Heat stress‐induced alterations of antioxidants in the freshwater fish Channa punctata Bloch

The effect of elevated temperature on the antioxidants in the freshwater fish Channa punctata was investigated. Fish stressed with an elevated temperature of 12° C, range ± 1° C over the ambient temperature for 3 h showed a significant ( P  < 0·05–0·01) reduction in the levels of antioxidants: reduced glutathione (GSH) and non‐protein thiols. Activity of glutathione reductase was also reduced in all the tissues (liver, kidney and gills) after 3 h of heat stress and 24 h recovery. Catalase (CAT) showed enhanced activity in liver in both the conditions while gills and kidney showed a decreased CAT activity. Glutathione S‐transferase (GST) activity in kidney and liver decreased significantly ( P  < 0·05–0·01) after 3 h of heat stress. At 24 h GST activity showed a tendency to normalize in all the tissues along with a concomitant increase in the GSH level in the kidney. Total and protein thiols in heat stressed fish when matched with controls, showed significant ( P  < 0·05) reduction in the kidney only with a transient increase in liver and gills. Heat shock also induced lipid peroxidation in 3 h heat‐treated and recovery groups when compared with controls. Elevated temperature therefore resulted in tissue specific and time‐dependent alterations of antioxidants in the fish. It also induced lipid peroxidation in various tissues.     

Goldfish exposure to cobalt enhances hemoglobin level and triggers tissue-specific elevation of antioxidant defenses in gills, heart and spleen

Cobalt ions can enhance the generation of reactive oxygen species (ROS), which may be the reason for cobalt toxicity. This study aimed to determine whether Co(2+) toxicity in goldfish is related to induced oxidative stress in gills, heart and spleen, and to assess responses of antioxidant systems. Exposure of goldfish to 50, 100 and 150 mg L(-1) of Co(2+) for 96 h elevated total hemoglobin in blood by 23, 44 and 78%, respectively. In gills, cobalt exposure enhanced lipid peroxide levels and activities of primary antioxidant enzymes; superoxide dismutase (SOD) rose by 125% and glutathione peroxidase (GPx) increased by 53-296%. Glutathione-S-transferase (GST) activity also increased by 117-157% and glucose-6-phosphate dehydrogenase (G6PDH) enhanced by 46-96%. Heart showed limited effects of fish exposure to 50 or 100 mg L(-1) of Co(2+), but the exposure to 150 mg L(-1) of Co(2+) elevated concentrations of lipid peroxides by 123% and activities of GPx by 98% and SOD by 208%. The most substantial effects of goldfish exposure to Co(2+) were observed in spleen: a decrease in total protein concentration by 44-60% and high molecular mass thiols by 59-82%, reduced activities of catalase by 24-58% and GR by 25-68%, whereas the level of low molecular mass thiols increased by 153-279% and activities of GPx, GST, G6PDH were enhanced by 114-120%, 192-769%, and 256-581%, respectively. The data show that fish exposure to 50-150 mg L(-1) of Co(2+) elevates blood hemoglobin level, mimicking effects of hypoxia, and causes the activation of defense systems against ROS.     

Modulatory effect of copper on nonenzymatic antioxidants in freshwater fish Channa punctatus (Bloch.)

Effect of the low level of copper exposure on nonenzymatic antioxidants was studied in a freshwater fish Channa punctatus (Bloch.). Fish were exposed to cupric chloride at the concentration of 10 ppb for 4 wk (28 d) in a static culture condition. Copper significantly (p < 0.001) increased the serum ceruloplasmin level and total iron-binding capacity. A significant (p < 0.05) increase in reduced glutathione level was recorded in all of the tissues. With regard to nonprotein thiols, copper decreased their level in the liver, but increased it in the gill. The protein-bound thiols remained unaltered except for an increase in the liver. Metallothionein (MT) induction was observed in liver only. Copper exposure had no significant effect on the ascorbic acid level and induced no lipid peroxidation over control values. It is suggested that by modulating the ceruloplasmin level, copper indirectly protects the fish, as it facilitates conversion of pro-oxidant iron to nonoxidant iron. It also induces an array of antioxidants that may be beneficial to fish in the case of oxidative stress resulting from chemical pollutants.     

Investigation of Cadmium-Induced Genotoxicity and Oxidative Stress Response in Indian Major Carp,Labeo rohita

We investigated genotoxicity and oxidative stress in the gills of Labeo rohita exposed to 33.6, 67.1, and 100.6 mg L^–1of cadmium chloride at 96 h. Genotoxicity was assessed using single cell gel electrophoresis whereas oxidative stress was monitored through lipid peroxidation induction and antioxidant response parameters, namely reduced glutathione (GSH), glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, and catalase (CAT) activities. Significant (p < .05) effect of both concentration and time of exposure was observed on the extent of DNA damage in treated fish. Similarly, malondialdehyde content, level of GSH, and activities of antioxidant enzymes were significantly elevated in treated groups, except CAT. The increased DNA damage and lipid peroxidation (LPO) content along with fluctuation in antioxidant defense system in fish indicated the interaction of cadmium (Cd) with DNA repair processes and production of reactive oxygen species. Thus, Cd is liable for induction of LPO, alteration of antioxidant defenses, and DNA damage in gills of L. rohita.     

Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium

Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. Oxidative damage and antioxidant properties have been studied in liver and gills of the European bullhead (Cottus gobio) subjected to cadmium (CdCl(2) at nominal concentrations of 0.01 and 1mg/L) for 4 days at either 15°C or 21°C. First, exposure to 1mg Cd/L induced a high mortality rate (67%) in fish held at 21°C. Regarding the antioxidant enzymes, exposure to 0.01 mg Cd/L significantly increased the activity of superoxide dismutase (SOD) and decreased the activity of glutathione reductase (GR) in liver, independently of heat stress. In gills, exposure to 21°C resulted in a significantly increased activity of glutathione peroxidase (GPx), whereas the activity of glutathione S-transferase (GST) was significantly reduced as compared to fish exposed to 15°C. Furthermore, regardless of Cd stress, exposure to elevated temperature resulted in a significant decrease of lipid peroxidation (LPO) level in liver and in a significant increase in the activity of chymotrypsin-like 20S proteasome in both studied tissues of C. gobio. Overall, the present results indicated that elevated temperature and cadmium exposure independently influenced the antioxidant defense system in bullhead with clear tissue-specific and stress-specific antioxidant responses. Further, elevated temperature affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues.     

Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

Deltamethrin-induced oxidative stress and biochemical changes in tissues and blood of catfish (Clarias gariepinus): antioxidant defense and role of alpha-tocopherol

ABSTRACT: BACKGROUND: The pyrethroid class of insecticides, including deltamethrin, is being used as substitutes for organochlorines and organophosphates in pest-control programs because of their low environmental persistence and toxicity. This study was aimed to investigate the impact of commonly used pesticides (deltamethrin) on the blood and tissue oxidative stress level in catfish (Clarias gariepinus); in addition to the protective effect of alpha-tocopherol on deltamethrin induced oxidative stress. METHODS: Catfish were divided into three groups, 1st control group include 20 fish divided into two tanks each one contain 10 fish, 2nd deltamethrin group, where Fish exposed to deltamethrin in a concentration (0.75ug/l) and 3rd Vitamin E group, Fish exposed to deltamethrin and vitamin E at a dose of 12ug/l for successive 4 days. Serum, liver, kidney and Gills were collected for biochemical assays. Tissue oxidative stress biomarkers malondialdhyde (MDA) and catalase activity in liver, kidney and gills tissues, serum liver enzymes (ALT and AST), serum albumin, total protein, urea and creatinine were analysed. RESULTS: Our results showed that 48 h. exposure to 0.75 ug/l deltamethrin significantly (p<0.05) increased lipid peroxidation (MDA) in the liver, kidney and gills while catalase activity was significantly decreased in the same tissues. This accompanied by significant increase in serum ALT, AST activity, urea and creatinine and a marked decrease in serum albumin and total proteins. CONCLUSIONS: It could be concluded that deltamethrin is highly toxic to catfish even in very low concentration (0.75 ug/l). Moreover the effect of deltamethrin was pronounced in the liver of catfish in comparison with kidneys and gills. Moreover fish antioxidants and oxidative stress could be used as biomarkers for aquatic pollution, thus helping in the diagnosis of pollution. Adminstration of 12 ug/l alpha-tocopherol restored the quantified tissue and serum parameters, so supplementation of alpha-tocopherol consider an effective way to counter the toxicity of deltamethrin in the catfish.     

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.     

The effects of selenium on oxidative stress biomarkers in the freshwater characid fish matrinxã, Brycon cephalus (Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BR (methyl parathion)

Methyl parathion (MP), an organophosphate widely applied in agriculture and aquaculture, induces oxidative stress due to free radical generation and changes in the antioxidant defense system. The antioxidant roles of selenium (Se) were evaluated in Brycon cephalus exposed to 2 mg L(-1) of Folisuper 600 BR (MP commercial formulation - MPc, 600 g L(-1)) for 96 h. Catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) levels in the gills, white muscle and liver were evaluated in fish fed on diets containing 0 or 1.5 mg Se kg(-1) for 8 weeks. In fish treated with a Se-free diet, the MPc exposure increased SOD and CAT activities in all tissues. However, the GPx activity decreased in white muscle and gills whereas no alterations were observed in the liver. MPc also increased GST activity in all tissues with a concurrent decrease in GSH levels. LPO values increased in white muscle and gills and did not change in liver after MPc exposure. A Se-supplemented diet reversed these findings, preventing increases in LPO levels and concurrent decreases in GPx activity in gills and white muscle. Similarly, GSH levels were maintained in all tissue after MPc exposure. These results suggest that dietary Se supplementation protects cells against MPc-induced oxidative stress.     

Heat Shock Protein 60 (HSP60) Response of <Emphasis Type="Italic">Plationus patulus</Emphasis> (Rotifera: Monogononta) to Combined Exposures of Arsenic and Heavy Metals

Organisms produce stress proteins as a response to natural and anthropogenic environmental changes. Induction of stress proteins has been reported in a variety of aquatic organisms, including rotifers, exposed to pollutants. Past studies on stress protein responses of rotifers have focused on exposure to single toxicants. In this study the rotifer Plationus patulus was exposed singly and in combination to various concentrations of As, Cr, Cu, Ni, Pb, and Zn. Following exposure, total protein was quantified (Bradford method) and stress protein 60 (HSP60) was identified using Western blotting. P. patulus induced HSP60 as a response to single exposures to Cr, Cu, Ni, Pb and Zn. HSP60 expression was increased (2 fold) in rotifers exposed to these single elements at both low and high concentrations as compared to unexposed rotifers. Arsenic exposure resulted in a 2 fold decrease in HSP induction. In rotifers exposed to metal mixtures, HSP60 was induced by the presence of As–Zn, As–Cr–Cu–Pb, As–Cr–Cu, As–Cr–Cu–Ni and As–Cr–Cu–Ni–Pb combinations in the media. HSP60 response to As and heavy metals toxicity depends on the type and number of elements present in the media as well as their concentrations and length of the exposure time.     

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.     

Quantitative RT-PCR analysis and immunohistochemical localization of HSP70 in sea bass Dicentrarchus labrax exposed to transport stress

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.     

The hepatic stress protein (hsp70) response to interacting abiotic parameters in fish exposed to various levels of pollution

Two indigenous fish species, brown trout (Salmo trutta f. fario) and stone loach(Barbatula barbatula) were exposed tocomplex stressors (mixtures of environmentalpollutants) in laboratory and semi-fieldexperiments (aquaria connected to stream water)and in field studies. As a biomarker of effect,the level of the 70 kD heat shock protein(hsp70) was quantified in the liver of troutand loach. Laboratory experiments withdifferent pollutant mixtures did not mimic thehsp70-inducing or inhibiting potential of fieldconditions, whereas effects of long-termexposure in the bypass systems showed asignificant correlation with effects recordedin feral fish. Laboratory as well as semi-fieldstudies revealed the stress response to followan optimum curve, resulting in a maximum hsp70level under stress but rather low hsp70 levelswhen stressors (chemicals, high temperature)become too severe. Consequently, the hsp70level in the liver of both species was highlyseason-dependent with two peaks in late springand fall, and rather low hsp70 levels insummer, particularly in fish exposed to waterand sediment of the complexly polluted stream.In winter, the low hsp70 level of lab controlswas elevated by exposure to natural streamwater only, but elevation did not occur undercontrol conditions independent of apre-exposure to polluted streamwater two months earlier. Despite the highvariability of the hsp70 level within one yearand among five subsequent years, patternanalysis indicated the prevailing importance ofwater temperature on stress protein response.Temperature alone, however, could not explainthe regularly observed low summer levels inhsp70. Non-linear regression analysis on labcontrols revealed an optimum temperature(T_opt) for the highest hsp70 level forboth fish species. In both investigated streams, thechemical influence led to a decrease in thehsp70 level only when T_opt was surpassedby the ambient temperature at the same time.Otherwise, the chemical impact resulted in anelevated hsp70 level relative to the control.The study demonstrated the suitability of hsp70stress protein levels to integrate the responsedynamics of several different stressors and,therefore, to effectively function as abiomarker for the integrated effect of allenvironmental stressors acting on an organism(not only of chemical pollution). Rathercomplex kinetics of hsp70 elevation anddecrease should be taken into consideration.     

Hymenolepis diminuta: Activity of anti-oxidant enzymes in different parts of rat gastrointestinal tract

The aim of this study was to assess the intensity of oxidative stress by measuring levels of lipid peroxidation products in the duodenum, jejunum and colon of rats infected with Hymenolepis diminuta and evaluate the effectiveness of protection against oxidative stress by measuring the glutathione levels and activity of anti-oxidant enzymes: superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase.In exposed rats we observed a significant increase of lipid peroxidation products in the duodenum and jejunum. A significant decrease in superoxide dismutase activity in all the examined parts of the digestive tract was observed. Additionally, rats from 16 to 40 days post H. diminuta infection (dpi) had a decreased catalase activity in the colon, while at 60 dpi it increased. The glutathione peroxidase activity increased significantly in the colon at 60 dpi. The increase in glutathione reductase activity was observed in the colon in rats 60 dpi. There was a lack of changes in the levels of glutathione in the duodenum and a significant increase in its concentration in the jejunum and colon from 40 to 60 dpi and from 16 to 40 dpi, respectively. In this study we observed altered activity of anti-oxidant enzymes and glutathione level in experimental hymenolepidosis, as a consequence of oxidative stress. It may indicate a decrease in the efficiency of intestinal protection against oxidative stress induced by the presence of the parasite. The imbalance between oxidant and anti-oxidant processes may play a major role in pathology associated with hymenolepidosis.     

Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature

Oxidative damage and antioxidant properties have been studied in Perna viridis subjected to short-term exposure to Hg along with temperature (72h) and long-term temperature exposures (14 days) as pollution biomarkers. The elevated thiobarbituric acid reactive substances (TBA-RS) levels observed in gills and digestive gland under exposure to Hg, individually and combined with temperature, as also long-term temperature stress have been assigned to the oxidative damage resulting in lipid peroxidation (LPX). Increased activities of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) both in gills and digestive glands under long-term exposures to temperatures are more prominent to heat rather than cold stress suggesting activation of physiological mechanism to scavenge the ROS produced during heat stress. Also decreased values of reduced glutathione (GSH) on long exposures to temperature stress indicate utilisation of this antioxidant, either to scavenge oxiradicals or act in combination with other enzymes, was more than its production capacity under heat stress. The results suggest that temperature variation does alter the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sublethal effects of pollution.     

Interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus)

The present study was to investigate the interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus). The fish were exposed to four total ammonia nitrogen (0, 5, 20, 40 mg/L TAN) and two stocking density. After 96 h of exposure, blood, gill, and liver samples were collected to measure biochemical parameters and mRNA levels of immune-related genes. The results showed that co-exposure to high TAN (20 and 40 mg/L) and high density significantly increased plasma sodium (Na⁺), gill Na⁺/K⁺-ATPase activity and mRNA levels. Following individual and combined exposure to high TAN and high density, the heat shock protein 70 (HSP 70), HSP 90, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) genes expression were obviously higher than their control. Conversely, the lysozyme (LZM) and hepcidin mRNA levels were down-regulated in liver of fish exposed to high TAN alone and combination of high TAN-density. Moreover, glutathione S-transferase (GST) mRNA levels significantly increased in treatments with individual high TAN and high density, but decreased in high TAN-density co-exposed fish. Overall, ion homeostasis and immune status were adversely influenced in high exposed turbot under high density.