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Reductions in CYP1A expression and hydrophobic DNA adducts in liver neoplasms of English sole (Pleuronectes vetulus): Further support for the ‘resistant hepatocyte’ model of hepatocarcinogenesis
Marine Environmental Research,
PII:
SOl41-1136(98)00010-S
Vol.46,No. 1-5, pp. 197-202,1998 Publishedby ElsevierScienceLtd Printedin Great Britain 0141-1136/98 $19.00+0.00
ELSEVIER
Reductions in CYPlA Expression and Hydrophobic DNA Adducts in Liver Neoplasms of English Sole (Pleuronectes vetulus): Further Support for the ‘Resistant Hepatocyte’ Model of Hepatocarcinogenesis
M. S. Myers, B. L. French, W. L. Reichert, M. L. Willis, B. F. Anulacion, T. K. Collier and J. E. Stein Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, Washington 98 112, USA
ABSTRACT Our recent studies have investigated the applicability of the ‘resistance to cytotoxicity’ paradigm for chemically induced hepatocarcinogenesis in rats and mice to liver neoplasia in wild English sole (Pleuronectes vetulus). Sole resident at polycyclic aromatic hydrocarbon (PAH)-contaminated sites, such as the Duwamish Waterway in Puget Sound, Washington, exhibit high prevalences of hepatic neoplasms and precursor lesions related to the histogenesis of neoplasms. Previous immunohistochemical studies in English sole show a consistent reduction of CYPlA expression, localized with a polyclonal antibody to Atlantic cod CYPIA, in hepatic neoplasms and most preneoplastic foci of cellular alteration. The present study utilized immunohistochemical localization and quantitation of CYPIA expression by image analysis, linked with quantitation of hydrophobic DNA adducts by the 32P-postlabeling method, in hepatocellular neoplasms as compared to matched samples of adjacent non-neoplastic liver tissue from the same fish. Allfish were from the Duwamish Waterway in Seattle, Washington. In the eight neoplasms assessed (four hepatocellular adenomas, four hepatocellular carcinomas), there was a signljicant and nearly parallel reduction in DNA adduct concentrations (.56-90%) and levels of CYPlA expression (3-99%) as compared to the adjacent non-neoplastic liver tissue. These jindings are consistent with the hypothesis that neoplastic hepatocytes in English sole possess a ‘resistant’ phenotype in which there is a reduced capacity for CYPlA-mediated activation of genotoxic PAHs to their toxic and carcinogenic intermediates, and a consequent reduction in the formation of covalent, hydrophobic DNA adducts from these reactive intermediates. Published by Elsevier Science Ltd 197
198
M. S. Myers
et al.
English sole from the Duwamish Waterway in Puget Sound, Washington, are affected with high prevalences of toxicopathic hepatic lesions, including hepatocellular, cholangiocellular, and mixed hepato/cholangiocellular neoplasms (Myers et al., 1987). These lesions in wild sole have consistently been statistically associated with exposure to polycyclic aromatic hydrocarbons (PAHs) in sediments from this and other urban estuaries near Seattle, Washington (Myers et al., 1991). Among the PAHs in these sediments are compounds genotoxic and hepatocarcinogenic to mammals and fish, such as benzo(a)pyrene (BaP). Moreover, preneoplastic foci of cellular alteration have been induced in sole injected with either an extract of a PAH-contaminated sediment, or with BaP (Schiewe et al., 1991). The DNA adduct profiles, determined by 32P-postlabeling, of hepatic DNA in sole from the Duwamish Waterway, contain a diagonal zone where DNA adducts of PAHs are shown to chromatograph (Varanasi et al., 1989). Taken together, these findings strongly support the hypothesis that environmental exposure to PAHs in English sole is a major etiologic factor in the development of hepatic neoplasms and lesions related to their histogenesis. Isozymes of the CYPl family in mammals and teleosts are inducible by, and metabolize, numerous xenobiotics, and mediate the transformation of PAHs and certain chlorinated hydrocarbons (CHs) to reactive and cytotoxic or carcinogenic intermediates. In English sole, BaP is metabolized by the CYP system to the proximate carcinogen, BaP 7,8-dihydrodiol-9,10-epoxide (Varanasi et al., 1989) which can bind to DNA bases and cause mutations that represent a molecular lesion involved in initiation of carcinogenesis. The major hydrocarbon-inducible form of CYP in fish possesses immunological, catalytic and sequence similarities to isozymes in the mammalian CYPlA family, and has been induced in multiple fish species exposed to PAH-type inducers (Stegeman and Hahn, 1994). English sole captured from the Duwamish Waterway or injected with an extract of Duwamish Waterway sediment exhibit significantly elevated levels of aryl hydrocarbon hydroxylase (Collier et al., 1992), which is catalysed by the major PAH-inducible CYP isozyme in sole homologous to mammalian CYPlA (Varanasi et al., 1986). This isozyme in English sole and numerous other fish species cross-reacts with a polyclonal antibody to Atlantic cod CYPlA (Goksoyr et al., 1991), binding to a 5400&59000-dalton protein in western blots. Furthermore, recent immunohistochemical studies utilizing this antibody have shown dramatic and consistent reductions of CYPlA expression in cholangiocellular carcinomas, hepatocellular adenomas and carcinomas, and mixed hepatobiliary carcinomas in English sole (Myers et al., 1995), a pattern consistent with that seen in hepatic tumors in rodents (Roomi et al., 1985; Buchmann et al., 1987) and other fish species (van Veld et al., 1992; Parker et al., 1993). Reduced CYPlA expression in cells forming these neoplasms is hypothesized to confer an adaptive resistance to xenobiotic chemicals requiring CYP-mediated metabolic activation for their cyto- or genotoxic, effects (Roomi et al., 1985), consistent with the ‘resistance to cytotoxicity’ paradigm for mammalian hepatocarcinogenesis (Farber and Sarma, 1987). Recent studies in humans have shown significantly lower levels of hydrophobic DNA adducts in tumorous lung tissue (van Schooten et al., 1990) and colonic adenocarcinomas (Pfohl-Leszkowicz et al., 1995) compared to adjacent non-tumorous lung and colonic tissues. Our previous studies have demonstrated that hydrophobic DNA adducts in liver of English sole are a sensitive biomarker of environmental exposure to genotoxic PAHs. Adduct levels are significantly higher in English sole from PAH-contaminated sites such as the Duwamish Waterway (Stein et al., 1992), and are formed in a dose-responsive
DNA adducts, CYPlA
(43
i80-
Q ‘i
180-
5
140-
z
120’
=
loo-
f
80-
3
expression in Pleuronectes
1
0
199
non-tumor
W tumor
w-
Z!
4
5
8
7
fish # 0
q
normal parenchyma tumor
AB
12345
8
7
fish # Fig. 1. (a) Hydrophobic DNA adduct levels (nmol adducts/mol nucleotides) in matched samples of surrounding non-tumorous liver and liver neoplasms from adult English sole. (b) Per cent area of tissue staining positive for CYPlA expression in matched samples of surrounding non-tumorous liver and liver neoplasms from adult English sole, as determined by immunohistochemical localization followed by image analysis. Positive staining was determined by a color thresholding method (see text). HC = hepatocellular carcinoma; HA = hepatocellular adenoma; A = neoplasm A from fish No. 5 (HA); B = neoplasm B from fish No. 5 (HA); O=zero or near-zero value for CYPlA expression. manner following exposure to model PAHs bioactivated by CYPlA (French et al., 1996). Adduct levels are also statistically associated with the prevalence of, and are significant risk factors for, the occurrence of liver lesions involved in the early histogenesis of liver findings). Because DNA neoplasms in English sole (Myers et al., 1998; unpublished adducts are a relatively stable genotoxic effect mechanistically linked to CYPlA expres-
sion, we examined adduct levels in matched tissue samples of hepatic tumors and surrounding non-neoplastic tissue in English sole, in order to further examine the applicability of the ‘resistant hepatocyte’ paradigm of hepatocarcinogenesis in this teleost species.
M. S. Myers
200
et al.
n q HC
HA
HC
HC
DNA adducts CYPlA HA HA
A
P
HA
HC
B
t234667
fish # Fig. 2.
Per cent reduction in DNA adduct levels and CYPlA expression in liver neoplasms from adult English sole in comparison to matched samples of surrounding non-tumorous liver. HC = hepatocellular carcinoma; HA = hepatocellular adenoma; A = neoplasm A from fish No. 5 (HA); B = neoplasm B from fish No. 5 (HA).
Our objective was to immunohistochemically quantitate CYPlA expression and measure DNA adduct levels in eight histologically confirmed hepatic neoplasms and matched surrounding non-neoplastic liver in seven adult English sole from the Duwamish Waterway. DNA adducts were measured by the 32P-postlabeling method according to Reichert and French (1994). CYPlA expression was localized with polyclonal rabbit anti-Atlantic cod CYPlA IgG, using standard methods for immunohistochemical localization (a DABnickel chloride-peroxidase substrate solution was employed as the chromogen) in routinely fixed and paraffin-embedded tissues (Hussy et al., 1994). CYPlA expression was then semi-quantitated by computer-assisted image analysis, using a light microscope equipped with a 3CCD color video camera, linked to a 486 MB computer with OPTIMAS 4.1 image analysis software (Optimas Corp., Edmonds, Washington, USA), utilizing a color thresholding criterion (Anulacion et al., 1998). Images were captured using a x40 objective, and applying the threshold criterion, mean per cent area positively stained for CYPlA in tumor tissue and surrounding non-tumourous parenchyma was computed. For each specimen and tissue type (tumor, non-tumor), at least 12 fields of 0.002-0.01 mm2 each (composed of hepatic parenchyma only) were analyzed for per cent area expressing CYPlA positivity, as defined by the threshold. The proportional reduction in CYPlA expression in tumor tissue as compared to surrounding non-tumorous parenchyma was calculated and then compared to the proportional reduction in DNA adduct levels for that specimen. The DNA adduct analyses (Fig. l(a)) showed a dramatic and significant reduction (p < 0.0001, paired t-test) in all eight of the hepatic neoplasms from seven fish. Similarly, in seven of the eight hepatic neoplasms semi-quantitated for CYPlA expression (Fig. l(b)), there was a marked and significant reduction (p < 0.0001, ANOVA) in CYPl A-associated staining. In a single hepatocellular adenoma (fish No. 6, Figs l(b) and 2), there was little
DNA adducts, CYPIA
expression in Pleuronectes
201
measurable change in CYPlA expression. Adduct levels in the neoplasms showed proportional reductions of 56-90% as compared to the matched non-tumor liver tissue samples, with nearly parallel reductions of 9O-100% for CYPlA expression in the tumors (Fig. 2). There was no difference in proportional reduction for either DNA adducts or CYPlA expression between the adenomas and carcinomas. The reduction in CYPlA expression corresponds well with the reduction in DNA adduct levels in these hepatic neoplasms in English sole, although the relationship was not linear or completely consistent. A number of factors other than CYPlA expression may affect DNA adduct concentrations in hepatic neoplasms, including: (i) dilution of adduct levels by cell proliferation in neoplasms, since adducts formed following PAH exposure, metabolism and binding to DNA bases are not duplicated during replicative DNA synthesis; (ii) presence of preneoplastic focal lesions in the ‘normal’ tissue sample, where adduct levels might be lower than in totally normal liver; (iii) differential repair of adducts within neoplasms and non-neoplastic tissues; (iv) differential expression of Phase II enzymes such as gamma-glutamyl transpeptidase, glutathione-S-transferase, and UDPglucuronyl transferase, where increases would tend to reduce adduct formation; (v) differential expression of other P450 isozymes or proteins involved in multidrug resistance (MDR); (vi) differential carcinogen dose reaching neoplastic and non-neoplastic tissue; and (vii) formation of some adducts mediated via pathways other than CYPlA or involving chemicals not activated by CYPlA. In summary, the findings in this limited dataset showing nearly parallel reductions in DNA adducts and CYPlA expression in English sole liver neoplasms are consistent with the position that neoplastic hepatocytes in this species possess a ‘resistant’ phenotype in which there is: (i) a reduced capacity to activate PAHs and other compounds whose metabolism is mediated via CYPlA to their toxic and carcinogenic intermediates; and (ii) a consequent reduction in the formation of hydrophobic DNA adducts from these reactive intermediates formed via CYPlA bioactivation.
ACKNOWLEDGEMENTS The authors would like to thank Dr Anders Gokssyr of the University of Bergen, Norway, for providing the polyclonal antibody to Atlantic cod CYPlA; Cindy Bucher for assistance in the image analysis procedures, and Lyndal Johnson and Dan Lomax for manuscript review.
REFERENCES Anulacion, B. F., Myers, M. S., Willis, M. L. and Collier, T. K. (1998) Marine Environmental Research 46( l-5), 7-l 1. Buchmann, A., Schwarz, M. and Schmitt, R. et al. (1987) Cancer Research 47,2911-2918. Collier, T. K., Singh, S. V., Awasthi, Y. C. and Varanasi, U. (1992) Toxicology Applied Pharmacology 113, 319-324.
Farber, E. and Sarma, D. S. R. (1987) Laboratory Investigations 56, 4-22. French, B. L., Reichert, W. L., Horn, T., Nishimoto, M., Sanborn, H. R. and Stein, J. E. (1996) Aquatic Toxicology 36, 1-16.
Gokssyr, A., Andersson, T. and Buhler, D. R. et al. (1991) Fish Physiology Biochemistry 9, 1-13.
M. S. Myers et al.
202
Husey, A.-M., Myers, M. S., Willis, M. J., Collier, T. K., Celander, M. and Gokssyr,
A. (1994)
Toxicology Applied Pharmacology 129, 294-308.
Myers, M. S., Rhodes, L. D. and McCain, B. B. (1987) Journal of the National Cancer Institute 78, 333-363.
Myers, M. S., Landahl,
J. T., Krahn,
M. M. and McCain,
B. B. (1991) Environmental Health
Perspectives 90, 7-15.
Myers, M. S., Willis, M. J., Hussy, A.-M., Goksoyr, A. and Collier, T. K. (1995) Marine Environmental Research 39, 283-288.
Myers, M. S., Johnson, L. L., Tom, T., Collier, T. K., Stein, J. E. and Varanasi, U. (1998) Marine Environmental Research 45, 4767.
Parker, L. M., Lauren, D. J., Hammock, B. D., Winder, B. and Hinton, D. E. (1993) Carcinogenesis 14,211-217.
Pfohl-Leszkowicz, A., Grosse, Y. and Carriere, V. et al. (1995) Cancer Research 55, 5611-5616. Reichert, W. L. and French, B. (1994) U.S. Dep. Commer., NOAA Tech. Memo. NMFS-NWFSC14, 89 pp. Roomi, M. W., Ho, R. K., Sarma, D. S. R. and Farber, E. (1985) Cancer Research 45, 564-571. Schiewe, M. H., Weber, D. D. and Myers, M. S. et al. (1991) Canadian Journal of Fisheries and Aquatic Sciences 48, 175&1760. Stegeman, J. J. and Hahn, M. E. (1994) In: Aquatic Toxicology: Molecular, Biochemical and Cellular Perspectives, eds D. C. Malins and G. K. Ostrander, pp. 87-206. Lewis Publishers, CRC Press. Stein, J. E., Collier, T. K., Reichert, W. L., Casillas, E., Horn, T. and Varanasi, U. (1992) Environmental Toxicology Chemistry 11, 701-714. van Schooten, F. J., Hillebrand, M. J. X. and van Leeuwen, F. E. et al. (1990) Carcinogenesis 11, 1677-1681. van Veld, P. A., Vogelbein, W. K. and Smolowitz, R. M. et al. (1992) Carcinogenesis 13, 505-507. Varanasi, U., Collier, T. K., Williams, D. E. and Buhler, D. R. (1986) Biochemical Pharmacology 35, 2967-297 1.
Varanasi, U., Reichert, W. L. and Stein, J. E. (1989) Cancer Research 49, 1171-l 177.
PII:
SOl41-1136(98)00010-S
Vol.46,No. 1-5, pp. 197-202,1998 Publishedby ElsevierScienceLtd Printedin Great Britain 0141-1136/98 $19.00+0.00
ELSEVIER
Reductions in CYPlA Expression and Hydrophobic DNA Adducts in Liver Neoplasms of English Sole (Pleuronectes vetulus): Further Support for the ‘Resistant Hepatocyte’ Model of Hepatocarcinogenesis
M. S. Myers, B. L. French, W. L. Reichert, M. L. Willis, B. F. Anulacion, T. K. Collier and J. E. Stein Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, Washington 98 112, USA
ABSTRACT Our recent studies have investigated the applicability of the ‘resistance to cytotoxicity’ paradigm for chemically induced hepatocarcinogenesis in rats and mice to liver neoplasia in wild English sole (Pleuronectes vetulus). Sole resident at polycyclic aromatic hydrocarbon (PAH)-contaminated sites, such as the Duwamish Waterway in Puget Sound, Washington, exhibit high prevalences of hepatic neoplasms and precursor lesions related to the histogenesis of neoplasms. Previous immunohistochemical studies in English sole show a consistent reduction of CYPlA expression, localized with a polyclonal antibody to Atlantic cod CYPIA, in hepatic neoplasms and most preneoplastic foci of cellular alteration. The present study utilized immunohistochemical localization and quantitation of CYPIA expression by image analysis, linked with quantitation of hydrophobic DNA adducts by the 32P-postlabeling method, in hepatocellular neoplasms as compared to matched samples of adjacent non-neoplastic liver tissue from the same fish. Allfish were from the Duwamish Waterway in Seattle, Washington. In the eight neoplasms assessed (four hepatocellular adenomas, four hepatocellular carcinomas), there was a signljicant and nearly parallel reduction in DNA adduct concentrations (.56-90%) and levels of CYPlA expression (3-99%) as compared to the adjacent non-neoplastic liver tissue. These jindings are consistent with the hypothesis that neoplastic hepatocytes in English sole possess a ‘resistant’ phenotype in which there is a reduced capacity for CYPlA-mediated activation of genotoxic PAHs to their toxic and carcinogenic intermediates, and a consequent reduction in the formation of covalent, hydrophobic DNA adducts from these reactive intermediates. Published by Elsevier Science Ltd 197
198
M. S. Myers
et al.
English sole from the Duwamish Waterway in Puget Sound, Washington, are affected with high prevalences of toxicopathic hepatic lesions, including hepatocellular, cholangiocellular, and mixed hepato/cholangiocellular neoplasms (Myers et al., 1987). These lesions in wild sole have consistently been statistically associated with exposure to polycyclic aromatic hydrocarbons (PAHs) in sediments from this and other urban estuaries near Seattle, Washington (Myers et al., 1991). Among the PAHs in these sediments are compounds genotoxic and hepatocarcinogenic to mammals and fish, such as benzo(a)pyrene (BaP). Moreover, preneoplastic foci of cellular alteration have been induced in sole injected with either an extract of a PAH-contaminated sediment, or with BaP (Schiewe et al., 1991). The DNA adduct profiles, determined by 32P-postlabeling, of hepatic DNA in sole from the Duwamish Waterway, contain a diagonal zone where DNA adducts of PAHs are shown to chromatograph (Varanasi et al., 1989). Taken together, these findings strongly support the hypothesis that environmental exposure to PAHs in English sole is a major etiologic factor in the development of hepatic neoplasms and lesions related to their histogenesis. Isozymes of the CYPl family in mammals and teleosts are inducible by, and metabolize, numerous xenobiotics, and mediate the transformation of PAHs and certain chlorinated hydrocarbons (CHs) to reactive and cytotoxic or carcinogenic intermediates. In English sole, BaP is metabolized by the CYP system to the proximate carcinogen, BaP 7,8-dihydrodiol-9,10-epoxide (Varanasi et al., 1989) which can bind to DNA bases and cause mutations that represent a molecular lesion involved in initiation of carcinogenesis. The major hydrocarbon-inducible form of CYP in fish possesses immunological, catalytic and sequence similarities to isozymes in the mammalian CYPlA family, and has been induced in multiple fish species exposed to PAH-type inducers (Stegeman and Hahn, 1994). English sole captured from the Duwamish Waterway or injected with an extract of Duwamish Waterway sediment exhibit significantly elevated levels of aryl hydrocarbon hydroxylase (Collier et al., 1992), which is catalysed by the major PAH-inducible CYP isozyme in sole homologous to mammalian CYPlA (Varanasi et al., 1986). This isozyme in English sole and numerous other fish species cross-reacts with a polyclonal antibody to Atlantic cod CYPlA (Goksoyr et al., 1991), binding to a 5400&59000-dalton protein in western blots. Furthermore, recent immunohistochemical studies utilizing this antibody have shown dramatic and consistent reductions of CYPlA expression in cholangiocellular carcinomas, hepatocellular adenomas and carcinomas, and mixed hepatobiliary carcinomas in English sole (Myers et al., 1995), a pattern consistent with that seen in hepatic tumors in rodents (Roomi et al., 1985; Buchmann et al., 1987) and other fish species (van Veld et al., 1992; Parker et al., 1993). Reduced CYPlA expression in cells forming these neoplasms is hypothesized to confer an adaptive resistance to xenobiotic chemicals requiring CYP-mediated metabolic activation for their cyto- or genotoxic, effects (Roomi et al., 1985), consistent with the ‘resistance to cytotoxicity’ paradigm for mammalian hepatocarcinogenesis (Farber and Sarma, 1987). Recent studies in humans have shown significantly lower levels of hydrophobic DNA adducts in tumorous lung tissue (van Schooten et al., 1990) and colonic adenocarcinomas (Pfohl-Leszkowicz et al., 1995) compared to adjacent non-tumorous lung and colonic tissues. Our previous studies have demonstrated that hydrophobic DNA adducts in liver of English sole are a sensitive biomarker of environmental exposure to genotoxic PAHs. Adduct levels are significantly higher in English sole from PAH-contaminated sites such as the Duwamish Waterway (Stein et al., 1992), and are formed in a dose-responsive
DNA adducts, CYPlA
(43
i80-
Q ‘i
180-
5
140-
z
120’
=
loo-
f
80-
3
expression in Pleuronectes
1
0
199
non-tumor
W tumor
w-
Z!
4
5
8
7
fish # 0
q
normal parenchyma tumor
AB
12345
8
7
fish # Fig. 1. (a) Hydrophobic DNA adduct levels (nmol adducts/mol nucleotides) in matched samples of surrounding non-tumorous liver and liver neoplasms from adult English sole. (b) Per cent area of tissue staining positive for CYPlA expression in matched samples of surrounding non-tumorous liver and liver neoplasms from adult English sole, as determined by immunohistochemical localization followed by image analysis. Positive staining was determined by a color thresholding method (see text). HC = hepatocellular carcinoma; HA = hepatocellular adenoma; A = neoplasm A from fish No. 5 (HA); B = neoplasm B from fish No. 5 (HA); O=zero or near-zero value for CYPlA expression. manner following exposure to model PAHs bioactivated by CYPlA (French et al., 1996). Adduct levels are also statistically associated with the prevalence of, and are significant risk factors for, the occurrence of liver lesions involved in the early histogenesis of liver findings). Because DNA neoplasms in English sole (Myers et al., 1998; unpublished adducts are a relatively stable genotoxic effect mechanistically linked to CYPlA expres-
sion, we examined adduct levels in matched tissue samples of hepatic tumors and surrounding non-neoplastic tissue in English sole, in order to further examine the applicability of the ‘resistant hepatocyte’ paradigm of hepatocarcinogenesis in this teleost species.
M. S. Myers
200
et al.
n q HC
HA
HC
HC
DNA adducts CYPlA HA HA
A
P
HA
HC
B
t234667
fish # Fig. 2.
Per cent reduction in DNA adduct levels and CYPlA expression in liver neoplasms from adult English sole in comparison to matched samples of surrounding non-tumorous liver. HC = hepatocellular carcinoma; HA = hepatocellular adenoma; A = neoplasm A from fish No. 5 (HA); B = neoplasm B from fish No. 5 (HA).
Our objective was to immunohistochemically quantitate CYPlA expression and measure DNA adduct levels in eight histologically confirmed hepatic neoplasms and matched surrounding non-neoplastic liver in seven adult English sole from the Duwamish Waterway. DNA adducts were measured by the 32P-postlabeling method according to Reichert and French (1994). CYPlA expression was localized with polyclonal rabbit anti-Atlantic cod CYPlA IgG, using standard methods for immunohistochemical localization (a DABnickel chloride-peroxidase substrate solution was employed as the chromogen) in routinely fixed and paraffin-embedded tissues (Hussy et al., 1994). CYPlA expression was then semi-quantitated by computer-assisted image analysis, using a light microscope equipped with a 3CCD color video camera, linked to a 486 MB computer with OPTIMAS 4.1 image analysis software (Optimas Corp., Edmonds, Washington, USA), utilizing a color thresholding criterion (Anulacion et al., 1998). Images were captured using a x40 objective, and applying the threshold criterion, mean per cent area positively stained for CYPlA in tumor tissue and surrounding non-tumourous parenchyma was computed. For each specimen and tissue type (tumor, non-tumor), at least 12 fields of 0.002-0.01 mm2 each (composed of hepatic parenchyma only) were analyzed for per cent area expressing CYPlA positivity, as defined by the threshold. The proportional reduction in CYPlA expression in tumor tissue as compared to surrounding non-tumorous parenchyma was calculated and then compared to the proportional reduction in DNA adduct levels for that specimen. The DNA adduct analyses (Fig. l(a)) showed a dramatic and significant reduction (p < 0.0001, paired t-test) in all eight of the hepatic neoplasms from seven fish. Similarly, in seven of the eight hepatic neoplasms semi-quantitated for CYPlA expression (Fig. l(b)), there was a marked and significant reduction (p < 0.0001, ANOVA) in CYPl A-associated staining. In a single hepatocellular adenoma (fish No. 6, Figs l(b) and 2), there was little
DNA adducts, CYPIA
expression in Pleuronectes
201
measurable change in CYPlA expression. Adduct levels in the neoplasms showed proportional reductions of 56-90% as compared to the matched non-tumor liver tissue samples, with nearly parallel reductions of 9O-100% for CYPlA expression in the tumors (Fig. 2). There was no difference in proportional reduction for either DNA adducts or CYPlA expression between the adenomas and carcinomas. The reduction in CYPlA expression corresponds well with the reduction in DNA adduct levels in these hepatic neoplasms in English sole, although the relationship was not linear or completely consistent. A number of factors other than CYPlA expression may affect DNA adduct concentrations in hepatic neoplasms, including: (i) dilution of adduct levels by cell proliferation in neoplasms, since adducts formed following PAH exposure, metabolism and binding to DNA bases are not duplicated during replicative DNA synthesis; (ii) presence of preneoplastic focal lesions in the ‘normal’ tissue sample, where adduct levels might be lower than in totally normal liver; (iii) differential repair of adducts within neoplasms and non-neoplastic tissues; (iv) differential expression of Phase II enzymes such as gamma-glutamyl transpeptidase, glutathione-S-transferase, and UDPglucuronyl transferase, where increases would tend to reduce adduct formation; (v) differential expression of other P450 isozymes or proteins involved in multidrug resistance (MDR); (vi) differential carcinogen dose reaching neoplastic and non-neoplastic tissue; and (vii) formation of some adducts mediated via pathways other than CYPlA or involving chemicals not activated by CYPlA. In summary, the findings in this limited dataset showing nearly parallel reductions in DNA adducts and CYPlA expression in English sole liver neoplasms are consistent with the position that neoplastic hepatocytes in this species possess a ‘resistant’ phenotype in which there is: (i) a reduced capacity to activate PAHs and other compounds whose metabolism is mediated via CYPlA to their toxic and carcinogenic intermediates; and (ii) a consequent reduction in the formation of hydrophobic DNA adducts from these reactive intermediates formed via CYPlA bioactivation.
ACKNOWLEDGEMENTS The authors would like to thank Dr Anders Gokssyr of the University of Bergen, Norway, for providing the polyclonal antibody to Atlantic cod CYPlA; Cindy Bucher for assistance in the image analysis procedures, and Lyndal Johnson and Dan Lomax for manuscript review.
REFERENCES Anulacion, B. F., Myers, M. S., Willis, M. L. and Collier, T. K. (1998) Marine Environmental Research 46( l-5), 7-l 1. Buchmann, A., Schwarz, M. and Schmitt, R. et al. (1987) Cancer Research 47,2911-2918. Collier, T. K., Singh, S. V., Awasthi, Y. C. and Varanasi, U. (1992) Toxicology Applied Pharmacology 113, 319-324.
Farber, E. and Sarma, D. S. R. (1987) Laboratory Investigations 56, 4-22. French, B. L., Reichert, W. L., Horn, T., Nishimoto, M., Sanborn, H. R. and Stein, J. E. (1996) Aquatic Toxicology 36, 1-16.
Gokssyr, A., Andersson, T. and Buhler, D. R. et al. (1991) Fish Physiology Biochemistry 9, 1-13.
M. S. Myers et al.
202
Husey, A.-M., Myers, M. S., Willis, M. J., Collier, T. K., Celander, M. and Gokssyr,
A. (1994)
Toxicology Applied Pharmacology 129, 294-308.
Myers, M. S., Rhodes, L. D. and McCain, B. B. (1987) Journal of the National Cancer Institute 78, 333-363.
Myers, M. S., Landahl,
J. T., Krahn,
M. M. and McCain,
B. B. (1991) Environmental Health
Perspectives 90, 7-15.
Myers, M. S., Willis, M. J., Hussy, A.-M., Goksoyr, A. and Collier, T. K. (1995) Marine Environmental Research 39, 283-288.
Myers, M. S., Johnson, L. L., Tom, T., Collier, T. K., Stein, J. E. and Varanasi, U. (1998) Marine Environmental Research 45, 4767.
Parker, L. M., Lauren, D. J., Hammock, B. D., Winder, B. and Hinton, D. E. (1993) Carcinogenesis 14,211-217.
Pfohl-Leszkowicz, A., Grosse, Y. and Carriere, V. et al. (1995) Cancer Research 55, 5611-5616. Reichert, W. L. and French, B. (1994) U.S. Dep. Commer., NOAA Tech. Memo. NMFS-NWFSC14, 89 pp. Roomi, M. W., Ho, R. K., Sarma, D. S. R. and Farber, E. (1985) Cancer Research 45, 564-571. Schiewe, M. H., Weber, D. D. and Myers, M. S. et al. (1991) Canadian Journal of Fisheries and Aquatic Sciences 48, 175&1760. Stegeman, J. J. and Hahn, M. E. (1994) In: Aquatic Toxicology: Molecular, Biochemical and Cellular Perspectives, eds D. C. Malins and G. K. Ostrander, pp. 87-206. Lewis Publishers, CRC Press. Stein, J. E., Collier, T. K., Reichert, W. L., Casillas, E., Horn, T. and Varanasi, U. (1992) Environmental Toxicology Chemistry 11, 701-714. van Schooten, F. J., Hillebrand, M. J. X. and van Leeuwen, F. E. et al. (1990) Carcinogenesis 11, 1677-1681. van Veld, P. A., Vogelbein, W. K. and Smolowitz, R. M. et al. (1992) Carcinogenesis 13, 505-507. Varanasi, U., Collier, T. K., Williams, D. E. and Buhler, D. R. (1986) Biochemical Pharmacology 35, 2967-297 1.
Varanasi, U., Reichert, W. L. and Stein, J. E. (1989) Cancer Research 49, 1171-l 177.