., 2000
): (1). It uses human endometrial tissue, rather than mouse or primate tissue; (2) it allows experimental manipulation of the tissue prior to the induction of endometriosis; (3) it uses endometrium alone, without myometrium, to more accurately mimic endometriosis; (4) it allows the natural attachment of endometrium to peritoneal surfaces without suturing; and (5) it allows the biochemical effects of in vitro exposure to be correlated with the development of endometriotic lesions. Using this human model of endometriosis, Bruner-Tran et al. (1999) demonstrated that in vitro exposure of human endometrial explants to TCDD promoted the development of endometriosis by enhancing tissue metalloproteinase expression. The expression of cytochrome P450 biomarkers of TCDD exposure have not been previously examined in the human endometrial explant culture model of Osteen et al. (1994) or in the model of Bruner et al. (1997, 1999) and Bruner-Tran et al. (1999).
In humans, at least 25 distinct cytochrome P450 enzymes have been identified (Guengerich, 1994) and at least 60 are estimated to be expressed (McKinnon and McManus, 1996
). Dioxin-inducible cytochrome P450 enzymes, including CYP1A1, CYP1A2, and CYP1B1, catalyze the metabolic activation of a wide range of chemical carcinogens. In addition to their role in the metabolism of environmental pollutants, CYP1A1, CYP1A2, and CYP1B1 have other significant biological/toxicological functions. For example, CYP1A1 and CYP1B1 are capable of modulating endocrine function by metabolizing 17ß-estradiol to the 2- and 4-hydroxylated derivatives, respectively (Hayes et al., 1996
; Safe, 1995
). In contrast to CYP1A2, which is generally considered a liver-specific enzyme, CYP1A1 and CYP1B1 are ubiquitously expressed in most human tissues. Although the constitutive expression of CYP1A1 and CYP1B1 is generally low, these enzymes are readily inducible in a number of tissues, including liver, lung, pulmonary alveolar macrophages, lymphocytes, epidermal keratinocytes, prostate, mammary tissue, placenta, and endometrium (Drahushuk et al., 1998
; Hakkola et al., 1997
; Omiecinski et al., 1990
; Rannug et al., 1995
; Sesardic et al., 1990
; Shimada et al., 1992
, 1996
; Sutter et al., 1994
; Vadlamuri et al., 1998
; Wong et al., 1986
). Since the induction of these cytochrome P450 enzymes is a sensitive response (Trischer et al., 1992), they are commonly employed as biomarkers for exposure, effect, and susceptibility to TCDD and other dioxin-like environmental contaminants.
In the current study, the CYP1B1 protein exhibited substantial constitutive expression in uncultured human endometrial biopsies, newly established explant cultures (2030 h with 3%FBS medium), and explants cultured for an additional 72 h in serum-free medium without TCDD exposure. However, EROD activity, CYP1A1 mRNA, and CYP1B1 mRNA were undetectable or minimal in cultures without TCDD exposure. Previous studies, using quantitative reverse transcriptasepolymerase chain reaction, rather than Northern blotting, demonstrated substantial constitutive expression of CYP1B1 mRNA in eutopic human endometrium (Bulun et al., 2000; Hakkola et al., 1997
; Igarashi et al., 1999
), while CYP1A1 mRNA levels were undetectable (Hukkanen et al., 1998
) or minimal (Bulun et al., 2000
), as in the current report.
To the knowledge of the authors, the present study provides the first report on the localization of CYP1B1 protein in human endometrium. In this report, CYP1B1 protein was localized throughout the cytoplasm of epithelial glands in an uncultured fresh secretory phase endometrial biopsy (Fig. 7A) and in explants established in culture from the same tissue for 2030 h in the presence of E2:P4 (Fig. 7C
). In a previous study, AhR protein was distinctly present in the apical portion of the glands, while AhR mRNA was distributed throughout the cytoplasm (Küchenhoff et al., 1999
). In the present study, the surface epithelial membrane of the fresh biopsy contained only occasional patches of CYP1B1. In contrast, the cultured explants contained prominent levels of CYP1B1 in the newly formed surface epithelial membrane, suggesting that CYP1B1 protein expression is associated with the tissue remodeling process. This hypothesis needs to be tested further in cultures under different hormonal conditions from both proliferative and secretory phase specimens.
A dose-response experiment (Fig. 2) demonstrated that the expression of CYP1A1 mRNA, CYP1B1 mRNA, and EROD activity was highest in explants cultured for 24 h in the presence of 10 nM TCDD. Thus, 10 nM TCDD was used in all other experiments in this report. A dose-dependent induction of CYP1A1 was also observed in rat and human liver slices maintained in dynamic organ culture, with 10 nM TCDD producing maximal induction (Drahushuk et al. 1998
, 1999
). Bruner-Tran et al. (1999) also used 10 nM TCDD in a study in which proliferative phase human endometrial explants were cultured for 24 h with or without TCDD exposure in the presence of hormones, then injected into nude mice. This level of TCDD enhanced E2-mediated tissue metalloproteinase expression and endometriotic lesion growth, and completely blocked P4-induced suppression of these events (Bruner-Tran et al., 1999
).
A time-course study (Fig. 3) demonstrated that exposure to 10 nM TCDD for 24 h resulted in a time-dependent increase in CYP1A1 and CYP1B1 mRNA for up to 72 h following initial TCDD exposure, while EROD activity continued to increase for up to 120 h after initial exposure. Similarly, in TCDD-exposed rat and human liver slices maintained in dynamic organ culture, CYP1A1 protein and EROD activity increased with time over a 96-h incubation period (Drahushuk et al., 1998
, 1999
), although only the initial 24 h was with TCDD.
Bruner-Tran et al. (1999; Discussion) observed that the human endometrial explant cultures begin to break down after 45 days, and routinely used or analyzed the cultures after 2448 h. In the current study, the cultures were not studied beyond 5 days. Due to the limited availability of human tissue, CYP1A1 and CYP1B1 mRNA expression were routinely measured 24 h after initial TCDD exposure, and EROD activity and CYP1B1 protein expression were routinely measured 72 h after initial TCDD exposure. Constitutive expression was measured under the same conditions without TCDD exposure.
Under these conditions, all 4 biomarkers were significantly induced by 10 nM TCDD (Fig. 4). The average induction of EROD activity was 35-fold (range = 3.5- to 137-fold), while the average induction of CYP1B1 protein was only 1.6-fold (range = 1.1- to 2.5-fold) above the already high constitutive expression. The degree of induction could not be measured for CYP1A1 mRNA and CYP1B1 mRNA because of the undetectable levels of constitutive expression. A recent study by Bulun et al. (2000) demonstrated that the level of CYP1A1 mRNA expression was 8.7-fold higher in endometriotic tissues than in paired eutopic endometrium, suggesting that expression of this dioxin-responsive gene may be associated with endometriosis.
In the current study, the immunohistochemical localization of CYP1B1 protein in secretory phase explants exposed to 10 nM TCDD in the presence of E2:P4 (Fig. 7D and 7E) was similar to that of newly established explants before TCDD exposure (Fig. 7C
). CYP1B1 was localized in the cytoplasm of the epithelial glands and in the newly formed surface epithelial membrane for up to 120 h after exposure.
In contrast to the consistent detection of both constitutive and TCDD-induced expression of CYP1B1 protein, CYP1A1 protein could not be routinely detected, even after TCDD exposure. For CYP1A1 protein, an antibody against CYP1A1 protein from marine fish scup (Park et al., 1986) was used for both Western blotting and immunohistochemistry, while for CYP1B1 protein, an antibody against mouse CYP1B1 protein (Pottenger et al., 1991
) was used for Western blotting, and an antibody against a peptide of human CYP1B1 (Walker et al., 1998
) was found to be optimal for immunohistochemistry. The limit of detection of human CYP1A1 by Western immunoblot was from 0.025 to 0.05 pmol of recombinant human CYP1A1. While this antibody is very sensitive in detecting CYP1A1 in microsomal protein from human liver (Drahushuk et al., 1998
), it is less sensitive in detecting CYP1A1 in whole lysates of tissue. Whole lysates of human endometrium were used because the limited sample size prohibited the routine preparation of microsomal samples from human endometrium. CYP1A1 was detected in microsomal samples obtained from a larger sample of human endometrium, cultured with and without TCDD. This single experiment showed an increase in CYP1A1 protein with TCDD exposure (results not shown). This limited finding suggests that while CYP1A1 is present at low levels in cultured human endometrium, it was not able to be routinely detected, since whole lysates were used in Western blot studies.
Despite the apparent disparity in the levels of CYP1A1 and CYP1B1 proteins, preliminary measurements on the levels of CYP1A1 and CYP1B1 mRNA measured by quantitative RT-PCR demonstrated similar levels of CYP1A1 and CYP1B1 mRNA in human endometrial explants. Using modifications of the methods of Drahushuk et al. (1998) and Spencer et al. (1999), the molecules of CYP1A1 and CYP1B1 mRNA/femtogram total RNA were determined in explants cultured at 2 different time points. At 24 h after initial TCDD exposure, the number of molecules was 2.513 for CYP1A1 (n = 2) and 7.433 (n = 3) for CYP1B1, and at 72 h after initial TCDD exposure, the number of molecules was 60100 for CYP1A1 (n = 2) and 2060 (n = 3) for CYP1B1 (results not shown). The observation that TCDD induces a similar level of expression of CYP1A1 and CYP1B1 mRNA, with the detection of only CYP1B1 protein, suggests that the translation of CYP1A1 mRNA may be diminished relative to CYP1B1 in human endometrial explants. If the translation of CYP1A1 mRNA is diminished, the EROD activity in human endometrium may be predominantly due to CYP1B1 activity rather than CYP1A1 activity. A previous study (Shimada et al., 1997) demonstrated that recombinant human CYP1B1 protein in yeast microsomes exhibited about one-tenth the EROD activity of human CYP1A1 protein in a reconstituted system isolated from the membranes of E. coli in which the cDNA was expressed.
In the present study, the levels of CYP1A1 protein and EROD activity might be diminished because of the presence of E2 in the cultures. In a previous study in a human endometrial adenocarcinoma epithelial cell line (ECC-1), 10 nM E2 reduced TCDD-induced 7-ethoxycoumarin-O-deethylase (ECOD) activity by 75% and produced a similar decrease in CYP1A1 mRNA levels (Ricci et al., 1999a). The same concentration of E2 also reduced TCDD-induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in a mouse endometrial stromal cell line (E041; Stols and Iannaccone, 1985) and reduced TCDD-induced ECOD activity in MCF-7 human breast cancer cells (Ricci et al., 1999a
). However, in contrast to the E2-mediated decrease in TCDD-induced CYP1A1 mRNA levels in ECC-1 cultures, E2 did not affect the induction of CYP1B1 mRNA (Ricci et al., 1999a
). Evidence was presented that E2 controlled the TCDD-induced expression of CYP1A1 at the transcriptional level by decreasing available nuclear factor-1 (NF-1), a transcription factor that interacts with both the Ah and estrogen receptors (Ricci et al., 1999a
). The CYP1B1 gene does not have NF-1 binding sites (Wo et al., 1997
) and this was thought to be the reason for the differential effect of E2 on the TCDD-induced expression of CYP1A1 and CYP1B1 mRNA in ECC-1 cultures (Ricci et al., 1999a
).
In the current study, no differences were observed in the expression of CYP1A1 mRNA, CYP1B1 mRNA, EROD activity, and CYP1B1 protein with the 2 different hormone treatments (10 nM E2 and 1:500 nM, E2:P4) in human endometrial explant cultures, either with or without TCDD exposure. In contrast to the 75% reduction in TCDD-induced ECOD activity by 10 nM E2 in ECC-1 cultures, 10 nM P4 caused a 20% increase in this activity (Ricci et al., 1999a). In the same cultures, TCDD reduced estrogen receptor-mediated transcription by 50% (Ricci, et al., 1999b
), an effect reciprocal to the effect of E2 on TCDD-induced gene expression (Ricci, et al., 1999b
). TCDD also reduced estrogen receptor levels by 40%, but a reciprocal effect of E2 on AhR levels was not evaluated in this study (Ricci, et al., 1999b
). The studies of Ricci et al. (1999a,b) clearly demonstrate differential interactions between the various hormones and TCDD. In the current study, the effect of hormones on TCDD-induced gene expression could not be thoroughly evaluated because of the limited availability of specimens. Also, no specimens were treated with TCDD in the absence of hormones in order to establish a hormone-free baseline for TCDD responsiveness. Further work is needed on the human endometrial explant culture model to evaluate the interactions between the hormones and TCDD.
In addition to variations in the hormone treatment used in culture, the current study used endometrium specimens from both the proliferative and secretory phases. Proliferative phase specimens from donors at cycle days 113 were treated with E2 and proliferative phase specimens from donors at cycle days 14 or higher (longer than average cycles) were treated with E2:P4, while all secretory-phase specimens were treated with E2:P4. The hormonal treatment of the proliferative-phase specimens varied because the phase of the endometrial specimens could not be confirmed until the pathology report was available 2 weeks after specimen collection. Thus, during culture set up, specimens from women at cycle days 113 were presumed to be in the proliferative phase and were treated with E2, and specimens from women at cycle days 14 or higher were presumed to be in the secretory phase and were treated with E2:P4. The intent of the hormone treatment was to mimic the hormonal milieu of the specimen donor. In the current study, the effects of hormonal treatment on cytochrome P450 expression in proliferative phase specimens could not be assessed because too few specimens were treated with E2 compared to E2:P4 and, also, because the cycle lengths were not comparable for the 2 treatment regimens.
Although the effect of hormonal treatment could not be evaluated for either phase of the tissue, the effects of cycle phase on the expression of EROD activity (8 proliferative and 8 secretory; Fig. 6B) and CYP1B1 protein (6 proliferative and 5 secretory) (data not shown) could be evaluated for the E2:P4-treatment group. In these specimens, TCDD-induced EROD activity was significantly higher in proliferative-phase specimens. A similar trend was observed in constitutively expressed, but not TCDD-induced, CYP1B1 protein, but did not reach the level of statistical significance (p = 0.09). Pitt et al. (unpublished data) observed that ARNT weakly correlated with cycle phase in human endometrial explants cultured with E2:P4, being marginally higher in the proliferative phase. In uncultured human endometrium, neither ARNT nor AhR mRNA (Igarashi et al., 1999
) nor CYP1B1 mRNA (Bulun et al., 2000
; Igarashi et al., 1999
) was related to the phase of the cycle, while maximum expression of AhR protein was reported to occur at the time of ovulation (Küchenhoff et al., 1999
).
In addition to the effect of cycle phase on TCDD-induced EROD activity in E2:P4-treated human endometrial explant cultures, TCDD-induced EROD activity was related to the age of the endometrial tissue donor (Fig. 6A). Tissues from donors of age 30 years and under had statistically higher TCDD-induced EROD activities than tissues from older donors. However, the limited number of available specimens prevented the effects of age and cycle phase from being distinguished. In this study, both younger age and proliferative phase were associated with higher TCDD-induced EROD activity. None of the other biomarkers were related to age or cycle phase.
While higher levels of TCDD-induced EROD activity were observed in E2:P4-treated human endometrial explants prepared from younger donors, Pitt et al. (unpublished data) observed lower levels of AhR and ARNT mRNA in explants similarly cultured from younger donors, both with and without TCDD exposure. In contrast to Pitt et al. (unpublished data.) and analogous to the present study, Küchenhoff et al. (1999) observed higher levels of AhR protein in younger donors of uncultured endometrial biopsy tissue. Pitt et al. (unpublished data) suggested that a downregulation in the translation of AhR mRNA with age may account for the lower levels of AhR protein in the Küchenhoff et al. (1999) study.
Aside from age and cycle phase, none of the other covariates (donor birth control pill use, smoking history, or endometriosis status) were related to the expression of CYP1A1 mRNA, CYP1B1 mRNA, EROD activity, or CYP1B1 protein in the current study. Previous studies on uncultured endometrium also did not observe any differences in the expression of CYP1A1 mRNA (Bulun et al., 2000) or CYP1B1 mRNA (Bulun et al., 2000
; Igarashi et al., 1999
) in eutopic endometrium from women with and without endometriosis. Similarly, Igarashi et al. (1999) and Pitt et al. (unpublished data) did not detect any endometriosis-related differences in the expression of AhR or ARNT mRNAs in uncultured endometrium or human endometrial explants, respectively.
Although no significant endometriosis-related differences were observed for any of the biomarkers in the current study, the power of the study to detect disease-specific change was limited by small sample size and variability in the cycle phase of the tissues. The real value of the current study is that it demonstrates the viability and TCDD responsiveness of the human endometrial explant culture model and its usefulness for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS, NIH.
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