Itro using gel retardation analysis (Figure 1A, B). All of the DMSO 4EGI-1 custom synthesis extracts stimulated high amounts AhR:DNA complex formation, and most ethanol extracts were also active (particularly those of newspaper and all rubber products tested), stimulating AhR DNA binding to 60?0 of that of a maximally activating concentration of TCDD. It should be noted that similar materials from other suppliers/manufacturers have also been examined in this assay with comparable results (data not shown), 1655472 indicating that the AhR 25033180 agonist activity of extracts of these materials is not specific to a single supplier. We previously detected AhR agonists in ethanol and DMSO extracts of newspaper ink and newspapers from throughout the world [25], although attempts to identify the responsible chemicals have not yet been successful (data not shown). While AhR agonists are typically very hydrophobic, the high degree of AhR transformation and DNA binding observed after incubation of hepatic cytosol with the water extract of printed newspaper and to a lesser extent by that of the red rubber band (Figure 1A, B) indicate the existence of novel water soluble AhR agonists. Taken together, the presence of AhR agonist activity in DMSO, ethanol and water extracts suggests the existence of AhR agonists with a variety of physicochemical characteristics in the tested commercial and consumer product extracts. Since the ability of a compound or extract to induce AhR transformation in vitro does not always correlate with its ability toactivate the AhR signal transduction pathway [12], we next examined the ability of these extracts to induce gene expression in a guinea pig adenocarcinoma cell line containing a stably transfected Licochalcone-A biological activity AhR-responsive luciferase reporter gene [15] (Figure 1C). The ability of the extracts to induce AhR-dependent gene expression in these cells compared well with their ability to stimulate guinea pig AhR transformation and DNA binding in vitro (compare Figures 1B and 1C). Interestingly, while the ethanol extracts were either equipotent to or less potent than the DMSO extracts in the DNA binding assays (Figure 1B), luciferase reporter gene induction by ethanol extracts was greater than that of DMSO extracts of the same material (Figure 1C) and suggests that ethanol extracts a different subset of AhR agonists from the materials that have a greater affinity for the AhR and/or produces a more efficacious induction response. Interestingly, the magnitude of reporter gene induction by the ethanol extracts of newspaper (sample 1) and rubber products (samples 5?) was also considerably greater than that obtained with a maximally inducing concentration of TCDD. “Superinduction” of AhR-dependent gene expression by selected chemicals and crude extracts of environmental samples has been previously reported by several laboratories. While the exact molecular mechanisms responsible for the effect have not been elucidated, it has been attributed previously to cross-talk between the AhR and components of cell signaling (i.e. protein kinase C) and protein degradation pathways [26?9]. Similar to the DNA binding assay results, these analyses also revealed that water extracts of newspaper and select rubber products (cell scraper and black stopper, samples 5 and 8, respectively) contain polar AhR agonists that can activate AhRdependent gene expression in intact cells. Examination of the ability of DMSO and ETOH extracts to compete directly with [3H]TCDD for binding to the guinea pig.Itro using gel retardation analysis (Figure 1A, B). All of the DMSO extracts stimulated high amounts AhR:DNA complex formation, and most ethanol extracts were also active (particularly those of newspaper and all rubber products tested), stimulating AhR DNA binding to 60?0 of that of a maximally activating concentration of TCDD. It should be noted that similar materials from other suppliers/manufacturers have also been examined in this assay with comparable results (data not shown), 1655472 indicating that the AhR 25033180 agonist activity of extracts of these materials is not specific to a single supplier. We previously detected AhR agonists in ethanol and DMSO extracts of newspaper ink and newspapers from throughout the world [25], although attempts to identify the responsible chemicals have not yet been successful (data not shown). While AhR agonists are typically very hydrophobic, the high degree of AhR transformation and DNA binding observed after incubation of hepatic cytosol with the water extract of printed newspaper and to a lesser extent by that of the red rubber band (Figure 1A, B) indicate the existence of novel water soluble AhR agonists. Taken together, the presence of AhR agonist activity in DMSO, ethanol and water extracts suggests the existence of AhR agonists with a variety of physicochemical characteristics in the tested commercial and consumer product extracts. Since the ability of a compound or extract to induce AhR transformation in vitro does not always correlate with its ability toactivate the AhR signal transduction pathway [12], we next examined the ability of these extracts to induce gene expression in a guinea pig adenocarcinoma cell line containing a stably transfected AhR-responsive luciferase reporter gene [15] (Figure 1C). The ability of the extracts to induce AhR-dependent gene expression in these cells compared well with their ability to stimulate guinea pig AhR transformation and DNA binding in vitro (compare Figures 1B and 1C). Interestingly, while the ethanol extracts were either equipotent to or less potent than the DMSO extracts in the DNA binding assays (Figure 1B), luciferase reporter gene induction by ethanol extracts was greater than that of DMSO extracts of the same material (Figure 1C) and suggests that ethanol extracts a different subset of AhR agonists from the materials that have a greater affinity for the AhR and/or produces a more efficacious induction response. Interestingly, the magnitude of reporter gene induction by the ethanol extracts of newspaper (sample 1) and rubber products (samples 5?) was also considerably greater than that obtained with a maximally inducing concentration of TCDD. “Superinduction” of AhR-dependent gene expression by selected chemicals and crude extracts of environmental samples has been previously reported by several laboratories. While the exact molecular mechanisms responsible for the effect have not been elucidated, it has been attributed previously to cross-talk between the AhR and components of cell signaling (i.e. protein kinase C) and protein degradation pathways [26?9]. Similar to the DNA binding assay results, these analyses also revealed that water extracts of newspaper and select rubber products (cell scraper and black stopper, samples 5 and 8, respectively) contain polar AhR agonists that can activate AhRdependent gene expression in intact cells. Examination of the ability of DMSO and ETOH extracts to compete directly with [3H]TCDD for binding to the guinea pig.
