Variation in genotoxic susceptibility and biomarker

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14590 Environ Sci Pollut Res 2016 23 14589 14599, of the country A recently conducted nationwide wastewater low concentrations Goksoyr et al 1991 The micronucleus. assessment showed that the total wastewater supply in assay was initially developed for application in mammals. Pakistan is 4 6 106 m3 day and a total of 7 85 million cubic but has now been subsequently modified for use in fish. meters per day of wastewater 30 of the total is used for Micronucleus assay is one of the best biomarkers that clearly. irrigating an area of 32 500 ha It has also been estimated that correlate with pollution load Bar ien et al 2013 The aim. 64 of the total wastewater is disposed of either into rivers or of this study was to assess the genotoxic potential of Catla. into the Arabian Sea Similarly 400 000 m3 day wastewater is catla and Cirrhinus mrigala as data regarding the genotoxic. additionally added into canals These practices threaten both and mutagenic nature of composite heavy metals on fish. human health and the environment at downstream and more which is lacking from natural aquatic ecosystem. importantly reduce the effective availability of Pakistan s al Furthermore the study aimed to evaluate the suitability. ready short water supplies Ensink et al 2004 Indiscriminate and sensitivity of blood cells through the comet and micro. disposal of sewage and industrial effluent has seriously affect nucleus assays of two selected fish species indwelling a. ed the quality of surface water heavy metal contaminated aquatic ecosystem as a reliable. Aquatic animals have an important role as bioassays to indicator of DNA damage and water pollution load. monitor water bodies for pollution Changes in the ge. nome caused by genotoxic pollutants can lead to muta. tions and DNA damaging agents require continuous Materials and methods. monitoring and detection Villela et al 2006 There is a. need to develop the molecular basis that can mark the Study area. effects of environmental pollutants for a wide range of. organisms Ostling and Johanson 1984 introduced a The polluted water of the Chenab River has led to the extinc. microgel electrophoresis technique for the detection of tion and decrease in population of different species of fishes in. DNA damage in a single cell Singh et al 1988 used almost 190 km of its length The industrial and sewage wastes. the same technique under alkaline conditions pH 13 of Faisalabad city are disposed into the Rivers Chenab and. in order to detect the DNA damage in individual cells Ravi Chenab River receives a vast amount of these industrial. the so called comet assay This assay presents advantages and municipal wastes from the eastern and southern parts of. in comparison to other genotoxicity assays in that it is Faisalabad city through the Chakbandi Drain This polluted. sensitive enough to detect low levels of DNA damage water contains large amounts of toxic chemicals from a variety. and requires only a small number of cells per sample of industries such as textiles chemical and pharmaceutical. Tice et al 2000 Another of the most promising and industries tanneries and sugar mills etc well sufficient to. popular assays is the micronucleus MN test This tech reduce the water productivity of Chenab River by changing. nique is a marker of cytogenetic damage mainly caused the water quality parameters necessary for the growth of. by aneugenic or clastogenic compounds The assessment aquatic flora and fauna This detrimental change in water qual. of cytogenetic damage is a very important assay for the ity has reduced the population of many fish species including. identification of pollution threats in aquatic environments Indian major carps The freshwater fish species C mrigala. Dixon et al 2002 and C catla collected from highly polluted areas were ana. Genotoxic pollution in aquatic environments has led to lyzed for DNA damage. the development of many different test systems Water and. sediment samples could be tested for water quality parame Sampling of fish species. ters and biological systems such as bacteria yeast aquatic. animals and plants Unfortunately the river system in Fish were harvested from Chenab River in Pakistan through. Pakistan has been dangerously exposed to toxic industrial its 190 km length upstream of Trimu Head This part of the. effluents and various kinds of pollutants from urban and river is highly polluted due to the disposal of sewage and. agricultural areas These toxicants adversely affect not only industrial waste through the Chakbandi Drain at latitude. the ecosystems of water bodies but also human health 31 570 and longitude 72 534 Fig 1 Three polluted sites. Recently research interest on the use of bioindicators and viz Wara Thatta Muhammad Shah R1 Bela Reta R2 and. biomarkers for investigation of the mutagenic effects of pol Bandimahni Beg R3 were selected along the length of. lution environmental monitoring is developing El Chenab River after receiving polluted water from the. Shehawi et al 2007 For this purpose fishes are suitable Chakbandi Drain Two upstream sites Libhan Wala U1. organisms Landolt and Kocan 1983 because they are sus and Thali U2 were selected as the upstream sites before. ceptible to a variety of pollutants and play different and entering the drain into Chenab River in Tehsil and District. important roles in heterotrophic web undergoing bioaccumu Jhang for the comparison of wild fish Apart from during the. lation They respond to a variety of mutagens even at very rainy season this polluted water flows through Chenab River. Environ Sci Pollut Res 2016 23 14589 14599 14591,Fig 1 Percent DNA damage in. Catla catla and Cirrhina mrigala,type and site interactions. up to Trimu Head Dragnets and gill nets were used to harvest Comet assay. seven fish of each species from three different sites R1 R2. and R3 of the river upstream to this area with farmed fish Immediately after blood sampling a small amount of blood. being used as the control Farmed fish with toxicants supplied 40 L was diluted with phosphate buffered saline and stored. to consumers was designated as the Bpositive control and in ice The comet assay was performed on erythrocytes fol. toxicant free fish was designated as the control fish The lowing the technique of Singh et al 1988 with slight mod. weight of the recovered fish ranged from 500 to 880 g All ifications Cavalcante et al 2008 Lysis 1 h at 4 C in a lysis. the fish specimens were freshened out in running buffer DNA unwinding 30 min in the dark in an electropho. dechlorinated tap water Venous blood was collected from resis buffer Electrophoresis 20 min 300 mA 25 V. the caudal vein of each fish in heparin coated tubes Neutralization three washes for 5 min each in buffer Slides. were fixed with absolute ethanol for 10 min and kept under. Water analysis refrigeration until cytological analyses Slides were stained. with ethidium bromide and analyzed under a fluorescent mi. Water samples were taken from the river at every point from croscope The length of DNA migration measured DNA dam. which fish were harvested and these were then analyzed for age which was visually determined in 250 randomly selected. selected heavy metals and other water quality parameters as cells as 50 per slide for each fish DNA damage was by Comet. defined by the Environmental Protection Agency of Pakistan Score V5 and classified into five classes head diameter DNA. and by Boyd 1981 The selected heavy metals analyzed were in tail tail length tail moment and olive tail moment based. tin Sb chromium Cr lead Pb zinc Zn manganese Mn on the comet tail length and DNA damage. cupper Cu cadmium Cd and mercury Hg The concen, tration of each metal was detected by using heavy metal kits Micronucleus assay. and according to APHA 1998 by using Hitachi polarized. Zeeman Atomic Absorption Spectrophotometer 2000 series Fresh blood was smeared on the slides which were then air. The blanks and calibration standard solution were also ana dried before being fixed in cold Corney fixative for 5 min. lyzed in the same way as for the samples The instrument cal After fixing the slides were stained in aqueous 10 Giemsa. ibration standards were made by diluting standard 1000 ppm for 30 min Five fish were analyzed for a total of 25 000. supplied by Merck Germany A known 1000 mg L concentra erythrocytes fish sample The frequencies of micronuclei in. tion of all the abovementioned metal solutions was prepared the erythrocytes were detected using a binocular microscope. from their salts All reagents used were of analytical grade The under T1200x magnification Erythrocytes with intact cellular. percent recoveries in all the cases were within the acceptable and nuclear membranes were scored using the same criteria as. limits of 70 120 as per regulatory guidelines in previous studies Bombail et al 2001 Serrano Garcia and. 14592 Environ Sci Pollut Res 2016 23 14589 14599, Montero Montoya 2001 Nuclear abnormalities NAs in the significant differences in head diameter were reported be.
erythrocytes were scored according to Cavas and Ergene tween fish collected from polluted waters and those collected. Gozukara 2005 and classified as in Carrasco et al 1990 upstream indicating the extent of damage The differences. found between farmed and upstream fish were not significant. Statistical analysis however In respect to tail length for the polluted sites R1 and. R3 significant differences in tail length were reported com. Data were statistically analyzed by one way analysis of vari pared to farmed and upstream fish For the polluted site R2. ance The results are represented as the mean standard devi however these differences were not significant In the case of. ation Variance was considered significant at p 0 05 All sta DNA in the tail for site R1 significant differences were re. tistical analyses were performed using the program SPSS 9 for ported between farmed and polluted fish 4 423 and 17 330. the PC respectively whereas the differences between farmed and. upstream fish were not significant Fig 2 For site R2 signif. icant differences were obtained for the positive control and all. Results other types but only non significant differences were reported. between fish collected from farmed polluted and upstream. Comet assay waters 3 914 11 529 and 11 477 respectively For site. R3 significant differences were obtained for fish collected. The comet assay or single cell gel electrophoresis is a rapid from polluted and upstream waters 35 991 and 13 026. and sensitive technique that detects DNA strand breaks mea respectively but the differences between fish collected from. suring the migration of DNA from immobilized individual farmed and upstream waters were not significant 5 63 1 44. cell nuclei Fairbairn et al 1995 DNA damage was by and 13 023 respectively Table 2 In the case of tail mo. Comet Score V5 and classified into five classes head diame ment significant differences were reported only for fish from. ter DNA in tail tail length tail moment and olive tail mo polluted and upstream waters whereas the differences be. ment based on the comet tail length and DNA damage Two tween fish from farmed and upstream waters were non. fish species viz C catla a surface feeder and C mrigala a significant. bottom feeder were analyzed for DNA damage All water In the case of head diameter in the comet assay of blood. quality parameters were found to be far beyond the permissi from C mrigala collected from site R1 there were significant. ble limits Table 1 and significant p 0 05 DNA damage p 0 05 differences between the farmed polluted and up. was observed in C mrigala Fig 1 In respect to the comet stream area fish Fig 3 Similar findings were observed in the. assay of C catla specimens taken from sites R1 R2 and R3 case of the comet tail in the comet assay Table 3. Table 1 Comparison of the,means of water quality parameters Sites. in Chenab River mean SE Cadmium mg L Copper mg L Manganese mg L Zinc mg L. R1 0 139 0 012C 0 907 0 212E 01 59 0 150C 00 215 0 036E. R2 0 135 0 013C 0 863 0 211EF 01 53 0 138C 00 207 0 036 F. R3 0 130 0 014CD 0 826 0 203 F 01 36 0 139D 00 206 0 035 F. Lead mg L Chromium mg L Tin mg L Mercury mg L, R1 1 501 0 151C 0 349 0 051D 0 304 0 037D 00 996 0 033BC. R2 1 348 0 120D 0 289 0 040E 0 273 0 030DE 01 013 0 017BC. R3 1 298 0 121D 0 246 0 032 F 0 261 0 030E 00 893 0 012CD. Phenols mg L Sulfates mg L BOD mg L COD mg L, R1 01 67 0 145E 264 79 47 230D 70 64 2 33 F 146 43 13 61 F. R2 01 48 0 121 F 250 36 47 271E 61 70 1 88G 135 00 13 40G. R3 01 32 0 135G 246 07 45 679E 50 88 1 44H 124 07 13 87G. pH TDS mg L Salinity mg L Conductivity mS m, R1 10 37 0 053CD 1597 64 221 95E 1392 86 153 16E 02 25 0 258E. R2 10 28 0 019D 1475 43 220 16 F 1250 00 145 16 F 02 11 0 269 F. R3 10 06 0 044E 1214 43 237 61G 921 43 137 15G 01 70 0 309G. Means sharing the same letter in a row or in a column are statistically non significant p 0 05 R1 R3 polluted. experimental sites in the river, BOD biochemical oxygen demand COD chemical oxygen demand.
Environ Sci Pollut Res 2016 23 14589 14599 14593,Fig 2 Comparative analyses of. the comet assays of blood from,Catla catla collected from. different environments, In the case of the percent DNA in the tail a significant Micronucleus assay. difference p 0 05 was reported for farmed and polluted area. fish a significant difference was also found in respect to pol The frequency of the micronucleus in polluted and control. luted and upstream area fish but the differences between the C. Variation in genotoxic susceptibility and biomarker responses in Cirrhinus mrigala and Catla catla from different ecological niches of the Chenab River Bilal Hussain 1 2 amp Tayyaba Sultana1 amp Salma Sultana 1 amp Shahid Mahboob1 3 amp K A Al Ghanim3 amp Shahid Nadeem4 Received 2 August 2015 Accepted 4 April 2016 Published online 12 April 2016 Springer Verlag Berlin Heidelberg 20

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