Chiral Polychlorinated Biphenyls Are Biotransformed

Nov 20, 2008 - Polar Environmental Centre, Norwegian Institute of Air. Research, Tromsø ... Winnipeg, Winnipeg Manitoba R3B 2E9. Received August 9, 2...
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Environ. Sci. Technol. 2009, 43, 114–121

Chiral Polychlorinated Biphenyls Are Biotransformed Enantioselectively by Mammalian Cytochrome P-450 Isozymes to Form Hydroxylated Metabolites N I C H O L A S A . W A R N E R , †,‡ JONATHAN W. MARTIN,§ AND C H A R L E S S . W O N G * ,‡,| Polar Environmental Centre, Norwegian Institute of Air Research, Tromsø NO-9296 Norway, Department of Chemistry, University of Alberta Edmonton, Alberta T6G 2G2, Canada, Department of Laboratory Medicine and Pathology, University of Alberta Edmonton, Alberta T6G 2G3, Canada, and Richardson College for the Environment, Environmental Studies Program and Department of Chemistry, University of Winnipeg, Winnipeg Manitoba R3B 2E9

Received August 9, 2008. Revised manuscript received October 14, 2008. Accepted October 21, 2008.

In vitro incubations of purified rat cytochrome P-450 (CYP) 2B1 and human CYP 2B6 were performed to determine if CYP isozymes biotransform polychlorinated biphenyls (PCBs) enantioselectively. Enantioselective metabolism of chiral PCBs 45, 84, 91, 95, 132, and 136 and production of hydroxylated PCB metabolites (OH-PCBs) were observed, while no changes in PCB 183 atropisomer composition were observed for either isozyme. Enantiomer fractions (EFs) of parent PCBs, individually incubated as racemates at 25 ng/mL initial concentration, with rat CYP 2B1 ranged from 0.353 to 0.822. Enantioselectivity was also observed for PCBs 45 (EF ) 0.437) and 132 (EF ) 0.537) incubated at that concentration with human CYP 2B6. Both atropisomers of chiral PCBs appeared to be biotransformed simultaneously by rat CYP 2B1, except for (+)-PCB 132, but at different rates. Hydroxylated PCBs were identified using gas chromatography-high resolution mass spectrometry for all chiral PCBs enantioselectively transformed by CYPs. These metabolites did not correspond to any commercially available authentic standards, supporting the hypothesis that many unidentified OH-PCBs detected in wildlife may have arisen from in vivo biotransformation of chiral PCBs. A rough estimate suggested that more than half of the total congener metabolized by rat CYP 2B1 was converted to OH-PCBs. Similar concentration decreases were observed for congeners incubated with human CYP 2B6, but less OHPCBs were formed. Formation of OH-PCBs via an enantioselective OH insertion mechanism was suggested, and may be a source of the unidentified OH-PCBs currently found in the environment. * Corresponding author phone: +1-204-786-9335; fax: +1-204775-2114; e-mail: [email protected]. † Norwegian Institute of Air Research. ‡ Department of Chemistry, University of Alberta. § Department of Laboratory Medicine and Pathology, University of Alberta. | University of Winnipeg. 114

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Introduction Environmental concentrations of polychlorinated biphenyls (PCBs) have slowly declined over time as a result of regulations on their production and use. However, PCBs remain abundant environmental contaminants because of their recalcitrance. Food web biomagnification of PCBs (1-3) presents an increased toxicological risk to higher trophic level organisms. While such biota may have greater metabolic capacity to eliminate accumulated PCBs through biotransformation, this is not necessarily a detoxification step because persistent bioactivated metabolites may be formed (4). Phase I biotransformation of PCBs includes reaction with the cytochrome P-450 (CYP) enzyme system (4) to form hydroxylated PCBs (OH-PCBs), which may be conjugated and excreted due to their increased polarity compared to the parent compounds. However, some OH-PCBs persist in blood plasma (5-7) due to their structural similarity to thyroid hormones. This allows OH-PCBs to bind with high affinity to the thyroid transport protein transthyretin (8) and potentially cause hormone disruption as well as cytotoxic and thyroidogenic effects. In recent years, enantiospecific biomonitoring has provided insight into the biotransformation capacity of organisms toward chiral PCBs (i.e., atropisomers) (9-13). Biological processes favoring one enantiomer may cause significant enantiomer enrichment and may thus influence toxicological risks if one enantiomer is inherently more toxic (14). Such enantiomer enrichment in organisms has mainly been attributed to metabolism, but other biological processes, including selective protein binding (15, 16) and tissue transport (17), can also influence enantiomer compositions in biota. Thus, the mechanisms controlling PCB enantiomer enrichment remain poorly understood. While enantioselective biotransformation of PCBs is likely the dominant means of enantiomer enrichment (9-13, 18), this has not been demonstrated unambiguously, and little effort has been made to date to identify the specific metabolites arising from individual chiral PCB congeners, rather than from congener mixtures (16, 18-21). Kania-Korwel et al. recently demonstrated enantiospecific binding of PCB 136 to rodent microsomal CYP isozymes, but the extent of biotransformation and product identification was not examined (22). While many chiral PCBs are biotransformed through the mercapturic acid pathway to form methylsulfonylPCBmetabolites(MeSO2-PCBs)(16,19-21,23), little attention has been paid to whether OH-PCB metabolites may also form from chiral congeners. Most of the OH-PCBs detected in environmental samples remain unidentified (24, 25) due to lack of appropriate commercial standards. Given laboratory and field observations of nonracemic chiral PCBs in invertebrates (13), fish (11, 26), birds (12, 27), and mammals (9, 10, 12, 23, 28-33), biotransformation of chiral PCBs may be a source of the unidentified OH-PCBs observed. This hypothesis has not yet been tested to date. Our objectives were to use purified mammalian CYP isozymes to determine unambiguously if chiral PCBs could be biotransformed enantioselectively and whether OH-PCBs were significant products. Specifically, we used rat CYP 2B1 and human CYP 2B6 isozymes because the chlorine substitution pattern of most chiral PCBs makes them preferred mammalian substrates for CYP2B (34-36). Understanding the factors controlling stereospecific transformation of chiral PCBs and formation of their hydroxylated metabolites in a simple in vitro system will aid in understanding the biological 10.1021/es802237u CCC: $40.75

 2009 American Chemical Society

Published on Web 11/20/2008

fate of chiral PCBs in vivo, and in helping to identify the source of OH-PCBs in wildlife.

Materials and Methods Chemicals and Reagents. Rat CYP 2B1 and human 2B6 isozymes, insect cell control supersomes (P450 reductase and Cytochrome b5) and NADPH regeneration solutions (solution A: 26.1 mM NADP+, 66 mM glucose-6-phosphate, 66 mM MgCl2; solution B: 40 U/mL glucose-6-phosphate dehydrogenase, 5 mM sodium citrate) were purchased from BD Biosciences (San Jose, CA). Racemic chiral PCBs 45, 84, 91, 95, 132, 136, 149, 183, and recovery and internal standards PCBs 30, 159, and 204 (>99% purity) were obtained from Accustandard (West Haven, CT). The chiral congeners were chosen as they are present in Aroclor and other commercial PCB mixtures (37), and have been found in nonracemic proportions in laboratory biotransformation studies (13, 14, 18, 22, 26, 32, 38, 39) and in the environment (9-12, 27, 28, 30, 31, 33, 40-42). Acetone solutions of chiral PCBs (5 µg/mL) were prepared as single congeners or mixtures. Mass-labeled hydroxylated PCBs (4-hydroxy-[13C12]-PCB 29; 4-hydroxy-[13C12]-PCB 61; 4-hydroxy-[13C12]-PCB 120; 4-hydroxy-[13C12]-PCB 159; 4-hydroxy-[13C12]-PCB 172; 4-hydroxy[13C12]-PCB 189) were obtained from Wellington Laboratories (Guelph, ON, Canada) as recovery standards for metabolite extraction (chemical purities >98%, isotopic purities >99%). In Vitro Biotransformation Experiments. In vitro assays contained 25 pmol of CYP, 50 µL of solution A, 10 µL of solution B, and 915 µL of 110 mM K3PO4 (pH 7.4). The amount of CYP used was determined through preliminary in vitro screening incubations with mixtures of chiral PCB congeners, hereafter referred to as “mixture screens”, at varying CYP amounts of 10, 20, and 40 pmol and incubation periods of 10-60 minutes. Definitive assays, hereafter referred to as “single-congener assays”, were conducted with 25 pmol CYP in 30 min incubations with individual racemic congeners (25 ng) at 37.5 °C in 1 mL (i.e., concentrations for tetra-, penta-, hexa-, and heptachlorobiphenyls of 85.6, 76.6, 69.3, and 63.2 nM, respectively). Incubations were terminated with 1 mL of ice-cold methanol and immediately extracted. Control incubations were run identically, except 75 pmol of insect control supersomes, which contain no CYP, were used, equivalent to the total protein content of CYP incubations. Extraction Procedure. Methods for extraction of PCBs and OH-PCBs were previously described (6). Briefly, 20 ng each of PCBs 30, 204, and the mass-labeled OH-PCBs were added as internal standards to incubations after reaction termination. Incubations were acidified with HCl and extracted with 6 mL of 1:1 methyl-t-butyl ether (MTBE)/ hexane. The organic phase was collected, solvent exchanged to hexane, and partitioned with 6 mL of 1 M KOH to separate OH-PCBs from neutral analytes. The aqueous fraction, containing OH-PCBs, was then acidified and back-extracted into 1:1 MTBE/hexane. This organic phase was collected, solvent exchanged to hexane, and OH-PCBs methylated with diazomethane to their respective methoxy-PCBs (MeO-PCBs). The neutral fraction, containing parent PCBs, was purified using an acidified silica gel column (3 g, 22% H2SO4) and eluted with 20 mL of 15% (v/v) dichloromethane (DCM)/ hexane. The MeO-PCB fraction was purified using a 5 g, 22% H2SO4 silica column, and eluted with 50 mL of 1:1 DCM/ hexane. Both fractions were solvent exchanged to hexane, concentrated to ca. 200 µL, and fortified with 50 ng of PCB 159 as internal standard. Instrumental Analysis. Analytes were quantified using an Agilent 5890 (Mississauga, ON, Canada) gas chromatograph equipped with an electron capture detector (GC/ECD). Separation of PCBs was performed on a DB-XLB column (30 m × 0.25 mm × 0.5 µm, J&W Scientific, Folsom CA) as previously reported (37). Briefly, 2 µL samples were splitless

TABLE 1. Calculated and Measured Mass-to-Charge Ratios (m/ z) of Methoxy-PCBs (MeO-PCBs) from Single-Congener Assays of Chiral PCBs by Gas Chromatography-High Resolution Mass Spectrometry relative retention timea

measured m/z

integrated m/z range

MeO-PCB 45

0.6797

319.93292 319.93243-319.93343 321.92998 321.92948-321.93048 323.92703 323.92653-323.92753

MeO-PCB 84

0.9298

353.89395 353.89345-353.89445 355.89100 355.89050-355.89150 357.88805 357.88755-357.88855

MeO-PCB 91

0.8886

353.89395 353.89345-353.89445 355.89100 355.89050-355.89150 357.88805 357.88755-357.88855

MeO-PCB 95 (1)b

0.8477

353.89395 353.89345-353.89445 355.89100 355.89050-355.89150 357.88805 357.88755-357.88855

MeO-PCB 95 (2)b

0.8627

353.89395 353.89345-353.89445 355.89100 355.89050-355.89150 357.88805 357.88755-357.88855

MeO-PCB 132

1.3555

387.85498 387.85448-387.85548 389.85203 389.85153-389.85253 391.84908 391.84858-391.84958

MeO-PCB 136

1.0448

387.85498 387.85448-387.85548 389.85203 389.85153-389.85253 391.84908 391.84858-391.84958

a Relative retention time is normalized to that of MeO-PCB 120. b MeO-PCB 95 (1) and (2) refer to first-eluted and second-eluted peaks corresponding to masses of a pentachlorinated MeO-PCB detected in PCB 95 rat CYP 2B1 assay.

injected at 250 °C with He carrier at 27.5 psi constant pressure. The oven temperature was initially 100 °C for 1 min, ramped at 2.5 °C/min to 293 °C, and held for 2 min. The detector temperature was 320 °C with N2 as makeup gas. The MeOPCB fraction was also separated using DB-XLB with 15 psi He constant pressure at initial oven temperature 100 °C for 2 min, ramped at 20 °C min to 240 °C held for 25 min, ramped at 10 °C/min to 275 °C held for 14 min. To confirm analyte identity, selected MeO-PCB fractions were analyzed by gas chromatography/high resolution mass spectrometry (GC/HRMS) using an Agilent 6890 GC interfaced to a Kratos M-50 magnetic sector high resolution mass spectrometer (Manchester, UK) with similar GC conditions except for He carrier at constant 34 cm/s flow rate. Single ion monitoring in electron impact mode (70 eV) with a mass resolution of 10 000 was used to scan for unknown peaks with mass-to-charge ratios (m/z) corresponding to molecular ions of MeO-PCBs (Table 1). Enantiomer analysis was performed with GC/MS, using several enantioselective stationary phases as previously reported (43): Cyclosil-B (30 m × 0.25 mm × 0.25 µm, J&W Scientific), Chirasil-Dex (30 m × 0.25 mm × 0.25 µm, Varian, Palo Alto, CA), and BGB-172 (30 m × 0.25 mm × 0.18 µm, BGB Analytik, Adliswil, Switzerland). Enantiomer compositions are reported as enantiomer fractions (EFs) (44), defined as the ratio of the (+)-enantiomer concentration to the concentration of both atropisomers for congeners with known elution order (i.e., PCBs 84, 132, 136, and 149), or as the first-eluted enantiomer concentration on a given stationary phase over the concentration of both atropisomers when elution order was unknown (i.e., PCBs 45 on CyclosilB, 91 and 95 on Chirasil-Dex, and PCB 183 on BGB-172). VOL. 43, NO. 1, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Racemic standards had EFs varying from 0.497 to 0.503, with a mean EF for all congeners of 0.500. We thus used a conservative 95% confidence interval of ( 0.032 previously established (11) for racemic EF values to determine if EFs were statistically nonracemic. Recoveries of PCB 30s and 204 were 80 ( 20% and 93 ( 14%, respectively, and PCB concentrations were corrected based on PCB 204 recoveries. Mass-labeled derivatized OHPCB recoveries were dependent upon degree of chlorination, with lower recoveries for lower chlorinated OH-PCBs: 52 ( 27% (MeO-PCB 29), 48 ( 19% (MeO-PCB 61), 78 ( 13% (MeOPCB 120), 81 ( 18% (MeO-PCB 159), 84 ( 16% (MeO-PCB 172), and 79 ( 16% (MeO-PCB 189). These recoveries are consistent with previously reported OH-PCB extraction efficiencies using this extraction technique (6, 7, 25).

Results and Discussion Congener Mixture Screens with Rat CYP 2B1 and Human CYP 2B6. In the mixture screens, rat CYP 2B1 preferentially eliminated E2-PCB 45, (-)-PCB 84, E1-PCB 91, E2-PCB 95, (-)-PCB 132, and (+)-PCB 136 at all rat isozyme concentrations and incubation times investigated. These observations are consistent with preferential depletion of E2-PCB 95 and (-)-PCB 132 in adipose, skin, liver, and lung of rats exposed in vivo (23, 32). The similarity in enantioselectivity for these two congeners suggests that biotransformation by CYP 2B1 is the process responsible for observed enantiomer compositions in rats. Of the remaining two congeners, PCB 183 remained racemic. This observation is consistent with its greater recalcitrance given its higher degree of chlorination (45) and lack of vicinal meta, para H atoms, which make the other PCBs amenable to CYP 2B activity (34-36). No significant enantiomer enrichment was observed for PCB 149 in any rat CYP 2B1 mixture screens except at 40 pmol rat CYP for 40 min (EF ) 0.464), suggesting that at there may have been competition from other congeners at lower rat CYP concentrations. The slope of PCB 149 concentration versus time was statistically different from zero (P < 0.05) over the 60 min incubation period. Thus, PCB 149 was stereoselectively eliminated, but at a much slower rate than other congeners. These observations are generally consistent with studies finding nonstereoselective PCB 149 elimination by rat hepatocytes. However, 5-MeSO2-PCB 149 was eliminated stereoselectively by hepatocytes (40) and in vivo (16). It should be noted that MeSO2-PCBs arise from PCB arene oxide intermediates, which react through the mercapturic acid pathway to generate MeSO2-PCBs (4). It is possible that the nonracemic amounts of MeSO2-PCBs observed in vivo (16, 19-21, 23) is due to enantioselectivity in some of these downstream processes, and not necessarily in the initial CYP attack. Alternatively, selective transport across cell membranes or selective protein binding may also be responsible for the enantiomer enrichment of this metabolite (16), which is not possible in our experiments. Preferential depletion of E1-PCB 45 within 30 min was observed in the mixture screen using 20 pmol of human CYP 2B6. No enrichment was observed in control screens. While concentrations of PCBs 91 and 132 decreased under longer incubation times, only PCB 45 was eliminated enantioselectively, suggesting nonenantioselective elimination of the former two congeners by human CYP 2B6. These results may be attributable to preferential binding of one enantiomer to the CYP active site (22). Single-Congener Assays. Single-congener assays were conducted to eliminate competition for the enzyme active site among congeners, and to determine if OH-PCBs were produced during enantioselective elimination of individual chiral PCBs. Focus was placed on congeners that showed 116

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TABLE 2. Enantiomer Fractions (EFs) of Chiral PCB Congeners for Racemic Standards, Control Assay, And Single-Congener Assays Using Rat CYP 2B1 and hHuman CYP 2B6, Using 25 pmol/mL CYP in 30 min Incubations at 37.5°C with 25 ng/mL Initial Concentrations of Individual Racemic Congenersa PCB 45 PCB 84 PCB 91 PCB 95 PCB 132 PCB 136 standards control CYP 2B1

standards control CYP 2B6

rat CYP 2B1 0.497 0.503 0.497 0.493 0.503 (0.006 (0.002 (0.001 (0.001 (0.004 0.498 0.494 0.501 0.497 0.495 0.822 0.551 0.353 0.616 0.619

0.499 (0.006 0.501 0.437

human CYP 2B6 0.504 (0.012 0.501 0.492

0.503 (0.002 0.497 0.415

0.500 (0.006 0.511 0.537

a n ) 3 for standards except for PCBs 91 and 132 in human CYP 2B6 where n ) 4. One assay each was done for each PCB for each CYP treatment.

enantioselective elimination in our screens (i.e., PCBs 45, 84, 91, 95, 132, and 136). Rat CYP 2B1 eliminated the same PCB congeners in singlecongener assays as in the rat mixture screens, and in the same enantioselective manner (Table 2). The direction of enantiomer enrichment we observed for PCBs 84, 95, and 132 is consistent with that found in vivo for mice (38) and rats (23, 32), and was the same for PCBs 91, 95, and 132 as that previously found in porpoises (29) and seals (12, 28, 41). These observations suggest that these species may share similar CYP 2B-like detoxification mechanisms, and is supported by findings of enzymes and activities similar to CYP 2B within ringed seals (46) and other pinnipeds (47). Similar enantiomer patterns were observed in sharks (9), although several species showed racemic proportions (42), indicating that species-specific differences in metabolism dietary intake may also play a role in accumulated PCB enantiomer residues (11). Kania-Korwel et al. (22) found that (+)-PCB 136 preferentially bound to rat and mouse microsomes in vitro compared to (-)-PCB 136. They suggested that this binding may have led to enrichment of (+)-PCB 136 in mice in vivo, from either sequestration of the bound CYP complex or from more rapid biotransformation of (-)-PCB 136 due to its weaker binding to rat CYPs. In contrast, we found preferential elimination, not enrichment, of (+)-PCB 136 by rat CYP 2B6 (Table 2). It is unclear why our results contrast those of KaniaKorwel et al. (22). However, it should be noted that microsomes contain a variety of other enzymes, such as other CYP 2B or 3A isozymes not investigated here, which may also act upon PCBs enantioselectively. Enantioselective biotransformation in single-congener assays by human CYP 2B6 was not as extensive as in the rat 2B1 single-congener assays, as only E1-PCB 45 and (-)-PCB 132 were enantioselectively eliminated (Table 2). It is not certain why enantioselectivity was observed in the PCB 132 single-congener assay with human CYP 2B6, but not in the mixture screens with this isozyme. It is possible that significant competition among congeners existed among congeners for the active site in the human CYP 2B6 mixture screen. Our results are consistent with reported enrichment of (+)-PCB 132 in human liver (30), and (+)-PCB 132 and PCB 95 in human breast milk (33), suggesting that enantioselective CYP metabolism could be the responsible mechanism. In contrast, E2-PCB 91 was enriched in Spanish breast milk (33), and PCBs 84, 95, 149, and 183 were also nonracemic with no obvious pattern (33). While enantiomer enrichment of E1-PCB 95 and (-)-PCB 149 has been reported in cadaver

FIGURE 1. Metabolite fractions of control and rat 2B1 assays for chiral PCBs analyzed by GC-ECD. Mass-labeled derivatized standards: MeO-PCB 29 (peak no. 1); MeO-PCB 61 (peak no. 2); MeO-PCB 120 (peak no. 3); MeO-PCB 187 (peak no. 5); MeO-PCB 159 (peak no. 6); MeO-PCB 172 (peak no. 7). PCB 159 (internal standard) (peak no. 4); pentachlorinated MeO-PCB (circled peaks) (peaks no. 8-10). livers (30) and feces (31), most other measurements of these two congeners in human feces were racemic in volunteers fed food with racemic background residues (31), consistent with our results. As previously noted for rats, other potentially enantioselective enzyme activities may exist in vivo in humans, although human CYP 3A may be nonenantioselective toward PCBs (48). Interestingly, enrichment of PCB 45 differed between rat CYP 2B1 (EF > 0.5) and human CYP 2B6 (EF < 0.5), indicating that different enantiomer preferences exist for these two

similar isozymes. This result is not unexpected, as speciesdependent differences in enantiomer enrichment have previously been found even among closely related species. Enrichment of (-)-PCB 149 was observed in harbor and gray seals, whereas (+)-PCB 149 was enriched in Baikal seals, and no enrichment was found in Caspian seals (28). Similar species-dependent enantiomer compositions for PCB 91 and chlordanes were also observed in seabirds of the Northwater Polynya (12, 49). Rat CYP 2B1 produced more nonracemic proportions of PCB 45 than did human CYP 2B6 (Table 2). VOL. 43, NO. 1, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Recovery-corrected concentrations (ng/mL, sum of both atropisomers) (9) and enantiomer fractions (EFs) (2) vs time (minutes) for single-congener assays of chiral PCB congeners incubated with 25 pmol of rat CYP 2B1 enzyme. Chlorine substitution patterns (ring 1-ring 2) are noted beside each congener name. Dotted lines around racemic EF of 0.500 represent the 95% confidence interval of racemic EF precision ((0.032). Pseudo first order rate constants, correlation coefficients (r2), and P-values of regressions also shown for individual atropisomers ((+) and (-) for PCBs 84, 132, and 136; first-eluted, E1 and second-eluted, E2 for PCB 45 on Cyclosil-B and PCBs 91 and 95 on Chirasil-Dex) with significant changes in EF over time (* indicates P < 0.05). One possible explanation is that rat CYP 2B1 is known to catalyze direct meta-insertion of a hydroxyl group to the PCB biphenyl core (50), a process our results suggest is enantioselective, whereas the same process in human CYP 2B6 is much slower and thus would produce less enantioselectivity under similar reaction conditions. This hypothesis is expounded further below in our discussion of OH-PCB identification. Identification of OH-PCBs. To determine if OH-PCBs were formed, MeO-PCB fractions from single-congener assays were analyzed by GC/ECD. This detector was chosen as it is sensitive to halogenated compounds, a necessity given the lack of commercial standards of OH-PCBs from chiral congeners. Peaks not present in control assays were observed in rat CYP 2B1 MeO-PCB fractions from PCB 91 and PCB 95 assays (Figure 1). Thus, these peaks were due to formation 118

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of PCB metabolites, not artifacts from the incubation matrices. Metabolite identity was confirmed by GC/HRMS (Table 1) to correspond to the expected exact masses of the parent ions of pentachlorinated MeO-PCBs (i.e., derivatized OH-PCBs). For the rat single-congener assay with PCB 95, two peaks were detected with the same exact mass as a pentachlorinated MeO-PCB (Figure 1), indicating the formation of two OH-PCBs from the metabolism of this one congener. This result is consistent with the fact that PCB 95 has two unsubstituted meta-para vicinal H sites available, one or both of which could be oxidized by CYP 2B (34, 35) to form several different OH-PCB congeners. The reason why only one OH-PCB was found in the PCB 91 single-congener assays may be because any others were below detection levels, or perhaps because this congener only has one open meta-para site. Based on the latter explanation, we should

also expect to see the formation of two OH-PCBs for PCB 84, which also has two open meta-para sites, but only one pentachlorinated MeO-PCB was detected for this congener. Thus, regioselectivity is a factor in determining how many and which OH-PCBs are formed by PCB metabolism (12) using rat CYP 2B1. While formation of OH-PCBs from PCB 136 has been previously demonstrated in vitro (51), our study is the first to our knowledge to provide evidence that a number of other chiral PCBs are transformed enantioselectively by CYPs to form OH-PCBs (Table 1). While we did not demonstrate directly that the OH-PCBs were formed enantioselectively (i.e., EFs for OH-PCBs were not determined), a rough mass balance was estimated as indirect evidence. We lacked authentic OH-PCB standards for the identified products, thus we estimated OH-PCB concentrations using the closest eluting OH-PCB standard of the same homologue group in GC/ECD chromatograms. While crude, this analysis suggested that 64-96% of the observed parent congener concentration decreases could be attributed to observed OH-PCBs. It is important to note that this attribution is a maximum value. Our analysis did not account for the possibility that covalent adducts can be formed between reactive PCB intermediates and microsomal proteins (52), nor for possible OH-PCB biotransformation to diOH-PCBs (51) which we did not measure. Nonetheless, we conclude from this study that OH-PCB production from chiral PCBs can be a major fate in mammals. Taken together with the fact that the parent PCBs were biotransformed enantioselectively, OH-PCBs were likely produced in nonracemic quantities, as was demonstrated recently in vivo in rats (53). Rat CYP 2B1 can form OH-PCBs via hydroxy group insertion, particularly at the meta position (50, 54), and via epoxidation to an arene oxide intermediate followed by epoxide opening (4). The former is the major pathway in rats (4, 50, 54). While we are unable to determine the exact structures of the OH-PCBs formed, given the small quantities formed in our experiments, it is likely that at least some of them are meta-OH substituted (4). However, it is not clear what the extent of OH-PCB production is in biota, because PCBs may also be converted to other metabolites (e.g., arene oxides to methylsulfonyl metabolites) and OH-PCBs can be further biotransformed (4). For human CYP 2B6, unknown peaks detected in the MeOPCB fractions of PCB 45, 91, and 132 single-congener assays corresponded to exact masses and retention time ranges expected for tetra-, penta-, and hexachlorinated MeO-PCBs (Table 1), confirming that OH-PCBs were formed. The integrated GC/ECD areas for OH-PCBs produced by human CYP 2B6 were 5-10 times lower than for OH-PCBs produced by rat CYP 2B1, while concentration decreases of parent congeners were comparable for both sets of isozymes. It is possible that less OH-PCB was produced by human CYP 2B6 compared to rat CYP 2B1, because the former may be more likely to produce arene oxides, which can remain stable for several weeks (4). Because no epoxidase was present in our incubations, less epoxide ring opening occurred to form the subsequent OH-PCB in the time scale of our experiments. In addition, PCB arene oxides are known to bind to microsomal proteins (52), so these intermediates may be sequestered prior to OH-PCB formation. Regardless, it is clear that the different enantiomer signatures (Figure 2) indicate that stereoselectivity plays some role in the mechanistic differences between PCB biotransformation between rat and human CYPs. Many of the OH-PCBs detected in recent studies cannot be identified due to the lack of commercially available standards (6, 7, 18, 24, 55, 56). Unidentified di- to hexachlorinated OH-PCBs were predominant in bottlenose dolphins (7), and unidentified penta- and hexachlorinated OH-PCBs made up much of the OH-PCB burden in cetacean brain

tissue (56). Our results show that the formation of tetra-, penta-, and hexachlorinated OH-PCBs from enantioselective metabolism of chiral PCBs may be the source of many unknown OH-PCBs observed in laboratory and field studies. Chiral PCB Enantiomer Biotransformation Kinetics. Four general routes have been proposed for enantioselective biotransformation (57). First, two enantioselective enzymes, or variants of the same enzyme, each convert one of the enantiomers but at different rates. Second, both enantiomers are biotransformed by one enzyme at the same time at different rates. Third, one enantiomer is preferentially biotransformed first, then the other enantiomer is biotransformed after the first is gone. Finally, one enantiomer is enantioselectively biotransformed by one enzyme and the other is racemized by an isomerase. The first and fourth routes are ruled out in our study. All incubations used only a single enzyme, none of which are considered isomerases. In addition, the detection of a large proportion of metabolites seems to minimize the potential importance of racemization. This leaves only the second and third scenarios as possible routes of CYP-mediated enantioselective PCB biotransformation. In single-congener assays, labile chiral PCBs decreased in concentration and increased in enantiomer enrichment over time (Figure 2), with PCB 45 showing the most rapid kinetics (Figure 2). This observation is consistent with past studies, in which elimination rates for PCBs in rats decreased with higher degrees of chlorination (45). For all atropisomers investigated, the rate constants were significantly different from zero except for (+)-PCB 132, suggesting that only (-)PCB 132 was biotransformed. Norstro¨m et al. (23) found formation of both meta- and para-substituted MeSO2-PCB 132 in rats exposed in vivo to (+)-PCB 132, but at much lower concentrations compared to rats exposed to (-)-PCB 132. It is possible that (-)-PCB 132 was preferentially hydroxylated by rats, but (+)-PCB 132 preferentially formed arene oxides eventually converted into methyl sulfones. While this hypothesis is consistent with our results, it remains to be proven in future work. Rate constants among individual atropisomers of chiral PCB congeners were not statistically different, except for PCBs 95 and 132 (Figure 2), indicating that the individual atropisomers for these congeners were metabolized at different rates. These results suggest that the route by which enantiomer enrichment occurred was due to different rates of metabolism of the individual atropisomers, which is supported by other findings of enantioselective metabolism within rats (23). While a thorough investigation of Michaelis-Menten kinetics for PCB atropisomers would provide much more insight into the stereochemistry of PCB metabolism, this is beyond the scope of this study and is the topic of future research. Nonetheless, this is the first time to the best of our knowledge that individual rates of metabolism have been quantified for individual PCB atropisomers, shown to be unambiguously biotransformed at different rates by a single isozyme.

Acknowledgments We thank Don Morgan for help with GC/HRMS analysis, Christina Wolinski for technical assistance, and the Natural Sciences and Engineering Research Council of Canada for funding. Additional funding to CSW was provided by the 2003 Early Career Award for Applied Ecological Research, cosponsored by the Society of Environmental Toxicology and Chemistry and the American Chemistry Council, and the Canada Research Chairs Program.

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