Analysis of the Ah Receptor Signal Transduction Pathway (Michael S. Denison, Jane M. Rohers, S. Renee Rushing. Carol L. Jones, Selwyna C. Tetangico, and Sharon Heath-Pagliuso, University of California, Davis, California).The protocols in this unit will allow researchers to detect the Ah receptor and characterize its functional activities (i.e., ligand binding, transformation and DNA binding, and gene expression) in their biological test system and to use these methods to detect chemical and biochemical events that affect this signaling system.

This paper reports the synthesis of haptens and the generation and preliminary evaluation of polyclonal antibodies for the detection of dioxins such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) by ELISA (enzyme-linked immunosorbent assay). These novel haptens contain unsaturation between the halogenated dibenzo-p-dioxin ring system and the protein to which it is conjugated, presenting a rigid handle structure. The substitution pattern is identical with or similar to that of TCDD (i.e., 2,3,7,8- or 1,2,3,7,8-). Finally, the haptens lack polar groups for hydrogen bonding. In direct binding assays using the new polyclonal antibodies there was excellent recognition of hapten−protein conjugates, including recognition of those hapten conjugates that were not used as immunogens (i.e., assay systems heterologous in hapten structure). These haptens do elicit selective immune responses in rabbits. Their evaluation in an ELISA format demonstrated the usefulness of these haptens for the detection of dioxins. An IC50 of 0.8 ng/well (16 ng/mL) was observed for an unoptimized system that used 2,3,7-trichloro-8-methyldibenzo-p-dioxin as an analytical surrogate standard. Keywords: TCDD; dioxin; immunoassay; polyclonal antibodies; hapten synthesis; polychlorinated hydrocarbons

We have used human ovarian carcinoma BG-1 cells to determine which steps in the pathway of estrogen signaling are disrupted by the aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We report that inhibition of estrogen signaling occurs between 7 and 18 h after TCDD treatment and that this effect is not caused by a decrease in estradiol concentration. TCDD decreased estrogen receptor (ER) levels in cells grown in standard medium; however, in estrogen-stripped medium, ER (but not AhR) levels were dramatically reduced (ϳ7-fold) but were not decreased further by TCDD. Because the absolute level of estradiol inducibility and inhibition by TCDD was similar in either medium, decreases in ER are not responsible for the antiestrogenic effect. The AhR also did not bind to the estrogen-responsive element (ERE) in vitro, and ERE binding by nuclear ER complexes was not decreased by TCDD, indicating that the effect of TCDD does not involve direct competition between the AhR and ER for DNA binding. However, inhibition of protein synthesis by cycloheximide blocked the TCDD-induced inhibition of ER-dependent gene expression. Overall, our results are consistent with the action of a TCDD-induced protein at a step(s) after ER-DNA binding, most likely at the level of gene transcription.

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