We tested whether multicomponent mixtures of xenoestrogens would produce significant effects when each component was combined at concentrations below its individual NOEC or EC01 level. The estrogenic effects of eight chemicals of environmental relevance, including hydroxylated PCBs, benzophenones, parabenes, bisphenol A, and genistein, were recorded using a recombinant yeast estrogen screen (YES). To ensure that no chemical contributed disproportionately to the overall combination effect, a mixture was prepared at a mixture ratio proportional to the potency of each individual component. The performance of four approaches for the calculation of additive combination effects (concentration addition, toxicity equivalency factors, effect summation, and independent action) was compared. Experimental testing of the predictions revealed that concentration addition and its application, the toxicity equivalency factor approach, were valid methods for the calculation of additive mixture effects. There was excellent agreement between prediction and observation. In contrast, independent action and effect summation led to clear underestimations of the experimentally observed responses. Crucially, there were substantial mixture effects even though each chemical was present at levels well below its NOEC and EC01. We conclude that estrogenic agents are able to act together to produce significant effects when combined at concentrations below their NOECs. Our results highlight the limitations of the traditional focus on the effects of single agents. Hazard assessments that ignore the possibility of joint action of estrogenic chemicals will almost certainly lead to significant underestimations of risk.

The low potency of many man-made estrogenic chemicals, so-called xenoestrogens, has been used to suggest that risks arising from exposure to individual chemicals are negligible. Another argument used to dismiss concerns of health effects is that endogenous steroidal estrogens are too potent for xenoestrogens to contribute significantly to estrogenic effects. Using a yeast reporter gene assay with the human estrogen receptoralpha, we tested these ideas experimentally by assessing the ability of a combination of 11 xenoestrogens to affect the actions of 17ss-estradiol. Significantly, each xenoestrogen was present at a level well below its no-observed-effect concentration (NOEC). To derive accurate descriptions of low effects, we recorded concentration-response relationships for each xenoestrogen and for 17ss-estradiol. We used these data to predict entire concentration-response curves of mixtures of xenoestrogens with 17ss-estradiol, assuming additive combination effects. Over a large range of concentrations, the experimentally observed responses decisively confirmed the model predictions. The combined additive effect of the 11 xenoestrogens led to a dramatic enhancement of the hormone's action, even when each single agent was present below its NOEC. Our results show that not even sub-NOEC levels of xenoestrogens can be considered to be without effect on potent steroidal estrogens when they act in concert with a large number of similarly acting chemicals. It remains to be seen to what degree these effects can be neutralized by environmental chemicals with antiestrogenic activity. Nevertheless, potential human and wildlife responses induced by additive combination effects of xenoestrogens deserve serious consideration.

The assessment of mixture effects of estrogenic agents is regarded as an issue of high priority by many governmental agencies and expert decision-making bodies all over the world. However, the few mixture studies published so far have suffered from conceptual and experimental problems and are considered to be inconclusive. Here, we report the results of assessments of two-, three- and four-component mixtures of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol, all compounds with well-documented estrogenic activity. Extensive concentration-response analyses with the single agents were carried out using a recombinant yeast screen (yeast estrogen screen, YES). Based on the activity of the single agents in the YES assay we calculated predictions of entire concentration-response curves for mixtures of our chosen test agents assuming additive combination effects. For this purpose we employed the models of concentration addition and independent action, both well-established models for the calculation of mixture effects. Experimental concentration-response analyses revealed good agreement between predicted and observed mixture effects in all cases. Our results show that the combined effect of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol in the YES assay does not deviate from expected additivity. We consider both reference models as useful tools for the assessment of combination effects of multiple mixtures of xenoestrogens.

We tested whether bisphenol A (BPA) or o,p'-DDT, when combined with 17beta-estradiol (E2), would contribute to the overall mixture effect using a yeast reporter gene assay, the yeast estrogen screen. Following comprehensive concentration-response analyses of the single agents, the pharmacologically well-founded models of concentration addition and independent action were used to predict entire concentration-response relationships for mixtures of the agents with a variety of fixed mixture ratios, assuming additivity. For molar mixture ratios proportional to the levels normally found in human tissues (i.e., below 1:5000, E2:BPA or o,p'-DDT), these predictions suggest that the effects of individual xenoestrogens are too weak to create an impact on the actions of steroidal hormones. However, at mixture ratios more in favor of the xenoestrogens, a significant contribution to the overall mixture effect was predicted. The predictions were tested experimentally. The observed combined effects of mixtures of E2 with either BPA or o,p'-DDT did not deviate from the additivity expectation. On combining E2 with either BPA or o,p'-DDT at approximately equieffective concentrations corresponding to molar mixture ratios between 1:20,000 and 1:100,000 (E2:BPA or o,p'-DDT), substantial modulations of the effects of E2 became discernible. The assumption that weak xenoestrogens are generally unable to create an impact upon the already strong effects of endogenous steroidal estrogens is not supported by our observations. Our studies indicate that the potential health implication of additive combination effects between xenoestrogens and steroidal estrogens deserve serious consideration.

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An intriguing deviation from expected additivity is reported with mixtures containing 17beta-estradiol, 17alpha-ethinylestradiol, genistein, bisphenol A, 4-nonylphenol, and 4-tert-octylphenol. The effect of these chemicals on the proliferation of estrogen-dependent MCF-7 human breast cancer cells (the E-SCREEN) was measured. Data variance-component analyses, carried out to optimize the assay for mixture studies, showed that between-experiment variability was the dominant source of data variation. Adoption of a data-normalization procedure reduced the impact of this variability and allowed the pooling of historical E-SCREEN data. Concentration-response relationships for all six chemicals were recorded and utilized to calculate predictions of their joint effects by employing the model of concentration addition. Surprisingly, the observed combination effects of the mixture fell short of the additivity expectations, indicating weak antagonism. Experimental or prediction errors were ruled out as possible explanations for this deviation, which suggested that it might be the result of interactions between mixture components. With the aim of identifying the responsible components, mixtures were designed by excluding one or more of the chemicals from the original six-component mixture, and the resulting combination effects were assessed. These permutation studies allowed us to conclude thatthe presence of 4-nonylphenol and 4-tert-octylphenol is associated with the antagonisms observed with the six-component mixture and thus negatively affected the predictability of mixture effects. Future mixture studies utilizing the E-SCREEN with endocrine disrupters that also exhibit toxicity or growth-inhibitory effects will have to take account of the possibility that such interactions might compromise the predictability of estrogenic combination effects.

Long-term exposure to steroidal estrogens is a key factor contributing to increases in the risk of developing breast cancer. Proposed mechanisms include receptor-activated increases in the rate of cell proliferation leading to the accumulation of genetic damage resulting from reading errors, and the production of DNA damage by species arising from metabolism of 17beta-estradiol (E2) resulting in mutations. In support of the second mechanism, catechol metabolites of E2 induce DNA damage in vitro. In the present study, utilizing the single-cell gel electrophoresis (Comet) assay, we observed increases in the number of single-strand breaks in estrogen receptor alpha-positive (MCF-7) and -negative (MDA-MB-231) breast cancer cells exposed to E2 (for 24 hr) or 4-hydroxy-17beta-estradiol (4-OH-E2; for 2 hr). The concentrations of 4-OH-E2 sufficient to induce these effects were approximately 100 nM, substantially lower than reported previously. The catechol 2-hydroxy-17beta-estradiol (2-OH-E2) also induced strand breaks. 2-OH-E2, often referred to as an improbable carcinogen in humans, is not a major metabolite of E2 in the breast; however, our findings show that it is as DNA-damaging as 4-OH-E2. Formamidopyrimidine glycosylase posttreatment of E2-, 4-OH-E2-, and 2-OH-E2-exposed MCF-7 cells led to an up to sixfold increase in mean tail moment, suggesting that oxidative DNA damage was formed. Comet formation could be partially attenuated by coincubation with dimethylsulfoxide, attributing a small DNA-damaging role to oxyradicals emanating from catechol redox cycling. Similar findings were obtained with MDA-MB-231 cells, indicating that estrogen receptor status is not relevant to these effects. Our observations show that exposure to E2 adds to the oxidative load of cells, and this may contribute to genomic instability.

In the last few years, significant advances have been made toward understanding the joint action of endocrine disrupting chemicals (EDCs). A number of studies have demonstrated that the combined effects of different types of EDCs (e.g., estrogenic, antiandrogenic, or thyroid-disrupting agents) can be predicted by the model of concentration addition (CA). However, there is still limited information on the effects of mixtures of large numbers of chemicals with varied structural features, which are more representative of realistic human exposure scenarios. The work presented here aims at filling this gap. Using a breast cancer cell proliferation assay (E-Screen), we assessed the joint effects of five mixtures, containing between 3 and 16 estrogenic agents, including compounds as diverse as steroidal hormones (endogenous and synthetic), pesticides, cosmetic additives, and phytoestrogens. CA was employed to predict mixture effects, which were then compared with experimental outcomes. The effects of two of the mixtures tested were additive, being accurately predicted by CA. However, for the three other mixtures, CA slightly overestimated the experimental observations. In view of these results, we hypothesized that the deviations were due to increased metabolism of steroidal estrogens in the mixture setting. We investigated this by testing the impact of two such mixtures on the activation and expression of steroidal estrogen metabolizing enzymes, such as cytochrome P450 (CYP) 1A1, CYP 1B1, and CYP 3A4. Activation of CYP 1B1 and, consequently, a reduction in the levels of steroidal estrogens in the mixture could contribute to the shortfall from the additivity prediction that we observed.