WV, KA Thayer, BM Judy, JA Taylor, EM Curran and FS vom Saal. 2003.
Large effects from small exposures. I. Mechanisms for endocrine
disrupting chemicals with estrogenic activity. Environmental
Health Perspectives 111:994-1006.
paper examines the soft underbelly of regulatory toxicology: the
fact that current testing procedures are vulnerable to dramatic
under-estimations of health risks because of molecular
details of how endocrine disruption of estrogenic signaling actually
works. Fixing these mistakes may require strengthening of
exposure standards by factors of 10,000x or greater, for
endocrine disrupting compounds.
the mechanistic information concerning hormone action that
we review here had been considered, the currently accepted
practice of only testing very high doses to predict effects
of doses thousands or even millions of times lower would
have been recognized to be inappropriate. The result
would have been that doses of EDCs, such as methoxychlor and
bisphenol A, far below those currently being described as
"safe" would, in fact, have been predicted to produce
biological responses, and much lower doses would have been
et al. reach this conclusion by careful review of a series
of issues and findings in toxicological research. It is not a simple
argument, but its implications are so important that carefully exploring
their paper is worthwhile. They concentrate on contaminants that
disrupt estrogen signaling, what they call "endocrine-disrupting
chemicals with estrogenic activity," or EEDCs. The conclusions,
however, are relevant to other chemical signaling systems, including
but not limited to other hormones.
core of the argument is four-fold:
hormones like 17ß-estradiol
work at extremely low concentration levels, far beneath the levels
at which all hormone receptors would be bound by the hormone.
Once all receptors are bound, further increases in the natural
hormone can't increase the response of the system, at least via
the interaction of the hormone and its receptor.
may be thousands of times less powerful than estradiol itself,
but are present at sufficient concentrations to alter the percentage
of occupied receptors.
testing of EEDCs for toxicological impact involve exposures that
are thousands of times higher than the level at which all receptors
are bound. At these levels, therefore, even large
changes in EEDC concentration cannot alter the percentage of bound
receptors, and therefore any effect of EEDCs at those
levels cannot be due to signaling through estrogen receptors.
tests are explicitly designed to observe EEDC impacts at concentration
levels in which the estrogenicity of the EEDC is comparable in
potency to the level at which the natural hormone operates normally
(i.e., extremely low levels of estrogenicity), it will be impossible
to observe EEDC-mediated estrogenic effects. Traditional
testing at high levels by definition cannot reveal those effects.
follows a more detailed exploration of this paper. Note also a link
to an analysis of one key set of experiments presented within
this larger paper.
et al. begin by distinguishing between three different
levels of hormone (or hormone mimic/antagonist).
lowest is the physiological level (graph
below). This is the level at which hormones are naturally
found in the body. Importantly, what matters is not the total
amount of the hormone but instead the small fraction of total
hormone that is free in blood serum: the free hormone concentration.
Typically, most of the hormone is bound to plasma proteins or
joined to other substances and thus not available for binding
with receptors. Free hormone levels are astoundingly low. For
example, in mouse and rat fetuses during development, the level
of free estradiol in serum is less than 1 picogram per
milliliter. While that is less than one part
per trillion, experiments
confirm that minor variations in that level alter the course of
above this is the toxicological level. This is the range
of doses that cause cell death, mutation, weight loss, etc.,
and is the level used in standard toxicological testing. It is
a high level.
finally, they consider the environmental level. This
is the level at which a contaminant is in the blood. High exposures
result in environmental levels reaching toxicological levels.
Lower exposures may or may not place the contaminant's concentration
at a level at which it is within the range, correcting for the
strength of its receptor affinity, of natural hormones' physiological
figure (adapted from Welshons et al.) shows the
proliferation response of MCF-7
cells to estradiol over a wide range of exposure levels,
from parts per quadrillion on the far left to parts per
million on the far right. Response is displayed as a percent
of the proliferation seen in the control group. Note that
the X-axis is logarithmic.
this experiment, the "physiological level" for estradiol
runs from parts per quadrillion to low parts per trillion. The "toxicological
level" is 1,000-10,000 times higher, high parts per billion
to parts per million. And in a sequence
of experiments, Welshons et al. demonstrate that the
response within the physiological level is mediated by estradiol
binding with the estrogen receptor, while the toxicological
response does not involve the estrogen receptor.
analysis rests crucially upon the fact that the biochemical pathway
that allows changes in hormone concentration to lead to gene activation
(or suppression) depends upon the hormone binding with its receptor,
then initiating a sequence of events that finally involves a gene
being activated or suppressed when the receptor/hormone complex
binds with specific DNA sequences.
within a given cell there are a finite number of intracellular receptors,
the density of which vary from tissue to tissue. Up to a point,
the more hormone the greater the number of bound receptors, and
the more receptors that are bound, the greater the strength of the
signal. But because there are a finite number of receptors,
the biochemical signal for gene activation cannot increase beyond
a maximum which is reached when all receptors are occupied.
This can be seen in the graphs below.
concept of receptor
occupancy is key: imagine a cell with a given number of estrogen
receptors. Receptor occupancy reflects the portion of that number
that have been bound to estrogen, or to a contaminant capable of
binding to the receptor like DES or bisphenol A.
et al. argue that regulatory toxicology works at such a
high range of EEDC concentration that for compounds like bisphenol
A, DES and other estrogenic contaminants, all available receptors
must necessarily be occupied at the levels used in traditional experiments.
Hence any variation in responsivity to dose can't be because of
more or less receptor occupancy. That will only be seen much lower
on the dose-response curve.
implication of this is that within dose ranges used by regulatory
toxicology, variations in response caused by variations in dose
aren't mediated by the estrogen receptor but via some other mechanism.
In other words, by accident of design, regulatory testing
never tests for receptor-mediated endocrine disruption.
Only low dose testing built explicitly to test within the range
of EEDC concentration where receptor occupancy can vary can observe
do they get there?
consider the relationship between hormone (or contaminant) concentration,
receptor occupancy, and estrogenic response.
low EEDC doses, not all receptors are occupied. Increases
in EEDC lead to increases in receptor occupancy, up to 100%
occupancy. Any increase in EEDC above that level cannot lead
to a greater estrogenic response mediated by the estrogen
receptor, because no more receptors are available to be bound.
from Welshons et al. Table 2)
the more EEDC, the greater the response, up to a point.
also turns out that cellular mechanisms further strengthen this
pattern. They amplify the sensitivity of the system at low levels
and suppress sensitivity at high levels. The capacity of the system
to respond to increasing levels of hormone (or contaminant) diminishes
long before receptor occupancy is complete. Or as Welshons and his
coauthors state: "response saturates well before receptor occupancy
is evident in the graph to the right, based on theoretical
calculations in their first table. The response to estradiol
measured as a % of maximum cell proliferation reaches saturation
at approximately 50% saturation of receptors.
from Welshons et al. Table 2
important point to note is the extraordinary sensitivity to extremely
low levels of estradiol. While these calculations are based upon
theory, they rest upon well established principles of the
biochemical interactions between hormones and their receptors.
et al. emphasize the importance of the fact that the system
is much more sensitive to changes in hormone levels when only a
small portion of receptors are occupied. In
the graph above, it is evident that the greatest sensitivity occurs
at receptor occupancy levels beneath 10%, where the rise is roughly
linear and sharpest. The curve reaches a plateau not far above 10%
occupancy. In fact, according to Welshons et al., over 90% of the
proliferative response to estradiol occurs before receptors reach
does this mean for regulatory toxicology?
challenges for regulatory toxicology that these observations present
are stark. According to the authors, "at the dose ranges of
EEDCs used in current toxicity testing, chemical are likely to be
present within target cells many orders of magnitude above their
for estrogen receptors. Within this dose range, changes in hormone
density cannot have a detectable effect on receptor occupancy, because
all receptors are saturated at 100% and no additional binding, which
is required to result in an increase in response, can be observed.
No primary hormonal effects can be observed in response
to changes within this high dose range, only secondary effects not
mediated by estrogen receptors."
other words, countless regulatory tests for safety have been conducted
in which there was literally no chance of detecting low
level effects mediated by hormone receptors.
et al. go on to argue forcefully and elegantly that the
use of linear extrapolations from high dose testing to low dose
risk analysis will lead to significant underestimates of risk. They
develop a specific example for bisphenol
A and show that using a linear model to predict low level effects
from high level experiments would erroneously conclude that a bisphenol
level of approximately 0.8 parts per billion would result in negligible
receptor binding and thus no response, when in fact it would fall
precisely within the range of bisphenol A concentration where one
should expect maximum sensitivity because of the receptor occupancy
issues outlined above.
one adds the additional complication of a non-monotonic dose response
curve (as for example, shown above), caused
by a combination of low level signal disruption and high level toxicity,
the underestimation of risk only becomes more extreme.
an understated tone, they conclude: ""The potential for
error inherent in drawing strong positive conclusions [i.e., that
the chemical is safe] from purely negative data has clearly not
been appreciated by some toxicologists as well as regulators responsible
for assessing this information." ... "Responses to low
doses of EEDCs should be determined by testing a much wider range
of doses than the 50-fold range common in toxicological studies
today, including doses in the environmentally relevant range, and
by accounting for all sources of estrogenic activity (endogenous
and exogenous) and their interactive effects."
important corollary of this analysis is what it says about another
facet of endocrine disruption. As noted elsewhere, inverted-U or
non-monotonic dose response curves
are found commonly in studies of EDCs. A common question asked upon
first hearing about these dose-response curves is: "Does that
mean that some range of higher doses is acceptable because within
that range, the response appears to be no different than seen in
answer to this is clearly no. At that higher range of doses, all
receptors are bound. This means the system is prevented from responding
appropriately to variations in natural hormone levels.