Basdeo Kushwaha

Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis.

Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridylphosphorothioate) is one of the organophosphate pesticides widely used in agricultural practices throughout world and irreversible inhibitor of cholinesterase in all animal species. Limited efforts have been made to study acute genotoxic effects of chlorpyrifos (CPF) in different tissues of fish using genotoxic biomarkers. Therefore, the present investigation was aimed to study the induction of DNA damage by CPF in freshwater teleost fish Channapunctatus using micronucleus assay (MN assay) and alkaline single-cell gel electrophoresis (comet assay). The value of LC(50) - 96 h of CPF was determined as 811.98 microgl(-1) for C. punctatus, in a semi-static system and on the basis of LC(50) value three acute concentrations viz., 203, 406 and 609 microgl(-1) were determined. The fishes were exposed to the different concentrations of CPF for 96 h and samplings were done at regular intervals 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. It was found that the micronucleus induction was highest on 96 h at all concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the lymphocyte and gill cells. This study explored the combined use of micronucleus assay and comet assay for in vivo laboratory studies using fresh water fish for screening the genotoxic potential of xenobiotics.

Toxicity of the Herbicide Atrazine: Effects on Lipid Peroxidation and Activities of Antioxidant Enzymes in the Freshwater Fish Channa Punctatus (Bloch)

The present study was undertaken to evaluate the toxicity and effects of a commercial formulation of the herbicide atrazine (Rasayanzine) on lipid peroxidation and antioxidant enzyme system in the freshwater air breathing fish Channa punctatus. The 12, 24, 48, 72 and 96 h LC50 of atrazine, calculated by probit analysis, were determined to be 77.091, 64.053, 49.100, 44.412 and 42.381 mg·L−1, respectively, in a semi static system with significant difference (p < 0.05) in LC10–90 values obtained for different times of exposure. In addition to concentration and time dependent decrease in mortality rate, stress signs in the form of behavioral changes were also observed in response to the test chemical. In fish exposed for 15 days to different sublethal concentrations of the herbicide (1/4 LC50 = ∼10.600 mg·L−1, 1/8 LC50 = ∼5.300 mg·L−1 and 1/10 LC50 = ∼4.238 mg·L−1) induction of oxidative stress in the liver was evidence by increased lipid peroxidation levels. The antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) responded positively in a concentration dependent pattern, thus, suggesting the use of these antioxidants as potential biomarkers of toxicity associated with contaminations exposure in freshwater fishes.

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 LC(50) - 96 h of CPF was estimated for the fish in a semi-static system. On this basis of LC(50) 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 microg/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.

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 96h LC(50), estimated by probit analysis in a semi-static bioassay experiment, was 0.268 mg l(-1). Based on the LC(50) value, three sub-lethal concentrations of carbosulfan (1/4th LC(50)= approximately 67 microg l(-1), 1/2nd LC(50)= approximately 134 microg l(-1) and 3/4th LC(50)= approximately 201 microg l(-1)) were selected and fishes were exposed to the said concentrations for 96h and the samplings were done at regular intervals of 24h 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 96h 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.

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.

DNA damage and oxidative stress modulatory effects of glyphosate-based herbicide in freshwater fish, Channa punctatus.

The present study was undertaken to evaluate the genotoxic and oxidative stress modulatory effects of commercial formulation of glyphosate-based herbicide (Roundup(®)) in freshwater fish Channa punctatus. Three sublethal test concentrations of the herbicide viz., SL-I (1/10th of LC50=∼3.25mgL(-1)), SL-II (1/8th of LC50=∼4.07mgL(-1)) and SL-III (1/5th of LC50=∼6.51mgL(-1)) were calculated using 96-LC50 value and the test specimens were exposed to these concentrations. Blood and gill cells of the exposed specimens were sampled on day 1, 7, 14, 21, 28 and 35 to examine the DNA damage using comet assay and to assess the alteration in lipid peroxidation and antioxidant enzymes activities. The highest DNA damage was observed on day 14 at all test concentrations followed by gradual non-linear decline. Induction of oxidative stress in the blood and gill cells were evidenced by increased lipid peroxidation level, while antioxidants namely superoxide dismutase, catalase and glutathione reductase responded in a concentration-dependent manner. The results supported the integrated use of comet and antioxidant assays in determining the toxicity of water pollutants which could be used as part of monitoring programs.

Genotoxicity and antioxidant enzyme activity induced by hexavalent chromium in Cyprinus carpio after in vivo exposure.

Abstract Fish, being an important native of the aquatic ecosystem, are exposed to multipollution states and are therefore considered as model organisms for ecotoxicological studies of aquatic pollutants, including metal toxicity. We investigated oxidative stress (OS) in liver, kidney and gill tissues through antioxidant enzyme activities and genotoxicity induced in whole blood and gill tissues through comet assay and micronucleus (MN) test in Cyprinus carpio after 96-hour in vivo static exposure to potassium dichromate at three sublethal (SL) test concentrations, including SL-I [93.95 mg/L, i.e. one quarter of half-maximal lethal concentration (LC50)], SL-II (187.9 mg/L, i.e. one half of LC50), and SL-III (281.85 mg/L, i.e. three quarters of LC50), along with a control. The 96-hour LC50 value for potassium dichromate was estimated to be 375.8 mg/L in a static system in the test species. Tissues samples were collected at 24, 48, 72 and 96 hours postexposure. Results indicated that the exposed fish experienced OS as characterized by significant (p < 0.05) variation in antioxidant enzyme activities, as compared to the control. Activities of superoxide dismutase and glutathione peroxidase increased, whereas activity of catalase decreased with the progression of the experiment. The mean percent DNA damage in comet tail and MN induction in gills and whole blood showed a concentration-dependent increase up to 96-hour exposure. The findings of this study would be helpful in organ-specific risk assessment of Cr(VI)-induced OS and genotoxicity in fishes.

Genotoxic and Mutagenic Assessment of Hexavalent Chromium in Fish Following In Vivo Chronic Exposure

ABSTRACT Chromium is a well-documented carcinogen. To evaluate the genotoxic potential of hexavalent chromium on an aquatic bio-system, freshwater murrel fish (Channa punctatus) were exposed to potassium dichromate. The 96-h LC50 for potassium dichromate was 61.80 mg/L for the test fish in a static system. On the basis of the 96-h LC50, fish were exposed to sublethal concentrations of the test chemical. Fish exposed to the test chemical were sampled on days 1, 7, 14, 21, and 28 post-exposure and blood and gill cells were collected. Significantly (p < .05) higher DNA damage in both lymphocyte and gillcells and micronuclei formation in whole blood was observed at different test concentrations and sampling times of the test chemical as compared to control fish. The mean% tail DNA in the comet tail assay showed a concentration-dependent increase and the maximum% tail DNA was observed on day 7 of exposure in both cells. A similar trend was also observed in micronuclei induction in blood with maximum induction on day 21. Hexavalent chromium showed genotoxic potential in chronic exposure of C. punctatus, and the micronucleus test and the comet assay are the methods for sensitive and rapid detection of the genetic effects.

Population distribution of 45S and 5S rDNA in golden mahseer, Tor putitora: population-specific FISH marker

Chromosomal locations of major 45S and minor 5S ribosomal DNAs (rDNAs) and organization of 5S rRNA genes were analysed in five different populations of golden mahseers (Tor putitora) using fluorescence in situ hybridization (FISH) and Southern blot hybridization. All five populations of T. putitora (2n = 100) showed a similar type of macro-karyotype composed of 12 metacentric, 22 submetacentric, 14 subtelocentric and 52 telocentric chromosomes. Analysis of active nucleolar organizer regions (NORs) by silver staining did not show any differences in number and chromosomal position in different populations. But FISH data showed significant difference between the populations, four of the five populations showed six 18S (three pairs) and two 5S (one pair) signals with positional polymorphism, while one population showed eight 18S and four 5S signals, respectively. Southern blot data confirms that 5S rDNA clusters present on two different chromosome pairs in Kosi river population contain non-transcribed spacers (NTS) of same length. In the present study, simultaneous localization of 45S and 5S rDNA by in situ hybridization helped us to develop the discrete population-specific markers in different geographically isolated populations of T. putitora.

In situ assessment of genotoxic and mutagenic potential of polluted river water in Channa punctatus and Mystus vittatus

River Gomti, a tributary of river Ganga in northern India, is being polluted due to indiscriminate disposal of domestic sewage and industrial wastes that contain genotoxic chemicals. The study was conducted to evaluate the genotoxic potential of polluted water of river Gomti in two fish species, namely Channa punctatus and Mystus vittatus. The fishes were exposed in situ in nylon cages to the polluted water of river Gomti fixed near a distillery outlet located in Lucknow. The induction of DNA damage and micronuclei were determined in blood erythrocytes using comet assay and micronucleus test, respectively. The induction in micronuclei frequencies and DNA damage were found to be significantly elevated (p < 0.01) in exposed specimens after 3 days post-exposure as compared to the control, i.e. from laboratory-acclimatized fish specimens. The comparison of DNA damage between the two species indicated that C. punctatus is more sensitive to aquatic pollutants. Thus, this fish could be used as a bio-indicator of genotoxicity for bio-monitoring of water bodies. The results further revealed that the river Gomti is being contaminated with potential genotoxic and mutagenic chemicals produced from industrial and domestic activities; therefore, immediate measures are needed to reduce the inflow of pollutants in the river.