Environmental Health Perspectives 105, Supplement 3, March 1997

Predicting Health Effects of Exposures to Compounds with EstrogenicActivity: Methodological Issues

Ruthann Rudel

Silent Spring Institute, Newton, Massachusetts

Abstract

Many substances are active in in vitro tests for estrogenic activity,but data from multigenerational and other toxicity studies are not availablefor many of those substances. Controversy has arisen, therefore, concerningthe likelihood of adverse health effects. Based on a toxic equivalencefactor risk assessment approach, some researchers have concluded that exposureto environmental estrogens is not associated with estrogen receptor (ER)-mediatedhealth effects. Their rationale cites the low potency of these compoundsin in vitro assays relative to estradiol, and the widespread exposureto pharmaceutical, endogenous, and dietary estrogens. This reasoning relieson two assumptions: that the relative estrogenic potency in in vitroassays is predictive of the relative potency for the most sensitive invivo estrogenic effect; and that all estrogens act via the same mechanismto produce the most sensitive in vivo estrogenic effect. Experimentaldata reviewed here suggest that these assumptions may be inappropriatebecause diversity in both mechanism and effect exists for estrogenic compounds.Examples include variations in ER-ligand binding to estrogen response elements,time course of nuclear ER accumulation, patterns of gene activation, andother mechanistic characteristics that are not reflected in many invitro assays, but may have significance for ER-mediated in vivoeffects. In light of these data, this report identifies emerging methodologicalissues in risk assessment for estrogenic compounds: the need to addressdifferences in in vivo end points of concern and the associatedmechanisms; pharmacokinetics; the crucial role of timing and duration ofexposure; interactions; and non-ER-mediated activities of estrogenic compounds.-- Environ Health Perspect 105(Suppl 3):655-663 (1997)

Key words: estrogen, environmental estrogen, phytoestrogen, dose response,risk assessment, mechanism, pharmacokinetics

This paper was presented in part at the Workshop on Hormones,Hormone Metabolism, Environment, and Breast Cancer held 28-29 September1995 in New Orleans, Louisiana. Manuscript received at EHP 6 June1996; manuscript accepted 22 August 1996.

I would like to acknowledge D. Davis and S. Swedis fortheir thoughtful suggestions on the work described here. Our research onthis topic has been supported by the Massachusetts Department of PublicHealth as part of the Cape Cod Breast Cancer and Environment Study andby the Susan G. Komen Breast Cancer Foundation, Boston Race for the Cure.

Address correspondence to R. Rudel, Silent Spring Institute,29 Crafts Street, Newton, MA 02158. Telephone: (617) 332-4288, ext. 14.Fax: (617) 332-4284. E-mail:rudel@silent.shore.net

Abbreviations used: DES, diethylstilbestrol; DMS, dimethylstilbestrol;ER, estrogen receptor; ERE, estrogen response element; GJIC, gap-junctionalintercellular communication; GnRH, gonadotropin-releasing hormone; OP,4-tert-octylphenol; PCBs, polychlorinated biphenyls; SDN-POA, sexuallydimorphic nucleus in the preoptic area of the hypothalamus; SHBG, sex hormone-bindingglobulin; 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEF,toxic equivalence factor.

Introduction

A number of plant-derived and anthropogenic compounds are now knownto have estrogenic activity (1-3). These compounds include plant-deriveddietary constituents as well as plasticizers, surfactants, constituentsof detergents, pesticides, and a variety of other chemicals (1,4-6). Identificationof estrogenic activity is primarily based on results of in vitroassays that rely on indicators such as estrogen receptor (ER) binding,gene transcription, or cell proliferation, or on short-term in vivoassays such as uterine growth bioassays (7).

It is well known that estrogenic activity may be observed due to directeffects of a compound binding the ER and inducing gene transcription, andto indirect effects such as induction of enzymes involved in metabolismor synthesis of endogenous estrogens (8,9), effects on binding of endogenousestrogens to hormone binding proteins (10), effects on ER regulation (11),and others. Estrogenic compounds have also been reported to have othereffects, such as involvement in other cell signaling pathways that maystimulate receptor-mediated estrogen action, e.g., protein kinase C (2,12,13),inhibition of microtubule polymerization (14), or inhibition of gap-junctionalintercellular communication (GJIC) (15).

Exposure to estrogenic chemicals could be substantial because of theirwidespread use, yet data from sensitive in vivo studies are notavailable for many of them. Reproductive toxicity studies in animals aretypically required for pesticides and can detect certain endocrine systemeffects; however, many chemicals in common use have not been screened forendocrine activity or examined in reproductive toxicity studies. Data frommultigenerational toxicity studies in animals that allow identificationof developmental effects are available for relatively few chemicals. Furthermore,new protocols for conducting multigenerational studies have identifiedmore sensitive end points for endocrine effects on development than havebeen utilized in traditional protocols (16-18).

Public concern about the potential health effects of exposure to environmentalpollutants with estrogenic activity, often referred to as environmentalestrogens, has created pressure for scientists to make predictions aboutthe significance of current exposures to environmental estrogens in theabsence of good information on exposure, effects, or dose response (19-21).Predicting human health effects of exposure to estrogenic compounds involvessynthesizing assay information on the activities of a broad spectrum ofestrogenic compounds to which humans are simultaneously exposed, includingendogenous and pharmaceutical estrogens, as well as phytoestrogens andenvironmental estrogens. Many types of compounds have been characterizedas having estrogenic activity; predicting health effects associated withone class (environmental estrogens) requires consideration of the activitiesof, and interactions with, the other classes of estrogenic compounds.

To date, predictions of human health effects of exposure to these compoundshave covered a wide range. Some researchers have hypothesized that theremay be an association with serious health effects such as breast cancerand fertility (3,22-25). Others, however, have concluded that exposureto environmental estrogens will not be associated with any estrogen-mediatedadverse health effects in humans because of the low potency of those compoundsin the in vitro assays relative to endogenous estradiol, the highlevels of circulating endogenous estrogen, and the fact that so many plant-derivedestrogenic compounds are present at high concentrations in food (26). Althoughthese factors are important to consider in predicting the health effectsof exposure to compounds with estrogenic activity, the conclusion thatenvironmental estrogens will not be associated with adverse health effectsrelies on several assumptions that must be carefully examined (27). Reviewof these assumptions brings into focus a number of considerations thatan empirically grounded risk assessment methodology for estrogenic compoundsmust address. These include the need to address differences between estrogeniccompounds in in vivo end points of concern and the associated mechanisms;the impact of pharmacokinetics on dose to target tissue and type of responseobserved; the crucial role of timing and duration of exposure in determiningtype and severity of response; the adequate consideration of non-ER-mediatedactivities of estrogenic compounds; and the effects of mixtures, includinginteractions between endogenous hormones and substances that affect endocrinefunction.

Toxic Equivalence Factors for Risk Assessment of Estrogenic Compounds

Because of the paucity of comprehensive multigenerational toxicity studiesfor estrogenic compounds, the conclusion that exposure to environmentalestrogens will not be associated with adverse health effects (26) relieson a toxic equivalence factor (TEF) risk assessment based on extrapolationfrom relative potency for ER-mediated activity in in vitro screeningassays. The TEF risk assessment strategy was developed as part of the riskassessment for dioxin so that the in vivo toxic potency for thedioxin congeners could be expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD) activity, based on relative potency in producing enzymeinduction in in vitro assays (28).

In the case of dioxin, use of the TEF approach was predicated on thesignificant finding that the chemical has to bind to a receptor beforecausing any toxic effects (29). In addition, many chronic studies had beendone to characterize the most sensitive end points for 2,3,7,8-TCDD, andto demonstrate similarity in mechanism and effects of 2,3,7,8-TCDD andother dioxin congeners (28,29). Use of the TEF for estrogenic activityrelies similarly on two assumptions: that the relative estrogenic potencyin in vitro assays for all compounds is predictive of the relativepotency for the most sensitive in vivo estrogenic effect, and thatall estrogens act via the same mechanism (e.g., binding ER) to producethe most sensitive in vivo effect.

One implication of making those assumptions for estrogenic compoundsis the prediction that all estrogenic compounds, like the dioxin congeners,have a similar spectrum of ER-mediated effects and so are essentially interchangeable.In other words, diethylstilbestrol (DES), genistein, 17ß-estradiol,chlordecone, nonylphenol, and other estrogenic compounds (1) are assumedto induce similar effects via the ER; and those effects are assumed torepresent the most sensitive effects in vivo. These assumptionsalso allow the prediction that a high dose of a weak estrogen is equivalentto a low dose of a potent estrogen because, according to the TEF rationale,the compounds would differ from each other only in the dose required toproduce the effects.

This article highlights emerging methodological issues in risk assessmentfor estrogenic compounds. It presents experimental data from in vitroand in vivo studies that suggest that the TEF assumptions may notbe appropriate for all estrogenic compounds, and that additional invivo and in vitro data are needed before such an approach canbe applied with confidence. In particular, it considers mechanisms by whichsteroid receptors cause a diverse spectrum of effects depending on ligand,target tissue, timing of dose, presence of other stimuli, and other factors(30,31). It is possible that the factors responsible for this diversityof mechanism and effect may be important in differentiating the in vivoactivities of estrogenic compounds. These considerations also suggest thatthe in vitro assays used to develop relative potency estimates havenot been demonstrated to be representative of the mechanism that will beimportant for predicting the most sensitive in vivo effects.

Experimental data presented here also highlight examples in which timingor duration of dose, rather than simply size of dose, determines the magnitudeand type of effect. The data also demonstrate the impact of pharmacokineticson timing of dose, and consequently the impact of pharmacokinetics on typeof response, rather than just size or duration of response. These factorsare also important to consider when using a TEF approach to extrapolatefrom in vitro to in vivo effects.

In addition, many estrogenic compounds have multiple activities, bothER- and non-ER mediated (above). Although most chemicals have multiplemechanisms of toxicity, these may be particularly important to considerfor estrogenic compounds because activity at more than one point in thesteroid signaling system may not be identified in in vitro studiesbut may have profound implications in vivo.

Finally, although the question of how to fashion risk assessment techniquesto address exposures to mixtures of chemicals has been discussed for sometime, it may be a particularly important component of risk assessment forhormonally active compounds. This is important so that interactions betweenexogenous and endogenous hormonally active compounds can be considered,and so that synergistic or antagonistic interactions between estrogenic/antiestrogeniccompounds can be addressed (22), as well as those between compounds withactivity in more than one component of the endocrine system, such as estrogensand antiandrogens (32).

Diversity in Mechanisms and Effects of Estrogenic Compounds

The use of the TEF approach for risk assessment for estrogenic compoundsrelies on the assumption that all estrogens produce a similar spectrumof ER-mediated responses for the most sensitive in vivo effectsand differ only in their potency relative to the reference compound. Exposureto environmental or pharmaceutical estrogens or phytoestrogens is assumedto be an extension of the exposure to endogenous estrogens; all estrogensare assumed to interact with the ER to increase or decrease biologicalresponses in the same way as endogenous estrogens.

There are examples in the literature of the induction of somewhat differenteffects by different estrogenic compounds (30,31,33-43). Although it iswell known that estrogenic compounds act on biological materials in differentways and have multiple effects, this information is reviewed here becauseof its relevance to risk assessment for these compounds. Specifically,these reports illustrate some of the difficulties in predicting in vivohealth effects based on in vitro screening assays for estrogenicactivity.

For example, the phytoestrogen coumestrol, a constituent of soy, hasbeen established as an estrogen in MCF-7 cells (1) and immature rat uterus(33). In many assays, coumestrol acts very much like a typical estrogen.On the other hand, researchers have reported that coumestrol in ovariectomizedrats behaved as an atypical estrogen. It has been known for many yearsthat coumestrol has mixed estrogen agonist and antagonist activities (34,35).In more recent experiments, it increased uterine wet and dry weights butdid not cause depletion of cytosolic ER, accumulation of nuclear ER, oruterine hyperplasia and DNA synthesis, all characteristic estrogen responses(36). Lack of uterine DNA synthesis was observed in these experiments evenafter multiple injections of coumestrol, indicating that coumestrol wasnot like estriol or other short-acting or weak endogenous estrogens, whichhave been shown to mimic more potent estrogens following chronic dosing.Whitten et al. (37) investigated the influence of dietary coumestrol onestradiol action in the rat uterus and found that coumestrol acted additivelywith estradiol for some end points (increased uterine weight and decreasedcytosolic ER binding) in these experiments but also dampened estradiol'sinduction of progestin receptors, uterine protein, and nuclear estrogenreceptor binding (37). Thus, in this system coumestrol's activities donot appear to be identical to those of estradiol, even after multiple doses;and coumestrol modulates the activity of endogenous estradiol.

In the male mouse treated neonatally with DES, adult treatment with17ß-estradiol induces prostatic metaplasia while adult treatmentwith coumestrol or soy does not induce metaplasia or prevent 17ß-estradiol-inducedmetaplasia (38). Makela et al. refer to coumestrol as a partial estrogenagonist in this system because it weakly induced c-fos expression,though estrogenic effects of coumestrol were generally missing in adultmale neo-DES mice (38). These researchers also found that male mice feda soy diet from fertilization onward and neonatally treated with DES hadreduced dysplasia in the prostate compared with mice not fed soy diets;this effect was attributed to antiestrogenic activity of soy (39). In summary,coumestrol appears to be quite similar to 17ß-estradiol in some systemsunder some conditions (e.g., MCF-7 cells, immature rat uterus), but actsas an incomplete estrogen or even modulates and dampens the effects ofendogenous estrogens in other systems (male mouse, female rat). These variationsin activity cannot necessarily be predicted from in vitro assays,but may have implications for in vivo response.

In another study, the effect of prenatal exposure to genistein, alsoa component of soy, was compared with that of DES and estradiol in rats(40). All animals except DES-treated females had smaller anogenital distancethan controls. Genistein-treated females had decreased volume of sexuallydimorphic nucleus in the preoptic area of the hypothalamus (SDN-POA) whileDES and estradiol-treated females had increased SDN-POA volume comparedto controls. In addition, genistein had a unique effect in that it delayedpuberty onset. DES (but not estradiol or genistein) increased the incidenceof atypical vaginal cycles (40). Thus genistein, DES, and estradiol inthis model induced different effects. The researchers also noted that theeffects of prenatal and neonatal exposure to genistein were inconsistent.In previous experiments involving neonatal exposure to genistein, castratedfemale rats had decreased pituitary responsiveness to gonadotropin-releasinghormone (GnRH) and enlarged SDN-POA volume. However, prenatal genisteinexposure in this experiment did not affect pituitary responsiveness toGnRH and caused a nonsignificant decrease in SDN-POA volume (40).

Although very little research has been done to compare activities ofendogenous with environmental estrogens, one recent study compared theeffects of two environmental estrogens, 4-tert-octylphenol (OP)and methoxychlor, in immature female rats (41). Both compounds acceleratedvaginal opening. Methoxychlor exposure increased uterine weight but OPdid not. In ovariectomized rats, 17ß-estradiol and methoxychlor administeredfor 7 days induced uterine growth, vaginal opening, and elevated pituitaryprolactin, while OP induced vaginal opening but did not stimulate uterinegrowth or pituitary prolactin (41). Thus, OP appears to exhibit a differentspectrum of estrogenic responses than either estradiol or methoxychlor.

Although all the compounds discussed in these examples of variationin effects of estrogenic compounds were shown in the respective studiesto bind to the ER, and most have been shown to induce cell proliferationin MCF-7 cells (1), there are many potential explanations for the differenteffects observed. For example, different pharmacokinetics, induction ofenzymes involved in endogenous estrogen synthesis or metabolism, or other,unrelated effects of test chemicals may have accounted for the differentresponses observed. Timing of dose during development also seems to bean important modulator of estrogen action. These experiments involved complexphysiologic responses involving multiple receptor-mediated processes, andsufficient data are not available to characterize the mechanisms responsiblefor the differences. However, the major point to note is that differencesin responses to estrogenic compounds occur that could not be predictedby the TEF risk assessment approach that relies on relative in vitropotencies for a single effect.

Much more information is available on differences between activitiesof endogenous and pharmaceutical estrogens, such as DES. These experimentsprovide insight and support for the idea that different ER ligands mayproduce different patterns of ER-associated gene activation. A recent paperby Stancel et al. (31) discusses their hypothesis and that of others indicatingthat different estrogens may exhibit selective patterns in the activationof estrogen-responsive genes. These researchers report that the estrogenresponse element (ERE) for different genes varies in sequence and in locationrelative to the gene (31). They suggest that the EREs of different genesmay be selectively or differentially activated by different ER-ligand complexes,and this may be responsible for the differential response patterns producedby different estrogens.

In support of this hypothesis, Stancel et al. (31) review a number ofreports that suggest differential activation of EREs by different estrogens.Korach et al. (42) found that DES and a series of DES analogs, all of whichbound to the mouse uterine ER with high affinity and caused nuclear ERretention and occupancy similar to that of estradiol, differed in theirability to induce responses such as induction of uterine DNA synthesisand stimulation of uterine glucose 6-phosphate dehydrogenase. These researchersspeculate that the chemical nature of the ligand-receptor complex may influenceits activity at different genetic sites. In addition, VanderKuur et al.(43) found that the relationship between ER binding affinity, nuclear bindingof the ER complex, and induction of progesterone receptor was not consistentfor a series of estradiol analogs examined.

Stancel et al. (31) discuss the toxicological implications of this proposedparadigm with respect to environmental and other estrogens, noting thatdifferent conformations of ligand-ER could activate or repress genes differentlythan endogenous hormone and thus produce an imbalanced estrogenic response.For example, consider the possibility that estradiol produces hypotheticalgene products A, B, and C in a 1:1:1 concentration ratio and another estrogenproduces only products A and B and produces them in a ratio of 1:2. Becausethe signal from the two estrogens differs, it is possible that disruptionof cell function could result that is not predicted by the effect of aslightly increased dose of estradiol, which in this hypothetical examplewould be expected to produce slightly more of A, B, and C in a 1:1:1 ratio.Thus, some estrogens could produce a state of estrogenization that is qualitativelydifferent from that produced by endogenous hormone (rather than simplya quantitative extension of estradiol) and that perhaps cannot be compensatedfor by homeostatic mechanisms (31).

The examples cited here illustrate the difficulty of predicting thespecific in vivo estrogen-related activities of compounds identifiedas estrogenic in screening assays. The many mechanisms by which steroidreceptors bind a diverse set of ligands and produce an even more diversespectrum of responses has been discussed by Fuller (30), who reviewed mechanismsby which specificity and diversity are generated at each step in the fundamentalprocess of gene regulation by steroid receptors. Because these variousmechanisms of creating diverse responses to signals involving a set ofsimilar steroid hormone receptors are likely to be active in modulatingthe effects of various estrogenic ligands (above), they need to be consideredin predicting the effects of exposure on human health.

Can Screening Assays Help Predict in Vivo No-Effect Levels?

Although these examples demonstrate that it is difficult to predictin vivo estrogenic effects at doses that are high enough to produceeffects, it is worth considering whether relative potency in in vitroor short-term in vivo screening assays for ER-mediated activitycould be useful in identifying a threshold dose, or no-effect level, forthe activity of estrogenic compounds. A number of issues that would beimportant to address are briefly mentioned here. First, it would be necessaryto identify the most sensitive in vivo effect and the conditionsin which an in vitro test should be done to ensure that the relevantmechanism is captured. In other words, the normal parameters of the steroidsignaling system in vivo, at critically sensitive times, would haveto be understood, so that predictions could be made as to what level ofperturbation to that signaling system would have no adverse effect. Inevaluating whether a particular exposure to an ER ligand would result inbinding and any subsequent effect, one would have to consider the manyin vivo factors that would impact the ability of the ligand in questionto bind the ER. Such factors might include sensitivity of target tissue,presence of another ligand, amount of receptor available, serum bindingproteins, and others. Second, the recent reports of striking synergisticeffects of multiple ER ligands applied as mixtures (22) suggest other importantfactors involved in regulating ER activity that need to be explored beforethe activity of a single ligand in an in vitro screening assay providesa basis for predicting in vivo estrogenic effects or lack of them.

In addition, consider the hypothesis of Stancel and others (31) thatER ligands vary in the specific pattern of genes activated. The in vivoend point associated with a test compound that activates one set of genesmay be different from that expected from 17ß-estradiol. Because theTEF approach is useful only when the most sensitive in vivo effectis similar for the test and reference compounds, it would no longer bevalid to compare the potency of test compounds with the potency of estradiol.Furthermore, the in vitro assay in which relative potency is establishedwould have to represent a mechanism relevant to the in vivo effectof concern, which in this hypothetical example would be a pattern of geneactivation related to the end point of concern caused by the test chemical,not by estradiol.

Finally, biological systems closely regulate steroid hormone and receptorlevels through multiple and complex mechanisms (30). Endogenous estrogenshave multiple activities in this signaling system, so it is not surprisingthat the structural characteristics of some compounds that bind ER mayalso result in those compounds being active in other estrogen-sensitiveprocesses, such as inhibition of enzymes involved in synthesis of endogenousestrogen (8). Most sensitive effects in vivo may occur due to compoundsacting by multiple mechanisms in approximately the same dose range. Thein vivo significance of these multiple activities is difficult topredict based on the study of one end point, such as ER binding. For example,polychlorinated biphenyls (PCBs) not only mimic thyroid hormones but mayalso bind thyroid hormone receptors in the pituitary, thus blocking thyroid-stimulatinghormone release and inhibiting a mechanism that would compensate for reducedhormone levels (44). It is well known that different estrogenic compoundsact through different mechanisms and produce multiple and varied effects.We are reviewing this information here because it needs to be consideredwhen predicting in vivo health effects based on extrapolation fromscreening tests for estrogenic activity.

ER Dynamics: Nuclear Accumulation and Impact of Pharmacokinetics

The time course of nuclear accumulation of the ER-ligand complex hasbeen studied in the context of efforts to understand mechanisms of estrogen-inducedresponses such as uterine hyperplasia (45-49). Generally, researchers usinga mouse or rat uterine assay have shown a biphasic increase in nuclearER levels to follow treatment with 17ß-estradiol or DES. Increasein uterine wet weight follows the time course of the biphasic nuclear ERincrease, with only the second phase described as "true uterine growth"due to increased cellular DNA synthesis. The first increase in nuclearER accumulation and uterine weight is shown 1 to 3 hr after dose, and thesecond increase 7 to 9 hr after dose. Weak estrogens show the early responsephase only, although multiple doses of some weak endogenous estrogens mimicthe effect of more potent estrogens (7). Nuclear ER levels return to approximatelycontrol levels about 10 hr after treatment with estradiol (46). CytosolicER levels decrease as nuclear ER levels increase and ultimately increaseabove control levels (36,46).

Limited work has been done in these model systems to examine the timecourse of ER nuclear accumulation following treatment with a variety ofestrogens. Katzenellenbogen et al. (50) evaluated temporal relationshipsbetween estrogen receptor binding and uterine growth for DES and stilbestrolderivatives. These researchers and others noted that increased retentionof nuclear receptors correlated with prolonged elevation of uterine weightand stimulation of deoxyglucose metabolism, or true uterine growth. Theimportant role of pharmacokinetic factors in modulating the estrogeniceffect of a weak estrogen is apparent from their study. The weak estrogendimethylstilbestrol (DMS) and its dimethylether DMS-(OMe)₂,which does not bind ER, were tested in the assay. Acting like a typicalweak estrogen, DMS induced short-term but not long-term nuclear accumulationof ER and uterine growth. The DMS-(OMe)₂, which had to be metabolicallyactivated to the weakly estrogenic DMS, induced long-term increases innuclear ER and uterine growth because metabolic activation proceeded ata rate that simulated chronic dosing (50). Repeated dosing with some weakestrogens can cause long-term increases in uterine growth such as thosecaused by more potent estrogens (above). Thus, the pharmacokinetics ofestrogens can modify not only the duration of a response, but also thetype of estrogenic response observed.

Patterns of ER localization after treatment with coumestrol in similarassays shows some conflicting but interesting differences from ER localizationafter estradiol, estriol, or DES treatment. While Whitten et al. (33) foundthat coumestrol in the diet of immature rats increased nuclear ER concentrations,Markaverich et al. (36) reported that coumestrol failed to cause substantialnuclear accumulation of ER in ovariectomized rats, although it did causeincrease in uterine wet and dry weights. Markaverich et al. (36) show aslight increase in nuclear ER after coumestrol treatment, with levels returningto control levels in less than 5 hr and then decreasing slightly belowcontrol levels through 24 hr. Treatment with a higher dose of coumestroldid not modify this pattern or increase nuclear ER, indicating that coumestrolwas not able to function as a more potent estrogen in this system. Estradiolstimulation, on the other hand, caused a significant increase in nuclearER that returned to control levels by 5 hr after treatment. Coumestrolappeared to induce long-term increases in cytosolic ER, which increasedslowly but continuously over the 24 hr during which measurements were made.

In an experiment looking at coumestrol modulation of estradiol actionin the rat uterus, Whitten et al. (37) found that animals that receivedcoumestrol along with physiologic doses of estradiol for 90 hr had lowerlevels of nuclear ER than controls. When these animals were challengedwith a single estradiol dose, the coumestrol-treated animals produced asmaller increase in nuclear ER than controls (37). Although there is conflictingevidence about whether coumestrol alone substantially increases nuclearER (33,36), it does appear that a coumestrol diet diminished the nuclearER accumulation after estradiol treatment (37). In another example of thedifferential abilities of various estrogens to induce nuclear accumulation,Martin et al. (51) reported that in MCF-7 cells, genistein and coumestrol(both soy derivatives) were less effective at translocating ER to the nucleusthan zearalenol (a mycotoxin) and estradiol.

Hammond et al. (52) reported that the organochlorine pesticide chlordeconeis estrogenic and interacts with rat uterine estrogen receptors. In experimentscomparing estradiol and chlordecone, nuclear ER levels increased quicklyfollowing estradiol treatment and then decreased to nearly control levelsby 12 hr posttreatment. Chlordecone, on the other hand, increased nuclearER slowly, reaching maximum at 36 hr and maintaining that level throughthe end of the experiment at 48 hr. The long half-life of chlordecone,therefore, appears to moderate the relative potency of the compound invivo, but may also moderate the qualitative estrogenic effect due tothe potential importance of the time course of ER-ligand activity in thenucleus in determining the nature of the estrogenic response. For example,pharmacokinetics of the ER-ligand complex could affect the length of timethat expression of some genes remain elevated (31).

In another example of how different estrogens exhibit different pharmacokineticswith respect to nuclear accumulation of ER, administration of o,p'-DDTto immature female rats caused translocation of ER to the nucleus thatwas maximal 3 hr after treatment; estradiol in this system caused maximalnuclear ER 1 hr after treatment (53). In experiments comparing the timecourse of uterine weight increases in rats following treatment with amsonicacid and DES, both test compounds induced an extended increase relativeto estriol (6). Amsonic acid is an optical brightening agent that was testedfor estrogenic activity after reports of sexual impotence among exposedfactory workers (6).

It has been observed that the duration of nuclear ER accumulation affectsthe response observed, although this relationship has been explored onlyfor a few endogenous and pharmaceutical estrogens (45-50). The fact thatcertain phytoestrogens and environmental estrogens show variation in timingof nuclear ER accumulation (33,36,37,52,53) strongly suggest that it maybe important to consider timing and duration of dosing in determining effect.This is important because it is not clear that the effects of increasingthe duration of nuclear accumulation of activated ER are equivalent tothe effects of simply increasing the dose of estradiol. Pharmacokineticconsiderations are also important, therefore, in determining not only doseto target tissue, but potentially also in characterizing the end pointexpected.

Consideration of Effects on Estrogen Synthesis, Metabolism, and Bioavailability

Because estrogen synthesis and metabolism in vivo are regulatedby many factors including endogenous estrogen, it is not surprising thatexogenous estrogens often also affect these regulatory mechanisms (8,23).In attempting to predict health effects of exposure to xenobiotic compounds,it is important to consider the ability of a compound to alter the endogenoushormone environment by influencing synthesis or metabolism of endogenousestrogens (and other endogenous steroids). Of course, these effects involvemechanisms that do not necessarily involve binding ER. Some compounds causechanges in levels of cytochrome P450 enzymes that are involved in estrogenmetabolism (indirect estrogenic effects) (9,23,54), while other compoundsthat induce ER-mediated gene transcription also affect synthesis or metabolismof estradiol (8). Effects on the endogenous hormone environment cannotbe accounted for by assuming that the consequences of exposure to exogenousestrogens are simply an extension of exposure to endogenous hormone, becausethe effects on hormone synthesis and metabolism vary among estrogenic compounds.Although it is not surprising that any compounds have multiple effects,consideration of multiple effects for estrogenic compounds may be particularlyimportant because of the resulting difficulty of predicting interferencewith normal signaling processes.

For example, Bradlow et al. (23) have shown that exposure to a numberof compounds, many of which are estrogenic, can affect the metabolism of17ß-estradiol by shifting the ratio of two metabolites, 2-hydroxyestroneand 16-hydroxyestrone.This effect may be important for predicting health effects because the16-hydroxy metaboliteis genotoxic and a potent estrogen, while the 2-hydroxyestrone metaboliteis not reported to be genotoxic and is only very weakly estrogenic (55).Others have reported that the plant-derived flavonoid quercetin, whichincreases the severity of estradiol-induced tumorigenesis in hamster kidney,operates by increasing the formation of the catechol estradiol metabolite4-hydroxyestradiol, which may undergo redox cycling and generate free radicals(54).

Makela et al. (8) reported that several plant estrogens, including coumestroland genistein, reduce the conversion of estrone to 17ß-estradiolby inhibiting the estrogen-specific enzyme 17ß-hydroxysteroid oxidoreductaseType 1 in vitro, but zearalenone and DES did not inhibit this enzyme.Thus, the phytoestrogens coumestrol and genistein, which have been reportedby some as incomplete estrogens incapable of inducing all the effects of17ß-estradiol (36,40), may also decrease availability of active endogenousestrogen by inhibiting its synthesis. These types of differences in thecombination of ER-mediated and other estrogen-related effects of exogenouscompounds may have significant impacts on their potential health effects.

Differences in the bioavailability of compounds in vivo and effectsof estrogenic compounds on the bioavailability of endogenous estrogensare additional factors that will modulate toxicity in vivo. Thesefactors should also be considered in predictions of health effects of exposureto these compounds. One of the most important modulators of the availabilityof endogenous estrogens may be the serum-binding proteins like sex hormone-bindingglobulin (SHBG). While this protein can modulate the availability of endogenousestrogens, in most cases its ability to modulate the availability of exogenousestrogens remains to be explored. SHBG appears not to bind many environmentalestrogens (16,56), but estrogenic compounds may affect SHBG binding toendogenous estrogens (57). This observation offers another mechanism bywhich exogenous estrogens may modulate endogenous estrogen activity.

The importance of the steroid hormone microenvironment within cellshas been recognized and mechanisms of regulation of enzymes involved inestrogen metabolism and synthesis are being explored at the level of thetarget tissue (58-61). The effects of xenobiotics on systemic estrogenregulation may be different from their effects on estrogen regulation inthe target tissue. For example, the cytochrome P450 enzymes that are inducibleby different xenobiotics vary among tissues, which means that a compoundmay have different effects on endogenous estrogen metabolism in the liverand the breast, for example (61-64). Thus, it is important to considerthe questions of synthesis, metabolism, and bioavailability at the levelof the cell and target tissue, as well as at the systemic level. Of course,pharmacokinetics are also important in determining dose to target tissue.Concentrations of lipophilic chemicals in mammary adipose tissue, for example,may be much higher than serum concentrations, and exposure presented asbody burden is often substantially different from exposure presented asdaily intake due to pharmacokinetic considerations.

Other Effects of Estrogens and Effects of Mixtures

Environmental estrogens present a special challenge for risk assessmentbecause they have the potential to be active in many different ways. Effectsat multiple points in a signaling system in vivo may be difficultto predict from in vitro or short-term in vivo tests, asdemonstrated for PCBs and thyroid hormones, discussed earlier (44).

In addition, researchers have reported the ability of DES, some stilbeneestrogens, and the common environmental estrogen bisphenol-A, to inhibitmicrotubule formation (14). Endogenous estrogens and phytoestrogens testedin this cell-free assay did not have that effect. Inhibition of microtubulesin intact cells may lead to the induction of micronuclei and aneuploidy,which may play a role in estrogen-mediated carcinogenesis (14).

The idea that estrogens may regulate cellular function at sites otherthan specific gene-regulating receptors has been explored recently (13,65).Plasma membrane-resident forms of ER have been proposed to explain observationsof cellular responses to estrogen that occur within minutes and so cannotbe explained through gene transcription (65). In addition, other researchershave shown that chemicals that activate peptide growth factor signalingsystems, such as protein kinase-C activators, can also induce ERE-dependenttranscription (12). These researchers showed that a protein kinase-C activatoracted synergistically with 17ß-estradiol to induce ERE-dependenttranscription. They also showed that epidermal growth factor, which producesestrogenlike effects in the mouse reproductive tract, increases levelsof nuclear ER (12). These researchers note that the potential health effectsassociated with exposure to exogenous estrogens may also be observed followingexposure to chemicals that could activate peptide growth factor signalingsystems.

Recently researchers showed that dieldrin, DDT, and toxaphene, all ofwhich have been reported to be estrogenic, inhibited GJIC in normal humanbreast epitheliel cells in a dose responsive manner. Effects of these compoundswere additive, with subthreshold doses of individual compounds being effectivewhen combined (15). Many tumor promotors have the ability to inhibit GJIC.It is hypothesized that inhibition of GJIC may release initiated cellsfrom suppressing effects of signals passing from surrounding normal cells(15).

The effects of mixtures of compounds may be particularly striking forestrogenic and other hormonally active compounds, and thus particularlyimportant for risk assessment. As illustrated above, certain phytoestrogensmodulate the activity of endogenous estrogens (37). In addition, recentreports of synergistic activity of some environmental estrogens in vitro[(22); A Soto, personal communication], as well as information on the presenceof environmental and dietary antiestrogens (26,66), suggest that interactionscould be important.

Screening and exposure characterization needs to be comprehensive enoughto identify all kinds of biological activity (67). Although o,p'-DDTwas reported to be estrogenic before 1970 (68), the potent antiandrogenicactivity of p,p'-DDE was not reported until 1995 (32). In addition,because activity of estrogen is also modulated by other hormones, suchas progesterone (69), it is important that these biological activitiesare considered in a comprehensive manner.

Conclusions

The challenge of predicting health effects of exposures to estrogeniccompounds is daunting because of the current limitations in our understanding.Chemicals have not been routinely screened for these endocrine activitiesbefore being introduced to commerce, and so the significance of currentlevels of exposure to environmental estrogens, or other hormonally activecompounds, is unclear. In addition, data from multigenerational or othersensitive toxicity studies are not available for most compounds to provideinformation on hazard identification and dose response. The goal of thisarticle is to suggest that it is simplistic to generalize that the effectsof all estrogenic compounds can be predicted by assuming that their invivo effects will necessarily be extensions of the effects of 17ß-estradiol,based on a screening test for estrogenic activity. Screening tests areuseful to identify compounds for further study, but must be used with cautionto predict health effects or no-effect levels. Although it is not clearwhether current levels of exposure to estrogenic or other hormonally activecompounds in the environment are associated with health effects, it ispremature to dismiss exposure to environmental estrogens as a concern forhuman health effects based on relative in vitro potency.

A substantial body of experimental data provides insight into differencesamong estrogenic compounds in terms of mechanisms of action and end points.For example, data suggest variations between compounds in ER-ligand bindingto EREs (31), time course of nuclear ER accumulation (36,52), patternsof gene activation (38), and other mechanistic characteristics. These andother data presented here suggest that the assumption that relative potencyin in vitro screening assays is representative of relative potencyfor the most sensitive ER-mediated in vivo effect has not been demonstratedto be accurate.

Current toxicity testing protocols may not be adequate to identify endocrineeffects, and may need to be expanded to accommodate the special challengesof risk assessment for estrogenic compounds. For example, for some estrogen-mediatedend points the dose response curve is such that high-dose experiments arenot likely to be predictive of low dose effects (16,56). Therefore, itmay be necessary to broaden testing protocols to look at an extended dose-responsecurve. It may also be appropriate to modify protocols to evaluate an enlargedspectrum of end points, including more sensitive end points like delayeddevelopmental or behavioral effects. For example, Vom Saal et al. (16)found territorial behavior in male mice affected by prenatal exposure to0.001 mg/day of DES or 1 mg/day of o,p'-DDT; and Chapin et al. (17)have developed new protocols for testing a variety of endocrine, immune,and neurological effects of certain pesticides.

It is well known that timing of exposure has a substantial impact onthe dose required to induce an effect. Testing protocols need to identifythe most sensitive periods for exposure and to follow up for latency andmultigenerational effects. In addition, timing of exposure can affect thetype of response observed. For example, neonatal exposure of rats to genisteinproduced an enlarged SDN-POA, while prenatal exposure decreased SDN-POAvolume (40). Duration of dosing also has an important impact on patternsof nuclear accumulation of ER and resulting effects (50), so pharmacokineticsand dosing regimes have an impact on the qualitative as well as quantitativenature of the response. Thus, consideration of time as a third axis onthe dose-response curve may be particularly important for endocrine effects.The time axis could incorporate information on when in the lifecycle ofthe organism exposure occurs, as well as duration of exposure of the targettissue. The experimental data reviewed in this paper provide examples ofthe importance of both these factors in determining the toxicological endpoints observed.

Risk assessment for estrogenic compounds must consider, among otherfactors, the diversity in effects observed between classes of estrogensin various animal models, the importance of pharmacokinetics, timing, andduration of exposure in modulating the spectrum of toxicological end points,the diverse (ER- and non-ER-mediated) activities of many estrogenic compounds,and the interactions between multiple compounds to which individuals aresimultaneously exposed, including interactions between exogenous and endogenousfactors. Emerging questions about risk assessment techniques for hormonallyactive compounds, therefore, may require new methods.

We propose that a focused research strategy be developed to investigatethe mechanisms of action, diversity of effects, and pharmacokinetics ofendocrine disrupters. This research should integrate the study of endogenous,synthetic, anthropogenic, and phytoestrogens in a focused program thatwill not only increase our understanding of potential health effects associatedwith exposure to these compounds in diet and the environment, but willprovide insight into the role of endogenous hormones in breast cancer andother major health concerns.

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