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Byline: F. Ambreen and M. Javed


The present study was undertaken to examine the DNA damage induced by binary metal mixture (cadmium-lead) in peripheral blood erythrocytes of freshwater carp, Cyprinus carpio by using Comet bioassay. The 96-hr LC50 value of mixture was estimated for 180-old fingerlings of Cyprinus carpio in a static system and then four sub-lethal concentrations viz. SL-I (10.42mgL-1), SL-II (7.81mgL-1), SL-III (6.25mgL-1) and SL-IV (5.21mgL-1) were calculated and fish were exposed to these concentrations, separately in glass aquaria for 30 days along with negative and positive control at constant water temperature (30C), pH (7.75) and total hardness (225mgL-1). Peripheral erythrocytes were sampled after 30 day exposure for DNA damage assessment. Statistically significant effects were observed at sub-lethal concentrations in-terms of percentage of DNA damage, cumulative tail lengths (u m) and genetic damage index.

Concentration dependent response was observed in fish erythrocytes with induction of maximum DNA damage, due to positive control, followed by at highest concentration (SL-I) of mixture. This study also concluded that comet bioassay can be used for in-vivo experiments, using fish as a model for the screening of genotoxic and mutagenic pollutants in aquatic environment.

Key words: Carp, Comet bioassay, DNA damage, Metal Mixture, Sub-lethal.


Pollution of aquatic environment due to heavy metals not only endangers the physiology and survival of the organisms but can also induce genetic alterations which lead to mutation and cancer (Russo et al., 2004). Naturally, heavy metals present in water due to various processes such as weathering and erosion (Damodara et al., 2008) but anthropogenic activities are the main cause of heavy metal pollution in the aquatic environments. Some metals are essential for living organisms in only trace amounts (e.g. cobalt, copper and zinc, iron) while others like cadmium, lead, arsenic, mercury and chromium have no significant biological roles (Javed, 2012; Ambreen et al., 2015). Cadmium is a non-essential heavy metal with carcinogenic, teratogenic and mutagenic effect on aquatic organisms. Integrity of freshwater ecosystems is heavily threatened by cadmium due to greater sensitivity of aquatic organisms than that of mammals (Burger, 2008; Jia et al., 2011).

Among different heavy metals lead is most common in earth crust, used for thousands of years. Inorganic and organic lead enters in the environment through several different ways and poses diverse effect on organisms. Lead can mimick the essential elements like magnesium, iron, calcium and zinc, increased the incorporation of erroneous nucleotides in which it implicated as a co-carcinogen and effect on DNA repairing mechanisms (Godwin, 2001; Frascasso et al., 2002).

Although each metal have unique mechanism of toxicity but there are some common mechanism which include mimicry, adduct formation with DNA or protein and oxidative damage. Generation of reactive oxygen species is induced by heavy metals in their ionic forms resulting in oxidative modifications of DNA, inducing aberrant gene expression and carcinogenesis (Ballatori, 2002; Baselt, 2004). Cocktails of compounds create huge problem as toxicity of a mixture is not easily linked to individual toxicities of components in the mixture (Fernandez-Alba et al, 2001). Much work on acute toxicity of individual metals for fish was done by many scientists (Abdullah et al., 2007; Azmat et al., 2012; Javed, 2012) however, the genotoxic effects of metals in the form of mixture are scant in the literature. Therefore, present study was aimed to evaluate the DNA damage in freshwater carp, Cyprinus carpio after 30-day exposure to sub-lethal concentrations of cadmium and lead mixture.


Experimental Fish and Chemicals: The fingerlings of freshwater common carp, Cyprinus carpio were purchased from the local outlets and transported to Fisheries Research Farms, University of Agriculture, Faisalabad, Pakistan. Fish fingerlings of 180-day old (similar weight) were acclimatized under laboratory conditions in cemented tanks for two weeks prior to start the experiment and fed with pelleted diet twice a day (34% Digestible Protein and 3 Kcal/g Digestible Energy).

Appropriate quantity of technical grade CdCl2 and PbCl2 were separately dissolved in deionized water for stock solutions preparation while binary mixture of both metals was prepared by its further dilution on ions equivalence basis (1: 1 ratio).

Comet Bioassay: Healthy fingerlings of Cyprinus carpio were exposed to four test concentrations (10.42, 7.81, 6.25 and 5.21mgL-1) separately, in glass aquaria having seventy litter water capacity. These exposure concentrations were selected on the basis of 96-hr LC50 value (31.25mgL-1) of cadmium-lead mixture on test animal. Based on this 96-hr LC50 value four sub-lethal concentrations viz. SL-I (1/3rd of LC50), SL-II (1/4th of LC50), SL-III (1/5th of LC50) and SL-IV (1/6th of LC50) were calculated and used for in vivo genotoxicity experiments. Simultaneously, one group of fish was maintained in metal mixture free water considered as "Negative Control" while cyclophosphamide was used as "Positive Control". The fish were fed daily with small quantity of nutritious food (34% Digestible Protein and 3 Kcal/g Digestible Energy). Water temperature (30 C), pH (7.75) and total hardness (225mgL-1) were kept constant throughout the experiment.

The peripheral blood erythrocytes were sampled after 30 day exposure to four different test concentrations and controls, and subjected to Comet bioassay. Whole experiment was conducted with three replications for each sub-lethal concentration. Peripheral blood erythrocytes were sampled from caudal vein of fish, immediately transferred in eppendorf and initially treated with anticoagulants (Kousar and Javed, 2015). Comet bioassay was performed as three layer procedure, followed by lysis, electrophoresis and staining by using methods of Singh et al. (1988). Two slides per specimen were prepared, scored randomly and analyzed by using an image analysis system attached to Epi-Fluorescence microscope (N-400M, American Scope; USA) equipped with light source of mercury short arc reflector lamp filters for ethidium bromide at 400X magnification and low lux camera (MD-800, USA).

The length of DNA migration in the comet tail is an estimate of DNA damage. The DNA damage was quantified by visual classification of cells into the five categories "comets" corresponding to the tail lengths as undamaged (Type 0); low level damage (Type I); medium level damage (Type II); high level damage: (Type III) and complete damage (Type IV). The cells with no DNA damage possess intact nuclei without a tail, while cells having damaged DNA showed comet like appearance. The extent of DNA damage was examined as the mean percentage of cells with medium, high and complete damaged DNA, which was calculated as the sum of cells with Types II+ III + IV. TriTek CometScore TM software was used to measure the comet tail length of damaged cells (Jose et al., 2011) and cumulative tail length (u m) was obtained by adding the tail length of all examined cells (n = 50/replicate).

From the arbitrary values assigned to the different categories (from Type=0 to Type IV) a genetic damage index (GDI) was calculated by using the following formula:


Statistical Analyses of Data: Means were compared for statistical differences through Tukey's Student Newnan-Keul test by using the MSTATC computer software (Steel et al., 1996). A p-value less than 0.05 were considered as statistically significant.


Table 1 shows the variable frequency of undamaged nuclei (Type 0), damaged nuclei (Type I to IV), percentage of damaged cells (Type II+III+IV), cumulative tail lengths and genetic damage indices at four sub-lethal concentrations of metal mixture induced in the peripheral erythrocytes of Cyprinus cario along with negative and positive control after 30-day exposure period. Among all test treatments, proportions of Type 0 cells were observed higher in negative control (92.673.06%) while same was lower due to SL-II concentration. Percentage of Type I damaged cells were observed maximum due to SL-III concentration exposure as evident from their mean value of 52.671.15%. Percentage of Type II and Type III damaged cells were observed maximum at SL-II and positive control as compared to negative control.

Regarding different treatments, Type IV damaged nuclei in peripheral blood erythrocytes of Cyprinus carpio followed the sequence: positive control > SL-I > SL-II > SL-III [greater than or equal to] SL-IV > negative control. However, Type IV damaged cells at SL-III and SL-IV did not vary significantly at pless than 0.05.

The extent of DNA damage was examined as the mean percentage of cells with medium, high and complete damaged DNA, calculated as sum of Type II, III and IV. Statistically significant (pless than 0.05) DNA damage was observed during whole exposure period due to different test concentrations. Regarding different treatments (negative control, positive control, SL-I, SL-II, SL-III and SL-IV) the DNA damage frequency was observed significantly higher (pless than 0.05) due to positive control, followed by that of SL-I, SL-II, SL-III, SL-IV and negative control indicating dose/concentration dependent DNA damage. However, among four test concentrations, SL-III and SL-IV showed non-significant differences for percentage of damaged cells induction in peripheral erythrocytes of Cyprinus carpio. Cyprinus carpio also respond differently towards DNA damage induction determined in terms of cumulative tail length of comets (u m).

Cumulative tail length of comets, induced due to different concentrations of metal mixture, negative and positive controls, ranged between the mean values of 3.44 to 132.12u m with statistically significant differences at pless than 0.05. Among different treatments, positive control caused significantly maximum genetic damage index of 1.740.04, followed by that of SL-I, SL-II, SL-III and SL-IV. However, least GDI value was observed in specimens of negative control group.

Table 1. DNA damage in peripheral erythrocytes of Cyprinus carpio exposed for 30-days to cadmium-lead mixture.


###Damaged Nuclei (%)###*Damaged###**CTL###***

Treatments###Concentrations###Nuclei (%)###GDI

###Cells (%)###(um)


Negative Control###0.00###92.673.06a###7.333.06f###0.000.00e###0.000.00e###0.000.00e###0.000.00e###3.440.06f###0.070.03f

Positive Control###CP (20ugg-1)###36.004.00bc###11.333.06ef###17.333.06bc###13.331.15a###22.002.00a###52.671.15a###132.120.11a###1.740.04a






During present experiment, peripheral blood erythrocytes of carp (Cyprinus carpio) showed concomitant increase in DNA damage with increase in metallic ion concentrations. Similarly, Cok et al. (2011) observed significant DNA damage in common carp, Cyprinus carpio, sampled from natural environment by using comet bioassay in terms of tail lengths (u m), tail intensity (%) and tail moment. Low concentration of cadmium can induce oxidative stress in Cyprinus carpio. DNA damage percentage, tail length and tail moment were significantly increased even at 0.41mgL-1 of cadmium exposure in common carp as observed by Jia et al. (2011). Concentration dependent increase in DNA damage in the blood cells of Oreochromis mossambicus was also observed upon metal exposure (Ahmed et al., 2011a) as compared to control. Kousar and Javed (2015) also reported DNA damage in peripheral blood erythrocytes of four fish species, under 30-day exposure to metals.

Statistically significant extent of DNA damage in terms of percentage of damage cells, cumulative tail lengths of comets and genetic damage index due to different sub-lethal concentrations of heavy metals were observed in their study. Significantly larger tail lengths of comets in peripheral erythrocytes of Cobitis elongate due to toxicity of industrial effluents were also observed by Kopjar et al. (2008). Similarly, Pereira et al. (2013) observed genotoxic potential of cadmium and aluminum for zebra fish, which exhibited significantly (pless than 0.05) higher DNA damage due to aluminum as compared to cadmium. Present results are in accordance with Ahmed et al. (2010) who also observed concentration dependent DNA damage at 2mgL-1, followed by that of 1.0 and 0.1mgL-1 of cadmium exposure in freshwater climbing perch, Anabas testudineus. Similarly, Ahmed et al. (2011b) observed genetic damage due to exposure of three sub-lethal concentrations of lead (0.1, 1.0 and 2.0mgL-1).

They also observed concentration dependent DNA damage in freshwater climbing perch in terms of different comet parameters viz. %age of DNA in comet tail and %age of DNA in comet heads. Cestari et al. (2004) and Ferraro et al. (2004) reported that breakage of single strand is actually the main reason of lead induced DNA damage. Significantly higher DNA damage in blood cells observed during present study can be comparable with results of Kousar and Javed (2014) that also worked with blood cells of carps and reported significant (pless than 0.05) DNA damage due to heavy metals.

Direct relationships of heavy metals viz. cadmium, lead, arsenic and copper toxicities with DNA strand breakage were also observed by Costa et al. (2008) in Solea senegalensis. In study performed by Matsumoto et al. (2006) a significant increase in the level of DNA strand breaks was observed in the peripheral blood erythrocytes of Oreochromis niloticus exposed to chromium. Similarly, concentration/dose dependent increase in DNA damage in Misgurnus anguillicaudatus (loach) after exposure to Pb+Cd+Zn mixture was also observed by Zhang et al. (2008). Concentration dependent DNA damage as observed during present study is also supported by Chandra and Khuda-Bukhsh (2004) who found dose dependent DNA aberrations in Oreochromis mossambicus. Genotoxicity of heavy metals is mainly related to accumulation of free radicals, clastogenic process or simultaneously to clastogenic and aneugenic action in fish (Nepomuceno et al., 1997).

DNA damage induced by mixture of cadmium and lead suggested a serious concern towards their potential danger to the survival of carp in natural environment.

Acknowledgement: The author is grateful to the Higher Education Commission Pakistan for providing funds under the Indigenous Ph.D. Fellowship Program to complete this work as a part of Ph.D. Research.


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Publication:Journal of Animal and Plant Sciences
Date:Jun 30, 2016

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