Aryl Hydrocarbon Receptor-Activating Polychlorinated Biphenyls and Their Hydroxylated Metabolites Induce Cell Proliferation in Contact-Inhibited Rat Liver Epithelial Cells

Jan Vondrácek*,{dagger},1, Miroslav Machala{dagger}, Vítezslav Bryja{ddagger},§, Katerina Chramostová*, Pavel Krcr{dagger}, Cornelia Dietrich, Ales Hampl{ddagger},§,|| and Alois Kozubík*

* Laboratory of Cytokinetics, Institute of Biophysics, 612 65 Brno, Czech Republic; {dagger} Department of Chemistry and Toxicology, Veterinary Research Institute, 621 32 Brno, Czech Republic; {ddagger} Center of Cell Therapy and Tissue Repair, Charles University, 150 06 Prague, Czech Republic; § Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic; Institute of Toxicology, Johannes Gutenberg-University, 55131 Mainz, Germany; and || Laboratory of Molecular Embryology, Mendel University Brno, 613 00 Brno, Czech Republic

1 To whom correspondence should be addressed at Laboratory of Cytokinetics, Institute of Biophysics, Královopolská 135, 612 65 Brno, Czech Republic. E-mail: vondracek{at}ibp.cz

Received August 13, 2004; accepted October 7, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) exhibit tumor-promoting effects in experimental animals. We investigated effects of six model PCB congeners and hydroxylated PCB metabolites on proliferation of contact-inhibited rat liver epithelial WB-F344 cells. The ‘dioxin-like’ PCB congeners, PCB 126, PCB 105, and 4'-OH-PCB 79, a metabolite of the planar PCB 77 congener, induced cell proliferation in a concentration-dependent manner. In contrast, the ‘non-dioxin-like’ compounds that are not aryl hydrocarbon receptor (AhR) agonists, PCB 47, PCB 153, and 4-OH-PCB 187, an abundant noncoplanar PCB metabolite, had no effect on cell proliferation at concentrations up to 10 µM. The concentrations of dioxin-like PCBs leading to cell proliferation corresponded with the levels inducing the expression of cytochrome P450 1A1 mRNA, suggesting that the release from contact inhibition was associated with AhR activation. The effects of PCB 126 and PCB 153 on expression of proteins controlling G0/G1-S-phase transition and S-phase progression were compared. Only PCB 126 was found to upregulate cyclin A and D2 protein levels, and to increase both total cyclin-dependent kinase 2 (cdk2) and cyclin A/cdk2 complex activities. Despite the observed upregulation of cyclin D2, no increase in cdk4 activity was observed. The expression of cdk inhibitor p27Kip1 was not affected by either PCB 126 or PCB 153. These results suggest that dioxin-like PCBs can induce cell proliferation of contact-inhibited rat liver epithelial cells by increasing cyclin A protein levels, a process that then leads to upregulation of cyclin A/cdk2 activity and initiation of DNA replication. This mechanism could be involved in tumor-promoting effects of dioxin-like PCBs.

Key Words: cell proliferation; tumor promotion; contact inhibition; PCBs; liver epithelial cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are a group of structurally diverse and persistent environmental pollutants that became widely distributed throughout various environmental compartments as complex mixtures. Although their production has been banned, environmental PCB levels can still be very high, especially at the sites of their production and industrial use (Cogliano, 1998Go). The biological effects of individual PCB congeners strongly depend on the number and position of chlorine atoms. While the non-ortho-substituted coplanar PCBs are known to elicit a set of adverse ‘dioxin-like’ effects associated with the activation of the aryl hydrocarbon receptor (AhR) (van den Berg et al., 1998Go), di-ortho-substituted PCBs exhibit a different spectrum of toxic modes of action (Hansen, 1998Go; Robertson and Hansen, 2001Go). Both groups of compounds are carcinogenic to laboratory animals, and PCB mixtures have been classified as possible human carcinogens (IARC, 1987Go). Several studies have implicated low-molecular-weight PCBs as potential tumor-initiating compounds that may either induce oxidative DNA damage or formation of DNA adducts (Espandiari et al., 2003Go; Oakley et al., 1996Go). However, it has been extensively documented in various two-stage liver carcinogenesis models that PCB mixtures, as well as individual PCB congeners, are effective at promoting both gross tumors and putative preneoplastic lesions in rodent liver (reviewed in Glauert et al., [2001]Go).

The tumor-promoting activity of PCBs has been suggested to be associated with their capacity to either directly or indirectly activate signal transduction pathways leading to increased cell proliferation, inhibition of negative growth control and programmed cell death, or inhibition of gap junctional intercellular communication (GJIC) (Glauert et al., 2001Go). The effects of dioxin-like PCBs are considered to be related predominantly to their capacity to activate genes regulated by AhR (Safe, 1994Go); however, the exact mechanisms of their action still remain elusive. Several studies have demonstrated the capability of AhR ligands to stimulate cell proliferation and to inhibit apoptosis in liver; however, opposite effects have also been reported (Puga et al., 2002Go; Schwarz et al., 2000Go; Tharappel et al., 2002Go; Wölfle et al., 1993Go). Activation of AhR might also play a role in inhibition of GJIC that is observed in hepatoma cells or in hepatocytes (De Haan et al., 1994Go; Hemming et al., 1991Go). However, neither dioxins, known as efficient liver tumor promoters, nor dioxin-like PCBs are able to suppress GJIC in rat liver epithelial "stem-like" WB-F344 cells (Hemming et al., 1991Go; Machala et al., 2003Go). This cell line, isolated from the liver of an adult male Fischer 344 rat (Tsao et al., 1984Go), is an in vitro model of oval cells, small oval-shaped epithelial cells that are considered to be liver progenitor cells. The oval cells can give rise to both hepatocytes and biliary epithelial cells, and they might play a significant role in hepatocarcinogenesis (Alison, 2003Go; Dumble et al., 2002Go; Roskams et al., 2003Go). In humans, both dedifferentiation of mature hepatocytes and maturation arrest of progenitor cells have been suggested to lead to hepatocellular carcinomas (Roskams et al., 2003Go). Therefore, the liver progenitor cells might represent a potential target for tumor-promoting chemicals.

The rate of proliferation of most non-transformed adherent cells decreases with increased cell density as they become arrested in G1 phase of the cell cycle, which is a phenomenon known as contact inhibition (Dietrich et al., 1997Go; Levenberg et al., 1999Go). The loss of contact inhibition can lead to deregulated growth and is often associated with malignant transformation (Tsukita et al., 1993Go). A release from contact inhibition is a mechanism suggested to be an important part of effects of tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (Oesch et al., 1988Go). Interestingly, both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and AhR-activating polycyclic aromatic hydrocarbons have been found to interfere with mechanisms of contact inhibition in rat liver epithelial cells (Chramostová et al., 2004Go; Dietrich et al., 2002Go; Köhle et al., 1999Go). Contact inhibition usually leads to decreased expression of cyclins involved in regulation of G1-S phase transition and early S phase, which in turn decreases the activities of the cyclin-dependent kinases (cdks) in rat liver epithelial cells (Dietrich et al., 2002Go). Tetrachlorodibenzo-p-dioxin, a powerful liver tumor promoter, has been reported to upregulate cyclin A expression in confluent WB-F344 cells, and that upregulation has been associated with increased cdk2 activity. Therefore, the cyclin A/cdk2 complex activity could play a pivotal role in the TCDD-induced release of confluent cells from contact inhibition (Dietrich et al., 2002Go). The increased activity of cytochrome P450 1A1 (CYP1A1), one of the AhR-regulated genes, has been reported to correlate with induction of cell proliferation by dioxins in WB-F344 cells (Köhle et al., 1999Go).

Taken together, AhR might be involved in the TCDD-induced release from contact inhibition. Because dioxin-like PCBs are potent AhR ligands, with PCB 126 being considered only 10 times less effective than TCDD itself (van den Berg et al., 1998Go), the AhR-activating PCB congeners might disrupt contact inhibition in rat liver stem-like cells, and thus contribute to tumor promotion. The present study aimed to verify the hypothesis that dioxin-like PCBs and their hydroxylated PCB derivatives could stimulate a release of rat liver stem-like cells from contact inhibition by a mechanism that would involve modulation of expression and/or activity of proteins involved in the control of G1-S phase transition and early S phase progression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. 2,2',4,4'-Tetrachlorobiphenyl (PCB 47), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) were purchased from Promochem (Wesel, Germany). The hydroxylated PCBs 4'-OH-3,3',4,5'-tetrachlorobiphenyl (4'-OH-PCB 79) and 4-OH-2,2',3,4',5,5',6-heptachlorobiphenyl (4-OH-PCB 187) were kindly provided by Drs. L. W. Robertson and H.-J. Lehmler (University of Iowa, Iowa City, IA) and Dr. Å. Bergman (Stockholm University, Stockholm, Sweden), respectively. The structures of test compounds are shown in Figure 1. Tetrachlorodibenzo-p-dioxin was purchased from Cambridge Isotope Laboratories (Andover, MA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored in the dark. Mouse monoclonal antibody to mouse cyclin D1 (sc-450), rabbit polyclonal antibodies to cyclin A (sc-751), cyclin E (sc-481) and cdk2 (sc-163), and goat polyclonal antibody to cdk4 (sc-601-G) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to mouse p27 (K25020) was purchased from Transduction Laboratories (Lexington, KY). Mouse monoclonal antibodies against cyclin A (Ab-1, E23) and cyclin D2 (Ab-4, DCS-3.1, and DCS-5.2) were obtained from Neomarkers (Fremont, CA). Mouse monoclonal antibody against a C-terminal part of human cyclin D3, which cross-reacts with the mouse homologue (DCS-22), was generously provided by Dr. J. Lukas (Danish Cancer Society, Copenhagen, Denmark). Mouse monoclonal antibody against ß-actin and horseradish peroxidase–conjugated anti-immunoglobulins were from Sigma-Aldrich (Prague, Czech Republic). Polyvinylidene difluoride (PVDF) membrane Hybond-P and chemiluminescence detection reagents (ECLPlus) were supplied by Amersham (Amersham, Aylesbury, UK). All other chemicals were provided by Sigma-Aldrich.



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FIG. 1. Chemical structures of and hydroxylated polychlorinated biphenyls (PCB) derivatives under study.

 
Assessment of cell proliferation and cell cycle distribution. WB-F344 rat liver epithelial cells, originally isolated in the laboratory of Joe W. Grisham, National Cancer Institute, Bethesda, MD (Tsao et al., 1984Go), were kindly provided by James E. Trosko and Brad L. Upham (Michigan State University, East Lansing, MI). Cells were grown in modified Eagle's Minimum Essential Medium (Sigma-Aldrich, Prague, Czech Republic) with 50% increased concentrations of nonessential amino acids, and supplemented with 1 mM sodium pyruvate, 10 mM HEPES, and 5% heat-inactivated fetal bovine serum (Sigma-Aldrich). The cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. Cells were routinely maintained in 75 cm2 flasks and subcultured twice a week. Only the cells at passage levels 15–22 were used throughout the study. The proliferative effects of PCBs on confluent WB-F344 cells were determined as described previously (Chramostová et al., 2004Go). Briefly, cells were seeded at an initial concentration of 30,000 cells per cm2 in 4-well cell-culture plates (Nunc, Roskilde, Denmark) and grown until they reached an approximate confluency. The cells were exposed to tested compounds dissolved in DMSO for 72 h. The final concentration of DMSO did not exceed 0.1% (v/v) in any of the samples. The medium with tested compounds was changed daily to ensure that the cells would receive an adequate amount of nutrients to enable proliferation at high cell densities. Following the exposure, the medium was removed, cells were harvested with trypsin and counted with a Coulter Counter (Model ZM, Coulter Electronics, Luton, UK). Cells were then washed with phosphate-buffered saline (PBS), and fixed in 70% ethanol at 4°C overnight. Fixed cells were washed once with PBS and resuspended in 0.5 ml of Vindelov solution (1 M Tris-HCl – pH 8.0; 0.1 % Triton X-100, v/v; 10 mM NaCl; propidium iodide 50 µg/ml; RNAse A 50 Kunitz units/ml) (Vindelov, 1977Go) and incubated at 37°C for 30 min. Cells were analyzed on FACSCalibur, using 488-nm (15 mW) air-cooled argon-ion laser for propidium iodide excitation, and CELLQuestTM software for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME).

Western blot analyses and detection of cdk2 activity. Confluent WB-F344 cells grown on 60-mm-diameter cell culture dishes were exposed for 48 h to tested compounds or 0.1% DMSO (vehicle). Proteins were extracted for 30 min in ice-cold lysis buffer (50 mM Tris/HCl [pH 7.4], 150 mM sodium chloride, 0.5% Nonidet P-40, 1 mM EDTA, 0.1 mM dithiothreitol, 50 mM sodium fluoride, 8 mM ß-glycerolphosphate, 100 mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml tosylphenylalanine chloromethane). The extracts were cleared by centrifugation at 15,000 g for 15 min at 4°C and stored at –80°C until use. Concentrations of total protein were determined using a DC Protein Assay Kit (Bio-Rad Laboratories, Prague, Czech Republic). For kinase assays, the extracts (150 µg of total protein per reaction) were first subjected to initial absorption with protein G agarose beads and then incubated with appropriate antibodies (sc-163 for cdk2, sc-751 for cyclin A, and sc-601-G for cdk4) for 1 h in an ice bath. Immunoprecipitates were collected on protein G agarose beads by overnight rotation, washed three times with lysis buffer, and twice with kinase assay buffer (50 mM HEPES, pH 7.5; 10 mM MgCl2; 10 mM MnCl2; 8 mM ß-glycerolphosphate; 1 mM dithiothreitol). The cdk2 kinase reactions were carried out for 30 min at 37°C in a total volume of 25 µl in kinase assay buffer supplemented with 100 µg/ml histone H1 (type III-S) and 40 µCi/ml [32P] ATP. Cdk4 kinase assay was performed with 80 µg/ml GST-pRb (a gift from Dr. J. Lukas) and 40 µCi/ml 32P-ATP. Reactions were terminated by mixing with 2x Laemmli sample buffer, and each total reaction mix was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Densitometry was performed using AIDA Image Analyzer software (raytest Isotopenmeßgeräte, Starubenhardt, Germany).

For Western blotting, equal amounts of total protein were subjected to 10% SDS PAGE, electrotransferred onto a PVDF membrane, immunodetected using appropriate primary and secondary antibodies, and visualized by ECLPlus reagent (Amersham, Little Chalfont, UK) according to the manufacturer's instructions. When required, membranes were stripped in 62.5 mM Tris/HCl pH 6.8, 2% SDS, and 100 mM ß-mercaptoethanol, washed, and reblotted with another antibody. After immunodetection, each membrane was stained by amidoblack to confirm equal protein loading.

Real-time RT-PCR for quantification of CYP1A mRNA. Total RNA was isolated from cells using the RNeasy mini kit (Qiagen, Valencia, CA) including treatment with DNase I (Qiagen). The amplifications of the samples were carried out in a final volume of 20 µl in a reaction mixture containing 10 µL of QuantiTect Probe RT-PCR Master Mix, 0.2 µl of QuantiTect RT Mix (Qiagen), 2 µl of solution of primers and probe, 5.8 µl of water, and 2 µl of sample. The final concentrations of primers and probe were 1.0 µM and 0.2 µM, respectively. The probes were labeled with a 5' FAM reporter and a 3' BHQ 1 quencher. The amplifications were run on the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the following program: reverse transcription at 50°C for 20 min and initial activation step at 95°C for 15 min, followed by 45 cycles at 95°C for 0 s and 60°C for 60 s. The primers and probe for rat CYP1A1 (Lake et al., 2003Go) were: forward 5'-TGAGTTTGGGGAGGTTACTGGTT-3', reverse 5'-TGAAGGCATC CAGGGAAGAGT-3', probe 5'-ATACCCAGCTGACTTCATTCCTATCCTCCGTT-3'. The primers and probe for the reference gene porphobilinogen deaminase (EC 4.3.1.8) were: forward 5'-CCCAACCTGGAATTCAAGAGTATTCG-3, reverse 5'-TTCCTCTGGG TGCAAAATCT-GGCC-3', probe 5'-CCTCAACACCCGCCTTCGGAAGCT-3'.

Statistical analysis. Data were expressed as means ± S.D. and analyzed by Student's t-test, or by analysis of variance (ANOVA), followed by Dunnett's test. A p value of less than 0.05 was considered to be significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Proliferative Activity of PCBs and OH-PCBs Corresponds with Their Ability to Activate AhR
To investigate effects of PCBs on contact inhibition in confluent WB-F344 cells, the compounds that are representative of di-ortho-substituted (PCB47 and 153), mono-ortho-substituted (PCB 105) and non-ortho-substituted (PCB 126) congeners were selected. The study also included determination of effects of 4-OH-PCB 187, a highly abundant and persistent hydroxylated noncoplanar PCB derivative found in mammalian blood (Fangström et al., 2002Go), and 4'-OH-PCB 79, a planar metabolite of the non-ortho-substituted PCB 77 congener.

As reported previously, WB-F344 cells seeded at 30,000 cells per cm2 show a significant drop in proliferation after 72 h of incubation, which corresponds with a marked decrease in percentage of S phase cells (Chramostová et al., 2004Go). However, when cultivated in the presence of PCB 126, PCB 105, and 4'-OH-PCB 79 for an additional 72 h, a significant concentration-dependent increase in percentage of cells in S phase was observed in confluent WB-F344 cells, with PCB 126 being effective at a concentration as low as 100 pM (Fig. 2). These results corresponded with significantly increased cell numbers that were found for all three compounds (Fig. 3). PCB 126 did not exert a general proliferative stimulus because it had no effect on proliferation of WB-F344 cells cultivated at subconfluent densities (data not shown), and this suggested that its effects on confluent cells are due to the release from contact inhibition. Contrary to the above results, none of the other three compounds, carrying two or more chlorines at ortho positions, had any effect either on cell numbers or on cell cycle (Figs. 2 and 3).



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FIG. 2. Modulation of the cell cycle by selected PCBs and their hydroxylated derivatives in WB-F344 cells. Cells were treated with tested compounds and collected as described in the Materials and Methods. Control cells were treated with solvent (0.1% DMSO) and 5 nM TCDD was used as a positive control. The percentage of cells in the S phase is expressed as the mean ± S.D. of three independent experiments run in duplicate. ANOVA followed by Dunnett's test were used to assess statistical significance of results. *A significant difference between control and treated samples (p < 0.05). **A significant difference between control and treated samples (p < 0.01).

 


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FIG. 3. Modulation of cell proliferation by selected PCBs and their hydroxylated derivatives in WB-F344 cells. Cells were treated with tested compounds and collected as described in the Materials and Methods. Control cells were treated with solvent (0.1% DMSO), and 5 nM TCDD was used as a positive control. Cell numbers (open bars, left axis) are expressed as means ± S.D. of at least three independent experiments run in duplicate. ANOVA followed by Dunnett's test were used to assess statistical significance of results. *A significant difference between control and treated samples (P < 0.05). **A significant difference between control and treated samples (p < 0.01).

 
To determine whether the proliferative activity was associated with the capacity of PCBs to act as AhR agonists, we compared their effects on cell proliferation and cell cycle with induction of CYP1A1 mRNA expression. There is only limited information on inducibility of AhR-regulated transcripts, such as CYP1A1 in rat oval cells. It has been suggested that WB-F344 cells may not contain inducible CYP1A1 activity (Herrmann et al., 2002Go). However, both we and others have found that AhR ligands, such as polycyclic aromatic hydrocarbons or polychlorinated dibenzo-p-dioxins, are capable of inducing AhR-dependent 7-ethoxyresorufin O-deethylase activity in WB-F344 cells (Chramostová et al., 2004Go; Köhle et al., 1999Go). As outlined in Figure 4, the concentration-dependent induction of CYP1A1 by PCB 126 in WB-F344 cells corresponded with induction of cell proliferation (Figs. 2 and 3). PCB126, PCB 105, and 4'-OH-PCB 79 induced both a significant increase in percentage of cells in S phase and an increased CYP1A1 mRNA expression when applied at a concentration of 1 µM. In contrast, the non-dioxin-like PCB 47, PCB 153, or 4-OH-PCB 187 had no effect on CYP1A1 mRNA levels (Fig. 4).



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FIG. 4. Induction of cytochrome p450 (CYP) 1A1 mRNA by PCBs in WB-F344 cells. (A) PCB 126 induced CYP1A1 mRNA expression in WB-F344 cells in a dose-dependent manner. Cells were treated with the indicated concentrations of PCB 126 or DMSO as a solvent control for 24 h. Total RNA was isolated and quantitative real-time RT-PCR was performed as described in the Materials and Methods. One representative experiment is shown out of three independent experiments. (B) Induction of CYP1A1 mRNA expression following the 24-h treatment of WB-F344 cells with PCBs (1 µM). (C) Expression of the reference gene porphobilinogen deaminase in samples analyzed in parts A and B.

 
Induction of Cell Proliferation by PCB 126 Is Associated with Increased Cyclin A Expression and Increased Cyclin A/Cdk2 Activity
It has been reported that contact inhibition of WB-F344 cells leads to downregulation of cyclin D1 and D2, decreased cyclin D2/cdk 4 activity, downregulation of cyclin A, decreased cyclin A/cdk2 activity, and accumulation of p27Kip1 (Dietrich et al., 2002Go). The TCDD-induced release from contact inhibition has been shown to be associated with increased expression of D-type cyclins and cyclin A, and with a significant increase of cyclin A/cdk2 activity (Dietrich et al., 2002Go). Therefore, we compared the effects of PCB 126, which was found to be an efficient inducer of cell proliferation, with those of PCB 153, which had no effect on proliferation of confluent WB-F344 cells or on expression of proteins associated with control of G1-S phase transition and S phase progression. The confluent cells were exposed to the test compounds for 48 h. This time interval has been reported to be optimal for detection of expression and activities of cell cycle regulatory proteins in this cell model (Dietrich et al., 2002Go). As shown in Figure 5, PCB 126 was found to upregulate cyclin A expression, which was similar to the effect of TCDD. Both TCDD and PCB 126 also induced a significant increase in cyclin D2 protein levels. In contrast, the levels of p27Kip1, a major cdk2 inhibitor known to be involved in contact inhibition of cell growth and S phase entry, remained unaffected by either TCDD or PCB 126. PCB 126 had no effect or only marginal on the expression of cyclins D1, D3, and E, or on protein levels of cdk2 and 4.



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FIG. 5. Comparison of the effects of PCB 126 and PCB 153 on expression of proteins involved in control of G1-S phase transition. Confluent WB-F344 cells were treated with DMSO (solvent control), TCDD (1 nM), PCB 126 (100 nM and 1 µM) and PCB 153 (1 µM and 10 µM) for 48 h. Cell lysates were prepared as described in the Materials and Methods and subjected to Western blot analysis. The results are representative of three independent experiments. ANOVA followed by Dunnett's test were used to assess statistical significance of results. The results of densitometry shown in parallel represent means ± S.D. of three independent experiments. *A significant difference between control and treated samples (p < 0.05).

 
Because the levels of cyclin A and D were significantly increased by PCB 126 treatment, we next examined the activities of cdks, which depend on the association with the above cyclins. As outlined in Figure 6A, PCB 126 induced approximately a twofold increase in total cdk2 activity. The level of cdk2 activity in untreated cells was very low, almost at background levels (data not shown), suggesting that cdk2 activity is strongly inhibited in confluent WB-F344 cells. The levels of cyclin A/cdk2 complex activity were significantly increased by both TCDD and PCB 126 treatment (Fig. 6B). In contrast, PCB 153 did not significantly affect cyclin A/cdk2 activity. Cdk4 activity in confluent cells was very low, and it was not altered by treatment with either PCB 126 or PCB 153 (Fig. 6A).



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FIG. 6. Effects of PCB 126 and 153 on cdk2 and cdk4 activities. (A) PCB 126 increased cdk2 activity, while it has no effect on cdk4 activity. (B) PCB126 increased cyclin A/cdk2 activity in confluent WB-F344 cells. Cells were treated with 5 nM TCDD (positive control), PCB 126, and PCB 153, or with DMSO (solvent control). Immunoprecipitation was performed using anti-cyclin A, anti-cdk4, and anti-cdk2 antibodies. Kinase activity of the immunoprecipitates was measured in the presence of [{gamma}-32P]ATP using histone H1 (cdk2) or pRb (cdk4) as a substrate. The figure shows one representative experiment of three independent experiments. The results of densitometry shown in parallel represent means ± S.D. of three independent experiments. Student's t-test or ANOVA followed by Dunnett's test were used to assess statistical significance of results. *A significant difference between control and treated samples (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls are known to exert tumor-promoting activity in liver, and it is conceivable that they might employ multiple tumor promoting mechanisms, because both dioxin-like and non-dioxin-like PCBs can act as hepatic tumor promoters (Glauert et al., 2001Go; Safe, 1994Go). The tumor-promoting activity of PCBs could be associated with their capacity to alter signal transduction pathways controlling cell proliferation and programmed cell death (reviewed in Glauert et al. [2001]Go). The effects of dioxin-like PCBs are considered to be predominantly related to activation of AhR. Several studies have demonstrated the capability of AhR ligands to modulate cell proliferation and/or to inhibit apoptosis in the liver; however, opposite effects have also been reported from various cellular models (Puga et al., 2002Go; Schwarz et al., 2000Go; Tharappel et al., 2002Go; Wölfle et al., 1993Go). Activation of AhR might also play a role in inhibition of GJIC (De Haan et al., 1994Go; Herrmann et al., 2002Go). The non-dioxin-like PCBs have been reported to inhibit apoptosis in rat hepatocytes or block GJIC in rat liver epithelial cells (Bohnenberger et al., 2001Go; Hemming et al., 1991Go; Machala et al., 2003Go). However, little is known about the role of PCBs in regulation of proliferation of hepatic progenitor cells, which may represent a potential target for tumor promoters. It has been shown that both TCDD and the AhR-activating polycyclic aromatic hydrocarbons induce a release from contact inhibition in rat liver epithelial stem-like WB-F344 cells (Dietrich et al., 2002Go; Chramostová et al., 2004Go). Therefore, we hypothesized that PCBs or their derivatives that act as AhR ligands might alter the normal control of cell proliferation via this mechanism.

As shown in Figures 2 and 3, PCB 126, which is considered to be the most potent AhR agonist among coplanar PCBs (Glauert et al., 2001Go; van den Berg et al. 1998Go), was found to increase both cell numbers and percentage of S phase cells within the concentration range of 100 pM to 10 µM. These concentrations corresponded well with the levels of PCB 126 that were found to induce CYP1A1 mRNA expression in WB-F344 cells (Fig. 4). Similarly, the mono-ortho-substituted congener PCB 105 was found to increase CYP1A1 mRNA levels, as well as to increase the percentage of S phase cells and/or cell numbers in this cell model. Both PCB 126 and PCB 105 have been reported to induce promotion of altered hepatic foci (Dean et al., 2002Go; Haag-Grönlund et al., 1998Go; Hemming et al., 1995Go). In contrast, PCB 47 and PCB 153 had no effect on cell proliferation at concentrations up to 10 µM, nor did either compound induce CYP1A1 mRNA expression. To our knowledge, this is the first evidence that AhR-activating PCB congeners can increase cell proliferation of contact-inhibited liver epithelial cells. Such a mode of action could participate in the tumor-promoting effects of these PCBs. The concentrations of dioxin-like PCBs that induced cell proliferation in the present study correspond with the levels of PCBs used for various in vivo tumor-promotion studies in rats (Glauert et al., 2001Go; van den Berg et al., 1998Go). Polychlorinated biphenyls are known to be present at nanomolar levels in human blood (Kimbrough 1995Go); however, their concentrations can be significantly higher in other tissues. Significantly higher concentrations have also been found in individuals living in PCB-contaminated regions (Pavúk et al., 2004Go).

The effects of PCBs within an organism can be further modified by formation of active hydroxylated metabolites. Hydroxylated PCBs (OH-PCBs) have been reported to disrupt estrogen and thyroid hormone signaling, or vitamin A transport (Connor et al., 1997Go; Kramer et al., 1997Go; Moore et al., 1997Go). Several highly chlorinated OH-PCBs have been reported to persist and accumulate in animal tissues at high concentrations (Fangström et al., 2002Go). However, besides the fact that OH-PCBs can inhibit GJIC in vitro, little is known about their role in tumor promotion (Machala et al., 2004Go; Satoh et al., 2003Go). In the present study we found that the hydroxylated derivative of non-ortho-substituted PCB 77 and 4'-OH-PCB 79 can induce both the AhR-mediated induction of CYP1A1 mRNA expression and a release from contact inhibition in WB-F344 cells. In contrast, the noncoplanar hydroxylated PCB metabolite 4-OH-PCB 187 did not affect either CYP1A1 mRNA expression or cell proliferation.

Disruption of normal control of the cell cycle is one of the important steps in carcinogenesis (Malumbres and Barbacid, 2001Go). Progression through the cell cycle is controlled by a sequential activation of cyclin/cdk complexes (Sherr, 2000Go). The D-type cyclins associate with cdk4 or cdk6, and they play an important role during both early and late G1 phase of the cell cycle. Cdk2 associates with cyclins E and A, which act at the G1-S phase transition and during S phase entry and progression, respectively (Ekholm and Reed, 2000Go; Sherr, 2000Go). Inhibition of cdk2 activity is known to be involved in maintenance of contact inhibition, and upregulation of its activity has been linked to the onset of proliferation (Chen et al., 2000Go). TCDD has been shown both to upregulate cyclin A levels and to increase cdk2 activity in confluent rat liver epithelial cells (Dietrich et al., 2002Go). This suggested that this mechanism might be activated by dioxin-like PCBs as well. We found that PCB 126, a model ‘dioxin-like’ PCB congener, can increase the expression of cyclin A and the activity of the cyclin A/cdk2 complex. In contrast, PCB 153 had no effect either on cyclin A levels or on cdk2 activity. Neither compound affected cdk4 activity, although PCB 126 increased cyclin D2 expression (Figs. 5 and 6). Although cdk4 activity was not increased, an increase in cyclin A expression and cyclin A/cdk2 activity alone could be sufficient to induce G1-S phase transition. It has been shown that the downregulation of cdk2 activity can be causative for the cell cycle inhibition, while the ectopic expression of cyclin A can restore cell cycle progression into S phase (Strobeck et al., 2000Go). The exact mechanism of PCB action in WB-F344 cells, leading to an increase in cdk2 activity, remains unclear. The dioxin-like compounds could activate several early-response genes involved in the regulation of cell proliferation, e.g., c-fos or c-jun, both of which have been shown to be upregulated by AhR ligands, or they could alter cell-signaling pathways stimulating cell proliferation, such as mitogen-activated protein kinases (Puga et al., 2002Go; Schwarz et al., 2000Go; Tharappel et al., 2002Go; Wölfle et al., 1993Go). It has been speculated that an increase in c-Src activity is responsible for the proliferative effects of TCDD on contact-inhibited WB-F344 cells (Köhle et al., 1999Go). AhR ligands have also been suggested to increase the activity of extracellular signal-regulated kinases (Tan et al., 2002Go). However, recent data suggest that neither c-Src nor ERK1/2 is involved in the effects of TCDD on cell proliferation in this in vitro model (Hölper et al., in pressGo).

Cyclin-dependent kinase 2 activity is controlled not only by its association with cyclin E and A, but also by cdk inhibitors, namely p27Kip1, which is an important regulator of contact inhibition in various cell types (Polyak et al., 1994Go). This inhibitor has been shown to be upregulated in confluent WB-F344; yet, it is also known to be upregulated by TCDD in rat hepatoma cells in an AhR-dependent manner (Dietrich et al., 2002Go; Kolluri et al., 1999Go). We found in the present study that release from contact inhibition induced either by TCDD or by PCB 126 is not associated with a decrease in p27Kip1 expression, suggesting that it is not involved in their effects on proliferation of confluent cells. Nevertheless, it cannot be excluded that the high levels of p27Kip1protein in cells released from contact inhibition by TCDD or by PCB 126 are also maintained through AhR-induced expression of this cdk inhibitor.

Taken together, our data show that dioxin-like PCBs can release rat liver epithelial cells from contact inhibition by increasing cyclin A protein levels, which leads to upregulation of cyclin A/cdk2 activity. This effect correlates with induction of CYP1A1 expression, suggesting that AhR activation is involved. Disruption of cell cycle control in liver progenitor cells might play a role in the tumor-promoting effects of individual PCBs or their mixtures. Future studies should aim to describe in more detail the mechanisms responsible for this effect, as well as its in vivo significance.


    ACKNOWLEDGMENTS
 
The authors thank Drs. L. Robertson and H.-J. Lehmler (University of Iowa) for providing 4'-OH-PCB 79, and Dr. Å. Bergman for providing 4-OH-PCB 187. This study was supported by grant 525/03/1527 from the Czech Science Foundation and by the Research Plan of the Academy of Sciences of the Czech Republic under grant. Z 5004920.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alison, M. R. (2003). Characterization of the differentiation capacity of rat-derived hepatic stem cells. Semin. Liver Dis. 23, 325–336.[CrossRef][ISI][Medline]

Bohnenberger, S., Wagner, B., Schmitz, H. J., and Schrenk, D. (2001). Inhibition of apoptosis in rat hepatocytes treated with "non-dioxin-like" polychlorinated biphenyls. Carcinogenesis 22, 1601–1606.[Abstract/Free Full Text]

Chen, D., Walsh, K., and Wang, J. (2000). Regulation of cdk2 activity in endothelial cells that are inhibited from growth by cell contact. Arterioscler. Thromb. Vasc. Biol. 20, 629–635.[Abstract/Free Full Text]

Chramostová, K., Vondrácek, J., Sindlerová, L., Vojtesek, B., Kozubík, A., and Machala, M. (2004). Polycyclic aromatic hydrocarbons modulate cell proliferation in rat hepatic epithelial stem-like WB-F344 cells. Toxicol. Appl. Pharmacol. 196, 136–148.[CrossRef][ISI][Medline]

Cogliano, V. J. (1998). Assessing the cancer risk from environmental PCBs. Environ. Health Perspect. 106, 317–323.[ISI][Medline]

Connor, K., Ramamoorthy, K., Moore, M., Mustain, M., Chen, I., Safe, S., Zacharewski, T., Gillesby, B., Joyeux, A., and Balaguer, P. (1997). Hydroxylated polychlorinated biphenyls (PCBs) as estrogens and antiestrogens: structure-activity relationships. Toxicol. Appl. Pharmacol. 145, 111–123.[CrossRef][ISI][Medline]

De Haan, L. H., Simons, J. W., Bos, A. T., Aarts, J. M., Denison, M. S., and Brouwer, A. (1994). Inhibition of intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like PCBs in mouse hepatoma cells (Hepa1c1c7): involvement of the Ah receptor. Toxicol. Appl. Pharmacol. 129, 283–293.[CrossRef][ISI][Medline]

Dean, C. E., Jr., Benjamin, S. A., Chubb, L. S., Tessari, J. D., and Keefe, T. J. (2002). Nonadditive hepatic tumor promoting effects by a mixture of two structurally different polychlorinated biphenyls in female rat livers. Toxicol. Sci. 66, 54–61.[Abstract/Free Full Text]

Dietrich, C., Faust, D., Budt, S., Moskwa, M., Kunz, A., Bock, K. W., and Oesch, F. (2002). 2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent release from contact inhibition in WB-F344 cells: involvement of cyclin A. Toxicol. Appl. Pharmacol. 183, 117–126.[CrossRef][ISI][Medline]

Dietrich, C., Wallenfang, K., Oesch, F., and Wieser, R. (1997). Differences in the mechanisms of growth control in contact-inhibited and serum-deprived human fibroblasts. Oncogene 15, 2743–2747.[CrossRef][ISI][Medline]

Dumble, M. L., Croager, E. J., Yeoh, G. C., and Quail, E. A. (2002). Generation and characterization of p53 null transformed hepatic progenitor cells: Oval cells give rise to hepatocellular carcinoma. Carcinogenesis 23, 435–445.[Abstract/Free Full Text]

Ekholm, S. V., and Reed, S. I. (2000). Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle. Curr. Opin. Cell Biol. 12, 676–684.[CrossRef][ISI][Medline]

Espandiari, P., Glauert, H. P., Lehmler, H. J., Lee, E. Y., Srinivasan, C., and Robertson, L. W. (2003). Polychlorinated biphenyls as initiators in liver carcinogenesis: Resistant hepatocyte model. Toxicol. Appl. Pharmacol. 186, 55–62.[CrossRef][ISI][Medline]

Fangström, B., Athanasiadou, M., Grandjean, P., Weihe, P., and Bergman, Å. (2002). Hydroxylated PCB metabolites and PCBs in serum from pregnant Faroese women. Environ. Health Perspect. 110, 895–899.[ISI][Medline]

Glauert, H. P., Robertson, L. W., and Silberhorn, E. M. (2001). PCBs and tumor promotion. In Recent Advances in the Environmental Toxicology and Health Effects of PCBs (L. W. Robertson and L. Hansen, Eds.), pp. 355–371. The University Press of Kentucky, Lexington, KY.

Haag-Grönlund, M., Johansson, N., Fransson-Steen, R., Hakansson, H., Scheu, G., and Wärngård, L. (1998). Interactive effects of three structurally different polychlorinated biphenyls in a rat liver tumor promotion bioassay. Toxicol. Appl. Pharmacol. 152, 153–165.[CrossRef][ISI][Medline]

Hansen, L. G. (1998). Stepping backward to improve assessment of PCB congener toxicities. Environ. Health Perspect. 106 (Suppl 1), 171–189.[ISI][Medline]

Hemming, H., Bager, Y., Flodström, S., Nordgren, I., Kronevi, T., Ahlborg, U. G., and Warngard, L. (1995). Liver tumour promoting activity of 3,4,5,3',4'-pentachlorobiphenyl and its interaction with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Eur. J. Pharmacol. 292, 241–249.[Medline]

Hemming, H., Wärngård, L., and Ahlborg, U. G. (1991). Inhibition of dye transfer in rat liver WB cell culture by polychlorinated biphenyls. Pharmacol. Toxicol. 69, 416–420.[ISI][Medline]

Herrmann, S., Seidelin, M., Bisgaard, H. C., and Vang, O. (2002). Indolo[3,2-b]carbazole inhibits gap junctional intercellular communication in rat primary hepatocytes and acts as a potential tumor promoter. Carcinogenesis 23, 1861–1868.[Abstract/Free Full Text]

Hölper, P., Faust, D., Oesch, F., and Dietrich, C. (2004). Evaluation of the role of c-Src and ERK in TCDD-dependent release from contact-inhibition in WB-F344 cells. Arch. Toxicol. in press.

IARC (International Agency for Research in Cancer). (1987). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Overall Evaluations of Carcinogenicity. Suppl.7. IARC Press, Lyon, France.

Kimbrough, R. D. (1995). Polychlorinated biphenyls (PCBs) and human health: An update. Crit. Rev. Toxicol. 25, 133–163.[ISI][Medline]

Köhle, C., Gschaidmeier, H., Lauth, D., Topell, S., Zitzer, H., and Bock, K. W. (1999). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)–mediated membrane translocation of c-Src protein kinase in liver WB-F344 cells. Arch. Toxicol. 73, 152–158.[CrossRef][ISI][Medline]

Kolluri, S. K., Weiss, C., Koff, A., and Gottlicher, M. (1999). p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells. Genes Dev. 13, 1742–1753.[Abstract/Free Full Text]

Kramer, V. J., Helferich, W. G., Bergman, A., Klasson-Wehler, E., and Giesy, J. P. (1997). Hydroxylated polychlorinated biphenyl metabolites are anti-estrogenic in a stably transfected human breast adenocarcinoma (MCF7) cell line. Toxicol. Appl. Pharmacol. 144, 363–376.[CrossRef][ISI][Medline]

Lake, B. G., Meredith, C., Scott, M. P., Renwick, A. B., and Price, R. J. (2003). Use of cultured precision-cut rat lung slices to study the in vitro induction of pulmonary cytochrome P450 forms. Xenobiotica 33, 691–702.[CrossRef][ISI][Medline]

Levenberg, S., Yarden, A., Kam, Z., and Geiger, B. (1999). p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry. Oncogene 18, 869–876.[CrossRef][ISI][Medline]

Machala, M., Bláha, L., Lehmler, H. J., Plísková, M., Májková, Z., Kapplová, P., Sovadinová, I., Vondrácek, J., Malmberg, T., and Robertson, L. W. (2004). 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. Chem. Res. Toxicol. 17, 340–347.[CrossRef][ISI][Medline]

Machala, M., Bláha, L., Vondrácek, J., Trosko, J. E., Scott, J., and Upham, B. L. (2003). Inhibition of gap junctional intercellular communication by noncoplanar polychlorinated biphenyls: inhibitory potencies and screening for potential mode(s) of action. Toxicol. Sci. 76, 102–111.[Abstract/Free Full Text]

Malumbres, M., and Barbacid, M. (2001). To cycle or not to cycle: A critical decision in cancer. Nat. Rev. Cancer 1, 222–231.[CrossRef][Medline]

Moore, M., Mustain, M., Daniel, K., Chen, I., Safe, S., Zacharewski, T., Gillesby, B., Joyeux, A., and Balaguer, P. (1997). Antiestrogenic activity of hydroxylated polychlorinated biphenyl congeners identified in human serum. Toxicol. Appl. Pharmacol. 142, 160–168.[CrossRef][ISI][Medline]

Oakley, G. G., Devanaboyina, U., Robertson, L. W., and Gupta, R. C. (1996). Oxidative DNA damage induced by activation of polychlorinated biphenyls (PCBs): Implications for PCB-induced oxidative stress in breast cancer. Chem. Res. Toxicol. 9, 1285–1292.[CrossRef][ISI][Medline]

Oesch, F., Schäfer, A., and Wieser, R. J. (1988). 12-O-tetradecanoylphorbol-13-acetate releases human diploid fibroblasts from contact-dependent inhibition of growth. Carcinogenesis 9, 1319–1322.[Abstract]

Pavúk, M., Cerhan, J. R., Lynch, C. F., Schecter, A., Petrík, J., Chovancová, J., and Kocan, A. (2004). Environmental exposure to PCBs and cancer incidence in eastern Slovakia. Chemosphere 54, 1509–1520.[CrossRef][ISI][Medline]

Polyak, K., Kato, J. Y., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M., and Koff, A. (1994). p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 8, 9–22.[Abstract]

Puga, A., Xia, Y., and Elferink, C. (2002). Role of the aryl hydrocarbon receptor in cell cycle regulation. Chem. Biol. Interact. 141, 117–1130.[CrossRef][ISI][Medline]

Robertson, L. W., and Hansen, L. (2001). Recent Advances in the Environmental Toxicology and Health Effects of PCBs. The University Press of Kentucky, Lexington, KY.

Roskams, T. A., Libbrecht, L., and Desmet, V. J. (2003). Progenitor cells in diseased human liver. Semin. Liver Dis. 23, 385–396.[CrossRef][ISI][Medline]

Safe, S. H. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol. 24, 87–149.[ISI][Medline]

Satoh, A. Y., Trosko, J. E., and Masten, S. J. (2003). Epigenetic toxicity of hydroxylated biphenyls and hydroxylated polychlorinated biphenyls on normal rat liver epithelial cells. Environ. Sci. Technol. 37, 2727–2733.[CrossRef][ISI][Medline]

Sherr, C. J. (2000). The Pezcoller Lecture: Cancer cell cycles revisited. Cancer Res. 60, 3689–3695.[Abstract/Free Full Text]

Schwarz, M., Buchmann, A., Stinchcombe, S., Kalkuhl, A., and Bock, K. (2000). Ah receptor ligands and tumor promotion: Survival of neoplastic cells. Toxicol Lett 112–113, 69–77.[ISI]

Strobeck, M. W., Fribourg, A. F., Puga, A., and Knudsen, E. S. (2000). Restoration of retinoblastoma mediated signaling to Cdk2 results in cell cycle arrest. Oncogene 19, 1857–1867.[CrossRef][ISI][Medline]

Tan, Z., Chang, X., Puga, A., and Xia, Y. (2002). Activation of mitogen-activated protein kinases (MAPKs) by aromatic hydrocarbons: role in the regulation of aryl hydrocarbon receptor (AHR) function. Biochem. Pharmacol. 64, 771–780.[CrossRef][ISI][Medline]

Tharappel, J. C., Lee, E. Y., Robertson, L. W., Spear, B. T., and Glauert, H. P. (2002). Regulation of cell proliferation, apoptosis, and transcription factor activities during the promotion of liver carcinogenesis by polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 179, 172–184.[CrossRef][ISI][Medline]

Tsao, M. S., Smith, J. D., Nelson, K. G., and Grisham, J. W. (1984). A diploid epithelial cell line from normal adult rat liver with phenotypic properties of ‘oval’ cells. Exp. Cell Res. 154, 38–52.[ISI][Medline]

Tsukita, S., Itoh, M., Nagafuchi, A., and Yonemura, S. (1993). Submembranous junctional plaque proteins include potential tumor suppressor molecules. J. Cell Biol. 123, 1049–1053.[ISI][Medline]

van den Berg, M., Birnbaum, L., Bosveld, A. T., Brunstrom, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., et al. (1998). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 106, 775–792.[ISI][Medline]

Vindelov, L. L. (1977). Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei. Virchows Arch. B. Cell. Pathol. 24, 227–242.[ISI]

Wölfle, D., Becker, E., and Schmutte, C. (1993). Growth stimulation of primary rat hepatocytes by 2,3,7,8- tetrachlorodibenzo-p-dioxin. Cell Biol. Toxicol. 9, 15–31.[ISI][Medline]