Journal of Nanomedicine & Nanotechnology

Abstract

In the food and fertilizer industries, zinc oxide nanoparticles (ZnO-NPs) are frequently utilized. In our investigation, rats received oral administration of ZnO NPs with a particle size of 30 ±5 nm once daily at doses of 100, 200, 300, 400, and 500 mg/kg for ten weeks in order to assess the genotoxic effect. Impacts on hematological markers, genotoxic impact, and growth were investigated. The results indicated that ZnO-NPs significantly reduced body weight gain, red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit value (HCT), and platelet count (PLT), while increasing white blood cell (WBC), mean capsular volume (MCV), mean capsular hemoglobin (MCH), and mean capsular hemoglobin concentration (MCHC) in the treated rats. Our data show a dosage-dependent increase in DNA fragmentation for both the comet assay and the micronuclei test, which was supported by an increase in the proportion of DNA that was tail-containing, the length and intensity of DNA tails, and the tail moment, especially at the dose of 500 mg/kg. The findings revealed an increase in the number of micronucleated cells.

Keywords

Rats; zinc oxide nanoparticle; Hematology; Micronuclei; Comet assay.

INTRODUCTION

Small substances with at least one dimension between one and one hundred nanometers are known as nanoparticles (NPs). Due to their small size and large surface area, nanoparticles play a key role in all aspects of modern life [1]. By shrinking matter to a scale of 1 to 100 nm, nanotechnology is a significant area of innovation in the industry [2]. The food sector, drug delivery, diagnosis, cosmetics, and several sunscreens are just a few industries where nanoparticles (NPs) are used [3]. ZnO nanoparticles are exposed to the human body more and more as their use increases. Inhalation, cutaneous contact, and ingestion are the three main ways that ZnO nanoparticles are exposed to the body [4]. When ZnO nanoparticles are exposed to the body through any method, they can reach the circulatory system and spread throughout the entire body through it [5]. When ZnO nanoparticles enter the body, they are small enough to penetrate cells right away and are subsequently internalized by the cells either as free Zn2+ ions or as nanoparticles [6].

Micronuclei are reliable indicators of genotoxic exposure in both humans and animals, and scoring them has been widely utilized to detect probable genotoxic substances [7]. The micronucleus test is a crucial in vivo cytogenetic screening method for identifying newly produced structural chromosomal abnormalities in bone marrow cells [8]. Chromosome abnormalities in mouse bone marrow and the micronucleus test have both been used extensively to clarify the connection between food and mutagenesis. Micronuclei can be utilized as a mutation index because they are an indicator of irreversible DNA loss. Wistar rats had decreased red cell counts, liver lesions, and hepatocyte inflammation during them feeding on ZnO NPs at dose of 2000 mg/kg. According to diet-borne ZnO NPs can change the molecular structure and morphology of the blood, intestine, liver, and kidneys. These NPs may display unpredictable genotoxic features through direct interaction with genetic material or by indirect DNA damage brought on by reactive oxygen species due to their tiny size and increased surface area combined with physiochemical properties like charged surfaces. Chromosome abnormalities in mouse bone marrow and the micronucleus test have both been used extensively to clarify the association between food and mutagenesis. Micronuclei can be utilized as a mutation index because they are an indicator of permanent DNA loss (9). A greater understanding of the genotoxic potential of ZnO NPs in an animal model is needed in consideration of the rapidly expanding field of nanotechnology and increasing exposure to nanoparticles. The objective of the current study was to assess the acute oral toxicity of ZnO-NPs at dosages of 100, 200, 300, 400, and 500 mg/ kg. ZnO NPs' genotoxic effects as determined by hematology tests and comet assays.

MATERIALS AND METHODS

Chemicals

Zinc Oxide Nanoparticles (ZnO-NPs) were obtained from NanoTech Egypt Company (City of 6 October, Egypt). The manufacturer's datasheet indicates that ZnO nanoparticles have a size of 30±5 nm. Chemicals were bought from Bio Diagnostic Company and Human Company for the quantitative measurement of several biochemical and hematological parameters (Egypt).

Preparation of ZnONPs suspension

The ZnO NPs participles (30±5 nm) were dispersed in distilled water (10 mg/mL) and the suspension was solicited at 230 V for 20 minutes using ultra-sonic cleaner sonicator (Branson ultrasonic corporation, Danbury, Connecticut, USA) at room temperature. The suspension was stirred on vortex agitator immediately before administration in different dosages (100,200,300,400 and 500mg/ kg).

Experimental design

Thirty Sprague-Dawley male rats (weighing 140-160g and 8–10 weeks old) were obtained from Rapitco farm Company, Giza, Egypt. Animals were housed (five/cage) in universal polypropylene cages. After the adaptation period, the thirty rats were divided into six groups (Five rats per group). Group 1 (G1) was termed as control group and in which rats fed a standard synthetic diet and had free access of water ad libitum, while animals in the other five groups were given zinc oxide nanoparticles by oral gavage at different concentrations for a period of 10 weeks. Group 2 (G2) in which rats received 100 mg/kg body weight ZnONPs suspension orally once per day, Group 3 (G3) in which rats received 200 mg/ kg ZnO-NPs suspension orally once per day, Group 4 (G4) in which rats received 300 mg/kg body weight ZnONPs suspension orally once per day, Group 5 (G5) in which rats received 400 mg/ kg body weight ZnONPs suspension orally once per day and Group 6 (G6) in which rats received 500 mg/kg body weight ZnONPs suspension orally once per day.

Hematological Examination

Each rat in each group had blood drawn from the retinal vein for hematological analysis using EDTA (Ethylene Diamine Tetra Acetic Acid). Using a hematological analyzer, an entire blood image was taken for each group (MEDONIC, S.E 12613, Sweden). The analysis included an erythrogram with the following components: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell count (RBC), hematocrit (HCT), hemoglobin (Hb) concentration and red cell indices, white blood cell count (WBC), and their differential (lymphocytes, granulocytes, and monocytes), and platelet count (PLT).

Genotoxicity Assay and Micronuclei Assay

According to Schmid (8), the micronuclei (MN) test and the scoring of micro nucleated polychromatic erythrocytes (MnPCEs) were conducted. For scoring from coded slides and determining the ratio of polychromatic erythrocytes (PCEs) / normochromatic erythrocytes (NCEs), a minimum of 2000 polychromatic erythrocytes (PCEs) per animal were utilized. For the purpose of estimating the MnPCEs, the slides were examined under 100 oil immersions.

Comet Assay

The single cell approach typical of cytogenetic assays is combined with the convenience of utilizing biochemical techniques for detecting DNA single strand breaks (frank strand breaks and incomplete excision repair sites), alkali-labile sites, and crosslinking in the single cell gel electrophoresis (SCGE)/comet assay, developed. The Animal Reproduction Research Institute (ARRI), located near Giza, Egypt, conducted a comet assay. Comet assay was performed in Animal Reproduction Research Institute (ARRI), in Giza, Egypt.

Statistical Analysis

Using SPSS-PC software experimental data were statistically examined for the quantitative variables, namely body weight gain and MnPCEs. Probability values less than 0.05 (P<0.05) were regarded as statistically significant.

RESULTS AND DISCUSSION

Animal Observation

The observation during this study showed that no serve toxicity signs such as diarrhea or hair loss. Furthermore, no mortality was observed related to different doses of ZnO-NPs administration (100, 200,300, 400 and500 mg/kg body weight). Also no behavioral changes were observed related to be orally administered to rats.

Effect of ZnO-NPs on the Body Weight Gain of Rats

The effects of various ZnO-NPs concentrations on the rat body weight gain throughout the period of the entire experiment are displayed in [Table 1]. Data revealed a significantly lower average rate of rat body weight gain when compared to the control group (p <0.05). The current study's findings regarding the reduction in body weight gain were in agreement with those of who demonstrated that rats administered ZnO NPs by gavage at doses of 0, 100, 200, and 400 mg/kg/day experienced a decrease in body weight as a result of decreased food consumption. High doses of ZnO NPs in diet can have toxicological effects, but the decrease in body weight at 5000 mg/kg ZnO NPs may help explain why the relative weights of the pancreas, brain, and lung have increased in response to ZnO NPs (16). Nano-ZnOs at 50 and 500 mg/kg was demonstrated increased body weight but decreased body weight was showed at 5000 mg/kg. Decrease of body weight may be attributed to anabolic metabolism in body of treated animals, or as a result of anti-digestion effect, or due to the loose of appetite in treated animals as a result of nanoparticles administration [9]. Data represent the means ± SE of 5 animals per group. P < 0.05 compared to control.