Human Liver Microsome-Mediated Metabolism of Brominated

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Chem. Res. Toxicol. 2009, 22, 1802–1809

Human Liver Microsome-Mediated Metabolism of Brominated Diphenyl Ethers 47, 99, and 153 and Identification of Their Major Metabolites Sara J. Lupton,† Barbara P. McGarrigle,‡ James R. Olson,*,‡ Troy D. Wood,† and Diana S. Aga*,† Department of Chemistry, UniVersity at Buffalo, State UniVersity of New York, Buffalo, New York 14260, and Department of Pharmacology and Toxicology, UniVersity at Buffalo, State UniVersity of New York, 3435 Main Street, Buffalo, New York 14214 ReceiVed June 24, 2009

While the metabolism and excretion of polybrominated diphenyl ethers (PBDEs) have been reported in rodents, PBDE metabolism in humans has only recently been investigated. In this present study, individual human liver microsomes were incubated for 120 min with radiolabeled and nonradiolabeled BDE 47, 99, or 153 to determine their relative degrees of metabolism and to identify the structures of metabolites formed. Radiolabeled samples were analyzed using high-performance liquid chromatography/ radiochemical detection, while nonradiolabeled samples were analyzed with and without derivatization using gas chromatography/mass spectrometry. Results from radiolabeled incubations demonstrated that human liver microsomes metabolized BDEs 47 and 99 but not BDE 153. Differences in the extent of BDE metabolism by the three individual liver specimens used in the study were observed. BDE 47 metabolized to a dihydroxylated BDE 47 and 2,4-dibromophenol, while BDE 99 metabolized to a dihydroxylated BDE 99, 2,4,5-tribromophenol and 1,3-dibromobenzene. This study showed that BDEs 47 and 99 are metabolized by human liver microsomes with relatively large interindividual differences. Results of this study could provide one explanation for the high bioaccumulation rate of BDE 153 in humans. Introduction Polybrominated diphenyl ethers (PBDEs)1 are a class of environmental contaminants of great concern because of their potential to cause deleterious health effects to humans and wildlife. The similarities in the physicochemical properties between PBDEs and polychlorinated biphenyls (PCBs)1 lead to the concern that exposure to PBDEs could lead to similar adverse health effects caused by PCBs, including disruption of the endocrine system, neurodevelopmental delays, and carcinogenic effects, to name a few (1, 2). Inhalation and ingestion of PBDE-containing particles as well as ingestion of contaminated food products are the primary routes of exposure to PBDEs (3). Because PBDEs are bioaccumulative and persistent in humans, they can be transferred from mothers to offspring through the placenta and breastfeeding, posing risks to the fetus and developing children (4, 5). Three mixtures of PBDEs were produced worldwide, pentaBDE, octaBDE, and decaBDE. However, the lower congener mixtures are more bioaccumulative and persistent (6) and are still found at increasing levels in the environment despite the fact that the decaBDE mixture is the only mixture still being used in the United States. One thing that is poorly understood * To whom correspondence should be addressed. (D.S.A.) Tel: 716-6454220. Fax: 716-645-6963. E-mail: [email protected]. (J.O.) Tel: 716829-2319. Fax: 716-829-2901. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Pharmacology and Toxicology. 1 PBDEs, polybrominated diphenyl ethers; PCBs, polychlorinated biphenyls; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC/ RCD, high-performance liquid chromatography/radiochemical detection; ACN, acetonitrile.

to date is how the ratio of the main congeners in commercial mixtures changes, as observed in environmental samples, which in turn are different than the ratios found in humans. In many investigations where environmental samples, such as household air, dust, and fish, have been analyzed for PBDEs, one common trend is easily observed; BDE 153 is the least abundant congener among BDEs 47, 99, and 153. It is at least 4-fold lower in concentration than BDE 99. On the other hand, BDE 47 is at least approximately equal to or more concentrated than BDE 99 (3, 7-10). These ratios in environmental samples differ from those of the commercial mixtures where BDE 99 is approximately two times more concentrated than BDE 47 (6, 11). In contrast, BDE 99 in humans is generally the least concentrated, relative to BDE 47 and BDE 153, or is approximately equal to BDE 153 (3-5, 8, 12-15). These differences between the PBDE ratios in human samples, environmental samples, and commercial mixtures may indicate variability in uptake and metabolism of the various BDE congeners. While several studies on the PBDE metabolism in laboratory animals have been reported, studies on human metabolism of PBDEs are very limited. To date, there are two studies that reported in vitro PBDE metabolism in human microsomes and S9 fractions (16, 17). In addition, a few studies have reported the occurrence of PBDE metabolites in human plasma (5, 18, 19). In this present study, human liver microsomes from three different individual specimens and a pooled sample from 15 donors were used to determine the metabolism of radiolabeled and nonradiolabeled BDEs 47, 99, or 153 (Figure 1). The goals of this study are to determine the relative extent of PBDE metabolism by human liver microsomes from different individu-

10.1021/tx900215u CCC: $40.75  2009 American Chemical Society Published on Web 10/16/2009

Human Hepatic Metabolism of BDEs 47, 99, and 153

Figure 1. Structures of BDE congeners 47, 99, and 153.

als focusing on phase I metabolism, mainly by CYPs, and to identify the metabolites formed. Knowledge on the identities and persistence of initial metabolites formed in the body is important because hydroxylated PBDE metabolites are proposed to play a significant role in disrupting the thyroid function due to the resemblance of these metabolites to the thyroid hormones. Therefore, hydroxylated PBDEs are postulated to have the ability to bind to hormone transport proteins (20). Accumulation of metabolites may therefore contribute to the endocrine-disrupting effects of PBDEs. The information on the extent of formation of hydroxylated metabolites is key to understanding the complete risks associated with PBDE bioaccumulation and metabolism in humans and wildlife.

Materials and Methods Human Liver Microsome Incubations and Extraction. Specimens of human liver found unsuitable for transplantation were procured from Tissue Transformation Technologies (Edison, NJ) (specimens A and B) or obtained from Upstate New York Transplant Services (Buffalo, NY) (specimen C). In addition, a pooled sample of human liver microsomes from 15 donors was obtained from CellzDirect (Durham, NC). The human liver samples were homogenized in a 10 mM Tris/ 250 mM sucrose buffer (pH 7.4), with 1 g of liver to 5 mL of buffer. After centrifugation at 9000g (4 °C), the supernatant was centrifuged at 100000g (4 °C) for 1 h. The microsomal pellet was resuspended in a 10 mM Tris/250 mM sucrose buffer with 20% glycerol and 1 mM EDTA using 1 mL/g of tissue. The CYP content

Chem. Res. Toxicol., Vol. 22, No. 11, 2009 1803 was measured by the method of Omura and Sato (21) using a molar extinction coefficient of 0.091 M-1 cm-1. Microsomes were divided into aliquots and stored at -80 °C. For microsomal incubations, the assay buffer consisted of 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),1 6 mM magnesium chloride, 0.8 mg/mL bovine serum albumin (BSA) as a brominated diphenyl ether (BDE) carrier, and 1 mM NADPH. The assay volume was 1 mL with 0.5 mg of microsomal protein and contained either 10 or 20 µM 14C radiolabeled or nonradiolabeled BDE 47, 99, or 153 (Figure 1). Nonradiolabeled BDE neat standards were purchased from Accustandard, Inc. (New Haven, CT). The assay was initiated with NADPH, incubated for 2 h, and quenched by transferring tubes to an ice bath. Samples were then frozen and stored at -20 °C pending analysis. Control samples consisting of BDE incubations in the absence of microsomal proteins were also prepared in duplicate to determine any abiotic transformation of BDEs during incubations that might be present. Human liver microsome specimens incubated with no BDEs in duplicate were also analyzed for residual PBDEs or metabolites that may have been present in the human samples. After incubation, the 1 mL samples were thawed and transferred into 50 mL glass centrifuge tubes. Proteins were denatured with 0.5 mL of 6 M hydrochloric acid. After protein denaturation, 3 mL of a 75/25 v/v solution of hexane/methylene chloride was added for extraction. The mixture was vortexed and centrifuged, and the organic layer was removed. A second addition of 3 mL of a 50/50 v/v solution of hexane/methylene chloride was also added for extraction. After the solution was vortexed and centrifuged, the organic layer was removed and combined with the previous organic layer. The extract was evaporated to dryness under a stream of nitrogen and reconstituted in 0.5 mL of acetonitrile (ACN)1 for high-performance liquid chromatography/radiochemical detection (HPLC/RCD)1 analysis. To determine the relative in vitro rate of metabolism of BDEs 47, 99, and 153, human liver microsomes were incubated with cold BDE standards as described above for 30, 60, and 120 min. For all nonradioactive incubations, the extracts were prepared similar to the radiolabeled samples up to the evaporation step. Then, after the extracts were evaporated to approximately 0.4 mL under a stream of nitrogen, each sample was split into two for analysis by GC/MS with and without derivatization. Derivatization was needed to allow detection of any hydroxylated or carboxylated metabolites by GC/MS. Extraction recoveries were determined from microsome samples by spiking with known concentrations of 2,4,5-tribromophenol (2,4,5-TBP) (Accustandard, Inc., New Haven, CT), a 13C-labeled 6-hydroxy-2,2′,4,4′-tetrabromo diphenyl ether (6-OH BDE 47) (Wellington Laboratories, Guelph, ON, Canada), BDE 47 (Accustandard, Inc.), and BDE 99 (Accustandard, Inc.) into microsome samples after incubation, allowed to evaporate, and then extracted and derivatized as previously described. Average extraction recoveries (n ) 4) for 2,4,5-TBP, 6-OH BDE 47, BDE 47, and BDE 99 were 49.7 ((9.6), 107.7 ((10.1), 109.1 ((11.0), and 112.1% ((16.2), respectively (Figure S1 of the Supporting Information). Analysis by HPLC/RCD. Analysis for radioactive incubations was performed with HPLC/RCD. Reversed phase separation was achieved on a C-18 Betabasic column (100 mm × 2.1 mm, 3 µm particle size) (Thermo Scienctific, Waltham, MA) using a Thermo Scientific Surveyor HPLC with a gradient of ACN (solvent A) and 10 mM ammonium acetate in water (solvent B), starting at 50/50 A/B and increasing strength to 70% solvent A over 5 min. Finally, the column was kept at 70/30 A/B for 45 min, which allowed for the elution of all compounds. Detection was accomplished using liquid scintillation counting. The IN/US Systems, Inc., β-Ram detector (Tampa, FL) used a flow through cell with a volume of 0.5 mL and a 3:1 scintillation fluid (Ecoscint, National Diagnostics, Atlanta, GA) to eluent ratio. Derivatization of BDE Metabolites and Identification by GC/MS. After extraction of cold incubations, methylation of the hydroxyl groups present in the metabolites was accomplished by using trimethylsilyl diazomethane (Pfaltz and Bauer, Inc., Water-

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bury, CT). The reaction was performed over 18 h in a mixture of 0.1 mL of methanol, 0.05 mL of hexane, 0.2 mL of extract, and 0.25 mL of trimethylsilyl diazomethane. After derivatization, samples were evaporated to dryness using a slow stream of nitrogen and reconstituted in 0.2 mL of hexane. The underivatized and derivatized extracts were analyzed using a GC/MSD system (GC 6890/5973 MSD, Agilent Technologies, Inc., Santa Clara, CA) with electron impact (EI) ionization. Separation was performed on a RTX-5MS column (40 m × 0.25 mm i.d. × 0.25 µm thickness) (Restek U.S., Bellefonte, PA). The inlet temperature was 250 °C, and the injection was performed in pulsed splitless mode. The oven initial temperature was 50 °C and held for 1 min. The temperature was ramped to 200 at 30 °C/min, and a second ramp was performed to 250 at 4 °C/min and held for 10 min. Finally, the oven was ramped to 300 at 30 °C/min and held for 4 min to give a total run time of 34.17 min. The auxiliary temperature for the MSD was 300 °C. A full scan to determine initial mass to charge (m/z) ratios was performed with the MSD from m/z 100 to 700, after which selected ion monitoring (SIM) was used for GC/MS analysis. Quantification and qualifier ions are provided in Table S1 of the Supporting Information for metabolites and parent compounds.

Results Radioactive Incubations. Both BDEs 47 and 99 were metabolized by each of the human liver microsome specimens from individual donors, as can be seen in the radiochromatograms in Figure 2a,b, respectively. In contrast, none of the human liver microsomes employed had detectable amounts of metabolites for BDE 153 in either radiolabeled incubations (Figure 2c) or nonradiolabeled incubations, indicating that BDE 153 is recalcitrant within this experimental system. Therefore, it is reasonable to assume that BDE 153 has high potential to bioaccumulate in humans. Figure 2a is a chromatogram of the extract from 14C-labeled BDE 47 incubation using specimen A. The peak that elutes at 24.5 min corresponds to unmetabolized BDE 47, while the peak eluting at 7.5 min is the major metabolite formed, which is 31% of the total radioactivity in the chromatogram. The metabolite peak corresponds to the dihydroxylated BDE 47 as confirmed by GC/MS. The mass spectrum for this metabolite is presented in the Supporting Information (Figure S2). The BDE 47 incubations with specimens B and C showed a metabolite at the same retention time but at relatively lower amounts as compared to that observed in specimen A. For example, specimen B and specimen C converted only 9 and 12% of BDE 47 to the metabolites, respectively (Table 1). Figure 2b is the radiochromatogram of the extract from the 14 C-labeled BDE 99 incubation that used specimen A. BDE 99 is observed at 33.5 min, and a major metabolite is observed at 9.5 min. The metabolite corresponds to a dihydroxylated BDE 99, also confirmed by GC/MS (Figure S4 of the Supporting Information). This metabolite peak accounts for 52% of the total counts in the radiochromatogram for specimen A. Specimens B and C, when incubated with BDE 99, also had a metabolite peak at the same retention time. For specimens B and C, the metabolite peak represented only 12 and 13% of the total counts in the radiochromatograms, respectively (Table 1). Qualitative Analysis of Nonradioactive Incubations for Metabolite Identification. Microsomes from specimens A-C were incubated with nonradiolabeled BDEs 47, 99, and 153 for purposes of identifying the metabolites formed using GC/MS. Analysis by GC/MS was performed with and without derivatization using trimethylsilyl diazomethane to detect hydroxylated metabolites. Note that hydroxylated BDEs cannot be detected in their underivatized form using GC/MS because of their poor thermal stability and low volatility. Because of the lack of

Lupton et al.

reference standards for the dihydroxylated metabolites, quantification was not possible in this study using the nonradiolabeled BDEs. Unfortunately, the GC/MS instrument used in this study is not certified for injection with radiolabeled compounds. Therefore, collection of radiolabeled metabolites from the HPLC column, followed by analysis using GC/MS, was not an option in this study. Two metabolites were observed for BDE 47 (Figures S2 and S3 of the Supporting Information), and three metabolites were observed for BDE 99 (Figures S4 and S5 of the Supporting Information). A small GC/MS signal corresponding to 1,3dibromobenzene was observed in the BDE 99 incubations. However, 1,3-dibromobenzene was not observed in the BDE 47 incubations, probably due to the loss of this metabolite during sample extraction or evaporation. As expected, on the basis of earlier observations using radiolabeled BDEs, incubations with nonradiolabeled BDE 153 did not show detectable metabolites by GC/MS. No residual BDEs were observed in controls that contained only microsomes. Also, no abiotic transformation of BDEs 47, 99, or 153 was observed in controls containing only BDEs and no microsomes. The derivatized extract from specimen A incubated with BDE 47 had a metabolite at 20.41 min and at 6.39 min in GC/MS. The corresponding mass spectrum for the peak at 20.41 min is seen in Figure S2 of the Supporting Information, which shows a mass/charge (m/z) of 546 and an isotope pattern characteristic of a tetrabrominated compound. This molecular ion (m/z of 546) corresponds to the dimethoxy BDE 47 derivative, formed by the derivatization of two hydroxyl groups on the BDE 47 metabolite into the methoxy forms. The derivatized extract from specimen A also had a metabolite at 6.39 min, which corresponds to 2,4-dibromophenol. The mass spectrum for the derivatized extract is seen in Figure S3 of the Supporting Information where the m/z of 266 corresponds to the methoxy derivative. This metabolite is an indication that a cleavage in the diphenyl ether bond has occurred; however, no debromination from the phenyl rings was observed. The fact that two metabolites were detected by GC/MS but only one major radioactive metabolite peak was observed in the chromatograms from the HPLC/RCD can be explained by the lower detection limits afforded by GC/MS. Three metabolites were observed in the extract from specimen A incubated with BDE 99. A metabolite at 25.79 min was observed in the derivatized extract with m/z 626 corresponding to a dimethoxylated BDE 99 (Figure S4 of the Supporting Information), which means that a dihydroxylated BDE metabolite was formed. Also, a metabolite with m/z 344 at 7.84 min showing a tribrominated isotope pattern was observed in the derivatized extract. This mass corresponds to a 2,4,5-tribromophenol after derivatization. The identity of this metabolite was confirmed by comparing retention time and full MS spectrum with a reference standard of 2,4,5-tribromophenol using GC/MS. Again, this metabolic product indicates the cleavage of the diphenyl ether bond but not debromination of the phenyl rings. In addition, the 1,3-dibromobenzene was observed in small abundance but only in the BDE 99 incubations. On the basis of relative peak areas in the GC/MS, it can be inferred that the dihydroxylated metabolites in the derivatized extracts from BDEs 47 and 99 correspond to the major metabolites observed in the radiochromatograms from BDEs 47 and 99, respectively. However, because reference standards for dihydroxylated BDE 99 or 47 are not available, it is not possible to quantify these metabolites at this time. Nevertheless,

Human Hepatic Metabolism of BDEs 47, 99, and 153

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Figure 2. (a) Radioactive chromatogram following 2 h of incubation of 14C-BDE 47 with human liver microsome specimen A. BDE 47 was observed at 24.5 min, and a metabolite was detected at 7.5 min. (b) Radioactive chromatogram following 2 h of incubation of 14C-BDE 99 with human liver microsome specimen A. BDE 99 was observed at 33.5 min, and a metabolite was detected at 9.5 min. (c) Radioactive chromatogram following 2 h of incubation of 14C-BDE 153 with human liver microsome specimen A. BDE 153 was observed at 45.5 min; however, no metabolites were detected.

it is known that the ionization efficiency in GC/MS with EI does not change significantly between compounds of increasing bromination (22). Therefore, it would be reasonable to assume that the dihydroxylated BDE 99 (7 765 872 area) has a higher concentration than 2,4,5-tribromophenol (2 814 385 corrected

area for extraction recovery) if we consider that the compounds would ionize very similarly in GC/MS. For more quantitative studies in the future, it will be important to synthesize the dihydroxylated BDE metabolites as reference standards to allow absolute quantification.

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Table 1. Metabolism of 14C-BDEs by Human Liver Microsomesa CYP content of specimen

donor information

BDE 47b

BDE 99b

BDE 153b

A, 0.283 nmol/mg

female, 21 years nonsmoker occ. marijuana and ethanol use male, 20 years smoked 1/2 ppd for 4 years social ethanol use female, 51 years no information available

parent, 59% major metab, 31% minor metab, 10% parent, 90% major metab, 9% minor metab, 1% parent, 88% major metab, 12% minor metab, 0%

parent, 40% major metab, 52% minor metab, 8% parent, 84% major metab, 12% minor metab, 4% parent, 85% major metab, 13% minor metab, 2%

parent, 100%

B, 0.173 nmol/mg C, 0.297 nmol/mg

parent, 100% parent, 100%

a CYP content, cytochrome P450 concentration in human liver microsome specimens (nmol/mg); Occ., occasional; ppd, packs per day; and metab, metabolite. b Percentage of PBDE-derived radioactivity converted to major and minor metabolites following a 2 h incubation with human liver microsomes (0.5 mg/mL).

Figure 3. Comparison of the relative rates of metabolism of unlabeled BDEs 47 and 99 by human liver microsome specimens A, B, and C and a pooled microsomal specimen from 15 donors. Relative metabolism is expressed as the metabolite peak area/nmol CYP/2 h. Values are the mean ( SD of three incubations.

Relative Metabolism of BDEs by Individual and Pooled Human Microsomes. Microsomal incubations with BDEs 47 and 99 were conducted for 30, 60, and 120 min using specimens A-C and a pooled hepatic microsome specimen from 15 donors. The formation of metabolites increased linearly from 30 to 120 min for BDEs 47 and 99 in all four microsome specimens. Therefore, 120 min was used as the optimal incubation time for the analysis of the relative rates of metabolite formation. The relative formations of BDEs 47 and 99 metabolites were expressed per nmol of cytochrome P450, to account for the differences in CYP content between the microsome specimens. Figure 3 shows that there is a difference in the relative rates of BDE metabolism, with specimen A having a higher rate of metabolite formation than the other three specimens. Analysis of variances between samples showed that differences in the results between incubations are statistically significant from each other for BDE 47 (p ) 0.0004) and BDE 99 (p < 0.05).

Discussion While previous studies have assessed the metabolism and disposition of these compounds in rats and mice (16, 23-26), this study is one of the few recent reports that describe the qualitative metabolism of BDEs 47, 99, and 153 congeners by human liver microsomes (16, 17). Sanders et al. (25) proposed that rats and mice metabolize BDE 47 in the liver through several different pathways, including cytochrome P450s (CYPs), and identified a dihydroxylated PBDE in bile. For this reason, we chose to specifically look at human liver microsomes to understand the oxidative metabolism of BDEs 47, 99, and 153.

Despite the limitations of using human liver microsomes as compared to S9 liver fractions due to the fewer types of enzymes available in liver microsomes, its use provides a clear understanding of the role of P450s in PBDE metabolism. The dihydroxylated metabolites of BDEs 47 and 99 in human liver microsomes were identified in this present study. However, the specific location of hydroxylation was not determined because of the limited amount of formed metabolite to allow further characterization. The dihydroxylated metabolites have been observed in a previous metabolism study using human liver microsomes (16). However, dihydroxylated metabolites were not observed in the study that used S9 liver fractions possibly due to the longer incubation time (2 h as compared to 48 h) and the presence of other enzymes available for metabolism (17). Differences in the incubation times probably resulted in further metabolism of the dihydroxylated metabolites to monohydroxylated metabolites, the latter of which were not observed in this study. It has been speculated that the large interindividual differences in PBDE concentrations and congener patterns in humans could in part be attributed to the variability in enzyme metabolic activities between humans. Evidence to support the interindividual differences in the relative in vitro rate of hepatic metabolism of BDEs 47 and 99 is illustrated in Figure 3; however, more studies on larger populations need to be completed. These data were normalized for the variability in total CYP content between individuals. Specimen A metabolized BDEs 47 and 99 more readily than the other microsome specimens, which is consistent with the

Human Hepatic Metabolism of BDEs 47, 99, and 153 Table 2. Average Ratios of BDE 47, BDE 99, and BDE 153 in Human and Environmental Samples from North America in the Literature as Well as Average Ratios in the Commercial PentaBDE Mixture

b

refs

sample

BDE 47:BDE 99:BDE 153

8 8 8 4 4 12 13 13 5 5 5 14 15 3 7 7 7 8 9 10 3 11

adult blood breast milk breast tissue maternal blood fetal blood plasma maternal blood breast milk maternal blood breast milk fetal liver breast milk serum breast milk personal aira bedroom aira main living area aira outdoor aira house dustb house dustb house dustb commercial pentaBDE

2.0:1:1.1 8.5:1.9:1 4.5:1.6:1 4.5:1:1.2 3.9:1:2.2 6:1:1 4.9:1:3.4 4.8:1:2 4.4:1.9:1 6.4:2.1:1 10:4.3:1 4.9:1:1.6 4.1:1:1.1 1.5:1:1.1 26.4:12.9:1 39.5:16.7:1 41.5:17.2:1 13.7:9.4:1 6.7:9.4:1 2.3:3.8:1 26.5:26.9:1 7.0:8.9:1

a Air samples include particle-bound BDEs and gas-phase BDEs. Dust samples include particle-bound BDEs only.

relative amounts of metabolites formed using the radioactive BDEs 47 and 99 (Table 1). PBDE congener patterns found in humans from North America show BDE 47 as being the most abundant congener, as summarized in Table 2 (1.5-6 times more abundant than BDE 99) (3-5, 8, 12-15). One could get a general impression that the ratio of BDE 47:BDE 99 is higher in human samples than in environmental samples. For example, the study by Hites (8) shows that in breast tissue the ratio is 2.8:1, in breast milk the ratio is 4.5:1, and in blood the ratio is 2.0:1, while in air the ratio is 1.5:1. While Allen et al. (7) reported that BDE 47 is also the most abundant congener in air samples (2.0-2.4 times higher than BDE 99), no comparison with human samples was reported in this study. In dust samples, it appears that BDE 47 is lower or equal to BDE 99 (0.6-1 times BDE 99) (3, 9, 10). Therefore, if one considers the BDE 47 to 99 ratios shown in Table 2, these ratios are slightly larger in human tissues (1.5-6 times) than in environmental samples (0.6-2.4 times) (Table 2) (3-5, 7-10, 12-15). For a more quantitative comparison of intake and metabolism of BDEs, more data that examine BDE levels in the human blood and the environments that they are living in (side-by-side comparison) are needed. In this study, in which equal concentrations of radiolabeled BDE 47, 99, or 153 were used to investigate metabolism by human liver microsomes, it was observed that the relative rate of BDE 99 metabolism was slightly faster than the metabolism of BDE 47. Qualitatively, this metabolic pattern is consistent with that observed for the metabolism in rats and mice (23-26). The fact that BDE 153 was not metabolized by any of the three human liver microsomes could explain the well-known, but poorly understood, bioaccumulation of this particular congener in humans. The recalcitrance of BDE 153 results in a relatively higher proportion of BDE 153 in plasma and in breast milk as compared to its relative proportion in environmental samples (Table 2), even to the point where BDE 153 concentrations become higher than BDE 47 and BDE 99 in approximately 10% of the population (15). It should be noted that the bromine substitution for BDE 153 (Figure 1) has no unsubstituted

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adjacent carbons, which is necessary for the formation of the arene oxide during metabolism by CYPs (27). This indicates that CYPs might not be capable of metabolizing BDE 153 through the normal pathway. For example, Weiss et al. (28) observed high concentrations (0.29-4.7 ng/g lipids) of BDE 153, which were comparable to the concentrations observed for BDE 47 in human sera. This observation is not consistent with the relative composition of PBDEs found in environmental samples and commercial mixtures (3, 6-11). Oral exposure of rodents showed lower uptake efficiency for BDE 153 as compared to BDEs 47 and 99, indicating that preferential uptake is not the reason for the increased proportion of BDE 153 in plasma and tissue (23-26, 29). While Weiss et al. (28) proposed debromination of BDE 209 as the source of BDE 153, no evidence to support this speculation had been presented. A study by Huwe and Smith (30) with rats observed debromination of BDE 209, but not to BDE 153, which also contradicts the debromination hypothesis as a source of BDE 153. However, debromination of other congeners could result in BDE 153 or other congeners. The results of our present study provide evidence that the increase in the relative retention of BDE 153 in humans may be due to its resistance to enzymatic metabolism in the liver by CYPs. It is also likely that BDE 153 possibly has a longer half-life in tissue and the environment due to its chemical and physical properties. Two other important observations with significant implications can be drawn from the results of our study. First, the formation of dihydroxylated metabolites from BDEs 47 and 99 suggests the initial formation of an arene oxide by CYPs (27). This in turn indicates that biotransformation pathways for BDEs 47 and 99 in humans produce reactive metabolites that have not been detected previously. Other reactions of the parent compounds or metabolites can lead to cleavage of the diphenyl ether bond and cause the formation of brominated phenols. These metabolites have been detected in humans, rats, and mice that have been exposed to PBDEs (16, 17, 23-26). Previous studies have focused mainly on the analysis of monohydroxylated PBDEs, which are the main metabolites observed in fish blood (31, 32), mice (23-26), and human plasma and tissue (18, 19). It has been speculated that debromination and hydroxylation of BDE 99 may result in the formation of hydroxylated tetra-BDE (24). However, no debrominated BDE 47, BDE 99, or BDE 153 metabolites were detected by GC/ MS following incubation with human liver microsomes. This is possible due to the fact that microsomes do not contain the enzymes capable of debromination, such as deiodinases (30). However, instead of debromination, we see cleavage of the diphenyl ether bond, producing a brominated phenol, which is consistent with the Stapleton et al. study (17). Overall, the results of this study suggest that the biotransformation of PBDEs in humans is mediated by P450 enzymes, as revealed by the formation of dihydroxylated metabolites and the observed cleavage of the ether bond, which are characteristic of oxygenase activity. Future studies are needed to identify the specific human P450 enzymes that are primarily responsible for the metabolism of PBDEs, as well as including studies using the S9 fraction to include other enzymatic pathways for PBDE metabolism. Also, reference standards will be included in future experiments to quantify kinetics of metabolite formation. It was shown previously that CYP 2B6 from humans metabolized PCB 153 (33); however, to the best of our knowledge, this observation has not been confirmed by others. Other studies have shown that CYP 2B11 from dogs and CYP 2B1 from rats are able to

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metabolize PCB 153 as well (34, 35). It is also thought that the CYP 2B form will metabolize PBDEs, which would be a phenobarbital-inducible form (1). Dioxin-like coplanar PCBs are known to induce CYPs 1A1 and 1B1 (36). PBDEs are also known to moderately induce CYP 1A1 and CYP 1A2 (1, 2). However, the induction of CYP 1A1 is thought to be due to planar compound impurities such as dioxins that are present in the commercial mixture (37). Interindividual genetic and environment-induced variability in CYP activities may in part be responsible for the wide range of levels of PBDEs detected in humans as well as exposure to these compounds. The identification of PBDE metabolites and potential biotransformation pathways in humans will not only assist efforts to better understand factors regulating the disposition and retention of these contaminants but also the potential toxicity of PBDE metabolites. Finally, the results of this study provide one possible explanation as to why the congener pattern of PBDEs in human samples does not completely reflect the congener pattern in the environment to which humans are exposed. For instance, in Table 2, environmental samples show that BDE 153 is the least concentrated congener of BDEs 47, 99, and 153; however, that is not the case for concentrations in humans, where BDE 99 is often found to be the least concentrated congener. This indicated that there are possible differences in the uptake or metabolism of these congeners; however, previous studies have shown that uptake of BDE 153 is less than that of BDEs 47 and 99 (23-26, 29). Hence, the increase in BDE 153 levels in humans indicates resistance to metabolism. However, some increase could also be due to BDE 153 being present in two commercial mixtures, the pentaBDE and octaBDE mixtures (11). On the other hand, BDE 47 has a larger uptake efficiency than 99 (23-26, 29); therefore, both uptake and metabolism could explain the relative concentrations of these congeners in humans. However, differences in the rate of metabolism between BDE 47 and BDE 99 observed in this study indicate that metabolism possibly plays a more important role. It would be important to further examine if this difference in BDE metabolism is the major reason that could explain the relative change in the ratio of BDE 47:BDE 99 between humans and environmental samples in the same environmental settings. Acknowledgment. Thanks to Dr. Michael Sanders of the Chemistry Group within the Laboratory of Pharmacology at the National Institute of Environmental Health SciencessNational Institutes of Health (NIEHS-NIH) for the radiolabeled BDEs 47, 99, and 153. Supporting Information Available: Table of quantification and qualifier ions for GC/MS, a graph representation of extraction recoveries, and mass spectra for all metabolites observed in human liver microsome incubations. This material is available free of charge via the Internet at http:// pubs.acs.org.

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(4) (5)

(6) (7)

(8) (9) (10)

(11)

(12)

(13) (14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

References (1) McDonald, T. A. (2002) A perspective on the potential health risks of PBDEs. Chemosphere 46, 745–755. (2) Siddiqi, M. A., Laessig, R. H., and Reed, K. D. (2003) Polybrominated diphenyl ethers (PBDEs): New pollutants-old diseases. Clin. Med. Res. 1, 281–290. (3) Wu, N., Herrmann, T., Paepke, O., Tickner, J., Hale, R., Harvey, E., La Guardia, M., McClean, M. D., and Webster, T. F. (2007) Human exposure to PBDEs: Associations of PBDE body burdens with food

(22)

(23)

(24)

consumption and house dust concentrations. EnViron. Sci. Technol. 41, 1584–1589. Mazdai, A., Dodder, N. G., Abernathy, M. P., Hites, R. A., and Bigsby, R. M. (2003) Polybrominated diphenyl ethers in maternal and fetal blood samples. EnViron. Health Perspect. 111, 1249–1252. Schecter, A., Johnson-Welch, S., Tung, K. C., Harris, T. R., Papke, O., and Rosen, R. (2007) Polybrominated diphenyl ether (PBDE) levels in livers of U.S. Human fetuses and newborns. J. Toxicol. EnViron. Health, Part A 70, 1–6. Birnbaum, L. S., and Staskal, D. F. (2004) Brominated flame retardants: Cause for concern. EnViron. Health Perspect. 112, 9–17. Allen, J. G., McClean, M. D., Stapleton, H. M., Nelson, J. W., and Webster, T. F. (2007) Personal exposure to polybrominated diphenyl ethers (PBDEs) in residential indoor air. EnViron. Sci. Technol. 41, 4574–4579. Hites, R. A. (2004) Polybrominated diphenyl ethers in the environment and in people: A meta-analysis of concentrations. EnViron. Sci. Technol. 38, 945–956. Stapleton, H. M., Dodder, N. G., Offenberg, J. H., Schantz, M. M., and Wise, S. A. (2005) Polybrominated diphenyl ethers in house dust and clothes dryer lint. EnViron. Sci. Technol. 39, 925–931. Wilford, B. H., Shoeib, M., Harner, T., Zhu, J., and Jones, K. C. (2005) Polybrominated diphenyl ethers in indoor dust in Ottawa, Canada: Implications for sources and exposure. EnViron. Sci. Technol. 39, 7027–7035. La Guardia, M. J., Hale, R. C., and Harvey, E. (2006) Detailed polybrominated diphenyl ether (PBDE) congener composition of the widely used penta-, octa-, and deca-PBDE technical flame-retardant mixtures. EnViron. Sci. Technol. 40, 6247–6254. Sandanger, T. M., Sinotte, M., Dumas, P., Marchand, M., Sandau, C. D., Pereg, D., Berube, S., Brisson, J., and Ayotte, P. (2007) Plasma concentrations of selected organobromine compounds and polychlorinated biphenyls in postmenopausal women of Quebec, Canada. EnViron. Health Perspect. 115, 1429–1434. Schecter, A., Papke, O., Harris, T. R., and Tung, K. C. (2006) Partitioning of polybrominated diphenyl ether (PBDE) congeners in Human blood and milk. Toxicol. EnViron. Chem. 88, 319–324. She, J., Holden, A., Sharp, M., Tanner, M., Williams-Derry, C., and Hooper, K. (2007) Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in breast milk from the Pacific Northwest. Chemosphere 67, S307–S317. Sjodin, A., Wong, L.-Y., Jones Richard, S., Park, A., Zhang, Y., Hodge, C., Dipietro, E., McClure, C., Turner, W., Needham Larry, L., and Patterson Donald, G., Jr. (2008) Serum concentrations of polybrominated diphenyl ethers (PBDEs) and polybrominated biphenyl (PBB) in the United States population: 2003-2004. EnViron. Sci. Technol. 42, 1377–1384. Cheng, S. W., Randall, K., and Kotchevar, A. T. (2008) In vitro metabolism studies of polybrominated diphenyl ethers using rat and Human liver microsomes. Am. J. Biochem. Biotechnol. 4, 295– 303. Stapleton, H. M., Kelly, S. M., Pei, R., Letcher, R. J., and Gunsch, C. (2009) Metabolism of polybrominated diphenyl ethers (PBDEs) by human hepatocytes in vitro. EnViron. Health Perspect. 117, 197– 202. Athanasiadou, M., Cuadra, S. N., Marsh, G., Bergman, A., and Jakobsson, K. (2008) Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated PBDE metabolites in young Humans from Managua, Nicaragua. EnViron. Health Perspect. 116, 400– 408. Qiu, X., Bigsby, R. M., and Hites, R. A. (2009) Hydroxylated metabolites of polybrominated diphenyl ethers in Human blood samples from the United States. EnViron. Health Perspect. 117, 93– 98. Meerts, I. A. T. M., Van Zanden, J. J., Luijks, E. A. C., Van LeeuwenBol, I., Marsh, G., Jakobsson, E., Bergman, A., and Brouwer, A. (2000) Potent competitive interactions of some brominated flame retardants and related compounds with Human transthyretin in vitro. Toxicol. Sci. 56, 95–104. Omura, T., and Sato, R. (1964) Carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370–2378. Ikonomou, M. G., and Rayne, S. (2002) Chromatographic and ionization properties of polybrominated diphenyl ethers using GC/ high-resolution MS with metastable atom bombardment and electron impact ionization. Anal. Chem. 74, 5263–5272. Chen, L. J., Lebetkin, E. H., Sanders, J. M., and Burka, L. T. (2006) Metabolism and disposition of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE99) following a single or repeated administration to rats or mice. Xenobiotica 36, 515–534. Qiu, X., Mercado-Feliciano, M., Bigsby, R. M., and Hites, R. A. (2007) Measurement of polybrominated diphenyl ethers and metabolites in

Human Hepatic Metabolism of BDEs 47, 99, and 153

(25)

(26)

(27) (28)

(29)

(30)

(31)

mouse plasma after exposure to a commercial pentabromodiphenyl ether mixture. EnViron. Health Perspect. 115, 1052–1058. Sanders, J. M., Chen, L. J., Lebetkin, E. H., and Burka, L. T. 2006a Metabolism and disposition of 2,2′,4,4′- tetrabromodiphenyl ether following administration of single or multiple doses to rats and mice. Xenobiotica 36, 103–117. Sanders, J. M., Lebetkin, E. H., Chen, L. J., and Burka, L. T. 2006b Disposition of 2,2′,4,4′,5,5′-hexabromodiphenyl ether (BDE153) and its interaction with other polybrominated diphenyl ethers (PBDEs) in rodents. Xenobiotica 36, 824–837. Boyd, D. R., and Sharma, N. D. (1996) The changing face of arene oxide-oxepine chemistry. Chem. Soc. ReV. 25, 289–296. Weiss, J., Wallin, E., Axmon, A., Joensson, B. A. G., Aakesson, H., Janak, K., Hagmar, L., and Bergman, A. (2006) Hydroxy-PCBs, PBDEs, and HBCDDs in serum from an elderly population of Swedish fishermen’s wives and associations with bone density. EnViron. Sci. Technol. 40, 6282–6289. Burreau, S., Axelman, J., Broman, D., and Jakobsson, E. (1997) Dietary uptake in pike (Esox lucius) of some polychlorinated biphenyls, polychlorinated naphthalenes and polybrominated diphenyl ethers administered in natural diet. EnViron. Toxicol. Chem. 16, 2508–2513. Huwe, J. K., and Smith, D. J. (2007) Accumulation, whole-body depletion, and debromination of decabromodiphenyl ether in male sprague-dawley rats following dietary exposure. EnViron. Sci. Technol. 41, 2371–2377. Letcher, R. J., D’Sa, I., Valters, K., Marsh, G., Li, H., Bennett, E., and Alaee, M. (2003) Polybrominated diphenyl ethers and hydroxy-

Chem. Res. Toxicol., Vol. 22, No. 11, 2009 1809

(32)

(33)

(34)

(35)

(36) (37)

lated and methoxylated analogues in Detroit River fish. Organohalogen Compd. 61, 29–32. Marsh, G., Athanasiadou, M., Bergman, A., and Asplund, L. (2004) Identification of hydroxylated and methoxylated polybrominated diphenyl ethers in Baltic Sea salmon (Salmo salar) blood. EnViron. Sci. Technol. 38, 10–18. Ariyoshi, N., Oguri, K., Koga, N., Yoshimura, H., and Funae, Y. (1995) Metabolism of highly persistent PCB congener, 2,4,5,2′,4′,5′-hexachlorobiphenyl, by Human CYP2B6. Biochem. Biophys. Res. Commun. 212, 455–460. Ariyoshi, N., Koga, N., Oguri, K., and Yoshimura, H. (1992) Metabolism of 2,4,5,2′,4′,5′-hexachlorobiphenyl with liver microsomes of phenobarbital-treated dog; the possible formation of PCB 2,3-arene oxide intermediate. Xenobiotica 22, 1275–1290. Duignan, D. B., Sipes, I. G., Leonard, T. B., and Halpert, J. R. (1987) Purification and characterization of the dog hepatic cytochrome P-450 isozyme responsible for the metabolism of 2,2′,4,4′,5,5′-hexachlorobiphenyl. Arch. Biochem. Biophys. 255, 290–303. Rendic, S. (2002) Summary of information on human CYP enzymes: Human P450 metabolism data. Drug Metab. ReV. 34, 83–448. Kuiper, R. V., Murk, A. J., Leonards, P. E. G., Grinwis, G. C. M., van den Berg, M., and Vos, J. G. (2006) In vivo and in vitro Ahreceptor activation by commercial and fractionated pentabromodiphenylether using zebrafish (Danio rerio) and the DR-CALUX assay. Aquat. Toxicol. 79, 366–375.

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