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Subchronic effects of different doses of Zinc oxide nanoparticle on reproductive organs of female rats: An experimental study

Background Zinc performs many biochemical and physiological functions; however, toxicological studies demonstrate that Nano-zinc oxide has harmful effects on human health and environmental species in high concentrations. Objective The aim of this study was to investigate the toxicity of zinc oxide nanoparticles on reproductive tissues of female rat. Materials and Methods Eighty female Wistar adult rats weighing 180–200 gr, divided into eight groups (n= 10 in each group) including control, sham (treated with saline), and six groups injected with different doses of zinc oxide nanoparticle with 10–30 nanometer size (4, 8, 25, 50, 100, and 200 mg/kg) twice a week for four weeks. At the end of the study, the rats were bled and slaughtered; the Ovary and Uterus were taken for histopathology studies and blood samples were transferred to the laboratory for biochemical analysis. Results Microscopic diagnoses in ovary tissue were included; increase in the corpus luteum, follicular cysts, inflammatory cells infiltration and fibrosis. Histopathological changes in ovary in a dose-dependent manner. In uterus tissue the lesions consisted; epithelial destruction, hyperplasia of endometrial glands. The Estrogen and Progesterone level in the serum of rats increased in low doses and reduced in a dose-dependent manner at high doses. Conclusion The results of the current study proved the toxicity of zinc oxide nanoparticles on the ovary and uterus organs at high concentrations, so further investigation is needed to reduce these effects.


Introduction
Heavy metals are relatively dense materials that exist in natural constituents of earth crust (1). In recent years, human activities such as industrial chemicals production and mining have increased the amounts of heavy metals into the environment and there has been increasing concern about their effects (1). In particular, zinc oxide (ZnO) nanoparticles (NPs) are commonly used to manufacturing products such as cosmetics, ceramics, plastics, glass, cement, rubber, grease, paints, pomade, glue and drug (2). The nanoparticle distribution in the environment depends on the properties of the metal including particle size, shape, surface characteristics, and various environmental factors (3). The evaluation of nanomaterials safety is of great importance for the industrial applications (4).
It is also generally accepted that some of these metals are essentially required to retain various biochemical and physiological functions for living organisms in low amount, but toxic when they exceed their threshold concentrations (5). Many researchers investigated the potential side effect of Nano-sized particles and reported that prolonged exposure to environmental pollutants such as zinc can cause adverse health effects and harmful impacts on sexual function (1,6). Exposure ZnO NPs during the organogenesis stage indicated poor embryonic development in mice. (7). Also ZnO dissolution has been shown to be contributing to the acute or chronic toxicity of aquatic organisms (8). Heavy metals can be combined through food ingestion and inhalation and can influence the hypothalamic-pituitary-ovarian axis and play a role in the human fertility as a certain environmental factor (9). Mice treated with silica and titanium dioxide NPs had small uterus and fetuses (7). Jo and colleagues showed that exposure to Exposure to ZnO NPs prior to and during gestation could compromise the health of pregnant females and their fetuses. (8).
In addition heavy metals accumulation reduce progesterone and estradiol output. Pregnancies that occur despite a high heavy metal body load are at a greater risk of abortion, fetal deformity, premature birth and placental insufficiency (10).
The aim of this study was to investigate toxicity of ZnO NPs on reproductive tissues of female rat.

Experimental design
In this Experimental study, the animals were divided into eight different experimental groups of 10 animals each: control: normal and apparently healthy rats that did not take any type of foodstuff.
All other animals were received intraperitoneally (IP) twice a week for four weeks (1/2 ml for any animal) including: Group sham were given 10% normal saline body weight and the treatment group was given, respectively, 4 Table I

Body weight and hormonal assay
At the end of the fourth week, the rats were anesthetized with intraperitoneal injection of xylazine and ketamine (20 and 60 mg/kg body weight, respectively, Alfasan, Woerden, Holland). Blood samples (3-5 cc) were taken from the heart of all the rats. Serum Estrogen and Progesterone hormones level was measured by using Elisa kit protocol (Sigma Aldrich, USA).

Evaluation of malondialdehyde (MDA)
Ovary homogenates were prepared by mixing

Ethical consideration
The experimental design adhered to the guidelines and was approved by the Animal Ethics Committee of Islamic Azad University, Babol branch, Babol, Iran.

Statistical analysis
The data were analyzed using one-way analysis of variance, followed by post-hoc Duncan test by SPSS software version 22. A p< 0.05 was considered statistically significant.

Results
Histopathological findings in ovary tissue included: Hyperemia, increase the corpus luteum, follicular cysts, inflammatory cells infiltration, and fibrosis (  Table III. MDA levels increased dose-dependently in the treated group in ovary. There is a significant difference between treated groups at doses greater than 25 mg/kg (p< 0.0001) (Table III).   Note: Data presented as mean ± SE; , , , , : Different letters a, b and c, etc., to show statistically significant differences between the groups.

Discussion
ZnO NPs is functional in many industries includ- Degeneration of uterine and decrease in primary follicles of ovaries were observed after sodium arsenite treatment. (15). Another study reported that the ovaries of Ni NPs treatment rats after gavage were associated with the infiltration of inflammatory cells, congestion, and ovarian lymphocytosis (16).
Jo and colleagues reported that the uterus of adult female rats orally exposed to ZnO nanomaterials (500 mg/kg) showed pathological lesions such as localized necrosis, inflammation, thrombosis, and histiocyte infiltration (8 Oxidation protein products' level in rat kidney and increased MDA level in ovarian rats (19). In a study reported that following administration, Cd, as and Hg, serum MDA levels increased time and dose dependent. (20).
In the study of lipid peroxidation, MDA content in plasma, liver, and pancreas of exposed rats to Zinc was higher than that of ad libitum values (21). Chattopadhyay and co-worker showed that arsenic exposure led to a significant decrease of peroxidase activities in ovary and uterus. (15).
A study showed that cadmium causes oxidative stress in kidney (19). The results of this study con-  (25). In another study, Estrogen decreased after gavage administration of Ni NPs in adult rat (16).
Low levels of ZnO NPs through stimulation of the glands cause increase in the Estrogen secretion.
But high levels of ZnO NPs because of destructive effects on the ovary can cause decrease in the Estrogen secretion. It has been suggested that NPs have a greater risk of toxicity than larger particle (26), although the production and usage of these materials are increasing, the available information on the toxicological features about them are still incomplete.

Conclusion
ZnO NPs can enter animal and human tissues via air inhalation or food and manual handling; and they get absorbed in the tissues such as ovary.
So because of the various usage and the possible harmful effects of ZnO NPs, investigating its toxicity is critical especially in mammalian cells. Due to changes in Estrogen and Progesterone and in the absence of lesions, it seems that low doses have positive effects.