Over the last few years, increasing evidence has become available that some brominated flame retardants (BFRs) may have endocrine-disrupting (ED) potencies. The goal of the current study was to perform a systematic in vitro screening of the ED potencies of BFRs (1) to elucidate possible modes of action of BFRs in man and wildlife and (2) to classify BFRs with similar profiles of ED potencies. A test set of 27 individual BFRs were selected, consisting of 19 polybrominated diphenyl ether congeners, tetrabromobisphenol-A, hexabromocyclododecane, 2,4,6-tribromophenol, ortho-hydroxylated brominated diphenyl ether 47, and tetrabromobisphenol-A-bis(2,3)dibromopropyl ether. All BFRs were tested for their potency to interact with the arylhydrocarbon receptor, androgen receptor (AR), progesterone receptor (PR), and estrogen receptor. In addition, all BFRs were tested for their potency to inhibit estradiol (sulfation by estradiol sulfotransferase (E2SULT), to interfere with thyroid hormone 3,3',5-triiodothyronine (T3)-mediated cell proliferation, and to compete with T3-precursor thyroxine for binding to the plasma transport protein transthyretin (TTR). The results of the in vitro screening indicated that BFRs have ED potencies, some of which had not or only marginally been described before (AR antagonism, PR antagonism, E2SULT inhibition, and potentiation of T3-mediated effects). For some BFRs, the potency to induce AR antagonism, E2SULT inhibition, and TTR competition was higher than for natural ligands or clinical drugs used as positive controls. Based on their similarity in ED profiles, BFRs were classified into five different clusters. These findings support further investigation of the potential ED effects of these environmentally relevant BFRs in man and wildlife.

In this study, the endocrine-disrupting (ED) potency of metabolites from brominated flame retardants (BFRs) was determined. Metabolites were obtained by incubating single-parent compound BFRs with phenobarbital-induced rat liver microsomes. Incubation extracts were tested in seven in vitro bioassays for their potency to compete with thyroxine for binding to transthyretin (TTR), to inhibit estradiol-sulfotransferase (E2SULT), to interact with thyroid hormone-mediated cell proliferation, and to (in-)activate the androgen, progesterone, estrogen, or aryl hydrocarbon receptor. For most BFRs, TTR-binding potencies, and to a lesser extent E2SULT-inhibiting potencies, significantly increased after biotransformation. Microsomal incubation had less pronounced effects on other ED modes of action, due to low biotransformation efficiency and background activities determined in control incubations without BFRs. Moreover, cell-based bioassays suffered from cytotoxicity from metabolites of lower-brominated polybrominated diphenyl ethers. For the environmentally relevant 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), six hydroxylated metabolites were identified. Individual metabolites had TTR-binding and E2SULT-inhibiting potencies 160-1600 and 2.2-220 times higher than BDE-47 itself, whereas their combined potencies in a realistic mixture were well predicted via concentration addition. In combination with other environmentally relevant hydroxylated organohalogens acting on TTR-binding and E2SULT inhibition, internal exposure to BFR metabolites may significantly contribute to the overall risk of endocrine disruption.

Commercial hexabromocyclododecane (HBCD) is a highproduction-volume flame-retardant applied in polystyrene foams. It contains three stereoisomers, of which γ-HBCD always dominates. Here we report on the levels of HBCD in blubber of harbor porpoise and common dolphin from different European seas. The highest total (Σ)-HBCD levels were measured in harbor porpoises stranded on the Irish and Scottish coasts of the Irish Sea (median concentration 2.9 µg (g of lipid) -1 ) and the northwest coast of Scotland (median concentration 5.1 µg (g of lipid) -1 ). The median levels in other areas were, for the harbor porpoise south coast of Ireland, 1.2 µg (g of lipid) -1 , for the coasts of The Netherlands, Belgium, and France north of Calais (southern North Sea), 1.1 µg (g of lipid) -1 , for the east coast of Scotland (northern North Sea), 0.77 µg (g of lipid) -1 , and, for Galicia (Spain), 0.1 µg (g of lipid) -1 . The median levels for the common dolphin were, for west coast of Ireland, 0.9 µg (g of lipid) -1 , for the French coast of the English Channel between Normandy and Brest, 0.4 µg (g of lipid) -1 , and, for Galicia, 0.2 µg (g of lipid) -1 . A subset of 10 harbor porpoise and 9 common dolphin blubber samples representing all areas were analyzed by LC/MS to determine the diastereomeric composition of their HBCD residues. All samples showed exclusively the peak of R-HBCD. To test if biotransformation by the cytochrome P450 system could explain the observed compositional difference with technical HBCD mixtures, a number of in vitro assays with microsomal preparations of liver were carried out. We had to revert to material stored at -80°C from laboratory rats and a fresh harbor seal found dead in the Dutch Wadden Sea, since such liver samples of cetaceans were not in our possession. The in vitro assays showed that -and γ-HBCDs were indeed significantly metabolized when incubated in the presence of NADPH as electron donor, compared to a set of reference samples which were identical except for the addition of NADPH. In contrast, the peak of R-HBCD did not decrease significantly in the presence of NADPH. In separate microsomal assays with -and γ-HBCDs, new peaks of brominated compounds (signal at m/z ) 79 or 81) with masses of [M + O] were formed only when NADPH was added. This confirms the process of cytochrome P450 mediated biotransformation. Although rat and harbor seal belong to different families of the mammalia than the cetaceans, we propose that biotransformation by the cytochrome P450 system is also the most likely process to explain the exclusive accumulation of R-HBCD in harbor porpoise and common dolphin.