Inhibition of Gap-Junctional Intercellular Communication by Environmentally Occurring Polycyclic Aromatic Hydrocarbons

Ludek Bláha*,{dagger}, Petra Kapplová*,{dagger}, Jan Vondrácek*,{ddagger}, Brad Upham§ and Miroslav Machala*,1

* Veterinary Research Institute, Hudcova 70, CZ-62132 Brno, Czech Republic; {dagger} Research Center for Atmospheric and Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk University, CZ-63700 Brno, Czech Republic; {ddagger} Institute of Biophysics, Czech Academy of Sciences, CZ-61265 Brno, Czech Republic; and § Department of Pediatrics and Human Development and the National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824

Received July 14, 2001; accepted October 5, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons (PAHs) are a broad class of ubiquitous environmental pollutants with known or suspected carcinogenic properties. Tumor promotion is a cell-proliferative step of cancer that requires the removal of cells from growth suppression via the inhibition of gap-junctional intercellular communication (GJIC). Inhibition of GJIC measured with an in vitro WB-F344 rat liver epithelial cell system was used to assess the relative potencies of 13 PAHs suggested by the U.S. Environmental Protection Agency (EPA) as the principal contaminants and 22 other PAHs, most of them identified in environmental samples. Maximal inhibition of GJIC was detected after 30 min of exposure, followed by a recovery in intercellular communication after an additional 30 min of exposure, suggesting a transient character of inhibition. Although µM concentrations of PAHs were required to reach the inhibition level equal to the model tumor promoter phorbol 12-myristate 13-acetate (IC50 = 8 nM), 12 of the PAHs under study were found to be strong inhibitors of GJIC (strongest effects were observed with fluoranthene, picene, 5-methylchrysene and nine additional PAHs). The other nine PAHs, including benzo[a]pyrene, inhibited GJIC only up to 50–75% of the control level. Interestingly, several high molecular weight PAHs with known strong carcinogenic properties possessed only weak (dibenzopyrenes) or no inhibition potency (dibenzofluoranthenes, naphtho[2,3-a]pyrene and benzo[a]perylene). Based on the IC50 values related to the reference PAH benzo[a]pyrene, we suggested arbitrary values of inhibition equivalency factors (GJIC-IEFs) ranging from 0 (noninhibiting PAHs) to 10.0 (strongest inhibitors), suitable for the purposes of environmental risk assessment.

Key Words: gap-junctional intercellular communication (GJIC); polycyclic aromatic hydrocarbons (PAHs); nongenotoxic carcinogenicity; tumor promotion; in vitro.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemically induced carcinogenesis involves 3 operationally defined stages: initiation, promotion, and tumor progression. While the first step (tumor initiation) is known to involve mutagenic/genotoxic events leading to DNA damage (Trosko, 1997Go), during the further stages of carcinogenesis the initiated cells undergo a series of reversible, nongenotoxic events (also called "epigenetic"), which lead to their independence from the homeostatic control mechanisms of a tissue and result in uncontrolled proliferation of initiated cells (Trosko et al., 1998Go).

The downregulation of gap junctional intercellular communication (GJIC) by tumor promoting compounds is considered to be a critical step in the removal of a cell from growth suppression (Trosko and Ruch, 1998Go; Upham et al., 1998Go; Yamasaki et al., 1995Go). Gap junctions are channels formed by connexin proteins that permit small regulatory molecules and ions <1000 molecular weight (e.g., glutathione, cAMP, Ca2+, inositoltriphosphate, etc.) to pass directly between adjacent cells (Loewenstein, 1987Go; Upham et al., 1997bGo). GJIC has been linked to the regulation of development, cellular proliferation, differentiation, and apoptosis (Trosko and Goodman, 1994Go; Trosko and Ruch, 1998Go; Yamasaki et al., 1995Go). Downregulation of GJIC by chronic exposure to toxicants has been suggested as playing an important role in the tumor-promoting steps of cancer. Strong correlations between the results of in vivo two-stage carcinogenesis tests and in vitro GJIC inhibition assays were found for a number of known tumor promoters, such as phorbol 12-myristate 13-acetate (PMA) (Fitzgerald and Yamasaki, 1990Go) and several organochlorine compounds (Baker et al., 1995Go; Flodström et al., 1988Go; Ren et al., 1998Go; Sai et al., 1998Go; Warngard et al., 1985Go, 1989Go), using rat liver WB-F344 epithelial cells, hamster V79 fibroblasts, or primary rat hepatocytes as model systems. A structure activity-relationship model on GJIC inhibiting activity resulted in a very strong concordance between experimental and predicted results (Rosenkranz et al., 1997Go) and indicated that inhibition of GJIC is linked to the carcinogenic process in rodents (Rosenkranz et al., 2000Go). Therefore, a potency of a given chemical to inhibit GJIC in vitro can be assumed to be a representative marker of tumor-promoting properties for a majority of known classes of tumor promoters.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and food contaminants formed mainly during the incomplete combustion of organic materials. High concentrations of PAHs were found in various environmental samples and complex mixtures, such as air particulate matter, soil, river, and marine sediments, petroleum products, and tar or tobacco smoke (Marvin et al., 1999Go; WHO, 1998Go). Their presence in the environment is of concern, since many of them are suspected of being strong mutagens and carcinogens (Delistraty, 1997Go; Durant et al., 1996Go, 1999Go). In vivo tests have shown that many PAHs induce tumors in rodents (IARC, 1983Go). Until recently, the studies on the carcinogenicity of PAHs focused almost exclusively on genotoxic events. However, PAHs have been shown to cause adverse nongenotoxic effects, such as aryl hydrocarbon receptor (AhR)-mediated activation of genes (Bols et al., 1999Go; Clemons et al., 1998Go; Machala et al.Go, in press; Piskorska-Pliszczynska et al., 1986Go; Willett et al., 1997Go), perturbation of Ca2+ levels (Tannheimer et al., 1997Go), activation of mitogen-activated protein kinase (MAPK)-mediated intracellular signaling (Rummel et al., 1999Go), and inhibition of GJIC (Upham et al., 1994Go, 1998Go). PAHs form an extremely heterogeneous class of individual chemicals numbering in the hundreds, but it is practical to routinely monitor only a few selected PAHs in complex environmental matrices. The U.S. EPA requires monitoring of 16 so-called priority PAHs (Callahan, 1979Go). However, the selection of monitored PAHs does not sufficiently reflect the real toxicity of complex mixtures (mutagenicity, AhR-mediated toxicity, or tumor-promoting activity). Therefore, in the present study, we compared the potencies of 35 PAHs to inhibit GJIC using an in vitro rat liver epithelial cell system, which included U.S. EPA priority PAHs as well as some high molecular weight PAHs, most of them detected in various environmental samples (Machala et al., 2001Go; Marvin, 1999Go). Based on the relative potencies (REPs) of individual PAHs expressed as a ratio of the IC50 of the reference compound (benzo[a]pyrene) and the IC50 of individual PAHs, arbitrary inhibition equivalency factors (GJIC-IEFs) were suggested in this study for the purposes of toxicity estimation of complex samples and environmental risk assessment. Such values allow us to characterize individual PAHs of ecotoxicological importance by multiplying the IEF value with the concentration of the respective compound. The concept of GJIC-IEFs is similar to a widely accepted approach using toxic equivalency factors (TEFs) for assessment of dioxin-like toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds (van den Berg et al., 1998Go), and the TEF approach for risk assessment of carcinogenic PAHs (Delistraty, 1997Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Fluorene (CAS No. 86-73-7, purity 99.9%), anthracene (CAS No. 120-12-7, purity 99.9%), phenanthrene (CAS No. 85-01-8, purity 98%), fluoranthene (CAS No. 206-44-0, purity 99.9%), pyrene (CAS No. 129-00-0, purity 99.9%), benz[a]anthracene (CAS No. 86-73-7, purity 99.9%), chrysene (CAS No. 218-01-9, purity 99.9%), benzo[b]fluoranthene (CAS No. 205-99-2, purity 99.9%), benzo[j]fluoranthene (CAS No. 205–82–3, purity 99.9%), benzo[k]fluoranthene (CAS No. 207-08-9, purity 99.9%), benzo[a]pyrene (CAS No. 50-32-8, purity 99.9%), indeno[1,2,3-cd]pyrene (CAS No. 193-39-5, purity 99.9%), dibenz[ac]anthracene (CAS No. 21-58-7, purity 99.9%), dibenz[a,h]anthracene (CAS No. 53-70-3, purity 99.9%), benzo[ghi]perylene (CAS No. 191-24-2, purity 99.9%), perylene (CAS No. 198-55-0, purity 97%), and picene (CAS No. 213-46-7, purity 99.9%) were supplied by Ehrenstorfer (Augsburg, Germany). Benzo[a]perylene (CAS No. 19-85-5, purity 99.0%), benzo[c]phenanthrene (CAS No. 195-19-7, purity 99.9%), cyclopenta[cd]pyrene (CAS No. 27208-37-3, purity 99.8%), dibenz[a,j]anthracene (CAS No. 224-41-9, purity 99.9%), dibenzo[a,e]fluoranthene (CAS No. 5385-75-1, purity 99.4%), dibenzo[a,k]fluoranthene (CAS No. 84030-79-5, purity 99.8%), dibenzo[a,e]pyrene (CAS No. 192-65-4, purity 99.8%), dibenzo[a,h]pyrene (CAS No. 189-64-0, purity 99.8%), dibenzo[a,i]pyrene (CAS No. 189-55-9, purity 99.9%), dibenzo[a,l]pyrene (CAS No. 191-30-0, purity 99.8%), 7,12-dimethylbenz[a]anthracene (CAS No. 57-97-6, purity 99.8%), 1-methylpyrene (CAS No. 2381-21-7, purity 99.5%), 5-methylchrysene (CAS No. 3697-24-3, purity 99.8%), and triphenylene (CAS No. 217-59-4, purity 98%) were purchased from Promochem (Wesel, Germany). 4H-cyclopenta[def]phenantrene (CAS No. 203-64-5, purity 97%), naphtho[2,3-a]pyrene (CAS No. 196-42-9, purity 99.5%), coronene (CAS No. 191-07-01, purity 97%) and benzo[e]pyrene (CAS No. 192-97-2, purity 99%) were purchased from Sigma-Aldrich Chemie (Schnelldorf, Germany). HPLC gradient grade acetonitrile and methanol, were purchased from Merck (Merck, Darmstadt, Germany). The other organic solvents used were for organic trace analysis. Ultrapure water was obtained from a Milli-Q UF Plus water system (Millipore, Molsheim, France). The other chemicals used were of the highest purity available. Stock solutions were prepared in dimethylsulfoxide (DMSO) and stored in the dark.

The chemical structures of PAHs under study, with the exception of known U.S. EPA priority PAHs, are presented in Figure 1Go.



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FIG. 1. Chemical structures of the studied polycyclic aromatic hydrocarbons (PAHs). The structures of known PAHs suggested by U.S. EPA as the principal contaminants are not shown.

 
GJIC inhibition assay.
WB-F344 rat liver epithelial cell line (Tsao et al., 1984Go) were cultured in modified Eagle's Minimum Essential Medium (Sigma-Aldrich, Prague, Czech Republic) with 50% increased concentrations of essential and nonessential amino acids, and supplemented with pyruvate (110 mg/l), 10 mM HEPES, and 5% fetal bovine serum (Sigma-Aldrich, Prague). Confluent cells, grown in 24-well plates, were exposed to PAHs (up to 100 µM concentration), PMA (positive control), or DMSO (negative control) for 15, 30, or 60 min. The concentrations of DMSO did not exceed 0.5% v/v. After the exposure, a modified protocol of scrape-loading (El-Fouly et al., 1987Go) was used to assess GJIC. The cells were washed twice with 0.5x PBS (phosphate-buffered saline solution), fluorescent dye was added (lucifer yellow, 0.05% w/v in PBS), and the cells were scraped using a surgical steel blade. After 4 min, the cells were washed twice by 0.5x PBS and fixed with 4% formaldehyde (v/v). The migration of the dye from the scrape line was measured with an epifluorescence microscope (Nikon, Inc., Japan). Three independent experiments were carried out in duplicate; at least 3 scrapes per well were evaluated. Cytotoxicity was assessed by a conventional MTT assay (Mosmann, 1983Go). No apparent toxic effects of PAHs were observed within concentrations and exposure periods under study.

Data analyses.
The ratio of GJIC inhibition related to the negative control was evaluated and expressed in percentage (fraction of control, FOC). Nonparametric statistical methods were used for the data analysis. Kruskal-Wallis ANOVA followed by the Mann-Whitney test were used for the assessment of significance, and p values of less than 0.05 were considered statistically significant. The concentrations of reference toxicants (PMA and benzo[a]pyrene) and concentrations of the PAHs under study causing 50% inhibition of GJIC (IC50) were determined by the nonlinear logit regression, and 95% confidence intervals for IC50 were estimated; relative error did not exceed 15%. The values of relative inhibition potencies (REPs) were based on calculated ratios of reference IC50 (benzo[a]pyrene) vs. the IC50 of the respective PAH under study.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Three exposure periods (15, 30, and 60 min) were selected, based on previously reported time dependence of WB cells' GJIC responses to PMA and several PAHs (Rummel et al., 1999Go; Weis et al., 1998Go) Temporal changes within 60 min after addition of PMA or PAHs are shown in Figure 2Go. The reference tumor promoter (PMA) and the PAHs showed characteristic rapid inhibitory effect on GJIC (strongest being at 15 and 30 min). Inhibition was transient with recovery occurring 60 min after initial exposure to PAHs. Recovery from the inhibition of GJIC was maintained, even after 2- and 6-h exposure periods (data not shown). Noninhibiting PAHs had no effect on GJIC during a 15- to 60-min exposure up to the maximal concentration used (100 µM). To characterize and compare the in vitro tumor-promoting potency of PAHs, a 30-min exposure period was selected for further comparisons. Dose-response curves were plotted for the individual PAHs tested. Inhibitory effects of all the PAHs under study on GJIC after a 30-min exposure are summarized in Table 1Go.



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FIG. 2. Inhibition of GJIC in the WB F344 cells exposed to model inhibitor (PMA) and selected PAHs after three exposure periods (15, 30, and 60 min). Error bars represent mean values (± SD) of at least 3 independent experiments performed in duplicate. Fla, fluoranthene; 1-MePy, 1-methylpyrene; B[a]P, benzo[a]pyrene; B[ghi]Per, benzo[ghi]perylene; B[e]P, benzo[e]pyrene.

 

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TABLE 1 Inhibition of Gap-Junctional Intercellular Communication in WB-F344 Cells Exposed to Model Tumor Promoter Phorbol-12-myristate-13-acetate or to 35 Polycyclic Aromatic Hydrocarbons
 
Twenty-one PAHs showed significant concentration-dependent inhibition of GJIC within tested concentrations (Fig. 3Go). Two different patterns in the inhibition of GJIC were observed: Rapid, almost 100% inhibition of GJIC (2–3% FOC) was observed when cells were treated with fluorene, phenanthrene, fluoranthene (U.S. EPA priority PAHs) and cyclopenta[def]phenanthrene, cyclopenta[cd]pyrene, benzo[c]phenanthrene, 7,12-dimethylbenz[a]anthracene, 1-methylpyrene, 5-methylchrysene, and picene. The only exception within this group was pyrene and dibenz[a,c]anthracene, which reached a plateau at a 70% inhibition level (i.e., 30% FOC, see Figs. 3A–3CGo). The strongest effects were observed with fluoranthene, picene and 5-methylchrysene (IC50 ranged from 9 to 13 µM, Table 1Go).



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FIG. 3. Concentration-dependent inhibition of GJIC by PAHs in the WB F344 cells (30 min exposure). (A) U.S. EPA priority PAHs with strong inhibitory effects; (B, C) other PAHs with strong inhibitory effects; (D) U.S. EPA priority PAHs with weak inhibitory effects; (E) other PAHs with weak inhibitory effects. Error bars eliminated for legibility; coefficient of variance did not exceed 15%.

 
Exposure to benzo[a]pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[ghi]perylene (belonging among the U.S. EPA priority PAHs) and dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene and triphenylene also led to significant inhibition of GJIC, but the maximum inhibition was not observed at the highest doses tested. Inhibition ranged from 50–75% FOC at the highest doses tested (Figs. 3D and 3EGo). For these compounds, the IC50 value could not be estimated. The strongest inhibitor within this group of compounds was benzo[a]pyrene, selected as the reference compound for further comparisons.

Nine PAHs showing no significant inhibitory effects on GJIC included anthracene, benzo[k]fluoranthene, dibenzo[a,h]anthracene (U.S. EPA priority PAHs), as well as dibenzo[a,i]pyrene, dibenz[a,j]anthracene, benzo[j]fluoranthene, benzo[e]pyrene, perylene, and coronene. Due to the overlap of fluorescence spectra of the other 5 compounds (indeno[1,2,3-cd]pyrene, naphtho[2,3-a]pyrene, benzo[a]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,k]fluoranthene) with the fluorescence of lucifer yellow, it was not possible to estimate the effect of these compounds at the highest tested concentration (100 µM). However, none of the compounds showed significant inhibitory activity at 50 µM concentration. Thus, these were considered as noninhibiting compounds of GJIC in the WB-F344 cells.

Based on our results, relative inhibition potencies were calculated for all the PAHs under study, as the ratio of the IC50 of the reference PAH benzo[a]pyrene and IC50 of each respective PAH. The values of IC50 and calculated ratios (REPs) are summarized in Table 1Go. Based on the REP values, the GJIC-IEFs for the purposes of risk assessment were arbitrarily chosen (Table 1Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the mutagenicity of PAHs has been studied extensively as the major mechanism of their carcinogenicity (IARC, 1983Go; WHO, 1998Go), nongenotoxic effects of PAHs may also play a role (Ashby and Tennant, 1991Go). For example, although no evidence of tumor-initiating activity of fluoranthene was observed in experimental animals (IARC, 1983Go; Stocker et al., 1996Go), or in a mutagenicity bioassay using human transgenic cells (Durant et al., 1996Go), this PAH increased lung and liver tumors in mouse neonates (Wang and Busby, 1993Go).

Several nongenotoxic effects of PAHs, which could play a role in carcinogenesis, were reported. These include strong Ah receptor-mediated activities of PAHs (Bols et al., 1999Go; Clemons et al., 1998Go; Machala, 1996Go; Machala et al., 2001Go; Piskorska-Pliszczynska et al., 1986Go; Willett et al., 1997Go), estrogenic receptor-mediated activities, i.e., agonistic activation of estrogenic receptor and/or antiestrogenic effects associated with AhR activation (Clemons et al., 1998Go; Safe et al., 1998Go), oxidative stress (Burczynski et al., 1999Go), modulation of intracellular signal transduction pathways involving release of Ca2+ (Tannheimer et al., 1997Go), and MAPK activation (Rummel et al., 1999Go). The known carcinogenic, genotoxic, and AhR-mediated potencies of PAHs are presented in Table 1Go, in order to compare them with the inhibitory effects of individual compounds on GJIC.

Of all the in vitro methods for detecting tumor-promoting activity, the assays for GJIC inhibition seem to have the best predictive power (Autrup and Dragsted, 1987Go; Rosenkranz et al., 2000Go, 1997Go). In previous reports (Ghoshal et al., 1999Go; Upham et al., 1994Go; Weis et al., 1998Go) several low molecular weight PAHs (such as fluorene, phenanthrene, fluoranthene, and their methylated derivatives) have been shown to elicit a strong downregulation of GJIC in the WB-F344 cells, while pyrene, benzo[a]pyrene and benzo[e]pyrene induced only a partial inhibition within micromolar exposure concentrations. However, a number of PAHs that are prevalent in environmental samples have not been included in those studies. Therefore, in the present study, the inhibitory potencies of a broader set of PAHs were investigated.

Exposure of WB-F344 cells to relatively smaller molecules (such as fluorene, phenanthrene, fluoranthene, pyrene and others) resulted in higher than 70% inhibition of GJIC (up to 98% inhibition), while the other nine PAHs (mostly higher molecular weight compounds, such as benzo[a]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene and others) caused only 25–50% inhibition of GJIC where a plateau in the response was reached (exposure to higher concentration did not further inhibit GJIC). Since pyrene and dibenz[a,c]anthracene caused higher than 50% inhibition and elicited relatively low IC50 values, these compounds seemed to belong to the group of strong inhibitors (Fig. 3Go, Table 1Go). No inhibition up to 100 µM was observed for the remaining PAHs. Therefore, we classified these compounds as noninhibitors of GJIC in the cellular model used (Table 1Go).

The results showed that several PAHs considered to possess a nonmutagenic or low mutagenic activity in human and bacterial in vitro models, such as phenanthrene, fluoranthene, pyrene, benzo[c]phenanthrene and picene (Delistraty, 1997Go; Durant et al., 1996Go), belong among the most potent inhibitors of GJIC. Other compounds that are usually not analysed during routine monitoring procedures, such as cyclopenta[cd]pyrene and 5-methylchrysene (both known to be potent mutagens) were found to be also potent inhibitors of GJIC. On the other hand, some of the strong mutagens and carcinogens, such as dibenzopyrenes and dibenzofluoranthenes, showed very low or no inhibition of GJIC. Taken together, in agreement with the results of Rosenkranz et al. (2000), no apparent correlations were found between the reported genotoxic potencies of the PAHs and their potencies to inhibit GJIC in vitro (see Table 1Go). Similarly, we have previously shown that the data on the Ah receptor-mediated potencies of PAHs also did not correlate with their mutagenic properties (Machala et al., 2001Go). The results suggest an urgent need to study carcinogenicity of PAHs, not only by means of mutagenicity or AhR-mediated toxicity in a single assay, but also in using test systems on GJIC inhibition and/or other important nongenotoxic modes of toxic action.

Similar to time- and dose-dependent GJIC inhibition previously reported for several PAHs (Ghoshal et al., 1999Go; Weis et al., 1998Go), responses to the exposures of 21 GJIC-inhibiting PAHs in our study were observed to have a transient character. Strong inhibitory effects were found within 30 min, while a recovery of GJIC was observed after prolonged exposure periods (1–4 h). Several model tumor promoters such as PMA also produce only a transient inhibition of GJIC (Kanemitsu et al., 1993Go). Despite the transient character of this process, the inhibition of GJIC is currently considered to be a suitable in vitro biomarker of tumor promoting potency of tested compounds.

PMA and PAHs inhibited GJIC at significantly different concentrations (IC50 of PMA was 8 nM, while PAHs inhibited within micromolar concentrations after a 30-min exposure). Interestingly, estimated IC50 values of individual strongly inhibiting PAHs in this study did not differ more than one order of concentration range (10–50 µM). Similar micromolar concentrations were previously reported for GJIC inhibiting methylated and chlorinated PAHs (Rummel et al., 1999Go; Weis et al., 1998Go), and also for persistent chlorinated tumor promoters such as DDT (Fransson et al., 1990Go) and lindane (Upham et al., 1997aGo). However, the concentrations of PAHs required to inhibit GJIC could approach the levels known from human exposure data (WHO, 1998Go).

With respect to rapid occurrence of GJIC inhibition, the mechanism of action of these compounds is probably at the posttranslational level, as suggested by Rummel et al. (1999). One of the possible mechanisms of GJIC control is phosphorylation of connexins. Several protein kinases, such as protein kinase C (PKC), mitogen-activated protein kinases (MAPK), or tyrosine kinases are known to be involved in this process (Lampe and Lau, 2000Go). Although PKC-mediated phosphorylation of connexin is supposed to be a major mechanism by which PMA disrupts GJIC (Lampe and Lau, 2000Go; Madhukar et al., 1996Go), the mode of action of PAHs may be different. It has been shown, that PAHs induce MAPK activation downstream of GJIC inhibition (Rummel et al., 1999Go). Another mechanism may involve phospholipase-induced release of arachidonic acid metabolites after PAH exposure (Upham et al., 2000Go). In conclusion, the molecular mechanisms involved in the inhibition of GJIC by PAHs are still poorly understood and additional studies are required to elucidate this process.

Several structure-activity relationships have been described, based on the previous studies on downregulation of GJIC by PAHs (Rummel et al., 1999Go; Upham et al., 1994Go, 1998Go; Weis et al., 1998Go). The methylated and chlorinated PAH derivatives that had bay-like regions were more inhibitory than the PAH-counterparts that did not contain the angular pocket of the bay-like region, which had been formed by either a methyl or a chlorine group, and the 3-ringed PAHs appeared to possess the higher inhibition when compared to 2-, 4-, and 5-ringed PAHs. In the present study, the following structural dependencies were apparent and corresponded to previous findings discussed above: PAHs with higher molecular masses and higher lipophility (Kow values) elicited low or negligible inhibition activity; stronger GJIC inhibition potency of bay region-forming or bay-like PAHs (methylated derivatives) was confirmed. In contrast, some higher molecular mass PAHs with bay-like regions (picene and dibenz[a,c]anthracene) showed strong inhibitory effect. Thus, several physicochemical characteristics seem to affect inhibitory potencies of individual PAHs.

In this study, IC50 values were estimated for individual compounds, inhibitory potencies (REPs) related to a reference PAH such as benzo[a]pyrene were calculated, and arbitrary inhibitory equivalency factors (GJIC-IEFs) were suggested (Tab. 1). Arbitrary IEFs values 10.0 and 5.0 were chosen for strongest inhibitors, 1.0 and 0.5 were attributed to weak GJIC-inhibitors.

The similar arbitrary approach has been proposed formerly for the formulation of genotoxic equivalents of PAHs (Nisbet and LaGoy, 1992Go). This concept is based on the similar toxic-equivalency-factor (TEF) approach that is used in environmental and health risk assessment of various toxic pollutants. The approach is generally accepted for persistent dioxin-like chemicals (van den Berg et al., 1998Go). For PAHs, the genotoxicity equivalency factors relative to benzo[a]pyrene (Delistraty, 1997Go; Nisbet and LaGoy, 1992Go) and AhR-mediated activity equivalency factors relative to TCDD or benzo[a]pyrene (Clemons et al., 1998Go; Delistraty, 1997Go; Jones and Anderson, 1999Go; Machala et al., 2001Go; Willett et al., 1997Go) have been previously suggested. Specific TEFs (based on multiple in vivo and in vitro studies) or REPs/IEFs (derived from a single assay) are multiplied by concentrations measured in complex samples. Calculated values representing "benzo[a]pyrene equivalents" can be used for comparative studies of promotional and carcinogenic effects, as well as ecological risk assessment of PAHs and their mixtures. Such comparison of environmental significance of individual compounds or complex mixtures is generally accepted (WHO, 1998Go).

In a previous study, high concentrations of some strong GJIC inhibitors, such as fluoranthene, pyrene and picene (up to 779, 1.033, and 382 ng/g dry weight, respectively), were found in river sediments contaminated by PAHs (Machala et al., 2001Go). Due to their high levels in the environment and high relative potencies to inhibit GJIC, these PAHs may contribute most significantly to the promotional potency of complex environmental mixtures.

In conclusion, our results indicate that many environmentally important PAHs are potent in vitro inhibitors of GJIC in rat liver epithelial cell line WB-F344. Inhibition of GJIC seems to be an important mode of action of a series of PAHs, especially for those with lower molecular mass. On the other hand, when considering complete carcinogenic effects of other PAHs, showing only weak or no GJIC inhibitory properties, such as dibenzopyrenes and dibenzofluoranthenes, then other mechanisms could be involved in the tumor promoting effects. Suggested arbitrary equivalency factors (GJIC-IEFs) should serve for evaluation of promotional activity of complex environmental samples contaminated with PAHs. Although the GJIC inhibition assay remains the most suitable in vitro system to detect potential tumor promoters, it would be beneficial to combine the GJIC results with assays detecting other important, nongenotoxic modes of action, such as modulation of intracellular signals leading to an increased cell proliferation and survival.


    ACKNOWLEDGMENTS
 
The authors would like to acknowledge the Grant Agency of the Czech Republic (Grant No. 525/00/D101) and Czech National Agency for Agricultural Research (Grant No. Q0194) for the funding of this research. Authors wish to thank Dr. J. Turánek (VRI Brno, Czech Republic) for help with introduction of the method for GJIC detection.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +420-5-41321229. E-mail: machala{at}vri.cz. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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