Common commercial and consumer products contain activators of the aryl hydrocarbon (dioxin) receptor

Abstract

Activation of the Ah receptor (AhR) by halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), can produce a wide variety of toxic and biological effects. While recent studies have shown that the AhR can bind and be activated by structurally diverse chemicals, how widespread of these AhR agonists are in environmental, biological and synthetic materials remains to be determined. Using AhR-based assays, we demonstrate the presence of potent AhR agonists in a variety of common commercial and consumer items. Solvent extracts of paper, rubber and plastic products contain chemicals that can bind to and stimulate AhR DNA binding and/or AhR-dependent gene expression in hepatic cytosol, cultured cell lines, human epidermis and zebrafish embryos. In contrast to TCDD and other persistent dioxin-like HAHs, activation of AhR-dependent gene expression by these extracts was transient, suggesting that the agonists are metabolically labile. Solvent extracts of rubber products produce AhR-dependent developmental toxicity in zebrafish in vivo, and inhibition of expression of the metabolic enzyme CYP1A, significantly increased their toxic potency. Although the identity of the responsible AhR-active chemicals and their toxicological impact remain to be determined, our data demonstrate that AhR active chemicals are widely distributed in everyday products.

Figure 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 protein-DNA (AhR∶ARNT∶DRE) complex in the gel retardation assay and results are representative of three individual experiments. (B) The amount of ligand-activated 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 ± 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 µl/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 ± 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 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 20°C, and specific binding of [³H]TCDD was determined by hydroxyapatite binding. Values represent the mean ± 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.

Figure 2. Effect of extracts of commercial and consumer products on CYP1A1 mRNA levels in mouse hepatoma cells.

Hepa1c1c7 cells were incubated with DMSO (10 µl/ml), TCDD (1 nM in DMSO) or the indicated extract (20 µl/ml) for 3.5 hr at 37°C, mRNA was extracted, subjected to RT-PCR and the resulting products were visualized by agarose gel electrophoresis. PCR amplification of CYP1A1 and ribosomal S15 (the loading control) from these samples was carried out as described in Materials and Methods and the results shown are representative of two separate experiments.

Figure 3. Induction of AhR-dependent luciferase reporter gene activity in stably transfected mouse, rat and human hepatoma cells by extracts of commercial and consumer products.

Recombinant mouse (H1L1.1c2), rat (H4L1.1c4) and human (HG21.1c3) hepatoma cell lines were incubated with the indicated extract (10 µl/ml) for 4 hr and luciferase activity determined as described in Material and methods. Values are expressed as a percentage of the maximal luciferase induction by TCDD and represent the mean ± SD of triplicate determinations. The results shown are representative of duplicate experiments and those values significantly greater than that of solvent alone (p≤0.05 as determined by the students T-test) are indicated by an asterisk.

Figure 4. Effect of extracts of newspaper and rubber on human skin CYP1A1 mRNA, embryonic zebrafish CYP1A-dependent EROD activity and zebrafish development.

(A) Human skin was incubated with the indicated extract (newsprint (NP) or rubber stopper (RUB) at 1% final concentration) overnight at 37°C, mRNA was isolated and transcribed into cDNA and quantitated by real time PCR. Values are expressed as the mean ± SD of 4 (TCDD) or 6 (extract) individual skin samples. All values were significantly different from those of DMSO controls (set to 1) at p<0.05 as determined by one-way ANOVA using Stata/SE9.2 software for Windows with Bonferroni corrections. (B) Newly fertilized zebrafish embryos were exposed for 96 h to DMSO (0.02% v/v), newspaper (NP) extract (1∶5,000 dilution), or rubber (RUB) stopper (1∶5,000 or 1∶20,000 dilution) added to the water and some also injected with 2 pumps of 1× Danio embryo water or embryo water containing 0.15 mM CYP1A-morpholino; additional embryos were exposed to the AhR agonist beta-naphthoflavone (BNF, 1 µg/L) as the positive control for the same period. Hatched larvae were collected and analyzed for EROD activity. EROD values are expressed as the mean ± SE of 5 embryos, where the asterisk indicates those values significantly different from the DMSO control at p<0.05 as determined by Student's t-test. (C) The hatched larvae treated with extracts as in Figure 5B were examined for deformities by brightfield microscopy.

Figure 5. Estrogen reporter stimulation by rubber extracts.

Human ovarian BG1Luc4E2 cells, containing a stably transfected estrogen receptor responsive reporter, were treated with the indicated extracts as previously described (Rogers and Denison, 2000). Induction of luciferase is shown in comparison with maximal induction by 17β-estradiol (1 nM in ethanol) after incubation for 24 h. Values represent the mean ± SD of at least triplicate incubations and the results shown are representative of three individual experiments. Values significantly different from solvent alone (p≤0.05 as determined by the students T-test) are indicated by an asterisk.