Biotransformation of Hexabromocyclododecanes (HBCDs) - American

Article pubs.acs.org/est

Biotransformation of Hexabromocyclododecanes (HBCDs) with LinBAn HCH-Converting Bacterial Enzyme Norbert V. Heeb,†,* Daniel Zindel,†,‡ Birgit Geueke,§ Hans-Peter E. Kohler,§ and Peter Lienemann‡ Laboratory for Analytical Chemistry, Swiss Federal Laboratories for Materials Testing and Research, Empa, Ü berlandstrasse 129, CH-8600 Dübendorf, Switzerland. ‡ Institute of Chemistry and Biological Chemistry, Reidbach, ZHAW, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland. § Eawag, Swiss Federal Institute of Aquatic Science and Technology, Ü berlandstrasse 133, CH-8600 Dübendorf, Switzerland. †

S Supporting Information *

ABSTRACT: Hexabromocyclododecanes (HBCDs) and hexachlorocyclohexanes (HCHs) are polyhalogenated hydrocarbons with similar stereochemistry. Both classes of compounds are considered biologically persistent and bioaccumulating pollutants. In 2009, the major HCH stereoisomers came under regulation of the Stockholm convention. Despite their persistence, HCHs are susceptible to bacterial biotransformations. Here we show that LinB, an HCHconverting haloalkane dehalogenase from Sphingobium indicum B90A, is also able to transform HBCDs. Racemic mixtures of α-, β-, and γ-HBCDs were exposed to LinB under various conditions. All stereoisomers were converted, but (−)α-, (+)β-, and (+)γ-HBCDs were transformed faster by LinB than their enantiomers. The enantiomeric excess increased to 8 ± 4%, 27 ± 1%, and 20 ± 2% in 32 h comparable to values of 7.1%, 27.0%, and 22.9% as obtained from respective kinetic models. Initially formed pentabromocyclododecanols (PBCDOHs) were further transformed to tetrabromocyclododecadiols (TBCDDOHs). At least, seven mono- and five dihydroxylated products were distinguished by LC-MS so far. The widespread occurrence of HCHs has led to the evolution of bacterial degradation pathways for such compounds. It remains to be shown if LinB-catalyzed HBCD transformations in vitro can also be observed in vivo, for example, in contaminated soils or in other words if such HBCD biotransformations are important environmental processes.

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grade mixtures followed by α- and β-HBCDs (7a/b, 6a/b) with proportions of about 81%, 12%, and 6%, respectively, and δand ε-HBCDs, both meso forms, account for 0.5% and 0.3%.6 Not only the stereochemistry of HCHs and HBCDs is comparable, but also some of their physicochemical properties such as the octanol/water partitioning coefficients. Log Kow values vary from 3.72 to 4.14 and from 5.38 to 5.80 for different HCH and HBCD stereoisomers.7,8 HCHs are ubiquitous in the environment. Production and use has been banned in 52 and restricted in 33 countries.9 Since 2009, α-, β-, and γ-HCHs are regulated under the Stockholm Convention for persistent organic pollutants.10 HCHs and HBCDs are high production volume chemicals (>10 000 t/y). The global HCH production peaked in 1980 at a volume of >400 000 t/y, but was lowered to approximately 20 000 t/y in 1995.11 HBCDs are currently produced at volumes above 16 000 t/y. They are widely used as flameretardants for plastic materials such as polystyrenes and textiles.12 HBCDs are now ubiquitous as well and found in

INTRODUCTION HBCDs Bear Striking Resemblance to HCHs. Although hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs) have different applicationsInsect control and flame protection, respectivelyboth are polyhalogenated, lipophilic hydrocarbons containing six secondary halogen groups (CHX) and both are persistent, bioaccumulating, and toxic pollutants. The industrial production of HCHs was accomplished by photocatalyzed chlorination of benzene. Six stereogenic centers are formed and nine stereoisomers, seven meso forms and one pair of enantiomers have to be expected (Supporting Information (SI) Figure S1, 1−8). Technical-grade HCH contains both α-enantiomers (7a/b) as racemic mixture and the β-, γ-, δ-, and ε-stereoisomers (8, 5, 6, 4) at proportions of 53−70%, 3−14%, 10−18%, 6−10%, and 1−5%, respectively.1−5 γ-HCH (lindane) is the biologically active compound, which was applied in pure form from the 80ies.1−5 On the other hand, HBCDs are produced industrially by bromination of 1,5,9-cyclododecatrienes (CDTs). Six stereocenters are formed as well, but with fewer elements of symmetry, 16 different stereoisomers are expected, four meso forms and six pairs of enantiomers.6 The structures of all 16 HBCD stereoisomers (1−10) are given in SI Figure S2. The γHBCD enantiomers (8a/b) are most prominent in technical© 2012 American Chemical Society

Received: Revised: Accepted: Published: 6566

December 23, 2011 May 9, 2012 May 11, 2012 May 11, 2012 dx.doi.org/10.1021/es2046487 | Environ. Sci. Technol. 2012, 46, 6566−6574

Environmental Science & Technology

Article

sediments, wildlife, and humans.13−17 Levels in Swiss lake sediments, which can be considered as environmental archives, increased over the years.18,19 An increase of the HBCD flux by a factor of 5 was observed from 1970 to 2000.18 HBCDs were even detected in remote mountain- or glacial-lake sediments.19 HBCDs are considered as persistent, bioaccumulating, and toxic and are included in the European Chemicals Bureau PBT list.20 Biotransformation of HCHs. Despite their persistence and their slow degradation in the environment, the number of microorganisms that are able to transform HCHs continues to increase.21,22 Three Sphingobium strains, found in geographically distant countries such as Japan, India, and France, are proof for the global distribution of such HCH degraders.23 Especially the first two enzymes of the HCH degradation pathway, the HCH dehydrochlorinase LinA and the haloalkane dehalogenase LinB, were investigated in detail.24−27 The X-ray structures of LinA and LinB from S. japonicum UT26 are published and reaction mechanisms were proposed for both enzymes.28−30 LinB from Sphingobium indicum B90A converts a range of HCH isomers, heptachlorocyclohexanes, pentachlorocyclohexenes, and tetrachlorocyclohexadienes to mono- and sometimes to dihydroxylated metabolites (Figure 1). In general, it has broad substrate specificity and also dehalogenates brominated and iodated compounds.31

60 (Merck, Darmstadt, Germany) with n-hexane/dichloromethane mixtures (Merck, 19/1, 14/1, 9/1, 4/1 vol./vol.) as described elsewhere.6,32 Diastereomerically pure materials were obtained by repetitive crystallization from dichloromethane/nhexane mixtures.6,32 These materials are now also available commercially. An acetonitrile solution of enantiomerically pure (1S,2S,5S,6R,9R,10S)-isobutoxy-(2,5,6,9,10-pentabromocyclododecane was used as internal standard.33 HPLC-grade methanol (Biosolve, Valkenswaard, Netherlands), acetonitrile (ROMIL, Cambridge, United Kingdom), and water (Merck) were used for reversed- and chiral-phase LC. Respective chromatographic methods are described below. Glycine and tris(hyroxymethyl)amino methane (tris) were used as buffer materials (Sigma-Aldrich, Buchs, Switzerland). LinB Cloning, Protein Expression and Purification. Codon-optimized linB that was cloned into pDEST17, was obtained from John Oakeshott (CSIRO, Canberra, Australia). After transformation into E. coli BL21 AI, a fed-batch fermentation was carried out to produce 6X His-tagged LinB as described elsewhere.34 The feed was started after 15.5 h and the cells were induced with 2 g/L of L-(+)-arabinose after 17.5 h. After a total fermentation time of 20.7 h, a volume of 1.6 L was reached and 100 g of cells (wet weight) were harvested by centrifugation, frozen in liquid nitrogen and stored at −20 °C. For protein purification, 3 g of E. coli BL21 AI harboring 6X His-tagged LinB were suspended in 12 mL of equilibrium buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 7.5) and disrupted by ultrasonification under constant cooling on ice. LinB was purified on a Ni-nitrilotriacetic acid superflow cartridge (5 mL; Qiagen, Hombrechtikon, Switzerland) according to the previously published procedure.35 Protein concentration was measured with the Bio-Rad protein assay (Bio-Rad, Reinach, Switzerland) and bovine serum albumin (Sigma-Aldrich, Buchs, Switzerland) was used as standard. Enzymatic Incubation, Workup, Chemical Analysis. Three series of exposure experiments were performed with varying concentrations of enzyme, substrates, modifiers, and exposure times as described below. Aliquots of acetonitrile solutions of racemic α-, β-, and γ-HBCD were transferred to brown glass vials and concentrated to dryness with a stream of N2. The residues were taken up in acetone and mixed with buffer (glycine 192 mM, tris 25 mM pH 8.3) and enzyme to final volumes of 0.5 or 1.0 mL. Nominal concentrations of LinB were 100 and 200 mg/L, those of individual HBCD stereoisomers were 50 and 1 mg/L (78 and 1.56 μM) with modifier concentrations of 5 and 10 vol %. Samples were shaken at 100 rounds per minute at room temperature. The exposure was stopped after 0, 4, 8, 16, and 32 h by addition of ethyl acetate (0.5 mL). Internal standard (10 μL) was added to samples with HBCD concentrations of 1.56 μM. After liquid− liquid extraction with dichloromethane (3 × 0.5 mL), organic extracts were combined and concentrated to dryness with N2. Residues were dissolved in methanol/water (75/25%) and stored at 4 °C until analyzed. Separation of HBCDs and their transformation products was proceeded by LC (Spectra System P4000, Thermo Separation Products, San Jose, CA) on a C18-reversed-phase column (C18RP, 125 mm × 4 mm, 5 μm, 100 Å, Nucleosil 100−5, Macherey-Nagel, Oensingen, Switzerland) with a methanol− water gradient (70% methanol for 5 min, 70−98% in 15 min, 98% for 4 min) at a flow rate of 1 mL/min. Separation of enantiomers was achieved with a permethylated-β-cyclodextrin

Figure 1. LinB-catalyzed dehalogenation of 1,2,3,4,5,6-hexachlorocyclohexanes (HCHs) and 1,2,5,6,9,10-hexabromocyclododecanes (HBCDs). LinB catalyzes a stepwise transformation of these polyhalogenated alicyclic hydrocarbons to hydroxylated and dihydroxylated products.

Based on the structural and physicochemical similarities of HCHs and HBCDs, we hypothesized that comparable enzymatic transformations may be elicited when exposing HBCDs to HCH-converting enzymes. Herein we report on the biotransformation of α-, β-, and γ-HBCDs by LinB from Sphingobium indicum B90A, a strain that originated from contaminated soils of a former HCH production site. The formation of several first- and second-generation transformation products is described and some insights in the selectivity and the kinetics of these new HBCD transformation pathways are given.

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EXPERIMENTAL SECTION Materials, Chemicals, Buffers. Racemic α-, β-, and γHBCD materials have been isolated from a low-melting, technical grade HBCD mixture (Saytex HP-900 , mp =168− 184 °C) by normal phase liquid chromatography (LC) on silica 6567

dx.doi.org/10.1021/es2046487 | Environ. Sci. Technol. 2012, 46, 6566−6574


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Figure 2. Stereoisomer pattern of HBCDs, PBCDOHs, and TBCDDOHs after incubation (24 h, RT) of racemic mixtures of α-, β-, and γ-HBCDs (Co = 78 μM each stereoisomer) with LinB (100 mg/L, glycine 192 mM, tris 25 mM, pH 8.3, acetone 5%). Chromatograms from reversed- (RP, left) and chiral-phase columns (CP, right) are shown.

chiral-phase column (PM-β-CP, 200 mm × 4 mm, 5 μm, Nucleodex 5, Macherey-Nagel) at a flow rate of 0.9 mL/min with a methanol/water gradient (75−85% methanol in 20 min, 85−98% in 10 min, 98% for 3 min). Mass spectrometric analyses of LC-effluents were performed on a triple stage quadrupole mass spectrometer (TSQ 7000, Thermo Finnigan, San Jose, CA) with atmospheric pressure chemical ionization. All compounds of interest were detected in selective ion monitoring mode (SIM), recording the three most prominent anions of the respective chloride-adduct clusters [M +Cl]− at a corona current of 5 μA, an octapol potential of 0 V and heated capillary and vaporizer temperatures of 150 and 400 °C, respectively. HBCDs were monitored at m/z: 678.8, 680.8, and 682.8, PBCDOHs at 612.7, 614.7, and 616.7 and TBCDDOHs at 548.8, 550.8, and 552.8. Those transformation products detected at signal-to-noise ratios >5 and separable chromatographically were labeled and reported in SI Table S1. Potentially, other transformation products, either coeluting or present at lower quantities, might be found in these samples. The quantification of different stereoisomers is biased to some degree by differing MS responses at different chromatographic

retention times. Such problems can appear under CI-MS conditions where adduct ions are monitored and steep chromatographic gradients with fast changing solvent mixtures are used. To minimize such effects, individual isotope-labeled stereoisomers can be used, some are now commercially available for certain HBCD stereoisomers. Herein, we have not compensated for MS response differences of enantiomers which were small. Under the given LC-MS conditions, ratios of MS responses for (−)α/(+)α-, (−)β/(+)β-, and (+)γ/(−)γHBCDs were 0.985 ± 0.075, 1.005 ± 0.017, and 1.072 ± 0.067, respectively. Quality Assurance, Blanks, Controls. At a pH of 8.3, a base-catalyzed hydrolysis of HBCDs cannot be excluded a priori. Consequently, control samples with identical substrate, modifier, and buffer concentrations but without enzyme were exposed in parallel. No indications for an abiotic transformation of HBCDs or the formation of PBCDOHs and TBCDDOHs were found for the examined control samples under the same exposure conditions. Mass traces for PBCDOHs and TBCDDOHs remained at the noise level of blank samples. Thus one can conclude that those hydroxylation products 6568



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Figure 3. Transformation products formed during 24 h incubation of racemic mixtures of α-(left), β- (middle), and γ-HBCDs (right, Co = 78 μM each stereoisomer) with LinB (100 mg/L, glycine 192 mM, tris 25 mM, pH 8.3, acetone 5%). Major transformation products are labeled. The respective chromatographic data is given in SI Table S2.

catalytic triad. In a first step, a halogen atom is substituted by the aspartate residue to form an alkyl ester, which subsequently is hydrolyzed to a dehalogenated alcohol. Figure 1 displays the transformation of HCHs to pentachlorocyclohexanols and further to tetrachlorocyclohexadiols as well as those of HBCDs which are presumably converted to PBCDOHs and TBCDDOHs as discussed below. As a positive control for every incubation experiment, activity of LinB was measured with β-HCH as the substrate at pH 8.3. The enzyme tolerated 5 and 10 vol % of acetone that was used as a modifier to increase the substrate solubility without substantial decrease in enzyme activity. The expected HCH metabolites could be detect in all experiments showing that LinB indeed was active under these conditions.26 Figure 2 displays mass traces of the most prominent chloride adduct ions [M+Cl]− of racemic mixtures of α-, β-, and γ-

detected were obtained from enzyme-catalyzed transformations, but not from spontaneous hydrolysis reactions in the buffer. As a positive control, the degradation of β-HCH was measured over the complete reaction time under identical assay conditions, but with an elevated substrate concentration of 250 μg/mL, proving that the enzyme stayed active during exposure. β-HCH degradation and formation of hydroxylated metabolites were analyzed by GC-MS.26

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RESULTS AND DISCUSSION LinB-Catalyzed Transformations of β-HCH and α-, βand γ-HBCDs. The HCH-converting enzyme LinB (EC number 3.8.1.5) belongs to the family of the haloalkane dehalogenases. In its native form, it consists of a 296 amino acid long chain. The active site includes aspartate (Asp108), histidine (His272), and glutamate (Glu132) which form a 6569



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HBCDs partially separated on a C18-reversed-phase column (RP, upper left) and on a permethylated β-cyclodextrin chiralphase column (CP, upper right). Chemical structures of the investigated HBCDs are highlighted in SI Figure S2. The six HBCD stereoisomers could be resolved on the CP column with (−)forms eluting ahead of (+)forms in case of α- and βHBCDs and the (+)form eluting before the (−)form in case of γ-HBCDs. As expected, only three diastereoisomers can be distinguished on the RP column (Figure 2 left). Other chromatographic conditions relying on ternary solvent mixtures have been used to separate HBCDs.36−39 The use of acetonitrile as a cosolvent improves the separation but suppresses the chloride-adduct formation. The binary methanol−water system used herein gives better MS response but poorer chromatographic separation. Figure 2 also displays the three most prominent chloride adduct ions [M+Cl]− of PBCDOHs and TBCDDOHs that were produced upon incubation of HBCDs with LinB. On the RP column, at least 5 PBCDOHs and 4 TBCDDOHs could be distinguished. Surprisingly, the same number of PBCDOHs at comparable intensities was observed on the CP column. This indicates that the enzymatic transformation of HBCDs must be stereoselective. Otherwise racemic mixtures of pairs of enantiomers would be expected for these products as well. In a second series of experiments, pairs of HBCD enantiomers were separately incubated with LinB to study the substrate and product specificity. Figure 3 displays mass traces of the LinB transformation products of racemic mixtures of α- (left), β- (middle), and γ-HBCDs (right). Again, very few mono- and dihydroxylated products were formed after 24 h. Major transformation products, which appeared at signal-tonoise ratios >5 and at the three diagnostic m/z ratios, were named according to their parent compound. At least, seven PBCDOHs and five TBCDDOHs were distinguishable so far. Two PBCDOHs (P1α, P2α) and one TBCDDOH (T1α) were obtained from α-HBCDs. Three PBCDOHs (P1β, P2β, P3β) and two TBCDDOHs (T1β, T2β) were formed from β-HBCDs, and two PBCDOHs (P1γ, P2γ) and two TBCDDOHs (T1γ, T2γ) appeared during γ-HBCD transformations. MS signal intensities are in accordance with the expected values at m/z 612.7(89.7%), 614.7(100%), 616.7(61.2%) for PBCDOHs and at m/z 548.8(100%), 550.8(80%), 552.8(60.8%) for TBCDDOHs. Other stereoisomers, either coeluting or formed in smaller quantities may be found in these samples as well. SI Table S1 lists chromatographic retention times of the transformation products as they elute from reversed- and chiralphase columns. Retention times relative to the one of (−)γHBCD are reported too. All transformation products eluted ahead of the parent HBCDs, both from the RP- and the CPcolumn. This indicates that dihydroxylated compounds are more polar than monohydroxylated ones and the latter are more polar than HBCDs. From these findings, one must conclude that LinB indeed is transforming α-, β-, and γ-HBCDs to products postulated in Figure 1. Selectivity and Kinetics of LinB-Catalyzed HBCD Transformations. To further investigate the selectivity and the kinetics of these transformations, three time series experiments were performed, in which 50× smaller quantities of racemic α-, β-, and γ-HBCD mixtures were incubated with LinB. At these lower substrate concentrations (Co = 1.56 μM) substantial HBCD conversion is observed and changes of enantiomer ratios become visible (SI Figure S3). Figure 4 displays trends for the conversion of both β-HBCD

Figure 4. Kinetics of the LinB-catalyzed transformation of racemic mixtures of β-HBCDs (Co = 1.56 μM each stereoisomer). Samples were exposed to LinB (100 mg/L, glycine 192 mM, tris 25 mM, pH 8.3, acetone 10%) for 0, 4, 8, 16, and 32 h, respectively. Signal intensities relative to an internal standard are plotted versus time for HBCDs (top), PBCDOHs (middle) and TBCDDOHs (bottom). Individual stereoisomers and sum values are shown together with the respective stereoisomer pattern.

enantiomers and the formation of hydroxylated and dihydroxylated products. Ratios of peak areas of individual compounds and of the internal standard (A(compound)/A(IS)) are plotted for 0, 4, 8, 16, and 32 h of exposure (100 mg/L LinB, 10% acetone). About 1/3 of the β-HBCDs (sum) were transformed in 32 h. PBCDOH levels (sum) rapidly increased in the first 8 h but leveled off toward the end of the experiment. The formation of TBCDDOHs (sum) was slow at the beginning, but increased after 8 h. A delayed formation of TBCDDOHs is in accordance with a stepwise mechanism as postulated in 6570



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Figure 1, with PBCDOHs as first- and TBCDDOHs as secondgeneration products. A stepwise hydrolytic dechlorination has also been observed for β-HCH.26,40,41 Trends for individual stereoisomers differ considerably. Whereas about half of (+)β-HBCD is transformed in 32 h, only about 1/6 of (−)β-HBCD is consumed in the same time period. Consequently, the enantiomeric pattern changes during the course of the reaction. The enantiomeric fraction of (−)βHBCD increased up to 64% during exposure (Figure 4 upper right) corresponding to an increase of the enantiomeric excess (EE) to 27 ± 1% (Figure 5 middle right). One can conclude that LinB preferentially converts (+)β-HBCD leading to a relative enrichment of (−)β-HBCD. Nevertheless, both

enantiomers are converted by LinB under the given conditions. Looking at the PBCDOH pattern (Figure 4, middle), it can be noticed that the three detected products are formed immediately, but the proportion of P2β decreases over time. A maximal P2β concentration was observed at about 16 h. We assume that this product is the preferred precursor for the subsequent transformation to one or both TBCDDOHs that appear later. Both TBCDDOHs are formed simultaneously but the stereoisomer patterns change as well over time with a decreasing T1β proportion toward the end of the incubation. Not only the conversion of β-HBCD enantiomers is selective to some extent, but also the transformation of the hydroxylated products. Consequently, different mixtures of substrates and products are obtained for various exposure times. For example, starting with a racemic mixture of β-HBCDs, three PBCDOHs (P1β, P2β, P3β) are formed in the first few hours. One or two of them are converted further to two TBCDDOHs (T1β, T2β). The kinetics of such a multicompound system is rather complex and influenced by several factors. Enzymatic transformations are typically of mixed order and conversion rates depend on both, enzyme and substrate concentrations. Indeed, we could show that an increase of the enzyme concentration also increased the β-HBCD conversion rates. So far, LinB exposure experiments were performed in presence of a modifier, with HBCD concentrations above their water solubilities. An increase of the acetone concentration from 5 to 10 vol % also increased the HBCD transformation rate. As noticed, LinB is selective with respect to the substrates converted and the products formed. When starting with a mixture of six HBCD stereoisomers, at least 7 first- and 5 second-generation products were obtained (Figure 2). Starting with only two stereoisomers, either a racemic mixture of α-, β-, or γ-HBCDs, reduces the complexity, at least at an early stage, with only few products formed. Figure 5 (left diagrams) contains plots of the relative substrate concentrations (C t /C o ) of both α-, β-, and γ-HBCD enantiomers over time. Despite some scatter in the data, a second-order kinetics was found to best fit the experimental data. SI Table S2 reports the apparent second-order rate constants (kapp) in L g−1 s−1. The conversion of both α-HBCDs was slower than those of both β-HBCDs and of (+)γ-HBCD. Differences among both α-HBCD enantiomers were small, resulting in a moderate increase of the enantiomeric excess (EE) up to 8 ± 4% after 32 h (Figure 5, upper right). Transformations of β- and γ-HBCDs were more enantioselective. The respective enantiomeric excess increased to 27 ± 1% and to 20 ± 2% in 32 h. Comparable values of 7.1%, 27.0%, and 22.9% were obtained from the applied kinetic model (Figure 5, solid lines). In other words, (−)α-, (+)β-, and (+)γHBCDs are converted about 1.4, 3.4, and 8.6 times faster than their enantiomers (SI Table S2). SI Figure S3 displays CPchromatograms after 0, 8, and 32 h of exposure. It has to be mentioned that the MS response for two enantiomers eluting at different retention times can vary due to differing ionization efficiencies. This can be corrected by the use of isotope-labeled internal standards for both enantiomers. We have not compensated for such effects but under the given LC-MS conditions, responses of both enantiomers on average varied only by 2, 0, and 7% for α-, β-, and γ-HBCDs. Furthermore, we determined trends of Ct/Co for single stereoisomers. From the respective rate constants we deduced time trends of the enantiomeric excess as well. These data are not biased by differing MS responses and were found to give comparable

Figure 5. Comparison of LinB-catalyzed transformations of racemic mixtures of α- (top), β- (middle), and γ-HBCDs (bottom). HBCDs (Co = 1.56 μM each stereoisomer) were exposed to LinB (100 mg/L, glycine 192 mM, tris 25 mM, pH 8.3, acetone 10%) for 0, 4, 8, 16, and 32 h. Relative concentrations (Ct/Co) are plotted for the left- (×) and right-handed forms (□). Apparent second-order rate constants were deduced from plots of Co/Ct versus time. The regression lines follow the equation Co/Ct = kapp × Co × t + 1. Solid lines represent time trends deduced from the second-order kinetic model. Time trends of the measured (bars) and modeled (lines) enantiomeric excess (EE) are also given. Measured EE values are not corrected for differing MS responses of both enantiomers. 6571



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The findings presented herein indicate that the similarities between these two classes of compounds are even further reaching. LinB, a bacterial enzyme that presumably has evolved in HCH-contaminated environments, is well-suited to transform HBCDs. Thus, not only structural and physicochemical properties of HCHs and HBCDs are similar, but also their reactivities and their LinB-catalyzed transformations follow similar pathways.

results (Figure 5, right). In other words, the deduced secondorder kinetic models matched both, changes of Ct/Co ratios over time and changes of the enantiomeric excess. This preliminary kinetic study documents that the LinB-catalyzed conversion of HBCDs is enantioselective to some degree, best seen for transformations of β- and γ-HBCDs. Environmental Perspectives. A prolonged exposure of a biological system with increased loads of environmental contaminants, in this case with HCHs, can trigger the evolution of microorganisms to gain the ability to metabolize such pollutants. Nevertheless, biotransformations are typically slow and do not lead to a complete HCH decontamination of sites with deposited HCH waste of several thousand tons.5,22 However, the bacterial strain Sphingobium indicum B90A, isolated from contaminated soils of such a HCH production plant, expresses several enzymes that dehalogenate HCHs, or in other words, break carbon-chlorine bonds. We hypothesized that such enzymes may also cleave carbon−bromine bonds in HBCDs with a similar mode of action. Indeed, our findings show that LinB transforms all of the examined HBCDs. Transformations of (+)β- and (+)γ-HBCDs are fastest under the given conditions. One might expect that stereoisomer patterns of HBCDs would also change, if similar bacterial biotransformations occur in the environment. The hydroxylation of HBCDs is stereoselective, with so far seven of the 64 possible PBCDOH stereoisomers formed. A stepwise transformation of HBCDs to mono- and dihydroxylated products was observed. First attempts were made to analyze the selectivity and the kinetics of these transformations, but additional work is needed to elucidate the exact course and stereochemistry of these reaction cascades. To our knowledge PBCDOHs have not been found in the environment so far. We recently found that they are byproducts in technical HBCD mixtures and flame-proofed polystyrenes.42 Unsaturated hydroxylated pentabromocyclododecenes have been detected in exposed wistar rats together with hydroxylated HBCDs.43 The latter are also found in harbor seals44 and are formed in vitro through cytochrome P450-mediated hydroxylations.45 Herein we report that the bacterial enzyme LinB from Sphingobium indicum B90A is able to convert HBCDs to PBCDOHs and to TBCDDOHs in vitro. In principle, such bacteria or the respective enzymes might also be used for bioremediation of HBCD-contaminated sites. Some attempts were already made to us such an approach for HCHbioremediation.22 However, several questions have to be answered in advance. It has to be shown that Spingomonadacea expressing LinB are also able to convert HBCDs under environmental conditions, for example, in soils of contaminated sites it there are any. It is not clear, if HBCDs are bioavailable for bacteria under such conditions. Furthermore, it should be clarified if the various biotransformation products also contribute to the overall toxicity, persistence, and bioaccumulation potential. Whether these reactions finally lead to a mineralization of HBCDs or increase the spectrum of metabolites remains to be shown. The structural similarities of HBCDs and HCHs are striking and have been discussed in the past. We postulated that the fate of individual HBCD stereoisomers in the environment might differ, as observed before for HCHs.46 By now, it has been proven that environmental stereoisomer pattern of HBCDs vary considerably.47−49 The comparison of HCHs and HBCDs provoked a critical comment50 and a response at that time.51

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1 and S2 display configurations of all HCH and HBCD stereoisomers. Figure S3 shows chromatograms of LinB-exposed pairs of HBCD enantiomers at different times. Tables S1 and S2 report chromatographic retention times of the newly formed transformation products and the apparent second-order rate constants of HBCD conversions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +41 58 765 4257; fax: +41 58 765 4041; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge the financial support of the Swiss Federal Office for the Environment.

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REFERENCES

(1) Buser, H.-R.; Müller, M. D. Isomer and enantioselective degradation of hexachlorocyclohexane isomers in sewage sludge under anaerobic conditions. Envrion. Sci. Technol. 1995, 29, 664−672. (2) Willett, K. L.; Ulrich, E. M.; Hites, R. A. Differential toxicity and environmental fates of hexachlorocyclohexane isomers. Environ. Sci. Technol. 1998, 32, 2197−2207. (3) Report of the Persistent Organic Pollutants Review Committee on the Work of Its Second Meeting. Risk Profile on Lindane, UNEP/POPS/ POPRC.2/17/Add.4, 21; United Nations Environment Programme, November 2006. (4) Shen, L.; Wania, F.; Lei, Y. D.; Teixeira, C.; Muir, D. C. G.; Bidleman, T. F. Hexachlorocyclohexanes in the North American atmosphere. Environ. Sci. Technol. 2004, 38, 965−975. (5) Vijgen, J.; Abhilash, P. C.; Li, Y. F.; Lal, R.; Forter, M.; Torres, J.; Singh, N.; Yunus, M.; Tian, C.; Schäff er, A.; Weber, R. Hexachlorocyclohexane (HCH) as new Stockholm Convention POPs-a global perspective on the management of Lindane and its waste isomers. Environ. Sci. Pollut. Res. Int. 2011, 182, 152−162. (6) Heeb, N. V.; Schweizer, W. B.; Kohler, M.; Gerecke, A. C. Structure elucidation of hexabromocyclododecanesA class of compounds with a complex stereochemistry. Chemosphere 2005, 61, 65−73. (7) National Toxicology Program, Department of Health and Human Services. Lindane, Hexachlorocyclohexane (Technical Grade), And Other Hexachlorocyclohexane Isomers. Report on Carcinogens. 2011. http://ntp.niehs.nih.gov/ntp/roc/twelfth/profiles/Lindane.pdf (accessed December 2011). (8) Marvin, C. G.; Tomy, G. T.; Armitage, J. M.; Arnot, A. A.; McCarty, L.; Covaci, A.; Palace, V. Hexabromocyclododecane: Current understanding of chemistry, environmental fate, and toxicology and implications for global management. Environ. Sci. Technol. 2011, 24, 8613−8623. 6572



Article

(9) Commission for Environmental Cooperation, (NAAEC), The North American Regional Action Plan (NARAP) on Lindane and Other Hexachlorocyclohexane (HCH) Isomers, 2006. http://www. cec.org/Storage/36/2797_NARAP_on_Lindane_and_Other_ Hexachlorocyclohexane_Isomers.pdf (accessed). (10) Report of the Conference of the Parties of the Stockholm Convention on Persistent Organic Pollutants on the Work of Its Fourth Meeting, UNEP/POPS/COP.4/38 8; United Nations Environment Programme, May 2009. (11) Li, Y. F. Global technical hexachlorocyclohexane usage and its contamination consequences in the environment: from 1948 to 1997. Sci. Total Environ. 1999, 232, 121−158. (12) Alaee, M.; Arias, P.; Sjödin, A.; Bergman, A. An overview of commercially used brominated flame retardants, their applications, their use patterns in different countries/regions and possible modes of release. Environ. Int. 2003, 29, 683−689. (13) de Wit, C. A. An overview of brominated flame retardants in the environment. Chemosphere 2002, 46, 583−624. (14) Covaci, A.; Gerecke, A. C.; Law, R. J.; Voorspoels, S.; Kohler, M.; Heeb, N. V.; Leslie, H.; Allchin, C. R.; De Boer, J. Hexabromocyclododecanes (HBCDs) in the environment and humans: A review. Environ. Sci. Technol. 2006, 40, 3679−3688. (15) de Wit, C. A.; Alaee, M.; Muir, D. C. C. Levels and trends of brominated flame retardants in the Arctic. Chemosphere 2006, 64, 209−233. (16) Sellström, U.; Bignert, A.; Kierkegaard, A.; Häggberg, L.; de Wit, C.; Olsson, M.; Jansson, B. Temporal trend studies on tetra- and pentabrominated diphenyl ethers and hexabromocyclododecane in guillemot egg from the Baltic sea. Environ. Sci. Technol. 2003, 37, 5496−5501. (17) Marvin, C. H.; Tomy, G. T.; Alaee, M.; MacInnis, G. Distribution of hexabromocyclododecanes in Detroit River suspended sediments. Chemosphere 2006, 64, 268−275. (18) Kohler, M.; Zennegg, M.; Bogdal, C.; Gerecke, A. C.; Schmid, P.; Heeb, N. V.; Sturm, M.; Vonmont, H.; Kohler, H.-P.; Giger, W. Temporal trends, congener patterns, and sources of octa-, nona-, and decabromodiphenyl ethers (PBDE) and hexabromocyclododecanes (HBCD) in Swiss lake sediments. Environ. Sci. Technol. 2008, 42, 6378−6384. (19) Bogdal, C.; Schmid, P.; Zennegg, M.; Blüthgen, N.; Anselmetti, F. S.; Kohler, M.; Heeb, N. V.; Scheringer, M.; Hungerbühler, K. Temporal trends of hexabromocyclododecanes in urban, rural and remote lakes in Switzerland. Organohalogen Compd. 2010, 72, 338− 341. (20) European Chemicals Bureau. Identification of PBT and VPVB substances. http://ecb.jrc.ec.europa.eu/documents/PBT_ EVALUATION/PBT_sum058_CAS_25637-99-4.pdf (accessed November 2011). (21) Lal, R.; Dogra, C.; Malhotra, S.; Sharma, P.; Pal, R. Diversity, distribution and divergence of lin genes in hexachlorocyclohexanedegrading sphingomonads. Trends Biotechnol. 2006, 24, 121−130. (22) Lal, R.; Pandey, G.; Sharma, P.; Kumari, K.; Malhotra, S.; Pandey, R.; Raina, V.; Kohler, H.-P-E.; Holliger, C.; Jackson, C.; Oakeshott, J. G. Biochemistry of microbial degradation of hexachlorocyclohexane and prospects for bioremediation. Microbiol. Mol. Biol. Rev. 2010, 74, 58−80. (23) Pal, R.; Bala, S.; Dadhwal, M.; Kumar, M.; Dhingra, G.; Prakash, O.; Prabagaran, S. R.; Shivaji, S.; Cullum, J.; Holliger, C.; Lal, R. Hexachlorocyclohexane-degrading bacterial strains Sphingomonas paucimobilis B90A, UT26 and Sp+, having similar lin genes, represent three distinct species, Sphingobium indicum sp. nov., Sphingobium japonicum sp. nov. and Sphingobium f rancense sp. nov., and reclassification of [Sphingomonas] chungbukensis as Sphingobium chungbukense comb. nov. Int. J. Syst. Evol. Microbiol. 2005, 55, 1965− 1972. (24) Nagata, Y.; Miyauchi, K.; Takagi, M. Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26. J. Ind. Microbiol. Biotechnol. 1999, 23, 380−390.

(25) Suar, M.; Hauser, A.; Poiger, T.; Buser, H.-R.; Müller, M. D.; Droga, C.; Raina, V.; Holliger, C.; van der Meer, J. R.; Lal, R.; Kohler, H.-P. E. Enantioselective transformation of α-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A. Appl. Environ. Microbiol. 2005, 12, 8514−8518. (26) Raina, V.; Hauser, A.; Buser, H.-R.; Rentsch, D.; Sharma, P.; Lal, R.; Holliger, C.; Kohler, H.-P. E. Hydroxylated metabolites of β- and δhexachlorocyclohexane: Bacterial formation, stereochemical configuration, and occurrence in groundwater at a former production site. Environ. Sci. Technol. 2007, 41, 4291−4298. (27) Raina, V.; Rentsch, D.; Geiger, T.; Sharma, P.; Buser, H. R.; Holliger, C.; Lal, R.; Kohler, H.-P. E. New metabolites in the degradation of alpha- and gamma-hexachlorocyclohexane (HCH): pentachlorocyclohexenes are hydroxylated to cyclohexenols and cyclohexenediols by the haloalkane dehalogenase LinB from Sphingobium indicum B90A. J. Agric. Food Chem. 2008, 15, 6594−6603. (28) Marek, J.; Vevodova, J.; Smatanova, I. K.; Nagata, Y.; Svensson, L. A.; Newmann, J.; Takagi, M.; Damborsky, J. Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26. Biochemistry 2000, 46, 14082−14086. (29) Oakley, A. J.; Klvaňa, M.; Otyepka, M.; Nagata, Y.; Wilce, M. C. J.; Damborský, J. Crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 Å resolution: Dynamics of catalytic residues. Biochemistry 2004, 43, 870−878. (30) Okai, M.; Kubota, K.; Fukuda, M.; Nagata, Y.; Nagata, K.; Tanokura, M. Crystal structure of γ-hexachlorocyclohexane dehydrochlorinase LinA from Sphingobium japonicum UT26. J. Mol. Biol. 2010, 403 (2), 260−269. (31) Kmunicek, J.; Hynkova, K.; Jedlicka, T.; Nagata, Y.; Negri, A.; Gago, F.; Wade, R. C.; Damborsky, J. Quantitative analysis of substrate specificity of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26. Biochemistry 2005, 44, 3390−3401. (32) Heeb, N. V.; Schweizer, W. B.; Mattrel, P.; Haag, R.; Gerecke, A. C.; Kohler, M.; Schmid, P.; Zennegg, M.; Wolfensberger, M. Solidstate conformations and absolute configurations of (+) and (−) α-, β-, and γ-hexabromocyclododecanes (HBCDs). Chemosphere 2007, 68, 940−950. (33) Heeb, N. V.; Graf, H.; Schweizer, W. B.; Heeb, M.; Lienemann, P. Crystal structure of δ-isobutoxypentabromocyclododecanes, kinetics and selectivity of their isomerization during thermal treatment of flame-proofed polystyrenes. Chemosphere 2011, 83, 1568−1574. (34) Geueke, B.; Heck, T.; Limbach, M.; Seebach, D.; Kohler, H. P. Bacterial β-peptidyl aminopeptidases with unique substrate specificities for β- and mixed β/α-oligopeptides. FEBS J. 2006, 273, 5261−5272. (35) Bala, K.; Geueke, B.; Miska, M.; Rentsch, D.; Poiger, T.; Dadhwal, M.; Lal, R.; Holliger, C.; Kohler, H.-P. Enzymatic conversion of ε-hexachlorocyclohexane and a heptachlorocyclohexane isomer, two neglected components of technical hexachlorocyclohexane. Environ. Sci. Technol. 2012, 46, 4051−4058. (36) Tomy, G. T.; Halldorson, T.; Danell, R.; Law, K.; Arsenault, G.; Alaee, M.; MacInnis, G.; Marvin, C. H. Refinements to the diastereoisomer-specific method for the analysis of hexabromocyclododecane. Rapid Commun. Mass Spectrom. 2005, 19, 2819−2826. (37) Covaci, A.; Voorspoels, S.; Ramos, L.; Neels, H.; Blust, R. Recent divelopments in the analysis of brominated flame retardants and brominated natural compounds. J. Chromatogr., A 2007, 1153, 145−171. (38) Yu, Z.; Chen, L.; Mai, B.; Wu, M.; Sheng, G.; Fu, J.; Peng, P. Diastereoisomer and enantiomer-specific profiles of hexabromocaclododecanes in the atmosphere of an urban city in South China. Environ. Sci. Technol. 2008, 42, 3996−4001. (39) Harrad, S.; Abdallah, M. A.; Covaci, A. Causes of variability in concentrations and diastereomer patterns of hexabromocyclododecanes in indoor dust. Environ. Int. 2009, 35, 573−579. (40) Kumar, M.; Chaudhary, P.; Dwivedi, M.; Kumar, R.; Paul, D.; Jain, R. K.; Garg, S. K.; Kumar, A.. Enhanced biodegradation of β- and δ-hexachlorocyclohexane in the presence of α- and γ-isomers in contaminated soils. Environ. Sci. Technol. 2005, 39, 4005−4011. 6573



Article

(41) Sharma, P.; Raina, V.; Kumari, R.; Malhotra, S.; Dogra, C.; Kumari, H.; Kohler, H.-P. E.; Buser, H.-R.; Holliger, C.; Lal, R. Haloalkane dehalogenase LinB is responsible for β- and δhexachlorocyclohexane transformation in Sphingobium indicum B90A. Appl. Environ. Microbiol. 2006, 72, 5720−5727. (42) Heeb, N. V.; Zindel, D.; Schweizer, W. B.; Lienemann, P. 2,5,6,9,10-Pentabromocyclododecanols (PBCDOHs): A new class of HBCD transformation products. Chemosphere 2012, 88, 655−662. (43) Brandsma, S. H.; van der Ven, L. T. M.; de Boer, J.; Leonards, P. E. G. Identification of hydroxylated metabolites of hexabromocyclododecanes in wildlife and 28-days exposed wistar rats. Environ. Sci. Technol. 2009, 43, 6058−6063. (44) Zegers, B. N.; Mets, A.; van Bommel, R.; Minkenberg, C.; Hamers, T.; Kamstra, J. H.; Pierce, G. J.; Boon, J. P. Levels of hexabromocyclododecane in harbour porpoises and common dolphins from Western European seas, with evidence for stereoisomer-specific biotransformation by cytochrome P450. Environ. Sci. Technol. 2005, 39, 2095−2100. (45) Esslinger, S.; Becker, R.; Maul, R.; Nehls, I. Hexabromocyclododecane enantiomers: Microsomal degradation and patterns of hydroxylated metabolites. Environ. Sci. Technol. 2011, 45, 3938−3944. (46) Law, R. J.; Kohler, M.; Heeb, N. V.; Gerecke, A. C.; Schmid, P.; Voorspoels, S.; Covaci, A.; Becher, G.; Janak, K.; Thomsen, C. Hexabromocyclododecanes challenge scientists and regulators. Environ. Sci. Technol. 2005, 39, 281A−287A. (47) Tomy, G. T.; Budakowski, W.; Halldorson, T.; Whittle, D. M.; Keir, M. J.; Marvin, C.; Macinnis, G.; Alaee, M. Biomagnification of alpha- and gamma-hexabromocyclododecane isomers in a Lake Ontario food web. Environ. Sci. Technol. 2004, 38, 2298−2303. (48) Janak, K.; Covaci, A.; Voorspoels, S.; Becher, G. Hexabromocyclododecane (HBCD) in marine species from the Western Scheldt Estuary: Diastereomer- and enantiomer-specific accumulation. Environ. Sci. Technol. 2005, 39, 1987−1994. (49) Peck, A. M.; Pugh, R. S.; Moors, A.; Ellisor, M. B.; Porter, B. J.; Becker, P. R.; Kucklick, J. R. Hexabromocyclododecane in white-sided dolphins: Temporal trend and stereoisomer distribution in tissues. Environ. Sci. Technol. 2008, 42, 2650−2655. (50) Drohmann, D. HBCD: Facts and insinuations. Environ. Sci. Technol. 2006, 40, 1. (51) Law, R. J.; Kohler, M.; Heeb, N. V.; Gerecke, A. C.; Schmid, P.; Voorspoels, S.; Covaci, A.; Becher, G.; Janak, K.; Thomsen, C. Response. Environ. Sci. Technol. 2006, 40, 2.

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Biotransformation of Hexabromocyclododecanes (HBCDs) - American

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