Biochimica et Biophysica Acta 1830 (2013) 2256–2266
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Cellular internalization and stress response of ingested amorphous silicananoparticles in the midgut of Drosophila melanogaster
Ashutosh Pandey a, Swati Chandra a, Lalit Kumar Singh Chauhan b,Gopeshwar Narayan c, Debapratim Kar Chowdhuri a,⁎a Embryotoxicology Section, CSIR-Indian Institute of Toxicology Research, Lucknow 226001, Uttar Pradesh, Indiab Electron Microscopy Section, CSIR-Indian Institute of Toxicology Research, Lucknow 226001, Uttar Pradesh, Indiac Department of Molecular and Human Genetics, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India
Background: Amorphous silica nanoparticles (aSNPs) are used for various applications including food indus-try. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the mid-gut cells of an organism. The study aims to examine cellular uptake of aSNPs (b30 nm) in the midgut ofDrosophila melanogaster (Oregon R+) owing to similarities between the midgut tissue of this organism andhuman and subsequently cellular stress response generated by these nanoparticles.Methods: Third instar larvae of D. melanogasterwere exposed orally to 1–100 μg/mL of aSNPs for 12–36 h andoxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bro-mide staining), cellular internalization (TEM) and apoptosis endpoints.Results: A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in aconcentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 alongwith caspases activation, membrane destabilization and mitochondrial membrane potential loss was also ob-served. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles
and by direct membrane penetration.Conclusion: aSNPs after their internalization in the midgut cells of exposed Drosophila larvae showmembranedestabilization along with increased cellular stress and cell death.General significance: Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thustheir industrial use as a food-additive may raise concern to human health.
With an increase in the use of nanomaterials (NMs) in industrial andbiomedical applications in recent years, concern has been raised onentry of the NMs in the environment and subsequently, their possibleeffects on human health. The unique physico-chemical properties ofNMs such as particle size, agglomeration state, size distribution, shape,
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crystal structure, chemical composition, surface chemistry, surfacecharge and porosity supplemented by altered dimensional configura-tion and high surface area to volume ratiomay account for their hazard-ous effects on biological systems [1,2].
Engineered nanoparticles (ENPs) comprise wide range of NMswhich are synthesized by molecular level engineering to acquireunique properties for their vast applications. Among the ENPs, silicananoparticles are one of the most important and most utilized NMs.Amorphous silica nanoparticles (aSNPs), characterized by theirnon-crystalline structure are produced in different sizes along with dif-ferent surface modifications with relative ease. This has led to the ex-tensive use of aSNPs for diverse applications that include agriculture,food and animal-feed additives [3].
aSNPs can enter into an organism including human through all pos-sible routes [2]. Since aSNPs are used in food products following theirapproval by FDA as a food additive [4], concern has been raised ontheir exposure to an organism through ingestion. Earlier, different ex-posure routes such as intra-tracheal instillation [5], inhalation [6] or in-travenous injection [7]were used for in vivo studies on aSNPs in rats andmice. Limited studies on aSNPs through oral route are available [8,9],
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however, with inadequate characterization of nanoparticles (NPs) anddose level [10]. Dekkers et al. [3] analyzed several food products withadded silica (E551) for the presence of silica NPs, their particle size(30–200 nm) and concentration (0.3-33.0 mg nanosilica/day) usingexperimental analytical data. Further, theoretical approach towardrisk assessment of NMs in food [included in guidelines of EuropeanFood Safety Authority (EFSA, 2011)] [11] was put forth to find whetherNPs get absorbed in gastrointestinal (GI) tract in dissolved form or inparticulate form [10]. In a recent study, Peters et al. [12] performed invitro digestion of food products (instant soup, coffee creamer, pancake)having synthetic amorphous silica as food additives and demonstratedexposure of human intestinalwall to nano-sized silica particles. Howev-er, studies on internalization/absorption of aSNPs from GI tract aftertheir entry by oral route are not available.
NPs–membrane interactions are relevant in the context of NPsmediated cellular injury and hence gaining insights on the adverse ef-fects of NPs on membrane perturbation is pertinent. aSNPs wereshown to penetrate the platelet surface membrane causing distur-bance in calcium ion channels [13] and also to cause decrease inmembrane fluidity in exposed mouse macrophage cells [14]. Pattaniet al. [15] showed membrane perturbation by chitosan NPs by usingphospholipid vesicles as a model membrane while Valant et al. [16]showed cell membrane destabilization potential of nanotized TiO2,ZnO and fullerenes (C60) in a terrestrial invertebrate, Porcellio scaberafter their ingestion. However, studies on membrane destabilizationby ingested aSNPs in GI tract are not available.
Reactive oxygen species (ROS) mediated oxidative stress (OS) hasbeen correlated with NPs mediated cellular damage [1,17]. A numberof in vitro studies are now available on the mechanism of cellular stressagainst aSNPs in the context of their size. Reduced level of glutathione(GSH) and increased malondialdehyde level indicating lipid peroxida-tion (LPO) and membrane damage were correlated with OS inducedby aSNPs (15 nm) in A549 cells [18]. GSH depletion in aSNPs (30 nm)exposed mouse keratinocytes [19] and increased ROS generation anddepletion in GSH level in exposed human hepatic cells [20] were alsoreported. Yang et al. [21] observed size dependent hazard (smaller thesize greater the hazard) of aSNPs in human hepatoma (HepG2) cellsconcordant with previous reports [19,20,22]. Summarizing these stud-ies, it is nowevident that OS induced cellular adversities of aSNPs are as-sociated with their smaller size. However, data on cellular stressresponse after oral exposure of aSNPs is lacking.
Under various stressful conditions like exposure to xenobiotics,free radicals and elevated temperature, cells set up their defense byexpressing heat shock genes (hsg) which after their translation pro-duce heat shock proteins (Hsps) [23]. While temperature shock wasused as the primary inducer to demonstrate the elevated levels ofHsps in Drosophila [24], later, diverse groups of stressors were foundto induce a number of hsgs and hence a broader term stress proteins(Sps)was coined [25]. Under adverse conditions, Hspsmaintain the un-folded proteins prior to their assembly into multi-molecular complexesin cytosol and guide the newly synthesized as well as denatured pro-teins to accomplish their appropriate conformation, thereby protectingthe cells from further damage [26]. Among the families of Hsps, hsp70 isthe most conserved across the taxa and commonly expressed. Hsp70executes functions mainly as a chaperone facilitating the refolding ofpolypeptides and dissolution of insoluble aggregates by binding tohydrophobic surfaces of denatured proteins [27]. Earlier studiesfrom this laboratory showed that elevated levels of Hsp70 againstexposure to various environmental stressors such as, pesticides andfungicides [28,29] and industrial runoffs [30], thereby establishinghsp70 expression as a first tier indicator of cellular stress [30].Along with hsp70, other Hsps, such as small heat shock proteins(sHsps) having molecular mass 12–42 kDa, also play importantroles in cellular defense [31]. sHsps have been reported to formlarge oligomers which get phosphorylated under stress conditionsand subsequently dissociate from the oligomers and bind to
non-native proteins [32]. They interact with a number of proteinsto protect cells from OS [33], and apoptosis [34].
The present study therefore is aimed to examine the internaliza-tion of aSNPs (b30 nm) in the midgut tissue of the digestive tract ofDrosophila after their oral exposure and subsequently cellular stressresponse generated by these particles. Midgut tissue of Drosophilashares similarity with that of human intestine. Moreover, Drosophilaand human have similarities in food passage, tissue, anatomy andphysiological functions of intestine and its development. Both themammalian gut and Drosophilamidgut comprise an epithelial mono-layer of enterocytes having cytoplasmic extensions and microvilli toincrease the cell surface area facing gut lumen [35].
With the environment being the ultimate sink for the disposal ofchemicals, organisms inhabiting different compartments of the envi-ronment are expected to be exposed to chemicals/materials. Duringthe past decade, alternative animal models especially the lower eukary-otemodel organisms have gained importance in the context of studyingadverse effects of environmental chemicals/materials/contaminants[36]. Drosophila, an insect model, with well-documented genetics anddevelopmental biology and high degree of homology of its genes withthat of higher mammals, is the closest invertebrate to the humans andhas been used for toxicological studies [37] and for studying human dis-eases [38]. It raises fewer ethical concerns and falls within the recom-mendations of the European Centre for the Validation of AlternativeMethods (ECVAM), aimed to prop up the scientific and regulatory ac-ceptance of alternative methods that are important in the field of bio-logical science and toward reducing, refining and replacing the use oflaboratory animals [39].
2. Materials and methods
2.1. Fly strain and culture
Wild type D. melanogaster (Oregon R+) strain was used for thestudy. Flies and larvae were reared at 24±1 °C on standard Drosophiladiet containing agar-agar, maize powder, sugar, yeast, nepagin(methyl-p-hydroxy benzoate) and propionic acid. Additional yeast sus-pension was provided for healthy growth of the organism.
2.2. NPs and their characterization by transmission electron microscopy(TEM) and dynamic light scattering (DLS)
aSNPs (20–30 nm, purity>99%, specific surface area>600 m2/g)(NANOSHEL LLC, Wilmington DE, USA) were used. The morphologyand average size of aSNPs were analyzed by TEM after preparing thesample by drop coating aSNPs suspension (50 μg/mL in MilliQ water)on carbon-coated copper TEM grids. Films on the TEM grids wereallowed to dry. Measurement of aSNPs was performed on a FEI TecnaiG2 sprit Twin Transmission Electron Microscope equipped with a CCDCamera (Netherlands) and at an accelerating voltage of 80 kV. Averagesize of aSNPs (n=30) was measured from three independently pre-pared grids. Hydrodynamic size of the aSNPs was determined usingDLS method in a Zetasizer (Nano-ZS, Model ZEN3600) equipped withthe 4 mW, 633 nm laser (Malvern instruments Ltd., UK). aSNPs weresuspended in Milli-Q water and 5% sucrose solution (used as mediumfor aSNPs exposure) at a concentration of 200 μg/mL and probe sonicat-ed at 30 W for 10 min before the DLSmeasurement. DLSmeasurementswere carried out thrice with three replicates prepared from indepen-dent pools.
2.3. Treatment schedule
Three different concentrations (1, 10, 100 μg/mL) of aSNPs wereused.We essentially followed the exposure regimen as described previ-ously [40]. Briefly, third instar larvae (68–92 h) of Oregon R+ were ex-posed to different concentrations of aSNPs dispersed in 5% sucrose
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solution (20 μL sucrose solution/10 larvae) for 12–36 h. Control groupreceived 5% sucrose solution without aSNPs.
2.4. Temperature shock treatment
Healthy third instar larvae of Oregon R+ were given temperatureshock (37±1 °C) for 1 h as described previously [41]. Temperatureshock was used as a positive control for the induction of hsgs.
2.5. Reverse transcriptase-PCR (RT-PCR) analysis of hsgs in the midguttissue of third instar Oregon R+ larvae
Total RNA from control and treated third instar larvae of Oregon R+
was extracted using TRI reagent (Ambion, Austin, TX, USA) and reversetranscribed into cDNA using cDNA synthesis kit (Fermentas, MD, USA)essentially following the manufacturer's instructions. Forward and re-verse primers for hsp83, hsp70, hsp60, hsp27, hsp26, hsp23, hsp22 andgapdh (Table 1) were synthesized as previously described [42]. Follow-ing PCR, the amplicons were separated on an 1.5% agarose gelcontaining ethidium bromide at 5 V cm−1 and visualized under aVERSA DOC Imaging System Model 1000 (Bio-Rad, Hercules, CA, USA).The intensity of the bands was quantified by Quantity One software ofBio-Rad, CA, USA. For comparison, data were presented in terms of rel-ative intensity, calculated by normalization using gapdh as the internalcontrol. Each experiment was carried out thrice with three replicatesprepared from independent pools.
2.6. Assay of OS markers
To evaluate the oxidative damage caused by the aSNPs, ROS genera-tion, superoxide dismutase (SOD), catalase (CAT) and glutathioneS-transferase (GST) activities, glutathione (GSH) content, protein car-bonyl (PC) content and lipid peroxidation (LPO) product were assayedin control and exposed third instar larvae of Oregon R+. Except for theROS measurement for which single cell suspension was used, rest ofthe abovementioned assayswere carried out in 10% tissue homogenate.Each experiment was carried out thrice with three replicates preparedfrom independent pools.
2.6.1. Preparation of tissue homogenateLarval midgut tissue homogenate was prepared essentially follow-
ing a method as described previously [42]. The homogenate wascentrifuged at 10,000 ×g and the supernatant was used for differentassays and protein estimation.
2.6.2. Preparation of single cell suspensionMidgut tissues of 15 larvae from control and treated groupswere in-
cubated in collagenase (0.5 mg/ml) for 15 min at 24±1 °C. The cellswere passed through a nylon mesh (85 μm) and were washed with
Table 1Genes and their primer sequences used in RT-PCR amplification.
phosphate buffered saline (PBS) to remove collagenase. Finally, thecells were processed for measurement of ROS generation.
2.6.3. Measurement of ROS
Intracellular ROS generation in the midgut cells of control andtreated larvae was estimated by flowcytometry using a dye, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA; Sigma Chemicals,St. Louise, MO, USA) [42,43]. Data were analyzed using cell quest soft-ware (Mac OS 8.6) and 10,000 events were counted per sample.
SOD activity was estimated as described by Nishikimi et al. [44]with minor modifications as reported by Singh et al. [42]. One unitof enzyme activity is defined as enzyme concentration required forinhibiting chromogen production (optical density 560 nm) by 50%in 1 min under assay condition and the data were expressed as thespecific activity in units/min/mg larval protein.
2.6.5. Catalase (CAT) (H2O2:H2O2 oxidoreductase EC 1.11.1.6)CAT activity in the control and treated larvaewasmeasured as de-
scribed previously [45]. Enzyme activity was expressed as μmol H2O2
decomposed/min/mg/larval protein.
2.6.6. Glutathione S-transferase (GST, EC 2.5.1.18)Glutathione S-transferase (GST) activity was determined by the
method of Habig et al. [46]. The enzyme activity was calculated asnmol CDNB reduced/min/mg larval protein.
2.6.7. Glutathione (GSH) contentGSH content in the control and exposed larvae was quantified using
Elmann's reagent [47]. The assay was performed essentially following apreviously describedmethod [42]. GSH content was expressed in termsof nmol/mg larval protein.
2.6.8. Assay for lipid peroxidation (LPO)Malonyl dialdehyde (MDA) content as a measure of LPO was
assayed using tetraethoxypropane as an external standard [48]. Lipidperoxide level was expressed in terms of nmoles MDA formed/h/mglarval protein.
2.6.9. Determination of protein carbonyl (PC) contentWemeasured PC content in control and treated groups as described
previously [49]. In brief, absorption of di-nitro phenyl hydrazine(DNPH) was determined at 370 nm using 2 M HCl as a blank. The re-sults were expressed as nmoles of DNPH incorporated mg−1 protein.
2.6.10. Protein estimationProtein concentrationwas determined by themethod of Lowry et al.
[50] using Folin reagent and bovine serum albumin (BSA) as thestandard.
2.7. Determination of mitochondrial membrane potential (MMP)
Depolarization of mitochondrial membranes was analyzed by fluo-rochrome 5, 5′, 6, 6′-Tetrachloro-1, 1′, 3, 3′-tetraethyl benzimidazolylcarbocyanine iodide (JC-1) (Invitrogen, USA) following a previouslypublished procedure [51]. Results were expressed as the percentage ofdepolarized cells. Each experiment was carried out thrice with threereplicates prepared from independent pools.
2.8. Colorimetric assay for IETD- and DEVD-ase activities
Caspase activities (as apoptotic endpoint marker) weremeasuredin control and treated larvae using synthetic tetrapeptide substrates
Fig. 1. Characterization of aSNPs by TEM. Transmission electron microscopic images ofaSNPs showing (A) dispersion inwaterwith smaller aggregates and (B)morphology. Rep-resentative image is from the grid prepared from one of the three independent pools. Datarepresent are mean±SD of aSNPs size (n=30) from three independently prepared grids.
Table 2Dynamic light scattering (DLS) measurements of aSNPs showing hydrodynamic sizeand polydispersity index (PDI) in water and 5% sucrose solution.
Dispersion medium Average hydrodynamicsize (nm)
Polydispersity Index (PDI)
MilliQ water 138.63±7.81 0.2615% sucrose solution 148.14±9.96 0.267
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against each enzyme essentially following a previously describedmethod [52]. Each experiment was carried out thrice with three rep-licates prepared from independent pools.
2.9. Electron microscopy in larval gut tissue
Midgut tissues dissected from third instar larvae of control and NPstreated groups were fixed in a solution of 2% glutaraldehyde and 4%paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2), post-fixed in2% osmium tetroxide, dehydrated and embedded in Araldite 502 (TedPella, Inc., Redding, CA, USA). Ultrathin sections (70–90 nm thick,using an Ultra Cut UCT ultra-microtome, Leica) of the blocks (preparedfrom three independent pools) were picked up on copper grids, stainedwith uranyl acetate and lead citrate, and analyzed on a FEI Tecnai G2sprit Twin Transmission Electron Microscope equipped with CCD Cam-era (Netherlands) at 80 kV.
2.10. Acridine orange (AO)/Ethidium Bromide (EB) staining for mem-brane stability in midgut cells of Oregon R+ larvae
Cell membrane destabilization was measured following a recentlydescribed method [53]. Isolated gut tissues from control and aSNPs ex-posed groups were stained with a mixture of AO and EB for 5 min andobserved on a Leica TCS SPE confocal microscope (Nussloch, Germany).Cell membrane integrity was assessed visually by counting the EBstained cells from three independent pools with three replicates(30 larvae/replicate). Scale of membrane destabilization was set asnormal (b5%), medium (b95%) and severe (>95%).
2.11. Statistical analysis
Statistical significance of the mean values for different parame-ters was monitored in control and exposed larvae using ANOVAfollowed by post hoc tests after ascertaining the homogeneity of var-iance and normality of data. For multiple comparisons One-way andtwo-way ANOVA were followed by Dunnett and Bonferroni's test re-spectively. Each endpoint was considered as dependent variablewhile concentration and time of exposure as independent variables.Statistical significance level was ascribed as *Pb0.05 or **Pb0.001.Pearson's correlations were calculated and then linear regressionanalysis was carried out. Prism computer program (GraphPad ver-sion 5.0, San Diego, CA, USA) was used for the statistical analysis.
3. Results
During the course of study, no overt signs of toxicity were ob-served in the exposed organisms.
3.1. Characterization of NPs
TEM analysis showed spherical aSNPs (average particle size of27.28±4.1 nm) dispersed with small aggregates (Fig. 1). The averagehydrodynamic size of aSNPs by DLS measurement was found to be138.63±7.81 nm inwater and 148.14±9.96 nm in 5% sucrose solution(Table 2). Size distribution by intensity of the aSNPs in both themediumis presented in Fig. 2.
3.2. aSNPs induced hsp70 and hsp22 expression in exposed third instarlarvae of D. melanogaster (Oregon R+)
To examine aSNPs-induced expression of selected stress genes in theexposed Oregon R+ larvae, semi-quantitative RT-PCR assay for hsp83,hsp70, hsp60, hsp27, hsp26, hsp23 and hsp22 was carried out. We ob-served a non-significant (P>0.05) change in the expression of hsp83,hsp60, hsp27 and hsp26 in the larvae exposed to 1–100 μg/mL aSNPsfor 12–36 h as compared to their respective control. A significant
(Pb0.05; Pb0.001) concentration-dependent increase in the expressionof hsp70 and hsp22was observed in the exposed larvae after 36 h (72.5and 78% increase in the expression of hsp70 and hsp22 at 100 μg/mLaSNPs after 36 h respectively) (Fig. 3).
3.3. Elevated ROS generation in aSNPs exposed third instar larvae ofOregon R+
Fig. 4 shows flowcytometricmeasurement of ROS generation in con-trol and aSNPs-exposed larvae. A concentration- and time-dependentsignificant (Pb0.05; Pb0.001) increase in ROS generationwas observedin the exposed larvae (3.5-fold increase in ROS generation in 100 μg/mLaSNP treated larvae after 36 h).
3.4. Effect of aSNPs on anti-oxidant markers in exposed third instarOregon R+ larvae
Fig. 5A shows SOD activity in control and aSNPs-exposed larvae. Weobserved a non-significant (P>0.05) change in SOD activity in 1 μg/mLaSNPs-treated larvae throughout the exposure periods while atime-dependent significant (Pb0.05; Pb0.001) increase in the enzymeactivity was observed in 100 μg/mL aSNPs-treated group (44.5% in-crease in the enzyme activity at 100 μg/mL aSNPs concentration after36 h). A similar trend was observed for CAT activity and PC content inthe exposed larvae (41.4 and 42.8% increase in CAT activity and PC
Fig. 2. Characterization of aSNPs by DLS method. DLS measurements showing size intensity curves in (C) water and (D) 5% sucrose solution. Data represent are mean±SD of hy-drodynamic size from three independent measurements.
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content at 100 μg/mL aSNPs after 36 h respectively) (Fig. 5B–C). A sig-nificant (Pb0.05; Pb0.001) concentration- and time-dependent reduc-tion in GSH content was observed in the exposed larvaewithmaximumreduction in GSH content (53%) at 100 μg/mL aSNPs after 36 h (Fig. 5D).We observed a significantly increased MDA content and GST activity inthe exposed larvae in a concentration- and time-dependent manner(Fig. 5E–F).
3.5. Uptake and distribution of aSNPs in gut epithelial cells of exposedOregon R+ larvae
Ultrastructural morphology in control cells show distinct mito-chondria, Golgi bodies, smooth and rough endoplasmic reticulum,fewer vacuoles along with intact microvilli. Deviation from the con-trol cells was evident in 100 μg/mL aSNPs treated organism as re-vealed by damaged mitochondria (membrane and cristae damage),increased vacuolization and shrink and damaged microvilli. aSNPsand their aggregates were found to be entrapped in endocytic vesi-cles, accumulated in mitochondria and internalized after their pene-tration through cell membrane. More distinctly, aSNPs were found
Fig. 3. RT-PCR analysis for mRNA expression of hsp70 and hsp22. (A) Agarose gel images andaSNP treated third instar larvae of D. melanogaster for 12–36 h. Data represent are mean±Splate] of three independent experiments with three replicates. Significance is ascribed as *P
to be dispersed in cytoplasm in the form of small aggregates(Fig. 6A–H).
3.6. Membrane stability in midgut cells of exposed third instar Oregon R+
larvae
To confirm our observation of cell membrane andmicrovilli damagein the exposed larvae by TEM, AO/EB staining was performed in controland treated group to examine membrane destabilization. We observedsignificant (Pb0.05; Pb0.001) membrane destabilization of gut cells(>5% EB stained nuclei) in a concentration dependent manner (severedestabilization in 17% of the larvae exposed to 100 μg/mL aSNPs for36 h) (Fig. 7).
3.7. Loss of mitochondrial membrane potential (MMP) in midgut cells ofexposed third instar Oregon R+ larvae
Disintegrated mitochondrial structure along with aSNPs depositionin mitochondria observed by TEM prompted us to examine the effectof aSNPs on MMP in the exposed larvae. A significant (Pb0.05;
(B) densitometric representation of mRNA expression of hsp70 and hsp22 in control andD of relative intensity [relative to internal control (gapdh), originating from same tem-b0.05 or **Pb0.001 as compared to control. PC, positive control.
Fig. 4. Estimation of intracellular ROS. ROS generation in control and aSNPs treatedthird instar larvae of D. melanogaster for 12–36 h. Data represent are mean±SD ofthree independent experiments made in three replicates. Significance is ascribed as*Pb0.05 or **Pb0.001 as compared to control.
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Pb0.001) concentration-dependent loss of MMP (expressed as percentof depolarized cells) was observed in the cells of exposed larvae after36 h in comparison to control (Fig. 8).
Fig. 5. Measurement of OS endpoints. (A–B) Superoxide dismutase (SOD) and catalase (C(MDA) contents and (F) glutathione S-transferase (GST) activity in control and aSNP exposthree independent experiments made in three replicates. Significance is ascribed as *Pb0.0
3.8. IETD- andDEVD-ase activities in exposed third instar Oregon R+ larvae
To investigate aSNPs-induced apoptosis in the exposed organism,we measured IETD- and DEVD-ase activities as markers of apoptosisin control and treated group after 36 h. Both these enzyme activitieswere found to be increased in the exposed larvae in a concentration de-pendent manner (3- and 2.5-folds increase in IETD- and DEVD-ase ac-tivities at 100 μg/mL aSNPs concentration) (Fig. 9).
3.9. Correlation among different stress parameters
A correlation was drawn among different endpoints relevant toaSNPs-induced adverse effects in the exposed organism (Table 3). A sig-nificant negative correlation (r=−0.987; Pb0.001) was observed be-tween MDA content (lipid peroxidation) and GSH content. Further,GSH content was found to be negatively correlated (r=0.969;Pb0.001) with ROS generation. A significant positive correlation wasdrawn between ROS generation and hsp70 (r=0.996; Pb0.001), MMP(r=0.995; Pb0.001), ROS vs. hsp22 (r=0.996; Pb0.001) and ROS vs.DEVDase activity (r=0.969; Pb0.001).
AT) activities, (C–E) protein carbonyl (PC), glutathione (GSH) and malondialdehydeed third instar larvae of D. melanogaster for 12–36 h. Data represent are mean±SD of5 or **Pb0.001 as compared to control.
Fig. 6. TEM analysis of midgut tissue. TEM images of midgut tissue of control and aSNP exposed third instar larvae of D. melanogaster for 36 h showing intracellular organization in(A) control and (B) treated, morphology of microvilli in (C) control and (D) treated (E) aSNPs aggregates in cytoplasm, (F) mitochondrial disintegration, (G) endocytic uptake and(H) direct invasion of aSNPs aggregates inside the cell. M, mitochondria; V, vesicles; N, nucleus; MV, microvilli. Arrows indicate the presence of aSNPs.
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Fig. 7. Membrane destabilization in midgut cells. (A) Graphical representation and(B) confocal images showing AO/EB staining in the midgut cells of control and aSNPsexposed third instar larvae of D. melanogaster after 36 h. Red and white arrows denoteAO and EB stained nuclei respectively. Data represent are mean±SD of three indepen-dent experiments made in three replicates. Significance is ascribed as *Pb0.05 or**Pb0.001 as compared to control.
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4. Discussion
The present in vivo study was carried out to examine absorption/internalization of ingested aSNPs in cells lining the midgut and alsocellular stress response in exposed D. melanogaster larvae.
Physiochemical properties of NPs are essential to be characterizedbefore they are evaluated for the adverse effects [19,54] and this ismore so due to alteration in their properties viz. dispersity, agglomera-tion and aggregation in different mediums. We used aSNPs of b30 nm,average particle size of which was further confirmed by TEM analysis.During the state of suspension, aSNPs exhibitedminimum precipitationand aggregation which was apparent from the correlograms in waterand 5% sucrose solution as the end of curves were almost smooth(please see S1). According to the emerging picture about NPs adversi-ties, several in vitro studies had documented that smaller NPs, with a di-ameter of ≤10 nm, are consistently more detrimental to the biologicalsystems than the larger analogues, i.e., >100 nm [20–22]. Taking thisinto consideration, aSNPs of b30 nm were chosen for the study.
Excessive ROS generation and decreased antioxidant capacity inan organism have been suggested to play role in the toxicity of NPs[55,56]. In agreement with the earlier studies, we also observed sig-nificant generation of ROS in the aSNPs exposed organism along with
Fig. 8. Measurement of MMP in midgut cells. Alteration in MMP in control and aSNPstreated third instar larvae of D. melanogaster for 36 h. Data represent are mean±SDof three independent experiments made in three replicates. Significance is ascribedas *Pb0.05 or **Pb0.001 as compared to control.
induction of OS such as significant alteration in LPO, GSH content,anti-oxidant enzyme activities (SOD, CAT) and PC content. We deter-mined the generation ROS in control and in aSNPs exposed organismby flowcytometry using a dye DCF-DA. Its use is recommended as asuitable probe for the measurement of total ROS generation withproper methodological considerations to avoid technical pitfalls[57]. However, direct reaction of this dye with cytochrome c and cy-toplasmic oxidases or peroxidases and self ROS generation by thisdye are also reported [58]. Thus, possibility of overestimating thegeneration of ROS in the present experimental set up cannot beruled out. Significantly enhanced SOD and CAT activities observed inthe exposed organism may be correlated with the activation of cellulardefense to abate the adverse effect of free radicals [59]. Utilization ofGSH for scavenging free peroxide radicals in the aSNPs exposed organ-ism was reflected by a significant negative correlation drawn betweenMDA content and GSH content (YGSH=−7.34XMDA+56.42; r=0.987; Pb0.001). Similarly, a decline in anti-oxidant level in the exposedorganismmay be correlated with an increased free radical/ROS state asevident by a significant negative correlation drawn between GSH con-tent and ROS (YROS=−0.861XGSH+41.89; r=0.969; Pb0.001).
Parallel to the activation of anti-oxidant defense system in a xenobi-otic exposed organism, another mode of cellular defense operates, thatis induction of hsgs. Increased expression of hsgs occurs as a defensivemean through non-specific mechanisms of toxicity that involves thegeneration of abnormal proteins and alteration of cellular functions.We, therefore, tested this possibility by measuring the induction of se-lected hsgs viz. hsp83, hsp70, hsp60, hsp27, hsp26, hsp23 and hsp22.Among these hsgs, only hsp70 and hsp22were found to be significantlyinduced in the aSNPs exposed organism. Hsp70 has been reported as anearly indicator of cellular stress induced by several environmentalchemicals [31] including NPs [55]. We also observed a significant in-crease in the induction of hsp70 in the exposed organism reflecting cel-lular stress caused by the aSNPs. ROS levels have been documentedearlier to play an important role in the regulation of gene expressionby activating transcription factors that, in turn, canmodulate the induc-tion of proteins involved in cellular response to environmental condi-tions [60]. Therefore, it is likely that ROS generated in the exposedorganism would activate transcription factors that can trigger the in-duction of hsps, viz., hsp70. This was further corroborated when a posi-tive correlation was drawn between hsp70 expression and ROS levels(YHSP70=1.548XROS+25.48; r=0.995; Pb0.001).
Screening of only one particular class of stress protein may not beadequate for identifying a sensitive bioindicator against a broad rangeof pollutants due to different levels of expression of different familiesof stress proteins against different agents [61,62]. Studies from this labas well by others reported induction of other hsgs either parallel tothe induction of hsp70 or without the significant induction of hsp70 bydifferent environmental chemicals [37,42,63]. In this context, we ob-served a significant induction of hsp22 along with hsp70. Parallel tothe positive correlation drawn between hsp70 and ROS levels, we alsoobserved a positive correlation drawn between hsp22 and ROS levels(YHSP22=2.051XROS+23.44; r=0.995, Pb0.001) in the aSNPs exposedgroups. Morrow et al. [64] demonstrated an increased expression ofhsp22 and its association with mitochondrial damage in Drosophila. Inthis study too, significant loss of MMP as demonstrated byflowcytometry and mitochondrial membrane disintegration by TEMfurther indicates that hsp22 induction might have a role in such cellularresponses in the exposed organism.
Excess OS generated in an organism after xenobiotics exposure canlead to cell death [65,66]. Parallel to the increased OS generated inaSNPs exposed Drosophila, we observed a significant increase in IETD-and DEVD-ase activities (corresponding to Caspase −9 and 3) concur-rent with a significant change in MMP indicating increased cell deathin the organism. This was further corroborated when we drew a posi-tive correlation between OS endpoints and IETD- and DEVD-ase activi-ties measured in this study (YDEVDase=0.002XROS+0.017; please see
Fig. 9. Colorimetric estimation of caspases activity. (A) IETDase (caspase 9) and (B) DEVDase (caspase 3) activities in control and aSNPs treated third instar larvae of D. melanogasterfor 36 h. Data represent are mean±SD of three independent experiments made in three replicates. Significance is ascribed as *Pb0.05 or **Pb0.001 as compared to control.
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S2). Hsps have been reported to have both pro- and anti-apoptoticproperties [67]. While Hsp70 has been reported to be largelyanti-apoptotic, Hsp22 has been suggested to possess both pro- andanti-apoptotic properties depending on the cell types [68]. Significantincrease in hsp70 and hsp22 expression observed in the aSNPs exposedorganism in the context of increased cell death might be a consequenceof subtle balance in the expression of these two hsgs under the presentexperimental condition. At present, we are unable to critically evaluatethe role of each hsg on apoptotic cell death in the exposed organism.
Membranes as the biological boundary of cells facilitate the uptake/internalization of different molecules inside cells while endocytosis is aphysiological process for engulfing themolecules/substances and isme-diated by the formation of endocytic vesicles (endosomes). Theentrapped molecules are eventually localized in intracellular environ-ment. NPs of varying size and compositionmay be taken up by differentendocytic mechanisms ending up in different intracellular traffickingpathways [69]. Earlier reports have shown internalization of NPsthrough the process of endocytosis [70,71]. Thus, release of these NPsfrom the endosomes into cytoplasm [72] and subsequently, their expo-sure to intracellular moieties and organelles remains a possibility. Weobserved entrapped aSNPs inside the endocytic vesicles in aggregatedform in the midgut cells of exposed Drosophila. In agreement with ear-lier in vitro reports [70,73], we also observed the dispersion of aSNPs inthe cytoplasm of cells of the exposed organism by TEM analysis. Thus,internalization of aSNPs inside the cells taking place through direct pen-etration of cell membrane along with endocytic uptake cannot be ruledout.
To gain insight on the mechanism of NPs-induced cellular stress,earlier studies using either model membrane systems (lipid bilayer)or cell lines or in vivomodel systems revealed NPsmediatedmembranedamage through particle-membrane interaction [14,16,18]. Membranedisintegration due to exposure of NPs and their aggregates can lead to
Table 3Correlation among different stress endpoints.
the damage of proteins and lipid molecules. We hypothesized thataSNPs induced cellular stress in the exposed organism could be associ-ated with membrane damage caused by these particles. Our observa-tion of abnormal morphology (thinning and shortening in size) ofmicrovilli of themidgut of exposedDrosophila by TEMalongwithmem-brane destabilization as evident by positive AO/EB staining in the mid-gut cells of exposed organism at the highest tested concentration ofaSNPs further confirms the above possibility. It is tempting to speculatethat physical obstruction to the membrane by aggregates may happenas the concentration of aSNPs increase (1–100 μg/mL) in the exposedorganism leading to severe membrane destabilization. Among differentcellular organelles, mitochondria are responsible for intracellular ROSmetabolism [74]. Safeguarding of ROS homeostasis in mitochondria isreported to be dependent on respiratory chain andmembrane potential[75]. Our observation of aSNPs accumulation in mitochondria alongwith a significant loss ofMMP in the exposed organism raises a possibil-ity of mitochondria mediated ROS generation leading to cellular stressin the organism. Since we have used collagenase to make a suspensionof dissociated cells for assaying MMP and ROS, a possibility that the en-zyme influencing stability of the cellular membranes of the aSNPs ex-posed organism cannot be ruled out. To the best of our knowledge,this is the first report on aSNPs induced cellular stress response vis avis their cellular internalization using a well accepted in vivo modelDrosophila which has minimum ethical concern.
5. Conclusion
Taken together, the study suggests a negative impact of ingestedaSNPs (b30 nm) on the midgut tissues of exposed Drosophila alongwith elucidation of possible mechanism of cellular stress response(Fig. 10). The study also recommends the use of Drosophila as an invivo model for studying adverse effects of NPs which might havewider applications in the context of similarities existing between themidgut tissue of this organism and that in higher organisms.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbagen.2012.10.001.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
The authors are grateful to Dr. K.C. Gupta, Director, CSIR-Indian Insti-tute of Toxicology Research, Lucknow for support and for Oregon R+
stock by Drosophila Stock Centre, Bloomington, USA. Financial
Fig. 10. Schematic illustration of aSNPs-uptake and cellular response. Schematic model showing the mechanism of internalization of aSNPs and cellular stress evoked by these NPsin midgut cells of exposed Drosophila larvae. aSNPs, amorphous silica nanoparticles; PM, plasma membrane; EV, endocytic vesicle; LPO, lipid peroxidation GSH, glutathione; ROS,reactive oxygen species; MT, mitochondria; MMP, mitochondrial membrane potential; HSPs, heat shock proteins; SOD, superoxide dismutase; CAT, catalase.
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assistance to AP as JRF and SRF [20-6/2008(ii)EU-IV], SC as JRF [20-12/2009(ii)EU-IV] from University Grants Commission (UGC), New Delhiand to DKC from Council of Scientific of Industrial Research (CSIR),NewDelhi (SIP-08 and NWP-34) is thankfully acknowledged. IITR com-munication number 3061.
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