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[Cancer Research 59, 4776-4780, October 1, 1999]
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[Cancer Research 59, 4776-4780, October 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Addition of Peroxisome Proliferator-activated Receptor {alpha} to Guinea Pig Hepatocytes Confers Increased Responsiveness to Peroxisome Proliferators

Neil Macdonald, Peter R. Holden and Ruth A. Roberts1

AstraZeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, United Kingdom


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
The fibrate drugs, such as nafenopin and fenofibrate, show efficacy in hyperlipidemias but cause peroxisome proliferation and liver tumors in rats and mice via nongenotoxic mechanisms. However, humans and guinea pigs appear refractory to these adverse effects. The peroxisome proliferator (PP)-activated receptor {alpha} (PPAR{alpha}) mediates the effects of PPs by heterodimerizing with the retinoid X receptor (RXR) to bind to DNA at PP response elements (PPREs) upstream of PP-regulated genes, such as acyl-CoA oxidase. Hepatic expression of PPAR{alpha} in guinea pigs and humans is low, suggesting that species differences in response to PPs may be due at least in part to quantity of PPAR{alpha}. To test this hypothesis, we introduced mouse PPAR{alpha} and its heterodimerization partner, RXR{alpha}, into guinea pig hepatocytes by transient transfection and determined responsiveness to the PP nafenopin by cyanide-insensitive palmitoyl-CoA oxidation (CIPCO). Expression of the mRNA for mouse PPAR{alpha} in transfected guinea pig hepatocytes was verified using species-specific PCR. In guinea pig hepatocytes transfected with control plasmids and treated with 50 µM nafenopin in the absence or presence of the RXR ligand, 9-cis-retinoic acid (5 µM) gave only a 1.7 ± 1.5- or 3.3 ± 1.5-fold induction in CIPCO, respectively. However, addition of ligands to hepatocytes co-transfected with both mPPAR{alpha} and RXR gave a strong induction of CIPCO (14.8 ± 8.6-fold). Mouse, human, and guinea pig PPAR{alpha} showed equivalent function in the CIPCO assays. Thus, quantity of PPAR{alpha} plays a significant role in the lack of response to PPs in guinea pigs. In humans, however, lack of PPAR{alpha} may be only one factor dictating lack of response because recent data show that the human acyl-CoA oxidase gene lacks a functional PP response element.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
PPs2 are a class of nongenotoxic rodent hepatocarcinogens that includes industrial plasticizers; fibrate hypolipidemic drugs, such as nafenopin; and certain chlorinated solvents (1, 2, 3, 4, 5) . In mice and rats, treatment with PPs results in hepatic peroxisome proliferation, increased hepatocyte DNA synthesis, suppression of hepatocyte apoptosis, liver enlargement, and hepatocarcinoma (reviewed in Refs. 3 , 6 , and 7 ). In addition, PPs up-regulate transcription of enzymes involved in the ß-oxidation of long-chain fatty acids, such as ACO, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and thiolase, as well as genes of the cytochrome P4504A family (8, 9, 10) . In contrast, species such as guinea pigs and humans are considered to be nonresponsive to the adverse effects of PPs associated with increased ß-oxidation and peroxisome proliferation (3 , 7 , 11 , 12) . In guinea pigs, there is no increased DNA synthesis or liver enlargement and only a small increase in peroxisome proliferation, and peroxisomal ß-oxidation enzyme activity is weak (12) even at very high PP concentrations (13) . Similarly, cultured human hepatocytes are refractory to the adverse effects of PPs (14) because the induction of peroxisomal ß-oxidation by PPs weak (15) or absent (15) , and PPs cannot induce DNA synthesis (reviewed in Ref. 11 ) or suppress apoptosis (16) . In addition, there appears to be no increased risk of liver cancer in patients receiving fibrate PP drug therapy (17) .

The PPAR{alpha} was originally cloned from mouse liver (18) and was shown to mediate the pleiotropic effects of PPs in rodents, such as enzyme induction, peroxisome proliferation, and hepatocarcinogenesis (8 , 9 , 19, 20, 21, 22) . PPAR{alpha} is activated by hypolipidemic drugs but also by natural ligands, such as fatty acids and eicosanoids (23 , 24) . Activated PPAR{alpha} binds to DNA as a heterodimer with the RXR at direct repeat 1 (DR1) elements (degenerate AGGTCA direct repeats spaced by 1 bp) that comprise two degenerate direct AGGTCA repeats spaced by 1 bp, termed PPREs (25 , 26) . PPREs have been identified in the promoter regions of a number of genes that are transcriptionally regulated by PPs (8 , 9 , 19 , 25 , 27, 28, 29, 30) . However, the presence of an active PPRE in the promoter region of a particular gene from one species does not necessarily infer the presence of an active PPRE in the equivalent gene in all species (31) . For example, the rat ACO promoter contains an active PPRE (9) , but the human ACO promoter displays sequence differences and lacks activity (32 , 33) .

Although human and guinea pig hepatocytes are refractory to the adverse effects of PPs, cDNAs encoding for a functional, full-length PPAR{alpha} have been isolated both from guinea pig (34 , 35) and human liver (10 , 36) . The hPPAR{alpha}, gpPPAR{alpha}, and mPPAR{alpha} display comparable activity in reporter gene assays using a minimal PPRE from the rat ACO promoter (34) . However, humans and guinea pigs show around 10-fold lower hepatic expression of PPAR{alpha} when compared with responsive species, such as rats and mice (Refs. 34 , 37 , and 38 ; reviewed in Ref. 39 ), and, at least in humans, the pool of active PPAR{alpha} may be depleted due to expression of alternatively spliced PPAR{alpha} mRNA lacking exon 6 that leads to a truncated, inactive PPAR{alpha} (30 , 38) . This suggests that the quantity of functional PPAR{alpha} may represent an important aspect of species differences in response. To test this, we used transient transfection to increase the level of PPAR{alpha} and RXR in guinea pig hepatocytes in vitro and determined responsiveness to the PP nafenopin, using CIPCO as an end point. CIPCO is an established and robust indicator of peroxisome proliferation. Expression of functional PPAR{alpha} was verified by species-specific PCR for mouse PPAR{alpha} and by reporter gene assay using a rat ACO PPRE promoter-luciferase reporter construct (9 , 32) . Key experiments were repeated with hPPAR{alpha} and gpPPAR{alpha} to determine whether the ability of PPAR{alpha} to confer increased responsiveness was dependent on the species origin of the receptor. The data presented suggest that species differences in quantity of PPAR{alpha} plays a role in the lack of response to the PP class of nongenotoxic rodent hepatocarcinogens.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Reagents.
Nafenopin was a gift from Ciba-Geigy (Basel, Switzerland). N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate and S-palmitoyl-CoA were from Roche Molecular Biochemicals. Trypsin was from Life Technologies, Inc. (Paisley, United Kingdom). 9-cis-Retinoic acid was purchased from Sigma. Galacto-Light Plus ß-galactocidase assay reagent was from Tropix Inc. Luciferin luciferase assay reagent was from Promega. All tissue culture plastics were from Nunc. All other materials were purchased from Flow, Life Technologies, Inc., or Sigma.

Plasmid Constructions.
The ß-galactocidase expression plasmid pCMV.LacZ was obtained from Clontech (Basingstoke, United Kingdom). pcDNA3 was from R+D Systems (Oxon, United Kingdom). hPPAR{alpha} cDNA was a gift from Dr. F. Gonzalez (National Cancer Institute, Bethesda, MD), and mRXR{alpha} cDNA was a gift from Prof. Pierre Chambon. The plasmids pCMV.mPPAR{alpha}, pAco(-581/-471).G.Luc (30) , pCMV.gpPPAR{alpha}, pCMV.hPPAR{alpha} (34) , and pCMV.mRXR{alpha} (40) have been described previously.

Culture and Transient Transfection of Primary Hepatocytes.
Hepatocytes were isolated from male Dunkin-Hartley guinea pigs by collagenase perfusion as described previously (41) . Viability was determined by trypan blue exclusion. All cell preparations had a viability of 70–95% on isolation. Freshly isolated hepatocytes were diluted to 2 x 106 cells/ml in FCS-free L-15 medium supplemented with 10% tryptose phosphate broth, 10 µg/ml insulin, 1 mM hydrocortisone, 2 mM L-glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, and 340 µM vitamin C. Hepatocytes (2 x 106) were transfected in suspension using 12 µg of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate and a plasmid cocktail containing 6.0 µg of pCMV.PPAR{alpha} or pcDNA3, 6.0 µg of pCMV.mRXR{alpha} or pcDNA3, 1.0 µg of pCMV.LacZ, and 1.0 µg of pAco(-581/-471)G.Luc. After transfection, hepatocytes were placed in 25-cm2 flasks containing 3 ml of L-15 medium supplemented as described above plus 10% fetal bovine serum. Untransfected control hepatocytes were placed in culture immediately after dilution. Cultures were maintained at 37°C in a humidified atmosphere, and the medium was changed after 5 h. After 24 h, fresh medium was added containing either DMF, 5 µM 9-cis-retinoic acid, 50 µM nafenopin, or 5 µM 9-cis-retinoic acid/50 µM nafenopin. Cells were cultured in the presence of ligands for a further 72 h, and the medium was changed every 24 h. In all of these experiments, the transfection efficiency for guinea pig hepatocytes, as estimated by 5-bromo-4-chloro-3-indolyl-{alpha}-D-galactopyranoside staining for ß-galactosidase activity (see Ref. 42 ), was between 7% and 12% (data not shown).

Species-specific PCR Detection of Transfected mPPAR{alpha}.
Total RNA was isolated from transfected and untransfected guinea pig hepatocytes 24 h after transfection using Total RNA Isolation Reagent (Advanced Biotechnologies, Epsom, Surrey, United Kingdom). To eliminate transfected plasmid DNA from the RNA samples, poly(A)+ RNA was isolated from the total RNA samples using Miltenyi Biotec mRNA isolation kit following the manufacturer’s instructions. Fifty ng of each poly(A)+ RNA sample, as well as 50 ng of mouse hepatocyte total RNA, were reverse-transcribed using the Amersham Pharmacia Biotech First Strand cDNA synthesis kit (33-µl reaction) following the manufacturer’s instructions. Three µl of each cDNA synthesis reaction, 10 ng of poly(A)+ RNA that had not undergone reverse transcription, and a RNA free reverse transcription reaction mixture were used as PCR templates with primers that were species specific for mPPAR{alpha} [sense, 5'-CGCCAGCACGGACGAGT-3' (bases 261–277 of the mPPAR{alpha} coding region); antisense, 5'-AAAAGGCGGGTTGTTGC-3' (bases 701–717 of the mPPAR{alpha} coding region)]. PCR conditions were as follows: 50 pmol of each primer, 200 µM dNTPs, 3 mM MgCl2, 1 unit of Taq (Promega); reaction volume, 50 µl. Cycles were as follows: 94°C for 1 min; 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min; and 72°C for 3 min. Ten µl of each PCR reaction were run on a 2% agarose gel containing ethidium bromide. Gels were photographed under UV illumination using the Imager (Appligene) gel documentation system.

Reporter Gene and CIPCO Assays.
Medium was discarded and the monolayers washed once with 2 ml of PBS. Cells were scraped into 1 ml of ice-cold PBS that was subsequently split into a 200-µl and an 800-µl aliquot prior to pelleting cells by centrifugation at 2000 rpm for 2 min. The pellet from the 800-µl aliquot was resuspended in N-tris(hydroxy-methyl)methyl-2-aminoethanesulfonic acid, disrupted by sonication, and stored at -70°C for CIPCO and protein assays. The pellet from the 200-µl aliquot was resuspended in 100 µl of lysis buffer (25 mM Tris·phosphate, 2 mM DTT, 5 mM 1,2-diaminocyclohexane N,N,N',N''-tetraacetic acid, 5% glycerol, 0.01% Triton X-100 in dH2O) and stored at -70°C for ß-galactocidase and luciferase assays. CIPCO assays were carried out as described previously (43) with some modifications. The assay medium contained 60 mM Tris-HCl, pH 8.3, 50 µM CoA, 370 µM NAD+, 94 mM nicotinamide, 2.8 mM DTT, 2 mM KCN, 12.5 µg/ml BSA (fatty acid free), 100 µg/ml flavin adenosine dinucleotide, 50 µg/ml palmitoyl-CoA. Protein concentration in the CIPCO assay aliquot of each sample was assessed using the Bradford protein assay reagent (Bio-Rad) following the manufacturer’s instructions. ß-Galactosidase and luciferase activity were assayed as described previously (40) . Briefly, ß-galactosidase activity was determined by incubating 10 µl of cell extract with Galacto-Light Plus reagent (Tropix Inc.) according to the manufacturer’s instructions. Luciferase activity was determined by incubating a 40-µl aliquot of cell extract with luciferin reagent (Promega) according to the manufacturers instructions. Luciferase activity for each plate was determined and normalized for ß-galactosidase activity. CIPCO activity was determined per plate and expressed as nmol of NAD+ reduced/min/mg of protein, normalized for ß-galactosidase activity. Data points are the mean of three determinations.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
The PP Nafenopin Induces ß-Oxidation of Long-Chain Fatty Acids in Rat but not in Guinea Pig Primary Hepatocytes.
Fig. 1Citation shows that the PP nafenopin (50 µM) gave a strong induction of ß-oxidation of long-chain fatty acids in rat but not in guinea pig hepatocytes. In the rat, a 20-fold induction was seen as determined by CIPCO, a convenient and robust marker of peroxisome proliferation and associated ß-oxidation. In contrast, in hepatocytes from the guinea pig, there was only a small induction of CIPCO activity (2.6 ± 0.6-fold). This is consistent with previous reports, in which PPs cause a marked induction of CIPCO in rat liver in vivo but only a small induction in guinea pig liver at equivalent plasmatic levels of the hypolipidemic PP ciprofibrate (44) . These data confirm species differences in CIPCO reported in vivo and confirm our in vitro system as a useful model for testing the hypothesis that addition of PPAR{alpha} can confer increased responsiveness to PPs.



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  		Fig. 1. Induction of ß-oxidation by 50 µM nafenopin in rat but not in guinea pig hepatocytes. Long-chain fatty acid ß-oxidation is measured by CIPCO activity per mg of hepatocyte protein. Data are expressed as fold induction over solvent (DMF) control. Columns, mean of triplicate results; bars, SD.

 
Mouse PPAR{alpha} mRNA Is Expressed and Can Activate a PPRE Reporter Plasmid Co-transfected into Guinea Pig Hepatocytes.
Constitutively, guinea pig hepatocytes express only a fraction of the PPAR{alpha} seen in the mouse or rat (34) , suggesting that low levels of PPAR{alpha} may explain their lack of response to the adverse effects of PPs, such as peroxisome proliferation and ß-oxidation. We wished to test this hypothesis by introducing a mouse PPAR{alpha} expression vector into guinea pig hepatocytes and evaluating the effects on the expression of endogenous ß-oxidation genes. First, we established that mouse PPAR{alpha} mRNA was being expressed after transfection of the mouse PPAR{alpha} expression into guinea pigs hepatocytes (Fig. 2)Citation . Species-specific PCR for mPPAR{alpha} detected a strong band at the expected size (456 bp) in cDNA from mouse hepatocytes and from mPPAR{alpha}-transfected guinea pig hepatocytes but not in untransfected guinea pig hepatocytes.



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  		Fig. 2. Expression of mouse PPAR{alpha} mRNA in guinea pig hepatocytes after transfection. Mouse PPAR{alpha}-specific PCR of reverse-transcribed poly(A)+ RNA from transfected (T; lane 2) and untransfected (UT; lane 1) guinea pig hepatocytes. - (lane 3) depicts the control (no RNA reverse transcription), and con (lane 4) depicts the same poly(A)+ RNA as in lane 2 (T) but with the reverse transcription step omitted. Mu (lane 5) depicts the positive control (reverse-transcribed mouse total RNA).

 
Next, we wished to determine that the vector we were introducing was expressing sufficient PPAR{alpha} to give increased PP-dependent PPRE-mediated gene expression using the rat ACO minimal PPRE reporter construct. Fig. 3aCitation shows that the PPAR{alpha} ligand nafenopin was unable to activate transcription in control transfections but caused a 6-fold increase in reporter gene activity after addition of mPPAR{alpha}.



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  		Fig. 3. Addition of mPPAR{alpha} to guinea pig hepatocytes activates transcription of the rat acyl-CoA-luciferase reporter plasmid and confers increased CIPCO activity. a, ligand-dependent transcriptional activation of the rat ACO reporter plasmid by mPPAR{alpha}. prACO (-581/-471).G.Luc reporter plasmid (1.0 µg) was transfected into 2 x 106 guinea pig hepatocytes together with pcDNA3.LacZ (1.0 µg) as a transfection control in either the presence or absence of receptor expression vector (pCMV.mPPAR{alpha}; 6 µg) and 50 µM nafenopin (naf). Data are shown as fold induction over DMF/pcDNA3 control of luciferase, corrected for transfection efficiency. b, ligand-dependent increase in CIPCO activity by addition of mPPAR{alpha} in the presence of mRXR{alpha}. pCMV.PPAR{alpha} (6.0 µg) and/or pCMV.mRXR{alpha} (6.0 µg) was transfected into 2 x 106 guinea pig hepatocytes together with pcDNA3.LacZ (1.0 µg) in either the presence or absence of 50 µM nafenopin (naf) and/or 9-cis-retinoic acid (5 µM). Data are shown as fold induction over DMF/pcDNA3 control (column 1) of CIPCO, corrected for transfection efficiency. Columns, mean of triplicate results; bars, SD.

 
Introduction of mPPAR{alpha}/RXR into Guinea Pig Hepatocytes Confers Increased PP Responsiveness in Assays of ß-Oxidation.
Having established introduction of sufficient PPAR{alpha} to activate reporter gene expression, we examined the induction of ß-oxidation by nafenopin in guinea pig hepatocytes with and without transfected mPPAR{alpha} and its heterodimerization partner, RXR (Fig. 3b)Citation . The ß-oxidation of long-chain fatty acids was determined by CIPCO activity. In the absence of both receptor expression plasmids, the addition of nafenopin (column 5), 9-cis-retinoic acid (column 2), or both ligands (column 6) had only a weak effect on CIPCO. When RXR was transfected alone, either with or without 9-cis-retinoic acid, again there was no effect or only a weak effect on CIPCO upon addition of nafenopin (columns 8 and 7, respectively). When PPAR{alpha} was transfected alone, there was some induction by nafenopin both with (column 14) and without (column 13) 9-cis-retinoic acid, although there was no induction of CIPCO on addition of 9-cis-retinoic acid in the absence of nafenopin (column 10), suggesting the presence of a small amount of 9-cis-retinoic acid but very little natural PPAR{alpha} ligand (24) . When RXR was co-transfected with PPAR{alpha}, the level of CIPCO was quite high, even in the absence of nafenopin (column 12), but there was little stimulation of CIPCO on addition of PPAR{alpha} ligand alone (column 14). However, in the presence of both receptors and both ligands (column 16), there was a large induction of CIPCO activity. Thus, the addition of mPPAR{alpha} to guinea pig hepatocytes in the presence of appropriate co-factors confers on guinea pig hepatocytes the ability to respond to PPs, as monitored by endogenous enzyme activity. This pattern of ligand dependent PPAR{alpha} and RXR effects is comparable to that seen in rACO.PPRE reporter gene assays using cell lines (26) .

The nafenopin-dependent CIPCO induction seen in transfected guinea pig hepatocytes was marked under optimal conditions but was not as consistent nor as strong as that seen in untransfected rat hepatocytes (Fig. 1)Citation . This may be because the guinea pig hepatocytes can mount only a partial response to PPs even in the presence of sufficient receptor. However, it seems more likely that the lower level of CIPCO in transfected guinea pig hepatocytes reflects the low transfection efficiency seen in primary hepatocytes.

PPAR{alpha} from Nonresponsive Species also Increase PP Responsiveness of Guinea Pig Hepatocytes.
Having shown that increasing the quantity of PPAR{alpha} expressed in guinea pig hepatocytes altered increased their PP responsiveness, we next determined whether there were qualitative differences between PPAR{alpha} cloned from PP nonresponsive and responsive species played in response to PPs. First, we compared the ability of mPPAR{alpha}, gpPPAR{alpha}, and hPPAR{alpha} to increase ligand-dependent induction of a PPRE reporter plasmid (Fig. 4a)Citation under the optimal conditions (co-addition of RXR and 9-cis-retinoic acid) defined previously. In the absence of any PPAR{alpha} expression plasmid, the addition of nafenopin had no effect. However, as seen previously, the addition of nafenopin in the presence of mPPAR{alpha} gave a 3-fold induction of reporter gene activity. Similarly, transfection of PPAR{alpha} from either guinea pig or human into guinea pig hepatocytes also was able to increase nafenopin-dependent activation of a co-transfected reporter plasmid. Thus, PPAR{alpha} was functional in the activation of a reporter gene, irrespective of the species from which it was cloned. Next, we compared the ability of mPPAR{alpha}, gpPPAR{alpha}, and hPPAR{alpha}, co-transfected with RXR{alpha}, to increase nafenopin-dependent induction of ß-oxidation in guinea pig hepatocytes over that seen in control transfected hepatocytes. Again, all three PPAR{alpha}s gave similar increases in ligand-dependent ß-oxidation induction in guinea pig hepatocytes (Fig. 4b)Citation .



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  		Fig. 4. Addition of mPPAR{alpha}, hPPAR{alpha}, or gpPPAR{alpha} to guinea pig hepatocytes activates transcription of the rat acyl-CoA-luciferase reporter plasmid and confers increased CIPCO activity. a, ligand-dependent transcriptional activation of the rat ACO reporter plasmid by mPPAR{alpha}, hPPAR{alpha}, or gpPPAR{alpha} under conditions found to be optimal for mPPAR{alpha} activity (see Fig. 3Citation ). The mPPAR{alpha}, hPPAR{alpha}, or gpPPAR{alpha} receptor expression vectors (6.0 µg) were transfected into 2 x 106 guinea pig hepatocytes together with prACO(-581/-471).G.Luc reporter plasmid (1.0 µg), pcDNA3.LacZ (1.0 µg), and pCMV.mRXR{alpha} (6 µg) in the presence of 9-cis-retinoic acid (5 µM) and in the presence or absence of 50 µM nafenopin. Data are shown as fold induction over DMF/pcDNA3 control of luciferase, corrected for transfection efficiency. b, ligand-dependent increase in CIPCO activity caused by addition of mPPAR{alpha}, gpPPAR{alpha}, or hPPAR{alpha} in the presence of mRXR{alpha}. pCMV.PPAR{alpha} (6.0 µg) and/or pCMV.mRXR{alpha} (6.0 µg) was transfected into 2 x 106 guinea pig hepatocytes together with pcDNA3.LacZ (1.0 µg) in the presence of 9-cis-retinoic acid (5 µM) and in the presence or absence of 50 µM nafenopin (naf). Data are shown as fold induction over DMF/pcDNA3 control (column 1) of CIPCO, corrected for transfection efficiency. Columns, mean of triplicate results; bars, SD.

 
It has been suggested that the lack of an adverse response to PPs in hepatocytes from nonresponsive species may be due to insufficient levels of PPAR{alpha}. However, some human genes clearly can respond to PPs because the activation of PPAR{alpha} by the hypolipidemic fibrate PPs forms the basis of their clinical use (29 , 45 , 46) . Thus, human and guinea pig hepatocytes may express sufficient PPAR{alpha} to activate the genes associated with hypolipidemia but insufficient PPAR{alpha} to activate the full response seen in rat and mice, such as peroxisome proliferation, ß-oxidation, growth perturbation, and cancer. Here, we have shown that transient transfection of additional PPAR{alpha} along with its dimerization partner RXR{alpha} into guinea pig hepatocytes is sufficient to increase PP responsiveness as assayed by CIPCO activity. In addition, mPPAR{alpha}, gpPPAR{alpha}, and hPPAR{alpha} were equally able to activate genes controlled by PPREs, either in the context of a simple reporter plasmid or within a more complex native response element. These data provide evidence that, at least in guinea pigs, quantity of PPAR{alpha} plays a significant role in the reduced ß-oxidation response to PPs. Although quantity of PPAR{alpha} may also contribute to the lack of response in humans, the PPRE in the promoter region of the ACO gene is inactive in humans (32 , 33) . Because this is the first and rate-limiting enzyme in the ß-oxidation pathway, human hepatocytes would continue to be refractory to PP induction of ß-oxidation even after overexpression of PPAR{alpha}.

As well as variations in levels of PPAR{alpha} and the structure of PPREs, there may be additional factors that contribute to species differences in response to PPs. The promoter regions of PP-responsive genes can contain negative regulatory elements that are indirectly switched off by ligand dependent PPAR{alpha} activation (31) . Also, other nuclear receptors, such as HNF-4{alpha} and COUP-TF, are able to bind to the rat ACO PPRE and suppress PP-dependent gene activation (47) . Specifically, HNF-4{alpha} shares the ability of PPAR{alpha} to bind to the rat ACO PPRE (47) but gives only a fraction of the reporter gene expression. In addition, both COUP-TF and HNF-4{alpha} show saturable dose-dependent suppression of activation of PPRE reporter plasmid by PPAR{alpha} in the presence of PPs (47) . Thus, species differences both in the hypolidemic and in the adverse effects of PPs may be dictated in part by levels of PPAR{alpha}, but also by diversity in the PPREs of specific genes, and by expression of other direct repeat 1 (DR1) binding nuclear receptors, such as HNF-4{alpha} and COUP-TF. Suppression by negative regulatory elements together with competition with other nuclear receptors for PPRE binding may constitute the molecular basis of a threshold for PPAR{alpha} activation at PPREs.

In summary, the data presented here suggest that species differences in quantity of PPAR{alpha} plays a role in the lack of an adverse response in guinea pigs to the PP class of nongenotoxic rodent hepatocarcinogens. In humans, lower expression of PPAR{alpha} provides one explanation for a lack of response, in addition to the lack of activity of PPREs upstream of key genes, such as ACO (32 , 33) .


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Cancer Biology Group, AstraZeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, United Kingdom. Phone: 1625-516413; Fax: 1625-582897; E-mail: ruth.roberts{at}CTL.astrazeneca.com Back

2 The abbreviations used are: PP, peroxisome proliferator; ACO, acyl-CoA oxidase; PPAR{alpha}, peroxisome proliferator-activated receptor {alpha}; RXR, retinoid X receptor; PPRE, PPAR{alpha} response element; HNF-4{alpha}, hepatocyte nuclear factor 4{alpha}; COUP-TF, chicken ovalbumin upstream promotor-transcription factor; CIPCO, cyanide-insensitive palmitoyl CoA oxidase; DMF, dimethyl formamide; hPPAR{alpha}, human PPAR{alpha}; mPPAR{alpha}, mouse PPAR{alpha}; gpPPAR{alpha}, guinea pig PPAR{alpha}; CMV, cytomegalovirus. Back

Received 3/29/99. Accepted 8/17/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

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