WY-14,643 Research Articles

Evidence for the involvement of xenobiotic-responsive nuclear receptors in transcriptional effects upon perfluoroalkyl acid exposure in diverse species

Humans and ecological species have been found to have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotypic effects similar to peroxisome proliferator chemicals (PPC) that work through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα). Recent studies indicate that along with PPARα other nuclear receptors are required for transcriptional changes in the mouse liver after PFOA exposure including the constitutive activated receptor (CAR) and pregnane X receptor (PXR) that regulate xenobiotic metabolizing enzymes (XME). To determine the potential role of CAR/PXR in mediating effects of PFAAs in rat liver, we performed a meta-analysis of transcript profiles from published studies in which rats were exposed to PFOA or PFOS. We compared the profiles to those produced by exposure to prototypical activators of CAR, (phenobarbital (PB)), PXR (pregnenolone 16 alpha-carbonitrile (PCN)), or PPARα (WY-14,643 (WY)). As expected, PFOA and PFOS elicited transcript profile signatures that included many known PPARα target genes. Numerous XME genes were also altered by PFOA and PFOS but not WY. These genes exhibited expression changes shared with PB or PCN. Reexamination of the transcript profiles from the livers of chicken or fish exposed to PFAAs indicated that PPARα, CAR, and PXR orthologs were not activated. Our results indicate that PFAAs under these experimental conditions activate PPARα, CAR, and PXR in rats but not chicken and fish. Lastly, we discuss evidence that human populations with greater CAR expression have lower body burdens of PFAAs.

Effects of peroxisome proliferator-activated receptor α (PPARα) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells

Effect of peroxisome proliferator-activated receptor α (PPARα) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.

Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for the Involvement of Nuclear Receptors PPARα and CAR

A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha). To determine the role of PPAR alpha in mediating PFOA transcriptional events, we compared the transcript profiles of the livers of wild-type or PPAR alpha-null mice exposed to PFOA or the PPAR alpha agonist WY-14,643 (WY). After 7 days of exposure, 85% or 99.7% of the genes altered by PFOA or WY exposure, respectively were dependent on PPAR alpha. The PPAR alpha-independent genes regulated by PFOA included those involved in lipid homeostasis and xenobiotic metabolism. Many of the lipid homeostasis genes including acyl-CoA oxidase (Acox1) were also regulated by WY in a PPAR alpha-dependent manner. The increased expression of these genes in PPAR alpha-null mice may be partly due to increases in PPAR gamma expression upon PFOA exposure. Many of the identified xenobiotic metabolism genes are known to be under control of the nuclear receptor CAR (constitutive activated/androstane receptor) and the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). There was excellent correlation between the transcript profile of PPAR alpha-independent PFOA genes and those of activators of CAR including phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) but not those regulated by the Nrf2 activator, dithiol-3-thione. These results indicate that PFOA alters most genes in wild-type mouse liver through PPAR alpha, but that a subset of genes are regulated by CAR and possibly PPAR gamma in the PPAR alpha-null mouse.

Induction of UGT1A genes by the fibrate WY‐14643 in Tg‐UGT1 mice and identification of peroxisome proliferator activated receptor (PPAR) responsive elements on the UGT1A1 and UGT1A4 promoters

The UGT1 enzymes are an essential part of drug metabolism and detoxification. A transgenic mouse model expressing all nine functional UGT1A genes was created (Tg-UGT1) and we have shown that the enzymes are induced by many of the nuclear receptors. The PPARs are activated by hypolipidemic fibrate drugs. The fibrate drug WY-14643 (WY) was administered orally to Tg-UGT1 mice and induced UGT1A1, 1A3, 1A4, 1A6, and 1A9. A DR1 element was identified in the UGT1A1 enhancer: serial deletion of the enhancer and mutation of the DR1 element identified it as essential for WY induction of UGT1A1. Induction of UGT1A4 protein was observed extrahepatically following WY treatment. The 5kb UGT1A4 enhancer was cloned into a reporter vector. Serial deletion and transfection in LS180 cells identified a region of the promoter responsive to WY. A concatenated DR1 element within this region was induced by WY. These results show that UGT1A genes are induced following PPARα activation by fibrates via DR1 elements on the 3′ enhancers. Induction of these genes may impact the metabolism of these and other drugs and affect drug-drug interactions in patients on fibrate therapy. (Supported by USPHS Grants ES10337, GM49135)

Activation of Peroxisome Proliferator–Activated Receptor Alpha Enhances Apoptosis in the Mouse Liver

Chronic exposure to peroxisome proliferators (PPs) leads to increased incidence of liver tumors in rodents. Liver tumor induction is thought to require increased hepatocyte proliferation and suppression of apoptosis. Transcript profiling showed increased expression of proapoptotic genes and decreased expression of antiapoptotic genes in the livers of mice exposed to the PP WY-14,643 (WY). We tested the hypothesis that prior exposure to WY would increase susceptibility to apoptosis inducers such as Jo2, an antibody which activates the Fas (Apo-1/CD95) death pathway. When compared to their untreated counterparts, wild-type mice pretreated with WY exhibited increased caspase-3 activation and hepatocyte apoptosis following challenge with Jo2. Livers from WY-treated peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice were resistant to the effects of Jo2. In the absence of Jo2 and detectable apoptosis, wild-type mice treated with WY exhibited increases in the activated form of caspase-9. As caspase-9 is a component of the apoptosome, we examined the expression of upstream effectors of apoptosome activity including members of the Bcl-2 family. The levels of the antiapoptotic Mcl-1 transcript and protein were significantly decreased by PPs. PPARalpha-null mice were also resistant to another treatment (concanavalin A) that induces hepatocyte apoptosis. These results (1) indicate that PPARalpha activation increases sensitivity of the liver to apoptosis and (2) identify a mechanism by which PPARalpha could serve as a pharmacological target in diseases where apoptosis is a contributing feature.

Inhibition of Hepatocarcinogenesis by the Deletion of the p50 Subunit of NF-κB in Mice Administered the Peroxisome Proliferator Wy-14,643

Wy-14,643 (WY) is a hypolipidemic drug that induces hepatic peroxisome proliferation and tumors in rodents. We previously showed that peroxisome proliferators increase NF-kappaB DNA binding activity in rats, mice, and hepatoma cell lines, and that mice deficient in the p50 subunit of NF-kappaB had much lower cell proliferation in response to the peroxisome proliferator ciprofibrate. In this study we examined the promotion of hepatocarcinogenesis by WY in the p50 knockout (-/-) mice. The p50 -/- and wild type mice were first administered diethylnitrosamine (DEN) as an initiating agent. Mice were then fed a control diet or a diet containing 0.05% WY for 38 weeks. Wild-type mice receiving DEN only developed a low incidence of tumors, and the majority of wild-type mice receiving both DEN and WY developed tumors. However, no tumors were seen in any of the p50 -/- mice. Cell proliferation and apoptosis were measured in hepatocytes by BrdU labeling and the TUNEL assay, respectively. Treatment with DEN + WY increased both cell proliferation and apoptosis in both the wild-type and p50 -/- mice; DEN treatment alone has no effect. In the DEN/WY-treated mice, cell proliferation and apoptosis were slightly lower in the p50 -/- mice than in the wild-type mice. These data demonstrate that NF-kappaB is involved in the promotion of hepatic tumors by the peroxisome proliferator WY; however, the difference in tumor incidence could not be attributed to alterations in either cell proliferation or apoptosis.

Toxic Effects of Fumonisin in Mouse Liver Are Independent of the Peroxisome Proliferator-Activated Receptor α

Fumonisin mycotoxins occur worldwide in corn and corn-based foods. Fumonisin B1 (FB1) is a rodent liver carcinogen and suspected human carcinogen. It inhibits ceramide synthase and increases tissue sphinganine (Sa) and sphingosine (So) concentrations. Events linking disruption of sphingolipid metabolism and fumonisin toxicity are not fully understood; however, Sa and So were shown to bind mouse recombinant peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the role of PPARalpha in fumonisin hepatotoxicity in vivo, wild-type (WT) and PPARalpha-null mice were fed control diets or diets containing 300 ppm FB1, Fusarium verticillioides culture material (CM) providing 300 ppm FB1, or 500 ppm of the peroxisome proliferator WY-14,643 (WY) for 1 week. WY-fed WT mice exhibited hepatomegaly, an effect not found in WY-fed PPARalpha-null mice, and WY did not change liver sphingoid base concentrations in either strain. Hepatotoxicity found in FB1- and CM-fed WT and PPARalpha-null mice was similar, qualitatively different from that found in WY-treated animals, and characterized by increased Sa concentration, apoptosis, and cell proliferation. Transcript profiling using oligonucleotide arrays showed that CM and FB1 elicited similar expression patterns of genes involved in cell proliferation, signal transduction, and glutathione metabolism that were different from that altered by WY. Real-time RT-PCR analysis of gene expression demonstrated PPARalpha-dependence of lipid metabolism gene expression in WY-treated mice, whereas PPARalpha-independent alterations of genes in lipid metabolism, and other categories, were found in CM- and FB1-fed mice. Together, these findings demonstrate that FB1- and CM-induced hepatotoxicity in mice does not require PPARalpha.

The Transcriptional Response to a Peroxisome Proliferator-activated Receptor α Agonist Includes Increased Expression of Proteome Maintenance Genes

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha), in addition to regulating lipid homeostasis, controls the level of tissue damage after chemical or physical stress. To determine the role of PPARalpha in oxidative stress responses, we examined damage after exposure to chemicals that increase oxidative stress in wild-type or PPARalpha-null mice. Primary hepatocytes from wild-type but not PPARalpha-null mice pretreated with the PPAR pan-agonist WY-14,643 (WY) were protected from damage to cadmium and paraquat. The livers from intact wild-type but not PPARalpha-null mice were more resistant to damage after carbon tetrachloride treatment. To determine the molecular basis of the protection by PPARalpha, we identified by transcript profiling genes whose expression was altered by a 7-day exposure to WY in wild-type and PPARalpha-null mice. Of the 815 genes regulated by WY in wild-type mice (p < or = 0.001; > or =1.5-fold or < or =-1.5-fold), only two genes were regulated similarly by WY in PPARalpha-null mice. WY increased expression of stress modifier genes that maintain the health of the proteome, including those that prevent protein aggregation (heat stress-inducible chaperones) and eliminate damaged proteins (proteasome components). Although the induction of proteasomal genes significantly overlapped with those regulated by 1,2-dithiole-3-thione, an activator of oxidant-inducible Nrf2, WY increased expression of proteasomal genes independently of Nrf2. Thus, PPARalpha controls the vast majority of gene expression changes after exposure to WY in the mouse liver and protects the liver from oxidant-induced damage, possibly through regulation of a distinct set of proteome maintenance genes.

PPARα ligands activate antioxidant enzymes and suppress hepatic fibrosis in rats

Oxidative stress is a major pathogenetic factor in hepatic fibrosis. Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor which is known to affect oxidative stress and PPARα ligands may have rescue effects on hepatic fibrosis. We tested this hypothesis using rat thioacetamide (TAA) models of liver cirrhosis. Rats were given intraperitoneal injection of TAA and treated with a diet containing one of the two PPARα ligands, Wy-14,643 (WY) or fenofibrate. WY treatment dramatically reduced hepatic fibrosis and also prevented the inhibition catalase of mRNA expression caused by TAA. Correspondingly, catalase activity increased in the TAA+WY group but decreased in the control TAA group. The antifibrotic action of fenofibrate in the TAA model was comparable with that of WY. PPARα ligands have an antifibrotic action in the rat TAA model of liver cirrhosis, probably due to an antioxidant effect of enhanced catalase expression and activity in the liver.

PPAR alpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.

Previous studies demonstrated that during cisplatin-induced acute renal failure, there is a significant reduction in proximal tubule fatty acid oxidation. We now report on the effects of peroxisome proliferator-activated receptor-alpha (PPAR alpha) ligand Wy-14643 (WY) on the abnormalities of medium chain fatty acid oxidation and pyruvate dehydrogenase complex (PDC) activity in kidney tissue of cisplatin-treated mice. Cisplatin causes a significant reduction in mRNA levels and enzyme activity of mitochondrial medium chain acyl-CoA dehydrogenase (MCAD). PPAR alpha ligand WY ameliorated cisplatin-induced acute renal failure and prevented cisplatin-induced reduction of mRNA levels and enzyme activity of MCAD. In contrast, in cisplatin-treated PPAR alpha null mice, WY did not protect kidney function and did not reverse cisplatin-induced decreased expression of MCAD. Cisplatin inhibited renal PDC activity before the development of acute tubular necrosis, and PDC inhibition was reversed by pretreatment with PPAR alpha agonist WY. Cisplatin also induced increased mRNA and protein levels of pyruvate dehydrogenase kinase-4 (PDK4), and PPAR alpha ligand WY prevented cisplatin-induced increased expression of PDK4 protein levels in wild-type mice. We conclude that PPAR alpha agonists have therapeutic potential for cisplatin-induced acute renal failure. Use of PPAR alpha ligands prevents acute tubular necrosis by ameliorating cisplatin-induced inhibition of two distinct metabolic processes, MCAD-mediated fatty acid oxidation and PDC activity.

Studies on the induction of rat hepatic CYP1A, CYP2B, CYP3A and CYP4A subfamily form mRNAs in vivo and in vitro using precision-cut rat liver slices

1. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan®) was used to examine the induction of some selected rat hepatic cyto-chrome P450 (CYP) forms in vivo and in vitro using cultured precision-cut liver slices. 2. TaqMan primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1, CYP2B1/2, CYP3A1, CYP3A2 and CYP4A1 mRNAs. 3. To characterize the responsiveness of the rat CYP mRNA TaqMan primers and probe sets, rats were treated in vivo with a single intraperitoneal dose of 500 mg kg − 1 Aroclor 1254 (ARO) and with four daily oral doses of either 50 mg kg − 1 day − 1 dexamethasone (DEX) or 75 mg kg − 1 day − 1 methylclofenapate (MCP). Treatment with ARO produced 22 600-, 5480-, 648-, 52-, 47- and 9-fold increases in levels of CYP1A1, CYP2B1, CYP2B1/2, CYP1A2, CYP3A1 and CYP3A2 mRNA, respectively. DEX treatment produced 97-, 24-, 8- and 4-fold increases, respectively, in CYP3A1, CYP2B1, CYP2B1/2 and CYP3A2 mRNA levels, and MCP produced 339-, 126- and 25-fold increases, respectively, in CYP4A1, CYP2B1 and CYP2B1/2 mRNA levels. All three CYP inducers also increased microsomal CYP content and produced corresponding increases in CYP1A, CYP2B, CYP3A and CYP4A form marker enzyme activities. 4. Rat liver slices were cultured for 6 and 24 h in medium containing 0.1 µ M insulin and 0.1 µ M DEX, and also for 24 h in medium containing only 0.1 µ M insulin (DEX-free medium). Liver slices were cultured in control medium or in medium containing either 10 µ M β -naphthoflavone (BNF), 10 µ g ml − 1 ARO, 500 µ M sodium phenobarbitone (NaPB), 20 µ M pregnenolone-16 α -carbonitrile (PCN), 50 µ M Wy-14,643 (WY) or 50 µ M MCP. 5. With the exception of the effect of BNF on CYP1A1 mRNA levels, the induction of all the CYP mRNAs studied was greater after 24- than after 6-h treatment. Generally, the magnitude of induction of CYP mRNA levels was greater after 24 h in liver slices cultured in DEX-free than in DEX-supplemented medium. 6. Treatment of liver slices with BNF and ARO for 24 h in DEX-free medium produced 21- and 35-fold increases, respectively, and 38- and 37-fold increases, respectively, in CYP1A1 and CYP1A2 mRNA levels. NaPB, PCN, WY and MCP did not increase either CYP1A1 or CYP1A2 mRNA levels. 7. After 24 h, levels of CYP2B1/2 mRNA were increased 18-, 20-, 9-, 16- and 13-fold by treatment with ARO, NaPB, PCN, WY and MCP, respectively. PCN also produced 56- and 4-fold increases, respectively, in CYP3A1 and CYP3A2 mRNA levels. 8. Treatment with WY and MCP for 24 h produced 437- and 186-fold increases, respectively, in levels of CYP4A1 mRNA. None of the other CYP inducers studied had any effect on CYP4A1 mRNA levels. 9. The results demonstrate the utility of cultured precision-cut liver slices as an in vitro model system to evaluate the effects of xenobiotics on rat CYP1A, CYP2B, CYP3A and CYP4A form mRNA levels.

Use of a quantitative real-time reverse transcription-polymerase chain reaction method to study the induction of CYP1A, CYP2B and CYP4A forms in precision-cut rat liver slices

1. The aim was to employ real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) technology (TaqMan ®) to examine the induction of some selected cytochrome P450 (CYP) forms in precision-cut rat liver slices. 2. Taqman ® primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1 and CYP4A1 forms. 3. Rat liver slices were cultured in control medium or medium containing either 10 µg ml -1 Aroclor 1254 (ARO), 500µM sodium phenobarbitone (NaPB) or 50µM Wy-14,643 (WY) for 3, 6 and 24 h. 4. Compared with control liver slices, treatment with ARO for 3 and 6 h produced 24- and 184-fold increases, respectively, in CYP1A1 mRNA levels, and after 24h produced an 85-fold increase in CYP1A2 mRNA levels. Levels of CYP1A1 and CYP1A2 mRNA were not markedly affected by NaPB and WY. 5. Treatment with ARO and PB for 24 h produced 10.6- and 23.8-fold increases, respectively, in CYP2B1 mRNA. Levels of CYP2B1 mRNA were not markedly affected by WY. 6. Treatment with WY, but not ARO and NaPB, for 24h produced a 20.4-fold increase in levels of CYP4A1 mRNA. 7. These results demonstrate that cultured liver slices may be used to evaluate the effect of xenobiotics on CYP form mRNA levels.

Altered expression of the carboxylesterases ES-4 and ES-10 by peroxisome proliferator chemicals

The nonspecific carboxylesterases (EC.3.1.1.1) are a large group of enzymes that play important roles in the metabolism of foreign xenobiotics and endogenous lipids, including activators of the peroxisome proliferator-activated receptor α, a nuclear receptor that is the central mediator of peroxisome proliferator (PP) effects in the rodent liver. A number of reports have demonstrated that PP exposure leads to alterations in levels of carboxylesterases in the liver. In this study, we determined by Western blot analysis whether exposure to diverse PP results in alteration of expression of two highly expressed microsomal carboxylesterases. Chronic exposure to the PP WY-14,643 (WY) and gemfibrozil (GEM), but not di- n-butyl phthalate (DBP), led to decreases in ES-4 in male rat livers. ES-4 was increased in female rat livers treated with GEM. WY exposure led to decreases in ES-10 in male and female rat livers. ES-10 was increased in female rats treated with DBP. Compared with other end points that are altered within days after PP exposure, the downregulation of ES-4 and ES-10 by WY was considerably slower, occurring between 1 and 5 weeks of exposure. Decreased expression of ES-4 was observed at doses of WY or GEM as low as 10 or 8000 ppm, respectively, whereas decreased expression of ES-10 was more resistant to changes by any PP occurring only with WY at doses as low as 50 ppm. After chronic exposure to WY or diethylhexyl phthalate in wild-type mice, kidney, but not liver, expression of ES-4 and ES-10 was downregulated. These decreases in kidney ES expression were not observed in PPARα-null mice lacking a functional PPARα gene, demonstrating the importance of this transcription factor in these changes. These studies demonstrate that ES protein expression is under complex control by PP that is sex- and compound-dependent. These results lend support to the hypothesis that PP exposure leads to a reprogramming of expression of enzymes important in the metabolism of PPARα activators.

Species differences in hepatic peroxisome proliferation, cell replication and transforming growth factor-β1 gene expression in the rat, Syrian hamster and guinea pig

The objective of this study was to evaluate species differences in the hepatic effects of three potent rodent peroxisome proliferators, namely methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY), particularly with respect to effects on replicative DNA synthesis and transforming growth factor-β1 (TGF-β1) gene expression. Male Sprague–Dawley rats, Syrian hamsters and Dunkin–Hartley guinea pigs were given daily oral doses of 0 (corn oil) and 75 mg/kg MCP for periods of 6 and 21 days. Syrian hamsters and guinea pigs were also treated with 25 mg/kg CIP and 25 mg/kg WY. Relative liver weights were significantly increased in peroxisome proliferator-treated rats and Syrian hamsters, but not in guinea pigs. Hepatic peroxisomal (palmitoyl–CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities and CYP4A isoform mRNA levels were significantly increased in rats and Syrian hamsters, whereas only minor effects were observed in the guinea pig. Replicative DNA synthesis was studied by implanting 7-day osmotic pumps containing 5-bromo-2′-deoxyuridine during study days −1 to 6 and 14 to 21. Hepatocyte labelling index values were increased by MCP in the rat, but neither MCP, CIP nor WY produced any significant effect on replicative DNA synthesis in the Syrian hamster and guinea pig. MCP treatment increased TGF-β1 and insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor gene expression in the rat. In the Syrian hamster, effects on TGF-β1 and IGFII/Man6P receptor gene expression were also observed in some instances, whereas TGF-β1 mRNA levels were essentially unchanged in the guinea pig. These results provide further evidence for marked species differences in response to rodent peroxisome proliferators. While peroxisome proliferators produce a wide spectrum of effects in rat liver, other species such as the Syrian hamster and guinea pig are less responsive and in the case of some endpoints (e.g., cell replication) may be refractory.

Hepatic expression of acute-phase protein genes during carcinogenesis induced by peroxisome proliferators.

Concern exists regarding peroxisome proliferator (PP) xenobiotic exposure because many PPs are potent hepatocarcinogens in rodents. The mechanism of carcinogenicity induced by PPs is atypical compared with those of other hepatocarcinogens in that the former appears to involve alterations in expression of PP-activated receptor (PPAR) target genes rather than direct mutagenicity. To begin to identify some of these genes, we used differential display to compare mRNA expression between hepatic adenomas and adjacent non-tumor liver from rats fed the potent PP Wy-14643 (WY) for 78 wk. Here, we report increased expression of the acute-phase protein (APP) gene alpha-1 antitrypsin (AT) and decreased expression of alpha2-urinary globulin in the tumors. Similar changes were seen in hepatic adenomas induced by a diethylnitrosamine and phenobarbital protocol, indicating a lack of specificity for PP-induced tumors. Additional APP genes, including ceruloplasmin, haptoglobin, beta-fibrinogen, and alpha1-acid glycoprotein were also upregulated in WY-induced tumors but were downregulated in the livers of rats administered a different PP for 13 wk. Mice treated with either WY or di(2-ethylhexyl) phthalate for 3 wk had decreased hepatic AT expression but increased expression of ceruloplasmin and haptoglobin. PPARalpha-null mice showed no hepatic APP gene alteration after PP treatment but had higher basal expression than did wild-type controls. We conclude that PPARalpha activation by several different PPs leads to dysregulation of hepatic APP gene expression in rats and mice. This dysregulation may indicate alterations in cytokine signaling networks regulating both APP gene expression and hepatocellular proliferation.

Alteration of protein kinase C isoform-specific expression during rat hepatocarcinogenesis after exposure to the peroxisome proliferator WY-14,643

The role of protein kinase C (PKC) isoforms in mediating peroxisome proliferator chemical- (PPC) induced hepatocarcinogenesis was examined. After an acute gavage exposure to WY-14,643 (WY) membrane-bound PKCδ and cytosolic PKCβ decreased, whereas the expression of the other isoforms was not altered. After a 13-week chronic exposure, membrane-bound PKCβ, δ and ζ levels decreased. In WY-induced hepatocellular adenomas, PKCα was increased, and PKCβ was further decreased in membrane fractions. These results, taken together with previous studies, indicate that alterations in PKCα, β and δ isoforms, which regulate mitogenesis, could play important roles in perpetuating the high cell proliferative rate in PPC-induced hepatocellular adenomas.

NADPH oxidase is the source of oxidants for activation of NF-κB by the peroxisome proliferator WY-14,643 (WY)

NADPH oxidase is the source of oxidants for activation of NF-κB by the peroxisome proliferator WY-14,643 (WY)

Peroxisome proliferators alter the expression of estrogen-metabolizing enzymes

Exposure to some peroxisome proliferator chemicals (PPC) leads to toxic effects on sex organ function possibly by alterations of steroid hormone metabolism. A systematic search for genes whose mRNA levels are modulated by the PPC WY-14643 (WY) was carried out in rat liver, a site of steroid hormone metabolism. The sequence of one up-regulated cDNA (2480 bp) was predicted to encode a protein of 735 amino acids with 82% identity to the porcine 17β-hydroxysteroid dehydrogenase type IV (HSD IV) originally isolated as a 17β-estradiol dehydrogenase. The rat HSD IV was localized to peroxisomes and was regulated by diverse PPC by two distinct mechanisms. Induction of HSD IV and acyl-CoA oxidase (ACO) proteins in rat liver at different treatment times and concentrations of gemfibrozil (GEM) and di- n-butyl phthalate (DBP) were almost identical, suggesting that HSD IV mRNA induction involves the peroxisome proliferator-activated receptor α, a regulator of ACO. In contrast, HSD IV protein levels were only weakly induced by WY, a strong inducer of ACO protein, even though the levels of both HSD IV and ACO mRNA were strongly stimulated by WY. Thus HSD IV protein levels were uniquely regulated pretranslationally by WY. In addition to HSD IV we also identified the male-specific α 2u-globulin as a PPC down-regulated gene. This prompted us to examine the expression of another male-specific gene, CYP2C11, that catalyzes the hydroxylations of estradiol at the 2 and 16α positions. Cyp2C11 protein expression in rat liver was either decreased or completely abolished after a 3-week treatment by GEM or WY, respectively. Decreased expression of enzymes which inactivate estradiol including Cyp2C11, and the reported increased expression of aromatase may explain why male rats exposed to diverse PPC have higher serum estradiol levels. These higher estradiol levels in male rats have been thought to be mechanistically linked to Leydig cell hyperplasia and adenomas. Increased conversion of estradiol to the less active estrone by HSD IV induction may explain how exposure to the phthalate di-(2-ethylhexyl) phthalate leads to decreases in serum estradiol levels and suppression of ovulation in female rats.

The Peroxisome Proliferators WY-14,643 and Methylclofenapate Induce Hepatocyte Ploidy Alterations and Ploidy-Specific DNA Synthesis in F344 Rats

WY-14,643 (WY) and methylclofenapate (MCP) are peroxisome proliferators (PP) and hepatocarcinogens in rats. MCP causes hepatic polyploidization and preferentially induces replicative DNA synthesis in binucleate tetraploid hepatocytes (2 × 2N) in young Alpk:AP rats. To compare the effect of WY and MCP on hepatocyte ploidy and ploidy-specific DNA synthesis, male F344 rats were fed WY (0.1% in diet) or gavaged with MCP (25 mg/kg/day in corn oil) for 2, 5, or 10 days. Four rats per treatment group (including corn oil and diet control groups) were euthanized and the livers perfused at each time point. To identify cells undergoing DNA synthesis, all animals received BrdU by continuous infusion for 2 or 5 days prior to euthanasia. Hepatocyte ploidy and DNA synthesis were determined using one- or two-parameter flow cytometry. Averages ± SEM for adult male F344 rats as a percentage of total hepatocytes for each ploidy subclass are 2N = 3.4 ± 0.7%, 4N = 69.9 ± 1.9%, 2 × 2N = 14.4 ± 2.4%, 8N = 2.2 ± 0.4%, and 2 × 4N = 9.6 ± 0.9%. Significant alterations were not induced in the proportions of 2 × 2N or 4N ploidy subclasses by WY or MCP at any time point. However, WY caused increases in 8N hepatocytes at 2, 5, and 10 days (2 days, 5.2% vs 2.2% for controls; 5 days, 7.0% vs 3.1% for controls; 10 days, 6.4% vs 3.6% for controls) as did MCP at 5 and 10 days (5 days, 6.3% vs 2.5% for controls; 10 days, 5.3% vs 2.9% for controls). In addition, a majority of BrdU-containing hepatocytes were 4N following 5 and 10 days of WY and MCP [34.3% (WY) and 16.8% (MCP) vs 1.8% and 1.1% for controls, respectively, for 2 × 2N (5 days) as a percentage of total hepatocytes]. Hepatocytes with intermediary DNA content (between tetraploid and octaploid) from MCP- and WY-treated rats were predominantly mononuclear, the percentage of binucleate hepatocytes being similar to or less than the percentage of binucleate cells within the total tetraploid hepatocyte population. These data suggest that polyploidization is induced by PP and induction of S-phase by WY and MCP occurs primarily in 4N hepatocytes in mature F344 rats and not within 2 × 2N hepatocytes. Identification of a ploidy subpopulation at risk for tumor development in rodents is essential for clarifying the role of cell replication in risk assessment studies of PP.

Effect of some peroxisome proliferators on transforming growth factor-beta 1 gene expression and insulin-like growth factor II/mannose-6-phosphate receptor gene expression in rat liver.

Male Sprague-Dawley rats were given daily oral doses of either corn oil (control), 80 mg/kg nafenopin (NAF), 50 mg/kg methylclofenapate (MCP), 50 mg/kg Wy-14,643 (WY) or 250 mg/kg clofibric acid (CA) for 7 days. All four compounds increased relative liver weight and produced hepatic peroxisome proliferation as assessed by induction of both peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities. RNA was extracted from liver samples and analysed for expression of transforming growth factor-beta 1 (TGF-beta 1) and the insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor (which may be involved in transporting latent TGF-beta 1 into hepatocytes). TGF-beta 1 mRNA levels were increased to 151-178% of control by all four compounds, whereas NAF, MCP and WY, but not CA, increased IGFII/Man6P receptor mRNA levels to 195-209% of control. The induction of TGF-beta 1 and IGFII/Man6P receptor expression by short term treatment with peroxisome proliferators may represent an adaptive response to limit the initial hyperplastic effects of such compounds.