APPENDIX
II-CA: Myers, et al, “Does the Dose Make
the Poison?” Environmental Health News,
This appendix is copied from:
http://www.endocrinedisruption.com
Then click on Power of Poison.
http://www.endocrinedisruption.com/files/2007-04-30_does_the_dose_make_the_poison.pdf
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
cost.
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
1 of 6
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
asymptote.
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'
assumption.
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
compounds
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
different doses.
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
2 of 6
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
doses?
A frequent response from people
seeing a
dose-response curve like that
above for the first
time is to ask
'Does this mean higher doses are
safer?'
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
affected.
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
designed for.
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.
3 of 6
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
male form.
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
people 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
-10
molar). By the
time the dose rose to
10 parts per
billion (10
-8
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
lower levels.
4 of 6
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.
5 of 6
Resources
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:
185-192.
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.
6 of 6