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
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ABSTRACT |
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Key Words: glutathione S-transferase; altered foci; immunohistochemistry; cholangiocellular carcinoma; brown bullhead; liver; neoplasia.
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INTRODUCTION |
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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., 1991). 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., 1998
). 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., 1991
). 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., 1990
). In contrast, Parker et al. (1993)
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., 1998; Sikka et al., 1990
; Steward, et al., 1990
). Sediment and tissue concentrations of organochlorines, polychlorinated biphenyls (PCBs), and PAHs have all been correlated with hepatic neoplasia in bullheads (Baumann et al., 1990
, 1991
, 1996
, 1998
); however, sediment PAHs are the strongest correlates with chemical-associated neoplasia (Baumann et al., 1991
, 1996
; Smith et al., 1994
). Bullheads rapidly metabolize benzo[a]pyrene (B[a]P) to reactive intermediates that covalently bind liver DNA (Sikka et al., 1990
; Steward et al., 1990
), a process that is augmented by treatment with the CYP1A-inducing agent ß-napthoflavone (ß-NF) (Ploch et al., 1998
). 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., 2000
). 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.
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MATERIALS AND METHODS |
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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., 1996; Smith et al., 1994
). 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., 2000) and channel catfish intestinal GST pi (James et al., 1998
). 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., 2000
). 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., 1998
) and a pi-like GST in brown bullhead liver (Henson et al., 2001
). 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., 1998
), 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 (45 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, 1996). 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. 1990; Hampton et al., 1985
1988
, 1989
; Myers et al., 1987
). 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.
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RESULTS |
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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., 1997). 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, 1996
).
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DISCUSSION |
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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., 1990, 1991
, 1996
; Smith et al., 1994
). 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., 1996
; Myers et al., 1987
). In contrast, eosinophilic, basophilic, clear cell, and vacuolated foci are observed in medaka (Oryzias latipes) exposed to diethylnitrosamine (Boorman et al., 1997
) as well as in brown bullheads exposed to chemical pollutants (Baumann et al., 1996
). 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., 1990
, 1991
), 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., 1996) and other fish species such as white suckers (Hayes et al., 1990
) 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., 1996
; Hayes et al., 1990
). 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., 1990
). 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., 1990
). 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., 2001). 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., 1998
). In rats, hepatic GST pi isozyme rGSTP1-1 exhibits particularly high activity toward another procarcinogenic PAH metabolite, (+)-7ß, 8
-dihydroxy-9
, 10
oxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (Robertson et al., 1986
). 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., 2001
). 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., 1998). 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., 1991
). 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., 2002), and extensive GST catalytic activity is present in rat hepatic nuclear fractions (Rogers et al., 2002
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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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