This document “Revised Draft Detailed Review Paper on Aromatase” has been prepared for Dr. Gary E. Timm, Work Assignment Manager, U.S. Environmental Protection Agency, Endocrine Disruptor Screening Program, Washington, D.C. 20004, by BATTELLE, 505 King Avenue, Columbus, Ohio 43201, February 25 2002. EPA contract number 68-W-01-023, work assignment 2-7.

2 REVISED DRAFT DETAILED REVIEW PAPER

ON

AROMATASE

EPA CONTRACT NUMBER 68-W-01-023 WORK ASSIGNMENT 2-7

February 25, 2002

PREPARED FOR

GARY E. TIMM WORK ASSIGNMENT MANAGER U.S. ENVIRONMENTAL PROTECTION AGENCY ENDOCRINE DISRUPTOR SCREENING PROGRAM WASHINGTON, D.C.

BATTELLE 505 KING AVENUE COLUMBUS, OH 43201 TABLE OF CONTENTS Page

EXECUTIVE SUMMARY ...... 1

2.0 INTRODUCTION ...... 3 2.1 DEVELOPING AND IMPLEMENTING THE ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP) ...... 3 2.2 THE VALIDATION PROCESS ...... 3 2.3 PURPOSE OF THE REVIEW ON THE AROMATASE ASSAY ...... 5 2.4 METHODOLOGY USED IN THE ANALYSIS ...... 5 2.4.1 Literature Search ...... 5 2.4.2 Databases Searched ...... 5 2.4.3 Methods and Sources for Acquiring Additional Information ...... 6 2.5 DEFINITIONS (E.G., METHOD, PROCEDURE, TECHNIQUE, PROTOCOL) ...... 7

3.0 BACKGROUND ON AROMATASE ...... 9 3.1 ENDOCRINOLOGY OF ESTROGENS AND ROLE OF AROMATASE ...... 9 2.1.1 Female Sex Hormones ...... 9 2.1.2 Female Reproductive Cycle ...... 9 2.1.3 Steroidogenesis ...... 10 2.1.4 Estrogen Action ...... 13 3.2 AROMATASE - GENERAL DESCRIPTION ...... 14 2.2.1 Biochemistry and Molecular Biology ...... 14 2.2.2 Aromatase in Other Species ...... 17 2.2.3 Significance of Alterations of Aromatase ...... 17

4.0 SCIENTIFIC BASIS OF THE AROMATASE ASSAY ...... 19 4.1 METHODS FOR MEASURING AROMATASE ACTIVITY ...... 19 4.2 IN VITRO AROMATASE ASSAYS TO DETECT ENDOCRINE DISRUPTORS ...... 20 4.2.1 Human Placental Microsomal Aromatase Assay ...... 20 4.2.2 Human Cell Culture Systems for Measuring Aromatase Activity ...... 22 4.2.3 Other In Vitro Mammalian Cell Systems for Measuring Aromatase Activity ...... 24 4.3 IN VIVO STUDIES OF AROMATASE TO DETECT ENDOCRINE DISRUPTORS .... 24 4.4 QSAR APPROACHES TO PREDICTING AROMATASE INHIBITION ...... 27 4.5 ADDITIONAL INFORMATION FROM INTERVIEWEES ...... 28 4.6 SUMMARY OF DOCUMENTS REVIEWED AND CONTACT INFORMATION FOR INVESTIGATORS ...... 29

5.0 CANDIDATE PROTOCOLS FOR AN IN VITRO ASSAY ...... 30 5.1 CANDIDATE ASSAY A - PLACENTAL MICROSOMAL AROMATASE ...... 31 5.1.1 Flow Diagram of the Human Placental Microsomal Aromatase Assay .... 31 5.1.2 Description of the Placental Microsomal Aromatase Assay ...... 32 5.1.3 Strengths of Placental Microsomal Aromatase Assay ...... 35 5.1.4 Weaknesses And/or Limitations of Placental Microsomal Aromatase Assay ...... 35 5.1.5 Test Method Performance and Test Method Reliability ...... 36

Battelle Draft i February 25, 2002 5.2 CANDIDATE ASSAY B - AROMATASE ACTIVITY IN CELL LINES ...... 38 5.2.1 Flow Diagram of the Aromatase Assay in JEG-3 Cells ...... 38 5.2.2 Description of the Aromatase Assay in Cell Lines ...... 39 5.2.3 Strengths of the Aromatase Assay in Cell Lines ...... 41 5.2.4 Weaknesses And/or Limitations of the Aromatase Assay in Cell Lines . . . 42 5.2.5 Test Method Performance and Test Method Reliability ...... 43

6.0 IMPLEMENTATION CONSIDERATIONS ...... 45 6.1 ESTABLISHMENT OF ASSAY ...... 45 6.2 COST/TIME REQUIRED ...... 45 6.3 ANIMAL WELFARE CONSIDERATIONS ...... 46 6.4 HUMAN SUBJECTS CONSIDERATIONS ...... 46 6.5 ASSURANCE OF BIOLOGICAL TEST (IN VITRO MODEL) VIGOR AND STABILITY ...... 46

7.0 RECOMMENDED PROTOCOL ...... 47

8.0 DEVELOPMENTAL STATUS OF THE ASSAY ...... 49 8.1 CURRENT STATUS ...... 49 8.2 RECOMMENDATION FOR PRE-VALIDATION STUDIES ...... 49 8.3 RECOMMENDATION FOR VALIDATION STUDIES ...... 49

9.0 REFERENCES ...... 50

APPENDIX A MANUSCRIPTS REVIEWED (IN ALPHABETICAL ORDER) ...... A-1

APPENDIX B INVESTIGATORS CONTACTED AND QUESTIONNAIRE ...... B-1

LIST OF FIGURES

Figure 1. Trophic Hormones and Steroidogenesis ...... 11 Figure 2. Biosynthesis of Sex Hormones ...... 12 Figure 3. General Mechanism of Steroid Hormone Action ...... 13 Figure 4. Reaction Mechanism for Estrogen Biosynthesis by Aromatase ...... 15

Figure 5. IC50 Values for Selected ...... 22

Figure 6. IC50 Values for Selected ...... 23

LIST OF TABLES

Table 1. Comparison of Aromatase Inhibition in In Vitro Assays ...... 30

Table 2. Comparison of IC50 Values for Flavonoids ...... 37 Table 3. Comparison of the Two Candidate Assays ...... 43

Battelle Draft ii February 25, 2002 1 DETAILED REVIEW PAPER 2 AROMATASE AND ENDOCRINE DISRUPTORS 3 4 1.0 EXECUTIVE SUMMARY 5 6 The Food Quality Protection Act of 1996 was enacted by Congress to authorize the EPA 7 to implement a screening program on pesticides and other chemicals found in food or water 8 sources for endocrine effects in humans. Thus, the United States Environmental Protection 9 Agency (U.S. EPA) is implementing an Endocrine Disruptor Screening Program (EDSP). In this 10 program, comprehensive toxicological and ecotoxicological screens and tests are being 11 developed for identifying and characterizing the endocrine effects of various environmental 12 contaminants, industrial chemicals, and pesticides. The program’s aim is to develop a two-tiered 13 approach, e.g., a combination of in vitro and in vivo mammalian and ecotoxicological screens 14 (Tier 1) and a set of in vivo tests (Tier 2) for identifying and characterizing endocrine effects of 15 pesticides, industrial chemicals, and environmental contaminants. Validation of the individual 16 screens and tests is required, and the Endocrine Disruptor Methods Validation Subcommittee 17 (EDMVS) will provide advice and counsel on the validation assays. 18 19 Estrogens are sex steroid hormones that are necessary for female reproduction and affect 20 the development of secondary sex characteristics of females. Estrogens are biosynthesized from 21 cholesterol by a series of enzymatic steps, with the last step involving the conversion of 22 androgens into estrogens by the enzyme aromatase. Estrogen biosynthesis occurs primarily in 23 the ovary in mature, premenopausal women. During pregnancy, the placenta is the main source 24 of estrogen biosynthesis and pathways for production change. Small amounts of these hormones 25 are also synthesized by the testes in the male and by the adrenal cortex, the hypothalamus, and 26 the anterior pituitary in both sexes. The major source of estrogens in both postmenopausal 27 women and men occurs in extraglandular sites, particularly in adipose tissue. One potential 28 endocrine target for environmental chemicals is the enzyme aromatase, which catalyzes the 29 biosynthesis of estrogens. An aromatase assay is proposed as one of the Tier 1 Screening Battery 30 Alternate Methods. A detailed literature review on aromatase was performed and encompassed 31 (1) searching the literature databases, (2) contacting individuals to obtain information on 32 unpublished research, and (3) an evaluation of the literature and personal communications. 33 34 Aromatase is a cytochrome P450 enzyme complex responsible for estrogen biosynthesis 35 and converts androgens, such as and , into the estrogens 36 and . Aromatase is present in the ovary, placenta, uterus, testis, brain, and extraglandular

37 adipose tissues. Two proteins, cytochrome P450arom and NADPH-cytochrome P450 reductase, 38 are necessary for enzymatic activity, and the enzyme complex is localized in the smooth 39 endoplasmic reticulum. The aromatase gene, designated CYP19, encodes the cytochrome

40 P450arom and consists of 10 exons, with the exact size of the gene exceeding 70 kilobases. 41 Aromatase is found in breast tissue, and the importance of intratumoral aromatase and local 42 estrogen production is being unraveled. Effective aromatase inhibitors have been developed as 43 therapeutic agents for estrogen-dependent breast cancer to reduce the growth stimulatory effects

Battelle Draft 1 February 25, 2002 1 of estrogens in breast cancer. Investigations on the development of aromatase inhibitors began 2 in the 1970's and have expanded greatly in the past three decades. 3 4 An in vitro aromatase assay could easily be utilized as an alternative screening method in 5 the Tier 1 Screening Battery to assess the potential effects of various environmental toxicants on 6 aromatase activity. Both in vitro subcellular (microsomal) assays and cell-based assays are 7 available for measuring aromatase activity. The in vitro subcellular assay using human placental 8 microsomes, is commonly used to evaluate the ability of pharmaceuticals and environmental 9 chemicals to inhibit aromatase activity. In addition, human JEG-3 and JAR choriocarcinoma cell 10 culture lines, originally isolated from cytotrophoblasts of malignant placental tissues, have been 11 used as in vitro systems for measuring the effects of compounds on aromatase activity. These cell 12 lines are also utilized for investigations on the effects of agents in placental toxicology. 13 14 Numerous flavonoids and related phytoestrogen derivatives have been extensively 15 evaluated for their ability to inhibit aromatase activity for two primary reasons: (1) these plant 16 natural products can serve as possible leads for the development of new aromatase 17 inhibitors; and (2) humans and other animals are exposed to these agents through the diet. In

18 general, the flavonoids and related analogs demonstrate aromatase inhibition with IC50 values in 19 the micromolar range; however, these compounds lack both the potency and specificity of 20 aromatase inhibitors developed for breast cancer therapy. Several pesticides have also 21 demonstrated inhibition of aromatase activity in the human placental microsomal assay system,

22 with IC50 values for aromatase inhibition ranging from 0.04 µM to greater than 50 µM. 23 24 The human placental microsomal aromatase assay is a straight forward, inexpensive, and 25 rapid in vitro assay for measuring the effects of chemicals on aromatase activity. Aromatase 26 inhibition of endocrine disruptors have also been evaluated in intact cell systems such as the 27 JEG-3 or JAR choriocarcinoma cell culture lines. In general, the inhibitory effects on the 28 aromatase activity are similar between the two in vitro assays for a wide variety of compounds. 29 Advantages of a cell culture assay are that it can provide information on membrane transport 30 abilities of the test agent, and the assay can provide information on the effect of the test agent on 31 the expression of the aromatase protein, i.e., if the test compound will induce or suppress the 32 production of CYP 19 mRNA and/or new aromatase protein. 33 34 The human placental microsomal aromatase assay is recommended as the in vitro 35 aromatase screening assay to be included in the Tier 1 Screening Battery Alternate Methods. 36 This assay will detect environmental toxicants that possess the ability to inhibit aromatase 37 activity. The overall protocol for the assay is straightforward and uses a radiometric assay 38 measuring the release of tritiated water from the substrate [1β-3H]-androstenedione. A 39 preliminary screening assay can be performed at concentrations of environmental toxicant at 0.1, 40 1.0, and 10.0 µM in quadruplicate. For compounds exhibiting 10% inhibitory activity or more, a 41 full dose-response studies will be performed using concentrations ranging from 1.0 nM (1.0 x 10­ 42 9 M) to 1.0 mM (1.0 x 10-3 M). 43

Battelle Draft 2 February 25, 2002 1 2.0 INTRODUCTION 2 3 2.1 DEVELOPING AND IMPLEMENTING THE ENDOCRINE DISRUPTOR SCREENING 4 PROGRAM (EDSP) 5 6 In 1996, the passage of the two laws, the Food Quality Protection Act (FQPA) and 7 Amendments to the Safe Drinking Water Act (SDWA) mandated the United States 8 Environmental Protection Agency (U.S. EPA) to screen chemicals found in drinking water 9 sources or food to determine whether they possess estrogenic or other endocrine activity (Federal 10 Register, 2001). Pursuant to this goal, the U. S. EPA is required to “develop a screening 11 program, using appropriate validated test systems and other scientifically relevant information, 12 to determine whether certain substances may have an effect in humans that is similar to an effect 13 produced by a naturally occurring estrogen, or other such endocrine effect...” (Federal Register, 14 2001). The U.S. EPA established the Endocrine Disruptor Screening and Testing Advisory 15 Committee (EDSTAC), to provide recommendations regarding a strategy for developing a 16 testing paradigm for compounds that may have activities similar to naturally-occurring 17 hormones. Following the recommendations made by EDSTAC in its final report (EDSTAC, 18 1998), the EPA established the Endocrine Disruptor Screening Program (EDSP). The program’s 19 aim is to develop a two-tiered approach, e.g., a combination of in vitro and in vivo mammalian 20 and ecotoxicological screens (Tier 1) and a set of in vivo tests (Tier 2) for identifying and 21 characterizing endocrine effects of pesticides, industrial chemicals, and environmental 22 contaminants. To date, the U.S. EPA has implemented the program on two fronts: 1) the 23 development of the Endocrine Disruptor Priority Setting Database, and the approach that will be 24 used to establish priorities for screening compounds, and 2) prevalidation and validation studies 25 of some of the Tier1 and Tier 2 assays that are likely to be included in the testing battery. The 26 Endocrine Disruptor Methods Validation Subcommittee (EDMVS) has been set up to advise and 27 review new and ongoing work in the validation of these assays. 28 29 2.2 THE VALIDATION PROCESS 30 31 The U.S. EPA (and EDMVS) has chosen to follow the validation process established by 32 the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), 33 of which the U.S. EPA was a charter member, for validation of the EDSP screening and testing 34 methods. ICCVAM was established by the National Institute of Environmental Health Sciences 35 as a standing interagency committee to aid in the validation, acceptance, and harmonization of 36 test methods designed to reduce animals use, refine procedures involving animals to make them 37 less stressful, and to replace animal tests wherever appropriate (Federal Register, 2001). To this 38 end, ICCVAM defined a flexible, adaptatable framework for test method validation that was 39 applicable to conventional and alternate methods, and could be applied to the needs of different 40 agencies and regulatory processes. The purpose of validation is to establish the reliability and 41 relevance of a test method with respect to a specific use. The process is science-driven, and 42 addresses the scientific principles of objectivity and experimental design (NIEHS, 1997). In 43 addition, as stated in the ICCVAM report “A test is considered validated when its performance 44 characteristics, advantages, and limitations have been adequately determined for a specific

Battelle Draft 3 February 25, 2002 1 purpose.” (NIEHS, 1997). The framework for the validation process by ICCVAM (NIEHS, 2 1997) is outlined below. 3 4 ICCVAM Test Method Validation Process 5 6 I. Test Development IV. Test Validation 7 8 II. Prevalidation/Test Optimization A. Form steering committee/ management team 9 Define purpose of validation study 10 A. Preliminary planning Design study 11 1. Define basis and purpose of test Select participating laboratories 12 2. Develop protocol Establish management evaluation and oversight 13 3. Develop control values procedures 14 4. Develop data/outcome prediction model 15 B. Pretest procedures 16 B. Activities Implement data recordkeeping procedures 17 Select reference chemicals 18 1. Qualify and train laboratories Code and distribute reference chemicals 19 2. Measure intra- and interlaboratory reproducibility 20 3. Identify limitations of test C. Test coded chemicals 21 Measure interlaboratory performance 22 III. Determine Readiness for Validation Compile and evaluate data 23 24 A. Analyze test development and prevalidation data D. Evaluate test 25 Analyze and summarize test results 26 B. Standardize protocol Challenge data with prediction model 27 Peer review of protocol and data 28 Accept, revise, or reject model V. Submission of Test for Regulatory Approval

A. Prepare report

B. Make supporting data available

C. Prepare results for publication.

29 30 A test is considered validated when its performance characteristics, advantages, and 31 limitations have been adequately determined for a specific purpose. The measurement of a test’s 32 reliability and relevance are independent stages in the validation of a test method, and both are 33 required. Reliability is an objective measure of a method’s intra- and interlaboratory 34 reproducibility. If the test is not sufficiently reliable, it cannot be used for its intended purpose. 35 Alternatively, if the test is not relevant, or of questionable relevance to the biological effect of 36 interest, or if it is not an appropriate measure of the effect, its reliability is academic. The 37 relevance of a test may be linked to the mechanism of the toxic effect it measures and to its 38 proposed uses (NIEHS, 1997). The studies conducted will be used to develop, standardize, and 39 validate methods, prepare appropriate documents for peer review of the methods, and develop 40 technical guidance and test guidelines in support of EDSP. 41 42

Battelle Draft 4 February 25, 2002 1 2.3 PURPOSE OF THE REVIEW ON THE AROMATASE ASSAY 2 3 One potential endocrine target for environmental chemicals is the enzyme aromatase. An 4 aromatase assay is proposed as one of the Tier 1 Screening Battery Alternate Methods. If the 20­ 5 day pubertal female assay is not selected as a primary Tier 1 Screening Method, the 20-day male 6 pubertal assay will be used as an alternative. The aromatase assay would be used to complement 7 the male pubertal assay. An in vitro aromatase assay could easily be utilized to assess the effects 8 of various environmental toxicants on aromatase activity. Environmental chemicals and various 9 natural products can inhibit aromatase activity through a direct alteration of the catalytic activity 10 of the enzyme, resulting in a rapid decrease in the levels of estrogens. In other cases, 11 environmental chemicals have been reported to influence estrogen synthesis through a 12 suppression/induction of aromatase protein levels via alterations in CYP19 mRNA or aromatase 13 protein synthesis. 14 15 The purpose of Work Assignment 1 – 7 is to prepare a Detailed Review Paper (DRP) to 16 survey and investigate the status of in vitro assay methodologies based upon human placental 17 aromatase. 18 19 2.4 METHODOLOGY USED IN THE ANALYSIS 20 21 2.4.1 Literature Search 22 23 The literature review encompassed (1) searching the literature databases, (2) contacting 24 individuals to obtain additional information, and (3) an evaluation of the literature and personal 25 communications. 26 27 2.4.2 Databases Searched 28 29 The following databases were searched: 30 31 Database Literature coverage 32 MedLine / PubMed biomedical literature 33 Biological Abstracts biological literature 34 Chemical Abstracts chemical literature and patents 35 Toxline toxicological literature 36 DART developmental & reproductive toxicology 37 CancerLit cancer literature and abstracts 38 IPA pharmaceutical literature 39 40 The search strategies involved keyword searching and author searching strategies. The 41 words or phrases selected are those keywords that are indexed by the database, and the search 42 strategies of the databases utilize “expanded” or “exploded” searches with the indexed 43 keywords. Each database was first searched using the keyword aromatase to identify all 44 references for aromatase (for example, 3899 references on aromatase retrieved in MedLine using

Battelle Draft 5 February 25, 2002 1 PubMed). Then, this subset of references on aromatase was searched individually by using the 2 Boolean operator “and” with the other keywords or phrases. The other keywords or phrases 3 utilized were: 4 5 endocrine disruptors, flavonoids, isoflavonoids, phytoestrogens, dioxins, 6 herbicides, pesticides, PAHs (or polycyclic aromatic hydrocarbons), 7 PCBs (or polychlorinated biphenyls) 8 9 The retrieved searches were then downloaded electronically either as ASCII files or 10 downloaded directly into Reference Manager for Windows, Version 9 (ISI ResearchSoft, 11 Berkeley, CA). Initially, each database search, e.g., “aromatase and [other keyword],” were 12 stored as individual database files. 13 14 Each individual search on a literature database was then combined into a Reference 15 Manager file designated by the name of the particular literature database. For example, each of 16 the individual searches on a PubMed (MedLine) database was first combined into a Reference 17 Manager file designated pubmed search.rmd and pubmed search.rmx. Duplicate references were 18 removed by the software program. The literature databases were then combined into a single, 19 large database designated combined searches.rmd and combined searches.rmx. The software 20 program will not recognize accurately duplicates from different literature databases (due to 21 different formatting of the database providers), so duplicate references appear in this single 22 combined file. These duplicates were sorted manually. The final result was a combined 23 database of over 400 references. 24 25 2.4.3 Methods and Sources for Acquiring Additional Information 26 27 Additional information can include research presented at recent meetings and research 28 currently on-going in various research laboratories. Information on the research presented at 29 meetings may be available through the abstracts (if published), and some abstracts are available 30 in certain electronic databases. In the latter case, these presentations and abstracts were retrieved 31 in the database searches. Presentations and abstracts not available in public databases can be 32 retrieved electronically by attendees or by members of the organization sponsoring the meeting 33 (e.g., American Association for Cancer Research, Endocrine Society, American Chemical 34 Society) or can be examined manually. Abstracts from recent meetings of American Association 35 for Cancer Research, Endocrine Society, and American Chemical Society were reviewed and 36 abstracts identified. 37 38 Research currently on-going in various research laboratories can only be obtained by 39 contacting the researchers or laboratory directors. Such individuals include researchers and/or 40 authors in the field of aromatase pharmacology and toxicology identified from relevant 41 publications. Other scientists involved in similar work can also be identified based upon 42 examination of the participants of relevant symposiums, panels, and committees. At best, 43 clarification of study design, interpretation, and data analysis of studies may be obtained through 44 interviews with these scientists. It is not likely that detailed methodologies and results will be

Battelle Draft 6 February 25, 2002 1 obtained through this interview process, due to confidentiality issues with both scientists in 2 commercial firms and with academic researchers. 3 Similar methods, sources, and approaches were used to identify and acquire unpublished 4 material and to identify and interview principal investigators in the field. Investigators were 5 selected based upon the number and the quality of recent relevant publications (study design, 6 quality of methods, chemicals tested, data analysis) and the individuals reputation 7 (national/international reputation in area, author of reviews and/or critical documents). The 8 following individuals were contacted and provided responses. 9 10 Investigator Expertise 11 Shiuan Chen, Ph.D. Biochemistry and molecular biology of aromatase; 12 Division of Immunology structure-function studies; aromatase inhibition by 13 Beckman Research Institute, City of Hope flavonoids 14 1450 E. Duarte Road 15 Duarte, CA 91006, U.S.A. 16 E-mail: [email protected] 17 Rajeshwar R. Tekmal, Ph.D. Molecular biology of aromatase; development of 18 Department of Ob/Gyn transgenic mice expressing aromatase 19 Emory University School of Medicine 20 4219 WMB, 1639 Pierce Drive 21 Atlanta, GA 30322, U.S.A. 22 E-mail: [email protected] 23 Edwin D. Lephart, Ph.D. Brain aromatase; flavonoids and aromatase 24 633 WIDB, Neuroscience Center 25 Brigham Young University 26 Provo, UT 84602, U.S.A. 27 E-mail: [email protected] 28 Sari Mäkelä, Ph.D. and Risto Santti, Ph.D. Biochemistry and pharmacology of aromatase; 29 University of Turku aromatase inhibition by flavonoids; endocrine 30 Institute of Biomedicine and Medicity Research disruptors 31 Laboratory 32 Turku, FINLAND 33 E-mail: [email protected] 34 35 36 The individuals were contacted by phone or e-mail. The title and objectives/goals of this 37 particular work assignment and a brief description of the two study designs under consideration 38 (human placental microsomal aromatase assay, human choriocarcinoma cell culture assay) were 39 presented . The interview questions and responses are presented in Appendix B. 40 41 2.5 DEFINITIONS 42 43 The definitions of terms that will be utilized throughout this DRP are presented here: 44 45 46 placental aromatase aromatase activity present in the placenta 47 ovarian aromatase aromatase activity present in the ovary

Battelle Draft 7 February 25, 2002 1 brain aromatase aromatase activity present in the brain 2 microsomes aromatase subcellular fraction consisting of the endoplasmic reticulum of the cell; isolated following cell fractionation and differential centrifugation methods

3 cytochrome P450arom the heme protein in the aromatase complex 4 NADPH-cytochrome P450 reductase the protein in the aromatase complex that provides reducing equivalents for the enzyme reaction 5 aromatase protein complex two protein complex in the endoplasmic reticulum that consists of the

cytochrome P450arom and the NADPH-cytochrome P450 reductase

6 CYP19 the gene expressing the cytochrome P450arom protein 3 7 aromatase radiometric assay (also aromatase enzyme assay that measures the release of H2O from 3 3 8 referred to as H2O assay) [1$- H]-androstenedione 9 aromatase product isolation assay aromatase enzyme assay that measures the formation of estrogen products from androgen precursors

10 JEG-3 cell a transformed human choriocarcinoma cell line, ATCC no. HTB-36 11 JAR cell a transformed human choriocarcinoma cell line, ATCC no. HTB-144 12 13 14

Battelle Draft 8 February 25, 2002 1 3.0 BACKGROUND ON AROMATASE 2 3 3.1 ENDOCRINOLOGY OF ESTROGENS AND ROLE OF AROMATASE 4 5 3.1.1 Female Sex Hormones 6 7 Estrogens are sex steroid hormones that are necessary for female reproduction and affect 8 the development of secondary sex characteristics of females. The other principal class of female 9 sex steroid hormones is progestins, which are also essential for female reproduction. Chemically,

10 the naturally occurring estrogens are C18 and have in common an unsaturated A ring 11 (which is planar) with a resulting phenolic function in the 3 position that aids in separation and 12 purification from nonphenolic substances. The most potent endogenous estrogen is estradiol.

13 The naturally occurring progestins are C21 steroids and have in common a 3-keto-4-ene structure 14 in the A ring and a ketone at the C-21 position. The most potent endogenous progestin is 15 progesterone. A brief summary of estrogens, the reproductive cycle, steroidogenesis and 16 hormone action are presented here. More detailed information can be obtained from recent 17 treatise.1-5 18 19 OH O 20 21 22 23 HO O 24 25 Estradiol Progesterone 26 27 28 3.1.2 Female Reproductive Cycle 29 30 Among the key events in the female reproductive process is ovulation, which is regulated 31 by the endocrine and central nervous systems.3 In humans, sexual maturation, or the period in 32 which cyclic menstrual bleeding begins to occur, is reached between the ages of 10 and 17; the 33 average age is 13. The period of irregular menstrual cycles before the cessation of menses 34 (usually between the ages of 40 and 50) is commonly known as menopause. 35 36 The female reproductive cycle in humans and other primates is controlled through an 37 integrated system involving the hypothalamus, pituitary gland, ovary, and reproductive tract. The 38 hypothalamus exerts its action on the pituitary gland through a decapeptide called luteinizing 39 hormone-releasing hormone (LHRH), also referred to as gonadotropin-releasing hormone 40 (GnRH), which is released by the hypothalamus and initiates the release of gonadotropins, 41 follicle stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary. The two 42 main gonadotropins, FSH and LH, regulate the ovary and its production of sex hormones. As the 43 name implies, FSH promotes the initial development of the immature graafian follicle in the 44 ovary. This hormone cannot induce ovulation but must work in conjunction with LH. The

Battelle Draft 9 February 25, 2002 1 combined effect is to promote follicle growth and increase the biosynthesis and secretion of 2 estrogens. Through negative feedback on the brain and pituitary, estrogens inhibit production of 3 FSH and stimulate output of LH. The level of LH rises to a sharp peak at midpoint in the 4 menstrual cycle and acts on the mature follicle to bring about ovulation; LH levels are low 5 during the menses. In contrast, FSH reaches its high level during menses, falls to a low level 6 during and after ovulation, and then increases again toward the onset of menses. Once ovulation 7 has taken place, LH induces luteinization of the ruptured follicle, which leads to corpus luteum 8 formation. After luteinization has been initiated, there is an increase in progesterone from the 9 developing corpus luteum, which in turn suppresses production of LH. Once the corpus luteum is 10 complete, it begins to degenerate toward menses, and the levels of progesterone and estrogen 11 decline. 12 13 The endometrium, a component of the genital tract, passes through different phases, 14 which depend on the steroid hormones secreted by the ovary. During the development of the 15 follicle, which takes approximately 10 days and is referred to as the follicular phase, the 16 endometrium undergoes proliferation owing to estrogenic stimulation. The luteal phase follows 17 ovulation, lasts about 14 days, and ends at menses. During the luteal phase, the endometrium 18 shows secretory activity, and cell proliferation declines. In the absence of pregnancy, the levels 19 of estrogen and progesterone decline; this leads to sloughing of the endometrium. This, together 20 with the flow of interstitial blood through the vagina, is called menses and lasts for 4 to 6 days. 21 Because the estrogen and progesterone levels are now low, the hypothalamus releases more 22 LHRH, and the cycle begins again. The reproductive cycle in the female extends from the onset 23 of menses to the next period of menses, with a regular interval varying from 20 to 35 days; the 24 average length is 28 days. 25 26 If pregnancy occurs, the menstrual cycle is interrupted because of the release of a fourth 27 gonadotropin. In human pregnancy, the gonadotropin produced by the placenta is referred to as 28 human chorionic gonadotropin (hCG). hCG maintains and prolongs the life of the corpus luteum. 29 The hCG level in the urine rises to the point where it can be detected after 14 days and reaches a 30 maximum around the seventh week of pregnancy. After this peak, the hCG concentration falls to 31 a constant level, which is maintained throughout pregnancy. The corpus luteum, because of 32 HCG stimulation, provides an adequate level of estrogens and progestins to maintain pregnancy 33 during the first nine weeks. After this period, the placenta can secrete the required level of 34 estrogen and progestational hormones to maintain pregnancy. The levels of estrogen and 35 progesterone increase during pregnancy and finally reach their maximal concentrations a few 36 days before parturition. Because the level of HCG in the urine rises rapidly after conception, it 37 has served as the basis for many pregnancy tests. 38 39 3.1.3 Steroidogenesis 40 41 Estrogens are biosynthesized from cholesterol, primarily in the ovary in mature, 42 premenopausal women.1 During pregnancy, the placenta is the main source of estrogen 43 biosynthesis and pathways for production change. Small amounts of these hormones are also 44 synthesized by the testes in the male and by the adrenal cortex, the hypothalamus, and the

Battelle Draft 10 February 25, 2002 1 anterior pituitary in both sexes. The major source of estrogens in both postmenopausal women 2 and men occurs in extraglandular sites, particularly in adipose tissue. 3 4 In endocrine tissues, cholesterol is the steroid that is stored and is converted to estrogen, 5 progesterone, or androgen when the tissue is stimulated by a gonadotropic hormone. The major 6 pathways for the biosynthesis of sex steroid hormones are summarized in Figure 1. In the ovary, 7 FSH acts on the preovulatory follicle to stimulate the biosynthesis of estrogens. The thecal cells 8 of the preovulatory follicle convert cholesterol into androgens, whereas the granulosa cells 9 convert androgens to estrogens. After ovulation, LH acts on the corpus luteum to stimulate both 10 estrogen and progesterone biosynthesis and secretion. 11

trophic hormone plasma membrane

γ β α α AC ATP GDP GTP GTP GDP

mitochrondia cAMP + PPi (+) P450 scc StAR protein kinase A pregnenolone (+) lipid droplet cholesterol esterase cholesterol cholesterol esters

other steroids endoplasmic reticulum 12 13 Figure 1. Trophic Hormones and Steroidogenesis 14 15 Cholesterol is converted by side chain cleavage into pregnenolone, which can be 16 converted to progesterone or by several steps to the ring A aromatic system found in estrogens 17 (Figure 2). Pregnenolone is converted by 17α-hydroxylase to 17α-hydroxypregnenolone. The

18 next biosynthetic step involves the cleavage of the C17-C20 carbon-carbon bond and is referred to 19 as the 17-20-lyase reaction. This step is catalyzed by the same cytochrome P450 enzyme 20 complex, 17α-hydroxylase. The 17α-hydroxylase is found in the endoplasmic reticulum 21 membrane and is comprised of the cytochrome P45017α protein and the ubiquitous NADPH- 22 cytochrome P450 reductase. Cytochrome P45017α is expressed by the gene CYP17 found on 23 chromosome 10 in humans. Loss of the 17β-acetyl side chain of 17α-hydroxypregnenolone

24 affords the C19 intermediate (3β-hydroxyandrost-5-en-17-one, DHEA). 25 DHEA is converted by 5-ene-3β-hydroxysteroid dehydrogenase/3-oxosteroid-4,5-isomerase to

Battelle Draft 11 February 25, 2002 1 the 17-ketosteroid, androstenedione (androst-4-ene-3,17-dione). This compound is 2 interconvertible with the reduced 17β-hydroxy intermediate (testosterone) by 17β-hydroxy­ 3 steroid dehydrogenase. Loss of the C-19 angular methyl group and aromatization of the A ring of 4 testosterone or androstenedione results in 17β-estradiol or estrone, respectively. 17β-Estradiol

O

a HO HO cholesterol pregnenolone b O c O OH

O HO progesterone 17α -hydroxypregnenolone

c O d O OH

O HO 17 α -hydroxyprogesterone dehydroepiandrosterone

b d O e OH

O HO androstenedione e f b O OH

HO O estrone testosterone e f OH

HO estradiol

Figure 2. Biosynthesis of Sex Steroid Hormones

Enzyme activities: (a) side chain cleavage, (b) 3β-hydroxysteroid 5 dehydrogenase/4,5-isomerase, (c) 17α-hydroxylase, (d) 17,20-lyase, (e) 17β-hydroxysteroid dehydrogenase, (f) aromatase.

Battelle Draft 12 February 25, 2002 1 and estrone are metabolically interconvertible, catalyzed by estradiol dehydrogenase. 2 3 4 3.1.4 Estrogen Action 5 6 The biochemical mechanism of estrogen action is the regulation of gene expression and 7 subsequent induction of protein biosynthesis via specific, high affinity estrogen receptors,2 as 8 illustrated in Figure 3. These receptors are soluble intracellular proteins that can both bind 9 steroid ligands with high affinity and act as ligand-dependent transcriptional factors via 10 interaction with specific deoxyribonucleic acid (DNA) sites and other proteins associated with 11 the chromatin. Receptor binding sites for estradiol are located in the nucleus of target cells and -11 -10 12 exhibit both high affinity (KD = 10 to 10 M) and high specificity. When estradiol binds to the 13 , a conformational change of the estradiol-receptor complex occurs and steroid- 14 receptor complex undergoes homodimerization. The steroid-receptor homodimers interact with 15 particular regions of the cellular DNA, referred to as hormone-responsive elements (HRE) or 16 estrogen responsive elements (EREs) present in the promoter region of responsive genes. 17 Binding of the nuclear steroid-receptor complexes to DNA and interaction with various nuclear 18 transcriptional factors initiate the transcription of the gene to produce messenger ribonucleic acid 19 (mRNA). The elevated mRNA levels result in increased protein synthesis in the endoplasmic 20 reticulum. These proteins include enzymes, receptors, and secreted factors that subsequently 21 result in the steroid hormonal response regulating cell function, growth, differentiation and 22 playing central roles in normal physiological processes, as well as in many important diseases. 23 Initially, a single protein termed the estrogen receptor was believed to be responsible for all the 24 actions of estrogens. Recently, a second estrogen receptor has been identified, and the two 25 estrogen receptors are now referred to as ERα (representing the classical ER, the first ER 26 studied) and ERβ (the second ER identified). The two receptors differ in size and tissue 27 distribution. The predominant estrogen receptor in the female reproductive tract and mammary 28 glands is ERα, whereas ERβ is the primary estrogen receptor in vascular endothelial cells, bone, 29 and male prostate tissues. Estradiol has similar affinities for both ERα and ERβ, whereas certain 30 nonsteroidal estrogenic compounds and antiestrogens have differing affinities between ERα and 31 ERβ, with slightly higher affinities for ERβ.

Steroid (S)

S S S R + R R mRNA

S HRE R

hormonal proteins response

autocrine & paracrine effects 32

Battelle Draft 13 February 25, 2002 1 2 Figure 3. General Mechanism of Steroid Hormone Action 3 4 Estrogens act on many tissues in the female, such as those of the reproductive tract, 5 breast, and CNS. In the female, the primary physiological action of estrogens 6 is to stimulate the development of secondary sex tissues. The growth and development of tissues 7 in the reproductive tract of animals, in terms of actual weight gained, are not seen for as long as 8 16 hours after administration of the estrogen, although some biochemical processes in the cell 9 are affected immediately. The growth response produced in the uterus by estrogens is temporary, 10 and the maintenance of such growth requires the hormone to be available almost continuously. 11 The initial growth induced by the estrogen is therefore of limited duration, and atrophy of the 12 uterus occurs if the hormone is withdrawn. Another physiologic effect of estrogens, observed 13 one hour after their administration, is edema in the uterus. During this period, vasodilation of the 14 uterine pre- and postcapillary arterioles occurs, and there is an increase in permeability to plasma 15 proteins. These effects appear to occur predominantly in the endometrium and not to any great 16 extent in the myometrium. 17 18 Another target of estrogens is breast tissue. Estrogens can stimulate the proliferation of 19 breast cells and promote the growth of hormone-dependent mammary carcinoma. Because the 20 breast is the primary site for cancer in women, considerable research has been focused on 21 understanding breast cancer and the factors that influence its growth. Estradiol will stimulate 22 gene expression and the production of several proteins in breast cancer cells via the estrogen 23 receptor mechanism. These proteins include both intracellular proteins important for breast cell 24 function and growth, and secreted proteins that can influence tumor growth and metastasis. 25 26 Finally, estrogens are also important for male sexual development and reproductive 27 function.6,7 Estrogens, produced locally by aromatase in the Sertoli cells, are important for 28 spermatogenesis in rats, and aromatase has been detected in human testis.8 Estrogen receptors are 29 present in male reproductive tissues such as Sertoli cells, Leydig cells, epididymis, and accessory 30 sex organs.7 During gestational development of the rodent, the local aromatization of 31 testosterone to estrogen within the brain, plays a key role in the structural organization of the 32 hypothalamus which can effect sexual differentiation the male brain.9,10 Although there is 33 currently no definitive evidence to demonstrate analogous effects of estrogen on masculization 34 of the human brain during gestational development, it is likely that estrogen and androgens play 35 a critical role in producing the sexual dimophism of the primate.11 36 37 3.2 AROMATASE - GENERAL DESCRIPTION 38 39 3.2.1 Biochemistry and Molecular Biology 40 41 Estradiol is the most potent endogenous estrogen in humans. Estradiol is biosynthesized 42 from androgens by the cytochrome P450 enzyme complex called aromatase. This enzyme was 43 first reported in human placental tissues by K. J. Ryan in 1959.12 In humans, aromatase is present 44 in the ovaries of premenopausal women, in the placenta of pregnant women, and in the

Battelle Draft 14 February 25, 2002 1 peripheral adipose tissues of women and of men, and these tissues are the major sources for 2 estrogens circulating in the blood stream.13 Aromatase is also important for the local tissue 3 production of estrogens. Aromatase activity has been demonstrated in breast tissue in vitro.14-17 4 Furthermore, expression of aromatase is highest in or near breast tumor sites,15,18 various regions 5 in the brain of both men and women contain the enzyme aromatase,10,19the male testis, and bone 6 in men and women.20,21 7 8 Aromatase is the enzyme complex responsible for the conversion of androgens to 9 estrogens during steroidogenesis.22 The enzyme complex is bound in the endoplasmic reticulum 22,23 10 of the cell and is comprised of two major proteins. One protein is cytochrome P450arom, a 11 hemoprotein that converts C19 steroids (androgens) into C18 steroids (estrogens) containing a 12 phenolic A ring. The second protein is NADPH-cytochrome P450 reductase, which transfers

13 reducing equivalents to cytochrome P450arom. Three moles of NADPH and three moles of 14 oxygen are utilized in the conversion of one mole of substrate into one mole of estrogen product 15 (Figure 4). Aromatization of androstenedione, the preferred substrate, proceeds via three 16 successive oxidation steps, with the first two being hydroxylations of the angular C-19 methyl 17 group. The final oxidation step, whose mechanism remains for complete elucidation, proceeds 18 with the aromatization of the A ring and loss of the C-19 carbon atom as formic acid. 19 20 O O O O HO 2 HO O2 HO

NADPH NADPH O O O

androstenedione 19-hydroxyandrostenedione 19,19-dihydroxyandrostenedione

- H2O +3Fe O O O O - HCOOH O O HO 2 O

- H O 2 NADPH HO O O estrone peroxy enzyme 19-oxoandrostenedione intermediate

Figure 4. Reaction Mechanism for Estrogen Biosynthesis by Aromatase

Battelle Draft 15 February 25, 2002 1 Over the past two decades, knowledge of the biochemistry, molecular biology, and 2 regulation of aromatase has increased greatly. The aromatase gene, designated CYP19, encodes

3 the cytochrome P450arom and consists of 10 exons, with the exact size of the gene exceeding 70 4 kilobases.22 The gene is located on chromosome 15q21.1. The full length cDNA of 3.4 5 kilobases encodes for a protein of 503 amino acids with a molecular weight of approximately 6 55,000 daltons. The regulation of aromatase is complex in various tissues, and several tissue­ 7 specific promoter regions have been identified upstream from the CYP19 gene.24-26 These tissue­ 8 specific promoters include promoter P1.1, P1.3, P1.4, and PII. The PII promoter is utilized in the 9 ovary and in breast cancer tissues, and it contains a cAMP response element. Promoters P1.3 and 10 P1.4 are the primary promoter used in extraglandular sites such as adipose tissue and are 11 responsive to glucocorticoids and cytokines such as IL-1β, IL-6 and TNFα. Promoter P1.1 is the 12 promoter used in placental tissues and its regulation is still under extensive investigations.27 13 14 Aromatase is found in breast tissue, and the importance of intratumoral aromatase and 15 local estrogen production is being unraveled.15,28 29 Aromatase has been measured in the stromal 16 cell component of normal breast and breast tumors, but the enzyme has also been detected in the 17 breast epithelial cells in vitro. 14,18,29-31 Furthermore, expression of aromatase is highest in or near 18 breast tumor sites.29 The exact cellular location(s) of aromatase must await more rigorous 19 analysis by several labs with a new monoclonal antibody now being developed and evaluated.32

20 The increased expression of aromatase cytochrome P450arom observed in breast cancer tissues 21 was recently associated with a switch in the major promoter region utilized in gene expression, 22 and promoter II is the predominant promoter used in breast cancer tissues.33,34 As a result of the 23 use of the alternate promoter, the regulation of estrogen biosynthesis switches from one 24 controlled primarily by glucocorticoids and cytokines to a promoter regulated through cAMP­ 33 25 mediated pathways. The prostaglandin PGE2 increases intracellular cAMP levels and 26 stimulates estrogen biosynthesis, whereas other autocrine factors such as IL-1β do not appear to 35 27 act via PGE2. 28

29 Local production of PGE2 via the cyclooxygenase isozymes (constitutive COX-1 isozyme 30 and inducible COX-2 isozyme) can influence estrogen biosynthesis and estrogen-dependent 31 breast cancer. Recent studies suggest a relationship between CYP19 gene expression and the 32 expression of COX genes.36 Gene expressions of CYP19, COX-1, and COX-2 were performed 33 in 20 human breast cancer specimens and in 5 normal control breast tissue samples. A positive 34 correlation was observed between CYP19 expression and the greater extent of breast cancer 35 cellularity, in agreement with literature reports showing that aromatase levels were higher in 36 tumors than in normal tissue. Furthermore, a positive linear correlation was observed between 37 COX-2 expression and the level of pathology in each sample, and a linear regression analysis 38 using a bivariate model shows a strong linear association between CYP19 expression and the 39 sum of COX-1 and COX-2 expression.36 This significant relationship between the aromatase and 40 cyclooxygenase enzyme systems suggests that autocrine and paracrine mechanisms may be 41 involved in hormone-dependent breast cancer development via growth stimulation from local 42 estrogen biosynthesis. 43

Battelle Draft 16 February 25, 2002 1 Effective aromatase inhibitors have been developed as therapeutic agents for estrogen­ 2 dependent breast cancer to reduce the growth stimulatory effects of estrogens in breast cancer. 3 Investigations on the development of aromatase inhibitors began in the 1970's and have 4 expanded greatly in the past three decades. Research summaries of aromatase inhibitors have 5 been presented at international aromatase conferences32,37-40 and several reviews have also been 6 published.41-50 7 8 3.2.2 Aromatase in Other Species 9 10 In addition to the extensive investigations on the importance of human aromatase in 11 normal physiology and in cancer, this enzyme complex has also been well studied in other 12 mammalian species (rodents, cows, pigs, horses) and in non-mammalian vertebrates as well. In 13 general, these investigations have focused on identifying the presence of aromatase in various 14 tissues, comparative endocrinology of aromatase, and examination of the importance of 15 aromatase in the physiology of various species. In many species, aromatase expression and/or 16 activity is restricted to the gonads and the brain. Placental aromatase is found only in primates, 17 cattle, horses, and pigs. Aromatase in the adult rat ovary is regulated by the gonadotropins, FSH 18 and LH, and by prolactin51,52 and is induced by elevation in cAMP levels and subsequent 19 activation of protein kinase A.53 Rat testicular tissues also contain aromatase54 and convert 20 androgens into estrogens locally for spermatogenesis. The importance of aromatase in the brain 21 was first demonstrated in vivo in rodents,55,55 and this conversion of androgens to estrogens in the 22 brain is responsible for neural cell differentiation.56 Aromatase is present in both bovine ovary 23 and bovine placenta57 and is under complex regulation.58,59 Pigs express two distinct aromatases, 24 one in gonadal tissues and the other in placental tissues.60,61 Aromatase has also been identified 25 in gonadal and brain tissues in many non-mammalian vertebrates as well. Numerous species of 26 fish have aromatase activity,62-64 and higher levels are often found in the brain compared to the 27 gonads.63 Recently, two distinct aromatases have been reported in fish.63,65,66 Various reptiles 28 also have aromatase in the gonads and the brain.67,68 29 30 3.2.3 Significance of Alterations in Aromatase 31 32 The levels of estradiol, the most potent endogenous estrogen in humans, are critical for 33 female reproduction and other hormonal effects in females. The levels of estrogens are 34 controlled by the extent of estrogen biosynthesis from androgens by the enzyme aromatase. 35 Alterations in the amount of aromatase present or in the catalytic activity of the enzyme will alter 36 the levels of estrogens in tissues and dramatically disrupt estrogen hormone action. Inhibition of 37 aromatase alters the catalytic activity of the enzyme and results in a rapid decrease in the levels 38 of estrogens. This mechanism of enzyme inhibition is the reason for the therapeutic 39 effectiveness of aromatase inhibitors in the treatment of estrogen-dependent breast cancer and 40 illustrates the importance of estrogen levels to estrogen action. Suppression or induction of the 41 aromatase protein levels also can dramatically influence the subsequent levels of estrogens in 42 tissues and effect hormone action. Environmental agents, toxicants, and various natural products 43 can act via aromatase inhibition and/or alteration in aromatase protein levels to result in altered 44 levels of estrogen and function as endocrine disruptors. Aromatase deficiencies in humans and

Battelle Draft 17 February 25, 2002 1 the aromatase knockout (ArKO) mice illustrate dramatically the importance of aromatase.27 In 2 females with aromatase deficiency (currently a total of 8 patients worldwide), newborns have 3 psuedohermaphroditism and, at puberty, these patients have primary amenorrhea, lack of breast 4 development, hypogonadism, and cystic ovaries. Two males with aromatase deficiency have 5 been reported, and these individuals exhibit tall stature due to failure of epiphyseal fusion, 6 osteopenia, and infertility. Female ArKO mice exhibit infertility and underdeveloped uteri and 7 mammary glands, and male ArKO mice have impaired spermatogenesis and enlarged 8 prostates.69,70 Both male and female ArKO mice have excessive intra-abdominal fat and elevated 9 serum lipid levels,71 and both have excessive long bone growth and osteopenia.72

Battelle Draft 18 February 25, 2002 1 4.0 SCIENTIFIC BASIS OF THE AROMATASE ASSAY 2 3 4.1 METHODS FOR MEASURING AROMATASE ACTIVITY 4 5 The method employed for measuring aromatase activity in vitro and in vivo is critical. 6 This assay endpoint, i.e., measurement of aromatase activity, must be accurate and reproducible. 7 The endpoint is accurate when the assay measurement is in agreement with the accepted 8 reference value. The endpoint is reproducible when the same findings occur under the same 9 conditions within a single laboratory (intra-laboratory) and among other laboratories (inter­ 10 laboratory). The two methods that are utilized extensively for the determination of aromatase 11 activity in both in vitro and in vivo assays are the product isolation method and the radiometric 12 method. 13 14 The most rigorous method is the product isolation technique. The method involves 15 administration of a substrate such as androstenedione or testosterone, incubation or treatment for 16 a designated time, isolation of the estrogen products formed, and measurement of the amount of 17 estrogens. One common method uses radiolabeled substrate (either 3H or 14C) in the aqueous 18 incubation medium. At the end of the incubation or treatment period, isolation of the 19 radiolabeled steroids is accomplished by organic solvent extraction techniques. The radiolabeled 20 substrate and products are separated using either thin layer chromatography or high-pressure 21 liquid chromatography (HPLC) and analysis of the quantity of estrogen products formed using 22 liquid scintillation counting. This method is best suited for in vitro assays using subcellular 23 enzyme preparations, tissue incubations, or cell culture systems. Variations of the product 24 isolation assay method include unlabeled substrate (or endogenous substrate in an in vivo study) 25 and other methods for measurement and quantification. These other measurement methods 26 include mass spectrometry, radioimmunoassays (RIA), or enzyme-linked immunoassays (EIA). 27 28 The radiometric method is also utilized for in vitro measurements of aromatase activity. 3 29 This method is also referred to as the H2O assay. The level of aromatase activity is determined 3 3 30 by measuring the amount of H2O released from [1β- H]-androstenedione substrate. The basis of 31 this assay is that the aromatization of the A-ring catalyzed by aromatase involves the 32 stereospecific cleavage of the covalent bond between the carbon atom at position 1 and the 33 hydrogen on the β face of the steroid ring system. The procedures for this method are similar to 34 the product isolation method but do not involve any chromatography to separate steroid substrate 3 35 and products. Rather, the amount of H2O released is measured in the aqueous phase following 36 rigorous extractions with organic solvents and/or dextran-coated charcoal. This method is very 37 straightforward and more rapid. However, the results of the radiometric method must be 38 confirmed for the specific aromatase assay conditions using the product isolation assay before 39 extensive use. This radiometric method has been confirmed for in vitro assays using human 40 placental microsomes, ovarian microsomes and tissues, cell culture systems (including human 41 breast cancer cells and human choriocarcinoma cells), and isolated human breast or ovarian 42 cells. This comparison of the results from the radiometric method with the product isolation 43 assay is critical because other biochemical pathways that involve extensive androgen metabolism 44 without aromatization of the androgen can produce false positive measurements. Examples are

Battelle Draft 19 February 25, 2002 3 1 determinations of H2O released by liver or prostate cells, tissues, or tissue homogenates, which 2 results in much higher activity measurements than is observed with a product isolation method as 3 a result of androgen metabolism, and presence of a 19-hydroxylase activity in adrenal tissues 4 which does not result in A ring aromatization and formation of estrogens.73 5 6 Recently, a fluorescence aromatase assay was described using a fluorometric substrate O ­ 7 benzylfluorescein benzyl ester, also referred to as dibenzylfluorescein (DBF).74 In this report, 8 recombinant human aromatase in microsomal preparations from stably-transfected insect cells 9 was the source of enyzme.75 DBF was a substrate for aromatase and was converted into the 10 metabolite, fluorescein, which was measured using an excitation wavelength of 485 nm and an

11 emission wavelength of 538 nm. The apparent Km for DBF was reported as 188 nM and the -1 12 apparent Vmax was 0.32 min . For comparison, apparent Km for testosterone was 43 nM and the -1 13 apparent Vmax was 8.4 min . Kinetic data for androstenedione, the preferred substrate for human 14 aromatase, was not reported. Based upon the comparison of enzyme kinetic values, DBF is a

15 poor substrate for aromatase. The IC50 values for several agents were reported, and in several 16 cases, the IC50 values were quite different than reported literature values using the radiometric 3 17 method measuring H2O released. 18 19 Aromatase assay activity is typically reported as velocities or rates, examples include 20 descriptions such as nmol product formed per mg microsomal protein per assay minute, pmol 21 product formed per 106 cells, or pmol product formed per mg tissue. Also, inhibition data is 22 reported in table or graphical format as % inhibition, with a definition of 100%. For

23 biochemical enzyme assays, the kinetic parameters of Vmax for the enzyme rate, Km for the 24 substrate, Ki for an inhibitor should also be reported. Catalytic efficiencies, measured by Vmax/Km 25 also provide valuable information. In pharmacological inhibition assays, the IC50 values should 26 be reported. 27 28 4.2 IN VITRO AROMATASE ASSAYS TO DETECT ENDOCRINE DISRUPTORS 29 30 This section is subdivided according to aromatase assay method used (i.e., placental 31 microsomal assay, cell line assays, in vitro mammalian cell systems, and in vivo studies) and 32 includes discussions on the chemical natures of the endocrine disruptors. 33 34 4.2.1 Human Placental Microsomal Aromatase Assay 35 36 This is one of the most common assays used for measuring the inhibition of aromatase 37 activity. This assay has been utilized to evaluate the ability of various endocrine disruptors to 38 inhibit aromatase. The source of the aromatase is a microsomal preparation isolated from human 39 term placenta. Normal placenta is obtained after delivery at a local hospital, is delivered on ice 40 to the laboratory, and then processed at 4°C following a standard procedure. The microsomal 41 preparation, is isolated by differential centrifugation procedures, with the final centrifugation at 42 105,000 x g for one hour. This microsomal preparation consists of the endoplasmic reticulum

43 membrane of the cell and contains the membrane-bound cytochrome P450arom and the NADPH­ 44 cytochrome P450 reductase. For the enzyme assay, the microsomal preparation is resuspended in

Battelle Draft 20 February 25, 2002 1 phosphate buffer, pH 7.4. Complete enzyme activity requires the addition of either NADPH or 2 an NADPH-generating system, and the activity is measured using either the product isolation 3 method or the radiometric method. These assay methods have been utilized routinely for the 4 determination of the presence of aromatase in tissues since the first report of aromatase by K.J. 5 Ryan.12 The highest tissue levels of human aromatase are present in term placenta and the 6 placenta is discarded after birth, thus providing a rich and inexpensive source of the enzyme. 7 Consequently, the human placental microsomal aromatase assay is used extensively in academic 8 labs and pharmaceutical firms as the initial biological evaluation for potential steroidal and 9 nonsteroidal aromatase inhibitors (see discussion in Section 2.2.1). 10 11 Numerous flavonoids and related phytoestrogen derivatives have been extensively 12 evaluated for aromatase inhibition for two primary reasons: (1) these plant natural products can 13 serve as possible leads for the development of new nonsteroidal aromatase inhibitors and (2) 14 humans and other animals are exposed to these agents through the diet. The class of flavonoids 15 encompasses the flavones, isoflavones, flavanones, and flavonols, all being derived chemically 16 from the benzopyranone nucleus. These natural products have demonstrated numerous 17 biological activities, interact with various enzymes and receptor systems of pharmacological 18 significance, and have been potential applications in both cancer therapy and in cancer 19 chemoprevention. Considerable interest in flavonoids in breast cancer has been stimulated by the 20 hypothesis that these natural products, present in soy and in rye flour, are dietary factors that 21 may be responsible for the lower incidence of breast cancer in women from certain regions of the 22 world, e.g., Japan and Finland.76 23 24 The initial reports of aromatase inhibition by flavonoids and flavones appeared in the 25 mid-1980's from the lab of L.E. Vickery,77,78 and the synthetic benzoflavone analogs were more 26 potent than naturally occurring flavones or flavonoids in the human placental microsomal assay 27 system. Several research groups reported aromatase inhibition by various flavonoids using the 28 human placental microsomal assay system.76,79-86 In general, the flavonoids and related analogs

29 demonstrate aromatase inhibition with IC50 values in the micromolar range; however, these 30 compounds lack both the potency and specificity of aromatase inhibitors developed for breast

31 cancer therapy. Figure 5 provides the chemical structures of selected flavonoids and their IC50 32 values. Flavonoids have also been examined for aromatase inhibitory activity in other biological 33 systems as well. Aromatase inhibition by flavonoids has been observed in human 34 preadipocytes,87,88 in breast cancer cell systems,89 in a transformed yeast cell system,90 and in fish 35 ovarian microsomal assays.91,92 The relative inhibitory activity is similar to that observed with 36 the human placental microsomal aromatase assay. 37 38 Several pesticides have also demonstrated inhibition of aromatase activity in the human 93 39 placental microsomal assay system. This study reported IC50 values for aromatase inhibition 40 ranging from 0.04 µM to greater than 50 µM. , imazalil, propioconazole, ,

41 triadimenol, and triadimefon inhibited placental aromatase with IC50 values of 0.04, 0.34, 6.5, 10,

42 21 and 32 µM, respectively (Figure 6). Further information and comparisons of IC50 values is 43 provided later in Tables 1 and 2. 44

Battelle Draft 21 February 25, 2002 O O O

O O HO O

α-naphthoflavone flavone 7-hydroxyflavone IC50 = 0.07 µM IC50 = 8.0 µM IC50 = 1.0 µM

OH O OH O O OH

HO O HO O O

OH chrysin quercitin OH flavanone IC = 0.5 µM 50 IC50 = 12.0 µM IC50 = 8.0 µM

OH O OH O O

HO O HO O O

OH OH OCH3 7-methoxyflavanone IC = 2.9 µM IC = 9.2 µM 50 50 IC50 = 2.6 µM

Figure 5. IC50 values for selected flavonoids

1 2 4.2.2 Human Cell Culture Systems for Measuring Aromatase Activity 3 4 Human JEG-3 and JAR choriocarcinoma cell culture lines, originally isolated from 5 cytotrophoblasts from malignant placental tissues, have been used as in vitro systems for 6 measuring the effects of compounds on aromatase activity. These cell lines are also utilized for 7 investigations on the effects of agents in placental toxicology.94 The cells express a large amount 8 of aromatase activity and have been used extensively in academic labs and pharmaceutical firms 9 for the biological evaluation for potential steroidal and nonsteroidal aromatase inhibitors.95-98

10 The JEG-3 and JAR cells are cultured at 37°C and 5% CO2 atmosphere in standard DMEM 11 media and supplemented with 10% fetal calf serum, and aromatase activity is measured in intact 12 cells using either the radiometric method (most common) or the product isolation method . 13 14 Various organochlorine contaminants have been examined in the JEG-3 and/or JAR cell 15 line systems. The contaminants 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5­ 16 pentachlorobiphenyl (PCB126) caused concentration-dependent decreases in the aromatase 99 17 activity of up to 4.9-fold in the JEG-3 cells. The IC50 values for aromatase inhibition were 52 18 pM and 13 nM for TCDD, and 75 nM and 48 nM for PCB126 in the presence and absence of

Battelle Draft 22 February 25, 2002 1 serum, respectively. These studies were further extended to twenty-one organochlorine PCB 2 derivatives tested in both JEG-3 and JAR cell lines.100 Aromatase inhibition was observed, but

Battelle Draft 23 February 25, 2002 Cl N Cl

OH Cl O O Cl O O N

Cl N O N OH N N N N

fenarimol tridimefon tridimenol IC = 21.0 µM IC50 = 10.0 µM IC50 = 32.0 µM 50

O O Cl Cl O N N N N Cl Cl N

imazalil propioconazole IC = 0.34 µM 50 IC50 = 6.5 µM

Figure 6. IC50 values for selected pesticides

1 cytotoxicity was also significant. These observations led to the conclusion by the authors that 2 the JEG-3 and JAR cells “appear too sensitive” to the cytotoxic effects of organochlorines for 3 use in evaluating effects on aromatase activity. The fungicide fenarimol was also evaluated in 93 4 JEG-3 cells, exhibiting an IC50 value for aromatase inhibition of 2.0 µM. In general, the 5 results on aromatase inhibition for a wide variety of chemical compounds that have been 6 obtained from an intact cell system such as the JEG-3 or JAR cell culture lines are similar to the 7 results from the human placental microsomal system. 8 9 Two reports have recently been published on evaluation of environmental contaminants 10 in other human cell culture systems. Yang and Chen have evaluated the organochlorine 11 pesticides toxaphene and chlordane in a transfected SK-BR-3 breast cancer cell system and 12 reported a decrease in aromatase expression, possibly through antagonism of the ERRα-1 13 transcription factor.101 The H295R human adrenocortical carcinoma cell line has been used by 14 Sanderson and colleagues to examine the effect the 2-chloro-s-triazine herbicides.102 In this 15 report, , simazine, and propazine at concentrations of 0-30 µM resulted in dose­ 16 dependent induction of aromatase activity by about 2.5-fold in H295R cells. In another study by 17 these researchers,103 diindolylmethane (DIM) and analogs, formed in the stomach by an acid­ 18 catalyzed condensation product of indole-3-carbinol, induced aromatase activity approximately 19 2-fold increase at 10 mM in H295R cells. Recently, Sanderson and colleagues reported a 20 significantly less pronounced induction of aromatase by atrazine, simazine, propazine, and 21 metabolites in JEG-3 cells and no induction of aromatase in MCF-7 cells.104 These studies 22 further illustrate the tissue-specific nature of CYP19 expression and aromatase levels.

Battelle Draft 24 February 25, 2002 1 4.2.3 Other In Vitro Mammalian Cell Systems for Measuring Aromatase Activity 2 3 Isolated ovaries, ovarian follicles, ovarian granulosa cells, and ovarian microsomes from 4 various animal species (including humans) have also been utilized to examine the effects of 5 various environmental contaminants on aromatase activity. These in vitro ovarian cell systems 6 have been extensively used for decades in reproductive endocrinology studies.105 Two types of 7 investigations are generally performed that involve the measurement of ovarian aromatase 8 activity. One type of study are those in which the ovaries are isolated from animals treated with 9 a particular agent and then aromatase activity is measured. These assays will be discussed in 10 detail in the next section (Section 3.4). The second type of study involves using ovarian 11 microsomes or ovarian cells in a culture system for an in vitro evaluation of the effects of 12 compounds on aromatase inhibition. Odum and Ashby examined nonsteroidal aromatase

13 inhibitors anastrozole, fadrozole, and letrozole in rat ovarian microsomes, found IC50 values 14 similar to those reported from human placental microsomes, and observed much lower aromatase 15 activities in the rat ovarian microsomes.106 Human granulosa cells isolated from follicles were 16 treated with constituents from cigarette smoke, and the smoke extracts produced dose-dependent 17 inhibition of granulosa cell aromatase activity at the 10 µM to 10 mM concentration range.107 18 Human granulosa cells were also utilized by Moran et al.108 to demonstrated that TCDD does not 19 affect aromatase activity but does lower estradiol production by affecting the availability of 20 androgen precursor. Laskey and colleagues109 studied steroid production by cultured ovarian 21 fragments as a method for assessment of toxicant-induced alterations in ovarian steroidogenesis 22 in Sprague-Dawley rats. Although these researchers did not examine any environmental agents,

23 they did show that the drug aminoglutethimide produced similar inhibitory activity (IC50 of 2.43 24 mM) as observed in cell culture systems. The polycyclic aromatic hydrocarbons (PAHs), 20­ 25 methylcholanthrene and β-naphthoflavone, weakly inhibited aromatase activity in ovarian 26 follicles from coho salmon,110 and several PAHs also demonstrated weak inhibition of aromatase 27 in in vitro incubations with ovaries from flounder.111 In a recent report, phthalates suppressed 28 CYP19 mRNA levels and aromatase activity in rat granulosa cell cultures.112 29 30 4.3 IN VIVO STUDIES OF AROMATASE TO DETECT ENDOCRINE DISRUPTORS 31 32 Evaluation of the in vivo effects of endocrine disruptors on aromatase are more 33 complicated, and interpretation of results can be problematic for several reasons. The sensitivity 34 of a species or an individual to an endocrine disruptor may be related to circulating 35 concentrations of gonadal steroid hormones. One important contributor to these hormonal levels 36 in the total organism is the enzyme aromatase. In general, ovarian aromatase is the major source 37 of circulating estrogens in adults. On the other hand, extraglandular sources of estrogen can be 38 important in certain species (such as aromatase in adipose tissues in humans). Alterations in 39 estrogen biosynthesis locally in a tissue may produce significant effects on estrogen levels within 40 that tissue while not altering overall circulating hormone levels.113 Additionally, alterations in 41 estrogen levels in vivo are also influenced by the levels and cyclicity of pituitary gonadtropins. 42 Therefore, it is critical to differentiate effects via aromatase inhibition and effects via disruption 43 of the hypothalamus-pituitary-ovary axis. 44

Battelle Draft 25 February 25, 2002 1 Nonetheless, the hazards of potential endocrine disruptors in the environment can not be 2 determined by in vitro experiments alone. Results from screening assay can guide further 3 examination of the compounds’ potential for inducing endocrine disruption after in vivo 4 exposure. Studies to evaluate the effect of endocrine disruptors have been conducted in an 5 extensive number of both mammalian and nonmammalian species. 6 7 Administration of potent aromatase inhibitors (such as the drugs anastrozole, letrozole, 8 and exemestane developed for breast cancer therapy) to adult female cycling rats can result in an 9 inhibition of estrogen biosynthesis, decreases in plasma concentrations of estrogens, a disruption 10 of ovarian cyclicity, a reduction in uterine weight and changes in serum LH.114-117 11 12 Studies have been conducted in mammalian species to evaluate the effect of endocrine 13 disruptors on the reproductive parameters of both males and females. Several pesticides, 14 including fenarimol, imazalil, prochloraz, and triadimefon, have found to inhibit aromatase in 15 vivo. In a three-generation study with imazalil in rats, no reproductive effects were found , 16 although, a trend towards a lower number of live births at 80 mg/kg level was observed.118 In a 17 multigenerational study, rats were fed doses up to 31.2 mg prochloraz per kg per day in the 18 diet.119 In males receiving the highest dose, a prolonged aggression was noted.In two-generation 19 studies in rats, fenarimol (6.5 mg/kg) caused reduced fertility and a reduction in live-born litter 20 size. Parturition in females was also reduced.120 Reproduction studies in in different species 21 showed that fenarimol had adverse effects on reproductive parameters and hormone levels in rats 22 and mice, but not in guinea pigs or rabbits. Mechanistic studies showed that fenarimol inhibits 23 male sex differentiation in the CNS by inhibiting CYP19 aromatase activity and therefore the 24 synthesis of estradiol.121,122 Triadimefon was given to rats in their diet for two generations at 25 levels of 2.5 or 90 mg /kg/day. Reproductive performance was impaired at the high level. The 26 male/female ratio was reduced in the F2 generation and F1 males showed reduced mating and 27 elevated testosterone, whereas FSH levels were unchanged.123 A two-generation reproduction 28 study in rats using triadimenol was conducted using levels of 1,4, and 20 mg/kg/day and no 29 reproductive endpoints were affected.124 In other studies, a dose of 120 mg/kg/day gave rise to an 30 increased rate of resorption of foetuses.124 In long term studies with rabbits receiving the 31 propioconazole, no effects were observed on the classical parameters of reproductive 32 toxicity (Report from the Danish Environmental Protection Agency). In summary, aromatase 33 inhibitors might in some cases be able to affect hormone levels in vivo in rodents, but a general 34 reproductive toxicity cannot be predicted solely from inhibition of aromatase in vitro.93 35 36 Averal et al. 125 have suggested that the cytoplasm of sperm may be the target of action of 37 cytotoxic drugs. It has also been hypothesized that the cytoplasmic droplet (small amount of 38 cytoplasm adhered to the middle section of mature sperm) has a role in the aromatization of 39 androgen into estrogen, which has a role in the regulation of male reproductive tract function.126 40 The droplet is shed before ejaculation, after fulfilling its role, and then sperm obtain optimal 41 motility.This should occur by the time the sperm reach the cauda epididymis. In a recent study 42 by Akbarsha et al.127 adult male rats receiving ursolic acid, andrographolide, malathion and 43 dichlorvos showed increased numbers of sperm with retained droplets compared to the control 44 values. Sperm were obtained from the distal cauda epididymis and control treated animals had no

Battelle Draft 26 February 25, 2002 1 sperm with retained droplets compared to 65-95% retained droplets in treated males. Motility of 2 the sperm from treated males was also impaired or completely zero. This may provide an 3 interesting approach to studying the relationship of aromatase in males and its relationship to 4 reproductive toxicity of compounds. 5 6 Extensive research has shown the implication of steroids in gonadal sex differentiation of 7 non-mammalian vertebrates. Treatments of larvae or embryos with sex steroid hormones have 8 led to various degrees of sex reversal in fishes, amphibians, reptiles, and birds. This effect has 9 been observed in species with temperature-dependent sex determination and those with 10 genotypic sex determination. During the last two decades, the phenomena of imposex has been 11 described in more than 120 species world-wide. Researchers failed to find a difference in the 12 aromatase activity in the gastropod Bolinus brandaris in tributyltin polluted sites compared to 13 non-polluted sites on the Mediterranean coast. There were, however, significant reductions in 14 estradiol titers and an increased testosterone/estradiol ratio.128 15 16 In turtles, aromatase activity strongly correlates with ovarian differentiation.129 17 Willingham and Crews130 have shown that even 0.4 ng of estradiol applied to the eggshells of the 18 red-eared slider turtle will affect sexual development during embryogenesis. Only 0.2 % of the 19 hormone is believed to end up in the actual embryo. These researchers also found that 20 compounds produced different results when tested individually vs in mixtures. Aroclor 1242 21 significantly altered aromatase activity levels in the brain but not in the adrenal-kidney-gonad 22 complex during a crucial developmental period but did increase the aromatase activity in this 23 complex just prior to hatch. This may emphasize the importance of testing during different 24 periods of development. Sex is determined by the temperature of the incubating egg and the 25 exogenous application of steroid hormones or steroidogenic enzyme inhibitors can reproduce 26 temperature’s effects.131 At 26 °C male hatchlings are produced, but if estrogen is applied to the 27 eggshell, females result. At 29.4 °C, females are produced, but after applying an aromatase 28 inhibitor , males are produced. The aromatase activity during development can also be used as a 29 marker since its activity exhibits temperature dependence and it appears to be an enzyme critical 30 to thermosensitive sex determination and is capable of modification by extrinsic factors. These 31 embryonic systems of many fish, amphibians, and reptiles may be more sensitive to the low 32 doses of ED which may be present in the environment. Hundreds of eggs may be used in these 33 studies so that statistical power of the study is increased above that of mammalian studies. 34 35 Studies by Crain et al.,132 have shown that juvenile female alligators from Lake Apopka, 36 a lake historically contaminated with a number of persistent organochlorines, have a 37 significantly lower mean gonadal-adrenal-mesonephros (GAM) aromatase activity when 38 compared to females from Lake Woodruff, a control lake. In juvenile female alligators treated in 39 a laboratory setting, atrazine induced GAM aromatase activity, while 2,4-dichlorophenoxyacetic 40 acid (2,4-D) had no effect. In in ovo experiments, aromatase activity in gonadal tissues from 41 hatchlings incubated at male-producing temperatures (33°C) was elevated by atrazine, while no 42 effect on aromatase activity was observed at female-producing temperatures (30°C). 43 Additionally, no alteration in hepatic aromatase activities was observed in either sex.133 44

Battelle Draft 27 February 25, 2002 1 The presence and effect of naturally occurring and synthetic aromatase inhibitors are not 2 well studied. Shilling et al.,92 have investigated the ability of 4-hydroxyandrostenedione, 3 letrozole and clotrimazole (an imidazole fungicide) to inhibit aromatase activity in vivo by 4 blocking DHEA induced vitellogenesis in the rainbow trout, Oncorhynchus mykiss. Although 5 these agents were able to inhibit ovarian aromatase activity in vitro, neither letrozole nor 6 clotrimazole blocked vitellogenin production or estradiol synthesis at doses up to 1000 ppm. 7 This lack of observed in vivo activity may be due to species differences in pharmacokinetics or 8 to high variability between individual fish, probably due to the lack of inbreeding compared to 9 most mammalian models used for testing. In an experiment involving the feeding of lake trout to 10 rats, the aromatase activity was lowered in all groups compared to the control.134 Total thyroxine 11 and estrogen were not significantly different in these offspring of dosed dams from that of 12 controls. 13 14 4.4 QSAR APPROACHES TO PREDICTING AROMATASE INHIBITION 15 16 Aromatase has not been crystallized to date, and thus no x-ray crystal structure is 17 available for a detailed analysis of the enzyme active site. As a result, two general approaches 18 have been used to study the enzyme active site of aromatase and aromatase inhibitors ­ 19 computational analysis techniques based upon ligand-based molecular modeling and based upon 20 protein homology modeling. The development of both steroidal and nonsteroidal aromatase 21 inhibitors during the 1980's and 1990's provided initial structure-activity relationships and 22 enabled the application of ligand-based molecular modeling. Early studies focused on energy 23 minimization calculations, conformational analysis, molecular volume calculations, and 24 pharmacophore mapping of aromatase inhibitors.135-137 138,139 Analysis of the steroidal aromatase 25 inhibitors identified restricted sites (e.g., surrounding the steroid A ring) and accessible sites for 26 structural modifications (e.g., lipophilic pocket at 7α-position). Analysis of nonsteroidal agents 27 identified key components as a heterocyclic ring (proposed interaction with heme iron) and 28 lipophilic aromatic ring (mimic of steroidal backbone). Recently, the Comparative Molecular 29 Field Analysis (COMFA) 3D-QSAR method was applied to the analysis of nonsteroidal 30 aromatase inhibitors, correlating inhibitory activity with steric field values.140,141 31 32 Protein homology modeling analysis of aromatase has involved modeling the aromatase 33 cytochrome P450 based upon sequence comparisons with bacterial cytochrome P450's that have

34 been crystallized, particularly cytochrome P450cam, cytochrome P450terp, and cytochrome P450BM­ 80,142,143 35 3, and site-directed mutational studies. These models have identified the active site of the 36 enzyme located80near the heme-binding region and the I-helix, with the carboxyl-terminal 37 residues of helix F and the N- terminal residues of helix G contributing to the structure of the 38 active site. A report on the comparisons of the results of protein homology modeling analysis 39 with COMFA ligand-based molecular modeling revealed several mismatches between the 40 modeling approaches.141 In summary, further modeling approaches are needed before docking 41 and in silico screening methods can be applied. Particularly, the accuracy of protein homology 42 modeling cannot be easily determined until a crystal structure of aromatase (or other closely 43 similar human cytochrome P450s) is solved. Similarly, ligand-based modeling and COMFA are

Battelle Draft 28 February 25, 2002 1 derived from a finite number of aromatase inhibitors and do not probe the entire active site 2 space. 3 4 4.5 ADDITIONAL INFORMATION FROM INTERVIEWEES 5 6 Investigators were initially contacted via e-mail in late June 2001 or early July 2001 7 describing the EPA contract and the title and objectives/goals of this particular work assignment. 8 A document was attached to the e-mail describing in more detail the objectives/goals of this 9 particular work assignment, discussing the two study designs under consideration (human 10 placental microsomal aromatase assay, human choriocarcinoma cell culture assay), and listing 11 questions. Follow-up telephone calls and/or e-mail messages were placed to the investigators in 12 the U.S. Follow-up e-mail messages were sent to investigators in Europe. The original e-mail 13 document containing the questions and the responses are presented in Appendix B. 14 15 Discussions were held with Dr. Shiuan Chen (USA), Dr. Raj Tekmal (USA), Dr. Edwin 16 Lephart (USA), and Dr. Sari Mäkelä (Finland). All of the investigators indicated that an in vitro 17 assay for aromatase inhibition would be useful as a high-throughput screening method for 18 identifying compounds as potential endocrine disruptors for subsequent evaluation. The 19 advantages of the two assays (the placental microsomal aromatase assay and the human cell 20 aromatase assay) include the availability of specific radiolabeled substrate, use of standard 21 conditions and methodologies, and the ease of performing the assays. Human placenta can be 22 obtained and the microsomal preparations are straightforward. The microsomal assay may show 23 inhibitory activity for compounds that are not capable of crossing the cell membrane and 24 entering the cell. The JEG-3 cells are commercially available, and use of this assay allows for 25 evaluation of issues related to cell membrane transport. 26 27 Both Dr. Chen and Dr. Mäkelä discussed alternative cell-based assays using stably 28 transfected cell culture systems exhibiting increased aromatase expression. Such cell lines have 29 been generated by several research groups and exhibit less variation in the aromatase activity. 30 Dr. Chen has used both transfected MCF-7 breast cancer cells, transfected SK-BR-3 breast 31 cancer cells, and transfected CHO cells.101 He has also generated cell lines expressing mutant 32 aromatase,81 and he recently has expressed aromatase from other species (not human) in CHO 33 cells (unpublished data from submitted manuscript). These transfected cell systems are used by 34 the investigators as research tools for investigations on the role of aromatase, and the transfected 35 cells are not available for large scale production and distribution at this time. 36 37 Concerns were raised about the sole use of in vitro assays. Activity in vitro does not 38 necessarily indicate activity in vivo. It is difficult to correlate in vitro assay activity and activity 39 in vivo. Dr. Mäkelä described this concern in her response, and a very recent publication by this 40 research group further emphasizes the matter.144 Compounds showing activity in the in vitro 41 assay must be further tested in vivo. Using intact animals, it is relatively difficult and/or time­ 42 consuming to detect aromatase inhibition reliably. Agents producing weak inhibition are not 43 typically identified by measuring changes in circulating estrogen and/or androgen 44 concentrations. Weak inhibitors more likely to act locally in estrogen target tissues are not so

Battelle Draft 29 February 25, 2002 1 easily detected. Also, measurement of small changes in serum estrogen and/or androgen 2 concentrations is difficult. In vivo testing may be more easily performed using transgenic 3 animals overexpressing aromatase gene, as they display very prominent reproductive phenotypes 4 that are relatively easily detected (e.g. cryptorchidism and gynecomastia). Both Dr. Tekmal and 5 Dr. Mäkelä have developed and used such animals.145-147 Also, nude mice injected with MCF-7 6 cells (or other estrogen-responsive cancer cells) overexpressing aromatase gene may be useful 7 here. 8 9 Dr. Lephardt and Dr. Mäkelä also discussed the need for in vivo endpoints, such as 10 effects on ovarian function, on testicular function, on gonadotropin secretion patterns, on the 11 onset of puberty, on mammary gland development and tumorigenesis, on prostate gland 12 development and tumorigenesis, on reproductive and sexual behavior, and on bone strength and 13 osteoporosis. 14 15 Finally, several important questions related to the use of placental aromatase were raised 16 by interviewees. Aromatase activity is regulated in a tissue-specific fashion using various 17 mechanisms that differ in the tissue sites. What are the reasons to use placental tissue/cells in the 18 assay, and what are the reasons that only placental tissue sources are being tested? There may be 19 an endocrine effect on aromatase by endocrine disrupters (in other tissue sites with weak 20 aromatase expression) that would not be picked up in the human placental assay. 21 22 4.6 SUMMARY OF DOCUMENTS REVIEWED AND CONTACT INFORMATION FOR 23 INVESTIGATORS 24 25 Appendix A lists the documents reviewed (in alphabetical order), and a detailed 26 electronic database of references is provided in Reference Manager format. Also provided in 27 Appendix B is a list of the principal investigators and/or authors contacted, including addresses, 28 phone numbers, and e-mail addresses. Copies of e-mail responses are also included in 29 Appendix B.

Battelle Draft 30 February 25, 2002 1 5.0 CANDIDATE PROTOCOLS FOR AN IN VITRO ASSAY 2 3 As described in Sections 3.3 and 3.4, numerous assay systems have been utilized for the 4 examination of the effects of exogenous agents, such as endocrine disruptors, on inhibition of 5 aromatase activity. The human placental microsomal assay is one of the most common assays 6 used for measuring aromatase inhibition. The human placental microsomal aromatase assay is a 7 straightforward, inexpensive, and rapid in vitro assay for measuring the effects on chemicals on 8 aromatase activity. The tissue source, normal human term placenta, is readily available as 9 discarded tissues. The human placental microsomal assay has been utilized to evaluate the 10 ability of various endocrine disruptors to inhibit aromatase. For these reasons, the human 11 placental microsomal assay has been selected as one of the candidate protocols for consideration 12 as an in vitro assay in the Tier 1 Screening Battery Alternate Methods. 13 14 Aromatase inhibition of endocrine disruptors have also been evaluated in intact cell 15 systems such as the JEG-3 or JAR choriocarcinoma cell culture lines; in general, the results for 16 a wide variety of chemical compounds were similar to the results obtained from the human 17 placental microsomal system. Advantages of a whole cell-based assay is that it can provide 18 information on membrane transport abilities of the test agent and the assay can provide 19 information on the effect of the test agent on the expression of the aromatase protein, i.e., if the 20 test compound will induce or suppress the production of new aromatase protein. The 21 choriocarcinoma cell-based aromatase assay has been selected as a second candidate protocol for 22 consideration as an in vitro assay in the Tier 1 Screening Battery Alternate Methods. 23 24 Both the human placental aromatase assay and the JEG-3 or JAR choriocarcinoma cells 25 have been used in the development of potent aromatase inhibitors for breast cancer therapy. 26 Table 1 provides comparative data from these two in vitro assays for nonsteroidal aromatase 27 inhibitors,148,149 several derivatives,144 and the pesticide fenarimol.93 28 29 Table 1. Comparison of Aromatase Inhibition in In Vitro Assays 30

31 Compound IC50 from human IC50 from 32 placental microsomal assay (µM) choriocarcinoma cell line assay (µM) 33 Therapeutic Agents 34 Aminoglutethimide 1.90 15.8 35 Letrozole 0.011 0.070 36 Anastrozole 0.023 0.990 37 Fadrozole 0.005 0.070 38 39 Flavanoid Derivatives 40 Flavone 8.0 >100 41 7-Hydroxyflavone 1.0 0.35 42 Chrysin 0.5 0.5 43 Apigenin 2.9 0.18 44 Naringenin 9.2 1.4 45 46 Pesticide 47 Fenarimol 10.0 2.0 48

Battelle Draft 31 February 25, 2002 1 5.1 CANDIDATE ASSAY A - PLACENTAL MICROSOMAL AROMATASE 2 3 5.1.1 Flow Diagram of the Human Placental Microsomal Aromatase Assay

Addition of the following into 16x100 mm test Preparation of human tubes: placental microsomal pellet

0.1 mL propylene glycol 50.0 nM 3H-androstenedione (0.1 µCi) x.x µM test compound 1.7 mM NADP+ Suspension of placental 2.8 mM glucose-6-phosphate microsomes and dilution to 1.0 u glucose-6-phosphate dehydrogenase ~0.1 mg/mL in phosphate buffer

Dilute to 1.0 ml volume with phosphate buffer, pH 7.4, and warm to 37°C Warm microsomes to 37°C

Add 1.0 mL placental microsomal suspension to each test tube

Incubate at 37°C for 30 min. in shaking water bath

Add 2.0 mL of CHCl3 to quench enzyme reaction; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

Transfer aqueous layer to a new test tube;

add 2.0 mL of CHCl3 to quench enzyme reaction; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

Transfer aqueous layer to a new test tube;

add 2.0 mL of CHCl3 to quench enzyme reaction; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

Transfer aqueous layer to a capped vial; transfer 0.5 mL to a LSC vial; add 5.0 mL LSC solution; count in LSC

4 5.1.2 Description of the Placental Microsomal Aromatase Assay

Battelle Draft 32 February 25, 2002 1 Source of Placental Tissue, Methods for Preparation of Microsomes. Normal 2 placenta is obtained after delivery from a local hospital, packed in ice and forwarded 3 immediately to the laboratory. While keeping the placenta at 4°C , the membrane and fibrous 4 material are dissected and removed, and the tissue is then homogenized in a buffer (2:1, w:v) 5 containing 0.25 M sucrose, 0.05 M sodium phosphate, pH 7.0, and 0.04 . The 6 microsomal preparation, is isolated by differential centrifugation procedures. The tissue 7 homogenate is centrifuged at 10,000 x g for 30 minutes; the supernatant is then centrifuged at 8 105,000 x g for one hour to obtain the crude microsomal pellet. The supernatant is removed, and 9 the microsomal pellet is resuspended in 0.1 M sodium phosphate buffer, pH 7.4, and 10 recentrifuged at 105,000 x g for one hour to wash the microsomes. This washing procedure is 11 repeated one additional time. The twice-washed microsomal pellet is resuspended in 0.1 M 12 sodium phosphate buffer, pH 7.4, containing 0.25 M sucrose, 20% glycerol, and 0.05 mM 13 dithiothreitol, and then frozen at -80°C until needed. Under these storage conditions, the 14 microsomal suspension retains aromatase activity for more than one year. For the enzyme assay, 15 the microsomal suspension is thawed and resuspended in 0.1 M sodium phosphate buffer, pH 16 7.4, at the appropriate microsomal protein concentration determined using standard methods. 17 Alternatively, a recombinant human aromatase in microsomal preparations from stably­ 18 transfected insect cells is commercially available.75 19 20 Chemical/product Evaluated. The chemical and/or product to be evaluated will be fully 21 characterized prior to use in the aromatase assay. The chemical and/or product name will be 22 identified and, if a mixture, information on all components will be provided. The CAS registry 23 number, chemical and/or product class, and supplier/source will also be identified. 24 Physical/chemical characteristics directly related to test performance (e.g., water and lipid

25 solubility, pH, pKa, etc.), stability of test material in test medium, and purity will also be 26 provided. 27 28 Method of Exposure. A stock solution of the chemical and/or product to be evaluated 29 will be dissolved in 0.1 M sodium phosphate buffer, pH 7.4. If the test chemical is insoluble in 30 aqueous solution, then the chemical will be dissolved in ethanol or DMSO and diluted to the 31 appropriate concentration in 0.1 M sodium phosphate buffer, pH 7.4, with the amount of ethanol 32 or DMSO less than 0.5%. 33 34 Dose Selection Procedures and Number of Replicates. A screening assay to 35 determine initial aromatase inhibition will be performed at concentrations of 0.1, 1.0, and 10 µM 36 in quadruplicate. For compounds exhibiting 10% inhibitory activity or more, full dose-response 37 studies will be performed. Full dose-response studies of the chemical and/or product will be 38 performed at doses from 1.0 nM (1.0 x 10-9 M) to 1.0 mM (1.0 x 10-3 M). Accurate

39 determination of IC50 values for steep dose-response curves may require that the doses tested 40 differ by 0.5 log units (i.e., log values of -9.0, -8.5, -8.0, -7.5, 41 etc.), and the number of replicates to be used is four. Solubility of lipophilic agents may limit 42 the concentration range in the assays to 1.0 or 10.0 µM. 43 44 Controls. Both positive and negative controls are required. Negative controls (blank)

Battelle Draft 33 February 25, 2002 1 consist of the use of boiled diluted microsomal suspension. Incubations with the substrate only 2 is a positive control and identifies the maximum aromatase activity present under the particular 3 enzyme assay conditions. Under these assay conditions using a frozen microsomal preparation 4 as the enzyme source, the range of aromatase activity is typically 40 to 70 pmol (10 to 19 ng) 5 product formed per mg microsomal protein per minute. Equally important in assays is the 6 inclusion of reference compounds, such as a potent steroidal inhibitor, such as 4­ 7 hydroxyandrostenedione, exemestane, or a 7α-substituted androstenedione, or a potent 8 nonsteroidal inhibitor such as anastrozole or letrozole. The concentration of the potent 9 will be 0.1 µM. 10 11 Test Conditions. Incubations will be performed in 16x100 mm test tubes maintained at 12 37°C in a shaking water bath. An aliquot (100 µL) of propylene glycol will be added to the 13 tubes to serve as a co-solvent. The substrate, [1β-3H]-androstenedione (0.1 µCi, 50 nM), and the 14 inhibitor at test concentrations will be added to the tubes. An NADPH-generating system 15 comprised of NADP+ (1.7 mM), glucose-6-phosphate (2.8 mM) and glucose-6-phosphate 16 dehydrogenase (1.0 units) will be added to each tube. The tubes will be placed at 37°C in the 17 water bath for five minutes. The assay will begin with the addition of the diluted placental 18 microsomal suspension (~0.1 mg microsomal protein/mL). The total volume will be 2.0 mL, and 19 the tubes will be incubated for 30 minutes. The incubations will be stopped by the addition of 20 chloroform (2.0 mL) and the tubes vortexed for 30 seconds. The tubes will then be centrifuged 21 for 10 minutes at 500 rpm. The aqueous layers will be transferred to new test tubes and 22 extracted again with chloroform (2.0 mL). This extraction procedure will be performed one 23 additional time. The aqueous layers will be transferred to vials, and an aliquot (0.5 mL) will be 24 removed and transferred to a 7.0 mL liquid scintillation counting vial. Liquid scintillation 25 solution (emulsion-type, 5.0 mL) will be added to each counting vial and shaken to mix the 3 3 26 solution. The amount of H2O in each counting vial will be determined using a H counting 27 protocol available on a standard liquid scintillation counter. 28 3 29 Endpoint Measured. The amount of H2O formed in each assay tube will be determined 30 from the 3H counts per minute (cpm) detected by the counter. The amount of disintegrations per 31 minute (dpm) will be calculated by dividing the cpm by the 3H counting efficiency for the liquid 32 scintillation counter (determined using certified sealed 3H standards). The average dpm value of 33 blank (negative control) will be subtracted from the dpm value of each tube to give net dpm of 3 34 H2O detected. 35 3 36 Data Collected. The total amount of H2O formed in each assay tube will be calculated 3 37 from the net dpm of H2O detected by LSC, corrected for the volume of the aliquot sampled. 38 The amount of estrogen product formed in the microsomal assay is determined by dividing the 3 3 39 total amount of H2O formed by the specific activity of the [1β- H]-androstenedione substrate 40 (expressed in nmol/dpm). The velocity of the enzyme reaction is expressed in nmol mg protein-1 41 min-1 and is calculated by dividing the amount of estrogen formed by the product of mg 42 microsomal protein used times 30 minutes. 43 44

Battelle Draft 34 February 25, 2002 1 Incubations with the substrate only is a positive control and identifies the maximum 2 aromatase activity or maximum amount of estrogen formed (100% activity) present under the 3 particular enzyme assay conditions. The percentage of enzyme activity for each sample is 4 calculated by dividing the amount of estrogen formed in the sample by the maximum amount of 5 estrogen formed in the positive control. The percentage of aromatase inhibition is determined by 6 subtracting the percentage enzyme activity in each sample from 100%. 7 8 Quality Assurance Guidelines. Quality assurance (QA) guidelines should be established 9 to ensure that the assay is running at optimal conditions. Factors that influence optimal assay 10 conditions include sufficient substrate concentrations, sufficient cofactor concentrations, 11 microsomal protein concentrations, test agent solubility, positive controls, and methods to detect 12 inter-assay variability. 13 14 Known False Negatives, Known False Positives. Using the proper negative and 15 positive controls, the only false positive for aromatase inhibition would be the decrease in 16 enzyme activity as the result of enzyme degradation produced by a test chemical, such as an 17 organic solvent. 18 19 Sensitivity of the Assay, Lowest Level of Detection. The microsomal aromatase assay 20 can measure as low as 0.20 pmol of estrogen formed per mg microsomal protein per minute. The 21 minimum limit of aromatase inhibition that can be measured is a difference of 0.20 pmol of 22 estrogen formed per mg microsomal protein per minute. 23 24 Statistical Methods. The means, standard errs, and standard deviations are calculated 25 for each concentration of test compound. The unpaired student t-test is used to determine 26 statistically significant differences with a p value of >0.05 between test compound and positive 27 controls. The full dose-response studies will be analyzed by nonlinear regression analysis, and

28 dose-response curves will be generated. The IC50 values are determined from dose-response 29 studies, and the log IC50 values, standard err of the log IC50 values, and the correlation coefficient 30 will also be obtained in this analysis. Standard nonlinear regression analysis of sigmoidal dose­ 31 response curves are available on commercial software packages such as SigmaPlot or GraphPad 32 Prism. 33

34 Decision Criteria. The activity of a test compound will be classified based upon its IC50 35 value. Common convention in the literature is comparison of the IC50 value for a particular agent

36 relative to the IC50 value for aminoglutethimide (range from 1.0 to 8.0 µM). For an approximate 37 comparison of agents, the following terms are frequently used by investigators in the field: 38 39 no activity no aromatase inhibition measured

40 weak activity IC50 value of greater than 20 µM

41 moderate activity IC50 value between 1 and 20 µM

42 strong activity IC50 value less than 1 µM 43 44

Battelle Draft 35 February 25, 2002 1 5.1.3 Strengths of Placental Microsomal Aromatase Assay 2 3 The human placental microsomal aromatase assay is one of the most common in vitro 4 assays used for measuring aromatase and aromatase inhibition because of its reliability, 5 reproducibility, and ease of use. The overall protocol of the assay is straightforward for 6 individuals with undergraduate laboratory experiences in biology, biochemistry, and/or 7 molecular biology. 8 9 The route of administration/exposure of the test compounds involve dissolving an 10 accurate amount of the compound in ethanol or DMSO and pipetting into appropriate test tubes. 11 The recommended dosing period or duration is 30 minutes. 12 13 The measurements of endpoints directly determines the amount of aromatase activity 14 present and the amount of inhibition. The assay involves the use of liquid scintillation counting, 15 which provides excellent efficiency (50%) for the detection of 3H radioisotope used in this assay. 16 17 5.1.4 Weaknesses And/or Limitations of Placental Microsomal Aromatase Assay 18 19 This in vitro enzyme assay is limited to measuring the inhibitory effects of the test agent 20 on aromatase enzymatic activity. As described, it is not an enzyme kinetic assay and will not 21 determine the mechanism of aromatase inhibition, i.e., competitive, noncompetitive, or 22 irreversible. Also, it will not provide any information on the effect of the test agent on the 23 expression of the aromatase protein, i.e., if the test compound will induce or suppress the 24 production of new aromatase protein. 25 26 The microsomal fraction of cells is comprised of the smooth endoplasmic reticulum 27 membrane, which also contains other steroid metabolizing enzymes (such as 17β-hydroxysteroid 28 dehydrogenase) and other cytochrome P450 enzymes. The test compounds may influence 29 (inhibit) the activity of these “competing” enzymes, and cause indirect changes in the level of 30 aromatization without any interaction with aromatase enzyme. 31 32 A limitation of the assay may arise regarding route of administration/exposure of the test 33 compounds if the test compound is insoluble in aqueous solutions and sparingly soluble in 34 ethanol or DMSO. This solubility problem may limit the doses used in the dose-response 35 studies. 36 37 Another limitation is the protein degradation and the loss of aromatase enzymatic 38 activity. This can be minimized by storage of isolated placental microsomes in small aliquots at 39 -80 °C, with the level of enzyme activity stable for approximately 6 to 12 months. 40 41 The assay does utilize radioactivity (3H) and the limitations of the use of radioactive 42 materials exist (licensing, record-keeping, personnel training, storage, disposal, etc.). 43 44 A final potential limitation is the access and availability of human term placenta as a 45 tissue source. Use of discarded human tissues falls under regulations for research involving

Battelle Draft 36 February 25, 2002 1 human subjects, and thus the research protocols require approval from an institutional human 2 subjects review committee. Additionally, access to such tissues may be limited to certain 3 institutions (e.g., universities but not industry) depending upon hospital policies, and access 4 requires a nearby hospital with a delivery ward. 5 6 Variability in the levels of aromatase cytochrome P450 per mg microsomal protein and in 7 levels of overall cytochrome P450 per mg microsomal protein among placental tissues exist and 8 can also be a concern. Examples include patient heterogeneity and differences between placental 9 tissues obtained from smokers vs. non-smokers. No aromatase isozymes exist in humans, and 10 only a few mutations of the enzyme have been reported in humans. 11 12 5.1.5 Test Method Performance and Test Method Reliability 13 14 An active test compound is evaluated in a full dose-response study (1.0 nM to 1.0 mM) 15 with quadruplicate samples at each dose concentration tested. The full dose-response studies 16 will be analyzed by nonlinear regression analysis, and dose-response curves will be generated.

17 The IC50 values are determined from dose-response studies, and the log IC50 values, standard err 18 of the log IC50 values, and the correlation coefficient will also be obtained in this analysis. 19 20 This assay endpoint, i.e., measurement of aromatase activity, is accurate and 21 reproducible. The human placental microsomal aromatase assay is one of the most common in 22 vitro assays used for measuring aromatase and aromatase inhibition.25-38 The assay has been 23 utilized extensively and performed reproducibly since the late 1970's. It continues to be used in 24 academic labs and pharmaceutical firms as the initial biological evaluation for potential steroidal 25 and nonsteroidal aromatase inhibitors. The endpoint of the measurement of aromatase activity by 3 26 the radiometric assay ( H2O method) is accurate as the assay measurement and is in agreement 27 with the product isolation method. 28 29 The relative inhibitory activities of aromatase inhibitors in the human placental

30 microsomal assay are similar between various laboratories. However, the actual IC50 values vary 31 from lab to lab due to variable experiment conditions employed in the particular laboratory. The

32 table below illustrates the variability in the IC50 values for aminoglutethimide and for several 33 flavonoids examined in five different laboratories using the human placental microsomal 34 aromatase assays. Experimental conditions varied greatly among the five laboratories, and key 35 differences were observed in substrate concentrations (from 13 nM to 875 nM) and microsomal 36 protein concentrations (from 0.05 mg/mL to 10 mg/mL). Since both the substrate 37 androstenedione and many of the flavonoids are lipophilic, such variability in microsomal lipid 38 membrane preparations in the assay can significant affect the actual concentrations of substrate

39 and inhibitor and result in varying IC50 values. 40

Battelle Draft 37 February 25, 2002 1 Table 2. Comparisons of IC50 Values for Flavonoids 2 3 compound LeBail et al. Wang et Pelissero et Jeong et al. Chen et 200086 al. 199488 al. 199691 199983 al. 1997150 4 aminoglutethimide* 1.0 µM 5.0 µM 130.0 µM 1.6 µM 5.5 µM 5 7,8-dihydroxyflavone 8.0 µM 8.8 µM 55.0 µM 6 chrysin 0.7 µM 4.4 µM 11.0 µM 7 17.0 µM 25.0 µM 8 flavone 48.0 µM 375.0 µM 9 flavanone 250.0 µM 34.8 µM 10

11 *Aminoglutethimide is used as a known inhibitor for comparisons, with IC50 values typically ranging 12 from 1.0 to 10.0 µM. 13 14

Battelle Draft 38 February 25, 2002 1 5.2 CANDIDATE ASSAY B - AROMATASE ACTIVITY IN CELL LINES 2 3 5.2.1 Flow Diagram of the Aromatase Assay in JEG-3 Cells 4 5

JEG-3 cells in 12-well plates with DMEM/F-12 media containing 10% FCS and Preparation of supplemented media containing: gentamycin (20 mg/mL)

100.0 nM 3H-androstenedione (0.2 µCi) x.x µM test compound Remove media

Add media (2.0 mL) containing substrate and test compound to each well

Incubate at 37°C for 2 hours in 5% CO2 atmosphere

Transfer media to 16x100 mL test tube; add 2.0 mL of CHCl3; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

Transfer aqueous layer to a new test tube;

add 2.0 mL of CHCl3 to quench enzyme reaction; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

Transfer aqueous layer to a new test tube;

add 2.0 mL of CHCl3 to quench enzyme reaction; vortex for 30 seconds; centrifuge for 10 minutes at 500 rpm

Transfer aqueous layer to a capped vial; transfer 0.5 mL to a LSC vial; add 5.0 mL LSC solution; count in LSC

Battelle Draft 39 February 25, 2002 1 5.2.2 Description of the Aromatase Assay in Cell Lines 2 3 Source of Cell Lines. Human JEG-3 cells, ATCC no. HTB-36, are obtained from 4 American Tissue Culture Collection. The cells are maintained in DMEM/F-12 media 5 supplemented with 10% heat-inactivated fetal calf serum and gentamycin (20 mg/mL) and

6 incubated at 37°C in a 5% CO2 atmosphere. For the determination of aromatase inhibition, JEG­ 7 3 cells are seeded into 12-well culture plates at a concentration of 50,000 cells per well in 2.0 mL

8 of supplemented DMEM/F-12 media. The cells are incubated at 37°C in a 5% CO2 atmosphere 9 for 2 days, reaching approximately 40-50% confluency. 10 11 Chemical/product Evaluated. The chemical and/or product to be evaluated will be fully 12 characterized prior to use in the aromatase assay. The chemical and/or product name will be 13 identified and, if a mixture, information on all components will be provided. The CAS registry 14 number, chemical and/or product class, and supplier/source will also be identified. 15 Physical/chemical characteristics directly related to test performance (e.g., water and lipid

16 solubility, pH, pKa, etc.), stability of test material in test medium, and purity will also be 17 provided. 18 19 Method of Exposure. A stock solution of the chemical and/or product to be evaluated 20 will be dissolved in 0.1 M sodium phosphate buffer, pH 7.4. If the test chemical is insoluble in 21 aqueous solution, then the chemical will be dissolved in ethanol or DMSO and diluted to the 22 appropriate concentration in 0.1 M sodium phosphate buffer, pH 7.4, such that the amount of 23 ethanol or DMSO is less than 0.5%. 24 25 Dose Selection Procedures and Number of Replicates. A screening assay to determine 26 initial aromatase inhibition will be performed at concentrations of 0.1, 1.0, and 10.0 µM in 27 quadruplicate. For compounds exhibiting 10% inhibitory activity or more, full dose-response 28 studies will be performed. Full dose-response studies of the chemical and/or product will be 29 performed at doses from 1.0 nM (1.0 x 10-9 M) to 1.0 mM (1.0 x 10-3 M). Accurate

30 determination of IC50 values for steep dose-response curves may require that the doses tested 31 differ by 0.5 log units (i.e., log values of -9.0, -8.5, -8.0, -7.5, etc.), and the number of replicates 32 to be used is four. Solubility of lipophilic agents may limit the concentration range in the assays 33 to 1.0 or 10.0 µM. 34 35 Controls. Both positive and negative controls are required. Negative controls (blank) 36 consist of the media only (no cells). Incubations with the substrate only is a positive control and 37 identifies the maximum aromatase activity present under the particular enzyme assay conditions. 38 Under these assay conditions, the range of aromatase activity is typically 20.0 to 25.0 pmol (5.4 39 to 6.8 ng) product formed per mg cellular protein per hour. Equally important in assays is the 40 inclusion of reference compounds, such as a potent steroidal inhibitor, such as 4­ 41 hydroxyandrostenedione, exemestane, or a 7α-substituted androstenedione, or a potent 42 nonsteroidal inhibitor such as anastrozole or letrozole. The concentration of the potent 43 aromatase inhibitor will be 0.1 µM. 44

Battelle Draft 40 February 25, 2002 1 Test Conditions. At the beginning of the experiment, the JEG-3 cells in 12-well plates 2 are taken from the cell culture incubator and the media removed. Fresh supplemented media (2.0 3 mL) is added to the wells along with the substrate, [1β-3H]-androstenedione (0.2 µCi, 100 nM), 4 and the inhibitor at test concentrations will be added to the tubes. The cells are then incubated at

5 37°C in a 5% CO2 atmosphere for 2 hours. After 2 hours, the media is transferred to 16x100 mm 6 test tubes, chloroform (2.0 mL) is added to each tube, and the tubes vortexed for 30 seconds. 7 The tubes will then be centrifuged for 10 minutes at 500 rpm. The aqueous layers will be 8 transferred to new test tubes and extracted again with chloroform (2.0 mL). This extraction 9 procedure will be performed one additional time. The aqueous layers will be transferred to vials, 10 and an aliquot (0.5 mL) will be removed and transferred to a 7.0 mL liquid scintillation counting 11 vial. Liquid scintillation solution (emulsion-type, 5.0 mL) will be added to each counting vial 3 12 and shaken to mix the solution. The amount of H2O in each counting vial will be determined 13 using a 3H counting protocol available on a standard liquid scintillation counter. 14 3 15 Endpoint Measured. The amount of H2O formed in each assay tube will be determined 16 from the 3H counts per minute (cpm) detected by the counter. The amount of disintegrations per 17 minute (dpm) will be calculated by dividing the cpm by the 3H counting efficiency for the liquid 18 scintillation counter (determined using certified sealed 3H standards). The average dpm value of 19 blank (negative control) will be subtracted from the dpm value of each tube to give net dpm of 3 20 H2O detected. 21 3 22 Data Collected. The total amount of H2O formed in each assay tube will be calculated 3 23 from the net dpm of H2O detected by LSC, corrected for the volume of the aliquot sampled. The 24 amount of estrogen product formed in the cell-based assay is determined by dividing the total 3 3 25 amount of H2O formed by the specific activity of the [1β- H]-androstenedione substrate 26 (expressed in nmol/dpm). The velocity of the enzyme reaction is expressed in nmol per well. 27 28 Incubations with the substrate only is a positive control and identifies the maximum 29 aromatase activity or maximum amount of estrogen formed (100% activity) present under the 30 particular enzyme assay conditions. The percentage of enzyme activity for each sample is 31 calculated by dividing the amount of estrogen formed in the sample by the maximum amount of 32 estrogen formed in the positive control. The percentage of aromatase inhibition is determined by 33 subtracting the percentage enzyme activity in each sample from 100%. 34 35 Quality Assurance Guidelines. Quality assurance (QA) guidelines should be established 36 to ensure that the assay is running at optimal conditions. Factors that influence optimal assay 37 conditions include sufficient substrate concentrations, number of viable cells, test agent 38 solubility, positive controls, and methods to detect inter-assay variability. 39 40 Known False Negatives, Known False Positives. Using the proper negative and 41 positive controls, the only false positive for aromatase inhibition would be the decrease in 42 enzyme activity as the result of cell cytotoxicity or cell death, such as was observed with certain 43 organochlorines described in Section 3.3.2. 44

Battelle Draft 41 February 25, 2002 1 Sensitivity of the Assay, Lowest Level of Detection. The aromatase activity in JEG-3 2 cells can measure as low as 0.20 pmol of estrogen formed per mg cellular protein per hour. The 3 minimum limit of aromatase inhibition that can be measured is a difference of 0.20 pmol of 4 estrogen formed per mg cellular protein per hour. 5 6 Statistical Methods. The means, standard errs, and standard deviations are calculated 7 for each concentration of test compound. The unpaired student t-test is used to determine 8 statistically significant differences with a p value of >0.05 between test compound and positive 9 controls. The full dose-response studies will be analyzed by nonlinear regression analysis, and

10 dose-response curves will be generated. The IC50 values are determined from dose-response 11 studies, and the log IC50 values, standard err of the log IC50 values, and the correlation coefficient 12 will also be obtained in this analysis. Standard nonlinear regression analysis of sigmoidal dose­ 13 response curves are available on commercial software packages such as SigmaPlot or GraphPad. 14

15 Decision Criteria. The activity of a test compound will be classified based upon its IC50 16 value. Common convention in the literature is comparison of the IC50 value for a particular agent

17 relative to the IC50 value for aminoglutethimide (range from 1.0 to 8.0 µM). For an approximate 18 comparison of agents, the following terms are frequently used by investigators in the field: 19 20 no activity no aromatase inhibition measured

21 weak activity IC50 value of greater than 20 µM

22 moderate activity IC50 value between 1 and 20 µM

23 strong activity IC50 value less than 1 µM 24 25 5.2.3 Strengths of the Aromatase Assay in Cell Lines 26 27 Human JEG-3 choriocarcinoma cell culture lines have been used as in vitro systems for 28 determination of the effects of compounds on aromatase activity and for investigations on the 29 effects of agents in placental toxicology. The assay involves standard mammalian cell culture 30 techniques and can be performed reliably by individuals with cell biology and culturing 31 experiences. 32 33 The route of administration/exposure of the test compounds involve dissolving an 34 accurate amount of the compound in ethanol or DMSO and pipetting into appropriate test tubes. 35 The recommended dosing period or duration is 2 hours, but this can be adjusted improve 36 sensitivity or minimize degradation of test agents. The measurements of endpoints directly 37 determines the amount of aromatase activity present and the amount of inhibition. 38 39 The assay involves the use of liquid scintillation counting, which provides excellent 40 sensitivity for this assay. One advantage of this cell culture assay is that it can provide 41 information on the effect of the test agent on the expression of the aromatase protein, i.e., if the 42 test compound will induce or suppress the production of new aromatase protein. 43

Battelle Draft 42 February 25, 2002 1 5.2.4 Weaknesses And/or Limitations of the Aromatase Assay in Cell Lines 2 3 This in vitro cell culture assay is limited to measuring the effect of the test agent on 4 aromatase enzymatic activity. It is not an enzyme kinetic assay and will not determine the 5 mechanism of aromatase inhibition, i.e., competitive, noncompetitive, or irreversible. 6 7 Aromatase activity in JEG-3 cells varies markedly during the growth phase, particularly 8 at confluence. Also, other steroid metabolizing enzymes are present in JEG-3 cells. The labeled 9 substrate may be metabolized by other enzymes, thus not allowing its aromatization (i.e. the 10 amount of substrate available for aromatase may be modulated by other enzymes). The test 11 compounds may influence (inhibit) the activity of these “competing” enzymes, and cause 12 indirect changes in the level of aromatization without any interaction with aromatase enzyme. 13 14 Cytotoxicity from the test agents is also a concern and may limit the use of this method in 15 evaluating effects of certain compounds on aromatase activity. An apparent decrease in 16 aromatase activity may arise from cytotoxic effects of the agents, resulting in lower numbers of 17 JEG-3 cells compared to untreated or control-treated cells. 18 19 This assay utilizes various cell culture techniques, and thus is limited to laboratories 20 capable of performing studies with mammalian cell cultures. Essential equipment needed

21 include laminar flow hoods, CO2 incubators, microscopes, liquid nitrogen storage containers, 22 autoclaves, media storage and/or media preparation facilities, etc. 23 24 A limitation of the assay may arise regarding route of administration/exposure of the test 25 compounds if the test compound is insoluble in aqueous solutions and sparingly soluble in 26 ethanol or DMSO. This solubility problem may limit the doses used in the dose-response 27 studies. Additionally, test compounds that are too hydrophilic may not readily pass through 28 biological membranes, whereas highly lipophilic compounds could be preferentially retain in the 29 membrane and insufficient intracellular levels be attained . 30 31 Another limitation is that possible metabolism or other transformations of test 32 compounds may occur within intact cells used in this assay, and thus aromatase inhibition may 33 be due to a metabolite and not the parent agent. On the other hand, this may provide additional 34 information of possible activity of metabolites. 35 36 As stated earlier under strengths, this cell culture assay is that it can provide information 37 on the effect of the test agent on the expression of the aromatase protein. However, since CYP19 38 expression is tissue specific, the JEG-3 cells may represent induction or suppression of CYP19 39 expression only under the control of the placental promoter I.1. 40 41 The assay utilizes the JEG-3 human choriocarcinoma cell line available from the 42 American Type Culture Collection (ATCC), and the catalog number for the JEG-3 cells is ATCC 43 HTB 36. The alternative cell line, JAR human choriocarcinoma cell line, is also obtained from 44 ATCC, and the catalog number for the JEG-3 cells is ATCC HTB 144. In order to obtain these 45 cells, an investigator must complete the required ATCC forms and agree to ATCC guidelines

Battelle Draft 43 February 25, 2002 1 regarding use of the cells. These guidelines include (1) the line will be used for research 2 purposes only, (2) cell lines and their products shall not be sold or used for commercial purposes, 3 and (3) cells will not be distributed further to third parties for sale or use for commercial 4 purposes. These restrictions from ATCC may be a significant limitation related to use of the cell 5 lines as a screening assay system in commercial settings. Further clarification from ATCC will 6 be necessary if this protocol is selected. 7 8 The assay does utilize radioactivity (3H) and the limitations of the use of radioactive 9 materials exist (licensing, record-keeping, personnel training, storage, disposal, etc.). 10 11 5.2.5 Test Method Performance and Test Method Reliability 12 13 An active test compound is evaluated in a full dose-response study (1.0 nM to 1.0 mM) 14 with quadruplicate samples at each dose concentration tested. The full dose-response studies 15 will be analyzed by nonlinear regression analysis, and dose-response curves will be generated.

16 The IC50 values are determined from dose-response studies, and the log IC50 values, standard err 17 of the log IC50 values, and the correlation coefficient will also be obtained in this analysis. 18 19 This assay endpoint, i.e., measurement of aromatase activity, is accurate and 20 reproducible. The JEG-3 cell culture assay is a common in vitro assays used for measuring 21 aromatase and aromatase inhibition.25-38 The assay has been utilized extensively and performed 22 reproducibly since the late 1980's. It continues to be used in academic labs and pharmaceutical 23 firms for aromatase inhibition and for investigations on the effects of agents in placental 24 toxicology. The endpoint of the measurement of aromatase activity by the radiometric assay 3 25 ( H2O method) is accurate as the assay measurement and is in agreement with the product 26 isolation method. 27 28 The relative inhibitory activities of aromatase inhibitors in JEG-3 cell culture assay are 29 similar to the activities observed in human placental microsomal assays. However, the actual

30 IC50 values can vary from lab to lab due to variable experiment conditions, such as substrate 31 concentrations, cell number, and culture conditions, employed in the particular laboratory. 32 33 Table 3. Comparison of the Two Candidate Assays 34 35 Placental Microsomal Aromatase Assay Aromatase Activity in JEG-3 Cells 36 37 Strengths: Strengths: 38 Common assay in use for over 40 years Standard mammalian cell culture techniques used 39 Straightforward enzyme assay Available cell line from ATCC 40 Directly measures inhibitory activity of test compounds Excellent sensitivity using 3H radioisotope 41 Readily availability of placental tissues Can measure ability of test compounds to induce or to 42 Excellent sensitivity using 3H radioisotope suppress aromatase mRNA or protein levels 43 44 Weaknesses: Weaknesses: 45 Does not determine mechanism of inhibition Does not determine mechanism of inhibition 46 Does not measure ability of test compounds to induce Aromatase activity varies markedly during cell 47 or suppress enzyme levels growth phase

Battelle Draft 44 February 25, 2002 1 Microsomal preparations contain other enzymes, Cytotoxicity of test compounds to the cells 2 including other cytochrome P450s Other enzymes present in cells that metabolize substrate 3 Requires solubility of test compounds in enzyme Other enzymes present in cells that can metabolize test 4 solution compounds 5 Activity altered by protein degradation Regulation of aromatase mRNA or protein levels is 6 Patient variability of cytochrome P450 tissue-specific, thus, any induction or suppression by 7 content in microsomes test compounds may not represent other tissues 8 Limitations with the use of radioactive materials Limitations with the use of radioactive materials 9 Potential limitation of ATCC cell line for commercial use 10 Requires solubility of test compounds in enzyme in 11 solution 12 Requires test compounds to permeate cell membrane 13 14

Battelle Draft 45 February 25, 2002 1 6.0 IMPLEMENTATION CONSIDERATIONS 2 3 6.1 ESTABLISHMENT OF ASSAY 4 5 Candidate Assay A - placental microsomal aromatase: The assay is rather 6 straightforward to establish. Standard equipment in a biochemistry laboratory are needed. This 7 equipment includes a low speed (clinical) bench-top centrifuge, a high speed centrifuge, an 8 ultracentrifuge, a shaking constant-temperature water bath, pH meter, and a liquid scintillation 9 counter. General materials and supplies include a mechanical homogenizer, glassware, test 10 tubes, vials, [1β-3H]-androstenedione, biochemicals (androstenedione, NADP+, glucose-6­ 11 phosphate, glucose-6-phosphate dehydrogenase), pipettes, buffers, reagents, and organic 12 solvents. Personnel performing the assays would require training in general 13 biology/biochemistry techniques, in enzyme assay methods, and in the use of radioactive 14 materials. 15 16 Candidate Assay B - aromatase assay in cell lines: The assay is rather straightforward 17 to establish. Standard equipment in a cell biology laboratory are needed. This equipment

18 includes laminar flow hoods, CO2 incubators, microscopes, liquid nitrogen storage containers, 19 autoclaves, media storage and/or media preparation facilities, a low speed (clinical) bench-top 20 centrifuge, a high speed centrifuge, a constant-temperature water bath, pH meter, and a liquid 21 scintillation counter. General materials and supplies include a mechanical homogenizer, 22 glassware, cell culture plates, vials, [1β-3H]-androstenedione, androstenedione, DMEM/F-12 23 media, fetal calf serum, gentamycin, pipettes, buffers, reagents, and organic solvents. Personnel 24 performing the assays would require training in general cell biology, cell culturing/sterile 25 techniques, and in the use of radioactive materials. 26 27 6.2 COST/TIME REQUIRED 28 29 Candidate Assay A - placental microsomal aromatase: The time required to establish 30 the assay in a properly equipped biochemistry laboratory would involve one to two months. 31 Each assay, from initiation to data analysis, requires approximately 12-16 hours of labor, and up 32 to 60-100 samples (15-25 doses in quadruplicate) can performed in one assay by one 33 experienced individual. Microsomal preparations from one placenta provide enough material for 34 2 - 3 months. The recurring costs for operation of the assay (excluding personnel and 35 equipment) are approximately $2,000 to $3,000 per month for radioactive materials, LSC 36 supplies, biochemical reagents, and general supplies. 37 38 Candidate Assay B - aromatase assay in cell lines: The time required to establish the 39 assay in a properly equipped biochemistry laboratory would involve three to four months. Cell 40 cultures must be maintained on a daily basis, and cells are plated for an assay 3 to 4 days in 41 advance. Each assay, from initiation to data analysis, requires approximately 12-16 hours of 42 labor, and up to 60-100 samples (15-25 doses in quadruplicate) can performed in one assay by 43 one experienced individual. The recurring costs for operation of the assay (excluding personnel 44 and equipment) are approximately $4,000 to $6,000 per month for radioactive materials, LSC

Battelle Draft 46 February 25, 2002 1 supplies, cell culture media and sera, culture plasticware, and general supplies.. 2 3 6.3 ANIMAL WELFARE CONSIDERATIONS 4 5 The human placental microsomal aromatase assay uses an enzyme preparation obtained 6 from human term placenta. The cell culture assay uses a stable human choriocarcinoma cell line, 7 JEG-3 cells. The benefit of these assays are that no animals are utilized. For comparison, 8 microsomal preparations from adult rat ovarian tissues typically provide approximately 1.5 mg 9 of microsomal protein per ovary. The level of aromatase activity in rat ovarian microsomes is 10 approximately 6.0 pmol per mg microsomal protein per minute, which is 10% of the activity 11 present in human placental microsomes. One adult female rat would provide ovarian microsomal 12 preparations sufficient for approximately three or four assay tubes, the equivalent of one dose in 13 quadruplicate. Thus, a screening assay for one chemical test compound would use 5 female rats, 14 and a full dose-response curve for one chemical test compound would use a minimum of 10 15 female rats. 16 17 6.4 HUMAN SUBJECTS CONSIDERATIONS 18 19 The human placental microsomal aromatase assay uses an enzyme preparation obtained 20 from normal human term placenta, tissue that is discarded after delivery. Use of discarded 21 human tissues falls under regulations for research involving human subjects, and thus the 22 research protocols require approval from an institutional human subjects review committee. 23 24 6.5 ASSURANCE OF BIOLOGICAL TEST (IN VITRO MODEL) VIGOR AND STABILITY 25 26 As stated earlier, these two assays (the human placental microsomal aromatase assay and 27 the JEG-3 cell culture assay) are common in vitro assays used for measuring aromatase and 28 aromatase inhibition because of their reliability, reproducibility, and ease of use. However, the 29 assays are in vitro tests that specifically measure the ability of test agents to inhibit aromatase 30 activity. The assessment of risk of human exposure to potential endocrine disruptors in the 31 environment can not be determined by in vitro experiments alone. Such screening results can 32 alert us to the need for further examination of the compounds’ potential for inducing endocrine 33 disruption after in vivo exposure. This analysis should also include any knowledge of human 34 exposure from use of the compounds to provide a proper risk assessment. 35

Battelle Draft 47 February 25, 2002 1 7.0 RECOMMENDED PROTOCOL 2 3 The human placental microsomal aromatase assay is recommended as the initial in vitro 4 aromatase screening assay to be one of the Tier 1 Screening Battery Alternate Methods. This 5 assay will enable the assessment of the effects of various environmental toxicants as an 6 alternative screening method for possible inclusion in the Tier 1 Screening Battery. The human 7 placental microsomal aromatase assay is recommended based upon ease in establishing the 8 assay, use of standard biochemistry laboratory equipment, ready access to the source tissues, and 9 standard level of training in general biology and/or biochemistry techniques for personnel 10 performing the assays. Also, use of the human placental microsomal aromatase assay has an 11 advantage over the use of the JEG-3 cell based assay since the use of JEG-3 cells must adhere to 12 ATCC guidelines (e.g., research purposes only and no use for commercial purposes). Although 13 this cell culture assay can provide information on the effect of the test agent on the expression of 14 CYP19 mRNA and aromatase protein, the JEG-3 cells may represent induction or suppression of 15 CYP19 expression only under the control of the placental promoter I.1. Since CYP19 expression 16 is tissue specific, studies in JEG-3 cells will not provide information on induction or suppression 17 of CYP19 mRNA and aromatase protein in other critical tissues such as ovary, breast, brain, 18 adipose, and bone. 19 20 The overall protocol of the assay is straightforward, and the initial screening assay to 21 determine initial aromatase inhibition will be performed at concentrations of environmental 22 toxicant at 0.1, 1.0, and 10.0 µM in quadruplicate. For compounds exhibiting 10% inhibitory 23 activity or more, full dose-response studies will be performed. Full dose-response studies of the 24 chemical and/or product will be performed at doses from 1.0 nM (1.0 x 10-9 M) to 1.0 mM (1.0 x 25 10-3 M). The doses tested differ by 0.5 log units (i.e., log values of -9.0, -8.5, -8.0, -7.5, etc.), 26 and the number of replicates to be used is four. The route of administration/exposure of the test 27 compounds involve dissolving an accurate amount of the compound in ethanol or DMSO and 28 pipetting into appropriate test tubes. Incubations will be performed in 16x100 mm test tubes 29 maintained at 37°C in a shaking water bath. An aliquot (100 µL) of propylene glycol will be 30 added to the tubes to serve as a co-solvent. The substrate, [1β-3H]-androstenedione (0.1 µCi, 50 31 nM), and the inhibitor at test concentrations will be added to the tubes. An NADPH-generating 32 system comprised of NADP+ (1.7 mM), glucose-6-phosphate (2.8 mM) and glucose-6-phosphate 33 dehydrogenase (1.0 units) will be added to each tube. The tubes will be placed at 37°C in the 34 water bath for five minutes. The assay will begin with the addition of the diluted placental 35 microsomal suspension (~0.1 mg microsomal protein/mL). The total volume will be 2.0 mL, and 36 the tubes will be incubated for 30 minutes. The incubations will be stopped by the addition of 37 chloroform (2.0 mL) and the tubes vortexed for 30 seconds. The tubes will then be centrifuged 38 for 10 minutes at 500 rpm. The aqueous layers will be transferred to new test tubes and 39 extracted again with chloroform (2.0 mL). This extraction procedure will be performed one 40 additional time. The aqueous layers will be transferred to vials, and an aliquot (0.5 mL) will be 41 removed and transferred to a 7.0 mL liquid scintillation counting vial. Liquid scintillation 42 solution (emulsion-type, 5.0 mL) will be added to each counting vial and shaken to mix the 3 3 43 solution. The amount of H2O in each counting vial will be determined using a H counting 44 protocol available on a standard liquid scintillation counter.

Battelle Draft 48 February 25, 2002 1 Both positive and negative controls are required. Negative controls (blank) consist of the 2 media only (no cells). Incubations with the substrate only is a positive control and identifies the 3 maximum aromatase activity present under the particular enzyme assay conditions. Under these 4 assay conditions, the range of aromatase activity is typically 20.0 to 25.0 pmol (5.4 to 6.8 ng) 5 product formed per mg cellular protein per hour. Equally important in assays is the inclusion of 6 reference compounds, such as a potent steroidal inhibitor, such as 4-hydroxyandrostenedione, 7 exemestane, or a 7α-substituted androstenedione, or a potent nonsteroidal inhibitor such as 8 aminoglutethimide, anastrozole, or letrozole. 9 10

Battelle Draft 49 February 25, 2002 1 8.0 DEVELOPMENTAL STATUS OF THE ASSAY 2 3 8.1 CURRENT STATUS 4 5 The various endpoints included in the proposed assay have been evaluated in other 6 studies. However, the protocol itself has not been validated. Pending a final decision on the 7 study design, the protocol would be ready to enter the pre-validation phase. 8 9 8.2 RECOMMENDATION FOR PRE-VALIDATION STUDIES 10 11 Pre-validation studies following the ICCVAM validation process should be initiated. 12 Pre-validation studies should include evaluation of 1-3 chemicals. It is recommended that the 13 study design be performed using known aromatase inhibitors as positive control compounds, 14 such as a potent steroidal inhibitor, such as 4-hydroxyandrostenedione, exemestane, or a 7α ­ 15 substituted androstenedione, or a potent nonsteroidal inhibitor such as aminoglutethimide, 16 anastrozole, or letrozole. As cited earlier in Section 4.1.5, the concentrations of substrate and

17 microsomal protein used in the assays can influence the IC50 values obtained. Therefore, pre­ 18 validation studies should be performed to identify the optimal concentrations of substrate, 19 microsomal protein, and co-factors (i.e., the NADPH-generating system). 20 21 8.3 RECOMMENDATION FOR VALIDATION STUDIES 22 23 Validation of the study design through interlaboratory comparisons should be conducted 24 with compounds - aromatase inhibitors and test agents as putative endocrine disruptors. 25 26 27 28 Prepared by: 29 30 Robert W. Brueggemeier, Ph.D., The Ohio State University, Columbus, OH 31 Carol S. Sloan, M.S., Research Triangle Institute, Research Triangle Park, NC 32

Battelle Draft 50 February 25, 2002 1 9.0 REFERENCES 2

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2 110. Afonso, L. O., Campbell, P. M., Iwama, G. K., Devlin, R. H., and Donaldson, E. M. The 3 effect of the aromatase inhibitor fadrozole and two polynuclear aromatic hydrocarbons on 4 sex steroid secretion by ovarian follicles of coho salmon. Gen.Comp Endocrinol., 106: 5 169-174, 1997.

6 111. Rocha Monteiro, P. R., Reis-Henriques, M. A., and Coimbra, J. Polycyclic aromatic 7 hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder (Platichthys flesus 8 L.). Aquatic.Toxicol., 48: 549-559, 2000.

9 112. Lovekamp, T. N. and Davis, B. J. Mono-(2-ethylhexyl) phthalate suppresses aromatase 10 transcript levels and estradiol production in cultured rat granulosa cells. 11 Toxicol.Appl.Pharmacol., 172: 217-224, 2001.

12 113. Miller, W. R. and O'Neill, J. The importance of local synthesis of estrogen within the 13 breast. Steroids, 50: 537-548, 1987.

14 114. Simpson, E. R. Aromatase 2000: Fifth International Aromatase Conference. 2000. 11-3­ 15 2000.

16 115. Santen, R. J. Aromatase Conference: Future Perspectives. Steroids, 50: 1-665, 1987.

17 116. Brodie, A. M., Brodie, H. J., Callard, G., Robinson, C. H., Roselli, C., and Santen, R. J. 18 Proceedings of the Third International Aromatase Conference. J.Steroid 19 Biochem.Mol.Biol., 44: 321-696, 1993.

20 117. Simpson, E. R., Bhatnagar, A., Brodie, A. M., Brueggemeier, R. W., Chen, S., DeCoster, 21 R., Dowsett, M., Harada, N., Naftolin, F., Osawa, Y., Plourde, P., Reed, M. J., and 22 Santen, R. J. Proceedings of the Fourth International Aromatase Conference. J.Steroid 23 Biochem.Mol.Biol., 61: 107-425, 1997.

24 118. WHO. Pesticide residues in food- 1977 evaluations. Part II-Toxicology. WHO/IPCS/10 25 Sup, 283-303. 1977. World Health Organization, Geneva. International Programme on 26 Chemical Safety.

27 119. WHO. Pesticide residues in food- 1983 evaluations. Part II-Toxicology. WHO/IPCS/61, 28 366-442. 1983. World Health Organization, Geneva. International Programme on 29 Chemical Safety.

30 120. WHO. Pesticide residues in food- 1995 evaluations. Part II-Toxicology. 31 WHO/IPCS/96.48, 75-104. 1995. World Health Organization, Geneva. International 32 Programme on Chemical Safety.

33 121. Hirsch, K. S., Adams, E. R., Hoffman, D. G., Markham, J. K., and Owen, N. V. Studies 34 to elucidate the mechanism of fenarimol-induced infertility in male rats.

Battelle Draft 60 February 25, 2002 1 Toxicol.Appl.Pharmacol., 86: 391-399, 1986.

2 122. Hirsch, K. S., Weaver, D. E., Black, L. J., Falcone, J. F., and MacLusky, N. J. Inhibition 3 of central nervous system aromatase activity: a mechanism for fenarimol-induced 4 infertility in the male rat. Toxicol.Appl.Pharmacol., 91: 235-245, 1987.

5 123. WHO. Pesticide residues in food- 1985 evaluations. Part II-Toxicology. 6 WHO/IPCS/72.2, 199-202. 1985. World Health Organization, Geneva. International 7 Programme on Chemical Safety.

8 124. WHO. Pesticide residues in food- 1989 evaluations. Part II-Toxicology. 9 WHO/IPCS/100.2, 215-235. 1989. World Health Organization, Geneva. International 10 Programme on Chemical Safety.

11 125. Averal, H. I., Stanley, A., Murugaian, P., Palanisamy, M., and Akbarsha, M. A. Specific 12 effect of vincristine on epididymis. Indian J.Exp.Biol., 34: 53-56, 1996.

13 126. Hess, R. A., Bunick, D., and Bahr, J. M. Sperm, a source of estrogen. Environ.Health 14 Perspect., 103 Suppl 7: 59-62, 1995.

15 127. Akbarsha, M. A., Latha, P. N., and Murugaian, P. Retention of cytoplasmic droplet by rat 16 cauda epididymal spermatozoa after treatment with cytotoxic and xenobiotic agents. 17 J.Reprod.Fertil., 120: 385-390, 2000.

18 128. Morcillo, Y. and Porte, C. Evidence of endocrine disruption in the imposex-affected 19 gastropod Bolinus brandaris. Environ.Res., 81: 349-354, 1999.

20 129. Desvages, G. and Pieau, C. Aromatase activity in gonads of turtle embryos as a function 21 of the incubation temperature of eggs. J.Steroid Biochem.Mol.Biol., 41: 851-853, 1992.

22 130. Willingham, E. and Crews, D. The red-eared slider turtle: An animal model for the study 23 of low doses and mixtures. American Zoologist, 40: 421-428, 2000.

24 131. Crews, D. Temperature-dependent sex determination: the interplay of steroid hormones 25 and temperature. Zoolog.Sci., 13: 1-13, 1996.

26 132. Crain, D. A., Guillette, L. J., Jr., Rooney, A. A., and Pickford, D. B. Alterations in 27 steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and 28 experimentally to environmental contaminants. Environ.Health Perspect., 105: 528-533, 29 1997.

30 133. Crain, D. A., Spiteri, I. D., and Guillette, L. J., Jr. The functional and structural 31 observations of the neonatal reproductive system of alligators exposed in ovo to atrazine, 32 2,4-D, or estradiol. Toxicol.Ind.Health, 15: 180-185, 1999.

33 134. Gerstenberger, S. L., Heimler, I., Smies, R., Hutz, R. J., Dasmahapatra, A. K., Tripoli, V.,

Battelle Draft 61 February 25, 2002 1 and Dellinger, J. A. Minimal endocrine alterations in rodents after consumption of lake 2 trout (Salvelinus namaycush). Arch.Environ.Contam Toxicol., 38: 371-376, 2000.

3 135. Li, P. K. and Brueggemeier, R. W. 7-substituted steroidal aromatase inhibitors: structure­ 4 activity relationships and molecular modeling. J.Enzyme Inhib., 4: 113-120, 1990.

5 136. Furet, P., Batzl, C., Bhatnagar, A., Francotte, E., Rihs, G., and Lang, M. Aromatase 6 inhibitors: synthesis, biological activity, and binding mode of azole-type compounds. 7 J.Med.Chem., 36: 1393-1400, 1993.

8 137. Ahmed, S., Davis, P. J., and Owen, C. P. Molecular modeling study of the binding of 9 inhibitors of aromatase to the cytochrome P-450 heme. Drug Design and Discovery, 14: 10 91-102, 1996.

11 138. Laughton, C. A., McKenna, R., Neidle, S., Jarman, M., McCague, R., and Rowlands, M. 12 G. Crystallographic and molecular modeling studies on 3-ethyl-3-(4- pyridyl)piperidine­ 13 2,6-dione and its butyl analogue, inhibitors of mammalian aromatase. Comparison with 14 natural substrates: prediction of enantioselectivity for N-alkyl derivatives. J.Med.Chem., 15 33 : 2673-2679, 1990.

16 139. Nagy, P. I., Tokarski, J., and Hopfinger, A. J. Molecular shape and QSAR analyses of a 17 family of substituted dichlorodiphenyl aromatase inhibitors. J.Chem.Inf.Comput.Sci., 34: 18 1190-1197, 1994.

19 140. Recanatini, M. and Cavalli, A. Comparative molecular field analysis of non-steroidal 20 aromatase inhibitors: an extended model for two different structural classes. 21 Bioorg.Med.Chem., 6: 377-388, 1998.

22 141. Cavalli, A., Greco, G., Novellino, E., and Recanatini, M. Linking CoMFA and protein 23 homology models of enzyme-inhibitor interactions: an application to non-steroidal 24 aromatase inhibitors. Bioorg.Med.Chem., 8: 2771-2780, 2000.

25 142. Graham-Lorence, S., Khalil, M. W., Lorence, M. C., Mendelson, C. R., and Simpson, E. 26 R. Structure-function relationships of human aromatase cytochrome P-450 using 27 molecular modeling and site-directed mutagenesis. J.Biol.Chem., 266: 11939-11946, 28 1991.

29 143. Laughton, C. A., Zvelebil, M. J., and Neidle, S. A detailed molecular model for human 30 aromatase. J.Steroid Biochem.Mol.Biol., 44: 399-407, 1993.

31 144. Saarinen, N., Joshi, S. C., Ahotupa, M., Li, X., Ammala, J., Makela, S., and Santti, R. No 32 evidence for the in vivo activity of aromatase-inhibiting flavonoids. J.Steroid 33 Biochem.Mol.Biol., 78: 231-239, 2001.

34 145. Tekmal, R. R., Ramachandra, N., Gubba, S., Durgam, V. R., Mantione, J., Toda, K., 35 Shizuta, Y., and Dillehay, D. L. Overexpression of int-5/aromatase in mammary glands

Battelle Draft 62 February 25, 2002 1 of transgenic mice results in the induction of hyperplasia and nuclear abnormalities. 2 Cancer Res., 56: 3180-3185, 1996.

3 146. Kirma, N., Gill, K., Mandava, U., and Tekmal, R. R. Overexpression of aromatase leads 4 to hyperplasia and changes in the expression of genes involved in apoptosis, cell cycle, 5 growth, and tumor suppressor functions in the mammary glands of transgenic mice. 6 Cancer Res., 61: 1910-1918, 2001.

7 147. Li, X., Nokkala, E., Yan, W., Streng, T., Saarinen, N., Warri, A., Huhtaniemi, I., Santti, 8 R., Makela, S., and Poutanen, M. Altered structure and function of reproductive organs in 9 transgenic male mice overexpressing human aromatase. Endocrinology, 142: 2435-2442, 10 2001.

11 148. Yue, W. and Brodie, A. M. Mechanisms of the actions of aromatase inhibitors 4­ 12 hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell 13 culture. J.Steroid Biochem.Mol.Biol., 63: 317-328, 1997.

14 149. Bhatnagar, A. S., Brodie, A. M., Long, B. J., Evans, D. B., and Miller, W. R. Intracellular 15 aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors. 16 J.Steroid Biochem.Mol.Biol., 76: 199-202, 2001.

17 150. Chen, S., Kao, Y. C., and Laughton, C. A. Binding characteristics of aromatase inhibitors 18 and phytoestrogens to human aromatase. J.Steroid Biochem.Mol.Biol., 61: 107-115, 19 1997.

20

Battelle Draft 63 February 25, 2002 1 APPENDIX A 2 3 MANUSCRIPTS REVIEWED AND CITED (IN ALPHABETICAL ORDER) 4 5 6 Adlercreutz, H., Bannwart, C., Wahala, K., Makela, T., Brunow, G., Hase, T., Arosemena, P. J., 7 Kellis, J. T., Jr., and Vickery, L. E. Inhibition of human aromatase by mammalian lignans and 8 isoflavonoid phytoestrogens. J.Steroid Biochem.Mol.Biol., 44: 147-153, 1993.

9 Afonso, L. O., Campbell, P. M., Iwama, G. K., Devlin, R. H., and Donaldson, E. M. The effect 10 of the aromatase inhibitor fadrozole and two polynuclear aromatic hydrocarbons on sex steroid 11 secretion by ovarian follicles of coho salmon. Gen.Comp Endocrinol., 106: 169-174, 1997.

12 Ahmed, S., Davis, P. J., and Owen, C. P. Molecular modeling study of the binding of inhibitors 13 of aromatase to the cytochrome P-450 heme. Drug Design and Discovery, 14: 91-102, 1996.

14 Akbarsha, M. A., Latha, P. N., and Murugaian, P. Retention of cytoplasmic droplet by rat cauda 15 epididymal spermatozoa after treatment with cytotoxic and xenobiotic agents. J.Reprod.Fertil., 16 120: 385-390, 2000.

17 Albrecht, E. D. and Pepe, G. J. Placental steroid hormone biosynthesis in primate pregnancy. 18 Endocr.Rev., 11: 124-150, 1990.

19 Averal, H. I., Stanley, A., Murugaian, P., Palanisamy, M., and Akbarsha, M. A. Specific effect of 20 vincristine on epididymis. Indian J.Exp.Biol., 34: 53-56, 1996.

21 Banting, L., Smith, H. J., James, M., Jones, G., Nazareth, W., Nicholls, P. J., Hewlins, M. J., and 22 Rowlands, M. G. Structure-activity relationships for non-steroidal inhibitors of aromatase. 23 J.Enzyme Inhib., 2: 215-229, 1988.

24 Banting, L., Nicholls, P. J., Shaw, M. A., and Smith, H. J. Recent developments in aromatase 25 inhibition as a potential treatment for oestrogen-dependent breast cancer. Prog.Med.Chem., 26: 26 253-298, 1989.

27 Barbieri, R. L., McShane, P. M., and Ryan, K. J. Constituents of cigarette smoke inhibit human 28 granulosa cell aromatase. Fertil.Steril., 46: 232-236, 1986.

29 Bhatnagar, A. S., Brodie, A. M., Long, B. J., Evans, D. B., and Miller, W. R. Intracellular 30 aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors. J.Steroid 31 Biochem.Mol.Biol., 76: 199-202, 2001.

32 Brodie, A. M., Brodie, H. J., Callard, G., Robinson, C. H., Roselli, C., and Santen, R. J. 33 Proceedings of the Third International Aromatase Conference. J.Steroid Biochem.Mol.Biol., 44: 34 321-696, 1993.

Battelle Draft A-1 February 25, 2002 1 Brodie, A. M. and Njar, V. C. Aromatase inhibitors and breast cancer. Semin.Oncol., 23: 10-20, 2 1996.

3 Brodie, A., Lu, Q., and Long, B. Aromatase and its inhibitors. J.Steroid Biochem.Mol.Biol., 69: 4 205-210, 1999.

5 Brodie, A., Inkster, S., and Yue, W. Aromatase expression in the human male. Mol.Cell 6 Endocrinol., 178: 23-28, 2001.

7 Brueggemeier, R. W. Biochemical and molecular aspects of aromatase. J.Enzyme Inhib., 4: 101­ 8 111, 1990.

9 Brueggemeier, R. W. and Katlic, N. E. Aromatase inhibition by an enzyme-activated irreversible 10 inhibitor in human carcinoma cell cultures. Cancer Res., 50: 3652-3656, 1990.

11 Brueggemeier, R. W., Katlic, N. E., Kenreigh, C. A., and Li, P. K. Aromatase inhibition by 7­ 12 substituted steroids in human choriocarcinoma cell culture. J.Steroid Biochem.Mol.Biol., 41: 85­ 13 90, 1992.

14 Brueggemeier, R. W. Aromatase inhibitors--mechanisms of steroidal inhibitors. Breast Cancer 15 Res.Treat., 30: 31-42, 1994.

16 Brueggemeier, R. W., Gilbert, N. E., Gu, X., O'Reilly, J. M., and Lovely, C. J. Aromatase 17 inhibition in JAr choriocarcinoma cells by 7alpha- arylaliphatic androgens. J.Steroid 18 Biochem.Mol.Biol., 61: 73-77, 1997.

19 Brueggemeier, R. W., Quinn, A. L., Parrett, M. L., Joarder, F. S., Harris, R. E., and Robertson, F. 20 M. Correlation of aromatase and cyclooxygenase gene expression in human breast cancer 21 specimens. Cancer Lett., 140: 27-35, 1999.

22 Bulun, S. E., Price, T. M., Aitken, J., Mahendroo, M. S., and Simpson, E. R. A link between 23 breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue 24 aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after 25 reverse transcription. J.Clin.Endocrinol.Metab, 77: 1622-1628, 1993.

26 Callard, G. V., Petro, Z., and Ryan, K. J. Identification of aromatase in the reptilian brain. 27 Endocrinology, 100: 1214-1218, 1977.

28 Callard, G. V. and Tchoudakova, A. Evolutionary and functional significance of two CYP19 29 genes differentially expressed in brain and ovary of goldfish. J.Steroid Biochem.Mol.Biol., 61: 30 387-392, 1997.

31 Campbell, D. R. and Kurzer, M. S. Flavonoid inhibition of aromatase enzyme activity in human 32 preadipocytes. J.Steroid Biochem.Mol.Biol., 46: 381-388, 1993.

Battelle Draft A-2 February 25, 2002 1 Cavalli, A., Greco, G., Novellino, E., and Recanatini, M. Linking CoMFA and protein homology 2 models of enzyme-inhibitor interactions: an application to non-steroidal aromatase inhibitors. 3 Bioorg.Med.Chem., 8: 2771-2780, 2000.

4 Chen, S., Kao, Y. C., and Laughton, C. A. Binding characteristics of aromatase inhibitors and 5 phytoestrogens to human aromatase. J.Steroid Biochem.Mol.Biol., 61: 107-115, 1997.

6 Chen, S., Sun, X. Z., Kao, Y. C., Kwon, A., and Eng, E. Suppression of breast cancer cell growth 7 with grape juice. Pharmaceutical Biology (Netherlands), 36: 53-61, 1998.

8 Chiang, E. F., Yan, Y. L., Guiguen, Y., Postlethwait, J., and Chung, B. Two Cyp19 (P450 9 Aromatase) Genes on Duplicated Zebrafish Chromosomes Are Expressed in Ovary or Brain. 10 Mol.Biol.Evol., 18: 542-550, 2001.

11 Cole, P. A. and Robinson, C. H. Mechanism and inhibition of cytochrome P-450 aromatase. 12 J.Med.Chem., 33: 2933-2942, 1990.

13 Conley, A., Corbin, J., Smith, T., Hinshelwood, M., Liu, Z., and Simpson, E. Porcine 14 aromatases: studies on tissue-specific, functionally distinct isozymes from a single gene? 15 J.Steroid Biochem.Mol.Biol., 61: 407-413, 1997.

16 Cooke, B., Hegstrom, C. D., Villeneuve, L. S., and Breedlove, S. M. Sexual differentiation of the 17 vertebrate brain: principles and mechanisms. Front Neuroendocrinol., 19: 323-362, 1998.

18 Corbin, C. J., Trant, J. M., and Conley, A. J. Porcine gonadal and placental isozymes of 19 aromatase cytochrome P450: sub-cellular distribution and support by NADPH-cytochrome P450 20 reductase. Mol.Cell Endocrinol., 172: 115-124, 2001.

21 Covey, D. F. Aromatase Inhibitors: Specific Inhibitors of Oestrogen Biosynthesis. In D. Berg 22 and M. Plempel (eds.), Sterol Biosynthesis Inhibitors, pp. 534-571. Chichester, UK: Ellis 23 Horwood, 1988.

24 Crain, D. A., Guillette, L. J., Jr., Rooney, A. A., and Pickford, D. B. Alterations in 25 steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to 26 environmental contaminants. Environ.Health Perspect., 105: 528-533, 1997.

27 Crain, D. A., Spiteri, I. D., and Guillette, L. J., Jr. The functional and structural observations of 28 the neonatal reproductive system of alligators exposed in ovo to atrazine, 2,4-D, or estradiol. 29 Toxicol.Ind.Health, 15: 180-185, 1999.

30 Crews, D. Temperature-dependent sex determination: the interplay of steroid hormones and 31 temperature. Zoolog.Sci., 13: 1-13, 1996.

32 Desvages, G. and Pieau, C. Aromatase activity in gonads of turtle embryos as a function of the 33 incubation temperature of eggs. J.Steroid Biochem.Mol.Biol., 41: 851-853, 1992.

Battelle Draft A-3 February 25, 2002 1 Dowsett, M., Macaulay, V., Gledhill, J., Ryde, C., Nicholls, J., Ashworth, A., McKinna, J. A., 2 and Smith, I. E. Control of aromatase in breast cancer cells and its importance for tumor growth. 3 J.Steroid Biochem.Mol.Biol., 44: 605-609, 1993.

4 Drenth, H. J., Bouwman, C. A., Seinen, W., and van den, B. M. Effects of some persistent 5 halogenated environmental contaminants on aromatase (CYP19) activity in the human 6 choriocarcinoma cell line JEG-3. Toxicol.Appl.Pharmacol., 148: 50-55, 1998.

7 Fisher, C. R., Graves, K. H., Parlow, A. F., and Simpson, E. R. Characterization of mice 8 deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. 9 Proc.Natl.Acad.Sci.U.S.A, 95: 6965-6970, 1998.

10 Furet, P., Batzl, C., Bhatnagar, A., Francotte, E., Rihs, G., and Lang, M. Aromatase inhibitors: 11 synthesis, biological activity, and binding mode of azole-type compounds. J.Med.Chem., 36: 12 1393-1400, 1993.

13 Gerstenberger, S. L., Heimler, I., Smies, R., Hutz, R. J., Dasmahapatra, A. K., Tripoli, V., and 14 Dellinger, J. A. Minimal endocrine alterations in rodents after consumption of lake trout 15 (Salvelinus namaycush). Arch.Environ.Contam Toxicol., 38: 371-376, 2000.

16 Graham-Lorence, S., Khalil, M. W., Lorence, M. C., Mendelson, C. R., and Simpson, E. R. 17 Structure-function relationships of human aromatase cytochrome P-450 using molecular 18 modeling and site-directed mutagenesis. J.Biol.Chem., 266: 11939-11946, 1991.

19 Gross, T. S. and Williams, W. F. In vitro steroid synthesis by the placenta of cows during late 20 gestation and at parturition. J.Reprod.Fertil. 83, 565-573. 1988.

21 Harvey, H. A., Lipton, A., and Santen, R. J. Aromatase: New Perspectives for Breast Cancer. 22 Cancer Res., 42: 3261s-3467s, 1982.

23 Hess, R. A., Bunick, D., and Bahr, J. M. Sperm, a source of estrogen. Environ.Health Perspect., 24 103 Suppl 7: 59-62, 1995.

25 Hickey, G. J., Krasnow, J. S., Beattie, W. G., and Richards, J. S. Aromatase cytochrome P450 in 26 rat ovarian granulosa cells before and after luteinization: adenosine 3',5'-monophosphate­ 27 dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5' 28 genomic DNA. Mol.Endocrinol., 4: 3-12, 1990.

29 Hinshelwood, M. M., Liu, Z., Conley, A. J., and Simpson, E. R. Demonstration of tissue-specific 30 promoters in nonprimate species that express aromatase P450 in placentae. Biol.Reprod., 53: 31 1151-1159, 1995.

32 Hinshelwood, M. M., Dodson, M. M., Sun, T., and Simpson, E. R. Regulation of aromatase 33 expression in the ovary and placenta: a comparison between two species. J.Steroid 34 Biochem.Mol.Biol., 61 : 399-405, 1997.

Battelle Draft A-4 February 25, 2002 1 Hirsch, K. S., Adams, E. R., Hoffman, D. G., Markham, J. K., and Owen, N. V. Studies to 2 elucidate the mechanism of fenarimol-induced infertility in male rats. Toxicol.Appl.Pharmacol., 3 86: 391-399, 1986.

4 Hirsch, K. S., Weaver, D. E., Black, L. J., Falcone, J. F., and MacLusky, N. J. Inhibition of 5 central nervous system aromatase activity: a mechanism for fenarimol-induced infertility in the 6 male rat. Toxicol.Appl.Pharmacol., 91: 235-245, 1987.

7 Hughes, R., Timmermans, P., and Schrey, M. P. Regulation of arachidonic acid metabolism, 8 aromatase activity and growth in human breast cancer cells by interleukin-1beta and phorbol 9 ester: dissociation of a mediatory role for prostaglandin E2 in the autocrine control of cell 10 function. Int.J.Cancer, 67: 684-689, 1996.

11 Ibrahim, A. R. and Abul-Hajj, Y. J. Aromatase inhibition by flavonoids. J.Steroid 12 Biochem.Mol.Biol., 37: 257-260, 1990.

13 James, V. H., McNeill, J. M., Lai, L. C., Newton, C. J., Ghilchik, M. W., and Reed, M. J. 14 Aromatase activity in normal breast and breast tumor tissues: in vivo and in vitro studies. 15 Steroids, 50: 269-279, 1987.

16 Jeong, H. J., Shin, Y. G., Kim, I. H., and Pezzuto, J. M. Inhibition of aromatase activity by 17 flavonoids. Arch.Pharm.Res., 22: 309-312, 1999.

18 Jeyasuria, P., Roosenburg, W. M., and Place, A. R. Role of P-450 aromatase in sex 19 determination of the diamondback terrapin, Malaclemys terrapin. J.Exp.Zool., 270: 95-111, 20 1994.

21 Johnston, J. O. and Metcalf, B. W. Aromatase: A Target Enzyme in Breast Cancer. In P. Sunkara 22 (ed.), Novel Approaches to Cancer Chemotherapy, pp. 307-328. New York: Academic Press, 23 1984.

24 Jones, M. E., Thorburn, A. W., Britt, K. L., Hewitt, K. N., Wreford, N. G., Proietto, J., Oz, O. K., 25 Leury, B. J., Robertson, K. M., Yao, S., and Simpson, E. R. Aromatase-deficient (ArKO) mice 26 have a phenotype of increased adiposity. Proc.Natl.Acad.Sci.U.S.A, 97: 12735-12740, 2000.

27 Kao, Y. C., Zhou, C., Sherman, M., Laughton, C. A., and Chen, S. Molecular basis of the 28 inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: 29 A site-directed mutagenesis study. Environ.Health Perspect., 106: 85-92, 1998.

30 Kellis, J. T., Jr. and Vickery, L. E. Inhibition of human estrogen synthetase (aromatase) by 31 flavones. Science, 225: 1032-1034, 1984.

32 Kellis, J. T., Jr., Nesnow, S., and Vickery, L. E. Inhibition of aromatase cytochrome P-450 33 (estrogen synthetase) by derivatives of alpha-naphthoflavone. Biochem.Pharmacol., 35: 2887­ 34 2891, 1986.

Battelle Draft A-5 February 25, 2002 1 Kellis, J. T., Jr. and Vickery, L. E. Purification and characterization of human placental 2 aromatase cytochrome P-450. J.Biol.Chem., 262: 4413-4420, 1987.

3 Kirma, N., Gill, K., Mandava, U., and Tekmal, R. R. Overexpression of aromatase leads to 4 hyperplasia and changes in the expression of genes involved in apoptosis, cell cycle, growth, and 5 tumor suppressor functions in the mammary glands of transgenic mice. Cancer Res., 61: 1910­ 6 1918, 2001.

7 Kishida, M. and Callard, G. V. Distinct cytochrome P450 aromatase isoforms in zebrafish 8 (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early 9 development. Endocrinology, 142: 740-750, 2001.

10 Laskey, J. W., Berman, E., and Ferrell, J. M. The use of cultured ovarian fragments to assess 11 toxicant alterations in steroidogenesis in the Sprague-Dawley rat. Reprod.Toxicol., 9: 131-141, 12 1995.

13 Laughton, C. A., Zvelebil, M. J., and Neidle, S. A detailed molecular model for human 14 aromatase. J.Steroid Biochem.Mol.Biol., 44: 399-407, 1993.

15 Laughton, C. A., McKenna, R., Neidle, S., Jarman, M., McCague, R., and Rowlands, M. G. 16 Crystallographic and molecular modeling studies on 3-ethyl-3-(4- pyridyl)piperidine-2,6-dione 17 and its butyl analogue, inhibitors of mammalian aromatase. Comparison with natural substrates: 18 prediction of enantioselectivity for N-alkyl derivatives. J.Med.Chem., 33 : 2673-2679, 1990.

19 Letcher, R. J., van, H., I, Drenth, H. J., Norstrom, R. J., Bergman, A., Safe, S., Pieters, R., and 20 van den, B. M. Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in 21 human placental JEG-3 and JAR choriocarcinoma cells. Toxicol.Appl.Pharmacol., 160: 10-20, 22 1999.

23 Le Bail, J. C., Laroche, T., Marre-Fournier, F., and Habrioux, G. Aromatase and 17beta­ 24 hydroxysteroid dehydrogenase inhibition by flavonoids. Cancer Lett., 133: 101-106, 1998.

25 Le Bail, J. C., Aubourg, L., and Habrioux, G. Effects of pinostrobin on estrogen metabolism and 26 estrogen receptor transactivation. Cancer Lett., 156: 37-44, 2000.

27 Le Bail, J. C., Champavier, Y., Chulia, A. J., and Habrioux, G. Effects of phytoestrogens on 28 aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer 29 cells. Life Sci., 66: 1281-1291, 2000.

30 Le Bail, J. C., Pouget, C., Fagnere, C., Basly, J. P., Chulia, A. J., and Habrioux, G. Chalcones are 31 potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities. Life Sci., 68: 32 751-761, 2001.

33 Lephart, E. D. Molecular aspects of brain aromatase cytochrome P450. J.Steroid 34 Biochem.Mol.Biol., 61: 375-380, 1997.

Battelle Draft A-6 February 25, 2002 1 Lephart, E. D. A review of brain aromatase cytochrome P450. Brain Res.Brain Res.Rev., 22: 1­ 2 26, 1996.

3 Lephart, E. D. Molecular aspects of brain aromatase cytochrome P450. J.Steroid 4 Biochem.Mol.Biol., 61: 375-380, 1997.

5 Lephart, E. D. and Simpson, E. R. Assay of aromatase activity. Methods Enzymol., 206: 477­ 6 483, 1991.

7 Li, P. K. and Brueggemeier, R. W. 7-substituted steroidal aromatase inhibitors: structure-activity 8 relationships and molecular modeling. J.Enzyme Inhib., 4: 113-120, 1990.

9 Li, X., Nokkala, E., Yan, W., Streng, T., Saarinen, N., Warri, A., Huhtaniemi, I., Santti, R., 10 Makela, S., and Poutanen, M. Altered structure and function of reproductive organs in transgenic 11 male mice overexpressing human aromatase. Endocrinology, 142: 2435-2442, 2001.

12 Lombardi, G., Zarrilli, S., Colao, A., Paesano, L., Di Somma, C., Rossi, F., and De Rosa, M. 13 Estrogens and health in males. Mol.Cell Endocrinol., 178: 51-55, 2001.

14 Lovekamp, T. N. and Davis, B. J. Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript 15 levels and estradiol production in cultured rat granulosa cells. Toxicol.Appl.Pharmacol., 172: 16 217-224, 2001.

17 Mak, P., Cruz, F. D., and Chen, S. A yeast screen system for aromatase inhibitors and ligands for 18 androgen receptor: yeast cells transformed with aromatase and androgen receptor. 19 Environ.Health Perspect., 107: 855-860, 1999.

20 Marshall, J. C. Hormonal Regulation of the Menstrual Cycle and Mechanism of Ovulation. In L. 21 J. DeGroot, J. L. Jameson, H. G. Burger, D. L. Loriaux, J. C. Marshall, S. Melmed, W. D. Odell, 22 J. T. Jr. Potts, and A. H. Rubenstein (eds.), Endocrinology, Fourth Edition, pp. 2073-2085. 23 Philadelphia: W.B. Saunders Company, 2001.

24 McNamara, J., Stocker, P., Miller, V. P., Patten, C. J., Stresser, D. M., and Crespi, C. L. CYP19 25 (aromatase): Characterization of the recombinant enzyme and its role in the biotransformation of 26 xenobiotics. www.gentest.com/products/pdf/post_016.pdf , 1999.

27 McPhaul, M. J., Herbst, M. A., Matsumine, H., Young, M., and Lephart, E. D. Diverse 28 mechanisms of control of aromatase gene expression. J.Steroid Biochem.Mol.Biol., 44: 341-346, 29 1993.

30 Means, G. D., Kilgore, M. W., Mahendroo, M. S., Mendelson, C. R., and Simpson, E. R. Tissue­ 31 specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and 32 fetal tissues. Mol.Endocrinol., 5: 2005-2013, 1991.

33 Miller, W. R., Mullen, P., Sourdaine, P., Watson, C., Dixon, J. M., and Telford, J. Regulation of 34 aromatase activity within the breast. J.Steroid Biochem.Mol.Biol., 61: 193-202, 1997.

Battelle Draft A-7 February 25, 2002 1 Miller, W. R. and O'Neill, J. The importance of local synthesis of estrogen within the breast. 2 Steroids, 50: 537-548, 1987.

3 Moran, F. M., Conley, A. J., Corbin, C. J., Enan, E., VandeVoort, C., Overstreet, J. W., and 4 Lasley, B. L. 2,3,7,8-tetrachlorodibenzo-p-dioxin decreases estradiol production without altering 5 the enzyme activity of cytochrome P450 aromatase of human luteinized granulosa cells in vitro. 6 Biol.Reprod., 62: 1102-1108, 2000.

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11 Naftolin, F. Brain aromatization of androgens. J.Reprod.Med., 39: 257-261, 1994.

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15 Odum, J. and Ashby, J. Detection of aromatase inhibitors in vitro using rat ovary microsomes. 16 Toxicol.In Vitro, in press, 2001.

17 Oz, O. K., Zerwekh, J. E., Fisher, C., Graves, K., Nanu, L., Millsaps, R., and Simpson, E. R. 18 Bone has a sexually dimorphic response to aromatase deficiency. J.Bone Miner.Res., 15: 507­ 19 514, 2000.

20 Pasmanik, M., Schlinger, B. A., and Callard, G. V. In vivo steroid regulation of aromatase and 5­ 21 alpha-reductase activities in bnrain and pituitary of goldfish. Gen.Comp Endocrinol. 71, 175­ 22 182. 1988.

23 Pelissero, C., Lenczowski, M. J., Chinzi, D., Davail-Cuisset, B., Sumpter, J. P., and Fostier, A. 24 Effects of flavonoids on aromatase activity, an in vitro study. J.Steroid Biochem.Mol.Biol., 57: 25 215-223, 1996.

26 Quinn, A. L., Burak, W. E., Jr., and Brueggemeier, R. W. Effects of matrix components on 27 aromatase activity in breast stromal cells in culture. J.Steroid Biochem.Mol.Biol., 70: 249-256, 28 1999.

29 Recanatini, M. and Cavalli, A. Comparative molecular field analysis of non-steroidal aromatase 30 inhibitors: an extended model for two different structural classes. Bioorg.Med.Chem., 6: 377­ 31 388, 1998.

32 Reed, M. J., Owen, A. M., Lai, L. C., Coldham, N. G., Ghilchik, M. W., Shaikh, N. A., and 33 James, V. H. In situ oestrone synthesis in normal breast and breast tumour tissues: effect of 34 treatment with 4-hydroxyandrostenedione. Int.J.Cancer, 44: 233-237, 1989.

Battelle Draft A-8 February 25, 2002 1 Reed, M. J., Topping, L., Coldham, N. G., Purohit, A., Ghilchik, M. W., and James, V. H. 2 Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors. 3 J.Steroid Biochem.Mol.Biol., 44: 589-596, 1993.

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5 Robertson, K. M., O'Donnell, L., Jones, M. E., Meachem, S. J., Boon, W. C., Fisher, C. R., 6 Graves, K. H., McLachlan, R. I., and Simpson, E. R. Impairment of spermatogenesis in mice 7 lacking a functional aromatase (cyp 19) gene. Proc.Natl.Acad.Sci.U.S.A, 96: 7986-7991, 1999.

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22 Sanderson, J. T., Seinen, W., Giesy, J. P., and van den, B. M. 2-Chloro-s-triazine herbicides 23 induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel 24 mechanism for estrogenicity? Toxicol.Sci., 54: 121-127, 2000.

25 Sanderson, J. T., Slobbe, L., Lansbergen, G. W., Safe, S., and van den, B. M. 2,3,7,8­ 26 Tetrachlorodibenzo-p-dioxin and Diindolylmethanes Differentially Induce Cytochrome P450 27 1A1, 1B1, and 19 in H295R Human Adrenocortical Carcinoma Cells. Toxicol.Sci., 61: 40-48, 28 2001.

29 Sanderson, J. T., Letcher, R. J., Heneweer, M., Giesy, J. P., and van den, B. M. Effects of chloro­ 30 s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on 31 vitellogenin production in male carp hepatocytes. Environ.Health Perspect., 109: 1027-1031, 32 2001.

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Battelle Draft A-9 February 25, 2002 1 Santen, R. J. and Harvey, H. A. Use of aromatase inhibitors in breast carcinoma. Endocr.Relat 2 Cancer, 6: 75-92, 1999.

3 Shilling, A. D., Carlson, D. B., and Williams, D. E. Rainbow trout, Oncorhynchus mykiss, as a 4 model for aromatase inhibition. J.Steroid Biochem.Mol.Biol., 70: 89-95, 1999.

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10 Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., 11 Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., Michael, M. D., and . Aromatase 12 cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr.Rev., 15: 342-355, 13 1994.

14 Simpson, E. R., Bhatnagar, A., Brodie, A. M., Brueggemeier, R. W., Chen, S., DeCoster, R., 15 Dowsett, M., Harada, N., Naftolin, F., Osawa, Y., Plourde, P., Reed, M. J., and Santen, R. J. 16 Proceedings of the Fourth International Aromatase Conference. J.Steroid Biochem.Mol.Biol., 61: 17 107-425, 1997.

18 Simpson, E. R., Zhao, Y., Agarwal, V. R., Michael, M. D., Bulun, S. E., Hinshelwood, M. M., 19 Graham-Lorence, S., Sun, T., Fisher, C. R., Qin, K., and Mendelson, C. R. Aromatase expression 20 in health and disease. Recent Prog.Horm.Res., 52: 185-213, 1997.

21 Simpson, E., Jones, M., Davis, S., and Rubin, G. Do intracrine mechanisms regulate aromatase 22 expression? J.Steroid Biochem.Mol.Biol., 69: 447-452, 1999

23 Simpson, E. R. Aromatase 2000: Fifth International Aromatase Conference. 2000. 11-3-2000.

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27 Simpson, E. R., Clyne, C., Rubin, G., Boon, W. C., Robertson, K., Britt, K., Speed, C., and 28 Jones, M. Aromatase - A Brief Overview. Annu.Rev.Physiol, 64: 93-127, 2002.

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33 Stresser, D. M., Turner, S. D., McNamara, J., Stocker, P., Miller, V. P., Crespi, C. L., and Patten, 34 C. J. A high-throughput screen to identify inhibitors of aromatase (CYP19). Anal.Biochem., 284:

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Battelle Draft A-11 February 25, 2002 1 Willingham, E. and Crews, D. The red-eared slider turtle: An animal model for the study of low 2 doses and mixtures. American Zoologist, 40: 421-428, 2000.

3 Yang, C. and Chen, S. Two organochlorine pesticides, toxaphene and chlordane, are antagonists 4 for estrogen-related receptor alpha-1 orphan receptor. Cancer Res., 59: 4519-4524, 1999.

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8 Zhao, Y., Agarwal, V. R., Mendelson, C. R., and Simpson, E. R. Estrogen biosynthesis proximal 9 to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of 10 the CYP19 (aromatase) gene. Endocrinology, 137: 5739-5742, 1996.

11 Zhou, C., Zhou, D., Esteban, J., Murai, J., Siiteri, P. K., Wilczynski, S., and Chen, S. Aromatase 12 gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger 13 RNA by reverse transcription- polymerase chain reaction. J.Steroid Biochem.Mol.Biol., 59: 163­ 14 171, 1996.

Battelle Draft A-12 February 25, 2002 1 APPENDIX B 2 3 INVESTIGATORS CONTACTED AND QUESTIONNAIRE 4 5 Investigators contacted 6 7 Investigator Expertise

8 Shiuan Chen, Ph.D. Biochemistry and molecular biology of aromatase; 9 Division of Immunology structure-function studies; aromatase inhibition by 10 Beckman Research Institute, City of Hope flavonoids 11 1450 E. Duarte Road 12 Duarte, CA 91006, U.S.A. 13 E-mail: [email protected]

14 Rajeshwar R. Tekmal, Ph.D. Molecular biology of aromatase; development of 15 Department of Ob/Gyn transgenic mice expressing aromatase 16 Emory University School of Medicine 17 4219 WMB, 1639 Pierce Drive 18 Atlanta, GA 30322, U.S.A. 19 E-mail: [email protected]

20 Edwin D. Lephart, Ph.D. Brain aromatase; flavonoids and aromatase 21 633 WIDB, Neuroscience Center 22 Brigham Young University 23 Provo, UT 84602, U.S.A. 24 E-mail: [email protected]

25 Gerard Habrioux, Ph.D. Biochemistry and pharmacology of aromatase; 26 Laboratory of Biochemistry aromatase inhibition by flavonoids 27 Faculty of Pharmacy 28 2 rue du Dr. Marcland 29 87025 Limoges Cedex, FRANCE 30 E-mail: [email protected]

31 Sari Mäkelä, Ph.D., and Risto Santti, Ph.D. Biochemistry and pharmacology of aromatase; 32 University of Turku aromatase inhibition by flavonoids; endocrine disruptors 33 Institute of Biomedicine and Medicity Research 34 Laboratory 35 Turku, FINLAND 36 E-mail: [email protected]

37 A.M. Vinnggaard, Ph.D. Endocrine toxicology; endocrine disruptors 38 Institute of Food Safety and Toxicology 39 Division of Biochemistry and Molecular Biology 40 Danish Veterinary and Food Administration 41 Morkhoj Bygade 19 42 DK-2860 Soborg, DENMARK 43 E-mail: [email protected]

44 Martin van den Berg, Ph.D. Endocrine toxicology; endocrine disruptors 45 Research Institute of Toxicology 46 Utrecht University 47 P.O. Box 80.176, Yaleaan 2 48 Utrecht 3508 TD, The NETHERLANDS 49 E-mail: [email protected]

Battelle Draft B-1 February 25, 2002 1 Questionnaire sent via e-mail 2 3 Questionnaire: Aromatase as a possible target for endocrine disrupters and use of an in vitro 4 aromatase assay for screening chemicals 5 6 From: Bob Brueggemeier, The Ohio State University (E-mail: 7 [email protected]) 8 9 The United States Environmental Protection Agency (EPA) is implementing an 10 Endocrine Disruptor Screening Program (EDSP). In this program, comprehensive toxicological 11 and ecotoxicological screens and tests are being developed for identifying and characterizing the 12 endocrine effects of various environmental contaminants, industrial chemicals, and pesticides. 13 One potential endocrine target for environmental chemicals is the enzyme aromatase, the 14 cytochrome P450 enzyme complex responsible for estrogen biosynthesis. The EPA is 15 considering employing an in vitro aromatase assay as an alternative for possible inclusion in the 16 Tier 1 Screening Battery. Battelle Research Institute, Columbus, Ohio, has the EPA contract for 17 reviewing, establishing, and performing validation studies on the toxicological and 18 ecotoxicological screens and tests. I am serving as a consultant to Battelle because of my 19 research expertise in aromatase and aromatase inhibitors. 20 21 Two in vitro aromatase assays being evaluated for their use in measuring endocrine disrupters 22 that affect estrogen biosynthesis are: 23 24 Human Placental Microsomal Aromatase assay: Determines the effects of environmental 25 chemicals/toxicants on aromatase activity in human placental microsomal incubations by 3 3 26 measuring inhibition of the release of H2O from [1β- H]-androstenedione. Initial screening 27 bioassays will determine %inhibition, and IC50 values and/or Ki values will be determined for 28 active test compounds. 29 30 Human JEG-3 (or JAR) choriocarcinoma cell culture assay: Determines the effects of 31 environmental chemicals/toxicants on aromatase activity in choriocarcinoma cell cultures by 3 3 32 measuring inhibition of the release of H2O from [1β- H]-androstenedione. Initial screening 33 bioassays will determine %inhibition, and IC50 values will be determined for active test 34 compounds. 35 36 Part of the process of reviewing relevant literature includes contacting experts in aromatase 37 pharmacology and toxicology. We would appreciate your comments to the following interview 38 questions: 39 40 • What are the strengths/weaknesses of the study designs under consideration? 41 • Based on your expertise and experience, what endpoints will be most appropriate? 42 43 • What changes would you recommend to the study designs under consideration and why? 44 45 • If appropriate, what statistical methods would you employ or suggest that we employ? 46 • Based on your experience, what chemicals, routes, duration, and doses should we use to

Battelle Draft B-2 February 25, 2002 1 validate the assay? 2 3 • Can you suggest published references (yours or others) to aid us in our study and endpoint 4 selection? If so, which? 5 6 • Do you have any unpublished data relevant to these assays that you would be willing to 7 share? If so, are there any restrictions? 8 9 • Would you be willing/like to be involved in the study progress, results, and interpretation? 10 11 • Is there anyone else you think we should contact? If so, whom? Can we mention your name 12 when we contact him/her? 13 14 Responses received 15 16 • Dr. Shiuan Chen (USA) met with Dr. Brueggemeier at the 2001 Hormonal Carcinogenesis 17 Gordon Conference held July 8-13, 2001 and provided his verbal responses to the questions 18 and additional input. 19 20 • Dr. Raj Tekmal (USA) met with Dr. Brueggemeier at the 2001 Hormonal Carcinogenesis 21 Gordon Conference held July 8-13, 2001 and provided his verbal responses to the questions 22 and additional input. 23 24 • Dr. Edwin Lephart (USA) sent an response to the questions on August 10, 2001. He also 25 provided additional information as a result of his recent visit to the Reproductive 26 Endocrinology Section, EPA, Research Triangle Park, NC. 27 28 • Dr. Sari Mäkelä (Finland) met with Dr. Brueggemeier at the 2001 Hormonal Carcinogenesis 29 Gordon Conference held July 8-13, 2001. She also talked with Dr. Risto Santti upon her 30 return to Finland and sent an e-mail response on August 22, 2001. 31 32 • Dr. Gerard Habrioux (France) did not respond. 33 34 • Dr. A.M. Vinnggaard (Denmark) did not respond. 35 36 • Dr. Martin van den Berg (Netherlands) did not respond. 37 38 E-mail Responses from Dr. Edwin Lephardt are indicated in bold print 39 40 What are the strengths/weaknesses of the study designs under consideration? 41 42 Strengths- ease of availability of substrate and assay conditions. 43 Weakness- androstenedione is a weak androgen in very low circulating levels with 44 relatively low affinity to the aromatase enzyme vs. that of testosterone, etc. 45 46 Based on your expertise and experience, what endpoints will be most appropriate?

Battelle Draft B-3 February 25, 2002 1 2 Parameters- Reproductive and behavioral.; Reproductive- female, puberty onset, 3 cyclicity, gonadotrophin levels and steroid hormone levels; Male, gonadotrophin levels 4 and steroid hormone levels (especially circulating estradiol levels); Female and Male­ 5 reproductive behaviors 6 7 What changes would you recommend to the study designs under consideration and why? 8 9 What are the reasons to use placental tissue/cells in each assay? Aromatase activity is 10 regulated by various mechanisms, especially in brain, bone, etc., that is different than 11 other tissue sites. Also, what are the reasons that only placental tissue sources are being 12 tested? There may be an endocrine effect on aromatase by endocrine disrupters (in 13 other tissue sites with weak aromatase expression) that would not be picked up in the 14 human placental assay. 15 16 If appropriate, what statistical methods would you employ or suggest that we employ? 17 18 This is a difficult question to address without knowing more about the design of the 19 experiment (i.e. number of different tissues assayed, affinity and saturating 20 concentration of substrate, etc.). 21 22 Based on your experience, what chemicals, routes, duration, and doses should we use to validate 23 the assay? 24 25 For endocrine disrupters, circulating levels should be determined or estimated, then 26 doses should be established. Routes should simulate oral (in a food matrix or 27 nutritional capsule) or skin cream/patch, etc. 28 29 Can you suggest published references (yours or others) to aid us in our study and endpoint 30 selection? If so, which? 31 32 Please search: KDR Setchell, H Adlercruetz, S. Barnes 33 34 Do you have any unpublished data relevant to these assays that you would be willing to share? 35 If so, are there any restrictions? 36 37 We have several endocrine parameters that we have measured, but I do not feel 38 comfortable sharing this information, since I don’t know how this information will be 39 used. 40 41 Would you be willing/like to be involved in the study progress, results, and interpretation? 42 43 Sure 44 45 Is there anyone else you think we should contact? If so, whom? Can we mention your name 46 when we contact him/her?

Battelle Draft B-4 February 25, 2002 1 2 [No answer provided.] 3

Battelle Draft B-5 February 25, 2002 1 E-mail Responses from Dr. Sari Mäkelä are indicated in bold print 2 3 Strengths and weaknesses of the in vitro study designs 4 5 Strengths: 6 Suitable for high-throughput screening 7 JEG-3 cells are available commercially 8 Human placenta can be obtained easily 9 10 Weaknesses: 11 Activity in vitro does not necessarily indicate activity in vivo. It is difficult to 12 determine how high activity in in vitro assay is required to indicate activity in vivo (i.e. 13 the level of inhibitory activity in vitro required, for the compound to be classified as a 14 putative aromatase inhibitor, and selected for further testing). 15 16 In placental microsome assay compounds that are not capable to enter the cell 17 may show activity (“false positive result”). 18 19 Aromatase activity in JEG-3 cells varies markedly, depending on the growth 20 phase (confluency). This causes variation between individual experiments. 21 22 Compounds that modulate aromatization by influencing the expression of 23 aromatase gene/protein will most likely not be recognized in these assays. 24 25 In JEG-3 cells other steroid metabolizing enzymes are present, and the labeled 26 substrate may be metabolized by other enzymes, thus not allowing its aromatization 27 (i.e. the amount of substrate available for aromatase may be modulated by other 28 enzymes). The test compounds may influence (inhibit) the activity of these “competing” 29 enzymes, and cause indirect changes in the level of aromatization without any 30 interaction with aromatase enzyme. 31 32 33 Most appropriate endpoints (In general? Not necessarily in this order) 34 35 testicular function 36 ovarian function 37 onset of puberty 38 mammary gland development, growth and tumorigenesis 39 prostate gland development, growth and tumorigenesis 40 gonadotropin secretion patterns 41 sexual behavior 42 bone strength / osteoporosis (?) 43 44 Suggested changes to the study designs under consideration: 45

Battelle Draft B-6 February 25, 2002 1 Use of cells stably transfected with human aromatase, instead of JEG-3 cells. 2 Such cell lines have been generated by several research groups, and (at least in our 3 hands) they are easier to work with, as there is less variation in the aromatase activity. 4 Validation of the assay: 5 6 Use of one or two well characterized aromatase inhibitors (e.g. letrozole), proven 7 to be active in vivo, as a positive control, is necessary. 8 9 It would be nice to include a model EDC, proven to be aromatase inhibitor in 10 vitro and in vivo, in the assays as another positive control. However, such compound 11 may not be available, as none of the natural or manmade compounds showing 12 aromatase inhibitory activity in vitro (such as chrysin and apigenin), have been shown 13 to act as aromatase inhibitors in vivo. 14 15 Compounds showing activity in the in vitro assay must be further tested in vivo. 16 Using intact animals, it is relatively difficult and/or time-consuming to detect aromatase 17 inhibition reliably. In order to observe marked changes in circulating estrogen and/or 18 androgen concentrations, potent inhibitors are needed. Weak inhibitors more likely to 19 act locally in estrogen target tissues are not so easily detected. Furthermore, 20 measurement of small changes in serum estrogen and/or androgen concentrations is not 21 easy (in particular, measurement of estrogen concentrations in male serum is very 22 challenging). In vivo testing may be more easily performed using transgenic animals 23 overexpressing aromatase gene (two models published so far), as they display very 24 prominent reproductive phenotypes that are relatively easily detected (e.g. 25 cryptorchidism and gynecomastia). Also, nude mice injected with MCF-7 cells (or other 26 estrogen-responsive cancer cells) overexpressing aromatase gene may be useful here. 27 28 29 Recent references 30 31 Li X, Nokkala E, Yan W, Streng T, Saarinen N, Warri A, Huhtaniemi I, Santti R, 32 Mäkelä S, Poutanen M: Altered structure and function of reproductive organs in 33 transgenic male mice overexpressing human aromatase. Endocrinology 142: 2435-2442, 34 2001. 35 36 Saarinen N, Joshi SC, Ahotupa M, Li X, Ämmälä J, Mäkelä S, Santti R: No evidence 37 for the in vivo activity of aromatase-inhibiting flavonoids. J Steroid Biochem Mol Biol, 38 in press. 39 40 41 Unpublished data: 42 43 We have recently tested the effects of a synthetic aromatase inhibitor, finrozole, in our 44 aromatase-overexpressing mouse model. It appears that the phenotype in males can be 45 reversed by long-term aromatase inhibitor treatment. Similar experiments with some 46 natural polyphenolics that are active in vitro, are ongoing.

Battelle Draft B-7 February 25, 2002