scientific studies of mixtures and synergy
||Hayes, TB, P Case, S Chui, D Chung, C Haefele, K Haston, M Lee, VP Mai, Y Marjuoa, J Parker and M Tsui 2006. Pesticide mixtures, endocrine disruption, and amphibian declines: Are we underestimating the impact? Environmental Health Perspectives, in press.
A study of pesticide effects on frog tadpoles finds unexpectedly that a mixture of 9 pesticides, each at environmentally-relevant levels (0.1 ppb), undermines the tadpoles' defenses against a bacterial infection. 35% of animals exposed to the mixture died compared to 4% of those treated with pesticides one at a time. Of those that survived, 70% of the animals exposed to mixtures developed bacterial infections whereas none of the controls or animals exposed to one pesticide at a time showed similar symptoms. More...
N, E Silva and A Kortenkamp. 2002. Combining Xenoestrogens
at Levels below Individual No-Observed-Effect Concentrations Dramatically
Enhances Steroid Hormone Action. Environmental Health
research from England shows conclusively that mixtures matter. Publishing
in the journal of the U.S. National Institute of Environmental Health
Sciences, Rajapakse et al. reveal that even minute quantities
of "xenoestrogens" --levels beneath the concentrations
at which they appear to have effects individually, combine in mixtures
with one another and natural estrogen, 17ß-estradiol, to dramatically
increase the impact of the natural hormone. They are able to show
that the effective is additive, not synergistic. Their results
definitively refute the oft-heard criticism that xenoestrogens are
so weak and dilute compared to natural hormones that they can't
possible have an effect. More...
MF, J Jaeger and W Porter. 2002. Developmental Toxicity
of a Commercial Herbicide Mixture in Mice: I. Effects on Embryo
Implantation and Litter Size. Environmental Health Perspectives
et al. report that a commonly-used commercial herbicide
mixture of 2,4-D reduces the litter size of mice
exposed to low, environmentally-relevant doses. They found the largest
reductions in litter size at the lowest dosage level used,
which corresponded to EPA's "reference dose," the concentration
calculated on the basis of experiments to be sufficiently low to
avoid adverse health effects. This was one-seventh of the "maximum
contaminant level" allowed in drinking water by EPA standards.
N, D Ong and A Kortenkamp. 2001. Defining the Impact of Weakly
Estrogenic Chemicals on the Action of Steroidal Estrogens. Toxicological
Sciences 60: 296-304.
an elegant set of experiments, Rajapkse et al. demonstrate
that the additive interactions of weak xeno-estrogens significantly
change cellular responses to 17ß-estradiol. Their results
effectively eliminate the argument that xeno-estrogens can't
be important because they are only "weak estrogens."
J, N Rajapakse, M Wilkins, and A Kortenkamp 2000. Prediction
and Assessment of the Effects of Mixtures of Four Xenoestrogens.
Health Perspectives 108:983-987.
et al. examined the ability of several estrogen mimics to
act alone and then in combination with one another. They used used
four well-established estrogen mimics, o,pī-DDT,
genistein, 4-nonylphenol, and 4-n-octylphenol, in cell-culture
experiments measuring the strength of response provoked by binding
with the human estrogen receptor. They found that the results of
mixtures could be predicted from careful testing of the behavior
of compounds individually. More...
WP, JW Jaeger and IH Carlson. 1999. Endocrine, immune and behavioral
effects of aldicarb (carbamate), atrazine (triazine) and nitrate
(fertilizer) mixtures at groundwater concentrations. Toxicology
and Industrial Health 15: 133-150.
et al. examined the impacts on adult mice of different combinations
of two pesticides and a fertilizer at levels and in mixtures typical
of drinking water in the mid-West United States. They found that
while mice exposed to the compounds one at a time showed little
impact, combinations altered behavior and immune system function.
More on their research findings...
on the inadequacy of current regulatory approaches.
JM, E Willingham, CT Osborn, III, T Rhen and David Crews. 1999. Developmental
synergism of steroidal estrogens in sex determination. Environmental
Health Perspectives 107:93-97.
et al. demonstrate that weak natural estrogens interact synergistically
with a more powerful estrogen and alter sex in developing turtles.
Their experiments were done on sex determination in turtles, a process
normally controlled through the influence of incubation temperature
on hormonal metabolism and/or sensitivity. In their test species
(the red slider), eggs incubated at constant temperatures beneath
28.6°C normally develop as males while those incubated above
29.6°C develop as females.
these experiments they observed the impact of various concentrations
of three estrogens (estrone, 17ß-estradiol and estriol) on
sex determination, first determining the impacts of exposure to
single estrogens and then to combinations. They found that estrone
and 17ß-estradiol alone had significant but weak impacts,
while estriol alone displayed much greater potency. They then combined
the chemicals in different combinations, predicting the degree of
sex reversal based on the assumption that the effects should be
additive. They found that two of these combinations (17ß-estradiol
and estriol, estrone and estriol) were significantly more powerful
together than would be expected on the basis of simple additive
study "gives credence to the proposition that strong natural
estrogens at low doses may synergize with low doses of weak natural
and man-made estrogens."
DO, KF Arcaro, B Bush, W Niemi, S Pang and DD Vakharia. 1998. Human
health and chemical mixtures: an overview. Environmental
Health Perspectives 106 Supplement 6:1263-1270.
this review, Carpenter et al. conclude: "Unlike laboratory
animals, people are rarely exposed to a single hazardous chemical.
However, most of the information documenting adverse human health
effects from environmental and occupational contaminants has come
from studies focused on exposure to single chemicals... Two or more
compounds may show additive, antagonistic, or synergistic interactions
or may act on totally different systems and thus not interact. Furthermore,
even a single chemical may have multiple effects and affect more
than one organ system. Effects may vary with age, and metabolites
[breakdown products] may have totally different actions from the
N., K. P. Cantor, A Blair, D Bush, JW Brock, K Helzlsouer, SH Zahm,
LL Needham, GR Pearson, RN Hoover, GW Comstock, PT Strickland 1997.
A nested case-control study
of non-Hodgkin lymphoma and serum organochlorine residues.
The Lancet 350 (July 26): 240-244.
et al. found that a powerful interaction between levels of PCB exposure
and exposure to Epstein Barr virus in affecting the risk of people
to non-Hodgkin's Lymphoma (NHL), a hormonally-based cancer that
has been increasing steadily within the US. Without elevated exposures
to PCB, exposure to the virus did not appear to raise the risk of
NHL. Without elevated exposure to the virus, PCB exposure had only
a modest effect. But elevated levels of both risk factors dramatically
increased the risk to NHL, by over 20-fold.
paper is especially important because it focuses attention on potential
synergies between infectious disease agents and contamination.
More on their research findings...
JA. 1997. Synergistic effect of environmental estrogens:
report withdrawn. Science 277:459-463.
this publication, John McLachlan withdrew the study
published in June 1996 showing dramatic synergy among weakly estrogenic
pesticides. After several papers had challenged the replicability
of the research, McLachlan attempted unsuccessfully, over a period
of months, to replicate his lab's results. His published acknowledgement
stimulated a series of highly critical articles in venues like
Forbes Magazine and the Wall
Street Journal , which attempted to use this one failed experiment
to claim that the entire issue of endocrine disruption was therefore
bogus. These contorted attacks tried to argue that all
synergy was disproven and that this allayed any concern about endocrine
got it wrong: synergy had already been demonstrated by others
(and has again), and endocrine disruption is a concern even without
synergy. In fact, Our Stolen Future was written and sent
to press before McLachlan even began the experiments that he eventually
withdrew. The science of endocrine disruption rests on a vast base
of experimental and epidemiological research. It does not rise or
fall on one paper. More...
SF, DM Klotz, BM Collins, PM Vonier, LJ Guillette, Jr., and JA McLachlan.
1996. Synergistic activation of estrogen receptor with combinations
of environmental chemicals. Science 272:1489-1492.
now-withdrawn study, Arnold et al. found 150- to 1600-fold
synergistic interactions among weakly estrogenic pesticides using
a test system in which yeast cells were genetically engineered to
contain functional human estrogen receptors.
striking thing about these findings with the same yeast screening
method isn't the existence of synergistic effects, but their reported
magnitude. Within a year of the study's publication, several publications
reported unsuccessful efforts to replicate these results. Ultimately,
as noted above, McLachlan confirmed that his own laboratory could
not replicate the magnitude of synergistic interactions.
Birgelen, APJM, KM Fase, J van der Kolk, H Poiger, A Brouwer, W Seinen
and M van den Berg. 1996. Synergistic effect of 2,2',4,4',5,5'-Hexachlorobiphenyl
and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on hepatic porphyrin
levels in the rat. Environmental
Health Perspectives 104:550-557.
Birgelen et al. report dramatic synergistic interaction between
a PCB congener, PCB 153, and dioxin. They studied the impact of
several PCB congeners and dioxin on hepatic porphyrin accumulat
ion in rats. PCB 153 by itself caused no increase in accumulation.
Maximum increases for dioxin alone were about twice control levels.
But combining PCB 153 and dioxin caused increases 800 times above
SF and JA McLachlan. 1996. Synergistic
signals in the environment. Environmental Health Perspectives.
article reviews evidence about synergy and integrates information
about environmental signaling involving several systems, including
chemicals, temperature and oxygen stress. The authors concludes
that "biological synergy ... may be more common than previously
thought. Furthermore, it is clear that environmental signals can
produce synergistic biological responses."
PM, D Andrew Crain, JA McLachlan, LJ Guillette, Jr., and SF Arnold.
1996. Interaction of Environmental Chemicals with the Estrogen
and Progesterone Receptors from the Oviduct of the American Alligator.
with the alligator estrogen receptor (aER), Vonier et al. find
that several environmental chemicals which individually do not interact
with the estrogen receptor nonetheless inhibit 17ß-estradiol
when applied in combination with one another.
toxaphene, dieldrin and chlordane alone had no effect on 17ß-estradiol
binding. When combined, however, with a mixture of other chemicals
(which had already reduced binding by 40%), these four together
reduced total binding by another 14%.
of DDT metabolies reduced estradiol binding to aER in an additive
fashion, whereas combinations of chemicals in Lake Apopka inhibited
binding in a greater than additive manner." Their experiments "suggest
that combinations of chemicals found in the alligator eggs can inhibit
binding of 17ß-estradiol by 60% at environmental concentrations"
(i.e., levels of contamination measured in the wild are within the
dose range used in these experiments which produced significant
impacts). This paper also documents the ability of some environmental
chemicals to interact with the alligator progesterone
Birgelen, APJM, KM Fase, J van der Kolk, H Poiger, A Brouwer, W Seinen,
and M van den Berg. 1996. Synergistic Effect of 2,2',4,4',5,5'-Hexachlorobiphenyl
and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Hepatic Porphyrin Levels
in the Rat. Environmental
Health Perspectives 104(5):550-557.
porphyrin accumulation has been observed in people following a hexachlorobenzene
poisoning in SE Turkey in the late 1960s, in industrial workers
producing certain herbicides, in the aftermath of the Seveso, Italy,
dioxin accident and in the Taiwanese Yu-Cheng PCB poisoning incident.
It has also been observed under field conditions (Herring gulls
in the Great Lakes) and laboratory experiments following exposure
to dioxin and related compounds. van Birgelen et al. studied
the effect of PCBs and dioxins on hepatic porphyrin accumulation
in rats in a lab diet experiment. They found a dose-dependent increase
in hepatic porphyrin accumulation after administration of the contaminants:
"this study clearly shows that porphyrins accumulate in the liver
after subchronic treatment with TCDD (dioxin), PCB 126, or PCB 156
Birgelen et al. report that PCB 153 by itself caused no increase
in accumulation. Maximum increases for dioxin alone were about twice
control levels. But combining PCB 153 and dioxin caused increases
800 times above control levels.
M and LG Hansen. 1996. Enzyme Induction and Acute Endocrine Effects
in Prepubertal Female Rats Receiving Environmental PCB/PCDF/PCDD Mixtures.
Health Perspectives 104(7):712-722.
study measures real-world mixtures of contaminants from air, subsurface
soil and superficial dust around a toxic landfill in southern Illinois.
It then asks how well the standard "toxic equivalency factors" predict
biological responses in experiments with pre-pubertal rats when exposed
to mixtures extracted from the landfill.
study demonstrated that the environmental mixtures containing mainly
PCBs with significant proportions of PCDFs caused both TCDD (dioxin)-like
and non-TCDD-like effects.. after a short exposure."
the risk assessment of an environmental mixture focuses only on
the TCDD-like compounds in the mixture, the important endocrine-disrupting
effects of a mixture could be underestimated."
P, E Eilertsen, E Einarsdottir, A Haugen, V Skaug and S Ovrebo. 1995.
Fertility in mice after prenatal exposure to benzo[a]pyrene and
inorganic lead. Environmental
Health Perspectives 103(6): 588-590.
et al. found a synergistic interaction between inorganic lead and
benzo[a]pyrene impairing fertility of mice that had been exposed in
the womb. Mice exposed to both lead and BaP had significantly longer
gestation periods. Data also suggested that the compounds together
further reduced number of offspring, number of litters and litter
A, KL Chung and C Sonnenschein. 1994. The Pesticides Endosulfan,
Toxaphene, and Dieldrin Have Estrogenic Effects on Human Estrogen-Sensitive
Health Perspectives 102(4): 380-383.
et al. report here that endosulfan, toxaphene and dieldrin
are estrogenic. The experiments they summarize used the "E-screen"
assay developed by Soto and Sonnenschein, which tests the ability
of compounds to simulate estrogen's capacity to stimulate proliferation
of human MCF7 breast cancer cells in vitro.
report also presents results on mixtures of the compounds. In
the mixture experiment Soto et al. compare the stimulative
effects of 10 compounds, each present in the mixture at a level
at which by itself it has no apparent effect. By themselves, they
have no effect. But in combination, the mixture of these 10 compounds
has a strongly proliferative impact. They conclude that "measuring
the total estrogenic burden due to environmental contaminants may
be more meaningful than assessing exposure by measuring the levels
of each of the known xenoestrogens."