Biomedical subjects

T R Zacharewski

Publications and source records attributed to T R Zacharewski.

At least 19 recordsLinked to original sources

PubMed2003

Identification of temporal patterns of gene expression in the uteri of immature, ovariectomized mice following exposure to ethynylestradiol.

Estrogen induction of uterine wet weight provides an excellent model to investigate relationships between changes in global gene expression and well-characterized physiological responses. In this study, time course microarray GeneChip data were analyzed using a novel approach to identify temporal changes in uterine gene expression following treatment of immature ovariectomized C57BL/6 mice with 0.1 mg/kg 17alpha-ethynylestradiol. Functional gene annotation information from public databases facilitated the association of changes in gene expression with physiological outcomes, which allowed detailed mechanistic inferences to be drawn regarding cell cycle control and proliferation, transcription and translation, structural tissue remodeling, and immunologic responses. These systematic approaches confirm previously established responses, identify novel estrogen-regulated transcriptional effects, and disclose the coordinated activation of multiple modes of action that support the uterotrophic response elicited by estrogen. In particular, it was possible to elucidate the physiological significance of the dramatic induction of arginase, a classic estrogenic response, by elucidating its mechanistic relevance and delineating the role of arginine and ornithine utilization in the estrogen-stimulated induction of uterine wet weight.

Animals↗

PubMed2003

Effect of human dietary exposure levels of genistein during gestation and lactation on long-term reproductive development and sperm quality in mice.

The objective of the present study was to determine the long-term reproductive effects of gestational and lactational exposure (0, 0.1, 0.5, 2.5 and 10 mg/kg/day) to genistein on male mice at levels comparable to or greater than human dietary exposures. Testicular growth, sperm count and motility, and sperm fertilizing ability in vitro was assessed in male offspring on postnatal days (PND) 105 and 315. Selected genes were also examined by real-time PCR to determine whether genistein caused changes in gene expression similar to those previously observed with diethylstilbestrol (DES). No significant treatment-related effects on male offspring body weight, anogenital distance, seminal vesicle weight or testis weight were observed. There were also no significant effects on sperm count, the percent of motile sperm or the number of motile sperm at any age. The in vitro fertilizing ability of epididymal sperm was increased significantly in the high-dose group approximately 17% (P < 0.001) on PND 105 and 315. The results indicate that developmental exposure of mice to genistein at human exposure levels does not induce adverse effects on sperm quality or changes in testicular gene expression similar to DES.

Animals↗

PubMed2002

Ability of structurally diverse natural products and synthetic chemicals to induce gene expression mediated by estrogen receptors from various species.

The ability of 14 structurally diverse estrogenic compounds to induce reporter gene expression mediated by estrogen receptors (ERs) from different species was examined. MCF-7 cells were transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and Gal4-ER chimeric receptors containing the D, E and F domains of the human alpha (Gal4-hERalphadef), mouse alpha (Gal4-mERalphadef), mouse beta (Gal4-mERbetadef), chicken (Gal4-cERalphadef), green anole (Gal4-aERalphadef), Xenopus (Gal4-xERdef) or rainbow trout alpha ERs (Gal4-rtERalphadef). The efficacy of 17beta-estradiol (E2) in inducing reporter gene expression was similar among the different constructs overall, with EC(50) values ranging from 0.05 to 0.7nM. However, Gal4-rtERalphadef had an EC(50) value at 37 degrees C of 28nM, though at 20 degrees C an EC(50) value of 1nM was observed. Despite a similar response to E2 treatment among the ERs, many differences were observed in the magnitude of the response to other structurally diverse chemicals. For example, coumestrol induced Gal4-mERbetadef- and Gal4-aERdef-mediated reporter gene expression 164- and 8-fold greater, respectively, than mediated with the other Gal4-ERs. As well, in contrast to results with other Gal4-ERs, alpha-zearalenol consistently induced Gal4-rtERalphadef-mediated reporter gene activity at lower concentrations than did E2. Overall, the results demonstrate that selected estrogenic compounds exhibit a differential ability to induce reporter gene activity mediated by ERs from different vertebrate species. These data also highlight the importance of incubation temperature when examining rtERalpha-mediated activity.

Animals↗

PubMed2002

GP3: GenePix post-processing program for automated analysis of raw microarray data.

UNLABELLED: Here we describe an automated and customizable program to correct, filter and normalize raw microarray data captured using GenePix, a commonly used microarray image analysis application. Files can be processed individually or in batch mode for increased throughput. User defined inputs specify the stringency of data filtering and the method and conditions of normalization. The output includes gene summaries for replicate spots and descriptive statistics for each experiment. The source code (Perl) can also be adapted to handle raw data output from other image analysis applications. AVAILABILITY: http://bch.msu.edu/~zacharet/microarray/GP3.html

Algorithms↗

PubMed2002

Estrogen receptor-mediated actions of polyphenolic catechins in vivo and in vitro.

Recent investigations have demonstrated that polyphenolic catechins inhibit breast cancer cell proliferation and tumor growth. However, the ER-mediated effects of the three predominant catechins (EGCG, ECG, and EGC) have not been extensively examined in vitro or in vivo. Therefore, EGCG, ECG, and EGC were examined for their ability to compete with [(3)H]-17beta-estradiol ([(3)H]-E(2)) for binding to ERalpha and ERbeta and to elicit reporter gene activity in MCF-7 human breast cancer cells transiently transfected with either chimeric ERalpha or ERbeta. EGCG and ECG displaced [(3)H]-E(2) from GST-hERalphadef (D, E, and F domains of human ERalpha fused to GST) or from full-length human ERbeta. Additionally, only EGCG elicited Gal4-hERalphadef and Gal4-mERbetadef-mediated reporter gene expression (EC(50) values: 28 and 19 micro M, respectively) in MCF-7 cells cotransfected with a Gal4-regulated luciferase reporter gene. In cotreatment experiments, EGCG (1-50 micro M) and ECG (1 micro M) decreased E(2)-induced (1 nM) ERbeta-mediated gene expression 35-50%. In vivo, no catechin induced estrogenic responses (uterine weight or uterine peroxidase activity) in immature C57BL/6 mice. However, when mice were cotreated with E(2) (10 micro g/kg/day, 3 days) and either EGCG (30 and 50 mg/kg/day, 3 days) or ECG (50 mg/kg/day, 3 days), uterine peroxidase activity was increased 2.3-fold above that elicited by E(2) alone. In conclusion, EGCG and ECG bind to ERalpha and ERbeta, but only EGCG elicited ER-mediated gene expression in vitro. However, both of these compounds moderately increased E(2)-inducible responses in vivo.

Animals↗

PubMed2001

Reciprocal mutagenesis between human alpha(L349, M528) and rainbow trout (M317, I496) estrogen receptor residues demonstrates their importance in ligand binding and gene expression at different temperatures.

Several fish proteins exhibit compromised function at temperatures outside of their normal physiological range. In this study, the effect of temperature on the ligand binding and the transactivation abilities of the rainbow trout estrogen receptor (rtER) and human estrogen receptor alpha (hER alpha) were examined. Saturation analysis and gene expression assays, using GST-ER and Gal4-ER fusion proteins consisting of the D, E and F domains of human (hER alpha def) and rainbow trout (rtERdef) receptors, show that GST-rtERdef E2 binding affinity and transactivation ability decrease with increasing temperature. A comparison of the amino acid sequence differences between their ligand binding pockets identified two conservative amino acid residue substitutions in rtER (M317, I496) and hER alpha (L349, M528). The effect of these substitutions on ligand binding and transactivation were examined by constructing reciprocal mutants, which effectively exchanged the binding pockets between rtER and hER alpha. The rtERdef M317L:I496M double mutant exhibited increased E2 binding affinity and transactivation ability at higher temperatures, and displayed hER alpha phenotypic behavior for the phytoestrogen, coumestrol. The hER alpha def L349M:M528I double mutant also exhibited a modest trend towards adopting the rtER phenotype. These studies demonstrate that conservative changes in residue hydrophobicity and volume can significantly affect ER ligand binding and transactivation ability in a temperature-dependent manner. The lack of a complete exchange of phenotypes between rtER and hER alpha indicates that factors outside of the ligand binding pocket are also involved.

Amino Acid Sequence↗

PubMed2001

Interaction of PAH-related compounds with the alpha and beta isoforms of the estrogen receptor.

The ability of several 4- and 5-ring polycyclic aromatic hydrocarbons (PAHs), heterocyclic PAHs, and their monohydroxy derivatives to interact with the estrogen receptor (ER) alpha and beta isoforms was examined. Only compounds possessing a hydroxyl group were able to compete with 3H-labeled 17beta-estradiol (E2) for binding to either a glutathione-S-transferase and human ERalpha D, E, and F domain fusion protein (GST-hERalphadef) or to the full-length human ERbeta. Competitive binding was comparable for both isoforms, with IC(50) values ranging from 20 to 300 nM (E2 IC(50) approximately 3 nM). However, several compounds were able to induce reporter gene expression preferentially through mERbeta, using MCF-7 cells transiently transfected with either a Gal4-human ERalphadef or Gal4-mouse ERbetadef construct, as well as a Gal4-regulated reporter. These data extend the number and type of PAH-related compounds capable of interacting with ERalpha and ERbeta, and provides additional evidence that even though some compounds may possess a similar affinity for both ER isoforms, the capacity for transcriptional activation can still be isoform-specific.

Animals↗

PubMed2001

Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones.

This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62. 2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1% of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9% of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel pre-screening, colony isolation and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted. When this is not possible, pre-screening non-sequence verified clones with agarose gel electrophoresis provides an inexpensive and efficient method to eliminate contaminated clones from the probe set.

Animals↗

PubMed2001

In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors alpha and beta.

The estrogenic activities of bisphenol A (BPA) and its major metabolite BPA glucuronide (BPA-G) were assessed in a number of in vitro and in vivo assays. BPA competed with [3H]-17beta-estradiol (E2) for binding to mouse uterine cytosol ER, a glutathione S-transferase (GST)-human ER D, E, and F domain fusion protein (GST-hERalphadef) and full-length recombinant hERbeta. The IC(50) values for E2 were similar for all three receptor preparations, whereas BPA competed more effectively for binding to hERbeta (0.96 microM) than to either mouse uterine cytosol ER (26 microM) or GST-hERalphadef (36 microM). In contrast, BPA-G did not competitively displace [3H]E2 from any of the ER preparations. In MCF-7 cells transiently transfected with Gal4-hERalphadef or Gal4-hERbetadef, BPA induced reporter gene activity with comparable EC(50) values (71 and 39 microM, respectively). No significant induction of reporter gene activity was seen for BPA-G. Cotreatment studies showed that concentrations of (10 microM) BPA and BPA-G did not antagonize E2-induced luciferase mediated through either Gal4-hERalphadef or Gal4-hERbetadef. In vivo, the uterotropic effect of gavage or subcutaneous (sc) administration of 0.002-800 mg of BPA/kg of body weight/day for three consecutive days was examined in immature rats. Dose-related estrogenic effects on the rat uterus were observed at oral doses of 200 and 800 mg/kg and at sc doses of 10, 100, and 800 mg/kg. These results demonstrate that BPA competes more effectively for binding to ERbeta, but induces ERalpha- and ERbeta-mediated gene expression with comparable efficacy. In contrast, BPA-G did not exhibit any in vitro estrogenic activity. In addition, there was a clear route dependency on the ability of BPA to induce estrogenic responses in vivo.

Animals↗

PubMed2001

Hydroxylated benzo[a]pyrene metabolites are responsible for in vitro estrogen receptor-mediated gene expression induced by benzo[a]pyrene, but do not elicit uterotrophic effects in vivo.

The estrogenic activities of benzo[a]pyrene (B[a]P) and 10 metabolites (1, 3-, 7-, and 9-hydroxy-B[a]P; 4,5-, 7,8-, and 9,10-dihydrodihydroxy-B[a]P; and 1,6-, 3,6-, and 6,12-B[a]P-dione) were investigated. In vitro, B[a]P did not displace tritiated 17beta-estradiol ([3H]E2) from either a bacterially expressed fusion protein consisting of glutathione-S:-transferase linked to the D, E, and F domains of human ERalpha (GST-hERalphadef), or from full-length human ERbeta (hERbeta) at concentrations as high as 60 microM. However, 10 microM B[a]P demonstrated partial agonist activity in human Gal4-ERalphadef and mouse Gal4-ERbetadef reporter gene assays in transiently transfected MCF-7 cells, relative to 10 nM E2. 1-, 3-, 7-, and 9-hydroxy-B[a]P were found to bind to both receptor isoforms, each showing a higher affinity for the beta isoform. At 10 microM the four monohydroxylated metabolites were able to induce Gal4-hERalphadef- and Gal4-mERbetadef-mediated reporter gene expression to levels 20-100% of that caused by 10 nM E2, suggesting that these metabolites, and not the parent compound, induced reporter gene expression following B[a]P treatment of transiently transfected MCF-7 cells. In addition, the effect of B[a]P on two estrogen-inducible end points, uterine weight and lactoferrin mRNA levels, was determined in ovariectomized DBA/2 and C57BL/6 mice. Neither orally administered B[a]P at doses as high as 10 mg/kg body weight nor subcutaneously injected 3- or 9-hydroxy-B[a]P at doses as high as 20 mg/kg induced effects on uterine wet weight or uterine lactoferrin mRNA levels in either strain. These data suggest that B[a]P metabolites that are estrogenic at high concentrations in vitro do not induce estrogenic effects in the mouse uterus.

Animals↗

PubMed2001

Challenges and limitations of gene expression profiling in mechanistic and predictive toxicology.

RNA and protein expression profiling technologies have revolutionized how toxicologists can study the molecular basis of adverse effects of chemicals and drugs. It is expected that these new technologies will afford efficient and high-throughput means to delineate mechanisms of action and predict toxicity of unknown agents. To reach these goals, a more thorough understanding of the constraints of the methodology is needed to design genome-scale studies and to interpret the vast amount of data collected. This paper addresses some of the limitations and uncertainties of gene expression profiling in mechanistic and predictive toxicology with respect to the expectations of toxicogenomics. The challenges associated with interpreting information from large-scale gene expression experiments in vivo is also discussed.

Animals↗

PubMed2000

Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17 beta or alpha-zearalenol.

Estrogen receptor-mediated induction of zona radiata (ZR) and vitellogenin (VTG) mRNA and protein in rainbow trout (Oncorhynchus mykiss) was compared to assess their utility as biomarkers for exposure to estrogenic compounds. Partial sequences of rainbow trout ZR and beta-actin were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers based on conserved regions across a number of species. A 549 bp fragment of the rainbow trout ZR-gene showed a high degree of amino acid sequence identity to that of salmon (77%), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp beta-actin fragment was approximately 100% identical to sequences from several species. Real-time PCR was used to quantify the induction of ZR-gene and VTG in rainbow trout liver after in vivo exposure to estradiol-17 beta (E(2)) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or alpha-zearalenol (alpha-ZEA) (0.1, 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) showed that ZR and VTG were induced in both the liver and the plasma after a single injection of E(2) or alpha-ZEA. ZR was more responsive to low levels of E(2) and alpha-ZEA than VTG, and real-time PCR was shown to be more sensitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensitive biomarker for assessing estrogenic activity.

Actins↗

PubMed2000

Interactions between human plasma sex hormone-binding globulin and xenobiotic ligands.

Human sex hormone-binding globulin (SHBG) binds sex steroids with high affinity. In plasma, the number of SHBG steroid-binding sites far exceeds the molar concentrations of sex steroids, and will accommodate other ligands such as phytoestrogens and fatty acids. We have therefore developed a screening assay to identify ligands for SHBG, which exist in our diet or environment. This assay allows the binding of potential ligands to SHBG to be assessed under physiological conditions, and is sensitive to the effects of plasma constituents. Several classes of endocrine active compounds were tested, including hydroxy-polychlorinated biphenyls (HO-PCBs), phthalate esters, monoesters, chlorinated pesticides, as well as synthetic estrogens and phytoestrogens. The relative binding affinities (RBAs) of various compounds to SHBG were determined in competitive displacement assays, by comparison with 17 beta-estradiol (RBA=100). Synthetic estrogens bound SHBG with RBAs of 0.4 (ethinylestradiol)-0.2 (diethylstilbestrol), while some phytoestrogens bound with RBAs of 0.12 (coumestrol)-0.04 (naringenin). Many compounds did not bind to SHBG with sufficient affinity to allow RBA measurements, and these include: several phytoestrogens, such as genistein and kaempferol, polychlorinated biphenyls, phthalate esters and monoesters. Of nine HO-PCB congeners tested only 4-OH-2', 3', 4', 5'-tetraCB and 4-OH-2, 2', 3', 4', 5'-pentaCB bound SHBG in undiluted serum with RBAs of 0.05 and 0.11. Although all test compounds bound to SHBG with much lower affinity than endogenous sex steroids, these interactions may be physiologically relevant in situations where plasma SHBG levels are high and endogenous sex steroid levels are low, such as in pre-pubertal children and women taking oral contraceptives.

Environmental Pollutants↗

PubMed2000

Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-alpha reporter gene assay.

A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations > or = 1 microM produced responses comparable to that of 0.1 nM 17beta-estradiol (E2). The ER antagonist ICI 182,780 (ICI) completely inhibited the response to both E2 and B[a]P, indicating that the responses were ER-mediated. However, 2 microM alpha-napthoflavone (alpha-NF), an Ah receptor antagonist and P450 inhibitor, also decreased the response to B[a]P but not to E2. Analysis of the profile of B[a]P metabolites in the transfected MCF-7 cultures indicated that alpha-NF inhibited the production of the 3- and 9-hydroxy (3-OH and 9-OH), as well as the 7, 8- and 9,10-dihydroxy (7,8-OH and 9,10-OH) B[a]P species. In the ER-alpha reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 microM and 0.7 microM, respectively. The 9,10-OH metabolite exhibited minimal activity in the assay. These responses were inhibited by ICI for both the 3-OH and the 9-OH species; however, alpha-NF inhibited only the response to the 9-OH metabolite. The 7,8-OH metabolite did not exhibit significant estrogenic activity. Furthermore, 7,8-OH B[a]P displayed observable cytotoxicity at concentrations > or = 10(-7) M. This cytotoxic response was completely inhibited by alpha-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites.

Benzo(a)pyrene↗

PubMed1999

pS2 (TFF1) levels in human breast cancer tumor samples: correlation with clinical and histological prognostic markers.

The expression of pS2 (TFF1) has been previously shown to identify patients with improved response to anti-hormonal therapy and more favorable outcome. In the current study, 100 human breast carcinoma samples obtained from the Manitoba Breast Tumor Bank were analyzed for pS2 mRNA using a quantitative, competitive reverse transcriptase-polymerase chain reaction (qcRT-PCR) assay. A pS2/beta-actin cut-off criterion of 0.010 was established to classify tumors as either pS2 positive or pS2 negative. pS2 mRNA levels were positively associated with both ER and PR, with the majority of ER+ (59%) and PR+ (60%) tumors also being positive for pS2. In addition, a significant linear correlation was observed between the amount of pS2 mRNA and ER (p < 0.0001) and PR (p < 0.0001) protein. pS2 mRNA levels also exhibited an inverse association with tumor size and histological grade, consistent with the observation that pS2 is primarily expressed in small (T < 2.0 cm), but well differentiated tumors (Grades I and II). No associations were observed with tumor cell type, patient age, or lymph node status. The strong correlation displayed between pS2 and a number of currently used breast cancer prognostic markers supports the clinical use of pS2 to further assess tumor status and patient outcome.

Actins↗

PubMed1998

N,N-Dimethylformamide modulates acid extrusion from murine hepatoma cells.

N,N-Dimethylformamide (DMF) affects cellular differentiation, causes hepatotoxicity and gastric irritation, and may be carcinogenic. Since these processes involve changes in cellular pH homeostasis, we investigated the effects of DMF on H+ extrusion and cytosolic pH (pHi) of mouse hepatoma cells (Hepa 1C1C7). Extracellular pH was monitored using a silicon-based sensor system (Cytosensor microphysiometer) and pHi was monitored by fluorescence spectrophotometry. Superfusion of cells with DMF (0.25 to 0.5 M) suppressed the extracellular acidification rate (ECAR) below baseline. Following washout of DMF there was a rapid, concentration-dependent, prolonged overshoot of ECAR above baseline rates. Removal of extracellular Na+ or superfusion with amiloride abolished the overshoot in acidification rate, indicating involvement of Na+/H+ exchange. The overshoot was dependent on extracellular glucose, suggesting that it arises from an increase in metabolic acid production. Fluorescence measurements showed that DMF did not change pHi. Furthermore, DMF did not alter the rate of pHi recovery of cells acid loaded using nigericin, indicating that DMF does not directly alter Na+/H+ exchange activity in these cells. In summary, these data suggest that suppression of acidification rate by DMF is likely due to decreased metabolic acid production. Washout of DMF is then accompanied by increased glucose metabolism and H+ efflux via Na+/H+ exchange. It is possible that alterations in H+ production and transport contribute to the hepatotoxicity of DMF and its effects on cellular differentiation.

Amiloride↗

PubMed1998

Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters.

The estrogenic activities of eight phthalate esters (i.e., di-2-ethylhexyl, di-n-butyl (DBP), butylbenzyl (BBP), di-hexyl (DHP), diiso-heptyl, di-n-octyl, diiso-nonyl, diiso-decyl) were investigated in vitro using estrogen receptor (ER) competitive ligand-binding and mammalian- and yeast-based gene expression assays. In vivo, their effects on uterine wet weight and vaginal cell cornification using ovariectomized Sprague-Dawley rats were assessed. DBP, BBP, and DHP weakly competed with 17 beta-estradiol (E2) for binding to the ER in competitive ligand-binding assays. In gene expression assays using MCF-7 cells transiently transfected with the Gal4-human estrogen receptor construct, Gal4-HEGO, and the Gal4-regulated luciferase reporter gene, 17m5-G-Luc, 10 microM DBP, BBP, or DHP exhibited 36, 42, and 20% activity, respectively, when compared to the 100% response observed with 10 nM E2. Only BBP was found to induce luciferase activity (32%) in HeLa cells stably transfected with Gal4-HEGO and 17m5-G-Luc constructs and to impart minimal ER-mediated viability to the E2-dependent recombinant yeast strain, PL3, on selective medium. No significant responses were observed with the other phthalate esters in any of the in vitro assays. In vivo, none of the eight phthalate esters reproducibly induced significant increases in uterine wet weight in immature ovariectomized Sprague-Dawley rats treated with oral doses of 20, 200, or 2000 mg/kg of phthalate ester. In addition, treatment with phthalate esters at the same doses did not affect the degree of vaginal epithelial cell cornification in mature ovariectomized rats. These results indicate that only selected phthalate esters (i.e., DBP, BBP, and DHP) exhibit weak ER-mediated activity in some in vitro assays at high concentrations but none of the eight phthalate esters elicited in vivo estrogenic responses based upon results obtained from uterotrophic and vaginal cornification assays.

Animals↗

PubMed1997

Identification of a motif within the 5' regulatory region of pS2 which is responsible for AP-1 binding and TCDD-mediated suppression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds modulate several endocrine systems by altering hormone synthesis, enhancing ligand metabolism, and down-regulating receptor levels/binding activity. Previous studies have demonstrated that TCDD inhibits the 17beta-estradiol (E2)-induction of pS2, a human breast cancer prognostic marker. This inhibition occurs at the gene expression level and is Ah receptor (AhR)-mediated. Analysis of the 5' regulatory region has identified three motifs which resemble dioxin response element (DRE) core sequences. pS2-regulated luciferase deletion constructs identified the DRE-like motif located at -527 to -514 as being required for TCDD-mediated suppression. A point mutation within this core motif (T-518C) abolished the inhibition by TCDD while UV-induced protein-DNA cross-linking and competitive gel retardation assays demonstrated AhR complex binding to this motif. Further study of this region also revealed an adjacent putative AP-1 site, diverging by one base pair from the consensus sequence. Gel retardation assays using TPA-treated MCF-7 cell nuclear extracts showed an induced complex binding to the AP-1-like site. Competition studies and antibody supershifts confirmed that the retarded complex consists of AP-1-like proteins. pS2-regulated luciferase constructs containing mutations specific to the AP-1-like motif greatly diminished the inducibility in response to E2. These results suggest that an interaction between AhR complexes and AP-1-like proteins may be responsible for TCDD-mediated inhibition of E2-induced pS2 expression.

Base Sequence↗