Computational Toxicological Investigation on the Mechanism and

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Computational Toxicological Investigation on the Mechanism and Pathways of Xenobiotics Metabolized by Cytochrome P450: A Case of BDE-47 Xingbao Wang,†,‡ Yong Wang,§ Jingwen Chen,*,† Yuqin Ma,‡ Jing Zhou,† and Zhiqiang Fu† †

Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China ‡ School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022, China § State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China S Supporting Information *

ABSTRACT: Understanding the transformation mechanism and products of xenobiotics catalyzed by cytochrome P450 enzymes (CYPs) is vital to risk assessment. By density functional theory computation with the B3LYP functional, we simulated the reaction of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) catalyzed by the active species of CYPs (Compound I). The enzymatic and aqueous environments were simulated by the polarizable continuum model. The results reveal that the addition of Compound I to BDE-47 is the rate-determining step. The addition of Compound I to the ipso and nonsubstituted C atoms forms tetrahedral σ-adducts that further transform into epoxides. Hydroxylation of the epoxides leads to hydroxylated polybrominated diphenyl ethers and 2,4-dibromophenol. The addition to the Br-substituted C2 and C4 atoms has a higher barrier than addition to the nonsubstituted C atoms, forming phenoxide and cyclohexadienone which subsequently undergo debromination/hydroxylation. A novel mechanism was identified in which the approach of Compound I to C2 led to formation of a phenoxide and an expelled Br− ion. The predicted products were consistent with the metabolites identified by others. As a first attempt to simulate the enzymatic transformation of a polycyclic compound, this study may enlighten a computational method to predict the biotransformation of xenobiotics catalyzed by CYPs.

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INTRODUCTION Humans encounter a wide range of xenobiotics with potentially harmful consequences.1 Xenobiotics can be biotransformed, and biotransformation occurs mostly in the liver.2 Among the various transformation reactions, the oxidation of xenobiotics catalyzed by the cytochrome P450 enzymes (CYPs) plays an important role. Generally, the oxidative transformation serves to detoxify the body as more water-soluble compounds are produced, facilitating enhanced excretion of xenobiotic molecules.3 However, the metabolism can also transform xenobiotics into more reactive and more toxic compounds. For example, polybrominated diphenyl ethers (PBDEs), a class of widely used additive flame retardants for which the environmental level increased exponentially,4−6 were supposed or observed to be biotransformed to hydroxylated PBDEs (HO-PBDEs) by CYPs.7−9 HO-PBDEs were observed to have the potential to disrupt estrogen and thyroid hormone effects.10−13 Thus, understanding the CYPs metabolic mechanism and pathways is vital for predicting conversions of xenobiotics, and hence for accurate risk assessment. © 2012 American Chemical Society

Most previous studies rely heavily on in vivo or in vitro tests to probe the CYPs metabolic reactions.7−9,14 In vivo testing may violate the “3R principles” (Replacing animal testing, Reducing the number of animals required, and Refinement of techniques so that the distress or pain of animals is avoided or minimized) for the ethics of animal tests.15 The experimental methods cannot fully explain the reaction mechanism as the transition states (TSs) and intermediates (IMs) are difficult to identify due to their high reactivity and short lifetime.16,17 Furthermore, the experimental methods are generally laborious, time-consuming, costly, and equipment-dependent, and thus cannot meet the need of risk assessment for newly synthesized compounds prior to large-scale production and commercialization.18 Therefore, there is an urgent need to develop computational toxicology approaches (in silico) to investigate the CYPs metabolic reactions of xenobiotics. Received: Revised: Accepted: Published: 5126

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Computational toxicology is a growing research area that is melding advances in chemistry and molecular biology with modeling and computational science in order to increase the predictive power of the field of toxicology.19−21 In 2005, the United States Environmental Protection Agency established the National Center for Computational Toxicology, which was designed to understand and safeguard public health and the environment by computational methods.20 To date, several in silico tools have been developed or are in the state of development, among which the virtual liver model is a major research focus.17,19,20,22 Computational toxicology on CYPs deserves particular attention because CYPs contribute extensively to the metabolism of xenobiotics.3,16,23,24 According to experimental and computational evidence, the major part of a large variety of reactions catalyzed by CYPs involves the same active species: a high-valent oxoiron species located at the heme pocket, known as Compound I (Cpd I) as verified in spectroscopic studies.25 Benzene hydroxylation by Cpd I has been intensively studied by the density functional theory (DFT) computation, due to the concerns on the reaction mechanism and toxicity of its metabolites.3,24,26,27 The DFT computation and experimental studies showed that the reaction proceeds by an initial attack of Cpd I on the π-system to generate σ-complexes, followed by benzene epoxidation or the proton-shuttle mechanism.24,28 The subsequent rearrangement leads to phenol products. Besten et al.29 found that CYPs mediated oxidation of pentafluorophenol led to tetrafluorobenzoquinone as a primary product; and Hackett et al.16 identified a novel mechanism by DFT calculation, in which perhalogenated benzenes were transformed directly into tetrahaloquinones. All the previous studies indicate that quantum chemical computations based on DFT well explain the metabolism of xenobiotics catalyzed by Cpd I. However, to date few studies have been reported on the Cpd I catalyzed transformation of polycyclic compounds such as PBDEs. It is the purpose of this study to probe the Cpd I catalyzed reaction mechanism and pathways of PBDEs through DFT computations. 2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) was selected as a model compound for three reasons: (a) BDE47 is dominant in commercial PBDE products and has been widely detected in the environment;5,30 (b) in vivo and in vitro data for the biotransformation of BDE-47 are available, which can be employed to verify the computation results;7−9,14 and (c) the molecular C2 symmetry of BDE-47 can reduce the computational load.31

Figure 1. Orbital occupation of Cpd I.

only considered the doublet state, as the previous calculations on Cpd I oxidation of benzene or substituted benzenes showed that the energy barriers for Cpd I in the quartet state were higher than in the doublet state by ca. 3.0 kcal/mol.3,24,36 DFT Calculation. Based on previous computational studies on benzene and perhalogenated benzenes,3,16,24,26,27 we proposed that the reaction process for BDE-47 includes 4 steps (Figure 2): (a) addition of Cpd I to BDE-47 to form different intermediates (IMs) or product complexes (PCs), (b) rearrangement of the IMs, (c) proton catalyzed rearrangement of the epoxides, and (d) reduction of the PCs. The reactions (a) and (b) occur in the enzymatic environment,24,27 (c) occurs in the aquatic environment, and (d) occurs in both.16 For the Cpd I catalyzed reaction processes (a) and (b), the spin-unrestricted hybrid density functional UB3LYP was employed with two basis sets: (I) the LACVP basis set on iron and the 6-31G basis set on the remaining atoms (BS-I in brief) for geometry optimization, and (II) the LACV3P+* basis set on iron and the 6-311+G** basis set on all the other atoms (BS-II) for single-point energy calculations. Geometries for all the species were fully optimized without symmetry constraints. The profile of the potential energy surface (PES) was constructed at the UB3LYP/BS-II//UB3LYP/BS-I level. These functional and basis sets have been fully tested and proved to be reliable.32−34 Bulk polarity effects simulating the protein environment were evaluated using the polarizable continuum model (PCM)37 solvation model with a dielectric constant of ε = 5.7.24 Group spin density (ρ) for an open-shell system38 and charge (Q) were estimated by Mulliken population analysis39 at the UB3LYP/BS-I level. We further considered the dispersion effect using the empirical B3LYP-D formula,40,41 and the details are provided in the Supporting Information (SI). To evaluate the reaction process (c), i.e., the reactions of epoxide rearrangement and cleavage of the diphenyl ether bond, the B3LYP/6-311G* was employed for geometry optimization, and the high level B3LYP/6-311+G** was used for single point energy calculations. The polar nonenzymatic aqueous environment (ε = 78.4) was evaluated with the PCM model, as was done in previous studies.32,33,42 To assess the tendency of the PCs toward reduction, the vertical and adiabatic electron affinities (VEA and AEA) were computed. VEA is the difference between the electronic energies of the optimized neutral geometry and the anion at the neutral geometry, and AEA is the difference in electronic energies of the optimized neutral geometry and optimized

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COMPUTATIONAL METHODS Model System. The iron-oxo porphyrin without side chains but with a thiolate axial ligand Fe4+O2−(C20N4H12)−(SH)− was used to model the oxidized active site of Cpd I, as was also done by other researchers.24,32,33 Comparison of the results from the combined quantum and molecular mechanical (QM/ MM) method that simulates reactions in the protein environment with the results from pure quantum mechanics with the model system revealed that the current model represents the real CYP enzymes quite well.34 The electronic ground state of Cpd I involves three unpaired electrons, two spin-up (α) electrons on the π* orbitals of the iron-oxo unit and one spin-up/down (α/β) electron in an a2u orbital on the porphyrin macrocycle (Figure 1), giving rise to two energetically closely lying spin states, high spin quartet state (S = 3/2) and low spin doublet state (S = 1/2).32,35 We 5127

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Figure 2. Possible reaction processes for BDE-47.

Figure 3. Geometries for the transition states of Cpd I addition to BDE-47 and energy barriers (kcal/mol) calculated in gas phase (ΔE‡gas) and solvation environment (ΔE‡sol) with ε = 5.7 at the UB3LYP/BS-II//UB3LYP/BS-I level. Bond distances and angles are shown in Angstroms (Å) and degrees (°), respectively. ρ and Q stand for the group spin density and charge on the BDE-47 moiety, respectively.

anion geometry.43 The employed functional and basis set were the same as those in the computation of the epoxide rearrangement. The solvation effects were included using the PCM model with ε = 5.7 and ε = 78.4 to mimic the hydrophobic interior of the P450 active site and the nonenzymatic aqueous environment, respectively.16 All the calculations were carried out with the Gaussian 09 suite of the programs.44 Transition states (TSs) were obtained in a two-step procedure: (a) First, an energy profile was generated by geometry scans. A series of calculations were performed in which one of the structural parameters was fixed to a certain value, while all the other parameters were optimized

to their most favorable values. The process was performed automatically in the redundant internal coordinate system. (b) Then, the obtained TS geometry was fully optimized.26 The TSs were characterized by a single imaginary frequency for the correct mode. The stable species (minima) were verified to all have real frequencies only.

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RESULTS AND DISCUSSION Addition of Cpd I. Due to the C2 symmetry,31 only one phenyl of BDE-47 was considered in the computation. There are 6 possible positions to be approached by Cpd I: the ipso C atom (C1), the bromo-substituted C atoms (C2 and C4), and 5128

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Figure 4. Geometries for the intermediates/product complexes and the calculated reaction enthalpies (ΔHsol, kcal/mol) in solvation environment (ε = 5.7) at the UB3LYP/BS-II//UB3LYP/BS-I level. The bond distances and angles are shown in Angstroms (Å) and degrees (°), respectively. ρ and Q are group spin density and charge, respectively.

leads to formation of a phenoxide (PCC2) and an expelled Br− ion, for which the mechanism is novel and has not been observed in previous studies. For PCC2 and PCC4, the distances between the Fe and O atoms are observably longer than those of the σ-adducts. The calculated reaction enthalpies (Figure 4) show that the formation of the IMs is endothermic, while the formation of the PCs is very exothermic. The spin density (ρ) of an open-shell system is the total electron density of the spin-up (α) electrons minus the total electron density of the spin-down (β) electrons.38 The calculated ρ and Q values (Figure 3) reveal that the BDE-47 moiety has a moderate radical cation character in the transition states. Even though the tetrahedral intermediates were not formed in PCC2 and PCC4, inspection of the ρ values for TSC2 and TSC4 reveals electronic structures similar to the other TSs and those for hydroxylation of benzene.27 ρ = 0.93 (≈ 1) for phenoxide of PCC2, proving the radical nature of the moiety. ρ = 0 for cyclohexadienone of PCC4, indicating that there are no unpaired electrons. Moreover, small positive charge still accumulates on the phenoxide and cyclohexadienone moieties. For IMC3 and IMC5, ρ(BDE-47) ≈ 0, and Q(BDE-47) are 0.32 and 0.45, respectively, implying the σ-adducts have cationic natures. IMC1 and IMC6 still have hybrid cationic/radicalar natures, with spin density as well as charge residing on the BDE-47 moiety. Rearrangement of the IMs to Form Epoxides. The intermediates IMC1, IMC3, IMC5, and IMC6 can further undergo rearrangement (ring closure and cleavage of the O−Fe bonds) reactions. Six epoxides (1,2-, 2,3-, 3,4-, 4,5-, 5,6- and 1,6epoxide) can be formed through 8 transition states (1,2-, 1,6-, 3,2-, 3,4-, 5,4-, 5,6- 6,5- and 6,1-TS), for which the computed ΔE‡gas/ΔE‡sol values are 1.1/0.4, 1.9/0.9, 4.1/5.5, 2.2/3.2, 0.9/ 1.8, −0.8/−0.4, 2.5/2.4, and −1.0/−1.0 kcal/mol, and the reaction enthalpies relative to the IMs are 12.0, 11.6, 13.6, 13.9, 13.8, 14.7, 15.5, and 16.5 kcal/mol, respectively. Thus, although

the C atoms adjacent to H atoms (C3, C5, and C6). The addition of Cpd I to the C atoms leads to the corresponding transition states (TSs) and intermediates (IMs) successively, for which the optimized structures are shown in Figures 3 and 4, respectively. We computed the energy barriers (ΔE‡) in both the gasphase (ΔE‡gas) and the solvation environment (ΔE‡sol). The results (Figure 3) indicate that the medium has marginal effects on ΔE‡. The medium also has weak effects on the ΔE‡ value of benzene hydroxylation catalyzed by Cpd I.24 According to ΔE‡, the approach of Cpd I to the nonsubstituted C atoms (C3, C5, and C6) is the most favorable. The addition of Cpd I to C1, C2, and C4 atoms has higher ΔE‡ values. The ΔE‡gas values for TSC3, TSC5, and TSC6 are comparable to those of benzene calculated by the same method.24,26,27 As shown in Figure 3, for TSC1 TSC2, TSC3, and TSC4, the phenyl rings are parallel with the porphyrin plane (face-on). However, for TSC5 and TSC6, the phenyls are almost perpendicular to the porphyrin ring (side-on). For the sideon addition that forms the tetrahedral σ-adducts in which the O atoms of the reactive heme FeO species are bound to the C atoms of the phenyl (TSC5 and TSC6), the lengths between the attacked C and the O atoms of Cpd I are larger than those of the face-on additions (TSC1 and TSC3). TSC4 is “earlier” than TSC2, as the C−O distance for TSC4 is longer than that of TSC2.3 Nevertheless, the angles of C−O−Fe are similar for TSC2 and TSC4. The formed intermediates IMC1, IMC3, IMC5, and IMC6 are tetrahedral intermediate σ-adducts (Figure 4). Benzene hydroxylation catalyzed by Cpd I also forms the tetrahedral intermediate σ-adducts.3,24,26 The approach of Cpd I to C4 leads to a cyclohexadienone product complex (PCC4), followed by a Br atom shift from C4 to C5. The concerted reaction was also observed in the oxidative dehalogenation of hexachlorobenzene by Cpd I.16 However, the approach of Cpd I to C2 5129

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Figure 5. Schematic representation of proton catalyzed rearrangement of three representative hydroxyl-cyclohexadienyl cations. The ΔE‡sol values (kcal/mol) were calculated in aqueous medium (ε = 78.4).

benzene in a realistic enzyme environment showed that the NIH shift is a less favorable pathway for the side-on approach. However, the NIH shift can occur for the face-on orientation.3 All the above calculation results indicate that the epoxides are obligatory products for the rearrangement of the tetrahedral intermediates. The attack of Cpd I on the π-system is the rate limiting step for the reaction of BDE-47 catalyzed by Cpd I, although the electron donation to the heme is usually believed to be the rate-limiting step in the whole enzymatic cycle.32,46 Proton Catalyzed Rearrangement of the Epoxides. The 6 epoxides (1,2-, 2,3-, 3,4-, 4,5-, 5,6-, and 1,6-epoxide) subsequently undergo proton catalyzed rearrangement reactions, leading to 6 hydroxyl-cyclohexadienyl cations. Hydroxylation of the epoxides occurs via protonation by a sequestered hydronium ion or an array of water molecules held by a side chain amino acid.24 The calculation shows that the protonated epoxides cannot be located as minima on the PES as their rings open by a barrierless process. Borosky and Laali47,48 computed the ring-opening reactions of polycyclic aromatic hydrocarbon epoxides, and also found that the protonated epoxides open with a barrierless process. The formed hydroxyl-cyclohexadienyl cations can be classified into 3 kinds: −OH attached to the nonsubstituted C atoms, to the ipso C atom, and to the bromo-substituted C atoms. We further computed the rearrangement of three representative hydroxyl-cyclohexadienyl cations (#1, #2, and #3) shown in Figure 5. More stabilized protonated cyclohexadienones #4− #7 resulted from intramolecular rearrangement of #1 and #2

the formation of the IMs is endothermic, the whole enzymatic reaction processes is exothermic. Again, the medium shows marginal effects on the ΔE‡ values. The energy barriers for the reactions are very low relative to the approach of Cpd I to BDE-47 or even barrierless (ΔE‡ < 0). The face-on adducts (IMC1 and IMC3) rearrange with barriers slightly higher than those for the side-on adducts (IMC5 and IMC6). The observation is similar to the finding for epoxidation of benzene.3,26 We performed several geometry scans to know whether the side-on adducts (IMC5 and IMC6) reacted via the proton shuttle mechanism, i.e., the hydrogen atom at the tetrahedral carbon is abstracted by one of the nitrogen atoms on the porphyrin.24 The scans show the epoxides form first. Formation of the epoxides was followed by high-energy pathways that did not yield the desired products. The details of the scans are shown in Figure S1. These results show that the mechanism of proton shuttle did not occur for IMC5 and IMC6, although it was observed for the side-on adducts of benzene hydroxylation.3,24,26 The calculation results also show that the NIH shift mechanism, i.e., the migration of an atom from the site of tetrahedral carbon to the adjacent carbon atoms,45 does not occur in the rearrangement of the IMs. As an example, the computed ΔE‡gas values for H atom transfer from C3 of IMC3 to the ortho C2 or C4 atoms are as high as 16.3 and 17.2 kcal/ mol, respectively, which means that this transfer cannot yield the desired products. Previous studies on the hydroxylation of 5130

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Table 1. Vertical and Adiabatic Electron Affinities (VEA and AEA) (eV) of Phenoxide Radical and Cyclohexadienone

a

The geometries were optimized on the B3LYP/6-311G(d) level.

followed by a NIH shift. From the computed ΔE‡sol values (Figure 5), it can be concluded that the Br atoms more easily migrate to the adjacent carbons than the H atoms. The protonated cyclohexadienones #4−#7 further collapsed to form the corresponding hydroxylated products (4-HO-BDE-42, 4′HO-BDE-49, and 5-HO-BDE-47) by proton loss.49 The hydroxyl-cyclohexadienyl cation #2 can also collapse to form 5-HO-BDE-47 directly by proton loss. According to Coxon et al.,49 the NIH pathway is more energetically favored than the direct proton loss pathway. Our results show that the −OH addition on the C1 position (Figure 5) leads to breaking of the ether bond with a very low energy barrier. The fission of the ether bond to form heterolytic products #8 and #9 is favored by ca. 3.0 kcal/mol over formation of the homolytic products #10 and #11. The cationic product #8 may undergo further reactions to form corresponding quinone or hydroxylation product. Reduction of the Phenoxide Radical and Cyclohexadienone. The computed VEA and AEA values for the formed phenoxide radical and cyclohexadienone are listed in Table 1. The VEA values for the two species in the gas phase are high,16 indicating that they have significant potential for reduction. The VEA values are further increased in the enzymatic and aqueous environment, implying that the two species can effectively capture electrons from the reduced iron porphyrin or the physiological environment.16 Indeed, geometry optimization of the cyclohexadienone radical anion resulted in the expulsion of Br− to form a phenoxide radical. The VEA and AEA values indicate that the capture of an electron by the cyclohexadienone is a very exothermic expulsion of Br− (1.30−1.45 eV). The resulting phenoxide radicals could further capture electrons to form anionic compounds, and the anionic compounds could combine with protons to form hydroxylated products such as 2′-HO-BDE-28 and 4′-HO-BDE-90. Toxicological Implications. The calculations indicate that the reaction pathways of BDE-47 catalyzed by Cpd I include hydroxylation, cleavage of the ether bond, and debromination/ hydroxylation, leading to possible metabolites that include 4HO-BDE-42, 4′-HO-BDE-49 and 5-HO-BDE-47, 2,4-dibromophenol, and debromination/hydroxylation products such as 2′HO-BDE-28 and 4′-HO-BDE-90. The epoxides are responsible for the production of hydroxylated products and bromophenols. Marsh et al.50 identified 6 hydroxylated tetrabrominated diphenyl ethers and 3 hydroxylated tribrominated diphenyl ethers from feces of BDE-47 exposed rats. Erratico et al. incubated BDE-47 with hepatic microsomes from phenobarbi-

tal-treated rats and identified 5 HO-PBDE metabolites. Qiu et al.9 measured metabolites in mouse plasma after exposure to a commercial PBDE mixture (DE-71 consisting of BDE-47, 85, 99, 100, 153, 154) and detected several HO-PBDEs and 2,4dibromophenol as products. They also detected bromophenols and HO-PBDEs in human blood samples,8 and concluded that 2,4-dibromophenol is a metabolite of BDE-47, and HO-PBDEs are formed through hydroxylation and debromination/hydroxylation reactions. Stapleton et al.7 determined metabolism of PBDEs by human hepatocytes in vitro and identified 2,4,5tribromophenol and two HO-PBDEs as metabolites of BDE-99. Table S2 compares the metabolites predicted in the current study with those identified in the in vivo and in vitro tests. It can be concluded that the identified metabolites support the current computational results on BDE-47 transformation pathways and products. It deserves mentioning that the pattern of predicted metabolites can deviate from the experiments due to the following reasons: (a) There are a number of isoforms of CYPs that have their own native protein environments.2,23 Although all the isoforms share the same active species that was used in the current and in other calculations, each isoform has a different selectivity in oxidation of substrates.23 The current DFT computation is powerless to exactly consider the effects of the protein environments because of the limit of computational capacity. (b) In almost all the experiments, a mixture of different isoforms of CYPs was exposed to PBDEs including BDE-47.7,9,14,50 (c) In some experimental studies, the commercial mixture of PBDEs was employed.8,9 Rietjens et al.28 found that regioselectivity of CYPs catalyzed hydroxylation of fluorobenzene could be predicted by frontier orbital substrate characteristics. As sterical hindrance of -Br and phenoxide is far more significant than -F substituents,28 further in vivo testing should be performed to determine the metabolite pattern for a specific PBDE congener so as to know whether the frontier orbital characteristics can predict the metabolite distribution of the PBDE congener. In summary, this study was successful in unveiling the principal mechanism, electronic structure information, complete reaction pathway, and metabolite type of BDE-47 transformation catalyzed by Cpd I. DFT calculation is an important tool to probe the biotransformation mechanism of PBDEs catalyzed by Cpd I. The current study may enlighten a computational toxicological method to predict the transformation pathways and products of xenobiotics catalyzed by CYPs. 5131

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bioaccumulative hydroxylated PBDE metabolites in young humans from Managua, Nicaragua. Environ. Health Perspect. 2008, 116 (3), 400−408. (14) Erratico, C. A.; Moffatt, S. C.; Bandiera, S. M. Comparative oxidative metabolism of BDE-47 and BDE-99 by rat hepatic microsomes. Toxicol. Sci. 2011, 123 (1), 37−47. (15) Kroeger, M. How omics technologies can contribute to the ’3R’ principles by introducing new strategies in animal testing. Trends Biotechnol. 2006, 24 (8), 343−346. (16) Hackett, J. C.; Sanan, T. T.; Hadad, C. M. Oxidative dehalogenation of perhalogenated benzenes by cytochrome P450 Compound I. Biochemistry 2007, 46 (20), 5924−5940. (17) Zhou, J.; Chen, J. W.; Liang, C. H.; Xie, Q.; Wang, Y. N.; Zhang, S. Y.; Qiao, X. L.; Li, X. H. Quantum chemical investigation on the mechanism and kinetics of PBDE photooxidation by ·OH: A case study for BDE-15. Environ. Sci. Technol. 2010, 45 (11), 4839−4845. (18) Blotevogel, J.; Borch, T.; Desyaterik, Y.; Mayeno, A. N.; Sale, T. C. Quantum chemical prediction of redox reactivity and degradation pathways for aqueous phase contaminants: An example with HMPA. Environ. Sci. Technol. 2010, 44 (15), 5868−5874. (19) Rusyn, I.; Daston, G. P. Computational toxicology: Realizing the promise of the toxicity testing in the 21st century. Environ. Health Perspect. 2010, 118 (8), 1047−1050. (20) Kavlock, R. J.; Ankley, G.; Blancato, J.; Breen, M.; Conolly, R.; Dix, D.; Houck, K.; Hubal, E.; Judson, R.; Rabinowitz, J.; Richard, A.; Setzer, R. W.; Shah, I.; Villeneuve, D.; Weber, E. Computational toxicology - A state of the science mini review. Toxicol. Sci. 2008, 103 (1), 14−27. (21) Benfenati, E.; Gini, G. Computational predictive programs (expert systems) in toxicology. Toxicology 1997, 119 (3), 213−225. (22) Johnson, D. E.; Wolfgang, G. H. I. Computational toxicology and virtual development in drug design. Abstr. Pap. Am. Chem. Soc. 2000, 220, U197−U197. (23) Shaik, S.; Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel, W. P450 enzymes: Their structure, reactivity, and selectivity-modeled by QM/MM calculations. Chem. Rev. 2010, 110 (2), 949−1017. (24) de Visser, S. P.; Shaik, S. A proton-shuttle mechanism mediated by the porphyrin in benzene hydroxylation by cytochrome P450 enzymes. J. Am. Chem. Soc. 2003, 125 (24), 7413−7424. (25) Rittle, J.; Green, M. T. Cytochrome P450 compound I: Capture, characterization, and C-H bond activation kinetics. Science 2010, 330 (6006), 933−937. (26) Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Mechanism and structure-reactivity relationships for aromatic hydroxylation by cytochrome P450. Org. Biomol. Chem. 2004, 2 (20), 2998−3005. (27) Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Aromatic hydroxylation by cytochrome P450: Model calculations of mechanism and substituent effects. J. Am. Chem. Soc. 2003, 125 (49), 15004−15005. (28) Rietjens, I. M. C. M.; Soffers, A. E. M. F.; Veeger, C.; Vervoort, J. Regioselectivity of cytochrome P-450 catalyzed hydroxylation of fluorobenzenes predicted by calculated frontier orbital substrate characteristics. Biochemistry 1993, 32 (18), 4801−4812. (29) Besten, C. D.; Bladeren, P. J. V.; Duizer, E.; Vervoort, J.; Rietjens, I. M. C. M. Cytochrome P450-mediated oxidation of pentafluorophenol to tetrafluorobenzoquinone as the primary reaction product. Chem. Res. Toxicol. 1993, 6 (5), 674−680. (30) Castorina, R.; Bradman, A.; Sjodin, A.; Fenster, L.; Jones, R. S.; Harley, K. G.; Eisen, E. A.; Eskenazi, B. Determinants of serum polybrominated diphenyl ether (PBDE) levels among pregnant women in the chamacos cohort. Environ. Sci. Technol. 2011, 45 (15), 6553−6560. (31) Zeng, X.; Freeman, P. K.; Vasil’ev, Y. V.; Voinov, V. G.; Simonich, S. L.; Barofsky, D. F. Theoretical calculation of thermodynamic properties of polybrominated diphenyl ethers. J. Chem. Eng. Data 2005, 50 (5), 1548−1556.

ASSOCIATED CONTENT

S Supporting Information *

Table of B3LYP-D energy barriers; table listing metabolites; tables with absolute energies, relative energies, group spin densities, group charges, and Cartesian coordinates for all reaction species; figure of geometry scan results, and PES profiles. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-411-84706269; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Dr. W. Peijnenburg (RIVM) for improving the manuscript. The study was supported by the National Natural Science Foundation of China (21137001, 20890113, 20977014), the High-tech Research and Development Program of China (2010AA065105), the Fundamental Research Funds for the Central University and the Program for Changjiang Scholars and Innovative Research Team in University (IRT0813).

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