Effect of bromobenzene and O-bromophenol on kidney and liver of English sole (Parophrys vetulus)

1. English sole (Parophrys vetulus) were injected intraperitoneally with a single dose of 9.8 mmol bromobenzene/kg of fish or 1.9 mmol O-bromophenol/kg of fish, both known renal toxicants in mammals. 2. Kidney, liver, gill spleen, intestines, heart and blood samples were subsequently obtained up to 48 hr post-injection for determination of microscopic lesions, concentrations of selected tissue antioxidants (glutathione and ascorbic acid), and selected serum parameters. 3. Bromobenzene and O-bromophenol were both found to be hepatotoxic in English sole, as indicated by the presence of hepatocellular coagulation necrosis and fatty change in the liver, altered glutathione and ascorbic acid levels in liver tissue, elevated serum aspartate aminotransferase and alkaline phosphatase activity and increased serum glucose and triglyceride levels. 4. No evidence of nephrotoxicity was found in English sole exposed to either toxicant. 5. It is concluded that bromobenzene and O-bromophenol cannot be used as model nephrotoxicants but can be used as hepatotoxicants in English sole.     

Oxidative DNA damage in tissues of English sole (Parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Formation and persistence of benzo[a]pyrene-diolepoxide-DNA adducts in liver of English sole (Parophrys vetulus)

The formation of DNA adducts from the carcinogenic environmental pollutant benzo[a]pyrene (BaP) was investigated in liver of English sole (Parophrys vetulus), a fish species that exhibits a high prevalence of liver neoplasms in several polycyclic aromatic hydrocarbon (PAH)-contaminated areas of Puget Sound, WA. Analysis by the 32P-postlabeling assay of hepatic DNA digests from English sole exposed parenterally to BaP showed the presence of BaP-diol epoxide (BaPDE)-DNA adducts. When English sole were injected with 2-15 mg BaP/kg body wt., one major adduct was detected and was identified as the anti-BaPDE-DNA adduct. Moreover, in English sole sampled at 1, 28 and 60 days post-exposure to 15 mg BaP/kg body wt., there was no significant change in the level of the anti-BaPDE-DNA adduct. The autoradiographs of 32P-labeled hepatic DNA digests from fish exposed to 100 mg BaP/kg body wt. showed an elongated spot suggesting the presence of more than one adduct. Chromatography on large polyethyleneimine sheets (20 x 20 cm) showed 2 spots with the same chromatographic characteristics as those of syn- and anti-BaPDE-deoxyguanosine adduct standards. Mild acid hydrolysis of hepatic DNA of English sole, exposed to 100 mg BaP/kg body wt., also revealed the presence of tetrols derived from both anti- and syn-BaPDE, thus confirming the presence of syn- and anti-BaPDE. In fish exposed to 2-100 mg BaP/kg body wt., a linear (0.996) dose response for anti-BaPDE-DNA adduct formation was observed. The results from this study offer the first direct evidence for the formation of the suspected ultimate carcinogen, BaPDE, in liver of English sole exposed to BaP in vivo and thus further support the hypothesis that exposure to PAHs is an important factor in the etiology of hepatic neoplasms in English sole from contaminated sites.     

Effects of low temperature on the development of the microsporidan Glugea stephani in English sole (Parophrys vetulus)

Glugea stephani requires temperatures above 15 C for development in juvenile pleuronectid flatfishes in Yaquina Bay, Oregon. The effect of low temperature (10 C) on the development of recently established parasites was tested experimentally in juvenile English sole (Parophrys vetulus). Low temperature arrested parasite development, but did not kill the protozoan which resumed development on return to 19-20 C after as long as 42 days at 10 C. No parasites detectable with the light microscope were found in fish examined after 70 days at 10 C. Although most juvenile English sole move permanently from the estuary to cooler ocean waters in fall and do not contribute to the continuation of the parasite life cycle, the cycle may be maintained by low numbers of English sole that overwinter in the estuary.     

Multiplication of Trypanosoma pacifica (Euglenozoa: Kinetoplastea) in English sole, Parophrys vetulus, from Oregon coastal waters

Multiplication of Trypanosoma pacifica was common in the fish host from observations of live flagellates and Giemsa-stained blood smears. Multiplication began with the elongation of the kinetoplast, thickening of the posterior portion of the body, and appearance of a new flagellum near the kinetoplast. The new flagellum was very rigid when less than 3 microm in length, but it became flexible as it elongated. When the new flagellum was approximately 12 microm in length, cell division began and the kinetoplast also began to divide. The timing of nuclear division was variable. Generally, it did not occur until division of the kinetoplast had begun, but occasionally binucleate individuals were observed before cell or kinetoplast division was apparent. As division continued, 1 nucleus migrated past the dividing kinetoplast into the future daughter trypanosome. Finally, the kinetoplast completed division and the trypanosomes separated. Cell division was unequal, with the daughter trypanosome being smaller than the parent and with a more weakly developed undulating membrane.     

Intertidal recruitment and feeding in relation to optimal utilization of nursery areas by juvenile English sole (Parophrys vetulus: Pleuronectidae)

Synopsis Juvenile Parophrys vetulus 19–102 mm were collected on an intertidal flat in Humboldt Bay during 1976–1977. Recently metamorphosed fish that fed almost exclusively on harpacticoid copepods and other epibenthic crustaceans were found in large numbers during spring and early summer. A feeding transition occurred among fish 50–65 mm in length, and infaunal polychaetes were the dominant prey of fish greater than 65 mm. Increases in the feeding niche width and average size of prey items, as well as a decrease in the number of prey items per stomach, accompanied growth.The disappearance of English sole from intertidal areas in early fall at an average size of 82 mm and their subsequent residence in subtidal channels until a size of 140 mm suggest a size-depth segregation within the nursery ground. The advantages of such a distribution are discussed in terms of optimal foraging and reduction of intra- and interspecific competition.     

Epizootic characteristics of hepatic and renal lesions in English sole, Parophrys vetulus, from Puget Sound

English sole, Parophrys vetulus , from eight locations within Puget Sound, U.S.A. were examined for histological abnormalities of the liver and kidney. A total of 1083 fish were collected between February 1979 and June 1984 in nine separate sampling periods. Idiopathic lesions, i.e. lesions of unknown cause, were classified into eight hepatic and four renal lesion categories. The significance of certain potential risk factors associated with lesion prevalences, including age, gender, site of capture, season of capture and year of capture, were assessed by logistic regression. The odds ratios for the significant risk factors were also calculated. Site of capture and age of the examined fish were found to be the most important factors for most of the observed lesion types, e.g., hepatic neoplasms and foci of cellular alteration, whereas year and season of capture were significant influences for only a few lesion types, e.g., specific and nonspecific degenerative/necrotic lesions of the liver. Gender of examined fish was not found to be a significant factor for any of the lesion types. These results emphasize the importance of including certain potential influences, such as age and season of capture, in the analysis of field-collected organ pathology data.     

A new species of marine leech (Hirudinea: Piscicolidae) from the north-eastern Pacific Ocean, parasitic on the English sole, Parophrys vetulus Girard

The marine piscicolid leech Calliobdella knightjonesi sp. nov. from the Oregon coast is described and illustrated. Mature individuals do not exceed 10 mm in length, have faint reddish-brown segmental pigment bands, small pulsatile vesicles, six pairs of testisacs and a well-developed seminal receptacle connected to the posterior portion of the bursa by paired tissue strands. The leech lacks eyes and ocelli.     

Xenobiotic metabolizing enzymes in spawning English sole (Parophrys vetulus) exposed to organic-solvent extracts of marine sediments from contaminated and reference areas

Female English sole (Parophrys vetulus) within 1-2 days of spawning were exposed by i.m. injection to organic-solvent extracts of marine sediments at the following doses: Eagle Harbor (EHSE, contaminated site)--6.8 mg aromatic hydrocarbons (AHs)/kg body wt; Duwamish Waterway (DSE, contaminated site)--0.52 mg AHs and 0.040 mg chlorinated hydrocarbons (CHs)/kg body wt; Hood Canal (HCSE, reference site)--0.00090 mg AHs/kg body wt. Hepatic aryl hydrocarbon hydroxylase (AHH) activity, measured at spawning, was induced 10-, 23-and 2-fold by EHSE, DSE and HCSE, respectively, compared to sham and vehicle controls. Hepatic glutathione-S-transferase and epoxide hydrolase activities were not affected by any treatment. EHSE, but not DSE or HCSE, inhibited spawning (P less than 0.01) in 36% of the exposed fish and hepatic AHH activity in the non-spawning fish was significantly (P less than 0.05) higher than in the fish that did spawn. These results suggest a potential for reproductive toxicity in benthic fish after exposure to sediment-associated contaminants.     

Isolation and characterization of four major neutral glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel branched lacto-ganglio-iso-globo hybrid structure

We have previously reported on carbohydrate structures of the major acidic glycosphingolipids from the liver of the English sole, Parophrys vetulus (Ostrander, G. K., Levery, S. B., Hakomori, S., and Holmes, E. H. (1988) J. Biol. Chem. 263, 3103-3110). We have now isolated four major neutral glycosphingolipids from English sole liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, and by fast atom bombardment-mass spectrometry. In addition to neutral glycosphingolipids with known structures (CMH, lactosylceramide, and Gg3), a major polar neutral glycosphingolipid was isolated and characterized as having the following novel structure: (formula; see text) The compound represents a novel hybrid of neolacto-, ganglio-, and iso-globo-series glycosphingolipid structures, a combination not previously encountered. Furthermore, the linkage Fuc alpha 1----3GalNac has not been previously reported for a glycosphingolipid. The relationship of these structural elements to known glycoconjugates is discussed.     

Isolation and characterization of the major acidic glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel ganglioside with a Forssman antigen determinant

Acidic glycosphingolipids of the liver of English sole, Parophrys vetulus, have been isolated and characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, and by direct probe electron-impact and fast atom bombardment mass spectrometry. In addition to the acidic glycosphingolipids with known structures (sulfatide, GM4, GM3, GM2, and GD1a), two fractions of a major monosialosylganglioside with TLC mobility slower than GM1 were isolated and characterized as having the following structure. (Formula:q see text). The structure represents a novel combination of a terminal Forssman disaccharide (GalNAc alpha 1----3GalNAc beta 1----3R) and a GM1 ganglioside core linked together. The identity of the terminal Forssman disaccharide was further established by TLC immunostaining with an anti-Forssman monoclonal antibody. This antibody showed strongly positive staining of the ganglioside only after removal of the sialic acid. Thus, the II3NeuAc residue inhibited antibody binding to the terminal disaccharide unit. Analysis of the ceramide moieties of both fractions indicated a predominance of 16:0, 22:1, 22:0, and 24:1 fatty acids in the faster migrating form and 16:0, 18:0, and 18:1 in the slower form in combination with d18:1 sphingosine.     

Optimization of assay conditions for, and the selected tissue distribution of, alanine aminotransferase and aspartate aminotransferase of English sole, Parophrys vetulus Girard

Optimization of assay conditions for serum alanine aminotransferase (ALAT, GPT) and aspartate aminotransferase (ASAT, GOT) are described for English sole, Parophrys vetulus . Although most conditions for measurement of these fish enzymes parallel conditions in mammals, the optimal pH is lower for both these enzymes. Liver represents a potentially large source of ASAT activity, potential sources for ALAT activity are liver and kidney. As in mammals, the use of these enzymes as indicators of fish liver and kidney dysfunction may be warranted.     

Molecular detection and phylogenetic placement of a microsporidian from English sole (Pleuronectes vetulus) affected by X-cell pseudotumors

Flatfish tissue samples exhibiting X-cell pseudotumors were tested with a number of ribosomal DNA (rDNA) general primers in polymerase chain reactions (PCRs). Microsporidian primers resulted in the amplification of an rDNA fragment and molecular phylogenetic analysis indicated that although the organism did not relate closely with any current microsporidian genera, it was most similar to Nucleospora salmonis and branched within the Enterocytozoonidae. Re-examination of the original tissues used for DNA extractions revealed the presence of putative microsporidian spores in PCR-positive samples. These observations reiterate the highly sensitive diagnostic feature of PCR, allowing detection of organisms overlooked by conventional methods and demonstrate the occurrence of rare, coinfecting organisms.     

Protective effects of selenium-glutathione-enriched probiotics on CCl4-induced liver fibrosis

Hepatic fibrosis is a common pathological basis of liver cirrhosis and hepatocellular carcinomas. So, prevention and treatment of liver fibrosis is one of the crucial therapeutic goals in hepatology. Organic selenium, glutathione or probiotics supplementation could ameliorate hepatic fibrosis, respectively. The purpose of this study is to develop a novel selenium-glutathione-enriched probiotics (SGP) and to investigate its protective effect on CCl₄-induced liver fibrosis in rats. Yeast strains with the high-yield glutathione were isolated and identified by analysis of 26S ribosomal DNA sequences. The fermentation parameters of SGP were optimized through single-factor, Plackett–Burman (PB) design and response surface methodology (RSM). The final SGP contained 38.4 μg/g of organic selenium, 34.1 mg/g of intracellular glutathione, approximately 1×10¹⁰ CFU/g live Saccharomyces cerevisiae and 1×10¹² CFU/g live Lactobacillus acidophilus. SGP had better protective effects on liver fibrosis than selenium, glutathione or probiotics, respectively. The hepatic silent information regulator 1 (SIRT1) level was down-regulated and oxidative stress, endoplasmic reticulum (ER) stress, inflammation and phosphorylated MAPK was increased in CCl₄-treated rats. However, SGP can significantly reverse these changes caused by CCl₄. Our findings suggest that SGP was effective in attenuating liver fibrosis by the activation of SIRT1 signaling and attenuating hepatic oxidative stress, ER stress, inflammation and MAPK signaling.     

Mathematical modelling of liver regeneration after intoxication with CCl(4)

Liver regeneration is a complex process, having evolved to protect animals from the consequences of liver loss caused by food toxins. In this study, we established a mathematical spatial-temporal model of the liver lobule regenerating after CCl(4) intoxication. The aim of modelling the regeneration process by matching experimental observations with those from a mathematical model is to gain a better understanding of the process and to recognize which parameters are relevant for specific phenomena. In order to set up a realistic minimal model, we first reconstructed a schematised liver lobule after determination of: (i) the mean number of hepatocytes between the central vein and the periphery of the lobule, (ii) the mean size of the hepatocytes and (iii) the mean number of hepatocyte columns in the inner, midzonal and peripheral ring of the lobule. In a next step, we determined the time course of cell death and BrdU incorporation after intoxication of male Sprague Dawley rats with CCl(4), thereby differentiating between inner, midzonal and peripheral hepatocytes. These parameters were used to construct a model. The basic unit of this model is the individual cell. The detailed behaviour of the cells is studied, controlled by the model parameters: (1) probability of cell division at defined positions of the lobule at a given time, (2) "coordinated cell orientation", i.e., the ability of the cells to align during the regeneration process into columns towards the central vein of a liver lobule, (3) cell cycle duration, (4) the migration activity and (5) the polarity of the hepatocytes resulting in polar cell-cell adhesion between them. In a schematised lobule, the model shows that CCl(4) initially induced cell death of a pericentral ring of hepatocytes, followed by a wave of proliferation that starts in the surviving hepatocytes next to the inner ring of dead cells and continues to the peripheral hepatocytes, finally restoring the characteristic micro-architecture of the lobule in a 7-day process. This model was used to systematically analyze the influence of parameters 1-5. Interestingly, coordinated cell orientation and cell polarity were identified to be the most critical parameters. Elimination led to destruction of the characteristic micro-architecture of the lobule and to a high degree of disorder characterized by hexagonal cell structures. Our model suggests that the ability of hepatocytes to realign after cell division by a process of coordinated cell orientation (model parameter 2) in combination with cell polarity (model parameter 5) may be at least as critical as hepatocyte proliferation (model parameter 1) itself.     

Effect of Asteracantha longifolia Nees. against CCl4 induced liver dysfunction in rat

Significant recovery after treatment with the whole plant slurry of A.longifolia Nees. was observed in plasma AST, ALT and cholesterol levels in CCl4 induced hepatotoxic rats. This was amply supported by electron micrographs, which indicated normalization of cytoarchitecture of mitochondria and endoplasmic reticulum. The results suggest that the slurry of the plant is useful as a liver tonic.