Glutathione S-Transferase Expression in Pollution-Associated Hepatic Lesions of Brown Bullheads (Ameiurus nebulosus) from the Cuyahoga River, Cleveland, Ohio

K. L. Henson and E. P. Gallagher1

Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611-0885

Received January 7, 2004; accepted March 15, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In rodents, overexpression of glutathione S-transferase pi is a characteristic feature of foci of cellular alteration (FCA) and neoplastic liver lesions induced by genotoxic chemicals. Alterations of glutathione S-transferase (GST) expression in hepatic lesions have also been reported in fish exposed to environmental carcinogens, and cellular GST expression may be an important determinant of growth and progression of chemical-associated liver tumors in certain fish species. In the present study, GST expression was examined in hepatic lesions of brown bullheads (n = 44) from the Cuyahoga River, a highly industrialized site located in Cleveland, Ohio. GST proteins were detected by immunohistochemistry and polyclonal antibodies that recognize either two major bullhead GST proteins or a pi-like GST isoform. Hepatic lesions were present in 70% of the fish and included biliary hyperplasia and biliary fibrosis; eosinophilic, basophilic, clear cell, and vacuolated FCA; and biliary neoplasms. Eosinophilic FCA and biliary tumors were the most prevalent preneoplastic and neoplastic lesions. GST expression in hyperplastic biliary tissue, FCA and tumors did not markedly differ from that of surrounding normal hepatocytes or biliary epithelium. Some hepatocytes within eosinophilic FCA had decreased GST expression. A complete absence of GST immunoreactive protein was not observed in any lesion, and there were no marked differences when comparing GST pi to overall GST expression. Our results indicate that GST expression in hepatic lesions of brown bullhead exposed to environmental carcinogens does not significantly differ from that in surrounding normal cells and is therefore not a useful predictor of environmental carcinogenesis in this species. Furthermore, the regulation and expression of GST pi in bullhead hepatocarcinogenesis appears to differ markedly from that during hepatocarcinogenesis in rats and some other fish species.

Key Words: glutathione S-transferase; altered foci; immunohistochemistry; cholangiocellular carcinoma; brown bullhead; liver; neoplasia.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemical-associated hepatic neoplasia in rodents and fish occurs through a stepwise progression characterized by the presence of discrete hepatocellular foci of cellular alteration (FCA) and neoplasms (Bailey et al., 1996Go; Farber, 1980Go; Farber and Sarma, 1987Go; Stanton, 1965Go). In rats, chemical-associated FCA and neoplasms express elevated levels and activity of certain phase II detoxification enzymes and decreased levels and activity of cytochrome P450 s (Farber, 1990Go). In particular, overexpression of pi-class glutathione S-transferase isoenzyme P1-1 (rGSTP1-1) is a characteristic feature of rat preneoplastic and neoplastic liver lesions induced by genotoxic chemicals, and GST pi overexpression is often used as a neoplastic marker in rat chemical carcinogenesis studies (Sato, 1989Go; Tatematsu et al., 1988Go). The altered enzyme profile of FCA and neoplasms is thought to provide a growth advantage to clonally altered, preneoplastic cells through decreased bioactivation and increased detoxification of cytotoxic and carcinogenic chemicals (Farber, 1980Go). Subsequent carcinogen exposure results in a preferential anti-mitotic effect on surrounding, nonaltered cells due to increased bioactivation of the carcinogen(s) by the normal cells. Thus, nonaltered cells do not respond to mitogenic stimuli, whereas proliferation occurs in the altered cells.

Unlike rats, in which GST pi overexpression is a consistent feature of chemically induced FCA and neoplasms, GST expression in hepatic lesions of fish during chemical carcinogenesis appears to be highly variable. For example, immunoreactive GST expression in some hepatic FCA and in all hepatocellular and biliary neoplasms was decreased in white suckers (Catostomus commersoni) exposed to sediments containing high levels of polycyclic aromatic hydrocarbons (PAHs) (Stalker et al., 1991Go). Similarly, expression of GST-A, a theta-like GST, is reduced in extrafocal hepatocytes and in eosinophilic FCA of pollutant-exposed European flounder (Platichthys flesus L) (Köhler et al., 1998Go). In contrast, GST catalytic activities in hepatic lesions of mummichog (Fundulus heteroclitus) from a PAH-contaminated site did not differ from GST activities measured in surrounding normal liver (Van Veld et al., 1991Go). Laboratory studies of GST expression in fish hepatic lesions during carcinogenesis are also highly variable. For example, the majority of hepatic FCA and tumors in rainbow trout (Oncorhynchus mykiss) exposed to 1, 2-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) were GST deficient, although GST induction was observed in some small FCA (Kirby et al., 1990Go). In contrast, Parker et al. (1993)Go reported increased GST activity in mixed hepato- and cholangiocellular carcinomas of AFB1-exposed trout. Ultimately, GST expression during neoplastic development in fish is likely to vary among aquatic species and, possibly, with the initiating agent.

Among the aquatic species, brown bullhead catfish (Ameiurus nebulosus) exhibit an extremely high sensitivity to environmental carcinogens (Leadley et al., 1998Go; Sikka et al., 1990Go; Steward, et al., 1990Go). Sediment and tissue concentrations of organochlorines, polychlorinated biphenyls (PCBs), and PAHs have all been correlated with hepatic neoplasia in bullheads (Baumann et al., 1990Go, 1991Go, 1996Go, 1998Go); however, sediment PAHs are the strongest correlates with chemical-associated neoplasia (Baumann et al., 1991Go, 1996Go; Smith et al., 1994Go). Bullheads rapidly metabolize benzo[a]pyrene (B[a]P) to reactive intermediates that covalently bind liver DNA (Sikka et al., 1990Go; Steward et al., 1990Go), a process that is augmented by treatment with the CYP1A-inducing agent ß-napthoflavone (ß-NF) (Ploch et al., 1998Go). Although little is known regarding the ability of phase II enzymes to detoxify PAH-reactive intermediates in bullheads, we have observed that brown bullheads exhibit high rates of hepatic GST catalytic activity and a high catalytic efficiency for detoxification of a diverse array of electrophilic GST substrates (Gallagher et al., 2000Go). Conceivably, cellular alterations of bullhead GST expression during carcinogenesis could be modifying factors in the development and progression of contaminant-associated neoplasia. At minimum, GST alterations in bullheads from contaminant sites could be potentially used as a diagnostic indicator of cellular transformation during environmental carcinogenesis. The present study was initiated to determine GST enzyme expression in pollution-associated liver lesions of brown bullheads and to specifically determine if bullhead GST pi expression could be used as a diagnostic indicator of chemical-induced neoplasia.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Polyoxyethylenesorbitan monolaurate (Tween-20) and {delta}-methacryloxypropyltrimethoxysilane (Silane) were obtained from Sigma Chemical Co. (St. Louis, MO). HistoMark® Biotin Streptavidin Kit (10% normal goat serum, biotinylated goat anti-rabbit secondary antibody, and streptavidin-alkaline phosphatase) and HistoMark® RED kit (6-bromo-2-hydroxy-3-napthoic acid substrate, PhThaloRED chromogen, contrast BLUE hematoxylin counterstain) were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Xylene, 10% neutral buffered formalin, and buffer components were obtained from Fisher Scientific (Pittsburgh, PA).

Animals and treatment. Adult brown bullheads greater than 3 years of age were captured in June 1999 from the Cuyahoga River, a heavily industrialized site in Cleveland, Ohio, as part of a U.S. Geological Survey study of contamination in Great Lakes tributaries. Fish were collected from two sites along the river with previous documentation of elevated sediment-associated PAHs associated with histopathological abnormalities in brown bullheads (Baumann et al., 1996Go; Smith et al., 1994Go). The first site of fish collection (harbor) encompassed both sides of the river as it entered the Cleveland Harbor and included an embayment adjacent to the southwest end of Burke Lakefront Airport. The second site (upper river, UR) was 6 to 8 km from the river mouth and was above the dredged area of the river and several steel processing plants. At necropsy, samples of normal and grossly abnormal liver tissue from a subset of these fish (n = 44) were collected in 10% neutral buffered formalin and embedded in paraffin. Sections were stained routinely with hematoxylin and eosin or by a three-step immunohistochemical method for GST protein expression (see below). Livers from adult brown bullheads of similar ages to those samples from the Cuyahoga River were collected from nonpolluted reference sites (Trout Lake, FL and Lake Harris, FL) for use as reference comparisons of histological features and GST immunoreactivity studies.

Antibodies and Western blotting analysis. Cytosolic proteins (100 µg/lane) from reference site bullhead liver were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 1.5 mm 12% polyacrylamide gel. Total cytosolic GST protein was isolated from reference bullhead liver using a GST-affinity purification spin column containing Sephadex G-50 according to manufacturer's directions (MicroSpinTM G-50, Amersham Pharmacia Biotech, Piscataway, NJ). Immunoreactive GST proteins in brown bullhead liver were detected using polyclonal antibodies raised against affinity-purified GST proteins from striped bass (Morone saxitilis) liver (Gallagher et al., 2000Go) and channel catfish intestinal GST pi (James et al., 1998Go). The striped bass GST antibody recognizes two major GST-like proteins in brown bullhead liver with minimal cross-reactivity to other non-GST cytosolic proteins (Gallagher et al., 2000Go). The channel catfish GST pi antibody specifically recognizes channel catfish intestinal GST isoform with high sequence homology to mammalian pi-class GSTs (James et al., 1998Go) and a pi-like GST in brown bullhead liver (Henson et al., 2001Go). In Western blotting experiments, cytosolic protein from channel catfish liver was used as a positive control for the presence of GST pi, and hepatic cytosolic protein from English sole (Pleuronectes vetulus), an aquatic species that does not express a pi-like GST (Gallagher et al., 1998Go), was used as a negative control. Liver cytosolic proteins (100 µg/lane) were fractionated by SDS-PAGE using 15% polyacrylamide gels followed by transfer to polyvinylidene difluoride membranes (Immuno-Blot PVDF, Bio-Rad Laboratories, Hercules, CA). Nonspecific binding was blocked by overnight incubation with 5% nonfat dried milk in TBS-T buffer (20 mM Tris, 0.9% (w/v) NaCl, pH 7.6 containing 0.1% Tween 20). Following three rinses in TBS-T, the blots were incubated with the catfish GST pi antibody for 3 h at room temperature. The blots were then rinsed in TBS-T and incubated with goat anti-rabbit horseradish peroxidase conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA). Protein-bound antibody was detected with enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech Inc., Piscataway, NJ) and detected by autoradiography.

Immunohistochemistry. Livers were serially sectioned (4–5 µm) and mounted on electrostatically charged slides (Fisherbrand Superfrost Plus, Fisher Scientific, Pittsburgh, PA) or Silane-treated slides for immunohistochemistry. These sections were deparaffinized in three sequential xylene washes and rehydrated in graded alcohol baths. Following two rinses in distilled water, target antigens were unmasked with 0.1 M sodium citrate buffer (0.1 M citric acid/0.1 M sodium citrate, pH 6.0) and microwave heating (Hofman, 1996Go). The sections were then rinsed for 5 min in TBS-T and TBS (20 mM TBS, 0.9% NaCl, pH 7.6) buffers. Unless otherwise noted, all subsequent slide incubations were performed in a humidified chamber at room temperature and followed by a 5-min rinse in TBS-T and TBS. After antigen retrieval, 10% normal goat serum was applied to the sections for 15 min to block nonspecific binding. Sections were then incubated overnight at 4°C with the fish GST primary antibodies diluted 1:100 in 1% normal goat serum and TBS-T. For each sample, two negative control sections were incubated separately with buffer and preimmune rabbit serum. Slides were incubated with biotinylated goat anti-rabbit secondary antibody for 30 min followed by application of the streptavidin-alkaline phosphatase label for 20 min. The substrate/chromogen solution was prepared according to manufacturer's directions and applied to the sections. Color development was monitored by visual inspection and stopped by rinsing in distilled water. Sections were counterstained with hematoxylin.

Histological and immunohistochemical evaluation of brown bullhead liver. Classification of hepatic lesions identified on histological examination of hematoxylin- and eosin-stained sections was based upon criteria previously described in the literature (Baumann et al. 1990Go; Hampton et al., 1985Go 1988Go, 1989Go; Myers et al., 1987Go). Expression of liver GST protein was determined by light microscopic evaluation of immunostained sections. GST expression in hepatic lesions was qualitatively assessed as normal, clearly increased, or clearly decreased, by comparing intensity of positive staining to surrounding nonaffected hepatocytes or bile ducts, as appropriate.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Western Blotting of Bullhead Cytosolic Proteins Using Fish GST Antibodies
SDS PAGE analysis of bullhead liver cytosolic proteins and GSH-affinity purified cytosolic GST revealed the presence of highly expressed subunits of approximately 28 kDa (Fig. 1A). As observed in Figure 1B, Western blotting of cytosolic proteins with an antibody raised against total striped bass GST revealed the presence of two strongly stained GST subunits in the 27–30 kDa range. A subsequent Western blotting experiment using the channel catfish pi-like GST antisera revealed that one of the GST subunits (lower subunit) strongly and specifically cross-reacted to catfish GST pi antisera. Cross-reactivity of either the striped bass GST antibody or the catfish GST pi class antibody with other non-GST proteins in bullhead liver cytosol was not observed.



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FIG. 1. SDS-PAGE and Western blotting of brown bullhead cytosolic proteins. (A) 12% SDS PAGE of 20 µg total cytosolic proteins (lane 1) and 10 µg GSH-affinity purified liver GST (lane 2) from brown bullhead. GST protein was isolated from cytosolic fractions using GST purification spin columns as described in Materials and Methods. (B) Western blot showing cross-reactivity of brown bullhead cytosolic GST proteins (100 µg) against affinity-purified striped bass GST polyclonal antisera, lane 1 male bullhead, lane 2 female bullhead. (C) Western blot analysis of 100 µg brown bullhead cytosolic GST proteins using a channel catfish GST pi polyclonal antibody. Lane (1) female brown bullhead; lane (2–3) male brown bullheads.

 
Histology and GST Expression in Normal Brown Bullhead Liver
The histological appearance of control brown bullhead liver was similar to descriptions of fish hepatic histology in the literature (Baumann et al., 1990Go; Boorman et al., 1997Go; Hampton et al., 1985Go, 1988Go, 1989Go; Myers et al., 1987Go). Moderate to large amounts of hepatocellular cytoplasmic vacuolation compatible with glycogen was a notable feature in the majority of these fish. Immunohistochemical staining of livers from control bullheads using the striped bass GST antibody resulted in diffuse cytoplasmic staining of moderate intensity within both hepatocytes and biliary epithelium. Immunostaining with the channel catfish GST pi antibody also resulted in diffuse mild-to-moderate positive cytoplasmic staining of hepatocytes and biliary epithelium. No immunostaining was observed using the preimmune sera. Immunoreactive GST expression was observed in some hepatocyte nuclei using the striped bass GST antibody and also using the channel catfish GST antibody. However, no consistent discernable pattern was observed in regards to GST nuclear staining either in extrafocal hepatocytes or hepatocellular FCA. In general, the intensity of positive staining with the channel catfish GST antibody was less in both hepatocytes and biliary epithelium as compared to the staining observed using the striped bass GST antibody (data not shown).

Histology and GST Expression in Cuyahoga River Brown Bullhead Liver
The histological appearance of livers from Cuyahoga River bullheads was similar to those bullheads from unpolluted sites. However, liver lesions were consistently present in the Cuyahoga River bullheads that were not seen in reference bullheads. Of 44 fish examined, 20/28 (71%) animals from Cuyahoga River harbor exhibited histopathological lesions, 11/16 animals (69%) from the upper Cuyahoga River exhibited lesions, and 31/44 (70%) of all animals examined exhibited lesions.

A notable pathological feature was the presence of moderate-to-severe bile duct hyperplasia with fibrosis in all of the Cuyahoga bullhead livers examined. A few biliary trematode parasites (E. Greiner, personal communication) associated with mild-to-moderate fibrosis were observed in 3 of 44 Cuyahoga bullhead livers. As observed in Table 1, a total of 58 hepatocellular FCA and 13 biliary neoplasms from 44 Cuyahoga bullheads were examined immunohistochemically. Hepatocellular FCA included eosinophilic, basophilic, clear cell, and vacuolated types (Table 1). Neoplastic lesions were exclusively of biliary origin with an almost equal distribution between benign cholangiomas and malignant cholangiocarcinomas (Table 1). Cholangiomas were characterized by discrete clusters of well-differentiated ducts with stromal proliferation that resulted in mild compression of surrounding hepatocytes (Fig. 2A). Although a continuum of hyperplastic biliary epithelium through benign neoplasia made differentiation of these two lesions difficult in some sections, the presence of compression or expansion into adjacent parenchyma was used to differentiate cholangiomas from biliary hyperplasia (Boorman et al., 1997Go). Cholangiocarcinomas were composed of invasive ductular epithelium that exhibited cellular and nuclear atypia, increased mitotic activity, and abundant stromal proliferation (Fig. 3A). Although no hepatocellular neoplasms were positively identified, differentiation between a few FCA and hepatocellular adenoma was equivocal. In these cases, a final classification of FCA was made based upon an absence of sharply demarcated borders, compression of adjacent parenchyma, and alterations in hepatic structure and orientation (Hofman, 1996Go).


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TABLE 1 GST expression in hepatocellular foci of cellular alteration (FCA) and liver tumors in brown bullhead (Ameiurus nebulosus)

 


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FIG. 2. (A) Biliary adenoma (cholangioma) in section of brown bullhead liver. Hematoxylin and eosin. Bar = 100 µm. (B) Immunohistochemical staining of section as in (A) with striped bass GST antibody. Streptavidin-biotin-alkaline phosphatase-method. Bar = 25 µm. (C) Immunohistochemical staining with channel catfish GST antibody. Streptavidin-biotin-alkaline phosphatase-method. Bar = 25 µm. Intensity of positive immunostaining with the two antibodies similar in cytoplasm of neoplastic biliary ducts and adjacent nonaffected tissue. No positive staining is present within the associated connective tissue proliferation.

 


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FIG. 3. (A) Locally invasive bile duct carcinoma (cholangiocarcinoma) in section of liver from brown bullhead. Hematoxylin and eosin. Bar = 100 µm. (B) Immunohistochemical staining of section as in (A) with striped bass GST antibody. Streptavidin-biotin-alkaline phosphatase-method. Bar = 25 µm. (C) Immunohistochemical staining of section as in (A) with channel catfish GST antibody. Streptavidin-biotin-alkaline phosphatase-method. Bar = 25 µm. Intensity of positive immunostaining with two antibodies is similar in cytoplasm of neoplastic biliary ducts and adjacent nonaffected tissue. No positive staining is present within the associated connective tissue proliferation.

 
GST proteins in extrafocal hepatocytes and biliary epithelium from the Cuyahoga River bullheads were evident using either of the two fish GST antibodies. Immunoreactive GST expression in hyperplastic bile ducts as detected by the two antibodies was similar to surrounding hepatocytes and nonhyperplastic biliary epithelium. Diffuse mild-to-moderate cytoplasmic staining was also observed in the majority of hepatocellular FCA and in all biliary neoplasms in the presence of the fish GST antibodies (Table 2, Figs. 2B–2C through 4B–4C). A qualitative examination of FCA in all Cuyahoga bullheads indicated that GST expression was not markedly different in these hepatic lesions compared to surrounding tissue (Figs. 2B–2C through 4B–4C). However, some hepatocellular eosinophilic FCA exhibited variably decreased positive staining with both GST antibodies (Figs. 4B–4C). Although decreased GST staining was present in some hepatocytes within these lesions, a complete absence of GST expression was not observed in any cells. No clear induction of GST expression was observed in any altered foci or neoplasms relative to GST expression in surrounding normal tissues.



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FIG. 4. (A) Discrete hepatocellular foci of alteration (eosinophilic type) in section of liver from brown bullhead. Hematoxylin and eosin. Bar = 100 µm. (B) Immunohistochemical of section as in (A) with striped bass GST antibody. Streptavidin-biotin-alkaline phosphatase-method. Bar = 25 µm. (C) Immunohistochemical staining of section as in (A) with channel catfish GST antibody. Streptavidin-biotin-alkaline phosphatase-method. Bar = 25 µm. Arrows indicate decreased immunoreactive GST in some hepatocytes within the altered lesion.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we have examined the expression of hepatic GST in lesions of brown bullheads from the Cuyahoga River. The Cuyahoga River was selected as the site for collection of samples due to the well-documented history of PAH contamination and high prevalence of hepatocellular and biliary neoplasms in the resident bullhead population. Previous studies have reported a 45% or greater prevalence of hepatic preneoplastic lesions and neoplasms of bullheads collected from the Cuyahoga River as compared to 4% or less prevalence of similar lesions in bullhead from uncontaminated sites in the same geographic area (Baumann et al., 1991Go; Smith et al., 1994Go). These studies have also shown that the presence of bullhead liver neoplasms has been historically associated with elevated PAH concentrations in Cuyahoga River sediments and bullhead tissues (Baumann et al., 1991Go; Smith et al., 1994Go). Despite remediation efforts on sites along the Cuyahoga River, analysis of sediments collected from the sites of fish collection for this study indicates that PAH concentrations remain elevated as compared to the relatively unpolluted reference site (Huron River, S. Smith, personal communication). However, PAH concentrations in Cuyahoga River sediments collected in June 1999 have clearly decreased when compared to sediment PAHs from a similar survey conducted in April 1987 (Smith et al., 1994Go). In addition to PAH, Cuyahoga River sediments contain a myriad of other compounds including nitroaromatics, aromatic hydrocarbons, metals, PCBs, and historical-use organochlorine pesticides. Thus, although PAH constitute an important contaminant class at this site that has been mechanistically linked to tumorogenesis, it is likely that the etiology of liver cancer in these fish involves coexposure to several toxicologically significant groups of environmental chemicals.

The type and frequency of hepatocellular FCA and neoplasms observed in the fish sampled in the present study are consistent with those of previous studies (Baumann et al., 1990Go, 1991Go, 1996Go; Smith et al., 1994Go). In general, the significance of hepatocellular FCA as markers of chemical-induced preneoplastic lesions appears to vary in certain fish species. For example, basophilic foci are considered most predictive of hepatocellular carcinoma in rainbow trout, whereas eosinophilic and basophilic foci are both associated with liver neoplasia in English sole exposed to carcinogenic sediments (Bailey et al., 1996Go; Myers et al., 1987Go). In contrast, eosinophilic, basophilic, clear cell, and vacuolated foci are observed in medaka (Oryzias latipes) exposed to diethylnitrosamine (Boorman et al., 1997Go) as well as in brown bullheads exposed to chemical pollutants (Baumann et al., 1996Go). Our observations support the hypothesis that the presence of hepatic FCA is reflective of pollutant exposure in Cuyahoga River bullheads. Although hepatocellular neoplasms have been reported in wild bullheads exposed to pollutants (Baumann et al., 1990Go, 1991Go), we could not confirm the presence of these lesions in our samples. The lack of hepatocellular neoplasms could be related to the ongoing remediation efforts along the Cuyahoga River, sample differences, or criteria used for histological interpretation.

The predominance of biliary neoplasms among liver lesions in this study is consistent with previous studies in bullheads (Baumann et al., 1996Go) and other fish species such as white suckers (Hayes et al., 1990Go) from polluted sites. Although biliary neoplasia is often observed in bullheads and white suckers from contaminated field sites, the presence of biliary disease (i.e., cholangiohepatitis and biliary hyperplasia) has also been reported in these species when sampled from unpolluted, as well as contaminated, sites (Baumann et al., 1996Go; Hayes et al., 1990Go). Thus, the significance of biliary hyperplasia and fibrosis as biomarkers of contaminant-induced lesions in wild bullheads is uncertain. In addition to environmental chemicals, underlying bile duct disease related to parasitism may also contribute to the predominance of biliary neoplasia in fish inhabiting polluted ecosystems. For example, treatment of white suckers with obstructive biliary disease with B[a]P resulted in a preferential colocalization of immunodetectable B[a]P-DNA adducts with hyperplastic bile ducts, and were rarely seen in hepatocytes (Hayes et al., 1990Go). These findings prompted the suggestion that bile duct disease may promote biliary neoplasia following exposure to chemical carcinogens due to increased cellular proliferation or alterations in biotransformation, biliary excretion, or DNA repair (Hayes et al., 1990Go). Similar mechanisms could contribute to the high sensitivity of wild bullheads from PAH contaminated sites to the development of biliary neoplasia.

GST expression in Cuyahoga bullhead lesions analyzed in the present study was similar to surrounding tissue, although a few eosinophilic FCA had variably decreased GST expression. Our results were consistent whether we used the striped bass overall GST antibody or the catfish pi antibody. In a recent laboratory study, the levels of bullhead liver GST pi protein and catalytic activity were not affected by exposure to the prototypical GST inducing agent, ethoxyquin, despite treatment-related increases in GST activity toward 1-chloro-2,4-dinitrobenzene (Henson et al., 2001Go). Thus, it is possible that bullhead GST pi expression is not readily modulated by chemical exposure or during cellular transformation. The pi-like GST recognized by the catfish GST antibody has high activity toward benzo[a]pyrene 4,5-oxide, a procarcinogenic metabolite produced during the oxidative metabolism of BaP (James et al., 1998Go). In rats, hepatic GST pi isozyme rGSTP1-1 exhibits particularly high activity toward another procarcinogenic PAH metabolite, (+)-7ß, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}–oxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (Robertson et al., 1986Go). Although we have not characterized bullhead GST isozyme activity toward carcinogens, we have observed that bullhead liver cytosolic preparations rapidly conjugate benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), the ultimate carcinogen of BaP metabolism, via GST (Henson et al., 2001Go). Thus, a lack of induction or decreased GST pi expression in bullhead liver may increase the sensitivity of hepatocytes or biliary epithelial cells to the initiating effects of PAH-contaminated sediments, thereby promoting pollutant-associated tumor formation.

Our studies do not support the use of total GST or GST pi expression as diagnostic cellular markers of environmental carcinogenesis in bullheads. When viewed collectively, our results and those of others indicate that cytosolic GST activity and GST protein expression in chemical-induced hepatic lesions in fish appears to vary according to species. In those instances where fish liver GST expression is increased in chemical hepatocarcinogenesis, it has been suggested that elevated GST may promote tumor growth through increased cytoprotection (Köhler et al., 1998Go). However, it is also possible that decreased GST expression in certain fish FCA and neoplasms may promote progression of altered and neoplastic lesions through decreased detoxification and subsequent increased susceptibility to the effects of genotoxic and carcinogenic compounds (Stalker et al., 1991Go). The variable decrease in immunoreactive GST expression observed in a few of the eosinophilic hepatocellular FCA in the present study is more supportive of the latter hypothesis. However, we cannot evaluate the role of decreased GST expression in hepatocellular neoplastic progression in bullheads due to the absence of advanced hepatocellular neoplasms in our samples. At minimum, our results suggest that neither loss nor induction of GST expression appears to be a significant component of neoplastic progression in bullheads, especially in regards to the predisposition to biliary neoplasia. However, it is important to note that results do not rule out altered expression of other potential bullhead GST isoenzyme(s) that may be present but expressed at comparatively low levels and not recognized by our antibodies.

Interestingly, immunoreactive GST expression was observed in some hepatocyte nuclei from nonexposed and Cuyahoga River bullheads, using the striped bass GST antibody and, to a lesser extent, the channel catfish GST antibody. However, no consistent discernable pattern was observed in regards to GST nuclear staining either in extrafocal hepatocytes or hepatocellular FCA. In this regard, there is little information available on the presence of nuclear GST expression in fish. In mice, theta class mGSTT1 is present in pericentral hepatocytes (Sherratt et al., 2002Go), and extensive GST catalytic activity is present in rat hepatic nuclear fractions (Rogers et al., 2002Go). If the presence of nuclear GST isoforms are confirmed in bullhead liver, it will be important to determine their substrate specificity toward epoxide carcinogens as well as their potential modulation during environmental chemical exposure. Such information will help shed light on the precise role of GST in protecting against environmental carcinogenesis in this species.

In summary, the results of this study indicate that overall expression of hepatic GST, as well as pi-like GST, are not markedly altered in bullhead catfish during chemical carcinogenesis. Thus, brown bullheads appear to differ significantly from rats in that overexpression of GST pi is not a useful marker of preneoplastic or neoplastic lesions in hepatocarcinogenesis. The expression and function of fish hepatic GSTs in neoplastic development following exposure to environmental carcinogens is likely to vary among species. Our results do not rule out alterations in expression of other phase II detoxification enzymes such as UDP-glucuronyl transferases or epoxide hydrolases and/or bioactivating cytochrome 450 s as factors in the sensitivity of brown bullheads to environmental carcinogens. Further studies, including controlled laboratory exposures to known carcinogens, are needed to more clearly define the role of GSTs and other chemical biotransformation enzymes in the formation and progression of hepatic lesions during fish environmental carcinogenesis.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Paul Baumann and Dr. Steve Smith for their help with collecting the Cuyahoga bullheads, and Mr. Mike Mittner and William Johnson of the Florida Game and Freshwater Fish Commission for providing reference site fish. The assistance of Drs. Bernard Gadagbui and Margaret James in providing the channel catfish GST pi antisera was greatly appreciated, as well as Drs. Michael Lame' and Hank Segall of the University of Davis for providing the striped bass GST antisera. The technical assistance of Joan Yonchek and Betty Hall is also acknowledged. This work was supported in part by N.I.H. Superfund Basic Sciences Program project P42 ES07375, which included a Superfund Basic Research Program Graduate Assistantship Award to K.L.H.


    NOTES
 

1 To whom correspondence should be addressed at Department of Environmental and Occupational Health Sceinces, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, Was 98105-6099. E-mail: evang3{at}u.washingtion.edu.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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