Ovarian Toxicology

TARGET ORGAN TOXICOLOGY SERIES Series Editors A.Wallace Hayes, John A.Thomas, and Donald E.Gardner OVARIAN TOXICOLOGY Patricia B.Hoyer, editor, 248 pp., 2004 CARDIOVASCULAR TOXICOLOGY, THIRD EDITION Daniel Acosta, Jr., editor, 616 pp., 2001 NUTRITIONAL TOXICOLOGY, SECOND EDITION Frank N.Kotsonis and Maureen A.Mackey, editors, 480 pp., 2001 TOXICOLOGY OF SKIN Howard I.Maibach, editor, 558 pp., 2000 TOXICOLOGY OF THE LUNG, THIRD EDITION Donald E.Gardner, James D.Crapo, and Roger O.McClellan, editors, 668 pp., 1999 NEUROTOXICOLOGY, SECOND EDITION Hugh A.Tilson and G.Jean Harry, editors, 386 pp., 1999 TOXICANT-RECEPTORINTERACTIONS: MODULATION OF SIGNAL TRANSDUCTIONS AND GENE EXPRESSION Michael S.Denison and William G.Helferich, editors, 256 pp., 1998 TOXICOLOGY OF THE LIVER, SECOND EDITION Gabriel L.Plaa and William R.Hewitt, editors, 444 pp., 1997 FREE RADICAL TOXICOLOGY Kendall B.Wallace, editor, 454 pp., 1997 ENDOCRINE TOXICOLOGY, SECOND EDITION Raphael J.Witorsch, editor, 336 pp., 1995 CARCINOGENESIS Michael P.Waalkes and Jerrold M.Ward, editors, 496 pp., 1994 (Continued) DEVELOPMENTAL TOXICOLOGY, SECOND EDITION Carole A.Kimmel and Judy Buelke-Sam, editors, 496 pp., 1994 IMMUNOTOXICOLOGY AND IMMUNOPHARMACOLOGY, SECOND EDITION Jack H.Dean, Michael I.Luster, Albert E.Munson, and Ian Kimber, editors, 784 pp., 1994

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NUTRITIONAL TOXICOLOGY Frank N.Kotsonis, Maureen A.Mackey, and Jerry J.Hjelle, editors, 336 pp., 1994 TOXICOLOGY OF THE KIDNEY, SECOND EDITION Jerry B.Hook and Robin J.Goldstein, editors, 576 pp., 1993 OPHTHALMIC TOXICOLOGY George C.Y.Chiou, editor, 352 pp., 1992 TOXICOLOGY OF THE BLOOD AND BONE MARROW Richard D.Irons, editor, 192 pp., 1985 TOXICOLOGY OF THE EYE, EAR, AND OTHER SPECIAL SENSES A.Wallace Hayes, editor, 264 pp., 1985 CUTANEOUS TOXICITY Victor A.Drill and Paul Lazar, editors, 288 pp., 1984

Target Organ Toxicology Series

Ovarian Toxicology Edited by Patricia B.Hoyer Department of Physiology The University of Arizona Tucson

Boca Raton London New York Washington, D.C.

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Library of Congress Cataloging-in-Publication Data Catalog record is available from the Library of Congress This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. Visit the CRC Press Web site at www.crcpress.com © 2004 by CRC Press LLC No claim to original U.S. Government works ISBN 0-203-50923-4 Master e-book ISBN

ISBN 0-203-57157-6 (Adobe eReader Format) International Standard Book Number 0-415-28795-2 (Print Edition)

CONTENTS

Contributors

vii

Foreword

xii

1

Ovarian physiology DIANE SUTER

1

2

Ovarian toxicity in small pre-antral follicles PATRICIA B.HOYER

17

3

Ovarian toxicity caused by pesticides CHRISTINA BORGEEST KIMBERLY P.MILLER DRAGANA TOMIC JODI A.FLAWS

41

4

Ovarian toxicity caused by endocrine disruptors PAUL F.TERRANOVA AND KARL K.ROZMAN

62

5

Phthalate toxicity in the ovary FRIEDERIKE C.L.JAYESTARA LOVEKAMP-SWAN AND BARBARA J.DAVIS

87

6

Hormonal control of ovarian function following chlorotriazine exposure: effect on reproductive function and mammary gland tumor development RALPH L.COOPER SUSAN C.LAWS MICHAEL G.NAROTSKY JEROME M.GOLDMAN AND TAMMY E.STOKER

96

7

The role of ovarian metabolism in chemical-induced ovarian injury ELLEN A.CANNADY AND I.GLENN SIPES

116

8

Placental induction of ovarian toxicity JENNIFER L.MARCINKIEWICZ

135

9

Chemoresistance in human ovarian cancer: possible roles of Xlinked inhibitor of apoptosis protein (XIAP) CHAO WU XIAO XIAOJUAN YAN HIROMASA SASAKI FUMIKAZU KOTSUJI AND BENJAMIN K.TSANG

150

vi

10

The epidemiology of ovarian cancer: the role of reproductive factors and environmental chemical exposure KATHRYN COE

175

11

Assessment of toxicant-induced alterations in ovarian steroidogenesis: a methodological overview JEROME M.GOLDMAN SUSAN C.LAWS AND RALPH L.COOPER

206

Index

227

CONTRIBUTORS

Christina Borgeest Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 Ellen A.Cannady Eli Lilly and Company Drug Disposition Indianapolis, Indiana 46285 Kathryn Coe Arizona Cancer Center P.O. Box 245024 The University of Arizona Tucson, AZ 85724–5024 Ralph L.Cooper Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Barbara J.Davis National Institute of Environmental Health Sciences (NIEHS) Laboratory of Women’s Health (LWH) 111 TW Alexander Drive P.O. Box 12233 Mail Drop B3–06 Research Triangle Park NC 27709

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USA Jodi A.Flaws Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 USA Jerome M.Goldman Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Patricia B.Hoyer Professor Department of Physiology Arizona Health Sciences Center The University of Arizona Tucson, AZ 85724 Friederike C.L.Jayes National Institute of Environmental Health Sciences (NIEHS) Laboratory of Women’s Health (LWH) 111 TW Alexander Drive P.O. Box 12233 Mail Drop B3–06 Research Triangle Park NC 27709 USA Fumikazu Kotsuji Department of Obstetrics & Gynecology Fukui Medical University Yoshidagun Fukui Japan 910–1193 Susan C.Laws Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park

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NC 27711 USA Tara Lovekamp-Swan National Institute of Environmental Health Sciences (NIEHS) Laboratory of Women’s Health (LWH) 111 TW Alexander Drive P.O. Box 12233 Mail Drop B3–06 Research Triangle Park NC 27709 USA Jennifer L.Marcinkiewicz Department of Biological Sciences Kent State University Kent, OH 44242–0001 USA Kimberly P.Miller Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 USA Michael G.Narotsky Endocrinology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Karl K.Rozman Department of Pharmacology, Toxicology and Therapeutics University of Kansas Medical Center Kansas City Kansas 66160 Section of Environmental Toxicology GSF-Institute für Toxikologie Neuherberg Germany Hiromasa Sasaki Department of Obstetrics & Gynecology Fukui Medical University

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Yoshidagun Fukui Japan 910–1193 I. Glenn Sipes Department of Pharmacology The University of Arizona Yucson, AZ 85724 USA Tammy E.Stoker Gamete and Early Embryo Biology Branch Reproductive Toxicology Division National Health and Environmental Effects Research Laboratory United States Environmental Protection Agency Research Triangle Park NC 27711 USA Diane Suter Department of Biology Loyola University Chicago Chicago, IL 60626 USA Paul F.Terranova Center for Reproductive Sciences Department of Molecular & Integrative Physiology University of Kansas Medical Center Kansas City KS 66160 USA Dragana Tomic Department of Epidemiology and Preventive Medicine Division of Gender Based Epidemiology University of Maryland School of Medicine 660 West Redwood Street Baltimore, MD 21201 Benjamin K.Tsang Reproductive Biology Unit and Division of Gynecologic Oncology Department of Obstetrics & Gynecology and Cellular & Molecular Medicine University of Ottawa Ottawa Health Research Institute The Ottawa Hospital (Civic Campus) Ottawa Ontario Canada K1Y Y 4E9

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Reprint Request: Benjamin K. Tsang, PhD Ottawa Health Research Institute 725 Parkdale Avenue Ottawa, Ontario Canada K1Y Y 4E9 Chao Wu Xiao Reproductive Biology Unit and Division of Gynecologic Oncology Department of Obstetrics & Gynecology and Cellular & Molecular Medicine University of Ottawa Ottawa Health Research Institute The Ottawa Hospital (Civic Campus) Ottawa Ontario Canada K1Y Y 4E9 Nutrition Research Division Food Directorate Health Products and Food Branch Health Canada 2203C Banting Research Centre Ottawa ON Canada K1A A 0L2 Xiaojuan Yan Reproductive Biology Unit and Division of Gynecologic Oncology Department of Obstetrics & Gynecology and Cellular & Molecular Medicine University of Ottawa Ottawa Health Research Institute The Ottawa Hospital (Civic Campus) Ottawa Ontario Canada K1Y Y 4E9

FOREWORD

In recent years in the USA, there has been an increase in the number of working women, and a tendency for women to postpone the start of a family. These trends have heightened an awareness of the impact of environmental chemicals in the workplace on reproductive function. A variety of considerations can affect fertility in women who are older when beginning a family, and women with fertility problems may not discover them until their reproductive life span is waning. In addition to a generally reduced quality of oocytes with age, more years of exposure to environmental influences can also have a potential effect. A better understanding of the effect of these chemicals on ovarian function is of particular importance since the ovary is critical to normal reproduction. Reproductive function in women can be compromised by exposure to toxic chemicals. Reproductive toxicants can act via direct alterations in steroid hormone production (ovary) or by interference with steroid hormone action (hypothalamus, pituitary, reproductive tract). Alternatively, the effects of ovarian toxicants can result from ovarian failure caused by extensive oocyte destruction. As a result of extensive follicular damage, neuroendocrine feedback is disrupted, and circulating levels of the gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) rise. Therefore, follicle destruction can ultimately disrupt endocrine balance by causing a reduction in ovarian production of estrogen and progesterone, and an elevation in FSH and LH. This book represents a compilation of chapters prepared by researchers who have substantially contributed to our understanding of the impact of xenobiotics and environmental factors on ovarian function. Additionally, issues associated with epidemiology and risk assessment testing as regards the ovary have also been addressed. It is hoped that this volume will prove equally interesting and helpful to scientists in the academic, industrial and regulatory settings. Patricia B.Hoyer, PhD Editor

1 OVARIAN PHYSIOLOGY Diane Suter

INTRODUCTION It is the hope of the author of this chapter that, for readers with expertise in toxicology, some detail or concept will spark a light to illuminate an intersection between ovarian physiology and toxicology. To that end, this chapter is broad but not particularly deep. The reviews and books cited at the end are excellent sources for those seeking more on a particular topic (Stouffer, 1987; Adashi and Leung, 1993; Hsueh and Schomberg, 1993; Fauser et al., 1999). DEVELOPMENT OF THE OVARY Molecular biology The ovary and the testis develop from the same primordial tissue. Only a few genes of importance in their embryonic development have been identified. The most famous is Sry, located only on the Y chromosome and therefore not present in genetic females. Its gene product is the testis-determining factor. Its functional counterpart is Dax-1, located on the short arm of the X chromosome. Its gene product can be thought of as the ovary-determining factor. SRY and DAX-1 proteins are mutually antagonistic. Two active copies of Dax-1 produce enough DAX-1 protein to stimulate the indifferent gonad to become an ovary, even in the case of abnormal presence of Sry. So, in the presence of no Sry and one or more develops into an ovary. If one Sry gene and one or fewer copies of Dax-1 are Dax-1, or in the presence of two Dax-1 genes, regardless of Sry, the indifferent gonad present, the indifferent gonad develops into a testis. The SRY protein is a transcription factor that acts as a switch, turning on expression of genes that cause embryonic differentiation of the testis. The DAX-1 protein encodes an orphan member of the nuclear receptor superfamily and may be a repressor of transcription. In the male, Sry and Dax-1 are both expressed in the genital ridge immediately before sexual differentiation, but Dax-1 expression

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soon ceases. In the female, Dax-1 is expressed and likely suppresses the activity of a variety of genes involved in testis formation. In addition, it acts to direct the development of granulosa cells, the first ovarian cells to develop, without which the other ovarian somatic cells and the oogonia cannot survive. The X-chromosome homolog of Sry is sox3. Its function is unknown, but mutations seem to be lethal, as no sox3¯ females have been reported. The only two other genes known to be involved in ovarian differentiation are encoded at the steel and white spotting loci in the mouse. The white spotting locus is thought to be the same as c-kit locus, a proto-oncogene. The protein encoded by this gene is the receptor for stem cell factor, the gene product of the steel locus. These two genes are necessary for survival of germ cells. Morphology The source of the somatic cells of the ovary is the genital ridge, a thickening of the mesoderm on the posterior wall of the celom. The genital ridge has a cortex and a medulla. In most species, the mature ovary is primarily of cortical origin, and the testis of medullary origin. During the indifferent stage, the medulla grows short spaghetti-like protrusions called cords or primary sex cords. In the testis, these develop, but in the ovary, they degenerate into the connective tissue of the ovarian hilus, the stalk that attaches the ovary to the body wall. In the cortex, a set of secondary cords develop. These secondary cords become the somatic cells of the ovary. Instead of retaining their cord-like appearance, the cords subdivide into clusters of cells. About this time, the germ cells arrive. The germ cells originate in a region of the embryo completely separate from the somatic cells. They originate in the yolk sac and are first apparent after about 1 month in the human embryo and 7 days in the mouse. They migrate first to the hindgut and then to the genital ridge, mitosing along the way. This mitosis has an interesting variation, however, in that cytokinesis is not completed, so the germ cells become clusters of interconnected oogonia. They arrive at the ovary at about 6 weeks in the human, and at about mid-gestation in the mouse. When they get there, a close association forms between the clusters of oogonia and the clusters of secondary sex cord cells. The secondary sex cord cells then completely engulf each individual oogonium with a single layer of cells, breaking the cytoplasmic connections between the oogonia and forming the nascent primordial follicles. A basement membrane, or membrana propria, is already present outside the layer of somatic cells. The germ cells are diploid at this point and are called primary oocytes. This packaging process is complete around the time of birth in rodents and about the end of the first trimester in humans. Once the primordial follicles form, the enclosed oocytes begin meiosis. Recombination occurs and the oocytes hypertrophy. The oocytes progress through meiosis I until the diplotene stage of prophase I. At that point, meiosis I is arrested and remains so until that particular oocyte is ovulated, weeks or

OVARIAN PHYSIOLOGY 3

decades after puberty. Thus, as long as they are in the ovary, the germ cells are diploid primary oocytes. ANATOMY In the mature ovary, all follicles are in the cortex and therefore near the surface of the ovary. An interesting anatomical exception is the horse, in which the cortex inverts and is largely surrounded by medulla. Only a small region of the surface of the equine ovary is made of cortex, and this is the only region of the surface from which ovulation can occur. As female reproductive cycles follow one after the other in overlapping waves, all of the structures described below are likely to appear simultaneously in a mature ovary. The least mature follicular structure is the primordial follicle, comprising a diploid primary oocyte arrested in diplotene I, a single surrounding layer of squamous granulosa cells, and a membrana propria surrounding the granulosa cells. No blood vessels traverse the membrana propria, even in the most mature follicles. The next stage in maturation of a follicle is the primary follicle. The granulosa cells are cuboidal rather than squamous, and a non-cellular layer, the zona pellucida, appears between the primary oocyte and the granulosa cells. The granulosa cells, however, send cytoplasmic processes through the zona pellucida, which touch microvilli on the primary oocyte and form gap junctions with the oocyte. The granulosa cells and the oocyte maintain constant biochemical contact with each other via these gap junctions throughout follicular development. In secondary follicles, also called pre-antral follicles, the granulosa cells divide and organize themselves into multiple layers surrounding the oocyte. A third cell type, the thecal cell, is recruited into the structure. Thecal cells, which differentiate from ovarian stromal cells, are spindle-shaped and surround the granulosa cells, outside the membrana propria. The thecal cells also proliferate, and substantial angiogenesis occurs, making the follicle a well-vascularized structure. The primary oocyte hypertrophies. The next stage of development results in a tertiary follicle, also called a Graafian or antral follicle. The granulosa cells further differentiate into four subspecialties. The corona radiata is the single layer of granulosa cells immediately adjacent to the zona pellucida. These are the granulosa cells that maintain gap junctions with the primary oocyte. Surrounding the corona radiata is the cumulus oophorus, a 3–4-cell-thick layer of granulosa cells that adhere closely to the corona radiata. At ovulation, the cells of the corona radiata and cumulus oophorus leave the ovary and travel down the oviduct along with the oocyte. The intercellular adhesions of the more distal granulosa cells begin to break down, and the granulosa cells secrete a serum-like fluid into the interstices. The fluid, called follicular fluid or liquor folliculi, accumulates and creates one large fluid-filled chamber called the antrum. In addition to water, electrolytes and serum proteins,

4 DIANE SUTER

the follicular fluid contains high concentrations of steroid and protein hormones secreted by the granulosa cells. The third variety of granulosa cell, the mural cells, cling to the membrana propria and maintain their intercellular adhesions, creating a sphere of granulosa cells surrounding the antrum and the oocyte/ corona radiata/cumulus oophorus complex. That complex does not float free in the antrum, but is attached to the mural granulosa cells by the fourth variety of granulosa cells, the stalk. The thecal cells comprise two subtypes, the theca interna and the theca externa. The theca interna has the cytoplasmic ultrastructure of steroidogenic cells, featuring many lipid droplets and extensive smooth endoplasmic reticulum. Not all follicles grow to maturity. Most, in fact, become atretic follicles. Atresia can occur at any of the above stages of development. Atretic follicles are defined anatomically by an irregular follicular shape, eosinophilia of the ooplasm, wrinkling of the oocyte’s nuclear membrane and degeneration of the granulosa cells by apoptosis. The dying granulosa cells can be identified with light-level histology by their pyknotic appearance, characterized by a shrunken, degenerating nucleus with dense, formless chromatin. They can also be identified with modern apoptosis-detecting kits. The number of apoptotic granulosa cells present at any one time, however, is very low, less than 1 percent. Thecal cells do not appear apoptotic. It is thought that they dedifferentiate back into stromal cells and then redifferentiate into thecal cells as other follicles enter the growth phase. When a follicle ovulates, only the oocyte, corona radiata and cumulus oophorus leave the ovary. The mural granulosa cells and the thecal cells stay behind. At the moment of ovulation, the follicular wall and its extensive vasculature break open. This process creates a transient structure, the corpus hemorrhagicum, a collapsed follicle in which the antrum has filled with blood. The presence of corpora hemorrhagica can be used as an assay to quantitate ovulatory sites in a recent ovulation. The corpus hemorrhagicum rapidly redifferentiates into a new structure, the corpus luteum. The name means “yellow body” in Latin, but in many species the corpora lutea are pink. Like the mature follicle, the corpus luteum is a temporary structure, enduring 2–21 days, depending on the species. It is a solid structure with no antrum. One corpus luteum appears per ovulating follicle. Although a more histologically homogeneous structure than the follicle, it does have more than one cell type. The two main functional cell types are defined by size. Large luteal cells, somewhat arbitrarily defined as being greater than 20 µm in diameter, have the ultrastructure of metabolically active cells: considerably dense rough and smooth endoplasmic reticulum, an extensive Golgi apparatus, and many electron-dense granules. The small luteal cells lack electron-dense granules but do have much smooth endoplasmic reticulum. At the end of its prescribed life span, the corpus luteum degenerates via apoptosis. The evidence of its past existence persists for some days in the form of a corpus albicans. This lingering structure appears white due to retention of connective tissue between degenerated cells.

OVARIAN PHYSIOLOGY 5

A structure indicative of a pathological condition is the ovarian cyst, an antral follicle that neither ovulated nor underwent atresia at its appointed hour. Having a structure similar to healthy antral follicles except enlarged, often considerably so, ovarian cysts can be visualized via ultrasound. They cause acyclicity and lower back pain in humans, and because they secrete excess androgens, they also cause hirsutism and acne. The rest of the ovary is made up of stromal cells, and vascular and neural elements. Stromal cells are undifferentiated mesenchymal cells supporting the rest of the structures in the ovary. They are thought also to differentiate into thecal and luteal cells. Like all endocrine tissues, the ovary is highly vascularized. The exception is inside the membrana propria, where there are no blood vessels. In a mature follicle, a dense sphere of blood vessels, sort of a vascular “buckyball,” surrounds the membrana propria but does not traverse it. Despite this lack of vasculature, molecules such as glycoprotein hormones do reach the granulosa cells. Nonetheless, anything reaching the oocyte must pass first through the cells of the corona radiata. The innervation of the ovary is primarily sensory and sympathetic, with a modest amount of parasympathetic input. Stromal, follicular and luteal cells are innervated as well as the vascular smooth muscle. The innervation arises from the ovarian plexus and superior ovarian nerve. ENDOCRINOLOGY AND BIOCHEMISTRY Receptors and signal transduction Chemical messengers acting on or emanating from the ovary can have endocrine, paracrine or autocrine effects. Endocrine effects are those occurring at a distant site from where the messenger is secreted. Paracrine effects are those occurring at close range, on cells near the source of secretion. Autocrine effects occur at pointblank range, on the very cell from which a messenger molecule was secreted. Regardless of the range of action, hormones, growth factors and cytokines all exert their intracellular effects via signal transduction mechanisms. Most signal transduction mechanisms identified to date are active in the ovary. For an excellent review, see Findlay (1994). Two gonadotrophic glycoprotein hormones of the anterior pituitary, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are essential for ovarian function. Their receptors are a specialized subfamily of the superfamily of seven transmembrane domain G-protein-coupled receptors. They activate cAMP-protein kinase A pathways and inositol phosphate-protein kinase C pathways. The genes for these receptors have been cloned for several species. Along with the gene for the thyroid-stimulating hormone receptor, the DNA sequences have much similarity, indicating recent evolutionary divergence. The genes for the LH and FSH receptors are located very close to each other (in

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humans, on chromosome 2). Unlike others in the G-protein-coupled receptor family, these genes are large and have several introns. Also unlike others in the family, the extracellular N-terminal portion of the protein is very long and is the site of ligand binding. The transmembrane domains in the C-terminal half of the receptor protein are the sites of G-protein association. Receptors for LH are located in the theca interna, the late-stage granulosa cell, and the corpus luteum. Receptors for FSH are located in the granulosa cell. Receptors for the gonadotropins are internalized and down-regulated by their ligand. Some recycling of receptors occurs. The signal transduction pathways for steroid hormones are strikingly different from those for protein hormones. The receptors for steroid hormones belong to a superfamily of ligand-activated transcription factors. These receptors all contain an individualized ligand-binding domain, a highly conserved DNA-binding domain, an unconserved hinge region between them and an unconserved Nterminus. They are synthesized in the rough endoplasmic reticulum like other proteins and remain in an inactive state by virtue of their association with the heat shock protein (hsp90). They are translocated to the nucleus either before or after ligand binding, depending on the particular steroid. Ligand binding causes a conformational shift that induces dissociation of hsp90 and association of two identical receptorligand complexes into a homodimer. The homodimer, along with other requisite factors, binds to specific regions of DNA and induces their transcription. Steroids have endocrine, paracrine and autocrine actions, and most cells of mature follicles and corpora lutea express steroid receptors. Many growth factors are important in ovarian physiology. Receptors for many of these growth factors are tyrosine kinases (epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, nerve growth factor and ckit). Others are serine-threonine kinases (transforming growth factor β inhibin, activin). Receptors for prolactin are present in the corpus luteum and follicular thecal cells. They are in the same family as receptors for growth hormone, interleukins and erythropoietin. They have no intrinsic kinase activity but activate a soluble cytoplasmic kinase system known as Janus kinase signal transducers and activators of transcription (JAK-STAT). Steroids Soluble factors were identified as important mediators of reproductive function by Berthold in 1849, in the first controlled endocrinology experiment. These factors eventually proved to be steroids, small, lipid-soluble molecules with four common interlocking carbon rings and individualized residual groups that confer hormone specificity. They include the progestins, the estrogens and the androgens. Progestins, most notably progesterone, are 21-carbon steroids, synthesized in the follicle (mostly by the theca interna, but also by granulosa cells) and the corpus luteum. Androgens, including testosterone, are 19-carbon steroids. They are considered to be primarily male hormones, but they are

OVARIAN PHYSIOLOGY 7

synthesized in the ovary as obligate intermediates for the estrogens. Estrogens, most notably estradiol, are 18-carbon steroids synthesized primarily by granulosa cells, although the primate corpus luteum does synthesize estradiol. Detailed diagrams of steroidogenesis are available in any good endocrinology or reproductive biology text, such as Griffin and Ojeda (2000), Hadley (2000) or Johnson and Everitt (2000). Cholesterol is the synthetic precursor of steroids. Steroidogenesis comprises a progressive loss of carbons in a series of hydroxylation reactions. The enzymes involved in this process are in the superfamily of cytochrome P450 enzymes, including their associated electron-transport systems. The ovary can synthesize cholesterol from the simple 2-carbon molecule acetate, but the ovary gets most of its cholesterol by endocytosis of cholesterol from the blood. Granulosa cells have low-density lipoprotein (LDL) receptors in their plasma membranes and take up LDL-bound cholesterol via endocytosis. The cholesterol is esterified and stored in lipid droplets in the cytoplasm. The first step in steroidogenesis is the conversion of cholesterol to pregnenolone, catalyzed by the cholesterol side-chain cleavage enzyme. It is the rate-limiting step in steroidogenesis and P450scc is expressed in all steroidogenic cells. This enzyme is located on the inner mitochondrial membrane, and thus cholesterol must be transported across both mitochondrial membranes to initiate steroidogenesis. The steroidogenic acute regulatory (StAR) protein is responsible for chaperoning cholesterol across the mitochondrial membranes. StAR protein is short-lived (although its mRNA is not), conferring cycloheximidesensitivity upon steroidogenesis. The next enzyme in the steroidogenic pathway is 3β-hydroxysteroid dehydrogenase/∆5 → ∆4-isomerase, which catalyzes the conversion of pregnenolone to progesterone. Progestins, both pregnenolone and progesterone, are converted irreversibly to 19-carbon androgens by 17α-hydroxylase/17–20lyase (P45017α), which clips off a 2-carbon side chain. Androgens are converted irreversibly to estrogens by aromatase (P450aro), whicl cyclizes the A-ring, removing one carbon in the process. Another steroidogenic enzyme that merits mention is 17β-hydroxysteroid dehydrogenase/oxidoreductase, which catalyzes conversion of androstenedione to testosterone, a more potent androgen. It also catalyzes the conversion of estrone to estradiol, a more potent estrogen. Finally, 5α-reductase converts testosterone to 5α-dihydrotestosterone, an extremely potent androgen. The expression of 5α-reductase is inversely proportional to the expression of aromatase throughout the cycle. It serves as an alternative pathway for metabolism of androgens, rather than aromatization to estrogens. Furthermore, 5α-DHT may be luteolytic. The main steroidal product of the follicle is estradiol. Its synthesis is described by the two-cell, two-gonadotropin theory of steroidogenesis in the follicle. According to this well-supported theory, the theca interna expresses LH receptors, side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase and

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17α hydroxylase/ 17,20 lyase, but not FSH receptors or aromatase. Granulosa cells express FSH receptors, side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase and high levels of aromatase. The thecal cells synthesize androgens from cholesterol, which diffuse across the membrana propria into the granulosa cells, where they are aromatized into estradiol. This theory is supported by autoradiographic studies demonstrating the localization of radiolabeled LH to thecal cells and of FSH to granulosa cells. It is also supported by immunocytochemistry and in situ hybridization studies showing the localization of steroidogenic enzymes to the above mentioned cell types. Because of the presence of the membrana propria, it is possible to do a reasonably thorough job of separating thecal from granulosa cells in vitro, and subsequent incubation studies also support the idea that these two cell types work together to synthesize estradiol in the follicle. Proteins Protein hormones synthesized and secreted by the ovary include oxytocin and relaxin. Oxytocin is synthesized by the corpus luteum and participates in luteolysis by virtue of its ability to inhibit synthesis of progesterone. Relaxin is important in pregnancy. It increases endometrial aromatase activity, decreases myometrial contractility, induces cervical ripening and dilatation, and serves as a mammary gland growth factor. Other important ovarian proteins include the proteins of the zona pellucida, which are essential for proper fertilization. Proteins ZP-2 and ZP-3 are synthesized by the oocyte. ZP-3 is the sperm receptor and activates the acrosome reaction. Both are glycosylated and both are modified in the zona reaction to prevent polyspermy. Many of the proteins and peptides synthesized by the ovary are growth factors or cytokines, locally produced factors that exert autocrine and paracrine effects on proliferation and differentiation of ovarian structures. The number of these factors known to be expressed in the ovary is long and continues to grow. An excellent summary is presented in Findlay (1994). Foremost among them are the proteins of the inhibin family. Varying dimeric combinations of three protein subunits make either inhibin or activin. One of the three subunits has high sequence similarity to mullerian-inhibiting substance and transforming growth factor (TGF) β. Inhibin and activin are synthesized by granulosa cells and luteal cells. They behave in classical endocrine fashion: inhibin reduces secretion of FSH from the anterior pituitary and activin stimulates secretion of FSH. A third ovarian protein, follistatin, binds to activin, thereby inactivating it in the local ovarian environment. A theory of the paracrine effects of inhibin, activin and follistatin has yet to solidify. The second most well-characterized ovarian growth factor is insulin-like growth factor-1 (IGF-1). The ovary is one of the major producers of IGF-1 (human granulosa cells, however, secrete IGF-2 rather than IGF-1). It is secreted

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by granulosa and thecal cells. Its sites of action include the granulosa, thecal and luteal cells, as well as the oocyte. It acts synergistically with FSH to promote follicular growth. FSH increases expression of IGF-1 receptors. This synergy first stimulates granulosa cell differentiation, then estrogen and progesterone synthesis, and finally late expression of LH receptors on granulosa cells. It exerts a classical sulfation effect, stimulating production of heparan and dematan sulfate, possibly important for building the physical infrastructure of the antral follicle. In addition, thecal and granulosa cells also secrete several IGF-binding proteins (IGFBPs). Their production is inhibited by FSH. Atretic follicles do not produce IGF-1, suggesting that IGF-1 is necessary for follicular selection. Thus, FSH, IGFs and IGFBPs participate in a physiological balancing act that, depending on the particular balance, will result in maturation or atresia. Several growth factors have been demonstrated to play clear roles in ovarian function, even though their biochemistry and molecular biology is not as well characterized as those of inhibin or IGF-1. One of these is c-kit, mentioned above as being vital for the survival and migration of primordial germ cells. A second growth factor known to be essential to ovarian development is nerve growth factor (NGF). NGF stimulates innervation during development and perhaps maintains the strength of innervation during adulthood. It is synthesized by granulosa and thecal cells. Its receptors are located on these follicular cells as well as on innervating fibers. Ovarian cells and innervating fibers interact in stimulating follicular growth. A third well-characterized factor is maturation promoting factor (MPF), known to be the central trigger for resumption of meiosis at ovulation. It is a ubiquitous protein, made in the oocyte as somatic tissues, and is highly conserved across the animal kingdom. It consists of two components: a 34 Kd protein homologous to the cdc2+ gene product and cyclin B. Several other growth factors are known to be essential for ovarian function, but well-supported theories of their roles are not yet available. One is epidermal growth factor (EGF). EGF itself is not produced by the ovary, but granulosa cells do produce transforming growth factor (TGF) α and express TGFα receptors, which also bind EGF. These factors promote proliferation of granulosa and thecal cells, and are angiogenic. Furthermore, they inhibit expression of FSH receptors in granulosa cells, thus prolonging mitosis and delaying differentiation of granulosa cells. These facts suggest they play a role in the early growth phase of follicular development. A second in this category is basic fibroblast growth factor (FGF), which is produced by granulosa cells and stimulates cell proliferation. A counterpart to EGF/TGFα is TGFβ. It is synthesized by theca, granulosa and oocytes. It promotes differentiation rather than proliferation of granulosa cells. A cytokine known to be produced by the ovary is interleukin-1 (IL-1). IL-1, its receptor, and its natural antagonist are synthesized locally in the ovary. They appear in thecal cells before ovulation. An inflammation mediator, it is thought to be involved in ovulation, which may be an inflammatory-like process. A second factor that may behave as a cytokine in the ovary is tumor necrosis factor

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(TNF) α. It is synthesized by local macrophages and other immune cells, and also by granulosa cells and oocytes. Its roles are unclear: both growth-promoting and cell-killing effects have been observed. It promotes angiogenesis and inhibits steroidogenesis, suggesting a role in early follicular growth. In the corpus luteum, it inhibits steroidogenesis and induces prostaglandin F2α synthesis, suggesting a role in luteolysis. Enzymes The renin/angiotensin system is a classic endocrine system for regulation of blood pressure, as described in any basic physiology text. The ovary locally produces renin, angiotensinogen and angiotensin-converting enzyme. Angiotensin II is a potent vasoconstrictor and as such may promote atresia. It also, however, stimulates ovulation in mature follicles and promotes angiogenesis in corpora lutea. Two other enzymes, collagenase and plasminogen activator, are critical components of ovulation. They are synthesized by granulosa cells and break down the intercellular connective tissue in the follicle, reducing the tensile strength of the follicular wall at the time of ovulation. Eicosanoids Of the prostaglandins (PGs), PGF2α and PGE2 are the most important in the ovary. PGE2 is synthesized in the human ovary and is luteotrophic. PGF2α is synthesized in the follicle and is an important part of the triggering mechanism for ovulation. It also plays an important role in luteolysis in non-primates. THE OVARIAN CYCLE The ovary, via all of the hormones and other factors mentioned above, performs an intricate dance that creates reproductive cycles in females. Other than the ovary, the important participants in the dance are the anterior pituitary and hypothalamus. Protein hormones from the anterior pituitary—LH, FSH and prolactin—exert gonadotrophic effects on the ovary. Ovarian hormones— estradiol, progesterone, inhibin and activin—act in turn on the hypothalamus and anterior pituitary in negative and positive feedback fashions to create a cyclical pattern of follicular development, ovulation, luteal development and luteolysis. Follicular growth The first step in the dance is early follicular growth, during which many follicles develop from primordial follicles containing a single layer of squamous granulosa cells, to early antral follicles. There is some lack of uniformity of nomenclature here. Some biologists use the word “recruitment” to refer to this

OVARIAN PHYSIOLOGY 11

early period of growth, which can occur at any time, including before puberty. Some use it to mean a later process by which a small number of early antral follicles develop to mature antral follicles. I will follow the latter convention. In humans, unlike most other mammals, early follicular growth is quite protracted and begins several cycles prior to maturation of these follicles. This growth period is characterized primarily by proliferation of granulosa cells. Granulosa cells can divide in vitro in the absence of gonadotropins as long as intraovarian growth factors such as EGF, IGFs, bFGF, TNFα and TGFα are present. In vivo, however, experimental manipulation of gonadotropins suggests that tonic levels of FSH do play at least a facilitatory role in early mitosis in the granulosa cells of primary follicles. As would be predicted from the permissive role of FSH, the granulosa cells acquire FSH receptors and also steroid receptors. The granulosa cells divide until they are about 6–7 layers thick. The zona pellucida appears, next to the oocyte, and ZP-2 and ZP-3 are expressed. Angiogenesis begins even before theca cells appear. Later, there is extensive angiogenesis in the space between the granulosa and thecal cells. The follicular cells are mitotic and not yet differentiated. They do not yet express high levels of steroidogenic enzymes. The antrum begins to form. The corona radiata and cumulus oophorus remain tightly associated with the oocyte, surrounded by follicular fluid but attached to the mural granulosa cells by the stalk. The oocyte hypertrophies throughout this period but remains arrested in prophase I. Recruitment The second step is follicular recruitment, the entrance of some of the early antral follicles into a pool of pre-ovulatory follicles of which one or several, depending on the species, will ovulate. It is not known why, in any given cycle, some growing follicles are recruited and others are reserved for future rounds of recruitment. This process may be a stochastic one in which the follicles that are, by chance, the furthest along developmentally put the remaining follicles into a holding pattern until the more mature ones ovulate or become atretic. Many more follicles are recruited than are ovulated. Recruitment begins with negative feedback. As the ovary nears the end of the luteal phase from the previous cycle, circulating steroids and inhibin drop. This releases FSH from negative feedback. FSH levels rise and stimulate the transition of a cohort of follicles from the early antral to the mature stage. This transition is characterized first by the appearance of thecal cells and the acquisition of LH receptors by thecal cells. Expression of steroidogenic enzymes and a vastly expanded steroidogenic capability arises. Levels of estradiol in the blood rise. Estradiol and FSH act synergistically to promote this stage of maturation, including differentiation of granulosa and thecal cells, characterized by less mitosis, more steroidogenesis and more expression of gonadotropin receptors. Late in this step, expression of LH receptors by granulosa cells occurs. The appearance of LH receptors on granulosa cells is critical for the processes of luteinization and ovulation.

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Selection The third step is follicular selection, determination of which follicle (for monovulatory species such as humans) or follicles (for multiple-ovulating species such as rats) will ovulate. Timing is everything here. For example, monovulatory species like humans have a dominant (i.e., largest) follicle at any given day during follicular development, but it is not the same follicle every day. At some point, a critical series of events culminate in selection of one follicle (or a few, in multiple ovulators) and atresia for the rest. The acquisition of significant numbers of FSH receptors must coincide with a transient rise in FSH levels early in the follicular phase of the cycle. For follicles that are behind the times in generating FSH receptors, follicular atresia occurs. Those follicles that acquire enough FSH receptors respond to FSH by secreting estradiol and inhibin, which in turn inhibits FSH secretion, withdrawing trophic support from other recruited follicles. Again, the result is atresia for the also-rans. The synergy of endocrine FSH and paracrine/ autocrine estradiol allows the selected follicle(s) to continue to grow, despite the withdrawal of FSH’s gonadotrophic support, through enhanced sensitivity to FSH. The acquisition of LH receptors is also critical for successful selection. Having more LH receptors on granulosa cells than do other recruited follicles allows accelerated development, since LH and FSH, both acting through cAMP, have an additive stimulatory effect on granulosa cells that have both kinds of receptors. This synergy is enhanced by angiogenesis as well, as the follicles with the richest vascularization will be exposed to more circulating gonadotropins than other follicles. Atresia Not all Graafian follicles ovulate. For any follicle that does not ovulate, the fate is atresia, an apoptotic process. Atresia occurs even before birth in primordial follicles. It slows at puberty, but continues throughout reproductive life. Atresia is a phenomenon primarily of granulosa cells and oocytes. Thecal cells probably are able to dedifferentiate and redifferentiate into thecal cells in other follicles. Atretic follicles are poorly vascularized, and the lack of delivery of oxygen, nutrients and gonadotrophic factors as the volume of the growing follicle increases, may trigger apoptosis. Protein synthesis declines. The degeneration of the oocyte and pyknosis of the granulosa cells described in the anatomy section occur, and the dying cells are engulfed by invading macrophages. The mechanism is not well understood. Atretic follicles have a high ratio of testosterone:estradiol, suggesting that initiation of aromatase expression is essential for rescue from atresia. Growth factors implicated in the process include IGF-binding proteins and activin. Production of activin would tip the local environment away from inhibin (since inhibin and activin are assembled of the same subunits), and IGF-binding proteins would block the proliferative effect of IGF.

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Ovulation Ovulation relies on a positive feedback interaction between the ovary and the hypothalamic-pituitary axis to bring about the pre-ovulatory gonadotropin surge. Surges of LH and FSH trigger a cascade of events that result in meiotic maturation of the oocyte and physical rupture of the wall of the follicle. The ultimate, and physically obvious, step in meiotic maturation is germinal vesicle breakdown (GVB) or dissolution of the nuclear membrane that must occur in order for homologous chromosomes to segregate in completion of meiosis I. Microscopic observation of GVB is often used as an assay for meiotic maturation. The immature follicle contains a brake, a meiosis-inhibiting factor, the identity of which is not yet certain. A primary part of this braking mechanism is probably sufficiently high levels of cAMP. There is a complex signal transduction cascade that takes the oocyte to GVB. It begins with the pre-ovulatory surge of LH stimulating the granulosa cells. The granulosa cells then decrease their production of GVB-inhibiting substances and increase their production of GVB-stimulating substance(s). In Xenopus laevis, the stimulatory substance is progesterone. In mammals, the identity of the stimulatory factor from the granulosa cells is not certain. Subsequent early changes in the oocyte include increased cytoplasmic calcium, decreased cytoplasmic cAMP and increased cytoplasmic pH. Intermediate events have not all been delineated yet. The final signal transduction, however, is known to be increased MPF, a molecule known to be important in the cell cycle of all cells. It stimulates the transition from G2phase to M-phase, dissolution of the nuclear envelope, phosphorylation of histones that causes chromosome condensation and formation of the spindle apparatus. The first polar body is extruded, bringing the reduction division to completion. The oocyte proceeds to meiosis metaphase II and then enters a second meiotic arrest. Thus, after ovulation, the oocyte becomes a secondary oocyte. The secondary oocyte contains the cytoplasmic mRNAs that will be active in the early stages of development of the conceptus. Proceeding simultaneously with the above chemical process is the physical process of rupture of the follicular wall, necessary for transportation of the oocyte into the oviduct. An increased follicular volume occurs, but no increased intrafollicular pressure. The follicular wall distends as intercellular connections weaken. There is an increased permeability of blood vessels, leakage of erythrocytes and edema. Wall rupture is essentially an enzymatic process involving hydrolysis of intercellular proteins such as collagen. The components of the lytic system, including a plasmin-activating system, are all in place in a relatively mature follicle, but held in check by plasminogen activator inhibitor type 1 (PAI-1), present in granulosa cells. PAI-1 is suppressed by FSH during the pre-ovulatory surge. Also, LH and FSH stimulate prostaglandin synthesis, and prostaglandins in turn stimulate activation of collagenase and plasmin, and promote the vascular effects.

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A third aspect of ovulation is cytoplasmic maturation, the synthesis and storage of many proteins essential for early growth and development of the conceptus. Luteinization and luteal function Meanwhile, back in the ovary, a marvelous transformation of the follicular cells left behind occurs. Late in the process of follicular recruitment, granulosa cells acquire LH receptors. At ovulation, the oocyte, corona radiata and cumulus oophorus leave the ovary, but the rest of the granulosa cells and thecal cells remain. At ovulation, the membrana propria breaks down, allowing mixing of the granulosa and thecal compartments. Further angiogenesis occurs, including invasion of blood vessels into the granulosa layer. Expression of steroidogenic enzymes shifts to favor progesterone production, although the primate corpus luteum does produce significant amounts of estradiol. A new structure, the corpus luteum, is born. The corpus luteum has two cell types—large and small. A 20µm diameter is the arbitrary size cut-off between them. Large cells probably come from granulosa cells and small cells from thecal. Later, though, small cells likely become large cells, at least in some species. The number of small cells increases during the luteal phase. Small cells do not seem to undergo mitosis, however, suggesting that some small cells may arise from differentiating fibroblasts. The large cells are of greater secretory activity. They have all the cellular apparatus necessary to do so, including smooth and rough endoplasmic reticulum, Golgi and electron-dense granules. They make progesterone, oxytocin, relaxin and inhibin. In most species, only small cells respond to LH, even though both cells have LH receptors. Other cell types include vascular epithelial cells, immune and other blood cells, connective tissue cells and smooth muscle cells. The corpus luteum is a transient organ. It is kept alive by a luteotrophic complex of differing composition in different species. The presence of LH is an absolute requirement in most species, including primates. LH levels are in fact quite low during the luteal phase, due to progesterone’s negative feedback effects. The corpus luteum is, however, exquisitely sensitive to LH, due to the high concentration of high-affinity receptors for LH. Prolactin is an important component of the luteotrophic complex in some species. It is more important in rodents than in primates. In particular, it maintains the early corpus luteum, triggered by a neuroendocrine reflex arc initiated by the male’s stimulation of sensory neurons on the flanks of the female during mating. Progesterone is luteotrophic, as is estradiol in many species. Luteolysis The demise of this transient organ, called luteolysis, is an apoptotic process. It involves cessation of steroidogenesis and death of most luteal cells. An old saying is that “in most species, the corpus luteum is murdered by the uterus, but

OVARIAN PHYSIOLOGY 15

in primates, the corpus luteum commits suicide”. In primates, there is not an active luteolytic factor that anyone has found yet. Diminishing LH levels over the course of the luteal phase do occur, but this change does not seem to be sufficient for luteolysis. The primate corpus luteum becomes refractory to LH, but exactly how this occurs is not clear. Nonetheless, the combination of ebbing LH and reduced luteal sensitivity to LH brings about the demise of the primate corpus luteum. In non-primates, particularly ungulates, an early step in luteolysis is the loss of granules from large luteal cells, probably indicating the release of oxytocin. Ovarian oxytocin stimulates secretion of uterine PGF2α. PGF2α travels to the ipsilateral ovary and causes vasoconstriction and also directly inhibits progesterone production. Prostaglandin effects are mediated primarily via the large cells. PGF2α binds to large cells and inhibits their production of progesterone by a protein kinase C-mediated process. Some factors must also act in a paracrine fashion to inhibit progesterone production in the small cells as well. Increased cytoplasmic calcium in response to PGF2α starts the apoptotic process. Maternal recognition of pregnancy Pregnancy in mammals is absolutely dependent upon a steady source of progesterone. In some species, the placenta performs this function, but even in those species, another source of progesterone must be found until the placenta has time to develop. That source of progesterone is the corpus luteum. Hence, the conceptus must somehow signal the maternal physiology of its presence, lest luteolysis occur and cause spontaneous abortion. In the primate and a few other species, this phenomenon is accomplished by the conceptus’ secretion of a chorionic gonadotropin. This molecule is closely evolutionarily related to LH, binds to LH receptors in the corpus luteum, and causes maintenance of luteal function in early pregnancy in the absence of adequate luteotrophic support from LH itself. In some non-primates, including many ungulates, the conceptus secretes interferon-τ, which binds specifically to PGF2α. This binding process sequesters PGF2α in the uterus, preventing it from traveling to the ovary and triggering the luteolytic cascade. In many small animals such as rats and mice, gestation is short, and the reproductive cycle is extremely short. The maternal physiology, figuratively speaking, does not have time to wait around for the conceptus to get physically large enough to signal its presence before it has to make a decision about whether to go on to the next cycle. The evolutionary solution to this problem is pseudopregnancy, a state of luteal maintenance triggered by the physical act of mating. In response to coitus, the maternal anterior pituitary secretes pulsatile prolactin, which acts as a luteotrophic factor for 11 days. At that point, the placentae are large enough to secrete enough progesterone on their own to support the pregnancy for the remainder of gestation.

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CONCLUSION The cyclic nature of ovarian function entails involvement of myriad hormones, growth factors, signal transduction pathways and organismal feedback systems. As such, it is particularly vulnerable to toxic compounds, which need impact upon only one of these factors to disrupt ovarian function. To toxicologists, however, it is more than a vulnerable target: its intricacy and accessibility make it a valuable model for examining the impact of many toxic compounds. REFERENCES AND RECOMMENDED TEXTS Adashi, E.Y. and Leung, P.C.K. (eds) (1993) The Ovary. Comprehensive Endocrinology Revised Series, L.Martini (editor-in-chief). New York, NY: Raven Press. Fauser, B.C.J.M., Rutherford., A.J., Strauss III, J.F. and Van Steirteghem, A. (eds) (1999) Molecular Biology in Reproductive Medicine. New York: Parthenon Publishing Group. Findlay, J.K. (ed.) (1994) Molecular Biology of the Female Reproductive System. San Diego, CA: Academic Press. Griffin, J.E. and Ojeda, S.R. (2000) Textbook of Endocrine Physiology, 4th edn. New York, NY: Oxford University Press. Hadley, M.A. (2000) Endocrinology, 5th edn. Upper Saddle River, NJ: Prentice Hall. Hsueh, A.J.W. and Schomberg, D.W. (eds) (1993) Ovarian Cell Interactions: Genes to Physiology, Serono Symposia, USA, New York, NY: Springer-Verlag. Johnson, M.H. and Everitt, B.J. (2000) Essential Reproduction, 5th edn. Malden, MA: Blackwell Science. Stouffer, R.L. (ed.) (1987) The Primate Ovary, New York, NY: Plenum Press.

2 OVARIAN TOXICITY IN SMALL PREANTRAL FOLLICLES Patricia B.Hoyer

FOLLICULAR DEVELOPMENT Development and maturation of oocytes occurs within ovarian follicles. Successful ovulation requires appropriate follicular development, during which the follicle has passed through a number of distinct developmental stages (Hirshfield, 1991). Throughout the life of a mammalian female preceding each ovarian cycle, some follicles are selected to develop to maturity for ovulation and potential fertilization. The most immature stage of follicular development is termed primordial. This is the stage at which follicles first appear in the ovary of a developing female fetus. Development of a primordial (25 µm diameter) to an ovulatory follicle involves transitions through several stages as a pre-antral follicle (25–250 µm diameter; primordial, primary, secondary) and later as an antral follicle (>250 µm diameter; early antral and pre-ovulatory). The stages of follicular development toward ovulation involve a continuum of events, each providing further maturation of the follicular cells (Figure 2.1). Upon receipt of an as yet unknown signal for development, the primordial follicle is activated and becomes a primary follicle. As the follicle develops, there is proliferation of the granulosa cells surrounding the oocyte and acquisition of a layer of theca interna cells surrounding the granulosa layer. Follicles progress from the primary stage to the secondary stage when multiple layers of granulosa cells are present around the oocyte. When the follicle develops sufficiently, an antrum (fluidfilled space) develops within the granulosa cell layer. The antral follicle continues to grow, and at its most mature stage prior to ovulation is known as a Graaffian (pre-ovulatory) follicle. Resumption of meiosis in the oocyte occurs only at the time of impending ovulation. The exact mechanism for selection of a follicle for ovulation is not understood, but is believed to be under intra-ovarian control (Richards, 1980).

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Figure 2.1 Development of ovarian follicles. Primordial follicles are activated to grow and develop from primary through secondary and antral stages of follicular development until ovulation. Most follicles degenerate by the process of atresia before reaching the ovulatory stage. Those follicles which do ovulate luteinize to become corpora lutea which support pregnancy if fertilization occurs (Hoyer and Devine, 2001, with permission from CRC Press).

Fetal development Because of the nature of fetal development of ovarian follicles, women are born with a set number of oocytes, which cannot be regenerated later in life. During fetal development, primordial germ cells that are formed invade the indifferent gonad and undergo rapid hyperplasia. By one month of embryonic life, primordial germ cells have become established in the genital ridge, and oogonia proliferate by mitosis (Hoyer and Devine, 2001). During the period of mitotic proliferation of germ cells, somatic cells and supporting connective tissue develop and gradually become interspersed among the oogonia (Hoyer, 1997). Oogonia develop into oocytes in synchronous waves, once they stop dividing and enter into the first meiotic division. During this period, oocytes grow slowly and proceed to the diplotene stage of the meiotic prophase. The oocyte does not fully complete the first meiotic division at that time but becomes arrested until ovulation, if this should occur. Therefore, the number of oocytes at the time of birth is finite and comprises the total germ cell pool available to a female throughout her life. Around the time of birth, small individual oocytes within the ovary become surrounded by a few flattened somatic cells (pre-granulosa cells), and a basement membrane to form primordial follicles. Association of the granulosa cells with the oocyte at all times is critical for maintenance of viability, and follicle growth and development (Buccione et al., 1990).

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 19

Primordial follicles Primordial follicles consist of a small oocyte surrounded by a single layer of fusiform-shaped granulosa cells. The small oocyte contained in these follicles is arrested in prophase of the first meiotic division, is non-dividing as follicles develop, and does not resume meiotic division until just prior to ovulation. Primordial follicles provide the pool for recruitment of developing follicles; therefore, they are a fundamental reproductive unit within the ovary. In humans, 1–2 pre-ovulatory follicles develop approximately every 28 days, whereas, in rats, 6–12 follicles develop every 4–5 days (Richards, 1980). The primordial follicles from which pre-ovulatory follicles develop are mainly located near the ovarian cortex and represent the pool for recruitment. Regular waves of the onset of development of primordial follicles is a continuous process from birth, until ovarian senescence occurs (Richards, 1980). Primary follicles Some primordial follicles leave the quiescent state as soon as they are formed, and some are dormant for months or years. The first sign of oocyte growth in primordial follicles is alteration of surrounding squamous (flattened) granulosa cells into cuboidal-shaped cells (Hirshfield, 1991). Once the follicle makes this transition from primordial to primary, other structural changes occur such as development of the zona pellucida and acquisition of the theca layer. The zona pellucida is composed of a glycoprotein matrix to provide protection for the oocyte as well as to provide attachments for the specialized inner layer of granulosa cells, known as cumulus cells. At this stage, another layer of specialized somatic cells begin to proliferate and form a shell outside the basement membrane enclosing the oocyte and granulosa cell layer. These cells appear in concentric rings surrounding the follicle and are designated theca interna cells. Theca cells provide two important functions: (1) attachment of arterioles for the development of an independent blood supply and (2) secretion of progestins and androgens to regulate follicle development (Hirshfield, 1991). Secondary and antral follicles The oocyte contained in a secondary follicle has a large, spherical nucleus (germinal vesicle) that grows in proportion to the growth of the oocyte, and the follicular cells continue to proliferate and form a second layer around the oocyte. As the secondary follicle continues to develop, the layers of granulosa cells surrounding the oocyte increase rapidly to reach follicular diameters of up to 250 µm. Gap junctions are formed between individual cells in the granulosa cell layer to facilitate intercellular transport of nutrients and metabolites to the oocyte (Hoyer, 1997). At that point an antrum begins to form within the granulosa cell layer and this fluid-filled cavity enlarges as the final stages of follicular

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development are reached. The number of follicles that reach the antral stage is quite small. In women, only one follicle per menstrual cycle is usually chosen as the dominant follicle destined for ovulation. A number of autocrine, paracrine and endocrine signals begin to influence follicular development in these stages. At that point, an antrum begins to form within the granulosa cell layer and this fluid-filled cavity enlarges as the final stages of follicular development are reached. The number of follicles that reach the antral stage is quite small. In women, only one follicle per menstrual cycle is usually chosen as the dominant follicle destined for ovulation. The somatic cells acquire receptors for follicle stimulating hormone (FSH) to enhance follicle growth, and they develop steroidogenic capacity for synthesis of androgens, estrogens and progesterone. Collectively, primordial, primary and secondary follicles are less than 250 µm in diameter and are referred to as pre-antral follicles, with primordial and primary being considered small pre-antral follicles. Atresia Throughout the reproductive life span of a female, the total number of primordial follicles selected to develop for ovulation is small compared to the total population. Instead, the vast majority are lost to attrition in various stages of development by a process called atresia. Ovarian content of oocytes is dynamic and fluctuates with age. The total number of oocytes peaks during embryonic development. In humans, the peak number of oocytes ever present, about seven million, occurs at five months gestation, at birth the number has dropped to two million, 250,000–400,000 at puberty, and no functional oocytes remain at menopause (Hirshfield, 1991; Hoyer, 1997). During the lifetime of a woman, ovulation only accounts for 400–600 oocytes. Therefore, of the primordial follicles a female is born with, greater than 99 percent will be lost by atresia at various stages of development. CONSEQUENCES OF FOLLICULAR DESTRUCTION Follicle-specific effects For chemicals that destroy ovarian follicles, the stage of development at which the follicle is targeted determines the impact that exposure to the chemical will have on reproduction (Hoyer and Sipes, 1996). Damage to large growing or antral follicles can cause a disruption of cyclicity by impacting on ovarian steroid production and ovulation. Chemicals which selectively damage large growing or antral follicles only temporarily interrupt reproductive function because these follicles can be replaced by recruitment from the greater pool of primordial follicles. Thus, these chemicals produce a readily reversible form of infertility that is manifest relatively soon after exposure. Conversely, chemicals which

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 21

extensively destroy oocytes contained in primordial and primary follicles can cause permanent infertility and premature ovarian failure (early menopause in women) since once a primordial follicle is destroyed, it cannot be replaced. Destruction of oocytes contained in primordial follicles may have a delayed effect on reproduction until such a time that recruitment for the number of growing and antral follicles can no longer be supported (Generoso et al., 1971; Hooser et al., 1994). Table 2.1 Long-term effects of 30d dosing of female B6C3F1 mice with VCHa,b

Notes a Summarized from Hooser et al. (1994). With permission from Annual Review of Pharmacology and Toxicology. b Data expressed as % control mice. c Day after onset of dosing (30d). d Determined by vaginal cytology. e Different from control mice, p < 0.05.

Although direct destruction of small ovarian pre-antral follicles may not immediately alter circulating hormone levels, the long-term result is disruption of negative feedback at the level of the hypothalamus and pituitary in response to loss of ovarian hormone regulation. FSH produced in the anterior pituitary regulates follicular development, and is under a negative feedback regulation of release by ovarian hormones, such as estrogen, progesterone and inhibin (Hedge et al., 1987). A primary effect at the ovarian level might cause a later disruption of this regulatory axis and FSH levels should increase due to loss of negative feedback from the ovarian hormones. In a long-term study, female B6C3F1 mice were treated with the occupational chemical, 4-vinylcyclohexene (VCH) for 30 days (age 28–58 days) and then observed for one year (Hooser et al., 1994; Table 2.1). Although a greater than 90 percent loss of oocytes in small follicles was measured by 30 days, FSH levels were only first observed to be increased above control animals at 240 days. Therefore, ovarian changes preceded the rise in circulating FSH levels. Also at 240 days, vaginal cytology still displayed evidence of ovarian cyclicity in VCH-treated mice. However, by 360 days (from the onset of 30 days of dosing), unlike control animals, complete ovarian failure had resulted in VCH-treated mice, as determined by increased circulating levels of FSH, lack of cyclicity, the complete absence of ovarian follicular or luteal structures and marked ovarian atrophy. From this study, it was concluded that the

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ovarian failure that occurs long after cessation of dosing with VCH is an indirect consequence resulting from extensive depletion of small, pre-antral follicles. Menopause Ultimately, if the ovary is depleted of primordial follicles, ovarian failure occurs. Ovarian failure (menopause in women) is associated with the cessation of ovarian cyclicity. The average age of menopause in the United States is 51, and this is a direct consequence of depletion of the follicular reserve. Menopause has been associated with a variety of health problems in women. These include increased incidences of osteoporosis, cardiovascular disease, depression and Alzheimer’s disease (Christensen et al., 1980; Paganini-Hill and Henderson, 1994; Sowers and La Pietra, 1995; Oparil, 1999; Dhar, 2001). Additionally, in laboratory animals, premature ovarian failure is associated in the long term with an increased incidence of ovarian neoplasms (Hoyer and Sipes, 1996). A variety of environmental factors have been highly correlated with early menopause in women (Beverson et al., 1986; Cooper et al., 1995). Therefore, as a woman ages, her overall health is significantly affected by the onset of menopause, and this can be further impacted by environmental factors to which she has been exposed. Because of these health risks, the subject of this chapter is chemicals that can cause premature ovarian failure by destruction of small ovarian follicles. OVOTOXICITY IN SMALL PRE-ANTRAL FOLLICLES Ionizing radiation Rapidly dividing primordial germ cells and oogonia present during fetal development in all species are highly sensitive to destruction (Dobson and Felton, 1983). Destruction of oocytes contained in ovarian primordial follicles can be caused by a variety of environmental chemicals (Hoyer and Sipes, 1996). Additionally, exposure to irradiation is known to produce rapid destruction of oocytes contained in primordial follicles, followed by increased follicular atresia, stromal hypertrophy and loss of ovarian weight (Dobson and Felton, 1983). In animal studies, Mattison and Schulman (1980) noted that pre-natal exposure to ionizing radiation also affects the number of oocytes and reproductive capacity of female offspring. More recently it was shown in pre-pubertal female mice that exposure to irradiation caused degeneration of ovarian primordial and primary follicles by causing apoptosis in granulosa cells, oocytes or both (Lee et al., 2000). These effects are suspected in humans because of reports of amenorrhea and sterility in women undergoing therapeutic irradiation (Chapman, 1983; Damewood and Grochow, 1986).

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 23

CHEMOTHERAPEUTIC AGENTS Cylcophosphamide (CPA) is an alkylating chemotherapeutic agent. Since their early use as antineoplastic drugs, alkylating agents have been associated with ovarian failure. Animal studies have confirmed this effect by observing follicle destruction. CPA significantly reduced the number of primordial and antral follicles in C57BL/6N and DBA/2N mice and Sprague-Dawley (SD) rats (Shiromizu et al., 1984; Plowchalk and Mattison, 1991). CPA is a precursor for the active form of the drug that must be bioactivated to exert its functional activity (Brock, 1967). The active metabolites include 4-hydroxy-CPA, aldophosphamide and phosphoramide mustard, which is thought to be the active anticancer agent (Boddy and Ratain, 1997; Boddy and Yule, 2000). Phosphoramide mustard is also thought to be responsible for the ovarian follicle destruction caused by CPA (Plowchalk and Mattison, 1991; Anderson et al., 1995). The ovary does not appear to metabolize CPA, rather metabolism is thought to occur in the liver with uptake of the reactive metabolites from the blood (Anderson et al., 1995). Miller and Cole (1970) studied the ovaries in mice treated with CPA in low doses for 1 year, and found reduced numbers of oocytes (especially primordial) and corpora lutea with no effect on other tissues such as the kidney, spleen, thymus or lymph nodes. Estrous cyclicity was destroyed and cysts/tumors were observed in the ovarian germinal epithelium. In a short-term study, susceptibility to CPA was greatest in primordial follicles in exposed C57BL/6N and D2 mice and SD rats (Shiromizu et al., 1984). Accordingly, in a 5–6-week study in Balb/c mice, CPA destroyed primordial follicles but left larger follicles and ovulation, mating and pregnancy rates intact (Meirow et al., 1999). Plowchalk and Mattison (1992) observed a time- and dose-dependent relationship between CPA and ovarian toxicity by looking at changes in ovarian structure and function. In C57BL/6N mice given a single i.p. injection of CPA (75, 200, or 500mg/kg), primordial follicle numbers were significantly reduced to 73, 42 and 38 percent of controls, respectively. The loss of primordial follicles was essentially complete in three days, and the estimated ED50 (concentration that produced 50 percent follicle loss) was 122mg/kg body weight. From these results it appears that premature ovarian failure in women treated with CPA is likely to be via destruction of primordial follicles. Interestingly, in a study in rats, it was shown that a single injection of CPA caused damage in growing and antral follicles, but spared an effect on primordial follicles, because cyclicity was disrupted within a week, but had been restored within 2 weeks of the one-time exposure (Jarrell et al., 1991). Therefore, the impact of CPA on ovarian follicles may be species dependent.

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Polycyclic aromatic hydrocarbons Many animal studies have demonstrated ovotoxic effects of polycyclic aromatic hydrocarbons (PAH). Cigarette smoke contains high levels of the PAHs, 9:10dimethyl-1:2-benzanthracene(DMBA),benzo[a]pyrene (BaP) and 3methylcholanthrene (3-MC) (Mattison and Thorgeirsson, 1978; Vahakangas et al., 1985). Krarup (1967, 1969) reported that DMBA depletes oocytes and produces ovarian tumors in mice. Subsequently, these effects have also been reported for 3-MC and BaP. In mouse studies, oocyte destruction was shown to occur in response to these three chemicals (Mattison, 1979). The three PAHs destroyed oocytes in small follicles in SD rats and in D2 and B6 mice, within 14 days following a single i.p. injection (Mattison, 1979). Under these conditions, mice were more susceptible to ovotoxicity than rats. Oocyte destruction in primordial and primary follicles was observed in mice treated with DMBA, BaP and 3-MC (Mattison and Thorgeirsson, 1979). The relative toxicities for primordial follicles were DMBA > 3MC > BaP. Furthermore, a direct relationship between the dose of PAHs and destruction of primordial follicles has been shown in the mouse ovary. Daily oral exposure during pregnancy in mice between 7 and 16 days of gestation with high doses of BaP also caused complete sterility of the female offspring (Mackenzie and Angevine, 1981). Pregnant mice exposed to a lower dose (10 mg BaP/kg) gave birth to offspring with severely compromised fertility. In both studies the litters of exposed mothers were smaller in number and size, compared with controls. PAHs are not directly ovotoxic, but require metabolic activation to reactive metabolites. Ovarian enzymes involved in the biotransformation (i.e. aryl hydrocarbon hydroxylase and epoxide hydrolase) of PAHs have been identified in mice humans (Bengtsson et al., 1988). Therefore, oocyte destruction by PAHs may and rats (Mattison et al., 1983), monkeys (Bengtsson and Mattison, 1989) and involve distribution of the parent compound to the ovary where ovarian enzymes metabolize the compound to reactive intermediates (Mattison et al., 1983). These metabolites are capable of covalent binding to macromolecules such as DNA, RNA and protein (Sims and Grover, 1974). However, the direct intracellular target for ovotoxicity has not been determined. B6 mice were more susceptible to BaP than D2 mice (Mattison and Nightingale, 1980); yet, both strains were equally susceptible to the arene oxide metabolite of BaP (Sims and Grover, 1974). Furthermore, inhibition of PAH metabolism with αnaphthoflavone prevented oocyte destruction observed in mice (Mattison et al., 1983). These observations indicate the PAHs must undergo bioactivation to arene oxides to produce their ovotoxic effects.

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 25

Occupational chemicals 1, 3-Butadiene 1,3-Butadiene (BD) is an industrial compound used in the synthesis of polymers, resins and plastics (IARC, 1986). BD is also released as a by-product from the production of plastics and is found in automobile exhaust, gasoline vapors and cigarette smoke (NTP, 1984). BD and the related olefins, isoprene and styrene are also released during the manufacture of synthetic rubber and thermoplastic resins and the estimated annual occupational exposure of US employees is 3,700– 1,000,000 people (IARC, 1986). These chemicals have also been reported in cigarette smoke and automobile exhaust. Chronic animal inhalation studies have shown that carcinogenesis caused by BD is higher in mice than rats. At lower doses, female mice exposed daily by inhalation for up to two years exhibited ovarian atrophy, granulosa cell hyperplasia and benign and malignant granulosa cell tumors (Melnick et al., 1990). Thus, ovarian effects of BD appear to occur at lower concentrations than are required to produce effects in other tissues. Because of the possibility of epoxidation of these butadiene-related compounds, they have the potential to be ovotoxic and carcinogenic. Metabolites of BD were used in one study in female B6C3F1 mice dosed daily for 30 days. 1, 3-Butadiene monoepoxide (BMO, 1.43 mmole/kg) depleted small follicles by 98 percent and growing follicles by 87 percent compared with control animals (Doerr et al., 1995). At 0.14 mmole/kg 1,3-butadiene diepoxide (BDE) depleted small follicles by 85 percent and growing follicles by 63 percent. The results of this study support that a diepoxide formed in the metabolism of BD is more potent at inducing follicle loss. Additionally, isoprene was reported to be ovotoxic, whereas, styrene and its monoepoxide did not reduce mouse ovarian follicle numbers (Doerr et al., 1995). The results of these studies demonstrated direct ovarian targeting of the ovary by the diepoxide of BD and provided evidence that this is the ovotoxic form of the chemical. 4-Vinylcyclohexene (VCH) VCH is a dimer of BD which forms spontaneously in the manufacture of butadiene (Keller et al., 1997). VCH is also used as an intermediate in the manufacture of flame retardants and plasticizers, as well as a solvent in the manufacture of VCH diepoxide (VCD). Exposure to VCH is likely to be in the occupational and/ or industrial setting. Workers could potentially be exposed to VCH during the production of BD-based rubber, rubber vulcanization in the manufacture of shoe soles, tires and other rubber products, as well as in the manufacture of flame retardants and insecticides. However, production policies in these venues have mandated that these chemical processes are performed in closed vessels. Thus, human exposure to VCH is limited, with the exception of accidental spills and leaks (IARC, 1994). Nonetheless, air concentrations of VCH

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have been measured in the workplace. For instance, levels of 0.03-0.21 mg/m3 were measured in an Italian shoe plant, levels of 0-0.003 mg/m3 were measured in an Italian tire factory, and levels of 0.24-0.43 mg/m3 were measured in an American tire curing room (Rappaport and Fraser, 1977; IARC, 1994). After these short-term air concentration studies, the American Conference of Industrial Hygienists in 1992 established 0.4 mg/m3 as a threshold value for VCH exposure in an 8-hour period. Currently there are no epidemiological studies to relate human exposures to adverse effects. However, in animal studies, ovarian damage caused by VCH and its related epoxide metabolites has been demonstrated by a variety of exposure routes, including dermal, oral, inhalation, and intraperitoneal injection (NTP, 1989; Smith et al., 1990; Grizzle et al., 1994; Bevan et al., 1996). It is, therefore, important to consider the potential risks for human exposure. The occupational chemical, VCH and its diepoxide metabolite, VCD, have been well characterized for their ability to cause selective loss of primordial and primary ovarian follicles in mice and rats (Smith et al., 1990; Springer et al., 1996a; Kao et al., 1999). This selective follicle loss has been induced in neonatal rats (post-natal day 4), rats that were immature at the onset of dosing (d28), and in adult rats (d58) (Flaws et al., 1994; Devine et al., 2002). But, the studies have largely characterized this follicle loss in d28 rats. The manner in which VCD exerts such a selective, targeted response is the subject of ongoing research. However, it is felt that this, in part, is because it affects meiotically arrested cells in small follicles that are directly exposed to circulating distribution of the chemical. Earlier structure-activity studies determined that the diepoxide metabolite of VCH, VCD, is the active form for inducing follicle loss in both mice and rats (Smith et al., 1990; Doerr et al., 1995). 2-Bromopropane One of the most recently identified ovarian toxicants is 2-bromopropane (2BP) (NTP, 2002). Concern was raised over the toxicity of this chemical in 1996 due to adverse reproductive problems in workers in a Korean factory (Kim et al., 1996). Subsequent clinical investigations in Korea as well as experimental research in animals demonstrated adverse effects on hematopoiesis and the reproductive system in both males and females, in humans and rats (Park et al., 1997; Takeuchi et al., 1997). 2BP has been used as a cleaning agent, an intermediate for various chemical syntheses, and a replacement for chlorofluorocarbons. Because of the concerns raised in the Korean factory, 2BP use has been limited to some extent in the US. However, it is also a contaminant of 1-bromopropane which is widely used as a solvent for the manufacture of a variety of chemicals in the industrial setting (NTP, 2002). Human exposures, therefore, would predominantly occur in industrial settings and could be through either dermal contact or inhalation. Data on environmental levels for humans or animals are not available (NTP, 2002). However, the similarity of effects

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 27

observed in humans and in experimental animals provides greater credibility to 2BP-induced reproductive effects. There is evidence that male and female germ cells are the ultimate targets of 2BP. Reduced numbers of early stages of spermatogonia (stage 1) were determined to be the initial morphological alteration in response to a single subcutaneous injection of 1355 mg/kg 2BP administered to rats (Omura et al., 1999). Similarly, Yu et al. (1999) demonstrated that primordial follicles of rats were the first ovarian follicle stage to be affected by a single 8-hour inhalation exposure to 3000 ppm 2BP. Additionally, loss of ovarian follicles of all types was seen in female offspring of SD rats when mothers were exposed during gestation and lactation (Kang, 2002). To date, neither the mechanism nor the cellular or molecular target by which 2BP induces germ cell loss has been elucidated. Other ovotoxic agents The alkylating agents 1,4-di(methanesulfonoxy)-butane (Myleran), trimethylenemelamin (TEM) and isopropyl methanesulfonate (IMS) have been shown to destroy oocytes in small follicles in SECXC57BL/F1 mice following a single i.p. injection (Generoso et al., 1971). This destruction was observed within three days of dosing with TEM and IMS, and within 14 days with Myleran. Daily oral administration of nitrofurazone over two years caused ovarian lesions including development of benign mixed tumors and granulosa cell tumors in mice (Kari et al., 1989). The results of an in vitro mutagenicity study in E.coli using a number of industrial and laboratory chemicals demonstrated a high correlation between alkylating activity and increased mutagenicity (Hemminki et al., 1980). In addition to the chemicals discussed so far, Dobson and Felton (1983) reported a variety of other compounds that were capable of producing significant primordial follicle loss in mice. These chemicals included methyl and ethyl methanesulfonate, busulfan and urethane. Additionally, of a number of fungal toxins and antibiotics tested, procarbazine HCl and 4-nitroquinoline-1-oxide were ovotoxic. Finally, dibromochloropropane, urethane, N-ethyl-N-nitrosourea and bleomycin demonstrated primordial follicle killing, with bleomycin being the most potent. In general, all of these ovotoxic chemicals are also known to possess mutageniccarcinogenic effects. Thus, these studies have further provided a correlation between ovotoxicity and subsequent development of tumorigenesis. How these two events are linked is not clearly understood at this time. The polychlorinated biphenyl compound, 3,3,4,4-tetrachlorobephenyl (TCB), has been shown to be teratogenic in the mouse and embryolethal in the rat, as well as having transplacental ovarian toxicity in the mouse (Ronnback, 1991). Follicles in all stages of development were reduced 40-50 percent in female offspring at 28d of age when mice were exposed in utero on day 13 of gestation. Interestingly, during a 5-month period of testing, this extent of follicular damage did not adversely affect reproductive function in these offspring. Finally, the chlorinated organic chemical hexachlorobenzene has been shown to cause

28 PATRICIA B.HOYER

destruction of primordial follicles in cynomolgus monkeys at doses that did not produce evidence of systemic or hepatic effects (Jarrell et al., 1993). MECHANISMS OF OVOTOXICITY How ovotoxic effects of environmental chemicals are produced is generally not well understood, but might be due to one of several possible mechanisms. Oocyte destruction can result from a toxic chemical directly impairing oocyte viability. Conversely, because oocytes at all stages of follicular development are surrounded by granulosa cells, these mechanisms might also be indirect, involving alterations within the granulosa cell, which compromise its ability to maintain viability in the oocyte (Buccione et al., 1990). Lastly, environmental chemicals might cause follicle loss by accelerating the overall rate of atresia, the normal mechanism by which the majority of follicles degenerate during development. In determining mechanisms involved in ovotoxicity, it is first important to determine whether selective or distinct follicular populations are targeted. In studies investigating the mechanism(s) by which VCD is ovotoxic, rats were dosed daily for 30 days. Morphological evaluation of ovaries from control and treated rats revealed that significant loss of small follicles had occurred in follicles in the primordial, primary and secondary stages (Figure 2.2). However, at a shorter time of dosing, reduced numbers of only primordial and primary follicles were seen on day 15, yet the number of secondary follicles was unchanged. Thus, these findings supported that primordial and primary follicles are directly targeted by VCD and that the loss of secondary follicles seen on day 30 was the result of fewer primordial and primary follicles from which to recruit. Two long-term studies were conducted in mice and rats (age d28) which were dosed daily for 30d with VCH (mice) or VCD (rats) and evaluated at different time points for up to 360d after the onset of dosing for effects on reproductive function (Hooser et al., 1994; Mayer et al., 2002). These studies established that premature ovarian failure could be induced in mice and rats as a result of the selective depletion of the primordial follicle pool. Taken together, the reports related to mechanisms of cell death during ovotoxicity suggest that dose and duration of exposure impact the outcome. This provides further rationale for designing animal studies using low-dose repeated exposure to more closely mimic the nature of human exposures. Cell death The ultimate event associated with follicular destruction is cell death. This is the natural fate of the majority of ovarian follicles because only a selected few follicles that develop will ever be ovulated (Hirshfield, 1991). This process, called atresia, can occur in follicles in all stages of development and can be morphologically distinguished from healthy follicles. Follicular atresia in rats

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 29

Figure 2.2 Reductions of small follicle numbers by repeated dosing with 4vinylcyclohexene diepoxide (VCD). Female Fischer 344 rats (age d28) were dosed daily with vehicle control (open bars) or VCD (80 mg/kg, i.p., closed bars) for 15 days or 30 days. Ovaries were collected and processed for histological counting of primordial, primary and secondary follicles. *p<0.05 different from control (n = 5/group) (Hoyer and Devine, 2001, with permission from CRC Press).

has been shown to occur via a mechanism of physiological cell death, apoptosis (Tilly et al., 1991). Thus, morphological changes of a cell undergoing atresia are those characteristic of apoptosis. Apoptosis is used by many tissues to delete unwanted cells by a non-inflammatory mechanism (Wyllie et al., 1980). Therefore, this form of cell death is physiological and likely to go undetected by the organism. This is in distinct contrast to cell death by necrosis, which usually occurs in response to injury and elicits an inflammatory response in the surrounding tissue. Apoptosis and necrosis can be distinguished by morphological criteria, and the most reliable distinction between apoptotic and necrotic mechanisms of cell death still resides in morphological evaluation at the ultrastructural level (Payne et al., 1995). In recent years there has been an increase in the investigation of apoptotic cell death following treatment with toxic chemicals (Corcoran et al., 1994). A number of reports have provided examples of xenobiotic-induced apoptosis. However, little is known about the types of cell death induced by reproductive toxicants in the ovary. In a study by Mattison (1980), the ovotoxic effects of three PAHs, DMBA, 3-methylcholanthrene (3-MC) and BaP were described as morphological changes in primordial follicles more consistent with necrosis.

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These effects were observed in mice following administration of a single dose. The changes caused by 3-MC and BaP were seen in the oocyte, in the absence of visible effects in the associated granulosa cells. However, DMBA produced more visible toxicity by destroying oocytes and follicles more extensively and disrupting ovarian architecture. Ultrastructural evidence consistent with increased atresia in small pre-antral follicles has also been reported. 3-MC produced a destruction of oocytes in mice that resembled the physiological process of atresia (Gulyas and Mattison, 1979). This was also observed in ovaries collected from rats dosed daily for 10 days with the occupational chemical, VCD (Mayer et al., 2002). Under these conditions, there was no evidence of necrosis, such as cellular swelling or infiltration of macrophages in ovaries from treated rats. In other studies investigating ovotoxicity in rats and mice, morphological evidence consistent with both types of cell death has been reported. Ovaries collected from mice exposed to a relatively high dose of CPA (500 mg/kg) demonstrated necrotic damage in oocytes contained in primordial follicles (Swartz and Mattison, 1985; Plowchalk and Mattison, 1992). This effect was specific for the oocyte because surrounding granulosa cells appeared unchanged. Conversely, atretic changes in primordial follicles were reported at lower doses (100mg/kg). Taken together, these results suggest that mild cellular damage can induce physiological cell death, apoptosis, whereas, more severe damage results in passive cell death, necrosis (Corcoran et al., 1994). It has been determined by a number of observations that VCD-induced follicle loss is by acceleration of the natural process of atresia (apoptosis). One observation that VCD-induced follicle loss is the result of interactions with the atretic process came from a study in which the effect of a single dose of VCD in rats was evaluated (Borman et al., 1999). Twenty-four hours following a single dose there was an increase in percentage of healthy appearing ovarian primary follicles, relative to those measured in animals given vehicle control. The importance of this observation was evident 15 days later. Compared with controls, there were more primary follicles in animals treated with a single dose of VCD. This provided evidence that whereas, repeated daily dosing with VCD causes follicle loss via acceleration of atresia, a single dose protects against the normal rate of atresia. Sites of cellular damage In general, intracellular sites targeted by ovotoxic chemicals have not been identified. Compounds known to contain epoxide moieties (or which are capable of bioactivation by epoxidation) have been shown to affect ovarian function in laboratory animals. Many of these compounds lead to induction of ovarian tumors following long-term exposures. These carcinogens include 1,3-BD and its derivatives (Miller and Boorman, 1990; Mehlman and Legator, 1991), and VCH and its derivatives (NTP, 1986, 1989; Collins et al., 1987; Chhabra et al., 1990).

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 31

Many epoxidated compounds have been associated with increased mutagenicity in in vitro bacterial assays (Hoyer and Sipes, 1996). The ability of epoxides to produce DNA adducts and induce sister chromatin exchanges has also demonstrated effects at the molecular level (Hoyer and Sipes, 1996). However, whether DNA damage is the event that initiates ovotoxicity has not been determined for these chemicals. It has been proposed that plasma membrane damage is more highly correlated with ovotoxicity than DNA damage (Dobson and Felton, 1983). This observation was supported by comparing alkylating properties with genetic activity in a variety of epoxide-containing chemicals (Turchi et al., 1981). Thus, the cellular event(s) initiated directly by ovotoxic chemicals may be at the level of proteins involved in signaling pathways or regulatory mechanisms associated with cell death/viability determination, rather than as a direct result of DNA damage. Effects on gene expression and cell signaling The effect of ovotoxic exposures on ovarian gene expression has not been widely studied to date. Perhaps the most extensive characterization of the impact of ovotoxic chemicals on gene expression has been conducted using rats exposed to the occupational chemical, VCD. Changes in intracellular pathways associated with apoptosis have been measured in follicles undergoing VCD-induced follicle loss. Thus, the results of these studies have provided additional support that ovotoxicity is via apoptosis. Elevated levels of mRNA encoding the cell death enhancer gene, bax (elevated in apoptosis) were measured in isolated fractions of small pre-antral follicles collected from VCD-dosed rats (Springer et al., 1996b). This effect was specific for the small follicles targeted by VCD and was not seen in large pre-antral follicles or hepatocytes (non-target tissues). Other reported effects of VCD on intracellular signaling pathways have included responses in members of the Bcl-2 associated family of proto-oncogenes: increased expression of pro-apoptotic Bad; translocation of Bclx-L from mitochondria to the cytosol; increased ratio of Bax/Bclx-L on the mitochondrial membrane; leakage of cytochrome C from mitochondria to the cytosol; and increased expression and activation of the caspase cascade involving the executioner protease, caspase-3 (Hu et al., 2001a,b). This signaling pathway as a site of regulation of VCD-induced ovotoxicity was supported by a recent observation in transgenic mice (Takai et al., 2003). In that study, there was a reduction in the ability of VCD to reduce primordial and primary follicle numbers in bax, and caspase-2- and caspase-3-deficient mice. Additionally, it has been demonstrated that pro-apoptotic members of the MAPK kinase family are also activated in VCD-induced apoptosis (Hu et al., 2002).

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PREDICTION OF HUMAN RISK The level of exposure to an environmental chemical required to produce ovarian damage is of particular importance. It is under rare, accidental circumstances that large groups of individuals are acutely exposed to toxic levels of reproductive toxicants, and the effects of these exposures can usually be detected and evaluated. However, the possible effects of chronic exposure to low levels of reproductive toxicants are more difficult to determine because of the potential for additive or cumulative effects that might be produced. Therefore, fertility problems produced by environmental exposures may go unrecognized for years, but might manifest as early menopause and/or still later development of ovarian cancer. Thus, it is these potential types of exposures that are of particular concern. Chemotherapy Now that cancer patients are living longer, the toxic effects of chemotherapeutic drugs on the health and quality of life of these survivors have become important issues. Since the beginning of antineoplastic therapy to treat a variety of diseases and malignancies, the ability of these agents to produce ovarian failure has been documented. This effect has been described in patients being treated with CPA, nitrogen mustard, chlorambulcil or vinblastine (Sobrinho et al., 1971; Chapman, 1983; Damewood and Grochow, 1986; Wayne et al., 2002). These observations in humans have motivated a variety of studies with CPA in rodents to better elucidate its mechanism of ovotoxicity. From the results of these animal studies, it appears that premature ovarian failure in women treated with CPA is likely to be via destruction of primordial follicles. Cigarette smoking Epidemiological studies conducted over the last four decades have demonstrated a relationship between smoking and impaired fertility. Cigarette smoke is a wellknown reproductive toxicant. One study reported that rates of pregnancy were reduced to 57 percent in heavy smokers and 75 percent in light smokers when compared with non-smokers; furthermore, smokers required one year longer to conceive than did non-smokers (Baird and Wilcox, 1985). Women smokers have also been reported to experience a one to four year earlier age at the onset of menopause (Jick et al., 1977; Beverson et al., 1986). Thus, a significant amount of data exist to demonstrate a relationship between smoking and an impact on ovarian function. Cigarette smoke is a complex mixture of alkaloids (nicotine), PAH, nitroso compounds, aromatic amines and protein pyrolysates, many of which are carcinogenic (Stedman, 1968). Smoking women have been shown to have significantly decreased follicular levels of estradiol, compared with non-smokers

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 33

(VanVoorhis et al., 1992). Furthermore, extracts of cigarette smoke significantly decreased estradiol secretion by human granulosa cells in culture (Barbieri et al., 1986). Thus, these effects may relate to the infertility associated with cigarette smoking. However, because of the logical association between early menopause and oocyte destruction, some of the effects of cigarette smoke on fertility are likely to be due to destruction of primordial follicles as has been reported by PAHs in animal studies (Mattison and Thorgeirsson, 1979). Of additional concern is the finding in animal studies that exposure of mice in utero to cigarette smoke resulted in a reduced number of ovarian primordial follicles in female offspring (Vahakangas et al., 1985). Mode of exposure The level of exposure to an environmental chemical required to produce ovarian damage is of particular importance. The possible effects of chronic exposure to low levels of reproductive toxicants may go unrecognized for years, due to the potential for additive or cumulative effects that might be produced. Because of their insidious nature, these types of exposures can cause "silent" damage and are of the most concern. This is particularly important when the target cells are of a non-renewing type (ovarian follicles). Several studies have addressed the issue of toxicity as a function of the dosing regimen in mice. Primordial follicle destruction is known to result from dosing of mice and rats with the widely studied PAHs (DMBA, 3-MC, B[a]P), contaminants in cigarette smoke and automobile exhaust. Because these chemicals destroy primordial follicles in laboratory animals, it is likely that they contribute to the early menopause in women smokers. Earlier studies examined ovotoxic effects of these PAHs caused by a single high dose. The extent of primordial follicle loss following this high-dose exposure in mice was reported to be about 50 percent within 1-2 days (Mattison and Thorgeirsson, 1979). In a subsequent study, mice given single intraperitoneal (i.p.) doses of the PAH, BaP ranging from 1-100 mg/kg demonstrated an ED50 of 15 mg/kg for oocyte destruction in B6 mice (Mattison et al., 1983). Interestingly, significant oocyte destruction was demonstrated following a single high dose of BaP (100mg/kg), whereas, the same level of oocyte loss was observed with a low dose (10mg/kg) given daily for 10 days (Mattison and Nightingale, 1980). This observation provides support for a cumulative ovotoxic effect of chronic exposures to low doses. Because repeated low-dose exposure is a more likely source of toxicity in women, another study was undertaken to evaluate the effects of lower doses of these chemicals. Female mice were exposed repeatedly to doses of the PAHs, sufficient to cause 50 percent loss of primordial follicles after 15 days of daily dosing (Borman et al., 2000). Calculating an ovotoxic index using the doses required to cause 50 percent follicle destruction in both studies, it was determined that relative to a single high-dose exposure, repeated low-dose exposure was more ovotoxic by a 250 (DMBA), 120 (3-MC), or 2

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Table 2.2 Comparison of the OI (ED50, mmol/kg × days of dosing) for a single high dose (1×) versus repeated low doses (15×) in B6 mice

Source: From Borman et al. (2000). With permission from Toxicology and Applied Pharmacology. Notes a 80 mg/kg; Mattison and Thorgeirsson, 1979. Lowest OI = greatest toxicity.

(BaP) times greater extent (Table 2.2). Thus, these results demonstrate that animal studies designed to more closely mimic human types of exposures may reveal surprising and disturbing insights as to realistic risk. SUMMARY AND CONCLUSIONS In summary, environmental chemicals that impact ovarian function can directly disrupt endocrine balance by decreasing production of ovarian hormones and interfering with ovulation. These effects are rather immediate, target large antral follicles and can be reversed once there is no longer exposure to the chemical.On the other hand, ovarian function can be impaired by exposure to chemicals that destroy small pre-antral follicles. This produces an indirect disruption of endocrine balance, once hormonal feedback mechanisms have been affected. The manifestation of this type of ovarian toxicity is delayed until irreversible ovarian failure (menopause) has occurred. This specific type of ovotoxicity is of particular concern in women because of the health risks known to be associated with menopause. Future research should be aimed at understanding specific mechanisms of ovotoxicity and improving our ability to predict human risk from the wide variety of exposures to these chemicals in the environment. LITERATURE CITED Anderson, D., Bishop, J.B., Garner, R.C., Ostrodky-Wegman, P. and Selby, P.B. (1995) Cyclophosphamide: review of its mutagenicity for an assessment of potential germ cell risks, Mutat. Res., 330:115-81. Baird, D.D. and Wilcox, A.J. (1985) Cigarette smoking associated with delayed conception, J. Am. Med. Assoc., 253:2979-83. Barbieri, R.L., McShane, P.M. and Ryan, K.J. (1986) Constituents of cigarette smoke inhibit human granulosa cell aromatase, Fertil Steril., 46:232-36.

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Bengtsson, M. and Mattison, D.R. (1989) Gondatropin-dependent metabolism of 7,12dimethylbenz(a)anthracene in the ovary of rhesus monkey, Biochem. Pharmacol., 38: 1869-72. Bengtsson, M., Hamberger, L. and Rydstrom, J. (1988) Metabolism of 7,12-diemthylbenz (a)anthracene by different types of cells in the human ovary, Xenobiotica, 18: 1255-70. Bevan, C, Stadler, J.C., Elliot, G.S., Frame, S.R., Baldwin, J.K., Leung, H.W., Moran, E. and Panepinto, A.S. (1996) Subchronic toxicity of 4-vinylcyclohexene in rats and mice by inhalation exposure, Fundam. Appl. Toxicol., 32:1-10. Beverson, R.B., Sandler, D.P., Wilcox, A.J., Schreinemachhers, D., Shore, D.L. and Weinberg, C. (1986) Effect of passive exposure to smoking on age at natural menopause, Br. Med. J., 293:792. Boddy, A.V. and Ratain, M.J. (1997) Pharmacogenetics in cancer etiology and chemotherapy, Clin. Cancer Res., 3:1025-30. Boddy, A.V. and Yule, S.M. (2000) Metabolism and pharmacokinetics of oxazaphosphorines, Clin. Pharmacol., 38:291-304. Borman, S.M., VanDePol, B.J., Kao, S.W., Thompson, K.E., Sipes, I.G. and Hoyer, P.B. (1999) A single dose of the ovotoxicant 4-vinylcyclohexene diepoxide is protective in rat primary ovarian follicles, Toxicol. Appl. Pharmacol., 158:244-52. Borman, S.M., Christian, P.J., Sipes, I.G. and Hoyer, P.B. (2000) Ovotoxicity in female fischer rats and B6 mice induced by low-dose exposure to three polycyclic aromatic hydrocarbons: comparison through calculation of an ovotoxic index, Toxicol. Appl. Pharmacol., 167:191-98. Brock, N. (1967) Pharmacologic characterization of cyclophosphamide (NSC-26271) and cyclophosphamide metabolites, Cancer Chemother. Rep., 51:315-25. Buccione, R., Schroeder, A.S. and Eppig, J.J. (1990) Interactions between somatic cells and germ cells throughout mammalian oogenesis, Biol Reprod., 43:543-47. Chapman, R.M. (1983) Gonadal injury resulting from chemotherapy, Am. J. Ind. Med., 4: 149–61. Chhabra, R.S., Huff, J., Haseman, J., Jokinen, M.P. and Hetjnancek, M. (1990) Dermal toxicity and carcinogenicity of 4-vinyl-1-cyclohexene diepoxide in Fischer rats and B6C3F1 mice, Fund. Appl. Toxicol., 14:752–63. Christensen, C, Christensen, M.S., McNair, P.L., Hagen, C, Stocklund, K.E. and Transbol, I. (1980) Prevention of early menopause bone loss: conducted 2-year study, Eur. J. Clin. Invest., 10:273–79. Collins, J.J., Motali, R.J. and Manus, A.G. (1987) Toxicological evaluation of 4-vinylcyclohexene. II. Induction of ovarian tumors in female B6C3F1 mice by chronic oral administration of 4-vinylcyclohexene, J. Toxicol. Environ. Health, 21:507–24. Cooper, G.S., Baird, D.D., Hulka, B.S., Weinberg, C.R., Savitz, D.A. and Hughes, C.L. (1995) Follicle stimulating hormone concenterations in relation to active and passive smoking, Obstet. Gynecol., 85:407–11. Corcoran, G.B., Fix, L., Jones, D.P., Moslen, M.T., Oberhammer, F.A. and Buttyan, R. (1994) Apoptosis: molecular control points in toxicology, Toxicol. Appl. Pharmacol., 128:169–81. Dhar, H.L. (2001) Aging, health, and society,J. Assoc. Physicians India, 49:1012–20. Damewood, M.D. and Grochow, L.B. (1986) Prospects for fertility after chemotherapy or radiation for neoplastic disease, Fert. Steril., 45:443–59.

36 PATRICIA B.HOYER

Devine, P.J., Sipes, I.G., Skinner, M.K. and Hoyer, P.B. (2002) Characterization of a rat in vitro ovarian culture system to study the ovarian toxicant 4-vinylcyclohexene diepoxide, Toxicol. Appl. Pharmacol, 184:107–15. Dobson, R.L. and Felton, J.S. (1983) Female germ cell loss from radiation and chemical exposures, Am. J. Indust. Med., 4:175–90. Doerr, J.K., Hooser, S.B., Smith, B.J. and Sipes, I.G. (1995) Ovarian toxicity of 4-vinylcyclohexene and related olefins in B6C3F1 mice: role of diepoxides, Chem. Res. Toxicol., 8:963–69. Flaws, J.A., Doerr, J.K., Sipes, I.G. and Hoyer, P.B. (1994) Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide, Reprod. Toxicol., 8:509– 14. Generoso, W., Stout, S.K. and Huff, S.W. (1971) Effects of alkylating chemicals on reproductive capacity of adult female mice, Mut. Res., 13:172–84. Grizzle, T.B., George, J.D., Fail, P.A., Seely, J.C. and Heindel, J.J. (1994) Reproductive effects of 4-vinylcyclohexene in Swiss mice assessed by a continuous breeding protocol, Fundam. Appl. Toxicol., 22:122–29. Gulyas, B.J. and Mattison, D.R. (1979) Degeneration of mouse oocytes in response to polycyclic aromatic hydrocarbons, Anatom. Rec., 193:863–82. Hedge, G.A., Colby, H.D. and Goodman, R.L. (1987) Clinical Endocrine Physiology, Philadelphia: Saunders, p. 189. Hemminki, K., Falck, K. and Vainio, H. (1980) Comparison of alkylation rates and mutagenicity of directly acting industrial and laboratory chemicals, Arch. Toxicol., 46:277–85. Hirshfield, A.N. (1991) Development of follicles in the mammalian ovary, Int. Rev. Cytol., 124:43–101. Hooser, S.B., Douds, D.A., Hoyer, P.B. and Sipes, I.G. (1994) Long term ovarian and hormonal alterations due to the ovotoxin, 4-vinylcyclohexene, Reprod. Toxicol., 8: 315–23. Hoyer, P.B. (1997) Female reproductive toxicology—introduction and overview. In: I.G. Sipes, C.A. McQueen and J.A. Gandolfi (eds) Comprehensive Toxicology, Oxford, England: Elsevier Pub, Vol. 10. Hoyer, P.B. and Devine, P.J. (2001) Endocrinology and toxicology: the female reproductive system. In: M.J.Derelanko and M.A.Hollinger (eds) Handbook of Toxicology, 2nd edn, Boca Raton, FL: CRC Press. Hoyer, P.B. and Sipes, I.G. (1996) Assessment of follicle destruction in chemical-induced ovarian toxicity, Annu. Rev. Pharmacol Toxicol., 36:307–31. Hu, X.M., Christian, P.J., Sipes, I.G. and Hoyer, P.B. (200 la) Expression and redistribution of cellular bad, bax and bcl-xl protein is associated with VCD-induced ovotoxicity in rats, Biol Reprod., 65:1489–95. Hu, X.M., Christian, P.J., Thompson, K.E., Sipes, I.G. and Hoyer, P.B. (2001b) Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the Caspase cascades, Biol. Reprod., 65:87–93. Hu, X.M., Flaws, J.A., Sipes, I.G. and Hoyer, P.B. (2002) Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats, Biol. Reprod., 67:718–24. IARC (International Agency for Research on Cancer) (1986) 1,3-Butadiene, Lyon, France, World Health Organization, IARC Monogr. Eval Carcinog. Risks Hum., 39:155.

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 37

IARC (International Agency for Research on Cancer) (1994) Some industrial chemicals, IARC Monogr. Eval Carcinog. Risks Hum., France: Lyon, 60. Jarrell, J.F., Bodo, L., Young Lai, E.V., Barr, R.D. and O’Connell, G.J. (1991) The shortterm reproductive toxicity of cyclophosphamide in the female rat, Reprod. Toxicol., 5:481–85. Jarrell, J.F., McMahon, A., Villeneuve, D., Franklin, C, Singh, A., Valli, V.E. and Bartlett, S. (1993) Hexachlorobenzene toxicity in the monkey primordial germ cell without induced porphyria, Reprod. Toxicol., 7:41–47. Jick, H., Porter, J. and Morrison, A.S. (1977) Relation between smoking and age of natural menopause, Lancet, 1:1354–55. Kang, K.S., Li, G.X., Che, J.H. and Lee, Y.S. (2002) Impairment of male rat reproductive function in F1 offspring from dams exposed to 2-bromopropane during gestation and lactation, Reprod. Toxicol., 16:151–59. Kao, S.W., Sipes, I.G. and Hoyer, P.B. (1999) Early effects of ovotoxicity induced by 4vinylcyclohexene diepoxide in rats and mice, Reprod. Toxicol., 13:67–75. Kari, F.W., Huff, J.E., Leininger, J., Haseman, J.K. and Eustis, S.L. (1989) Toxicity and carcinogenicity of nitrofurazone in F344/N rats and B6C3F1 mice, Food Chem. Tox., 27:129–37. Keller, D.A., Carpenter, S.C., Cagen, S.Z. and Reitman, F.A. (1997) In vitro metabolism of 4-vinylcyclohexene in rat and mouse liver, lung, and ovary, Toxicol. Appl. Pharmacol., 144:36–44. Kim, Y., Jung, K., Hwang, T., Jung, G., Kim, H., Park, J., Kim, J., Park, J., Park, D., Park, S., Choi, K. and Moon, Y. (1996) Hematopoietic and reproductive hazards of Korean electronic workers exposed to solvents containing 2-bromopropane, Scand. J. Work Environ. Health., 22:387–91. Krarup, T. (1967) 9:10-Dimethyl-1:2-benozanthracene induced ovarian tumors in mice, Acta Path. Micro. Scand., 70:241–48. Krarup, T. (1969) Oocyte destruction and ovarian tumorigenesis after direct application of a chemical carcinogen (9:10-dimethyl-1:2benzanthrene) to the mouse ovary, Int. J. Canc., 4:61–75. Lee, C.J., Park, H.H., Do, B.R., Yoon, Y. and Kim, J.K. (2000) Natural and radiationinduced degeneration of primordial and primary follicles in mouse ovary, Anim. Reprod. Sci., 59:109–17. Mackenzie, K.M. and Angevine, D.M. (1981) Infertility in mice exposed in utero to benzo (a)pyrene, Biol. Reprod., 24:183–91. Mattison, D.R. (1979) Difference in sensitivity of rat and mouse primordial oocytes to destruction by polycyclic aromatic hydrocarbons, Chem. Biol. Interactions, 28:133– 37. Mattison, D.R. (1980) Morphology of oocyte and follicle destruction by polycyclic aromatic hydrocarbons in mice, Toxicol. Appl. Pharmacol., 53:249–59. Mattison, D.R. and Nightingale, M.R. (1980) The biochemical and genetic characteristics of murine ovarian aryl hydrocarbon (benzo(a)pyrene)hydroxylase activity and its relationship to primordial oocyte destruction by polycyclic aromatic hydrocarbons, Toxicol. Appl Pharmacol., 56:399–408. Mattison, D.R. and Schulman, J.D. (1980) How xenobiotic compounds can destroy oocytes, Contemp. Obstet Gynecol., 15:157. Mattison, D.R. and Thorgeirsson, S.S. (1978) Smoking and industrial pollution, and their effects on menopause and ovarian cancer, Lancet, 1:187–88.

38 PATRICIA B.HOYER

Mattison, D.R. and Thorgeirsson, S.S. (1979) Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic hydrocarbons in mice, Canc. Res., 39:3471–75. Mattison, D.R., Shiromizu, K. and Nightingale, M.S. (1983) Oocyte destruction by polycyclic aromatic hydrocarbons, Am. J. Ind. Med., 4:191–202. Mayer, L.P., Pearsall, N.A., Christian, P.J., Devine, P.J., Payne, C.M., McCuskey, M.K., Marion, S.L., Sipes, I.G. and Hoyer, P.B. (2002) Long-term effects of ovarian follicular depletion in rats by 4-vinylcyclohexene diepoxide, Reprod. Toxicol., 16: 775–81. Mehlman, M.A. and Legator, M.S. (1991) Dangerous and cancer-causing properties of products and chemicals in the oil refining and petrochemical industry. Part II: Carcinogenicity mutagenicity and developmental toxicity of 1,3-butadiene, Toxicol. Indust. Health, 7:207–20. Meirow, D., Lewis, H., Nugent, D. and Epstein, M. (1999) Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide: clinical importance and proposed accurate investigative tool, Hum. Reprod., 14:1903–07. Melnick, R.L., Huff, J., Chou, B.J. and Miller, R.A. (1990) Carcinogenicity of 1,3butadiene in C57BL/6 X C3HF1 mice at low exposure concentrations, Cancer Res., 50:6592–99. Miller, R.A. and Boorman, G.A. (1990) Morphology of neoplastic lesions induced by 1, 3 butadiene in B6C3F1 mice, Environ. Health Perspec., 86:37–48. Miller, J.J. and Cole, L.J. (1970) Changes in mouse ovaries after prolonged treatment with cyclophosphamide, Proc. Soc. Exp. Biol Med., 133:190–93. NTP (National Toxicology Program) (1984)Toxicology and carcinogenesis studies of 1,3butadiene (CAS No. 106–99–0) in B6C3F1 mice (inhalation studies), NTP Technical Report, 288:1–111. NTP (National Toxicology Program) (1986) Toxicology and carcinogenesis studies of 4vinyl-cyclohexene in F344/N rats and B6C3F1 mice, NTP Technical Report, 303. NTP (National Toxicology Program) (1989) Toxicology and carcinogenesis studies of 4vinyl-1-cyclohexene diepoxide in F344/N rats and B6C3F1 mice, NTP Technical Report, 362. NTP (National Toxicology Program) (2002) NTP-CERHR expert panel report on the reproductive and developmental toxicity of 2-bromopropane, NTP Technical Report, 1–46. Omura, M., Romero, Y., Zhao, M. and Inoue, N. (1999) Histopathological evidence that spermatogonia are the target cells of 2-bromopropane, Toxicol. Lett., 104:19–26. Oparil, S. (1999) Hormones and vasoprotection, Hypertension, 33:170–76. Paganini-Hill, A. and Henderson, V.W. (1994) Estrogen deficiency and risk of Alzheimer’s disease in women, Am. J. Epidemiol., 140:256–61. Park, J., Kim, Y., Park, D., Choi, K., Park, S. and Moon, Y. (1997) An outbreak of hematopoietic and reproductive disorders due to solvents containing 2-bromopropane in an electronic factory, South Korea: epidemiological survey, J. Occup. Health, 39: 138–43. Payne, C.M., Bernstein, C. and Bernstein, H. (1995) Apoptosis overview emphasizing the role of oxidative stress, DNA damage and signal-transduction pathways, Leuk. Lymphoma, 19:43–93. Plowchalk, D.R. and Mattison, D.R. (1991) Phosphoramide mustard is responsible for the ovarian toxicity of cyclophosphamide, Tox. Appl. Pharm., 107:472–81.

OVARIAN TOXICITY IN SMALL PRE-ANTRAL FOLLICLES 39

Plowchalk, D.R. and Mattison, D.R. (1992) Reproductive toxicity of cyclophosphamide in the C57GBL/6N mouse. 1. Effects on ovarian structure and function, Reprod. Toxicol., 6:411–21. Rappaport, S.M. and Fraser, D.A. (1977) Air sampling and analysis in rubber vulcanization area, Amer. Hyg. Assoc. J., 38:205–10. Richards, J.S. (1980) Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation, Physiol. Rev., 60:51–89. Ronnback, C. (1991) Effect of 3,3',4,4'-tetrachlorobiphenyl (TCB) on ovaries of foetal mice, Pharm. Tox., 69:340. Shiromizu, K., Thorgeirsson, S.S. and Mattison, D.R. (1984) Effect of cyclophosphamide on oocyte and follicle number in Sprague Dawley rats, C57BL/6N and DBA/2N mice, Ped. Pharm., 4:213–21. Sims, P. and Grover, P.L. (1974) Epoxides in PAH metabolism and carcinogenesis, Adv. Canc. Res., 20:165–74. Smith, B.J., Mattison, D.R. and Sipes, I.G. (1990) The role of epoxidation in 4-vinylcyclohexene-induced ovarian toxicity, Toxicol. Appl. Pharmacol., 105:372–81. Sobrinho, L.G., Levine, R.A. and DeConti, R.C. (1971) Amenorrhea in patients with Hodgkins disease treated with antineoplastic agents, Am. J. Obstet. Gynecol., 109: 135–39. Sowers, M.R. and La Pietra, M.T. (1995) Menopause: its epidemiology and potential association with chronic diseases, Epidemiol Rev., 17:287–302. Springer, L.N., McAsey, M.E., Flaws, J.A., Tilly, J.L., Sipes, I.G. and Hoyer, P.B. (1996a) Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats, Toxicol. Appl Pharmacol., 139:394–401. Springer, L.N., Tilly, J.L., Sipes, I.G. and Hoyer, P.B. (1996b) Enhanced expression of bax in small preantral follicles during 4-vinylcyclohexene diepoxide-induced ovotoxicity in the rat, Toxicol. Appl. Pharmacol., 139:402–10. Stedman, R.L. (1968) The chemical composition of tobacco and tobacco smoke, Chem. Rev., 68:153–207. Swartz, W.J. and Mattison, D.R. (1985) Benzo(a)pyrene inhibits ovulation in C57BL/6N mice, Anatom. Rec., 212:268–76. Takai, Y., Canning, J., Perez, G.I., Pru, J.K., Schlezinger, J.J., Sherr, D.H., Kolesnick, R.N., Yuan, J., Flavell, R.A., Korsmeyer, S.J. and Tilly, J.L. (2003) Bax, caspase-2, and caspase-3 are required for ovarian follicle loss caused by 4-vinylcyclohexene diepoxide exposure of female mice in vivo, Endocrinology, 144:69–74. Takeuchi, Y., Ichihara, G. and Kamijima, M. (1997) A review of toxicity of 2bromopropane: mainly on its reproductive toxicity, J. Occup. Health., 39:191. Tilly, J.L., Kowalski, K.I., Johnson, A.L. and Hsueh, A.J.W. (1991) Involvement of apoptosis in ovarian follicular atresia and post-ovulatory regression, Endocrinology, 129:2799–801. Turchi, G., Bonatti, S., Citti, L., Gervasi, P.G. and Abbondandolo, A. (1981) Alkylating properties and genetic activity of 4-vinylcyclohexene metabolites and structurally related epoxides, Mut. Res., 83:419–30. Vahakangas, K., Rajaniemi, H. and Pelkonen, O. (1985) Ovarian toxicity of cigarette smoke exposure during pregnancy in mice, Toxicol., Lett., 25:75–80. VanVoorhis, B.J., Syrop, C.H., Hammit, D.H., Dunn, M.S. and Snyder, G.D. (1992) Effects of smoking on ovulation induction for assisted reproductive techniques, Fertil. and Steril, 58:981–85.

40 PATRICIA B.HOYER

Wayne, G.L., Fairley, K.F., Hobbs, J.B. and Martin, F.I.R. (2002) Cyclophosphamideinduced ovarian failure, N. Engl J. Med., 289:1159–62. Wyllie, A.H., Kerr, J.F.R. and Currie, A.R. (1980) Cell death: the significance of apoptosis, Int. Rev. Cytol., 68:251–306. Yu, X.Z., Kamijima, M., Ichihara, G., Li, W., Kitoh, J., Xie, Z., Shibata, E., Hisanaga, N. and Takeuchi, Y. (1999) 2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats, Toxicol., Appl Pharmacol., 159: 185–93.

3 OVARIAN TOXICITY CAUSED BY PESTICIDES Christina Borgeest, Kimberly P.Miller, Dragana Tomic and Jodi A.Flaws

INTRODUCTION Pesticides are defined as any substance or mixture of substances that prevent, destroy, repel, or mitigate any pest (US EPA, 1997). Pests might include insects, weeds, fungi or other harmful mircoorganisms. Pesticides are an important part of our farming system, and it is not likely that their use will be discontinued anytime soon (Ecobichon, 1996). Although pesticides have an important role in maintaining crop health, it is thought that no pesticide is completely non-toxic (Ecobichon, 1996). Humans are exposed to pesticides occupationally, in accidental poisonings and through the environment (Durham, 1965). There is also documentation of wildlife being unintentionally exposed through the environment (Burkhart et al., 2000; Guillette, 2000; Loeffler et al, 2001). Pesticides are non-specific in both the species and the tissues they target (Ecobichon, 1996). For this reason, some pesticides that are intended to attack the nervous system are also ovarian toxicants. As an example, the pesticide methoxychlor, in spite of its classification as a nervous system toxicant, has been found to be an ovarian toxicant (Bal, 1984; Martinez and Swartz, 1991, 1992; Swartz and Corkern, 1992; Eroschenko et al., 1995, 1997; Chapin et al., 1997; Swartz and Eroschenko, 1998; US EPA, 2001; Okazaki et al, 2001; Borgeest et al., 2002). Many pesticides have been classified as endocrine disrupters (EDCs). EDCs are natural or synthetic chemicals that mimic, enhance (agonists), or inhibit (antagonists) endogenous hormones (US EPA, 1997). Since the ovary is a major producer and target of many endogenous hormones, any pesticide that acts as an EDC could potentially harm the ovary. Pesticides that damage the ovary are of particular concern because normal ovarian function is critical for female fertility and adequate hormone production (Hirshfield, 1991). Pesticides may damage the ovary directly by attacking the follicles, the corpora lutea or the ovarian surface epithelium (Bal, 1984; Babineau et al., 1991; Eroschenko et al., 1995; Swartz and Eroschenko, 1998; Beard and Rawlings, 1999; Okazaki et al., 2001).

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Alternatively, they may damage the ovary indirectly by altering the levels of hormones that regulate the development and/or function of the ovary (Rattner et al., 1984; Ateia et al., 1990). The mechanisms by which many pesticides exert their effects on the ovary are not understood, and as there are many types of pesticides, there are most likely many mechanisms by which they exert their effects. This chapter will focus on the effects of pesticides on the ovary by first providing background information on the ovary and then by providing information on the major classes of pesticides that have been shown to affect the ovary. After an assessment of normal ovarian function, this chapter has been divided into sections on different classes of pesticides, namely organochlorine insecticides, organophosphate insecticides, herbicides, and fungicides. This chapter focuses on these chemical classes due to the number of well-documented examples of exposure to them in the environment. Many organochlorines were banned in the 1970s and replaced by organophosphates. In addition, the herbicides make up a rapidly growing category of pesticides in use, while fungicides are important in fighting mycotoxins in food. NORMAL OVARIAN FUNCTION Mammals are born with a finite number of primordial follicles. These follicles consist of an oocyte surrounded by approximately four fusiform granulosa cells (Figure 3.1). Some primordial follicles are selected to grow to the next stage, the

Figure 3.1 Stages of ovarian follicle growth. This schematic shows the normal stages of ovarian follicles beginning with the oocyte, and primordial follicles and the growth of these follicles to the pre-ovulatory stage.

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primary follicle. Follicles in this stage are characterized by a slightly larger oocyte surrounded by 6–8 square-shaped granulosa cells (Figure 3.1) (Hirshfield, 1997). Following the primary stage, follicles grow to the pre-antral stage, which is characterized by an oocyte surrounded by 2–4 layers of granulosa cells and the beginnings of a thecal cell layer that surrounds the granulosa cell layer (Figure 3.1). At the pre-antral stage, an antrum is either nascent or absent. The antrum is a fluid-filled cavity that contains steroid-binding proteins and high levels of estrogen and progesterone (Hirshfield, 1997). The granulosa and thecal cells serve to maintain the health of the oocyte and to produce hormones such as estrogens, which are required for fertility (Hirshfield, 1997). The pre-antral follicles then grow into antral follicles, which contain the oocyte surrounded by greater than or equal to five layers of granulosa cells, an antrum, and at least two distinct layers of thecal cells (Figure 3.1) (Hirshfield, 1997). The final stage of development is called the pre-ovulatory stage (Figure 3.1). Follicles in this stage contain a ripe oocyte surrounded by an inner layer of granulosa cells (cumulus layer), a large antral space, an outer layer of granulosa cells (membrana granulosa), and at least two distinct thecal layers. Follicles must grow to the preovulatory stage to be capable of releasing eggs for fertilization. While these descriptions represent ovarian structure in the mammal, it should be noted that some basic structural and functional similarities exist between mammalian and non-mammalian vertebrate ovaries (Guraya, 1976). In the lifetime of a female, many primoridal follicles grow to the pre-ovulatory stage; however, the vast majority of follicles (over 99 percent) die once they reach the pre-antral and antral stages via a process called atresia (Hirshfield, 1997). Atresia is thought to involve a form of programmed cell death known as apoptosis. Several studies have shown that ovarian follicles undergo atresia via apoptosis in numerous in vivo and in vitro systems (Hsueh et al, 1996). Because over 99 percent of ovarian follicles undergo atresia, any pesticide that hastens this process could have potentially devastating effects on fertility by causing premature ovarian failure. The overall damage to the ovary and its implications on reproductive health depends on the type of follicle affected by the pesticide. For example, administration of a single dose of a pesticide that destroys primordial follicles would eventually result in permanent sterility because the primordial follicle pool is finite and non-renewable. These types of exposures are of concern because they can go unnoticed as normal cycling might continue until the remaining larger follicles have been depleted from the ovary through ovulation or atresia. Exposure to a single dose of a pesticide that targets primary follicles may result in permanent infertility if all of the primordial follicles are destroyed and none become available to advance to the pre-ovulatory stage. If sufficient primary follicles remain and the pesticide exposure is removed, temporary infertility might result because primary follicles would have a chance to grow to larger stages. The situation would be similar in the case of a single exposure to a pesticide that targets pre-antral, antral, or preovulatory follicles. Namely, this

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could result in permanent infertility if insufficient numbers of small follicles remain to advance to the pre-ovulatory stage, or temporary infertility followed by normal cycling if enough small follicles are available to grow to the ovulatory stage (Hirshfield, 1997). Follicles are not the only possible targets within the ovary. The ovary also contains another potential target of pesticides, namely the corpus luteum (CL). After the egg has been released from the pre-ovulatory follicle, the granulosa and theca cells of the ruptured follicle differentiate to form the CL (Davis and Rueda, 2002). This is a transient gland that is responsible for the production of progesterone, a hormone that helps to establish and maintain pregnancy if an ovulated egg becomes fertilized (Davis and Rueda, 2002). If pregnancy occurs, the CL will remain and continue to synthesize progesterone (Davis and Rueda, 2002). If pesticide exposure targets cells within the CL, it is possible that production of progesterone may be compromised and thus the ability to establish and maintain pregnancy may be impaired. Another possible target of pesticides is a single layer of endothelial cells that envelop the mammalian ovary called the ovarian surface epithelium (OSE). The OSE is a dynamic tissue that can undergo proliferation and apoptosis, and is the site where human ovulation occurs (Murdoch and McDonnel, 2002). Some studies suggest that multiple disrupters of the OSE that occur as a result of ovulation may pre-dispose this tissue to cancer (Murdoch and McDonnel, 2002). This is supported by studies showing reduced risk of ovarian cancer among women using oral contraceptives (The Centers for Disease Control, 1983). By suppressing ovulation, it is proposed that less damage to the OSE occurs, reducing DNA damage, characteristic of cancer initiation and progression. Pesticides that damage the OSE may inhibit ovulation or lead to ovarian cancers. Finally, pesticides can cause direct damage to the ovary or indirect damage by affecting hormone levels. The ovary manufactures steroid hormones, most importantly, estrogen and progesterone. Estrogen and progesterone production is under feedback control from the hypothalamus and the pituitary (Figure 3.2). Estrogens are vital for the maintenance of reproduction and cyclicity (Findlay et al., 2001), while progesterone is vital for the maintenance of pregnancy and is regulated by estrogen (Spencer and Bazer, 2002). The hypothalamus synthesizes and secretes gonadotropin-releasing hormone, which binds to receptors on the anterior pituitary and stimulates it to synthesize and secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH are important for follicle growth and ovulation, and bind to receptors in the ovary which stimulate it to synthesize estrogen and progesterone (Hillier, 1994). Estrogen and progesterone then feed back to the pituitary to regulate FSH and LH (Lingappa and Farey, 2000). If pesticides act as estrogen mimics, they could potentially alter the delicate hormonal balance that makes up the hypothalamic-pituitary axis.

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Figure 3.2 The hypothalamic-pituitary-ovarian axis. This schematic diagram shows the negative feedback system between the ovary, the hypothalamus and the pituitary. The hypothalamus releases gonadotropin-releasing hormone (GnRH), which binds to receptors on the anterior pituitary and causes the release of FSH and LH. FSH and LH bind to receptors on the antral follicles and stimulate the production of steroid hormones. Two important steroid hormones, estrogen and progesterone, feed back negatively to the hypothalamus and the pituitary, and stop production of GnRH, FSH, and LH.

ORGANOCHLORINE INSECTICIDES Organochlorine pesticides are chlorine-containing hydrocarbons, such as 1,1,1trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), methoxychlor, aldrin, dieldrin, endrin, heptachlor, chlordane, lindane, kepone, and mirex (Ecobichon, 1996). These pesticides are known for their chemical stability and lipid solubility (Ecobichon, 1996). Their use has been restricted in the United States, but they are still used widely throughout the world, and newer generations of organochlorine pesticides are still used in the US (Cummings, 1997). Furthermore, organochlorines that have been banned in North America still persist in the environment (Campagna et al., 2001). The organochlorine pesticide, DDT, which was banned in the 1970s, is a good example of a stable and persistent chemical that has been found to accumulate in the ovaries of various species (Hellou et al., 1993; Jarrell et al., 1993b Brim et al., 2001; Furusawa, 2002) underscoring its persistence in the environment and the food chain. Its accumulation in the ovaries also suggests that the chemical is a potential ovarian toxicant. Nevertheless, there are some studies that suggest that the levels are too low to affect reproductive outcomes. For instance, in humans, metabolites of DDT have been detected in the follicular fluid of women seeking treatment for infertility and have been associated with failure to conceive

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(Younglai et al., 2002). Another study, however, found that the presence of small amounts of organochlorine compounds (including DDT) in the follicular fluid did not affect the outcome of in vitro fertilization (Jarrell et al., 1993b). In hens, DDT was found to accumulate in the ovary, oviduct, and egg yolk (Furusawa, 2002), but some studies suggest that these levels may be within tolerance limits (Furusawa and Morita, 2000). Finally, in fish, a derivative of DDT, p, p´-dichlordiphenyl-dichloroethylene (DDE), was found to accumulate in the ovaries of the striped bass (Morone saxatilis) (Brim et al., 2001). However, in another study performed in a different species of fish, Gadus morhua, the concentrations of organochlorines in the ovaries were considered to be too low to affect reproduction (Hellou et al., 1993). In laboratory studies, DDT has been shown to affect the ovary. In one study, the eggs of Leghorn chickens were exposed to DDT at a single time-point and incubated for 12 days (Swartz, 1984). The ovaries of exposed embryos had histological abnormalities, namely distended medullary cords (Swartz, 1984). In addition, the ovarian stroma showed increased alkaline phosphatase activity, which is a measure of phosphorylation, and suggests that the cell-signaling pathways are activated by DDT (Swartz, 1984). Even the cockroach, a species clearly intended for poisoning, was found to suffer ovarian damage at sub-lethal concentrations of DDT (Jain and Bhide, 1990). Methoxychlor is a good model chemical for the effects of organochlorines on the ovary. Methoxychlor has been found to reduce ovarian weights (Martinez and Swartz, 1991; Eroschenko et al., 1995), the number of corpora lutea (Bal, 1984; Eroschenko et al., 1995; Chapin et al., 1997; Swartz and Eroschenko, 1998; Okazaki et al, 2001), and ovulation rates (Eroschenko et al., 1997) in rodents. Methoxychlor has also been found to increase follicular atresia in the ovaries of rodents (Martinez and Swartz, 1991; Swartz and Corkern, 1992; Borgeest et al, 2002). Another study found that methoxychlor increased lipid accumulation in the thecal and interstitial cells (Martinez and Swartz, 1992). Finally, a recent study found that methoxychlor caused a thickening of the OSE (Borgeest et al, 2002). This latest finding is an illustration of how the same chemical can have a very different effect on the ovary, i.e., methoxychlor causes ovarian antral follicles to die, while simultaneously causing an apparent proliferation of the OSE. Other organochlorine pesticides also have been shown to affect the ovary. The organochlorine pesticide kepone, banned in the United States after an industrial accident in the 1970s, has been shown to increase follicular atresia in rodents (Swartz and Mall, 1989; Borgeest et al, 2002). Treatment of albino rats with dieldrin caused an infiltration of lymphocytes in the antral follicles, presumably due to injury of these follicles by the chemical with subsequent immune response (Ateia et al., 1990). A study in ducks found that the ovaries of animals treated with lindane had fewer antral follicles than controls and that the follicles that were present had an atrophic thecal layer (Chakravarty et al, 1986). Finally, a second study in ducks treated with lindane found that the eggs were significantly

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smaller with thinner eggshells than those of control ducks (Chakravarty and Lahiri, 1986). Other studies have shown that organochlorine pesticides affect ovulation, which could either be caused by a direct effect on the ovary (e.g., damage to the pre-ovulatory follicles and the OSE) or an indirect effect on the ovary via disruption of the hypothalamic-pituitary-ovarian axis. A study conducted in the rabbit model found that animals treated with lindane had a reduced ovulation rate (Lindenau et al., 1994). Mirex, an organochlorine insecticide, reduced ovulation rates in immature rats by 40–80 percent compared to controls depending on the dose levels. In this case, the authors concluded that the ovary was not the only target, but rather the hypothalamic-pituitary-ovarian axis was also targeted because administration of human chorionic gonadotropin following mirex treatment reversed this result (Fuller and Draper, 1975). Finally, dieldrin caused a decrease in FSH and LH levels in albino rats, an event thought to lead to problems with ovulation (Ateia et al, 1990). In non-mammals, interference with vitellogenesis (manufacture of vitellogenin or yolk protein) is another commonly observed effect of organochlorine insecticides. A study in ducks found that exposure to lindane caused a significant reduction in laying frequencies and vitellogenin levels in the liver, plasma, and ovary (Chakravarty et al., 1986). Chakravarty et al. suggested that lindane damages egg laying in ducks by inducing estradiol insufficiency, leading to a breakdown in the production of vitellogenin, thus delaying ovulation (Chakravarty et al., 1986). Similarly, cessation of vitellogenesis was cited as the cause for ovarian failure in cockroaches after treatment with DDT (Jain and Bhide, 1990). Organochlorines are also thought to interact with the estrogen receptor (ER) and interfere with estradiol and progesterone production (Pickford and Morris, 1999; Waters et al., 2001). Estrogen acts by binding to the estrogen receptor, so it is reasonable to hypothesize that organochlorines that produce estrogenic effects might also be acting through the estrogen receptor; a kind of guilt by association. A recent study by Waters et al. (2001), used cDNA microarrays and real time polymerase chain reaction (rt-PCR) to show that the estrogenic methoxychlor metabolite, 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), but not estradiol, induced ERβ in the mouse ovary. In addition, this study demonstrated that HPTE reverses the induction of cathepsin B by estradiol in the ovary. Cathepsin is an enzyme that catalyzes the hydrolysis of peptide bonds, so in this case, HPTE interfered with the action of estradiol. Organochlorines can also work through an ER-independent pathway (Pickford and Morris, 1999; Crellin et al., 2001). The pesticide methoxychlor was found to inhibit progesterone-induced oocyte maturation; however, this action was not blocked by the pure anti-estrogen, ICI 182,780 (Pickford and Morris, 1999). Since ICI 182,780 blocks ER binding, the authors concluded that methoxychlor was inducing an endocrine-disrupting effect through a non-ER mechanism. An in vitro study in porcine granulosa cells shed some light on how organochlorines

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might affect progesterone synthesis without interacting with the ER (Crellin et al., 2001). The authors found that DDE alters the expression of the P450-cholesterol side-chain cleavage enzyme (P450scc), leading to a potentiation of protein kinase A activators on progesterone synthesis (Crellin et al, 2001). Treatment of female Sprague-Dawley (SD) rats with heptachlor, another organochlorine, caused a decrease in blood progesterone and estrogen levels (Oduma et al., 1995). In this same experiment, production of progesterone was altered in a doserelated manner, namely progesterone levels increased at lower doses of heptachlor and increased further at higher doses of heptachlor (Oduma et al., 1995). Organochlorine insecticides may also cause metabolic changes that affect the ovary indirectly. A study in SD rats found that even in the absence of ovarian histopathologic changes, the ovary showed changes in glucose metabolism when exposed to photomirex, a breakdown product of mirex (Todoroff et al., 1998). Since the ovary is a major producer of estrogen, pesticides could potentially interfere with estrogen metabolism. While no study is known to examine this in the ovary, exposure of MCF-7 breast cancer cells to organochlorine pesticides significantly increased production of 16 α-hydroxyestrone, a potent estrogen thought to be tumorigenic and genotoxic (Bradlow et al, 1995). This study suggests that an organochlorine may not be mimicking estrogen per se, but rather inhibiting the breakdown and inactivation of estrogen, or promoting the production of genotoxic estrogen metabolites, thus increasing the bioavailability of harmful endogenous estrogens. Organochlorine insecticides might act through cell-signaling pathways, although very little research has yet examined the effects of pesticides on the cell-signaling pathways within the ovary itself. One study in chick embryos found that acid phosphatase activity in the ovary was drastically reduced after exposure to DDT, suggesting that it might be interfering with cellular phoshorylation and therefore, cell signaling (Swartz, 1984). As mentioned previously, DDE alters the expression of the P450scc enzyme leading to an increase in progesterone synthesis (Crellin et al., 1999). This study actually suggests several possible mechanisms, including interference with a metabolic enzyme, which leads to changes in cell-signaling molecules and a downstream effect on hormone production. One study examined the effects of the organochlorine pesticides, DDE and methoxychlor, on steroidogenesis in porcine and Chinese hamster ovary cells (Chedrese and Feyles, 2001). The authors suggested that DDE may inhibit the generation of cAMP in ovarian cells, an important second messenger in cell-signaling pathways, while methoxychlor may act through a mechanism distal to cAMP generation. These data indicate that these chemicals may modulate the expression of cAMP-regulated genes in the ovary by affecting the cAMP-signaling pathway. Juberg et al. (1995) observed that the chlorinated insecticide 1,1-dichloro-2,2-bis(chlorophenyl)ethane, a well-known DDT isomer, caused a significant increase in intracellular free calcium, another important second messenger in cell signaling, in cultured rat myometrial smooth

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muscle cells. Although this study was not done in the ovary, it suggests that organochlorines and their metabolites may be interacting with cell-signaling pathways, which are conserved among different tissues and cell types. ORGANOPHOSPHATE INSECTICIDES The persistence of the organochlorine pesticides in the environment and in lipophilic tissues led to the development of pesticides that were more biodegradable. Organophosphate insecticides were found to be less chemically stable than the organochlorines, and for that reason, do not accumulate as readily in the environment (Ecobichon, 1996). Nevertheless, the organophosphates are in general more overtly toxic than the organochlorines, and are in fact a major culprit in accidental poisonings (Ecobichon, 1996). Furthermore, though considered to be less persistent than organochlorine insecticides, they have been known to accumulate in the ovary (Piao et al, 1997). Organophosphates also have been shown to damage the ovaries of various species. The organophosphate insecticide, monocrotophos, was found to decrease ovarian weights, as well as reduce the numbers of various follicle types in the mouse ovary (Rao and Kaliwal, 2002). The authors also observed an increase in the number of atretic follicles at the selected doses. The organophosphate insectide, parathion, has been found to decrease the number of healthy follicles in rats (Dhondup and Kaliwal, 1997). In an avian species, the bobwhite quail, parathion was found to inhibit follicular development and decrease egg production (Rattner et al., 1982). The fish Tilapia leucosticta suffered extensive atresia in the ovaries after exposure to the organophosphate, lebaycid, making the fish unable to spawn for up to 9 weeks (Kling, 1981). Organophosphates appear to interact with the hypothalamic-pituitary-ovarian axis to reduce gonadotropin levels (Singh and Singh, 1981; Rattner et al., 1982). A reduction in gonadotropins could harm the ovary indirectly by causing atresia and reduced fertility. In the bobwhite quail, parathion reduced plasma LH concentrations (Rattner et al., 1982). In the freshwater catfish, Heteropneustes fossilis (Bloch), exposure to the organophosphates, aldrin and parathion, reduced serum and pituitary levels of both FSH and LH (Singh and Singh, 1981). HERBICIDES Insecticides are not the only pesticides capable of damaging the ovary. The herbicides, designed to inhibit photosynthesis and eliminate unwanted plant growth, can also cause ovarian damage. Development of herbicides began in the 1930s (Gysin and Knuesli, 1960; Jager, 1983). Highly toxic chemicals were used as first generation herbicides and included sulfuric acid, iron sulfate, copper sulfate, sodium chlorate and arsenic trioxide (Ecobichon, 1996), but these chemicals were far too non-specific for the unwanted crop or plant. Second generation herbicides proved more specific, however, they were still toxic to

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mammals (Ecobichon, 1996). Although the newest herbicides are thought to be less toxic than first and second generation herbicides, these chemicals still possess many carcinogenic, mutagenic, and teratogenic properties, affecting both human and animal life (Ecobichon, 1996). The primary routes of exposure are dermal contact and inhalation, with chemicals exhibiting a range of acute and chronic toxicities (Ecobichon, 1996). Chlorotriazines, such as atrazine, simazine, and cyanazine, were first introduced as broad-spectrum herbicides in the 1950s (US EPA, 1994; Eldridge et al, 1994b). Nearly 27 million kilograms of atrazine is used annually on corn and other crops in the United States, and springtime atrazine levels in aquatic tributaries of the US reach higher levels than at other times of the year (Withgott, 2002). In the environment, atrazine is a persistent chemical found in soil residues (Goh et al., 1993), environmental and drinking water (Gojmerac et al., 1994; Vidacek et al, 1994), and crops (Norris and Fong, 1983). Physiological changes in the ovaries and circulating steroid hormone levels in mammals are observed following atrazine exposure. Gojmerac and coworkers (1996) studied the effects of subacute exposure to atrazine on female pigs undergoing intensive breeding. The authors showed that low doses of atrazine caused an apparent persistence of corpora lutea and an inhibition of ovarian function. Multiple ovarian follicular cysts were found upon examination of the morphology of the porcine ovary following subacute exposure to atrazine. In addition, atrazine increased serum progesterone levels and decreased serum 17βestradiol concentrations at 24 and 48 hours before the onset of the next predicted estrus (Gojmerac et al., 1996). In a study of SD and Fischer 344 female rats, atrazine and simazine caused significant reductions in ovarian and uterine weights, along with decreased circulating estradiol (Eldridge et al, 1994a). These studies suggest that atrazine causes damage to the ovary and interferes with endogenous estrogen levels. Damage to the ovary by herbicides may lead to adverse reproductive outcomes. Altered cyclicity is a commonly observed effect following atrazine exposure (Cooper et al, 1996; Gojmerac et al, 1996; Eldridge et al, 1999). An illustration of this is seen in the previously cited study in pigs (Gojmerac et al, 1996). In addition to causing ovarian damage, atrazine disrupted cyclicity, thereby prolonging estrus and causing a cessation of ovulation (Gojmerac et al., 1996). The authors concluded that the observed failure in cycling, subsequent ovulation and cystic ovarian degeneration were due to the fact that unruptured follicles had become cystic (Gojmerac et al., 1996). In a study on female Fischer 344 rats, atrazine disrupted the estrous cycle by causing an extended period of vaginal diestrus (Simic et al., 1994). This resulted in a decrease in successful matings, even when unexposed males were used in the breeding experiments (Simic et al, 1994). In another study using SD and Long-Evans-hooded rat strains, exposure to an acute dose of atrazine disrupted regular estrous cycles, while subchronic exposure induced an early agerelated cessation of cycling, characterized by constant vaginal estrus (Cooper et al, 1996; Eldridge et al, 1999).

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At high doses, atrazine has been shown to cause irregular cycles, repetitive pseudopregnancies, atrophied ovaries, induced anestrus and prolonged vaginal diestrus (Cooper et al., 1996). Pseudopregnancies were induced when atrazine was administered on the day of proestrus; however, there was no effect on ovulation at this time (Cooper et al., 2000). The effects of herbicides on the amphibian population are of additional interest because these animals live near potential agricultural runoff sites. In laboratory studies of Xenopus laevis (African clawed frog) exposed to atrazine, male tadpoles developed extra gonads and became hermaphrodites (Hayes et al, 2002). At 30-fold lower than the Environmental Protection Agency’s safe drinking water level standard, 16–20 percent of the exposed frogs developed up to six gonads (both ovaries and testes). The investigators hypothesized that the mechanism of action of atrazine in the frogs involved an ability of the chemical to activate aromatase, an enzyme that converts androgens to estrogens, resulting in a greater production of estrogens than testosterone, hence hermaphroditic tendencies (Hayes et al, 2002). Interestingly, no effect of atrazine was found on mortality, growth rate or external appearance, and it is unclear whether the abnormalities resulting in hermaphroditic frogs affected reproductive abilities (Withgott, 2002). More applicable to the wild frog species in North America are studies examining the Northern leopard frog. Compared to their laboratory counterparts, these wild frogs showed increased endocrine damage in areas of high atrazine levels with respect to unexposed frogs (Hayes et al, 2002). In another study examining Xenopus laevis female tadpoles, primary germ cells were reduced by 20 percent following a 48-hour exposure to atrazine (TaveraMendoza et al., 2002). Additionally, an increase in atresia of both primary and secondary oogonia was observed (Tavera-Mendoza et al, 2002). Other chemicals developed for herbicidal use have considerable toxicity in a variety of organs; however, few have been evaluated or have shown potential for ovarian toxicity. Chemical classes of herbicides that have been found to elicit ovarian toxicity include chlorophenoxy compounds (2,4-dichlorophenoxyacetic acid), chlorinated benzenes, cyclohexanes (lindane), dinitroanilines (trifluralin), carbamates (chlorpropham, cycloate), amides (pentanochlor) and urea derivatives (chloroxurone) (Kosanke et al, 1988; Charles et al., 1996; Rawlings et al., 1998). The herbicide, 2,4-dichlorophenoxyacetic acid, more commonly known as 2,4D, is used to control the growth of broadleaf and woody plants, and has been shown to exhibit ovarian toxicity. This herbicide also was used extensively in the Vietnam War as 50 percent of the defoliant Agent Orange (Ecobichon, 1996). In studies examining the ovarian toxicity of this chemical, subchronic doses of 2,4D resulted in decreased ovarian weights in rats (Charles et al, 1996). In the snail, other herbicides, including chlorpropham, chloroxurone, cycloate, propanil, simazine, and terbutryne (chlorotriazine) delayed the time of egg maturation and increased the amount of non-viable embryos in freshwater snails exposed to these herbicides at low concentrations (Kosanke et al., 1988).

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Mixtures of herbicides have been evaluated for ovarian toxicity. Heindel et al. (1994) examined the ovarian effects of mixtures of herbicides used in modern agricultural practices that contribute to groundwater contamination in both California and Iowa. The California mixture contained aldicarb, atrazine, dibromochloropropane, 1,2-dichloropropane, ethylene dibromide, simazine, and ammonium nitrate, whereas the Iowa mixture contained alachlor, atrazine, cyanazine, metolachlor, metribuzin, and ammonium nitrate. While these mixtures were prepared at 100 times the median level found in groundwater in each of these areas, neither caused treatment-related effects on fertility, reproductive performance, embryo/ fetal toxicity or fetal malformations in either mice or rats. This, however, does not negate the fact that each of these chemicals individually could possess ovarian toxicity on their own. As a mixture, toxic effects may be added and/or subtracted depending on the chemicals making up the mixture and how they interact with one another. There is a paucity of data on the mechanism of actions of herbicidal damage to the ovary. There could be direct toxicity to ovarian tissues, causing atresia in follicles, or a disruption of the hypothalamic-pituitary-ovarian axis leading to an alteration of gonadotropins and/or ovarian hormones. Future studies need to be conducted to assess the effects of herbicides on the ovary by follicle counts, corpora lutea measurements and determination of percent atretic follicles to gain more information as to which ovarian structures are most vulnerable. A study looking at ovulation capacity might also reveal whether the disruption of the estrous cycles of these rodents is due to an effect on the ovary (e.g., disruption of ovarian hormone production) or rather a direct effect on the vaginal cytology. FUNGICIDES Finally, we move our discussion to the fungicides, a class of pesticides that control fungal infestations of crops. Fungi can be particularly problematic in tropical climates and agricultural-based regions (Ecobichon, 1996). For example, aflatoxins are particularly potent hepatocarcinogens in humans as well as rodents (Kotsonis et al., 1996), and are found in various crops including edible nuts, oil seeds, and grains (Kotsonis et al, 1996). As with the other classes of pesticides discussed herein, the fungicides have been found to damage the ovary. Although many fungicides were predicted to have a low toxicity in the mammalian population, studies indicate that 90 percent of fungicides are carcinogenic in animal models, and that 75 million pounds of these carcinogenic compounds are used agriculturally per year (Ecobichon, 1996). Chemical classes of fungicides include organomercurials, phthalimides, chlorinated organics, and dithiocarbamates (Ecobichon, 1996). The latter two classes have been confirmed as mammalian reproductive toxicants and will be discussed here. Hexachlorobenzene (HCB) is classified as a chlorinated organic compound that is no longer used commercially as a fungicide because of its known toxic properties. One factor that led to its discontinuation was an epidemic of HCB

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poisoning in Turkey in the 1950s, where nearly 4,000 people who had consumed treated grain products experienced extreme dermal toxicities (Ecobichon, 1996). Despite the ban on HCB use as a fungicide, it is still produced as a by-product of commercial chlorination processes. In a variety of female animal species, exposure to HCB at low levels induced morphological and functional changes in the ovary (Babineau et al, 1991; Foster et al., 1992a,b). Common toxicities from chronic administration of HCB (30 days) include morphological ovarian changes, abnormal estrous cyclicity, follicular damage in small follicle types, OSE injury, increased atresia in antral follicles, reduced estradiol (E2) levels and reduced number of ova (Babineau et al., 1991; Sims et al., 1991; Bourque et al, 1995; Alvarez et al, 2000). HCB is a highly lipophilic compound and has been found in follicular fluid, as well as outside of the ovary in periovarian fat (Trapp et al., 1984; Foster et al., 1993; Jarrell et al., 1993b). HCB has been shown to cause ovarian damage in the rat model. For example, in one study, chronic HCB caused an alteration in ovarian responses in the female Wistar rat, including irregular and abnormal cycling, characterized by extended periods of estrus and reduced number of ova (Alvarez et al., 2000). In SD rats, granulosa lutein cells showed changes in the smooth endoplasmic reticulum (SER) and Golgi complexes, along with prominent free polysomes (MacPhee et al., 1993). The SER appeared to be dilated as a result of hyperactivity, and the authors hypothesized that HCB may be upregulating the synthetic activity of the granulosa lutein cells to produce additional hormones as a response to injury since SER participates in steroid hormone synthesis (MacPhee et al., 1993). HCB also has been shown to alter hormonal status in rats. For instance, HCB caused lowered serum E2 levels, but did not change progesterone levels (Alvarez et al., 2000). Strain-specific effects of HCB cannot be overlooked, however, since opposing results of estrogen/progesterone levels have been shown in Wistar rats. After treatment, Wistar rats had significantly elevated progesterone levels, but serum concentrations of E2 were unchanged (Foster et al., 1992b). Primate studies have also looked at the effects of HCB on the ovary. In the Rhesus monkey model, the corpora lutea of HCB-treated animals were not receptive to gonadotropin stimulation nor were they capable of steroidogenesis (Iatropoulos et al, 1976). Over a more extended period of time (13 weeks), treated ovaries exhibited altered follicle structure, characterized by internal lesions, and condensed mitochondria within the ova and follicular cells. In addition, ooplasm herniation, follicular cell degeneration, and abnormal spaces between follicular cells were noted. The population of primary follicles appeared to be the major target of HCB in this study (Bourque et al., 1995); however, other studies have shown that the primordial germ cell population is also a target of toxicity (Jarrell et al., 1993a). In addition to follicular toxicity, HCB has a toxic effect on the cellular structure of the OSE in primate ovaries. Low doses of HCB have been shown to cause visible signs of surface epithelial cell

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degeneration, including a tall columnar shape, irregular outline, and numerous lysosomes and vesicles within the cytoplasm. High doses have been shown to advance stages of degeneration of the OSE (Babineau et al., 1991). HCB also has been shown to lower estrogen levels in primates without lowering FSH and LH levels (Muller et al, 1978). Dithiocarbamates are a widely used subset of fungicidal chemicals that have been found to affect the ovary. Their low acute toxicity and high oral LD50 values make them a popular fungicide; however, their carcinogenic and teratogenic properties have been found to exist at more chronic exposures (Ecobichon, 1996). High doses of dithiocarbamates have been shown to decrease ovarian hypertrophy, the number of estrous cycles and the duration of each phase of the estrous cycle (Mahadevaswami et al, 2000). Dithiocarbamates also have been shown to decrease the number of healthy follicles and to increase the number of atretic follicles. This overall increase in ovarian atresia may be due to a direct effect on the ovary and/or the hypothalamic-pituitary-ovarian axis (Mahadevaswami et al, 2000). In addition, dithiocarbamates have been shown to cause a decrease in protein, glycogen, total lipid, phospholipid, and neutral lipid levels in the ovary, which the authors suggested could be due to reduced lipid synthesis because of tissue damage or increased catabolism of these biomolecules (Mahadevaswami et al, 2000). High doses of mancozeb and maneb, both dithiocarbamates, are teratogenic in rats, though not in mice, demonstrating that the effects of these fungicides can be species-specific (Larsson et al, 1976). A single exposure to the dithiocarbamate fungicide, thiram, delays ovulation if the animal is treated during the LH surge during vaginal proestrus (Stoker et al., 1993). However, it does not appear to decrease the number of ova shed during ovulation. On gestation day 7, embryos implanted normally, remained stable at gestation day 11 despite impaired development, but a reduction in embryo survival at gestation day 20 was evident. A single dose of thiram at the afternoon period of LH surge was capable of decreasing the number of live births distinctly through disabling the pituitary LH release and delaying ovulation by a period of 24 hours (Stoker et al, 2001). There are several possible mechanisms of action regarding the effects of fungicides on the ovary. Some studies suggest fungicides may alter estrogen metabolism, thereby causing ovarian toxicity (Alvarez et al., 2000). For example, HCB induces cytochrome P450 enzymes, IA1 and IA2, that contribute to the metabolism of E2 and levels of estrogen receptors (Hahn et al., 1989). Furthermore, HCB binds to the aryl hydrocarbon receptor (AhR) (Hahn et al, 1989), and the AhR interacts with the ER; so there is a possibility of modulation of E2 levels through binding to ER via an AhR-mediated pathway (Alvarez et al., 2000). Another possible mechanism of action of herbicides is to act as antiandrogens, hence creating an “estrogenic environment” (Sohoni and Sumpter 1998). For example, the fungicide, vinclozolin was found to antagonize the androgen receptor (AR) in a yeast-based assay (Sohoni and Sumpter, 1998). Although this in vitro assay was not performed in ovarian tissue, these results

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suggest that herbicides may be acting to block AR antagonists as well as ER agonists. CONCLUSION Pesticides continue to be developed and their use will likely continue for many years to come, for they serve an important purpose in agriculture, namely, they enable us to produce large quantities of food at reasonable prices (Ecobichon, 1996). Yet their effects on the ovary are by and large not well understood. Indeed, much of the literature covering the effects of pesticides on the ovary focuses on a limited number of compounds. It is important to perform more studies to uncover the mechanisms of action of these chemicals if we ever hope to prevent or combat their potentially toxic effects. In addition, we need to broaden the scope of our testing of potential reproductive hazards to mixtures of different types of compounds and to include those pesticides that have not been studied. Wildlife may be vulnerable to pesticide exposure through agricultural runoff, but humans may be affected as well since pesticide residues have been found in the follicular fluid of sub-fertile and infertile women (Younglai et al., 2002; Jarrell et al., 1993b). Finally, it is important to establish whether exposure to pesticides presents a true reproductive health risk to wildlife and humans so that we can reassess how these chemicals are regulated. Ultimately, the goal is to understand their effects so that we may begin to address any damage that has been done and prevent any future damage. REFERENCES Alvarez, L., Randi, A., Alvarez, P., Piroli, G., Chamson-Reig, A.,Lux-Lantos, V. and Kleiman de Pisarev, D. (2000) Reproductive effects of hexachlorobenzene in female rats, J. Appl. Toxicol., 20(1):81–87. Ateia, M.M., Zaki, A.A. and Korayem, W.I. (1990) Toxic effect of dieldrin on gonadotrophin levels (FSH and LH) in serum of mature female albino rats, Arch. Exp. Veterinarmed, 44(3):357–60. Babineau, K.A., Singh, A., Jarrell, J.F. and Villeneuve, D.C. (1991) Surface epithelium of the ovary following oral administration of hexachlorobenzene to the monkey, J. Submicrosc. Cytol Pathol., 23(3):457–64. Bal, H.S. (1984) Effect of methoxychlor on reproductive systems of the rat, Proc. Soc. Exp. Biol Med., 176(2): 187–96. Beard, A.P. and Rawlings, N.C. (1999) Thyroid function and effects on reproduction in ewes exposed to the organochlorine pesticides lindane or pentachlorophenol (PCP) from conception, J. Toxicol. Environ. Health A, 58(8):509–30. Borgeest, C, Symonds, D., Mayer, L.P., Hoyer, P.B. and Flaws, J.A. (2002) Methoxychlor may cause ovarian follicular atresia and proliferation of the ovarian epithelium in the mouse, Toxicol. Sci., 68(2):473–78.

56 OVARIAN TOXICITY CAUSED BY PESTICIDES

Bourque, A.C., Singh, A., Lakhanpal, N., McMahon, A. and Foster, W.G. (1995) Ultrastructural changes in ovarian follicles of monkeys administered hexachlorobenzene, Am. J.Vet. Res., 56(12): 1673–77. Bradlow, H.L., Davis, D.L., Lin, G., Sepkovic, D. and Tiwari, R. (1995) Effects of pesticides on the ratio of 16 alpha/2-hydroxyestrone: a biologic marker of breast cancer risk, Environ. Health Perspect., 103(Suppl 7): 147–50. Brim, M.S., Alam, S.K. and Jenkins, L.G. (2001) Organochlorine pesticides and heavy metals in muscle and ovaries of Gulf coast striped bass (Morone saxatilis) from the Apalachicola River, Florida, USA, J. Environ. Sci. Health B, 36(1): 15–27. Burkhart, J.G., Ankley, G., Bell, H., Carpenter, H., Fort, D., Gardiner, D., Gardner, H., Hale, R., Helgen, J.C., Jepson, P., Johnson, D., Lannoo, M., Lee, D., Lary, J., Levey, R., Magner, J., Meteyer, C., Shelby, M.D. and Lucier, G. (2000) Strategies for assessing the implications of malformed frogs for environmental health, Environ. Health Perspect., 108(1):83–90. Campagna, C., Sirard, M.A., Ayotte, P. and Bailey, J.L. (2001) Impaired maturation, fertiliztion, and embryonic development of porcine oocytes following exposure to an environmentally relevant organochlorine mixture, Biol Reprod., 65(2):554–60. Centers for Disease Control, the (1983) Oral contraceptive use and the risk of ovarian cancer. The Centers for Disease Control Cancer and Steroid Hormone Study, JAMA, 249(12):1596–99. Chakravarty, S. and Lahiri, P. (1986) Effect of lindane on eggshell characteristics and calcium level in the domestic duck, Toxicology, 42(2–3):245–58. Chakravarty, S., Mandal, A. and Lahiri, P. (1986) Effect of lindane on clutch size and level of egg yolk protein in domestic duck, Toxicology, 39(1):93–103. Chapin, R.E., Harris, M.W., Davis, B.J., Ward, S.M., Wilson, R.E., Mauney, M.A., Lockhart, A.C., Smialowicz, R.J., Moser, V.C., Burka, L.T. and Collins, B.J. (1997) The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function, Fundam. Appl Toxicol., 40(1): 138–57. Charles, J.M., Cunny, H.C., Wilson, R.D. and Bus, J.S. (1996) Comparative subchronic studies on 2,4-dichlorophenoxyacetic acid, amine, and ester in rats, Fundam. Appl. Toxicol., 33(2):161–65. Chedrese, P.J. and Feyles, F. (2001) The diverse mechanism of action of dichlorodiphenyldichloroethylene (DDE) and methoxychlor in ovarian cells in vitro, Reprod. Toxicol., 15(6):693–98. Cooper, R.L., Stoker, T.E., Goldman, J.M., Parrish, M.B. and Tyrey, L. (1996) Effect of atrazine on ovarian function in the rat, Reprod. Toxicol., 10(4):257–64. Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M. and McElroy, W.K. (2000), Atrazine disrupts the hypothalamic control of pituitary-ovarian function, Toxicol. Sci., 53(2): 297–307. Crellin, N.K., Rodway, M.R., Swan, C.L., Gillio-Meina, C. and Chedrese, P.J. (1999) Dichlorodiphenyldichloroethylene potentiates the effect of protein kinase A pathway activators on progesterone synthesis in cultured porcine granulosa cells, Biol. Reprod., 61(4):1099–103. Crellin, N.K., Kang, H.G., Swan, C.L. and Chedrese, P.J. (2001) Inhibition of basal and stimulated progesterone synthesis by dichlorodiphenyldichloroethylene and methoxychlor in a stable pig granulosa cell line, Reproduction, 121(3):485–92. Cummings, A.M. (1997) Methoxychlor as a model for environmental estrogens, Crit. Rev. Toxicol., 27(4):367–79.

BORGEEST ET AL. 57

Davis, J.S. and Rueda, B.R. (2002) The corpus luteum: an ovarian structure with maternal instincts and suicidal tendencies, Front. Biosci., 7:d1946–78 1949–78. Dhondup, P. and Kaliwal, B.B. (1997) Inhibition of ovarian compensatory hypertrophy by the administration of methyl parathion in hemicastrated albino rats, Reprod. Toxicol., 11(1):77–84. Durham, W.F. (1965) Pesticide exposure levels in man and animals, Arch. Environ. Health, 10:842–46. Ecobichon, D.J. (1996) Toxic effects of pesticides. In: C.D. Klaassen (ed.) Casarett and Doull’s Toxicology: The Basic Science of Poisons, New York: McGraw-Hill, pp. 643–89. Eldridge, J.C., Fleenor-Heyser, D.G., Extrom, P.C., Wetzel, L.T., Breckenridge, C.B., Gillis, J.H., Luempert, L.G. 3rd and Stevens, J.T. (1994a) Short-term effects of chlorotriazines on estrus in female Sprague Dawley and Fischer 344 rats, J. Toxicol. Environ. Health, 43(2): 155–67. Eldridge, J.C., Tennant, M.K., Wetzel, L.T., Breckenridge, C.B. and Stevens, J.T. (1994b) Factors affecting mammary tumor incidence in chlorotriazine-treated female rats: hormonal properties, dosage, and animal strain, Environ. Health Perspect., 102 (Suppl 11): 29–36. Eldridge, J.C., Wetzel, L.T., Stevens, J.T. and Simpkins, J.W. (1999) The mammary tumor response in triazine-treated female rats: a threshold-mediated interaction with strain and species-specific reproductive senescence, Steroids, 64 (9):672–78. Eroschenko, V.P., Abuel-Atta, A.A. and Grober, M.S. (1995) Neonatal exposures to technical methoxychlor alters ovaries in adult mice, Reprod. Toxicol., 9 (4): 379–87. Eroschenko, V.P., Swartz, W.J. and Ford, L.C. (1997) Decreased superovulation in adult mice following neonatal exposures to technical methoxychlor, Reprod. Toxicol., 11 (6):807–14. Findlay, J.K., Britt, K., Kerr, J.B., O’Donnell, L., Jones, M.E., Drummond, A.E. and Simpson, E.R. (2001) The road to ovulation: the role of oestrogens, Reprod. Fertil. Dev., 13 (7–8):543–47. Foster, W.G., McMahon, A., Villeneuve, D.C. and Jarrell, J.F. (1992a) Hexachlorobenzene (HCB) suppresses circulating progesterone concentrations during the luteal phase in cynomolgus monkeys, J. Appl. Toxicol., 12 (1): 13–17. Foster, W.G., Pentick, J.A., McMahon, A. and Lecavalier, P.R. (1992b) Ovarian toxicity of hexachlorobenzene (HCB) in the superovulated female rat, J. Biochem. Toxicol., 7 (1): 1–4. Foster, W.G., Pentick, J.A., McMahon, A. and Lecavalier, P.R. (1993) Body distribution and endocrine toxicity of hexachlorobenzene (HCB) in the female rat, J. Appl. Toxicol., 13 (2):79–83. Fuller, G.B. and Draper, S.W. (1975) Effect of mirex on induced ovulation in immature rats (38550), Proc. Soc. Exp. Biol Med., 148 (2):414–17. Furusawa, N. (2002) Transferring and distributing profiles of p, p´-(DDT) in egg-forming tissues and eggs of laying hens following a single oral administration, J. Vet. Med. A. Physiol Pathol Clin. Med., 49 (6):334–36. Furusawa, N. and Morita, Y. (2000) Polluting profiles of dieldrin and DDTs in laying hens of Osaka, Japan, J. Vet. Med. B Infect. Dis. Vet. Public Health, 47 (7):511–15. Goh, K.S., Weaver, D.J., Hsu, J., Richman, S.J., Tran, D. and Barry, T.A. (1993) ELISA regulatory application: compliance monitoring of simazine and atrazine in California soils, Bull. Environ. Contam. Toxicol., 51 (3):333–40.

58 OVARIAN TOXICITY CAUSED BY PESTICIDES

Gojmerac, T., Kartal, B., Zuric, M. and Zidar, V. (1994) Use of immunoassay (ELISA) in determination of s-triazine herbicide in drinking water, Vet. Stanica, 25:75–80. Gojmerac, T., Kartal, B., Curic, S., Zuric, M., Kusevic, S. and Cvetnic, Z. (1996) Serum biochemical changes associated with cystic ovarian degeneration in pigs after atrazine treatment, Toxicol. Lett., 85 (1):9–15. Guillette, L.J. Jr (2000) Organochlorine pesticides as endocrine disruptors in wildlife, Cent. Eur. J. Public Health, 8 (Suppl):34–35. Guraya, S.S. (1976) Recent advances in the morphology, histochemistry, and biochemistry of steroid-synthesizing cellular sites in the nonmammalian vertebrate ovary, Int. Rev. Cytol., 44:365–409. Gysin, H. and Knuesli, E. (1960) Chemistry and herbicidal properties of triazine derivatives. In: R. Metcalf (ed.) Advances in Pest Control Research, New York: Wiley (Interscience): Vol. III, pp. 289–358. Hahn, M.E., Goldstein, J.A., Linko, P. and Gasiewicz, T.A. (1989) Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo, Arch. Biochem. Biophys., 270(l):344–55. Hayes, T.B., Collins, A., Lee, M., Mendoza, M., Noriega, N., Stuart, A.A. and Vonk, A. (2002) Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses, Proc. Natl. Acad. Sci. USA, 99 (8):5476–80. Heindel, J.J., Chapin, R.E., Gulati, D.K., George, J.D., Price, C.J., Marr, M.C., Myers, C.B., Barnes, L.H., Fail, P.A., Grizzle, T.B., Schwetz, B.A. and Yang, R.S.H. (1994) Assessment of the reproductive and developmental toxicity of pesticide/fertilizer mixtures based on confirmed pesticide contamination in California and Iowa groundwater, Fundam. Appl. Toxicol., 22 (4):605–21. Hellou, J., Warren, W.G. and Payne, J.F. (1993) Organochlorines including polychlorinated biphenyls in muscle, liver, and ovaries of cod, Gadus morhua, Arch. Environ. Contam. Toxicol., 25 (4):497–505. Hillier, S.G. (1994) Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis, Hum. Reprod., 9(2): 188–91. Hirshfield, A.N. (1991) Development of follicles in the mammalian ovary, Int. Rev. Cytol., 124:43–101. Hirshfield, A.N. (1997) Overview of ovarian follicular development: considerations for the toxicologist, Environ. Mol. Mutagen, 29(1): 10–15. Hsueh, A.J.W., Eisenhauer, K., Chun, S.J., Hsu, S.Y. and Billig, H. (1996) Gonadal cell apoptosis, Recent Prog. Horm. Res., 51:433–55, discussion 455–56. Iatropoulos, M.J., Hobson, W., Knauf, V. and Adams, H.P. (1976) Morphological effects of hexachlorobenzene toxicity in female Rhesus monkeys, Toxicol. Appl. Pharmacol., 37 (3):433–44. Jager, G. (1983) Herbicides. In: K.H. Buchel (ed.) Chemistry of Pesticides, New York: Wiley, pp. 322–93. Jain, K. and Bhide, M. (1990) Comparative studies of ovarian histopathology and the mating behaviour of Periplaneta americana, Linn. (Orthoptera) treated with sublethal concentrations of BHC and DDT, Folia Morphol (Praha), 38 (4):344–51. Jarrell, J.F., McMahon, A., Villeneuve, D., Franklin, C, Singh, A., Valli, V.E. and Bartlett, S. (1993a) Hexachlorobenzene toxicity in the monkey primordial germ cell without induced porphyria, Reprod. Toxicol., 7 (1):41–47.

BORGEEST ET AL. 59

Jarrell, J.F., Villeneuve, D., Franklin, C, Bartlett, S., Wrixon, W., Kohut, J. and Zouves C.G. (1993b) Contamination of human ovarian follicular fluid and serum by chlorinated organic compounds in three Canadian cities, CMAJ, 148 (8): 1321–27. Juberg, D.R., Stuenkel, E.L. and Loch-Caruso, R. (1995) Dde chlorinated insecticide 1,1dichloro-2,2-bis (4-chlorophenyl)ethane (p,p'-DDD) increases intracellular calcium in rat myometrial smooth muscle cells, Toxicol. Appl. Pharmacol., 135 (1): 147–55. Kling, D. (1981) Total atresia of the ovaries of Tilapia leucosticta (Cichlidae) after intoxication with the insecticide Lebaycid, Experientia, 37 (l):73–74. Kosanke, G.J., Schwippert, W.W. and Beneke, T.W. (1988) The impairment of mobility and development in freshwater snails (Physa fontinalis and Lymnaea stagnalis) caused by herbicides, Comp. Biochem. Physiol. C, 90 (2):373–79. Kotsonis, F.N., Burdock, G.A. and Flamm, W.G. (1996) Food toxicology. In: C.D. Klaassen (ed.), Casarett and Doull’s Toxicology: The Basic Science of Poisons, New York: McGraw-Hill: pp. 909–49. Larsson, K.S., Arnander, C., Cekanova, E. and Kjellberg, M. (1976) Studies of teratogenic effects of the dithiocarbamates maneb, mancozeb, and propineb, Teratology, 14 (2): 171–83. Lindenau, A., Fischer, B., Seiler, P. and Beier, H.M. (1994) Effects of persistent chlorinated hydrocarbons on reproductive tissues in female rabbits, Hum. Reprod., 9 (5):772–80. Lingappa, V.R. and Farey, K. (2000) Physiology of the female reproductive system. In: J. Dolan et al. (eds) Physiological Medicine, New York: McGraw-Hill, pp. 685–736. Loeffler, I.K., Stocum, D.L., Fallon, J.F. and Meteyer, C.U. (2001) Leaping lopsided: a review of the current hypotheses regarding etiologies of limb malformations in frogs, Anat. Rec., 265 (5):228–45. MacPhee, I.J., Singh, A., Wright, G.M., Foster, W.G. and LeBlanc, N.N. (1993) Ultrastructure of granulosa lutein cells from rats fed hexachlorobenzene, Histol Histophathol., 8 (1):35–40. Mahadevaswami, M.P., Jadaramkunti, U.C., Hiremath, M.B. and Kaliwal, B.B. (2000) Effect of mancozeb on ovarian compensatory hypertrophy and biochemical constituents in hemicastrated albino rat, Reprod. Toxicol., 14 (2): 127–34. Martinez, E.M. and Swartz, W.J. (1991) Effects of methoxychlor on the reproductive system of the adult female mouse. 1. Gross and histologic observations, Reprod. Toxicol., 5 (2): 139–47. Martinez, E.M. and Swartz, W.J. (1992) Effects of methoxychlor on the reproductive system of the adult female mouse. 2. Ultrastructural observations, Reprod. Toxicol., 6 (1):93–98. Muller, W.F., Hobson, W., Fuller, G.B., Knauf, W., Coulston, F. and Korte, F. (1978) Endocrine effects of chlorinated hydrocarbons in rhesus monkeys, Ecotoxicol. Environ. Saf., 2 (2): 161–72. Murdoch, W.J. and McDonnel, A.C. (2002) Roles of the ovarian surface epithelium in ovulation and carcinogenesis, Reproduction, 123 (6):743–50. Norris, R.F. and Fong, J.L. (1983) Localization of atrazine in corn (Zea mays), oat (Avena staiva) and kidney beans (Phaseolus vulgaris) leaf cells, Weed Sci., 31:664–71. Oduma, J.A., Wango, E.O., Oduor-Okelo, D., Makawiti, D.W. and Odongo, H. (1995) In vivo and in vitro effects of graded doses of the pesticide heptachlor on female sex steroid hormone production in rats, Comp. Biochem. Physiol C Pharmacol Toxicol. Endocrinol., 111 (2): 191–96.

60 OVARIAN TOXICITY CAUSED BY PESTICIDES

Okazaki, K., Okazaki, S., Nishimura, S., Nakamura, H., Kitamura, Y., Hatayama, K., Nakamura, A., Tsuda, T., Katsumata, T., Nishikawa, A. and Hirose, M. (2001) A repeated 28-day oral dose toxicity study of methoxychlor in rats, based on the enhanced OECD test guideline 407' for screening endocrine-disrupting chemicals, Arch. Toxicol., 75 (9):513–21. Piao, F.Y., Xie, X.K., Kitabatake, M. and Yamauchi, T. (1997) Transfer of leptophos in hen eggs and tissues of embryonic rats, J. Toxicol. Sci., 22 (2):99–109. Pickford, D.B. and Morris, I.D. (1999) Effects of endocrine-disrupting contaminants on amphibian oogenesis: methoxychlor inhibits progesterone-induced maturation of Xenopus laevis oocytes in vitro, Environ. Health Perspect., 107 (4):285–92. Rao, R.P. and Kaliwal, B.B. (2002) Monocrotophos induced dysfunction on estrous cycle and follicular development in mice, Ind. Health, 40 (3):237–44. Rattner, B.A., Eroschenko, V.P., Fox, G.A., Fry, D.M. and Gorsline, J. (1984) Avian endocrine responses to environmental pollutants, J. Exp. Zool., 232 (3):683–89. Rattner, B.A., Sileo, L. and Scanes, C.G. (1982) Oviposition and the plasma concentrations of LH, progesterone and corticosterone in bobwhite quail (Colinus virginianus), J. Reprod. Fertil., 66 (1): 147–55. Rawlings, N.C., Cook, S.J. and Waldbillig, D. (1998) Effects of the pesticides carbofuran, chlorpyrifos, dimethoate, lindane, triallate, trifluralin, 2,4-D, and pentachlorophenol on the metabolic endocrine and reproductive endocrine system in ewes, J. Toxicol. Environ. Health A, 54 (l):21–36. Simic, B., Kniewald, J. and Kniewald, Z. (1994) Effects of atrazine on reproductive performance in the rat, J. Appl Toxicol., 14 (6):401–404. Sims, D.E., Singh, A., Donald, A., Jarrell, J. and Villeneuve, D.C. (1991) Alteration of primate ovary surface epithelium by exposure to hexachlorobenzene: a quantitative study, Histol Histophathol., 6(4):525–29. Singh, H. and Singh, T.P. (1981) Effect of parathion and aldrin on survival, ovarian 32Puptake and gonadotrophic potency in a freshwater catfish, heteropneustes fossilis (Bloch), Endokrinologie, 77 (2): 173–78. Sohoni, P. and Sumpter, J.P. (1998) Several environmental oestrogens are also antiandrogens, J. Endocrinol., 158 (3):327–39. Spencer, T.E. and Bazer, F.W. (2002) Biology of progesterone action during pregnancy recognition and maintenance of pregnancy, Front. Biosci., 7:d1879–98. Stoker, T.E., Goldman, J.M. and Cooper, R.L. (1993) The dithiocarbamate fungicide thiram disrupts the hormonal control of ovulation in the female rat, Reprod. Toxicol., 7(3):211–18. Stoker, T.E., Goldman, J.M. and Cooper, R.L. (2001) Delayed ovulation and pregnancy outcome: effect of environmental toxicants on the neuroendocrine control of the ovary, Environ. Toxicol. Pharmacol., 9 (3): 117–29. Swartz, W.J. (1984) Effects of 1,1-bis (p-chlorophenyl)-2,2,2-trichloroethane (DDT) on gonadal development in the chick embryo: a histological and histochemical study, Environ. Res., 35 (2):333–45. Swartz, W.J. and Corkern, M. (1992) Effects of methoxychlor treatment of pregnant mice on female offspring of the treated and subsequent pregnancies, Reprod. Toxicol., 6 (5):431–37. Swartz, W.J. and Eroschenko, V.P. (1998) Neonatal exposure to technical methoxychlor alters pregnancy outcome in female mice, Reprod. Toxicol., 12(6): 565–73.

BORGEEST ET AL. 61

Swartz, W.J. and Mall, G.M. (1989) Chlordecone-induced follicular toxicity in mouse ovaries, Reprod. Toxicol., 3 (3):203–06. Tavera-Mendoza, L., Ruby, S., Brousseau, P., Fournier, M., Cyr, D. and Marcogliese, D. (2002) Response of the amphibian tadpole Xenopus laevis to atrazine sexual differentiation of the ovary, Environ. Toxicol. Chem., 21 (6): 1264–67. Todoroff, E.C., Sevcik, M., Villeneuve, D.C., Foster, W.G. and Jarrell, J.F. (1998) The effect of photomirex on the in vitro perfused ovary of the rat, Reprod. Toxicol., 12 (3):305–16. Trapp, M., Baukloh, V., Bohnet, G. and Heeschen, W. (1984) Pollutants in human follicular fluid, Fertil Steril., 42 (1): 146–48. US EPA (US Environmental Protection Agency) (1994) Atrazine, simazine and cyanazine: notice of initiation of special review, Fed. Reg., 59:60412–43. US EPA (US Environmental Protection Agency) (1997) Special report on environmental endocrine disruption: an effects assessment and analysis, EPA/630/R-96/012. US EPA (US Environmental Protection Agency) (2001) Consumer factsheet on methoxychlor, EPA Office of Water. Vidacek, Z., Drevenkar, V., Husnjak, S., Sraka, M. and Karavidovic, P. (1994) Nitrates, pesticides and heavy metals in the soils and water of the tenitory drained by the Karasica and Vucica river system., Proceeding of the Meeting Agriculture and Water Management, Bizovacke Toplice, Croatia, November, 211–22. Waters, K.M., Safe, S. and Gaido, K.W. (2001) Differential gene expression in response to methoxychlor and estradiol through ERalpha, ERbeta and AR in reproductive tissues of female mice, Toxicol. Sci., 63 (1):47–56. Withgott, J. (2002) Amphibian decline. Ubiquitous herbicide emasculates frogs, Science, 296 (5567):447–48. Younglai, E.V., Foster, W.G., Hughes, E.G., Trim, K. and Jarrell, J.F. (2002) Levels of environmental contaminants in human follicular fluid, serum, and seminal plasma of couples undergoing in vitro fertilization, Arch. Environ. Contam. Toxicol., 43 (l):121– 26.

4 OVARIAN TOXICITY CAUSED BY ENDOCRINE DISRUPTORS Paul F.Terranova and Karl K.Rozman

DEFINITION An ovarian endocrine disruptor (ED) is an exogenous chemical agent that alters the hormonal function of the ovaries. EDs include environmental toxicants, drugs and other chemicals, and hormones. EDs may act directly on the ovary, alter ovarian hormone receptors and/or signal transduction of numerous ovarian growth regulators such as gonadotropins, steroids and growth factors. EDs may also act indirectly by altering secretion of gonadotropins and ovary-related growth regulators emanating from other organs. The possibility exists that EDs may perturb neural networks and change vascular input to the ovary, but these aspects have not yet been investigated. The potential direct and indirect actions of EDs on the ovary are shown in Figure 4.1. At any stage of development an ovarian ED may alter the synthesis or elimination, secretion, transport, binding, or other actions of ovarian hormones or hormones which maintain the function of the ovary. A comprehensive review of EDs on numerous organs can be found in an EPA report from 1997 (Crisp et al., 1997, 1998). Reviews pertinent to ovarian endocrine disruption are listed in Table 4.1. Classification of EDs relevant to ovarian toxicology categorized by their direct and/or indirect effects For the purpose of this review, EDs are classified as acting directly and/or indirectly on the ovary. However, EDs may also be classified into several categories based on their chemical structure, use and mode of entry into the environment. For example, herbicides such as the chlorotriazines are known inhibitors of LH secretion resulting in a block or delay of ovulation (Cooper et al., 2000). Thus, according to our classification, chlorotriazines induce ovarian toxicity and endocrine disruption through indirect means at the level of the hypothalamic-pituitary axis. Polychlorinated aromatic hydrocarbons such as dioxins, polychlorinated biphenyls and furans are aryl hydrocarbon receptor

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Figure 4.1 Potential direct and indirect actions of endocrine disruptors (EDs) on the ovary. Table 4.1 A list of reviews related to ovarian endocrine disruption in the last 5 years.

(AhR) agonists and environmental contaminants, which enter the environment as unwanted industrial by-products. AhR agonists are known to disrupt the rat estrous cycle, block the LH surge, and delay ovulation by acting on the hypothalamic-pituitary axis and also by acting directly on the ovary (Petroff et

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al., 2001). Dietary constituents such as indole-3-carbinol (I3C), whose metabolites are weak agonists of the AhR, block ovulation similar to that of the potent AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Gao et al, 2002). Drugs, such as barbiturates including pentobarbital and phenobarbital, may block the LH surge at the level of the hypothalamus (Butcher et al., 1975; Terranova and Ascanio, 1982) and lead to a delay in ovulation similar to the herbicidal chlorotriazines (Cooper et al., 2000) and some of the polychlorinated hydrocarbons including TCDD, other polyhalogenated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) (Gao et al, 2000a; Ushinohama et al, 2001). Polycyclic aromatic hydrocarbons (PAHs) are EDs originating from cigarette smoking and combustion of fuel. Destruction of oocytes and ovarian failure occur in PAH-treated mice, and cigarette smoking is associated with early menopause in women. Also PAHs were shown to destroy primordial follicles in mice (Mattison and Nightingale, 1980; Matikainen et al, 2001). Thus, PAHs alter follicular development and steroidogenesis leading to premature ovarian aging resulting in disrupted cycles and premature ovarian failure. The progestational agent, diethylstilbestrol (DES), which was used to prevent spontaneous abortion in the mid-twentieth century, is considered an ED. DES alters the secretion of pituitary gonadotropins at the hypothalamic level, has direct ovarian actions, and as a result alters the estrous cycle, puberty and fertility (Hendry et al, 2002). Direct effects of EDs on the ovary include effects on follicular development and ovulation by reducing LH and FSH receptors on growing follicles and by destroying primordial follicles. The mechanisms of these direct ovarian actions are unclear, but it is known that induction of apoptosis of small follicles (including primordial) is related to the apoptotic gene Bax (Hoyer et al., 2001; Matikainen et al., 2001). MODELS OF ED Various models have been used to test for endocrine disruption of ovarian function in mice, rodents, primates as well as other species. The most widely used models in academia, industry and government are acute or chronic exposure coupled with analyses of vaginal estrous cycles of rats (Crisp et al., 1997, 1998). Vaginal smears are performed daily on rats or mice over a 14–21-day period to reveal regular cycling with a 4–5-day periodicity. After exhibiting three or more consecutive 4–5-day cycles, the putative ED is given by the chosen route to the animal, and monitoring of the estrous cycle is continued. Disruption of the cycle is detected statistically by analyzing the number of cycles completed before and after treatment. Other parameters such as the number of days spent on proestrus, estrus and diestrus are also useful in these analyses. Another ovarian ED model is the immature gonadotropin-primed rat (Petroff et al., 2001). This model was chosen since the immature rat of -25 days of age

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exhibits a large population of small follicles in its ovary but is devoid of corpora lutea. The first spontaneous ovulation does not occur until -35 days of age. On day 25 of age, animals are given 5 IU equine chorionic gonadotropin (eCG) to induce synchronized development of a group of growing follicles that will reach the pre-ovulatory stage -48 hours later. At 52 hours after eCG administration, 10 IU human chorionic gonadotropin is given to simulate an LH surge and to induce follicular rupture (ovulation) ~12 hours after injection. On the morning following ovulation (72 hours after eCG), the ovaries and oviducts are assessed for the number of corpora lutea and ova shed, respectively. In addition, relevant ovarian hormones such as progesterone, estradiol, LH and FSH are measured. Usually, hormone levels in sera exhibit dose-dependent responses when EDs are administered (Gao et al., 2000a, 2002). Other models include (1) treatment of pregnant dams with EDs during the latter half of pregnancy that alters development of the fetal ovary (Theobald and Peterson, 1997), (2) acute or chronic treatment of neonatal rodent pups (Hendry et al, 2002) and (3) chronic treatment of adult mice and rats (Hoyer et al., 2001). EFFECTS OF EDS ON OVARIAN FUNCTION Ah receptor agonists as multiple organ EDs Multiple organ EDs refer to the numerous reports that AhR agonists block ovulation at least at two sites, the level of the hypothalamus and ovary. The PCDDs, PCDFs and PCBs as well as dietary AhR agonists, such as indole-3carbinol (I3C), apparently act in this manner. The hypothalamus exhibits AhR (Kainu et al., 1995; Huang et al., 2000; Petersen et al, 2000; Hays et al, 2002). However, it is unclear whether Ah receptors exist on neurons regulating the signaling involved in the LH and FSH surges on proestrus. Evidence of an ovarian Ah receptor exists in rats, mice, pigs, humans and macaques (Mattison and Nightingale, 1980; Enan et al, 1996a; Chaffin et al., 1999,2000; Benedict et al, 2000; Gregoraszczuk, 2002; Mizuyachi et al., 2002). Several polychlorinated aromatic hydrocarbons (PAHs), which have been tested for endocrine disrupting activity, include the PCDDs, PCDFs and PCBs (Gao et al., 2000b). Those compounds have been shown to reduce ovarian weights dosedependently in the immature gonadotropin-primed rat model (Petroff et al., 2001) and in other models with chronic exposure (Cummings et al, 1996). Additional studies revealed that TCDD altered estrous cycles in which ovulation was blocked (Li et al, 1995a,b; Cummings et al., 1996). Histological assessment of the ovaries of TCDDtreated rats in adult cycling models or in the immature rat model revealed that the ovaries were smaller than controls on the morning after expected ovulation, with few to no corpora lutea and large unruptured preovulatory follicles (see Figure 4.2 and Petroff et al., 2001). In addition, serum concentrations of FSH, LH, progesterone and estradiol were dose-dependently

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Figure 4.2 Effects of TCDD on ovarian morphology in the immature rat. TCDD (32µg/kg BW) was given p.o. on day 24 of age. eCG was given on day 25 to stimulate follicular development. Ovaries were collected for histology on the morning of day 25. Permission from Elsevier.

altered with some of the hormones increasing and others decreasing. For example, blockage of ovulation (dose-dependent reduction of the number of ova shed) was associated with a stepwise increase in the serum concentrations of estradiol (E2) and a decrease in LH, FSH and progesterone (P4) on the morning of expected estrus. Interestingly, in the immature TCDD-treated rat, synchronous yet premature LH and FSH surges occurred within 24 hours after injection of eCG when the surges were not expected until after 58 hours (Figure 4.3). The hormonal data provided clear evidence of endocrine disruption. Similar blockage of ovulation was observed with PCDFs, PCBs, and I3C indicating the likelihood of similar, if not identical modes of action (Gao et al., 2000a, 2002). TCDD administered to pregnant (in utero exposure) and lactating rats altered follicular development as evidenced by a reduction in the number of antral and pre-antral follicles during later stages of life (Heimler et al., 1998b). Currently, it is unknown if in utero exposure to TCDD acts locally on the ovary and/or on hypothalamo-pituitary control of LH and FSH secretion. Moreover lactation represents a very efficient transfer of TCDD to the pups (Li et al., 1995c). Therefore, the contribution of in utero exposure via lactation to this effect is not entirely clear. Using the immature gonadotropin-primed model, direct effects of TCDD on the ovary have been observed in hypophysectomized and intact rats (Li et al., 1995b; Petroff et al, 2000; Roby, 2001). Hypophysectomized immature rats at

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Figure 4.3 A diagram of the effects of polychlorinated dibenzo-p-dioxins (PCDDs) on follicular and hormonal changes in the eCG-treated immature rat. FSH, follicle stimulating hormone; LH, luteinizing hormone. Permission from Elsevier.

-25 days of age were treated with TCDD the day prior to administration of eCG. Approximately 52 hours after eCG, hCG was administered to induce ovulation. The number of ova shed was reduced by TCDD apparently by a direct inhibitory action on the ovary and was independent of pituitary action since the effect was also observed when the pituitary had been removed (Li et al, 1995b). Petroff et al. (2000) also demonstrated direct inhibitory effects of TCDD on the ovary in both pituitary intact and hypophysectomized immature rats treated with gonadotropins. TCDD injected directly into the ovarian bursa reduced ovulation. This effect was overcome by the administration of exogenous estrogen indicating that TCDD’s inhibitory action on the rats ovary is probably due to its antiestrogenic actions. Moreover, specific effects of TCDD on follicular development and ovarian gonadotropin receptors have also been reported in the immature eCG-treated hypophysectomized rat (Roby, 2001). Follicular development was retarded by 52 hours after injecting eCG in the TCDD-treated hypophysectomized immature rat model as revealed by a reduction in the number of follicles greater than 350 microns in diameter (Figure 4.4). This coincided with low serum concentrations of estradiol (Roby, 2001). Also at that time point hCG (LH) binding was lower in granulosal and thecal cells of TCDDtreated rats as well as lower FSH binding in granulosal cells only. These results were corroborated by an extremely low number of ova shed in response to an

68 PAUL F.TERRANOVA AND KARL K.ROZMAN

Figure 4.4 The size and number of healthy antral follicles present in ovaries from control and TCDD-treated rats 52 hours after administration of eCG. Follicle numbers were obtained from one ovary in six controls and seven TCDD-treated animals. Data are the mean ± SEM. *, p≤0.05 TCDD versus control within the same diameter group. Permission from The Endocrine Society.

ovulation-inducing dose of hCG (10 IU) when compared to controls (~9 ova in controls versus 0.6 ova in TCDD-treated rats). Lower ovarian cAMP concentrations than controls at 5 hours after hCG is also consistent with these findings. Thus, TCDD altered the growing follicles at a critical stage (>350 microns in diameter) to respond to eCG, a hormone with FSH and LH-like activities. Clearly at the time of hCG injection, the pre-ovulatory follicles were not of the same quality as controls (lower LH and FSH binding). Previous studies indicated that TCDD may interfere with follicular rupture and that may very well be the case (Petroff et al, 2001). However, the subnormal pre-ovulatory follicles at the time of hCG injection must be also considered as a possible explanation for the reduced number of ova shed. Administration of TCDD after the pre-ovulatory follicles had matured may provide insights as to whether or not this environmental toxicant interferes with follicular rupture as well. A large volume of data indicates a major role of estradiol in enhancing follicular development in the rat (Richards, 1980; Farookhi and Desjardins, 1986; Richards et al, 1987; Tonetta and diZerega, 1989; Wang and Greenwald, 1993; Robker and Richards, 1998; Drummond and Findlay, 1999). Several studies have also demonstrated antiestrogenic properties of TCDD in nonovarian tissues (Astroff and Safe, 1988, 1990; Romkes and Safe, 1988; Kharat and Saatcioglus, 1996; Tian et al, 1998a, b). Whether or not the growing stage of follicles (>350 microns) also depends on estradiol for further development is unknown. However, since such follicles’ response to eCG was altered, it is conceivable that the antiestrogenic properties of TCDD were responsible for this effect, which would suggest a role for estradiol in this critical growing stage of the follicle.

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Direct effects of TCDD on gonadotropin-stimulated ovarian steroidogenesis in the rat are unclear because different studies reported a variety of effects. In vitro, TCDD decreased FSH-stimulated granulosal aromatase activity and reduced CYP450scc and aromtase mRNA in rat granulosa cells (Dasmahapatra et al., 2000). However, a report using enriched populations of granulosa and thecainterstitial cells as well as whole ovarian dispersates (mixtures of thecainterstitial cells, granulosa cells and other ovarian cell types) from immature rats revealed no in vitro effect of TCDD on basal and FSH- and LH-stimulated steroidogenesis (Son et al, 1999). Two studies reported in vitro effects of TCDD on FSH (Hirakawa et al, 2000a) and LH (Hirakawa et al, 2000b) receptors in rat granulosa cells. In vitro, FSH increased FSH and LH receptor mRNA and both were reduced dose-dependently by TCDD. 8-Bromo-cAMP also increased FSH receptor mRNA in granulosa cells and this was inhibited by TCDD indicating a post-cAMP site of TCDD action. The rate of FSH receptor mRNA gene transcription was also reduced by TCDD, but the stability of the FSH receptor mRNA was not affected. However, the stability of LH receptor mRNA was decreased. In vitro effects of TCDD have been consistently observed on human granulosallutein cells collected from patients undergoing in vitro fertilization (Enan et al., 1996a,b). TCDD decreased protein kinase A activity, and progesterone and estradiol secretion. Another study also confirmed that TCDD reduced estradiol secretion in the human granulosa luteal cell model; however, with additional culture of the cells and addition of an androgen precursor, the reduced estradiol returned to control values (Heimler et al., 1998). A summary of the in vitro effects of TCDD on granulosa cells is given in Table 4.2. Another potential site of TCDD action is the hypothalamic-pituitary axis since the LH and FSH surges are blocked in adults (Li et al., 1995a,b) and in the immature gonadotropin-primed model (Petroff et al, 2001). However, the mechanisms by which TCDD blocks the LH/FSH surges are still unclear. Circulating levels of estradiol in the intact gonadotropin-primed immature female rat are similar to control levels prior to the LH surge (Gao et al., 2000a). It is well known that estradiol during the pre-ovulatory period acts as a critical positive feedback hormone stimulating the secretion of LH and FSH to “surge” levels that ultimately induce rupture of the pre-ovulatory follicle (s). If TCDD is indeed acting as an antiestrogen, then it likely reduces the effectiveness of estradiol during this critical positive feedback period. Indeed this appears to be the case since an exogenous long-acting estrogen, estradiol cypionate, overcame the inhibitory effects of TCDD in blocking of the LH and FSH surges (Gao et al., 2001). In fact, a circulating estradiol level 8–10 times higher than normal had to be maintained in the preovulatory period in order to overcome the inhibitory effects of TCDD. There was no demonstrable inhibitory effect of TCDD on the pituitary since exogenous gonadotropin-releasing hormone (GnRH) induced significant secretion of LH and FSH in TCDD-treated rats during the pre-ovulatory period (Gao et al., 2000b).Although exogenous GnRH significantly increased LH

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Table 4.2 In vitro effects of TCDD on granulosa cells

and FSH secretion beyond control values, ovulation was only partially restored indicating that TCDD also had a direct inhibitory effect on the ovary. Indole-3-carbinol (I3C), a component of cruciferous vegetables, is a weak agonist of the AhR (Bjeldanes et al., 1991) although other studies have reported antagonistic activity towards the AhR (Chen et al., 1996). A recent report using an immature gonadotropin-primed rat model for inducing synchronized follicular development and ovulation was used to test I3C’s ability to block ovulation (Gao et al., 2002). Immature rats were given daily doses of I3C ranging from 0 to 1.5 g/kg/day. The initial dose of I3C was given one day prior to 5IU equine chorionic gonadotropin. I3C blocked ovulation dose-dependently at the expected time and this coincided with reduced concentrations of LH and FSH in sera at the time of the expected proestrus surges. Administration of hCG that mimicked a normal pre-ovulatory surge of LH partially restored ovulation indicating that I3C may have had direct effects on the ovaries in blocking ovulation in addition to its ability to block the surges of LH and FSH. I3C given by gavage is subjected to acidic conditions in the stomach and is converted to various oligomers that are thought to mediate the biological effects of I3C (DeKruif et al, 1991; Wortelboer et al, 1992). One of the oligomers, 3,3'diindolylmethane (DIM) has structural similarities to TCDD (Cashman et al, 1999), is capable of binding to the Ah receptor, and inducing cytochrome P450 (Vang et al., 1990; Bjeldanes et al, 1991; Jellnick et al, 1993). However, DIM had no effect on ovulation in the immature rat model (Gao et al., 2002). Thus, the similarity in actions between I3C and TCDD may be the result of their capability to bind to the Ah receptor, but a lack of effect by an AhR agonist such as DIM raises questions about the generalizability of the AhR hypothesis. I3C and its oligomers exhibit antiestrogenic activity like TCDD in a variety of assays (Kociba et al, 1978; Wattenberg and Loub, 1978; Stoewsand et al, 1988; Bradlow et al, 1991; Gierthy et al, 1993; Grubbs et al, 1995), although some of its reaction products can bind to the estrogen receptor exhibiting weak estrogenic activity (Liu et al., 1994; Riby et al, 2000). Unlike some of the I3C metabolites, TCDD does not bind to the estrogen receptor but it may interfere with signal transduction of estrogens (Safe, 1995). Nevertheless, an antiestrogenic action of I3C may account for its ability to block the LH surge on expected proestrus. It is

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Figure 4.5 Direct and indirect effects of Ah receptor agonists hypothalamicpituitary axis and ovary leading to blockage of ovulation.

on

the

clear that the inhibitory action of TCDD is due to its antiestrogenicity since a long acting estrogen, estradiol cypionate, effectively overcomes the ability of TCDD to block the LH surge (Gao et al, 2001) but this is not known for I3C. Direct and indirect effects of Ah receptor agonists on the hypothalamic-pituitary axis and ovary leading to blockage of ovulation are shown in Figure 4.5. Tamoxifen (TAM), a well-known antiestrogen, with high affinity to bind to the estrogen receptor blocked ovulation very similar to that of TCDD and I3C in the gonadotropin-primed immature rat model (Gao et al., 2002). However, unlike with I3C and TCDD, exogenous hCG given on expected proestrus completely overcame the inhibitory effects of TAM on ovulation (Gao et al., 2002) or TCDD (Petroff et al, 2001). Thus, it appears that the effects of TAM occur solely at the level of the hypothalamic-hypophyseal axis whereas I3C and TCDD, each, blocked ovulation at the level of the ovary as well as at the hypothalamichypophyseal axis. Atrazine, a neuroendocrine-ovarian axis disruptor Atrazine, a chloro-5-triazine herbicide, has been studied extensively as an ED because of its wide spread use in the United States. It has been reported that atrazine increases the incidence of spontaneous mammary tumors in female Sprague-Dawley (SD) rats (Stevens et al., 1994). The original reports of endocrine disruption by atrazine appeared in 1994, and attempted to explain the differential effects in inducing mammary tumors in SD rats but not in Fischer 344 rats (Eldridge et al., 1994; Stevens et al., 1994; Wetzel et al, 1994). In short-

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term studies, atrazine and simazine, each by itself, were given daily for 2 weeks at 100 or 300mg/kg BW to SD and Fischer 344 female rats (Eldridge et al., 1994). Treatments reduced ovarian weights and serum estradiol levels. SD rats exhibited a lengthening of the estrous cycle with an increase in the number of days in vaginal estrous and a decrease in the number of days in diestrus. Interestingly, Fischer 344 rats also exhibited an increase in the length of the estrous cycle, but there was an increase in the number of day s in diestrus with a reduction in vaginal estrus. It was concluded that prolonged blockage of ovulation associated with continuous secretion of estradiol in the SD rat led to increased secretion of prolactin, which in turn led to mammary gland tumors. SD rats have a high rate of spontaneous mammary tumors. Fischer 344 rats exhibiting prolonged diestrus mimicked pseudopregnancy resulting in activated corpora lutea with prolonged progesterone secretion; however, these rats are not prone to develop mammary tumors (Cutts and Noble, 1964). In fact, differences in reproductive aging of these two strains of rats were noted. SD rats enter constant estrus as they age whereas Fischer 344 rats exhibit repeated pseudopregnancies. Chronic administration of atrazine in the diet also resulted in a lengthening of the estrous cycle and the number of days in estrus in SD rats (Wetzel et al, 1994). In Fischer rats, however, although estrous cycles were slightly prolonged by atrazine there was no effect on serum concentrations of progesterone and estradiol (Wetzel et al., 1994). Exposure of the SD rat to maximally tolerated doses (or higher) of atrazine and simazine (both chloro-S-triazines) for 2 years altered the neuroendocrine axis, prolonged cycles and induced pathology in the mammary gland. The changes in hormones and the prolonged estrous cycles were similar to natural aging in the SD rat except they were more pronounced in the simazine-treated groups. Thus those studies revealed that chloro-S-triazines had detrimental effects on the female reproductive system in various strains of rats, and disrupted neuroendocrine control of ovarian function. In 1996, Cooper et al. found that high daily doses of atrazine (150mg/kg BW by gavage) over 21 consecutive days altered estrous cycles in Long Evans (LE)hooded and SD rats in favor of sustained diestrus, i.e., pseudopregnancy. A higher dose (300 mg/kg) of atrazine led to ovarian regression and an anestrous smear. This study confirmed the endocrine-disrupting effects of atrazine on ovarian function and extended the observations to an additional strain of rat. Later studies targeted the central nervous system, specifically the hypothalamicpituitary axis, as a potential site for endocrine disruption. Thus, it was proposed that atrazine increased dopamine and reduced norepinephrine concentrations in the hypothalamus (Cooper et al., 1998), because atrazine was shown to have direct effects on catecholamine neurons in vitro leading to alteration in catecholamine synthesis (Das et al, 2000, 2001). These alterations are likely to reduce the ability of estrogen to induce an LH surge. This was investigated using ovariectomized estrogen-treated SD and LE-hooded rats with spontaneous surges of LH and prolactin (Cooper et al., 2000). In this study, atrazine (50–300 mg/kg/

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day) was administered by gavage for 1, 3 or 21 days. A single dose of atrazine (300 mg/kg) suppressed the LH and prolactin surges in ovariectomized LE rats but not in SD rats. Three hundred mg/kg given on proestrus did not block ovulation but it did induce pseudopregnancy in the majority of LE rats. SD and LE rats responded to atrazine in a dose-dependent manner after 21 days of treatment with reduced LH and prolactin surges. The results of these studies indicated clearly that neuro-endocrine control of LH and prolactin was altered and continued treatment led to altered cycles in both strains of rats. These data are consistent with the hypothesis that atrazine reduces GnRH pulse frequency similar to that observed in aging rats and that this is caused by continuous exposure to estrogen (Cooper et al, 2000). However, atrazine does not bind to the estrogen receptor (Tennant et al, 1994; Connor et al, 1996), requiring other mechanisms to account for this action. Atrazine appears to alter regulation of the CNS component of the hypothalamic-pituitary axis and not the pituitary, since atrazine did not affect prolactin secretion when a pituitary graft was placed under the kidney capsule (Cooper et al, 2000). In addition, exogenous GnRH restored LH secretion in atrazine-treated rats to a level comparable to normal surge levels indicating that atrazine likely reduced GnRH secretion without altering pituitary gonadotrophs (Cooper et al., 2000). Lastly, secretion of LH and prolactin was unaffected by perifusion of pituitaries with atrazine in vitro (Cooper et al., 2000). Pre-pubertal female rats given atrazine on days 22–41 post-natally exhibited a delay in vaginal opening associated with the first ovulation (Laws et al., 2000). However, when treatment was stopped, vaginal opening occurred within 3–4 days but vaginal estrous cyclicity was irregular during the following 15 days. Thereafter, estrous cycles resumed as normal. In fact, high doses of atrazine prevented vaginal opening as long as it was administered. When the high-dose treatment was withdrawn, normal vaginal cycles eventually resumed. These data (Laws et al., 2000) are consistent with the inhibition of LH and prolactin secretion in adult rats in which cycles were prolonged (Cooper et al., 2000). In summary, atrazine appears to increase dopamine and reduce norepinephrine concentrations in the hypothalamus leading to a decrease in GnRH release and as a result to reduced LH secretion (Figure 4.6). The lack of LH secretion provides a satisfactory explanation for the disrupted cycles, delayed vaginal opening, and a blockage of ovulation (Das et al, 2000, 2001), although secretion of FSH with low levels of LH allows follicular development and estradiol secretion to continue. 2-Bromopropane (2BP), an ovarian axis ED It is clear that 2BP is an ED based on its ability to disrupt the menstrual cycles in humans, rat vaginal cycles and gonadotropin-induced ovulation in mice; all are endocrine-controlled events. Thus, the effects of 2BP are reviewed here to elucidate the mechanisms of endocrine disruption. 2BP is very volatile, permeable to the skin, and is used as a substitute for Freon 113 (trichlorotrifluoroethane),

74 PAUL F.TERRANOVA AND KARL K.ROZMAN

Figure 4.6 Hypothetical mechanism by which atrazine blocks ovulation in rats.

which contributes to the reduction of the stratospheric ozone levels. The use of Freon 113 has been curtailed because of this detrimental effect on the environment. 2BP has also been used as a substitute for trichloroethylene for the extraction of asphalt mixtures. Detrimental effects of 2BP on humans were first reported in 1996 (Kim et al., 1996a,b). The incidents occurred in 1995 in South Korea. Industrial workers who were exposed to 2BP at an electronic factory in Korea exhibited signs of reproductive and hematopoietic toxicity (Kim et al., 1996a,b; Maeng and Yu, 1997; Park et al., 1997). Exposure occurred in the cleansing portion of the tactile switch assembly area where levels of 2BP in the air were ~12 ppm as estimated by simulation studies. However, short-term exposure to much higher levels likely occurred inside the hoods of the cleaning baths with concentrations reaching ~4,100 ppm in the air during simulation studies. Often the heads of the workers were put inside the hood of the cleaning baths and they also dipped bare hands into the cleaning solution containing 2BP. No type of protective equipment (masks and gloves) was used by the exposed workers. Of twenty-five women exposed to 2BP, sixteen exhibited signs of ovarian dysfunction as evidenced by amenorrhea and serum concentrations of FSH greater than 40 mIU/ml (range: 28–137 mIU; normal serum FSH is 10mIU/ml for the follicular phase and premature ovarian failure is considered at >20 mIU/ ml). Only sera from three women were analyzed for estradiol and the values (<13.6pg/ml) were compatible with premature ovarian failure. In addition, ten of sixteen women complained of hot flashes. Progesterone withdrawal (a test of positive estrogen action on the uterus) was not observed in women with amenorrhea (16/16 tested). Serum concentrations of LH and prolactin were normal. The apparent exposure occurred for a period of 4–16 months to a cleaning solution containing 97.4 percent 2BP, 0.01 percent 1,1,1trichloroethane, 0.33 percent n-heptane, and 0.2 percent 1,2-dibromopropane

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(Park et al, 1997). Two workers who quit their job due to hematopoietic problems recovered normal health apparently due to the cessation of exposure to the cleaning solution containing mainly 2BP. The initial study describing effects of 2BP in female rats was that of Kamijima et al. (1997). Adult Wistar rats were treated by inhalation exposure for 9 consecutive weeks for 8 hours per day (2–10 PM) with doses of 0, 100, 300 and l,000ppm. In the high-dose group, mobility, muscle tone and weight gain were reduced but were unaffected in the other groups. As early as the second week of exposure the estrous cycles were altered in the high-dose group as evidenced by prolonged diestrus or estrus leading to a decreased number of normal cycles. In the 300 ppm group, prolonged diestrus was observed beginning at the seventh week of exposure with an average cycle of -12 days; normally the length of a rat cycle is 4–5 days. Exposure of rats to 100-ppm 2BP did not alter the number of cycles compared to controls. Ovarian and uterine weights decreased in the highdose group at the end of the study with diestrous uterine weights being also lowered in the 300-ppm dosage group as well. Ovarian histology revealed extensive follicular atresia with reduced numbers of growing and antral follicles in the high-dose group, which exhibited constant estrus. In fact, few viable oocytes, numerous cystic follicles with thin layers of granulosa cells and a lack of newly formed corpora lutea were observed in the ovaries of the sustained estrous rats in the high-dose group. In the 300 and l,000ppm treatment groups with constant diestrus, antral follicles and corpora lutea were present although the number of follicles was reduced. LH and FSH concentrations in sera were not altered by 2BP based on the statistical analyses; however, the serum concentrations of LH tended to decrease and FSH tended to increase dose dependently. These studies indicated that 2BP may have direct effects on the ovary since LH and FSH levels were in a range which would not be expected to result in a major cycle disruption. The reason(s) for the constant diestrus and estrus observed in the rats is (are) unknown. Constant diestrus is usually associated with activated corpora lutea leading to increased progesterone secretion, which may be related to some stress exhibited by the rats in response to the high doses of 2BP since the mobility of the rats was reduced. Possibly prolactin levels in the sera were increased which would induce persistent functional corpora lutea following ovulation. The duration of the prolonged cycles in the high-dose group was -12 days which is similar to the length of pseudopregnancy in the rat. The reason for constant estrus is also not known but may be related to blockage of ovulation. The authors noted that estrogen secretion may have been reduced since uterine weights on diestrus were reduced by 2BP (Kamijima et al., 1997). Thus, 2BP may have reduced ovarian estrogen secretion sufficiently to cause insufficient activation of the hypothalamic-pituitary axis in which case a lack of LH would have prevented ovulation (Kamijima et al., 1997). A study using gonadotropin-treated mice investigated potential direct effects of 2BP on ovulation (Sekiguchi and Honma, 1998). Adult mice were given 2BP (0, 500, 1,000 and 2,000 mg/kg BW) i.p. eight times at intervals of 2–3 days for 17

76 PAUL F.TERRANOVA AND KARL K.ROZMAN

days. Follicular development was initiated by 10 IU equine chorionic gonadotropin (eCG) on day 15 of 2BP treatment, and ovulation was induced by an injection of 10 IU human chorionic gonadotropin (hCG) on day 17. On the next day (day 18), the number of ova within the oviducts was determined. Approximately 52 ova were found in control mice. A dose-dependent decrease was observed (86, 45 and 11 percent of controls) in rats treated with 500, 1,000 and 2, 000 mg of 2BP/kg BW, respectively. Since this method of ovulation induction is independent of the pituitary, it appears that 2BP may have interfered with the responsiveness of the ovary to the exogenous chorionic gonadotropins. However, since ovulation induction was not initiated until near the end of 2BP treatment, the number of follicles could have been depleted by 2BP prior to gonadotropin treatment since there is evidence of increased follicular atresia by 2BP in adult rat models (Kamijima et al., 1997). Thus, the pool of growing follicles from which to recruit in response to exogenous eCG may have been reduced in 2BP-treated mice. An alternative explanation would be to postulate the accumulation of a metabolite of 2BP with a prolonged half-life such as bromide (t1/2~5–10 days), which then could have exerted the toxicity on follicular development. In fact, this is the more likely explanation unless a single low dose of 2BP would also cause an effect on the primordial follicles. The need for the dosing regimen (i.p. injections eight times during 17 days) applied is a strong indication for an important role of kinetics in the reproductive toxicity of 2BP. The primary target cell of 2BP in the ovary appears to be the oocyte of the primordial follicle as determined by early alterations in ovarian histology and a quantitative assessment of follicular development in the rat (Yu et al., 1999). An increase in the number of atretic primordial follicles (containing evidence of apoptosis by in situ terminal deoxynucleotidyl transferase assays) was observed in response to 2BP. Adult female rats were given 0-, 100-, 300-, and 1,000-ppm 2BP for 8 hours per day over 9 weeks. In the mid- and high-dose groups, ovaries were small with few primordial, growing, and antral follicles and few or no corpora lutea. All three categories of follicles were reduced by 2BP compared to controls. Animals exposed to 100-ppm 2BP appeared histologically normal compared to controls, but quantitative assessment revealed statistically fewer primordial and growing follicles compared to controls. The number of antral follicles in the 100-ppm group, although fewer in number, was not statistically different from controls. Simultaneous studies of effects of the same doses of 2BP on vaginal estrous patterns revealed disruption of cycles at 300 and l,000ppm but not at 100ppm, indicating that follicular development was more sensitive than vaginal smears in detecting effects of 2BP. In addition, a time course was conducted on days 1, 3, 5 and 17 after a single 8-hour exposure to 3,000 ppm 2BP (Yu et al, 1999). The symmetry of the oocyte and nucleus was abnormal at day 5 after exposure and this effect remained and even progressed with more distortion and shrinkage through day 17. The latter coincided with in situ end labeling staining for DNA strand breaks. The number of primordial follicles was also reduced by 3,000-ppm 2BP by day 17. Granulosa

OVARIAN TOXICITY CAUSED BY ENDOCRINE DISRUPTORS 77

cells of growing and antral follicles appeared unaffected by 5 and 17 days after exposure to 2BP and their numbers were also unaffected during this time period. Assuming the correctness of the bromide hypothesis due to generation of high steady state concentrations of bromide as a result of reaction with glutathione, inhalation of 100-ppm 2BP is equivalent to about 4–5 mg/kg/day of bromide. The allowable daily intake of bromide (ADI) is 1 mg/kg/day (International Program on Chemical Safety). The rat and human no observable effect level (NOEL) with regard to neurophysiological or endocrinological effects was estimated to be 12 and 9 mg/kg/day, respectively. This is in good agreement with 4–5 mg/kg/day being at or near the threshold dose, since the effect on primordial follicles was not significant at this dose, but quite so at 300 ppm of 2BP corresponding to 12–15mg/ kg/day of bromide. These data indicate that, if the bromide hypothesis is confirmed, there is very little if any species difference between human and rats regarding the reproductive toxicity of 2BP. This view is compatible with a similar bromide half-life in humans and rats. EDs that destroy growing and large antral follicles may cause only transient alteration in cycles once the ED is withdrawn. The latter is supported by previous studies using cyclophosphamide (Generoso et al, 1971; Mattison and Schulman, 1980), TCDD (Ushinohama et al, 2001) and barbiturates (Butcher et al, 1975). On the other hand, EDs that destroy primordial follicles, may have a “delayed” effect on estrous cycles since it may take -60 days for a primordial follicle to eventually ovulate (Hirshfield, 1994). For example, treatment with 4vinyl-cyclohexene (VCH) does not cause cycle disruption until several months after initiation of treatment (Hooser et al., 1994). This delayed effect may manifest itself by (1) reduction of the number of ova shed even though estrous cycles are normal; thus there is a “silent” effect on the quality of the cycle, (2) disrupted cycles (long cycles as with 2BP described above) due to the lack of estrogenproducing follicles and failure of an LH surge and (3) premature cessation of cycles due to depletion of the growing pool of follicles. A hypothetical mechanism of 2-bromopropane as an ED is shown in Figure 4.7. Further evidence of these phenomena exists in women exposed to 2BP (Kim et al., 1996a,b; Park et al, 1997). First, a period of 4–16 months from time of exposure and subsequent onset of amenorrhea was evident. In a two-year followup study of the sixteen intoxicated female workers exposed to 2BP, only two patients did not resume normal ovarian function. Four of six patients exhibited ovarian fibrosis as determined by laparoscopic analysis of the ovaries in situ and by histological analysis of ovarian biopsies. Also, follicular development in the exposed human was suppressed and was similar to that in rats exposed to 2BP (Koh et al., 1998). Concerning 2BP, quantitative assessment of ovarian follicular development may be a more sensitive endpoint of ovarian toxicology than estrous cycle disruptio n (Yu et al., 1999). This is not an uncommon observation as others have also observed this with various toxicants including benzo(a)pyrene,

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Figure 4.7 Hypothetical mechanism by which 2-bromopropane blocks ovulation in rats.

cyclophosphamide and VCH (Takizawa et al, 1984; Smith et al, 1990; Plowchalk et al, 1993; Bolon et al, 1997). A contaminant of the cleaning fluid contained in the Korean factory was 1,2dichloropropane (DCP). Thus, DCP was subsequently tested in female rats for reproductive toxicity (Sekiguchi et al., 2002). Because 1-bromopropane (1BP) is an isomer of 2BP, it was also tested in the same study. Adult female rats (F344) were exposed via inhalation daily for 8 hours for 3 weeks to 0, 50, 200, and l, 000ppm of either 1BP, 2BP or DCP. No effects of 1BP and 2BP on vaginal estrous cycles and ovulation were observed during the 3-week study. However DCP induced long estrous cycles and reduced the number of ova shed in a doserelated manner. Thus, it appears that DCP may be a more potent toxicant than 1BP and 2BP. These results are not inconsistent with reports of effects of 2BP after longer exposures (9 weeks) (Kamijima et al, 1997). SUMMARY (a) An ovarian ED is an exogenous chemical agent which alters the functioning of the ovaries. EDs include environmental toxicants, chemicals, drugs and hormones. EDs may act directly or indirectly in perturbing ovarian function. EDs acting directly may alter ovarian function by altering ovarian hormone receptors and signal transduction of numerous ovarian growth regulators such as gonadotropins, steroids and growth factors. Indirect actions of EDs

OVARIAN TOXICITY CAUSED BY ENDOCRINE DISRUPTORS 79

may alter secretion of gonadotropins and ovarian-related growth regulators emanating from other organs. (b) EDs can be classified as acting directly and/or indirectly on the ovary. However, EDs may also be classified into several categories based on their chemical structure, use and mode of entry into the environment. Examples of EDs discussed in detail in this chapter are TCDD, atrazine, I3C and 2bromopropane. (c) Ah receptor agonists disrupt the estrous cycle and block ovulation by postponing the LH surge by one or more days and by directly altering ovarian follicular responses to gonadotropins. Similar actions have been shown for furans, polychlorinated dibenzodioxins, PCBs and I3C. Reduction of ovarian FSH and LH receptors by TCDD may account for most of its direct actions on the ovary and its antiestrogenic action may account for the ability to block the LH surge. (d) Atrazine may block ovulation by increasing hypothalamic dopamine and decreasing norepinephrine which disrupts GnRH secretion and prevents the LH surge. (e) 2BP disrupts the menstrual cycle in women characterized by high serum concentrations of FSH, hot flashes and amenorrhea. In rodent models, 2BP appears to have a direct destructive action on the primordial follicles that eventually leads to alterations in the estrous cycle. In conclusion, EDs can exert their reproductive toxicity by means of dynamicsor kinetics, whichever process has the longer half-life. Destruction of primordialfollicles is an irreversible dynamic effect with an infinite half-life, because destroyedprimordial follicles cannot be replaced. For such compounds the kinetic half-lifeis entirely irrelevant. The consequence of this type of action is reduced reproductive life span. If the kinetic half-life of a compound is longer than the dynamichalf-life (e.g. effect on ovarian rupture by TCDD: 4–5 days versus 20 days),then this will drive the effect, and reproduction will be affected as long as theconcentration of the chemical is above the threshold dose for this particulareffect. This type of action is only dependent on the half-life of the compound. Suchchemicals will not affect reproductive life span even though they may inhibitovulation. REFERENCES Astroff, B. and Safe, S. (1988) Comparative antiestrogenic activities of 2,3,7,8tetrachlorodibenzo-p-dioxin and 6-methyl-1,3,8-trichlorodibenzofuran in the female rat, Toxicol. Appl. Pharmacol, 95:435–43. Astroff, B. and Safe, S. (1990) 2,3,7,8-Tetrachlordibenzo-p-dioxin as an antisestrogen: effect on rat uterine peroxidase activity, Biochem. Pharmacol., 39:485–88.

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Benedict, J., Lin, T., Loeffler, I., Peterson, R. and Flaws, J. (2000) Physiological role of the aryl hydrocarbon receptor in mouse ovary development, Toxicol. Sci., 56:382– 88. Bjeldanes, L., Kim, J., Grose, K., Bartholomew, J. and Bradfield, C. (1991) Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlordibenzo-p-dioxin, Proc. Natl. Acad. Sci., 88:9543–47. Bolon, B., Bucci, T., Warbritton, A., Chen, J., Mattison, D. and Heindel, J. (1997) Differential follicle counts as a screen for chemically induced ovarian toxicity in mice: results from continuous breeding bioassays, Fundam. Appl. Toxicol., 39:1–10. Bradlow, H., Michnovicz, J., Telang, N. and Osborne, M. (1991) Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice, Carcino-genesis, 12:1571–74. Butcher, R., Collins, W. and Fugo, N. (1975) Altered secretion of gonadotropins and steroids resulting from delayed ovulation in the rat, Endocrinology, 96:576–86. Cashman, J., Xiong, Y., Lin, H., Verhagen, H., van Poppel, G., van Bladeren, P., LarsenSu, S. and Williams, D. (1999) In vitro and in vivo inhibition of human flavincontaining monooxygenase form 3 (FMO3) in the presence of dietary indoles, Biochem. Pharmacol., 58:1047–55. Chaffin, C, Stouffer, R. and Duffy, D. (1999) Gonadotropin and steroid regulation of steroid receptor and aryl hydrocarbon receptor messenger ribonucleic acid in macaque granulosa cells during the periovulatory interval, Endocrinology, 140:4753– 60. Chaffin, C, Trewin, A. and Hutz, R. (2000) Estrous cycle-dependent changes in the expression of aromatic hydrocarbon receptor (AHR) and AHR-nuclear translocator (ARNT) mRNAs in the rat ovary and liver, Chem. Biol Interact., 124:205–16. Chen, I., Safe, S. and Bjeldanes, L. (1996) Indole-3-carbinol and diindolyl-methane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells, Biochem. Pharmacol., 51:1069–76. Connor, K., Howell, J., Chen, I., Kiu, H., Berhane, K., Sciarretta, C, Safe, S. and Zacharewski, T. (1996) Failure of cholor-S-traizine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro, Fundam. Appl. Toxicol., 30:93–101. Cooper, R., Stoker, T., Goldman, J., Parrish, M. and Tyrey, L. (1996) Effect of atrazine on ovarian function in the rat, Reprod. Toxicol., 10:257–64. Cooper, R., Stoker, T., McElroy, W. and Hein, J. (1998) Atrazine (ATR) disrupts hypothalamic catecholamines and pituitary function, The Toxicologist, 42:160. Cooper, R., Stoker, T., Tyrey, L., Goldman, J. and McElroy, W. (2000) Atrazine disrupts the hypothalamic control of pituitary-ovarian function, Toxicol. Sci., 53:297–307. Crisp, T., Clegg, E. and Cooper, R. (1997) Special report on environmental endocrine disruption: an effects assessment and analysis, EPA/630/R-96/012, www.epa.gov/ ORD/ WebPubs/endocrine/endocrine.pdf. Crisp, T., Clegg, E., Cooper, R., Wood, W., Anderson, D., Baetcke, K., Hoffmann, J., Morrow, M., Rodier, D., Schaeffer, J., Touart, L., Zeeman, M. and Patel, Y. (1998) Environmental endocrine disruption: an effects assessment and analysis, Environ. Health Perspect., 106 (Suppl 1): 11–56.

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Cummings, A., Metcalf, J. and Birnbaum, L. (1996) Promotion of endometriosis by 2,3,7, 8-tetrachlorodibenzo-p-dioxin in rats and mice: time-dose dependence and species comparison, Toxicol. Appl. Pharmacol., 138:131–39. Cutts, J. and Noble, R. (1964) Estrone-induced mammary tumors in the rat I. Inducation and behavior of tumors, Cancer Res., 24:1116–23. Das, P., McElroy, W. and Cooper, R. (2000) Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro, Toxicol. Sci., 56:324–31. Das, P., McElroy, W. and Cooper, R. (2001) Alteration of catecholamines in pheochromocytoma (PC12) cells in vitro by the metabolites of chlorotriazine herbicide, Toxicol. Sci., 59:127–37. Dasmahapatra, A., Wimpee, B., Trewin, A., Wimpee, C., Ghorai, J. and Hutz, R. (2000) Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptasepolymerase chain reaction assay, Mol. Cell Endocrinol., 164:5–18. DeKruif, C., Marsman, J., Venekamp, J., Falke, H., Noordhoek, J., Blaauboer B., and Wortelboer, H. (1991) Structure elucidation of acid reaction products of indole-3carbinol: detection in vivo and enzyme induction in vitro, Chem. Biol Interact., 80: 303–15. Drummond, A. and Findlay, J. (1999) The role of estrogen in folliculogenesis, Mol. Cell Endocrinol., 151:57–64. Eldridge, J., Fleenor-Heyser, D., Extrom, P., Wetzel, L., Breckenridge, C., Gillis, J., Luempert, L.G. and Stevens, J. (1994) Short-term effects of chlorotriazines on estrus in female Sprague-Dawley and Fischer 344 rats, J. Toxicol. Environ. Health, 43:155– 67. Enan, E., Lasley, B., Stewart, D., Overstreet, J. and Vandevoort, C. (1996a) 2, 3, 7, 8Tetrachlorodibenzo-p-dioxin (TCDD) modulates function of human luteinizing granulosa cells via cAMP signaling and early reduction of glucose transporting activity, Reprod. Toxicol., 10:191–98. Enan, E., Moran, F., VandeVoort, C., Stewart, D., Overstreet, J. and Lasley, B. (1996b) Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cultured human luteinized granulosa cells, Reprod. Toxicol., 10:497–508. Farookhi, R. and Desjardins, J. (1986) Luteinizing hormone receptor induction in dispersed granulosa cells requires estrogen, Mol. Cell Endocrinol., 47:13–24. Gao, X., Petroff, B., Rozman, K. and Terranova, P. (2000a) Gonadotropin-releasing hormone (GnRH) partially reverses the inhibitory effect of 2,3,7,8tetrachlorodibenzop-dioxin on ovulation in the immature gonadotropin-treated rat, Toxicology, 147:15–22. Gao, X., Terranova, P. and Rozman, K. (2000b) Effects of polychlorinated dibenzofurans, biphenyls, and their mixture with dibenzo-p-dioxins on ovulation in the gonadotropinprimed immature rat: support for the toxic equivalency concept, Toxicol. Appl. Pharmacol., 163:115–24. Gao, X., Mizuyachi, K., Terranova, P. and Rozman, K. (2001) 2,3,7,8Tetrachlorodibenzop-dioxin decreases responsiveness of the hypothalamus to estradiol as a feedback inducer of preovulatory gonadotropin secretion in the immature gonadotropin-primed rat, Toxicol. Appl. Pharmacol., 170:181–90.

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Gao, X., Petroff, B., Oluola, O., Georg, G., Terranova, P. and Rozman, K. (2002) Endocrine disruption by indole-3-carbinol and tamoxifen: blockage of ovulation, Toxicol. Appl. Pharmacol., 183:179–88. Generoso, W., Stout, S. and Huff, S. (1971) Effects of alkylating chemicals on reproductive capacity of adult female mice, Mutat. Res., 13:172–78. Gierthy, J., Bennett, J., Bradley, L. and Cutler, D. (1993) Correlation of in vitro and in vivo growth suppression of MCF-7 human breast cancer by 2,3,7,8-tetrachlordibenzopdioxin, Cancer Res., 53:3149–53. Gregoraszczuk, E. (2002) Dioxin exposure and porcine reproductive hormonal activity, Cad. Saude Publica, 18:453–62. Grubbs, C, Steele, V., Casebolt, T., Juliana, M., Eto, I., Whitaker, L., Dragnev, K., Kelloff, G. and Lubet, R. (1995) Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol, Anticancer Res., 15:709–16. Hays, L., Carpenter, C. and Petersen, S. (2002) Evidence that GABAergic neurons in the preoptic area of the rat brain are targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin during development, Environ. Health Perspect., 110 (Suppl 3):369–76. Heimler, I., Rawlins, R., Owen, H. and Hutz, R. (1998a) Dioxin perturbs, in a dose- and time-dependent fashion, steroid secretion, and induces apoptosis of human luteinized granulosa cells, Endocrinology, 139:4373–79. Heimler, I., Trewin, A., Chaffin, C, Rawlins, R. and Hutz, R. (1998b) Modulation of ovarian follicle maturation and effects on apoptotic cell death in Holtzman rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally, Reprod, Toxicol., 12:69–73. Hendry, W.J., Sheehan, D., Khan, S. and May, J. (2002) Developing a laboratory animal model for perinatal endocrine disruption: the hamster chronicles, Exp. Biol Med., 227:709–23. Hirakawa, T., Minegishi, T., Abe, K., Kishi, H., Ibuki, Y. and Miyamoto, K. (2000a) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of luteinizing hormone receptors during cell differentiation in cultured granulosa cells, Arch. Biochem. Biophys., 375:371–76. Hirakawa, T., Minegishi, T., Abe, K., Kishi, H., Inoue, K., Ibuki, Y. and Miyamoto, K. (2000b) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of folliclestimulating hormone receptors during cell differentiation in cultured granulosa cells, Endocrinology, 141:1470–76. Hirshfield, A. (1994) Relationship between the supply of primordial follicles and the onset of follicle growth in rats, Biol Reprod., 50:421–28. Hooser, S., Douds, D., DeMerell, D., Hoyer, P. and Sipes, I. (1994) Long-term ovarian and gonadotropin changes in mice exposed to 4-vinylcyclohexene, Reprod. Toxicol., 8:315–23. Hoyer, P. (2001) Reproductive toxicology: current and future directions, Biochem. Pharmacol., 62:1557–64. Hoyer, P., Devine, P., Hu, X., Thompson, K. and Sipes, I. (2001) Ovarian toxicity of 4vinylcyclohexene diepoxide: a mechanistic model, Toxicol. Pathol., 21:91–99. Huang, P., Rannug, A., Ahlbom, E., Hakansson, H. and Ceccatelli, S. (2000) Effect of 2,3, 7,8-tetrachlorodibenzo-p-dioxin On the expression of cytochrome P450 1A1, the aryl hydrocarbon receptor, and the aryl hydrocarbon receptor nuclear translocator in rat brain and pituitary, Toxicol. Appl. Pharmacol., 169:159–67.

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Jellnick, P., Forkert, P., Riddick, D., Okey, A., Michnovicz, J. and Bradlow, H. (1993) Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation, Biochem. Pharmacol., 45:1129–36. Kainu, T., Gustafsson, J. and Pelto-Huikko, M. (1995) The dioxin receptor and its nuclear translocator (Arnt) in the rat brain, Neuroreport, 6:2557–60. Kamijima, M., Ichihara, G., Kitoh, J., Tsukamura, H., Maeda, K., Yu, X., Xie, Z., Nakajima, T., Asaeda, N., Hisanaga, N. and Takeuchi, Y. (1997) Ovarian toxicity of 2-bromopropane in the non-pregnant female rat, J. Occup. Health, 39:144–49. Kharat, I. and Saatcioglus, F. (1996) Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzopdioxin are mediated by direct transcriptional interference with the liganded estrogen receptor, J. Biol Chem., 271:10533–37. Kim, H., Chung, Y., Yi, K., Kim, J. and Yu, I. (1996a) LC50 of 2-bromopropane, Ind. Health, 34:403–07. Kim, Y., Jung, K., Hwang, T., Jung, G., Kim, J., Park, J., Park, D., Park, S., Choi, K. and Moon, Y. (1996b) Hematopoietic and reproductive hazards of Korean electronic workers exposed to solvents containing 2-bromopropane, Scand. J. Work. Environ. Health, 22:387–91. Kociba, R., Keyes, D., Beyer, J., Carreon, R., Wade, C, Dittenber, D., Kalnins, R., Frauson, L., Park, C., Barnard, S., Hummel, R. and Humiston, C. (1978) Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-pdioxin in rats, Toxicol. Appl. Pharmacol., 46:279–303. Koh, J., Kim, C., Hong, S. Lee, K., Kim, Y., Kim, O. and Kim, G. (1998) Primary ovarian failure caused by a solvent containing 2-bromopropane, Eur. J. Endocrinol., 36:297– 99. Laws, S., Ferrell, J., Stoker, T., Schmid, J. and Cooper, R. (2000) The effects of atrazine on female Wistar rats: an evaluation of the protocol for assessing pubertal development and thyroid function, Toxicol. Sci., 58:366–76. Li, X., Johnson, D. and Rozman, K. (1995a) Effects of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) on estrous cyclicity and ovulation in female Sprague-Dawley rats, Toxicol. Lett., 78:219–22. Li, X., Johnson, D. and Rozman, K. (1995b) Reproductive effects of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation, hormonal regulation, and possible mechanism(s), Toxicol. Appl. Pharmacol., 133:321–27. Li, X., Weber, L. and Rozman, K. (1995c) Toxicokinetics of 2,3,7,8-tetrachlorodibenzopdioxin in female Sprague-Dawley rats including placental and lactational transfer to fetuses and neonates, Fundam. Appl Toxicol., 27:70–76. Liu, H., Wormke, M., Safe, S. and Bjeldanes, L. (1994) A dietary-derived factor that exhibits both antiestrogenic and estrogenic activity, J. Natl. Cancer Inst., 86:1758– 65. Maeng, S. and Yu, I. (1997) Mutagenicity of 2-bromopropane, Ind. Health, 35:87–95. Matikainen, T., Perez, G., Jurisicova, A., Pru, J., Schlezinger, J., Ryu, H., Laine, J., Sakai, T., Korsmeyer, S., Casper, R., Sherr, D. and Tilly, J. (2001) Aromatic hydrocarbon receptordriven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals, Nat. Genet., 28:355–60. Mattison, D. and Nightingale, M. (1980) The biochemical and genetic characteristics of murine ovarian aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity and its relationship to primordial oocyte destruction by polycyclic aromatic hydrocarbons, Toxicol. Appl Pharmacol., 56:399–408.

84 PAUL F.TERRANOVA AND KARL K.ROZMAN

Mattison, D. and Schulman, J. (1980) How xenobiotic compounds can destroy oocytes, Contemp. Obstet. Gynecol., 15:157–69. Mizuyachi, K., Son, D., Rozman, K. and Terranova, P. (2002) Alteration in ovarian gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin: reduction of cyclooxygenase-2 in the blockage of ovulation, Reprod. Toxicol., 16:299–307. Park, J., Kim, Y., Park, D., Choi, K., Park, S. and Moon, Y. (1997) An outbreak of hematopoietic and reproductive disorders due to solvents containing 2-bromopropane in an electronic factory, South Korea: epidemiological survey, J. Occup. Health, 39: 138–43. Petersen, S., Curran, M., Marconi, S., Carpenter, C., Lubbers, L. and McAbee, M. (2000) Distribution of mRNAs encoding the aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator, and aryl hydrocarbon receptor nuclear translocator-2 in the rat brain and brainstem, J. Comp. Neurol., 427:428–39. Petroff, B., Gao, X., Rozman, K. and Terranova, P. (2000) Interaction of estradiol and 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an ovulation model: evidence for systemic potentiation and local ovarian effects, Reprod. Toxicol., 14:247–55. Petroff, B., Roby, K., Gao, X., Son, D., Williams, S., Johnson, D., Rozman, K. and Terranova, P. (2001) A review of mechanisms controlling ovulation with implications for the anovulatory effects of polychlorinated dibenzo-p-dioxins in rodents, Toxicology, 158:91–107. Plowchalk, D., Smith, B. and Mattison, D. (1993) Assessment of toxicity to the ovary using follicle quantitation and morphometrics, Female Reproductive Toxicology, R.C.A.J. Heindel. San Diego: Academic Press, pp. 57–68. Riby, J., Feng, C., Chang, Y., Schaldach, C, Firestone, G. and Bjeldanes, L. (2000) The major cyclic trimeric product of indole-3-carbinol is a strong agonist of the estrogen receptor signaling pathway, Biochemistry, 39:910–18. Richards, J. (1980) Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation, Physiol Rev., 60:51–89. Richards, J., Jahnsen, T., Hedin, L., Lifka, J., Ratoosh, S., Durica, J. and Goldring, N. (1987) Ovarian follicular development: from physiology to molecular biology, Recent Prog. Horm. Res., 43:231–70. Robker, R. and Richards, J. (1998) Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip-1, Mol. Endocrinol. 12:924–40. Roby, K. (2001) Alterations in follicle development, steroidogenesis, and gonadotropin receptor binding in a model of ovulatory blockade, Endocrinology, 142:2328–35. Romkes, M. and Safe, S. (1988) Comparative activities of 2,3,7,8-tetrachlorodibenzo-pdioxin and progesterone as antiestrogens in the female rat uterus, Toxicol. Appl Pharmacol, 92:368–80. Safe, S. (1995) Modulation of gene expression and endocrine response pathways by 2,3,7, 8-tetrachlorodibenzo-p-dioxin and related compounds, Pharmacol Ther. 67:247–81. Sekiguchi, S. and Honma, T. (1998) Influence of 2-bromopropane on reproductive system 2-bromopropane inhibits forced ovulation in mice, Ind. Health, 36:297–99. Sekiguchi, S., Suda, M, Zhai, Y. and Honma, T. (2002) Effects of 1-bromopropane, 2bromopropane, and 1,2-dichloropropane on the estrous cycle and ovulation in F344 rats, Toxicol. Lett., 126:41–49. Smith, B., Mattison, D. and Sipes, I. (1990) The role of epoxidation in 4vinylcyclohexene ovarian toxicity, Toxicol. Appl. Pharmacol., 105:372–81.

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Son, D., Ushinohama, K., Gao, X., Taylor, C., Roby, K., Rozman, K. and Terranova, P. (1999) 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, Reprod. Toxicol., 13:521–30. Stevens, J., Breckenridge, C, Wetzel, L., Gillis, J., Luempert, L.G. and Eldridge, J. (1994) Hypothesis for mammary tumorigenesis in Sprague-Dawley rats exposed to certain triazine herbicides, /. Toxicol. Environ. Health, 43:139–53. Stoewsand, G., Anderson, J. and Munson, L. (1988) Protective effect of dietary Brussels sprouts against mammary carcinogenesis in Sprague-Dawley rats, Cancer Lett., 39: 199–207. Takizawa, K., Yagi, H., Jerina, D. and Mattison, D. (1984) Murine strain differences in ovotoxicity following intraovarian injection in benzo(a)pyrene, (+)-(7R, 8S)-oxide, (-)-(7R,8S)-dihydrodiol, or (+)-(7R, 8S)-diol-(9S, 10R)-epoxide-2, Cancer Res., 44: 2571–76. Tennant, M., Hill, D., Eldridge, J., Wetzel, L., Breckenridge, C. and Stevens, J. (1994) Chloro-S-triazine antagonism of estrogen action: limited interaction with estrogen receptor binding, J. Toxicol. Environ. Health, 43:197–211. Terranova, P. and Ascanio, L. (1982) Alterations of ovarian steroidogenesis induced by ovulatory delay in immature rats treated with pregnant mare serum gonadotropin, Biol. Reprod., 26:129–39. Theobald, H. and Peterson, R. (1997) In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-rho-dioxin: effects on development of the male and female reproductive system of the mouse, Toxicol. Appl. Pharmacol., 145:124–35. Tian, Y., Ke, S., Thomas, T., Meeker, R. and Gallo, M. (1998a) Regulation of estrogen receptor mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin as measured by competitive RT-PCR, /. Biochem. Mol. Toxicol., 12:71–77. Tian, Y., Ke, S., Thomas, T., Meeker, R. and Gallo, M. (1998b) Transcriptional suppression of estrogen receptor gene expression by 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), /. Steroid Biochem. Mol. Toxicol., 67:17–24. Tonetta, S. and diZerega, G. (1989) Intragonadal regulation of follicular maturation, Endocr. Rev., 10:205–29. Ushinohama, K., Son, D., Roby, K., Rozman, K. and Terranova, P. (2001) Impaired ovulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature rats treated with equine chorionic gonadotropin, Reprod. Toxicol., 15:275–80. Vang, O., Jensen, M. and Autrup, H. (1990) Induction of cytochrome P4501A1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone, Carcinogenesis, 11:1259– 263. Wang, X.-N. and Greenwald, G. (1993) Hypophysectomy of the cyclic mouse: I. Effects on follicular genesis, oocyte growth, and follicle-stimulating hormone and human chorionic gonadotropin receptors, Biol. Reprod., 48:585–94. Wattenberg, L. and Loub, W. (1978) Inhibition of polycyclic aromatic hydnrcarboninduced neoplasia by naturally occurring indoles, Cancer Res., 38:1410–13. Wetzel, L., Luempert, L.G., Breckenridge, C, Tisdel, M, Stevens, J., Thakur, A., Extrom, P. and Eldridge, J. (1994) Chronic effects of atrazine on estrus and mammary tumor formation in female Sprague-Dawley and Fischer 344 rats, J. Toxicol. Environ. Health, 43:169–82.

86 PAUL F.TERRANOVA AND KARL K.ROZMAN

Wortelboer, H., DeKruif, C, van Iersel, A., Falke, H., Noordhoek, J. and Blaauboer, B. (1992) Acid reaction products of indole-3-carbinol and their effects on cytochrome P450 and phase II enzymes in rat and monkey hepatocytes, Biochem. Pharmacol., 43: 1439–47. Yu, X., Kamijima, M., Ichihara, G., Li, W., Kitoh, J., Xie, Z., Shibata, E., Hisanaga, N. and Takeuchi, Y. (1999) 2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats, Toxicol. Appl. Pharmacol., 159: 185–93.

5 PHTHALATE TOXICITY IN THE OVARY Friederike C.L.Jayes, Tara Lovekamp-Swan and Barbara J.Davis

INTRODUCTION Phthalates are found in polyvinyl chloride (PVC) piping and numerous consumer products such as perfumes, cosmetics, nail polish, insect repellents, detergents, and PVC-plastics (flexible PVC) including soft squeeze children’s toys, vinyl shower curtains, medical tubing, and gloves. In these products, they are most commonly used as plasticizers and solvents, and they easily leach into the environment over the lifetime of the products. Globally, over 18 billion pounds of phthalates are used each year (Blount et al., 2000), and the production volume of di-(2-ethylhexyl) phthalate (DEHP) alone in 1999 was estimated to be 2 million tons (cited in CERHR, 2000). Considering the high production volumes and the type of products involved, women have a unique exposure profile to phthalates and are exposed to these compounds through ingestion, inhalation, and dermal exposure on a daily basis. Research efforts in our laboratory examine how phthalates affect the female reproductive system in animal models to provide insights into the potential health effects of these chemicals in women. DEHP and its active metabolite mono-(2ethylhexyl) phthalate (MEHP) are classified as female and male reproductive toxicants and carcinogens in rodent studies, and are considered a potential hazard to human reproductive health (CERHR, 2000). Here we review our work and the work of others studying the mechanism for the effect of DEHP/MEHP in the ovary. OVARY AS A TARGET Our previous work demonstrated that the target organ of DEHP in the female reproductive tract is the ovary, and that the toxicity of DEHP manifests as a failure to increase serum estradiol to a level that is sufficient to trigger ovulation in adult, cycling rats (Davis et al, 1994a). During the natural cycle, ovarian follicles are stimulated by follicle-stimulating hormone (FSH) to grow into mature, antral (“Graafian”) follicles, at which time they secrete increasing amounts of estradiol

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Figure 5.1 Development of ovarian follicles and underlying serum hormone profiles in adult, cycling rats. Top panel: During the normal estrous cycle, follicles are recruited (RF) by FSH, and grow to become antral (AF) and Graafian follicles (GF). The granulosa cells in these growing follicles are the main source of estrogen production. This increase in estrogen triggers the release of LH and FSH surges from the pituitary, which induce ovulation of the mature Graafian follicles, and recruitment of follicles for the next cycle. The ovulated follicle forms the progesterone secreting corpus luteum (CL), which will regress if the animal does not become pregnant. Bottom panel: If DEHP is fed during the phase of follicular growth, granulosa cells fail to acquire the ability to produce estradiol and the pituitary will not release a surge of LH, resulting in anovulation and cystic follicles (CF) instead of corpora lutea.

to trigger a surge of luteinizing hormone (LH) for ovulation to ensue. Because DEHP suppresses the rise in estradiol, no LH surge occurs, there are no ovulations, the cycle is prolonged and follicles become cystic (Figure 5.1). However, ovulation can be induced after exogenous LH is administered, and the granulosa cells are able to differentiate into luteal cells (Davis et al., 1994a).

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MECHANISMS OF TOXICITY To determine the molecular mechanism by which DEHP suppresses estradiol, we examined the effects of MEHP (the active metabolite of DEHP) in primary cultures of rat granulosa cells (Davis et al., 1994b). This is a useful system to study hormone production in vitro since these cells respond to FSH by inducing steroidogenic enzymes and granulosa cell differentiation (Erickson, 1983). The hormonal effects of FSH in primary granulosa cell cultures have striking similarities to normal biochemical events in vivo (Erickson, 1983; Hsueh et al, 1984). In this system, MEHP (50–100 µM) inhibited FSH-stimulated cAMP and progesterone production (Treinen et al., 1990). The decrease in progesterone was prevented by providing cAMP analogues or pregnenolone, the precursor of progesterone. In contrast, MEHP inhibited FSH-stimulated estradiol production even in the presence of its precursor testosterone, and stimulation with the nondegradable analogue of cAMP, 8-br-cAMP, did not prevent the inhibitory effect of MEHP (Davis et al, 1994b). Therefore, the effect of MEHP on estradiol is independent from the effect on FSH-stimulated cAMP and progesterone production in the granulosa cell. As the rat granulosa cell is devoid of the 17αhydroxylase/17,20-lyase enzyme complex and requires an outside source of androgens to produce estradiol, the post-cAMP effect of MEHP on estradiol production involves the aromatase enzyme itself. Indeed, MEHP decreased the maximum velocity of aromatase without acting as an enzyme inhibitor (Davis et al., 1994b). Thus, MEHP alters the levels or availability of aromatase in the granulosa cell either by decreasing synthesis or increasing degradation of the enzyme. More recently we determined that MEHP decreased aromatase mRNA levels, which correlated with decreased protein levels in a dose-dependent manner in rat granulosa cells (Lovekamp and Davis, 2001). This MEHP effect on aromatase gene expression appears to be fairly specific because MEHP did not alter basal transcript levels of the cholesterol side-chain cleavage enzyme (P450scc) in the granulosa cell. Further, 8-br-cAMP treatment did not override MEHP suppression of aromatase, but P450scc mRNAwas very sensitive to stimulation by 8-br-cAMP even in the presence of MEHP (Lovekamp and Davis, 2001). These observations further support our hypothesis that MEHP suppresses aromatase and estradiol independent of FSH-stimulated cAMP. ROLE OF PEROXISOME PROLIFERATOR ACTIVATED RECEPTORS (PPARs) We queried how MEHP affects aromatase gene transcription. DEHP is a known peroxisome proliferator and liver carcinogen, and others showed that MEHP alters gene expression by activating PPARs. PPARs are ligand activated transcription factors and key regulators of lipid metabolism and cell differentiation (Maloney and Waxman, 1999). The liver toxicity of DEHP is

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dependent on PPARα, as mice lacking this receptor did not develop liver tumors or induce fatty acid metabolizing enzymes in response to DEHP (Macdonald et al., 2001; Ward et al., 1998). MEHP activated PPARα in cell transactivation assays (Corton et al, 2000; Hasmall et al, 2000) and increased PPARα-mediated gene expression in the liver (Fan et al, 1998; Macdonald et al, 2001). However, MEHP can work through pathways other than PPARα in some tissues, because testicular toxicity was maintained in PPARα null mice treated with DEHP (Ward et al, 1998). Indeed, MEHP activation of PPARγ in transactivation assays (Maloney and Waxman, 1999) suggests that this isoform of PPAR may be responsible for DEHP-mediated toxicity in tissues other than the liver (Maloney and Waxman, 1999; Ward et al., 1998). Therefore, we set out to investigate whether the toxicity of MEHP in granulosa cells is mediated through the PPARs. All three isoforms of PPAR are expressed in the rat ovary (Braissant et al., 1996), and we confirmed their presence in our cultured rat granulosa cells by RTPCR. Others showed that PPARγ was highly expressed in pre-ovulatory granulosa cells and was downregulated after ovulation, which suggests it may be involved in the differentiation of estrogen-producing granulosa cells to progesterone-producing luteal cells (Komar et al, 2001; Lohrke et al., 1998). PPARγ ligands such as troglitazone and 15-deoxy prostaglandin J2 (15d-PGJ2) inhibited aromatase expression level and activity in human breast adipose stromal cells (Rubin et al., 2000). The PPARγ ligand troglitazone inhibited aromatase activity and mRNA levels in human ovarian granulosa cells (Mu et al., 2000). Combined treatment with both troglitazone and a retinoic X receptor (RXR) ligand resulted in a synergistic decrease of aromatase, suggesting that this effect is mediated by the PPARγ:RXR heterodimer (Mu et al, 2000). Based on these data, we hypothesized that MEHP decreases aromatase through activation of PPARα and PPARγ in the rat granulosa cell. To address this hypothesis, we compared the effects of MEHP (50µM), the PPARα-specific ligand GW 327647 (2 µM) and the PPARγ ligands troglitazone (1 µM), 15d-PGJ2 (1 µM) and GW 347845 (2 µM) in rat ovarian granulosa cells (Lovekamp-Swan et al, 2003). Similar to MEHP, these PPARα and PPARγ agonists all decreased estradiol production and aromatase RNA message levels to 50–70% of controls (Figure 5.2). The involvement of both PPAR subtypes (α and γ) in the suppression of ovarian aromatase by MEHP was further illustrated employing the PPARγ-selective antagonist, GR 259662 (2µM) and the PPARαspecific endpoint 17β-HSD IV mRNA. This PPARγ antagonist, partially blocked the effect of MEHP on aromatase mRNA, completely blocked the effect of the PPARγ-selective agonist GW 347845 and did not alter the effect of the PPARαspecific ligand GW 327647 (2 µM) on aromatase mRNA (Figure 5.3). Furthermore, the PPARγ antagonist by itself did not alter 17β-HSD IV mRNA and did not attenuate the MEHP induced, PPARα-mediated increase in 17β-HSD IV mRNA (Figure 5.4). These observations lead to the conclusion that MEHP affects granulosa cell aromatase through PPARγ- and PPARα-specific pathways (Lovekamp-Swan et al, 2003).

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Figure 5.2 MEHP and PPARs reduce aromatase mRNA expression in cultured rat granulosa cells. Granulosa cells were isolated from immature PMSG-primed rats and cultured in testosterone and FSH-containing medium in the presence of different PPAR activators (see text). Aromatase mRNA was measured using real time RT-PCR. * Indicates p<0.05 compared to control (DMSO) (Lovekamp-Swan et al., 2003, with permission from Elsevier).

EFFECTS ON GENE EXPRESSION Given that MEHP mediates changes through PPARs and that PPARs are involved in differentiation and metabolism, we hypothesized that other genes involved in toxicant metabolism may be under the control of PPAR activation in the granulosa cell. We tested this in cultured granulosa cells treated with MEHP or PPAR-specific agonists and antagonists (Lovekamp-Swan et al., 2003). RTPCR determined that AhR (aryl hydrocarbon receptor), CYP1B1, epoxide hydrolase, and 17β-HSD IV, genes which are generally thought of as involved in xenobiotic activation and metabolism (Murray et al., 2001), were all significantly increased in the granulosa cell by 50 µM MEHP. Heart-fatty-acid-binding protein (H-FABP) was also induced by MEHP. Cholesterol side chain cleavage enzyme (P450scc) was not changed by MEHP, consistent with previously published studies in vitro (Lovekamp and Davis, 2001). The PPARα-selective ligand GW 327647 significantly increased mRNA for the same five genes and

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Figure 5.3 The PPARγ antagonist GR 259662 attenuates the MEHP-induced reduction of aromatase mRNA in cultured rat granulosa cells. Cells were cultured for 48 hours with MEHP (50 µM), the PPARγ ligand GW 347845 (2 µM), or the PPARα-specific ligand GW 327647 (2 (µM) alone or in combination with the PPARγ-selective antagonist GR 259662 (2µM). mRNA was quantified by real time RT-PCR. * Indicates p<0.05 compared to control (DMSO). ** Indicates p < 0.05 compared to MEHP alone (Lovekamp-Swan et al, 2003, with permission from Elsevier).

also did not alter P450scc mRNA. The PPARγ-selective ligand GW 347845 significantly increased only H-FABP. These studies demonstrate that MEHP has both PPARγ- and PPARα-mediated effects on gene expression in the granulosa cell, resulting in altered metabolism and differentiation. Only PPARα activation induced estradiol metabolizing enzymes such as 17β-HSD IV, which metabolizes estradiol to the less active estrone; CYP1B1, which metabolizes 17β-estradiol to 4-hydroxyestradiol (Murray et al., 2001), and epoxide hydrolase, which appears indirectly involved in estrogen production in human luteal cells (Hattori et al, 2000). Induction of these enzymes likely contributes to further decreases in estradiol levels and increases estrone levels produced by granulosa cells in vitro and in vivo. Notably, activation of either pathway (α or γ) suppressed aromatase mRNA and

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Figure 5.4 The PPARγ antagonist GR 259662 does not block the MEHP-induced increase in 17β-HSD IV mRNA in cultured rat granulosa cells. * Indicates p<0.05 compared to control (DMSO) (Lovekamp-Swan et al., 2003, with permission from Elsevier).

estradiol levels, and induced H-FABP, a protein that is altered after hCG induction of ovulation (Leo et al., 2001). Because altered gene expression and changes in metabolism are crucial in reprogramming the granulosa cell during differentiation, PPARα may be as important as PPARγ in this process. Our observations suggest that PPARα as well as PPARγ function as regulators of metabolism and differentiation in the granulosa cell, and MEHP toxicity is mediated through changes in gene expression affecting steroidogenesis, steroid metabolism and prematurely starting the process of differentiation through these pathways. SUMMARY The phthalate DEHP reduces ovarian production of estradiol through activation and suppression of key genes, most importantly aromatase. MEHP, its active metabolite, employs both PPARα and PPARγ pathways to regulate genes in

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granulosa cell differentiation and metabolism, and activation of the two PPAR pathways has both redundant and isoform-specific effects on gene expression in the granulosa cell. We suggest that activation of PPAR pathways may be the trigger that suppresses aromatase at ovulation under normal physiological conditions. By activating PPAR pathways before sufficient estradiol has been produced to trigger the LH surge, MEHP alters the programming of granulosa cell differentiation. Additionally, the induction of AhR and CYP1B1 transcripts by PPARα agonists, including MEHP, is a novel finding and leads to new hypotheses about the regulation and function of these genes in the ovary. These studies characterize a molecular mechanism by which DEHP, a ubiquitous environmental contaminant, causes female reproductive toxicity, and provide a model to understand actions of female reproductive toxicants and a foundation for understanding human health effects. REFERENCES Blount, B.C., Milgram, K.E., Silva, M.J., Malek, N.A., Reidy, J.A., Needham, L.L. and Brock, J.W. (2000) Quantitative detection of eight phthalate metabolites in human urine using HPLC-APCI-MS/MS, Anal. Chem., 72:4127–34. Braissant, O., Foufelle, F., Scotto, C, Dauca, M. and Wahli, W. (1996) Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat, Endocrinology, 137: 354–66. CERHR (Center for the Evaluation of Risks to Human Reproduction) (2000) NTPCERHR Expert Panel Report on Di (2~ethylhexyl) Phthalate, Alexandria, VA: Science International, Inc. Corton, J.C., Lapinskas, P.J. and Gonzalez, F.J. (2000) Central role of PPARalpha in the mechanism of action of hepatocarcinogenic peroxisome proliferators, Mutat. Res., 448:139–51. Davis, B.J., Maronpot, R.R. and Heindel, J.J. (1994a) Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats, Toxicol. Appl. Pharmacol., 128: 216–23. Davis, B.J., Weaver, R., Gaines, L.J. and Heindel, J.J. (1994b) Mono-(2-ethylhexyl) phthalate suppresses estradiol production independent of FSH-cAMP stimulation in rat granulosa cells, Toxicol. Appl Pharmacol, 128:224–28. Erickson, G.F. (1983) Primary cultures of ovarian cells in serum-free medium as models of hormone-dependent differentiation, Mol. Cell Endocrinol., 29:21–49. Fan, L.Q., Cattley, R.C. and Corton, J.C. (1998) Tissue-specific induction of 17 betahydroxysteroid dehydrogenase type IV by peroxisome proliferator chemicals is dependent on the peroxisome proliferator-activated receptor alpha, J. Endocrinol., 158:237–46. Hasmall, S.C., James, N.H., Macdonald, N., Soames, A.R. and Roberts, R.A. (2000) Species differences in response to diethylhexylphthalate: suppression of apoptosis, induction of DNA synthesis and peroxisome proliferator activated receptor alpha-mediated gene expression, Arch. Toxicol., 74:85–91.

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Hattori, N., Fujiwara, H., Maeda, M., Fujii, S. and Ueda, M. (2000) Epoxide hydrolase affects estrogen production in the human ovary, Endocrinology, 141:3353–65. Hsueh, A.J., Adashi, E.Y., Jones, P.B. and Welsh, T.H., Jr (1984) Hormonal regulation of the differentiation of cultured ovarian granulosa cells, Endocr. Rev., 5:76–127. Komar, C.M., Braissant, O., Wahli, W. and Curry, T.E. Jr (2001) Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period, Endocrinology, 142:4831–38. Leo, C.P., Pisarska, M.D. and Hsueh, A.J. (2001) DNA array analysis of changes in preovulatory gene expression in the rat ovary, Biol Reprod., 65:269–76. Lohrke, B., Viergutz, T., Shahi, S.K., Pohland, R., Wollenhaupt, K., Goldammer, T., Walzel, H. and Kanitz, W. (1998) Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells, J. Endocrinol., 159:429–39. Lovekamp, T.N. and Davis, B.J. (2001) Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells, Toxicol. Appl. Pharmacol., 172:217–24. Lovekamp-Swan, T., Jetton, A. and Davis, B.J. (2003) Dual activation of PPARα and PPARγ by mono- (2-ethylhexyl) phthalate alters aromatase, estradiol and differentiation in ovarian granulosa cells, Mol. Cell. Endocrinol., 201:133–41. Macdonald, N., Chevalier, S., Tonge, R., Davison, M., Rowlinson, R., Young, J., Rayner, S. and Roberts, R. (2001) Quantitative proteomic analysis of mouse liver response to the peroxisome proliferator diethylhexylphthalate (DEHP), Arch. Toxicol., 75:415– 24. Maloney, E.K. and Waxman, D.J. (1999) trans-Activation of PPARalpha and PPARgamma by structurally diverse environmental chemicals, Toxicol. Appl. Pharmacol., 161: 209–18. Mu, Y.M., Yanase, T., Nishi, Y., Waseda, N., Oda, T., Tanaka, A., Takayanagi, R. and Nawata, H. (2000) Insulin sensitizer, troglitazone, directly inhibits aromatase activity in human ovarian granulosa cells, Biochem. Biophys. Res. Commun., 271:710–13. Murray, G.I., Melvin, W.T., Greenlee, W.F. and Burke, M.D. (2001) Regulation, function, and tissue-specific expression of cytochrome P450 CYP1B1, Annu. Rev. Pharmacol. Toxicol., 41:297–316. Rubin, G.L., Zhao, Y., Kalus, A.M. and Simpson, E.R. (2000) Peroxisome proliferatoractivated receptor gamma ligands inhibit estrogen biosynthesis in human breast adipose tissue: possible implications for breast cancer therapy, Cancer Res., 60:1604–08. Treinen, K.A., Dodson, W.C. and Heindel, J.J. (1990) Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono (2-ethylhexyl) phthalate in rat granulosa cell cultures, Toxicol. Appl. Pharmacol., 106:334–40. Ward, J.M., Peters, J.M., Perella, C.M. and Gonzalez, F.J. (1998) Receptor and nonreceptormediated organ-specific toxicity of di(2-ethylhexyl)phthalate (DEHP) in peroxisome proliferator-activated receptor alpha-null mice, Toxicol. Pathol., 26:240– 46.

6 HORMONAL CONTROL OF OVARIAN FUNCTION FOLLOWING CHLOROTRIAZINE EXPOSURE: EFFECT ON REPRODUCTIVE FUNCTION AND MAMMARY GLAND TUMOR DEVELOPMENT Ralph L.Cooper, Susan C.Laws, Michael G.Narotsky, Jerome M.Goldman and Tammy E.Stoker INTRODUCTION Atrazine was introduced as a broad-spectrum herbicide in the 1950s to control annual grasses and broadleaf weeds (Eldridge et al., 1994). This herbicide remains one of the most widely used agricultural products worldwide (Gressel et al., 1984; Stevens and Sumner, 1991). The total annual use of atrazine within the United States is approximately 76 million pounds of active ingredient. Crops with the highest percentage of acres treated are field corn (75 percent), sugarcane (76 percent), sorghum (59 percent) and sweet corn (50–58 percent). In terms of pounds applied, corn (86 percent), sorghum (10 percent) and sugarcane (3 percent) account for the greatest use. Less than 1 percent of atrazine applied is for forestry, turf and other uses. In plants, the principal mode of action (MOA) of chlorotriazines is to inhibit photosynthesis by preventing electron transfer at the reducing site of photosynthesis complex II in the chloroplasts (Gysin and Knuesli, 1960). In this chapter, the evidence that atrazine is a reproductive toxicant in the female rat will be reviewed by first examining the data that support a correlation between mammary gland tumor development and atrazine exposure. These are the data that raised concern about potential human health effects. Next, the hypothesized neuroendocrine MOA through which atrazine induces mammary tumors will be discussed. Finally we will present an overview of the work demonstrating that atrazine, as well as several other chlorotriazines and related metabolites, can alter the neuroendocrine control of the ovary. BACKGROUND: ATRAZINE LEADS TO PREMATURE MAMMARY TUMORS IN THE FEMALE RAT It is interesting that when atrazine and several related chlorotriazines (simazine, cyanazine, propazine, etc.) were examined for their potential reproductive effects using standard risk assessment protocols or guideline studies, they were reported

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to be negative. Giknis (1989) reported that atrazine at doses of 0, 1, 5 and 75 mg/ kg/day was not teratogenic. Early studies investigating the potential reproductive and developmental effects of atrazine did identify reduced ossification in the offspring of dams dosed from gestational day (GD) 6–15 (100mg/kg/day or greater); however, no other effects were noted. In a two-generation reproductive study, rats were administered atrazine in the diet at concentrations ranging from 0 to 500 ppm. Although reduced body weights at weaning were reported in the F1 offspring receiving 500 ppm atrazine (approximately 28–30 mg/kg/day), all other parameters measured were negative suggesting that atrazine posed no reproductive hazard. It should be emphasized that these studies did not include an evaluation of the more sensitive endpoints (i.e. onset of puberty, vaginal cyclicity, etc.) indicative of endocrine disruption that were recently added to the standard tests (Cooper and Goldman, 1999). Furthermore, as ovarian cyclicity in the adult through middle-aged female was not examined, the now wellestablished effect of atrazine on reproductive aging was not detected. A number of studies have evaluated the potential of atrazine and other chlorotriazines to induce tumors, either benign or malignant, in rats and mice (Simpkins et al., 1998). The majority of these indicate that atrazine is not mutagenic or genotoxic (Hauswirth and Wetzel, 1998). However, it was reported that dietary exposure to atrazine (400 ppm, approximately 22.5 mg/kg/day) or the related triazine, simazine, led to an earlier onset and greater incidence of mammary tumors in Sprague-Dawley (SD) rats (Stevens et al, 1994; Wetzel et al, 1994). In light of these observations, atrazine, simazine and cyanazine were considered by the US EPA to be “possible” or category “C" carcinogens (US EPA, 2002, for review). Because atrazine was neither mutagenic nor genotoxic, it was initially hypothesized that the tumors were developing because these compounds were estrogenic (Stevens et al., 1994; Wetzel et al., 1994). However, atrazine does not appear to possess estrogenic properties. Atrazine does not bind to the estrogen receptor (Tennant et al., 1994; Conner et al., 1996) nor does it induce uterine growth when injected into ovariectomized female rats (Cooper et al., 1995). In fact, current data on this compound indicate that it possesses some antiestrogenic properties. For example, estrogens consistently lead to increased prolactin synthesis and release, whereas more recent studies clearly show that atrazine has an opposite effect on prolactin (Stoker et al, 1999; Cooper et al., 2000). Also, atrazine attenuates the estrogen-induced uptake of tritiated thymidine in the uterus and at higher doses reduces the estrogen-induced increase in uterine weight (Tennant et al., 1994). Since neither atrazine nor simazine appeared to be mutagenic or genotoxic and since there was no evidence that this class of herbicides were estrogenic, it was proposed that chlorotriazine administration promotes mammary tumor development by inducing a premature ovarian senescence and thus creates the endocrine milieu conducive to tumor growth (Stevens et al, 1994; Eldridge et al, 1998). Several recent studies support this hypothesis and as a result have shifted the classification of the

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chlorotriazines from possible human carcinogens to unlikely human carcinogens. In this hypothesis, atrazine’s effect on ovarian function and reproductive aging is the pivotal link in its MOA on tumor development. Finally, these studies have clearly established the chlorotriazines as endocrine disruptors mediated through their direct effect on the brain which then disrupts pituitary hormone secretion. This hypothesis is explored further below by first describing the relationship between normal ovarian aging and mammary tumor development and then the effect of atrazine on pituitary-ovarian function and reproductive aging. MAMMARY GLAND TUMORS AND ALTERED OVARIAN CYCLES IN THE AGING RAT Mammary gland tumors are the most common type of spontaneous neoplasm observed in the laboratory rat. Most mammary neoplasms are benign tumors of epithelial origin usually classified histopathologically as fibroadenomas, adenomas or fibromas (Greaves, 1990). Although spontaneous mammary tumors in female rats are extremely common, such neoplasms in male rats are rarely observed. This sex pre-disposition for the genesis of these neoplasms provided the earliest empirical evidence which led inductively to the concept that the female’s pituitary-ovarian hormones are important in the genesis of these neoplasms (Noble and Cutts, 1959). The two most important hormones for development and growth of mammary tumors in rats are prolactin and estrogen. Ovarian estradiol stimulates the secretion of prolactin and acts with prolactin directly on the mammary tissues to promote tumorogenesis. Estrogen alone can neither induce nor maintain growth of mammary tumors in the absence of the pituitary, but prolactin may have a limited capacity to induce and maintain mammary tumor growth in the absence of the ovary (Meites, 1972). As the pituitary-ovarian hormonal milieu present in the female rat is the primary causative factor in the development of mammary gland tumors, it is not surprising that the hormonal environment that develops as it ages is conducive to the development of mammary gland tumors. With advancing age, the female of some rat strains normally undergoes a transition from regular ovarian cycles to an acyclic pattern of “persistent” or “constant” estrus (e.g., Long-Evans (LE) (Cooper and Walker, 1979), SD (Eldridge et al., 1999)). In these strains, this transition occurs prior to the first year of age and is related to a disruption in both the timing and amplitude of the pre-ovulatory surge of luteinizing hormone (LH) (Cooper et al, 1980). The ovaries of the constant-estrus female contain many large follicles (i.e., polyfollicular ovaries) but no corpora lutea (CL) (Huang and Meites, 1975). These follicles continue to secrete estradiol, while progesterone secretion is minimal (Huang et al., 1978). Furthermore, as estrogen increases pituitary prolactin synthesis and secretion, the concentration of this hormone is also elevated in the constant estrous female. Thus the pattern of hormone secretion present in the aged female (unopposed estradiol and enhanced prolactin) is precisely the hormonal milieu that has been

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shown to facilitate the development of mammary gland tumors in aging rats (Cooper, 1983). While the pattern of tumor development in the aging female SD (Eldridge et al, 1999) and LE (Cooper and Walker, 1979) is closely linked to the onset of constant estrus, there are other endocrine profiles that are also associated with the promotion of mammary tumors. For example, mammary tumors (primarily fibroadenomas) have also been linked to the hormonal environment present in the repetitively pseudopregnant animal. In these females, serum estradiol concentrations are like those observed in the constant-estrus female, and daily serum prolactin pulses are observable (both diurnal and nocturnal) (Damassa et al, 1980). However, serum progesterone is also elevated in these females, similar to that observed in early pregnancy (Huang and Meites, 1975; Huang et al., 1978). Thus, Welsch et al. (1970a,b) reported that inducing repetitive pseudopregnacies (by medial-basal hypothalamic lesions or ectopic pituitary transplants) in SD females increased the incidence of mammary tumors. Again, this effect has been attributed primarily to the increased exposure to endogenous prolactin. The relationship between mammary gland tumor development and reproductive senescence was further established in studies that extended the female rat’s reproductive life span. For example, a loss in hypothalamic catecholaminergic (norepinephrine and dopamine) function with age has been attributed to the loss of the LH surge which is necessary to maintain cycles. If the female rat is treated with catecholaminergic agonists such as L-tyrosine (Cooper and Walker, 1979) or L-dopa (Cotzias et al, 1977), the ovulatory surge of LH, ovarian cycles and reproductive senescence is delayed. Importantly, there is a corresponding delay in the development of mammary gland tumors. In these studies, the effect of treatment with agents that enhance catecholaminergic neurotransmission on LH secretion and subsequent ovarian function was the primary focus. However, as indicated above, prolactin secretion during the animal’s life span has also been associated with mammary tumor development. Further support for the role of prolactin in the age-associated development of mammary tumors was provided by Nagasawa and Morii (1982) who delayed the onset of these tumors in SD females by treatment with 2-bromo-α-ergocryptine mesylate, a dopamine receptor agonist and potent suppressor of pituitary prolactin secretion. However, whether or not this delay in tumor development was a direct result of decreased prolactin secretion per se or the fact that such treatment may extend the reproductive life of the female should be considered. In summary, these studies show that the development of mammary tumors in the rat is closely associated with the onset of reproductive senescence in the rat and that reproductive (ovarian) aging can be modified. The studies examining the effect of atrazine on aging within the reproductive system of the female rat are discussed in the next section.

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EFFECT OF ATRAZINE ON REPRODUCTIVE AGING The above evidence clearly establishes a link between the development of mammary gland tumors and the pituitary-ovarian hormonal environment during reproductive senescence. In particular, age-related changes in ovarian cyclicity are known to be a primary factor in the development of such tumors. Thus, if atrazine or related chlorotriazines alter the rate of aging within the pituitaryovarian axis in the female rat, this effect could represent the primary MOA through which the premature occurrence of mammary tumors develops. Recently, Eldridge et al. (1999) reported that dietary exposure to atrazine (400 ppm, approximately 22.5 mg/kg) beginning at 8 weeks of age resulted in the early onset of persistent estrus in SD females (>50 percent of animals by 26 weeks on test). Age-matched controls also displayed persistent estrus, but to a lesser degree. The relationship between the age-dependent loss of estrous cycles and the onset of mammary gland tumors in these animals is depicted in Figure 6.1. It is noteworthy that Eldridge et al. (1999) also reported that this same dietary exposure (400 ppm) as well as exposure by oral gavage (200 mg/kg/day) to the 7– 8-week-old female resulted in prolonged vaginal diestrus. Previously, Cooper et al. (1996a) reported that doses of 75–300 mg/kg to both LE and SD females induced extended diestrus and repetitive pseudopregnancies. Thus, both the induction of pseudopregnancies (observed after short-term high-dose atrazine) and premature persistent or constant estrus are consistent with the hypothesis that atrazine exposure will induce an altered endocrine profile that is consistent with the development of mammary tumors. This role of altered pituitary-ovarian hormone secretion and altered cyclicity to the development of mammary tumors wa s further demonstrated by the fact that ovariectomy (at 8 weeks of age)

Figure 6.1 Effect of dietary atrazine on reproductive aging and mammary gland tumor development in the female SD rat. Reproductive senescence (left panel) was defined as an increase in the periods of persistent vaginal estrus. Tumor incidence (left panel) was determined by weekly examination of individual females (based on Eldridge et al, 1999).

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completely eliminated the development of tumors in animals exposed to atrazine at 400 ppm. Whereas the development of constant vaginal estrus is the normal pattern of aging in the LE and SD female, this is not the case in the Fischer 344 (F344) female. In F344 rats, reproductive senescence is characterized by the spontaneous development of repetitive pseudopregnancies. Stevens et al. (1994) reported that dietary atrazine exposure to F 344 female produced a treatment-related lengthening of the diestrous periods. Notably, in this strain, atrazine did not result in an increased incidence of mammary tumors in the aging females. These investigators argued that this pattern of change in vaginal cytology and the pattern of hormone secretion associated with pseudopregnancy explained the lack of tumor development in the F344. Thus, in contrast to the SD female, which had polycystic ovaries and constant estradiol and prolactin stimulation, the F344 females had ovaries that contained many CL and secreted substantial amounts of progesterone in addition to estradiol and prolactin. Whether this explanation of the strain difference in tumor response is indeed due to differences in the pattern of ovarian hormone secretion remains to be determined. However, repetitive pseudopregnancies are clearly associated with an early onset of mammary tumors in other rat strains. For example, in the SD strain, the induction of repetitive pseudopregnancies (by hypothalamic lesions or ectopic pituitaries) has been reported to cause an increase in mammary tumors (Welsch et al., 1970b). Thus, it is not the pseudopregnant condition present in the F344 versus the constant estrus condition present in the SD female after atrazine exposure that was responsible for the differences in tumor incidence, but rather the F344 female’s general resistance to the development of mammary gland tumors as the F344 is generally less likely to develop mammary tumors regardless of ovarian status or toxicant treatment (Cutts and Noble, 1964). Thus, although there is a clear strain difference, the failure of atrazine to induce tumors in the F344 rat did not occur because the animal’s pituitary-ovarian axis was unaffected, but because this strain is more resistant to mammary tumor development in general. BRAIN AS THE SITE OF ACTION OF ATRAZINE’S EFFECT ON OVARIAN FUNCTION The above observations demonstrate that atrazine alters the rate of reproductive senescence and brings about a premature change in the female’s ovarian hormone miliu. Many of these changes suggested that this herbicide alters ovarian function through an action of the chlorotriazine on the brain and/or pituitary. In fact, pilot studies (Cooper et al., 1996b) suggested that atrazine inhibits the ovulatory surge of LH and that this disruption of LH secretion would explain why altered ovarian function occurs in response to this herbicide. To better characterize the effect of atrazine on pituitary hormone secretion, we examined the effect of atrazine on the estrogen-induced LH and prolactin surges

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in the ovariectomized young-adult female using both LE and SD females. These two strains were examined because we noted previously that the disruptive effects of atrazine on ovarian function in the LE female were somewhat greater than that observed in similarly treated SD rats (Cooper et al., 1996a). In this study, ovariectomized, estrogen-primed females were exposed to atrazine (50– 300 mg/kg) for either 1, 3 or 21 days. One dose of atrazine (300mg/kg) suppressed the LH and prolactin surges in ovariectomized LE, but not SD, females (Figure 6.2). Three days of dosing with atrazine (50–300 mg/kg) suppressed the estrogen-induced LH and prolactin surges in ovariectomized LE females in a dose-dependent manner, but this same treatment was without effect on serum LH in SD females (Figure 6.3). Prolactin secretion was decreased at 300 mg in the SD female. The estrogen-induced surges of both pituitary hormones were suppressed by atrazine (75–300 mg/kg/day) in a dose-dependent manner in females of both strains evaluated after 21 days of treatment (Figure 6.4). We had shown previously that daily exposure to atrazine in doses ranging from 75 to 300mg/kg disrupted ovarian cyclicity in these two strains of rats (Cooper et al., 1996a). In this study we also examined the effect of a single exposure to atrazine on ovulation and subsequent vaginal cycling. A single dose of atrazine at 300mg/kg administered to intact LE females on the day of vaginal proestrus was without effect on ovulation but did induce pseudopregnancy in seven of nine females. A dose of 300mg/kg was without effect on ovulation or cyclicity in the SD female, and changes in ovulation or cyclicity were not noted at the lower doses of atrazine in the LE female. These results demonstrate that atrazine suppresses the estrogen-induced LH and prolactin surges providing, in part, an explanation for the previously observed effect of atrazine on ovarian cyclicity in these two strains of rats. To determine whether these effects on the hormonal control of ovarian function were due to a direct effect on the pituitary itself, or mediated through a change in hypothalamic control of pituitary secretion, we conducted two additional experiments. In the first experiment, we demonstrated that the atrazine-induced suppression of the LH surge could be reversed if the animals were subsequently given an intravenous dose of GnRH. This indicated that the pituitary gonadotrophs were still responsive to GnRH stimulation and that LH release by the gonadotrophs was not impaired (Cooper et al., 2000). In the second experiment, we examined the potential direct effect of atrazine on the pituitary lactotrophs. Female rats were hypophysectomized using a transaural approach. The pituitary fragments were then implanted beneath the kidney capsule. This procedure removes the pituitary from the prolactin-inhibiting influences of the hypothalamus and results in a tonic hyperprolactinemia (Cooper et al., 2000). Thus, any compound-induced change in prolactin secretion would result from a direct effect of the compound on the transplanted pituitary tissue itself and not mediated by any substances of CNS origin. In this study, we found that atrazine was without effect on prolactin secretion, again indicating that any change in

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pituitary hormone secretion is likely the result of an indirect action of atrazine on the pituitary. The fact that atrazine inhibits the estrogen-induced surge of LH would support the hypothesis that the chlorotriazines bring about changes in the neuroendocrine control of ovarian function similar to those known to occur prior to the loss of ovarian cycling in the aging rat. In most rat strains (LE and SD included), reproductive senescence develops by 1 year of age and is characterized by the appearance of persistent or constant estrus, a condition in which the vaginal smear remains cornified and the ovaries are polyfollicular without CL (Cooper et al, 1986; Everett, 1989). There is a general agreement that the underlying neuroendocrine events responsible for the loss of ovarian cycling result from changes within the CNS that lead to a decrease in the amplitude and a delay in the onset of the proestrous LH surge (van der Schoot, 1976; Cooper et al, 1980). These alterations in the pre-ovulatory surge of LH are thought to result from an age-associated reduction in the frequency of the GnRH pulses (Scarbrough and Wise, 1990). We reported elsewhere that atrazine can reduce GnRH pulse frequency (Tyrey et al, 1996). The age-dependent changes in pulsatile GnRH release are reported to result from the cumulative, lifetime, exposure to endogenous estrogen (Brawer et al., 1980), as the regulation of GnRH in the older rat is affected only minimally if the female is ovariectomized at an early age (Scarbrough and Wise, 1991). Furthermore, age-related changes in ovarian cycles can be restored with centrally acting pharmacological agents (e.g., catecholamine precursors) known to enhance GnRH and LH activity in rats (Watkins et al., 1975; Linnoila and Cooper, 1976; Forman et al, 1980) and mice (Flurkey et al, 1987). Thus, the present studies indicate that this herbicide can bring about changes in LH secretion that are similar to those observed during reproductive aging in the female rat. CHLOROTRIAZINES AND REPRODUCTIVE FUNCTION The previous discussion clearly indicates that atrazine alters the neuroendocrine control of ovarian function in at least two strains of rats. As noted above (see background), it is interesting that earlier reproductive/developmental studies of atrazine failed to recognize the potential of this compound as a reproductive toxicant even when the doses tested were greater than those used in our current studies. To a large extent, the failure to identify the reproductive effects of atrazine was due to the endpoints assessed in the early studies. For example, an assessment of ovarian cycles was not required, nor were measurements of pubertal development. This prompted us to re-examine the effect of atrazine on a number of reproductive developmental endpoints. Pregnancy initiation and pregnancy maintenance In the female rat, the initiation and maintenance of CL function is regulated by

Figure 6.2 Effect of a single treatment (oral gavage) of atrazine administered at time 12:30 hours on the estrogen-induced LH (top) and prolactin (bottom) surge in LE and SD. Rats maintained on a 14 hours-light: 10 hours-dark schedule (lights off at 1900 hours). The zero hour sample was taken at 1300 hours and last sample at 1900 hours (Cooper et al., 2000, with permission from Oxford University Press).

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Figure 6.3 Effect of a three daily treatments (oral gavage) of atrazine administered at time zero on the estrogen-induced LH (top) and prolactin (bottom) surge in LE and SD. See Figure 6.2 for details (Cooper et al., 2000, with permission from Oxford University Press).

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Figure 6.4 Atrazine exposure for 21 days. Three days prior to sampling, the females were ovariectomized and implanted with a silastic-estrogen filled capsule. Animals were killed at 1700 hours. The serum concentrations (top panels) of both LH (left) and prolactin (right) were decreased in LE (light bars) and SD (black bars) rats. Pituitary LH concentration (lower left panel) was not altered; however, pituitary prolactin concentration was significantly increased in both strains after atrazine exposure (Cooper et al., 2000, with permission from Oxford University Press).

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pituitary and placental hormone secretion. During early pregnancy, pituitary prolactin plays a key role in rescuing the CL and subsequently maintaining CL progesterone secretion. In mid-pregnancy, LH plays a primary role in maintaining progesterone secretion. Later in gestation, placental lactogen becomes the primary stimulant for luteal progesterone. We hypothesized that atrazine herbicide would inhibit implantation by disrupting the twice-daily surges of prolactin essential for establishing pregnancy and implantation. In a study examining several different rat strains, Cummings et al. (2000) reported that 100 and 200 mg/kg/day atrazine, dosed prior to the nocturnal prolactin surge (2 hours prior to lights on), showed an increase in percent pre-implantation loss which would be consistent with an effect of atrazine on prolactin secretion. Narotsky et al. (2001) examined the effect of atrazine exposure on pregnancy maintenance. Since chlorotriazines were shown to affect the ovulatory surge of LH, it was hypothesized that these chemicals might also alter LH secretion during the rat’s LH-dependent period in mid-gestation, thereby disrupting pregnancy. They found that atrazine does indeed cause pregnancy loss (i.e., fulllitter resorption) in the F344 rat when administered on gestation days 6–10 (encompassing the LH-dependent period), but failed to disrupt pregnancy when it was administered on days 11–15 (after the LH-dependent period) (Figure 6.5). In view of the strain specificity of atrazine’s tumorogenicity (i.e., SD rats are sensitive, but F344 rats are not), Narotsky et al. (2001) also compared three rat strains for their susceptibility to atrazine-induced pregnancy loss. They found that the F344, SD and LE strains were similarly sensitive at 200mg/kg, but that only the F344 strain was sensitive at 50 or 100mg/kg. Thus, in sharp contrast to the previous reports that this herbicide is effective only in the SD rat, these data indicate that regarding pregnancy loss, the F344 strain is most sensitive.

Figure 6.5 Pregnancy loss (full liter resorption) following atrazine exposure (200mg/kg/ day) to pregnant F344 rats. Dosing with atrazine during GD 6–10 caused a significant number of full-litter resorptions. The same dose on GD 11–15 was without effect. The number above each bar represents the number with full liter resorption/total observed (based on Narotsky et al., 2001).

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As part of our investigation of its MOA, we also evaluated atrazine’s effects on gestational progesterone levels in the F344 rat. During the LH-dependent period, LH is required to maintain pregnancy by stimulating luteal secretion of progesterone. Pregnancy loss was associated with progressively reduced progesterone levels resulting in marked reductions by GD 11. Although a direct ovarian effect remains possible, these findings are consistent with reduced secretion of LH, and may reflect an LH-mediated mechanism. Finally, in addition to atrazine, Narotsky et al. (2002) evaluated three chlorinated metabolites (diaminochlorotriazine (DACT), deethylatrazine, deisopropylatrazine) and one dechlorinated plant metabolite (hydroxyatrazine) for their ability to cause pregnancy loss in F344 rats. All four of these degradation by-products disrupted pregnancy with DACT being the most potent; on a molar basis, DACT was similar to atrazine in potency, whereas hydroxyatrazine was the least potent. These findings provide insights into the role of metabolism in atrazine-induced pregnancy loss and in the structureactivity relationships of this class of compounds. Furthermore, since these degradation by-products have all been detected in water supplies or on food, these data also provide valuable hazard-identification information toward the risk assessment of these environmental chemicals. Atrazine’s effect on pregnancy maintenance is very similar to that seen for bromodichloromethane (BDCM), a structurally unrelated contaminant of drinking water. Using very similar research protocols, we have shown that both drinking water contaminants cause pregnancy loss in F344 rats when exposed during the LH-dependent period, but not afterwards (Bielmeier et al, 2001). For both atrazine and BDCM, the SD rat is less sensitive than the F344 strain. Also, pregnancy loss is associated with reduced progesterone levels. Unlike BDCM, however, atrazine has additional adverse consequences of gestational exposure. Whereas BDCM-induced pregnancy loss appears to be an all-or-none effect, dams of surviving chlorotriazine-exposed litters may show delays in parturition, and their pups have increased post-natal mortality. In summary, the chlorotriazines’ disruptive effects on pregnancy maintenance in the rat are consistent with their proposed CNS-mediated MOA, and heighten concerns about the potential health effects for this class of pesticide. Atrazine and suckling-induced prolactin release The observation that atrazine inhibited the estrogen-induced prolactin surge in the ovariectomized female suggested that this compound may also modify suckling-induced prolactin release in the nursing dam, an effect that could ultimately modify milk production. Stoker et al. (1999) evaluated the effect of atrazine on prolactin secretion during the early lactation in the female Wistar rat. In this study, dams were dosed twice daily (6.25–50mg/kg) on post-natal days (PNDs) one through four. On PND 4, the pups were removed from the dam at

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0900 hours and the dam fitted with a cardiac catheter. Four hours after removal, the pups were returned to the dam to nurse for 45 minutes. Basal and sucklinginduced prolactin concentrations in the dam’s serum were then determined (Figure 6.6) from blood samples collected at 10-minute intervals. In this study, prolactin secretion was not altered by the twice-daily dose of 6.25 mg/kg dose. However, doses above 12.5 mg/kg reduced serum prolactin concentrations in the dam. The suppression of prolactin by the 50 mg/kg twice-daily dose was associated with a decrease in pup weight following nursing that was likely attributed to either impaired milk production or milk let-down as evidenced by the reduced amount of milk in the pups’ stomach following the nursing period. Importantly, the dams in all dose groups displayed normal maternal behavior (pup retrieval, crouching and grooming pups) during the course of this experiment. These observations demonstrate that even brief post-natal exposure to atrazine will dramatically affect prolactin secretion. In addition to the obvious effects on body weight and milk production observed at the highest dose, limiting the amount of prolactin availability to the pup was shown to have adverse effects on the offspring later in life (Stoker et al, 1999). For example, Stoker et al. (1999) showed an increased incidence of prostatitis in the adult male offspring of atrazineexposed dams and provided evidence that this effect was the result of decreased suckling-induced prolactin in the dams. One may also speculate that other adverse outcomes related to altered prolactin might occur in the offspring of these dams, such as pubertal development and altered immune functions. Pubertal development Because atrazine was found to alter pituitary hormone secretion in the adult female, we hypothesized that this herbicide would also affect pubertal development in the rat. To examine this hypothesis, the effects of atrazine and selected metabolites on the onset of puberty were evaluated in male (Stoker et al., 2000a, 2002) and female (Laws et al., 2000) Wistar rats. In these studies, we used the recently published protocols for the assessment of pubertal development and thyroid function in juvenile male and female rats (Goldman et al., 2000; Stoker et al., 2000b). These in vivo protocols are under consideration by the US EPA as part of a Tier I Screening Battery for the Agency’s Endocrine Disruptors Screening Program (www.epa.gov/scipoly/oscpendo/index.htm). Using these protocols female rats are gavaged from PND 22 to 41, whereas the males are dosed from PND 23 to 53. This dosing regimen encompasses the critical periods of sexual maturation for both sexes, and allows for the detection of environmental chemicals that display antithyroid, estrogenic, antiestrogenic (estrogen receptor- or steroid-enzyme-mediated) activity, or alter puberty via changes in hypothalamic function or pituitary LH, follicle-stimulating hormone, prolactin or growth hormone. During dosing, growth and pubertal indices (vaginal opening in the female, pre-putial separation in the male) are examined.

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Figure 6.6 Effect of atrazine on suckling induced prolactin release. Dams were dosed for 4 days with atrazine. On the fourth day, the pups were separated from the dam for 4 hours. Serum prolactin values were determined 60 and 30 minutes prior to placing the pubs with the dam and at every 10 minutes thereafter (based on Stoker et al., 1999).

At necropsy, several reproductive tissues sensitive to changes in gonadal steroids are examined (weight at necropsy and histopathology). Summaries of data obtained from the male and female pubertal studies are shown in Tables 6.1 and 6.2. Atrazine induced a delay in the onset of puberty in both sexes (Table 6.1). The no observed effect level (NOEL) was lower for the males (6.25 mg/kg/d) as compared to the females (25 mg/kg/d). After vaginal opening, irregular estrous cycles were also observed in the females receiving 50 mg/kg/day or greater. Atrazine exposure also led to a number of differences in the weight of some reproductive tissues in both sexes. Importantly, the differences noted in the tissue weights and external markers of pubertal development occurred at doses that were without effects on the animal’s body weight. Thus, as anticipated from earlier studies that detected effects of atrazine on LH and prolactin following exposure to adult females, these data demonstrate that this herbicide can modify pubertal development. In addition, as observed in the pregnancy maintenance studies described above, the primary chlorinated metabolites of atrazine were also effective in altering pubertal development in both the male (Stoker et al, 2002) and female (Laws et al, 2002). In these studies, the main chlorinated metabolite, diamino-s-chlorotriazine (DACT), was as potent as atrazine (Table 6.2).

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Table 6.1 Summary of endpoints observed in the male and female pubertal studies using atrazine

Notes a Range of doses with significant effects (p < 0.05) as compared with control. b Original data reported in Stoker et al. (2000a) and Laws et al. (2000).

Table 6.2 Comparison of the NOELs (as molar equivalents of atrazine) for the delay in the onset of puberty in male and female Wistar ratsa,b

Notes a Doses for metabolites are reported as molar equivalents of the atrazine dose (AED). Actual doses of each metabolite were as follows: DIA (10.4mg/kg/d, AED=12.5mg/kg/d), DEA (10.8 mg/kg/d, AED=12.5 mg/kg/d), DACT (4.4 mg/kg/d, AED=6.25 mg/kg/kg; 16. 9 mg/kg, AED=25 mg/kg/d), OH-ATR (183 mg/kg/d, AED=200 mg/kg/d). b Data reported in Stoker et al. (2000b, 2002) and Laws et al. (2000, 2002).

Summary The studies reviewed in this manuscript demonstrate that atrazine, the related chlorotriazine herbicides and the chlorinated metabolites of these compounds disrupt the neuroendocrine control of ovarian function. The primary site of action of atrazine has been shown to be at the level of the hypothalamus. Prolonged atrazine exposure in the female rat appears to accelerate aging within the brain-

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pituitary-ovarian axis. This premature reproductive senescence (i.e., constant estrus) establishes the hormonal milieu conducive to the development of mammary gland tumors. As the causative factors associated with reproductive aging in the rat (impaired hypothalamic function) and human (depletion of primary follicles) are dramatically different, the possibility that a similar process may occur in women is remote. However, because the hypothalamic regulation of LH and prolactin secretion in the rat and human are similar, it is likely that the chlorotriazine herbicides could influence the secretion of these important pituitary hormones in humans. Importantly, the likelihood that the human would be exposed to these herbicides at the concentrations used in the rodent studies reviewed in this chapter appears remote (US EPA, 2002). However, there are numerous chlorotriazine herbicides in use, and these compounds produce metabolites that are similar to those of atrazine. Atrazine, other chlorotriazine herbicides, as well as their metabolites, are persistent in the groundwater (US EPA, 2002). Thus, the likelihood that humans may be exposed to these compounds in cumulative or combined manner, at concentrations that exceed the maximum coricentration levels (MCL) for atrazine alone, must be considered. This chapter has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policy of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. REFERENCES Bielmeier, S.R., Best, D.S., Guidici, D.L. and Narotsky, M.G. (2001) Pregnancy loss in the rat caused by bromodichloromethane, Toxicol. Sci., 59:309–15. Brawer, J.R., Schipper, H. and Naftolin, F. (1980) Ovary-dependent degeneration in the hypothalamic arcuate nucleus, Endocrinology, 107:274–79. Connor, K., Howell, J., Chen, I., Liu, H., Berhane, K., Sciarretta, C, Safe, S. and Zacharewski, T. (1996) Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro, Fundam. Appli. Toxicol., 30:93–101. Cooper, R.L. (1983) Pharmacological and dietary manipulations of reproductive aging in the rat. Significance to central nervous system aging. In: R.F. Walker and R.L. Cooper (eds) Clinical and Experimental Intervention of the Aging Process, New York: Marcel Dekker, pp. 27–44. Cooper, R.L. and Goldman, J.M. (1999) Vaginal Cytology. In: G. Daston and C. Kimmel (eds) An Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment, Washington: International Life Sciences Institute/Health & Environmental Sciences Institute, pp. 42–56. Cooper, R.L. and Walker, R.F. (1979) Potential therapeutic consequences of agedependent changes in brain physiology, Interdiscip. Top. Gerontol., 15:54–76.

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Cooper, R.L., Conn, P.M. and Walker, R.F. (1980) Characterization of the LH surge in middle-aged female rats, Biol Reprod., 23:611–15. Cooper, R.L., Goldman, J.M. and Rehnberg, G.L. (1986) Neuroendocrine control of reproductive function in the aging female rodent, Journal of the American Geriatrics Society, 34:735–51. Cooper, R.L., Parrish, M.B., McElroy, W.K., Rehnberg, G.L., Hein, J.F., Goldman, J.M., Stoker, T.E. and Tyrey, T.E. (1995) Effect of atrazine on the hormonal control of the ovary, The Toxicologist, 15:294. Cooper, R.L., Stoker, T.E., Goldman, J.M., Parrish, M.B. and Tyrey, L. (1996a) Effect of atrazine on ovarian function in the rat, Reprod. Toxicol., 10:257–64. Cooper, R.L., Stoker, T.E., Goldman, J.M., Hein, J.F. and Tyrey, L. (1996b) Atrazine disrupts hypothalamic control of pituitary-ovarian function, The Toxicologist, 30:66. Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M. and McElroy, W.K. (2000) Atrazine disrupts hypothalamic control of pituitary-ovarian function, Toxicol. Sci., 53:297– 307. Cotzias, C.G., Miller, S.T., Tang, T.C. and Papavasiliou, P.S. (1977) Levodopa, fertility, and longevity, Science, 196:549–50. Cummings, A.M., Rhodes, B.E. and Cooper, R.L. (2000) Effect of atrazine on implantation and early pregnancy in four strains of rats, Toxicol. Sci., 58:135–43. Cutts, J.H. and Noble, R.L. (1964) Estrone-induced mammary tumors in the rat: I. induction and behavior of tumors, Cancer Res., 24:1116–23. Damassa, D.A., Gilman, D.P., Lu, K.H., Judd, H.L. and Sawyer, C.H. (1980) The twentyfour hour pattern of prolactin secretion in aging female rats, Biol Reprod., 22:571–75. Eldridge, J.C., Fleenor-Heyser, D.G., Extrom, P.C., Wetzel, L.T., Breckenridge, C.B., Gillis, J.H., Luempert, L.G. and Stevens, J.T. (1994) Short-term effects of chlorotriazines on estrus in female Sprague-Dawley and Fischer 344 rats, J. Toxicol. Environ. Health, 43:155–67. Eldridge, J.C., McConnell, R.F., Wetzel, L.T. and Tisdel, M.O. (1998) Appearance of mammary tumors in atrazine-treated female rats: probable mode of action involving strain-related control of ovulation and estrous cycling. In: L.G. Ballantine, J.E. McFarland and D.S. Hackett (eds) Triazine herbicides: Risk assessment, Washington, DC: American Chemical Society, pp. 413–24. Eldridge, J.C., Wetzel, L.T. and Tyrey, L. (1999) Estrous cycle patterns of SpragueDawley rats during acute and chronic atrazine administration, Reprod. Toxicol., 13: 491–99. Everett, J.W. (1989) Neurobiology of Reproduction in the Female Rat, Springer-Verlag, New York. Flurkey, K., Randall, P.K., Sinha, Y.N., Ermini, M. and Finch, C.E. (1987) Transient shortening of estrous cycles in aging C57BL/6J mice: effect of spontaneous pseudopregnancy, progesterone, 1-dihydroxyphenylalanine, and hydergine, Biol Reprod., 36:949–59. Forman, L.J., Sonntag, W.E., Miki, N. and Meites, J. (1980) Maintenance by L-dopa treatment of estrous cycles and LH response to estrogen in aging female rats, Exp. Aging Res., 6:547–54. Giknis, M.L.A. (1989) Ciba-Geigy Pharmaceuticals SEF Project No. MIN 832110, unpublished data. EPA acceptable. Greaves, P. (1990) Mammary gland, In: Histopathology of preclinical toxicity studies: Interpretation and relevance on drug safety evaluation, Amsterdam: Elsevier.

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Gressel, J., Ammon, H.U., Fogelfors, H., Kay, Q.O.N. and Kees, H. (1984) Discovery and distribution of herbicide-resistant weeds outside of North America. In: H.M. LeBaron and J. Gessel (eds) Herbicide Resistance in Plants, New York: John Wiley & Sons, pp. 31–46. Goldman, J.M., Laws, S.C., Balchak, S.K., Cooper, R.L. and Kavlock, R.J. (2000) Endocrine disrupting chemicals: Prepubertal exposures and effects on sexual maturation and thryroid activity in the female rat. A review of the EDSTAC recommendations, Crit. Rev. Toxicol., 30:135–96. Gysin, H. and Knuesli, E. (1960) Chemistry and herbicidal properties of triazine derivatives. In: R. Metcalf (ed.) Advances in Pest Control Research, New York: Wiley (Interscience), Vol. III, pp. 289–358. Hauswirth, J.W. and Wetzel, L.T (1998) Toxicity characteristics of 2-chlorotriazines atrazine and simazine. In L.G. Ballantine, J.E. McFarland and D.S. Hackett (eds) Triazine Herbicides: Risk Assessment, Washington, DC: American Chemical Society, pp. 370–83. Huang, H.H. and Meites, J. (1975) Reproductive capacity in aging female rats, Neuroendocrinology, 7:289–95. Huang, H.H., Steger, R.W., Bruni, J. and Meites, J. (1978) Changes in patterns of sex steroid and gonadotropin secretion in aging female rats, Endocrinology, 103:1855– 59. Laws, S.C., Ferrell, J.M., Stoker, T.E., Schmid, J. and Cooper, R.L. (2000) The effect of atrazine on puberty in female Wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid function, Toxicol. Sci., 58:366–76. Laws, S.C., Ferrell, J.M., Stoker, T.E. and Cooper, R.L. (2002) Pubertal development in female Wistar rats following exposure to propazine and atrazine metabolites, diamino-s-chlorotriazine and hydroxyatrazine, The Toxicologist, 66:1-S, 343. Linnoila, M. and Cooper, R.L. (1976) Reinstatement of vaginal cycles in aged female rats, J. Pharmacol and Exp. Ther., 199:477–82. Meites, J. (1972) Relation of prolactin and estrogen to mammary tumorigenesis in the rat, J. Nat. Cancer Inst., 48:1217–24. Nagasawa, H. and Morii, S. (1982) Inhibition by early treatment with bromocriptine blocks spontaneous mammary tumor development in the rat with no side effects, Acta Endocrinol., 101:51–55. Narotsky, M.G., Best, D.S., Guidici, D.L. and Cooper, R.L. (2001) Strain comparisons of atrazine-induced pregnancy loss in the rat, Reprod. Toxicol., 15:61–69. Narotsky, M.G., Best, D.S., Bielmeier, S.R., Spangler, S.A. and Cooper, R.L. (2002) Pregnancy loss and delayed parturition caused by atrazine and its metabolites in F344 rats, Biol Reprod., 66 (Suppl 1):215–16. Noble, R.L. and Cutts, J.H. (1959) Mammary tumors of the rat: a review, Cancer Res., 1125–39. Scarbrough, K. and Wise, P.M. (1990) Age-related changes in pulsatile luteinizing hormone release precede the transition to estrous acyclicity and depend upon estrous cycle history, Endocrinology, 126:884–90. Scarbrough, K. and Wise, P.M. (1991) Diurnal rhythmicity of norepinephrine activity associated with the estradiol-stimulated luteinizing hormone surge: effect of age and long-term ovariectomy on hemispheric asymmetry, Biol Reprod., 44:769–75. Simkins, J.W., Eldridge, J.C. and Wetzel, L.T. (1998) Role of strain-specific reproductive patterns in the appearance of mammary tumors in atrazine-treated rats. In: L.G.

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Ballantine, J.E. McFarland and D.S. Hackett (eds) Triazine Herbicides: Risk Assessment, Washington, DC: Oxford University Press, pp. 399–413. Stevens, J.T. and Sumner, D.D. (1991) Herbicides. In: W.J. Hayes and E.R. Laws (eds) Handbook of Pesticide Toxicology, New York: Academic Press, Vol. 3, pp. 1317– 408. Stevens, J.T., Breckenridge, C.B., Wetzel, L.T., Gillis, J.H., Luempert, L.G. and Eldridge, J.C. (1994) Hypothesis for mammary tumorigenesis in Sprague Dawley rats exposed to certain triazine herbicides, J. Toxicol. Environ. Health, 43:139–53. Stoker, T.E., Robinette, C.L. and Cooper, R.L. (1999) Maternal exposure to atrazine during lactation suppresses suckling-induced prolactin release and results in prostatitis in the adult offspring, Toxicol. Sci., 52:68–79. Stoker, T.E., Laws, S.C., Guidici, D. and Cooper, R.L. (2000a) The effects of atrazine on puberty and thryroid function in the male Wistar rat: An evaluation of a protocol for the assessment of pubertal development and thyroid function, Toxicol. Sci., 58:50–59. Stoker, T.E., Parks, L.G., Gray, L.E. and Cooper, R.L. (2000b) Effects of endocrine disrupting chemicals on puberty in the male rat: A review of the EDSTAC recommendations., Crit. Rev. Toxicol., 30:197–252. Stoker, T.E., Guidici, D.L., Laws, S.C. and Cooper, R.L. (2002) The effects of atrazine metabolites on puberty and thyroid function in the male Wistar rat, Toxicol. Sci., 67: 198–206. Tennant, M.K., Hill, D.S., Eldridge, J.C., Wetzel, L.T., Breckenridge, C.B. and Stevens, J.T. (1994) Chloro-s-triazine antagonism of estrogen action: limited interaction with estrogen receptor binding, J. Toxicol. Environ. Health, 43:197–211. Tyrey, L., Cooper, R.L., Stoker, T.E. and Hein, J.F. (1996) Atrazine suppression of LH secretion in the rat, Presented at the Third Annual NHEERL Symposium on Susceptibility and Risk, Durham, NC. US EPA (2002) Revised Human Health Risk Assessment: Atrazine, http://www.epa.gov/ pesticides/reregistration/atrazine/. van der Schoot, P. (1976) Changing pro-oestrous surges of luteinizing hormone in ageing 5-day cyclic rats, J. Endocrinol, 69:287–88. Watkins, B.E., McKay, D.W. and Riegle, G.D. (1975) L-dopa effects on serum LH and prolactin in old and young female rats, Neuroendocrinology, 19:331–38. Welsch, C.W., Nagasawa, H. and Meites, J. (1970a) Increased incidence of spontaneous mammary tumors in female rats with induced hypothalamic lesions, Cancer Res., 30: 2310–13. Welsch, C.W., Jenkins, T.W. and Meites, J. (1970b) Increased incidence of mammary tumors in the female rat grafted with multiple pituitaries, Cancer Res., 30:1024–29. Wetzel, L.T., Leumpert-III, L.G., Breckenridge, C.B., Tisdel, M.O. and Stevens, J.T. (1994) Chronic effects of atrazine on estrus and mammary tumor formation in female Sprague-Dawley and Fischer 344 rats, J. Toxicol. Environ. Health, 43:169–72.

7 THE ROLE OF OVARIAN METABOLISM IN CHEMICAL-INDUCED OVARIAN INJURY Ellen A.Cannady and I.Glenn Sipes

INTRODUCTION Biotransformation overview Humans are exposed by a variety of routes to a vast array of xenobiotics. A number of physiological systems have evolved to eliminate them from the body. Some hydrophilic chemicals are directly excreted from the body unchanged, while the majority of lipophilic chemicals are metabolized prior to elimination. Metabolism, or biotransformation, is an all inclusive term that includes a multitude of bio-chemical processes, which are largely enzymatic in nature. The enzymology of biotransformation reactions is divided into two phases. Phase I metabolism refers to those biochemical reactions that in general, are oxidative in nature, and either expose or add a functional group to a xenobiotic. This process in many cases makes the compound more water soluble, which facilitates excretion and/or subsequent phase II conjugation. Phase I reactions may also produce metabolites that are chemically reactive. These reactive metabolites may result in tissue injury associated with administration of the parent chemical. Many phase I reactions are mediated by the cytochrome P450 (CYP) superfamily of enzymes, as well as by oxidases, esterases, amidases and flavin-containing monooxygenases (FMO). Phase II metabolism refers to those biochemical processes that generally result in conjugation of a functional group (often a phase I-derived metabolite), to ‘small molecular weight endogenous molecules, by glutathione transferases, UDPglucuronosyltransferases, acetyltransferases, and sulfotransferases, as well as epoxide hydrolases. With few exceptions, phase II metabolism inactivates, or detoxifies, more reactive chemicals. Phase II metabolism also results in an increased ability to transport the chemical, as a conjugate, across membranes to aid in the ultimate elimination of the compound from the body (Figure 7.1). The balance between phase I and phase II biochemical reactions is important in determining the ultimate fate of a compound. For instance, CYP-mediated metabolism can produce either more

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Figure 7.1 Biotransformation schematic. Assimilation of phase I and phase II metabolism and common enzymes involved. Imbalances in bioactivation and detoxification of reactive metabolites often lead to toxic insult (Sipes and Gandolfi, 1991).

water-soluble, non-toxic metabolites or electrophilic metabolites that are highly reactive and potentially toxic (Wrighton and Stevens, 1992). Thus, depending on the nature of the xenobiotic, CYP may be responsible for either bioactivation or detoxification reactions. EXTRAHEPATIC METABOLISM Although the liver is the primary organ involved in the biotransformation of xenobiotics, extrahepatic organs can also contribute to the bioactivation and/or detoxification of such compounds (Parkinson, 1996). Various methodologies, including immunoblotting techniques and in vitro incubations with the tissue/ enzyme and chemical of interest, have demonstrated the presence of CYP isoforms, epoxide hydrolases, glutathione S-transferases, UDPglucuronosyltransferases, sulfotransferases, N-acetyltransferases and methyltransferases in a variety of tissues. The human tissue distribution of these enzymes is broad, including the gastrointestinal tract mucosa, kidney, lung, brain, skin, testis, and ovary, to name a few (Oesh et al., 1977; Mukhtar et al., 1978; Dannan and Guengerich, 1982; Krishna and Klotz, 1994). This chapter focuses on the ovarian metabolism of exogenous chemicals and its potential ramifications in chemically induced ovarian injury.

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THE OVARY AS A TARGET ORGAN As discussed in previous chapters, the mammalian ovary contains a finite number of follicles in different stages of development that cannot be regenerated. Understanding this concept is crucial when one considers toxic chemicals and drugs that can cause follicle loss. Depending on the follicle population targeted by such agents, a myriad of effects can occur, varying from temporary infertility (via destruction of antral follicles) to premature ovarian failure (via complete loss of primordial follicles). Determining mechanisms of ovarian toxicity can be complicated because toxicity can be attributed to species, strain, and target tissue specificities. Differences in dispositional and metabolic parameters, both hepatic and extrahepatic, may also affect the resulting ovarian toxicity. Therefore, each of these factors must be evaluated separately and as a whole. Although hepatic metabolism of compounds usually governs their disposition and elimination, one must consider the involvement of ovarian metabolism in chemically induced ovarian injury. Additionally, in some disease states, such as hepatic cirrhosis or cancer, certain metabolic pathways in the liver may not function properly. This can result in higher systemic exposure, allowing other metabolic pathways to predominate, and in some instances involve extrahepatic tissues, including the ovary, in the metabolism of compounds that normally would have been cleared by the liver. Such metabolism could include bioactivation of a chemical, further bioactivation of a circulating metabolite, or a decreased ability to detoxify reactive/ toxic metabolites. Taken together, ovarian metabolism may play a significant role in ovotoxicity caused by xenobiotics. BIOTRANSFORMATION ENZYMES IN THE OVARY Mukhtar et al. (1978) were among the first researchers to identify the presence of metabolic enzymes in the ovary. Their studies evaluated the post-natal development of microsomal CYP, microsomal epoxide hydrolase (mEH), and glutathione S-transferase in microsomes and cytosol obtained from rat whole ovarian homogenates. Total CYP content, determined from carbon monoxidebinding spectra (Omura and Sato, 1964), was approximately 0.02 nmol/mg of microsomal protein in ovaries from rats 12 days of age (Mukhtar et al, 1978). It gradually increased to a maximum of approximately 0.07 nmol/mg of microsomal protein at 60 days of age. Utilizing benzo(a)pyrene(B(a)P)-4,5-oxide as a substrate, ovarian mEH activity was determined to be 0.3, 0.85, or 0.7 nmol/ min/mg of microsomal protein on days 12, 40, or 60 of age. Styrene-7,8-oxide and B(a)P-4,5-oxide were used as substrates to determine cytosolic glutathione-Stransferase activity. On day 12, enzyme activity was similar for both substrates (approximately 20 nmol/min/mg of cytosolic protein). Additionally, the patterns of glutathione-S-transferase activity were similar for both substrates, in which activity peaked at 35 days of age, then gradually decreased and remained constant by 140 days of age. However, specific activity utilizing styrene oxide as

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a substrate was greater compared to B(a)P oxide at every time point. These studies demonstrated that several enzymes important in the biotransformation of xenobiotics are present and functional in the rat ovary. Thus, these enzymes may contribute to the metabolism of a variety of agents in the ovary. EXPRESSION OF PHASE I ENZYMES IN THE RAT OVARY Over the past few years, as multiple isoforms for the CYP enzymes were discovered, the identity of the isoforms in the ovary became of particular interest. Studies by Bengtsson et al. (1990) evaluated the identity of several CYP450 enzymes in the rat ovary. In ovarian microsomes obtained from control rats, Western blotting revealed that CYP1A1, CYP2A1, CYP2A2, CYP2B1, CYP2B2, and CYP3A were not present in sufficient amounts to play a significant role in polycyclic aromatic hydrocarbon (PAH) metabolism, at least in the noninduced state. However, with continued exposure to chemicals, these enzymes are induced and may contribute to the bioactivation and ultimate ovarian toxicity of the PAHs. Although their involvement in PAH metabolism is not known, several CYP2C isoforms have been detected in rat ovarian microsomes. Interestingly, this subfamily of enzymes is thought to be involved in metabolism of xenobiotics, as well as endogenous steroids and arachidonic acid (Karara et al., 1993; Van Voorhis et al., 1993). More recently, Dasmahapatra et al. (2001, 2002) evaluated expression of CYP1A1 and CYP1B1 in the rat ovary. In the absence of stimulus, mRNA encoding CYP1A1 was expressed at a lower level than CYP1B1 (approximately 15-fold higher than CYP1A1) in ovarian granulosa cells obtained from rats. This observation further supported the conclusions made by Bengtsson et al. (1990) that CYP1A1 is not constitutively expressed in the ovary. However, following in vivo pre-treatment with 2,3,7,8-tetrachlorodibenzo-/?-dioxin (TCDD), a known inducer of CYP1A1 and CYP1B1, mRNA levels for both CYP1A1 and CYP1B1 were increased. Induction of mRNA encoding CYP1A1 was more significant, compared to CYP1B1. Following in vitro incubation with TCDD, induction of mRNA encoding both enzymes was maintained for 48 hours (CYP1A1) and 6 hours (CYP1B1) (Dasmahapatra et al, 2001). Previous in vitro studies had shown an increase in estrogen receptor-β (ER-β) mRNA following CYP1B1 induction by TCDD. Therefore, Dasmahapatra et al. (2002) also evaluated expression of CYP1A1 and CYP1B1 at various time points throughout the estrous cycle to determine the hormonal influences on this expression. mRNA encoding CYP1A1 A1 was undetectable at all times evaluated. Interestingly, CYP1B1 was significantly increased (approximately 5-fold) on the evening of pro-estrus and decreased on the morning of estrus. ER-β mRNA remained unchanged during these times. Although not clearly understood, these results suggest that there may be common mechanisms/factors governing the estrous cycle and expression of

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CYP1B1 in the rat ovary (Bengtsson and Rydstrom, 1983; Bengtsson et al, 1987; Dasmahapatra et al, 2002). EXPRESSION OF PHASE II ENZYMES IN THE RAT OVARY Other investigators have evaluated phase II enzymes in the rat ovary. mEH has been evaluated in different sizes of pre-antral follicles from rat ovaries. Springer et al. (1996) evaluated the mRNA expression of mEH by reverse transcriptasepolymerase chain reaction. mRNA encoding mEH was present in all sizes of pre-antral follicles evaluated (small pre-antral, 25–100 µm; large preantral, 100–250 µm) from vehicle-treated rats. Following 10 d of repeated daily dosing with the ovotoxic chemical, 4-vinylcyclohexene diepoxide (VCD), mEH expression was increased (252 percent above control) only in small pre-antral follicles. Since repeated exposure to VCD causes selective loss of primordial and small primary follicles, which are included in the small pre-antral fraction of follicles, this increase in mEH expression suggested that those vulnerable follicles were increasing their ability to detoxify VCD, in an attempt to escape VCD-induced follicle destruction. As previously mentioned, Mukhtar et al. (1978) identified the presence of glutathione-S-transferase in microsomes and cytosol obtained from rat whole ovarian homogenates. Studies by Maser et al. (1992) evaluated carbonyl reductase activity in rat ovaries utilizing the substrate metyrapone. Cytosolic enzyme activity was 15-fold and 12-fold greater in the ovary, compared to the liver, for Wistar and Sprague-Dawley rats, respectively. This suggests that ovarian metabolism may be important in the metabolism of carbonyl compounds that have “escaped their metabolic conversion by the liver.” Bostrom et al. (2000) measured functional activities for UDP-glucuronosyltransferases and sulfotransferases in cultured ovarian cells. Utilizing 1-naphthol as a substrate, various conjugated products were observed, with the glucuronide product being the major metabolite produced. Although several investigators have evaluated phase II metabolic enzymes in the rat ovary, these studies are unfortunately limited. As a result, the phase II metabolic enzymes are less characterized than the phase I metabolic enzymes. PAHs An example of ovarian metabolism in the rat ovary Ovarian metabolism is thought to play a critical role in the ovotoxic and carcino genic effects of several PAHs, including B(a)P, 7,12-dimethylbenzanthracene (DMBA), and 3-methylcholanthrene (3-MC). Since bioactivation of these compounds is required to form the metabolites responsible for tissue injury,

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Figure 7.2 Prototypical metabolic scheme for the polycyclic aromatic hydrocarbon, DMBA. The parent compound, DMBA, is bioactivated via cytochromes P450 and mEH to form the ultimate ovotoxic metabolite, DMBA-3, 4-diol-1, 2-epoxide (Miyata et al., 1999).

the response of the ovary may be dependent on metabolism (Figure 7.2). Studies by Jull et al. (1968) supported the role of ovarian metabolism in the toxicity of DMB A because when whole ovaries were treated with DMB A in vitro and subsequently transplanted into ovariectomized mice that were not previously exposed to DMBA, granulosa cell tumors formed in those ovaries. Subsequent studies revealed the formation of ovarian granulosa cell tumors in mice following exposure to B(a)P and 3-MC (Jull, 1973). Mattison and Thorgeirsson (1977)

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further evaluated the in vivo ovotoxic effects of 3-MC in mice. Their studies revealed a correlation between induction of ovarian aryl hydrocarbon hydroxylase activity and a decrease in the number of ovarian primordial follicles. When they compared primordial follicle loss following in vivo treatment with B(a)P, DMBA, or 3-MC, the most potent chemical in terms of follicle destruction, was DMBA, followed by 3-MC and B(a)P. These effects were doseand time-dependent. Additionally, follicle loss was inhibited when αnaphthoflavone (ANF), a known CYP inhibitor (Weibel et al., 1971), was coadministered with a PAH. However, these studies did not directly elucidate the role of ovarian metabolism in the resulting ovotoxicity. Thus, Shiromizu and Mattison (1984) investigated the effects of intraovarian injection of B(a)P on primordial follicle number and ovarian aryl hydrocarbon hydroxylase activity. Following unilateral injection of B(a)P into one ovary in mice, follicle destruction was only evident in the B(a)P-treated ovary. This primordial follicle destruction was dose- and time-dependent. Follicle loss was also inhibited when mice were pre-treated with ANF, via the i.p. route of administration. Additionally, aryl hydrocarbon hydroxylase activity was induced in those ovaries injected with B(a)P, relative to the other ovary that was injected with corn oil, serving as the vehicle control. These studies provided direct evidence for the role of ovarian metabolism (bioactivation) in B(a)P-induced ovotoxicity. Interestingly, the ovotoxic effects following PAH exposure demonstrate marked species variation in sensitivity. Following PAH exposure in mice, primordial follicles are destroyed and granulosa cell tumors develop after complete oocyte destruction (Jull, 1973). However, rats are more resistant to both follicle loss and tumor formation. This difference in species specificity is thought to be due to differences in hepatic and/or ovarian metabolism, since most PAHs are not directly toxic and thus require metabolic activation to reactive metabolites. Mattison (1979) suggested that the “decreased oocyte toxicity in the rat may reflect differences in monooxygenases, epoxide hydrases, or transferases.” Results of studies utilizing B(a)P as a substrate suggested that rat and mouse ovarian monooxygenases differ in the relative amounts of products formed. For instance, ovarian aryl hydrocarbon hydroxylase activity (measured in the S9 fraction) in naïve mice and rats was 7.0 and 3.1 pmol/min/ mg of protein, respectively. Furthermore, when B(a)P metabolism was directly evaluated, by quantification of metabolites formed, B(a)P metabolism was 3-fold greater in the mouse compared to the rat. Following in vivo treatment with 3MC, activity was induced in both species; however, activity was still greater in the mouse compared to the rat (19.6 versus 10.6 pmol/min/mg of protein, respectively). Pre-treatment with 3-MC induced ovarian B(a)P metabolism in both species; however, the mouse ovarian S9 enzymes still exhibited a 2.5 times greater capacity to metabolize B(a)P compared to the rat. Since the suspected ovotoxic and carcinogenic metabolite of B(a)P is the diol-epoxide metabolite (7, 8-diol-9,10-oxide), which requires both epoxide hydrase and monooxygenase activity, the differences in species sensitivity are not controlled by the activity of

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a single factor. Rather, there are likely a number of factors that result in differences in the bioactivation or detoxification of PAHs. Additionally, since multiple isoforms exist for both the monooxygenases and epoxide hydrases, it is not surprising that there are inherent species differences. Thus, it is likely the mouse and rat ovary generate different B(a)P metabolic profiles. Mattison et al. (1979) concluded “the lower rate of production of the 7,8-diol by rat ovarian monooxygenase is consistent with the greater resistance of the rat ovary to PAH ovotoxicity and ovarian carcinogenicity.” Although differences in metabolic enzymes in the ovary may partially explain differences in the species specificity to the PAHs, Mattison (1979) also suggests that other factors may be involved. For instance, mice are about ten times more susceptible to primordial oocyte destruction by ionizing radiation, than the rat. This difference in susceptibility is thought to be due to differences in oocyte chromatin, which is more diffusely organized in the mouse compared to the rat, thus making mouse oocytes more sensitive to destruction by ionizing radiation (Mattison, 1979). Genetic differences in various strains of the same species may also affect the outcome following PAH exposure. Studies by Mattison and Nightingale (1980) showed that C57BL/6N (B6N) mice were sensitive to the ovotoxic effects of PAHs, whereas DBA/2N (D2N) mice were resistant. These results were supported by a previous study in which Mattison and Thorgeirsson (1977) measured aryl hydrocarbon hydroxylase activity in ovaries from B6N and D2N mice following in vivo pre-treatment with 3-MC. 3-MC induced ovarian aryl hydrocarbon hydroxylase activity by 2–3-fold in B6N mice compared to control. No effect was seen in D2N mice following in vivo exposure to 3-MC. Mattison and colleagues (1979) concluded that the basic differences in sensitivity to oocyte destruction by the PAHs were due to differences in the balance between bioactivation and detoxification, as well as differences in repairing damaged DNA in oocytes. Studies by Bengtsson et al. (1983), further characterized the relative contribution of ovarian metabolism of B(a)P in the rat ovary, compared to metabolism by the liver and adrenal gland. Metabolite patterns, determined by HPLC, were similar in the ovary and adrenal gland. Both organs produced similar amounts of 9,10-diol and 7,8-diol, while the adrenal gland produced more of the 3-hydroxy and 9-hydroxy metabolites than the ovary. The quinone metabolites, produced by the liver, were not observed in the ovary or adrenal gland. These results further demonstrate the capacity of the ovary to bioactivate PAHs. Additionally, the ovary may be more sensitive to the effects of these chemicals, compared to the liver, possibly making the contribution of ovarian metabolism even more important. To further elucidate the regulatory mechanism of DMBA hydroxylase in the rat ovary, Bengtsson et al. (1987) evaluated several endogenous factors. In vivo administration of 17β-estradiol (E2) increased aryl hydrocarbon hydroxylase activity in ovarian microsomes (thought to be primarily composed of granulosa and theca cells), likely through induction. The synthetic estrogen,

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diethylstilbestrol (DES) also increased aryl hydrocarbon hydroxylase activity. Additionally, subcutaneous administration of the gonadotropins FSH and LH, as well as pregnant mare serum gonadotropin (PMSG), which stimulates both FSH and LH receptors, induced this enzyme activity via increased proliferation of the granulosa and theca cells. This finding suggests that most of the enzyme activity is confined to the large, antral follicles. Progesterone, prolactin, and growth hormone had no effect. Ovarian aryl hydrocarbon hydroxylase activity was only partially blocked in ovarian microsomes obtained from rats that were treated in vivo with the E2 antagonist, tamoxifen, which inhibits cell proliferation. Thus, there may be several mechanisms governing the regulation of aryl hydrocarbon hydroxylase activity in the rat ovary, or the rat ovary may contain multiple enzyme isoforms of the same or different CYP family (Bengtsson et al, 1987, 1992). Regardless, these intrinsic factors complicate our understanding of DMBA metabolism in the ovary, as well as the ovarian metabolism of other PAHs and xenobiotics. EXPRESSION OF METABOLIC ENZYMES IN THE MOUSE OVARY Recent studies have evaluated the ovarian expression of metabolic enzymes in species other than the rat. Using in situ hybridization, Dey et al. (1999) reported expression of mRNA encoding CYP1A1 in the mouse ovary, following pretreatment with the inducer, 3-MC. Expression appeared to be greater in the medulla than the cortex region of the ovary. Constitutive expression of either CYP1A1 or CYP1A2 was not detected. Other studies in the mouse ovary have evaluated the presence of mRNA, total protein, and functional protein for CYP2E1, CYP2A, CYP2B, and mEH (Cannady et al., 2002, 2003). These studies were unique in that enzyme expression (mRNA and total protein) was evaluated in distinct compartments of ovarian follicles and interstitial cells. mRNA analysis via realtime reverse transcriptase-polymerase chain reaction revealed that the enzyme isoforms evaluated were present in all follicle types and interstitial cells (Table 7.1). Total protein distribution, as evaluated by immunostaining and confocal microscopy, showed the presence of metabolic enzyme proteins in different sizes of follicles and interstitial cells in the mouse ovary. Enzymes were distributed in oocytes, granulosa cells, theca cells, and interstitial cells. Interestingly, there was a high level of protein in the interstitial cells for all enzymes evaluated. This suggests an important metabolic function for the interstitial cells, a cell type whose function has largely remained unclear. Studies utilizing model substrates (p-nitrophenol, coumarin, 7-ethoxy-4-trifluormethyl coumarin, cis-stilbene oxide) further determined that CYP2E1, CYP2B, and mEH were functional in the mouse ovary. However, due to assay detection limitations, only mEH activity could be evaluated in distinct ovarian compartments (Cannady et al., 2002, 2003).

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Table 7.1 mRNA expression of CYP450 isoforms and mEH in distinct ovarian fractions from untreated mice

Notes a Arbitrary values expressed as a ratio of mRNA: 18S rRNA. b Different from CYP2E1 interstitial tissue (p < 0.05). c Different from mEH in other fractions (p < 0.05).

4-VINYLCYCLOHEXENE (VCH) AS A MODEL OVARIAN TOXICANT Effects on biotransformation enzymes in the mouse ovary The role of ovarian metabolism in PAH-induced ovotoxicity and/or carcinogenicity forces one to consider the role of ovarian metabolism for other ovotoxic compounds, such as VCH (Figure 7.3). Evaluating the role of ovarian metabolism in VCH-induced ovotoxicity is interesting for several reasons. First, this industrial intermediate used in the manufacture of flame retardants, insecticides, and plasticizers, causes ovarian toxicity in mice, but not rats. However, the VCH-monoepoxides and the diepoxide metabolite, VCD, the ultimate ovotoxic metabolite, are ovotoxic in both species (Smith et al., 1990a,b; Doerr et al., 1995). This species-specific response may be explained by differences in metabolism in which the mouse may exhibit a greater ability to bioactivate VCH (likely via hepatic CYP isoforms 2A, 2B, and 2E1) and a lesser ability to detoxify VCD (via mEH) (Smith et al, 1990c; Keller et al., 1997; Doerr-Stevens et al, 1999). Additionally, VCH selectively targets and destroys the small pre-antral (primordial and primary) follicles. Therefore, utilizing VCH as a model compound to evaluate the role of ovarian metabolism is ideal, since it is not a general ovarian toxicant, affecting all follicle types. Studies by Cannady et al. (2002, 2003) suggested that repeated exposure to VCH (15 d) had varying effects on enzyme expression (mRNA and total protein) and functional activities for CYP2A, CYP2B, CYP2E1, and mEH, in distinct ovarian fractions (small pre-antral follicles, 25–100 µm; large pre-antral follicles, 100–250 µm; antral follicles, >250 µm; interstitial tissue) or whole ovarian homogenates obtained from mice. Repeated daily dosing with VCH increased mRNA encoding various metabolic enzymes in the targeted small preantral follicles, compared to control (Table 7.2). VCH dosing also increased (p<0.05) mRNA encoding CYP2E1 in non-targeted antral follicles (168 percent

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Figure 7.3 Proposed scheme for hepatic metabolism of VCH. The parent compound, VCH, is bioactivated via cytochromes P450 to form the mono- and di-epoxide metabolites. VCD is the ultimate ovarian toxicant. Hydrolysis by microsomal epoxide hydrolase results in detoxification (Keller et al, 1997).

above control) and CYP2A in interstitial tissue (207 percent above control), suggesting that the vulnerable population of follicles is not necessarily responsible for either the bioactivation of VCH or the detoxification of VCD. Instead, non-targeted ovarian compartments have the enzymatic machinery to participate in metabolic reactions associated with VCH/VCD-induced ovarian toxicity. VCH dosing altered immunostaining intensity in interstitial cells for CYP2E1 and CYP2A, in granulosa cells of small pre-antral and antral follicles for CYP2B, and in theca cells for mEH (Cannady et al, 2002, 2003). Pretreatment of mice with VCH increased functional activity for CYP2E1 (p < 0.05) in whole ovarian homogenates, as determined by the hydroxylation of pnitrophenol (149 percent above control). Specific activity for mEH, measured by the hydrolysis of [3H]-cis-stilbene oxide, was increased in small pre-antral follicles following VCH dosing (381 percent above control). Although the

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Table 7.2 Effect of VCH dosing on mRNA expression of metabolic enzymes in small preantral follicles from micea

Notes a VCH dosing regimen: 7.4 mmol/kg/d; 15d. b Values expressed as a ratio of CYP450 mRNA: 18S ribosomal RNA. c Different from control (p < 0.05).

relative contribution of ovarian metabolism, compared to hepatic metabolism, in VCH-induced ovotoxicity is not known, these studies have demonstrated that the mouse ovary has the capacity to be involved in both bioactivation of VCH and the subsequent detoxification of its epoxides. Additionally, an important finding was the large metabolic potential for the interstitial cells. These highly vascularized cells, that are likely exposed to toxicants present in the circulation, could be the first line of defense against chemical insult in the ovary. In other words, one of their functions may be to protect the finite population of follicles from blood-borne chemicals. OVARIAN EXPRESSION OF BIOTRANSFORMATION ENZYMES IN OTHER SPECIES Although less characterized than in rodents, the expression of xenobioticmetabolizing enzymes has also been studied in ovaries from other species. Leighton et al. (1995) evaluated the expression of CYP1A1 in porcine granulosa cells. Gene expression was only observed in cultured primary granulosa cells obtained from developing, not immature, follicles. However, in the presence of 3MC, mRNA encoding CYP1A1 was induced in cultured primary granulosa cells obtained from both developing and immature follicles. Expression of CYP1A1 was also detected in growing, but not immature, follicles in vivo. Taken together, the authors concluded that CYP1A1 is developmentally regulated. Studies by Zaphiropoulos et al. (1995) further evaluated expression of several CYP2C isoforms at the mRNA level in porcine ovaries. Based on structural identity, two forms were identified. Unfortunately, it is not known how this information correlates to levels of the corresponding proteins, or whether these isoforms participate in xenobiotic metabolism. Information regarding expression of biotransformation enzymes in ovaries from non-human primates is lacking.

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EXPRESSION OF METABOLIC ENZYMES IN THE HUMAN OVARY As is evident in this review, there are several differences in ovarian metabolic capabilities across species. Additionally, the available information is not all inclusive. Since in many toxicology studies, researchers must extrapolate results obtained from rodent studies to predict what may happen in humans following exposure to a variety of chemicals, it would be beneficial to add human data to this species comparison. In the past several years, a number of investigators have characterized the metabolic enzymes present in the human ovary. By immunohistochemical analysis, McFadyen et al. (2001) evaluated the presence of CYP1B1 protein in primary and metastatic ovarian cancer. Interestingly, CYP1B1 staining was increased in 92 percent of the cancerous ovarian tissues that were investigated. There was a strong correlation between the presence of CYP1B1 and both stages of ovarian cancer. CYP1B1 could not be detected in normal, non-cancerous ovaries. However, this remains controversial, since Muskhelishvili et al. (2001) also evaluated mRNA and total protein expression of CYP1B1 in human ovary by in situ hybridization and immunohistochemistry, respectively. Both message and protein were present in normal ovary, with protein being localized in cell nuclei. Yokose et al. (1999) examined localization of CYP2C and CYP3A in a variety of non-neoplastic and neoplastic ovarian tissues. Immunohistochemical analysis revealed staining for CYP3A in the corpus luteum of non-neoplastic ovaries. Klose et al. (1999) have detected the presence of mRNA encoding CYP2C8 and CYP2C9 in human ovary. Recent studies evaluated mEH in the human ovary. By immunohistochemical analysis, Coller et al. (2001) showed that mEH is highly expressed throughout the ovary. Additional studies by Hattori et al. (2000) detected mEH in granulosa cells and theca cells, also by immunohistochemistry. In vitro studies utilizing the mEH inhibitor, l,2-epoxy-3,3,3-trichloropropane, caused a dose-dependent decrease in the production of 17β-estradiol. Thus, mEH may also be involved in different ovarian steroidogenic pathways. Most importantly, however, these findings support that xenobiotic-metabolizing enzymes are expressed in the human ovary and may play important roles in human ovarian function. Further characterization of metabolic enzyme profiles in humans would prove useful. mRNA expression, total protein, and functional activity could be determined in different cell types of the human ovary, since samples can be obtained from in vitro fertilization clinics. Furthermore, in vitro metabolism of a variety of compounds could be monitored by metabolite formation and identification. Such human information would prove invaluable, as relative risks to women exposed to a variety of chemicals in the workplace and environment could be determined.

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Figure 7.4 Schematic representing impact of ovarian metabolism on toxicity. Ovarian toxicity may be governed by presence/absence of metabolic enzymes necessary to metabolize chemicals to toxic metabolites or to deactivate toxic metabolites. (A) Toxic metabolite X is biotransformed in the liver and delivered to the ovary via the circulation. The toxic metabolite can be directly toxic to specific follicle populations or undergo subsequent ovarian metabolism to more toxic or less toxic products. (B) Compound X is biotransformed to toxic metabolite by susceptible follicle. (C) The metabolite of compound X is toxic to primordial follicles. Metabolism occurs in non-targeted ovarian compartments. (D) Toxic metabolite X is biotransformed to inactive metabolite, thus protecting from toxic insult.

MECHANISMS OF OVARIAN METABOLISM IN CHEMICALLY INDUCED OVOTOXICITY Whether an ovotoxic chemical must be biotransformed to the toxic metabolite or not, metabolic enzymes (involved in bioactivation and detoxification reactions) present in different ovarian follicle and cell types, may ultimately play a role in whether this toxic insult will or will not occur (Figure 7.4). Conceptually, there are four basic metabolic models that can be used to describe ovotoxicity. In the first model (Figure 7.4A), the xenobiotic is either directly toxic to the ovary or is metabolized in the liver to the toxic metabolite, prior to presentation to the ovary via the circulation. Toxicity occurs after delivery of the toxicant to the ovary. An example of a xenobiotic that is directly toxic to the ovary is VCD (Smith et al., 1990a,b; Doerr et al., 1995). A compound that requires hepatic bioactivation prior to causing ovotoxicity is VCH, which is epoxidated to form the ultimate ovotoxicant, VCD (Smith et al, 1990a,b; Doerr et al, 1995).

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The second and third model resulting in ovotoxicity require ovarian bioactivation of the chemical to the toxic metabolite (Figure 7.4B,C). This biotransformation can occur in either targeted or non-targeted follicle/cell populations. For example, a compound may only be destructive to secondary follicles, due to the presence, or lack of, metabolic activity only in this population of follicles. This selectivity makes the secondary follicles more vulnerable to insult compared to other follicle types. However, on the other hand, a non-targeted population of cells or follicles can also be involved in metabolic reactions. For instance, a mature antral follicle could metabolize a compound to the toxic metabolite, even though the metabolite is specifically toxic to primordial follicles, not antral follicles or other cell types. Due to the close proximity of metabolite formation, the active metabolite could travel (via diffusion) to the nearby primordial follicles, resulting in toxicity. Examples of compounds that must be bioactivated in the ovary to cause ovarian injury include the PAHs. Currently, it is not known whether ovarian bioactivation occurs in the targeted or non-targeted follicles. Although the mechanism of ovarian toxicity is not completely elucidated, the model ovotoxicant, VCH, or the monoepoxide metabolites of VCH, may also be bioactivated in the ovary to the ultimate diepoxide metabolite, likely by nontargeted interstitial cells (Cannady et al., 2003). The fourth model of ovarian metabolism involves detoxification of an ovotoxicant. In this scenario, a targeted or non-targeted follicle/cell population could biotransform a chemical to a non-toxic species (Figure 7.4D). For instance, a primordial follicle could detoxify a compound, thus protecting itself from toxic insult. Likewise, an antral follicle could detoxify an ovotoxicant, thereby protecting the vulnerable primordial follicles. VCD is an example of a compound that is detoxified by mEH in the targeted primordial follicles, in an attempt to escape follicle destruction (Springer et al, 1996; Cannady et al, 2002). Taken together, there are various metabolic scenarios in the biotransformation and bioactivation of ovarian toxicants. Furthermore, the heterogeneity of the ovary presents many complexities when one considers metabolic pathways. Understanding the metabolic potential of the ovary, and more specifically that of the different populations of follicles within the ovary, will help explain such mechanisms and provide insight to reproductive toxicologists. CONCLUSION In conclusion, a variety of biotransformation enzymes are present in ovaries from several species. These enzyme systems (both bioactivation and detoxification) need further characterization at the message, protein, and activity levels to provide a greater understanding of regulation in different species. Additionally, the impact of a variety of environmental factors on these enzyme systems needs to be evaluated. The resulting knowledge will help researchers evaluate both regulatory mechanisms of ovarian toxicity and extrahepatic drug metabolism.

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Such information would serve to better elucidate the metabolic role of the ovary in responding to xenobiotic exposures. ACKNOWLEDGEMENTS The authors would like to thank Dr John-Michael Sauer and Ms Karla Hayes for their editing and administrative assistance. REFERENCES Bengtsson, M. and Rydstrom, J. (1983) Regulation of carcinogen metabolism in the rat ovary by the estrous cycle and gonadotropin, Science, 219:1437–38. Bengtsson, M., Montelius, J., Mankowitz, L. and Rydstrom, J. (1983) Metabolism of polycyclic aromatic hydrocarbons in the rat ovary, Biochem. Pharmacol., 32:129– 36. Bengtsson, M., Dong, Y., Mattison, D.R. and Rydstrom, J. (1987) Mechanisms of regulation of rat ovarian 7,12 dimethylbenz(a)anthracene hydroxylase, Chem. Biol. Interact., 63:15–27. Bengtsson, M., Hallberg, E., Georgellis, A. and Rydstrom, J. (1990) On the identity of xenobiotic-metabolizing form(s) of cytochrome P-450 in endocrine organs, Cancer Lett., 52:235–41. Bengtsson, M., Reinholt, F.P. and Rydstrom, J. (1992) Cellular localization and hormonal regulation of 7,12 dimethylbenz(a)anthracene mono-oxygenase activity in the rat ovary, Toxicology, 71:203–22. Bostrom, M., Becedas, L. and DePierre, J.W. (2000) Conjugation of 1-naphthol in primary cell cultures of rat ovarian cells, Chem. Biol Interact., 124:103–18. Cannady, E.A., Dyer, C.A., Christian, P.J., Sipes, I.G. and Hoyer, P.B. (2002) Expression and activity of microsomal epoxide hydrolase in follicles isolated from mouse ovaries, Toxicol. Sci., 68:24–31. Cannady, E.A., Dyer, C.A., Christian, P.J., Sipes, I.G. and Hoyer, P.B. (2003) Expression and activity of cytochromes P450 2E1, 2A, and 2B in the mouse ovary: the effect of 4-vinylcyclohexene and its diepoxide metabolite, Toxicol. Sci., 73:423–30. Coller, J.K., Fritz, P., Zanger, U.M., Siegle, I., Eichelbaum, M., Kroemer, H.K. and Murdter, T.E. (2001) Distribution of microsomal epoxide hydrolase in humans: an immunohistochemical study in normal tissues, and benign and malignant tumors, Histochem. J., 33:329–36. Dannan, G.A. and Guengerich, F.P. (1982) Immunochemical comparison and quantitation of microsomal flavin-containing monooxygenases in various hog, mouse, rat, rabbit, dog, and human tissues, Mol. Pharmacol., 22:787–94. Dasmahapatra, A.K., Wimpee, B.A.B., Trewin, A.L. and Hutz, R.J. (2001) 2,3,7,8Tetrachlorodibenzo-p-dioxin increases steady state estrogen receptor-β mRNA levels after Cyp 1A1 and Cyp 1B1 induction in rat granulosa cells in vitro, Mol. Cell Endrocrinol., 182:39–48. Dasmahapatra, A.K., Trewin, A.L. and Hutz, R.J. (2002) Estrous cycle-regulated expression of Cyp 1B1 mRNA in the rat ovary, Comp. Biochem. Physiol B Biochem. Mol. Biol., 133:127–34.

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Dey, A., Jones, J.E. and Nebert, D.W. (1999) Tissue- and cell type-specific expression of cytochrome P450 1 A1 and cytochrome P450 1A2 mRNA in the mouse localized in situ hybridization, Biochem. Pharmacol., 58:525–37. Doerr, J.K., Hooser, S.B., Smith, B.J. and Sipes, I.G. (1995) Ovarian toxicity of 4-vinylcyclohexene and related olefins in B6C3F1 mice: role of diepoxides, Chem. Res. Toxicol., 8:963–69. Doerr-Stevens, J.K., Liu, J., Stevens, G.J., Kraner, J.C., Fontaine, S.M., Halpert, J.R. and Sipes, I.G. (1999) Induction of cytochrome P-450 enzymes after repeated exposure to 4-vinylcyclohexene in B6C3F1 mice, Drug Metab. Dispos., 27:281–87. Hattori, N., Fujiwara, H., Maeda, M., Fujii, S. and Ueda, M. (2000) Epoxide hydrolase affects estrogen production in the human ovary, Endocrinology, 141:3353–65. Jull, J.W. (1973) Ovarian tumorigenesis, Methods Cancer Res., 7:131–86. Jull, J.W., Hawryluk, A. and Russell, A. (1968) Mechanism of induction of ovarian tumors in the mouse by 7,12 dimethylbenz(a)anthracene: tumor induction in organ culture, J. Natl. Cancer Inst., 40:687–706. Karara, A., Makita, K., Jacobson, H.R., Falck, J.R., Guengerich, F.P., DuBois, R.N. and Capdevila, J.H. (1993) Molecular cloning, expression, and enzymatic characterization of the rat kidney cytochrome P-450 arachidonic acid epoxygenase, /. Biol. Chem., 268:13565–70. Keller, D.A., Carpenter, S.C., Cagen, S.Z. and Reitman, F.A. (1997) In vitro metabolism of 4-vinylcyclohexene in rat and mouse liver, lung, and ovary, Toxicol. Appl Pharmacol., 144:36–44. Klose, T.S., Blaisdell, J.A. and Goldstein, J.A. (1999) Gene structure of cyp 2C8 and extrahepatic distribution of the human cyp2Cs, /. Biochem. Mol. Toxicol., 13:289– 95. Krishna, D.R. and Klotz, U. (1994) Extrahepatic metabolism of drugs in humans, Clin. Pharmacokinet., 26:144–60. Leighton, J.K., Canning, S., Guthrie, H.D. and Hammond, J.M. (1995) Expression of cytochrome P450 1A1, an estrogen hydroxylase, in ovarian granulosa cells is developmentally regulated, J. Steroid Biochem. Mol. Biol., 52:351–56. Maser, E., Hoffmann, J.G., Friebertshauser, J. and Netter, K.J. (1992) High carbonyl reductase activity in adrenal gland and ovary emphasizes its role in carbonyl compound detoxication, Toxicology, 74:45–56. Mattison, D.R. and Nightingale, M.R. (1980) The biochemical and genetic characteristics of murine ovarian aryl hydrocarbon (benzo(a)pyrene) hydroxylase activity and its relationship to primordial oocyte destruction by polycyclic aromatic hydrocarbons, Toxicol. Appl. Pharmacol., 56:399–408. Mattison, D.R. and Thorgeirsson, S.S. (1977) Genetic differences in mouse ovarian metabolism of benzo(a)pyrene and oocyte toxicity, Biochem. Pharmacol., 26:909–12. Mattison, D.R. (1979) Difference in sensitivity of rat and mouse primordial oocytes to destruction by polycyclic aromatic hydrocarbons, Chem. Biol. Interact., 28:133–37. Mattison, D.R., West, D.M. and Menard, R.H. (1979) Differences in benzo(a)pyrene metabolic profile in rat and mouse ovary, Biochem. Pharmacol., 28:2101–04. McFadyen, M.C., Cruickshank, M.E., Miller, I.D., McLeod, H.L., Melvin, W.T., Haites, N.E., Parkin, D. and Murray, G.I. (2001) Cytochrome P450 cyp1B1 over-expression in primary and metastatic ovarian cancer, Br. J. Cancer, 85:242–46. Miyata, M., Kudo, G., Lee, Y., Yang, T.J., Gelboin, H.V., Fernandez-Salguero, P., Kimura, S. and Gonzalez, F.J. (1999) Targeted disruption of the microsomal

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epoxide hydrolase gene: microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12 dimethylbenz(a)anthracene, J. Biol Chem., 274:23963– 68. Mukhtar, H., Philpot, R.M. and Bend, J.R. (1978) The postnatal development of microsomal epoxide hydrase, cytosolic glutathione S-transferases, and mitochondrial and microsomal cytochrome P450 in adrenals and ovaries of female rats, Drug Metab. Dispos., 6:577–83. Muskhelishvili, L., Thompson, P.A., Kusewitt, D.F., Wang, C. and Kadlubar, F.F. (2001) In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues, /. Histochem. Cytochem., 49:229–36. Oesch, F., Glatt, H. and Schimassmann, H. (1977) The apparent ubiquity of epoxide hydratase in rat organs, Biochem. Pharmacol., 26:603–07. Omura, T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes, J. Biol Chem., 239:2370–85. Parkinson, A. (1996) Biotransformation of xenobiotics. In: C. Klaassen (ed.), Casarett and Doull’s Toxicology: The Basic Science of Poisons, McGraw-Hill, New York, pp. 113–86. Shiromizu, K. and Mattison, D.R. (1984) The effect of intraovarian injection of benzo(a) pyrene on primordial oocyte number and ovarian aryl hydrocarbon [benzo(a)pyrene] hydroxylase activity, Toxicol. Appl. Pharmacol., 76:18–25. Sipes, I.G. and Gandolfi, A.J. (1991) Biotransformation of toxicants. In: M.A. Amdur, J. Doull and C. Klaassen (eds), Casarett and Doull’s Toxicology: The Basic Science of Poisons, Elmsford, New York: Pergamon Press, Inc., pp. 88–126. Smith, B.J., Carter, D.E. and Sipes, I.G. (1990a) Comparison of the disposition and in vitro metabolism of 4-vinylcyclohexene in the female mouse and rat, Toxicol. Appl. Pharmacol., 105:364–71. Smith, B.J., Mattison, D.R. and Sipes, I.G. (1990b) The role of epoxidation in 4-vinylcyclohexene-induced ovarian toxicity, Toxicol. Appl. Pharmacol., 105:372–81. Smith, B.J., Sipes, I.G., Stevens, J.C. and Halpert, J.R. (1990c) The biochemical basis for the species difference in hepatic microsomal 4-vinylcyclohexene epoxidation between female mice and rats, Carcinogenesis, 11:1951–57. Springer, L.N., McAsey, M.E., Flaws, J.A., Tilly, J.L., Sipes, I.G. and Hoyer, P.B. (1996) Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats, Toxicol. Appl Pharmacol., 139:394–401. Van Voorhis, B.J., Dunn, M.S., Falck, J.R., Bhatt, R.K., VanRollins, M. and Snyder, G.D. (1993) Metabolism of arachidonic acid to epoxyeicosatrienoic acids by human granulosa cells may mediate steroidogenesis, J. Clin. Endocrinol. Metab., 76:1555– 59. Weibel, F.J., Leutz, J.C., Diamond, L. and Gelboin, H.V. (1971) Aryl hydrocarbon (benzo (a)pyrene) hydroxylase in microsomes from rat tissues: differential inhibition and stimulation by benzoflavones and organic solvents, Arch. Biochem. Biophys., 144:78– 86. Wrighton, S.A. and Stevens, J.C. (1992) The human hepatic cytochromes P450 involved in drug metabolism, Crit. Rev. Toxicol., 22:1–21. Yokose, T., Doy, M., Taniguchi, T., Shimada, T., Kakiki, M, Horie, T., Matsuzaki, Y. and Mukai, K. (1999) Immunohistochemical study of cytochrome P450 2C and 3A in human non-neoplastic and neoplastic tissues, Virchows Arch., 434:401–11.

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Zaphiropoulos, P.G., Skantz, A., Eliasson, M. and Bengtsson-Ahlberg, M. (1995) Cytochrome P450 genes expressed in porcine ovaries: identification of novel forms, evidence for gene conversion, and evolutionary relationships, Biochem. Biophys. Res. Commun., 212:433–41.

8 PLACENTAL INDUCTION OF OVARIAN TOXICITY Jennifer L.Marcinkiewicz

INTRODUCTION The ovary plays a pivotal role, not only in continuation of the species, but also in producing hormones such as estradiol, that are important in maintaining good physical and mental health. It is therefore increasingly important to understand the environmental factors that adversely impact ovarian development and function. It is even more important as we live in a society that is increasingly industrialized, with greater opportunity for exposure to environmental pollutants, occupational hazards, pharmaceutical agents and food additives. Most of our attention has heretofore been focused upon direct hazards to ovarian function, but serious effects on offspring by a maternal route of exposure also exist. In some ways, the potential for adverse impact may be even greater via transplacental effects due to effects occurring during critical “windows” of fetal development. OVERVIEW OF OVARIAN DEVELOPMENT AND FUNCTION Ovarian development is a complex process which involves intricate intra-cellular communication. Several of the developmental milestones have previously been reviewed (Mauleon, 1978; Zamboni, 1989; Findlay, 1991; Hirshfield, 1991). The first challenge in development is migration of primordial germ cells from the hindgut region of the yolk sac to the indifferent genital ridge. After the germ cells reach the genital ridge they continue to undergo proliferation, while somatic cells are also rapidly dividing. In the absence of a Y chromosome, the cortical regions of the indifferent gonad proliferate to become ovarian somatic tissue. Additionally, the mesonephric epithelium forms cords termed the rete ovarii (Byskov and Lintern-Moore, 1973; Peters and McNatty, 1980). Oogonia continue to proliferate until they enter into meiosis (embryonic day 17 in rodents; 3 months in human fetus (Peters and McNatty, 1980)) at which point

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they are considered to be oocytes. Meiosis is arrested in the first meiotic prophase and does not resume until the oocyte undergoes the ovulatory surge of luteinizing hormone (LH). Assembly of the primordial follicle occurs in the human fetus at 4.5 months gestational age (Peters and McNatty, 1980); whereas in the rat and mouse, primordial follicles are not observed until a day after birth (Beaumont and Mandl, 1962; Ueno et al, 1989; Hirshfield, 1991). During the interval between meiotic arrest and primordial follicle assembly, another important event occurs, namely oocyte atresia. This short period of time encompasses the most significant attrition of female germ cells, with 60–70% oocyte loss reported in rodents (Beaumont and Mandl, 1962) and a 7-fold reduction in germ cells in humans (Tilly, 1998). Loss of oocytes also occurs after puberty during each reproductive cycle, but this loss pales in comparison with the fetal/neonatal attrition rate. Once the follicle/oocyte pool is depleted, ovarian senescence (menopause in humans) ensues (Gosden and Faddy, 1998). Primordial follicles are initially recruited into the growing pool by the action of intraovarian factors (reviewed by Kezele et al., 2002). Further growth of follicles containing multiple layers of granulosa cells and a theca layer, requires the involvement of the gonadotropins, follicle-stimulating hormone (FSH) and LH (reviewed in McGee and Hsueh, 2000). The gonadotropins, in concert with intraovarian growth factors, such as IGF-I, ultimately produce a mature Graafian follicle. The maturing follicle differentiates so that it produces high concentrations of estradiol, which acts to further stimulate follicle growth and to trigger the LH surge. The LH surge serves several purposes: (1) resumption of meiosis in the oocyte; (2) luteinization of the follicle; and (3) rupture of the follicle. The granulosa and theca cells remaining in the ovary after ovulation differentiate into functional luteal cells. LH remains important in stimulating luteal steroid production. Thus, the cyclic recruitment of follicles depends on a functional hypothalamic-pituitary axis and on female sexual differentiation of the hypothalamus. Ovarian toxicology may be viewed either in the strictest sense, focusing on direct effects on the ovary, or more broadly by examining effects on other targets that have a subsequent negative impact upon the ovary. Several studies have shown deleterious effects of environmental chemicals on the population of primordial follicles/oocytes; however in many cases the mechanisms have not yet been elucidated, and frequently these studies have only examined post-natal administration of compounds. These chemicals may act either by inducing apoptosis or necrosis or by interfering with the process of primordial follicle development. Other direct effects on the ovary include ovotoxicity, changes in follicular growth, impaired ovulation, alterations in steroidogenic pathways and increased incidence of ovarian cancer. Indirect effects that would likely result in an adverse effect on the ovary include actions on the hypothalamus, pituitary or peripheral nervous system.

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MATERNAL-FETAL TRANSFER Transplacental induction of ovarian toxicity depends upon the timing of administration of the toxicant and the amount of toxicant ultimately reaching the fetus. Because maternal and fetal circulation is discontinuous, transplacental transfer is governed to a large extent by the transport mechanisms that exist in the various layers of cells comprising the placenta. In humans and most laboratory animal models (rats, mice, hamsters, and guinea pigs), the placental structure is hemochorial (Faber and Thornburg, 1983; reviewed in Page, 1993). Hemochorial placentae are those in which maternal blood “bathes” the outermost layer of the trophoblast in such a way that substances in maternal blood cross the trophoblast layer(s) and pass through the fetal capillary endothelium to reach the fetal circulation. In humans, there is a single layer of syncytial trophoblast cells (hemomonochorial), whereas in rodents there are two layers of syncytial trophoblast cells in addition to a nonsyncytial layer of trophoblast cells (hemotrichorial). The overall shape of placentae in humans and rodents is discoid, or circular in shape. Placental structure and blood circulation influence transfer between fetal and maternal circulation (reviewed in Page, 1993; Clarke, 1997). The thickness of the placenta presents an obstacle that acts to decrease the rate of transfer across the placenta; however the amount of transfer is positively influenced by an increase in placental surface area. Increased placental blood flow also stimulates transfer. Placental blood flow is influenced by the degree of vascularization of the placenta, blood volume, cardiac output, and radius of the placental blood vessels. For example, exposure of the pregnant animal to a vasoactive compound is likely to alter placental blood flow and transplacental transfer. Furthermore, transfer of toxicants across the placenta is highly dependent upon the characteristics of the particular chemical to be transported (reviewed in Page, 1993; Clarke, 1997). Of particular importance are lipid solubility, charge, and molecular weight. Lipophilic compounds diffuse across membranes, following their concentration gradient between maternal and fetal blood. Small, uncharged, hydrophilic compounds are also generally able to diffuse across membranes. Other compounds cross the membrane via specific transport processes, such as carrier-mediated transport or endocytosis. In carrier-mediated transport mechanisms (active transport or facilitated diffusion), transfer is limited by availability of specific carrier proteins in placental membranes. Transfer of a substance between fetal and maternal blood is also highly dependent upon the concentration of that substance in maternal blood. Maternal concentrations of a toxicant depend upon the balance between absorption, metabolism, and elimination (reviewed in Clarke, 1997). There are several potential routes of exposure, namely through ingestion, dermal inhalation and direct introduction into the blood (e.g. injections of medications, etc.). Metabolism may render a given compound less active, or in some cases such as 4-vinylcyclohexanc (VCH) (Hoyer et al., 2001), actually increases the toxic

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effects of the chemical. Drugs are enzymatically altered by metabolism in the gastrointestinal system, the liver, lungs, and placenta, as well as other sites. Primary routes of elimination are excretion in urine or in feces via a biliary route. Many chemicals are also passed into the milk during lactation. This is particularly important as it provides another route for maternal transfer of toxicant to offspring. Almost all physiological systems undergo significant changes during pregnancy, altering the absorption and elimination of toxicants in the mother. Examples include changes in the cardiovascular system, kidney, gastrointestinal system, and adipose tissue (Clarke, 1997). This makes it extremely challenging to build pharmacokinetic models of tissue distribution. EVIDENCE FOR TRANSPLACENTAL INDUCTION OF OVARIAN TOXICITY Estrogenic compounds Estrogenic compounds are either closely related estrogenic steroids, such as estrone estriol, and ethinyl estradiol (found in many oral contraceptives) or are non-steroidogenic compounds that have the ability to bind and activate the estrogen receptor, such as many pesticides (methoxychlor), phytoestrogens (genistein), and plasticizers (bisphenol A) (De Rosa et al., 1998). Diethylstilbestrol (DES) is an estrogenic steroid that was administered in the 1940s through the 1960s to prevent miscarriage (reviewed in Hendry et al, 2002). In 1971, Herbst et al. first described an increased incidence of vaginal clear-cell adenocarcinoma in women pre-natally exposed to DES. As is well known today, this tragic medical error has resulted in both female and male offspring suffering an increased risk of various types of cancer, reproductive tract abnormalities and infertility (reviewed in Hendry et al., 2002). Adverse effects of pre-natal administration of DES on the ovary were first reported in the late 1970s and early 1980s (Napalkov and Anisimov, 1979; Newbold and McLachlan, 1979; Newbold et al., 1983; Haney et al., 1984). Ovarian consequences of pre-natal exposure to DES include a higher incidence of ovarian cysts and tumors (Napalkov and Anisimov, 1979; Newbold and McLachlan, 1979; Newbold et al, 1983; Kitamura et al, 1999), and polyovular follicles (Iguchi and Takasugi, 1986; Iguchi et al, 1986, 1990; Hendry et al, 2002). The presence of polyovular follicles and ovarian cysts, accompanied by few corpora lutea (Wordinger and Highman, 1984; Hendry et al., 2002), points to ovulation failure. The ovarian consequences of pre-natal DES exposure, coupled with developmental defects in the reproductive tract likely account for much of the infertility of exposed women. Methoxychlor is a chlorinated insecticide that has been used extensively on food crops (in Chapin et al., 1997). Post-natal exposure to methoxychlor disrupts estrous cycles and fertility of female rats (Gray et al, 1989). Pre-natal exposure of

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mice (Swartz and Corkern, 1992) and rats (Chapin et al., 1997) to methoxyclor also appears to significantly impact many parameters of female reproduction in exposed offspring. Ovarian effects include increased incidence of follicular atresia (Swartz and Corkern, 1992), decreased number of corpora lutea, cystic follicles, and decreased progesterone concentrations on estrus (Chapin et al., 1997). The decreased numbers of corpora lutea (and consequently low progesterone on estrus) were attributed to ovulatory failure rather than inhibition of follicle growth, since estradiol concentrations on proestrus in exposed animals were not different from controls (Chapin et al, 1997). Bisphenol A is a monomer that is used in plastic manufacture and that is said to leach from plastics and resins (Rubin et al., 2001) thus providing a significant route of oral exposure in humans. Most studies of the pre-natal effects of this compound seem to have focused on the uterotrophic aspects of estrogen action; however there is some evidence for ovarian toxicity as well. In rats exposed in utero (gestation day 6 through term) and throughout lactation, approximately 80% of bisphenol A-exposed pups exhibited abnormal estrous cycles (Rubin et al., 2001). Gestational/lactational exposure to bisphenol A also significantly suppressed plasma LH concentrations after long-term ovariectomy, suggesting alterations of anterior pituitary function. Exposure to bisphenol A in mice on gestation days 10–18 caused a decreased number of corpora lutea in ovaries at 30 days after birth (Suzuki et al, 2002), suggesting either an inhibition of ovulation or a decrease in the number of antral follicles formed. Overall, the estrogenic compounds often appear to inhibit ovulation, impair pituitary function and reduce overall female fertility. Polyaromatic hydrocarbons (PAHs) The group of chemicals termed PAHs include both halogenated and nonhalogenated compounds that bind to the intracellular aryl hydrocarbon receptor (AhR). The AhR is a member of the per-arnt-sim (PAS) family of proteins and acts as a transcription factor for genes containing a dioxin-response element (reviewed in Safe, 2001). Prior to ligand binding, the AhR is localized within the cytoplasm bound to two heat shock proteins (hsp90). Upon ligand binding, there is dissociation of the heat shock proteins and association of another protein, arnt (aryl hydrocarbon nuclear translocation) protein. The ligand-receptor complex then binds to target DNA and alters gene transcription. The AhR is known to target many genes that are estrogen responsive and it has often been characterized as an “anti-estrogen.” Activation of the AhR also initiates transcription of the P450 drug-metabolizing enzymes such as Cyp1a1 (reviewed in Ma, 2001). Many ligands that bind to the AhR have profound effects on primordial follicle numbers (discussed below), an effect that fits well with the observation that genetic deletion of the AhR causes a significant increase in the number of primordial follicles observed shortly after birth (Benedict et al., 2000; Robles et al., 2000).

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Non-halogenated PAHs Several polycyclic hydrocarbon compounds are known to be ovotoxic, including 9,10-dimethyl-1,2-benzanthracene (DMBA), 3-methylcholanthrene and benzo(a) pyrene (B(a)P) (Mattison, 1979; Mattison et al, 1980). Several previous studies have demonstrated transplacental transfer of B(a)P (Mackenzie and Angevine, 1981; Legraverend et al, 1984; Neubert and Papken, 1988; McCabe and Flynn, 1990; Rodriguez et al., 1999). B(a)P is a persistent bioaccumulating toxin that is listed as a priority chemical by the EPA. It is generated by combustion of fuel, coal and cigarettes, and is found in fish and meats that have been smoked or grilled. B(a)P destroys primordial follicles whether administered post-natally (Mattison and Thorgeirsson, 1977; Mattison et al, 1980; Mattison and Nightingale, 1982; Shirmizu and Mattison, 1984) or in utero (Mackenzie and Angevine, 1981). Total sterility was noted in 97 percent of mice exposed to B(a) P in utero and ovaries exhibited a high degree of hypoplasia (Mackenzie and Angevine, 1981). In addition, a strong correlation has been made between cigarette smoking (which contains B(a)P) and advanced onset of menopause (Cramer and Xu, 1996; Harlow and Signorello, 2000). The ability of a specific AhR ligand to induce ovotoxicity appears to be related to AhR-stimulated transcription of the pro-apoptotic protein, Bax (Matikainen et al., 2001). Bax knockout mice have a 3-fold increase in the number of oocytes/ primordial follicles in the early neo-natal period (Perez et al., 1999), indicating that Bax is involved in pre-natal/neo-natal oocyte apoptosis. Bax knockout mice also appeared to have a delay in the onset of reproductive senescence, as demonstrated by abundant growing follicles and evidence of steroid-induced uterine growth late in life, compared to wild-type counterparts with atrophied ovaries and uteri. The bax promoter contains two dioxin-response elements, and treatment with DMBA significantly increases Bax expression and apoptosis (Matikainen et al., 2001). Furthermore, this action of DMBA is inhibited when ovaries are co-treated with the AhR antagonist, α-napthoflavone. Curiously, dioxin and other polychlorinated aromatic hydrocarbons do not induce oocyte apoptosis, although they bind the AhR with high affinity (Flaws et al., 1997; Salisbury and Marcinkiewicz, 2002). Careful examination of the dioxin response elements in the bax promoter showed a single nucleotide difference between the bax promoter sequences and the sequence necessary for the dioxin-AhR complex to initiate gene transcription (Matikainen et al, 2001). Taken together these studies provide strong evidence that the ovotoxic effect of PAHs depends on AhR-induced bax gene expression. Halogenated aromatic hydrocarbons (HAHs) HAHs include the PCDDs (polychlorinated dibenzo-p-dioxins), the PCDFs (polychlorinated dibenzofurans), and the PCBs (polychlorinated biphenyls). The PCDDs, PCDFs, and PCBs are widespread environmental contaminants that bio-

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accumulate due to their lipophilic properties and stability (Poland and Knutson, 1982). The transplacental effects of PCBs have not been extensively investigated, so this review will focus on the effects of the PCDDs and PCDFs. PCDDs and PCDFs were previously introduced into the environment as a result of insecticide and herbicide use and in the manufacture of paper pulp (Poland and Knutson, 1982). Furthermore, dioxin was found in high concentrations in Agent Orange, a defoliant used in the Vietnam War. Currently the greatest contribution to dioxin accumulation results from municipal solid waste incineration (Schecter et al., 1995). PCDDs and PCDFs have been found in wildlife species (Giesy et al, 1999) and within human tissue and milk (Schecter et al., 1995; Vartiainen et al, 1998; Nakagawa et al, 1999). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the dioxin with the highest affinity for the AhR and the highest degree of toxicity within the PCDDs (Bandiera et al, 1984; Hebert et al., 1990). TCDD has been shown to be a potent disrupter of developmental and reproductive systems in numerous species (reviewed in Birnbaum, 1995), and we have recently demonstrated similar effects of PCDF on estrous cycles and ovulation (Salisbury and Marcinkiewicz, 2002). 2,3,4,7,8-PCDF is considered the most active of the polychlorinated dibenzofurans with the highest binding affinity for the AhR (Bandiera et al., 1984; Hebert et al, 1990) and PCDF is thought to act by the same mechanism because of its binding and activation of the AhR (reviewed in Hankinson, 1995). The AhR has been localized within numerous tissues of many species, including rat (Chaffin and Hutz, 1997; Robles et al, 2000, personal observation) and primate ovarian tissue, and in human granulosa cells (Chaffin et al., 1996). In the rat ovary, AhR is localized in oocytes, granulosa, and theca cells. Two primary in vivo models have been utilized to study reproductive effects of TCDD: (1) an acute toxicity model in which immature or cycling animals are treated post-natally and effects are measured shortly thereafter and (2) an in utero and lactational (IUL) exposure model in which pregnant animals are treated with TCDD and effects are observed in their pups. In addition, many investigators have also studied the effects of TCDD on isolated ovarian cells in vitro. This review will focus on the IUL effects of TCDD on the ovary. The demonstration of significant effects of TCDD on pups after maternal administration strongly supports the hypothesis that TCDD is transferred to pups either across the placenta or through the milk. In two separate toxicokinetic studies, radiolabeled TCDD was administered as a single oral dose to rats during pregnancy (Li et al., 1995; Hurst et al., 1998) and the distribution to fetuses/pups was determined over time. Lactational transfer was clearly demonstrated when significant amounts of TCDD were measured in rat pups cross-fostered from untreated dams to treated dams (Li et al., 1995). In an early study of transplacental effects of TCDD on the female reproductive system, Gray and Ostby (1995) found that pre-natal administration of TCDD on gestational day 10 or 15 induced malformations of external genitalia, characterized by partial clefting of the phallus and an incomplete vaginal opening

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characterized by a persistent thread of tissue extending across the vaginal orifice. In addition to causing malformations of the reproductive tract, fetal exposure to TCDD also disrupts normal processes of female reproduction. For example, TCDD exposure disrupts estrous cycles in offspring (Gray and Ostby, 1995; Salisbury and Marcinkiewicz, 2002), also inducing constant estrus at an young age (Gray and Ostby, 1995, personal observation). The premature aging was also apparent by the reduction in lifetime fertility of TCDD-exposed females. When control and TCDD-exposed animals were placed in a continuous breeding protocol, the reduction in fertility was readily apparent when only 17 percent of TCDD-exposed animals were able to produce a fifth consecutive litter of pups. This is in striking contrast to the 51 percent of control animals producing a fifth litter of pups (Gray and Ostby, 1995). It is possible that the reproductive deficits observed after IUL exposure to TCDD could arise either from direct effects of TCDD on the ovary or from effects at the level of the hypothalamus or pituitary. IUL exposure to TCDD reduces serum estradiol concentrations during the peripubertal period (Chaffin et al., 1997). Furthermore, this reduction in estradiol is accompanied by a significant reduction in the numbers of growing follicles, particularly those reaching maximal size (>100,000 µm2) (Heimler et al, 1998; Salisbury and Marcinkiewicz, 2002). It does not appear as though the reduction in follicle growth or estradiol secretion is due to an inhibitory effect of TCDD on gonadotropin concentrations. Chaffin et al. (1997) measured serum LH and FSH in animals gestationally/lactationally exposed to TCDD and found no effect of TCDD on gonadotropin concentrations. In addition, we have examined follicle development in rats exposed to TCDD (IUL) in a gonadotropin-primed immature rat model (Salisbury and Marcinkiewicz, 2002). In this study, exposed rats treated with exogenous gonadotropin had significantly fewer pre-ovulatory follicles 48 hours after PMSG injection than their control counterparts. IUL exposure to TCDD also significantly reduced serum estradiol levels 48 hours after PMSG. Possible mechanisms of impaired follicle growth could include decreased responsiveness to FSH or a reduction in intraovarian growth factors that contribute to follicular growth. For example, Hirakawa et al. (2000) showed that isolated granulosa cells co-treated with FSH and TCDD had fewer FSH receptors than granulosa cells treated with FSH alone. The decrease in follicle growth could also be a consequence of the antiestrogenic properties of TCDD, such as the reduction in ovarian estrogen receptor expression observed after a single i.p. injection of TCDD in a mature mouse (Tian et al., 1998). Additional halogenated compounds Several other halogenated compounds are toxic to primordial follicles: hexachlorobenzene (HCB) (Jarrell et al, 1993; Bourque et al., 1995; Alvarez et al, 2000), 2-bromopropane (Koh et al., 1998; Yu et al., 1999) and the water disinfection by-product, dibromoacetic acid (DBA) (Bodensteiner et al., 2000).

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In humans, HCB is found in ovarian follicular fluid (Jarrell et al., 1993), and there have been incidents of very high exposure in Turkish women in the 1950s after eating contaminated grain (Jarrell et al., 1998). HCB is known to induce follicle toxicity in monkeys (Jarrell et al., 1993) and rats (Foster et al., 1992; Alvarez et al., 2000); however, it is currently unknown whether HCB induces transplacental ovarian toxicity. The reproductive toxicity of 2-bromopropane was observed in 1995 when electronic factory workers exposed to this solvent exhibited a striking cluster of amenorrhea and azoospermia (Kim et al., 1996). Further studies demonstrated that inhalation of 2-bromopropane depletes the ovary of primordial follicles (Koh et al, 1998; Yu et al, 1999). 2-Bromopropane was shown to exert similar ovarian toxicity in rats exposed to 2-bromopropane during gestation and lactation (Kang et al., 2002). In this study, rats were injected from gestation day 6 through 20 days of lactation. At the highest dose (1215 mg/kg/day), significant reductions in primordial, growing, and antral follicles were observed. DBA is a water disinfection by-product that has been shown to exert adverse effects on testis function and primordial follicles when administered to pregnant rabbits (Bodensteiner et al., 2000) or rats (Klinefelter et al, 2000) during gestation/lactation. No other follicle sizes were affected and the inhibition of primordial follicle number appeared to be dose dependent. Other compounds Lead is a heavy metal that is widely dispersed in the environment that continues to present a public health problem. It is associated with increased incidence of miscarriage and disordered menstrual cycles (reviewed in Taupeau et al, 2001) in women with high exposure rates. Mice exposed to lead in utero on gestation day 8 had fewer primordial follicles than control mice (Wide, 1985). In addition, prenatal exposure of rats to lead in utero and during lactation significantly decreased ovarian hCG and FSH binding in pre-pubertal, pubertal, and cycling rats (Wiebe et al, 1988). Direct exposure of 5–8-week-old mice to lead also decreased primordial follicles and increased atresia of antral follicles (Taupeau et al., 2001) indicating that lead may induce ovarian toxicity during both pre-natal and postnatal time periods. Busulfan (1,4-butanediol dimethanesulfate) is an alkylating agent used to treat various forms of cancer, such as leukemia. Midgestation (day 13 or 14) administration of busulfan results in widespread destruction of primordial germ cells/ oogonia and neo-natal rats have ovaries that are nearly devoid of both oocytes and primordial follicles (Hemsworth and Jackson, 1963; Merchant-Larios, 1976; Reddoch et al, 1986; Gray et al, 1993). Congo Red is a diazo dye based on benzidine. When pregnant mice were treated orally on gestation days 8–12, their female offspring were subfertile (Gray et al., 1992). A closer examination of their ovaries showed that ovarian atrophy was pronounced by and that 30 percent of exposed animals lacked

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maturing follicles at 170 days of age. A high percentage of ovaries (60 percent) also showed evidence of ovarian cysts at this age. CONCLUDING REMARKS The ovary is clearly the target of many types of toxic insult during pregnancy. Many compounds are able to cross the placenta to reach the fetus during critical windows of development. Specific ovarian consequences include ovotoxicity, ovulation failure, suppressed follicular growth and steroidogenesis, and cyst formation. It is imperative that we further explore the nature of these agents, their mechanism of action and the specific dangers they pose to health of the environment and to human reproduction. REFERENCES Alvarez, L., Randi, A., Alvarez, P., Piroli, G., Chamson-Reig, A., Lux-Lantos, V. and Kleiman de Pisarev, D. (2000) Reproductive effects of hexachlorobenzene in female rats, J. Appl Toxicol., 20:81–87. Bandiera, S., Sawyer, T., Romkes, M., Zmudzka, B., Safe, L., Mason, G., Keys, B. and Safe, S. (1984) Polychlorinated dibenzofurans (PCDF): effects of structure on binding to the 2,3,7,8-TCDD cytosolic receptor protein, AHH induction and toxicity, Toxicology, 32:131–44. Beaumont, H. and Mandl, A. (1962) A quantitative and cytological study of oogonia and oocytes in the foetal and neonatal rat, Proc. Roy. Soc. B, 155:557–79. Benedict, J.C., Lin, T.M., Loeffler, I.K., Peterson, R.E. and Flaws, J.A. (2000) Physiological role of the aryl hydrocarbon receptor in mouse ovary development, Toxicol. Sci., 2:382–88. Birnbaum, L.S. (1995) Developmental effects of dioxins and related endocrine disrupting chemicals, Toxicol. Lett., 82–83:743–50. Bodensteiner, K., Veeramachaneni, D.N.R., Klinefelter, G.R., Kane, C.M., Higuchi, T.T., Moeller, C.L. and Sawyer, H.R. (2000) Chronic exposure to dibromoacetic acid, a water disinfection by-product, diminishes primordial follicles in the rabbit, abstract presented at the Symposium on Gender Differences in Reprod. Biol. Toxicol., Tucson, AZ, November. Bourque, A.C., Singh, A., Lakhanpal, N., McMahon, A. and Foster, W.G. (1995) Ultrastmctural changes in ovarian follicles of monkey administered hexachlorobenzene, Am. J. Vet. Res., 56:1673–77. Byskov, A. and Lintern-Moore, S. (1973) Follicle formation in the immature mouse ovary: the role of the rete ovarii, /. Anat., 116:207–17. Chaffin, C.L. and Hutz, R.J. (1997) Regulation of the aromatic hydrocarbon receptor (AHR) by in utero and lactational exposure to 2,3,7,8-tetrachlorodizenzo-p-dioxin (TCDD), J. Reprod. Dev., 43:47–51. Chaffin, C.L., Heimler, I., Rawlins, R.G., Wimpee, B.A., Sommer, C. and Hutz, R.J. (1996) Estrogen receptor and the aromatic hydrocarbon receptor in the primate ovary, Endocrine, 3:315–21.

JENNIFER L.MARCINKIEWICZ 145

Chaffin, C.L., Trewin, A.L., Watanbe, G., Taya, K. and Hutz, R.J. (1997) Alternations to the pituitary-gonadal axis in the peripubertal female rat exposed in utero and through lactation to 2,3,7,8-tetra-chlorodibenzo-p-dioxin, Biol Reprod., 56:1498–502. Chapin, R.E., Harris, M.W., Davis, B.J., Ward, S.M., Wilson, R.E., Mauney, M.A., Lockhart, A.C., Smialowicz, R.J., Moser, V.C., Burka, L.T., Collins, B.J. (1997) The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function, Fundam. Appl Toxicol., 40:138–57. Clarke, D.O. (1997) Pharmacokinetic and structure-activity considerations. In: K. Boekelheide, R.E. Chapin, P.B. Hoyer, C. Harris (eds) Comprehensive Toxicology, Reproductive and Endocrine Toxicology, New York, NY: Elsevier Science, Vol. 10. Cramer, D.W. and Xu, H. (1996) Predicting age at menopause, Maturitas, 23:319–26. De Rosa, C., Richter, P., Pohl, H. and Jones, D.E. (1998) Environmental exposures that affect the endocrine system: Public health implications, /. Toxicol. Environ. Health, Part B, 1:3–26. Faber, J.J. and Thornburg, K.L. (1983) Placental Physiology: Structure and Function of Fetomaternal Exchange, New York, NY: Raven Press. Findlay, J. (1991) The ovary, Bailliere’s Clin. Endo. Metab., 5:755–69. Flaws, J.A., Sommer, R.J., Silbergeld, E.K., Peterson, R.E. and Hirshfield, A.N. (1997) In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces genital dysmorphogenesis in the female rat, Toxicol. Appl. Pharmacol., 147: 351–62. Foster, W.G., Pentick, J.A., McMahon, A. and Lecavalier, P.R. (1992) Ovarian toxicity of hexachlorobenzene (HCB) in the superovulated female rat, /. Biochem. Toxicol., 7:1– 4. Giesy, J.P., Kannan, K., Kubitz, J.A., Williams, L.L. and Zabik, M.J. (1999) Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in muscle and eggs of salmonid fishes from the great lakes, Arch. Environ. Contam. Toxicol., 36:432–46. Gosden, R. and Faddy, M. (1998) Biological bases of premature ovarian failure, Reprod. Fertil Dev., 10:73–78. Gray, L.E. and Ostby, J.S. (1995) In utero 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology and function in female rat offspring, Toxicol. Appl. Pharmacol., 133:285–94. Gray, L.E., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., Goldman, J., Slott, V. and Laskey, J. (1989) A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat, Fundam. Appl. Toxicol., 12:92–108. Gray, L.E., Ostby, J.S., Kavlock, R.J. and Marshall, R. (1992) Gonadal effects of fetal exposure to the azo dye Congo Red in mice: infertility in female but not male offspring, Fundam. Appl Toxicol., 19:411–22. Gray, L.E., Ostby, J.S. and Marshall, E.R. (1993) Gestational busulfan (B) alters CNS, testicular, and ovarian development and fertility in LE hooded rat offspring, The Toxicologist, 13:75–79. Hankinson, O. (1995) The aryl hydrocarbon receptor complex, Annu. Rev. Pharmacol Toxicol., 35:307–40. Haney, A.F., Newbold, R.R. and McLachlan, J.A. (1984) Prenatal diethylstilbestrol exposure in the mouse: effects on ovarian histology and steroidogenesis in vitro, Biol. Reprod., 30:471–78.

146 PLACENTAL INDUCTION OF OVARIAN TOXICITY

Harlow, B. and Signorello, L. (2000) Factors associated with early menopause, Maturitas, 35:3–9. Hebert, C.D., Harris, M.W., Elwell, M.R. and Birnbaum, L.S. (1990) Relative toxicity and tumor-promoting ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8pentachlorodibenzofuran (PCDF) and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) in hairless mice, Toxicol. Appl Pharmcol, 102:362–77. Heimler, I., Trewin, A.L., Chaffin, C.L., Rawlins, R.G. and Hutz, R.J. (1998) Modulation of ovarian follicle maturation and the effects on apoptotic cell death in Holtzman rats exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally, Reprod. Toxicol., 12:69–73. Hemsworth, B.N. and Jackson, H. (1963) Effect of busulfan on the developing ovary in the rat, / Reprod. Fertil., 6:187–94. Hendry, W.J., Sheehan, D.M., Khan, S.A. and May, J.V. (2002) Developing a laboratory animal model for perinatal endocrine disruption: the hamster chronicles, Exp. Biol. Med., 227:709–23. Herbst, A.L., Ulfelder, H. and Poskanzer, D.C. (1971) Adenocarcinoma of the vagina: association of maternal stilbestrol therapy with tumor appearance in young women, N. Engl. J. Med., 284:878–81. Hirakawa, T., Minegishi, T., Abe, K., Kishi, H., Inoue, K., Ibuki, Y. and Miyamoto, K. (2000) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of folliclestimulating hormone receptors during cell differentiation in cultured granulosa cells, Endocrinology, 141:1470–76. Hirshfield, A. (1991) Development of follicles in the mammalian ovary, Int Rev. Cytol., 124:43–101. Hoyer, P.B., Cannady, E.A., Kroeger, N.A. and Sipes, I.G. (2001) Mechanisms of ovotoxicity induced by environmental chemicals: 4-vinylcyclohexene diepoxide as a model chemical, Adv. Exp. Med. Biol., 500:73–81. Hurst, C.H., Abbott, B.D., DeVito, M.J., Birnbaum, L.S. (1998) 2,3,7,8Tetrachlorodizenzo-p-dioxin in pregnant Long Evans rats: disposition to maternal and embryo/fetal tissues, Toxicol., Sci., 45:129–36. Iguchi, T. and Takasugi, N. (1986) Polyovular follicles in the ovary of immature mice exposed prenatally to diethylstilbestrol, Anat. Embryol., 175:53–55. Iguchi, T., Takasugi, N., Bern, H.A. and Mills, K.T. (1986) Frequent occurrence of polyovular follicles in ovaries of mice exposed neonatally to diethylstilbestrol, Teratology, 34:29–35. Iguchi, T., Fukazawa, Y., Uesugi, Y. and Takasugi, N. (1990) Polyovular follicles in mouse ovaries exposed neonatally to diethylstilbestrol in vivo and in vitro, Biol. Reprod., 43:478–84. Jarrell, J.F., McMahon, A., Villeneuve, D. and Franklin, C. (1993) Hexachlorobenzene toxicity in the monkey primordial germ cell without induced porphyria, Reprod. Toxicol., 7:41–47. Jarrell, J.F., Villeneuve, D., Franklin, C. and Bartlett, S. (1993) Contamination of human ovarian follicular fluid and serum by chlorinated organic compounds in three Canadian cities, CMAJ, 148:1321–27. Jarrell, J., Gocmen, A., Foster, W., Brant, R., Chan, S. and Sevcik, M. (1998) Evaluation of reproductive outcomes in women inadvertently exposed to hexachlorobenzene in southeastern turkey in the 1950’s, Reprod. Toxicol., 12:469–76.

JENNIFER L.MARCINKIEWICZ 147

Kang, K.-S., Li, G.-X., Che, J.-H. and, Lee Y.-S. (2002) Impairment of male rat reproductive function in F1 offspring from dams exposed to 2-bromopropane during gestation and lactation, Reprod. Toxicol., 16:151–59. Kezele, P., Nilsson, E. and Skinner, M.K. (2002) Cell-cell interactions in primordial follicle assembly and development, Front. Biol., 7:d1946–78 1990–96. Kim, Y.H., Jung, K., Jung, G., Kim, H., Park, J., Kim, J., Park, J., Park, D., Park, S., Choi, K. and Moon, Y. (1996) Hematopoietic and reproductive hazards of Korean electronic workers exposed to solvents containing 2-bromopropane, Scand. J. Work Environ. Health, 22:387–91. Kitamura, T., Nishimura, S., Sasahara, K., Yoshida, M., Ando, J., Takahashi, M., Shirai, T. and Maekawa, A. (1999) Transplacental administration of diethylstilbestrol (DES) causes lesions in female reproductive organs of Donryu rats, including endometrial neoplasia, Cancer Lett., 141:219–28. Klinefelter, G.R., Strader, L., Suarez, J., Roberts, N., Holmes, M., and Mole, L. (2000) Dibromoacetic acid, a drinking water disinfection by-product, alters male reproductive development and fertility, Biol Reprod. 62 (Suppl 1), 454. Koh, J.M., Kim C.H., Hong, S.K., Lee, K.U., Kim, Y.T., Kim, O.J. and Kim, G.S. (1998) Primary ovarian failure caused by a solvent containing 2-bromopropane, Eur. J. Endocrinol., 138:554–56. Legraverend, C, Guenthner, T.M. and Nebert, D.W. (1984) Importance of the route of administration for genetic differences in benzo(a)pyrene-induced in utero toxicity and teratogenicity, Teratology, 29:35–41. Li, X., Johnson, D.C. and Rozman, K.K. (1995) Reproductive effects of 2,3,7,8,-Tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation, hormonal regulation, and possible mechanisms, Toxicol. Appl. Pharmacol., 133:321–27. Ma, Q. (2001) Induction of CYP1a1. The AhR/DRE paradigm: transcription, receptor regulation, and expanding biological roles, Curr. Drug Metab., 2:149–64. Mackenzie, K. and Angevine, D. (1981) Infertility in mice exposed in utero to benzo(a) pyrene, Biol Reprod., 24:183–91. Matikainen, T., Perez, G.I., Jurisicova, A., Pru, J.K., Schlezinger, J.J., Ryu, H.Y., Laine, J., Sakai, T., Korsmeyer, S.J., Casper, R.F., Sherr, D.H. and Tilly, J.L. (2001) Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals, Nat. Genet., 28:355–60. Mattison, D.R. (1979) Difference in sensitivity of rat and mouse primordial oocytes to destruction by polycyclic aromatic hydrocarbons, Chem. Biol Interact., 28:133–37. Mattison, D. and Nightingale, M. (1982) Oocyte destruction by polycyclic aromatic hydrocarbons is not linked to the inducibility of ovarian aryl hydrocarbon (benzo(a) pyrene) hydroxylase activity in (DBA/2N×C57BL/6N)F1 X DBA/2N backcross mice, Ped. Pharmacol., 2:11–21. Mattison, D. and Thorgeirsson, S. (1977) Genetic differences in mouse ovarian metabolism of benzo(a)pyrene and oocyte toxicity, Biochem. Pharmacol., 26:909–12. Mattison, D.R., White, N.B. and Nightingale, M.R. (1980) The effect of benzo(a)pyrene on fertility, primordial oocyte number, and ovarian response to pregnant mare’s serum gonadotropin, Ped. Pharmacol., 1:143–51. Mauleon, P. (1978) Ovarian development in young mammals. In: D.B. Creighton, G.R. Foxcroft, N.B. Haynes, and G.E. Lamming (eds) Control of Ovulation, London, UK: Butterworths.

148 PLACENTAL INDUCTION OF OVARIAN TOXICITY

McCabe, D. and Flynn, E. (1990) Deposition of low dose benzo(a)pyrene into fetal tissue: influence of protein binding, Teratology, 41:85–95. McGee, E. and Hsueh, A. (2000) Initial and cyclic recruitment of ovarian follicles, Endocr. Rev., 21:200–14. Merchant-Larios, H. (1976) The role of germ cells in the morphogenesis and cytodifferentiation of the rat ovary. In: N. Muller-Berat (ed.), Progress in Differentiation Research, New York, NY, North-Holland: Amsterdam Publishing Co. Nakagawa, R., Hirakaawa, H., Matsueda, T. and Nagayama, J. (1999) Maternal body burden of organochloride pesticides and dioxins, J. of AOAC Int., 82:716–24. Napalkov, N.P. and Anisimov, V.N. (1979) Transplacental effect of diethylstilbestrol in female rats, Cancer Lett., 6:107–14. Neubert, D. and Papken, S. (1988) Transfer of benzo(a)pyrene into mouse embryos and fetuses, Arch. Toxicol., 62:236–39. Newbold, R.R. and McLachlan, J.A. (1979) Ovarian abnormalities in mice following prenatal exposure to diethylstilbestrol, Proceedings of the American Association of Cancer Research, 20:103–12. Newbold, R.R., Bullock, B.C. and McLachlan, J.A. (1983) Exposure to diethylstilbestrol during pregnancy permanently alters the ovary and oviduct, Biol. Reprod., 28:735– 44. Page, K.R. (1993) The Physiology of the Human Placenta, London: University College London Press. Perez, G.I., Robles, R., Knudson, C.M., Flaws, J.A., Korsmeyer, S.J. and Tilly, J.L. (1999) Prolongation of ovarian lifespan into advanced chronological age by Baxdeficiency, Nat. Genet., 21:200–03. Peters, H. and McNatty, K. (1980) The Ovary, Berkeley: University of California Press. Poland, A. and Knutson, J.C. (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity, Ann. Rev. Pharmacol Toxicol., 22:517–54. Reddoch, R.B., Pelletier, R.M., Barbe, G.J. and Armstrong, D.T. (1986) Lack of ovarian responsiveness to gonadotropic hormones in infantile rats sterilized with busulfan, Endocrinology, 119:879–86. Robles, R., Morita, Y., Mann, K.K., Perez, G.I., Yang, S., Matikainen, T., Sherr, D.H. and Tilly, J.L. (2000) The aryl hydrocarbon receptor, a basic helix-loop-helix transcription factor of the PAS gene family, is required for normal ovarian germ cell dynamics in the mouse, Endocrinology, 141:450–53. Rodriguez, J.W., Kirlin, W.G., Wirsiy, Y.G., Matheravidathu, S., Hodge, T.W. and Urso, P. (1999) Maternal exposure to benzo(a)pyrene alters development of T lymphocytes in offspring, Immunopharmacol. Immunotoxicol., 21:379–96. Rubin, B.S., Murray, M.K., Damassa, D.A., King, J.C. and Soto, A.M. (2001) Perinatal exposure to low doses of bisphenol A affects body weight, patterns of estrous cyclicity, and plasma LH levels, Environ. Health Perspect., 109:675–80. Safe, S. (2001) Molecular biology of the Ah Receptor and its role in carcinogenesis, Toxicol. Lett., 120:1–7. Salisbury, T.B. Marcinkiewicz, J.L. (2002) In utero and lactational exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-pentachlorodibenzofuran reduces growth and disrupts reproductive parameters in female rats, Biol Reprod., 66:1621–26.

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Schecter, A., Papke, O., Ball, M., Lis, A. and Brandt-Rauf, P. (1995) Dioxin concentrations in the blood of workers at municipal waste incinerators, Occup. Environ. Med., 52:385–87. Shirmizu, K. and Mattison, D. (1984) The effect of intraovarian injection of benzo(a) pyrene on primordial oocyte number and ovarian aryl hydrocarbon [benzo(a)pyrene] hydroxylase activity, Toxicol. Appl Pharmacol., 76:18–25. Suzuki, A., Sugihara, A., Uchida, K., Sato, T., Ohta, Y., Katsu, Y., Watanabe, H. and Iguchi, T. (2002) Developmental effects of perinatal exposure to bisphenol-A and diethylstilbestrol on reproductive organs in female mice, Reprod. Toxicol., 16:107– 16. Swartz, W.J. and Corkern, M. (1992) Effects of methoxychlor treatment of pregnant mice on female offspring of the treated and subsequent pregnancies, Reprod. Toxicol., 6: 431–37. Taupeau, C., Poupon, J., Nome, F. and Lefevre, B. (2001) Lead accumulation in the mouse ovary after treatment-induced follicular atresia, Reprod. Toxicol., 15:385–91. Tian, Y., Ke, S., Thomas, T., Meeker, R.J. and Gallo, M.A. (1998) Transcriptional suppression of estrogen receptor gene expression by 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), J. Steroid Biochem. Mol. Biol, 67:17–24. Tilly, J.L. (1998) Molecular and genetic basis of normal and toxicant-induced apoptosis in female germ cells, Toxicol. Lett., 102–03:497–501. Ueno, S., Takahashi, M., Manganaro, T.F., Ragin, R.C. and Donahoe, P.K. (1989) Cellular localization of mullerian inhibiting substance in the developing rat ovary, Endocrinology, 124:1000–06. Vartiainen, T., Jaakkola, J., Saarikoski, S. and Tuomisto, J. (1998) Birth weight and sex of children and the correlation to the body burden of PCDDs/PCDFs and PCBs of the mother, Environ. Health Perspect., 106:61–66. Wide, M. (1985) Lead exposure on critical days of fetal life affects fertility in the female mouse, Teratology, 32:375–80. Wiebe, J.P., Barr, K.J. and Buckingham, K.D. (1988) Effect of prenatal and neonatal exposure to lead on gonadotropin receptors and steroidogenesis in rat ovaries, J. Toxicol. Environ. Health, 24:461–76. Wordinger, R.J. and Highman, B. (1984) Histology and ultrastructure of the adult mouse ovary following a single prenatal exposure to diethylstilbestrol, Virchows Arch. Part /?., 45:241–53. Yu, X., Kamijima, M., Ichihara, G., Li, W., Kitoh, J., Xie, Z., Shibata, E., Hisanaga, N. and Takeuchi, Y. (1999) 2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats, Toxicol. Appl. Pharmacol. 159: 185–93. Zamboni, L. (1989) Overview of embryological and fetal development of the ovary and testis. In: A.N. Hirshfield (ed.), Growth Factors and the Ovary, New York: Plenum Press.

9 CHEMORESISTANCE IN HUMAN OVARIAN CANCER: POSSIBLE ROLES OF X-LINKED INHIBITOR OF APOPTOSIS PROTEIN (XIAP) Chao Wu Xiao, Xiaojuan Yan, Hiromasa Sasaki, Fumikazu Kotsuji and Benjamin K. Tsang

INTRODUCTION Ovarian cancer is the most lethal gynecological cancer in the Western World and ranks fourth among the most common female cancers (Landis et al., 1999). Approximately 23,000 women are diagnosed with, and ~14,000 women die from ovarian cancer annually in the United States (Landis et al., 1999). Chemotherapy and cytoreductive surgery are current standard modalities of treatment for ovarian cancer. Platinum derivatives (e.g., cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin), carboplatin) and paclitaxel are the first-line chemotherapeutic agents for this treatment. However, development of resistance to chemotherapy is the major concern in treating ovarian cancer patients. Multiple mechanisms, including decreased drug accumulation, increased levels of the intracellular thiols, and increased DNA repair have been implicated in cisplatin resistance, but recent evidence suggests that decreased induction of proapoptotic factors (Fas/ FasL) and increased intracellular levels of the antiapoptotic factor, X-linked inhibitor of apoptosis protein (XIAP) may be key determinants in chemoresistance in ovarian cancer. This chapter reviews the current understanding of the mechanisms involved in the development of chemoresistance and specially focuses on the possible roles of XIAP in chemoresistance in human ovarian cancer. INVOLVEMENT OF APOPTOSIS IN CHEMOTHERAPY Cisplatin is one of the most effective chemotherapeutic agents for treating ovarian cancer. The cytotoxicity of cisplatin is believed to be due to the formation of DNA adducts, which include DNA-protein cross-links, DNA monoadducts, interstrand, and intrastrand DNA cross-links. It has been shown that 1, 2-intrastrand d(GpG), and d(ApG) cross-links account for 65 and 25 percent, respectively, of the cisplatin adducts formed in vitro (Eastman, 1986; Fichtinger-Schepman et al., 1985). The intrastrand cisplatin cross-link produces

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a severe local distortion in the DNA double helix, leading to unwinding and kinking (Bellon et al, 1991). It was originally believed that cytotoxicity of chemotherapeutic agents was the result of inhibition of DNA synthesis (Harder and Rosenberg, 1970; Howle and Gale, 1970). However, evidence from Sorenson’s group has shown that inhibition of DNA synthesis is not a critical step in cis-DDP-induced cytotoxicity (Sorenson and Eastman, 1988). Analysis of the relationship between inhibition of DNA synthesis and toxicity as well as DNA repair and cell-cycle progression in Chinese hamster ovary cells showed that DNA repair-deficient cells were hypersensitive to cis-DDP and markedly arrested in the G2-phase, and that cells were killed at concentrations lower than those required to inhibit DNA synthesis (Sorenson and Eastman, 1988). In contrast, DNA repair proficient cells survive at concentrations of cis-DDP high enough to inhibit DNA synthesis and arrest the cells in S-phase. This suggested that cell death induced by cis-DDP is not due to the inhibition of DNA synthesis. The analysis of cell death induced by cis-DDP (Barry et al., 1990; Sorenson et al., 1990) reveals DNA fragmentation into multimers of 180 base pairs, consistent with internucleosomal cleavage of chromatin by an endonuclease, followed by loss of membrane integrity and cell shrinkage. Furthermore, both cell death and DNA fragmentation were inhibited by cycloheximide, indicating the requirement for new protein synthesis. These findings suggest that activation of signal transduction pathway for apoptosis is involved in the cis-DDP-induced cell death (Wyllie et al., 1980). However, the signal transduction mechanism that links DNA damage to the cell death pathway is still not completely understood. APOPTOSIS AND SIGNAL TRANSDUCTION Apoptosis is a physiological cell-suicide process crucial for maintaining appropriate cell number and tissue organization (Frisch and Francis, 1994; Jacobson et al., 1997). Abnormal inhibition of apoptosis can lead to cancer and autoimmune disease, whereas excessive cell death has been implicated in neurodegenerative disorders (Thompson, 1995). The execution of cellular apoptosis involves the activation of a cascade of intracellular proteases belonging to the caspase protease family (Thornberry and Lazebnik, 1998). Caspases are initially synthesized as inactive precursors which, upon receipt of an apoptotic signal, are processed into mature forms composed of a tetramer of two large and two small subunits (Nunez et al., 1998). The initiator caspases (caspase-8, -9, and -10) are activated through intrinsic autocatalytic activity in conjunction with other proteins to form apoptosomes (Green, 1998). The activated upstream initiator caspases activate downstream effector caspases (caspase-3, -6, and -7) (Li et al., 1997; Nunez et al., 1998; Sun et al., 1999b). Executioner caspases cleave specific cellular substrate proteins, facilitating the demise of the cell (Cryns and Yuan, 1998).

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Three distinct pathways leading to apoptosis have been demonstrated. The extrinsic pathway involves ligation of death receptors such as Fas or tumor necrosis factor receptor-1 (TNFR1) by their ligands, resulting in the recruitment of procaspase-8 or procaspase-10 to the receptor complex through the adaptor Fasassociated death domain (FADD) (Nunez et al, 1998). Following oligomerization, procaspase-8 or procaspase-10 is self-activated presumably through induced proximity of procaspase molecules (Yang et al., 1998). These active enzymes then cleave their downstream targets, which include effector caspases, such as caspase-3, -6, and -7. A second pathway involves the release of mitochondrial cytochrome c into the cytosol upon cellular stress (Hakem et al., 1998; Kuida et al., 1998; Slee et al., 1999; Woo et al, 1998). In the presence of dATP/ATP, cytochrome c binds to apoptotic protease-activating factor-1 (Apaf-1), which then oligomerizes and binds procaspase-9 to form an apoptosome (Cain et al., 2000; Green, 1998; Sun et al., 1999b). Apaf-1 contains at least three functional domains: (i) an N-terminal caspase-activation recruitment domain, which binds the prodomain of caspase-9, (ii) a CED-4 domain required for Apaf-1 self-oligomerization, and (iii) a series of C-terminal WD-40 repeats thought to mediate protein-protein interactions (Zou et al., 1999). Apaf-1 oligomerization allows proximity and enzymatic self-activation of bound caspase-9 (Liu et al., 2002). In native cell lysates, Apaf-1 oligomerizes into a ~700 kD complex and, in addition to processed caspase-9, contains fully processed caspase-3 and -7 (p17 and p12 subunits) (Cain et al., 1999). Thus, the initial processing of effector caspases by caspase-9 and their subsequent autocatalytic processing appears to take place within the apoptosome (Bratton et al., 2001). A third apoptotic pathway involves the endoplasmic reticulum (ER) and is activated upon ER stress. While the mechanism of apoptotic induction via this pathway is not well understood, it is believed to be mediated via caspase-12 activation (Aridor and Balch, 1999; de Bruin et al, 2002; Nakagawa et al, 2000; Pepling and Spradling, 2001). THE POSSIBLE MECHANISMS RESPONSIBLE FOR CHEMORESISTANCE Alterations in the regulation of apoptosis may contribute both to the pathogenesis and the development of chemoresistance in ovarian cancer. Resistance to chemotherapy has been associated with decreased susceptibility to apoptosis, introducing the possibility that cell death determinants may influence the outcome of treatment (Dive and Wyllie, 1993). Several possible mechanisms have been suggested to explain the drug resistance. These are: (a) decreased drug accumulation, (b) increased levels of the intracellular thiols, and (c) increased DNA repair (Godwin et al., 1992; Johnson et al., 1994, 1997). Decreased intracellular accumulation of a drug is an important factor in the development of cisplatin resistance. Both increased efflux and decreased uptake of drugs can reduce the net accumulation of drugs in the cells. Two membrane

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proteins that may be associated with the uptake and efflux of cisplatin have been identified in the cisplatin-resistant cells with decreased drug accumulation. Decreased expression of a 48-kD protein may be responsible for the uptake of drug (Bernal et al., 1990) and an increased expression of a 200-kD glycoprotein may be related to the efflux of the drug (Kawai et al., 1990). Several other genes including MDR1, MRP1, MRP2, and LRP have been shown to be responsible for drug transportation (Cole and Deeley, 1993; Riordan et al., 1985; Scheffer et al., 1995). MDR1 and MRP1 function as a drug efflux pump (Cole et al., 1994; Ueda et al., 1987), and MRP2 has been found to be overexpressed in a number of cisplatin-resistant cell lines (Kool et al., 1997; Taniguchi et al., 1996) and might also act as a drug efflux pump. The 110-kD LRP is frequently overexpressed in multidrug resistance cells, and has an important role in transporting drug from nuclei to the cytoplasm and confer multidrug resistance in vitro (Izquierdo et al., 1996). It has been demonstrated that LRP expression in advanced ovarian carcinoma is an indicator of poor response to cisplatin-based chemotherapy (Izquierdo et al., 1995). Recently, the copper-transporting P-type adenosine triphosphatase gene has been reported to be responsible for cisplatin resistance in human prostate cells (Komatsu et al., 2000) and ovarian carcinoma (Nakayama et al., 2002). Glutathione (GSH) or γ-glutamylcysteinylglycine is a tripeptide thiol. As a potent nucleophile, GSH reacts with alkylating agents and cisplatin. The reaction of GSH and cisplatin forms a GSH-platinum complex that is then eliminated from the cell by an ATP-dependent glutathione S-conjugate export pump (Ishikawa and Ali, 1993). GSH may protect cells by intercepting reactive platinum complexes before they react with DNA and also by supporting DNA repair, possibly by stabilizing repair enzymes such as DNA polymerase α or by promoting the formation of deoxyribonucleotides (Lai et al, 1989). Increased glutathione levels have been found in some cisplatin-resistant cells (Godwin et al, 1992; Hamilton et al, 1985). Increased DNA repair plays an important role in chemoresistance to platinumbased compounds (Park et al., 2001). Repair of cisplatin adducts occurs primarily by the nucleotide excision repair (NER) pathway, one of the major DNA repair systems in mammalian cells. NER is the only known mechanism in mammalian cells for removing bulky, helix-distorting DNA adducts produced by platinum agents (Reardon and Sancar, 1998). Cisplatin-resistant cell lines show increased levels of DNA repair, as measured by the loss of platinum adducts (Eastman and Schulte, 1988; Parker et al, 1991), DNA repair synthesis (Lai et al, 1988; Masuda et al., 1988), and reactivation of cisplatin-damaged plasmid DNA (Chu and Chang, 1990; Sheibani et al., 1989). When the removal of specific cisplatin adducts was measured, the most significant observation was the enhanced repair of d(GpG) adducts in the resistant cells (Eastman and Schulte, 1988). Normal cells repair cisplatin interstrand cross-links preferentially in transcriptionally active genes, and resistant cells showed increases in this preferential repair (Zhen et al., 1992).

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Multiple genes are involved in the NER pathway. Among them, ERCC1 encodes a protein which contains a helix-turn-helix motif characteristic of DNAbinding proteins. This protein may be involved in the recognition of cisplatin damage and serves as an excision nuclease. ERCC1 has been shown to be expressed at levels of 2.6-fold higher in clinically resistant tumors than in sensitive tumors (Dabholkar et al., 1992). Another DNA repair gene, ERCC2 encodes the Xeroderma pigmentosum group D (XPD) protein, a helicase. XPD protein is a component of transcription factor TFIIH (Coin et al., 1998; Sung et al., 1993). Mutations of XPD protein such as a nucleotide substitution can change the repair capacity (Park et al., 2001). Xeroderma pigmentosum group E-binding protein is another DNA repair protein and has been increased 5-fold in the tumor cell lines selected in cisplatin (Chu and Chang, 1990). Drug resistance of a particular cell may involve several possible mechanisms. For example, the cisplatin-resistant ovarian cancer cell A2780-CP70 is known to have increased drug efflux and glutathione levels, as well as DNA repair activity (Masuda et al., 1988; Parker et al., 1991; Zhen et al., 1992). However, DNA repair is almost always enhanced in resistant cells while drug uptake, efflux, glutathione level may remain unchanged. In addition, more and more evidence indicates that the development of chemoresistance in human ovarian cancer cells may be related to the decreased induction of pro-apoptotic factors (Fas/FasL system) and increased levels of anti-apoptotic factors (such as IAPs). FAS/FASL SYSTEM AND CHEMORESISTANCE Fas antigen (Fas) is a 45-kD cysteine-rich transmembrane glycoprotein belonging to the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily (Itoh et al., 1991). Fas ligand (FasL) is a 40-kD type II transmembrane protein belonging to the TNF family. Upon ligand binding, Fas induces apoptosis in mammalian cells (Suda and Nagata, 1994). The expression of Fas has been reported in ovarian cancer cell lines (Wakahara et al., 1997) and shown to be upregulated by chemotherapeutic agents (Muller et al., 1997; Uslu et al., 1996). Failure in the induction of either Fas or FasL expression may contribute to the resistance of ovarian cancer cells to cisplatin treatment. In vitro studies from our laboratory have demonstrated that cisplatin induced the expression of cell-associated Fas and FasL, soluble FasL and apoptosis in cisplatin-sensitive cell lines (OV2008 and A2780s). In contrast, while cisplatin effectively increased cell-associated Fas protein content in the resistant variant C13*, it failed to upregulate FasL (cell-associated and soluble forms) and to induce apoptosis, irrespective of concentration and duration of cisplatin treatment (Schneiderman et al., 1999). Interestingly, spent media from cultures of cisplatintreated OV2008 cells were effective in inducing apoptosis in C13* cells, a phenomenon attenuated by the presence of an antagonistic Fas monoclonal antibody. In addition, cisplatin induced a concentration- and timedependent cleavage and activation of procaspase-3 and that of caspase-8 in the

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sensitive (OV2008) but not resistant (C13*) cells, suggesting a functional Fas is present in the sensitive but not resistant cancer cells. These data suggest that the expression of both Fas and FasL is required for Fas-mediated apoptosis, the soluble FasL present in the spent media was biologically active, and the lack of response of resistant cells (C13*) to cisplatin may in part be due to the failure of the cells to express FasL induced by the anticancer agent (Schneiderman et al., 1999). Moreover, in the resistant A2780cp cells, neither Fas nor FasL upregulation was evident in the presence of the chemotherapeutic agent, and apoptosis remained low compared to its sensitive counterpart (Schneiderman et al., 1999). A significant interaction between cisplatin and agonistic Fas monoclonal antibody (which binds and activates Fas) was observed in the apoptotic response when the OV2008 and C13* cells were challenged with both agents. These results not only indicate that the cisplatin-induced Fas receptors were functional but also support the notion that combined immunotherapy and chemotherapy (i.e. agonistic Fas antibody plus cisplatin) may provide added benefits in the treatment of both chemo-sensitive and -resistant ovarian tumors. However, preliminary studies from our laboratory have demonstrated that co-treatment with an antagonistic Fas antibody (to block Fas receptor) was effective in blocking cisplatin-induced apoptosis in only 30 percent of the OV2008 cells. These findings indicate that the induction of Fas/FasL system expression alone cannot fully account for the pro-apoptotic action of cisplatin, and that other cell death or survival pathways may be involved. X-LINKED INHIBITOR OF APOPTOSIS PROTEIN (XIAP) AND CHEMORESISTANCE Direct inhibition of caspases Inhibitors of apoptosis proteins (IAPs) were originally found in baculoviruses where they function to keep the host cells alive while the viruses replicate (Birnbaum et al, 1994; Crook et al, 1993). Five IAPs have been identified in mammalian cells: neuronal apoptosis inhibitory protein (NAIP) (Roy et al., 1995), X-linked IAP (XIAP; also called hILP, MIHA, or cIAP3) (Duckett et al., 1996; Uren et al., 1996), human IAP-1 (HIAP-1; also call cIAP2) (Duckett et al., 1996; Liston et al, 1996; Uren et al., 1996), human IAP-2 (HIAP-2; also called cIAP1) (Liston et al., 1996; Rothe et al, 1995a; Uren et al, 1996), survivin (Ambrosini et al., 1997), and Livin (Kasof and Gomes, 2001). All mammalian IAPs identified to date possess the N-terminal repeats named baculovirus IAP repeats (BIRs) required for biologic function. In addition, XIAP, HIAP-1, and HIAP-2 contain another C-terminal RING-zinc finger domain, believed to be involved in protein-protein interaction. The BIR domain consists of three tandem repeats of 70 amino acids motif (Clem and Miller, 1994).

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IAPs play important roles in regulating programmed cell death in a variety of organisms (Deveraux and Reed, 1999; Miller, 1999). XIAP, HIAP-1, and HIAP-2 have been shown to be potent direct inhibitors of cell death proteases, caspase-3, -7, and -9 (Deveraux et al., 1997, 1998; Roy et al., 1997). Among the above caspaseinhibiting IAPs, XIAP has the most potent anti-apoptotic effect in cells with the lowest Ki for purified caspase-3 and -7 in vitro (Deveraux et al., 1997; Deveraux and Reed, 1999). Structure-function analysis of XIAP has shown that the RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas-induced apoptosis when expressed in cells. Further dissection of the XIAP protein has demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. Overexpression of the BIR2 domain in cells partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of procaspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings indicate that BIR2 is the minimal caspase-inhibitory domain of XIAP, and that a single BIR domain can be sufficient for binding and inhibiting caspases (Takahashi et al., 1998), whereas an XIAP fragment encompassing the third BIR domain (BIR3) and the RING finger domain specifically inhibits caspase-9 (Deveraux et al., 1999; Sun et al., 2000). A recent report of the nuclear magnetic resonance solution-structure of XIAP-BIR2 domain revealed that it consists of a three-stranded antiparallel βsheet and four α-helices, resembling a classical zinc finger motif (Sun et al., 1999a). Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were shown to be essential for inhibiting caspase-3 and caspase-7, and the BIR2 itself interacts with the NH2-terminal region of caspase-7 (Sun et al., 1999a; Suzuki et al., 2001a). Moreover, it was suggested that these residues might bind to the active site and play an essential role in caspase inhibition, whereas the BIR2 domain might interact with an adjacent site on caspase-3 and play a supportive role (Sun et al., 1999a; Suzuki et al., 2001a). Caspase-3 and -7 were inhibited by XIAP through different mechanisms: caspase-3 is inhibited by active site-directed mechanisms, whereas caspase-7 is inhibited by both active site-directed and non-competitive mechanisms. Furthermore, recent studies have shown that XIAP can act as an ubiquitinprotein ligase for caspase-3 and promote the degradation of caspase-3, but not procaspase-3, in living cells. This enhances the anti-apoptotic effect of XIAP (Suzuki et al., 2001b). XIAP is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATPactivated lysates. It associates with oligomerized Apaf-1 and/or processed caspase-9 and influences the activation of caspase-3 produced within the apoptosome and sequesters it within the complex (Bratton et al., 2001). Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within

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the apoptosome and by preventing release of active caspase-3 from the complex (Bratton et al., 2001). Role of XIAP in determining chemosensitivity The anti-apoptotic effect of mammalian IAPs has been previously shown. Overexpression of IAPs, including Naip, XIAP, HIAP-1, and HIAP-2 protects Chinese hamster ovary and RAT-1 cells from apoptosis triggered by menadione, a potent inducer of free radicals, or by growth factor withdrawal (Liston et al., 1996). Overexpression of XIAP or HIAP-2 protected HeLa cells from apoptosis induced by transient transfection of pro-interleukin-1ß-converting enzyme (Uren et al., 1996) and XIAP has been shown to suppress Sindbis virus-induced apoptosis (Duckett et al., 1996). The results from immunohistochemical studies on ovarian carcinomas have shown that XIAP and HIAP-2 immunoreactivities were high in the proliferative cells and low in the apoptotic ones, and that the differences in the expression of XIAP were more prominent between the proliferative and non-proliferative region (Li et al., 2001). The presence of IAPs in ovarian cancer tissue and their distribution are consistent with their possible roles in the regulation of apoptosis and proliferation. In addition, cisplatin treatment decreased XIAP protein content in chemo-sensitive (OV2008 and A2780s) but not -resistant (C13* and A2780cp, respectively) ovarian cancer cell lines. Interestingly, although HIAP-2 is present in both pairs of the cell lines, its content in the A2780 cell lines was not significantly affected by cisplatin and decreased to a lesser extent than XIAP in OV2008, suggesting a less important role for HIAP-2 in ovarian tumor biology (Li et al., 2001). Furthermore, downregulation of XIAP with adenoviral antisense cDNA infection in the cisplatin-sensitive cells in the absence of cisplatin decreased XIAP protein content and induced apoptosis. More importantly, cisplatin-induced apoptosis in cisplatin-sensitive cells was suppressed by overexpression of XIAP by adenoviral XIAP sense infection, suggesting that XIAP may be an important determinant in chemosensitivity in ovarian cancer cells (Li et al., 2001). Role of XIAP in the modulation of p53 and MDM2 A tumor suppressor, p53, acts as a “guardian of the genome” (Lane, 1992) and serves as a G1 cell-cycle checkpoint (Kuerbitz et al., 1992). It plays a central role in the cellular response to DNA damage stimuli from both endogenous and exogenous sources and protects cells against tumorigenesis. Activation of p53 results in a cell-cycle delay which may allow DNA repair before replication or mitosis (Hartwell and Kastan, 1994) or induction of apoptotic cell death as a means of eliminating irreparably damaged cells. Alterations of the p53 gene, either as a result of point mutations and deletions, or as a result of protein stabilization without any obvious genetic change, are the most frequent abnormalities found

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in human malignancies. Major advances have been made in understanding the complex role p53 plays in the regulation of cell-cycle progression and apoptosis (Levine, 1997). MDM2 is an oncoprotein that binds to p53 and facilitates ubiquitin-mediated degradation of the tumor suppressor protein (Honda et al., 1997). It has been reported that a decrease in MDM2 content stabilizes p53, whereas the opposite is true when the oncoprotein is overexpressed (Momand et al., 1992). MDM2 is cleaved during apoptosis, and the involvement of caspase-3 has been suggested (Chen et al., 1997; Erhardt et al., 1997). The role of XIAP in the control of cellular p53 content by maintaining MDM2 integrity (Barak et al., 1993; Wu et al., 1993b) has recently been investigated in our laboratory (Sasaki et al., 2000). We examined the influence of XIAP downregulation by adenoviral XIAP antisense expression on wild-type p53 (C13*) and mutated p53 (A2780cp) cisplatin-resistant human ovarian surface epithelial cancer cell lines. Our studies showed that XIAP downregulation is associated with significant cleavage of procaspase-3 and MDM2, p53 accumulation, and increased apoptosis in wild type p53-expressing C13* cells, as well as sensitization of the cells to the cytotoxic action of cisplatin (Figure 9.1). An identical MDM2 cleavage pattern was noted when cell lysate was treated with recombinantactivated human caspase-3 in vitro. However, XIAP downregulation alone failed to induce apoptosis in A2780cp cells, a p53 mutated cisplatinresistant ovarian cancer cell line. Restoration of this mutant with wild-type p53 by p53 sense infection resulted in the successful induction of apoptotic cell death by antisense XIAP expression (Figure 9.2). This indicates XIAP plays a role in the regulation of cellular p53 levels and suggests that caspase-3-mediated MDM2 processing may be an additional regulatory point for the anti-apoptotic protein in promoting survival of wild type p53-expressing human ovarian cancer cells. Cisplatin resistance is due in part to the failure of the chemotherapeutic agent to downregulate XIAP and to induce apoptosis. Furthermore, the ability of antisense XIAP expression to induce apoptotic cell death is dependent on the status of p53. XIAP and focal adhesion kinase (FAK) processing Cell adhesion is an important cell-survival determinant, and disruption of integrin-mediated signal transduction may be involved in anchorage-dependent cell death. Apoptosis was originally defined on the basis of morphological characteristics, including the dissociation of the cell from its neighboring cells or extracellular matrix (ECM) (Kerr et al., 1994). The loss of cell to cell or cell to matrix contact itself also induces apoptosis, a process often known as anoikis (Frisch and Francis, 1994). Focal adhesion kinase (FAK) is a 125-kD nonreceptor protein tyrosine kinase and is believed to be important for integrinmediated cell adhesion. The anti-apoptotic action of FAK is mediated via its binding either to other signal transduction molecules required for activation of

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Figure 9.1 Influence of XIAP downregulation on MDM2 cleavage and p53 protein content and of caspase-3 on MDM2 processing. (1) C13* cells were infected with various concentrations of adenoviral LacZ or Antisense XIAP (MOI=5, 10, or 20). Representative Western blots for MDM2 cleavage and p53 (A) are presented. Densitometric analysis for p53 content is shown in (C). (2) MDM2 cleavages in C13* whole-cell lysates after incubation with caspase-3 or -7 in the absence (B) or presence ((D) 25 µM) of DEVD are shown: 60-kD MDM2 isoform (closed arrow head), 30-kD fragment (open arrow head) (Sasaki et al., 2000).

the Ras mitogenactivated protein (MAP) kinase cascade (Schlaepfer and Hunter, 1998) and of PKB/Akt through phosphoinositide 3-kinase (Khwaja et al., 1997) or cytoskeletal proteins in the formation of focal adhesion. It has been demonstrated that FAK suppressed anchorage-dependent apoptosis (Frisch et al., 1996) and that inhibition of FAK-induced apoptosis (Hungerford et al., 1996) and reduced cell motility (Ilic et al, 1995). FAK is proteolytically cleaved during the induction of apoptosis and caspases may be involved (Crouch et al., 1996). FAK processing and the regulation of cell morphology have been suggested to play a role in cisplatin-induced apoptosis. Our results have shown that exposure of human ovarian epithelial cancer cells to cisplatin induced procaspase-3 and FAK cleavage, cell detachment from the growth surface and apoptosis in a temporally related and concentration-dependent manner (Sasaki et al, 2002). Addition of recombinant active caspase-3 but not caspase-7 to whole cell lysate elicited a pattern of FAK cleavage identical to that observed in cisplatin-induced apoptosis. The synthetic caspase inhibitors (ZVAD-fmk and DEVD-fmk) significantly decreased FAK cleavage induced by exogenous active caspase-3 (but not caspase-7) and cisplatin, respectively, suggesting the observed FAK processing was caspase-3 specific. The cisplatin-induced FAK processing, morphological changes and apoptosis were attenuated by overexpression of

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Figure 9.2 Influence of XIAP downregulation and/or wild type p53 restoration on apoptosis in A2780-cp (p53 mutated). (A) A2780-cp cells were infected with adenoviral LacZ (open circle) or antisense XIAP (filled circle) (MOI=0, 50, 100, or 200; 72 hours; a and b) or sense p53 (filled circle; MOI=0, 5, 10, 20, or 40; 48 hours; c and d). Apoptosis is shown in a and c. Panels b and d show representative Western blots for XIAP and p53, respectively. (B) Synergistic effect of adenoviral wild type p53 sense (MOI=10) and XIAP antisense (MOI=100; 72 hours) expression on apoptosis. (C) Concentration effects of sense p53 (MOI=0, 5, 10, or 20) on LacZ (open circle), and antisense XIAP (filled circle)-induced apoptosis in A2780-cp (Sasaki et al., 2000).

XIAP (Figures 9.3 and 9.4). However, overexpression of XIAP cDNA-deleted BIR domains (a functional motif for caspase inhibition) failed to exert these responses. Deletion of the RING-zinc finger from XIAP resulted in a

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Figure 9.3 Inhibition of cisplatin-induced cell detachment and apoptosis by XIAP overexpression. Detached cells (A) and apoptotic cells (B) were counted after incubation with medium alone (control) or with adenovirus carrying fulllength XIAP sense cDNA or LacZ for 48 hours prior to cisplatin treatment (10µM, 24 hours). XIAP protein contents before cisplatin treatment after 48 hours culture with medium alone (control), LacZ, or sense XIAP adenoviruses are shown in the inset to panel A (Sasaki et al., 2002).

potentiation of cisplatin-induced apoptosis, suggesting the possibility that this domain may have a modulatory role in the cellular function of XIAP (Sasaki et al., 2002). These findings indicate that XIAP plays a critical role not only in the regulation of apoptosis in ovarian cancer but also in the regulation of cell adhesion by modulating caspase-3-mediated FAK processing. It is possible that the cisplatin resistance conferred by XIAP may in part be due to the maintenance of FAK integrity, and thus of the integrin cell-survival pathway, through caspase-3 inhibition.

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Figure 9.4 Inhibition of cisplatin-induced FAK cleavage by XIAP overexpression. (A, B) Representative Western blot and densitometric analysis, respectively, of intact FAK protein content in attached cells treated with medium alone (control) or LacZ or sense XIAP adenoviruses for 48 hours following cisplatin treatment (10µM). (C, D) Representative Western blot and densitometric analysis, respectively, of intact FAK and FAK fragment contents in cells detached from the growth surface after the same treatment (Sasaki et al., 2002).

NUCLEAR FACTOR-κB-MEDIATED FLIP EXPRESSION AND RESISTANCE TO TNFα TNFα is a pleiotropic cytokine that can induce differentiation, proliferation, and apoptosis in many cell types (Andreani et al., 1991; Baker and Reddy, 1996) and has been suggested to play an important role in the biology of ovarian cancer and tumorigenesis. Ovarian tumor cells produce macrophage colony-stimulating factor, a potent chemoattractant for monocytes which secrete TNFα. TNFα concentrations are significantly increased in ovarian cancer patients (Hassan et al., 1999) and the levels of TNFα expression are positively correlated with tumor grade (Naylor et al, 1993). TNFα has selective cytolytic activity against some but not all tumor cells (Takeyama et al., 1991). The resistance of human epithelial tumor cells to TNFα appears to be associated with the expression of this cytokine (Spriggs et al., 1988; Takeyama et al., 1991; Wu et al, 1993a, 1994) and to be controlled by a protein-synthesis-dependent mechanism (Massad et al, 1991). The actions of TNFα are mediated by its two receptors, TNFR1 and TNFR2 (Fiers, 1991; Loetscher et al, 1990; Smith et al, 1990). TNFR1 contains an

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intracellular death domain required for induction of apoptosis and is coupled to an NFκB activation pathway. Binding of TNFα to its receptors activates caspase-8 and -3 (Boldin et al, 1996; Chinnaiyan et al., 1996; Medema et al., 1997; Yang et al., 1998) as well as induces IκB phosphorylation and degradation, and activates NFκB (Berberich et al, 1994; Kruppa et al, 1992; Laegreid et al, 1994; Rothe et al., 1994, 1995b; Sarma et al., 1995). NFκB activation regulates the expression of a number of genes involved in the modulation of TNFα-induced apoptosis, such as IAPs (Erl et al, 1999; Stehlik et al., 1998; Xiao et al., 2001) and flice-like inhibitory protein (FLIP) (Kreuz et al., 2001; Micheau et al., 2001; Xiao et al, 2002). FLIP is a Fas-associated death domain (FADD)-binding suppressor of apoptosis and present in two spliced isoforms, long (FLIPL) and short (FLIPS) (Irmler et al., 1997). Both isoforms contain two death effector domains, a structure resembling the N-terminal half of caspase-8 (Goltsev et al, 1997; Hu et al, 1997; Thome et al., 1997). In addition, FLIPL isoform has an inactive caspase-like domain. FLIP is recruited to the death-inducing signaling complex through the adaptor molecule, FADD, thereby preventing the recruitment of caspase-8 into the complex and subsequent caspase-8 activation, and suppressing apoptosis (Hu et al., 1997; Irmler et al., 1997; Srinivasula et al., 1999). Our recent data have demonstrated that FLIPS plays a key role in conferring cellular resistance to the cytotoxic action of TNFα in human ovarian surface epithelial cancer cells (Xiao et al., 2003). TNFα alone is unable to induce apoptosis but, in the presence of the protein synthesis inhibitor cycloheximide (CHX), significantly increases the number of apoptotic cells in vitro. While TNFα induces the expression of FLIPS but not of FLIPL in those cells in the concentration- and time-dependent manner, lowering FLIPS levels by antisense expression facilitated the pro-apoptotic action of the cytokine. This indicates that increased expression of FLIPS in response to TNFα challenge prevents downstream death signaling by the cytokine in this cell type. This contention is consistent with the current observations that while TNFα alone had no effect on caspase-8 cleavage in OV2008 cells, cotreatment of the cells with CHX and the cytokine resulted in significant cleavage of this caspase and increased apoptosis, which could be prevented by sense FLIPS cDNA expression (Xiao et al., 2003). Although our previous studies (Xiao et al., 2001) have shown that XIAP is important in determining the apoptotic responsiveness of rat ovarian granulosa cells to TNFα, this intracellular survival protein appears to play a minimal role, if any, in conferring resistance of the human ovarian cancer cells (OV2008, A2780-S, and OVCAR-3) to the cytotoxic action of the cytokine. In this latter context, TNFα failed to increase XIAP content in the ovarian cancer cells. However, it is of interest to note that, in the presence of protein synthesis inhibitor CHX, TNFα induced XIAP cleavage in OV2008 cells, a process sensitive to the presence of the caspase inhibitors ZVAD and DEVD (Xiao et al., 2003). These findings, together with the observations that cleavage of XIAP produces an N-terminal BIR-2 fragment with reduced ability to inhibit caspase-3

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and suppress apoptosis (Deveraux et al., 1999) support the concept that the caspase-3-mediated decrease in XIAP content may be involved in the execution of apoptosis in ovarian cancer cells in response to TNFα. SUMMARY Although the mechanisms of chemoresistance appear to be multifactorial, XIAP may be the key target of cisplatin action in human ovarian cancer. Failure of the chemotherapeutic agent to downregulate the survival gene in ovarian cancer cells may be the major determining factor in the development of chemoresistance. XIAP may be a novel target for gene therapy of human ovarian epithelial cancer. Generation and identification of potent tumor cell-specific XIAP inhibitors will significantly improve the efficacy of chemotherapy and reduce the dosages of chemotherapeutic agents required and minimize their side effects. Especially it will help overcome the chemoresistance. Developing a tumor cell-specific vector or expression system to downregulate XIAP content or function alone or in combination with wild-type p53 sense (dependent on p53 status) may offer another new approach for the treatment of the chemoresistant cancer. ACKNOWLEDGEMENTS 1 This work was supported in part by a grant from Canadian Institutes of Health Research (MOP-15691; B.K.T.) and a Grant-in-Aid for International Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (Joint Research #10044255; F.K. & B.K.T.). We thank the Canadian Institutes of Health Research and the Japan-North America Medical Exchange Foundation for award of post-doctoral fellowships to C.W.X. and H.S., respectively. 2 Figures 9.1 and 9.2 were reprinted from Cancer Research, Vol. 60, Sasaki, H., Sheng, Y., Kotsuji, F., Tsang, B.K., Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells, 5659–66, 2000, with permission from American Association for Cancer Research. 3 Figures 9.3 and 9.4 were reprinted from Gynecologic Oncology, Vol. 85, Sasaki, H., Kotsuji, F., Tsang, B.K., Caspase 3-mediated focal adhesion kinase processing in human ovarian cancer cells: possible regulation by Xlinked inhibitor of apoptosis protein, 339–50, 2002, with permission from Elsevier Science. REFERENCES Ambrosini, G., Adida, C. and Altieri, D.C. (1997) A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma, Nat. Med., 3:917–21.

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Andreani, C.L., Payne, D.W., Packman, J.N., Resnick, C.E., Hurwitz, A. and Adashi, E.Y. (1991) Cytokine-mediated regulation of ovarian function. Tumor necrosis factor alpha inhibits gonadotropin-supported ovarian androgen biosynthesis, J. Biol Chem., 266:6761–66. Aridor, M. and Balch, W.E. (1999) Integration of endoplasmic reticulum signaling in health and disease, Nat. Med., 5:745–51. Baker, S.J. and Reddy, E.P. (1996) Transducers of life and death: TNF receptor superfamily and associated proteins, Oncogene, 12:1–9. Barak, Y., Juven, T., Haffner, R. and Oren, M. (1993) mdm2 expression is induced by wild type p53 activity, EMBO J., 12:461–68. Barry, M.A., Behnke, C.A. and Eastman, A. (1990) Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia, Biochem. Pharmacol., 40:2353–62. Bellon, S.F., Coleman, J.H. and Lippard, S.J. (1991) DNA unwinding produced by sitespecific intrastrand cross-links of the antitumor drug cisdiamminedichloroplatinum(II), Biochemistry, 30:8026–35. Berberich, I., Shu, G.L. and Clark, E.A. (1994) Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B, J. Immunol., 53:4357–66. Bernal, S.D., Speak, J.A., Boeheim, K., Dreyfuss, A.I., Wright, J.E., Teicher, B.A., Rosowsky, A., Tsao, S.W. and Wong, Y.C. (1990) Reduced membrane protein associated with resistance of human squamous carcinoma cells to methotrexate and cisplatinum, Mol. Cell Biochem., 95:61–70. Birnbaum, M.J., Clem, R.J. and Miller, L.K. (1994) An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs, J. Virol., 68:2521–28. Boldin, M.P., Goncharov, T.M., Goltsev, Y.V. and Wallach, D. (1996) Involvement of MACH, a novel MORTl/FADD-interacting protease, in Fas/APO-1- and TNF receptorinduced cell death, Cell, 85:803–15. Bratton, S.B., Walker, G., Srinivasula, S.M., Sun, X.M., Butterworth, M., Alnemri, E.S. and Cohen, G.M. (2001) Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes, EMBO J., 20:998–1009. Cain, K., Brown, D.G., Langlais, C. and Cohen, G.M. (1999) Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex, J. Biol Chem., 274:22686–92. Cain, K., Bratton, S.B., Langlais, C, Walker, G., Brown, D.G., Sun, X.M. and Cohen, G.M. (2000) Apaf-1 oligomerizes into biologically active approximately 700-kDa and inactive approximately 1.4-MDa apoptosome complexes, J. Biol Chem., 275: 6067–70. Chen, L., Marechal, V., Moreau, J., Levine, A.J. and Chen, J. (1997) Proteolytic cleavage of the mdm2 oncoprotein during apoptosis, J. Biol. Chem., 272:22966–73. Chinnaiyan, A.M., Tepper, C.G., Seldin, M.F., O’Rourke, K., Kischkel, F.C., Hellbardt, S., Krammer, P.H., Peter, M.E. and Dixit, V.M. (1996) FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis, J. Biol Chem., 271:4961–65. Chu, G. and Chang, E. (1990) Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA, Proc. Natl. Acad. Sci. USA, 87:3324–28. Clem, R.J. and Miller, L.K. (1994) Control of programmed cell death by the baculovirus genes p35 and iap, Mol. Cell Biol., 14:5212–22.

166 CHAO WU XIAO ET AL.

Coin, F., Marinoni, J.C., Rodolfo, C, Fribourg, S., Pedrini, A.M. and Egly, J.M. (1998) Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH, Nat. Genet., 20:184–88. Cole, S.P. and Deeley, R.G. (1993) Multidrug resistance-associated protein: sequence correction, Science, 260:879. Cole, S.P., Sparks, K.E., Fraser, K., Loe, D.W., Grant, C.E., Wilson, G.M. and Deeley, R.G. (1994) Pharmacological characterization of multidrug resistant MRPtransfected human tumor cells, Cancer Res., 54:5902–10. Crook, N.E., Clem, R.J. and Miller, L.K. (1993) An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif, J. Virol., 67:2168–74. Crouch, D.H., Fincham, V.J. and Frame, M.C. (1996) Targeted proteolysis of the focal adhesion kinase pp125 FAK during c-MYC-induced apoptosis is suppressed by integrin signalling, Oncogene, 12:2689–96. Cryns, V. and Yuan, J. (1998) Proteases to die for, Genes. Dev., 12:1551–70. Dabholkar, M., Bostick, B., Weber, C, Bohr, V.A., Egwuagu, C. and Reed, E. (1992) ERCC1 and ERCC2 expression in malignant tissues from ovarian cancer patients, J. Natl. Cancer Inst., 84:1512–17. de Bruin, J.P., Dorland, M., Spek, E.R., Posthuma, G., van Haaften, M., Looman, C.W. and te Velde, E.R. (2002) Ultrastructure of the resting ovarian follicle pool in healthy young women, Biol Reprod., 66:1151–60. Deveraux, Q.L. and Reed, J.C. (1999) IAP family proteins—suppressors of apoptosis, Genes Dev., 13:239–52. Deveraux, Q.L., Takahashi, R., Salvesen, G.S. and Reed, J.C. (1997) X-linked IAP is a direct inhibitor of cell-death proteases, Nature, 388:300–04. Deveraux, Q.L., Roy, N., Stennicke, H.R., Van Arsdale, T., Zhou, Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S. and Reed, J.C. (1998) IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases, EMBO J., 17:2215–23. Deveraux, Q., Leo, E., Stennicke, H.R., Welsh, K., Salvesen, G.S. and Reed, J.C. (1999) Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases, EMBO J., 18:5242–51. Dive, C. and Wyllie, A.H. (1993) Apoptosis and cancer chemotherapy. In: J.A. Hickman and T.R. Tritton (eds), Cancer Chemotherapy, Oxford, United Kingdom: Blackwell, pp. 21–56. Duckett, C.S., Nava, V.E., Gedrich, R.W., Clem, R.J., Van, D., Gilfillan, M.C., Shiels, H., Hardwick, J.M. and Thompson, C.B. (1996) A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors, EMBO J., 15: 2685–94. Eastman, A. (1986) Reevaluation of interaction of cis-dichloro(ethylenediamine)platinum (II) with DNA, Biochemistry, 25:3912–15. Eastman, A. and Schulte, N. (1988) Enhanced DNA repair as a mechanism of resistance to cis-diamminedichloroplatinum (II), Biochemistry, 27:4730–34. Erhardt, P., Tomaselli, K.J. and Cooper, G.M. (1997) Identification of the MDM2 oncoprotein as a substrate for CPP32-like apoptotic proteases, J. Biol Chem., 272: 5049–52. Erl, W., Hansson, G.K., de Martin, R., Draude, G., Weber, K.S. and Weber, C. (1999) Nuclear factor-kappa B regulates induction of apoptosis and inhibitor of apoptosis protein-1 expression in vascular smooth muscle cells, Circ. Res., 84:668–77.

CHEMORESISTANCE IN HUMAN OVARIAN CANCER 167

Fichtinger-Schepman, A.M., van der Veer, J.L., den Hartog, J.H., Lohman, P.H. and Reedijk, J. (1985) Adducts of the antitumor drug cis-diamminedichloroplatinum (II) with DNA: formation, identification, and quantitation, Biochemistry, 24:707–13. Fiers, W. (1991) Tumor necrosis factor. Characterization at the molecular, cellular and in vivo level, FEBS Lett., 285:199–212. Frisch, S.M. and Francis, H. (1994) Disruption of epithelial cell-matrix interactions induces apoptosis, J. Cell Biol., 124:619–26. Frisch, S.M., Vuori, K., Ruoslahti, E. and Chan, H. (1996) Control of adhesion-dependent cell survival by focal adhesion kinase, J. Cell Biol, 134:793–99. Godwin, A.K., Meister, A., Dwyer, P.J., Huang, C.S., Hamilton, T.C. and Anderson, M.E. (1992) High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis, Proc. Natl. Acad. Sci. USA, 89:3070–74. Goltsev, Y.V., Kovalenko, A.V., Arnold, E., Varfolomeev, E.E., Brodianskii, V.M. and Wallach, D. (1997) CASH, a novel caspase homologue with death effector domains, J. Biol. Chem., 272:19641–44. Green, D.R. (1998) Apoptotic pathways: the roads to ruin, Cell, 94:695–98. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M, Soengas, M.S., Elia, A., de la Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M. and Mak, T.W. (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo, Cell, 94:339–52. Hamilton, T.C., Winker, M.A., Louie, K.G., Batist, G., Behrens, B.C., Tsuruo, T., Grotzinger, K.R., McKoy, W.M., Young, R.C. and Ozols, R.F. (1985) Augmentation of adriamycin, melphalan, and cisplatin cytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion, Biochem. Pharmacol., 34:2583–86. Harder, H.C. and Rosenberg, B. (1970) Inhibitory effects of anti-tumor platinum compounds on DNA, RNA and protein syntheses in mammalian cells in vitro, Int. J.Cancer, 6:207–16. Hartwell, L.H. and Kastan, M.B. (1994) Cell cycle control and cancer, Science, 266:1821– 28. Hassan, M.I., Kassim, S.K., Saeda, L., Laban, M. and Khalifa, A. (1999) Ovarian cancerinduced immunosuppression: relationship to tumor necrosis factor-alpha (TNFalpha) release from ovarian tissue, Anticancer Res., 19:5657–62. Honda, R., Tanaka, H. and Yasuda, H. (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53, FEBS Lett., 420:25–27. Howle, J.A. and Gale, G.R. (1970) Cis-dichlorodiammineplatinum(II). Persistent and selective inhibition of deoxyribonucleic acid synthesis in vivo, Biochem. Pharmacol., 19:2757–62. Hu, S., Vincenz, C., Ni, J., Gentz, R. and Dixit, V.M. (1997) I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis, J. Biol Chem., 272: 17255–57. Hungerford, J.E., Compton, M.T., Matter, M.L., Hoffstrom, B.G. and Otey, C.A. (1996) Inhibition of pp125FAK in cultured fibroblasts results in apoptosis, J. Cell Biol., 135: 1383–90. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M. and Yamamoto, T. (1995) Reduced cell motility and

168 CHAO WU XIAO ET AL.

enhanced focal adhesion contact formation in cells from FAK-deficient mice, Nature, 311:539–44. Irmler, M, Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J.L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L.E. and Tschopp, J. (1997) Inhibition of death receptor signals by cellular FLIP (see comments), Nature, 388:190–95. Ishikawa, T. and Ali, O. (1993) Glutathione-associated cis-diamminedichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione-platinum complex and its biological significance, J.Biol Chem., 268:20116–25. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y. and Nagata, S. (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis, Cell, 66:233–43. Izquierdo, M.A., van der Zee, A.G., Vermorken, J.B., van der Valk, P., Belien, J.A., Giaccone, G., Scheffer, G.L., Flens, M.J., Pinedo, H.M. and Kenemans, P. (1995) Drug resistanceassociated marker Lrp for prediction of response to chemotherapy and prognoses in advanced ovarian carcinoma, J.Natl. Cancer Inst., 87:1230–37. Izquierdo, M.A., Scheffer, G.L., Flens, M.J., Giaccone, G., Broxterman, H.J., Meijer, C.J., van der Valk, P. and Scheper, R.J. (1996) Broad distribution of the multidrug resistance-related vault lung resistance protein in normal human tissues and tumors, Am. J.Pathol., 148:877–87. Jacobson, M.D., Weil, M. and Raff, M.C. (1997) Programmed cell death in animal development, Cell, 88:347–54. Johnson, S.W., Swiggard, P.A., Handel, L.M., Brennan, J.M., Godwin, A.K., Ozols, R.F. and Hamilton, T.C. (1994) Relationship between platinum-DNA adduct formation and removal and cisplatin cytotoxicity in cisplatin-sensitive and -resistant human ovarian cancer cells, Cancer Res., 54:5911–16. Johnson, S.W., Laub, P.B., Beesley, J.S., Ozols, R.F. and Hamilton, T.C. (1997) Increased platinum-DNA damage tolerance is associated with cisplatin resistance and crossresistance to various chemotherapeutic agents in unrelated human ovarian cancer cell lines, Cancer Res., 57:850–56. Kasof, G.M. and Gomes, B.C. (2001) Livin, a novel inhibitor of apoptosis protein family member, J.Biol. Chem., 276:3238–46. Kawai, K., Kamatani, N., Georges, E. and Ling, V. (1990) Identification of a membrane glycoprotein overexpressed in murine lymphoma sublines resistant to cisdiamminedichloroplatinum(II), J.Biol Chem., 265:13137–42. Kerr, J.F., Winterford, C.M. and Harmon, B.V. (1994) Apoptosis. Its significance in cancer and cancer therapy, Cancer, 73:2013–26. Khwaja, A., Rodriguez, V., Wennstrom, S., Warne, P.H. and Downward, J. (1997) Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway, EMBO /., 16:2783–93. Komatsu, M., Sumizawa, T., Mutoh, M., Chen, Z.S., Terada, K., Furukawa, T., Yang, X.L., Gao, H., Miura, N., Sugiyama, T. and Akiyama, S. (2000) Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance, Cancer Res., 60:1312–16. Kool, M., de Haas, M., Scheffer, G.L., Scheper, R.J., van Eijk, M.J., Juijn, J.A., Baas, F. and Borst, P. (1997) Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and

CHEMORESISTANCE IN HUMAN OVARIAN CANCER 169

MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines, Cancer Res., 57:3537–47. Kreuz, S., Siegmund, D., Scheurich, P. and Wajant, H. (2001) NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling, Mol. Cell. Biol., 21:3964–73. Kruppa, G., Thoma, B., Machleidt, T., Wiegmann, K. and Kronke, M. (1992) Inhibition of tumor necrosis factor (TNF)-mediated NF-kappa B activation by selective blockade of the human 55-kDa TNF receptor, J.Immunol., 148:3152–57. Kuerbitz, S.J., Plunkett, B.S., Walsh, W.V. and Kastan, M.B. (1992) Wild-type p53 is a cell cycle checkpoint determinant following irradiation, Proc. Natl. Acad. Sci. USA, 89:7491–95. Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Karasuyama, H., Su, M.S., Rakic, P. and Flavell, R.A. (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9, Cell, 94:325–37. Laegreid, A., Medvedev, A., Nonstad, U., Bombara, M.P., Ranges, G., Sundan, A. and Espevik, T. (1994) Tumor necrosis factor receptor p75 mediates cell-specific activation of nuclear factor kappa B and induction of human cytomegalovirus enhancer, J. Biol Chem., 269:7785–91. Lai, G.M., Ozols, R.F., Smyth, J.F., Young, R.C. and Hamilton, T.C. (1988) Enhanced DNA repair and resistance to cisplatin in human ovarian cancer, Biochem. Pharmacol., 37:4597–600. Lai, G.M., Ozols, R.F., Young, R.C. and Hamilton, T.C. (1989) Effect of glutathione on DNA repair in cisplatin-resistant human ovarian cancer cell lines, J. Natl. Cancer Inst., 81:535–39. Landis, S.H., Murray, T., Bolden, S. and Wingo, P.A. (1999) Cancer statistics, CA Cancer J. Clin., 49:8–31. Lane, D.P. (1992) Cancer. p53, guardian of the genome, Nature, 358:15–16. Levine, A.J. (1997) p53, the cellular gatekeeper for growth and division, Cell, 88:323–31. Li, J., Feng, Q., Kim, J.M., Schneiderman, D., Liston, P., Li, M., Vanderhyden, B., Faught, W., Fung, M.F., Senterman, M., Korneluk, R.G. and Tsang, B.K. (2001) Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins, Endocrinology, 142:370–80. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. and Wang, X. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade, Cell, 91:479–89. Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., Cherton, H., Farahani, R., McLean, M., Ikeda, J.E., MacKenzie, A. and Korneluk, R.G. (1996) Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes, Nature, 379:349– 53. Liu, J.R., Opipari, A.W., Tan, L., Jiang, Y., Zhang, Y., Tang, H. and Nunez, G. (2002) Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance, Cancer Res., 62:924–31. Loetscher, H., Pan, Y.C., Lahm, H.W., Gentz, R., Brockhaus, M., Tabuchi, H. and Lesslauer, W. (1990) Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor, Cell, 61:351–59. Massad, L.S., Mutch, D.G., Kao, M.S., Powell, C.B. and Collins, J.L. (1991) Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor alpha and

170 CHAO WU XIAO ET AL.

interferon gamma in cell lines derived from gynecological malignancies, Cancer Immunol. Immunother., 33:183–88. Masuda, H., Ozols, R.F., Lai, G.M., Fojo, A., Rothenberg, M. and Hamilton, T.C. (1988) Increased DNA repair as a mechanism of acquired resistance to cisdiamminedichloroplatinum (II) in human ovarian cancer cell lines, Cancer Res., 48:5713–16. Medema, J.P., Scaffidi, C., Kischkel, F.C., Shevchenko, A., Mann, M., Krammer, P.H. and Peter, M.E. (1997) FLICE is activated by association with the CD95 deathinducing signaling complex (DISC), EMBO J., 16:2794–804. Micheau, O., Lens, S., Gaide, O., Alevizopoulos, K. and Tschopp, J. (2001) NF-kappaB signals induce the expression of c-FLIP, Mol. Cell Biol., 21:5299–305. Miller, L.K. (1999) An exegesis of IAPs: salvation and surprises from BIR motifs, Trends Cell Biol., 9:323–28. Momand, J., Zambetti, G.P., Olson, D.C., George, D. and Levine, A.J. (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation, Cell, 69:1237–45. Muller, M., Strand, S., Hug, H., Heinemann, E.M., Walczak, H., Hofmann, W.J., Stremmel, W., Krammer, P.H. and Galle, P.R. (1997) Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53, J. Clin. Invest., 99:403–13. Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A. and Yuan, J. (2000) Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta, Nature, 403:98–103. Nakayama, K., Kanzaki, A., Ogawa, K., Miyazaki, K., Neamati, N. and Takebayashi, Y. (2002) Copper-transporting P-type adenosine triphosphatase (ATP7B) as a cisplatin based chemoresistance marker in ovarian carcinoma: comparative analysis with expression of MDR1, MRP1, MRP2, LRP and BCRP is expressed in human breast carcinoma, Int. J. Cancer, 101:488–95. Naylor, M.S., Stamp, G.W., Foulkes, W.D., Eccles, D. and Balkwill, F.R. (1993) Tumor necrosis factor and its receptors in human ovarian cancer. Potential role in disease progression, J. Clin. Invest., 91:2194–206. Nunez, G., Benedict, M.A., Hu, Y. and Inohara, N. (1998) Caspases: the proteases of the apoptotic pathway, Oncogene, 17:3237–45. Park, D.J., Stoehlmacher, J., Zhang, W., Tsao, W., Groshen, S. and Lenz, H.J. (2001) A Xeroderma pigmentosum group D gene polymorphism predicts clinical outcome to platinum-based chemotherapy in patients with advanced colorectal cancer, Cancer Res., 61:8654–58. Parker, R.J., Eastman, A., Bostick, B. and Reed, E. (1991) Acquired cisplatin resistance in human ovarian cancer cells is associated with enhanced repair of cisplatin-DNA lesions and reduced drug accumulation, J.Clin. Invest., 87:772–77. Pepling, M.E. and Spradling, A.C. (2001) Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles, Dev. Biol., 234:339–51. Reardon, J.T. and Sancar, A. (1998) Molecular mechanism of nucleotide excision repair in mammalian cells. In: M. Dizdaroglu and A. Karakaya (eds), Advances in DNA damage and repair, New York: Plenum Publishing Corp., pp. 377–93. Riordan, J.R., Deuchars, K., Kartner, N., Alon, N., Trent, J. and Ling, V. (1985) Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines, Nature, 316:817–19.

CHEMORESISTANCE IN HUMAN OVARIAN CANCER 171

Rothe, M., Wong, S.C., Henzel, W.J. and Goeddel, D.V. (1994) A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor, Cell, 78:681–92. Rothe, M., Pan, M.G., Henzel, W.J., Ayres, T.M. and Goeddel, D.V. (1995a) The TNFR2TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins, Cell, 83:1243–52. Rothe, M., Sarma, V., Dixit, V.M. and Goeddel, D.V. (1995b) TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40, Science, 269:1424–27. Roy, N., Mahadevan, M.S., McLean, M., Shutler, G., Yaraghi, Z., Farahani, R., Baird, S., Besner, J., Lefebvre, C. and Kang, X. (1995) The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy, Cell, 80:167–78. Roy, N., Deveraux, Q.L., Takahashi, R., Salvesen, G.S. and Reed, J.C. (1997) The cIAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases, EMBO J., 16: 6914–25. Sarma, V., Lin, Z., Clark, L., Rust, B.M., Tewari, M., Noelle, R.J. and Dixit, V.M. (1995) Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis, J. Biol Chem., 270:12343–46. Sasaki, H., Sheng, Y., Kotsuji, F. and Tsang, B.K. (2000) Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells, Cancer Res., 60:5659–66. Sasaki, H., Kotsuji, F. and Tsang, B.K. (2002) Caspase 3-mediated focal adhesion kinase processing in human ovarian cancer cells: possible regulation by X-linked inhibitor of apoptosis protein, Gynecol. Oncol., 85:339–50. Scheffer, G.L., Wijngaard, P.L., Flens, M.J., Izquierdo, M.A., Slovak, M.L., Pinedo, H.M., Meijer, C.J., Clevers, H.C. and Scheper, R.J. (1995) The drug resistancerelated protein LRP is the human major vault protein, Nat. Med., 1:578–82. Schlaepfer, D.D. and Hunter, T. (1998) Integrin signalling and tyrosine phosphorylation: just the FAKs?, Trends. Cell Biol., 8:151–57. Schneiderman, D., Kim, J.M., Senterman, M. and Tsang, B.K. (1999) Sustained suppression of Fas ligand expression in cisplatin-resistant human surface epithelial cancer cells, Apoptosis, 4:1–11. Sheibani, N., Jennerwein, M.M. and Eastman, A. (1989) DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): host cell reactivation of damaged plasmid DNA, Biochemistry, 28:3120–24. Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Casiano, C.A., Newmeyer, D.D., Wang, H.G., Reed, J.C., Nicholson, D.W., Alnemri, E.S., Green, D.R. and Martin, S.J. (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner, J. Cell Biol., 144:281–92. Smith, C.A., Davis, T., Anderson, D., Solam, L., Beckmann, M.P., Jerzy, R., Dower, S.K., Cosman, D. and Goodwin, R.G. (1990) A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins, Science, 248:1019–23. Sorenson, C.M. and Eastman, A. (1988) Influence of cis-diamminedichloroplatinum(II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells, Cancer Res., 48:6703–07.

172 CHAO WU XIAO ET AL.

Sorenson, C.M., Barry, M.A. and Eastman, A. (1990) Analysis of events associated with cell cycle arrest at G2 phase and cell death induced by cisplatin, J. Natl. Cancer Inst., 82:749–55. Spriggs, D.R., Imamura, K., Rodriguez, C., Sariban, E. and Kufe, D.W. (1988) Tumor necrosis factor expression in human epithelial tumor cell lines, J.Clin. Invest., 81: 455–60. Srinivasula, S.M., Ahmad, M., Lin, J.H., Poyet, J.L., Fernandes-Alnemri, T., Tsichlis, P.N. and Alnemri, E.S. (1999) CLAP, a novel caspase recruitment domain-containing protein in the tumor necrosis factor receptor pathway, regulates NF-kappaB activation and apoptosis, J.Biol Chem., 274:17946–54. Stehlik, C., de Martin, R., Kumabashiri, I., Schmid, J.A., Binder, B.R. and Lipp, J. (1998) Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis, J. Exp. Med., 188:211–16. Suda, T. and Nagata, S. (1994) Purification and characterization of the Fas-ligand that induces apoptosis, J. Exp. Med., 179:873–79. Sun, C., Cai, M., Gunasekera, A.H., Meadows, R.P., Wang, H., Chen, J., Zhang, H., Wu, W., Xu, N., Ng, S.C. and Fesik, S.W. (1999a) NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP, Nature, 401:818–22. Sun, X.M., MacFarlane, M., Zhuang, J., Wolf, B.B., Green, D.R. and Cohen, G.M. (1999b) Distinct caspase cascades are initiated in receptor-mediated and chemicalinduced apoptosis, J.Biol Chem., 274:5053–60. Sun, C, Cai, M., Meadows, R.P., Xu, N., Gunasekera, A.H., Herrmann, J., Wu, J.C. and Fesik, S.W. (2000) NMR structure and mutagenesis of the third Bir domain of the inhibitor of apoptosis protein XIAP, J.Biol Chem., 275:33777–81. Sung, P., Bailly, V., Weber, C, Thompson, L.H., Prakash, L. and Prakash, S. (1993) Human xeroderma pigmentosum group D gene encodes a DNA helicase, Nature, 365:852–55. Suzuki, Y., Nakabayashi, Y., Nakata, K., Reed, J.C. and Takahashi, R. (2001a) X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes, J.Biol. Chem., 276:27058–63. Suzuki, Y., Nakabayashi, Y. and Takahashi, R. (2001b) Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death, Proc. Natl. Acad. Sci. USA, 98:8662–67. Takahashi, R., Deveraux, Q., Tamm, I., Welsh, K., Assa-Munt, N., Salvesen, G.S. and Reed, J.C. (1998) A single BIR domain of XIAP sufficient for inhibiting caspases. J.Biol Chem., 273:7787–90. Takeyama, H., Wakamiya, N., Hara, C., Arthur, K., Niloff, J., Kufe, D., Sakarai, K. and Spriggs, D. (1991) Tumor necrosis factor expression by human ovarian carcinoma in vivo, Cancer Res., 51:4476–80. Taniguchi, K., Wada, M, Kohno, K., Nakamura, T., Kawabe, T., Kawakami, M., Kagotani, K., Okumura, K., Akiyama, S. and Kuwano, M. (1996) A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation, Cancer Res., 56:4124–29. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J.L., Schroter, M., Scaffidi, C., Krammer, P.H.,

CHEMORESISTANCE IN HUMAN OVARIAN CANCER 173

Peter, M.E. and Tschopp, J. (1997) Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors, Nature, 386:517–21. Thompson, C.B. (1995) Apoptosis in the pathogenesis and treatment of disease, Science, 267:1456–62. Thornberry, N.A. and Lazebnik, Y. (1998) Caspases: enemies within, Science, 281:1312– 16. Ueda, K., Cardarelli, C, Gottesman, M.M. and Pastan, I. (1987) Expression of a fulllength cDNA for the human “MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine, Proc. Natl. Acad. Sci. USA, 84:3004–08. Uren, A.G., Pakusch, M., Hawkins, C.J., Puls, K.L. and Vaux, D.L. (1996) Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors, Proc. Natl. Acad. Sci. USA, 93:4974–78. Uslu, R., Jewett, A. and Bonavida, B. (1996) Sensitization of human ovarian tumor cells by subtoxic CDDP to anti-fas antibody-mediated cytotoxicity and apoptosis, Gynecol. Oncol., 62:282–91. Wakahara, Y., Nawa, A., Okamoto, T., Hayakawa, A., Kikkawa, F., Suganama, N., Wakahara, F. and Tomoda, Y. (1997) Combination effect of anti-Fas antibody and chemotherapeutic drugs in ovarian cancer cells in vitro, Oncology, 54:48–54. Woo, M., Hakem, R., Soengas, M.S., Duncan, G.S., Shahinian, A., Kagi, D., Hakem, A., McCurrach, M., Khoo, W., Kaufman, S.A., Senaldi, G., Howard, T., Lowe, S.W. and Mak, T.W. (1998) Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes, Genes Dev., 12:806–19. Wu, S., Boyer, C.M., Whitaker, R.S., Berchuck, A., Wiener, J.R., Weinberg, J.B. and Bast, R.C. (1993a) Tumor necrosis factor alpha as an autocrine and paracrine growth factor for ovarian cancer: monokine induction of tumor cell proliferation and tumor necrosis factor alpha expression, Cancer Res., 53:1939–44. Wu, X., Bayle, J.H., Olson, D. and Levine, A.J. (1993b) The p53-mdm-2 autoregulatory feedback loop, Genes Dev., 7:1126–32. Wu, S., Meeker, W.A., Wiener, J.R., Berchuck, A., Bast, R.C. and Boyer, C.M. (1994) Transfection of ovarian cancer cells with tumor necrosis factor-alpha (TNF-alpha) antisense mRNA abolishes the proliferative response to interleukin-1 (IL-1) but not TNF-alpha, Gynecol Oncol., 53:59–63. Wyllie, A.H., Kerr, J.F. and Currie, A.R. (1980) Cell death: the significance of apoptosis, Int. Rev. Cytol., 68:251–306. Xiao, C.W., Ash, K. and Tsang, B.K. (2001) Nuclear factor-kappaB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor alpha-induced apoptosis, Endocrinology, 142:557–63. Xiao, C.W., Asselin, E. and Tsang, B.K. (2002) Nuclear factor kappaB-mediated induction of Flice-like inhibitory protein prevents tumor necrosis factor alphainduced apoptosis in rat granulosa cells, Biol Reprod., 67:436–41. Xiao, C.W., Yan, X., Li, Y., Reddy, S. and Tsang, B.K. (2003) Resistance of human ovarian cancer cells to TNF-alpha is a consequence of NF-KappaB-mediated induction of FLICE-like inhibitory protein, Endocrinology, 144:623–30. Yang, X., Chang, H.Y. and Baltimore, D. (1998) Autoproteolytic activation of procaspases by oligomerization, Mol. Cell, 1:319–25.

174 CHAO WU XIAO ET AL.

Zhen, W., Link, C.J., Connor, P.M., Reed, E., Parker, R., Howell, S.B. and Bohr, V.A. (1992) Increased gene-specific repair of cisplatin interstrand cross-links in cisplatinresistant human ovarian cancer cell lines, Mol. Cell Biol., 12:3689–98. Zou, H., Li, Y., Liu, X. and Wang, X. (1999) An APAF-l.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9, J. Biol. Chem., 274: 11549–56.

10 THE EPIDEMIOLOGY OF OVARIAN CANCER: THE ROLE OF REPRODUCTIVE FACTORS AND ENVIRONMENTAL CHEMICAL EXPOSURE Kathryn Coe

INTRODUCTION Ovarian cancer, as it is clinically silent until it becomes well established and has many chromosomal abnormalities already present, is a deadly, insidious disease (Murdoch and McDonnel, 2002). Consequently, an understanding of ovarian cancer is crucial and epidemiology offers some hints—biological and environmental/cultural—that, when considered together, can help us increase our understanding of this disease. Many cases of ovarian cancer are sporadic, lacking any known risk factors. The known risk factors, however, include such things as being a female, growing older, family history of ovarian cancer, personal history of cancer (e.g., endometrial, colon or breast cancer), hyperthyroidism, exposure to xenobiotic agents, such as talcum powder used on the perineum; and reproductive history, including early onset of menstruation (before age 12), irregular menstrual cycles, never having given birth or having a first child after age 30, menopause after age 50, use of hormone replacement therapy (HRT) and use of fertility drugs especially without achieving pregnancy. Many of these risk factors offer clinicians little help in anticipating who is likely to get ovarian cancer or how to prevent it. The most clinically significant risk factors include being a woman, growing older, having had breast cancer and having a first-degree relative (mother, sister or daughter) who has been diagnosed with ovarian cancer. The other risk factors seem to have only a weak association with ovarian cancer, occur too infrequently, or are physiologic events (e.g., nulliparity, refractory infertility) that are not easily amenable to intervention. Further, the factors that seem to protect women from getting ovarian cancer (e.g., race, multiparity, tubal ligation or hysterectomy, oral contraceptive use (OCU), regular intake of aspirin) are not readily modifiable. Other protective factors, while theoretically modifiable through health education (e.g., breastfeeding) programs, have not proven to be easy to modify. In this chapter it is assumed that ovarian cancer is a multifactorial, multidimensional disease. A consideration of epidemiology, genetics, exposure

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to xenobiotic agents, ovarian development and human evolution are all necessary elements if we are to increase our understanding of ovarian cancer etiology and the chain of interacting and sequential events that make it more likely that one female will experience ovarian cancer. Changes in DNA are responsible for the progression toward and development of ovarian cancer. These changes accumulate over a lifetime of exposures and are the result of multiple events. Although the identification of ovotoxins specific to humans is difficult, just as their effects are difficult to predict or evaluate (Hoyer, 1997), it seems clear that variable effects (short term or long term) are likely to result from different forms of exposure, length of exposure and the particular genetic, developmental and reproductive history of the organism during the period in which exposure occurs. All these factors can influence whether or not tissue and cells are susceptible to the action of ovotoxins, whether a normal cell is converted to a cancerous one and whether or not the converted cells begin to proliferate. WHAT IS OVARIAN CANCER? Most vertebrate females, including humans, have two ovaries that are held in place by a membrane on each side of the uterus. These ovaries produce the ova or female reproductive cells, and secrete the steroid hormones, estrogen, during the follicular or pre-ovulatory phase of a woman’s cycle, and progesterone, during the luteal or post-ovulatory phase. These hormones control the development of the sexual organs and the secondary sexual characteristics and their cessation is associated with the atrophy of these organs. Ovarian cancer is not a single disease; there are 30 types and subtypes of ovarian malignancies, each with its own histopathologic appearance, biologic behavior and possible etiology (Hildreth et al., 1981). All ovarian malignancies, however, are categorized into three major groups: epithelial tumors, germ cell tumors and sex cord-stromal cell tumors in the connective cells. Mutations in key target genes alter the normal processes that help a cell regulate its fate. These mutations are responsible for the progression of a normal, well-behaved cell into a cancerous cell, the descendants of which will all be cancer cells. The accumulated mutations divide, obstruct, invade and destroy normal tissue architecture and these converted cells acquire properties that allow them to escape the normal biological defenses and controls (Collins et al., 1996). Xenobiotics are agents that cause cell change. Ovotoxicants are those xenobiotic agents that negatively target the ovaries, affecting their functioning and perhaps leading to infertility, a risk factor for ovarian cancer. Carcinogens are xenobiotic agents that cause a statistically significant increase in incidence of neoplasm. These agents can be physical, biological, such as viruses, or chemical, such as complex hydrocarbons, aromatic amines, certain metals and chemicals that occur naturally in molds, plants and hormones (Prescott and Flexer, 1982; Russo et al, 1992).

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Carcinogens appear to alter the DNA of normal cells, thus modifying the content or flow of genetic information coded in the DNA’s base sequence. Reactive electrophilic intermediates or carbonium ions appear to be formed during the metabolism of carcinogens. It is the reaction of these highly reactive intermediates with cellular components that may initiate the chain of events that results in cancer (Russo et al., 1992). Susceptibility of the cell to undergo malignant transformation seems to be correlated with the rate of DNA synthesis and cell proliferation and cellular competency in DNA excision repair (Russo et al., 1992). The ultimate carcinogenicity of any compound seems to depend upon two factors: level of metabolites in target tissue available for interaction with critical cellular nucleophiles and presence of susceptible or non-susceptible tissue (Russo et al., 1992). Developmental stages involving cell proliferation may be crucial, as cell proliferation is the driving force of tumor growth; carcinogens induce a greater response in dividing than in resting cells (Eaton et al., 1988; Russo et al., 1992). HOW COMMON IS OVARIAN CANCER? The Surveillance, Epidemiology and End Results (SEER), which is funded by the National Cancer Institute, collects and publishes cancer incidence and survival data from eleven population-based cancer registries and three supplemental registries covering approximately 14 percent of the US population. According to data from the SEER program, ovarian cancer is the fifth most commonly diagnosed cancer in women in the US, excluding non-melanoma skin cancers. It accounts for about 23,000 cases each year or about 4 percent of all annually diagnosed female cancers (Cannistra, 1983; American Cancer Society (ACS), 2002). The average lifetime risk for developing ovarian cancer in the US is about 1 in 70 (Cannistra, 1983). Ovarian cancer that begins on the surface of the ovary (epithelial ovarian carcinoma or EOC) is the most common type; 85–90 percent of ovarian cancer originates in the ovarian surface epithelium (Bai et al, 2000). Ovarian cancer, that begins in the egg-producing cells (germ cell tumors) and in the supportive tissue surrounding the ovaries (stromal tumors), is rare. Carcinosarcoma of the ovary, defined by the presence of malignant epithelial and mesenchymal elements, is very rare, accounting for fewer than 1 percent of ovarian malignancies. IS OVARIAN CANCER CURABLE? It has been estimated that in the year 2002, approximately 14,000 women would die of ovarian cancer (ACS, 2002). A number of prognostic factors influence ovarian cancer survival: stage of disease at diagnosis, age at diagnosis, type of chemotherapeutic agents and the type of surgical treatment and specialty of the surgeon (McGuire et al., 2002). If diagnosed in its early stages, ovarian cancer is curable in a high percentage of patients. However, as ovarian cancer is often

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asymptomatic in its early stages, most patients (roughly 75 percent) have advanced disease at the time of diagnosis (ACS, 2002). When a multimodality approach is used to treat ovarian cancer, five-year survival is as follows: Stage I, 93 percent; Stage II, 70 percent; Stage III, 37 percent and Stage IV, 25 percent (Holschneider and Berek, 2000). By and large, however, only about 18 percent of women who present with advanced disease survive five years or more after diagnosis (see Schwartz and Taylor, 1995; Tortolero et al., 1995). Ovarian cancer is the fifth most frequent cause of cancer death in US women (ACS, 2002), accounting for more deaths than any other cancer of the reproductive tract. Over the past 20 years, ovarian cancer mortality has remained more or less constant despite the introduction of new chemotherapy agents and interest in early stage disease screening. Overall survival rates are static at about 30 percent (Edmondson and Monaghan, 2001). RISK AND PROTECTIVE FACTORS FOR OVARIAN CANCER Geography Incidence of ovarian cancer varies internationally, with low incidence in Japan and higher rates among women of North America and northern Europe (Holschneider and Berek, 2000; McLaughlin, 2002). In northern Europe, as well as in the United States, ovarian cancer represents the third most frequent cancer of the female genital tract (Runnebaum and Stickeler, 2001). Data from cancer registries indicate that epithelial ovarian cancer rates are higher in industrialized nations, with the exception of Japan, and lower in non-industrialized parts of the world, such as Sub-Saharan Africa and China (Katchy and Briggs, 1992; Holschneider and Berek, 2000). To some extent, these geographic patterns are correlated with family size (Daly, 1992). Age Ovarian cancer is a disease of aging (ACS, 2002). Incidence starts rising in the late teenage years and gradually increases with age. After age 40, incidence and mortality rise sharply with increasing age. Maximum incidence occurs in the 80– 84-year old age group, with an incidence of 61.8 per 100,000 women (Edmondson and Monaghan, 2001). Reproductive history While certain reproductive factors, such as multiparity and breastfeeding, seem to offer protection against ovarian cancer, others, including early onset of

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menstruation, nulliparity and having a first child after age 30, seem to increase a woman’s risk. • Late childbirth: Women who experience their first childbirth at 25 or younger have a decreased risk for getting ovarian cancer (Daly and Obrams, 1998). An even higher risk of ovarian cancer is associated with first birth after age 35 (Negri et al., 1991). • Nulliparity: Nulliparous women experience a higher risk for ovarian cancer (Negri et al., 1991). Nulliparous women who have a family history of ovarian cancer may be at particularly high risk. In a prospective cohort study of 31, 377 Iowa women, aged 55–69, nulliparous women who had a family history of ovarian cancer were at much higher risk than were parous ones (Relative risk (RR)=2.7, 95 percent CI= 1.1–6.6) (Vachon et al, 2002). Similar results were observed when the family history included first and second-degree relatives with breast or ovarian cancer, or a first- or second-degree relative with ovarian cancer only. Nulliparity may be voluntary or involuntary. Refractory infertility refers to unexplained infertility, as occurs when a woman experiences no pregnancies and is sexually active, yet has no history of birth control usage. Women who have had trouble getting pregnant seem to be at increased risk for ovarian cancer. Ness et al. (2002), who pooled interview data on infertility and fertility drug use from eight case-control studies conducted in the US, Denmark, Canada and Australia, found that nulligravid women who attempted to become pregnant for more than five years, compared with nulligravid women who attempted to become pregnant for less than one year, experienced a 2.67-fold increased risk of getting ovarian cancer. • Multiple births: Based upon theoretical models, it has been predicted that women with a history of multiple births (twins, triplets, etc.) should be at increased risk of epithelial ovarian cancer. The scant evidence available, however, seems to suggest that these women actually may be at lower risk. Whiteman et al. (2000), in a study that pooled data from eight separate studies focusing on multiple births and risk of epithelial ovarian cancer (2,859 parous women with EOC and 7,434 controls), found no evidence that parous women with non-mucinous ovarian cancer were more likely to have a history of multiple births than were other parous women. • Multiparity: Of the several reproductive factors known to influence the risk of ovarian cancer, the strongest and most frequently studied is the number of full-term pregnancies. Multiparity appears to significantly decrease a women’s risk of getting ovarian cancer (Daly and Obrams, 1998). The Nurses Cohort Study of 121,700 women, who have been studied prospectively since 1976, found that the effect of parity has been to reduce ovarian cancer risk (OR=0.84, 95 percent CI= 0.77–0.91 for each pregnancy) (Hankinson et al., 1995). John et al. (1993) summarized the results of 12 US case control studies and found that a single-term pregnancy had a significant effect on ovarian

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•

•

•

•

cancer risk (OR=0.47, 95 percent CI). Risk decreased as the number of pregnancies increased, such that after six-term pregnancies, the OR is reduced to 0.29, with 95 percent CI of 0.20–0.42. Risk declines by about 15 percent for each additional full-term pregnancy (Risch et al., 1994). A low pregnancy rate is apparently a cause, not a consequence, of ovarian pathology leading to ovarian cancer (Berg and Lampe, 1981). Whereas incomplete pregnancies and a history of OCU appear to reduce risk, a Danish case control study found no relationship between ovarian cancer and miscarriages, induced abortions or ectopic pregnancies (Mosgaard et al., 1997). Pregnancy involves a series of immunological, metabolic, vascular and endocrine-regulating processes, inducing both anovulation and suppression of pituitary gonadotropins as well as immunosuppression. While we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities, maternal immunosuppression during pregnancy seems to be necessary for mammalian maternal tolerance of the fetus (Burton et al., 2001; Fessler, 2002). Pregnancy is associated with a progressive suppression of cell-mediated immunity (Taylor et al., 2002) and this immunosuppression makes the mother vulnerable to pathogens. While pregnancy is said to be a strong protective factor, it actually may have a dual effect on risk: increasing short-term risk of breast and ovarian cancer, while conferring long-term protection (Lambe et al., 1994). Breastfeeding: When John et al. (1993) reviewed US studies of ovarian cancer, they separated the effects of breastfeeding from pregnancy. They found that breastfeeding had a small protective effect (OR=0.81, 95 percent CI=0.68–0.95). Prolonged breastfeeding may confer additional protection (Whittemore, 1994, but see Rosenblatt and Thomas, 1993). Early menarche: Early menarche has been associated with increased risk of ovarian cancer in studies conducted in the US (Wu et al, 1988), Pakistan (Malik, 2002), Italy (Chiaffarino et al, 2001), Norway (Kvale et al, 1991), China (Shu et al, 1989), but not in Qatar (Ejeckam et al, 1994). Later age at menopause: Approximately 36 million women in the US are in the post-menopausal phase of life, having experienced spontaneous cessation of menses between the ages of 47 and 55 (Friedlander, 2002). Those women who enter menopause in the later ages are at higher risk for ovarian cancer (see Hildreth et al., 1981; Malik, 2002). Ovarian cancer risk increases as a woman ages; the majority of ovarian cancer cases are diagnosed after menopause. Tubal ligation and prior hysterectomy: Epidemiologic studies suggest that tubal ligation and prior hysterectomy may decrease the risk of ovarian cancer. In a case control study, Rosenblatt and Thomas (1996) found that the possible protective effect of tubal ligation was greatest in women of parity less than four and that the protective effect was only for clear cell and endometrioid tumors. When Cramer and Xu (1995) combined data from two case control

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studies, they found that both tubal ligation and prior hysterectomy were protective and that those women who had previously undergone either surgery, 20 years or more before, had the lowest risk of ovarian cancer. Family history The idea that cancer might be inherited is not a new one, and it now is common knowledge that a family history of ovarian cancer, especially if two or more firstdegree relatives on either the father’s side or the mother’s side have been affected, is the most important risk factor. These familial ovarian cancers tend to occur at an early age, before 50 years, and tend to be advanced serous epithelial cancers. In 1994, the gene BRCA1 was discovered, with BRCA2 discovered the following year. These genes normally work to prevent breast and ovarian cancer; however, in some cases we can inherit a mutated or altered form of BRCA1 or BRCA2 from either parent. This mutation interferes with the normal activity of the gene, making individuals more susceptible to both breast and ovarian cancer. Individuals with one of these gene mutations have a higher risk of developing breast and ovarian cancers and may also pass that gene mutation on to his or her children. Researchers have identified three ways in which ovarian cancer can be inherited. The syndromes include the site-specific ovarian cancer syndrome, the breast/ ovarian cancer syndromes (involving mutation in either the BRCA1 or BRCA2 genes) and the lynch syndrome II, which applies to women with female or male relatives who have had non-polyposis-related colorectal and endometrial cancer (see Murdoch and McDonnel, 2002). Mutations at other loci, including p53, may explain remaining inherited ovarian cancer cases (Sellers et al., 1993). Although mutations in BRCA1 and BRCA2 genes are implicated in ovarian cancer risk, it is not yet clear as how these mutations differ in their relative contributions to ovarian cancer. Further, while early researchers assumed that women who inherited the BRCA genes were almost certain to get breast or ovarian cancer, more recent research indicates that these mutations are considerably less frequent than initially was assumed, in women with modest family history of ovarian cancer (see Foulkes and Narod, 1995; Nussbaum Cohen, 1997). The genes seem to work differently in different environments; the same mutations may behave differently in different populations of women. A number of factors (e.g., reproductive history, hormone therapy, diet, smoking and the presence of other genes which, for example, control the metabolism of hormones) modify the effect of any gene in determining the final outcome. All cancers are ultimately determined by a combination of genetic and environmental factors.

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Ethnicity and ancestry Researchers have known for many years that certain diseases occur more commonly in certain ethnic groups, just as they do in certain families or lineages. Researchers often assume that ethnicity is a predictive factor because individuals sharing ethnicity share certain cultural behaviors, such as religious practices, that inhibit or promote health. Another possibility, however, is that ethnicity is a predictor of health because we inherit our ethnicity from our ancestors. Those sharing ethnicity, thus, are co-descendants of a common ancestor from whom they have inherited both genes and traditions (Coe, 2003). It may be those individuals who are of greatest theoretical interest. “Ethnicity,” van den Berghe (1981) writes, “is common descent…” (p. 16). He continues, …the notion that ethnicity has something to do with kinship or ‘blood’ is not new. Indeed descent seems to be, implicitly and very often explicitly, the essential element of the definition of those... that go under a wide variety of labels: tribe, band, horde, deme, ethnic group, race, nation and nationality (van den Berghe, 1981, p. 15). Nagel (1996) echoed this thinking, writing that ethnicity is popularly viewed as biological, a feature of ancestry and genetics. Although ethnicity has a genetic element we should not ignore its traditional or cultural dimensions. Crews and Bindon (1991, p. 42) propose that ethnic groups are identifiable by “differences in physiology, genetics, beliefs and life-style…” In 1992, incidence rates in US Hispanic, American Indian and African American women were approximately 40 percent lower than those for White women. These differences can be at least partially explained by reproductive histories, which in turn are influenced by culture (Daly, 1992). Women of Ashkenazi Jewish ancestry are reported to be at greater risk for ovarian cancer (see Daly and Obrams, 1998; Koifman and Koifman, 2001), because they appear to be more likely to have inherited and to carry mutations such as BRCA1 (Steinberg et al., 1998). Women who are more traditional, regardless of country of residence, share certain values of kinship and motherhood that may put them at lower risk, perhaps related to larger completed family size. Immigrants to Britain from Africa and the Caribbean (Grulich et al., 1992) are at lower risk. A retrospective study conducted in Thrace, Greece (n = 57) compared prevalence of two ethnic groups: Christian Orthodox and Muslims. They found that the ovarian cancer incidence was 6.8/100,000 for Christian Orthodox and 1.3/100,000 for Muslims. Christians were more westernized and it was to this westernization that the authors attributed increased risk of ovarian cancer (Anastasiadis et al., 2000). • Ethnicity and poverty: Poverty, which is associated with ethnicity in the US, as well as various parts of the world, may be a risk factor for a number of

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chronic diseases, including ovarian cancer, perhaps through an association with a higher risk lifestyle—more smoking, poor nutrition, higher alcohol consumption and exposure to certain infectious agents. Ethnicity, when associated with poverty, also may be related to increases in ovarian cancer mortality (Averette et al, 1995; McGuire et al, 2002). While a number of studies have shown an association of increased risk for ovarian cancer and poverty, other studies have not shown this association. Low-income Puerto Rican women living in Connecticut had very low standardized incidence rate for invasive ovarian cancer (Polednak, 1992). In a study conducted in Scotland of 1,387 women with ovarian cancer, researchers found no consistent evidence that patients from deprived communities presented with more advanced disease (Brewster et al., 2001). • Ethnicity and physical or sexual abuse: Physical or sexual abuse may be related to increased risk of ovarian cancer, perhaps through their effect on ovarian function, primarily if that abuse occurred during childhood or the teenage years (Allsworth et al, 2001). Researchers found a slight positive association of violence during early childhood and the teenage years with high follicle-stimulating hormone (Allsworth et al., 2001). These researchers concluded that abuse may affect ovarian function, with the potential to lead to an altered age at the perimenopausal transition. This neuroendrocrine disruption, in turn, can affect ovarian cancer risk. • Ethnicity and environmental exposures: Increased risk of ovarian cancer among ethnic minority populations also may be related to increases in environmental exposures (e.g., some forms of radiation or chemicals and pesticides) and/or infectious diseases (e.g., human immunodeficiency virus (HIV) or human papilomavirus (HPV)) that are known carcinogens. Further, some ethnic traditions encourage the use of chemicals that may be carcinogenic for cultural reasons. One example would be sprinkling of mercury around the house for religious reasons. • Ethnicity and diet: Diet, which may be a key ingredient of culture and ethnicity, is a risk factor for ovarian cancer. A number of studies have proposed that high dietary fat intake is associated with increased risk of epithelial ovarian cancer; this conclusion, however, remains speculative due to the fact that the mechanism by which dietary fat, or even stored fat, increased risk is unknown. The effect of fat may be independent or it may act primarily though an influence on hormonal status. Dietary fat consumption appears to affect enteric reabsorption of steroid hormones by influencing the intestinal flora (Mansfield, 1993). In 2001, Huncharek and Kupelnick conducted a meta-analysis that examined the association between high versus low dietary fat intake and risk of EOC. These researchers, in their summary of eight observational studies that had enrolled a total of 6,689 subjects, concluded that high dietary fat intake appears to represent a significant risk factor of the development of ovarian cancer.

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A study conducted in Zhejiang, China of 254 patients with histologically confirmed EOC (and 652 controls) investigated whether dietary factors had an etiological association with ovarian cancer (Zhang et al, 2002). Controlling for demographic, lifestyle, familial factors, hormonal status, family history and total energy intake, ovarian cancer risk decreased with increasing consumption of vegetables and fruits and increased with high intakes of animal fat and salted vegetables. Highest risk appeared to increase among women preferring fat, fried, cured and smoked foods. Diet, due to the chemical contamination of food, may also be associated with increased risk of certain reproductive problems such as endometriosis, that increase ovarian cancer risk (Schafer and Kegley, 2002). In many parts of the world, the food supply is contaminated with persistent organic pollutants (PCPs), which include chlordane, dieldrin, DDT (and its main metabolite, DDE), aldrin, endrin, heptachlor, hexachlorobenzene, mirex and toxaphene (Schafer and Kegley, 2002). PCP chemicals persist in the environment for many years, travel long distances in air and water, and concentrate in fatty tissues and bioacumulate as they move up the food chain. Dietary sugar consumption may be related to increased risk of ovarian cancer based on evidence supporting that galactose might be toxic to ovarian germ cells (Cramer et al., 2000). In populations with a high dietary intake of lactose and in which individuals lack the enzyme galactose-1-phosphate uridyltransferase (GALT), galactose and its metabolites accumulate in the ovary. If galactose raises gonadotropin levels, the ovarian epithelium may proliferate. Cramer et al. (2000) concluded, based on their study of 563 women with newly diagnosed epithelial ovarian cancer and 523 controls, that certain genetic or biochemical features of galactose metabolism may influence disease risk for particular types of ovarian cancer. Coffee consumption may increase risk of ovarian cancer. Whittemore et al. (1988) found that when adjusted for smoking, women who had consumed coffee for more than 40 years, were at 3.4 times greater risk of getting ovarian cancer than were women who had never consumed coffee. Each additional ten years of coffee drinking conferred an 11 percent increase in risk. No clear trends in risk were associated with increasing frequency of consumption. Tea, which is consumed on a daily basis in many parts of the world, may decrease risk for ovarian cancer. According to a case control study conducted in China by Zhang et al. (2002), the risk of ovarian cancer for tea consumption declined with increasing frequency and duration of overall tea • Ethnicity and obesity: While obesity is more common in westernized than consumption. The mechanism whereby tea reduced risk is unclear. nonwesternized societies, women in some ethnic groups are more likely to be obese. In the US, more than half of adult African American women, according to analyses of the NHANES data (National Health and Nutrition Examination Survey, which is conducted by the National Center for Health Statistics), were

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obese (Flegal et al., 2002). Obesity and body fat distribution may increase risk for ovarian cancer, perhaps because they seem to increase estrogen levels. Further, where the body fat is stored also may be important in ovarian cancer risk. Women who have a high waist/hip ratio that is associated with obesity, and a family history of ovarian cancer, experience a 4.83-fold increased risk (95 percent CI = 1.55–15.1) (Sellers et al, 1993). Further, the age when a woman is overweight may be important. Researchers who analyzed data from 1,09,445 females who participated in the Nurses’ Health Study found that women who were overweight as teenagers may be more likely to develop pre-menopausal ovarian cancer (Fairfield et al, 2002). These researchers controlled for other factors, such as smoking, OCU, family history, number of pregnancies, age at first menstrual period and history of tubal ligation. OVARIAN CANCER AND EXOGENOUS HORMONES Steroid hormone exposure is known to influence ovarian cancer (Spurdle et al., 2002) and a specific role for estrogens has been suspected in ovarian cancer etiology (Bai et al., 2000). Estrogens may act as promoters in the carcinogenic process and occasionally their metabolites may act as antihormones or have other physiologic effects (Lipsett, 1979). During fetal life, exposure to environmental chemicals with inherent estrogenic activity may lead to female reproductive function impairment. Epidemiological and experimental data point to a relationship between environmental estrogens and the development of polycystic ovaries (Gotz et al., 2001). As oral contraceptives suppress ovulation, they may reduce risk for ovarian cancer. The Norwegian Women and Cancer Cohort Study followed 102,443 women aged 31–70 from 1991 to 1997. In the 1999 follow-up analysis, 171 of the women (of the 96,355 who were eligible) had developed ovarian cancer. The study concluded that the risk of ovarian cancer decreased with the use of oral contraceptives (p for trend p < 0.0001 for ovarian cancer) (Kumle et al., 2003). In Australia, a study of 794 women (and 853 controls) examined the effects of oral contraceptive use, after controlling for estimated number of ovulatory cycles. The protective effect of oral contraceptive use appeared to be multiplicative. There was a 7 percent decrease in relative risk per year (95 percent confidence interval (CI)=4–9 percent) that persisted beyond 15 years of exposure. Even use for up to 1 year may have a greater than predicted effect (odds ratio=0.57; 95 percent CI=0.40–0.82), and use before the first pregnancy may be additionally beneficial (odds ratio=0.95; 95 percent CI=0.87–1.03, adjusted for overall duration of use) (Siskind et al., 2000). Even for women with pathogenic mutations in the BRCA1 and BRCA2 genes, the use of oral contraceptives might reduce the risk of ovarian cancer (Narod et al., 1998). Walker (2002), in her population-based case control study of 767 women, found that 4–8 years of OCU might substantially reduce (by 50 percent) the risk

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of ovarian cancer by age 70 in women with a family history of the disease. It is possible that lactose users (consuming more than 11 g per day) may be the most likely to benefit from OCU and that the benefit may be greatest when OCU occurs after age 30 (Harlow et al., 1991). Significant controversy surrounds the relationships among infertility, fertility drug use and the risk of ovarian cancer (Sit et al., 2002). Ness et al. (2002) pooled interview data on infertility and fertility drug use from eight case control studies conducted between 1989 and 1999 in the US, Denmark, Canada and Australia (N= 5,207 cases and 7,705 controls). Among nulliparous, subfertile women, neither use of any fertility drug nor use of fertility drug for more than 12 months was associated with ovarian cancer risk. These data suggested that specific biological causes of infertility might play a role in overall risk for ovarian cancer. Fertility drugs, however, may not increase risk. Rossing et al. (1994), however, examined the risk of ovarian tumors in a cohort of 3,837 women evaluated for infertility. Computer linkage with a population-based tumor registry was used to identify women in whom tumors were identified. These researchers found that the prolonged use of clomiphene, an ovulation-induction agent, may increase the risk of a borderline or invasive ovarian tumor for both the women with infertility due to ovulatory abnormalities and those women with infertility due to other causes. Ovarian tumors, however, were roughly twice as likely to develop in the women with ovulatory abnormalities. HRT and estrogen replacement therapy (ERT) also may be related to ovarian cancer risk. While the link between ovarian cancer and HRT has been inconsistent (see Sit et al., 2002), this inconsistency may be related to the fact that estrogen formulations in HRT vary in their effects on estrogen-sensitive target tissues, such as the ovary. Rodriguez and colleagues (2001) studied 211, 581 healthy, non-hysterectomized, post-menopausal women in the US who had taken oral HRT after age 35. The women were followed for 14 years; 944 of them died of ovarian cancer. Researchers compared ovarian cancer mortality with the effect of HRT among women who had never used treatment, those using treatment at baseline and previous but not current users, and for total accumulated years of treatment use. Risk of ovarian cancer mortality was reported to be higher in users at baseline and slightly higher for previous users than never users. Risk doubled with duration of use ten years or more; however, only 66 of the 944 women who died of ovarian cancer had used HRT for that long and most of them took unopposed estrogen (ERT) in the 1970s and early 1980s, when powerful synthetic estrogens were in common usage. Long-term, high-dose unopposed ERT might increase the risk of ovarian cancer (see also Drew, 2001). Lacey et al. (2002), in their study of 44,241 post-menopausal women, also found that women who used estrogen-only replacement therapy, particularly those who used it for 10 years or longer, were at significantly increased risk of ovarian cancer. Women who used short-term estrogen-progestin-only

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replacement therapy did not experience increased risk. Riman et al. (2002) in their study of 655 histologically verified incident case patients with epithelial ovarian cancer and 3,899 randomly selected controls, found that increased risk of EOC was elevated among ever users as compared with never users of both ERT and HRT, and that risks were elevated for serous, mucinous and endometroid subtypes. Women who experienced the greatest risk were those who had used hormones for more than 10 years. They concluded that ever users of ERT and HRTsp (sequentially added progestins), but not HRTcp (continuously added progestins) may be at increased risk of EOC. Diethylstilbestrol (DES) is a synthetic hormone used as a treatment for symptoms associated with menopause, menstrual disorders, post-partum breast engorgement, primary ovarian failure and chemotherapy of advanced breast cancer. From 1940 to 1971, DES was used as a treatment to prevent spontaneous abortions in humans. In experimental animals, there is evidence for the carcinogenicity of DES. Although no studies were found that directly linked DES to ovarian cancer in humans, it has been linked to ovarian cancer in guinea pigs (Silva et al., 1998) and it also has been shown to promote ovarian failure and menstrual disorders, which may increase risk for ovarian cancer. OVARIAN CANCER AND EXPOSURE TO XENOBIOTIC AGENTS For millennia, humans have used chemicals, including heavy metals, to promote health. However, since the Industrial Revolution, the production of heavy metals such as lead, copper and zinc has increased exponentially, as have the emissions that occur during their processing. Contamination of ground water and air, through the production process and pesticide use, spread rapidly and may contain carcinogenic substances ranging from metals, to pesticides, PCBs, vinyl chloride, carbon tetrachloride, leaded gasoline, nitrous and sulfur gases to hydrocarbons, carbon monoxide and other chemicals. Chemicals can increase risk for ovarian cancer when they act directly on the cell, converting the normal cell to a cancerous one or indirectly by disrupting ovarian function. Animal studies have shown that a variety of toxicants target ovarian follicles for destruction; the consequences of the destruction depend on the type of follicle targeted (Thompson, 2001). In animal studies, the destruction of follicles can result in ovarian failure and can enhance the development of ovarian neoplasms (Hoyer, 1997). The disruption of ovarian function, as the ovaries are central to hormonal activity in the human body, can have a significant effect on women’s reproductive and endocrine health, possibly leading to any of several hormone and aging-related diseases, including birth defects, infertility, menopause and cancer. If exposure occurs during puberty, a young woman’s ovaries might lack appropriate ovarian stimulation for oocyte development and ovulation (Hoyer, 1997). Menarche may be delayed; sterility, which is associated with increased risk for ovarian cancer, may result.

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Chemicals that we use voluntarily, such as certain chemicals found in talcum powder or cigarette smoke, may increase risk for ovarian cancer while other chemicals, such as those found in certain pain medications, may decrease risk. Cosmetic talc applied to undergarments, sanitary napkins, diaphragms or directly on the perineum is one exposure consistently associated with increased risk of ovarian cancer (Daly and Obrams, 1998). Exposure to cosmetic talc prior to a first live birth may be more harmful than exposure following the first live birth (Cramer et al., 1999). Harlow et al. (1992) found that risk may be highest among women who apply talc directly on the body as a powder, who used it for more than ten years, or who used it on a daily basis. The subgroup at highest risk was comprised of women who estimated that they had made more than 10,000 applications during their reproductive years (OR=2.8, 95 percent CI=1.4–5.4). The greatest risk was associated with invasive serous tumors (OR=1.70, CI=1.22 and 2.39). Talc application may increase risk because ovarian cancer is similar to mesotheliomas and talc has a chemical relationship to asbestos, a known cause of mesotheliomas (Cramer et al., 1982), which are histologically similar to EOC (Parmley and Woodruff, 1979). Talcum powder is now required to be asbestosfree; tests of the cancer-causing potential of these asbestos-free talcs have yet to be conducted. Chemicals found in cigarette smoke, animal studies indicate, destroy both large and small follicles (Mattison, 1980). Among humans, a number of studies have found that women who have ever smoked a cigarette are at increased risk of developing ovarian cancer. Implicated in this increased risk is class of compounds found in the smoke associated with cigarettes (polycyclic aromatic hydrocarbons, PAHs). PAHs appear not only to have toxic effects on the oocytes, but they also promote premature elevation of gonadotropins and stimulation of the ovaries (Marchbanks et al., 2000). Although women who smoked enter menopause 1–5 years earlier than women who do not smoke, thus decreasing their risk (Mattison, 1982), these women are less fertile, by 57–75%, than non-smoking women (Baird and Wilcox, 1985). Infertility increases risk of ovarian cancer. In an Australian study of 794 women with histologically confirmed EOC, aged 18–79 years, and 855 randomly selected controls from the same geographic areas, Green et al. (2001) found that cigarette smoking was a risk factor for ovarian cancer, especially mucinous and borderline mucinous types. Women who ever smoked cigarettes were more likely to develop ovarian cancer than women who had never smoked (OR=1.5, 95 percent CI= 1.2–1.9). A recent study conducted in the US, found that the OR of mucinous tumors for current smokers was significantly elevated, regardless of the age when the women began smoking or the number of years they had smoked (Marchbanks et al., 2000). Compounding the problem is the fact that cigarette smoke acts synergistically with air, food and water-born carcinogens to further increase cancer risk.

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While many xenobiotic agents damage cells, some agents may protect cells. Drake and Becker (2002) found that regular intake of aspirin may reduce risk of ovarian cancer. While low doses of aspirin had little effect on cell growth in the OVCAR-3 human ovarian adenocarcenoma cell line, doses of five millimoles per liter retarded ovarian cancer cell growth by 68 percent. These researchers hypothesized that aspirin may work by reducing levels of cyclooxygenase, an enzyme often found in high concentrations in colon, breast and stomach cancers. Aspirin appears to block the HER-2/neu proto-oncogene, over-expression of which has been found in ovarian carcinoma. A population-based case control study of 563 women with EOC and 523 controls, found no significant association between ovarian cancer and ibuprofen use, a modest inverse association with aspirin use and a significant protective association between paracetamol use and ovarian cancer (Cramer et al., 1998). The protective benefit was more apparent for women who used paracetamol on a daily basis, who had used it for more then 10 years, or who had accumulated more than 20 tablet years (number of tablets per day×years of use). Paracetamol, like estradiol, contains a phenol ring and an acetyl group, as does progesterone. This suggests that it has possible sex-steroid agonist and antagonist properties or, in other words, an antigonadotropic effect. Individuals working in a number of occupations are at higher risk for exposure to xenobiotics. These individuals work in the manufacture of rubber tires, flame retardants, insecticides, or as smelter workers, battery makers, ceramics makers, ship burners, construction workers, steel workers, printers, shoe makers, laboratory technicians and health professionals, firefighters and gas station employees. Xenobiotics associated with occupational or environmental exposure are outlined in Table 10.1. Occupational exposure of aerospace workers to trichloroethylene was measured using a job exposure index with four categories ranging from no to high exposure (Morgan et al., 2000). The researchers observed elevated rates of ovarian cancer for those women with peak exposure (medium-high levels). RR=2.74; 95 percent CI= 2.14–23.54. In Italy, a cohort mortality study of 487 rock salt workers, who experienced high exposure to dusts and chrysotile asbestos, found two cases of malignant ovarian cancer, as against 0.42 cases expected on the basis of the regional rates (Tarchi et al, 1994). A study conducted of working Finnish women (N=892,591) found elevated risks for ovarian cancer associated with aromatic hydrocarbon solvents (SIR=1.3; CI=1.0–1.7–2.7), leather dust (1.4; 0.7–4.1), man-made vitreous fibers (1.3; 0.9– 1.8) and high levels of asbestos (1.3; 0.9–1.8), diesel (1.7; 0.7–4.1) and gasoline (1.5; 1.0–2.0) (Vasama-Neuvonen et al., 1999). In another study conducted in Finland, women working in the pulp and paper industry also experienced excess risk of ovarian cancer (SIR=1.5, 95 percent CI= 1.07–2.09). Although the exposure was unknown, these women were exposed to

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various work-related agents such as talc, microbes and paper dust of various kinds (Langseth and Anderson, 1999). In a study of Swedish women (1,670,517 who were followed from 1971 to 1989), Poisson regression was used to estimate RR of ovarian cancer by occupation (Shields et al., 2002). Although this study did not control for reproductive history, a total of 9,591 ovarian cancer cases were identified. The jobs associated with increased risk of ovarian cancer included dry cleaning, telegraph and telephone work, paper packaging, and graphic and printing work. Hairdressers were not at increased risk. Researchers suggested that increased risk was associated with organic dusts, aromatic amines, and aliphatic and aromatic hydrocarbons. Women working in the two large printing plants in Moscow (3,473 women with 47,791 person years) were assessed by job (compositors, press operators and bookbinders) (Bulbulyan et al., 1999). This study found that ovarian cancer was significantly elevated among bookbinders (12 observed, 4.2 expected). Suspected agents were benzene, which is used in bookbinding, and the talc fillers that were used in the paper. Benzene also may cause menstrual irregularities, which, in turn, influence fertility. In Beijing, China over 3,000 women working in a large petrochemical company were questioned regarding their menstrual cycles (Thurston et al., 2000). Researchers found that the odds of having an abnormal cycle (short or long) increased with years of exposure to benzene. While pesticides are designed to kill or repel organisms, a problem influencing our understanding of the effect of pesticides, indeed of many airborne contaminants, is that exposure is so difficult to measure. The length, continuity and repetitiveness of the exposure, the intensity of the exposure, along with other factors, all must be considered. One study conducted in Costa Rica’s 81 counties (Wesseling et al., 1999), using data drawn from the cancer registry (1981–1993), the 1984 population census, the 1984 agricultural census and a national pesticide data set, found that in urban regions increased risk was observed for ovarian cancer, as well as lung, colorectal, breast, uterus, prostate, testis, kidney and bladder cancers. Atrazine is a chemical that is heavily used in certain sectors of agriculture; it is a corn herbicide. In female Sprague-Dawley (SD) rats, high doses of atrazine, by interfering with normal hormonal functioning, are associated with an increased incidence and earlier appearance of carcinomas (Cancer Assessment Review Committee, 2000). In humans, a possible association between atrazine exposure and ovarian cancer has been reported in a few epidemiologic studies. Donna et al. (1989) found that women with previous exposure to triazine herbicides showed a 2–3-fold risk of epithelial ovarian cancer as compared with unexposured women. The EPA, however, which questioned the use of this herbicide, concluded that there is no supporting evidence or a sound argument of biological plausibility that cancers may have resulted from exposure to atrazine. A cancer assessment review committee established by the EPA categorized

Table 10.1 Chemicals associated with reproductive problems or ovarian cancer

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atrazine as not likely to be carcinogenic in humans. A recent study conducted in several counties and Area Developmental Districts in Kentucky found no association between ovarian cancer risk and exposure to atrazine (HopenhaynRich et al., 2002). The World Health Organization’s International Agency for Research on Cancer (IARC) categorizes atrazine as not classifiable as to carcinogenicity to humans and places it in the same cancer risk category as tea, rubbing alcohol and talc (http://www.fb.org/issues/analysis/ Triazine_Update.html). Lead is an ancient metal once added, and at some costs to health, to wine to sweeten its taste (Nriagu, 1996). Today, lead is added to gasoline around the world; virtually all of the lead in gasoline is emitted into the environment in fine particulate form through vehicle exhaust, causing widespread contamination of air, dust and soil. Lead is an established animal carcinogen (Landrigan et al., 2000) and has been classified as a possible human carcinogen (group 2B) (Silbergeld et al., 2000). Lead also has toxic effects on reproductive function in both males and females. Mercury also affects female fertility, perhaps by inducing cellular or genetic damage in the ovary (BBC News, 9/23/2002). Radiation injury For over a century, radiation has been a serious concern to individuals because, once inhaled or absorbed into the lungs, radiation harms living cells by causing abnormal cell function and structure. In westernized counties such as the US, we are constantly exposed to background radiation (nuclear power plants and weapons testing, consumer products, medical X-rays, etc.). The health effects of radiation are numerous. Researchers have found a statistically significant increase in malignant ovarian neoplasms among survivors of Hiroshima and Nagasaki. The age-adjusted rates show a significant increase in rates with increased exposure and they show that the radiation effect was higher in the age group that was younger at the time of the bombing (Tukuoka et al., 1987). They found that the minimum latent period for radiation-induced ovarian cancer was approximately 15–20 years. Irradiation has not been confirmed to be a risk factor for ovarian cancer. There have been sporadic case reports of the development of carcinosarcomas of the cervix, vagina and extragenital areas and perhaps of the ovary, after previous pelvic irradiation (Wei et al., 2001). THEORETICAL APPROACHES TO OVARIAN CANCER RISK Three different hypotheses have been put forth to explain ovarian cancer risk. One hypothesis attributes increased risk of ovarian cancer to an increased number of uninterrupted lifetime ovulations and their effects on the epithelium (Fathalla, 1971). The second hypothesis argues that circulating levels of pituitary gonadotropins, suppressed during pregnancy and OCU, increase malignancy

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risk. The third hypothesis focuses on risk related to environmental exposure that might lead to inflammation of the ovarian epithelium (Ness et al., 2000). While each of these hypotheses can account for some of the patterns of risk associated with ovarian cancer, none can account for a large number of those risks. In this chapter, an alternative hypothesis is outlined which proposes that understanding of human evolution is necessary if we wish to increase our understanding of ovarian cancer. More specifically, increased risk of ovarian cancer is related to exposure to xenobiotic agents that are novel in human evolutionary history. As humans have only recently been exposed to these agents, natural selection has not had time to produce defenses against them. Further, risk is related to reproductive factors associated with differences between the reproductive sequence common to our distant hominid ancestresses, who spent much of their lifetime after puberty either pregnant or lactating, and those observed today in modern females living in westernized societies. The first hypothesis, which attempts to explain increased risk for ovarian cancer by focusing on uninterrupted ovulation over the lifetime, is based upon the assumption that ovulation is hard work for the female body and that some sequelae of ovulation increase the risk of neoplasia as the ovarian surface epithelium is repeatedly involved in the process of ovulation with inadequate physiologic rest (Fathalla, 1971). The repetitive trauma to the ovarian surface that is associated with ovulation may result in cellular proliferation, epithelial entrapment and, ultimately, transformation of epithelial inclusion cysts within the ovarian stroma leading to the genesis of ovarian cancer. This theory, which is now being tested using experimental approaches (e.g., Hamilton et al., 1998), can help explain why events that interrupt the constant cycle of ovulations— pregnancies, breastfeeding and OCU—are associated with decreased risk. Perhaps supporting the incessant ovulation hypothesis are studies of the p53 gene, a gene that encodes a protein that signals cells to commit programmed apoptosis when their growth patterns are irregular (as in cancer). Normally, p53 blocks cell division when a cell has sustained DNA damage. If the p53 gene is disabled through cell stress, as it occurs in repeated ovulation, damaged cells can proliferate and p53-positive epithelial ovarian cancer can result (Schildkraut et al., 1997). The second hypothesis attributes increased risk of ovarian cancer to elevations in hormones associated with ovulation, such as pituitary gonadotropin levels acting in concert with estrogen (Gardiner, 1961; Stadel, 1975). Experiments in animals suggest that an excess of gonadotropin and stromal stimulation may result in increased risk for ovarian cancer by disturbing normal feedback inhibition between ovary and pituitary or by destroying ovarian follicles (Cramer and Welch, 1983). In humans, gonadotropin elevation may be a result of mechanisms that cause primary ovarian failure, such as exposure to chemicals or metabolites that are toxic to follicles. A critical event in the transformation of cells is the entrapment of surface epithelium in inclusion cysts, followed by stimulation of the entrapped epithelium by estrogen or estrogen precursors, particularly if levels

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of gonadotropins (LH and FSH) are high (Cramer and Welch, 1983). This hypothesis, while difficult to test (e.g., modalities inhibiting ovulation do so by inhibiting gonadotropin release), can help explain why pregnancy decreases risk and why fertility drugs, which can increase both the number of ovulations and the hormone levels associated with ovulation, are associated with increased risk. The third hypothesis proposes that risk is related to inflammation in the ovarian epithelium resulting from exposure to environmental factors. Inflammation involves rapid cell division, DNA excision and repair, oxidative stress and high concentrations of cytokines and prostaglandins, all of which are established promoters of mutagenesis (Ames et al., 1995). This hypothesis can explain why suppression of ovulation, tubal ligation and hysterectomy decrease risk: they cut off the pathway from the lower to upper genital tract, thereby keeping inflammatory substances from ascending to the ovarian epithelium. It also can explain why exposure to asbestos and use of talcum powder increase risk; they all initiate local inflammation (Ness et al., 2000). An evolutionary approach to health, the alternative hypothesis, is based upon the assumption that modern human anatomy and physiology were designed by natural selection acting within ancestral populations. The assumption underlying an evolutionary approach is that the aim of life is survival and reproduction, as it is only through reproduction that the genotypes that underlie our physical traits (our phenotypes) are transmitted from one generation to the next. Our complex bodies were designed to perform certain functions that ultimately promoted survival and reproduction in particular environments, namely the environment in which our distant ancestors lived. Disease and even symptoms of illness can be viewed as either adaptations (fever and hypoferremia, for example, may have been designed by natural selection to serve as a defense against infection) (Nesse and Williams, 1994) or as pathologies (as in the case of ovarian cancer) that is related to differences—environmental or behavioral changes—that distinguish modern populations of humans from ancestral ones. While fever, on average, may have helped our ancestors survive and subsequently reproduce, as they were better able to fight off infection, ovarian cancer, particularly to the extent it results in the death of women in their reproductive prime, clearly did not. Evolutionary approaches distinguish proximate causes from ultimate ones. A proximate explanation for a disease focuses on such things as the genetic instructions provided by DNA, the release of a particular hormone, or the particular physiological reaction to a xenobiotic agent. An ultimate or evolutionary explanation would ask why the DNA specifies the trait in the first place. Proximate and ultimate explanations are not seen as contradictory, they are just different levels of explanation, each of which can contribute to the testing of the other level. For millions of years, during much of human evolutionary history, our ancestors were hunter-gathers. Given how slowly natural selection works, this is the environment in which the traits we possess as modern humans were selected. Our bodies, in other words, were designed for a hunter-gatherer lifestyle and an

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understanding of that lifestyle is crucial to an understanding of our health. Based upon our studies of living hunter-gatherers, whose lifestyle best approximates that of these distant ancestors, the lifestyle was simple and highly social. The level of physical activity was high, driven by the need to migrate following wild game and to gather the uncultivated plant foods that comprised the simple diet. Diets, typically, were low in fat: the meat of game animals has seven times less saturated fat than that of domesticated animals (Eaton et al., 1988). Rarely were refined carbohydrates or foods such as honey consumed. Humans were exposed to few xenobiotic agents beyond those found in natural foods or in the smoke that came from the fires they used to keep warm and cook meat. When we closely examine the ancestral lifestyle, it is also clear that the reproductive life of modern females differs from that of our distant ancestresses (Coe and Steadman, 1995). Our ancestresses married early, soon after puberty, and then experienced a reproductive developmental sequence that involved, with few interruptions, a series of pregnancies and lactation. Menstruation, rather than being a monthly event, was infrequent (women were constantly pregnant or lactating); women were rarely exposed to the hormones associated with menses. As the biological aim of life is survival and reproduction, natural selection will select good reproducers, women who are fecund. These women have been designed by natural selection to have strong immune systems and to be protected from disease in their reproductive prime. Natural selection, however, could not act on women who were infertile and although it may seem harsh, these infertile women, like women who have passed menopause, would be, in nature, more expendable. Their importance would lie in any contributions that they made to the survival and reproduction of close kin, with whom they shared some, but not all genes. Beginning about 10,000 years ago, with the domestication of plants and plant processing, radical changes in diet and levels of physical activity occurred. Later, the industrial revolution and the stream of technological invention and related byproducts would have complex effects on the biosphere and on human biology. Further, as women entered the workplace, their reproductive patterns began to change. They began to produce fewer offspring, and were less likely to nurse them. In the workplace, they also were exposed to chemicals that their distant ancestresses were not. Increases in the chronic and deadly “diseases of civilization”—including ovarian cancer—may be associated with these behavioral and environmental changes (Eaton et al., 1988). This evolutionary hypothesis can explain why women in westernized societies have higher rates of ovarian cancer, compared with women in non-westernized societies. As reproductive patterns begin to change in non-westernized women, we should see corresponding increases in breast and ovarian cancer rates, even if those countries do not become industrialized, as air contamination from industrialized areas can be widely distributed. This hypothesis also can help explain why high fat diets might increase risk, as does exposure to xenobiobic agents that were unknown in the distant past. It also can explain why women

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who are infertile may be at high risk, and why women who delay reproduction and/or are nulliparous also experience a higher risk for ovarian cancer than do multifarious women. It also can explain why lower risk for ovarian cancer is experienced by women who follow the ancestral reproductive sequence (begin childbearing early, soon after the initiation of menses, are multiparous and breastfeed their children for prolonged periods of time). While none of this information may be useful for an educational intervention aimed at educating women about protecting themselves from ovarian cancer, as women are unlikely to change their reproductive patterns, what this does suggest is that researchers need to continue their focus on women who are infertile or who have passed menopause, attempting to identify why these women are at high risk, and take a look at what happens during pregnancy and lactation that may protect a woman from ovarian cancer. In the case of breast cancer, pregnancy and lactation promote the differentiation of the breast, shifting cells from the proliferating to the resting stage, and producing cells that have more efficient DNA repair, and that produce less-polar metabolites (Russo et al, 1992). One might predict that similar events in the ovaries would protect these same women from ovarian cancer. REFERENCES Allsworth, J., Zierler, S., Krieger, N. and Harlow, B. (2001) Ovarian function in late reproductive years in relation to lifetime experiences of abuse, Epidemiology, 12 (6): 676–81. American Cancer Society (2002) How Many Women Get Ovarian Cancer (http:// www.cancer.org/eprise/main/docroot/CRI/CRI_2x?sitearea=CRI&dt=33). Ames, B., Gold, L. and Wilett, W. (1995) The causes and prevention of cancer, Proc. Natl. Acad. Sci., 92:5258–65. Anastasiadis, P., Koutlaki, N. and Liberis, V. (2000) Trends in epidemiology of cervical cancer in Thrace, Greece, Int. J. Gynaecol. Obstet., 68(1):59–60. Averette, H., Janicek, M. and Menck, H. (1995) The National Cancer Data Base report on ovarian cancer, Cancer, 76:1096–103. Bai, W., Oliveros-Saunders, B., Wang, Q., Acevedo-Duncan, M., Nicosia, S. and Bondy, C. (2000) Estrogen stimulation of ovarian surface epithelial cell proliferation, In Vitro Cell. Dev. Biol., 36(10):657–66. Baird, D. and Wilcox, A. (1985) Cigarette smoking associated with delayed conception, /. Am. Med. Assoc., 253:2979–83. Berg, J. and Lampe, J. (1981) High-risk factors in gynecologic cancer, Cancer, 15;48 (Suppl 2):429–41. Borman, S., Christian, P., Sipes, I. and Hoyer, P. (2000) Ovotoxicity in female Fischer rats and B6 mice induced by low-dose exposure to three polycyclic aromatic hydrocarbons: comparison through calculation of an ovotoxic index, Toxicol. Appl. Pharmacol, 15;167 (3):191–98. Brewster, D., Thomson, D., Hole, D., Black, R., Stroner, P. and Gillis, C. (2001) Relation between socioeconomic status and tumour stage in patients with breast, colorectal,

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ovarian and lung cancer: results from four national, population based studies, Br. Med. J., 322 (7290):830. Bulbulyan, M., Ilychova, S., Zahm, S., Astashevsky, S. and Zaridze, D. (1999) Cancer mortality among women in the Russian printing industry, Am. J. Ind. Med., 36 (1): 166–71. Burton, J., Madsen, S., Yao, J., Sipkovsky, S. and Coussens, P. (2001) An immunogenomics approach to understanding periparturient immunosuppression and mastitis susceptibility in dairy cows, Acta Vet. Scand., 42 (3):407–24. Cancer Assessment Review Committee (CARC) (2000) Atrazine: Evaluation of Carcinogenic Potential United States Environmental Protection Agency, Health Effects Division, Washington DC HED Document number 014431. Cannistra, S. (1983) Cancer of the ovary, N.Engl J.Med., 329 (21): 1550–59. Chiaffarino, F., Pelucchi, C., Parazzini, F., Negri, E., Franceschi, S., Talamini, R., Conti, E., Montella, M. and La Vecchia, C. (2001) Reproductive and hormonal factors and ovarian cancer, Ann. Oncol, 12 (3):337–41. Coe, K. (2003) The Ancestress Hypothesis, Newark, NJ: Rutgers University Press. Coe, K. and Steadman, L. (1995) The human breast and the ancestral reproductive cycle: An inquiry into the etiology of breast cancer, Human Nat., 6(3): 197–220. Collins, F., Klausner, R. and Olden, K. (6 March 1996) Cancer, Genetics and the Environment. Presentation made before the Senate Committee on Labor and Human Resources. Cramer, D. and Welch, W. (1983) Determinants of ovarian cancer risk. II. Inferences regarding pathogenesis, J. Natl. Cancer Inst., 71:717–21. Cramer, D., Welch, W., Scully, R. and Wojciechowski, C. (1982) Ovarian cancer and talc: A case-control study, Cancer, 50 (2):372–76. Cramer, D., Harlow, B., Titus-Ernstoff, L., Bohlke, K., Welch, W. and Greenberg, E. (1998) Over-the-counter analgesics and the risk of ovarian cancer, The Lancet, 351: 104–97. Cramer, D., Liberman, R., Titus-Ernstoff, L., Welch, W., Greenberg, R., Baron, J. and Harlow, B. (1999) Genital talc exposure and risk of ovarian cancer, J. Cancer, 81: 351–56. Cramer, D., Greenberg, E., Titus-Ernstoff, L., Liberman, R., Welch, W., Li, E. and Ng, W. (2000) A case-control study of galactose consumption and metabolism in relation to ovarian cancer, Cancer Epidemiol Biomarkers Prev., 9 (1):95–101. Cramer, D.W. and Xu, H. (1995) Epidemiologic evidence for uterine growth factors in the pathogenesis of ovarian cancer, Ann. Epidemiol., 5 (4):312–14. Crews, D. and Bindon, J. (1991) Ethnicity as a taxonomic tool in biomedical and biosocial research, Ethn. and Dis., 1:42–49. Daly, M. (1992) The epidemiology of ovarian cancer, Hematol. Oncol. Clin. of North Am., 6 (4):729–38. Daly, M. and Obrams, G. (1998) Epidemiology and risk assessment for ovarian cancer, Semin. Oncol., 25 (3):255–64. Doerr, J., Hollis, E. and Sipes, I. (1996) Species difference in the ovarian toxicity of 1,3butadiene epoxides in B6C3F1 mice and Sprague-Dawley rates, Toxicology, 113(1– 3):128–36. Donna, A., Crosignani, P., Robutti, R., Betta, P., Bocca, R., Mriani, N.O., Farrario, R., Fissi, R. and Berrino, F. (1989) Triazine herbicides and ovarian epithelian neoplasms, Scand. J. Work Environ. Health, 15:47–53.

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Drake, J. and Becker, J. (2002) Aspirin-induced inhibition of ovarian tumor cell growth, Obstet. Gynecol., 100 (4):677–82. Drew, S. (2001) Oestrogen replacement therapy and ovarian cancer, The Lancet, 358: 1910. Eaton, S., Shostak, M. and Konner, M. (1988) The Paleolithic Prescription, New York, NY: Harper & Row. Edmondson, R. and Monaghan, J. (2001) The epidemiology of ovarian cancer, Intl. J. Gynecol Cancer, 11 (6):423–33. Ejeckam, G., Abdulla, F., el-Sakka, M., Dauleh, W. and Haseeb, F. (1994) Gynaecological malignancies in Qatar, East Afr. Med. J., 71 (12):777–81. Fairfield, K., Willett, W., Rosner, B., Manson, J., Speizer, F. and Hankinson, S. (2002) Obesity, weight gain and ovarian cancer, Obstet. Gynecol., 100(2):288–96. Fathalla, M. (1971) Incessant ovulation, a factor in ovarian neoplasia, The Lancet, 2:163. Fessler, M.D.T. (2002) An evolutionary perspective on pregnancy sickness and meat consumption, Curr. Anthropol., 43(1): 19–61. Flegal, K., Carroll, M., Ogden, C. and Johnson, C. (2002) Prevalence and trends in obesity among US adults, 1999–2000, J. Am. Med. Assoc., 288(14): 1723–27. Foulkes, W. and Narod, S. (1995) Hereditary breast and ovarian cancer: epidemiology, genetics, screening and predictive testing, Clin. Investig. Med., 18(6):473–83. Friedlander, A. (2002) The physiology, medical management and oral implications of menopause, J. Am. Dent. Assoc., 133(1):73–81. Gardiner, J. (1961) Tumorigenesis in transplanted irradiated and nonirradiated ovaries, J. Natl. Cancer Inst., 26:829–54. Gotz, F., Thieme, S. and Dorner, G. (2001) Female infertility-effect of perinatal xenoestrogen exposure on reproductive functions in animals and humans, Folia Histochem. Cytobiol., 39(Suppl 2):40–43. Green, A., Purdie, D., Bain, C, Siskind, V. and Webb, P. (2001) Cancer Causes Control, 12(8):713–19. Groot de Restropo, Sicard, D. and Torres, M. (2000) DNA damage and repair in cells of lead exposed people, Am. J. Ind. Med., 38:330–34. Grulich, A., Swerdlow, A., Head, J. and Marmot, M. (1992) Cancer mortality in African and Caribbean migrants to England and Wales, Br. J. Cancer, 66(5):905–11. Hamilton, T., Penault-Llorca, F. and Dauplat, J. (1998) Natural history of ovarian adenocarcinomas: from epidemiology to experimentation, J. Contracept. Fertil. Sex, 26(11): 800–04, November. Hankinson, S., Colditz, G., Hunter, D., et al. (1995) A prospective study of reproductive factors and risks of epithelian ovarian cancer, Cancer, 75:284–90. Harlow, B., Cramer, D., Geller, J., Willett, W., Bell, D. and Welch, W. (1991) The influence of lactose consumption on the association of oral contraceptive use and ovarian cancer risk, Am. J. Epidemiol., 134(5):445–53. Harlow, B., Cramer, D., Bell, D. and Welch, W. (1992) Perineal exposure to talc and ovarian cancer, Obstet. Gynecol., 80(1): 19–26. Hartge, P. and Stewart, P. (1994) Occupation and ovarian cancer: a case-control study in the Washington, DC, metropolitan area, 1978–1981, J. Occup. Environ. Med., 36(8): 924–927. Heller, D., Gordon, R., Westhoff, C. and Gerber, S. (1996) Am. J. Ind. Med., 29(5):435– 39.

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Hildreth, N., Kelsey, J., LiVolsi, V., Fischer, D., Holford, T., Mostow, P. and White, C. (1981) An epidemiologic study of epithelial carcinoma of the ovary, Am. J. Epidemiol., 114(3):398–405. Holschneider, C. and Berek, J. (2000) Ovarian cancer: Epidemiology, biology and prognostic factors, Semin. Surg. Oncol., 19(1):3–10. Hopenhayn-Rich, C., Stump, M. and Browning, S. (2002) Regional assessment of atrazine exposure and incidence of breast and ovarian cancers in Kentucky, Arch. Environ. Contam. Toxicol., 42:127–36. Hoyer, P. (1997) Female reproductive toxicology: Introduction and overview, In: K. Boekelheide, R. Chapin, P. Hoyer and P. Harris (eds), Comprehensive Toxicology, Oxford, Pergamon, pp. 249–54. John, E., Whittemore, A., Harris, R. and Itnyre, J. (1993) Characteristics relating to ovarian cancer risk: collaborative analysis of seven U.S. case-control studies, J. Natl. Cancer Inst., 85(2): 142–47. Katchy, J. and Briggs, N. (1992) Clinical and pathological features of ovarian tumours in Rivers state of Nigeria, East Afr. Med. /., 69(8):456–59. Koifman, S. and Koifman, R. (2001) Breast cancer mortality among Ashmenazi Jewish women in Sao Paulo and Porto Alegre, Brazil, Breast Cancer Res., 3(4):270–75. Kumle, M., Alsaker, E. and Lund, E. (2003) Use of oral contraceptives and risk of cancer, a cohort study, Tidsskr Nor Laegeforen, 12; 123(12): 1653–56. Kvale, G., Heuch, I. and Nilssen, S. (1991) Reproductive factors and cancers of the breast and genital organs—are the different cancer sites similarly affected?, Cancer Detect. Prev., 15(5):369–77. Lacey, J., Mink, P., Lubin, J., Sherman, E., Troisi, R., Hartge, P., Schatzkin, A. and Schairer, C. (2002) Menopausal hormone replacement therapy and the risk of ovarian cancer, J. Am. Med. Assoc., 288(3):334–41. Lambe, M., Hsieh, D., Trichopoulos, A., Ekbom, M., Pavia, M. and Adami, H. (1994) Transient increase in the risk of breast cancer after giving birth, N. Engl. J. Med., 331 (1):5–9. Landrigan, P., Bofetta, P. and Apostoli, P. (2000) The reproductive toxicity and carcinogenicity of lead: A critical review,. Am J. Ind. Med., 38:231–43. Langseth, H. and Andersen, A. (1999) Cancer incidence among women in the Norwegan pulp and paper industry, Am. J. Ind. Med., 36(1): 108–13. Lindgren, P., Backstrom, T., Cajander, S., Damber, M., Mahlck, C, Zhu, D. and Olofsson, J. (2002) The pattern of estradiol and progesterone differs in serum and tissue of benign and malignant ovarian tumors, Int. J. Oncol., 21(3):583–89. Lipsett, M.B. (1979) Interaction of drugs, hormones and nutrition in the causes of cancer, Cancer, 43(Suppl 5): 1967–81. Malik, I. (2002) A prospective study of clinico-pathelogical features of epithelial ovarian cancer in Pakistan, J. Pak. Med. Assoc., 52(4): 155–58. Mansfield, C. (1993) A review of the etiology of breast cancer, J. Am. Med. Assoc., 85(3): 217–21. Marchbanks, P.l. Wilson, H., Bastos, E., Cramer, D., Schildkraut, J. and Peterson, H. (2000) Cigarette smoking and epithelial ovarian cancer by histologic type. Obstet. Gynecol., 95(2):255–60. Mattison, D. (1980) Morphology of oocytes and follicle desteruction by polycyclic aromatic hydrocarbons in mice, Toxicol. Appl. Pharmacol., 52(2):249–59.

THE EPIDEMIOLOGY OF OVARIAN CANCER 203

Mattison, D. (1982) The effects of smoking on fertility from gametogenesis to implantation, Environ. Res., 28(2):410–33. McGuire, V.I., Herrinton, L. and Whittemore, A. (2002) Race, epithelial ovarian cancer survival and membership in a large health maintenance organization, Epidemiology, 13(2):231–34. McLaughlin, R.S. (2002) Health Concerns Fact Sheets: Ovarian Cancer, 30 July 2002, http://www.emcom.ca/health/ovarian.shtml. Morgan, R., Kelsh, M., Zhao, K. and Heringer, S. (2000) Mortality of aerospace workers exposed to trichloroethylene, Epidemiology, 9(4):424–31. Mosgaard, B., Lidegaard, O., Kjaer, S., Shlou, G. and Anderson, A. (1997) Infertility, fertility drugs and invasive ovarian cancer: a case-control study, Fertil. Steril., 67: 1005–12. Murdoch, W. and McDonnel, A. (2002) Roles of the ovarian surface epithelium in ovulation and carcinogenesis, Reproduction, 123:743–50. Nagel, J. (1996) American Indian Ethnic Renewal: Red Power and the Resurgence of Identity and Culture, New York: Oxford University Press. Narod, S., Risch, H., Moslehi, R., Dorum, A., et al. (1998) Oral contraceptives and the risk of hereditary ovarian cancer, N. Engl J. Med., 339(7):424–71. Negri, E., Franceschi, S., Tzonou, A., Booth, M., La Vecchia, C., Parazzini, F., Beral, V., Boyle, P. and Trichopoulos, D. (1991) Pooled analysis of 3 European case-control studies: I. Reproductive factors and risk of epithelial ovarian cancer, Intl. J. Cancer, 49(1):50–56. Ness, R. and Cottreau, C. (1999) Possible role of ovarian epithelian inflammation in ovarian cancer, J. Natl. Cancer Inst., 91(17): 1459–67. Ness, R., Grisso, J., Cottreau, C., Klapper, J., Vergona, R., Wheeler, J., Margan, M. and Schlesselman, J. (2000) Factors related to inflammation of the ovarian epithelium and risk of ovarian cancer, Epidemiology, 11(2):111–17. Ness, R., Cramer, D., Goodman, M., Kruger Kjaer, S., Mallin, K., Mosgaard, B., Purdie, D., Risch, H., Vergona, R. and Wu, A. (2002) Infertility, fertility drugs and ovarian cancer: A pooled analysis of case-control studies, Am. J. Epidemiol., 155(3):217–24. Nesse, R. and Williams, G. (1994) Why We Get Sick: The New Science of Darwinian Medicine, New York: Times Books Random House. Nriagu, J. (1996) A history of global metal pollution, Science, 272(5259) (April 12), 223. Nussbaum Cohen, D. (1997) Ashkenazi breast-cancer gene now seen as less of risk, Jewish Bulletin, 5:23. Pacchierotti, F., Adler, I., Anderson, D., Brinkworth, M., Demopoulos, N., Lahdetie, J., Osterman-Golkar, S., Peltonen, K., Russo, A., Tates, A. and Waters, R. (1998) Genetic effects of 1,3-butadiene and associated risk for heritable damage, Mut. Res./ DNA Repair, 397(l):93–115. Parmley, T. and Woodruff, J. (1979) The ovarian mesothelioma, Am. J. Obstet. Gynecol., 120:234–41. Polednak, A. (1992) Cancer incidence in the Puerto Rican-born population of Connecticut, Cancer, 70(5): 1172–75. Prescott, D. and Flexer, A1. (1982) Cancer: The Misguided Cell, 2nd edn, Sunderland, Massachusetts: Sinauer Associates. Riman, T., Dickman, P., Nilsson, S., Correia, N., Nordlinder, H., Magnusson, C. and Persson, I. (2002) Risk factors for invasive epithelian ovarian cancer: results from a Swedish case-control study, Am. J. Epidemiol., 156(4):363–73.

204 KATHRYN COE

Risch, H., Marrett, L. and Howe, G. (1994) Parity, contraception, infertility and the risk of epithelial ovarian cancer, Am. J. Epidemiol., 140(7):585–97. Rodriguez, C, Patel, A., Calle, E., et al. (2001) EstroGen replacement therapy and ovarian cancer mortality in a large prospective study of US women, J. Am. Med. Assoc., 285: 1460–65. Rosenblatt, K. and Thomas, D. (1993) Lactation and the risk of epithelial ovarian cancer. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives, Intl J. Epidemiol., 22(2): 192–97. Rosenblatt, K. and Thomas, D. (1996) Reduced risk of ovarian cancer in women with tubal ligation or hysterectomy, Cancer Epidemiol., Biomarkers Prev., 5(11):933. Rossing, M., Daling, J., Weiss, N., Moore, D. and Self, S. (1994) Ovarian tumors in a cohort of infertile women, N. Engl J. Med., 331(12):771–76. Runnebaum, I. and Stickeler, E. (2001) Epidemiological and molecular aspects of ovarian cancer risk, J. Cancer Res. Clin. Oncol., 127(2):73–79. Russo, J., River, R. and Russo, I. (1992) Influence of age and parity on the development of the human breast, Breast Cancer Res. Treat., 23:211–18. Schafer, K. and Kegley, S. (2002) Persistent toxic chemicals in the US food supply, J. Epidemiol. Community Health, 56:813–17. Schildkraut, J., Bastos, E. and Berchuck, A. (1997) Relationship between lifetime ovulatory cycles and overexpression of mutant p53 in epithelial ovarian cancer, J. Natl. Cancer Inst., 89(13):906–07. Schwartz, P. and Taylor, K. (1995) Is early detection of ovarian-cancer possible?, Ann. Med., 27(5):519–28. Sellers, T., Gapstur, S., Potter, J., Hushi, L., Bostick, R. and Folsom, A. (1993) Association of body fat distribution and family histories of breast and ovarian cancer with risk of postmenopausal breast cancer, Am. J. Epidemiol., 138(1):799–803. Shields, T., Gridley, G., Moradi, T., Adami, J., Plato, N. and Dosemeci, M. (2002) Occupation exposures and the risk of ovarian cancer in Sweden, Am. J. Ind. Med., 42: 200–13. Shu, X., Brinton, L., Gao, Y. and Yuan, J. (1989) Population-based case-control study of ovarian cancer in Shanghai, Cancer Res., 49(13):3670–74. Silbergeld, E., Waalkes, M. and Rice, J. (2000) Lead as a carcinogen: experimental evidence and mechanisms of action, Am. J. Ind. Med., 39:316–23. Silva, E.G., Tornos, C, Deavers, M., Kaisman, K., Gray, K. and Gershenson, D. (1998) Induction of epithelial neoplasms in the ovaries of guinea pigs by estrogenic stimulation, Gynecol Oncol., 71(2):240–46. Siskind, V., Green, A., Bain, C. and Purdie, D. (2000) Beyond ovulation: oral contraceptives and epithelial ovarian cancer, Epidemiology, 11(2): 106–10. Sit, A., Modugno, F., Weissfeld, J.L., Berga, S.L. and Ness, R.B. (2002) Hormone replacement therapy formulations and risk of epithelial ovarian carcinoma: comments, Gynecol Oncol., 86(2):112–18. Spurdle, A., Goodwin, B., Hodgson, E., Hopper, J., Chen, X., Prudie, D., McCredie, M., Giles, C, Chenevix-Trench, G. and Liddle, C. (2002) The CYP3A*1B polymorphism has no functional significance and is not associated with risk of breast or ovarian cancer, Pharmacogenetics, 12(5):355–66. Stadel, B. (1975) The etiology and prevention of ovarian cancer, Am. J. Obstet. Gynecol., 123:772–74.

THE EPIDEMIOLOGY OF OVARIAN CANCER 205

Steinberg, K., Pernarelli, J., Marcus, M., Khoury, M., Schildkarut, J. and Marchbanks, P. (1998) Increased risk for familial ovarian cancer among Jewish women: A population-based case-control study, Genet. Epidemiol., 15:51–59. See also www.cdd.gov/genomics/ info/reports/research/o_cancer.html. Tarchi, M., Orsi, D., Comba, P., DeSantis, M., Pirastu, R., Battista, G. and Valiana, M. (1994) Cohort mortality study of rock-salt works in Italy, Am. J. Ind. Med., 25(2): 251–56. Taylor, D., Sullivan, S., Eblen, A. and Gercel-Taylor, C. (2002) Modulation of T-cell CD3-zeta chain expression during normal pregnancy, J. Reprod. Immunol., 54(1): 15–31. Thompson, K. (2001) Involvement of the Estrogen Receptor and Aryl Hydrocarbon Receptor in 4-Vinlycyclohexene Diepoxide-Induced Ovotoxicity. Dissertation, University of Arizona. Thurston, S., Ryan, L., Christiani, D., Snow, R., Carlson, J., You, L., Cui, S., Ma, G., Wang, L., Huang, Y. and Xu, X. (2000) Petrochemical exposure and menstrual disturbances, Am. J. Ind. Med., 38:555–64. Tokuoka, S., Kawai, K., Shimizu, Y., Inai, K., Ohe, K., Fukijura, T. and Kato, H. (1987) Malignant and benign ovarian neoplasms among atomic bomb survivors, Hiroshima and Nagasaki, 1950–1980, J. Am. Cancer Inst., 79(1):47–57. Tortolero Luna, G. and Mitchell, M. (1995) The epidemiology of ovarian cancer, J. Cell Biochem., 23:200–07. Vachon, C., Mink, P., Janney, C., Sellers, T., Cerhan, J., Hartman, L. and Folsom, A. (2002) Association of parity and ovarian risk by family history of breast or ovarian cancer in a population-based study of postmenopausal women, Epidemiology, 9:66– 71. van den Berghe, P. (1981) The Ethnic Phenomenon, New York: Elsevier. Vasama-Neuvonen, K., Pukkala, E., Paakkulainen, H., Mutanen, P., Weiderpass, E., Boffetta, P., Shen, N., Kauppinen, T., Vainio, H. and Partanen, T. (1999) Ovarian cancer and occupational exposures in Finland, Am. J. Ind. Med., 36(l):83–89. Walker, G. (2002) Family history of cancer, oral contraceptive use and ovarian cancer risk, Am. J. Obstet. Gynecol, 186:8–14. Wei, L., Huang, C, Cheng, S., Chen, C. and Hsieh, C. (2001) Carcinosarcoma of ovary associated with previous radiotherapy, Intl J. Gynecol Cancer, 11:81–84. Wesseling, C, Antich, D., Hogstedt, C, Rodrigues, A. and Ahlbom, A. (1999) Geographical difference of cancer incidence in relation to environmental and occupational pesticide exposure, Int. J. Epidemiol., 28:365–74. Whiteman, D., Michael, F., Murphy, G., Cook, L., Cramer, D., et al. (2000) Multiple births and risk of epithelial ovarian cancer, /. Natl. Cancer Inst., 92(14): 1172. Whittemore, A. (1994) Characteristics relating to ovarian cancer risk: implications for prevention and detection, Gynecol Oncol., 55(3 Pt 2): 15–19. Whittemore, A., Wu, M., Paffenbarger, R., Sarles, D., Kampert, J., Grosser, S., Jung, D., Ballon, S. and Hendrickson, M. (1988) Personal and environmental characteristics related to epithelial ovarian cancer. II, Am. J. Epidemiol., 128(6): 1228–40. Wu, M., Whittemore, A., Paffenbarger, R., Sarles, D., et al. (1988) Personal and environmental characteristics related to epithelial ovarian cancer. I. Reproductive and menstrual events and oral contraceptive use, Am. J. Epidemiol., 128(6): 1216–27. Zhang, M., Binns, C.W., Lee, A.H. (2002) Tea consumption and ovarian cancer risk: A case-control study in China, Cancer Epidemiol, Biomarkers Prev., 11(8):713–18.

11 ASSESSMENT OF TOXICANTINDUCED ALTERATIONS IN OVARIAN STEROIDOGENESIS: A METHODOLOGICAL OVERVIEW Jerome M.Goldman, Susan C.Laws and Ralph L.Cooper

INTRODUCTION Ovaries exist as the principal component of the female hypothalamicpituitarygonadal axis. The population of ovarian follicles contains the lifetime repository of oocytes, and the synthesis and secretion of sex steroids by the follicular cells participate with other components of the axis to regulate the reproductive cycle and broadly stimulate responsive tissues throughout the body. Consequently, the impact of xenobiotics that can directly interfere with oocyte release and ovarian endocrine production has been a growing concern in environmental toxicology. The focus of present chapter will be on those methods that have been employed to assess the influence of toxicant exposure on the endocrine processes critical to normal reproductive activity. SYNTHESIS OF THE SEX STEROID HORMONES The principal bioactive mammalian sex steroid hormones, progesterone, testosterone, dihydrotestosterone, estradiol, estrone and estriol, are all formed from a source of cholesterol that can be either dietary or synthesized from acetate de novo. Cholesterol is converted to pregnenolone within the inner mitochondrial membrane by the catalytic action of cytochrome P450 side chain cleavage enzyme (P450scc, CYP11A1). For this to occur, cholesterol must be transported from the outer portion of the membrane to the inner region. It is a critical segment of the pathway and the rate-limiting step in steroidogenesis because the aqueous nature of the mitochondrial inter-membrane space presents a barrier to hydrophobic cholesterol. It now appears that two principal proteins are involved in the process. Steroidogenic acute regulatory (StAR) protein serves to mobilize cholesterol into mitochondrial membrane contact sites (e.g., Wang et al., 1998). While the exact mechanisms underlying the transport through the membrane are still not clear, it has been suggested that StAR serves to transfer the cholesterol to a second protein, the membrane-spanning peripheral benzodiazepine receptor,

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Figure 11.1 The ovarian steroidogenic pathway from cholesterol to estradiol. The relevant enzymes are italicized and the abbreviations defined, along with their cytochrome P450 designations, in the box at the lower left. Shaded areas identify the delta 4 and 5 segments of the pathway. StAR—steroidogenic acute regulatory protein, PBR—peripheral benzodiazepine receptor, 17β-HSD—17β-hydroxysteroid dehydrogenase, 5α-DHT—5αdihydrotestosterone.

which then carries it to the inner mitochondrial region (Papadopoulos et al, 1997; West et al., 2001) for conversion to pregnenolone. Two enzymes can then use pregnenolone as a substrate, each taking it into a different path on the way to androstenedione (Figure 11.1). 3β-hydroxysteroid dehydrogenase/A5–4 isomerase (3β-HSD) catalyzes the production of progesterone from pregnenolone (the delta 4 pathway). Progesterone is then converted, via a 17hydroxyprogesterone intermediate, to androstenedione. Alternatively, the action of 17α-hydroxylase/17,20-lyase (CYP17) will generate 17-hydroxypregnenolone and dihydroepiandrosterone (DHEA) (the delta 5 pathway) before converging with the delta 4 pathway at androstenedione. Androstenedione can then serve as the substrate for either P450aromatase (CYP19) or 17β-HSD to produce, respectively, estrone or testosterone. 17β-HSD will then convert estrone to estradiol, while the production of estradiol from testosterone will be catalyzed by P450aromatase. There is a species specificity in the preferential use of the delta 4 or delta 5 pathways. Delta 4 is used by rat (Weusten et al., 1987), ferret (Kintner and Mead, 1983), mare and some macaques (Weusten et al., 1990), whereas the

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delta 5 pathway predominates in humans (Weusten et al., 1987), rabbits, dogs and cows (Fortune, 1986). However, the predominance of one pathway over the other may vary with the cell type and be affected by the conditions of stimulation. In the chicken ovary, for example, both theca and granulosa cells were reported to have functional delta 4 and delta 5 pathways, although the theca layer showed a preferential use of the delta 5 route, while in granulosa cells delta 4 was predominant (Lee et al., 1998). Also, in hamster pre-ovulatory follicles under stimulation with luteinizing hormone (LH), there appears in theca cells to be a shift from delta 5 to delta 4 as the major metabolic pathway (Makris et al., 1983). Follicular steroidogenesis had commonly been viewed as a compartmentalized process, whereby androgens produced by the theca cells are provided to the granulosa cells for aromatization to estradiol (e.g., Fortune and Armstrong, 1977; Moor, 1977; Armstrong et al., 1981; Tsang et al., 1982). However, as touched upon above, both thecal and granulosa cells, along with stromal tissue, have all been found to possess the capacity to generate progestins, androgens and estrogens (McNatty et al., 1979; Soendoro et al., 1993), so that the relationship among the cell types in the process of steroid synthesis may be more interactive under conditions of gonadotropin stimulation than simply unidirectional. ASSESSMENT OF STEROID PRODUCTION Assessments of the impact of xenobiotic exposure on ovarian steroidogenesis have been carried out using a number of different approaches, both in vivo and in vitro. Although toxicological evaluations have been primarily concerned with alterations in estradiol and progesterone, determinations of estrone, pregnenolone and even testosterone can be informative. For the purposes of this discussion, the approaches are categorized according to the paired combination of exposure and sampling: in vivo exposure/in vivo sampling, in vitro exposure/in vitro sampling, or in vivo exposure followed by in vitro sampling. Each combination has both advantages and limitations (Table 11.1), and the choice of method will often depend on a variety of factors, including the type of information that is desired (hazard identification or mechanistic data), sensitivity, cost and technical difficulty. In vivo exposures/in vivo sampling The simplest approach to evaluating the influence of toxicant exposure on ovarian steroidogenesis is a straightforward measurement of circulating concentrations of the steroid hormones at a known point in the ovarian cycle. For toxicity testing, such measures will provide a comprehensive evaluation of the whole endocrine system as a unit and incorporate a broad range of mechanisms responsive to toxic insult.

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Table 11.1 Comparative advantages and limitations of different exposure/sampling paradigms

Consequently, the approach may not speak to any direct effects on ovarian toxicity, and any alterations in circulating steroid concentrations may be a reflection of a primary impact elsewhere within the hypothalamic-pituitaryovarian axis. Nevertheless, in vivo assessments can account for toxicant absorption, distribution, metabolism and excretion (ADME), factors which in vitro exposures cannot. While a determination of circulating steroid concentrations may provide a general indication of ovarian toxicity, the approach

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can be enhanced by assessing the ability of the ovaries to respond to some type of direct challenge. LH and human chorionic gonadotropin will both bind to ovarian LH receptors and stimulate steroid synthesis. Serial blood samples can then be collected and ovarian steroid concentrations measured. Microdialysis One of the newer applications for the in vivo assessment of extracellular materials is microdialysis. A growing number of publications have used the technique in investigations of regional secretory changes in the brains of both freely moving and sedated animals. The approach essentially is a sampling technique that uses a probe implanted into the tissue of interest. Sampling occurs from the extracellular space through a dialysis membrane that is permeable to water and small molecules (Figure 11.2). Using a physiologically compatible fluid, the probe is fed by an inflow line to a segment of dialysis tubing. As the fluid is slowly pumped through the microdialysis probe, the solution becomes equilibrated with the surrounding extracellular tissue fluid. After a period of time, it will then contain a representative proportion of the tissue fluid’s molecules which is collected through an outflow line. Molecules smaller than the pore size may permeate into the perfusion medium following the concentration gradient, while molecules larger than the membrane cut-off (e.g., proteins, enzymes) will be excluded. Although the technique has been employed most frequently in investigations of regional brain secretory activity, a small number of studies have adapted it for use in other tissues, including liver (Yokel et al., 1991), muscle (Okuda et al., 1992), lung (Eisenberg et al., 1993), adipose tissue (Tsuda et al, 2002), adrenal gland (Voigt et al, 1994), pituitary (Ishikawa et al, 1997) and testis (Cheng et al., 2001). While ovarian microdialysis has been employed more often with larger animals (under both in vivo [Acosta et al., 2000] and in vitro conditions [Acosta et al., 1998]) for which the surgical procedures are less technically demanding, Hirsch and colleagues (1993) have used fenestrated microdialysis tubing to measure collagenase activity from fluid sampled beneath the ovarian bursa in freely moving rats. For such studies in soft tissue, newer, more flexible probes have since become commercially available (Zuo et al., 1995). Nevertheless, its application in assessments of steroid secretion still has limitations, since such hydrophobic compounds do not easily diffuse across the dialysis membrane and recoveries in the dialysate can be relatively low. Jarry et al. (1990) estimated progesterone and estradiol recoveries with a 4–5 mm segment of acrylic copolymer dialysis tubing (molecular cut-off- 50 K) to be about 1 percent. Although the recovery of neurotransmitters from implanted brain microdialysis probes of similar composition is about 7–8-fold greater (Kendrick, 1991), the technique can still provide important information about the endocrine response of ovarian tissue to stimulation (Jarry et al., 1990; Miyamoto and Schams, 1991; Robinson, 1995). Moreover, since dialysis across the membrane is bidirectional,

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Figure 11.2 Representation of in vivo rat ovarian microdialysis, showing a segment of dialysis tubing threaded through the interior of the ovary and connected to teflon inflow and outflow tubing which can be exteriorized at the nape of the neck. The approach has also been used to sample steroids from extirpated ovarian tissue maintained in vitro.

it is possible in freely moving animals to incorporate a targeted exposure of ovarian tissue to a toxicant of interest along with a corresponding responsive sampling of secreted material. Certain restrictions would apply here, including any membrane limitations on the passage of a toxicant (e.g., molecular size cut-off, compound solubility). It should also be noted that an important pre-condition for

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the employment of this approach in any assessment of ovarian steroid secretion is the minimization of steroid adsorbance to the outflow tubing. For various types of plastic tubing, this is particularly true for progesterone (Bruning et al, 1981; Brännström et al, 1987), but has been markedly diminished by the incorporation of polytetrafluoroethylene (PTFE) or Teflon® tubing into the system. In vitro exposure/in vitro sampling For investigations of toxic alterations in steroid production, in vitro methods offer a number of advantages, including response specificity, an increased sensitivity to low exposure concentrations, reductions in the amounts of toxicant material required and lower costs compared to in vivo approaches. However, in terms of toxicity testing for risk assessment, the absence of mechanisms for toxicant ADME can often yield both false positive and false negative results. For example, a compound may not typically reach the ovaries in effective concentrations, or it may be rapidly excreted or metabolically inactivated, factors which may present false positives when extrapolating from in vitro assessments. On the other hand, if the effectiveness of an administered compound requires a process of metabolic activation, strictly in vitro assessments may result in a false negative. Nevertheless, when there exists previous evidence that metabolic activation is not required, or there is information about a compound’s metabolism in vivo, in vitro approaches in toxicity testing can be quite informative. A variety of methods have been commonly employed to assess the secretory capacity of ovarian tissue under baseline and stimulated conditions, ranging from whole organ culture to purified cell preparations. Each has distinct advantages and limitations for use in the assessment of toxicant insult (Table 11.2). Whole organ perfusion/perifusion The in vitro approach that most closely retains both the functional and cytoarchitectural integrity of the in vivo ovary is a whole organ perfusion. The ovary is removed with its arterial supply and venous drainage vessels intact. The ovarian vasculature is cannulated and a nutrient medium (Medium 199, for example, supplemented with bovine serum albumin and an antibiotic to retard bacterial contamination) is used both to bath and perfuse the organ. Ovarian cytoarchitectural integrity is retained, allowing for normal interactions among the different cell types. The in vitro system used to bath the ovary can be structured as either closed or open. In a closed system, the medium is recirculated, and any sampling performed over time will be a product of current and prior accumulation of secreted hormone. A schematic of such a system is presented in Figure 11.3. In an open system, no recirculation occurs, and sampling will reflect a temporal hormonal release. Any potential feedback effects are minimized,

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Table 11.2 Comparative advantages and limitations of type of ovarian material employed

although at least in the case of recirculated progesterone from perfused rabbit ovaries, such alterations do not appear to be a factor (Dharmarajan et al, 1988). An open system does, however, require a much greater volume of medium over the course of incubation. The complexity of the system, be it open or closed, will likely limit the number of cannulated whole ovarian perfusions that can be conducted at any one time. On the other hand, use of the ovary’s own vascular network allows both oxygenated medium and a toxicant of interest to reach throughout the preparation. Steroid secretion (and ovulation (e.g., Lambertsen et al., 1976; Koos et al., 1984)) can then be assessed under baseline or stimulated conditions. A less technically demanding approach to the evaluation of steroid production in whole ovaries has involved the use of perifusion. As opposed to perfusion, employing the ovarian vascular network, perifusion is characterized by the passage of nutrient medium around the ovary. Consequently, penetration of the material and oxygen takes place at the periphery, meaning that the utility of the approach will be limited to smaller ovaries in order to avoid interior necrosis and a consistency in toxicant exposure and steroid secretion. Alternatively, minced

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Figure 11.3 Schematic drawing of an apparatus designed for the perfusion of whole rat ovaries in a closed system using recirculated, oxygenated medium (Janson et al., 1982; Brännström et al., 1987).

fragments of tissue can be perifused or employed under static conditions in simple incubation. Perifusion systems can range from the relatively simple to the more complex depending on the degree of automation and control exercised over the various experimental parameters. As discussed above, non-steroid-adsorbing PTFE tubing should be used wherever possible. Two examples of such systems can be found in Zimmerman et al. (1985) and Peluso and Pappalarda (1993). As a rule, the use of ovarian cultures from adult cycling females would be expected to show a greater degree of variability in steroid secretion than preparations employing more homogeneous cell populations. Ovaries taken for culture without regard to the stage of the adult estrous cycle may exhibit considerable intragroup variation. There is improved consistency in ovarian secretion when ovaries are obtained at the same time in the cycle (Laskey et al., 1995) or on a specific day of gestation (Powlin et al., 1998), but the follicular and luteal heterogeneity may still show sufficient variability to mask modest toxicant-induced alterations in steroid release. On the other hand, as a shortterm screen for toxicology testing with sampling conducted over a period of

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Figure 11.4 An example of cAMP- and hCG-stimulated progesterone secretion (ng/ minute/ CL) from single perifused rat corpora lutea (in medium 199, n=9) removed from rats on day 6 of an induced pseudopregnancy. Shaded areas represent 30 minute exposures to either dibutyryl-cAMP or hCG, as indicated.

hours, the approach appears to be capable of detecting marked alterations in secretion. Follicular/luteal incubations The use of ovarian follicular or luteal cultures has a number of advantages over the minced tissue approach. In addition to the more homogenous nature of the preparation, gaseous and nutrient penetration are improved. Assessments of steroid production can be done in short-term culture using both static incubation (e.g., Nakamura et al., 1990; Brännström et al, 1993; Szoltys, 1995; Kishi and Greenwald, 1999) or perifusion (e.g., Hedin et al, 1983). An example of progesterone release from perifused corpora lutea taken from rats on day 6 of an induced pseudopregnancy is shown in Figure 11.4. For follicular preparations, survival over the course of several days has also allowed evaluations of maturational changes in a number of species (e.g., Roy and Treacy, 1993; Hirao et al., 1994; Cain et al, 1995; McGee et al, 1997). Nevertheless, follicular or luteal size does impose some physical constraints in culture. Diffusion gradient limitations and the absence of a luteal or follicular thecal blood supply can result in a degree of interior hypoxia. However, in short-term culture, normal hypoxic

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conditions within the follicular interior (Gosden and Byatt-Smith, 1986) may mean that there is a broadened window of tolerance to various levels of oxygen availability. For longer incubations over the course of several days, healthy appearing follicles showing progressive in vitro development in a number of species have been reported using oxygen concentrations of 20–95 percent (e.g., Fainstat, 1968; Cain et al., 1995; Smitz et al., 1996). Data on the impact of lower concentrations (down to 5 percent) are inconsistent, with some reports showing negligible adverse effects (e.g., Cecconi et al, 1999), while others have found various impairments (Boland et al, 1994; Smitz et al, 1996; Kaidi et al, 1998; Hu et al., 2001). As is frequently the case for this type of work, immature mammalian test species are primed after weaning with gonadotropins to induce maturation of an original cohort of follicles. After removal of the ovaries, these follicles can be isolated by enzymatic or physical methods. The use of collagenase treatment will dissociate greater numbers of large follicles (and small follicles), but with an erosion of the thecal layer and possible damage to the basement membrane (Hartshorne, 1997). Physical dissection will retain the thecal layer and avoid effects on the basement membrane, but the number of follicles that can be obtained is much smaller and the possibility of damage during isolation is a concern. The adaptation of whole/minced rat ovarian preparations (e.g., Laskey and Berman, 1993; Laskey et al, 1995; Piasek and Laskey, 1999) or follicular cultures (e.g., Yu et al., 2001; Goldman and Murr, 2002) for the assessment of alterations in steroid production has been successfully used as a screening approach to toxicant hazard identification. Moreover, the use of precursor supplementation can often provide additional information about the particular site(s) of toxicant impact along the steroidogenic pathway. For example, inclusion of pregnenolone in preparations of rat pre-ovulatory follicles exposed to the drinking water disinfection by-product dibromoacetic acid demonstrated that an observed suppression in progesterone secretion was not due to an alteration in 3β-HSD activity (Figures 11.5A and B). Under non-stimulated conditions, the suppression appeared, however, to be linked to an effect on StAR, since supplementation with the cholesterol analog 22R-hydroxycholesterol was able to by-pass the mitochondrial StAR transport system and eliminate the significant fall in progesterone (Figure 11.5C) (Goldman and Murr, 2002). Isolated cells For investigations of xenobiotic and endogenous biochemical effects on ovarian steroidogenesis, the use of cell cultures has long been the most common in vitro approach. Although the architectural integrity present in whole organ or tissue preparations is lost, homogeneous cell populations can provide an improved uniformity of response to exposure and permit comparisons across experiments and laboratories.

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Figure 11.5 Progesterone secretion from pre-ovulatory follicles exposed in vitro over the course of 24 hours to the drinking water disinfection by-product, dibromoacetic acid (DBA, 50 µg/ml). (A) Comparison of control versus DBA-exposed follicular pairs. (B) Effects of supplementation of the incubation medium with 1 µg/ml pregnenolone (Pregn), demonstrating viability of the DBA A-treated follicular preparation and lack of an effect of the compound on the 3β-HSD catalyzed conversion of Pregn to progesterone. (C) Supplementation of the two treatment groups with the cholesterol analog, 22R-hydroxycholesterol (22R-HC) which by-passed the involvement of StAR in intramitochondrial transport and elevated progesterone production, indicating an effect of DBA on StARlinked cholesterol transfer (Goldman and Murr, 2002).

A variety of different media formulations, both serum-supplemented and serum-free, have been successfully used for investigations of in vitro follicular cell steroidogenesis. And while the inclusion of a serum source (e.g., fetal calf serum) will provide various hormones and growth factors which may help cells survive outside of their particular tissues, factors inhibitory to growth and steroid production (Orly et al., 1980) may also be present. Moreover, a significant proportion of the medium then becomes undefined chemically. For this reason, most investigators have preferred the use of defined, serum-free formulations when optimal conditions for cell growth and survival are demonstrated. Many employed for studies of ovarian cell steroidogenesis have been extensions of Eagle’s minimal essential medium, including Dulbecco’s enriched modification, Ham’s F-12, McCoy’s 5A and Medium 199, under an atmosphere of 5 percent CO2. The isolation of granulosa cells is not difficult, and they can be dislodged from an incised follicle by agitation in culture or by flushing the follicular interior with a small amount of incubation medium. If desired, further purification to

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eliminate contaminant red blood cells can be carried out by centrifuging through a Percoll layer (Magoffin, 1993a). For theca-interstitial cell isolation from small ovaries, both granulosa and theca can be separated from a collagenase-dispersed ovarian cell preparation in a Percoll gradient (Magoffin, 1993b). Cell viability in sample aliquots can then be determined (see below) and plated cells evaluated for a steroidogenic response to toxicant treatment. In vivo exposure/in vitro sampling This type of experimental design, commonly termed ex vivo, has been used to circumvent the ADME limitations of in vitro exposures discussed above, although often such assessments have not been the principal focus but were included as an ancillary component of a larger in vivo toxicological evaluation. The approach does take advantage of additional strengths afforded by an in vivo treatment paradigm, permitting a variety of exposures in either F0 or multigenerational designs. However, it is possible that once the tissue is placed in culture medium and sampling begins, the impact on steroid production may change. An effect may be lost if the compound is particularly water soluble and is readily washed from the tissue. One such example was observed following treatment in vivo with another disinfection by-product, bromodichloromethane (Bielmeier et al., 2003). Corpora luteal and serum progesterone from rat dams on the ninth day of pregnancy following 4 days of exposure showed a significant suppression compared to controls. However, when corpora lutea from these animals were assessed in vitro, progesterone release was elevated under either hCG-stimulated or non-stimulated conditions. This was possibly due to a disinhibitory phenomenon resulting from a washout of the compound in culture. A similar discrepancy between data from ex vivo and in vitro exposures of testicular Leydig cells to ammonium perfluorooctanoate has also been reported by Biegel et al. (1995). ASSESSMENTS OF VIABILITY In any evaluation of steroidogenesis under either ex vivo or in vitro paradigms, it is essential that some determination of cell/tissue viability be conducted, since reductions in steroid secretion could be a reflection of general toxicant-induced cell death and not a focal alteration in steroidogenesis. Indications of viability in cell preparations can be relatively straightforward and include dye exclusion using trypan blue (e.g., Best et al., 1994; McCluskey et al., 1999) or propidium iodide (Slemmer et al., 2002), dye inclusion with fluorescein diacetate (e.g., Hutz et al., 1985; Nyberg et al., 1993), lactic dehydrogenase (LDH) leakage (e.g., Mathison et al., 1984), ATP bioluminescence (Connor et al., 1994) and reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reduction) (e.g., Iselt et al, 1989; Best et al, 1994; Morgan, 1998). While data obtained using different assays may yield somewhat

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disparate results, depending on the nature of the cell preparations and the culture conditions employed (Coco-Martin et al., 1992; Petty et al, 1995; da Costa et al., 1999), LDH release, trypan blue exclusion and tetrazolium dye reduction have all been used to assess ovarian cell viability (e.g., Hurwitz et al., 1992; Best and Hill, 1995; Dave et al, 1997). For whole ovarian and tissue cultures, the accessibility of dyes to all regions of the preparation (especially the most interior areas) may be uneven and mitigate against using them for assessments of viability. On the other hand, the release of LDH by dying cells into culture media is a useful alternative, and these assessments using pre-packaged kits are not difficult. However, for this type of determination, collected media should not have been previously frozen which would cause a denaturation of the enzyme and affect the accuracy of measurements. Alternative approaches would be the above-mentioned ATP bioluminescence assay or possibly an assessment of cytokine release, although for the latter, one would have to be reasonably certain that the compound under investigation would not typically trigger a cytokine response apart from an effect on cell toxicity. Finally, an additional option involves histological processing of tissue post-incubation for an assessment of cell morphology, which could be a more expensive alternative. SCREENING FOR TOXICANT-INDUCED EFFECTS ON STEROIDOGENESIS Concerns over the potential effects of environmental contaminants on endocrine mechanisms of regulation led the US Congress to charge the Environmental Protection Agency to provide recommendations for a screening and testing program that would serve to identify compounds termed “endocrine disrupting chemicals” or “endocrine disruptors.” Once optimized and validated, the battery of tests would be made available for widespread screening for endocrinedisrupting activity in pesticides, commercial chemicals and environmental contaminants. In addition to screening procedures to identify chemical estrogenicity/androgenicity, effects on steroid-receptor binding and mammalian pubertal maturation, assays to characterize xenobiotic effects on steroidogeneis were included. The selection of a screen for steroidogenesis would be based on a variety of criteria, including predictiveness, sensitivity, variability, ease of use, extent of animal usage, cost, time requirements and metabolic activation. Realistically, each potential approach would then have a combination of strengths and limitations, and its utility in the assessment of biological risk will be as one component of a comprehensive battery of tests. In vitro assays sampling steroid release from both male and female gonadal tissue were originally considered for selection. The initial recommendation was for a minced rat testis screen (Gray et al., 1997) for alterations in testosterone secretion that would be coupled with a determination of aromatase activity (predictive of a

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potentialinduced suppression in ovarian estradiol production). While immunoassays are commonly the procedures of choice to measure testosterone (or progesterone and estradiol) concentrations, a tritiated water assay has frequently been employed for P450arom determinations from homogenates or microsomal preparations from aromatase-containing tissues as ovaries, testes, brain or placenta. It measures the amount of 3H2O released during incubation with [1β-3H]-androstenedione (or 3H-testosterone) and does not require any separation of steroid substrate from product. One mole of 3H2O is released during the aromatization of one mole of [1β-3H]-androstenedione to estrone (or 3H-testosterone to estradiol) (Lephart and Simpson, 1991). Both the sliced testis and aromatase approaches are currently undergoing independent evaluation, along with an alternative method using a stabilized steroidogenically active cell line that would combine the two different assessments. A variety of such cell lines have been used to elucidate the roles of constituent biochemicals and the impact of xenobiotic compounds on the steroidogenic pathway, including those derived from rodent (MA-10, R2C, H540, mLTC-1) and human (KGN) gonadal tumors, human choriocarcinoma (JEG-3, JAR) and mammary carcinoma (MCF-7) cells. Each has its own composite of characteristics that can make it useful for investigations of one segment or another of the pathway. For example, the above-mentioned rodent cell lines were derived from mouse and rat Leydig cell tumors and have been useful in investigations of early steps in steroidogenesis. The JEG-3 (Krekels et al., 1991), JAR (Bellino et al, 1978) and KGN (Nishi et al., 2001) lines display relatively high basal levels of aromatase activity, which would be more advantageous for evaluations of chemicals that might have an inhibitory effect. One cell line in particular may have added value as a screen for a toxicant impact along the entire pathway. H295R cells, although not of gonadal origin and derived from a human adrenocortical tumor (Rainey et al., 1994), contain all steroidogenic enzymes from P450scc to P450arom (Logie et al., 1999) and could potenially serve as a surrogate for in vitro evaluations of ovarian toxicity. They have already been employed in a number of reports that have shown toxicant-induced shifts in the activity of various P450 enzymes (Sanderson et al., 2000, 2001, 2002). Their utility as part of an endocrine disruptor screening battery will ultimately depend on an evaluation of the criteria mentioned above. Although not mentioned previously, the emergence of microarray technology has offered one of the more promising approaches to the assessment of toxicantinduced alterations in steroid production. For in vitro exposures, detection of early differential expression of those genes associated with steroid synthesis may both serve as a sensitive predictor of alterations in product formation and establish chemical-specific patterns of gene expression that can be used to characterize the molecular mechanisms that lead to toxicity. While the approach has the capacity to reach beyond a determination of whether or not a compound is able to elicit a toxic response, it is still being shaped as a useful tool in predictive toxicology. It is important in these stages of methods development

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to couple such toxicogenomic applications with both studies in steroid synthesis and well-established functional toxicological evaluations. ACKNOWLEDGMENTS Dr Michael Narotsky contributed valuable comments on an earlier version of the manuscript, and we thank him for his input. The assistance that Ashley Murr provided in proofreading the manuscript is also greatly appreciated. We also wish to thank Dr Pat Hoyer for her input and patience in awaiting a final draft for submission. This chapter has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policy of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. REFERENCES Acosta, T.J., Miyamoto, A., Ozawa, T., Wijayagunawardane, M.P.B. and Sato, K. (1998) Local release of steroid hormones, prostaglandin E2, and endothelin-1 from bovine mature follicles in vitro: Effects of luteinizing homone, endothelin-1, and cytokines, Biol Reprod., 59:437–43. Acosta, T.J., Ozawa, T., Kobayashi, S., Hayashi, K., Ohtani, M., Kraetzl, W.D., Sato, K., Schams, D. and Miyamoto, A. (2000) Periovulatory changes in the local release of vasoactive peptides, prostaglandin F2α, and steroid hormones from bovine mature follicles in vivo, Biol Reprod., 63:1253–61. Armstrong, D.T., Weiss, T.J., Selstam, G. and Seamark, R.F. (1981) Hormonal and cellular interactions in follicular steroid biosynthesis by the sheep ovary, /. Reprod. Fertil Suppl, 30:143–54. Bellino, F.L., Hussa, R.O. and Osawa, Y. (1978) Estrogen synthetase in choriocarcinoma cell culture. Stimulation by dibutyryl cyclic adenosine monophosphate and theophylline, Steroids, 32:37–44. Best, C.L. and Hill, J.A. (1995) Interleukin-1 alpha and -beta modulation of luteinized human granulosa cell oestrogen and progesterone biosynthesis, Hum. Reprod., 10: 3206–10. Best, C.L., Pudney, J., Anderson, D.J. and Hill, J.A. (1994) Modulation of human granulosa cell steroid production in vitro by tumor necrosis factor alpha: implications of white blood cells in culture, Obstet. Gynecol., 84:121–27. Biegel, L.B., Liu, R.C., Hurtt, M.E. and Cook, J.C. (1995) Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies, Toxicol. Appl. Pharmacol., 134:18–25. Bielmeier, S.R., Murr, A.S., Best, D.S., Goldman, J.M. and Narotsky, M.G. (2003) Effect of bromodichloromethane (BDCM) on ex vivo luteal function in the F344 rat during pregnancy, Toxicol. Sci., 72 (S-1):26 (abstract).

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Boland, N.I., Humpherson, P.G., Leese, H.J. and Gosden, R.G. (1994) Characterization of follicular energy metabolism, Hum. Reprod., 9:604–09. Brännström, M., Johansson, B.M., Sogn, J. and Janson, P.O. (1987) Characterization of an in vitro perfused rat ovary model: ovulation rate, oocyte maturation, steroidogenesis and influence of PMSG priming, Acta Physiol Scand., 130:107–14. Brännström, M., Wang, L. and Norman, R.J. (1993) Effects of cytokines on prostaglandin production and steroidogenesis of incubated preovulatory follicles of the rat, Biol. Reprod., 48:165–71. Bruning, P.F., Jonker, K.M. and Boerema-Baan, A.W. (1981) Adsorption of steroid hormones by plastic tubing, J. Steroid Biochem., 14:533–39. Cain, L., Chatterjee, S. and Collings, T.J. (1995) In vitro folliculogenesis of rat preantral follicles, Endocrinology, 136:3369–77. Cecconi, S., Barboni, B., Coccia, M. and Mattioli, M. (1999) In vitro development of sheep preantral follicles, Biol. Reprod., 60:594–601. Cheng, C.L., Yang, C.R., Yang, D.Y., Kuo, J.S., Huang, Y.F. and Cheng, F.C. (2001) Simultaneous monitoring of extracellular glucose, pyruvate, and lactate in rat testis during ischemia: a microdialysis study, Urolog. Res., 29:272–77. Coco-Martin, J.M., Oberink, J.W., van der Velden-de Groot, T.A. and Beuvery, E.C. (1992) Viability measurements of hybridoma cells in suspension cultures, Cytotechnology, 8:57–64. Connor, J.P., Buller, R.E. and Conn, P.M. (1994) Effects of GnRH analogs on six ovarian cancer cell lines in culture, Gynecol Oncol., 54:80–86. da Costa, A.O., de Assis, M.C., Marques, E.A. and Plotkowski, M.C. (1999) Comparative analysis of three methods to assess viability of mammalian cells in culture, Biocell, 23:65–72. Dave, S., Farrance, D.P. and Whitehead, S.A. (1997) Evidence that nitric oxide inhibits steroidogenesis in cultured granulosa cells, Clin. Sci. (Lond.), 92:277–84. Dharmarajan, A.M., Yoshimura, Y., Sueoka, K., Atlas, S.J., Dubin, N.H., Ewing, L.L., Zirkin, B.R. and Wallach, E.E. (1988) Progesterone secretion by corpora lutea of the isolated perfused rabbit ovary during pseudopregnancy, Biol. Reprod., 38:1137–43. Eisenberg, E.J., Conzentino, P., Eickhoff, W.M. and Cundy, K.C. (1993) Pharmacokinetic measurement of drugs in lung epithelial lining fluid by microdialysis: aminoglycoside antibodies in rat bronchi, J. Pharmacol. Toxicol. Meth., 29:93–98. Fainstat, T. (1968) Organ culture of postnatal rat ovaries in chemically defined medium, Fertil. Steril., 19:317–38. Fortune, J.E. (1986) Bovine theca and granulosa cells interact to promote androgen production, Biol. Reprod., 35:292–99. Fortune, J.E. and Armstrong, D.T. (1977) Androgen production by theca and granulosa isolated from proestrous rat follicles, Endocrinology, 100:1341–47. Goldman, J.M. and Murr, A.S. (2002) Alterations in ovarian follicular progesterone secretion by elevated exposures to the drinking water disinfection by-product dibromoacetic acid: Examination of the potential sites(s) of impact along the steroidogenic pathway, Toxicology, 171:83–93. Gosden, R.G. and Byatt-Smith, J.G. (1986) Oxygen concentration gradient across the ovarian follicular epithelium: model, predictions and implications, Hum. Reprod., 1: 65–68. Gray, L.E. Jr, Kelce, W.R., Wiese, T., Tyl, R., Gaido, K., Cook, J., Klinefelter, G., Desaulniers, D., Wilson, E., Zacharewski, T., Waller, C., Foster, P., Laskey, J., Reel,

JEROME M. GOLDMAN ET AL. 223

J., Giesy, J., Laws, S., McLachlan, J., Breslin, W., Cooper, R., Di Giulio, R., Johnson, R., Purdy, R., Mihaich, E., Safe, S. and Colborn, T. (1997) Endocrine screening methods workshop report: detection of estrogenic and androgenic hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme mechanisms, Reprod. Toxicol., 11:719–50. Hartshorne, G.M. (1997) In vitro culture of ovarian follicles, Rev. Reprod., 2:94–104. Hedin, L., Ekholm, C. and Hillensjo, T. (1983) Dose-related effects of luteinizing hormone on the pattern of steroidogenesis and cyclic adenosine monophosphate release in superfused preovulatory rat follicles, Biol Reprod., 29:895–904. Hirao, Y., Nagai, T., Kubo, M., Miyano, T., Miyake, M. and Kato, S. (1994) In vitro growth and maturation of pig oocytes, J. Reprod. Fertil., 100:333–39. Hirsch, B., Leonhardt, S., Jarry, H., Reich, R., Tsafriri, A. and Wuttke, W. (1993) In vivo measurement of rat ovarian collagenolytic activities, Endocrinology, 133:2761–65. Hu, Y., Betzendahl, I., Cortvrindt, R., Smitz, J. and Eichenlaub-Ritter, U. (2001) Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from preantral follicle culture, Hum. Reprod., 16:737–48. Hurwitz, A., Hernandez, E.R., Payne, D.W., Kharmarajan, A.M. and Adashi, E.Y. (1992) Interleukin-1 is both morphogenic and cytotoxic to cultures rat ovarian cells: obligatory role for heterologous, contact-independent cell-cell interaction, Endocrinology, 131:1643–49. Hutz, R.J., DeMayo, F.J. and Dukelow, W.R. (1985) The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus auratus, Stain Technol., 60:163–67. Iselt, M., Holtei, W. and Hilgard, P. (1989) The tetrazolium dye assay for rapid in vitro assessment of cytotoxicity, Arzneimittelforschung, 39:747–49. Ishikawa, M., Mizobuchi, M, Takahashi, H., Bando, H. and Saito, S. (1997) Somatostatin release as measured by in vivo microdialysis: circadian variation and effect of prolonged food deprivation, Brain Res., 749:226–31. Janson, P.O., LeMaire, W.J., Kallfelt, B., Holmes, P.V., Cajander, S., Bjersing, L., Wiqvist, N. and Ahren, K. (1982) The study of ovulation in the isolated perfused rabbit ovary. I. Methodology and pattern of steroidogenesis, Biol Reprod., 26:456– 65. Jarry, H., Einspanier, A., Kanngieber, L., Dietrich, M., Pitzel, L., Holtz, W. and Wuttke, W. (1990) Release and effects of oxytocin on estradiol and progesterone secretion in porcine corpora lutea as measured by an in vivo microdialysis system Endocrinology, 126:2350–58. Kaidi, S., Donnay, I., Van Langendonckt, A., Dessy, F. and Massip, A. (1998) Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification, Anim. Reprod. Sci., 52:39–50. Kendrick, K.M. (1991) In vivo measurments of amino acid, monoamine and neuropeptide release using microdialysis. In: B. Greenstein (ed.), Neuroendocrine Research Methods, Chur, Switzerland: Harwood Academic Publishers, Vol. 1, pp. 249–78. Kintner, P.J. and Mead, R.A. (1983) Steroid metabolism in the corpus luteum of the ferret, Biol Reprod., 29:1121–27. Kishi, H. and Greenwald, G.S. (1999) In vitro steroidogenesis by dissociated rat follicles, primary to antral, before and after injection of equine chorionic gonadotropin, Biol Reprod., 61:1177–83.

224 ASSESSMENT OF TOXICANT-INDUCED ALTERATIONS

Koos, R.D., Jaccarino, F.J., Magaril, R.A. and LeMaire, W.J. (1984) Perfusion of the rat ovary in vitro: methodology, induction of ovulation, and pattern of steroidogenesis, Biol. Reprod., 30:1135–41. Krekels, M.D., Wouters, W., De Coster, R., Van Ginckel, R., Leonaers, A. and Janssen, P.A. (1991) Aromatase in the human choriocarcinoma JEG-3: inhibition by R 76 713 in cultured cells and in tumors grown in nude mice, J. Steroid Biochem. Mol. Biol., 38:415–22. Lambertsen, C.J. Jr, Greenbaum, D.F., Wright, K.H. and Wallach, E.E. (1976) In vitro studies of ovulation in the perfused rabbit ovary, Fertil Steril., 27:178–87. Laskey, J.W. and Berman, E. (1993) Steroidogenic assessment using ovary culture in cycling rats: effects of bis (2-diethylhexyl)phthalate on ovarian steroid production, Reprod. Toxicol., 7:25–33. Laskey, J.W., Berman, E. and Ferrell, J.M. (1995) The use of cultured ovarian fragments to assess toxicant alterations in steroidogenesis in the Sprague-Dawley rat, Reprod. Toxicol., 9:131–41. Lee, K.A., Volentine, K.K. and Bahr, J.M. (1998) Two steroidogenic pathways present in the chicken ovary: theca layer prefers delta 5 pathway and granulosa layer prefers delta 4 pathway, Domest. Anim. Endocrinol., 15:1–8. Lephart, E.D. and Simpson, E.R. (1991) Assay of aromatase activity, Methods Enzymol., 206:477–83. Logie, A., Boulle, N., Gaston, V., Perin, L., Boudou, P., Le Bouc, Y. and Gicquel, C. (1999) Autocrine role of IGF-II in proliferation of human adrenocortical carcinoma NCI H295R cell line, J. Mol. Endocrinol., 23:23–32. Magoffin, D.A. (1993a) Human granulosa and granulosa luteal cells in culture. In: J.J. Heindel and R.E. Chapin (eds), Female Reproductive Toxicology (Methods in Toxicology), San Diego: Academic Press, Vol. 3B, pp. 289–94. Magoffin, D.A. (1993b) Rat thecal-interstitial cells in culture. In: J.J. Heindel and R.E. Chapin (eds), Female Reproductive Toxicology (Methods in Toxicology), San Diego: Academic Press, Vol. 3B, pp. 320–29. Makris, A., Olsen, D. and Ryan, K.J. (1983) Significance of the delta 5 and delta 4 steroidogenic pathways in the hamster preovulatory follicle, Steroids, 42:641–51. Mathison, J.C., LaForest, A.C. and Ulevitch, R.J. (1984) Properties and requirements for production of a macrophage product which suppresses steroid production by adrenocortical cells, Infect. Immun., 45:360–66. McCluskey, S., Hall, M., Stanton, C. and Devery, R. (1999) Alpha-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells, Mol. Cell Biochem., 194:217–25. McGee, E., Spears, N., Minami, S., Hsu, S.-Y., Chun, S.-Y., Billig, H. and Hsueh, J.W. (1997) Preantrol ovarian follicles in serum-free culture: suppression of apoptosis after activation of the cyclic guanosine 3',5'-monophosphate pathway and stimulation of growth and differentiation by follicle-stimulating hormone, Endocrinology, 138: 2417–24. McNatty, K.P., Makris, A., DeGrazia, C., Osathanondh, R. and Ryan, K.J. (1979) The production of progesterone, androgens, and estrogens by granulosa cells, theca tissue, and stromal tissue from human ovaries in vitro, J.Clin. Endocrinol Metab., 49:687– 99. Miyamoto, A. and Schams, D. (1991) Oxytocin stimulates progesterone release from microdialyzed bovine corpus luteum in vitro, Biol Reprod., 44:1163–70.

JEROME M. GOLDMAN ET AL. 225

Moor, R.M. (1977) Sites of steroid production in ovine Graafian follicles in culture, J.Endocrinol., 73:143–50. Morgan, D.M. (1998) Tetrazolium (MTT) assay for cellular viability and activity, Methods Mol. Biol., 79:179–83. Nakamura, Y., Kato, H. and Terranova, P.F. (1990) Interkeukin-1α increases thecal progesterone production of preovulatory follicles in cyclic hamsters, Biol Reprod., 43:169–73. Nishi, Y., Yanase, T., Mu, Y., Oba, K., Ichino, I., Saito, M., Nomura, M., Mukasa, C., Okabe, T., Goto, K., Takayanagi, R., Kashimura, Y., Haji, M. and Nawata, H. (2001) Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor, Endocrinology, 142:437–45. Nyberg, S.L., Shatford, R.A., Payne, W.D., Hu, W.S. and Cerra, F.B. (1993) Staining with fluorescein diacetate correlates with hepatocyte function, Biotech. Histochem., 68:56– 63. Okuda, C, Sawa, T., Harada, M, Murakami, T., Matsuda, T. and Tanaka, Y. (1992) Lactate in rat skeletal muscle after hemorrhage measured by microdialysis probe calibrated in situ, Am. J.Physiol., 263:E1035–39. Orly, J., Sato, G. and Erickson, G.F. (1980) Serum suppresses the expression of hormonally induced functions in cultured granulosa cells, Cell, 20:817–27. Papadopoulos, V., Amri, H., Li, H., Boujrad, N., Vidic, B. and Garnier, M. (1997) Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line, J. Biol Chem., 272:32129–35. Peluso, J.J. and Pappalarda, A. (1993) Ovarian perifusion culture: A tool to assess ovarian toxicity. In: J.J. Heindel and R.E. Chapin (eds), Female Reproductive Toxicology (Methods in Toxicology), San Diego: Academic Press, Vol. 3B, pp.180–93. Petty, R.D., Sutherland, L.A., Hunter, E.M. and Cree, I.A. (1995) Comparison of MTT and ATP-based assays for the measurement of viable cell number, J. Biolumin. Chemolumin., 10:29–34. Piasek, M. and Laskey, J.W. (1999) Effects of in vitro cadmium exposure on ovarian steroidogenesis in rats, J. Appl. Toxicol., 19:211–17. Powlin, S.S., Cook, J.C., Novak, S. and O’Conner, J.C. (1998) Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier 1 screening battery, Toxicol. Sci., 46:61–74. Rainey, W.E., Bird, I.M. and Mason, J.L (1994) The NCI-H295 cell line: a pluripotent model for human adrenocortical studies, Mol. Cell. Endocrinol., 100:45–50. Robinson, J.E. (1995) Microdialysis: A novel tool for research in the reproductive system, Biol Reprod., 52:237–45. Roy, S.K. and Treacy, B.J. (1993) Isolation and long-term culture of human preantral follicles, Fertil Steril., 59:783–90. Sanderson, J.T., Seinen, W., Giesy, J.P. and van den Berg, M. (2000) 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity?, Toxicol., Sci., 54:121–27. Sanderson, J.T., Slobbe, L., Lansbergen, G.W., Safe, S. and van den Berg, M. (2001) 2,3, 7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells, Toxicol. Sci., 61:40–48.

226 ASSESSMENT OF TOXICANT-INDUCED ALTERATIONS

Sanderson, J.T., Boerma, J., Lansbergen, G.W. and van den Berg, M. (2002) Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells, Toxicol. Appl Pharmacol., 182:44– 54. Slemmer, J.E., Matser, E.J., De Zeeuw, C.I. and Weber, J.T. (2002) Repeated mild injury causes cumulative damage to hippocampal cells, Brain, 125:2699–709. Smitz, J., Cortvrindt, R. and Van Steirteghem, A.C. (1996) Normal oxygen atmosphere is essential for the solitary long-term culture of early preantral follicles, Mol. Reprod. Dev., 45:466–75. Soendoro, T., Diamond, M.P., Pepperell, J.R. and Naftolin, F. (1993) The in vitro perifused rat ovary: III. Interrelationship of the follicular and stromal compartments on steroid release, Gynecol Endocrinol., 7:1–6. Szoltys, M. (1995) The pattern of steroid secretion by perfused preovulatory rat follicles, J. Physiol Pharmacol, 46:87–95. Tsang, B.K., Moon, Y.S. and Armstrong, D.T. (1982) Estradiol-17beta and androgen secretion by isolated porcine ovarian follicular cells in vitro, Can. J. Physiol. Pharmacol., 60:1112–18. Tsuda, K., Yoshimatsu, H., Niijima, A., Chiba, S., Okeda, T. and Sakata, T. (2002) Hypothalamic histamine neurons activate lipolysis in rat adipose tissue, Exp. Biol. Med., 227:208–13. Voigt, J.P., Kaufmann, G., Hirsch, B., Leonhardt, S., Jarry, H., Oehme, P. and Wuttke, W. (1994) In vivo effect of intraadrenal nicotine and substance P application on rat adrenal medullary catecholamine secretion, Exp. Clin. Endocrinol., 102:111–17. Wang, X., Liu, Z., Eimerl, S., Timberg, R., Weiss, A.M., Orly, J. and Stocco, D.M. (1998) Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer, Endocrinology, 139:3903–12. West, L.A., Horvat, R.D., Roess, D.A., Barisas, B.G., Juengel, J.L. and Niswender, G.D. (2001) Steroidogenid acute regulatory protein and peripheral-type benzodiazepine receptor associate at the mitochondrial membrane, Endocrinology, 142:502–05. Weusten, J.J., Smals, A.G., Hofman, J.A., Kloppenborg, P.W. and Benraad, T.J. (1987) Early time sequence in pregnenolone metabolism to testosterone in homogenates of human and rat testis, Endocrinology, 120:1909–13. Weusten, J.J., van der Wouw, M.P., Smals, A.G., Hofman, J.A., Kloppenborg, P.W. and Benraad, T.J. (1990) Differential metabolism of pregnenolone by testicular homogenates of human and two species of macaques. Lack of synthesis of the human sex pheromone precursor 5,16-androstadien-3 beta-ol in nonhuman primates, Horm. Metab. Res., 22:619–21. Yokel, R.A., Lidums, V., McNamara, P.J. and Ungerstedt, U. (1991) Aluminum distribution into brain and liver of rats and rabbits following intravenous aluminum lactate or citrate: a microdialysis study, Toxicol. Appl. Pharmacol., 107:153–63. Yu, C.C., Chen, W.Y. and Li, P.S. (2001) Protein phosphatase inhibitor canthardin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles, Life Sci., 70:57–72. Zimmerman, R.C., Wun, W.-S., Tcholakian, R.K., Rodriguez-Rigau, L.J., Braendle, W. and Steinberger, E. (1985) In vitro steroid secretion by intact bovine ovarian follicles in a superfusion system, Horm. Metabol. Res., 17:458–63. Zuo, H., Ye, M. and Davies, M.I. (1995) The linear probe: A flexible choice for in vivo microdialysis sampling in soft tissues, Curr. Separations, 14:54–57.

INDEX

Note: Authors’ articles appear where page numbers are bold. α-naphthoflavone (ANF) 24, 122 [3H]-cis-stilbene oxide 125, 126 1,1,1 -trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) 44, 45, 47, 74, 191 l,2-dichloropropane (DCP) 51, 78 1,3-butadiene (BD) 24, 25, 191 1-bromopropane (1BP) 26, 78 1-naphthol 120 2,2-bis-(p-hydroxyphenyl)-1,1,1trichloroethane (HPTE) 47 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 64, 65, 66, 67, 68, 69, 70, 71, 78, 79, 118, 141, 142 2,3,7,8-tetrachlorodibenzo-p-dioxin 141 2,4-dichlorophenoxyacetic acid 51 see also herbicides 2-bromopropane (2BP), an ovarian axis ED 26, 73, 77, 78, 142, 143 blocking ovulation 77 3,3,4,4-tetrachlorobephenyl (TCB) 27 3,3'-diindolylmethane (DIM) 70 3β-hydroxysteroid dehydrogenase/A5–4 isomerase (3β-HSD) 7, 206 3-methyl-cholanthrene (3-MC) 23 4-vinylcyclohexene (VCH) 25–6, 28, 77, 126 model ovarian toxicant 125 effects on biotransformation enzymes 125–5 9:10-dimethyl-1 :2-benzanthracene (DMBA) 23, 24, 29, 32, 33, 120, 121, 122, 123, 124, 139, 192 ACS see American Cancer Society

Additional halogenated compounds 142–40 AhR see aryl hydrocarbon receptor aldrin 44, 49, 184 American Cancer Society (ACS) 176 American Conference of Industrial Hygieniest 25 anterior pituitary 5, 8, 10, 15, 21, 44, 45, 138 follicle-stimulating hormone 5, 7, 8, 9, 10, 11, 21, 44, 45, 47, 49, 53, 64, 74, 78, 87, 88, 90, 123, 142, 143, 196 luteinizing hormone 5, 7, 8, 10, 44, 49, 53, 54, 61, 72, 77, 86, 93, 97, 98, 100, 101, 112, 123, 135, 138, 142, 196, 207, 209 prolactin 6, 10, 15, 71, 96 antral follicle see tertiary follicle antrum 3, 4, 11, 16, 18, 20, 42 Apaf-1 151, 155 functional domains 151 apoptosis 4, 12, 21, 29, 30, 31, 42, 44, 64, 76, 135, 139, 149, 150, 151, 153, 196 in chemotherapy 149 -detecting kits 4 pathways leading to 151 protease-activating factor-1 (Apaf-1) 151 regulation of 151 role of inhibitors of apoptosis proteins 155–4 signal transduction 150–9 aryl hydrocarbon receptor (AhR) 54, 61, 90, 138 227

228 INDEX

atrazine 49, 50, 51, 71, 72, 73, 78, 95, 96, 98, 190, 194 as herbicide 95 causing pre-mature mammary tumors 96 effect on ovarian function 100 effect on reproductive aging 99 effects and metabolites 110 exposure on pregnancy maintenance 107 leads to pre-mature mammary tumors 96–6 neuroendocrine-ovarian axis disruptor 71, 74 2BP 74 EDs 76 South Korea 74 Wistar rats 74 on ovarian function 100 on reproductive aging 99 pituitary hormone secretion 100 pregnancy loss 106 pubertal development 110 reproductive aging 99 reproductive function 100 reproductive toxicant in rats 95 and suckling-induced prolactin release 107–8 tumor development 96 see also xenobiotics; chlorotriazine atresia 12, 135 atretic follicles 4, 8, 12, 49, 52, 53 basement membrane/membrana propria 2, 17, 18, 213, 215 BDE see butadiene diolepoxide benzo[a]pyrene (BaP) 23, 192 Berthold 6 biotransformation enzymes 24, 115, 116, 117, 118, 125, 126, 127, 129 ovarian expression of 126 in porcine 126 in rodents 126 overview 115 phase I metabolism 115 phase II metabolism 115

in the ovary, 117 BMO see butadiene monoepoxide Borgeest, Christina 40 bromo-dichloromethane (BDCM) 107 busulfan 143 butadiene diolepoxide (BDE) 191 butadiene monoepoxide (BMO) 25, 191 cAMP-protein kinase A pathways 5 Cannady, Ellen A. 115 carbamates 51 carboplatin 149 carcinosarcoma 176 cell adhesion 158 cell death 28, 29, 30, 31, 42, 150, 151, 154, 155, 157, 158, 217 see also apoptosis; atresia; ovotoxicty effects cell transactivation assays 90 cellular damage, sites of 30 Chao Wu Xiao 149 chemical exposure, mode of 32 chemoresistance 149, 151, 152, 163 fas/fasl systems and 153 mechanisms responsible for 151 and XIAP 154 chemotherapeutic agents 21, 31, 149, 150, 153, 154, 158, 163, 177 anticancer agent 23 balb/c mice 23 C57BL/6N and DBA/2N mice 23 cyclophosphamide 21 cytotoxicity of 150 dose-dependent relationship 23 Sprague-Dawley (SD) 23 chemotherapy 31 involvement of apoptosis 149 chloro-S-triazine see atrazine chlorotriazine see EDs as endocrine mediators 97 pregnancy initiation and maintenance 106 and reproductive function atrazine and suckling-induced prolactin release 107–8

INDEX 229

pregnancy initiation and pregnancy maintenance 106–6 pubertal development 110 role in tumor development 110 chlorpropham 51 cholesterol 6, 7, 47, 88, 90, 205, 206, 215, 216 chromosomes xii, 2, 5, 12, 13, 134 X chromosome xii, 2 Y chromosome xii, 134 CHX see cycloheximide cigarette smoking see occupational chemicals cis-DDP/cisplatin 149 cis-DDP-induced cytotoxicity 150 cis-diamminedichloroplatinum(II) 149 cisplastin cytotoxicity of 149 interaction between agonistic fas monoclonal antibody 154 reaction of GSH and 152 role in apoptosis 153 CL see corpus luteum Coe, Kathryn 174 Congo Red Cooper, Ralph L. 95 corona radiata 3, 4, 5, 11, 13 corpus albicans 4 corpus hemorrhagicum 4 corpus luteum (CL) (“yellow body”) 4, 5, 6, 8, 9, 13, 14, 15, 44, 87, 127 CPA see cylcophosphamide cumulus cells 18 cumulus oophorus 3 cycloheximide (CHX) 7, 150, 162 cylcophosphamide (CPA) 21 CYP see cytochrome P450 CYP1A1 118, 121, 124, 126, 138 CYP1B1 91, 93, 118, 121, 127 cytochrome P450 (CYP) 54, 115, 117, 118, 124, 125, 205 DACT see diaminochlorotriazine Davis, Barbara J. 86 Dax-1/DAX-1 xii DDE see p, p´-dichlordipehenyldiciclhorethylene

DDT see 1,1,1 -trichloro-2,2-bis(pchlorophenyl)ethane in cockroach 46–6 in fish 46 in hens 45, 48 in humans 45 Leghorn chickens 46 DEB see diepoxybutane DEHP 86, 87, 88, 90, 93 liver toxicity of 88 mechanism of suppressing estradiol 87, 88 ovary-target, organ of 86 DES see diethylstilbestrol di-(2-ethylhexyl) phthalate (DEHP) 86 diaminochlorotriazine (DACT) 107 dieldrin 44, 47, 184 see also organochlorine diepoxybutane (DEB) 191 diethylstilbestrol (DES) 64 DMB A-3,4-diol-1,2-epoxide 121 DNA synthesis cytotoxicity of chemotherapeutic agents 150 inhibition 150 dopamine function 72, 73, 78, 98 eCG see equine chorionic gonadotropin ECM see extracellular matrix EDCs see endocrine disruptors EDs: Ah receptor agonists as multiple organ 65 AhR agonists 65 direct and/or indirect effects 61 drugs 64 effects on ovarian function 65 immature rats 66 models of 64 PAHs 65 in utero 66 in vitro effects of FSH and LH 69 EGF see epidermal growth factor eicosanoids 10 endocrine disrupting chemicals see endocrine disruptors endocrine disruptors (EDCs) 40, 218

230 INDEX

definition 61 endocrinology and biochemistry 0 enzymes 6, 7, 9, 10, 11, 13, 24, 54, 88, 90, 91, 115, 116, 117, 118, 120, 122, 123, 124, 125, 127, 128, 129, 138, 151, 152, 191, 206, 209, 219 angiotensin-converting enzyme 9 angiotensinogen 9 collagenase and plasminogen activator 10 renin 9 epidermal growth factor (EGF) 6, 9 equine chorionic gonadotropin (eCG) 64, 70 estradiol 6, 7, 8, 11, 12, 13, 14, 32, 45, 47, 50, 52, 65, 67, 68, 69, 70, 71, 72, 73, 74, 86, 87, 88, 90, 91, 92, 93, 97, 98, 100, 110, 134, 135, 138, 142, 205, 206, 207, 209, 219 estrogenic compounds 138, 138 bisphenol A 138 DES 138 methoxychlor 138 extracellular matrix (ECM) 158 extrahepatic metabolism 116, 117, 129 FADD see fas-associated death domain FAK see focal adhesion kinase fas antigen (fas) expression of 153 fas-associated death domain (FADD) 151, 162 fas ligand (FasL) 153 fas see tumor necrosis factor receptor-1 Fas/FasL system and chemoresistance 153– 2 FasL see fas ligand fetal development 17 FGF see fibroblast growth factor fibroblast growth factor (FGF) 6, 9 flavin-containing monooxygenases (FMO) 115 Flaws, Jodi A. 40 flice-like inhibitory protein (FLIP) 161, 162 FMO see flavin-containing monooxygenases

focal adhesion kinase (FAK) 158, 159, 160, 161, 163 follicle-specific effects 20 follicle-stimulating hormone (FSH) 5, 44, 86, 110, 183 follicular destruction, consequences of B6C3F1 mice 21 VCH-mice 21 development Graafian follicle 16 mammals 16 primodial development 16 growth granulosa cells 10 in vitro 10 in vivo 10 human 10 recruitment estradiol 11 FSH 11 mammals 11 selection human 11 follicular fluid/liquor folliculi 3 follistatin 8 FSH see follicle-stimulating hormone Fumikazu Kotsuji 149 fungicides 42, 52 dithocarbamates 53 hexachlorobenzene 52 Gadus morhua 46 galactose-1 -phosphate uridyltransferase (GALT) 184 GALT see galactose-1-phosphate uridyltransferase gene and cell signaling 30 effects on 90 preventing cancer BRCA1 180 BRCA2 180 germ cells 2, 9, 17, 21, 26, 51, 134, 135, 184 germinal vesicle breakdown (GVB) 12, 13

INDEX 231

glutathione (GSH)/ γglutamylcysteinylglycine see also tripeptide thiol Goldman, Jerome M. 95, 205 gonadotrophic glycoprotein hormones 5, 10, 11 gonadotropin-releasing hormone (GnRH) 44, 69 G-protein-coupled receptors 5 Graafian follicle see tertiary follicle granulosa cells 2, 42, 42, 47, 68, 69, 75, 86, 118, 121, 122, 125, 126, 126, 127, 135, 141, 142, 162, 207, 217 GSH see glutathione halogenated aromatic hydrocarbons (HAHs) 139–9 paper pulp 141 HCB see hexachlorobenzene hCG see human chorionic gonadotropin heat shock protein (hsp90) 6, 138 hepatic metabolism of VCH 126 herbicides see EDs amphibians 50 chlorotriazines 49 physiological changes in mammals 50 primary routes 49 in snail 51 Xenopus laevis 50 Heteropneustes fossilis 49 hexachlorobenzene (HCB) 27, 52, 184 Hiromasa Sasaki 149 HIV see human immunodeficiency virus hormone replacement therapy (HRT) 174 Hoyer, Patricia B. 16 HPV see human papilomavirus HRT see hormone replacement therapy human chorionic gonadotropin (hCG) 47, 65, 75, 209 human immunodeficiency virus (HIV) 183 human papilomavirus (HPV) 183 human risk, prediction of 31 hypothalamus 10, 21, 44, 45, 64, 65, 73, 101, 110, 124, 135, 142 I3C see indole-3-carbinol IAPs see inhibitors of apoptosis proteins

IGF-binding proteins (IGFBPs) 8 IGFBP see IGF-binding proteins IMS see isopropyl methanesulfonate indole-3-carbinol (I3C) 63, 65, 66, 70, 71, 78 see also EDs inhibitors of apoptosis proteins (IAPs) 154, 155, 157, 158, 162 anti-apoptotic effect 157 inositol phosphate-protein kinase C 5 insulin-like growth factor-1 (IGF-1) 6, 8 interleukin-1 (IL-1) 9 intraovarian factors, action 135 ionizing radiation 2, 123 animal studies 21 irradiation 21 isopropyl methanesulfonate (IMS) 26 Janus kinase signal transducers (JAKSTAT) 6 Jayes, Friederike C.L. 86 lactic dehydrogenase (LDH) 217, 218 large luteal cells 4 Laws, Susan C. 95, 205 LDH see lactic dehydrogenase Lead 143, 191, 194, 195 leuteotrophic complex 14 LH see luteinizing hormone lindane 45, 46, 47, 51 see also organochlorine long (FLIPL) and short (FLIPs) 162 Lovekamp-Swan, Tara 86 low-density lipoprotein (LDL) receptors 7 luteinization and luteal function 13–14 luteinizing hormone (LH) 5, 44, 67, 87, 97, 105, 135, 207 luteolysis 8, 9, 10, 14, 15 mammary gland tumors 71, 97, 98, 99, 100, 110 altered ovarian cycle 97 pituitary-ovarian hormonal environment 99 Marcinkiewicz, Jennifer L. 134 maternal-fetal transfer 136

232 INDEX

hemochorial placentae 136 placental structure and blood circulation 136 of substance 136 of toxicants 134 maternal recognition of pregnancy chorionic gonadotropin 15 non-primates 15 pseudopregnancy 15 rats and mice 15 maturation promoting factor (MPF) 9 MDM2 157, 158, 159 mEH see microsomal epoxide hydrolase MEHP see metabolite mono-(2-ethylhexyl) phthalate aromatase gene transcription 88 effect on estradiol 88 PPARγ- and PPARα-mediated effects on gene expression 91–1 menopause 20, 21, 31, 32, 33, 34, 64, 135, 139, 174, 179, 187, 188, 192, 198 environmental factors 21 mesotheliomas 188 metabolic enzymes 117, 120, 123, 124, 125, 126, 127, 128 human ovary 127 mouse ovary 124 metabolite mono-(2-ethylhexyl) phthalate (MEHP) 86 methoxychlor 40, 44, 46, 47, 48, 138 see also ovotoxicity; organochlorine microsomal epoxide hydrolase (mEH) 117, 126 Miller, Kimberley P. 40 Mirex 45, 47, 48, 184 see also organochlorine MPF see maturation promoting factor mural cells 3 mutation 2, 153, 157, 175, 180, 182, 185, 191 Narotsky, Michael G. 95 National Cancer Institute 176 nerve growth factor (NGF) 6, 9, 153 NGF see nerve growth factor NHANES data 184

no observable effect level (NOEL) 76 non-halogenated PAHs AhR ligand 139 benzo(a) pyrene (B(a)P) 139 9,10-dimethyl-1,2-benzanthracene 139 norepinephrine 72, 73, 78, 98 nuclear factor-κB-mediated flip expression and resistance to TNFα 161–61 nucleotide excision repair (NER) pathway 152 occupational chemicals 24, 32 OCU see oral contraceptive use oral contraceptive use (OCU) 174, 185 organochlorine, effects of cell-signalling pathway 48 in cockroaches 46 DDT 45 ducks 46 in fish 46 in hens 45 histopathological changes 48 in human 45 metabolic changes 48 rabbit model 47 in rodents 46 organophosphate insecticides 48 OSE see ovarian surface epithelium ovarian aging see reproductive aging ovarian cancer 31, 44, 127, 135, 149, 151, 153, 155, 157, 158, 160, 161, 162, 163, 174 clomiphene 186 diethylstilbestrol (DES) 187 and exogenous hormones 185 exposure to xenobiotic agents 187 aspirin 188 cigarette smoke 188 heavy metals 187 PAHs 188 paracetamol 189 talcum powder 187 HRT and estrogen replacement therapy 186 results from immunohistochemical studies 157 risk factors 174

INDEX 233

steroid hormone 185 use of oral contraceptives 185 ovarian cancer, risk factors for age 177 ethnicity and ancestry 180 and diet 183 and environmental exposure 183 and obesity 184 and physical or sexual abuse 183 poverty 182 family history 180 BRCA1 180 BRCA2 180 geography 177 reproductive history 178 breastfeeding 179 early menarche 179 late childbirth 178 later age at menopause 179 multiparity 178 multiple births 178 nulliparity 178 tubal ligation and prior hysterectomy 179–7 theoretical approaches to 195 evolutionary approach to health 197 hormone elevation 196 inflammation of ovarian epithelium 196 uninterrupted ovulation 196 ovarian cyst 4 ovarian development and function 9, 175 in humans 135 oocyte atresia 135 rete ovarii 134 in rodents 135 ovarian function 5, 9, 15, 30, 32, 33, 34, 40, 42, 50, 64, 65, 72, 77, 78, 95, 97, 100, 101, 106, 127, 134, 183, 187 pre-ovulatory stage 42 see also atresia; FSH; LH; OSE ovarian hilus 2 ovarian hormones 10, 21, 33, 52, 61, 97 activin 6, 8, 10, 12

estradiol 6, 7, 8, 10, 11, 12, 13, 14, 32, 45, 47, 50, 65, 67, 68, 69, 71, 72, 74, 87, 88, 90, 91, 92, 93, 97, 98, 100, 110, 127, 134, 135, 138, 142, 189, 205, 206, 207, 209, 219 inhibin 6, 8, 9, 10, 11, 12, 14, 21, 45 progesterone 6, 7, 8, 10, 13, 14, 15, 20, 21, 42, 44, 45, 47, 48, 50, 53, 65, 69, 72, 74, 75, 87, 88, 98, 100, 107, 107, 124, 138, 175, 189, 205, 206, 207,209, 211,214,215–16 ovarian malignancies 175 ovarian material, advantages and limitations, type of 213 ovarian metabolism 116, 117, 120, 126, 128 in chemically induced ovotoxicity 128– 8 on toxicity, impact of 128 ovarian surface epithelium (OSE) 40, 44, 176, 196 ovarian toxicity, transplacental induction of 138 ovary anatomy of 3 as a target organ 86, 117 development of the xii morphology 2 ovotoxic agents 26 E. coli 27 in mice 27 monkeys 27 mutagenic carcinogenic effects 27 in vitro 27 ovotoxicity mechanisms of 27 rats and mice 28 in small pre-antral follicles 21 ovulation 3, 4, 9, 10, 11, 12, 16, 17, 20, 23, 42, 44, 46, 47, 52, 54, 61, 63, 64, 65, 66, 67, 70, 71, 72, 73, 75, 78, 86, 87, 90, 92, 93, 101, 135, 138, 138, 141, 185, 186, 187, 190, 195, 196, 213 meiotic maturation 11 GVB 11 Xenopus laevis 11 oxytocin 8, 14

234 INDEX

p, p´-dichlordipehenyl-diciclhorethylene (DDE) 46, 47, 48, 184, 191 PAHs see polychlorinated aromatic hydrocarbons ovarian metabolism in the rat ovary 120–22 see also EDs PAI-1 see plasminogen activator inhibitor type xii paracrine or autocrine effects 5 PCBs see polychlorinated biphenyls PCDDs see polyhalogenated dibenzo-pdioxins per-arnt-sim (PAS) family of proteins 138 perfusion in rat ovary 213 peroxisome proliferator activated receptors (PPARs), role of 88–9 pesticides 40, 138, 187, 190, 194, 218 chlordane 45, 184 endrin 45, 184 heptachlor 45, 47, 47, 184 kepone 45, 46 lindane 45, 46, 47 mirex 45, 47, 48, 184 PGs see prostaglandins phase I enzymes 115, 116, 118, 120 in the rat ovary 118 CYP enzyme 118 phase II enzymes 115, 116, 120 in the rat ovary 120 in vivo 122 PAH exposure 122 transcriptase-polymerase chain reaction 120 photomirex 48 pituitary hormone secretion 97, 100, 101, 110 atrazine effect 100 placental lactogen 107 plasminogen activator inhibitor type 1 (PAI-1) 13 platinum derivatives 149 polyaromatic hydrocarbons (PAHs) 138 polychlorinated biphenyls (PCBs) 61, 64, 139 polycyclic aromatic hydrocarbons (PAHs) 23, 64, 120, 188, 192 biotransformation 23

2-bromopropane 26 1,3-butadiene 24–5 effects of EDs on ovarian function 65– 7 exposure to xenobiotic agents 187 in mice 24 non-halogenated 139 occupational chemicals 24 4-vinylcyclohexene (VCH) 25–6 polyhalogenated dibenzo-p-dioxins (PCDDs) 64 polytetrafluoroethylene (PTFE) or Teflon® tubing 211 polyvinyl chloride (PVC) 86 PPAR pathways in differentiation and metabolism 90 role of MEHP 88 pregnancy 8, 15, 17, 23, 24, 32, 44, 65, 98, 106, 107, 107, 110, 138, 141, 174, 179, 185, 195, 196, 198 atrazine exposure on 107–6 chlorotriazine initiation and maintenance 106–6 maternal recognition of see maternal recognition of pregnancy pregnancy loss 107 atrazine induced 106 progesterone levels in 107 pregnant mare serum gonadotropin (PMSG) 90, 123, 142 primary follicles 10, 18, 20, 21, 24, 28, 30, 42, 53, 110, 120, 125 primary oocytes 2, 3 primary sex cords or cords 2 primordial follicles 2, 10, 17, 18, 20, 21, 21, 23, 24, 26, 27, 29, 30, 32, 33, 42, 42, 64, 76, 77, 78, 79, 82, 122, 128, 129, 135, 138, 139, 142, 143 pro-interleukin-1 β-converting enzyme 157 progesterone 6, 7, 8, 10, 13, 14, 15, 20, 21, 42, 44, 45, 47, 48, 50, 53, 65, 69, 72, 73, 75, 88, 97, 98, 100, 107, 107, 124, 138, 175, 189, 205, 207, 209, 211, 214, 215, 216, 217 secretion from pre-ovulatory follicles 216 prolactin receptors 6

INDEX 235

prostaglandins (PGs) 9, 10, 13, 14, 90, 196 proteins xii, 3, 6, 8, 12, 13, 30, 42, 125, 126, 136, 138, 150, 151, 152, 153, 154, 159, 193, 205, 209 PTFE see polytetrafluoroethylene radiation injury 195 Ras mitogen-activated protein (MAP) 158 real time polymerase chain reaction (rtPCR) 47 receptors and signal transduction 5 relaxin 8, 14 reproductive aging cancer 189 see also xenobiotics and function atrazine effect on 99 chlorotriazines and 106 pregnancy initiation and maintenance 106 Rozman, Karl K. 61 secondary and antral follicles 18 gap junctions 18 secondary follicles/pre-antral follicles 3 secondary sex cord cell 2 SER see smooth endoplasmic reticulum serine-threonine kinases 6 sex steroid hormones, synthesis of the 189, 205 cholesterol 205 side-chain cleavage enzyme 7 Sipes, I. Glenn 115 small luteal cells 4 smooth endoplasmic reticulum (SER) 4, 53 sox3 xii Sprague-Dawley (SD) rats 23, 47, 71, 96, 120, 190, 194 Sry/SRY xii stalk 4 steroid production, assessment of follicular/luteal incubations 214 isolated cells 215 microdialysis 209 organ perfusion/perifusion 211

steroidogenesis, toxicant-induced effects on 218–17 steroidogenic acute regulatory (StAR) protein 7, 205 steroidogenic pathway 7–8 steroids 3, 6, 10, 20, 42, 44, 45, 50, 53, 92, 110, 135, 138, 139, 175, 183, 185,205–16 androgens 4, 6, 7, 18, 20, 45, 51, 54, 69, 88, 207, 218 testosterone 6, 7, 12, 51, 88, 90, 206, 207, 219 estrogens 6, 7, 8, 20, 21, 42, 44, 47, 48, 50, 51, 53, 54, 67, 69, 70, 72, 73, 74, 75, 87, 90, 91, 96, 97, 101, 103, 118, 123, 138, 142, 175, 184, 186, 196 estradiol 6, 7, 8, 10, 11, 12, 13, 32, 45, 47, 50, 64, 65, 68, 69, 70, 71, 73, 74, 86, 87, 88, 90, 91, 92, 93, 98, 100, 110, 134, 135, 138, 138, 142, 189, 205, 206, 207, 209, 219 progestins 6 progesterone 6, 7, 8, 10, 13, 14, 15, 20, 21, 42, 44, 45, 47, 48, 50, 53, 65, 69, 72, 74, 75, 87, 88, 98, 100, 107, 107, 124, 138, 175, 189, 205, 206, 207, 209, 211, 214,215– 16 Stoker, Tammy E. 95 stromal cells 5 Surveillance, Epidemiology and End Results (SEER) 176 Suter, Diane xii Tamoxifen (TAM) 70, 124 see also EDs TCDD see 2,3,7,8-tetrachlorodibenzop-dioxin inhibitory effect of 142 on granulosa cells, in vitro effects of 69 reproductive effects of 141 significant effects on pups 141 transplacental effects 141 Terranova, Paul F. 61

236 INDEX

tertiary follicle 3, 12, 87, 135 tetrazolium dye 218 TGF see transforming growth factor theca interna and the theca externa 4 thecal cell 3, 18 Tilapia leucostica 49 TNFR1 see tumor necrosis factor receptor-1 Tomic, Dragana 40 toxicant exposure, advantages and limitations of 208 toxicity mechanisms 88 transforming growth factor (TGF)α/β 6, 8, 9 trimethylen-emelamin (TEM) 26 tripeptide thiol 152 trophoblast cell inhuman 136 in rodents 136 tumor development 96 age-associated 98 caused by atrazine and cholrotrazine 96–6 leading to mammary gland tumors 97–7 supressor role of 157 tumor necrosis factor (TNF)α 9, 153 tumor necrosis factor receptor-1 (TNFR1) 162 tyrosine kinases 6 UDP-glucuronosyltransferases 120 in utero and lactational (IUL) 141 vascular and neural elements 5 VCH diepoxide (VCD) 25 VCH see 4-vinyl-cyclohexene white spotting locus xii World Health Organization’s International Agency for Research on Cancer (IARC) 194 xenobiotics 115, 126, 128, 174, 175, 187, 195, 197, 207, 208, 215, 218, 219

associated with reproductive cancer asbestos 190 atrazine 190 benzene 190 1,3-butadiene 191 DDT 191 lead 191 mercury 192 methylmercury 192 polycyclic aromatic hydrocarbons 192 talc 193 induced apoptosis 29 metabolism 127 Xenopus laevis 51 Xeroderma pigmentosum group D (XPD) protein 153 Xiaojuan Yan 149 X-linked inhibitor of apoptosis protein (XIAP) and chemoresistance and FAK processing 158 and focal adhesion kinase (FAK) processing 158 direct inhibition of caspases 154 role in determining chemosenstivity 157 role in modulation of p53 and MDM2 157 yolk sac 2, 134 zona pellucida 3, 8, 11, 18 zona reaction 8