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Protective effect of magnesium against lead and cadmium-induced liver injury in swiss albino rats
*Corresponding author E-mail address: ezzat.awad@meduniwien.ac.at (E. Awad)
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Received: ,
Accepted: ,
Abstract
The present study was planned to elucidate the therapeutic impact of magnesium as an anti-apoptotic and anti-inflammatory agent, as well as a mediator in various biological and metabolic processes, in protecting liver tissues from the cellular toxicity of multiple chemicals, including heavy metals such as lead (Pb) and cadmium (Cd). The Swiss albino rats were categorized into six treatment groups (n=5). The first group was the control (CN), which received no treatment. The second (Mg), third (Pb), and fourth (Cd) groups were treated with magnesium chloride, lead acetate, and cadmium chloride, respectively, at a dose of 1 mg/kg/day for 15 days. The fifth (Cd + Mg) and sixth groups (Pb + Mg) were given cadmium chloride and lead acetate at 1 mg/kg/day for 15 days, followed by treatment with magnesium chloride (0.5 mg/kg) for 15 days. After the treatment, the animals were sacrificed to retrieve liver and serum samples for biochemical and histological evaluation. The data analysis revealed that both heavy metals, cadmium (Cd) and lead (Pb), induced severe liver injury, as evidenced by higher serum enzymatic activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), and lactate dehydrogenase (LDH). At the same time, lipid peroxidation Malondialdehyde (MDA) indices were significantly elevated, along with markedly decreased levels of the non-enzymatic antioxidant glutathione (GSH) in the tissue samples compared to the control. Administration of magnesium at a dose of 0.5 mg/kg significantly reversed most of the serum hepatic biomarker enzymes towards the control levels. Additionally, magnesium significantly reduced lipid peroxidation activity and restored the GSH level in the liver. Furthermore, the histopathological deformations in the liver induced by heavy metals also tended towards restoration, resembling the normal histological architecture of the tissue. Notably, Mg countered Cd-induced toxicity more effectively than Pb. Hence, magnesium acts as a protective element in mitigating the degenerative hepatotoxic effects of lead and cadmium.
Keywords
Heavy metals
Hepatoprotective
Histopathological
Magnesium
Oxidative stress
1. Introduction
Environmental pollution directly threatens all forms of life, including humankind. Heavy metals like lead (Pb) and cadmium (Cd) are released into the environment from various sources, including industrial and municipal wastes, the burning of fossil fuels, ceramic glazes, paints, cosmetic items, processed food, contaminated water, and batteries (Lv et al., 2023). Both metals are considered toxic due to their adverse effect on the respiratory and digestive systems (Zwolak 2020). Fadl et al. (2021) reported that these metals were not only detected in trace amounts in urine, stool, and other excretory products but also as remnants in various body organs, thereby impacting molecular and cellular activities (Fadl et al., 2021). Various studies have explained that the toxicity of lead and cadmium results from their longer half-lives, affecting the liver as the primary target organ of their accumulation, followed by the cortex and medullary region of the kidney (Abo-EL-Sooud et al., 2023). Both metals are known to cause structural and functional damage to the target cells, disrupting various biochemical processes and resulting in cytotoxicity in the liver and kidneys. The metals have been reported to elevate oxidative stress by generating a bulk amount of reactive oxygen species (ROS), resulting in DNA damage (Haj-Khlifa et al., 2024).
Besides being an accessory digestive organ, the liver plays a pivotal role in the oxidation of compounds through its biotransforming enzymes under xenobiotic metabolism. It participates in the primary immune response and is considered the major organ for detoxifying all chemicals that enter the body through food and drugs (Ebaid et al., 2021). Magnesium is an essential metal that acts as a comediator in various biochemical processes involving macromolecules (carbohydrates, lipids, and proteins) metabolism. The metal also acts as a cofactor in over 300 enzyme systems and regulates diverse biochemical reactions involved in protein synthesis, muscle and nerve function, blood sugar, and pressure regulation (Jahnen-Dechent and Ketteler, 2012). Additionally, the metal is involved in glycolysis and oxidative phosphorylation, resulting in energy production at the cellular level. It is also essential for bone formation, action potential across cell membranes, nucleic acid, and glutathione (GSH) synthesis (Kouadria et al., 2020). Furthermore, it has been reported to be a potent antioxidant and anti-inflammatory agent (Yüce, 2023). Several studies have reported the ameliorative effect of moderate levels of magnesium, both alone and in combination with calcium, against lead (Pb) and cadmium (Cd)-induced hepatic and nephrotoxicity (Dabak et al., 2016).
Magnesium has an exceptionally low health hazard profile, with a NOAEL of 1000 mg/kg/day for males and 500 mg/kg/day for females. The present study aims to investigate the alleviative effect of Mg on Pb- and Cd-induced liver injury in Swiss albino rats.
2. Materials and Methods
2.1 Materials
All the reagents and chemicals used in the study were of analytical grade and were procured from Sigma Aldrich (St. Louis, MO, USA), BDH chemicals (Atlanta, GA, USA), and Roche (Basel, Switzerland). Commercial kits were purchased from Quimica Clinica Aplicada S.A. (QCA, Amposta, Spain) for biochemical analysis.
2.2 Animal studies
Thirty adult male rats of the Swiss Albino strain (100–120 g; 2-3 months old) were procured from the Animal House of the Department of Zoology (King Saud University, Riyadh). The animals were housed in large cages with free access to a standard pellet diet and water (Alhazza et al., 2020), maintained at a temperature of 23±2 °C and a 12-h day/night cycle, as previously described (Hassan et al., 2019).
The rats were randomly distributed into six experimental groups (n=5) as follows:
Group 1 (CN): Normal healthy rats without any treatment.
Group 2 (Mg): Rats treated with magnesium chloride (1 mg/kg/day) for 15 days.
Group 3 (Pb): Treated with lead (II) acetate (1 mg/kg/day) for 15 days (Draid et al., 2016)
Group 4 (Cd): Treated with cadmium chloride (1 mg/kg/day) for 15 days (Ojo et al., 2023)
Group 5 (Pb + Mg): Treated with lead (II) acetate (1 mg/kg/day) for 15 days) followed by magnesium chloride (0.5 mg/kg/day) for 15 days. Group 6 (Cd + Mg): Treated with cadmium chloride (1 mg/kg/day) for 15 days) followed by magnesium chloride (0.5 mg/kg/day) for 15 days.
All test compounds were administered intraperitoneally (i.p.) to rats using a 1 mL syringe (BD Science, USA). The animals were euthanized as described by the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020 Ed.). All animal handling procedures were carried out in accordance with the guidelines of the Departmental Ethical Committee (Department of Zoology, KSU), with approval number KSU-SE-20-69.
2.3 Biochemical analysis
2.3.1 Measurement of liver function markers
Liver toxicity markers- aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured in serum samples that were quantified by commercial kits as per the manufacturer’s protocols (QCA, Spain) following the company’s instructions.
2.3.2 Measurement of liver toxicity markers
γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), and total bilirubin were chosen as toxicity markers that were quantified using commercial kits according to the manufacturer’s protocols (QCA, Spain).
2.3.3 Measurement of redox parameters
Total reduced GSH and malondialdehyde (MDA) were that are quantified by commercial kits as per manufacturer’s protocols (Hassan et al., 2019).
2.4 Liver histological examination
After being stored in 10% neutral buffered formalin, the samples were wrapped in paraffin. Their 6-μm paraffin sections were processed for Hematoxylin-eosin (H&E) staining, following the protocol of Bancroft and Layton (2012).
2.5 Morphometric analysis
The histomorphometric parameters, such as the hepatocytes’ diameters and density, sinusoidal spaces’ diameters, density besides fibrosis, and necrosis intensity, were calculated by image processing using ‘ImageJ’ software according to the standard procedures (Gewehr et al., 2021, Li et al., 2022). The results were analyzed by SPSS to compare the five experimental groups.
2.6 Statistical analysis
The statistical data analysis was executed using SPSS 25 (SPSS Corp., Armonk, NY, USA). The analysis of variance was one-way (Hachmeriyan et al., 2022), followed by a Bonferroni post-hoc test. The result was expressed as the average value plus or minus the standard deviation (mean ± STD). Statistical significance was defined as a p-value of less than 0.05.
3. Results
3.1 Biochemical observations
3.1.1 Effect on liver function markers
3.1.1.1 ALT
The treatment groups 2 (Mg–treated),3 (Pb-treated), and 4 (Cd-treated) showed extensive elevation in ALT activity by 104.05%, 302.29%, and 256.08% in comparison to the control group 1 (Fig. 1). However, administration of Mg in the rats priorly treated with Pb and Cd (groups 5 and 6) showed a decrease in activity by 20.12% and 10.84 % compared to groups 3 and 4, respectively (Fig. 1a).
- Bar charts showing the biochemical analysis of liver samples from six experimental groups as Control; Mg treated; Pb treated; Cd treated, Mg+Cd treated, and Mg+Pb treated expressed as Mean ± STDV. Where (a) ALT, (b) AST, (c) GGT, (d) GSH, (e) LDH and (f) MDA, among six experimental groups: Means within the same parameter and not sharing a common superscript symbol(s) differ significantly at p < 0.05.
3.1.1.2 AST
Groups 2, 3, and 4 demonstrated an increase in the activity by 112.02%, 285%, and 208.10%, respectively, compared to the control, while groups 5 and 6 showed a decline in activity by 17.16% and 10%, respectively, compared to groups 3 and 4 (Fig. 1b).
3.1.2 Effect on liver toxicity markers
3.1.2.1 GGT
The animal groups 2, 3, and 4 exhibited an increase in GGT activity by 83.75%, 464.38%, and 314.45%, respectively, as compared to group 1; however, groups 5 and 6 showed a dip by 32.42% and 24.20% in comparison to groups 3 and 4, respectively (Fig. 1c).
3.1.2.2 LDH
The treatment groups- 2, 3, and 4 showed an elevation in LDH activity by 68.90%, 588.38%, and 500.81% in comparison to the control group 1. However, administration of Mg in groups 5 and 6 showed a decrease in activity by 63.64% and 51.04% compared to groups 3 and 4, respectively (Fig. 1e).
3.1.3 Effect on redox parameters
3.1.3.1 MDA
Groups 2, 3, and 4 displayed an increase in their level by 53.79%, 185.51%, and 133.79% compared to the control, while groups 5 and 6 showed a decline in level by 15.70% and 10.91%, respectively, compared to groups 3 and 4 (Fig. 1f).
3.1.3.2 GSH
The treatment groups 2, 3, and 4 demonstrated a decline in their level by 31.25%, 41.27%, and 32.54% compared to the control. However, the combination groups 5 and 6 showed replenishments in their level by 18.07% and 12.33%, respectively, as compared to groups 3 and 4, respectively (Fig. 1d).
3.2 Histological and morphometric observations
Normally, the liver is considered a primary metabolite parenchymal organ with distinguishing hexagonal lobulation in its structure. The microscopic examination revealed that the mesothelium layer originated from the peritoneum stratum covering the outer surface of the hepatic lobes (Fig. 2); the capsule was observed to be intact in the control liver (Figs. 2a and b). Also, the central vein was partially located mostly at the center of the lobule. In addition, the hepatic parenchyma was organized in polyhedral hepatocytes with prominent nuclei and granular cytoplasm. They radiated peripherally from the central vein, outside the portal triad in strips terminated with hepatic cords, constructing regular sinusoidal spaces. They were arranged in lines terminated with hepatic sinusoidal cords draining to the central vein, as illustrated in Figs. 2(a, b, and e). Additionally, there were Kupffer cells, which are considered monocyte derivatives with immune phagocytosis activity, dispersed in the sinusoidal spaces, as shown in Figs. 2(b, d, and e). On the other hand, at each terminal corner of the hexagonal plate, the portal triad was constructed by circulating portal vein, bile duct, and hepatic artery through a thick layer of loose stromal connective tissue (Figs. 2c and d).
- Photomicrograph of the histological section of a control liver (a, b, c, d & e) showing the capsular region (Cp) with mesothelium cells (mes), the hepatocytes (H) with prominent nucleus (PN) and granular cytoplasm (Gc) around the central vein (Cv) in cords named with hepatic cords (Hc) separated by sinusoidal spaces (Si) in cords called hepatic sinusoid cords (Hsc). The hepatic cords are separated from each other by hepatic sinusoidal cords (Hsc). Kupffer cells (Ku) and erythrocytes (Ery) dispersed among hepatocytes. The portal triad (PT) is constituted of the portal vein (PV), bile duct (Bd), and hepatic artery (Ar). (H&E X, A &C: 100 µm & B, D& E: 400 µm).
The liver sections from rats administered magnesium (Mg) exhibited a non-significant (P > 0.05) constant pattern of histological features compared to the control, except for slight alterations in the hepatic parenchyma (Fig. 3-7) The hepatic lobules were generally apparent like the control ones, with slight shrinkage (P<0.05) in the hepatocyte diameter (1.33±0.01µm2) less than the standard size. This, in turn, significantly affected their density per unit area (P < 0.05) more than the normal range (1.39 ± 0.015 cell/µm2), further crowding the sinusoidal spaces (Figs. 3a-c). The hepatocyte population showed a pathological sign-angiectasis of sinusoidal spaces through the erythrocyte dilation (Figs. 4a and c). Histologically, there were common features between control and magnesium-treated hepatic sections from the capsular layer passing with the central vein reaching to the terminal corner where the portal triad was located (Figs. 4b-e).
- Bar charts showing the morphometric analysis of histological parameters of liver tissue which (a) Diameter of hepatocytes, (b) Density of hepatocytes, (c) Diameter of sinusoidal spaces, (d) Density of sinusoidal spaces, (e) Diameter of central vein, (f) Density of kupffer cells, among six experimental groups: Control; Mg liver; Pb liver; Cd liver, Mg+Cd liver, and Mg+Pb liver. Values are represented as Mean ± STDV. Means within the same parameter and not sharing a common superscript symbol(s) differ significantly at p < 0.05, and values that are recorded with non-significance difference (n.s). Mg: Magnesium chloride, Pb: Lead acetate, Cd: Cadmium chloride, Cd+ Mg: Combination of Cd with Mg, Pb+ Mg: Combination of Pb with Mg.
- Photomicrograph of the histological section of a Mg liver (a-e) showing the capsular region (Cp) with mesothelium cells (mes), the hepatocytes (H) with prominent nucleus (PN) and granular cytoplasm (Gc) around the central vein (Cv) in cords named with hepatic cords (Hc) separated by sinusoidal spaces (Si) in cords called hepatic sinusoid cords (Hsc). The hepatic cords are separated from each other by hepatic sinusoidal cords (Hsc). Kupffer cells (Ku) and endothelial cells (En), erythrocytes (Ery) dispersed among hepatocytes. The portal triad (PT) is constituted of the portal vein (PV), bile duct (Bd), and hepatic artery (Ar). Angiectasis (Ag) was described. (H&E X, (a) 100 μm & (c-e) 400 μm).
- Bar charts showing the morphometric analysis of histological parameters of liver tissue which (a) intensity of necrosis, (b) intensity of congestion, (c) intensity of fibrosis, among six experimental groups: Control; Mg liver; Pb liver; Cd liver, Mg+Cd liver, and Mg+Pb liver. Values are represented as Mean ± STDV & n = 10 animals. Means within the same parameter and not sharing a common superscript symbol(s) differ significantly at p < 0.05, and values that are recorded with non-significance difference (n.s). Mg: Magnesium chloride, Pb: Lead acetate, Cd: Cadmium chloride, Cd+ Mg: Combination of Cd with Mg, Pb+ Mg: Combination of Pb with Mg.
- Photomicrograph of the histological section of a Pb liver (a-f) showing the capsular region (Cp) with mesothelium cells (mes), the hepatocytes (H) around the central vein (Cv) in cords named with hepatic cords (Hc) separated by sinusoidal spaces (Si) in cords called hepatic sinusoid cords (Hsc) filled with erythrocytes (Ery). The hepatic cords are separated from each other by hepatic sinusoidal cords (Hsc), and Kupffer cells (Ku). The portal triad (PT) is constituted of the portal vein (Pv), bile duct (Bd), and hepatic artery (Ar). Angiectasis (Ag), Congestion (Con), fibrosis (Fb), necrosis (Nc), inflammatory cells (inf), and hyperplasia of the biliary duct (Hy B) were manifested. (H&E X, (a-c) 100 μm & (b, d & e) 400 μm).
- Photomicrograph of the histological section of a Cd liver (a-e) showing the capsular region (Cp), the hepatocytes (H) around the central vein (Cv) separated by sinusoidal spaces (Si), and Kupffer cells (Ku). Amyloid deposition (Am), Basophilic foci (Fo), degenerated hepatocytes (Dg H), Congestion (Con), glycogen accumulation (G), fibrosis (Fb), necrosis (Nc), and hyperplasia of bile ducts (Hy B) and per biliary fibrosis (PFB) were explained. (H & E X, (a) 100 μm & (b, c, d & e) 400 μm).
In the third group, (Pb)-exposed rat liver exhibited a significant deformation in its general histological architecture, such as dispersion of organization of hepatic cords (Figs. 3a and b), along with a significant (P<0.01) relative diminution of hepatocyte diameter (1.03±0.02 µm2) less than the control diameter (1.69±0.04 µm2). Subsequently, the hepatocyte population was found to be significantly reduced (P < 0.01) (0.46 ± 0.03 cell/µm2) with a narrower range than the standard population (Figs. 3a and b). There were also malformation alterations in the sinusoidal spaces, with narrower diameters and higher densities than the control section (1.78±0.04 µm2), which were significantly (P < 0.01) illustrated (Figs. 3c and d). The sinusoidal alteration in their diameter might have resulted from the nonsignificant (P>0.05) increase in the central vein diameter (6691.42±216.74 µm2), more than normal. However, the hyperplasia of Kupffer cells might also be attributed to this construction with a density (0.57±0.02 cell/µm2) that is more than the normal range, besides the dispersion of some inflammatory cells, as manifested (Figs. 3e and f). The extent of cytotoxicity of this heavy metal is evident from the higher occurrence of necrosis (2.19±0.05%) and fibrosis (6.16±0.19%), which is significantly (Figs. 4d and f). In addition, the congestion intensity (10.05±0.47%) as well as hyperplasia of the bile duct were also observed (Figs. 5a-c and Figs. 6a-c). Additionally, the hepatocyte population exhibited angiectasis findings in the dilated sinusoidal channels that were filled with RBCs (Figs. 6b and e).
The fourth group treated with cadmium (Cd) exhibited a higher level of cytotoxicity and diffusion of the hepatic cord, leading to disorganization, with the capsular region being delaminated (Figs. 7a and b). The cellular toxicity caused dysfunction in the cells, evidenced by glycogen accumulation and a reduction in hepatocytes’ diameter (0.37±0.02 µm2) and density (0.27±0.027 cell/µm2), compared to the control (Figs. 3a and b). Additionally, the microscopic examination revealed several atrophic features, including amyloid deposition and the spreading of degenerated hepatocytes alongside necrotic cells. Consequently, the previous deformations contributed to the formation of liver foci, represented by preneoplastic lesions, which are dispersed among hepatocytes and sinusoidal spaces (Figs. 7a-c). Hyperplasia of bile ducts around the portal vein was also observed, associated with perbiliary fibrosis (Figs. 7d and e). Therefore, sinusoidal abnormalities exposed severe constriction in their diameter (0.53±0.04 µm2), lower than control ones and wide dispersion (1.48±0.05 cell/µm2) in their density during cadmium intake, as has been illustrated in (Figs. 3a and b). Several atrophic markers were raised significantly (P<0.001) after cadmium intake, like hyperplasia of Kupffer cells in the diseased tissue (2.26±0.11 cell/µm2) more than the normal cell density (Fig. 3f). The present observations revealed that the elevation in cellular intensity for necrosis (1.2±0.11%), congestive hepatopathy (9.66±0.60%) and fibrosis (3.10±0.12%) in a significant way (P<0.001), were the primary reasons for widening of the central vein (11254.75±388.17 µm2) more than the control and Mg-treated ones (Figs. 3e and 5a-c).
Finally, the administration of Mg in the rats pre-exposed to Cd and Pb showed significant improvement in their overall health status. However, they also exhibited some principal atrophic signs such as necrosis, congestion, and fibrosis at low resolution (P< 0.01) to a non-significant extent (P>0.05) relative to the control ones (Figs. 5a-c). Nevertheless, there was a significant variation (P< 0.001) in recovery to the normal levels between two malformed hepatic lobules with hepatocyte diameter and density, sinusoidal diameter and density, and diameter of central vein and Kupffer density as demonstrated in Figs. 3(a-f). Notably, supplementation of Mg improved Pb-elicited toxicity more than that of Cd. On the other hand, Mg could not alleviate amyloidosis and mild lymphoid infiltration in hepatic lobules, besides the dispersion of necrotic hepatocytes. Moreover, the hyperplasia of bile ducts, hepatic foci, and glycogen accumulation were still observed after treatment with Mg in the Cd group (Figs. 8a-e). On the other hand, the liver of the Pb-treated group, after administration of Mg, revealed higher recovery, with the hepatic lobules and hepatocytes returning to a radiating pattern from the central vein to the portal triad, appearing without any atrophic markers. Additionally, the capsular region appeared healthier and relatively free of amyloidosis (Figs. 9a-f).
- Photomicrograph of the histological section of a Cd with Mg liver (a-e) showing the capsular region (Cp), the hepatocytes (H) around the central vein (Cv) separated by sinusoidal spaces (Si), and Kupffer cells (Ku). Portal vein (PV), bile ducts (Bd), and hepatic artery (Ar) constituted the portal triad. Amyloid deposition (Am), Basophilic foci (Fo), inflammatory lymphocytes (Ly), Congestion (Con), glycogen accumulation (G), fibrosis (Fb), necrosis (Nc), and hyperplasia of bile ducts (Hy B) were demonstrated. (H&E X, (a, b & c) 100 μm and (d & e) 400 μm).
- Photomicrograph of the histological section of a Pb with Mg liver (a-f) showing the capsular region (Cp), the hepatocytes (H) form hepatic cords (Hc) around the central vein (Cv) separated by sinusoidal spaces (Si) that form hepatic sinusoid cords (Hsc) that filled with erythrocytes (Ery), and Kupffer cells (Ku). The portal vein (PV), bile ducts (Bd), and hepatic artery (Ar) constituted a portal triad. Amyloid deposition (Am), Congestion (Con), and necrosis (Nc) were illustrated. (H & E X, (a & c) 100 μm & (b, e, d & f) 400 μm).
4. Discussion
Heavy metals like Cd and Pb have been classified as highly toxic elements. They are responsible for disruption in physiology and metabolism, including deterioration of many key proteins and enzymes. In contrast, light metals such as Zn and Mg have been documented to mitigate the toxicity of various heavy metals and chemical toxicants (Kouadria et al., 2020). The current study displays the curative efficacy of magnesium in lead- and cadmium-induced hepatotoxicity in rats. The biochemical results clearly show this notion in the present study. Moreover, detailed histological analysis of liver sections confirms this.
The control liver parenchyma exhibited normal organization of hepatic cords, radiating from the central vein towards the portal triad, separated from each other by sinusoidal capillaries. In the case of lead and cadmium-treated groups, the organization of hepatocytes was disoriented, and the hepatocytes were arranged diffusely. A similar diffusion of hepatocytes was recorded in the cadmium-administered liver (Kouadria et al., 2020). Additionally, a similar disorganization of hepatic lobules was observed in vivo with lead acetate (Haouas et al., 2014). However, magnesium supplementation (Munir et al., 2021) exhibited a better response against Pb-induced toxicity than cadmium, attributed to magnesium’s capacity to displace Pb particles from the binding sites in the key proteins. The normal hepatocytes appeared to have a polygonal shape and a centrally spheroid nucleus with faintly granular cytoplasm that was exposed to a little significant shrinkage in diameter after being administered Mg. Hitherto, relatively low distortions were observed in the diameter and density of sinusoid spaces. A similar observation was reported by Akkoca et al. (2019), previously showing that magnesium sulfate caused hepatocytes and sinusoid spaces in the prevention of excess water accumulation inside cells through the equilibrium between sodium and potassium ions. On the contrary, Pb and Cd administration caused significant hepatic deformation, including a severe decline in the diameter and density of the hepatocytes. Notably, Cd elicited more hepatic toxic insults than Pb; however, comparable changes were observed in the sinusoidal spaces reported (Ebokaiwe et al., 2024), indicating cadmium-induced hepatic and splenic degeneration through cellular toxicity, which negatively impacts cellular proliferation, differentiation, apoptosis, and other cellular activities. In the present study, magnesium supplementation ameliorated Pb-induced hepatotoxicity with greater efficacy than Cd. A few non-significant features, such as hepatocytes with normal size and density, along with sinusoid spaces resulting from defects in cellular proliferation and apoptosis, were observed less frequently in Pb-treated rats than in Cd-treated rats. Further, in separate studies, Flora et al. (2008) and Velarde et al. (2023) confirmed that the powerful antioxidant activity of Mg ameliorated Pb-induced toxicity by restoring the redox status of Pb and reducing chronic inflammation and apoptosis behavior.
On the other hand, Kupffer cells proliferated significantly in Cd-treated rats, as evidenced by hepatic histopathology, more than in Pb-treated ones, with a higher significance rate. Earlier, an increase in the Kupffer cell density has been reported in hepatic injury induced by 15 mg/Bw of Cd (Orororo and Asagba, 2022). The toxicity of Cd arises from the generation of excessive ROS, leading to lipid peroxidation and chronic inflammation in the area around the portal triad and central vein, with mild lymphoid infiltration, resulting in the proliferation of Kupffer cells (Dabak et al., 2016). Hitherto, the present study demonstrates that Mg acts as a hepatoprotective agent in mitigating the cellular toxicity of lead and cadmium, along with a reduction in inflammatory cell infiltration, resulting in a decrease in Kupffer cell density that tends towards the normal range (Dabak et al., 2018). They further documented that Mg was far more effective than Ca in decreasing the Kupffer density in the rats pre-administered with Cd and Pb.
It is worth noting that the atrophic histopathological signs exhibited during Pb and Cd intake varied between the two groups. The Pb-treated rats showed hepatic lobules expressing angiectasis beside congestion, whereas Cd-treated ones exhibited basophilic foci and amyloid deposition in their hepatic lobules. Both groups exhibited a proliferation of bile ducts surrounding the portal vein, congestion, necrosis, and fibrosis to significant levels compared to normal rates. Similar pathological atrophic signs have been reported for Pb, Ni, and Cd cellular toxicity, and previous diffusion of oxidative stress status in the whole hepatic lobules (Noor et al., 2022). Hence, Mg has been applauded for normalizing the histopathology of chemicals and toxicants that significantly abused the liver in many studies (Liu et al., 2019; Bravo et al., 2023). Bhattacharya (2022) also demonstrated that Mg can substantially reverse heavy metals-induced pathological alterations by significantly neutralizing ROS.
The histological findings in the present study also agree with the biochemical data. The biomarkers for assessing Pb and Cd-induced hepatic injury reverse the heavy metal-induced elevation of ALT and AST activity in serum samples to a significant extent. Previous studies have shown similar effects attributed to the heavy metal-induced damage in the target organs, which leads to the leakage of these enzymes into the bloodstream due to cellular damage, altered membrane permeability, and liver injury (Đukić-Ćosić et al., 2020). Some studies suggest that Mg can significantly alleviate lead and cadmium-induced tissue damage (Zhai et al., 2015). Many previous studies have reported a decline in the leakage of serum hepatoxicity markers due to the stabilization of cell membranes following treatment with a combination of magnesium and zinc (Samir et al., 2012; Kouadria et al., 2020). A similar trend was observed for the activity of hepatotoxicity markers- GGT and LDH in the present study. After magnesium administration, their level significantly came down to the normal in the bloodstream. Researchers (Renugadevi and Prabu, 2010; Renu et al., 2021) have reported a similar alleviative property of plant extract, naringenin, in normalizing GGT and LDH activities in cadmium-induced injury in vivo. Cadmium and lead-induced cellular toxicity occur via an elevation in oxidative stress status in vivo, leading to cellular damage and increased lipid peroxidation, which in turn increases MDA levels (Hassan et al., 2010). In the present investigation, supplementation of Mg decreased the MDA level in the rats pre-treated with Cd and Pb. Moreover, the major cellular reductant, GSH, was also replenished after Mg administration in the rats treated with heavy metals. In contrast, applying magnesium as a therapeutic agent showed no variation from contaminated tissue with heavy metals, either lead or cadmium. In separate studies, Ebaid et al. (2014) and Noor et al. (2022) have indicated similar results in Cd-treated rats.
5. Conclusions
The present study reveals that Pb is more hepatotoxic than Cd, as evidenced by the deterioration of the hepatic lobule’s histoarchitecture. The current investigation establishes the hepatoprotective effect of magnesium, which possesses potent antioxidant properties and serves as a corrective mineral to restore histopathological deteriorations elicited by Cd and Pb. The metal significantly alleviates the pathological features associated with heavy metals, including hepatocyte degeneration, necrosis, and inflammation. However, Mg showed a stronger hepatoprotection in Cd-treated rats than that of Pb. Further studies are warranted to retrieve the exact mechanism involved.
Acknowledgement
The authors would like to thank Ongoing Research Funding Program, (ORFFT-2025-053-1), King Saud University, Riyadh, Saudi Arabia for financial support.
CRediT authorship contribution statement
Iftekhar Hassan and Hossam Ebaid designed the study. Jameel H. Al-Tamimi and Iftekhar Hassan conducted animal treatment and biochemical analysis with Altaaf Khan. Zeinab Abdelfteh conducted detailed histological analysis. Ibrahim M. Alhazza provided all lab facilities and permission. Ezzat M. Awad helped in troubleshooting during experimentation and analysis. Zeinab Abdelfteh and Iftekhar Hassan drafted the manuscript.
Declaration of competing interest
The authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper.
Data availability
Data will be made available on reasonable request to the corresponding author.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of Artificial Intelligence (AI)-Assisted Technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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