Biotransformation of the flame retardant tetrabromo-bisphenol A by human and rat sub-cellular liver fractions
Introduction
Brominated flame retardants (BFR) are widely used in the manufacture of industrial equipment and consumer goods in order to prevent or minimise fire damage. Tetrabromo-bisphenol A (TBBPA) is the largest selling BFR (WHO, 1997) with a world-wide demand currently estimated over 120 000 tons/year (Arias, 2001, BSEF, 2004). TBBPA can be used either as an additive (physically mixed with polymers) or a reactive (polymerized) BFR to produce resins, high impact polystyrene and adhesives (WHO, 1995). It is primarily used as a reactive flame retardant in the manufacture of epoxy resins (Hyötyläinen and Hartonen, 2002). The presence of TBBPA in environmental samples was first reported in Japan, in river sediments (Watanabe et al., 1983). It was later found in sediments in Sweden (Sellström and Jansson, 1995) and in municipal sewage sludge in Canada, Sweden and the USA (Lee and Peart, 2002, Oberg et al., 2002, Quade et al., 2003). Human exposure to TBBPA was demonstrated by analyses carried out on plasma samples from electronics dismantling plant workers in Sweden and Norway (Hagmar et al., 2000, Thomsen et al., 2002). Significant amounts of this BFR have also been found in human plasma with no direct relation to occupational exposure (Thomsen et al., 2001, Thomsen et al., 2002, Hayama et al., 2004), as well as tribromo-BPA (Thomsen et al., 2001, Thomsen et al., 2002). The latter could be a metabolite produced by the enzymatic de-halogenation of TBBPA, or may be the result of a direct exposure. Indeed, tribromo-BPA, bisphenol A (BPA), monobromo-BPA and dibromo-BPA are all potential biodegradation products of TBBPA in the environment (Arbeli and Ronen, 2003).
Current data about the metabolic fate of TBBPA in animal models is limited, and though its half-life in human has been examined (Hagmar et al., 2000), no studies directly dealing with the metabolism of TBBPA in humans have been published so far. The formation of 3 different biliary conjugates was demonstrated in vitro in rats dosed TBBPA by an oral route (Hakk et al., 2000, Hakk and Letcher, 2003). In another study, it was stated that tribromo-BPA was a major metabolite of TBBPA in rat (Szymanska et al., 2001). TBBPA is the brominated analogue of the xeno-estrogen BPA, which undergoes in vivo and in vitro oxidative metabolism in addition to conjugation reactions (Zalko et al., 2003, Jaeg et al., 2004). For TBBPA, the balance between these different pathways is unknown and so is the relevance of rat as a model animal for the understanding of the fate of TBBPA in humans. On this basis, we started the present work to study the in vitro comparative biotransformation of TBBPA, using rat and human liver sub-cellular fractions. Both microsomes and S9 fractions were selected. Radio-labelled TBBPA was synthesised and analytical systems enabling the separation of TBBPA and its main metabolites were developed in order to achieve the separation of putative debrominated analogues of TBBPA and characterise the structure of TBBPA metabolites produced by the oxidative or conjugative biotransformation of this BFR.
Section snippets
Chemicals
Radio-labelled TBBPA [2,2-bis(3,5-dibromo-4-hydroxyphenyl)propane] was synthesised from ring-[14C]-BPA (Moravek Biochemicals, CA, USA; radio-purity: >99%, specific activity: 7.4 GBq mmol−1) according to Susãn et al. (1978): 4.4 equivalents of bromine were added to 6.29 MBq [14C]-BPA and 600 μg unlabelled BPA (Sigma Aldrich, Saint Quentin Fallavier, France; purity: >99%) in 200 μl ethanol/water (1:1 v/v); after 1 h at room temperature, the reaction was quenched with sodium bisulfite, extracted with
Radio-HPLC profiling of TBBPA incubations
Incubation of TBBPA with liver microsomes. Radio-HPLC profiles corresponding to the analyses of 20 μM TBBPA incubations are shown in Fig. 1, for female rat (A) and human (B). Using HPLC system I, the retention time (Rt) of TBBPA was 29.2 min. Two major metabolite peaks were observed. The more hydrophilic peak (M3) had a Rt of 12.9, while M7 (Rt: 36 min) was less hydrophilic than TBBPA. Additional peaks eluting before TBBPA were observed (M1, M5, M6) but none of them accounted for more than 1% of
Discussion
The comparative in vitro study of TBBPA biotransformation in rat and human demonstrates that this BFR is extensively metabolised by oxidative as well as conjugative enzyme-dependent pathways. High-grade purity [14C]-TBBPA enabled us to identify several metabolites present in small amounts in incubation supernatants. Neither rat nor human liver sub-cellular fractions were able to debrominate TBBPA, unless these compounds were produced in very small amounts and could not be detected.
In vivo
Acknowledgements
We wish to thank F. Blas-y-Estrada and R. Gazel for excellent technical support and express our appreciation for the financial support provided by the “Agence Française de Sécurité Sanitaire Environnementale” (AFSSE grant RD-2004-011).
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