Deviation from Additivity with Estrogenic Mixtures Containing 4-Nonylphenol and 4-tert-Octylphenol Detected in the E-SCREEN Assay

Abstract

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.

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase ( cyp19a1b ) in zebrafish specific in vitro and in vivo bioassays

“…Results from these studies demonstrate that binary or multi-component mixtures of ER agonists generally act in an additive manner on the expression of ER-regulated genes in both brain and liver (Thorpe et al 2001, Rajapakse et al 2004, Lin and Janz 2006, Kortenkamp 2007, Petersen and Tollefsen 2011, Petersen et al 2013. In some cases, some deviations from additivity can be observed, especially with infra-additive effects, both in in vitro and in vivo experiments (Rajapakse et al 2004, Lin and Janz 2006, Petersen and Tollefsen 2011, Petersen et al 2013).…”

“…In some cases, some deviations from additivity can be observed, especially with infra-additive effects, both in in vitro and in vivo experiments (Rajapakse et al 2004, Lin and Janz 2006, Petersen and Tollefsen 2011, Petersen et al 2013). …”

Complex contaminant exposure in cetaceans: A comparative E‐Screen analysis of bottlenose dolphin blubber and mixtures of four persistent organic pollutants

“…Tested individually at their predetermined LOECs, PCBs 138 and 180 were not significantly antiestrogenic, which may be attributed to the high intraexperimental variability observed in E-Screen assay [26]. However, when combined the binary mixture of PCB 138 and 180 induced a significant antiestrogenic response.…”

“…The NPE value describes the efficacy of the test mixture relative to E 2 and reduces interexperimental variability [26].…”

Combined effects of estrogenic chemicals with the same mode of action using an estrogen receptor binding bioassay

“…Although the paper was retracted the following year because the result was lack of reproducibility in other laboratories (McLachlan, 1997), it led to academic concern and intense follow-up research. There have been lots of researches to study the effects of estrogenic mixtures on organisms ranging from recombinant yeasts and estrogen-sensitive cells (Arnold et al, 1997;Payne et al, 2001;Rajapakse et al, 2004;Silva et al, 2011Silva et al, , 2002Suzuki et al, 2001) to vertebrates such as fish and mice (Brian et al, 2007;Ramamoorthy et al, 1997;Ribeiro et al, 2012;Sun et al, 2009;Thorpe et al, 2005;Tinwell and Ashby, 2004). Among these test organisms, the recombinant human ER gene yeast obtained wide application because it provided not only fast and sensitive detection but also single MoA and ToA.…”