Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Modulates the Pathophysiology of Endometriosis in the Cynomolgus Monkey

Jack Z. Yang*, Sanjay K. Agarwal{dagger} and Warren G. Foster*,{ddagger},1

* Environmental and Occupational Toxicology Division, Health Protection Branch, Department of Health, Ottawa, Ontario, Canada; and {dagger} Department of Obstetrics and Gynecology and {ddagger} Center for Women's Health, Cedars-Sinai Medical Center, Los Angeles, California 90048

Received February 4, 2000; accepted April 11, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An increase in the incidence and severity of endometriosis following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was a serendipitous finding in a reproductive toxicology study in rhesus monkeys. The purpose of this study was to investigate the effects of subchronic exposure to TCDD on the survival and growth of surgically implanted endometrial fragments. Endometrial fragments of equal size (4 x 1 mm2) were auto-transplanted to the pelvic cavity of nulliparous cynomolgus monkeys (Macaca fascicularis, n = 23), who were divided into 4 treatment groups and dosed 5 days a week with gelatin capsules containing 0, 1, 5, or 25 ng/kg body weight of TCDD mixed with glucose. Endometrial implant survival was monitored by laparoscopy at intervals of 1, 3, and 6 months. Animals were euthanized at 12 months of treatment in the early to mid luteal phase and the maximal and minimal endometrial implant diameter was measured. Both the maximal and minimal diameters were significantly reduced in the 0.71-ng/kg/day-TCDD dose group, compared to controls, whereas the survival rate was unaffected (20 vs. 16%, respectively). In contrast, exposure to 3.57 and 17.86 ng/kg/day TCDD for 1 year resulted in a significantly higher survival rate of implants (26.7% and 33.3% respectively vs. 16.0%) and significantly larger diameter implants in the 17.86-ng/kg/day dose group only, compared to the control group. Treatment had no effect on circulating gonadal steroid levels or menstrual cycle characteristics. It is concluded that TCDD facilitates the survival of endometrial implants and exerts a bimodal effect on endometrial implant growth.

Key Words: endometriosis; cynomolgus monkey; toxicology; laparoscopy; 2,3,7,8-tetrachlorodibenzo-p-dioxin; interleukin-6; interleukin-6sR..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial glands and stroma outside the uterine cavity. It is a common gynecologic disorder affecting approximately 14% of women of all reproductive ages (Vercillini et al., 1995Go) as well as a major cause of infertility (Chedid et al., 1995Go). Although retrograde menstruation or bleeding into the peritoneal cavity during menstruation is widely accepted as a major contributing factor in the pathogenesis of this disease, it is a common phenomenon even in women without endometriosis (Halme et al., 1984Go). Hence, factors other than retrograde menstruation are thought to contribute to the development and progression of endometriosis.

Chemical contaminants have been inculpated in the pathobiology of endometriosis as a result of a series of animal and epidemiology studies. The notion that environmental contaminants are involved in the pathophysiology of endometriosis gained momentum with the demonstration (Rier et al., 1993Go) that the incidence and severity of spontaneous endometriosis was increased in rhesus monkeys following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a ubiquitous contaminant produced during the manufacture of chlorophenols and phenoxy herbicides (Firestone et al., 1972Go; Lilienfield and Gallo., 1989Go), waste incineration (Alexandrou and Pawliszyn, 1989Go; Rappe et al., 1979Go) and the bleaching of pulp for paper (EPA, 1987Go). The mean adipose tissue level for the American population has been reported (Orban et al., 1994Go; Schecter et al., 1994aGo,bGo) to be between 5.2 and 5.4 ng/kg. Adipose tissue levels in individuals exposed, either through the workplace or as a result of accidental exposure, range between 10 and 141 ng/kg (Schecter et al., 1994bGo). In women with both endometriosis and measurable levels of TCDD in whole blood, the non-lipid-adjusted concentration of TCDD was between 0.7 and 1.2 ng/kg (Mayani et al., 1997Go). Two epidemiology studies (Gerhard and Runnebaum, 1992Go; Mayani et al., 1997Go) suggest a potential positive association between endometriosis and exposure to environmental contaminants while others have been unable to find a positive association (Boyd et al., 1995Go; Lebel et al., 1998Go). Hence, the human data neither confirm nor refute the hypothesis that environmental contaminants play a role in the pathobiology of endometriosis. Consequently, the role of TCDD in the pathophysiology of endometriosis remains highly controversial.

The objective of this study was to test the hypothesis that subchronic exposure to TCDD facilitates the survival and growth of surgically induced endometriosis in monkeys. The dose levels used in the current study (1, 5, and 25 ng/kg, 5 days/week) were selected so as to be comparable with those of a previous study in rhesus monkeys (Rier et al., 1993Go). Finally, surgical induction of endometriosis was employed in the present study since prior studies (Fanton and Golden, 1991Go; Rier et al., 1993Go) have shown that the development of spontaneous endometriosis in the monkey, induced by either TCDD or radiation exposure, involves a substantial time delay of between 6 and 7 years.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Nulliparous cynomolgus monkeys (Macaca fascicularis, n = 23) between the ages of 7 and 10 years of age were selected from 36 monkeys available for the study. All of these monkeys were colony-bred at the Health Protection Branch, Health Canada, Ottawa, Ontario, Canada. The selection criteria included (1) at least 3 regular menstrual cycles prior to surgical manipulations; and (2) body weight greater than 3 kg and less than 5 kg in order to minimize potential variation in results from differences in pharmocokinetics of the administered dose in obese vs. lean animals. Any animals with a history of pelvic or abdominal surgery, pregnancy, or evidence of existing endometriosis were excluded from the study.

Monkeys were randomly assigned to 1 of 4 treatment groups, control (n = 5), 1, 5, and 25-ng/kg TCDD (n = 6, each). Doses were prepared for each animal by emulsifying TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, Lot number 129–404, AccuStandard, New Haven, CT., purity > 99%) in saline and a 20 % Tween solution (Bio-Rad Laboratories, Mississauga, ON.) at a concentration of 1000 µg/ml TCDD, thus forming a stock emulsion. Serial dilutions were prepared and TCDD was added to glucose-filled capsules to yield appropriate doses for each animal, based on their monthly body weight. Animals assigned to the control group received gelatin capsules containing glucose only. The monkeys received 1 capsule 5 days per week, and hence, the actual delivered doses were 0.71, 3.57, and 17.86 ng/kg/day, respectively.

All monkeys were housed in individual cages in light- and temperature-controlled rooms with lights on 6:00 A.M. to 6:00 P.M., with a temperature of 20–22°C. Access to food (Purina monkey chow; Ralston-Purina, St. Louis, MO) and water was on an ad libitum basis. The diets were supplemented with fruits and vegetables. All animal husbandry practices and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Health Protection Branch and followed the guidelines of the Canadian Council of Animal Care.

The experimental design is summarized in Figure 1Go. Briefly, menstrual cycle characteristics were monitored daily to insure that all animals were cycling normally before and throughout the study. Time zero was assigned to the month in which endometriosis was induced by auto-transplantation of endometrial strips, and TCDD treatment was initiated the morning after surgical induction of endometriosis. Laparoscopies were performed in all groups at 1, 3, and 6 months to monitor the survival of endometrial implants and to obtain peritoneal fluid for determination of interleukin-6 (IL-6) and interleukin-6 soluble receptor (IL-6sR) concentrations and immunotyping. However, the amount of peritoneal fluid present in our monkeys was insufficient for assay purposes. Therefore, the peritoneal cavity was flushed with 150 ml of body temperature isotonic saline; 10 min later, the fluid was collected for flow cytometric analyses (data not shown). Blood was collected at the time of laparoscopies for routine hematology and at 6 and 12 months for assessment of circulating levels of the endometrial growth regulatory cytokine IL-6 and IL-6sR. Serum gonadal steroid levels were determined at the time of necropsy. All monkeys were euthanized at 12 months of treatment in the early to mid luteal phase to minimize the influence of menstrual cycle on circulating gonadal steroid levels.



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FIG. 1. Schematic representation of the experimental design employed in this study. All manipulations are indicated above, with outcome measures indicated below the time line. Routine hematology was performed to monitor the animals for signs of general toxicity. Monkeys were also monitored daily for menstrual cycle characteristics. Endometriosis was induced by auto-transplantation of endometrium on days 12–14 of the cycle following 3 months of regular menstrual cycle characteristics, and TCDD treatment was started on the day after surgery. Laparoscopies were performed at 1, 3, and 6 months after induction of endometriosis to monitor the viability of endometrial implants. All monkeys were euthanized during the early to mid luteal phase of the menstrual cycle to minimize variation in outcome measures from changes in circulating gonadal steroids.

 
Anesthesia
Animals were fasted overnight, given glycopyrrolate (0.015 mg/kg, IM, Wyeth-Ayerst Canada, Inc., Montreal, Canada) 20 min before surgery, followed by an intramuscular injection of ketamine (10 mg/kg, Pittman-Moore, Middlesex, UK) for induction of general anesthesia. Animals were intubated and maintained under isoflurane anesthesia.

Induction of Endometriosis
Endometriosis was induced on days 12–14 of the menstrual cycle by auto-transplantation of endometrial strips to multiple abdominal sites. This time interval was selected because the endometrium in these animals has achieved maximal thickness by this stage of the menstrual cycle. Briefly, a mid-line abdominal incision was made, the uterus was exposed, and an anterior-vertical fundal hysterotomy made. The endometrium was dissected from the myometrium on both sides of the hysterotomy incision site only. Endometrial strips were removed and placed in warm sterile saline and the uterus was closed.

The endometrial strips were cut into 6 small pieces of equal size (4 x 1 mm2). Five of the 6 endometrial explants were auto-transplanted with a single 8–0 nylon micro-suture stitch (Ethicon Inc., Johnson and Johnson Co., Somerville, NJ) to 5 sites: the uterine fundus (UF), left and right ovary (LO and RO), and the posterior surface of the left and right broad ligament (LBL and RBL). These sites were chosen on the basis that in the human, the most frequent locations of endometriosis are the ovary and Douglas's pouch. During surgical induction of endometriosis, one uterine strip from each monkey was immediately preserved in 10% buffered formalin and processed for histology to verify the presence of endometrial tissues. The abdomen was closed and the animals were allowed to recover.

Laparoscopy
A pneumoperitoneum was created in the anesthetized monkey and a pediatric laparoscope (Wolf, Germany) was inserted through the trocar sheath (Wolf, Germany) which was connected to a video camera (OTV-S2 Olympus Camera, AR-TF2, Japan). A laparoscopy was performed at 1, 3, and 6 months to evaluate the survival of endometrial implants. Blood was collected for hematology at each laparoscopy. No measurements of implant size were made at this time.

Necropsy
Monkeys were euthanized in the early to mid luteal phase of the cycle in all cases, with the exception of 4 acyclic monkeys (one each from control and the low-dose group and 2 from the moderate-dose group). Euthanasia was achieved by a 2.0-ml intravenous injection of pentobarbitol (240 mg/ml Euthanyl, M.T.C. Pharmaceuticals, Cambridge, Ont.), exsanguination and perfusion fixation with 10% buffered formalin. Endometrial implants were examined in situ and photographed, before being dissected free of surrounding tissue. Vernier calipers were used to obtain both a maximum and minimum diameter (recorded to the nearest 0.5 mm) for each implant. All surviving endometrial implants were placed in fresh fixative and processed for routine histology. Tissues collected for histology were processed through a graded series of alcohol and xylene solutions and embedded in paraffin. Four- to 5-ml thick representative sections were collected and stained with hematoxylin and eosin for histological analysis.

Concentration of Interleukin-6 (IL-6) and Interleukin-6 Soluble Receptor (IL-6 sR) in the Sera
Since IL-6 is a growth regulatory cytokine for the endometrium, and dysregulation of this system has been demonstrated in women with endometriosis (Rier et al., 1995aGo), we elected to explore the effect of TCDD treatment on circulating levels of IL-6 and IL-6sR. The concentrations of IL-6 and IL-6sR were quantified in the sera obtained at 6 and 12 months after starting exposure to TCDD, using commercially available sandwich enzyme immunoassays for the human (Quantikine IL-6 and IL-6sR ELISAs, R&D Systems, Minneapolis, MN). The detection limits of these assays were 0.7 pg/mL for IL-6, and 7 pg/mL for IL-6sR, and all samples were assayed on the same day. The intra-assay coefficients of variation were less than 9% and 11%, respectively.

Circulating Levels of Estradiol (E2) and Progesterone (P4)
Circulating levels of E2 and P4 were determined using commercial double antibody radioimmunoassay kits (DPC Los Angeles, CA.). The detection limit was 1.4 pg/mL for E2 and 1.2 ng/mL for P4. All samples were assayed on the same day and the intra-assay coefficients of variation were less than 7% and 9%, respectively.

Data Analysis
All surgical and analytical procedures were conducted with the treatment group of each animal or tissue unknown to the evaluator and technician. Effect of TCDD on survival of endometrial implants over time was evaluated by repeated measures ANOVA. Treatment effects on implant diameter, serum hormones, IL-6 and IL-6sR were examined for using 1-way analysis of variance (ANOVA). Within-group comparisons were tested for by the Student-Newman-Keuls method for multiple comparisons. All calculations were done with Sigma Stat software (Jandel Scientific, San Rafael, CA). Differences were considered significant at the p < 0.05 level.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Signs of general toxicity such as from routine hematology, food consumption, body weight, daily activity level, and changes in interactions with caretaker personnel were examined, and no differences were found among the treatment groups. In addition, TCDD treatment had no effect on circulating levels of gonadal steroids measured at necropsy (Table 1Go). Similarly, no differences in the number of menstrual cycles, menstrual-cycle length, and bleeding interval were detected (Table 1Go).


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TABLE 1 Effect of TCDD Treatment on Mean Circulating Levels of Estradiol and Progesterone, and Menstrual Cycle Characteristics in Monkeys with Surgically Induced Endometriosis
 
Endometrial implants were found on at least one site in all 23 monkeys at the first laparoscopy. The lesions had re-established vascularization and appeared variously as pink or white cysts as well as dark-colored lesions. Endometrial implant induction success rates were site-dependent. Induction success rates were: uterus, 100%; left ovary, 96.0%; right ovary, 91.0%; and broad ligament, 78.0%.

Follow-up laparoscopies revealed that there was a continuous loss of endometrial implant over time in each dose group. At 1, 3, and 6 months of study, the decrease in the number of surviving endometrial implants was not significantly different among the various dose groups, until 12 months of study, when a significantly higher rate of survival of endometrial implants was observed in the 3.57 and 17.86-ng/kg/day dose groups compared to that of controls (Fig. 2Go). No significant difference was found between the 0.71 ng/kg/day dose group and animals of the control group. At 12 months after surgical induction of endometriosis, regardless of dose level, the highest survival rate of endometrial implants was found on the ovaries. All lesions disappeared from the left broad ligament, whereas 2 on the right broad ligament and one on the uterine fundus survived.



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FIG. 2. Percentage of endometrial implants surviving vs. time in monkeys with surgical induction of endometriosis. Five endometrial fragments of equal size (4 x 1 mm2) were auto-transplanted to various sites in the pelvis of each monkey. Each animal was treated with TCDD at doses of 0, 0.71, 3.57, and 17.86 ng/kg/day over the course of one year; n = 5 for controls, n = 6 for the 3 dose groups (low, medium, and high, respectively). *Indicates significance vs. controls at the p < 0.05 level.

 
Surviving implants were of irregular shapes and surrounded by adhesions, making it difficult to both preserve the tissue for histopathology and obtain accurate measurements of length, width, and depth or to determine an implant volume. Therefore, we elected to measure the maximum and minimum diameter of the implants as this, in our opinion, provided the best reflection of implant size at the time of necropsy. There was a dose-related divergence in the growth response of endometrial implants to TCDD. Both the maximum and minimum implant diameters in the 17.86-ng/kg/day-dose group were significantly larger than that for the comparable measures in the control group (Fig. 3Go). In contrast, the maximum and minimum implant diameters in the 0.71 ng/kg/day dose group were significantly smaller than that of the control group whereas TCDD had no effect on implant diameters in the 3.57 ng/kg/day dose group compared to controls.



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FIG. 3. Mean ± SEM of maximum and minimum endometrial implant diameters at necropsy in monkeys with surgical induction of endometriosis and treated with TCDD at doses of at 0, 0.71, 3.57, and 17.86 ng/kg/day over the course of one year; n = 5 for controls, n = 6 for the 3 dose groups (low, medium, and high, respectively). *Indicates significance vs. controls at the p < 0.05 level.

 
Histological analysis revealed that endometrial glands and stromal cells were present in all of the surviving implants. Representative photomicrographs from each of the control and TCDD dose groups are depicted in Figure 4Go, panels A–D, respectively. Endometrial implants in animals from the control group were characterized by tortuous endometrial glands surrounded by an abundant stroma (Fig. 4AGo). The glandular lumen was lined by crowded columnar epithelial cells and the glands were separated by abundant stroma. Large quantities of inflammatory infiltrates were also found in sections from animals in the control group. Endometriotic cysts in animals from the 0.71-ng/kg/day-TCDD dose group were lined with squamous epithelial cells surrounded by a loose stroma and fibrous connective tissue (Fig. 4BGo). Sections from endometriotic cysts of monkeys treated with 3.57 ng/kg/day of TCDD possessed highly tortuous endometrial glands with cuboidal epithelial cells and an abundant stroma, which resembled the control group (Fig. 4CGo). Sections examined from monkeys treated with the 17.86 ng/kg/day of TCDD possessed cystic endometrial glands that were more frequently observed in this dose group than in any of the other groups (Fig. 4DGo). The glands were lined with crowded columnar epithelial cells separated by densely packed small stromal cells. These cysts were fluid filled and contained many inflammatory cells, including macrophages.



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FIG. 4. Representative photomicrographs of endometrial implants from animals dosed with TCDD at 0, 0.71, 3.57, and 17.86 ng/kg/day. (A) Endometrial glands in a control animal were tortuous, as suggested by various sizes and shapes of these glands. Crowded columnar epithelial cells line the open lumen of the glands. Large quantities of inflammatory infiltrates were also found on the section. (B) Endometriotic cysts were observed in the low-dose group (0.71 ng/kg/day TCDD) with a suppressed epithelial lining. (C) Sections from the moderate-dose group (3.57 ng/kg/day TCDD) were characterized by highly tortuous endometrial glands. Nuclei were located at various positions within epithelial cells. (D) Cystic endometrial glands were observed in sections from animals of the high (17.86 ng/kg/day)-dose group. Epithelial cells lining the lumen of the glands were columnar, and nuclei were displaced from their basal location and found centrally and apically positioned in these cells. Magnification x300.

 
Circulating levels of IL-6 were significantly decreased in monkeys exposed to 17.86 ng/kg/day of TCDD at both 6 and 12 months, compared to the control group. In contrast, the concentrations of IL-6sR in the sera were significantly elevated in the animals exposed to 3.57 and 17.86 ng/kg/day of TCDD at 6 months and only in the animals exposed to the high-dose levels at 12 months (Fig. 5Go).



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FIG. 5. Mean ± SEM circulating levels of (A) interleukin-6 (IL-6) and (B) interleukin-6sR (IL-6sR) for animals in each TCDD dose group (0, 0.71, 3.57, and 17.86 ng/kg/day; n = 5 for controls, n = 6 for the 3 dose groups (low, medium and high, respectively) at 6 and 12 months. *Indicates significance vs. controls at the p < 0.05 level.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hypothesis that TCDD exposure facilitates the survival and growth of endometrial implants was tested using surgical induction of endometriosis in a non-human primate model. Our results demonstrate that TCDD exposure, at levels representative of human adipose tissue levels, exerts a bimodal effect on endometrial implant growth. Furthermore, the rate of endometrial implant resorption in monkeys with surgically induced endometriosis was retarded by TCDD treatment in a dose-dependent manner. TCDD treatment also induced dysregulation of the endometrial growth regulatory cytokine IL-6 and its soluble receptor IL-6sR. Taken together, these data reveal that TCDD, at the higher concentrations used in this study, facilitates the survival and growth of endometriotic lesions, whereas the lowest dose used inhibited the growth of these lesions.

Surgical induction of endometriosis was employed as a model in this study. The laparoscopic and histological examination of the lesions revealed that the endometriotic implants were similar in appearance to endometriosis found in humans (Ueki et al., 1995Go) and that the success rate of endometriosis induction is in agreement with previous reports (Canis et al., 1995Go; Mann et al., 1986Go). Histologic examination of implants demonstrated the presence of endometrial glands and stroma in all of the sections from all animals.

Laparoscopies revealed a continuous trend of endometrial implant resorption over the course of study, in each dose group. The ovaries, in contrast to other anatomical implant sites, favored endometrial implant survival at all time points during the study. The site variation of survival rates of endometriotic implants may be due to various factors such as access to blood supply, the hormonal milieu, as well as the local action of growth factors and cytokines. Spontaneous disappearance of surgically induced endometriosis has previously been documented, even in the presence of endogenous steroids (Canis et al., 1995Go; Mann et al., 1986Go). Our data suggest that TCDD, at a dose level of 3.57 and 17.86 ng/kg/day, acts to facilitate the survival and growth of endometrial implants. We were surprised to discover that the low dose of TCDD (0.17 ng/kg/day) was inhibitory, as shown by a decrease in both the maximum and minimum implant diameters when compared to controls, whereas, a dose of TCDD representative of occupational or accidental exposure induced an increase in lesion diameters when compared to the control group. The inhibitory effect of the low dose of TCDD on implant growth is supported by the histological appearance of these implants, which were cystic, with a limited amount of surviving stroma, no surviving glands, and fibrotic changes. Our results appear to both confirm and extend, as well as to contradict an earlier report in which TCDD was shown to induce a dose-dependent increase in the incidence and severity of endometriosis in rhesus monkeys, using exposures of 5 and 25 ng/kg/day (Rier et al., 1993Go). Divergence of our findings from those of Rier and colleagues (1993) may reflect differences in the doses of TCDD used. Specifically, inhibitory effects of TCDD were found only in the 0.71-ng/kg/day-dose group of the present study, a dose not used in the prior study (Rier et al, 1993Go). Both the 3.57 and 17.86 ng/kg/day of the TCDD dose groups facilitated the survival of the implants, whereas only the 17.86-ng/kg/day-TCDD dose induced an increase in the maximum and minimum diameters of endometriotic implants, which extends the findings in the rhesus monkey (Rier et al., 1993Go).

The mechanism(s) of TCDD action on endometrial implants are unknown. TCDD is widely recognized as an anti-estrogen, as shown by decreased circulating levels of gonadal steroids in monkeys (Barsotti et al., 1979Go) and decreased concentrations of the estrogen (DeVito et al., 1994Go) and epidermal growth factor receptors in rodents (Madhukar et al., 1984Go). However, no effect of TCDD on the expression of hepatic and uterine estrogen receptors has also been reported in mice (DeVito et al., 1992Go). Furthermore, the dose level required to induce suppression of gonadal steroids (500 ppt for 6 months, Barsotti et al, 1979) was far in excess of the dose used in the present study. Since there was no effect of treatments in the present study on the circulating levels of either E2 or P4 we propose that in all dose groups the ectopic endometrial implants received a similar exposure to these steroids. However, since a TCDD-induced decrease in estrogen-receptor expression or post-receptor signaling in ectopic endometrium was not studied, such an effect of TCDD cannot be excluded.

TCDD is an immunosuppressive compound in mice, as demonstrated by decreased T-lymphocyte activity (Hill, 1992Go; House et al., 1990Go; Mann et al, 1986Go; Rier et al., 1995bGo). Although TCDD is without effect on macrophage and NK-cell activity (House et al., 1990Go), it does adversely affect leukocyte production of cytokines known to participate in the regulation of uterine physiology (Taylor et al., 1990Go; Hoglen et al., 1992Go). Furthermore, peritoneal leukocytes are reported to be in an activated state in women with endometriosis compared to women without endometriosis (Hill, 1988). Therefore, we speculate that TCDD treatment (3.57 and 17.86 ng/kg/day) in the current study enhanced the survival of endometriotic implants through effects upon peritoneal leukocytes and endometrial stromal cells. Increased peritoneal fluid levels of tumor necrosis factor-alpha (TNF-{alpha}) and interleukin-1 (IL-1) have been demonstrated in women with endometriosis relative to women without endometriosis (Eisermann et al., 1988Go; Taketani et al., 1992Go). IL-1ß is a target gene for TCDD (Sutter et al., 1991Go) whereas TNF-{alpha} sensitizes and activates phagocytic cells (Tabibzadeh, 1991Go; Tabibzadeh et al., 1989Go) as well as inducing the production of IL-6 by endometrial stromal cells (Alsarif et al., 1994Go). IL-6 is a growth factor derived from multiple cell types including leukocytes, fibroblasts, keratinocytes, endothelial cells (Van Snick, 1990Go) and endometrial stromal cells (Rier et al., 1995aGo). IL-6 is involved in the mediation of inflammatory responses and remodeling of the endometrium where it is growth inhibitory for normal uterine endometrial stromal cells (Rier et al., 1995aGo; Tabibzadeh et al., 1989Go). In the present study, increased levels of IL-6 and suppressed levels for IL-6sR in the sera of the animals that received 17.86 ng/kg/day of TCDD suggest a possible role of this cytokine in the pathophysiology of endometriosis. The peritoneal fluid levels of the endometrial growth regulatory cytokine IL-6 and production of interleukin-6-soluble receptor (IL-6sR) by endometrial stromal cells were shown to be elevated and suppressed, respectively, in women with endometriosis compared to a control group of women without endometriosis (Rier et al., 1995aGo). Peritoneal fluid levels in cynomolgus monkeys were found to be insufficient for our assay requirements. Hence peripheral blood, although remote to the site of the endometrial implants, was collected for these determinations. Our results demonstrate that the mechanisms regulating the production and interaction of IL-6 and its receptor IL-6sR are affected by TCDD treatment; this would obviate the endometrial growth inhibitory effect of IL-6.

The role of man-made chemicals in the pathophysiology of endometriosis remains a controversial topic. A positive association has been described previously (Gerhard and Runnebaum, 1992Go; Mayani et al., 1997Go) while no-association has also been described by others (Boyd et al., 1995Go; Lebel et al., 1998Go). Investigations in non-human primates suggest that exposure to environmental hazards such as radiation (Fanton and Golden, 1991Go) or TCDD (Rier et al., 1993Go) may be etiologic factors in the development of endometriosis. This study was not designed to examine the effects of TCDD as a causative factor in endometriosis but our results do support the hypothesis that TCDD facilitates the survival and, depending on exposure level, the growth of endometrial implants, and thus it may play a role in the pathobiology of this disease. The bimodal effect of TCDD on the diameter of endometrial implants may in part account for the discrepant findings in the human literature regarding a link between exposure and endometriosis.

In summary, TCDD exerts a bimodal effect on both the maximal and minimal diameter of endometriotic implants. At dose levels representative of occupational or accidental exposure, survival and growth of endometrial implants was facilitated, whereas the low dose of TCDD inhibited the growth of endometrial implants. Therefore, we conclude that TCDD, at dose levels representative of human adipose tissue levels, affects endometrial implant survival and growth.


    ACKNOWLEDGMENTS
 
This work was supported by the Bioregional Health Effects Program, Department of Health, Canada (Project BCH-341206). The authors also gratefully acknowledge the technical assistance provided by the Animal Resources Division staff during the conduct of this study.


    NOTES
 
1 To whom correspondence should be addressed at 135 Beverly Module 1, Center for Women's Health, Burns and Allen Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048. Fax: (310) 423-0305. E-mail: Warren.Foster{at}cshs.org. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alexandrou, N., and Pawliszyn, J. (1989). Superficial fluid extraction for the rapid determination of polychlorinated dibenzo-p-dioxins and dibenzofurans in municipal incinerator fly ash. Anal. Chem. 61, 2770–2776.[ISI]

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Canis, M., Chassagne, J., Dechelotte, P., Bassil, S., Wattiez, A., Pouly, J. L., Mage, G., and Bruhat, M. A. (1995). Experimental endometriosis: Spontaneous evolution of surgically induced peritoneal implants in rhesus monkeys. In Progress in the Management of Endometriosis (E. M. Coutinho, P. Spinola, and L. H. de Moura, Eds.), pp. 245–253. The Parthenon Publishing Group, New York.

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