Sub-acute Effects of α-Fe 2 O 3 Nanoparticles on Some Biochemical Parameters in Mice

The goal of the study was to find out the toxic effect of daily treatment with α-Fe 2 O 3 nanoparticles for 14 and 28 days on some biochemical indicators in mice by measuring ferritin, transferrin, cholinesterase enzyme activity, the concentration of Caspase-3, and the concentration of glutathione and malondehyde in brain and liver tissues at doses of 75, 150 and 300 mg/kg. α-Fe2O3 nanoparticles at the dose of 75 mg/kg caused a significant decrease in the activity of the acetyl cholinesterase enzyme in the brain and liver after 14 days of treatment, compared with the control group and the doses of α-Fe 2 O 3 (75, 150, and 300 mg/kg) led to a significant decrease in the activity of the acetyl cholinesterase enzyme in the brain after 28 days of treatment. Fe 2 O 3 at a dose of 75 mg/kg on the 14th day and at a dose of 150 mg/kg on the 28 th day of treatment resulted in a significant increase in the caspase-3 enzyme in the brain compared to the control group. While the doses of 150 and 300 mg/kg for 28 days led to a significant increase in transferrin concentration compared with the control group, The α-Fe 2 O 3 nanoparticles at doses of 75, 150, and 300 mg/kg for 28 days caused a significant decrease in the concentration of glutathione in the liver and brain tissues compared with the control group, accompanied by a significant increase in the concentration of malondehyde in the brain and liver tissues at doses of 150 and 300 mg/kg of body weight. We conclude that repeated exposure to α - Fe 2 O 3 nanoparticles has toxic effects on vital organs such as the brain and liver, represented by a decrease in the concentration of acetylcholinesterase and its ability to induce oxidative stress through a decrease in glutathione concentration and an increase in malondehyde concentration in mice .


INTRODUCTION
Nanotechnology has huge potential in magnetic resonance imaging and early disease recognition, with specific nano-scale agents being adapted for molecular imaging in the context of magnetic resonance imaging, ultrasound, optical imaging, and X-ray imaging. Magnetic nanoparticles can also be widely used in drug delivery, gene delivery, and targeting (Indira and Lakshmi, 2010) and as iron supplements for anaemic patients (Lu et al., 2010). Iron is an essential cofactor for respiratory chain enzymes as well as for DNA synthesis and cell proliferation (Rouault and Tong, 2008). Iron oxide nanoparticles (IONPS) are able to cross the bloodbrain barrier and reach the brain (Wang et al., 2010) and magnetic nanoparticles must be biocompatible and biodegradable with a size ranging from 10 to 100 nanometers because nanoparticles smaller than 10 nanometers are excreted by the kidneys and particles larger than 200 nanometers do not pass through the cell membrane easily and can stimulate the immune system as foreign elements and be excreted outside the body (Gupta and Gupta, 2005).
The biomedical applications of iron oxide magnetic nanoparticles are broader than others due to their biocompatibility, high stability, and ease of use. One of its most important uses is drug delivery (Liang et al., 2008;Espanani et al., 2013). Because the use of these particles in various industries has increased human exposure to them, the investigation of nanoparticles role in cell growth and survival is of greater importance (Zhu et al., 2009). Human skin, lungs, and gastrointestinal tracts are the most common entry routes for nanoparticles and their pathogens (Buzea et al., 2007). Airborne nanoparticles have high mobility and can be easily inhaled through the respiratory tract (Yeh et al., 2012). One of the most common adverse effects of nanoparticles is the emergence of reactive oxygen species (ROS). It is almost certain that all studied nanoparticles produce reactive oxygen species, and it is the main mechanism of nanoparticle toxicity that can lead to enlargement and apoptosis in human and animal tissues ( Miura and Shinohara 2009).
The use of nanoparticles, including iron oxide (Fe2O3), has recently increased in various fields, including industrial and medical applications and the production of nano-based drugs. Our current study aimed to reveal the toxic effects of iron oxide nanoparticles (α-Fe 2 O 3 , 20-40nm) on some biochemical parameters in mice.

Ethical Approval
In accordance with institutional policies on the care and use of animals in research, we received official clearance for the study protocol from the Committee of Postgraduate Studies at the College of Medicine, University of Mosul, Iraq.

Animals
Forty mice (males and females), white, of Swiss origin, that were raised in the laboratories of the College of Veterinary Medicine at the University of Mosul. The weights of these mice ranged between 25-35 g. The mice were raised in laboratory conditions characterized by a light cycle distributed over 12 hours of light and 12 hours of darkness, and the temperature of the animal house was 22 ± 2 degrees C • . Laboratory food was provided from local factories for the production of animal feed in the city of Mosul.

Preparation of iron oxide particles Fe2O3
The (α-Fe 2 O 3 ) nanoparticles are characterized by a reddish-brown color, a round shape, a purity of 98 +%, and a size of partials 20-40 nm. www.usnano.com, the required weight of α-Fe 2 O 3 nanoparticles were mixed with distilled water at a volume of administration 10 ml/kg of body weight with an ultrasonic 50 Hz device to facilitate the dispersion of particles with water and facilitate swallowing by mice (Kumari et al., 2012;Dhakshinamoorthy et al., 2017).

Experimental design
Forty mice were divided into four groups, 10 /group, and they were administration as follows: A-The first (control) group were given distilled water at a volume of 10 ml / kg of body weight orally. B-The second group were given 75 mg/kg iron oxide nanoparticles (α-Fe 2 O 3 ) orally. C-The third group was given 150 mg/kg iron oxide nanoparticles (α-Fe 2 O 3 ) orally D-The fourth group was given 300 mg/kg iron oxide nanoparticles (α-Fe 2 O 3 ) orally.
After blood was collected half of mice in each group were killed at the fourteenth in order to isolated brain and liver tissues. The same process was repeated at the 28 th day: Solutions used: Buffer phosphate solution with a pH of 8.1 and prepared as follows: 0.309 g of sodium barbital, 0.040 g of dihydrogen potassium phosphate and 8.768 g of sodium chloride were added in 225 ml of distilled water, then the pH of the final solution was adjusted to 8.1 Using hydrochloric acid (0.1 M) and measuring the pH with a pH meter, complete the volume to 250 ml by adding distilled water. The aqueous solution of acetylcholine iodide 7.1% was prepared as follows: 0.375 mg of acetylcholine iodide was added in 5 ml of distilled water, and prepared on the same measurement day.
3-Caspase-3 activity in the brain and liver was estimated by using ELISA kit (SunLong Biotech, China), since the optical density is measured by the optical spectrum at the wavelength of 450 nm. The optical density value is proportional to the Casp-3 concentration and the Casp-3 concentration in the samples can be calculated by comparing the OD of the samples to the standard curve.

4-In addition Glutathion and Malondyhayde
concentration in brain and liver were measured using modified Ellmann method (James, 1982) and method of (Ohkawa et al., 1979), respectilly .

Statistical analysis
The results were analyzed statistically using the test of variance One way analysis of variance and Two way analysis of variance, then the results were subjected to the Least significant difference test based on the Sigma plot statistical analysis program, and the level of significant difference for all tests was at the (p) probability level less than 0.05.

1-Effects of α-Fe 2 O 3 nanoparticles at doses
(75, 150, and 300 mg/kg) on the serum transferrin and ferritin concentration of mice treated on the 14 th and 28 th day The treatment of mice with 150 and 300 mg/kg of α-Fe 2 O 3 nanoparticles for 28 days led to a significant elevation in transferrin concentration compared with the control group (table 1). While there was no significant change in ferritin concentration in mice's serum (table 2).

2.Effects of α-Fe2O3 nanoparticals on the cholinesterase activity in the brain and liver by an electrometric method
Treatment of mice with 75,150,300 mg/kg of α-Fe 2 O 3 resulted in a significant decrease in the cholinesterase activity of brain and liver tissues after 14 days of treatment, compared to the control group, as shown by the inhibition rates (34.92, 33.33, and 47.46%) in the brain and (38.46, 30.76, and 25 % ), respectively, in the liver (table 3).  Treatment of mice with 75, 150, and 300 mg/kg of α-Fe 2 O 3 resulted in a significant decrease in the cholinesterase activity of the brain after 28 days of treatment, compared to the control group, as shown by the inhibition rates of 36.49, 33.78, and 31.08%, respectively, while the rate of inhibition in the liver was 45.68%, caused by the 300 mg/kg dose of α-Fe 2 O 3 table (4).

Effects of α-Fe 2 O 3 nanoparticles at doses (75, 150 and 300 mg/kg) at the 14 and 28 day of treatment on the activity of the caspase-3 enzyme in the brain and liver of mice
Treatment of mice with 75 mg/kg of α-Fe 2 O 3 resulted in a significant elevation in the caspase-3 concentration of brain tissue after 14 days of treatment, compared to the control group (table 5). The dose of 150 mg/kg after 28 days of treatment caused a significant elevation in the concentration of brain tissue compared to the control group table (6). Mean ± SE with various letters in the column means significant at a level of (P ≤ 0.05). Mean ± SE with various letters in the column means significant at a level of (P ≤ 0.05).

Effects of α-Fe 2 O 3 nanoparticles at doses (75, 150 and 300 mg/kg) at the 14 and 28 day of dosing on the concentration of glutathione and malondyhyde in the brain and liver of mice
Treatment mice with 75,150,300 mg/kg of α-Fe2O3 resulted in a significant decrease in the concentration of glutathione in brain and liver after14 and 28 days of treatment, compared to the control group table (7,8).  Treatment mice with 75,150,300 mg/kg of α-Fe 2 O 3 resulted in a significant elevation in the concentration of malondyhed in brain and liver after14 and 28 days of treatment, compared to the control group table (9, 10).

DISCUSSION
Magnetic nanoparticles have recently attracted great interest due to their unique physical and chemical properties and potential applications in various biomedical fields (Lu et al., 2010). Among these magnetic nanoparticles, iron oxide nanoparticles, which are characterized by their compatibility, are bioavailable, injectable, and may have a high rate of accumulation in target tissue (Ito et al., 2005).
Ferritin, in its classic and complex role, is the storage protein for excess iron within living cells that is not used immediately (Jacobs and Worwood, 1975). The amount of ferritin ranges from one-third to three-quarters of the amount of iron in the brain (Octave et al., 1983) . There are several reasons to increase serum ferritin, including liver cirrhosis, cancer, and hereditary diseases caused by deficiency of ferritin protein (Wang et al., 2010) . An increase in iron concentration does not necessarily mean an increase in ferritin concentration (Loeffler et al., 1995). Previous research indicates that serum ferritin leaks from damaged cells , including liver cell damage, in other words, an increase in ferritin concentration results from cellular damage, (Dominguez-Versa et al., 2010) and it is associated with the formation of free radicals, which results in more cellular damage (Kell and Pretorius, 2014).
Our results are consistent with the previous studies by Kell and Pretorius, (2014) which showed an increase in iron levels in the brains of Alzheimer's patients compared to normal ferritin levels, i.e., an increase in iron levels without a concomitant change in the level of ferritin (Halliwell, 1992;2001).
Transferrin is a transporter protein in the blood (Briley-Saebo et al., 2004). In the case of saturation of the blood with iron, the haemoglobin stock in the blood increases with an increase in the level of transferrin in the serum. A previous study showed an increase in haemoglobin and transferrin in rats treated with iron oxide nanoparticles in drinking water for ten days (Elshemy, 2018). These results are consistent with our results, which showed an increase in the level of transferrin at doses of 150 and 300 mg/kg after 28 days of dosing with Fe 2 O 3 .
The chemical activity of the acetylcholineesterase enzyme is demonstrated through the breakdown of the neurotransmitter acetylcholine into choline and acetic acid (Romano et al., 2015). An Inhibiting the activity of this enzyme leads to the accumulation of the neurotransmitter acetylcholine in the nerve endings, causing the signs of cholinergic poisoning, muscarinic, nicotinic, and the central nervous system signs (Lotti, 2010), and due to the great importance of this enzyme, its activity was measured in the tissues of the brain and liver, as it was found that when iron oxide particles Fe 2 O 3 -were given in doses (75, 150, and 300) mg/kg by oral dose, the treatment 14 days there was a significant decrease in the enzyme activity in the brain tissue of the treated mice compared with the control group, and a significant decrease in the enzyme activity in the brain tissue of the treated mice on 28 days compared with the control group for all doses. Exposure to iron oxide nanoparticles Fe 2 O 3 leads to neuronal degeneration and affects neurobehavior and signs of toxicity (Wang et al., 2007;Zhu et al., 2009;Dhakshinamoorthy et al.,2017). Our results were consistent with a previous study in which rats were given iron oxide particles at a dose of (500,1000,200) mg/kg for 60 days. (Kumari et al.,2013 ).
The nanoparticles produce reactive oxygen species (ROS), which is the main mechanism that can lead to apoptosis and DNA damage in mice exposed to iron oxide nanoparticles (Dhakshinamoorthy et al.,  2017). Our results are consistent with what the researchers stated, who indicated that giving iron oxide nanoparticles led to an increase in the level of the caspase-3 enzyme in the mice's brain. The repeated administration of iron oxide nanoparticles leads to the accumulation of iron in the brain, causing the destruction of cellular structures and the fragmentation of DNA. (Dhakshinamoorthy et al.,  2017). These results can be explained by the fact that apoptosis increases in a dose-dependent manner (Liu et al., 2018). Our results indicated that there was no significant effect on the level of the caspase-3 enzyme in liver cells treated for 28 days. The reason is that the liver is one of the dominant organs for the removal of nanoparticles, and it is the first and largest line for phagocytosis of nanoparticles after the spleen (Lee et al., 2010); this coincides with the study of Voss et al., (2021).
Nanoparticles pose adverse effects such as oxidative stress, because they may generate oxidants and have the potential to stimulate the generation of ROS. This defect caused by nanoparticles causes, directly or indirectly, the synthesis of free radicals that may lead to cell toxicity (Nel et al., 2006) and due to a defect in the cell's vital antioxidant defences, the most important of which are glutathione, while measuring it in body tissues such as the brain and liver, it was inferred that oxidative stress occurred, with an imbalance in the level of antioxidant enzymes and glutathione, as these enzymes help in detecting and diagnosing the occurrence of oxidative stress, as the level of glutathione decreases and is accompanied by an increase in the level of malondialdehyde (Dalle-Donne et al., 2006). Our results indicate that high and continuous doses of iron oxide nanoparticles had an effect on the levels of glutathione and malondialdehyde in brain and liver tissue, and this is consistent with a previous study conducted on rats (Reddy et al., 2017).

CONCLUSION
From the current study, it is concluded that α-Fe 2 O 3 nanoparticles have toxic effects that depend on dose and duration of exposure, and show through transferrin, cholinesterase, and caspase-3 levels, as well as their effects on oxidative stress induction.

Conflicts of interest
The writers certify that they have no relationships that could give rise to conflicts of interest.