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Chemosphere 104 (2014) 134–140
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Chemosphere
journal homepage: www.elsevier .com/locate /chemosphere
Sensitivity of freshwater pulmonate snail Lymnaea luteola L., to silvernanoparticles
0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.10.081
⇑ Corresponding author. Address: Department of Zoology, College of Science, KingSaud University, BOX 2455, Riyadh 11451, Saudi Arabia. Tel.: +966 558904621.
E-mail addresses: [email protected], [email protected] (D. Ali).
Daoud Ali a,⇑, Phool Gend Yadav b, Sudhir Kumar b, Huma Ali c, Saud Alarifi a, Abdul Halim Harrath a
a Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabiab Department of Zoology, University of Lucknow, Lucknow, Indiac Department of Chemistry, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India
h i g h l i g h t s
�We provide new information about ecotoxicity of silver nanoparticles on freshwater snail Lymnaea luteola.� Silver nanoparticles induced oxidative stress in freshwater snail Lymnaea luteola.� We observed that silver nanoparticles induced genotoxicity in freshwater snail Lymnaea luteola.
a r t i c l e i n f o
Article history:Received 20 June 2013Received in revised form 12 September2013Accepted 29 October 2013Available online 2 December 2013
Keywords:Silver nanoparticlesOxidative stressDNA damageComet assay: Lymnaea luteola L
a b s t r a c t
Toxicity of nanoparticles depends on many factors including size, shape, chemical composition, surfacearea and surface charge. Silver nanoparticles (AgNPs) are likely to enter the aquatic ecosystems becauseof their multiple applications and pose a health concern for humans and aquatic species. Therefore, weused a freshwater snail Lymnaea luteola L (L. luteola) to investigate the acute toxicity and genotoxicityof AgNPs in a static-renewal system for 96 h. AgNPs caused molluscicidal activity in L. luteola, with 96-h median lethal concentrations (LC50) (48.10 lg L�1). We have observed that AgNPs (36 lg L�1) eliciteda significant (p < 0.01) reduction in glutathione, glutathione-s-transferase and glutathione peroxidasewith a concomitant increase in malondialdehyde level and catalase in digestive gland of L. luteola. How-ever, a significant (p < 0.01) induction in DNA damage was observed by the alkaline single cell gel elec-trophoresis in digestive gland cells treated with AgNPs for 24 and 96 h. These results demonstrate thatsilver nanoparticles are lethal to freshwater snail L. luteola. The oxidative stress biomarkers and cometassay can successfully be used as sensitive tools of aquatic pollution biomonitoring.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The environmental risk of silver nanoparticles (AgNPs) is acurrent and highly topical focus of concern. AgNPs antibacterialproperties are being exploited in a rapidly growing range of con-sumer goods (http://www.nanotechnology.org). Release of AgNPsinto the aquatic environment from this emerging nanotechnologyis inevitable (Mueller and Nowack, 2008). Ionic silver (Ag+) is ex-tremely toxic to bacteria, phytoplankton, invertebrates, and fish(Liau et al., 1997; Ratte, 1999; Croteau et al., 2011). Interestingly,the physical properties of nanomaterials often deviate dramaticallyfrom the properties of the bulk materials. However, the sameproperties that make these particles exciting in technology andconsumer markets also make them public health concerns. Becauseof their small sizes, nanoparticles are more likely to infiltrate
biological systems where larger molecules could not (Moore,2006) and diffuse through cell membranes (Lin et al., 2010). Theecotoxicity of AgNPs in freshwater snail is unclear and relativelyunexplored.
Metal oxide nanoparticles toxicity has been documented in thefreshwater microalga, Daphnia magna and zebrafish. Lee et al.(2007) reported that citrate-capped AgNPs (0.04–0.71 nM) aretransported inside developing zebrafish embryos, eliciting defor-mities and causing death. Some studies have demonstrated thetoxicity of AgNPs to bacteria (Yoon et al., 2007), suggesting thatthe antimicrobial effects of silver may be detrimental to aquaticecosystems. AgNPs is steadily released into the water fromcommercial clothing (specifically, socks) by washing (Benn andWesterhoff, 2008). Therefore, it is important to assess the toxicityof AgNPs in aquatic ecosytems. Gastropod organisms are ubiqui-tous in the aquatic ecosystem and are considered good bioindica-tors of contaminants in view of their wide geographicdistribution, relatively sedentary life habit, and their easy availabil-ity. Moreover, most of the ecotoxicity studies on snails has been
D. Ali et al. / Chemosphere 104 (2014) 134–140 135
conducted using organism level end-points; such as mortality andgrowth (Crane et al., 2002), with only a few studies have been per-formed that include genotoxic endpoints. In snails, pollutants aretransferred by blood cells to the digestive gland, which is one ofthe major target tissues of accumulation. Reactive oxygen species(ROS) and the resulting oxidative stress to living organisms havebeen reported to be related with the pollution-mediated mecha-nism of toxicity, which can damage macromolecules, such asDNA, proteins, and membranes. Antioxidant biomarkers are ableto eliminate the highly reactive intermediate ROS induced by pol-lutants to maintain cell homeostasis, and they include antioxidantenzymes and antioxidant nonenzymatic scavengers, e.g., superox-ide dismutase (SOD), catalase (CAT), glutathione S-transferases(GST), and reduced glutathione (GSH). Malonyldialdehyde (MDA)is an end point of lipid peroxidation; therefore, the formation ofMDA is regarded as a general indicator of lipid peroxidation. SODand Catalase (CAT) activities play important role in the antioxidantprotection of invertebrates (Livingstone, 2001). GST is a family ofphase II detoxification enzymes that catalyze the conjugation ofglutathione to a wide variety of endogenous and exogenous elec-trophilic compounds, such as therapeutic drugs and environmentaltoxins (Hayes et al., 2005). Oxidative stress may manifest as dam-age to tissue macromolecules, including proteins and DNA (Di Giu-lio et al., 1989). In addition, DNA damage is considered to be abiomarker of genotoxicity induced by noxious substances, andthe comet assay has recently been considered a robust ecotoxico-logical tool with which to assess DNA damage at the individual celllevel.
We aimed to assess the acute toxicity of AgNPs to freshwatersnail Lymnaea luteola, which is an important component of river,ponds and lakes of South East Asian freshwater ecosystems. Ourresults provide critical information to regulatory agencies andindustry to determine the need for monitoring and regulationregarding AgNPs.
2. Materials and methods
2.1. Chemicals
Silver nanoparticles (AgNPs) (Product No. 736465 and APS:650 nm), ethylene diamine tetra acetic disodium salt, dimethylsulphoxide, thiobarbituric acid (TBA), trichloroacetic acid (TCA)were purchased from M/s. Sigma (St. Louis, MO). All other chemi-cals used were of the highest purity available from commercialsources.
2.2. Snail collection and culture
Individuals of adult snail of similar size and weight were care-fully collected from non-contaminated artificial fish culture pondsand transferred to the laboratory. They were maintained in glassaquaria. Snails were acclimatized to laboratory conditions for2 weeks before experimentation, at temperature 21 ± 1 �C and feddaily ad libitum with thoroughly washed freshwater green aquaticplant (Marsilia sp.) leaves. They had an average wet weight of490 mg (range, 310–640 mg) and shell length 21 mm (range 18–24.8 mm).
2.3. Characterization of AgNPs
AgNPs was suspended in deionized water at a concentration of1 mg mL�1, and then sonicated using a sonicator bath at room tem-perature for 10 min at 40 W to form a homogeneous suspension.For hydrodynamic size, sonicated AgNPs stock solution(1 mg mL�1) was diluted to 1–80 lg mL�1 working solutions.
Hydrodynamic size and zeta potential of the AgNPs suspension inwater were measured by dynamic light scattering (Zeta sizer-HTMalvern Instrument, Worcestershire, U.K.). Transmission electronmicroscopy (FETEM, JEM-2100F, JEOL Inc.) was used to character-ize the size and shape of AgNPs at an accelerating voltage of15 kV and 200 kV.
2.4. Determination of silver ion in test water
Silver ion (Ag+) concentration was determined in exposed solu-tions with flame atomic absorption spectroscopy (GBC Avanta Ver2.01). Exposure concentrations (10, 40, 100 lg L�1) was acidified,to pH 2, by adding 150 lL concentrated nitric acid to 20 mL of sam-ple. The Ag+ concentration analysis was conducted within 96 h ofsample preparation.
2.5. Determination of sub lethal concentrations
The acute toxicity bioassay to determine the LC50-96 h value ofAgNPs was conducted in a static-renewal system. The acute bioas-say procedure was based on standard methods (APHA et al., 2005).A stock solution of AgNPs (1 mg mL�1) was prepared in deionizedwater.
A set of 10 acclimatized L. luteola specimens was randomly ex-posed to each of the seven AgNPs target concentrations (0, 1, 5, 10,20, 40, 60, and 100 lg L�1) in transparent polystyrene beakers of1000 mL test water and the experiment was repeated twice to ob-tain the LC50-96 h value of the test AgNPs for the freshwater snail L.luteola. Photoperiod was controlled to simulate the natural day:light cycle (12 h:12 h). Fluorescent light with two 48 W lampwas used as light source.
The LC50-96 h value (48.10 lg L�1) of AgNPs for L. luteola wasdetermined by using the probit analysis method as described byFinney (1971). On the basis of LC50-96 h value, the four test con-centrations of AgNPs viz., dose I (1/12th of LC50 = �4.01 lg mL�1),dose II (1/4nd of LC50 = �12.03 lg mL�1), dose III (1/2th ofLC50 = �24.05 lg mL�1) and dose VI (3/4th of LC50 = �36.08lg mL�1) were determined.
2.6. In vivo exposure of AgNPs and isolation of digestive gland cells
The L. luteola were exposed to the four aforementioned test con-centrations of AgNPs in a static-renewal system. The exposure wascontinued up to 96 h and isolation of digestive gland cells wasdone at intervals of 24 and 96 h at the rate of five snails per dura-tion. The snails maintained in tap water were considered as nega-tive control.
The physicochemical properties of test water, namely tempera-ture, pH, total conductivity, dissolved oxygen and total hardnesswere analyzed by standard methods (APHA et al., 2005).
At each sampling duration, the digestive glands of the exposedsnails were quickly removed, washed with ice-cold saline (0.9%),and cleaned from accessory connective adipose tissues. Digestivegland tissues from each group were weighed and homogenizedin 10 vol. ice-cold saline solution (w/v ratio) using a polytronhomogenizer for one min. The homogenates were centrifuged at5000 rpm for 30 min at 4 �C. The supernatants were used for themeasurement of catalase, glutathione peroxidase (GPx), glutathi-one-S-transferase (GST) activities, and lipid peroxidation (LPO)level and glutathione (GSH) content.
2.7. Lipid peroxidation levels
The concentration of malondialdehyde (MDA) as a marker ofLPO was determined according to the method of Nair and Turner(1984). Briefly, 0.33 mL of digestive gland homogenate was mixed
136 D. Ali et al. / Chemosphere 104 (2014) 134–140
well with 3 mL of thiobarbituric acid (TBA) reagent that wasfreshly prepared by mixing 1 vol. of 0.8% TBA to 3 vol. of 20%trichloroacetic acid. The mixture was incubated for 50 min in aboiling water bath. After cooling, the mixture was centrifuged at3000 rpm for 10 min. The MDA level was measured spectrophoto-metrically (Varian-Cary 300 Bio) at 532 nm and the results areexpressed as n moles of MDA per mg of wet tissue.
2.8. Reduced GSH contents
Glutathione content as a nonenzymatic antioxidant was mea-sured according to Owens and Belcher (1965) at 412 nm. The assaymixture consisted of 0.1 mL of the homogenate, 1.5 mL of 0.5 Mphosphate buffer, pH 8.0, followed by 0.4 mL of 3% metaphosphoricacid and 30 lL 50, 5-dithio-bis-(2-nitrobenzoic acid) (DTNB)(0.01 M). The amount of reduced GSH present in the digestiveglands sample in terms of lg per gram of wet tissue was calculatedafter calibration against the standard curve of GSH.
2.9. Determination of catalase activity
Catalase activity was measured following the decrease of absor-bance at 240 nm due to H2O2 consumption (Beers and Sizer, 1952).The reaction mixture consisted of 1 mL of 12.5 mM hydrogen per-oxide (substrate), 2 mL of 66.7 mM phosphate buffer, pH 7.0, andan aliquot amount of the supernatant. Catalase activity was ex-pressed as units per gram of wet tissue. The unit of catalase isthe amount of enzyme that liberates half of the peroxide oxygenfrom the hydrogen peroxide solution of any concentration in100 s at 25 �C.
2.10. Determination of glutathione peroxidase activity
Glutathione peroxidase (GPx) activity was assayed according tothe method described by Chiu et al. (1976). The assay mixture con-sisted of 2.6 mL of 0.4 M Tris–HCl buffer, pH8.9, 100 lL of thesupernatant, 100 lL of 1 mM GSH, 100 lL of 0.05% cumene hydro-peroxide, and 100 lL of 0.01 M Ellman’s reagent (DTNB). The mix-ture was vortexed and incubated at 25 �C for 5 min. GPx activitywas monitored at 412 nm and expressed as optical density (O.D.)per gram of protein per min.
2.11. Determination glutathione-S-transferase activity
Glutathione-S-transferase (GST) activity was assessed by themethod of Vessey and Boyer (1984) using 1-chloro-2, 4, dinitro-benzene (CDNB) as a substrate. The reaction mixture contained0.2 mL of 4 mM GSH, 20 lL of 0.25 mM CDNB, 20 lL of superna-tant, and 2.76 mL of 0.1 M phosphate buffer, pH 7.0, in a finalvolume of 3.0 mL. The formation of the CDNB-GSH conjugate wasevaluated by monitoring the increase in absorbance at 340 nm. Re-sults are expressed as O.D. per gram of protein per min.
2.12. Determination of DNA strand breakage
The comet assay was performed as a three layer procedure(Singh et al., 1988) with slight modification in which conventionalmicroscopic slides were used (Ali et al., 2008). The digestive glandsof the exposed snails were quickly removed, washed with ice-coldsaline (Ca2+ Mg2+ free), and cleaned from accessory connective adi-pose tissues. The tissue was cut into small pieces using scissors andfinally homogenized to obtain single-cell suspension. The cell sus-pension was centrifuged at 3000 rpm at 4 �C for five min and thecell pellet was finally suspended in chilled phosphate buffer salinefor comet assay. Viability of cells was evaluated by trypan blueexclusion method (Anderson et al., 1994). The samples showing
cell viability higher than 84% were further processed for comet as-say. In brief, about 15 lL of cell suspension (approx. 20000 cells)was mixed with 85 lL of 0.5% low melting point agarose and lay-ered on one end of afrosted plain glass slide, pre-coated with alayer of 200 lL normal agarose (1%). There after, it was coveredwith a third layer of 100 lL low melting-point agarose. After solid-ification of the gel, the slides were immersed in lysing solution(2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris pH 10 with 10% DMSOand 1% Triton X-100 added fresh) overnight at 4 �C. For positivecontrol, the digestive gland cells were treated ex vivo with100 lM H2O2 for ten min at 4 �C. The slides were then placed ina horizontal gel electrophoresis unit. Fresh cold alkaline electro-phoresis buffer (300 mM NaOH, 1 mM Na2EDTA and 0.2% DMSO,pH 13.5) was poured into the chamber and left for 20 min at 4 �Cfor DNA unwinding and conversion of alkali-labile sites to single-strand breaks. Electrophoresis was carried out using the same solu-tion at 4 �C for 20 min, using 15 V (0.8 V cm�1) and 300 mA. Theslides were neutralized gently with 0.4 M tris buffer at pH 7.5and stained with 75 lL ethidium bromide (20 lg mL�1). Two slideswere prepared from each specimens and 50 cells per slide (100cells per concentration) were scored randomly and analyzed usingan image analysis system (Komet-5.0, Kinetic Imaging, LiverpoolU.K.) attached to fluorescent microscope (DMLB, Leica, Germany)equipped with appropriate filters. The parameters selected forquantification of DNA damage in the digestive gland cells werepercent tail DNA (i.e.% Tail DNA = 100-% Head DNA) and olive tailmoment (OTM; arbitrary units, the products of the distance ofDNA migration from the body of the nuclear core and the total frac-tion of DNA in the tail) as determined by the software.
2.13. Estimation of protein
The total protein content was measured by the Bradford (1976)method using Bradford reagent (Sigma–Aldrich, USA) and bovineserum albumin as the standard.
2.14. Statistical analysis
At least three independent experiments were carried out eachevaluation. Data were expressed as mean (±SE) and analyzed byone-way analysis of variance (ANOVA). The p-value less than0.01 was considered statistically significant.
3. Results
3.1. Silver nanoparticles
Fig. 1A showed the typical TEM image of the AgNPs and themajority of the particles were in spherical shape with smooth sur-face. The average diameter of the AgNPs was calculated from mea-suring over 100 particles in random fields of TEM view. Theaverage TEM diameter of AgNPs was 32.40 ± 2.60 nm (Fig. 1B).The average hydrodynamic size and zeta potential of AgNPs inwater determined by DLS were 260.5 ± 26 nm and �12.5 mV,respectively.
3.2. Physicochemical analysis of the test water
The water temperature varied from 24.5 to 28.3 �C and pH val-ues ranged from 7.03 to 8.20. The dissolved oxygen concentrationwas normal, varying from 6.0 to 8.4 mg L�1, during experimentalperiod. The conductivity of the water ranged from 243 to298 lM cm�1 and chloride from 45 to 56.8 lg mL�1. The totalhardness ranged from 160 to181 lg mL�1 and total alkalinity from260 to 290 lg mL�1 as CaCO3.
Fig. 1. Characterization of silver nanoparticles (A) TEM image and (B) the size distribution histogram generated by using TEM image.
Table 1Concentration of Ag+ in exposed concentrations ofAgNPs.
Ag NPs (lg L�1) Calculated Ag+ (lg L�1)
Control >0.00110 0.5540 0.166100 2.47
D. Ali et al. / Chemosphere 104 (2014) 134–140 137
3.3. Release of Ag ion concentration
AgNPs has tendency to precipitate from solution and settled atbottom of test beaker. Silver ion concentrations in test water were0.55 lg L�1 at lowest and 2.470 lg L�1 at highest exposure concen-tration (Table 1).
Fig. 2. (a) Levels of GSH and (b) lipid peroxides (LPO) in of L. luteola after exposureto different concentrations of silver nanoparticles for 24 h and 96 h. Each valuerepresents the mean ± S.E. of three experiments. *p < 0.01 vs. control.
3.4. Lethality
AgNPs were lethal to L. luteola with LC50-96 h, approximately48.10 lg L�1 (data not shown). We have used three-fourth of thedetermined LC50-96 h as the highest exposure concentration ofAgNPs to L. luteola.
Fig. 3. Catalase activity in digestive gland of L. luteola snails after exposure todifferent concentrations of silver nanoparticles for 24 h and 96 h. Each valuerepresents the mean ± S.E. of three experiments. *p < 0.01 vs. control.
3.5. AgNPs induced oxidative stress
The LPO level, GSH content and CAT, GPx and GST activities inthe digestive gland of AgNPs exposed snails were investigatedand illustrated (Figs. 2–5).
At sublethal concentration of AgNPs exposure to the snailsexhibited significant elevation (p < 0.01) in MDA content comparedto the control, which indicates increased LPO level. It was observedthat MDA content was elevated in concentration and time depen-dent manner (Fig. 2b).
GSH level was significantly (P < 0.01) reduced in time and dosedependent manner (Fig. 2a). The maximum reduction in GSH levelwas observed in snail at the highest concentration of AgNPs(36 lg L�1) at 96 h (Fig. 2a). Catalase activity in digestive gland ofsnail was found to be increased significantly (p < 0.01) at differentexposed dose of AgNPs when compared with control group (Fig. 3).The maximum increased catalase activity was observed at36 lg L�1 AgNPs exposure.
Different doses of AgNPs caused significant (p < 0.01) reductionin GPx and GST activities in the digestive gland of snails (Figs. 4 and5). GPx activity was highly reduced by AgNPs at the highest con-centration (36 lg L�1). The highest reduction in GST activity wasobserved at the highest concentration of AgNPs at 96 h (Fig. 5).
3.6. DNA damage
The DNA damage was measured as% tail DNA and olive tail mo-ment in the control as well as exposed cells. The exposed cells todifferent doses of AgNPs, exhibited significantly (p > 0.01) higherDNA damage in cells than those of control groups (Fig. 6). The grad-ual nonlinear increase in DNA damage was observed in cells asdose and time exposure of AgNPs increased and the highest DNAdamage was recorded at 24 lg L�1 AgNPs treated snails for 96 h(Fig. 6a–d).
Fig. 4. Glutathione peroxidase (GPx) activity in digestive gland of L. luteola afterexposure to different concentrations of silver nanoparticles for 24 h and 96 h. Eachvalue represents the mean ± S.E. of three experiments. *p < 0.01 vs. control.
Fig. 5. Glutathione-S-transferase (GST) activity in digestive gland of L. luteola afterexposure to different concentrations of silver nanoparticles for 24 h and 96 h. Eachvalue represents the mean ± S.E. of three experiments. *p < 0.01 vs. control.
138 D. Ali et al. / Chemosphere 104 (2014) 134–140
4. Discussion
The application of nanotechnology has been recently extendedin the areas of medicine, biotechnology, materials and processdevelopment, energy and environment. AgNPs are used exten-sively in clothing, water purification, baby products (e.g. nipples
Fig. 6. DNA damage in digestive gland of L. luteola after exposure to different concentrati(arbitrary unit), (c) control cell and (d) exposed cell. Each value represents the mean ± S
and bottles) personal care products (e.g. shampoos, toothpastes,deodorants, etc.), bedding and appliances (e.g. washing machines,humidifiers and refrigerators) (Woodrow Wilson InternationalCenter for Scholars, 2008). The major reason of it is use is its anti-fungal and antimicrobial properties. However, AgNPs are knowntoxic nanomaterials to bacteria and fungi; its toxicity to freshwater snail is under-investigated and consequently poorly under-stood. AgNPs may induce deleterious effects in aquatic life after re-lease into aquatic environment. The high bioavailability of Agobserved in a predatory snail (Cheung and Wang, 2005), but arelower in marine herbivores (Wang et al., 1995). Dallinger (1994)has reported that freshwater snails are sentinel model to monitoraquatic pollution, so it is important to study AgNPs mechanismsof toxicity that induced oxidative damage and the state of their de-fence systems by the snails. We have observed that AgNPs inducedoxidative stress and DNA damage in digestive gland of L. luteolasnail as dose and time dependent manner. The digestive gland(hepatopancreas) of gastropod molluscs is the key organ of metab-olism and it is concerned with the production of digestive en-zymes, absorption of nutrients, endocytosis of food substances,food storage, and excretion (Dallinger et al., 2002). It has been re-ported as a major site of xenobiotic, oxy-radical generating andbiotransformation enzymes (Livingstone et al., 1992). Pollutantsaccumulation through different routes are transported by bloodcells to the digestive gland, which also represents the main targetorgan for metabolic and detoxification processes (Beeby and Rich-mond, 2002; Regoli et al., 2005). So in the present study we haveused the digestive gland tissue to investigate the biochemical re-sponses to AgNPs. Croteau et al. (2011) reported that silver bioac-cumulation dynamics in a freshwater invertebrate after aqueousand dietary exposures to AgNPs and Ag+. A general pathway of tox-icity for many environmental pollutants is mediated by theenhancement of intracellular ROS, which modulate the occurrenceof cell damage (Regoli et al., 2002) via initiation and propagation oflipid peroxidation (Gutteridge, 1995). Lipid peroxidation is a com-plex process in which polyunsaturated fatty acids in the biologicalmembrane system undergo changes by chain reactions and form li-pid hydro peroxides, which decompose double bonds of unsatu-rated fatty acids and disrupt membrane lipid (Gutteridge, 1995).The estimation of malondialdehyde content (an index of lipid per-oxidation) provides a relative measure of the potential for AgNPs to
ons of silver nanoparticles for 24 h and 96 h of. (a) % Tail DNA, (b) olive tail moment.E. of three experiments. *p < 0.01 vs. control.
D. Ali et al. / Chemosphere 104 (2014) 134–140 139
cause oxidative injury. Nusetti et al. (2001) reported that LPO lev-els increased during exposure to metals in several organisms. Inthe present study, significantly elevation of lipid peroxidation lev-els in the digestive gland of AgNPs exposed L. luteola indicate thatsome cell damage might have occurred. Our result accorded withresults of Viarengo et al. (1990) study in which lipid peroxidationlevel increased in mussel tissues due to copper metal exposure.Glutathione is a tripeptide nonenzymatic antioxidant with a singlecysteine residue and constitutes an important pathway of the anti-oxidant and detoxification defenses. Chemical compounds, such astrace metals, are biotransformed to a conjugate of GSH. It is also acofactor of many enzymes catalyzing the detoxification and excre-tion of several toxicants, which will be destroyed in the cytosolicand mitochondrial compartments by GPx in the presence of GSH(Doyotte et al., 1997). The decrease in glutathione concentrationsin our study might be attributed to the intensification of turnoverbetween reduced and oxidized glutathione under the conditions,which cause increased consumption of this peptide for the synthe-sis of heavy metal-binding proteins, like metallothioneins. A de-crease in glutathione content in the digestive gland appears to bea common response of molluscs to metal exposure (Regoli andPrincipato, 1995). In this study, we observed a decrease in GSHcontent accompanied by the elevation of LPO levels. Through catal-yzation by glutathione peroxidase, GSH can eliminate H2O2 and li-pid hydro peroxide (Ahmed, 2005); therefore, a negativerelationship between MDA and GSH was a reasonable result.
GSH is one of the most important factors protecting from oxida-tive attacks by reactive oxygen species such as lipid peroxidation,because GSH acts as a reducing agent and free-radical trapperand is known to be a cofactor substrate and/or GSH-related en-zymes (Verma et al., 2007). SOD catalyzes the dismutation reactionof the superoxide anion radical, O2�, to form the less-reactivemolecular oxygen, and CAT converts H2O2 to H2O and O2 to preventoxidative stress and maintain cell homeostasis. Increased SOD andCAT activities indicated that O2� and H2O2 were formed during thefreshwater snails’ exposure to contaminant (Zheng et al., 2013).
The antioxidant catalase is an extremely important componentof intracellular and antioxidant defenses of organisms (Jamil,2001). It reduces the H2O2 into water and oxygen to prevent oxida-tive stress and for maintaining cell homeostasis. Many studies havefound varying responses of catalase to increased metal exposures,with some organisms exhibiting increased activity, others exhibit-ing decreased activity, and still others showing no catalase re-sponse at all (Regoli et al., 1998). In the present study, weobserved that catalase activity was significantly increased; thisdata suggests that the increase in antioxidant defenses would bedue to enhanced oxygen free radicals production, which couldstimulate antioxidant activities (Torres et al., 2002) to cope withthis increased oxidative stress and protect the cells from damage.The obtained results are in accordance with the findings of Almeidaet al. (2004), who found that catalase activity was increased inmussels after exposure to lead.
Glutathione peroxidase (GPx) is the most important peroxidasefor the detoxification of hydro peroxides (Orbea et al., 2000). How-ever, the decreased activities of GPx might be due to over produc-tion of ROS, especially O�2 , by the AgNPs and depletion of itssubstrate level (GSH). It is also reported that under high rates offree radicals input, enzyme inactivation prevails and the enzymaticactivities are reduced, leading to autocatalysis of oxidative damageprocess (Escobar et al., 1996).
The role of GST is to conjugate tripeptide glutathione with elec-trophilic and other xenobiotic. GST activity inhibition could haveoccurred either through direct action of the metal on the enzymeor indirectly via the production of ROS that interact directly withthe enzyme, depletion of its substrate (GSH), and/or downregulation of GST genes through different mechanisms (Roling
and Baldwin, 2006). This explanation might be the reason for theGST activity decrease that was caused in the present study in thecase of snails exposed to AgNPs. Similarly, Regoli et al. (1997)showed that Cu resulted in a significant reduction in the activityGST in aquatic animals. Changes in the levels of antioxidants havebeen proposed as biomarkers of a contaminant-mediated pro oxi-dant challenge in a variety of invertebrates (Regoli et al., 2002).Genotoxicity is considered one of the most important toxic end-points in most chemical toxicity testing and risk assessment; how-ever, little is known about the genotoxicity of AgNPs, especiallytowards aquatic organisms L. luteola. The results of the comet assaysuggested that AgNPs may provoke DNA damage in L. luteola. It isalso observed DNA damaging effects, in which oxidative stress maybe attributed as one of the probable cause. Reactive oxygen speciesis known to react with DNA molecule causing damage to purineand pyrimidine bases as well as DNA backbone. Another importantoutcome of reactive oxygen species generation, DNA damageresulting from any of these probable mechanisms may trigger sig-nal transduction pathways leading to apoptosis or cause interfer-ences with normal cellular processes thereby causing cell death.
The results of this investigation may demonstrate ecologicalimplications of AgNPs release in aquatic ecosystems. Our resultsprovide critical information to regulatory agencies and industryto determine the need for monitoring and regulation regardingAgNPs.
Acknowledgement
The authors extend their appreciation to the Deanship of Scien-tific Research at King Saud University for funding this workthrough the research group project No. RGP-VPP-164.
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