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Monitoring DNA Damage in Gills of Freshwater Mussels (Anodonta anatina) Exposed to Heavy Metals.

Byline: Muhammad Sohail, Muhammad Naeem Khan, Naureen Aziz Qureshi and Abdul Shakoor Chaudhry


Study was carried out to investigate the genotoxic effect of different levels of heavy metals on gill cells of freshwater mussels (Anodonta anatina), a sentinel species in aquatic environment. Freshwater mussels were exposed to none (0ug L-1), low (120 ug L-1), medium (240 ug L-1) and high (360 ug L-1) levels of lead (Pb), chromium (Cr) and copper (Cu) alone and in combinations (Pb + Cr + Cu) for 15 days under laboratory conditions. Gill cells of mussels were used to determine the DNA damage by comet assay. The tail DNA (%), comet tail length and olive tail moment (OTM) were the parameters selected to detect DNA damage.

Low doses (120 ug L-1) of each metal induced significantly higher levels of DNA strands breaks as compared to medium dose (240 ug L-1) and very low levels of DNA damaged was observed at high dose (360 ug L-1). Cu and Pb showed significantly higher value of % of tail DNA (56.74+-1.81, 47.36+-1.23) and comet tail length (41.30+-0.758, 49.15+-1.90), respectively, as compared to Cr and combined metal exposure (Pb + Cu + Cr). The lowest levels of DNA damage for all the parameters were observed in combined metal treatment. Genotoxic effect of metals on freshwater mussels is very important to assess the aquatic health and could be suggested as biomarker. It is concluded that the Cu and Pb induced more DNA damage as compared to Cr and combined metal exposure (Pb + Cu + Cr). Moreover, our results showed that the low dose treatment of metals have more genotoxic effect as compared to the medium and high doses.

Key words

DNA damage, Freshwater mussels, He avy metals, Comet assay.


Freshwater ecosystems such as lakes and rivers are very essential resources because they contribute in biodiversity, regulation of climate, managing floods and to meet the demands of drinking water (Ra et al., 2011; Hansen, 2012). The biodiversity of freshwater ponds are mainly depends on the variety of free floating organisms such as phytoplankton and zooplankton (Sohail et al., 2014). Unfortunately, freshwater system are continually polluted due to increasing anthropogenic activities such as urbanization, industrialization, agricultural expansion, and manipulation of mineral resources which cause a severe damage especially in less develop countries (Thevenon et al., 2013; Zan et al., 2012). Among the other harms, the risks of toxic metals in freshwater bodies cause adverse effect to the health of ecosystem (UNEP, 2011). The exposure to these metals can also induce harmful effects in organisms such as interruption in homeostasis and destruction in their DNA (Tsangaris et al., 2010).

Few of the metals such as copper and zinc are required for metabolic processal though could be lethal at higher concentrations. Some metals prove dangerous even at very minute concentrations (e.g., mercury, cadmium, and lead) and are suggested as harmful metals for aquatic life because of their perseverance as describe by Turkmen et al. (2008). Lead is one of the more common and persistent environmental pollutants. Lead has no valuable function for the living organism and is said to be non-essential metal (Johannesson, 2002). It can also accumulate in the tissues and it interferes with the other bio elements such as Ca, Zn and Cu which cause a variety of serious disorders (Berrahal et al., 2011).

Many invertebrates found in aquatic ecosystem are sensitive to contaminants and could accumulate heavy metals form their surrounding water (Reinecke et al., 2003). Soegianto et al. (1999) reported that the concentration of copper in unpolluted water is 85% in all the cases.

Comet assay

The protocol of comet assay was used according to Singh et al. (1988). Ethidium bromide (2 ug/ml) was used for staining the slide and then examined with fluorescence microscope at magnification power of x400. The scoring of microscopic images was carried out by using computer software Comet IV. Three slides were prepared from each sample and the images of 20 cells were scored from each slide. Olive tail moment (product of tail length and the fraction of total DNA in the tail), comet tail length and amount of DNA in comet tail were the parameters assessed for DNA damaging.

Statistical analysis

Analysis of variance (ANOVA) was applied on data to analyse the variation among comet parameters for different doses of heavy metals. Tukey pairwise comparison test was used to assess the difference between control and treatment groups and statistical significance was defined at p<0.05. Minitab 17 software and Microsoft excel were used for all the statistical analysis.


There were significant differences between treatment groups at all concentration as compared to controls except the combined treatment at low and medium dose and Cr alone at higher dose (Table I). The control levels were the same for all metals so had same value as mentioned in Table I. The value of tail DNA percentage, tail length and olive tail moment decreased from lower to higher doses of metals but still the values at high doses were significantly higher than the controls except the chromium and combined treatments which returned to the control level. The comet image of gill cells of freshwater mussels collected from the control tank showed no damages in DNA (Fig. 1A) whereas damage was observed in all treatment groups of various heavy metals (Fig. 1B-E).

For % of tail DNA highest values were observed in Cu-exposed mussels (56.74+-1.81) as compared to Pb (47.36+-1.23), Cr (43.0+-1.31) and combined treatment (25.94+-1.16) at low dose (120 ug L-1).The least value of tail DNA was noticed at high dose (360 ug L-1) such as for Cu (20.89+-0.55), Pb (10.88+0.73), Cr (7.94+-0.71) and combined (8.16+-0.98) treatments. There were no significant differences between the Pb and Cr at low concentration (120 ug L-1) but both were significantly different from Cu and combined treatment (Pb+Cu+Cr) at the same concentration. The values in combined metal exposure at medium concentration (240 ug L-1) was significantly lower than all the other treatments at the same concentration (p Cu > Cr > Pb + Cu + Cr (Table I). As usual the value of comet tail length was also decreasing with increasing the concentration of metals. The lowest values of tail length were found at higher dose (360 ug L-1) of all metals exposure such as Cu (16.05+-0.73), Pb (9.00+-0.67), Cr (8.30+-0.54) and combined-metal exposure (7.20+-0.53) (Table I).

The value of olive tail moment (OTM) also varied among the different doses of metals. Cu and Pb exposure showed significantly higher value of OTM (13.48+-0.44, 12.91+-0.45, respectively) at the low doses (p0.05) (Table I). Cr induced low DNA damage than Cu and Pb but higher than the combined metal treatment at all the doses for all the parameters (Table I).


Cu and Pb both induced high DNA damage in gill cells as compared to Cr and combined exposure of metals. Pb showed slightly higher value for the tail length than Cu at low dose as difference in means was 7.85+-1.92 (n=20) but in case of % of tail DNA Cu shows more value than Pb and mean difference was 9.38+-2.01 (n=20) at the same dose. For the OTM at the same dose the little difference was observed in between Cu and Pb but again more values were observed in Cu-exposed mussels and difference in means was 0.92+-0.65 (n=20). The combined metals exposure induced very low damaging in DNA as showed in all observed parameters. Vincent-Hubert et al. (2011) compared the haemocytes and gills of mussels for comet assay study and concluded that the gills have more sensitivity for the contaminants as compared to the haemocytes.

Our results resemble to previous study where the mussels were exposed to the Nano and ionic forms of copper and significant difference was observed in copper exposed mussel as compared to controls and it was also stated that the higher damage was observed in mussel exposed to ionic form of Cu (Gomes et al., 2013).

Bolognesi et al. (1999) also showed similar results where the mussels were exposed to different concentrations of Cu and high levels of DNA damage were observed at low concentration of Cu (40 ug L-1). Al-Subiai et al. (2011) reported the effect of Cu on marine mussels and suggested that the high dose of Cu was toxic to animal and 100% mortality was observed at 100 ug L-1 which is contradictory to our results where no mortality was observed in freshwater mussels at any dose of copper. Our finding are in line with a previous study where mussels were exposed to low and high doses of polycyclic aromatic hydrocarbon and higher levels of DNA strands breaks were observed in mussels which were exposed to the low doses of polycyclic aromatic hydrocarbon (Large et al., 2002).

Freshwater mussels were exposed to various doses of Pb to evaluate the genotoxic effects and significantly high DNA strands breaks was observed at low dose 50 ug L-1 of lead and very little DNA damage was found at high dose of 500 ug L-1 (Black et al., 1996).

Cr and combined treatments (Pb + Cu + Cr) showed the sametrends; the low level of DNA breaks was observed as the doses were increased. The combined exposure of metals showed very low value in DNA damage than the metals individually which showed some synchronized effects of metals. It is unclear why the combined treatment of heavy metal induced low level of DNA damage but one of the previous study also showed the similar results where the mussels were exposed to the a combination of Cu, Cd and Hg and low level of DNA damage were observed in mussels that were exposed to the combination of metals, studied by Bolognesi et al. (1999). Heavy metals have been shown to induce changes in the metabolism of organism and enhanced the reactive oxygen species which generated oxidative stress and induced DNA damage (Gaetke and Chow, 2003).

It is previously mentioned that the metals and nanoparticles have direct effect on DNA in nucleus and occasionally during cell division which can cause DNA damage (Bhatt and Tripathi, 2011; Singh et al., 2009; Karlsson, 2010).

Many previous studies had been conducted on freshwater and marine mussels to assess the genotoxic effect of heavy metals and found deleterious effects. Cr is the major industrial discharge that put adverse effects on animal tissues (Wahlberg and Skog, 1965). The large concentration of Cr was observed in aquatic environment and it can greatly accumulate in tissues of aquatic animals which may cause defects at molecular level (Fatima and Usmani, 2013; Taweel et al., 2011). The sufficient amount of lead also accumulated in aquatic animals even though at minute levels in aquatic environment (Vinodhini and Narayan, 2008; Abdel-Baki et al., 2011). Pb is noxious even in very small concentration because Pb can mimic other essential elements such as calcium, magnesium and zinc which inflict deleterious effects on enzyme activity (Jennette, 1981) and also prove carcinogenic (Fracasso et al., 2002).

The genotoxic effects of Pb on freshwater mussels were also reported by Black et al. (1996). Emmanouil et al. (2007) reported that the Cr has more adverse effects and damaged DNA strands at the tissue concentration of [greater than or equal to]2.70 ug/g wet weight under laboratory conditions.

Cr has been significantly correlated with the DNA strand breaks in the mussels collected from the wild (Rank et al., 2005). Many previous studies reported the significantly higher levels of DNA damage in aquatic organism collected from contaminated water with metals (Frenzilli et al., 2001; Nacci et al., 2002; Steinert et al., 1998). This is in accordance with our findings where the DNA damage detected with the Comet assay seem to be correlated with the comparative heavy metal concentrations in the surrounding environment. Al-Subiai et al. (2011) reported the genotoxic effects of Cu on the DNA strand breaks of bivalve mussels.

Induction of DNA damaging has also been reported in numerous other studies where animals were exposed to different heavy metals such as mercury, cadmium, Cr, Cu and Pb (Hartmann and Speit, 1994; Hayashi et al., 2000). Several previous studies also reported that the 50% or higher level of DNA strand breaks in cells of aquatic animals including mussels were linked to the different chemicals exposed in laboratory or in polluted environment (Frenzilli et al., 2001, 2004; Regoli et al., 2005; Machella et al., 2006; Gorbi et al., 2008). Heavy metals are harmful for aquatic life and in present study the specific effect of metals at the specific concentrations was evaluated on sub-cellular level of bivalves.


It is concluded that the Cu and Pb induced higher levels of DNA damage compared to Cr and combined metal exposure. Furthermore our findings showed that the low dose (120ug L-1) of metal concentrations had more genotoxic effect compared to the medium (240 ug L-1) and high (360 ug L-1) doses. Gills are suggested to be the best target organ to assess the genotoxic effects and the freshwater mussels (Anodonta anatina) are considered as one of the key species for bio-monitoring studies.


This work was funded by Higher Education Commission (HEC) Pakistan under International Research Support Initiative Program (IRSIP) which enabled the first author to attend the Newcastle University, UK.

Statement of conflict of interest

Authors have declared no conflict of interest.


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Author:Sohail, Muhammad; Khan, Muhammad Naeem; Qureshi, Naureen Aziz; Chaudhry, Abdul Shakoor
Publication:Pakistan Journal of Zoology
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Date:Feb 28, 2017
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