Effect of 2,2',4,4',5,5'-Hexachlorobiphenyl (PCB-153) on Hepatocyte Proliferation and Apoptosis in Mice Deficient in the p50 Subunit of the Transcription Factor NF-{kappa}B

Zijing Lu*,{ddagger}, Eun Y. Lee*,{dagger},{ddagger}, Larry W. Robertson*,{ddagger},1, Howard P. Glauert*,{ddagger},§ and Brett T. Spear*,{dagger},{ddagger},2

* Graduate Center for Nutritional Sciences, {dagger} Department of Pathology and Laboratory Medicine, {ddagger} Graduate Center for Toxicology, § Department of Nutrition and Food Science, and Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536

Received January 21, 2004; accepted May 18, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are a group of synthetic chemicals that induce and promote liver tumors in rodents. We previously showed hepatic nuclear factor kappaB (NF-{kappa}B) activation and increased hepatocyte proliferation in PCB-treated rats. In this study, the role of NF-{kappa}B in hepatocyte proliferation and apoptosis after PCB administration was analyzed in wild-type mice and in mice deficient in the NF-{kappa}B p50 subunit (p50–/–). In a 2-day study, mice received a single intraperitoneal (ip) injection of corn oil or PCB-153. Hepatic NF-{kappa}B DNA binding activity and cell proliferation were increased by PCB-153 in wild-type mice but not in p50–/– mice. In a 21-day study, mice received six ip injections of corn oil or PCB-153 (twice weekly for 3 weeks) and were euthanized 4 days after the last injection. In this study, NF-{kappa}B DNA binding activity was not increased after PCB-153 treatment in wild-type or p50–/– mice. Cell proliferation was significantly increased in the wild-type mice treated with PCB-153; in the p50–/– mice treated with PCB-153, cell proliferation was greater than in untreated mice but less than in wild-type mice treated with PCB-153. The livers of p50–/– mice showed greater apoptosis than those of wild-type mice; PCB-153 decreased apoptosis in p50–/– mice, with higher inhibition in the 21-day study than in the 2-day study. RNase protection assays indicated that PCB-153 decreased the mRNA level of cyclin A2, B1, B2, and C in the 2-day study, but not in the 21-day study; however, it did not affect cyclin D1 and D2 mRNA levels at either time point. Cyclin D1 protein levels were not affected by PCB-153. Taken together, these data indicate that the absence of the NF-{kappa}B p50 subunit alters the proliferative and apoptotic changes in mouse liver in the response to PCB-153.

Key Words: PCBs; NF-{kappa}B; hepatocytes; apoptosis; cell proliferation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) comprise a group of 209 synthetic isomers and congeners that differ in the position and number of chloride atoms in the biphenyl structure. Because of their physical properties, chemical stability, and fire-retardant properties, the use of PCBs was once widespread. Their stability and lipophilicity contributed to their wide distribution in the environment and a tendency to bioaccumulate and bioconcentrate in the food chain. Once it was determined that PCBs represented a health hazard to animals and humans, they were removed from the market (Safe, 1994Go; Safe et al., 1985Go). Toxicity studies have shown that PCB mixtures can cause cancer in experimental animals (Mayes et al., 1998Go), and that PCBs have tumor-promoting activity (Glauert et al., 2001Go; Silberhorn et al., 1990Go).

Although PCBs are known to bind to a variety of receptors (Robertson and Hansen, 2001Go), they have been traditionally divided into two groups according to the positions of chlorine atoms. Coplanar PCB congeners substituted in both para positions and at least two meta positions, but not in any of the ortho positions, may assume a more coplanar configuration, similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and they bind strongly to the Ah receptor (AhR) (Bandiera et al., 1982Go); binding to the AhR induces expression of AhR-regulated genes including cytochrome P450 1A1 and 1A2 (CYP1A1, CYP1A2; Parkinson et al., 1983Go). On the other hand, PCB congeners with chlorine substitution in two ortho positions are non-coplanar and have low affinity for the AhR, but they induce a battery of drug-metabolizing enzymes, including cytochrome P450 2B1 and 2B2 (CYP2B1, CYP2B2), in a pattern that is similar to phenobarbital (Denomme et al., 1983Go; Parkinson et al., 1983Go).

In rodents pretreated with a genotoxic carcinogen, PCBs promote the development of liver cancer (Glauert et al., 2001Go), although the exact mechanisms of this promotion are still unknown. Both co-planar PCBs, such as 3,3',4,4'-tetrachlorobiphenyl (PCB-77) and 3,3'4,4',5-pentachlorobiphenyl (PCB-126), and ortho-substituted PCBs, such as 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) have promoting activity in rodent liver carcinogenesis (Glauert et al., 2001Go). A number of mechanisms have been investigated, including direct effects on signal transduction pathways, induction of oxidative stress, effects on vitamin A metabolism, and effects on intercellular communication (Glauert et al., 2001Go). Recently, hepatic tumor promoters such as peroxisome proliferators and phenobarbital have been found to activate the nuclear transcription factor NF-{kappa}B (Li et al., 1996aGo, 1996bGo, 2000Go; Nilakantan et al., 1998Go), which regulates the expression of cell proliferation–related and apoptosis-related genes (Li and Stark, 2002Go).

In hepatocytes, the major form of NF-{kappa}B is a heterodimer consisting of 50-kDa (p50) and 65-kDa (p65/relA) subunits that is complexed to an inhibitory subunit (I{kappa}B) in the cytoplasm. Upon stimulation, I{kappa}B is phosphorylated, polyubiquitinated, and degraded, allowing translocation of NF-{kappa}B into the nucleus (Li and Stark, 2002Go). Among the NF-{kappa}B–regulated genes, cyclin D1 controls the cell cycle checkpoint at G1/S phase (Guttridge et al., 1999Go; Hinz et al., 1999Go), and NF-{kappa}B is required for the initiation of cyclin D1 transcription and hyperphosphorylation of retinoblastoma protein (pRB), which lead cells to progress through G1 and entry into S phase (Biswas et al., 2000Go; Henry et al., 2000Go; Joyce et al., 1999Go; Kaltschmidt et al., 1999Go). NF-{kappa}B also regulates expression of certain anti-apoptotic genes, such as the inhibitor of apoptosis proteins (IAPs) (Wang et al., 1998Go) and Bcl-2/Bcl-xL (Lee et al., 1999Go; Tamatani et al., 1999Go; Wang et al., 1999Go). Knockout studies revealed that the p65 subunit of NF-{kappa}B is essential for hepatocytes to survive tumor necrosis factor alpha (TNF-{alpha})–induced apoptosis, because deficiency in p65 results in embryonic lethality from hepatocyte apoptosis (Beg et al., 1995Go). Inhibition of NF-{kappa}B activation by an I{kappa}B{alpha} superrepressor results in extensive hepatocyte apoptosis after partial hepatectomy (Iimuro et al., 1998Go). Although p65 null mice die during embryogenesis, p50–/– mice are viable (Sha et al., 1995Go), thus providing a model to study the role of the p65/p50 dimer in the regulation of cell proliferation and cell apoptotic death in the liver. Hepatocyte apoptosis was increased in p50–/– mice, and cell proliferation in response to the peroxisome proliferator ciprofibrate was inhibited (Tharappel et al., 2003Go). However, DNA synthesis and liver regeneration after partial hepatectomy or carbon tetrachloride treatment were not affected by the absence of the p50 subunit, nor was the hepatic inflammatory response after ischemia/reperfusion (DeAngelis et al., 2001Go; Kato et al., 2002Go). In addition, B cells lacking p50 show decreased activity in response to the lymphocyte proliferation response (LPS) (Sha et al., 1995Go; Snapper et al., 1996Go).

Our previous studies indicated that PCB-153, a non-coplanar PCB congener, activated hepatic NF-{kappa}B after a single injection (Lu et al., 2003Go) or during the promotion stage of hepatocarcinogenesis (Tharappel et al., 2002Go). In addition, hepatocyte proliferation was induced by a single injection of PCB-153 (Lu et al., 2003Go), which correlated with hepatic NF-{kappa}B activation in the same animals. Thus, the purpose of this study was to use p50–/– mice to determine the role of NF-{kappa}B in PCB-induced cell proliferation. The effects on apoptotic cell death after treatment with PCB-153 in these p50–/– mice were also studied.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) was synthesized and characterized as described previously (Schramm et al., 1985Go). Vitamin E–stripped corn oil (Acros Organics, Morris Plains, New Jersey), Alzet osmotic pumps (model 1003D, Alza Scientific Products, Palo Alto, CA), anti 5-bromo-2'-deoxyuridine (BrdU) antibody (Becton Dickinson, San Jose, CA), Antigen Retrieval Citra solution (BioGenex, San Ramon, CA), and the In situ cell death detection assay kit (Intergen, Purchase, NY) were obtained from sources indicated. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Experimental design. Eight-week-old male B6.129 mice homozygous for the NF-{kappa}B (p50) mutation and wild-type controls were obtained from our breeding colony (originally purchased from the Jackson Laboratory, Bar Harbor, ME). Mice were housed 2–4 animals per cage in a temperature-controlled and light-controlled room and fed an unrefined diet (#5001, Purina Mills, St. Louis, MO) and water ad libitum. In a 2-day study, 16 p50–/– mice and 16 wild-type littermates were first injected ip with either corn oil (5 p50–/– mice and 6 wild-type mice) or PCB-153 (300 µmol/kg; 11 p50–/– mice and 10 wild-type mice) and then euthanized 2 days later. One p50–/– mouse (PCB-153 group) and two wild-type mice (one in the corn oil group and one in the PCB-153 group) died before the end of the study. All mice were injected subcutaneously (sc) with BrdU (100 mg/kg) 2 h before euthanasia.

Then, in a 21-day study, mice received ip injections of corn oil (10 wild-type mice and 7 p50–/– mice) or PCB-153 (100 µmol/kg, 10 wild-type mice and 7 p50–/– mice) on days 0, 3, 7, 10, 14, and 17 and were euthanized on day 21; before the end of the study, 4 p50–/– mice (2 in the control group and 2 in the PCB-153 group) and 2 wild-type mice (PCB-153 group) died. Three days before euthanasia mice were surgically implanted with osmotic pumps containing BrdU (20 mg/ml, 10 µl/h). The liver was removed from each mouse, and pieces of each liver were fixed in 10% formalin and processed for histology. The remaining liver was snap frozen in liquid N2 and stored at –80°C.

Nuclear extract preparation. Nuclear extracts were prepared from frozen liver tissue by a modification of the method of Deryckere and Gannon (Deryckere and Gannon, 1994Go). Approximately 250 mg of tissue was homogenized in 5 ml of buffer [0.6% IGEPAL CA-630; 150 mM NaCl; 10 mM Hepes-KOH, pH 7.9; 1 mM EDTA; and 0.5 mM phenylmethylsulfonyl fluoride (PMFS)] using a Dounce homogenizer. The homogenate was centrifuged at 270 x g for 30 s. The supernatant was transferred into a new tube, incubated on ice for 5 min, and then centrifuged again at 3,000 x g for 20 min. The supernatant was discarded and the pellet was resuspended with 1 ml homogenization buffer, and then transferred into a microcentrifuge tube and centrifuged at 3,000 x g for 5 min. Again, the supernatant was discarded and the pellet was resuspended in 100 µl buffer (20 mM Hepes-KOH, pH 7.9; 420 mM NaCl; 1.2 mM MgCl2; 0.2 mM EDTA; 0.5 mM PMSF; 0.5 mM dithiothreitol; 2 mM benzamidine; 5 µg/ml aprotinin; 5 µg/ml leupeptin; 5 µg/ml pepstatin A; and 25% glycerol), incubated on ice for 1 h and then centrifuged at 11,000 x g for 10 min. The resulting supernatant was then divided into aliquots and stored at –80°C. One aliquot was diluted in PBS to measure the protein concentration using the BCA method (Pierce, Rockford, IL).

Electrophoretic mobility shift assay. Nuclear extract (5µg) from each liver was incubated with 0.5 mg poly (dI-dC) in binding buffer (50 mM KCl; 10 mM Hepes-KOH, pH 7.9; 6.5 mM dithiothreitol; and 10% glycerol) on ice for 5 min, and then for 15 min at room temperature after the addition of radiolabeled probe [20,000 counts per min (cpm)]. The NF-{kappa}B oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was obtained from Promega (Madison, WI) and end-labeled using T4 polynucleotide kinase. After incubation, samples were resolved by electrophoresis on a 7% polyacrylamide gel at 180 volts for 2 h using 0.5x TBE as the running buffer. After the electrophoresis, gels were dried under vacuum and exposed to a phosphorimage screen. The radioactivity was counted with a Storm phosphorimaging system (Amersham Biosciences, Piscataway, NJ).

BrdU immunohistochemical staining. The paraffin-embedded liver tissues were sectioned, stained with an anti-BrdU antibody, and counter-stained with hematoxylin. The staining was carried out using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA), according to the protocol provided by the manufacturer. Cells that had incorporated BrdU were easily identified as those with brown nuclei. At least 3000 hepatocellular nuclei per slide (1000 in each of three lobes) were counted in random fields, and the labeling index was expressed as a percentage of the number of labeled hepatocyte nuclei out of the total number of hepatocyte nuclei counted.

In situ cell death detection assay. The liver tissue processed for paraffin sections was used for the detection of apoptotic hepatocytes by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The assay was carried out using an in situ cell death detection assay kit (Intergen, Purchase, NY), according to the protocol provided by the manufacturer. At least 3000 hepatocyte nuclei were counted as described above, and the apoptotic index was expressed as a percentage of the number of labeled nuclei out of the total number of nuclei counted.

Western blotting. For analysis of IKB kinases (IKK) and I{kappa}B proteins, frozen liver pieces were diluted 1:4 with lysis buffer (1% Nonidet P-40, 0.1% SDS (sodium dodecyl sulfate), 0.1 mg/ml PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, and 1x PBS) and homogenized with an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH). The homogenates were centrifuged at 10,000 x g for 20 min, and the supernatants were collected and then centrifuged at 100,000 x g for 1 h; the supernatants (cytosolic fraction) were then collected and divided into aliquots. For the cyclin D1 protein, frozen liver tissues were homogenized in a buffer containing 50 mM Tris-HCl, pH 7.4 l; 150 mM NaCl; 1% IGEPAL; 0.1% SDS; 0.5% sodium deoxycholate; 50 mM NaF; 1 mM Na2VO3; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; 5 µg/ml aprotinin; 5 µg/ml leupeptin; and 5 µg/ml pepstatin A. The homogenates were centrifuged at 12,000 rpm for 15 min, and the supernatants were divided into aliquots and stored at –80°C. Protein concentrations of all samples were determined using the BCA method (Pierce). The samples were denatured by boiling for 5 min in 2x gel-loading buffer (17.3% glycerol; 1.25 M ß-mercaptoethanol' 5.2% SDS; 0.22 M Tris, pH 6.8; and 1–2 mg bromophenol blue). For each sample, 25 µg of the protein was electrophoresed on an 8.5% separating gel and 4% stacking gel at 175 V for 1 h. The proteins in the gels were transferred to nitrocellulose membranes (Life Technologies, Carlsbad, CA) at 100 V for 1 h. The membranes were incubated with a blocking buffer (5% fat-free dry instant powdered milk, 1 mM Tris-base, 15 mM NaCl, and 0.05% Tween-20) for 1 h at room temperature with shaking. The primary and secondary antibodies were diluted in the blocking buffer and incubated with the membrane for 1 h with shaking at room temperature. The membranes were washed three times with a wash buffer (1 mM Tris-base, 15 mM NaCl, and 0.05% Tween-20) between the primary and secondary antibodies. All primary and secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA), and the SuperSignal Chemiluminescent Substrate Kit (Pierce) was used to detect proteins.

RNA isolation and ribonuclease protection assays (RPA). Frozen liver tissues were homogenized in 1 ml Trizol reagent (Life Technologies) according to the manufacturer's instructions. The concentration and purity of RNA were determined by absorption at 260 nm and 280 nm using a spectrophotometer. The RPA were carried out using the RiboQuant RNase Protection Assay Kit from Becton Dickinson Pharmingen (San Diego, CA), according to the protocol provided by manufacturer. The RNA template used was MCYC-1 RiboQuant Mouse Cyclin Multi-Probe Template Set (Becton Dickinson Pharmingen).

Statistical analysis. Results were first analyzed by a two-way analysis of variance—ANOVA (the two factors being genotype and PCB treatment). If a significant interaction was observed, individual differences between means were determined using Tukey's post hoc test. The results were reported as means ± standard error of mean (SEM).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body Weight and Liver Weight
Wild-type and p50–/– mice were treated with corn oil alone or with PCB-153 in corn oil for 2 days or 21 days. In the 2-day study, there were no significant differences in body weights or liver weight among all groups of mice (Table 1). In the 21-day study, the body weights of p50–/– mice were significantly lower than those of wild-type mice. Despite this finding, the p50–/– mice were alert and healthy, and they did not exhibit any signs of stress. PCB-153 treatment significantly increased the liver weights and relative liver weights (percentage of body weight) in the 21-day study, both in the wild-type mice and the p50–/– mice. The liver weight to body weight ratio was also increased in the p50–/– mice, in both the untreated group and the PCB-153– treated group.


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Table 1 Effect of PCB-153 on the Body and Liver Weights of p50–/– Mice and Wild-Type Mice in Two Studies of 2 Days and 32 Days

 
Hepatic NF-{kappa}B Activity
Our previous studies have shown that a single dose of PCB-153 caused a transient increase in hepatic DNA binding activity of NF-{kappa}B, with the highest activity at 2 days posttreatment (Lu et al., 2003Go). In the present study, electrophoretic mobility shift assays (EMSAs) were performed with liver nuclear extracts and incubated with a radiolabeled NF-{kappa}B DNA probe to monitor changes in NF-{kappa}B activity. In the 2-day study, the hepatic DNA binding activity of NF-{kappa}B was significantly increased by PCB-153 in wild-type mice, whereas no NF-{kappa}B induction was seen in the p50–/– mice (Fig. 1). Despite the NF-{kappa}B induction seen in PCB-treated wild-type mice, the protein levels of IkBß and I{kappa}B kinases (IKK{alpha}, IKKß, IKK{gamma}) were not changed by PCB treatment (data not shown).



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FIG. 1. Effect of PCB-153 on the hepatic DNA binding activity of nuclear factor kappaB (NF-{kappa}B) in the 2-day study in wild-type and p50–/– mice. A. Electrophoretic mobility shift assays (EMSAs) were performed using a radiolabeled probe for NF-{kappa}B (Promega) with liver nuclear extracts from mice treated with corn oil, or PCB-153 (300 µmol/kg). Each lane contains extracts from a single mouse (5 µg). B. Net radioactive counts of NF-{kappa}B bands from the EMSA data shown in A. Quantitation of the specific NF-{kappa}B band was determined by subtracting background counts from the counts in each NF-{kappa}B band. *Value was significantly different from the other 3 groups by Tukey's post hoc test (p < 0.05); n = 3.

 
In the 21-day study, the hepatic DNA binding activity of NF-{kappa}B was higher in wild-type mice than in p50–/– mice (Fig. 2), but there was no difference in NF-{kappa}B activity between PCB-treated animals and corn oil–treated controls. As in the 2-day study, the protein levels of I{kappa}Bß and I{kappa}B kinases (IKK{alpha}, IKKß, IKK{gamma}) were not changed by PCB treatment (data not shown).



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FIG. 2. Effect of PCB-153 on the hepatic DNA binding activity of NF-{kappa}B in the 21-day study in wild-type and p50–/– mice. A. EMSAs were performed using a radiolabeled probe for NF-{kappa}B (Promega) with liver nuclear extracts from mice treated with corn oil or PCB-153 (100 µmol/kg, twice a week for 3 weeks). Each lane contained extracts from a single mouse (5 µg). B. Net radioactive counts of NF-{kappa}B bands from the EMSA data shown in A. Quantitation of the specific NF-{kappa}B band was determined by subtracting background counts from the count in each NF-{kappa}B band. *Values in wild-type mice were significantly different from those in p50–/– mice by ANOVA (p < 0.05); n = 3.

 
Hepatocyte Proliferation and Apoptosis
Our previous studies showed that a single dose of PCB-153 could cause a transient increase in cell proliferation, with the highest increase at 2 days posttreatment (Lu et al. 2003Go). In the 2-day study, hepatocyte proliferation in wild-type mice treated with PCB-153 was 193% of that in control mice, but this percentage was not statistically significant; in p50–/– mice PCB-153 had no effect (Fig. 3). In the 21-day study, PCB-153 treatment increased hepatocyte proliferation significantly in the wild-type mice (p < 0.05); cell proliferation in the PCB-treated p50–/– mice was intermediate between untreated p50–/– mice and PCB-treated wild-type mice, but was not significantly different from either group (Fig. 4).



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FIG. 3. Effect of PCB-153 on hepatocyte proliferation in wild-type and p50–/– mice in the 2-day study. Mice were administrated BrdU by sc injection 2 hours before euthanasia. Tissue sections were immunohistochemically stained for BrdU, and the labeling index was determined.

 


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FIG. 4. Effect of PCB-153 on hepatocyte proliferation in wild-type and p50–/– mice in the 21-day study. Mice were administrated BrdU by a 3-day infusion using Alzet osmotic pumps. Tissue sections were immunohistochemically stained for BrdU, and the labeling index was determined. Groups with different letters are significantly different, by Tukey's test (p < 0.05).

 
Apoptosis in hepatocytes was measured by the TUNEL assay. The apoptotic index was significantly higher (p < 0.05) in p50–/– mice than in wild-type mice at both time points (Figs. 5 and 6). This increase was significantly decreased by PCB-153 (p < 0.05) in the 21-day study (Fig. 6).



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FIG. 5. Effect of PCB-153 on hepatocyte apoptosis in wild-type and p50–/– mice in the 2-day study. Liver sections were immunostained using the TUNEL method, and an apoptotic index was determined for each mouse. *Values in p50–/– mice were significantly higher (p < 0.05) than those in wild-type mice by ANOVA with no significant interaction.

 


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FIG. 6. Effect of PCB-153 on hepatocyte apoptosis in wild-type and p50–/– mice in the 21-day study. Liver sections were immunostained using the TUNEL method, and an apoptotic index was determined for each mouse. Groups with different letters are significantly different, by Tukey's test (p < 0.05).

 
mRNA and Protein Levels of Cyclins
The cell cycle control system is regulated by the activity of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) (Bartek et al., 1996Go; Beijersbergen and Bernards, 1996Go). The mRNA levels of cyclins A2, B1, B2, C, D1, and D2 in liver were measured using RPAs (Fig. 7). For all the cyclins analyzed, there were no significant differences between wild-type mice and p50–/– mice at either time point. PCB-153 significantly decreased cyclin A2, B1, B2, and C mRNA levels in the 2-day study in both p50–/– and wild-type mice (p < 0.05), but it did not decrease levels of cyclin D1 or D2; in the 21-day study, the mRNA levels of all cyclins were not significantly different among groups.



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FIG. 7. Effect of PCB-153 on hepatic cyclin mRNA expression in p50–/– mice and wild-type mice. RNase protection assays were carried out as described in Materials and Methods with RNA from mice treated with corn oil or PCB-153 (one injection of 300 µmol/kg in the 2-day study, and six injections of 100 µmol/kg in the 21-day study). Each lane contained RNA (15 µg) from a single mouse. The radioactivity of each cyclin band was counted with a Storm phosphorimaging system (Amersham Biosciences). A. RPA in 2-day study. B. RPA in 21-day study. Results are expressed as the percentage of the control (L32 gene). Numbers are shown as the mean ± SEM with n = 3 for each group. *Values are significantly different from respective corn oil–treated controls (p < 0.05)

 
The cyclin D1 levels were measured using Western blotting. Compared to wild-type mice, p50–/– mice had less cyclin D1 protein in the liver (Fig. 8). However, cyclin D1 protein levels in the livers of the two groups of mice were not changed by PCB-153 at either time point.



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FIG. 8. Protein level of cyclin D1 in the 2-day and 21-day studies as determined by Western blotting. Each lane contains 25 µg protein.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have indicated that PCB mixtures and specific PCB congeners can increase cell proliferation and inhibit apoptosis in rodent liver or hepatocytes (Bohnenberger et al., 2001Go; Haag-Gronlund et al., 2000Go; Kolaja et al., 2000Go; Tharappel et al., 2002Go; Whysner and Wang, 2001Go). Three compounds, 2,3',4,4',5-pentachlorobiphenyl (PCB-118), 3,3',4,4'- tetrachlorobiphenyl (PCB-77), and Aroclor 1254, a PCB mixture, have all been found to induce hepatocyte proliferation during hepatocarcinogenesis (Haag-Gronlund et al., 2000Go; Kolaja et al., 2000Go; Tharappel et al., 2002Go; Whysner and Wang, 2001Go). Previous work by our laboratory also showed an increase in hepatocyte proliferation after a single dose of PCB-153 in rats (Lu et al. 2003Go). The purpose of the present study was to determine whether NF-{kappa}B regulated hepatocyte proliferation and inhibition of apoptosis after PCB treatment by using mice deficient in the NF-{kappa}B p50 subunit.

In the 2-day study, PCB-153 increased the hepatic DNA binding activity of NF-{kappa}B in wild-type mice, but not in p50–/– mice, consistent with the idea that p50 is important for full NF-{kappa}B activity in the liver. The levels of IKK and I{kappa}B proteins were not changed by PCB-153, although I{kappa}B{alpha} protein levels were lower in p50–/– livers than in wild-type livers. Unlike the 2-day study, hepatic NF-{kappa}B activity in wild-type mice was not induced by PCB-153 in the 21-day study. The increased NF-{kappa}B activity in corn oil–treated wild-type mice may be due to increased stress caused by the multiple ip injections.

In the 2-day study, PCB-153 increased hepatocyte proliferation approximately twofold in wild-type mice but not in p50–/– mice, indicating that NF-{kappa}B (p50/p65) is important in PCB-153–induced cell proliferation. An increase in cell proliferation was seen in both the wild-type and p50–/– mice after 21 days of PCB treatment, although the increased proliferation was greater in the wild-type animals. An increase in p65 or other NF-{kappa}B family members, decreased amounts of I{kappa}B{alpha}, or alterations in other signal transduction pathways in p50–/– mice could compensate for the lack of p50 and explain the increase in DNA synthesis at the later time point. Studies have shown normal liver regeneration in p50–/– mice after partial hepatectomy or carbon tetrachloride administration (DeAngelis et al., 2001Go), and it was suggested that this was due to a compensatory increase in the p65 NF-{kappa}B subunit.

Hepatocytes typically proliferate at a very low rate, yet increased cellular proliferation can occur in response to physical, infectious, or toxic injury (Kitamura et al., 1998Go). The cell cycle control system is regulated by the expression of cyclins, CDKs, and CKIs (Bartek et al., 1996Go; Beijersbergen and Bernards, 1996Go). In the present study, we compared the changes in the mRNA level of cyclin A2, B1, B2, C, D1, and D2. There was no significant change in the cyclin D1 mRNA level between wild-type and p50–/– livers, or after PCB-153 treatments, but there was less cyclin D1 protein in p50–/– livers than in the wild-type livers. This lack of concomitant changes between cyclin D1 mRNA and protein levels has been described early in regenerating liver after partial hepatectomy (Albrecht et al., 1995Go) and in adult rat liver (Awad and Gruppuso, 2000Go). Cyclin D1 mRNA was highly induced in rat liver after partial hepatectomy, whereas protein levels changed less than twofold and did not parallel changes in the mRNA (Albrecht et al., 1995Go). In adult rat livers, cyclin D1 mRNA levels are present at a higher level than in fetal livers, whereas the protein is not detectable in adult livers, suggesting posttranscriptional regulation (Awad and Gruppuso, 2000Go). How the cyclin D1 protein level was decreased while the mRNA level remained unchanged in the p50–/– livers is unclear, but our data do suggest a role for posttranslational control of cyclin D1 in hepatocyte growth regulation.

The suppression of apoptosis is another mechanism by which nongenotoxic hepatocarcinogens may induce cancer. Phenobarbital and non-coplanar PCBs, both of which are tumor promoters, have been shown to inhibit UV-induced apoptosis in primary hepatocytes (Bohnenberger et al., 2001Go). Tharappel et al. have shown inhibition of apoptosis by PCB-153 in placental glutathione S-transferase (PGST)-positive hepatocytes in an initiation-promotion study (Tharappel et al., 2002Go). Salvi and Toniello have shown that PCBs inhibit the mitochondrial permeability transition and the consequent release of cytochrome c from mitochondria (Salvi and Toninello, 2001Go). Considering the pivotal role of cytochrome c in caspase activation (Liu et al., 1996Go; Wang, 2001Go), inhibition of its release would indicate an interruption of the apoptotic pathway. However, little is known about the function of the p50 NF-{kappa}B subunit in the regulation of apoptotic pathways in hepatocytes. In the studies by DeAngelis et al. (2001)Go, in which Fas antibody was used to activate Fas receptor–mediated apoptosis, p50–/– livers showed a small increase in apoptosis compared to wild-type mice. Unfortunately, apoptosis in untreated livers was not reported in that study. In our study, we showed that apoptosis in the liver was increased in p50–/– mice compared to wild-type mice regardless of PCB-153 treatment. Although this increase in spontaneous apoptosis was not as severe as that seen in the p65-deficient mice, in which massive hepatic apoptosis leads to embryonic death (Beg et al., 1995Go), it demonstrates the importance of NF-{kappa}B (p50/p65) as an anti-apoptotic mediator. PCB-153 lowered apoptosis in p50–/– mice but not in wild-type mice. Because the apoptotic index in control wild-type mice was very low, it would be difficult to detect a PCB-153 effect unless large numbers of mice were used. The inhibition of apoptosis by PCB-153 in the p50–/– mice is likely related to an alteration in a p50-independent signal transduction pathway.

In summary, our data support the hypothesis that NF-{kappa}B activation plays a critical role in regulation of cell proliferation in liver in response to PCB-153 treatment. Apoptosis was increased in p50–/– mice; PCB-153 inhibited this increase, implying that the inhibition of apoptosis may be an important property of non-coplanar PCBs. These results raise the possibility that NF-{kappa}B may contribute to the tumor-promoting activity of non-coplanar PCB congeners, including PCB-153. Further studies will be required to determine the molecular mechanisms of these NF-{kappa}B–mediated effects.


    ACKNOWLEDGMENTS
 
We are grateful to Job Tharappel, Karen Calfee-Mason, and Petruta Bunaciu for their assistance with animal treatment and tissue collection. We thank Hans-Joachim Lehmler, of the Synthesis Core of the University of Kentucky Superfund Basic Research Program (ES07380), for chemically synthesizing the PCB-153 used in this study. This research was supported by grant ES07380 from the National Institute of Environmental Health Sciences (NIEHS) and by the Kentucky Agricultural Experiment Station. Z. Lu was supported by the Training Core of the NIEHS Superfund Basic Research Program (ES07380), by a Kentucky Opportunity Fellowship from the Graduate School of the University of Kentucky, and by a Dissertation Enhancement Award from the Graduate School of the University of Kentucky.


    NOTES
 
1 Present address: University of Iowa, Department of Occupational and Environmental Health, Iowa City, IA 52242–5000. Back

2 To whom correspondence should be addressed at Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 50536-0298. Fax: 859-257-8994. E-mail: bspear{at}uky.edu.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Albrecht, J. H., Hu, M. Y., and Cerra, F. B. (1995). Distinct patterns of cyclin D1 regulation in models of liver regeneration and human liver. Biochem. Biophys. Res. Commun. 209, 648–55.[CrossRef][ISI][Medline]

Awad, M. M., and Gruppuso, P. A. (2000). Cell cycle control during liver development in the rat: Evidence indicating a role for cyclin D1 posttranscriptional regulation. Cell Growth Differ. 11, 325–334.[Abstract/Free Full Text]

Bandiera, S., Safe, S., and Okey, A. B. (1982). Binding of polychlorinated biphenyls classified as either phenobarbitone-, 3-methylcholanthrene- or mixed-type inducers to cytosolic Ah receptor. Chem. Biol. Interact. 39, 259–77.[CrossRef][ISI][Medline]

Bartek, J., Bartkova, J., and Lukas, J. (1996). The retinoblastoma protein pathway and the restriction point. Curr. Opin. Cell Biol. 8, 805–814.[CrossRef][ISI][Medline]

Beg, A., Sha, W., Bronson, R., Ghosh, S., and Baltimore, D. (1995). Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376, 167–170.[CrossRef][ISI][Medline]

Beijersbergen, R. L., and Bernards, R. (1996). Cell cycle regulation by the retinoblastoma family of growth inhibitory proteins. Biochim. Biophys. Acta 1287, 103–120.[CrossRef][ISI][Medline]

Biswas, D. K., Cruz, A. P., Gansberger, E., and Pardee, A. B. (2000). Epidermal growth factor–induced nuclear factor kappa B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 97, 8542–8547.[Abstract/Free Full Text]

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]

DeAngelis, R. A., Kovalovich, K., Cressman, D. E., and Taub, R. (2001). Normal liver regeneration in p50/nuclear factor kappaB1 knockout mice. Hepatology 33, 915–924.[CrossRef][ISI][Medline]

Denomme, M. A., Bandiera, S., Lambert, I., Copp, L., Safe, L., and Safe, S. (1983). Polychlorinated biphenyls as phenobarbitone-type inducers of microsomal enzymes. Structure–activity relationships for a series of 2,4-dichloro-substituted congeners. Biochem. Pharmacol. 32, 2955–2963.[CrossRef][ISI][Medline]

Deryckere, F., and Gannon, F. (1994). A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16, 405.[ISI][Medline]

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

Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G., and Baldwin, A. S., Jr. (1999). NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol. Cell. Biol. 19, 5785–5799.[Abstract/Free Full Text]

Haag-Gronlund, M., Conolly, R., Scheu, G., Warngard, L., and Fransson-Steen, R. (2000). Analysis of rat liver foci growth with a quantitative two-cell model after treatment with 2,4,5,3',4'-pentachlorobiphenyl. Toxicol. Sci. 57, 32–42.[Abstract/Free Full Text]

Henry, D. O., Moskalenko, S. A., Kaur, K. J., Fu, M., Pestell, R. G., Camonis, J. H., and White, M. A. (2000). Ral GTPases contribute to regulation of cyclin D1 through activation of NF-kappaB. Mol. Cell. Biol. 20, 8084–8092.[Abstract/Free Full Text]

Hinz, M., Krappmann, D., Eichten, A., Heder, A., Scheidereit, C., and Strauss, M. (1999). NF-kappaB function in growth control: Regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol. Cell. Biol. 19, 2690–2698.[Abstract/Free Full Text]

Iimuro, Y., Nishiura, T., Hellerbrand, C., Behrns, K. E., Schoonhoven, R., Grisham, J. W., and Brenner, D. A. (1998). NFkappaB prevents apoptosis and liver dysfunction during liver regeneration. J. Clin. Invest. 101, 802–811.[Abstract/Free Full Text]

Joyce, D., Bouzahzah, B., Fu, M., Albanese, C., D'Amico, M., Steer, J., Klein, J. U., Lee, R. J., Segall, J. E., Westwick, et al. (1999). Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway. J. Biol. Chem. 274, 25245–25249.[Abstract/Free Full Text]

Kaltschmidt, B., Kaltschmidt, C., Hehner, S. P., Droge, W., and Schmitz, M. L. (1999). Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators. Oncogene 18, 3213–3225.[CrossRef][ISI][Medline]

Kato, A., Edwards, M. J., and Lentsch, A. B. (2002). Gene deletion of NF-kappa B p50 does not alter the hepatic inflammatory response to ischemia/reperfusion. J. Hepatol. 37, 48–55.[CrossRef][ISI][Medline]

Kitamura, T., Watanabe, S., and Sato, N. (1998). Liver regeneration, liver cancers and cyclins. J. Gastroenterol. Hepatol. 13(Suppl), S96–S99.[ISI][Medline]

Kolaja, K. L., Engelken, D. T., and Klaassen, C. D. (2000). Inhibition of gap-junctional-intercellular communication in intact rat liver by nongenotoxic hepatocarcinogens. Toxicology 146, 15–22.[CrossRef][ISI][Medline]

Lee, H. H., Dadgostar, H., Cheng, Q., Shu, J., and Cheng, G. (1999). NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc. Natl. Acad. Sci. U. S. A. 96, 9136–9141.[Abstract/Free Full Text]

Li, X., and Stark, G. R. (2002). NFkappaB-dependent signaling pathways. Exp. Hematol. 30, 285–296.[CrossRef][ISI][Medline]

Li, Y., Glauert, H. P., and Spear, B. T. (2000). Activation of nuclear factor-kappaB by the peroxisome proliferator ciprofibrate in H4IIEC3 rat hepatoma cells and its inhibition by the antioxidants N-acetylcysteine and vitamin E. Biochem. Pharmacol. 59, 427–434.[CrossRef][ISI][Medline]

Li, Y., Leung, L. K., Glauert, H. P., and Spear, B. T. (1996a). Treatment of rats with the peroxisome proliferator ciprofibrate results in increased liver NF-kappaB activity. Carcinogenesis 17, 2305–2309.[Abstract]

Li, Y., Leung, L. K., Spear, B. T., and Glauert, H. P. (1996b). Activation of hepatic NF-kappaB by phenobarbital in rats. Biochem. Biophys. Res. Commun. 229, 982–989.[CrossRef][ISI][Medline]

Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996). Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 86, 147–157.[ISI][Medline]

Lu, Z., Tharappel, J. C., Lee, E. Y., Robertson, L. W., Spear, B. T., and Glauert, H. P. (2003). Effect of a single dose of polychlorinated biphenyls on hepatic cell proliferation and the DNA binding activity of NF-kappaB and AP-1 in rats. Mol. Carcinogen. 37, 171–180.[CrossRef][ISI][Medline]

Mayes, B. A., McConnell, E. E., Neal, B. H., Brunner, M. J., Hamilton, S. B., Sullivan, T. M., Peters, A. C., Ryan, M. J., Toft, J. D., Singer, et al. (1998). Comparative carcinogenicity in Sprague-Dawley rats of the polychlorinated biphenyl mixtures Aroclors 1016, 1242, 1254, and 1260. Toxicol. Sci. 41, 62–76.[Abstract]

Nilakantan, V., Spear, B. T., and Glauert, H. P. (1998). Liver-specific catalase expression in transgenic mice inhibits NF-kappaB activation and DNA synthesis induced by the peroxisome proliferator ciprofibrate. Carcinogenesis 19, 631–637.[Abstract]

Parkinson, A., Safe, S. H., Robertson, L. W., Thomas, P. E., Ryan, D. E., Reik, L. M., and Levin, W. (1983). Immunochemical quantitation of cytochrome P-450 isozymes and epoxide hydrolase in liver microsomes from polychlorinated or polybrominated biphenyl-treated rats. A study of structure–activity relationships. J. Biol. Chem. 258, 5967–5976.[Free Full Text]

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

Safe, S., Bandiera, S., Sawyer, T., Robertson, L., Safe, L., Parkinson, A., Thomas, P. E., Ryan, D. E., Reik, L. M., Levin, W., Denomme, M. A., and Fujita, T. (1985). PCBs: Structure-function relationships and mechanism of action. Environ. Health Perspect. 60, 47–56.

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]

Salvi, M., and Toninello, A. (2001). Aroclor 1254 inhibits the mitochondrial permeability transition and release of cytochrome c: A possible mechanism for its in vivo toxicity. Toxicol. Appl. Pharmacol. 176, 92–100.[CrossRef][ISI][Medline]

Schramm, H., Robertson, L. W., and Oesch, F. (1985). Differential regulation of hepatic glutathione transferase and glutathione peroxidase activities in the rat. Biochem. Pharmacol. 34, 3735–3759.[CrossRef][ISI][Medline]

Sha, W. C., Liou, H. C., Tuomanen, E. I., and Baltimore, D. (1995). Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 80, 321–330.[ISI][Medline]

Silberhorn, E. M., Glauert, H. P., and Robertson, L. W. (1990). Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs. Crit. Rev. Toxicol. 20, 440–496.[Medline]

Snapper, C. M., Zelazowski, P., Rosas, F. R., Kehry, M. R., Tian, M., Baltimore, D., and Sha, W. C. (1996). B cells from p50/NF-kappa B knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching. J. Immunol. 156, 183–191.[Abstract]

Tamatani, M., Che, Y., Matsuzaki, H., Ogawa, S., Okado, H., Miyake, S., Mizuno, T., and Tohyama, M. (1999). Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFkappaB activation in primary hippocampal neurons. J. Biol. Chem. 274, 8531–8538.[Abstract/Free Full Text]

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]

Tharappel, J. C., Nalca, A., Owens, A. B., Ghabrial, L., Konz, E. C., Glauert, H. P., and Spear, B. T. (2003). Cell proliferation and apoptosis are altered in mice deficient in the NF-kB p50 subunit after treatment with the peroxisome proliferator ciprofibrate. Toxicol. Sci. 75, 300–308.[Abstract/Free Full Text]

Wang, C. Y., Guttridge, D. C., Mayo, M. W., and Baldwin, A. S. J. (1999). NF-kappaB induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol. Cell. Biol. 19, 5923–5929.[Abstract/Free Full Text]

Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin, A. S., Jr. (1998). NF-kappaB antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c- IAP2 to suppress caspase-8 activation. Science 281, 1680–1683.[Abstract/Free Full Text]

Wang, X. (2001). The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922–2933.[Free Full Text]

Whysner, J., and Wang, C. X. (2001). Hepatocellular iron accumulation and increased cell proliferation in polychlorinated biphenyl-exposed Sprague-Dawley rats and the development of hepatocarcinogenesis. Toxicol. Sci. 62, 36–45.[Abstract/Free Full Text]