Biotransformation of 2,2â²,4,4â²-Tetrabromodiphenyl Ether (BDE-47

Article pubs.acs.org/crt

Biotransformation of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) by Human Liver Microsomes: Identification of Cytochrome P450 2B6 as the Major Enzyme Involved Claudio A. Erratico, András Szeitz, and Stelvio M. Bandiera* Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 S Supporting Information *

ABSTRACT: Polybrominated diphenyl ethers (PBDEs) were widely used flame retardants that have become persistent environmental pollutants. In the present study, we investigated the in vitro oxidative metabolism of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), a major PBDE detected in human tissue and environmental samples. Biotransformation of BDE47 by pooled and individual human liver microsomes and by human recombinant cytochrome P450 (P450) enzymes was assessed using a liquid chromatography/tandem mass spectrometry-based method. Of the nine hydroxylated metabolites of BDE-47 produced by human liver microsomes, seven metabolites were identified using authentic standards. A monohydroxy-tetrabrominated and a dihydroxy-tetrabrominated metabolite remain unidentified. Kinetic analysis of the rates of metabolite formation revealed that the major metabolites were 5-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE47), and possibly the unidentified monohydroxy-tetrabrominated metabolite. Among the human recombinant P450 enzymes tested, P450 2B6 was the most active enzyme in the formation of the hydroxylated metabolites of BDE-47. Moreover, the formation of all metabolites of BDE-47 by pooled human liver microsomes was inhibited by a P450 2B6-specific antibody and was highly correlated with P450 2B6-mediated activity in single donor liver microsomes indicating that P450 2B6 was the major P450 responsible for the biotransformation of BDE-47. Additional experiments involving the incubation of liver microsomes with individual monohydroxy-tetrabrominated metabolites in place of BDE-47 demonstrated that 2,4-dibromophenol was a product of BDE-47 and several primary metabolites, but the dihydroxy-tetrabrominated metabolite was not formed by sequential hydroxylation of any of the monohydroxy-tetrabrominated metabolites tested. The present study provides a comprehensive characterization of the oxidative metabolism of BDE-47 by human liver microsomes and P450 2B6.

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umbilical cord blood8−10 and is the dominant PBDE congener in human plasma and serum.11−14 Furthermore, BDE-47 levels are higher in human samples from North America than from Europe and Asia because of the more extensive use of the Penta-BDE mixture in North America.8,9 Human exposure is a health concern because evidence from animal studies suggests that treatment with BDE-47 during development can alter thyroid hormone homeostasis and produce neurobehavioral disturbances. For example, circulating thyroxine levels were decreased in rats and mice following oral exposure to 18 mg of BDE-47/kg body weight/day for 14 days.15 Decreased ovarian weight and alterations in folliculogenesis were noted in female rats at postnatal day 38 following exposure on gestational day 6 to 140 or 700 μg of BDE-47/kg body weight,16 respectively. Moreover, increased locomotor activity was observed in rats that were developmentally treated with BDE-47,17 and hyperactivity and delayed development of

INTRODUCTION Polybrominated diphenyl ethers (PBDEs) were used as additive flame retardant chemicals on a variety of industrial and commercial products for more than 30 years. PBDEs were formulated as commercial mixtures known as Penta-, Octa-, and Deca-BDEs, according to their average bromine content. The Penta-BDE mixture consisted mostly of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) and was predominantly used in North America.1 Because of environmental and human health concerns, the manufacture of the Penta-BDE mixture was voluntarily discontinued by the major U.S. manufacturer2 and in the European Union in 2004.3,4 Because PBDEs were blended with, rather than chemically bound to, polymers during the manufacture of textiles, polyurethane foam, plastics, and other products,5 PBDEs tend to leach out of these products over time.6 As a consequence, PBDEs are found in air, water, household dust, fish, birds, mammals, and people.7 More specifically, BDE-47 has been detected in human serum, adipose tissue, breast milk, and © 2013 American Chemical Society

Received: December 27, 2012 Published: March 28, 2013 721

dx.doi.org/10.1021/tx300522u | Chem. Res. Toxicol. 2013, 26, 721−731

Chemical Research in Toxicology

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phenyl ether (5-OH-BDE-47), 6-OH-2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE-47), 4′-OH-2,2′,4,5′-tetrabromodiphenyl ether (4′-OH-BDE-49), and 4′-OH-2,2′,4,6′-tetrachlorobiphenyl (4′-OHCB-50; neat, 99.9% purity) were obtained from AccuStandard (New Haven, CT). Note: there are 209 possible PBDE congeners, which are numbered BDE-1 to BDE-209. Further information about PBDE nomenclature is available in the literature.33 2,4-Dibromophenol (2,4DBP) and NADPH were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). Hydrochloric acid, sodium hydroxide, and organic solvents (HPLC grade or better) were purchased from Fisher Scientific (Ottawa, Ontario, Canada). Ultrapure water was prepared using a Millipore Milli-Q system (Billerica, MA). Pooled human liver microsomes (mixed gender, n=50) were purchased from Xenotech (Lenexa, Kansas). Single donor human liver microsomes were purchased from Xenotech (H0426, H0435, H0442, H0444, and H0455) and BD Biosciences (HG95, HH13, HH18, and HH837; Oakville, Ontario, Canada). Baculovirus-insect cell microsomes containing expressed human P450 enzyme (P450 1A1, P450 1A2, P450 1B1, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1, P450 3A4, or P450 3A5) coexpressed with human P450-oxidoreductase or with human P450-oxidoreductase and human Cytochrome b 5 (BD SUPERSOMES Enzymes) were purchased from BD Biosciences. Human P450 2B6 selective mouse monoclonal antibody (MAB-2B6) was obtained from BD Biosciences. Polyclonal antibody against rat epoxide hydrolase was raised in female New Zealand rabbits immunized with the electrophoretically homogeneous protein, which was purified from Long Evans rats using the method described by Ryan et al.34 IgG was purified from a pool of heat-inactivated high-titer sera obtained from multiple bleedings from several rabbits. The specificity of the antibody relative to purified enzymes and hepatic microsomal proteins was assessed using enzyme-linked immunosorbent assays and immunoblot analysis. Rabbit antiepoxide hydrolase reacted with rat, mouse and human epoxide hydrolase but not with rat, mouse, or human P450 enzymes or other microsomal proteins. BDE-47 Biotransformation Assay. The in vitro biotransformation assay for BDE-47 was conducted as previously described.35 Briefly, reaction mixtures containing 50 mM potassium phosphate buffer with 3 mM magnesium chloride (pH 7.4), human liver microsomes (0.1 mg/mL final protein concentration), and BDE-47 (0.5 to 200 μM final concentration), in a volume of 0.99 mL, were preincubated for 5 min in a shaking water bath at 37 °C. Reactions were initiated by addition of 0.01 mL of NADPH solution (1 mM final concentration) and terminated after 5 min by addition of 1 mL of icecold 0.5 M sodium hydroxide. Internal standard (4′-OH-CB-50) was then added to each tube to give a final concentration of 0.5 μM. Blank and negative control samples in which BDE-47, hepatic microsomes, or NADPH was omitted from the reaction mixture were routinely included in each assay. Hydroxylated metabolites were extracted as previously described.35 Using pooled human liver microsomes, preliminary experiments were conducted to determine linearity of product formation with respect to incubation time and hepatic microsomal protein concentration. Unless indicated otherwise, samples were prepared in duplicate and replicate experiments were conducted on three different days. For incubations with human recombinant P450 enzymes, reaction mixtures contained 50 mM potassium phosphate buffer with 3 mM magnesium chloride (pH 7.4), individual P450 enzymes (at a concentration of 10 pmoles of recombinant P450/mL), and BDE-47 (100 μM final concentration). The incubation time was 10 min. Additional experiments were performed with human recombinant P450 2B6 to determine linearity of product formation with respect to incubation time and P450 concentration. On the basis of the results obtained, metabolite formation was measured over a range of BDE-47 concentrations (0.5 to 200 μM final concentration) using human recombinant P450 2B6 at 5 pmol/mL and an incubation time of 5 min. Insect cell control microsomes containing expressed human P450-oxidoreductase were used as a control at an equivalent amount of protein (50 μg). Incubations with recombinant P450 enzymes were performed on three different days using single samples.

neuromotor function were observed in mice following a single treatment with 10 or 30 mg of BDE-47/kg body weight on postnatal day 10.18,19 In addition, BDE-47 disrupted the release of vasopressin, a neuropeptide that regulates blood volume and osmolality, from rat hypothalamic neurosecretory cells in vitro.20 Several hydroxylated PBDEs have been shown to be more potent than PBDE congeners as disruptors of thyroid activity in vitro.21−27 Oxidative biotransformation can be an important determinant of the toxicological effects and bioaccumulation of PBDEs. Additional information regarding the in vitro hepatic biotransformation of BDE-47 in humans would be beneficial. Two hydroxylated metabolites, 2,4-dibromophenol (2,4-DBP) and an unidentified dihydroxy-tetrabrominated PBDE, were detected following the incubation of human liver microsomes with BDE-47 for 2 h.28,29 More recently, three different hydroxylated metabolites (namely, 5-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (5-OH-BDE-47) and 6-hydroxy-2,2′,4,4′tetrabromodiphenyl ether (6-OH-BDE-47), and an unidentified dihydroxy-tetrabrominated PBDE were detected when human primary hepatocytes were incubated with BDE-47 for 24 h.30 In another study, three hydroxylated metabolites (namely, 3hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (3-OH-BDE-47), 5-OH-BDE-47, and 6-OH-BDE-47) were produced when human liver microsomes were incubated with BDE-47 for 60 min.31 In the same study, four additional metabolites, namely, 4-hydroxy-2,2′,3,4′-tetrabromodiphenyl ether (4-OH-BDE-42), 4′-hydroxy-2,2′,4,5′-tetrabromodiphenyl ether (4′-OH-BDE49), a monohydroxy-tetrabrominated-PBDE (tentatively identified as 2′-OH-BDE-66), and an unidentified dihydroxytetrabrominated-PBDE metabolite were produced by human recombinant P4502B6.31 Monohydroxy-PBDEs were also detected in human blood samples of women and children who were environmentally12−14 or occupationally11 exposed to PBDE mixtures. Taken together, these studies suggest that BDE-47 is biotransformed to hydroxylated PBDEs by human liver in vitro and in vivo, and that BDE-47 is metabolized by recombinant P450 2B6. By comparison, BDE-99 was recently shown to be metabolized by human liver microsomes and P450 2B6 to 10 hydroxylated metabolites.32 The aims of the present study were to identify the hydroxylated metabolites of BDE-47 formed by human liver microsomes using authentic standards, to measure their rates of formation and to identify the P450 enzymes involved in metabolite formation. Using an improved liquid chromatography/tandem mass spectrometry-based assay, we identified seven hydroxylated metabolites of BDE-47, quantified the rates of formation of four metabolites, and conducted enzyme kinetic analysis to obtain apparent Km and Vmax values associated with metabolite formation. A combined approach involving a panel of human recombinant P450 enzymes, P450 2B6-specific inhibitory antibody, and correlation analysis with single donor liver microsomal incubations was used to assess the contribution of individual P450 enzymes to BDE-47 biotransformation. In addition, possible mechanisms of BDE-47 secondary metabolite formation were investigated.

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EXPERIMENTAL PROCEDURES

Chemicals and Reagents. BDE-47 (neat, ≥ 99.2% purity), 4′hydroxy-2,2′,4-tribromodiphenyl ether (4′-OH-BDE-17), 2′-hydroxy2,4,4′-tribromodiphenyl ether (2′-OH-BDE-28), 4-hydroxy-2,2′,3,4′tetrabromodiphenyl ether (4-OH-BDE-42), 3-OH-2,2′,4,4′-tetrabromodiphenyl ether (3-OH-BDE-47), 5-OH-2,2′,4,4′-tetrabromodi722



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Figure 1. Chemical structures of hydroxylated metabolites of BDE-47 formed by human liver microsomes. General structures for M1 and M2 are shown. To investigate whether 2,4-DBP, 4′-OH-BDE-17, 2′-OH-BDE-28, and the dihydroxy-tetrabrominated metabolite were produced directly from BDE-47 or from primary hydroxylated metabolites of BDE-47, pooled human liver microsomes were incubated with 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, or 4′-OH-BDE-49 (5 to100 nM, final concentrations) instead of BDE-47. In addition, pooled human liver microsomes were incubated with with BDE-47 (25 μM) plus increasing concentrations of 4-OH-BDE-42 or 4′-OH-BDE-49 (0 to 150 nM). Samples were prepared in duplicate and one experiment was conducted. Antibody Inhibition Experiments. Reaction mixtures containing pooled human liver microsomes (0.1 mg/mL) in 50 mM potassium phosphate buffer with 3 mM magnesium chloride (pH 7.4) were preincubated for 10 min in a shaking water bath at 37 °C with various amounts of mouse antihuman P450 2B6 ascites fluid or control mouse serum (0.5 to 7.5 μL of ascites/mg microsomal protein), or with various amounts of rabbit antirat microsomal epoxide hydrolase IgG or rabbit control IgG (0.1 to 2.0 mg IgG/mg microsomal protein). BDE47 at 50 or 10 μM (final concentration) was then added and the mixtures were incubated for further 5 min at 37 °C. A BDE-47 concentration that was approximately equal to the apparent Km value(s) associated with the formation of the metabolites of BDE-47 of interest (i.e., 50 μM for monohydroxy-tetrabrominated or 10 μM for the dihydroxy-tetrabrominated metabolite) was selected for these

experiments so that the effect of the antibody would be more apparent. Reactions were initiated by addition of NADPH (1 mM final concentration), allowed to proceed for 5 min, and stopped by addition of 1.0 mL of ice-cold 0.5 M sodium hydroxide. Hydroxylated metabolites were extracted as previously described.35 Samples were prepared in duplicate and two separate experiments were conducted. Analytical Method. Hydroxylated metabolites of BDE-47 were identified and quantified using an improved version of a previously validated ultra performance liquid chromatography−mass spectrometry method.35 Improvements include a modified elution program, a longer column, and ultra high performance liquid chromatography coupled with tandem mass spectrometry analysis (UHPLC/MS/MS). The UHPLC/MS/MS system consisted of an Agilent 1290 Infinity UHPLC coupled with an AB Sciex QTrap 5500 hybrid linear ion-trap triple quadrupole mass spectrometer equipped with a Turbo Spray source (Concord, Ontario, Canada). Chromatographic separation of 2,4-DBP, 4′-OH-BDE-17, 2′-OH-BDE-28, 4-OH-BDE-42, 3-OHBDE-47, 5-OH-BDE-47, 6-OH-BDE-47, 4′-OH-BDE-49, M1, M2, and 4′-OH-CB-50 (internal standard) was achieved using a Waters Acquity UPLC BEH130 C18 column (150 × 2.1 mm, 1.7 μm particle size) and the following mobile phase composition: water with 0.1% formic acid (A), methanol with 0.1% formic acid (B). The elution was isocratic with 70% B for 45 min at a flow rate of 0.2 mL/min. The injection volume was 15 μL. The mass spectrometer was operated in 723



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electrospray negative ionization mode with the ion spray voltage of −4500 V, curtain gas 20, nebulizing gas 18, desolvation gas 30 units, and temperature 300 °C. The analytes were detected in multiple reaction monitoring mode and identified by comparison of their retention time and the isotopic mass to charge transition (parent/ daughter ions) values with those of authentic standards (Table S1, Supporting Information). Range, linearity, and limit of quantification values (Table S2, Supporting Information) were determined as reported previously36 using 1.5.2 software (Concord, Ontario, Canada). Quality Control. A calibration curve and quality control samples were prepared with each set of unknown samples to assess the linearity, accuracy, and precision values of the assay as previously described.36 Hepatic microsomes diluted in 50 mM potassium phosphate buffer (0.1 mg/mL final protein concentration) were spiked with authentic metabolite standards for 2,4-DBP, 4′-OH-BDE17, 2′-OH-BDE-28, 4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 6OH-BDE-47, and 4′-OH-BDE-49 at 2.5, 5.0, 10, 25, 50, 100, 250, and 500 nM (final concentrations) to prepare calibration samples and at 7.5, 80, and 400 nM (final concentrations) to obtain quality control samples (QC-Low, QC-Mid, and QC-High, respectively). The acceptance criteria were assessed on an interday basis as previously described.36 Interday accuracy and precision values are reported in the Supporting Information file. Data Analysis. Metabolite formation as a function of substrate concentration was analyzed by nonlinear regression analysis using the SigmaPlot Enzyme Kinetics Module (v.1.1, Systat Software Inc., Richmond, CA). Apparent Km, Ki, and Vmax values were calculated using the Michaelis−Menten equation, the Hill equation, or the substrate inhibition equation37 depending on which equation best-fit the data. Several criteria including sum of squares, standard deviation of residuals, Akaike Information Criterion (corrected for limited sample set), and visual inspection of the fit were considered in selecting the best-fit equation for each data set.

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RESULTS Identification of BDE-47 Metabolites. Biotransformation of BDE-47 by pooled human liver microsomes was assessed using a protein concentration of 0.1 mg/mL, an incubation time of 5 min, and a BDE-47 concentration of 0.5 to 200 μM. Rates of metabolite formation were determined to be linear with respect to the incubation time and protein concentration used. Under these conditions, nine metabolites were routinely detected. Seven metabolites were identified as 2,4-DBP, 4′-OHBDE-17, 2′-OH-BDE-28, 4-OH-BDE-42, 5-OH-BDE-47, 6OH-BDE-47, and 4′-OH-BDE-49 using authentic standards (Figure 1). A peak with m/z transition values corresponding to a monohydroxy-tetrabrominated-PBDE (M1) was detected, but its retention time did not match those of the available authentic standards. Similarly, a peak with m/z transition values corresponding to a dihydroxy-tetrabrominated-PBDE (M2) was detected but not identified because of the lack of an authentic standard. Formation of 3-OH-BDE-47 was investigated but could not be detected. A representative chromatogram showing the hydroxylated metabolites of BDE-47 is presented in Figure 2. Metabolite formation was not observed when BDE-47, hepatic microsomes, or NADPH was omitted from the reaction mixture suggesting the involvement of P450 enzymes. Kinetic Analysis of Hydroxylated BDE-47 Metabolite Formation by Human Liver Microsomes. After the establishment of initial rate conditions, metabolite formation rates were measured over a range of BDE-47 concentrations (0.5 to 200 μM) using pooled liver microsomes to determine values for enzyme kinetic parameters. Formation of 5-OHBDE-47, 6-OH-BDE-47, 4-OH-BDE-42, 4′-OH-BDE-49, and

Figure 2. Representative UHPLC/MS/MS chromatograms of the hydroxylated metabolites of BDE-47 formed by pooled human liver microsomes: monohydroxy-tetrabrominated metabolites (A), the dihydroxy-tetrabrominated metabolite (B), monohydroxy-tribrominated metabolites (C), and 2,4-dibromophenol (D). Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-47 (100 μM) for 5 min. Hydroxylated metabolites were detected with the mass spectrometer in electrospray negative mode and identified by comparison of the transition values (reported on each panel above) and retention times obtained with those of the authentic standards.

M1 was best described by Michaelis−Menten kinetics, whereas the formation of M2 was best described by the substrate inhibition kinetic model (Figure 3). Kinetic analyses of 4′OHBDE-17 and 2′-OH-BDE-28 formation were not conducted because levels of these metabolites were below the limit of quantification over much of the BDE-47 concentration range tested (data not shown). Formation of 2,4-DBP was observed only at BDE-47 concentration greater than 10 μM and was not adequately described by any of the kinetic models tested. Therefore, no kinetic parameter values associated with 2,4-DBP formation were generated. Apparent Vmax, Km, and Ki values, calculated using the Michaelis−Menten or substrate-inhibition equations, as appropriate, are listed in Table 1. The highest apparent Vmax values were obtained for 5-OH-BDE-47 and 6-OH-BDE-47, indicating that these were the major hydroxylated metabolites of BDE-47 produced by human liver microsomes. On the basis of relative metabolite peak size (Figure 2), we suggest that M1 is a major metabolite also. The apparent Km values suggest that 4′-OHBDE-49 and M2 are preferentially formed at low BDE-47 concentrations. 724



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Figure 3. Enzyme kinetic profiles of 5-OH-BDE-47 (A), 6-OH-BDE-47 (B), 4-OH-BDE-42 (C), 4′-OH-BDE-49 (D), M1 (D), and M2 (F) formation by human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-47 (0.5 to 200 μM) for 5 min. Data points are the mean ± SD of three separate experiments. Lines represent rates of metabolite formation modeled by nonlinear regression analyses. The insets depict Eadie−Hofstee plots. Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values (n = 3). Because 2,4-DBP, 4′-OH-BDE-17, and 2′-OH-BDE-28 were only quantifiable at the highest BDE-47 concentrations used, their enzyme kinetic profiles could not be determined.

Table 1. Apparent Vmax, Km, and Ki Values for the Formation of Hydroxylated Metabolites of BDE-47 by Human Liver Microsomesa BDE-47 metabolite 4-OHBDE-42 5-OHBDE-47 6-OHBDE-47 4′-OHBDE-49 M1 M2

apparent Vmax (pmol/min/mg protein)

apparent Vmax (response/min/ mg protein)

apparent Km (μM)

14 ± 4.1

44 ± 42

27 ± 3.9

36 ± 15

23 ± 3.4

25 ± 6.3

13 ± 3.0

10 ± 2.9 5.7 ± 1.9 0.55 ± 0.13

23 ± 3.9 6.6 ± 4.3

active enzyme and catalyzed the conversion of BDE-47 to all nine hydroxylated metabolites (Figure 4). Formation of 5-OHBDE-47 and 6-OH-BDE-47 was also catalyzed by recombinant P450 3A4 but at a rate that was 0.93) values were obtained

apparent Ki (μM)

86 ± 42

Values represent the mean ± SD of three independent experiments. Rates of M1 and M2 formation could not be expressed as pmol/min/ mg protein because of the lack of authentic standards and are reported as response/min/mg protein.

a

Identification of the P450 Enzymes Involved in the Oxidative Biotransformation of BDE-47. To identify the human P450 enzymes responsible for the formation of the hydroxylated metabolites, BDE-47 was incubated with 12 recombinant P450 enzymes individually. Among the human recombinant P450 enzymes tested, P450 2B6 was the most 725



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Figure 4. Rates of formation of 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, 4′-OH-BDE-49, M1, and M2 following the incubation of BDE-47 with twelve human recombinant P450 enzymes. Individual human recombinant P450 enzymes (10 pmol/mL) or an equivalent amount of oxidoreductase (OR, 50 μg) were incubated with BDE-47 (100 μM) for 5 min. Data points are the mean ± SD of three separate experiments. 2,4DBP, 4′-OH-BDE-17, and 2′-OH-BDE-28 were also formed by recombinant P450 2B6 only but were not included in the figure for the sake of clarity.

Figure 5. Effect of mouse antihuman P450 2B6 ascites on the formation of 5-OH-BDE-47 (A), 6-OH-BDE-47 (B), 4-OH-BDE-42 (C), 4′-OHBDE-49 (D), M1 (D), and M2 (F). Pooled human liver microsomes (0.1 mg/mL) were preincubated for 10 min with various amounts of anti-P450 2B6 ascite fluid (open symbols) or control mouse serum (closed symbols). BDE-47 (10 or 50 μM) was then added, and the reaction mixtures were incubated for a further 5 min. Data are expressed as the percent of the activity measured with no antibody. Data points represent the average of two experiments.

oxidative biotransformation of BDE-47 by human liver microsomes. Kinetic Analysis of Metabolite Formation by Recombinant P450 2B6. Rates of metabolite formation were determined over a BDE-47 concentration range of 0.5 to 200 μM using 5 pmol of P450 2B6/mL and an incubation time of 5 min (Figure S1, Supporting Information). Under these assay conditions, metabolite formation was determined to be first order with respect to incubation time and protein concen-

when rates of BDE-47 hydroxylated metabolite formation were compared with rates of 4-OH-bupropion formation (Table 3). In contrast, smaller values for the correlation coefficient (0.26 to 0.66) were obtained when rates of BDE-47 hydroxylated metabolite formation were compared with P450 1A2, P450 2C9, P450 2C19, P450 2E1, and P450 3A4 marker enzyme activities (Table 3), providing further evidence that P450 2B6 was the P450 enzyme predominantly responsible for the 726



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Table 2. Rates of Formation of Metabolites of BDE-47 by Single Donor Human Liver Microsomesa rates of identified metabolite formation (pmol/min/mg protein) BDE-47 metabolite

HG95

HH837

HH13

HH18

H0435

2,4-DBP 2′-OH-BDE-17 4-OH-BDE-28 4-OH-BDE-42 5-OH-BDE-47 6-OH-BDE-47 4′-OH-BDE-49

1.8 BDLb BDLb BDLb 2.4 2.3 1.7

36 3.4 4.6 20 68 67 56

BDE-47 metabolite

HG95

HH837

HH13

HH18

H0435

H0426

M1 M2

1.3 0.023

71 1.8

31 0.42

21 0.68

3.7 0.063

120 4.3

14 14 2.5 1.1 2.3 1.2 10 8.0 32 24 29 22 25 16 rates of unidentified metabolite

H0426

H0455

H0442

H0444

26 2.3 2.9 14 52 46 35

38 5.0 6.4 37 94 92 87

H0455

H0442

H0444

22 0.69

45 1.6

100 1.1

2.3 60 11 0.32 5.4 1.3 BDLb 6.9 1.5 0.75 43 7.8 3.7 140 20 3.7 120 20 3.0 97 16 formation (response/min/mg protein)

Single donor human liver microsomes (0.1 mg/mL) were incubated with BDE-47 (100 μM) for 5 min as described in the Experimental Procedures section. bBDL, below detection limit.

a

BDE-28 formation was not undertaken because the amounts of metabolite formed were often below the limit of quantification. The kinetic models did not provide a satisfactory fit with the data for 2,4-DBP and M2 formation (data not shown). Therefore, no kinetic parameter values were generated for these metabolites. Investigation into the Mechanism of M2 and 2,4-DBP Formation. To investigate whether the dihydroxy-tetrabrominated metabolite of BDE-47 (M2) was formed by two sequential hydroxylation steps, pooled human liver microsomes were incubated with 4-OH-BDE-42, 5-OH-BDE-47, 6-OHBDE-47, or 4′-OH-BDE-49, instead of BDE-47. Formation of M2 was not detected when any of the monohydroxytetrabrominated metabolites was used as substrate (data not shown), indicating that M2 is not a secondary metabolite of BDE-47. We also assessed the effect of antiepoxide hydrolase on the formation of M2 from BDE-47 to determine if M2 was produced via a stable epoxide intermediate. No difference in rates of M2 formation was observed when BDE-47 was incubated with pooled human liver microsomes in the presence of varying amounts of antiepoxide hydrolase or rabbit IgG (data not shown), demonstrating that epoxide hydrolase was not involved in M2 formation. To determine if 2,4-DBP, 4′-OH-BDE-17, and 2′-OH-BDE28 were formed as secondary metabolites of BDE-47, pooled human liver microsomes were incubated with 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, or 4′-OH-BDE-49, instead of BDE-47. Formation of 4′-OH-BDE-17 or 2′-OH-BDE-28 was not detected when 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE47, or 4′-OH-BDE-49 were used as substrate (data not shown). Formation of 2,4-DBP, however, was observed when human liver microsomes were incubated with 4-OH-BDE-42, 5-OHBDE-47, 6-OH-BDE-47, or 4′-OH-BDE-49 (Figure 6A). Further experiments showed that 2,4-DBP was also formed when human liver microsomes were incubated with BDE-47 together with 4-OH-BDE-42 or 4′-OH-BDE-49 (Figure 6B).

Table 3. Correlation Analysis of Rates of Formation of Hydroxylated Metabolites of BDE-47 and P450-Mediated Marker Activities Using Single Donor Human Liver Microsomesa correlation coefficient (r) values BDE-47 metabolite

P450 1A2b

P450 2B6c

P450 2C9d

P450 2C19e

P450 2E1f

P450 3A4g

2,4-DBP 4′-OH-BDE17 2′-OH-BDE28 4-OH-BDE42 5-OH-BDE47 6-OH-BDE47 4′-OH-BDE49 M1 M2

0.52 0.36

0.99 0.95

0.56 0.52

0.82 0.62

0.59 0.80

0.58 0.33

0.36

0.95

0.56

0.58

0.81

0.26

0.36

0.96

0.56

0.64

0.82

0.36

0.45

0.99

0.56

0.77

0.68

0.50

0.43

0.99

0.59

0.74

0.71

0.49

0.37

0.97

0.62

0.66

0.77

0.43

0.39 0.61

0.98 0.93

0.62 0.37

0.68 0.96

0.75 0.33

0.45 0.66

a

Marker activity for P450 2B6 was experimentally determined as described in the Experimental Procedures section. Marker activity values for the other P450 enzymes were provided by the vendors of the single donor human liver microsomes. bP450 1A2 marker activity: phenacethin O-deethylation. cP450 2B6 marker activity: bupropion 4hydroxylation. dP450 2C9 marker activity: diclofenac 4′-hydroxylation. e P450 2C19 marker activity: (S)-mephenytoin 4′-hydroxylation. fP450 2E1 marker activity: chlorzoxazone 6-hydroxylation. gP450 3A4 marker activity: testosterone 6β-hydroxylation.

tration. Formation of 6-OH-BDE-47 and M1 was best described by the Michaelis−Menten model, which was the same model that best described the formation of these metabolites by human hepatic microsomes. In contrast, formation of 4-OH-BDE-42, 5-OH-BDE-47, and 4′-OH-BDE49 by recombinant P450 2B6 was best described by the substrate inhibition model. Values of Vmax, Km, and Ki for metabolite formation by recombinant P450 2B6 are listed in Table S4 (Supporting Information). Consistent with the results obtained with pooled human liver microsomes, 5-OH-BDE-47 and 6-OH-BDE-47 were the major hydroxylated metabolites, and M1 was the major unidentified hydroxylated metabolite of BDE-47 produced by recombinant P450 2B6, as indicated by Vmax values. Kinetic analysis of 4′-OH-BDE-17 and 2′-OH-

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DISCUSSION In the present study, the oxidative metabolism of BDE-47 by human liver microsomes and human recombinant P450 enzymes was characterized in terms of identification and quantification of the hydroxylated metabolites formed, identification of the P450 enzymes involved in BDE-47 metabolism, and determination of the kinetic parameters 727



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Figure 6. Formation of 2,4-DBP following the incubation of pooled human liver microsomes (0.1 mg/mL) for 5 min with (A) the four hydroxylated metabolites of BDE-47 (5 to 100 nM) and (B) with BDE-47 (25 μM) plus increasing concentrations (25 to 150 nM) of 4-OH-BDE-42 or 4′-OHBDE-49.

hydroxylated metabolites of BDE-47 produced by human liver microsomes and human recombinant P450 2B6. Differences in the experimental design adopted and the sensitivity of the analytical methods used can account for differences in metabolite detection between our study and previous studies. All of the hydroxylated metabolites of BDE-47 identified in the present study were detected in human serum and plasma samples,11−14 indicating a good agreement between in vitro metabolism and in vivo biomonitoring studies. In most of the human plasma and serum samples analyzed, concentrations of 5-OH-BDE-47 and 6-OH-BDE-47 were greater than those of 4OH-BDE-42 and 4′-OH-BDE-49, which, in turn, were equal to or greater than those of 4′-OH-BDE-17 and 2′-OH-BDE-28. These findings are consistent with our quantitative analysis showing that 5-OH-BDE-47 and 6-OH-BDE-47 are major metabolites, 4-OH-BDE-42 and 4′-OH-BDE-49 are intermediate metabolites, and 4′-OH-BDE-17 and 2′-OH-BDE-28 are minor metabolites of BDE-47 in human liver microsomes. Experiments involving a panel of 12 human recombinant P450 enzymes, a monoclonal mouse anti-P450 2B6, and single donor human liver microsomes identified P450 2B6 as the P450 enzyme predominantly involved in the in vitro oxidative metabolism of BDE-47, which is consistent with a recent study.31 Therefore, P450 2B6 can catalyze oxidation at ortho, meta, and para positions (with or without NIH-shift of a bromine atom), dihydroxylation, O-dealkylation and, to a minor extent, oxidative debromination of BDE-47, suggesting that the active site of P450 2B6 can accommodate BDE-47 in various orientations, resulting in the oxidation of different carbon atoms of BDE-47. The predominant role of P450 2B6 in the oxidative biotransformation of BDE-99 to multiple metabolites by human liver microsomes was recently reported32 suggesting that P450 2B6 is a versatile catalyst in the oxidative metabolism of BDE-47 and BDE-99. Human hepatic expression of P450 2B6 is influenced by genetic and environmental factors and is highly variable. Interindividual variability in human P450 2B6 protein levels and catalytic activities is well known.38,40,41 This variability is consistent with differences of up to 50-fold in rates of formation of hydroxylated metabolites of BDE-47 that were observed in the present study among single donor human liver microsomes (Table 2). Gender and ethnic differences affecting P450 2B6 protein levels and catalytic activity have been reported and can contribute to interindividual variability of P4502B6-mediated metabolism.42 In addition, P450 2B6 is a highly polymorphic enzyme with 37 alleles and more than 100 single nucleotype polymorphic variants (http://www.cypalleles.ki.se/cyp2b6. htm). Some P450 2B6 variants have decreased catalytic activity

associated with metabolite formation. Collectively, the results show that nine hydroxylated metabolites were formed when human liver microsomes were incubated with BDE-47 and that P450 2B6 was the predominant P450 enzyme responsible for their formation. Formation of 5-OH-BDE-47 and 6-OH-BDE-47 indicates that BDE-47 was oxidatively metabolized via oxygen insertion at an unsubstituted carbon atom. Formation of 4-OH-BDE-42 and 4′-OH-BDE-49 shows that BDE-47 was also oxidized via oxygen insertion at a substituted carbon atom accompanied by a NIH-shift mechanism. O-Dealkylation of BDE-47 results in the formation of 2,4-DBP. Although 2,4-DBP is a primary and secondary metabolite of BDE-47, it was not a major metabolite of BDE-47. In contrast, O-dealkylation is a more important mechanism in the oxidative metabolism of BDE-99 by human liver microsomes, 32 suggesting that small differences in the PBDE structure (i.e., the presence or absence of a bromine on carbon 5) can affect the mechanism of oxidative metabolism in vitro. Formation of 4′-OH-BDE-17 and 2′-OH-BDE-28 shows that BDE-47 is oxidatively debrominated by P450. However, 4′OH-BDE-17 and 2′-OH-BDE-28 are minor BDE-47 metabolites, suggesting that oxidative debromination is a minor pathway for human liver microsomes. A dihydroxy-tetrabrominated metabolite was also formed when human liver microsomes were incubated with BDE-47 but not with primary metabolites of BDE-47. Moreover, antiepoxide hydrolase had no effect on the formation of the di-OH-tetrabrominatedPBDE metabolite suggesting that a stable epoxide intermediate was not involved in its formation and that it was produced directly from BDE-47 by P450-catalyzed dihydroxylation. Previous studies of BDE-47 biotransformation in vitro described the formation of many of the same hydroxylated metabolites by human hepatic preparations as identified in the present study. Formation of 2,4-DBP, 3-OH-BDE-47, 5-OHBDE-47, 6-OH-BDE-47, and an unidentified dihydroxytetrabrominated metabolite by human liver microsomes28,29,31 or cultured human hepatocytes30 has been reported. Moreover, formation of 4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 6OH-BDE-47, 4′-OH-BDE-49, a monohydroxy-tetrabrominated-PBDE (tentatively identified as 2′-OH-BDE-66), and an unidentified dihydroxy-tetrabrominated-PBDE metabolite by human recombinant P4502B6 was reported recently.31 The present study demonstrated the formation of 4′-OH-BDE-17, 2′-OH-BDE-28, and an unidentified monohydroxy-tetrabrominated PBDE (M1), along with 2,4-DBP, 4-OH-BDE-42, 5-OHBDE-47, 6-OH-BDE-47, 4′-OH-BDE49, and an unidentified dihydroxy-tetrabrominated PBDE, but not 3-OH-BDE-47, as 728



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compared to that of the wild type enzyme.43−46 Moreover, P450 2B6 is an inducible enzyme, at least in vitro. Increased P450 2B6 expression has been reported in human primary hepatocytes that were treated with phenobarbital, dexamethasone, or rifampicin.47,48 The large interindividual variability in P450 2B6 expression has implications for BDE-47 bioaccumulation and metabolism and is reflected in the interindividual variability in hydroxylated PBDE concentrations in human plasma and serum samples.11−14 Compared to our previous biotransformation study of BDE47 by rat liver microsomes,37 a larger number of hydroxylated metabolites of BDE-47 was formed by human than rat liver microsomes. The two major hydroxylated metabolites produced by human liver microsomes (namely, 5-OH-BDE47 and 6-OH-BDE-47) were minor metabolites of BDE-47 in rat liver microsomes. A major hydroxylated metabolite of BDE47 produced by rat liver microsomes, namely, 3-OH-BDE-47, was below the limit of detection in incubations containing human liver microsomes. Rates of BDE-47 metabolite formation were greater for human liver microsomes (i.e., Vmax values ranged between 13.0 and 27.3) than liver microsomes from corn oil-treated rats (i.e., Vmax values less than 0.25 or 0.5 pmol/min/mg protein, depending on the metabolite).37 In addition, P450 1A1, 2A2, and 3A1 were the most active rat P450 enzymes in the metabolism of BDE-47, whereas P450 2B6 was the predominant P450 enzyme responsible for BDE47 hydroxylated metabolite formation by human liver microsomes. Interspecies differences in the oxidative metabolism of BDE-99 by rat and human liver microsomes were also reported32,37 and suggest that in vitro metabolism of PBDEs should be assessed in a species-specific manner. Oxidative metabolism of BDE-47 in humans can be of toxicological concern due to disruption of thyroid hormone homeostasis. Several OH-BDEs, including 4-OH-BDE-42, 5OH-BDE-47, 6-OH-BDE-47, and 4′-OH-BDE-49 exhibited 250- to 5,000-times greater potency compared to that of BDE47 for in vitro inhibition of thyroxine (T4) binding to human transthyretin, a major T4 blood carrier.21−25 In a related in vitro study, the combined potency of hydroxylated metabolites of BDE-47 for binding to rat transthyretin was shown to be well predicted according to the concentration-addition model.22 Some hydroxylated metabolites of BDE-47 are also in vitro agonists of the human thyroid receptor β. For example, 4-OHBDE-42, 5-OH-BDE-47, 6-OH-BDE-47, and 4′-OH-BDE-49 exhibited 70- to 25,000-times greater activity than the parent compound, BDE-47, in a T3-mediated transcription assay using yeast transfected with human thyroid receptor β.26,27 In vivo thyroid toxicity of PBDEs and OH-BDEs in humans is currently unknown. However, results from in vitro studies such as those discussed above suggest that hydroxylated metabolites of BDE-47 might be more active, by more than one mechanism, than BDE-47 in disrupting thyroid hormone homeostasis in vivo. Therefore, the in vivo oxidative metabolism of PBDEs could be an important determinant of PBDE toxicity in humans. Decreased thyroid function is a particular concern during pregnancy because thyroid hormone insufficiency is associated with neurodevelopmental effects in children.49 In conclusion, oxidative metabolism of BDE-47 by human liver microsomes was thoroughly characterized for the first time. Nine hydroxylated metabolites were formed, with 5-OHBDE-47 and 6-OH-BDE-47 being the major metabolites. Formation of all the nine hydroxylated metabolites of BDE47 was catalyzed by P450 2B6. All the hydroxylated metabolites

of BDE-47 identified in our study have been measured in human plasma and serum samples, suggesting that the oxidative metabolism of BDE-47 occurs in vivo and that it can be reliably predicted by in vitro metabolism studies using human liver microsomes. Moreover, the present data set provides evidence that several hydroxy-tetrabrominated PBDEs, which exhibited disruption of thyroid homeostasis in vitro, are of metabolic origin, suggesting that hepatic metabolism of BDE-47 and possibly other PBDEs might play an important role in explaining PBDE thyroid toxicity in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

Details of the analytical method, including mass spectrometry key settings, determination of the limit of quantification and quality control values, together with kinetic profiles of the formation of hydroxylated metabolites of BDE-47 by human recombinant P450 2B6 and the corresponding Vmax, Km, and Ki. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Westbrook Mall, Vancouver, British Columbia, Canada V6T 1Z3. Tel: 1-604-822-3815. Fax: 1-604-822-3035. E-mail: [email protected]. Funding

This work was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada [RGPIN 138733-10 to S.M.B.]. A graduate student fellowship was provided by The University of British Columbia to C.A.E. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS PBDEs, polybrominated diphenyl ethers; 2,4-DBP, 2,4dibromophenol; 4′-OH-BDE-17, 4′-hydroxy-2,2′,4-tribromodiphenyl ether; 2′-OH-BDE-28, 2′-hydroxy-2,4,4′-tribromodiphenyl ether; 4-OH-BDE-42, 4-hydroxy-2,2′,3,4′-tetrabromodiphenyl ether; 3-OH-BDE-47, 3-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether; 5-OH-BDE-47, 5-hydroxy-2,2′,4,4′tetrabromodiphenyl ether; 6-OH-BDE-47, 6-hydroxy-2,2′,4,4′tetrabromodiphenyl ether; 4′-OH-BDE-49, 4′-hydroxy2,2′,4,5′-tetrabromodiphenyl ether; 4′-OH-CB-50, 4′-hydroxy2,2′,4,6′-tetrachlorobiphenyl; BDE-47, 2,2′,4,4′-tetrabromodiphenyl ether; BDE-99, 2,2′,4,4′,5-pentabromodiphenyl ether; IC50, inhibitory concentration of 50% P450 cytochrome P450; MS, mass spectrometry; M1, unidentified metabolite no.1; M2, unidentified metabolite no. 2; UHPLC, ultra high performance liquid chromatography

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REFERENCES

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Biotransformation of 2,2â²,4,4â²-Tetrabromodiphenyl Ether (BDE-47

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