Interactions of polybrominated diphenyl ethers with the aryl hydrocarbon receptor pathway.

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use as additives in various consumer products. Structural similarities of PBDEs with other polyhalogenated aromatic hydrocarbons that show affinity for the aryl hydrocarbon receptor (AhR), such as some polychlorinated biphenyls, raised concerns about their possible dioxin-like properties. We studied the ability of environmentally relevant PBDEs (BDE-47, -99, -100, -153, -154, and -183) and the "planar" congener BDE-77 to bind and/or activate the AhR in stably transfected rodent hepatoma cell lines with an AhR-responsive enhanced green fluorescent protein (AhR-EGFP) reporter gene (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma). 7-Ethoxyresorufin-O-deethylation (EROD) was used as a marker for CYP1A1 activity. Dose- and bromination-specific inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced responses was measured by their ability to inhibit the induction of AhR-EGFP expression and EROD activity. Individual exposure to these PBDEs did not result in any increase in induction of AhR-EGFP or CYP1A1 activity. The lower brominated PBDEs showed the strongest inhibitory effect on TCDD-induced activities in both cell lines. While the highest brominated PBDE tested, BDE-183, inhibited EROD activity, it did not affect the induction of AhR-EGFP expression. Similar findings were observed after exposing stably transfected human hepatoma (xenobiotic response element [XRE]-HepG2) cells to these PBDEs, resulting in a small but statically significant agonistic effect on XRE-driven luciferase activity. Co-exposure with TCDD resulted again in antagonistic effects, confirming that the inhibitory effect of these PBDEs on TCDD-induced responses was not only due to direct interaction at receptor level but also at DNA-binding level. This antagonism was confirmed for BDE-99 in HepG2 cells transiently transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. In addition, a chromatin immunoprecipitation assay further confirmed that BDE-99 could bind to the AhR and activate the AhR nuclear translocation and dioxin responsive element (DRE) binding in the context of the CYP1A1 promoter. However, the transactivation function of the BDE-99-activated AhR seems to be very weak. These combined results suggest that PBDEs do bind but not activate the AhR-AhR nuclear translocator protein-XRE complex.

Antagonism of TCDD-induced ethoxyresorufin-O-deethylation activity by polybrominated diphenyl ethers (PBDEs) in primary cynomolgus monkey (Macaca fascicularis) hepatocytes.

Polybrominated diphenyl ethers (PBDEs) are widespread environmental pollutants, and the levels of certain congeners have been increasing in biota and abiota in recent decades. Some PBDEs are lipophilic and persistent, resulting in bioaccumulation in the environment. Their structural similarity to other polyhalogenated aromatic hydrocarbons (PHAHs) such as polychlorinated biphenyls (PCBs) has raised concerns that PBDEs might act as agonists for the aryl hydrocarbon receptor (AhR). Recent studies in our laboratory with human and rat cell lines indicated no AhR mediated CYP1A1 induction for PBDEs. However, an earlier in vitro study by Van der Burght et al. (1999) [Van der Burght, A.S., Clijsters, P.J., Horbach, G.J., Andersson, P.L., Tysklind, M., van den Berg, M., 1999. Structure-dependent induction of CYP1A by polychlorinated biphenyls in hepatocytes of cynomolgus monkeys (Macaca fascicularis). Toxicol. Appl. Pharmacol. 155, 13-23] indicated that in cynomolgus monkey (M. fascicularis) hepatocytes PCBs with a non-planar configuration could induce CYP1A. As PBDEs show a structural similarity with non-planar (ortho substituted) PCBs, our present study focused on the possible CYP1A induction by PBDEs (BDE-47, -99, -100, -153, -154, -183, and -77) in individual preparations (n=4) of primary hepatocytes of cynomolgus monkeys (M. fascicularis). 7-Ethoxyresorufin-O-deethylase (EROD) was used as a marker for CYP1A-mediated catalytic activity. Cells were exposed for 48 h to various PBDE concentrations (0.01-10 microM), positive controls 2,3,7,8-TCDD (0.001-2.5 nM) and PCB-126 (0.01-10nM), and negative control (DMSO vehicle alone). No statistically significant induction of CYP1A was observed in the hepatocytes after 48 h of exposure to all environmentally relevant PBDEs. After exposing hepatocytes to PBDEs in combination with TCDD, a concentration-dependent decrease in TCDD-induced EROD activity was observed. All PBDEs tested showed a similar reduction in each of four experiments, though quantitative differences were observed. The observed antagonism of TCDD-induced EROD activity by PBDEs occurred in both male (n=3) and female (n=1) hepatocytes and was not due to catalytic inhibition of EROD activity or cytotoxicity. However, based on the results of this study we do not expect these antagonistic effects of PBDEs on CYP1A induction at environmental relevant levels, since these in vitro interactive effects with TCDD were observed only at relatively high concentrations that are normally not seen, e.g. in the human body.

Determination of in vitro relative potency (REP) values for mono-ortho polychlorinated biphenyls after purification with active charcoal.

The TEF system for dioxin-like compounds has included assignment of TEF values for mono-ortho polychlorinated biphenyls (MO-PCBs). Small traces of aryl hydrocarbon receptor (AhR)-active impurities could result in artifactually higher relative potency (REP) values. MO-PCBs -105, -118, -156, and -167 were purified on an active charcoal column to remove AhR agonists that could be present as impurities. Activation or inhibition of AhR-dependent gene expression by purified MO-PCBs was studied in stably transfected cell lines (H1G1.1c3 mouse, H4G1.1c2 rat hepatoma), containing an AhR-responsive (AhR-EGFP) reporter gene. In addition, EROD activity was used as marker for CYP1A1 activity in these cell lines. MO-PCBs -105, -118, -156 induced AhR-EGFP expression in both rodent cell lines, with PCB-156 (10microM) being most effectively; inducing gene expression to approximately 27% of TCDD (mouse cells) and 62.5+/-3.4% (rat cells) of TCDD. This concurred with increased EROD activity in both cell lines to maxima of 20.5+/-1.5% and 68+/-3.2% of TCDD, respectively. No induction was observed for PCB-167. In the H1G1.1c3 mouse cells, PCB-105, -118 and -156 (10microM) significantly reduced TCDD-induced AhR-EGFP expression to 50.9+/-2.9%, 58.3+/-2.2% and 70.8+/-1.3% of TCDD. Reduced EROD activity was also observed, of 39.3+/-2.8%, 67+/-5% and 48.3+/-4% compared to TCDD. PCB-167 did not result in significant reduction. In rat cells, only PCB-156 resulted in significant decrease in TCDD-induced AhR-EGFP expression of 35%, suggesting species differences play a role. Our results suggest that purification of MO-PCBs is an essential step in determining accurate REP values, and could very likely lead to lower TEF values than those presently assigned by the WHO.

Effects of polybrominated diphenyl ethers on basal and TCDD-induced ethoxyresorufin activity and cytochrome P450-1A1 expression in MCF-7, HepG2, and H4IIE cells.

Polybrominated diphenylethers (PBDEs) are used as additive flame-retardants in consumer products to reduce the chances of ignition and burning. Levels of certain PBDE congeners have been increasing in fish, wildlife, and human tissues during the last decades. Some PBDEs are lipophilic and persistent, resulting in bioaccumulation in the environment. The structural similarity of PBDEs to other polyhalogenated aromatic hydrocarbons such as PCBs, has raised concerns that PBDEs might act as agonists for the aryl hydrocarbon receptor (AhR). To study the possible AhR-mediated effects of the environmentally relevant PBDEs (BDE47, 77, 99, 100, 153, 154, 183, 209), the induction of cytochrome P450-1A1 (CYP1A1) was studied in human breast carcinoma (MCF-7), human hepatocellular carcinoma (HepG2), and rat hepatoma (H4IIE) cells. 7-Ethoxyresorufin-O-deethylase (EROD) was used as a marker for CYP1A1 activity. Cells were exposed for 72 h to various PBDE concentrations (0.01-10 microM). Positive controls were 2,3,7,8-TCDD (0.001-2.5 nM) and PCB126 (0.01-10 nM). None of these PBDEs was capable of inducing EROD activity; this was confirmed by real time RT-PCR for CYP1A1 mRNA. However, in cells exposed to PBDEs in combination with TCDD, a concentration-dependent decrease in TCDD-induced EROD activity occurred. Co-exposure of BDE153 (10 muM) and a maximally inducing concentration of TCDD (1 nM) reduced EROD activity to 49% of the maximum induction by TCDD alone. All tested PBDEs showed similar effects in each cell line, though quantitative differences were observed. The observed decrease in CYP1A1 activity was not due to PBDE-dependent catalytic inhibition of EROD activity or cytotoxicity, nor were decreased CYP1A1 mRNA levels observed. However, inhibition of luciferase induction in mouse (Hepa) and rat (H4IIE) hepatoma cells containing a stably transfected AhR-responsive luciferase reporter gene, suggests that BDE77 is a weak AhR antagonist or partial agonist.