Catalytic activity and immunochemical quantification of hepatic cytochrome P-450 in β-naphthoflavone and isosafrol treated rainbow trout (Oncorhynchus mykiss)

In addition to catalytical assays, immunochemical techniques have recently been employed to measure induction of the cytochrome P-450 (P450) monooxygenase system in fish with polyaromatic hydrocarbons (PAH). In the present study, polyclonal antibodies were raised against rainbow trout P450IA1. Levels of rainbow trout P450IA1 determined using protein blotting- and ELISA procedures were compared with levels of 7-ethoxyresorufin-O-deethylase (7-EROD) activity in liver microsomes from rainbow trout. These comparisons showed that values of P450A1 were positively correlated (r=0.99 and r=0.97) with 7-EROD activities. In addition, the effects of isosafrol (ISF) or -naphthoflavone (NF) treatments on P450 levels in rainbow trout liver were investigated using immunochemical and catalytical methods. ISF treatment induced 7-EROD activity as well as 7-methoxycoumarin-O-demethylase-, 7-ethoxycoumarin-O-deethylase-, 7-propoxy-coumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities, although to a lesser extent, compared with the NF treatment. In contrast, immunochemical quantification of rainbow trout P450IA1 protein revealed a slightly different pattern. ISF appeared to be a weak inducer of P450IA1 in rainbow trout compared with NF. In addition, the degree of inhibition of 7-alkoxycoumarin-O-dealkylase activities in ISF microsomes differed from that measured in control- and NF microsomes. The discrepancies between catalytic and immunochemical estimates of rainbow trout P450IA1 in ISF treated fish in addition to differencs between specific inhibitory pattern by specific polyclonal antibodies raised against rainbow trout P450IA1, indicate that important differences exists between the responses induced by NF- and ISF treatments in the rainbow trout liver.Part of this work was presented at the 6th International Conference on Biochemistry and Biophysics of Cytochrome P-450, Vienna, Austria, July 3–8, 1988.     

Are dietary genistein and equol potent enhancers of eicosapentaenoic acid and docosahexaenoic acid levels in rainbow trout (Oncorhynchus mykiss)?

Phytoestrogens are putatively able to enhance the biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but have also been shown to affect fish growth dose dependently. The aim of the present study was to identify a concentration for the phytoestrogen genistein and the phytoestrogen metabolite equol that further increases the endogenous biosynthesis of EPA and DHA without impairing fish growth. Juvenile rainbow trout (87.2 ± 0.3 g) were fed seven diets on a fixed ratio for 8 weeks. A vegetable oil‐based diet served as a control diet and was supplemented with equol (EQ) and genistein (G), respectively, at 0.1%, 0.2% and 0.3% of feed dry matter (1, 2 and 3). Growth and nutrient composition of whole body homogenates were not affected by dietary treatments. EPA and DHA levels in liver, fillet and whole body samples were not significantly increased by EQ and G diets. Fish fed EQ diets showed dose dependently increased liver weights and C18:0 liver levels, indicating estrogen‐like effects at increased dietary dosages. In conclusion, the utilization of equol and genistein in plant oil‐based diets in order to enhance the biosynthesis of EPA and DHA seems not reasonable in rainbow trout.     

Comparison of 3,5,3′-triiodo-L-thyronine and L-thyroxine absorption from the intestinal lumen of the fasted rainbow trout,Oncorhynchus mykiss

The absorptions of 3,5,3-triiodo-L-thyronine (T₃) and L-thyroxine (T₄) from the intestinal lumen of the rainbow trout were compared in vivo. Tracer doses of [¹²⁵I]T₄ (⁺T₄) or [¹²⁵I]T₃ (^*T₃) were injected through an anal cannula into the duodenum of trout fasted for 3 days at 12°C, and radioactivity was measured in blood and tissues at 4–48 h. ^*T₃ was removed more extensively than ^*T₄ from the intestinal lumen and more radioactivity was absorbed into the blood and tissues of u+T₃-injected trout than ^*T₄-injected trout. HPLC analysis showed that a high proportion of the radioactivity in the plasma, liver, kidney and intestinal lumen of ^*T₃-injected trout remained as the parent ^*T₃. However, in ^*T₄-injected trout most plasma radioactivity was in the form of ¹²⁵I^–, and by 24 h a high proportion of luminal radioactivity was ¹²⁵I^–. By 48 h, over 4% of the injected ^*T₃ and 1% of the injected ^*T₄ dose resided in the gall bladder, primarily as derivatives of ^*T₃ or ^*T₄. We conclude that T₃ is absorbed more effectively than T₄ from the intestinal lumen of fasted trout, indicating the potential for an enterohepatic T₃ cycle.     

Cortisol does not affect hepatic α- and β-adrenoceptor properties in rainbow trout (Oncorhynchus mykiss)

Distribution and function of hepatic - and -adrenoceptors were examined in rainbow trout (Oncorhynchus mykiss) injected with slow release hydrogenated coconut oil implants alone (sham) or containing cortisol. - and -Adrenoceptors were assayed on purified hepatic membranes 10–14 days post-implantation using ³H-prazosin () and ³H-CGP (). At 10–14 days, plasma cortisol values were significantly elevated to approximately 220 compared with 35.0 ng ml^-1 in cortisol implanted vs. sham trout. No significant differences were found between any of the experimental groups for either the affinity (K_d) or maximal number of binding sites (B_max) for either receptor type. Epinephrine significantly stimulated glucose release from hepatocytes isolated from sham injected trout, but not from cortisol-treated fish. Epinephrine-induced glucose release was blocked by both - and -antagonists. These studies do not support the hypothesis that rainbow trout exposed to chronic cortisol alter properties of hepatic adrenoceptors.     

Characterization of hepatic and extrahepatic glutathione S-transferases in rainbow trout (Oncorhynchus mykiss) and their induction by 3,3′,4,4′-tetrachlorobiphenyl

Liver, kidney, gill and olfactory epithelium cytosolic fractions of rainbow trout (Oncorhynchus mykiss) were examined for glutathione S-transferase (GST) contents. Proteins retained on a glutathione (GSH)-affinity matrix were separated as monomers by reversed-phase HPLC and characterized by immunoblotting, mass spectrometry and partial amino acid sequence. For each organ concerned, a specific pattern of these proteins was determined and appeared similar for liver and kidney on one hand, and for gill and olfactory epithelium on the other hand. It was confirmed that the prominent hepatic GST is a class enzyme, also constitutively expressed as a major isoform in the four organs studied. Moreover, a class variant and two new class GST subunits were characterized in minor fractions. An unknown protein, which was found major in gills and olfactory epithelium, exhibited some characteristics of class GSTs. Occurrence of possible GSH-adduct formation observed on two distinct monomers in specific experimental conditions is discussed. These results and methods were used to investigate the effect of 3,3,4,4-tetrachlorobiphenyl (TCB), a polychlorinated biphenyl (PCB), on GST expression in trout liver. From HPLC-profiling, significant co-induction of the major class and the two minor class GST subunits was observed in trout after waterborne exposure to TCB which was followed by a slight increase in 1-chloro-2,4-dinitrobenzene (CDNB) activity. The present work allows qualitative evaluation of the specific detoxification potential of rainbow trout. The use of HPLC-profiling of GSTs as a possible tool for the biomonitoring of polluted aquatic environment is suggested.     

Temporal changes in plasma thyroid hormone,growth hormone and free fatty acid concentrations,and hepatic 5′-monodeiodinase activity,lipid and protein content during chronic fasting and re-feeding in rainbow trout (Oncorhynchus mykiss)

Temporal changes in growth, plasma thyroid hormone, cortisol, growth hormone (GH) and non-esterified fatty acid (NEFA) concentrations, hepatic T₃ content and hepatic 5-monodeiodinase activity were measured in rainbow trout (Oncorhynchus mykiss) subjected to a sustained fast for up to eight weeks, and during a four-week re-feeding period. The purpose of the study was to examine aspects of the endocrine control of energy partitioning processes characteristic of short-term (acute; fasting) and long-term (chronic; starvation) food-deprivation states in fish, and to explore the role of the thyroid hormones, cortisol and GH in the energy repartitioning that takes place during an acute anabolic (re-feeding) state following chronic food deprivation.Differences in growth rate between fed and fasted groups were evident after two weeks, but significant weight loss by the fasted groups was not evident until between four and six weeks into the fast. Hepatosomatic indices (HSIs) were significantly reduced in the fasted fish within seven days, and as early as two days in one study; recovery of the HSI in fasted fish was evident within three days of re-feeding. Liver protein content (expressed as % wet weight) was consistently depressed in the fasted fish in only one of the three studies. Liver total lipid content (expressed as % wet weight) was depressed in the fasted fish within two days of food deprivation. Because of the rapid and sustained decrease in the HSI of fasted fish, the hepatic total protein and lipid reserves, when considered on a body weight basis, were markedly lowered within the first few days of the fast. Plasma GH concentrations exhibited a bi-modal pattern of change, with a transient fall in levels, followed by a sustained increase in fasted fish. The indicators of interrenal activity were suggestive of a depressed pituitary-interrenal axis in fasted animals; plasma cortisol levels were elevated to levels of fed animals within one day of re-feeding. The indicators of thyroid hormone economy (plasma thyroid hormone levels, liver triiodothyronine content, hepatic 5-monodeiodinase (MD) activity, thyroid epithelial cell height) were similarly indicative of a depressed pituitary-thyroid axis in fasted animals, with recovery to levels of the fed animals within one week. Despite the compensatory changes in accumulation of reserves (as indicated by a compensatory increase in HSI), there were no apparent compensatory changes in any of the endocrine parameters evident during the re-feeding period.     

Effects of chronic exposure to γ-HCH (Lindane) on brain serotonergic and gabaergic systems,and serum cortisol and thyroxine levels of rainbow trout,Oncorhynchus mykiss

Rainbow trout (Oncorhynchus mykiss) were implanted intraperitoneally with 0.5 ml.100 g^–1 body weight of coconut oil alone (controls) or coconut oil contaning 0.05 mg of -HCH (Lindane). After 18 days, changes in brain serotonin and GABA metabolism, as well as in serum cortisol and thyroxine levels, were measured. A lower final body weight was observed in -HCH treated fish when compared with control fish. No significant differences were found for serum thyroxine levels between control and treated fish, but a significantly higher cortisol level was found in the -HCH-implanted trout. Although GABA levels did not differ significantly in any brain region in the two treatment groups, the activity of the serotonergic system was significantly altered by the pesticide in both the hypothalamus and the telencephalon.     

Tartrate resistant acid phosphatase as a marker for scale resorption in rainbow trout,Oncorhynchus mykiss: effects of estradiol-17β treatment and refeeding

In teleosts, a considerable part of the body calcium is found in the scales. Associated with the scales are osteoblasts and osteoclasts, and during periods of high calcium demand such as during sexual maturation or starvation, the scales can be resorbed and thereby act as an internal calcium reservoir. In mammalian bone tissue, the activity of an acid phosphatase (ACP) isoenzyme, tartrate resistant acid phosphatase (TRACP), can be used as a marker for osteoclastic activity. In the present study, an evaluation of TRACP as a marker for osteoclastic activity in teleost scales has been performed. ACP and TRACP was histologically localized at resorption sites around the edge of the scales as well as at resorption holes in the scales. The optimal conditions for biochemical measurements of ACP and TRACP activity were found to be pH 5.3, 10 mM paranitrophenylphosphate, incubated for 30 min at room temperature, and 10 mM tartrate added when required. Using TRACP as a marker, estradiol-17 (E₂) was found to increase the proportion of scales being resorbed, as well as the number and size of resorption sites per scale. Also, the scales of E₂-treated fish showed weaker staining for calcium. Together, the obtained data indicate that estradiol-17 induces osteoclastic activity in teleost scales, resulting in increased resorption of the scales. A period of refeeding following a period of starvation did not have detectable effects on the scale osteoclastic activity and scale resorption.     

Influence of Ulva meal on growth, feed utilization, and body composition of juvenile Nile tilapia (Oreochromis niloticus) at two levels of dietary lipid

A nutrition trial was conducted to investigate the effects of dietary lipid levels and supplemental Ulva meal on growth performance, feed efficiency, nutrient utilization, and body composition of juvenile Nile tilapia, Oreochromis niloticus. Four isonitrogenous (CP 40%) diets containing 0% and 5% Ulva meal were formulated to contain 10% (low-lipid; LL) and 20% (high-lipid; HL) crude lipid. Triplicate groups of fish (~10 g) were fed to apparent satiation three times daily for 16 weeks. Fish fed 5% Ulva meal showed an increased growth performance (P < 0.05) compared with fish fed non-Ulva supplemented diets, irrespective of dietary lipid level. In particular, the incorporation of Ulva meal improved specific growth rate (SGR), feed conversion ratio (FCR), and protein efficiency ratio (PER). Feeding fish 5% Ulva meal diets resulted in significantly lower carcass lipid content. The results indicate that 5% inclusion of Ulva meal at both dietary lipid levels improves growth performance, feed efficiency, nutrient utilization, and body composition of Nile tilapia.     

Effects of dietary lipid levels on growth,feed utilization,body composition,fatty acid profiles and antioxidant parameters of juvenile chu’s croaker <Emphasis Type="Italic">Nibea coibor</Emphasis>

The present study aimed at determining the growth performance, feed utilization, body composition and antioxidant status of chu’s croaker (Nibea coibor) juveniles fed with increasing levels of dietary lipid: 6 % (D6), 9 % (D9), 12 % (D12) and 15 % (D15). Each diet was assigned to triplicate groups of fish in a total of 12 floating pens (300 fish, 25 fish per pen). After a 49-day growth trial, survival rate was not affected. D12 and D15 led to significantly higher specific growth rate (SGR). Fish fed D12 showed the highest protein efficiency ratio (PER), protein and lipid retention efficiencies (PER and LRE, respectively). The hepatosomatic and viscerosomatic indexes (HIS and VSI, respectively) increased, while feed conversion ratio (FCR) and feed intake (FI) decreased. Body protein, ash and muscle lipid contents were not significantly affected, but significantly higher body and liver lipid were noticed in fish fed with D15. Monounsaturated fatty acid (MUFA) was found to decrease compared to the experimental diets in muscle, while high unsaturated fatty acid (HUFA) was selectively accumulated in all treatments. Serum alanine transaminase (ALT, 14.13–22.53 U/L), aspartate transaminase (AST, 34.31–51.25 U/L), cholesterol (CHO, 2.02–3.03 mmol/L) and triglyceride (TG, 5.61–8.50 mmol/L) were correlated with increasing dietary lipids. Liver malate dehydrogenase (MDA, 3.32–6.67 mmol/L) and superoxide dismutase (SOD, 42.69–52.86 U/mg prot) increased with dietary lipids, while total antioxidant capacity (t-AOC, 1.08–3.50 U/mg prot) decreased. Polynomial regression analyses between SGR and dietary lipid levels showed that the optimal dietary lipid requirement of chu’s croaker is 12.9 % of dry matter.     

The changes in digestive enzymes and hormones of gilthead seabream larvae (Sparus aurata, L 1758) fed on Artemia nauplii enriched with free methionine

Variations in digestive enzymes and hormones during the larval development of gilthead seabream (Sparus aurata) fed on live prey (Artemia nauplii) enriched with free methionine were investigated for 16 days (from day 24 to day 40). Prior to initiation of the experiment, newly hatched larvae were transferred from incubators to fiber glass tanks (300 l) with black walls and fed with same diets until day 24. Each experiment was performed in triplicate. In the experimental group, the content of the free methionine in the Artemia nauplii was increased by adding a 5.3 mM free methionine solution to the culture water during a 16-h enrichment period. The larvae of both the control and enriched-methionine groups were sampled four times, with 4-day intervals between samplings, during a 16-day period. The larvae in the control group had a significantly lower growth than those of the methionine group at the end of the study (P < 0.05). The highest trypsin activity and leucine aminopeptidase N/leucine–alanine peptidase ratios were observed in the control group. A significant difference between bombesin activities in the treatment groups was not found at 5th minute after the initiation of feeding (P > 0.05), but they were significant at 15th minute post-initiation of feeding (P < 0.05). A significant difference between the cholecystokinin levels of the treatment groups was found (P < 0.05).     

An enzyme linked immunosorbant assay (ELISA) for testosterone, estradiol, and 17,20β-dihydroxy-4-pregenen-3-one using acetylcholinesterase as tracer: application to measurement of diel patterns in rainbow trout (Oncorhynchus mykiss)

A simple and rapid Enzyme Linked ImmunoSorbant Assay (ELISA) is described and validated for testosterone, estradiol, and 17,20-dihydroxy-4-pregnen-3-one (17,20P). A general procedure for preparation of the acetylcholinesterase labeled steroid is described which is applicable to any steroid. Use of acetylcholinesterase tracer increased the sensitivity of assay so that reliable measurements of each steroid could be achieved with only 10 l of plasma. The ELISA was applied to measurement of all three steroids every hour for over 24 hours in a female trout using cannulation of the dorsal aorta. This high sampling frequency revealed several short-term (<2 h) episodic pulses of testosterone and estradiol.