Histopathological effects of zinc oxide (ZnO) nanoparticles on skin and muscle tissues of rats
Sepideh Arbabi-Bidgoli
1
(
Department of Toxicology, Faculty of Pharmacy, Islamic Azad University-Pharmaceutical Sciences Branch, (IAUPS), Tehran, Iran.
)
Parisa Saberi-Hasanabadi
2
(
Department of Toxicology, Faculty of Pharmacy, Islamic Azad University-Pharmaceutical Sciences Branch, (IAUPS), Tehran, Iran.
)
Keywords:
Abstract :
Histopathological effects of zinc oxide (ZnO) nanoparticles on skin and muscle tissues of rats
Abstract
Although ZnO nanoparticles possess novel properties that make them available to a vast range of applications, the questions regarding their safety arise when it comes in contact with the biological systems (such as skin, lungs and tissues). In this study, we evaluated the possible toxicology effects of different dosage of zinc oxide nanoparticles (with quantities of 25, 50 and 100 mg/kg of nano solution) in three treatments for four consecutive weeks on skin and muscle tissues of rats (n= 40; weight: 150-200 g) compared with the control samples and the results were analyzed by one-way analysis of variance (ANOVA).
The results showed that the use of zinc oxide did not have much effect on skin and skeletal muscle tissue of rats. Although, we did not find any harm to the skin and musculoskeletal system in the use of different dosage of zinc oxide nanoparticles in this study, but we can not ignore the reports of the sensitivity of cells and tissues to potentially cytotoxic nanoparticles. Therefore, it is suggested that further, wider and stronger research should be paid on the complex mechanism of toxicity of zinc oxide nanoparticles in living systems.
Keywords: Zinc oxide nanoparticles; Toxicology; Rats; Skin and Tissues Histopathology.
1. Introduction
Nanotechnology has become the forefront of research in the past decade. With the advent of this emerging field, wide varieties of nanoparticles with exciting characteristics are manufactured and are used for a broad range of applications especially in pharmacology and food branches [1]. Because of the special properties including small size and high specific surface area, the biological safety of nanomaterials has received wide attention.
Zinc oxide nanoparticles have been used in a variety of products including semiconductors, catalysts, and paints. These particles are increasingly found in consumer products, such as sunscreen, because of their strong UV absorption especially in case of ZnO [8, 11]. Products containing ZnO nanoparticles may release Zn ions, which may be translocated from the environment into the human blood circulation and accumulate in skin, muscle tissues, potentially leading to toxicity in human organs [14]. Limited studies have dealt with the potential dangerous aspects of nanoproducts on ecosystem. One major reason for this scarcity is difficulty of identifying the effects of nanoparticles using currently available test methods [17].
Although nanoparticles possess novel properties that make them available to a vast range of applications, the questions regarding their safety arise when it comes in contact with the biological systems [2]. Despite the fact that these compounds are safe, studies reported under certain conditions adverse effects occurred cells. It is well documented that toxic effects of nanoparticles can dramatically increases by reduction of their size and increasing their concentration [17]. Thus it is essential to study toxicity of these materials in living organisms.
In vivo studies explored different routes of exposure of ZnO nanoparticles [2, 5]. In a study, toxicities symptoms including oxidative stress were observed after bronchoalveolar lavage fluid was collected in ZnO nanoparticles in treated rats [4]. In other study, with application of zinc oxid nanoparticles in three different dosages (75, 180, and 360 mg/kg) for 28 days, dermal collagen content of skin were decreased in treated rats [3]. In other study, in order to compare the absorption rate of nano and micro-sized particles after oral administration, Zn levels in serum of rats were investigated. The results showed that nano-sized particles were higher compared to the micro-sized treated group in serum [5]. While numerous studies has been carried out on the toxic effects of ZnO on living tissues of animals, studies regarding histopathological effects of this substance on skin and muscle tissues of rats is limited.
As a result of the substantial amount of production and usage of ZnO nanoparticles in pharmaceutical branches, it is necessary to evaluate their potential toxic effects on skin and muscle tissues in biological conditions. Moreover, investigations regarding the possibility of susceptible subgroups are also critical to better understand the neurotoxicity of ZnO nanoparticles, as well as to guide exposure standards. In this study, we aimed to investigate the effects of the impact of ZnO nanoparticles with three different doses (25, 50 and 100 mg) for 28 days on skin and muscle tissues of rats.
2. Materials and Methods
2.1. Preparation of a nanoparticle solution
The nanoparticle used in this experiment was purchased from Nanosany Company (Mashhad, Iran). Their specification was a nearly spherical white crystalline with particles in the range of 10 to 30 nm with a purity percentage of 99%. Then, the amount of 25, 50 and 100 mg of the solution was prepared by diluting with 0.9% normal saline solution in Milli-Q water. The characteristics and the image of its electron microscope (TEM) are presented in Table 1 and Figure 1, respectively.
2.2. Experimental animals
Rats from the Animal Breeding Center, located in the North of the country were prepared. The rates weighing 150–200g were maintained in a 12-hr light/dark cycle at a temperature of 22±1 °C with free access to standard pellet diet and water. Animal experiments were carried out according to the guidelines of Institute Animal Ethics Committee regulations approved by Committee for the purpose of control and supervision of experiments on rats. Individual rats were identified with picric acid marks. In addition to this, each rat cage was identified by labels having details such as experiment number, name, animal number(s) and date of experiment. All the animals were acclimatized for a period of 5 days before initiation of experiment.
2.3. Treatments with ZnO nanoparticles
Rates were randomly divided into 4 groups as below:
2.3.1. Control group (A): In this group, rats fed normally with water and food and did not receive any nano solution or drug.
2.3.2. First experimental group (B): Rats in this group received 0.5 ml of 25 mg/kg of zinc oxide solution in the form of intraperitoneal, dermal and oral intravenous injection twice a week.
2.3.3. Second experimental group (C): Rats in this group received intraperitoneal, dermal and oral injections of 0.5 ml of 50 mg/kg nanoparticle solution twice a week.
2.3.4. Third experimental group (D): Rats in this group received intraperitoneal, dermal and oral injections of 0.5 ml of 100 mg/kg nanoparticles solution twice a week.
All rats were examined for the onset of any immediate signs of toxicity and body changes were recorded for 28 continuous days. At the end of all experimental stages, treated samples were taken from skin and muscle tissues of rats and were transferred to the laboratory for digestion, atomic absorption and histopathologic tests.
2.4. Skin and muscle tissue sampling
Skin and muscle tissues sampling were collected to assess histological changes. Other pretreatments (tissue collection from the rats, fixation, special processes such as decalcification, tissue trimming, cassetting, processing, embedding, sectioning, deparaffinization and rehydration, staining, and cover slipping) were performed in according to Samrot et al., (2017) method [13].
2.5. Statistical Analysis
Statistical differences between the control and treated samples were examined with the aid of one-way ANOVA test followed by SPSS 18.0KO (SPSS Inc., Chicago, IL, USA).
3. Results
Size of the nanoparticles is one of the critical factors responsible for nanoparticles property and their mechanism of cellular interaction. Thus in the present study, the nanoparticles size has been studied by TEM analysis. Although the data available from the manufacturer indicated that the primary particle size of the ZnO nanoparticles was < 50 nm, the characterization of ZnO nanoparticles was also detected in our laboratory. The TEM images demonstrated that ZnO nanoparticles were within 10–30 nm in size (Table 1), and the majority of the particles comprised a polygonal shape with smooth surfaces (Figure 1).
Table 1. The ZnO nanoparticle characteristics
Parameters Nanoparticle type Nanoparticle size Specific surface Percentage of purity Appearance color Real density |
ZnO nanoparticle 10-30 nm 20-60 m2 99% Milky white 5.606 g/cm3 |
|
Figure 1. TEM image of ZnO nanoparticle.
3.1. The histopathological effects of ZnO nanoparticles on rat skin
The natural structure of the rat skin was visible in four groups and its building was preserved in all three layers of epidermis, dermal and hypodermic. During the study period, skin treatment with ZnO nanoparticles did not cause any adverse effects because no statistically significant differences in three layers of skin gain were observed between the ZnO-treated and control rats. Further, no abnormal skin clinical signs and behaviors were detected in both the control and treated groups (data not shown). Exposure to the most dosage of ZnO nano particles solution for 28 days generally didn't cause to significant changes in the skin of rats (see Figure 2).
(b): 25 mg/kg of nano solution |
(a): Control sample |
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(d): 100 mg/kg of nano solution |
(c): 50 mg/kg of nano solution |
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Figure 2. Histopathological changes in skin of treated rats with different dosages of ZnO nanoparticles (25, 50 and 100 mg/kg, respectively) as compared with control sample (a) ×100.
3.2. The histopathological effects on muscle tissues
The effect of feature changes of muscle tissues was determined in different dosages of ZnO nanoparticles. As shown in Figure 3, no change in statues was observed in tissues sample of treated rats following of the low and high dosage of ZnO nanoparticles solution. In all of groups, muscle cells tissues were healthy and their dark and clear lines were visible. In these groups, the nuclei were located below the sarcolemma and their dark and clear lines were visible (see Fig 3).
(b): 25 mg/kg |
(a): Control sample |
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(c): 50 mg/kg |
(d): 100 mg/kg |
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Figure 3. Histopathological changes in muscle tissues of treated rats with ZnO nanoparticles. Rats were treated with 25, 50 and 100 mg/kg-ZnO nano particles, repectively as compared with control sample (a) ×400.
4. Discussion
As ZnO nanoparticles are used increasingly in various commercial products as well as in biological and medical applications, it is more important than ever to study their possible toxicological effects on humans. Our study showed that the use various dosages of (25, 50 and 100 mg/kg) zinc oxide nanoparticles in treated rats did not have much effect on their skin and skeletal muscle tissues. Recently, a few in vivo toxicity tests of ZnO nanoparticles showed that toxicity appeared only in relatively high-dosage groups when rats were treated by oral administration [12, 14].
Other exposure routes including skin or inhalation produced different results according to the exposure routes [4, 15]. When rats were treated orally with 50 or 300 mg/kg ZnO nanoparticles once a day for 14 days, ZnO nanoparticles accumulated in liver and led to cellular damage.
A study showed that some male wistar rats treated with the zinc oxide nanoparticles presented tissue necrosis. These complications were attributed to improper injection techniques or inaccurate after-treatment management, besides the intrinsic characteristics of the local environment. The evidence of tissue necrosis was linked to external events whereas it could have been related to an endogenous reaction based on inflammatory response. The presence of inflammation after the intratesticular injection of 200 mM zinc oxide nanoparticles was reported, and anti-inflammatory drugs were prescribed to minimize this side effect in male wistar rats [6, 7].
The aim of this study is to evaluate the zinc-based nanoparticles toxicity based on the OESD guidelines that is currently used to evaluate the toxicity of chemicals. Of course, in these treatments, application of different dosage of nanoparticles used was more than their ordered actual dose. It seems that since the surface of nanoparticles is high, their major effects depend on their surface to their molecular mass. Moreover, different nanoparticles with different physicochemical properties have different toxicological effects [7, 8].
Different studies on the toxicological effects of nanoparticles have indicated that these particles act with the similar oxidative toxicological mechanisms and these effects are dependent on their physicochemical properties such as specific surface area, the properties of the metal concerned, as well as nanoparticle size. Furthermore, different exposure route, such as oral administration, dermal contact, and inhalation have various specific toxicological impacts [7, 9, 16].
In a study, a comparison was made between the effects injection of different dosages of nano titanium oxide particles in different sizes on health through long-term inhalation of these particles. The results showed that smaller nano titanium oxide particles tended to produce more pulmonary tonsils than coarser ones [16].
Therefore, according to the obtained results of this study, we suggest that ZnO nanoparticles have not destructive effects on skin and muscle tissues of rat's bio system in used dosages. With regards to previous studies, it was confirmed that ZnO nanoparticles have toxicity effects on the some organs [9, 10]. Understanding the toxicity mechanism of ZnO nanoparticles in bio organisms need to be more evaluated in different laboratory conditions.
Finally, because ZnO nanoparticle is a complex system with no identifiable mechanism in different conditions, the best way to move forward is with a multidisciplinary approach incorporating results from emerging imaging techniques, biochemical, pharmacologic and genetic studies in order to better understand the molecular basis of this nanoparticle.
5. Conclusion
Rats were treated with ZnO nanoparticles by three different exposure routes, oral administration, skin and intravenous injection. In current study, treated rats with various dosages of zinc oxide nanoparticles didn’t exhibited significant histopathological changes regarding to different histological parameters. In summary, histopathological observations found in our study indicated that ZnO nanoparticles have not cytotoxic actions on skin rats system and doesn't induce damage to muscle tissue. Nowadays, due to the development of nanotechnology and wide uses of these nanoparticles in food and pharmaceutical industries, medicine and biology, there is wide exposure of this material to humans and the environment.
Therefore, these results provide useful information for designing proper metal nanoparticles in order to better understand their pathophysiological mechanisms for delivery of bioactive molecular and other nutraceuticals in food and pharmaceutical products.
6. Acknowledgments
The authors are grateful to the technical support of Islamic Azad University-Pharmaceutical Sciences Branch (IAUPS), Tehran, Iran.
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