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Assessing potential harmful effects of CdSe quantum dots by using Drosophila melanogaster as in vivo model
Science of the Total Environment 530–531 (2015) 66–75
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Assessing potential harmful effects of CdSe quantum dots by using Drosophila melanogaster as in vivo model Mohamed Alaraby a,b, Esref Demir c, Alba Hernández a,d, Ricard Marcos a,d,⁎ a
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Spain Sohag University, Faculty of Sciences, Zoology Department, 82524-Campus, Sohag, Egypt Akdeniz University, Faculty of Sciences, Department of Biology, 07058-Campus, Antalya, Turkey d CIBER Epidemiología y Salud Pública, ISCIII, Madrid, Spain b c
H I G H L I G H T S • • • • •
CdSe QDs were able to cross the intestinal barrier of Drosophila. Elevated ROS induction was detected in larval hemocytes. Changes in the expression of Hsps and p53 genes were observed. Primary DNA damage was induced by CdSe QDs in hemocytes. Overall, CdSe QD effects were milder than those induced by CdCl2.
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Article history: Received 25 March 2015 Received in revised form 12 May 2015 Accepted 17 May 2015 Available online xxxx Editor: D. Barcelo Keywords: Drosophila melanogaster CdSe QDs Hemocytes Comet assay Wing-spot assay
a b s t r a c t Since CdSe QDs are increasingly used in medical and pharmaceutical sciences careful and systematic studies to determine their biosafety are needed. Since in vivo studies produce relevant information complementing in vitro data, we promote the use of Drosophila melanogaster as a suitable in vivo model to detect toxic and genotoxic effects associated with CdSe QD exposure. Taking into account the potential release of cadmium ions, QD effects were compared with those obtained with CdCl2. Results showed that CdSe QDs penetrate the intestinal barrier of the larvae reaching the hemolymph, interacting with hemocytes, and inducing dose/time dependent significant genotoxic effects, as determined by the comet assay. Elevated ROS production, QD biodegradation, and significant disturbance in the conserved Hsps, antioxidant and p53 genes were also observed. Overall, QD effects were milder than those induced by CdCl2 suggesting the role of Cd released ions in the observed harmful effects of Cd based QDs. To reduce the observed side-effects of Cd based QDs biocompatible coats would be required to avoid cadmium's undesirable effects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The semiconductors Quantum Dots (QDs) constitute a generation of nanomaterials characterized by their small size (1–10 nm), containing about 200–10,000 atoms, and having invaluable optical, chemical, electrical and magnetic properties (Niemeyer, 2001). They are increasingly used in medical and pharmaceutical sciences due to their high volume ratio that enable them to conjugate with multiple ligands (GeszkeMoritz and Moritz, 2013) and proved to be useful in imaging probes in various tumors (Mashinchian et al., 2014). In spite of the interesting ⁎ Corresponding author at: Grup de Mutagènesi, Department de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès (Barcelona), Spain. E-mail address: [email protected] (R. Marcos).
http://dx.doi.org/10.1016/j.scitotenv.2015.05.069 0048-9697/© 2015 Elsevier B.V. All rights reserved.
properties of QDs, there are doubts about their potential harmful health effects. These doubts are related to the fact that these materials contain heavy metals such as Cd, As, Zn, and Pb. The toxic properties of QDs depend on several parameters including composition, size, surface coating, charge, and period/route of exposure. In particular CdSe QDs have two well-known toxic elements cadmium and selenium that can produce harmful effects to many cell types. Cadmium ions (Cd2 +) are well known as probable carcinogens and can penetrate through the blood–brain barrier and placenta, accumulating in the brain, liver, kidney and even in bone tissue (Luevano and Damodaran, 2014). Selenium, although it is an essential nutrient for humans due to its importance for many cellular processes also poses toxic potential (Zwolak and Zaporowska, 2012) mainly above homeostatic requirements (Zhang et al., 2014). In vitro toxicity of QDs has been widely reported (Chang et al., 2006; Chen et al., 2012; Nagy
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et al., 2012; Pathakoti et al., 2013), including DNA damage (Liang et al., 2007; Aye et al., 2013). Although in vitro physiological models can give an initial estimation of toxicity profiles, these in vitro approaches are unable to match the exact complex biological interplay taking place in vivo (Patri et al., 2005). In this context, some in vivo studies have reported severe effects from exposure to Cd QDs (Khalil et al., 2011; Galeone et al., 2012; Ambrosone et al., 2012; Jackson et al., 2012). In contrast there are some other studies showing that QDs do not cause significant toxicity in the course of long-term studies in Sprague–Dawley rats (Hauck et al., 2010), in monkeys (Ye et al., 2012) or in fish (Blickley et al., 2014). Differences among studies can be associated with the composition of QDs as well as with the type of shell coating, organism used or type of biomarkers used. Understanding the relative toxicities of different modes of nanoparticle exposure, as compared with their dissolved metal ions, are emerging areas in ecotoxicology (Jackson et al., 2012). In this context it must be remembered that Drosophila melanogaster as an in vivo organism has proved to be a qualified model in detecting potential harmful effects of nanomaterials (Pompa et al., 2011a,2011b; Vecchio, 2014; Ong et al., 2015; Alaraby et al., 2014). In fact Drosophila has already been successfully used to detect the toxic effect of CdSe/ ZnS QDs (Galeone et al., 2012). The current study contains a systematic study on the potential adverse effects of CdSe QDs in Drosophila after exposure during larval stage development. To differentiate the potential effects attributed to Cd ions, treatments aiming to evaluate CdSe QD effects were carried out in parallel with those using CdCl2 as model of Cd ionic release form. 2. Materials and methods 2.1. Quantum dots CdSe QDs (Lumidot™ CdSe) with 3–3.5 nm size have an absorption at 535–545 nm and fluorescent at λem 555–565 nm, the ionic form of cadmium (CdCl2) and all the other compounds used in the different tests were supplied by Sigma Chemical Co. (St. Louis, MO). Transmission electron microscopy (TEM, Jeol 1400), Dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) methodologies were used to determine size, morphology, distribution diameter and charge respectively. DLS and LDV measurements were carried out in a Malvern ZetasizerNano-ZS zen3600 (Malvern, UK) instrument. An energydispersive X-ray spectroscopy (EDX) spectrum was used to analyze CdSe QD chemical composition by using TEM (200 kV) Jeol 2011 (Jeol Ltd, Tokyo, Japan). 2.2. Exposure Three different D. melanogaster strains were used in this study: the wild-type Canton-S, the multiple-wing-hairs and the flare-3 strains. The last two strains were used for the wing-spot assay and carried the wing-markers namely multiple-wing-hairs (mwh), and flare-3 (flr3), respectively. To determine the survival rate (egg-to-adult) eggs from Canton-S individuals were platted in food vials containing 4 g of instant medium previously wetted with 10 mL of different doses (0.0, 1, 5, 25, 100, 500 μM) of both Cd forms until adult emergence (Alaraby et al., 2014). Final concentrations were 0.48, 2.4, 12, 48 and 240 μg/g food for CdSe QDs and 0.46, 2.3, 11.5, 46 and 230 μg/g food for CdCl2. Morphological abnormalities were also determined in emerged adults. Thus, one hundred adults from the highest tested dose (500 μM) were carefully investigated using a stereo-microscope to detect the presence of morphological changes at head, thorax, abdomen, wings and legs. 2.3. CdSe QD internalization To verify the QDs uptake throughout the intestinal barrier its presence in the hemolymph and hemocytes was determined. The
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hemolymph is the fluid existing in the opened circulatory system of Drosophila with a blood-like function. It is the fate of digested food and, inside it, immune phagocyte cells (hemocytes) are distributed which have a role similar to mammals' leucocytes. To detect the internalization of CdSe QDs in hemolymph and hemocytes, hemolymph samples were collected following a method previously described (Carmona et al., 2011). Briefly chilled 96 ± 2-h-old larvae were removed from food media, washed in water, and dried. The cuticle from 20–30 larvae was disrupted with two fine forceps and the hemolymph and the circulating hemocytes were collected in Schneider's medium containing 0.07% phenylthiourea (PTU). The presence of CdSe QDs in the hemolymph and hemocytes was determined by confocal microscopy. 2.4. Gene expression The expression of different genes Hsp70 (NCBI: NM_169441.2), Hsp83 (NCBI: NM_001274433.1), Catalase (CAT, NCBI: NM_080483.3), Super oxide dismutase (SOD, NCBI: NM_057577.3) and p53 (NCBI: NM_ 206544.2) in treated and untreated larvae were detected. Third-instar larvae in groups of 30 larvae (~50 mg) were homogenized in TRIzol® Reagent (Invitrogen, Carlsbad, CA), and RNA was extracted according to the manufacturer's procedures. RNase-free DNase I (DNA-freeTM kit; Ambion, Paisley, UK) was used to remove DNA contamination. Nanodrop was used to determine the quantity of RNA in each sample. cDNA was synthesized using 1 μg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) following the manufacturer's instructions and stored at − 20 °C until further use. The resulting cDNA was subjected to real-time RT-PCR analysis on a Light Cycler 480 (Roche, Basel, Switzerland) to determine the relative expression of the selected genes, using β-actin as the housekeeping control. For each one of the selected genes, 20 μL of reaction volume were used containing 1 μL of cDNA (400 ng/μL) mixed with 10 μL of 2 × SYBER Green mix, 2 μL of 10 μM gene specific primer mix and 7 μL of water. Reaction conditions for all genes were: pre-incubation for 5 min at 95 °C, 1 cycle, and the amplification was repeated 45 times (10 s at 95 °C, 15 s at 61 °C, 72 °C for 25 s). 2.5. Measurement of reactive oxygen species (ROS) The presence of intracellular ROS was measured in hemocytes using the 6-carboxy-2,7′-dichlorodihydro-fluorescein diacetate (DCFH-DA) assay. Hemocytes collected according to the previous protocol were exposed to 5 μM DCFH-DA for 30 min at 24 °C. The fluorescence in cells was investigated using a fluorescent microscope with an excitation of 485 nm and an emission of 530 nm (green filter). ImageJ program was used for the quantitative evaluation of fluorescent images from both control and treated larvae. 2.6. Genotoxicity experiments 2.6.1. The wing-spot assay The wing-spot test was carried out as previously described by our group (Marcos and Carmona, 2013). Briefly, virgin females of the flr3 strain were mated to mwh males and eggs resulting from this cross were collected for 8-h periods in culture bottles containing standard medium. The emerging 3-day-old larvae were transferred to plastic vials with 4 g of Drosophila instant medium (Carolina Biological Supply Co., Burlington, NC) prepared with the different concentrations (0.0, 5, 25, 100, 500 μM) of CdSe QDs or CdCl2. Ethyl methanesulfonate (EMS, 1 mM) was used as positive control. Larvae were fed on this medium until pupation. The surviving adults were collected from the treatment vials and stored in 70% ethanol. Afterwards, 80 wings were carefully removed and mounted in Faure's solution on microscope slides of each dose treatment. Wings were scored at 400× magnification for detecting mutant clones.
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2.6.2. Comet assay To determine DNA damage, third-instar larvae (72 ± 4 h old) were placed in plastic vials containing 4 g of Drosophila instant medium, pre-wetted with 10 mL of different concentrations (0.0, 25, 100, 500 μM) of CdSe QDs or CdCl2 for 24 ± 2 h. Distilled water and 4 mM EMS were used as negative and positive controls, respectively. All experiments were performed at 25 ± 1 °C and at ~ 60% relative humidity. D. melanogaster hemocytes were collected according to Carmona et al. (2011). Ten microliters of cell samples (~ 10,000 cells) were carefully resuspended in 90 μL of 0.75% LMA at 37 °C, mixed and dropped in triplicate on the hydrophilic surface of Gelbond film (GBF). The GBFs were left for 2–3 min at 4 °C and immersed in cold, freshly made lysis solution (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, 1% Triton X-100 and 1% N-lauroylsarcosinate, pH 10) for 1 h at 4 °C in a dark chamber. To avoid additional DNA damage the next steps were performed under dim light. GBFs were washed for 5 min with cold electrophoresis buffer (0.001 M EDTA, 0.3 M NaOH, pH 13.2) and directly placed for 25 min in a horizontal gelelectrophoresis tank filled with cold electrophoresis buffer to allow DNA unwinding. Electrophoresis was carried out in the same buffer for 20 min at 20 V and 300 mA. The unwinding and electrophoresis were done at 4 °C. After electrophoresis the GBFs were neutralized with two washes for 5 min with PBS followed by 1 min in distal water. The GBFs were rinsed in 100% ethanol for 5–10 min, then they were left to dry for (~ 2 h) before being stained with 25 mL of TE-buffer pH 7.5 containing 10,000 × diluted SYBERGold fluorochrome for 20 min. Finally the GBFs were washed with water to remove excess staining and allowed to dry. The images were examined at 400 × magnification with a Komet 5.5 Image-Analysis System (Kinetic Imaging Ltd., Liverpool, UK) fitted with an Olympus BX50 fluorescence microscope equipped with a 480–550-nm wideband excitation filter and a 590-nm barrier filter. Triplicates of 100 randomly selected cells were analyzed per treatment. The percentage of DNA in the tail (% DNA tail) was used to measure DNA damage. Results were considered statistically significant at P b 0.05. All data were presented in arithmetic mean ± standard error.
3.2. QD internalization D. melanogaster is a good model to demonstrate the uptake and internalization of CdSe QDs via translocation throughout the intestinal barrier, passing towards the internal compartment (fluid hemolymph). In this study CdSe QDs succeeded in crossing the intestinal barrier, reached the hemolymph (analogous to mammalian blood) and interacted with hemocytes (immune cells analogous to mammalian leucocytes). TEM images indicate the presence of CdSe QD aggregates in both microvilli and cytoplasm of midgut cells (Fig. 2A and B). The confirmation that these aggregates correspond to CdSe QD aggregates was demonstrated by EDX methodologies (Fig. 2C). The engulfed QDs in hemocytes and their distribution in hemolymph samples have been detected using confocal microscopy. Fig. 3 indicates QD aggregates in the hemolymph as well as some hemocytes showing internalized QDs. Optical spectra showed maximum emission at 540 nm for CdSe QDs in dispersion (Fig. 4A) and at 560 nm for up-taken QDs (Fig. 4B), the differences of wave length maximum emission and the broadness of fluorescent peaks of the up-taken Cd QD peaks, indicate a change in their physico-chemical characteristics. 3.3. Reactive oxygen species (ROS) in hemocytes Reactive oxygen species (ROS) are considered the main factor behind the severe effects induced in cells. ROS induction in the hemocytes of third instar larvae exposed to CdSe QDs and Cd ions was evaluated and normalized with untreated controls. ROS production detection is based on the conversion of unfluorescent 6-carboxy2,7′-dichlorodihydro-fluorescein diacetate (DCFH-DA) to fluorescent oxidized DCF inside the cells, which can be investigated using a fluorescent microscope. Our results indicated that CdSe QD exposure elevates ROS production, especially at 500 μM exposure (P = 0.06), close to the significant level; on the other hand, ionic Cd induced dose dependent ROS production and significant elevation, especially at higher doses (Fig. 5). 3.4. Gene expression
2.7. Statistical analysis Data were calculated as mean ± standard error (SE). The statistical analysis of SMART assay was performed according to Frei and Würgler (1988) using MICROSTA program, at probability levels α = β = 0.05 (Kastenbaum and Bowman, 1970). Before the statistical analysis of the remaining data the normality was checked with the Kolmogorov– Smirnov and Shapiro–Wilk test, and the homogeneity of variance with Levene's test. Data following normal distribution and equal variance was analyzed with one-way ANOVA followed by post hoc, multiple comparisons (comet assay and the expression of some genes). Data with unequal variance or skewed distribution was analyzed with nonparametric analysis “Mann–Whitney U test” (ROS production, viability and the expression of some genes). Significant differences were considered at P ≤ 0.05.
Cells respond to any external effect in a systematic and coordinated manner at molecular level via tier cascades. One of the first tiers involved in early response, corresponds to Hsps gene family (conserved genes). At the same time genes related to antioxidant defense are also considered as cellular alarms to any change in free radical balance; and p53 gene, involved in DNA repair and genomic integrity, is a safe factor for cell health. In this context changes in the expression of Hsp70, Hsp83, CAT, SOD and p53 genes were studied as indicators of stress induced by CdSe QDs. These effects were compared with those detected after CdCl2 exposure. Results demonstrate that third instar Drosophila larvae exposed to CdSe QDs (Fig. 6A), and to CdCl2 (Fig. 6B), showed high transcriptional activity for all studied genes with respect to untreated larvae. Significant effects in gene expression already appear at lower doses, but more clearly after exposure to the higher doses. This means that Cd, regardless of its form, induces significant disturbances to cells that can be detected at molecular level.
3. Results 3.5. Toxicity/genotoxicity studies 3.1. CdSe QD physico-chemical characterization The physico-chemical characterization of CdSe QDs is summarized in Fig. 1(A–E). TEM images showed that CdSe QDs have a narrow size average (3.0 ± 0.43 nm), and the average of hydrodynamic radius and zeta potential of CdSe QDs were 91.61 ± 0.23 nm and − 17.0 ± 1.8 mV, respectively. Optical characteristics of CdSe QDs according to the manufacturer are presented in (D), and EDX spectra showing its chemical composition (E).
The toxic effects of CdSe QDs were determined measuring changes in the egg-to-adult viability. Significant effects were observed at high doses, especially at 500 μM, whereas CdCl2 induced significant toxicity at lower doses (1 μM) as shown in Fig. 7. It is noteworthy that both forms of Cd didn't elicit any morphological malformations in emerged Drosophila adults' structures like the head, thorax, abdomen, wings and legs as determined at the highest concentration tested (500 μM) (data not shown).
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Fig. 1. Characterization of CdSe-QDs. (A) Size frequency of CdSe-QDs, as observed by TEM, gives a mean ± SD value of 3.0 ± 0.43 nm. (B) Typical TEM images of CdSe-QDs. (C) The CdSe-QD diameter in suspension (DLS) and zeta potential (LDV) characterization reported values of 91.61 ± 0.23 nm and −17.0 ± 1.8 mV, respectively. (D) Absorption–emission spectra of CdSeQDs according to the manufacturer, (E) EDX spectrum shows the chemical composition of CdSe-QDs in the dispersion solution.
In regard to the genotoxic effects induced by Cd based QDs two known genotoxic assays were used, one detecting the induction of mutagenic and/or recombinagenic effects (wing-spot assay) and the other measuring DNA strand breaks (comet assay). The data obtained in these genotoxicity assays are indicated in Fig. 8. As observed, neither CdSe QD
nor CdCl2 exposure was able to induce mutagenic/recombinogenic effects (Fig. 8A). Although a significant increase was observed after exposure to 25 μM of CdSe QDs these slight effects must be considered as marginal. In contrast, the results obtained in the comet assay clearly showed significant dose-dependent increases in the percentage of
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Fig. 2. CdSe-QD uptake throughout the intestinal barrier. CdSe-QD aggregates are observed inside the cytoplasm and microvilli of midgut cells as indicated by arrows (A) and (B). Energy dispersive X-rays microanalysis (EDX) spectrum showing the presence of Cd in the CdSe-QD aggregates internalized into the midgut cells of third-instar larvae (C).
DNA strand breaks at the two higher doses of CdSe QDs tested and all doses of CdCl2 (Fig. 8B).
4. Discussion There is no doubt about the benefits that the use of quantum dots provides to medical and pharmaceutical sciences, which can be described as revolutionary. Nevertheless, since these new materials contain heavy metals, a deep knowledge of their potential harmful effects is urgently required. One of the most used QDs is based on cadmium (CdSe) which is known to be a very toxic and carcinogenic element. This study was designed to understand the biological effects of CdSe QDs related only to its core composition reducing the interference with other factors such as coating and ligand ones. We used D. melanogaster as a reliable in vivo model and the obtained effects were compared with those observed with ionic cadmium form. Our results showed the induction of toxic and genotoxic effects in addition to changes in the levels of oxidative stress and in the expression of different genes involved in stress response. In general, the harmful effects observed after QD exposure were lower than those generated by ionic cadmium.
When egg-to-adult toxicity was taken into account we observed that significant toxic effects of QDs appeared only at higher doses, compared to ionic Cd form that induced significant toxicity at lower ones. This can be attributed to differences in Cd nature whether as a free ion or as bound compound (Yeh et al., 2011). These results would confirm the results obtained in a long-term exposure experiment showing that Cd based QDs significantly reduced Drosophila life span (Galeone et al., 2012). Since the biological effects of any agent depend on its uptake via different biological barriers, we have been able to detect the presence of QDs not only in midgut cells but also in the hemolymph which is the general compartment of any material passing through the intestinal barrier (Alaraby et al., 2014). CdSe QDs have been detected in hemocytes in agreement with previous results reporting increased rate of apoptosis in hemocytes (Galeone et al., 2012). Hemocytes are a suitable model to demonstrate oxidative stress induction (Alaraby et al., 2014) as well as innate immune response and DNA damage in vivo (Irving et al., 2005; Cherry and Silverman, 2006; Carmona et al., 2011). Our results showed that QDs increase ROS production in Drosophila hemocytes although to a lesser extent than after exposure to CdCl2. Changes of intracellular ROS levels are assumed to be due to the semiconducting properties of CdSe as gap and valence/ conduction band energy levels (Ekimov and Onushchenko, 1984). On
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Fig. 3. CdSe-QDs detected in the hemolymph and hemocytes of Drosophila melanogaster using a confocal microscope. Representative fluorescent images of third-instar D. melanogaster larvae hemolymph showed CdSe-QDs distributed inside the hemolymph (A) and (B) and inside the hemocytes (C, D, E and F), scale bar 20 μm.
the other hand, the number of atoms on the QDs' surface increases rapidly with the decrease of the QDs' particle size leading to generation of many dangling bonds that are prone to combine with other atoms to become saturated and stable, hence the surface of QDs has a high chemical reactivity (Zhan and Tang, 2014). Although the toxic effects observed in Drosophila after QD exposure were related to ROS elevation (Galeone et al., 2012) ROS induction by QDs in human cultured cells is strongly related to the nature of the included ligands and the presence of free Cd2+ (Nagy et al., 2012). The proposal that ROS elevation is free Cd2+ dependent (Wang et al., 2009; Xu et al., 2011; Son et al., 2011), completely agrees with our results with CdCl2. To address the molecular response to QD exposure, we have checked potential changes of expression in different types of genes related to general stress, antioxidant protection and DNA damage response. Drosophila succeeded in previous studies to demonstrate changes in gene
expression as response to NPs exposure (Ahamed et al., 2010; Siddique et al., 2014; Alaraby et al., 2014) including QDs (Brunetti et al., 2013). Up-regulation of Hsp genes is important to counteract proteotoxic effects via chaperoning proteins during synthesis, folding, assembly and degradation and give preliminary information on the potential exposure to foreign substances. Chang et al. (2006) postulated that CdSe-core QD suppressed Hsp90 in human cells and, accordingly, we have observed similar results related to expression of Hsp70 and Hsp83 in Drosophila. Antioxidant capacity of cells is the first defense line facing any change in the reactive radical balance. Drosophila showed antioxidant response to the exposure to Cd QDs through significantly expression of SOD mRNA, especially at highest dose. This would agree with those results obtained with CdSe QDs in solution (Ipe et al., 2005), and in the epithelial A549 (human lung carcinoma) and the neuronal SH SY5Y (human neuroblastoma) cell lines (Brunetti et al., 2013).
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Fig. 4. Optical spectra showed maximum emission at 540 nm for CdSe-QDs in dispersion (A) and at 560 nm for up-taken QDs (B).
In addition, our results with CdCl2 showed significant expression of CAT at all doses, while SOD expression significantly inhibited at 5 μM and not effected at the two higher doses. The inhibition effect of cadmium on SOD might be due to protein conformation changes by interacting with the enzyme (Casalino et al., 2002). High doses of cadmium can affect the repair of oxidative DNA damage by down-regulating p53 enzyme levels (Casalino et al., 2002) and, possibly, affecting also SOD activity. Our results with CdCl2 also support those previously reported in neuronal and epidermal cells (Xu et al., 2011; Son et al., 2011) where the levels of ROS and SOD were significantly elevated. The cell guardian p53 plays a key role as a hub in cellular genotoxic stress response by acting as a transcription factor to elicit
cellular functions of DNA damage and repair, cell cycle arrest, and apoptosis. P53 is normally accumulated in the nucleus and converted into an active DNA-binding form to control several sets of genes to prevent the proliferation of DNA damaged cells (Inoue et al., 2005). Over-expression of p53 has already been observed after Drosophila larva exposure to Cd QDs (Brunetti et al., 2013), similar to our study. On the contrary irregular significant expression at the two highest doses of CdCl2 was observed which would agree with the results observed in zebra fish (Tang et al., 2013). In human breast MCF-7 cancer cells Cd2 + exposure results in impaired p53 functions such as conformational changes, loss of DNA binding activity, downregulation of transcriptional activity and inhibition of gamma-
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Concentration (µM) Fig. 5. ROS production in hemocytes of third-instar of untreated (0.0) and treated larvae with different doses (5, 25, 100 and 500 μM) of CdSe-QDs and CdCl2. Hemocytes were incubated with 5 μM DCFH-DA at 24 °C for 30 min and observed using fluorescent microscopy. The fluorescence intensity of hemocytes was quantified by ImageJ analysis. 0.5 mM H2O2 was used as positive control. *P b 0.05, **P b 0.01, ***P b 0.001 compared to control.
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Concentration (µM) Fig. 6. The expression of Hsp70, Hsp83, CAT, SOD and p53 in third-instar larvae induced by exposure to different doses of CdSe-QDs (A) and CdCl2 (B). The expressions were normalized using β-actin and are presented compared to control values. Data represent the mean ± standard error of the mean. *P b 0.05, **P b 0.01 compared to negative control.
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Concentration (µM) Fig. 7. Toxicity of CdSe-QDs and CdCl2 in D. melanogaster. Toxicity effects were measured as loss of viability (egg-to-adult survival) related to the control values. *P b 0.05, **P b 0.0 compared to control.
radiation induced DNA damage responses (Meplan et al., 1999). Thus, the expression of p53 might depend on the amount of free Cd2 + which of course differs between Cd QDs and CdCl2 exposure. Cd QDs continually biodegrade and, as consequence, free Cd2 + is continuously released and their size become smaller during the degradation process (Kwon et al., 2012; Sabella et al., 2014). As consequence the fluorescence emission shifts from red to blue and the excitation fluorescence peak becomes broader (Tsoi et al., 2012) which agrees with our results. Although our results showed that the fluorescence emission slightly shifts from blue to red there is a clear broadness of the fluorescence peak of the up-taken Cd QDs indicating their biodegradation. QDs may be degraded after several days in the living cells (Aillon et al., 2009) and this may occur due to low pH or oxidation of QD surface (Khalil et al., 2011). In our study QDs were administered orally to Drosophila larvae and the transit throughout the gastric tract can contribute to the QDs' degradation process via low pH conditions. Although our results showed that both Cd forms failed to induce somatic mutation and/or recombination they were able to demonstrate
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Fig. 8. Genotoxicity of CdSe-QDs and CdCl2 using the wing-spot (A) and comet (B) assays. In (A) the frequency of total spots induced after the treatment of third-instar larvae with different doses (0.0, 5, 25, 100 and 500 μM) of CdSe-QDs and CdCl2 are indicated. EMS (1 mM) was used as positive control, *P b 0.05. In (B) results indicate the % of DNA tail induced after larval exposure with different doses (0.0, 25, 100 and 500 μM) of CdSe-QDs and CdCl2 for exposures lasting 24 h. EMS (4 mM) was used as positive control. *P b 0.05, **P b 0.01 ***P b 0.001.
the induction of primary DNA damage by means of the comet assay, indicating a potential genotoxic risk associated with Cd QD exposure. This would confirm that the comet assay is more sensitive than the wing-spot assay (Alaraby et al., 2014). In fact, non-genotoxic effects of CdCl2 in Drosophila using the wing-spot test were observed early by our group (Rizki et al., 2004). Indirect genotoxicity of QDs in Drosophila has already been determined by means of the tunnel assay (Galeone et al., 2012) and positive effects of CdCl2 have been reported in midgut cells (Shukla et al., 2011) and in hemocytes of spiders fed with Drosophila treated with CdCl2 (Stalmach et al., 2014). No genotoxic effects of Cd QDs were observed in bacteria but positive induction was detected in CHO cells by means of the comet and micronucleus assays (Aye et al., 2013). In the comet assay both Cd QDs and CdCl2 showed a significant dose-dependent increase in DNA strand breaks regardless of their chemical composition which would indicate that the observed effects are related to the presence of Cd2+ ions. Although a direct relationship between DNA damage and Cd2 + has been observed the underlying mechanism is not exactly clear. Cadmium has been demonstrated to amplify the intensity of damage by interfering with the DNA repair NER pathway (Candéias et al., 2010), or might be related to single strand breaks in DNA through direct cadmium–DNA interactions, as well as by
the action of incision nucleases and/or DNA-glycosylase during DNA repair (Katsumiti et al., 2014). 5. Conclusions The obtained results support D. melanogaster as a suitable in vivo model to determine the potential adverse effects of Cd based QDs. In this in vivo model we have been able to demonstrate that Cd based QDs are toxic, and induce oxidative stress and deregulation of gene expression as well as damage to DNA. The higher effects observed when CdCl2 were used indicate that such effects are related to the presence of Cd2+ ions. The increased use of QDs may affect human health after short- and long-term exposures. Thus, to reduce their side effects, biocompatible coats are required to avoid cadmium's undesirable effects. This would agree with the pilot study of Ye et al. (2012) where Rhesus macaques injected with phospholipid micelle-encapsulated CdSe/CdS/ ZnS quantum dots did not exhibit evidence of toxicity. Conflict of interest statement The authors declare that there is no conflict of interest.
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Acknowledgments This investigation has been partially supported by the Generalitat de Catalunya (CIRIT, 2014SGR-202), Barcelona (Spain). M. Alaraby held a fellowship from the Cultural Affairs Sector and Missions (Ministry of Higher Education), Egypt. E. Demir was supported by a postdoctoral fellowship from the Scientific and Technological Research Council of Turkey (TUBITAK), Science Fellowships and Grant Programs Department (BIDEB), 2219-International Post Doctoral Research Fellowship Program, Ankara (Turkey). References Ahamed, M., Posgai, R., Gorey, T.J., Nielsen, M., Hussain, S.M., Rowe, J.J., 2010. Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster. Toxicol. Appl. Pharmacol. 242, 263–269. Aillon, K.L., Xie, Y., El-Gendy, N., Berkland, C.J., Laird, Forrest M., 2009. Effects of nanomaterial physicochemical properties on in vivo toxicity. Adv. Drug Deliv. Rev. 61, 457–466. 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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Assessing potential harmful effects of CdSe quantum dots by using Drosophila melanogaster as in vivo model Mohamed Alaraby a,b, Esref Demir c, Alba Hernández a,d, Ricard Marcos a,d,⁎ a
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Spain Sohag University, Faculty of Sciences, Zoology Department, 82524-Campus, Sohag, Egypt Akdeniz University, Faculty of Sciences, Department of Biology, 07058-Campus, Antalya, Turkey d CIBER Epidemiología y Salud Pública, ISCIII, Madrid, Spain b c
H I G H L I G H T S • • • • •
CdSe QDs were able to cross the intestinal barrier of Drosophila. Elevated ROS induction was detected in larval hemocytes. Changes in the expression of Hsps and p53 genes were observed. Primary DNA damage was induced by CdSe QDs in hemocytes. Overall, CdSe QD effects were milder than those induced by CdCl2.
a r t i c l e
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Article history: Received 25 March 2015 Received in revised form 12 May 2015 Accepted 17 May 2015 Available online xxxx Editor: D. Barcelo Keywords: Drosophila melanogaster CdSe QDs Hemocytes Comet assay Wing-spot assay
a b s t r a c t Since CdSe QDs are increasingly used in medical and pharmaceutical sciences careful and systematic studies to determine their biosafety are needed. Since in vivo studies produce relevant information complementing in vitro data, we promote the use of Drosophila melanogaster as a suitable in vivo model to detect toxic and genotoxic effects associated with CdSe QD exposure. Taking into account the potential release of cadmium ions, QD effects were compared with those obtained with CdCl2. Results showed that CdSe QDs penetrate the intestinal barrier of the larvae reaching the hemolymph, interacting with hemocytes, and inducing dose/time dependent significant genotoxic effects, as determined by the comet assay. Elevated ROS production, QD biodegradation, and significant disturbance in the conserved Hsps, antioxidant and p53 genes were also observed. Overall, QD effects were milder than those induced by CdCl2 suggesting the role of Cd released ions in the observed harmful effects of Cd based QDs. To reduce the observed side-effects of Cd based QDs biocompatible coats would be required to avoid cadmium's undesirable effects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The semiconductors Quantum Dots (QDs) constitute a generation of nanomaterials characterized by their small size (1–10 nm), containing about 200–10,000 atoms, and having invaluable optical, chemical, electrical and magnetic properties (Niemeyer, 2001). They are increasingly used in medical and pharmaceutical sciences due to their high volume ratio that enable them to conjugate with multiple ligands (GeszkeMoritz and Moritz, 2013) and proved to be useful in imaging probes in various tumors (Mashinchian et al., 2014). In spite of the interesting ⁎ Corresponding author at: Grup de Mutagènesi, Department de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès (Barcelona), Spain. E-mail address: [email protected] (R. Marcos).
http://dx.doi.org/10.1016/j.scitotenv.2015.05.069 0048-9697/© 2015 Elsevier B.V. All rights reserved.
properties of QDs, there are doubts about their potential harmful health effects. These doubts are related to the fact that these materials contain heavy metals such as Cd, As, Zn, and Pb. The toxic properties of QDs depend on several parameters including composition, size, surface coating, charge, and period/route of exposure. In particular CdSe QDs have two well-known toxic elements cadmium and selenium that can produce harmful effects to many cell types. Cadmium ions (Cd2 +) are well known as probable carcinogens and can penetrate through the blood–brain barrier and placenta, accumulating in the brain, liver, kidney and even in bone tissue (Luevano and Damodaran, 2014). Selenium, although it is an essential nutrient for humans due to its importance for many cellular processes also poses toxic potential (Zwolak and Zaporowska, 2012) mainly above homeostatic requirements (Zhang et al., 2014). In vitro toxicity of QDs has been widely reported (Chang et al., 2006; Chen et al., 2012; Nagy
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et al., 2012; Pathakoti et al., 2013), including DNA damage (Liang et al., 2007; Aye et al., 2013). Although in vitro physiological models can give an initial estimation of toxicity profiles, these in vitro approaches are unable to match the exact complex biological interplay taking place in vivo (Patri et al., 2005). In this context, some in vivo studies have reported severe effects from exposure to Cd QDs (Khalil et al., 2011; Galeone et al., 2012; Ambrosone et al., 2012; Jackson et al., 2012). In contrast there are some other studies showing that QDs do not cause significant toxicity in the course of long-term studies in Sprague–Dawley rats (Hauck et al., 2010), in monkeys (Ye et al., 2012) or in fish (Blickley et al., 2014). Differences among studies can be associated with the composition of QDs as well as with the type of shell coating, organism used or type of biomarkers used. Understanding the relative toxicities of different modes of nanoparticle exposure, as compared with their dissolved metal ions, are emerging areas in ecotoxicology (Jackson et al., 2012). In this context it must be remembered that Drosophila melanogaster as an in vivo organism has proved to be a qualified model in detecting potential harmful effects of nanomaterials (Pompa et al., 2011a,2011b; Vecchio, 2014; Ong et al., 2015; Alaraby et al., 2014). In fact Drosophila has already been successfully used to detect the toxic effect of CdSe/ ZnS QDs (Galeone et al., 2012). The current study contains a systematic study on the potential adverse effects of CdSe QDs in Drosophila after exposure during larval stage development. To differentiate the potential effects attributed to Cd ions, treatments aiming to evaluate CdSe QD effects were carried out in parallel with those using CdCl2 as model of Cd ionic release form. 2. Materials and methods 2.1. Quantum dots CdSe QDs (Lumidot™ CdSe) with 3–3.5 nm size have an absorption at 535–545 nm and fluorescent at λem 555–565 nm, the ionic form of cadmium (CdCl2) and all the other compounds used in the different tests were supplied by Sigma Chemical Co. (St. Louis, MO). Transmission electron microscopy (TEM, Jeol 1400), Dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) methodologies were used to determine size, morphology, distribution diameter and charge respectively. DLS and LDV measurements were carried out in a Malvern ZetasizerNano-ZS zen3600 (Malvern, UK) instrument. An energydispersive X-ray spectroscopy (EDX) spectrum was used to analyze CdSe QD chemical composition by using TEM (200 kV) Jeol 2011 (Jeol Ltd, Tokyo, Japan). 2.2. Exposure Three different D. melanogaster strains were used in this study: the wild-type Canton-S, the multiple-wing-hairs and the flare-3 strains. The last two strains were used for the wing-spot assay and carried the wing-markers namely multiple-wing-hairs (mwh), and flare-3 (flr3), respectively. To determine the survival rate (egg-to-adult) eggs from Canton-S individuals were platted in food vials containing 4 g of instant medium previously wetted with 10 mL of different doses (0.0, 1, 5, 25, 100, 500 μM) of both Cd forms until adult emergence (Alaraby et al., 2014). Final concentrations were 0.48, 2.4, 12, 48 and 240 μg/g food for CdSe QDs and 0.46, 2.3, 11.5, 46 and 230 μg/g food for CdCl2. Morphological abnormalities were also determined in emerged adults. Thus, one hundred adults from the highest tested dose (500 μM) were carefully investigated using a stereo-microscope to detect the presence of morphological changes at head, thorax, abdomen, wings and legs. 2.3. CdSe QD internalization To verify the QDs uptake throughout the intestinal barrier its presence in the hemolymph and hemocytes was determined. The
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hemolymph is the fluid existing in the opened circulatory system of Drosophila with a blood-like function. It is the fate of digested food and, inside it, immune phagocyte cells (hemocytes) are distributed which have a role similar to mammals' leucocytes. To detect the internalization of CdSe QDs in hemolymph and hemocytes, hemolymph samples were collected following a method previously described (Carmona et al., 2011). Briefly chilled 96 ± 2-h-old larvae were removed from food media, washed in water, and dried. The cuticle from 20–30 larvae was disrupted with two fine forceps and the hemolymph and the circulating hemocytes were collected in Schneider's medium containing 0.07% phenylthiourea (PTU). The presence of CdSe QDs in the hemolymph and hemocytes was determined by confocal microscopy. 2.4. Gene expression The expression of different genes Hsp70 (NCBI: NM_169441.2), Hsp83 (NCBI: NM_001274433.1), Catalase (CAT, NCBI: NM_080483.3), Super oxide dismutase (SOD, NCBI: NM_057577.3) and p53 (NCBI: NM_ 206544.2) in treated and untreated larvae were detected. Third-instar larvae in groups of 30 larvae (~50 mg) were homogenized in TRIzol® Reagent (Invitrogen, Carlsbad, CA), and RNA was extracted according to the manufacturer's procedures. RNase-free DNase I (DNA-freeTM kit; Ambion, Paisley, UK) was used to remove DNA contamination. Nanodrop was used to determine the quantity of RNA in each sample. cDNA was synthesized using 1 μg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) following the manufacturer's instructions and stored at − 20 °C until further use. The resulting cDNA was subjected to real-time RT-PCR analysis on a Light Cycler 480 (Roche, Basel, Switzerland) to determine the relative expression of the selected genes, using β-actin as the housekeeping control. For each one of the selected genes, 20 μL of reaction volume were used containing 1 μL of cDNA (400 ng/μL) mixed with 10 μL of 2 × SYBER Green mix, 2 μL of 10 μM gene specific primer mix and 7 μL of water. Reaction conditions for all genes were: pre-incubation for 5 min at 95 °C, 1 cycle, and the amplification was repeated 45 times (10 s at 95 °C, 15 s at 61 °C, 72 °C for 25 s). 2.5. Measurement of reactive oxygen species (ROS) The presence of intracellular ROS was measured in hemocytes using the 6-carboxy-2,7′-dichlorodihydro-fluorescein diacetate (DCFH-DA) assay. Hemocytes collected according to the previous protocol were exposed to 5 μM DCFH-DA for 30 min at 24 °C. The fluorescence in cells was investigated using a fluorescent microscope with an excitation of 485 nm and an emission of 530 nm (green filter). ImageJ program was used for the quantitative evaluation of fluorescent images from both control and treated larvae. 2.6. Genotoxicity experiments 2.6.1. The wing-spot assay The wing-spot test was carried out as previously described by our group (Marcos and Carmona, 2013). Briefly, virgin females of the flr3 strain were mated to mwh males and eggs resulting from this cross were collected for 8-h periods in culture bottles containing standard medium. The emerging 3-day-old larvae were transferred to plastic vials with 4 g of Drosophila instant medium (Carolina Biological Supply Co., Burlington, NC) prepared with the different concentrations (0.0, 5, 25, 100, 500 μM) of CdSe QDs or CdCl2. Ethyl methanesulfonate (EMS, 1 mM) was used as positive control. Larvae were fed on this medium until pupation. The surviving adults were collected from the treatment vials and stored in 70% ethanol. Afterwards, 80 wings were carefully removed and mounted in Faure's solution on microscope slides of each dose treatment. Wings were scored at 400× magnification for detecting mutant clones.
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2.6.2. Comet assay To determine DNA damage, third-instar larvae (72 ± 4 h old) were placed in plastic vials containing 4 g of Drosophila instant medium, pre-wetted with 10 mL of different concentrations (0.0, 25, 100, 500 μM) of CdSe QDs or CdCl2 for 24 ± 2 h. Distilled water and 4 mM EMS were used as negative and positive controls, respectively. All experiments were performed at 25 ± 1 °C and at ~ 60% relative humidity. D. melanogaster hemocytes were collected according to Carmona et al. (2011). Ten microliters of cell samples (~ 10,000 cells) were carefully resuspended in 90 μL of 0.75% LMA at 37 °C, mixed and dropped in triplicate on the hydrophilic surface of Gelbond film (GBF). The GBFs were left for 2–3 min at 4 °C and immersed in cold, freshly made lysis solution (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, 1% Triton X-100 and 1% N-lauroylsarcosinate, pH 10) for 1 h at 4 °C in a dark chamber. To avoid additional DNA damage the next steps were performed under dim light. GBFs were washed for 5 min with cold electrophoresis buffer (0.001 M EDTA, 0.3 M NaOH, pH 13.2) and directly placed for 25 min in a horizontal gelelectrophoresis tank filled with cold electrophoresis buffer to allow DNA unwinding. Electrophoresis was carried out in the same buffer for 20 min at 20 V and 300 mA. The unwinding and electrophoresis were done at 4 °C. After electrophoresis the GBFs were neutralized with two washes for 5 min with PBS followed by 1 min in distal water. The GBFs were rinsed in 100% ethanol for 5–10 min, then they were left to dry for (~ 2 h) before being stained with 25 mL of TE-buffer pH 7.5 containing 10,000 × diluted SYBERGold fluorochrome for 20 min. Finally the GBFs were washed with water to remove excess staining and allowed to dry. The images were examined at 400 × magnification with a Komet 5.5 Image-Analysis System (Kinetic Imaging Ltd., Liverpool, UK) fitted with an Olympus BX50 fluorescence microscope equipped with a 480–550-nm wideband excitation filter and a 590-nm barrier filter. Triplicates of 100 randomly selected cells were analyzed per treatment. The percentage of DNA in the tail (% DNA tail) was used to measure DNA damage. Results were considered statistically significant at P b 0.05. All data were presented in arithmetic mean ± standard error.
3.2. QD internalization D. melanogaster is a good model to demonstrate the uptake and internalization of CdSe QDs via translocation throughout the intestinal barrier, passing towards the internal compartment (fluid hemolymph). In this study CdSe QDs succeeded in crossing the intestinal barrier, reached the hemolymph (analogous to mammalian blood) and interacted with hemocytes (immune cells analogous to mammalian leucocytes). TEM images indicate the presence of CdSe QD aggregates in both microvilli and cytoplasm of midgut cells (Fig. 2A and B). The confirmation that these aggregates correspond to CdSe QD aggregates was demonstrated by EDX methodologies (Fig. 2C). The engulfed QDs in hemocytes and their distribution in hemolymph samples have been detected using confocal microscopy. Fig. 3 indicates QD aggregates in the hemolymph as well as some hemocytes showing internalized QDs. Optical spectra showed maximum emission at 540 nm for CdSe QDs in dispersion (Fig. 4A) and at 560 nm for up-taken QDs (Fig. 4B), the differences of wave length maximum emission and the broadness of fluorescent peaks of the up-taken Cd QD peaks, indicate a change in their physico-chemical characteristics. 3.3. Reactive oxygen species (ROS) in hemocytes Reactive oxygen species (ROS) are considered the main factor behind the severe effects induced in cells. ROS induction in the hemocytes of third instar larvae exposed to CdSe QDs and Cd ions was evaluated and normalized with untreated controls. ROS production detection is based on the conversion of unfluorescent 6-carboxy2,7′-dichlorodihydro-fluorescein diacetate (DCFH-DA) to fluorescent oxidized DCF inside the cells, which can be investigated using a fluorescent microscope. Our results indicated that CdSe QD exposure elevates ROS production, especially at 500 μM exposure (P = 0.06), close to the significant level; on the other hand, ionic Cd induced dose dependent ROS production and significant elevation, especially at higher doses (Fig. 5). 3.4. Gene expression
2.7. Statistical analysis Data were calculated as mean ± standard error (SE). The statistical analysis of SMART assay was performed according to Frei and Würgler (1988) using MICROSTA program, at probability levels α = β = 0.05 (Kastenbaum and Bowman, 1970). Before the statistical analysis of the remaining data the normality was checked with the Kolmogorov– Smirnov and Shapiro–Wilk test, and the homogeneity of variance with Levene's test. Data following normal distribution and equal variance was analyzed with one-way ANOVA followed by post hoc, multiple comparisons (comet assay and the expression of some genes). Data with unequal variance or skewed distribution was analyzed with nonparametric analysis “Mann–Whitney U test” (ROS production, viability and the expression of some genes). Significant differences were considered at P ≤ 0.05.
Cells respond to any external effect in a systematic and coordinated manner at molecular level via tier cascades. One of the first tiers involved in early response, corresponds to Hsps gene family (conserved genes). At the same time genes related to antioxidant defense are also considered as cellular alarms to any change in free radical balance; and p53 gene, involved in DNA repair and genomic integrity, is a safe factor for cell health. In this context changes in the expression of Hsp70, Hsp83, CAT, SOD and p53 genes were studied as indicators of stress induced by CdSe QDs. These effects were compared with those detected after CdCl2 exposure. Results demonstrate that third instar Drosophila larvae exposed to CdSe QDs (Fig. 6A), and to CdCl2 (Fig. 6B), showed high transcriptional activity for all studied genes with respect to untreated larvae. Significant effects in gene expression already appear at lower doses, but more clearly after exposure to the higher doses. This means that Cd, regardless of its form, induces significant disturbances to cells that can be detected at molecular level.
3. Results 3.5. Toxicity/genotoxicity studies 3.1. CdSe QD physico-chemical characterization The physico-chemical characterization of CdSe QDs is summarized in Fig. 1(A–E). TEM images showed that CdSe QDs have a narrow size average (3.0 ± 0.43 nm), and the average of hydrodynamic radius and zeta potential of CdSe QDs were 91.61 ± 0.23 nm and − 17.0 ± 1.8 mV, respectively. Optical characteristics of CdSe QDs according to the manufacturer are presented in (D), and EDX spectra showing its chemical composition (E).
The toxic effects of CdSe QDs were determined measuring changes in the egg-to-adult viability. Significant effects were observed at high doses, especially at 500 μM, whereas CdCl2 induced significant toxicity at lower doses (1 μM) as shown in Fig. 7. It is noteworthy that both forms of Cd didn't elicit any morphological malformations in emerged Drosophila adults' structures like the head, thorax, abdomen, wings and legs as determined at the highest concentration tested (500 μM) (data not shown).
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Fig. 1. Characterization of CdSe-QDs. (A) Size frequency of CdSe-QDs, as observed by TEM, gives a mean ± SD value of 3.0 ± 0.43 nm. (B) Typical TEM images of CdSe-QDs. (C) The CdSe-QD diameter in suspension (DLS) and zeta potential (LDV) characterization reported values of 91.61 ± 0.23 nm and −17.0 ± 1.8 mV, respectively. (D) Absorption–emission spectra of CdSeQDs according to the manufacturer, (E) EDX spectrum shows the chemical composition of CdSe-QDs in the dispersion solution.
In regard to the genotoxic effects induced by Cd based QDs two known genotoxic assays were used, one detecting the induction of mutagenic and/or recombinagenic effects (wing-spot assay) and the other measuring DNA strand breaks (comet assay). The data obtained in these genotoxicity assays are indicated in Fig. 8. As observed, neither CdSe QD
nor CdCl2 exposure was able to induce mutagenic/recombinogenic effects (Fig. 8A). Although a significant increase was observed after exposure to 25 μM of CdSe QDs these slight effects must be considered as marginal. In contrast, the results obtained in the comet assay clearly showed significant dose-dependent increases in the percentage of
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A
B
C
Fig. 2. CdSe-QD uptake throughout the intestinal barrier. CdSe-QD aggregates are observed inside the cytoplasm and microvilli of midgut cells as indicated by arrows (A) and (B). Energy dispersive X-rays microanalysis (EDX) spectrum showing the presence of Cd in the CdSe-QD aggregates internalized into the midgut cells of third-instar larvae (C).
DNA strand breaks at the two higher doses of CdSe QDs tested and all doses of CdCl2 (Fig. 8B).
4. Discussion There is no doubt about the benefits that the use of quantum dots provides to medical and pharmaceutical sciences, which can be described as revolutionary. Nevertheless, since these new materials contain heavy metals, a deep knowledge of their potential harmful effects is urgently required. One of the most used QDs is based on cadmium (CdSe) which is known to be a very toxic and carcinogenic element. This study was designed to understand the biological effects of CdSe QDs related only to its core composition reducing the interference with other factors such as coating and ligand ones. We used D. melanogaster as a reliable in vivo model and the obtained effects were compared with those observed with ionic cadmium form. Our results showed the induction of toxic and genotoxic effects in addition to changes in the levels of oxidative stress and in the expression of different genes involved in stress response. In general, the harmful effects observed after QD exposure were lower than those generated by ionic cadmium.
When egg-to-adult toxicity was taken into account we observed that significant toxic effects of QDs appeared only at higher doses, compared to ionic Cd form that induced significant toxicity at lower ones. This can be attributed to differences in Cd nature whether as a free ion or as bound compound (Yeh et al., 2011). These results would confirm the results obtained in a long-term exposure experiment showing that Cd based QDs significantly reduced Drosophila life span (Galeone et al., 2012). Since the biological effects of any agent depend on its uptake via different biological barriers, we have been able to detect the presence of QDs not only in midgut cells but also in the hemolymph which is the general compartment of any material passing through the intestinal barrier (Alaraby et al., 2014). CdSe QDs have been detected in hemocytes in agreement with previous results reporting increased rate of apoptosis in hemocytes (Galeone et al., 2012). Hemocytes are a suitable model to demonstrate oxidative stress induction (Alaraby et al., 2014) as well as innate immune response and DNA damage in vivo (Irving et al., 2005; Cherry and Silverman, 2006; Carmona et al., 2011). Our results showed that QDs increase ROS production in Drosophila hemocytes although to a lesser extent than after exposure to CdCl2. Changes of intracellular ROS levels are assumed to be due to the semiconducting properties of CdSe as gap and valence/ conduction band energy levels (Ekimov and Onushchenko, 1984). On
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Fig. 3. CdSe-QDs detected in the hemolymph and hemocytes of Drosophila melanogaster using a confocal microscope. Representative fluorescent images of third-instar D. melanogaster larvae hemolymph showed CdSe-QDs distributed inside the hemolymph (A) and (B) and inside the hemocytes (C, D, E and F), scale bar 20 μm.
the other hand, the number of atoms on the QDs' surface increases rapidly with the decrease of the QDs' particle size leading to generation of many dangling bonds that are prone to combine with other atoms to become saturated and stable, hence the surface of QDs has a high chemical reactivity (Zhan and Tang, 2014). Although the toxic effects observed in Drosophila after QD exposure were related to ROS elevation (Galeone et al., 2012) ROS induction by QDs in human cultured cells is strongly related to the nature of the included ligands and the presence of free Cd2+ (Nagy et al., 2012). The proposal that ROS elevation is free Cd2+ dependent (Wang et al., 2009; Xu et al., 2011; Son et al., 2011), completely agrees with our results with CdCl2. To address the molecular response to QD exposure, we have checked potential changes of expression in different types of genes related to general stress, antioxidant protection and DNA damage response. Drosophila succeeded in previous studies to demonstrate changes in gene
expression as response to NPs exposure (Ahamed et al., 2010; Siddique et al., 2014; Alaraby et al., 2014) including QDs (Brunetti et al., 2013). Up-regulation of Hsp genes is important to counteract proteotoxic effects via chaperoning proteins during synthesis, folding, assembly and degradation and give preliminary information on the potential exposure to foreign substances. Chang et al. (2006) postulated that CdSe-core QD suppressed Hsp90 in human cells and, accordingly, we have observed similar results related to expression of Hsp70 and Hsp83 in Drosophila. Antioxidant capacity of cells is the first defense line facing any change in the reactive radical balance. Drosophila showed antioxidant response to the exposure to Cd QDs through significantly expression of SOD mRNA, especially at highest dose. This would agree with those results obtained with CdSe QDs in solution (Ipe et al., 2005), and in the epithelial A549 (human lung carcinoma) and the neuronal SH SY5Y (human neuroblastoma) cell lines (Brunetti et al., 2013).
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Fig. 4. Optical spectra showed maximum emission at 540 nm for CdSe-QDs in dispersion (A) and at 560 nm for up-taken QDs (B).
In addition, our results with CdCl2 showed significant expression of CAT at all doses, while SOD expression significantly inhibited at 5 μM and not effected at the two higher doses. The inhibition effect of cadmium on SOD might be due to protein conformation changes by interacting with the enzyme (Casalino et al., 2002). High doses of cadmium can affect the repair of oxidative DNA damage by down-regulating p53 enzyme levels (Casalino et al., 2002) and, possibly, affecting also SOD activity. Our results with CdCl2 also support those previously reported in neuronal and epidermal cells (Xu et al., 2011; Son et al., 2011) where the levels of ROS and SOD were significantly elevated. The cell guardian p53 plays a key role as a hub in cellular genotoxic stress response by acting as a transcription factor to elicit
cellular functions of DNA damage and repair, cell cycle arrest, and apoptosis. P53 is normally accumulated in the nucleus and converted into an active DNA-binding form to control several sets of genes to prevent the proliferation of DNA damaged cells (Inoue et al., 2005). Over-expression of p53 has already been observed after Drosophila larva exposure to Cd QDs (Brunetti et al., 2013), similar to our study. On the contrary irregular significant expression at the two highest doses of CdCl2 was observed which would agree with the results observed in zebra fish (Tang et al., 2013). In human breast MCF-7 cancer cells Cd2 + exposure results in impaired p53 functions such as conformational changes, loss of DNA binding activity, downregulation of transcriptional activity and inhibition of gamma-
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Concentration (µM) Fig. 5. ROS production in hemocytes of third-instar of untreated (0.0) and treated larvae with different doses (5, 25, 100 and 500 μM) of CdSe-QDs and CdCl2. Hemocytes were incubated with 5 μM DCFH-DA at 24 °C for 30 min and observed using fluorescent microscopy. The fluorescence intensity of hemocytes was quantified by ImageJ analysis. 0.5 mM H2O2 was used as positive control. *P b 0.05, **P b 0.01, ***P b 0.001 compared to control.
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Concentration (µM) Fig. 6. The expression of Hsp70, Hsp83, CAT, SOD and p53 in third-instar larvae induced by exposure to different doses of CdSe-QDs (A) and CdCl2 (B). The expressions were normalized using β-actin and are presented compared to control values. Data represent the mean ± standard error of the mean. *P b 0.05, **P b 0.01 compared to negative control.
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Concentration (µM) Fig. 7. Toxicity of CdSe-QDs and CdCl2 in D. melanogaster. Toxicity effects were measured as loss of viability (egg-to-adult survival) related to the control values. *P b 0.05, **P b 0.0 compared to control.
radiation induced DNA damage responses (Meplan et al., 1999). Thus, the expression of p53 might depend on the amount of free Cd2 + which of course differs between Cd QDs and CdCl2 exposure. Cd QDs continually biodegrade and, as consequence, free Cd2 + is continuously released and their size become smaller during the degradation process (Kwon et al., 2012; Sabella et al., 2014). As consequence the fluorescence emission shifts from red to blue and the excitation fluorescence peak becomes broader (Tsoi et al., 2012) which agrees with our results. Although our results showed that the fluorescence emission slightly shifts from blue to red there is a clear broadness of the fluorescence peak of the up-taken Cd QDs indicating their biodegradation. QDs may be degraded after several days in the living cells (Aillon et al., 2009) and this may occur due to low pH or oxidation of QD surface (Khalil et al., 2011). In our study QDs were administered orally to Drosophila larvae and the transit throughout the gastric tract can contribute to the QDs' degradation process via low pH conditions. Although our results showed that both Cd forms failed to induce somatic mutation and/or recombination they were able to demonstrate
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Fig. 8. Genotoxicity of CdSe-QDs and CdCl2 using the wing-spot (A) and comet (B) assays. In (A) the frequency of total spots induced after the treatment of third-instar larvae with different doses (0.0, 5, 25, 100 and 500 μM) of CdSe-QDs and CdCl2 are indicated. EMS (1 mM) was used as positive control, *P b 0.05. In (B) results indicate the % of DNA tail induced after larval exposure with different doses (0.0, 25, 100 and 500 μM) of CdSe-QDs and CdCl2 for exposures lasting 24 h. EMS (4 mM) was used as positive control. *P b 0.05, **P b 0.01 ***P b 0.001.
the induction of primary DNA damage by means of the comet assay, indicating a potential genotoxic risk associated with Cd QD exposure. This would confirm that the comet assay is more sensitive than the wing-spot assay (Alaraby et al., 2014). In fact, non-genotoxic effects of CdCl2 in Drosophila using the wing-spot test were observed early by our group (Rizki et al., 2004). Indirect genotoxicity of QDs in Drosophila has already been determined by means of the tunnel assay (Galeone et al., 2012) and positive effects of CdCl2 have been reported in midgut cells (Shukla et al., 2011) and in hemocytes of spiders fed with Drosophila treated with CdCl2 (Stalmach et al., 2014). No genotoxic effects of Cd QDs were observed in bacteria but positive induction was detected in CHO cells by means of the comet and micronucleus assays (Aye et al., 2013). In the comet assay both Cd QDs and CdCl2 showed a significant dose-dependent increase in DNA strand breaks regardless of their chemical composition which would indicate that the observed effects are related to the presence of Cd2+ ions. Although a direct relationship between DNA damage and Cd2 + has been observed the underlying mechanism is not exactly clear. Cadmium has been demonstrated to amplify the intensity of damage by interfering with the DNA repair NER pathway (Candéias et al., 2010), or might be related to single strand breaks in DNA through direct cadmium–DNA interactions, as well as by
the action of incision nucleases and/or DNA-glycosylase during DNA repair (Katsumiti et al., 2014). 5. Conclusions The obtained results support D. melanogaster as a suitable in vivo model to determine the potential adverse effects of Cd based QDs. In this in vivo model we have been able to demonstrate that Cd based QDs are toxic, and induce oxidative stress and deregulation of gene expression as well as damage to DNA. The higher effects observed when CdCl2 were used indicate that such effects are related to the presence of Cd2+ ions. The increased use of QDs may affect human health after short- and long-term exposures. Thus, to reduce their side effects, biocompatible coats are required to avoid cadmium's undesirable effects. This would agree with the pilot study of Ye et al. (2012) where Rhesus macaques injected with phospholipid micelle-encapsulated CdSe/CdS/ ZnS quantum dots did not exhibit evidence of toxicity. Conflict of interest statement The authors declare that there is no conflict of interest.
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