Induction of oxidative stress, DNA damage and apoptosis in mouse liver after sub-acute oral exposure to zinc oxide nanoparticles

Abstract

Zinc oxide (ZnO) nanoparticles are finding applications in a wide range of products including cosmetics, food packaging, imaging, etc. This increases the likelihood of human exposure to these nanoparticles through dermal, inhalation and oral routes. Presently, the majority of the studies concerning ZnO nanoparticle toxicity have been conducted using in vitro systems which lack the complex cell–cell, cell–matrix interactions and hormonal effects found in the in vivo scenario. The present in vivo study in mice was aimed at investigating the oral toxicity of ZnO nanoparticles. Our results showed a significant accumulation of nanoparticles in the liver leading to cellular injury after sub-acute oral exposure of ZnO nanoparticles (300 mg/kg) for 14 consecutive days. This was evident by the elevated alanine aminotransferase (ALT) and alkaline phosphatase (ALP) serum levels and pathological lesions in the liver. ZnO nanoparticles were also found to induce oxidative stress indicated by an increase in lipid peroxidation. The DNA damage in the liver and kidney cells of mice was evaluated by the Fpg-modified Comet assay which revealed a significant (p < 0.05) increase in the Fpg-specific DNA lesions in liver indicating oxidative stress as the cause of DNA damage. The TUNEL assay revealed an induction of apoptosis in the liver of mice exposed to ZnO nanoparticles compared to the control. Our results conclusively demonstrate that sub-acute oral exposure to ZnO nanoparticles in mice leads to an accumulation of nanoparticles in the liver causing oxidative stress mediated DNA damage and apoptosis. These results also suggest the need for a complete risk assessment of any new engineered nanoparticle before its arrival into the consumer market.

Introduction

Nanotechnology is now making headway in different spheres of human lives. This is leading to a diverse array of products with applications in diagnosis, drug delivery, food industry, paints, electronics, sports, environmental cleanup, cosmetics, sunscreens, etc. [1], [2]. At the same time, the novel and useful properties possessed by these engineered nanomaterials can lead to unpredictable outcomes in terms of their interactions with biological systems. Therefore, it is necessary to understand and assess the potential toxicity of engineered nanomaterials to avoid their adverse effects on environmental and human health.

Among the varieties of engineered nanoparticles being used today, zinc oxide (ZnO) nanoparticles are one of the most widely used in consumer products. They are extensively used in cosmetics and sunscreens because of their efficient UV absorption properties without scattering the visible light. This makes them transparent and more aesthetically acceptable as compared to their bulk counterpart [3]. ZnO nanoparticles are being used in the food industry as additives and in packaging due to their antimicrobial properties [4], [5]. They are also being explored for their potential use as fungicides in agriculture [6] and as anticancer drugs and imaging in biomedical applications [7], [8].

The increased production and use of ZnO nanoparticles enhances the probability of exposure in occupational and environmental settings. This has culminated in some studies investigating the toxicity of zinc oxide nanoparticles in different biological systems such as bacteria [9], [10] and mammalian cells [11]. In mammalian cells, the toxic effects of ZnO nanoparticles such as membrane injury, inflammatory response, DNA damage and apoptosis have been demonstrated [12], [13], [14], [15], [16]. The majority of these studies have been conducted using in vitro systems. However, unlike the in vivo systems, the complex cell–cell and cell–matrix interactions as well as the diversity of cell types and hormonal effects are not present in cultured cells. Studying the long-term chronic effects of the test compound is also not possible without in vivo experiments. The importance of the in vivo studies in the area of nanomaterial toxicology has already been highlighted [17].

There are no existing guidelines or standard methodologies for risk assessment of nanomaterials. However, the “committees on toxicity (COT), mutagenicity (COM) and carcinogenicity (COC) of chemicals in food, consumer products and the environment, UK” have suggested extrapolating the in vitro nanotoxicity findings to in vivo experiments and confirm the results [18]. The same committees (COT, COM and COC) have also advised on the importance of considering appropriate routes of exposure in the in vivo experiments. In case of engineered nanoparticles, the exposure and uptake can occur through different routes. For ZnO nanoparticles, the oral exposure and uptake through the gastrointestinal route needs to be considered. This is keeping in view the fact that ZnO nanoparticles can be ingested directly when used in food, food packaging, drug delivery and cosmetics. Workers involved in the synthesis of ZnO nanoparticles can be exposed by unintentional hand-to-mouth transfer of nanomaterials. When discharged accidentally into the environment, these nanoparticles may enter the human body through the food chain. In addition, some of the nanoparticles can be swallowed into and reach the gastrointestinal tract when they are expelled from the mucociliary system of the lungs after inhalation [19], [20]. Nanoparticles could be then translocated from the lumen of the intestinal tract and blood into different organs like the liver.

Earlier studies have shown that nano-forms of different particles are more toxic than their micro-counterparts after acute exposure via the oral route [21], [22], [23]. Biodistribution experiments have revealed liver, kidney and spleen as the target organs for engineered nanoparticles after uptake by the gastrointestinal tract [21], [23], [24]. For ZnO nanoparticles, only a few in vivo studies are available and most of them are focused on respiratory tract exposure [24], [25]. A previous study evaluated the acute oral toxicity of ZnO nanoparticles at a high dose range (1–5 g/kg body weight) and found a decrease in damage to the liver, spleen and pancreas with an increase in nanoparticle dose [25]. The spleen showed enlargement of the splenic corpuscle while an infiltration of inflammatory cells was observed in the pancreas interstice. The authors attributed the decrease in damage at higher doses to increased agglomeration and suggested the need to study ZnO nanoparticle induced oral toxicity at low doses. Moreover, these studies revolve around the general toxicity parameters and have not considered the genotoxic potential of ZnO nanoparticles, nor investigated their mechanism of toxicity.

Therefore, the present study was undertaken to investigate the sub-acute oral toxicity of ZnO nanoparticles including the genotoxicity and the mechanism of toxicity involved. For this we exposed mice to ZnO nanoparticles via the oral route and the distribution of nanoparticles in different tissues was investigated. The effects of nanoparticles on body/tissue weight and serum biochemical parameters were also examined. Additionally, we analyzed the genotoxic effects of these particles by the in vivo Comet assay and organ damage by histopathology. To understand the mechanism of cell death, we measured oxidative damage by lipid peroxidation and the Fpg-modified Comet assay. The TUNEL assay was also done in the target organ to understand the mode of cell death. A schematic of the experimental design has been given in Fig. 1.