April 30, 2007
Does 'the dose make the poison?'
Extensive results challenge a core assumption in toxicology

by Pete Myers, Ph.D. and Wendy Hessler
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.              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).

Low-level exposure to DES causes obesity in mice

Photograph from Retha Newbold, NIEHS.



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.
Toxicology testing assumes 'the dose makes the poison.'

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.

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)
and non-monotonic (right) dose-response curves. Monotonic can either
be linear or non-linear. 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).

Types of dose-response curves



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 in
sensitivity 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 a part per million), then the refence 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
diffferent doses.

The graph below 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).
        Estradiol causes non-monotonic dose response curve

Figure adapted from Welshons et al. 2003

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 estrogen-
responsive 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.

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.


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.

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.

In 2006, a team of German researchers published a vivid example of how
traditional toxicological 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 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 Germany

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.

DEHP causes non-monotonic dose response curveAdapted from Andrade et
al. 2006.



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.
Immune sensitivity follows NMDRC

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.

DEHP causes nonmonotonic immune response


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.

BPA and DDE both have nonmonotonic effects on prolactin secretion


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 calcium influx into 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.

Nonmonotonic changes in response to hexachlorobenzene           Ralph et al.
discovered that prostate cells respond in a non-monotonic fashion to
exposure to the organochlorine pesticide hexachlorobenzene (HCB). High
levels suppress androgenic activity of the cells relative to controls
(red line), 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.

BPA affects prostate tumor proliferation nonmonotonically
                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.




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): Non-monotonic dose-response and low
dose 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.

Wozniak, AL, NN Bulayeva and CS Watson. 2005. Xenoestrogens at
Picomolar to Nanomolar Concentrations Trigger Membrane Estrogen
Receptor-alpha-Mediated Ca++ Fluxes and Prolactin Release in GH3/B6
Pituitary Tumor Cells. Environmental Health Perspectives 113:431-439.


Non-monotonic Dose-Response

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Search

30 April Does 'the dose make the poison?' Extensive results from
studies of endocrine-disrupting compounds indicate that toxicological
testing can no longer assume high dose results predict the effect of
low doses. Environmental Health News.

24 April Tracking the chemicals in us. Large-scale measurements of
contaminants in human tissues-- biomonitoring -- has led to a
revolution in how chemicals are assessed, to new opportunities for
disease prevention, and to recognition of adverse effects of endocrine
disrupton. Chemical & Engineering News.

14 April New studies link health problems to toxic chemicals. Some
provocative new studies link prostate cancer and asthma to exposure to
tiny amounts of pollutants. Living On Earth.

7 April 'Inherently toxic' chemical faces its future. Bisphenol A is
ingested by practically everyone in Canada who eats canned foods or
drinks from a can or hard plastic water bottles. Now a controversy is
raging over the safety of widespread public exposure to the chemical,
which is known to act like a synthetic female sex hormone. Toronto
Globe and Mail, Ontario.

3 April Environmental estrogens increase the intensity of immune
reactions. Scientists report in a new study that six environmental
contaminants which act like the hormone estrogen increase the speed
and intensity of immune reactions in human and mouse cells.
Environmental Health News.



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