Differential Activation of Hepatic NF-{kappa}B in Rats and Hamsters by the Peroxisome Proliferators Wy-14,643, Gemfibrozil, and Dibutyl Phthalate

Job C. Tharappel*,{dagger}, Michael L. Cunningham{ddagger}, Brett T. Spear§ and Howard P. Glauert*,{dagger},1

* Graduate Center for Nutritional Sciences and {dagger} Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40506-0054; {ddagger} National Institute for Environmental Health Sciences, Research Triangle Park, North Carolina; and § Department of Microbiology and Immunology and Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, Kentucky 40536-0298.

Received December 27, 2000; accepted March 14, 2001

ABSTRACT

Nuclear factor-{kappa}B (NF-{kappa}B) is an oxidative stress-activated transcription factor involved in the regulation of cell proliferation and apoptosis. We found previously that the peroxisome proliferator ciprofibrate activates NF-{kappa}B in the livers of rats and mice. These species are sensitive to the hepatocarcinogenic effects of peroxisome proliferators, whereas other species such as Syrian hamsters are not. In the present study we examined the effects of 3 different peroxisome proliferators on NF-{kappa}B activation in rats and Syrian hamsters. The peroxisome proliferators Wy-14,643, gemfibrozil, and dibutyl phthalate were administered to animals for 6, 34, or 90 days. NF-{kappa}B activity was determined using electrophoretic mobility-shift assays and confirmed using supershift assays. Wy-14,643 increased the DNA binding activity of NF-{kappa}B at all 3 time points in rats and produced the highest activation of the 3 chemicals tested. Gemfibrozil and dibutyl phthalate increased NF-{kappa}B activation to a lesser extent in rats and not at all times. There were no differences in hepatic NF-{kappa}B levels between control hamsters and hamsters treated with any of the peroxisome proliferators. This study demonstrates species-specific differences in hepatic NF-{kappa}B activation by peroxisome proliferators.

Key Words: NF-{kappa}B activation; rats; hamsters; Wy-14,643; gemfibrozil; dibutyl phthalate.

Peroxisome proliferators comprise a diverse group of chemicals that include hypolipidemic drugs such as nafenopin, Wy-14,643 and clofibrate, and industrial chemicals such as plasticizers and chlorinated solvents (Rao and Reddy, 1987Go). These compounds have numerous effects when administered to rats and mice, effects which are seen primarily in the liver and to a lesser extent in the kidney (Lake, 1995Go; Rao and Reddy, 1987Go). Peroxisome proliferators increase both the size and number of peroxisomes and increase the activities of several peroxisomal enzymes, including those involved in the ß-oxidation of long-chain fatty acids (Lake, 1995Go; Rao and Reddy, 1987Go). Increased levels of these enzymes are due to the ability of peroxisome proliferators to activate peroxisome proliferator activated receptors (PPARs) which can then bind DNA and increase the transcription of target genes (Corton et al., 2000Go). Increased levels of peroxisomal enzymes can lead to the production of reactive oxygen species (ROS), which can damage various cellular components (Yeldandi et al., 2000Go). Peroxisome proliferators can also lead to increased proliferation and decreased apoptosis in hepatocytes (Roberts et al., 2000Go). In addition, even though peroxisome proliferators are non-genotoxic, long-term administration of these compounds can result in hepatocarcinogenesis (Rao and Reddy, 1987Go). It has been proposed that oxidative DNA damage, caused by increased ROS, could contribute to the carcinogenic properties of peroxisome proliferators (Yeldandi et al., 2000Go). However, increased proliferation or decreased apoptosis could also contribute to the transforming potential of these compounds.

The induction of peroxisomes and peroxisomal enzymes seen in rats and mice is not present in all species. Humans, non-human primates, dogs, and guinea pigs are generally not responsive to these effects of peroxisome proliferators. The response in Syrian hamsters is intermediate between that seen in rats and mice and in these other species (Elcombe and Mitchell, 1986Go; Gray and de la Iglesia, 1984Go; Lake, 1995Go; Lake et al., 1984Go, 1989Go; Oesch et al., 1988Go). Wy-14,643 produced a long-term increase in cell proliferation in rats, whereas this agent had no effect in hamsters; nafenopin did not increase proliferation in either species (Lake et al., 1993Go). In in vitro studies, the peroxisome proliferators nafenopin, methyl clofenapate, and Wy-14,643 induced cell proliferation in rat and mouse hepatocytes, whereas no effect was seen in hamster or guinea pig hepatocytes (James and Roberts, 1995Go, 1996Go). In contrast, peroxisome proliferators suppressed apoptosis in cultured hepatocytes from both responsive and non-responsive species (James and Roberts, 1996Go). The carcinogenicity of peroxisome proliferators has been examined in two species other than rats and mice: in Syrian hamsters, the administration of Wy-14,643 or nafenopin for up to 60 weeks did not induce tumors (Lake et al., 1993Go), and in marmosets, the administration of clofibrate for 6 years did not increase tumor incidence (Tucker and Orton, 1993Go).

The transcription factor nuclear factor-{kappa}B (NF-{kappa}B) can be activated in many different cell types by a variety of stimuli (Wulczyn et al., 1996Go). NF-{kappa}B consists of 2 subunits (p50 and p65), and under normal conditions is primarily in the cytoplasm as an inactive complex non-covalently linked to an inhibitory subunit called I{kappa}B (Verma et al., 1995Go). Activation occurs primarily through the phosphorylation and subsequent degradation of I{kappa}B, which allows NF-{kappa}B to enter the nucleus and activate the transcription of target genes (Verma et al., 1995Go), including those that regulate cell proliferation and apoptosis (Beg et al., 1995Go; Fitzgerald et al., 1995Go). Reactive oxygen species (ROS), including H2O2, can activate NF-{kappa}B (Meyer et al., 1993Go), while antioxidants such as vitamin E can block NF-{kappa}B activation (Liu et al., 1995Go).

We have shown that the peroxisome proliferator ciprofibrate activates NF-{kappa}B in the livers of rats and mice and in cultured rat hepatoma cells (Li et al., 1996Go, 2000aGo; Nilakantan et al., 1998Go). This activation was inhibited in cultured cells treated with vitamin E or N-acetylcysteine, suggesting that ROS are involved in ciprofibrate-mediated NF-{kappa}B activation (Li et al., 2000aGo).

The involvement of NF-{kappa}B in cell proliferation and apoptosis suggests that this factor may contribute to the response to peroxisome proliferators. Therefore, we have investigated NF-{kappa}B activation in the livers of rats and Syrian hamsters, since these 2 species are responsive or resistant to peroxisome proliferator-induced hepatocarcinogenesis, respectively. Animals were treated for 6, 34, or 90 days with the peroxisome proliferators Wy-14,643, gemfibrozil, or dibutyl phthalate. Our data indicate that treatment with these compounds resulted in elevated hepatic NF-{kappa}B levels in rats at all or most of the time points tested, with Wy-14,643 being the most efficacious. Conversely, none of these compounds led to NF-{kappa}B activation in hamsters. Thus, the differential response to peroxisome proliferators in these 2 species is also seen at the level of NF-{kappa}B activation.

MATERIALS AND METHODS

Chemicals.
Wyeth-14,643 was obtained by custom synthesis from Chemsyn Science Laboratories (Lenexa, KS). Gemfibrozil was purchased from Sigma Chemical Company (St. Louis, MO). Dibutyl phthalate was obtained from Research Triangle Institute (Research Triangle Park, NC). All chemicals were determined to be >99% pure.

Experimental design.
This study was carried out with tissues provided by the National Toxicology Program, Research Triangle Park, NC. Male Sprague-Dawley rats (4–6 weeks old) were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Male Syrian hamsters (4–6 weeks old) were obtained from Frederick Cancer Research and Development Center (Frederick, MD). After a quarantine period of 12–18 days, animals were administered the test agents that were mixed in the NTP-2000 unrefined diet (Ziegler Brothers Inc., Gardners, PA). Animals were fed control diets or two doses of the following peroxisome proliferators: Wy-14,643 at 50 and 500 ppm in both species; gemfibrozil at 1000 and 16,000 ppm in rats and 6000 and 24,000 ppm in hamsters; dibutyl phthalate at 5000 and 20,000 ppm in both species. Final concentrations were determined to be within 10% of the target concentration. Animals were administered these agents for 6, 34, or 90 days, at which time they were killed by overexposure to carbon dioxide. Livers were frozen in liquid nitrogen and then stored at –70°C.

Blood samples were collected only at the 90-day time point, and from a separate set of animals. Animals were anesthetized with a mixture of carbon dioxide and oxygen, and blood was then collected by cardiac puncture into tubes containing EDTA. The white-cell fraction was separated from plasma and red cells and stored at –70°C.

Electrophoretic mobility shift assays.
Nuclear extracts were made from frozen liver tissue as described (Deryckere and Gannon, 1994Go) and stored in aliquots at –80°C. Protein concentrations were determined using the BCA assay (Pierce, Rockford IL). The NF-{kappa}B oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC; Promega, Madison, WI) was endlabeled with {gamma}32P using polynucleotide kinase. For EMSAs, 5 µg of protein from each sample were incubated in binding buffer (50 mM KCl, 10 mM HEPES pH 7.9, 6.5 mM DTT, 10% glycerol) with 0.5 µg poly (dI:dC) for 10 min on ice and then for 20 min. at room temperature with 1 ng (approximately 20,000 cpm) of the radiolabeled NF-{kappa}B probe. For antibody supershift assays, 4 µg of rabbit anti-p50, anti-p65, or preimmune serum (Santa Cruz Biotech, Santa Cruz, CA) was also added. After incubations, the samples were resolved on a 7% non-denaturing polyacrylamide gel in 0.5 µ TBE buffer at 150 V for 2 h. The gels were subsequently dried and autoradiographed. The autoradiographs were also analyzed using an Ambis analytic imaging system (Ambis, San Diego, CA).

Statistics.
The data from the EMSA quantitation were analyzed by 2-way analysis of variance and Bonferroni's test. Results were considered significant at p < 0.05.

RESULTS

We previously showed that mice and rats exhibit increased hepatic NF-{kappa}B DNA binding activity in response to treatment with the peroxisome proliferator ciprofibrate (Li et al., 1996Go; Nilakantan et al., 1998Go). To further investigate this relationship, we have extended our study in rats to 3 additional peroxisome proliferators: Wy-14,643, gemfibrozil, and dibutylphthalate. We have also analyzed NF-{kappa}B activation after administering these 3 chemicals to hamsters, which, in contrast to rats, do not develop hepatic tumors after peroxisome proliferator treatment (Lake et al., 1993Go).

To monitor changes in NF-{kappa}B activity, we performed EMSAs with nuclear extracts prepared from frozen liver tissues. Three animals were used in each data group. Untreated control rat livers contained low but detectable levels of NF-{kappa}B (Fig. 1Go). All 3 peroxisome proliferators increased hepatic NF-{kappa}B DNA binding activity in rats, although the extent of activation was variable. Wy-14,643 was the most efficacious activator of NF-{kappa}B. At all 3 time points, rats fed both the 50 and 500 ppm doses had significantly increased NF-{kappa}B DNA binding activity when compared to control rats (Figs. 1 and 2GoGo). At 6 days, rats fed the highest dose of Wy-14,643 had about a 4-fold increase in NF-{kappa}B activation compared to controls; after 34 and 90 days the DNA binding activity was increased about 3-fold.



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FIG. 1. NF-{kappa}B activity in liver nuclear extracts of rats and hamsters treated with peroxisome proliferators. Animals were untreated (control) or treated with a low or high doses of Wy-14,643, gemfibrozil, and dibutyl phthalate or 6, 34, or 90 days. EMSAs were performed using a radiolabeled NF-{kappa}B binding site with 5 µg of nuclear proteins. The first lane (+) contains extracts from Hela cells which were used as a positive control. The second lane (C) contains Hela extracts with a 50-fold molar excess of unlabeled NF-{kappa}B oligonucleotide. The subsequent lanes contain liver nuclear extracts from a single animal as labeled.

 


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FIG. 2. Quantitation of NF-{kappa}B activity in animals treated with Wy-14,643. Autoradiographs shown in Figure 1Go were analyzed using densitometry. The averages for each of the 6 groups of animals are shown. The different letters indicate significant differences (p <= 0.05) caused by Wy-14,643 treatment within each species; asterisks indicate a species difference for that dietary level of Wy-14,643.

 
Gemfibrozil was a less potent activator of NF-{kappa}B than Wy-14,643 (Figs. 1 and 3GoGo). A 2-fold increase in NF-{kappa}B DNA binding activity was seen in rats that had been treated with gemfibrozil for 6 days (low and high dose) or 90 days (low dose); no increase in NF-{kappa}B was seen in the 34-day group. However, only rats treated with the low dose of gemfibrozil for 90 days showed a statistically significant difference (p < 0.05) in NF-{kappa}B DNA binding activity when compared to the untreated controls.



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FIG. 3. Quantitation of NF-{kappa}B activity in animals treated with gemfibrozil. Autoradiographs shown in Figure 1Go were analyzed as described in Figure 2Go. The different letters indicate significant differences (p <= 0.05) caused by gemfibrozil treatment within each species; asterisks indicate a species difference for that dietary level of gemfibrozil.

 
In rats treated with low or high doses of dibutyl phthalate, there were no significant differences in NF-{kappa}B levels in rats treated for 6 days when compared to the untreated controls (Figs. 1 and 4GoGo). Rats treated for 34 days showed a significant activation of NF-{kappa}B at both the low and high dose. At the 90-day time point, only the rats administered the low dose of dibutyl phthalate had a significant increase in NF-{kappa}B activation when compared to non-treated control rats.



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FIG. 4. Quantitation of NF-{kappa}B activity in animals treated with dibutyl phthalate. Autoradiographs shown in Figure 1Go were analyzed as described in Figure 2Go. The different letters indicate significant differences (p <= 0.05) caused by dibutyl phthalate treatment within each species; asterisks indicate a species difference for that dietary level of dibutyl phthalate.

 
The specificity of rat NF-{kappa}B binding was confirmed using supershift assays with liver extracts that were pooled from the Wy-14,643 (high dose), 90-day samples (Fig. 5Go). The NF-{kappa}B complex was unaffected by the presence of preimmune serum (PIS). The anti-p50 antibody supershifted a majority of the NF-{kappa}B and resulted in the appearance of a supershifted complex (*); a similar result was seen with the anti-p65 antibody. Addition of both the p50 and p65 antibodies together led to a near complete loss of the NF-{kappa}B band. These results confirm that the complex that bound the radiolabeled NF-{kappa}B fragment did contain both p50 and p65.



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FIG. 5. Supershift assays to confirm NF-{kappa}B activity in rat hepatic nuclear extracts. EMSAs were performed with the radiolabeled NF-{kappa}B probe and 5 µg of liver nuclear extracts from a rat treated with Wy-14,643 for 90 days. The probe was incubated with nuclear extract alone (Control) or with pre-immune serum (PIS), anti-p50 antibody (p50), anti-p65 antibody (p65), or both anti-p50 and -p65 antibodies (Both). The asterisk (*) denotes the supershifted complex.

 
NF-{kappa}B activity, as measured by EMSA, was undetectable in the livers of hamsters fed a control diet (Fig. 1Go); these levels were usually significantly lower than NF-{kappa}B levels in control rat livers (Figs. 2–4GoGoGo). In addition, there were no statistically significant increases in hamster liver NF-{kappa}B levels with any of the 3 peroxisome proliferators that were used at either low or high doses (Figs. 1–4GoGoGoGo). These results indicate that hepatic NF-{kappa}B is not activated in hamsters after treatment with peroxisome proliferators.

The apparent lack of DNA binding activity in hamster-liver samples could be due to the inability of hamster NF-{kappa}B to bind the NF-{kappa}B oligonucleotide. To test this possibility, we examined NF-{kappa}B DNA binding activity in white-blood-cell extracts obtained from control hamsters after 90 days of treatment (Fig. 6Go). EMSAs demonstrated the presence of 2 bands in extracts from these peripheral blood cells. To confirm that these bands contained NF-{kappa}B, we performed supershift assays with anti-p50 and anti-p65 antibodies. The addition of pre-immune serum (PIS) had no effect on any of the bands. The addition of the anti-p50 antibody resulted in the presence of a new band (*) and a dramatic reduction in the lower mobility band. Addition of the anti-p65 antibody supershifted both bands; a similar result was seen with when both the anti-p50 and anti-p65 antibodies were used together. These studies with white-blood-cell extracts demonstrate that hamster NF-{kappa}B is capable of binding the radiolabeled NF-{kappa}B probe. This indicates that the lack of NF-{kappa}B activity seen in hamster liver extracts is due to low levels of nuclear NF-{kappa}B in these samples, rather than to a lack of affinity for the NF-{kappa}B oligonucleotide used in this study.



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FIG. 6. Supershift assays to confirm NF-{kappa}B activity in hamster nuclear extracts. EMSAs were performed with the radiolabeled NF-{kappa}B probe and 5 µg of nuclear extracts that were prepared from white blood cells collected and pooled from control hamsters. The probe was incubated with nuclear extract alone (Control) or with pre-immune serum (PIS), anti-p50 antibody (p50), anti-p65 antibody (p65), or both anti-p50 and -p65 antibodies (Both). The asterisk (*) denotes the supershifted complex.

 
DISCUSSION

We previously demonstrated that ciprofibrate can activate NF-{kappa}B in rats and mice (Li et al., 1996Go; Nilakantan et al., 1998Go). Here we have shown that 3 additional peroxisome proliferators, Wy-14,643, gemfibrozil, and dibutyl phthalate, can increase hepatic NF-{kappa}B DNA binding activity in rats. These data suggest that peroxisome proliferators as a class can activate NF-{kappa}B in the liver.

The extent of NF-{kappa}B activation correlates with the ability of these 3 chemicals to induce hepatic tumors and long-term cell proliferation. Of the 3 peroxisome proliferators tested here, Wy-14,643 was the most potent and dibutyl phthalate the least potent activator of NF-{kappa}B in rats. Wy-14,643 is effective at inducing hepatocellular carcinomas in rats, producing tumors in nearly all animals after 14–16 months (Lake et al., 1993Go; Reddy et al., 1979Go). Gemfibrozil was found to induce hepatic tumors at a lower incidence in male rats at 18 and 24 months, but did not do so in female rats (Fitzgerald et al., 1981Go). Although dibutyl phthalate has not been tested in a rodent 2-year carcinogenesis bioassay, other phthalate esters such as diethylhexyl phthalate induce only a low tumor incidence and only at very high doses (6000 and 12,000 ppm in the diet) (NTP, 1982Go). In the present study, Wy-14,643 was more efficacious than gemfibrozil or dibutyl phthalate in activating NF-{kappa}B, indicating that the carcinogenicity of these peroxisome proliferators generally correlates with the extent of NF-{kappa}B activation. The lack of carcinogenicity of Wy-14,643 in Syrian hamsters (Lake et al., 1993Go) also correlates very well with its inability to activate NF-{kappa}B in this species. Cell proliferation rates in response to peroxisome proliferators in rats and hamsters were also quantified as part of the present NTP study (Durnford et al., 1998; Cunningham et al., unpublished data) and another study (Lake et al., 1993Go). Both Wy-14,643 and gemfibrozil increased hepatocyte proliferation in rats but not in hamsters; this change in proliferation correlated well with the species difference in NF-{kappa}B activation. The peroxisome proliferator nafenopin, which was not tested in this study, did not influence long-term cell proliferation in either rats or hamsters, although it is carcinogenic in rats and mice (Eacho et al., 1991Go; Lake et al., 1993Go; Reddy et al., 1976Go; Reddy and Rao, 1977Go).

In rats and mice, peroxisome proliferators substantially increase the activity of the hydrogen peroxide (H2O2)-generating enzyme fatty acyl CoA oxidase (FAO) while not affecting or decreasing the activities of the hydrogen peroxide-metabolizing enzymes catalase and glutathione peroxidase, respectively (Rao and Reddy, 1987Go). We have proposed that this oxidant/antioxidant imbalance is responsible for NF-{kappa}B activation by peroxisome proliferators (Li et al., 1996Go). In support of this notion, increased FAO levels in Cos-1 cells can activate NF-{kappa}B (Li et al., 2000bGo). Moreover, ciprofibrate-mediated NF-{kappa}B activation is inhibited in rat hepatoma cells by the antioxidants N-acetylcysteine and vitamin E and in transgenic mice by catalase overexpression (Li et al., 2000aGo; Nilakantan et al., 1998Go). The 3 peroxisome proliferators used in this study efficaciously induced FAO activity in rats (Durnford et al., 1998; Cunningham et al., unpublished data). In hamsters, both gemfibrozil and dibutyl phthalate induced less than a 2-fold increase in FAO activity (Durnford et al., 1998; Cunningham et al., unpublished data). In contrast, Wy-14,643 produced a large increase in hepatic FAO activity in both rats and hamsters, which would presumably lead to elevated H2O2 levels in both species (Durnford et al., 1998; Cunningham et al., unpublished data). After Wy-14,643 treatment, catalase was slightly induced in rats and slightly decreased in hamsters (Durnford et al., 1998; Cunningham et al., unpublished data). However, we have found that basal levels of glutathione S-transferase, glutathione reductase, selenium-dependent glutathione peroxidase, and DT-diaphorase are much higher in the livers of hamsters than of rats (O'Brien et al., 2001aGo,bGo). Moreover, hepatic selenium-dependent glutathione peroxidase activity and glutathione peroxidase 1 mRNA levels were increased in hamsters but not in rats after Wy-14,643 treatment (O'Brien et al., 2001aGo). This indicates that an increased capacity for detoxifying excess H2O2 in hamsters could account for the lack of NF-{kappa}B activation after peroxisome proliferator treatment.

In other models of liver stress, NF-{kappa}B activation was rapid and transient (Cressman et al., 1994Go; Kovalovich et al., 2000Go). In contrast, we have found that NF-{kappa}B DNA binding activity continued to be elevated for the entire 90-day study with Wy-14,643 and gemfibrozil. The I{kappa}B{alpha} gene itself is one target of NF-{kappa}B, resulting in a feedback inhibition that down-regulates NF-{kappa}B (Pahl, 1999Go). Therefore, it is of some interest that NF-{kappa}B remained active for an extended period of time in rats. Whether this feedback loop is blocked or some other mechanism leads to the persistent NF-{kappa}B activation will require further study. Regardless of the mechanism, these results indicate that NF-{kappa}B target genes may also be up-regulated for extended periods. This suggests that NF-{kappa}B may contribute to the long-term effects of peroxisome proliferators, including the ability of these agents to cause hepatocellular carcinomas.

In conclusion, this study demonstrated a difference between rats and hamsters in hepatic NF-{kappa}B activation by 3 peroxisome proliferators. This difference correlates with the induction of hepatic tumors and long-term cell proliferation in these 2 species. This correlation, along with the persistent activation of NF-{kappa}B in rats over 90 days, suggests a potential role for NF-{kappa}B in peroxisome proliferator-mediated carcinogenesis. Further studies will be needed to explore this possibility.

ACKNOWLEDGMENTS

This work was supported by NCI grants CA-74147 and CA-01688 and by the Kentucky Agricultural Experiment Station. J.C.T. was supported by an NIEHS training grant (ES-07266).

NOTES

1 To whom correspondence should be addressed at the University of Kentucky, Graduate Center for Nutritional Sciences, 222 Funkhouser Building, Lexington, KY 40506–0054. Fax: (859) 323-0061. E-mail: hglauert{at}pop.uky.edu. Back

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