By selected chemicals and crude extracts of environmental Homotaurine 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 hepatic cytosolic AhR revealed that all of the extracts, except for the DMSO extracts of paper products (i.e., yellow pad, blue paper towel and business card), could competitively bind to the AhR and are thus full agonists (Figure 1D). Little or no competitive binding was observed with the water extracts (data not shown). The ability of the DMSO and water extracts of paper products to directly stimulate AhR transformation and DNA binding as well as AhRdependent luciferase induction, but to show little or no competitive ligand binding activity, suggests that they have relatively low 1454585-06-8 affinity for the AhR and thus are not able to compete effectively with the high affinity ligand [3H]TCDD. We previously observed this phenomenon with other weak AhR agonists [6,8,11]. The induction response was also characterized with respect to incubation time, effect on endogenous CYP1A1 and effectiveness in several species. First, mouse hepatoma CALUX cell luciferase induction response was compared at 4 hours versus 24 hours of incubation (Figure S2). The lower luciferase activity evident at the later time point is consistent with the AhR agonists present in the extracts as being metabolically labile. Additionally, since the AhR agonist activity/potency of our DMSO extracts was not reduced if the vials containing the extracts were left open for a day, the reduction in gene induction over time was unlikely to be due to evaporative loss of the AhR agonists during incubation (data not shown). In contrast, we observed little or no loss of luciferase induction potency of these extracts when they were stored at room temperature in the dark for up to one year (data not shown), indicating that these agonists are chemically stable. Second, the ability of DMSO and ETOH extracts to stimulate expression 1516647 of an endogenous AhR-responsive gene (CYP1A1) was confirmed by demonstrating an increase in mRNA levels in mouse hepatoma (hepa1c1c7) cells using RT-PCR. Incubation of cells with DMSO or ETOH extracts (1:100 (v/v) dilution) of rubber products,Commercial/Consumer Products Contain AhR AgonistsFigure 1. Activation of the AhR and AhR-dependent signal transduction pathway by DMSO, ETOH and water extracts of commercial and consumer products. The products used in these studies were (1) newspaper (black print section only); (2) business card; (3) blue paper towel; (4) yellow pad; (5) cell scraper; (6) black rubber O-ring; (7) black rubber stopper; (8) red rubber-band. (A) Stimulation of AhR transformation and DNA binding by extracts of the 
indicated commercial and consumer products in vitro. The arrow indicates the position of the ligand-activated proteinDNA (AhR:ARNT:DRE) complex in the gel retardation assay and res.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 hepatic cytosolic AhR revealed that all of the extracts, except for the DMSO extracts of paper products (i.e., yellow pad, blue paper towel and business card), could competitively bind to the AhR and are thus full agonists (Figure 1D). Little or no competitive binding was observed with the water extracts (data not shown). The ability of the DMSO and water extracts of paper products to directly stimulate AhR transformation and DNA binding as well as AhRdependent luciferase induction, but to show little or no competitive ligand binding activity, suggests that they have relatively low affinity for the AhR and thus are not able to compete effectively with the high affinity ligand [3H]TCDD. We previously observed this phenomenon with other weak AhR agonists [6,8,11]. The induction response was also characterized with respect to incubation time, effect on endogenous CYP1A1 and effectiveness in several species. First, mouse hepatoma CALUX cell luciferase induction response was compared at 4 hours versus 24 hours of incubation (Figure S2). The lower luciferase activity evident at the later time point is consistent with the AhR agonists present in the extracts as being metabolically labile. Additionally, since the AhR agonist activity/potency of our DMSO extracts was not reduced if the vials containing the extracts were left open for 
a day, the reduction in gene induction over time was unlikely to be due to evaporative loss of the AhR agonists during incubation (data not shown). In contrast, we observed little or no loss of luciferase induction potency of these extracts when they were stored at room temperature in the dark for up to one year (data not shown), indicating that these agonists are chemically stable. Second, the ability of DMSO and ETOH extracts to stimulate expression 1516647 of an endogenous AhR-responsive gene (CYP1A1) was confirmed by demonstrating an increase in mRNA levels in mouse hepatoma (hepa1c1c7) cells using RT-PCR. Incubation of cells with DMSO or ETOH extracts (1:100 (v/v) dilution) of rubber products,Commercial/Consumer Products Contain AhR AgonistsFigure 1. Activation of the AhR and AhR-dependent signal transduction pathway by DMSO, ETOH and water extracts of commercial and consumer products. The products used in these studies were (1) newspaper (black print section only); (2) business card; (3) blue paper towel; (4) yellow pad; (5) cell scraper; (6) black rubber O-ring; (7) black rubber stopper; (8) red rubber-band. (A) Stimulation of AhR transformation and DNA binding by extracts of the indicated commercial and consumer products in vitro. The arrow indicates the position of the ligand-activated proteinDNA (AhR:ARNT:DRE) complex in the gel retardation assay and res.