II (also see introduction)
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 doi:10.1289/ehp.5494
page explores one component of the publication
by Welshons et al., specifically their analysis of
the different mechanisms involved in inducing changes in MCF-7 breast
cancer cell proliferation through exposure to estradiol.
central finding of these experiments is that low level responses
to estradiol are mediated by hormone binding to the estrogen receptor,
whereas toxicological responses to high level exposures
take place independently of binding with the estrogen receptor.
That's important because standard toxicological testing is designed
in a way that Welshons et al. conclude cannot detect estrogen
receptor mediated responses. In other words, countless tests
of potentially estrogenic substances have been done without any
possibility of revealing whether or not they can interfere with
natural estrogen signaling.
implication is that regulatory testing is full of false
negatives: conclusions that contaminants have no effects when in
fact they do.
did Welshons and his coworkers do? When exposed to estrogenic
cells increase their rate of cell division and proliferate.
Welshons et al. studied this proliferation response over
a wide range of doses of 17ß-estradiol,
both by itself and in combination with two other types of chemicals,
synthetic estrogens and anti-estrogens.
of estradiol ranged from the control, 0 estradiol, through exposures
at the parts per quadrillion, parts per trillion, parts per billion,
and parts per million range. At the highest doses used, estradiol
other chemicals were diethylstilbestrol (DES), a synthetic estrogen,
at 3 parts per trillion, and two different anti-estrogens, raloxifene
and ICI 182,780, at doses sufficient to eliminate any
response mediated by the estrogen receptor.
used two forms of the MCF-7 cell. One was the standard MCF-7, with
estrogen receptors. The other was a variant of MCF-7 called C4-12-5
cells. These have been genetically engineered to lack estrogen receptors.
Hence any response to estradiol (or any other chemical) in the C4-12-5
cells cannot be mediated by estrogen receptors.
did they find?
shown in the graph to the right, experiments with estradiol
alone in the normal MCF-7 cells (with estrogen receptors) revealed
a strongly non-monotonic shape, a classic inverted U.
they exposed the C4-12-5 variant of MCF-7 cells to the same
range of estradiol (left), they observed no enhancement of
proliferation at low levels of exposure. The high level decrease
in proliferation to levels beneath the control response, however,
a third experiment (right), they found that the presence of
the synthetic estrogen DES at 3 ppt was sufficient to eliminate
the low dose, estrogen receptor mediated response to estradiol,
but that it had no effect on the high dose response.
crucial point to note here is the difference in shape of the first
graph compared to the other two. Without the estrogen
receptor (second graph), or
in the presence of a contaminant that is estrogenic (third
graph), the low level response of the system is eliminated.
et al. carried out a crucial additional set of experiments
that serve as standard controls and positive
controls. These controls allowed the researchers to separate
responses mediated by the estrogen receptors from those that acted
via some other (unspecified) mechanism. Without these controls
and without explicit testing at extremely low levels (parts per
trillion and below), the experiments would have indicated that estradiol
itself is not estrogenic!
the reality is that standard toxicological testing requires neither
a full set of controls nor experiments at relevant low doses.
controls employed by Welshons et al. confirmed that:
MCF-7 cell system used in these experiments is responsive to estrogens
and the response can be suppressed by anti-estrogens. This is
important to show because, while in principal this cell system
is known to be responsive to estrogens, it is possible that the
specific procedures and materials used by Welshons et al.
could have contaminated the experiment with another estrogen that
would have rendered the system insensitive to further hormonal
stimulation. Inadvertent contamination can lead to false
the cell line lacking estrogen receptors, C4-12-5, estradiol did
not elicit a response at low levels and the anti-estrogens did
not inhibit the response at high levels. The clear implication
is that estrogen receptors are necessary for the low-level response
but not necessary for the high level response.
perhaps the most telling positive control (right), purposeful
low level contamination of the experiment by an estrogenic
compound, DES, eliminated responsiveness to estradiol. Adding
an anti-estrogen reduced the response, indicating that an
estrogen (DES) was present in the background.
In this case, the "control" is the MCF-7 system
plus 3 ppt DES. By adding the anti-estrogen (blocking DES)
plus a higher but non-cytotoxic amount of estradiol they
show that DES's impact was via the same mechanism as estradiol.
combination of experiments and controls illustrates how easy it
would be to mistakenly classify estradiol—and by implication
other estrogenic substances—as non-estrogenic at low levels
in the first experiment (top graph)
with functional estrogen receptors, estradiol had both a low-dose
effect and a high dose effect. By showing in the second experiment
(second graph) that the low-dose effect was
not present in the absence of estrogen receptors, they prove that
the low dose response is mediated by that receptor. This conclusion
is strengthened further by the third experiment (third
graph), which shows that contamination by another estrogen,
DES, also eliminates the low dose response. This is because at the
level of DES used, all estrogen receptors are already occupied even
prior to the addition of estradiol. Hence adding estradiol cannot
increase the proliferation response because it is already at its
by using anti-estrogens in their positive controls (fourth
graph), they show that the effect of the DES contamination can
be removed, decreasing the proliferative response from 100% of the
"control" to roughly 40%.
does it mean? This seemingly simple and logical set of
experiments raise huge questions about basic assumptions and procedures
used in regulatory testing. Two issues loom large:
testing takes place at high levels and is assumed to allow extrapolation
to low levels. These experiments show that for estrogens, not
only is extrapolation inappropriate, the mechanisms
mediating high-level vs. low-level responses are different at
the molecular level. Estrogen toxicity (high level) is not the
same as estrogen signal disruption (low level). Tests appropriate
for examining estrogen toxicity reveal nothing about signal disruption
at physiologically relevent levels.
contamination by estrogenic substances can obscure estrogen signaling
responses, making them impossible to detect unless extensive negative
and positive controls are included in the experimental procedure.
Such contamination is highly likely: from food (phytoestrogens
and other contaminants) and from caging. For example, Howdeshell
et al. report that polycarbonate caging creates the
opportunity for estrogenizing animal subjects because it leaches
bisphenol A even at room temperatures. It is highly likely that
many experiments within the published literature have been misled
by this sort of contamination, leading to spurious conclusions.
together, these two factors imply that for toxicological testing
of estrogenic substances, the literature is highly likely to be
rife with false
et al. provide more detail about one
specific example of a recent false negative in the toxicological
literature, the failure of industry scientists to replicate findings
that low level bisphenol A exposure in the womb leads to prostate
enlargement in mice.