Assessment of Heavy Metals Induced Genotoxicity in the Fish by Using Single Cell Gel Electrophoresis

Abstract

A research work on the assessment of heavy metals induced genotoxicity in the fish by using single cell gel electrophoresis was carried out in two phases (1) acute toxicity of metals to the fish (2) genotoxic impacts of heavy metals on the fish. Acute toxicity of aluminium, arsenic, manganese, Mn+Al, Mn+As, Al+As and Mn+Al+As mixtures (MM) was calculated in respect of 96-h LC50 and the lethal concentrations for the three species of fish viz. Labeo rohita, Cyprinus carpio and Oreochromis niloticus at constant levels of water temperature (26°C), pH (7.60) and hardness (240mgL-1) with three replications for each treatment. Accumulation of the metals in body organs viz. liver, gill, kidney, heart, muscle, intestine, bone, fin and skin of the three fish species was also determined at 96-h LC50 and the lethal concentration exposures. During second phase, the three species of fish were exposed, separately, to 1/2, 1/3, 1/4 and 1/5th of their respective 96-hr LC50, for eight weeks at controlled laboratory conditions. During 8 week exposure, each fish species was analyzed, periodically, for their peripheral erythrocyte DNA damage concerning percentage of damaged cells, cumulative comets tail lengths and the genetic damage index (GDI). After chronic exposure of individual metals/MM, the organs viz. liver, gills, kidney, heart, muscles, intestine, bones, fins, skin and blood of each fish species were examined for the accumulation of their specific metals. During both acute toxicity and genotoxicity trials, the physico-chemical characteristics of water were analyzed on 12 hourly basis for each test concentration. Probit analysis method was used to determine the acute toxicity of metals with 95% confidence intervals. Analysis of variance and Tukey’s tests were applied to determine statistical differences existing among various variables. Regression and correlations among selected variables were also computed. Among individual metals, arsenic was significantly increased toxic to fish, following aluminium and manganese. The three species of fish showed significantly higher sensitivity towards tertiary mixture of Mn+Al+As, followed by As, Al+As, Mn+Al, Al and Mn. Significantly variable tendencies of three fish species for their tolerance limits against individual metals and metals mixture were accredited to physiological variances and their species-specific manner to interact with metals of different nature. This also predicts differential abilities of three fish species to produce metallothionein to normalize the metal load through depuration from the body and protect it against their detrimental effects while living in metals contaminated waters. Among three fish species, Labeo rohita appeared significantly more sensitive to the toxicity of both individual metals and metals mixtures while Oreochromis niloticus showed significantly least sensitivity. The toxic effects of metals mixtures on the fish appeared significantly more severe as compared to the individual metals, depending upon the specific composition of metals in a mixture, concentration and duration of exposure. However, exposure of manganese, aluminum and arsenic in a mixture form presented additive effects towards sensitivity of all the three species of fish. Exposure of the fish to individual metals and MMs caused significantly variable accumulation of metals into their bodies at 96-hr LC50 and the lethal concentrations. However, manganese accumulation was significantly higher while As exhibited significantly least potential of amassing in all three species of fish. Overall abilities of the three species of fish to amass metals into their body followed the specific order: Oreochromis niloticus > Cyrinus carpio > Labeo XIX rohita with statistically significant variations. Fish organs showed significantly variable ability to accumulate metals also. In general, kidney, liver and gills appeared active organs to bioaccumulate metals while skin, intestine, fins, bone and muscles showed the least tendency to amass metals. In general, the bioaccumulation of metals in the body organs of the three species of fish followed a particular order: kidneys > liver > gill > heart > blood > skin > intestine > fins > bones > muscle. The chronic exposure of Al caused significantly greater accumulation in body organs of fish while arsenic amassing was significantly least. The three species of fish viz. Labeo rohita, Cyrinus carpio and Oreochromis niloticus exhibited significantly variable sensitivity towards different concentrations of metals/MMs at 1/2th, 1/3rd, 1/4th and 1/5th of LC50 (concentrations) to cause DNA damage into their peripheral blood erythrocytes, measured with respect to damage cells (%), GDI and cumulative comets tail lengths during 56 days chronic exposure period. The exposure of Mn+Al+As mixture induced significantly greater damage to fish erythrocyte nuclei in terms of average percentage of damaged cells (37.83±1.35%), genetic damage index (1.32±0.05) and cumulative tail length of comets (145.37±4.64μm) while manganese exposure exhibited significantly lower damage as 17.85±0.93%, 0.88±0.04 and 68.95±1.87μm, respectively. The overall sensitivities of three fish species to individual metals and MMs, to induce DNA damage, followed the order: Labeo rohita > Cyrinus carpio > Oreochromis niloticus. The exposure of metals/MMs at 1/2th of LC50 induced significantly higher DNA damage to nuclei in the peripheral erythrocytes of the three species of fish. Moreover, DNA damage increased concomitantly with the increase in both exposure duration and the concentration of metallic ions indicating ROS generation and also the inhibition of the DNA repair that would cause genomic instability and oxidation stress in the fish as metals can also act through redox cycle to induce ROS which possibly cause DNA strand breakage and hence Mn+Al+As mixture appeared significantly more toxic than the other metals/MMs studied during this investigation. The difference in genotoxicity, caused by various metals in three fish species, was dependent upon the extent of metals accumulation in the fish body. Three fish species showed concentration and duration dependent (p<0.05) linear increase in DNA damage under exposure of both individual metals and metals mixture that followed the order: Labeo rohita > Cyrinus carpio > Oreochromis niloticus. The extent of DNA damage in the fish body showed significantly direct dependence on metal species and its exposure duration. Therefore,comet assay appeared as a sensitive technique for the detection of DNA damage in peripheral blood erythrocytes of the Labeo rohita, Cyrinus carpio and Oreochromis niloticus that can serve as biological indicators of metal's pollution in aquatic ecosystems of Pakistan.