Toxicological Sciences 55, 107-115 (2000)
Copyright © 2000 by the Society of Toxicology
In Vitro Toxicology and Alternative Testing |
Ah Receptor-Based Chemical Screening Bioassays: Application and Limitations for the Detection of Ah Receptor Agonists
Department of Environmental Toxicology, University of California, Davis, California 95616
Received April 14, 1999; accepted December 22, 1999
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biologic and toxicologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we utilized two AhR-dependent bioassay systems as screening tools to identify novel AhR agonists and to detect the presence of AhR agonists in sample extracts. These assays measure the ability of a chemical to activate AhR DNA binding in vitro (GRAB bioassay) or AhR-dependent (luciferase) gene expression in cultured cells (CALUX bioassay). Known AhR agonists (halogenated and nonhalogenated aromatic hydrocarbons) were positive in both assays, whereas the AhR antagonist
-naphthoflavone exhibited agonist activity only in the GRAB assay. In vitro GRAB analysis has identified several imidazoline receptor ligands and ß-carbolines as AhR agonists and also revealed the presence of AhR agonist activity in crude DMSO extracts of commercial newspapers. In contrast to their positive activity in the GRAB assay, the majority of these chemicals/extracts were only weakly active or inactive in the cell-based CALUX assay. Our results not only reveal that the ability of a chemical to activate the AhR in vitro does not necessarily correlate with its ability to induce gene expression in intact cells, but the high level of false positives obtained with the GRAB assay clearly demonstrates its inability to accurately identify AhR agonists or agonist activity. Screening of unknown chemicals, chemical classes, and samples for AhR agonist activity will require the use of intact cell bioassays. Key Words: Ah receptor; 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; CALUX; halogenated aromatic hydrocarbon; imidazoline.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates many of the toxic and biologic effects of halogenated aromatic hydrocarbons (HAHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) (Denison et al., 1998a
; Fernandez-Salguero et al., 1996; Mimura et al., 1997; Safe, 1990; Schmidt and Bradfield, 1996). Mechanistically, the AhR functions in a manner similar to that of the steroid hormone receptors, although they are clearly members of distinct families of transcription factors (Denison et. al., 1998a; Schmidt and Bradfield, 1996; Whitlock et. al., 1996). Following ligand (TCDD) binding, the cytosolic ligand:AhR complex dissociates from its associated protein subunits, accumulates within the nucleus, and dimerizes with the Ah receptor nuclear translocator (Arnt) protein, with the resulting conversion of the heteromeric complex into its high-affinity DNA binding form (Denison et al., 1988, 1998a; Hankinson, 1995; Probst et al., 1993; Whitlock et al., 1996). The binding of the ligand:AhR:Arnt complex to its specific recognition site, the dioxin responsive element (DRE), stimulates transcription through an adjacent promoter and gene (Denison et. al., 1988). The lack of TCDD toxicity in AhR knockout mice (Fernandez-Salguero et al., 1996; Mimura et al., 1997; Staples et al., 1998), combined with the ability of the AhR to act as a ligand-dependent transcription factor, indicates that the AhR mediates the toxic and biologic effects of TCDD.
The best-characterized high affinity ligands for the AhR include a variety of HAHs such as polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls, as well as numerous polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene, 3-methylcholanthrene, benzoflavones, and other chemicals (Denison et al., 1998c; Gillner et al., 1993; Kafafi et al., 1993; Safe, 1990; Waller and McKinney, 1995). Extensive modeling analysis of HAH and PAH ligands using various techniques provide some insight into physical and chemical characteristics of high-affinity AhR ligands as well as the AhR ligand binding site (Kafafi et al., 1992; Waller and McKinney, 1995). Although review of the literature has identified a large number of chemicals that can bind to the AhR and/or induce AhR-dependent gene expression, the physiochemical and structural properties of many of these chemicals deviate significantly from the currently defined structural requirements for AhR ligands (Denison et al., 1998c). This information is important, especially since the identity of endogenous ligands that activate the AhR at physiologic concentrations remains a major unresolved issue. Knowledge of the spectrum of chemicals and chemical classes that can bind to and activate the AhR would provide an avenue for the identification and characterization of such ligands. Rapid and sensitive AhR-based screening bioassays are valuable to more fully characterize the ligand-binding specificity of the AhR, to identify known AhR ligands (HAHs and PAHs) present in extracts of environmental and biologic samples, and to identify novel chemicals that can activate the AhR signaling system to produce biologic and toxicologic responses.
Although not all of the HAH-inducible biologic responses are directly related to HAH toxicity, they are nevertheless quite specific indicators of exposure to this class of compounds. Consequently, knowledge of the AhR-dependent mechanism of action has permitted development of bioassays for the detection of TCDD and HAHs and for the identification and characterization of other AhR ligands. These assays either measure (1) the direct binding of chemicals to the AhR in vitro (Bradfield and Poland, 1988; Heath-Pagliuso et al., 1998; Phelan et al., 1998), (2) transformation2 and nuclear accumulation of the AhR using tissue extracts (i.e., DNA binding [Heath-Pagliuso et al., 1998; Phelan et al., 1998]), (3) or specific biological responses in cells in culture (i.e., keratinization [Knutson and Poland, 1980]), (4) gene expression or enzyme induction (Garrison et al., 1996; Heath-Pagliuso et al., 1998; Kennedy et al., 1993; Phelan et al., 1998; Safe et al., 1989), or (5) porphyrin accumulation (Kennedy et al., 1993). The induction of EROD activity has been extensively used for these purposes (Giesy et al., 1994; Kennedy et al., 1993; Safe, 1990). However, the ability of many AhR ligands to act as substrates for CYP1A1 results in competitive inhibition of EROD activity and/or inactivation of the enzyme, thus underestimating the inducing potency or potential of a chemical or chemical mixture (Hahn, 1994).
Because of inherent problems with traditional analytical methods and other bioassay techniques, we previously developed two AhR-dependent recombinant bioassays for use in the detection, characterization, and evaluation of AhR agonists and/or antagonists. The gel retardation of AhR binding (GRAB) assay measures the ability of a chemical or chemical mixture to stimulate AhR transformation and DNA binding in vitro (Denison and Yao, 1991; Heath-Pagliuso et al., 1998; Helferich and Denison, 1991; Phelan et al., 1998), whereas the chemically activated luciferase expression (CALUX) assay measures the ability of the chemical(s) to activate AhR-dependent gene expression in cells in culture (Garrison et al., 1996; Heath-Pagliuso et al., 1998; Phelan et al., 1998). Activation of these bioassay systems occurs in a time-, dose-, chemical-, and AhR-dependent manner (Denison and Yao, 1991; Denison et al., 1998c; Garrison et al., 1996). Given the potential utility of these AhR-based bioassays for large- scale chemical and environmental screening, further characterization of these systems has been conducted.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
TCDD and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were obtained from Dr. S. Safe (Texas A&M University), 3,3',4,4',5-pentachlorobiphenyl (PCB) was purchased from AccuStandard (New Haven, CT) and [
32P]ATP from New England Nuclear. Polybrene, benzo[a]anthracene (BA), ß-naphthoflavone (BNF), -naphthflavone (ANF), and 3-methylcholanthrene (3MC) were obtained from Aldrich Chemical Co. (Milwaukee, WI), and the imidazoline receptor ligands (idazoxan, guanabenz, phentolamine, and epinephrine) and ß-carbolines (norharman, harman, harmine, harmol, harmalol, harmaline, and harman-1,2,3,4-tetrahydro-3-carboxylic acid [HTCA]) from Sigma Chemical Co. (Milwaukee, WI). Fluorescamine was purchased from Molecular Probes (Eugene, OR), and molecular reagents were from New England Biolabs (Beverly, MA).
Animals and preparation of cytosol.
Male Hartley guinea pigs (250–300 g) were obtained from Charles River Breeding Laboratories (Wilmington, DE) and were exposed to 12 h of light and 12 h of dark daily and 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. Protein concentrations were determined by the method of Bradford (1976), using bovine serum albumin as the standard.
Preparation of crude newspaper extracts.
DMSO extracts of four different commercial newspapers (designated NP 1–4) were prepared for bioassay analysis. Approximately 0.1 g of minced newsprint (omitting colored printing) from each newspaper was deposited into individual Teflon-lined screw-cap tubes followed by the addition of 10 volumes of DMSO. The capped sample tube was allowed to stand overnight (16 h) followed by transfer of the crude solvent extract into a clean Teflon-capped vial and an aliquot (20 µl/ml for GRAB and CALUX) of the extracts tested in the bioassays.
Synthetic oligonucleotides and GRAB analysis.
A complementary pair of synthetic oligonucleotides containing the sequence 5'-GATCTGGCTCTTCTCACGCAACTCCG-3' and 5'-GATCCGGAGTTGCGTGAGAAGAGCCA-3' (corresponding to mouse DRE3) were synthesized, purified, annealed, and radiolabeled with [32P]ATP as described (Denison et al., 1988a) and referred to as the [32P]DRE. Guinea pig hepatic cytosol (62.5 µl at 8 mg protein/ml) was incubated with DMSO (20 µl/ml), TCDD (20 nM), the indicated chemical (100 µM), or newspaper extract (2.5 µl) for 2 h at 20°C, followed by electrophoretic separation of protein complexes and autoradiography of the dried gels as we have described in detail (Bank et al., 1995). The final incubation mixture contained 80 µg protein, 225 ng poly (dI•dC), ~1 ng (100,000 c.p.m.) [32P]DRE, 25 mM HEPES, 1 mM EDTA, 1 mM DTT, 10% glycerol (v/v), and 80 mM KCl (Bank et al., 1995). Quantitation of the amount of induced protein–DNA complex formed following incubation with each sample was carried out using a Molecular Dynamics PhosphorImager SI. The amount of [32P]DRE specifically bound in the induced complex was estimated by measuring the amount of radioactivity in the induced protein–DNA complex and subtracting the amount of radioactivity present in the same position in the control (DMSO-treated) sample lane. The difference in radioactivity between these samples represents the amount of inducible AhR:[32P]DNA complex and was expressed relative to that produced using TCDD.
Cell culture, chemical treatment, and CALUX analysis.
The CALUX assay utilizes recombinant mouse hepatoma (H1L1.1c2) cells that we previously generated and that were grown and maintained as described (Garrison et al., 1996). These cells, derived from the Hepa1c1c7 line, were engineered to contain a stably integrated DRE-driven firefly luciferase reporter gene plasmid whose transcriptional activation occurs in a time-, dose-, ligand-, and AhR-dependent manner (Garrison et al., 1996). For chemical treatment, H1L1.1c2 cells were grown in 24-well tissue culture plates and incubated with DMSO (10 µl/ml), TCDD (1 nM), or the indicated chemical or extracts for 4 h at 37°C. The final DMSO concentration in all wells was 5 µl in 500 µl of media. After incubation, the cells were lysed with 100 µl of Promega cell lysis reagent and luciferase activity in an aliquot (50 µl) determined using a Dynatech ML3000 Microplate Luminometer with automatic injection of Promega stabilized luciferase reagent. Luciferase activity, normalized to sample protein concentration determined using the fluorescamine protein assay (Lorenzen and Kennedy, 1993) and bovine serum albumin as the standard, was calculated as relative light units (RLU) per milligram of protein and expressed relative to that induced by TCDD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CALUX and GRAB Analysis
To examine the ability of chemicals and sample extracts to stimulate AhR transformation and DNA binding and to induce AhR-dependent gene expression, we used both the CALUX cell culture reporter gene assay and the in vitro GRAB DNA binding assay. We have previously demonstrated that incubation of H1L1.1c2 cells with TCDD and related AhR agonists for 4 h results in a significant induction of luciferase activity (Denison et al., 1998b
; Garrison et al., 1996; Heath-Pagliuso et al., 1998; Phelan et al., 1998). The GRAB bioassay measures the ability of a chemical to stimulate AhR transformation and DNA binding in vitro (Denison and Yao, 1991; Helferich and Denison, 1991; Henry et al., 1994; Probst et al., 1993). A comparison of the dose-dependence and relative sensitivity of the CALUX and GRAB assays for detecting TCDD is shown in Figure 1. These results reveal that the CALUX bioassay is approximately 10 times more sensitive than the GRAB assay (EC50 of ~2 x 10–11 M compared to ~1.5 x 10–10 M, respectively). Moreover, these and other results (data not shown) indicate that the CALUX assay also has a slightly lower minimal detection limit (~1 x 10–12 M as compared to ~1–5 x 10–12 M for the GRAB assay). Given its slightly greater sensitivity, rapidity (~5 h for CALUX compared to ~16 h for GRAB) and ease of analysis, the CALUX bioassay is generally used for first-tier screening.
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Analysis of HAHs and PAHs
As described above, the CALUX and GRAB assays are sensitive bioassay systems for the detection of TCDD and related chemicals. In initial positive control studies, we compared the ability of several known HAH and PAH ligands for the AhR to activate both bioassay systems. Exposure of H1L1.1c2 cells to the indicated concentration of TCDD, TCDF, PCB, 3MC, or BNF induced luciferase activity by 35- to 40-fold (Fig. 2A
); ANF, a partial AhR antagonist (Gasiewicz and Rucci, 1991; Santostefano et al., 1993), induced luciferase about 3-fold. A qualitatively similar profile of response was also observed using GRAB analysis (Fig. 2B), where relatively comparable levels of induced protein–DNA (ligand:AhR:DRE) complex was observed for all chemicals, including ANF. This latter result is interesting, as our results (Fig. 2A) revealed little induction of reporter gene activity by ANF. These results suggest that the partial AhR antagonist activity of ANF must occur at a step distinct from AhR transformation and DNA binding. In addition, the ANF results also highlight a potential limitation of these assays, in that screening using only an in vitro AhR-based assay (such as GRAB) would result in false-positive results. Overall, these positive control results are consistent with previously published observations on the ability of each of these chemicals to bind to the AhR and induce gene expression (Denison and Wilkinson, 1985; Poland and Knutson, 1982; Safe, 1990; Santostefano et al., 1993), and they confirm the utility of these assays for detection of AhR ligands/agonists.
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Application of CALUX and GRAB to Identify Novel AhR Agonists
Given the rapidity and sensitivity of the CALUX and GRAB bioassays, these systems are amenable for use as screening procedures for detection of novel classes of AhR ligands/agonists. Using this approach we have begun to screen large numbers of chemicals and have recently demonstrated that carbaryl, bilirubin, tryptamine, and indole acetic acid are also ligands for and activators of the AhR (Denison et al., 1998b
; Heath-Pagliuso et al., 1998; Phelan et al., 1998). In order to demonstrate the utility of the bioassays to detect novel classes of AhR ligands, we have examined whether several imidazoline receptor ligands (specifically, idazoxan, guanabenz, phentolamine, and epinephrine (Fig. 3A) (Regunathan and Reis, 1996) and ß-carbolines (Fig. 4A) could activate the AhR and AhR-dependent gene expression. Significant luciferase induction was observed following incubation with 100 µM guanabenz, moderate to low levels of induction by idazoxan and epinephrine, and no significant induction by phentolamine (Fig. 3B). In fact, phentolamine treatment resulted in a significant reduction in luciferase activity below that observed with DMSO. Although the reason for this decreased luciferase activity remains to be determined, we observed no overt phentolamine-dependent cell death during the 4-h incubation period. GRAB analysis of these chemicals revealed relatively high levels of induced AhR:DRE complex following incubation with idazoxan and phentolamine, and moderate complex formation with epinephrine and guanabenz (Fig. 3C). The phentolamine results are more typical of that observed with AhR antagonists (Fig. 2
; Ziccardi and Denison, unpublished observations). In contrast to the HAH/PAH results, the imidazoline receptor ligand results reveal a striking lack of correlation in response between these AhR-dependent bioassay systems. The results also identify selected imidazoline receptor ligands as a new class of AhR agonists, albeit weak.
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Another class of chemicals examined using these bioassays includes several ß-carbolines (de Meester, 1995
). The structure of this class of chemicals (Fig. 4A) resembles that of dibenzofurans, indoles, and heterocyclic amines, all chemicals known to be AhR ligands and/or CYP1A1 substrates (Bjeldanes et al., 1991; Denison et al., 1998c; Gillner et al., 1993; Heath-Pagliuso et al., 1998; Miller, 1997). In fact, there is evidence to suggest that ß-carbolines may be substrates for CYP1A1 (Vahakangas and Pelkonen, 1979). Of the ß-carbolines tested, only HTCA treatment resulted in a significant induction of luciferase activity over control (DMSO) levels (Fig. 4B). In contrast to the CALUX results, relatively high levels of induced AhR:DRE complex were observed in the GRAB assay with harmaline, harman, harmine, and norharman, less complex formation was observed with HTCA and harmalol, and no protein–DNA complex with harmol. These results, like those with the imidazoline receptor ligands, clearly demonstrate a dramatic discrepancy between the ability of a chemical to activate AhR transformation and DNA binding (GRAB) and its ability to induce gene expression (CALUX).
Application of CALUX and GRAB Assays to Screen Extracts Containing Unknown Chemical Mixtures for AhR Agonists
A major application of these screening bioassays is for the detection and relative quantitation of HAHs, PAHs, and other AhR agonists present in extracts of various matrices. In fact, the induction of AhR-dependent gene expression (primarily that of CYP1A1 and EROD activity) has been extensively used to detect the presence of HAHs in sample extracts containing complex mixtures of chemicals (Giesy et al., 1994; Kennedy et al., 1996; Safe, 1990). In order to demonstrate the utility of these bioassays for widespread rapid screening of complex mixtures for the presence of AhR agonists, we have recently begun examining solvent extracts of a variety of commercial and consumer products (Rogers et al., 1997). As shown in Figure 5A, incubation of H1L1.1c2 cells with crude DMSO extracts of four commercially available newspapers (NPs) resulted in an elevation in luciferase activity by NP1, NP2, and NP4, although only NP1 induced activity significantly above control (DMSO) levels. These results suggest that these extracts may contain low levels of and/or low-affinity AhR agonists. However, GRAB analysis of the same samples (Fig. 5B) revealed that all newspaper extracts induced AhR:DRE complex formation, with NP1, NP2, NP3, and NP4 inducing 91%, 86%, 41%, and 12%, respectively, of that induced by TCDD. These results reveal a significant discrepancy between the ability of these two bioassays to detect AhR agonists and again demonstrates that the in vitro bioassay alone is not a good predictor of the ability of a chemical or chemical extract to activate the AhR and AhR-dependent gene expression in intact cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Numerous bioassay systems based on the AhR-dependent mechanism have been developed (Bradfield and Poland, 1988
; Denison and Yao, 1991; Garrison et al., 1996; Kennedy et al., 1993; Knutson and Poland, 1980; Postlind et al., 1993; Safe, 1990). These bioassays not only allow identification and detection of HAHs and PAHs in samples and sample extracts by their ability to activate the AhR, but they can be used to estimate the relative biologic potency of these samples. In addition, the AhR bioassays have application for the identification of novel AhR ligands and they can be used as a screening assay in analytical separation and identification of active ingredients (AhR agonists) in complex mixtures. We have recently begun to use these bioassays as screening tools in an attempt to identify endogenous physiologic chemicals that can activate the AhR as well as other novel classes of AhR ligands/ agonists that are distinctly different from typical AhR ligands. In fact, numerous chemicals that can bind to and/or activate the AhR, yet whose structures are divergent from the classical HAH/PAH AhR ligands, have been observed using these approaches (Cheung et al., 1996; Daujat et al., 1996; Denison et al., 1998b; Heath-Pagliuso et al., 1998; Lee et al., 1996; Lesca et al., 1995; Phelan et al., 1998). The CALUX bioassay system described here is a significant improvement over the existing AhR-based bioassay systems because of its increased rapidity, sensitivity, and lack of reporter gene inhibition by the inducing chemical. The rapidity of analysis (4 h) should also allow detection of agonists that might normally be inactivated or converted to non-AhR binding forms by metabolism in longer-term incubations.
Utilizing these two bioassays, we have observed that several imidazoline receptor ligands (epinephrine, idazoxan, and guanbenz) appear to represent a new class of AhR ligands/agonists, albeit weak. Moreover, epinephrine, like bilirubin, biliverdin, and tryptamine, is another endogenous physiological chemical that acts as a weak AhR agonist. The ability of phentolamine to stimulate AhR transformation and DNA binding in vitro in the GRAB assay, yet not induce gene expression in the CALUX assay (but actually suppress constitutive luciferase activity), suggests that this chemical can act as an AhR antagonist, as we have observed similar results with the partial other AhR antagonists (Ziccardi and Denison, unpublished observations). Alternatively, it is possible that phentolamine is an agonist of the guinea pig AhR (used in the GRAB assay) and an antagonist of the mouse AhR (CALUX assay). Although this is a possibility given our previous observation of a species-specific difference in ligand-binding specificity of the antagonist 2,5,2',5'-TCB between guinea pig and mouse AhR (Aarts et al., 1995; Denison and Wilkinson, 1985; Garrison et al., 1996), no chemical that is an AhR antagonist in one species and an agonist in another has yet been identified. Additional structure-activity relationship studies on this novel class of AhR agonists should provide more insight into the specific chemical substituents important for AhR transformation by imidazoline receptor ligands as well as expanding the list of structurally diverse chemicals that can activate the AhR.
The ability of chemicals alone or within complex mixtures to transform the AhR into its DNA binding form is expected to be strongly correlated with its ability to induce AhR-dependent gene expression in intact cells. Although studies using HAHs and PAHs have almost always been consistent with this conclusion (Fig. 2) (Garrison et al., 1996; Giesy et al., 1994; Safe, 1990), some of the results presented here (Figs. 3–5) and those previously reported (Daujat et al., 1996; Gradelet et al., 1997; Lesca et al., 1995; Rogers et al., 1997; Schafer et al., 1993) indicate that this correlation does not hold for all chemicals or chemical mixtures. In our studies, the imidazoline receptor agonists, ß-carbolines, and crude newspaper extracts resulted in significant transformation in the in vitro GRAB assay (i.e., stimulated AhR transformation and DNA binding), whereas relatively low levels of induction were observed in the cell-based CALUX gene expression assay. There are many possible explanations as to why a chemical may be positive in one AhR-dependent assay but not the other. One major difference in bioassay response could result from a difference in the accessibility of the chemical to the AhR. In the GRAB assay, the chemical has direct access to the AhR in the in vitro incubation conditions, whereas in the CALUX assay the inducer must be able to cross biologic membranes effectively and then bind to the cytosolic AhR. Sequestration of the chemical by serum proteins and membranes and/or the inability of the chemical to readily traverse the cellular membrane will reduce the CALUX bioassay response. In addition to these barriers, the chemical must be able to survive cellular metabolic degradation processes in order to activate the AhR and induce gene expression within the time frame of the bioassay. AhR agonists that are metabolically labile or at relatively low concentrations could give positive GRAB results, yet may be weak or negative in the CALUX bioassay. Although the CALUX bioassay is rapid (4 h) the chemical can still undergo a significant amount of metabolic degradation during this period. Given that species-specific differences in ligand-binding specificity have been reported (Aarts et al., 1995; Denison et al., 1995; Denison et al., 1986; Denison and Wilkinson, 1985; Garrison et al., 1996), differences in bioassay responses could also be related to species-specific differences in the two bioassay systems as described above. The low level of responsiveness and relative insensitivity of the guinea pig CALUX system we previously developed has precluded our use of this cell line for this analysis. We are currently generating other stably transfected guinea pig cell lines for such studies. In addition, although we expect species-specific differences in AhR ligand binding specificity to exist, there are few documented examples of striking differences in AhR ligand binding specificity between mammalian species. We believe that the dramatic differences in response between the GRAB and CALUX bioassay results are due primarily to factors other than species-specific differences in ligand binding activity between guinea pigs and mice. The inability of a chemical to activate AhR-dependent gene expression in intact cells, yet activate in vitro, may be a function of the relative affinity of the chemical for the AhR, in addition to the above factors. These considerations rationalize why the PAHs and HAHs show a good correlation between GRAB and CALUX. PAHs/HAHs are high-affinity AhR ligands that passively diffuse through the cell membrane due to their high degree of lipophilicity and, in the case of HAHs, are also resistant to metabolism. These features clearly contribute to the biologic potency of these chemicals.
Chemicals may also be identified that may be active in the CALUX assay yet inactive in the GRAB assay. Those that fall into this category might require cellular conversion to forms that bind to the AhR. It is also possible that a chemical may be a very weak AhR ligand, and thus relatively inactive in in vitro experiments, but is of moderate potency in inducing gene expression in intact cells. Bilirubin and biliverdin are two chemicals of this type (Phelan et al., 1998) in that they bind to the AhR and stimulate AhR DNA binding very weakly but are significantly more potent in intact cells. The reason for this difference in potency in the two assays remains to be determined. It is also possible that a given chemical can activate luciferase expression in the CALUX bioassay by an AhR-independent mechanism(s). Although we view this as less likely, confirmation of the role of the AhR in induction in the CALUX assay could easily be established by confirming the presence of activated/transformed AhR in the nucleus of treated, but not untreated, cells by immunohistochemical techniques or by GRAB analysis using nuclear extracts from these cells.
The CALUX bioassay has numerous advantages over current AhR-based bioassay systems for detection of AhR agonists. Our results also clearly demonstrate that the GRAB bioassay is inappropriate for use as a screening bioassay to detect AhR agonists. The high level of false positives with the GRAB assay would not provide any insight into whether the chemical(s) of interest is actually an AhR agonist. Although these and other AhR-based bioassays can be used to address specific questions when used individually (such as ligand binding ability or ability to induce gene expression) they also have some limitations for large-scale screening applications. The CALUX assay provides a sensitive, relatively quantitative measure of the ability of a chemical to enter the cell, survive metabolism, and activate reporter gene expression, but it does not directly assess or confirm the AhR-dependent nature of this response nor the ability of the chemical to bind with the AhR. Confirmation of the AhR dependence of the response can be confirmed using a DRE-luciferase transfected cell line that lacks a functional AhR signal system. This system is currently under development. Analysis using the GRAB assay can provide information as to the ability of a chemical to stimulate transformation of the AhR into its DNA binding form, but it provides no information as to whether it will actually induce AhR-dependent gene expression in intact cells. However, the ability of chemicals and chemical extracts to activate the AhR in vitro but not in intact cells, as shown in our results, is a clear major limitation of GRAB or AhR ligand binding assays for screening of unknown sample extracts for dioxins and related HAHs. To complicate this even more, we have recently observed that many chemical solvents and chromatographic matrices contain substances that are positive in GRAB but not CALUX assays (Denison, unpublished observation), and that GRAB- but not CALUX-positive results have been obtained using DMSO-, ethanol-, and water-soluble extracts of a large number of commercial and consumer products (Rogers et al., 1997). Consequently, use of GRAB or ligand binding assays as environmental screening tools will clearly result in large numbers of non-HAH false positives and, as such, they are inappropriate for use in chemical screening procedures. Clearly, the preferred approach for identification of dioxins and related HAHs in extracts containing unknown mixtures of chemicals is initial screening using the CALUX (or a related cell-based bioassay). Use of the CALUX bioassay approach will allow identification of novel AhR ligands (as has already been done) as well as detection of the presence of bioactive AhR ligands in complex mixtures of chemicals with fewer false positives.
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ACKNOWLEDGMENTS |
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We thank Dr. Steve Safe for the TCDD and TCDF and Drs. Robert Rice and Reen Wu for critical evaluation of this manuscript. This work was supported by the National Institutes of Environmental Health Sciences (ES07865 and NIEHS Environmental Toxicology Training Grant (ES07059)), a Superfund Basic Research Grant (ES04699) and the California Agriculture Experiment Station.
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NOTES |
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1 To whom correspondence should be addressed at Department of Environmental Toxicology, Meyer Hall, One Shields Avenue, University of California, Davis, CA 95616. Fax: (530) 752-3394. E-mail: msdenison{at}ucdavis.edu.
2 In this report, we have defined transformation as the process by which the TCDD:AhR complex is converted into a form that can bind to DNA with high affinity.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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