Mouse Hepatoma Hepa-1c1c7 Cells Research Articles

Induction of hepatic thioredoxin reductase activity by sulforaphane, both in Hepa1c1c7 cells and in male Fisher 344 rats

Sulforaphane (SF), a glucosinolate-derived isothiocyanate found in cruciferous vegetables, is considered an anticarcinogenic component in broccoli. Sulforaphane induces a battery of detoxification enzymes, including quinone reductase (QR). Induction is thought to be mediated through a common regulatory region termed the antioxidant response element (ARE). To test the hypothesis that the antioxidant selenoprotein thioredoxin reductase (TR) may be induced as part of this coordinated host-defense response to dietary anticarcinogenic compounds, TR activity was measured in livers of rats pair-fed diets containing SF and/or broccoli (n = 6/group). At the doses used, neither SF nor broccoli alone significantly elevated TR activity, whereas treatments containing both broccoli and SF caused a significant increase in TR activity. Glutathione peroxidase (GSH-Px), a second selenium-dependant enzyme with antioxidant activity, was downregulated in rats fed both SF and broccoli, compared to the control diet. A second experiment, using mouse hepatoma Hepa1c1c7 cells, tested whether an interaction exists between selenium (Se) and SF in TR inducibility, since Se is known to induce TR activity. Selenium (2.5 μM) plus SF (2.0 μM) caused significantly greater TR activity than either treatment alone. All treatments with added Se or SF caused significantly greater TR activities than no Se or SF treatment. Glutathione peroxidase activity was elevated by Se, but not by SF. These data suggest that TR, known to be regulated by Se, is also upregulated as part of a host response to the dietary anticarcinogen SF, a trait not shared by another Se-dependent enzyme, GSH-Px.

Differential responses from seven mammalian cell lines to the treatments of detoxifying enzyme inducers

Cell-based models have been used extensively in screening novel bioactive chemical entities. In this study, seven well-established mammalian cell lines, which have different origins, were utilized to compare their responses to the treatments of three detoxifying enzyme inducers, tert-butylhydroquinone (tBHQ), β-naphthoflavone (β-NF), and sulforaphane (SUL), which are potential chemopreventive compounds. The enzymatic activities of glutathione s-transferase (GST), NAD(P)H:quinone oxidoreductase (QR), aldehyde reductase (AR), and glutathione reductase (GR) were measured by kinetics methods using UV-Vis spectroscopy, and analyzed statistically by Student's t-test. Among these mammalian cell lines, the mouse hepatoma Hepa1c1c7 cells were the most robust and sensitive cells, which had higher basal as well as upregulated enzymatic activities. In human cell lines, the prostate LNCaP and hepatic HepG2 cells were also very responsive to the inducers. The results suggested that different cell lines responded differently to individual detoxifying gene inducer, and the selection of appropriate cell line is important for screening potential chemopreventive agents.

Antiproliferative effect of antioxidant polyphenols from grape in murine Hepa-1c1c7.

Grapes and wine contain high concentrations of polyphenolic compounds. Although their cancer protective effect has been well documented, their activity as anticarcinogens should be cautiously considered since the molecular bases of action and their applicability to human cancer prevention are still unclear. We studied the antioxidant/antiradical activity and the antiproliferative effect in vitro of different polyphenolic mixtures, extracted from grapes and fractionated through RP-HPLC. The polyphenolic fractions were chemically characterized and their antioxidant/antiradical activity was determined by the DPPH assay. Mouse hepatoma Hepa-1c1c7 cells were used to study the cell growth inhibition capacity of these fractions by MTT assay. Their capacity of altering cell cycle and possible induction of apoptosis was examined using FACS analysis. The original polyphenolic fraction OW, which contained gallic acid (GA), (+)-catechin (Cat), (-)-epicatechin (Ec), glycosylated flavonols (F) and procyanidin oligomers was fractionated into fraction I, composed of monomers and small oligomers, and fraction II that included flavonols and procyanidin oligomers of higher molecular weight. The three polyphenolic fractions tested showed quite similar antiradical activity, although fraction I was the most potent antiradical agent (lowest ED(50) value: 9 microg). Fraction II was the least potent cell growth inhibitor (highest IC(50) value: 100 microg/ml) but showed the strongest effect on the cell cycle of Hepa-1c1c7, inducing apoptosis in those cells. The original fraction OW was demonstrated to have the most potent cell growth inhibition effect (lowest IC(50) value: 43 microg/ml). However, it only appeared to alter cell cycle of Hepa-1c1c7 at concentrations higher than its IC(50) and did not induce apoptosis in those cells. A similar effect on cell cycle and apoptosis was encountered for fraction I. The polyphenolic fractions tested in this study were potent antiradical agents and exerted an antiproliferative effect in mouse hepatoma Hepa-1c1c7 cells; the fraction with the highest degree of polimerization and galloylation (fraction II) had the most influence on the cell cycle and induction of apoptosis on Hepa-1c1c7.

Constituents of Musa x paradisiaca cultivar with the potential to induce the phase II enzyme, quinone reductase.

A new bicyclic diarylheptanoid, rel-(3S,4aR,10bR)-8-hydroxy-3-(4-hydroxyphenyl)-9-methoxy-4a,5,6,10b-tetrahydro-3H-naphtho[2,1-b]pyran (1), as well as four known compounds, 1,2-dihydro-1,2,3-trihydroxy-9-(4-methoxyphenyl)phenalene (2), hydroxyanigorufone (3), 2-(4-hydroxyphenyl)naphthalic anhydride (4), and 1,7-bis(4-hydroxyphenyl)hepta-4(E),6(E)-dien-3-one (5), were isolated from an ethyl acetate-soluble fraction of the methanol extract of the fruits of Musa x paradisiaca cultivar, using a bioassay based on the induction of quinone reductase (QR) in cultured Hepa1c1c7 mouse hepatoma cells to monitor chromatographic fractionation. The structure and relative stereochemistry of compound 1 were elucidated unambiguously by one- and two-dimensional NMR experiments ((1)H NMR, (13)C NMR, DEPT, COSY, HMQC, HMBC, and NOESY) and single-crystal X-ray diffraction analysis. Isolates 1-5 were evaluated for their potential cancer chemopreventive properties utilizing an in vitro assay to determine quinone reductase induction and a mouse mammary organ culture assay.

Cell-type specificity of ectonucleotidase expression and upregulation by 2,3,7,8-tetrachlorodibenzo- p-dioxin

We report here that induction of ectoATPase by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is cell-type specific and not a generalized response to aryl hydrocarbon ( Ah) receptor activation. TCDD increased [ 14C]-ATP and -ADP metabolism in two mouse hepatoma lines, Hepa1c1c7 and Hepa1-6 cells, but not in human hepatoma HepG2 or HuH-7 cells, human umbilical vein endothelial cells (HUVEC), chick hepatoma (LMH) cells, or chick primary hepatocytes or cardiac myocytes, even though all of those cell types were Ah receptor-responsive, as evidenced by cytochrome P4501A induction. To determine whether the differences in ectonucleotidase responsiveness to TCDD might be related to differences in cell-type ectonucleotidase expression, ATP and ADP metabolite patterns, the products of several classes of ectonucleotidases including ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectophosphodiesterase/pyrophosphatases (E-NPP enzymes) and ectoalkaline phosphatase activities were examined. Those patterns, together with results of enzyme assays, Western blotting, or semiquantitative RT–PCR show that NTPDase2 is the main ectonucleotidase for murine and human hepatoma cells, NTPDase3 for chick hepatocytes and LMH cells, and an E-NPP enzyme for chick cardiac myocytes. Evidence for NTPDase2 expression was lacking in all cells except the mouse and human hepatoma cells. TCDD increased expression of the NTPDase2 gene but only in the mouse and not in the human hepatoma cells. TCDD did not increase NTPDase3, NTPDase1, E-NPP, or alkaline phosphatase in any of the cell types examined. The failure of TCDD to increase ATP metabolism in HUVEC, chick LMH cells, hepatocytes, and cardiac myocytes can be attributed to their lack of NTPDase2 expression, while the increase in ATP metabolism by TCDD in the mouse but not the human hepatoma cells can be explained by differences in TCDD effects on mouse and human hepatoma NTPDase2 gene expression. In addition to characterizing effects of TCDD on ectonucleotidases, these studies reveal major differences in the complements of ectonucleotidases present in different cell types. It is likely that such differences are important for cell-specific susceptibility to extracellular nucleotide toxicity and responses to purinergic signaling.

Effects of o, p′-DDT on the 2,3,7,8-tetrachlorodibenzo- p-dioxin-inducible CYP1A1 expression in murine Hepa-1c1c7 cells

Cultured mouse hepatoma Hepa-1c1c7 cells were treated with o, p′-DDT and/or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) to assess the role of o, p′-DDT on CYP1A1 expression. o, p′-DDT alone did not affect CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. In contrast, TCDD-inducible EROD activities were markedly reduced on concomitant treatment with TCDD and o, p′-DDT in a dose-dependent manner. Treatment with ICI 182.780, an estrogen-receptor antagonist, did not affect the suppressive effects of o, p′-DDT on TCDD-inducible EROD activity. TCDD-inducible CYP1A1 mRNA levels were markedly suppressed on treatment with TCDD and o, p′-DDT, and this was consistent with their effects on EROD activity. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and an electrophoretic mobility shift assay revealed that o, p′-DDT reduced the transformation of the aryl hydrocarbons (Ah) receptor to a form capable of specifically binding to the DRE sequence in the promoter region of the CYP1A1 gene. These results suggest that the downregulation of CYP1A1 gene expression by o, p′-DDT in Hepa-1c1c7 cells might be an antagonism of the DRE binding potential of the nuclear Ah receptor but is not mediated through the estradiol receptor.

Development of a Green Fluorescent Protein-Based Cell Bioassay for the Rapid and Inexpensive Detection and Characterization of Ah Receptor Agonists

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of a variety of chemicals. Although halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs, respectively) represent the highest affinity and most toxic ligands, recent studies have demonstrated that the AhR can be activated by chemicals with structures distinctly different from HAHs/PAHs. In order to identify and characterize novel AhR ligands, we developed a rapid and inexpensive high-throughput screening bioassay based on the ability of AhR agonists to induce an HAH/PAH-responsive, enhanced green fluorescent protein (EGFP) reporter gene in a stably transfected mouse hepatoma (Hepa1c1c7) cell line. EGFP induction in the resulting recombinant cell line, H1G1.1c3, is sensitive (with a minimal 1-pM detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin, the most potent AhR ligand), and it responds to HAHs and PAHs in a time-, dose-, and chemical-specific manner. Application of this bioassay was demonstrated by the rapid characterization of the relative inducing potency of a series of previously uncharacterized dioxin surrogates. This bioassay system has numerous advantages over currently available AhR-based bioassays including increased rapidity and ease of use, low reagent cost, and application for high-throughput screening.

Induction of quinone reductase activity by stilbene analogs in mouse Hepa 1c1c7 cells.

Based on the potential cancer chemopreventive activity of resveratrol, a trihydroxystilbene with the induction of quinone reductase activity, this study was designed to determine if stilbene-related compounds were inducers of phase II detoxifying metabolic enzyme quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cells. Among the thirteen compounds tested, several compounds including 3,4,5,3',5'-pentamethoxy-trans-stilbene were found to potentially induce QR activity in this cell line. In addition, substitution with 3-thiofurane ring instead of phenyl ring in the stilbene skeleton also exhibited potential induction of QR activity. This result will give primary information to design the potential inducers of QR activity in the stilbene analogs.

Induction of Cytochrome P450 1A1 Gene Expression by a Vitamin K3 Analog in Mouse Hepatoma Hepa-1c1c7 Cells

Induction of Cytochrome P450 1A1 Gene Expression by a Vitamin K3 Analog in Mouse Hepatoma Hepa-1c1c7 Cells

Tryphostin AG879, a tyrosine kinase inhibitor: prevention of transcriptional activation of the electrophile and the aromatic hydrocarbon response elements

To investigate a possible role of phosphorylation in the signal transduction pathways responsible for transcriptional regulation of drug-metabolizing enzymes, we tested seven specific tyrosine kinase inhibitors (tyrphostins) for their effects on NAD(P)H:quinone oxidoreductase-1 (NQO1) mRNA levels in mouse hepatoma Hepa-1c1c7 (Hepa-1) cells and chose to study AG879 further. The potent electrophile tert-butylhydroquinone (tBHQ) is known to activate NQO1 gene transcription via the electrophile response element (EPRE). Among the tyrphostins tested, tyrphostin AG879 was unique in preventing the accumulation of tBHQ-induced NQO1 mRNA; this effect was dependent on the AG879 dose and was also sensitive to the time when AG879 was added relative to the beginning of tBHQ treatment. 2,3,7,8-Tetrachlorodibenzo- p-dioxin (dioxin; TCDD) is known to activate Cyp1a1 gene transcription by way of aromatic hydrocarbon response elements (AHREs). We found that AG879 also prevents, to a lesser extent, the AHRE-mediated induction of CYP1A1 and NQO1 mRNA by dioxin. Zinc or cadmium is known to activate metallothionein ( Mt1) gene transcription via the metal response element (MRE). AG879 induced MT1 mRNA, and AG879 did not block zinc- or cadmium-induced MT1 mRNA, indicating that the effects of AG879 on NQO1 or CYP1A1 mRNA levels cannot be generalized to all transcripts. Using transient transfection of EPRE-, AHRE-, or MRE-driven luciferase reporter gene constructs in Hepa-1 cells, we showed that the inhibitory effects of AG879 occurred at the level of EPRE- and AHRE-mediated transcription, but that AG879 did not affect the MRE-driven transcriptional response. These data suggest that AG879 might inhibit an unknown tyrosine kinase(s) whose activity is essential for EPRE- and AHRE-mediated trans-activation of certain mammalian genes. These results also indicate that some sharing of common signal transduction pathways might exist in the regulation of genes involved in drug metabolism that also respond to oxidative stress.

Down-Regulation of Murine Cyp1a-1 in Mouse Hepatoma Hepa-1c1c7 Cells by Bisphenol A

Cultured mouse hepatoma Hepa-1c1c7 cells were treated with either bisphenol A or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or in combination to assess the role of bisphenol A in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cyp1a-1, as determined by analysis of 7-ethoxyresorufin O-deethylase (EROD) activities. Bisphenol A alone did not affect the activity of Cyp1a-1-specific EROD; in contrast, TCDD-induced EROD activities were markedly reduced in the concomitant treatment of TCDD and bisphenol A in a dose-dependent manner. Treatment with tamoxifen, an antiestrogen that acts through the estrogen receptor, did not affect the suppressive effects of bisphenol A on TCDD-induced EROD activity. TCDD-induced Cyp1a-1 mRNA levels were markedly suppressed in the concomitant treatment of TCDD and bisphenol A consistent with their effects on EROD activity. Transient transfection assay using dioxin-response element (DRE)-linked luciferase revealed that bisphenol A reduced transformation of the aryl hydrocarbons (Ah) receptor to a form capable of specifically binding to the DRE sequence in the promoter of the Cyp1a-1 gene. These results suggest the down-regulation of the Cyp1a-1 gene expression by bisphenol A in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear Ah receptor but not mediated through estradiol receptor.

Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells

The objective of this study was to determine if prenylchalcones (open C-ring flavonoids) and prenylflavanones from hops and beer are inducers of quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cell line. All the prenylchalcones and prenylflavanones tested were found to induce QR but not CYP1A1 in this cell line. In contrast, the synthetic chalcone, chalconaringenin, and the flavanone, naringenin, with no prenyl or geranyl groups, were ineffective in inducing QR. The hop chalcones, xanthohumol and dehydrocycloxanthohumol hydrate, also induced QR in the Ah-receptor-defective mutant cell line, Hepa 1c1c7 bp rc1. Thus, the prenylflavonoids represent a new class of monofunctional inducers of QR.

Differential gene expression in wild-type and arnt-defective mouse hepatoma (Hepa1c1c7) cells.

The aryl hydrocarbon nuclear translocator (Arnt) is a basic helix-loop-helix (per/Arnt/Ahr/sim) PAS-containing protein that can heterodimerize with the aryl hydrocarbon receptor (AhR), the hypoxia-inducible factor-1 alpha, and other PAS-containing proteins to form transcriptionally active complexes. To identify the genes whose expression is modulated by Arnt, we used the technique of differential display to compare the expression of genes in wild-type and Arnt-defective (BPRc1) mouse hepatoma (Hepa1c1c7) cells. Here we report two gene products whose expression was reduced in BPRc1 cells (a WW domain-binding, protein-like factor and one unknown gene product) when compared to wild-type cells, and two that were elevated (Steel factor and a serpin-like protein). Comparison of the relative expression of these gene products between two independently-derived, Arnt-defective cell lines, as well as in BPRc1 cells in which Arnt expression was restored by a stably integrated Arnt-expression plasmid, revealed that each gene was expressed in an Arnt-independent manner. Our results clearly demonstrate that gene expression in the variant cell clones is distinctly different from that of the parental wild-type Hepa1c1c7 cells from which they were derived and involves genes in addition to, and unrelated to, that of Arnt. The identification of these differentially expressed gene products suggests that caution should be exercised when using these variant cell lines to confirm the role of the AhR/Arnt-signaling pathway in a given cellular response.

Metabolism-Based Polycyclic Aromatic Acetylene Inhibition of CYP1B1 in 10T1/2 Cells Potentiates Aryl Hydrocarbon Receptor Activity

We have used polycyclic aromatic hydrocarbon (PAH) alkyne metabolism-based inhibitors to test whether CYP1B1 metabolism is linked to aryl hydrocarbon receptor (AhR) activation in mouse embryo fibroblasts (MEF). 1-ethynylpyrene (1EP) selectively inactivated CYP1B1 dimethylbenzanthracene (DMBA) metabolism in C3H10T1/2 MEFs; whereas 1-(1-propynyl)pyrene (1PP) preferentially inhibited CYP1A1 activity in Hepa-1c1c7 mouse hepatoma cells (Hepa). In each cell type >90% inhibition of DMBA metabolism after 1 h treatment with each inhibitor (0.1 μM) was progressively reversed and then increased to levels seen with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induction (fourfold stimulation). It was found that 0.1 μM 1EP and 1PP maximally induce CYP1B1 and CYP1A1 mRNA levels in10T1/2 and Hepa cells, respectively, after 6 h. 1-Ethylpyrene (EtP), which lacks the activatable acetylene moiety, was far less effective as an inhibitor and as an inducer. AhR activation is essential for 1EP induction as evidenced by the use of AhR antagonists and AhR-deficient MEFs and absence of induction following inhibition of DMBA metabolism with carbon monoxide (CO). Inhibition of CYP1B1 was linked to enhanced AhR activation even at early stages prior to significant ligand depletion. 1EP and EtP were similarly effective in stimulating AhR nuclear translocation, though 5–10 times slower compared with TCDD, and produced no significant down-regulation of the AhR. TCDD activated AhR/Arnt complex formation with an oligonucleotide xenobiotic response element far more extensively than 1EP or EtP, even at concentrations of 1EP that increased CYP1B1 mRNA to similar levels. CO did not influence these responses to EtP, event hough CO treatment potentiated EtP induction of CYP1B1 mRNA. These differences suggest a fundamental difference between PAH/AhR and TCDD/AhR complexes where CYP1B1 metabolic activity regulates the potency, rather than the formation of the AhR/Arnt complex.

Regulation of GLUT1 Gene Transcription by the Serine/Threonine Kinase Akt1

We used mouse hepatoma (Hepa1c1c7) cells to study the role of the serine/threonine kinase Akt in the induction of GLUT1 gene expression. In order to selectively turn on the Akt kinase cascade, we expressed a hydroxytamoxifen-regulatable form of Akt (myristoylated Akt1 estrogen receptor chimera (MER-Akt1)) in the Hepa1c1c7 cells; we verified that hydroxytamoxifen stimulates MER-Akt1 activity to a similar extent as the activation of endogenous Akt by insulin. Our studies reveal that stimulation of MER-Akt1 by hydroxytamoxifen induces GLUT1 mRNA and protein accumulation to levels comparable to that induced by insulin; therefore, activation of the Akt cascade suffices to induce GLUT1 gene expression in this cell system. Furthermore, expression of a kinase-inactive Akt mutant partially inhibits the response of the GLUT1 gene to insulin. Additional studies reveal that the induction of GLUT1 mRNA by Akt and by insulin reflects increased mRNA synthesis and not decreased mRNA degradation. Our findings imply that the GLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1 gene expression in this system.

Suppressive effects of α-Hederin on 2,3,7,8-tetrachlorodibenzo- p-dioxin-mediated murine Cyp1a-1 expression in the mouse hepatoma Hepa-1c1c7 cells

Cultured mouse hepatoma cell line Hepa-1c1c7 cells were treated with α-Hederin to assess the role of α-Hederin in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induced Cyp1a-1, as indicated by analysis of 7-ethoxyresorufin O-deethylation (EROD) activity and Cyp1a-1 protein. When α-Hederin and TCDD were both added to cultures, TCDD-inducible EROD activity was greatly suppressed by α-Hederin in a dose-dependent manner. TCDD-induced Cyp1a-1 protein and mRNA levels were markedly reduced in the concomitant treatment of TCDD and α-Hederin consistent with EROD activity. Electrophoretic mobility shift assay using nuclear extraction of cells revealed that α-Hederin reduced transformation of the Ah receptor to a form capable of specifically binding to an oligonucleotide containing a dioxin-response element (DRE) sequence of the Cyp1a-1 gene. These results suggest that the suppressive effect of α-Hederin on TCDD-induced Cyp1a-1 gene expression in Hepa-1c1c7 cells might be an antagonist of the DNA binding potential of a nuclear Ah receptor.

Diluted Mainstream Cigarette Smoke Condensates Activate Estrogen Receptor and Aryl Hydrocarbon Receptor-Mediated Gene Transcription

Background: Epidemiological data indicate that in females cigarette smoking exerts antiestrogenic effects that manifest clinically in an increased incidence of osteoporosis, earlier menopause, increased spot bleeding, and decreased risk of endometrial cancer for female smokers. The molecular mechanism of this effect is unclear; however, decreased serum estrogen levels in female smokers have been correlated with increased concentrations of the metabolite 2-hydroxyestrogen in females who smoke. Induction of estrogen metabolizing enzymes, CYP1A1 and 1A2, is one mechanism by which increased 2-hydroxyestrogen concentrations may occur. It has also been suggested that the estrogen receptor (ER) may contribute to this anti-estrogenic effect by binding to antagonist(s) in cigarette smoke. Methods: Gel retardation analysis was employed to determine if diluted mainstream cigarette smoke condensates (DMCSCs) could activate the aryl hydrocarbon receptor (AhR). AhR-regulated ethoxyresorufin-O-deethylase (EROD) activity and dioxin response element (DRE)-mediated luciferase induction were assessed in Hepa1c1c7 mouse hepatoma cells. A competitive ligand binding assay was utilized to determine if DMCSCs could bind to the ER. ER-dependent luciferase activity was assessed in MCF-7 cells. Results: In gel retardation assays, DMCSCs induced a protein–DNA complex when incubated with a radiolabeled wild-type DRE oligonucleotide. The complex was effectively competed by excess unlabeled DRE but not by excess unlabeled mutant DRE. In Hepa1c1c7 mouse hepatoma cells transiently transfected with a DRE-regulated luciferase reporter gene, pGudluc1.1, treatment with DMCSCs resulted in a 23- and 25-fold increase in luciferase activity (P<0.01) and an 8.5- and 10.5-fold (P<0.01) induction in EROD activity, respectively. DMCSCs completely displaced bound tritiated E2 from the ER in a dose-dependent manner and induced ER-regulated luciferase activity significantly 6-fold (P<0.01), representing 86% of the maximal induction observed with E2. Conclusions: DMCSCs can bind to and transcriptionally activate the AhR and ER nuclear receptors and cause induction of DRE- and ER-regulated genes. Further study is required to identify the specific compound(s) responsible for these activities.

Suppressive effects of estradiol on 2,3,7,8-tetrachlorodibenzo- p-dioxin-mediated transcriptional activation of murine Cyp1a-1 in mouse hepatoma Hepa 1c1c7 cells

Cultured mouse hepatoma Hepa 1c1c7 cells were treated with either estradiol or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) or in combination to assess the role of estradiol in the process of Cyp1a-1 induction. Estradiol at a concentration as high as 1 μM slightly increased the activity of Cyp1a-1-specific 7-ethoxyresorufin O-deethylase (EROD); in contrast, TCDD-induced EROD activity and Cyp1a-1 mRNA levels were markedly reduced in the concomitant treatment of TCDD and estradiol in a dose-dependent manner. Treatment with tamoxifen, an anti-estrogen which acts through the estrogen receptor, did not affect the suppressive effects of estradiol on TCDD-induced EROD activity. Electrophoretic mobility shift assay using nuclear extract of cells revealed that estradiol reduced transformation of the Ah receptor to the form capable of specifically binding to an oligonucleotide containing dioxin-response element (DRE) sequence. Consistent with this, estradiol decreased TCDD-induced increased chloramphenicol acetyltransferase (CAT) activity from a DRE-containing CAT reporter plasmid after transient transfection into the cells. The levels of the cytosolic [ 3H]TCDD-Ah receptor complex were reduced by estradiol in competitive Ah receptor binding assay using [ 3H]TCDD. This study demonstrated that estradiol acts as an antagonist to TCDD and can regulate Cyp1a-1 expression in an Ah receptor-dependent manner but not through estradiol receptor in Hepa 1c1c7 cells.

Ah Receptor and Estrogen Receptor-Dependent Modulation of Gene Expression by Extracts of Diesel Exhaust Particles

The ability of a methylene chloride extract of diesel exhaust particle (EDEP) to activate the aryl hydrocarbon receptor (AhR), bind to and activate the estrogen receptor (ER), and induce gene expression mediated via these nuclear receptors was examined in Hepa1c1c7 mouse hepatoma and MCF-7 human breast cancer cells. EDEP was able to induce a protein–DNA complex by gel retardation assays using a [γ-32P]dATP-labeled dioxin response element (DRE). This complex could be effectively competed by a 150-fold excess of unlabeled DRE but not by a 150-fold excess of unlabeled mutated DRE. In Hepa1c1c7 cells that were transiently transfected with a DRE-regulated luciferase reporter gene, 4.6 ng/μl EDEP treatment for 24 h resulted in a 22-fold induction of luciferase activity. In the same cell line, ethoxyresorufin-O-deethylase activity was significantly induced 20-fold following 24 h treatment with 4.6 ng/μl EDEP. Using a competitive ligand binding assay, EDEP displaced bound tritiated E2 from the rat uterine ER in a dose-dependent manner with an IC50of approximately 100 ng/μl compared to the IC50of E2, which was approximately 4.4×10−4ng/μl (1.6 nM). In MCF-7 human breast cancer cells transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and a chimeric ER (Gal4-HEG0), treatment with 4.6 ng/μl EDEP for 24 h resulted in a three-fold increase in luciferase activity (P<0.01) compared with the seven-fold increase observed with E2. This study demonstrates that EDEP is able to activate the AhR and ER and induce transcription of reporter genes regulated by these receptors' DNA response elements. Further study is required to identify the individual compound(s) responsible for the observed activity.

Glucocorticoids Repress Transcription from a Negative Glucocorticoid Response Element Recognized by Two Homeodomain-containing Proteins, Pbx and Oct-1

Several studies have established that the prolactin (PRL) gene is expressed not only in lactotrophs and somatotrophs of the anterior pituitary but, albeit to a lesser extent, in non-pituitary cells like human thymocytes, decidualized endometrium, mammary glands during lactation, and some human non-pituitary cell lines. Despite the requirement in the pituitary for the pituitary-specific transcription factor Pit-1/GHF-1 for PRL expression, the expression in non-pituitary cells occurs in the absence of Pit-1/GHF-1 and can be repressed by glucocorticoids. This prompted us to investigate the transcription factors in non-pituitary cells which are involved in controlling expression and glucocorticoid repression of a previously characterized negative glucocorticoid response element from the bovine prolactin gene (PRL3 nGRE). Here we have demonstrated that non-pituitary cells (COS-7 and mouse hepatoma Hepa1c1c7 cells) conferred increased expression via the PRL3 nGRE mainly because of the binding of the ubiquitously expressed POU-homeodomain-containing octamer transcription factor-1 (Oct-1) to an AT-rich sequence present in the PRL3 sequence. However, full transcriptional activity required the binding of a second ubiquitously expressed homeodomain-containing protein, Pbx, previously shown to bind cooperatively with several homeotic selector proteins. The Pbx binding site in the PRL3 nGRE, located just upstream of the Oct-1 binding site, showed a strong sequence similarity with known Pbx binding sites and bound Pbx with an affinity similar to that of other established Pbx target sequences. Interestingly, both Oct-1 and Pbx binding to the PRL3 nGRE were found to be required for glucocorticoid repression. Addition of in vitro translated glucocorticoid receptor DNA binding domain to the nuclear extract prevented Oct-1 and Pbx from binding to the PRL element. The involvement of the homeobox protein Pbx in glucocorticoid repression via an nGRE identifies a new role for this protein.