Decabromobiphenyl (PBB-209) Activates the Aryl Hydrocarbon

Chem. Res. Toxicol. 2008, 21, 643–658

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Decabromobiphenyl (PBB-209) Activates the Aryl Hydrocarbon Receptor While Decachlorobiphenyl (PCB-209) Is Inactive: Experimental Evidence and Computational Rationalization of the Different Behavior of Some Halogenated Biphenyls Mercedes Alonso,† Susana Casado,‡ Carlos Miranda,† José V. Tarazona,‡ José M. Navas,*,‡ and Bernardo Herradón*,† Instituto de Química Orgánica General, CSIC, Juan de la CierVa 3, E-28006 Madrid, Spain, and Departamento de Medio Ambiente, INIA, Ctra. de la Coruña, km. 7,5, E-28040 Madrid, Spain ReceiVed October 4, 2007

In rat H4IIE cells permanently transfected with a luciferase gene under the control of AhR, incubation with PBB-209 led to a statistically significant increase of luminescence. In this system, PCB-209 only caused a small induction of luciferase activity. In a fish cell line, only PBB-209 was able to provoke an induction of ethoxyresorufin-O-deethylase activity. Ligand binding to the AhR was studied by means of a cell-free in vitro system in which the activation of AhR is very unlikely to occur without ligand binding. None of the biphenyls studied provoked any activation of AhR in this system. To rationalize the results and to get insight into the molecular mechanism of activation of AhR by PBB-209 as compared with PCB-209, a comprehensive computational study was carried out on these congeners as well as on PCB126 and PCB-169, two potent AhR activators through ligand binding. The calculations include (i) conformational analysis and dipole moments of each conformer, (ii) aromaticity indices, (iii) molecular electrostatic potentials, (iv) quadrupole moments, (v) electronic and reactivity descriptors, and (vi) dissociation energies of C-Cl and C-Br bonds in model aromatic compounds. It was found that some molecular features of PBB-209, such as the electrostatic potential (EP) and EP-derived descriptors (Politzer’s parameters), indicate that PBB-209 is more similar to PCB-126 and PCB-169 than to PCB209, which share quite similar geometries based on the substitution pattern. The similarity between PBB209, PCB-126, and PCB-169 seems to hint that these three compounds can share, at least partially, similar mechanisms of activation of AhR. It is unquestionable that PCB-126 and PCB-169 directly bind AhR and PBB-209 does not. We hypothesize that there are several simultaneous mechanisms for activation of AhR, and the most active compounds act for more than one mechanism. Introduction One of the main concerns of modern society is the analysis, treatment, and fate of persistent organic pollutants. As a class of compounds, the halogenated arenes are frequently found in the environment (1), broadly distributed in a variety of systems * To whom correspondence should be addressed. (J.M.N.) Fax: +34913572293. E-mail: [email protected]. (B.H.) Fax: +34915644853. E-mail: [email protected]. † CSIC. ‡ INIA. 1 Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; ASE, aromatic stabilization energy; BDE, bond dissociation energy; CM, control medium; CS, control solvent; CYP1A, cytochrome P4501A; DMSO, dimethyl sulfoxide; DR-CALUX, dioxin-responsive chemically activated luciferase gene expression; DRE, dioxin-responsive elements; EC50, effective concentration 50; EP, electrostatic potential; EROD, 7-ethoxyresorufin-O-deethylase; GRAB, gel retardation of AhR binding; H-bond, hydrogen bond; HOMA, harmonic oscillator model of aromaticity; HOMO, highest occupied molecular orbital; IUPAC, International Union of Pure and Applied Chemistry; LOEC, lowest observed effect concentration; LU, luminiscence unit; LUMO, lowest unoccupied molecular orbital; R-MEM, minimum essential medium; MEP, molecular electrostatic potential; MSEP, molecular surface electrostatic potential; βNF, β-naphthoflavone; NICS, nucleus-independent chemical shifts; PAHs, polycyclic aromatic hydrocarbons; PBB, polybrominated biphenyl; PBS, phosphate buffer saline; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzo-p-dioxin; PCDF, polychlorinated dibenzofuran; PHA, polyhalogenated aromatic compound; PKC, protein kinase C; QM, quadrupole moment; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ZPE, zero-point energy.

(atmosphere, water, and food). Because of their lipophilic/ hydrophobic nature, they accumulate in the adipose tissue and are difficult to metabolize and remove, causing long-term pernicious effects (2, 3). Although considerable progress has been achieved in aspects such as the analysis and determination of toxic effects, basic research has to be done to understand the molecular mechanism of toxicity, which can be useful to find methods for remediation. Halogenated biphenyls, namely, chlorinated (PCBs)1 and brominated (PBBs), represent an important group of compounds widely used in the fabrication of diverse products. Since their detection in several environmental samples (4–6), exhaustive studies related to their physical, chemical, and toxicological profiles have been done (7–10). There exist 209 different congeners of each PBB and PCB, depending on the number of halogen atoms and on their relative position in the molecule (Figure 1). PCBs have been used as dielectric and heat transfer fluids, plasticizers, wax extenders, and flame retardants since the 1920s. The widespread presence of PCBs in the environment and two poisoning incidents in Japan (Yusho) and Taiwan (Yu Cheng) led to the ban of these substances in industrialized countries in the 1970s (11). However, PCBs are still present in a high number of old electrical devices that would need special recycling processes. Some brominated derivatives, including PBBs, have

10.1021/tx700362u CCC: $40.75  2008 American Chemical Society Published on Web 03/01/2008

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Figure 1. Generic structures of PBBs and PCBs and of the compounds studied herein.

been added or applied to combustible materials as flame retardants to increase their fire resistance (12, 13) since the 1970s. However, their persistence and widespread presence in biota, together with some cases of accidental contamination of animal feed, have raised environmental and health concerns (14) so that its production and use have been progressively banned in the European Union (15, 16) and in United States (17). Because of their more prolonged use, most of the research on the toxicity of these xenobiotics has been done with PCBs (18). Some adverse effects of PCBs include neurotoxicity (19), carcinogenesis (10, 20), and reproductive alterations (21) as well as several actions on the endocrine pathways by different mechanisms (22). Although some exceptions have been described (23–26), most of the harmful effects of PCBs have been linked to binding to the aryl hydrocarbon receptor (AhR). On the other hand, PBBs have been scarcely studied from a biological point of view, and because of the similarity with PCBs, the activation of AhR has also been proposed as the mechanism to trigger toxicity (27). The AhR is a ligand-activated transcription factor present in the cytosol as a multiprotein complex (28–32). After binding to a cognate ligand, the AhR complex translocates to the nucleus, dissociates from the complex, and dimerizes with the AhR nuclear translocator (ARNT). This heterodimer is able to recognize specific DNA sequences, the dioxin-responsive elements (DRE), upstream of target genes, increasing their transcription rates (33, 34). One of these genes is that of the cytochrome P4501A (CYP1A), which plays an important role in phase I detoxification processes (35–37). It has been traditionally assumed that typical AhR ligands share several structural features: They are hydrophobic, polycyclic aromatic compounds having a planar structure (1, 18).2 According to this hypothesis, the prototypical AhR ligand is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), whose toxic effects are preceded by specific and high affinity binding to this receptor. A structural feature of biphenyl derivatives (such as PCBs) is the potential rotation around the C-C single bond, which 2 Most of the early results on the activation of the AhR pointed to the fact that the binding site was strict on the magnitude of the third dimension (thickness), being able to accommodate only flat molecules. In 1995, Waller and McKinney, through a QSAR study, postulated that AhR has a rectangular binding site with maximal dimensions 14.0 Å × 12.0 Å × 5.0 Å; however, the third dimension of this proposed binding site is big enough to accommodate a huge variety of organic molecules, which is contradictory to early experiments. The topography of this site was deduced from results on the biological activity of coplanar molecules (38).

Alonso et al.

provides different conformers. The energetic accessible conformers depend on the substitution patterns, principally on the ortho positions. On analyzing the putative conformations, we can highlight two extremes, namely, the planar conformers (dihedral angle ) 0 or 180°) and the perpendicular conformer (dihedral angle ) 90°). According to the proposed model of binding to AhR, only PCBs having a coplanar conformation can be an AhR activator. This assumption was confirmed by early experiments that showed that PCBs without ortho substitution are CYP1A inducers and that the ortho-substituted PCBs are weak or noninducers of CYP1A (18). However, more recent computational studies have shown that some PCBs, considered as typical AhR ligands, are not planar molecules (39–41). In addition, it has been demonstrated that compounds having very diverse structures (including nonplanar) are found to be activators of AhR (42). In this context, previous studies in our group indicated that some imidazole derivatives, for which a planar conformation was energetically inaccessible, were able to induce CYP1A presumably after AhR activation (43–45). Another case especially interesting is carbaryl, which is a naphthalene-derived carbamate, whose most stable conformer is not coplanar (with a dihedral angle between the naphthyl and the carbamoyl fragments of ca. 45°), but the energy necessary to achieve a planar conformation can be provided by a favorable hydrogen bond (H-bond) with the receptor (46). The results from our and other groups indicate that the activation of the AhR is more complex than previously devised, and besides the ground state of a molecule, we have to consider the potential interactions with the receptor. To this end, several electronic and electrostatic characteristics of the potential activator, such as the capacity to form an H-bond, the dipole moment, the quadrupole moment (QM), the polarizability, and the electrostatic potential (EP), have to be taken in count. Additionally, other paths for activation of AhR through phosphorylation by kinases (47–49)3 can be operative as a parallel route for activation. Moreover, the biological activity can be due to a metabolite and not directly to the exogenous chemical, which complicates the analysis of the results (51, 52). The interaction of PCBs and PBBs with AhR is highly dependent on the chlorination pattern of the congener, which, in turn, influences its geometry (possibility to take a planar conformation) as well as its electrostatic properties. The perhalogenated biphenyls PBB-209 and PCB-209 (Figure 1) do not fulfill the a priori structural features to be AhR ligands or activators. Because of the presence of halogen atoms in the four ortho-positions, it is anticipated that both phenyl rings in these compounds will have a dihedral angle of ca. 90° and a high rotational barrier around the biphenyl linkage, preventing these molecules from assuming a planar conformation. Strikingly, in a series of preliminary experiments in our laboratory, in which these compounds were intended to be used as negative controls, activation of AhR by PBB-209 was detected. These results prompted us to perform a thorough study with the objective to determine the ability of this substance, as compared with PCB-209, to induce in vitro some processes normally dependent on AhR activation and to establish, by means of computational calculations, the molecular characteristics as well as the reactivity profile that could explain this particular behavior. For this purpose, the dioxin-responsive 3 One important candidate for this specific phosphorylation is protein kinase C (PKC). However, its contribution to a direct regulation of the AhR was later challenged, as inhibitors for specific isoforms of the PKC did not block the CYP1A induction after TCDD exposure; see ref 50.

ActiVation of the Aryl Hydrocarbon Receptor by PBB-209

chemically activated luciferase gene expression (DR-CALUX, Bio Detection Systems, Amsterdam, The Netherlands) system has been particularly useful, since it accurately and very rapidly detects the activation of the AhR, with independence of the molecular mechanisms underlying this activation (53, 54).

Experimental Procedures and Theoretical Background Chemicals. Polyhalogenated biphenyls PBB-209 and PCB-209 were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany); β-naphthoflavone (βNF, used as a model ligand of the AhR) was obtained from Sigma (St. Louis, MO). Stock solutions of these compounds were prepared by dissolving them in dimethyl sulfoxide (DMSO, Sigma). All of these chemicals can be considered hazardous and should be handled carefully. Cell Culture and Xenobiotic Exposure. The DR-CALUX bioassay performed in this study was based on the use of a rat hepatoma (H4IIE) cell line stably transfected with a construct containing the luciferase reporter gene under direct control of DRE (BioDetection Systems, Amsterdam, The Netherlands). The induction of luminescence was directly related to AhR activation. Cells were maintained in R-MEM (minimum essential medium, Cambrex, North Brunswick, NJ) with phenol red and supplemented with 10% fetal bovine serum (Cambrex), 1% 2 mM L-glutamine (Cambrex), and penicilline-streptomycine (10 U/mL to 10 µg/mL, respectively, Cambrex). Cells were grown at 37 °C with 5% CO2 in a humidified incubator. For the assay, cells grown in bottles were trypsinised and plated in 96 well plates at a density of 2.5 × 104 cells per well in 100 µL. After 24 h, cells were exposed to different concentrations of xenobiotics, and after 24 h, cells were washed with phosphate buffer saline (PBS), and the luminescence was measured by means of the Steady Glo luminescence kit (Promega, Madison, WI) in a Genios luminometer (Tecan, Männendorf, Switzerland). The rainbow trout RTL-W1 cell line was grown as originally outlined (55). Cells were maintained in Leibovitz’s L-15 cell culture medium (Cambrex) supplemented with 5% fetal bovine serum (Cambrex) and penicillin-streptomycin (20 U/mL to 20 µg/mL, respectively, Cambrex) in 75 cm2 Nunclon tissue culture flasks (Nunc, Roskilde, Denmark) at 20 °C. Cells were detached from confluent flasks using trypsin (Sigma), seeded in 96 well Falcon plates (Becton Dickinson, Oxnard, CA) at a density of 20000 cells in 200 µL culture medium per well, and allowed to grow to confluency for 24 h. After that, the medium was substituted with new medium containing the corresponding concentrations of xenobiotics. After 48 h of treatment, the medium was removed, cells were washed with PBS (pH 7.5), and the plates were introduced at -80 °C where they were maintained for 2 h until analysis of ethoxyresorufin-O-deethylase (EROD, an enzymatic activity dependent on CYP1A) and protein following the methodology previously described (56). Both biphenyl derivatives used in this study showed a low solubility in DMSO. To make the data comparable, it was decided to prepare similar solutions of both compounds and to increase the maximum concentration of DMSO in the medium, to reach the highest possible concentrations of both compounds. In the DRCALUX system, it was observed that treatment with DMSO alone induced a soft increase of luminescence at a percentage of 1%, although no statistically significant differences (P < 0.05) were reached with respect to control cells (incubated without DMSO). The luminescence measured at DMSO concentrations from 0.1 to 0.5% was similar to the basal luminescence measured in controls. Taking these results into consideration, it was decided to use a constant DMSO concentration of 0.4% in all concentrations of xenobiotics in the DR-CALUX cells. In the case of the RTL-W1 cells, no effect of DMSO was observed, and it was decided to increase its concentration to a maximum of 2% to reach a concentration of 100 µM for PCB-209 and PBB-209. Ah-Immunoassay. The ability of PBB-209 and PCB-209 to interact with the AhR in a ligand-binding manner was assessed by means of the Ah-Immunoassay (Biosense, Bergen, Norway). In this

Chem. Res. Toxicol., Vol. 21, No. 3, 2008 645 assay, cognate ligands of the AhR were incubated with a cytoplasmic extract of guinea pig liver cells together with ARNT. Because this was not a cell system and cellular membranes were not present, alternative pathways for AhR activation different from ligand binding were very unlikely. After activation, the formed AhR-ARNT complexes interacted with DRE in oligonucleotides bound to the well surfaces of the plate. These complexes remained bound after washing and were evident by means of rabbit polyclonal anti-ARNT antibodies and the later use of mouse monoclonal antirabbit antibodies coupled to a specific enzyme. After incubation of xenobiotics according to manufacturer instructions, absorbance was measured at 405 nm in a plate spectrometer (Genios, Tecan). Statistical Analysis. Results obtained in the biological systems were expressed as means ( standard errors of the mean (SE). Exposures to the xenobiotics were carried out in triplicate in at least three independent experiments. Statistical analysis of the data was done using Sigma Stat software from Jandel Scientific (San Rafael, CA). The program automatically determined the normality of the data using a Kolmogorov Smirnoff test and the homogeneity of variances by checking the variability about the group means. Differences between treated and control groups were tested with oneway repeated measurements analysis of variance (RM ANOVA) with P < 0.05 followed by Dunnett’s test. The estimation of the concentration–response function and the calculation of the effective concentration 50 (EC50, i.e., the concentration of the xenobiotic that produced a 50% of the maximum effect measured) were done using Sigma Plot 8.0 software from Jandel Scientific. Dose-response curves were fitted to a regression model for a sigmoid curve (eq 1).

y ) min + {max ⁄ [1 + (x ⁄ EC50)b]}

(1)

where max is the maximal response observed (asymptotic value), b is the slope of the curve, and min is the minimal response. The concentrations producing the maximal observable effect (Cmax) and the LOEC (lowest observed effect concentration, i.e., the lowest concentration that provokes any effect significantly different than that of controls) were also calculated. Computational Studies. The calculations were done on a Dell Precision 450 (Round Rock, TX) workstation with a double 1.40 GHz Intel Xeon processor running Microsoft Windows XP and on a Compaq HPC 320 supercomputer (Palo Alto, CA, Cluster of eight SMP servers with 32 1 GHz Alpha EV68 processors) at the Centro de Supercomputación de Galicia (CESGA). All of the computational simulations were carried out in a vacuum using the Gaussian 03 suite of programs (57), with the hybrid Hartree–Fock/density functional methodology (58) employing the three-parameter B3LYP functional (59–61). Previous studies showed that the experimental rotational barriers and vibrational frequencies of some biphenyl derivatives and PCBs were accurately reproduced computationally (62, 63) and that similar results were obtained using either time-consuming basis sets [6-311++G(d,p) and cc-pVTZ] or a simpler basis set [6-31G(d,p)] (64). Therefore, we used two more slightly modest basis sets [6-31+G(d,p) and 6-311G(d,p)] to find which one better describes these kinds of compounds, especially PBBs. The potential energy curves, which provide the relative energy vs the aryl-aryl dihedral angle φ, were computed optimizing the structure at different angle φ between 0 and 180° with intervals of 30°. The orbital energies, the molecular electrostatic potential (MEPs), the dipole moment (µ), the QM, and the polarizability were calculated for the optimized geometries, according to standard procedures implemented in the Gaussian 03 program. The molecular surfaces electrostatic potentials (MSEPs) were represented using gOpenMol program (65, 66; web address, http://www.csc.fi/gopenmol; last accessed October 1, 2007). The MEPs were plotted on an isoelectronic density surface of 0.002 e bohr-3. The nucleus-independent chemical shift (NICS) is a useful aromaticity criterion. It is defined as the negative value of the absolute magnetic shielding computed at the ring center or at

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Alonso et al. n

V)

∑

1 V(ri) n i)1

(4)

R

∑

Figure 2. Orientation of the molecule and coordinate origin used to calculate the QM and the polarizability.

V+ )

1 V +(rj) R j)1

another interesting point of the system (67, 68). These parameters have been calculated at the ring center determined by the nonweighted average of the heavy atom coordinates (NICS), 1 Å above it [NICS(1)], and 1 Å below it [NICS(-1)] by the GIAO/B3LYP/ 6-311G(d,p) method (69) as implemented in Gaussian 03. The NICS(1) values computed 1 Å above and below the molecular plane are considered to better reflect the π-electron effects (70). Rings with highly negative values of NICS, NICS(1), and NICS(-1) are quantified as aromatic, whereas those with positive values are antiaromatic. The most useful aromaticity descriptor based on structural features is the harmonic oscillator model of aromaticity (HOMA) index, defined by Kruszewski and Krygowski by eq 2 (71).

V- )

1 V -(rk) β k)1

n

HOMA ) 1 -

R (R - Ri)2 n i)1 opt

∑

(2)

where n is the number of bonds taken into the summation and R is an empirical constant fixed to give HOMA ) 0 for a model nonaromatic system and HOMA ) 1 for a system with all bonds equal to an optimal value Ropt, assumed to be realized for a fully aromatic system. Ri is the running bond length. The dissociation energies of the C-X bonds of halogenated benzenes were computed using the B3LYP hybrid functional with the 6-31G(d), 6-31+G(d,p), and 6-311G(d,p) basis sets and corrected by the zero-point energy (ZPE) of the three chemical species involved.

(5)

β

∑

(6)

Because in the large majority of neutral molecules, the parameters V, V+, and V- are quite similar (slightly positive for V and V+ and slightly negative for V-), they alone would not be useful to establish correlations with properties. One more representative descriptor is the average of positive and negative potential, which is estimated by the average deviation, Π (eq 7), which represents the extent of the local polarity or internal charge separation, even in molecules with null dipole moments. Although Π reflects the charge separation, there are other parameters that are more sensitive to the extreme values of potential. Thus, Politzer and Murray described the positive (σ+2), negative (σ-2), and total (σtot2) variances of V(r), obtained by eq 8. Finally, for a better quantification of the electrostatic balance degree, the relative strength of the positive and negative surfaces, the term ν is included (eq 9), which reaches a maximum value of 0.25 when σ+2 and σ-2 are equal. n

Π)

∑

1 |V(ri) - V| n i)1

(7)

R

σtot2 ) σ+2 + σ-2 )

∑

1 [V +(rj) - V +]2 + R j)1 β

∑

1 [V -(rk) - V -]2 (8) β k)1

Theoretical Basis of the Computed Properties Molecular Electrostatic Potential (MEP) and Statistical Analysis of the MEP on a Molecular Surface. The MEP [V(r)] at the point r is a representation of the electrostatic interaction energy between a molecule and a test charge of magnitude e (that is, a proton) placed at that point, supposing that the molecule is not polarized by the test charge (72, 73). The MEP is calculated by eq 3.

V(r) )

Z

F(r′)

∑ |RA -A r| - ∫ |r - r′| dr′

(3)

A

where ZA is the charge on nucleus A, located at RA, which is considered to be a point charge and where the second term arises from the electron density of the molecule, F(r′), which can be obtained computationally (74) or experimentally (75). Useful information can be extracted from the values of the EP on a molecular surface following the procedure developed by Politzer, Murray, and co-workers (76). The method consists of performing a statistical analysis of the MEP surface, yielding several mathematical descriptors that later are correlated with a property (either chemicophysical or biological activity). Politzer’s descriptors are defined below. Some of these parameters are straightforward derived from the MEP, like Vmax, the most positive value of EP; Vmin, the most negative value of EP; and Vring, the most positive value of EP on the aromatic ring. Other descriptors are average values, such that of the potential (V), of the positive values (V+), and of the negative (V-) potentials, which are calculated according to eqs 4-6, respectively, where n, R, and β are the number of points i, j, and k taken from the grid used to compute the potential.

ν)

σ+2σ-2

(9)

[σtot2]2

QM. This property is a tensor magnitude (77), whose value depends on the coordinate origin and on the orientation of the molecule (Figure 2). The components of the QMs in the xyzcoordinate system were obtained from the QMs evaluated at the center of mass (Qcm) and by applying a similarity transformation as indicated by eqs 10 and 11.4 [Q] ) [TQcmT -1]

(

)(

)

Qxx Qxy Qzx 1 0 0 Qxy Qyy Qyz ) 0 cos(θ) -sen(θ) × Qzx Qyz Qzz 0 sen(θ) cos(θ)

(

Qcm Qcm Qcm xx xy zx Qcm Qcm Qcm xy yy yz cm Qcm Qcm Qzz zx yz

)(

(10)

)

1 0 0 0 cos(θ) sen(θ) (11) 0 -sen(θ) cos(θ)

Reactivity Descriptors. The theoretical basis for the reactivity descriptors has been amply developed elsewhere (78). The chemical hardness (η) is a concept frequently used to explain stability and reactivity (79–81). The absolute hardness at a constant external potential υ(r) has been quantified by Parr and Pearson according to eq 12, where E is the electronic energy 4 A full description on the coordinate transformation and the calculation of the QM is included as Supporting Information.


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and N is the total number of electrons. Hardness η gauges the resistance of the chemical potential to change the number of electrons.

η)

( )

1 ∂2E 2 ∂N2

(12)

υ(r)

Assuming a quadratic relation between E and N, the chemical hardness η can be calculated from the vertical ionization potential I and the vertical electron affinity A according to the finite difference approximation (eq 13).

η ) (I - A) ⁄ 2

(13)

I and A are determined from the electronic energies of the systems having N - 1, N, and N + 1 electrons (eqs 14 and 15).

I ) EN-1 - EN

(14)

A ) EN - EN+1

(15)

The global molecular softness S is inversely proportional to hardness S (eq 16); the higher the softness is, the higher the reactivity is.

S)

( ) ∂2N ∂E2

)

υ(r)

1 1 ) 2η (I - A)

(16)

Many studies have related softness S with polarizability R in a chemical system. Polarizability is a tensor magnitude that measures the facility with which the electron distribution is distorted in the presence of an external electric field. This property can be a significant factor in the interaction between molecules, especially if they have null permanent dipole moments, as is the case of many activators of AhR (82–84). Polarizability is a manifestation of the linear response of the electron density in the presence of an infinitesimal electric field F, and it is mathematically expressed by a second-rank variation in the energy (eq 17), where a and b are the three-dimensional coordinates x, y, and z. Polarizability, as a tensor, has several components, and it is usually calculated as the average of the principal components of the 3 × 3 matrix (eq 18).

Ra,b )

(

∂2E ∂Fa∂Fb

)

(17)

where a, b ) x, y, and z.

R ) (Rxx + Ryy + Rzz) ⁄ 3

Results (18)

The global electrophilic index ω is defined by Parr et al. by eq 19 (85).

ω ) µp2 ⁄ 2η ) (I + A)2 ⁄ 4(I - A)

Figure 3. Dose-dependent induction of luciferase activity in the dioxinresponsive, chemically activated luciferase (DR-CALUX) bioassay after 48 h of treatment with different concentrations of βNF, PBB-209, and PCB-209. Asterisks indicate significant differences (P < 0.05) with respect to control solvent (CS) cells. Means ( SE of four different experiments are represented.

(19)

where µp is the chemical potential, which is related to the ionization potential I and the electron affinity A by µp ) -(I + A)/2. The electrophilicity index (ω) measures the tendency of a molecule to react with an electron-donating center as well as the capacity to be reduced by accepting electrons. It can help to understand the biological activity of many compounds, including toxic effects mediated by AhR and/or P450 (86). From the definition, the electron affinity A and the electrophilicity index ω are related, although while A gauges the capacity to take a single electron, ω measures the energetic stabilization of a ligand due to the maximum electron flux between donor and acceptor, which can be different to one.

Effect of PCB-209 and PBB-209 on the Induction of DRE-Reporter Gene Expression and EROD Activity. The exposure of DR-CALUX cells to increasing concentrations of βNF (n ) 4) provoked a concomitant increase of luminescence (Figure 3) indicating a trans-activation of the DRE reporter gene. Significant differences with respect to solvent controls were observed for βNF concentrations of 2.75 µM (LOEC) and above (P < 0.05), and the EC50 for βNF was calculated as 3.57 µM. The maximal induction appeared at 22 µM βNF, reaching approximately 23000 arbitrary luminescence units (LU). In the case of PBB-209 (n ) 4), a significant (P < 0.05) increase of luminescence was observed for the concentrations 10 and 20 µM, for which the luminescence values were approximately 4000 and 6000 LU, respectively. For PCB-209 (n ) 4), a significant difference (P < 0.05) with respect to the controls was observed at 20 µM, although in this case the difference was not so marked, with values of approximately 2000 and 2800 LU in controls and 20 µM PCB-209, respectively.

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Figure 5. Increase of absorbance with the concentration detected altered incubation of βNF in the Ah-Immunoassay. PBB-209 and PCB-209 did not provoke any effect in this assay, suggesting that both compounds are not able to bind the receptor.

Figure 4. EROD activity measured in the RTL-W1 cell line after exposure for 48 h to different concentrations of βNF, PBB-209, and PCB-209. Asterisks indicate significant differences (P < 0.05) with respect to CS. Means ( SE are represented (n ) 4 for βNF, 2 for PBB-209, and 3 for PCB-209).

The induction of EROD activity in RTL-W1 cells was confirmed by the dose–response curve obtained after incubation with βNF (n ) 5). Significant increments of EROD activity with respect to the controls were observed at βNF concentrations of 0.006 µM and above, and the EC50 was 2 nM. The maximal value of EROD activity observed was 14.56 pmol/mg/min and appeared at 98 nM βNF. On this hand, PBB-209 (n ) 2) provoked a sustained increase of EROD activity to reach maximal values (3.81 pmol/mg/min) at a concentration of 100 µM. No effect of PCB-209 (n ) 3) on EROD activity was detected at any of the concentrations used (from 0.75 to 100 µM). Although the percentage of DMSO in the culture medium reached a maximal of 2%, no effect of the solvent alone was observed on the EROD activity, as it can be deduced from the lack of activity in the PCB-209 treatment (Figure 4). Activation of AhR by βNF, PCB-209, and PBB-209 in the Ah-Immunoassay. Results obtained for the different xenobiotics in the Ah-Immunoassay are shown in Figure 5. Incubation of increasing concentrations of βNF in this assay provoked a concomitant increase of absorbance with an EC50 of 1.97 µM. The maximal effect (1.74 of absorbance) was observed at a concentration of 44 µM. When this test was

applied to the PBB-209 and PCB-209, no response was detected at any concentrations tested, from 0.15 to 20 µM. Computational Studies. The computational study was prompted by the fact that many molecular features, both in the ground and in transition states, can help to understand the mechanism of activation of the receptor. In general, the research on the bioactivity of aromatic compounds requires a complete set of chemical and physicochemical properties. Although these data might be obtained experimentally (87, 88), this approach is hampered by the elevated toxicity of some of them. Therefore, a viable alternative is the computational one. With the objective to get insight into the molecular mechanism of activation of AhR by PBB-209 as compared with PCB209, a comprehensive computational study was carried out. In some cases, the computational properties of PCB-126 and PCB169, two potent AhR activators through ligand binding (89), were also calculated for comparative purposes. The calculations include (i) conformational analysis and dipole moments of each conformer, (ii) aromaticity indices, (iii) MEP, (iv) QMs, (v) electronic and reactivity descriptors, and (vi) dissociation energies of C-Cl and C-Br bonds in model aromatic compounds. Conformational Analysis, Dipole Moment, and Rotational Barrier Energy. In the particular case of the biphenyl derivatives, its interaction with AhR is highly dependent on the geometry of the congener, especially the torsion angle around the aryl fragments. Biphenyl, the parent compound without substituents, has been computationally (41) and experimentally (90, 91) studied, having a dihedral angle between 0 and 44°, which represents a compromise between the resonance stabilization between the two aromatic fragments and the steric destabilization of a coplanar conformation. The substitution of hydrogen atoms with halogen atoms in the ortho positions forces the phenyl rings to adopt a conformation with a larger dihedral angle. Most of the previous structural studies on halogenated biphenyls have been carried out with PCBs (39–41, 62–64, 92). The toxicity associated to the congeners is dependent upon both the degree of chlorination and the position of the halogens atoms. Non-ortho-substituted congeners that could take a planar conformation present the highest affinity to the AhR and the highest toxicity level. However, the di-ortho-halogenated compounds exhibit a different spectrum of toxic modes of action (93). The geometries of the conformational minima as well as of the planar conformer have been calculated as indicated above. In the four compounds studied, the planar (or nearly planar) conformations are transition states on the corresponding potential energy surfaces as assessed by the analysis of the harmonic


Figure 6. B3LYP/6-311G(d,p) optimized geometries of the minima (left) and transition states (right) of (a) PBB-209, (b) PCB-209, (c) PC-B169, and (d) PCB-126.

vibrational frequencies, where the single imaginary frequency corresponds to the rotation around the aryl-aryl bond. Figure 6 shows the most stable and the transition state conformers of the four compounds studied. Table 1 depicts the dihedral angle φ between the two aromatic rings of the most stable conformer and the transition state of PBB-209, PCB-209, PCB-169, and PCB-126 along with the rotational barriers calculated at both B3LYP/6-31+G(d,p) and B3LYP/6-311G(d,p). The dipole moment µ of each conformation is also included. A selection of the main geometrical parameters of the computed and experimental (X-ray diffraction analysis) of PCB-209 and PBB-209 is included in the Supporting Information. To assess how the energy varies with the dihedral angle, the potential energy curves (relative energy ∆E vs dihedral angle φ) for PCB-209 and PBB-209 were calculated at B3LYP/6-31G+(d,p) and B3LYP/6-311G(d,p) in Gaussian03. The results at B3LYP/6-311G(d,p) are shown in Figure 7. It is found that the curves are symmetrical around the global minima (φ ) 90°) and relatively flat between 60 and 120°, with an energy difference relative to the minima of 3.4 kcal mol-1 for PCB-209 and 4.7 kcal mol-1 for PBB-


209. These results indicate that PCB-209 and PBB-209 have a limited mobility between dihedral angles of 60–120° and undoubtedly show that the coplanar (or nearly coplanar structures) are very unstable.5 Aromaticity Indices. Aromaticity is a fundamental property of organic compounds. It refers to the existence of some properties similar to those of benzene, that is, electron cyclic delocalization with energetic stabilization (94, 95). Aromaticity is a concept that allows classifying organic compounds in three wide groups: aromatic, nonaromatic, and antiaromatic. Because aromaticity is not and observable, it has to be defined by convention with a scale relative to a reference compound (usually benzene). Although some attempts have been done to quantify aromaticity, this goal has not been achieved (96). It is likely that the aromatic character of organic compounds influences the noncovalent interactions of organic molecules, including the interaction with target biomacromolecules and, therefore, the biological activity. The local aromaticity of each ring of the different conformations of PBB-209, PCB-209, PCB-169, and PCB-126 was calculated employing several aromaticity indices, namely, a structural index, HOMA (71), and three magnetic indices, NICS, NICS(1), and NICS(-1), which were measured at the center of the ring (as the mean of the coordinates of the heavy atoms) at 1 Å above the ring and 1 Å below the ring, respectively (68).6 Other frequently used indices, such as aromatic stabilization energy (ASE) (97) and the magnetic susceptibility exaltation (98), were not measured due to the absence of suitable homodesmotic or isodesmotic reactions. The computed aromaticity indices are collected in Table 2, along with the mean values of the dihedral angles of the two benzene rings, denoted as |CCCC|. The deviation from planarity of an aromatic ring is a structural measurement of aromaticity: The higher the planarity is, the higher the π-electron delocalization and the higher the aromaticity are. Electrostatic Properties. Independently of the mechanism of AhR activation (i.e., ligand binding, participation of other proteins such as kinases or by a metabolite), it is evident that, grounded on the principles of molecular recognition between biologically active compounds and target biomacromolecules (99), an interaction between two components has to be produced. This interaction can be of several types (100), although the most important types are dispersion and electrostatic. The dispersion energy is due to the interaction between instantaneous dipoles generated in the molecules, and they are mostly dominated by the polarizability of the participating molecules. This property will be discussed in the next section. On the other hand, electrostatic interactions, including some hydrogen bonds (101), are essential to rationalize the biological activity of organic compounds (102). They act at both short and large ranges, and its strength depends on the distance, the relative orientation of the interacting species, and the electrostatic features of the molecules. The activators of AhR are aromatic compounds, which are able to participate in a huge variety of intermolecular interactions, including arene-arene, weak hydrogen bond, arene-cation, arene-heteroatom, and so on (103–106); many of them can be classified as electrostatic 5 The differences between the energies shown in Table 1 and the energies shown in Figure 7 are due to the different methodologies employed in the two calculations (full vs partial optimizations). The results in Table 1, corresponding to the full optimization calculation, are more accurate. Figure 7 shows graphically which regions of the conformational spaces of PCB209 and PBB-209 are energetically available. 6 Aromaticy indices calculated at the 6-31+G(d,p) level of theory are included in the Supporting Information.

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Table 1. Torsion Angle (φ), Energy Differences (∆E and ∆G in kcal mol-1) between the Most Stable Conformation and the Planar Structure (TS) and Dipole Moment (µ in Debyes) of Each Compound 6-31+G(d,p) compound

conformer

φ

PBB-209

minima TS minima TS Minima TS minima TS

90.0 -23.7 90.0 6.0 39.7 0.0 39.8 0.0

PCB-209 PCB-169 PCB-126 a

∆Ea

6-311G(d,p) ∆G

65.37 (56.43)b

66.14 (59.06)b

b

b

55.93 (56.37) 1.44

58.60 (59.09) 3.39

1.44

3.21

µ

φ

0.00 0.44 0.00 0.00 0.00 0.00 1.42 1.43

90.0 -23.3 90.0 5.8 39.5 0.0 39.9 0.0

∆Ea

∆G

58.48

60.96

55.98

59.07

1.33

3.30

1.36

3.17

µ 0.00 0.45 0.00 0.00 0.00 0.00 1.48 1.50

b

Zero-point and thermal corrections (at 298.15 K) included. Energy barriers calculated at B3LYP/6-31G(d) are in parentheses.

Figure 7. Plot of energy vs dihedral angles of the different conformers of PBB-209 (2) and PCB-209 (b). Each conformation was generated by 30° rotation around the aryl-aryl bond. The calculations were carried out at the B3LYP/6-311G(d,p) theory level.

interactions. The electrostatic features of organic molecules can be gauged by the electrostatic moments and the EP. Molecular Electrostatic Potential. The MEPs of PBB-209, PCB-209, PCB-169, and PCB-126 are calculated to help us understand the different biological activities. Once the MEPs are computed, they are mapped on an isodensity surface of 0.002 e bohr-3, yielding the molecular surface electrostatic potential (MSEP) represented in Figure 8 for the most stable conformer of each PBB-209, PCB-209, PCB-169, and PCB-126. On the other hand, the statistical analysis of the values of the MEP on an isodensity surface, as explained previously, can give useful numerical information on charge distribution in a molecule. This goal has been achieved using Politzer’s parameters as defined above. The calculated Politzer’s parameters from the MEPs of PBB-209, PCB-209, PCB-169, and PCB-126 are shown in Table 3. QM and Polarizability (r) Tensors. Because of the symmetry of the molecule, many halogenated biphenyls have null or very low dipole moments (see Table 1), and this property can not be used to differentiate the biological activity of the congeners. When the dipole moment is null, we can recourse to higher electrostatic moments to explain differences in behavior. Because the potential of a interaction with higher order electrostatic moments decreases rapidly with the order of the moment, the most significant electrostatic moment is the lower non-null, that is, the QM for PCBs and PBBs. QM measures the deviation from spherical symmetry of a global charge distribution (107). Because of the substitution pattern, many aromatic compounds have low dipole moments, but they possess high QMs, and this feature influences their properties, including intermolecular interactions (108, 109). The computed components of the traceless QM tensors of PBB-209, PCB-209, PCB-169, and PCB-126 (in their most stable conformations) are indicated in Table 4. Because the coordinate reference system used for calculating the QMs and

the polarizability tensor R is the same, the components of R are also included in Table 4. Polarizability is more related to the reactivity pattern, as will be discussed below. Reactivity Descriptors Based on the Electronic Structures of the Molecules. The theoretical basis of the reactivity descriptors has been indicated in the previous section. Using the equations indicated therein, several global descriptors of reactivity have been calculated for PBB-209, PCB-209, PCB-169, and PCB-126 in both the most stable conformation and in the transition state. They include highest occupied molecular orbital (HOMO) energy (HOMO), lowest unoccupied molecular orbital (LUMO) energy (LUMO), and their differences (∆EHOMO–LUMO), ionization potential (I), electron affinity (A), Parr-Pearson absolute hardness (η), electrophilic global index (ω), and polarizability (R) (Table 5). Dissociation Energy of C-Cl and C-Br Bonds. The ability of an exogenous chemical to activate AhR can be influenced by its capacity to form a radical. With the aim to compare the facility of PCBs and PBBs to generate radicals through C-X bond breaking, we have computationally studied the unimolecular reaction indicated in Figure 9, using six derivatives of benzene (1–6) as model compounds. Others goals of this study were to assess the relative influence of the number of halogens and their positions as well as to learn which computational basis set gives the best performance. Previous results showed that the B3LYP hybrid functional is suitable for these kinds of compounds (110). The reactions have been modeled following the methodology reported in the Experimental Procedures section. The computed results along with the experimental ones, when available, are collected in Table 6. The results show that the 6-31+G(d,p) basis set does not correctly describe the bromine atom, and more satisfactory results are obtained with the 6-311G(d,p) basis set. It must be remarked that the experimental values are enthalpies at 298 K (∆H298), and the computed values are obtained without considering thermal effects. Table 7 collects the BDEs calculated, at the 6-311G(d,p) level, as the enthalpy differences between products and reactants, according to eq 20. On considering thermal effects, the differences between experimental and theoretical results diminish, although still a slight underestimation is observed in the computational method.

BDE (R-X) ) H298°(R) + H298°(X) - H298°(RX) (20) Discussion Comparison of the Biological Activity of PBB-209 and PCB-209. The study of the biological activity of PBB-209 and PCB-209 has been focused on the susceptibility of AhR to be activated by these compounds. In a recent report (27), a recombinant mouse hepatoma (Hepa1c1c7) cell line stably transfected with DRE-luciferase vectors was used to study AhR



Table 2. Aromaticity Indices of the Minima and Transition States (TS) of PBB-209, PCB-209, PCB-169, and PCB-126 minima compound

a

HOMA c

PBB-209 PCB-209 PCB-169 PCB1-26d C6H2Cl3 C6H3Cl2

0.949 (0.937/0.950) 0.964 (0.969)c 0.976 0.976 0.982

TS a

a

b

a

NICS

NICS(1)

NICS (-1)

|CCCC|

HOMA

NICS

NICS(1)a

NICS (-1)a

|CCCC|b

-9.3 -10.3 -9.5

-9.1 -9.6 -10.0

-9.1 -9.6 -10.0

0.0 0.0 0.1

0.798 0.801 0.974

-7.2 -8.5 -8.7

-9.0 -9.7 -9.4

-7.4 -8.3 -9.4

17.8 17.5 0.0

-9.5 -9.3

-9.9 -10.2

-9.9 -10.2

0.1 0.1

0.974 0.978

-8.6 -8.5

-9.3 -9.6

-9.3 -9.6

0.0 0.0

a NICS, NICS(1), and NICS(-1) are in ppm. b |CCCC| is the average dihedral angle of the phenyl rings. c HOMA values of the single crystal X-ray diffraction structures of PCB-209, 2,2′,4,4′,6,6′-HPBB (PBB-155), and 2,2′,6,6′-HPBB (PBB-54) are given in parentheses. d The aromatic indices of the two different rings of PCB-126 are listed below and denoted as C6H2Cl3 and C6H3Cl2, respectively.

Figure 8. MSEPs of the most stable conformers of PBB-209, PCB-209, PCB-126, and PCB-169. The values of the EPs (in kcal mol-1) are indicated in the scale.

Table 3. Politzer’s Descriptors Obtained from the Statistical Analysis of the MEPa compound

Vmax

Vmin

Vring

V

V+

V-

Π

σ+2

σ-2

σtot2

ν

PBB-209 PCB-209 PCB-169 PCB-126

26.5 23.7 34.4 32.1

-7.0 -4.5 -8.4 -11.5

13.3 19.1 12.3 11.3/9.4

2.60 3.89 6.72 6.68

8.70 7.62 10.21 11.60

-3.89 -2.22 -3.79 -4.31

6.41 5.56 6.31 8.27

3.2 1.4 6.1 9.7

48.2 35.6 54.0 76.7

51.4 37.0 60.1 86.4

0.058 0.035 0.091 0.099

a

Vmax, Vmin, Vring, V, V+, V-, and Π are in kcal mol-1; σ+2, σ-2, and σtot2 are in (kcal mol-1)2; and ν is dimensionless.

Table 4. Components of the Polarizability r and QM Traceless Tensor Q (in Atomic Units) compound

Rxx

PBB-209 PCB-209 PCB-169 PCB-126

415.78 336.82 322.06 310.62

Ryy

Rzz

R

Qxx

Qyy

Qzz

259.23 259.23 311.41 2.78 -1.39 -1.39 203.60 203.60 248.01 0.79 -0.39 -0.39 202.38 106.00 210.15 -10.34 6.62 3.73 186.54 94.13 197.10 -11.34 7.23 4.12

activation. In this system, PBB-209 at concentrations between 1 and 10 µM provoked a steady induction of luciferase activity after 20 h of incubation with an EC10 (defined in this case as the concentration of PBB eliciting a 10% of the maximal response caused by TCDD) of 2.6 µM. These results are similar to those observed in the present study, although we detected the maximal induction of luciferase at 20 µM PBB-209, and they suggest the ability of PBB-209 to activate DRE in the transfected

cells. These results are corroborated by the induction of EROD activity. It is now accepted that induction of P450 and EROD activity is dependent on AhR activation (111), although a variety of mechanisms have been suggested, apart from ligand binding, to explain the activation of the receptor, from the action of metabolites to phosphorylation/dephosphorylation processes. Brown et al. (27) also tested the ability of PBB-209 to activate the AhR in vitro in a gel retardation assay (GRAB) using guinea pig hepatic cytosol. These authors state that in this system, binding of the AhR to an oligonucleotide containing its specific DNA binding site (the DRE) occurs only if the chemical can activate the AhR as observed with TCDD. In the present study, we used Ah-Immunoassay, which also utilizes guinea pig hepatic cytosol as a source of AhR, to observe any in vitro activation of the AhR. While both Ah-Immunoassay and gel retardation assay using cytosol detect the interaction of activated AhR with

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Table 5. Electronic Reactivity Descriptors of PBB-209, PCB-209, PCB-169, and PCB-126 Calculated at the B3LYP/6-311G(d,p) Level of Theorya compound

HOMO

∆EHOMO–LUMO

LUMO

I

A

η

ω

R

8.50 8.80 8.54 8.43

1.30 0.66 0.80 0.59

3.60 4.07 3.87 3.92

3.33 2.75 2.81 2.60

311.41 248.01 210.15 197.10

7.89 6.30 8.47 8.32

2.72 2.65 0.87 0.81

2.58 1.83 3.80 3.75

5.45 5.47 2.99 2.79

342.49 279.86 214.72 201.67

minima PBB-209 PCB-209 PCB-169 PCB-126

-7.16 -7.47 -7.13 -6.99

-2.48 -1.99 -2.22 -2.04

4.68 5.48 4.91 4.96

PBB-209 PCB-209 PCB-169 PCB-126

-6.66 -6.94 -7.03 -6.88

-3.94 -3.95 -2.43 -2.26

2.72 2.99 4.60 4.61

TS

HOMO, LUMO, ∆EHOMO–LUMO, I, A, η, and ω are given in eV, and R is given in atomic units.

a

Figure 9. Homolytic bond dissociation reaction and compounds used in the computational modeling.

Table 6. Calculated and Experimental Bond Dissociation Energies (BDEs) (in kcal mol-1) for Polyhalogenated Benzenes Calculated with the 6-311G(d,p) and 6-31+G(d,p) Basis Sets (Latter in Parentheses) compound

a

1

2

expa

3

1 2 3

BDE 88.7 (88.5) 85.6 (85.7) 82.8 (83.0)

C-Cl for the site 88.7 (88.5) 88.7 (88.5) 85.1 (85.1) 85.6 (85.7) 82.8 (83.0) 82.8 (83.0)

4 5 6

BDE 77.3 (86.2) 72.9 (85.8) 67.5 (81.2)

C-Br for the site 77.3 (86.2) 77.3 (86.2) 71.7 (85.3) 72.9 (85.8) 67.5 (81.2) 67.5 (81.2)

94.5/95.5

79.2/82

The experimental data for 1 and 4 were taken from ref 110.

Table 7. BDEs (T ) 298 K) (as Enthalpies, in kcal mol-1) for Polyhalogenated Benzenes Calculated at the B3LYP/ 6-311G(d,p) Level of theory (Eq 20) compound

a

1

2

3

1 2 3

BDE C-Cl for the site 89.5 89.5 89.5 86.4 85.8 86.4 83.6 83.6 83.6

4 5 6

BDE C-Br for the site 77.9 77.9 77.9 73.5 72.3 73.5 68.0 68.0 68.0

expa 94.5/95.5

79.2/82

The experimental data for 1 and 4 were taken from reference (110).

DRE, the advantage of the Ah-Immunoassay is that it permits one to assay several concentrations of the test compound. In this manner, a dependence of the response with respect to the concentration of the chemical can be observed in a straightforward way. Our results differ from those of Brown, since they detect in GRAB an activation of the AhR after incubation with PBB-209 that we were unable to observe at the PBB-209 concentrations used in the present study. This is not the first

time that divergent results have been found when the direct activation of the receptor is studied. In all cases, divergences have been associated with the different cellular fraction used in GRAB or in competition assays. However, a report by Denison and co-workers compared the results on AhR activation using either GRAB or CALUX assays, concluding that the latter are more reliable (112). In any case, these experimental results are supported for the extensive computational study reported herein and which is discussed in the next paragraphs. Conformational Analysis and Dipole Moment. As a consequence of the elevated repulsion between the four halogen atoms at ortho positions, the lower energy conformations of PBB-209 and PCB-209 have perpendicular phenyl rings (90° dihedral angles). On the other hand, the most toxic congeners, PCB-126 and PCB-169, which are nonsubstituted at the ortho positions, have conformations with dihedral angles of ca. 40° in agreement with the postulates indicated above (Figure 6). The activation energy of the rotation around the aryl-aryl bond is over 56 kcal mol-1 for PCB-209 and more than 65 kcal mol-1 for PBB-209. Because the transition states are not coplanar structures, these are even higher in energy. These results indicate that the existence of a coplanar structure in PBB209 and PCB-209 can be completely discarded. On the other hand, the energy difference between the most stable and the coplanar conformation of PCB-126 and PCB-169 is lower than 2 kcal mol-1, which indicates that the planar structure is accessible for these congeners, and they can bind to AhR through a coplanar structure. It is interesting to note that while the three tested basis sets gave essentially the same result for the rotational barriers of the chlorinated derivatives, there is a clear dependence on the basis sets in the rotation barriers for PBB-209. These results along with the BDEs of C-Br indicate that the 6-31G(d) and 6-31G+(d,p) are not conveniently parametrized for bromine. The high Coulombic repulsion and steric hindrance of the halogen atoms at the ortho positions cause the transition state of the conformational changes in PBB-209 and PCB-209 to be quite distorted, and the phenyl rings are not plane. As expected, these deviations from planarity of the aromatic rings are larger in PBB-209 than in PCB-209, which contribute to a decrease in the aromaticity of the rings on passing from the ground to the transition state (see below). Because of the high symmetry of the molecules studied, the dipole moment of each conformation is null or quite low (Table 1). These compounds are known to cause long-term pernicious effects that are likely due to the fact that they accumulate in adipose tissue and are not metabolized. We have previously hypothesized (41) that the low dipole moment of the most toxic congeners is responsible of this tendency to accumulate in adipose tissue.


Aromaticity. All of the aromaticity indices indicate that PBB209 is slightly less aromatic than PCB-209, both in the ground and in the transition state conformations (Table 2). On comparing the data of the three PCBs, it is observed that the aromaticity of the ring decreases on increasing the number of chlorine atoms, except for the values of NICS, where an inverse trend is observed. It is quite well-established that the NICS values, as a measure at the center of a ring, are influenced by the σ-framework and by the in-plane component of the tensor (113). Therefore, except for the value of NICS, all of the criteria give the same aromaticity order: PCB-126 > PCB-169 > PCB-209 > PBB-209. All of the aromaticity criteria reveal a substantial diminution of the local aromaticity on passing from the minima to the transition state in PBB-209 and PCB-209. This fact is also corroborated by the mean dihedral angle in the aromatic rings. The phenyl rings of the transition states of PBB-209 and PCB-209 are quite distorted, and as a result, the values of NICS(1) and NICS(-1) for each congener are different. On the other hand, the aromaticity of PCB-169 and PCB126 is essentially the same on passing from the minima to the transition state. Because aromaticity leads to a high thermodynamic stability, the present results confirm that the transition state conformations of PBB-209 and PCB-209 are quite unstable species, and these chemicals are not able to achieve a coplanar conformation. Although the studied compounds herein are too few to draw general conclusions, the found differences in aromaticity between these halogenated biphenyls as well as the changes of aromaticity between conformations might be used, in conjunction with others, as descriptors of toxicity. To the best of our knowledge, this is the first report on this issue. Molecular Electrostatic Potential (MEP). The MEP is a useful tool that has been employed to understand a variety of chemical and physical properties (114–116), solvent effects (117, 118), chemical reactivities (119–121), molecular similarities (122), and intermolecular interactions (123–126). Additionally, the MEP is an indicator of the charge distribution in a molecule (127) where the regions with higher negative values of V(r) are richer in electron density, which is a suitable parameter to gauge the basicity and nucleophilicity of a molecule (128). For the purpose of the present research, it must be mentioned that the MEP has been used to rationalize the interaction between a biological active compound and its biomacromolecular target (129). In the field of the activators of AhR, the electrostatic properties of molecules have been used to explain some results. Thus, the MEPs of some polychlorinated dibenzodioxins have been used to rationalize their toxicities (130, 131). On the other hand, our group has computed the MEPs of 209 chlorinated biphenyls, finding that their toxicities can be correlated with the pattern of EP, where the most active congeners have positive values at the aromatic rings and highly negative values at the chlorine atoms (41). The MEP has been mapped on an isodensity surface, generating the corresponding MSEP (Figure 8). An advantage of the MSEPs is that it allows a rapid visual comparison of the EP pattern of several molecules. From a qualitative point of view, it is observed that the PCB-126 and PCB-169, the most active congeners, have a suitable (nearly) planar conformation and similar electrostatic regions: Chlorine atoms have high negative EPs, and both aromatic rings are slightly positive EPs. On the other side, PCB-209, the completely inactive congener,


has nearly neutral EPs on the chlorine atoms.7 Finally, the brominated analogue PBB-209 shows an intermediate situation: slightly negative EPs on the bromine atoms and medium-positive EPs on the aromatic rings. The difference in the EPs on the rings for PCB-209 and PBB-209 can be explained by the lower electronegativity of bromine than chlorine.8 The positive and negative regions of the EP on a molecular surface give a pictorial view of the polarities, which play an important role in intermolecular interactions. In a series of pioneering articles, the group of Politzer and Murray has shown that it is possible to extract useful quantitative information by a global analysis of the EP features on an isodensity molecular surface (76, 123), which have been used to link microscopic EP to macroscopic properties (133) and, more recently, to rationalize the biological activity of organic compounds (134). Politzer’s descriptors obtained from the MEPs of PBB-209, PCB-209, PCB-169, and PCB-126 (Table 3) give interesting results. Thus, except for V, which has a limited significance, the values of the descriptors for PBB-209 are between those of PCB-209 and those of the most active congeners, PCB-126 and PCB-169, and sometimes (Vmin, Vring, V-, σ+2, σ-2, and σtot2) much closer to PCB-126 and PCB-169. This numerical analysis corroborates the electrostatic similarity between PBB-209 and PCB-126 and PCB-169 despite the high conformational differences, and it confirms that the electrostatic features of xenobiotics are key factors in the activation of AhR. QM. As commented above, the QM is a useful property to analyze the charge distribution in halogenated biphenyls, especially for those having null or low dipole moments. In the field of AhR ligands, Mhin et al. have shown that the electrostatic properties are important to rationalize the toxicity of 76 polychlorinated dibenzo-p-dioxins (PCDDs), finding that all of the active congeners share a unique pattern of charge distribution, which can be quantitatively described by the QM (135). In a related work, Hirokawa et al. have reported the relationship of QM and toxicity of polychlorinated dibenzofuran (PCDFs) (136). These authors have related the sign of the components Qxx, Qyy, and Qzz with the binding affinity to AhR, reporting that the most active PCDDs and PCDFs have negative values of the Qxx component and positive values of the Qyy and Qzz components. As observed in the results of Table 4, the two most active congeners, PCB-126 and PCB-169, share the same pattern that the most active PCDDs and PCDFs (ligands of group I according to Mhin’s nomenclature), and as observed with these last kind of compounds, the highest activity is observed in the compound (PCB-126) having the larger absolute values of the components. On the other hand, both PCB-209 and PBB-209 have positive values of Qxx and negative values of Qyy and Qzz (ligands of group IV), which corresponds to the PCDDs and PCDFs that do not bind AhR directly. In accordance with Mhin et al., it is plausible that the binding site of AhR, besides having strict steric requirements, possesses electrostatic/polarity characteristics where the most active compounds can interact. Reactivity Descriptors Based on the Electronic Structures of the Molecules. Reactivity can be gauged based on thermodynamic principles, geometry, and electronic structure. The latter 7 The strongly positive value at the halogen atoms in the axis of the C-X bond is due to the nuclear contribution of the halogen atom. Recently, Politzer et al. (132) have recognized the importance of this positive regions (σ-hole) in intermolecular interactions (halogen bonding). Ongoing research in our group is trying to correlate positive values of the EP of the whole set of PCBs, previously reported (41), with the biological activity. 8 For a discussion between the EP on an aromatic ring and the electronegativity of the substituents, see ref (41).

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approach can be readily implemented within the framework of density functional theory (78). In the area of potential AhR activators, global and local reactivity descriptors have been used to analyze and to predict the reactive sites of several dibenzop-dioxins (137) and to explain the toxicity differences between PCB-126 and PCB-25 (2,2′,5,5′-tetrachlorobiphenyl) (138). All of the reactivity descriptors (Table 5) indicate that PBB209 is more reactive than PCB-209, having lower hardness η, higher polarizability R, and ionization potential I. Additionally, the brominated biphenyl possesses higher values of electron affinity A and electrophilicity ω, indicating a higher reactivity in reactions with electron-rich centers as well as an elevated tendency to be reduced through one-electron processes. In any case, these features can explain that, although neither PCB209 nor PBB-209 can directly bind the AhR, the brominated congener is more likely to be transformed in a compound able to activate AhR. Previously, Arulmozhiraja et al. have postulated that the toxic effects of PCDDs and PCDFs are partially due to the capacity of these compounds to act as electron acceptor (137); this hypothesis can be useful to explain the different behaviors of PCB-209 and PBB-209. The chemical and biochemical inertness of PCB-209 is also manifested by the energy difference between HOMO and LUMO, which is the highest of all of the studied compounds. When the reactivity descriptors of PBB-209 are compared with those of the most toxic congeners PCB-126 and PCB-169, it is observed that PBB-209 has higher softness, possesses a much higher polarizability (also due to the higher molecular volume of PBB-209), greater electron affinity and electrophilicity, and lower energy difference between HOMO and LUMO orbitals, having similar ionization potentials. All of these characteristics clearly indicate that PBB-209 is, from a chemical point of view, more reactive than PCB-126, PCB-169, and PCB209, and this higher reactivity can also be translated to a biological system, where PBB-209 can suffer a prior transformation to an activator of the AhR. On the other hand, the unfavorable geometry of PCB-209 along with its chemical inertness explains why this compound is unable to activate AhR. From the data in Table 5, it is observed that all of the coplanar (or nearly coplanar for PBB-209 and PCB-209) structures are more reactive than the noncoplanar structures, which is in agreement with reported results for PCB-25 (137). As expected, on the basis of the more profound change in geometry, the differences in the reactivity descriptors are higher for the decahalogenated biphenyls than for PCB-126 and PCB-169. Dissociation Energy of C-Cl and C-Br Bonds. The results obtained with the model compounds 1–6 (Tables 6 and 7) provide useful information on the feasibility of unimolecular reactions for PCBs and PBBs. The results of BDEs and reactivity descriptors are complementary, since the latter are applied to polar reactions,9 while the former model the chemical behavior in one-electron processes, generating a radical able to activate the AhR. The feasibility of the reaction has been gauged through the determination of the BDEs. In a previous report, Cioslowski et al. (139) calculated the BDEs of C-H and C-Cl of benzene and all of their chlorinated derivatives, showing that the BDEs of the C-Cl decrease and the C-H increase on augmenting the number of Cl atoms. Our results indicate that the abstraction of a bromine atom is easier than that of a chlorine atom (Tables 6 and 7). Additionally, it is observed that the facility of radical generation is slightly 9

Reactions where the studied compounds can gain or lose one or more electrons (that is, in oxidation/reduction as well as in electrophile/nucleophile reactions).

Alonso et al.

dependent on the position of the halogen atom. More interesting for the purpose of comparing PBB-209 and PCB-209 is the fact that the dissociation energies of the C-X bond decrease considerably on augmenting the number of halogen atoms, and this effect is more pronounced for the polybrominated analogue. As a consequence, the generation of radicals from hexabromobenzene is over 15 kcal mol-1 less energy demanding than for hexachlorobenzene. Therefore, we can affirm that the generation of radicals would be easier for PBB-209 than for PCB-209, and this dissimilarity can influence the different activation of the AhR.10

Conclusion The activation of the AhR by decabromobiphenyl (PBB-209), which from a geometrical point of view is totally unable to fit on the proposed binding site of the receptor, discards the generally accepted view that the AhR is only activated by planar molecules and raises the question on the possible mechanisms of AhR activation. Because such activation is an initial step in the biological activity of many toxicants and pharmaceuticals, the understanding of the molecular features of the AhR activators is an important issue to deal with. Thus, although PBB-209 and PCB-209 share similar geometries and QM patterns, two properties likely to influence the direct binding to AhR, their diverse biological behaviors can be explained by differences in the reactivity and EP. On one hand, all the reactivity parameters that we have computed, both in polar and radical reactions, show that PBB209 is much more reactive than PCB-209, which opens the possibility that PBB-209 activates AhR through a mechanism involving a previous metabolic transformation. Additionally, it is noteworthy that some molecular features of PBB-209, such as the EP and EP-derived descriptors (Politzer’s parameters), indicate that PBB-209 is more similar to PCB-126 and PCB-169 than to PCB-209, which share quite similar geometries based on the substitution pattern. It is wellestablished that the electrostatic interaction plays a key role in the recognition process between a small molecule and its target biomacromolecule and that the MEP is a valuable property to relate structure and toxicity. The electrostatic similarity between PBB-209, PCB-126, and PCB-169 seems to hint that these three compounds can share, at least partially, similar mechanisms of activation of AhR. It is unquestionable that PCB-126 and PCB169 directly bind AhR and that PBB-209 does not; these results suggest that there are several simultaneous mechanisms for activation of AhR and the most active compounds could act for more than one mechanism. Considering the results presented herein, it must be emphasized that any structure–activity study of AhR and P450 activators should contain a comprehensive set of molecular properties, which include, as in the present article, geometrical data and dynamical features, electrostatic properties (dipole and higher-order moments, EP, and polarizability), aromaticity indices, reactivity descriptors, and studies on potential reaction mechanisms. Acknowledgment. This work was taken in part from the projected Ph.D. thesis of M.A. and S.C. This paper is dedicated to Professor Miguel Yus (University of Alicante) on the occasion 10 The computed results are the expected results based on chemical reasons. The dissociation energy of the C-Br bond is still too high to be feasible in vivo without the involvement of an enzymatic system. The objectives of the present study have been indicated in the Results section.


of his 60th birthday. We thank the Spanish Ministry of Education and Science (Projects CTQ2004-01978 and CTQ20076481/BQU) and INIA (Project RTA 2006-00022) for financial support and the Centro de Supercomputación de Galicia (CESGA) for the use of the Compaq HPC 320 supercomputer. M.A. thanks the Spanish Ministry of Education and Science for a FPU fellowship. Valuable comments from the reviewers are also acknowledged. Supporting Information Available: Cartesian coordinates of the optimized geometries of the minima and transition states, experimental and calculated bond lengths and dihedral angles, aromaticity indices, Politzer’s descriptors, results (MSEPs, reactivity descriptors, and BDEs) calculated at the B3LYP/631+G(d,p) theory for PBB-209, PCB-209, PCB-169, and PCB126, and calculation of the quadrupole moment. This information is available free of charge via the Internet at http://pubs.acs.org.

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