Assessment of heavy metals and its impact on DNA fragmentation in different fish species

This study was conducted to assess water pollution by examining DNA fragmentation in selected fish organs (kidney, liver, gills, and muscle tissue) from Wallago attu , Sperata sarwari , Vulgaris vulgaris , and Labeo rohita collected from a known polluted section of the Chenab River, Pakistan, and from a control site. The fish were caught using a gill net and were assigned to three different weight groups (W1, W2, and W3) to study the degree of variation in DNA fragmentation in relation to body weight. In fish from the polluted site, DNA fragmentation was higher in kidney, liver, gills, and muscles, compared to the control. No significant DNA fragmentation was observed in fish collected from the control site. Highly significant (P < 0.01) relationship between body weight and DNA fragmentation was found in the organs of fish procured at the contaminated site. DNA fragmentation in body organs was found to be affected by the concentrations of lead, copper, nickel, and cadmium in W. attu , S. sarwari , L. rohita , and V. vulgarus harvested from Chenab River. DNA fragmentation in different freshwater fish species is therefore a reliable biomarker of water pollution.


Introduction
Successful aquaculture depends on a continuous supply with pollution free water because fish is more sensitive to water pollutants. Water can potentially be polluted with suspended solids, nutrients, heavy metals, organic matter, pesticides, and industrial chemicals (Boyd and Tucker, 1998). Rivers, streams, and other water bodies are often vulnerable to contaminants to organic and inorganic chemical toxicants, which has shown to produce detrimental effects on fauna and flora in aquatic ecosystem (Wood, 2001). The rapid industrialization and urbanization is one of the most factors for increase in water pollution and becoming a serious challenge for the environmentalists (Mayon et al., 2006). There is a dire need, to evaluate the effect of pollution on biomarkers such as DNA fragmentation (Hussain et al., 2016). The aquatic fauna and flora is exposed to the pesticides and heavy metals, which is main the main of genotoxicity in fish (Souza and Fontanetti, 2006;Santos et al., 2014;Saeedi Saravi and Shokrzadeh, 2013). The increasing loads of these toxicants pollute the freshwater ecosystem and pose serious human health risks (Zhao et al., 2014).
Fish are physiologically affected by pollutants in their environment, and fish abundance is typically a function of water quality (Aleya et al., 2007;Hued and Bistoni, 2005). Furthermore, fish, play key and important role in community structure in the food web. Previous results mentioned that trace metals may adversely affect […] cellular organelles and components such as nucleic and mitochondrial DNA; moreover, heavy metals may inhibit several enzymes involved in DNA damage repair which causes DNA damage and conformational changes that may lead to cell cycle modulation and apoptosis (Wang and Shi, 2001). DNA fragmentation changes have been associated with pollutant exposure in C. carpio, however, the patterns of DNA fragmentation seem to depend on tissue type. DNA extracted from fins showed a higher proportion of fragmentation than that of tissues (Salman et al., 2012). Liyan et al. (2005) suggested DNA fragmentation status is a reliable indicator of genotoxicity in aquatic organisms in polluted environments. The objective of this research work was (i) to investigate the effects of heavy metal exposure on DNA fragmentation; (ii) and to assess a potential correlation of fish weight; and (iii) DNA fragmentation in Wallago attu, Sperata sarwari, Valgarius valgarius, and Labeo rohita harvested from Chenab River.

Study area and selection criteria
Fish procured from the Chenab River near Chiniot, Pakistan, were used in this study. The city of Chiniot is located on the left bank of the Chenab River at a latitude of 31.7200° and a longitude of 72.9789°. Fish caught from Chenab River are a regionally popular source of food. Effluents from adjacent industries and domestic sources discharge into the Chakbandi drain and it adds into Chenab River.

Sample collection
Forty-eight (N=12) for each species samples of Wallago attu, Sperata sarwari, Vulgaris vulgaris, and Labeo rohita were procured from a polluted site of the Chenab River, and control fish were obtained from a commercial fish farm. The sampled live fish were transported to the laboratory and morphometric characteristics (body weight and length) were recorded. The weight and standard length of the fish species of the polluted and control site, respectively, were as follows: W. attu 750-1550 g and 18.5-22.7 cm, S. sarwari 500-1360 g and 14.2-45.1 cm, V. vulgaris 225-350 g and 9.6-12.6 cm, and L. rohita 150-950 g and 18.5-22.7 cm. Each fish species was categorized into three weight groups (W1, W2 and W3) as mentioned in Table 1.

Fish dissection and preservation
Each fish was dissected, and 10 g samples of kidney, liver, gills, and muscle were collected. Each sample was removed individually and placed in marked sterilized polyethylene bags for storage at -20 °C until DNA fragmentation analyses.

DNA fragmentation
DNA fragmentation was studied in the kidney, liver, gills, and muscle using a Bichrome Libra S12 UV/Vis spectrophotometer and following the method of Perandones et al. (1993).

Assessment of heavy metals
The aim of this study was also to evaluate heavy metals in the different fish organs, as this may be an early indicator of freshwater pollution in order to safeguard the quality of aquatic life to promote fish production. Heavy metals were analyzed using an Atomic Absorption Spectrophotometer (Hitachi Polarized Zeeman AAS, Z-8200, Japan) following the methods described in AOAC (1995). The concentrations of cadmium (Cd), copper (Cu), lead (Pb), and nickel (Ni) were measured.

Statistical analysis
Data are presented as means ± S.E. Statistical analyses were performed using a two-way analysis of variance followed by a Tukey's test. The correlation of weight and different organs regarding DNA fragmentation (in percent) and heavy metals was worked out.

Heavy metals in fish organs
In weight group W1, a maximum Pb level of 5.5 mg/kg was found in the liver of S. sarwari, whereas no Pb was found in liver, gills, and muscle of W. attu, and in muscle tissue of S. sarwari. In weight group W2, a maximum Pb level of 7.5 mg/kg was detected in the liver of S. sarwari, and no Pb was found in kidney and gills of W. attu, and in muscle tissue of S. sarwari. In category W3, a maximum Pb concentration of 8.5 mg/kg was detected in the liver Table 1. Categorization of experimental (Exp.) and control (Cont.) fish (mean weight in g) in three different weight categories.

Labeo rohita
Pollu. Cont. of S. sarwari, and no Pb was found in muscle tissue of S. sarwari ( Table 2). The maximum concentrations of Cu were 9.0, 8.5, and 6.5 mg/kg in the liver of S. sarwari, W. attu and V. vulgaris of weight group W3, W2, and W1, respectively. No Cu was detected in the muscle of S. sarwari and W. attu of weight groups W1, W2, and W3 ( Table 2).

Vulgaris vulgaris
In weight group W1, the highest Ni concentration of 4 mg/kg was found in the liver and kidney of L. rohita and no Ni was found in "liver and muscle tissue" of W. attu and L. rohita, and in gills of S. sarwari; the maximum Ni concentration of 5.5 mg/kg was detected in the liver of W. attu. In weight group W2, no Ni was detected in muscle tissue and gills of W. attu and S. sarwari. In weight group W3, the maximum Ni concentration of 8 mg/kg was found in the kidney of L. rohita, whereas no Ni detected in the liver of W. attu ( Table 2). The maximum Cd concentration in weight group W1 was 3.5 mg/kg and was found in the kidney of L. rohita. No Cd was detected in gills of W. attu, S. sarwari, and V. vulgaris and in muscle tissue of L. rohita and V. valgarius. In categories W2 and W3 the maximum Cd concentrations of 3.5 and 4.5 mg/kg, respectively, were found in the kidney of V. vulgaris. No Cd was detected in gills and muscle tissue of W. attu and in the gills of S. sarwari of weight group W3 ( Table 2).

Discussion
Water pollution affects the stability of freshwater ecosystem and can also induce genotoxicity in aquatic animals. Heavy metals, most frequently accumulate in freshwater ecosystems (Aanand et al., 2010;Omar et al., 2012). The main source of heavy metals in the area of the current study was exogenous due to either surface runoff and discharge of untreated effluents from nearby agriculture and industry (Authman and Abbas, 2007;Omar et al., 2012). The observed pollutants can cause genetic changes leading to detrimental mutations (Hussain et al., 2016). "DNA fragmentation" values recorded in the muscle of fish were within permissible limits to "that in the gills, kidney and skin". The results of our study showed a relationship of heavy metal levels in various tissues of fish and DNA fragmentation. The gills are a metabolically active organ in fish and can accumulate more heavy metals than other organs. Heavy metals catalyze reactions that generate reactive oxygen species, which may cause oxidative stress and damage to tissues and macromolecules such as DNA, proteins, and lipids. Our findings are in line with the results of other studies (Mahboob et al., 2011;Chaudhry and Jabeen, 2011;Hussain et al., 2016). These studies reported that this may be due to a repair mechanism induced in response to a reactive oxygen species that are produced by heavy metal or organic pollution. Jabeen and Chaudhry (2010) examined the muscle of L. rohita and O. mossambicus and reported that DNA integrity in muscle tissue was not affected by pollution, whereas DNA extracted from different organs showed severe damage, which corroborate with our findings. Gonzalez-Mille et al. (2010) reported the impact of heavy metals and other toxicants on muscle tissues of fish harvested from the Coatzacoalcos River, Mexico, and found an increase in DNA damage due to high genotoxin concentrations in the water. Almeida et al. (2005) mentioned comparatively higher concentration of metals in a river due to the deposition of waste from adjacent industrial areas. The findings of this study were also supported by Binelli et al. (2010) who reported that fluctuation pollution levels in rivers. DNA fragmentation, increased with fish weight in the examined fish species. Lima et al. (2006), Saeed and Shaker (2008) and Simone et al. (2000) reported more DNA damage in fish exposed to toxins for longer period. Liyan et al. (2005) proposed the use of DNA fragmentation as an assessment tool for heavy metal pollution. Analía et al. (2016) suggested the use of a multi-biomarker approach for the study of toxicological mechanisms caused by exposure to a combination of chromium and Pb.

Conclusions
DNA damage can be used as a biomarker of water pollution and may be used as an early warning for pollution monitoring in freshwater ecosystems.