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Absence of mutagenic and recombinagenic activity of multi-walled carbon nanotubes in the Drosophila wing-spot test and Allium cepa test
Ecotoxicology and Environmental Safety 99 (2014) 92–97
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Absence of mutagenic and recombinagenic activity of multi-walled carbon nanotubes in the Drosophila wing-spot test and Allium cepa test Laise Rodrigues de Andrade a, Arian Sandin Brito b, Anna Maria Gouvea de Souza Melero b, Hudson Zanin c, Helder José Ceragioli c, Vitor Baranauskas c, Kênya Silva Cunha a,n, Silvia Pierre Irazusta b,d a
Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil Faculdade de Tecnologia de Sorocaba, SP, Brazil c Faculdade de Engenharia Elétrica e Computação/Universidade Estadual de Campinas, SP, Brazil d Programa de Pós-Graduação/Centro Estadual de Educação Tecnológica Paula Souza, SP, Brazil b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 March 2013 Received in revised form 4 October 2013 Accepted 7 October 2013 Available online 1 November 2013
In order to assess the safety of the carbon nanotubes to human health and the environment, we investigated the potential toxicity and ability of multi-walled carbon nanotubes (NT), to induce DNA damage by employing the Allium cepa genotoxicity/mutagenicity test and the Somatic Mutation and Recombination Test (SMART) in the fruitfly, Drosophila melanogaster. The results demonstrated that NT did not significantly induce genotoxic or mutagenic effects in the Allium cepa test. All concentrations evaluated in the SMART assay showed survival rates higher than 90 percent, indicating the absence of chronic toxicity for NT. Furthermore, the various treatments showed no significant increase in the NT mutation and recombination frequencies in mwh/flr3 genotype compared to respective negative controls, demonstrating the absence of DNA damage caused by NT. & 2013 Elsevier Inc. All rights reserved.
Keywords: Nanoparticles Recombination Mutation Genotoxicity
1. Introduction Carbon nanotubes (CNTs) are cylinders constituted by one or several graphene layers with nanometer diameters (Iijima, 1991) and they are one of the most promising materials in nanotechnology. They exhibit remarkable physicochemical properties (i.e. high tensile strength, ultra-light weight, thermal and chemical stability, excellent semi-conductive electronic properties) that are useful in many industrial applications such as electronics, aerospace, construction, pharmaceutical and biomedical sciences (Asakura et al., 2010). Nevertheless, procedures for handling CNTs can result in aerosol release of these materials into the surroundings (Maynard et al., 2004), contaminating the air, groundwater, and soil (Helland et al., 2007; Di Sotto et al., 2009). Other issues related to environmental exposure should be considered: (i) the physical and chemical processes in the environmental compartments may alter the properties of CNTs, and consequently also influence their environmental fate and impact (Helland et al., 2007); (ii) CNTs may accumulate along the food chain becoming bioavailable to organisms, as they are possibly one of the least biodegradable man-made materials, they are lipophilic by nature and are totally
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insoluble in water in pristine form Wu et al. (2006); (iii) the CNTs relatively large surface area may also cause them to adhere to other molecules, potentially allowing them to pick up pollutants and transport them through the environment (Kleiner and Hogan, 2003). Humans, in turn, are more likely to be exposed occupationally or via consumer products and the environment. Recent research suggests that CNTs may pose risks to human health (Kisin et al., 2007; Karlsson et al., 2008; Patlolla et al., 2010). The CNTs structural characteristics, extreme aspect ratio, low specific density and low solubility make them similar to asbestos fiber, and consequently raising similar health concerns (Maynard et al., 2004). One of the most discussed mechanisms behind the health effects induced by nanoparticles (NPs) is their ability to cause oxidative stress (Li et al., 2003; Kato et al., 2013). This mechanism is believed to be important for the genotoxicity of manufactured NPs, and subsequent carcinogenic processes (Xia et al., 2006; Karlsson et al., 2008). However, little data is available in the scientific literature regarding the genotoxic, mutagenic and/or carcinogenic potential of different engineered nanoparticles (Landsiedel et al., 2009). This study aims to provide a reliable base of information on the biosafety of carbon nanotubes. Multi-walled carbon nanotubes (henceforth denoted NT) were evaluated for their potential toxicity and ability to induce DNA damage employing two in vivo tests: (i) Allium cepa test of genotoxicity/mutagenicity and (ii)
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Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. The A. cepa test assesses chromosome damage and disturbances in the mitotic cycle, facilitated by characteristically large chromosomes in a reduced number (2n ¼16; Fiskesjö, 1985), and it has been employed to study the genotoxic/mutagenic potential of a variety of NPs (Kumari et al., 2009; Ghosh et al., 2010; Klančnik et al., 2011; Panda et al., 2011; Ghodake et al., 2011). Another advantage of A. cepa test is its ability to expose the test organism directly to complex mixtures without previous treatment of the test sample (Rank and Nielsen, 1993; Rank, 2003). SMART is an in vivo sensitive assay based on the loss of heterozygosity (LOH) of two genetic markers involved in the metabolic pathways of wing hairs—multiple wing hairs (mwh) and flare3 (flr3). Genetic alterations induced in mitotically dividing cells of a developing wing disc, may give rise to clone(s) of mwh and/or flr3 cells which are visible on the wing surface of the adult fly (Graf et al., 1984; Andrade et al., 2004). The development of SMART has provided a sensitive, rapid and cheap assay, able to detect a wide variety of genetic alterations including somatic recombination, one of the main factors related to carcinogenesis.
The meristematic cells of onion roots were collected after a period of germination of 72 h in distilled water. Slides were prepared using cells incubated with stained NT. After 24 h of incubation the glass slides were fixed with four percent formaldehyde and washed with PBS. Nuclear material was stained using DAPI (1:100) during slide mounting. The slides were examined by confocal microscopy, using a Zeiss LSM 510 Meta microscope.
2. Materials and methods
2.4.1. Treatment procedure and statistical analysis Eggs from both ST cross progenies were collected for 8 h in culture bottles containing a solid agar base (five percent w/v agar in water) covered with a 5 mm layer of live baker's yeast supplemented with sucrose. After 727 4 h, third-instar larvae were collected by flotation in water. One hundred larvae were placed in bottles containing 0.9 g of mashed potatoes medium rehydrated with 3 mL of different concentration NT suspensions: 64, 160, 400 and 1000 mg/mL. These suspensions were prepared by dispersion of NT in distilled water using sonication for 15 min. Negative control (distilled water) and positive control (doxorubicin DXR 0.2 mM) were included in the experiments. DXR was obtained from Glenmark Farmaceutic Ltd. The nanotubes were tested in two independent experiments, performed at 25 1C and 65 percent relative humidity. The larvae were fed on this medium for the rest of their development (48 h). After the pupal stage, all surviving flies were collected from the treatment vials and stored in 70 percent ethanol. Subsequently, the wings of mwh/flr3 flies were removed and mounted on slides. Both the dorsal and ventral surfaces of the wings were analyzed at 400 magnification for the occurrence of mutant clones. The frequencies of spots per fly in the treated series and negative controls were compared for evaluation of genotoxic effects. Statistical significance was ascertained following a multiple decision procedure according to Frei and Würgler (1988; 1995). Hypothesis testing was done using a conditional binomial test according to Kastenbaum and Bowman (1970).
2.1. Characterization of NT Nanotubes were synthesized in the Laboratory of Nanoengineering and Diamond (NanoEng), Department of Semiconductors, Instruments and Photonics Faculty of Electrical and Computer Engineering at State University of Campinas (UNICAMP). The NT were synthesized using the method of chemical deposition from the vapor phase assisted hot-filament (HFCVD), grown on copper substrate, that had been covered with a film of polyaniline and catalyzed with nickel. The hydrocarbons used as carbon source were camphor and acetone (Grecco et al., 2011). NT were characterized by Raman spectroscopy using a Renishaw Invia microscopy system employing a He–Ne laser for excitation (λ ¼ 633 nm). Energydispersive X-ray spectroscopy (EDS) was used for elemental chemical characterization. Morphological analysis of the sample was made with FESEM (field emission scanning electron microscopy) using a JEOL JSM–6330 F operated at 5 kV and 8 μA and with HRTEM (high resolution transmission electron microscopy) using a JEOL 3010 operated at 300 kV and 120 μA. 2.2. Allium cepa test of genotoxicity/mutagenicity Chromosome aberration (CA) and micronucleus (MN) tests using A. cepa cells were performed according to a modified version of Caritá and Marin-Morales’ protocol (Caritá and Marin-Morales, 2008). One hundred onion seeds were germinated at room temperature (207 5 1C) in Petri dishes. Each dish was covered with filter paper and received 2 mL of NT at 20, 50 and 500 μg/mL in PBS (phosphate buffer saline), followed by sonication for 15 to 20 min. Ultra-pure water was used as a negative control and 1.68 ppm of trifluralin (herbicide) was used as a positive control, according to Fernandes et al. (2007; 2009). When the roots reached 2 cm in length (approximately five days after the beginning of the assay), they were collected and fixed in alcohol–acetic acid (3:1) for 24 h. The fixed tissue samples were subsequently washed in distilled water three times for 5 min. The roots were then hydrolyzed in HCl 1 M at 60 1C for 11 min, followed by three washes of 5 min in distilled water. Excess water was blotted using filter paper and the material was transferred to a dark area in containers with Schiff reagent (Lyon et al., 2002) for approximately 1 h. Slides were prepared using the meristematic and F1 regions and coverslips were mounted using a drop of two percent acetic carmine solution. For the CA analysis, aberrations within different cell divisions phases (metaphase, anaphase and telophase) were evaluated. Chromosomes, or lost fragments and bridges, were classified into just one category in order to evaluate the CA as a single endpoint, following the criteria used by Leme and Marin-Morales (2008). MN analysis was evaluated in the F1 region by counting the number of cells with MN. All categories were analyzed by counting 2500 cells per treatment (500 cells per slide, total of five slides). Statistical analysis was performed using the Student t test with a significance level of α ¼ 0.05.
2.4. Somatic mutation and recombination test (SMART) A standard cross was used, following the methods described in Graf et al. (1984): virgin females (flr3/In(3LR)TM3, rippsep l(3)89Aa bx34e e BdS) were crossed with mwh/mwh males, resulting larvae with two genotypes: (i) mwh þ /þ flr3 – trans-heterozygous for the recessive markers mwh and flr3 and (ii) mwh þ /TM3,BdS – heterozygous for TM3 balancer chromosome. The induction of mutation or recombination in the marker-heterozygous flies (mwh/flr3) produces two categories of mutant clones: (i) single spots, either mwh or flr3, resulting from point or chromosome mutations, as well as mitotic recombination between mwh and flr3 genes, and (ii) twin spots, consisting of both mwh and flr3 subclones, which originate exclusively from mitotic recombination between flr3 and the centromere. In the balancer-heterozygous genotype (mwh/TM3), mwh spots predominantly reflect somatic point mutations and chromosome mutations, since mitotic recombination involving the balancer chromosome and its structurally normal homologue is a lethal event. More detailed information on genetic symbols can be found in Lindsley and Zimm (1992).
3. Results 3.1. Characterization of NT A typical Raman spectrum of the NT particles is shown in Fig. 1 The spectrum can be divided into frequency regions of the first and second order. In the region of the first order there are two intense peaks at 1330 cm 1 and 1591 cm 1, corresponding to the peak disorder sp3 (line D) and the peak of highly oriented graphite sp2 E2g (line G). In the region of the second order, there is an intense peak at 2659 cm 1 corresponding to the second harmonic
2.3. Internalization of NT in the meristematic cells of onion roots The NT labeling was performed with the kit PKH26PCL (Sigma Aldrich, USA) as described by Kateb et al. (2007).
Fig. 1. Typical Raman spectrum of NT particles.
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of the line D (2 D), a small peak around 2918 cm 1, which corresponds to the sum of the frequencies of the lines D and G (D þG), and a small peak around 3218 cm 1, which corresponds to the second harmonic of the line G (2 G). The intensity of peak D is higher when compared to the corresponding G peak intensity, which does not indicate a high degree of order sp2 C–C. Fig. 2 shows a typical energy X-ray dispersive spectroscopy (EDS) analysis of the NT. The NT has an elemental purity of C (98.95 wt%) with smaller contaminating amounts of Ni (0.11 wt%) and Cu (0.94 wt%). Such contaminants are probably due to the NT fabrication process. Fig. 3 shows FESEM and HRTEM typical images of NT samples at different magnifications. Fig. 3A and B show FESEM representative images from entangled fiber-like morphologies. Further magnification by HRTEM shows the multi-wall structure of NT (Fig. 3C and D).
3.2. Allium cepa test of genotoxicity/mutagenicity NT produced chromosome aberration indexes (CAI) of 0.6070.40; 0.8070.37 and 1.070.31 for the concentrations of 20, 50 and 500 μg/ mL, respectively. The micronucleus indexes (MNI) were 0.2070.20; 0.4070.25 and 0.4070.25 for concentrations of 20, 50 and 500 μg/ mL, respectively. The positive control trifluralin herbicide produced a CAI of 1.6070.40 and a MNI of 2.070.45, that were significantly greater than the negative control (distilled water) (CAI¼ 0.270.2 and MNI¼0.470.4). The results demonstrate that NT, at the test concentrations, were not genotoxic or mutagenic in Allium cepa meristematic and F1 cells, as shown in Table 1.
3.3. Internalization of NT cells of Allium cepa root: Analysis by confocal microscopy Confocal microscopy of the meristematic cells from Allium cepa roots showed that NT were captured and internalized (Fig. 4). NT stained with PKH26PCL can be seen colored in red in the cytoplasm of cells whose nuclei were stained with DAPI for better contrast. Table 1 Chromossomic Aberration Index (CAI) and Micronuclei Index (MNI) in Allium cepa seeds. Compound (lg/mL)
Index Chromosomal Aberration (CAI)
NT Neg. Control 20 50 500 Pos. Controla a
Fig. 2. Typical curve of EDS analysis of NT particles.
n
0.20 0.60 0.80 1.00 1.60*
7 0.20 7 0.40 7 0.37 7 0.31 7 0.40
Micronuclei (MNI)
0.40 0.20 0.40 0.40 2.00*
7 0.40 7 0.20 7 0.25 7 0.25 7 0.45
Trifluralin (1.68 ppm). p o 0.05.
Fig. 3. (A) FESEM image of entangled NTs, scale bar of 1 μm; (B) FESEM magnified image of NT at the scale bar of 200 nm; (C) HRTEM image of NTs at the scale bar of 50 nm; (D) HRTEM image of NTs at the scale bar of 10 nm.
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Fig. 4. Confocal microscopy images: In (A) NT stained with PKH26PCL; (B) meristematic cells with nuclei stained with DAPI; (C) meristematic cells with NT in the cytoplasm (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Evaluation of the mutagenic and/or recombinogenic effects of the NT in somatic cells larvae proceeding from standard cross. Compound and genotype (lg/mL) No of flies (N) Spots per fly (n1 of Spots) statistical diagnosisa Small Single Spots (1-2 cells) m ¼2 Large Single Spots ( 4 2 cells) m¼5 Twin Spots m¼ 5 Total Spots m¼ 2 3
NT mwh/flr Neg. control 64 160 400 1000 Pos. controlb
80 80 80 80 80 40
0.51 (41) 0.63 (50) 0.54 (43) 0.69 (55) 0.59 (47) 1.05 (42) i
0.08 (06) 0.11 (09) i 0.10 (08)i 0.09 (07) i 0.10 (08) i 1.63 (65) þ
0.04 (03) 0.03 (02) 0.03 (02)0.03 (02) 0.03 (02) 1.30 (52) þ
0.63 (50) 0.76 (61) 0.66 (53) 0.80 (64) 0.71 (57) 3.98 (159) þ
a Statistical diagnosis according to Frei and Würgler (1988): þ, positive; , negative; i, inconclusive. m, factor of multiplication for evaluation of results significantly negatives. Levels of significance α ¼0.05. b Doxorubicin (0.2 mM).
3.4. Somatic mutation and recombination test (SMART) 3.4.1. Toxic effects The chronic toxicity of the NT was determined quantitatively in a pilot experiment, to establish the concentration range for the genotoxicity analysis. For this purpose, groups of 100 larvae were treated for 48 h with four different concentrations of NT. The concentrations selected to be used in the main experiment were based on two criteria: (i) concentrations were such that the number of survivor adults would be high enough to indicate that the NT do not compromise the development of treated larvae (over 90 percent in all cases), (ii) low enough to represent exposure conditions reported for humans either directly or indirectly via the environment (Ursini et al., 2012).
3.4.2. Genetic toxicity The results obtained from all NT concentrations analyzed showed negative responses, considering the total spots in the mwh/flr3 genotype (Table 2). No significant increase in mutation and recombination frequencies in the treated series was detected when compared to the negative control group. As such, it was not necessary to analyze the mwh/TM3 genotype flies.
4. Discussion The genotoxic, phytotoxic and mutagentic potential of carbon nanotubes was investigated using two tests: the Allium cepa test of genotoxicity/mutagenicity and the SMART. Our results with the
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Allium cepa test indicate that NT have no phytotoxic, genotoxic or mutagenic effects despite their internalization by the root cells. Although NT uptake was observed, it likely did not lead to a reaction with biomolecules and the subsequent induction of cellular stress and cellular damage. Negative results were also observed when the genotoxic effects of TiO2 nanoparticles were analyzed by recording the type and the frequency of chromosome aberrations and the presence of micronuclei in Allium cepa. The frequency of aberrations and the abundance of micronuclei induced by TiO2 nanoparticles were similar in range to those observed in the negative controls, irrespective of exposure duration and NPs concentrations (Klančnik et al., 2011). However, Ghosh et al. (2011) showed an association between the internalization of carbon nanotubes (MWCNT) and chromosomal aberrations, DNA fragmentation and apoptosis in roots cells of Allium cepa, in contrast with the data presented herein. Likewise, different NPs have demonstrated toxicity in the Allium cepa assay. Recently, it was demonstrated that ZnO nanoparticles can be a clastogenic/genotoxic and cytotoxic agent (Kumari et al., 2011). Ag nanoparticles (AgNP-P and AgNP-S) exhibited similar biological effects, causing a lesser extent of cytotoxicity and a greater extent of genotoxicity (Panda et al., 2011). Kumari et al. (2009) also suggested that Ag nanoparticles could penetrate into the plant system and impair stages of cell division causing chromatin bridge, stickiness, disturbed metaphase, multiple chromosomal breaks and cell disintegration. CoO and ZnO nanoparticles were demonstrated to have an effect on root elongation, cell morphology and adsorption potential in a hydroponic culture of A. cepa. It was demonstrated that the phytotoxic nature of these NPs causes damage because of their severe accumulation in both the cellular and the chromosomal modules (Ghodake et al., 2011). Demir et al. (2011) first proposed the SMART assay in D. melanogaster as a useful model for detecting the genotoxic potencial of NPs. The results presented in that study showed that different concentrations of silver nanoparticles (0.1–10 mM) were able to induce genotoxic activity in somatic cells of D. melanogaster mainly via induction of somatic recombination. The negative results obtained here using the SMART assay show that NT are not toxic and did not induce DNA damage in somatic cells of D. melanogaster, confirming previous reports in the literature of low toxicity risk for NT (Leeuw et al., 2007, Liu et al., 2009). Leeuw et al. (2007) demonstrated that the ingestion of single wall carbon nanotube (SWCNT) dispersed in culture medium (9 mg/g) by D. melanogaster larvae leads to systemic uptake and a distinct distribution in various tissues and internal organs without adverse effects on larval development and fertility of adult flies. The authors estimated that only a small fraction (10 8) of ingested nanotubes cross through the gut wall and become incorporated into tissues. Liu et al. (2009) showed, in turn, that the effects of nanotubes in D. melanogaster may depend mainly of the exposure route and material aggregation state. No effect was found on the stages of development of Drosophila larvae fed with different concentrations of SWCNT and MWCNT (100 and 1000 mg/g in food). However, dry exposure of adults to primary particles or small aggregates (o20 mm) led to whole-body coverage, resulting motor function loss and mortality. In this light, the lack of toxicity observed in this study may be a reflection of the exposure route. Although the intake of NT has not been quantified, black dots indicating the presence of the nanotubes in the intestine of the treated larvae could be seen within hours. Over time a noticeable decrease of black dots could be observed (data not shown), suggesting that the nanotubes were ingested, but possibly excreted, resulting in low bioavailability of NT in the larvae tissues. Adverse effects may occur even at low concentrations, without necessarily causing cell death. One of the most important adverse
effects is DNA damage (Karlsson, 2010). Although some CNTs studies have used D. melanogaster as the experimental organism (Leeuw et al., 2007; Liu et al., 2009), they did not address the possible effects NT may have on the genetic material of cells. To our knowledge, this study is the first to evaluate the genotoxic potential of NT in D. melanogaster. An inherent property of many nanomaterials is their hydrophobicity, and their consequent tendency to agglomerate in aqueous medium. Muller et al. (2008) suggested that the absence of doseresponse in experiments to evaluate the genotoxic potential of MWCNT in human epithelial cells (MCF-7) using the micronucleus test may be related to inhomogeneous distribution of the nanotubes agglomerates in the culture medium, which affects the amount of material delivered to target cells. Lindberg et al. (2009) also did not observe a dose-response effect in lung epithelial cells (BEAS-2B) treated with concentrations of 10, 60 and 100 μg/cm2 of a mixture of nanotubes containing 450 percent SWCNT. Mouchet et al. (2008) observed that elevated concentrations of double-walled nanotubes (10, 100 and 500 mg/L) did not induce increase in micronuclei frequency in erythrocytes of Xenopus laevis larvae. According to the authors, large agglomerates can be formed at high concentrations, which are not efficiently taken up by cells. Ursini et al. (2012) observed that large agglomerates of pristine MWCNTs damaged the cellular membrane and only nanotubes with minor dimensions or functionalized MWCNT (more dispersed and water soluble) are able to cross the undamaged membrane and interact with nuclear DNA. In mice MWCNTs were shown to induce genotoxic effects both in in vitro and in vivo tests, probably by mechanisms involving oxidative stress and inflammatory responses (Kato et al., 2013). Considering the evidence in the literature, we can raise the following hypotheses to explain the negative results obtained in the Allium cepa assay and SMART test: (i) under physiological conditions, NT form large agglomerates that are less efficiently internalized by cells or are not internalized at all, (ii) if internalized, large agglomerates are less reactive, as the percentage of atoms in the surface area is reduced inversely to increasing agglomerate size, (iii) NT may cause small changes in DNA that were not detected after cell divisions (i.e. the damages were repaired) and thus cannot be detected by SMART, or (iv) NT does not induce genetic changes, such as mutation and/or recombination due to its physicochemical properties. Finally, the purity of the nanomaterial being analyzed must be taken under consideration, as residual contaminating metals (such as Co, Fe, Ni and Mo) may be responsible for the genotoxicity responses observed rather than the actual nanomaterial itself, and its quantity may be dependent upon the synthesis procedure employed (Stern and McNeil, 2008; Singh et al., 2009). It is likely that carbon nanotubes with high purity such as the ones used here, may promote lesser generation of ROS (Kagan et al., 2006; Murray et al., 2009), and as such reduce the risk of DNA damage, which also can explain the absence of genotoxicity in this in vivo study.
5. Conclusion The results demonstrated that the new carbon nanotubes synthesized in the Laboratory of Nanoengineering and Diamond (UNICAMP) are not able to cause significant genotoxic effects in the Allium cepa test and SMART assay. Further investigation should be performed to determine their safety for humans and the environment.
Acknowledgments Trifluralin was kindly donated by Dr. M.A. Marin-Moralles, State University of São Paulo (UNESP-Rio Claro/Brazil). Laise Rodrigues
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Absence of mutagenic and recombinagenic activity of multi-walled carbon nanotubes in the Drosophila wing-spot test and Allium cepa test Laise Rodrigues de Andrade a, Arian Sandin Brito b, Anna Maria Gouvea de Souza Melero b, Hudson Zanin c, Helder José Ceragioli c, Vitor Baranauskas c, Kênya Silva Cunha a,n, Silvia Pierre Irazusta b,d a
Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil Faculdade de Tecnologia de Sorocaba, SP, Brazil c Faculdade de Engenharia Elétrica e Computação/Universidade Estadual de Campinas, SP, Brazil d Programa de Pós-Graduação/Centro Estadual de Educação Tecnológica Paula Souza, SP, Brazil b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 March 2013 Received in revised form 4 October 2013 Accepted 7 October 2013 Available online 1 November 2013
In order to assess the safety of the carbon nanotubes to human health and the environment, we investigated the potential toxicity and ability of multi-walled carbon nanotubes (NT), to induce DNA damage by employing the Allium cepa genotoxicity/mutagenicity test and the Somatic Mutation and Recombination Test (SMART) in the fruitfly, Drosophila melanogaster. The results demonstrated that NT did not significantly induce genotoxic or mutagenic effects in the Allium cepa test. All concentrations evaluated in the SMART assay showed survival rates higher than 90 percent, indicating the absence of chronic toxicity for NT. Furthermore, the various treatments showed no significant increase in the NT mutation and recombination frequencies in mwh/flr3 genotype compared to respective negative controls, demonstrating the absence of DNA damage caused by NT. & 2013 Elsevier Inc. All rights reserved.
Keywords: Nanoparticles Recombination Mutation Genotoxicity
1. Introduction Carbon nanotubes (CNTs) are cylinders constituted by one or several graphene layers with nanometer diameters (Iijima, 1991) and they are one of the most promising materials in nanotechnology. They exhibit remarkable physicochemical properties (i.e. high tensile strength, ultra-light weight, thermal and chemical stability, excellent semi-conductive electronic properties) that are useful in many industrial applications such as electronics, aerospace, construction, pharmaceutical and biomedical sciences (Asakura et al., 2010). Nevertheless, procedures for handling CNTs can result in aerosol release of these materials into the surroundings (Maynard et al., 2004), contaminating the air, groundwater, and soil (Helland et al., 2007; Di Sotto et al., 2009). Other issues related to environmental exposure should be considered: (i) the physical and chemical processes in the environmental compartments may alter the properties of CNTs, and consequently also influence their environmental fate and impact (Helland et al., 2007); (ii) CNTs may accumulate along the food chain becoming bioavailable to organisms, as they are possibly one of the least biodegradable man-made materials, they are lipophilic by nature and are totally
n
Corresponding author. Fax: þ55 6235211190. E-mail address: [email protected] (K. Silva Cunha).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.013
insoluble in water in pristine form Wu et al. (2006); (iii) the CNTs relatively large surface area may also cause them to adhere to other molecules, potentially allowing them to pick up pollutants and transport them through the environment (Kleiner and Hogan, 2003). Humans, in turn, are more likely to be exposed occupationally or via consumer products and the environment. Recent research suggests that CNTs may pose risks to human health (Kisin et al., 2007; Karlsson et al., 2008; Patlolla et al., 2010). The CNTs structural characteristics, extreme aspect ratio, low specific density and low solubility make them similar to asbestos fiber, and consequently raising similar health concerns (Maynard et al., 2004). One of the most discussed mechanisms behind the health effects induced by nanoparticles (NPs) is their ability to cause oxidative stress (Li et al., 2003; Kato et al., 2013). This mechanism is believed to be important for the genotoxicity of manufactured NPs, and subsequent carcinogenic processes (Xia et al., 2006; Karlsson et al., 2008). However, little data is available in the scientific literature regarding the genotoxic, mutagenic and/or carcinogenic potential of different engineered nanoparticles (Landsiedel et al., 2009). This study aims to provide a reliable base of information on the biosafety of carbon nanotubes. Multi-walled carbon nanotubes (henceforth denoted NT) were evaluated for their potential toxicity and ability to induce DNA damage employing two in vivo tests: (i) Allium cepa test of genotoxicity/mutagenicity and (ii)
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Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. The A. cepa test assesses chromosome damage and disturbances in the mitotic cycle, facilitated by characteristically large chromosomes in a reduced number (2n ¼16; Fiskesjö, 1985), and it has been employed to study the genotoxic/mutagenic potential of a variety of NPs (Kumari et al., 2009; Ghosh et al., 2010; Klančnik et al., 2011; Panda et al., 2011; Ghodake et al., 2011). Another advantage of A. cepa test is its ability to expose the test organism directly to complex mixtures without previous treatment of the test sample (Rank and Nielsen, 1993; Rank, 2003). SMART is an in vivo sensitive assay based on the loss of heterozygosity (LOH) of two genetic markers involved in the metabolic pathways of wing hairs—multiple wing hairs (mwh) and flare3 (flr3). Genetic alterations induced in mitotically dividing cells of a developing wing disc, may give rise to clone(s) of mwh and/or flr3 cells which are visible on the wing surface of the adult fly (Graf et al., 1984; Andrade et al., 2004). The development of SMART has provided a sensitive, rapid and cheap assay, able to detect a wide variety of genetic alterations including somatic recombination, one of the main factors related to carcinogenesis.
The meristematic cells of onion roots were collected after a period of germination of 72 h in distilled water. Slides were prepared using cells incubated with stained NT. After 24 h of incubation the glass slides were fixed with four percent formaldehyde and washed with PBS. Nuclear material was stained using DAPI (1:100) during slide mounting. The slides were examined by confocal microscopy, using a Zeiss LSM 510 Meta microscope.
2. Materials and methods
2.4.1. Treatment procedure and statistical analysis Eggs from both ST cross progenies were collected for 8 h in culture bottles containing a solid agar base (five percent w/v agar in water) covered with a 5 mm layer of live baker's yeast supplemented with sucrose. After 727 4 h, third-instar larvae were collected by flotation in water. One hundred larvae were placed in bottles containing 0.9 g of mashed potatoes medium rehydrated with 3 mL of different concentration NT suspensions: 64, 160, 400 and 1000 mg/mL. These suspensions were prepared by dispersion of NT in distilled water using sonication for 15 min. Negative control (distilled water) and positive control (doxorubicin DXR 0.2 mM) were included in the experiments. DXR was obtained from Glenmark Farmaceutic Ltd. The nanotubes were tested in two independent experiments, performed at 25 1C and 65 percent relative humidity. The larvae were fed on this medium for the rest of their development (48 h). After the pupal stage, all surviving flies were collected from the treatment vials and stored in 70 percent ethanol. Subsequently, the wings of mwh/flr3 flies were removed and mounted on slides. Both the dorsal and ventral surfaces of the wings were analyzed at 400 magnification for the occurrence of mutant clones. The frequencies of spots per fly in the treated series and negative controls were compared for evaluation of genotoxic effects. Statistical significance was ascertained following a multiple decision procedure according to Frei and Würgler (1988; 1995). Hypothesis testing was done using a conditional binomial test according to Kastenbaum and Bowman (1970).
2.1. Characterization of NT Nanotubes were synthesized in the Laboratory of Nanoengineering and Diamond (NanoEng), Department of Semiconductors, Instruments and Photonics Faculty of Electrical and Computer Engineering at State University of Campinas (UNICAMP). The NT were synthesized using the method of chemical deposition from the vapor phase assisted hot-filament (HFCVD), grown on copper substrate, that had been covered with a film of polyaniline and catalyzed with nickel. The hydrocarbons used as carbon source were camphor and acetone (Grecco et al., 2011). NT were characterized by Raman spectroscopy using a Renishaw Invia microscopy system employing a He–Ne laser for excitation (λ ¼ 633 nm). Energydispersive X-ray spectroscopy (EDS) was used for elemental chemical characterization. Morphological analysis of the sample was made with FESEM (field emission scanning electron microscopy) using a JEOL JSM–6330 F operated at 5 kV and 8 μA and with HRTEM (high resolution transmission electron microscopy) using a JEOL 3010 operated at 300 kV and 120 μA. 2.2. Allium cepa test of genotoxicity/mutagenicity Chromosome aberration (CA) and micronucleus (MN) tests using A. cepa cells were performed according to a modified version of Caritá and Marin-Morales’ protocol (Caritá and Marin-Morales, 2008). One hundred onion seeds were germinated at room temperature (207 5 1C) in Petri dishes. Each dish was covered with filter paper and received 2 mL of NT at 20, 50 and 500 μg/mL in PBS (phosphate buffer saline), followed by sonication for 15 to 20 min. Ultra-pure water was used as a negative control and 1.68 ppm of trifluralin (herbicide) was used as a positive control, according to Fernandes et al. (2007; 2009). When the roots reached 2 cm in length (approximately five days after the beginning of the assay), they were collected and fixed in alcohol–acetic acid (3:1) for 24 h. The fixed tissue samples were subsequently washed in distilled water three times for 5 min. The roots were then hydrolyzed in HCl 1 M at 60 1C for 11 min, followed by three washes of 5 min in distilled water. Excess water was blotted using filter paper and the material was transferred to a dark area in containers with Schiff reagent (Lyon et al., 2002) for approximately 1 h. Slides were prepared using the meristematic and F1 regions and coverslips were mounted using a drop of two percent acetic carmine solution. For the CA analysis, aberrations within different cell divisions phases (metaphase, anaphase and telophase) were evaluated. Chromosomes, or lost fragments and bridges, were classified into just one category in order to evaluate the CA as a single endpoint, following the criteria used by Leme and Marin-Morales (2008). MN analysis was evaluated in the F1 region by counting the number of cells with MN. All categories were analyzed by counting 2500 cells per treatment (500 cells per slide, total of five slides). Statistical analysis was performed using the Student t test with a significance level of α ¼ 0.05.
2.4. Somatic mutation and recombination test (SMART) A standard cross was used, following the methods described in Graf et al. (1984): virgin females (flr3/In(3LR)TM3, rippsep l(3)89Aa bx34e e BdS) were crossed with mwh/mwh males, resulting larvae with two genotypes: (i) mwh þ /þ flr3 – trans-heterozygous for the recessive markers mwh and flr3 and (ii) mwh þ /TM3,BdS – heterozygous for TM3 balancer chromosome. The induction of mutation or recombination in the marker-heterozygous flies (mwh/flr3) produces two categories of mutant clones: (i) single spots, either mwh or flr3, resulting from point or chromosome mutations, as well as mitotic recombination between mwh and flr3 genes, and (ii) twin spots, consisting of both mwh and flr3 subclones, which originate exclusively from mitotic recombination between flr3 and the centromere. In the balancer-heterozygous genotype (mwh/TM3), mwh spots predominantly reflect somatic point mutations and chromosome mutations, since mitotic recombination involving the balancer chromosome and its structurally normal homologue is a lethal event. More detailed information on genetic symbols can be found in Lindsley and Zimm (1992).
3. Results 3.1. Characterization of NT A typical Raman spectrum of the NT particles is shown in Fig. 1 The spectrum can be divided into frequency regions of the first and second order. In the region of the first order there are two intense peaks at 1330 cm 1 and 1591 cm 1, corresponding to the peak disorder sp3 (line D) and the peak of highly oriented graphite sp2 E2g (line G). In the region of the second order, there is an intense peak at 2659 cm 1 corresponding to the second harmonic
2.3. Internalization of NT in the meristematic cells of onion roots The NT labeling was performed with the kit PKH26PCL (Sigma Aldrich, USA) as described by Kateb et al. (2007).
Fig. 1. Typical Raman spectrum of NT particles.
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of the line D (2 D), a small peak around 2918 cm 1, which corresponds to the sum of the frequencies of the lines D and G (D þG), and a small peak around 3218 cm 1, which corresponds to the second harmonic of the line G (2 G). The intensity of peak D is higher when compared to the corresponding G peak intensity, which does not indicate a high degree of order sp2 C–C. Fig. 2 shows a typical energy X-ray dispersive spectroscopy (EDS) analysis of the NT. The NT has an elemental purity of C (98.95 wt%) with smaller contaminating amounts of Ni (0.11 wt%) and Cu (0.94 wt%). Such contaminants are probably due to the NT fabrication process. Fig. 3 shows FESEM and HRTEM typical images of NT samples at different magnifications. Fig. 3A and B show FESEM representative images from entangled fiber-like morphologies. Further magnification by HRTEM shows the multi-wall structure of NT (Fig. 3C and D).
3.2. Allium cepa test of genotoxicity/mutagenicity NT produced chromosome aberration indexes (CAI) of 0.6070.40; 0.8070.37 and 1.070.31 for the concentrations of 20, 50 and 500 μg/ mL, respectively. The micronucleus indexes (MNI) were 0.2070.20; 0.4070.25 and 0.4070.25 for concentrations of 20, 50 and 500 μg/ mL, respectively. The positive control trifluralin herbicide produced a CAI of 1.6070.40 and a MNI of 2.070.45, that were significantly greater than the negative control (distilled water) (CAI¼ 0.270.2 and MNI¼0.470.4). The results demonstrate that NT, at the test concentrations, were not genotoxic or mutagenic in Allium cepa meristematic and F1 cells, as shown in Table 1.
3.3. Internalization of NT cells of Allium cepa root: Analysis by confocal microscopy Confocal microscopy of the meristematic cells from Allium cepa roots showed that NT were captured and internalized (Fig. 4). NT stained with PKH26PCL can be seen colored in red in the cytoplasm of cells whose nuclei were stained with DAPI for better contrast. Table 1 Chromossomic Aberration Index (CAI) and Micronuclei Index (MNI) in Allium cepa seeds. Compound (lg/mL)
Index Chromosomal Aberration (CAI)
NT Neg. Control 20 50 500 Pos. Controla a
Fig. 2. Typical curve of EDS analysis of NT particles.
n
0.20 0.60 0.80 1.00 1.60*
7 0.20 7 0.40 7 0.37 7 0.31 7 0.40
Micronuclei (MNI)
0.40 0.20 0.40 0.40 2.00*
7 0.40 7 0.20 7 0.25 7 0.25 7 0.45
Trifluralin (1.68 ppm). p o 0.05.
Fig. 3. (A) FESEM image of entangled NTs, scale bar of 1 μm; (B) FESEM magnified image of NT at the scale bar of 200 nm; (C) HRTEM image of NTs at the scale bar of 50 nm; (D) HRTEM image of NTs at the scale bar of 10 nm.
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Fig. 4. Confocal microscopy images: In (A) NT stained with PKH26PCL; (B) meristematic cells with nuclei stained with DAPI; (C) meristematic cells with NT in the cytoplasm (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Evaluation of the mutagenic and/or recombinogenic effects of the NT in somatic cells larvae proceeding from standard cross. Compound and genotype (lg/mL) No of flies (N) Spots per fly (n1 of Spots) statistical diagnosisa Small Single Spots (1-2 cells) m ¼2 Large Single Spots ( 4 2 cells) m¼5 Twin Spots m¼ 5 Total Spots m¼ 2 3
NT mwh/flr Neg. control 64 160 400 1000 Pos. controlb
80 80 80 80 80 40
0.51 (41) 0.63 (50) 0.54 (43) 0.69 (55) 0.59 (47) 1.05 (42) i
0.08 (06) 0.11 (09) i 0.10 (08)i 0.09 (07) i 0.10 (08) i 1.63 (65) þ
0.04 (03) 0.03 (02) 0.03 (02)0.03 (02) 0.03 (02) 1.30 (52) þ
0.63 (50) 0.76 (61) 0.66 (53) 0.80 (64) 0.71 (57) 3.98 (159) þ
a Statistical diagnosis according to Frei and Würgler (1988): þ, positive; , negative; i, inconclusive. m, factor of multiplication for evaluation of results significantly negatives. Levels of significance α ¼0.05. b Doxorubicin (0.2 mM).
3.4. Somatic mutation and recombination test (SMART) 3.4.1. Toxic effects The chronic toxicity of the NT was determined quantitatively in a pilot experiment, to establish the concentration range for the genotoxicity analysis. For this purpose, groups of 100 larvae were treated for 48 h with four different concentrations of NT. The concentrations selected to be used in the main experiment were based on two criteria: (i) concentrations were such that the number of survivor adults would be high enough to indicate that the NT do not compromise the development of treated larvae (over 90 percent in all cases), (ii) low enough to represent exposure conditions reported for humans either directly or indirectly via the environment (Ursini et al., 2012).
3.4.2. Genetic toxicity The results obtained from all NT concentrations analyzed showed negative responses, considering the total spots in the mwh/flr3 genotype (Table 2). No significant increase in mutation and recombination frequencies in the treated series was detected when compared to the negative control group. As such, it was not necessary to analyze the mwh/TM3 genotype flies.
4. Discussion The genotoxic, phytotoxic and mutagentic potential of carbon nanotubes was investigated using two tests: the Allium cepa test of genotoxicity/mutagenicity and the SMART. Our results with the
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Allium cepa test indicate that NT have no phytotoxic, genotoxic or mutagenic effects despite their internalization by the root cells. Although NT uptake was observed, it likely did not lead to a reaction with biomolecules and the subsequent induction of cellular stress and cellular damage. Negative results were also observed when the genotoxic effects of TiO2 nanoparticles were analyzed by recording the type and the frequency of chromosome aberrations and the presence of micronuclei in Allium cepa. The frequency of aberrations and the abundance of micronuclei induced by TiO2 nanoparticles were similar in range to those observed in the negative controls, irrespective of exposure duration and NPs concentrations (Klančnik et al., 2011). However, Ghosh et al. (2011) showed an association between the internalization of carbon nanotubes (MWCNT) and chromosomal aberrations, DNA fragmentation and apoptosis in roots cells of Allium cepa, in contrast with the data presented herein. Likewise, different NPs have demonstrated toxicity in the Allium cepa assay. Recently, it was demonstrated that ZnO nanoparticles can be a clastogenic/genotoxic and cytotoxic agent (Kumari et al., 2011). Ag nanoparticles (AgNP-P and AgNP-S) exhibited similar biological effects, causing a lesser extent of cytotoxicity and a greater extent of genotoxicity (Panda et al., 2011). Kumari et al. (2009) also suggested that Ag nanoparticles could penetrate into the plant system and impair stages of cell division causing chromatin bridge, stickiness, disturbed metaphase, multiple chromosomal breaks and cell disintegration. CoO and ZnO nanoparticles were demonstrated to have an effect on root elongation, cell morphology and adsorption potential in a hydroponic culture of A. cepa. It was demonstrated that the phytotoxic nature of these NPs causes damage because of their severe accumulation in both the cellular and the chromosomal modules (Ghodake et al., 2011). Demir et al. (2011) first proposed the SMART assay in D. melanogaster as a useful model for detecting the genotoxic potencial of NPs. The results presented in that study showed that different concentrations of silver nanoparticles (0.1–10 mM) were able to induce genotoxic activity in somatic cells of D. melanogaster mainly via induction of somatic recombination. The negative results obtained here using the SMART assay show that NT are not toxic and did not induce DNA damage in somatic cells of D. melanogaster, confirming previous reports in the literature of low toxicity risk for NT (Leeuw et al., 2007, Liu et al., 2009). Leeuw et al. (2007) demonstrated that the ingestion of single wall carbon nanotube (SWCNT) dispersed in culture medium (9 mg/g) by D. melanogaster larvae leads to systemic uptake and a distinct distribution in various tissues and internal organs without adverse effects on larval development and fertility of adult flies. The authors estimated that only a small fraction (10 8) of ingested nanotubes cross through the gut wall and become incorporated into tissues. Liu et al. (2009) showed, in turn, that the effects of nanotubes in D. melanogaster may depend mainly of the exposure route and material aggregation state. No effect was found on the stages of development of Drosophila larvae fed with different concentrations of SWCNT and MWCNT (100 and 1000 mg/g in food). However, dry exposure of adults to primary particles or small aggregates (o20 mm) led to whole-body coverage, resulting motor function loss and mortality. In this light, the lack of toxicity observed in this study may be a reflection of the exposure route. Although the intake of NT has not been quantified, black dots indicating the presence of the nanotubes in the intestine of the treated larvae could be seen within hours. Over time a noticeable decrease of black dots could be observed (data not shown), suggesting that the nanotubes were ingested, but possibly excreted, resulting in low bioavailability of NT in the larvae tissues. Adverse effects may occur even at low concentrations, without necessarily causing cell death. One of the most important adverse
effects is DNA damage (Karlsson, 2010). Although some CNTs studies have used D. melanogaster as the experimental organism (Leeuw et al., 2007; Liu et al., 2009), they did not address the possible effects NT may have on the genetic material of cells. To our knowledge, this study is the first to evaluate the genotoxic potential of NT in D. melanogaster. An inherent property of many nanomaterials is their hydrophobicity, and their consequent tendency to agglomerate in aqueous medium. Muller et al. (2008) suggested that the absence of doseresponse in experiments to evaluate the genotoxic potential of MWCNT in human epithelial cells (MCF-7) using the micronucleus test may be related to inhomogeneous distribution of the nanotubes agglomerates in the culture medium, which affects the amount of material delivered to target cells. Lindberg et al. (2009) also did not observe a dose-response effect in lung epithelial cells (BEAS-2B) treated with concentrations of 10, 60 and 100 μg/cm2 of a mixture of nanotubes containing 450 percent SWCNT. Mouchet et al. (2008) observed that elevated concentrations of double-walled nanotubes (10, 100 and 500 mg/L) did not induce increase in micronuclei frequency in erythrocytes of Xenopus laevis larvae. According to the authors, large agglomerates can be formed at high concentrations, which are not efficiently taken up by cells. Ursini et al. (2012) observed that large agglomerates of pristine MWCNTs damaged the cellular membrane and only nanotubes with minor dimensions or functionalized MWCNT (more dispersed and water soluble) are able to cross the undamaged membrane and interact with nuclear DNA. In mice MWCNTs were shown to induce genotoxic effects both in in vitro and in vivo tests, probably by mechanisms involving oxidative stress and inflammatory responses (Kato et al., 2013). Considering the evidence in the literature, we can raise the following hypotheses to explain the negative results obtained in the Allium cepa assay and SMART test: (i) under physiological conditions, NT form large agglomerates that are less efficiently internalized by cells or are not internalized at all, (ii) if internalized, large agglomerates are less reactive, as the percentage of atoms in the surface area is reduced inversely to increasing agglomerate size, (iii) NT may cause small changes in DNA that were not detected after cell divisions (i.e. the damages were repaired) and thus cannot be detected by SMART, or (iv) NT does not induce genetic changes, such as mutation and/or recombination due to its physicochemical properties. Finally, the purity of the nanomaterial being analyzed must be taken under consideration, as residual contaminating metals (such as Co, Fe, Ni and Mo) may be responsible for the genotoxicity responses observed rather than the actual nanomaterial itself, and its quantity may be dependent upon the synthesis procedure employed (Stern and McNeil, 2008; Singh et al., 2009). It is likely that carbon nanotubes with high purity such as the ones used here, may promote lesser generation of ROS (Kagan et al., 2006; Murray et al., 2009), and as such reduce the risk of DNA damage, which also can explain the absence of genotoxicity in this in vivo study.
5. Conclusion The results demonstrated that the new carbon nanotubes synthesized in the Laboratory of Nanoengineering and Diamond (UNICAMP) are not able to cause significant genotoxic effects in the Allium cepa test and SMART assay. Further investigation should be performed to determine their safety for humans and the environment.
Acknowledgments Trifluralin was kindly donated by Dr. M.A. Marin-Moralles, State University of São Paulo (UNESP-Rio Claro/Brazil). Laise Rodrigues
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