Comparative Neurotoxicity Evaluation of Zinc Oxide Nanoparticles by Crawling Assay on Drosophila melanogaster

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1 Comparative Neurotoxicity Evaluation of Zinc Oxide Nanoparticles by Crawling Assay on Drosophila melanogaster Kritika Sood, Jasreen Kaur, Madhu Khatri* (Department of Biotechnology Engineering, UIET, Panjab University, Chandigarh, India) ABSTRACT This paper evaluates the neurotoxicity imposed by Zinc Oxide nanoparticles on Drosophila melanogaster larvae by conducting crawling assay, thereby exhibiting the affect that these nanoparticles have on the neuromuscular coordination of these larvae. Both green and chemically synthesized ZnO nanoparticles were studied during our research in order to assess and compare the neurotoxic effects caused by these nanoparticles on the crawling ability of these larvae. The influence of ZnO nanoparticles on the central nervous system (CNS) of these larvae were studied and can be related to the comparative neurotoxicity caused by green and chemically synthesized nanoparticles. The results show a notable increase in crawling ability of these larvae at concentration of 100µg/ml in both green and chemically synthesized ZnO NPs thereby indicating that a change in neuronal activity of these larvae occurred at this concentration that may have affected its neuromuscular coordination. However, further studies need to be carried out to quantify the changes occurring in larval CNS caused by these nanoparticles. Keywords- crawling assay, nanoparticles, neurotoxicity, zinc oxide, Drosophila melanogaster, larvae. I. INTRODUCTION Zinc oxide nanoparticles (ZnO NPs) has widespread uses in cosmetic preparations and daily healthcare items such as tooth paste and sunscreen lotions due to its optical and biochemical properties and as skin protectant by absorbing the ultraviolet sunburn rays. Textiles, wall paints, and building materials industries also use ZnO NPs in their ingredients. Because of their special properties including small size and high surface area/volume ratio, the biological safety of nanomaterials has been scrutinized over and again. Toxicity of ZnO NPs has been extensively studied in different type of animal systems and cell lines. The cytotoxicity of cell lines has also been found in many cell cultures such as macrophages[1], human lung epithelial cells[2] and CHO cells[3]. Results have shown that ZnO NPs reach various organs after systemic distribution, and manifest hazardous effects on the lungs, brain, blood, liver, kidney, stomach, pancreas, spleen, testis and heart[4][5][6][7][8]. Recently many researchers have provided evidence that NPs can cross the brain via blood-brain barrier (BBB) and along the olfactory nerve pathway and subsequently cause damage by the inducing oxidative stress, inflammatory responses, and cytotoxicity which is not case for macro or microparticles. It has also been reported that ZnO NPs could reach the brain after oral and nasal administration in animals and induce changes in the spatial learning and memory ability of rats by altering the synaptic plasticity[9] and interact diversely with plasma and brain protein thus causing toxic effects in both the blood and the brain[10]. Thus, the neurotoxicity caused by ZnO NPs is being assessed in this paper by using Drosophila melanogaster as a model organism. Drosophila has been extensively studied by neurobiologists due to its simple genetic structure, easy manipulation, high fecundity and low cost maintenance. Neurodegenerative diseases belong to one class of age-related, slowly progressing neurological diseases that include Alzheimer s disease (AD), Parkinson s disease (PD), Huntington s disease, and amyotrophic lateral sclerosis (ALS)[11]. In recent years, an increasing number of studies have indicated that particulate matter (PM) and NPs in the environment may pose many risk factors for neurodegenerative diseases[12]. 440 Kritika Sood, Jasreen Kaur, Madhu Khatri

2 Thus, we aim to evaluate the neurotoxicity caused by ZnO NPs synthesized by both green and chemical methods and compare these results using larval crawling ability as a parameter[13]. However, detailed studies need to be carried out to quantify the changes occurring in larval CNS caused by these nanoparticles. II. MATERIALS AND METHODS 1. Synthesis of Zinc Oxide nanoparticles by Green and Chemical Method Zinc Oxide nanoparticles were synthesized by two different methods- Chemical method and Green method. In chemical synthesis, zinc acetate dehydrate was allowed to react with 2M NaOH added dropwise until ph 12 at room temperature and kept at stirring for 2 hrs. In green synthesis, aqueous leaf extract of Corriandrum sativum was added dropwise to zinc acetate dehydrate and kept on stirring for 1 hr[14].next, 2M NaOH was added dropwise to the solution at room temperature until ph 12 is reached and kept at stirring for 2 hrs. The solution is then centrifuged at 9000 rpm for 20 min to extract the nanoparticles and washed with distilled water and ethanol in alternation 3-4 times to remove any kind of impurities. The obtained nanoparticles are then kept in hot air oven at 70 C temperature overnight for drying. The powder formed is collected and stored at a cool place away from direct sunlight for further characterization and use. 2. Characterisation of nanoparticles The nanoparticles so formed are characterized using HR-TEM, EDS, DLS and Zeta Sizer. The instrument used for HR-TEM (Figure1 and Figure 2) and EDS is DST funded and the analysis was done at NIPER, Mohali. The grid used was made of Copper coated with Carbon (as shown in figure 3 and figure 4). The DLS and zeta potential of particles (Table 1) were analysed using the zeta sizer (Malvern Instruments Ltd., UK). 3. Culturing of flies The flies used in this experiment were of strain W118 received as a kind gift from Dr. Lolitika Mandal, IISER- Mohali. The fly food consisted of maize powder, sugar, yeast, agar, nipagin and propionic acid. The flies were transferred to new bottles periodically and the stocks were maintained at 25 C. 4. Crawling Assay Third instar larvae (10 per concentration/ nanoparticle) were obtained and put in 5% sucrose solution[13] along with 500ul of each nanoparticle at different concentration and were allowed to feed for atleast 45 mins. Next, the flies were graded for their crawling ability using a petriplate in which graph paper was attached to the base and 2% agar was poured over it and allowed to harden. The number of 0.1X0.1 squares traversed by each larva per concentration per nanoparticle was recorded for 1 minute interval each. The total distance travelled was then calculated and results were shown graphically (figure 5). III. RESULTS Fig 1-HR-TEM images of chemical ZnO nanoparticles at 19000X (left) and 5000X (right) magnification 441 Kritika Sood, Jasreen Kaur, Madhu Khatri

3 Fig 2- HR-TEM images of green ZnO nanoparticles at 19000X (left)and 5000X(right) magnification Fig 3- EDX Results of Green ZnO nanoparticles Table 1- DLS, zeta potential and pdi results of ZnO nanoparticles NPs DLS (nm) Zeta potential PdI Result Quality (mv) Green ZnO Good Chemical ZnO Good 442 Kritika Sood, Jasreen Kaur, Madhu Khatri

4 Fig 4- EDX Results of Chemical ZnO nanoparticles Fig 5- Number of squares vs Concentration of nanoparticles graph. The average no. of squares crossed by larvae treated with Green ZnO (shown in blue) is greater than the average no. of squares crossed by larvae treated with Chemical ZnO (shown in red). Both nanoparticles show a notable hike in their crawling ability at 100 µg/ml and crossed approximately the same no. of squares at this concentration. 443 Kritika Sood, Jasreen Kaur, Madhu Khatri

5 IV. CONCLUSIONS AND DISCUSSIONS The HR-TEM results show that all of the ZnO nanoparticles synthesized by both green and chemical methods are within permissible limits of nm range. The DLS results show that the average size of green ZnO nanoparticles is 498 nm while that of chemical ZnO is 519 nm. The zeta potential of green ZnO is and that of chemical ZnO is which are both more than -25, indicating lesser aggregation. The polydispersity index (PdI) of green nanoparticles is while that of chemical ZnO NPs is 0.409, both are less than 1, showing less aggregation of nanoparticles, which is a good sign, signifying that the nanoparticles are monodispersed. The EDS results show that nanoparticles synthesized by both green and chemical methods are both 100% pure and the peaks for Cu and C are due to coating material of the grid used during analysis. The trend with increasing concentration of exposure of both green and chemically synthesized nanoparticles is the same i.e. it decreases upto a certain concentration, and upon reaching 100 µg/ml, it gives a notable hike and then decreases again until 300 µg/ml. This indicates that both these nanoparticles affect the larvae in a somewhat similar manner although the average no. of squares crossed by larvae in green ZnO is greater than chemically synthesized ZnO nanoparticles. The concentration of 100 µg/ml is somewhat pivotal and signifies a change in neuronal activity of the larvae thereby affecting its neuromuscular coordination and hence it s crawling ability. However, further studies need to be carried out to quantify and assess the changes observed with such behavior in these larvae. V. REFERENCES 1. Roy, R., Tripathi, A., Das, M. & Dwivedi, P. D. Cytotoxicity and uptake of zinc oxidenanoparticles leading to enhanced inflammatory cytokines levels in murine macrophages: comparison with bulk zinc oxide. J Biomed Nanotechnol. 7, (2011). 2. Hsiao, I. L. & Huang, Y. J. Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells. Sci Total Environ. 409, (2011). 3. Dufour, E. K., Kumaravel, T., Nohynek, G. J., Kirkland, D. & Toutain, H. Clasto-genicity, photo-clastogenicity or pseudo-photoclastogenicity: genotoxic effectof zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinesehamster ovary cells. Mutat Res. 607, (2006). 4. Vandebriel, R. J. & De Jong, W. H. A review of mammalian toxicity of ZnO nanoparticles. Nanotechnol Sci Appl. 5, (2012). 5. Cho, W. S. et al. Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes. Part Fibre Toxicol. 8, 27 (2011). 6. Cho, W. S. et al. Zeta potential and solubility to toxic ions asmechanisms of lung inflammation caused by metal/metal oxide nanoparticles. Toxicol Sci. 126, (2012). 7. Li, C. H. et al. Organ biodistribution, clearance, and genotoxicity of orally administered zinc oxide nanoparticles in mice. Nanotoxicology. 6, (2012). 8. Wang, B. et al. Acute toxicological impact of nano- and submicro-scaled zinc oxide powder on healthy adult mice. J Nanopart Res. 10, (2008). 9. Han, D., Tian, Y., Zhang, T., Ren, G. & Yang, Z. Nano-zinc oxide damages spatial cognition capability via overenhanced longterm potentiation in hippocampus of Wistar rats. Int J Nanomedicine. 6, (2011). 10. Shim, K. H., Hulme, J., Maeng, E. H., Kim, M. k. & An, S. S. Analysis of zinc oxide nanoparticles binding proteins in rat blood and brain homogenate. Int J Nanomedicine. 9, (2014). 11. Tian, L. et al. Neurotoxicity induced by zinc oxide nanoparticles: age-related differences and interaction. Sci. Rep. 5, 16117; doi: /srep16117 (2015). 12. Costa, L. G., Cole, T. B., Coburn, J., Chang, Y. C., Dao, K. & Roque, P. Neurotoxicants are in the air: convergence of human, animal, and in vitro studies on the effects of air pollution on the brain. Biomed Res Int. 2014, (2014). 13. Nichols CD, Becnel J, Pandey UB. Methods to assay Drosophila behavior. Journal of Visualized Experiments. 2012;61:e3795 doi: / One Pot Synthesis of Zinc Oxide Nanoparticles via Chemical and Green Method, Gnanasangeetha D.and SaralaThambavani D., Research Journal of Material Sciences, Vol. 1(7), 1-8, August (2013) 444 Kritika Sood, Jasreen Kaur, Madhu Khatri