Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 127-132, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-226 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: T. Atıcı, D. Altun Çolak (2020) The role of oleuropein against nano- composite toxicity in fruit fly: evidence for lifespan extension. Caryologia 73(3): 127-132. doi: 10.13128/caryologia-226 Received: April 24, 2020 Accepted: July 22, 2020 Published: December 31, 2020 Copyright: © 2020 T. Atıcı, D. Altun Çolak. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. The role of oleuropein against nanocomposite toxicity in fruit fly: evidence for lifespan extension Tuğba Atıcı1, Deniz Altun Çolak2,* 1 Institute of Natural and Applied Science, Erzincan Binali Yıldırım University, Erzincan, 24100 Turkey 2 Department of Biology, Faculty of Art and Science, Erzincan Binali Yıldırım University, Erzincan, 24100 Turkey * Corresponding author. E-mail: dnz_altun@yahoo.com Abstract. The effect of zinc oxide/titanium dioxide nanocomposite the lifespan of the fruit fly Drosophila melanogaster and the protective role of oleuropein, a strong anti- oxidant, against the zinc oxide/titanium dioxide were investigated. Chemicals prepared in different concentrations (0.005, 0.1, 0.5, and 1 g/L for zinc oxide/titanium dioxide; 0.1 mmol/L for oleuropein) have been separately applied to female and male popula- tions of D. melanogaster for the control and application groups. In both female and male populations, it has been observed that zinc oxide/titanium dioxide has decreased the lifespan and oleuropein has increased the lifespan according to the control group, depending on the concentration. These findings demonstrate the beneficial effect of oleuropein, suggested as a protective role in the prevention of zinc oxide/titanium dioxide induced developmental toxicity. Keywords: Drosophila melanogaster, lifespan, oleuropein, toxicity, zinc oxide/titanium dioxide nanoparticles. INTRODUCTION Nanotechnology has a great potential for applications in many fields advancements in nanotechnology are increasing rapidly (Karahalil 2013). So, this technology is appearing as a new research field that is investigating the potential risks of nanoparticles (NPs) on human and environmental health (Landsiedel et al. 2009). In this respect, many reports have demonstrated vari- ous effects of NPs exposure on different animals, plants, and microorganisms; depending on their species, growth conditions, NPs type, and exposure con- centrations. Also, the in vitro and in vivo studies using different experimen- tal models indicate that nanoparticles may cause genotoxic effects that involve oxidative stress and inflammation (Kang et al. 2008; Xu et al. 2009; Petković et al. 2011). However, the mechanisms involved in NPs-induced toxicity have not been clearly explained and are poorly studied in vivo. 128 Tuğba Atıcı, Deniz Altun Çolak Metal NPs are the most widely used among nano- particles. Titanium dioxide (TiO2) and zinc oxide (ZnO) NPs are of special concern since they used in food products, plastics, paper, drugs, cosmetics, sunscreens, paints, and medical materials and are two of the fast- est-growing product categories in the nanotechnology industry (Yeber et al. 2000). As a result of this wide- spread use, increased environmental release and a higher potential for human exposure will appear. The data from several in vitro studies demonstrate that TiO2NPs cause various adverse effects at the cellular level, such as oxi- dative stress and DNA damage (Wang et al. 2007). The aging process is generally described as the pro- gressive decline of homeostatic maintenance functions and physiological fitness (Sohal et al. 2002). Drosophila or fruit fly is commonly used for studying in the aging model system because the genetic background, devel- opmental processes, and some more aspects are well known. They are also highly similar to mammalians in terms of genetic structure (Çakır and Bozcuk 2000). The production of free radicals and other oxidants produced during aerobic respiration is the most reason for mecha- nistic explanations for aging links (Barja 2002). Oxida- tive stress is supposed to be an important mechanism underlying nanoparticle toxicity. Thus, it is believed that nanoparticle toxicity can be prevented by antioxidants (Nel et al. 2006; Mocan et al. 2010). From this point of view, the effects of zinc oxide/titanium dioxide (ZnO- TiO2) nanocomposite on the lifespan of D. melanogaster and the protective role of oleuropein (OLE, Olive Leaf Extract), a strong antioxidant, on these effects. MATERIALS AND METHODS Experimental organism Drosophila melanogaster (Diptera; Drosophilidae) Oregon R strain was reared in Standard Drosophila Medium (SDM) containing 15 g sucrose, 17 g cornmeal, 3 g agar-agar, and 9 g yeast in 360 mL distilled water within environmental chamber maintained at 25±1  °C and 40-60% relative humidity in darkness. Further addi- tion of 1 mL propionic acid as a preservative/fungicide was done. The flies used in the experiments were at the same age (1-3 days) and the females were virgins. Chemicals and solutions Oleuropein (98% pure; St. Louis, MO, USA) and tita- nium dioxide (99% pure, anatase, Steinheim, Germany) were purchased from Sigma Aldrich. ZnO-TiO2 (zinc oxide-titanium dioxide) nanoparticles were synthesized in the chemistry laboratory of Black Sea Technical Uni- versity. ZnO was loaded into the TiO2 photocatalyst with a 1% ratio. TiO2 catalyst (10 g) and water (10 mL) were mixed to have a slurry. ZnO (0.3355 g) was added to the slurry and calcined at 400 °C for 6 h. After this process, it was cooled in a desiccator and stored in a closed dark bottle. For the stock solutions, ZnOTiO2 NPs powder was dispersed in deionized water. Then, this solution was vortexed for 20 seconds, and sonicated for 30 min in an ultrasonic bath (Sonorex, Bandelin Electronic, Berlin, Germany) at a frequency of 60 kHz, to ensure uniform suspension of NPs. Finally, the nanoparticles concentra- tions were prepared by diluting the stock solution. Characterization of ZnOTiO2NPs ZnOTiO2NPs size distribution and morpholog y were represented by scanning electron microscope (SEM, (JEOL JSM 5600 LV, Tokyo, Japan) at the magnification of 100×. The hydrodynamic diameter was characterized by a master sizer (Malvern, Zetasizer ver. 7.02, Malvern Instruments Ltd, Worcestershire, UK) using the dynam- ic light scatter (DLS) technique. Lifespan assay The lifespan experiments were studied separately in female and male populations. For this purpose, about 100 individuals were collected from among the same aged (1-3 days) female and male flies which were not mated and obtained from the pupa. The gathered indi- viduals were then put into the empty culture bottles and left hungry for 2 hours before ZnOTiO2 and OLE appli- cations. For the application, two layers of blotting papers were placed into each culture vial; ZnOTiO2 and OLE in different concentrations (0.005, 0.1, 0.5, and 1 g/L for ZnOTiO2; 0.1 mmol/L for OLE) were absorbed into these papers. Afterward, the flies were put into these application vials and were left for 2 hours. After 2 hours, the individuals (separately female and male flies) were placed into the culture vials containing only SDM as 25 × 25. The experiments for both the control and applica- tion groups were started at the same time. All the vials were kept in appropriate thermal cabins. During the experiments, food was replaced with fresh food twice a week. The number of individuals was controlled both at the beginning and the end of each application day and the dead individuals were counted. The application was carried out until the last individual died. The experi- ments were repeated three times. 129Th e role of oleuropein against nanocomposite toxicity in fruit fl y: evidence for lifespan extension Statistical analyses Th e obtained data were analyzed with SPSS version 16.0 (Statistical Package for the Social Sciences Soft ware, SPSS, Chicago, IL). Th e mean lifespan values of the con- trol and application groups were subjected to Duncan’s one-way range test (p<0.05). RESULTS Characterization of ZnOTiO2NPs Th e size of the nanoparticles is an important param- eter that determines activity in biomedical applications. DLS is an analytical method that estimates the hydro- dynamic diameter while SEM is used for the estima- tion of the actual diameter of nanoparticles. DLS stud- ies revealed that the hydrodynamic diameter of ZnO- TiO2NPs was 42.5±1.2 nm. Th e morphological charac- terization of ZnOTiO2NPs was performed by SEM to visualize the actual particle size and the overall size dis- tribution (Figure 1). SEM image indicates that the nano- particles formed aggregates of diff erent sizes and these aggregations have a porous structure. Lifespan assay In this study, it was obser ved that ZnOTiO2, depending on the concentration, has decreased the lifes- pan of the male and female population according to the control group. It was also determined that OLE has increased the lifespan according to the control group. Th e maximum female lifespan of the control and appli- cation groups was observed for 78 days while the maxi- mum male lifespan belonging to the control and appli- cation groups was 76 days, respectively. Th e diff erence between the control and application groups is not statis- tically signifi cant (p>0.05) (Table 1). In the female population applied with ZnOTiO2, the maximum lifespan for the lowest concentration (0.005 g/L) was 70 days however for the highest concentration (1 g/L) the maximum lifespan was 54 days. Also, in the 0.1 and 0.5 g/L ZnOTiO2 application groups, the female maximum lifespan was 68 and 62 days, respectively (Figure 2). According to results, in the male population applied with ZnOTiO2, the maximum lifespan for the lowest concentration (0.005 g/L) was 71 days however for the highest concentration (1 g/L) the maximum lifespan was 51 days. Also, in the 0.1 and 0.5 g/L ZnOTiO2 applica- Figure 1. SEM images of ZnOTiO2NPs in dry form. Table 1. Th e longevity of male and female populations of D. melanogaster Experimental group (No) Female number Maximum lifespan Mean lifespan Probability level Male number Maximum lifespan Mean lifespan Probability level CONTROL - (1) 100 72 56.58±1.10 1-2* 4-7* 5-6* 100 74 55.79±1.12 1-2* 3-4* 3-7* 4-5* 4-7* 5-6* OLE (0.1 mmol/L)- (2) 100 78 57.64±1.23 100 76 55.51±1.17 ZnOTiO2 5 mg/L- (3) 100 70 49.48±1.45 100 71 44.12±1.84 0.1 g/L- (4) 100 68 45.17±1.59 100 66 41.03±1.85 0.5 g/L- (5) 100 62 35.41±1.29 100 60 39.11±1.70 1 g/L- (6) 100 54 34.19±1.65 100 51 35.50±1.45 ZnOTiO2 +OLE (1 g/L+0.1 mmol/L)- (7) 100 64 43.77±1.79 100 62 43.80±1.69 *Th e mean diff erence is not signifi cant at the 0.05 level. 130 Tuğba Atıcı, Deniz Altun Çolak tion groups, the male maximum lifespan was 66 and 60 days, respectively (Figure 3). Also in every group, it is determined that female individuals live longer in com- parison to male individuals (Table 1). DISCUSSION In recent years, studies about the toxic risks of NPs are increasing (Landsiedel et al. 2009; Yilmaz Öztürk 2019). First reports about the toxicity of some nano- particles show that they can affect biological systems at the organ, tissue, cellular, subcellular, and protein lev- els (Braydich-Stolle et al. 2009; Khajavi et al. 2019). In vivo toxicity studies have demonstrated that inhalation of TiO2 NPs causes pulmonary inflammation in rats and mice (Bermudez et al. 2004) and TiO2 NPs induce DNA damage and genetic instability in mice (Trouiller et al. 2009). Reeves et al. (2008) showed oxidative stress- related effects, including inf lammation, cytotoxicity, and genomic instability, either alone or in the presence of UVA irradiation, in mammalian studies. Also, zinc oxide nanoparticles have been reported to be cytotoxic and exhibited strong protein adsorption abilities (Horie et al. 2009). Toxicity of ZnO NPs on human bronchial epithelial cells was investigated and suggested that oxi- dative stress is a mechanism of toxicity (Heng et al. 2010). The production of free radicals has been supposed to be one of the primary mechanisms of NPs toxicity (Nel et al. 2006; Yang et al. 2009). It may result in oxi- dative stress, inf lammation, and consequent damage to proteins, membranes, and DNA (Bhabra et al. 2009; Hu et al. 2009). Thus, in our study, we investigated the toxic effects of ZnOTiO2 nanocomposite on the lifes- pan of D. melanogaster and the protective role of ole- uropein (OLE), a strong antioxidant. Oleuropein is the major phenolic constituent of olive leaves (Olea euro- paea) and is also present in the fruit and oil (van Acker et al. 1998). Many studies demonstrated that OLE has an antiinflammatory activities (Carluccio et al. 2003), free-radical scavenging properties (Le Tutour and Gue- don 1992; Manna et al. 1997) and inhibit the growth of different tumor cell types (Hamdi and Castellon 2005). In our preliminary study, it was determined that ZnO- TiO2NPs were relatively increased levels of total  oxidant status (TOS) and was decreased level of total  antioxi- dant capacity (TAC) compared to the control group. But, in a ZnOTiO2+OLE group, it was observed that vice ver- sa (Çolak et al. 2016). CONCLUSION These experiments provide evidence that ZnOTiO2 nanocomposite can shorten the lifespan of Drosophila which is partially or completely prevented by oleuropein. This means oxidative stress is the major contributor to ZnOTiO2 toxicity. 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