TOXICOLOGICAL SCIENCES 123(1), 37–47 (2011)

doi:10.1093/toxsci/kfr155

Advance Access publication June 14, 2011

Comparative Oxidative Metabolism of BDE-47 and BDE-99 by RatHepatic Microsomes

Claudio A. Erratico,1 Sarah C. Moffatt,1 and Stelvio M. Bandiera2

Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

1These authors contributed equally to this study.2To whom correspondence should be addressed at Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver,

British Columbia, Canada V6T 1Z3. Fax: þ1 (604) 822-3035. E-mail: bandiera@mail.ubc.ca.

Received March 4, 2011; accepted June 7, 2011

Polybrominated diphenyl ethers (PBDEs) are flame-retardant

chemicals that have become ubiquitous environmental pollutants.

2,2#,4,4#-Tetrabromodiphenyl ether (BDE-47) and 2,2#,4,4#,5-pentabromodiphenyl ether (BDE-99) are among themost prevalent

PBDEs detected in humans, wildlife, and abiotic environmental

matrices. The purpose of this study was to investigate the oxidative

metabolism of BDE-47 and BDE-99 in rat hepatic microsomes by

comparing metabolite formation rates, kinetic parameters associ-

ated with metabolite formation, and the effects of prototypical

cytochrome P450 (CYP) inducers. TheCYP enzymes involvedwere

also identified. Incubation of BDE-47 with hepatic microsomes

from phenobarbital-treated rats generated a total of five hydroxyl-

ated (OH-BDE) metabolites, among which 4#-hydroxy-2,2#,4,5#-tetrabromodiphenyl ether (4#-OH-BDE-49) and 3-hydroxy-2,2#,4,4#-tetrabromodiphenyl ether (3-OH-BDE-47) were the

major metabolites, as identified using authentic standards and

quantified by liquid chromatography/mass spectrometry. Incuba-

tions of BDE-99 with hepatic microsomes from dexamethasone-

treated rats produced a total of seven hydroxylated metabolites,

among which 4-hydroxy-2,2#,3,4#,5-pentabromodiphenyl ether

(4-OH-BDE-90) and 6#-hydroxy-2,2#,4,4#,5-pentabromodiphenyl

ether (6#-OH-BDE-99) were the major metabolites. Although the

overall rate of oxidative metabolism of BDE-99 by hepatic

microsomes was greater than that of BDE-47, para-hydroxylation

involving a National Institutes of Health shift mechanism

represented a major metabolic pathway for both PBDE congeners.

Among the rat recombinant CYP enzymes tested, CYP2A2 and

CYP3A1 were the most active in BDE-47 and BDE-99 metabolism,

respectively.However,CYP1A1exhibited the highest activity for 4#-OH-BDE-49 and 6#-OH-BDE-99 formation, and CYP3A1

exhibited the highest activity for 3-OH-BDE-47 and 4-OH-BDE-

90 formation. Collectively, the results demonstrate that oxidative

metabolism of BDE-47 and BDE-99 is mediated by distinct but

overlapping sets of CYP enzymes and represents a key process that

determines the bioaccumulation of BDE-47 and BDE-99 in

mammals.

Key Words: BDE-47; BDE-99; hydroxylated metabolites;

hepatic microsomes; cytochrome P450 enzymes.

Polybrominated diphenyl ethers (PBDEs), which have been

used as flame retardants on a variety of consumer products,

have become ubiquitous environmental pollutants. PentaBDE,

a commercial PBDE mixture that mainly contains 2,2#,4,4#-tetrabromodiphenyl ether (BDE-47) and 2,2#,4,4#,5-pentabro-

modiphenyl ether (BDE-99) (La Guardia et al., 2006), was

used predominantly in North America for application to

polyurethane foam, textiles, upholstery, and electronic compo-

nents (Hale et al., 2003). PBDEs are not chemically bound to

the products they are applied to and consequently are released

during manufacture, use, and disposal of these products (Hale

et al., 2002; Odusanya et al., 2009). As a result, PBDEs have

been detected in air, soil, and water and in biota ranging from

invertebrates to humans (Hale et al., 2006; Hites, 2004).

Although BDE-47 and BDE-99 are present at similar levels in

the PentaBDE mixture and in abiotic matrices, concentrations

of BDE-99 are lower than those of BDE-47 in biotic samples

(Hites, 2004). Different metabolism of BDE-47 versus BDE-99

could account for the greater bioaccumulation of BDE-47 than

BDE-99 in biota.

In mammals, oxidative metabolism of persistent organic

pollutants occurs mainly in the liver and produces polar

metabolites that are more easily excreted than the parent

compounds. The cytochrome P450 (CYP) enzymes represent

the predominant biotransformation system because CYPs

catalyze the first step in oxidative metabolism and, therefore,

can serve as a significant determinant of bioaccumulation of

pollutants such as PBDEs. Oxidative metabolism is of

toxicological interest because recent studies have reported that

hydroxylated PBDE (OH-BDE) metabolites have greater

biological activity than the parent compounds. For example,

several OH-BDEs exhibit higher in vitro binding affinities for

rat transthyretin (Hamers et al., 2008) and estrogen receptor

(Meerts et al., 2001; Mercado-Feliciano and Bigsby, 2008)

than PBDE congeners and also inhibit estradiol sulfotransferase

in rat hepatic microsomes (Hamers et al., 2008). These adverse

effects can contribute to the hormonal, reproductive, and

� The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For permissions, please email: journals.permissions@oup.com

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neurobehavioral toxicity observed in laboratory animals treated

with PBDEs (Kuriyama et al., 2005; Viberg et al., 2002; Zhou

et al., 2001).

Accumulated evidence from several studies demonstrates

that PBDEs are biotransformed to hydroxylated metabolites in

mammals. OH-BDEs were identified in blood samples of

women and children who were environmentally exposed to

PBDEs (Athanasiadou et al., 2008; Qiu et al., 2009). In

addition, hydroxylated metabolites have been detected in bile,

urine, and feces of rats and mice treated with BDE-47, BDE-

99, or PBDE mixtures (Chen et al., 2006; Hakk et al., 2002;

Malberg et al., 2005; Marsh et al., 2006; Orn and Klasson-

Wehler, 1998; Qiu et al., 2007; Staskal et al., 2006). More

recently, formation of hydroxylated metabolites of BDE-47 and

BDE-99 by rat and human hepatic preparations has been

reported (Dong et al., 2010; Erratico et al., 2010; Hamers et al.,2008; Lupton et al., 2009, 2010; Stapleton et al., 2009). For

example, three hydroxylated metabolites were detected when

BDE-99 was incubated with rat or human hepatocytes for 72 h

(Dong et al., 2010; Stapleton et al., 2009). Lupton et al. (2010)

detected two hydroxylated metabolites of BDE-47 and BDE-

99, following a 2-h incubation with human liver microsomes,

whereas Hamers et al. (2008) identified six hydroxylated

metabolites of BDE-47, following a 90-min incubation with rat

liver microsomes. Rates of metabolite formation were not

measured, and the CYP enzymes involved in metabolite

formation were not determined in these studies. Characteriza-

tion of hepatic BDE-47 and BDE-99 metabolism in vitro can

provide a better assessment of the role of metabolism as

a determinant of BDE-47 and BDE-99 bioaccumulation,

persistence, and toxicity.

The present study was conducted to characterize CYP-

mediated biotransformation of BDE-47 and BDE-99 by rat

hepatic microsomes. Using our recently developed liquid

chromatography (LC)/mass spectrometry (MS)-based assays

(Erratico et al., 2010; Moffatt et al., 2011), we quantified rates

of OH-BDE metabolite formation, measured kinetic parameters

associated with metabolite formation, and determined the

effects of prototypical CYP inducers. In addition, the hepatic

CYP enzymes responsible for BDE-47 and BDE-99 metabolite

formation were determined using a panel of recombinant CYP

enzymes.

MATERIALS AND METHODS

Chemicals and reagents. BDE-47 (neat, 98.8% or greater purity) and

BDE-99 (neat, 97.7% or greater purity) were purchased from Chiron

AS (Trondheim, Norway). 4-Hydroxy-2,2#,3,4#-tetrabromodiphenyl ether

(4-OH-BDE-42), 3-hydroxy-2,2#,4,4#-tetrabromodiphenyl ether (3-OH-BDE-

47), 5-hydroxy-2,2#,4,4#-tetrabromodiphenyl ether (5-OH-BDE-47),

6-hydroxy-2,2#,4,4#-tetrabromodiphenyl ether (6-OH-BDE-47), 4#-hydroxy-

2,2#,4,5#-tetrabromodiphenyl ether (4#-OH-BDE-49), 4-hydroxy-2,2#,3,4#,5-pentabromodiphenyl ether (4-OH-BDE-90), 5#-hydroxy-2,2#,4,4#,5-pentab

romodiphenyl ether (5#-OH-BDE-99), 6#-hydroxy-2,2#,4,4#,5-pentabromodi-

phenyl ether (6#-OH-BDE-99), 4#-hydroxy-2,2#,4,5,5#-pentabromodiphenyl

ether (4#-OH-BDE-101), and 2,4,5-tribromophenol (2,4,5-TBP) (10 or 50 lg/ml

in acetonitrile, 97.7% grade purity or higher) were obtained from AccuStandard

(New Haven, CT). 4#-Hydroxy-2,2#,4,6#-tetrachlorobiphenyl (4-OH-CB-50)

(neat, 99.9% purity) and 4-hydroxy-2#,3,4#,5,6#-pentachlorobiphenyl

(4-OH-CB-121) (neat, 100% purity), which served as the internal standards

for BDE-47 and BDE-99 biotransformation assay, respectively, were also

purchased from AccuStandard. 2-Hydroxy-2#,3,4,4#,5-pentabromodiphenyl

ether (2-OH-BDE-123; neat) was a generous gift from Dr R.J. Letcher

(Environment Canada, Ottawa, Ontario, Canada). Sodium phenobarbital (PB),

dexamethasone (DEX), and 3-methylcholanthrene (MC) were purchased from

Sigma-Aldrich Canada (Oakville, Ontario, Canada). Hydrochloric acid, sodium

hydroxide, and organic solvents (high performance liquid chromatography

grade or better) were purchased from Fisher Scientific (Ottawa, Ontario,

Canada). Ultra-pure water was obtained using a Millipore Milli-Q system

(Billerica, MA). Baculovirus-insect cell microsomes containing expressed rat CYP

enzyme (CYP1A1, CYP1A2, CYP2A1, CYP2A2, CYP2B1, CYP2C6, CYP2C11,

CYP2C12, CYP2C13, CYP2D1, CYP2D2, CYP2E1, CYP3A1, or CYP3A2)

coexpressed with rat CYP oxidoreductase or with rat CYP oxidoreductase and rat

cytochrome b5 (BD Supersomes) were purchased from BD Biosciences (Oakville,

Ontario, Canada). Baculovirus-insect cell control microsomes containing expressed

rat CYP oxidoreductase and rat cytochrome b5 were also purchased from BD

Biosciences.

Animal treatment and preparation of hepatic microsomes. Male Long-

Evans rats (7–8 weeks of age) were purchased from Charles River Canada

(Saint-Constant, Quebec, Canada). Rats were housed in pairs on corncob

bedding in polycarbonate cages (The Anderson’s, Maumee, OH) with free

access to water and food (Laboratory Rodent Diet; PMI Feeds Inc., Richmond,

IN). Animal quarters were maintained at a constant temperature (23�C) with

controlled light (14 h) and dark (10 h) cycles. Rats were cared for in accordance

with the principles and guidelines of the Canadian Council on Animal Care.

Rats (n ¼ 6–7) were treated with CYP inducers as follows: PB (dissolved in

PBS, 80 mg/kg/day), MC (dissolved in corn oil, 25 mg/kg/day), DEX

(dissolved in corn oil, 100 mg/kg/day), or vehicle (corn oil, 2 ml/kg/day). PB,

MC, and DEX are well-characterized inducers of CYP enzymes and were

chosen because they preferentially induce CYP2B, CYP1A, or CYP3A

enzymes, respectively, in rats, under the dosing regimen described (Edwards

et al., 2007; Hrycay and Bandiera, 2003; Ryan and Levin, 1990). Compounds

were administered by intraperitoneal injection for three consecutive days and

rats were killed by decapitation 24 h after the last treatment. Microsomes were

prepared from pooled livers as described previously (Thomas et al., 1983).

Microsomal pellets were suspended in 0.25M sucrose and aliquots were stored

at �80�C, until needed. Protein concentration was measured by the method of

Lowry et al. (1951) using bovine serum albumin as a standard. Total CYP

concentration and ethoxyresorufin dealkylase, benzyloxyresorufin dealkylase,

and benzyloxyquinoline dealkylase activities of the microsomal preparations

were measured, as reported in the Supplementary data.

BDE biotransformation assay. In vitro biotransformation assays for BDE-

47 and BDE-99 were performed as previously described (Erratico et al., 2010;

Moffatt et al., 2011). Briefly, reaction mixtures contained BDE-47 or BDE-99

(2.5–200lM), hepatic microsomal protein (0.1, 0.5, or 1.0 mg), and 50mM

potassium phosphate buffer containing 3mM magnesium chloride (pH 7.4) in

a final volume of 0.99 ml in a screw cap tube. Reaction mixtures were

preincubated for 5 min in a shaking water bath. Reactions were initiated by

addition of 0.01 ml of nicotinamide adenine dinucleotide 2’-phosphate reduced,

tetrasodium salt hydrate (NADPH) solution (1mM final concentration) and

terminated after 5 min for BDE-47 or 10 min for BDE-99 by addition of 1 ml

of ice-cold 0.5M sodium hydroxide. A fixed amount (10 ll) of internal standard

was then added to each tube (final concentration 5.0lM for 4-OH-CB-50

or 3.0lM for 4-OH-CB-121). Tubes were vortex mixed for 1 min and

subsequently heated for 10 min in a water bath at 70�C. After cooling to room

temperature, 2 ml of 6M HCl and 1 ml of isopropanol were added to each tube.

Tubes were vortex mixed again for 1 min. An aliquot (2.0 ml) of a mixture of

methyl tert-butyl ether:hexane (1:1 vol/vol) was then added to each tube. Tubes

38 ERRATICO, MOFFATT, AND BANDIERA

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were vortex mixed for 1 min and then spun in a centrifuge at 2500 revolutions

per minute for 5 min. The top organic layer was transferred to a set of clean

tubes and the extraction procedure was repeated two more times. The three

organic solvent extracts were pooled, dried under nitrogen, reconstituted in 250

ll of methanol, and filtered through a syringe filter (0.45 lm, polytetrafluoro-

ethylene membrane) into 300-ll HPLC vials.

Blank and negative control samples were included in each assay. Blank

samples did not contain substrate and NADPH, whereas negative control

samples were devoid of either NADPH or substrate. To determine if metabolite

formation was enzyme mediated, experiments were conducted using heat-

denatured microsomes. Hepatic microsome samples were boiled for 5 min in

assay buffer prior to use. To determine if metabolite formation was CYP

mediated, carbon monoxide, which is a classic nonspecific CYP inhibitor, was

bubbled for 2 min into an incubation mixture containing assay buffer,

microsomes, and NADPH prior to use.

Preliminary experiments were conducted to determine the most active

microsomal preparation for each substrate. This microsomal preparation was

then used to confirm that both substrate and cofactor concentrations used were

saturating. Samples were prepared in duplicate and three independent

experiments were conducted.

Incubations with rat recombinant CYP enzymes were carried as described

above except that reaction mixtures contained 30 pmoles of recombinant CYP

enzyme instead of rat hepatic microsomes. Insect cell control microsomes

containing expressed rat CYP oxidoreductase and cytochrome b5 were used as

blank controls at an equivalent amount of protein (0.30 mg). To ensure that

product formation was linear with respect to incubation time and CYP

concentration, preliminary experiments were performed using recombinant

CYP3A1 enzyme. Samples were prepared singly and three independent

experiments were performed.

Analytical methods. Formation of hydroxylated BDE metabolites was

analyzed by LC/MS as described previously (Erratico et al., 2010; Moffatt et al.,

forthcoming). Briefly, OH-BDE metabolites were resolved using a Waters

Acquity UPLC BEH C18 (2.1 3 100 mm, 1.7 lm) column and a Waters Acquity

Ultra Performance Liquid Chromatograph System (UPLC; Waters Corp.,

Milford, MA) consisting of a Binary Solvent Manager and Sample Manager.

To extend the validated method for OH-penta-BDEs to two newly available

standards (4#-OH-BDE-101 and 2-OH-BDE-123), a slightly modified elution

gradient was used in which the composition of the mobile phase (water:-

methanol) was increased linearly from 35:65 (vol/vol) to 25.5:74.5 (vol/vol)

between 0 and 38 min. At 38.1 min, methanol was increased to 100% and

maintained for 2 min. The column was then re-equilibrated with water:methanol

35:65 (vol/vol) for 3 min. Flow rate was maintained at 0.2 ml/min. Metabolites

were detected using a Waters Quattro Premier XE triple quadrupole mass

spectrometer (Waters Corp.) and quantified using calibration curves prepared as

previously described (Erratico et al., 2010; Moffatt et al., forthcoming). The mass

spectrometer was operated in negative electrospray ionization mode using single

ion recording mode. Waters MassLynx v4.1 software (Waters Corp.) was used

for data acquisition. Metabolites were identified by comparison of their retention

times and mass to charge ratio (m/z) values with those of authentic standards. The

following m/z values were used: OH-tri-BDEs m/z 421.3, OH-tetra-BDEs m/z500.3, OH-penta-BDEs m/z 578.4, and 2,4,5-TBP m/z 328.7. Because authentic

standards for di-OH-BDEs are not commercially available, m/z values used

to monitor the formation of di-OH-BDEs (di-OH-tetra-BDEs m/z 516.2 and di-

OH-penta-BDEs m/z 594.5) were derived from exact mass values and the

ionization system used. Internal standards for the BDE-47 and BDE-99

biotransformation assays (4-OH-CB-50 and 4-OH-CB-121, respectively) were

monitored at m/z 306.8 and 340.8, respectively. It should be noted that

the present assay was unable to detect nonhydroxylated metabolites or 2,

4-dibromophenol.

Quality control. Along with each set of unknown samples, several quality

control samples were freshly prepared to assess linearity, accuracy, precision,

and recovery values of the assays. To assess linearity, a calibration curve was

prepared by spiking hepatic microsomes (at the same protein concentration as

that used when measuring biotransformation) with OH-BDE authentic

standards. The acceptance criterion for linearity was R2 > 0.95. Accuracy,

precision, and recovery rates were determined using quality control samples

obtained by spiking hepatic microsomes with authentic standards at 30, 150,

and 700nM for OH-tetra-BDEs, at 25 and 500nM for OH-penta-BDEs, and at

0.250 and 5.0lM for 2,4,5-TBP. The acceptance criterion for accuracy was

percent deviation within ±20% of the nominal value, that for precision was

percent relative standard deviation <20%, and that for recovery rates was

values between 80 and 120%. All the acceptance criteria were assessed on an

interday basis. System suitability samples were also used to monitor the

performance of the UPLC/MS system as previously described (Erratico et al.,

2010; Moffatt et al., forthcoming).

Data analysis. Data were analyzed using the SigmaPlot Enzyme Kinetics

Module (v.1.1; Systat Software Inc., Richmond, CA). Metabolite formation as

a function of substrate concentration was analyzed by nonlinear regression

analysis. Apparent Km, K#, and Vmax values were generated using the

Michaelis–Menten equation (Equation 1), the Hill equation (Equation 2), or the

substrate inhibition kinetic equation (Equation 3).

v ¼ Vmax½S�Km þ ½S� ; ð1Þ

v ¼ Vmax½S�n

K# þ ½S�n ; ð2Þ

v ¼ Vmax

1þ Km=½S� þ ½S�=Ki; ð3Þ

where v is initial velocity of the reaction, Vmax is the maximal velocity, [S] is

the substrate concentration, K# is the Hill dissociation constant, n is the Hill

coefficient representing cooperativity in the reaction, Km is the Michaelis–

Menten constant, and Ki is the dissociation constant of substrate binding to the

inhibitory site. The goodness of the fit was assessed according to the following

criteria: residual analysis, coefficient of determination (R2), and Akaike

Information Criterion.

RESULTS

Kinetic Analysis of BDE-47 and BDE-99 Metabolite Formation

Biotransformation of BDE-47 and BDE-99 was assessed

using hepatic microsomes prepared from rats pretreated with

PB or DEX, respectively, because of the low metabolic activity

obtained with hepatic microsomes from corn oil-treated rats, as

discussed in greater detail in the next section.

Formation of hydroxylated metabolites of BDE-47 was

evaluated over a range of substrate concentrations (10–100lM)

using hepatic microsomes from PB-treated rats, an incubation

time of 5 min and a microsomal protein concentration of 1 mg/ml.

Under these conditions, metabolite formation was directly

proportional to incubation time and protein concentration (Moffatt

et al., 2011). Five hydroxylated metabolites, identified as 4-OH-

BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, and 4#-OH-BDE-49 were formed. A representative chromatogram

showing the hydroxylated metabolites of BDE-47 is presented

in the Supplementary Figure 1. Formation of OH-tri-BDEs or di-

OH-tetra-BDEs was not observed. To further characterize hepatic

microsomal BDE-47 biotransformation, metabolite formation was

quantified and analyzed with respect to kinetic parameters. As

shown in Figure 1, formation of 4-OH-BDE-42, 3-OH-BDE-47,

COMPARATIVE OXIDATIVE METABOLISM OF BDE-47 AND BDE-99 39

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and 4#-OH-BDE-49 exhibited sigmoidal kinetic profiles and

nonlinear Eadie–Hofstee plots, which signify atypical enzyme

kinetics. Formation of 5-OH-BDE-47 and 6-OH-BDE-47 was

below the limit of quantification at all substrate concen-

trations examined. Apparent K# and Vmax values were calcu-

lated for hepatic microsomal 4-OH-BDE-42, 3-OH-BDE-47, and

4#-OH-BDE-49 formation using the Hill equation (Equation 2).

A Hill coefficient value (n) >2, which is indicative of positive

cooperativity, was obtained for all BDE-47 metabolites quantified

(Fig. 1). The apparent Vmax value for 4#-OH-BDE-49 formation

was approximately 2.5 and seven times higher than the values for

3-OH-BDE-47 and 4-OH-BDE-42 formation, respectively, in-

dicating that 4#-OH-BDE-49 was the major hydroxylated

metabolite of BDE-47 produced by hepatic microsomes from

PB-treated rats. However, apparent K# values were similar

suggesting that all three metabolites would be produced at low

BDE-47 concentrations.

Formation of hydroxylated metabolites of BDE-99 was

similarly evaluated over a range of substrate concentrations

(2.5–200lM) using hepatic microsomes from DEX-treated

rats, an incubation time of 10 min and a microsomal protein

concentration of 0.1 mg/ml. Metabolite formation was

determined previously (Erratico et al., 2010) to be linearly

related to incubation time and protein concentration under

these conditions. Incubation of BDE-99 with liver micro-

somes from DEX-treated rats yielded seven hydroxylated

metabolites, six of which were identified as 2,4,5-TBP,

4-OH-BDE-90, 5#-OH-BDE-99, 6#-OH-BDE-99, 4#-OH-

BDE-101, and 2-OH-BDE-123. A seventh metabolite peak

(M1), corresponding to a OH-penta-BDE based on its m/z value,

was detected but could not be identified because its retention time

did not match that of any of the authentic standards. A repre-

sentative chromatogram showing the hydroxylated metabolites of

BDE-99 is presented in the Supplementary Figure 1. Metabolite

peaks corresponding to OH-tetra-BDEs or di-OH-penta-BDEs

were not detected. Formation of 4-OH-BDE-90, 5#-OH-BDE-99,

6#-OH-BDE-99, and M1 (due to the lack of an authentic standard,

the plot of M1 formation was prepared using relative peak area)

exhibited Michaelis–Menten kinetics, but linear Eadie–Hoftsee

plots were only obtained for 6#-OH-BDE-99 and M1 formation.

Nonlinear Eadie–Hoftsee plots were obtained for 4-OH-BDE-90

and 5#-OH-BDE-99 formation (Fig. 2). Formation of 2,4,5-TBP

was best described by a substrate inhibition kinetic model (Equa-

tion 3). Kinetic analysis of 4#-OH-BDE-101 and 2-OH-BDE-123

formation was not conducted because 4#-OH-BDE-101 and

2-OH-BDE-123 were formed in quantifiable amounts only at

substrate concentrations of 50lM or higher (data not shown).

Apparent Km and Vmax values for hepatic microsomal formation

of 4-OH-BDE-90, 5#-OH-BDE-99, 6#-OH-BDE-99, and M1

were calculated using the Michaelis–Menten equation (Equa-

tion 1), and apparent Km, Ki, and Vmax values for 2,4,5-TBP

formation were calculated using substrate inhibition equation

(Equation 3). The apparent Vmax value for 4-OH-BDE-90

formation was approximately 20 times higher than that for

5#-OH-BDE-99 or 6#-OH-BDE-99 formation and 46 times

greater than that for 2,4,5-TBP formation, indicating that 4-OH-

BDE-90 was the major hydroxylated metabolite of BDE-99

produced by hepatic microsomes from DEX-treated rats (Fig. 1).

Apparent Km values associated with the formation of 2,4,5-TBP

FIG. 1. Enzyme kinetic profiles of 4-OH-BDE-42 (A), 3-OH-BDE-47 (B),

and 4#-OH-BDE-49 (C) formation by rat hepatic microsomes. Rate of

metabolite formation was plotted as a function of substrate concentration

following a 5-min incubation of BDE-47 with hepatic microsomes (1.0 mg

protein/ml) from PB-treated rats. Data points are the mean ± SD of three

separate experiments. Lines represent rates of metabolite formation modeled by

nonlinear regression analysis. The insets depict Eadie–Hofstee plots. Error bars

are not shown on the insets to avoid obscuring the data points, which represent

mean values.

40 ERRATICO, MOFFATT, AND BANDIERA

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and 6#-OH-BDE-99 were lower than those for 4-OH-BDE-90 and

5#-OH-BDE-99 (Fig. 1), suggesting that 2,4,5-TBP and 6#-OH-

BDE-99 metabolites would be produced preferentially at low

BDE-99 concentrations.

The aggregate BDE-47 and BDE-99 metabolite yields, which

were estimated using Vmax values for quantifiable hydroxylated

metabolites, indicate that approximately 0.05% of BDE-47 and

0.5% of BDE-99 were converted to hydroxylated metabolites/

min/mg protein, under in vitro conditions. This calculation

indicates that BDE-47 and BDE-99 were metabolized relatively

slowly, and that BDE-99 undergoes hepatic microsomal

oxidative biotransformation more readily than BDE-47.

Metabolite formation was not observed when BDE-47 or

BDE-99 was incubated with boiled microsomal preparations,

indicating that metabolite formation was enzyme mediated. In

addition, metabolite formation was not observed when

NADPH or substrate (BDE-47 or BDE-99) was omitted from

the reaction mixture or when carbon monoxide was bubbled for

2 min into an incubation mixture prior to addition of substrate,

suggesting that metabolite formation was CYP mediated.

Effect of CYP Inducers on BDE-47 and BDE-99Biotransformation

To investigate the influence of CYP induction on oxidative

biotransformation of BDE-47 and BDE-99, rates of BDE-47

and BDE-99 metabolite formation were determined using

hepatic microsomes prepared from rats pretreated with corn oil,

DEX, MC, or PB. Based on the enzyme kinetic results reported

above, saturating substrate concentrations (BDE-47 50lM or

BDE-99 100lM) were used. Distinct metabolite profiles were

obtained for BDE-47 (Table 1) and BDE-99 (Table 2) with

each hepatic microsomal preparation. BDE-47 metabolites

were not detected with hepatic microsomes from corn oil-

treated rats even when the microsomal preparation was

incubated with BDE-47 for 60 min at 2 mg protein/ml. Two

metabolites, 4#-OH-BDE-49 and 4-OH-BDE-42, were detected

following incubation with hepatic microsomes from MC-

treated rats, but only 4#-OH-BDE-49 could be quantified

because 4-OH-BDE-42 was formed at a concentration below

the limit of quantification. Three metabolites, 3-OH-BDE-47,

5-OH-BDE-47, and 4#-OH-BDE-49, were formed at quantifi-

able levels by hepatic microsomes from DEX-treated rats, and

four metabolites, 4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-

47, and 4#-OH-BDE-49, were produced by hepatic microsomes

from PB-treated rats (Table 1). Under the experimental

FIG. 2. Enzyme kinetic profiles of 2,4,5-TBP (A), 4-OH-BDE-90 (B),

5#-OH-BDE-99 (C), 6#-OH-BDE-99 (D), and M1 (E) formation by rat hepatic

microsomes. Rate of metabolite formation was plotted as a function of substrate

concentration following a 10-min incubation of BDE-99 with hepatic

microsomes (0.1 mg protein/ml) from DEX-treated rats. Data points are the

mean ± SD of three separate experiments. Lines represent rates of metabolite

formation modeled by nonlinear regression analysis. The insets depict Eadie–

Hofstee plots. Error bars are not shown on the insets to avoid obscuring the data

points, which represent mean values.

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conditions used, 3-OH-BDE-47 and 4#-OH-BDE-49 were the

major metabolites produced by hepatic microsomes from DEX-

and PB-treated rats, respectively.

In comparison, three metabolites, 4-OH-BDE-90, 5#-OH-

BDE-99, and 6#-OH-BDE-99, were detected and quantified

when BDE-99 was incubated with hepatic microsomes from

corn oil- or MC-treated rats. The major metabolite produced

by hepatic microsomes from corn oil- or MC-treated rats was 4-

OH-BDE-90 or 6#-OH-BDE-99, respectively (Table 2). Seven

metabolites, 2,4,5-TBP, 4-OH-BDE-90, 5#-OH-BDE-99, 6’-

OH-BDE-99, 4’-OH-BDE-101, 2-OH-BDE-123, and M1, were

detected using hepatic microsomes from DEX- or PB-treated

rats. The major metabolite produced by both hepatic microsomal

preparations was 4-OH-BDE-90. On the basis of relative peak

areas, M1 formation was greater in hepatic microsomes from

DEX- than PB-treated rats and it appears to be a quantitatively

important metabolite in both microsomal preparations.

Biotransformation Activities of Recombinant CYP Enzymes

A panel of 14 recombinant rat CYP enzymes was used to

identify the individual CYP enzymes involved in the bio-

transformation of BDE-47 and BDE-99 (Fig. 3). Experiments

were initially conducted using CYP3A1 and a saturating

substrate concentration (50lM for BDE-47 or 100lM for

BDE-99) to ensure linearity of product formation with respect

to incubation time and CYP concentration. An incubation time

of 10 min and a recombinant CYP enzyme concentration of 30

pmol/ml were found to be optimal. Hydroxylated metabolites

were not detected when BDE-47 or BDE-99 was incubated

with control insect cell microsomes containing expressed rat

CYP oxidoreductase without CYP enzymes.

As shown in Figure 3A, relatively few recombinant rat CYP

enzymes were involved in the oxidative biotransformation of

BDE-47. Formation of 4-OH-BDE-42 and 6-OH-BDE-47 was

catalyzed solely by CYP2A2, whereas 3-OH-BDE-47 forma-

tion was mediated exclusively by CYP3A1. Formation of

5-OH-BDE-47 was catalyzed at a high rate by CYP2A2 and at

a lower rate by CYP3A1. Formation of 4#-OH-BDE-49 was

catalyzed by CYP1A1, CYP2B1, and CYP3A1. Under the

experimental conditions used, CYP2A2 was the most active

recombinant CYP enzyme in the biotransformation of BDE-47

and 5-OH-BDE-47 was the major metabolite produced.

TABLE 1

Effect of Treatment with CYP Inducers on Formation of Hydroxylated Metabolites of BDE-47 by Rat Hepatic Microsomes

Treatment

Rate of metabolite formation (pmol/min/mg protein)

4-OH-BDE-42 3-OH-BDE-47 5-OH-BDE-47 6-OH-BDE-47 4#-OH-BDE-49

Corn oil N.D.a N.D. N.D. N.D. N.D.

DEX N.D. 3.6 ± 0.3 1.6 ± 0.1 N.D. 1.5 ± 0.2

MC <LOQb N.D. N.D. N.D. 11 ± 2.2

PB 3.6 ± 0.2 6.8 ± 0.1 1.3 ± 0.04 <LOQ 17 ± 0.6

Note. Rat hepatic microsomes (1 mg/ml) were incubated with BDE-47 at saturating substrate concentrations (50lM) for 5 min as described in the Materials and

Methods section. Values represent the mean ± SD of three independent experiments.aN.D., not detected.b<LOQ, below limit of quantification.

TABLE 2

Effect of Treatment with CYP Inducers on Formation of Hydroxylated Metabolites of BDE-99 by Rat Hepatic Microsomes

Treatment

Rate of metabolite formation (pmol/min/mg protein)

2,4,5-TBP 4-OH-BDE-90 5#-OH-BDE-99 6#-OH-BDE-99 4#-OH-BDE-101 2-OH-BDE-123

Corn oil N.D.a 2.7 ± 1.6 0.3 ± 0.1 0.6 ± 0.4 N.D. N.D.

DEX 8.2 ± 2.4 323 ± 51.6 18 ± 3.8 12 ± 3.7 3.6 ± 1.1 3.7 ± 0.6

MC N.D. 6.0 ± 3.6 1.9 ± 0.7 20 ± 4.0 N.D. N.D.

PB 21 ± 1.4 76.5 ± 16 27 ± 5.4 27 ± 3.7 9.0 ± 1.8 7.5 ± 1.0

Note. Rat hepatic microsomes were incubated with BDE-99 at saturating substrate concentrations (100lM) for 10 min. A protein concentration of 0.5 mg/ml was

used with hepatic microsomes from corn oil-treated rats and a protein concentration of 0.1 mg/ml was used with hepatic microsomes from DEX-, MC-, or PB-

treated rats, as described in the Materials and Methods section. Values represent the mean ± SD of three independent experiments. M1 was detected in hepatic

microsomes from DEX- or PB-treated rats only. Rates of M1 formation could not be expressed as pmol/min/mg protein because of the lack of an authentic

standard. Using peak area count values, the rate of M1 formation was estimated to be eight times higher in hepatic microsomes from DEX-treated than PB-treated

rats.aN.D., not detected.

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Several recombinant CYP enzymes were involved in the

oxidative biotransformation of BDE-99 (Figs. 3B and 3C). For

example, formation of 2,4,5-TBP was catalyzed by 8 of the 14

CYP enzymes tested. Biotransformation of BDE-99 to 4-OH-

BDE-90 was catalyzed at a relatively high rate by CYP3A1.

Formation of 5#-OH-BDE-99 and 6#-OH-BDE-99 was cata-

lyzed by several enzymes including CYP1A1, CYP2A2,

CYP2B1, CYP3A1, and CYP3A2. Biotransformation of

BDE-99 to 4#-OH-BDE-101 was catalyzed at a low rate by

CYP1A2 and CYP2A2, and 2-OH-BDE-123 formation was

mediated predominantly by CYP1A1 and CYP2B1, also at

a low rate. CYP2C11 and CYP3A1 enzymes were the major

enzymes involved in M1 formation. Under the experimental

conditions used, CYP3A1 was the most active recom-

binant CYP enzyme in the biotransformation of BDE-99, and

4-OH-BDE-90 was the major metabolite produced. There was no

evidence for the formation of OH-tri-BDE or di-OH-tetra-BDE

metabolites of BDE-47 or of OH-tetra-BDE or di-OH-penta-BDE

metabolites of BDE-99.

DISCUSSION

In the present study, we analyzed the oxidative biotransforma-

tion of BDE-47 and BDE-99 by rat hepatic microsomes in terms of

hydroxylated metabolite formation rates, kinetic parameters

associated with metabolite formation, and the effects of prototypical

CYP inducers. In addition, the hepatic CYP enzymes responsible

for BDE-47 and BDE-99 metabolite formation were identified.

Collectively, the results demonstrate that BDE-47 and BDE-99

undergo congener-specific hepatic microsomal biotransformation

mediated by distinct but overlapping sets of CYP enzymes.

FIG. 3. Comparison of OH-BDE metabolite formation using a panel of rat recombinant CYP enzymes. (A) Rates of OH-BDE metabolite formation were

measured following a 10-min incubation of BDE-47 (50lM) with baculovirus-insect cell microsomes containing expressed rat CYP enzymes (30 pmol) or

oxidoreductase (OR). (B and C) Rates of OH-BDE metabolite formation were measured following a 10-min long incubation of BDE-99 (100lM) with

baculovirus-insect cell microsomes containing expressed rat CYP enzymes (30 pmol) or OR. Plots show the mean values ± SD of triplicate determinations.

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Among the BDE-47 metabolites detected, 4#-OH-BDE-49

was the major metabolite produced, as determined by rate of

formation, in hepatic microsomes from MC- and PB-treated

rats, and 3-OH-BDE-47 was the major metabolite produced in

hepatic microsomes from DEX-treated rats. These results

suggest that CYP1A and CYP2B enzymes, which are highly

induced by treatment with MC and PB (Edwards et al., 2007),

respectively, are involved in the hydroxylation of BDE-47 to

4#-OH-BDE-49, whereas CYP3A enzymes, which are induced

by treatment with DEX (Hrycay and Bandiera, 2003), play

a major role in 3-OH-BDE-47 formation. Among the BDE-99

metabolites detected, 4-OH-BDE-90 was the major metabolite

in hepatic microsomes from corn oil-, DEX-, and PB-pretreated

rats, whereas 6#-OH-BDE-99 was the major metabolite in

hepatic microsomes from MC-treated rats. The results suggest

that CYP2B and CYP3A enzymes are involved in 4-OH-BDE-

90 formation, whereas CYP1A enzymes are involved in

6#-OH-BDE-99 formation. CYP2B and CYP3A enzymes are

likely involved in the formation of 2,4,5-TBP, 4#-OH-BDE-101,

2-OH-BDE-123, and M1, as these metabolites were detected

only in hepatic microsomes from PB- and DEX-treated rats. The

contribution of individual CYP enzymes to hydroxylated

metabolite formation was confirmed by experiments with rat

recombinant CYP enzymes. More specifically, among the 14 CYP

enzymes tested, CYP1A1 and CYP3A1 exhibited the highest

activity for 4#-OH-BDE-49 and 3-OH-BDE-47 formation, re-

spectively, from BDE-47 (Fig. 3A). High rate of 4#-OH-BDE-49

formation in liver microsomes from PB-treated rats was due to

CYP2B1 and CYP3A1 activities (Fig. 3A), as PB is known to

induce CYP2B1 and CYP3A1 in rats (Hrycay and Bandiera

2003). The lack of formation of 4#-OH-BDE-49 in liver

microsomes from corn oil-treated rats is due to the low level

of expression of CYP1A1, CYP2B1, and CYP3A1 enzymes in

uninduced rat liver microsomes (Hrycay and Bandiera, 2003).

With BDE-99 as the substrate, CYP3A1 and CYP1A1 were the

most efficient catalysts of 4-OH-BDE-90 and 6-OH-BDE-99

formation, respectively (Figs. 3B and 3C).

Experiments with the recombinant CYP enzymes provided

additional insights into congener-specific differences in CYP-

mediated oxidation of BDE-47 and BDE-99. For example,

CYP3A1 preferentially catalyzed meta-hydroxylation of BDE-

47 and para-hydroxylation of BDE-99, CYP1A1 preferentially

hydroxylated BDE-47 at the para position but demonstrated

a preference for ortho-hydroxylation of BDE-99, and CYP2A2

preferentially catalyzed meta- and para-hydroxylation of BDE-

47 but only para-hydroxylation of BDE-99 (summarized in

Figs. 4 and 5). CYP3A1 catalyzed the formation of 4-OH-

BDE-90, 5#-OH-BDE-99, 6#-OH-BDE-99, and 2,4,5-TBP

demonstrating that a single CYP enzyme mediated ortho-,

meta-, and para-hydroxylation, as well as oxidative cleavage,

of BDE-99. Similarly, recombinant CYP2A2 catalyzed ortho-,

meta-, and para-hydroxylation of BDE-47.

Two major oxidative metabolites of BDE-47 and BDE-99

(4#-OH-BDE-49 and 4-OH-BDE-90) result from a National

Institutes of Health (NIH) shift of a bromine atom, possibly in

concert with formation of an arene oxide intermediate. Direct

oxygen insertion at an unsubstituted ortho- or meta- position of

BDE-47 and BDE-99 does not require formation of an arene

oxide intermediate but was less favorable, in most of the rat

liver microsomal preparations, than para-hydroxylation in-

volving the NIH shift mechanism. The NIH shift mechanism

was also more efficient than hydroxylation at the carbon atom

of the ether bond, which leads to dealkylation (i.e., formation

of 2,4,5-TBP from BDE-99). We did not observe formation of

OH-tri-BDE or OH-tetra-BDE metabolites of BDE-47 or BDE-

99, respectively (i.e., oxidative debromination), or of dihydroxy-

lated metabolites by either hepatic microsomes or recombinant

CYP enzymes, under the experimental conditions used.

FIG. 4. Scheme showing CYP-mediated biotransformation of BDE-47 in rat hepatic microsomes. For each hydroxylated metabolite, the major CYP

enzyme(s) involved are shown.

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The present study indicates that oxidative metabolism of

PBDEs by rat hepatic microsomes is relatively slow compared

with many other xenobiotics such as drugs and polyaromatic

hydrocarbons (pmoles vs. nmoles of product formed/min/mg

protein). The rate and extent of biotransformation of halogenated

aromatic compounds such as polychlorinated biphenyls and

PBDEs was thought to be dependent on the number and position

of halogen atoms in the molecule, so that the greater the number

of halogens, the slower the rate of metabolism (Bandiera, 2001).

In addition, the availability of neighboring unsubstituted carbon

atoms on one ring, especially at the meta- and para- positions,

was thought to facilitate metabolism (Bandiera, 2001). The

results of the present study are inconsistent with this paradigm

because oxidative metabolism of a penta-brominated diphenyl

ether (i.e., BDE-99) by rat hepatic microsomes was greater, in

terms of metabolite formation rates, than that of tetra-brominated

diphenyl ether (i.e., BDE-47). Moreover, hydroxylation of

the tri-brominated phenyl ring of BDE-99 was favored over

the dibrominated ring, and hydroxylation at the substituted

para position of BDE-99 was favored over the unsubstituted

ortho- and meta- positions.

The lower rate of BDE-47 metabolism by rat hepatic

microsomes compared with BDE-99 (Tables 1 and 2) can be

partially explained by differences in the CYP enzymes

involved (Fig. 3). The rate of xenobiotic metabolism depends

on the microsomal level and the intrinsic catalytic activity of

the individual CYP enzymes involved. Our data show that

BDE-47 was mainly metabolized by CYP1A1 and CYP2A2 at

a relatively high rate (Fig. 3A). However, CYP1A1 and

CYP2A2 together represent approximately 5%, or less, of total

CYP content in adult male rat liver microsomes (Thummel

et al., 1988). Although CYP1A1 is extensively induced by MC

treatment (Hrycay and Bandiera, 2003), CYP2A2 is not

induced by MC, PB, or DEX treatments (Matsunaga et al.,1988; Thummel et al., 1988). Conversely, BDE-99 is

metabolized by several CYP enzymes including CYP2B,

CYP2C, and CYP3A enzymes, which together represent 45–

70% of the total CYP content in adult male rat liver

microsomes (Bandiera, 2001; Ryan and Levin, 1990).

Furthermore, CYP3A1, which is induced by PB or DEX

treatment, exhibits higher intrinsic activity toward BDE-99

than BDE-47 (approximately 400 vs. 60 pmol/min/mg protein;

Fig. 3). Thus, differences in hepatic levels of individual CYP

enzymes can account for differences in hepatic microsomal

metabolite profiles and metabolite formation rates for BDE-47

and BDE-99. The lower in vivo excretion rates of BDE-99

compared with BDE-47 reported in mice (Staskal et al., 2006)

support the role of hepatic metabolism in explaining the

difference in bioaccumulation between BDE-99 and BDE-47 in

mammals (Hites, 2004).

The BDE-47 and BDE-99 metabolites quantified in the

present study are consistent with previous in vitro and in vivometabolism studies. All five hydroxylated metabolites of BDE-

47 detected in our study were identified as in vitro metabolites

of BDE-47 produced by rat hepatic microsomes (Hamers et al.,2008) and as in vivo metabolites of rats exposed to BDE-47 by

oral administration (Marsh et al., 2006). We did not observe

formation of 2#-OH-BDE-66 or of OH-tri-BDE metabolites of

BDE-47, which were identified in the study by Marsh et al.(2006). Of the seven hydroxylated metabolites of BDE-99

detected in our study, only two (i.e., 2,4,5-TBP and 5#-OH-

BDE-99) were identified in recent in vitro studies involving rat

and human hepatocytes incubated with BDE-99 (Dong et al.,2010; Stapleton et al., 2009). OH-BDE and di-OH-BDE

FIG. 5. Scheme showing CYP-mediated biotransformation of BDE-99 in rat hepatic microsomes. For each hydroxylated metabolite, the major CYP

enzyme(s) involved are shown.

COMPARATIVE OXIDATIVE METABOLISM OF BDE-47 AND BDE-99 45

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metabolites of BDE-47 and BDE-99 were also reported in

earlier in vivo rodent studies (Chen et al., 2006; Hakk et al.,2002; Staskal et al., 2006), but the lack of structure elucidation

precludes comparison with the OH-BDE metabolites identified

in our study. Differences in hepatic preparations and the

incubation conditions used may account for discrepancies in

metabolite profiles between our study and other studies.

Several of the OH-BDE metabolites identified in our study

are toxicologically relevant because in vitro and in vivo studies

have shown that they have greater biological activity than the

parent compound. For example, 4-OH-BDE-42, 3-OH-BDE-

47, and 4#-OH-BDE-49 exhibited greater potency than BDE-

47 in displacing 17b-estradiol from the estrogen receptor

(Mercado-Feliciano and Bigsby, 2008), inhibiting aromatase

and 17b-estradiol sulfotransferase activities (Canton et al.,2008; Hamers et al., 2008), binding to transthyretin (Hamers

et al., 2008), and altering intracellular calcium homeostasis

(Dingemans et al., 2008). Another metabolite of BDE-47, 6-

OH-BDE-47, was found to be acutely toxic in developing and

adult zebrafish through uncoupling oxidative phosphorylation

(Van Boxtel et al., 2008). In addition, 4-OH-BDE-90, 5#-OH-

BDE-99, and 6#-OH-BDE-99 exhibited significantly higher

thyroid hormone activities than BDE-99 or other PBDEs (Li

et al., 2010). Thus, oxidative metabolism can enhance PBDE

toxicity and should be an important consideration in the

toxicological risk assessment of PBDEs.

In conclusion, the oxidative metabolism of BDE-47 and

BDE-99 was thoroughly characterized in vitro, and the CYP

enzymes catalyzing the formation of BDE-47 and BDE-99

metabolites in rat liver microsomes were determined for the

first time. BDE-99 undergoes more extensive CYP-mediated

metabolism than BDE-47 because of the metabolic involve-

ment of a larger number of constitutively expressed CYP

enzymes that have higher catalytic activity for BDE-99 than

BDE-47. Extrapolation of our in vitro findings suggests that

BDE-99 is more likely to undergo hepatic CYP-mediated

oxidation in vivo, consistent with the relatively lower levels of

BDE-99 found in mammals. The present study strongly

suggests that CYP-mediated metabolism is a major determinant

of the bioaccumulation and toxicity of BDE-47 and BDE-99 in

rats and, possibly, in other mammalian species.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.

oxfordjournals.org/.

FUNDING

Natural Sciences and Engineering Research Council of Canada

(RGPIN 138733-01 to S.M.B.); A graduate student fellowship

from The University of British Columbia to C.A.E.

ACKNOWLEDGMENTS

The authors thank Mr Patrick Edwards for under-

taking development of the BDE-47 biotransformation assay,

Mr Andras Szeitz for technical help with the LC/MS analysis,

and Dr Robert J. Letcher, Environment Canada, for providing

the 2-OH-BDE-123 standard.

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