Toxicological Sciences 55, 69-77 (2000)
Copyright © 2000 by the Society of Toxicology
Endocrine Toxicology |
A Rapid and Sensitive Reporter Gene that Uses Green Fluorescent Protein Expression to Detect Chemicals with Estrogenic Activity
* Department of Anatomy and Public Health and
Department of Pathobiology, College of Veterinary Medicine, and
Department of Animal Science, Texas A&M University, College Station, Texas 77843
Received April 29, 1999; accepted November 19, 1999
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10–9 M 17ß-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000–3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10–12 M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0–10 M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10–7 M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2–4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17ß-estradiol.
Key Words: cellular steroid receptors; endocrine disruption; xenobiotics; transfection; MCF7-ERE cells; estrogenic activity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hormonally active xenobiotic compounds include a variety of environmental contaminants that may induce biochemical and physiological alterations in animals. Some cell function alterations in animals exposed to xenobiotics with steroid-like activity may be initiated by as yet undetermined, nongenomic mechanisms. However, the majority of data suggest that endocrine disruptive effects are due to the interaction of xenobiotics with one or more of the many cellular steroid receptors that regulate gene expression (Kelce et al., 1994, 1995; Korach et al., 1988; reviews by Barton and Anderson, 1998 and Sumpter et al., 1996). Estrogenic chemicals typically bind the estrogen receptor (ER), the ER-ligand complex subsequently interacts with estrogen response elements (ERE), and transcription factors are recruited to the ER-ligand/ERE complex, resulting in enhanced mRNA synthesis and gene expression. Endocrine agonist or antagonist activities of pollutants may lead to altered gene expression, due either to receptor-ligand interaction with response elements leading to enhanced or repressed mRNA synthesis, or chemical up- or down-regulation of receptor expression. Receptor/ligand binding causing changes in gene expression may lead to:
- Induction of protein synthesis resulting in altered metabolism of endobiotics such as physiologically occurring estrogens, or xenobiotics such as benzo[a]pyrene,
- Altered function of endocrine-regulated physiological systems, including sex determination, reproductive functions, and morphological defects of development, or
- Interactions that decrease both immune surveillance-associated protection against transformed cells and normal immune system responses to infectious agents.
These interacting phenomena of endocrine disruption may result in altered embryological development and birth defects, feminization of male offspring, diminished reproductive efficiency, and/or a chronic state of debilitation leading to deteriorating health and eventual death of the organism, even though concentrations of the offending chemicals may be quite low (Abbott et al., 1994
; Brouwer et al., 1995; Chen 1987; Colborn and Clement, 1992; Colborn et al., 1993; De Swart et al., 1993; Guillette et al., 1996; Hoffman 1992; Kelce et al., 1994, 1995; Roman and Peterson 1998; Roman et al., 1998).
Methods such as the E-screen procedure and uterotrophic assays have been used for several years to detect estrogenicity of chemicals. Newer methods, such as vitellogenin induction in fish cells (Smeets et al., 1999), are effective and relatively simple, but the most sensitive new methods are typically those employing reporter genes and expression systems (Legler et al., 1999) for determining the estrogenicity of chemicals. Relatively simple, direct assay methods that are rapid and inexpensive, but extremely sensitive, are needed for detecting and quantifying endocrine-mimetic activities of chemicals. We report here the development of a direct, effective, relatively simple method for detecting, quantifying, and studying endocrine disruptive chemicals in eukaryotic cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
MCF-7 cells were purchased from American Type Culture Collection (Rockville, MD). All media were from Gibco (Gaithersburg, MD). Fetal bovine serum was from Summit Biotechnology (Ft. Collins, CO). [3H]-Estradiol (40 Ci/mmole) was purchased from New England Nuclear. Other chemicals were from Sigma (St. Louis, MO) unless otherwise stated. Enzymes were from Boehringer Mannheim (Indianapolis, IN) unless otherwise stated.
Cell culture.
MCF-7 cells were grown in Minimal Essential Medium (MEM) with Earle's salts, non-essential amino acids, essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 µg/ml insulin, 0.1 mg/ml gentamycin sulfate, and 2.5 mg/l amphotericin B. Media were supplemented with 5% fetal bovine serum (FBS), and 5%, 10%, or 15% charcoal dextran-treated FBS (CDFBS). The cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air.
Charcoal dextran-stripped FBS.
A mixture of 0.5% charcoal and 0.05% dextran in 50 mM HEPES, pH 8.0, was agitated gently for 0.5 h. at 37°C. The slurry was centrifuged at 3000 rpm, 4°C, for 30 min, and the supernatant was removed and replaced with fetal bovine serum that had been heat-inactivated for 30 min at 56°C (Soto and Sonnenschein, 1984, 1985). The mixture was agitated gently for 3 h at 37°C and centrifuged at 3000 rpm, 4°C, for 1 h. The serum was carefully pipetted away from the charcoal pellet, filter-sterilized, and stored at –20°C until used.
Estrogen receptor binding.
Estrogen receptor was characterized in MCF-7 cells using a modification of the method described by Gasiewicz and Neal (1982). Briefly, ER was isolated by hydroxylapatite adsorption from cell lysates incubated with [3H]-E2 and quantified by liquid scintillation counting. MCF-7 cells were grown in 5% FBS for 4 days, rinsed once with 4% BSA in PBS, then returned to the incubator for 4 h in fresh 4% BSA/PBS to bind exogenous E2. Cells were then cultured 4 additional days in MEM containing 10% heat-inactivated charcoal dextran-stripped FBS (HICDFBS), Earle's salts, non-essential amino acids, essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 µg/ml insulin, 0.1 mg/ml gentamycin sulfate, and 2.5 mg/l amphotericin B, but without phenol red. Cells were harvested by trypsinization, washed with 10% glycerol in HED buffer (25 mM HEPES, pH 7.8, 1 mM dithiothreitol [DTT], 5 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 40 µg/ml soybean trypsin inhibitor, 1 mM EDTA), and resuspended in 10 ml HED at 2.5 x 107 cells/ml for one cycle of freeze-thawing, followed by homogenization at 4°C with 20 passes of a tight fitting pestle. Over 80% of the cells were lysed, as assessed by phase contrast microscopy using trypan blue. The cell lysate was diluted with 25 ml of 10% glycerol in HED and centrifuged at 105,000 x g for 1 h at 4°C. The protein content of the supernatant was determined, using the Bradford assay (Bio-Rad), to be 1.5 mg/ml. Mixtures containing the appropriate amount of [3H]-E2 ± a 200-fold excess of diethylstilbestrol (DES) were incubated with 2 ml of supernatant for 2 h at 20°C. After incubation, a 0.2 ml aliquot of each reaction mixture was removed to a scintillation vial to determine total radioactivity. Aliquots of 0.2 ml from the remaining reaction mixture were added to 0.25 ml of a slurry of DNA-grade hydroxylapatite (HAP) previously washed with HED containing 10% glycerol (but no protease inhibitors), pH 7.4, and resuspended in 2 vol of HED + 10% glycerol (HEDG). The mixtures were incubated for 30 min at 4°C with gentle shaking every 10 min, followed by extensive washing with 1 ml of HEDG + 1% Triton X-100. The HAP pellet, containing adsorbed estrogen receptor labeled with [3H]-E2, was resuspended in 1 ml of absolute ethanol and transferred to a scintillation vial. The centrifuge tube was rinsed with 1 ml of absolute ethanol, which was also added to the scintillation vial. Radioactivity was determined for each pellet, corrected for background and quench, and converted to moles of [3H]-E2 bound per mg of protein or moles of [3H]-E2 bound per ml of reaction mix. Specific ER binding was calculated by subtracting dpm obtained in the presence of 200-fold excess of DES (control) from dpm obtained in the absence of DES. Free [3H]-E2 was determined by subtracting bound [3H]-E2 from added [3H]-E2. The affinity constant (Kd) and binding site concentration (Bmax) were determined by Scatchard analysis. Assays were completed in quadruplicate with standard deviations less than 10%.
Plasmid preparation.
A 47-bp oligonucleotide containing 2 estradiol response elements (ERE) was synthesized at the DNA Core Laboratory (College of Veterinary Medicine, Texas A&M University). The 5'–3' (forward) sequence is as follows:
EcoR1 / first ERE / 20 bp spacer with a NotI site / 2nd ERE / Kpn1
5'-aattcGGTCACAGTGACCGCGGCCGCTCTACAGTCGACGGTCACAGTGACCgtac-3'
The 5'–3' and 3'–5' (reverse) oligonucleotide sequences were annealed by heating at 96°C with slow cooling of the mixture to room temperature. The PGK-GFP vector was digested with restriction enzymes Kpn 1 and Eco R1. The ERE oligonucleotide was phosphorylated with T4 polynucleotide kinase and the vector and oligonucleotide were ligated using the Rapid DNA Ligation Kit® (Boehringer Mannheim, Indianapolis, IN). The ligation product, containing a 5'-ERE-ERE-PGK-GFP-polyA-3' sequence, was transfected into XL2-Blue MRF®(Stratagene, LaJolla, CA) E. coli, which were grown on LB agar plates containing ampicillin. Plasmid mini-preparations were made using the method of Del Sal et al., (1989) with restriction mapping to confirm the plasmid cut sites. A large-scale plasmid preparation was done using the Plasmid Maxi kit (Qiagen, Santa Clarita, CA). The plasmid was linearized with Sca 1 and the restriction enzyme was inactivated by heating at 65°C for 15 min. The plasmid was concentrated using Microcon-30® spin concentrators (Amicon, Beverly, MA) at 500 x g for 60 min. Control plasmid, the PGK-GFP vector, was prepared and linearized in like manner.
Transfection and selection of green fluorescing cells.
MCF-7 cells in 10% FBS were fed 24 h and 4 h before the transfection procedure. Cells were harvested by trypsinization and placed into 0.8 ml of MEM at a density of 7.7 x 106/ml. The cell suspension was transferred to a sterile plastic cuvette along with 25 µg of the linearized plasmid. Sterile BTX electroporation electrodes were placed in the suspension and an electric current was applied at 250 µFad, 300 V for 0.9 sec. Cells were added to 30 ml of MEM containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 µg/ml insulin, 0.1 mg/ml gentamycin sulfate, and 2.5 mg/l Amphotericin B, and were returned to the incubator for 30 min. Cells were then diluted 1:500 and plated into 24-well plates (Corning, Corning, NY). After overnight incubation, cells were changed to selection medium (MEM with 5% charcoal dextran-treated FBS (CDFBS), non-essential amino acids, 1 nM E2, 2 mM glutamine, 1 mM sodium pyruvate, 1 µg/ml insulin, 0.1 mg/ml gentamycin sulfate, and 2.5 mg/l amphotericin B). The cells were left undisturbed for 7–10 days.
Green fluorescent colonies were observed using an Olympus IMT-2 inverted fluorescence microscope equipped with a 100-watt mercury light and a triple band pass filter cube for detection of red, green, or blue fluorescence (RGB). The fluorescent images were digitized with a color charge-coupled device (CCD) camera (Optronics Engineering, Goleta, CA) and viewed on a Trinitron color video monitor (Sony). The image was processed using RGB signal technology and transferred to a computer, where it was analyzed using Image-Pro Plus software (Media Cybernetics, Silver Springs, MD).
Colonies with a mixture of fluorescent and non-fluorescent cells were trypsinized and subcloned at a ratio of 1:50. After twenty days, those wells which contained enriched populations of green fluorescent cells were trypsinized and resuspended in sterile PBS at 1.5 x 107 cells/ml. Fluorescent and non-fluorescent cells were separated using a FACSCalibur fluorescence activated (FACS) cell sorter (Becton Dickinson, San Jose, CA) and were plated into 24-well plates in medium with 15% charcoal dextran-treated FBS. Wells containing colonies highly enriched in cells expressing GFP were harvested, and the colonies were expanded and designated as MCF7-ERE. Cells derived from colonies containing GFP without the ERE sequences were not fluorescent in the second passage and were not selected by the FACS procedure.
Growth curve.
The MCF-7 growth rate was determined in MEM supplemented with 5% FBS. MCF7-ERE growth rate was determined in selection medium (1 nM E2 with 5% CDFBS). Cells were seeded into 60-mm culture dishes at 5 x 104 cells per dish and allowed to settle in growth medium for 4 h before changing the medium to the experimental formula. At this time the number of cells attached to the dish was determined and reported as day 0. Cells were counted daily in triplicate, with a medium change every 2 days. Doubling time was determined from the following relationship:
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Concentration curves of estradiol and estrogenic chemicals.
Wells of transfected cells selected for GFP expression using FACS were harvested and grown in MEM without phenol red and supplemented with 10% CDFBS, non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 µg/ml insulin, 0.1 mg/ml gentamycin sulfate, and 2.5 mg/l amphotericin B (GFP medium). At confluency, the cells were harvested and plated in GFP medium onto sterile 25 x 75 x 1-mm glass slides that had been treated overnight with CDFBS. After attaching to the glass slides, cells were held in GFP medium for 24 h; E2 or chemicals to be evaluated for estrogenic activity (genistein, o,p'-DDT, nonylphenol, methoxychlor, dieldrin, or lindane) were added to the cells as indicated, and cells were held another 24 h prior to assay. Estradiol for this study was encapsulated in cyclodextrin to improve its water solubility (Sigma). In control cells, only cyclodextrin was present at either 1 or 100 µM, equivalent to the amount of cyclodextrin present with 3 x 10–8 M estradiol.
Cells were examined by phase contrast and fluorescence microscopy at 150x, and GFP fluorescence was quantified and recorded using color charge-coupled camera technology and computerized analysis. The data are reported as fluorescence units (FU)/1000 cells expressing GFP above background level.
Inhibition of GFP expression by 4-hydroxytamoxifen.
MCF7-ERE cells were plated onto sterile slides at 5 x 104/ml in MEM without phenol red and containing 5% HICDFBS, essential and non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 µg/ml insulin, 0.1 mg/ml gentamycin sulfate, and 2.5 mg/l amphotericin B. After 3 days, the slides were incubated overnight with 4% BSA/PBS and experimental media containing 10% HICDFBS were added as indicated after 6 days. Fluorescence was assayed after 40 h.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Specific [3H]-E2 binding to MCF-7 ER was linear over a concentration range of 3 x 10–11 to 10–9 M (Fig. 1A
). At E2 concentrations higher than ~5 x 10–9 M, binding plateaued, declining slightly between 3 x 10–8 and 3 x 10–7 M E2. Scatchard analysis of the binding isotherm showed a dissociation constant (Kd) of 0.29 x 10–9 M and a maximum binding (Bmax) of 63.2 x 10–12 M, equivalent to 3.8 x 104 binding sites per cell (Fig. 1B).
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Transfection of ERE-PGK-GFP into MCF-7 cells initially resulted in a mixed cell population containing about 2% fluorescent cells. Attempts to enrich these cells by dilution cloning met with little success because MCF-7 cells tend to clump, even after several passes through a pipet. In addition, many of the cells lost fluorescence capacity after 1 or 2 passages. This instability was also observed in cells that received the plasmid (PGK-GFP) with no ERE enhancer sequences.
One colony of transfected cells was observed in which increased levels of fluorescent cells were noted. This colony was harvested by trypsinization and plated at a 1:50 dilution into 24-well plates. After attachment and growth for 2 days, all of the wells contained 5–10% fluorescent cells. During this procedure, we observed that the green fluorescence was preserved in suspended trypsinized cells over a period of at least several h.
Three wells with confluent E2-induced cells were harvested to yield 7.5 x 106 cells, which were resuspended in sterile PBS. Fluorescent and non-fluorescent cells were separated by fluorescence-activated cell sorting, with about 5% of the cells having 2000–3000 times higher fluorescence than the rest (Fig. 2). Fluorescent cells were collected in tubes containing charcoal dextran-treated FBS, resuspended in MEM containing 1 nM E2 and an additional 15% CDFBS, and allowed to grow undisturbed for 14 days. Three of the wells contained greater than 95% fluorescent cells, which were harvested and combined as MCF7-ERE.
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The growth rate of MCF7-ERE in GFP medium with 10–9 M estradiol and 5% CDFBS was slightly lower than MCF-7 cells in 5% FBS and MEM, but the cell numbers did not differ significantly until day 8 (Fig. 3
). There was significant loss of cells in both cell lines during the seeding operation, probably because not enough time was allowed for the cells to firmly attach before removing the seeding medium. Therefore, the growth rates shown may reflect to some extent the capacity for MCF7-ERE to rapidly attach to the culture dish. This characteristic was not different from the parent MCF-7 plating efficiency.
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The expression of GFP in MCF7-ERE is dependent on the presence of E2. Phase contrast and fluorescent images of the cells show that green fluorescent protein was not present in the absence of E2 (Figs. 4A and 4B
). A 24-h treatment with 10–10 M E2 induced expression of GFP in virtually all of the cells (Figs. 4C and 4D). The induction of GFP expression in MCF7-ERE cells by E2 was quite sensitive (Fig. 5), increasing from basal levels without E2 (basal fluorescence levels could not be distinguished from induced levels at about 3 x 10–13 M E2) to about twice basal at 10–12 M E2, and reached a maximum expression at 3 x 10–10 M E2. At 10–9 M E2 GFP expression was only slightly less than maximal, but at concentrations higher than 10–9 M GFP expression declined significantly. Concentrations of E2 above 10–7 M inhibited the growth of MCF7-ERE cells and were not used for this dose response curve. Morphological characteristics of cells and cell density were not visibly affected at the levels of E2 reported here. In most treatment groups, the range of fluorescence values was similar between cells.
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The involvement of ER in GFP expression is indicated by capacity of the E2 antagonist, 4-hydroxytamoxifen (TFN-OH) to inhibit E2-dependent induction of fluorescence by binding to ER (Fig. 6
). Addition of increasing amounts of TFN-OH decreased GFP expression in a dose-dependent manner. The decrease from maximal E2 induction of GFP expression, shown in Figure 6 as an unconnected data point, was apparent at 10–10 M TFN-OH and continued with increased TFN-OH concentrations. The decrease was linear as TFN-OH concentrations were increased between 10–9 and 10–7 M.
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We conducted pilot experiments to determine if estrogenic compounds could be detected by the ERE-PGK-GFP reporter gene system, using lindane, a non-estrogen, and 5 differentially estrogenic chemicals (Fig. 7
). Genistein, a plant hormone, induced GFP to levels equivalent to those induced by E2, but required a concentration about 100-fold higher for maximal induction. Genistein, o,p'-DDT and nonylphenol all induced GFP expression in the 10–7–10–8 M range. Methoxychlor exhibited maximal GFP induction at concentrations about 1000-fold higher than E2, while dieldrin showed maximal induction of GFP at about 5000-fold higher concentrations than E2, and lindane did not induce GFP expression above basal levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We present a rapid, direct, sensitive, and quantitative assay to detect natural estrogens and estrogen-mimetic chemicals. MCF-7 cells transfected with a plasmid carrying an ERE-PGK-GFP sequence exhibited linearly increased expression of GFP from a 2x basal level at 10–12 M E2 to maximal expression at 3 x 10–10 M E2. As the E2 concentration increased above 3 x 10–10 M, GFP expression plateaued and then declined. The induction of GFP expression was inhibited in a dose-dependent manner by TFN-OH, indicating that E2 binding to ER is essential for expression of the reporter gene (Lerner and Jordan, 1990
). Test chemicals with known estrogenic vs. non-estrogenic characteristics were readily distinguished by the reporter gene system and quantified by fluorescence microscopy.
The superfamily of steroid hormone receptors includes cellular proteins that bind estrogens, androgens, glucocorticoids, progestins, vitamins A and D, and thyroid hormone. They exert exquisite regulation of gene expression essential for maintenance of normal physiological function and good health (Fuller, 1991; Lieberman, 1997; O'Malley and Tsai, 1992; Truss and Beato, 1993). Animals, both terrestrial and aquatic, may be chronically exposed to a variety of industrial and agricultural chemicals as environmental contaminants that may bind one or more of the steroid receptor proteins, or may bind the Ah receptor regulating cytochrome P450-mediated processes that are interactive with hormone synthesis and/or metabolism. While a large body of data exists on acutely toxic effects of substituted polycyclic (PAH) and/or aromatic (AH) hydrocarbons and related organochlorines (OC), there are few data on the physiological effects of chronic exposure to sub-acute levels of plasticizers, detergents, pesticides, herbicides, AH, PAH, and other OC that bind cellular receptors and are potentially endocrine interactive in man or other animals. In addition, few rapid, practical, readily quantifiable, and inexpensive methods exist to routinely screen water and soil samples for the presence of chemical contaminants in order to identify sources of chronic exposure to endocrine disruptive agents.
A direct cause and effect relationship has been drawn between exposure to a variety of chemicals, such as plasticizers, phenolics, detergents, AH, PAH, and OC, and the initiation of altered physiological responses in humans and laboratory animals (Abbott et al., 1994; Colborn et al., 1993; Constable and Hatch, 1985; Jackson and Halbert, 1974; Kashimoto and Miyata, 1987; Lawrence et al., 1985). These responses may include weight loss, thymic atrophy and impaired immune function, hepatotoxicity, fetal toxicity and developmental disorders, loss of reproductive efficiency, and increased incidence of cancer (Poland and Knutson, 1982; Safe, 1990). These responses differ dramatically between individual humans and between inbred strains of animals. They are dependent, at least in part, on their endocrine receptors and endocrine-regulated cell functions, and on their capacity for induction of enzymes such as the cytochromes P450 that metabolize hydrocarbons (Whitlock, 1986; Santostefano et al., 1998) and interact to one degree or another in the expression of endocrine-regulated genes. While acute administration of endocrine disruptive chemicals has begun to be well investigated (Faqi et al., 1998; Rier et al., 1995; Vickers et al., 1989; Zacharewski et al., 1994), very little is known about how humans and other mammals react to chronic, low-level environmental hydrocarbon exposure, or whether the bioaccumulation of environmental contaminants increases the pharmacologically effective dose of endocrine-disruptive chemicals to which an animal is exposed. Available data do, however, show that stressed mammals metabolize fat reserves and release bioaccumulated chemicals that readily cross the placental barrier and affect fetal cells, that lactating females offload large quantities of bioaccumulated lipophilic chemicals in milk, and that nursing young may have extremely high serum levels of chemicals relative to their actual environmental levels (Ridgway and Reddy, 1995; Tanabe et al., 1982).
A reporter gene system capable of detecting the presence of chemicals with estrogen-like activity requires a host cell that expresses the ER or can be transfected with a vector expressing ER. The cell line MCF-7 was chosen for this study because it expresses ER and a number of other steroid receptors. Numerous investigators have reported the Kd of MCF-7 ER for E2 to be about 10–10 M. A minimal E2 concentration of 10–9 M was therefore selected for initial screening of MCF7-ERE cells. This E2 concentration was close to the beginning of the plateau region of the ER-E2 binding curve, and was high enough to ensure reasonable binding without completely saturating the receptors. Chronic treatment of cells with saturating levels of E2 has been reported to down-regulate receptor expression. Such down-regulation would have required MCF7-ERE cells to depend upon de novo protein synthesis to respond to a challenge by exogenous E2, and may explain the rapid drop in GFP expression seen above an E2 concentration of about 10–8 M. In addition, the decline in gene expression with increased E2 has been suggested by Levenson and Jordan (1994) to be a function of excessive E2 binding to ER producing transcriptional interference. The principle of transcriptional interference has been previously shown for ER and glucocorticoid receptor- or progesterone receptor-mediated responses, but in those instances, ER was transiently overexpressed (Meyer et al., 1989; Watts and King, 1994). Similarly, Kushner et al., (1990) reported the construction of a cell line that overexpressed ER and, in the presence of physiological E2, stopped growing. In the absence of a selectable marker, selection of MCF7-ERE cells in constant concentrations of E2 during long-term growth would not be possible under saturating conditions. By using E2 at an arbitrary concentration of 10–9 M, we selected for cells that were not significantly E2-inhibited. By selecting for green fluorescent cells after 2 weeks, we also chose cells that were not sensitive to down-regulation of ER by E2.
A comparison of the ERE-PGK-GFP expression system in MCF-7 cells with other in vitro assay systems indicates the ERE-PGK-GFP system to be significantly more sensitive than the recently reported vitellogenin (Vtg) expression system of Smeets et al. (1999), and approximately the same sensitivity as that of the ERCALUX assay reported by Legler et al. (1999). When the ERE-PGK-GFP expression system was compared with the E-screen and transiently reporter gene-transfected HeLa and MCF-7 methods, the method reported here had about the same sensitivity for DDT and dieldrin as the E-screen, slightly less sensitivity for nonylphenol detection than the E-screen, and more sensitivity for genistein detection than the E-screen method. The ERE-PGK-GFP method was typically 2 log units more sensitive for each chemical than the methods using transiently transfected human cells (Table 1). We show a lowest observed effective concentration (LOEC) of 10–12 M E2 for GFP expression, while Smeets et al. reported an LOEC of 2x10–9 M E2 for Vtg expression and Legler et al. reported a luciferase expression detection limit of about 0.5 x 10–12 M E2. The ERE-regulated luciferase system also included use of a co-transfected plasmid sequence expressing the ligand binding domain of ER linked to the DNA binding domain of the yeast transcription factor Gal4 (Legler et al., 1999). The ERE-PGK-GFP maximal expression occurred at about 3 x 10–10 M E2, while the luciferase maximal expression reported by Legler et al. occurred at about 3 x 10–11 M E2. The LOEC for ERE-PGK-GFP was about10–12 M E2, while Legler et al. reported a minimal E2 detection limit of 0.5 x 10–12 M. At E2 concentrations between 10–12 and 10–13 M, the potential exists for very small methodological errors to make an enormous expression difference. The lower limits of these methods are, therefore, functionally similar. The differences in the 2 methods appear to be in the relative complexity of the assays.
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The plasmid-introduced ERE-PGK-GFP sequence was linearly expressed in MCF7-ERE cells between E2 concentrations of 10–12 and 3 x 10–10 M, with expression declining from the maximum as the E2 concentration was increased to 10–7 M. The attenuation of GFP expression by TFN-OH indicates the essential involvement of ER in gene expression. The expression vector system detected the following chemicals listed in their order of apparent estrogenicity as methoxychlor > dieldrin = o,p'DDT > genistein > nonylphenol > lindane. Maximal GFP expression was induced by methoxychlor, the most potent of the chemicals, at a concentration approximately 1000-fold higher than E2, and by nonylphenol, the least potent of the estrogenic chemicals tested here, at more than 10,000-fold higher than E2. This expression system has the potential to detect estrogenic chemicals in environmental samples and to quantify estrogenic activities of environmental and industrial chemicals compared with a standard curve of GFP expression induced by estradiol or other induction standards.
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ACKNOWLEDGMENTS |
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This work was supported in part by U.S. EPA award 430891, a NOAA Sea Grant award 404227, and an award from the Texas Agricultural Experiment Station.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (409) 847-8981. E-mail: dbusbee{at}cvm.tamu.edu.
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