ARTICLE pubs.acs.org/est
Hexabromocyclododecane Enantiomers: Microsomal Degradation and Patterns of Hydroxylated Metabolites Susanne Esslinger, Roland Becker,* Ronald Maul, and Irene Nehls BAM Federal Institute for Materials Research and Testing, Richard-Willst€atter-Strasse 11, 12489 Berlin, Germany
bS Supporting Information ABSTRACT: The degradation of the enantiomers of R-, β-, and γ-hexabromocyclododecane (HBCD) by phase I metabolism was investigated using induced rat liver microsomes. HBCD isomers were quantified using HPLC-MS/MS (ESI) after separation on a combination of a reversed phase and a chiral analytical column. The degradation of all six isomers followed first-order kinetics and the estimated half-lives ranged from 6.3 min for both β-HBCD enantiomers to 32.3 min in case of (þ)-γ-HBCD. (þ)-R- and ()γ-HBCD displayed significantly shorter half-lives than their corresponding antipodes. It could be shown that this degradation led to a significant enrichment of the first eluting enantiomers ()-R- and (þ)-γ-HBCD. Individual patterns of mono- and dihydroxylated derivatives obtained from each R- and γ-HBCD enantiomer were seen to be distinctly characteristic. The patterns of monohydroxylated HBCD derivatives detected in liver and muscle tissues of pollack, mackerel and in herring gull eggs were largely similar to those observed in the in vitro experiments with rat liver microsomes. This enabled individual hydroxy-HBCDs to be assigned to their respective parent HBCD enantiomers.
’ INTRODUCTION The brominated flame retardant hexabromocyclododecane (HBCD) is an important additive widely used in expanded and extruded polystyrene (EPS/XPS), high impact polystyrene (HIPS), and in polymer dispersion for textiles.1 Aside from its beneficial effects as flame retardant, HBCD is known to be persistent, bioaccumulative and undergoes long-range environmental transport. As a consequence HBCD has been found widely distributed in the environment24 and receives growing awareness as trace pollutant. Commercially available HBCD consists mainly of three diastereomeric pairs of enantiomers, (()-R-, β-, and γ-HBCD (R: 112%, β: 1013%, γ: 7589%)57 along with minor amounts of the δ- and the ε-diastereomer.6,8 While the γ-diastereomer dominates the technical mixture R-HBCD is the dominating diastereomer in biota samples.2,3,914 Furthermore, available data from various biota indicate the enrichment of either the ()- or the (þ)-enantiomer of R-HBCD in different species of crustaceans,9 fish,1012 birds,10,13,14 and marine mammals.9 To date, little is known about the metabolism of HBCD. Zegers et al.15 and Huthala et al.16 reported the incubation of HBCD diastereomers with liver microsomes and observed a shift in the diastereomeric pattern toward R-HBCD. These findings are an indication of a potential enrichment of R-HBCD and the formation of metabolites, which were identified as monohydroxyHBCDs. Two monohydroxylated metabolites eluting prior to the parent compound β-HBCD and two monohydroxy-HBCDs were formed from γ-HBCD.16 In addition, one monohydroxy-HBCD originating from γ-HBCD was detected in residues of blubber samples from harbor porpoises (Phocoena phocoena) and interpreted as evidence for diastereomer-specific metabolisation.15 r 2011 American Chemical Society
A number of unidentified polar metabolites were found in blood, bile, and urine of female adult mice exposed to γ-HBCD.17 The degradation product pentabromocyclododecene (PBCD) has been observed in office dust samples,18 chicken eggs,19 and whitefish (Coregonus lavaretus).20 Two isomers of tetrabromocyclododecadienes (TBCDs) were found in office dust samples.18 TBCDs were also detected in sediment samples from English lakes in concentrations between 72 and 810 pg g1 dw (dry weight)20 along with PBCD (n.d. 220 pg g1 dw). Furthermore, Brandsma et al.21 found monohydroxy-HBCDs in tern eggs (Sterna hirundo) and seal blubber (Phoca vitulina). Wistar rats, which had been exposed to the technical mixture of HBCD for 28 days, displayed diverse monohydroxylated derivatives of HBCD, PBCD, and TBCD as well as dihydroxylated derivatives of HBCD and PBCD.21 The objective of this study was to systematically compare the behavior of individual HBCD stereoisomers during phase I metabolism using a microsome model. First, the relative degradation rates of the enantiomers of R-, β-, and γ-HBCD in this model were investigated and discussed. Second, the patterns of hydroxylated metabolites derived from each individual HBCD stereoisomer in vitro were characterized. On this basis, it was attempted to assign the hydroxy-HBCDs found in selected biota as derivative of their respective parent HBCD stereoisomers.
Received: November 25, 2010 Accepted: March 30, 2011 Revised: March 9, 2011 Published: April 07, 2011 3938
dx.doi.org/10.1021/es1039584 | Environ. Sci. Technol. 2011, 45, 3938–3944
Environmental Science & Technology
ARTICLE
’ MATERIALS AND METHODS Separation of HBCD Enantiomers. The separation of HBCD enantiomers was performed using a Varian HPLC-system (Varian Deutschland GmbH, Darmstadt, Germany) consisting of two PrepStar SD-1 pumps, a ProStar 410 HPLC autosampler, a ProStar 335 HPLC diode array detector (DAD) and a ProStar 701 fraction collector. The procedure was described in detail elsewhere22 and absolute configurations of enantiomers were assigned as reported earlier.23 Microsomal Incubations. Rat liver microsomes from male Spargue-Dawley rats preinduced with phenobarbital/β-naphtoflavon were obtained from the Center for Cardiovascular Research (Charite - Universit€atsmedizin Berlin, Germany). Incubations of the induced hepatic microsomes with individual enantiomers, diastereomers and a mixture of R-, β-, and γ-HBCD were conducted in microcentrifuge tubes at 37 °C. For each time series on reaction kinetics and metabolite identification constant amounts of the respective HBCD stereoisomer cHBCD in a range between 0.0614 and 0.950 μM were used. A mixture consisting of potassium phosphate buffer (pH 7.4, including 0.1 mol MgCl2), HBCD (dissolved in DMSO) and microsomal suspension was preincubated for 3 min at 37 °C. The reaction was initiated thereafter by the addition of a 0.67 mM NADPH solution. The reaction was run for 0, 5, 10, 20, 30, 40, and 50 min and stopped by adding ethyl acetate (about 4 °C) to the incubation mixture, which was then extracted three times with ethyl acetate using a Vortex shaker (Heidolph, Schwabach, Germany). The combined organic phases were evaporated using a gentle stream of nitrogen and redissolved in methanol for HPLC-MS/MS analysis. HPLC-ESI()-MS/MS. HBCD stereoisomers and their hydroxylated metabolites were determined on an Agilent 1200 series HPLC binary pump system (Agilent Technologies, Waldbronn, Germany), coupled with an API 4000 Q-Trap high performance hybrid triple quadrupole/linear ion trap mass spectrometer from Applied Biosystems/MDS SCIEX (Foster City, California/Concord, Ontario, Canada). A combination of a Zorbax XDB-C18 (double end-capped, pore size: 80 Å, 1.8 μm particle size, 150 4.6 mm, Agilent Technologies, Waldbronn, Germany) and a chiral NUCLEODEX β-PM (pore size: 100 Å, 5 μm particle size, 200 4.6 mm, Macherey-Nagel GmbH & Co, D€uren, Germany) analytical column was maintained at 15 °C and the mobile phase was set to a mixture of 10 mM ammonium acetate buffer (A) and acetonitrile:methanol (90:10, v:v) (B) at a flow rate of 600 μL min1. The elution program started with an initial composition of 15% A and was ramped to 12% A in 3 min. Subsequently it was held for 19 min, followed by an equilibration time (8 min) to return to starting conditions for the next run. The injection volume was 10 μL. The HPLCMS/MS run time was 30 min per sample. Native and labeled R-, β-, and γ-HBCD were purchased from Wellington Laboratories, Inc. (Ontario, Canada). The SRM (single reaction monitoring) transitions monitored for native HBCDs were 640.6 f 79.0 (quantifier), 640.6 f 81.0 (qualifier) and 652.6 f 79.0 for the 13C12 labeled HBCDs, respectively. The transitions of 656.7 f 79.0 and 672.6 f 79.0, respectively, were established to monitor mono- and dihydroxy-HBCDs. The first and third quadrupoles were set to unit resolution. Source parameters and used MS-software are described elsewhere.22 Biota Samples. Eggs of herring gull (Larus argentatus), collected from the islands Trischen (n = 120) and Mellum
Figure 1. Degradation of the enantiomers of R-, β-, and γ-HBCD during incubation with rat liver microsomes.
(n = 120) in the German Wadden Sea and from the island Heuwiese (n = 75) near the German Baltic Sea coast were sampled in the year 2000 for the German Environmental Specimen Bank (ESB). Directly after sampling the whole egg contents were pooled and the material was stored as a milled powder in subsamples of approximately 10 g in the gas phase above liquid nitrogen24 at the ESB. The R-HBCD levels in the egg pools ranged between 74.80 ( 2.55 (Trischen) and 106.86 ( 3.89 ng g1 lipid weight (lw) (Mellum), whereas β- and γ-HBCD played a subordinate role.13 Furthermore, pollack (Pollachius pollachius) and mackerel (Scomber scombrus) were caught (n = 3) in September 2009 in the Eikelandsfjorden (60°140 1500 N, 5°430 1800 E) located in the southwestern part of Norway. The fish were eviscerated and heads, scales, and skins were detached before being cut into fillets. The fillets and livers were stored immediately at 20 °C. All samples have previously been analyzed enantiomer-specifically for HBCD using a method described elsewhere.22 HBCD contents in the fillets were 10.06 ( 1.15 (Pollack) and 16.05 ( 1.54 ng g1 lw (mackerel), whereas contents in the livers ranged between 38.86 ( 5.92 (mackerel) and 48.11 ( 4.80 ng g1 lw (pollack). Quality Assurance. Each specific incubation was performed in duplicate. Blank runs without HBCD and reaction controls without NADPH were conducted in each sequence to monitor background levels of HBCD and to ensure the functionality of the enzymatic system. For quantification, incubation mixtures 3939
dx.doi.org/10.1021/es1039584 |Environ. Sci. Technol. 2011, 45, 3938–3944
Environmental Science & Technology
ARTICLE
With half-lives of 6.3 min, β-HBCD displays the most rapid disappearance and ()-β- and (þ)-β-HBCD do not seem to differ from each other. Concerning R- and γ-HBCD, in both cases the first eluting enantiomer (()-R and (þ)-γ) displays a significantly longer half-life during microsomal incubation than its corresponding enantiomer. It should be noted that these rates were observed likewise regardless if pure enantiomers or racemates of the three diastereomers were used as substrates and were satisfactorily reproducible using constant conditions (e.g., temperature, batch of microsomes, concentration of HBCD and incubation time). This relative degradation of the enantiomers during incubation of racemic R-, β-, and γ-HBCD is detailed in the Supporting Information (SI). The observed rates certainly depend on the specific characteristics of the biological system used in the experiments and should therefore be assessed in relation to each rather than as absolute values. The data suggest that phase I metabolism might affect the chiral signatures of HBCD diastereomers in biota. Indeed, recent reports on the chiral signature of R-HBCD in biota reveal an enrichment of the ()-enantiomer in a number of fish from Europe,1012 in herring gull eggs from the German coast14 and in various marine mammals from the Canadian Arctic.9 On the other hand, the enrichment of (þ)-R-HBCD was observed in certain fish from Europe,10,11 birds from Scandinavia10 and China,14 and in shrimps and clams from the Canadian Arctic.9 This suggests that the chiral signature of R-HBCD tends to be species-dependent. To our knowledge, only two in vitro investigations revealing details on the relative stability of HBCD stereoisomers against phase I metabolism are available.15,16 Zegers et al. 2005 incubated pure HBCD diastereomers and a 1:1:1 mixture of the three diastereomers with liver microsomes of wistar rats and harbor porpoises15 and observed that R-HBCD displayed the lowest degradation rate. Huthala et al. presented similar results from hepatocyte and microsome assays.16 Therefore, both studies provide evidence that the phenomenon of different diastereomeric patterns of HBCDs in the technical mixture and in biota might be due to a faster degradation of the γ-diastereomer
(including blank runs and control samples) were spiked gravimetrically controlled with 20 μL of a methanolic solution containing 450 ng g1 of each 13C12-labeled R-, β-, and γ-HBCD diastereomer after the reaction was stopped. This resulted in an absolute content of 3.56 ng for each 13C12-labeled R-, β-, and γ-HBCD enantiomer. Quality control of HPLC analyses was done by repeated injections of solvent blanks (methanol). Each sample was injected twice. The identity of each of the six investigated HBCD stereoisomers was confirmed by retention time and two SRM transitions (quantifier, qualifier) as demanded by the European Commission decision (2002).25 For the quantification of the stereoisomers an external calibration (9 points) in the range from 5.98 to 307 ng g1 was performed.
’ RESULTS AND DISCUSSION Degradation Kinetics. Pure enantiomers of R- and γ-HBCD and the racemate of β-HBCD were separately incubated between 5 and 50 min and the resulting time series are depicted in Figure 1. Degradations followed first order kinetics as seen from the linear relationships between ln cHBCD and incubation time t (Table 1) as well as between starting and remaining HBCD concentration after a fixed incubation period (see SI, Figure S1). Rate constants and half-lives collected in Table 1 indicate significantly different levels of resistance of the individual stereoisomers against phase I metabolism.
Table 1. Degradation Rate Constants (k), Half Lives (t1/2) of HBCD Isomers after Incubation with Rat Liver Microsomes HBCD isomer
k (min1)
t1/2 (min)
R2a
pb
()-R (þ)-R
0.028 0.049
24.4 14.1
0.9973 0.9933
