Induction of Glutathione S-transferase Activity and Protein Expression in Brown Bullhead (Ameiurus nebulosus) Liver by Ethoxyquin

Kristin L. Henson, Gregory Stauffer,1 and Evan P. Gallagher,2

Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida

Received January 26, 2001; accepted April 3, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inducibility of hepatic cytosolic glutathione S-transferases (GSTs) was examined in brown bullheads, a freshwater fish that is highly susceptible to hepatic neoplasia following exposure to carcinogen-contaminated sediments. Juvenile bullheads were fed a semi-purified antioxidant-free diet supplemented with ethoxyquin (0.5% w/w dissolved in 3% corn oil), a prototypical rodent GST-inducing agent, twice daily for 14 days. Control bullheads received the antioxidant-free diet supplemented with corn oil (3% w/w). A significant increase (1.6-fold, p <= 0.01) in hepatic cytosolic GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the ethoxyquin-treated bullheads relative to control fish. A trend toward increased GST-NBC activity was observed in the ethoxyquin-treated fish (1.2-fold, p = 0.06), whereas no treatment-related effects were observed on GST activities toward ethacrynic acid (ECA). In contrast, GST activity toward (±)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) was repressed in affinity-purified cytosolic fractions prepared from ethoxyquin-treated bullheads relative to control bullheads. Silver staining and densitometric analysis of isoelectric-focused, affinity-purified GST proteins revealed increased expression of two basic GST-like isoforms in ethoxyquin-treated fish. In summary, exposure to ethoxyquin increases brown bullhead GST-CDNB catalytic activity and hepatic cationic GST protein expression. However, the increase in overall GST-CDNB activity by ethoxyquin is associated with repression of GST-BPDE activity, suggesting differential effects on hepatic bullhead GST isoforms by ethoxyquin. The potential repression of bullhead GST isoforms that conjugate the carcinogenic metabolites of PAH metabolism under conditions of environmental chemical exposure could be a contributing factor in the sensitivity of bullheads to pollutant-associated neoplasia.

Key Words: glutathione S-transferase; liver; enzyme induction; brown bullheads; ethoxyquin; isoelectric focusing..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The glutathione S-transferases (GSTs, EC 2.5.1.18) represent a supergene family of phase II enzymes that provide cellular protection against the toxic effects of a variety of environmental chemicals. Currently, mammalian cytosolicGST isoenzymes comprise seven gene families (alpha, mu, pi, theta, sigma, zeta, and omega) and are classified based upon protein sequence homology (Board et al., 1997Go, 2000Go; Hayes and Pulford, 1995Go). Mechanisms of detoxification by GSTs involve catalytic substrate conjugation and oxidant reduction with reduced glutathione (GSH) (Mannervik and Danielson, 1988Go). Most mammalian species express multiple GST isoenzymes with overlapping substrate specificities (Mannervik and Danielson, 1988Go). Because certain GST isoenzymes possess high catalytic activity toward epoxide carcinogens and other highly reactive chemical contaminants, variations in expression or activity of specific GST isoenzymes may markedly affect resistance or sensitivity to chemical toxicities (Eaton and Gallagher, 1994Go; Hayes et al., 1993Go; Hayes and Pulford, 1995Go). For example, the selective resistance of mice to the hepatocarcinogenic effects of the dietary carcinogen aflatoxin B1 (AFB1) is associated with high constitutive expression of an alpha class GST protein with high catalytic activity toward AFB1-8,9-exo-epoxide (AFBO) (Eaton and Gallagher, 1994Go). Rats are sensitive to AFB1 hepatocarcinogenesis, but become resistant when fed the potent dietary GST-inducer ethoxyquin, largely due to a marked induction of GST activity toward AFBO (Buetler et al., 1995Go; Hayes et al., 1993Go). The ethoxyquin-conferred resistance to AFB1 in rats is due to induction of an alpha class GST isoform (rGSTA5) homologous to the mouse form that effectively conjugates AFBO (Hayes et al., 1993Go).

Currently, relatively little is known regarding the inducibility of hepatic GST activity in fish. Several studies have reported increased fish GST activity toward 1-chloro-2,4-dinitrobenzene (GST-CDNB activity) following exposure to inducing agents in the laboratory (Celander et al., 1993Go; Leaver et al., 1992Go; Petrivalsky et al., 1997Go) or in fish inhabiting polluted environments (Otto and Moon, 1996Go; Van Veld et al., 1991Go). Typically, a modest induction (2-fold or less) of overall GST-CDNB activity is observed under these conditions. However, studies of GST-CDNB activities in fish may be complicated by variations in diet, water temperature, gender, and reproductive cycling (McFarland et al., 1999Go; Swain and Melius, 1984Go; Vigano et al., 1993). Furthermore, GST-CDNB activity represents an integration of the activity of multiple isoforms and treatment effects on GST isoforms may not always be distinguishable by analysis of GST-CDNB activity. The importance in ascertaining effects of inducing agents on multiple fish GST isoforms is underscored by the fact that selective modulation of those GST isoforms with high specific activity toward environmental toxicants or their metabolites may be missed if GST-CDNB activity is the only endpoint. Ultimately, modulation of key GST isoforms that contribute to the conjugation of environmental agents or their metabolites will be the critical determinants of chemical susceptibility.

We are currently investigating the role of GST isozyme expression in the sensitivity of brown bullhead catfish (Ameiurus nebulosus) to environmental carcinogenesis. Brown bullheads are a benthic freshwater fish that develop a variety of liver tumors when exposed to sediment carcinogens. Furthermore, bullheads often exhibit the greatest tumor burden among other species inhabiting degraded environments (Baumann and Harshbarger, 1998Go; Baumann et al., 1990Go, 1991Go, 1996Go; Leadley et al., 1998Go; Smith et al., 1994Go). Sediment and tissue concentrations of polycyclic aromatic hydrocarbons (PAHs) are strongly associated with bullhead liver tumorigenesis (Baumann and Harshbarger, 1998Go; Baumann et al., 1991Go, 1996Go; Leadley et al., 1998Go). As in mammalian species, metabolic activation and the formation of electrophilic intermediates that covalently bind DNA appears to be a primary mechanism of PAH carcinogenesis in brown bullheads (Maccubbin et al., 1990Go; Sikka et al., 1990Go; Steward et al., 1990Go; Swain and Melius, 1984Go). Because GSTs play a critical role in the detoxification of PAH epoxides (Coles and Ketterer, 1990Go), variations in constitutive or inducible GST activity and expression may play a role in the sensitivity of bullheads to PAH-associated neoplasia in the wild. In the present study we have characterized the inducibility of brown bullhead GST following exposure to dietary ethoxyquin (EQ) under controlled laboratory conditions. Our approach was to determine treatment effects on GST catalytic activities toward 4 different GST substrates, including a carcinogenic PAH, and to examine potential effects on GST protein expression by isoelectric focusing of affinity-purified GST proteins. The results of our study demonstrate that ethoxyquin is an effective inducer of bullhead GST-CDNB activity and cationic GST protein expression. However, ethoxyquin may repress the expression of other GST isoforms that are expressed at low levels but have high catalytic activity toward PAH epoxides such as BPDE.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Ethoxyquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline), 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid (ECA), 4-nitrobenzyl chloride (NBC), reduced glutathione (GSH), dithiothreitol (DTT), phenyl methyl sulfonamide (PMSF), Coomassie® brilliant blue R-250, bicinchoninic acid, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). Racemic (±)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository (Bethesda, MD). Acrylamide, N, N, N', N'-tetra-methyl-ethylenediamine (TEMED), and ammonium persulfate were obtained from Bio-Rad Laboratories (Hercules, CA). Other reagents were purchased from Fisher Scientific (Pittsburgh, PA) and were of reagent grade quality or better.

Animals and treatments.
Juvenile brown bullheads (approximately 2 months in age) were raised in captivity at the University of Florida Aquatic Toxicology Facility. These fish were hatched from egg masses of natural spawns of brown bullheads captured in Florida. The experimental juvenile bullheads were maintained under conditions of a natural photoperiod in 113L aquaria receiving flow-through dechlorinated water at a temperature of 22 ± 3°C. After a 1-month acclimation period, the fish were weaned onto a semi-purified, antioxidant-free catfish diet composed of 31% protein, 5% fat, 3.5% fiber, 4.9% ash, 27% pepsin, 0.1% vitamin mix, 0.1% mineral mix, and 0.1% Stay-C (Formulation #5599500; Zeigler Brothers, Gardeners, PA). The fish were fed the antioxidant-free diet to satiation twice daily for at least 3 months prior to start of the experiment. The antioxidant-free diet was treated with ethoxyquin (0.5% w/w dissolved in 3% corn oil) or corn oil (3% w/w) alone. The treated feed was kept frozen and protected from light for the duration of the experiment. Due to the lack of controlled GST induction studies in fish, the dosage and exposure time was based on protocols used in rodent GST induction studies (Benson et al., 1978Go; Spencer et al., 1990Go). Fish were fed either ethoxyquin-treated feed or corn oil-treated feed (controls) to satiation twice daily for 14 days. At the end of the exposure period, control fish (n = 9) and ethoxyquin-treated fish (n = 11) were sacrificed by severing of the spinal cord. Livers were excised, rinsed in ice-cold, phosphate-buffered saline (PBS), weighed, immediately frozen in liquid nitrogen, and stored at –80°C.

Tissue preparation.
Prior to homogenization, livers were briefly thawed and weighed. Due to the large number of experimental endpoints and relatively small liver size of juvenile bullheads (20–30 g), it was often necessary to pool 2 bullhead livers within each treatment group. A total of 6-aggregate liver samples were sub-fractionated from the control (n = 6) and from the ethoxyquin-treated (n = 6) bullheads. All steps were carried out at 4°C on ice. The liver tissues were homogenized in 0.25 M sucrose, 10 mM Tris, 1 mM EDTA, 0.2 mM DTT, and 0.1 mM PMSF (pH 7.4) using a Teflon homogenizer, prior to centrifugation at 10,000 x g for 10 min at 4°C. Cytosolic fractions were prepared from the 10,000 x g supernatants by further centrifugation at 100,000 x g for 60 min. The resulting hepatic cytosolic fractions were stored at –80°C prior to enzymatic analysis. Cytosolic protein concentrations were determined by the Bio-Rad protein assay for a 96-well plate reader (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as the standard (Bradford, 1976Go).

Analysis of GST catalytic activities.
Cytosolic GST activity toward CDNB, ECA, and NBC was determined by the spectrophotometric assays of Habig and Jakoby (1981), as modified for a 96-well plate reader (Gallagher et al., 2000Go). Substrate concentrations for the activity assays were as follows: 1 mM CDNB, 0.2 mM ECA, and 0.25 mM NBC. GST catalytic activity toward BPDE was determined using the HPLC method of Ramsdell and Eaton (1990), as modified for analysis for fish cytosolic GST (Gallagher et al., 1996Go). All GST catalytic activity assays were carried out in triplicate at 30°C and were corrected for non-enzymatic activity.

SDS-PAGE and affinity purification of brown bullhead liver GSTs.
Cytosolic proteins (100 µg/lane) from control and ethoxyquin-treated bullhead liver were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) using a 1.5 mm 15% polyacrylamide gel (Laemmli, 1970Go). Coomassie® brilliant blue was used to detect total protein from control and treated fish. GST proteins were isolated from the control and ethoxyquin-treated samples using affinity purification spin columns containing Sephadex G-50, according to manufacturer's directions (MicroSpinTM G-50, Amersham Pharmacia Biotech, Piscataway, NJ). Liver cytosolic fractions from 3 control and 3 ethoxyquin-treated fish were pooled to provide adequate volumes of control and ethoxyquin-treated samples for affinity purification and analysis. Briefly, after equilibration with PBS, 400 µl of the control and treated liver cytosolic fractions containing 5 mM DTT and 0.5 mM PMSF were applied to the purification columns. The columns were mixed gently at room temperature for 10 min, followed by centrifugation at 400 x g for 1 min. The columns were then washed twice with PBS and centrifuged at 400 x g for 1 min, followed by elution of GST proteins with 200 µl of 10 mM Tris, 23 mM GSH, 200 mM NaCl, 1 mM DTT, and 0.1 mM PMSF (pH 7.4).

Composition of the elution buffer was based upon freshwater fish GST purification protocols reported in the literature (Dominey et al., 1991Go; James et al., 1998Go). Elution of GST protein was repeated with 100 µl of elution buffer and the eluates from each step were pooled. Eluates were dialyzed for 48 h, with a change after the first 12 h, against 1 liter of 40 mM NaPO4 with 0.5 mM DTT (pH 7.4, 4°C), using a micro dialyzer system (QuixSepTM, Membrane Filtration Products, San Antonio, TX). Protein concentrations of the affinity-purified samples were determined by the bicinchoninic-acid assay with bovine serum albumin as the standard (Smith et al., 1985Go). Purification of GST protein was confirmed by SDS-PAGE of eluted protein (10 µg/lane) using a 12% Tris-glycine polyacrylamide gel (Ready Gel, Bio-Rad Laboratories, Hercules, CA).

Isoelectric focusing of affinity-purified bullhead liver GSTs.
Affinity-purified control and ethoxyquin-treated samples were subjected to isoelectric focusing (IEF) analysis to separate the bullhead liver GST isoforms by isoelectric points (pI). Control and EQ-treated proteins (0.9 µg/lane) were loaded in a template placed on the surface of an agarose IEF Isogel (pH 3–10; FMC BioProducts, Rockland, ME), 2 cm from the cathode end, with 3 µl methyl red as an anolytic dye marker (pI 3.8). Isoelectric (pI) standards ranging from pH 3.6 to pH 10.2 (FMC BioProducts, Rockland, ME) were also loaded onto the template. Isoelectric focusing was performed at 15°C with a flat-bed electrophoresis unit equipped with 3000 xi power supply (Bio-Rad Laboratories, Hercules, CA) using 1 M NaOH as catholyte and 0.5 M acetic acid as anolyte at constant 25 watts with limiting 1000 voltage. Focusing was obtained at approximately 40 min. The IEF gel was stained with silver to visualize separated proteins. The pI value of each unknown protein band was determined from a standard curve derived by plotting the distance from the cathode end to each pI standard band (x-axis) against the pH value (y-axis). The silver stained IEF gel was scanned and densitometric analyses were used to quantify the expression of the separated GST proteins (Adobe Photoshop 5.5, San Jose, CA).

Statistical analysis.
Catalytic activity values reported for GST activities toward CDNB, ECA, and NBC represent the mean ± standard error (SEM) of triplicate reactions for n = 6 samples for each group. GST activity values for control and EQ-treated groups were tested for equality of variance, using the F-test. Differences in GST activities among control and treated groups were then assessed for significance using an unpaired two-tailed Student's t-test (p <= 0.05) and Statview 4.5 software (Abacus Concepts, Berkeley, CA). GST-BPDE catalytic activity values represent the mean ± SEM of triplicate reactions for pooled affinity-purified control and EQ-treated samples, and therefore were not assessed for statistically significant differences between the 2 groups.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 14-day dietary exposure of brown bullheads to 0.5% ethoxyquin was generally well tolerated and did not evoke any overt signs of toxicity. Furthermore, no differences in body or liver weights were observed between the 2 groups during the exposure.

The effects of dietary ethoxyquin exposure on hepatic cytosolic GST catalytic activities toward CDNB, ECA, and NBC are shown in Figure 1Go. Ethoxyquin treatment resulted in a significant (p < 0.01), 1.6-fold increase in bullhead liver GST activity toward CDNB (Fig. 1AGo). A trend toward increased GST-NBC activity (p = 0.06) was also observed in the ethoxyquin-treated fish, whereas there were no treatment-related effects on mean GST activities toward ECA (Figs. 1B and 1CGo). To avoid analytical problems associated with detection of the BPDE-GSH conjugate, hepatic GST proteins from control and ethoxyquin-treated liver cytosolic fractions were subjected to affinity purification prior to analyzing for effects of ethoxyquin exposure on GST-BPDE activities. As observed in Figure 2Go, SDS–PAGE analysis of the affinity-purified GST fractions yielded a single band of approximately 27 kDa, indicating that the fractions were relatively free of contaminating proteins. In addition, the majority of the GST activity was retained and eluted from the GSH-affinity columns (Table 1Go). GST-CDNB activity in the affinity-purified GST from ethoxyquin-treated bullheads was 2.8-fold greater than observed in the affinity-purified GST fraction from control bullhead livers (Table 1Go). As observed in Table 1Go, GST-BPDE activity in the ethoxyquin-treated, affinity-purified fraction was actually decreased to 21% that of controls. The ratios of specific GST activities toward BPDE relative to CDNB are also shown in Table 1Go. When normalized to overall GST-CDNB activity, the relative BPDE/CDNB activity ratios (GST-BPDE/GST-CDNB x 1000) were 13-fold higher in control bullheads as compared to ethoxyquin-treated bullheads (Table 1Go). These ratios reflect the poor predictive value of CDNB as a surrogate GST substrate for epoxide carcinogens such as BPDE.



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FIG. 1. Effect of 14-day dietary ethoxyquin (EQ) exposure on brown bullhead liver cytosolic GST catalytic activities toward GST substrates (A) CDNB, (B) ECA, (C) NBC, and (D) BPDE. Data represent the mean (SEM) of catalytic activity from 6 control and 6 EQ-treated samples for GST-CDNB, GST-ECA, and GST-NBC activities. Statistically significant treatment effects are indicated at *p <= 0.05. GST-BPDE activities were conducted on pooled affinity-purified fractions and were not subjected to statistical analysis.

 


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FIG. 2. Twelve percent SDS-PAGE of affinity-purified liver GST from control (CO) and ethoxyquin treated (EQ) brown bullhead. GST protein was isolated from cytosolic fractions of control and EQ-treated bullheads as described in Materials and Methods. Lanes (1) brown bullhead cytosolic protein (20 µg); (2) eluate from control fish (10 µg); (3) molecular weight marker; (4) brown bullhead cytosolic protein (20 µg); (5) eluate from EQ-treated fish (10 µg).

 

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TABLE 1 Glutathione S-transferase Activity toward 1-Choro-2,4-dintrobenzene (CDNB) and Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) in Affinity-Purified Fractions from Control and Ethoxyquin-Treated Brown Bullheads
 
As observed in Table 1Go, the total cytosolic protein content of bullheads fed ethoxyquin was similar to that observed in control animals. In addition, SDS–PAGE and Western-blotting analysis of cytosolic proteins isolated from control and ethoxyquin-treated bullheads did not reveal any marked alterations in the expression of cytosolic proteins between the 2 groups (data not shown). However, traditional 1-dimensional PAGE and Western-blotting studies may not reveal subtle differences in GST isoenzyme expression due to the fact that the subunits may share similar molecular weights which are not resolved by 1-dimensional analysis. Accordingly, potential treatment-related effects on GST isozyme expression were characterized by isoelectric focusing of the affinity-purified proteins. Silver staining of the IEF gel revealed GST isoforms with pIs of 9.5 (minor basic isoform) and 9.7 (major basic isoform) in lanes containing control and ethoxyquin-treated affinity-purified fractions (Fig. 3Go). Densitometric analysis of the silver-stained IEF gel demonstrated that protein expression of the minor and major basic GST isoforms was increased 2.5-fold and 1.4-fold, respectively, in ethoxyquin-treated fish (Fig. 3Go).



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FIG. 3. Silver stain of isoelectric-focused, affinity-purified liver GST from control (CO) and ethoxyquin-treated (EQ) brown bullhead (0.9 µg/lane). Lanes (1) pI markers; (2) control fish; (3) EQ-treated fish. Right figure represents densitometric analysis of major and minor basic GST-like proteins in control and ethoxyquin-treated fish. Bars represent the mean densitometry units per mg of isoelectric-focused protein within control (pool of 3 samples) and treated groups (pool of 3 samples).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Laboratory studies of GST induction in fish have typically been conducted in conjunction with studies of CYP1A induction and have therefore involved Ah-receptor inducers, such as 3-methylcholanthrene (3-MC) and ß-napthoflavone (ß-NF, reviewed in George, 1994). However, studies of GST induction in rodents indicate that antioxidant compounds such as ethoxyquin and butylated hydroxyanisole (BHA) are more effective inducers of GST expression than are Ah receptor agonists (Buetler et al., 1995Go, Hayes and Pulford, 1995Go). Furthermore, ethoxyquin is one of the most potent inducers of rat GST among the known inducing agents (Buetler et al., 1995Go). Accordingly, ethoxyquin was a logical selection as a GST-inducing agent for the present study involving brown bullheads. As compared to intraperitoneal injections, the use of dietary agents is more environmentally relevant, since environmental agents that modulate biotransformation enzyme activity are primarily ingested through waterborne or dietary exposure. Ethoxyquin can be added directly to feed, therefore reducing the stress associated with capture and injection. However, because antioxidants such as ethoxyquin are frequently added to fish feed as preservatives, an antioxidant-free catfish diet was used in the study in order to avoid diet-related enzyme induction that could potentially confound interpretation of experimental data.

The level of induction in GST-CDNB activity in bullhead liver (1.6-fold) reported here is consistent with previous studies of GST induction in other aquatic species. For example, treatment of rainbow trout (Oncorhynchus mykiss) with various dosages of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,2-bis(p-chlorophenyl)-1,1-dichloroethane (p,p'-DDE) resulted in 1.2- to 1.5-fold increases of GST activity toward CDNB (Petrivalsky et al., 1997Go). Similarly, approximately 2-fold inductions of GST-CDNB activity have been reported in trout exposed to ß-NF (Celander et al., 1993Go), and in plaice (Pleuronectes platessa) exposed to BHA or trans-stilbene oxide (Leaver et al., 1992Go). Interestingly, studies of GST induction in rodents exposed to ethoxyquin and other antioxidant inducers typically reveal a 1.5- to 3-fold induction of GST-CDNB activity, which is similar to GST-induction responses observed in fish (Buetler et al., 1995Go; Derbel, et al., 1993Go; Plumb et al., 1996Go). However, substantially higher induction of GST catalytic activity can occur for those particular GST subunits such as alpha class isozymes, that are highly responsive to ethoxyquin induction (Buetler et al., 1995Go, Pulford and Hayes, 1995).

Our results indicate that the treatment-related increase in GST-CDNB activity was likely due to increased synthesis of GST proteins. In this regard, ethoxyquin is one of a group of synthetic antioxidants that induce transcription of a number of phase II detoxification enzymes in rodents (Prochaska and Talalay, 1988Go). Antioxidant inducing agents possess electrophilic centers associated with production of intracellular signals that target key promoter elements resulting in increased transcription of antioxidant-responsive genes (Li and Jaiswal, 1992Go; Rushmore et al., 1991Go). Promoter sequences targeted by antioxidant-inducing agents include antioxidant response elements (AREs) and electrophile response elements (EpREs), which confer inducibility by the monofunctional phenolic antioxidants to several rat GST and other phase II genes (Bergelson et al., 1994Go; Li and Jaiswal, 1992Go; Rushmore et al., 1991Go). Although studies of the molecular mechanisms of GST induction in fish are lacking, Leaver and George identified 4 ARE consensus sequences in the promoter region of GST-A, a theta-like GST of plaice (Leaver and George, 1996Go). It is likely that the plaice ARE-like sequences are functional, since a 2-fold inducibility of plaice liver GST-CDNB activity occurs following exposure to the monofunctional inducers BHA and trans-stilbene oxide (Leaver et al., 1992Go). Thus, the ethoxyquin-associated induction of GST-CDNB activity observed in the present study suggests that at least some bullhead GST isoforms may be responsive to antioxidants via ARE-like promoter elements. The growing body of studies of GST induction in fish suggests that ARE-mediated GST gene induction may be conserved among many fish species.

Isoelectric focusing of affinity-purified GST proteins from the livers of control and ethoxyquin-treated bullhead revealed the presence of two basic GST-like proteins, which appear to be the predominant GST isoforms in brown bullhead liver. The presence of basic GST isoforms in bullheads is consistent with isoelectric focusing studies of affinity-purified intestinal GSTs in channel catfish, whose predominant liver GST is a pi-like protein that exhibits an isoelectric point of 7.9 (James et al., 1998Go). In addition, the presence of two predominant hepatic GST proteins in bullheads is supported by our earlier report demonstrating the presence of at least two major GST isozymes in bullhead liver (Gallagher et al., 2000Go). Although the majority of liver GST isozymes characterized in vertebrates conjugate CDNB with GSH, the GST subunits exhibit unique biochemical, enzymatic, and kinetic characteristics, which ultimately results in distinctive isozyme substrate specificities (Mannervik and Danielson, 1988). GoAccordingly, variations in GST-CDNB activity are often not representative of actual changes in activity or expression of individual GST isozymes, and thus may not reflect modulation of particular isoforms with high specific activity toward GST substrates other than CDNB (Buetler et al., 1995Go; Hayes and Pulford, 1995Go; Ramsdell and Eaton, 1990Go). Therefore, it was necessary in the present study to examine the inducibility of bullhead hepatic GST activity using structurally divergent reference substrates. In mammals, NBC is primarily a rat GST theta class specific substrate whereas ECA is predominantly a pi class substrate, although rodent and human alpha class isoenzymes have some activity toward this compound (Hayes and Pulford, 1995Go). In fish, pi-like GST proteins have also been shown to exhibit high activity toward ECA (Celander et al., 1993Go; James et al., 1998Go). The significant induction of GST-CDNB activity with lack of an effect on GST-ECA, as observed in this study, is suggestive of induction of GST isoform(s) with high specific activity toward CDNB relative to ECA. However, it is possible that the mild increases in GST activity observed toward NBC were associated with the initiation of an induction response or a decline in activity from an induction occurring earlier during the exposure period.

In addition to determination of GST catalytic activity toward reference substrates, GST-BPDE activity in affinity-purified cytosolic fractions from control and ethoxyquin-treated bullheads was determined as a measure of inducibility toward a relevant environmental carcinogen. The induction of bullhead GST-CDNB activity by ethoxyquin, with a concurrent decrease in GST-BPDE activity, is suggestive of differential regulation of GST isoforms with high CDNB activity, but low activity toward BPDE. Similarly, treatment of mice with BHA does not significantly increase total liver cytosolic GST activity toward BPDE as compared to CDNB, a process due to preferential induction of mice GST isoforms that are relatively inefficient at conjugating BPDE (Ramsdell and Eaton, 1990Go). In general, rodent cytosolic GST-CDNB activity may not correlate with GST-BPDE in vitro, due to the high rate of BPDE conjugation by GST isoenzymes (e.g., alpha and pi classes) with relatively low CDNB activity (Ramsdell and Eaton, 1990Go). A related occurrence, the induction of GST-CDNB activity with repression of specific GST isoforms, has also been reported in fish. For example, treatment of plaice with 3-MC elicits an increase in GST-CDNB activity, but causes a decrease in GST-A protein and GST-A mRNA expression (Leaver et al., 1992Go). The increased CDNB activity in the presence of decreased GST-A expression was attributed to induction of plaice GST isoforms other than GST-A (George, 1994Go). We are currently conducting 2-dimensional analysis of GST proteins from control and ethoxyquin-treated bullheads to determine treatment effects on other potential minor GST isoforms that may have high specific activity toward BPDE.

The ability of bullheads to modulate GST should theoretically facilitate the detoxification and clearance of certain environmental chemicals that are substrates for bullhead GST. The lack of elevated GST activity toward ECA (primarily a pi class GST substrate) in conjunction with the lack of increased GST pi-protein expression in EQ-treated fish suggest that brown bullhead liver pi class GSTs may not be inducible. In rats, pi class GSTs have high activity toward the carcinogenic PAH metabolite, (+)-7ß, 8{alpha}-dihydroxy-9{alpha},10{alpha}-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (Robertson et al., 1986Go). Thus it is possible that the ability of bullheads to clear the products of oxidative PAH metabolism via GST is not affected by exposure to environmental or dietary inducing agents. However, our results do not rule out the potential modulation of minor bullhead GST isoform(s) with high activity toward the carcinogenic products of PAH metabolism other than BPDE that may not be detected by measurement of total cytosolic GST-BPDE activity. In this regard, preliminary data from 2-dimensional electrophoresis of brown bullhead affinity-purified cytosolic fractions indicate the presence of at least 2 additional minor isoforms with near neutral isoelectric points, which were not detected on the silver-stained IEF gel (data not shown). Ultimately, the characterization of constitutive and inducible GST isoenzyme expression toward carcinogenic substrates in the environment will shed light on the role of these enzymes in the susceptibility of brown bullheads to environmental hepatocarcinogenesis.


    ACKNOWLEDGMENTS
 
The authors would like to thank Mike Mittner of the Florida Game and Freshwater Fish Commission for providing the juvenile bullheads. We would also like to thank Dr. Nancy Denslow, Marjorie Chow, and John Munson for their technical assistance. This research was supported by a NIEHS Superfund Basic Sciences Program Grant (P42 ES07375). K.L.H. was supported in part through the University of Florida Superfund Basic Research Program Graduate Assistantship Award.


    NOTES
 
1 Present address: Jurassic Fish, Inc., P.O. Box 1018, Bartow, FL 33831. Back

2 To whom correspondence should be addressed at P.O. Box 110885, Department of Physiological Sciences, University of Florida, Gainesville, FL. 32611–0885. Fax: (352) 392-4707. E-mail: gallaghere{at}mail.vetmed.ufl.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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