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Molecular orbital studies on brominated diphenyl ethers. Part II—reactivity and quantitative structure–activity (property) relationships
Chemosphere 59 (2005) 1043–1057 www.elsevier.com/locate/chemosphere
Molecular orbital studies on brominated diphenyl ethers. Part II—reactivity and quantitative structure–activity (property) relationships ˚ ke Bergman c, Eva Jakobsson c, Jiwei Hu a, Lars Eriksson b, A d Erkki Kolehmainen , Juha Knuutinen d, Reijo Suontamo d, Xionghui Wei a
a,*
Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, P.R. China b Department of Structural Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden c Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden d Department of Chemistry, University of Jyva¨skyla¨, SF-40351 Jyva¨skyla¨, Finland Received 28 April 2003; received in revised form 21 September 2004; accepted 12 November 2004
Abstract Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and are increasingly turning up in the environment. Their structural similarities to polychlorinated biphenyls and thyroid hormones suggest they may be a risk to human health. The present study examines the reactivity of brominated diphenyl ethers (BDEs) on the basis of the electronic structures as calculated by semiempirical AM1 self-consistent field molecular orbital (SCF-MO) method. Frontier orbital energies were used to elucidate the reactivity of BDEs in electrophilic, nucleophilic and photolytic reactions. From an examination of the frontier electron densities, the regioselectivity, or orientation, of metabolic reactions of BDEs was predicted. Furthermore, satisfactory quantitative structure–activity (property) relationship (QSAR and QSPR) models were derived to calculate gas chromatographic and ultraviolet spectral properties and luciferase induction activities from the AM1-computed electronic parameters. 2004 Elsevier Ltd. All rights reserved. Keywords: Brominated diphenyl ethers; Molecular orbital studies; Reactivity; QSARs; QSPRs
1. Introduction Polybrominated diphenyl ethers (PBDEs) (Fig. 1) are used in large quantities as flame retardants in computers, TV sets, textiles and cars. Today they are ubiquitous in the environment. Since PBDEs and their metabolites are in general persistent, bioaccumulative and structu-
*
Corresponding author. Tel./fax: +86 10 62751529. E-mail address: [email protected] (X. Wei).
rally related to polychlorinated biphenyl (PCBs) and thyroid hormones, their increasing levels in the environment pose a potential danger to human health in thyroid hormone disruption, neurobehavioral deficits and carcinogenicity (World Health Organization, 1994; Paasivirta, 2000; McDonald, 2002). With the limited experimental data available (McDonald, 2002), theoretical evaluation of physicochemical properties of PBDEs such as conformational properties, reactivity and lipophilicity can significantly contribute to overall fate and risk assessments. The conformational properties of PBDEs,
0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.11.029
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Fig. 1. Backbone of polybrominate diphenyl ethers and definition of torsional angles u1 and u2.
which are reported in other paper (Hu et al., in press), create a basis for the present investigation of reactivity and other critical properties. According to Lewis et al. (1993), the chemical reactivity of xenobiotics may to some extent be related to their rates of metabolism and degradation and thus to metabolic activation and deactivation processes. Although PBDEs in general are chemically stable, the reactivities and thereby the metabolites of their congeners may vary considerably. Characterization of their chemical reactivity is also vital for understanding the adduct formation of PBDE congeners with macromolecules. Use of frontier orbital theory (Fukui et al., 1954) to describe reactions of aromatic compounds is well established. According to this theory, frontier orbital characteristics of reacting molecules, namely the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), determine the kinetics of a chemical reaction. In principle, frontier orbital characteristics, which are static indices, should be applied with caution in elucidating reactivities because this theory only describes the early stage of interactions between molecular orbitals of reactants. Ultimately, the reactivity depends on the relevant transition states formed from the reactants, not on the unperturbed reactants themselves. Locating the transition states (firstorder saddle points) in the potential energy surface (PES) of a reaction is a computationally expensive task, however, and meaningful only for that specific reaction. Use of static indices is probably a better choice for the present study, therefore, which aims at a general elucidation of the reactivity of PBDEs. A number of previous studies have indeed shown the efficacy of frontier orbital theory in addressing toxicological and environmental problems. For example, the frontier orbital characteristics and other electronic parameters calculated at AM1, PM3 or MNDO selfconsistent field molecular orbital (SCF-MO) level of theory have been shown to correlate with or relate to the chemical or biological properties of several classes of organic compounds, including polychlorinated dibenzop-dioxins and dibenzofurans (PCDDs and PCDFs), polychlorinated biphenyls (PCBs), polybrominated (polychlorinated) diphenyl ethers (PBDEs and PCDEs) and polycyclic aromatic hydrocarbons (Koester and Hites,
1988; Tuppurainen et al., 1991, 1992; Tysklind et al., 1992; Vervoort et al., 1992; Rietjens et al., 1993, 1995; Poso et al., 1993; Nevalainen and Kolehmainen, 1994; Cnubben et al., 1994; Andersson et al., 1996; Harju et al., 2002; Chen et al., 2003a,b; Kitti et al., 2003). Both AM1 and PM3 are commonly used semiempirical SCF-MO methods at present and have basically identical quantum mechanical model based on Hartree–Fock theory. Since their parameterization approaches are different, however, one method may be more suitable than the other for certain type of systems and problems. It is repeated applications that determine which method is a better choice. Generally speaking, AM1 can describe chemistry (e.g. electronic structure) better than PM3, while PM3 may in turn give more accurate data (e.g. heat of formation) in some cases. AM1 is therefore applied in the present study in order to better reveal the chemistry of PBDEs. The objectives of this investigation were, on the basis of AM1-calculated electronic structures, to elucidate the reactivity of brominated diphenyl ethers (BDEs) and account for the chemical and toxicological properties including mass spectral characteristics, chromatographic and ultraviolet spectral properties and luciferase induction activities. Further, quantitative structure–activity (property) relationship (QSAR or QSPR) models were developed through statistical analysis (multilinear regression) of datasets of experimental properties and datasets of computed electronic parameters. The study examines 48 BDE congeners (Table 1) and two thyroid hormone analogues, ‘‘CH3-T4’’ and ‘‘CH3-T3’’ (Fig. 2).
2. Methods The semiempirical calculations in vacuum using an AM1 Hamiltonian (Dewar et al., 1985) with the Mopac package (public domain version 6.00 by Coolidge and Stewart, 1990) were carried out on a Digital Unix/ DEC-3000/600 AlphaStation and several personal computers with Intel · 86 architecture processors. The C–C ˚ , C–O bond lengths bond lengths were started at 1.40 A ˚ ˚ , and C–Br bond at 1.39 A, C–H bond lengths at 1.10 A ˚ . The geometry optimizations were lengths at 1.87 A achieved applying the Broyden–Fletcher–Goldfarb– Shanno (BFGS) quasi-Newton method, which is incorporated into the Mopac program. All calculations are based on the closed-shell RHF-SCF-MO method except in the cases where open-shell UHF-SCF-MO computations were performed to calculate heats of formation for cation radicals of BDEs (Table 3), which were used for the prediction of fragmentation patterns in the mass spectra. For all factor analysis and regression analysis, SPSS software was used in this study. The relative retention times (RRTs) of the 19 BDEs (Table 1) were determined on a Varian 3400 gas chro-
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Table 1 Structures, maximum luciferase induction activities, GC RRTs and wavelengths of maximum UV absorption of 48 BDEs BDE no.
Structure (bromination)
Luciferase induction (%)a
RRT1b
RRT2
RRT3
RRT4
RRT5
CPSil-8
HP-1701
SP-2380
SB-Smectic
XTI-5
1 2 3 7 8 10 12 13 15 17 25 28 30 32 33 35 37 45 46 47 49 50 51 53 54 66 71 75 77 85 99 100 104 116 119 128 138 140 153 154 155 166 181 183 186 190 204 209
2 3 4 2,4 2,4 0 2,6 3,4 3,4 0 4,4 0 2,2 0 ,4 2,3 0 ,4 2,4,4 0 2,4,6 2,4 0 ,6 2 0 ,3,4 3,3 0 ,4 3,4,4 0 2,2 0 ,3,6 2,2 0 ,3,6 0 2,2 0 ,4,4 0 2,2 0 ,4,5 0 2,2 0 ,4,6 2,2 0 ,4,6 0 2,2 0 ,5,6 0 2,2 0 ,6,6 0 2,3 0 ,4,4 0 2,3 0 ,4 0 ,6 2,4,4 0 ,6 3,3 0 ,4,4 0 2,2 0 ,3,4,4 0 2,2 0 ,4,4 0 ,5 2,2 0 ,4,4 0 ,6 2,2 0 ,4,6,6 0 2,3,4,5,6 2,3 0 ,4,4 0 ,6 2,2 0 ,3,3 0 ,4 2,2 0 ,3,4,4 0 ,5 0 2,2 0 ,3,4,4 0 ,6 0 2,2 0 ,4,4 0 ,5,5 0 2,2 0 ,4,4 0 ,5,6 0 2,2 0 ,4,4 0 ,6,6 0 2,3,4,4 0 ,5,6 2,2 0 ,3,4,4 0 ,5,6 2,2 0 ,3,4,4 0 ,5 0 ,6 2,2 0 ,3,,4,5,6,6 0 2,3,3 0 ,4,4 0 ,5,6 2,2 0 ,3,,4,4 0 ,5,6,6 0 Deca-
– – – – – – – – 7.5 – – 8.4 9.6 20.7 – – – – – 3.3 – – 10.1 – – – 6.3 13.2 12.8 28.3 24.1 8.0 – – 26.9 – 9.7 – 34.2 – – 65.8 – – – 50.3 – –
– – – 0.400 0.411 – 0.415 0.415 0.424 0.506 0.506 0.519 0.468 0.496 0.520 0.528 0.540 – – 0.627 – – 0.605 – – 0.641 0.614 0.600 0.667 0.787 0.735 0.711 – 0.750 0.719 – 0.944 – 0.864 – – – – 1.061 – – – –
– – – 0.385 0.400 – 0.400 0.402 0.412 0.496 0.493 0.508 0.447 0.487 0.510 0.516 0.529 – – 0.612 – – 0.594 – – 0.628 0.602 0.580 0.654 0.794 0.720 0.690 – 0.732 0.699 – 0.986 – 0.869 – – – – 1.107 – – – –
– – – 0.474 0.505 – 0.494 0.500 0.530 0.629 0.616 0.651 0.529 0.609 0.642 0.650 0.677 – – 0.797 – – 0.766 – – 0.838 0.767 0.723 0.886 1.128 0.953 0.884 – 0.976 0.897 – 1.494 – 1.182 – – – – 1.546 – – – –
– – – 0.437 0.460 – 0.420 0.477 0.530 0.598 0.597 0.663 0.494 0.567 0.622 0.655 0.765 – – 0.882 – – 0.785 – – 0.941 0.792 0.748 1.076 1.407 1.109 1.023 – 1.161 1.048 – 1.646 – 1.354 – – – – 1.673 – – – –
0.806 0.815 0.831 1.031 1.055 0.988 – 1.070 1.084 1.241 1.241 1.267 1.171 1.226 – – – – – 1.433 1.400 – 1.394 – – – – – – – – 1.555 – 1.610 – – – – – – – 1.823 – – – – – –
UV (kmax, nm)c 200.0 200.0 200.0 205.2 202.0 205.0 204.8 200.0 203.2 203.2 203.2 201.4 210.4 204.6 203.8 207.4 203.4 – – 204.4 – – 211.8 – – 205.8 205.4 210.8 202.6 – – 210.6 – 225.6 209.2 – – 213.2 – 211.4 – 226.4 227.6 – – 226.8 – –
a Maximum luciferase induction cited from Meerts et al. (1998a). Maximal luciferase induction (100%) was obtained with 100 pM TCDD in a recombinant H4IIE rat hepatoma cell line. b RRT1 (low-polar column), RRT2 (medium polar column), RRT3 (very polar column) and RRT4 (medium polar column) are cited from Sjo¨din et al. (1998). RRT5 were determined in the present work. c Cited from Marsh et al. (1999).
matograph equipped with XTI-5 fused silica capillary column (30 m · 0.25 mm, 0.25 lm film (Restek) thick-
ness) and quadrupole TSQ 700 Finnigan MAT MSD (electron ionization at 70 eV and ion source temperature
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in press), the BDEs with intraring C2 symmetry axis from C1 to C4 and C1 0 to C4 0 , i.e., BDE-3, 10, 15, 30, 32, 54, 75, 104, 116, 155, 186, 204 and 209 and ‘‘CH3-T4’’, have only single energy minimum, similarly to BDE-166. For BDE-1, 2, 7, 8, 12, 13, 28, 37, 46, 50, 53, 71, 100, 119, 140, 154, 181 and 190 and ‘‘CH3-T3’’, there are two different energy minima as for BDE-51. For BDE-47, 77 and 153, three energy minima were identified for each congener. And for BDE-17, 25, 33, 35, 45, 49, 66, 85, 99, 128, 138 and 183, four energy minima were obtained. These results are compatible with those of AM1 studies on PCDEs (Nevalainen and Rissanen, 1994). For clarity, only the parameters of the global minima (lowest energy conformations) are given for the 51 diphenyl ethers including 48 BDEs listed in Table 2. On the basis of the ortho-substitution patterns the following three types of global minima (conformation) were identified: (i) twist type for the eight BDEs with no bromine substituents at the ortho-positions (namely BDE-2, 3, 12, 13, 15, 35, 37, and 77), which are more or less similar to diphenyl ether; (ii) skew type for the seven congeners with 2,6-di-ortho-substitution (namely BDE-10, 30, 32, 75, 116, 166, and 190), in which one phenyl ring is close to perpendicular to the C–O–C central plane and the other lies approximately in the plane; (iii) another twist type for the remaining 33 congeners, in which u1 and u2 generally differ from each other. 3.1. Dipole moments, polarizabilities and QSPR models for predicting gas chromatographic properties
Fig. 2. (a) Thyroxine (T4); (b) 3,3 0 ,5-triiodothyronine (T3); (c) ‘‘CH3-T4’’ and (d) ‘‘CH3-T3’’.
140). The temperature program for the GC column was 100 C (1 min); 10 C/min (22 min); 320 C (22 min). Helium was used as the carrier gas and the temperature of injector was 280 C. The relative retention times were referenced to PCB-53.
3. Results and discussion Conformations of diphenyl ethers are described by the torsional angles (u1 and u2) between the C–O–C plane and planes of the phenyl rings. The angles are defined as positive when the rotation is clockwise looking down the C4–C1 and C4 0 –C1 0 axes toward the oxygen (Fig. 1). According to conformational analysis of PBDEs based on calculated contour energy maps (Hu et al.,
The dipole moment (l) of a molecule reflects its structural symmetry. Calculated dipole moments of the lowest energy conformation of the BDEs studied vary widely, from around zero to 4.16 D (Table 2). Values are 1.83 D for 4-bromodiphenyl ether (BDE-3) and 0.04 D for 4,4 0 -dibromodiphenyl ether (BDE-15). The latter is the smallest value calculated and indicates the high structural symmetry of BDE-15. The previously determined experimental values are 1.57 D and 0.60 D for BDE-3 and BDE-15 respectively (Anderson and Smyth, 1965). The fairly wide discrepancy between the theoretical and experimental values may be ascribed to the conformational freedom of these two non-orthocongeners. Their structures oscillate widely about the energy minima and the moments calculated for single minima do not necessarily well reflect their time-averaged values. In addition, different energy minima always give different values of dipole moments. To illustrate this, Table 3 presents the dipole moments, l (along with other electronic properties), of different minima of several congeners. The results confirm that the calculated moments of BDEs are not a reliable descriptor for their physicochemical properties. Molecular polarizability measures the ability of the electrons in a molecule to move in response to an exter-
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Table 2 AM1 calculated electronic properties of 51 diphenyl ethers including 48 BDEs BDE no.
DH (kJ/mol)
EHOMO (eV)
ELUMO (eV)
DE (eV)
l (Debye)
˚ 3) a (A
u1()
u2()
Diphenyl ether 1 2 3 7 8 10 12 13 15 17 25 28 30 32 33 35 37 45 46 47 49 50 51 53 54 66 71 75 77 85 99 100 104 116 119 128 138 140 153 154 155 166 181 183 186 190 204 209 ‘‘CH3-T3’’ ‘‘CH4-T4’’
98.521 131.624 120.796 118.621 154.959 151.168 167.168 143.921 141.607 139.808 189.878 177.109 175.343 192.807 186.996 175.987 165.594 166.988 228.204 226.672 211.141 212.275 226.974 227.066 224.593 242.157 200.497 211.819 213.208 191.757 239.588 237.496 252.471 267.040 252.651 238.492 268.337 266.328 278.353 264.893 276.257 292.474 273.362 310.265 305.972 327.105 299.173 353.038 414.216 132.114 211.510
8.954 9.139 9.139 9.066 9.265 9.183 9.187 9.221 9.219 9.173 9.350 9.441 9.301 9.304 9.216 9.335 9.301 9.345 9.387 9.464 9.416 9.484 9.414 9.415 9.410 9.470 9.435 9.360 9.326 9.426 9.522 9.527 9.518 9.537 9.445 9.463 9.581 9.585 9.567 9.599 9.549 9.613 9.436 9.569 9.596 9.694 9.561 9.746 9.872 8.961 9.135
0.171 0.200 0.127 0.136 0.545 0.344 0.429 0.400 0.343 0.403 0.443 0.646 0.694 0.794 0.561 0.418 0.613 0.532 0.839 0.594 0.765 0.716 0.849 0.529 0.595 0.423 0.760 0.631 0.924 0.762 0.839 0.850 0.801 0.711 1.197 0.990 0.990 0.991 1.047 1.104 1.050 0.900 1.308 1.348 1.255 1.123 1.349 1.217 1.343 0.484 0.580
9.125 8.939 9.012 8.930 8.719 8.839 8.758 8.821 8.876 8.770 8.907 8.795 8.607 8.510 8.655 8.917 8.688 8.813 8.448 8.870 8.651 8.768 8.565 8.886 8.815 9.047 8.675 8.729 8.402 8.664 8.683 8.677 8.717 8.826 8.248 8.473 8.591 8.594 8.520 8.495 8.499 8.713 8.128 8.221 8.341 8.571 8.212 8.529 8.529 8.477 8.555
1.245 1.889 2.361 1.831 1.317 2.695 2.111 2.996 1.438 0.036 1.427 0.730 1.316 1.290 2.876 2.569 1.230 2.464 2.856 4.163 2.028 1.064 2.520 1.906 2.287 2.200 1.448 2.668 1.420 1.195 2.820 1.378 0.779 2.201 1.549 1.392 2.800 1.588 3.000 0.629 1.574 1.154 0.531 1.580 1.246 2.671 0.785 1.232 0.635 1.488 2.421
16.602 17.788 18.325 18.385 19.609 19.552 19.276 20.045 20.135 20.238 20.903 21.083 21.419 21.150 21.032 21.008 21.936 21.570 22.484 22.453 23.013 22.779 22.686 22.442 22.443 22.128 22.854 22.457 22.947 23.650 24.635 24.585 24.376 24.022 24.480 24.373 26.138 26.058 26.164 26.357 26.134 25.964 26.318 27.906 27.777 27.334 27.746 29.326 32.715 24.667 27.002
37.0 108.1 34.7 26.1 113.0 104.9 87.6 25.0 44.8 36.4 135.6 110.4 108.5 95.2 93.5 173.9 36.1 28.9 77.6 24.3 25.8 71.3 77.7 139.8 20.3 55.0 110.9 96.7 94.0 38.3 156.0 156.8 131.5 53.2 83.9 93.0 72.2 73.5 159.0 72.9 164.7 54.7 92.4 80.6 82.6 56.3 93.8 57.2 55.0 89.7 87.7
37.1 19.7 40.0 47.6 25.0 14.9 9.3 49.7 29.5 36.5 55.1 23.8 18.6 2.5 0.1 92.0 37.3 45.0 25.9 79.2 73.0 153.5 25.9 60.4 81.3 54.8 20.7 4.4 1.2 148.7 72.0 74.1 51.1 55.9 15.2 0.8 156.9 156.4 78.6 155.7 104.8 53.6 1.7 161.2 164.9 54.3 0.6 52.4 55.4 6.4 10.3
nal electric field. As shown in Fig. 3, the calculated average molecular polarizabilities (a) (Table 2) correlate significantly with the relative retention times (RRTs) in GC (Table 1) determined in the present work for a XTI-5
column (non-polar) (R2 = 0.987) and in earlier work for four columns of different polarity (R2 = 0.973 for CPSil-8, 0.953 for HP-1701, 0.912 for SP-2380 and 0.932 for SB-Smectic) (Sjo¨din et al., 1998). Correlation
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Table 3 AM1 calculated electronic properties for different minima of seven diphenyl ethers BDE no.
DHf (kJ/mol)
EHOMO (eV)
ELUMO (eV)
l (Debye)
˚ 3) a (A
u1()
u2()
99
237.496 238.266 239.781 268.337 270.341 266.328 266.889 268.475 264.893 266.680 276.257 279.274 305.972 308.838 132.114 132.938
9.527 9.507 9.547 9.581 9.593 9.585 9.609 9.168 9.599 9.650 9.549 9.626 9.596 9.678 8.961 8.984
0.850 1.047 0.881 0.990 0.866 0.991 1.112 0.952 1.104 1.004 1.050 0.951 1.255 1.099 0.484 0.516
1.378 1.476 1.053 2.800 1.640 1.588 2.168 1.456 0.629 0.427 1.574 1.212 1.246 0.899 1.488 4.818
24.585 24.779 24.567 26.138 25.698 26.058 26.430 26.042 26.357 26.395 26.134 26.175 27.777 27.790 24.667 24.976
156.8 73.0 53.5 72.2 53.7 73.5 155.4 53.8 72.9 53.1 164.7 49.3 82.6 59.8 89.7 102.4
74.1 155.8 52.9 156.9 53.3 156.4 73.0 52.6 155.7 53.9 104.8 58.1 164.9 47.7 6.4 166.8
128 138
153 154 183 ‘‘CH3-T3’’
of the polarizabilities was best with the RRTs determined in the low polarity column (CPSil-8) (R2 = 0.976), compatible with the result for our non-polar XTI-5 column. These models are basically consistent with the multivariate QSPR models developed recently by Harju et al. (2002) on the same data based on 40 physicochemical descriptors, which give R2 value 0.991, 0.984, 0.985 and 0.977 for these columns, respectively. Our results are reasonable since the dispersion forces, which are closely related to a, should be dominant in the interactions of non-polar solute molecules with non-polar liquid phase molecules. The correlations are evidently poorer for the other three columns with higher polarity, because some other forces, such as the dipoleinduced–dipole-interaction, may play a more important role in those intermolecular interactions. It is thus apparent from these results that the average molecular polarizability is a useful descriptor for predicting such molecular properties as lipophilicity (LgKow) of these compounds. It is expected that LgKow increases with a within a certain rang for BDEs, however, this rule ceases to hold at high molecular weights, for the highly brominated BDEs are neither lipophilic nor hydrophilic. This is in principle consistent with an optimal PLS model developed by Chen et al. (2003a) for predicting LgKoa (octanol–air partition coefficient) of PBDEs based on 18 theoretical molecular structure descriptors, which gives the cross validated Q2 cum value 0.975, showing high predictive power. 3.2. Heats of formation and relation to thermodynamic stability and mass spectral characteristics AM1 calculations give heats of formation (DHf) as a measure of the relative thermodynamic stabilities of
compounds, which are only comparable, however, when molecules are of equivalent atomic composition (isometric). As can be seen in Table 2, DHf of the BDEs increases with the number of bromine substituents. With equivalent bromination degree, DHf increases with the number of ortho-bromine substituents, which means that thermodynamic stability is higher for BDEs with fewer ortho-substituents. For example, BDE-77 (with no ortho-substitution) is 50.4 kJ/mol more stable than BDE-54 (with tetra-ortho-substitution). The other tetraortho-congeners such as BDE-104, 155 and 186 were also calculated to have distinctly high heats of formation. The higher energy of these congeners apparently comes from the stronger ortho-interaction. The extreme example is decabromodiphenyl ether, another congener with tetra-ortho-substitution, for which an exceptionally high heat of formation (414.2 kJ/mol) is calculated. This theoretical result indicates a relatively low thermodynamic stability for decaBDE and conforms to its widespread use as a flame retardant. Flame retardants should easily decompose so as to interrupt the radical chain reactions taking place in combustion. The easy decomposition of decaBDE, in addition to its strong absorption to particles (sediments and soils), may also partly explain why it is relatively rarely encountered in the environment despite its large-scale use. These findings are further compatible with a wellknown characteristic of mass spectra of PBDEs (Marsh et al., 1999) as well as PCDEs (Nevalainen et al., 1994; Nevalainen, 1995). Namely, the ortho-congeners generally give rise to (M-2Br)+ or (M-2Cl)+ fragments as base peaks rather than to the molecular ions that generally appear as the base peaks of non-ortho-congeners. Perhaps, the molecular ions of the ortho-congeners are less stable than those of the non-ortho-congeners in parallel
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Fig. 3. Correlations between average molecular polarizabilities and RRTs of BDEs for five GC columns.
with the stabilities of their parent compounds. To test this postulation, heats of formation were calculated for the molecular ions of six dibrominated and eight tetrabrominated congeners. For the dibrominated congeners, the molecular ions of the ortho-substituted congeners BDE-7, 8 and 10 were found to have considerably higher energies (average DHf = 959.0 kJ/mol) than those of the non-ortho-congeners BDE-12, 13 and 15 (average DHf = 950.7 kJ/mol). For the tetrabrominated congeners, the heats of formation for the molecular ions of tetra-ortho congener BDE-54 (DHf = 1067.3 kJ/mol) and tri-ortho BDE-45, 46, 50, 51 and 53 (average DHf = 1045.7 kJ/mol) are all clearly higher than those for diortho BDE-75 (DHf = 1028.6 kJ/mol) and non-ortho BDE-77 (DHf = 1019.7 kJ/mol); the energy difference between the molecular ions of BDE-54 and 77 is as high as 47.5 kJ/mol. These results may also help explain mechanism and kinetics of free radical reactions of these compounds, such as reaction with OH radicals in air. 3.3. Frontier orbital energies and their relevance to kinetics of electrophilic and nucleophilic reactions Having seen above the low total thermodynamic stability of PBDEs relative to their chlorinated analogues, we can reasonably assume, even without examining free energy changes of respective metabolism reactions, that PBDEs should at least have a similar tendency to their
chlorinated analogues to undergo well-known metabolic reactions. What we are concerned with here, therefore, is not the thermodynamics but the kinetics of the reactions. A major metabolic pathway of halogenated aromatics, such as PCBs, has been postulated to proceed through an arene oxide intermediate by oxidation under catalysis of a cytochrome P-450 (CYP). The intermediate is then attacked by water to generate the hydroxylated compounds and some relevant conjugates, and by a glutathione under catalysis of glutathione S-transferase (GST) to generate mercapturic conjugates and ultimately methyl sulfonyl metabolites (Hutzinger et al., 1974; Safe, 1994). This mechanism should also be applicable to PBDEs. Frontier orbital energies were found to correlate with the kinetics of enzyme catalyzed reactions of several classes of halogenated aromatic compounds (Vervoort et al., 1992; Cnubben et al., 1994; Rietjies et al., 1995). It is not clear to what degree the chemical reactivity determines the metabolism in the case of BDEs, however, since other factors such as lipophilicity and steric factors may also play important roles. According to our AM1 calculations, the energies of the lowest unoccupied molecular orbital (ELUMO) and the energies of the highest occupied molecular orbital (EHOMO) of the BDEs tend to decrease with an increasing number of bromine substituents (Table 2). Following the frontier orbital theory, the higher brominated diphenyl ethers
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should be less reactive towards electrophiles such as CYPs, but more reactive towards nucleophiles. These results are consistent with the basic chemical rule that the chlorination or bromination of organic molecules will deactivate them for further electrophilic reactions. Recently, in an in vitro bioassay with hepatic microsomes from fish, 4,4 0 -dibromobiphenyl (PBB-15) was found to be subjected to CYP oxidation, while the other higher PBB and PBDE congeners showed no indication of this biotransformation (de Boer et al., 1998). Also, 2,2 0 ,4,4 0 tetrabromodiphenyl ether (BDE-47) was shown to ¨ rn slowly metabolize through hydroxylation in rats (O and Klasson-Wehler, 1998). Reactivity appeared to be decisive for the rate of metabolism of these brominated aromatics. However, LgKow and size of these compounds, which are closely related to EHOMO, can also be important factors since the two properties might give differences in their affinity to CYPs. Comparing our data to those of PCDDs, PCDFs, PCBs and PCDEs, we see that the HOMO energies of PCBs, PCDFs and PCDEs are similar to each other, while those of PCDDs are considerably higher. The LUMO energies are similar for all compounds (Nevalainen and Kolehmainen, 1994; Koester and Hites, 1988). These findings may indicate that PCDDs are generally more reactive towards electrophiles such as CYP, albeit this sort of comparison is not theoretically justified. An experiment with rainbow trouts did give much shorter
biological half lives for PCDDs than for PCBs and PCDEs (Niimi, 1986). 3.4. Frontier orbital energy gaps (dE), QSPR model for predicting ultraviolet (UV) spectral properties, and photolytic reactions The energy gaps (dE)(Table 2) between LUMO and HOMO of BDEs, which are calculated from ELUMO and EHOMO, correlate adequately (R2 = 0.734) with the wavelengths at maximum UV absorbance (kmax) of these compounds (Marsh et al., 1999) (Fig. 4). It was reported that dE relates to or correlates with phototoxicity and kinetics of photolysis for polycyclic aromatic hydrocarbons (PAHs) (Mekenyan et al., 1994; Chen et al., 1996) and with the receptor binding affinities of PCDDs, PCDFs and PCDEs (Nevalainen and Kolehmainen, 1994). As a general trend, dE of the BDEs decreases with increasing degree of bromination, which means that an electronic transition from the HOMO (ground state) to LUMO (excited state) in the higher BDEs proceeds more easily with increasing bromination. Reactions such as photolysis and formation of PBDFs may take place as one of possible route for energy decay of BDE molecules excited through the absorption of solar energy. In the higher BDEs, moreover, there are plenty of comparatively labile Br–C bonds that are considerably weaker than the corre-
Fig. 4. Correlation between frontier energy gaps and wavelengths at maximum UV absorbance of BDEs.
J. Hu et al. / Chemosphere 59 (2005) 1043–1057
sponding C–Cl bonds. Cleavage of such Br–C bonds is expected to be an easier route for energy decay than corresponding cleavage in their chlorinated analogues. The labiality of C–Br bonds could account for experimental results in which decaBDE was transformed to lower BDE congeners in the different matrices under UV radiation or sunlight (Watanabe et al., 1986; So¨derstro¨m et al., 2004). That, in addition to factors such as rather large molecular size and weight, may be an important reason why deca- and other higher BDEs are so rarely bioavailable in the environment. In addition to its connection to UV spectra, according to Pearson (1986), dE also indicates hardness of molecule. The tetra-ortho-congeners such as BDE-54, 104, 155 and 186 were calculated to give clearly higher dE values (Table 2) than the corresponding congeners with tri-, di- and non-ortho-substituents, probably indicating restricted rotation of the phenyl groups around the ether bond. The restricted rotation prevents effective delocalisation of p electrons on the phenyl groups and thus increases energy gap (dE) between LUMO and HOMO. These molecules also gave relatively low molecular polarizabilities, because molecules with high hardness are not easy to be polarized. Contrarily, the di-orthoand tri-ortho-congeners show no clear differences from mono- and non-ortho-congeners and may thus have no rotational restriction in these molecules. These results are consistent with those from our conformational analysis (Hu et al., in press).
3.5. QSAR models for predicting luciferase induction activity We set up three QSAR models for calculating the luciferase induction activity (I) of BDEs. These models show that the induction activity increases with increasing electrophilicity or electron affinity (ELUMO), with decreasing hardness (dE), or with increasing average molecular polarizability (a) Model 1 I ¼ 0:528ð0:106ÞELUMO 0:244ð0:093Þ n = 17, R2 = 0.625, SE = 0.108, F = 24.991, p = 0.000 Model 2 I ¼ 0:693ð0:139ÞdE þ 6:147ð1:195Þ n = 17, R2 = 0.623, SE = 0.108, F = 24.761, p = 0.000 Model 3 I ¼ 0:055ð0:015Þa 1:107ð0:353Þ n = 17, R2 = 0.479, SE = 0.127, F = 13.767, p = 0.002 Here R2 is the square of correlation coefficient, S.E. is the standard error of estimate, F is the F value for analysis of variance and p is the significance level.
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The models were obtained by a linear regression using as the dependent a data set of the measured luciferase induction activity values of 17 BDEs, obtained in a recombinant H4IIE rat hepatoma cell bioassay (Meerts et al., 1998a). A plot of the measured vs calculated (Model 1) luciferase induction activities is presented in Fig. 5. Since, in general, the independent variables themselves such as ELUMO, EHOMO, dE and a are strongly inter-correlated, probably owing to the similar dependence on the molecular wave function (Table 4), only one-parameter equations are possible if without special treatment of data such as application of factor and PLS analysis. Although these models are far from complete, a general trend is apparent: ELUMO or dE and a are major determinants for the luciferase induction activity (ELUMO and dE are closely related). These results are fairly compatible with the findings for related organochloro compounds (Albro and McKinney, 1981; Lewis et al., 1989, 1993; Nevalainen and Kolehmainen, 1994). These models indicate that higher BDEs generally have higher activities. The models also show that tetra-orthocongeners, with relatively high dE value and low a values owing to restricted rotation, have relatively low activities, while other congeners with non-, di- and tri-orthosubstituents show no clear differences in the activity. Harju et al. (2002) recently developed a multivariate (PLS) model (R2 = 0.618) using 40 descriptors to predict the luciferase activity for PBDEs. In the present study, a kind of factor analysis, principle component analysis (PCA) (Tysklind et al., 1992; Chen et al., 1998), was performed to extract orthogonal principal components (PCs). PLS and PCA are both projection methods, and PLS make projections of two related matrixes of descriptors and responses simultaneously while PCA only makes projection of matrix of descriptors. The descriptors are EHOMO (eh), ELUMO (el), average molecular polarizability (pl), dipole moment (dp) and energy gap (de) between LUMO and HOMO. Two significant PCs, which explain 65% and 22% of the variance respectively, were extracted and their loadings plot is shown in Fig. 6. PC1 is dependent on the size-related parameters EHOMO, ELUMO, dE and a, while PC2 is mainly dependent on dipole moment, a structure/symmetry parameter. Using stepwise multiple linear regressions, Model 4 for calculating the luciferase induction activity (I) was established based on the resulting principle factor scores (Fa1 and Fa2) for individual BDE compounds. In this model, Fa1 is only the significant variable (p = 0.000) since less important Fa2 (p = 0.343) was excluded in the stepwise regressions. This model is consistent with Models 1–3 and with our result in Chapter 3.1 that dipole moment is not a good descriptor for properties of PBDEs. The present models and previous result by Harju et al. (2002) have all shown that physicochemical parameters of the BDEs can only account for
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Fig. 5. Observed vs calculated luciferase induction activities of BDEs in H4IIE rat hepatoma cell line.
Table 4 Correlation matrix of independent variables (calculated from 17 cases)
Induc (I) EHOMO ELUMO DE Polar (a) Dipole (l)
Induc (I)
EHOMO
ELUMO
DE
Polar (a)
Dipole (l)
1.000 0.386 0.791 0.789 0.692 0.287
1.000 0.674 0.237 0.897 0.093
1.000 0.877 0.887 0.340
1.000 0.584 0.386
1.000 0.248
1.000
approximately 60% variation of the luciferase induction activity. That may mean that this biochemical process may involve complex mechanisms and more tests are therefore needed to clarify them. In addition, uncertainty in luciferase bioassay may also give a significant contribution to the uncertainty of the model. Model 4 I ¼ 0:130ð0:029ÞFa1 þ 0:200ð0:028Þ 2
n = 17, R = 0.579, SE = 0.114, F = 20.589, p = 0.000 Further efforts were also made to improve significance of the models. From Fig. 5, BDE-15, 32 and 138 appeared to be outliers. If these three outliers were excluded, all four models were improved in terms of R2 values (R2 for Model 1 = 0.803, R2 for Model
2 = 0.625, R2 for Model 3 = 0.699 and R2 for Model 4 = 0.768). These new models are however not justified because we cannot interpret those outliers based on the information presently available. 3.6. Frontier electron densities and regioselectivity of electrophilic and nucleophilic reactions A study on regioselectivity (orientation) of hydroxylation of BDEs under the catalysis of a CYP could be expected to be useful in elucidating their toxic effects. The regioselectivity can be predicted from familiar chemical principles or by examining frontier orbital densities. In general, an enzymatic hydroxylation of BDEs may take place on the phenyl ring with lower halogenation because it is less deactivated by the halogen substituents; a GST, on the other hand, may react with the ring with
J. Hu et al. / Chemosphere 59 (2005) 1043–1057
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Fig. 6. Component plot.
higher halogenation. This is in agreement with the result of a previous metabolic study with PCDEs, in which both 4-chloro- and 2,4-dichlorodiphenyl ether were hydroxylated at 4 0 -position (Becker et al., 1991). Examination of the frontier electron densities may give a quantitative prediction of the regioselectivity of enzymatic reactions. Our calculations indicate that the distribution pattern of frontier orbital densities of BDEs having a skew conformation is rather different from that of BDEs having a twist conformation. According to the equation given by Fukui et al. (1954), the frontier electron densities can be calculated from HOMO and HOMO-1 characteristics and LUMO and LUMO + 1 characteristics. However, we found that the energy gaps between the LUMO + 1 and LUMO in some cases are too small for the equation to give reasonable results. Following Poso et al. (1993), we therefore consider only HOMO and LUMO in calculating the frontier electron densities (Table 5). Variation in electronic properties of the phenyl rings with change in conformation of PCDEs was well recognized through a large number of NMR experiments (Hu et al., 1994; Nevalainen, 1995). The present calculations in fact show another direct proof for this variation. In the molecules with skew conformation, the HOMO orbital densities are almost completely distributed over the oxygen and carbon atoms of the phenyl ring that is coplanar with the C–O–C plane (u1 = 0). The LUMO density, on the other hand, is distributed almost totally
over the carbons of the other phenyl ring that is perpendicular to the C–O–C plane (u2 = 90). This may indicate that electrophilic attacks, for example by CYP, would preferably take place on the carbons of the phenyl ring coplanar with the C–O–C plane, whereas nucleophilic attacks would be favored on the carbons of the other phenyl ring (Rietjens et al., 1993). In molecules with twist conformation, the frontier orbital densities are more uniformly distributed and their reactivity is difficult to predict. On the whole, it appears that the congeners with skew conformation may be more reactive than the corresponding congeners with twist conformation owing to their highly non-uniform frontier electron distribution. These findings are also applicable to ‘‘CH3-T3’’ and ‘‘CH4-T4’’, which have a skew conformation. It needs to be emphasized that the above predictions of the reactivity of BDEs were made on the basis of frontier orbital characteristics alone, and should be considered only as a tentative indication of the orientation of reactions associated with these compounds. The coulombic characteristics of BDEs and the steric effects arising from the interaction between BDEs and other reactants may greatly affect their reactivity as well. 3.7. Thyroid hormone analogues It is suspected that BDEs may have thyroid hormone-like activity since thyroid hormones, e.g. T3 and
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Table 5 Frontier electron densities and atomic charges on carbons and oxygen of five diphenyl ethers with a skew conformation BDE no.
C1
C2
C3
C4
C5
C6
O
C1 0
C2 0
C3 0
C4 0
C5 0
C6 0
10
HD LD Q
0.00 0.03 0.10
0.00 0.56 0.18
0.00 0.41 0.09
0.00 0.02 0.13
0.00 0.62 0.09
0.00 0.30 0.17
0.26 0.00 0.15
0.48 0.00 0.07
0.20 0.00 0.18
0.13 0.00 0.10
0.55 0.00 0.16
0.07 0.00 0.10
0.26 0.00 0.14
30
HD LD Q
0.00 0.69 0.11
0.00 0.17 0.18
0.00 0.15 0.07
0.00 0.55 0.18
0.00 0.14 0.07
0.00 0.18 0.18
0.26 0.00 0.15
0.48 0.03 0.07
0.19 0.00 0.18
0.14 0.00 0.10
0.56 0.00 0.16
0.07 0.00 0.10
0.26 0.00 0.14
75
HD LD Q
0.00 0.69 0.10
0.00 0.17 0.18
0.00 0.15 0.07
0.00 0.55 0.17
0.00 0.13 0.07
0.00 0.18 0.18
0.21 0.00 0.15
0.39 0.03 0.09
0.15 0.00 0.19
0.14 0.00 0.07
0.49 0.00 0.20
0.09 0.00 0.08
0.18 0.00 0.14
‘‘T3’’
HD LD Q
0.00 0.69 0.10
0.00 0.17 0.27
0.00 0.13 0.08
0.00 0.54 0.07
0.00 0.09 0.08
0.00 0.23 0.27
0.19 0.00 0.15
0.42 0.03 0.04
0.08 0.00 0.12
0.24 0.00 0.26
0.41 0.00 0.09
0.11 0.00 0.19
0.18 0.00 0.08
‘‘T4’’
HD LD Q
0.00 0.67 0.09
0.00 0.17 0.27
0.00 0.12 0.08
0.00 0.52 0.07
0.00 0.08 0.08
0.00 0.22 0.27
0.19 0.00 0.15
0.41 0.08 0.03
0.08 0.00 0.10
0.22 0.00 0.27
0.38 0.00 0.13
0.13 0.00 0.33
0.18 0.00 0.06
HD: electron density of HOMO. The summation proceeded over the atomic orbitals (s, px, py, pz) according to Poso et al. (1993); LD: electron density of LUMO; Q: atomic charge; ‘‘T3’’: ‘‘CH3-T3’’; ‘‘T4’’: ‘‘CH3-T4’’.
T4, include iodated diphenyl ethers as their hydrophobic moiety. It was of interest to study the conformational and electronic properties of T3 and T4 as well, and compare them with those of BDEs. The AM1 calculations were performed for two molecules in which the thyronine moiety of T3 and T4 was exchanged for a methyl group (‘‘CH3-T4’’ and ‘‘CH3-T3’’). Both molecules were found to have lowest energy conformations of skew type similar to those of the corresponding BDEs and the frontier orbital energies are comparable as well (Table 2). These comparisons have shown a good resemblance between T4/T3 analogues and the corresponding BDEs as far as conformational properties and reactivity are concerned. Experimental results showed that substances such as hydroxylated PCBs and pentachlorophenol can strongly bind to transthyretin (TTR), one of the thyroid hormone transport proteins (De la Paz et al., 1992; Lans et al., 1993). Other results indicated that the rat microsomal formation of BDE metabolites is of high affinity to TTR (Murk, Personal communication; Meerts et al., 1998b). There was evidence to suggest, moreover, that BDEs can be metabolized to their hydroxylated ana¨ rn and Klasson-Wehler, 1998). It looks likely, logues (O therefore, that BDEs impose toxic effects on animals through the formation of hydroxylated metabolites that then bind to TTR and other proteins. In an attempt at clarification, some hydroxy-BDEs were synthesized and indeed found to bind to the thyroid receptors TR-a1 and TR-b (Marsh et al., 1998). Our theoretical calculations are in reasonable agreement with these preliminary experimental results.
4. Conclusions The reactivity and lipophilicity of PBDEs were elucidated on the basis of AM1-calculated electronic structures and satisfactory QSAR and QSPR models were derived. The results can provide a solid theoretical basis for assessing the impact of PBDEs on the environment. In general, the structures of BDE metabolites are difficult to determine in metabolic assay: they are present in only trace amount in complex mixtures and the required model compounds are frequently lacking. Calculated frontier orbital characteristics allow us to predict which structures are of importance in metabolic reactions and hence give a priori direction for metabolic assays. According to Di-region theory (Dai, 2000; Dai et al., 2002, 2003), metabolic reactions may activate some aromatic compounds to become dangerous carcinogens binding to DNA. From that perspective, the present investigation can be considered to further our understanding of carcinogenisis of aromatic compounds and may assist the development of novel anticancer drugs (such as therapeutic agents that selectively damage cancer cell DNA by crosslinking DNA duplex) (Wei, 1997; Dai, 2000). The calculated average molecular polarizabilities of BDEs, shown here to be valid descriptors for the QSPR models of gas chromatographic properties, might also be useful in modeling the accumulation, transportation and distribution of these compounds in the environment (Mackay, 1991; Southwood et al., 1998; Palm et al., 2002). Normally, before such modeling can be undertaken, large amount of data such as partition coeffi-
J. Hu et al. / Chemosphere 59 (2005) 1043–1057
cients, solubility and fugacity need to be determined experimentally and this procedure is usually very time consuming. Establishment of relevant QSPR models by the present methods offer more economical way to obtain partition data and can thereby facilitate the application of the environmental modeling to PBDEs. Because of complexity of the natural environment and the limitations of the AM1 method, it is unrealistic to think that these semiempirical calculations could account for everything occurring to PBDEs in the environment. Firstly, the present computations do not consider various intermolecular interactions in real environmental matrix such as solvent effects. Secondly, this semiempirical level of theory is a further approximation (e.g. employing core approximation) to first principle (or ab initio level of theory) that is derived from several critical simplifications including Born–Oppenheimer, non-relativistic and Hartree–Fock approximations. We believe, nevertheless, that the valuable information presently obtained from fundamental quantum mechanics will make our experiments more productive, well oriented and more insightful. Finally, some of the methods and results of the present investigation may be readily extended to other dioxin-like compounds and, if so, this investigation will contribute to understanding the chemistry of the whole class of these dangerous pollutants. Acknowledgment Dr. Juha Hyo¨tyla¨inen, Department of Chemistry, University of Jyva¨skyla¨, is gratefully acknowledged for his useful comments on this manuscript. References Albro, P.W., McKinney, J.D., 1981. The relationship between polarizability of polychlorinated biphenyls and their induction of mixed function oxidase activity. Chem. Biol. Interact. 34, 373–378. Anderson, J.E., Smyth, C.P., 1965. Microwave absorption and molecular structure in liquid. LX. Intramolecular relaxation mechanisms in aromatic ethers and several related molecules. J. Chem. Phys. 42, 473–478. Andersson, P., Haglund, P., Rappe, C., Tysklind, M., 1996. Ultraviolet absorption characteristics and calculated semiempirical parameters as chemical descriptors in multivariate modeling of polychlorinated biphenyls. J. Chemom. 10, 171–185. Becker, M., Philips, T., Safe, S., 1991. Polychlorinated diphenyl ethers—a review. Toxicol. Environ. Chem. 33, 189–200. Chen, J.W., Kong, L.R., Zhu, C.M., Huang, Q.G., Wang, L.S., 1996. Correlation between photolysis rate constants of polycyclic aromatic hydrocarbons and frontier molecular orbital energy. Chemosphere 33, 1143–1150. Chen, J.W., Peijnenburg, W.J.G.M., Wang, L.S., 1998. Using PM3 Hamiltonian, factor analysis and regression analysis in
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Wei, X.H., 1997. Mechanisms of carcinogenesis or anticancer effects and synthesis of anticancer effects and synthesis of anticancer agents-synthesis of derivative 1,2 diaminomethylbenzeneplatinum complexes in the light of Di-region theory (in Chinese). Ph.D. Thesis, Peking University, China. World Health Organization (WHO), 1994. Environmental Health Criteria 162: Brominated Diphenyl Ethers.
Molecular orbital studies on brominated diphenyl ethers. Part II—reactivity and quantitative structure–activity (property) relationships ˚ ke Bergman c, Eva Jakobsson c, Jiwei Hu a, Lars Eriksson b, A d Erkki Kolehmainen , Juha Knuutinen d, Reijo Suontamo d, Xionghui Wei a
a,*
Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, 100871 Beijing, P.R. China b Department of Structural Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden c Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden d Department of Chemistry, University of Jyva¨skyla¨, SF-40351 Jyva¨skyla¨, Finland Received 28 April 2003; received in revised form 21 September 2004; accepted 12 November 2004
Abstract Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and are increasingly turning up in the environment. Their structural similarities to polychlorinated biphenyls and thyroid hormones suggest they may be a risk to human health. The present study examines the reactivity of brominated diphenyl ethers (BDEs) on the basis of the electronic structures as calculated by semiempirical AM1 self-consistent field molecular orbital (SCF-MO) method. Frontier orbital energies were used to elucidate the reactivity of BDEs in electrophilic, nucleophilic and photolytic reactions. From an examination of the frontier electron densities, the regioselectivity, or orientation, of metabolic reactions of BDEs was predicted. Furthermore, satisfactory quantitative structure–activity (property) relationship (QSAR and QSPR) models were derived to calculate gas chromatographic and ultraviolet spectral properties and luciferase induction activities from the AM1-computed electronic parameters. 2004 Elsevier Ltd. All rights reserved. Keywords: Brominated diphenyl ethers; Molecular orbital studies; Reactivity; QSARs; QSPRs
1. Introduction Polybrominated diphenyl ethers (PBDEs) (Fig. 1) are used in large quantities as flame retardants in computers, TV sets, textiles and cars. Today they are ubiquitous in the environment. Since PBDEs and their metabolites are in general persistent, bioaccumulative and structu-
*
Corresponding author. Tel./fax: +86 10 62751529. E-mail address: [email protected] (X. Wei).
rally related to polychlorinated biphenyl (PCBs) and thyroid hormones, their increasing levels in the environment pose a potential danger to human health in thyroid hormone disruption, neurobehavioral deficits and carcinogenicity (World Health Organization, 1994; Paasivirta, 2000; McDonald, 2002). With the limited experimental data available (McDonald, 2002), theoretical evaluation of physicochemical properties of PBDEs such as conformational properties, reactivity and lipophilicity can significantly contribute to overall fate and risk assessments. The conformational properties of PBDEs,
0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.11.029
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Fig. 1. Backbone of polybrominate diphenyl ethers and definition of torsional angles u1 and u2.
which are reported in other paper (Hu et al., in press), create a basis for the present investigation of reactivity and other critical properties. According to Lewis et al. (1993), the chemical reactivity of xenobiotics may to some extent be related to their rates of metabolism and degradation and thus to metabolic activation and deactivation processes. Although PBDEs in general are chemically stable, the reactivities and thereby the metabolites of their congeners may vary considerably. Characterization of their chemical reactivity is also vital for understanding the adduct formation of PBDE congeners with macromolecules. Use of frontier orbital theory (Fukui et al., 1954) to describe reactions of aromatic compounds is well established. According to this theory, frontier orbital characteristics of reacting molecules, namely the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), determine the kinetics of a chemical reaction. In principle, frontier orbital characteristics, which are static indices, should be applied with caution in elucidating reactivities because this theory only describes the early stage of interactions between molecular orbitals of reactants. Ultimately, the reactivity depends on the relevant transition states formed from the reactants, not on the unperturbed reactants themselves. Locating the transition states (firstorder saddle points) in the potential energy surface (PES) of a reaction is a computationally expensive task, however, and meaningful only for that specific reaction. Use of static indices is probably a better choice for the present study, therefore, which aims at a general elucidation of the reactivity of PBDEs. A number of previous studies have indeed shown the efficacy of frontier orbital theory in addressing toxicological and environmental problems. For example, the frontier orbital characteristics and other electronic parameters calculated at AM1, PM3 or MNDO selfconsistent field molecular orbital (SCF-MO) level of theory have been shown to correlate with or relate to the chemical or biological properties of several classes of organic compounds, including polychlorinated dibenzop-dioxins and dibenzofurans (PCDDs and PCDFs), polychlorinated biphenyls (PCBs), polybrominated (polychlorinated) diphenyl ethers (PBDEs and PCDEs) and polycyclic aromatic hydrocarbons (Koester and Hites,
1988; Tuppurainen et al., 1991, 1992; Tysklind et al., 1992; Vervoort et al., 1992; Rietjens et al., 1993, 1995; Poso et al., 1993; Nevalainen and Kolehmainen, 1994; Cnubben et al., 1994; Andersson et al., 1996; Harju et al., 2002; Chen et al., 2003a,b; Kitti et al., 2003). Both AM1 and PM3 are commonly used semiempirical SCF-MO methods at present and have basically identical quantum mechanical model based on Hartree–Fock theory. Since their parameterization approaches are different, however, one method may be more suitable than the other for certain type of systems and problems. It is repeated applications that determine which method is a better choice. Generally speaking, AM1 can describe chemistry (e.g. electronic structure) better than PM3, while PM3 may in turn give more accurate data (e.g. heat of formation) in some cases. AM1 is therefore applied in the present study in order to better reveal the chemistry of PBDEs. The objectives of this investigation were, on the basis of AM1-calculated electronic structures, to elucidate the reactivity of brominated diphenyl ethers (BDEs) and account for the chemical and toxicological properties including mass spectral characteristics, chromatographic and ultraviolet spectral properties and luciferase induction activities. Further, quantitative structure–activity (property) relationship (QSAR or QSPR) models were developed through statistical analysis (multilinear regression) of datasets of experimental properties and datasets of computed electronic parameters. The study examines 48 BDE congeners (Table 1) and two thyroid hormone analogues, ‘‘CH3-T4’’ and ‘‘CH3-T3’’ (Fig. 2).
2. Methods The semiempirical calculations in vacuum using an AM1 Hamiltonian (Dewar et al., 1985) with the Mopac package (public domain version 6.00 by Coolidge and Stewart, 1990) were carried out on a Digital Unix/ DEC-3000/600 AlphaStation and several personal computers with Intel · 86 architecture processors. The C–C ˚ , C–O bond lengths bond lengths were started at 1.40 A ˚ ˚ , and C–Br bond at 1.39 A, C–H bond lengths at 1.10 A ˚ . The geometry optimizations were lengths at 1.87 A achieved applying the Broyden–Fletcher–Goldfarb– Shanno (BFGS) quasi-Newton method, which is incorporated into the Mopac program. All calculations are based on the closed-shell RHF-SCF-MO method except in the cases where open-shell UHF-SCF-MO computations were performed to calculate heats of formation for cation radicals of BDEs (Table 3), which were used for the prediction of fragmentation patterns in the mass spectra. For all factor analysis and regression analysis, SPSS software was used in this study. The relative retention times (RRTs) of the 19 BDEs (Table 1) were determined on a Varian 3400 gas chro-
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Table 1 Structures, maximum luciferase induction activities, GC RRTs and wavelengths of maximum UV absorption of 48 BDEs BDE no.
Structure (bromination)
Luciferase induction (%)a
RRT1b
RRT2
RRT3
RRT4
RRT5
CPSil-8
HP-1701
SP-2380
SB-Smectic
XTI-5
1 2 3 7 8 10 12 13 15 17 25 28 30 32 33 35 37 45 46 47 49 50 51 53 54 66 71 75 77 85 99 100 104 116 119 128 138 140 153 154 155 166 181 183 186 190 204 209
2 3 4 2,4 2,4 0 2,6 3,4 3,4 0 4,4 0 2,2 0 ,4 2,3 0 ,4 2,4,4 0 2,4,6 2,4 0 ,6 2 0 ,3,4 3,3 0 ,4 3,4,4 0 2,2 0 ,3,6 2,2 0 ,3,6 0 2,2 0 ,4,4 0 2,2 0 ,4,5 0 2,2 0 ,4,6 2,2 0 ,4,6 0 2,2 0 ,5,6 0 2,2 0 ,6,6 0 2,3 0 ,4,4 0 2,3 0 ,4 0 ,6 2,4,4 0 ,6 3,3 0 ,4,4 0 2,2 0 ,3,4,4 0 2,2 0 ,4,4 0 ,5 2,2 0 ,4,4 0 ,6 2,2 0 ,4,6,6 0 2,3,4,5,6 2,3 0 ,4,4 0 ,6 2,2 0 ,3,3 0 ,4 2,2 0 ,3,4,4 0 ,5 0 2,2 0 ,3,4,4 0 ,6 0 2,2 0 ,4,4 0 ,5,5 0 2,2 0 ,4,4 0 ,5,6 0 2,2 0 ,4,4 0 ,6,6 0 2,3,4,4 0 ,5,6 2,2 0 ,3,4,4 0 ,5,6 2,2 0 ,3,4,4 0 ,5 0 ,6 2,2 0 ,3,,4,5,6,6 0 2,3,3 0 ,4,4 0 ,5,6 2,2 0 ,3,,4,4 0 ,5,6,6 0 Deca-
– – – – – – – – 7.5 – – 8.4 9.6 20.7 – – – – – 3.3 – – 10.1 – – – 6.3 13.2 12.8 28.3 24.1 8.0 – – 26.9 – 9.7 – 34.2 – – 65.8 – – – 50.3 – –
– – – 0.400 0.411 – 0.415 0.415 0.424 0.506 0.506 0.519 0.468 0.496 0.520 0.528 0.540 – – 0.627 – – 0.605 – – 0.641 0.614 0.600 0.667 0.787 0.735 0.711 – 0.750 0.719 – 0.944 – 0.864 – – – – 1.061 – – – –
– – – 0.385 0.400 – 0.400 0.402 0.412 0.496 0.493 0.508 0.447 0.487 0.510 0.516 0.529 – – 0.612 – – 0.594 – – 0.628 0.602 0.580 0.654 0.794 0.720 0.690 – 0.732 0.699 – 0.986 – 0.869 – – – – 1.107 – – – –
– – – 0.474 0.505 – 0.494 0.500 0.530 0.629 0.616 0.651 0.529 0.609 0.642 0.650 0.677 – – 0.797 – – 0.766 – – 0.838 0.767 0.723 0.886 1.128 0.953 0.884 – 0.976 0.897 – 1.494 – 1.182 – – – – 1.546 – – – –
– – – 0.437 0.460 – 0.420 0.477 0.530 0.598 0.597 0.663 0.494 0.567 0.622 0.655 0.765 – – 0.882 – – 0.785 – – 0.941 0.792 0.748 1.076 1.407 1.109 1.023 – 1.161 1.048 – 1.646 – 1.354 – – – – 1.673 – – – –
0.806 0.815 0.831 1.031 1.055 0.988 – 1.070 1.084 1.241 1.241 1.267 1.171 1.226 – – – – – 1.433 1.400 – 1.394 – – – – – – – – 1.555 – 1.610 – – – – – – – 1.823 – – – – – –
UV (kmax, nm)c 200.0 200.0 200.0 205.2 202.0 205.0 204.8 200.0 203.2 203.2 203.2 201.4 210.4 204.6 203.8 207.4 203.4 – – 204.4 – – 211.8 – – 205.8 205.4 210.8 202.6 – – 210.6 – 225.6 209.2 – – 213.2 – 211.4 – 226.4 227.6 – – 226.8 – –
a Maximum luciferase induction cited from Meerts et al. (1998a). Maximal luciferase induction (100%) was obtained with 100 pM TCDD in a recombinant H4IIE rat hepatoma cell line. b RRT1 (low-polar column), RRT2 (medium polar column), RRT3 (very polar column) and RRT4 (medium polar column) are cited from Sjo¨din et al. (1998). RRT5 were determined in the present work. c Cited from Marsh et al. (1999).
matograph equipped with XTI-5 fused silica capillary column (30 m · 0.25 mm, 0.25 lm film (Restek) thick-
ness) and quadrupole TSQ 700 Finnigan MAT MSD (electron ionization at 70 eV and ion source temperature
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in press), the BDEs with intraring C2 symmetry axis from C1 to C4 and C1 0 to C4 0 , i.e., BDE-3, 10, 15, 30, 32, 54, 75, 104, 116, 155, 186, 204 and 209 and ‘‘CH3-T4’’, have only single energy minimum, similarly to BDE-166. For BDE-1, 2, 7, 8, 12, 13, 28, 37, 46, 50, 53, 71, 100, 119, 140, 154, 181 and 190 and ‘‘CH3-T3’’, there are two different energy minima as for BDE-51. For BDE-47, 77 and 153, three energy minima were identified for each congener. And for BDE-17, 25, 33, 35, 45, 49, 66, 85, 99, 128, 138 and 183, four energy minima were obtained. These results are compatible with those of AM1 studies on PCDEs (Nevalainen and Rissanen, 1994). For clarity, only the parameters of the global minima (lowest energy conformations) are given for the 51 diphenyl ethers including 48 BDEs listed in Table 2. On the basis of the ortho-substitution patterns the following three types of global minima (conformation) were identified: (i) twist type for the eight BDEs with no bromine substituents at the ortho-positions (namely BDE-2, 3, 12, 13, 15, 35, 37, and 77), which are more or less similar to diphenyl ether; (ii) skew type for the seven congeners with 2,6-di-ortho-substitution (namely BDE-10, 30, 32, 75, 116, 166, and 190), in which one phenyl ring is close to perpendicular to the C–O–C central plane and the other lies approximately in the plane; (iii) another twist type for the remaining 33 congeners, in which u1 and u2 generally differ from each other. 3.1. Dipole moments, polarizabilities and QSPR models for predicting gas chromatographic properties
Fig. 2. (a) Thyroxine (T4); (b) 3,3 0 ,5-triiodothyronine (T3); (c) ‘‘CH3-T4’’ and (d) ‘‘CH3-T3’’.
140). The temperature program for the GC column was 100 C (1 min); 10 C/min (22 min); 320 C (22 min). Helium was used as the carrier gas and the temperature of injector was 280 C. The relative retention times were referenced to PCB-53.
3. Results and discussion Conformations of diphenyl ethers are described by the torsional angles (u1 and u2) between the C–O–C plane and planes of the phenyl rings. The angles are defined as positive when the rotation is clockwise looking down the C4–C1 and C4 0 –C1 0 axes toward the oxygen (Fig. 1). According to conformational analysis of PBDEs based on calculated contour energy maps (Hu et al.,
The dipole moment (l) of a molecule reflects its structural symmetry. Calculated dipole moments of the lowest energy conformation of the BDEs studied vary widely, from around zero to 4.16 D (Table 2). Values are 1.83 D for 4-bromodiphenyl ether (BDE-3) and 0.04 D for 4,4 0 -dibromodiphenyl ether (BDE-15). The latter is the smallest value calculated and indicates the high structural symmetry of BDE-15. The previously determined experimental values are 1.57 D and 0.60 D for BDE-3 and BDE-15 respectively (Anderson and Smyth, 1965). The fairly wide discrepancy between the theoretical and experimental values may be ascribed to the conformational freedom of these two non-orthocongeners. Their structures oscillate widely about the energy minima and the moments calculated for single minima do not necessarily well reflect their time-averaged values. In addition, different energy minima always give different values of dipole moments. To illustrate this, Table 3 presents the dipole moments, l (along with other electronic properties), of different minima of several congeners. The results confirm that the calculated moments of BDEs are not a reliable descriptor for their physicochemical properties. Molecular polarizability measures the ability of the electrons in a molecule to move in response to an exter-
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Table 2 AM1 calculated electronic properties of 51 diphenyl ethers including 48 BDEs BDE no.
DH (kJ/mol)
EHOMO (eV)
ELUMO (eV)
DE (eV)
l (Debye)
˚ 3) a (A
u1()
u2()
Diphenyl ether 1 2 3 7 8 10 12 13 15 17 25 28 30 32 33 35 37 45 46 47 49 50 51 53 54 66 71 75 77 85 99 100 104 116 119 128 138 140 153 154 155 166 181 183 186 190 204 209 ‘‘CH3-T3’’ ‘‘CH4-T4’’
98.521 131.624 120.796 118.621 154.959 151.168 167.168 143.921 141.607 139.808 189.878 177.109 175.343 192.807 186.996 175.987 165.594 166.988 228.204 226.672 211.141 212.275 226.974 227.066 224.593 242.157 200.497 211.819 213.208 191.757 239.588 237.496 252.471 267.040 252.651 238.492 268.337 266.328 278.353 264.893 276.257 292.474 273.362 310.265 305.972 327.105 299.173 353.038 414.216 132.114 211.510
8.954 9.139 9.139 9.066 9.265 9.183 9.187 9.221 9.219 9.173 9.350 9.441 9.301 9.304 9.216 9.335 9.301 9.345 9.387 9.464 9.416 9.484 9.414 9.415 9.410 9.470 9.435 9.360 9.326 9.426 9.522 9.527 9.518 9.537 9.445 9.463 9.581 9.585 9.567 9.599 9.549 9.613 9.436 9.569 9.596 9.694 9.561 9.746 9.872 8.961 9.135
0.171 0.200 0.127 0.136 0.545 0.344 0.429 0.400 0.343 0.403 0.443 0.646 0.694 0.794 0.561 0.418 0.613 0.532 0.839 0.594 0.765 0.716 0.849 0.529 0.595 0.423 0.760 0.631 0.924 0.762 0.839 0.850 0.801 0.711 1.197 0.990 0.990 0.991 1.047 1.104 1.050 0.900 1.308 1.348 1.255 1.123 1.349 1.217 1.343 0.484 0.580
9.125 8.939 9.012 8.930 8.719 8.839 8.758 8.821 8.876 8.770 8.907 8.795 8.607 8.510 8.655 8.917 8.688 8.813 8.448 8.870 8.651 8.768 8.565 8.886 8.815 9.047 8.675 8.729 8.402 8.664 8.683 8.677 8.717 8.826 8.248 8.473 8.591 8.594 8.520 8.495 8.499 8.713 8.128 8.221 8.341 8.571 8.212 8.529 8.529 8.477 8.555
1.245 1.889 2.361 1.831 1.317 2.695 2.111 2.996 1.438 0.036 1.427 0.730 1.316 1.290 2.876 2.569 1.230 2.464 2.856 4.163 2.028 1.064 2.520 1.906 2.287 2.200 1.448 2.668 1.420 1.195 2.820 1.378 0.779 2.201 1.549 1.392 2.800 1.588 3.000 0.629 1.574 1.154 0.531 1.580 1.246 2.671 0.785 1.232 0.635 1.488 2.421
16.602 17.788 18.325 18.385 19.609 19.552 19.276 20.045 20.135 20.238 20.903 21.083 21.419 21.150 21.032 21.008 21.936 21.570 22.484 22.453 23.013 22.779 22.686 22.442 22.443 22.128 22.854 22.457 22.947 23.650 24.635 24.585 24.376 24.022 24.480 24.373 26.138 26.058 26.164 26.357 26.134 25.964 26.318 27.906 27.777 27.334 27.746 29.326 32.715 24.667 27.002
37.0 108.1 34.7 26.1 113.0 104.9 87.6 25.0 44.8 36.4 135.6 110.4 108.5 95.2 93.5 173.9 36.1 28.9 77.6 24.3 25.8 71.3 77.7 139.8 20.3 55.0 110.9 96.7 94.0 38.3 156.0 156.8 131.5 53.2 83.9 93.0 72.2 73.5 159.0 72.9 164.7 54.7 92.4 80.6 82.6 56.3 93.8 57.2 55.0 89.7 87.7
37.1 19.7 40.0 47.6 25.0 14.9 9.3 49.7 29.5 36.5 55.1 23.8 18.6 2.5 0.1 92.0 37.3 45.0 25.9 79.2 73.0 153.5 25.9 60.4 81.3 54.8 20.7 4.4 1.2 148.7 72.0 74.1 51.1 55.9 15.2 0.8 156.9 156.4 78.6 155.7 104.8 53.6 1.7 161.2 164.9 54.3 0.6 52.4 55.4 6.4 10.3
nal electric field. As shown in Fig. 3, the calculated average molecular polarizabilities (a) (Table 2) correlate significantly with the relative retention times (RRTs) in GC (Table 1) determined in the present work for a XTI-5
column (non-polar) (R2 = 0.987) and in earlier work for four columns of different polarity (R2 = 0.973 for CPSil-8, 0.953 for HP-1701, 0.912 for SP-2380 and 0.932 for SB-Smectic) (Sjo¨din et al., 1998). Correlation
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Table 3 AM1 calculated electronic properties for different minima of seven diphenyl ethers BDE no.
DHf (kJ/mol)
EHOMO (eV)
ELUMO (eV)
l (Debye)
˚ 3) a (A
u1()
u2()
99
237.496 238.266 239.781 268.337 270.341 266.328 266.889 268.475 264.893 266.680 276.257 279.274 305.972 308.838 132.114 132.938
9.527 9.507 9.547 9.581 9.593 9.585 9.609 9.168 9.599 9.650 9.549 9.626 9.596 9.678 8.961 8.984
0.850 1.047 0.881 0.990 0.866 0.991 1.112 0.952 1.104 1.004 1.050 0.951 1.255 1.099 0.484 0.516
1.378 1.476 1.053 2.800 1.640 1.588 2.168 1.456 0.629 0.427 1.574 1.212 1.246 0.899 1.488 4.818
24.585 24.779 24.567 26.138 25.698 26.058 26.430 26.042 26.357 26.395 26.134 26.175 27.777 27.790 24.667 24.976
156.8 73.0 53.5 72.2 53.7 73.5 155.4 53.8 72.9 53.1 164.7 49.3 82.6 59.8 89.7 102.4
74.1 155.8 52.9 156.9 53.3 156.4 73.0 52.6 155.7 53.9 104.8 58.1 164.9 47.7 6.4 166.8
128 138
153 154 183 ‘‘CH3-T3’’
of the polarizabilities was best with the RRTs determined in the low polarity column (CPSil-8) (R2 = 0.976), compatible with the result for our non-polar XTI-5 column. These models are basically consistent with the multivariate QSPR models developed recently by Harju et al. (2002) on the same data based on 40 physicochemical descriptors, which give R2 value 0.991, 0.984, 0.985 and 0.977 for these columns, respectively. Our results are reasonable since the dispersion forces, which are closely related to a, should be dominant in the interactions of non-polar solute molecules with non-polar liquid phase molecules. The correlations are evidently poorer for the other three columns with higher polarity, because some other forces, such as the dipoleinduced–dipole-interaction, may play a more important role in those intermolecular interactions. It is thus apparent from these results that the average molecular polarizability is a useful descriptor for predicting such molecular properties as lipophilicity (LgKow) of these compounds. It is expected that LgKow increases with a within a certain rang for BDEs, however, this rule ceases to hold at high molecular weights, for the highly brominated BDEs are neither lipophilic nor hydrophilic. This is in principle consistent with an optimal PLS model developed by Chen et al. (2003a) for predicting LgKoa (octanol–air partition coefficient) of PBDEs based on 18 theoretical molecular structure descriptors, which gives the cross validated Q2 cum value 0.975, showing high predictive power. 3.2. Heats of formation and relation to thermodynamic stability and mass spectral characteristics AM1 calculations give heats of formation (DHf) as a measure of the relative thermodynamic stabilities of
compounds, which are only comparable, however, when molecules are of equivalent atomic composition (isometric). As can be seen in Table 2, DHf of the BDEs increases with the number of bromine substituents. With equivalent bromination degree, DHf increases with the number of ortho-bromine substituents, which means that thermodynamic stability is higher for BDEs with fewer ortho-substituents. For example, BDE-77 (with no ortho-substitution) is 50.4 kJ/mol more stable than BDE-54 (with tetra-ortho-substitution). The other tetraortho-congeners such as BDE-104, 155 and 186 were also calculated to have distinctly high heats of formation. The higher energy of these congeners apparently comes from the stronger ortho-interaction. The extreme example is decabromodiphenyl ether, another congener with tetra-ortho-substitution, for which an exceptionally high heat of formation (414.2 kJ/mol) is calculated. This theoretical result indicates a relatively low thermodynamic stability for decaBDE and conforms to its widespread use as a flame retardant. Flame retardants should easily decompose so as to interrupt the radical chain reactions taking place in combustion. The easy decomposition of decaBDE, in addition to its strong absorption to particles (sediments and soils), may also partly explain why it is relatively rarely encountered in the environment despite its large-scale use. These findings are further compatible with a wellknown characteristic of mass spectra of PBDEs (Marsh et al., 1999) as well as PCDEs (Nevalainen et al., 1994; Nevalainen, 1995). Namely, the ortho-congeners generally give rise to (M-2Br)+ or (M-2Cl)+ fragments as base peaks rather than to the molecular ions that generally appear as the base peaks of non-ortho-congeners. Perhaps, the molecular ions of the ortho-congeners are less stable than those of the non-ortho-congeners in parallel
J. Hu et al. / Chemosphere 59 (2005) 1043–1057
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Fig. 3. Correlations between average molecular polarizabilities and RRTs of BDEs for five GC columns.
with the stabilities of their parent compounds. To test this postulation, heats of formation were calculated for the molecular ions of six dibrominated and eight tetrabrominated congeners. For the dibrominated congeners, the molecular ions of the ortho-substituted congeners BDE-7, 8 and 10 were found to have considerably higher energies (average DHf = 959.0 kJ/mol) than those of the non-ortho-congeners BDE-12, 13 and 15 (average DHf = 950.7 kJ/mol). For the tetrabrominated congeners, the heats of formation for the molecular ions of tetra-ortho congener BDE-54 (DHf = 1067.3 kJ/mol) and tri-ortho BDE-45, 46, 50, 51 and 53 (average DHf = 1045.7 kJ/mol) are all clearly higher than those for diortho BDE-75 (DHf = 1028.6 kJ/mol) and non-ortho BDE-77 (DHf = 1019.7 kJ/mol); the energy difference between the molecular ions of BDE-54 and 77 is as high as 47.5 kJ/mol. These results may also help explain mechanism and kinetics of free radical reactions of these compounds, such as reaction with OH radicals in air. 3.3. Frontier orbital energies and their relevance to kinetics of electrophilic and nucleophilic reactions Having seen above the low total thermodynamic stability of PBDEs relative to their chlorinated analogues, we can reasonably assume, even without examining free energy changes of respective metabolism reactions, that PBDEs should at least have a similar tendency to their
chlorinated analogues to undergo well-known metabolic reactions. What we are concerned with here, therefore, is not the thermodynamics but the kinetics of the reactions. A major metabolic pathway of halogenated aromatics, such as PCBs, has been postulated to proceed through an arene oxide intermediate by oxidation under catalysis of a cytochrome P-450 (CYP). The intermediate is then attacked by water to generate the hydroxylated compounds and some relevant conjugates, and by a glutathione under catalysis of glutathione S-transferase (GST) to generate mercapturic conjugates and ultimately methyl sulfonyl metabolites (Hutzinger et al., 1974; Safe, 1994). This mechanism should also be applicable to PBDEs. Frontier orbital energies were found to correlate with the kinetics of enzyme catalyzed reactions of several classes of halogenated aromatic compounds (Vervoort et al., 1992; Cnubben et al., 1994; Rietjies et al., 1995). It is not clear to what degree the chemical reactivity determines the metabolism in the case of BDEs, however, since other factors such as lipophilicity and steric factors may also play important roles. According to our AM1 calculations, the energies of the lowest unoccupied molecular orbital (ELUMO) and the energies of the highest occupied molecular orbital (EHOMO) of the BDEs tend to decrease with an increasing number of bromine substituents (Table 2). Following the frontier orbital theory, the higher brominated diphenyl ethers
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should be less reactive towards electrophiles such as CYPs, but more reactive towards nucleophiles. These results are consistent with the basic chemical rule that the chlorination or bromination of organic molecules will deactivate them for further electrophilic reactions. Recently, in an in vitro bioassay with hepatic microsomes from fish, 4,4 0 -dibromobiphenyl (PBB-15) was found to be subjected to CYP oxidation, while the other higher PBB and PBDE congeners showed no indication of this biotransformation (de Boer et al., 1998). Also, 2,2 0 ,4,4 0 tetrabromodiphenyl ether (BDE-47) was shown to ¨ rn slowly metabolize through hydroxylation in rats (O and Klasson-Wehler, 1998). Reactivity appeared to be decisive for the rate of metabolism of these brominated aromatics. However, LgKow and size of these compounds, which are closely related to EHOMO, can also be important factors since the two properties might give differences in their affinity to CYPs. Comparing our data to those of PCDDs, PCDFs, PCBs and PCDEs, we see that the HOMO energies of PCBs, PCDFs and PCDEs are similar to each other, while those of PCDDs are considerably higher. The LUMO energies are similar for all compounds (Nevalainen and Kolehmainen, 1994; Koester and Hites, 1988). These findings may indicate that PCDDs are generally more reactive towards electrophiles such as CYP, albeit this sort of comparison is not theoretically justified. An experiment with rainbow trouts did give much shorter
biological half lives for PCDDs than for PCBs and PCDEs (Niimi, 1986). 3.4. Frontier orbital energy gaps (dE), QSPR model for predicting ultraviolet (UV) spectral properties, and photolytic reactions The energy gaps (dE)(Table 2) between LUMO and HOMO of BDEs, which are calculated from ELUMO and EHOMO, correlate adequately (R2 = 0.734) with the wavelengths at maximum UV absorbance (kmax) of these compounds (Marsh et al., 1999) (Fig. 4). It was reported that dE relates to or correlates with phototoxicity and kinetics of photolysis for polycyclic aromatic hydrocarbons (PAHs) (Mekenyan et al., 1994; Chen et al., 1996) and with the receptor binding affinities of PCDDs, PCDFs and PCDEs (Nevalainen and Kolehmainen, 1994). As a general trend, dE of the BDEs decreases with increasing degree of bromination, which means that an electronic transition from the HOMO (ground state) to LUMO (excited state) in the higher BDEs proceeds more easily with increasing bromination. Reactions such as photolysis and formation of PBDFs may take place as one of possible route for energy decay of BDE molecules excited through the absorption of solar energy. In the higher BDEs, moreover, there are plenty of comparatively labile Br–C bonds that are considerably weaker than the corre-
Fig. 4. Correlation between frontier energy gaps and wavelengths at maximum UV absorbance of BDEs.
J. Hu et al. / Chemosphere 59 (2005) 1043–1057
sponding C–Cl bonds. Cleavage of such Br–C bonds is expected to be an easier route for energy decay than corresponding cleavage in their chlorinated analogues. The labiality of C–Br bonds could account for experimental results in which decaBDE was transformed to lower BDE congeners in the different matrices under UV radiation or sunlight (Watanabe et al., 1986; So¨derstro¨m et al., 2004). That, in addition to factors such as rather large molecular size and weight, may be an important reason why deca- and other higher BDEs are so rarely bioavailable in the environment. In addition to its connection to UV spectra, according to Pearson (1986), dE also indicates hardness of molecule. The tetra-ortho-congeners such as BDE-54, 104, 155 and 186 were calculated to give clearly higher dE values (Table 2) than the corresponding congeners with tri-, di- and non-ortho-substituents, probably indicating restricted rotation of the phenyl groups around the ether bond. The restricted rotation prevents effective delocalisation of p electrons on the phenyl groups and thus increases energy gap (dE) between LUMO and HOMO. These molecules also gave relatively low molecular polarizabilities, because molecules with high hardness are not easy to be polarized. Contrarily, the di-orthoand tri-ortho-congeners show no clear differences from mono- and non-ortho-congeners and may thus have no rotational restriction in these molecules. These results are consistent with those from our conformational analysis (Hu et al., in press).
3.5. QSAR models for predicting luciferase induction activity We set up three QSAR models for calculating the luciferase induction activity (I) of BDEs. These models show that the induction activity increases with increasing electrophilicity or electron affinity (ELUMO), with decreasing hardness (dE), or with increasing average molecular polarizability (a) Model 1 I ¼ 0:528ð0:106ÞELUMO 0:244ð0:093Þ n = 17, R2 = 0.625, SE = 0.108, F = 24.991, p = 0.000 Model 2 I ¼ 0:693ð0:139ÞdE þ 6:147ð1:195Þ n = 17, R2 = 0.623, SE = 0.108, F = 24.761, p = 0.000 Model 3 I ¼ 0:055ð0:015Þa 1:107ð0:353Þ n = 17, R2 = 0.479, SE = 0.127, F = 13.767, p = 0.002 Here R2 is the square of correlation coefficient, S.E. is the standard error of estimate, F is the F value for analysis of variance and p is the significance level.
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The models were obtained by a linear regression using as the dependent a data set of the measured luciferase induction activity values of 17 BDEs, obtained in a recombinant H4IIE rat hepatoma cell bioassay (Meerts et al., 1998a). A plot of the measured vs calculated (Model 1) luciferase induction activities is presented in Fig. 5. Since, in general, the independent variables themselves such as ELUMO, EHOMO, dE and a are strongly inter-correlated, probably owing to the similar dependence on the molecular wave function (Table 4), only one-parameter equations are possible if without special treatment of data such as application of factor and PLS analysis. Although these models are far from complete, a general trend is apparent: ELUMO or dE and a are major determinants for the luciferase induction activity (ELUMO and dE are closely related). These results are fairly compatible with the findings for related organochloro compounds (Albro and McKinney, 1981; Lewis et al., 1989, 1993; Nevalainen and Kolehmainen, 1994). These models indicate that higher BDEs generally have higher activities. The models also show that tetra-orthocongeners, with relatively high dE value and low a values owing to restricted rotation, have relatively low activities, while other congeners with non-, di- and tri-orthosubstituents show no clear differences in the activity. Harju et al. (2002) recently developed a multivariate (PLS) model (R2 = 0.618) using 40 descriptors to predict the luciferase activity for PBDEs. In the present study, a kind of factor analysis, principle component analysis (PCA) (Tysklind et al., 1992; Chen et al., 1998), was performed to extract orthogonal principal components (PCs). PLS and PCA are both projection methods, and PLS make projections of two related matrixes of descriptors and responses simultaneously while PCA only makes projection of matrix of descriptors. The descriptors are EHOMO (eh), ELUMO (el), average molecular polarizability (pl), dipole moment (dp) and energy gap (de) between LUMO and HOMO. Two significant PCs, which explain 65% and 22% of the variance respectively, were extracted and their loadings plot is shown in Fig. 6. PC1 is dependent on the size-related parameters EHOMO, ELUMO, dE and a, while PC2 is mainly dependent on dipole moment, a structure/symmetry parameter. Using stepwise multiple linear regressions, Model 4 for calculating the luciferase induction activity (I) was established based on the resulting principle factor scores (Fa1 and Fa2) for individual BDE compounds. In this model, Fa1 is only the significant variable (p = 0.000) since less important Fa2 (p = 0.343) was excluded in the stepwise regressions. This model is consistent with Models 1–3 and with our result in Chapter 3.1 that dipole moment is not a good descriptor for properties of PBDEs. The present models and previous result by Harju et al. (2002) have all shown that physicochemical parameters of the BDEs can only account for
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Fig. 5. Observed vs calculated luciferase induction activities of BDEs in H4IIE rat hepatoma cell line.
Table 4 Correlation matrix of independent variables (calculated from 17 cases)
Induc (I) EHOMO ELUMO DE Polar (a) Dipole (l)
Induc (I)
EHOMO
ELUMO
DE
Polar (a)
Dipole (l)
1.000 0.386 0.791 0.789 0.692 0.287
1.000 0.674 0.237 0.897 0.093
1.000 0.877 0.887 0.340
1.000 0.584 0.386
1.000 0.248
1.000
approximately 60% variation of the luciferase induction activity. That may mean that this biochemical process may involve complex mechanisms and more tests are therefore needed to clarify them. In addition, uncertainty in luciferase bioassay may also give a significant contribution to the uncertainty of the model. Model 4 I ¼ 0:130ð0:029ÞFa1 þ 0:200ð0:028Þ 2
n = 17, R = 0.579, SE = 0.114, F = 20.589, p = 0.000 Further efforts were also made to improve significance of the models. From Fig. 5, BDE-15, 32 and 138 appeared to be outliers. If these three outliers were excluded, all four models were improved in terms of R2 values (R2 for Model 1 = 0.803, R2 for Model
2 = 0.625, R2 for Model 3 = 0.699 and R2 for Model 4 = 0.768). These new models are however not justified because we cannot interpret those outliers based on the information presently available. 3.6. Frontier electron densities and regioselectivity of electrophilic and nucleophilic reactions A study on regioselectivity (orientation) of hydroxylation of BDEs under the catalysis of a CYP could be expected to be useful in elucidating their toxic effects. The regioselectivity can be predicted from familiar chemical principles or by examining frontier orbital densities. In general, an enzymatic hydroxylation of BDEs may take place on the phenyl ring with lower halogenation because it is less deactivated by the halogen substituents; a GST, on the other hand, may react with the ring with
J. Hu et al. / Chemosphere 59 (2005) 1043–1057
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Fig. 6. Component plot.
higher halogenation. This is in agreement with the result of a previous metabolic study with PCDEs, in which both 4-chloro- and 2,4-dichlorodiphenyl ether were hydroxylated at 4 0 -position (Becker et al., 1991). Examination of the frontier electron densities may give a quantitative prediction of the regioselectivity of enzymatic reactions. Our calculations indicate that the distribution pattern of frontier orbital densities of BDEs having a skew conformation is rather different from that of BDEs having a twist conformation. According to the equation given by Fukui et al. (1954), the frontier electron densities can be calculated from HOMO and HOMO-1 characteristics and LUMO and LUMO + 1 characteristics. However, we found that the energy gaps between the LUMO + 1 and LUMO in some cases are too small for the equation to give reasonable results. Following Poso et al. (1993), we therefore consider only HOMO and LUMO in calculating the frontier electron densities (Table 5). Variation in electronic properties of the phenyl rings with change in conformation of PCDEs was well recognized through a large number of NMR experiments (Hu et al., 1994; Nevalainen, 1995). The present calculations in fact show another direct proof for this variation. In the molecules with skew conformation, the HOMO orbital densities are almost completely distributed over the oxygen and carbon atoms of the phenyl ring that is coplanar with the C–O–C plane (u1 = 0). The LUMO density, on the other hand, is distributed almost totally
over the carbons of the other phenyl ring that is perpendicular to the C–O–C plane (u2 = 90). This may indicate that electrophilic attacks, for example by CYP, would preferably take place on the carbons of the phenyl ring coplanar with the C–O–C plane, whereas nucleophilic attacks would be favored on the carbons of the other phenyl ring (Rietjens et al., 1993). In molecules with twist conformation, the frontier orbital densities are more uniformly distributed and their reactivity is difficult to predict. On the whole, it appears that the congeners with skew conformation may be more reactive than the corresponding congeners with twist conformation owing to their highly non-uniform frontier electron distribution. These findings are also applicable to ‘‘CH3-T3’’ and ‘‘CH4-T4’’, which have a skew conformation. It needs to be emphasized that the above predictions of the reactivity of BDEs were made on the basis of frontier orbital characteristics alone, and should be considered only as a tentative indication of the orientation of reactions associated with these compounds. The coulombic characteristics of BDEs and the steric effects arising from the interaction between BDEs and other reactants may greatly affect their reactivity as well. 3.7. Thyroid hormone analogues It is suspected that BDEs may have thyroid hormone-like activity since thyroid hormones, e.g. T3 and
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Table 5 Frontier electron densities and atomic charges on carbons and oxygen of five diphenyl ethers with a skew conformation BDE no.
C1
C2
C3
C4
C5
C6
O
C1 0
C2 0
C3 0
C4 0
C5 0
C6 0
10
HD LD Q
0.00 0.03 0.10
0.00 0.56 0.18
0.00 0.41 0.09
0.00 0.02 0.13
0.00 0.62 0.09
0.00 0.30 0.17
0.26 0.00 0.15
0.48 0.00 0.07
0.20 0.00 0.18
0.13 0.00 0.10
0.55 0.00 0.16
0.07 0.00 0.10
0.26 0.00 0.14
30
HD LD Q
0.00 0.69 0.11
0.00 0.17 0.18
0.00 0.15 0.07
0.00 0.55 0.18
0.00 0.14 0.07
0.00 0.18 0.18
0.26 0.00 0.15
0.48 0.03 0.07
0.19 0.00 0.18
0.14 0.00 0.10
0.56 0.00 0.16
0.07 0.00 0.10
0.26 0.00 0.14
75
HD LD Q
0.00 0.69 0.10
0.00 0.17 0.18
0.00 0.15 0.07
0.00 0.55 0.17
0.00 0.13 0.07
0.00 0.18 0.18
0.21 0.00 0.15
0.39 0.03 0.09
0.15 0.00 0.19
0.14 0.00 0.07
0.49 0.00 0.20
0.09 0.00 0.08
0.18 0.00 0.14
‘‘T3’’
HD LD Q
0.00 0.69 0.10
0.00 0.17 0.27
0.00 0.13 0.08
0.00 0.54 0.07
0.00 0.09 0.08
0.00 0.23 0.27
0.19 0.00 0.15
0.42 0.03 0.04
0.08 0.00 0.12
0.24 0.00 0.26
0.41 0.00 0.09
0.11 0.00 0.19
0.18 0.00 0.08
‘‘T4’’
HD LD Q
0.00 0.67 0.09
0.00 0.17 0.27
0.00 0.12 0.08
0.00 0.52 0.07
0.00 0.08 0.08
0.00 0.22 0.27
0.19 0.00 0.15
0.41 0.08 0.03
0.08 0.00 0.10
0.22 0.00 0.27
0.38 0.00 0.13
0.13 0.00 0.33
0.18 0.00 0.06
HD: electron density of HOMO. The summation proceeded over the atomic orbitals (s, px, py, pz) according to Poso et al. (1993); LD: electron density of LUMO; Q: atomic charge; ‘‘T3’’: ‘‘CH3-T3’’; ‘‘T4’’: ‘‘CH3-T4’’.
T4, include iodated diphenyl ethers as their hydrophobic moiety. It was of interest to study the conformational and electronic properties of T3 and T4 as well, and compare them with those of BDEs. The AM1 calculations were performed for two molecules in which the thyronine moiety of T3 and T4 was exchanged for a methyl group (‘‘CH3-T4’’ and ‘‘CH3-T3’’). Both molecules were found to have lowest energy conformations of skew type similar to those of the corresponding BDEs and the frontier orbital energies are comparable as well (Table 2). These comparisons have shown a good resemblance between T4/T3 analogues and the corresponding BDEs as far as conformational properties and reactivity are concerned. Experimental results showed that substances such as hydroxylated PCBs and pentachlorophenol can strongly bind to transthyretin (TTR), one of the thyroid hormone transport proteins (De la Paz et al., 1992; Lans et al., 1993). Other results indicated that the rat microsomal formation of BDE metabolites is of high affinity to TTR (Murk, Personal communication; Meerts et al., 1998b). There was evidence to suggest, moreover, that BDEs can be metabolized to their hydroxylated ana¨ rn and Klasson-Wehler, 1998). It looks likely, logues (O therefore, that BDEs impose toxic effects on animals through the formation of hydroxylated metabolites that then bind to TTR and other proteins. In an attempt at clarification, some hydroxy-BDEs were synthesized and indeed found to bind to the thyroid receptors TR-a1 and TR-b (Marsh et al., 1998). Our theoretical calculations are in reasonable agreement with these preliminary experimental results.
4. Conclusions The reactivity and lipophilicity of PBDEs were elucidated on the basis of AM1-calculated electronic structures and satisfactory QSAR and QSPR models were derived. The results can provide a solid theoretical basis for assessing the impact of PBDEs on the environment. In general, the structures of BDE metabolites are difficult to determine in metabolic assay: they are present in only trace amount in complex mixtures and the required model compounds are frequently lacking. Calculated frontier orbital characteristics allow us to predict which structures are of importance in metabolic reactions and hence give a priori direction for metabolic assays. According to Di-region theory (Dai, 2000; Dai et al., 2002, 2003), metabolic reactions may activate some aromatic compounds to become dangerous carcinogens binding to DNA. From that perspective, the present investigation can be considered to further our understanding of carcinogenisis of aromatic compounds and may assist the development of novel anticancer drugs (such as therapeutic agents that selectively damage cancer cell DNA by crosslinking DNA duplex) (Wei, 1997; Dai, 2000). The calculated average molecular polarizabilities of BDEs, shown here to be valid descriptors for the QSPR models of gas chromatographic properties, might also be useful in modeling the accumulation, transportation and distribution of these compounds in the environment (Mackay, 1991; Southwood et al., 1998; Palm et al., 2002). Normally, before such modeling can be undertaken, large amount of data such as partition coeffi-
J. Hu et al. / Chemosphere 59 (2005) 1043–1057
cients, solubility and fugacity need to be determined experimentally and this procedure is usually very time consuming. Establishment of relevant QSPR models by the present methods offer more economical way to obtain partition data and can thereby facilitate the application of the environmental modeling to PBDEs. Because of complexity of the natural environment and the limitations of the AM1 method, it is unrealistic to think that these semiempirical calculations could account for everything occurring to PBDEs in the environment. Firstly, the present computations do not consider various intermolecular interactions in real environmental matrix such as solvent effects. Secondly, this semiempirical level of theory is a further approximation (e.g. employing core approximation) to first principle (or ab initio level of theory) that is derived from several critical simplifications including Born–Oppenheimer, non-relativistic and Hartree–Fock approximations. We believe, nevertheless, that the valuable information presently obtained from fundamental quantum mechanics will make our experiments more productive, well oriented and more insightful. Finally, some of the methods and results of the present investigation may be readily extended to other dioxin-like compounds and, if so, this investigation will contribute to understanding the chemistry of the whole class of these dangerous pollutants. Acknowledgment Dr. Juha Hyo¨tyla¨inen, Department of Chemistry, University of Jyva¨skyla¨, is gratefully acknowledged for his useful comments on this manuscript. References Albro, P.W., McKinney, J.D., 1981. The relationship between polarizability of polychlorinated biphenyls and their induction of mixed function oxidase activity. Chem. Biol. Interact. 34, 373–378. Anderson, J.E., Smyth, C.P., 1965. Microwave absorption and molecular structure in liquid. LX. Intramolecular relaxation mechanisms in aromatic ethers and several related molecules. J. Chem. Phys. 42, 473–478. Andersson, P., Haglund, P., Rappe, C., Tysklind, M., 1996. Ultraviolet absorption characteristics and calculated semiempirical parameters as chemical descriptors in multivariate modeling of polychlorinated biphenyls. J. Chemom. 10, 171–185. Becker, M., Philips, T., Safe, S., 1991. Polychlorinated diphenyl ethers—a review. Toxicol. Environ. Chem. 33, 189–200. Chen, J.W., Kong, L.R., Zhu, C.M., Huang, Q.G., Wang, L.S., 1996. Correlation between photolysis rate constants of polycyclic aromatic hydrocarbons and frontier molecular orbital energy. Chemosphere 33, 1143–1150. Chen, J.W., Peijnenburg, W.J.G.M., Wang, L.S., 1998. Using PM3 Hamiltonian, factor analysis and regression analysis in
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