Modulation of Ah receptor and CYP1A1 expression by alpha-naphthoflavone in rainbow trout hepatocytes

The objective of this study was to evaluate whether alpha-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout (Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10(-9)-10(-5) M), while beta-naphthoflavone (BNF 10(-10)-10(-6) M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased ( approximately 50%) BNF-induced AhR protein expression (only at 10(-9) M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10(-7) M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.     

Toxic effect of heavy metals on ovarian deformities,apoptotic changes,oxidative stress,and steroid hormones in rainbow trout

BackgroundAs is well known, the pollution in the aquatic environment in which fish grow has a direct impact on aquaculture practices. Pollution in aquatic systems because of multiple adverse effects on fish metabolic processes, especially the reproductive systems.AimThe goal of this study was to assess the severity of pollution impact in two different hatcheries, Verinag hatchery, Site 1 (S1) and Panzath hatchery, Site 2 (S2) in Anantnag region, using histopathological, ultrastructural, oxidative stress, genotoxic, and hormonal analysis in rainbow trout gonad (ovary).M&M: Fish were collected between May 2018 and April 2019 from two locations, Verinag hatchery (S1) and Panzath hatchery (S2), which were affected by heavy metals.ResultsThe histological and ultrastructural examination of rainbow trout ovaries from the Verinag hatchery (S1) revealed normal structure in growing oocytes in rainbow trout at various stages based on morphological features while the fish ovaries in the Panzath hatchery (S2) showed various deformities and irregularly shaped oocytes. The surfaces of some of these oocytes were wrinkled, rough, or distorted. Apoptotic studies revealed that the frequency of apoptotic cells collected from S2 water was significantly increased in ovarian cells (P < 0.05). The activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were found to be increased in fish collected from S1 but decreased in fish collected from S2. In S2 caught fish, malondialdehyde (MDA) levels were found to increase gradually, and the degree of heavy metal stress was positively correlated (p < 0.05). The comet assay was used to determine the induction of DNA damage in ovarian cells. The induction of DNA damage was found to be significantly higher (p < 0.05) in S2 fish specimens compared to fish from S1. On comparing the DNA damage of the rainbow trout from the two sampling sites, it was revealed that the fish is much more sensitive to aquatic contaminants. Regarding steroid hormones, higher levels of progesterone and estrogen were reported in the fish samples collected from S1 as compared to S2 captured fish.ConclusionIn conclusion, the comparative study of fish from two different sites viz. Verinag hatchery (S1) and Panzath hatchery (S2) revealed that S2 sampled fish suffered more heavy metal damage, including cellular deformities, apoptosis, oxidative damage, and altered steroid hormones.     

Diurnal rhythms of plasma growth hormone,somatostatin, thyroid hormones,cortisol and glucose concentrations in rainbow trout,Oncorhynchus mykiss,during progressive food deprivation

Abstract

The diurnal patterns of changes in plasma cortisol, growth hormone (GH), somatostatin‐14 (SRIF), thyroid hormones (L‐thyroxine, T₄ and triiodo‐L‐thyronine, T₃) and glucose were investigated in rainbow trout, Oncorhynchus mykiss, both during single meal‐feeding and during a progressive fast of 13 weeks. All measured variables exhibited a diurnal pattern in fed rainbow trout, most of which appeared to be correlated with the time of feeding (30–60 min after the onset of light), while additional changes, associated with the scotophase were also found for cortisol. Although fasting had no affect on mean daily plasma cortisol or SRIF concentrations, there was a progressive increase in mean daily plasma GH concentrations and a progressive decrease in mean daily plasma thyroid hormone and glucose concentrations associated with fasting. However, for GH, significant changes were not evident until week 10 of the fast. In addition, fasting appeared to phase‐shift the diurnal patterns of plasma GH, cortisol and glucose concentrations; the consequence of such a shift is that conclusions as to the effects of fasting, if based on a single time of sampling, may be flawed.     

Modulation of catecholamine storage and release by the pituitary-interrenal axis in the rainbow trout,Oncorhynchus mykiss

This study examined the effects of pituitary-interrenal hormones on catecholamine storage and release in the rainbow trout Oncorhynchus mykiss. An extract of trout pituitary elicited the release of adrenaline, but not noradrenaline, using an in situ perfusion preparation. A variety of doses of adrenocorticotropic hormone (2–2000 mU) caused the release of both catecholamines in situ which was unaffected by pre-treatment with the ganglion blocker, hexamethonium, or the serotonergic receptor antagonist, methysergide, but was abolished in calcium-free media. Intra-arterial injections of adrenocorticotrophic hormone in vivo caused an elevation of plasma adrenaline but not noradrenaline levels. Injections of cortisol in situ did not elicit catecholamine release. Trout given an intraperitoneal implant of cortisol (50 mg·kg^-1 body weight) had significantly higher plasma cortisol concentrations when compared to controls after 7 days of implantation. Increases in the levels of stored catecholamines were observed in various regions of the kidney and posterior cardinal vein following 3 and 7 days of cortisol treatment. The ability of the chromaffin cells to release catecholamines in response to cholinergic stimulation was assessed in situ after 7 days of treatment. Basal (non-stimulated) adrenaline outflowing perfusate levels were greater in the cortisol-treated fish. Cortisol treatment increased the responsiveness of the catecholamine release process to low doses of the cholinoceptor agonist carbachol. Three or 7 days of cortisol treatment did not alter the in vitro activity of the enzyme phenylethanolamine-N-methyl-transferase. The results of this study demonstrate that interactions within the pituitary-adrenal axis can influence both catecholamine storage and release in the rainbow trout.Abbreviations ACTH adrenocorticotropic hormone - AK anterior third of the kidney - APCV anterior third of the PCV - HPLC high performance liquid chromatography - MK middle third of the kidney - M1 maximum value - MPCV middle third of the PCV - MS222 ethyl-aminobenzoate - P1 pre value - PCA perchloric acid - PCV posterior cardinal vein - PK posterior third of the kidney - PNMT phenylethanolamine-N-methyltransferase - PPCV posterior third of the PCV - rbc red blood cells - SEM standard error of the mean - TK total kidney (i.e. the sum of the AK, MK, and PK) - TPCV total PCV (i.e. the sum of the APCV, MPCV and PPCV)     

Effects of arginine on pancreatic hormones and hepatic metabolism in rainbow trout

Arginine (Arg), injected intraperitoneally into rainbow trout (Oncorhynchus mykiss), increases plasma concentrations of glucagon, glucagon-like peptide-1 (GLP-1), and insulin by three- to 10-fold. Resulting ratios of glucagon and GLP-1 over insulin are unchanged in 20-d food-deprived fish (saline, 1.28 vs. Arg, 0.93; not significant) while slightly increased in feeding trout (saline, 0.70 vs. Arg, 0.92; P<0.05). In food-deprived juveniles, Arg injection leads to significant decreases in plasma fatty acids (saline, 1.65 mM L(-1) vs. Arg, 1.09 mM L(-1); P<0.05) and increases in glycogen phosphorylase total activity (saline, 3.7 units g(-1) vs. Arg, 4.6 units g(-1); P<0.05) and degree of phosphorylation (saline, 1.7 units g(-1) vs. Arg, 2.33 units g(-1); P<0.05). Plasma and liver glucose and liver enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, phosphoenolpyruvate carboxykinase, lactate dehydrogenase, and malic enzyme) are unaffected. Otherwise, fish show the changes in plasma metabolites expected with food deprivation. Arg injection into feeding fish results in decreases in plasma fatty acids, liver glycogen, and glucose, while liver glucose 6-phosphate concentrations increase. Hepatocytes isolated from feeding fish injected with Arg 2 h previously show significantly lower rates of lactate oxidation than controls (85% of control), while rates of gluconeogenesis and hormonal responses to mammalian glucagon and GLP-1 remain unchanged. Rates of lactate oxidation and gluconeogenesis are significantly decreased by 5%-10% on treatment with porcine insulin. Complete immunoneutralization of insulin with rabbit antisalmon insulin serum decreases hepatic glucose 6-phosphate concentrations and abolishes the Arg-dependent effects on glycogen phosphorylase. It appears that short-term increases in pancreatic hormones cause only minor metabolic readjustments in the relatively short time frame covered in these experiments. Surprisingly, complete removal of insulin does not have immediate altering or detrimental effects on key metabolites and metabolic pathways, even if glucagon and GLP-1 concentrations are concurrently several-fold higher than usual. Our data clearly show the dual role of Arg in fish metabolism.     

Plasma cortisol concentrations before and after social stress in rainbow trout and brown trout

Two related experiments examined the relationship between plasma cortisol concentrations and the development of social hierarchies in fish. In the first, rainbow trout, Oncorhynchus mykiss, and brown trout, Salmo trutta, were observed for dominance interactions when confined within single-species pairs for 4, 48, or 168 h. Subordinate members of a pair exhibited significantly higher cortisol concentrations than dominant and single fish, but the pattern of cortisol elevation differed between the two species, being quicker to rise and increasing to a higher level in rainbow trout. Cortisol concentrations were correlated with behavioural measurements; the more subordinate the behaviour exhibited by a fish, the higher its cortisol concentration. Social stress was a chronic stressor, and no acclimation to social status occurred during the week. In the second experiment, measurements of plasma cortisol were made before pairing of rainbow trout and then after 48 h of confinement in pairs. Subordinate fish demonstrated significantly higher concentrations of plasma cortisol both before and after social stress. It therefore appears that in addition to cortisol being elevated during periods of social stress, an association may exist between initial cortisol levels and the likelihood of a fish becoming subordinate.     

Carbohydrate energy reserves and effects of food deprivation in male and female rainbow trout

We investigated the effects of nutritional state on carbohydrate, lipid, and protein stores in the heart, liver, and white skeletal muscle of male and female rainbow trout. For fed animals we also partitioned glycogen into fractions based on acid solubility. Fish (10-14 months-old, ~400-500 g) were held at 14 °C and either fed (1% of body weight, every other day) or deprived of food for 14 days. Under fed conditions, glycogen was increased 54% in ventricles from males compared with females, and elevated in the liver (87%) and white muscle (70%) in sexually-maturing versus immature males. Acid soluble glycogen predominated over the acid insoluble fraction in all tissues and was similar between sexes. Food deprivation 1) selectively reduced glycogen and free glucose in male ventricles by ~30%, and 2) did not change glycogen in the liver or white muscle, or triglyceride, protein or water levels in any tissues for both sexes. These data highlight sex differences in teleost cardiac stores and the metabolism of carbohydrates, and contrast with mammals where cardiac glycogen increases during fasting and acid insoluble glycogen is a significant fraction. Increased glycogen in the hearts of male rainbow trout appears to pre-empt sex-specific cardiac growth while storage of acid soluble glycogen may reflect a novel strategy for efficient synthesis and mobilization of glycogen in fishes.     

Exhaustive exercise and the cellular stress response in rainbow trout, Oncorhynchus mykiss

The goal of this study was to examine the cellular response to exhaustive exercise in male and female rainbow trout to determine if HSPs are involved in the early stages of the recovery process. Levels of HSPs and key metabolic parameters were measured in white muscle, heart plasma, and blood plasma throughout 6 h of recovery from exhaustive burst exercise. Plasma creatine kinase (CK) was also quantified as an indicator of exercise-induced tissue damage. The observed trends in ATP and lactate were consistent with established patterns of exhaustion and the beginnings of metabolic recovery. However, no upregulation of hsp70, hsp30, or hsp90 was evident in heart or muscle tissue of males or females, and plasma CK measurements suggest that tissue damage was minimal. Our results indicate that hsp70, hsp30, and hsp90 are not part of the early recovery process from burst exercise in fish, perhaps due to the maintenance of core temperatures as well as a lack of exercise-induced tissue damage.     

Exercise training and the stress response in rainbow trout, Salmo gairdneri Richardson

Plasma levels of catecholamines, cortisol, and glucose were monitored in rainbow trout during a 6-week forced swimming exercise programme. Compared to resting non-exercised controls, resting trained fish had lower levels of epinephrine, norephinephrine, cortisol, and glucose during the last 3 weeks of training. Initially, trained fish that were swimming had higher levels of epinephrine than resting trained fish. After 2 weeks of exercise, swimming did not significantly elevate epinephrine levels in trained fish. Glucose levels were consistently greater in swimming fish than in resting fish. At the end of the training period, exercised trout had lower (15–20%) oxygen consumption rates while resting or swimming than unexercised fish.
After a 5-month forced swimming exercise programme plasma levels of catecholamines and glucose were monitored in trained and untrained cannulated rainbow trout after 2 min of mild agitation. Trained fish showed an immediate (within 1 min) increase in the levels of epinephrine, but not norepinephrine and a delayed (within 15 min) increase in the levels of plasma glucose. Epinephrine levels returned to pre-stress levels within 15 min. Untrained fish had no significant increase in the plasma levels of norepinephrine, epinephrine, or glucose.     

The effects of stress on the association between hsp70 and the glucocorticoid receptor in rainbow trout

The purpose of this study was to characterize the association between hepatic heat shock protein 70 (hsp70) and the glucocorticoid receptor in rainbow trout that were exposed to heat stress, cortisol, and beta-naphthoflavone. This study is the first to document that the glucocorticoid receptor complex in rainbow trout hepatic tissues contains hsp70. Heat stress significantly increased levels of total cellular hsp70, and by discerning the association of hsp70 with the glucocorticoid receptor, we demonstrated that heat stress significantly increased the amount of hsp70 not bound to the glucocorticoid receptor, while significantly decreasing the amount of hsp70 bound to the glucocorticoid receptor. By calculating the ratio of hsp70 bound to the glucocorticoid receptor, to the total number of glucocorticoid receptors, stress (heat stress and cortisol-treatment) promoted the association of hsp70 with the glucocorticoid receptor. These findings demonstrate a functional and structural link between hsp70 and the glucocorticoid receptor in rainbow trout, and raise questions regarding the existence of a complex, interrelated stress response that spans all levels of biological organization within the whole animal.     

Effects of autonomic blockade on acute thermal tolerance and cardioventilatory performance in rainbow trout,Oncorhynchus mykiss

Predicted future increases in global temperature may impose challenges for ectothermic animals like fish, but the physiological mechanisms determining the critical thermal maximum (CT_max) are not well understood. One hypothesis suggests that impaired cardiac performance, limited by oxygen supply, is an important underlying mechanism. Since vagal bradycardia is suggested to improve cardiac oxygenation and adrenergic stimulation may improve cardiac contractility and protect cardiac function at high temperatures, we predicted that pharmacological blockade of cardiac autonomic control would lower CT_max. Rainbow trout was instrumented with a flow probe and a ventilation catheter for cardioventilatory recordings and exposed to an acute thermal challenge until CT_max following selective pharmacological blockade of muscarinic or β-adrenergic receptors.Contrary to our prediction, CT_max (~26 °C) was unchanged between treatments. While β-adrenergic blockade reduced heart rate it did not impair cardiac stroke volume across temperatures suggesting that compensatory increases in cardiac filling pressure may serve to maintain cardiac output. While warming resulted in significant tachycardia and increased cardiac output, a high cholinergic tone on the heart was observed at temperatures approaching CT_max. This may represent a mechanism to maintain scope for heart rate and possibly to improve myocardial contractility and oxygen supply at high temperatures. This is the first study evaluating the importance of autonomic cardiac control on thermal tolerance in fish. While no effects on CT_max were observed, this study raises important questions about the underlying mechanisms determining thermal tolerance limits in ectothermic animals.     

Relationship between stress, feeding and plasma ghrelin levels in rainbow trout, Oncorhynchus mykiss

Sexually immature rainbow trout were acclimated to small-volume (1 m³) holding tanks and then exposed to short-term stress to examine the relationship between feeding, stress, plasma ghrelin levels and other plasma stress parameters. Plasma ghrelin levels showed an increase 24 h after a single feed, plasma lactate and glucose levels decreased over the same period and plasma cortisol levels were low and constant. One hour of confinement stress resulted in elevations of plasma cortisol, glucose and lactate and depression of plasma ghrelin levels. In a separate experiment, 2 h of confinement stress also depressed feeding immediately after stress, concomitant with increases in plasma cortisol, lactate and glucose; however, in this case there was no change in plasma ghrelin concentrations. A repeat of the 2-h confinement experiment using fish that had not been acclimated to small-volume holding tanks produced a more marked elevation in plasma cortisol and a stronger suppression of feeding post-stress but in this case also, there was no change in plasma ghrelin levels. The results of this study confirm that feeding in rainbow trout is suppressed by confinement stress although the effect is transitory in this domesticated stock. Similar to that in other fishes, plasma ghrelin levels appear to be modulated by feeding status and may be influenced by stress, suggesting an orexigenic role for ghrelin in rainbow trout.     

Modulation of cutaneous cold receptor function by electrolytes, hormones and thermal adaptation.

The response properties of feline cold receptors were analyzed under control conditions, during conditions of altered external calcium concentrations and during application of menthol, catecholamines and ouabain. Afferent activity was extracellularly recorded from cold fibres of an isolated preparation of the tongue. Reduced calcium levels (0.5 mM) generally enhanced and elevated calcium levels (5.0 mM) suppressed cold fibre activity. The effects of menthol (10(-5) M) on cold receptors were qualitatively similar to those of reduced calcium. Application of adrenaline and noradrenaline (10(-6) M) were predominantly inhibiting. In cold receptors, the mean discharge rate is determined by the frequency of an oscillating receptor process and the probability of each cycle of this process to initiate afferent impulses. All measures mainly affected the probability of impulse generation rather than the oscillation frequency. Application of ouabain (10(-6) M) resulted in excitatory responses, caused by an increase of both probability of impulse generation and frequency of the oscillating receptor process. It is concluded that cold receptor function is based on a specific combination of common neuronal elements rather than on specific sensory processes.     

Oxygen uptake by rainbow trout blood, Salmo gairdneri

Oxygen uptake rate by rainbow trout red cells was measured in the temperature range 5° to 40° C. Between 5° and 20° C there was a steady increase in rate and at 20° C a maximum rate of 2.5 μl O₂/min/ml red cells occurred, followed by a decline in the rate as the temperature increased. A morphological investigation of the red cell was conducted and possible functions of the nucleus in fish red blood cells are discussed.     

ACTH does not mediate divergent stress responsiveness in rainbow trout.

Two lines of rainbow trout selected for high (HR) and low (LR) responsiveness to a standardised confinement stressor displayed a sustained divergence in plasma cortisol levels during a 3-h period of confinement (max.: HR: 167+/-13 ng ml(-1); LR: 103+/-8 ng ml(-1); P<0.001). However, no significant difference in plasma ACTH levels was evident (max: HR: 153+/-9 pg ml(-1); LR: 142+/-7 pg ml(-1)). Dexamethasone (DEX) was administered to HR and LR fish to block endogenous adrenocorticotropin (ACTH) release. Administration of a weight-adjusted dose of ACTH to the DEX-blocked fish elevated plasma cortisol levels to a significantly greater extent in HR (233+/-24 ng ml(-1)) than LR (122+/-14 ng ml(-1)) fish (P<0.001). Plasma cortisol levels in DEX-blocked HR and LR fish after sham injection were low but also significantly different (HR: 6.7+/-1 ng ml(-1); LR: 2.2+/-0.2 ng ml(-1); P<0.001). These results indicate that modulation of cortisol responsiveness to stressors in HR and LR fish resides, at least in part, downstream of the hypothalamic-pituitary axis.