Arachidonate Products and CYP1A in Dioxin Toxicity

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DESCRIPTION (provided by applicant): Binding and activation of the Ah receptor (AhR) is required for the environmental toxin 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD, dioxin) to produce its toxic effects. TCDD toxicity includes a lethal wasting syndrome and cardiovascular dysfunction, but it is not known how TCDD activation of the AhR produces those effects. This research seeks to understand how TCDD activation of the AhR leads to suppression of gluconeogenesis, energy failure and death and to identify contributions of cytochrome P450 (CYP) 1A enzymes, the major transcriptional products of AhR activation, to the toxicity. The following hypothesis will be tested: (1) Transcriptional and posttranslational effects of TCDD on signaling pathways converging on PGC1?, i.e. Akt, AMPK, PKA and Sirt1, contribute to suppression of gluconeogensis by TCDD. PGC1? governs transcription of PEPCK and glucose 6-phosphatase, regulators of flux through the gluconeogenic pathway; (2) Suppression of gluconeogenesis forces reliance on lipids for energy but also limits availability of lipids as a fuel source; (3) Effects of TCDD on CYP-dependent metabolism of the membrane lipid arachidonic acid (aa) contribute to energy failure in liver and heart. The research will follow up on discoveries under prior funding periods of the grant that TCDD causes cardiac contractile dysfunction and increases hepatic formation of CYP-dependent aa epoxides, EETs, and decreases formation of 20-HETE, aa products with major physiologic cardiovascular regulatory effects, to examine their involvement in nutrient metabolism. TCDD will be used as a tool to learn how AhR activation and changes in CYP-dependent aa metabolism can lead to physiologic disturbances in glucose and lipid metabolism involved in the wasting syndrome (specific aim (SA) 1) and in ion channel and cardiac muscle function (SA2). Studies in SA2 will also determine whether increased production of EETs in liver affect the heart and are cardioprotective or cardiotoxic. The chick embryo close to hatching will continue to be used as the major model based on its track record in studying TCDD toxicity, its similarity to humans with respect to aa metabolism and its special utility for this research in permitting hepatic metabolic effects to be studied independently of confounding effects of food intake. Findings will be confirmed in mammalian cells. Definitive evidence for or against a role of CYP1A in AhR effects will be sought by silencing or overexpressing CYP1A4 and CYP1A5 in chick embryo hepatocytes and by a novel molecular approach in which sense and antisense CYP1A gene constructs in retroviral vectors will be targeted to liver or heart in chick embryos at early stages of development and the effects examined at a later stages. This research is expected to show that CYP1A enzymes contribute to metabolic and cardiovascular regulatory pathways, to improve understanding about relationships between glucose and lipid metabolism and the role of the AhR in regulating physiologic and pathologic responses to changes in nutrient availability, and to have implications for common related diseases, cardiovascular disease and diabetes. Relevance: Responses to changes in nutrient supply and metabolism contribute to aging and common human diseases including diabetes, cardiovascular disease and cancer. The environmental toxin, TCDD, by the single action of binding to the aryl hydrocarbon receptor (AhR), which is present in all our cells, initiates a lethal wasting syndrome characterized by a failure to synthesize glucose and leading to energy failure and death. By learning how TCDD activation of the AhR produces massive dysregulation of nutrient responses we expect to learn more about how the body normally orchestrates responses to changing levels of nutrients and how normal regulatory processes can spin out of control, with implications for common human diseases as well as normal physiology.