Abstract
Environmental researchers were taken by surprise
in recent years by the numbers of "feminized"
males found among several wildlife species. The
effects were finally traced to pesticides and other
chemicals that behave like estrogens--but don't
in any way look like them. Scientists are coming
to appreciate that chemicals don't have to resemble
estrogen to mimic them. Estrogen mimetics act by
interfering with steps in the endogenous chemical-signaling
pathway through which estrogens normally work. The
estrogen story helps to define a new class of environmental
hazard. Historically, potentially harmful environmental
agents have been evaluated for their direct effects
on DNA. The estrogen mimetics indicate that chemicals
can alter normal cellular activities by providing
an unnatural signal to a normal signalling pathway.
This work also points to the possibility that other
chemical agents in the environment may simulate
the effects of other classes of signaling molecules,
such as neurotransmitters or growth factors, for
example.
Caption: Yeast-estrogen response
system used by the authors gives scientists the
opportunity to study the whole-cell response to
natural and synthetic estrogens in a real-world
context. Yeast cells do not normally manufacture
estrogen receptors, so the system is constructed
by transferring the gene for the human estrogen
receptor into a yeast cell (a), which can then manufacture
the receptor proteins (b). In addition, a reporter
gene is transferred into the yeast cell. The reporter
gene contains an estrogen-response element, so it
senses the presence of an estrogen bound receptor
and reports it in a color-coded fashion. The yeast
cell containing the transferred genes, a transformed
yeast cell, turns blue to indicate an estrogenic
response in the presence of natural (c) or synthetic
estrogens. Some environmental compounds act as antiestrogens,
substances that bind to the estrogen receptor but
fail to activate an estrogenic response. An antiestrogen
can in some cases displace estrogens bound to the
receptor and curtail the estrogenic response (d),
turning the cell from blue to white.
Section 1
In many ways, the story of the pesticide DDT is
the story of America's attitude toward synthetic
chemicals in the environment. DDT was the first
of many new pesticides that people hoped would improve
the quality of their lives, but gradually it became
clear that such progress often had a cost. DDT was
one of the first environmental chemical agents to
be banned in the United States. Scientists are still
seeking a full understanding of how it came to have
broad and unexpected environmental and health effects.
First
synthesized in 1874, DDT took on its modern role
in the late 1930s, when the Swiss chemist Paul Muller
recognized its potential as an insecticide. It was
perceived to be so beneficial for public health
and military hygiene (mostly as a delousing agent),
in fact, that Muller was awarded the 1948 Nobel
Prize for Medicine and Physiology. In spite of occasional
reports that stirred concern about potential health
effects, it was used copiously in the United States
and around the world as an agricultural pesticide
and a malaria-control agent (a function for which
it continues to be used today in some developing
countries).
As America began to closely scrutinize technology
in general--and ecological agents in particular--in
the late 1960s, people took a closer look at DDT.
Early in that decade scientists had noticed a decrease
in certain bird populations in Europe. Eventually
this decrease was linked to the use of various pesticides,
among them DDT. By the early 1970s the use of DDT
had been banned in the U.S. and in many European
countries.
Recently, the DDT story has taken on a new dimension,
alerting scientists to a novel class of potential
interactions between environmental agents and living
creatures. Since the early 1980s, reports have been
surfacing of "feminized" wildlife that
have been exposed to certain chemicals in the environment.
One recent report by Louis Guillette and his group
at the University of Florida received a great deal
of publicity when it linked DDT exposure with a
growing population in a Florida lake of male alligators
whose penises were smaller than those of normal
males. DDT, as it turns out, can act in the body
like endogenous estrogen. Or, as in the case of
the alligators, its breakdown products may be estrogens
or even a compound that blocks the effects of androgens,
the male sex hormones.
This case and others suggest to biologists that
synthetic compounds in the environment can mimic
in animals the actions of natural signaling molecules,
such as hormones and growth factors. But what has
been particularly surprising about this discovery
is that the synthetic compounds in no way resemble
the chemical structure of the natural hormones or
growth factors. The classification of environmental
chemicals that mimic endogenous signaling molecules
opens up a whole new field of toxicology--environmental
signaling. It now appears that the toxicity of some
environmental pollutants may be the result of a
"natural" signal being sent by an "unnatural"
signaling molecule.
To be sure, environmental pollutants have been studied
intensely for the past 30 or so years. But the focus
of most of those studies has been on the potential
of toxins to cause genetic abnormalities by damaging
DNA directly. Environmental hormones, on the other
hand, do not alter genes themselves, but may change
the way they are expressed.
Recent reports, not only of feminized wildlife,
but also of the possibility of a precipitous fall
in sperm counts of people and of the rise in hormone-related
cancers, such as breast cancer, have brought popular
attention to environmental hormones--estrogen, in
particular. But the so-called ecoestrogens may be
only the most obvious of the chemical mimics in
the environment. Observations of the effects of
environmental estrogens are paving the way for what
will undoubtedly turn out to be a larger phenomenon
of environmental signaling. We believe that environmental
estrogens are the paradigm for a new understanding
of the health effects of external signals in the
environment.
Section 2
Accidental Estrogens
The observation that DDT could behave like estrogen
provided a framework for understanding that chemicals
not specifically designed to possess hormonal activity
may in fact have it. For example, a 1975 spill of
Kepone, a chemical used in the manufacture of the
pesticide Mirex, resulted in a lowered sperm count
in men exposed to the chemical. Since natural estrogens
given to men decrease their sperm count, some investigators
guessed that Kepone might also be estrogenic. Subsequent
studies designed to test chemicals for estrogen-like
activities confirmed that indeed Kepone was a weak
estrogen, but an estrogen nevertheless. The chemical
structure of Kepone is even farther removed from
estradiol-the main form of estrogen found in people--than
is DDT.
The
structural differences between natural estrogens
and estrogens in the environment were sufficiently
puzzling to scientists that they convened a national
meeting in 1979 to discuss the subject. Investigators
working on many aspects of estrogen biology and
chemistry met to solve the "estrogen problem."
At the time, the information available led investigators
to expect the number of possible estrogenic chemicals
to be fairly limited. At the meeting, the potential
for adverse effects on human health was considered,
but at the time, there were few examples. Nevertheless,
elegant studies on Kepone in Richard Palmiter's
laboratory at the University of Washington at Seattle
suggested that the effects of environmental hormones
might be more widespread than initially thought.
His laboratory demonstrated that a chemical such
as Kepone, which has no structural resemblance to
an estrogen, could activate some of the same very
specific estrogen-associated genes that the natural
hormone activates in the oviduct of chickens. Although
Kepone was much weaker as an estrogen, the functional
similarity of two widely disparate chemicals was
striking.
Figure
4. Many ecoestrogens also bind the estrogen
receptor. Yet a comparison of the structures of
estradiol and those of the ecoestrogens shows how
physically dissimilar they are. For this reason,
it is difficult to understand how so many differently
shaped keys can fit the same lock. Standard receptor-binding
assays therefore may not always give accurate information
about the potential strength of an environmental
estrogen.
Whereas the discovery of environmental estrogens
was news to scientists in the middle and late 1970s,
the world did have some experience with synthetic
estrogens. This came in the form of diethylstilbestrol,
or DES. DES was produced in 1938 in London by Sir
Charles Dodds and was the first synthetic agent
specifically designed to have estrogenic activity.
Like many of the environmental estrogens, DES is
not structurally similar to natural estrogens. This
landmark study in pharmacology provided an early
demonstration that compounds of diverse structures
could exhibit similar biological functions.
DES also taught scientists other lessons about the
possible toxic effects of estrogens. Because of
its growth-promoting effects, DES was used for decades
as a growth stimulant in cattle. It also had obvious
clinical applications. As early as 1948, it was
used to prevent miscarriages in women. In 1971 the
drug became associated with a rare form of vaginal
cancer called clear-cell adenocarcinoma detected
in some of the adolescent daughters of women who
had taken DES. In addition, the drug brought about
cellular changes in the vagina or Fallopian tubes
of female offspring, as well as structural changes
in the uterus. Studies on this important clinical
problem resulted in animal models demonstrating
the effects of estrogens on the sexual development
of both male and female mammals. DES was the first
documented example of a human "transplacental"
carcinogen--that is, a chemical, which when given
to the mother, causes cancer in her daughter. The
clinical and experimental studies surrounding the
DES findings in the 1970s and 1980s, gave scientists
a new appreciation for the effects of potent synthetic
estrogens on the development of the reproductive
system and on subsequent adult health.
Estrogens also have effects on male genital development.
As adults, male mice exposed in utero to DES had
a higher-than-average frequency of undescended testicles,
testicular cancer, sperm abnormalities and prostate
disease. Some of these outcomes were also reported
for men exposed in utero to DES. Even though these
men had more genital-tract abnormalities as adults,
the most recent studies suggest there is no loss
of fertility. The doses of DES required to cause
malformations of the male reproductive tract were
almost the same in mice and men.
The extensive research on the effects of DES in
mice and people served as a model for predicting
the possible outcomes associated with estrogens
of any source in many species and formed the basis
for identifying chemicals in the environment that
elicit changes similar to DES. The effects of such
a potent estrogen actually set the standard for
judging the activities, as well as outcomes, of
other chemicals acting like estrogens. The fact
that DES feminized the development of laboratory
animals provided insight into what ecoestrogens
might do to the development of many other species;
almost a kind of guidebook to outcomes. It also
suggested an upper limit on effects, since DES is
much more potent than any single ecoestrogen.
Section 3
One Lock, Many Keys
Studies on DES, natural estrogens and early pesticides
such as DDT have taught scientists that natural
estrogens play an important role in the normal growth
or function of many organs, including breast, bone,
liver, the organs of the reproductive system and
the cardiovascular system. Thanks to modern biochemistry
and molecular biology, scientists can now also work
out the details of how these compounds affect the
cells of target organs.
Estrogens, indeed all hormones, are chemical signals,
and as such are important links in the body's internal
communication system, helping cells in various organs
to sense and respond to changing physiological circumstances.
Endogenous estrogens are steroid hormones, produced
from cholesterol in the ovaries of females and the
testes of males (and possibly, the adrenal corticex
in both sexes) in response to signals from the brain
and other organs. Estrogens are secreted into the
blood, where they are carried to the cells of target
organs, such as the breasts and reproductive organs.
Figure
3. Natural estrogens, such as estradiol,
bring about their cellular effects by altering the
expression of particular genes in target cells.
Estradiol is soluble in the lipids that make up
the membranes surrounding cells and their nuclei,
so the hormone can pass unfacilitated into the nucleus.
Once inside, estradiol binds with an estrogen receptor,
a protein molecule dissolved in the aqueous nuclear
medium. Estradiol fits into a specific site on the
receptor, much as a key fits a lock. Although estradiol
can probably enter many cell types, only those with
estrogen receptors can respond. Two occupied receptors
join together to form a dimer, which then attaches
to a regulatory site called the estrogen response
element, or ERE, on a gene. The ERE is located within
the gene's "on" switch, its promoter.
The occupied and dimerized receptor molecules interact
with proteins associated with transcription factors
that are attached to the promoter and thus regulate
gene expression. Either gene expression is turned
on, or its level is modulated. In some cases, gene
expression can be suppressed when estrogens bind
their receptor.
In the case of most chemical signal molecules, elaborate
systems are required to admit the signal molecules
into target cells. This is not necessary for endogenous
estrogen, which is soluble in fats, such as the
lipids that make up the membranes surrounding cells.
Estrogen can therefore pass unaided through the
cellular membrane. Once inside the cell, estrogens
can also easily cross the membrane into the nucleus,
the compartment that contains the cell's DNA. Inside
the nucleus, estrogen binds to a protein, called
an estrogen receptor, which is dissolved in the
aqueous nuclear medium. The estrogen-receptor complex
can then bind to the regulatory regions of specific
genes and, by this, alter the way they are expressed.
The complex can either activate or repress gene
expression completely, or it can alter the level
at which a gene is expressed, the overall result
being the a change in cell programming. Among the
genes regulated by the binding of the estrogen-receptor
complex is the gene encoding the receptor for another
hormone, progesterone, as well as genes encoding
several growth factors and their receptors.
Because estrogens can easily enter many cells, and
probably their nuclei, it is at first difficult
to understand why only certain cells respond to
the hormone. The answer lies with the estrogen receptor
and groups of proteins that are associated with
it. Only some cells contain these proteins, and
those are the only ones that can respond to estrogen.
This provides the definition of a "target"
cell.
In the simplest terms, the estrogen receptor can
be thought of as a lock for which estrogen is a
key. The analogy implies a unique fit of one key
to one lock. However, research has taught biochemists
that is a somewhat unrealistic view of the interaction
between a receptor and its chemical key, called
a ligand. In reality, many physically dissimilar
keys seem to fit the same lock. In some cases, the
lock opens; one has an estrogen mimic. In other
cases, the key blocks the lock, and one has an antiestrogen.
The discovery that so many keys seem to open the
estrogen lock--to a greater or lesser extent--suggests
that the lock mechanism is looser, or the keys interact
with each other to a greater degree, than previously
thought. For many years, scientific locksmiths have
focused on understanding how the endogenous estrogen
key turns the receptor lock. Now with the help of
antiestrogens--compounds that bind the receptor
but fail for some reason to turn the lock or elicit
a physiological response-and more recently with
environmental estrogens, they are coming to understand
more about the lock mechanism.
Section 4
The Estrogen Receptor
The estrogen receptor is a large protein with different
regions that each performs a different function.
The function of one of the regions is to recognize
and bind endogenous estrogen. Another segment helps
the receptor-ligand complex bind to DNA. The exact
regulatory site on the DNA to which the estrogen
receptor-ligand complex binds is called an estrogen-response
element, or ERE. Once the complex binds to DNA,
particular sites on the estrogen receptor allow
the complex to interact with proteins attached to
an adjacent regulatory site on the gene, called
the promoter. The estrogen receptor is a transcription
factor and interacts with the promoter-bound proteins
to somehow bring about a change in the gene's expression--either
to activate or suppress gene expression, or to change
the level at which the gene is expressed. It is
believed that the longer the receptor-ligand complex
remains attached to the ERE, the longer the complex
modulates gene activity. Once the complex is removed
from the ERE, gene regulation also ceases. Although
the steps leading to the initiation of an estrogen
response have been extensively studied, little is
currently known about how the response is turned
off.
It is likely that some environmental estrogens bring
about their estrogen response by replacing endogenous
estrogens in the signaling pathway. For some, this
means direct interaction with the estrogen receptor.
Most of the ecoestrogens tested, however, bind the
receptor with only a fraction of the strength--anywhere
from one-fiftieth to one-ten-thousandth--of the
natural hormones. But, as it turns out, receptor
binding is only one factor of many that predict
how well an ecoestrogen mimics the physiological
response of the natural hormone.
Although ecoestrogens may be more weakly binding
than natural estrogens, they may be more effective
at gaining access to the receptor, or they may block
the natural hormone's access. For example, some
estrogens may be able to bind to proteins other
than the estrogen receptor. Some of these proteins,
such as serum albumin, sex-hormone-binding globulin
and alpha fetoprotein, are dissolved in the blood
serum, the extracellular fluid bathing the cell.
As a result, the actual concentration of an estrogen-like
chemical found inside the cell is a function, in
part, of the affinity the chemical has for proteins
outside the cell. If the chemical binds very strongly
to extracellular proteins, fewer molecules move
inside the cell. If, on the other hand, the chemical
in question has a greater binding affinity for the
estrogen receptor, more of it is found inside the
cell than out. Because different estrogenic chemicals
differ significantly in their binding affinities
for extracellular proteins, their intracellular
concentrations vary accordingly.
The natural hormone, estradiol, exhibits extensive
binding to extracellular proteins, whereas the synthetic
hormone, DES, has little affinity for them. Thus
at an equivalent concentration in the blood, more
DES enters the cell than does estradiol. In effect,
DES is a functionally more efficient estrogen than
is the natural hormone.
In simple terms, one may ask whether ecoestrogens
are more DES-like or more estradiol-like in their
binding to extra cellular constituents.We have recently
shown that some representative ecoestrogens, including
octophenol and o,p'-DDT, do not bind appreciably
to serum proteins in people or in alligators. These
compounds may thus be more physiologically active
than their estrogen-receptor binding characteristics
alone would predict.
Many studies on ecoestrogens are conducted in the
laboratory and look at the effects on cells and
receptors of these chemicals as they exist in the
environment. But scientists know that the body's
own metabolism can alter these chemicals, possibly
to even more potent forms inside the body. Over
the years, different groups have found that in some
cases metabolism converts a nonestrogenic substance
to one that has hormonal activity. This is true,
for example, for certain polycyclic aromatic hydrocarbons,
which become estrogenic after a hydroxyl group is
added to them metabolically. Hydroxyl groups also
enhance the estrogenic activity of polychlorinated
biphenyls, popularly known as PCBs, which are common
environmental contaminants.
In general, hydroxylation seems to enhance the affinity
a chemical has for the estrogen receptor. Recently
it was shown by investigators in Sweden that the
hydroxylated form of PCBs is retained more than
the unhydroxylated form within the serum of seals
and people. The relative estrogenicity of this group
of chemicals is not yet known, and Steve Safe at
Texas A & M has raised the possibility that
some of them are inactive, whereas others may be
actually antiestrogenic.
In some ecoestrogens, a chlorine atom is found in
positions that would normally become hydroxylated.
This suggests that the estrogen receptor may recognize
a variety of chemical species. Chlorinated chemicals,
such as DDT and its relatives, tend to persist the
longest, both in the body and in the ecosystem.
The half-lives of DDT-associated molecules are estimated
by some to be as high as 50 years. Moreover, DDT
and related compounds are part of a global system
in which compounds move through the atmosphere from
one ecosystem to another. Thus these compounds may
persist within an individual or a population, or
they may persist globally. The widespread and persistent
nature of some hormonally active compounds demands
improved methods for their detection, removal (where
possible) or prevention (where necessary).
Section 5
Cellular Litmus Test
The test routinely used to determine the presence
and relative strength of ecoestrogens has been to
assay a compound's binding affinity for the estrogen
receptor. As we have pointed out, this approach
excludes many mitigating factors that ultimately
determine how a substance will act within a cell.
In our laboratory we are developing what we believe
to be a more informative system--one that we hope
will yield greater detail about the molecular interactions
of ecoestrogens inside and outside cells.
To construct our system, we added an estrogen receptor
to a simple cell that did not contain one before.
We did that by transferring the gene for the human
estrogen receptor into a yeast cell, which has many
similarities to a human cell in its structure, molecular
biology and biochemistry. This process is referred
to as transformation. The transformed yeast cell
now produces, or expresses, the estrogen receptor.
In addition to the estrogen receptor gene, we also
inserted what is called a reporter gene, one that
in this case senses the action of estrogen in the
cell and reports it in a color-coded fashion, by
producing a blue-colored product. If a certain chemical
has estrogenic activity, the transformed yeast cell
turns blue; in the absence of activity, the cell
remains white.
This assay is also useful for detecting antiestrogens.
The cell is first exposed to estradiol, which turns
the cell blue. The introduction of an antiestrogen,
which blocks estrogenic activity, causes the blue
color to disappear.
Figure 5. Yeast-estrogen response
system used by the authors gives scientists the
opportunity to study the whole-cell response to
natural and synthetic estrogens in a real-world
context. Yeast cells do not normally manufacture
estrogen receptors, so the system is constructed
by transferring the gene for the human estrogen
receptor into a yeast cell (a), which can then manufacture
the receptor proteins (b). In addition, a reporter
gene is transferred into the yeast cell. The reporter
gene contains an estrogen-response element, so it
senses the presence of an estrogen bound receptor
and reports it in a color-coded fashion. The yeast
cell containing the transferred genes, a transformed
yeast cell, turns blue to indicate an estrogenic
response in the presence of natural (c) or synthetic
estrogens. Some environmental compounds act as antiestrogens,
substances that bind to the estrogen receptor but
fail to activate an estrogenic response. An antiestrogen
can in some cases displace estrogens bound to the
receptor and curtail the estrogenic response (d),
turning the cell from blue to white.
The model not only demonstrates the utility of a
simple cellular system for understanding the activity
of environmental estrogens, it can also be modified
to study particular mechanisms or to help screen
chemicals for their functional activities. In fact,
similar systems can be constructed to study a number
of classes of signaling molecules. The flexibility
of this system led us three years ago to propose
a new approach for detecting biologically active
chemicals in general. For example, one can introduce
the androgen receptor into mammalian or even yeast
cells and then use them to assess the androgenicity
of a substance. Very recently this exact approach
was taken by Bill Kelce at the U.S. Environmental
Protection Agency and Betty Wilson at the University
of North Carolina, with the result that an antiandrogenic
chemical was identified, providing the first example
of a steroid-hormone-like activity outside of the
estrogen-antiestrogen family.
In the future, the yeast-estrogen system can be
used to identify chemicals in the environment having
activities that mimic other hormones and biomolecules,
such as progestins, glucocorticoids or retinoids,
among others, and to assess their potential adverse
or beneficial effects. This experimental system
allows scientists to approximate a comprehensive
cellular response to biologically active molecules
and to assess the effects of the expression of drug-metabolizing
enzymes, specific serum proteins or growth factors
and their receptors. One can think of this as a
"Lego" system approach to model-cell building
because it permits scientists to make a stepwise
reconstruction of an overall hormone-signaling system.
Figure 6. Transformed yeast cells
yield information about extracellular as well as
intracellular events affecting the potency of the
estrogenic response. Estrogens can bind to proteins
in the blood serum surrounding a cell. If the estrogen
has a greater affinity for these external proteins
than it does for the estrogen receptor, fewer molecules
actually enter the cell. In a comparison between
estradiol and an ecoestrogen, for example, the estradiol
molecule has a greater affinity for serum proteins
(a) than does an ecoestrogen (b). Even though the
two estrogens are present in the same concentration,
more ecoestrogen molecules enter the transformed
yeast cell than do estradiol molecules. As a result,
the ecoestrogen produces a stronger than expected
estrogenic response, indicated by the deeper blue
color of the cell, more closely approaching the
natural estrogen.
The "Lego," or interconnecting building
block strategy, has already helped us discover new
aspects of the estrogen-signaling system. For example,
it was generally assumed that one molecule of endogenous
estrogen binds one receptor molecule. Using the
yeast system, we were able to determine that, in
some cases, introducing two ecoestrogens produced
a response greater than the simple sum of that produced
by each molecule. In other words, certain combinations
of these chemicals work synergistically to produce
a result greater than would be expected from the
sum of their inputs.
We studied four weakly estrogenic pesticides--dieldrin,
endosulfan, toxaphene and chlordane--in our yeast
model, and showed that, indeed, they produce a very
low-level response when were tested singly. However,
when we tested combinations of these chemicals,
the estrogenicity jumped by 160 to 1,600 times their
individual potencies.
On a molecular level, there are several possible
explanations for this. One is that the chemicals
may physically combine to form an estrogen-like
molecule. Another is that various ecoestrogens and
natural estrogens bind to one or both of the receptor
subunits that join together to form a functional
receptor pair or dimer. A third possibility, which
is the one we favor, is that the estrogen receptor
has two or more interactive binding sites, a situation
that may build flexibility and control into the
response system. It may also account for the great
structural diversity of estrogen-like molecules.
In fact, Elwood Jensen, one of the "fathers"
of estrogen-receptor research, recently proposed
a second binding site on the receptor that recognizes
antiestrogens. The question of multiple binding
sites on the estrogen receptor may be important
in endogenous as well as exogenous signaling pathways.
Figure
7. Surprising synergy of some ecoestrogens
was recently demonstrated in the authors' laboratory
using the yeast-estrogen system. Each of two ecoestrogens
acting alone caused a very weak estrogenic response,
as indicated by the pale blue color of the transformed
yeast cells (a and b ). Acting together,
the two ecoestrogens produce an estrogenic response
greater than the sum of the individual responses
(c ). In fact, the combined response can range
from 160 to 1,600 times the individual responses.
Our work with the yeast system has helped us expand
our understanding of the subtle relations between
ligands and the estrogen receptor. It may also inadvertently
reveal aspects of the endogenous estrogen system
that we had previously not known. For example, the
levels of synergy we have observed may come about
when two protein molecules bind together to act
as a unit. In one hypothetical scenario, after an
estrogen molecule binds to its receptor, the complex
binds to a second, unoccupied estrogen receptor.
Once bound, the unoccupied receptor molecule changes
it shape in such a way as to make it easier for
an estrogen molecule to bind to it than it than
to an unattached receptor molecule. The two receptor
molecules together, referred to as a dimer, could
then bind to the ERE and modulate gene expression.
Ecoestrogens may influence the dimerization process
itself, facilitating the binding of receptor molecules
to each other. There may be multiple binding sites
for ecoestrogens on a receptor molecule, or receptor
dimerization may create new binding sites for ecoestrogens.
Hence, ecoestrogen binding might enhance dimerization,
and dimerization might in turn enhance ecoestrogen
binding. As a result, the ecoestrogens end up being
more potent together than the binding of any one
alone would indicate.
In a sense, the entire receptor-ligand complex itself
becomes a signaling molecule. The complex effectively
becomes a ligand for the estrogen response element.
In this way a hierarchy of signals within signals
is set up. At the simplest level, there is the intramolecular
interaction between the ligand and the receptor,
followed by the interaction between that signal
and the DNA. The nature of these interactions and
their subsequent outcomes depends on the original
estrogen-like molecule. That is, each ecoestrogen
binds to the receptor in a slightly different fashion,
giving the overall ligand-receptor complex a different
shape. Under different circumstances, different
areas of the receptor molecule may be exposed or
hidden, or its charge may be altered. All of these
subtle modifications determine how the complex interacts
with DNA. One complex may stimulate a high level
of gene expression; another complex may stimulate
a lower level or may repress gene expression altogether.
It is even possible that different complexes can
modulate the activity of different genes entirely,
which, of course, profoundly affects subsequent
cellular activities.
Figure
8. At least two models may be developed
to describe the binding relationship between ecoestrogens
and the estrogen receptor. In the first, an estrogen,
either natural or synthetic, binds to a single site
on the receptor, possibly the site for endogenous
estrogen (left ). Based on their recent data,
the authors propose a second model in which ecoestrogens
act synergistically. In this hypothetical model,
each binds to distinct sites on the same molecule
(right ), one of which may be the endogenous
estrogen binding site. The authors are exploring
whether estrogen binding regulates other sites on
the receptor, called activation functions, which
interact with transcription factors on the promoter.
Synergistic ecoestrogen binding may increase the
interaction between activation functions and transcription
factors. The outcome is increased gene expression
and a more vigorous estrogenic response than is
produced by either two estrogens acting at a single
site or a single ecoestrogen acting alone.
Section 6
Other Pathways
Research in our lab and many others has started
to suggest that estrogens may exert some additional
influence through signaling pathways other than
the one directly involving the estrogen receptor.
There is, for example, evidence to suggest that
estrogens act, in part, through signaling pathways
usually activated by growth factors such as epidermal
growth factor (EGF), transforming growth factor
alpha (TGFa) or insulin-like growth factor (IGF).
Growth factors, unlike steroid hormones, are not
fat-soluble, and therefore do not pass unaided through
lipid membranes. Instead, a protein receptor must
be present in the membrane for the factor to have
an effect in a particular cell. These receptor molecules
span the length of the membrane, with the growth-factor
binding site located on the external membrane face.
The growth factor does not actually have to enter
the cell in order to activate the signaling pathway.
Rather, when the factor binds the external portion
of the receptor molecule, changes take place on
the portion of the receptor that lies inside the
cell. These changes initiate a biochemical chain
reaction, where each molecule in the pathway is
stimulated to activate the next molecular signal
until the final signal results in some sort of cellular
activity. One of the endpoints of various growth-factor
signaling cascades is, apparently, the estrogen
receptor.
This raises the possibility that some ecoestrogens
may bring about their effect by interacting with
a growth factor or with the appropriate growth-factor
receptor to change the activity of the estrogen
receptor. One possible outcome of such a signal
may be to alter the binding activity of other ecoestrogens
to the estrogen receptor. If this is the case, nature,
by regulating estrogen action through hierarchies
of signals, has provided additional possibilities
for environmental mimicry. It is getting hard to
tell the dancers from the dance.
Section 7
Ecoestrogens in Sickness and in Health
Since it now appears that ecoestrogens can bring
about many of the same effects as the endogenous
hormone, scientists must consider the consequences
of exposure to these compounds on the health of
people and animals. Since endogenous and pharmaceutical
estrogens are associated with various diseases and
dysfunctions, including breast and endometrial cancer,
lactation suppression, endometriosis and uterine
fibroids, the possibility that ecoestrogens may
also be associated with these disorders must be
considered.
So far, studies linking ecoestrogenic chemicals
in the blood to breast cancer have been equivocal--some
have shown an association, but others could not
find any of significance. Two studies, one in North
Carolina, the other in Mexico by the epidemiologist
Walter Rogan, demonstrated that estrogenic pesticides
decreased the length of time women breast fed their
infants, suggesting an estrogen-related suppression
of lactation.
Ecoestrogens are not only potentially harmful to
adults; they may also affect developing embryos,
sometimes with lifelong consequences. The known
effects of prenatal exposure to DES on sperm production
later in life have led to the hypothesis that exposure
to environmental hormones early in life may be partly
to blame for a reported decrease in semen quality
worldwide. The decline in semen quality first described
in 1992 by Niels Skakkebaek in Copenhagen as well
as any role for environmental factors, however,
remains highly controversial.
Although the effects of ecoestrogens on human health
remain, for the most part, uncertain, stronger reasons
for concern have been found in other species. Scientists
have seen the harmful effects of ecoestrogens both
in natural settings and in laboratory studies. In
one case, male fish living in polluted water produced
abnormally high amounts of vitellogenin, the egg-yolk
protein normally found only in female fish that
are laying eggs. This therefore strongly suggests
that the males had been exposed to some kind of
estrogen-like molecule. In another study conducted
by Stephen A. Bortone of the University of West
Florida in Pensacola, female fish were masculinized
following exposure to environmental wastes.
Alligators living in Florida's Lake Apopka, which
had been extensively contaminated with DDT-related
compounds and other agricultural chemicals, experienced
a sharp population decline following the contamination.
Subsequent studies by Louis Guillette have shown
less than half the normal levels of the male sex
hormone, testosterone, were present in the blood
of the males. These data, along with the observed
reduction in the size of these animals' genitals,
lead to the conclusion that the alligators were
"feminized."
Important work done by D. Michael Frye and his colleagues
at the University of California at Davis showed
that sea-gull eggs exposed to DDT developed as females,
no matter what their genetic sex. This was one of
the early works demonstrating the feminizing effects
of environmental chemicals.
More recently, in collaboration with David Crews
and Judy Bergeron at the University of Texas, we
showed the developmental consequences of exposing
turtles to estrogenic chemicals. Normally, sexual
differentiation in turtles is dependent on the temperature
at which the eggs develop. Eggs incubated at 31
degrees Celsius become females; eggs incubated at
26 degrees Celsius become male. Eggs incubated at
the male-producing temperature, however, develop
as females when they are exposed to natural estrogen.
The same effect was produced when the eggs were
exposed to estrogenic PCBs. Strikingly, as with
our molecular biological studies in yeast cells,
we could demonstrate a synergistic effect of ecoestrogens
on sex reversal in turtles. Taken altogether, these
field and laboratory studies strongly suggest that
ecoestrogens are capable of altering sexual development
in a manner consistent with their hormonal activity
and in some cases the hormonal activity of mixtures
of chemicals is greater than additive.
This knowledge provides the basis for the new science
of environmental signaling. It seems that biological
mimicry is a defense strategy adopted by some plants
and fungi that may inadvertently be exhibited by
classes of pesticides and other synthetic chemicals.
Unlike the rational synthesis of DES as a synthetic
hormone, there is little evidence that pesticides
and other industrial chemicals that have hormonal
activity were synthesized for this purpose; nor
does it appear that estrogenicity was related to
the way in which pesticides worked on pests. Nevertheless,
as we have shown, environmental chemicals may function
as signals, implying that they must interact with
a particular cellular receptor and thus demonstrate
some degree of inherent specificity. The outcomes
of these interactions then become reasonably predictable.
We have seen that many natural and synthetic compounds
in the environment can function as estrogens or
antiestrogens. The recent interest in environmental
chemicals as estrogens has stimulated thinking about
how synthetic chemicals may interact with biological
systems. The demonstration that an environmental
chemical can function as an antiandrogen portends
that there may be more hormonally active chemicals
in the ecosystem. It also suggests approaches to
look for other unintentional environmental signals.
It is possible that other environmental signaling
molecules are mimicking hormones, neurotransmitters,
growth factors, or other important biological functions.
The work with ecoestrogens certainly raises these
possibilities. But the work on estrogenic agents
also gives us experimental methods for approaching
this possibility. The work also provides new insights
into the mechanism of estrogen action itself and
points the way to a new understanding of the relationship
between people and their chemical environment at
a cellular level. The more we comprehend the mechanisim,
the better able we are to predict and, where possible,
prevent adverse effects.
Acknowledgment
The authors thank Dr. Louis Guillette, University
of Florida, for generously providing photographs
to illustrate the influences of ecoestrogens on
wildlife.
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© American Scientist, September-October
1996 
Hormonal
disorders