Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na+/K+ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions.     

Accumulation of zinc in rainbow trout (Oncorhynchus mykiss) after waterborne and dietary exposure

An acromegalic patient with nontoxic autonomous goiter was sequentially treated with octreotide and bromocriptine. Before therapy, serum GH, PRL and insulin-like growth factor-I (IGF-I) levels were increased. Free T3 and free T4 were within the normal range with suppressed TSH levels, whereas 123Iodine-uptake of thyroid was 5.6% after 24 h. During treatment with octreotide and bromocriptine, serum GH, PRL, and IGF-I became normal and free T3 and free T4 were slightly but significantly decreased, but TSH levels remained very low. After thyroidectomy, thyroglobulin, free T3 and free T4 were further decreased, and the TSH levels were recovered to normal. These findings suggested that octreotide and bromocriptine inhibit the release of thyroid hormones from the autonomous thyroid gland directly or indirectly through the decline in IGF-I.     

Characterization of biliary metabolites of 4-n-nonylphenol in rainbow trout (Oncorhynchus mykiss)

1. [R-2,6-3H]-4-n-nonylphenol was synthesized and a single dose (5 mg, 1850 KBq) orally administered to rainbow trout. After 48 h, the radioactivity present in the bile amounted 5.5%. More than ten biliary metabolites were separated by hplc and collected for subsequent mass spectrometry analysis. The metabolic profile was totally modified by beta-glucuronidase hydrolysis, showing that most of the metabolites were glucuronic acid conjugates. 2. Conjugated metabolites were identified by lc-ms analysis and their aglycones were analysed by gc-ms analysis as TMS and acetyl derivatives. 3. The major metabolite accounted for 52+/-11% of the biliary radioactivity and was identified as nonylphenol-glucuronide. 4. Nonylphenol was hydroxylated at both omega and omega-1 positions of the alkyl chain, giving 9-hydroxynonylphenol and 8-hydroxynonylphenol. 5. 9-Hydroxynonylphenol was oxidized to the corresponding acid, and subsequently beta-oxidized, yielding 7-(4-hydroxyphenyl)heptanoic acid, 5-(4-hydroxyphenyl)pentanoic acid, 3-(4-hydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)-2-propenoic acid.     

Glucose dehydrogenase in beef (Bos taurus) and rainbow trout (Oncorhynchus mykiss) liver: a comparative study

The monomer molecular mass of glucose dehydrogenase (GDH, EC 1.1.1.47) from rainbow trout liver and beef liver were estimated to be 90 kDa for both enzymes, by electrophoresis in the presence of Na-dodecyl-SO4 (SDS). The 90-kDa proteins were partially degraded to about 60 kDa when purified with a delayed procedure without protease inhibitors. Tryptic cleavage of the 90-kDa proteins gave fragments of about 60 kDa and 30 kDa, being similar for trout and beef GDH. Isoelectric points, kinetic and thermodynamic properties of the two enzymes are markedly different. Triton X-100 stimulated and stabilized the reactions catalysed by the purified enzymes.     

Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss)

The distribution of glucocorticoid receptor-expressing cells was studied in the forebrain of the rainbow trout by means of antibodies produced against a fusion protein made of the NH2-terminal fragment of the rainbow trout glucocorticoid receptor fused in frame with glutathione-S-transferase. The results indicate that glucocorticoid receptor-expressing cells are located in many brain regions from the telencephalon to the spinal cord, with the highest density in the neuroendocrine component of the brain, the preoptic region and the mediobasal hypothalamus, and in the periventricular zone of the optic tectum. In virtually all cases, the labeling was located in the nucleus of the cells, although on very rare occasions, a slight labeling of the cytoplasm was detected. Concerning the preoptic region, the most striking feature was the high density of glucocorticoid receptors in the magnocellular preoptic nucleus, known to contain corticotrophin-releasing factor (CRF)-, vasotocin-, and isotocin-expressing cells. Colocalization experiments showed that 100% of the CRF-immunoreactive neurons in the preoptic nucleus express glucocorticoid receptors. In the mediobasal hypothalamus, the highest expression was found in the nucleus lateralis tuberis and parts of the nucleus recessus lateralis. Concerning the pituitary, the glucocorticoid receptor was consistently found in the rostral pars distalis, with the exception of the prolactin cells, and in the proximal pars distalis, which in trout contains thyrotrophs, gonadotrophs, and somatotrophs. In the hindbrain, expression of glucocorticoid receptors were localized mainly in the periventricular regions.     

Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide

The sensitivity of Vibrio anguillarum to the bactericidal effect of rainbow trout serum was investigated with different strains of serogroups O1 and O2a, which are the most frequently found serogroups in clinical outbreaks of vibriosis. All of the V. anguillarum strains were able to activate complement in rainbow trout serum, but smooth strains of V. anguillarum serogroup O1 were resistant to complement-mediated killing in the absence of specific antibodies. In the case of V. anguillarum serogroup O2a strains, 80% of the analyzed strains were resistant to rainbow trout serum even when specific antibodies were present. Analysis of the lipopolysaccharide structures of the tested V. anguillarum strains showed a positive correlation between the O-antigen size of the lipopolysaccharide and resistance to serum killing. The classical complement pathway was responsible for the antibody-dependent serum killing of susceptible V. anguillarum strains. When serum-resistant V. anguillarum serogroup O2a strains were grown in glucose-enriched Lennox L broth, they produced lipopolysaccharide molecules with fewer high-molecular-weight O-antigen units than did strains grown in broth without the addition of glucose. Strains grown in glucose-enriched medium became sensitive to rainbow trout serum killing, indicating that the high-molecular-weight O-antigen side chains prevented the activated complement from damaging the bacterium.     

Pigmentation of the rainbow trout (Oncorhynchus mykiss) with oil-extracted astaxanthin from the langostilla (Pleuroncodes planipes)

The purpose of this contribution is to inform about the activities of the local ethics commission established at one of our largest teaching (University) hospitals. In 1995 the following priorities were recognized: studying international ethical standards of research involving human subjects and strictly adhering to them; bilingual administrative agenda in Czech and English; enforcing the patients' rights in health care and emphasizing them in education of medics and other students of health related disciplines; stimulating effective communication between doctors, other health care workers and lay persons in the commission as well as between the members of the commission and authors of research projects applying for ethical evaluation. Since 1993 the commission has been revising and storing in the archives more than 600 research projects. In 1996, 181 new research projects were reviewed, which is 35% more than in 1995. Approval was granted in 154 cases (85.08%), 3 cases (1.66%) were rejected, 15 cases (8.29%) approved on condition of complying with the recommendations of the commission, 6 cases (3.31%) recommended for another commission, and in 3 cases reviewing the project was postponed for lack of necessary data. The members of the commission agree that the future activities should lead to forming a broader group of consultants of the commission and other professional and lay people interested in clinical ethics. According to their meaning, legislation concerning ethical commissions in the Czech Republic, including material and financial support and administrative help, is urgently needed.     

CYP4T1--a cytochrome P450 expressed in rainbow trout (Oncorhynchus mykiss) liver

Total RNA isolated from a rainbow trout (Oncorhynchus mykiss) liver was subjected to RT/PCR using degenerate primers designed from homologous regions amongst cytochrome P450 CYP4 proteins. PCR amplification resulted in a single electrophoretic band which was excised, purified and sequenced directly, using cycle sequencing. The deduced protein sequence demonstrated the closest amino acid identity to rabbit CYP4B1 (54.6%) and rat CYP4B2 (55.4%). Phylogenic analysis of this sequence was found to be significantly different to any other CYP4 sequence and has been named CYP4T1. This represents the first CYP4 family member to be identified in an aquatic vertebrate.     

Reproductive success of female rainbow trout (Oncorhynchus mykiss) in response to graded dietary ascorbyl monophosphate levels

Ascorbic acid is an essential nutrient in rainbow trout diets and has been shown to play an important role in fish reproduction. Recommended dietary levels are based on immature fish, and the specific requirements for brood stock are unknown. To establish the optimum dietary level for mature rainbow trout, six graded levels of ascorbyl-2-monophosphate were fed to groups of female fish over a period of 10 mo until spawning. Increasing dietary levels of ascorbyl monophosphate resulted in significantly increased ascorbic acid concentrations in liver, kidney, ovaries, and ovulated eggs. Liver and egg concentrations were saturable at 109.3 and 266.6 micrograms ascorbic acid/g tissue, respectively. Tissue saturation levels of 83.7% and 91.2%, respectively, were reached at the highest dietary level (870 mg ascorbyl monophosphate/kg diet) tested. Both fecundity and embryo survival increased significantly with dietary ascorbyl monophosphate levels. The results indicated that the present National Research Council recommended dietary level of 50 mg ascorbic acid/kg diet for rainbow trout is inadequate for brood stock fish. An amount 8 times higher is necessary to optimize tissue ascorbic acid levels and achieve maximum reproductive success.     

Toxicological and kinetic study of musk xylene in rainbow trout, Oncorhynchus mykiss

In a prospective study of 6301 surgical patients in a university hospital, we examined the strength of association between ASA physical status classification and perioperative risk factors, and postoperative outcome, using both univariate analysis and calculation of the odds ratio of the risk of developing a postoperative complication by means of a logistic regression model. Univariate analysis showed a significant correlation (P < 0.05) between ASA class and perioperative variables (intraoperative blood loss, duration of postoperative ventilation and duration of intensive care stay), postoperative complications and mortality rate. Univariate analysis of individual preoperative risk factors demonstrated their importance in the development of postoperative complications in the related organ systems. Estimating the increased risk odds ratio for single variables, we found that the risk of complication was influenced mainly by ASA class IV (risk odds ratio = 4.2) and ASA class III (risk odds ratio = 2.2). We conclude that ASA physical status classification was a predictor of postoperative outcome.     

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.     

Hepatic cellular distribution of cytochrome P-450 IA1 in rainbow trout (Oncorhynchus mykiss): an immunohisto- and cytochemical study

Monoclonal antibody 1-12-3 reactive against scup (Stenotomus chrysops) cytochrome P450 E (a teleost CYP IA1) has been used to immunohistochemically localize CYP IA1 within hepatocytes and presumably sinusoidal endothelial and biliary epithelial cells of scup and trout. The goal of the present study was to extend immunohistochemical studies to the ultrastructural level determining intracellular locations of CYP IA1 in fish liver. Juvenile trout (5-10 g) were given i.p. injections once (50 micrograms/g b beta-naphthoflavone in cod liver oil; 0.5-ml injectate volume). After 5 days, livers were fixed (0.25% glutaraldehyde) via vascular in situ perfusion, removed, cut in 100-microns slices, infiltrated, and embedded in LR White monomer. Ultrathin sections of exposed livers were incubated in monoclonal antibody 1-12-3, rabbit anti-mouse IgG, and protein G colloidal gold. Membranes of granular endoplasmic reticulum in perinuclear regions of hepatocytes were consistently labeled. In addition, hepatocyte plasma membrane, particularly microvilli at bile canaliculi, was labeled. Biliary epithelial cells were labeled on luminal plasma membrane surrounding biliary passageway. Plasma membrane facing sinusoid and immediately subjacent cytoplasm was labeled in endothelial cells. Presence of CYP IA1 in sinusoidal endothelium could contribute to detoxication and/or bioactivation of blood borne chemicals. Granular endoplasmic reticulum was not uniformly labeled in hepatocytes. Rather, distribution seemed sequestered within highly specific regions and not dispersed along all membrane surfaces. Localization within biliary epithelial cells could signify potential of this cell type to bioactivate polycyclic aromatic hydrocarbons and may explain the common finding of biliary as well as hepatocytic tumors of trout liver.     

Oxygen consumption of juvenile rainbow trout (Oncorhynchus mykiss) exposed to sublethal concentrations of 1,2,4,5-tetrachlorobenzene and tetrachloroguaiacol

Dietary intake of highly polyunsaturated fats represents a major source of lipid hydroperoxides in the intestinal lumen. Under conditions of high peroxide intake, excessive concentrations of lipid hydroperoxides can persist in the gut lumen and contribute to impairment of mucosal GSH-dependent detoxication pathways, enterocyte dysfunction independent of cell injury, and development of gut pathologies, including cancer. This paper summarizes our current knowledge of the determinants of intestinal lipid hydroperoxide metabolism and of the physiological and biochemical processes in lipid peroxide-mediated changes in intestinal redox status, regulation of mucosal thiol and antioxidant balance and control of intestinal cell turnover. This discussion is pertinent to understanding dietary peroxides and thiol redox balance in intestinal physiology and pathophysiology and the potential benefit of oral GSH in preserving metabolic integrity of the intestinal epithelium.     

Direct effects of the pineal hormone melatonin on testosterone synthesis of Leydig cells in Djungarian hamsters (Phodopus sungorus) in vitro

Leydig cells of adult Djungarian hamsters, stimulated with luteinizing hormone (LH), were co-incubated with melatonin at various concentrations in a primary culture system. Testosterone secretion was only affected by melatonin when cells were stimulated with LH. Maximal suppression was observed at low doses of LH (0.5 ng/ml). These effects are at least partially mediated through the adenylate cyclase system, since melatonin was able to reduce forskolin-stimulated testosterone secretion. These results indicate that the time between pulses of LH can be considered to be most highly effective for tonic melatonin actions.     

Purification and partial characterization of 76 kDa transglutaminase in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss

Transglutaminase (TGase), responsible for crosslinking between proteins, is known to be localized exclusively in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss, and probably participates in the post-fertilization chorion hardening. We purified the TGase from unfertilized egg chorions by sequential chromatography using SP-Sepharose, Q-Sepharose, and TSK-gel G3000SWXL columns. The purified enzyme was a monomeric protein having the molecular mass of 76 kDa. It promoted incorporation of monodansyl-cadaverine into chorion protein and catalyzed the polymerization of chorion subunit proteins. The effect of various reagents suggested that the chorion TGase is a Ca2+-dependent SH-enzyme similar to the well-characterized TGases of various animals. The highest activity was observed at pH 6.0. The amines examined in the present study inhibited the TGase activity of the purified enzyme. However, they did not necessarily cause effective inhibition of its activity. These properties of the chorion TGase were essentially consistent with our previous observations on polymerization of chorion proteins, resulting in chorion hardening. We compared the amino acid composition of the purified TGase with those of the previously characterized TGases of fishes, such as chum salmon and red sea bream. The results suggest that the chorion 76 kDa TGase is not homologous with those liver TGases in terms of amino acid composition.     

Regulation of the intracellular concentration of free calcium ions in pinealocytes of the rainbow trout and the rat

Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout pinealocytes and in rat pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout pinealocyte. In rat pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in pinealocytes are complex and differ considerably between poikilothermic and mammalian species.     

Effect of a supplemental Aspergillus niger phytase on the utilization of plant phosphorus by rainbow trout (Oncorhynchus mykiss)

Effects of a supplemental Aspergillus niger-phytase on digestibility and utilization of dietary phosphorus (P) were studied in three experiments with rainbow trout. P concentration in the diets was 4.8 and 5.8 g/kg DM, respectively. The P contained in the diet originated solely from plants, mainly soy-products. Digestibility of P was studied using the stripping method and hydrochloride insoluble ash as marker. Utilization was studied in growth trials by use of the comparative body analysis. At a water temperature of 15 degrees C, both digestibility and utilization of P were increased from 25 to 57% and from 17 to 49%, respectively when 1000 U/kg phytase were supplemented. Feed consumption and gain of trout were significantly increased. At a water temperature of 10 degrees C, utilization of P was also increased from 6 to 25%. However, feed consumption and gain of trout were very low at this water temperature and not influenced by the supplemental phytase.     

Evaluation of the critical body burden concept based on inorganic and organic mercury toxicity to rainbow trout (Oncorhynchus mykiss)

Subadult rainbow trout (Oncorhynchus mykiss) were exposed to four waterborne concentrations each of 64-426 microg/L mercuric chloride (HgCl2) and 4-34 microg/L methylmercury chloride (CH3HgCl) until death to evaluate the critical body burden concept. Mean days to death for fish exposed to the highest and lowest concentrations of HgCl2 were 1 and 58 d, and 2 and > 100 d for fish exposed to CH3HgCl. Time to death was an important factor that influenced Hg tissue concentration, and was most evident among fish that died within a few days of exposure. Critical body burdens for Hg could be difficult to establish at the tissue level because no threshold concentrations were clearly indicated among the liver, kidney, spleen, brain, muscle, and gill that were monitored in this study. A critical burden for Hg was derived on a whole body basis for Hg in its organic form. An evaluation of this and other studies suggests whole body concentrations of 10-20 mg/kg Hg could be lethal to fish. Extrapolation from other studies indicate whole body concentrations of 1-5 mg/kg Hg could have chronic effects on fish and possibly other aquatic organisms. This concept could be used to assess the toxicological significance of chemical concentrations that are monitored in feral aquatic organisms. The tissue-based approach appears to have some advantages over current assessment protocols that focus on waterborne concentrations.