Toxicological Sciences 56, 95-104 (2000)
Copyright © 2000 by the Society of Toxicology
Potent Competitive Interactions of Some Brominated Flame Retardants and Related Compounds with Human Transthyretin in Vitro
* Toxicology Group, Department of Food Technology and Nutritional Sciences, Wageningen University and Research Center, Tuinlaan 5, 6703 HE Wageningen, The Netherlands;
Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, Stockholm, Sweden; and
Institute of Environmental Studies, Free University of Amsterdam, Amsterdam, The Netherlands
Received January 4, 2000; accepted February 23, 2000
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ABSTRACT |
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Brominated flame retardants such as polybrominated diphenyl ethers (PBDEs), pentabromophenol (PBP), and tetrabromobisphenol A (TBBPA) are produced in large quantities for use in electronic equipment, plastics, and building materials. Because these compounds have some structural resemblance to the thyroid hormone thyroxine (T4), it was suggested that they may interfere with thyroid hormone metabolism and transport, e.g., by competition with T4 on transthyretin (TTR). In the present study, we investigated the possible interaction of several brominated flame retardants with T4 binding to TTR in an in vitro competitive binding assay, using human TTR and 125 I-T4 as the displaceable radioligand. Compounds were tested in at least eight different concentrations ranging from 1.95 to 500 nM. In addition, we investigated the structural requirements of these and related ligands for competitive binding to TTR. We were able to show very potent competition binding for TBBPA and PBP (10.6- and 7.1-fold stronger than the natural ligand T4, respectively). PBDEs were able to compete with T4-TTR binding only after metabolic conversion by induced rat liver microsomes, suggesting an important role for hydroxylation. Brominated bisphenols with a high degree of bromination appeared to be more efficient competitors, whereas chlorinated bisphenols were less potent compared to their brominated analogues. These results indicate that brominated flame retardants, especially the brominated phenols and tetrabromobisphenol A, are very potent competitors for T4 binding to human transthyretin in vitro and may have effects on thyroid hormone homeostasis in vivo comparable to the thyroid-disrupting effects of PCBs.
Key Words: brominated flame retardants; transthyretin; structure-activity relationship.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well established that several classes of environmental contaminants can affect thyroid gland morphology and hormonal status (for reviews see Brouwer et al., 1998; Brucker-Davis, 1998), but the exact mechanisms of interference are not fully understood. There are at least three different levels at which organohalogen contaminants are known to interact with the thyroid hormone system: at the thyroid gland, in thyroid hormone metabolism, and with thyroid hormone transport proteins. A number of chemicals have been reported to bind to transthyretin (TTR), one of the thyroid hormone-binding transport proteins in plasma of vertebrate species. In particular, metabolites of the polyhalogenated aromatic hydrocarbons (PHAHs) such as hydroxylated polychlorinated biphenyls (HO-PCBs), hydroxylated polychlorinated dibenzo-p-dioxins (HO-PCDDs), and pentachlorophenol (PCP) have been shown to bind to TTR in in vitro and/or in vivo studies (Brouwer et al., 1988
; Lans et al., 1993; McKinney and Waller, 1994; van den Berg et al., 1991; van Raaij et al., 1991). It is hypothesized that the binding of chemicals to TTR, thereby displacing the natural ligand 3,3',5,5'-tetraiodothyronine (thyroxine, T4), leads to an increase in the clearance of T4 and a decrease in serum T4 concentrations (Darnerud et al., 1996), a common feature in animals that have been exposed to PHAHs (Brouwer et al., 1998; Brucker-Davis, 1998).The research on chemicals binding to transthyretin has been focused mainly on the polychlorinated dibenzo-p-dioxins (PCDDs) and biphenyls (PCBs), i.e., compounds that have been banned or are under control measures for further environmental reduction. Hydroxylated PCBs, especially those with a hydroxy group on meta or para positions with one or more adjacent halogen substituents, have been shown to be potent ligands for TTR (Lans et al., 1993; Rickenbacher et al., 1986) because of their structural resemblance with thyroxine. Other organohalogen compounds that are extensively used at the moment, particularly the brominated flame retardants tetrabromobisphenol A (TBBPA) and polybrominated diphenyl ethers (PBDEs) (TemaNord, 1998), show an even closer structural relationship to thyroxine than the PCBs. Therefore, the possibility exists that these brominated flame retardants interact with TTR and other aspects of thyroid hormone metabolism. Of the brominated flame retardants in use today, about one-third are polybrominated diphenyl ethers (PBDEs), another one-third are the tetrabromobisphenol A and derivatives, and the last third is composed of a variety of bromine-containing products, including polybrominated biphenyls (PBBs) (OECD, 1994). The production volume of TBBPA in 1995 was approximately 60,000 tonnes per year (WHO, 1995). Its primary use is as a reactive intermediate in the production of flame-retarded epoxy resins used in printed circuit boards (WHO, 1995). PBDEs are extensively used as flame retardants in plastics, paints, electrical components, and synthetic textiles (WHO, 1994). They have been produced in large quantities since the 1980s, mostly as commercial mixtures such as Bromkal 70-5DE. TBBPA has been detected in sediment samples in Japan in concentrations of 0.5–140 µg/kg dry weight (Watanabe et al., 1983), but is not normally detected in environmental biologic samples (WHO, 1995). PBDEs have been found in various biotic samples, such as fish-eating birds and marine mammals (Jansson et al., 1987), shellfish and sediment (Haglund et al., 1997), and even in human blood (Sjödin et al., 1999) and breast milk (Meironyté et al., 1999). So far, the PBDE concentrations detected in wildlife and humans are lower than the concentrations of PCBs. However, Meironyté et al. (1999) showed that PBDE concentrations in human milk sampled in Sweden from 1972 to 1998 increased from 0.07 to 4.02 ng/g lipid weight. Hydroxylated and methoxylated PBDEs (HO-PBDEs and MeO-PBDEs) have also been detected in various biotic samples from the Baltic Sea (Asplund et al., 1999; Haglund et al., 1997). Concentrations of the HO-PBDEs in blood plasma from Baltic salmons were estimated to be about 30–50 ng/g lipid weight, similar to concentrations of the major PBDEs in these samples (Asplund et al., 1999).Because of the high production volume of brominated flame retardants, the presence of these compounds in biotic samples, and their close structural resemblance with thyroxine, we examined the ability of several of these compounds and their metabolites (in the case of PBDEs) to bind to human TTR by an in vitro T4-TTR competition binding assay (Lans et al., 1993). Because no hydroxylated PBDEs were available at the time for this study, a method was designed to include microsomal activation in the T4-TTR competition binding assay. For comparison, three synthesized hydroxylated PBDEs with structural resemblance to the thyroid hormones 3,5-diiodothyronine (T2), 3,3',5-triiodothyronine (T3), and 3,3',5,5'-tetraiodothyronine (thyroxine, T4) were also tested for their potency to compete with T4-TTR binding. The resulting structure-activity relationships were compared with the known structure-activity relationships of related (especially chlorinated) compounds. |
MATERIALS AND METHODS |
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Chemicals.
All chemicals were of > 98% purity unless otherwise stated. 2,4,6-Tribromoaniline, 2,4-dibromophenol (2,4-DBP; 95%), 2,3,5,6-tetrabromo-p-xylene, 2,3,4,5,6-pentabromotoluene, brominated bisphenol A diglycidyl ether, tetrabromobisphenol A (TBBPA, 97%), tetrachlorobisphenol A (TCBPA), pentabromophenol (PBP, 96%), bisphenol A (97%), 4-phenoxy-phenol, and hexabromobenzene were obtained from Aldrich Chemical Company (Bornem, Belgium). Bisphenol A diglycidylether, bisphenol A bis(2,3-dihydroxypropyl)ether, bisphenol A bis(3-chloro-2-hydroxypropyl)ether, 2,4,6-tribromophenol (TBP), and phenobarbital (PB) were purchased from Fluka Chemie (Buchs, Switzerland). 2-Hydroxy-2',4,4'-trichlorodiphenyl ether was from Ultra Science (N. Kingstown, RI).Pure PBDE congeners (> 98% pure) were synthesized as described elsewhere (Marsh et al., 1999
; Örn et al., 1996). Three hydroxylated brominated diphenyl ethers, 4-(2,4,6-tribromophenoxy)phenol, 2-bromo-4-(2,4,6-tribromophenoxy)phenol and 2,6-dibromo-4-(2,4,6-tribromophenoxy)phenol, were synthesized as described by Marsh et al. (1998) and were at least 99% pure. Monobromobisphenol A (MBBPA, containing 96.5% monobromobisphenol A and 3.5% dibromobisphenol A), dibromobisphenol A (diBBPA, containing 99.4% dibromo- and 0.6% tribromobisphenol A), and tribromobisphenol A (triBBPA, 100% pure) were synthesized by bromination of bisphenol A using bromine in acetic acid at room temperature (Sara Rahm, unpublished).125I-L-3',5'-Thyroxine (spec. act. 46 µCi/µg) was purchased from Orange Medical (Tilburg, The Netherlands). Human prealbumin (transthyretin, TTR, 98% pure), pure clofibrate (CLOF), and 3,3',5,5'-L-thyroxine (T4) were obtained from Sigma Chemical Company, St. Louis, MO. Tris, saccharose, methanol, ethanol, dichloromethane, and diisopropyl ether (all analytical grade) were from Merck Chemical Company (Darmstadt, Germany). Biogel P-6DG desalting gel was obtained from Bio-Rad Laboratories (Richmond, CA). ß-Naphthoflavone (ß-NF), EDTA, and dimethylsulfoxide (99.9% pure) were obtained from Janssen Chimica (Geel, Belgium). NADPH was obtained from Boehringer (Mannheim, Germany).Bisphenol A and mono- and dibromobisphenol A were dissolved in ethanol and stored at –20°C. Only 2,3,5,6-tetrabromo-p-xylene and 2,3,4,5,6-pentabromotoluene were dissolved in dichloromethane, because they were not soluble in ethanol or dimethylsulfoxide (DMSO). All other compounds were dissolved in DMSO.
Preparation of microsomes.
Nine male Wistar WU rats (14 weeks of age) were purchased from Charles River (Sulzfeld, Germany) and allowed to acclimatize for 2 weeks. They were kept in an artificial light-dark cycle (06:00 lights on, 18:00 h lights off), with room temperature at 21 ± 1°C and humidity at 50 ± 10%. Animals were provided rat chow (Hope Farms, Woerden, The Netherlands) and tap water ad libitum. To induce microsomes, three rats per group that were naïve to chemical treatment were pretreated with ß-naphthoflavone (ß-NF, three daily ip injections of 30 mg/kg body weight dissolved in corn oil), phenobarbital (PB, 0.1% w/v in the drinking water for 7 days), or clofibrate (CLOF, four daily oral administrations of 200 mg/kg bw). One day after the last treatment, the rats were sacrificed under ether anesthesia and the livers were removed. All procedures were approved by the Animal Welfare Committee of Wageningen University. Livers of rats from each treatment group were pooled and homogenized in ice-cold 0.1 M Tris-HCl buffer, pH 7.5 (3 ml/g liver), containing 0.25 M sucrose, using a Potter-Elvehjem tube and Teflon pestle. The homogenate was centrifuged for 30 min at 9000 x g (4°C). The resulting supernatant was centrifuged at 105,000 x g and 4°C for 90 min. The microsomal pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.5). Microsomes were stored in aliquots of 1 ml at –80°C until use. Protein concentrations were determined using the Bio-Rad Coomassie blue assay (Bio-Rad, Richmond, CA), using BSA as a standard (Bradford, 1976).
Metabolism of PBDEs in vitro.
As almost no hydroxylated PBDEs have been synthesized so far, 17 PBDE congeners were metabolized by incubation with induced hepatic microsomes, as described for PCBs (Morse et al., 1995) with slight modifications. Briefly, 10 µM of each PBDE congener was incubated with 1 mg/ml hepatic microsomes in a 0.1 M Tris-HCl buffer (pH 7.5) in a total volume of 2 ml. After preincubation for 2 min in a shaking water bath at 37°C, the reaction was initiated with NADPH (1 mM). Metabolism was stopped after 30 min by the addition of 2 ml ice-cold methanol. After centrifugation, the supernatants were extracted twice with 2 ml diisopropyl ether by vortexing for 30 s, centrifugation at 1000 x g for 5 min, and then removal of the diisopropyl ether phase. The ether extracts were pooled, dried under nitrogen, and stored at 4°C until further analysis (but not longer than 1 week). Control incubations were carried out by performing identical incubations with the PBDE without the addition of NADPH. For determining the possible background of T4-TTR competition by microsomal extracts, incubations were also carried out without the addition of a PBDE to the microsomes. The extracts were dried by evaporation, and residues were dissolved in 50 µl methanol prior to the T4-TTR competition binding studies.
In vitro T4-TTR competition binding studies.
The analysis of the capacity of various compounds to compete with T4 binding to TTR was performed as described previously (Lans et al., 1993), with modifications. The assay mixture was a 0.1 M Tris-HCl buffer (pH 8.0) containing 0.1 M NaCl and 0.1 mM EDTA, 30 nM human TTR, a mixture of 125I- labeled and unlabeled T4 (70,000 cpm, 55 nM), and competitors (cold T4, pure compounds or extracts) with increasing concentrations (at least eight different concentrations), in a total volume of 200 µl. Control incubations contained 5 µl ethanol, methanol, DMSO, or dichloromethane (depending on the solvent used) instead of competitor. Total 125I-radioactivity added to each of the incubation mixtures was checked by gamma counting (Multi Prias, Packard Instrument Co., Meriden, CT). The incubation mixtures were allowed to reach binding equilibrium overnight at 4°C. After incubation, protein-bound and free 125I-T4 were separated on 1-ml Biogel P-6DG columns and spin-force eluted with 200 µl Tris-HCl buffer (1 min at 100 x g in a precooled centrifuge, Difuge, Hereaus) to reduce transit time on the column (about 30 s) in order to minimize possible dissociation of the complex (Somack et al., 1982). Radioactivity was determined in the eluate containing the protein-bound 125I-T4 fraction and compared to control incubations. Nonspecific binding was also determined in each series of experiments by addition of 10 µM cold T4 and was less than 10%.In the case of pure compounds, the competitors were first diluted in 0.1 M Tris-HCl buffer (pH 8.0, containing 0.1 M NaCl, 1 mM EDTA) and added to the assay mixture in a concentration series from 10–9 to 10–4 M. To study possible competition binding of PBDE metabolites, extracts of microsomal incubations were diluted 0, 3, or 9 times in methanol and 5 µl was added to the incubation mixture as competitor. The maximum concentration of metabolites formed could thus be no more than 250 nM with 100% conversion. The maximum percentage of solvent in the assay mixture did not exceed 0.5%. Control incubations with microsomal extracts without PBDEs were used to determine possible background competition, whereas microsomal extracts from incubations with PBDEs but without NADPH were used to determine possible competition by the parent compound itself.Competition binding curves for pure compounds were made by plotting relative 125I-T4 protein binding (% of control) against concentration competitor. For microsomal extracts, competition binding curves were made by plotting relative 125I-T4 protein binding (% of control, with control incubations of microsomes set to 100%) against the dilution factor, as no reference PBDE-metabolites are presently available.
Analysis of binding data.
Calculation of binding parameters was performed with the LIGAND-PC program from Munson and Rodbard (1980) (obtained from Dr. K. J. van den Berg, TNO Nutrition, Zeist, The Netherlands). Goodness-of-fit-statistics (R2s) were always higher than 0.95. Relative potencies of competitors compared to T4 were calculated by dividing IC50 (T4) by IC50 (competitor). All analyses were performed with data of at least three different experiments performed in duplicates. Data are given as means ± standard deviations (SD).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brominated Flame Retardants and Related Compounds
The structures of chemicals used in the T4-TTR binding experiments are given in Table 1
. Table 2 shows the binding affinity constants, IC50 values, percentage competition reached at the highest tested concentration, and the relative potencies compared to the natural ligand T4 of the non-PBDE brominated flame retardants and structural analogues measured in this study. In the case of the brominated phenols, it can be observed that an increased level of bromination, such as with 2,4,6-TBP and PBP, results in stronger competitors for binding to TTR (relative potencies of 1.2 and 7.1 compared to T4, respectively, Table 2). The maximum competition reached by these compounds at 500 nM exceeded that of T4 at the same concentration (Table 2). In contrast, the relative potency of 2,4-DBP (0.06) was almost 17 times lower than T4, and the maximum T4-TTR binding competition was only 50 ± 1.8% at a concentration of 25 µM. The binding affinity constants (Ka) also showed that phenols with an increasing amount of bromine substitution had increasing binding affinities to TTR. However, brominated flame retardants with another substituent on the para position, e.g., NH2 (in the case of 2,4,6-tribromoaniline) or CH3 (in the case of 2,3,5,6-tetrabromo-p-xylene or 2,3,4,5,6-pentabromotoluene) did not show any interaction with TTR (Table 2).The most potent competitor of the tested phenolic organohalogen compounds was TBBPA (relative potency of 10.6, competition of 96.5 ± 0.1% at 500 nM, Table 2). In addition, for the bisphenol A analogues, the TTR binding potency increased with a higher level of bromination. The potency of triBBPA was 18 times less compared to TBBPA, whereas no or only slight competition was observed with di-, mono- and nonbrominated bisphenol A (approximately 19, 11 and 7% competition reached at 500 nM, respectively, Table 2). Interestingly, replacing the bromine atoms by chlorine atoms in the bisphenol A core structure (e.g., tetrabromobisphenol A versus tetrachlorobisphenol A [TCBPA]) resulted in an almost 14 times lower TTR-binding competition potency (IC50 values of TCBPA and TBBPA were 106.8 ± 10.3 and 7.7 ± 0.9, respectively). The competition binding curves of diBBPA, triBBPA, TBBPA, and TCBPA are given in Figure 1.
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T4-TTR Binding Competition with Microsomal Extracts of PBDEs
In total, 17 different PBDE congeners (see Table 3
for their structure) were tested before and after incubation with differently induced hepatic microsomes for T4-TTR competition binding potency by their possible metabolites formed. Because no reference PBDE metabolites were available at the time of this study, the competition potency of microsomal extracts could be investigated only by dilution technique. In Figure 2, a representative example of the T4-TTR competition binding by microsomal extracts is given. No competition of T4-TTR was observed with control microsomal incubations without NADPH (Fig. 2 [A and B], triangles) or without PBDE (Fig. 2 [A and B], circles), indicating that microsomes did not cause background competition and parent PBDEs were not able to bind to TTR.Incubation of PBDEs with PB microsomes (mostly P450 2B enriched) in the presence of NADPH resulted in the formation of metabolites that were able to compete with T4 binding to TTR, with the exception of incubation extracts from 2,4',6-triBDE (BDE 32); 2,2',3,4,4',5'-hexaBDE (BDE 138, Fig. 2B); 2,2',4,4',5,5'-hexaBDE (BDE 153); and 2,3,3',4,4',5,6-heptaBDE (BDE 190) (Table 3). P450 1A, P450 2B, and P450 4A3 enriched microsomes all catalyzed the formation of TTR-binding metabolites from 2,4,6-triBDE (BDE 30); 2,3',4',6-tetraBDE (BDE 71); 2,4,4',6-tetraBDE (BDE 75); and 3,3',4,4'-tetraBDE (BDE 77).
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Hydroxylated PBDEs (HO-PBDEs)
Three pure hydroxylated PBDEs (HO-PBDEs), synthesized for their structural resemblance with the thyroid hormones 3,5-diiodothyronine (3,5-T2), 3,3',5-triiodothyronine (T3), and 3,3',5,5'-tetraiodothyronine (T4), were tested in the T4-TTR competition binding assay (Table 4
, Fig. 3). The relative potencies showed that the T4-like (2,6-dibromo-4-[2,4,6-tribromophenoxy]phenol) and T3-like (2-bromo-4-[2,4,6-tribromophenoxy]phenol) HO-PBDEs were 1.42- and 1.22-fold more potent, respectively, than T4, and the percentage competition at 500 nM exceeded that of the natural ligand (Table 4). 4-Phenoxy-phenol and 2-hydroxy-2',4,4'-trichlorodiphenyl ether showed no interaction with human TTR.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results presented in this study clearly demonstrate for the first time that hydroxylated brominated flame retardants of several different classes are able to bind to human transthyretin in vitro, some with extremely high potency, e.g., TBBPA and PBP. This is an important finding, as brominated flame retardants are used extensively at present for a large variety of applications and can be detected in wildlife and humans (Bergman et al., 1999
; Meironyté et al., 1999; WHO, 1997). The results of this paper thus indicate the possible capability of a large group of particularly brominated industrial chemicals to interfere with and potentially disrupt the thyroid hormone transport and metabolism.The structure-affinity data of brominated bisphenols that can be deduced from this study are in good agreement with previous studies on several industrial chemicals, such as the chlorinated benzenes and their hydroxylated metabolites (den Besten et al., 1991 and van den Berg, 1990), or the hydroxylated PCBs (Brouwer et al., 1990; Brouwer and van den Berg, 1986; Cheek et al., 1999; Lans et al., 1993; Rickenbacher et al., 1986). First, the degree of bromine substitution appeared to play a crucial role in the binding potency, as bisphenols with a lesser degree of bromination showed lower or no competitive binding to TTR. These results are consistent with earlier studies performed with chlorinated phenols, showing an increased interaction of higher chlorinated phenols with transthyretin as compared to lower chlorinated phenols (den Besten et al., 1991; van den Berg, 1990). Second, the nature of the halogen substitution also plays a major role in the binding affinity of the compounds to TTR. TBBPA was the most potent competitor in this study (relative potency of 10.6 compared to the natural ligand), whereas TCBPA, with the only structural difference being the bromine atoms replaced by chlorine atoms, competed with T4-TTR binding with lesser potency than TBBPA. Higher binding potency of brominated analogues over chlorinated ones was also observed for PBP (relative potency 7.14, this study) as compared to PCP (relative potency of 1.74 [van den Berg, 1990] and 2.50 [den Besten et al., 1991]).Third, comparison of the relative potencies of TBBPA versus triBBPA versus diBBPA and PBP versus 2,4,6-TBP versus 2,4-DBP indicates that hydroxylation at the para position with one but preferably two adjacent halogen substituents, which is proposed to be the prerequisite for binding of hydroxylated PCBs to human transthyretin (Lans et al., 1993), is also an essential requirement in the binding of the brominated bisphenols to TTR. It is hypothesized that these lateral (3,3',5,5') halogens can occupy the binding pockets of TTR normally occupied by the diiodophenolic ring of the thyroxine molecule, as has been shown for 4,4'-(OH)2-3,3',5,5'-tetrachlorobiphenyl (Lans, 1995) and proposed for 3,3',4,4',5,5'-hexachlorobiphenyl (Rickenbacher et al., 1986). On the contrary, hydroxylation is not always a prerequisite for binding, as several parent PCBs have also been shown to interact with human TTR (Chauhan et al., 1998; Cheek et al., 1999; McKinney and Waller, 1994; Meerts, unpublished results; Rickenbacher et al., 1986). This is further substantiated by earlier findings on the existence of different binding modes of T4 to TTR, e.g., a forward mode with the phenolic ring pointing towards the center in the TTR binding site, and a reversed mode with the phenolic ring positioned towards the mouth of the channel entrance (De la Paz et al., 1992). In addition, our recent observations based on X-ray crystallography data on organohalogen-TTR complexes showed that the hydroxy group in PBP and TBP was not essential for binding to TTR (Ghosh et al., 2000). The mode of binding of these latter compounds to TTR differs from the binding of other organohalogen compounds identified so far and will be described in detail elsewhere (Ghosh et al., 2000). This different binding may explain the similar potency of, e.g., the single-ring structure PBP and the much larger double-ring structure TBBPA.Of the 17 PBDEs examined in this study, none of the parent compounds competed with T4-TTR binding. In this case, metabolic conversion is most likely essential for the capability of PBDEs to displace 125I-T4 from TTR. The potency of a PBDE to compete with T4 on TTR appeared to be both congener- and metabolic enzyme-specific. CYP 2B-enriched liver microsomes were able to catalyze the formation of PBDE metabolites that showed T4-TTR competition binding potency. Almost none of the higher brominated diphenyl ethers were capable of displacing T4 from TTR after microsomal incubation. Two explanations are possible for this observation, i.e., higher brominated diphenyl ethers were not metabolized by the differently enriched microsomal preparations, or the metabolites formed were not able to compete with T4 for binding to TTR. Further studies will be focused on the elucidation of the chemical identity of these PBDE metabolites. The results with the synthetic HO-PBDEs resembling the thyroid hormones are in good agreement with the competitive binding with T4 on TTR and other structural analogues (Andrea et al., 1980). However, the small difference in binding affinities between the T4-like and T3-like HO-PBDEs (the binding affinity of the T3-like HO-BDE is a factor 1.1 smaller compared to the T4-like) cannot be explained at the moment. Comparing the binding affinities of the natural ligands T4 and T3 (3.5 x 107 and 3.2 x 106 M–1 respectively, i.e., a factor 11 difference [Andrea et al., 1980]), one would expect the affinity of the T3-like HO-BDE to differ from the T4-like in the same range. Further studies are necessary to determine the binding of these brominated thyroid hormone analogues to TTR in more detail.The interaction of brominated flame retardants with transthyretin may indicate interaction with other thyroid hormone-binding proteins such as enzymes involved in thyroid hormone metabolism. Hydroxylated PCBs with high affinity for TTR have been shown to interact with iodothyronine 5'-deiodinase (Adams et al., 1990; Lans, 1995; Rickenbacher et al., 1989) and iodothyronine sulfotransferase (Schuur et al., 1998). However, the interaction of hydroxylated PCBs with thyroxine-binding globulin (TBG), the major thyroid hormone transport protein in humans, is very rare, and affinities are 100-fold lower than T4 (Cheek et al., 1999; Lans et al., 1994). This may indicate that the impact of compounds binding to transthyretin is lower in humans and nonhuman primates that possess TBG as the major thyroxin carrier. However, the binding of HO-PCBs/HO-PBDEs to TTR may be involved in facilitated transfer of these compounds across the placenta and the blood-brain barrier, leading to relatively high levels in the fetus, and especially the fetal brain. Morse et al. (1996) showed a strong accumulation of the PCB metabolite 4-hydroxy-2,3,3',4',5-pentaCB (4-HO-pentaCB) in plasma and forebrain of fetuses 20 days of age after exposure of the dams to the commercial PCB-mixture Aroclor 1254 from gestation days 10 to 16. This accumulation could be explained by competition between the 4-HO-pentaCB and T4 for TTR binding, leading to a selective and facilitated transport of the metabolite over the placenta to the fetal compartment. The accompanying reduction in plasma T4 levels could be caused either by binding of the 4-HO-pentaCB to TTR in vivo and/or amplified biliary excretion of T4 due to induction of UDP glucuronosyltransferase (UDPGT) by Aroclor 1254 (Barter and Klaassen, 1992; Morse et al., 1996; van Birgelen et al. 1995). However, exposure of pregnant rats to the 4-HO-pentaCB alone resulted in decreased T4 levels in their fetuses without induction of UDP glucuronyltransferase (Meerts et al., manuscript in preparation), indicating that binding of a compound to TTR in vivo can result in lowered plasma levels of T4 in the rat.Surprisingly, TBBPA, which showed a high T4-TTR competitive interaction in vitro (this paper), showed no effects on thyroid hormone levels in fetuses 20 days of age after oral exposure of pregnant rats to 5 mg/kg body weight per day from gestation days 10 to 16 (Meerts et al., 1999). This may be explained by the poor absorption of TBBPA from the gastrointestinal tract in rats and its subsequently high fecal elimination after oral exposure (Meerts et al., 1999; WHO, 1994), or its fast metabolism, especially to a monoglucuronide, which is excreted in the bile (Larsen et al., 1998).Reduced serum total and free T4 levels were also reported in mice and rats treated with the commercial mixture Bromkal 70 (containing about 40% of tetraBDE) and the pure congener 2,2',4,4'-tetraBDE (dosage 250 mg [= 515 µmol]/kg body weight; Darnerud and Sinjari, 1996). The mechanism of this reduction was not investigated, but these results demonstrate that thyroid hormone homeostasis might also be a sensitive target of PBDEs (or metabolites) in vivo. In our study, 2,2',4,4'-tetraBDE itself was not able to bind to TTR in vitro. Metabolic conversion of 10 µM 2,2',4,4'-tetraBDE with CYP 2B-induced microsomes gave rise to metabolites that competed with T4 for binding to TTR by more than 60%. The concentration of these metabolites could maximally be 250 nM (with 100% conversion; see Materials and Methods section), but HPLC analysis revealed that only 10% of the total BDE-47 was metabolized (Meerts et al., unpublished results). Obtaining quantitative information about the potency of the formed metabolites is not possible, but our results strongly suggest that hydroxylated PBDEs are able to compete with thyroxine for TTR binding in vitro.In conclusion, some brominated bisphenols and hydroxylated PBDEs were found to interact with human transthyretin in vitro with high affinity. The structural requirements of the brominated compounds were similar to those observed for the chlorinated compounds studied so far and also for the natural ligand itself. The resemblance between these brominated phenolic compounds and hydroxylated PCBs is striking with respect to TTR interaction. This suggests that at least some components of these classes of brominated flame retardants may also interfere in the thyroid hormone system in vivo and may cause possible adverse health effects similar to PCBs. Further studies are aimed at investigating the impact of the findings presented in this paper on the in vivo situation.
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ACKNOWLEDGMENTS |
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The authors would like to thank Sara Rahm of the Wallenberg Laboratory, Stockholm University, for the synthesis of the mono-, di- and tribromobisphenol A. This research was financially supported by the European Commission, Environment and Climate Program (ENV4-CT96-0170).
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1 To whom correspondence should be addressed. Fax: +31-317-484931. E-mail: ilonka.meerts{at}algemeen.tox.wau.nl.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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