Ults are representative of three individual experiments. (B) The amount of ligandactivated protein-DNA complex formation from gel retardation experiments from part A was determined by phosphorimager analysis. Values are expressed as the percentage of maximal TCDD induction and represent the mean 6 SD of all DNA binding data compiled from duplicate gels from three individual experiments. Values significantly different from solvent alone (p#0.05 as determined by the students T-test) are indicated by an asterisk. (C) Induction of luciferase activity by individual extract in recombinant guinea pig (G16L1.1c8) cells. Cells were incubated with the indicated extract (10 ml/ml) for 4 h and luciferase activity determined as described in Materials and Methods. Values are expressed as a percentage of the maximal induction by TCDD and represent the mean 6 SD of triplicate determinations and those values significantly different from solvent alone (p#0.05 as determined by the students T-test) are indicated by an asterisk. The results shown are representative of three individual experiments. (D) Competitive binding between [3H]TCDD and the indicated DMSO or 11967625 ethanol extract for the guinea pig hepatic cytosolic AhR. Guinea pig cytosol was incubated with 2 nM [3H]TCDD in the absence or presence of the indicated extract for 2 h at 20uC, and specific binding of [3H]TCDD was determined by hydroxyapatite binding. Values represent the mean 6 SD of triplicate determinations and are representative of three individual experiments and those values significantly different from solvent alone (p#0.05 as determined by the students T-test) are indicated by an asterisk. 
doi:10.1371/journal.pone.0056860.gnewspaper, or yellow-pad paper for 3.5 hr resulted in increased CYP1A1 mRNA levels (Figure 2), and these results were inhibitor directly comparable with the ability of each extract to induce AhRdependent luciferase activity (Figure 1C). Finally, although these extracts could Epigenetics stimulate AhR-dependent luciferase gene induction in recombinant mouse, rat and human hepatoma cells (Figure 3), significant species differences in response to different extracts were also observed. Species-specific differences in ligand-selectivity of the AhR have been previously observed [3,30?2] and likely result from differences in the specificity and affinity of binding of ligands as well as from variations in the metabolic activity of each cell line. Taken together, our results indicate that chemicals present in these extracts can bind to the AhR, stimulate AhR DNA binding and consequently induce gene expression in vitro and incontinuous cell lines in culture. The physiological, biochemical and/or toxicological significance of these results would be strengthened by demonstrating their ability to activate the AhR in tissues and animals in vivo. Accordingly, we examined the ability of two of the most potent samples (DMSO extracts of newspaper and rubber stopper) to stimulate AhR-dependent CYP1A1 expression in normal human skin and in zebrafish in vivo. When fresh human foreskin was exposed in organ culture to TCDD or to DMSO extracts of newspaper or rubber stopper, real-time PCR revealed the ability of these extracts to increase CYP1A1 mRNA levels (Figure 4A), with the relative increase in mRNA comparable to their luciferase induction potency (Figure 1). These results demonstrate that the more potent DMSO extracts can increase expression of the endogenous AhR-responsiveCommercial/Consumer Products Contain.Ults are representative of three individual experiments. (B) The amount of ligandactivated protein-DNA complex formation from gel retardation experiments from part A was determined by phosphorimager analysis. Values are expressed as the percentage of maximal TCDD induction and represent the mean 6 SD of all DNA binding data compiled from duplicate gels from three individual experiments. Values significantly different from solvent alone (p#0.05 as determined by the students T-test) are indicated by an asterisk. (C) Induction of luciferase activity by individual extract in recombinant guinea pig (G16L1.1c8) cells. Cells were incubated with the indicated extract (10 ml/ml) for 4 h and luciferase activity determined as described in Materials and Methods. Values are expressed as a percentage of the maximal induction by TCDD and represent the mean 6 SD of triplicate determinations and those values significantly different from solvent alone (p#0.05 as determined by the students T-test) are indicated by an asterisk. The results shown are representative of three individual experiments. (D) Competitive binding between [3H]TCDD and the indicated DMSO or 11967625 ethanol extract for the guinea pig hepatic cytosolic AhR. Guinea pig cytosol was incubated with 2 nM [3H]TCDD in the absence or presence of the indicated extract for 2 h at 20uC, and specific binding of [3H]TCDD was determined by hydroxyapatite binding. Values represent the mean 6 SD of triplicate determinations and are representative of three individual experiments and those values 
significantly different from solvent alone (p#0.05 as determined by the students T-test) are indicated by an asterisk. doi:10.1371/journal.pone.0056860.gnewspaper, or yellow-pad paper for 3.5 hr resulted in increased CYP1A1 mRNA levels (Figure 2), and these results were directly comparable with the ability of each extract to induce AhRdependent luciferase activity (Figure 1C). Finally, although these extracts could stimulate AhR-dependent luciferase gene induction in recombinant mouse, rat and human hepatoma cells (Figure 3), significant species differences in response to different extracts were also observed. Species-specific differences in ligand-selectivity of the AhR have been previously observed [3,30?2] and likely result from differences in the specificity and affinity of binding of ligands as well as from variations in the metabolic activity of each cell line. Taken together, our results indicate that chemicals present in these extracts can bind to the AhR, stimulate AhR DNA binding and consequently induce gene expression in vitro and incontinuous cell lines in culture. The physiological, biochemical and/or toxicological significance of these results would be strengthened by demonstrating their ability to activate the AhR in tissues and animals in vivo. Accordingly, we examined the ability of two of the most potent samples (DMSO extracts of newspaper and rubber stopper) to stimulate AhR-dependent CYP1A1 expression in normal human skin and in zebrafish in vivo. When fresh human foreskin was exposed in organ culture to TCDD or to DMSO extracts of newspaper or rubber stopper, real-time PCR revealed the ability of these extracts to increase CYP1A1 mRNA levels (Figure 4A), with the relative increase in mRNA comparable to their luciferase induction potency (Figure 1). These results demonstrate that the more potent DMSO extracts can increase expression of the endogenous AhR-responsiveCommercial/Consumer Products Contain.
