For more information, see Introduction.

Prospective Study #1

Kennedy S. The genetics of endometriosis. J Reprod Med; VOL 43, ISS 3 Suppl, 1998, P263-8 (REF: 45). Author Address: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, England.

Endometriosis is probably a complex trait, like diabetes, hypertension or asthma, which implies that the disease is caused by an interaction between multiple genes and the environment. This paper reviews the evidence, in both human and nonhuman primates, that supports the theory of a genetic basis to endometriosis. The OXEGENE study, which aims to identify susceptibility genes in endometriosis through linkage analysis, is also described. DNA is being collected from families containing at least two sisters with surgically confirmed revised American Fertility Society stage III and IV disease to carry out a genome-wide search for susceptibility genes. Candidate genes involved in galactose metabolism and dioxin detoxification are also being analyzed. The identification of genes conferring susceptibility to endometriosis may lead to a better understanding of disease etiology and, in time, improved therapeutic strategies and diagnostic methods.

Prospective Study #2

BOYD JA, MOLECULAR GENETICS OF HUMAN GYNECOLOGIC PATHOLOGY Crisp Data Base National Institutes Of Health. Author Address: NIEHS, NIH

The goals of this project center around the molecular genetic analysis of pathologic conditions of the human uterus, including endometrial carcinoma, endometriosis, and uterine leiomyoma (fibroids). Related studies include the molecular genetic analyses of human and rodent cancers associated with exposure in utero to diethylstilbestrol, human latent prostatic carcinomas, and hereditary breast and ovarian cancers with emphasis on the BRCA1 susceptibility locus. Significant progress has been made in defining the relevant oncogenes and tumor suppressor genes involved in the pathobiology of endometrial carcinoma. Several genes and chromosomal loci have been found to be predominantly involved in either type I, estrogen-related tumors, or type II, nonestrogen-related tumors. Notable among these are a gene on chromosome 2p responsible for a replication error phenotype (type I tumors), and a novel locus on chromosome 14q that is highly correlated with death from disease (type II tumors). Studies on endometriosis have identified a novel endometrial cDNA clone that is recognized by serum antibodies in women with endometriosis; the sequencing, characterization, and determination of clinical utility of this gene is in progress. Further studies were initiated that will attempt to correlate serum levels of organochlorine toxicants (e.g., dioxin, PCBs) with the presence or extent of endometriosis in human subjects. Efforts were begun to clone and characterize genes involved in uterine leiomyoma.The most frequent cytogenetic abnormality in these tumors is a balanced translocation involving chromosomes 12 and 14. A large number of candidate cDNAs have been cloned from the 12q15 region, and their characterization and possible involvement in myometrial tumorigenesis are under study. An analysis of human and rodent tumors induced by DES exposure has found an absence of mutations in the P53, RAS, and WT-1 genes. Future studies are designed to identify other, perhaps novel genomic loci relevant to DES carcinogenesis, as well as the molecular pathway of DES-induced genital tract developmental anomalies.

Prospective Study #3

SPINK DC. ALTERATIONS IN ESTROGEN METABOLISM CAUSED BY EXPOSURE TO PCBS. Crisp Data Base National Institutes of Health. Author Address: UNIVERSITY AT ALBANY, SUNY, ONE UNIVERSITY PLACE, B242,RENSSELAER, NY 12144

The broad, long-term objective of this research is to determine the effects of exposure to polychlorinated biphenyls (PCBs) on the phase I and phase II metabolism of 17beta-estradiol (E2) in liver and extrahepatic tissues. Human exposure to PCBs may result in diverse endpoints such as impairment of intellectual development and reduced fertility. The alteration of estrogen metabolism by exposure to PCBs is a mechanism by which these xenobiotics may alter fertility, fecundity, and neuroendocrine development through disruption of normal estrogenic signal transduction. We hypothesize that, in a congener-specific manner, PCBs will increase the rates of E2 hydroxylation and conjugation in liver and in estrogen target tissues, and under some conditions aberrant estrogen metabolism will cause oxidative stress.

Our Specific Aims are:

(1) To determine the effects of exposure to PCBs (Aroclor 1248 and several individual congeners) on the rates cytochrome P450-catalyzed hydroxylation of E2. Microsomes prepared from liver, kidney,uterus, and brain of PCB- exposed female rates and from human-derived breast, liver, kidney, and uterine cells exposed to PCBs in vitro will be used for the E2 metabolism studies.

(2) To determine the effects of exposure to PCBs on the phase II metabolism of E2 in liver and extrahepatic tissues. Estrogen glucuronsyltransferase, T3 and T4 glucuronosyltransferase, and estrogen sulfotransferase activities will be determined with microsomes prepared from tissues of PCB-exposed female rats and with human-derived cells in vitro. It will be determined whether estrogens and the thyroid hormones, T3 and T4, are substrates for the same conjugating enzymes.

(3) To determine the effects of exposure to PCGs on the expression of cytochrome P450s of the CYP1A and the recently discovered CYP1B gene subfamilies. Immunochemical techniques, RNA blots, and the reverse transcriptase-polymerase chain reaction will be used to characterize gene expression in tissues of the female rat in vivo and in human breast, kidney, and endometrial cells in vitro.

(4) To determine the effects of PCB exposure and PCB-induced estrogen metabolism on oxidative stress in tissues of the female rat and in human breast cells in vitro. Lipid peroxides, DNA strand breaks, and the presence of 8-hydroxyguanine in DNA will be measured as evidence of oxidative stress.

Prospective Study #4

SHIVERICK KT. PLACENTAL/UTERINE EFFECTS OF CHLORINATED HYDROCARBONS. Crisp Data Base National Institutes of Health. Author Address: UNIVERSITY OF FLORIDA, 1600 ARCHER RD, BOX 100485, GAINESVILLE, FL 32610-0485

The proposed research will investigate mechanisms by which the chlorinated hydrocarbons 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) have disruptive effects on placental- uterine function. The hypothesis is that TCDD and PCBs alter placental- uterine paracrine and autocrine networks which are important for trophoblast invasiveness, proliferation and hormone secretion, as well as uterine cell growth. The major networks involve epidermal growth factor (EGF) and its receptor (EGF-R), transforming growth factors (TGFs)-alpha and -betas, the cytokine interleukin (IL) -1B and the protease inhibitor, plasminogen activator inhibitor (PAI). The PCB congeners 3,3',4,4'-tetrachloro and 2,2',4,4',5,5'-hexachloro, and the mixture Arochlor 1254 will be studied to represent different levels of AHH induction activity.

SPECIFIC AIM #1: Does exposure of cultured human placental trophoblstic cell lines to TCDD and PCBs alter expression of genes important for placental growth and endocrine function? Experiments will specifically evaluate: a) EGF receptor binding and kinase activity in choriocarcinoma JEG-3 and BeWo cell lines to determine whether there is down-regulation of receptors with altered cell proliferation or hormone secretion; b) The genes recognized to be regulated by TCDD in other human cell lines, including TGF-alpha, TGF-betas, IL-1B, and PAI; c) Induction of cytochrome P-450 1A1 and PCB metabolism to determine if there are dose- related effects in parallel with changes in EGF receptors and/or growth factor expression through the Ah receptor.

SPECIFIC AIM #2: Does exposure of cultured human endometrial adenocarcinoma cells to PCBs and TCDD alter expression of growth regulatory genes? Experiments will evaluate: a) EGF-R binding and kinase activity and EGF-stimulated cell proliferation; b) Estrogen receptor levels to determine whether the cellular response to estrogens is altered; c) Expression of TGF-alpha, TGF-betas, IL-1B, and PAI; d) Induction of cytochrome P450 1A1 and PCB metabolism for dose-response relationships with a), b) and c) above.

SPECIFIC AIM #3: In pregnant rats, does administration of TCDD and the respective PCBs alter expression of placental genes for P450 1A1,TGFs- alpha and -beta, IL-1B and PAI in correlation with feto-placental growth retardation? Experiments will evaluate: a) Feto-placental growth in late gestation; b) Expression of TGF-alpah, TGF-betas, IL-1B, and PAI in placenta and decidua; c) Cytochrome P450 1A1 induction and PCB metabolism for dose response relationships with a) and b) above.

SPECIFIC AIM #4: Is expression of placental amino acid transport systems altered in correlation with feto-placental growth retardation in pregnant rats treated with PCBs and TCDD? a) Amino acid transport via Systems B,+,y+,XAG-, and A will be examined in isolated vesicle preparations from the microvillous and basal membranes; b) mRNA levels will be examined utilizing cDNA probes to Systems y+ and XAG-; c) Cytochrome P450 1A1 induction for dos-response relationships with a) and b) above.

Prospective Study #5

ZHU BT. EFFECTS OF CIGARETTE SMOKING OR PCBS ON HUMAN ESTRADIOL. Crisp Data Base National Institutes of Health. Author Address: UNIVERSITY OF SOUTH CAROLINA, DEPART. OF BASIC PHARM SCIENCE, COLUMBIA, SC 29208

Earlier studies indicated that cigarette smokers have a decreased risk of uterine endometrial cancer and an increased risk of osteoporosis which are thought to be caused by an inhibitory effect of cigarette smoking on estrogen action. Some but not all studies have indicated that cigarette smokers have lower plasma levels of estradiol and enhanced systemic 2-hydroxylation of this estrogen. A major focus of this proposal is to characterize the effects of cigarette smoking on NADPH-dependent metabolism of estradiol to multiple hydroxylated and keto metabolites by human liver and, in particular, by extrahepatic estrogen target organs such as uterine endometrium and placenta. In addition, we will characterize the profile of multiple estradiol metabolites formed by placentas from women exposed accidentally to polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans which are potent inducers of microsomal monooxygenases. Our proposed studies should not only enhance our understanding of the antiestrogenic effect of cigarette smoking in women, but they should also provide us with useful information on the inducibility of cytochrome P450 enzymes that catalyzed the formation of various estradiol metabolites (including hormonally- active and/or genotoxic metabolites) in human liver as well as in human target organs for estrogen action. We plan to: (l).Determine the profile of NADPH-dependent estradiol metabolites formed by uterine endometrial microsomes from cigarette smokers and matched nonsmokers. (2).Determine the profile of NADPH-dependent estradiol metabolites formed by placental microsomes from cigarette smokers and matched nonsmokers. (3).Determine the profile of NADPH-dependent estradiol metabolites formed by liver microsomes from cigarette smokers and matched nonsmokers. (4).Determine the profile of NADPH-dependent estradiol metabolites formed by placental microsomes from women exposed accidentally to polychlorinated biphenyls and polychlorinated dibenzofurans. (5).Determine the effects of selective inhibitory antibodies to various cytochrome P450 isoforms on the pathways of estradiol metabolism observed in the studies described above. (6).Determine the effects of cigarette smoking on the concentration of unmetabolized estradiol in human uterine endometrium.

Prospective Study #6

Eskenazi B, Mocarelli P, Warner M, Samuels S, Vercellini P, Olive D, Needham L, Patterson D, Brambilla P. Seveso Women's Health Study: a study of the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive health. Chemosphere 2000 May-Jun;40(9-11):1247-53. Author Address: School of Public Health, University of California at Berkeley, 94720-7360, USA. mwarner@uclink4.berkeley.edu

Although reproductive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure have been reported in numerous investigations of animals, studies of this association in humans are limited. In 1976, an explosion in Seveso, Italy exposed the surrounding population to among the highest levels of TCDD recorded in humans. The relatively pure exposure to TCDD and the ability to quantify individual level TCDD exposure from sera collected in 1976 for the Seveso cohort affords a unique opportunity to evaluate the potential dose-response relationship between TCDD exposure and a spectrum of reproductive endpoints. The Seveso Women's Health Study (SWHS) is the first comprehensive study of the reproductive health of a human population exposed to TCDD. The primary objectives of the study are to investigate the relationship of TCDD and the following endpoints: (1) endometriosis; (2) menstrual cycle characteristics; (3) age at menarche; (4) birth outcomes of pregnancies conceived after 1976; (5) time to conception and clinical infertility; and (6) age at menopause. Included in the SWHS cohort are women who were 0-40 yr old in 1976, who have adequate stored sera collected between 1976 and 1980, and who resided in Zones A or B at the time of the accident. All women were interviewed extensively about their reproductive and pregnancy history and had a blood draw. For an eligible subset of women, a pelvic exam and transvaginal ultrasound were conducted and a menstrual diary was completed. More than 95% of the women were located 20 yr after the accident and roughly 80% of the cohort agreed to participate. Data collection was completed in July 1998, serum TCDD analysis of samples for analysis of endometriosis as a nested case-control study was completed in October 1998, and statistical analysis of these data should be completed in early 1999. Serum samples are now being analyzed in order to relate TCDD levels with the remaining reproductive outcomes.

Prospective Study #7

BUCK GM. PERSISTENT HALOGENATED ORGANICS AND ENDOMETRIOSIS RISK. Crisp Data Base National Institutes Of Health.Author Address: SUNY AT BUFFALO, 270 FARBER HALL, BUFFALO, NY 14214

Recent research findings suggest that endometriosis may be related to select environmental agents (e.g., dioxin). In particular, a significant correlation was reported for TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and severity of endometriosis in a colony of rhesus monkeys. As yet, little study of the relationship between exposure to halogenated organics and endometriosis in humans has been undertaken and that serves as the impetus for study. The long-term objectives of this study is to assess whether exposure to persistent halogenated organics, including dioxin, is involved in the aetiology of endometriosis. Five specific objectives have been developed: 1) to identify and elicit the cooperation of women with newly diagnosed endometriosis (cases) and women free of disease (controls); 2) to compare measured levels of persistent halogenated organics in fat and serum between cases and controls; 3) to assess biomarkers of exposure and effect related to dioxin-like compounds by measuring levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) in uterine endometrium and ectopic endometrial implants; 4) to validate the biomarkers by assessing CYP1A1 and CYP1B1 in tissues from the original colony of rhesus monkeys with known exposures to TCDD and laparoscopically-confirmed endometriosis; and 5) to assess endometriosis risk as a function of both biomarkers expression and measured exposure status. A case-control study design will be used to identify and recruit cases and controls from the population of women undergoing laparoscopic surgery at one University-affiliated hospital. Fifty women with newly-diagnosed endometriosis and 100 age-matched women undergoing interval tubal ligations and who are found to be free of endometriosis will comprise the study sample. A standardized operative alpha-sequencing inspection of the pelvis will be performed by surgeons for all women. All laparoscopic-confirmed endometriosis will be staged according to the revised American Fertility Society classification. Standardized in-home interviews will be conducted with all women; serum will be collected from women as well. Approximately 5 grams of omentum fat will be taken at the time of laparoscopy from all women for comprehensive exposure assessment. Among cases, biopsies will be taken from the endometrium and ectopic endometrial implants for biomarker assessment. The successful completion of this study will be enhanced by its multidisciplinary design, attention to sound epidemiologic methods and utilization of state-of-the-art exposure, biomarker and surgical techniques. As such, the proposed study shall help clarify the mechanisms underlying the development of endometriosis.

Prospective Study #8

RIER SE. DIOXIN EXPOSURE, IMMUNE ALTERATIONS AND ENDOMETRIOSIS Crisp Data Base National Institutes Of Health. Author Address: DARTMOUTH MEDICAL SCHOOL, 1 MEDICAL CENTER DR, LEBANON, NH 03756-0001

The link between TCDD or dioxin and endometriosis will be investigated in these studies. The phenotype and function of the immune cells in uterine and ectopic endometrium and peripheral blood from TCDD-treated rhesus monkeys will be related to expression of the aryl hydrocarbon receptor (AhR), cytochrome P4501A1 expression and serum levels of dioxin/furan compounds. The investigators have available a colony of female rhesus monkeys, both chronic TCDD exposed females and controls. The studies are designed to elucidate the role of chronic exposure to TCDD on the rhesus endometrium, leading to development of insight into the role of TCDD, development of biomarkers, therapeutic and diagnostic approaches. Uterine and ectopic endometrium samples from these monkeys will be characterized to compare leucocyte subpopulations, expression of specific activation markers and production of antibodies in TCDD-exposed females and controls. The phenotype, AhR and CYP1A1 expression and cytokine production the uterine endometrium (UTEM) and of PB leucocyte will be analyzed. In situ phenotypic analysis and intracellular staining for cytokine production and expression of AhR and CYP1A1 by specific cell types will be conducted.

Prospective Study #9

YEAMAN G. DIOXIN, MYELOID CELL FUNCTION AND HUMAN ENDOMETRIOSIS. Crisp Data Base National Institutes Of Health. Author Address: DARTMOUTH MEDICAL SCHOOL, 1 MEDICAL CENTER DRIVE, LEBANON, NH 03756-0001

Endometriosis, a disease of unknown etiology, is characterized by infertility, chronic pain and adhesion formation, and may affect up to 6 million women of reproductive-age in the USA. In spite of clinical reliance on endocrine based therapies, the role of sex steroid hormones in the growth of endometriotic tissue remains unclear. Normal uterine endometrium undergoes predictable histologic and biochemical changes in response to sex hormones throughout the menstrual cycle, but it is unknown whether this results from direct action on target cells or is exerted indirectly by soluble factors or cytokines produced within the endometrium. The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) alters the action of estrogen within reproductive organs and adversely affects immunocompetence. We have shown that TCDD exposure in the rhesus is associated with a dose-dependent increase in the incidence and severity of endometriosis. Additional studies demonstrated that the phenotype of peripheral blood leukocytes and in vitro secretion of cytokines by leukocytes from TCDD-exposed monkeys differed from unexposed animals more than 13 years after TCDD exposure was terminated. Immune cells are present within uterus and play a critical role in reproductive physiology and pathology. Furthermore, cytokines produced by activated leukocytes may participate in TCDD- mediated toxicity and in the pathogenesis of endometriosis. Using the unique resources of the NIH-funded Human Female Reproductive Tract Immunology Program Project (PP), the proposed studies will extend these findings from peripheral blood and animal models to the human uterine mucosal immune system. Using powerful new techniques developed for the PP, we will: determine the in vitro effects of TCDD exposure on sex hormone regulation of cytokine production and myeloid cell activation by cultured human endometrial tissue. These studies will give valuable insights into the role of a ubiquitous environmental toxin in the pathogenesis of endometriosis, and may ultimately lead to the development of biomarkers of immunotoxicity and novel therapeutic and diagnostic approaches to endometriosis. Furthermore, the techniques used in these studies will enable the future investigation of many potential environmental endocrine disruptors with respect to human reproductive disease pathogenesis at the interface between the immune and endocrine systems.

Prospective Study #10

BOFINGER DP. DIOXIN BIOMARKERS AND ENDOMETRIOSIS. Crisp Data Base National Institutes Of Health. Author Address: UB CLINICAL CENTER AA109, 462 GRIDER ST, BUFFALO, NY 14215

The overall aim of this project is to provide mechanistic data that will aid in understanding the role of dioxin exposure in the development of endometriosis. This proposal is based on the finding that rhesus monkeys exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) had a higher incidence of endometriosis than unexposed monkeys with the severity of disease increasing with environmentally relevant doses of TCDD (5 or 25 ppt/day). The focus of this project is to study the expression of mechanistically-based biomarkers of dioxin exposure, effect, and/or susceptibility in this colony of rhesus monkeys and in an in vitro human endometrial explant culture model. These preliminary results will be used together with the monkey tissue data to develop a full mechanism-based proposal to study the direct or indirect effects of TCDD on human endometrium and the role of dioxin exposure in the development of endometriosis.

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