* Wildlife Health Center and
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616; and
Department of Environmental Toxicology, University of California, Davis, California 95616
Received June 22, 1999; accepted November 11, 1999
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ABSTRACT |
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Key Words: HAH; PAH; Ah receptor; bioassay; blood; CALUX; TCDD.
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INTRODUCTION |
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In order to assess biologic responses of such compounds in complex systems better, much research effort has been directed toward the development of bioassays. Two classes of bioassays based on the AhR-dependent induction of CYP1A1 and its monoxygenase activity have been developed to detect animal exposure to PAHs and/or HAHs: those relying on in vivo metabolism in animals, and those measuring the induction of CYP systems in vitro. The first class, which includes techniques that examine in vivo responses in tissues [such as measurement of CYP1A1 induction by the quantification of ethoxyresorufin-o-deethylase (EROD) activity, aryl hydrocarbon hydroxylase (AHH) activity, or CYP1A1 mRNA levels], have been used widely to determine exposure of individuals (Burke and Mayer, 1974; Nebert and Gelboin, 1968
; Vanden Heuval et al., 1993
). However, this approach has several disadvantages that limit their widespread applicability as screening procedures, including species differences in response to HAHs/PAHs (Aarts et al., 1996
; Denison et al., 1995
; Denison and Wilkinson, 1985
). Additionally, the methods used to assess this type of activation often require either the euthanasia of the animal or the use of invasive surgical techniques for sample collection. More important, many CYP1A1-inducing chemicals (AhR ligands) are also substrates for this activity, thus resulting in competitive inhibition at high concentrations (Hahn et al., 1993
; Kennedy et al., 1993
), which would result in an underestimate of xenobiotic exposure.
Another class of bioassay, one that uses in vitro methods (through detection of enzymatic activity, Ah receptor binding, or the induction of reporter genes), has recently become more frequently used to evaluate exposure of animals or environments to HAHs/PAHs. The H4IIE rat hepatoma cell bioassay (Bradlaw and Casterline, 1979) has been used to detect HAHs/PAHs and provides an estimate of the biologic potency (expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents or TEQs) of compounds or complex mixtures (Tillitt et al., 1991
; Willett et al., 1997
). However, because this method also measures increases in EROD activity, it has many of the same limitations described above. The application of recombinant reporter plasmids such as the firefly luciferase gene (Denison et al., 1993
; Garrison et al., 1996
; Postlind et al., 1993
; Richter et al., 1997
) has proven to be a very effective method to detect these chemicals and to provide an approach to determine TEQs in a variety of matrices (Aarts et al., 1993
; Anderson et al., 1995
; Murk et al., 1996). The chemically activated luciferase expression (CALUX) bioassay system is extremely sensitive in directly detecting AhR-dependent potential of a variety of pure compounds and extracts of environmental and biologic matrices (Aarts et al., 1996
; Denison et al., 1996
, 1998b
; Heath-Pagliuso et al., 1998
; Murk et al., 1996a
,b
; Phelan et al., 1998
; Sanderson et al., 1996
; Ziccardi et al., 1997
).
Epidemiologic studies of the effects of HAHs and PAHs on human and wildlife populations require large-scale assessment of the level of exposure to these compounds and current analytical procedures are not amenable to large-scale screening procedures. Given the advantages of the CALUX assay, its modification to a high throughput screening system would have utility in these types of analyses. In order to maximize the utility of this method for use in these situations, however, analysis of readily accessible samples must be validated. In experimental studies, it has been noted that both PAHs and HAHs can be found in serum fractions at ng/g (or parts per billion) levels following acute exposure in laboratory animals (Abraham et al., 1988; Bartosek, 1984; Patterson et al., 1989
), thereby being a potentially valuable biologic matrix to analyze. Here we describe studies in which the CALUX bioassay system has been optimized for use in a 96-well microtiter plate format for the direct detection of HAHs and PAHs in small volumes of whole serum.
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MATERIALS AND METHODS |
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Recombinant cell line.
Mouse hepatoma (H1L1.1c2) cells, which were stably transfected with the PAH/HAH-inducible luciferase expression vector pGudLuc1.1, were described previously (Garrison et al., 1996). This vector contains the firefly luciferase gene under PAH/HAH-inducible control of four dioxin responsive elements (DREs) and induction of luciferase occurs in a time-, dose-, and AhR-dependent manner. These cells were grown in 100-mm cell culture plates (Corning Glass Works; Corning, NY) using sterile technique, maintained in alpha-minimum essential media (
-MEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin solution (Life Technologies, Gaithersburg, MD), and incubated at >80% humidity and 37°C.
General assay protocol.
Plates of stable cell clones (approximately 80100% confluent) were trypsinized and resuspended in 20 ml -MEM. An aliquot (200 µl) of the indicated cell suspension was added into rows two through eight of a sterile 96-well white CulturPlateTM (Packard Instruments) and plates were incubated for 24 h prior to ligand exposure, allowing cells to reach confluence. Prior to ligand addition, wells were washed with 100 µl of sterile 1X Dulbecco's phosphate-buffered saline (PBS), then exposed by the addition of either control serum [containing DMSO (1%) or TCDD (1 nM in DMSO)] or sample serum at the indicated concentration (in
-MEM supplanted with FBS). Plates were incubated for the indicated time at 37°C, after which cells were washed twice with PBS, 25 µl of 1X Lysis Buffer (Promega; Madison, WI) was added to each well and the plate placed on a plate shaker until cells were lysed (approximately 20 min). Luciferase activity was measured using an automated microplate luminometer (Dynatech ML3000; Chantilly, VA) in enhanced flash mode with the automatic injection of 50 µl of Promega stabilized luciferase reagent.
Correction for cell numbers (through protein quantification) was accomplished through the modification of an assay previously described (Kennedy et al., 1993). Bovine serum albumin (BSA) in lysis buffer was added to row one in differing concentrations ranging from 0 to 75 µg/well, then 100 µl of fluorescamine (Molecular Probes; Eugene, OR) in acetonitrile (at 500 µg/ml) was added to all wells. The subsequent fluorescence was quantified using a multiplate fluorometer (Fluostar, SLT) at 400 nm excitation and 460 nm emission wavelengths and results (in milligrams protein/well) were calculated by comparison to the standard curve.
Statistical methods.
Descriptive statistics and, where applicable, comparative analyses (Student t-test between samples and controls) were calculated using Excel (Microsoft, Redmond, WA). For TCDD, the median effective concentration (EC50) was calculated by fitting dose-response data to a logistic curve using Sigma Plot (SPSS, Chicago, IL) as previously described (Tillitt et al., 1991). Briefly, the regression equation used is as follows:
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RESULTS AND DISCUSSION |
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The CALUX bioassay has been used to determine the ability of natural and synthetic chemicals to activate the AhR system (Denison et al., 1998b; Heath-Pagliuso et al., 1998
; Phelan et al., 1998
) and to identify TCDD-like activity (i.e., AhR ligands/agonists) present in commercial and consumer products (Denison et al., 1996
) and in environmental and biologic matrices (Denison et al., 1996
; Ziccardi et al., 1997
). Studies in our lab, as well as those in our collaborator's, have indicated the feasibility of using the CALUX bioassay to detect HAHs/PAHs and other AhR agonists in serum extracts (Aarts et al., 1996
; Murk et al., 1996a
,b
, 1998
) and whole serum samples (Ziccardi et al., 1997
). Given these data, we sought to optimize this use of the bioassay as a screening tool for measurement of HAHs/PAHs and related chemicals in small volumes of whole serum.
Assay Optimization
In order to determine the minimal and optimal volume of serum needed for analysis, varying amounts of sterile goat serum (Gibco/BRL, Grand Island, NY) containing TCDD or DMSO were mixed with -MEM (with 10% FBS) to a final volume of 50 µl, and the induction of luciferase activity in H1L1.1c2 cells contained within 96-well microtiter plates was determined. The final TCDD and DMSO concentrations in all initial optimization experiments were maintained at 1 nM and 1%, respectively. Sterile goat serum was used in these experiments because we expected that this would better mimic those samples to be collected during population surveys, rather than FBS, which would lack a variety of protein factors (such as IgG) present in adult blood. Other advantages of using goat serum included the ready availability of large stocks of samples from a single lot, the ability to acquire samples from specific pathogen-free (SPF) herds, and fewer health and regulatory concerns in its use (vs the use of human serum).
Although the highest absolute luciferase activity was observed with 25% serum (Fig. 1), the greatest overall fold-induction was observed at 50% level and was due to a decreased basal activity at this concentration. Because of this finding, as well as the minimization of variation of estimates at this concentration, subsequent experiments were carried out at 50% serum. It should be noted, however, that because the standard culture media already contained 10% FBS, the final serum proportion at this concentration actually contained 55% total serum (50% sample serum plus 5% FBS).
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In order to correct for slight differences in cell numbers between wells, which would result in intraplate variability, a method to assess cell number postanalysis indirectly (through the assessment of protein concentrations within each well using fluorescamine) was optimized for use in this protocol. The fluorescamine protein assay has been previously used for rapid fluorescence-based estimation of protein concentrations in microplate assays (Heath-Pagliuso et al., 1998; Kennedy et al., 1993
; Phelan et al., 1998
; Sanderson et al., 1996
). In the CALUX bioassay, microplates are used directly after luminescent analysis, with 100 µl of a 500 µg/ml fluorescamine concentration in acetonitrile as the optimal volume and concentration (data not shown). Acetonitrile was selected as the solvent vehicle because it does not affect the polystyrene microtiter plate (unlike solvents such as acetone), and it also quenches the luminescence present within wells; therefore, the developing fluorescence is not confounded by reporter activity. After incubation for 15 min on a plate shaker, fluorescence is quantified using a multiplate fluorometer (Fluostar, SLT) at 400 nm excitation and 460 nm emission wavelengths. Protein concentrations (milligrams protein per well) were then calculated by comparison to a standard curve of 075 µg/well BSA assayed concurrently in rows of the same plate that are void of cells. Further characterization of this assay has shown that the results are linear up to 300 µg protein per well (data not shown), which is sufficient to detect the protein concentration of lysed cells present in this system. Overall, the application of these normalization/correction methods allows direct comparison of results within and among plates with a greater degree of confidence. The optimized protocol flow used in all subsequent studies is shown in Fig. 5
.
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Application of the CALUX Serum Bioassay for Screening of Wildlife Samples
As an example of the type of information that this bioassay system could afford biomedical researchers, a subset of sera from several different marine wildlife species (with unknown xenobiotic exposure) was acquired and analyzed using the optimized method described above (Fig. 9). Results from these analyses show a spectrum of luciferase activity that can be interpreted based roughly on the natural histories of each. Samples collected from black-footed albatross, a seabird that primarily resides at a distance from shore-based activities, had minimal luciferase activity. Double-crested cormorants, a species that has more interaction with the mainland (therefore more potential contact with xenobiotics) exhibited statistically significant levels of induction but at relatively low levels (on the order of picomolar equivalents of TCDD). The final species examined, the Southern sea otter (Enhydra lutris nereis), is a species at high risk of pollutant exposure due to its residing exclusively in the nearshore habitat, its preying on organisms known to bioaccumulate environmental toxicants, and its reliance on a high metabolic rate of activity and thick layer of hair for thermoregulation. These data indicate that the animals examined here have been exposed to relatively high levels of unknown pollutants (approximately equivalent to 0.1 nM TCDD). Although it is currently unknown what specific chemical(s) in these serum samples is responsible for the observed induction response, our results demonstrate that this bioassay system is a highly effective method for screening large numbers of potentially exposed individuals. Subsequent studies will focus on final validation of this bioassay approach by comparing CALUX induction results with those of analytical chemical analysis (GC/MS) of the same unknown sample. By comparison of the bioassay results with activity obtained from concurrent analysis of several concentrations of TCDD, overall induction equivalencies can be determined for a particular sample. Positive screening test results could then be followed by chemical analysis in order to identify the specific chemicals responsible for the induction response and to assess specific exposure in the individuals. It should be noted, however, that we have observed relatively high background activity in some serum samples from humans (Ziccardi et al., 1997
) and selected wildlife species, whereas serum from other species produces little or no background luciferase activity (Ziccardi et al., manuscript in preparation). Solvent extraction of serum eliminates this background activity (data not shown). These differences may be due to higher exposure of certain populations to AhR-inducing compounds or the presence of endogenous activators in the blood of species that exhibit high background activity. Studies are currently underway to identify the substance(s) present in these control serum samples responsible for activation of the reporter gene. Because this method utilizes an in vitro methodology to detect compounds of interest, interpretation of these data for predicting in vivo toxicologic effects for any species must be made carefully. Therefore, without a priori knowledge of background CALUX activity and endogenous AhR response in the population and species of interest, direct application of results may be limited.
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Although this assay technique has proven to be a valuable method to detect overall exposure to xenobiotic compounds, it cannot be used exclusively for diagnosis. Because of its lack of analytic specificity for individual compounds (due to the detection of any compound that binds and activates the AhR and AhR signal transduction pathway) and the potential for the presence of high basal levels of luciferase activity in some species, we believe that a primary application of this assay is as a rapid and inexpensive method for prescreening a large number of samples from selected species for the presence of AhR ligands. Once positive samples are identified, analytical chemical approaches such as GC/MS and/or HPLC can be used to identify the specific compounds or congeners. Work is currently underway, however, to further refine the methodology described here in order to separate the luciferase activity due to different classes of chemicals possibly present in serum. This combination of chemical and class-specific extraction procedures, coupled with the CALUX assay, will provide further information as to potential exposure to AhR ligands and activators in an inexpensive and relatively rapid manner.
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ACKNOWLEDGMENTS |
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NOTES |
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2 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.
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