Acquired Resistance to Ah Receptor Agonists in a Population of Atlantic Killifish (Fundulus heteroclitus) Inhabiting a Marine Superfund Site: In Vivo and in Vitro Studies on the Inducibility of Xenobiotic Metabolizing Enzymes

Susan M. Bello,1, Diana G. Franks, John J. Stegeman and Mark E. Hahn,2

Biology Department, MS#32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, Massachusetts 02543

Received June 22, 2000; accepted November 8, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
New Bedford Harbor (NBH), MA, is a federal Superfund site that is heavily contaminated with polychlorinated biphenyls (PCBs) and other halogenated aromatic hydrocarbons (HAHs), including some potent aryl hydrocarbon receptor (AhR) agonists. A population of Atlantic killifish (Fundulus heteroclitus) continues to inhabit this site, despite accumulating extraordinarily high concentrations of PCBs (272 µg/g dry weight). To determine if NBH killifish have developed resistance to HAHs that act through the AhR, we examined the inducibility of cytochrome P4501A1 (CYP1A1), UDP glucuronosyl transferase (UGT), and glutathione S-transferase (GST) in fish from NBH and a reference site, Scorton Creek (SC, Cape Cod, MA; PCB concentrations 0.177 µg/g dry weight). 2,3,7,8-Tetrachlorodibenzofuran (TCDF) induced CYP1A1 mRNA, protein, and activity in SC fish in all tissues examined (liver, heart, gut, gill, kidney, spleen, and gonad). In contrast, NBH fish expressed low levels of CYP1A1 and showed no induction of CYP1A1 mRNA, protein, or activity by TCDF, or induction that was lower in magnitude or required higher doses of inducer. p-Nitrophenol UGT activity was not induced by TCDF in either population, while GST activity with 1-chloro-2,4-dinitrobenzene as substrate was induced only in NBH fish in one experiment. Inducibility of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or ß-naphthoflavone (BNF) was measured in primary hepatocyte cultures prepared from SC and NBH fish. TCDD induced CYP1A1 activity (ethoxyresorufin O-deethylase) to the same degree in hepatocytes from both populations, demonstrating the functionality of the AhR signaling pathway in NBH fish. However, hepatocytes from NBH fish were 14-fold less sensitive to TCDD than were those from SC fish. The nonhalogenated AhR agonist BNF also induced CYP1A1 in cells from both populations, although with only a 3-fold difference in sensitivity (NBH < SC). These results indicate that chronic exposure to high levels of HAHs has led to a reduction in the sensitivity of NBH killifish to AhR agonists. The resistance is systemic and pretranslational, and exhibits compound-specific differences in magnitude. These findings suggest an alteration in the AhR signal transduction pathway in NBH fish.

Key Words: dioxin; Ah receptor; resistance; fish; cytochrome P4501A1; polychlorinated biphenyls (PCBs).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
New Bedford Harbor (NBH), MA, is a federal Superfund site contaminated with polychlorinated biphenyls (PCBs), polychlorinated dibenzofurans, polychlorinated dibenzo-p-dioxins, polynuclear aromatic hydrocarbons (PAHs), and heavy metals. PCB concentrations measured in NBH sediments have ranged from a few ppm in the lower harbor up to 100,000 ppm in the area closest to the source of PCB contamination (the "hot spot") (Weaver, 1984Go), with a mean in the hot spot of ~350 ppm (Lake et al., 1995Go). PCB body burdens of 1370 ppm dry weight (Lake et al., 1995Go) or 272 ppm dry weight (Bello, 1999Go) have been reported in Atlantic killifish (Fundulus heteroclitus) caught in and around this location. These are among the highest PCB concentrations ever measured in fish (Elskus et al., 1994Go).

Despite their high PCB body burdens, killifish inhabiting NBH express low levels of cytochrome P450 1A1 (CYP1A1) messenger RNA, protein, and activity equivalent to or less than expression in fish from a reference site, Scorton Creek (SC) (Bello, 1999Go). These findings suggest that the killifish inhabiting NBH may have developed resistance to some or all of the effects of PCBs and other halogenated aromatic hydrocarbons. Because killifish have restricted home ranges, are nonmigratory (Fritz et al., 1975Go; Lotrich, 1975Go), and their offspring are not readily dispersed (Kneib, 1986Go), their physiological and genetic characteristics are strongly influenced by local conditions. In addition, killifish populations are highly polymorphic (reviewed in Powers and Schulte, 1998) and can exhibit rapid evolutionary responses to changes in environmental variables (Mitton and Koehn, 1975Go; Powers et al., 1991Go). Consistent with this, killifish populations experiencing long-term exposure to high levels of toxic chemicals have been known to develop resistance to the toxic effects of those chemicals (Prince and Cooper, 1995aGo; Weis et al., 1981Go; Weis and Weis, 1989Go).

To investigate a possible HAH resistance of NBH killifish, we examined the responsiveness of the aryl hydrocarbon receptor (AhR) signaling pathway, using CYP1A1 induction (Whitlock, 1999Go) as a marker for its activation. The toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are dependent on the AhR, as demonstrated by studies in AhR-null mice (Fernandez-Salguero et al., 1996Go; Peters et al., 1999Go). Given the predominant role of the AhR in the toxicity of PHAHs in mammals, and the general similarity of mammalian and teleost AhR-dependent signaling pathways (Hahn, 1998aGo), it is likely that (1) the AhR mediates the toxicity of PHAHs in fish, and (2) an alteration in AhR-dependent signaling may be involved in the postulated PHAH resistance in NBH killifish.

F. heteroclitus possesses two distinct AhRs (Hahn et al., 1997Go; Karchner et al., 1999Go), and an AhR nuclear translocator (ARNT) protein (Powell et al., 1999Go). Killifish also express a single CYP1A1 form3 (Morrison et al., 1998Go), which is highly inducible, as demonstrated experimentally (Binder and Stegeman, 1980Go; Binder et al., 1985Go; Kloepper-Sams and Stegeman, 1989Go) and by comparison of fish from sites exhibiting different degrees of PCB or PAH contamination (Burns, 1976Go; Elskus and Stegeman, 1989Go). However, recent studies have shown that CYP1A1 inducibility in F. heteroclitus can vary, depending on prior exposure to aromatic hydrocarbons (reviewed in Hahn, 1998b). In these studies, CYP1A1 inducibility was impaired in killifish exposed for multiple generations to high levels of PAHs (Van Veld and Westbrook, 1995Go), TCDD (Prince and Cooper, 1995bGo), or PCBs (Elskus et al., 1999Go; Nacci et al., 1999Go).

In this paper, we explore the nature of the apparent PHAH resistance of NBH killifish by addressing several questions:

To deal with the first of these questions, we compared the responses of fish from NBH and SC after exposure to a potent AhR agonist, 2,3,7,8-tetrachlorodibenzofuran (TCDF). TCDF was chosen as the model inducer because previous experiments had shown it to be a potent inducer of CYP1A1 in marine teleosts (Hahn and Stegeman, 1994Go; Muir et al., 1992Go). We examined a suite of responses, including induction of CYP1A1 activity, protein, and mRNA, glutathione S-transferase (GST) activity, and UDP-glucuronosyl transferase (UGT) activity. These responses were chosen because they are mediated by the AhR signal transduction pathway in mammals (Nebert et al., 1993Go; Sutter and Greenlee, 1992Go).

To address the second and third questions, we examined the inducibility of ethoxyresorufin O-deethylase (EROD) activity in primary cultures of hepatocytes from NBH and SC fish. The use of cultured hepatocytes allowed us to test a wider range of agonist concentrations than was possible using whole fish. For this experiment, the halogenated AhR agonist TCDD and the nonhalogenated AhR agonist ß-naphthoflavone (BNF) were used. TCDD is the most commonly used model agonist for the AhR in mammals (Okey et al., 1994Go; Safe and Krishnan, 1995Go) and is a potent inducer of CYP1A1 in teleosts (Bend et al., 1974Go; Vodicnik et al., 1981Go). BNF is also an inducer of CYP1A1 in teleosts (Kloepper-Sams and Stegeman, 1989Go) as well as in mammals (Okey et al., 1994Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Material.
Frozen krill was purchased from MidJersey Pet Supply (Carteret, NJ). 2,3,7,8-TCDD (10 µg/ml in toluene, >98% purity) and 2,3,7,8-TCDF (>98% purity) were obtained from Ultra Scientific. Alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (secondary antibody) was purchased from Bio-Rad (Hercules, CA). Chemiluminescent substrates were purchased from Schleicher & Schuell (Keene, NH) and Tropix (Bedford, MA). Nonspecific antibody (UPC-10) was purchased from Organon Teknika (West Chester, PA). A universal immunoperoxidase kit (murine) was purchased from Signet Laboratories (Dedham, MA). [32P]dCTP was purchased from Dupont NEN (Boston, MA). RNA STAT-60TM was purchased from TEL-Test (Friendswood, TX). Permount, SDS, and acrylamide were purchased from Fisher (Pittsburgh, PA). All other chemicals were purchased from Sigma (St. Louis, MO).

Fish collection and maintenance.
Killifish were collected using minnow traps baited with dog food on the following dates: NBH (June 28–29, 1994 and July 12–14, 1995); SC (July 18, 1994 and August 17–18, 1995). Fish were maintained in 20°C flowing seawater and fed Tetramin® stapleflake and minced krill.

Experimental Treatment
General dosing procedure.
Fish were moved to 10-gallon experimental aquaria with 20°C flowing seawater, at least 2 days before the start of the experiment. On the first day of the experiment, fish were weighed and then injected intraperitoneally with 2,3,7,8-TCDF dissolved in corn oil, or with corn oil alone. Dosing solutions were made so fish received 5 µl/g body weight. After dosing, fish were held in static seawater (~20°C) and fed only Tetramin® stapleflake. After 7 days, fish were killed by cervical transection. Tissues for microsomes were put immediately in ice-cold AhR buffer (see below). Tissues for immunohistochemical (IHC) analysis were placed in 10% neutral buffered formalin. Tissues for RNA were snap frozen in liquid nitrogen and stored at –80°C until RNA preparation.

Experiment 1.
Female fish that had been held in the laboratory for 9 months were injected with 0, 0.06, 0.31, 1.5, or 7.6 nmol TCDF/kg. Each treatment group was held in a separate tank; for each site there were 4 control fish and 5 fish at each dose of TCDF (total of 24 fish per site). Whole livers were collected from individual fish for analysis of CYP1A1 activity and protein, GST activity, and UGT activity. Hearts, gills, gonads, and spleens were collected from individual fish for IHC analysis of CYP1A1 protein.

Experiment 2.
Male fish that had been held in the laboratory for 17 months were injected with 0 or 7.6 nmol TCDF/kg. Nine fish from each site were injected at each dose. After injection, fish from the same site treated with the same dose were randomly assigned to 1 of 3 tanks for a total of 3 fish per tank and 3 tanks per dose per site. There was one additional tank with 3 fish from NBH treated with 50 nmol TCDF/kg. Individual livers were sub-sampled into 3 portions, with the major portion (~2/3 total weight) used for analysis of CYP1A1 activity and protein, GST activity, and UGT activity (n = 9 for all groups, except n = 3 for the 50-nmol/kg group). Smaller liver portions were used individually for IHC analysis (n = 9 for each group, except n = 3 for the 50-nmol/kg group). Liver portions from 3 fish (in one tank) were pooled for RNA preparation (n = 3 pools for each group, except n = 1 pool for the 50-nmol/kg group). Extrahepatic tissues (heart, gill, intestine, kidney, spleen, and gonad) from one fish per tank were preserved for IHC analysis (n = 3 for each group, except n =1 for the 50-nmol/kg group). The extrahepatic organs from the other 2 fish in each tank were pooled for RNA preparation (n = 3 pools for all groups, except n = 1 pool for the 50-nmol/kg group).

Hepatocyte culture.
Female NBH and SC killifish were collected in July 1996 and held for 16 months in the laboratory before being used for isolation of hepatocytes in December of 1997. Fish were killed by cervical transsection and the livers from 7 fish (SC; total liver weight: 0.87 g) or 5 fish (NBH; total liver weight: 0.56 g) were each pooled and placed immediately into 50 ml of ice-cold Ca++-free Ringer's solution (0.18 M NaCl, 1.54 mM KCl, 6.49 mM 7H2O-MgSO4, 1.45 mM Na2HPO4, K3PO4, 0.08% [w:v] glucose, 2.0% [w:v] BSA, 0.5 mM EGTA, and 10 ml/l P/S/A [Sigma, Antibiotic-Antimycotic solution]). In a sterile hood, the livers were transferred to a petri dish with 20 ml fresh Ca++-free Ringer's solution, and were minced. The minced livers, without the Ringer's buffer, were transferred to sterile 50-ml centrifuge tubes and incubated for 20 min at room temperature in 30 ml trypsin/EDTA buffer (136 mM NaCl, 11 mM KCl, 0.35 mM KH2PO4, 0.21 mM Na2HPO4-7H2O, 0.24 nM phenol red, 0.003% penicillin, 0.005% streptomycin, 0.05% Trypsin, and 0.5 mM EDTA). To aid in digestion, the livers were gently homogenized with a Teflon-glass homogenizer. The resulting suspension was filtered through 4 layers of sterile cheesecloth into a sterile 50-ml centrifuge tube and then centrifuged for 5 min at 100 x g at 4°C. The resulting pellet was resuspended in 25 ml of MEM plus 10% calf serum and centrifuged for 5 min at 100 x g. This pellet was resuspended in 10 ml of MEM plus 10% calf serum and the concentration of cells determined with a hemacytometer. The hepatocytes were plated in 96-well plates at 4 x 105 cells/well and allowed to attach overnight. The following morning, the hepatocytes were exposed to TCDD (0.001–30 nM) or BNF (1–10,000 nM) in DMSO or to DMSO alone for 24 h, using methods described previously (Hahn et al., 1996Go). Three replicate wells were exposed at each concentration.

Microsome and cytosol preparation.
Tissues were homogenized in 9 ml of cold AhR buffer per gram of tissue. AhR buffer consists of 25 mM MOPS (pH 7.5) with 1 mM EDTA, 5 mM EGTA, 0.02% NaN3, 20 mM Na2MoO4, 10% (v:v) glycerol, 1 mM dithiothreitol, plus protease inhibitors (20 µM TLCK, 5 µg/ml leupeptin, 13 µg/ml aprotinin, 7 µg/ml pepstatin A, and 0.1 mM PMSF) (Hahn et al., 1994Go). Tissues were homogenized with a Teflon-glass homogenizer (10 passes); intestines and gills were minced with dissecting scissors prior to homogenization. Homogenates were centrifuged for 10 min at 750 x g and 10 min at 12,000 x g at 4°C. The supernatant was then centrifuged at 100,000 x g for 70 min at 4°C. This supernatant (cytosol) was removed and frozen in liquid N2. The pellet (microsomes) was resuspended in TEDG (0.05M Tris pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol) (Stegeman et al., 1979Go) and frozen in liquid N2. Total microsomal or cytosolic protein was measured fluorometrically (Lorenzen and Kennedy, 1993Go).

CYP1A1 activity.
CYP1A1 activity in microsomes was determined as EROD using a fluorometric, kinetic assay (Hahn et al., 1993Go). Assays were run in duplicate in 48-well plates with 2-µM 7-ethoxy resorufin (7-ER) and 1.0 mM NADPH (final concentrations). Samples were scanned once every minute for 15 min. A standard resorufin curve (ranging from 0 to 200 pmol of resorufin) accompanied each set of samples. EROD activity was calculated as pmol resorufin per min per mg total microsomal protein. EROD activity in individual wells was linear for at least 10 min; only this linear portion was used to calculate activities.

CYP1A1 activity in primary hepatocytes was determined using a fluorometric, stopped assay for EROD activity and total protein (Kennedy et al., 1995Go) with modifications (Hahn et al., 1996Go). Briefly, medium with inducers was removed and the cells were washed with phosphate-buffered saline (136 mM NaCl, 0.81 mM Na2HPO4, 0.15 mM KH2PO4, 0.27 mM KCl). Phosphate buffer (25 mM Na2HPO4-7H2O/NaH2PO4-H2O) and 7-ER (2 µM) were added to each sample well and incubated for 10 min at room temperature. The reaction was stopped with the addition of cold fluorescamine in acetonitrile (0.23 pM). The plates were then incubated for 15 min at room temperature and scanned. Protein (BSA) and resorufin standards were run on each plate.

GSH transferase (GST) activity.
The protocol described in Habig et al. (1974) as modified by Van Veld et al (1991) was followed, with modifications. Briefly, GST activity was measured by adding hepatic cytosol (0.02 to 0.14 mg total protein) to 1 ml of reaction buffer containing 1 mM 1-chloro-2,4-dinitrobenzene (CDNB), 100 mM Tris–HCl, and 1 mM reduced glutathione and then monitoring the change in absorbance for 1 min at 344 nM. GST activity was expressed as nmol of conjugate produced per min per mg total cytosolic protein.

UDP glucuronosyl transferase (UGT) activity.
The protocol described by Andersson et al (1985) was followed, with modifications. Briefly, microsomes (0.01 to 0.11 mg total protein) were added to 0.5 M KH2PO4 with p-nitrophenol (2.43 mg/50 ml) and digitonin (2 mg/ml), plus or minus uridine diphosphate glucuronic acid (UDPGA; 4 mg/ml). This mixture was incubated for 20 min at room temperature, shaking, in the dark, after which 0.45 ml of 3% trichloroacetic acid was added and the entire mix was centrifuged for 15 min at 4000 rpm. KOH (0.05 ml of 5M) was added to 0.375 ml of the supernatant and the absorbance was read at 400 nm. UGT activity was expressed as nmol of conjugate produced per minute per mg total microsomal protein.

Immunoblotting.
CYP1A1 protein content was measured with a chemiluminescent Western blot assay (Hahn et al., 1996Go) using monoclonal antibody (MAb) 1-12-3 against CYP1A1 from scup (Stenotomus chrysops) (Kloepper-Sams et al., 1987Go; Park et al., 1986Go). A standard curve (0.025 to 0.5 pmol CYP1A1) using scup microsomes with known CYP1A1 content was used to determine CYP1A1 protein in the NBH and SC samples. Samples were loaded so that they fell within the range of the standard curve. Multiple exposures were taken of each blot.

Northern blotting.
Total RNA was prepared from frozen tissues using TEL-TEST's RNA STAT-60TM. RNA (10 µg per lane) was run in 1% agarose/3.6% formaldehyde gels; equal loading of lanes was confirmed by ethidium bromide staining of gels prior to transfer. RNA was then transferred to a nylon membrane overnight, and probed with full-length 32P-labeled F. heteroclitus CYP1A1 cDNA (Morrison et al., 1998Go). Membranes were prehybridized for 1 h at 42°C in 50% formamide, 6x SSPE, 10x Denhardts, 0.1% SDS overnight at 42°C. Hybridization was performed overnight at 42°C in the same solution with the addition of the 32P-labeled cDNA probe. Membranes were washed at 65°C in 2x SSC, 0.1% SDS, for 30 min, followed by fluorography with Kodak XAR-5 film and one intensifying screen.

Immunohistochemistry.
IHC was done according to the methods of Smolowitz et al (1991), except that two 1-h incubations with 150 µl of MAb 1–12–3 (1.7 µg/ml in PBS/BSA) were used. Sections of induced and uninduced scup liver were run with each batch of sections as positive and negative controls, respectively. Matching sections were stained with a nonspecific IgG (purified mouse myeloma protein, UPC-10, Organon Teknika, West Chester, PA, 1.7 µg/ml in PBS/BSA) as a negative control. Sections were read blind and scored on 2 scales, occurrence (0 [no cells staining] to 3 [all cells staining]) and intensity (0 [no staining] to 5 [very dark red staining]). These two scores were multiplied for a final score ("staining index") of 0 to 15. Previous studies have shown the correspondence between this staining index and biochemical measurements of CYP1A protein (Hahn et al., 1993Go; Woodin et al., 1997Go).

Statistical Analyses
Data were analyzed by 2-way ANOVA comparing site and dose, followed by post hoc tests (Tukey's or modified Tukey's) to assess specific differences; details are provided in figure legends and table titles and notes. Statistical analyses were performed using Statistica (Statsoft, Inc.) and SigmaPlot (Jandel Scientific). For determination of EC50 values for induction of EROD in hepatocyte cultures, EROD data were fitted to a modified Gaussian function as described previously (Hahn et al., 1996Go; Kennedy et al., 1996Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Studies
The low levels of CYP1A1 expression despite high concentrations of PCBs in NBH killifish suggested that this population of fish has developed resistance to some AhR agonists (Bello, 1999Go). To test this hypothesis, we compared the biochemical responsiveness of NBH killifish and fish from the reference site, SC, to 2,3,7,8-TCDF, a potent AhR agonist. Two experiments were performed. The first experiment, conducted using female fish, examined the TCDF dose-response relationship for microsomal CYP1A1 activity and immunodetectable protein in liver, as well as CYP1A1 protein in selected extrahepatic tissues. In the second experiment, a single, high dose of TCDF was used to assess the inducibility of CYP1A1 protein and mRNA in liver and several other tissues. Hepatic Phase II enzyme activities were measured in both experiments.

Body and organ weights and microsomal yields.
There was no significant difference in body weight, liver somatic index (LSI), gonad somatic index (GSI), or microsomal yield between control and treated fish from the same site in either Experiment 1 (Table 1Go) or 2 (Table 2Go). In both experiments, fish from NBH had LSIs that were significantly higher, and GSIs that were significantly lower than those in fish from SC. Mean body weights of fish from NBH were significantly lower than those of fish from SC in Experiment 1. In Experiment 2 there was no difference in body weights between the sites.


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TABLE 1 Mean Body Weight, Liver Somatic Index (LSI), Gonad Somatic Index (GSI), and Microsomal Yield of Control and TCDF-Treated Killifish (Experiment 1)
 

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TABLE 2 Mean Body Weight, Liver Somatic Index (LSI), Gonad Somatic Index (GSI), and Microsomal Yield of Control and TCDF-Treated Killifish (Experiment 2)
 
CYP1A1.
In Experiment 1, female fish from SC responded to TCDF treatment with a dose-dependent increase in CYP1A1 expression. In the liver this was measured as increased EROD activity and CYP1A1 protein (Fig. 1Go). Using IHC analysis, induction of CYP1A1 protein was seen also in the vascular endothelium of the heart (Figs. 2A and 2BGo), gonad, spleen, and gill (Fig. 3Go, gill data not shown). In contrast, no induction was seen in the fish from NBH, by any measure, in any tissue, at doses up to 7.6 nmol TCDF/kg (Fig. 1Go, Figs. 2C and 2DGo, Fig. 3Go).



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FIG. 1. Induction of hepatic CYP1A1 (EROD) activity (A) and protein (B) in SC (open bars) and NBH (hatched bars) female killifish injected with corn oil, or the indicated doses of TCDF in Experiment 1. Each bar is the mean (± SE) of 4 (corn oil) or 5 (TCDF) fish. In A, EROD activities were significantly different between SC and NBH (p < 0.01, 2-way ANOVA comparing site and dose). *Significantly different from the control from the same site (p < 0.01, 2-way ANOVA comparing site and dose, followed by modified Tukey's test for unequal sample sizes).

 


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FIG. 2. CYP1A1 expression in the atria of SC and NBH female killifish injected with corn oil or 7.6 nmol TCDF/kg in Experiment 1. Immunohistochemical staining with MAb 1–12–3 was performed as described in Materials and Methods. Red color indicates cells positive for CYP1A1. (A) SC corn oil (B) SC TCDF (C) NBH corn oil (D) NBH TCDF. Scale bar, 1 µm. Arrowheads indicate endocardial cells.

 


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FIG. 3. CYP1A1 expression in the vascular endothelium of the atrium and ventricle (A) or spleen and gonad (B) of SC and NBH female killifish injected with corn oil or the indicated doses of TCDF in Experiment 1. CYP1A1 expression was determined by IHC. CYP1A1 staining index (range: 0 to 15) is the product of staining occurrence (scored from 0 to 3) and staining intensity (scored from 0 to 5). See Materials and Methods for further details. In A and B, IHC staining indices were significantly different between SC and NBH, for each tissue (p < 0.01, 2-way ANOVA comparing site and dose). *Significantly different from control fish from the same site (p < 0.01, 2-way ANOVA comparing site and dose, followed by modified Tukey's test for unequal sample sizes).

 
In Experiment 2, male fish from both sites had induced EROD activity, but the magnitude of induction and the dose required to achieve it differed (Table 3Go). In SC fish, EROD activity was induced significantly by 7.6 nmol TCDF/kg. EROD activity in the livers of fish from NBH was not significantly induced by this dose, but it was significantly induced by 50 nmol TCDF/kg. Despite the 7-fold greater dose, the EROD activity induced by this dose in NBH fish was 3-fold lower than that measured in SC fish treated with 7.6 nmol TCDF/kg (Table 3Go).


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TABLE 3 CYP1A1 Activity and Protein in SC and NBH Killifish Treated with 2,3,7,8-TCDF in Experiment 2
 
A slightly different pattern of response was observed when levels of CYP1A1 protein were measured (Table 3Go). Fish from SC treated with 7.6-nmol TCDF/kg had significantly elevated levels of CYP1A1 protein. NBH fish also had significantly higher levels of CYP1A1 protein in the 7.6 nmol/kg group as well as in the 50 nmol/kg group. However, the magnitude of the induced expression differed between sites. CYP1A1 levels induced by either dose in fish from NBH was 0.02 pmol/µg, equal only to the expression in control fish from SC, and much lower than that induced in SC fish (0.08 pmol/µg).

CYP1A1 mRNA was induced by TCDF in the livers of SC fish (Fig. 4Go, panel A, lanes 4–6 and 10–12), consistent with the strong induction of hepatic CYP1A1 activity and protein in these fish. However, there was variability in CYP1A1 mRNA levels among the pooled RNA samples, suggesting heterogeneity of response among individual fish. In contrast to the apparent induction seen in at least some SC fish, CYP1A1 mRNA was not induced in livers of NBH fish at any dose of TCDF (Fig. 4AGo, lanes 1–3 and 7–9).



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FIG. 4. Northern blots of total RNA from the liver (A), heart (B), kidney (C), and gill (D) of SC and NBH male killifish injected with corn oil or 7.6 nmol TCDF/kg in Experiment 2. Each lane contained total RNA (10 µg per lane) pooled from 3 fish (liver) or 2 fish (extrahepatic tissues); 3 pools per treatment were used for Northern blots with hybridization to killifish CYP1A1 cDNA probe, as described in Materials and Methods. Each lane represents RNA pooled from the same 2 or 3 individuals for each tissue. Lanes 1–3: NBH corn oil; lanes 4–6: SC corn oil; lanes 7–9: NBH TCDF (7.6 nmol/kg); lanes 10–12: SC TCDF (7.6 nmol/kg). No induction of CYP1A1 mRNA was evident in tissues of NBH fish treated with the 50 nmol TCDF/kg dose (not shown).

 
IHC analysis of CYP1A1 expression in extrahepatic tissues revealed differential inducibility like that seen in the liver. In the SC animals, CYP1A1 protein was induced in the vascular endothelium of the heart, gill, intestine, and kidney (Fig. 5Go) as well as in the gonad (not shown). In contrast, CYP1A1 protein expression was not induced in any tissue of NBH fish. Interestingly, as in fish collected from the field (Bello, 1999Go), fish from NBH and SC had moderate levels of CYP1A1 expression in the renal proximal tubules, regardless of treatment; the levels were slightly but significantly greater in SC fish than in NBH fish. Kidney was the only tissue in which fish from NBH consistently expressed CYP1A1. Overall, IHC analysis showed that TCDF treatment induced CYP1A1 protein expression in SC fish but not NBH fish.



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FIG. 5. IHC analysis of CYP1A1 expression in extrahepatic tissues (endothelium of atrium, ventricle, kidney, intestine, and gill; epithelial cells in kidney) of SC and NBH male killifish injected with corn oil or 7.6 nmol TCDF/kg in Experiment 2. CYP1A1 expression was determined by IHC. CYP1A1 staining index (range: 0 to 15) was calculated as described in Materials and Methods and the legend to Figure 3Go. For all tissues shown, site differences were statistically significant (p < 0.01, 2-way ANOVA comparing site and dose). Asterisks to the right of bars indicate that the value for TCDF-treated fish was significantly different from the control value for that tissue, at that site (p < 0.01, 2-way ANOVA comparing site and dose, followed by Tukey's test). There was no significant induction in any nonendothelial cell types of extrahepatic tissues (scores not shown). Abbreviations used: prox, proximal tubules; dist, distal tubules; endo, vascular endothelium; ud, no CYP1A1 protein was detected.

 
Measurement of CYP1A1 mRNA expression in extrahepatic tissues confirmed the results obtained with IHC. CYP1A1 mRNA was induced in the heart, kidney, gill, and intestine of SC fish (Figs. 4B–4DGo), although the magnitude of induction varied among pools of RNA from the same treatment group. CYP1A1 mRNA was not induced in the spleen or gonad of fish from SC (not shown). CYP1A1 mRNA was not induced in any tissue in fish from NBH, with the possible exception of heart (one of 3 pools).

GST.
Rates of GST activity in NBH and SC females (Experiment 1) did not change in response to TCDF treatment (Fig. 6AGo). Similarly, in Experiment 2 there was no change in rates of GST activity with TCDF treatment in the SC (male) fish. In the NBH male fish, however, there was a significant increase in GST activity with TCDF treatment (Fig. 6BGo).



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FIG. 6. Hepatic GST activity in SC (open bars) and NBH (hatched bars) killifish injected with corn oil or the indicated dose of TCDF. (A) Experiment 1: Bars are the mean (± SE) of 4 (corn oil) or 5 (TCDF) female fish per group. There were no significant differences in GST activity between TCDF doses or sites (2-way ANOVA comparing site and dose). (B) Experiment 2: Male killifish were treated with corn oil or 7.6 or 50 (NBH only) nmol TCDF/kg dissolved in corn oil. Bars are the mean (± SE) of 9 fish per group, except for the 50 nmol TCDF/kg, which is the mean (± SE) of 3 fish. There was no significant difference in GST activity between doses in SC fish. *There was a significant difference in GST activity between control and TCDF-treated NBH fish (p < 0.005, 2-way ANOVA comparing site and dose, followed by Tukey's test).

 
UGT.
In Experiment 1, UGT activity was variable in fish from both sites, but lacked any consistent trend related to TCDF dose (Fig. 7AGo). There was no significant difference in UGT activity between sites in this experiment. In Experiment 2, as in Experiment 1, there was no significant difference in UGT activity with TCDF treatment (Fig. 7BGo). There was a significant difference, however, between sites; fish from NBH had UGT activities that were approximately 60% of those of fish from SC.



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FIG. 7. Hepatic UGT activity in SC (open bars) and NBH (hatched bars) killifish injected with corn oil or the indicated dose of TCDF. (A) Experiment 1: Bars are the mean (± SE) of 4 (corn oil) or 5 (TCDF) female fish per group. There were no significant differences between sites or TCDF doses (2-way ANOVA comparing site and dose). (B) Experiment 2: Male killifish were treated with corn oil or 7.6 or 50 (NBH only) nmol TCDF/kg dissolved in corn oil. Bars are the mean (± SE) of 9 fish per group except for the 50 nmol TCDF/kg which is the mean (± SE) of 3 fish. There were no significant differences in UGT activity between doses in SC or NBH fish. There was a significant difference in UGT activity between sites when only the corn oil and 7.6 nmol TCDF/kg treatment groups were compared (p < 0.005, 2-way ANOVA comparing site and dose).

 
In Vitro Studies
The in vivo dosing studies described above did not reveal the magnitude of the difference in sensitivity to HAH between the NBH and SC killifish, nor did they allow us to ascertain whether the fish from NBH were capable of more than a minimal response. To address these questions, CYP1A1 inducibility was assessed in primary hepatocytes treated with TCDD. Primary hepatocyte cultures from both NBH and SC fish responded to TCDD treatment with increased EROD activities (Fig. 8AGo). There was no difference in the maximal EROD activity reached after TCDD treatment of hepatocytes from the two sites. However, the EC50 for TCDD in hepatocytes from NBH fish was 14-fold greater than the EC50 for TCDD in hepatocytes from SC fish (Table 4Go).



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FIG. 8. CYP1A1 (EROD) activity in primary cultures of hepatocytes from SC killifish (open symbols) or NBH killifish (filled symbols) treated with TCDD (A) or BNF (B). (A) Hepatocytes were treated with 2,3,7,8-TCDD (0.001 to 30 nM) and EROD activity was measured 24 h later. Duplicate plates were run for each site; each plate contained 3 replicate wells for each dose. Each set of symbols and its accompanying fitted curve represents results from one plate. (B) Hepatocytes were treated with BNF (1 to 10,000 nM), and EROD activity was measured 24 h later. EC50 values are listed in Table 4Go.

 

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TABLE 4 Sensitivity of Primary Cultures of Killifish Hepatocytes to CYP1A1 Induction by TCDD and BNF
 
To determine if the decreased sensitivity of NBH killifish to TCDD and TCDF is reflected in decreased sensitivity to a nonhalogenated AhR agonist, primary hepatocytes from SC and NBH fish were treated with BNF. As observed for TCDD, there was no difference between sites in the maximal EROD activity induced by BNF (Fig. 8BGo, Table 4Go). However, the EC50 for CYP1A1 induction in hepatocytes from NBH fish was 3-fold greater than that in hepatocytes from SC fish (Table 4Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Studies
CYP1A1.
In a previous study, we showed that killifish from NBH appear to be resistant to CYP1A1 induction by AhR agonists encountered in their environment (Bello, 1999Go). In the present work, we examined the responsiveness of NBH fish following laboratory treatment with model AhR agonists. The results of treatments with TCDF demonstrate that fish from NBH do not recover normal CYP1A1 inducibility even after prolonged periods (9–17 months) in clean water. As in the field studies (Bello, 1999Go), the lack of CYP1A1 inducibility in NBH fish was not limited to the liver, but was found in every tissue examined.

The reduced sensitivity of NBH fish to CYP1A1 induction was measured at multiple levels: catalytic function, immunodetectable protein, and messenger RNA. EROD activity does not always reflect the amount of CYP1A1 protein, since many CYP1A1 inducers also inhibit the induced enzyme (Gooch et al., 1989Go). Therefore, the lack of EROD inducibility in NBH fish was confirmed by measuring CYP1A1 protein, which was similarly nonresponsive. However, because CYP1A1 protein may also be lost due to oxidative inactivation at high doses of some PCBs (Gooch et al., 1989Go; Schlezinger et al., 1999Go), CYP1A1 mRNA content was also determined. The fact that resistance to CYP1A1 induction in NBH fish occurred at the level of mRNA suggests an effect on CYP1A1 gene transcription; nuclear run-on experiments would be needed to confirm this.

In this study, as in the previous study (Bello, 1999Go), the proximal tubules of the kidneys were the one cell type in which fish from NBH expressed readily detectable levels of CYP1A1 protein. Control and TCDF-treated animals from both sites were found to express similar, moderate levels of CYP1A1 in these cells. This pattern of expression has also been detected by others in F. heteroclitus adults (Van Veld et al., 1997Go) and larvae (Elskus et al., 1999Go), but not in pre-hatch embryos (Toomey et al., 2001Go). CYP1A1 mRNA was not detectable in whole kidneys from the same experiment in which moderate expression of CYP1A1 protein was found (Fig. 4Go); this could reflect the lower sensitivity of a whole-tissue assay (Northern blots) as compared to the localized expression revealed by IHC. Regardless, the constitutive expression of CYP1A1 (or a closely related CYP) in renal tubules of killifish suggests regulatory and (perhaps) functional differences as compared to CYP1A1 in other cells, and that these features are unaffected by the altered CYP1A1 inducibility in other tissues and cells of NBH fish.

Phase II enzymes.
We also examined the response of two phase II enzymes, GST and UGT. Some members of the GST and UGT gene families are part of the mammalian AhR gene battery (Nebert, 1989Go; Nebert et al., 1993Go). Although the existence of a similar gene battery under regulation of the AhR has not yet been established in fish, some studies have reported induction of GST and UGT by PHAH or PAH in fish (see below), suggesting that these activities might provide an additional measure of the relative responsiveness of NBH and SC fish. In Experiment 1, in which gravid female fish were exposed to multiple TCDF doses (0.06–7.6 nmol/kg), neither GST nor UGT activity differed significantly in relation to TCDF dose, in fish from either site. In Experiment 2, in which reproductively regressed male fish were exposed to TCDF (7.6 or 50 nmol/kg), a dose-dependent increase in GST activity occurred only in the fish from NBH, while UGT activity differed between sites but was not significantly induced by any dose of TCDF in SC or NBH fish. Such conflicting results are common when using the relatively nonspecific GST and UGT substrates CDNB and p-nitrophenol, respectively, in fish. Other studies in fish have found no change, induction, or suppression of GST or UGT activity after treatment with agents such as phenobarbital, PCBs, benzo[a]pyrene (Collier and Varanasi, 1991Go), 3-methylcholanthrene (Taysse et al., 1998Go), or 2,3,7,8-TCDD (Hektoen et al., 1994Go). Often the response of GST does not correspond to the response of UGT (Hitchman et al., 1995Go; Koponen et al., 1997Go; Pangrekar and Sikka, 1992Go). Both GST and UGT activity can be affected (either up- or down-regulated) by hormonal expression in mammals (Igarashi et al., 1984Go; Masmoudi et al., 1996Go) and in fish (Koivusaari et al., 1981Go; Sikoki et al., 1989Go; Leaver and George, 1996Go), suggesting that the different sex and reproductive status of the fish used in Experiments 1 and 2 may have affected these results.

In Vitro Studies
The use of primary hepatocyte cultures allowed us to evaluate a wider range and higher concentrations of inducers than was possible in vivo. This experiment produced 3 significant findings.

Importantly, CYP1A1 expression was inducible in hepatocytes from NBH fish, as indicated by the dose-dependent increase in EROD activity, and the maximal activity was the same as that found in hepatocytes from SC fish. Thus, the AhR signaling pathway is intact and functional in NBH fish. The finding of identical EROD maxima in SC and NBH hepatocytes differs somewhat from results of in vivo dosing experiments. Nacci et al. (1999) reported that NBH killifish embryos had significantly lower maximal EROD activities than embryos from a reference population (West Island) following waterborne exposure to CYP1A1 inducers. Similarly, in our in vivo experiments, levels of CYP1A1 in NBH fish did not approach the level measured in SC fish (Table 3Go). The experiments in cultured hepatocytes allowed us to evaluate the intrinsic responsiveness of the AhR signaling pathway in cells from NBH and SC fish, over a greater number and broader range of concentrations than could practically be used in vivo.

NBH hepatocyte cultures were less sensitive to CYP1A1 inducers than were SC hepatocytes. Because full dose-response curves were obtained for both populations, we could quantitatively determine the difference in sensitivity. The 14-fold difference in sensitivity to TCDD is similar to the difference between responsive and nonresponsive mice in sensitivity to induction of CYP1A1-dependent aryl hydrocarbon hydroxylase activity by TCDD, described more than 25 years ago (Poland et al., 1974Go; Poland and Glover, 1975Go). The magnitude of the difference in our hepatocyte experiments was somewhat less than the difference in responsiveness between NBH and reference fish seen by Nacci et al. (1999) based on induction of EROD by 3-methylcholanthrene or 3,3`,4,4`,5-pentachlorobiphenyl in live embryos (61- to 84-fold). The reasons for this difference are not known.

The hepatocyte experiments also revealed a distinction between the degree of resistance to the halogenated inducer TCDD (14-fold) and the nonhalogenated inducer BNF (3-fold). This could simply reflect a difference in the persistence of BNF between SC and NBH cultures, leading to a reduced concentration of this inducer in the SC cells compared to NBH cells. Rapid turnover of inducer is known to affect the apparent sensitivity to induction in cell culture (Riddick et al., 1994Go). However, a difference in responsiveness to halogenated versus nonhalogenated compounds similar to that seen here has been observed in PCB-exposed tomcod from the Hudson River (Courtenay et al., 1999Go; Wirgin et al., 1992Go). CYP1A1 mRNA in depurated Hudson River tomcod was highly induced by treatment with BNF or benzo[a]pyrene, but not by treatment with 3,3`,4,4`-tetrachlorobiphenyl or TCDD (Courtenay et al., 1999Go). The mechanism of the inducer-dependent difference in the magnitude of resistance in NBH killifish could involve altered expression or function of the two AhRs that we have identified in F. heteroclitus (Hahn et al., 1997Go; Karchner et al., 1999Go). Alternatively, a separate receptor, possibly related to the 4S PAH binding protein found in mammals (Bhat et al., 1997Go; Raha et al., 1995Go) and fish (Barton and Marletta, 1988Go), may be responsible for some, if not all, of the response to nonhalogenated compounds. However, we need not invoke multiple receptors to explain the differential responsiveness to TCDD and BNF. Alterations in a single receptor can result in ligand-specific changes in affinity, as demonstrated recently for a mutant form of peroxisome proliferator-activated receptor (Sarraf et al., 1999Go).

Mechanism of resistance.
Several populations of killifish resistant to AhR agonists have been reported, but in each case there are distinct features of the resistant phenotype. Prince and Cooper (1995a,b) described a dioxin-resistant population of killifish environmentally exposed to TCDD in Newark, NJ. In the Newark fish, as in NBH fish, CYP1A1 expression was not induced following treatment with TCDF or TCDD. However, while in our study hepatic CYP1A1 expression in untreated NBH fish was not significantly different from that in reference (SC) fish, basal hepatic CYP1A1 expression in untreated Newark killifish was significantly higher than in reference fish (Prince and Cooper, 1995bGo). A PAH-resistant population of killifish has been identified in a creosote contaminated site on the Elizabeth River (VA) (Van Veld and Westbrook, 1995Go). These fish also demonstrated a lack of CYP1A1 inducibility following exposure to a known CYP1A1 inducer, 3-methylcholanthrene (a nonhalogenated compound). However, CYP1A1 in the Elizabeth River fish was environmentally induced in both hepatic and extrahepatic tissues at certain times of the year. The variability in resistance phenotypes among different populations of chemically impacted killifish suggests that such resistance has occurred through different mechanisms or that the magnitude of resistance varies at each site. The actual mechanism(s) involved at any one site would likely depend on the type of chemical present and the inherent genetic variability of the initial population.

Acquired chemical resistance can occur through a variety of genetic and epigenetic mechanisms, many of which involve altered expression or functional properties of receptors (Taylor and Feyereisen, 1996Go; Hahn, 1998bGo). For example, point mutations in receptors for neurotransmitters or hormones are known to confer resistance to pesticides or hormones, respectively (Auchus and Fuqua, 1994Go; ffrench-Constant et al., 1993Go). A null mutant for a juvenile hormone-binding protein in Drosophila causes resistance to the insecticide methoprene (Wilson and Ashok, 1998Go). Interestingly, this protein, like the AhR, is a member of the bHLH-PAS family.

Our results provide some insight into the nature of the resistance in NBH killifish. In experiments with primary hepatocytes, the magnitude of the CYP1A1 induction response did not differ between NBH and SC fish, indicating that the AhR pathway is functional in the resistant fish. The differential sensitivity of NBH and SC fish to TCDD (in vitro) and TCDF (in vivo) is consistent with a possible difference in the ligand-binding affinity of their respective AhRs. A change in affinity could occur from mutation of a single base in an AhR gene. Such a mechanism is known from studies in inbred mice, in which a single amino acid difference in the AhR ligand-binding domain, resulting in a lower affinity AhR, is primarily responsible for the reduced sensitivity to PAHs and HAHs (Okey et al., 1989Go; Poland et al., 1994Go). Mutations in other portions of the AhR (Sun et al., 1997Go; Pohjanvirta et al., 1998Go) are also known or have been suggested to cause reduced TCDD sensitivity in rodent systems. Alterations in other components of the AhR pathway, such as ARNT (Pollenz et al., 1996Go; Wilson et al., 1997Go), coactivator proteins (Kumar et al., 1999Go; Nguyen et al., 1999Go; Xu et al., 1998Go), or CYP1A1 (Kimura et al., 1987Go), could also play a role in the resistance observed in NBH killifish. The expression of a mutant, catalytically altered form of CYP1A1 in fish from NBH is unlikely, given the close agreement of the maximal EROD activities in hepatocytes from the two sites, as well as the general agreement of CYP1A1 mRNA, protein, and activity measurements both within and between sites. We cannot rule out the possibility of an alteration in the CYP1A1 promoter that reduces transcriptional activation by ligand-activated AhR-ARNT complexes. However, because NBH fish are also less sensitive to the embryo-larval toxicity of 3,3`,4,4`,5-pentachlorobiphenyl (Nacci et al., 1999Go), the mechanism of PHAH resistance is not limited to CYP1A1 induction, but must be more general.

The existence of two forms of the AhR in killifish (Hahn et al., 1997Go; Karchner et al., 1999Go) suggests another possible mechanism of dioxin resistance of NBH fish. If the two AhRs possess differential ligand-binding affinities or other properties, altered expression of one form relative to the other could influence the sensitivity of response to TCDD and TCDF. Recently, we found increased expression of AhR1 mRNA in several tissues of NBH killifish, as compared to SC fish (Powell et al., 2000Go). However, this altered AhR1 expression was not heritable, raising questions about its relationship, if any, to the mechanism of resistance. Efforts to determine the possible roles of these two AhRs in the resistant phenotype are continuing.

Reduced sensitivity to AhR agonists following prolonged or repeated exposures to AhR agonists is also known to occur by nongenetic mechanisms. The AhR is down-regulated via proteosomal degradation (Davarinos and Pollenz, 1999Go) following exposure of mammals or mammalian cells to TCDD (Reick et al., 1994Go; Pollenz, 1996Go; Giannone et al., 1998Go; Pollenz et al., 1998Go). Whether AhR1 or AhR2 proteins are down-regulated in PHAH-exposed killifish is not known; such studies await the development of form-specific antibodies against these fish AhRs. Other nongenetic mechanisms could involve induction of proteins that repress AhR signaling, such as the AhR repressor (AhRR) recently identified in mice (Mimura et al., 1999Go), or other repressors (Watson et al., 1992Go; Boucher et al., 1993Go; Sterling and Bresnick, 1996Go; Walsh et al., 1996Go; Gradin et al., 1999Go). The expression of the mouse Ahrr gene is inducible by AhR agonists in some tissues, potentially reducing the sensitivity of those tissues to subsequent doses of dioxin-like compounds. It will be important to determine whether an AhRR homolog exists in killifish, and if its expression is altered in the NBH population.

Although nongenetic mechanisms could be operating to down-regulate the AhR pathway in NBH fish, results from studies using lab-reared offspring of NBH fish (Bello, 1999Go) and second generation embryos from NBH fish (Nacci et al., 1999Go) indicate that the reduced dioxin sensitivity found in fish from NBH is heritable, strongly suggesting that a genetic mechanism is responsible for this resistance. We have noticed a high degree of individual variability in inducibility among SC killifish (see Fig. 4Go). This suggests that there was sufficient genetic variability present in the NBH population prior to the introduction of the PCBs to allow for the existence of fish with resistant phenotypes and their selection by differential survival or differential reproductive success in the presence of high concentrations of PCBs.

Conclusions
We have shown that fish from NBH are resistant to CYP1A1 induction by halogenated, and to a lesser degree nonhalogenated, AhR agonists. This resistance is pre-translational, systemic, and persistent. While the mechanism of this resistance is not yet known, the presence of a functional CYP1A1 protein and the difference in sensitivity to halogenated versus nonhalogenated compounds suggest that a change (but not a loss of function) in the AhR signal transduction pathway, possibly involving one or both killifish AhRs, is responsible for the decreased sensitivity in NBH killifish. We are continuing to explore the nature and mechanism of this resistance (Powell et al., 2000Go). Studies of PHAH resistance in killifish may provide insight into mechanisms of differential chemical sensitivity that is known to occur among species, populations, individuals, or life stages. Such research will contribute to an understanding of gene-environment interactions and could lead to the development of useful biomarkers of dioxin susceptibility.


    ACKNOWLEDGMENTS
 
This work was supported in part by the Superfund Basic Research Program at Boston University (NIH Grant P42 ES07381), EPA grant R823889, an NSF predoctoral fellowship to S.M.B., and the WHOI Education Program. We are grateful to Drs. Wade Powell and Sibel Karchner for technical advice and for critical review of the manuscript. We thank Dr. Diane Nacci (U.S. EPA) for providing the NBH fish used for hepatocyte isolation, and Bruce Woodin for assistance with fish dissection. This is contribution No. 10213 from the Woods Hole Oceanographic Institution.


    NOTES
 
1 Present address: School of Pharmacy, Room 5104, University of Wisconsin, 425 N. Charter St., Madison, WI 53706–1508. Back

2 To whom correspondence should be addressed. Fax: (508) 457-2134. E-mail: mhahn{at}whoi.edu. Back

3 A single CYP1A form has been identified in Fundulus heteroclitus (Morrison et al., 1998Go). We refer to this form as "CYP1A1" because it is more closely related to mammalian CYP1A1 than to mammalian CYP1A2. For further discussion of CYP nomenclature in fishes, see Morrison et al. (1998), Nelson et al. (1996), and Stegeman and Hahn (1994). Back

This work was presented in part at the 1996 and 1998 annual meetings of the Society of Environmental Toxicology and Chemistry (Bello et al., 1996Go, 1998Go).


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