Genotoxic effects of metal pollution in two fish species, Oreochromis niloticus and Mugil cephalus, from highly degraded aquatic habitats

Mutation Research 746 (2012) 7–14

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Genotoxic effects of metal pollution in two fish species, Oreochromis niloticus and Mugil cephalus, from highly degraded aquatic habitats Wael A. Omar a,∗ , Khalid H. Zaghloul b , Amr A. Abdel-Khalek a , S. Abo-Hegab a a b

Department of Zoology, Faculty of Science, Cairo University, Egypt Department of Zoology, Faculty of Science, El-Fayoum University, Egypt

a r t i c l e

i n f o

Article history: Received 20 June 2011 Received in revised form 23 October 2011 Accepted 7 January 2012 Available online 20 March 2012 Keywords: Aquatic pollution Metal toxicity DNA damage Micronucleus test

a b s t r a c t In Egypt, Lake Qaroun and its neighbouring fish farms are in a serious environmental situation as a result of pollution by agricultural sewage and domestic non-treated discharges. The present study aims to evaluate genotoxic effects of toxic metals in cultured and wild Nile tilapia, Oreochromis niloticus and mullet, Mugil cephalus collected from these contaminated aquatic habitats, in comparison with fish from a non-polluted reference site. Heavy-metal concentrations (Cu2+ , Zn2+ , Pb2+ , Fe2+ and Mn2+ ) in water and sediment samples were recorded. The condition factor (CF) was taken as a general biomarker of the health of the fish, and genotoxicity assays such as the micronucleus (MN) test and a DNA-fragmentation assay were carried out on the fish species studied. In addition to micronuclei, other nuclear abnormalities (NA) were assessed in fish erythrocytes. Degradation of the studied aquatic habitats revealed speciesspecific effects. A significant decrease in CF values associated with a significant elevation in MN and NA frequencies was observed in fish collected from the polluted areas compared with those from the reference site. Moreover, mixed smearing and laddering of DNA fragments in gills and liver samples of both fish species collected from the polluted areas indicate an intricate pollution condition. Results of the present study show the significance of integrating a set of biomarkers to identify the effects of anthropogenic pollution. High concentrations of heavy metals have a potential genotoxic effects, and genotoxicity is possibly related to agricultural and domestic activities. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Increasing loads of industrial, agricultural and commercial chemicals discharged into aquatic habitats could pose a serious public health problem and a threat to the aquatic ecosystem [1]. This increases the interest in studies for the evaluation of the genotoxicity of polluted waters [2]. The exposure of aquatic organisms to a variety of genotoxic chemicals raises the question about the potential effects of exposure on the health status of both current and future aquatic populations [2,3]. As lakes are still-water bodies whose conditions deteriorate more rapidly by human activities compared with rivers, one of the most important threats to lakes due to human activities is pollution by heavy metals and other toxic substances [4]. Therefore, when assessing genotoxicity in fish, heavy metals represent an interesting group of elements due to their strong impact on stability of aquatic ecosystems, bioaccumulation in living organisms, toxicity persistence and tendency to accumulate in water and sediments [5].

∗ Corresponding author. Tel.: +20 201005127972; fax: +20 237746984. E-mail address: [email protected] (W.A. Omar). 1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2012.01.013

In Egypt, Lake Qaroun suffers from several environmental problems leading to a decrease in fish stock of the lake [6], thus the Egyptian government has motivated the development of aquaculture and intensification of culture methods along the banks of Lake Qaroun, especially for Oreochromis niloticus and Mugil cephalus [7]. Both Lake Qaroun and fish farms around it receive agricultural, domestic and sewage drainage water from the El-Fayoum province [8,9]. These discharges have serious effects on the water quality and on the health status of aquatic organisms. Although accumulation of heavy metals in the habitat studied here was previously reported by many authors [6–11], there is still a paucity of genotoxicological information regarding fish species living in such conditions. In recent years several studies have evaluated the impact of metals as genotoxic pollutants, by use of different endpoints [12,13]. The micronucleus (MN) test is a useful method to assess genotoxicity in aquatic environments [14]. Micronuclei arise from chromosomal fragments or whole chromosomes that are not incorporated into daughter nuclei at mitosis [15] and could be easily visualised in peripheral erythrocytes. In addition, there are some nuclear and cellular abnormalities that may be considered as genotoxic analogues of micronuclei, and may also be due to genotoxic agents [3,16].

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Furthermore, toxic heavy metals could result in injury to cells, which may die from necrosis and/or apoptosis [17]. Cells normally undergo apoptosis in response to mildly adverse conditions, whilst exposure to severe conditions will result in necrosis. Both processes are truly distinct and have important implications. A molecular hallmark of apoptosis is degradation of nuclear DNA into fragments with the size of an oligonucleosome, as a result of activation of endogenous endonucleases, recognised as a ‘DNA ladder’ on conventional agarose-gel electrophoresis [18]. In the present study the cytotoxic and genotoxic effects induced by environmental toxic metals in their complex state were assessed by use of several endpoints in two fish species: O. niloticus and M. cephalus along contaminated aquatic habitats of both Lake Qaroun and fish farms around it, compared with a model fish farm in the same province. So, the objective of this field study is to investigate the impacts of exposure to toxic metals on the genotoxicity of wild and cultured fish populations. 2. Materials and methods The present fieldwork was carried out on wild and cultured common fish species in Egypt: O. niloticus and M. cephalus facing differential environmental stresses. 2.1. Sites of collection The studied fish species were collected with the help of local fishermen from the following sites: Site 1 (reference site): Fish farm of the Faculty of Agriculture, El-Fayoum University; irrigated with a branch of the river Nile. Site 2: The south-west side of Lake Qaroun at the outlet of El-Wadi drainage canal. It is one of the main drainage canals that receives agricultural and sewage drainage water from the El-Fayoum province [11]. This site represents the wild habitat for the fish species studied. Site 3: Four fish farms at the southern side of Lake Qaroun, where tilapia and mullet are the common cultured fish species [7]. These farms depend on agricultural drainage water as water source [10]. 2.2. Sampling Water, sediments and fish samples were collected from the studied sites during the summer season in 2009. 2.2.1. Water sampling Duplicate water samples were taken with a water sampler from four localities in each of the studied sites between 10:00 and 12:00 a.m. at a depth of 30 cm below the water surface and stored at 4 ◦ C in clean 1000-ml sampling glass bottles according to Boyd [19]. 2.2.2. Sediment sampling Duplicates of four core samples of sediment up to 20 cm in length were taken from each sampling site with polyvinyl chloride (PVC) corers [20]. The corers were immediately sealed and stored at 4 ◦ C.

methanol for 10 min. Samples were stained with 10% Giemsa solution for 10 min, airdried and then prepared for permanent use. A total number of 2000 erythrocytes were examined for each specimen under a light microscope, with oil immersion at 1000× magnification. To minimise the technical variation, the blind scoring of micronuclei was performed on randomised and coded slides. Criteria described by Fenech et al. [24] were taken into consideration: the diameter of the MN should be less than one-third of that of the main nucleus, MN should be separated from or marginally overlapping with the main nucleus as long as there is clear identification of the nuclear boundary, and MN should have similar staining as the main nucleus. Other nuclear and cellular anomalies such as nuclear buds, erythrocytes bearing more than a single micronucleus, blebbed nuclei, lobed nuclei, notched nuclei, nuclear fragmentation, bi-nucleated erythrocytes, poly-nucleated erythrocytes, vacuolated nuclei, cariolysis, vacuolated cytoplasm, nuclear retraction and microcytes were recorded separately, on the basis of the criteria described by Da Silva Souza and Fontanetti [3]. Degenerated cells were discarded. 2.6. DNA fragmentation Frozen gills and liver samples were hashed into small pieces. Tissue was lysed by addition of DNA lysis buffer and incubated at 37 ◦ C for 1 h, followed by a 2-h incubation at 55 ◦ C in the presence of 100 ␮/ml proteinase-K, followed by addition of RNase A (10 ␮g/ml, 1 h at 37 ◦ C). DNA was then extracted from fragmented samples, separated on 1.8% (w/v) agarose gels and visualised with ethidium bromide (6 ␮g/ml) as described by Jones et al. [25]. Gels were illuminated with 300-nm UV light and a photographic record was made in order to detect the qualitative damage to genomic DNA. The densitometric analysis of low molecular-weight DNA fragments was performed by use of a Gel-Pro Analyzer, Version 3.1.00.00. 2.7. Statistical analyses The results were expressed as means ± S.E. Data were statistically analysed with the t-test, analyses of variance (F-test), and Duncan’s multiple-range test to evaluate the comparison between means at P < 0.05, using Statistical Analysis System, SAS, Version 9.1 [26].

3. Results 3.1. Concentration of heavy metals in water and sediments Concentrations of the heavy metals studied, viz. copper, zinc, lead, iron and manganese, are given in Table 1. There were highly significant differences (P < 0.01) in all metal concentrations between water and sediment samples. The concentrations of copper, zinc, lead and iron in water and sediments collected from Lake Qaroun were significantly higher than those of samples collected from other sites. The concentrations of manganese in water and sediment samples from site 3 were higher than those in samples from the other sites. In addition, the results affirmed that water and sediment samples collected from the reference site had significantly lower concentrations of all the heavy metals studied. 3.2. Condition factor

2.2.3. Fish sampling A total number of 48 fish of both species (16 fish from each site) were collected from the same localities where water and sediment samples were collected. The wet weight and total body length of the fish were measured, blood sampling was done, and the fish were then stored frozen for further investigation. 2.3. Determination of the concentration of metals in water and sediments Concentrations of heavy metals were determined by atomic absorption spectrophotometry (Model, PerkinElmer-2280) according to APHA [21]. Metals determined in this study were copper, zinc, lead, iron and manganese. 2.4. Condition factor (CF) CF was calculated as CF = Weight (g)/Length3 (cm3 ) × 100, according to Schreck and Moyle [22].

Table 2 presents means ± S.E. of wet weights and total body lengths of both fish species studied. For each species, significant differences were recorded among the study sites with a singlefactor ANOVA (F-valueslength = 28.0 and 0.24 for O. niloticus and M. cephalus, respectively and F-valuesweight = 40.0 and 27.0 for O. niloticus and M. cephalus, respectively). Table 2 shows that there were highly significant differences in CF values among the study sites. The highest values of CF for both fish species were observed in samples collected from the reference site. Conversely, fish collected from Lake Qaroun showed the lowest recorded CF values. 3.3. Micronucleus test

2.5. Micronucleus test MN frequency in erythrocytes was evaluated according to Fenech [23]. Immediately after sampling, a drop of blood was smeared on clean slides (three slides per fish), which were dried at room temperature and – after 24 h – fixed in 100%

This test was used to compare micronucleus frequencies and other nuclear and cellular abnormalities among fish samples. Different nuclear and cellular anomalies recorded in both fish species

Lake Qaroun < Qaroun fish farms < reference site. Moreover, the t-test between O. niloticus and M. cephalus for MN frequency in each of the studied sites showed highly significant differences (P < 0.01). 3.3.2. Other nuclear abnormalities Nuclear abnormalities are illustrated in Table 3. There was highly significant increase in the nuclear abnormalities considered at sites 2 and 3 compared with those at the reference site, except for poly-nucleated erythrocytes. 3.3.3. Cellular abnormalities Cellular abnormalities of both fish species are illustrated in Table 4. There was highly significant increase in the recorded cellular abnormalities at sites 2 and 3 compared with the reference site, except for microcytes of M. cephalus at the study sites (F-value). Generally, the t-test values showed that each species responded differently to the same pollution conditions at each site. 3.4. Analysis of DNA fragmentation

Data are represented as means of eight samples ± S.E. Means with the same capital letter in the same column for each parameter are not significantly different. ** Highly significant difference (P < 0.01).

Sediment Water Sediment

3.3.1. Micronucleus frequency There were highly significant differences in MN frequency of both fish species collected from the study sites (Table 3). Both fish species collected from Lake Qaroun and fish farms around it showed a significant increase in MN frequency compared with the fish sampled at the reference site. Generally, the clastogenic effect of the heavy metals studied was represented by the formation of micronuclei in the following order:

0.005 ± 0.001 C 1.33 ± 0.27 C 1.78 ± 0.30 A 369.76 ± 25.21 A 1.34 ± 0.39 B 313.05 ± 22.70 B 82** 820**

Water Sediment Water

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collected from the polluted aquatic habitats (Lake Qaroun and fish farms around it) are presented in Fig. 1.

0.004 ± 0.001 C 0.22 ± 0.05 C 0.21 ± 0.08 A 12.79 ± 4.34 A 0.13 ± 0.03 B 6.48 ± 2.74 B 31** 35** 1.83 ± 0.35 C 24.27 ± 3.72 A 13.66 ± 3.10 B 128**

Sediment Water

0.03 ± 0.007 C 0.38 ± 0.10 A 0.24 ± 0.08 B 37** 0.16 ± 0.04 C 6.18 ± 2.01 A 2.46 ± 0.79 B 47**

Sediment Water

0.015 ± 0.004 C 0.37 ± 0.11 A 0.19 ± 0.05 B 49** Site 1 (reference site) Site 2 (Lake Qaroun) Site 3 (Qaroun fish farms) F-value

Manganese Iron Lead Zinc Copper Locations

Table 1 Residual concentrations of heavy metals in water (mg/l) and sediments (mg/kg) collected from the study sites.

0.01 ± 0.004 C 0.93 ± 0.26 B 0.06 ± 0.01 B 65.96 ± 17.25 A 0.097 ± 0.01 A 69.77 ± 16.55 A 123** 63**

W.A. Omar et al. / Mutation Research 746 (2012) 7–14

The genomic DNA extracted from gills and liver samples of both fish species collected from the reference site, showed a very weakly stained smear pattern upon electrophoresis, with no evidence of DNA-ladder pattern (Fig. 2A). In contrast, the genomic DNA extracted from samples of both Lake Qaroun and the fish farms around it revealed internucleosomal fragmentation (ladder pattern) mixed with a smear-like pattern, which are generally regarded as a molecular hallmark of apoptosis and necrosis (Fig. 2B/C). The densitometric analysis of low molecular-weight DNA fragments extracted from gills and liver samples of both fish species is presented in Table 5. 4. Discussion Aquatic pollution not only affects the stability of aquatic bodies, but also causes developmental and genotoxic changes in aquatic inhabitants. 4.1. Concentration of heavy metals in water and sediments Among the important classes of pollutant, heavy metals are in the narrow window between their essentiality and toxicity [27]. The source of heavy metals at the study sites is mainly of exogenous origin, due to either agricultural influx and/or sewage via surrounding cultivated lands [28]. Moreover, the high concentration of the heavy metals studied in sediment samples results from precipitation of these metals from the water column under slightly elevated pH conditions recorded in the area, and from the adsorption of heavy metals onto organic matter and their settlement downwards. The high levels of heavy metals in both Lake Qaroun and neighbouring fish farms could be attributed to the agricultural and

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Fig. 1. Representative nuclear and cellular alterations recorded in erythrocytes of Oreochromis niloticus and Mugil cephalus. (A) micro-nucleated erythrocyte, (B) nuclear bud, (C and D) erythrocyte bearing more than a single micronucleus, (E) blebbed nucleus, (F) lobed nucleus, (G) notched nucleus, (H and I) nuclear fragmentation, (J) bi-nucleated erythrocyte, (K) poly-nucleated erythrocyte, (L) vacuolated nucleus, (M) cariolysis, (N) vacuolated cytoplasm, (O) nuclear retraction and (P) microcyte. 1000× magnification.

Fig. 2. Representative agarose-gel electrophoretograms of extracted DNA from liver and gills of Oreochromis niloticus and Mugil cephalus. (A) Site 1 (reference site), (B) site 2 (Lake Qaroun) and (C) site 3 (Qaroun fish farms).

W.A. Omar et al. / Mutation Research 746 (2012) 7–14

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Table 2 Body indices of Oreochromis niloticus and Mugil cephalus collected from the study sites. Locations

Site 1 (reference site) Site 2 (Lake Qaroun) Site 3 (Qaroun fish farms) F-value

Total body length (cm)

Wet weight (g)

O. n.

M. c.

O. n.

18.70 ± 0.29 A 15.85 ± 0.20 B 18.13 ± 0.33 A 28**

24.13 ± 0.21 A 25.05 ± 1.66 A 24.44 ± 0.15 A 0.24

134.35 ± 6.61 A 66.83 ± 3.47 C 109.80 ± 5.66 B 40**

Condition factor (CF) M. c. 151.88 ± 5.06 A 103.68 ± 6.25 B 143.68 ± 3.12 A 27**

O. n.

M. c.

2.06 ± 0.05 A 1.38 ± 0.10 C 1.83 ± 0.01 B 27**

1.06 ± 0.01 A 0.85 ± 0.01 C 0.98 ± 0.02 B 112**

Data are represented as means of eight samples ± S.E. Means with the same capital letter in the same column for each parameter are not significantly different. ** Highly significant difference (P < 0.01).

sewage drainage-water that passes directly to the lake from the El-Fayoum province through a system of twelve drainage canals. Bearing in mind that the lake is a closed basin and in view of extensive evaporation of water, the accumulation of chemical pollutants is expected to increase in all its components, especially during summer seasons where rates of evaporation reach maximum values. This is in agreement with studies of Mansour and Sidky [10] as well as Authman and Abbas [28]. Similarly, the increment of metals in the fish farms around the lake appeared as a result of the dependence of these fish farms on agricultural drainage and seepage from the neighbouring contaminated lake, which is already loaded by heavy metals from different sources [10]. According to the WHO [29], the guideline values for copper, lead and manganese in water are 2 mg/l, 0.01 mg/l and 0.4 mg/l, respectively, whilst no health-based guideline values have been proposed for zinc and iron in water. As indicated by Enserink et al. [30], metals in a mixture

may have additive chronic toxicity compared with their individual effect. Generally, variations in levels of the heavy metals studied at different sites could be attributed to the quality and quantity of the drainage water as well as a variety of interacting environmental factors, including climate, evaporation rate, land use and watersupply management practices for crop irrigation. 4.2. Condition factor Gross health indices, such as the condition factor CF, have been accepted as integrative indicators of general fish condition and can provide information on the ability of animals to tolerate environmental stresses. The CF is quite general and non-specific, but its low cost and simplicity still make it a valuable tool that indicates general effects of pollution in fish [31]. Therefore, CF can be estimated

Table 3 Frequency of micronuclei and other nuclear alterations recorded in erythrocytes of Oreochromis niloticus and Mugil cephalus. Site 1 (reference site)

Site 2 (Lake Qaroun)

Site 3 (Qaroun fish farms)

F-value

Micronucleus

O. n. M.c. tv

5.0 ± 0.50 C 2.75b ± 0.56 C 3.10**

34.50 ± 1.60 A 18.75b ± 2.66 A 5.10**

23.25 ± 1.40 B 10.0b ± 2.07 B 5.30**

143** 16**

Nuclear buds

O. n. M.c. tv

5.75a ± 0.56 B 3.50b ± 0.42 C 3.20**

23.0a ± 2.35 A 14.50b ± 1.27 A 3.20**

17.25a ± 1.72 A 8.50b ± 1.15 B 4.20**

26** 29**

Binucleated

O. n. M.c. tv

2.75a ± 0.41 C 1.25b ± 0.31 B 2.90*

10.75a ± 1.11 A 6.0b ± 0.96 A 3.20**

8.0a ± 0.71 B 5.50b ± 0.87 A 2.23*

26** 12**

More than single micronucleus

O. n. M.c. tv

0.25a ± 0.16 B 0.50a ± 0.20 B 1.0

8.75a ± 1.90 A 5.25a ± 0.82 A 1.70

2.75a ± 0.41 B 1.75a ± 0.41 B 1.70

15** 21**

Polynucleated

O. n. M.c. tv

0.50a ± 0.19 A 0.50a ± 0.33 A 0.01

0.25a ± 0.16 A 0.75a ± 0.31 A 1.41

0.25b ± 0.16 A 1.0a ± 0.27 A 2.39*

Nuclear fragmentation

O. n. M.c. tv

1.75a ± 0.41 B 1.25a ± 0.41 B 0.86

16.25a ± 2.70 A 15.75a ± 1.78 A 0.15

14.25a ± 2.77 A 13.50a ± 1.38 A 0.24

12** 35**

Notched nuclei

O. n. M.c. tv

3.25a ± 0.41 B 1.50b ± 0.42 B 3.0**

14.75a ± 1.93 A 8.25b ± 1.50 A 2.70*

12.50a ± 2.31 A 3.75b ± 0.41 B 3.70**

12** 14**

Lobed nuclei

O. n. M.c. tv

4.0a ± 0.53 C 3.75a ± 0.16 C 0.45

15.75a ± 1.45 A 8.75b ± 0.97 A 4.20**

12.0a ± 1.28 B 6.25b ± 0.41 B 4.30**

29** 16**

Blebbed nuclei

O. n. M.c. tv

5.25a ± 1.08 C 2.75a ± 0.94 B 1.75

13.0a ± 1.17 A 9.25b ± 0.31 A 3.10**

15.75a ± 1.42 B 11.50b ± 0.94 A 3.70**

26** 33**

a

a

Data are represented as means of eight samples ± S.E. tv = t-test values between Oreochromis niloticus (O. n.) and Mugil cephalus (M. c.) for each parameter in each site. Means with the same small superscript letter in the same column for each parameter are not significantly different. Means with the same capital letter in the same raw for each parameter are not significantly different. * Significant difference (P < 0.05). ** Highly significant difference (P < 0.01).

a

0.70 0.68

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Table 4 Frequency of cellular and cytoplasmic alterations recorded in erythrocytes of Oreochromis niloticus and Mugil cephalus. Site 1 (reference site)

Site 2 (Lake Qaroun)

Site 3 (Qaroun fish farms)

F-value

Microcytes

O. n. M.c. tv

4.75a ± 0.56 B 5.0a ± 0.53 A 0.32

7.50a ± 1.21 B 4.75a ± 1.26 A 1.57

16.38a ± 3.49 A 7.13b ± 0.30 A 2.64*

8** 2.6

Vacuolated nuclei

O. n. M.c. tv

2.25a ± 0.41 C 2.0a ± 0.53 B 0.37

18.50a ± 2.28 A 9.50b ± 1.32 A 3.42**

10.25a ± 1.01 B 8.0a ± 0.27 A 2.15

31** 22**

Cariolysis

O. n. M.c. tv

0.25a ± 0.16 C 0.25a ± 0.16 C 0.0

8.75a ± 1.47 A 6.50a ± 0.68 A 1.39

4.50a ± 0.87 B 3.0a ± 0.27 B 1.65

18** 52**

Vacuolated cytoplasm

O. n. M.c. tv

8.0a ± 1.16 B 7.0a ± 1.20 C 0.59

29.75a ± 3.60 A 32.0a ± 3.11 A 0.47

13.25a ± 2.16 B 16.25a ± 2.78 B 0.85

20** 25**

Nuclear retraction

O. n. M.c. tv

8.0a ± 0.93 B 4.50b ± 0.42 B 3.44**

14.0a ± 1.75 A 11.25a ± 0.98 A 1.37

15.75a ± 1.08 A 9.25b ± 1.37 A 3.71**

10** 12**

Data are represented as means of eight samples ± S.E. tv = t-test values between Oreochromis niloticus (O. n.) and Mugil cephalus (M. c.) for each parameter in each site. Means with the same small superscript letter in the same column for each parameter are not significantly different. Means with the same capital letter in the same raw for each parameter are not significantly different. * Significant difference (P < 0.05). ** Highly significant difference (P < 0.01).

for comparative purposes to assess the impact of environmental alterations on fish performance [32], but does not give information of specific responses to toxic substances in the media [33]. The low CF values for both fish species at sites 2 and 3 compared with those at the reference site are indicative of the impaired conditions and the higher environmental stress that are affecting fish. The current results strongly agree with those of Bonga and Lock [34], who reported that waterborne toxicants increase the permeability of gill epithelia towards water and ions and inhibit the ion-exchange activity of the so-called chloride cells, which regulate the response to a changing environment by responding to a rapid signal that stimulates chloride secretion. Thus the compensatory responses of the fish would significantly increase the energy required for maintenance of water and ion homeostasis; as a consequence, this will result in reduced growth. 4.3. Micronucleus test

to the type and concentration of pollutants in that location [2]. Our study confirmed that the two selected fish species O. niloticus and M. cephalus, had various degrees of sensitivity in monitoring genetic and clastogenic damage, as indicated by the variations in average numbers of micro-nucleated cells. Also, blood erythrocytes of O. niloticus were more susceptible to clastogenic damage and appeared to be more sensitive in MN formation than cells of M. cephalus. These results are supported by those of Palhares and Grisolia [35], who demonstrated that two closely related fish species can respond in completely different ways to a given genotoxic agent. This significant variation could be attributed to the toxicokinetics of the pollutant and to the speed of the haemopoietic cycle of the selected fish species [2]. Also, this scientific evidence could be related to an interspecies difference in metabolic competency and DNA repair, as well as in the MN expression and/or the different capability of the repair system in the kidneys that can recover the damage in erythrocytes [36].

4.3.1. Micronucleus frequency Current awareness of the potential hazards of heavy metals in the aquatic environment has stimulated much interest in the use of fish in the bio-monitoring of environmental carcinogens, teratogens and mutagens [31]. Chromosomal damage represented as micronucleus formation resulting from inefficient or incorrect DNA repair and/or from the physical presence of metals around the mitotic apparatus, is expressed during cell division and represents an index of accumulated genotoxic agents [2]. In our study, clear differences in MN frequencies between the study locations, displaying different levels of contamination, have been obtained for both fish species. The variation in MN frequency is dependent on the environmental stress and could be related

4.3.2. Other nuclear abnormalities The frequency of other nuclear abnormalities (NA) was considered as the least efficacious indicator of genotoxicity [37]. In contrast, such abnormalities have been used by various authors as good indicators of genotoxicity in fish [3,38]. Therefore, we accurately recorded these abnormalities to be used as indicators of genotoxic damage and complement the scoring of MN in this genotoxicity assessment. These nuclear protrusions and deformations could be attributed to the detrimental effects caused by clastogenic pollutants, which cause problems in chromosomal attachments or gene amplification. These abnormalities could also cause deformed nuclei as indicated by the presence of lobed, blebbed, bi-nucleated,

Table 5 Densitometric analysis of the fragmented DNA sampled from gills and liver of Oreochromis niloticus and Mugil cephalus. Locations

Fragmented DNA of the gills O. niloticus

Site 1 (reference site) Site 2 (Lake Qaroun) Site 3 (Qaroun fish farms)

116.22 ± 2.60 C 321.70 ± 4.82 B 367.26 ± 3.01 A

Fragmented DNA of the liver M. cephalus

O. niloticus

M. cephalus

51.64 ± 2.80 C 331.73 ± 1.76 B 370.65 ± 3.14 A

66.20 ± 11.56 B 235.84 ± 2.65 A 186.33 ± 35.42 A

50.83 ± 0.60 C 407.17 ± 1.88 A 177.30 ± 3.66 B

Data are represented as means of three gel runs in arbitrary units ± S.E. Means with the same capital letter in the same column for each parameter are not significantly different.

W.A. Omar et al. / Mutation Research 746 (2012) 7–14

notched, budded and other deformed nuclei during the elimination of amplified DNA from the nucleus [36,39].

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Conflict of interest There is no conflicting interest.

4.3.3. Cellular abnormalities Other recorded cellular anomalies such as microcytes, vacuolated nuclei, cariolysis, vacuolated cytoplasm and nuclear retraction reflect the consequences of cell injury, cell death and mitotic errors [2]. Some of these are accompanied by apoptosis and necrosis, which may result from DNA damage. Generally, Lake Qaroun was found to be highly contaminated at the level of the estimated heavy metals. In accordance with these results, the fish collected from the lake expressed the highest level of micronuclei and nuclear anomalies. These results support the facts demonstrated by Kligerman [2], who stated that fish inhabiting polluted waters have greater frequencies of micronuclei and other anomalies. 4.4. DNA fragmentation This endpoint was used to evaluate the complex genotoxic effects of the metals at the DNA level of different organs of both fish species studied. Different tissues are usually used for the determination of DNA damage, e.g., intestine, liver, kidneys, spleen and gills [40]. In the present study both liver – the organ of detoxification – and gills as the organs directly exposed to environmental pollutants, were chosen. Both liver and gills are metabolically active organs that accumulate more metals than other organs. Heavy metals catalyse reactions that generate reactive oxygen species such as hydrogen peroxide, superoxide anions and hydroxyl radicals, which may lead to environmental oxidative stress and to damage in tissues and macromolecules such as DNA, proteins and lipids [41,42]. In addition, free radicals could stimulate endogenous endonuclease activity [43]. A regulatory role of iron, copper and zinc in endonuclease activity and apoptosis has been suggested by several authors [44,45]. Therefore, the complexation of such metal ions leads to DNA fragmentation. DNA cleavage occurs at sites between nucleosomes and may lead to ladder pattern of fragmentation, which is a hallmark of apoptosis [46]. Furthermore, digestion of chromatin by proteases and endonucleases into a smear pattern – as the proteases obliterate the histones and expose the whole length of DNA to the nucleases – reflects a hallmark of necrosis [47]. Nevertheless, the present investigation shows mixed smearing and laddering of DNA fragments, which is most probably attributed to the nonspecific DNA-fragmentation process encountered during apoptosis. This result is in line with that of Razzaque [48], who stated that both fragmentation patterns may involve more than one mechanism leading to cell death and the combined effect of these events can induce apoptosis and/or necrotic cell death. 5. Conclusions and recommendations Pollution with metals posed a critical problem at the study sites, as reflected by the high metal concentrations recorded in water and sediment samples. Because of the continuous discharge to the aquatic habitats considered in this study, the concentrations of heavy metals may soon reach a dangerous level affecting the health of local human communities. Application of the micronucleus assay in fish erythrocytes and the DNA-fragmentation technique in the assessment of genotoxic pollutants, provide valuable biomarkers in field surveys, in monitoring studies and in comparing different levels of pollution. Therefore, this study strongly recommends the coordination of different efforts to rescue the polluted habitats from serious ecological problems using proper management and scientifically specialised research.

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