Toxicity of Hydroxylated and Quinoid PCB Metabolites: Inhibition of

In the present study, a series of 32 hydroxy- and dihydroxy-polychlorinated biphenyls (OH-PCBs) and PCB-derived quinones were prepared and evaluated f...
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Chem. Res. Toxicol. 2004, 17, 340-347

Toxicity of Hydroxylated and Quinoid PCB Metabolites: Inhibition of Gap Junctional Intercellular Communication and Activation of Aryl Hydrocarbon and Estrogen Receptors in Hepatic and Mammary Cells Miroslav Machala,*,† Ludeˇk Bla´ha,†,‡ Hans-Joachim Lehmler,§ Martina Plı´sˇkova´,†,‡ Zuzana Ma´jkova´,‡ Petra Kapplova´,† Iva Sovadinova´,‡ Jan Vondra´cˇek,†,| Tina Malmberg,⊥ and Larry W. Robertson§ Veterinary Research Institute, Brno, Czech Republic, RECETOX, Masaryk University, Brno, Czech Republic, Department of Occupational and Environmental Health, College of Public Health, The University of Iowa, Iowa City, Iowa, Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic, and Department of Environmental Chemistry, Stockholm University, Stockholm, Sweden Received July 2, 2003

In the present study, a series of 32 hydroxy- and dihydroxy-polychlorinated biphenyls (OHPCBs) and PCB-derived quinones were prepared and evaluated for their in vitro potencies to downregulate gap junctional intercellular communication (GJIC) and to activate the aryl hydrocarbon receptor (AhR) and the estrogen receptor R (ER) in well-established liver and mammary cell models. The rat liver epithelial cell line WB-F344 was used for in vitro determination of GJIC inhibition; the AhR-inducing activity was determined in the rat hepatoma H4IIE.Luc cells stably transfected with a luciferase reporter gene; ER-mediated activity was measured in two breast carcinoma cell lines, MVLN and T47D.Luc, stably transfected with luciferase under the control of estrogen responsive element. Acute inhibition of GJIC, potentially associated with tumor promotion, was detected after treatment with all OH-PCBs under study, with the persistent OH-PCBs being the strongest ones. Several compounds were found to significantly induce the AhR-mediated activity, including 4′-OHPCB 79, a metabolite of PCB 77, and 2-(4′-chloro)- and 2-(3′,4′-dichloro)-1,4-benzoquinones and 1,4-hydroquinones. Low molecular weight OH-PCBs, such as 3′-hydroxy, 4′-, and 3′,4′dihydroxy-4-chlorobiphenyl, elicited significant estrogenic activity and potentiated effect of 17βestradiol. Antiestrogenic potencies, determined in the presence of 17β-estradiol, were found for persistent 4-OH-PCB 187, 4-OH-PCB 146, and some low chlorinated PCB derivatives. However, no apparent association between induction of AhR activity and antiestrogenicity was observed. The majority of the OH-PCBs suppressed the 17β-estradiol response only at cytotoxic concentrations. Spearman’s rank correlations were calculated for these biological data and the physicochemical descriptors, hydrophobicity (log P), molar volume, pKa, log D, and dihedral angle. Significant correlations were found between potency to downregulate GJIC and log P and molar volume (R ) -0.7, p < 0.0001). Antiestrogenic effects were also negatively correlated with hydrophobicity and molar volume. No significant correlations among other biological end points and the physicochemical descriptors were observed for the entire set of compounds. These results show that oxygenated PCB metabolites are capable of multiple adverse effects, including gap junction inhibition, AhR-mediated activity, and (anti)estrogenicity. The inhibition of GJIC by OH-PCBs represents a novel mode of action of both the lower chlorinated and the persisting high molecular weight OH-PCBs.

Introduction PCBs1 are a major class of organic environmental contaminants whose persistence, transformation, and * To whom correspondence should be addressed. E-mail: machala@ vri.cz. † Veterinary Research Institute. ‡ Masaryk University. § The University of Iowa. | Czech Academy of Sciences. ⊥ Stockholm University. 1 Abbreviations: AhR, aryl hydrocarbon receptor; DMSO, dimethyl sulfoxide; ER, estrogenic receptor; GJIC, gap junctional intercellular communication; LOEC, lowest observed effect concentration; OH-PCBs, hydroxylated polychlorinated biphenyls; PCB, polychlorinated biphenyl; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

toxicology have been intensively studied since the 1970s. PCBs are known to elicit various adverse effects, including carcinogenicity, neuroendocrine, developmental and reproductive toxicity, and immunotoxicity. The relative toxic potencies of congeneric PCBs and/or their mixtures depend on the respective chemical characteristics, such as hydrophobicity and planarity (1-3). Routine analysis for PCBs does not consider the presence of PCB metabolites, even though recent studies have found that several high molecular weight hydroxylated PCB metabolites (OH-PCBs) are strongly and selectively accumulated in mammalian tissues, including human blood (4, 5). Lower molecular weight OH-PCBs

10.1021/tx030034v CCC: $27.50 © 2004 American Chemical Society Published on Web 02/03/2004

Toxicity of Hydroxylated PCB Metabolites

are usually considered nonpersistent; however, they may occur transiently during episodic exposures to PCBs (6) and their rise has been suggested in arctic areas (7). Hydroxy metabolites of many PCBs were found also in laboratory animals (8). The lower halogenated PCBs are more readily metabolized to monohydroxy derivatives and further to ortho- or para-dihydroxy metabolites and quinones; formation of these oxygenated compounds is accompanied by production of reactive oxygen species, oxidative DNA damage, and direct binding to glutathione and protein sulfhydryl groups (9-13). The modulation of thyroid and estrogen hormone levels, the alteration of the biotransformation enzymes sulfo- and glucuronosyl-transferases, and their binding to the thyroxine transport protein have all been suggested as contributing significantly to the endocrinedisrupting effects of PCBs and their metabolites (1416). (Anti)estrogenic actions of hydroxy-PCBs occurring via ERs have been reported as well (17-21). However, estrogenic or antiestrogenic activities reported in previous studies were variable and dependent on the selected cellular system. Other potential nongenotoxic modes of action have not as yet been determined. These include AhR-mediated activities and inhibition of GJIC, representing dioxin-like toxicity and one of the possible mechanisms of tumor promotion (22-24), respectively. In this study, we explore the hypothesis that PCB metabolites contribute significantly to the toxicity of PCBs. A series of model and environmentally occuring mono- and dihydroxylated PCBs and 2-(x′-chlorophenyl)1,4-benzoquinones and 1,4-hydroquinones were evaluated for their nongenotoxic potencies in hepatic and mammary in vitro cellular models, designed to detect potency to acutely inhibit GJIC and AhR- and ER-mediated activities.

Materials and Methods Chemicals. TCDD was supplied by Promochem (Wesel, Germany); phorbol 12-myristate 13-acetate, 17β-estradiol, cell culture media, and neutral red were purchased from SigmaAldrich (Prague, Czech Republic), and lucifer yellow was purchased from Molecular Probes (Eugene, OR). The other chemicals used were of the highest purity available. Synthesis of Test Compounds. The monohydroxylated and ortho-dihydroxylated PCBs were synthesized using the Suzuki coupling (25-27) or by the Cadogan coupling (5, 28). The PCB quinones were synthesized using the Meerwein arylation as described by Brassard et al. (29) from 1,4-benzoquinone and the respective chloroanilines (30). The 2,5-dihydroxy PCBs were obtained from the respective quinones by reduction with sodium dithionite (31). High molecular weight hydroxy-PCBs were prepared according to the Cadogan diaryl coupling reaction as described previously (5, 28). Purity of the synthesized chemicals was determined as previously described (25). Stock solutions were prepared in DMSO, maximally 14 days before their use in the assays, and stored in the dark. The chemical structures and nomenclature of the PCB derivatives used in this study are shown in Figure 1. Inhibition of GJIC. The acute inhibition of GJIC was determined in the rat liver epithelial cell line WB-F344 (33) by the scrape loading/dye transfer method, after 30 min treatment with a tested compound (34). The cells were washed twice with PBS solution, lucifer yellow was added (0.05% w/v in PBS), and the cells were scraped using a surgical steel blade. Following 2 min of the dye diffusion, the cells were washed with PBS and fixed in 4% (v/v) formaldehyde. The ratio of the gap junctional dye transfer from the scrape line was measured with an epifluorescence microscope (Nikon Inc., Japan). At least three

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 341 scrapes per well were evaluated in at least three independent experiments, carried out in duplicate. AhR-Mediated Activity. Activation of AhR after 24 h of exposure to oxygenated PCB metabolites was determined in the DR-CALUX assay using rat hepatoma H4IIE cells stably transfected with a luciferase reporter gene under the control of dioxin responsive enhancers (BioDetection Systems, Amsterdam, Netherlands) using experimental settings reported previously (32). Briefly, the cells grown in 96 well cell culture plates were 24 h after seeding (at 90-100% confluency) exposed to the tested or reference compounds (TCDD), dissolved in DMSO. Maximum concentrations of DMSO did not exceed 0.4% (v/v). Following the 24 h exposure, the cells were washed and luciferase was extracted with a low salt lysis buffer (10 mM Tris, 2 mM dithiothreitol, and 2 mM 1,2-diamin cyclic hexaneN,N,N′,N′-tetraacetic acid, pH 7.8), and the plates were frozen at -80 °C. Luciferase activity was determined with a luciferase kit (Labsystems, Oulu, Finland) on a microplate luminometer. Modulations of ER-Mediated Activity. The ER-mediated activity was determined in two cellular systems, using the MVLN cells (a human breast carcinoma MCF-7 variant stably transfected with luciferase reporter gene under control of estrogen responsive elements p-Vit-tk-luc, obtained from Dr. Michel Pons, Montpellier, France) (35) and human breast carcinoma T47D.Luc cell line stably transfected with pEREtataLuc reporter gene (ER-CALUX assay, BioDetection Systems) (36). Both types of assays were performed in 96 well plates; cells were incubated for 24 h in phenol red-free DMEM/F12 medium containing dextran/charcoal-stripped fetal bovine serum, and then, the medium was changed to a fresh one for another 24 h. Cells were treated for 24 h with tested compounds dissolved in fresh medium. Following exposure, cells were lysed and luciferase activity was determined with a luciferase kit as described for the H4IIE.Luc cells. For detection of antiestrogenic potencies of chemicals, the cells were exposed to test chemicals in the presence of estradiol concentrations corresponding to EC50 values in both systems. Cytotoxicity. Cytotoxic concentrations of tested compounds were determined by the conventional neutral red uptake assay (37) after 24 h of exposure in the H4IIE.Luc, T47D.Luc, and WB-F344 cells. Potential acute cytotoxic effects of hydroxylated and quinoid PCB metabolites on the WB-F344 cells were investigated by the neutral red release assay also after 30 min of exposure (38). Physicochemical Descriptors. The dihedral angles were calculated with MM2* using GB/SA water solvent continuum as implemented by MacroModel 5.0 (39). Estimates for pKa (an estimate of the composition of a mixture of molecules and ions), log P, and log D (estimates of the octanol/water partition coefficient of neutral molecules and a mixture of molecules and ions, respectively), as well as the molar volume, were obtained using the software program from Advanced Chemistry Development Inc. (Ontario, Canada) (25). Statistics and Data Analysis. In vitro experiments (each in three replicates) were repeated independently at least twice, and the results were pooled. All calculations were performed with Microsoft Excel, GraphPad Prism, or Statistica software packages. In the reporter gene assays, data were expressed as % of maximal luciferase induction of reference inducers (2,3,7,8TCDD for DR-CALUX or 17β-estradiol for ER-CALUX). Relative potencies of the individual compounds were calculated as a ratio of EC50 of the reference inducer and concentration of the compound inducing the same level of luciferase activity as the reference EC50. Log linear regression was used for data extrapolation. The ratio of GJIC inhibition related to the negative control was evaluated and expressed in % (fraction of control). Nonparametric Kruskal-Wallis ANOVA followed by the MannWhitney test was used for the assessment of significance (p values of less than 0.05 were considered statistically significant). The concentrations eliciting 50% inhibition of GJIC (IC50) were determined by logit regression.

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Figure 1. Chemical structures and nomenclature of the PCB derivatives used in the study. (A) Monohydroxylated PCB derivatives; (B) dihydroxy-PCBs and benzoquinones. Experimental LOEC was selected as a measure of cytotoxicity and antiestrogenicity of tested compounds. Experimental concentration of the compound eliciting statistically significant decrease in the measured parameter in comparison with negative control (viability of the cells exposed to 0.5% DMSO), or estrogenicity of 5 pM 17β-estradiol, was the experimental LOEC for cytotoxicity or antiestrogenicity, respectively. Statistical significance was tested by ANOVA followed by Duncan’s test. The correlations of in vitro effects with physicochemical parameters of individual compounds were evaluated using nonparametric robust Spearman’s rank coefficient (R).

Results Inhibition of GJIC. The potential effects of PCB derivatives that could contribute to the promotion of carcinogenesis were studied as inhibition of GJIC com-

munication in WB-F344 cell line after a short time (30 min) of pretreatment with an individual oxygenated PCB derivative. A series of these compounds were found to act as inhibitors of GJIC at micromolar concentrations (Table 1). Persistent OH-PCBs, especially 4-OH-PCB 187 and 4-OH-PCB 146, had the strongest inhibition potencies (Figure 2A), followed by some lower molecular weight OH-PCBs, including 3′,4′-dihydroxy-PCB 3 and 3′,4′dihydroxy-PCB 12 (Figure 2B). Other low molecular weight OH-PCBs and several PCB benzoquinones inhibited GJIC only at higher than 30 µM concentrations (Table 1). No acute cytotoxic effects of tested compounds were found after either 30 min or 24 h of exposure at concentrations up to 100 µM (data not shown).

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Table 1. Potencies of PCB Derivatives to Inhibit GJIC in the Rat Liver Epithelial WB-F344 Cells and to Induce AhR-Mediated Activity in the Rat Hepatoma H4IIELuc Cell Line inhibition of GJIC IC50 (µM) 2,3,7,8-TCDD phorbol-12-myristate-13-acetate 4-OH-PCB 107 4-OH-PCB 146 4-OH-PCB 187 2′-OH-PCB 3 3′-OH-PCB 3 4′-OH-PCB 3 4-OH-PCB 14 4-OH-PCB 34 4-OH-PCB 35 6′-OH-PCB 35 4-OH-PCB 36 4′-OH-PCB 36 6′-OH-PCB 36 4-OH-PCB 39 4′-OH-PCB 68 4′-OH-PCB 79 3′,4′-di(OH)PCB 3 3′,4′-di(OH)PCB 5 3′4′-di(OH)PCB 12 2-(2′-Cl-phenyl)-1,4-HQ 2-(3′-Cl-phenyl)-1,4-HQ 2-(4′-Cl-phenyl)-1,4-HQ 2-(3′,4′-di-Cl-phenyl)-1,4-HQ 2-(3′,5′-di-Cl-phenyl)-1,4-HQ 2-(2′,4′,5′-tri-Cl-phenyl)-1,4-HQ 2-(3′,4′,5′-tri-Cl-phenyl)-1,4-HQ 2-(2′-Cl-phenyl)-1,4-BQ 2-(3′-Cl-phenyl)-1,4-BQ 2-(4′-Cl-phenyl)-1,4-BQ 2-(3′,4′-di-Cl-phenyl)-1,4-BQ 2-(3′,5′-di-Cl-phenyl)-1,4-BQ 2-(3′,4′,5′-tri-Cl-phenyl)-1,4-BQ

8 × 10-3 20.0 13.2 8.7 NI 36.5 31.0 38.2 32.0 31.1 23.8 30.3 38.3 20.0 26.7 22.2 26.9 42.5 16.2 19.3 NI NI WI WI NI WI WI WI 69.8 73.2 49.0 37.1 42.1

AhR-mediated activity IC50 (µM) 1 × 10-5 NI NI NI 87.2 (1.2 × 10-7)a WI WI NI NI NI 8.3 (1.2 × 10-6) NI NI NI WI NI 6.3 (1.6 × 10-6) NI NI NI NI NI 3.6 (2.8 × 10-6) 0.8 (1.3 × 10-5) NI NI NI NI 69.1 (1.5 × 10-7) 0.3 (3.3 × 10-5) 1.0 (1.0 × 10-5) WI 7.1 (1.4 × 10-6)

a Induction potencies relative to 2,3,7,8-TCDD in parentheses; NI, no significant induction/inhibition; WI, weak induction/inhibition (20 >20 >20 >20 >20 20 10 >20 10 5 20 10 20 >20 20 20 20 20 10 2.5 5 5 5 20 5 5 5 5 5

20 >20 >20 20 20 50 35 50 35 20 20 10 20 20 35 10 50 15 10 50 20 15 10 15 50 2.5 35 25 10 10 20 15

a Cytotoxicity was determined by the 24 h neutral red uptake assay (see Materials and Methods). NI, no significant induction/inhibition; WI, weak induction/inhibition. Asterisks indicate additive induction effect of 17β-estradiol and respective compound. b ER-mediated activity (expressed as EC50 if available). c Maximal induction observed in T47D.Luc cells (% of 17β-estradiol). d Anti-ER, the LOEC suppressing significantly (p < 0.1) the 17β-estradiol response at a concentration of EC50. e LOEC significantly affecting viability of the T47D.luc cells (neutral red uptake assay after 24 h of exposure).

man’s rank correlations between the in vitro toxicological data and selected physicochemical parameters were calculated. Hydrophobicity (log P) and molar volume correlated with each other (Spearman’s R ) 0.9, p < 0.0001); correlations of these physicochemical descriptors with two biological endpoints, inhibition of GJIC and antiestrogenicity, were found to be significant. Calculated pKa, log D, and dihedral angle demonstrated a lower significance to all biological activities studied. Correlations for AhR- and ER-mediated effects were not evaluated due to low numbers of active compounds (N ) 9 and N ) 8, respectively), but the relatively weak AhR agonists apparently did not suppress ER-dependent gene expression.

Figure 4. ER-mediated activities of 4-OH-PCB 107 (circles) and 4′-OH-PCB 3 (triangles) in the MVLN (open symbols) and T47D.Luc (closed symbols) cell lines; comparison with the effect of 17β-estradiol (diamonds).

specific antiestrogenicity was the cause of the decline of the ER-mediated activity. Only 4-OH-PCB 187, 4-OHPCB 146, and 2-(2′,4′,5′-trichlorophenyl)-1,4-hydroquinone elicited a true antiestrogenic response (see Table 2). Structural Characteristics. Several physicochemical descriptors were estimated as described (25), and Spear-

For all compounds that downregulated GJIC (N ) 23), there were significant correlations between IC50 and both log P (Figure 6) and molar volume (data not shown); four outliers from a linear relationship were observed, namely, 3′,4′-dihydroxy-PCB 5, 3′,4′-dihydroxy-PCB 12, 2-(3′chlorophenyl)benzoquinone, and 2-(4′-chlorophenyl)benzoquinone. The correlations were significant also for the entire set of compounds (N ) 32) considering both weak and noninhibiting compounds (R < -0.7; p < 0.0001). Correlations of cytotoxicity and antiestrogenicity effects were observed only for some subsets of compounds. For example, antiestrogenicity was correlated with log P and

Toxicity of Hydroxylated PCB Metabolites

Figure 5. Additivity of ER-mediated responses after cotreatment of 17β-estradiol with 4-OH-PCB 107 (A) and 4′-OH-PCB 3 (B).

Figure 6. Correlations between potency of oxy-PCBs to inhibit GJIC and log P values (see Materials and Methods).

molar volume for 16 monohydroxy PCBs (N ) 16, R < -0.6, p < 0.01) but neither for other compound subsets nor for the entire set of compounds.

Discussion The availability of a large series of OH-PCBs and PCB quinones allowed us to investigate three major nongenotoxic adverse effects in well-established in vitro bioassays, the acute inhibition of GJIC, activation of AhR, and modulation of ER-dependent gene expression. Inhibition of GJIC has been shown to be strongly associated with in vivo tumor promotion and carcinogenicity of many xenobiotics (24). Therefore, inhibition potency of PCB derivatives was investigated in the rat liver epithelial WB-F344 cell line as a marker of one of potential modes of tumor-promoting activity. A majority of mono- and dihydroxy-PCBs inhibited GJIC in vitro in micromolar concentrations (Table 1). These concentrations were

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similar to reported effective doses of other environmental tumor-promoting pollutants such as PCBs (40), polycyclic aromatic hydrocarbons (34, 41), or organochlorine pesticides (42) and indicated a possible significant mode of action of OH-PCBs. The strongest inhibition potencies were found for persistent high molecular weight 4-OHPCB 187 and 4-OH-PCB 146. This is, as far as we know, the first indication of potential tumor promotional effects of OH-PCBs; however, a significance of this mechanism of action should be confirmed in other cellular models and in vivo. The AhR-inducing activities of both persistent polyhalogenated aromatic hydrocarbons and more readily metabolized planar compounds, such as polycyclic aromatic hydrocarbons, are well-known (1, 32). In this study, the AhR-inducing potencies of oxygenated PCB derivatives including OH-PCBs and PCB quinones were investigated in the DR-CALUX assay. A significant AhR-mediated activity of 4′-OH-PCB 79, a planar metabolite of PCB 77, and 6′-OH-PCB 35 (Table 1) was comparable to the potencies of weak nonsubstituted PCB inducers of this activity (22). Surprisingly, 2-(4′-chlorophenyl)- and 2-(3′,4′dichlorophenyl)-1,4-benzoquinones and hydroquinones were relatively potent inducers of AhR-mediated activity as well, with EC50 values between 0.3 and 3.6 µM. The high molecular weight OH-PCBs elicited no AhR-mediated activity. Several model and/or environmentally relevant OHPCBs have been previously reported to possess either estrogenic or antiestrogenic properties, and (anti)estrogenic effects of OH-PCBs remain controversial (17-21). Several low chlorinated OH-PCBs elicited a weak estrogenicity in the standardized yeast estrogen assay (21). High molecular weight OH-PCBs, including 4-OH-PCB 187, 4-OH-PCB 146, and 4-OH-PCB 107, were antiestrogenic in the luciferase gene transfected HeLa cells when cotreated with 17β-estradiol (18). In the present study, some of the low chlorinated monohydroxy- and dihydroxy-PCBs were found to be estrogenic in two human breast carcinoma cell lines, MVLN and T47D.Luc cells transfected stably with luciferase reporter constructs p-Vit-tk-luc and pEREtata-luc, respectively, with the order of the ER-mediated potencies decreasing as follows: 4′-HO-PCB 3 > 3′-HO-PCB 3 > 3′,4′-diOH-PCB 3 (Table 2). Interestingly, a significant 2-fold increase of maximum ER activation was observed after 4′-OH-PCB 3 treatment, when compared to 17β-estradiol response (Figure 4). Moreover, estrogenic PCB derivatives increased significantly the response of cells to 17β-estradiol (Figure 5). These findings might suggest a possible synergistic effect of OH-PCBs, which could be associated partially with a ligand-independent activation of ER; preliminary data documenting only a partial decrease of estrogenic response to 4-OH-PCB 3 when cotreated with ER antagonist ICI 182,780 (data not shown) seem to support this hypothesis. A similar effect was previously found also with some nonsubstituted polycyclic aromatic hydrocarbons (35). 4-HO-PCB 107 was weakly estrogenic at lower concentrations and increased 17β-estradiol response as well; its antiestrogenic effect was apparent at the doses close to the cytotoxic concentrations. Several AhR inducers, including 2-(3′,4′-dichlorophenyl)-1,4-HQ and 2-(3′,4′,5′-trichlorophenyl)-1,4-BQ, elicited weak in vitro antiestrogenicity when using lower than cytotoxic concentrations (2.5-5 µM). Suppression of ER-mediated activity and antiestrogenicity might in

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this case be related to inhibitory AhR-ER cross-talk (43). However, other AhR ligands (4′-OH-PCB 79, 6′-OH-PCB 35, 2-(4′-chlorophenyl)-1,4-BQ, and 2-(3′,4′-dichlorophenyl)-1,4-BQ) induced antiestrogenicity only at cytotoxic or near cytotoxic concentrations. Two persistent compounds, 4-OH-PCB 187 and 4-OH-PCB 146, as well as some other PCB derivatives, 6′-OH-PCB 36, 2-(2′-chlorophenyl)-1,4-HQ, and 2-(2′,4′,5′-trichlorophenyl)-1,4-HQ, showed a significant antiestrogenic activity without being cytotoxic at levels significantly reducing ER-medited activity. Structure-estrogenicity/antiestrogenicity relationships for high molecular weight 4-OH-PCBs have been reported by Connor et al. (19). Most of the tetra- and pentachlorinated OH-PCBs were antiestrogenic in both in vitro and in vivo bioassays using determination of ER-dependent luciferase reporter gene expression, cell proliferation, and uterine wet weight. However, a simple structure-activity relationship was not found. In another study, antiestrogenicity of 4-OH-PCB 107 and 4-OH-PCB 35 and several other PCB metabolites has been related to their effect on cell viability (20). This is in accordance with our findings that 4-OH-PCB 107 and a series of lower halogenated OH-PCBs as well as PCB hydroquinones and quinones decreased the response to estradiol only when cotreated at the doses close to the cytotoxic concentrations (Table 2). A correlation of the toxicological end points with selected physicochemical descriptors revealed that the size of the molecule (molar volume) and hydrophobicity (log P) are important parameters determining some of the studied biological activities. Correlations between log P (or molar volume) and selected in vitro effects in T47D.Luc cells (cytotoxicity and antiestrogenicity) were observed, but these were significant only for particular subsets of compounds. Such observations could indicate that other mechanisms might play a role in the antiestrogenic effects of PCB derivatives, particularly hydroquinones or benzoquinones. Inhibition of GJIC was significantly correlated with both log P and molar volume; compounds with higher log P and molar volumes had generally lower IC50 values (Figure 6). Although some compounds deviated from the linear relationship (four outliers), this could be attributed to other specific mechanisms not investigated, for example, possible modulation of intracellular serine/threonine protein kinase signaling, which can contribute to the inhibitory effect of some xenobiotics (42, 44). Apparent correlations between hydrophobicity/molecular volume parameters and potency to inhibit GJIC might also suggest a possible contribution of nonspecific disruption of plasma membrane physiology. A similar relationship between the potency to downregulate GJIC in vitro and hydrophobicity has been previously reported for polycyclic aromatic hydrocarbons (34, 41, 45). High levels of persistent higher molecular weight OHPCBs were reported in human and other mammalian blood samples (4, 46, 47). Both low molecular weight OHPCBs and PCB quinoid metabolites were detected in male rats (31), while metabolic activation of PCBs to quinones has been also reported (30). It has been documented that levels of some hydroxymetabolites of PCB congeners can make up to 10% as compared to the most prevalent PCB153 (4). This seems to suggest that OH-PCBs are present in blood at relatively high levels, which makes the effects of OH-PCBs observed at micromolar or sub-

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micromolar concentrations possibly relevant to both human and animal health. In conclusion, persistent OH-PCBs were found to inhibit GJIC, which in effect might play a role in tumor promotion. 4-OH-PCB 107 was weakly estrogenic, while 4-OH-PCB 187 and 4-OH-PCB 146 elicited antiestrogenicity. Also, several low molecular weight OH-PCBs belong among compounds that are weakly estrogenic and inhibit GJIC. 6′-OH-PCB 35 and 4′-OH-PCB 79, as well as several PCB benzoquinones and hydroquinones, are relatively potent AhR inducers; some of them acted also as antiestrogens in subcytotoxic concentrations. The data presented here show that both the low molecular weight PCB metabolites and the persistent HO-PCBs are capable of multiple nongenotoxic modes of action, including significant AhR-mediated activity, modulation of ERmediated responses, and inhibition of intercellular communication potentially associated with promotional effects. This multiplicity, and a possible synergism of modes of action of OH-PCBs, should be considered in risk assessment of the PCB derivatives.

Acknowledgment. We thank Prof. A. Bergman (Stockholm University, Sweden) for help with the preparation of high molecular weight OH-PCBs. This work was supported by the Czech Ministry of Agriculture (Grant No. QC0194) and the Czech Academy of Sciences (Project No. P1050128), by the University of Kentucky Superfund Basic Research Program (P42 ES 07380), by The University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH P30 ES05605, and by the NIH Research Grant No. D43 TW00621, funded by the Fogarty International Center, National Institute of Environmental Health Sciences, National Institute for Occupational Safety and Health, and the Agency for Toxic Substances and Disease Registry. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS or the NIH.

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