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1.
Toxicol Appl Pharmacol ; 238(2): 101-10, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-18486176

ABSTRACT

Endocrine disruptive compounds (EDC) alter hormone-stimulated, nuclear receptor-dependent physiological and developmental processes by a variety of mechanisms. One recently identified mode of endocrine disruption is through hormone sensitization, where the EDC modulates intracellular signaling pathways that control nuclear receptor function, thereby regulating receptor transcriptional activity indirectly. Methoxyacetic acid (MAA), the primary, active metabolite of the industrial solvent ethylene glycol monomethyl ether and a testicular toxicant, belongs to this EDC class. Modulation of nuclear receptor activity by MAA could contribute to the testicular toxicity associated with MAA exposure. In the present study, we evaluated the impact of MAA on the transcriptional activity of several nuclear receptors including the androgen receptor (AR), which plays a pivotal role in the development and maturation of spermatocytes. AR transcriptional activity is shown to be increased by MAA through a tyrosine kinase signaling pathway that involves PI3-kinase. In a combinatorial setting with AR antagonists, MAA potentiated the AR response without significantly altering the EC(50) for androgen responsiveness, partially alleviating the antagonistic effect of the anti-androgens. Finally, MAA treatment of TM3 mouse testicular Leydig cells markedly increased the expression of Cyp17a1 and Shbg while suppressing Igfbp3 expression by ~90%. Deregulation of these genes may alter androgen synthesis and action in a manner that contributes to MAA-induced testicular toxicity.


Subject(s)
Acetates/toxicity , Endocrine Disruptors/toxicity , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Animals , Cell Line , Extracellular Signal-Regulated MAP Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Leydig Cells/drug effects , Leydig Cells/metabolism , Male , Mice , Phosphatidylinositols/metabolism , Receptors, Androgen/drug effects , Testis/drug effects , Testis/metabolism
2.
Toxicol Appl Pharmacol ; 199(3): 266-74, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15364542

ABSTRACT

Phthalate esters, widely used as plasticizers in the manufacture of products made of polyvinyl chloride, induce reproductive and developmental toxicities in rodents. The mechanism that underlies these effects of phthalate exposure, including the potential role of members of the nuclear receptor superfamily, is not known. The present study investigates the effects of phthalates on the pregnane X receptor (PXR), which mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The ability of phthalate monoesters to activate PXR-mediated transcription was assayed in a HepG2 cell reporter assay following transfection with mouse PXR (mPXR), human PXR (hPXR), or the hPXR allelic variants V140M, D163G, and A370T. Mono-2-ethylhexyl phthalate (MEHP) increased the transcriptional activity of both mPXR and hPXR (5- and 15-fold, respectively) with EC50 values of 7-8 microM. mPXR and hPXR were also activated by monobenzyl phthalate (MBzP, up to 5- to 6-fold) but were unresponsive to monomethyl phthalate and mono-n-butyl phthalate (M(n)BP) at the highest concentrations tested (300 microM). hPXR-V140M and hPXR-A370T exhibited patterns of phthalate responses similar to the wild-type receptor. By contrast, hPXR-D163G was unresponsive to all phthalate monoesters tested. Further studies revealed that hPXR-D163G did respond to rifampicin, but required approximately 40-fold higher concentrations than wild-type receptor, suggesting that the ligand-binding domain D163G variant has impaired ligand-binding activity. The responsiveness of PXR to activation by phthalate monoesters demonstrated here suggests that these ubiquitous environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals.


Subject(s)
Diethylhexyl Phthalate/analogs & derivatives , Environmental Pollutants/pharmacology , Phthalic Acids/pharmacology , Plasticizers/pharmacology , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Steroid/agonists , Receptors, Steroid/physiology , Transcription, Genetic/drug effects , Animals , Cell Line , Diethylhexyl Phthalate/pharmacology , Dose-Response Relationship, Drug , Genes, Reporter/genetics , Humans , Ligands , Mice , Plasmids/genetics , Pregnane X Receptor , Rifampin/pharmacology , Species Specificity , Transcriptional Activation/genetics , Transfection
3.
J Immunol ; 173(5): 3165-77, 2004 Sep 01.
Article in English | MEDLINE | ID: mdl-15322177

ABSTRACT

The common commercial use of phthalate esters has resulted in significant human exposure to these bioactive compounds. The facts that phthalate ester metabolites, like endogenous PGs, are peroxisome proliferator-activated receptor (PPAR) agonists, and that PPARgamma agonists induce lymphocyte apoptosis suggest that phthalate esters are immunosuppressants that could act together with PGs to modulate early B cell development. In this study we examined the effects of a metabolite of one environmental phthalate, mono(2-ethylhexyl)phthalate (MEHP), and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), on developing B cells. MEHP inhibited [(3)H]thymidine incorporation by primary murine bone marrow B cells and a nontransformed murine pro/pre-B cell line (BU-11). Cotreatment with a retinoid X receptor alpha ligand, 9-cis-retinoic acid, decreased [(3)H]thymidine incorporation synergistically, thereby implicating activation of a PPARgamma-retinoid X receptor alpha complex. These results were similar to those obtained with the natural PPARgamma ligand 15d-PGJ(2). At moderate MEHP concentrations (25 or 100 microM for primary pro-B cells and a pro/pre-B cell line, respectively), inhibition of [(3)H]thymidine incorporation resulted primarily from apoptosis induction, whereas at lower concentrations, the inhibition probably reflected growth arrest without apoptosis. Cotreatment of bone marrow B cells with 15d-PGJ(2) and MEHP significantly enhanced the inhibition of [(3)H]thymidine incorporation seen with MEHP alone, potentially mimicking exposure in the bone marrow microenvironment where PG concentrations are high. Finally, MEHP- and 15d-PGJ(2)-induced death does not result from a decrease in NF-kappaB activation. These data demonstrate that environmental phthalates can cooperate with an endogenous ligand, 15d-PGJ(2), to inhibit proliferation of and induce apoptosis in developing bone marrow B cells, potentially via PPARgamma activation.


Subject(s)
Apoptosis/physiology , B-Lymphocytes/metabolism , Bone Marrow Cells/metabolism , Diethylhexyl Phthalate/analogs & derivatives , Diethylhexyl Phthalate/metabolism , Prostaglandin D2/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Transcription Factors/agonists , Tretinoin/metabolism , Alitretinoin , Animals , Cell Cycle/physiology , Cell Division/physiology , Ligands , Mice , Prostaglandin D2/analogs & derivatives , Thymidine/metabolism , Tritium/metabolism
4.
Toxicol Sci ; 80(1): 151-60, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15071170

ABSTRACT

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that activate target genes involved in lipid metabolism, energy homeostasis, and cell differentiation in response to diverse compounds, including environmental chemicals. The liver-expressed receptor PPARalpha mediates peroxisome proliferative responses associated with rodent hepatocarcinogenesis. Previous studies have established that certain perfluorooctanesulfonamide-based chemicals (PFOSAs) alter lipid metabolism, are hepatic peroxisome proliferators, and induce hepatocellular adenoma formation in rodents, suggesting that they activate PPARalpha. The present study investigates this question and characterizes the activation of mouse and human PPARalpha by PFOSAs. Perfluorooctanesulfonate (PFOS), an end-stage metabolite common to several PFOSAs, was found to activate both mouse and human PPARalpha in a COS-1 cell-based luciferase reporter trans-activation assay. Half-maximal activation (EC50) occurred at 13-15 microM PFOS, with no significant difference in the responsiveness of mouse and human PPARalpha. Mouse and human PPARalpha were activated by perfluorooctanesulfonamide (FOSA) over a similar concentration range; however, cellular toxicity precluded an accurate determination of EC50 values. Studies of 2-N-ethylperfluorooctanesulfonamido ethanol were less informative due to its insolubility. These findings were verified in an FAO rat hepatoma cell line that stably expresses PPARalpha, where the endogenous PPARalpha target genes peroxisomal bifunctional enzyme and peroxisomal 3-ketoacyl-CoA thiolase were activated up to approximately 10-20-fold by PFOS and FOSA. The interactions of PPARalpha with PFOS and FOSA, and the potential of these chemicals for activation of unique sets of downstream target genes, may help explain the diverse biological effects exhibited by PFOSAs and may aid in the evaluation of human and environmental risks associated with exposure to this important class of fluorochemicals.


Subject(s)
Alkanesulfonic Acids/toxicity , Fluorocarbons/toxicity , PPAR alpha/biosynthesis , Peroxisome Proliferators/pharmacology , Sulfonamides/toxicity , Alkanesulfonic Acids/chemistry , Animals , Blotting, Western , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Fluorocarbons/chemistry , Gene Expression Regulation , Genes, Reporter , Humans , Hydrocarbons, Fluorinated , Luciferases/genetics , Mice , PPAR alpha/genetics , Peroxisome Proliferators/chemistry , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Solubility , Sulfonamides/chemistry , Transcriptional Activation
5.
Toxicol Sci ; 74(2): 297-308, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12805656

ABSTRACT

Phthalate esters are widely used as plasticizers in the manufacture of products made of polyvinyl chloride. Mono-(2-ethylhexyl)-phthalate (MEHP) induces rodent hepatocarcinogenesis by a mechanism that involves activation of the nuclear transcription factor peroxisome proliferator-activated receptor-alpha (PPARalpha). MEHP also activates PPAR-gamma (PPARgamma), which contributes to adipocyte differentiation and insulin sensitization. Human exposure to other phthalate monoesters, including metabolites of di-n-butyl phthalate and butyl benzyl phthalate, is substantially higher than that of MEHP, prompting this investigation of their potential for PPAR activation, assayed in COS cells and in PPAR-responsive liver (PPARalpha) and adipocyte (PPARgamma) cell lines. Monobenzyl phthalate (MBzP) and mono-sec-butyl phthalate (MBuP) both increased the COS cell transcriptional activity of mouse PPARalpha, with effective concentration for half-maximal response (EC50) values of 21 and 63 microM, respectively. MBzP also activated human PPARalpha (EC50=30 microM) and mouse and human PPARgamma (EC50=75-100 microM). MEHP was a more potent PPAR activator than MBzP or MBuP, with mouse PPARalpha more sensitive to MEHP (EC50=0.6 microM) than human PPARalpha (EC50=3.2 microM). MEHP activation of PPARgamma required somewhat higher concentrations, EC50=10.1 microM (mouse PPARgamma) and 6.2 microM (human PPARgamma). No significant PPAR activation was observed with the monomethyl, mono-n-butyl, dimethyl, or diethyl esters of phthalic acid. PPARalpha activation was verified in FAO rat liver cells stably transfected with PPARalpha, where expression of several endogenous PPARalpha target genes was induced by MBzP, MBuP, and MEHP. Similarly, activation of endogenous PPARgamma target genes was evidenced for all three phthalates by the stimulation of PPARgamma-dependent adipogenesis in the 3T3-L1 cell differentiation model. These findings demonstrate the potential of environmental phthalate monoesters for activation of rodent and human PPARs and may help to elucidate the molecular basis for the adverse health effects proposed to be associated with human phthalate exposure.


Subject(s)
Diethylhexyl Phthalate/analogs & derivatives , Environmental Pollutants/pharmacology , Phthalic Acids/pharmacology , Receptors, Cytoplasmic and Nuclear/biosynthesis , Transcription Factors/biosynthesis , 3T3-L1 Cells/cytology , 3T3-L1 Cells/drug effects , 3T3-L1 Cells/metabolism , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Animals , COS Cells/cytology , COS Cells/drug effects , COS Cells/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Differentiation/drug effects , Cell Line, Tumor , Chlorocebus aethiops , Diethylhexyl Phthalate/pharmacology , Dose-Response Relationship, Drug , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Mice , Peroxisome Proliferators/pharmacology , RNA, Messenger/metabolism , Rats , Receptors, Cytoplasmic and Nuclear/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transfection
6.
Toxicol Sci ; 65(1): 87-98, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11752688

ABSTRACT

In female rats, in uteroexposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during critical periods of organogenesis causes a permanent thread of tissue, consisting of a core of mesenchyme surrounded by keratinized epithelia, across the vaginal opening. The objective of the current study was to determine the earliest time after exposure to TCDD during fetal development that morphological changes in the development of the lower reproductive tract could be detected. In addition, the spatio-temporal expression of several growth factors within the developing reproductive tract was investigated to provide insight into the mechanism of action involved in TCDD-induced vaginal thread formation. Pregnant rats received a single oral dose of 1.0 microg TCDD/kg on gestation day (GD) 15. Dams were sacrificed on GD 17, 18, 19, and 21 and individual reproductive tracts were isolated from female fetuses. As early as GD 18, TCDD produced distinct abnormalities in the female reproductive tract. The width of mesenchyme separating the Mullerian ducts was significantly greater in TCDD-exposed female GD 18 and 19 fetuses and the zone of unfused Mullerian ducts was substantially increased on GD 19 and 21. TCDD induced alterations within the developing reproductive tract in the subcellular and temporal expression of transforming growth factor-beta3 (TGF-beta3) and epidermal growth factor receptor (EGFR). DNA array analysis suggested effects on several genes expressed on GD 18 and 19.


Subject(s)
Genitalia, Female/abnormalities , Mesonephros/drug effects , Mullerian Ducts/drug effects , Polychlorinated Dibenzodioxins/toxicity , Urethra/drug effects , Abnormalities, Drug-Induced/etiology , Animals , Embryonic and Fetal Development/drug effects , Female , Gene Expression Regulation, Developmental , Genes, erbB-1/genetics , Genitalia, Female/metabolism , Immunohistochemistry , Laminin/genetics , Laminin/metabolism , Maternal Exposure/adverse effects , Mesoderm/drug effects , Mesonephros/embryology , Mullerian Ducts/embryology , Pregnancy , Rats , Rats, Long-Evans , Time Factors , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta3 , Urethra/embryology
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