Peroxisome Proliferator Chemicals Research Articles

PPARα-Dependent Activation of Cell Cycle Control and DNA Repair Genes in Hepatic Nonparenchymal Cells

Peroxisome proliferator-activated receptor-α (PPARα) mediates the diverse biological effects of peroxisome proliferator (PP) chemicals, including fatty acid catabolism, hepatomegaly, hepatocyte proliferation, and hepatocarcinogenesis in rodents. However, transgenic mice expressing a constitutively active PPARα in hepatocytes (VP16PPARα) do not develop hepatocellular carcinomas in spite of hepatocyte proliferation and hepatomegaly; this suggests that activation of genes in nonparenchymal cells may have a critical role in PP-induced carcinogenesis. VP16PPARα mice exhibited massive peroxisome proliferation and hepatomegaly as well as increased mortality upon Wy-14,643 treatment. Several genes involved in cell cycle or DNA damage repair, such as Chek1, Prkdc, Mcm, and Rad51, were significantly induced to a similar extent between wild-type and VP16PPARα mice after Wy-14,643 administration. This induction was completely abolished in Pparα-null mice, suggesting a PPARα-dependent pathway. These data revealed a DNA damage response signaling network as an early event upon PP treatment and provide novel putative mechanisms for PP-induced hepatocellular carcinoma.

Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor α

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPCs). One agonist of PPARα (WY-14,643) regulates responses in the mouse liver to chemical stress in part by altering expression of genes involved in proteome maintenance (PM) including protein chaperones in the heat shock protein (Hsp) family and proteasomal genes (Psm) involved in proteolysis. We hypothesized that other PPARα activators including diverse hypolipidemic and xenobiotic compounds also regulate PM genes in the rat and mouse liver. We examined the expression of PM genes in rat and mouse liver after exposure to 7 different PPCs (WY-14,643, clofibrate, fenofibrate, valproic acid, di-(2-ethylhexyl) phthalate, perfluorooctanoic acid, and perfluorooctane sulfonate) using Affymetrix microarrays. In rats and mice, 174 or 380 PM genes, respectively, were regulated by at least one PPC. The transcriptional changes were, for the most part, dependent on PPARα, as most changes were not observed in similarly treated PPARα-null mice and the changes were not consistently observed in rats treated with activators of the nuclear receptors CAR or PXR. In rats and mice, PM gene expression exhibited differences compared to typical direct targets of PPARα (e.g., Cyp4a family members). PM gene expression was usually delayed and in some cases, it was transient. Dose-response characterization of protein expression showed that Hsp86 and Hsp110 proteins were induced only at higher doses. These studies demonstrate that PPARα, activated by diverse PPC, regulates the expression of a large number of genes involved in protein folding and degradation and support an expanded role for PPARα in the regulation of genes that protect the proteome.

Characterization of Peroxisome Proliferator–Activated Receptor α—Independent Effects of PPARα Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate also Activates the Constitutive-Activated Receptor

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha. Recent studies indicate that the plasticizer di-(2-ethylhexyl) phthalate (DEHP) increased the incidence of liver tumors in PPARalpha-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPARalpha but dependent on other nuclear receptors, including the constitutive-activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate, and DEHP in rat liver and DEHP in mouse liver induced genes, including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and reverse transcription-PCR in the livers of DEHP-treated wild-type, PPARalpha-null, and CAR-null mice demonstrated that (1) most (approximately 94%) of the transcriptional changes induced by DEHP were PPARalpha-dependent, (2) many PPARalpha-independent genes overlapped with those regulated by PB, (3) induction of genes Cyp2b10, Cyp3a11, and metallothionine-1 by DEHP was CAR dependent but PPARalpha-independent, and (4) induction of a number of genes (Cyp8b1, Gstm4, and Gstm7) was independent of both CAR and PPARalpha. Our results indicate that exposure to PPARalpha activators including DEHP leads to activation of multiple nuclear receptors in the rodent liver.

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.

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.

Down-regulation of STAT5b transcriptional activity by ligand-activated peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is activated by a diverse group of acidic ligands, including many peroxisome proliferator chemicals present in the environment. Janus tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) signaling is activated by multiple cytokines and hormones and leads to the translocation of dimerized STAT proteins to the nucleus where they activate transcription of target genes. Previous studies have shown that growth hormone (GH)-activated STAT5b can inhibit PPAR-regulated transcription. Here, we show that this inhibitory cross-talk is mutual, and that GH-induced, STAT5b-dependent beta-casein promoter-luciferase reporter gene transcription can be inhibited up to approximately 80% by ligand-activated PPARalpha or PPARgamma. Dose-response experiments showed a direct relationship between the extent of PPAR activation and the degree of inhibition of STAT5-regulated transcription. PPAR did not block STAT5b tyrosine phosphorylation or inhibit DNA-binding activity. Both PPARs inhibited the transcriptional activity of a constitutively active STAT5b mutant, indicating that inhibition occurs downstream of the GH-stimulated STAT5 activation step. Transcriptionally inactive, dominant-negative PPAR mutants did not block STAT5b inhibition by wild-type PPAR, indicating that PPAR target gene transcription is not required. PPARalpha retained its STAT5b inhibitory activity in the presence of the histone deacetylase inhibitor trichostatin, indicating that enhanced histone deacetylase recruitment does not contribute to STAT5b inhibition. PPARalpha lacking the ligand-independent AF-1 trans-activation domain failed to inhibit STAT5b, highlighting the importance of the AF-1 region in STAT5-PPAR inhibitory cross-talk. These findings demonstrate the bidirectionality of cross-talk between the PPAR and STAT pathways and provide a mechanism whereby exposure to environmental chemical activators of PPAR can suppress expression of GH target genes.

Delayed liver regeneration in peroxisome proliferator-activated receptor-α-null mice

Peroxisome proliferator chemicals, acting via the peroxisome proliferator-activated receptor-α ( Pparα), are potent hepatic mitogens and carcinogens in mice and rats. To test whether Pparα is required for hepatic growth in response to other stimuli, we studied liver regeneration and hepatic gene expression following partial hepatectomy (PH) of wild-type and Pparα-null mice. Pparα-null mice had a 12- to 24-hour delay in liver regeneration associated with a delayed onset and lower peak magnitude of hepatocellular DNA synthesis. Furthermore, these mice had a 24-hour lag in the hepatic expression of the G 1/S checkpoint regulator genes Ccnd1 and cMyc and increased expression of the IL-1β cytokine gene. Hepatic expression of Ccnd1, cMyc, IL-1r1, and IL-6r was induced in wild-type mice, but not Pparα-null mice, after acute exposure to the potent Pparα agonist Wy-14,643, indicating a role for Pparα in regulating the expression of these genes. Expression of the fatty acid ω-hydroxylase gene Cyp4a14, a commonly used indicator gene for Pparα activation, was strongly induced in wild-type mice after hepatectomy, suggesting that altered hepatocyte lipid processing may also contribute to the impaired regeneration in mice lacking the Pparα gene. In conclusion, liver regeneration in Pparα-null mice is transiently impaired and is associated with altered expression of genes involved in cell cycle control, cytokine signaling, and fat metabolism. (H EPATOLOGY 2002;36:544-554.)

Elevated Basal Expression of Liver Peroxisomal β-Oxidation Enzymes and CYP4A Microsomal Fatty Acid ω-Hydroxylase in STAT5b−/− Mice: Cross-Talk in Vivo between Peroxisome Proliferator-Activated Receptor and Signal Transducer and Activator of Transcription Signaling Pathways

Long-term treatment of rodents with peroxisome proliferator chemicals, a group of structurally diverse nongenotoxic carcinogens, leads to liver cancer in a process dependent on the nuclear receptor peroxisome proliferator-activated receptor-α (PPARα). Previous in vitro studies have shown that growth hormone (GH) can inhibit PPARα-dependent gene expression by down-regulation of PPARα expression and by a novel inhibitory cross-talk involving the GH-activated transcription factor STAT5b. Presently, we evaluate the role of STAT5b in mediating these inhibitory actions of GH on PPAR function using a STATb-deficient mouse model. Protein levels of three PPARα-responsive peroxisomal β-oxidation pathway enzymes (fatty acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and l-bifunctional enzyme) were increased up to two- to threefold in STAT5b−/− relative to wild-type control mouse liver, as was the basal expression of two PPARα-regulated cytochrome P450 4A proteins. In contrast, protein levels of two PPARα-unresponsive peroxisomal enzymes, catalase and urate oxidase, were not affected by the loss of STAT5b. A corresponding increase in expression of fatty acyl-CoA oxidase and l-bifunctional enzyme mRNA, as well as PPARα mRNA, was observed in the STAT5b-deficient mice, suggesting a transcriptional mechanism for the observed increases. Although basal liver expression of PPARα and its target genes was thus elevated in STAT5b−/− mice, the clofibrate-induced level of enzyme expression was unaffected, suggesting that the inhibitory effects of STAT5b are overcome at high concentrations of PPARα activators. These findings support the hypothesis that GH and potentially other endogenous activators of STAT5b help to maintain liver PPARα function at a low basal level and may thereby moderate PPARα-dependent hepatocarcinogenesis and other responses stimulated by exposure to low levels of environmental chemicals of the peroxisome proliferator class.

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.

Trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals

A large number of industrial chemicals and environmental pollutants, including trichloroethylene (TCE), di(2-ethylhexyl)phthalate (DEHP), perfluorooctanoic acid (PFOA), and various phenoxyacetic acid herbicides, are nongenotoxic rodent hepatocarcinogens whose human health risk is uncertain. Rodent model studies have identified the receptor involved in the hepatotoxic and hepatocarcinogenic actions of these chemicals as peroxisome proliferator–activated receptor alpha (PPARα), a nuclear receptor that is highly expressed in liver. Humans exhibit a weak response to these peroxisome proliferator chemicals, which in part results from the relatively low level of PPARα expression in human liver. Cell transfection studies were carried out to investigate the interactions of peroxisome proliferator chemicals with PPARα, cloned from human and mouse, and with PPARγ, a PPAR isoform that is highly expressed in multiple human tissues and is an important regulator of physiological processes such as adipogenesis and hematopoiesis. With three environmental chemicals, TCE, perchloroethylene, and DEHP, PPARα was found to be activated by metabolites, but not by the parent chemical. A decreased sensitivity of human PPARα compared to mouse PPARα to trans-activation was observed with some (Wy-14,643, PFOA), but not other, peroxisome proliferators (TCE metabolites, trichloroacetate and dichloroacetate; and DEHP metabolites, mono[2-ethylhexyl]phthalate and 2-ethylhexanoic acid). Investigation of human and mouse PPARγ revealed the transcriptional activity of this receptor to be stimulated by mono(2-ethylhexyl)phthalate, a DEHP metabolite that induces developmental and reproductive organ toxicities in rodents. This finding suggests that PPARγ, which is highly expressed in human adipose tissue, where many lipophilic foreign chemicals tend to accumulate, as well as in colon, heart, liver, testis, spleen, and hematopoietic cells, may be a heretofore unrecognized target in human cells for a subset of industrial and environmental chemicals of the peroxisome proliferator class.

Cloning genes responsive to a hepatocarcinogenic peroxisome proliferator chemical reveals novel targets of regulation

To better understand the molecular basis of the hepatocyte proliferation and induction of hepatocellular adenomas by exposure to peroxisome proliferator chemicals (PPC), a systematic search for genes modulated by a PPC (WY-14643) in rat liver was carried out using the differential display technique. The fragments fell into two classes based on the time of initial and maximal induction by WY-14643. The class I genes (clones 5 and 30) were induced 3 h after a gavage exposure to WY-14643 with maximal expression at 24 h. The class II genes (clones 13 and 16) were induced after 24 h with maximal expression at 78 weeks. Expression of the class II genes was also increased after other treatments that cause cell proliferation. Clone 30 was identified as CYP4A2, previously shown to be regulated by PPC. Clone 13 was homologous to the mouse protein H gene, a component of the heterogeneous nuclear ribonucleoprotein particle important in mRNA splicing. Clone 16 was identified as cyclophilin-A, the receptor for the immunosuppressant drug cyclosporin A. The sequence of clone 5 was unique. These data demonstrate that WY-14643 increases the levels of a number of novel genes that are coordinately regulated with increases in chronic cell proliferation and fatty acid metabolism.

Down-Regulation of Cytochrome P450 2C Family Members and Positive Acute-Phase Response Gene Expression by Peroxisome Proliferator Chemicals

In this study, we show that peroxisome proliferator chemical (PPC) exposure leads to alterations in the expression of genes in rat liver regulated by the sex-specific growth hormone secretory pattern and induced during inflammation. Expression of the male-specific cytochrome P450 (P450) 2C11 and alpha2 urinary globulin (alpha2u) genes and the female-specific P450 2C12 gene was down-regulated by some PPC. Expression of P450 2C13, also under control by the sex-specific growth hormone secretory pattern, was not altered by PPC treatment, indicating that regulation of CYP2C family members does not involve perturbation of the growth hormone secretory pattern. In contrast to the increases in expression observed when inflammation was induced in male rats, two positive acute-phase response genes, alpha1-acid glycoprotein and beta-fibrinogen, were decreased by PPC exposure. The down-regulation of the P450 2C11 by WY-14,643 could be reproduced in cultured rat hepatocytes, indicating the down-regulation is a direct effect. Experiments in wild-type mice and mice that lacked a functional peroxisome proliferator-activated receptor-alpha gene showed that down-regulation by WY of alpha1-acid glycoprotein, beta-fibrinogen, and a mouse homologue of alpha2u was dependent on peroxisome proliferator-activated receptor-alpha expression. Our results demonstrate that PPC exposure leads to down-regulation of diverse liver-specific genes, including CYP2C family members important in hormonal homeostasis and acute-phase response genes important in inflammatory responses.

Tissue-specific induction of 17 beta-hydroxysteroid dehydrogenase type IV by peroxisome proliferator chemicals is dependent on the peroxisome proliferator-activated receptor alpha.

The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) family of proteins regulates the levels of the active 17 beta-hydroxy forms of sex steroids. The expression of 17 beta-HSD type IV is induced by peroxisome proliferator chemicals (PPC) in rat liver. In order to characterize more generally the impact of PPC on 17 beta-HSD expression, we determined (1) if expression of other members of the 17 beta-HSD family was coordinately induced by PPC exposure, (2) the tissues in which 17 beta-HSD was induced by PPC, and (3) whether the induction of 17 beta-HSD by PPC was dependent on the peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of PPC effects in the mouse liver. The mRNA levels of 17 beta-HSD I, II, and III were not altered in the liver, kidney, and testis or uterus of rats treated with PPC. The mRNA or 80 kDa a full-length protein levels of 17 beta-HSD IV were strongly induced in liver and kidney, but not induced in adrenals, brown fat, heart, testis, and uterus of rats treated with diverse PPC. In liver and kidneys from treated rats, additional proteins of 66 kDa, 56 kDa, and 32 kDa were also induced which reacted with the anti-17 beta-HSD IV antibodies and were most likely proteolytic fragments of 17 bega-HSD IV. Treatment of mice which lack a functional form of PPAR alpha with PPC, demonstrated that PPC-inducibility of 17 beta-HSD IV mRNA or the 80 kDa protein was dependent on PPAR alpha expression in liver and kidney. Our results demonstrate that 17 beta-HSD IV is induced by PPC through a PPAR alpha-dependent mechanism and support the hypothesis that exposure to PPC leads to alterations in sex steroid metabolism.

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.