Ile tires [13], prompted the present investigation to determine how widely distributed

Ile tires [13], prompted the present investigation to determine how widely distributed AhRactive chemicals are in common commercial and DprE1-IN-2 site Consumer products (rubber, plastic, paper, etc.). Given the documented ability of the AhR to respond to a wide range of exogenous and endogenous chemicals, the present work not only contributes to our understanding of the diversity and widespread nature of AhRagonists, but identifies putative sources of AhR ligands that 18325633 can complicate experimental studies of AhR signal transduction.Materials and Methods Chemicals and extractionsTCDD and [3H]TCDD (37 Ci/mmol) were from S. Safe (Texas A M University, College Station, TX), 2,3,7,8-tetrachlorodibenzofuran (TCDF) from Accustandard (New Haven, CT), [32P]ATP (6000 Ci/mmol) from Amersham (Arlington Heights, IL) and DMSO from Aldrich (St. Louis, MO). Commercial and consumer products were obtained from local department stores and laboratory product 307538-42-7 suppliers. The sources of the materials examined in detail are as follows: newspaper (Davis Enterprise, Davis, CA), business card (Kinkos, Davis, CA), blue paper towel (Georgia-Pacific professional), yellow legal writing pad (Universal Office Products, Waterford, NY), FisherBrand rubber cell scraper (Walter Stern, Inc., Port Washington, NY), black 0-ring (Danco Co., Irving, TX), FisherBrand black rubber stopper (Plasticoid, Elkton, MD), red rubber band (OfficeMax, Davis, CA). The indicated commercial and consumer products were finely diced with scissors and extracted for 24 hr in Teflon-capped glass tubes containing dimethylsulfoxide (DMSO), ethanol (ETOH, 95 ), or Milli-Q water using 1.5 ml of solvent for each gram of sample withCommercial/Consumer Products Contain AhR Agoniststhe exception of the paper products which were extracted with 9 volumes of solvent per gram of sample due to absorption of the solvent by the paper. After centrifugation, supernatants (extracts) were transferred into Teflon-capped glass vials and stored in the dark until use.Preparation of cytosol and DNA and ligand binding analysisMale Hartley guinea pig (500 g, Charles River Laboratories) hepatic cytosol was prepared and used in gel retardation analysis experiments to measure DNA binding of in vitro transformed AhR complexes and in hydroxyapatite assays to measure competitive [3H]TCDD ligand binding analysis as described in detail [14]. For gel retardation analysis, cytosol (8 mg protein/ml) was incubated with DMSO (20 ml/ml, final concentration), 20 nM TCDD or the indicated extract (20 ml/ml) for 2 hr at 20uC and ligand-activated protein-DNA complexes (AhR:ARNT (AhR nuclear translocator):DRE (dioxin responsive element)) were resolved in nondenaturing PAGE gels and quantitated using a Molecular Dynamics Phosphorimager [14]. The amount of ligand-activated AhR:DRE complex formation was expressed relative to that produced by TCDD. For ligand binding, cytosol (2 mg protein/ ml) was incubated with 2 nM [3H]TCDD in the absence or presence of 200 nM TCDF, DMSO (10 ml/ml, final concentration) or the indicated extract (10 ml/ml) for 2 hours in a room temperature water bath. [3H]TCDD binding in aliquots of the incubation (200 mL) was determined by HAP binding as previously described [14]. The total amount of [3H]TCDD specific binding was obtained by subtracting the non-specific binding ([3H]TCDD and TCDF) from the total binding ([3H]TCDD). The ability of a chemical(s) in a sample extract to bind to the AhR was indicated by its ability to competitively r.Ile tires [13], prompted the present investigation to determine how widely distributed AhRactive chemicals are in common commercial and consumer products (rubber, plastic, paper, etc.). Given the documented ability of the AhR to respond to a wide range of exogenous and endogenous chemicals, the present work not only contributes to our understanding of the diversity and widespread nature of AhRagonists, but identifies putative sources of AhR ligands that 18325633 can complicate experimental studies of AhR signal transduction.Materials and Methods Chemicals and extractionsTCDD and [3H]TCDD (37 Ci/mmol) were from S. Safe (Texas A M University, College Station, TX), 2,3,7,8-tetrachlorodibenzofuran (TCDF) from Accustandard (New Haven, CT), [32P]ATP (6000 Ci/mmol) from Amersham (Arlington Heights, IL) and DMSO from Aldrich (St. Louis, MO). Commercial and consumer products were obtained from local department stores and laboratory product suppliers. The sources of the materials examined in detail are as follows: newspaper (Davis Enterprise, Davis, CA), business card (Kinkos, Davis, CA), blue paper towel (Georgia-Pacific professional), yellow legal writing pad (Universal Office Products, Waterford, NY), FisherBrand rubber cell scraper (Walter Stern, Inc., Port Washington, NY), black 0-ring (Danco Co., Irving, TX), FisherBrand black rubber stopper (Plasticoid, Elkton, MD), red rubber band (OfficeMax, Davis, CA). The indicated commercial and consumer products were finely diced with scissors and extracted for 24 hr in Teflon-capped glass tubes containing dimethylsulfoxide (DMSO), ethanol (ETOH, 95 ), or Milli-Q water using 1.5 ml of solvent for each gram of sample withCommercial/Consumer Products Contain AhR Agoniststhe exception of the paper products which were extracted with 9 volumes of solvent per gram of sample due to absorption of the solvent by the paper. After centrifugation, supernatants (extracts) were transferred into Teflon-capped glass vials and stored in the dark until use.Preparation of cytosol and DNA and ligand binding analysisMale Hartley guinea pig (500 g, Charles River Laboratories) hepatic cytosol was prepared and used in gel retardation analysis experiments to measure DNA binding of in vitro transformed AhR complexes and in hydroxyapatite assays to measure competitive [3H]TCDD ligand binding analysis as described in detail [14]. For gel retardation analysis, cytosol (8 mg protein/ml) was incubated with DMSO (20 ml/ml, final concentration), 20 nM TCDD or the indicated extract (20 ml/ml) for 2 hr at 20uC and ligand-activated protein-DNA complexes (AhR:ARNT (AhR nuclear translocator):DRE (dioxin responsive element)) were resolved in nondenaturing PAGE gels and quantitated using a Molecular Dynamics Phosphorimager [14]. The amount of ligand-activated AhR:DRE complex formation was expressed relative to that produced by TCDD. For ligand binding, cytosol (2 mg protein/ ml) was incubated with 2 nM [3H]TCDD in the absence or presence of 200 nM TCDF, DMSO (10 ml/ml, final concentration) or the indicated extract (10 ml/ml) for 2 hours in a room temperature water bath. [3H]TCDD binding in aliquots of the incubation (200 mL) was determined by HAP binding as previously described [14]. The total amount of [3H]TCDD specific binding was obtained by subtracting the non-specific binding ([3H]TCDD and TCDF) from the total binding ([3H]TCDD). The ability of a chemical(s) in a sample extract to bind to the AhR was indicated by its ability to competitively r.