Environmental and Human Health Impacts of Spreading Oil and Gas Wastewater on Roads.

Thirteen states in the United States allow the spreading of O&G wastewaters on roads for deicing or dust suppression. In this study, the potential environmental and human health impacts of this practice are evaluated. Analyses of O&G wastewaters spread on roads in the northeastern, U.S. show that these wastewaters have salt, radioactivity, and organic contaminant concentrations often many times above drinking water standards. Bioassays also indicated that these wastewaters contain organic micropollutants that affected signaling pathways consistent with xenobiotic metabolism and caused toxicity to aquatic organisms like Daphnia magna. The potential toxicity of these wastewaters is a concern as lab experiments demonstrated that nearly all of the metals from these wastewaters leach from roads after rain events, likely reaching ground and surface water. Release of a known carcinogen (e.g., radium) from roads treated with O&G wastewaters has been largely ignored. In Pennsylvania from 2008 to 2014, spreading O&G wastewater on roads released over 4 times more radium to the environment (320 millicuries) than O&G wastewater treatment facilities and 200 times more radium than spill events. Currently, state-by-state regulations do not require radium analyses prior to treating roads with O&G wastewaters. Methods for reducing the potential impacts of spreading O&G wastewaters on roads are discussed.

Effects of a DASH-like diet containing lean beef on vascular health.

A DASH (dietary approaches to stop hypertension) dietary pattern rich in fruits and vegetables and low-fat dairy products with increased dietary protein provided primarily from plant protein sources decreases blood pressure. No studies, however, have evaluated the effects of a DASH-like diet with increased dietary protein from lean beef on blood pressure and vascular health. The aim of this study was to study the effect of DASH-like diets that provided different amounts of protein from lean beef (DASH 28 g beef per day; beef in an optimal lean diet (BOLD) 113 g beef per day; beef in an optimal lean diet plus additional protein (BOLD+) 153 g beef per day) on blood pressure, endothelial function and vascular reactivity versus a healthy American diet (HAD). Using a randomized, crossover study design, 36 normotensive participants (systolic blood pressure (SBP), 116 ± 3.6 mm Hg) were fed four isocaloric diets,: HAD (33% total fat, 12% saturated fatty acids (SFA), 17% protein (PRO), 20 g beef per day), DASH (27% total fat, 6% SFA, 18% PRO, 28 g beef per day), BOLD (28% total fat, 6% SFA, 19% PRO, 113 g beef per day) and BOLD+ (28% total fat, 6% SFA, 27% PRO, 153 g beef per day), for 5 weeks. SBP decreased (P<0.05) in subjects on the BOLD+ diet (111.4 ± 1.9 mm Hg) versus HAD (115.7 ± 1.9). There were no significant effects of the DASH and BOLD diets on SBP. Augmentation index (AI) was significantly reduced in participants on the BOLD diet (-4.1%). There were no significant effects of the dietary treatments on diastolic blood pressure or endothelial function (as measured by peripheral arterial tonometry). A moderate protein DASH-like diet including lean beef decreased SBP in normotensive individuals. The inclusion of lean beef in a heart healthy diet also reduced peripheral vascular constriction.

Characterization of Madin-Darby bovine kidney cell line for peroxisome proliferator-activated receptors: temporal response and sensitivity to fatty acids.

The peroxisome proliferator-activated receptors (PPAR) are critical for lipid metabolism, and many fatty acids are PPAR agonists. Madin-Darby bovine kidney (MDBK) cells were tested as an in vitro bovine model for PPAR activation, and preliminary evaluation of the effect of fatty acids on bovine PPAR was performed. Cells were treated with Wy-14643 (WY, specific PPARalpha agonist) and rosiglitazone (ROSI, specific PPARgamma agonist). The gene expression of specific PPARalpha-responsive genes such as carnitine palmitoyl transferase-1 (CPT1A) and acetyl coenzyme A oxidase (ACOX1) and of PPARgamma-responsive gene lipoprotein lipase (LPL) were analyzed using real-time reverse transcription PCR. It was found that CPT1A exhibited a significant increase in cells treated with WY, whereas the ACOX1 gene expression was not altered. The LPL gene expression showed an increase in response to ROSI. Interestingly, LPL was almost undetectable in MDBK cells not treated with ROSI. The potency of different fatty acids in activating PPARalpha as assessed by CPT1A mRNA abundance in MDBK cells was also tested. The mRNA of CPT1A (2.5- to 1.4-fold) was significantly increased by fatty acids in the order of palmitate > linolenate > linoleate > conjugated linoleate, and oleate. The results demonstrated MDBK cells to be responsive to PPAR agonists and thus a promising model to evaluate the role of PPAR in bovine cells. In addition, fatty acids were proven to have a different potency in modulating expression of CPT1A through PPARalpha.

Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: evidence for a PPAR gamma-dependent mechanism.

Conjugated linoleic acid (CLA) is a dietary fatty acid that has received considerable attention due to its unique properties in rodent models including anti-cancer, anti-atherogenic and anti-diabetic effects. The effects of CLA are similar to those seen with ligands for peroxisome proliferator-activated receptor (PPARs), most notably of the PPAR gamma subtype. With the recent observation of a role for PPAR gamma in regulation of immune responses, we suspected that CLA could affect immune function, in particular macrophage activity. The goal of our study was to examine whether this dietary fatty acid has anti-inflammatory properties similar to those reported for PPAR gamma activators such as 15-deoxy prostaglandin J(2) (PGJ(2)). In reporter assays, various CLA isomers activated PPAR gamma in RAW264.7 mouse macrophage (RAW) cells. CLA decreased the interferon-gamma (IFN gamma)-induced mRNA expression of mediators of inflammation including cyclooxygenase 2 (COX2), inducible NOS (iNOS), and tumor necrosis factor alpha (TNFalpha). Reporter assays also demonstrated reduced IFN gamma-stimulated transcriptional activity of the iNOS and COX2 promoters by CLA. Consequently, CLA decreased the production of PGE(2), TNFalpha and the inflammatory agent nitric oxide (NO) in RAW cells treated with IFN gamma. Other pro-inflammatory cytokines such as IL-1 beta and IL-6 were similarly decreased by CLA treatment of RAW cells. In addition, various CLA isomers induced HL60 cell differentiation along the monocytic lineage as assessed by measuring expression of the cell surface marker CD14. This differentiation process, as well as the regulation of iNOS and COX2 by 15dPGJ(2), is believed to involve PPAR gamma. Mutations of Leu(468) and Glu(471) to alanine in helix 12 of the ligand-binding domain of PPAR gamma resulted in a protein with strong dominant-negative activity (dnPPAR gamma). Transfecting dnPPAR gamma into RAW cells eliminated the ability of various CLA isomers to regulate the iNOS reporter construct. Taken together, these results suggest that CLA has anti-inflammatory properties that are mediated, at least in part, by the nuclear hormone receptor PPAR gamma.

The Q-rich subdomain of the human Ah receptor transactivation domain is required for dioxin-mediated transcriptional activity.

The aryl hydrocarbon receptor (AhR), a basic helix-loop-helix/Per-Arnt-Sim transcription factor, mediates many of the toxic and biological effects of the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which include the transcriptional activation of dioxin-responsive genes such as CYP1A1. Many aspects of this process are known; however, the mechanism of transcriptional activation and the proteins that are key to this process remain to be determined. The hAhR has a complex transactivation domain, composed of three potentially distinct subdomains. Deletional analysis of the hAhR transactivation domain indicates that removal of the P/S/T-rich subdomain enhances transcriptional activity, whereas the Q-rich subdomain is critical for hAhR transactivation potential, and the acidic subdomain by itself fails to activate a dioxin response element-driven reporter gene. Deletional analysis of the Q-rich subdomain identified a critical stretch of 23 amino acids between residues 666 and 688 of the hAhR, which are required for transactivation potential. Alanine scanning mutagenesis of this region identified a leucine residue (Leu-678), which is required for hAhR activity. Functional analysis of this point mutant revealed that it is capable of binding ligand, heterodimerization, and subsequent binding to dioxin response elements. Further, when hAhR/L678A and hAhR containing only the acidic subdomain were overexpressed they acted as dominant negative receptors and repressed wild-type hAhR activity. In addition, the hAhR/L678A failed to activate CYP1A1 gene transcription in transfected BP-8 cells and exhibited reduced binding to RIP140 in vitro. Thus, Leu-678 appears to be critical for efficient transactivation activity of the hAhR and appears to disrupt recruitment of co-regulators.

Inhibition of peroxisome-proliferator-activated receptor (PPAR)alpha by MK886.

Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may also indicate that PPAR alpha is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPAR alpha, making it the first compound identified to have such an effect.

Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARalpha.

We have previously shown that a mixture of dietary conjugated derivatives of linoleic acid (conjugated linoleic acid, CLA) induces peroxisome proliferator-responsive enzymes and modulates hepatic lipid metabolism in vivo. The present studies demonstrate that CLA is a high affinity ligand and activator of peroxisome proliferator-activated receptor alpha (PPARalpha) and induces accumulation of PPAR-responsive mRNAs in a rat hepatoma cell line. Using a scintillation proximity assay (SPA), CLA isomers were shown to be ligands for human PPARalpha with a rank order of potency of (9Z,11E)>(10E,12Z)>(9E,11E)> furan-CLA (IC(50) values from 140 nm to 400 nm). Levels of acyl-CoA oxidase (ACO), liver fatty acid-binding protein (L-FABP), and cytochrome P450IVA1 (CYP4A1) mRNA were induced by CLA in FaO hepatoma cells. Even though linoleate and CLA were incorporated into lipids of hepatoma cells to the same extent, linoleate had little or no effect on ACO, CYP4A1, or L-FABP mRNA. In agreement with its binding potency, (9Z,11E)-CLA was the most efficacious PPARalpha activator in the mouse PPARalpha-GAL4(UAS)(5)-CAT reporter system. These data indicate that CLA is a ligand and activator of PPARalpha and its effects on lipid metabolism may be attributed to transcriptional events associated with this nuclear receptor. Also, (9Z,11E)-CLA is one of the most avid fatty acids yet described as a PPARalpha ligand.

Peroxisome proliferator-activated receptors: a critical link among fatty acids, gene expression and carcinogenesis.

It has been known for many years that long-chain fatty acids derived from endogenous metabolism and/or nutrition can act as second messengers and regulators of cell signaling pathways. For example, fatty acids regulate the activity of protein kinase C (PKC) in a mechanism distinct from activation by diacylglycerol. Like PKC activators such as phorbol esters, essential fatty acids activate PKC and in doing so modulate the activity of growth factor receptors such as epidermal growth factor receptor (EGFR). Unsaturated fatty acids can inhibit GTPase activating protein, thereby quenching signals from p21-ras. These studies have shown that fatty acids can influence numerous signaling pathways and that these small lipophilic substances may be ancient second messengers. Fatty acids are also known modulators of the carcinogenic process, showing distinct tissue-specific pro- or anticancer effects. However, the reason for such a dichotomous effect on cellular processes has not been adequately described. In this article, the inclusion of a steroid hormone receptor-signaling pathway in mediating fatty acids' effects will be summarized. This signaling molecule has been deemed the peroxisome proliferator-activated receptor (PPAR) and has been extensively examined in regard to its response to xenobiotic, fatty acid-like chemicals (peroxisome proliferators, PP). PP, like fatty acids, activate PPAR and modulate tissue-specific responses. The goal of this review is to describe a potential role for PPAR in mediating the effects of fatty acids on gene expression, cell growth, differentiation and apoptosis.

Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis.

Peroxisome proliferators (PPs) are an important group of chemicals that include certain hypolipidemic drugs, plasticizers and pollutants. Many of these agents are known rodent liver tumor promoters and debate exists as to whether humans are at increased cancer risk following exposure to PPs. Research over the last decade has focused on determining the biochemical and molecular mechanisms by which peroxisome proliferators exert their effects, in the hope that this controversy will be settled. PPs regulate gene expression via a steroid hormone receptor, the peroxisome proliferator-activated receptor (PPAR). At least three subtypes of PPAR (alpha, beta and gamma) have been cloned from several species, including humans. These receptors have been implicated in tumor promotion, cellular differentiation, and apoptosis. In the present article, the current understanding of how PPARs are involved in tumorigenesis, and what this may mean to human risk assessment, will be discussed.

Identification of a novel peroxisome proliferator responsive cDNA isolated from rat hepatocytes as the zinc-finger protein ZFP-37.

The implementation of a rat hepatocyte model system and differential display-polymerase chain reaction resulted in the isolation of ZFP-37 as a peroxisome proliferator-responsive gene. In addition to being responsive to peroxisome proliferators, rat ZFP-37 (rZFP-37) mRNA accumulates rapidly after treating cells with several other hepatic tumor promoters, serum, and cycloheximide, indicating that this gene belongs to the immediate-early growth responsive gene family. Although rZFP-37 and mouse ZFP-37 (mZFP-37) are both members of the Krüppel-associated box and C2H2 zinc finger superfamily of proteins, there are several features that distinguish the two proteins. The primary protein sequences of rat and mouse ZFP-37 are highly conserved, especially within the region encoding the 12 C2H2 zinc finger motifs; however, a region believed to be involved in DNA binding in mZFP-37 is divergent in rZFP-37. Mouse ZFP-37 mRNA is expressed almost exclusively in testes and brain, whereas rZFP-37 mRNA is expressed in testes, brain, kidney, spleen, thymus, lung, and at low levels in liver. A major difference between regulation of ZFP-37 in the two species exists as rZFP-37 is induced, while mZFP-37 is repressed, in liver by the administration of the potent peroxisome proliferator Wy 14,643. Despite the fact that mZFP-37 is believed to be important in cell growth and differentiation in testes and brain, the pronounced differences in regulation of this gene in two closely related species preclude an extrapolation to rZFP-37's biological role. Nonetheless, the effects of tumor promoters and mitogens on its expression and the inclusion of rZFP-37 into the immediate-early growth gene families raise the possibility that this gene plays a role in hepatocyte proliferation and/or differentiation.

Differential expression of group I metabotropic glutamate receptors (mGluRs) in the rat pheochromocytoma cell line PC12: role of nerve growth factor and ras.

Glutamate treatment of PC12 cells has been shown to result in the accumulation of intracellular inositol phosphates suggesting the presence of glutamate metabotropic receptors (mGluRs) positively coupled to phospholipase C. The present study examined the expression of group I mGluRs (mGluR1 and mGluR5) in PC12 cells. Undifferentiated PC12 cells were found to express both mGluR5 mRNA and receptor protein by reverse transcription polymerase chain reaction (RT-PCR) and western blot techniques. However, mGluR1 mRNA was not detected in these cells and western blot analysis showed only faint mGluR1alpha immunoreactivity suggesting a very low level of mGluR1 expression. Nerve growth factor-induced differentiation of PC12 cells resulted in the induction of mGluR1alpha and mGluR1beta mRNA and mGluR1alpha protein. PC12 cells overexpressing dominant negative ras revealed that NGF-induced mGluR1 induction, but not mGluR5 expression, is dependent on ras pathway activation in these cells. These results suggest PC12 cells may be a useful model for investigating the regulation and expression of group I mGluR isoforms and their role in neuronal processes in vitro.

Comparison of dose-response relationships for induction of lipid metabolizing and growth regulatory genes by peroxisome proliferators in rat liver.

The regulation of gene expression via the peroxisome proliferator-activated receptor (PPAR) is believed to be critical in the effects of peroxisome proliferators on lipid metabolism and possibly in hepatocarcinogenesis. The involvement of PPAR in the peroxisome proliferator-mediated induction of fatty acid metabolizing genes such as acyl-CoA oxidase (ACO), fatty acid-binding protein (FABP), and cytochrome P450IVA1 (CYP4A1) has been clearly demonstrated. However, the induction by peroxisome proliferators of important growth regulatory genes such as c-myc has not been investigated extensively. In these studies we examined the dose-response relationships for the induction of mRNA for the PPAR-regulated and lipid metabolizing genes ACO, FABP, and CYP4A1 and compared them to the immediate early gene c-myc. Liver mRNA from rats fed various amounts of the peroxisome proliferator Wy14,643 for 13 weeks was utilized. The lipid metabolism and growth regulatory genes were induced by subchronic administration of Wy14,643 but to varying degrees and with different sensitivities. The lowest dose that resulted in a significant change in ACO and FABP expression was 10 ppm. The mRNA for CYP4A1 and c-myc was significantly affected at the lowest dose examined (5 ppm). Also, the maximal induction ranged from 10(5)-fold (CYP4A1) to less than 10-fold (FABP) relative to vehicle-treated animals. The accumulation of mRNA for ACO, FABP, and CYP4A1, but not c-myc, showed typical receptor-mediated dose-response relationships. The effects on gene expression were compared to rates of hepatic cell proliferation, a pertinent marker of tumor promotion and hepatocarcinogenesis. Surprisingly, ACO mRNA showed an excellent correlation (r2 = 0.9) while c-myc mRNA exhibited a poor correlation (r2 = 0.3) with cell proliferation in rat liver. Although the differences between the dose-response relationships of ACO and c-myc mRNA accumulation may suggest immediate early genes are not controlled by PPAR, evidence from PPARalpha null mice support this receptor in both lipid metabolism and growth regulatory genes. This study shows the complexity of responses mediated by peroxisome proliferators, with ACO being a good marker of PPAR-mediated events as well as cell proliferation, while c-myc, a known growth regulatory gene, was induced by Wy14,643 partially via PPAR but did not correlate well with cell proliferation.

Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat.

Conjugated linoleic acid (CLA) is a naturally occurring fatty acid which has anti-carcinogenic and anti-atherogenic properties. CLA activates PPAR alpha in liver, and shares functional similarities to ligands of PPAR gamma, the thiazolidinediones, which are potent insulin sensitizers. We provide the first evidence that CLA is able to normalize impaired glucose tolerance and improve hyperinsulinemia in the pre-diabetic ZDF rat. Additionally, dietary CLA increased steady state levels of aP2 mRNA in adipose tissue of fatty ZDF rats compared to controls, consistent with activation of PPAR gamma. The insulin sensitizing effects of CLA are due, at least in part, to activation of PPAR gamma since increasing levels of CLA induced a dose-dependent transactivation of PPAR gamma in CV-1 cells cotransfected with PPAR gamma and PPRE X 3-luciferase reporter construct. CLA effects on glucose tolerance and glucose homeostasis indicate that dietary CLA may prove to be an important therapy for the prevention and treatment of NIDDM.

The Nuclear Receptor Resource: a growing family.

Last year, the original Glucocorticoid Receptor Resource was expanded into a comprehensive project: the Nuclear Receptor Resource (NRR, http:// nrr.georgetown.edu/nrr/nrr.html ). The NRR has since been offering comprehensive information on nuclear receptor structure and function, as well as general facts of interest to the scientific community on meetings, funding and employment opportunities. The project now includes individual resources as part of a network which integrates information on glucocorticoid, androgen, mineralocorticoid, thyroid hormone, Vitamin D and peroxisome-proliferator activated receptors. Many investigators have joined the NRR network by filling the Who is who? form available in the NRR home page. This has facilitated communication among scientists in the field and dissemination of data nor otherwise published. Because several investigators have contacted NRR authors over the past few months asking for advice and materials for educational purposes, we have recently decided to include in our project an educational resource on nuclear receptors termed the 'Graphics Library'. The input and suggestions of NRR users do shape the future direction of the project, so we encourage user to give us feedback.

Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity.

Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an approximately 38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element-luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.

A 50 kilodalton protein associated with raf and pp60(v-src) protein kinases is a mammalian homolog of the cell cycle control protein cdc37.

Several oncogenic protein kinases including c-raf-1 and pp60(v-src) are known to directly interact with the 90 kDa heat shock protein (hsp90)/p50 complexes. Using a monoclonal antibody to detect p50 during a purification scheme, p50 was purified to homogeneity. Internal amino acid sequence information was obtained and used to clone a partial cDNA. Comparison of the p50 sequence to other cloned proteins revealed 89% homology with a glycosaminoglycan-binding protein and 54% homology with Drosophila cell cycle control protein (cdc) 37. Monoclonal and polyclonal antibodies were produced against a cleaved fusion protein that recognizes p50 with a high level of specificity. These antibodies recognize the 50 kDa protein present in c-raf-1 and pp60(v-src) complexes. No other proteins were recognized with these antibodies suggesting that p50 is a unique protein. Immunocytochemical visualization of p50 in NIH 3T3 cells indicates a primarily cytoplasmic localization around the nuclear membrane. A survey of p50 expression in murine tissues on a protein blot revealed the following relative levels of expression; thymus > spleen > brain > heart > kidney > liver > lung > skeletal muscle. These results link studies demonstrating complexation of certain kinases with hsp90/p50 in mammalian cells and a number of reports in yeast and Drosophila, demonstrating the importance of cdc37 in cell cycle and kinase function.

Isolation and characterization of a novel gene induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver.

The differential display technique was used to identify genes whose expression was regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of a novel sequence was up-regulated in a dose-dependent fashion in liver of Sprague-Dawley male rats exposed to both chronic and acute treatment with TCDD, as measured by densitometry of Northern blot analyses (P < 0.01). A rapid amplification of cDNA ends (RACE) procedure was used to isolate a 1.8 kb cDNA from a rat liver cDNA preparation. This cloned cDNA, called 25-Dx, was sequenced and found to encode a peptide of 223 amino acids. In control rats, the 25-Dx gene was expressed at high levels in lung and liver. A hydrophobic domain of 14 residues followed by a proline-rich domain, both located in the N-terminal region, showed 71% homology with the transmembrane domain of the precursor for the interleukin-6 receptor and a conserved consensus sequence found in the cytokine/growth factor/prolactin receptor superfamily respectively.

Perfluorodecanoic acid as a useful pharmacologic tool for the study of peroxisome proliferation.

The phenomena of peroxisome proliferation in rodent liver has received considerable attention due to its association with hepatocellular carcinoma. Chemicals that cause peroxisome proliferation include several structurally unrelated hypolipidemic drugs, phthalate esters and halogenated solvents. The mechanism by which peroxisome proliferators exert their beneficial (hypolipidemia) as well as their toxic (cancer) effects is still largely unknown. Perfluorodecanoic acid (PFDA) is a potent peroxisome proliferator in rodent liver that resembles other members of this chemical class in many aspects, including its effects on gene expression and fatty acid metabolism. However, there are many dissimilarities between PFDA and hypolipidemic peroxisome proliferators that have not been extensively explored. PFDA is unlike other peroxisome proliferators in parent compound metabolism, hypolipidemia, and tumor promotion. The present review article will discuss what is currently known about PFDA and how it may be utilized to dissect the mechanism of action of an important group of hypolipidemic drug and environmental pollutant, the peroxisome proliferators.

Regulation of peroxisome proliferator-activated receptor-alpha mRNA in rat liver.

Chemical-induced peroxisome proliferation in rodent liver is postulated to occur via activation of members of the steroid hormone receptor superfamily, the peroxisome proliferation-activated receptors (PPARs). In the present study, the expression of the predominant liver subtype PPAR alpha was examined and compared to that of acyl-CoA oxidase (ACO), a marker for peroxisome proliferation and a prototype for genes regulated via PPARs. Despite the induction of both mRNA species in vivo by the peroxisome proliferator perfluorodecanoic acid (PFDA), dose response and time course indicate PPAR alpha and ACO are not controlled similarly. Messenger RNA levels for ACO increased rapidly in rat liver and declined over the subsequent 7 days following PFDA administration, while PPAR alpha mRNA increased slower and remained elevated over this period. In addition, PPAR alpha mRNA accumulation in PFDA-treated rats appears to be due primarily to hypophagia as pair feeding and complete caloric restriction result in a large increase in the concentration of this messenger RNA. Nuclear run-on experiments in vivo suggest that, unlike ACO, PFDA as well as caloric restriction results in accumulation of PPAR alpha mRNA which cannot be explained solely by transcriptional activation. These data indicated that PPAR alpha mRNA accumulation has a very small peroxisome proliferator-dependent component and that other factors may be involved. A rat hepatoma cell line was examined to determine the direct effect on peroxisome proliferators on PPAR alpha mRNA. PPAR alpha and ACO mRNA levels were increased rapidly in the rat hepatoma cell line FaO after treatment with PFDA or the prototypical peroxisome proliferator Wy 14,643. In this cell line, PPAR alpha mRNA levels are not affected by glucagon or insulin and in addition to peroxisome proliferators are induced in this cell line by oleic acid and dexamethasone. The latter treatment had the greatest effect on PPAR alpha mRNA accumulation while having a minimal effect on ACO mRNA. Treatment of FaO cells with actinomycin D prior to Wy 14,643 abolished ACO and PPAR alpha mRNA accumulation, demonstrating that there must be a transcriptional component of the peroxisome proliferator response. Therefore, although PPAR alpha is responsive to peroxisome proliferators and direct effects are observed in cell cultures, mRNA accumulation in vivo is predominantly posttranscriptional, and endogenous regulators such as glucocorticoids may play critical roles in the tissue- and developmentally specific expression of this steroid hormone receptor.

Alterations in thyroid function in female Sprague-Dawley rats following chronic treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multisite carcinogen. Although the hepatocarcinogenic actions of TCDD have received the most attention, it has been demonstrated in several rodent carcinogenicity bioassays that TCDD causes a dose-related increase in thyroid follicular cell adenomas and carcinomas. The purpose of the present experiment was to investigate the dose-response relationship for thyroid function alterations in female Sprague-Dawley rats following chronic treatment with TCDD. TCDD was administered via oral gavage biweekly for 30 weeks at average daily equivalent doses of 0.1-125 ng/kg/day, thereby more than encompassing the dose range historically used in previous TCDD rodent bioassays. The endpoints examined include serum levels of thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH). In addition, the induction of the dioxin-responsive genes UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P450 1A1 (CYP1A1) in liver were measured using reverse-transcriptase-polymerase chain reaction (RT-PCR). In agreement with previous hypotheses, TCDD appears to alter thyroid function via a secondary mechanism, namely increased excretion of T4-glucuronide resulting from TCDD induction of UGT1. The observed follicular cell hyperplasia and hypertrophy are consistent with the observed elevated TSH levels and may represent the early stages in the progression of thyroid carcinogenesis. Therefore, TCDD induces alterations in thyroid hormone function, probably as a result of chronic perturbations of liver-pituitary-thyroid axis.