* Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Received July 24, 2003; accepted September 1, 2003
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ABSTRACT |
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Key Words: polybrominated diphenyl ethers; Ah receptor agonist and antagonist; dioxin-like activity; cytochrome P4501A1.
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INTRODUCTION |
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Three commercial PBDE mixtures are presently in use, known as deca-, octa-, and penta-BDE. Unlike technical PCB mixtures, PBDE mixtures contain only limited numbers of congeners. Penta-BDE comprises approximately 43% of 2,2',4,4',5-penta-BDE (PBDE-99), 27% of 2,2',4,4'-tetra-BDE (PBDE-47), 7% of 2,2',4,4',6-penta-BDE (PBDE-100), and small amounts of 2,2',4,4',5,5'-hexa-BDE (PBDE-153) and 2,2',4,4'5,6'-hexa-BDE (PBDE-154) (Sjödin et al., 1998). Octa-BDE contains 44% of 2,2,'3,4,4',5,6-hepta-BDE (PBDE 183) as the major congener (Dodder et al., 2002
) and deca-BDE contains almost exclusively fully substituted deca-BDE (PBDE-209). Congeners 47, 99, and 100 have been identified in every compartment of the environment, including air (Strandberg et al., 2001
); sewage sludge and sediment (Hale et al., 2001
; Sellstrom et al., 1999
); freshwater fish (Dodder et al., 2002
; Manchester-Neesvig et al., 2001
); seals, dolphins, and deep-sea whales (de Boer et al., 1998
; Ikonomou et al., 2002
; Sellstrom et al., 1993
; She et al., 2002
), as well as in human blood (Klasson-Wehler et al., 1997
; Sjödin et al., 2001
), adipose tissue (Meironyte-Guvenius et al., 2001
; She et al., 2002
), and breast milk (Noren and Meironyte, 2000
; Ryan and Patry, 2001
).
The acute toxicity of commercial PBDEs is low (e.g., the oral LD50 of penta-BDE is ~0.55 g/kg body weight in rats). They appear to be nonmutagenic on the basis of negative Ames reversion assay with Salmonella typhimurium and lack of chromosome aberrations (Darnerud et al., 2001; Hardy, 2002
). Their carcinogenicity is unknown. The most sensitive end points of PBDE toxicity observed to date are endocrine disruption, developmental neurotoxicity, and dioxin-like behavior (Brouwer et al., 2001
). Induction of hepatic microsomal cytochrome P4501A was seen upon exposure of Wistar rats to commercial penta-BDE mixture Bromkal 70 (von Meyerinck et al., 1990
). Decreased circulating thyroid hormone T4 concentration and induced hepatic EROD (ethoxyresorufin-O-deethylase) and PROD (pentoxyresorufin-O-deethylase) activities have been reported in both dams and offspring of rats after developmental exposure to commercial mixtures (Zhou et al., 2002
).
We recently demonstrated that individual PBDE congeners and mixtures interact with the cytosolic Ah receptor (AhR), with binding affinity at least two orders of magnitude less than that of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (Chen et al., 2001). Certain PBDE congeners induced CYP1A1-dependent EROD activity in cell cultures from fish, chicken, rat, and human. The induction curves showed increasing activity at low concentrations and decreases at higher concentrations, with maximal enzyme activity ~70% of that induced by TCDD.
The objectives of the present study were to assess the dioxin-like activity of PBDE congeners and to determine in vitro whether they act as AhR agonists or antagonists at each stage of the Ah receptor signal transduction pathway. Specific end points were the ability to activate cytosolic AhR to bind a synthetic oligonucleotide containing the consensus sequence of a dioxin response element (DRE) and the ability to induce both cytochrome P4501A1 mRNA and protein in primary rat hepatocytes.
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MATERIALS AND METHODS |
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Rodent hepatic cytosol.
Hepatic cytosol was prepared from immature male Sprague-Dawley rats (~100 g, six in a group) according to the method described in Chen et al. (2001). Briefly, rat livers were perfused with ice-cold HEGD buffer, excised, washed, and homogenized with HEGD buffer. The homogenate was spun at 9000 x g for 20 min at 4°C and then spun at 100,000 x g for 68 min in an ultracentrifuge at 4°C. The cytosol was stored in 1 ml aliquots at -70°C until required. The protein content of the cytosol was determined by the method of Bradford (1976)
using Bio-Rad Protein Assay Dye Reagent with bovine serum albumin (BSA) as a standard.
Electrophoretic mobility shift assay (EMSA).
EMSA was carried out according to the method described in Chen et al. (2001). Briefly, the 32-base pair oligonucleotides containing DRE consensus binding sequence 5'-TGCGTG-3' were [32P] end-labeled using
-[32P]-ATP by T4 polynucleotide kinase and annealed by being heated to 85°C and allowed to cool slowly to room temperature. The [32P]-labeled oligonucleotide was then purified on a NICK Sephadex G-50 spin column according to the manufacturers instruction and stored at -20°C before use. Then, 1 µl of [32P]-labeled oligonucleotide was taken for liquid scintillation counting.
Aliquots of 100 µl cytosol (16 mg protein/ml) were incubated with either 1 µl DMSO (control), 10 nM TCDD (reference), or various concentrations of PBDE or mixtures of TCDD and PBDE at 30°C for 2 h. Aliquots of 5 µl liganded cytosol were incubated at 23°C with 500 ng poly (dIdC) for 15 min; 1 µl of [32P]-oligonucleotide (~500,000 cpm/µl) was then added and the samples were mixed and incubated for another 15 min at 23°C. After mixing with bromophenol blue tracking dye, the samples were then electrophoresed in a 5% polyacrylamide gel in TBE buffer at 11 V/cm for 1 h. Gels were removed, sealed in plastic wrap, and exposed to radiographic film. Following overnight exposure at -20°C, the film was developed using Kodak Developer and Kodafix fixing solution (Eastman Kodak Co., Rochester, NY).
Isolation and culture of primary rat hepatocytes.
Primary rat hepatocytes were prepared by a protocol of Kreamer (Kreamer, 1986) with modification described in Chen et al. (2001)
. Briefly, the rat liver was first blanched by EGTA buffer and then perfused with media containing collagenase. The digested liver was then excised, rinsed, and desegregated in a sterile 150 mm petri dish. The cells were filtered with gauze and spun for 3 min at 50 x g. The pellet was resuspended in 25 ml attachment media (Williams medium E supplemented with 10 mM HEPES, 2 mM L-glutamine, and 10% fetal bovine serum) combined with 24 ml of percoll in Hanks balanced salt solution and spun at 50 x g for 10 min. After the enrichment by the iso-density percoll purification, the cells were washed, spun, and the pellets were resuspended in 3040 ml of attachment media. The cells were then counted using a hemocytometer. The viability of the cells was > 90% as assessed by trypan blue exclusion. The cells were inoculated (3 x 106 cells/3.0 ml attachment media) in polystyrene tissue culture dishes (60 mm; Corning, Corning, NY) precoated with collagen. After 2 h, the medium was aspirated away and 3.0 ml serum-free media (Williams medium E supplemented with 10 mM HEPES, 2 mM L-glutamine, 10 mM pyruvate, and 0.35 mM proline) were added to each plate. The cells were then incubated for 22 h at 37°C (95% air, 5% CO2). After 24 h of preincubation, the medium was refreshed, the cells were treated with various concentrations of TCDD or PBDE congeners dissolved in DMSO, and the cells were incubated for another 24 h. The vehicle concentration in each treatment was less than 0.5% (v/v).
RNA isolation and Northern blot analysis for CYP1A1 mRNA.
After treatment and incubation with TCDD or PBDE congeners for 24 h, the hepatocytes were rinsed with sterilized PBS buffer and lysed using TrizolTM reagent at -70°C. Total RNA was extracted with Trizol/chloroform and purified by cold isopropanol precipitation and 75% ethanol washing. The concentration and purity of RNA was measured by spectrophotometry using absorbance at 260/280 nm, with absorbance ratio A260/A280 between 1.6 to 2.0. Next, 10 µg of total RNA sample were electrophoresed in a 0.8% agarose gel containing 2.2 M formaldehyde. RNA integrity was assessed by the relative intensities of distinctly separated 28S and 18S rRNA bands visualized on ethidium bromidestained gels. Then, the RNA was blotted to a nylon membrane (HybondTM) using capillary transfer with 10x SSC (150 mM sodium citrate, 1.5M NaCl, pH 7.0) buffer. The RNA was cross-linked at 120 mJ/cm2 to the membrane using FB-UVXL-1000 UV Cross-Linker (Fisher Scientific, Fair Lawn, NJ) for 12 s.
The transferred membrane was prehybridized for 1 h at 42°C in 50% deionized formamide, 6x SSPE (0.9 M NaCl, 60 mM NaH2PO4, and 2 mM EDTA [pH 7.0]), 5x Denhardts (1% Ficoll 400, 1% polyvinylpyrrolidone [PVP], and 1% BSA), 1% sodium dodecyl sulfate (SDS) solution containing 40 µg/ml denatured salmon sperm DNA, and then hybridized with a random primer [32P]-labeled cDNA probe (Rediprime II labeling system) for 12 h. Human CYP1A1 cDNA (3.6 kilobase, Oxford Biomedical Research) was used for CYP1A1 detection. 7S cDNA (0.28 kilobase) used for loading control was supplied by A. Balmain (Onyx Pharmaceuticals, Richmond, CA) and excised from pBluescript/KS+ using BamH1 (Amersham Corp.) restriction enzyme digestion. Hybridized blots were washed by 2x SSC and 1% SDS buffer twice and then by 0.1x SSC and 0.1% SDS buffer at 55°C for 15 min. Blots were then exposed to radiographic film for autoradiography. Densitometry of the autoradiographs was used to quantitate the CYP1A1 mRNA level.
Western blot for CYP1A1 protein assay in primary rat hepatocytes.
Exposures were terminated by aspirating the media; then, the monolayer cells were rinsed twice with 2 ml warm PBS (10 mM potassium phosphate buffer, pH 7.4, containing 0.14 M NaCl). Cells were removed from the dishes by scraping in ice-cold PBS, pelleted, and resuspended in HEGD (20 mM HEPES, 1 mM EDTA, 10% [v/v] glycerol [pH 7.6], and 1 mM DTE added just before use). The cells were lysed by sonication and stored at -70°C. Microsomes were prepared from control and treated cultures. Sonicated lysates were centrifuged for 10 min at 10,000 x g and supernatants were further centrifuged at 100,000 x g for 60 min. The pellets were resuspended in 100 µl HEGD. The protein content of the microsome was determined by the method of Bradford (1976) using Bio-Rad Protein Assay Dye Reagent with bovine serum albumin (BSA) as a standard.
The cell microsomes were diluted to equivalent amounts of protein (1520 µg) in 16 µl and mixed with 4 µl sample buffer (60 mM Tris-HCl [pH 6.8], 25% glycerol, 2% sodium dodecyl sulfate [SDS], 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue). After denaturation by boiling for 5 min, polypeptides were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) with Tris-glycine buffer system at 100 V for 1.5 h. Separated proteins were transferred onto nitrocellulose membranes at 100 V for 2 h or 30 V overnight. Following transfer, the membrane was blotted with 5% skim milk for 1 h to block nonspecific binding sites. Goat antirat CYP1A1 polyclonal antibody (1:1000 dilution in 1% gelatin PBS) was applied to the membrane for 1 h. After washing with TTBS, the membrane was incubated with alkaline phosphatase (AP-) conjugated antigoat IgG (1:5000 dilution) for 1 h; then, the color was developed by the AP-conjugated substrate kit according to the manufacturers instructions. The blotted, dried membrane was then scanned and quantitated by densitometry using SigmaGel software.
Statistical analysis.
The major experiments were repeated at least three times and the results are expressed as means ± standard deviation. Statisticaldifferences (p < 0.05) between the groups were determined by one-tailed, paired Students t-test.
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RESULTS |
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Several cotreatments of primary rat hepatocytes with PBDEs and 0.1 nM TCDD were examined for their effect on CYP1A1 mRNA induction. PBDE congeners 47, 77, 85, 119, and penta-BDE mixture were studied separately over a range of concentrations; triplicate experiments of 10 µM of these representative PBDE congeners and of the penta mixture with 0.1 nM TCDD were also carried out (supplemental data Fig. B). In contrast with the foregoing results on DRE activation, none of the PBDEs significantly changed the induced mRNA level upon admixture with 0.1 nM TCDD (Fig. 6), probably due to their weaker competitive binding with Ah receptor and because a low level of activated nuclear AhR by TCDD suffices to maintain the transcription of the CYP1A1 gene (Okey et al., 1994
).
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CYP1A1 protein induction by PBDE congeners paralleled CYP1A1 mRNA induction (R2 = 0.90, supplemental data Fig. D). In both cases, TCDD and PBDEs 77, 119, and 126 gave a strong response while PBDEs 66, 85, 100, 153, 156, and 183 gave a moderate response, the latter showing maximal protein levels 5080% that of 1 nM TCDD (Fig. 8B). The commercial penta-BDE and octa-BDE mixtures were very weak CYP1A1 inducers; only 8% of the maximal CYP1A1 induction level was found for the penta-BDE mixture and about 20% for the octa-BDE mixture, both at the highest test concentration (supplemental data Fig. E). Induction of CYP1A1 by PBDEs 47 and 99 and by the commercial deca-BDE was negligible.
PBDEs 77, 85, and 119 behaved additively with 50 pM TCDD upon cotreatment in primary rat hepatocytes (PBDE 77 as example shown in supplemental data Fig. F). PBDE 47 and the penta-BDE commercial mixture acted as weak inhibitors of TCDD-induced formation of CYP1A1, but only at high concentrations (Fig. 9); the penta-BDE commercial mixture was more strongly inhibitory (Fig. 10
).
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DISCUSSION |
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Our results paint a consistent picture in which the congeners that bind most strongly to the AhR are also the strongest inducers of CYP1A1 mRNA and CYP1A1 protein (cf. Safe, 1990). An important finding is that environmentally prominent congeners, such as PBDEs 47 and 99, are among the least active with respect to dioxin-like behavior. AhR-binding affinity correlated with induction of both CYP1A1 mRNA (Fig. 11
, R2 = 0.84) and CYP1A1 protein (data not shown, R2 = 0.64). Between CYP1A1 mRNA and CYP1A1 protein induction we observed R2 = 0.90 (supplemental data Fig. D). A weak correlation was observed between AhR-binding affinity and AhR-DRE complex formation (R2 = 0.50). These results show that the production of CYP1A1 induced by PBDEs is AhR-mediated, as it is for numerous organochlorines, even though PBDEs do not readily adopt the planar conformation usually considered characteristic of AhR ligands (Chen et al., 2001
).
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An important objective was to determine whether PBDEs augment or suppress signal transduction due to TCDD. The most active congeners (PBDEs 77, 119, and 126) tended to enhance the behavior of a nonsaturating level of TCDD and to inhibit slightly the effect of a saturating concentration of TCDD. Weakly active and inactive congeners had little effect in combination with a nonsaturating level of TCDD but strongly inhibited a saturating concentration. These results are explicable in terms of target molecule antagonism, with the AhR as the target molecule (Petrulis and Bunce, 1999). The presence of a second active ligand such as PBDE 77 increased the concentration of transductionally active AhR molecules when there was insufficient TCDD to saturate all AhR-binding sites but had little effect when the concentration of TCDD was saturating. Less active congeners such as PBDE 47 did not increase the yield of transductionally active AhR in the presence of a nonsaturating concentration of TCDD and acted as antagonists when the AhR was saturated by TCDD.
To summarize the behavior of PBDEs at each stage of signal transduction, PBDE congeners bind the rat cytosolic AhR two to five orders of magnitude less strongly than TCDD, similar to mono-ortho-PCBs (cf. Safe, 1990). The behavior of the commercial mixtures is explicable in terms of the AhR affinities of their major constituents (Chen et al., 2001
). Concerning in vitro activation of AhR to a DRE-binding form, PBDE 119 was the only full agonist; it acted additively in combination with TCDD at both high and low concentrations. PBDEs 77 and 126, despite being stronger AhR ligands, were only partial agonists either alone or in combination with TCDD. PBDE 85 was exceptional; although it exhibited the highest affinity for the AhR, it did not activate the AhR toward DRE binding and completely inhibited DRE binding by TCDD. These results show that care must be taken in interpreting EMSA results: synthetic oligonucleotides that contain consensus-binding sequences are not equivalent to natural DNA. Factors neglected include cell uptake, metabolism, and the poorly understood transformation from cytoplasm to nucleus (Denison et al., 1999
). EMSA is also a rather insensitive end point (e.g., for TCDD, EMSA has EC50 300 pM compared with EC50 = 20 pM in the EROD assay).
PBDEs 77, 119, and 126 were full agonists with respect to CYP1A1 mRNA and protein induction; they did not antagonize the action of TCDD. PBDE congeners 100, 153, 156, and 183 were partial agonists towards these end points; they showed no antagonism with 0.1 nM TCDD in CYP1A1 mRNA induction or with 1 nM TCDD in CYP1A1 protein induction. PBDEs that bound weakly to the AhR failed to inhibit mRNA induction by TCDD, even at concentrations that would have occupied a substantial fraction of AhR, because a low level of activated nuclear AhR suffices to maintain the maximal transcription of the CYP1A1 gene (Okey et al., 1994). Because the mRNA was not inhibited, CYP1A1 protein induction by 1 nM TCDD was also not inhibited by PBDEs, the exceptions being PBDE 47 and penta-BDE (only at 10 µM).
The induction of CYP1A1 by PBDEs shows an apparently different response according to whether Western blotting or EROD activity is chosen as the end point (cf. Hahn et al., 1996, on the responses of 2,3,7,8-TCDF and PCBs 77 and 126 in fish cell culture). In Western blotting, plots of protein level versus inducer concentrations show typical saturation behavior, whereas EROD activity frequently displays biphasic behavior, as noted for PBDEs (Chen et al., 2001
) and many other inducers (Hahn et al., 1993
, 1996
; Kennedy et al., 1996
; Sawyer and Safe, 1982
; Verhallen et al., 1997
). Explanations for the decreased EROD activity at higher inducer concentrations include cytotoxicity, direct inhibition by the inducers, decreasing protein level of CYP1A, and impaired heme synthesis (Hahn et al., 1993
). We observed neither cytotoxicity nor decrease of immunodetectable CYP1A1 level for PBDEs at concentrations that inhibited the EROD reaction. The biphasic EROD induction curves found for PBDE congeners in primary rat hepatocytes can be explained in terms of competitive inhibition of EROD activity by PBDEs (cf. Petrulis et al., 2001
). The significance is that the most potent PBDE congeners (77, 119, and 126) only demonstrated 6075% activation of EROD activity even at the highest concentration; based on EROD data alone, they would be defined as partial agonists even though they are full agonists according to Western blot analysis. Competitive inhibition also explains the antagonism of EROD activity by PBDE congeners and TCDD, but not of CYP1A1 mRNA or CYP1A1 protein levels, in primary rat hepatocytes. Although EROD assays are much faster and more convenient than Western blot assays, they are unfortunately more difficult to interpret. Parallel to their weak induction of CYP1A1 protein, PBDE congeners induced luciferase activity only weakly compared with TCDD in H4IIE CALUX cells, a recombinant H4IIE rat hepatoma cell line stably transfected with an AhR-mediated luciferase reporter gene (Meerts et al., 1998
).
Penta-BDE and octa-BDE mixtures were inactive in AhR signal transduction except for weak CYP1A1 protein induction (820% that of TCDD). This is consistent with their modest EROD activity in vivo (von Meyerinck et al., 1990; Zhou et al., 2001
), although they showed no EROD activity in our cell cultures (Chen et al., 2001
). Deca-BDE neither bound the AhR (Chen et al., 2001
) nor induced hepatic enzyme activity (Carlson, 1980
; Zhou et al., 2001
) because of its very low water solubility.
We conclude by setting the dioxin-like toxicity of PBDEs in the context of risk assessment. Table 1 compares the EC50 values of PBDEs with those of TCDD for the end points discussed in this article and previously (Chen et al., 2001
). Relative induction potencies (REPs) of the most active PBDEs towards CYP1A1 are ~0.0001 compared with TCDD; these values are similar to some mono-ortho-PCBs and two orders of magnitude less than those of coplanar PCBs. For the environmentally prominent congeners like PBDE 47, REPs are essentially zero, indicating that PBDEs are no more than small contributors to the total TEQ "dioxin load." For example, from the data of Ryan and Patry (2001)
concerning PBDE concentrations in human milk, we calculate the contribution of PBDEs to be 0.0069 pg TEQ per gram lipid (Table 2
). Reported TEQs based on PCDD/Fs and PCBs are 30 pg TEQ/g lipid from Swedish human milk collected in 1996 (Noren and Meironyte, 2000
) and 17.4 and 9.2 pg TEQ/g lipid from Canadian human milk collected in 1992 and 2002, respectively (J. J. Ryan, personal communication). At the present time, PBDEs contribute negligibly to the TEQ due to halogenated aromatic hydrocarbons, although we caution that PBDE levels in the environment are rising while those of PCBs, PCDDs, and PCDFs are falling (cf. Rayne et al., 2003
). Moreover, our research has addressed only the dioxin-like behavior of PBDEs and does not exclude toxicities not mediated by the AhR, such as endocrine disruption and neurotoxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at the University of Guelph, Department of Chemistry and Biochemistry, Guelph, Ontario, Canada N1G 2W1. Fax: (519) 766-1499. E-mail: bunce{at}chembio.uoguelph.ca.
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