* Department of Environmental Toxicology, Meyer Hall, University of California, One Shields Avenue, Davis, California 95616;
Department of Entomology, University of California, Davis, California 95616; and
Cancer Research Center, University of California, Davis, California 95616
Received July 3, 2001; accepted November 5, 2001
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ABSTRACT |
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Key Words: dioxin; Ah receptor; green fluorescent protein; TCDD; HAH; GFP.
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INTRODUCTION |
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The widespread nature of TCDD and related chemicals and their ability to biomagnify in the food chain and produce adverse effects in humans and animals (Devito and Birnbaum, 1994; Giesy et al., 1994a
,b
; Mocarelli et al., 2000
) has generated considerable concern worldwide. Accordingly, numerous analytical techniques have been developed for the detection and quantification of these chemicals in environmental, biological, and food samples in order to assess their formation and sources, fate, transport, exposure, and body burdens. Quantitative extraction procedures coupled with high-resolution gas chromatography/mass spectrometry (HR GC/MS) have been developed to allow accurate detection of HAHs at the part-per-quadrillion level (Clement, 1991
; Stephens et al., 1992
). Although these instrumental analysis procedures provide an accurate measurement of these chemicals in sample extracts and are the "gold standard" for HAH analysis, the procedures are extremely costly, time consuming, have relatively low sample throughput, and require expensive and sophisticated equipment. These aspects are problematic, especially when the experimental design or monitoring program requires large numbers of samples to be screened. Although analytical chemical quantification of HAHs does not provide any information as to the toxic potency, the combination of these data with toxic equivalency factors (TEFs) for HAHs provides an avenue on which to estimate the potency of a complex HAH mixture (Safe, 1990
; Van den Berg et al., 1998
). In this method, the total dioxin-like activity is estimated by multiplying the concentration of a given HAH by its TEF, which is a factor expressing AhR-dependent toxicity relative to TCDD. The sum of the resulting values for all HAHs represents the toxic equivalency (TEQ) of the mixture. However, this approach has numerous limitations, including its inability to assess chemical interactions (synergism or antagonism) that can increase or decrease the potency of a given mixture. In addition, the established HRGC/MS procedures are specifically targeted to HAHs and do not allow for the identification of other novel chemicals and chemical classes that could activate the AhR and thus alter the toxic potency of a complex chemical mixture. Thus, the TEF approach could readily result in an underestimation or an overestimation of the potency of a complex mixture.
Given the specificity of the AhR for the majority of HAHs of concern, most currently developed bioassay systems take advantage of one or more aspects of the AhR-dependent mechanism of action (El-fouly et al., 1994; Garrison et al., 1996
; Murk et al., 1996
; Postlind et al., 1993
; Richter et al., 1997
; Sanderson et al., 1996
; Ziccardi et al., 2000
). These bioassay systems are gaining widespread use as rapid, low-cost, and sensitive screening methods for the detection and relative quantification of dioxin-like HAHs and PAHs in sample extracts. In addition, these systems have been utilized to identify and characterize novel chemicals and classes of chemicals that can activate the AhR (Denison et al., 1998c
; Heath-Pagliuso et al., 1998
; Phelan et al., 1998
; Seidel et al., 2000
). The most sensitive bioassay systems that have been developed to date are with recombinant cell lines that contain a stably transfected dioxin (AhR)-responsive firefly luciferase gene that we and other investigators have developed (Garrison et al., 1996
; Murk et al., 1996
; Postlind et al., 1993
; Richter et al., 1997
; Sanderson et al., 1996
; Ziccardi et al., 2000
). Treatment of these cells with TCDD and related HAHs and PAHs, as well as other AhR ligands, results in induction of reporter gene expression in a time-, dose-, AhR-, and chemical-specific manner. The level of reporter gene expression correlates with the total concentration of the TCDD-like AhR agonists in the sample. Although the firefly luciferase reporter gene contributes to the high degree of sensitivity of the assay, it has limitations with respect to our need for a rapid and inexpensive bioassay for high-throughput screening analysis. Here we describe the development, optimization, and characterization of a novel screening system that responds to AhR agonists with the induction of enhanced green fluorescent protein (EGFP). This new bioassay not only has the same sensitivity and chemical specificity as our previously described, luciferase-based cell bioassays, but it is easier, more rapid, and less expensive, and reporter gene activity can be measured in "real time."
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MATERIALS AND METHODS |
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Construction of GFP expression vector.
The expression vector pGreen1.1 (Fig. 1) was created by excising the 1846 base-pair (bp) Hind III fragment from the plasmid pGudLuc1.1 (Garrison et al., 1996
). This fragment contains the 480-bp dioxin-responsive domain from the mouse CYP1A1 gene inserted upstream of the mouse mammary tumor virus (MMTV) promoter and it confers dioxin responsiveness upon the MMTV promoter and adjacent reporter gene. This fragment was inserted into the Hind III site immediately upstream of the EGFP reporter gene in the plasmid pEGFP-1 (Clontech, Palo Alto, CA).
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Chemical treatment and measurement of EGFP.
In initial experiments, H1G1.1c3 cells were plated into 6-well culture plates and treated with DMSO (1% maximum final concentration), or TCDD (1 nM in DMSO) for 24 h at 37°C. Cells were harvested by scraping into lysis buffer (50 mM NaH2PO4, 10 mM TrisHCl pH 8, 200 mM NaCl), and the cells were lysed by repeated passage through a 27-gauge needle. Samples were centrifuged and the fluorescence of an aliquot of the supernatant was determined in a Perkin Elmer LS 50B fluorometer using excitation and emission wavelengths of 460 nm and 510 nm, respectively. For microtiter plate analysis of EGFP in intact cells, cells were plated into black, clear-bottomed 96-well tissue culture dishes (Corning, San Mateo, CA) at 75,000 cells per well and allowed to attach for 24 h. Selective media was then replaced with 100 µl of nonselective media containing the test chemical or DMSO (1% final solvent concentration). EGFP levels were measured in the intact cells (without removal of media) at the indicated time points, using a Fluostar microtiter plate fluorometer (Phoenix Research Products) with an excitation wavelength of 485 nm (25-nm bandwidth) and an emission wavelength of 515 nm (10-nm bandwidth). In order to normalize results between experiments, the instrument fluorescence gain setting was adjusted so that the level of EGFP induction by 1 nM TCDD produced a relative fluorescence of 9000 relative fluorescence units (RFUs). Samples were run in triplicate, and the fluorescent activity present in wells containing media only were subtracted from the fluorescent activity in all samples.
Fluorescence microscopy.
H1G1.1c3 cells were grown on 25-mm round cover slips for 24 h and then treated with DMSO or 1 nM TCDD for 48 h. To photograph the cells, the media was replaced with phosphate-buffered saline, and cell fluorescence was visualized using an Olympus BH2 fluorescence microscope with a 490-nm excitation filter and a 535-nm emission filter.
Measurement of luciferase activity in recombinant mouse hepatoma (H1L1.1c2) cells.
H1L1.1c2 cells, which contain a stably integrated, DRE-driven firefly luciferase reporter gene plasmid whose transcriptional activation occurs in a ligand- and AhR-dependent manner, were maintained as previously described (Garrison et al., 1996). These cells, grown in 24-well microplates, were incubated with DMSO (10 µl/ml), the indicated concentration of TCDD or surrogate in DMSO for 4 h at 37°C. After incubation, luciferase activity in cells in each well were determined, as we have previously described in detail (Denison et al., 1998b
). Luciferase activity was normalized to sample protein concentration using fluorescamine (Ziccardi et al., 2000
), with bovine serum albumin as the standard.
Animals and preparation of cytosol.
Male Hartley guinea pigs (250300 g), obtained from Charles River breeding laboratories (Wilmington, DE), were exposed to 12 h of light and 12 h of dark daily and were allowed free access to food and water. Hepatic cytosol was prepared in HEDG buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol) as described (Denison et al., 1986) and stored at 80°C until use. Protein concentrations were determined by the method of Bradford (1976), using bovine serum albumin as the standard.
Synthetic oligonucleotides and gel retardation analysis.
A complementary pair of synthetic oligonucleotides containing the sequence 5`-GATCTGGCTCTTCTCACGCAACTCCG-3` and 5`-GATCCGGAGTTGCGTGAGAAGAGCCA-3` (corresponding to the AhR binding site of DRE3 and designated as the DRE oligonucleotide) was synthesized, purified, annealed, and radiolabeled with [32P]ATP as described (Denison et al., 1988
). Gel retardation analysis of cytosolic AhR complexes transformed in vitro with TCDD (20 nM) or the indicated compound was carried out as previously described (Denison et al., 1998b
) and proteinDNA complexes were visualized by autoradiography. The amount of [32P]-labeled DRE present in the induced proteinDNA complex was determined using a Molecular Dynamics phosphorimager.
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RESULTS |
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In previous studies with the luciferase bioassay system, we have observed microtiter plate edge effects in white, clear-bottomed plates, wherein the activity of samples in the wells on the outer edge of the plate (rows A and H, and columns 1 and 12) was decreased compared to that of samples in the inner wells (data not shown). Since this phenomenon can result in an underestimation of actual reporter gene activity, we examined basal and 1 nM TCDD-induced activity in wells of black, clear-bottomed plates containing identical numbers of cells (data not shown). Although comparison of basal fluorescence revealed less than a 4% variation between any 2 wells in the plate, variation between wells in the induced plate were upwards of 1015%. We also observed a slight downward trend in fluorescence across the plate containing the uninduced cells (from rows A to H) with no apparent edge effect. In contrast, significant edge effects were observed in wells containing induced EGFP activity, presumably due to the higher activity, with some reduction in activity observed in rows A and H; induced EGFP activity in column 12 of all rows was consistently lower than that in all other wells in the same row. Although there is some variation between individual wells, we still utilize all wells on a plate for high-throughput screening analysis when the specific endpoint is simply to identify positive inducers. However, when accurate quantitative assessment of the relative inducing potency of a given chemical(s) is desired, these wells should not be used, and all comparisons are based on EC50 values estimated from dose response curves rather then simply comparison to the maximal level of induction. The reproducibility of the assay was then examined by comparing the level of EGFP induction by TCDD (1 nM) in 33 wells of cells in 3 different plates run on 3 successive days. The similarity of the results revealed a high degree of reproducibility between runs (the induction values expressed as a mean ± SD for these analyses were 5276 ± 308, 4404 ± 135, and 4199 ± 236).
We next examined the basal and TCDD-inducible level of EGFP fluorescence from cells in the microtiter plate. H1G1.1c3 cells were incubated with DMSO or TCDD (1 nM) for 24 h and the EGFP expression determined (Table 1). In order to ensure a high signal-to-background ratio and to normalize results between experiments, the fluorometer gain setting was adjusted so that maximal induction resulted in 9000 RFUs. TCDD treatment resulted in a 7-fold induction of fluorescence, while no change in fluorescence was observed in untransfected hepa1c1c7 cells. These data indicate that the increase in fluorescence is due to the induction of EGFP expression and not to a change in fluorescence of endogenous cellular component(s) in the cells themselves. In addition, these results indicate that approximately 60% of the background fluorescence comes from the medium. Subtraction of this background fluorescence (found with culture media only) from all samples revealed that TCDD treatment of H1G1.1c3 cells actually results in a 17-fold induction of EGFP expression (Table 1
). In all subsequent experiments, the background fluorescence from wells containing culture medium only is subtracted from all results to provide an accurate assessment of EGFP activity.
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DISCUSSION |
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We previously examined the utility of the unmodified form of GFP as a reporter gene for assay development. In our hands, this form of GFP was a poor reporter in that little fluorescence was detected in transfected mammalian cells (data not shown). Given the difficulty in measuring GFP and its slow rate of formation in mammalian cells, we focused our attention on the EGFP reporter gene, which has been modified by mutations to optimize it for expression in mammalian cells (Clontech technical manual, PT2040). The EGFP gene also contains site-directed mutations in the chromophore that result in a red-shifted excitation spectra and a 4 to 35-fold enhancement in fluorescence brightness over that of wild-type GFP (Cormack et al., 1996). Because EGFP is inherently fluorescent, it is not necessary to add enzyme substrates nor is it necessary to lyse the cells in order to measure reporter gene activity as is required for most common reporter genes such as luciferase, ß-galactosidase, alkaline phosphatase, and chloramphenicol acetyltransferase. This aspect contributes to reductions in both reagent and labor costs. In addition, the ease of measuring EGFP in intact cells also has applications for time-course studies as reporter gene expression can be measured in "real time," as is also the case for the secreted alkaline phosphatase and human growth hormone reporter genes. The one potential disadvantage of EGFP is its reduced sensitivity due to lack of signal amplification (i.e., the ability of a given reporter to amplify the detected signal by continuous enzymatic cleavage of substrate). However, this has not proven to be a problem for this bioassay and the sensitivity and dynamic range of detection are comparable to luciferase.
The simplicity, rapidity, and cost effectiveness of measurement is the primary attribute of EGFP as a reporter gene. As such, the H1G1.1c3 cells provide a bioassay system appropriate for the rapid screening of large numbers of samples for dioxin-like activity. For example, the H1G1.1c3 cells could be used for rapid analysis of quantitative structure activity relationships (QSAR) of a series of chemicals in order to determine their relative potency and to define structural elements important for ligand binding. In fact, we have begun to utilize these cells for the rapid screening of combinatorial chemical libraries, in order to identify novel AhR ligands or classes of ligands for subsequent QSAR analysis (Nagy et al., 2001). Environmental and product safety monitoring programs that also generate large numbers of samples will similarly benefit from this technology.
We demonstrate the suitability of these cells for chemical screening by determining the relative potency of a series of 17 previously uncharacterized dioxin surrogates to activate the AhR signal transduction pathway. To accomplish this, dose-response curves for each chemical were generated, which resulted in analysis of more than 200 individual measurements. Given the rapidity of the EGFP cell bioassay, it was possible for one person to complete these analyses in less than 3 h total time over a period of 3 days. Because assays are run concurrently, the number of samples that can be run in the same 3-day period can easily increase into the thousands. The lack of a requirement for expensive reagent addition also means that cost will not be a determining factor in deciding how many samples are to be screened (the cost for Promega luciferase reagent for a single 96-well assay is $0.20.3). In fact, the screening of the combinatorial chemical library, consisting of up to 12,090 chemicals, was completed by one person in our laboratory in less than one week. The total cost for this screening analysis using H1L1.1c2 cells would have been between $960 and $1400 for luciferase assay reagents alone. The reduction in person-hours needed for analysis by the EGFP, as compared to that of luciferase, is also a significant cost reduction. Thus, the H1G1.1c3 cells provide us with a relatively inexpensive and rapid high-throughput screening assay for AhR agonists, and it can provide useful mechanistic information regarding inducer stability when used in combination with gel retardation and luciferase cell bioassays.
There are numerous advantages to the EGFP cell bioassay as described above, but this screening system does have some minor limitations that can reduce assay utility. In contrast to our pGudLuc1.1-based H1L1.1c2 cell bioassay, which rapidly responds to AhR agonists (maximum induction at 4 h, Garrison et al., 1996), longer induction times are required for EGFP induction in H1G1.1c3 cells. Consequently for those chemicals or biological responses that produce a rapid or transient activation of the AhR, the EGFP cell bioassay may be of limited utility, and in this instance, the pGudLuc1.1-based AhR-screening bioassay would be the system of choice. However there are only a few examples of such transient responses, and the majority of known AhR agonists are relatively stable inducers. Another potential limitation of the EGFP reporter system comes from test chemicals or chemicals present in complex mixtures, which could interfere with EGFP detection by their ability either to quench EGFP fluorescence or to fluoresce at the same excitation/emission wavelengths as EGFP. Several acridine-containing compounds that we examined interfered with the EGFP bioassay due to their high fluorescence. These were subsequently analyzed using a luciferase-based bioassay.
We observed that when the H1G1.1c3 cell line was maintained under selective pressure, inducible expression of EGFP remained high. However, when the cells were maintained in nonselective media we observed a gradual loss in TCDD-inducible EGFP expression. Accordingly, cells are maintained under constant selective pressure. This loss of responsiveness may result from the relatively high constitutive EGFP expression found in these cells. Previous researchers have reported that NIH3T3 cells expressing high levels of a stably transfected EGFP gene gradually lose EGFP expression over a period of months when not maintained under selective pressure, even when the cells were selected from a single clonal colony (Zeyda et al., 1999). Although the reason for this loss is unknown, the relatively high levels of constitutive EGFP expression in H1G1.1c3 cells may put them at a competitive disadvantage, with high-expressing cells being outgrown by those with little or no constitutive EGFP. This reduction might also result from the instability of the plasmid vector at the genomic site of integration. Alternatively, it may be related to the reporter gene itself, since we have prepared cell lines stably transfected with a dioxin responsive luciferase construct (pGudLuc1.1) that have maintained their responsiveness for more than 10 years without any selective pressure. However, these cell lines express a much lower relative constitutive reporter gene expression than that of H1G1.1c3 cells. Analysis of additional EGFP stable cell clones may provide insight into this issue of instability.
Here we describe the development, optimization, and utilization of a recombinant cell line that responds to TCDD and other AhR agonists with the induction of EGFP expression in a time-, dose-, chemical- and AhR-dependent manner. The AhR-dependent nature of EGFP reporter gene induction and the rapidity of analysis have allowed for the detection and characterization of new AhR ligands. The H1G1.1c3 cell bioassay has the advantages of being rapid, low cost, and adaptable to high-throughput applications without sacrificing either sensitivity or selectivity. We therefore envision that this assay will be most suitable for adaptation to any application where cost or high sample numbers are an issue.
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ACKNOWLEDGMENTS |
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NOTES |
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