Relationship between aryl hydrocarbon receptor binding, induction of

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Chem. Res. Toxicol. 1993,6, 328-334

328

Relationship between Aryl Hydrocarbon Receptor Binding, Induction of Aryl Hydrocarbon Hydroxylase and 7-Ethoxyresorufin 0-Deethylase Enzymes, and Toxic Activities of Aromatic Xenobiotics in Animals. A New Model Sherif A. Kafafi,* Hussein Y. Afeefy,*l+Hakim K. Said, and Abdel G. Kafafi*J Department of Environmental Health Sciences, The Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205 Received October 7, 1992

A new mathematical model relating the affinities of aromatic xenobiotics for the aryl hydrocarbon receptor (AhR) to their potencies as aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin 0-deethylase inducers and toxic activities in animals is reported. Taking polychlorinated dibenzo-p-dioxins (PCDDs) as examples, the AHH activity of a PCDD relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is shown to be analytically related to corresponding relative affinities for AhR, and electronic energy gaps of PCDD and TCDD. (The electronic energy gap of a chemical is the difference between its ionization potential and electron affinity.) The reported model is capable of qualitatively explaining and quantitatively estimating potencies of PCDDs and related xenobiotics as AHH inducers in rat hepatoma H-4-11 E cells in culture. Therefore, a PCDD is expected to be a potent AHH inducer if its affinity for AhR is high and has a smaller energy gap than TCDD. In addition, it is shown that the derived equations for AHH induction by PCDDs apply equally well to 7-ethoxyresorufin 0-deethylase (EROD) activities; that is, there is a 1:l correspondence between AHH and EROD activities for PCDDs, in agreement with experimental findings. Furthermore, in harmony with experimental observations, AHH (and EROD) activities of PCDDs relative to TCDD parallel the corresponding toxic equivalency factors and AhR mediated in vivo toxicities of these xenobiotics in animals, such as thymic atrophy, body weight loss, and acute lethalities. Moreover, the developed methodology for AHH and EROD induction by PCDDs is shown to apply t o polychlorinated dibenzofurans, thus, eliminating cross-class comparison problem of traditional structure-activity studies. Last, it is demonstrated that trends in metabolic activation of polyhalogenated aromatics could be explained in terms of their binding to AhR and potencies as AHH inducers.

I. Introduction, The aryl hydrocarbon receptor (AhR)' is a cytosolic protein capable of binding a wide variety of aromatic xenobiotics (AXs) (1-4). It is known that AhR controls the induction of aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin 0-deethylase (EROD) and related enzymes, and several toxic endpoints in animals such as thymic atrophy, body weight loss, immunotoxicity, and acute lethality (1-7). The induction of AHH via AhR is important in metabolizing AXs into polar intermediates which in many cases could react with cellular macromolecules and cause cytotoxicityand cancer. However, despite their high potencies as AHH inducers, toxic polyhalogenated aromatics are poorly metabolized (3-7). That is,

* Authors to whom correspondence

should be addressed. Present address: The National Institute of Standards and Technology, Chemical Kinetics and Thermodynamics Division, Building 2221A260, Gaithersburg, MD 20899. Present address: Dynex International, Inc., 1194 Fairmeadow Trail, Oakville, ON L6M 2M7, Canada. I Abbreviations: AhR, aryl hydrocarbon receptor; AHH, aryl hydrocarbon hydroxylase; AX, aromatic xenobiotic; DD, dibenzo-p-dioxin; diC1EA, electron affinity; E C S O ( p ~aryl ~~), DD, dichlorodibenzo-p-dioxin; hydrocarbon hydroxylase activity of a polychlorinated dibenzo-p-dioxin; EROD, 7-ethoxyresorufii 0-deethylase; E,, energy gap; heC1-DD, hexachlorcdibenzo-p-dioxin; hpC1-DD,heptachlorodibenzo-p-dioxin;IP,ionization potential; L, lipophilicity; PCDD, polychlorinated dibenzo-p-dioxin; peC1DD, pentachlorodibenzo-p-dioxin; QSAR, quantitative structure-activity teC1relationship; S, entropy; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; DD, tetrachlorodibenzo-p-dioxin;trC1-DD, trichlorodibenzo-p-dioxin; TEF, toxic equivalency factor. +

the latter compounds are not bioactivated to toxic metabolites in order to elicit their responses. Therefore, unlike polycyclic aromatic hydrocarbons,the toxicities of potent polyhalgenated AXs are due to the parent compounds. Indeed, the results of in vivo and in vitro experiments demonstrated that a relationship exists between structure-AhR binding, structure-enzyme induction, and structure-toxicity for polyhalogenated aromatics (1-7). However, to date, this relationship has not been mathematically formulated in any form. Recently, Kafafi et al. (8, 9) reported a new model for AhR binding based on electron affinities, entropies, and lipophilicities of the ligands. The latter quantities were found to be key electronic and thermodynamic properties which control binding of AXs to the cytosolic protein. Unlike classical quantitative structure-activity relationship (QSAR) approaches,the reported AhR affinity model did not use multiple regression analysis in its formulation, was shown to be ,reliable for estimating the equilibrium dissociation constants of AXs-AhR complexes, explained the origin of corresponding QSARs in a physically consistent way, and was capable of overcoming the crossclass comparison inherent to traditional approaches. In this report, we derive quantitative relationships between binding affinities of polyhalogenated AXs to AhR and correspondingpotencies as AHH and EROD enzymes inducers and in vivo toxicities. Taking polychlorinated dibenzo-p-dioxins (PCDDs) as examples, we will first

0S93-228~193/2106-0328$04.0010 0 1993 American Chemical Society

Chem. Res. Toxicol., Vol. 6, No. 3, 1993 329

AHH, EROD,and Toxic Potencies of A X s

compute their AHH activities, E C ~ C in~rat, hepatomaH-4I1 E cells in culture. Then, we establish an AHH potency scale relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for these xenobiotics which correlates with some of their AhR mediated in vivo toxicities in animqls such as thymic atrophy, body weight loss, and acute lethality. In addition, we will show that the reported model for AHH induction by PCDDs could be used to estimate corresponding EROD activities, and acute lethalities, LDm, in animals. Furthermore, we demonstrate that the electronic and thermodynamic descriptors which quantify AHH and EROD inductions by PCDDs apply equally well to polychlorinated dibenzofurans (PCDFs),thus eliminating the cross class comparison problem of traditional QSARs. Moreover, we illustrate that metabolism of polyhalogenated aromatics could be explained in terms of their affinities for AhR and AHH induction potencies. Last, we emphasize the advantages of developed methodologies over classical QSARs.

11. Results and Discussion (i) Binding of PolyhalogenatedAromatics to AhR. The results of previous experimental and theoretical studies lead one to conclude that the interaction between AXs and AhR is of the stacking type where some electronic charge is transferred from the receptor to AX (8-11). Accordingly, consider the case of two aromatic xenobiotics, AX1 and AX2, at equilibrium with their AhR complexes, AX1-AhR and AXP-AhR, as given by eq 1. AX1-AhR

+ AX2 + AX2-.AhR + AX1

(1)

Kafafi et al. ( 4 9 ) showed that the equilibrium constant of reaction 1 is given by eq 2

K = exp(AS/R) exp(-AH/RT) exp(-AGow(Ax,,/RT)X exP(AGow(Ax,)/RT) (2) where, Kdl and Kd2 are equilibrium dissociation constants of AXl.-AhR and AXBe-AhR complexes, respectively. AH = EA(AX1) - EA(AX2), and EA(AX1) and EA(AX2) are the electron affinities of AX1 and AX2, respectively. AGow(~xl) and AGow(~x2) are free energies of partitioning of AX1 and AX2 between a lipophilic phase (octanol) and water. If AX1 is TCDD and AX2 is any other PCDD, we get eq 3 (8,9) Kd(PCDD)

--

[L(~CDD,/L(TCDD)I Kd(TCDD)exp(ASIR) exp W W R T ) (3) where, AEA and A S are given by eqs 4 and 5: AEA = (EATcDD - EApcDD)

(4)

(5) A S = (SPCDD - STCDD) As it is clear from eqs 3-5, a PCDD is expected to have high affinity for AhR if it is less lipophilic and has a higher EA and smaller entropy than TCDD (8, 9). Bradfield et al. (IO) suggested that ligand binding to AhR seems to conform to the model proposed for steroid hormone receptors in which (11) slow

fast

slow

L + AhR + L-AhR * La-AhR’ + L + AhR’ (6) where L is a ligand and L.-AhR’ is a transformed ligand-

Table I. Affinity of 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD) for Liver Aryl Hydrocarbon Receptors (AhRs) in Some Animal Species source of receDtor

Kd ( n M P

rat C57BLJ6 mice B6D2FlIJ mice hamster guinea pig gerbil chick embryo (7DI) monkey

0.22; 0.12; 0.44 0.52; 0.29 0.42 0.33; 0.22 0.16 0.20 0.20 0.26

~

_

_

_

_

Reference 13.

receptor species that can bind to DNA. Clearly, eqs 1and 6 are notably different. Accordingly, we ask the following question: “Do we need to account for the additional steps of eq 6 in the reported model?” Recent sudies by Reyes et al. (12) demonstrated that AhR is not a member of steroid hormones receptors superfamily and that initial binding of ligands to AhR is much slower than the transformation step (4,6,7,13). Since in kinetic studies, the rate-determining step of a reaction is always a slow one, and given that binding affinities of aromatic ligands to AhR obey the law of mass action (lo),we conclude that the initial binding step of polyhalogenated AXs to AhR controls their affinities for the protein and is a key determinant of their toxicities. In addition, the results of Bradfield et al. (10) studies showed that the time course of ligand-receptor dissociation is biphasic, suggesting two ligand-receptor species with different dissociation rate constants, eq 6. Since the ligand-receptor conformational change is a fast process, it is straightforward to verify that even by assuming that eq 6 is correct for the case of a single ligand binding to AhR, it will reduce to eq 1when two substrates are considered. Therefore, electron affinities, lipophilicities, and entropies of polyhalogenated aromatics are sufficient physicochemical properties for quantifying their affinities for AhR (8, 9). Moreover, it is known that different forms of AhRs exist in animals (4,6, 13). Despite differences in physical properties of AhRs, the results of binding affinity studies demonstrated that the nature of ligand binding sites in these proteins are likely to besimilar (4,13). Taking TCDD as an example, it is clear from Table I that the equilibrium dissociation constants of TCDD.-AhRs complexes in liver cytosols of several animal species are comparable; that is, TCDD is experiencing similar electrostatic interactions when it binds to any of these AhRs. Accordingly, there is no reason to believe that any other polyhalogenated aromatic compound will have a wide range of binding affinities to these cytosolic proteins. Therefore, the reported methodology is likely to be useful for predicting the affinities of polyhalogenated aromatics for liver cytosolic AhRs in these animals, and possibly other organs in mammals containing high concentrations of the protein. (ii) Relationship between AhR Binding, Enzyme Induction, and Toxic Activity. How can we relate the affinities of PCDDs for AhR to their potencies as AHH inducers? The results of in vivo and in vitro experiments demonstrated that a nonlinear relationship exists between structure-AhR binding and structure-enzyme induction for polyhalogenated AXs (1-7). That is, there is a poor linear correlation between AhR binding and corresponding enzyme induction for polyhalogenated aromatics (4, 7). Therefore, the latter relationship could not be quantified using traditional QSAR studies because

330 Chem. Res. Toaicol., Vol. 6, No. 3, 1993 Table 11. Calculated and Experimental Potencies of Some PCDDs and PCDFs as AHH Inducers in Rat Hepatoma H-4-11 E Cells in Culture

2,8-diCl-DD 2,3,7-trCl-DD 2,3,7,8-teCl-DD 1,3,7,8-teCl-DD 1,2,3,4,7-peCl-DD 1,2,4,7,8-peCl-DD 1,2,3,7,8-peCl-DD 1,2,3,4,7,8-heCl-DD 1,2,3,6,7,8-heCl-DD 1,2,3,4,6,7,8-hpCl-DD 2,3,7,8-teCl-DF 1,2,3,7-teCl-DF 2,3,4,7-teCl-DF 1,2,3,7,8-peCl-DF 2,3,4,7,8-peCl-DF 2,3,4,7,9-peCl-DF 1,2,3,4,7,8-heCl-DF 1,2,3,6,7,8-heCl-DF 2,3,4,6,7,8-heCl-DF

76.0 14.0 1.0 120.0 1000.0 1000.0 35.0 150.0 150.0 2600.0 1.0 25.6 2.0 26.4 2.1 26.4 27.6 27.6 27.6

0.24 0.12 0.00 0.09 0.02 0.02 -0.05 -0.06 -0.06 -0.13 0.00 0.13 0.05 -0.02 -0.03 -0.02 -0.03 -0.02 -0.03

1.4 X 1.7 X 7.2 X 4.0 X 1.7 X 1.7 X 2.2 X 6.2 X 6.2 X 5.2 X 3.9 x 2.5 X 6.5 X 4.4 x 2.3 X 4.4 x 3.0 X 4.6 X 1.2 X

lo-: lo-" lo-' lo-'

10-10 10-6 10-9 10-9

>LO X 3.6 X 7.2 X 5.9 X 6.6 X 2.1 X 1.1 X 2.1 X 1.5 X

25

y = 0.540 + 0 . 9 7 0 ~; R = 0.964

2

20-

.$ lo-' lo-" lo-'

lo-;

3.9 x 10-10 2.7 X 1.8 X 2.5 x 10-9 2.6 X 1O-Io 7.9 x 10-9 2.1 X 1.5 X 6.9 X

f

..

15-

51

ME ?

10-

57

'

1

'

'

'

'

I

It is interesting to note that despite the fairly high affinity of 2,B-dichloro-DD for AhR, it is the least potent 10-lo AHH inducer due to its large E,. On the other hand, See eqs 6 and 9 in ref 9. Note that X stands for furan or dioxin. 1,2,3,7,8-pentachloro-DD and 1,2,3,4,7,8-and 1,2,3,6,7,8T = 273 K. AHH potencies of PCDDs and PCDFs were calculated hexachloro-DDs have slightly smaller E,s than TCDD, from eqs 8 and 9, respectively. References 4 and 7. and yet, their potencies as AHH inducers are less than TCDD. Clearly, this is due to the lower affinities of these the multiple regression equations used in this approach congeners for AhR compared to TCDD. Furthermore, always assume a linear correlation between structural 1,2,3,4,6,7,8-heptachloro-DD has an affinity for AhR that descriptors of xenobiotics and corresponding biological is almost 3 orders of magnitude lower than TCDD. The activities (4, 7,13,14). However, recently Kobayashi et corresponding calculated AHH activity is only 10 times al. (15) found a correlation between logarithms of AHH less than TCDD due to the smaller E, of heptachloroactivities of selected PCDDs in the liver of chicken embryo congener compared to the latter. Therefore, high affinity and the difference between IP and EA of individual for AhR seems to be a necessary but not a sufficient congeners. Since most toxic PCDDs and related xenocondition for a PCDD to be a potent AHH inducer. In biotics are potent AHH inducers and have high affinities addition, it is important to note that although lipophifor the cytosolic protein (1-7), we hypothesize that the licities, electron affinities, and entropies of AXs are potency of a PCDD as an AHH inducer, EC~O(PCDD), has important descriptors of their affinities for AhR, electronic the following dependence on its affinity for AhR and IP energy gaps, the difference between ionization potentials and EA of PCDD and electron affinities, are key determinants of AHH EC50(PCDD) OC Kd(PCDD) exp[Eg(PCDD)/Rm (7) potencies. Polychlorinated dibenzofurans are structurally related Where = is the proportionality sign, E g ( p c ~=~ )IP(PCDD) to PCDDs. Therefore, we expect that the developed - E A ~ c D D1 and ) is known as the electronic energy gap (8, methodology for AHH induction by PCDDs should apply 15-17), R is the universal gas constant, and T the equally well to PCDFs. Accordingly, we write eq 9 temperature in kelvins. Since PCDDs are structurally related compounds, EC~O(PCDD) relative to EC~O(TCDD) takes -ECSO(PCDF) the form [Kd(PCDF)/Kd(TCDF)] EC50(TCDF) e x p ( M / R n (9) -EC,O(PCDD) where hE = E,(PcDF) - E g ( ~ c and ~ ~ the ) , other terms in eq [Kd(PCDD)/Kd(TCDD)] EC50(TCDD) e x p ( M / R n (8) 9 are similar to corresponding ones given by eq 8 for PCDDs. As shown in Table 11, the agreement between where AI3 = E,(PCDD) - E,(TcDD). As it is clear from eq 8, calculated and experimental potencies of PCDFs as AHH a PCDD is expected to be a potent AHH inducer if its inducers is fairly good. In addition, from Figure 1 it is affinity for AhR is high and has a smaller E, than TCDD. clear that there is a linear correlation between the natural The criteria for high affinity binding of ligands to AhR are logarithms of experimental and calculated EC50s for well documented in the literature (1-10). In Table 11, we PCDDs and PCDFs whose slope is close to unity and nearly compare calculated and in vitro potencies of PCDDs as passing by the origin. Therefore, we conclude that for AHH inducers in rat hepatoma H-4-11E cells in culture. any class of polyhalogenated AXs, electron affinities, Computation of the various terms of eq 8 using ab initio lipophilicities, entropies, and energy gaps of the ligands and AM1 quantum chemical methods was described are likely to be key electronic and thermodynamic propelsewhere (8, 9, 16, 17). As shown in Table 11, there is erties controlling their affinities for AhR as well as good agreement between calculated and experimental potencies as AHH inducers. Furthermore, it is clear that AHH activities for PCDDs. Therefore, we conclude that the reported model overcomes cross-class comparison the affinity of a PCDD for AhR is nonlinearly related to problem of classical QSARs on these AXs. its AHH potency. In addition, electron affinities, ionAs discussed above, traditional QSARs using linear ization potentials, lipophilicities, and entropies of PCDDs regression analysis on PCDDs, PCDFs, polychlorinated seem to be key electronic and thermodynamic properties bi-, tri-, and tetraphenyl, and corresponding polybromicontrolling AHH induction.

Chem. Res. Toxicol., Vol. 6, No. 3, 1993 331

AHH,EROD, and Toxic Potencies of AXs 25 1

y = 0.560

+ 0 . 9 7 0 ~; R = 0.941

Table IV. Comparison between Calculated AHH Potencies, Toxic Equivalency Factors (TEFs) in Animals, Average Thymic Atrophy (TA), and Average Body Weight Loss (BWL) in Rats of Some PCDDS and PCDFs Relative to TCDD

/

5

10

15

25

20

- Ln EC50 ;calculated Figure 2. P l o t of calculated -In EC5o of PCDDs a n d PCDFs for

AHH induction versus experimental EROD potencies in r a t hepatoma H-4-11 E cells in culture. Table 111. Calculated and Experimental EROD Potencies of Some PCDDs and PCDFs in Rat Hepatoma H-4-11 E Cells in Culture polyhalogenated AX

EROD potency (M) calculated" experimentalb

2,8-diCl-DD 2,3,7-trCl-DD 2,3,7,8-teCl-DD 1,3,7,8-teCl-DD 1,2,3,4,7-peCl-DD 1,2,4,7,8-peCl-DD 1,2,3,7,8-peCl-DD 1,2,3,4,7,8-heCl-DD 1,2,3,6,7,8-heCl-DD 1,2,3,4,6,7,8-hpCl-DD 2,3,7,8-teCl-DF 2,3,4,7-teCl-DF 1,2,3,7-teCl-DF 2,3,4,7,8-peCl-DF 2,3,4,7,9-peCl-DF 1,2,3,7,8-peCl-DF 1,2,3,4,7,8-heCl-DF 1,2,3,6,7,8-heCl-DF 2,3,4,6,7,8-heCl-DF

1.0x 1.1x 8.0 X 3.5 x 1.5 x 1.5 x 2.4 x 6.2 x 6.2 x 5.3 x 4.1 X 6.2 x 2.0 x 2.5 X 4.7 x 4.7 x 3.2 x 4.7 x 1.4 X

" See the equation in Figure

2. References 4 and 7.

10-4 10-7 lo-" 10-7 10-7 10-7 10-9 10-9 10-9 10-9 10-9 10-5 10-9 10-9 10-9 10-9

>LO x 1.4 x 1.9 x 3.2 x 8.2 x 1.1 x 1.7 X 4.1 x 1.2 x 3.9 x 1.5 X 6.3 x 1.3 X 5.8 x 3.1 X 3.4 x 1.2 x 5.8 X

10-4 10-7 10-10

10-7 10-7 10-8 10-9 10-9 10-10 10-5 10-9 10-10 10-9 10-lo

nated, polyiodinated, and mixed polyhalogenated analogues cannot be applied to quantify the relationship between AhR binding and AHH potencies ( 4 , 7 ) . Clearly, this is due to the fact that these biological processes are not linearly related, as the results of in vivo and in vitro experiments demonstrate ( 4 , 7 ) . However, Romkes et al. (14)found a linear correlation between AhR binding and corresponding AHH potencies in rat hepatoma cells for a series of 2-substitued-3,7,8-dibenzodioxins. In the latter work, the affinities of the majority of dioxins studied for AhR and corresponding AHH potencies varied by less than an order of magnitude. In addition, the methyl derivative and 2,3,7-trichloro-DD were treated as outliers and were not included in regression analysis. Therefore, the study by Romkes et al. (14) does not imply that there is a linear correlation between receptor binding and AHH potencies for these xenobiotics. The above mathematical model for AHH induction by PCDDs and PCDFs in rat hepatoma cells applies equally well to estimate their potencies as EROD inducers. As shown in Figure 2, there is a linear correlation between the natural logarithms of calculated potencies of PCDDs and PCDFs as AHH inducers in rat hepatoma cells and corresponding experimental EROD values. That is, the mechanisms of induction of AHH and EROD enzymes by

in vivo potencyb BWL TA

EC~orlY"r)/ ECSO(PCDXI~ TEFb

PCDX" 2,8-diCl-DD 2,3,7-trCl-DD 2,3,7,8-teCl-DD 1,3,7,8-teCl-DD 1,2,3,4,7-peCl-DD 1,2,4,7,8-peCl-DD 1,2,3,7,8-peCl-DD 1,2,3,4,7,8-heCl-DD 1,2,3,6,7,8-heCl-DD 1,2,3,4,6,7,8-hpCl-DD 2,3,7,8-teCl-DF 2,3,4,7-teCl-DF 1,2,3,7-teCl-DF 2,3,4,7,8-peCl-DF 2,3,4,7,9-peCI-DF 1,2,3,7,8-peCl-DF 1,2,3,4,7,8-heCl-DF 1,2,3,6,7,8-heCl-DF 2,3,4,6,7,8-heCl-DF

OC

0 1 0.0002 0.0004 0.0004 0.2 0.08 0.08 0.1 0.2 0.01 0 0.3 0.02 0.01 0.02 0.02 0.6

0 0 1 0.001 0.005 0.005 0.5 0.04 0.04 0.01 0.1 0.001 0.001 0.1 0.001 0.1 0.01 0.01 0.01

0 0 1 0.0004 0.001 0.001 0.08 0.03 0.03

0 0

1 0.0009 0.008 0.008 0.5 0.08 0.08 0.03 0.003 0.003 0.3 0.02 0.05 0.1 0.1 0.1

0.02 0.0006 0.0006 0.05 0.002 0.02 0.03

0.03 0.03

a The X stands for furan or dioxin. Reference 18. Value less than 0.00001.

Table V. Calculated and Experimental LD5os (pg/kg body weight) of Some PCDDs in Rats and Mice rat

mouse

PCDD

LD5&a1cp LDao(exp)b LD50(calc)~LD,50(exp)b 1.2 x 108 1.2 x 108 2.1 x 108

200' 1.7 x 105 1077 3173 3173 3173

8.5 X lo8

2.1 X 2.1 x >1.5 X 10; 3.7 X 200c 34c 5.2 X 337.5 183 825 539 1250 539 >1440 539

10' 10: 10:

>5 X lofi >1 x 106 >5 X lo6

10'

34' >1 X

lofi

Calculated fromeq 10; T = 310K. Reference 19. Average value.

PCDDs and PCDFs are similar and seem to be controlled by AhR, in agreement with experimental observations (4, 7). Furthermore, it is clear from Figures 1 and 2 that there is a 1:l correspondence between AHH and EROD induction by PCDDs and PCDFs. Accordingly, a polyhalogenated AX is expected to be a potent EROD inducer if its affinity for AhR is high and has a small energy gap compared to TCDD, Tables I1 and 111. In addition, as discussed above, we expect that high affinity binding to the cytosolic protein is a necessary but not a sufficient condition for a polyhalogenated aromatic to be a potent EROD inducer. Furthermore, the electronic and thermodynamic properties of PCDDs and PCDFs controlling their potencies as AHH inducers are the same descriptors that quantify EROD induction. The results of experimental studies showed that polyhalogenated AXs produce similar patterns of biochemical and toxic responses in animals (1-7) and that their potencies as AHH inducers correlate with AhR mediated toxicities in mammals (1-7, 13-15). In Table IV, we compare AHH activities of some PCDDs and PCDFs relative to TCDD with corresponding toxic equivalency factors (TEFs) in animals and the average thymic atrophy and average body weight loss in rats ( 1 4 ) . As shown in Table IV, there is good correspondence between relative AHH activities of PCDDs and PCDFs, TEFs, and in vivo potencies. The data in Table IV suggest that the mech-

332 Chem. Res. Toxicol., Vol. 6, No. 3, 1993

Kufufi et al. Table VI. Affinities for AhR, Potencies as AHH Inducers, and Estimated RBFs of Some PCDDs and PCDFs

/ Ci

E

10 -

y=

0.0443 + 0.814~; R = 0.98

y = - 0.892 + 1.039~; R e 0.98 C

4

0

1

Rat Mouse

I

0

10

20

Ln [LDM(PCDD)ILD50(TCDD)I;calc.

Figure 3. Plot of experimental versus calculated acute lethalities, pg/kg body weight, of PCDDs relative t o TCDD in rats and mice: W rat; 0 mouse.

anism of AHH induction by polyhalogenated aromatics seem to parallel the mechanism of toxicity of these xenobiotics in animals. Therefore, the reported model could be useful for quantifying AhR mediated biological and toxic activities of polyhalogenated AXs in animals. Moreover, since polyhalogenated AXs produce similar patterns of AhR mediated biological and toxic responses in animals (1-28), and AHH activities of a given class of polyhalogenated aromatics correlate with their toxicities, we expect that acute lethalities (15, 18-20), LD~os,of PCDDs and PCDFs in mammals could be estimated from eq 10

where X stands for furan or dioxin, and the AE term in eq 10 corresponds to those used in eq 8 and 9 for PCDDs and PCDFs, respectively. In Table V, we compare calculated and experimental lethalities of some PCDDs in rats and mice. We could not find sufficient LDbos for PCDFs in these two species. As shown in Table V, the agreement between estimated and in vivo lethalities is reasonable within less than a factor of 4. In addition, from Figure 3, it is clear that there is a linear correlation between calculated and experimental L D m for PCDDs. Therefore, acute lethalities of polyhalogenated aromatics in animals are likely to be mediated by AhR. In addition, the above physicochemical properties of polyhalogenated AXs are good quantitative descriptors of toxicological processes controlled by the cytosolic protein. It is interesting to note that despite differences in absolute lethalities of PCDDs in rats and mice, LDSO(PCDD) relative to TCDD seems to be similar in these two animal species. The latter observation holds fairly well for other mammals (18,19). It is straightforward to verify that the natural logarithms of LD~O(PCDD)S are expected to have a linear correlation with corresponding AHH and EROD potencies. Accordingly, a PCDD is expected to be more lethal than TCDD in an animal species if its affinity for AhR is high and has a small electronic energy gap. Having established quantitative relationships between receptor binding, enzymes induction, and some in vivo toxic endpoints, it is important to ask: Is there a relationship between the affinities of potyhalogenated AXs to AhR, potencies as AHH inducers, and their metabolic activation? we discuss this point in the next section.

polyhalogenated AhR AHH AX affinity (M)O potency (M)* RBFc 2,8-diCl-DD 7.6 X 1.4 X 25584.8 2,3,7-trCl-DD 1.4 X 1.7 X lo-' 168.7 2,3,7,8-teCl-DD 1.0 x 10-9 7.2 x 10-11 1.0 1,3,7,8-teCl-DD 1.2 x 10-7 4.0 x 10-7 46.3 1,2,3,4,7-peCl-DD 1.0 X 1.7 X lo-' 2.4 1,2,4,7,8-peCl-DD 1.0 x 10-6 1.7 x 10-7 2.4 1,2,3,7,8-peCl-DD 3.5 x 10-8 2.2 x 10-9 0.90 1,2,3,4,7,8-heCl-DD 1.5 X lo-' 6.2 X 10-9 0.60 1,2,3,6,7,8-heCl-DD 1.5 X 6.2 x 10-9 0.60 1,2,3,4,6,7,8-hpCl-DD 2.6 X 5.2 X 0.030 2,3,7,8-teCl-DF 2.5 x 10-9 3.9 x 10-10 2.2 2,3,4,7-teCl-DF 5.0 x 10-9 6.5 x 10-9 18.1 6.4 X 1,2,3,7-teCLDF 2.5 X 5425.3 2,3,4,7,8-peCl-DF 5.2 X 2.3 X 10-lo 0.60 2,3,4,7,9-peCl-DF 1.7 x 10-7 4.4 x 10-9 0.36 1,2,3,7,8-peCl-DF 6.6 X 4.4 X 0.93 1,2,3,4,7,8-heCl-DF 6.9 X 3.0 X 0.60 1,2,3,6,7,8-heCl-DF 6.9 X 4.6 X 0.93 2,3,4,6,7,8-heCl-DF 2.7 X 10-9 1.2 X 0.62 Calculated from eq 3. See ref 9. b Calculated from eq 8. Calculated from eq 11.

(iii) Role of AhR Binding and Enzyme Induction in Polyhalogenated Aromatics Metabolism. Taking PCDDs as examples, let us see how their affinities for AhR and potencies as AHH inducers might affect their metabolic activation. As discussed in sections i and ii, polyhalogenated AXs having higher electron affinities and lower entropies and lipophilicities than TCDD are expected to bind strongly to AhR, eq 3 (8,9). In addition, polyhalogenated aromatics whose energy gaps are smaller than that of TCDD and have high affinities for AhR were found to be potent AHH inducers, eq 8. Accordingly, let us define a simple function which we call relative bioactivation factor (RBF) by eq 11:

RBF = [ECSO(PCDD)/Kd(PCDD)]/[EC50(TCDD)/Kd(TCDD)] (11) Notice that eq 11is somewhat similar to eq 8. The reason for defining RBF as given by eq 11will become apparent from the following discussion. TCDD is highly resistant to metabolism in the majority of animal species, and is also the most toxic. With TCDD as the reference compound, its RBF value is thus equal to unity. Equation 11impliesthat bioactivation of a PCDD depends on its affinity for AhR and potency as AHH inducer. Therefore, dioxins having RBFs similar to that of TCDD are expected to be extremely difficult to metabolize by animals, Table VI, and thus are likely to be potent toxins. On the other hand, PCDDs whose RBFs are larger than that of TCDD are likely to be rapidly bioactivated, metabolized, and eliminated. As shown in Table VI, 2,B-dichloro-DD has a fairly high affinity for AhR, yet its estimated RBF is much greater than that of TCDD. Clearly, this is due to its low potency as AHH inducer and rapid metabolism. As the number of chlorine atoms increase, the corresponding RBFs of dioxins generally decrease, that is, metabolic activation of PCDDs become more difficult. It is interesting to note how RBFs of PCDDs depend on the positions of chlorine atoms. For example, while TCDD is not metabolized by the majority of animal species, 1,3,7,8-tetrachloro-DD is expected to be readily bioactivated. Similar to TCDD, 1,2,3,7,8pentachloro-DD, RBF = 0.9, is expected to be poorly metabolized in animals. Furthermore, despite the low

AHH, EROD, and Toxic Potencies of A X s

affinity of 1,2,3,4,6,7,8-heptachloro-DD for AhR and weaker potency as AHH inducer compared to TCDD, its RBF reflects that this congener is probably impossible to metabolize by mammalian species. The above arguments developed for PCDDs apply equally well to PCDFs. For example, 2,3,7,84etrachloroDF has an RBF = 2.2, Table VI. Clearly, this is due to its lower affinity for AhR and slighlty weaker inducing ability of AHH compared to TCDD, Table VI. Accordingly, we expect that 2,3,7,8-tetrachloro-DF to be poorly metabolized in animals. Similarly, penta- and hexachloroDFs listed in Table VI, RBFs slightly less than unity, are also expected to be resistant to bioactivation in animals. Do the above predictions based on AhR binding and enzyme induction conform with the results of metabolic studies on polyhalogenated AXs in animals? There is a strong correspondence between above RBFs of polyhalogenated aromatics and their metabolism in animals. Indeed, the results of in vivo experiments on laboratory animals showed that polyhalogenated AXs containing one to three halogen atoms are bioactivated and excreted fairly rapidly by animals (21-24).In addition, it is believed that metabolic activation of AXs to corresponding more polar hydroxylated products proceeds mainly via an arene oxide intermediate (21-24)and to a much lesser extent by direct insertion of a hydroxyl group into unhalogenated carbon atoms (21-24). Both routes of bioactivation become increasingly difficult as the number of halogen atoms attached to the aromatic moiety increase (21-24).These experimental observations conform fairly well with above predictions based on estimated RBFs of polyhalogenated aromatics. That is, metabolism of PCDDs, PCDFs, and related xenobiotics having adjacent unhalogenated carbon atoms proceed fairly rapidly, e.g. 2,8-dichloro-DD, 2,3,7-trichloro-DD, and 1,2,3,7-tetrachloro-DF. Notice that these compounds have RBFs >> 1, Table VI. On the other hand, the congeners that are highly resistant to metabolism, RBFs equal to or less than unity, are those lacking neighboring unsubstituted carbon atoms, and are heavily chlorinated, e.g. TCDD, 1,2,3,7,8pentachloro-DD, 1,2,3,4,7,8-hexachloro-DD, 1,2,3,4,7,8hexachloro-DF. Therefore, based on the above discussion, we conclude that RBFs are likely to be useful descriptors of metabolic activation of polyhalogenated aromatics in animals and could be used to identify the congeners that are highly resistant to metabolism. And even in cases where bioactivation of the xenobiotic takes place in vivo, relative AHH potencies are reliable descriptors of corresponding relative toxicities. Moreover, RBFs could be used to explain trends in toxicities of polyhalogenated AXs. Taking acute lethalities of PCDDs in animals as examples, it is clear from Table V that di- and trichloro-DDs have the highest acute lethalities among PCDDs in rats and mice. Similarly, 1,2,3,4-tetrachloro-DDalso has a fairly high LD50 in these animals. For these compounds, their RBFs >> 1,notice alsothat adjacent unsubstituted carbon atoms are available for metabolism to proceed. Accordingly, the high doses of these compounds required to cause deaths in rats and mice could be due to rapid metabolism, andlor their low affinities for AhR and weak potencies as AHH inducers. On the other hand, in harmony with experimental observations (21,23,241, bioactivation of heavily polyhalogenated aromatics lacking neighboring nonhalogenated carbon atoms is expected to become increasingly difficult.

Chem. Res. Toxicol., Vol. 6, No. 3, 1993 333

Notice that the latter compounds have RBF values near unity. As shown in Table V, TCDD, penta- and hexachloro-DDs have much lower LD50s than mono-, di-, and tripolychlorinated congeners. Therefore, in agreement with experimental observations (1-7), the toxicities of potent polyhalogenated aromatics are mainly due to the parent compounds. In addition, RBFs and AHH potencies of these classes of xenobiotics are likely to be useful indicators of AhR mediated biological and toxic responses in animals. (iv) Advantages of t h e New Methodologies over Classical QSARs. The reported models for AhR (8,9), AHH, EROD, metabolic activation, and toxic activities of polyhalogenated AXs have several advantages over classical QSARs (3-7, 10, 11). These are the following. (i) Multiple regression analysis was not used in deriving the working equations. To the authors’ knowledge, the reported methodologies are the first structure-activity studies that completely eliminated multiple regression analysis in their formulation and clearly showed that the affinities of polyhalogenated AXs to AhR are related to AHH and EROD potencies. (ii) The derived equations overcome the cross-class comparison problem inherent to classical QSARs. (iii) The models rely on physically measurable electronic and thermodynamic properties of ligands for developing the mathematical equations and, thus, could be tested experimentally. (iv) The working equations use one experimentally determined value for AhR, AHH, EROD, or LD50 of a given AX in a class to quantify the appropriate biologicaland/or toxic response(s) for other members in the class. It must be noted that for a given class of polyhalogenated AXs, the developed methodologies for receptor binding, enzymes induction, and toxic endpoints require one experimental value for each response to predict the corresponding values for other members in the class. That is, we used the affinity of TCDD for AhR as a stepping stone for predicting the dissociation constants of all PCDDs-.AhR complexes. Similarly, the potency of TCDD as AHH inducer in rat hepatoma cells in culture was used to estimate AHH activities of other PCDDs, etc. It is important to note that for a given class of halogenated xenobiotics, one can correlate their AHH activities with any other AhR mediated toxic response, such as thymic atrophy, body weight loss, immunotoxicity, and acute lethality (3-7). However, knowing AHH activities only are not sufficient for predicting other toxicological responses. (v) The models introduce to experimental scientists new criteria potencies for high affinity binding of ligands to AhR (8,9), as AHH and EROD inducers, metabolic activation, and in vivo toxicities. (vi) The results of in vivo, in vitro, and QSAR studies on receptor binding, enzymes induction, metabolic activation, and toxic potencies of AXs could be explained in a physically consistent way. (vii) AhR mediated biological and toxic responses could be quantified for other polyhalogenated AXs with minimal in vivo and in vitro experiments. Thus, the reported models could be helpful in reducing the number of experimental studies performed on laboratory animals and speeding up the risk assessment process of these environmental toxins in mammals. Future studies will apply the developed methodologies to different classes of Axe, e.g. polyhalogenated biphenyls and polycyclic aromatic hydrocarbons, and other AhR mediated responses in animals.

334 Chem. Res. Toxicol., Vol. 6, No. 3, 1993

Acknowledgment. The authors acknowledge various discussions with Professors R. J. Rubin and J. D. Groopman. This research was supported by the US.Environmental Protection Agency (R-817056-010) and the National Institutes of Health (ES03819). The authors thank the Johns Hopkins University School of Public Health Computer Center for the allocated computer time on the IBM-4381mainframe and the staff of Dynex International, Inc., for their assistance in performing the computations. Certain commercial instruments or computer programs are identified in this paper in order to specify adequately the research work. Such identification does not imply endorsement by the National Institute of Standards and Technology, nor does it imply that the equipment identified are the best available for the purpose.

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