Deviation from additivity with estrogenic mixtures containing 4-nonylphenol and 4-tert-octylphenol detected in the E-SCREEN assay

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 that the 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.