Igure S1) revealed that the majority of these extracts contained chemicals

Igure S1) revealed that the majority of these extracts contained chemicals that could stimulate transformation and DNA binding of guinea pig hepatic cytosolic AhR. In order to more fully characterize the AhR activity of chemicals contained in extracts of commercial and consumer products, we focused our analysis on extracts of a smaller subset of these materials, namely paper inhibitor products (newspaper, business cards, blue paper towel, yellow writing paper) and rubber products (cell scraper, black o-ring, black stopper and red rubber band). To characterize the AhR agonists contained in these materials, we extracted paper and rubber products with various solvents (DMSO, 12926553 ETOH, water) and tested the ability of the extracts to stimulate AhR transformation and DNA binding in vitro 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 inhibitor 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), indicating that the AhR 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 23727046 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.Igure S1) revealed that the majority of these extracts contained chemicals that could stimulate transformation and DNA binding of guinea pig hepatic cytosolic AhR. In order to more fully characterize the AhR activity of chemicals contained in extracts of commercial and consumer products, we focused our analysis on extracts of a smaller subset of these materials, namely paper products (newspaper, business cards, blue paper towel, yellow writing paper) and rubber products (cell scraper, black o-ring, black stopper and red rubber band). To characterize the AhR agonists contained in these materials, we extracted paper and rubber products with various solvents (DMSO, 12926553 ETOH, water) and tested the ability of the extracts to stimulate AhR transformation and DNA binding in vitro 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), indicating that the AhR 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 23727046 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.