Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure

Enzymatic and non-enzymatic antioxidants serve as an important biological defense against environmental oxidative stress. Information on antioxidant defense in fish is meager despite that fish are constantly exposed to a myriad of environmental stress including the oxidants. This study, therefore, assesses the activities of antioxidant enzymes viz., glutathione peroxidase, catalase and glutathione S-transferase and the non-enzymatic antioxidants viz., glutathione and metallothionein in various tissues of freshwater fish Channa punctatus (Bloch), in response to short-term and long-term exposures to paper mill effluent. The fish were exposed to the effluent at a concentration of 1.0% (v/v) for 15, 30, 60 and 90 days. The exposure caused a time-dependent increase in glutathione level (P < 0.001), activities of glutathione peroxidase (P < 0.05 to P < 0.001), glutathione S-transferase (P < 0.001) and a marginal initial decrease in catalase activity in the liver (P < 0.01 to P < 0.001). Metallothionein was induced in liver after 60 days of exposure. Two isoforms of metallothionein were detected. Catalase activity also increased 60 days afterwards. Antioxidant pattern was different in gill and kidney showing that liver was more resistant to oxidative damage as compared to gills and kidney. Our results demonstrate a pollutant-induced adaptive response in fish. In addition, levels of enzymatic and non-enzymatic tissue antioxidants may serve as surrogate markers of exposure to oxidant pollutants in fish.     

基于UPLC-Q-TOF/MS代谢组学探究虹鳟鳃对热应激的生理反应

全球气温日趋升高导致的水温上升可引起虹鳟(Oncorhynchus mykiss)代谢紊乱,为解析其在热应激下的代谢变化特征,研究基于UPLC-Q-TOF/MS代谢组学技术,探究了虹鳟鳃靶器官在高温暴露(20℃和24℃)及恢复到初始温度(14℃)下的代谢生理反应。研究结果表明,与对照组相比,在20℃、24℃高温组和14℃恢复组中分别鉴定出128、130和108种差异显著代谢物。在高温暴露下,亚油酸、花生四烯酸等与脂质代谢相关的代谢物及还原型辅酶Ⅱ(NADPH)、谷胱甘肽(GSH)、谷胱甘肽二硫化物(GSSG)等与细胞氧化还原状态相关的代谢物均发生显著改变。富集分析表明这些代谢物主要涉及虹鳟鳃的甘油磷脂代谢、鞘脂代谢、亚油酸代谢、花生四烯酸代谢、磷酸戊糖途径和谷胱甘肽代谢等代谢通路,但在温度恢复到14℃后,除鞘脂代谢外,其他代谢途径均未恢复至正常状态。上述结果表明,热激导致了虹鳟鳃靶器官的脂质代谢紊乱,可能导致鳃细胞膜的结构和功能的损伤,诱使鳃细胞出现炎症反应,并产生免疫应答。同时,虹鳟鳃细胞通过磷酸戊糖途径产生的NADPH来调节谷胱甘肽代谢中GSH/GSSG比值以提高细胞的抗氧化能力来...     

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss)

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 μg L⁻¹), propineb (3, 6 and 24 mg L⁻¹), and benomyl (2, 5 and 20 mg L⁻¹) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.     

Deltamethrin attenuates antioxidant defense system and induces the expression of heat shock protein 70 in rainbow trout

The current research aims to determine alterations in gene expression and enzymatic activity of fish antioxidant metabolism in response to pesticide administration. To this end, three different deltamethrin concentrations (0.25, 1, 2.5 μg/L) were administrated to rainbow trout (Oncorhynchus mykiss) at different time intervals (6, 12, 24, 48 and 72 h) in order to observe the influences of the pesticide on the activity of glutathione reductase, glucose 6-phosphate dehydrogenase, 6-ghosphogluconate dehydrogenase, and the expression of Hsp70 gene. We observed that the activities of the enzymes decreased with increasing deltamethrin concentrations and exposure time. The pesticide had more inhibitory effects on gill enzymes than those of muscle, liver and kidney. In addition, we detected that deltamethrin increased the expression of the stress-related protein Hsp70 with significant fold-chance values. The efficiency rate was 96.4% which is equal to 1.96 calculated via conversion formula used to calculate fold-chance value. We conclude that deltamethrin causes oxidative stress in fish both at protein and mRNA levels.     

Effects of continuous and episodic hyperoxia on stress and hepatic glutathione levels in one-summer-old rainbow trout (Oncorhynchus mykiss)

One-summer-old rainbow trout ( Oncorhynchus mykiss ) were exposed to continuous hyperoxia (173 ± 24%) and three hyperoxic/normoxic treatments for 14 days. Hepatic glutathione status as the indicator of oxidative stress, as well as classical stress indicators such as hemoglobin, hematocrit and plasma cortisol levels, were measured during normoxic, constantly hyperoxic and the following episodically hyperoxic oxygen treatment regimes: 12 h hyperoxia:12 h normoxia (12 HYP:12 NOR), 24 HYP:24 NOR and 48 HYP:24 NOR. Constant hyperoxia tended to shrink erythrocytes, but the 12 HYP:12 NOR treatment increased the number of erythrocytes and thus enhanced the oxygen carrying capacity of blood. Similarly, a trend toward an elevation in plasma cortisol concentrations was detected in 12 HYP:12 NOR treatment group. The finding that elevated hepatic glutathione (GSH) levels (P < 0.01), indicative of enhanced potential of the liver tissue to resist oxidative stress, coincided with elevated cortisol levels might suggest that in the 12 HYP:12 NOR treatment physiological processes were recruited to increase oxygen carrying capacity in blood and to elevate protection against oxyradicals. However, none of the episodic hyperoxia treatments or continuous hyperoxia caused mortality or resulted in better growth. These data indicate that continuous hyperoxia (173 ± 24%) and hyperoxic-normoxic treatments may be applied in intensive culture of rainbow trout provided that fish have at least 24 h in normoxia prior to the next bout of hyperoxia. Shorter recovery periods, like in a 12 HYP:12 NOR treatment, may result in the increased need of oxygen in tissues followed by an activation of glutathione dependent defence system against an increased oxygen load.     

Induction of metallothionein gene expression by cadmium and the retention of the toxic metal in the tissues of rainbow trout (Salmo gairdneri)

1. When rainbow trout were exposed to cadmium by intraperitoneal injection, there was a rapid (within 3hr) and significant (approx. 63%) loss of the metal from the whole bodies of the fish.2. Of the metal retained in the bodies of the fish (approx. 37% of the injected dose), more than 98% was accounted for collectively among the liver, kidney and gills.3. Subsequent maintenance of the rainbow trout in fresh water for up to 98 days post-metal administration, indicated that there was no further loss of the cadmium accumulated in the organs studied and that the distribution of the metal among the liver, kidney and gills remained unchanged over that period.4. During this 98-day period of maintenance of the fish, tissue concentrations of metallothionein-specific mRNA and metallothionein protein were quantified using riboprobe and ELISA systems respectively. Metallothionein-specific mRNA concentrations increased rapidly (within 24 hr) before falling back to levels similar to, or slightly greater than, those found in control animals. The concentration of metallothionein protein also increased significantly (within 3 days) then remained elevated thereafter.5. Throughout the experimental period, the concentrations of zinc and copper were also monitored in the liver, kidney and gills of the rainbow trout. The concentrations of each ion differed between each of the organs but did not change during the experiment.6. The induction of metallothionein gene expression by cadmium in the liver, kidney and gill of rainbow trout and the subsequent sequestration of the toxic metal is discussed with regard to the relative levels of these other essential metal ions.     

A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

Effect of arsenic (AsIII) on glutathione-dependent enzymes in liver and kidney of the freshwater fish Channa punctatus

The possible role of glutathione-dependent enzymes in the liver and kidney of the freshwater fish Channa punctatus has been studied after exposure to arsenic trioxide for different durations. Activities of glutathione-S-transferases, glutathione peroxidase, glutathione reductase, and catalase decreased in the liver and kidney as a result of the initial increase in arsenic concentration in the liver and kidney. However, during longer exposures, a decline in arsenic concentration corresponded with improved enzyme activity. Because arsenic manifests its toxicity by inducing oxidative stress, the antioxidant enzymes, especially the glutathione-dependent enzymes, play a protective role in arsenic toxicity.     

Dietary Ca inhibits waterborne Cd uptake in Cd-exposed rainbow trout, Oncorhynchusmykiss

The effects of chronic exposure to waterborne Cd and elevated dietary Ca, alone and in combination, were examined in juvenile rainbow trout, Oncorhynchusmykiss. Fish were chronically exposed to 0.05 (control) or 2.56 μg/l Cd [as Cd(NO₃)₂·4H₂O] and were fed 2% body mass/day of control (29.6 mg Ca/g) or Ca-supplemented trout food (52.8 mg Ca/g as CaCl₂·2H₂O). Cd accumulated mainly in gill, liver, and kidney. Waterborne Cd inhibited unidirectional Ca uptake from water into the gill and induced hypocalcemia in the plasma on day 40. Waterborne Cd also induced an elevated Ca concentration on day 20 in the gill tissue of trout fed the Ca-supplemented diet and a decreased Ca concentration on day 35 in the gills of trout fed the control diet. Dietary Ca protected against Cd accumulation in gill, liver, and kidney, but did not protect against the inhibition of Ca uptake into the gill or plasma hypocalcemia. When fed Ca-supplemented diet and exposed to waterborne Cd, fish showed 35% mortality, compared to 0–2% in control fish and in the Cd-exposed fish with normal Ca in the diet. Growth, on the other hand, was not affected by any treatment.     

Antioxidant defense of rainbow trout (Oncorhynchus mykiss) in relation to dietary n-3 highly unsaturated fatty acids and vitamin E contents

This study examined the modulation of the antioxidant status and related physiological changes in rainbow trout Oncorhynchus mykiss under different levels of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) and vitamin E. Six diets containing 0, 100 or 1000 mg alpha-tocopheryl acetate kg(-1) diet and 20% or 48% n-3 HUFA provided by normal fish oil or DHA concentrated fish oil, respectively, were fed to 100 g size fish for 15 weeks. Growth of fish fed vitamin E deficient diets under both levels of n-3 HUFA were slightly retarded, accompanied by a reduction of hematocrit values, an enlargement of liver and spleen, an elevation of lipid hydroperoxide in red blood cell and the antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase). Supplementation of vitamin E could protect the fish from these adverse effects; however the higher dose was no better compared to the moderate dose. The modulations were clearly seen in fish fed high n-3 HUFA (48%) since they were under greater oxidative stress as indicated by the markers, lipid hydroperoxide and 8-isoprostane. The increased activity of enzymes corresponds to physiological mechanisms combating the elevation of free radicals under oxidative stress and a dietary fatty acid profile-dependent moderate dose of vitamin E is all that is required to function as an effective antioxidant.     

Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver

In fish, as in other aerobic organisms, glutathione and glutathione-related enzymes are important components in the defences against oxidative stress. To study if hepatic glutathione levels and/or activities of glutathione-related enzymes can act as indicators of oxidative stress in fish, we injected rainbow trout (Oncorhynchus mykiss) intraperitoneally with paraquat (PQ), menadione (MD), naphthazarin (DHNQ), or beta-naphthoflavone (beta-NF), all known to cause a rise in reactive oxygen species (ROS). After 2 and 5 days of exposure, we measured the activities of hepatic glutathione peroxidase (GPox), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), and glutathione reductase (GR). We also measured total glutathione (tGSH) and oxidised glutathione (GSSG) in the liver of fish treated with PQ and MD. All chemicals caused an increase in GR activity after 5 days, which ranged from 160% in fish treated with beta-NF to 1,500% in fish treated with PQ. All chemicals except beta-NF caused moderate elevation in GST activity; GPox activity was lower in fish treated with DHNQ and MD, while GCS activity increased twofold in the fish treated with DHNQ, without being affected by beta-NF, PQ or MD. After 5 days of treatment with PQ or MD, tGSH content was elevated. Our findings demonstrated that of the parameters included in the study, GR activity was the most responsive to treatment with redox cycling compounds, indicating that GR activity is a promising biomarker of such compounds and possibly indicating oxidative stress in rainbow trout.     

A comparative study of antioxidant enzyme activities in freshwater and seawater-adapted rainbow trout.

Comparative studies were performed on the antioxidant enzyme activities and thiobarbituric acid reactive substance (TBARS) concentration in liver and red cells of two groups of rainbow trout (Oncorhynchus mykiss). The fish of the first group were cultured in freshwater and the others were adapted to sea-water by by being transferred from freshwater at 5-6 months of age. Catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were significantly higher in hepatic and extrahepatic tissues in both of the fish groups. Superoxide dismutase (SOD) activities were found lower in the seawater-adapted trout than in the freshwater-cultured trout. In both tissues, TBARS were found significantly higher in the seawater-adapted trout than in the freshwater trout. It was also observed that the red cells of the seawater-adapted trout were much more resistant to oxidative stress than the red cells of the freshwater-cultured trout. The results implicate that antioxidant capacities in the seawater-adapted trout and freshwater trout may be related to physical and chemical characteristics of the environment.     

Evaluating the toxicity of environmental concentrations of waterborne chromium (VI) to a model teleost, Oncorhynchus mykiss: a comparative study of in vivo and in vitro

Toxic effects of environmental concentrations (50, 100, and 200μg/L) of waterborne chromium (VI) were evaluated in rainbow trout by comparison of in vitro and in vivo assays. Multiple biomarkers were measured including oxidative stress indices and antioxidant response parameters in liver and brain, as well as Na(+)-K(+)-ATPase in gill. Superoxide dismutase (SOD) and glutathione reductase (GR) activities were significantly induced (1.54-fold and 1.37-fold, respectively) in fish brain in vivo, but no significant differences were observed in any other biomarker or in vivo test group. Oxidative stress was apparent in vitro as significantly higher levels of oxidative indices, with the highest induction of TBARS and CP found in brain at 200μg/L Cr(VI) (2.41-fold and 1.95-fold, respectively), and SOD and GR activities and reduced glutathione in brain were significantly inhibited (65%, 44%, and 36%, respectively). In vitro Na(+)-K(+)-ATPase activity in gill was also significantly inhibited at concentrations of 100 and 200μg/L (69% and 45%, respectively). Short-term exposure to environmental concentrations of Cr(VI) does not therefore evoke marked effects in fish in vivo. Based on the present results, a set of in vitro tests with tissue homogenate can be evoked more remarkable effects by the lower concentrations of Cr(VI) than in vivo, which could provide some useful information and might be a potential alternative approach for monitoring heavy metal pollution in aquatic environments. However, it needs more detailed studies in other area, such as hormonal response or genotoxicity, before these findings could be applied in the field investigation.     

The expression of immune-regulatory genes in rainbow trout, Oncorhynchus mykiss, during amoebic gill disease (AGD)

Amoebic gill disease (AGD) is an ectoparasitic disease caused by infection with the protozoan Neoparamoeba sp. and is characterised by epithelial hyperplasia that manifests as gill lesions. In order to examine the nature of the immune response to AGD, the expression of a range of immune-regulatory genes was examined in naïve uninfected rainbow trout, Oncorhynchus mykiss, and naïve rainbow trout subjected to a laboratory-induced AGD infection. The immune-regulatory genes examined were interleukin-1 beta isoform 1 (IL-1β1), tumour necrosis factor alpha isoforms 1 and 2 (TNF-α1, TNF-α2), interleukin-8 (IL-8), transforming growth factor beta isoform 1 (TGF-β1), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), major histocompatibility complex beta chain (MHC-II β-chain) and T-cell receptor beta chain (TCR β-chain). Immune-regulatory genes that were up/down-regulated in AGD-infected trout compared to uninfected controls at 0, 7, and 14 days post-inoculation (p.i.) in gill, liver and anterior kidney tissue were initially identified by means of semi-quantitative RT–PCR. Up/down-regulated immune-regulatory genes were subsequently quantitated and validated by real-time RT–PCR (qRT–PCR). The extent of AGD-associated pathology was consistent amongst all AGD-infected trout at 7 days p.i. and increased considerably by 14 days p.i. At both 7 and 14 days p.i. IL-1β1 and iNOS gene expression was significantly up-regulated in the gills, and IL-8 was significantly up-regulated in the liver of AGD-infected trout at 7 days p.i. These data demonstrate the involvement of the immune response to AGD at the molecular level, and indicate the importance of this response at the site of infection and the possible involvement of a systemic immune response.     

The effect of lycopene on oxytetracycline-induced oxidative stress and immunosuppression in rainbow trout (Oncorhynchus mykiss, W.)

The aim of this study was to determine the effects of lycopene on oxytetracycline (OTC)-induced oxidative stress and immunosuppression in rainbow trout. The experimental fish analysed in this study were divided into 6 different experimental groups. Group 1 was the control group, and groups 2, 3 and 4 received corn oil, lycopene and OTC, respectively, for 14 days. Group 5 received OTC for 14 days after lycopene pre-treatment for 14 days, while group 6 received OTC for 14 days before lycopene post-treatment for 14 days. Blood and tissue samples were collected at the end of the experiment and analysed for the oxidant-antioxidant status and changes in the immune response. There was a significant increase in the malondialdehyde level, which is an index of lipid peroxidation, and a decrease in superoxide dismutase, catalase, and glutathione peroxidase activity as well as a decrease in the glutathione level in the blood, liver, kidney and spleen of OTC-treated fish. Glutathione-S-transferase activity was significantly increased in the blood, liver, kidney and spleen samples of the group that received OTC alone. OTC also appeared to suppress specific and nonspecific immune system parameters, such as the haematocrit, leucocyte count, oxidative radical production (nitroblue tetrazolium activity), total plasma protein and immunoglobulin levels and phagocytic activity. Pre- and post-treatment with lycopene attenuated the OTC-induced oxidative stress by significantly decreasing the tissue malondialdehyde level. The superoxide dismutase, catalase and glutathione peroxidase activities as well as the glutathione levels were significantly increased with lycopene administration, while glutathione-S-transferase activity was significantly decreased. Lycopene administration was also associated with a significant increase in the OTC-suppressed immune system parameters in fish. Thus, the present results suggest that pre- and post-treatment with lycopene (10 mg per kg fish weight, delivered orally) may alleviate OTC-induced oxidative stress and immunosuppression.     

Effect of cellulase,phytase and pectinase supplementation on growth performance and nutrient digestibility of rainbow trout (Oncorhynchus mykiss,Walbaum 1792) fry fed diets containing canola meal

This experiment was conducted to evaluate the effects of supplementing exogenous enzymes on growth, feed conversion ratio (FCR) and apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fry diets containing 32% canola meal. Five experimental diets (including a control diet containing no enzymes) were prepared as isonitrogenous (44% crude protein) and isocaloric (4000 kcal DE kg¹). The four other diets contained either cellulase, phytase, pectinase or an enzyme mix (a mixture of cellulase, phytase and pectinase in the same ratio). The feeding trial was conducted in triplicate for 12 weeks in 15 tanks (100‐L). At the beginning of the experiment 20 rainbow trout fry (initial weight 1.23 g) were stocked into each tank. Mean water temperature in the rearing tanks was 11°C and water flow in each tank was 6 L min^?1. At the end of the experiment the growth parameters and FCR displayed no significant differences in enzyme supplementation (P > 0.05). In addition, no differences were observed in dry matter, protein, or lipid digestibility with enzyme supplementation (P > 0.05). The results of this study showed that the addition of pectinase, phytase, cellulase or an enzyme mix to a diet containing 32% canola meal had no effect on growth, feed efficiency or dry matter, protein, or lipid digestibility in rainbow trout fry.     

Rainbow trout (Oncorhynchus mykiss) exposed to oxygen supersaturation and handling stress: plasma cortisol and hepatic glutathione status

Three groups of one summer old rainbow trout were exposed for 22 days either to normoxia (100%) or moderate oxygen supersaturation; 120% and 140%. After the exposure, all groups were transported for three hours in hyperoxic conditions (123% O2) thus simultaneously experiencing density and handling stress. The recovery of rainbow trout to multiple stressors was measured in normoxic conditions. Moderate oxygen supersaturation did not have any negative effects on growth, feed conversion and blood hematology measured over 22 days. On the other hand, the combined effects of the stressful environment in the fish farm and oxygen supersaturation resulted in a 3-fold increase in plasma cortisol levels in those with 100% and 120% O2 supersaturation and a 2-fold increase in the 140% supersaturation group. Furthermore, the stress response after transportation was lowest in the 140% group 24 hours after recovery but highest after 70 hours. Moderate hyperoxia or transportation stress did not change glutathione concentrations in liver indicating that routine sampling does not affect hepatic glutathione status. Our results indicate that moderate O2 supersaturation (<140%) could be considered as feasible in cultivation of rainbow trout since no harmful effects were found.     

Ecotoxocological effects of short-term exposure to a human pharmaceutical Verapamil in juvenile rainbow trout (Oncorhynchus mykiss)

Verapamil (VRP) is a calcium channel blocker that is a highly prescribed compound and commonly present in aquatic environment, but the ecotoxicological effects of this pharmaceutical in fish have not been fully documented. In this study, the toxic effects of VRP were studied in juvenile rainbow trout, Oncorhynchus mykiss, by acute static bioassay. In the acute test, the median lethal concentration (LC50, 2.72 mg/L) was evaluated and the behavioral changes were obviously intensified with increasing VRP concentrations. Compared to the control, oxidative stress was observed in fish tissues with different levels after short-term exposure to sublethal concentrations (0.27 and 1.35 mg/L) of VRP. Activities of SOD and GPx in fish brain were induced at 0.27 mg/L VRP, but all the antioxidant enzymes (SOD, GPx and GR) in fish brain were decreased at 1.35 mg/L VRP. When compared to the control, all the antioxidant enzymes in gill were decreased in both treated groups, but there was no significant change in muscle. Additional, muscle DNA/RNA ratio in fish exposed at 1.35 mg/L VRP was significantly lower than that in the control. Furthermore, through chemometrics of all parameters measured in fish exposed to sublethal VRP concentrations using principal component analysis, two groups with 89.8% of total accumulated variance were distinguished. In short, the physiological and biochemical responses in of fish indicated that VRP-induced environmental stress; but according to VRP residual status in the natural environment, more long-term experiments at lower concentrations will be necessary in the future.     

Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated streams

Ultrastructural biomarkers in gill, liver, andkidney of brown trout (Salmo truttaf. fario) and stone loach (Barbatulabarbatula) were investigated over afive year period. The cellular damage of theorgans was assessed semi-quantitatively basedon a three-step classification ofultrastructural responses. Data obtained forfish exposed under semi-field conditions in twodifferently polluted test streams and in thelaboratory demonstrated that the ultrastructureof the organs can be correlated with differentpollutant exposure conditions. Cellular damagewas generally more severe in fish exposed to acomplexly polluted stream than in those exposedto a moderately polluted stream or to tap waterin the laboratory. Histopathological effects inliver and gill of trout were more pronounced inferal fish than in transplanted fish, whereasresponses in the kidney in both species, and inliver and gill of loach, were similar forintroduced and feral fish. In a laboratoryexperiment where trout were exposed todifferent mixtures of pollutants includingpesticides, PAH, and ammonia, only theultrastructure of kidney and liver showedsignificant differences between the threeexperimental set-ups. In a recovery experiment,where trout were transferred from thesemi-field condition back to the laboratory,ultrastructural investigations showed adifferential capacity of the respective organsto recover from stress under field conditions.Kidney and liver fully recovered after threemonths under control condition whereas gillsdemonstrated only partial recovery.     

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.