Liver Of Winter Flounder Research Articles

Toxics source reduction and sewage upgrades eliminated winter flounder liver neoplasia (1984-2017) from Boston Harbor, MA, USA.

Chemical carcinogen biomarkers can validate public investment in environmental remediation. A major factor driving the clean-up of Boston Harbor, MA, USA, induced by the federal Clean Water Act legislation of 1972, was the high prevalence of petroleum and halogenated aromatic hydrocarbon contaminant-associated liver neoplasia in winter flounder Pseudopleuronectes americanus in the harbor in the 1980s. In the present study, we examined the spatial and temporal relationships between the suspended solids and contaminants in the municipal sewage discharge, and liver neoplasia and histopathology in flounder, from 1987 to 2017. Toxics source reduction, sewage treatment, and sludge removal in the 1990s and outfall relocation offshore in 2000 enabled a decreasing prevalence of persistent toxic chemicals in flounder, effluent, and sediment, and consequent disappearance of liver neoplasia and reduction of neoplasm-associated, hydropically vacuolated biliary epithelial cells to background levels. This supports long-term investment in elimination and treatment of anthropogenic waste streams and the value of federal regulatory mandates to maintain and improve regional environmental quality.

Effects of starvation on liver microsomal P450 activity in juvenile Pleuronectes americanus

Recent work has produced evidence to support the existence of a cytochrome P450 CYP2E1-like isoform in the marine fish, Pleuronectes americanus (winter flounder) (Wall K, Crivello JF. Toxicol Appl Pharmacol 1998;151:98–104). Starvation has been previously demonstrated to induce CYP2E1 activity (assayed as chlorzozazone-6-hydroxylase activity) in mammals and this study was undertaken to determine the effects of starvation on liver chlorzozaxone-6-hydroxylase and ethoxy-resorufin- O-deethylase activity (a CYP1A1 activity) in juvenile winter flounder liver microsomes. A 2-week starvation period resulted in a statistically significant increase in liver microsomal protein, and a decrease in liver lipid and liver glycogen. Ethoxy-resorufin- O-deethylase activity (pmol/min/mg microsomal protein) was reduced with starvation, chlorzoxazone 6-hydroxylase activity (pmol/min per mg microsomal protein) initially decreased but then increased over controls. When these activities were expressed per gm/liver (to account for the starvation-induced changes in liver microsomal protein), chlorzoxazone 6-hydroxylase activity doubled over control during starvation but ethoxy-resorufin- O-deethylase was not significantly changed. The effects of starvation on liver microsomal chlorzoxazone 6-hydroxylase and ethoxy-resorufin- O-deethylase activities are discussed in the context of the impact of physiological states on the ability of fish to detoxify marine xenobiotics.

Associations between liver lesions in winter flounder (Pleuronectes americanus) and sediment chemical contaminants from north-east United States estuaries

Neoplastic diseases, particularly of bottom-dwelling fishes, are more prevalent in coastal areas than in areas that are relatively pristine. Although sediments in many urbanized estuaries contain high concentrations of contaminants, there is little evidence linking a specific organic or inorganic chemical to a particular liver lesion in winter flounder (Pleuronectes americanus), despite increasing study in recent years. Between 1984 and 1986, sediments and winter flounder were collected from 10 sites in the north-east United States ranging from grossly polluted to relatively unimpacted. Sediments were analysed for polycyclic aromatic hydrocarbons (PAHs), chlorinated pesticides, polychlorinated biphenyls (PCBs), and metals. Gross and microscopic pathological examinations were conducted on winter flounder liver sections. Factor and canonical correlation analyses were used to explore associations between biological and chemical measurements. Hepatitis, cholangitis, phlebitis, and macro-phage aggregate hyperplasia showed positive associations with low molecular weight, petroleum-derived PAHs (naphthalene, anthracene, 1-methylnaphthalene, 2-methylnaphthalene, 2,6-dimethylnaphthalene, acenaphthene, phenanthrene, bi-phenyl, fluorene, and 1-methylphenanthrene), the pesticides lindane, hexachloro-benzene, heptachlor epoxide, and o,p′-DDD, tri- to hexachlorobiphenyls, and chromium, cadmium, lead, thallium, and selenium, but were negatively associated with the pesticides o,p′-DDT, p,p′-DDT, p,p′-DDE, and mirex. Cytoplasmic hepatocellular vacuolation, cytoplasmic bile duct vacuolation, neoplasms, and pre-neoplasms showed positive associations with most PAHs measured, whether petroleum-derived or combustion-derived (benzo[a]pyrene, benzo[e]pyrene, benzo[a]anthracene, di-benzo[a,h]anthracene, perylene, chrysene, and fluoranthene), the pesticides dieldrin, trans-nonachlor and alpha-chlordane, and silver, copper, antimony, and tin, but no associations with PCBs were found. Coagulative necrosis, single cell necrosis and haemorrhagic necrosis showed positive associations with hepta- to nona-chlorobiphenyls and arsenic, zinc, nickel, and mercury, and negative associations with high molecular weight, combustion-derived PAHs and DDT compounds and metabolites.

Chlorzoxazone Metabolism by Winter Flounder Liver Microsomes: Evidence for Existence of a CYP2E1-like Isoform in Teleosts

Winter flounder (Pleuronectes americanus) liver microsomes metabolized chlorzoxazone (CZX), a CYP2E1 (cytochrome P450 2E1)- specific substrate, to a single product, 6-OH CZX. The liver microsomal fraction had the highest level of activity, which varied among fish, ranging from 60 to 220 pmol/mg/min. CZX metabolism was linearly related to microsomal protein and dependent on NADPH. The optimal pH range for this activity was from 7.2 to 8.0. Maximal activity was found at 18–22°C (154 ± 62 pmol/mg/min) but was detectable at all temperatures tested. CZX 6-hydroxylation displayed first-order kinetics as shown by Lineweaver–Burk plots. The average apparentKm, 280 μM, was higher than that reported for human liver microsomes but similar to theKmdetermined for human CYP2E1 expressed invacciniavirus.Vmaxvalues ranged from 65 to 141 pmoles/mg/min. The CYP2E1-specific inhibitors, diethyldithiocarbamate (DDC) and aniline, significantly reduced 6-OH CZX production by 80 and 35%, respectively. 7,8-Benzoflavone and ethoxyresorufin, both CYP1A-specific inhibitors, did not significantly inhibit CZX metabolism nor did trioleandomycin (TAO) a CYP3A-specific inhibitor. Diethyldithiocarbamate (DDC), at 50 μM, had no significant effect on winter flounder liver microsomal ethoxyresorufin-O-deethylase activity (EROD), a CYP1A-specific activity. 7,8-Benzoflavone, at 10 μM, inhibited EROD activity by 60%. This is the first report of CYP2E1 activity in teleost liver microsomes.

Stages of hydropic vacuolation in the liver of winter flounder Pleuronectes americanus from a chemically contaminated site

Stages of hydropic vacuolation in the liver of winter flounder Pleuronectes americanus from a chemically contaminated site

Induced Cytochrome P4501A in Winter Flounder,Pleuronectes americanus, from Offshore and Coastal Sites

Cytochrome P4501A (CYP1A), Aroclor 1254 (A1254), and 3,3′,4,4′-tetrachlorobiphenyl (TCB) were measured in liver of winter flounder, Pleuronectes americanus, from Boston Harbor, Mass., Hempsted Harbor, N.Y., Niantic, Conn., and an offshore site, Georges Bank. We also measured CYP1A content and activity in flounder from Passamaquoddy Bay, N.B. Concentrations of A1254 and TCB were the least in fish from Georges Bank (0.46 and 0.002 μg∙g dry weight−1, respectively); concentrations in fish from Boston, Niantic, and Hempsted ranged from 7.6 to 11.3 μg∙g−1and from 0.013 to 0.024 μg∙g−1. Immunodetected microsomal CYP1A contents (expressed as scup P450E equivalents) were 0.17 and 0.19 nmol∙mg−1in fish from Georges Bank and Passamaquoddy and 0.25–0.41 nmol∙mg−1in fish from Boston, Niantic, and Hempsted. Ethoxyresorufin-O-deethylase specific activities likewise were greater in fish from Boston, Niantic, and Hempsted (1.7–2.4 nmol∙min−1∙mg−1) than in fish from Georges Bank or Passamaquoddy (0.83 and 0.61 nmol∙min−1∙mg−1). CYP1A content and activity were correlated with hepatic concentrations of A1254 and TCB. These data, together with data reported in previous studies, indicate that strong induction of CYP1A protein occurs in winter flounder populations along most of the industrialized east coast and that induction of CYP1A is common, but less strong, at sites distant from the urban centers of the Northeast.

Elevated ornithine decarboxylase activity, polyamines and cell proliferation in neoplastic and vacuolated liver cells of winter flounder (Pleuronectes americanus)

Liver neoplasms, including hepatocellular and cholangiocellular tumors, commonly occur in winter flounder (Pleuronectes americanus) caught from some chemically contaminated areas such as Boston Harbor. Hydropically vacuolated cells, very often associated with neoplasia in winter flounder liver, appear to represent the first cellular abnormality in animals that later develop frank neoplasms. The proliferative capacity of hydropically vacuolated cells was studied by analyzing both ornithine decarboxylase (ODC) activity and bromodeoxyuridine (BrdU) labeling indices. Liver of winter flounder with vacuolated cellular lesions had ODC activity more than 5- to 12-fold greater than that in liver that lacked such vacuolation, whether caught from Boston Harbor or Georges Bank. Large focal areas of hydropically vacuolated cells dissected from severely affected livers had ODC activity as high or higher than surrounding parenchymal tissue. Significant elevations in hepatic polyamine levels and ratios of putrescine/spermidine were also present in all Boston Harbor animals studied, especially those exhibiting vacuolated cellular lesions, as compared to Georges Bank fish. BrdU labeling techniques indicate that hydropically vacuolated cells, along with perivacuolar small basophilic cells and neoplastic cholangiocytes, appear to have the capacity to synthesize DNA and undergo mitosis. The frequent association of hydropically vacuolated cells with hepatic neoplasia, along with high ODC activity and DNA synthesis capability, suggest that the vacuolated cells and/or perivacuolar basophilic cells may be integral to the development of some neoplastic phenotypes in winter flounder liver.

Bromodeoxyuridine uptake in hydropic vacuolation and neoplasms in winter flounder liver

Winter flounder ( Pleuronectes americanus) from Boston Harbor, Massachusett, undergo a progressive series of hepatic changes involving hydropic vacuolation of epithelial cells, biliary hyperplasia, and hepatocellular and cholangiocellular neoplasia. Severely affected fish often exhibit grossly visible lesions. To examine cell proliferation associated with these conditions, evidence for DNA synthesis was sought. Boston winter flounder livers were screened endoscopically to select fish with and without grossly visible hepatic lesions, and then injected intraperitoneally with bromodeoxyuridine (BrdU) 3 h before euthanasia. Incorporation of this nucleotide analog was visualized immunohistochemically in formalin-fixed, paraffin-embedded tissues. Basal epithelia in the gill and intestine stained strongly, as did renal hemopoietic epithelia. In contrast, renal tubular epithelia and cardiomyocytes did not stain. Normal appearing hepatocytes stained occasionally, whilst more frequent staining was observed in vacuolated and neoplastic hepatic epithelia. We conclude (1) that the BrdU assay as described here has potentially widespread application to the study of cell proliferation in fish tissues; (2) that vacuolated cells may actively proliferate in winter flounder liver from Boston Harbor; and (3) vacuolated cells may be involved in the neoplastic transformation of hepatic epithelia.

Sex differences in hepatic monooxygenases in winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) and regulation of P450 forms by estradiol

Details concerning the endogenous regulation of hepatic cytochrome P450 monooxygenases in teleosts, and the features of this regulation common among fish species, are poorly known. Gonadally mature female winter flounder (Pseudopleuronectes americanus) have been reported to have severalfold lower levels of microsomal cytochromes P450 and b5 and NADPH-cytochrome c reductase than do males (Stegeman and Woodin ('84) Mar. Environ. Res., 14:422-425). These strong sex differences prompted more detailed study of P450 regulation in winter flounder liver, and a comparison with sex differences in another marine teleost, scup (Stenotomus chrysops). Ethoxyresorufin O-deethylase (EROD) activity/nmol P450 was less in gonadally mature females than in males of both species. Immunoblot analysis with MAb 1-12-3 to P450E (the EROD catalyst) showed that the content of P450E counterpart was also much less in females of both species. Aminopyrine N-demethylase (APND) and testosterone 6 beta-hydroxylase (6 beta-OHase) activities per nmol P450 were higher in gonadally mature female than in mature male flounder, differences not seen in scup. Polyclonal antibodies to scup P450A were shown to detect proteins in a number of teleosts. The levels of anti-P450A cross-reacting protein were greater in mature female than in male flounder, but as with 6 beta-OHase activity, the content of this protein was not sexually differentiated in scup. Estradiol treatment of winter flounder depressed the rates of EROD, APND, 6 beta-OHase, and estradiol 2-OHase activities per mg protein, but APND and 6 beta-OHase activities per nmol P450 were unchanged. Thus, E2 promotes general decreases in some hepatic P450-catalyzed activities, but in achieving sex differences there is also specific regulation of the P450E counterpart, and possibly of the 6 beta-OHase (P450A?). Other factors, temporal or hormonal, can modify the effect of E2 treatment, and may contribute to the specific regulation of P450 forms in naturally maturing fish, and to species differences in this regulation.

Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues

Investigations into the precise role played by metallothionein (MT) in heavy-metal metabolism have been hampered by difficulties in positively identifying and quantifying MT in fish tissues. This study describes the development of an antisense MT RNA (cRNA) probe that will enable MT mRNA levels to be measured with a high degree of specificity and precision. Cadmium chloride administration induces the producton of MT mRNA in the liver and kidney of winter flounder (Pseudopleuronectes americanus). Poly(A)+ RNA purified from liver samples of winter flounder after cadmium chloride injections was used to construct a cDNA library. Several recombinant clones made complementary to MT mRNA were selected from this cDNA library by an oligonucleotide derived from the N-terminal amino acid sequence of winter flounder metallothionein. Sequence analysis of two of the cDNA inserts gave the structure of the entire 3′ untranslated region, a coding region corresponding to winter flounder MT and 49 nucleotides of the 5′ untranslated region. One of the flounder MT cDNAs, pWFMTC4, was subcloned into a RNA probe plasmid and transcribed to produce antisense MT RNA (cRNA). The MT cRNA was then used to detect the induction of MT mRNA production in the liver of winter flounder, following the administration of Cu2+, Zn2+, Cd2+, Pb2+, and Hg2+. The time required for the induction of hepatic MT mRNA by a single injection of Cd2+ was approximately 96 h. Dexamethasone did not induce an increase of MT mRNA in any of the winter flounder tissues examined (liver, kidney, heart, brain, intestinal scrape, and gill filament), whereas Cd2+ induced MT mRNA in all of the tissues except brain, where the constitutive level of expression was high.

Cellular distribution of cytochrome P-450E in winter flounder liver with degenerative and neoplastic disease

A characteristic hepatic pathology is highly prevalent in winter flounder ( Pseudopleuronectes americanus) from Boston Harbor. Here we report initial evaluation of the cellular expression of a cytochrome P-450E analog in normal and diseased winter flounder liver using immunohistochemical analysis with MAb 1-12-3, a monoclonal antibody specific for P-450E. Cytochrome P-450E (P-450IAI) is the polynuclear aromatic hydrocarbon (PAH)- and polychlorinated biphenyl-inducible form of P-450 from scup ( Stenotomus chrysops). MAb 1-12-3 was first applied to formalin-fixed, paraffin-embedded sections of control and 3,3,4,4-tetrachlorobiphenyl-treated scup liver using an indirect peroxidase staining technique to visualize antibody binding. The results demonstrated specific positive staining of hepatocytes, bile and pancreatic duct epithelium and endothelium for P-450E, confirming utility of MAb 1-12-3 in analysis of fixed tissue. Subsequent analysis of archival winter flounder liver also demonstrated positive staining of hepatocytes. No staining of abnormally vacuolated cells was present in any of the fish; however, nodules of such cells occasionally contained islands of intensely staining cells of unknown origin. The cellular distribution of P-450E demonstrated here indicates a metabolic heterogeneity which could influence the responses of different cell types to subsequent or continued exposure to PAH or other hepatotoxins.

Characterization of a cytosolic steroid-binding protein in the liver of the winter flounder, Pseudopleuronectes americanus

The winter flounder liver contains significant concentrations of estrogen binding protein(s) that fulfill some of the criteria normally assigned to receptor proteins, in that these proteins are saturated at low substrate concentrations (<5 n m) and have a high affinity for ligand ( K d approximately 6 × 10 −10 M for [ 3H]17β-estradiol ). Sedimentation analysis of the 17β-estradiol-binding protein complex on 5–20% sucrose density gradients detected an 8 S form of the binding protein under low salt conditions. Nonspecific sites sedimented in the 4 S region. These sedimentation characteristics are similar to those observed for the estrogen receptor and nonspecific binding sites in mammalian tissues. In contrast to mammalian estrogen receptor, which binds selectively to estrogens, the binding protein(s) investigated in our studies also binds other steroids. Competitive binding studies demonstrated that 10-fold excess of unlabeled testosterone and progesterone displaced 50 and 45% of [ 3H]17β-estradiol from the binding protein, respectively. These studies demonstrate the presence of steroid-binding protein in liver of female winter flounder which might play a role in regulating hepatic steroid hormone action.