Modulatory Role of Zeolite against Cadmium Chloride-Induced Renal Damage In Pregnant Rats and Their Fetuses

Cadmium (Cd) is a heavy metal and a significant inorganic toxin that is commonly found throughout the environment. The human body, as well as the kidneys, tend to accumulate Cd. Research conducted on both humans and animals suggests that exposure to cadmium can lead to significant skeletal damage, specifically osteoporosis. Based on the prevalence of cadmium in the environment and its frequent use, we studied the effect of its toxicity on pregnant rat and their fetuses and the effect of zeolite on them. The experimental rats were divided into 5 groups (n=8, each). The 1 st group received distilled water as a normal control group. The 2 nd received tween 80 and served as tween 80 group. The 3 rd was administrated with zeolite dissolved in tween 80 then in distilled water at a dose (100mg\kg\day). The 4 th was administrated with cadmium chloride at dose (30mg\kg\day). The 5 th was administrated with a combination of both zeolite and cadmium chloride. All groups were administered the materials orally from the 6 th day to the 15 th day of gestation. All animals were sacrificed at the end of gestation periods (on the 20 th day). Our obtained results showed adverse histological alterations in maternal and fetal kidneys. Also, CdCl 2 (cadmium chloride) induced remarkable changes in the levels of kidney functions. On the other hand, results showed incomplete ossification of the skull, metacarpals, and phalanges of the toes of fetuses’ skeletons. Zeolite administration markedly ameliorated the altered renal histological and physiological changes as well as the skeletal deformity in fetuses induced by cadmium.


INTRODUCTION
According to the industrial revolution that started in the 19 th century, there is an increase in heavy metals usage in today's industry.Heavy metals can easily enter the food chain because of their high resistance to environmental conditions.Due to the long biological half-life, slow metabolism, and low elimination rate of heavy metals, they can accumulate in the placenta, small intestine, liver and kidney, and cause toxic effects (Şensoy, 2023).
Cadmium, a heavy metal, is found in various locations and is highly toxic even at minimal levels of exposure.It has both immediate and long-term impacts on both human health and the environment.Widely Cd is used in industrial applications according to its unique chemical and physical properties (Krichah et al., 2003).The main routes of exposure to cadmium include inhalation of fumes and dust containing cadmium, accidental ingestion through contaminated hands and food, and passive/active smoking (Ali et al., 2023).
Cadmium builds up in the body's soft tissues and remains in the system for a long time, with a half-life of approximately 15-30 years in humans, mainly due to its slow elimination rate from the body (Henson and Chedrese, 2004).
Exposure to cadmium has been demonstrated to lead to the development of cancer and genetic mutations.(Waalkes et al., 2003;Huff et al., 2007).Cd has also been associated with a variety of harmful effects on reproductive organs.(Burukoglu and Bayçu, 2008;Aprioku et al., 2009).Cd has been demonstrated to disrupt ovarian functions, leading to ovulation failure and hindering implantation (Zhang et al., 2008;Obianime et al., 2011).
Furthermore, exposure to cadmium was found to cause skeletal demineralization by directly interacting with bone cells, reducing mineralization and inhibiting the formation of collagen and procollagen C-proteinases (Rahimzadeh et al., 2017).
The main targeted organ for cadmium accumulation is the kidney (Zhang et al., 2024).Chronic exposure to Cd causes severe nephrotoxicity in humans and animals.Renal dysfunction induced by cadmium may be due to proximal tubular damage affecting the passive paracellular pathway and a decrease in active transcellular ion transport (Jacquillet et al., 2007).Based on current understanding, renal tubular damage is likely the most significant health impact, involving various mechanisms such as essential element replacement, inflammatory response, autophagy, apoptosis, and epigenetic alterations (Järup et al., 1998, Zhang et al., 2024).
Zeolites are hydrogenated aluminium silicate minerals (Barbosa et al., 2016).They consist of interconnected tetrahedrons of aluminium oxide (AlO4) and silicon dioxide (SiO4) (Moshoeshoe et al., 2017).There are about 40 naturally occurring zeolites; the most commonly degraded form is clinoptilolite.Natural zeolites are formed in both volcanic and sedimentary rocks, according to the US Geological Survey (Bahgaat et al., 2020).On the other hand, approximately 150 synthetic zeolites have been created for particular uses, with the most famous being zeolite A, zeolites X, and Y. (Bahgaat et al., 2020).
Zeolites possess ion exchange and adsorption properties that can enhance the utilization of feed nitrogen in animal nutrition, decrease the occurrence of intestinal illnesses in young pigs and ruminants, manage the moisture and ammonia levels of animal manure, purify water in aquaculture circulatory incubators, supply fish farming with oxygenenriched air, and minimize the nitrogen level of feed and brood run-off waters (Papaioannou et al., 2005;El-Nile et al., 2021).
According to the properties of zeolites, they are used in large quantities in a variety of commercial processes.The biggest application of zeolites is the purification of water.Other applications include catalysis, adsorbents and many applications of natural zeolites, including soil improvers and nutrient release agents in horticulture (Weckhuysen and Yu, 2015).
Zeolites have the ability to act as host matrices for the containment and stabilization of metal clusters or nanoparticles, creating versatile composites with excellent properties.Thanks to these unrivaled properties, due to their high hydrothermal stability and costeffectiveness.Zeolites are extensively utilized as efficient catalysts, detergents, adsorbents, and ion exchangers in various chemical processes (Davis, 2002;Mintova et al., 2015).
Zeolites are interested in agriculture due to their main properties: high cation exchange, high adsorption capacity, and high water-holding capacity (Hedström, 2001).They improve soil properties by raising soil moisture and preventing hydraulic conductivity; also zeolites were used with fertilizers to help buffer soil pH (Cataldo et al., 2021).
Natural zeolites have beneficial effects on the body's detoxification, mineral metabolism, immune system, blood circulation, and digestion (Hecht 2010).According to a study carried out by Pavelić et al. (2001), natural clinoptilolite zeolites act as anticancer therapeutic agents in vivo animal study and tissue culture cells.They are administrated orally in mice and dogs to improve all over health state, elongation of life span, and decrease tumor size (Moshoeshoe et al., 2017).Synthetic zeolites have numerous benefits compared to natural ones.They are more efficient in radioactive waste removal from the environment (Lonin et al., 2015;Abdel Moamen et al., 2015), also they show a much higher capacity of adsorption for heavy metals like Cd 2+ , Pb 2+ , Zn 2+ , etc. (He et al., 2016;Kozera-Sucharda et al., 2020).Accordingly, this study aims to evaluate the possible ameliorative role of zeolites against the hazardous effects of CdCl2 on the kidneys of pregnant rats and their pups.

1-Experimental Animals:
The Albino rat (Rattus norvegicus) is an excellent choice for conducting experimental teratological studies due to its many advantages.It has a short gestation period of approximately 21 days.Additionally, rats are known for their genetic stability and exhibit a very low rate of spontaneous malformation (Kohn and Clifford, 2007;Modlinska and Pisula, 2020).Experimental animals (40) adult female albino rats, aged 12 weeks and weighing 160-190 g.They were mated with males; the vaginal smears were examined under light microscope.At the estrus phase in females, the existence of sperms in vaginal smears indicates the zero day of gestation.

2-Treatment and Doses:
Zeolite in a dose of 100 mg/kg/day was dissolved in tween eighty and then in distilled water (Saribeyoglu et al., 2011).Cadmium chloride at a dose of 30 mg/kg/day was dissolved in distilled water (Oluranti et al., 2021).

Experimental Design:
After mating, the pregnant rats were divided into 5 groups (n=8) according to the received substance as follows: Group 1: 8 rats received distilled water (normal control group) from the 6 th day to the 15 th of gestation.Group 2: 8 rats received tween 80 (control tween 80 group) from the 6 th day to the 15 th of gestation.Group 3: 8 rats received zeolite (dissolved in 1% tween 80 ) in a dose of 100 mg/kg per day from the 6 th day to the 15 th of gestation.Group 4: 8 rats received Cadmium Chloride (dissolved in distilled water) in a dose of 30 mg/kg/day from the 6 th day to the 15 th day of gestation.Group 5: 8 rats received zeolite concurrently with CdCl2 from the 6 th day to the 15 th day of gestation by the same doses as in groups 3&4.Animals of all groups were sacrificed at the end of gestation periods (on the 20 th day of gestation).Maternal kidneys and whole fetuses were quickly removed and collected to be used in our study.

4-Histological Examinations:
The maternal and fetal kidneys were extirpated, fixed in 10% formalin, dehydrated in ascending grades of ethyl alcohol, cleared in xylol, embedded in molten paraplast at 56°C, and cut at 5µ on rotator microtome.The paraffin sections were stained with haematoxylin and eosin (Drury and Wallington, 1980).Histopathological studies were conducted using light microscopy and photomicrographs were captured.

5-Biochemical Investigation:
Maternal blood samples were collected and centrifuged at 10000 xg for 10 minutes at 4°C.The resulting supernatant is serum.the serum should be stored and transported at -20°C or lower.Kidney function tests were measured to determine serum creatinine and urea levels by using a 96-well plate according to the method described by (REF).

6-Skeletal Staining And Examination:
1-Fetuses were carefully eviscerated.2-They were fixed in 95% ethyl alcohol for hardening of the specimens.3-Staining with double staining of fetal skeletons for cartilage (blue) and bone (red) according to the method described by Peters (1977).4-Fetuses were stained with alcian blue solution at first then treated with a relatively weak solution of potassium hydroxide which renders the muscles transparent then stained with Alizarin red solution.5.After staining, the specimens were kept in glycerin.The skeleton was examined under the dissecting binocular microscope to study any malformation and shortening in the bones of fetuses.

7-Statistical Analysis:
Results have been analyzed by the prism program version (5).Comparison between more than two different groups was carried out using the one-way analysis of variance (ANOVA) followed by Tukey-Kramer's Multiple Comparison Test (Armitage and Berry, 1987), where P<0.05 was considered significant.All the values were presented as means ± standard errors of the means (S.E.M.).

1-Histological Examination: 1.1-The Effect Of Zeolite And Cadmium Chloride On The Maternal Kidney: Control Groups:
The renal cortex was packed with renal (Malpighian) corpuscles, glomerular capillaries, Bowman's capsules, and urine space.Proximal convoluted tubules have a tiny lumen surrounded by cubical cells with round nuclei at the base of the tubule.The lumen of the distal convoluted tubules was large and bordered by simple cubical cells with sphere nuclei in the middle or at the apex of the tubule walls (Fig. 1 A and B) Zeolite Group: The most of renal glomeruli and tubules appeared normal.There was no lymphatic intrusion.The glomeruli showed normal and standard size inside Bowman's capsule (Fig. 1  C, D and E).

Cadmium Chloride Group:
It showed a glomerular disparity in size and shape, with some glomeruli contracting in size because of atrophied glomerular tuft and cystic appearance.Some glomeruli were seen to be vacuolated, fractured, and compacted.In the proximal and distal convoluted tubules, cell nuclei exhibited complete or partial damage, implying dissolution.Additionally, the nucleus has been observed to compact (pyknosis).Hydropic degeneration resulted in blurry development of the convoluted tubule endothelial lining cell.There was inflammatory cell infiltration in the injured tubules and renal corpuscles.Renal tubules were shown to have disrupted epithelial linings and desquamation.Within tubular cells, cellular remains were found (lumen cast) (Fig. 2).

Zeolite and Cadmium Chloride Group (Zeo+CdCl2) Group:
Some tubular epithelial cells in renal convoluted tubules exhibited moderate vacuolation and degenerative changes.The glomerulus is of normal size within Bowman's capsules with a normal volume of capsular space.Numerous glomeruli were atrophied.Few pyknotic nuclei are seen in only a few tubules and degeneration of the tubular hydropic membrane.There was no evidence of lymphatic invasion.There was no area of hemorrhage.Congestion was noted to be mild (Fig. 3).

1.2-The Effects on Fetal Kidney: Control Groups:
Bowman's capsule includes intact blood vessels glomerulus with clear capsular space.Renal tubules are normal and lined with simple epithelial cells and conspicuous spherical centrally located nuclei (Fig. 4 A and B).

Zeolite Group:
The examination showed standard renal tissue structure with the normal organization of Bowman's capsule, glomeruli, and renal tubules (Fig. 4 C and D).

Cadmium Chloride Group:
The fetal kidney showed obvious degeneration of the basement membrane enclosing the glomeruli and epithelial cells lining the renal tubules, as well as tubules that were destroyed.In the lining cells of the tubules, there was nuclei degradation, pyknotic nuclei, hazy swelling, and disintegration (Fig. 4 E and F).

Zeo+CdCl2 Group:
The examination showed a certain degree of recovery and restored the normal architecture.Most of Bowman's capsule and glomeruli appeared with the usual form and size, but others revealed atrophied glomeruli.Many renal tubules appeared normal configuration however other tubules revealed little extent of degeneration (Fig. 4 G, H and  I).

3-Skeletal study:
On the 20 th day of gestation, the cleared cartilage and bone preparations of control rat fetuses have been designated.The data showed well ossification of the skull (Fig. 7 panel 1, A and B), ribs (Fig. 7 panel 2, A and B), vertebrae, and hind limbs (Fig. 7 panel 3, A and  B).
In the fetuses treated with cadmium chloride (30mg/kg), skeleton showed lack ossification of skull (Fig. 7 panel 1, D), metacarpals and phalanges of toes were completely non-ossified, thoracic vertebrae were non-ossified and also its vertebral ribs (Fig. 7 panel 2, D), metatarsus and phalanges of the toes were completely non-ossified (Fig. 7 panel 3, D).On the other hand, the skeleton of fetuses treated with Zeo+CdCl2 showed mild ossification of skull (Fig. 7 panel 1, E), metacarpals and phalanges of the toes were mild ossified, thoracic vertebrae were mild and also its vertebral ribs (Fig. 7

DISCUSSION
The organs in charge of drug and xenobiotic metabolism are the kidney and liver.The liver and kidneys are first affected by chemical poisoning, then the other organs.Abdel-Daim et al. (2013) stated that the liver and kidney are chemically poisoned first, then the other organs.According to a study by Parzyck, et al., (1978), Cd exposure during pregnancy leads to several effects on kidney, blood pressure and essential elements rotation in the body, and also is the reason for other pathologies in the mothers and fetuses (Estrada et al., 2017).
In the present work and due to the administration of cadmium, maternal and fetal kidneys showed a glomerular disparity in size and shape, with some glomeruli contracting in size.Some glomeruli were seen to be vacuolated, fractured, and compacted.In renal tubules, cell nuclei exhibited complete or partial damage, implying dissolution.There was nuclei degradation, pyknotic nuclei, hazy swelling, and disintegration.There was inflammatory cell infiltration in the injured tubules and renal corpuscles.Renal tubules were shown to have disrupted epithelial linings and desquamation.Within tubular cells, cellular remains were found (lumen cast).That was found in previous studies by Brzóska et al. (2003) and Hamidian et al. (2020) which showed degeneration and hypertrophy of epithelial cells and dilation in glomeruli, pycnotic nuclei with focus necroses in urinary tubules and congestion of blood vessels.
Serum Creatinine and urea levels showed a significant increase in the dams that received CdCl2 as compared to the control group.Such findings agree with the previous reports (Karami et al., 2022;Badawy et al., 2023) which observed an increase in creatinine and urea levels at a dose (3 mg/kg) of CdCl2 administration in male Wister rats, also serum Creatinine and urea levels increased significantly compared to control groups at a dose (2 mg/kg) CdCl2 in male SD rats (Huang et al., 2022).
The skeletal examination of CdCl2 maternally treated fetuses observed a lack of ossification of the skull, metacarpals and phalanges of toes showed no signs of ossification, thoracic vertebrae were non-ossified, also its vertebral ribs.This was compatible with previous studies that showed Cd treatment during organogenesis leads to incomplete ossification of the skull, ribs and vertebrae, shortness of rib in some fetuses and reduced metacarpus ossification and cleft palate.(Salvatori et al (2004) and El-Sayed (2013) found that Cd treatment reduced metacarpus ossification and cleft palate.The most remarkable effects seen in the fetuses' skeleton of the Cd maternally treated group were delayed ossification of the head and vertebrae, as well as fused ribs and vertebrae (Argüelles, et al., 2013).The highest level of cadmium toxicity was found in the length of skeletons in the tibia, while the lowest was observed in the arm.In terms of the width of skeletons, the most toxic impact of cadmium was on the large diameter of the pelvis, with the lowest effect seen on the large diameter of the chest (Abdollahi, et al., 1998).
On the other hand, zeolite administration showed the normal histological structure of maternal and fetal kidneys.Most renal glomeruli and tubules appeared normal.There was no lymphatic intrusion.The glomeruli with normal and standard size inside Bowman's capsule.That was observed in the previous study by Sumer et al. (2022) Which found that kidneys of the zeolite group showed normal glomerulus and renal capsule and no pathological alterations in renal tubules.
Serum creatinine and urea levels recorded normal values in the zeolite group, as well as their levels decreased when the dams treated with zeolite with cadmium chloride which agreed with pablack and Zentek (2018) who found no effect on blood urea and creatinine concentration in dietary supplementation of zeolite.
Maternally treated fetuses with zeolite showed normal ossified bones.Zeo+CdCl2 maternally treated showed mild ossification of skull, metacarpals and phalanges of toes, vertebrae, and ribs.According to Zarrintaj et al. (2020), Zeolites are used in bone tissue

Fig. 7 :
Fig. 7: Photomacrographs of the Skeleton of Fetuses on the 20 th day of gestation: Panel 1 skull bones of (A) normal control group, (B) control tween 80 group and (C) zeolite group showed well ossification.(D) CdCl2 group showed a lack of ossification of the skull.(E) Zeo+CdCl2 group showed mild ossification of the skull.Panel 2 vertebral column and ribs bones of (A) normal control group, (B) control tween 80 group and (C) zeolite group showed complete ossification.(D) CdCl2 group showed non-ossification vertebral ribs.(E) Zeo+CCl2 group showed mild ossified vertebral ribs.Panel 3 hind limb and sacrum of (A) normal control group, (B) control tween 80 group and (C) zeolite group showed complete ossification.(D) CdCl2 group showed completely non-ossified metatarsus and phalanges of the toes.(E) Zeo+CdCl2 group showed completely weak ossified metatarsus and phalanges of the toes.