Neurotoxicological assessment of pendimethalin in freshwater fish Channa punctata Bloch

Over the years, indiscriminate usage of pesticides has resulted in situations which are not conducive for a good environment. In aquatic toxicology, fishes have been developed as established models for studying toxic responses of xenobiotics including pesticides. Pendimethalin (PD), an herbicide, is widely present in the aquatic environment, but little is known regarding its potential neurotoxicity in fish. The present study was conducted on Channa punctata Bloch exposed to sub-lethal doses of PD (0.5 and 0.8 ppb) for 96 h. The exposure resulted in alterations in epinephrine levels in the fish brain. Epinephrine levels decreased significantly in a dose dependent manner with increase in the PD exposure. A marked decrease in the activity of acetylcholinesterase along with reduction in Na⁺-K⁺-ATPase and monoamine oxidase activity was also observed. In comparison with the corresponding controls, the sub-lethal doses of PD also caused significant changes in the oxidative stress markers (lipid peroxidation and carbonyl derivatives of protein oxidative destruction levels) and antioxidant defenses (reduced glutathione levels, catalase, glutathione-s-transferase activity) in brain tissue. Our results reflect that a detailed investigation is warranted regarding the toxicity potential of PD.     

Multi-organ toxicological impact of fungicide propiconazole on biochemical and histological profile of freshwater fish Channa punctata Bloch

Fungicides are a pesticide that particularly kills or destroy fungi responsible for several diseases associated to humans and other living organisms. Assessment of toxic effects and mechanisms of fungicide action is important because humans and domesticated animals get exposed to these pesticides through a wide variety of applications. Several fungicides are being used at the large scale for the crop protection from the fungal invasion. Propiconazole (PCZ), a trazole-containing fungicide, is widely used in China and various Asian countries for food crop protection which made it easily to exposed to the aquatic system. Long term usage of PCZ may contaminate the water bodies, but its toxicity to aquatic organisms is not well studied. In this study, freshwater fish, Channa punctata Bloch was exposed to different sub-lethal concentrations of the fungicide, PCZ (0.5 and 5 ppm) for a period of 96 h. Various biochemical assays and histological alterations were measured to determine the organ toxicity caused by PCZ exposure particularly in liver, kidney and gills of the fishes. Compared to the control group, fish exposed to PCZ (96 h) showed marked dose dependent toxicity. The levels of lipid peroxidation (LPO) and protein carbonyls (PC), oxidative stress biomarker of liver, kidney and gills in the experimental group were significantly higher (P < 0.05 and P < 0.001) compared to the control group. Levels of reduced glutathione (GSH) and non-protein thiols (NP-SH) decreased significantly (P <0.05–0.001) in all analyzed intoxicated organs of the PCZ exposed fishes. Activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) in fungicide treated groups was significantly lowered (P < 0.05–0.001). In addition, histopathological examination in the organs showed significant changes like atrophy of primary and secondary gill lamellae, infiltrations, inflammation, hepatocyte degeneration, vacuolization and necrotic kidney. Thus, PCZ exposure altered the oxidative stress homeostasis and brought about histopathological changes which may serve as potential biomarkers of the PCZ toxicity in the laboratory set-up for potential risk assessment.     

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.     

In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctata Bloch

Mammalian metallothioneins (MT) have been reported to scavenge free radicals. There is no experimental evidence to show that fish MT has a similar property. In the present study cadmium-induced MT (Cd-MT) from the liver of an Indian freshwater fish Channa punctata Bloch was investigated for its free radical scavenging activity using three different in vitro assays. Exposure to cadmium chloride (0.2 mg/kg body weight; three doses on alternate days) resulted in a marked induction of Cd-MT in liver. Only a single isoform of Cd-MT was found to be induced. Molecular weight of Cd-MT was found to be 14 kDa as deduced by SDS-PAGE analysis. The purified Cd-MT effectively scavenged the following free radicals: superoxide radical (O2*-), 2,2'-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS*+) and 1,1-diphenyl-picrylhydrazyl radical (DPPH*). The radical scavenging effect was found to be concentration-dependent. Also, the purified MT exhibited an inhibitory effect on ferric nitrilotriacetate (Fe-NTA) induced oxidative DNA damage in vitro. The cysteine residues of MT are proposed to be the main candidate for its radical scavenging activity. Findings of the present study strongly suggest a free radical scavenging role for fish MT. Present study adds to the little existing knowledge about fish MT and its possible biological functions.     

Effects of exposure to multiple trace metals on biochemical, histological and ultrastructural features of gills of a freshwater fish, Channa punctata Bloch

The trace metals are frequently encountered as mixtures of essential and non-essential elements. Therefore, evaluation of their toxic effects individually does not offer a realistic estimate of their impact on biological processes. We studied effects of a mixture of four essential and toxic metals (Cu, Cd, Fe and Ni) on biochemical and morphological characteristics of the gills of a biomarker freshwater fish Channa punctata (Bloch) using environmentally relevant concentrations. Fish were exposed to metal mixture through tank water for 7, 15 and 30 days. Biochemical studies as well as light microscopy (LM) and scanning electron microscopy (SEM) revealed significant metal exposure-induced alterations in gills. Besides ultastructural changes, activities of antioxidant enzymes such catalase (CAT), glutathione S-transferase (GST) and superoxide dismutase (SOD) were significantly altered in the gills of exposed fish. The reduced glutathione (GSH) was significantly (p<0.001) decreased, while lipid peroxidation (LPO) was significantly (p<0.001) increased. The main alterations in general morphology of fish gills included spiking and fusion of secondary lamellae, formation of club-shaped filaments, and vacuolization and necrosis of filament epithelium in the interlamellar regions. SEM studies showed gradual increase of the density and apical surface area of the chloride cells and transformation of the surface structure of the pavement cells. The results of this study indicate adaptive as well a toxic responses in fish gills exposed to mixture of trace metals. Low concentrations of trace metal appear to compromise the antioxidant defense of gills. Lesions in the gill morphology caused by the effect of low concentrations of trace metals could lead to functional alterations and interference with fundamental processes such as maintenance of osmoregulation, gas exchange and xenobiotic metabolism in the exposed fish populations.     

Retinopetal neuronal system in the brain of an air-breathing teleost fish,Channa punctata

Summary Retinopetal neurons were visualised in the telencephalon and diencephalon of an air-breathing teleost fish, Channa punctata, following administration of cobaltous lysine to the optic nerve. The labelled perikarya (n=45–50) were always located on the side contralateral to the optic nerve that had received the neuronal tracer. The rostral-most back-filled cell bodies were located in the nucleus olfactoretinalis at the junction between the olfactory bulb and the telencephalon. In the area ventralis telencephali, two groups of telencephaloretinopetal neurons were identified near the ventral margin of the telencephalon. The rostral hypothalamus exhibited retrogradely labelled cells in three discrete areas of the lateral preoptic area, which was bordered medially by the nucleus praeopticus periventricularis and nucleus praeopticus, and laterally by the lateral forebrain bundle. In addition to a dorsal and a ventral group, a third population of neurons was located ventral to the lateral forebrain bundle adjacent to the optic tract. The dorsal group of neurons exhibited extensive collaterals; a few extended laterally towards the lateral forebrain bundle, whereas others ran into the dorsocentral area of the area dorsalis telencephali. A few processes extended via the anterior commissure into the telencephalon ipsilateral to the optic nerve that had been exposed to cobaltous lysine. However, the ventral cell group did not possess collaterals. In the diencephalon, retinopetal cells were visualised in the nucleus opticus dorsolateralis located in the pretectal area; these were the largest retinopetal perikarya of the brain. The caudal-most nucleus that possessed labelled somata was the retinothalamic nucleus; it contained the largest number of retinopetal cells. The limited number of widely distributed neurons in the forebrain, some with extensive collaterals, might participate in functional integration of different brain areas involved in feeding, which in this species is influenced largely by taste, not solely by vision.     

Evaluation of genotoxic and oxidative stress response to dimethoate in freshwater fish Channa punctatus (Bloch)

Abstract

Dimethoate is one of the organophosphate insecticides widely used in agriculture throughout the world and is an inhibitor of cholinesterase in animals. The objective of the present study was to detect oxidative stress and DNA damage induced by dimethoate in the freshwater fish Channa punctatus (C. punctatus). The LC₅₀-96 h value of technical grade dimethoate was estimated at 19.10 μg L^-1 in a semi-static system and, on the basis of the LC₅₀ value, three concentrations were determined. The fish were exposed to these concentrations of dimethoate for 96 h and samplings were done at 24 and 96 h for assessment of oxidative stress and DNA damage. After exposure to dimethoate, the level of superoxide dismutase declined while lipid peroxidation, glutathione, induction of micronucleus and DNA damage were increased in C. punctatus as the concentration and exposure time increased. Thus our results suggest that dimethoate induces genotoxic effects which invariably accompanied and correlated with oxidative stress.     

Bisphenol A induced toxicity in blood cells of freshwater fish Channa punctatus after acute exposure

The widespread use of bisphenol A (BPA) has led to its ubiquity in the natural environment. It is extensively incorporated into different industrial products and is associated with deleterious health effects on both public and wildlife. The current trial was conducted to determine the toxic potential of bisphenol A using various parameters viz haematological, biochemical, and cytological in freshwater fish Channa punctatus. For this purpose, fish were exposed to 1.81 mg/l (1/4 of LC₅₀) and 3.81 mg/l (1/2 of LC₅₀) of BPA along with positive (acetone) and negative controls (water) for 96 h. The blood samples were collected at 24, 48, 72, and 96 h post-exposure. Compared to the control group, fish after acute exposure to BPA showed a significant decrease in HB content, number of red blood cells, PCV values whereas a significant increase in WBCs count was recorded with an increase in the exposure period. Besides, oxidative stress (determined as malondialdehyde content) increased as BPA concentration increased. Further, the activity of different antioxidant enzymes like catalase, and superoxide dismutase decreased significantly after treatment. Results also showed significantly increased frequency of morphological alterations, nuclear changes, and increased DNA damage potential of BPA in red blood cells. Further structural analysis of erythrocytes in maximally damaged group using Scanning Electron Microscopy was performed. The study concludes that BPA exhibits genotoxic activity and oxidative stress could be one of the mechanisms leading to genetic toxicity.     

Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

The aim of the present study was to evaluate the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) in gill cells of freshwater fish Channa punctatus using single cell gel electrophoresis (SCGE)/Comet assay. The 96h LC(50) value of PFF (50% EC) was estimated for the fish species in a semistatic system and then three sub-lethal of LC(50) concentrations viz the sub-lethal 1, sub-lethal 2 and sub-lethal 3 concentrations were determined as 0.58ppb, 1.16ppb and 1.74ppb, respectively. The fish specimens were exposed to these concentrations of the pesticide and the gill tissue samplings were done on 24h, 48h, 72h and 96h post exposure for assessment of DNA damage in terms of percentage of DNA in comet tails. In general, a concentration dependent response was observed in the gill cells with induction of maximum DNA damage at the highest concentration of PFF. The results of the present investigation indicated that PFF could potentially induce genotoxic effect in fish, even in sub-lethal concentrations and SCGE as a sensitive and reliable tool for in vivo assessment of DNA damage caused by the genotoxic agents.     

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.     

Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain

The aim of the present study is to evaluate the oxidative damage in rats of different ages. Weaned rats of 25 g and adults of 300 g were used in groups of 6, a single i.p. dose of morphine sulfate of 3, 6 or 12 mg/kg was administered. All animals were sacrificed to measure GSH and 5-HT levels in brain by liquid chromatography, as well as Na⁺, K⁺-ATPase and total ATPase enzymatic activity. 5-HT levels decreased significantly (p<0.05) in adult animals that received 3 and 6 mg morphine. Na⁺, K⁺-ATPase activity increased significantly (p<0.05) in all groups of weaned animals. In adult animals, Na⁺, K⁺-ATPase and total ATPase partially diminished. GSH levels diminished significantly (p<0.05) both in weaned and in adult groups. The results indicate age-induced changes in cellular regulation and biochemical responses to oxidative stress induced by morphine.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resistance to oxidative stress in a freshwater fish Channa punctatus after exposure to inorganic arsenic

The biochemical toxicity of arsenic trioxide (As^III) in a freshwater edible fish Channa punctatus has been studied on exposures ranging from 7 to 90 d. The arsenic concentration increased exponentially in liver, kidney, gills, and muscles of fish up to 60 d of exposure to arsenic. However, arsenic concentration in these tissues declined at 90 d of exposure. This relationship between period of exposure and concentration of arsenic in selected tissues suggests an adaptive response of fish to arsenic. Furthermore, exposure to arsenic-induced lipid peroxidation in these organs increased initially at 7 d of exposure; however, it decreased up to 60 d of exposure but increased again at 90 d of treatment. Values of reduced glutathione (GSH) reflected the observations of lipid peroxidation. The role of GSH in this adaptive response has been discussed.     

A review of some physiological and toxicological responses of freshwater fish to acid stress

Synopsis Data relating to the specific effect of low pH on growth of freshwater fishes are ambiguous. Reproductive failure resulting from acid stress appears to be related to an upset in calcium metabolism and to faulty deposition of protein in developing oocytes. It appears that the ’no effect‘ level of pH depression for successful reproduction is around 6.5. Data on behaviorial responses of freshwater fish to acid stress and CO₂ are described. Most fish appear to be indifferent to pH within the range of approximately 10.5 to 5.5 and between 7.4 and 4.5 CO₂ appears to be the main directive factor. In cases of severe acid stress alteration of gill membranes and/or coagulation of gill mucus occurs and death due to hypoxia may result from a lengthening of the water-blood diffusion distance. Several reports agree that acid stress causes an upset of electrolyte homeostasis in fish but effects of low pH on osmotic permeability are largely lacking. Most hatcheryreared salmonids can tolerate pH 5.0 indefinitely but below this level the homeostatic electrolyte and osmotic regulatory mechanisms become inadequate. When fish are subjected to debilitating acid stress blood pH decreases possibly as the result of flux of H⁺ ions across gill membranes into the blood. This could change transepithelial potential and allow a blood, to-water diffusion of Na⁺ ions down an electrochemical gradient. Lowered ambient pH may interfere with gill calcium levels increasing permeability to both H⁺ and Na⁺ ions or an acidemia may occur as the result of a decrease in the excretion of metabolically produced H⁺ ions and CO₂. When the capacity of the buffer mechanisms is exceeded the blood pH drops and the capacity of hemoglobin to transport oxygen is decreased.     

The interactive effect of elevated temperature on deltamethrin-induced biochemical stress responses in Channa punctata Bloch

There are reports showing interactive effect of environmental factors with the toxic outcome of chemicals. We studied the interactive effect of elevated temperature as an abiotic stressor on deltamethrin-induced biochemical stress responses in a freshwater fish, Channa punctata Bloch. Heat stress (∼12 °C above ambient temperature for 3 h) and pesticide exposure (deltamethrin 0.75 ppb for 48 h) showed significant induction of heat shock protein-70 (HSP70) in liver, kidney and gills of fishes. Elevated temperature when followed by deltamethrin exposure showed synergistic effect showing a high level of HSP70 in liver and gills whereas response in the kidney was opposite. On the contrary, when deltamethrin exposure followed the heat stress, no significant difference was observed. Protein carbonylation was found to be more pronounced in heat-stressed group compared with control fish group. A significant increase in lipid peroxidation (LPO) was observed in different tissues of fish exposed to either of the stressors. In the kidney of fish exposed to heat stress followed by deltamethrin, LPO was relatively lower as compared to other treatments. Thiols content such as reduced glutathione (GSH), total thiols (T-SH), non-protein thiols (NP-SH) and protein thiols (P-SH) showed no consistent pattern in different tissues. In deltamethrin-exposed group that was subsequently exposed to heat stress, the GSH content was higher in liver and lower in both kidney and gills when compared with other groups. Alteration in the activities of antioxidant enzymes such as catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) was also observed when fish were exposed to heat stress and/or deltamethrin. Our study demonstrated that heat stress modulated biochemical stress responses in fish showing a tissue specific pattern. This implies that fish has the capacity to elicit differential response to exposure to abiotic stressors in order to reduce the systemic magnitude of stress which may otherwise lead to severe dysfunction of vital tissues.     

Changes in developmental profiles of brain gangliosides during ontogeny of a teleost fish (Sarotherodon mossambicus,Cichlidae)

Summary Ontogenetic changes of brain ganglioside concentration and composition have been followed in the teleost fishSarotherodon mossambicus Cichlidae) from the 1st day post hatching to the adult stage, in order to correlate these with findings in higher vertebrates. During the developmental period from hatching to the transition to free swimming, which comes along with maximum rate of synaptogenesis, a sharp rise in the brain ganglioside content occurs, which is mainly due to the trisialoganglioside GT1b. In the following phase of myelination (characterized in birds and mammals by an increase in GM1, GM1 and GM4) accretion of the novel and so far unidetified fraction (GM2) occurs, which is highly enriched in the brain stem. The results obtained are discussed with respect to gangliosides as useful biochemical markers for brain development and maturation in all vertebrates.     

Channa punctata brain metallothionein is a potent scavenger of superoxide radicals and prevents hydroxyl radical-induced in vitro DNA damage

Mammalian brain metallothioneins (MTs) have been shown to scavenge free radicals. However, a similar role for fish brain MT has not been established yet. Previously, we have reported that MT from the liver of a freshwater fish, Channa punctata Bloch, had free-radical-scavenging activity in vitro. In this study, we report on the induction of MT in brain and other tissues of C. punctata treated with a low concentration of zinc chloride. We partially purified MT (Zn-MT)-rich fraction from the brain and studied its free-radical-scavenging and DNA damage attenuating effects. Zinc exposure showed significant MT induction in brain, gill, kidney, and liver. C. punctata brain MT efficiently scavenged superoxide radicals and also attenuated hydroxyl radical-mediated DNA damage. These findings suggest that fish brain MT has a free-radical-scavenging activity, and its expression may be regulated in response to stress and chemical exposure. C. punctata has been identified as a potent biomarker fish species. It is suggested that this fish species may be a good model for the study of MTs with regard to their regulatory and biomarker functions.     

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.     

Effect of ammonia-stress on the total autolytic levels of proteins in tissues of an air-breathing fish,Channa punctatus (Bloch)

G season-specific effect of a sub-lethal ambient ammonium chloride concentration on the total autolysis of protein in different tissues of the Indian air-breathing murrel,Chauna punctatos (Bloch), has been demonstrated. While its effect on the autotytie levels of protein of different tissues (except white muscle) was marginal in the winter-adapted fish, the same in the summer-adapted fish was more pronounced. In general a reduction in the autolysic levels of the tissue protein of the ammonium chloride-stressed fish was observed. In certain tissues like white muscle of an ammonium chloride-stressed fish and gill of the ammonium hydroxide-stressed fish, augmented levels of autolysis were noticed. The present findings suggest that the accumulation of amino acids in different tissues of the ammonia-stressed fish, as observed earlier, can be explained best by mechanisms other than ammonia induced increased breakdown of proteins.