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Extramural Research

1999 Progress Report: Models Assessing Direct Effects of Dioxins and Related Compounds on the Ovary

EPA Grant Number: R826132
Title: Models Assessing Direct Effects of Dioxins and Related Compounds on the Ovary
Investigators: Terranova, Paul F.
Institution: University of Kansas Medical Center
EPA Project Officer: Deener, Kacee
Project Period: October 1, 1997 through September 30, 2000
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $597,855
RFA: Endocrine Disruptors (1997)
Research Category: Endocrine Disruptors , Economics and Decision Sciences

Description:

Objective:

The specific objectives of this project are to:

  1. Determine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on physiological responses of cultured theca-interstitial cells of immature hypophysectomized rats exposed either in vitro or in vivo to TCDD. Specifically, changes in gonadotropin receptor binding, second messenger, and steroid production will be evaluated.

  2. Determine the in vivo effects of TCDD on: follicular development in immature hypophysectomized rats exposed to physiologic doses of gonadotropin, the steroidogenic responses of the ovary to gonadotropin, and ovulation induction and corpora luteal function controlled by gonadotropin.

  3. Determine if other dioxins, furans, and polychlorinated biphenyls (PCBs) have the same mode of direct action on ovarian function using ovulation and steroidogenesis as biomarkers, and to demonstrate that these models can be used for the assessment of toxic equivalency factors (TEFs) of dioxins and related compounds.

Progress Summary:

Blockage of Ovulation by TCDD Without Affecting Ovarian Steroidogenesis: Lack of Direct and Indirect Effects of TCDD on Steroidogenesis in the Immature Rat. The main purpose of this study was to investigate the direct effect of TCDD on ovarian function including ovulation and steroidogenesis. In vivo effects of TCDD were investigated on ovulation and alteration of circulating and ovarian steroid hormones in immature hypophysectomized rats (IHRs) primed with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In addition, in vitro effects of TCDD on the steroidogenesis of granulosa cells (GCs), theca-interstitial cells (TICs), and whole ovarian dispersates derived from the ovaries of IHRs were investigated. In the ovulation model, rats were hypophysectomized on day 23 of age. On day 26, the IHRs were given 20 g TCDD/kg by gavage. The next day, eCG (10 IU) was injected subcutaneously to stimulate follicular development. At 52 hours after eCG injection, 10 IU hCG was given to induce ovulation. TCDD blocked ovulation and reduced ovarian weight in the IHRs. Concentrations of progesterone (P4), androstenedione (A4), and estradiol (E2) in sera and ovaries were not altered by TCDD at 12, 24, 48, and 72 hours after eCG injection. In isolated GCs from untreated IHRs, TCDD (0.1?100 nM) had no significant effect on P4 and E2 after stimulation by luteinizing hormone (LH) or follicle stimulating hormone (FSH). In TIC and whole ovarian dispersates containing GC, TIC, and other ovarian cells, TCDD (0.1?800 nM) had no effect on A4 and P4 secretion stimulated by LH. Using reverse transcriptase polymerase chain reaction (RT-PCR), aryl hydrocarbon receptor (AhR) mRNA was shown to be constitutively expressed in the whole ovaries of IHRs with maximum downregulation at 6 hours after TCDD (20 g/kg) administration. Ovarian CYP1A1 was induced maximally at 6 hours after TCDD administration, whereas CYP1B1 could not be detected. The induction of AhR-related genes by TCDD in the ovary implies the existence of AhR-mediated signal transduction pathways. In summary, these results indicate that TCDD does not block ovulation in IHRs by altering ovarian steroidogenesis. It appears that inhibition of ovulation by a direct action of TCDD on the ovary is due to processes related to follicular rupture.

Direct Effects of TCDD on Ovarian Epithelial Cells: Regulation by Estradiol Congeners. TCDD is a well known ubiquitous environmental pollutant and is a promoter of carcinogenesis. TCDD-induced ethoxyresorufin-o-deethylase (EROD) activity and CYP1A1 dose-dependently in a mouse ovarian epithelial cell line (ID8). Interestingly, congeners of 17 -estradiol (E2) increased dose-dependently the induction of EROD by TCDD in order of estrone (E1) > E2 > 4-hydroxyestradiol (4-OHE2) 2-hydroxyestradiol (2-OHE2), whereas estriol (E3) decreased EROD activity and CYP1A1 expression. Alpha-naphthoflavone and phenanthroline, AhR antagonists inhibited induction of EROD by TCDD. Progesterone and gonadotropins (FSH and LH) had no effect on the induction of EROD by TCDD. The E2 congeners did not affect the number of ID8 cells, as shown by similar DNA and protein concentrations in ID8 during 48 hours in culture. E2 increased dose-dependently TCDD-induced CYP1A1 protein and mRNA, whereas E3 in combination with TCDD decreased these parameters. E2 did not alter the luciferase activity induced by TCDD in cells transfected with a dioxin response element (DRE)-containing luciferase reporter, whereas E3 decreased its activity dose-dependently. The actions of E2 were blocked by a specific estradiol receptor (ER) antagonist, indicating the involvement of the ER. Although CYP1A1 mRNA was induced by TCDD, CYP1B1 mRNA was constitutively expressed and unaffected by TCDD. ER alpha mRNA was constitutively expressed in ID8 cells, but ER beta and progesterone receptor mRNAs were not present. In conclusion, the results indicate E2 congeners differentially regulate TCDD-inducible CYP1A1 expression and provide new insight into different mechanisms by which estrogen congeners regulate ovarian responses to TCDD.

Estradiol Enhances the Action of TCDD on Ovulation Blockade. Immature rats were treated with estradiol cypionate, (ECP, 0-2 mg/kg subcutaneously) followed by TCDD (0 or 10 mg/kg orally) 24 hours later. Follicular development was induced with eCG (5 or 10 IU subcutaneously) followed by an ovulatory dose of hCG (10 IU subcutaneously). Inhibition of ovulation by TCDD was potentiated by ECP in hypophysectomized but not intact rats. Only hypophysectomized rats exposed systemically to TCDD and ECP exhibited weight loss. Pair feeding mimicked the combined effects of TCDD and ECP in hypophysectomized rats. In another experiment, rats received ECP subcutaneously (0 or 2 mg/kg) and TCDD into the ovarian bursa (0 or 250 ng). Another group received TCDD orally (10 mg/kg) and ECP into the ovarian bursa (0 or 1.5 mg). Blockade of ovulation by systemic or local TCDD was alleviated by ECP pretreatment. Estrogen increased the systemic toxicity of TCDD in hypophysectomized rats while antagonizing its direct ovarian effects.

Toxic Equivalency, TCDD, and Ovulation. For the current experiments, the gonadotropin-primed immature female rat model was used to study the effect of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) on ovulation. Single doses of different PCDDs and their mixture were given orally to 23-day-old rats. Gonadotropin from Pregnant Mare's Serum (PMSG) was injected (5 IU) 24 hours later to induce follicular maturation. Rats were decapitated at various times after PMSG, blood was collected, and ovarian weight was measured. Serum concentrations of E2, P4, LH, FSH, and prolactin (PrL) were determined by radioimmunoassay. Ovulation was measured at 72 hours after injection of PMSG by counting ova flushed from oviducts. PCDDs dose-dependently decreased the number of ova per ovary and reduced ovarian weight gain induced by PMSG. The slopes of the dose response curves generated by individual PCDDs and/or their mixture were similar. PMSG-induced increase in serum E was enhanced on the day of expected ovulation by PCDDs; in contrast, serum P and FSH were decreased at that same time point. PCDDs also altered the temporal pattern of serum E2, FSH, and LH, but not that of PrL. Histologically, the effect of all three PCDDs consisted of ova trapped in preovulatory follicles and a lack of, or reduced number of, corpora lutea. The results indicate that the PCDDs, tested in the present model, have the same mode of action on ovulation and on the reproductive hormones (e.g., LH, FSH, progesterone, and estradiol). Furthermore, the dose responses of the individual congeners are parallel to each other and also to those of their equipotent mixture, which represents a validation of the toxic equivalency (TEQ) concept for one aspect of endocrine disruption, that is, for inhibition of ovulation.

PCBs, Furans, and Their Effects on Ovulation: Comparison with TCDD. The objective of the current experiment was to investigate the effect of other structurally related compounds, such as chlorinated furans and biphenyls, on ovulation and related hormonal endpoints. The gonadotropin-primed immature female rat model was used to study the effect of 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 3,3',4,4',5-pentachlorobiphenyl (PeCB), 2,2',5,5' tetrachlorobiphenyl (TCB), and their mixture with PCDDs on ovulation. Rats were dosed on day 23 of age at 09:00 hours with individual congeners (PeCDF, PeCB, and TCB) or a mixture of five compounds, which included TCDD, PeCDD, HxCDD, PeCDF, and PeCB. Equine chorionic gonadotropin (5 IU) was injected 24 hours later to induce follicular development. Blood and ovaries were harvested, and ovarian weights were determined at various times after eCG injection. Serum concentrations of 17 -estradiol (E), P4, LH, and FSH were determined by radioimmunoassay. At 72 hours after injection of eCG, the number of ova shed was measured by irrigating the ova from oviducts. The slopes of the dose responses for inhibition of ovulation generated by the individual PeCDF, PeCB, and/or their mixture with PCDDs were similar. PeCDF, PeCB, and the mixture increased serum concentrations of E at 72 hours after eCG injection, the day of expected ovulation; in contrast, serum P and FSH were decreased at that same time point. Only the high doses of TCDD, PeCDF, and PeCB blocked LH and FSH surges at 58 hours after eCG. Ovarian histology revealed that the effect of PeCDF, PeCB, and the mixture was very similar to that of PCDDs, consisting of ova trapped in preovulatory follicles and a lack of, or reduced number of, corpora lutea. Parallel dose responses of the individual congeners (PeCDF and PeCB) and their equipotent mixture with PCDDs support the TEQ concept for the blockage of ovulation. Thus, PCDDs, PCDFs, and PeCBs appear to block ovulation by the same or a very similar mechanism of action.

Gonadotropin Releasing Hormone Partially Overcomes the Blockade of Ovulation Induced by TCDD. Several of our studies have shown that TCDD has inhibitory effects on ovulation. This action may be the result of either direct effect(s) of TCDD on ovarian function, or of altered secretion of pituitary LH and FSH, which regulate ovarian follicular development and ovulation. To further evaluate the effects of TCDD on pituitary gonadotropins and their regulation, the potential role of gonadotropin releasing hormone (GnRH) was investigated in the current study. Immature (23-day old) female Sprague-Dawley rats were dosed with TCDD (32 g/kg) in corn oil or vehicle alone. Equine chorionic gonadotropin (eCG) was injected (5 IU, subcutaneously) 24 hours later to induce follicular development. Immediately prior to the expected time of the LH/FSH surge, 54 hours after eCG injection, half of TCDD- or corn oil-treated rats were injected with GnRH (2 g/ rat, subcutaneously). Blood and ovaries were collected at 54, 56, 58, 60, and 72 hours after eCG injection. Serum concentrations of E, P4, LH, and FSH were determined by radioimmunoassay. The number of ova shed was measured at 72 hours after injection of eCG by irrigating the ova from oviducts. TCDD inhibited ovulation by 70?80 percent (2?3 ova/rat). GnRH partially restored ovulation (6?7 ova/rat) in TCDD-treated rats without reversing its effect on ovarian weight reduction. The LH and FSH surges triggered by eCG injection in controls were completely abolished by TCDD at 56?60 hours after eCG injection; however, in rats treated with TCDD and GnRH, a huge LH/FSH surge occurred at 56?60 hours after eCG injection. GnRH alone enhanced E and P4 serum levels at 56?58 hours after eCG injection, but in rats treated with both TCDD and GnRH, E secretion was significantly inhibited at 58, 60, and 72 hours, whereas serum P4 was only decreased at 72 hours after eCG injection. Immortalized hypothalamic GnRH-producing neurons (GT1-7 cells) were cultured from 0 to 72 hours with TCDD (0?100 nM). GnRH accumulation and expression of the AhR gene were assessed. TCDD treatment slightly increased GnRH accumulation in these cells following 72 hours of culture with only the highest concentrations of TCDD; however, the AhR was undetectable in these cells by RT-PCR. The results indicate that exogenous GnRH given to TCDD treated-rats induces LH or FSH surges, but only partially restores the inhibitory effects of TCDD on ovulation.

Future Activities:

We will determine the in vivo effects of TCDD on: (1) follicular development in immature hypophysectomized rats exposed to physiologic doses of gonadotropin, and (2) ovulation induction and corpora luteal function controlled by gonadotropin. We also will be investigating the site of action of TCDD as the gonadotropin releasing hormone cells in the hypothalamus, because the LH surge is blocked by TCDD. In addition, we will be assessing molecular mechanisms by which TCDD blocks follicular rupture. We will be attempting to overcome the effects of TCDD on ovulation blockage with exogenous estrogen.


Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other project views: All 6 publications 6 publications in selected types All 6 journal articles

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Journal Article Gao X, Son DS, Terranova PF, Rozman KK. Toxic equivalency factors of polychlorinated dibenzo-p-dioxins in an ovulation model: validation of the toxic equivalency concept for one aspect of endocrine disruption. Toxicology and Applied Pharmacology 1999;157(2):107-116. R826132 (1999)
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  • Journal Article Gao X, Terranova PF, Rozman KK. Effects of polychlorinated dibenzofurans, biphenyls, and their mixture with dibenzo-p-dioxins on ovulation in the gonadotropin-primed immature rat: support for the toxic equivalency concept. Toxicology and Applied Pharmacology 2000;163(2):115-124. R826132 (1999)
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  • Journal Article Gao X, Petroff BK, Rozman KK, Terranova PF. Gonadotropin-releasing hormone (GnRH) partially reverses the inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on ovulation in the immature gonadotropin-treated rat. Toxicology 2000;147(1):15-22. R826132 (1999)
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  • Journal Article Petroff BK, Gao X, Rozman KK, Terranova, PF. Interaction of estradiol and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an ovulation model: evidence for systemic potentiation and local ovarian effects. Reproductive Toxicology 2000;14(3):247-255. R826132 (1999)
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  • Journal Article Petroff BK, Gao X, Ohshima KI, Shi FX, Son DS, Roby KF, Rozman KK, Watanabe G, Taya K, Terranova PF. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum inhibin concentrations and inhibin immunostaining during follicular development in female Sprague-Dawley rats. Reproductive Toxicology 2002;16(2):97-105. R826132 (1999)
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  • Journal Article Son DS, Ushinohama K, Gao X, Taylor CC, Roby KF, Rozman KK, Terranova PF. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro. Reproductive Toxicology 1999;13(6):521-530. R826132 (1999)
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  • Supplemental Keywords:

    dioxin, polychlorinated biphenyl, PCB, furan, ovary, toxicology., RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Toxics, Environmental Chemistry, Health Risk Assessment, pesticides, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Biochemistry, Physical Processes, Biology, Endocrine Disruptors - Human Health, adverse outcomes, biomarkers, embryo survival, endocrine disrupting chemicals, exposure, PCBs, steroid, 2, 3, 7, 8-Tetrachloro-dibenzo-p-dioxin (TCDD), chemical mixtures, animal models, developmental processes, furans, human exposure, PCB, mice, models, biological effects, dioxins, progesterone, human health risk

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