| 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 |
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