Treatment of rats with the peroxisome proliferator ciprofibrate results

Carcinogeneds vol.17 no. 11 pp.2305-2309, 1996

Treatment of rats with the peroxisome proliferator ciprofibrateresults in increased liver NF-KB activity

Y.Li1, L.K.Leung2, H.P.Glauert1^3 and B.T.Spear4*5

'Graduate Center for Toxicology, ^Nutritional Sciences Program,3Department of Nutrition and Food Science and 4Departments ofMicrobiology and Immunology and Pathology and Laboratory Medicine,University of Kentucky, College of Medicine, 800 Rose Street, Lexington,KY 40536-0084, USA5To whom correspondence should be addressed

Nuclear factor KB (NF-KB) is an important stress-inducedtranscription factor in many cell types, including hepato-cytes. Previous studies have shown that reactive oxygenspecies, including hydrogen peroxide, are potent activatorsof NF-KB. Peroxisome proliferators are a group of rodentchemical carcinogens that have been proposed to act byincreasing reactive oxygen in the liver. These results ledus to consider whether peroxisome proliferators wouldincrease NF-KB activity in the liver. Here we demonstratethat rats fed diets containing the peroxisome proliferatorciprofibrate exhibit increased hepatic NF-KB DNA-bindingactivity. This observation suggests that NF-KB may contri-bute, in part, to peroxisome proliferator-mediated changesin the liver.

Nuclear factor KB (NF-KB*) was first identified as an inducibletranscriptional regulator in B lymphocytes (1). Additionalstudies have shown that NF-KB is activated in many differentcell types by a variety of agents, suggesting that this factor isan important global transcriptional regulator which is inducedby numerous stimuli (2,3)- NF-KB is normally found in thecytosol as an inactive complex consisting of two subunits (p50and p65, although other members of the rel transcription factorfamily can also contribute to NF-KB complexes), which arebound to an inhibitory subunit (IKB; 4). Upon activation, N F -KB is released from IKB and translocates to the nucleus,where it binds its cognate DNA sequences and increases thetranscription of specific genes (2). Reactive oxygen inter-mediates, including hydrogen peroxide (H2O2), are potentactivators of NF-KB (5). Consistent with these data, anti-oxidants are effective inhibitors of NF-KB (6). These data haveled to the suggestion that NF-KB is an important reactiveoxygen-induced transcription factor (6).

One group of chemical carcinogens that have been proposedto act by increasing reactive oxygen are peroxisome pro-liferators. These chemicals, which induce hepatocellular carcin-omas in rodents, greatly increase the number and volume ofperoxisomes in the cell as well as several enzymes of lipidmetabolism (7). Among other changes, the enzymes of theperoxisomal (3-oxidation pathway, including fatty acyl CoAoxidase (FAO), which produce H2O2 as a by-product, arehighly induced. On the other hand, catalase, the peroxisomal

•Abbreviations: NF-KB, nuclear factor KB; H2O2, hydrogen peroxide; FAO,fatty acyl CoA oxidase; OHdG, 8-hydroxydeoxyguanosine; EMSA, electro-phoretic mobility shift assay; HNF-3, hepatocyte nuclear factor 3; PPAR,peroxisome proliferator activated receptor, PPRE, peroxisome proliferatorresponse element.

enzyme which detoxifies H2O2, is only slightly induced. It hastherefore been proposed that carcinogenesis by peroxisomeproliferators is due at least in part to overproduction of H2O2or other reactive oxygen species, which could then cause lipidperoxidation or oxidative DNA damage (7). Many investigatorshave examined whether peroxisome proliferators inducehepatic lipid peroxidation, mainly using malondialdehyde(thiobarbituric acid-reactive substances), conjugated dienes andlipofuscin as endpoints. Most studies saw no change inmalondialdehyde concentrations after treatment with peroxi-some proliferators, but others saw either a decrease or anincrease on at least one time point (8-13). After short termtreatment with peroxisome proliferators, some studies observeda decrease in conjugated diene concentrations, whereas otherssaw no change; in studies lasting 150 days or longer, however,conjugated diene concentrations were increased in many (butnot all) studies, particularly by the more efficacious and potentperoxisome proliferators (10,13—18). In studies that examinedlipofuscin, all peroxisome proliferators increased lipofuscinafter prolonged but not short-term administration (14,19-23).Studies of oxidative DNA damage have examined the oxidativeDNA adducts 8-hydroxydeoxyguanosine (OHdG) or hydroxy-methyldeoxyuridine. Several peroxisome proliferators havebeen shown to increase hepatic oxidative DNA damage in rats(24—29), but in other studies peroxisome proliferators werefound not to influence OHdG levels (8,30,31). Recently,Chu et al. (32) found that overexpressing FAO led to thetransformation of CV-1 African green monkey kidney cellsinto anchorage-independent cells that would develop intotumors in nude mice.

Regardless of the influence of peroxisome proliferators onlipid peroxidation and oxidative DNA damage, the well-documented increase in enzymes which produce H2O2 suggeststhat peroxisome proliferators may increase hepatic nuclearNF-KB levels. In addition, NF-KB activity is induced inregenerating liver following partial hepatectomy (33,34). Takentogether, these observations led us to investigate whetherperoxisome proliferators would activate NF-KB in the liver.To test this, rats were fed a diet containing 0.01% of theperoxisome proliferator ciprofibrate; control rats received thesame diet without ciprofibrate. The administration of cipro-fibrate did not affect body weights (data not shown). Ratswere killed by overexposure to carbon dioxide 3, 6 or 10 daysafter starting drug treatment. The livers were quickly removed,snap-frozen in liquid nitrogen, and stored at -80°C untilfurther use. To measure the efficacy of ciprofibrate treatment,we measured FAO enzyme levels (Figure 1) as described (35).Whereas basal FAO levels did not change over time in controlrats, a 10-fold increase in FAO was seen by day 3 in treatedrats. FAO levels showed a dramatic, steady increase with liverenzyme activity in day 10 rats nearly 20-fold greater than incontrol rats. Peroxisome proliferators are also known to inducea transient proliferation of hepatocytes shortly after theiradministration (7). Consistent with these data, [3H]thymidine

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Fig. 1. Temporal changes in FAO activity in the liver of rats in response tociprofibrate treatment. Rats were fed a diet with (dashed lines) or without(solid lines) ciprofibrate starting on day 0. Animals were killed at days 3, 6and 10. Each point represents data from eight rats. FAO activity wasmeasured using lauroyl CoA as the substrate.

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Fig. 2. Transient changes in DNA synthesis as measured by [3H]thymidineuptake in the livers of rats in response to ciprofibrate treatment. Rats werefed a diet with (dashed lines) or without (solid lines) ciprofibrate starting onday 0. Animals (8 per group) were killed at days 3, 6 and 10. DNAsynthesis was determined as described (57).

incorporation studies indicated that treated rats exhibited a3-fold increase in DNA synthesis at day 3 which declined tobaseline levels by day 10, whereas the level of [3H]thymidineincorporation in control rats was unchanged over the 10-dayperiod (Figure 2). These results are consistent with previousstudies (7,36) and indicate that the rats were responding to theciprofibrate.

NF-KB activation can be measured by the increased presenceof DNA binding activity in the nucleus. Therefore nuclearextracts were prepared from rat livers as described (37). NF-KBactivity was monitored using electrophoretic mobility shiftassays (EMSAs) with a radiolabeled probe containing twoNF-KB binding sites. Low levels of NF-KB were found in theliver nuclear extracts from control rats and remained unchangedover the 10-day period (Figure 3A). Three days after theinitiation of treatment, an increase in nuclear NF-KB DNAbinding activity was observed in treated versus control rats(Figure 3A). NF-KB levels continued to increase at 6 and 10

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Fig. 3. (A) Temporal pattern of NF-KB induction in response to ciprofibratefeeding. EMSAs were used to monitor the presence of NF-KB in rat livernuclei. Liver nuclear extracts were prepared from 0.5—1.0 g of frozen ratliver. A portion of the extract was used to determine the proteinconcentration by using the BCA assay (Pierce, Rockford, IL) and theremaining sample was aliquoted and stored at -80°C. A fragmentcontaining two NF-KB binding sites (5'-CAAGGGGACTTTCCATGGAT-CCAAGGGGACTTTCCATG) was flanked by GATC overhangs and clonedinto the BgUl site of a modified pUC9 vector (58). The NF-KB 2-mer wasexcised by digestion of the plasmid with Sail and Xho\, dephosphorylatedand acrylamide gel purified. For use as a radioactive probe, the NF-KB2-mer was end-labeled with [v-32P]ATP using polynucleotide kinase. ForEMSAs, nuclear extracts (10 |ig) were incubated on ice with 1 ng of theradiolabeled NF-KB binding site 2-mer and 1 ng of poly(dI:dC) as described(K.Li and B.Spear, submitted). After 30 min, samples were resolved onnon-denaturing 6% acrylamide gels which were subsequently dried andautoradiographed. Liver nuclear extracts were prepared from rats whichwere killed 3 (lanes 1, 2, 7 and 8), 6 (lanes 3, 4, 9 and 19) or 10 (lanes 5,6, 11 and 12) days after initiation of the diet. Lanes 1-6 (control)correspond to samples from rats receiving a normal diet; lanes 7-12 (+ciprofibrate) represent samples from rats eating a diet containing 0.01%ciprofibrate. Each lane represents a sample from a single rat. FP, free probe;NF-KB, DNA/NF-KB complexes. (B) Quantitation of the EMSA data shownin (A). Dried acrylamide gels used for the autoradiographs shown in (A)were analyzed using a radioanalytic imaging system (Ambis, San Diego,CA). Total c.p.m. in the different bands were determined; net c.p.m. wereobtained by subtracting background radioactivity. Each data point representsthe average of two rats in each sample group shown in (A).

days after treatment. Quantitative radioanalytic image analysisindicated that the level of induction was nearly 2-fold by day3 and increased to 4-fold by day 10 (Figure 3B). Hepatocyte

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nuclear factor 3 (HNF-3) is composed of a family of liver-enriched transcription factors that regulate the expression ofmany liver genes (38). EMSAs with a radiolabeled HNF-3binding motif derived from the region of the rat transthyretinpromoter showed that HNF-3 binding activity remainedunchanged over the 10-day period in both treated and controlrats (data not shown). This indicates that ciprofibrate does notlead to a global, but rather a more restricted increase intranscription factor activity.

To confirm that the ciprofibrate-induced complex in liverextracts contained NF-KB, several controls were performed.EMSAs were carried out in the presence of an excess ofunlabeled self (fragment containing two NF-KB sites) and non-self (size-matched unrelated DNA) fragments to determine ifthe complex was bound to the consensus NF-KB site(Figure 4A). The homologous fragment could effectivelycompete for DNA binding activity in a dose-dependent manner(lanes 4 and 5). The heterologous fragment did not diminishbinding to the radiolabeled probe (cf. lanes 1, 2 and 3),demonstrating specificity for the NF-KB site. In addition,EMSAs were performed with antibodies which react witheither the NF-KB p50 or p65 subunits (Figure 4B). Thepresence of pre-immune rabbit serum did not alter the NF-KBcomplex (cf. lanes 1 and 2). The addition of the anti-p50antiserum (lane 3) reduced the intensity of the complex labeledNF-KB and resulted in the presence of a new, supershiftedcomplex (labeled *). This novel complex presumably containsthe anti-p50 antibodies, resulting in a complex with a newmolecular mass. The anti-p65 antiserum did not give rise to anew complex, but did diminish the NF-KB complex (lane4). In this situation, the antibody has likely disrupted theNF-KB-DNA complex. While these results indicate that p50and p65 are components of the ciprofibrate-induced complex,they indicate that neither the p50 nor p65 antiserum completelyabolished the NF-KB complex. While these results do noteliminate the possibility that the antibodies are not sufficientlystrong or at high enough concentrations to bind all the proteinswithin the NF-KB complex, they raise the second possibilitythat the complexes include other members of the NF-KB familyof factors. Evidence exists for additional NF-KB members inthe liver (39).

Peroxisome proliferators are a class of compounds whichexert their effects in the liver, in part, through the activationof numerous genes, including enzymes of the peroxisomalp^oxidation pathway, which produces H2O2 as a by-product.That these genes are directly activated by the action ofnuclear hormone receptors (see below) does not eliminate thepossibility that additional mechanisms of transcriptional controlare involved in peroxisome proliferator-mediated changes in

Fig. 4. Specificity of NF-KB binding in EMSAs. Liver nuclear extracts wereused from rats that were treated with ciprofibrate for 10 days. EMSAs wereperformed as described in Figure 3. (A) Competition studies with non-radioactive DNA fragments to show specificity for the NF-KB consensusbinding site. Nuclear extracts were incubated in the absence (lane 1) orpresence of a 25-fold (lanes 2 and 4) or 50-fold (lanes 3 and 5) molarexcess of non-specific (NS, lanes 2 and 3) or specific (Self; lanes 4 and 5)unlabeled fragment prior to the addition of 1 ng of the radiolabeledfragment containing two copies of the NF-KB binding site. (B) EMSAswere performed with no antibodies Qane 1), non-specific rabbit antiserumGane 2), or rabbit antisera against p50 (lane 3) or p65 (lane 4). Antiserawere purchased from Santa Cruz Biotech, Inc. (Santa Cruz, CA). FP, freeprobe; NF-KB, DNA/NF-KB complexes; *, supershift complex observedwhen EMSAs were performed in the presence of anti-p50 antibodies.

gene expression. We have shown here that NF-KB is alsoinduced by peroxisome proliferators in the liver, suggestingthat this factor may be an important transcriptional regulator

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in response to peroxisome proliferator treatment. These resultsare consistent with the substantial evidence that reactive oxygenspecies are potent inducers of NF-KB activation (6).

An important component of peroxisome proliferator-medi-ated changes in gene expression are the peroxisome pro-liferator-activated receptors (PPARs), members of the nuclearhormone receptor family of transcriptional regulators. Thesefactors regulate genes through direct interactions with peroxi-some proliferator response elements (PPREs; 40). PPAR activ-ity is rapidly induced after treatment, leading to the rapidinduction of PPAR-regulated genes. As expected, many ofthese genes contain PPREs in their 5' regulatory region (41,42).If NF-KB was directly activated by peroxisome proliferatorsor PPARs, we would expect to see a substantial increase inNF-KB activity by day 3. However, the modest increase inNF-KB binding activity at day 3, with a continued increasebetween days 3 and 10 (Figure 3), suggests that NF-KBactivation is due to biochemical changes induced by peroxi-some proliferator-induced enzymes rather than a direct peroxi-some proliferator- or PPAR-mediated effect. We propose thatincreased levels of active oxygen are produced by peroxisomeproliferator-induced enzymes and lead to increased NF-KBactivity. The substantial increase in FAO in conjunction withthe modest increase in catalase activity may lead to higherH2O2 concentrations. If this notion is correct, increased levelsof catalase, the major peroxisomal H2O2-detoxifying enzyme,should reduce NF-KB induction. We have overexpressed cata-lase in the livers of transgenic mice (43) and are currentlyaddressing this issue using this animal system. Administrationof peroxisome proliferators also increases the activity of thecytochrome P-450 4A family. Active oxygen in the form ofsuperoxide or H2O2 could be released as a by-product fromcytochrome P-450 and thus could contribute to NF-KB activa-tion (44). Finally, the decrease of cellular antioxidants, suchas vitamins C and E, and antioxidant enzymes, such asglutathione peroxidase, by peroxisome proliferators may alsocontribute to increased levels of active oxygen in hepatocytes(8-10,12-14,16-18,45,46).

Alternatively, it is possible that the induction of NF-KB isrelated to some other biochemical changes. Several steps insignal transduction pathways are influenced by peroxisomeproliferators, including protein kinase C activity, free cytosoliccalcium concentrations and phosphorylation of the epidermalgrowth factor receptor (47—49). Whether these alterations canlead to increased activation of NF-KB is not known.

The consequences of increased NF-KB levels in hepatocytesare not known. NF-KB may contribute to changes in thetranscription of genes following peroxisome proliferator treat-ment. The cw-acting regulatory regions of several peroxisomeproliferator-induced genes have been cloned (50); analysis ofthese fragments, both in EMSAs and transient transfections,should elucidate whether these genes are regulated by NF-KB.Numerous other genes, including genes that are involved ingrowth control, are regulated by NF-KB (4). Several growth-and stress-related genes are also increased by peroxisomeproliferators, including c-myc, c-jun and cyclooxygenase-2(51-55). Whether the activation of these genes by peroxisomeproliferators is mediated by NF-KB is not known at present.

In addition to identifying the downstream targets of NF-KB,it will also be important to determine which cell types areproducing NF-KB in response to peroxisome proliferators.Although hepatocytes are the major cell population in theliver, other cell types are present, including Kupffer cells,

endothelial cells, Ito cells, bile duct cells and oval cells. NF-KBhas been shown to be present in both Kupffer cells andhepatocytes isolated from rat liver (56).

While the effects of NF-KB on lymphocytes are welldocumented, relatively little work has examined the role ofNF-KB in the liver. The induction of NF-KB after partialhepatectomy (33,34), during the 'acute phase' response (2)and by peroxisome proliferators (this paper) indicates thatNF-KB is an important stress-induced factor in the liver. Inaddition to the NF-KB induction in rat liver described here,we have also shown that peroxisome proliferators can induceNF-KB activity in cultured rat hepatoma H4ITEC3 cells treatedwith ciprofibrate (Y.Li, unpublished results). Interestingly,peroxisome proliferator insensitive human hepatoma HepG2cells do not show changes in NF-KB activity after ciprofibratetreatment. These cell culture systems should facilitate effortsto elucidate the basis of and subsequent consequences of N F -KB activation. These experiments are currently in progress.

Acknowledgements

The authors are grateful to Kelly Ke Li for advice about EMSAs and thankmembers of our laboratories for useful discussions. These studies weresupported by NIH grants ES-05815 and CA-01688, American Cancer SocietyInstitutional Grant 163 and by the Kentucky Agricultural Experiment Station(K.A.E.S. journal article no. 96-10-013).

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Received on March 14, 1996; revised on July 3, 1996; accepted on August14, 1996

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Treatment of rats with the peroxisome proliferator ciprofibrate results