*IL a Techniques for Detection of Estrogenicity Kenneth S. Korach and John A. McLachlan Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina Environ Health Perspect 103(Suppl 7):5-8 (1995)

The purpose of this workshop was to bring sequence has been cloned and can be used The gold standard at this time is still the together the collective experience of many to detect and measure estrogen response. mouse uterine bioassay, precisely because it disciplines to discuss and evaluate tech- The DNA sequence is closely analogous to replicates a living situation and incorporates niques for the detection of functional estro- the sequence of a glucocorticoid responsive the effects of , serum binding, genicity. Estrogenicity was first defined as a element with four base changes. and pharmacokinetics. However, a com- physiological response to a compound that Korach's group (1) cloned an estrogen- plete dose-response assay can be expensive induced estrus in vivo. The bioassay for responsive sequence and inserted it into a and time consuming; it is also a difficult induction of estrus developed into a utero- reporter vector. The construct became process for testing a large number ofchemi- tropic assay. The classical tissue response an estrogen "inducible promoter" linked to cals. In vitro assays may be cheaper, quicker, evaluated in this assay was an estrogen- bacterial chloramphenicol acetyltransferase and more reproducible; however, the induced increase in wet weight and tissue (CAT), which can be detected in a trans- difficulties already mentioned must be con- mass. Tissue response follows a time course fected cell to measure estrogen-related gene sidered and dealt with to allow their use in ofstimulation, including an early phase at 2 activity. When an ER-positive target cell exposure studies and possible risk assess- to 4 hr, followed by a second later phase. A such as MCF-7 is transfected with this ments. A question was posed by a number higher dose of compound generates a receptor-specific reporter-gene construct, ofparticipants: How do we come up with a stronger response. Weak estrogens exhibit CAT expression can be measured following way to design the best assays for screening the early response phase, but then the estrogen treatment. In a test against a non- water or other contaminated sources to response falls off; however, multiple low responsive (ER negative) cell, transfection determine estrogenic activity? doses of weak estrogens mimic the full with the receptor-specific reporter alone activity of strong estrogens. A host of does not lead to estrogen-induced CAT Proliferation Controls chemical and molecular biological factors expression. It must be transfected with ER The question that has guided the efforts of are involved in the uterotropic response. to get the hormonally induced CAT pro- Soto's laboratory in the area of prolifera- duction, demonstrating that the system is tion has been how to screen for a variety of Gene Activation specific for ER-mediated chemicals quickly (2). It is important to Hormone receptor mechanisms exist- and regulation. This system will respond to remember that environmental contami- both membrane and nuclear receptor path- treatment with stilbestrol estrogens with nants of widely diverse chemical structure ways-that affect gene regulation and CAT gene expression as well. Phospho- mimic estrogen actions. From a public interact with one another. Certain imaging of the CAT gene product can be safety concern, estrogenicity should be are specifically regulated by estrogen acting used for easy quantitation. tested before chemicals are released into through the estrogen receptor (ER). These Phytoestrogen compounds were tested the environment. effects appear to be mediated by the inter- with this ERE-CAT construct-transfection Screening should be based on the end action of the estrogen/ER complex with system. CAT stimulation by genistein, point of estrogenic action that has the estrogen response elements (EREs) in regu- coumestrol, and zearalenone was compared greatest physiological relevance. A crucial latory regions of those genes. For instance, with the stimulation produced by estradiol. end point can be based on the definition of the vitellogenin A2 estrogen-responsive CAT induction by genistein was about an estrogen promulgated by Roy Hertz in 20% that of estradiol; coumestrol activity 1985 (3): an estrogen is a substance that was about the same; zearalenone produced can elicit the mitotic stimulation of the tis- This paper summarizes the session "Detection of Estrogenicity" from the Symposium on Estrogens in about 1.7-fold stimulation compared to sues of the female genital tract; therefore, the Environment, III: Global Health Implications held estradiol. The addition of a commercially measuring cell proliferation is of key 9-11 January 1994 in Washington, DC. Manuscript available estrogen antagonist (ICI 164,384) importance in assessing estrogenicity. To received: March 15, 1995; manuscript accepted: April 4, 1995. resulted in almost complete inhibition of determine whether chemical "X" is an Address correspondence to Dr. Kenneth S. CAT induction by these compounds. It is estrogen, one must test its ability to induce Korach, NIEHS, P.O. Box 12233, Research Triangle important to keep in mind that the active proliferation of estrogen-responsive target Park, NC 27709. Telephone: (919) 541-3512. Fax: (919) 541-0696. compound may be a metabolite that the cells, even though not all estrogen responses Abbreviations used: ER, estrogen receptor; ERE, cell might not see in an in vitro assay but or target tissues respond with proliferation. estrogen response element; CAT, chloramphenicol which would show activity in vivo. Future The question of whether fish in lake acetyltransferase; SHBG, sex hormone-binding globu- studies different mutants of or in lin; E2, estradiol; VTG, vitellogenin; LF, ; pl, using the "Y birds region "Z" are exposed to isoelectric point; EGF, epidermal growth factor; CP, receptor can evaluate more precise regula- estrogenic xenobiotics can be answered at ; MA, microtubule assembly. tory mechanisms. an initial level of screening by examining

Environmental Health Perspectives 5 KORACH AND MCLACHLAN

endogenous vitellogenin (VTG) produc- McLachlan was concerned with the steroid-inducible ; if you expose an tion. When VTG levels are increased, the binding and whether they were oviparous vertebrate to estrogen, it will syn- next question is which chemicals caused still present in the charcoal-stripped serum. thesize VTG. VTG is synthesized in the the observed estrogenic effects? To answer It appears that the binding proteins are pre- liver under the control of estrogen. This this question, isolation and chemical analy- sent (sex hormone-binding usually occurs in females but can also be sis are required. When dealing with com- [SHBG] and serum ); however, a induced in males. VTG is taken up into the pounds that are not dassically described as significant amount of SHBG is denatured oocyte by receptor-mediated endocytosis. It estrogens, a bioassay is necessary to evalu- during heat inactivation of serum (final is present in the plasma of females several ate their estrogenicity. Estrogenicity cannot concentration in 10% charcoal-dextran months before ovulation and is related to be deduced solely from chemical structure. human serum is less than 3 nM (4). cholesterol and mineral transporters. In The classic method for measuring estro- Approximately 85% of estradiol is bound serum collected from brown bullhead, gen induction of cell proliferation is to to plasma proteins in this assay (5). Sullivan observed a massive induction of a determine the increase of mitotic indices of Screening for estrogens using males as a protein in tumor-bearing males and epithelia in rodents. As mentioned by model should not be done because estro- females, which is not present in control Korach, this is an accurate but labor-inten- gens are primarily defined by their ability females; this induction appeared to be the sive, time-consuming approach, totally to increase the mitotic activity of female result of an environmental insult. The unsuitable for the screening of large num- secondary sex organs. There are reports of induced protein was shown to be VTG by bers ofchemicals. effects on male fertility due to exposure to N-terminal sequencing. Soto et al. (2) have introduced a cell estrogens. In males, estrogens are believed For the purposes of a universal assay, proliferation assay termed the E-SCREEN to act via a negative feedback to inhibit VTG itself is hard to work with since it test. For this bioassay, MCF-7 breast can- gonadotropins; this results in the lowering varies in structure between species. So, the cer cells were chosen. These cells are gen- of androgen production by Leydig cells paradigm includes creation of monoclonal uine human estrogen-sensitive cells; they and inhibition ofspermatogenesis. to vertebrate VTG. Sullivan's remain quiescent when inoculated into There is no endogenous estrogen in this group screened first for antibodies that rec- ovariectomized hosts. They require the assay; we are measuring direct effects. An ognize both rainbow trout and striped bass presence of estrogen to grow as tumors in inhibitor could be an effector of an VTGs. The second screen was against VTG hosts. When MCF-7 cells are grown in estrogen response in a system where there from fish, amphibians, and mammals. A culture in medium supplemented with is a source of estrogens, such as in any ani- positive screening result from one clone nonestrogenic charcoal-stripped human mal model. Moreover, this assay can be included cross-reactivity to VTG from bass, serum, proliferation is prevented. When used as a first screen for estrogenicity; any perch, trout, tilapia, sturgeon, chicken, estrogen is added, the cells proliferate. The positive response would require further tuatara, and platypus; there was a question- E-SCREEN assay compares the cell yield study to determine an underlying mecha- able result in this assay for rartlesnake VTG achieved after 4 to 6 days of culture in nism of action. due to a high background. medium supplemented with 5 to 10% It was pointed out by McLachlan that Antibodies to specific portions of the charcoal-dextran stripped human serum in the 1930s CC14 was described as an molecule can be generated. It is possible to in the presence (positive control) or estrogen because of the enhanced estrogen- select conserved sequences, do structural absence (negative control) of estradiol and like sequelae of CC14 administration. It analysis, select a portion of the peptide that with diverse concentrations of xenobiotics was later learned that these estrogenlike seems likely to be on the outside in native suspected of being estrogenic. When cell effects occurred because of CC14 destruc- conformation, synthesize it and conjugate yield is examined over a wide range of test- tion of the liver, which resulted in it to carriers, generate antibodies, and compound concentrations, it is possible to decreased turnover and an elevation of the repeat first and second screens. The N-ter- distinguish agonists, partial agonists, and level of estradiol in the circulation. minal sequence of striped bass VTG shows inactive compounds from one another. An MCF-7 variant has been modified homology with various fish, amphibian, There is general agreement about cell yield to grow in serum-free and nonestrogenic and avian species. Demonstrated homol- being a more sensitive marker than gene medium by Briand et al. (6). This variant ogy was between 40 to 100%. Using VTG expression. A 6-fold increase can be mea- behaves similarly in the E-SCREEN assay and related proteins like von Willebrand sured in cell yields after 5 days of expo- when exposed to 10% charcoal-dextran factor, you can generate an assay that sure; no false positives and no false stripped human serum (estradiol [E2] would be applicable across species from negatives have been found among the test concentration in the medium is below fish to mammal. compounds of known estrogenicity. 0.001 pg/ml; this is below 4 fM or about The physiological significance of vitel- Methoxychlor, which requires metabolic 1/1000 of the dose needed for the smallest logenesis in males is not known. VTG activation, is positive in this assay; obvi- significant proliferative response). MCF-7 receptors in the testes and in muscles in ously, MCF-7 cells can provide the appro- cells are exposed to practically no estrogen males have been found, and VTG is found priate metabolic transformation. The in this "estrogenless" condition. in spermatocytes. VTG is a generic, E-SCREEN cell-yield assay is easy to per- ancient lipoprotein; it is not surprising that form, requiring only standard eukaryotic VFtellogenin as a Biomarker it is found in an abundance of tissues. The cell culture equipment plus an ELISA Could a universal assay for vertebrate vitel- VTG receptor might also be involved in reader or an electronic particle counter. logenins be used as a new test for environ- endocytosis ofother lipoproteins. For regulatory purposes, an assay that is mental estrogens? Sullivan described a biologically valid is needed; that is, the screen that defines an estrogen as something Lactoferrin as a Biomarker end point examined should have wide but that induces vitellogenesis [Heppell et al., What would be a good biomarker for unambiguous biological meaning. this volume; (7)]. Vitellogenin is a classic estrogenicity in mammals-similar to what

6 Environmental Health Perspectives TECHNIQUES FOR DETECTION OFESTROGENICITY

Sullivan described in fish or reptiles (see have been made with enterolactone. Are effect on microtubule assembly (MA) (9). Heppell et al., this volume). Ideally, one there any studies of effects of thyroxine or The assay for turbidity measures MA under would choose a natural product of the cells insulin? Could other hormones have an cell-free conditions. All stilbene estrogens that is sensitive to estrogenic compounds, effect? SHBG is a good marker in females. can bind and inhibit MA directly, but the one that is present in mammals-both Another protein marker is ceruloplasmin steroid estrogens and the phytoestrogen the humans and wildlife, and one that is con- (CP), a -containing protein. Treat- Metzler group has tested so far cannot bind venient to study, i.e., a well-characterized ment with a nonestrogenic oral contracep- or inhibit MA. Some steroid estrogens are estrogen-responsive protein or gene. Work tive stimulates CP, which is also increased inhibitory after metabolic activation. and studies described by Teng showed in pregnancy. Therefore, tubulin binding and MA dis- that, of the 30 to 40 total uterine proteins, Regarding estrogenicity, biology is a ruptions are not estrogenic markers per se. one band makes up approximately 15% of hierarchical science whereby questions However, all the estrogens tested were posi- the total; this band is lactoferrin (LF). As should be answered at the same hierarchi- tive in inducing micronuclei, an indication seen by immunolocalization, LF is made in cal level at which they are posed. Estrogens of chromosomal damage. Coumestrol, the response to diethylstilbestrol (DES) treat- were defined before the development of only phytoestrogen tested so far, was also ment. It is an iron-binding molecular biology and the discovery of the active in micronuclei induction. The with a molecular weight of 68 to 70 kDa. estrogen receptor. It is important to under- mechanism may be different from effects It is a basic protein with a pI of 9 to 10 stand mechanisms, but we are far from of compounds that are capable of binding belonging to the gene family. agreeing on them. In the meantime, estro- directly to tubulin and disrupting micro- Lactoferrin has 2.6 kb mRNA; the gene is gens are reliably defined by their effect on tubule assembly. Binding to tubulin itself 33 kb in the human and 22 kb in the the female genital tract. Moreover, estrogens is not a biomarker, but many estrogens can mouse. LF's biological function is antibac- exclusively induce cell proliferation in the cause chromosomal damage by different terial and antiviral; it also has a growth- E-SCREEN assay. These same estrogens mechanisms. stimulating factor activity. Low levels exist also induce progesterone receptor and dis- This introduces the concept of bipolar in biological fluids and wet surface mucosa, place estradiol from the estrogen receptor. characteristics of estrogens. Some of the whereas high levels exist in neutrophils, same compounds, which may be more or lactating mammary gland, and uterus. LF Toxicological Approaches less weak estrogens, may have different is regulated under various control mecha- Toxicological approaches rely on the chem- activities in spindle disruption or may nisms in different tissues. In the uterus, it ical attributes of estrogens and use the have other activities that have yet to be is inducible by estrogen. Looking at puta- study of the carcinogenic effects of DES as discovered. Consider, in addition, another tive regulatory elements of the mouse a background. Investigators have asked factor-time or life stage of exposure. lactoferrin gene promoter region, Teng's whether DES, acting as a carcinogen, 17a-Estradiol does not cause uterine group identified and characterized an estro- causes genetic damage. This approach has growth in an adult, but exposure during a gen response element (8). More recently, been tested by Metzler's laboratory (9). critical period of growth leads to manifold they found a response element that was One sort of genetic change that has been effects including aneuploidy. 17a-Estradiol responsive to cAMP and epidermal growth reported in many systems is aneuploidy or is a strong effector in neonatal or fetal tissue. factor (EGF) (C Teng, this volume). LF altered numbers following induction seems to be a complex interac- exposure to DES and other estrogens. Conclusion tion, not a simple one-way cause and Furthermore, it has been shown that DES At the end of the presentation and work- effect. The human LF gene has an ERE in has colchicinelike effects and disrupts shop, one of the participants posed two the same location, but it also has some dif- mitotic spindles. This suggests tubulin as a overlying problems. First, if we are con- ferent response elements. possible additional target for estrogen cerned about exposure to the male, the crit- As pointed out by Adlercreutz, SHBG action and aneuploidy as a measure ofsuch ical period is in utero. Second, how does the is another potential marker. SHBG action. Metzler showed there are at least male fetus protect itself against estrogens, increases when estrogens are given to two binding sites on the tubulin molecule: either maternal or from other sources? We women orally but not if they are given par- one is the colchicine-, which have no answer right now. We need an enterally; 200 nM estradiol is needed to get can also be bound by DES; another bind- overall picture of how we are exposed this response, but with a weak estrogen, ing site can be occupied by estradiol or by to estrogens, and we need a method to much more may be needed. Several tests DES but not by colchicine and has no determine our exact levels ofexposure.

REFERENCES 1. Makela S, Davis V, Tally WC, Korkman H, Salo L, Santti R, Princeton, NJ:Princeton Scientific Publishing, 1992;295-309. Vihko R, Korach KS. Dietary estrogens act through receptor 3. Hertz R The Estrogen Problem-Retrospect and Prospect. In: mediated processes and show no antiestrogenicity in cultured Estrogens in the Environment II (McLachlan JA, ed). New breast cancer cells. Environ Health Perspect 102:572-578 York:Elsevier, 1985;1-11. (1994). 4. Damassa DA, Lin TM, Sonnenschein C, Soto AM. Biological 2. Soto AM, Lin T-M, Justicia H, Silvia RM, Sonnenschein C. An effects of sex hormone-binding globulin on androgen-induced "in culture" bioassay to assess the estrogenicity of xenobiotics. proliferation and androgen metabolism in LNCaP prostate In: Chemically-induced Alterations in Sexual Development: cells. Endocrinology 129 1):75-84 (1991). The Wildlife/Human Connection (Colborn T, Clement C, eds). 5. Soto AM, Murai JT, Siiteri PK, Sonnenschein C. Control of

Volume 103, Supplement 7, October 1995 7 KORACH AND MCLACHLAN

cell proliferation: evidence for negative control on estrogen-sen- maturing under 6-, 9-, and 12-month photothermal cycles. sitive T47D human breast cancer cells. Cancer Res Gen Comp Endocrinol 94(1)122-134 (1994). 46(5):2271-2275 (1986). 8. Liu YH, Teng CT. Estrogen response module of the mouse 6. Petersen OW, van Deurs B, Nielsen KV, Madsen MW, lactoferrin gene contains overlapping chicken Laursen I, Balslev I, Briand P. Differential tumorigenicity of upstream promoter transcription factor and estrogen receptor two autologous human breast carcinoma cell lines, HMT- binding elements. Mol Endocrinol 6:355-364 (1992). 3909SI and HMT-3909S8; established in serum-free medium. 9. Epe B. Harttig UH, Schiffmann D, Metzler M. Microtubular Cancer Res 50(4):1257-1270 (1990). proteins as cellular targets for carcinogenic estrogens and other 7. Blythe WG, Helfrich LA, Sullivan CV. Sex carcinogens. Prog Clin Biol Res 318:345-351 (1989). and vitellogenin levels in striped bass (Morone saxatilis)

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