The effects of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) are similar to vitamin A abnormalities and TCDD induces loss of hepatic vitamin A esters that ultimately could produce frank vitamin A deficiency. It is unknown, however, whether a mechanism of TCDD toxicity is through interfering with the function of vitamin A, or whether TCDD merely interferes with vitamin A storage and has little direct affect on vitamin A function. The function of vitamin A would be disrupted by increasing or decreasing the steady-state concentration of retinoic acid and/or retinoic acid receptors (RAR, RXR), because retinoic acid is the vitamin A metabolite that directly supports most, if not all, known vitamin A-dependent processes. This work would test the hypothesis that TCDD causes functional vitamin A abnormalities by altering the steady- state concentrations of retinoic acid and/or RAR/RXR. Our preliminary data show that TCDD downregulates RAR-alpha mRNA, stimulates retinoic acid synthesis and depresses retinyl ester synthesis in the human hepatoma cell line HepG2. The first three specific aims are to determine the mechanisms in HepG2 cells of TCDD affects on: 1)RAR-alpha, RAR-beta and RXR-alpha gene expression; 2) RAR-alpha, RAR- beta and RXR-alpha protein levels and their affinities for retinoic acid; 3)the metabolism of retinoids, especially the reactions that would modulate specifically the steady-state concentrations of retinoic acid. The steady-state amounts, rates of transcription, and elimination t 1/2's of RAR/RXR mRNA will be determined by nuclear run-on transcription and Northern blot analyses. RAR/RXR concentrations and rates of syntheses and degradation will be obtained by immunoblotting. The affinities of RAR/RXR for retinoic acid and of the cellular retinol binding protein, CRBP, for retinol will be measured by competitive binding assays. HPLC assays will be used to study the conversions of retinol into retinoic acid and retinyl esters, and the rates of retinol and retinoic acid catabolism. Time-course and dose-response data will be generated and the ability of synthetic retinoids to prevent TCDD- mediated changes will be tested. A fourth specific aim is to measure the affects of TCDD on the steady-state concentrations of retinoic acid, RAR and RXR, the mRNA of RAR and RXR, and the specific activities of enzymes catalyzing retinoic acid synthesis and catabolism in male and female rat and male Syrian golden hamster tissues. The purposes are to compare these obligatory mediators of retinoid action in vivo among three groups of animals that respond differently to TCDD and to extend the results obtained with HepG2 cells to frequently-used animal models of TCDD effects.
