Tributyltin synergizes with 20-hydroxyecdysone to produce endocrine toxicity

One of the great challenges facing modern toxicology is in predicting the hazard associated with chemical mixtures. The development of effective means of predicting the toxicity of chemical mixtures requires an understanding of how chemicals interact to produce nonadditive outcomes (e.g., synergy). We hypothesized that tributyltin would elicit toxicity in daphnids (Daphnia magna) by exaggerating physiological responses to 20-hydroxyecdysone signaling via synergistic activation of the retinoid X receptor (RXR):ecdysteroid receptor (EcR) complex. Using reporter gene assays, we demonstrated that RXR, alone, is activated by a variety of ligands including tributyltin, whereas RXR:EcR heterodimers were not activated by tributyltin. However, tributyltin, in combination with the daphnid EcR ligand 20-hydroxyecdysone, caused concentration-dependent, synergistic activation of the RXR:EcR reporter. Electrophoretic mobility shift assays revealed that tributyltin did not enhance the activity of 20-hydroxyecdysone by increasing binding of the receptor complex to a DR-4 DNA-binding site. Exposure of daphnids to elevated concentrations of 20-hydroxyecdysone caused premature and incomplete ecdysis resulting in death. Tributyltin exaggerated this effect of exogenous 20-hydroxyecdysone. Further, exposure of daphnids to tributyltin enhanced the inductive effects of 20-hydroxyecdysone on expression of the 20-hydroxyecdysone-inducible gene HR3. Continuous, prolonged exposure of maternal daphnids to concentrations of tributyltin resulted in mortality concurrent with molting. Taken together, these results demonstrate that xenobiotics, such as tributyltin, can interact with RXR to influence gene expression regulated by the heterodimeric partner to RXR. The result of such interactions can be toxicity due to inappropriate or exaggerated hormonal signaling. The application of the in vitro/in vivo approach used in this study is discussed in relation to modeling of nonadditive interactions among constituents of chemical mixtures.