II-CA: Myers, et al, “Does the Dose Make
the Poison?” Environmental Health News,
This appendix is copied from:
Then click on Power of Poison.
The Endocrine Disruption Exchange
April 30, 2007.
Does 'the dose make the poison?'
Toxicology testing assumes 'the dose makes the poison.'
Photograph from Retha Newbold, NIEHS.
Measuring how much of a compound, called its dose, produces a response, usually some kind
of health effect, is difficult and time consuming. To understand how dose and effects are
linked, toxicologists expose animals, tissues, or cells to pollutants. They then examine how
the subject responds to the exposure.
The "dose makes the poison" is a
common adage in toxicology. It
implies that larger doses have greater
effects than smaller doses. That
makes common sense and it is the
core assumption underpinning all
regulatory testing. When "the dose
makes the poison," toxicologists can
safely assume that high dose tests will
reveal health problems that low dose
exposures might cause. High dose
tests are desirable because, the logic
goes, they not only will reveal low
dose effects, they will do so faster and
with greater reliability. Greater
reliability and speed also mean less
by Pete Myers, Ph.D. and Wendy Hessler While exposure in the womb to 100 parts
per billion of the estrogenic drug
diethylstilbestrol (DES) causes mice to
become scrawny as adults, exposure to a
much lower amount, 1 ppb, causes
grotesque obesity. This photograph
compares a control animal (left) to an
animal exposed to a very small amount of
DES in the womb (right).
The trouble is, some pollutants, drugs and natural substances don't adhere to this logic, as can
be seen in the photograph above. Instead, they cause different effects at different levels,
including impacts at low levels that do not occur at high doses. Sometimes the effects can even
be precisely the opposite at high vs. low. Because all regulatory testing has been designed
assuming that "the dose makes the poison," it is highly likely to have missed low dose effects,
and led to health standards that are too weak.
Extensive results challenge a core assumption in toxicology
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In standard toxicology, as the dose increases, so does the effect. Conversely as dose
decreases, so does its impact. This relationship is called a monotonic dose-response curve
because effects are either increasing or decreasing. In a monotonic curve, they never reverse
direction. It is akin to a dimmer switch and a lightbulb. The more electricity you let through by
turning the knob, the brighter the bulb gets.
The diagrams to the right present idealized forms of
monotonic (left) andnon-monotonic (right) doseresponse
curves. Monotonic can either be linear ornonlinear.
The key point is that the direction of the curve
never changes from positive to negative or vice-versa.
A monotonic curve can flatten, i.e., reach an
Non-monotonic curves, in contrast, change direction.
Over part of the curve, response increases with dose,
while over another portion it decreases as dose
increases. Non-monotonic curves are often called
'inverted-U' (upper) or 'U'(lower).
How toxicology tests are used to
develop health standards
Government agencies identify and
regulate dangerous substances assuming
that 'the dose makes the poison.'
To set exposure limits, three to five doses
of a substance are tested in the
laboratory. Toxicologist start at the
highest dose chosen and continue to lower
doses until they find the point where
effects are no longer detectable, that is,
the dose at which experimental animals
no longer differ from controls. This safe
dose - the lowest amount that poses an
acceptable risk - is called the 'no
observed adverse effect level,' or NOAEL.
Traditional toxicology guiding health
regulations rarely tests doses lower than
NOAEL due the 'dose makes the poison'
The final acceptable level for human
exposure--called the 'reference dose'—is
calculated from the NOAEL by adding a
series of safety factors. These safety
factors take into account uncertainties in
extrapolating animal research to human,
as well as differences insensitivity among
groups of people, and between kids and
adults. Thus if the NOAEL is found to be 1
milligram per kilogram of bodyweight per
day (which corresponds to apart per
million), then the reference dose might be
1 part per billion per day.
Blindsided by hormonally-active
While toxicologists have traditionally assumed
that the dose makes the poison,
endocrinologists --scientists who study the
action of hormones--have long known that
hormones can have different effects at
The graph to the right comes from a simple
study looking at the response of a gene inside
a cell as it is exposed to different amounts of
estradiol, the common form of the natural
human hormone, estrogen.
In the experiment, the scientists experimented
over an extremely wide range of doses, from
around 10 parts per quadrillion (ppq) to 10
parts per million (ppm).
Figure adapted from Welshons et al. 2003
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Most estradiol in human blood is bound up by
special proteins. When bound, it can't interact with
hormone receptors. Because that interaction is a
crucial step in the process that turns on estrogenresponsive
genes, bound estrogen doesn't turn on
genes. Only the unbound estrogen can, and its
concentration in human blood is normally in the
green zone of the graph, parts per quadrillion to low
parts per trillion.
As the dose of estradiol rises through the green
zone of the graph, the response increases. This
green zone is the range of concentrations over
which unbound estradiol is found in blood.
Initially, at just above 1 part per quadrillion, there's
no difference between the control (0 estradiol) and
the response to estradiol. As dose increases up to
just above 1 part per trillion, the response
increases. It then flattens out, over a wide range of
doses, all the way to 100 parts per billion. But once
it gets into the high-dose range, it drops, and by
just over 10 parts per million the system shuts
down, with no response whatsoever.
Could this mean higher
doses are safer than lower
A frequent response from people
dose-response curve like that
above for the first time is to ask
'Does this mean higher doses are
Emphatically, no. At the highest
doses used in this experiment, the
system was no longer able to
respond to estrogen signaling. That
means that crucial events under
the control of estrogen would not
occur. The consequences, for
example, of shutting off estrogen
signaling responses during
development would most likely be
catastrophic for the organism
What's happening? As estradiol increases in the low dose range, it is binding with receptors and
stimulating the responsive gene. This is what is supposed to happen over this dose range, the
range found naturally in people. However, as receptor occupancy increases above 10%, a
feedback loop cuts in, leading to a reduction in the availability of additional receptors.
As dose increases further, the effect of the feedback loop grows until no amount of additional
estradiol can increase the system's response. That produces the long flat portion of the graph,
from just over 1 ppt to 100 ppb.
As doses rise above 100 ppb, estradiol becomes overtly toxic to the cell and the system stops
responding completely, dropping even below the control level.
This dose-response curve dramatically violates the assumption that high dose experiments can
be used to predict low dose results. At high doses, estradiol shuts the system down. At low
doses it turns the system up. Over part of the dose range, response increases, while over
another part, it decreases. This curve is called a non-monotonic dose-response curve.
Consider this 'thought experiment.' Think again about that light bulb hooked up to a dimmer
switch, but instead of running it through your normal wiring (110 volts), plug it into the circuit
for the dryer (220 volts). When the dimmer is turned down, there's very little light coming
through. Turn it up and the light gets brighter. Turn it all the way up and the light bulb blows
up. All of a sudden, it's dark again. There was more voltage and current than the system was
With 'dose makes the poison' thinking dominating toxicology, traditional toxicologists didn't
pursue the possibility that there might be effects at levels far beneath those used in standard
experiments. No health standards incorporated the possibility. Over the past 15 years,
however, as scientists began to explore the impacts of endocrine disrupting compounds--
compounds that behave like hormones or interfere with hormone actions-- many examples of
non-monotonic dose response began to be published in scientific journals.
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In 2006, a team of German researchers published a vivid example of how traditional testing to
set health standards can miss low dose effects. Their work examined the effect of a phthalate
on the activity of an enzyme in the brain of developing male rats. This enzyme, aromatase,
converts testosterone to estrogen. Counter-intuitively, estrogen early in the life is necessary to
masculinize the brain of male mammals. If they don’t get enough, key parts of the brain that
normally differ between males and females will be more similar to the female form than the
In their experiment they exposed pregnant females to the phthalate DEHP, with different
groups exposed to an extremely wide range of doses. The highest dose used is one known to
cause reproductive damage to developing males without obviously harming the mother. The
lowest dose, 19,000 times beneath the high dose, was set at a level commonly observed in
Many cases of non-monotonic dose-response curves have now been published in research on
endocrine disruption. Below follow some recent examples. Because they are now being reported
frequently in research on the effects of endocrine-disrupting chemicals, it is clear that
regulatory toxicology can no longer safely assume that 'the dose makes the poison.' It is also
clear that the standard approaches used to develop estimates of safe exposure levels, by
basing their design on a false assumption, are likely to have set safety standards that are not
strong enough to protect public health.
Narita et al. report that a key step in
immune reactions, the release of
histamine and cytokines by mast cells,
is exacerbated by very low levels of
environmental contaminants, similar to
the effect of estradiol. These
experiments, done in cell culture, used
levels of the contaminants well within
the range of human exposure. The peak
response was seen at approximately 0.1
parts per billion (10
molar). By the
time the dose rose to 10 parts per
molar), the response
disappeared. This experiment was done
with mouse and human cells in culture.
Graph adapted from Narita et al.
Their results, seen to the right, show that
doses from 15 mg/kg/day to 405 mg/kg/day
(statistically significant in purple) cause an
increase in aromatase activity. Intermediate
doses (1.215 and 5 mg/kg/day) do not differ
from control (the blue horizontal line) But
lower doses suppress aromatase activity
(statistically significant in red, 0.134 and
0.405 mg/kg/day). As the research team
point out in their article, a regulatory test for
DEHP effects would not have gone below 5
mg/kg/day and therefore would have missed
the significant aromatase suppression at
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At doses far beneath the current EPA safe level,
Takano et al. found that the phthalate DEHP
increases the immune response of mice to a
common allergen. Clinical scores of an allergic
reaction were strongest at intermediate doses(4
and 20 µg). A dose of 100 µg (yellow line) was
no different than the control (purplish blue line).
Graph adapted from Takano et al.
Working with a suite of compounds that bind to
a newly discovered estrogen receptor on the
surface of the cell membrane, Wozniak et al.
found that cells and prolactin release (graphs to
left) follow markedly non-monotonic patterns.
Bisphenol A provoked responses at the lowest
dose tested, 0.23 parts per trillion. Bisphenol A
has been considered a weak estrogen because
its relatively binding affinity with the estrogen
receptor in the cell nucleus is much lower than
that of estradiol. In contrast, with this cell
membrane receptor, bisphenol A is just as
powerful as estradiol.
Graphs adapted from Wozniak et al.
Ralph et al. discovered that prostate cells
respond in anon-monotonic fashion to exposure
to the organochlorine pesticide
hexachlorobenzene (HCB). High levels suppress
androgenic activity of the cells relative to
controls (redline), whereas low levels enhance
androgenic activity. Their experiments with live
mice revealed that prostate weight in adult mice
also showed that high doses produced the
opposite effect of low doses.
Wetherill et al. found that a one nanomolar dose
of bisphenol A yields the strongest proliferation
response by prostate tumors in experiments
with cells. The impact of a dose 100-times
higher didn't differ from control.
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Andrade, AJM, SW Grande, CE Talsness, K Grote and I Chahoud. 2006. A dose–response study
following in utero and lactational exposure to di-(2-ethylhexyl)-phthalate (DEHP): Nonmonotonic
dose–response and lowdose effects on rat brain aromatase activity. Toxicology 227:
Narita, S, RM Goldblum, CS Watson, EG Brooks, DM Estes, EM Curran and T Midoro-Horiuti.
2007. Environmental Estrogens Induce Mast Cell Degranulation and Enhance IgE-mediated
Release of Allergic Mediators.Environmental Health Perspectives 115:48–52
Newbold, RR, E Padilla-Banks, RJ Snyder and WN Jefferson. 2005. Developmental Exposure to
Estrogenic Compounds and Obesity. Birth Defects Research (Part A) 73:478–480.
Ralph, JL, M-C Orgebin-Crist, J-J Lareyre and CC Nelson. 2003. Disruption of androgen
regulation in the prostate by the environmental contaminant hexachlorobenzene. Environmental
Health Perspectives 111:461-466
Takano, H, R Yanagisawa, K-I Inoue, T Ichinose, K Sadakano, and T Yoshikawa. 2006. Di-(2-
ehylhexyl) Phthalate Enhances Atopic Dermatitis-Like Skin Lesions in Mice. Environmental Health
Perspectives 114: 1266-1269.
Welshons, 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.
Wetherill, YB, CE Petre, KR Monk, A Puga, and KE Knudsen. 2002. The Xenoestrogen Bisphenol
A Induces Inappropriate Androgen Receptor Activation and Mitogenesis in Prostatic
Adenocarcinoma Cells. Molecular Cancer Therapeutics 1: 515–524.
Concentrations Trigger Membrane Estrogen Receptor-alpha-Mediated Ca++ Fluxes and
Prolactin Release in GH3/B6
Pituitary Tumor Cells. Environmental Health Perspectives 113:431-439.
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