A Stereological Study of the Toxic Effects of Cerium Oxide during Pregnancy on Kidney Tissues in Neonatal NMRI Mice

Background Both antioxidant and prooxidant activities have been previously reported for cerium oxide (CeO2). The aim of this study was to investigate the effects of CeO2 at different doses on changes in kidney tissues and markers in neonatal mice. Methods We randomly divided 30 pregnant NMRI mice into five groups (n = 6 per group)—a control group and four groups treated with intraperitoneal (i.p.) administration of different doses of CeO2 (10, 25, 80, or 250 mg/kg body weight (bw)) on gestation days (GD) 7 and GD14. At the end of the treatment period, we analyzed the kidney tissues and serum samples. The levels of two serum redox markers, malondialdehyde (MDA) and ferric reducing/antioxidant power (FRAP), were determined. Data were analyzed using one-way ANOVA and Tukey's test, and a P value of <0.05 was considered significant. Results The mean total volumes of the renal corpuscle, glomeruli, and Bowman's capsule membranes significantly increased, and there was a significant decrease in the mean total volume of Bowman's space in the high-dose CeO2 group compared to that in the control group. No statistically significant differences existed in the serum levels of MDA and FRAP in the treated and control groups. Conclusion Our results suggest that high doses of CeO2 impair fetal renal development in pregnant mice, which results in kidney damage. Therefore, CeO2 administration during pregnancy could have dose-dependent adverse effects on the developing kidneys in neonates.


Introduction
Cerium is the most abundant rare-earth metal and most active element in the lanthanide group. Cerium is a soft, ductile, and malleable metal with a color that ranges from irongray (commercial grade) to silver (pure form). Cerium compounds have the highest environmental activity compared to other members of the lanthanide group [1].
Cerium oxide (CeO 2 ) is the most commonly used commercial compound of cerium [2]. Cerium oxide lanthanides are widely used as catalysts, oxygen sensors, in the manufacture of solar/fuel cells, and polishing agents in various fields [3][4][5][6]. The unique properties of CeO 2 , especially its low toxicity and high reducibility, have increased the use of micro-and nanosized CeO 2 in various medical fields and led to significant advances in these fields [1]. The medical applications of CeO 2 are due to its antioxidant, anti-inflammatory, and antibacterial properties and its high angiogenic potential. Cerium oxide is used to assist with the healing of various tissues such as the bones, skin, cardiac, and nerves. Recently, the transfer of drugs and genes by CeO 2 nanoparticles and the use of CeO 2 as treatments for cancer and other diseases has received much attention [7,8].
Cerium, itself, has no properties and is not physiologically important for living organisms; however, soluble Ce 3+ salts (sulfate, nitrate, chloride, phosphate, and hydroxide) contain various properties that are of medical importance. Cerium oxide is a pale yellow-white powder with the chemical formula CeO 2 [9]. The autoregenerative cycle nature of CeO 2 is due to the presence of an enormous number of surface defects and its ability to switch between Ce 3+ and Ce 4+ oxidation states. The formation of an oxygen vacancy in CeO 2 is associated with reduced Ce 4+ and Ce 3+ oxidation. This property allows Ce to absorb or give off an electron from the active oxygen species, making them inactive and neutral, and indicates a key role in the ratio of the Ce 3+ /Ce 4+ oxide in the antioxidant activity of CeO 2 [9][10][11]. Cerium oxide is believed to function as a superoxide dismutase (SOD)/catalase mimetic [9,[12][13][14][15]. In an experiment on mice, the antioxidant properties of CeO 2 nanoparticles inhibited active oxygen species and its potential for the treatment of oxidative stress was reported [16]. Oxidative stress is an imbalance between reactive oxygen species (ROS) and antioxidants in the body [17]. Researchers propose that CeO 2 could be used to treat diseases associated with oxidative stress and inflammation [9,10,18,19]. This ability of the nanoscale to neutralize ROS from a pool of high concentration polymer ligands suggests that nanoscale activity may not decrease in the physiological environment, even when coated with a protein corona. A study on the distribution of inhaled CeO 2 nanoparticles in mice showed that the cells phagocytosed the nanoparticles [20,21].
Previous studies on the effects of CeO 2 in living organisms reported contradictory results. Some researchers reported that CeO 2 caused oxidative stress in mitochondria and hepatocellular damage [22], inflammation in tissues such as the kidneys and liver [23], and DNA damage in peripheral blood leukocytes (PBL) and liver cells [24]. Cerium oxide can also cause lung fibrosis [25] and angiogenesis [26].
In contrast, other researchers reported that CeO 2 could act as an antioxidant and be used for cancer prevention and treatment [27,28]. In another study, the optimum concentration (10 -3 -10 -9 M) of CeO 2 increased cell division of primary fetal fibroblasts in vitro [29]. The results of a study showed that CeO 2 nanoparticles reduced oxidative stress and inflammation in mice treated with diethylnitrosamine [30]. The protective effect of CeO 2 nanoparticles in preventing tissue damage and oxidative stress induced by diabetes in pregnant mice has been reported [31]. Existing synthetic protocols have the ability to obtain CeO 2 nanoparticles with different physical and chemical properties (shape, size, zeta potential, and cerium valence state). The synthesis method directly affects their biological activity [32]. The impact of these characteristics on toxicity, especially fetal toxicity, has not been elucidated.
The impact of a wide range of cerium nanoparticles stabilized by citrate on the growth of two-cell embryos was investigated. The results showed that the cerium nanoparticle concentrations had no toxic effects on fetal development [33]. Cerium oxide can cross the placenta and make its way to the liver, spleen, and lung tissues of adult, neonatal, and fetal mice, inducing tissue destruction and necrosis [34]. In addition, the results from our previous study have shown that high-dose CeO 2 can have a devastating effect on testicular tissue development in neonatal mice [35].
The kidneys play a key role in regulating the body's homeostasis and excreting waste products [36]. Metanephric development begins in humans during week five of gestation and in mice at embryonic day (E) 10.5 [37]. There is an enhanced chance for exposure to CeO 2 because of the increase in its various uses in daily life. Pregnant women are exposed to CeO 2 via the skin, inhalation, foods, and medicines.
Congenital anomalies of the kidneys are among the most important anomalies [38]. When pregnant mice are exposed to CeO 2 , these particles can cross the placenta and accumulate in the fetal organs [22][23][24]34]. CeO 2 may hinder embryonic development and may have possible demographic impacts [34]. Given the importance of kidney development during pregnancy and the postpartum period, the present study is aimed at comparing histological changes in neonatal kidneys after their mothers were exposed to different doses of a CeO 2 suspension during the gestational day (GD) 7 and GD14 of pregnancy.

Materials and Methods
2.1. Materials. Cerium (IV) oxide (CeO 2 ) powder that had a diameter < 5 μm, assay of 99.9% trace metal basis, and density of 7.13 g/ml at 25°C (lit.) was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). We prepared the different doses of CeO 2 in double-distilled (dd) water. Ultrasonic vibration (100 W, 30 kHz) was performed for 15 min before administration.
In this study, the selected doses were based on previous studies and doses lower than the lethal dose; 50% (LD50) were used for the animals [34,39]. Based on the contradictory results of previous studies, we selected various doses that ranged from low to high to detect dose-dependent effects in the laboratory animals. We performed our experiment based on the characteristics reported by the manufacturer of CeO 2 and previous experiments [1,34,[40][41][42].
The different doses of CeO 2 were prepared in doubledistilled (dd) water. Ultrasonic vibration (100 W, 30 kHz) was performed for 15 min before administration.

Animals and Experimental
Groups. We obtained adult NMRI mice (male : female ratio of 1 : 2) that had an average weight of 25-30 g from Pasteur Institute of Iran (Tehran, Iran). The animals were allowed to acclimate for one week under standard conditions that included a 12 : 12 h light/dark cycle with ad libitum access to food and water. Once acclimated to their new environment, the male and female mice were kept in a cage at a 1 : 2 ratio. The pregnant mice were placed in separate cages. The detection of a vaginal plug was considered to be gestation day (GD) 0. The pregnant mice were randomly divided into five groups (n = 6 per group): a control and four treatment groups. Mice in the 2 Oxidative Medicine and Cellular Longevity treatment groups received intraperitoneal (i.p.) injections of different doses of CeO 2 (10, 25, 80, or 250 mg/kg body weight (bw)) on GD7 and GD14. In this experiment, 15-day-old neonates were used for histological evaluation of kidney tissues and serum biochemical parameters. Changes in body weights and kidney tissue in 2-and 6-day-old neonates were evaluated.

Histological
Examinations of the Kidneys. The 15-day-old postpartum (dpp) offspring were weighed and anesthetized by chloroform. After dissection, blood samples were collected from the heart using a 1 cc syringe. The left kidneys from the mice were excised and rinsed with distilled water, weighed, and fixed for one week in a 10% formaldehyde solution. After tissue passage and paraffin block preparation, the paraffin blocks were sectioned into 5 μm sections with a microtome and subsequently stained with Heidenhain's Azan stain [43]. We randomly selected nine sections from each kidney to evaluate the histological parameters.

Kidney Volume.
We used the Cavalieri method to assess kidney volume [44]. First, we systematically selected 15 random tissue sections from all of the 5 μm sections at the same interval. The predesigned point probe was randomly uniform on the image of each of the sections, and the points encountered with the whole kidney image were counted.
The kidney volume was calculated in all the slices by using the following formula: where ∑ n i=1 P is the sum of the total points, "t" represents the thickness between selected sections, and "aðpÞ" is the level of the point probe.
Next, we calculated the cortex and medullary volumes. Tissues were chosen by regular, random sampling, and the average of 15 fields of view from each 5 μm section was assessed at 100x magnification by placing the point probe on each field.

Volumes of the Cortex, Medulla, and Cortex Components.
The total number of points that hit the probe with the entire field was ∑ n i=1 P total ; the whole number of the points that hit the probe in the cortex was ∑ n i=1 P cortex ; and the whole number of points that hit the probe in the medulla was ∑ n i=1 P medulla .
Volumetric density was calculated using the following formulas for the cortex and medulla: We separately estimated the volumes of the cortex and medulla by multiplying the volume density of each by the kidney volume in each neonatal mouse.
We estimated the volume of the components of the cortex, proximal tubule (PT), and distal tubule (DT), with the lumen and their epithelium, glomeruli, and interstitial tissue by systematic random sampling. An average of 15 fields of view from each 5 μm slide was assessed by placing a counting frame on each field. The total number of the points that hit the frame with the entire field of view was selected ð∑ n i=1 P total Þ, and the whole number of points that hit each component ð∑ n i=1 P x Þ was shown. The volume density was calculated using the following formula: where "x" represents the PT and DT, lumen, epithelium, interstitial tissue, and glomeruli. Then, using the following formula, we separately calculated the volumes of the PT and DT, lumen, epithelium, interstitial tissue, or glomeruli by multiplying the volume density of each in the cortical volume: In the above formula, "x" represents the PT, DT, lumen, epithelium, interstitial tissue, and glomeruli.
2.6. Volume of Bowman's Capsule and Space. In order to obtain the volume of the glomeruli components, we first compared the whole number of the points that hit the frame with these components ð∑ n i=1 P x Þ and the whole number of the points that hit the frame with each glomerulus ð∑ n i=1 P glomerulus Þ. The volumetric density of the glomerulus was calculated using the following formula: where "x" represents each of the components of the glomerulus (Bowman's capsule and space). Then, the volume of Bowman's capsule and space were calculated by multiplying the volume density of each component in the volume of the glomerulus as follows: where "x" represented any of the glomerulus components, namely, Bowman's capsule and space.

Length of Proximal Tubules (PT) and Distal Tubules (DT).
In order to calculate the length of the PT and DT from the 5 μm slides of kidney tissue at 400x magnification, we used systematic random sampling to select 15 fields of view. The counting probe was randomly placed on each of the microscopic fields of view, and the number of tubules 3 Oxidative Medicine and Cellular Longevity counted within the frame or those that collided with the reception lines was counted. The number of tubules that contacted the banned lines was not counted. Then, the longitudinal densities of the PT and DT were calculated from the following equation: where ∑Q i is the sum of selected tubules, a/f is the desired frame level at the texture scale, and ∑P i is the sum of the points of contact with the kidney tissue.
2.8. Serum Redox Markers. Blood samples were collected from 15-day-old neonatal hearts to estimate the serum redox markers, malondialdehyde (MDA) and ferric reducing/antioxidant power (FRAP).
2.9. Malondialdehyde (MDA) Levels. Buege and Aust's procedure was used to evaluate serum MDA levels. In this method, a solution that contained trichloroacetic acid (TCA; 15% g/ml), TBA (0.375%, g/ml), and hydrochloric acid (HCl, 25% normal) was prepared and the sera were combined in a 2 : 1 ratio and placed in a bain-marie for 15 min. The solution was placed in cold water and then centrifuged for 10 min. The absorbance of the solution was read using a spectrophotometer at a wavelength of 532 nm [45,46].

Ferric
Reducing/Antioxidant Power (FRAP) Assay. The FRAP assay was used to estimate the antioxidants. We combined 0.5 ml of serum with 1.5 ml of the reaction mixture. The degree of plasma regeneration is proportional to the concentration of this complex. At low pH, the reduction of the TPTZ-Fe 3+ complex in the form of ferrous (Fe 2+ ) creates a blue complex that has a maximum absorption of 593 nm. The degree of the regenerative capacity of the serum was measured by increasing the concentration of the above complex using a spectrophotometer. The FRAP assay directly evaluates the whole antioxidant power [47].
2.11. Statistical Analysis. Data were analyzed with SPSS 16 (Statistical Package for the Social Sciences), ANOVA, and Tukey's test. P values < 0.05 were considered statistically significant.

Histological Evaluation of the Kidney Tissues.
In the control group, we observed that the kidney tissues had a normal structure with regular tubules, cylindrical epithelial cells based on the basement membrane, lumen space, and normal glomeruli. In the group that received less than 250 mg/kg bw CeO 2 , the glomeruli were inflamed, and there was a significant increase in the volumes of the glomeruli and the membrane of the Bowman's capsule, along with a significant decrease in volume of Bowman's capsule space compared to the control group (P < 0:02). There were no statistically significant differences in the other groups treated with CeO 2 compared to the control group (Figure 1).

Body and Kidney
Weights. There were no statistically significant differences in body and kidney weights in the treatment and control groups (Table 1).

Volume of the Kidney, Cortex, Medulla, and Cortex
Components. A comparison of kidney volumes in the treatment and control groups showed a significant decrease in the group that received the 250 mg/kg bw CeO 2 dose (P < 0:02) ( Table 2). The cortex volume was significantly reduced in the group that received 250 mg/kg bw CeO 2 (P < 0:03) compared to that in the control group. There was no statistically significant difference in the other groups treated with CeO 2 compared to that in the control group. We also observed no statistically significant difference in medulla volume in the treatment groups compared to that in the control group.
There were significant increases in volume in the interstitial tissue (P < 0:01), renal corpuscle (P < 0:02), glomerulus (P < 0:02), and Bowman's capsule (P < 0:02) tissues in the 250 mg/kg bw CeO 2 group compared with those in the control group. The other treatment groups showed no significant volume changes in these tissues when compared with the control group (Table 3).
A significant decrease was observed in the volume of Bowman's space in the 250 mg/kg bw CeO 2 (P < 0:05) group compared with that in the control group; however, the other treatment groups did not significantly differ with the control group ( Table 3).
The volume of the PT and its epithelium (P < 0:04) and the PT lumen (P < 0:05) decreased significantly in the group that received 250 mg/kg bw CeO 2 compared to that in the control group (P < 0:05).
There was no significant difference between the volume of the DT and the epithelium and its lumen in the group that received 250 mg/kg bw of CeO 2 compared with that in the control group (Table 4).

Lengths of Proximal Tubules (PT) and Distal Tubules (DT).
There was no significant difference between the DT and PT lengths in the treatment groups compared to that in the control group (Table 5).

Biochemical
Evaluations. Statistical analysis of blood serum MDA showed no significant difference between treatment groups compared to the control group ( Figure 2). In addition, statistical analysis of blood serum total antioxidant capacity (TAC) showed no significant difference between treatment groups compared to the control group (Figure 3).

Discussion
In this study, we administered i.p. injections of different doses of a CeO 2 microparticle suspension to pregnant mice on GD7 and GD14 and examined their effects on neonatal mice kidney tissues by light microscopy. The selection of the CeO 2 micropowder for this experiment was based on previous studies in which the toxic effects of CeO 2 microparticles and their faster accumulation in the tissues of living organisms were confirmed [34,48]. Exposure of pregnant 4 Oxidative Medicine and Cellular Longevity    Determining the average tissue and body weights is an important indicator for assessing the toxic effects of a substance on the body. In this study, body and kidney weight changes in offspring were measured at 2, 6, and 15 dpp. The results indicated that there were no significant differences in body and kidney weights between the experimental and control groups (Table 1).
Previous findings have indicated that CeO 2 passes through the placenta [34]. In mice, the development of the metanephros is considered to begin at E10.5-11 and ends at 7-10 days after birth [49,50]. The kidney, like all other major organs of the body, is susceptible to exposure to a        Oxidative Medicine and Cellular Longevity wide range of chemicals during developmental periods. Regulated differentiation and proliferation of mesenchymal cells and urinary epidermal primordial cells cause nephrogenesis in the embryonic period [50]. Kidney development in mice is completed two weeks after birth [51]. Accordingly, we decided to investigate changes in kidney tissue 15 days after birth. Studies have also shown that CeO 2 can accumulate and cause inflammation in tissues such as the lungs, liver, and kidneys [23]. Cerium is capable of switching between the Ce 3+ and Ce 4+ states, which may aid the antioxidant property of CeO 2 . On the other hand, another investigation has shown that CeO 2 causes ROS formation, inflammation, and DNA loss [19]. The result of one study indicated that CeO 2 increased ROS formation and, consequently, induced oxidative damage in mitochondria [22].
Increased CeO 2 -induced ROS levels may be the cause of observed cellular damage and apoptosis. ROS production and oxidative stress might be due to the catalytic properties of CeO 2 , impaired mitochondrial function, or a combination of both mechanisms [24,42].
ROS is capable of reacting with proteins, lipids, and nucleic acids, leading to lipid oxidation in biological membranes and the effects of enzymatic processes such as ion pump activity and DNA damage, thereby inhibiting transcription, repair, and apoptosis [52,53]. As a result, lipid peroxidation destroys unsaturated fatty acids in the membranes [54]. This can be one reason for the decrease in cell volume and, ultimately, the decrease in kidney volume in the group that received 250 mg/kg bw CeO 2 compared to that in the control group (Table 2).
Increased glomeruli volume, as representative of the renal and functional units of the kidney, can compensate for lost glomeruli function, adapt to new conditions, and remove toxins from the body [55,56]. Glomeruli undergo hyperfiltration to control the conditions and maintain filtration, resulting in an increase in glomerular volume [57].
Oxidative stress contributes to kidney damage through several mechanisms. This primarily occurs through increased expression of the vascular endothelial growth factor (VEGF) gene in podocytes, endothelial cells, and renal mesangial cells that increase glomerular permeability and protein excretion through urine [58]. Growth factors increase the expression of collagen types I, III, IV, V, and VI, and the laminin and fibronectin proteins, which increases the extracellular matrix and thickening of the glomerular basement membrane [58,59].
It has been shown that oxygen free radicals play a major role in inflammation in kidney interstitial tissue [60,61]. Therefore, an increase in interstitial tissue volume in the group that received CeO 2 at a dose of 250 mg/kg bw compared to that in the control group (Table 3) might indicate inflammation caused by CeO 2 .
In the present study, the lumen space of the PT in the group that received 250 mg/kg bw CeO 2 was decreased compared to that in the control group (Table 4). This could be due to the destructive dose-dependent effect of CeO 2 on tubules and the presence of necrosis, an apoptotic margin of the PT epithelial cells, and swelling of the epithelial cells of the wall of the tubule. It can be concluded that the swelling of PT wall cells reduces the lumen spaces of tubules [23].
The data of this study showed no statistically significant difference between serum MDA and FRAP levels in the treatment groups compared with the control group ( Figure 3). Oxidative stress is due to reduced body resistance to oxidants and lower antioxidant levels in the blood. [22,62]. According to other studies, antioxidant capacity in vivo depends on many factors such as environmental conditions (diet, etc.) [63,64].
Studies of CeO 2 in different animals showed that the level of CeO 2 toxicity depended on the duration of exposure, tissue environment, and type of cell [19,65].
Previous research has suggested that the effect and toxicity of CeO 2 are closely related to the types of tissues and cells, as well as the type of animal and the duration of exposure [66][67][68].
Studies of the dose-dependent relationship of CeO 2 effects on living organisms are complex. In one study, ICR mice were treated by oral gavage with one of three doses (10, 20, or 40 mg/kg bw/day) for six weeks. The accumulation of Ce particles in the nuclei of liver cells and mitochondria had a direct relationship to the increased dose [22]. The inflammatory effects of CeO 2 nanoparticles were studied at different doses (2000, 3000, and 5000 mg/kg bw) administered daily for 14 days in CD-1 mice. The results did not show any relationship between the concentration used and toxic effects [23].
In the present study, animals exposed to the highest doses of CeO 2 (250 mg/kg bw) showed significantly different histological parameters from their control counterparts. Animals exposed to the lowest doses (10, 25, and 80 mg/kg bw) did not show significant differences in histological parameters compared to the control group. Studies on the effect of CeO 2 on living organisms in vivo and in vitro confirm our findings. Previous studies have shown that low-dose CeO 2 can be used to treat cancer and eye diseases and is a powerful 7 Oxidative Medicine and Cellular Longevity antioxidant [69,70]. Therefore, according to review data, low-dose CeO 2 may have beneficial and possibly protective effects.
According to histological data, the high dose of CeO 2 in this experiment (250 mg/kg bw) was not tolerable for the animals. This dose could lead to toxic effects and oxidative stress, as well as disruption in the development of kidney tissues in mice. The present study results indicated that the dose of CeO 2 could determine the presence of positive and negative effects from its various applications. However, additional research should be conducted to confirm these findings.

Conclusion
We observed significant increases in the mean total volume of the kidney, cortex, renal corpuscle, glomerulus, and membrane of Bowman's capsule and a significant decrease in the mean total volume of Bowman's space in the group that received 250 mg/kg bw of CeO 2 compared to that in the control group. Our data showed no statistically significant differences between serum MDA and TAC levels in the treatment and control groups. According to our experiment, the efficacy of CeO 2 on kidney development in neonatal mice was dose dependent. More studies should be conducted to investigate CeO 2 -induced renal damage in offspring exposed to CeO 2 in utero.  Total antioxidant capacity  TCA:  Trichloroacetic acid  TPTZ: 2,4,6-Tripyridyl-s-triazine TBA: Thiobarbituric acid TGF-1: Transforming growth factor-1 VEGF: Vascular endothelial growth factor V: Volume W: Weight.

Data Availability
No data were used to support this study.

Conflicts of Interest
There is no conflict of interest related to this research.