Research

Polychlorinated Biphenyls as Hormonally ActiveStructural AnaloguesJames D. McKinneyl and Chris L. Wailer21Health Effects Research Laboratory, U.S. Environmental Protection Agency, ResearchTriangle Park, NC 27711 USA; 2Center for Molecular Design, School of Medicine,Washington University, St. Louis, MO 63130 USA

There is growing concern (1) that a largenumber of man-made chemicals that havebeen released into the environment candisrupt the endocrine systems of animalsand humans. Among the chemicals of con-cern are the persistent, bioaccumulativeorganohalogen compounds that includesome pesticides and industrial chemicalssuch as polychlorinated aromatics, othersynthetic products, and some metals.Possible mechanisms by which these chem-

icals may interfere with endocrine functioninclude their ability to mimic natural hor-mones (both as agonists and antagonists),including recognition of their specificbinding sites, ability to react directly orindirectly with hormone structure to alterit, and ability to alter the pattern of hor-mone synthesis or modulate hormonereceptor numbers and affinities. The poly-chlorinated biphenyls (PCBs) are knownor suspected to express biological activitiesthrough most, if not all, of these mecha-nisms.

PCBs are mixtures of chlorinated aro-matic chemicals manufactured by chlorina-tion of biphenyl under suitable conditionsfor varying the percentage by weight ofchlorine incorporated. The chemical struc-ture (Fig. 1) of PCBs can be represented ina general way, where n,n' is the number ofchlorine atoms replacing hydrogen atoms ineach ring. A total of 209 different congenersare theoretically possible, but less than halfthat number appear to account for nearly allof the environmental contamination attrib-utable to PCBs. If potential toxicity, envi-ronmental prevalence, and relative abun-dance in animal tissues are considered ascriteria, the number of environmentallythreatening congeners decreases to fewerthan 50. Furthermore, fewer than 25 ofthese may account for most of the totalPCB burden in tissues of invertebrates, fish,birds, and mammals (2).

PCBs were discovered before the turnof the century, and their usefulness inindustry as, for example, dielectric andheat-exchange fluids with desirable physi-cal properties, was recognized early. Theirchemical resistance and neutral organicchemical nature have led to widespreaddistribution in the environment, includingfood webs. Now PCBs are almost entirelyrestricted to use in closed systems. Humanexposure to PCBs has resulted largely fromthe consumption of food, but occupationalexposure also results from inhalation andskin absorption. PCBs accumulate in thefatty tissues of humans and other animals.In turn, PCBs have caused toxic effects inhumans and animals. The skin and liverare major sites of pathologic change, andother targets include the gastrointestinaltract, the immune system, and the nervous

system. Studies in animals suggest thatsome PCB congeners may be carcinogenicand that they can promote the carcino-genicity of other chemicals. Such toxico-logical properties, both qualitatively andquantitatively, often are congener specificwith remarkable dependence on numberand position of chlorine substitutions.Structural patterns can thus be used to dis-criminate among PCB congeners on thebasis of toxic potential, if not entirely ontoxicity per se (3,4).

The toxic effects of PCBs appear toinvolve at least three types of basic mecha-nisms of action: 1) reversible interaction(binding) of the PCB with specific molecu-lar sites of action such as receptors,enzymes, etc., 2) irreversible covalent inter-action (binding) between the PCB (proba-bly a reactive metabolite) and target mole-cules (particularly macromolecules such asDNA and proteins), and 3) accumulationof highly lipid-soluble, metabolically stablePCBs in lipid-rich tissues or tissue com-partments. The same or different structuralproperties may be involved in the expres-sion of these toxic effects. Of particularinterest to this discussion is the use of

Figure 1. General structure for PCBs. o, m, p denoteortho, meta, and para positions, respectively.

Address correspondence to J.D. McKinney,Pharmacokinetics Branch (MD-74), Environ-mental Toxicology Division, Health EffectsResearch Laboratory, U.S. EPA, Research TrianglePark, NC 27711 USA. C.L. Waller is currently atthe Health Effects Research Laboratory, U.S. EPA,Research Triangle Park, NC.We thank our many co-workers for their contribu-tions over the years. The use of computer graphicsfacilities at the Center for Molecular Design,Washington University, St Louis, and similar facil-ities at the University of California at SanFrancisco and San Diego for molecular graphicsand modeling studies is also acknowledged.C.L.W. acknowledges the support ofNIH traininggrant T32HL07275. This manuscript has beenreviewed in accordance with the policy of theHealths Effects Research Laboratory, U.S. EPA,and approved for publication. Approval does notsignify that the contents necessarily reflect theviews and policies of the agency, nor does mentionof tradenames or commercial products constituteendorsement or recommendation for use.Received 12 November 1993; accepted 3 January1994.

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structure-activity relationship (SAR)approaches to study the reversible interac-tion ofPCB congeners with specific molec-ular sites in biological systems that normal-ly control or mediate hormonal activitiesin the body. In the study of SARs, thebasic philosophy is to draw conclusions byanalogy, assuming that similarity of PCBswith respect to certain chemical proper-ties/reactivities will result in similar biolog-ical responses (5). Thus, the problem isone of determining these properties andhow they are linked to specific biologicalactivities of interest.

PCB Chemical ReactivitiesUnderlying ToxicityPCB chemical reactivities are encoded with-in the chemical structure of the PCB mole-cule. Because chemical reactivities underliebiological activities (6), it is important tounderstand how structure determines theimportant reactivities and, in turn, howthese reactivities are linked to toxicity. Oneof the more important applications of SARapproaches is to help guide mechanisticresearch and permit testing of mechanistichypotheses (7). Simplifying and classifyingrelevant chemical reactivities can be animportant first approach to studying molec-ular mechanisms of toxicity.

Stacking Interactions andCoplanarity of StructureThe importance of coplanarity of PCBstructure (Fig. 2) in producing dioxinliketoxic effects has been recognized for sometime (8). The chemical reactivity (5) associ-ated with this structural feature is the abilityto undergo stacking-type molecular interac-tions with other planar aromatic ring sys-tems such as the heme system in hemopro-teins. Such stacking interactions betweenaromatic ring systems have also beenreferred to as a charge-transfer or donor-acceptor interaction and can be viewed in asimplistic sense as a "Velcro-type" interac-tion in which the planar rings facilitate the"sticking together" of the two pieces. Itshould be pointed out here that the term"coplanar" PCB as widely used in this con-text is a misnomer in the sense that allPCBs are basically non-coplanar in nature(8). The important distinguishing propertyof the non-ortho-substituted PCBs is thatthe increase in the conformational enthalpy(AH) required for the non-ortho-substitutedbiphenyl to achieve a coplanar state is suffi-ciently offset (outweighed) by the favorablechange in binding free energy (AG) associ-ated with formation of a stacking biphenyl-receptor complex. All ortho-substitutedPCBs are likely to undergo binding interac-tions in which a coplanar state is never fullyachieved.

Cleft-type Interactions and LateralChlorine SubstitutionLateral (meta, para substitutions) chlorina-tion (Fig. 2) has also been recognized(10-12) as an important structural featurein PCB binding interactions and toxicity.Halogen atoms like chlorine contain manyelectrons and are thus highly polarizable.Many appropriately placed polarizabilityinteractions in a protein-binding site canbe quite effective in binding a PCB mole-cule. The most polarizable chlorines arethose contained in the lateral positions ofthe PCB molecule. This can be envisionedas a molecular cleft-type interactionbetween the highly polarizable lateral halo-gen atoms and the hydrophobic interior ofthe cleft provided by amino acid sidechains that converge on the halogen sub-stituents. Similar binding interactionscould involve chlorines in nonlateral (orthosubstitutions) positions of the molecule,but there is not clear evidence that suchinteractions are important in PCB bindingand toxicity (3). An additional factor asso-ciated with the polarizable chlorines iswhen the number and positions of chlo-rines are favorable to driving a preferredpolarizability vector in the molecule (8).This is the case in the coplanar PCBs withlateral substitutions, where the preferredorientation is about the longest axis(through the para, para' positions) of the

molecule. This type of preferred polariz-ability can facilitate the stacking interac-tions referred to earlier by strengtheningthe charge-transfer interaction or in sim-ple, nonscientific terms, making it "sticki-er" (7). Both stacking and polarizabilityinteractions might also be possible for cer-tain stable metabolites of PCBs such astheir hydroxylated metabolites.

Areas of High Electron Density andOxidative MetabolismA third type of structural feature that maybe important in leading to a different typeof PCB toxicity is the availability of vicinalunsubstituted positions in the molecule(such as vacant meta, para positions) thatcan provide sites for oxidative metabolismto occur (13). In this case the chemicalreactivity in the molecule that may controlthe rate and regiospecificity of oxidativeattack involves areas of high electron densi-ty such as may occur at opposite ends ofthe polarization vector described above.Because reactive intermediary metabolitescan be formed in such a process, it is possi-ble that reactive metabolites can lead tocovalently bound residues with biomole-cules. Stable metabolites can have chemicalreactivity of their own and lead to similaror different kinds of binding and toxicityas seen with the parent PCBs (14). Othertypes of metabolites and metabolic process-

Figure 2. Wire- and space-filled models comparing coplanar (3,3',4,4',5,5'-HCB, upper right) and non-coplanar (2,2'4,4',6,6'-HCB, upper left) PCBs with a chlorinated diphenyl ether (2,3',4,4',6-CDE, lower left)and thyroxine (lower right; aligned by planar face of one or more rings; note sterically inaccessible planarstacking face in 2,2'4,4',6,6'-HCB and similar condition of diphenyl ehter system but with an accessibleplanar face). Carbon, green; hydrogen, white; oxygen, red; halogen, magenta.

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Figure 3. Space-filled stacking models comparing coplanar (3,3'4,4',5,5'-, upper left) and non-coplanar(2,3,4,4',5-, upper right) PCBs with dioxin (lower left) and chlorinated diphenyl ether (2,3',4,4',6-, lowerright) interactions with porphine ring system (note separation distance and planar geometrical extent ofstacking interaction involving planar faces).

Figure 4. Molecular surface representation of 3,3',4,4'5,5'-hexachlorobiphenyl (from docking, red) and thy-roxine complex (from X-ray, green) with binding domain (blue) of human protein prealbumin [comparecontacts with protein surface and importance of lateral chorines in filling binding pockets of thyroxine;see Rickenbacher et al. (11)].

es are possible and may be important inthe expression of certain forms of toxicitysuch as the reported association of chronicrespiratory effects with methylsulfonemetabolites of certain PCB congeners (3).

Although other structural features andreactivities of PCBs are possible, they havebeen less studied in an SAR framework.For example, certain pure phenobarbital(PB)-type inducing PCBs with ortho sub-stitutions cause hepatomegaly and exhibitactivity as promoters in short-term bioassaysfor carcinogenesis (3). The structural basisfor these activities is not clear, but they maybe more a function of specific substructuralfeatures than overall PCB structure consis-tent with the many diverse structuresknown to act as PB-type inducers.

Reactivity Models andPredictionsStacking ModelsIn attempting to understand the relation-ship between PCB structure, chemicalreactivity, and biological activity, theoreti-cal models for relevant biological activitiesbased on PCB molecular parameters havebeen sought (7). A planar (or energeticallyfavorable coplanar) aromatic ring system isthe primary structural feature that is foundin all known high-affinity ligands for theAh (or dioxin) receptor, which includeboth halogenated and nonhalogenated aro-matic compounds. Several lines of evidence(7,15-17) now suggest that an importantmolecular property determining receptorbinding and associated responses for thesecompounds is electron affinity. The depen-dence on this property can best beexplained in terms of compound electronacceptance in a charge-transfer or stacking-type receptor model. Such a model alsopermits one to readily visualize steric hin-drance to binding, as is the case for theortho-substituted PCBs (Fig. 3), and varia-tions in molecular size (e.g., comparePCBs with halogenated naphthalenes).The somewhat flexible but reasonablycoplanar dioxin molecule shows the closeststacking followed by the non-ortho- andortho-substituted PCBs. In general, thestacking model predicts increased bindingfor the class of chlorinated dioxins withincreased chlorination, but this trend hasnot been observed in in vitro experimentalbinding studies, possibly reflecting solubili-ty differences. There is evidence (18) forsuch a trend in in vivo absorption and tis-sue distribution studies in the rat. It isimportant to realize that a favorable, closestacking can occur in some aromatic com-pounds that are not totally coplanar buthave a sterically accessible planar face, as incertain chlorinated diphenyl ethers (Fig. 2and 3). This consideration bears directlyon the structural relationship of PCBs to

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Figure 5. Wire- and space-filled models comparing estrogenic PCB analog (4-OH, 2',4',6'-, bottom) withestradiol (top) (molecules aligned by common phenolic ring).

thyroid hormones, as discussed below.Other factors can play a role, such as com-pound solubilities and lipophilicities.Nevertheless, the receptor binding andassociated biological responses are bestexplained in terms of changes in stereoelec-tronic and thermodynamic properties ofligands. As applied to PCBs, ortho substitu-tion plays a major role in effecting changesin ligand binding (19,20). The relativebinding affinities to the Ah receptor can beviewed as a measure of the "stacking reac-tivity," which can be related directly todioxin (TCDD) and expressed as dioxinequivalents (3). This modeling work hasimplications in understanding the naturalrole and function of the Ah receptor.

Cleft-type ModelsAnother important structural property indetermining PCB dioxin -like toxicity is thepresence of lateral chlorine substitution inthe molecular structure. However, this isnot always a requirement for high-affinitybinding to the Ah receptor such as foundfor certain polynuclear aromatic hydrocar-bons (PAHs). As discussed above, thisstructural requirement for high toxicity sug-gests the involvement of other binding siteswith C-shaped molecular cavities or cleftswith viselike properties in the molecularrecognition process. A binding model ofthis type that has been extensively studied isthe prealbumin or transthyretin interactionmodel. The importance of lateral substitu-tions in binding both thyroid hormone ana-logues and PCBs has been well documented(10,11) (Fig. 4). Interestingly, this bindinginteraction model provides another struc-tural linkage between PCBs and thyroidhormones. In fact, there is a family of preal-

buminlike proteins apparently involved inregulating thyroid hormone action (21). Asfor stacking reactivity and Ah receptorbinding, relative binding affinities to preal-bumin can be viewed as a measure of the"lateral chlorine polarizability reactivity,"which can be related directly to thyroxineand expressed as thyroxine equivalents.PCB interactions with this family of pro-teins have potential biological consequencesas both agonists and antagonists.

Estrogen-Active Analog ModelWhile the stacking and cleft-type modelshave both pointed to a structural relation-ship of PCBs to thyroid hormones, otherwork (22) has suggested a structural rela-tionship to steroid hormones, particularlyestrogens. The steroidal estrogens are rela-tively rigid, polycylic molecules with pro-nounced asymmetry, and their binding tothe estrogen receptor generally reflects ahigh degree of stereospecificity. Severalclasses of nonsteroidal synthetic estrogenshave been based on phenyl-substitutedhydrocarbons (23) because of the structur-al relationship to the phenolic A ring inestradiol. Hydroxylated PCBs are oftenfound as metabolites of the parent PCBs(13). In this regard, we theorized that cer-tain hydroxylated PCBs (Fig. 5) that werealso conformationally restricted due toortho substitution might be effective inbinding the estrogen receptor. This provedto be the case in that the most active PCBanalogues where those that combined apara-hydroxyphenyl ring with some degreeof ortho substitution. However, it is notclear if there is some unique property ofthe hydroxyl group (such as hydrogenbonding) or if any other similar-sized

group (such as chlorine) could be substi-tuted. It is possible that increased orthochlorines in the PCB structure might alsoprovide important hydrophobic bulk struc-ture in binding the estrogen receptor, butthere was not any direct evidence for this.In additional analysis of these data (unpub-lished results), the solubility/lipophilicproperties of the PCB are also proving tobe important.

Although ortho substitution in PCBscan significantly lower the dioxinlike toxici-ty, it can potentially have the opposite effecton any estrogen-receptor-mediated toxicity.This PCB active analog modeling approachto estrogen receptor binding has implica-tions for understanding any associated toxi-city. As was suggested for other PCB reac-tivities and dioxin and thyroxine equiva-lents, it is possible to express this PCB reac-tivity in terms of estradiol equivalents.Possible Relationships toToxic EndpointsRelationship to ChlorinatedDiphenyl EthersAs mentioned earlier, the chlorinateddiphenyl ethers (CDEs) are an importanttest case for the structural requirements fordioxin like toxicity for PCBs. CDEs con-tain two phenyl rings as do PCBs but forenergetic reasons are unlikely to achievecoplanar states in their binding interac-tions, even in the absence of ortho substitu-tion. Any dioxinlike toxicity and associatedAh receptor binding are more likely toreflect molecular interactions involvingnon-coplanar states, especially with ortho-chlorine substituents. The main structuraldifference in the two classes of compoundsis the ether oxygen bridge (C-O-C angleof 1200) contained in the CDEs. In addi-tion to serving as structurally related modelcompounds for PCBs, the CDEs are alsoof interest as potential environmental cont-aminants, such as their association withused transformer fluid (24). For bothPCBs and CDEs, chlorine substitutionsprovide steric constraint that defines theminimum energy conformation of the aro-matic rings.

However, the effects on overall confor-mational properties and structure can bequite different in the context of having anaccessible planar face for a stacking-typeinteraction to occur as postulated for thedioxin-receptor-binding interaction. In thecase of PCBs, any degree of ortho substitu-tion substantially raises the energy barrierto internal rotation about the pivot bond(25), and it is likely that ortho-PCBs inter-act with the dioxin receptor in their non-planar minimum energy conformations(26). Therefore, to the extent that toxicityis truly mediated by coplanar conformers,ortho-PCBs are likely to be relatively non-

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Figure 6. Space-filled model showing the effects of ortho-chlorine substitution in a tri-ortho-substitutedchlorinated diphenyl ether. Note the relationship (in plane or in x-cloud) of chorines to stacking ring (asin Figure 3).

toxic. Lethality in guinea pigs may be anexample (10) of this because the nearlyisosteric 3,3',4,4',5,5'-hexachlorobiphenylis about 1/100 as toxic as dioxin (reflectingthe energy cost to achieve a coplanar state)and the structurally related 2,3,3',4,4'5,5'-heptachlorobiphenyl is relatively nontoxicat significantly larger (>10-fold) doses.

In the case of CDEs, the effects ofortho substitution on dioxin-receptor-bind-ing are likely to have an enhancing effect.Ortho substituents provide desirable mini-mum-energy conformations (Fig. 3)because the rings now approach a mutuallyperpendicular relationship with an accessi-ble planar face for entering into stackinginteractions. An additional enhancingeffect is realized because there is less energycost (more favorable binding free energyfrom freezing out rotatable bonds)required to position the more rigid bindinggroups. A similar effect of ortho sub-stituents in hydroxylated PCBs in enhanc-ing estrogen receptor binding was previ-ously described (22). Although other bind-ing modes are possible, they are somewhatless favored energetically, but in generalalso seriously limit the accessibility of theother phenyl ring system in entering intoother binding interactions such as thoseinvolving lateral substituents.

Recent work (27,28) has shown inter-esting differences in the structure-activityrelationships for CDEs as compared toPCBs. The evidence suggests that bothclasses of compounds can produce dioxin-like toxic effects mediated by binding tothe dioxin receptor. For example, non-ortho-but laterally substituted CDEs wereless toxic than their mono-ortho-substitut-

ed analogues, and similar results were alsoobserved for their enzyme-inducing poten-cies. Active congeners were also shown toinclude CDEs with multiple ortho-substi-tutions. These results are consistent withthe anticipated enhancing effects of orthosubstitution in CDEs on dioxin receptorbinding through a stacking molecularmechanism as previously predicted (7).

In addition, an interesting result wasfound (28) in certain highly chlorinatedCDEs that contain multiple ortho substitu-tions, especially in both rings. The poten-cies were shown to be similar to thosefound (27) for the non-ortho- and mono-ortho-substituted compounds previouslystudied. It is apparent that high degrees ofortho substitution in CDEs do not signifi-cantly diminish their Ah-receptor-mediat-ed responses as seen for the PCBs. It isproposed that in these cases increased con-formational restriction is brought about byadditional steric interactions between thering systems. The favored conformerswould place the adjacent ortho-chlorine inthe plane of the stacking ring (see Figure3), which could influence the nature of thestacking interaction. Highly chlorinateddiphenyl ethers, especially with highdegrees (three or four ortho-chlorines) ofortho substitution, would increase thepolarizability and steric rigidity of the sys-tem, both expected to further enhance astacking interaction. It is anticipated thatthe stacking ring would be the most highlysubstituted ring, although this is notalways clear (e.g., when both rings containthe same number of chlorines). However,high degrees of ortho substitution wouldnot only lead to chlorine atoms positioned

in the stacking plane but also to closethrough-space interactions of ortho- chlo-rine atoms with the face of the adjacentaromatic ring system (Fig. 6). This lattereffect can be relieved somewhat in theabsence of meta- chlorine substituents thatbuttress the adjacent ortho substituent(29). Both the in-plane and through-spaceintramolecular effects would weaken anyintermolecular stacking interaction.Nevertheless, it is anticipated that thestructure-activity relationships for thesetypes of molecules would be complex anddifficult to interpret. Ortho substituents inCDEs can have both qualitatively andquantitatively different effects on molecu-lar structure and reactivity as compared tothe effects seen in the PCBs.

The available SAR data on CDEs sup-port the involvement of a conformationallyrestricted diphenyl system with an accessi-ble planar face that can participate instacking interactions with protein bindingsites. This type of analysis of CDEs andmodel for active compounds further sup-port the proposal (20) for a stacking typeinteraction in dioxin receptor binding andshould be helpful in guiding the selectionof other congeners to test for the ability toproduce dioxinlike toxicity. The potentialtoxicity of members of this class of chlori-nated aromatic hydrocarbons needs furtherattention because hexa- to decachloro-diphenyl ethers, all of which are likely tohave high degrees of ortho substitution(wider range of active congeners possible),have been identified (30) in human adi-pose samples at concentrations as high as2000 pg/g, and they are known contami-nants of commercial products such as tech-nical-grade pentachlorophenol (31). Theclose structural relationship of active CDEsto thyroid hormones is also of considerableinterest and compatible with the possiblethyromimetic properties (7 of these com-pounds. Clearly, in the context of thisstacking model for binding, the non-ortho-substituted congeners do not take on spe-cial significance and, other things beingequal, may have reduced activity relative totheir ortho-substituted counterparts.Preliminary quantitative structure-activityrelationship (QSAR) studies (Waller C,and McKinney J, unpublished observa-tions) with the CDEs based on the stack-ing-model concept look promising inunderstanding the dioxin-receptor-mediat-ed responses of these chemicals. In a recentteratogenicity study (32) with nine CDEsin mice, 2,2',4,5,6'-penta- and 2,3',4',6-tetrachlorodiphenyl ether resulted in theloss of litters at relatively low doses. Bothof these CDEs contain one di-ortho-substi-tuted ring, and the other has lateral (meta,para) substituents, but they are not fullyortho substituted to maximize the steric

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interactions shown in Figure 6. Theseresults appear to be consistent with thestructural requirements of active congenerspredicted by our models.

Relationship to Thyroid HormonesWhat emerges from such analysis of SARresults is that these structurally relatedcompounds (PCBs and CDEs) can partici-pate in similar molecular recognitionprocesses in their potential interactionswith specific molecular sites in biologicalsystems in which the nature, accessibility,and polarizability of planar (or coplanar)face interactions can vary. The obviousnext step in this type of analysis is to com-pare these molecular recognition processeswith those that have been well documented(33,34) for thyroid hormone analogues asnatural halogenated diphenyl ether systemsof considerable biological importance. TheSAR literature for thyroid-hormone-bind-ing proteins points to two basic types ofbinding proteins: those that bind 3,5,3'-triiodo-L-thyronine (T3) in preference toL-thyroxine (T4) and those that bind T4 inpreference to T3. Interestingly, the SARsfor the T3 binding proteins seem todepend more on the structural propertiesof the inner (tyrosyl) ring, whereas theSARs for the T4 binding proteins dependmore on the outer (phenolic) ring proper-ties (Fig. 2). In this regard, it is interestingto note that the nuclear receptors for thy-roid hormones show considerable depen-dence of binding on a sterically con-strained inner ring brought about by the3,5- (ortho) substituents. We have furthershown (29) that these results can be rea-sonably explained using a donor-acceptoror stacking aromatic ring model as previ-ously described. In this model frameworkit is not surprising that biphenyl thyroidhormone analogues (which lack an etheroxygen bridge) are inactive (33) becausethey basically represent worse-case ortho-substituted PCBs. Binding ligands for thetriiodothyronine (T3) nuclear receptor andthe dioxin receptor may share commonmolecular recognition factors in the expres-sion of their binding activities.

In contrast, the lateral arrangement ofthe ortho-diiodophenolic structure in theouter ring of thyroid hormones is animportant feature characterizing the bind-ing of all three major transport proteins(globulin, prealbumin, and albumin), andis the sole major binding feature for albu-min. In fact, the T4 binding proteinsappeared to be ideally suited to bind lateralsubstituted chlorinated aromatic com-pounds such as the lateral (meta, para) sub-stituted PCBs. Molecular modeling andexperimental studies (10,11) now provideconsiderable support for this hypothesis.The best binders (four- to eightfold better

than T4) have only lateral chlorine sub-stituents, and additional ortho-chlorines donot lower binding much (two- to threefoldbetter than T4). One lateral chlorine (as inthe 2,4,6-pattern) is sufficient for apprecia-ble (about half T4) binding activity. Whenno chlorines (biphenyl) or all (2,2',6,6')ortho positions are filled, the compoundsshow no binding activity. As persistence inthe environment and in biological tissuesusually equates to a high degree of lateralsubstitution, it is anticipated that most, ifnot all, PCB residues found in human tis-sues are likely to effectively bind this fami-ly of proteins. It is also known (10,11,35)that hydroxy PCBs can bind this family ofproteins, which suggests that certain PCBhydroxylated metabolites could producethe same or similar responses associatedwith binding. Similar SAR results (11)were shown for a T4 nuclear-binding pro-tein (12) and the microsomal deiodinase(36) in liver tissue. Other proteins thatmay give similar results include tyrosinehydroxylase and the dopamine receptor.

An important consideration here is thatortho chlorination in PCBs does not signif-icantly affect this binding activity. Oneindirect effect of ortho substitution on thisbinding activity may be to effectivelyinhibit binding to the T3 family of bindingproteins that presumably require a stackinginteraction. Therefore, any biologicalresponses associated with this "laterality"type of binding activity (as in the prealbu-min interaction model) could be elicitedby a much larger number of PCB con-geners of environmental concern becausemost are ortho substituted to some degree.The recent finding of PCB neurotoxicty(37) with certain ortho but lightly substi-tuted congeners may fall into this category.Biochemical thyroid status at the cellularlevel has been shown (38) to depend notonly on the concentration of free T4 butalso on the concentration of prealbumin.Prealbumin is synthesized in the brain (aswell as the liver), where it plays a majorrole in transport of thyroid hormone to thecentral nervous system (39).

Relationship to RetinoidsAnother factor that may bear on some ofthe toxicological properties of PCBs relatesto evidence (40) suggesting a close associa-tion between thyroid hormone andretinoid transport, metabolism, and genetranscription. For example, prealbuminbinds to retinol-binding protein, and thisserves as both a transport and protectivemechanism for the smaller protein (41).Earlier findings (42) of prealbumin deple-tion in rat sera of dioxin-treated animalscoupled with findings (McKinney JD et al.unpublished observations) of a parallelincrease in retinol-binding protein and

retinol are consistent with this view andfurther support a role for thyroid-hor-mone-binding proteins in the mechanismof toxicity of these compounds. SAR stud-ies (43) on the effects of PCBs on retinoidconcentrations in serum and tissues indi-cate that there are at least two classes ofPCBs (i.e., the coplanar PCBs that affecthepatic, renal, and serum retinoids andother ortho-substituted PCBs that onlydecrease serum retinol). It is not clearwhether these effects are secondary to theeffects of PCBs on thyroid hormonemetabolism. These effects may also reflectdifferences in preferences for the two class-es of thyroid-hormone-binding proteinsdescribed earlier.

Relationship to EstradiolAnother class of hydroxylated PCBs (pre-sumed metabolites) have been shown (22)to bind specifically to the mouse uterineestrogen receptor and as such representpotential agonists/antagonists ligands forcertain estrogen receptor functions. Asdescribed earlier, this is believed to be asso-ciated with a close match of the phenolicring with the A-ring in estradiol andenhanced steroidlike structure broughtabout by the ortho substituents. Develop-mental and reproductive toxicity are possi-ble outcomes of interactions of this typewith steroid hormone receptors. For exam-ple, reproductive impairment in marinemammals has been directly attributed tofeeding on PCB-contaminated fish (44).The potential human health effects ofexposure to ortho-substituted PCBs andtheir hydroxylated metabolites should beconsidered in this light.

ConclusionsFrom a structural chemistry point of view,there are at least two distinct classes ofPCBs: the non-ortho- and ortho-substitutedcongeners. The important distinguishingfeature of the non-ortho-substituted PCBsis the energetic accessibility of the coplanarstate that can facilitate stacking interac-tions such as may occur in a receptor-bind-ing domain. The effects of ortho substitu-tion on PCB reactivity are less clear, butthey can include three basic types: hin-drance to stacking interactions, conforma-tional restriction and more rigid steroidlikestructure, and increased carbon-chlorinebond polarizability in nonlateral positions.Lateral chlorine substituents can be impor-tant in enhancing the binding properties ofboth classes of PCBs through polarizabilityinteractions.

For the non-ortho-substituted (copla-nar) congeners, most, if not all, of theimportant toxic effects we know aboutappear to be mediated by binding to thedioxin or Ah receptor. A simple stacking

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interaction model involving planar faces isoffered as a way to visualize the Ah-recep-tor-binding process, but it must be viewedas an oversimplication of a complex bind-ing interaction. Further support for thestacking model is derived from analysis ofthe important structural features of themore active dioxinlike chlorinateddiphenyl ethers. The activity of the chlori-nated diphenyl ethers also provides supportfor the thyromimetic properties of theseclasses of compounds. The prealbumininteraction model is offered as a way tovisualize the requirement for lateral sub-stituents in cleft-type binding interactions.The estrogen active analog modelingapproach offers one way of understandingthe importance of ortho substituents andconformational restriction in receptorbinding. For the ortho- substituted con-geners, the link to specific toxic endpointsare not well established, and it is likely thatmultiple mechanisms are involved. Tosome extent, quantitative differences inresponses appear to be due to variations inthe degree of coplanarity or conformation-al restriction (both dependent on degreesof ortho substitution) or accessibility of pla-nar faces and laterality that exists amongthe PCB congeners. Agonist and antago-nist effects may also depend on variationsand combinations of the important reactiv-ity properties (kinds and amount) amongthe congeners. There appear to be someareas of overlap between the two PCBclasses such as the weak dioxinlike proper-ties of certain mono-ortho-substitutedPCBs, but the significance of this is notfully understood. As ortho substitution inbiphenyls is expected to lower their dioxin-like binding properties in biological sys-tems, one might anticipate differences intheir toxicokinetic properties based on thedegree of ortho substitution (45).

As suggested earlier, PCBs may inter-fere with endocrine function in variousways. Their ability to directly mimic natur-al hormones (both as potentially potentand persistent agonists and antagonists)including recognition of their specificbinding sites is emerging as a guiding con-cept in the case of the thyroid and estro-genic steroid hormones. Indirect effectsmight include alterations in the receptornumber or affinity of other hormonesinvolved in multihormonal regulating sys-tems. For example, there is evidence (46)for a direct effect of thyroid hormone onthe hepatic synthesis of estrogen receptors.The antiestrogenic action of dioxin (4/)and possibly coplanar PCBs (48) in down-regulating estrogen receptors could berelated to their potential thyromimeticproperties. Toxicity could result from over-or underexpression of normal hormonalresponses that may be mediated by both

receptor and nonreceptor protein and asso-ciated DNA interactions. For example,recent work (49P) suggests that the humanestrogen receptor structural gene contains aDNA sequence that binds activated mouseand human Ah receptor.

The potential for reproductive anddevelopmental toxicity (50) is of particularconcern. Thyroid hormones are known tobe essential for brain development (51),and many investigators (52) have providedevidence demonstrating effects of hor-mones on immature brain cell maturation,myelinogenesis, protein and nucleic acidmetabolism, and electrical activity of thegrowing brain. In addition, there are anumber of well-documented examples ofthe regulation of single and multiple clus-ters of genes through multiple hormonalinteractions (53). Thyroidal, adrenal, andgonadal hormones are all necessary for thenormal differentiation of the nervous sys-tem and involved in the ultimate expres-sion of the target genes in several of thesemultihormonal regulating systems. Becausehumans are exposed to mixtures of PCBcongeners, it is likely that there are com-plex interactive biological effects.Understanding the biological properties ofPCBs and how they are determined by themolecular structure are important researchobjectives. Perhaps more direct molecularbiological approaches would be helpful inelucidating the involvement of both recep-tor and nonreceptor protein-mediatedresponses. In turn, such mechanisticinsight should be helpful in reducing thescientific uncertainty associated withhealth hazard/risk assessments associatedwith human exposures to PCBs and relatedcompounds.

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