Interactions between estrogenic chemicals in binary mixtures investigated using vitellogenin induction and factorial analysis

Considerable progress has been made in assessing the combined effects of chemicals, but the effect of mixtures remains one of the most daunting challenges in environmental toxicology. In this study, the effects of binary mixtures of estrogenic chemicals were investigated using plasma vitellogenin (VTG) induction in male adult Japanese medaka (Oryzias latipes) as the endpoint. We focused on whether or not the factorial design using rigorous statistical methods was appropriate for the identification of possible interactions. The estrogenic activities of 17β-estradiol (E2), 4-tert-nonylphenol (NP) and bisphenol A (BPA) were investigated in medaka following 14 d of exposure. Although all chemicals induced concentration-dependent increases in plasma VTG, a complete concentration–response curve was obtained only for E2, while partial curves were obtained for NP and BPA. Therefore, a 3×3 factorial design was employed to identify both the individual and interactive effects. A significant difference in mean plasma VTG induction was found when any chemical was considered alone. However, no significant interactions between E2 and NP, E2 and BPA, and NP and BPA were demonstrated. These results suggest that these three model estrogenic chemicals share a common mechanism for inducing VTG synthesis, and that no interactions occur when they act in combination. We also demonstrated the ability of this experimental design to detect interactions between binary mixtures, which will allow the assessment of biological effects of more complex mixtures in future research.