Di-n-butyl phthalate activates constitutive androstane receptor and pregnane X receptor and enhances the expression of steroid-metabolizing enzymes in the liver of rat fetuses.

The plasticizer di-n-butyl phthalate (DBP) is a reproductive toxicant in rodents. Exposure to DBP in utero at high doses alters early reproductive development in male rats. Di-n-butyl phthalate also affects hepatic and extrahepatic enzymes. The objectives of this study were to determine the responsiveness of steroid-metabolizing enzymes in fetal liver to DBP and to investigate the potential of DBP to activate nuclear receptors that regulate the expression of liver enzymes. Pregnant Sprague-Dawley rats were orally dosed with DBP at levels of 10, 50, or 500 mg/kg/day from gestation days 12 to 19; maternal and fetal liver samples were collected on day 19 for analyses. Increased protein and mRNA levels of CYP 2B1, CYP 3A1, and CYP 4A1 were found in both maternal and fetal liver in the 500-mg dose group. Di-n-butyl phthalate at high doses also caused an increase in the mRNA of hepatic estrogen sulfotransferase and UDP-glucuronosyltransferase 2B1 in the dams but not in the fetuses. Xenobiotic induction of CYP3A1 and 2B1 is known to be mediated by the nuclear hormone receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). In vitro transcriptional activation assays showed that DBP activates both PXR and CAR. The main DBP metabolite, mono-butyl-phthalate (MBP) did not interact strongly with either CAR or PXR. These data indicate that hepatic steroid- and xenobiotic-metabolizing enzymes are susceptible to DBP induction at the fetal stage; such effects on enzyme expression are likely mediated by xenobiotic-responsive transcriptional factors, including CAR and PXR. Our study shows that DBP is broadly reactive with multiple pathways involved in maintaining steroid and lipid homeostasis.

Constitutive expression of peroxisome proliferator-activated receptor alpha-regulated genes in dwarf mice.

Defects in growth hormone secretion or signaling in mice are associated with decreased body weights (dwarfism), increased longevity, increased resistance to stress, and decreases in factors that contribute to cardiovascular disease and cancer. Peroxisome proliferators (PP) alter a subset of these changes in wild-type mice through activation of the nuclear receptor family member PP-activated receptor alpha (PPARalpha). We tested the hypothesis that an overlap in the transcriptional programs between untreated dwarf mice and PP-treated wild-type mice underlies these similarities. Using transcript profiling, we observed a statistically significant overlap in the expression of genes differentially regulated in control Snell dwarf mice (Pit-1dw) compared with phenotypically normal heterozygote (+/dw) control mice and those altered by the PP 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid (WY-14,643) in +/dw mice. The genes included those involved in beta- and omega-oxidation of fatty acids (Acox1, Cyp4a10, Cyp4a14) and those involved in stress responses (the chaperonin, T-complex protein1epsilon) and cardiovascular disease (fibrinogen). The levels of some of these gene products were also altered in other dwarf mouse models, including Ames, Little, and growth hormone receptor-null mice. The constitutive increases in PPARalpha-regulated genes may be partly caused by increased expression of PPARalpha mRNA and protein as observed in the livers of control Snell dwarf mice. These results indicate that some of the beneficial effects associated with the dwarf phenotype may be caused by constitutive activation of PPARalpha and regulated genes.

Overlapping transcriptional programs regulated by the nuclear receptors peroxisome proliferator-activated receptor alpha, retinoid X receptor, and liver X receptor in mouse liver.

Lipid homeostasis is controlled in part by the nuclear receptors peroxisome proliferator (PP)-activated receptor alpha (PPARalpha) and liver X receptor (LXR) through regulation of genes involved in fatty acid and cholesterol metabolism. Exposure to agonists of retinoid X receptor (RXR), the obligate heterodimer partner of PPARalpha, and LXR results in responses that partially overlap with those of PP. To better understand the gene networks regulated by these nuclear receptors, transcript profiles were generated from the livers of wild-type and PPARalpha-null mice exposed to the RXR pan-agonist 3,7-dimethyl-6S,7S-methano, 7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]-2E,4E-heptadienoic acid (AGN194,204) or the PPAR pan-agonist WY-14,643 (WY; pirinixic acid) and compared with the profiles from the livers of wild-type and LXRalpha/LXRbeta-null mice after exposure to the LXR agonist N-(2,2,2-trifluoroethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethylethyl)phenyl] sulfonamide (T0901317). All 218 WY-regulated genes altered in wild-type mice required PPARalpha. Remarkably, approximately 80% of genes regulated by AGN194,204 required PPARalpha including cell-cycle genes, consistent with AGN-induced hepatocyte proliferation having both PPARalpha-dependent and -independent components. Overlaps of approximately 31 to 62% in the transcript profiles of WY, AGN194,204, and T0901317 required PPARalpha and LXRalpha/LXRbeta for statistical significance. Ofthe 50 overlapping genes regulated by T0901317 and WY, all but one were regulated in a similar direction. These results 1) identify new transcriptional targets of PPARalpha and RXR important in regulating lipid metabolism and liver homeostasis, 2) illustrate the importance of PPARalpha in regulation of gene expression by a prototypical PP and by an RXR agonist, and 3) provide support for an axis of PPARalpha-RXR-LXR in which agonists for each nuclear receptor regulate an overlapping set of genes in the mouse liver.

Gene expression profiling in spleens of deoxynivalenol-exposed mice: immediate early genes as primary targets.

Exposure to the trichothecene mycotoxin deoxynivalenol (DON) alters immune functions in vitro and in vivo. To gain further insight into DON's immunotoxic effects, microarrays were used to determine how acute exposure to this mycotoxin modulates gene expression profiles in murine spleen. B6C3F1 mice were treated orally with 25mg/kg body weight DON, and 2h later spleens were collected for macroarray analysis. Following normalization using a local linear regression model, expression of 116 out of 1176 genes was significantly altered compared to average expression levels in all treatment groups. When genes were arranged into an ontology tree to facilitate comparison of expression profiles between treatment groups, DON was found primarily to modulate genes associated with immunity, inflammation, and chemotaxis. Real-time polymerase chain reaction was used to confirm modulation for selected genes. DON was found to induce the cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and IL-11. In analogous fashion, DON upregulated expression of the chemokines macrophage inhibitory protein-2 (MIP-2), cytokine-induced chemoattractant protein-1 (CINC-1), monocyte chemoattractant protein (MCP)-1, MCP-3, and cytokine-responsive gene-2 (CRG-2). c-Fos, Fra-, c-Jun, and JunB, components of the activator protein-1 (AP-1) transcription factor complex, were induced by DON as well as another transcription factor, NR4A1. Four hydrolases were found to be upregulated by DON, including mitogen-activated protein kinase phosphatase 1 (MKP1), catalytic subunit beta isoform (CnAbeta), protein tyrosine phosphatase receptor type J (Ptprj), and protein tyrosine phosphatase nonreceptor type 8 (Ptpn8), whereas three other hydrolases, microsomal epoxide hydrolase (Eph) 1, histidine triad nucleotide binding protein (Hint), and proteosome subunit beta type 8 (Psmb8) were significantly decreased by the toxin. Finally, cysteine-rich protein 61 (CRP61) and heat-shock protein 40 (Hsp40), genes associated with signaling, were increased, while Jun kinase 2 (JNK2) was decreased. Taken together, data suggest that DON upregulated the expression of multiple immediate early genes, many of which are likely to contribute to the complex immunological effects reported for this and other trichothecenes.

Role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in responses to trichloroethylene and metabolites, trichloroacetate and dichloroacetate in mouse liver.

Trichloroethylene (TCE) is an industrial solvent and a widespread environmental contaminant. Induction of liver cancer in mice by TCE is thought to be mediated by two carcinogenic metabolites, dichloroacetate (DCA) and trichloroacetate (TCA). TCE is considered to be a relatively weak peroxisome proliferator (PP), a group of rodent hepatocarcinogens that cause adaptive responses in liver through the PP-activated receptor alpha (PPARalpha). The objectives of this study were to determine whether effects of TCE, TCA and DCA in the liver associated with carcinogenesis are mediated by PPARalpha. Male wild-type and PPARalpha-null mice were given TCE by gavage for 3 days or 3 weeks; TCA or DCA were given in the drinking water for 1 week. Increases in relative liver and kidney weights by TCE were dependent on PPARalpha whereas liver weight increases by DCA were PPARalpha-independent. Dose-dependent increases in hepatocyte proliferation observed in wild-type mice after TCE exposure as determined by BrdU-labeling of hepatocytes were PPARalpha-dependent. Transcript profiling using macroarrays containing approximately 1200 genes showed that 93% (40 out of 43) of all expression changes observed in wild-type mice upon TCE exposure were dependent on PPARalpha and included known targets of PP (Cyp4a12, epidermal growth factor receptor) and additional genes involved in cell growth. Increases in enzymes that catalyze beta- and omega-oxidation of fatty acids were dependent on PPARalpha after exposure to TCE, TCA or DCA. TCE altered a unique set of genes in the livers of PPARalpha-null mice compared to wild-type mice including those that respond to different forms of stress. These data support the hypothesis that PPARalpha plays a dominant role in mediating the effects associated with hepatocarcinogenesis upon TCE exposure.

Role of the peroxisome proliferator-activated receptor alpha in responses to diisononyl phthalate.

Diisononyl phthalate (DINP) is a compound widely used as a plasticizer in the production of polyvinyl chloride products. Chronic exposure to DINP leads to liver cancer in rats and mice. Many phthalates are considered to be relatively weak peroxisome proliferators (PP), a group of rodent hepatocarcinogens that cause a variety of adaptive responses in liver through the PP-activated receptor alpha (PPARalpha). The objectives of this study were to determine whether DINP-induced effects in the liver associated with carcinogenesis are mediated by PPARalpha and to identify novel gene targets of DINP. Male and female SV129 wild-type, SV129 PPARalpha-null, and B6C3F1 mice were administered DINP by gavage or in the feed. Transcript profile technology and reverse transcriptase (RT)-polymerase chain reaction (PCR) were used to identify gene targets. Dose-dependent increases in relative liver weights were dependent on PPARalpha in 10- or 12-week-old male and female mice and 30-week-old male mice. Female 30-week-old mice exhibited PPARalpha-independent increases in relative liver weights. Increases in hepatocyte proliferation, palmitoyl-CoA oxidase (PCO) activity, and levels of enzymes involved in beta- and omega-oxidation of fatty acids were shown to be dependent on PPARalpha. Five novel genes were shown to be altered in the livers of female wild-type mice after a 3-week exposure, but not in PPARalpha-null, mice. These genes included those involved in DNA repair and recombination (ATP-dependent helicase and Endonuclease III homolog), drug metabolism (Cyp2a4) and protein trafficking (FKBP-1, FKBP-13). An additional gene (Cyp2d9) was shown to be down-regulated in wild-type mice but up-regulated in PPARalpha-null mice indicating more complex regulation by PPARalpha and additional factors. These data support the hypothesis that PPARalpha plays a dominant role in mediating the effects associated with hepatocarcinogenesis after DINP exposure.