New scientific studies of mixtures and synergy

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...

Rajapakse, N, E Silva and A Kortenkamp. 2002. Combining Xenoestrogens at Levels below Individual No-Observed-Effect Concentrations Dramatically Enhances Steroid Hormone Action. Environmental Health Perspectives 110:917–921.

This 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...

Cavieres, 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 110: 1081-1085.

Cavieres 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. More...

Rajapakse, 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.

In 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." More...

Payne, J, N Rajapakse, M Wilkins, and A Kortenkamp 2000. Prediction and Assessment of the Effects of Mixtures of Four Xenoestrogens. Environmental Health Perspectives 108:983-987.

Payne 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,p´-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...

Porter, 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.

Porter 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...

They also comment on the inadequacy of current regulatory approaches.

Bergeron 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.

In 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 effects.

This 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."

In 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 parent compound."

Rothman, 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.

Rothman 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.

This paper is especially important because it focuses attention on potential synergies between infectious disease agents and contamination.

More on their research findings...

With 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 disruption.

They 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...

The 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.

van 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 control levels.

This 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."

Specifically, 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%.

"Mixtures 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 receptor.

Hepatic 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 in rats.

van 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.

"This 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."

"If 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."

Soto 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.

The 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."

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