Protein carbonyls: novel biomarkers of exposure to oxidative stress-inducing pesticides in freshwater fish Channa punctata (Bloch)

It has been established in mammalian system including humans that direct damage to proteins or chemical modification of amino acids in proteins during oxidative stress can give rise to protein carbonyls. Protein carbonyl induction, as a biomarker of oxidative stress was used in laboratory studies to assess the toxic effects of pesticides in freshwater fish, Channa punctata (Bloch), exposed to deltamethrin, endosulfan and paraquat. Protein carbonyls were measured in gills, kidney and liver. Significant (P<0.05-0.001) increase in protein carbonyls was observed in response to single 48h exposure to various pesticides in all the tissues. The time kinetics study involving deltamethrin (0.75μg/L) also showed a significant (P<0.05-0.001) induction of protein carbonyls in all the organs. The induction was significant (P<0.05-0.001) in all the durations of exposure (12h, 96h, 7 days, 14 days, 28 days). However, relatively pronounced induction was observed during shorter duration of exposure. The findings of the present investigation showed that deltamethrin had the maximum oxidative stress-inducing potential among the three pesticides used and gills are the most sensitive organs prone to oxidative damage. It is suggested that measurement of carbonyl groups may provide a convenient technique for detecting and quantifying oxidative modification of proteins during oxidative stress. The induction of protein carbonyl in fish was identified as a potentially useful biomarker of oxidative stress that warrants its application in the field investigations.     

Genotoxic and oxidative stress-inducing effects of deltamethrin in the erythrocytes of a freshwater biomarker fish species, Channa punctata Bloch

Deltamethrin, an alpha-cyano class of pyrethroid insecticide is used in insect pest control and antimalaria programs in several countries including India. Although various toxic manifestations of deltamethrin are reported in mammals, its ecotoxicologic dimensions are not adequately researched in ecologically and commercially important fishes. In this study, we report genotoxic effect of deltamethrin in a biomarker fish Channa punctata (Bloch). Adult fish were exposed to three concentrations of technical grade deltamethrin (0.4, 0.8, and 1.2 microg/L) for 48 and 72 h. Ethyl methane sulfonate was used as a positive control. Fish were analyzed for induction of micronucleus (MN), nuclear abnormalities (NAs), and oxidative stress biomarkers in erythrocytes. Deltamethrin significantly induced MN and NAs accompanied by increased lipid peroxidation. Activity of antioxidant enzyme superoxide dismutase was significantly decreased but an increase was observed in reduced glutathione level after 72 h of exposure. The NAs in exposed fish included blebbed, lobed and notched nuclei, and binucleated erythrocytes. Our findings suggest that oxidative stress may, in part, be contributing to deltamethrin-induced genotoxic damage to erythrocytes. Although MN induction is a nonspecific biomarker, it may provide an indication of pollution load of deltamethrin in the affected fish population when used as part of suite of other biomarkers.     

Inhibition of lymphocyte blastogenesis by serum leachates of fly ash

Samples of 8 ashes were leached with canine serum for 24 h to remove metal ions from the particle surfaces. The particles were removed by filtration, and the concentrations of 11 metal ions in the serum leachates were determined by atomic absorption spectrophotometry. The leachate samples were evaluated using the canine whole-blood lymphocyte stimulation test (WB/LST). The serum extracts of oil-related ashes were highly inhibitory, while lower biological activity was observed for the extracts of coal ashes. The observed inhibition in the WB/LST was correlated with the concentration of each metal ion using Kendall's rank correlation test. The highest correlations were observed for Mn and V. The results are compared with previous WB/LST studies on pure metal salts.     

Effect of acute hexavalent chromium exposure on pituitary–thyroid axis of a freshwater fish,Channa punctatus (Bloch)

Acute exposure to hexavalent chromium (10 mg L^?1, 20 mg L^?1, and 40 mg L^?1 potassium dichromate for 96 h) dose‐dependently affected the pituitary–thyroid axis of teleost, Channa punctatus. Significant hypertrophy of the thyroid follicle was observed in 20 mg L^?1 and 40 mg L^?1 groups; the follicular epithelium was however hypertrophied only in 40 mg L^?1 group. The colloid depletion in the lumen of thyroid follicle was evident in 20 mg L^?1 and 40 mg L^?1 groups. Serum thyroid hormones (thyroxine/T4 and triiodothyronine/T3) level increased significantly at both the higher doses. Increased immunointensity and significant hypertrophy of the pituitary thyrotrophs (anti TSHβ‐immunoreactive cells) was observed in both 20 mg L^?1 and 40 mg L^?1 chromium‐exposed fish. The increased thyroid hormones secretion observed in this study might be an adaptive response of the pituitary–thyroid axis under acute chromium‐induced stress condition to maintain homeostasis. The long‐term Cr(VI) exposures, however, may lead to attenuation/exhaustion of the pituitary–thyroid axis and pose serious threat to fish health and affect their population. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 621–627, 2015.     

Mutagenic and genotoxic assessment of atrazine-based herbicide to freshwater fish Channa punctatus (Bloch) using micronucleus test and single cell gel electrophoresis

The mutagenic and genotoxic effect of 'Rasayanzine', an atrazine herbicide, was carried out in fish Channa punctatus using micronucleus and single cell gel electrophoresis (SCGE) assays. Three sub-lethal concentrations of the test material viz., SL-I (1/5th LC(50)=～8.48 mgL(-1)), SL-II (1/8th LC(50)=～5.30 mgL(-1)) and SL-III (1/10th LC(50)=～4.24 mgL(-1)) were calculated using LC(50) value and the fish specimens were exposed to these concentrations. Erythrocyte and gill cells were sampled on day 0, 1, 3, 5, 7, 14, 21, 28 and 35 of exposure for assessment of micronuclei induction in blood erythrocytes and DNA damage using SCGE assay in both blood erythrocytes and gill cells. Significant effects (p<0.01) for both concentration and time of exposure were observed in treated fish. Micronuclei induction in erythrocytes was highest (8.4 mgL(-1)) on day 7 of exposure. The highest level of DNA damage in the SCGE was observed in both tissues on day 5 at all concentrations followed by gradual non-linear decline. This study further confirmed that the micronucleus and SCGE assays are useful in determining potential genotoxicity of water pollutants and might be appropriate as part of monitoring program.     

Ammonia-induced change in the hepatic glutathione level of an air-breathing freshwater teleost Channa punctatus (Bloch)

Exposure to ammonia (NH4OH) for 48 h resulted in greater than 50% depletion of the hepatic glutathione pool in Channa punctatus (Bloch). Removal of fish to ammonia free ambience induced a rapid and excessive resynthesis of glutathione within 24 h. This level, however, decreased within 5 days of return to ammonia-free fresh water. It is concluded that ammonia stress in a freshwater air-breathing teleost is counteracted by the glutathione system of detoxication of xenobiotics and resynthesis of glutathione is induced by short-term exposure to ammonia followed by transfer to ammonia-free water.     

Acute toxicity impacts of hexavalent chromium on behavior and histopathology of gill, kidney and liver of the freshwater fish, Channa punctatus (Bloch)

Alteration in behavioral patterns and histopathology of gill, kidney and liver were studied in Channa punctatus, after acute exposure to hexavalent chromium. The 96h LC(50) of chromium salt, potassium dichromate was determined to be 41.75mg/L. The exposed fish displayed erratic swimming and became lethargic. The changes in gills were characterized by epithelial hyperplasia, lamellar fusion, oedema, epithelial lifting, epithelial necrosis and desquamation, aneurism as well as curling of secondary lamellae. Hypertrophy of epithelial cells of renal tubules with reduced lumens, atrophy of the renal tubules, glomeruli contraction in the Bowman's capsules and necrosis of haematopoietic tissues were observed in the trunk kidney of exposed fish. The interrenal cells of the head kidney exhibited distinct hypertrophy and vacuolization. The liver hepatocytes showed cytoplasmic vacuolization with the lateral nuclei arrangement. Hepatocytes atrophy and increase in sinusoidal space were also observed. The result showed that acute chromium toxicity severely affects the vital organs and normal behavior which may be deleterious for fish populations.     

Impairments induced by sublethal doses of two pesticides in the ovaries of a freshwater teleost Channa striatus Bloch

Impairments induced by sublethal doses of two pesticides Carbaryl and Endosulfan have been studied on the ovaries of a freshwater teleost Channa striatus by exposing the fish for a period of 2 to 30 days during the spawning season. The pesticide exposure produced reduction in the number of oocytes, reduction in size and deformity in different stages of oocytes, damage to yolk vesicles in maturing and mature oocytes, increase in the number of atretic oocytes, development of interfollicular spaces, increase in the connective tissue of tunica albuginea, dilation of blood vessels and reduction in the gonadosomatic index. The histopathological effects were dependent on the dose, duration of exposure, and the type of pesticide. Sublethal doses of Endosulfan were more toxic than those of Carbaryl.     

Biochemical alteration in freshwater fish Channa punctatus due to latices of Euphorbia royleana and Jatropha gossypifolia

Exposure of fish over 96 h to 40 and 80% of LC(50) (24 h) of aqueous latex extracts of Euphorbia royleana and Jatropha gossypifolia of family Euphorbiaceae significantly altered the level of total protein, total free amino acids, nucleic acids, pyruvate, lactate, glycogen and as well as protease activity also in muscle, liver and gonadal tissue of the fish Channa punctatus. The alterations in all the biochemical parameters were significantly dose-dependent. Withdrawal study also shows that there is a partial recovery in the levels of glycogen, pyruvate, lactate and nucleic acids, but nearly complete recovery in total protein, total free amino acids level and protease activity in all the three tissues of the fish after the 7th day of the withdrawal of treatment, which supports the view that the plant product is safer in use as pesticides for control of common weed fishes in culture ponds.     

Alteration in the neurotransmitter levels in the brain of the freshwater snakehead fish (Channa punctatus) exposed to carbofuran

Channa punctatus a freshwater fish exposed to carbofuran (0.6 mg l^-1) for 15 days exhibited alterations in norepinephrine (NE), dopamine (DA) and serotonin (5-HT) levels. NE levels increased in the cerebellum and decreased in the medulla and cerebral cortex relative to the controls. Increased DA levels were observed in the cerebellum and medulla whereas DA levels decreased in the cerebral cortex relative to the controls. Depletion of 5-HT was noticed in the cerebellum, medulla and cerebral cortex relative to controls. The results suggested an overall decrease in the level of neurotransmitters in the cerebral cortex region of the brain. The neurotoxic effect of carbofuran was not uniform in the brain but was localized to the brain regions which regulate motor activity and behaviour in fish.     

Piscicidal and anti-acetylcholinesterase activity of Euphorbia royleana stem bark extracts against freshwater common predatory fish Channa punctatus

The present paper deals with the piscicidal activity of different solvent extracts of Euphorbia royleana stem bark against freshwater predatory fish Channa punctatus, which is commonly present in shrimp and carp culture ponds. The rank of order of toxicity of different solvent extract was: diethyl ether (LC(50) (96h) 31.76mg DW/L) > chloroform (LC(50) (96h) 56.26mg DW/L) > methanol (LC(50) (96h) 56.80mg DW/L) > acetone (LC(50) (96h) 65.77mg DW/L). There was a significant (P < 0.05) negative correlation between LC values and exposure periods, thus increase in exposure time. The LC(50) values of diethyl ether extract of E. royleana stem bark decrease from 81.78mg DW/L (24h) to 31.76mg DW/L (96h). Similar trend were also observed in case of chloroform, methanol and acetone extracts. Exposure of sub-lethal doses (40% and 80% of LC(50)) of the diethyl ether extract for 24h or 96h caused significant inhibition in the activity of enzyme acetylcholinesterase (AChE) in liver and muscle tissue of fish C. punctatus. There was a significant recovery observed in the activity of enzyme acetylcholinesterase in both the tissues of fish after 7th day of the withdrawal of treatment. Thus, the diethyl ether extract of E. royleana stem bark can be used for control of unwanted predatory fish C. punctatus from fish culture ponds. Their toxicological action is due to their anti-acetylcholinesterase enzyme activity, reversibility in their action is advantageous factor for their use as environmentally safe piscicide in aquatic bodies.     

Chronic toxic effects of the carbamate pesticide sevin on carbohydrate metabolism in a freshwater snakehead fish, Channa punctatus

The effect of exposure to a sublethal concentration of the carbamate pesticide, sevin (1.05 mg/l), on biochemical parameters of blood, liver and muscle, and enzyme activities in liver, kidney, intestine, brain, gills, and muscles of the freshwater teleost fish, Channa punctatus, was studied after 15, 30 and 60 days. The alterations produced were more marked after 30 and 60 days of exposure than after 15 days; fish were hyperglycaemic and hyperlactacidemic. Glycogen content of liver and muscles decreased, but lactic acid levels of the two tissues increased. In liver, muscles, brain and gills the lactate dehydrogenase (LDH) activity was higher in pesticide-exposed fish in comparison to control fish, but the same enzyme activity was inhibited in kidney and intestine. Decreased pyruvate dehydrogenase (PDH) activity occurred in all six tissues. Succinate dehydrogenase (SDH) activity decreased in muscle at the three time periods and after 30 and 60 days in liver and brain. However, in kidney and intestine the latter enzyme activity was elevated. The results suggest that anaerobic metabolism was favoured and aerobic oxidation of pyruvate was impaired in fish exposed to sevin.     

Mutagenic and genotoxic effects of carbosulfan in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Carbosulfan insecticide is widely used in agriculture and was recently proposed for treatment against pyrethroid-resistant mosquitoes. The mutagenic and genotoxic effect of carbosulfan was carried out in fish Channa punctatus using micronucleus (MN) test and comet assay. The 96 h LC₅₀, estimated by probit analysis in a semi-static bioassay experiment, was 0.268 mg l⁻¹. Based on the LC₅₀ value, three sub-lethal concentrations of carbosulfan (1/4th LC₅₀ = ∼67 μg l⁻¹, 1/2nd LC₅₀ = ∼134 μg l⁻¹ and 3/4th LC₅₀ = ∼201 μg l⁻¹) were selected and fishes were exposed to the said concentrations for 96 h and the samplings were done at regular intervals of 24 h for assessment of the MN frequencies and DNA damage. In general, significant effects (P < 0.01) from both concentrations and time of exposure were observed in exposed fishes. The MN induction was highest on 96 h at all the concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the erythrocyte and gill cells. This study confirmed that the comet and micronucleus assays are useful tools in determining potential genotoxicity of water pollutants and might be appropriate as a part of monitoring program.     

Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Chlorpyrifos (CPF) is the single largest selling agrochemical that has been widely detected in surface waters in India. The studies on long-term genotoxic effects of CPF in different tissues of fish using genotoxic biomarkers are limited. Therefore, in the present study DNA damage by CPF in freshwater fish Channapunctatus using micronucleus (MN) and comet assays was investigated. The LC₅₀ – 96 h of CPF was estimated for the fish in a semi-static system. On this basis of LC₅₀ value sublethal and nonlethal concentrations were determined. The DNA damage was measured in lymphocytes and gill cells as the percentage of DNA in comet tails and micronuclei were scored in erythrocytes of fishes exposed to above concentrations of CPF. In general, significant effects for both the concentrations and time of exposure were observed in treated fish. It was found that MN induction in the blood was highest on day 14 at 203.0 μg/l of CPF. The highest DNA damage was observed on day 5, followed by a gradual non-linear decline in the lymphocytes and gill cells. The study indicated MN and comet assays to be sensitive and rapid methods to detect mutagenicity and genotoxicity of CPF and other pollutants in fishes.     

Acute toxicity of synthetic pyrethroid cypermethrin to freshwater catfish Heteropneustes fossilis (Bloch)

Lethal concentrations of cypermethrin, dissolved either in water or acetone, were determined for freshwater catfish Heteropneustes fossilis at different hours of exposure by static bioassays. Up to 48 hours, there was no difference between LC(50) values of aqueous and acetone solublized cypermethrin. Seventy-two-hour LC(50) values of aqueous cypermethrin and acetone solublized cypermethrin to H. fossilis were 0.67 and 1.27 microg/L, respectively. Lethal values remained unchanged beyond 72 hours. The fish exposed to even lower concentration of cypermethrin (0.5 microg/L) showed hyperactivity.     

Effect of sublethal hexavalent chromium exposure on the pituitary-ovarian axis of a teleost, Channa punctatus (Bloch)

Exposure to sublethal hexavalent chromium (as 2 and 4 mg L^?1 potassium dichromate for 1 and 2 months) during late preparatory to mid prespawning phase of annual reproductive cycle severely affected the pituitary‐ovarian axis of a teleost Channa punctatus. Gonadosomatic index (GSI), ovarian histopathology, immunocytochemistry of the pituitary gonadotrophs (LHβ‐immunoreactive cells), and serum 17β‐estradiol level revealed distinct dose and duration‐dependent effects. Gonadosomatic index was declined. Diameter of ovary as well as ovarian follicles was reduced along with a distinct variation in the percentage of follicles. The greater percentage of previtellogenic follicles (stage I) and decline in vitellogenic ones (stage II and stage III) compared to control indicated the arrest of follicular development. The percentage of atretic follicles was also increased indicating toxic impact of metal on ovary. Decreased serum level of ovarian steroid 17β‐estradiol further indicated the same. Chromium interference of the pituitary was demonstrated as atrophy and less immunointensity of LH‐immunoreactive gonadotrophs. Thus, the hexavalent chromium impaired the pituitary‐ovarian axis affecting at the sites of both pituitary and ovary. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Acute spill‐mimicking exposure effect of hexavalent chromium on the pituitary‐ovarian axis of a teleost,Channa punctatus (Bloch)

Acute exposure to hexavalent chromium (as 10, 20, and 40 mg/L potassium dichromate for 96 h) adversely affected the pituitary‐ovarian axis of a teleost Channa punctatus. The toxic impact of metal exposure on fish ovary was revealed in the form of increased percentage of atretic follicles, significantly in 20 mg/L and 40 mg/L exposure groups. The follicular atresia mostly occurred in vitellogenic (stage II and stage III) oocytes. Reduction of serum level of 17β‐estradiol was also significant in 20 mg/L and 40 mg/L exposure groups. The increase of LH‐immunointensity of pituitary gonadotrophs (LHβ‐immunoreactive cells) and their hypertrophy was evident, significantly in fish of 40 mg/L exposed group. Thus, the present acute metal spill‐mimicking laboratory study clearly demonstrated that short‐term exposures to high doses of hexavalent chromium may disrupt reproduction of the fish and affect their population. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 733–739, 2014.