Synergistic and toxic cocktail effects of low dose endocrine disruptors – TOXSYN

Most current knowledge of endocrine disrupting chemicals (EDC) action is derived from data sets that use single molecule exposure, with few studies taking into account the more realistic situation where humans and wildlife are simultaneously and chronically exposed to low doses of multiple EDCs. The TOXSYN consortium will address the question of synergistic toxic effects and analyse how low dose combinations of EDCs affect physiology and homeostasis. We focus on two nuclear receptors directly implicated in xenobiotic responses: the pregnane X receptor (PXR) and peroxisome proliferator activated receptor gamma (PPARg), that both heterodimerize with the Retinoid X receptors (RXRs). The focus on these nuclear receptors is determined by the key observation that both have sufficiently large ligand-binding domains to accommodate two ligands simultaneously. We hypothesise that each signalling pathway is a front line candidate for activation by cocktails of endocrine disrupting compounds. A number of preliminary results support our hypothesis including:
• X ray crystallography on PPAR? ligand binding domain that shows simultaneous binding of two molecules of mono (2-ethylhexyl) phthalate (MEHP),
• in vitro tests that show synergistic activation effects of MEHP and perfluorooctanoic acid (PFOA) on human PPARg dependent-transcriptional activation.
These demonstrations of synergistic effects, lead TOXSYN to investigate the molecular basis and physiological consequences of combinatorial effects of low doses of emerging endocrine disruptors through PPAR?/RXR and on PXR/RXR signalling. Besides addressing PPAR for which we have strong preliminary data, we are extending our analysis to PXR signalling for two main reasons. First, like PPARg the ligand-binding pocket of PXR is large enough to accommodate simultaneously two molecules and second, PXR is one of the principal players in mediating the mammalian xenobiotic response. Notably, PXR plays a critical role in the regulation of phase I (CYP), phase II (conjugating), and phase III (ABC family transporters) detoxifying enzymes, co-ordinately regulating steroid, drug, and xenobiotic clearance in the liver and intestine.
TOXSYN brings together a highly qualified partnership, from the academic and biotech sectors, with state of the art expertise ranging from molecular and biochemical conformation assays to alternative testing models using cells and small model organisms (SMOs).
Our overall aim is to exploit our complementary leading-edge technologies to set-up a panel of structural tests and novel cell-based in vitro and in vivo, embryo-based assays for studying effects of low dose mixtures of EDCs. These mechanism-based assays will furnish extensive data sets on how low dose mixtures can interact with PPARg and PXR receptors at the molecular and biophysical levels (X-ray crystallography, binding affinity assays), cellular levels (high-throughput mammalian and non-mammalian in vitro assays) and whole organism levels (embryo-based SMOs, Zebrafish and the amphibian Xenopus leavis).
TOXSYN will:
• Identify EDC mixtures that exert synergistic or additive effects on PPARg-RXR activity
• Provide a detailed mechanistic view of these affects through structural studies
• Validate the physiological relevance of these mechanisms in vivo using reporter assays and tests of adipogenesis
• Determine whether these synergistic mechanisms also apply to PXR signalling
• Compare the data for each receptor and placing the results in their physiological and environmental contexts
Impact: Results from TOXSYN will improve current screening tests and strategies for understanding EDCs action. They will provide data on the molecular mechanisms underlying the physiological consequences of exposure to low-dose effects of mixtures. Their dissemination will help fill current knowledge gaps for regulators concerned with public health and environmental issues and drive new hypotheses for further research on combinatorial effects.