The protective role of resveratrol against zinc oxide induced nanotoxicity

Zinc oxide (ZnO) is a compound that has harmful effects as well as being used in many different areas. Numerous studies have been carried out to minimize the toxic effects of ZnO nanoparticles (NPs). In the present study, the protective role of resveratrol (RSV), a potent antioxidant polyphenol substance, was examined against ZnO-induced nanotoxicity on human pulmonary alveolar epithelial cells (HPAEpiC). In this context, the cytotoxic and genotoxic effects of different concentrations of RSV (5, 10, 20 mg/L) and ZnO NPs on the cells were measured alone and in combination. At the same time, the effects of aforementioned applications on the total antioxidant capacity (TAC) level in HPAEpiC were assessed. The results obtained showed that ZnO NPs alone significantly increased cytotoxicity and genotoxicity on cells compared to negative control (control (-)). In the experiments performed with RSV + ZnO NP combination, cytotoxic and genotoxic activity decreased at the level of p < 0.05 especially at 20 mg/L application of RSV. When the level of TAC in cells was examined, a concentration-dependent increase was detected between TAC and RSV. It was determined that ZnO NPs reduced the TAC level statistically (p < 0.05) in comparison with control (-). In conclusion, the present study revealed that RSV, a natural antioxidant, showed protective property against genotoxic and cytotoxic damage induced by ZnO NPs on HPAEpiC.


Introduction
Zinc oxide (ZnO) has very key properties.This compound has many industrial applications due to its unique properties such as high refractive index, high thermal conductivity, antibacterial property and low expansion coefficient (Moezzi et al., 2012).ZnO has proven itself in many sectors such as porcelain, ceramic, rubber, paint, cosmetics, paint, fertilizer, ink and flame retardant.ZnOcontaining creams are used to aid to the treatment of sunburn, insect bites and itchiness, rashes and skin irritations (Özgür et al., 2005).On the other hand, side effects of ZnO-containing products can not be overlooked.Especially cosmetic products containing ZnO nanomaterials have a significant risk of absorption.Toxicology studies have shown that nanomaterials can have various side effects on the central nervous system, the immune system and the lungs (Dwivedi et al., 2009;Zhao and Castranova, 2011).
The most important entry and target organ of the nanoparticles (NPs) in the human body is the lungs.It is well known that the lungs are easily exposed to NP materials (Kendall and Holgate, 2012).One of the main mechanisms of lung damage caused by NPs that are formed as a result of burning is the oxidative stress.Brown et al. (2001) reported that there is a significant correlation between the surface area of NPs and the inflammation caused by oxidative stress.It has also been determined that high-size NPs cause genotoxicity (Donaldson et al., 2010).Antioxidants, especially those of herbal origin, play an important role in reducing of oxidative stress and genetic damage in human body (Birben et al., 2012).Resveratrol (RSV) is one of these important natural components.RSV in the phytoalexin group is obtained as a secondary metabolite from different plants.The rate of this compound is especially high in Vitis vinifera L. bark (Savouret and Quesne, 2002).RSV with antioxidant activity inhibits the progression of vascular stiffness by inhibiting low density lipoprotein oxidation and reduces lipid peroxidation and reduces lipid peroxidation and production of reactive oxygen species (King et al., 2006).
Based on high antioxidant properties of RSV, in the present study, the toxicity induced by ZnO and the possible beneficial effect of RSV against ZnO-induced cyototoxicity and genotoxicity were investigated.

NP Synthesis and Characterization
ZnO NPs were synthesized by direct precipitation method using zinc nitrate and KOH as precursors as previously described (Ghorbani et al., 2015).For the synthesis, zinc nitrate hexahydrate (Zn(NO 3 ) 2 .6H 2 O) and KOH solutions were prepared using deionized water.The KOH was slowly added into Zn(NO 3 ) 2 .6H 2 O solution at room temperature under stirring, which resulted in the formation of a white suspension.The white product was centrifuged at 5000 rpm for 25 min and washed three times with distilled water, and one time with absolute alcohol.The obtained product was calcined at 500°C for 4 h.Characterization of NPs UV-Vis spectroscopy was used to prove the existence of nanoparticles.The NPs were characterized by X-ray diffraction (XRD).The average crystallite sizes of the ZnO NPs were calculated from the full width at half maximum of XRD peaks by using Debye-Scherrer's formula and were found to be between 50-100 nm.

Antioxidant Compound
RSV (Cas: 501-36-0, C 14 H 12 O 3 ) that used in this investigation as an antioxidant agent was obtained from Sigma-Aldrich, Germany.

Cell Cultures and Treatments
Human pulmonary alveolar epithelial cells (HPAEpiC) was provided from American Type Culture Collection (ATCC, USA).The cells were grown and maintained in Alveolar Epithelial Cell Medium (AEpiCM) consists of 500 ml of basal medium, 10 ml of fetal bovine serum, 5 ml of epithelial cell growth supplement and 5 ml of penicillin-streptomycin at 37°C in a 90% humidified incubator with 5% CO 2 .

Statistical Analyses
All the assays were carried out at least in triplicate measurements.Protective abilities of antioxidant agent were analyzed using variance (ANOVA) test followed by appropriate post-hoc test (Duncan test) and values with p < 0.05 were considered as significantly different.Hierarchical cluster analysis (HCA) with Ward's minimum variance method was utilized to investigate the similarities and dissimilarities among the cytotoxic effects.All analyses were performed using Statistical Package for Social Sciences (SPSS, version 21.0, IBM Corporation, Armonk, NY, USA).

Cytotoxicity activities
The results of the present study showed that control (+) caused maximum cytotoxicity (85.43%) on HPAEpiC.Next high cytotoxicity (51.79%) value belonged to ZnO NPs.The effects of RSV treatments on viability of the cells were high.Important statistical significances in the reduction of cell viabilities were found in the cultures concomitantly treated with RSV and ZnO NPs as compared to the group ZnO NPs treated alone.Cytotoxic effects of RSV 3 and RSV 3 + ZnO NPs applications were not statistically (p > 0.05) different (Figure 1).

Discussion
There are many studies showing the negative effects of NPs on humans (Tourinho et al., 2012;Fröhlich and Salar-Behzadi, 2014).It has been found that there is a relationship between NPs and respiratory system diseases such as lung cancers, obstructive and interstitial lung diseases (Bonner, 2010).Possible toxic effects of NPs on other organs and systems outside the respiratory system have also been investigated.The toxic effects on the central nervous system are another of these damages.The toxicity that NPs cause as a result of DNA damage disturbs the whole life balance (Glauert et al., 2008).Inhibition of the resulting toxic effects in different areas through natural products is one of the common areas of study (Emsen et al., 2016;Dogan et al., 2017).Especially natural antioxidant components are preferred in this process.Some of these components increase the antioxidant capacity by minimizing oxidative stressinduced damage (Karatas et al., 2015).RSV, one of these important compounds, has been used in many studies.RSV that is a compound in the phenolic structure and has strong antioxidant activity, is used for many treatment process.It was reported that RSV supplementation helped to prevent joint damage by reducing inflation in the body (Hao et al., 2017).It has also been shown that resveratrol may be effective in combating various cancer cells such as liver, breast, pancreas and prostate (Carter et al., 2014).
In the present study, 8-OH-dG level measured in the cells showed that ZnO NPs caused DNA damage in HPAEpiC.At the same time, cytotoxic effects of ZnO NPs were determined.It was detected that cytotoxic and genotoxic activities of these NPs were significantly high.However, RSV supplementation to cultures with ZnO NP increased the viabilities rate of the cells.As shown in dendogram in figure 2, RSV 3 + ZnO treatment was included with control (-) in one branch.So, RSV 3 was the most critical experiment in terms of reducing ZnO NP-induced cytotoxicity.Furthermore, ZnO NP-induced genotoxicity could be reduced significantly by the presence of different concentrations of RSV.In previous studies, it was reported that the cytotoxic and genotoxic activities increased in various cells such as MCF-7 (Elavarasan et al., 2017) and MDA-MB-231 human breast cancer cells (Roshini et al., 2017) treated with ZnO alone.In a research carried out on mouse testicular cells, it was pronounced that ZnO NPs induced apoptosis through DNA damage caused by reactive oxygen species (Han et al., 2016).There are ZnO NPs induced genotoxicity studies in the literature.Kononenko et al. (2017) revealed that ZnO NPs caused genotoxicity in vitro in the Madin-Darby canine kidney cells.On the other hand, some investigators reported that high oxidative stress caused by ZnO NPs in the cells generate oxidant injury, reactive oxygen species (ROS) and excitation of inflammation (Xia et al., 2008).The oxidant substances such as free radicals are inhibited through antioxidant compounds.The level of oxidative stress is reduced due to the increase of the antioxidant capacity in the cells (Birben et al., 2012).Our findings were in line with previous reports.The TAC in the tested cells was increased through a natural antioxidant component, RSV.
The findings of this investigation clearly indicated that RSV modulated ZnO-induced genetic damage in HPAEpiC due to its strong antioxidant and detoxifying nature.Therefore, natural antioxidants could be beneficial against heavy metal poisoning including zinc.

Figure 1 .
Figure 1.Viability rates in the HPAEpiC exposed to different treatments.Each value is expressed as mean ± standard deviation (n = 3).Values followed by different small letters differ significantly at p < 0.05 HCA was performed for cytotoxicity of control groups, RSV and ZnO NPs on HPAEpiC.Cytotoxic activities showed that the nine treatments can be divided into three groups (group A, B and C).Group A was larger than those of groups B and C. Control (+) was single treatment in group C and it had the most distant relationship with the

Figure 2 .
Figure 2. Dendrogram built from cytotoxic effects of different treatments tested on HPAEpiC3.2.Genotoxicity activitiesOxidative stress induced DNA damage emerged in HPAEpiC by RSV and ZnO NPs was measured with 8-OH-dG level occurring in the cells.It was observed that control (+), RSV 2 and RSV 3 applications significantly (p < 0.05) increased 8-OH-dG concentrations in the cells.RSV 1 was statistically (p > 0.05) indifferent from control (-).RSVs significantly decreased 8-OH-dG concentrations in ZnO NPs-treated HPAEpiC in a dose dependent manner (Figure3).

Figure 3 .
Figure 3. 8-OH-dG adducts in the HPAEpiC exposed to different treatments.Each value is expressed as mean ± standard deviation (n = 3).Values followed by different small letters differ significantly at p < 0.05

Figure 4 .
Figure 4. TAC levels in the HPAEpiC exposed to different treatments.Each value is expressed as mean ± standard deviation (n = 3).Values followed by different small letters differ significantly at p < 0.05