- Email: [email protected]
Chronic exposure of zinc oxide nanoparticles causes deviant phenotype in Drosophila melanogaster
Journal of Hazardous Materials 327 (2017) 180–186
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Chronic exposure of zinc oxide nanoparticles causes deviant phenotype in Drosophila melanogaster Avnika Singh Anand, Dipti N. Prasad, Shashi Bala Singh, Ekta Kohli ∗ Neurobiology Division, Defence Institute of Physiology and Allied Sciences, Delhi, 110054, India
g r a p h i c a l
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 19 September 2016 Received in revised form 20 December 2016 Accepted 22 December 2016 Available online 23 December 2016 Keywords: Genotoxicity Zinc oxide nanoparticles Drosophila melanogaster Risk assessment Nanotoxicology
a b s t r a c t Zinc oxide nanoparticles (ZnO NPs) are commonly used nanomaterials (NMs) with versatile applications from high-end technologies to household products. This pervasive utilisation has brought human in the close interface with nanoparticles (NPs), hence questioning their safety prior to usage is a must. In this study, we have assessed the effects of chronic exposure to ZnO NPs (<50 nm) on the model organism Drosophila melanogaster. Potential toxic effects were studied by evaluating longevity, climbing ability, oxidative stress and DNA fragmentation. Ensuing exposure, the F0 (parent), F1, F2, F3 and F4 generation flies were screened for the aberrant phenotype. Flies exposed to ZnO NPs showed distinctive phenotypic changes, like deformed segmented thorax and single or deformed wing, which were transmitted to the offspring’s in subsequent generations. The unique abnormal phenotype is evident of chronic toxicity induced by ZnO NPs, although appalling, it strongly emphasize the importance to understand NPs toxicity for safer use. © 2016 Elsevier B.V. All rights reserved.
Abbreviations: ROS, reactive oxidative species; ZnO, zinc oxide; AgNPs, silver nanoparticles; AuNPs, gold nanoparticles; NPs, nanoparticles; NMs, nanomaterials; TEM, transmission electron microscopy; DCF-DA, 2’,7’–dichlorofluorescin diacetate; DAPI, 4’,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; ANOVA, analysis of variance. ∗ Corresponding author at: Neurobiology Division, Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, Ministry of Defence, Lucknow Road, Timarpur, Delhi, India. E-mail addresses: [email protected], [email protected] (E. Kohli). http://dx.doi.org/10.1016/j.jhazmat.2016.12.040 0304-3894/© 2016 Elsevier B.V. All rights reserved.
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
181
1. Introduction
2.2. Characterisation of ZnO NPs
The ubiquitous utilisation of nanomaterials (NMs) has created lots of concerns for the living world, especially to human beings. At the nano level matter is governed by quantum mechanics and might possess distinct novel chemical and physical properties different from the molecular counterpart [1,2]. The nano size facilitates these particles to cross any biological barrier and intermingle with the different bio molecules. This nano-bio interference can result in detrimental biological effects like Reactive Oxidative Species (ROS) generation, cytotoxicity, genotoxicity and inflammatory responses [3–7]. The most detrimental is obliteration of the genetic material and erroneous genetic information transmitted to future generations. Zinc oxide nanoparticles (ZnO NPs) are widely used in cosmetics, sunscreens, UV radiation blockers, gas sensors, paints, ceramics, medical supplies and food additives [8–10] and thus come in direct contact with a large section of the population. Given the above, it is important to critically evaluate toxicity induced by these particles. In various in vivo and in vitro studies, ZnO NPs have been reported to be toxic; it has been shown to induce morphological modifications, oxidative stress, lipid peroxidation, mitochondrial dysfunction, cytotoxicity, chromosomal breakage and micronuclei formation [11–17]. ZnO NPs exposure also adversely affects the prokaryotic system as these particles were found to be mutagenic in bacterial assays [18]. ROS generation is a prime cause of ZnO NPs mediated toxicity which could subsequently lead to genotoxicity [19]. In contradiction, dissolution of ZnO NPs into Zn2+ ions can also lead to toxicity [20]. To evaluate the toxic effects of ZnO NPs in vivo, various experimental models have being used. Drosophila melanogaster is one of the most effective model organisms with several advantages like short lifespan, extensive DNA sequence homology with humans, large number of offspring and easy maintenance. Drosophotoxicology as a distinctive research area has been proposed because of suitability and remarkable contribution of this model in toxicological research [21–23]. Recent literature indicates that nanoparticles have adverse effects on flies. Nanomaterial mutated flies were first identified as a resultant of gold nanoparticles (AuNPs) mutagenicity. Silver nanoparticles (AgNPs) caused depigmentation in body colour due to altered copper homeostasis, carbon NPs altered physiology in terms of reduced body size, however no adverse effect were reported in case of gallium phosphate nano wires. Induction of Hsps, oxidative stress and apoptosis are other toxic effects on exposure of flies to NPs [24–27]. Not withstanding the above, toxicological consequences of ZnO NPs on acute and choric exposure is questionable. Cu-doped ZnO NPs showed no toxicity on acute exposure of flies for 4 days [28]. ZnO NPs were found not to be genotoxic on acute exposure in flies using wing spot assay, but elevated Hsp70 and p53 levels were reported which is indicative of their role in carcinogenesis. Currently in literature there are very few studies on long term exposure of ZnO NPs, the genotoxicity potential of ZnO NPs is reported weak as evident by mutation or recombinogenic effects [29–31]. Most of the current literature is focused on acute exposure to ZnO NPs; in this study we have made an attempt to examine chronic exposure to ZnO NPs emphasising on mutation resulting in adverse phenotypic changes in subsequent generations.
Physical characterisation of ZnO NPs was determined by dynamic light scattering and transmission electron microscopy. In order to determine the hydrodynamic diameter and zeta potential, dynamic light scattering and laser doppler velocimetry was performed with Malvern Instrument Zetasizer Nano-ZS (Malvern, U.S.A). Zinc oxide 6% Al doped nanopowder (< 50 nm) was dispersed in MilliQ water and ultra sonicated (Elmasonic S30/(H) water bath sonicator) at 37 kHz for 30 min at room temperature [17,20,30]. The dispersion was futher centrifuged at 10,000 rpm for 30 min, the supernatant obtained was added to Drosophila diet and acessed for uniform dispersion through TEM. A drop of Drosophila diet containing ZnO NPs was casted on 300 mesh carbon coated grid and vacuum dried [32]. The TEM images were collected using TECNAI G2 S-Twin HR-TEM (FEI Company) with an accelerating voltage of 200 KV to determine the size and shape of the ZnO NPs.
2. Experimental
2.3. Drosophila melanogaster strain and culture conditions D. melanogaster (Oregon R) strain was a gift from Dr. Abhay Sharma, IGIB, New Delhi, India. Flies were cultured on Drosophila diet (Himedia Pvt. Ltd. India) containing agar, cornmeal, dextrose anhydrous, sucrose, yeast extract and nipagin (methyl-phydroxybenzoate) was added as antifungal agent. Flies were reared in an incubator at 25 ± 1 ◦ C, 60 ± 5% humidity and 12 h day/night cycle. 2.4. ZnO NPs exposure Flies were exposed to ZnO NPs through ingestion method. NPs were dispersed in water and mixed with Drosophila diet to obtain the final concentration of 0.1 mM, 1 mM, and 10 mM [29]. Newly emerged male and virgin female flies were reared separately on normal and food containing 0.1 mM, 1 mM and 10 mM ZnO NPs. Flies were cultured on this diet throughout the entire life cycle from egg to egg stage. All the treatments were carried out in triplicates and repeated five times. Progeny flies (F1, F2, F3 and F4) were collected and screened for any phenotypic aberration; approximately 500 flies per treatment were screened per generation. 2.5. Longevity assay To study the effect of ZnO NPs on lifespan of flies, 20 flies (10 females and 10 males) were cultured on normal and NPs containing food in triplicates. Flies were transferred to fresh vial every fourth day, to ensure that the feeding surroundings and lifespan was not interrupted by the occurrence of larvae. During each transfer age, number of dead and live flies was recorded carefully [33]. Experiment was carried for 50 days until all flies were dead. Based on the data recorded, the survivorship curve was obtained. 2.6. Climbing assay In order to assess the locomotor ability, climbing assay was done as per Pendleton et al. [34]. Briefly, ten flies from both control and NPs feeded group were separately placed in vials and acclimatized for 15 min at room temperature. The tubes were gently tapped and climbing ability was calculated by counting the number of flies that reached above 7 cm in 10 s, the climbing ability was examined ten times for each treatment.
2.1. Chemicals 2.7. Haemocytes collection ZnO 6% Al doped with <50 nm average particle size NPs (CAS No. 1314-13-2, >97% Catalog No. 677450-5G) and all other chemicals were obtained from Sigma Chemical Co. Ltd. (USA).
Haemocytes were collected as per the method described by Irving et al. with slight modifications [35]. Briefly, third instar larvae
182
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
Fig. 1. Characterisation of ZnO NPs (A) particles size distribution measured through DLS technique (B) zeta potential.
were collected from food, rinsed with water and air dried. The cuticle of 20 larvae was disrupted with the help of forceps and haemolymph and haemocytes were collected in ice-cold PBS.
and blocking solution. The sample were then counter stained with DAPI and visualized by confocal microscopy. 2.10. Phenotypic variations
2.8. DCF-DA assay for measurement of oxidative stress Oxidative stress was measured through quantification of intracellular ROS in larvae haemocytes using the 6-carboxy-2, 7-dichlorodihydro-fluorescein diacetate (DCF-DA) assay. Haemocytes collected were exposed to 15 m DCFH-DA for 30 min at 37 ◦ C. Fluorescence was recorded at excitation of 485 nm and emission of 530 nm. The experimental set include larvae exposed to ZnO NPs at 0.1–10 mM concentration and 15 mM H2 O2 was used as a positive control.
2.9. TUNEL assay DNA damage was assessed through ApopTag Red In Situ Apoptosis Detection Kit (Catalog No., S7165, Millipore, USA). Briefly, 100 l of PBS containing haemocytes was spread on poly-l-lysine coated cover slips and left for 1hr for adhesion of the cells. 4% formaldehyde solution was added to the sample and incubated for 15 min at room temperature for fixation. The fixative was removed and cover slips were washed with 1% Tween 20 in PBS. After washing, the cover slips were again incubated at room temperature for 15 min in 0.3% Triton X-100 in PBS for permeabilization. Following equilibration, terminal deoxy transferase enzyme and modified dUTP was added and the sample was incubated at 37 ◦ C for 60 min. The cover slips were then washed with PBS and incubated in a humidified chamber for 30 min at room temperature with anti-digoxigenin conjugate
The F0 (parent), F1, F2, F3 and F4 generation flies of control and NPs exposed were carefully observed under stereo microscope (OLYMPUS Co. Japan) for any phenotypic changes. In each treatment, we screened approximately 500 files were screened. We randomly transferred 20 male and 20 female flies and cross mated for next generation flies. Each treatment was carried out in triplicates; in total 1500 flies were screened for each concentration of NPs in a particular generation. 2.11. Statistical analysis The data was statistically analysed through Graph Pad Prism software. Data represented was statistically analysed through oneway ANOVA, Dunnett’s test and arithmetic mean with standard deviation. 3. Results 3.1. Physio-chemical characterisation of ZnO NPs Prior to exposure, it was very crucial to characterise the NPs; as ZnO NPs were added to semi solid diet, it is essential to ensure uniform dispersion throughout for consistent exposure via ingestion method. We have used TEM to determine morphology, size and distribution of ZnO NPs in Drosophila diet. On TEM analysis ZnO NPs of an average size of 28 ± 5 nm was observed uniformly
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
Fig. 2. Climbing behaviour of flies monitored after seven days exposure to ZnO NPs recording after 10 s. Data analysed represent error bar as mean ± standard deviation; significance at ***P-value < 0.01.
183
Fig. 4. Estimation of ROS by DCF assay. The data represent relative fluorescence intensity units/mg protein normalised to control as 100% (error bar as mean ± standard deviation; significance at ***P-value < 0.01).
3.4. Reactive oxygen species in haemocytes Exposure to NPs results in an increased number of free radical thereby increasing the oxidative load. To assess oxidative load due to NPs exposure in Drosophila, we preferred circulating haemocytes in third instar larvae for experimental assay. Oxidative stress was measured by DCFDA assay, calculated as fluorescent units/mg of protein and expressed in percentage taking control as 100% as shown in Fig. 4. Significant increased ROS levels were observed in haemocytes with increasing concentration of ZnO NPs i.e. 0.1 mM (115.85 ± 3.8%), 1 mM (169.63 ± 6.0%) and 10 mM (182.92 ± 4.3%). 3.5. DNA fragmentation in haemocytes
Fig. 3. Survivorship curve of flies nurtured on normal and ZnO NPs containing food Percentage of flies survived on exposure ZnO NPs as compared to control. Data analysed represent error bar as mean ± standard deviation.
distributed (Supplementary material: Fig1). Most of the particles were spherical in shape and showed no agglomerations. To further evaluate the characteristics of the NPs, particle size distribution and zeta potential was estimated though dynamic light scattering. The mean hydrodynamic diameter and zeta potential was 243.8 ± 5 nm and −22 mv respectively with polydispersity index 0.2 (Fig1). These results showed that there was a uniform dispersion of particles with no aggregate formation. Subsequently, we then examined possible toxic effects of ZnO NPs by exposing flies to 0.1 mM, 1 mM and 10 mM concentration of NPs and measuring various biological parameter; climbing behaviour, life span, oxidative stress and DNA damage. 3.2. Effect of ZnO NPs exposure on climbing behaviour in flies Defective climbing ability in flies is an indication of impaired locomotory behaviour in flies. Significant difference was observed in climbing behaviour of test and control flies post seven days of NPs exposure. The climbing efficiency was highly impaired in the flies which were exposed to 1 mM NPs (73 ± 10.5%) as compared to control (91 ± 5.6%), 0.1 mM (91 ± 5.6%) and 10 mM (77 ± 9.48%) exposed groups (Fig. 2). 3.3. Effect of ZnO NPs exposure on life span of the flies To examine the effects of ZnO NPs on lifespan parent flies were continuously feed on food containing NPs. No significant effect on the lifespan was observed in test and control flies (Fig. 3).
Increased ROS might trigger DNA damage and mutations in flies exposed to NPs, thereafter DNA damage in circulating haemocytes was evaluated through TUNEL assay. The percentage of TUNEL positive cells increased with the increasing conc. of ZnO NPs from 0.1 mM to 10 mM (Fig5). There was significant increase in percentage of apoptotic cells in ZnO NPs treated sample 1 mM (50.53 ± 12.8%), 10 mM (68.31 ± 20.2%) as compared to control (6.96 ± 3.2%). These results clearly indicate increased oxidative stress and DNA fragmentation upon exposure to ZnO NPs. 3.6. Abnormal phenotype in the progeny To further evaluate the chronic effects of NPs in subsequent generation, F1, F2, F3 and F4 flies were screened for phenotypic changes. Most of the fly offspring’s showed wing deformation (mild phenotypic change), however, distinctive phenotypic alterations were observed in few (Supplementary material Fig. 2, Fig. 3). Moderate phenotypic changes which include deformed wings were observed, a severe phenotype of deformed thorax, duplication of notum and single wing was observed in 3 ± 0.4% of the population (Fig. 6). The aberrant phenotype become more severe in the offspring if the progeny were also exposed to the NPs. Fig. 7a shows representative image of a normal fly and demarcated change can be seen in rest of the flies. The thorax appeared to be pigmented and deformed at the notum and the right wing failed to develop (Fig. 7b). A similar phenotype was observed, (Fig. 7c) where the left wing failed to develop. Ventral nerve cord failed to develop, the thorax was completely deformed and left wing failed to develop. These distinctive severe mutagenic phenotypes observed viz. absence of single wings, segmented thorax and completely distorted thorax (Fig. 7d) demonstrated the genotoxic effects of prolong exposure to ZnO NPs. The F1 generation of flies includes both normal and defective flies. Also, when mutant female/male flies were crossed with normal female/male flies and reared on normal conditions, the F2 genera-
184
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
Fig. 5. Genotoxic effect of ZnO NPs estimated through TUNEL assay performed on third instar circulating hemocytes of D. melanogastor. (A) Negative control, (B) Positive control, (C) Control without NPs exposure, (D) 0.1 mM ZnO NPs exposure (E) 1 mM ZnO NPs exposure, (F) 10 mM ZnO NPs exposure. Data represent error bar as mean ± standard deviation, ***P-value < 0.01 as compared to control.
Fig. 6. Pie diagram showing percentage distribution of normal and variant phenotypes.
Fig. 7. Aberrant phenotype observed in the progeny from parent exposed to ZnO NPs. (A) normal phenotype of parent fly exposed to ZnO NPs for seven days, (B) phenotype of distorted and pigmented thorax, (C) severe phenotypic change; deformed thorax and single wing fly, (D) fly segmented thorax and single wing, (E) abnormal thorax and single wing phenotype observed in F4 generation.
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
tion showed both normal and abnormal flies. Moreover on crossing mutated flies among themselves, development was arrested at the pupa stage resulting in no progeny.
4. Discussion The intensive use of ZnO NPs in consumer products makes it crucial to understand toxicity induces by these particles. As reported by FDA different forms of ZnO particles are generally recognised as safe substance. ZnO NPs are generally reported to have weak impact on the genetic material on acute exposure [36]. However, ZnO NPs inducing genotoxicity is questionable. In this report we aimed to understand the genotoxicity induced by ZnO NPs on long term exposure. Commercially manufactured ZnO NPs ( < 50 nm) were used to expose flies through ingestion method. The particles size was ensured by DLS and TEM and no agglomeration of ZnO NPs was reported in drosophila diet. The zeta potential measured was −20 mv which ensure stability, no agglomeration, uniform dispersion and appropriate parameter for toxicological studies. To study the harmful effects of ZnO NPs, flies where exposed to NPs throughout their lifespan. D. melanogaster is an effective model to study interlization of ZnO NPs.These particles are effectively absorbed by the midgut cells. From the midgut these are reported to efficiently cross the intestinal barrier and accumulate in the circulating haemocyte of the haemolymph [29]. Haemocytes are directly exposed to any toxic substance circulating in the haemolymph and DNA damage at base level is also very low thus is an effective cell type for genotoxicological studies [37]. The prime mechanism of toxicity induced by NPs is through increased oxidative stress, ZnO NPs are reported to increase oxidative stress [19]. Third instar larvae haemocytes was used to evaluate the effect of NPs on oxidative stress. Increased oxidative load with increasing concentration of ZnO NPs clearly indicate toward oxidative stress triggered by these NPs. To further investigate any the damage to the genetic machinery we assess genotoxicity by TUNEL assay on haemocytes. TUNEL assay detect DNA damage by labelling fragmented DNA and is a widely accepted method for detection of apoptotic cell [24,38,39]. Through TUNEL assay apoptotic cells can be easily quantified as ratio of positive cells over total number of cells. The results clearly demonstrated significant DNA damage induced by ZnO NPs. The major potential hazard these particle can entail is compromised integrity of genetic material. Chronic exposure of ZnO NPs leads to DNA damage as indicated by the increasing percentage of tunel positive cells in TUNEL assay. DNA damage and failure in repair mechanism might result in tetratogenicity, mutation, carcinogenicity, reproductive toxicity and abnormal physical condition of subsequent generations. To the best of our knowledge, we are reporting for the first time severe aberrant phenotype in flies on chronic exposure to ZnO NPs. This study clearly shows that ZnO NPs causes phenotypic abnormalities which might be transmitted to the progeny; however no phenotypic abnormality is reported in parent flies. Development of the structured embryo to adult is complex mechanism with many unsolved natal queries. The complex but well-programmed schematic of various genes governs biological pathways which play an essential role to develop a specific morphology of body part [40]. As most of the phenotypic changes were restricted to the thorax and wings we speculate that this aberrant phenotype could be caused by genetic mutation in the genes regulating the development of wing, thorax and segment polarity. Another plausible mechanism can be release of metal ions by ZnO NPs thereby increasing the ROS overload, further resulting in DNA damage, the exact mechanism of which is yet to be understood in depth. However, the current study clearly indicate that these particles induce oxidative stress, genotoxicity and abnormal phenotype in the progeny.
185
5. Conclusion In conclusion, we have shown mutagenic effect of ZnO NPs via aberrant phenotype in the progeny. We made an attempt to draw attention towards the harmful effects imposed by the NPs, not only on us but also on generations to come. There prolonged use is roving us to the condition which could result in adversity in future. Inspite of important application of nanoparticles there genotoxicity is overlooked. It is crucial that the Janet facet of this upcoming technology is not ignored, and well define safety guideline and protocols are practiced prior to their use. Acknowledgements The work is supported by grants from DRDO,Govt of India (DIP-259). Anand A.S. is thankful to Department of Science and Technology, New Delhi for providing fellowship for the study. Authors are also thankful to Department of Zoology, Delhi University for confocal microscopy and All India Institute of Medical Sciences, New Delhi for TEM analysis in the current study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2016.12. 040. References [1] G.A. Silva, Neuroscience nanotechnology: progress, opportunities and challenges, Nat. Rev. Neurosci. 7 (2006) 65–74, http://dx.doi.org/10.1038/ nrn1827. [2] C. Medina, M.J. Santos-Martinez, A. Radomski, O.I. Corrigan, M.W. Radomski, Nanoparticles: pharmacological and toxicological significance, Br. J. Pharmacol. 150 (2007) 552–558, http://dx.doi.org/10.1038/sj.bjp.0707130. [3] G. Oberdörster, E. Oberdörster, J. Oberdörster, Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Env. Heal. Perspect. 113 (2005) 823–839, http://dx.doi.org/10.1289/ehp.7339. ´ Genotoxicity of metal nanoparticles: focus on in vivo [4] K. Klien, J. Godnic-Cvar, studies, Arh. Hig. Rada Toksikol. 63 (2012) 133–145, http://dx.doi.org/10. 2478/10004-1254-63-2012-2213. [5] S.J. Soenen, P. Rivera-Gil, J.-M. Montenegro, W.J. Parak, S.C. De Smedt, K. Braeckmans, Cellular toxicity of inorganic nanoparticles: common aspects and guidelines for improved nanotoxicity evaluation, Nano Today. 6 (2011) 446–465, http://dx.doi.org/10.1016/j.nantod.2011.08.001. [6] M.Y. Wani, M.A. Hashim, F. Nabi, M.A. Malik, Nanotoxicity: dimensional and morphological concerns, Adv. Phys. Chem. 15 (2011), http://dx.doi.org/10. 1155/2011/450912org/. [7] A. Nel, T. Xia, L. Mädler, N. Li, Toxic potential of materials at the nanolevel, Science 311 (5761) (2006) 622–627, http://dx.doi.org/10.1126/science. 1114397. [8] M.C. Yeber, J. Rodríguez, J. Freer, N. Durán, H.D. Mansilla, Photocatalytic degradation of cellulose bleaching effluent by supported TiO2 and ZnO, Chemosphere 41 (2000) 1193–1197, http://dx.doi.org/10.1016/S00456535(99)00551-2. [9] S. Hackenberg, F.-Z. Zimmermann, A. Scherzed, G. Friehs, K. Froelich, C. Ginzkey, C. Koehler, M. Burghartz, R. Hagen, N. Kleinsasser, Repetitive exposure to zinc oxide nanoparticles induces dna damage in human nasal mucosa mini organ cultures, Environ. Mol. Mutagen. 52 (2011) 582–589, http://dx.doi.org/10.1002/em.20661. [10] Y.-N. Chang, M. Zhang, L. Xia, J. Zhang, G. Xing, The toxic effects and mechanisms of CuO and ZnO nanoparticles, Materials (Basel) 5 (2012) 2850, http://dx.doi.org/10.3390/ma5122850. [11] D. Bhattacharya, C.R. Santra, A.N. Ghosh, P. Karmakar, Differential toxicity of rod and spherical zinc oxide nanoparticles on human peripheral blood mononuclear cells, J. Biomed. Nanotechnol. 10 (2014) 707–716, http://dx.doi. org/10.1166/jbn.2014.1744. [12] E. Demir, H. Akc¸a, B. Kaya, D. Burgucu, O. Tokgün, F. Turna, S. Aksakal, G. Vales, A. Creus, R. Marcos, Zinc oxide nanoparticles: genotoxicity, interactions with UV-light and cell-transforming potential, J. Hazard. Mater. 264 (2014) 420–429, http://dx.doi.org/10.1016/j.jhazmat.2013.11.043. [13] I.M. Kennedy, D. Wilson, A.I. Barakat, H.E.I.H.R. Committee, Uptake and inflammatory effects of nanoparticles in a human vascular endothelial cell line, Res. Rep. Health Eff. Inst. (2009) 3–32 http://www.ncbi.nlm.nih.gov/ pubmed/19552347. [14] R. Guan, T. Kang, F. Lu, Z. Zhang, H. Shen, M. Liu, Cytotoxicity, oxidative stress, and genotoxicity in human hepatocyte and embryonic kidney cells exposed to
186
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186 ZnO nanoparticles, Nanoscale Res. Lett. 7 (2012) 602, http://dx.doi.org/10. 1186/1556-276X-7-602. V. Valdiglesias, C. Costa, G. Kilic¸, S. Costa, E. Pásaro, B. Laffon, J.P. Teixeira, Neuronal cytotoxicity and genotoxicity induced by zinc oxide nanoparticles, Env. Int. 55 (2013) 92–100, http://dx.doi.org/10.1016/j.envint.2013.02.013. E. Demir, A. Creus, R. Marcos, Genotoxicity and D.N.A. repair processes of zinc oxide nanoparticles, J. Toxicol. Environ. Health A 77 (2014) 1292–1303, http:// dx.doi.org/10.1080/15287394.2014.935540. K. Jain, E. Kohli, D. Prasad, K. Kamal, S.M. Hussain, S.B. Singh, In vitro cytotoxicity assessment of metal oxide nanoparticles, Nanomed. Nanobiol. 1 (1) (2014) 10–19, http://dx.doi.org/10.1166/nmb.2014.1003. A. Kumar, A.K. Pandey, S.S. Singh, R. Shanker, A. Dhawan, Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells, Chemosphere 83 (2011) 1124–1132, http://dx.doi.org/10.1016/j. chemosphere.2011.01.025. V. Sharma, D. Anderson, A. Dhawan, Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2), Apoptosis 17 (2012) 852–870, http://dx.doi.org/10.1007/ s10495-012-0705.6. J.Y. Kwon, S.Y. Lee, P. Koedrith, J.Y. Lee, K.M. Kim, J.M. Oh, S.I. Yang, M.K. Kim, J.K. Lee, J. Jeong, E.H. Maeng, B.J. Lee, Y.R. Seo, Lack of genotoxic potential of ZnO nanoparticles in vitro and in vivo tests, Mutat. Res.—Genet. Toxicol. Environ. Mutagen. 761 (2014) 1–9, http://dx.doi.org/10.1016/j.mrgentox. 2014.01.005. M.D. Rand, Drosophotoxicology: the growing potential for Drosophila in neurotoxicology, Neurotoxicol. Teratol. 32 (2010) 74–83, http://dx.doi.org/10. 1016/j.ntt.2009.06.004. C. Ong, L.Y. Yung, Y. Cai, B.H. Bay, G.H. Baeg, Drosophila melanogaster as a model organism to study nanotoxicity, Nanotoxicology 9 (2015) 396–403, http://dx.doi.org/10.3109/17435390.2014.940405. M. Chifiriuc, A. Ratiu, M. Popa, A. Ecovoiu, Drosophotoxicology an emerging research area for assessing nanoparticles interaction with living organisms, Int. J. Mol. Sci. 17 (2016) 36, http://dx.doi.org/10.3390/ijms17020036. G. Vecchio, A. Galeone, V. Brunetti, G. Maiorano, L. Rizzello, S. Sabella, R. Cingolani, P.P. Pompa, Mutagenic effects of gold nanoparticles induce aberrant phenotypes in Drosophila melanogaster, Nanomedicine 8 (2012) 1–7, http://dx.doi.org/10.1016/j.nano.2011.11.001. N. Armstrong, M. Ramamoorthy, D. Lyon, K. Jones, A. Duttaroy, Mechanism of silver nanoparticles action on insect pigmentation reveals intervention of copper homeostasis, PLoS One 8 (2013) e53186, http://dx.doi.org/10.1371/ journal.pone.0053186. S.H. Lee, H.Y. Lee, E.J. Lee, D. Khang, K.J. Min, Effects of carbon nanofiber on physiology of Drosophila, Int. J. Nanomed. 10 (2015) 3687–3697, http://dx. doi.org/10.2147/IJN.S82637. K. Adolfsson, M. Schneider, G. Hammarin, U. Häcker, C.N. Prinz, Ingestion of gallium phosphide nanowires has no adverse effect on Drosophila tissue function, Nanotechnology 24 (2013) 285101, http://dx.doi.org/10.1088/09574484/24/28/285101. Y.H. Siddique, M. Haidari, W. Khan, A. Fatima, S. Jyoti, S. Khanam, F. Naz, Rahul, F. Ali, B.R. Singh, T. Beg, Mohibullah, A.H. Naqvi, Toxic potential of copper-doped ZnO nanoparticles in Drosophila melanogaster (Oregon R), Toxicol. Mech. Methods 25 (2015) 425–432, http://dx.doi.org/10.3109/ 15376516.2015.1045653.
[29] M. Alaraby, B. Annangi, A. Hernández, A. Creus, R. Marcos, A comprehensive study of the harmful effects of ZnO nanoparticles using Drosophila melanogaster as an in vivo model, J. Hazard. Mater. 296 (2015) 166–174, http://dx.doi.org/10.1016/j.jhazmat.2015.04.053. [30] E.R. Carmona, C. Inostroza-Blancheteau, L. Rubio, R. Marcos, Genotoxic and oxidative stress potential of nanosized and bulk zinc oxide particles in Drosophila melanogaster, Toxicol. Ind. Health (2015), http://dx.doi.org/10. 1177/0748233715599472. [31] M. É. de Reis, A.A. Alves de Rezende, D.V. Santos, P. Francielli de Oliveria, H.D. Nicolella, D.C. Tavares, A.C. Almeida Silva, N.O. Dantas, M.A. Spanó, Assessment of the genotoxic potential of two zinc oxide sources (amorphous and nanoparticles) using the in vitro micronucleus test and the in vivo wing somatic test, Food Chem. Toxicol. 84 (2015) 55–63, http://dx.doi.org/10.1016/ j.fct.2015.07.008. [32] G. Vecchio, A. Galeone, V. Brunetti, G. Maiorano, S. Sabella, R. Cingolani, P.P. Pompa, Concentration-dependent, size-independent toxicity of citrate capped AuNPs in Drosophila melanogaster, PLoS One 7 (2012) e29980, http://dx.doi. org/10.1371/journal.pone.0029980. [33] N.J. Linford, C. Bilgir, J. Ro, S.D. Pletcher, Measurement of lifespan in Drosophila melanogaster, J. Vis. Exp. (2013), http://dx.doi.org/10.3791/50068. [34] R.G. Pendleton, F. Parvez, M. Sayed, R. Hillman, Effects of pharmacological agents upon a transgenic model of Parkinson’s disease in Drosophila melanogaster, J. Pharmacol. Exp. Ther. 300 (2002) 91–96, http://dx.doi.org/10. 1124/jpet.300.1.91. [35] P. Irving, J.M. Ubeda, D. Doucet, L. Troxler, M. Lagueux, D. Zachary, J.A. Hoffmann, C. Hetru, M. Meister, New insights into Drosophila larval haemocyte functions through genome-wide analysis, Cell. Microbiol. (2005) 335–350, http://dx.doi.org/10.1111/j. 1462–5822.2004.00462.x. [36] S. Chibber, S.A. Ansari, R. Satar, New vision to CuO, ZnO, and TiO2 nanoparticles: their outcome and effects, J. Nanopart. Res. 15 (2013) 1–13, http://dx.doi.org/10.1007/s11051-013-1492-x. [37] E.R. Carmona, A. Creus, R. Marcos, Genotoxic effects of two nickel-compounds in somatic cells of Drosophila melanogaster, Mutat. Res./Genet. Toxicol. Environ. Mutagen. 718 (2011) 33–37, http://dx.doi.org/10.1016/j.mrgentox. 2010.10.008. [38] P.P. Pompa, G. Vecchio, A. Galeone, V. Brunetti, S. Sabella, G. Maiorano, A. Falqui, G. Bertoni, R. Cingolani, In vivo toxicity assessment of gold nanoparticles in Drosophila melanogaster, Nano Res. 4 (4) (2011) 405–413, http://dx.doi.org/10.1007/s12274-011-0095-z. [39] A. Galeone, G. Vecchio, M.A. Malvindi, V. Brunetti, R. Cingolani, P.P. Pompa, In vivo assessment of CdSe-ZnS quantumdots: coating dependent bioaccumulation and genotoxicity, Nanoscale 4 (2012) 6401–6407, http://dx. doi.org/10.1039/c2nr31826a. [40] A. Joshi, S. Chandrashekaran, R.P. Sharma, Mutants dissecting development and behaviour in Drosophila, Curr. Sci. 89 (2005) 341–352.
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Chronic exposure of zinc oxide nanoparticles causes deviant phenotype in Drosophila melanogaster Avnika Singh Anand, Dipti N. Prasad, Shashi Bala Singh, Ekta Kohli ∗ Neurobiology Division, Defence Institute of Physiology and Allied Sciences, Delhi, 110054, India
g r a p h i c a l
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 19 September 2016 Received in revised form 20 December 2016 Accepted 22 December 2016 Available online 23 December 2016 Keywords: Genotoxicity Zinc oxide nanoparticles Drosophila melanogaster Risk assessment Nanotoxicology
a b s t r a c t Zinc oxide nanoparticles (ZnO NPs) are commonly used nanomaterials (NMs) with versatile applications from high-end technologies to household products. This pervasive utilisation has brought human in the close interface with nanoparticles (NPs), hence questioning their safety prior to usage is a must. In this study, we have assessed the effects of chronic exposure to ZnO NPs (<50 nm) on the model organism Drosophila melanogaster. Potential toxic effects were studied by evaluating longevity, climbing ability, oxidative stress and DNA fragmentation. Ensuing exposure, the F0 (parent), F1, F2, F3 and F4 generation flies were screened for the aberrant phenotype. Flies exposed to ZnO NPs showed distinctive phenotypic changes, like deformed segmented thorax and single or deformed wing, which were transmitted to the offspring’s in subsequent generations. The unique abnormal phenotype is evident of chronic toxicity induced by ZnO NPs, although appalling, it strongly emphasize the importance to understand NPs toxicity for safer use. © 2016 Elsevier B.V. All rights reserved.
Abbreviations: ROS, reactive oxidative species; ZnO, zinc oxide; AgNPs, silver nanoparticles; AuNPs, gold nanoparticles; NPs, nanoparticles; NMs, nanomaterials; TEM, transmission electron microscopy; DCF-DA, 2’,7’–dichlorofluorescin diacetate; DAPI, 4’,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; ANOVA, analysis of variance. ∗ Corresponding author at: Neurobiology Division, Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, Ministry of Defence, Lucknow Road, Timarpur, Delhi, India. E-mail addresses: [email protected], [email protected] (E. Kohli). http://dx.doi.org/10.1016/j.jhazmat.2016.12.040 0304-3894/© 2016 Elsevier B.V. All rights reserved.
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
181
1. Introduction
2.2. Characterisation of ZnO NPs
The ubiquitous utilisation of nanomaterials (NMs) has created lots of concerns for the living world, especially to human beings. At the nano level matter is governed by quantum mechanics and might possess distinct novel chemical and physical properties different from the molecular counterpart [1,2]. The nano size facilitates these particles to cross any biological barrier and intermingle with the different bio molecules. This nano-bio interference can result in detrimental biological effects like Reactive Oxidative Species (ROS) generation, cytotoxicity, genotoxicity and inflammatory responses [3–7]. The most detrimental is obliteration of the genetic material and erroneous genetic information transmitted to future generations. Zinc oxide nanoparticles (ZnO NPs) are widely used in cosmetics, sunscreens, UV radiation blockers, gas sensors, paints, ceramics, medical supplies and food additives [8–10] and thus come in direct contact with a large section of the population. Given the above, it is important to critically evaluate toxicity induced by these particles. In various in vivo and in vitro studies, ZnO NPs have been reported to be toxic; it has been shown to induce morphological modifications, oxidative stress, lipid peroxidation, mitochondrial dysfunction, cytotoxicity, chromosomal breakage and micronuclei formation [11–17]. ZnO NPs exposure also adversely affects the prokaryotic system as these particles were found to be mutagenic in bacterial assays [18]. ROS generation is a prime cause of ZnO NPs mediated toxicity which could subsequently lead to genotoxicity [19]. In contradiction, dissolution of ZnO NPs into Zn2+ ions can also lead to toxicity [20]. To evaluate the toxic effects of ZnO NPs in vivo, various experimental models have being used. Drosophila melanogaster is one of the most effective model organisms with several advantages like short lifespan, extensive DNA sequence homology with humans, large number of offspring and easy maintenance. Drosophotoxicology as a distinctive research area has been proposed because of suitability and remarkable contribution of this model in toxicological research [21–23]. Recent literature indicates that nanoparticles have adverse effects on flies. Nanomaterial mutated flies were first identified as a resultant of gold nanoparticles (AuNPs) mutagenicity. Silver nanoparticles (AgNPs) caused depigmentation in body colour due to altered copper homeostasis, carbon NPs altered physiology in terms of reduced body size, however no adverse effect were reported in case of gallium phosphate nano wires. Induction of Hsps, oxidative stress and apoptosis are other toxic effects on exposure of flies to NPs [24–27]. Not withstanding the above, toxicological consequences of ZnO NPs on acute and choric exposure is questionable. Cu-doped ZnO NPs showed no toxicity on acute exposure of flies for 4 days [28]. ZnO NPs were found not to be genotoxic on acute exposure in flies using wing spot assay, but elevated Hsp70 and p53 levels were reported which is indicative of their role in carcinogenesis. Currently in literature there are very few studies on long term exposure of ZnO NPs, the genotoxicity potential of ZnO NPs is reported weak as evident by mutation or recombinogenic effects [29–31]. Most of the current literature is focused on acute exposure to ZnO NPs; in this study we have made an attempt to examine chronic exposure to ZnO NPs emphasising on mutation resulting in adverse phenotypic changes in subsequent generations.
Physical characterisation of ZnO NPs was determined by dynamic light scattering and transmission electron microscopy. In order to determine the hydrodynamic diameter and zeta potential, dynamic light scattering and laser doppler velocimetry was performed with Malvern Instrument Zetasizer Nano-ZS (Malvern, U.S.A). Zinc oxide 6% Al doped nanopowder (< 50 nm) was dispersed in MilliQ water and ultra sonicated (Elmasonic S30/(H) water bath sonicator) at 37 kHz for 30 min at room temperature [17,20,30]. The dispersion was futher centrifuged at 10,000 rpm for 30 min, the supernatant obtained was added to Drosophila diet and acessed for uniform dispersion through TEM. A drop of Drosophila diet containing ZnO NPs was casted on 300 mesh carbon coated grid and vacuum dried [32]. The TEM images were collected using TECNAI G2 S-Twin HR-TEM (FEI Company) with an accelerating voltage of 200 KV to determine the size and shape of the ZnO NPs.
2. Experimental
2.3. Drosophila melanogaster strain and culture conditions D. melanogaster (Oregon R) strain was a gift from Dr. Abhay Sharma, IGIB, New Delhi, India. Flies were cultured on Drosophila diet (Himedia Pvt. Ltd. India) containing agar, cornmeal, dextrose anhydrous, sucrose, yeast extract and nipagin (methyl-phydroxybenzoate) was added as antifungal agent. Flies were reared in an incubator at 25 ± 1 ◦ C, 60 ± 5% humidity and 12 h day/night cycle. 2.4. ZnO NPs exposure Flies were exposed to ZnO NPs through ingestion method. NPs were dispersed in water and mixed with Drosophila diet to obtain the final concentration of 0.1 mM, 1 mM, and 10 mM [29]. Newly emerged male and virgin female flies were reared separately on normal and food containing 0.1 mM, 1 mM and 10 mM ZnO NPs. Flies were cultured on this diet throughout the entire life cycle from egg to egg stage. All the treatments were carried out in triplicates and repeated five times. Progeny flies (F1, F2, F3 and F4) were collected and screened for any phenotypic aberration; approximately 500 flies per treatment were screened per generation. 2.5. Longevity assay To study the effect of ZnO NPs on lifespan of flies, 20 flies (10 females and 10 males) were cultured on normal and NPs containing food in triplicates. Flies were transferred to fresh vial every fourth day, to ensure that the feeding surroundings and lifespan was not interrupted by the occurrence of larvae. During each transfer age, number of dead and live flies was recorded carefully [33]. Experiment was carried for 50 days until all flies were dead. Based on the data recorded, the survivorship curve was obtained. 2.6. Climbing assay In order to assess the locomotor ability, climbing assay was done as per Pendleton et al. [34]. Briefly, ten flies from both control and NPs feeded group were separately placed in vials and acclimatized for 15 min at room temperature. The tubes were gently tapped and climbing ability was calculated by counting the number of flies that reached above 7 cm in 10 s, the climbing ability was examined ten times for each treatment.
2.1. Chemicals 2.7. Haemocytes collection ZnO 6% Al doped with <50 nm average particle size NPs (CAS No. 1314-13-2, >97% Catalog No. 677450-5G) and all other chemicals were obtained from Sigma Chemical Co. Ltd. (USA).
Haemocytes were collected as per the method described by Irving et al. with slight modifications [35]. Briefly, third instar larvae
182
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
Fig. 1. Characterisation of ZnO NPs (A) particles size distribution measured through DLS technique (B) zeta potential.
were collected from food, rinsed with water and air dried. The cuticle of 20 larvae was disrupted with the help of forceps and haemolymph and haemocytes were collected in ice-cold PBS.
and blocking solution. The sample were then counter stained with DAPI and visualized by confocal microscopy. 2.10. Phenotypic variations
2.8. DCF-DA assay for measurement of oxidative stress Oxidative stress was measured through quantification of intracellular ROS in larvae haemocytes using the 6-carboxy-2, 7-dichlorodihydro-fluorescein diacetate (DCF-DA) assay. Haemocytes collected were exposed to 15 m DCFH-DA for 30 min at 37 ◦ C. Fluorescence was recorded at excitation of 485 nm and emission of 530 nm. The experimental set include larvae exposed to ZnO NPs at 0.1–10 mM concentration and 15 mM H2 O2 was used as a positive control.
2.9. TUNEL assay DNA damage was assessed through ApopTag Red In Situ Apoptosis Detection Kit (Catalog No., S7165, Millipore, USA). Briefly, 100 l of PBS containing haemocytes was spread on poly-l-lysine coated cover slips and left for 1hr for adhesion of the cells. 4% formaldehyde solution was added to the sample and incubated for 15 min at room temperature for fixation. The fixative was removed and cover slips were washed with 1% Tween 20 in PBS. After washing, the cover slips were again incubated at room temperature for 15 min in 0.3% Triton X-100 in PBS for permeabilization. Following equilibration, terminal deoxy transferase enzyme and modified dUTP was added and the sample was incubated at 37 ◦ C for 60 min. The cover slips were then washed with PBS and incubated in a humidified chamber for 30 min at room temperature with anti-digoxigenin conjugate
The F0 (parent), F1, F2, F3 and F4 generation flies of control and NPs exposed were carefully observed under stereo microscope (OLYMPUS Co. Japan) for any phenotypic changes. In each treatment, we screened approximately 500 files were screened. We randomly transferred 20 male and 20 female flies and cross mated for next generation flies. Each treatment was carried out in triplicates; in total 1500 flies were screened for each concentration of NPs in a particular generation. 2.11. Statistical analysis The data was statistically analysed through Graph Pad Prism software. Data represented was statistically analysed through oneway ANOVA, Dunnett’s test and arithmetic mean with standard deviation. 3. Results 3.1. Physio-chemical characterisation of ZnO NPs Prior to exposure, it was very crucial to characterise the NPs; as ZnO NPs were added to semi solid diet, it is essential to ensure uniform dispersion throughout for consistent exposure via ingestion method. We have used TEM to determine morphology, size and distribution of ZnO NPs in Drosophila diet. On TEM analysis ZnO NPs of an average size of 28 ± 5 nm was observed uniformly
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
Fig. 2. Climbing behaviour of flies monitored after seven days exposure to ZnO NPs recording after 10 s. Data analysed represent error bar as mean ± standard deviation; significance at ***P-value < 0.01.
183
Fig. 4. Estimation of ROS by DCF assay. The data represent relative fluorescence intensity units/mg protein normalised to control as 100% (error bar as mean ± standard deviation; significance at ***P-value < 0.01).
3.4. Reactive oxygen species in haemocytes Exposure to NPs results in an increased number of free radical thereby increasing the oxidative load. To assess oxidative load due to NPs exposure in Drosophila, we preferred circulating haemocytes in third instar larvae for experimental assay. Oxidative stress was measured by DCFDA assay, calculated as fluorescent units/mg of protein and expressed in percentage taking control as 100% as shown in Fig. 4. Significant increased ROS levels were observed in haemocytes with increasing concentration of ZnO NPs i.e. 0.1 mM (115.85 ± 3.8%), 1 mM (169.63 ± 6.0%) and 10 mM (182.92 ± 4.3%). 3.5. DNA fragmentation in haemocytes
Fig. 3. Survivorship curve of flies nurtured on normal and ZnO NPs containing food Percentage of flies survived on exposure ZnO NPs as compared to control. Data analysed represent error bar as mean ± standard deviation.
distributed (Supplementary material: Fig1). Most of the particles were spherical in shape and showed no agglomerations. To further evaluate the characteristics of the NPs, particle size distribution and zeta potential was estimated though dynamic light scattering. The mean hydrodynamic diameter and zeta potential was 243.8 ± 5 nm and −22 mv respectively with polydispersity index 0.2 (Fig1). These results showed that there was a uniform dispersion of particles with no aggregate formation. Subsequently, we then examined possible toxic effects of ZnO NPs by exposing flies to 0.1 mM, 1 mM and 10 mM concentration of NPs and measuring various biological parameter; climbing behaviour, life span, oxidative stress and DNA damage. 3.2. Effect of ZnO NPs exposure on climbing behaviour in flies Defective climbing ability in flies is an indication of impaired locomotory behaviour in flies. Significant difference was observed in climbing behaviour of test and control flies post seven days of NPs exposure. The climbing efficiency was highly impaired in the flies which were exposed to 1 mM NPs (73 ± 10.5%) as compared to control (91 ± 5.6%), 0.1 mM (91 ± 5.6%) and 10 mM (77 ± 9.48%) exposed groups (Fig. 2). 3.3. Effect of ZnO NPs exposure on life span of the flies To examine the effects of ZnO NPs on lifespan parent flies were continuously feed on food containing NPs. No significant effect on the lifespan was observed in test and control flies (Fig. 3).
Increased ROS might trigger DNA damage and mutations in flies exposed to NPs, thereafter DNA damage in circulating haemocytes was evaluated through TUNEL assay. The percentage of TUNEL positive cells increased with the increasing conc. of ZnO NPs from 0.1 mM to 10 mM (Fig5). There was significant increase in percentage of apoptotic cells in ZnO NPs treated sample 1 mM (50.53 ± 12.8%), 10 mM (68.31 ± 20.2%) as compared to control (6.96 ± 3.2%). These results clearly indicate increased oxidative stress and DNA fragmentation upon exposure to ZnO NPs. 3.6. Abnormal phenotype in the progeny To further evaluate the chronic effects of NPs in subsequent generation, F1, F2, F3 and F4 flies were screened for phenotypic changes. Most of the fly offspring’s showed wing deformation (mild phenotypic change), however, distinctive phenotypic alterations were observed in few (Supplementary material Fig. 2, Fig. 3). Moderate phenotypic changes which include deformed wings were observed, a severe phenotype of deformed thorax, duplication of notum and single wing was observed in 3 ± 0.4% of the population (Fig. 6). The aberrant phenotype become more severe in the offspring if the progeny were also exposed to the NPs. Fig. 7a shows representative image of a normal fly and demarcated change can be seen in rest of the flies. The thorax appeared to be pigmented and deformed at the notum and the right wing failed to develop (Fig. 7b). A similar phenotype was observed, (Fig. 7c) where the left wing failed to develop. Ventral nerve cord failed to develop, the thorax was completely deformed and left wing failed to develop. These distinctive severe mutagenic phenotypes observed viz. absence of single wings, segmented thorax and completely distorted thorax (Fig. 7d) demonstrated the genotoxic effects of prolong exposure to ZnO NPs. The F1 generation of flies includes both normal and defective flies. Also, when mutant female/male flies were crossed with normal female/male flies and reared on normal conditions, the F2 genera-
184
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
Fig. 5. Genotoxic effect of ZnO NPs estimated through TUNEL assay performed on third instar circulating hemocytes of D. melanogastor. (A) Negative control, (B) Positive control, (C) Control without NPs exposure, (D) 0.1 mM ZnO NPs exposure (E) 1 mM ZnO NPs exposure, (F) 10 mM ZnO NPs exposure. Data represent error bar as mean ± standard deviation, ***P-value < 0.01 as compared to control.
Fig. 6. Pie diagram showing percentage distribution of normal and variant phenotypes.
Fig. 7. Aberrant phenotype observed in the progeny from parent exposed to ZnO NPs. (A) normal phenotype of parent fly exposed to ZnO NPs for seven days, (B) phenotype of distorted and pigmented thorax, (C) severe phenotypic change; deformed thorax and single wing fly, (D) fly segmented thorax and single wing, (E) abnormal thorax and single wing phenotype observed in F4 generation.
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186
tion showed both normal and abnormal flies. Moreover on crossing mutated flies among themselves, development was arrested at the pupa stage resulting in no progeny.
4. Discussion The intensive use of ZnO NPs in consumer products makes it crucial to understand toxicity induces by these particles. As reported by FDA different forms of ZnO particles are generally recognised as safe substance. ZnO NPs are generally reported to have weak impact on the genetic material on acute exposure [36]. However, ZnO NPs inducing genotoxicity is questionable. In this report we aimed to understand the genotoxicity induced by ZnO NPs on long term exposure. Commercially manufactured ZnO NPs ( < 50 nm) were used to expose flies through ingestion method. The particles size was ensured by DLS and TEM and no agglomeration of ZnO NPs was reported in drosophila diet. The zeta potential measured was −20 mv which ensure stability, no agglomeration, uniform dispersion and appropriate parameter for toxicological studies. To study the harmful effects of ZnO NPs, flies where exposed to NPs throughout their lifespan. D. melanogaster is an effective model to study interlization of ZnO NPs.These particles are effectively absorbed by the midgut cells. From the midgut these are reported to efficiently cross the intestinal barrier and accumulate in the circulating haemocyte of the haemolymph [29]. Haemocytes are directly exposed to any toxic substance circulating in the haemolymph and DNA damage at base level is also very low thus is an effective cell type for genotoxicological studies [37]. The prime mechanism of toxicity induced by NPs is through increased oxidative stress, ZnO NPs are reported to increase oxidative stress [19]. Third instar larvae haemocytes was used to evaluate the effect of NPs on oxidative stress. Increased oxidative load with increasing concentration of ZnO NPs clearly indicate toward oxidative stress triggered by these NPs. To further investigate any the damage to the genetic machinery we assess genotoxicity by TUNEL assay on haemocytes. TUNEL assay detect DNA damage by labelling fragmented DNA and is a widely accepted method for detection of apoptotic cell [24,38,39]. Through TUNEL assay apoptotic cells can be easily quantified as ratio of positive cells over total number of cells. The results clearly demonstrated significant DNA damage induced by ZnO NPs. The major potential hazard these particle can entail is compromised integrity of genetic material. Chronic exposure of ZnO NPs leads to DNA damage as indicated by the increasing percentage of tunel positive cells in TUNEL assay. DNA damage and failure in repair mechanism might result in tetratogenicity, mutation, carcinogenicity, reproductive toxicity and abnormal physical condition of subsequent generations. To the best of our knowledge, we are reporting for the first time severe aberrant phenotype in flies on chronic exposure to ZnO NPs. This study clearly shows that ZnO NPs causes phenotypic abnormalities which might be transmitted to the progeny; however no phenotypic abnormality is reported in parent flies. Development of the structured embryo to adult is complex mechanism with many unsolved natal queries. The complex but well-programmed schematic of various genes governs biological pathways which play an essential role to develop a specific morphology of body part [40]. As most of the phenotypic changes were restricted to the thorax and wings we speculate that this aberrant phenotype could be caused by genetic mutation in the genes regulating the development of wing, thorax and segment polarity. Another plausible mechanism can be release of metal ions by ZnO NPs thereby increasing the ROS overload, further resulting in DNA damage, the exact mechanism of which is yet to be understood in depth. However, the current study clearly indicate that these particles induce oxidative stress, genotoxicity and abnormal phenotype in the progeny.
185
5. Conclusion In conclusion, we have shown mutagenic effect of ZnO NPs via aberrant phenotype in the progeny. We made an attempt to draw attention towards the harmful effects imposed by the NPs, not only on us but also on generations to come. There prolonged use is roving us to the condition which could result in adversity in future. Inspite of important application of nanoparticles there genotoxicity is overlooked. It is crucial that the Janet facet of this upcoming technology is not ignored, and well define safety guideline and protocols are practiced prior to their use. Acknowledgements The work is supported by grants from DRDO,Govt of India (DIP-259). Anand A.S. is thankful to Department of Science and Technology, New Delhi for providing fellowship for the study. Authors are also thankful to Department of Zoology, Delhi University for confocal microscopy and All India Institute of Medical Sciences, New Delhi for TEM analysis in the current study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2016.12. 040. References [1] G.A. Silva, Neuroscience nanotechnology: progress, opportunities and challenges, Nat. Rev. Neurosci. 7 (2006) 65–74, http://dx.doi.org/10.1038/ nrn1827. [2] C. Medina, M.J. Santos-Martinez, A. Radomski, O.I. Corrigan, M.W. Radomski, Nanoparticles: pharmacological and toxicological significance, Br. J. Pharmacol. 150 (2007) 552–558, http://dx.doi.org/10.1038/sj.bjp.0707130. [3] G. Oberdörster, E. Oberdörster, J. Oberdörster, Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Env. Heal. Perspect. 113 (2005) 823–839, http://dx.doi.org/10.1289/ehp.7339. ´ Genotoxicity of metal nanoparticles: focus on in vivo [4] K. Klien, J. Godnic-Cvar, studies, Arh. Hig. Rada Toksikol. 63 (2012) 133–145, http://dx.doi.org/10. 2478/10004-1254-63-2012-2213. [5] S.J. Soenen, P. Rivera-Gil, J.-M. Montenegro, W.J. Parak, S.C. De Smedt, K. Braeckmans, Cellular toxicity of inorganic nanoparticles: common aspects and guidelines for improved nanotoxicity evaluation, Nano Today. 6 (2011) 446–465, http://dx.doi.org/10.1016/j.nantod.2011.08.001. [6] M.Y. Wani, M.A. Hashim, F. Nabi, M.A. Malik, Nanotoxicity: dimensional and morphological concerns, Adv. Phys. Chem. 15 (2011), http://dx.doi.org/10. 1155/2011/450912org/. [7] A. Nel, T. Xia, L. Mädler, N. Li, Toxic potential of materials at the nanolevel, Science 311 (5761) (2006) 622–627, http://dx.doi.org/10.1126/science. 1114397. [8] M.C. Yeber, J. Rodríguez, J. Freer, N. Durán, H.D. Mansilla, Photocatalytic degradation of cellulose bleaching effluent by supported TiO2 and ZnO, Chemosphere 41 (2000) 1193–1197, http://dx.doi.org/10.1016/S00456535(99)00551-2. [9] S. Hackenberg, F.-Z. Zimmermann, A. Scherzed, G. Friehs, K. Froelich, C. Ginzkey, C. Koehler, M. Burghartz, R. Hagen, N. Kleinsasser, Repetitive exposure to zinc oxide nanoparticles induces dna damage in human nasal mucosa mini organ cultures, Environ. Mol. Mutagen. 52 (2011) 582–589, http://dx.doi.org/10.1002/em.20661. [10] Y.-N. Chang, M. Zhang, L. Xia, J. Zhang, G. Xing, The toxic effects and mechanisms of CuO and ZnO nanoparticles, Materials (Basel) 5 (2012) 2850, http://dx.doi.org/10.3390/ma5122850. [11] D. Bhattacharya, C.R. Santra, A.N. Ghosh, P. Karmakar, Differential toxicity of rod and spherical zinc oxide nanoparticles on human peripheral blood mononuclear cells, J. Biomed. Nanotechnol. 10 (2014) 707–716, http://dx.doi. org/10.1166/jbn.2014.1744. [12] E. Demir, H. Akc¸a, B. Kaya, D. Burgucu, O. Tokgün, F. Turna, S. Aksakal, G. Vales, A. Creus, R. Marcos, Zinc oxide nanoparticles: genotoxicity, interactions with UV-light and cell-transforming potential, J. Hazard. Mater. 264 (2014) 420–429, http://dx.doi.org/10.1016/j.jhazmat.2013.11.043. [13] I.M. Kennedy, D. Wilson, A.I. Barakat, H.E.I.H.R. Committee, Uptake and inflammatory effects of nanoparticles in a human vascular endothelial cell line, Res. Rep. Health Eff. Inst. (2009) 3–32 http://www.ncbi.nlm.nih.gov/ pubmed/19552347. [14] R. Guan, T. Kang, F. Lu, Z. Zhang, H. Shen, M. Liu, Cytotoxicity, oxidative stress, and genotoxicity in human hepatocyte and embryonic kidney cells exposed to
186
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
A.S. Anand et al. / Journal of Hazardous Materials 327 (2017) 180–186 ZnO nanoparticles, Nanoscale Res. Lett. 7 (2012) 602, http://dx.doi.org/10. 1186/1556-276X-7-602. V. Valdiglesias, C. Costa, G. Kilic¸, S. Costa, E. Pásaro, B. Laffon, J.P. Teixeira, Neuronal cytotoxicity and genotoxicity induced by zinc oxide nanoparticles, Env. Int. 55 (2013) 92–100, http://dx.doi.org/10.1016/j.envint.2013.02.013. E. Demir, A. Creus, R. Marcos, Genotoxicity and D.N.A. repair processes of zinc oxide nanoparticles, J. Toxicol. Environ. Health A 77 (2014) 1292–1303, http:// dx.doi.org/10.1080/15287394.2014.935540. K. Jain, E. Kohli, D. Prasad, K. Kamal, S.M. Hussain, S.B. Singh, In vitro cytotoxicity assessment of metal oxide nanoparticles, Nanomed. Nanobiol. 1 (1) (2014) 10–19, http://dx.doi.org/10.1166/nmb.2014.1003. A. Kumar, A.K. Pandey, S.S. Singh, R. Shanker, A. Dhawan, Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells, Chemosphere 83 (2011) 1124–1132, http://dx.doi.org/10.1016/j. chemosphere.2011.01.025. V. Sharma, D. Anderson, A. Dhawan, Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2), Apoptosis 17 (2012) 852–870, http://dx.doi.org/10.1007/ s10495-012-0705.6. J.Y. Kwon, S.Y. Lee, P. Koedrith, J.Y. Lee, K.M. Kim, J.M. Oh, S.I. Yang, M.K. Kim, J.K. Lee, J. Jeong, E.H. Maeng, B.J. Lee, Y.R. Seo, Lack of genotoxic potential of ZnO nanoparticles in vitro and in vivo tests, Mutat. Res.—Genet. Toxicol. Environ. Mutagen. 761 (2014) 1–9, http://dx.doi.org/10.1016/j.mrgentox. 2014.01.005. M.D. Rand, Drosophotoxicology: the growing potential for Drosophila in neurotoxicology, Neurotoxicol. Teratol. 32 (2010) 74–83, http://dx.doi.org/10. 1016/j.ntt.2009.06.004. C. Ong, L.Y. Yung, Y. Cai, B.H. Bay, G.H. Baeg, Drosophila melanogaster as a model organism to study nanotoxicity, Nanotoxicology 9 (2015) 396–403, http://dx.doi.org/10.3109/17435390.2014.940405. M. Chifiriuc, A. Ratiu, M. Popa, A. Ecovoiu, Drosophotoxicology an emerging research area for assessing nanoparticles interaction with living organisms, Int. J. Mol. Sci. 17 (2016) 36, http://dx.doi.org/10.3390/ijms17020036. G. Vecchio, A. Galeone, V. Brunetti, G. Maiorano, L. Rizzello, S. Sabella, R. Cingolani, P.P. Pompa, Mutagenic effects of gold nanoparticles induce aberrant phenotypes in Drosophila melanogaster, Nanomedicine 8 (2012) 1–7, http://dx.doi.org/10.1016/j.nano.2011.11.001. N. Armstrong, M. Ramamoorthy, D. Lyon, K. Jones, A. Duttaroy, Mechanism of silver nanoparticles action on insect pigmentation reveals intervention of copper homeostasis, PLoS One 8 (2013) e53186, http://dx.doi.org/10.1371/ journal.pone.0053186. S.H. Lee, H.Y. Lee, E.J. Lee, D. Khang, K.J. Min, Effects of carbon nanofiber on physiology of Drosophila, Int. J. Nanomed. 10 (2015) 3687–3697, http://dx. doi.org/10.2147/IJN.S82637. K. Adolfsson, M. Schneider, G. Hammarin, U. Häcker, C.N. Prinz, Ingestion of gallium phosphide nanowires has no adverse effect on Drosophila tissue function, Nanotechnology 24 (2013) 285101, http://dx.doi.org/10.1088/09574484/24/28/285101. Y.H. Siddique, M. Haidari, W. Khan, A. Fatima, S. Jyoti, S. Khanam, F. Naz, Rahul, F. Ali, B.R. Singh, T. Beg, Mohibullah, A.H. Naqvi, Toxic potential of copper-doped ZnO nanoparticles in Drosophila melanogaster (Oregon R), Toxicol. Mech. Methods 25 (2015) 425–432, http://dx.doi.org/10.3109/ 15376516.2015.1045653.
[29] M. Alaraby, B. Annangi, A. Hernández, A. Creus, R. Marcos, A comprehensive study of the harmful effects of ZnO nanoparticles using Drosophila melanogaster as an in vivo model, J. Hazard. Mater. 296 (2015) 166–174, http://dx.doi.org/10.1016/j.jhazmat.2015.04.053. [30] E.R. Carmona, C. Inostroza-Blancheteau, L. Rubio, R. Marcos, Genotoxic and oxidative stress potential of nanosized and bulk zinc oxide particles in Drosophila melanogaster, Toxicol. Ind. Health (2015), http://dx.doi.org/10. 1177/0748233715599472. [31] M. É. de Reis, A.A. Alves de Rezende, D.V. Santos, P. Francielli de Oliveria, H.D. Nicolella, D.C. Tavares, A.C. Almeida Silva, N.O. Dantas, M.A. Spanó, Assessment of the genotoxic potential of two zinc oxide sources (amorphous and nanoparticles) using the in vitro micronucleus test and the in vivo wing somatic test, Food Chem. Toxicol. 84 (2015) 55–63, http://dx.doi.org/10.1016/ j.fct.2015.07.008. [32] G. Vecchio, A. Galeone, V. Brunetti, G. Maiorano, S. Sabella, R. Cingolani, P.P. Pompa, Concentration-dependent, size-independent toxicity of citrate capped AuNPs in Drosophila melanogaster, PLoS One 7 (2012) e29980, http://dx.doi. org/10.1371/journal.pone.0029980. [33] N.J. Linford, C. Bilgir, J. Ro, S.D. Pletcher, Measurement of lifespan in Drosophila melanogaster, J. Vis. Exp. (2013), http://dx.doi.org/10.3791/50068. [34] R.G. Pendleton, F. Parvez, M. Sayed, R. Hillman, Effects of pharmacological agents upon a transgenic model of Parkinson’s disease in Drosophila melanogaster, J. Pharmacol. Exp. Ther. 300 (2002) 91–96, http://dx.doi.org/10. 1124/jpet.300.1.91. [35] P. Irving, J.M. Ubeda, D. Doucet, L. Troxler, M. Lagueux, D. Zachary, J.A. Hoffmann, C. Hetru, M. Meister, New insights into Drosophila larval haemocyte functions through genome-wide analysis, Cell. Microbiol. (2005) 335–350, http://dx.doi.org/10.1111/j. 1462–5822.2004.00462.x. [36] S. Chibber, S.A. Ansari, R. Satar, New vision to CuO, ZnO, and TiO2 nanoparticles: their outcome and effects, J. Nanopart. Res. 15 (2013) 1–13, http://dx.doi.org/10.1007/s11051-013-1492-x. [37] E.R. Carmona, A. Creus, R. Marcos, Genotoxic effects of two nickel-compounds in somatic cells of Drosophila melanogaster, Mutat. Res./Genet. Toxicol. Environ. Mutagen. 718 (2011) 33–37, http://dx.doi.org/10.1016/j.mrgentox. 2010.10.008. [38] P.P. Pompa, G. Vecchio, A. Galeone, V. Brunetti, S. Sabella, G. Maiorano, A. Falqui, G. Bertoni, R. Cingolani, In vivo toxicity assessment of gold nanoparticles in Drosophila melanogaster, Nano Res. 4 (4) (2011) 405–413, http://dx.doi.org/10.1007/s12274-011-0095-z. [39] A. Galeone, G. Vecchio, M.A. Malvindi, V. Brunetti, R. Cingolani, P.P. Pompa, In vivo assessment of CdSe-ZnS quantumdots: coating dependent bioaccumulation and genotoxicity, Nanoscale 4 (2012) 6401–6407, http://dx. doi.org/10.1039/c2nr31826a. [40] A. Joshi, S. Chandrashekaran, R.P. Sharma, Mutants dissecting development and behaviour in Drosophila, Curr. Sci. 89 (2005) 341–352.