Int. J. Nanoparticles, Vol. 9, No. 1, 2016 41

Copyright © 2016 Inderscience Enterprises Ltd.

Genotoxicity detection following exposure to silver nanoparticles in African catfish (Clarias gariepinus)

Alaa El-Din H. Sayed Zoology Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt Fax: +020882342708 Email: alaa_h254@yahoo.com

Abstract: The aim of the present study is to evaluate the cytotoxic and genotoxic effects of silver nanoparticles (Ag-NPs) towards African catfish (Clarias gariepinus). Adult male catfish was exposed to 0, 25, 50, and 75 mgl–1 Ag-NPs for two weeks. Exposure to Ag-NPs exerted an increase in mortality rate and behavioural changes compared to control. A fluorescent microscopy examination was used to assess the cytotoxic effect. There was a 15-fold greater extent of apoptosis in erythrocytes of exposed fish to 75 mgl–1 compared to control fish. No significant differences in the extent of apoptosis were detected in 25 mgl–1 and 50 mgl–1 exposed fish. Also, the genotoxic effect of the tested compound was evaluated via micronucleus and DNA fragmentation assays. The micronucleated erythrocytes were observed as well as, DNA damage was recorded in the liver, kidney, gill, and muscles in all exposed groups and percentage elevated with the increase of Ag-NPs concentration. Overall, our results indicate that, Ag-NPs exhibited the both genotoxic and cytotoxic effects in African catfish.

Keywords: Ag-NPs; apoptosis; micronuclus; DNA; Clarias gariepinus.

Reference to this paper should be made as follows: Sayed, A.H. (2016) ‘Genotoxicity detection following exposure to silver nanoparticles in African catfish (Clarias gariepinus)’, Int. J. Nanoparticles, Vol. 9, No. 1, pp.41–53.

Biographical notes: Alaa El-Din H. Sayed is a Scientist and Researcher. He is currently working as a Lecturer at the Zoology Department, Faculty of Science, Assiut University, Egypt. His research interests relates to fish toxicology, environmental pollution, aquaculture and aquatic toxicology.

1 Introduction

Nanoparticles (NPs) are widely used to produce novel materials with unique physicochemical properties, which become an environmental concern in the event of unintended release into the environment. The increasing commercial application of NPs, with at least one dimension of ≤100 nm, is currently showing inventory listings of 1,317 nanotechnology-based consumer products in 30 countries (WoodrowWilsonDatabase,

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2011), while the production of engineered NPs is expected to reach approximately 60,000 tons in 2011 (Jovanović´ et al., 2011; Matranga and Corsi, 2012). The properties of NPs that contribute to biological perturbations strongly depend on their size, mineralogy, crystallinity, and surface reactivity which is directly connected to NP toxicity through redox reactions, production of oxygen or nitrogen free radicals, the dissolution of NPs, release of toxic ions, the sorption and transport of metal ions or xenobiotic pollutants (Bottero et al., 2011).

Choi et al. (2010) mentioned that, NPs may induce deterimental effects in aquatic systems as well as aquatic organisms. Despite their widespread use in medicine, cosmetics, renewable energies, electronic devices, and environmental remediation (Fabrega et al., 2011; Matranga and Corsi, 2012), there is limited data on the safety and toxicity of Ag-NPs especially those relating to its DNA interaction (Flower et al., 2012).

The effect levels of Ag-NPs range from µgl–1 to mgl–1 including the variety of aquatic organism type, particle type, and exposure conditions (Farmen et al., 2012; Hsin et al., 2008). Some studies aimed to investigate Ag-NPs genotoxicity, especially of biogenic NPs, resulting in an increase in reactive oxygen species (ROS) associated with DNA damage, apoptosis and necrosis (Lima et al., 2012).

Some of ecotoxicological studies have been conducted to study the effects of Ag-NPs in algae, bacteria invertebrates, fish, and human (Carlson et al., 2008; Gopinath et al., 2008; Govindasamy and Abdul Rahuman, 2012). However, some authors have been studied the effects of Ag-NPs on fishes (Choi et al., 2010; Griffitt et al., 2008), the toxicological action of nanoscale silver in the aquatic environment is largely unknown (Wu et al., 2010). Various toxic changes have been observed in NPs-exposed fish and embryos including lipid peroxidation, apoptosis, and changes in gene expression (Choi et al., 2010; Zhu et al., 2008). Apoptotic changes have been detected in Ag-NPs treated zebrafish (Choi et al., 2010). Many studies demonstrated that NPs have potential to induce ROS-mediated DNA damage and apoptosis (Ahamed et al., 2011, 2013; Ahmad et al., 2012). Apoptosis is induced by extracellular or intracellular signals, that trigger onset of signalling cascade with characteristics of biochemical and cytological signatures including nuclear condensation and DNA fragmentation (Ahmad et al., 2012; Gopinath et al., 2008).

Fish blood parameters are indicators of chemical toxicity and useful tools to study the effects of exposure of fish to toxins present in the aquatic environment (Mekkawy et al., 2011; Sayed et al., 2011, 2013). Because the blood has been recognised as the main route for a substance passage that enters the fish, we investigated the red blood cells (R.B.Cs) damage occurred by Ag-NPs using C. gariepinus as fish toxicological model (Sayed et al., 2015).

Farkas et al. (2011) has been recommended for further studies including endpoints such as ion regulatory capacities disturbance, alterations in cell morphology may deliver information concerning NPs effects on fish.

In the present study, the cytotoxic and genotoxic effects of Ag-NPs in African catfish were investigated by detection of apoptotic cell death, micronucleus in erythrocytes as well as DNA fragmentation in gill, liver, kidney and muscles.

Genotoxicity detection following exposure to silver nanoparticles 43

2 Materials and methods

2.1 Preparation and characterisation of Ag-NPs

The Ag-NPs were purchased from Sigma-Aldrich, St. Louis, MO, USA. The physical characteristics of the particles according to the manufactures data were; size (≤100 nm), purity (99.5%), trace metal basis, surface area (5.0 m2g–1), and density (10.49 gcc–1). According to Ghosh et al. (2012b), Ag-NPs were suspended in PBS and dispersed by ultrasonic vibrations (100 w. 30 khz) for 30 min. Filtering using a cellulose membrane (pore size 100 nm, advantec, Tokyo, Japan) to remove NPs aggregations. The concentration of the Ag-NPs stock suspension after filtration was 3975 mgl–1, that was very close to the concentration provided by the manufacturer (4000 mgl–1) measured by spectrometer (ICPE-9000, Tokyo, Japan). The stock suspension of Ag NPs was re-sonicated for 3 to 5 min before tests, and then it was diluted with deionised water to make final test concentrations according to the experimental design.

Energy filtering transmission electron microscopy (TEM) was used to examine the particle shape and size of the Ag-NPs. Twenty µL of the particle suspension were dried onto a 400 mesh carboncoated copper grid and electron micrographs [Figure 1(a)] were obtained using a Jeol JEM 1200 EX Transmission Electron Microscope at Electron microscope centre of Assiut University, Egypt at 80-120 kV. Also, the size distribution of the Ag- NPs was evaluated using a dynamic light scattering (DLS) spectrometer, DLS-7000 (Otsuka Electronics Co., Inc., Osaka, Japan). The main NP sizes distributed in the test medium were about 100 nm [Figure 1(b)].

Figure 1 (a) Micrograph of Ag-NPs in the test media using TEM (b) size distribution of particles based on number frequency (see online version for colours)

2.2 African catfish (C. gariepinus)

Adult male African catfish (C. gariepinus) (Burchell, 1822) were collected from the river Nile in July 2012. Fish were kept at approximately 28ºC with 12 hr: 12 hr light: dark cycle and held in a closed recirculating system (300 L) for six months to acclimatise to laboratory condition before to the experiment. During the acclimation period, 20% of the water in each recirculating system was replaced daily and were fed 5% body weight twice a day with commercial pellets (Ingredient as soybean meal (48% protein), cotton meal (41% protein), menhaden meal (61% protein), corn grain, wheat middlings, dicalcium

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phosphate, catfish vitamin and mineral mix and fat/oil). Fish measuring the average size about 36.7 ± 1.38 cm in length and the average weight about 424.4 ± 27.34 g in the prior day of exposure.

2.3 Experimental design

Following the acclimatisation period, the African catfish were examined to be free of external parasites (AFS-FHS 2003), the fish randomly assigned into four equal groups each containing 12 fish. Each group were maintained in 100 L glass aquaria and exposed to a graded series of Ag-NPs at 0, 25, 50 and 75 mgl–1 (Govindasamy and Abdul Rahuman, 2012) for two weeks in triplicates for each concentration. The doses selected for this study were based on the current guidelines and recommendations of bulk chemicals (OECD, 2012). The experimental media water was changed each day. Mortality was carefully recorded based on the Karber method (Yılmaz et al., 2004). The water temperature, pH, dissolved oxygen (DO) concentrations and electrical conductivity (EC) were measured daily as (23.2 ± 0.08ºC, pH: 6.8 ±11, DO: 6.5± 0.89 mgl–1 and EC: 260 ± 0.2 µmho.cm–1) respectively.

2.4 Sample collection

Drops of whole blood were directly smeared on clean grease free slides after two weeks for apoptosis and micronucleus test. The reminder of blood was into test tube without anticoagulant agent, then centrifuge at 5,000 rpm and finally isolated in Eppendorf and stored at –20ºC for future analysis). After fish dissecting, small pieces from gill, liver, kidney, and muscles were collected and stored at –80ºC for DNA analysis.

2.5 Determination of Ag bioaccumulation

Fish serum and water samples of media were collected. 1 ml of each serum and water media in 3 mL of concentrated HNO3, boiled for 45 min at 100ºC to break down the fish tissue and dissolve all the silver content. Then the mixture were cooled, diluted in 1,000 mL of deionised water, and the silver quantified using graphite furnace AA (GFAA) spectroscopy (Kennedy et al., 2010).

2.6 Detection of apoptosis

Apoptotic erythrocytes were detected using acridine orange (AO) stain (Cat. No. A1031), Life Technologies, 5791 Van Allen Way Carlsbad, CA 92008 USA). The modified protocol according to Darzynkiewicz (1990) was used to detect the apoptosis in RBCs as, after preparation of blood smears (6 samples from each group) on a clean glass slides (three slides from each sample), the slides were washed in 1X PBS (pH = 7.2). AO buffer (17 µgl–1 AO in 1X PBS buffer) was added to the slides for 30 minutes in the dark. Decolourisation process by washing the slides every 30 minutes with 1X PBS for four times. Fixation was in paraformaldehyde 4 % for five minutes. Finally, observation of cells (1,000 cells/group) under Zeiss Axioplan2 fluorescence microscope (X200) provided with a digital 3 CCD colour video camera (Sony, AVT-Horn).

Genotoxicity detection following exposure to silver nanoparticles 45

2.7 Micronucleus test

The blood smears were fixed in absolute methanol for 10 min after drying at room temperature. Slides were stained with Gimsa stain (Verma and Babu, 1989). Examined slides were selected based on staining quality, then coded, randomised and scored blindly. In each group 10,000 cells (a minimum of 1,000 per slide) were examined (Al-Sabti and Metcalfe, 1995) at 40× objective and 10× eyepiece for micronucleated erythrocytes in separate slides. The established criteria for identifying MN were strictly followed to ensure authentic scoring (Schmidt, 1975).

2.8 Measurement of DNA fragmentation

DNA fragmentation in gill, liver, kidney, and muscles of control and Ag-NPs exposed groups was determined by the procedure of Kurita-Ochiai et al. (1999) using UV-Vis spectrophotometer (Abbota Corporation, New Jersey, USA) at 600 nm against reagent blank. In brief, 100 mg of tissue (10% W/V) was added to 1 ml of buffer (102 mg Tris + 29 mg EDTA + 200 µ Triton in 100 mL distilled water). Then the mixture was incubated in ice for 10 min and centrifuged at 8,000 rpm for 10 min. The supernatant was used for the measurement of fragmented DNA while the pellet was used for the determination of intact DNA. For the measurement of fragmented DNA, 200 µ of supernatant was added to 200 µ of trichloroacetic acid (TCA). The total volume was centrifuged at 8,000 rpm for 10 min. After centrifugation, 50 µ supernatant or standard was added to 1 ml of diphenylamine reagent, boiled for 10 min in water bath and then cooled on ice. For the determination of intact DNA, the pellet was added to 1 ml buffer (102 mg Tris + 29 mg EDTA + 200 mg SDS in 100 ml distilled water). The mixture was heated in a water bath at 40ºC and centrifuged for 10 min at 5,000 rpm; 200 µ of supernatant was added to 200 µ of (TCA). The total volume was centrifuged at 5,000 rpm for 10 min. After centrifugation, 50 µ of supernatant was added to 1 ml of diphenylamine reagent, boiled for 10 min in a water bath and cooled on ice. The developing blue colour was measured at 575 or 600 nm against a blank (diphenylamine solution). The percentage of fragmented DNA was estimated by the following formula: percentage of fragmented DNA = fragmented DNA/ (fragmented + intact DNA) X 100.

2.9 Statistical analysis

The basic statistics (means, standard divisions, and ranges) were calculated. The pattern of variation was one-way analysis of variance using the SPSS package (SPSS, 1998) at the 0.05 significance level. The Tukey-HSD test was considered for multiple comparisons and designed to verify the frequency of apoptotic cells, MN incidence and mortality rate. Dunnett t-tests treat one group as a control and compare with all other groups against it.

2.10 Ethical statement

All experiments were carried out in accordance with the Egyptian laws and University guidelines for the care of experimental animals. All procedures of the current experiment have been approved by the Committee of the Faculty of Science of Assiut University, Egypt.

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3 Results

3.1 Behaviour and mortality rate

Normal colour and behaviour as (vigorous activity, static equilibrium, active swimming, normal gill movement and hanging horizontally in the water of fish) was observed in control group and group exposed to 25 mgl–1 Ag-NPs. Abnormal behaviour was observed at 50 mgl–1 and 75 mgl–1 Ag-NPs such as restlessness, sudden quick movements, air gulping, rolling movements, swimming on the back. However, the amount of mucus slightly increased with extensive pigmentation mainly on the dorsal side at 75 mgl–1 Ag-NPs. Reaction to excitation was manifested by sudden movement, very weak, and finally death. Mortality pattern for control and different Ag-NPs doses was reported where the increase in the mortality rate at P < 0.05 was as (0.0 ± 0.0, 0.0 ± 0.0, 0.027 ± 0.0278 and 0.055 ± 0.056) % in the control, 25, 50, and 75 mgl–1 Ag-NPs respectively.

3.2 Ag bioaccumulation

The present study showed that the concentration of the exposure dose had a dramatic effect on the Ag bioaccumulation during 14 days of exposure. The mean Ag level in the serum of fishes and water media exposed to Ag-NPs for 14 days was found to be concentration-dependent (Table 1). Table 1 Silver bioaccumulation concentration (mgl–1) in serum and water media (mean ± SD)

of African catfish exposed to different Ag-NP concentrations (25, 50 and 75 mgl–1) for 14 days, n = 12 fish/treatment

TreatmentsOrgan

Control 25 mgl–1 Ag-NPs 50 mgl–1 Ag- NPs 75 mgl–1 Ag-NPs

Fish serum 0.96 ± 0.208 (0.8–1.2) a

110.66 ± 11.37 (98–120) b

138.66 ± 8.32 (132–148) c

253.66 ± 28.43 (222–277) d

Water media 1.43 ± 0.152 (1.3–1.6) a

132 ± 7.2 (124–138) b

146 ± 6 (140–152) c

280.33 ± 6.02 (274–286) d

Note: Different letters indicates there is a significant difference at (p≤ 0.05).

3.3 Apoptosis detection

There was an increase in the percentage of apoptotic cells (erythrocytes) due to exposure of fishes to Ag-NPs versus those of controls (Figure 2). The increase was concentration-dependent and reaches its high level at the dose of 75 mgl–1 Ag-NPs. Statistically, this increase was significant between the Ag-NPs – exposed groups and the control one and within the three exposed groups (25, 50 and 75 mgl–1).

Figure 2 showed the apoptosis detection of blood smears of C. gariepinus in the control group [Figure 2(a)] and Ag-NPs exposed groups [Figure 2(b), 2(c) and 2(d)]. The apoptotic cells nuclei stained with AO were observed as light green under fluorescence microscope.

Genotoxicity detection following exposure to silver nanoparticles 47

Figure 2 Representative blood smears of adult African catfish (Clarias gariepinus) stained with (a) AO from control and exposed groups at (b) 25 (c) 50 and (d) 75 mgl–1 Ag-NPs showing the apoptotic cells (light green) (X400) (see online version for colours)

Figure 3 (a) Blood film of control catfish (Clarias gariepinus) (b) 75 mgl–1 Ag-NPs exposed showing the rounded shape of the nucleated erythrocytes (Er), altered erythrocytes (AEr), distinct micronucleus (Mn) (X400) (see online version for colours)

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3.4 Micronucleated erythrocytes formation

The percentage of micronucleated cells of control fish was 0.2 ± 0.42 % and this percentage increased significantly with increasing the dose of Ag-NPs as 1.6 ± 0.69%, 2.5 ± 0.97% and 4.2 ± 0.91% for 25, 50, and 75 mgl–1 Ag-NPs respectively. Also, Figure 3 showed the normal structure of the blood smears of the C. gariepinus that described in our pervious publications (Mekkawy et al., 2011; Sayed et al., 2013) where erythrocytes appeared normal and healthy [Figure 3(a)] in comparison with 75 mgl–1 Ag-NPs exposed group [Figure 3(b)]. The micronucleated cells were observed in all groups.

Figure 4 Values of the percentage of damage of DNA (%) in different organs [gills (G), liver (L), kidney (K) and muscles (M)] of Clarias gariepinus at different concentrations of Ag-NPs (see online version for colours)

Note: Different letters indicates there is a significant difference at (p ≤ 0.05).

3.5 DNA damage

DNA damage induced by Ag-NPs exhibited significant variability (p < 0.05) in C. gariepinus through organs (Figure 4). Kidney and gills recorded the highest percentage of DNA damage, almost followed by least DNA damage in liver and muscles. The pattern of DNA damage was recorded in all groups as G > K > L > M. DNA damage was found to be significantly different in each group between organs and there was a correlation (R = 0.968). However, in the other hand DNA damage has significant higher

Genotoxicity detection following exposure to silver nanoparticles 49

correlation with each organ (R = 0.973). Interaction between Ag-NPs dose and organ factors was evident (p < 0.0001). DNA damage was (21± 0.53) in gill for control group where it significantly increased with the increase of Ag-NPs doses as 22.81 ± 0.87, 26.13 ± 6.67 and 28.7 ± 0.51 for 25 mgl–1 Ag-NPs, 50 mgl–1 Ag-NPs and 75 mgl–1 Ag-NPs, respectively.

4 Discussion

To assess the risk of Ag-NPs, it is important to understand the impacts of other conditions (aquatic organism type, particle type, and exposure conditions) on its toxicity and mechanisms of toxicity. Although, Ag-NPs are being widely used as bactericidal agents in day to day life, their potential effects in aquatic organisms were not studied well (Vignesh et al., 2013). To evaluate the effects of Ag-NPs on catfish C. gariepinus, apoptosis detection, micronucleus test, mortality rate and DNA fragmentation were analysed. The obtained data demonstrate that Ag-NPs increased apoptosis induction and these results agree with that obtained by Christen et al. (2013). Also, it showed an increase in the mortality rate with the increase of Ag-NPs concentration. In this aspect, high mortality rate was found in 100 µgl–1 Ag-NPs exposed group for juvenile of Atlantic salmon after 48 h exposure (Farmen et al., 2012). Cell degeneration and necrosis o f the epithelial lining of secondary gill arches were observed in salmon exposed to 100 µgl–1 Ag-NPs showing the high mortality rate. The increase of mortality following 100 µgl–1 NPs exposure could be compatible with an added particle specific effect such as precipitation and deposition at the gill surface (Farmen et al., 2012).

In the present study, the measured concentrations of the Ag in both serum of fish and water were high, this is in agreement with the results of Farmen et al. (2012) where they found that Ag-NPs at 100 µgl–1 concentrations in natural soft waters were increased gill accumulation of Ag in Atlantic salmon and result in numerous physiological and biochemical responses indication of toxicity. Also, Salari et al. (2013) reported that, The bioaccumulation of Ag in the studied tissues was concentration-dependent. The current findings represent one of the first observations of erythrocytes alteration in C. gariepinus exposed to Ag-NPs. As oxidative stress may be a reason of causing cytotoxicity, the present investigation showed cytotoxicity in fish erythrocytes, and these results were agree with that of Ghosh et al. (2012a). The inflammation properties of NPs in fish were revealed in trout using toxicogenomics (Gagné et al., 2012). Also, previous literatures showed that Ag-NPs were cytotoxic (Ahmed et al., 2008; Ghosh et al., 2012b; Shin et al., 2007; Taju et al., 2014; Wise et al., 2010) to mammalian cells. Also, it has been reported that Ag-NPs accumulated in epithelial gill tissues (Farkas et al., 2011) causing cytotoxic effects for membrane stability. As the damage observed as side effects of Ag-NPs on erythrocytes of catfish in the present study, our results were agree with those results indicating the genotoxicity of Ag-NPs in blood cells of erythrocytes (Ghosh et al., 2012b). Morones et al. (2005) reported that silver ions may be attached to the bacteria plasma membrane leading to plasma lysis which inhibiting the bacterial cell membrane synthesis, by the same mechanism may be Ag-NPs altered the erythrocytes membranes leading different shapes of erythrocytes alterations. The toxic effects of Ag-NPs were observed in rainbow trout hepatocytes (Farkas et al., 2011). Previous studies using the same Ag-NPs concentrations showed many changes in liver, gill and skin (Govindasamy and Abdul Rahuman, 2012). All changes were reported in this study indicated the

50 A.H. Sayed

changes of genetic materials such as karyolysis, nuclear hypertrophy, and necrosis. Reduction in membrane integrity was recorded in trout primary hepatocytes (Farkas et al., 2011).

The percentage of micronuclei in the present study after exposure to Ag-NPs increased; this may be as DNA damage or genotoxicity. These results are agreed with those of Feng et al. (2000); they reported that Ag-NPs may penetrate the cell wall affecting DNA and protein synthesis. In contrast, Vignesh et al. (2013) has been reported that synthesised Ag-NPs do not affect the RNA, DNA ratio in L. lohita. Liver, muscles, kidney and gill were chosen as model organs, because of their important as target organs of NPs. It has been reported that Ag-NPs reaches the liver via uptake from gut and blood (Christen et al., 2013; Gaiser et al., 2012; Wu et al., 2010). Gagné et al. (2012) has been reported that exposure of trout to 0.06 µgl–1 Ag-NPs led to a 62% drop in DNA stand breaks. It has been reported that treatment of human blood cells with 50 and 100 µgl–1 Ag-NPs for 3 h resulted in DNA damage and they stated the DNA damage was resulted from cytotoxicity, apoptosis or necrosis (Flower et al., 2012). Bertram and Playle (2005) have been reported that accumulation of Ag-NPs or Ag ions on the gill epithelial structure and this may be led to direct diffusion from gill to blood causing cell abnormalities and increase apoptosis degree. Also, Rioux et al. (2006) mentioned that Ag-NPs- interaction with sulphur-phosphorus present interior of nucleic acid and protein of bacteria cell leads to cell division and mortality due to respiratory chain upsetting. Previous works have been reported metallic detoxification (MT) and oxidative stress after Ag-NPs exposure in Japanase medaka and trout (Chaea et al., 2009; Choi et al., 2010). So, the MT can initiate the production of ROS (Formigari et al., 2007; Gagné et al., 2012), and this led to apoptosis. It has been concluded the major reason for Ag-NPs include cell death is necrotic (Ghosh et al., 2012b).

In conclusion, based on the results obtained from the present study, it was found that exposure to 25–75 mgl–1 Ag-NPs produced significant adverse effects on the erythrocytes of catfish, presenting as apoptosis, morphological alterations and micronuclei observation. Also, the findings showed significant increases in mortality rate and genotoxicity in the liver, kidney, gill, and muscles. Further studies are still required to understand the toxicological mechanisms of Ag-NPs in fishes so that forth coming studies should shed new lights on the important activity of Ag-NPs, ultimately histopathology and other adverse impacts.

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