Elsevier

Toxicology Letters

Volume 199, Issue 3, 15 December 2010, Pages 389-397
Toxicology Letters

Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles

https://doi.org/10.1016/j.toxlet.2010.10.003Get rights and content

Abstract

With large-scale production and wide application of nanoscale ZnO, its health hazard has attracted extensive worldwide attention. In this study, cytotoxicity of different sized and shaped ZnO nanoparticles in mouse macrophage Ana-1 was investigated. And contribution of dissolved Zn2+ and ROS in toxicity of ZnO particles was analyzed. The results indicated that ZnO particles manifested dose-dependent toxic effect on Ana-1 cells without size-dependence, and the particles shape may impact cytotoxicity of ZnO particles. When the concentration of dissolved Zn2+ tended to equilibrium in the complete cell medium, the zinc ion concentration was approximately 10 μg/ml, inducing about 50% cell death, which was close to the cytotoxicity of ZnCl2 (IC50 = 13.33 μg Zn/ml). The Zn2+ concentration had significant correlations with cell viability and LDH level induced by the supernatant of ZnO particle suspensions (incubation at 37 °C for 24 h). Thus, the dissolved Zn2+ played the main role in toxic effect of ZnO particles. Moreover, ROS generation assays demonstrated that ZnO particles produced intrinsically a small quantity of ROS, intracellular ROS was mainly produced after ZnO particles or the dissolved Zn2+ entered into the cells. Although intracellular ROS had significant correlations with cell viability and LDH induced by ZnO particles, intracellular ROS may not be a major factor in cytotoxicity of ZnO nanoparticles, but the cytotoxic response.

Introduction

Nano scale ZnO has become a nano-functional material, widely concerned following carbon nanotubes. It is one of the materials with well-established production process and test method, and also is one of the materials of first commercial application. Since the Bayer Corporation (Bayer Co., Ltd.) first provided with nano-ZnO products in the market, various countries started the large-scale industrial production of nano-ZnO. Nano-ZnO is widely used in many fields, such as rubber manufacture, cosmetics, pigments, food additives, medicine, chemical fiber and electronics industries (Ji and Ye, 2008). However, theoretical study of nano-ZnO lags behind its production and application. And due to the traditional concept that zinc oxide is non-toxic, the toxicology study of nano-ZnO is farther behind the speed of its application development. In recent years, with the rise and development of nanotoxicology, health risks of nano-ZnO are gradually concerned. The study on the acute toxicological impact of ZnO nanoparticles on mice showed that the liver, spleen, heart, pancreas and bone are the target organs for 20 and 120 nm ZnO oral exposure (Wang et al., 2008). And exposures to nanoscale or fine-sized ZnO particles by intratracheal instillation produced potent but typical “metal fume fever”-like reversible inflammation (Sayes et al., 2009). Moreover, many in vitro studies also demonstrated that ZnO nanoparticles are toxic to mammalian cells. For example, ZnO nanoparticles induced toxicity in RAW264.7 and BEAS-2B cells, leading to the generation of reactive oxygen species (ROS), oxidant injury, excitation of inflammation, and cell death, and ZnO dissolution could happen in culture medium and endosomes (Xia et al., 2008). In addition, some studies have demonstrated that nano-ZnO is more toxic than other nanometer-scale structured metallic oxides (Jeng and Swanson, 2006, Lai et al., 2008, Horie et al., 2009).

However, there are still a lot of controversies on the toxicity of ZnO nanoparticles. Firstly, there is not a consistent conclusion on toxicity of ZnO nanoparticles and the impact of particle size on toxicity of ZnO nanoparticles. Some research showed that different sized ZnO nanoparticles had similar cytotoxicity on different cells, 24 h IC50 was about 10–20 μg/ml, and particle size had no effect on cytotoxicity (Lin et al., 2009, Deng et al., 2009, Yuan et al., 2010). Nair et al. found 100 μM (8.1 μg/ml) ZnO nanoparticles induced osteoblast cancer cells (MG-63) viability decrease to about 40%, and when particle size was under 350 nm, the size did not impact cytotoxicity, but nanoparticles were more toxic to MG-63 cell than microparticles (Nair et al., 2009). Reddy et al. reported ZnO nanoparticles (13 nm) still were not cytotoxic at 5 mmol/L (400 μg/ml), only at 10 mmol/L (800 μg/ml), ZnO nanoparticles caused 57% cell death. And cytotoxicity is limited to ZnO in the nanoscale size range as no significant effect of bulk ZnO powder was observed (Reddy et al., 2007, Hanley et al., 2008). Secondly, the effect of particle shape on cytotoxicity of ZnO nanoparticles is not yet clear. Lee et al. found spherical ZnO particles induced lower toxicity than rod-shaped ZnO particles (Lee et al., 2008). However in the study of Lee et al., ZnO exposure concentration was based on the bottom area of culture plate, if mass-based dosage conversed, the exposure concentration of rod ZnO particles was much higher than spherical ZnO particles. Nair et al. showed that rod ZnO nanoparticles were lower toxic than spherical ZnO nanoparticles, but rod ZnO nanoparticles were coated by starch and spherical ZnO nanoparticles were coated by poly ethylene glycol (PEG), starch and PEG may impact on toxicity of ZnO nanoparticles (Nair et al., 2009). In addition, now there are main two explanations on the toxicity mechanism of ZnO nanoparticles: zinc dissolution and oxidative damage, but the contribution of two sides is uncertain. Deng et al. found that ZnO nanoparticles and ZnCl2 had similar toxicity, and did not observe ZnO particles within cells with the TEM (Deng et al., 2009). Xia et al. also suggested that zinc ion played an important role in cytotoxicity of ZnO nanoparticles, and did not find ZnO nanoparticles in cells (Xia et al., 2008). However, Jeng and Swanson compared cytotoxicity of a variety of metal oxide nanoparticles on the Neuro-2A cell, and considered oxidative damage had an important effect on cytotoxicity (Jeng and Swanson, 2006). Lin et al. showed nanoparticles and microparticles of ZnO decreased A549 cell viability, and observed particles in cells with TEM. They still found toxicity of ZnO particles was reduced by antioxidant N-Acetylcysteine, this demonstrated toxicity of ZnO particles related with oxidative damage. Moreover, they considered that the low dissociation of ZnO in the cell culture medium (Ksp = 3.0 × 10−16) was not enough to cause cell toxicity. Thus, they inferred that oxidative damage induced by ZnO particles was due to sequential oxidation–reduction reactions occurring at ZnO particles surface to produce reactive species (Lin et al., 2009).

Macrophages were multifunctional immune cells. Their phagocytosis was a main pathway to clean up particles in vivo, and macrophages had a variety of stimulating rapid response capacities, played an important part in regulating the immune response and inflammation. ZnO nanoparticles were widely added to the feed as additives. Ding et al. showed that the oral exposure to low dosage of ZnO nanoparticles in mice increased the phagocytosis of mouse peritoneal macrophages (Ding et al., 2007). However, the oral exposure to high dosage of ZnO nanoparticles induced the edema and degeneration of hepatocytes, inflammation of pancreas and damages of stomach and spleen (Wang et al., 2008). Meanwhile, ZnO nanoparticles at high dose still may destroy the immune system. Especially, peritoneal macrophage had a close connection with pancreatitis (Ma et al., 2009). At present, the impact of ZnO nanoparticles on the immune system is not clear. Thus, in this study, we took mouse peritoneal macrophages Ana-1 cells as subject, investigated cytotoxicity of different sized and shaped ZnO particles, and analyzed the effect of the dissolved Zn2+ and ROS in cytotoxicity.

Section snippets

ZnO particles

Four different sized ZnO particles were supplied from commercial companies. Both fine-ZnO particles (<1 μm) were purchased from Hangzhou Wanjingxin Material Co. Ltd., China. 100 ± 10 nm and 30 ± 10 nm ZnO particles were purchased from Beijing Nachen Technology Co. Ltd., China. 10–30 nm ZnO particles were purchased from Shenzhen Nanuo Nanomaterials Corp., China. Visualize particles size and shape of ZnO particles was measured by transmission electron microscopy (TEM, H7650). Crystal structure of ZnO

Characterization of ZnO particles

The X-ray diffraction analysis (Fig. 1) clearly showed that all four types of ZnO particles were the hexagonal structure.

The TEM micrographs demonstrated the particle shapes and sizes (Fig. 2). Fine-ZnO, 100 nm ZnO and 30 nm ZnO were rod-shaped, with lengths of 341.75 ± 173.34 nm, 107.59 ± 38.44 nm and 70.89 ± 34.18 nm, diameters of 173.48 ± 72.73 nm, 60.21 ± 21.76 nm and 40.44 ± 12.51 nm, respectively. 10–30 nm ZnO was spherical with diameters of 18.76 ± 4.98 nm. Among them, the mean size of 30 nm ZnO was very

Cytotoxicity of ZnO particles

Much research has showed that ZnO nanoparticles resulted in cytotoxicity to many types of cell, such as human bronchial epithelial cells (BEAS-2B) (Huang et al., 2010), human lung epithelial cells (A549) (Lin et al., 2009, Karlsson et al., 2008, Horie et al., 2009), human keratinocyte HaCaT cells (Horie et al., 2009), human epidermal cell (A431) (Sharma et al., 2009), L2 rat lung epithelial cells and rat alveolar macrophages (Sayes et al., 2007, Sayes et al., 2009), the mouse macrophage cell

Conclusions

In summary, different sized ZnO particles induced slightly different cytotoxicity. The particles shape may impact on cytotoxicity of ZnO particles. And the dissolved Zn2+ equilibrium concentrations of ZnO particles in the complete cell medium were about 10 μg/ml, meanwhile, the supernatants induce 50% cell death. Thus, the dissolved Zn2+ played a main role in toxic effect of ZnO particles. Furthermore, ROS generation of ZnO particles under abiotic conditions is not enough to cause cytotoxicity.

Conflict of interest statement

None.

Acknowledgement

The authors are grateful to the financial support from the National Natural Science Foundation of China (30771771, 30800934 and 30901221) and the Natural Science Foudation of Tianjin (10JCZDJC17100).

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