Ten Years of Mixing Cocktails: A Review of Combination Effects of Endocrine-Disrupting Chemicals

It is often said that the effects of a combination of chemicals may be smaller or larger than the sum of the individual effects of all components. Without further justification, this is frequently taken to mean that the anticipated combination effect is accessible by calculating the simple arithmetic sum of the individual effects of all chemicals. The fallacy of this expectation has been discussed elsewhere (Berenbaum 1981; Kortenkamp and Altenburger 1998), but it becomes obvious when we consider, for example, 10 agents, each of which provokes 15% of a certain response. The anticipation that the resulting joint effect should be 10 × 15% = 150% turns out to be biologically impossible if the maximally inducible effect can only be 100%.

Thus, approaches are required that provide more reliable calculations of mixture effects, such that a reference point for the assessment of combination effects in terms of synergisms, additivity, and antagonism can be defined. For this purpose, two concepts are available: dose addition (often referred to as concentration addition) and independent action. These concepts are used depending on the presumed modes of action of the mixture components.

Dose addition is applied to mixtures of chemicals that exert their effects through similar modes of action. Examples include organophosphorus pesticides and polychlorinated dioxins (PCDDs) and polychlorinated furans (PCDFs). Because these chemicals interact with well-defined molecular targets, it is thought that one chemical can be replaced totally or in part by an equal fraction of an equieffective concentration of another without diminishing the overall combined effect (Loewe and Muischnek 1926). A widely used application of dose addition is the TEF/TEQ concept for the assessment of mixtures of PCDDs and PCDFs (Safe 1998; van den Berg et al. 1998). A great deal of the work on ED mixtures has utilized dose addition or concentration addition as the concept for calculating additivity expectations. Considering that the majority of mixture studies have been based on end points relatively close to the steps following hormone-receptor binding and activation, the choice of dose addition appears to be well-founded.

Independent action (also called “response addition”) is used for combinations of agents with diverse modes of action. By activating differing effector chains, every component of a mixture of dissimilarly acting chemicals is thought to provoke effects independent of all other agents that might also be present. The resulting combined effect can be calculated from the effects caused by the individual mixture components by adopting the statistical concept of independent events (Bliss 1939). Independent action (often confusingly also referred to as “response addition” or “effect multiplication”) has been used rarely for mixtures of EDs.

Both dose (or concentration) addition and independent action are able to account for saturation effects at higher effect doses and will not produce paradoxical predictions of supra-maximal combination effects such as in the example described above with 10 compounds, each inducing a 15% effect.

In this article, I will discuss work with the three most frequently studied hormone receptors: the estrogen receptor, the androgen receptor (AR), and the thyroid receptor. There is a rich literature concerning the aryl hydrocarbon receptor (AhR), which has been reviewed elsewhere (van den Berg et al. 1998) and will consequently not be dealt with here, but interactions between AhR agonists and other EDs will be considered.

Estrogenic chemicals have been the focus of most of the work on EDs, and it is not surprising that this group of substances has been the topic of the majority of ED mixture studies. Although the earlier efforts mainly employed binary mixtures (reviewed by Kortenkamp and Altenburger 1998), work carried out since 1998 has made significant contributions to the analysis of multicomponent mixtures containing 3, often 5, and up to 12 estrogenic chemicals. “Estrogenicity” can be defined in various ways. At the functional, physiologic level, the term denotes the ability of a chemical to evoke responses similar to those of 17β-estradiol (E₂), such as cornification of the vaginal epithelium and uterine cell proliferation. Of toxicologic concern is the role of estrogens in breast and ovarian cancer, and E₂ and synthetic estrogens are recognized human carcinogens. Advances in our understanding of the mode of action of estrogens have led to further definitions that refer to specific steps at various molecular levels. These definitions provide a way to structure the evidence on estrogen mixtures; thus, ”estrogenicity” can mean affinity to the estrogen receptor (ER-α or ER-β) (although this does not distinguish agonists from antagonists), the ability to activate expression of estrogen-dependent genes, or stimulation of cell proliferation of ER-competent cells. At present, no post-1998 multicomponent study with ER binding as the end point is available.

Androgens are key regulators of male sexual differentiation during in utero and early post-natal development. Chemicals that counteract androgen action at some stage in this period can lead to malformations of the reproductive tract. Changes in the anogenital distance, retained nipples, and alterations in the weight of sexual organs and accessory glands are frequently studied end points. These effects can arise through antagonism of androgens at the steroid receptor level and/or via suppression of testosterone synthesis in Leydig cells (Fisher 2004; Gray et al. 2001). Thus, anti-androgens can be defined narrowly as AR antagonists, but a broader definition in terms of counteracting the effects of androgens in a functional sense (which would include inhibition of uptake of testosterone precursors, and of testosterone synthesis steps) has also been proposed (Gray et al. 2001).

By applying the isobole method, which is another application of dose addition (Berenbaum 1981; Loewe and Muischnek 1926), Nellemann et al. (2003) found that procymidone and vinclozolin, both AR antagonists, additively inhibited testosterone binding to the AR. Administration of a 1:1 mixture of both fungizides to castrated, testosterone-treated male rats led to dose-additive alterations in reproductive organ weights, androgen levels, and AR-dependent gene expression. Birkhoj et al. (2004) extended the use of the isobole method to three-component mixtures of the pesticides deltamethrin, methiocarb, and prochloraz. An equimolar mixture of the three pesticides additively suppressed AR activation in vitro. When the authors gave a combination of these three chemicals with simazin and tribenuronmethyl to castrated testosterone-treated rats, weight changes of the adrenal gland and the levator ani, as well as alterations in gene expression of AR-associated genes were observed. The combination of all five chemicals showed effects that were not found for the individual pesticides, but whether these responses were additive could not be assessed.

A mixture of the AR antagonists procymidone and vinclozolin was evaluated in the Hershberger assay, where they acted additively in reducing ventral prostate and levator ani weights (Gray et al. 2001). A combination of procymidone and dibutyl phthalate, an inhibitor of androgen synthesis, significantly enhanced the occurrence of hypospadias in male offspring when given to pregnant rats during gestational days 14–18. Wolf et al. (2004) observed that vinclozolin and testosterone proprionate, two chemicals with opposing effects on male sexual differentiation, antagonized one another during sexual development of the male rat. A mixture of butyl benzyl phthalate, an inhibitor of testosterone synthesis, and linuron, an AR antagonist, decreased testosterone production and caused alterations of androgen-organized tissues in a dose-additive fashion (Hotchkiss et al. 2004). Jarfelt et al. (2005) studied changes in anogenital distance and retained nipples of male offspring of female rats treated with di-(2-ethylhexyl) phthalate and di-(2-ethylhexyl)adipate, but the effects of the mixture were not different from those of each chemical alone.

Compared with estrogens and antiandrogens, thyroid-disrupting chemicals are the least well studied EDs. It is therefore not surprising that few mixture studies exist using this kind of agents.

Thyroid-disrupting chemicals can alter structure and function of the thyroid gland, as well as the homeostasis of thyroid hormones, by interfering with associated regulatory enzymes. Changes in the circulating levels of thyroid hormones are often the consequence. A wide variety of chemicals are able to affect thyroid hormone levels in differing ways. PCDDs, PCDFs, and PCBs are thought to suppress circulating thyroid hormone levels by up-regulating hepatic enzymes that glucuronidate thyroxin (T₄). Most of the studies of thyroid-disrupting effects have analyzed the effects of mixtures without recording responses induced by individual mixture components; this complicates assessment of combination effects in terms of additivity, synergism, or antagonism. Wade et al. (2002) exposed rats to a combination of organochlorines and two heavy metals and analyzed effects on thyroid histopathology. Using the TCDD (2,3,7,8-tetrachloro-p-dibenzodioxin) equivalents method, Desaulniers et al. (2003) found that the effects of 16 PCBs, PCDDs, and PCDFs on circulating T₄ levels could be predicted well.

Crofton et al. (2005) presented an in-depth study of a mixture of 18 poly-halogenated hydrocarbons (2 PCDDs, 4 PCDFs, and 12 coplanar and noncoplanar PCBs) to investigate the hypothesis that their joint effect on reducing T₄ levels is dose additive. Young female rats were treated for 4 days with individual mixture components, and dose–response relationships with altered T₄ levels as the end point were recorded. This information was used to predict the dose-additive response to a mixture of all 18 chemicals. The mixture ratio was chosen to be proportional to the levels of the chemicals reported in breast milk, fish, and other human food sources. The dose-additivity model yielded anticipated effect doses that were higher by a factor of 2–3 than the observed responses. This deviation was statistically significant, and the joint effect of all polyhalogenated pollutants in this model can therefore be classed as synergistic. Nevertheless, the extent of underestimation of observed effects was small.

Taken together, there is good evidence that EDs produce combination effects in a dose-additive manner. This applies to a wide range of end points reflecting various hierarchical levels of hormone action in a variety of organisms. Where deviations from expected additivity occurred (Charles et al. 2002a, 2002b; Crofton et al. 2005; Rajapakse et al. 2004; Thorpe et al. 2001), the differences between anticipated and observed effects were small. Thus, it is safe to say that for regulatory purposes, the concept of dose addition is sufficiently accurate for predicting combination effects of groups of EDs with similar effects.

The reported deviations are nevertheless interesting from a conceptual view point. Toxicokinetic interactions such as differential activations of metabolizing enzymes in the mixtures may have played a role, and this requires further experimental study. For example, some estrogenic organochlorines may induce specific subsets of cytochrome P450 enzymes involved in steroid metabolism, thus leading to increased removal of steroidal estrogens from the mixture, with a certain loss of activity. This may explain the slightly lower-than-expected combination effects observed in the E-SCREEN assay by Rajapakse et al. (2004). Similar considerations may apply to the mixture of thyroid-disrupting chemicals analyzed by Crofton et al. (2005), where many diverse mechanisms are at play leading to reductions in circulating T₄ levels.

Comparatively little work has been performed with mixtures of different classes of EDs, such as estrogenic agents combined with anti-estrogenic chemicals, or EDs combined with other toxicants. In terms of design and data assessment, this type of study differs from those discussed so far, because not all components present in the mixture may induce the effect chosen for analysis. In these cases, a “modulatory” influence of toxicants on the effects of other chemicals is studied. It is important to realize that the magnitude of such effect modulations cannot be predicted by adopting additivity concepts such as concentration addition or independent action.

Perhaps the best-known example of effect modulation is the inhibitory effect of AhR agonists, such as PCDDs and co-planar PCBs, on the action of estrogenic chemicals. Although not estrogenic themselves, AhR agonists have been reported to suppress some E₂-induced responses, not by antagonizing hormone binding to the ER but by down-regulation of ER expression, induction of steroid-metabolizing enzyme systems such as CYP 1A1 and 1A2, and inhibiting various growth factors and cell cycle regulators (Chen et al. 2001, Reen et al. 2002, Safe 1998). There is a rich literature about the molecular biology underlying the interactions between dioxins and estrogens (Sone and Yonemoto 2002).

Somewhat misleadingly, the action of AhR agonists has been called antiestrogenic, when it is perhaps more appropriate to view them as disruptors of estrogen signaling. The dioxin TCDD has been reported to inhibit the estrogen-induced proliferation of uterine tissue in immature mice (Gallo et al. 1986) and to lead to diminuitions of ER levels in the liver and the uterus. Modulations of ER levels by TCDD were also described in rats (Astroff and Safe 1988; Romkes and Safe 1988; Romkes et al. 1987). Although down-regulation of ER expression by AhR agonists in cell models is not controversial, difficulties with reproducing the effects in rodents have led to questions about the relevance of antiestrogenic effects of AhR in vivo. White et al. (1995) examined the impact of TCDD on the keratinization of the vaginal epithelium and uterine proliferation in Sprague-Dawley rats induced by E₂, but they failed to observe any inhibitory effects of TCDD. Uterine ER and progesterone receptor levels were also not affected, although toxicity typical of TCDD (reductions in thymus weight, induction of hepatic CYP 1A1) occurred. Similarly, Desaulniers et al. (2003) did not observe an influence of a mixture of 16 AhR agonists (various PCDDs, PCDFs, and PCBs) on uterine growth stimulated by EE₂ in prepubertal female Sprague-Dawley rats. Although the reasons for these contradictory findings remain to be fully elucidated, Desaulniers et al. (2003) pointed to reports by Petroff et al. (2001) and Sarkar et al. (2000) of enhancements of TCDD-induced AhR expression and CYP 1A1 induction in the presence of E₂. This could explain the lack of antiestrogencity of AhR agonists in their study. White et al. (1995) even questioned the validity of ascribing a specific antiestrogenic property to TCDD in the rat. They pointed out that inhibitory actions of TCDD on E₂-induced effects reported by Safe and colleagues (Astroff and Safe 1988; Romkes and Safe 1988; Romkes et al. 1987) occurred at TCDD doses similar to the median lethal dose (LD₅₀) for the Sprague-Dawley and Long-Evan strains. Because TCDD induces a well-known wasting syndrome, it is conceivable that the antiestrogenicity of TCDD is in fact the result of such systemic toxicity, rather than due to specific effects opposing the action of the hormone. Thus, more work is required to evaluate whether disruption of estrogen signaling by AhR agonists occurs at realistic doses, and whether doses shown to interfere with estrogen-mediated biochemical effects, such as changes in gene expression, also lead to suppression of estrogen action with more apical end points, such as cell proliferation.

Epidermal growth factor (EGF) and insulin-like growth factor (IGF) are able to enhance estrogen signaling by inducing ER phosphorylation and other signaling events (Aronica and Katzenellenbogen 1993; Ignar-Trowbridge et al. 1996). These observations prompted Charles et al. (2002a) to study the impact of EGF and IGF on E₂-induced activation of ER in an MCF7 cell–based reporter gene system. Several combinations of all three agents were investigated and response surfaces recorded. Although EGF and IGF, on their own, did not promote gene transcription in this model, there were enhancements of the effects of E₂, mostly caused by EGF. These results indicate that the presence of growth factors may sensitize ER-competent cells to the action of the hormone, with significant consequences in terms of lowered effect thresholds. It remains to be seen whether similar effects also occur with estrogen-like environmental pollutants. Without a doubt, the potential for greater-than-additive interactions through interference with interacting signaling pathways deserves further attention and should be investigated systematically.

In the context of discussions about EDs, various often-conflicting definitions of the term “low dose” have been used. “Low dose” is variously taken to mean “doses lower than used normally in toxicity testing”; “doses that approach, or are equal to, those encountered by humans”; or “doses associated with low effects” (National Toxicology Program 2001; vom Saal and Hughes 2005). Not all of these definitions have proven useful in guiding experimental work aimed at investigating whether mixtures of EDs provoke effects at low doses. Many of the chemicals suspected of being EDs have not yet been subjected to toxicity testing and consequently, “doses lower than used normally in toxicity testing” are difficult to define. Similarly, the resolving power of most in vivo and many in vitro assays is insufficient to demonstrate effects of combinations of agents at doses approaching human exposure levels. For these reasons, low-dose levels in mixture studies were selected by adhering to the last of the above definitions [i.e., “low dose” in the sense of doses that produce low effects, usually around or below no observed effect levels (NOELs)]. Although such doses may be relatively large compared to human exposure levels, the relevant experimental studies provided valuable insights into the potential of EDs to act together at low doses.

The concept of dose addition implies that every effective agent in the mixture, at any dose, contributes more or less to the overall combination effect. Crucially, this also holds true when the individual doses are without effect. Thus, combination effects should also result from agents present at or even below effect thresholds, provided sufficiently large numbers of components sum up to a sufficiently high total effect dose. It may be helpful to illustrate these implications of dose addition by adopting a thought experiment first presented by Berenbaum (1981). Let us consider a large number of chemicals that by chance all exhibit the same sigmoidal dose–response curve. At small doses, the effect produced by one single component is too small to be distinguishable from untreated controls. However, the response expected from combining 10 chemicals at this same low dose is equivalent to the effect of a 10-fold higher dose, because all components are assumed to exhibit the same dose–response relationship. The procedure can be repeated with infinitesimally small doses below effect thresholds; as long as there are sufficiently high numbers of chemicals present simultaneously, combination effects should result.

The good agreement of ED mixture effects with dose addition makes it an attractive proposition to review whether these theory expectations are in line with experimental observation. Silva et al. (2002) assessed combinations of eight xenoestrogens in the yeast estrogen screen at concentrations of 50% of their no observed effect concentrations (NOECs) and observed responses of up to 40% of a maximal estrogenic effect. Using the same assay, Rajapakse et al. (2002) set out to investigate whether low levels of weak xenoestrogens would be able to modulate the effects of E₂. A combination of 11 xenoestrogens, all present at levels around their individual NOECs, led to a doubling of the effects of E₂. Tinwell and Ashby (2004) combined 8 estrogenic chemicals at doses that gave no significant uterotrophic responses when tested on their own. When administered together, quite strong uterotrophic effects were observed. The mixture experiments with 5 estrogenic chemicals in fathead minnows (Pimephales promelas) presented by Brian et al. (2005) also demonstrated combination effects at concentrations that individually did not induce vitellogenin synthesis. The 18 thyroid-disrupting chemicals chosen by Crofton et al. (2005) to analyze changes in T₄ levels were all present at doses equivalent to, or even below, their individual NOELs.

These examples clearly demonstrate that combinations of EDs with similar effects are able to act together at doses that when used alone do not lead to observable effects. The experimental evidence is in line with the assumptions of dose addition. Combination effects may result from cumulative exposure to EDs if they are present in sufficiently large numbers at levels equivalent to fractions of their individual NOELs. However, whether mixture effects will indeed occur is difficult to anticipate without comprehensive information about the levels and the identity of EDs in the environment and in human tissues. This is where one of the major challenges for mixture assessment currently lies: Our information about the occurrence of EDs in humans and the environment is patchy. Considerable efforts in mixture exposure assessment need to be made to fill these gaps; exposure assessment, and not hazard assessment, currently represents the “bottleneck” for making further progress in this important area.

It is evident that the traditional chemical-by-chemical approach to risk assessment is inadequate when dealing with EDs (and chemicals with other toxic profiles). The biological reality of combination effects from exposure to multiple agents at low doses highlights the potential for underestimating risks when mixture effects are not taken into account. This underlines the need to modify current risk assessment practice if humans and the environment are to be protected adequately from multiple exposures to EDs. As a first step in the direction of implementing better risk assessment, the idea of grouping EDs according to suitable similarity criteria suggests itself, as is already common practice with the group-wise assessment of AhR agonists such as PCDDs, PCDFs, and PCBs in the TEF/TEQ approach. For example, in a recent opinion paper, the European Scientific Committee on Toxicology, Ecotoxicology and the Environment (Commission of the European Communities 2004) pointed out that

As I have demonstrated in this article, comparatively little information exists concerning the ways in which EDs belonging to different classes may interact to produce combined effects. As a result, research efforts should be focused to pursue this area of study. For example, antiandrogens, including AR antagonists and inhibitors of steroid synthesis, should be combined with estrogenic agents that also possess antiandrogenic properties to allow the study of possible impacts on disruption of male sexual development in vivo. PCDDs, PCDFs, and PCBs are well-known disruptors of male sexual development, but very little is known about how they act together with antiandrogens and estrogens, and it is urgent to fill this gap.

Another knowledge gap that needs to be bridged concerns possible interactions between growth factors such as EGF and IGF with natural steroid hormones and xenoestrogens. A better understanding of the joint effects of these agents is required to evaluate environmental factors that are important in the formation of breast cancer. There is already evidence demonstrating a potential for synergism between steroid hormones and growth factors (Charles et al. 2002a), but studies building on this knowledge are required to evaluate potential interactions between growth factors and xenoestrogens. This area of investigation could be enhanced further by analyzing the role of signaling cross talk between natural dietary components that activate retinoid receptors and ER pathways.

Of particular interest are combinations of chemicals in which not all components produce the effect to be analyzed, but where some may significantly modulate the effects of others, without themselves being effective. By their very nature, the impact of such effect modulators will not be predictable quantitatively by employing the usual additivity expectations in mixture toxicology. However, it is necessary to explore whether the direction of such effect modulations can be anticipated in qualitative terms by analyzing interactions at the level of metabolism and transport. The approach taken could use existing databases for the visualization of complex gene and signaling networks as a mining and analytical tool for hypothesis generation (Ekins et al. 2006). Signaling nodes and interaction points identified in this way could then be targeted experimentally by gene expression profiling and proteomics techniques. This will also allow assessments of the usefulness of such data mining and experimental approaches for the grouping of EDs into classes with similar effect profiles.

Exposure assessment has revealed itself as another major limiting factor that is currently hampering progress with ED mixtures. Information exists about the concentrations of many individual chemicals in human tissues and in the environment, but data concerning the levels of multiple chemicals in the same samples are scarce. Put simply, we need to know whether women in agricultural areas in, say, Spain, who exhibit elevated levels of certain pesticides in their tissues also show high levels of phthalates from copious use of personal care products and cosmetics. To date, this information is not available, and it will require dedicated, targeted mixture exposure assessment strategies to fill this gap.

Perhaps the greatest challenge will be to develop ways in which concepts for mixture effect assessment can be used productively in epidemiology. Epidemiology has traditionally focused on defining the impact of single chemicals on disease outcomes, and only very few examples exist where the role of combinations of chemicals could be evaluated. As an example relevant to endocrine disruption, the shortcomings of traditional single-chemical epidemiology on elucidating causes for breast cancer have been previously discussed (Kortenkamp 2006). In order to develop viable approaches to solving this problem, the concerted efforts of mixture toxicology experts, exposure assessors, and epidemiologists will be required.

In the last 10 years, considerable progress has been made in assessing the effects of multi-component mixtures of EDs. Work has focused on mixtures with components belonging to certain classes of EDs, such as estrogenic, antiandrogenic, and thyroid-disrupting chemicals; these studies have demonstrated the usefulness of the concept of dose addition in anticipating combination effects. Good evidence is available to show that joint effects occur even when all mixture components are present at levels below doses that cause observable effects. In view of this evidence, the traditional chemical-by-chemical approach to risk assessment is hard to justify, and the ground is prepared to seriously consider group-wise regulation of EDs. In spite of serious shortcomings in our understanding of signaling cross talk between categories of EDs, we should group these chemicals according to their ability to induce similar effects (as opposed to similar mechanisms) until better mechanistic information in forthcoming. This modus operandi is only viable with a concurrent, targeted research program aimed at improving our understanding of ED mixtures. Future research should particularly focus on combinations of EDs that belong to different categories.

This article is part of the monograph “Endocrine Disruptors—Exposure Assessment, Novel End Points, and Low-Dose and Mixture Effects.”

This work is part of the European Union–supported EDEN project “Endocrine Disrupters: Exploring Novel Endpoints, Exposure, Low Dose- and Mixture-Effects in Humans, Aquatic Wildlife and Laboratory Animal” (QLK4-CT-2002-00603). Financial support from the European Commission is gratefully acknowledged.