Astrid-Mette Husøy

Immunochemical approaches to studies of CYP1A localization and induction by xenobiotics in fish

There is an increasing understanding that polynuclear aromatic hydrocarbons (PAHs) and organochlorine compounds (like polychlorinated biphenyls (PCBs), certain pesticides and dioxins) in the aquatic environment may lead to physiological and pathological effects such as immunological disturbances, effects on reproduction and development, and even neoplasms. Exposure to pollutants may have consequences at all levels in the biological organization, from the cellular level over effects on the individual organism, population, to the entire ecosystem. The cytochrome P450 system (CYP or P450) has an essential function in the biotransformation of endogenous and exogenous compounds. The fact that many different environmental pollutants induce de novo synthesis of cytochrome P450 1A (CYP1A) proteins in fish, gives these enzymes an interesting position in aquatic toxicology. Many investigations concerning the CYP1A system in fish have been performed over the last two decades, demonstrating its usefulness as a biomarker for aquatic pollution. A general overview of the biochemical and toxicological aspects concerning the cytochrome P450 system will be given here, followed by a more detailed description of CYP1A induction responses in fish. Ecotoxicological consequences of CYP1A induction and the use of immunochemical techniques for CYP1A detection as a biomarker in environmental monitoring will be discussed.

Accumulation and effects of aromatic and chlorinated hydrocarbons in juvenile Atlantic cod (Gadus morhua) caged in a polluted fjord (Sørfjorden, Norway)

Abstract Juvenile Atlantic cod were placed in net cages on the bottom at 20–30 m depth at four sites from the inner end of Sorfjorden (Hardanger, Western Norway), to the northern mouth of the fjord in October 1990. After 4 weeks the fish were killed, and liver samples were analyzed for aromatic and chlorinated hydrocarbons (PAHs and PCBs). Liver and gill were also analyzed for cytochrome P450 1A induction, a biomarker for exposure and effects of these contaminants, using catalytic measurements (7-ethoxyresorufin-O-deethylase, EROD) and immunodetection (P450 1A1-ELISA). The caging resulted in the accumulation of a number of PAHs, but very little PCBs, in the liver of the cod, with an inward gradient in the fjord. The gradient, although not dramatic in absolute terms, was parallelled by elevated levels of P450 1A1 when measured by EROD and, partly, by ELISA. The caging strategy seems to be a promising way to approach ecotoxicologically relevant problems, such as bioavailability of contaminants, biomarker responses in the field, and dose-response relationships, also under mixed contaminant situations.

Cellular localization of cytochrome P450 (CYP1A) induction and histology in Atlantic cod (Gadus morhua L.) and European flounder (Platichthys flesus) after environmental exposure to contaminants by caging in Sørfjorden, Norway

Abstract Immunohistochemical and histopathological studies were conducted in two marine teleosts, Atlantic cod ( Gadus morhua ) and European flounder ( Platichthys flesus ), caged for three months on contaminated sediments in a Norwegian fjord. Cellular localization of CYP1A was analyzed immunohistochemically in liver, intestine, heart, gill and kidney, as part of an extensive study that included a number of chemical and biological measurements. Both species exhibited marked CYP1A induction when caged at contaminated sites. CYP1A induction was observed in liver as well as in several extrahepatic organs, and with increased expression at the more contaminated sites. Staining was particularly strong in vascular endothelium. Induction responses were also observed in epithelial cells, including hepatocytes, biliary epithelial cells, mucosal epithelial cells in the intestine, and renal tubular epithelial cells. Histopathological examination of the liver did not reveal major cellular abnormalities. The immunohistochemical data indicate a strong relationship between CYP1A induction and exposure to sediment-associated industrial contaminants (polycyclic aromatic hydrocarbons). Interesting species differences in localization of CYP1A expression in various cell types in cod and flounder were demonstrated immunohistochemically.

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal β-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets

Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet. A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding. Rapeseed oil had no effect. Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet. Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet. Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet. The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose. Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h. In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal beta-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days. This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase, i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria. It is also conceivable that the mitochondrial beta-oxidation is proceeding before the enhancement of peroxisomal beta-oxidation.

Sixty minutes of normothermic ischemia in the rat liver: Correlation between adenine nucleotides and bile excretion

The effect of 60 min of normothermic liver ischemia on cellular levels of adenine nucleotides (ATP, ADP, and AMP), energy charge (EC), and bile excretion was studied. Following ischemia the concentration of ATP was reduced to 12% of preischemic and control values within the first 10 min and remained low during the remaining ischemic period. EC values were also low. During 120 min of reperfusion, ATP increased to 34% of the ATP level found in the control group. EC values increased immediately to reach values not significantly different from those of control animals. Bile flow was nonexistent during ischemia and increased during reperfusion. The increase paralleled those of ATP and EC. Bile flow seems to reflect the degree of liver ischemia and may be used as a functional parameter.

Application of a cytochrome P-450 IA1-ELISA in environmental monitoring and toxicological testing of fish

1. An enzyme-linked immunosorbent assay (ELISA) based on polyclonal rabbit anti-cod cytochrome P-450 IA1 IgG has been developed in our laboratory. 2. The antibodies employed in the ELISA demonstrate good cross-reactivity with the homologous protein in a number of other fish species, giving cross-reacting protein bands o 54-59 kDa in liver microsomes, as determined by Western blotting. 3. The ELISA technique has been used in numerous experiments with both field collected and laboratory exposed fish of different species, showing good correlation with contaminant exposure. 4. In some instances of PCB exposure where the classical P-450 IA1 monooxygenase assay 7-ethoxyresorufin O-deethylase (EROD) failed to reveal any induction, the ELISA technique demonstrated increased levels of P-450 IA1 protein, indicating inhibiting effects of the PCBs on EROD measurement. 5. In tissues like gill filaments, and in whole cod larvae, where EROD activity is barely detectable, if at all, the ELISA technique showed induction after exposure to a water soluble fraction (WSF) of North Sea crude oil. 6. The results reviewed indicate the usefulness of the ELISA technique to allow rapid screening of a large number of samples, and especially when catalytic measurement is difficult or impossible due to (a) small sample or tissue size, (b) loss of activities in bad storage conditions, or (c) presence of compounds inhibiting activity.

The cytochrome P450 1A1 response in fish: application of immunodetection in environmental monitoring and toxicological testing

We have developed polyclonal and, recently, monoclonal antibodies against the aromatic hydrocarbon-inducible P450 1A1 form purified from Atlantic cod (Gadus morhua). A simple, indirect enzyme-linked immunosorbent assay (ELISA) based on these antibodies has been developed in our laboratory and tested in numerous experiments with both field-collected and laboratory-exposed fish of different species. The exposure situations studied to date include complex mixtures such as polychlorinated biphenyls (PCBs), dioxins and water-soluble oil fractions, as well as defined compounds such as CB congeners, TCDD, and pesticides. Factors affecting the induction response, including sexual maturation and dietary factors, have also been investigated. The ELISA technique generally shows good correlation with contaminant exposure and catalytic measurements, but has also given new and important information in certain cases where catalytic measurements failed to reveal effects.

Immunohistochemical localization of cytochrome P4501A in multiple types of contaminant-associated hepatic lesions in English sole (Pleuronectes vetulus)

Abstract Variations in the expression of cytochrome P450 (CYP) isozymes in cells may affect the response of and consequent toxic effects in those cells during xenobiotic exposure. The expression of cytochrome P4501A (CYP1A), a major inducible CYP in teleosts, was examined immunohistochemically in multiple toxicopathic hepatic lesion types, including neoplasms and several types of preneoplastic foci of cellular alteration (FCA), in English sole ( Pleuronectes vetulus ) captured from the Duwamish Waterway, a highly contaminated estuary of Puget Sound, WA. Formalin or Bouins-fixed, paraffin-embedded tissue sections were stained with polyclonal rabbit anticod CYP1A IgG by the avidin-biotin peroxidase complex method; this antibody binds to both Atlantic cod ( Gadus morhua ) and English sole CYP1A in Western blotting and ELISA, and immunohistochemically-localizes CYP1A in hepatic and other tissues of cod exposed to β-naphthoflavone. CYP1A-associated staining intensity was lower in hepatocellular and cholangiocellular neoplasms, areas of biliary hyperplasia, and normal intrahepatic exocrine pancreas and bile ducts, as compared to histologically normal hepatocellular parenchyma. FCA of basophilic, eosinophilic and clear cell types showed variable staining intensities in comparison to background parenchymal staining, but generally showed reduced CYP1A-associated staining. Staining intensity for a single hemangiopericytic sarcoma showed highly reduced staining for CYP1A. These results, in fish naturally exposed to environmental toxicants, suggest a parallel response between teleosts and mammals with respect to CYP1A expression and resistance to cytotoxicity in cells composing several types of hepatic neoplasms and foci of cellular alteration.

Histopathological and immunohistochemical studies in roundnose grenadier (Coryphaenoides rupestris) in the Skagerrak, North Sea

Abstract The aim of this study was to investigate whether the large-scale distribution of anthropogenic compounds in the Skagerrak deep sea gives rise to histopathological and immunohistochemical effects in fish. For this purpose, roundnose grenadier, Coryphaenoides rupestris , were examined from the Skagerrak (exposed site) and from a locality outside the Faroe Islands (reference site). The cytoplasm of hepatocytes offish from both areas was dominated by vacuoles, i.e. storage of either lipid or glycogen. Melanomacrophage centres (MMC) were also found. The major histological difference between the two groups was the increase of MMC in fish from the Skagerrak site. This finding was statistically confirmed by morphometry. Analysis of EROD showed that the enzyme was induced in the liver of fish from the Skagerrak. Immunohistochemical localization of cytochrome P450 1A in liver indicated positive staining of both vascular endothelial cells and hepatocytes. The induction of these parameters in fish from the Skagerrak cannot be excluded as being an effect of a large-scale contamination of anthropogenic compounds in the area.

Ethionine-induced alterations of enzymes involved in lipid metabolism and their possible relationship to induction of fatty liver

Changes of enzymes involved in the hepatic metabolism of long-chain fatty acids (palmitoyl-CoA synthetase (EC 6.2.1.3), carnitine palmitoyltransferase (EC 6.2.1.3), glycerophosphate acyltransferase (EC 2.3.1.15)) in the liver of male rats were examined after ethionine exposure. Ethionine administration resulted in a dose- and time-dependent enhancement of the palmitoyl-CoA synthetase activity both in the mitochondrial, peroxisomal and microsomal fractions. The total carnitine palmitoyltransferase activity in the mitochondrial fraction was enhanced. Ethionine administration was also associated with dose- and time-dependent changes of the microsomal glycerophosphate acyltransferase activity, whereas the mitochondrial enzyme activity was marginally affected. The hepatic triacylglycerol content of the ethionine-treated animals was increased. Hepatic lipids were accumulated in large droplets. Serum triacylglycerol and cholesterol were decreased. In particular, the serum HDL-cholesterol level was lowered. The concentration of ATP in the liver decreased. Accumulation of the metabolic product S-adenosylethionine (AdoEth) was observed for the first 2 days of exposure followed by a fall in S-adenosylmethionine (Ado-Met) during the next 10 days. Linear regression analysis of ATP content versus AdoEth and AdoMet showed highly significant correlations. A significant correlation between the hepatic triacylglycerol and AdoEth content was also observed upon ethionine treatment. The data show that ethionine perturbs the hepatic lipid metabolism. Enhanced esterification of long-chain fatty acids, but not a simple reduction of their oxidation, might contribute to ethionine-induced fatty liver in addition to a block in secretion of lipoproteins and decreased protein synthesis.