The physiological stress response and oxidative stress biomarkers in rainbow trout and brook trout from selenium-impacted streams in a coal mining region.

Selenium (Se) is an essential element that can be toxic at concentrations slightly greater than those required for homeostasis. The main chronic toxic effects of Se in fish are teratogenic deformities, but Se can also activate the physiological stress response and redox cycle with reduced glutathione causing oxidative damage. Rainbow trout, Oncorhynchus mykiss, appear to be more sensitive to Se than brook trout, Salvelinus fontinalis. The objective of this study was to compare the physiological stress response (plasma cortisol, glucose, triiodothyronine, thyroxine, gill Na+/K+ ATPase, cortisol secretory capacity, K and liver somatic index) and oxidative stress biomarkers (liver GSH, GPx, lipid peroxidation, vitamin A and vitamin E) in rainbow trout (RNTR) and brook trout (BKTR) collected from reference and Se-exposed streams. The physiological stress response was not impaired (cortisol secretory capacity unchanged); although there were species differences in plasma cortisol and plasma glucose levels. Liver GSH, GPx and vitamin levels were higher in RNTR than BKTR, but lipid peroxidation levels were not different. The elevated GSH reserves may make RNTR more sensitive to Se-induced lipid peroxidation, but this may be offset by the RNTR's higher antioxidant (GPx and vitamin) levels. Species-specific biochemical differences may mediate differences in Se sensitivity and be used in aquatic Se risk assessments.

Homeoviscous adaptation and thermal compensation of sodium pump of trout erythrocytes.

The effects of thermal acclimation of trout on the transport activity and turnover number of the erythrocyte Na+ pump have been determined. Na+ pump activity was estimated by measuring the ouabain-sensitive K+ influx in Na(+)-loaded cells and the number of active pumps determined by Scatchard analysis of [3H]ouabain binding and by correlation of ouabain binding with pump inhibition. Cold acclimation was associated with an increase in pump activity of up to 60%, although the furosemide-sensitive and residual fluxes were unaffected. The number of ouabain binding sites was similar in both acclimation groups at approximately 21,000-23,000 sites/cell. This means that cold acclimation induced an increase in the transport turnover number of pump molecules from approximately 6 to 9 s-1. Cold acclimation was also associated with a decrease in membrane order as indicated by steady-state fluorescence polarization of the membrane probe, 1,3-diphenyl-1,3,5-hexatriene, with a homeoviscous efficacy of 25-41%. That membrane order may influence pump transport activity is supported by experiments on cholesterol supplementation, which caused both an increase in membrane order and a decrease in pump turnover number. The degree of pump compensation was dependent on the season, with greatest responses in the late spring and declining responses through to winter. By contrast, changes in membrane order were observed throughout the year. Expression of pump activity and erythropoiesis may vary throughout the seasonal cycle in complex ways that confuse the direct comparison study of cellular properties in a heterogeneous population of cells.

The influence of repeated stress on the release of melanin-concentrating hormone in the rainbow trout.

Melanin-concentrating hormone (MCH) is a neurohypophysial peptide that induces pigmentary pallor in teleosts and which is released when the fish are placed on a white background. An additional effect of the peptide is the depression of ACTH and hence cortisol secretion during moderate stress. The present work on rainbow trout shows that plasma MCH concentrations, while unaffected by a single stress, are raised by repeated stress (1 ml saline injected i.p. without anaesthesia) and remain high for several hours thereafter. The response to stress is observed only in white-adapted fish and not in fish kept in black-coloured tanks, when MCH release is normally low. Plasma concentrations of MCH vary diurnally but stress induces an equivalent incremental rise in plasma MCH, whether administered in the middle or towards the end of the photophase. The stress-induced rise in MCH concentrations is prevented by treatment with dexamethasone. The results support the suggestion that the modulatory effect of MCH on the hypothalamopituitary-interrenal axis of fish might be enhanced under conditions of stress.

Effects of recombinant human growth hormone and insulin-like growth factor 1 on body growth and blood metabolites in brook trout (Salvelinus fontinalis).

Groups of juvenile brook trout (Salvelinus fontinalis) were acclimated to 12.0-13.0 degrees dechlorified water and a photoperiod of 12 hr light: 12 hr dark. Recombinant human growth hormone (hGH) (10.0 micrograms/g body wt) or insulin-like growth factor 1 (hIGF-1), used in a wide range of dosages (0.001-10.0 micrograms/g body wt), were given weekly as intramuscular injections. The fish receiving hGH were already significantly heavier and longer than the saline-injected control fish after 3 weeks of treatment. In addition, a liver specific growth promoting effect of hGH was found. In contrast, hIGF-1 did not stimulate body growth in any dosage tested. The fish receiving the highest dosages of hIGF-1 were all seriously affected with retarded body growth and high mortality. A possible insulin-like activity of hIGF-1 was verified by measuring the plasma glucose and amino acid levels in brook trout after a single injection of hIGF-1 (2.0 micrograms/g body wt) or bovine insulin (0.01 IU/g body wt). Both hormones caused a reduction in both glucose and amino acid levels to 35% of the control levels 24-72 hr after injection. The results strongly suggest that hIGF-1 does not stimulate growth, but that in high dosages causes profound insulin-like effects in brook trout resulting in hypoglycemia and hypoaminoacidemia.

A new fluorescent test for cell vitality using calcofluor white M2R.

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.

Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta).

Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea trout (Salmo trutta) by estradiol-17 beta. A purification procedure for vitellogenin from trout plasma by precipitation with MgCl2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.63% in rainbow trout and 0.58% in sea trout vitellogenin. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.

Trout red blood cells treated with proteases fuse when placed on glass slides.

Treatment of trout red blood cells (RBC) with proteases and polyethylene glycol (PEG) either successively or concurrently caused cell fusion. Neither PEG nor protease treatment alone brought about the fusion of cells in suspension. However, incubation of RBC on glass slides with proteases caused extensive fusion.

Blood acid-base regulation during environmental hyperoxia in the rainbow trout (Salmo gairdneri).

Blood acid-base balance, blood gases, respiration, ventilation, and renal function were studied in the rainbow trout during and following sustained environmental hyperoxia (PIO2 = 3.50-650 Torr). Animals were chronically fitted with dorsal aortic cannulae for repetitive blood sampling, oral membranes for the measurement of ventilation, and bladder catheters for continuous urine collection. Hyperoxia caused a proportional increase in arterial O2 tension and a stable 60% reduction in ventilation volume (Vw), the latter mainly due to a decrease in ventilatory stroke volume. O2 consumption exhibited a short-term elevation. Arterial CO2 tension (PaCO2) rose within 1 h, causing an immediate drop in arterial pH (pHa), and continued to increase gradually thereafter, reaching a value 2-4x the normoxic control level after 96-192 h. Compensation of the associated acidosis by the accumulation of [HCO3-] in the blood plasma started within 5-6 h, and was complete by 48 h. Therefore, further compensation occurred simultaneously with the gradual rise in PaCO2. The kidney played an important active role in this compensation by preventing excretion of the accumulated [HCO3-]. Upon reinstitution of normoxia, PaCO2 dropped to control levels within 1 h, and restoration of blood acid-base status by reduction of [HCO3-] had commenced by this time. A complete return to control values occurred within 20 h. During hyperoxia, an experimental elevation of the depressed Vw above control normoxic levels caused only a minor and transient reduction in PaCO2 and no change in pHa, but injection of branchial vasodilator 1-isoprenaline (10 mumol/kg) produced a large drop in PaCO2 and rise in pHa. It is concluded that the rise in PaCO2 during hyperoxia is mainly due to internal diffusive and/or perfusive limitation associated with branchial vasoconstriction, rather than to external convective limitation associated with the decreased Vw.