Introduction

Recent years have witnessed great interest in the use of iron oxide nanoparticles (Fe₂O₃-NPs) in therapeutic and diagnostic applications in CNS (1). There is a lot of research on Fe₂O₃-NPs and their conjugates are used as magnetic resonance imaging (MRI) contrast agents (2). Possessing unique magnetic and paramagnetic properties, as well as their ability to cross the blood-brain barrier, α-Fe₂O₃-NPs are a promising neuroimaging (BBB) platform (3). For assessing CNS tumor lesions, reading, differential diagnosis, image-guided delivery in brain tumor treatment, detecting disease progression, monitoring therapies, and designing specialized treatment plans for CNS disorders (4-7). There is a lot of misinformation regarding the neurotoxic effects of α-Fe₂O₃-NPs on the CNS and their interference with neurobehavioral processes among these high-extending biomedical applications. Based on exposure to various NP concentrations, research in the last ten years has demonstrated the bioaccumulation of NPs and their detrimental consequences in important organs (8). Concerns regarding the biotoxicity of Fe₂O₃-NPs grow as their frequency of biological exposure grows. Fe₂O₃-NPs' clinical effects have been studied and determined to be safe; nevertheless, their molecular interactions with organ systems with repeated and prolonged use need to be examined (9). Since iron is a transition metal, it can easily go through the Fenton's reaction and control the oxidative stress that causes many neurodegenerative conditions in the brain (10). Despite the critical function of iron in healthy brain physiology and neural development, excessive buildup causes nervous system degeneration (11). In addition, it has been suggested that iron buildup may have a role in the initial stages of neurological memory problems like Alzheimer's disease (12). According to studies by authors (13-16), iron build up in the brain has been linked to oxidative stress, memory loss, impaired motor performance, and neurobehavioral dysfunction. The majority of neurodegenerative illnesses are characterized by a sequence of molecular processes, such as iron buildup, oxidative stress damage, and neuronal death, along with clinical signs such as impaired locomotion and memory loss (17). Exposure to Fe₂O₃-NPs has been shown to cause oxidative stress in the brain, which causes neuronal degeneration (18,19). Significant brain damage is brought on by elevated Fe₂O₃-NP accumulation in the basal ganglia of neuroferritinopathy patients (20). But, clarification of the effects of accumulating Fe₂O₃-NP buildup in the brain and locomotor is needed. It is necessary to lessen the neurotoxic effects of α-Fe₂O₃-NPs due to their high reactivity and the sensitivity of brain tissues to oxidative stress.

The aim of this research is to assess the toxic effects and processes that occur after α-Fe₂O₃- NP buildup in mice model brain system. This investigation looked at how α-Fe₂O₃-NPs affected brain and liver histology, and locomotor behavior.

Materials and methods

Ethical approve

All methods employed in investigations involving animals were upheld to the highest ethical standards by the facility or practice, where the tests were carried out UM.VET 2022.036.

Mice husbandry

We use 74 white male and female mice with body weights ranging from 25 to 35 g, ages between 60 and 90 days, housing them in an animal house.

Preparation of α-Fe₂O₃

Weights of nanoparticles supermagnetic α-Fe₂O₃ 98%, 20-40 nm produced by the American company, which were mixed and 10 ml per kilogram of distilled water is used to dissolve it, using ultrasonic cleaner at 20 Hz for 5 minutes at 4ºC to facilitate the dispersion of particles with water (21).

Determination of the acute oral LD₅₀ of α-Fe₂O₃-NPs based on the Dixon method

The first dose of α-Fe₂O₃-NPs was set at 10 g/kg for the nine mice that were employed (pilot study). One mouse was kidnapped and given a dose of 10 g/kg; the outcome (the mouse's survival or death) was then determined 24 hours after the dose. After the first change occurs, the dose of three mice individually was administered, taking into account the change in the dose up or down (22-25). The change in the dose amount must occur before the change in the outcome (the mouse's survival or its death) appears.

The effect of oral administering α-Fe₂O₃-NPs at different doses of the median LD₅₀ on acute toxicity events in mice

Five groups, each of five mice were created from a total of 25 animals. 10 ml/kg of distilled water was administered to the control group. The second group: the mice were dosed with nanoparticles α-Fe₂O₃ at a dose of 3.69 g/kg, which represents 25% of the LD₅₀. The third group: the mice were dosed with nanoparticles α-Fe₂O₃ at a dose of 7.37 g/kg, which represents 50% of the LD₅₀. The fourth group: the mice were dosed with nanoparticlesα- α-Fe₂O₃ at a dose of 11.06 g/kg, which represents 75% of the LD₅₀. The fifth group: the mice were dosed with nanoparticles α-Fe₂O₃ at a dose of 14.74 g/kg, which represents 100% of the LD₅₀. The mice received therapy, and they were observed for two hours after that, signs of intoxication were recorded, and the toxicity marks were calculated (26).

Sub-acute effects of repeated doses of nanoparticles α-Fe₂O₃

40 mice were divided into four groups randomly, each with 10 mice. For 28 days, the first group (the control group) received doses of distilled water at a rate of 10 ml per kilogram. In the second group, mice received 75 mg/kg of α-Fe₂O₃ nanoparticles. The third group received 150 mg/kg of α-Fe₂O₃ nanoparticles. The fourth group received 300 mg/kg of α-Fe₂O₃ nanoparticles. On the 7^th, 14^th, and 28^th days of dosing.

Open field activity test

To pass this test, you had to count how many squares mice crossed and how many times mice stood on back legs in three minutes (27,28).

Negative geotaxis test

This test shows neuromuscular balance and vestibular function in mice, and the negative geotropism test is performed using a rough wooden surface inclined at an angle of 45 degrees (29), as the head of the mouse is placed longitudinally down the wooden plank, and then according to the time required to rotate the mouse with its entire body 180 degrees toward the top of the inclined surface, and each rat is given a maximum of 60 seconds to turn completely (29).

Pocking head test

This test shows the extent of curiosity and movement of the treated mouse. This test was conducted using a circular plastic surface with a diameter of 30 cm with a small edge with a height of 10 cm containing 8 circular holes with a diameter of each hole 2 cm. The measurement begins by placing the mouse in the center of the perforated circular surface, then the number of times it enters it is calculated. Mouse head into the holes for 3 minutes (30).

Body weight

The weights of the mice were recorded on the 7^th, 14^th and 28^th days of dosing.

Histopathology

Animals exposed to α-Fe₂O₃ 30 were examined histopathological for the liver and brain in both control and treatment groups. Tissues were fixed in 10% neutral buffered formalin following sacrifice. After being embedded in paraffin blocks, the tissues were cut into sections with a microtome. Hematoxylin and eosin and the slide were used to stain 5 µm thick paraffin sections, the slides were examined with microscope at 400X (31,32).

Statistic evaluation

One-way ANOVA was used to examine the data. Its significance was assessed using the least significant test. The Fisher test was used to analyze the frequency data. Non-parametric data were evaluated using the Man Whitney test with a significance level of P≤ 0.05 (33).

Results

Determination of the acute oral LD₅₀ of nanoparticles α-Fe₂O₃ based on the Dixon method

The oral LD₅₀ of nanoparticles α-Fe₂O₃ was 14.74 g/kg, after administering various doses and the appearance of poisoning symptoms represented by lethargy, piloerection, rapid breathing, tremor, hypothermia, subcutaneous hemorrhage, and death (Table 1).

Table 1: The acute oral LD₅₀ of nanoparticles α-Fe₂O₃ in mice

O: Mouse alive for the past 24 hours. X: Mouse dead for the past 24 hours.

The effect of oral administering of nanoparticles α-Fe₂O₃ at different dose of LD₅₀ on acute toxicity

The administration of nanoparticles α-Fe₂O₃ in doses of 25, 50, 75, and 100% which are 3.68,7.37, 11.06, and 14.74 mg/kg, respectively, led to the appearance of the symptoms of toxicity in mice ranging from 20% to 100%, and the signs included lethargy, stillness of movement, rapid breathing, hypothermia, flatulence, tremor, piloerection, itching, subcutaneous hemorrhage, and the toxicity scores were 13, 17, 26, and 25, respectively (Table 2).

Table 2: Acute toxicity symptom elicited by nanoparticles α-Fe₂O₃ at different dose of LD₅₀ on acute toxicity

* Significant change from the control group at p < 0.05

Sub-acute effects of repeated doses of nanoparticles α-Fe₂O₃

After 7 days of treatment, oral administration of nanoparticles α-Fe₂O₃ at a dose of 75 mg/kg caused behavioral changes as evidenced by a significant increase in head pocking at doses of 75 and 150 mg/kg and a significant decline in open field activity (rearing and squares crossed) when compared to the control group, respectively (Table 3).

Table 3: Neurobehavioral measurements in mice treated with α-Fe₂O₃ Nanoparticles during 7 days of treatment

Values expressed as mean ± SE for 5 mice in each group. *Indicates significant change from the distilled water group at P<0.05.

The behavioral defect was caused by the oral administration of nanoparticles α-Fe₂O₃ at a dose of 300 mg/kg after 14 days of dosing, as evidenced by a significant increase in open field activity (squares crossed and raising) compared to the control group, a significant decrease in the time spent for the negative geotaxis test compared to the control group, and other behavioral changes (Table 4).

Table 4: Neurobehavioral measurements in mice treated with α-Fe₂O₃ Nanoparticles during 14 days of treatment

Values expressed as mean ± SE for 5 mice in each group. *Indicates significant change from the distilled water group at P<0.05. a indicate significant change from α-Fe₂O₃ treatment at P<0.05.

After 28 days of treatment, the oral administration of nanoparticles α-Fe₂O₃ at doses of 75 and 150 mg/kg resulted in behavioral defect as shown by a significant decline in open field activity (crossing squares and raising them) compared to the control group, a significant increase in the time spent performing negatively on geotaxis, and a significant decline in head pocking at dose 75 mg/kg, compared to the control group (Table 5).

Table 5: Neurobehavioral measurements in mice treated with α-Fe₂O₃ Nanoparticles during 28 days of treatment

Values expressed as mean ± SE for 5 mice in each group. *Indicates significant change from the distilled water group at P<0.05. a indicate significant change from α-Fe₂O₃ treatment at P<0.05.

Sub-acute effects of repeated doses of nanoparticles α-Fe₂O₃ on the body weight

 In mice, oral treatment with nanoparticles α-Fe₂O₃ at a concentration of 150,300 mg/kg body weight resulted in a notable rise in body weight on 7, 14 and 28 days (Table 6).

Table 6: The effect of repeated doses of α-Fe₂O₃ nanoparticles on the weights of mice for 7, 14 and 28 days

Values expressed as mean ± SE for 5 mice in each group. *Indicates significant change from the distilled water group at P<0.05.

Liver histopathological

The control group showed none of histopathological lesions (Figure 1). The oral administration of nanoparticles α-Fe₂O₃ at dose75mg/kg, caused histopathological defects after 7 days of the dosing, which is evidence by a mild vacuolar degeneration of hepatocytes, sinusoidal dilatation, mild vacuolar degeneration of hepatocytes, central vein congestion, sinusoidal dilatation (Figure 2). While the dose 150mg/kg causes vacuolar degeneration of hepatocytes, infiltration of inflammatory cells, central vein congestion, vacuolar degeneration of hepatocytes, central vein congestion (Figure 3). The dose 300 mg/kg causes mild cell swelling of hepatocytes, congestion of sinusoids, increase number of kupffer cells (Figure 4). Histological section of liver of 75mg/ kg after 14 days of treatment, mild vacuolar degeneration of hepatocytes, congestion of central vein, infiltration of inflammatory cells and increase in number of kupffer cells (Figure 5). While the dose 150 mg/kg after 14 days of treatment causes cell swelling of hepatocytes, congestion of sinusoids, and vacuolar degeneration (Figure 6). The dose 300 mg/kg after 14 days of treatment causes necrosis of hepatocytes, congestion of sinusoids and central vein, vacuolar degeneration, and increase in number of kupffer cells (Figure 7). In 28 days of treatment with nanoparticles α-Fe₂O₃ at dose75mg/kg causes sever vacuolar degeneration of hepatocytes, congestion of sinusoids and central vein, and necrosis (Figure 8). The dose 150 mg/kg after 28 days of treatment with the moderate vacuolar degeneration of hepatocytes, congestion of sinusoids and central vein, necrosis (Figure 9). The dose 300 mg/kg causes sinusoid dilatation (Figure 10).

Brain histopathological

The control group showed none of histopathological lesions (Figure 11). The oral administration of nanoparticles α-Fe₂O₃ at dose75mg/kg, causes histopathological defects after 7 days of the dosing, which is revised by a cortex with perivascular edema, vacuolization and satellitosis (Figure 12). The doses 150 and 300 mg/kg cause vacuolization, cytogenetic edema and neuronophagia (Figures 13 and 14). In 14 days of treatment with nanoparticles α-Fe₂O₃ at dose75, 150 and 300 mg/kg led to cortex with perivascular edema, vacuolization, gliosis, thrombus formation, cytogenetic edema, and satellitosis (Figures 15-17). The nanoparticles α-Fe₂O₃ at dose75,150 and 300 mg/kg after 28 days of treatment, which causes necrosis, gliosis, congestion of blood vessels, thrombus formation and neuronophagia (Figures 18-20).

Figure 1: The normal liver architectures are shown in the liver of mice (control group): (a) the central vein, (b) the hepatocytes, and (arrow) the sinusoids. 100x (H&E stain).

Figure 2: liver of mice dosing75mg /mg for 7days show (a) mild vacuolar degeneration of hepatocytes, (b) central vein congested, (arrow) sinusoidal dilatation. 400x (H&E stain).

Figure 3 liver of mice 150mg /mg dosing for 7days show (a) vacuolar degeneration of hepatocytes, (b) infiltration of inflammatory cells, (c) central vein congestion. 400x (H&E stain).

Figure 4: liver of mice dosing 300mg/mg for 7days show (a) mild cell swelling of hepatocytes, (arrow) increase number of Kupffer cells. 400x (H&E stain).

Figure 5: liver of mice dosing 75mg / kg for 14 days show (a) mild vacuolar degeneration of hepatocytes, (arrow) central vein congested, (c) infiltration of inflammatory cells, (arrow) increase number of kupffer cells. 400x (H&E stain).

Figure 6: liver of mice dosing 150mg/kg for 14 days show (a) cell swelling of hepatocytes, (arrow) sinusoids congested, and (b) vacuolar degeneration. 400x (H&E stain).

Figure 7: liver of mice dosing 300mg / kg for 14 days show (a) necrosis of hepatocytes, (black arrow) sinusoids congested and central vein, and (b) vacuolar degeneration, (yellow arrow) increase number of Kupffer cells. 400x (H&E stain).

Figure 8: liver of mice dosing 75mg/kg for 28 days show (a) sever vacuolar degeneration of hepatocytes, (b) sinusoids congested and central vein, (c) necrosis. 400x (H&E stain).

Figure 9: liver of mice dosing150mg/kg for 28 days show (a) moderate vacuolar degeneration of hepatocytes, (b) sinusoids congested and central vein, (c) necrosis. 400x (H&E stain).

Figure 10: liver of mice dosing 150mg/kg for 28 days show (a) mild vacuolar degeneration of hepatocytes, (b) sinusoids congested and central vein, and (arrow) sinusoid dilatation. 400x (H&E stain).

Figure 11: The normal brain mice showing normal architecture representing by neurons (A), glial cells (B) and blood vessels (C). 400x (H&E stain).

Figure 12: Brain of mice dosing 75 mg/kg for 7 days showing the cortex with perivascular edema (A), vacuolization (B) and satellitosis (C). 400x (H&E stain).

Figure 13: Brain of mice dosing 150 mg /kg for 7 days showing the cortex with perivascular edema (A), vacuolization (B) and cytogenetic edema (C). 400x (H&E stain).

Figure 14: Brain of mice dosing 300 mg/kg for 7 days showing the cortex with perivascular edema (A), vacuolization (B), cytogenetic edema (C) and neuronophagia (D). 400x (H&E stain).

Figure 15: Brain of mice dosing 75 mg /kg for 14 days showing the cortex with perivascular edema (A), vacuolization (B) and gliosis (C). 400x (H&E stain).

Figure 16: Brain of mice dosing 150 mg/kg for 14 days showing the cortex with perivascular edema (A), thrombus formation (B) and cytogenetic edema(C). 400x (H&E stain).

Figure 17: Brain of mice dosing 300 mg/kg for 14 days showing the cortex with perivascular edema (A), vacuolization (B), cytogenetic edema (C) and satellitosis (D). 400x (H&E stain).

Figure 18: Brain of mice dosing 75 mg/kg for 28 days showing the cortex with perivascular edema (A), vacuolization (B), cytogenetic edema (C) and necrosis (D). 400x (H&E stain).

Figure 19: Brain of mice dosing 150 mg/kg for 28 days showing the cortex with perivascular edema (A), congestion of blood vessels (B), vacuolization (C), cytogenetic edema (D) and gliosis (E). 400x (H&E stain).

Figure 20: Brain of mice dosing 300 mg for 28 days showing the cortex with perivascular edema (A), thrombus formation (B), vacuolization (C), cytogenetic edema (D) and neuronophagia (E). 400x (H&E stain).

Discussion           

Due to the limited information about the acute toxic effect of α-Fe₂O₃ nanoparticles in mice, the current study focused on determining the LD₅₀ of α-Fe₂O₃ nanoparticles for the first time and recording signs and scores of poisonings as well as its effects on the neurobehavioral and histological sections of the liver and brain of mice.

 In general, α-Fe₂O₃- NPs are thought to be secure, biocompatible, and non-poisoning substances. This is proven by our results by determining the acute oral LD₅₀ in mice, which was 14.7 g/kg, while the available data of uncoated iron oxide nanoparticles' LD₅₀ was estimated to be between 300 and 600 mg/kg (34). This number was increased to 2000-6000 mg/kg when the iron oxide nanoparticles were coated with stabilizing and biocompatible dextran molecules (34). This is based on how compounds are classified using the Globally Harmonized Classification System (GHS) that belongs to the unclassified toxicity, which means that α-Fe₂O₃ nanoparticles is rather safe in mice (35).

The highest toxic doses of α-Fe₂O₃ nanoparticles in mice causes symptoms of acute toxicity, such as lethargy, rapid respiration, subcutaneous hemorrhage, plioerection, tremor, flatulence, and itching. While the clinical toxic symptoms following acute oral administration in rat is dullness, irritation, and moribund signs (36). In humans, the toxic signs are nausea, vomiting, and flatulence (37). Also, they include headache, urticarial, pruritus, and back pain, chest pain, dyspnea, a skin rash, a drop in oxygen saturation, and anaphylactic shock (38).

The current work focuses on the numerous behavioral changes including the head pocking test (30), which reveals the negative effects on the brain and its cognitive function, the negative geotaxis test (29), which increases the duration of negative geotaxis performance due to a defect in the vestibular system, and the open-field activity test (28), which lowers motor activity due to central nervous system inhibition caused by repeated exposure to α-Fe₂O₃-nanoparticals for 28 days. Since Fe₂O₃-nanoparticals buildup has been proven to have negative effects on the CNS and aggravate neurodegenerative illnesses (18,19), much research is being done to shed light on the safety concerns of Fe₂O₃-nanoparticals exposure. According to De Lima et al. (16) and Budni et al. (39), oxidative stress mechanisms and apoptosis are linked to behavioral alterations (40). By producing free radicals, Fe₂O₃-nanoparticals cause oxidative stress and collapse the antioxidant defense system. The animals exposed to α-Fe₂O₃-nanoparticles repeatedly experience their entrance and buildup in the CNS due to BBB damage. Accumulated α-Fe₂O₃-nanoparticals interact with the important biomolecules, negatively affecting neuronal physiology and opening the door to alterations in behavior (40).

Similar to Kumari et al. and Manseel et al. (36,42) test groups subjected to low, medium, and high doses of α-Fe₂O₃ nanoparticles repeatedly till 28 days showed no reduction in body weight in the exposed mice. Wang et al. (43) reported considerable weight loss, gastro-intestinal damage, and severe discomfort in contrast to these observed results.

A considerable amount of microscopic damage was seen in all major organs (liver and brain) during a histopathological evaluation of the harvested tissues. Fe binding proteins such as transferrin and ferritin, which act as carriers of Fe to various tissues, appear to have an influence on the observed differential organ distribution pattern of Fe₂O₃ nanoparticles in a dose dependent. Wang et al. (44) also discovered accumulation of Fe₂O₃ nanoparticles at an oral dose of 600 mg/kg in the major organ systems of mice, supporting the aforementioned findings. Numerous reports have examined the poisoning of Fe₂O₃ nanoparticles (45,46). However, there is a discrepancy between the stated results. According to Kim et al. (47) Fe₂O₃ nanoparticles do not appear to be hazardous to mice. However, numerous other studies (48-50) have documented serious deleterious consequences linked to exposure to these Fe₂O₃ nanoparticles (47). In the current investigation, the majority of the organs, including the liver, exhibited histological alterations that were mostly caused by vascular congestion. Even when the α-Fe₂O₃ nanoparticles are tailored for tissue or organ specific targeting (e.g., tumors), liver is expected to contain the most efficient nanoparticle clearance system, which accounts for the observed vascular alterations in the highest dosage group (36,42).

Conclusion

The study findings suggest that mice exposure to α-Fe₂O₃ nanoparticles can cause neurodegeneration and behavioral abnormalities. It is discovered that the toxicity of α-Fe₂O₃ nanoparticles increases with concentration. As a result, it is possible to draw the further conclusion that doses and the duration of exposure to - α-Fe₂O₃ nanoparticles cause unfavorable histological alterations that could harm cellular functions. Given the quick development of α-Fe₂O₃-NP applications, the findings call for an understanding that is essential, not only for safety considerations, but also for their repeated biomedical applications in the future.

Acknowledgement

Sincere thanks are due to Department of Physiology, Biochemistry, and Pharmacology, and to College of Veterinary Medicine, University of Mosul.

Conflict of interests

The writers declare that they have no interests in opposition.