Amyloid beta levels and occludin depletion in low dose heavy metal mixture mediated cerebellar toxicity in Wistar rats

Abstract

Co-exposure to heavy metals can result in additive or synergistic toxicity in the brain, culminating in neurotoxicity. This study investigated the neurotoxic effects of a low-dose mixture of two toxic heavy metals—lead (Pb, 20 mg/kg) and aluminium (Al, 35 mg/kg)—and one essential metal, manganese (Mn, 0.564 mg/kg), on the cerebellum of rats. Animals were divided into five groups (n = 5) and orally treated for 90 days as follows: Group I received normal drinking water and served as the control; Group II received a heavy metal mixture of Pb (20 mg/kg), Al (35 mg/kg), and Mn (0.564 mg/kg) body weight; Group III received Pb (20 mg/kg) alone; Group IV received Al (35 mg/kg) alone; and Group V received Mn (0.564 mg/kg) alone. Chronic exposure to heavy metals resulted in a significant (p < 0.05) reduction in rotarod performance compared with the control group, indicating impaired cerebellar motor function. The low-dose heavy metal mixture significantly depressed antioxidant defences (p < 0.05), increased lipid peroxidation (p < 0.05), elevated amyloid-β peptide levels (Aβ₁–₄₀ and Aβ₁–₄₂) (p < 0.05), and markedly reduced occludin expression (p < 0.05) in the cerebellum relative to controls. These biochemical alterations were more pronounced in the mixture-exposed group than in animals treated with individual metals. Overall, chronic low-dose exposure to an environmentally relevant heavy metal mixture induces cerebellar neurotoxicity through enhanced amyloid-β accumulation and downregulation of occludin, a key tight junction protein, in adult male Wistar rats. These findings underscore the role of mixed metal exposure in amyloid-β dysregulation, tight junction disruption, and cerebellar dysfunction.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-42725-3.

Introduction

Human exposure to heavy metals is unfortunately inevitable due to constant and rapid urbanization and anthropogenic activities. Environmental contamination with heavy metals is posited as a possible factor in the development and progression of central nervous system related neurodegenerative disorders^1,2 and compromised neurocognitive function, particularly in adults³. Heavy metal toxicity has severe and long-term consequences on the brain, resulting in cognitive impairment. There has been an upsurge of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and prion’s disease in the recent past. It is currently reported that 5.3 million of US population is afflicted with AD with expected cases to reach 13.5 million by 2050⁴. Human studies showed that exposure to heavy metals during pregnancy may be associated with anxiety symptoms in childbearing woman and neurodevelopmental disorders in infants⁵.

Due to a protracted period of oil exploration and an upsurge in artisanal crude oil refining in the south-south region of Nigeria, lead (Pb), mercury (Hg), aluminum (Al) and manganese (Mn) are prevalent in this environment. Pb and Al are toxic and accumulate in both children and adult with disruption of normal physiological processes in various organs including the brain⁶. Lead is neurotoxic and crosses the blood-brain barrier (BBB). Its chronic exposure reduces antioxidants, increases oxidative stress, causes inflammation, morphologic damage, neurodegeneration and cognitive impairment in the brain^2,7. Al is a non-essential, neurotoxic metal implicated in the pathogenesis of neurodegenerative processes; mainly deposited in the hippocampus, cerebral cortex and cerebellum⁸. Al has proinflammatory and pro-apoptotic effects with the ability to up-regulate various cytokines. Al induced neuronal degeneration and death manifested as cognitive decline as observed in Morri’s water maze task⁹. Aluminium administration causes learning and memory deficits in rabbits and other experimental animals¹⁰. Mn is an essential metal, vital in proper human health maintenance and a cofactor in neurotransmitter synthesis and metabolism¹¹. In the case of chronic exposure, Mn is found in the brain, particularly in the motor control centres¹² and the hippocampus; can lead to toxic effects¹³. Mn causes neurodegenerative disorders in these regions, with symptoms that include memory impairment, cognitive slowing, and decreased learning capacity and mental flexibility^14,15. Toxic and sub-toxic levels of Mn may affect important cell signalling pathways that regulates cell survival, differentiation, and apoptosis, which are critically implicated in many neurodegenerative diseases^16–18.

Human exposure to heavy metals and their potential adverse effects on neurocognitive functions occur in both young and adult populations¹⁹. Cerebellum, hippocampus and prefrontal cortex are the parts of the brain critical for neurodevelopment prone to various heavy metal accumulation after crossing the blood brain barrier^2,20. Cerebellum plays a critical role in motor coordination and balance, aligning with the rotarod findings²¹. Cerebellum is particularly susceptible to intoxication and heavy metal poisoning including Pb, Al and Mn because it is the preferential site for their toxicity²². Al is largely deposited in the cerebellum and cortex and, implicated in pathogenesis of neurodegenerative diseases⁸. The cerebellum is increasingly recognized as vulnerable to amyloid pathology and oxidative injury, particularly in metal-induced neurotoxicity. Neurocognitive functions such as memory, attention, executive function, language, and visuospatial abilities are important in perception, processing and response to the environment²³. Heavy metal exposure can contribute to cognitive impairment through various mechanisms such as oxidative stress, inflammation, neurotransmitter disruption, disruption of blood–brain barrier, neuroinflammation, alteration of neurotrophic factors, impaired synaptic plasticity, accumulation in specific brain regions, epigenetic modifications and neurodegenerative pathways¹⁹.

The dysregulation of these factors can impair cognitive function²⁴. Heavy metals disrupt the balance of antioxidants, increase harmful reactive oxygen species (ROS) and impair mitochondrial function, disturbing cellular redox homeostasis²⁵. Memory impairment is reported to be related to altered brain oxidative stress status²⁶ and different antioxidants reduced oxidative damage in Alzheimer’s disease. It is reported that agents used to induce dementia-like conditions in rodent models usually cause oxidative damage, inflammation, apoptosis and neurochemical alteration ²⁷. A cascade of events ensues when excessive ROS is produced. Cellular damage stimulates inflammatory processes resulting in activation of transcription factors, synthesis and release of various inflammatory cytokines and mediators²⁸. Oxidative stress from metal exposure compromises the activity of the ubiquitin proteasome system, resulting in protein aggregation which disrupts cellular processes resulting in cell death²⁹. Nrf2 is a redox-sensitive transcription factor and closely related to many cell protection factors, which translocate to the nucleus to bind to antioxidant response element (ARE) under oxidative stress to induce expression of antioxidant genes, such as HO-1, NQO1 and GST³⁰. Nrf-2 interferes with beta amyloid and p-tau pathways³¹. Cyclooxygenase-2 (COX-2), an isoform of the cyclooxygenase enzyme family, along with COX-1 and COX-3, are involved in the synthesis of prostanoids (eicosanoid sub-class) from an essential fatty acid- arachidonic acid. COX-2 is expressed constitutively at post-synaptic membranes by groups of neurons in the prefrontal cortex, hippocampus and amygdala while it is inducible in other cell types and by inflammatory cytokines (IL-1, IL-2, and TNF-α) in the brain^32,33. COX-2 activity is important in neurogenesis and vital in tasks like synaptic plasticity, long-term potentiation, cognitive and behavioural functions³⁴. COX-2, predominantly present in neurons is important in regulating brain functions such as synaptic plasticity³⁵ though, its specific role in the hippocampus and cortex, which may be involved in cognitive functions, remains unclear³⁶. However, over-expression of COX-2 is associated with neurotoxicity in acute conditions, such as hypoxia/ischemia and seizures, enhancing glutamate excitotoxicity and inflammatory chronic diseases such as neurodegenerative diseases^34,35.

Humans’ exposure to heavy metals in the environment always occur as mixtures rather than singly and majority of them are neurotoxic³⁷. Heavy metals may contribute to progression of neurodegenerative diseases and, interactions among the components of a metal mixture may result in synergistic toxicity³⁸. Low level exposure to metals like Pb can disrupt normal central nervous system development and co-exposure to multiple metals can result in increased neurotoxicity compared to single metal exposures especially during early life³⁹. Miller et al.,⁴⁰ reported that the neurotoxicity of Hg at very low and otherwise safe levels is enhanced by co-exposure to Pb, Al, Mn, Cd, Zn, or Fe. Similarly, exposure to a nontoxic level of aluminium hydroxide markedly increased neuronal cell death when combined with Hg in an in vitro study⁴¹. Exposure to mixture of Al, Pb, Mn and Hg reduced the levels of antioxidants in the cerebral cortex and cerebellum, increased levels of MDA and NO; down-regulated cellular levels of Nrf2⁴².

Accumulation of Pb, Al and Mn in the brain is implicated in the pathophysiology of neurodegenerative disorders¹⁰ and so the consideration that animals exposed to low dose mixture of these metals can be used as animal model for neurological study. The collective impact of exposure to a mixture of heavy metals on neurocognitive function has yet to be explored^19,42 proposed that exposure to heavy metal mixture (Al, Pb, Mn and Hg) may promote the onset of Alzheimer disease as evinced by upregulated Caspase-3 levels, potentiated activity of acetylcholinesterase and decreased acetylcholine level with moderate histopathological alterations in cerebral cortex and cerebellum. Nevertheless, their study did not investigate the effect of low dose metal co-exposure on amyloidogenesis and neurotrophic factors in relation to oxidative damage in the brain. The effect of co-exposure to metals in the brain is imperative as the central nervous system is a common target organ for many environmentally toxic metals. Essential trace metals though necessary for normal cellular and enzyme activity, in their high levels together with other non-essential heavy metals and metalloids can damage biomolecules, impair cellular redox status, alter signal transduction, and cause protein misfolding, apoptosis, and malignant transformation⁴³. Mn-Al-Mn are prevalent environmental contaminants in the Niger Delta due to oil exploration and artisanal refining activities. Pb and Al are well-established neurotoxic metals, while Mn is an essential metal with neurotoxic effects at elevated or chronic exposure levels. These metals share common transport pathways (e.g., transferrin/DMT1), enabling potential synergistic neurotoxicity. This study hypothesized that chronic low-dose exposure to a mixture of aluminium, manganese, and lead induces cerebellar toxicity through amyloid-β accumulation and occludin depletion, resulting in functional impairment. Therefore, this study investigates the possible outcome of low dose metal mixture on oxidative damage, expression of transcription factors, amyloidogenesis, inflammation and apoptosis in the cerebellum of male Wistar rats.

Materials and methods

Chemicals and reagents

Analytical pure grade compounds of Lead acetate (Pb (CH₃COO)₂), aluminum (III) chloride (AlCl₃) and Manganese chloride (MnCl₂.4H₂O, ≥ 99.9%), were purchased from Sigma Chemical Co. (St. Louis, MO, USA); ELISA kits (Nrf-2, Catalog No. (E-EL-R1052), HO-1, Catalog No. (E-EL-R0488), BDNF Catalog No. (E-EL-R1235), COX − 2, Catalog No. (E-EL-R0792), Caspase-3 Catalog No. E-EL-R0160, Aβ₁₋₄₀, Catalog No: E-EL-R3030, Aβ₁₋₄₂ Catalog No: E-EL-R1402, Occludin Catalog No: (E-EL-H1073) were procured from Elabscience Biotechnology, Inc. Wuhan, China (Table S1).

Animals and ethics

Twenty – Five Young adult male Wistar rats (8–10 weeks old; 130–150 g) were used for this study and acclimatized for 2 weeks and housed at the Animal facility of the Department of Experimental Pharmacology and Toxicology, University of Port Harcourt, Rivers State. They were maintained at a 25 °C and humidity 55–60%, on a 12: 12 light dark cycle in well-ventilated standard polypropylene cages. The animals were allowed free access to animal feed and clean water ad libitum in adherence to the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) checklist⁴⁴. All experimental procedures were approved and performed according to the university committees’ guidelines for the use and care of animals. (UPH/CEREMAD/REC/MM188/003) in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science (NAS) and published by the National Institute of Health.

Animal welfare was monitored daily throughout the experimental period by trained personnel. Monitoring criteria included body weight changes, grooming behaviour, posture, locomotor activity, feeding and drinking patterns, and signs of distress (e.g., piloerection, lethargy, laboured breathing). Humane endpoints were predefined and included ≥ 20% body weight loss, severe lethargy, persistent anorexia, or any signs of severe pain or distress unresponsive to supportive care, at which point animals would be humanely euthanized. No invasive surgical or painful procedures were performed during the study. Animals were monitored daily for signs of discomfort or distress, and humane endpoints were predefined as described above. Routine analgesia was not required due to the non-invasive nature of the procedures; therefore, routine analgesia was not required.

Experiment

Animals were randomly assigned into experimental groups (n = 5 per group). Sample size was determined based on previous laboratory studies and published neurotoxicology literature demonstrating sufficient statistical power for biochemical and behavioral endpoints⁴⁵, while adhering to the principles of Replacement, Reduction, and Refinement (3Rs)⁴⁶. Rats received aluminum, manganese, lead, or their mixture orally at environmentally relevant low doses for 90 consecutive days. The 90-day exposure duration was selected to model sub-chronic to chronic environmental exposure consistent with OECD toxicological frameworks and cumulative neurotoxicity, amyloidogenesis, and oxidative stress following prolonged low-dose exposure as shown on Table 1:

Table 1.

Experimental design.

Experimental groups	Treatment
Group 1 (Control)	Drinking water alone
Group 2	Heavy metal mixtures (Pb 20 mg/kg, Al 35 mg/kg and Mn 0.564 mg/kg body weight)
Group 3	Pb 20 mg/kg body weight alone
Group 4	Al 35 mg/kg body weight alone
Group 5	Mn 0.564 mg/kg body weight alone

The following environmental relevant doses of Pb 20 mg/kg, Al 35 mg/kg and Mn 0.564 mg/kg body weight were chosen based on the previous reports of Yang et al., and³⁰ for Pb,^47,48 for Al and^49–51 for Mn, respectively. HMM were prepared separately as individual salt solutions in deionized water; one gram each of Pb (CH₃COO)₂), AlCl₃ and MnCl₂.4H₂O were separately dissolved in 100 ml deionized water in a 150 ml capacity airtight conical flask, stored in the refrigerator until needed. After 90 days of treatments, the rats were sacrificed under IP pentobarbitone (50 mg/kg) anaesthesia, sacrificed and the cerebellar tissue collected immediately for further experiments.

Metal concentrations in cerebellum

Cerebellum were weighed individually, frozen and freeze-dried for 24 h using Lyovac lyophilizer GT2e (Steris, Germany). The samples were weighed after drying, put into the polytetrafluoroethylene digestion tube and 65% nitric acid (Merck, Germany) was added to each sample to obtain a dilution factor (DF) of 10 (2.0–6.0 ml of nitric acid and 0.2–0.6 g of sample). Prepared samples were kept overnight to slow mineralization, and the samples were subsequently mineralized in a microwave oven (Mars Xpress 5, CEM USA). Concentrations of Pb, Al and Mn in the mineralized samples were determined using Atomic Absorption Spectrometer (AAS) 7000 (Shimadzu, Japan). Graphite furnace atomization (GF-AAS) was used for Pb and Al while flame atomization (F-AAS) was used for Mn and, analyses were done in triplicates according to⁵². The percentage RSD for GF-AAS and F-AAS analysis did not exceed 5 and 7% respectively. Atomic absorption spectrometry (AAS) was used to determine the concentrations of the metals Pb, Al, and Mn that bioaccumulated in the cerebellum after treatment.

Rotarod test

The animals were trained to walk on the accelerating rotarod (UgoBasile 47600, Milan, Italy) as previously described in our lab⁵³. The rotarod consisted of a cylinder with a diameter of 3 cm on which five animals could run at the same time, set apart by panels of adequate size ensuring they don’t see themselves. The speed of the rod was increased from 4 rpm to 40 rpm for 300 s. Thereafter the animals were placed back in their cages. As soon as the animal could not maintain its balance, it fell off the device and the time was recorded. In the first week of the experiment, the animals were trained to get used to the device and baseline values were recorded. Rotarod testing was conducted at baseline and subsequently at predefined weekly intervals. Daily training was limited to the acclimatization phase to avoid learning bias^54,55.

Biochemical analysis

The antioxidant enzyme activity of Superoxide dismutase (SOD) was assayed using the method by Misra and Fridovich⁵⁶ based on the principle that SOD has the capacity to inhibit the autoxidation of epinephrine at pH 10.2. Catalase (CAT) activity was determined using a slight modification of the Clairborne⁵⁷ technique. It is premised on the principle that catalase in the tissue will split hydrogen peroxide estimated at 240 nm using a spectrophotometer. Glutathione peroxidase (GPx) activity was determined using the method of Rotruck et al.⁵⁸ while the technique by Jollow et al.⁵⁹ was adopted for the evaluation of Reduced glutathione (GSH) activity.

The level of oxidative stress marker (malondialdehyde -MDA) was assayed using method of Ohkawa and Ohishi⁶⁰. This method is based on the principle that in acidic medium, MDA reacts with the chromogenic reagent, 2-thiobarbituric acid (TBA) with the formation of a pink coloured complex at 532 nm absorbance.

Immunosorbent assays in the Cerebellum

The transcription factors (Nrf-2, HO-1, BDNF), inflammation markers (COX − 2, caspase-3) amyloid precursor proteins (Aβ₁₋₄₀, Aβ₁₋₄₂) and occludin were evaluated using Enzymes linked immunosorbent assay (ELISA) kits. Triplicates of the assay was done with NM 9602 Microplate Reader according to the manufacturer’s instructions (Elab Science Biotechnology Company, (Beijing, China). The concentrations of biomarkers in the rats’ cerebellum were calculated by comparing the optic density of the samples to the standard curve. The ELISA calibration curves consisted of six non-zero standard concentrations covering the assay’s dynamic range; back-calculated concentrations met acceptance criteria (± 15% nominal, ± 20% at LLOQ) consistent with FDA/ICH bioanalytical guidelines. Duplicate wells (i.e., Intra-plate precision yielded CVs < 10%, and inter-assay CVs across three independent runs remained < 15%. Recovery was evaluated by spiking matrix-matched samples at low/mid/high levels, achieving 70–130% recovery. Each assay run incorporated QC samples and utilized Levey-Jennings plots with Westgard rules to ensure consistency; any runs failing QC were excluded. All validation and assay records were maintained per regulatory standards.

Statistical analysis

Data obtained were expressed as mean ± standard deviation (SD) and data were analysed with GraphPad^® Version 5 using one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test. Values were considered significant at p < 0.05.

Result

Bioaccumulation of Pb, Al and Mn (mg/kg) in the cerebellum

The bioaccumulation of Pb, Al and Mn in rat cerebellum after exposure to Pb, Al, Mn as individual metals and Pb-Al-Mn mixture is shown in Table 2. There was no significant difference in the bioaccumulation (0.37 ± 0.051 mg/kg) of Pb after exposure to Pb (20 mg /kg) as an individual metal and when it was administered as a mixture, (Pb (20 mg /kg), Al (35 mg / kg) Mn (0.56 mg/ kg)). There were, however, significant differences in the bioaccumulation of Al and Mn after exposure as individual metals and when it was given as a mixture. Al bioaccumulation was higher and Mn was lower in the metal mixture (Pb (20 mg /kg), Al (35 mg / kg) Mn (0.56 mg/ kg)) group.

Table 2.

Bioaccumulation of Pb, Al and Mn (mg/kg) in the cerebellum of rats after exposure to Pb, Al, Mn and Pb-Al-Mn tertiary mixture.

Group.	Pb	Al	Mn
Control De-ionized water	-	-	-
Pb (20 mg /kg)	0.37 ± 0.051	-	.
Al (35 mg / kg)	-	0.26 ± 0.06*	-
Mn (0.56 mg/ kg)	-	-	0.50 ± 0.09*
Metal mixture	0.38 ± 0.021	0.9 ± 0.05	0.17 ± 0.05
(Pb, Al and Mn)

Data are expressed as mean ± SD. (n = 5). One-way ANOVA was used to test for significant, followed by post hoc test for multiple comparisons of means: DF (4; 5), F = 4.583. *p-value = 0.01or p < 0.05 was considered statistically significant.

Effect of Pb, Al and Mn as individual metals Vs Pb, Al, Mn and Pb-Al-Mn tertiary mixture on rota-rod performance test

Pb, Al and Mn whether as individual metals or as tertiary mixture significantly reduced rotarod performance test of the animals compared to the control as shown on Fig. 1. There was no significant between of the test animal rotarod performance for the individual metals when compared with the HMM.

Fig. 1.

Time (sec) the animals spent on the rotating rod after the administration of lead (Pb), aluminum (Al), manganese (Mn) and tertiary metal (loid) mixture on a rotarod apparatus Values are expressed as means ± SD. *p-value = 0.01or p < 0.05 was considered statistically significant.

Effect of heavy metals on antioxidant and lipid peroxidation in the cerebellum

Figure 2 depicts the effect of HMM alone and individual metals (i.e., Pb, Al and Mn) on the antioxidant enzymes activities (GPx, CAT, and SOD) as well as GSH level in the cerebellar cortex of rats. The exposure to HMM and individual metal alone caused significant decrease in GPx, CAT and SOD activities and GSH level in the cerebellar cortex of the treated rats when compared to the control. Moreover, exposure to HMM also resulted in significant decrease in GPx and CAT when compared to Pb, Al, and Mn alone. Furthermore, Fig. 2 depicts the effects of HMM alone and individual metals (i.e., Pb, Al and Mn) on oxidative stress marker (LPO) in the cerebellar cortex of rats. Exposure of rats to MM alone caused a significant increase in LPO level in the cerebellar cortex of rats when compared to the control.

Fig. 2.

Effect of heavy metals on antioxidants and oxidative stress marker in the cerebellum. ^a Significantly different compared to control; ^b significantly different compared to HMM; ^c significantly different compared to Pb. *p-value = 0.01or p < 0.05 was considered statistically significant.

Effect of Heavy metals on transcription factors Nrf-2, HO-1 and BDNF in the cerebellum

Figure 3 depicts the effect of HMM alone and individual metals (i.e., Pb, Al and Mn) on Nrf-2, HO-1 and BDNF levels in the cerebellar cortex of rats. The exposure to HMM and individual metal alone caused significant (p < 0.05) increase in Nrf-2 and BDNF levels and significant (p < 0.05) decrease in HO-1 level in the cerebellar cortex of the treated rats when compared to the control. The neurotrophic factor BDNF was significantly reduced in the Pb, and Al only treated groups compared to the control (Fig. 3). HMOX-1 was significantly down regulated in metal mixture, Pb and Mn only treated groups compared to control. The neurotrophic factor BDNF was significantly reduced in the Pb and Al only treated groups compared to the control (Fig. 3).

Fig. 3.

Effect of Heavy metals on transcription factors Nrf-2, HMOX-1 and BDNF in the cerebellum. ^a Significantly different compared to control; ^c significantly different compared to Pb. *p-value = 0.01or, p < 0.05 was considered statistically significant.

Effect of heavy metals on COX-2 and Caspase-3 in the cerebellum

Figure 4 depicts the effect of HMM alone and individual metals (i.e., Pb, Al and Mn) on neuroinflammation marker (COX-2) and apoptotic marker (caspase-3) in the cerebellar cortex of rats. The exposure to HMM and individual metal alone caused significant (p < 0.05) increase in COX-2 and Caspase levels in the cerebellar cortex of the treated rats when compared to the control.

Fig. 4.

Effect of Heavy metals on COX 2 and Caspase-3 in the cerebellum. ^a Significantly different compared to control; ^b significantly different compared to HMM *p-value = 0.01or or p < 0.05 was considered statistically significant.

Effect of heavy metals on amyloid precursor proteins in the cerebellum

Figure 5 depict the effect of HMM alone and individual metals (i.e., Pb, Al and Mn) on amyloid precursor proteins (Aβ-40 and Aβ-42) in the cerebellar cortex of rats. The protein expression of Aβ-40 and Aβ-42 were significantly (p ≤ 0.05) upregulated in the rats cerebellar following exposure to HMM and individual metals alone when compared with the control. There was a significant (p < 0.01) increase in Aβ₁₋₄₀ and Aβ₁₋₄₂ formation in the metal mixture and single metal treated groups compared to the control (Fig. 5).

Fig. 5.

Effect of Heavy metals on amyloid precursor proteins in the cerebellum. ^a Significantly different compared to control; ^b significantly different compared to HMM; ^c significantly different compared to Pb; ^d significantly different compared to Al* p-value = 0.01or or p < 0.05 was considered statistically significant.

Effect of heavy metals on occludin in the cerebellum

Figure 6 depict the effect of HMM alone and individual metals (i.e., Pb, Al and Mn) on occludin in the cerebellar cortex of rats. Exposure to the heavy metal mixture resulted in a significant (p < 0.001) reduction in occludin levels in the cerebellar cortex compared to the control and single-metal treated groups (Fig. 6).

Fig. 6.

Effect of heavy metals on occludin proteins in the cerebellum. ^a Significantly different compared to control. *p-value=0.05.was considered statistically significant. There was a significant (p < 0.001) reduction in occludin level in the metal mixture treated group compared to the control and single metal only treated groups (Fig. 6).

Effect of heavy metals on cerebellar architecture

Photomicrograph (X400) of toluidine blue stained section of the cerebellar tissue after the administration of de-ionized water, showing a normal cerebellum with no structural changes in the Pia mater (PM), gray matter (GM), Outer molecular layer (ML) inner granular layers (GL) and white matter (WM). The cerebellum is the area of the hindbrain that controls motor movement coordination, balance, and equilibrium and muscle tone. Panel 2. Photomicrograph (X 400) of Toluidine blue-stained cerebellar tissue section of the (Pb + Al+Mn) treated group, showing severe neuronal loss with the Purkinje and granule layers appearing thinner and vacuolated. There are pyknotic granular cells undergoing necrosis (arrow), with dense dark blue plaques. Panel 3: Photomicrograph (X400) after treatment with Pb only, showed disruption of normal architecture of the cerebellar tissue and moderate neuronal loss. There are pyknotic granular cells undergoing necrosis (arrows) with dense, dark blue plaques. Panel 4: Al only treated group photomicrograph (X400) of the cerebellar tissue section. Showed disruption of the cerebellar layer with focal areas mild neuronal loss. There is neuronal degeneration with pyknotic neuronal nuclei and irregular arrangement of Purkinje cells. Panel 5: Mn only treated group photomicrograph (X400). Showed mild neuronal loss.

Discussion

Heavy metals are present as mixtures in the environment where humans are routinely exposed to these mixtures. Therefore, in this work, we studied the possible mechanism of induced neurotoxicity of low dose metal mixtures (Pb, Al and Mn) in the cerebellum using a rat model. Concurrent cerebellar accumulation of Al and depletion of Mn as shown in this study disrupts metal homeostasis via shared transport systems, for example both Al and Mn utilize shared the transferrin/transferrin receptor (Tf–TfR) and divalent metal transporter 1 (DMT1) pathways to cross the blood–brain barrier^61,62. Also, Al–Tf complexes can down‑regulate TfR and reduce Mn uptake, therefore, compromises antioxidant and mitochondrial resilience, and perturbs neurotransmitter regulation⁶³. This synergistic dysregulation exacerbates neuronal vulnerability and may contribute to the pathogenesis of neurodegenerative disorders⁶⁴.

The formation of ROS is a common phenomenon in normal cellular metabolism though, tightly regulated by the cellular antioxidant system. Oxidative stress occurs due to an imbalance between antioxidant defense and free radicals (ROS and RNS) in favour of the overproduction of free radicals. Redox homeostasis is maintained by both enzymatic and non-enzymatic antioxidants. This study showed that low dose heavy metal mixture affected the redox homeostasis in the cerebellum as seen by depletion of antioxidants SOD, CAT and GSH activities. Malondialdehyde was significantly elevated in the metal mixture exposed groups compared to the control group. Other studies also observed that low dose metal mixtures (Pb, He, Al and Mn) and (Pb, Cd and Hg) reduced antioxidants levels and increased lipid peroxidation in the brain of rats^42,65. The decreased antioxidant enzymes may be the result of heavy metal affinity for sulfhydryl groups restricting its ability to scavenge ROS (Virgolin and Aschner, 2021). The decreased levels of peroxisomal enzyme catalase (CAT) may have affected H₂O₂ removal that increased lipid peroxidation, which can lead to neuronal cell death. GSH is the main non-enzymatic antioxidant that catalyses H₂O₂ reduction⁶⁶. SOD is a major antioxidant enzyme that could remove ROS, protects cells from the oxidative damage of many biological macromolecules⁶⁷. So, the decreased SOD activity observed in this study would increase free radicals, which in turn cause the production of toxic factors/oxidative stress, further confirmed by the reduced GSH levels in cerebellum of metal mixture exposed rats.

Environmental heavy metals can bioaccumulate in the general population and cause various neurobehavioral deficits^68,69. Chronic individual heavy metal exposure has been reported to impair motor coordination using a rota-rod test in male rats^70,71. Although there is a plethora of reports on behavioral effects after single heavy metal exposure; there is sparsity of data heavy metal mixture exposure. One study reported that a mixture of heavy metals induced synergistic neurotoxicity, leading to impairments in neurobehavioral functions⁷². The present study suggests that the exposure to heavy metal mixture can collectively cause the impairment of motor function. Mixed exposure to metals may affect the processes of absorption, distribution, metabolism, and excretion of each metal, leading to metal–metal interactions, such as competition, antagonism, and synergism^68,73.

The cerebellum coordinates movement via control of diverse muscular systems to ensure fluency, synchrony and precision of different muscles. In a synchronous precision, the cerebellum acquires ceaseless feedback information about all forms of movements whether intended or actual. Cerebellar atrophy is often accompanied by different forms of ataxia and an unbalanced and tottering gait⁷⁴. In the present study, heavy metal either singly or as cocktail mixture -exposed rats exhibited significant motor deficits. This observation is like previous reports by Bazrgar and co-workers⁷⁵. It has previously been shown that low dose heavy exposure in rats impacts on the body burden of heavy metals^75,76 as seen in the present study with significant higher cerebellar concentrations of Pb, Al and Mn than control unexposed rats. The bioaccumulated heavy metals is thought to be retained for a long time even after exposure given the long half lives of these heavy metals. Consequently, the spatial and temporal patterns of the cerebellar heavy metal bioaccumulation precipitate neurobehavioral abnormalities associated with toxic heavy metal exposure.

Pb and Al have been shown to decrease GSH as the result of the inhibition of thiols groups, reducing the antioxidant enzymes activities, substituting Ca²⁺, Fe²⁺, or Zn²⁺ or altering the integrity, permeability and functionality of membranes, favouring lipid peroxidation⁷⁷. The marked increased level of malondialdehyde (MDA) in the metal mixture group compared to single metal exposure could possibly be due to additive toxicity of the heavy metals. The decreased antioxidant levels in the cerebrum increased the level of ROS production that could possibly lead to mitochondrial damage of neurons in the cerebrum. During oxidative stress, neurons undergo dendritic alterations characterized by dendritic contraction, which results in perturbations in neuronal connectivity⁷⁸. Oxidative stress also adversely affects other cellular components of the CNS like astrocytes, microglia and oligodendrocytes; their perturbations are implicated in a few neurological disorders. Nrf2 is a redox-sensitive transcription factor and closely related to many cell protection factors, which translocate to the nucleus to bind to antioxidant response element (ARE) under oxidative stress to induce expression of antioxidant genes, such as HO-1, NQO1 and GST^28,30. It was observed that their Nrf2 in metal mixture rats did not significantly differ from the control. However, the antioxidant gene, HO-1 expression was decreased in the metal mixture compared to control. HO-1 plays an important role in preventing ROS formation and oxidative injury by maintaining cellular redox homeostasis²⁷. Hmox-1 is a stress-induced enzyme, which exerts cytoprotective effect against inflammatory responses through inhibiting inflammation mediators^79,80. The increased level of MDA observed in this study contributed to oxidative injury and the down-regulated HO-1 expression implies that the neurons were HO-1 suppression despite unchanged Nrf-2 likely involves transcriptional repression (e.g., Bach1, NF-κB) or post-transcriptional mechanisms, decoupling it from Nrf-2’s status⁸¹. The reduced levels of occludin, in the cerebellum seem to be linked to synergistic oxido-inflammatory pathways activated by mixed metal exposure⁴⁵.

Oxidative stress is a common cause of neuronal cell loss in various neurodegenerative disorders and considered an important player in the pathophysiology of Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis and Huntington’s disease⁸². Oxidative stress leads to perturbation of many cellular functions which is highly detrimental to neuronal survival, contributing to the aetiology of neurodegenerative diseases⁸³.

Brain-derived neurotrophic factor (BDNF) is a major player in neuroplasticity, neurogenesis, and neuroprotection in the brain⁸⁴ and have been reported to be beneficial in the recovery and the prevention of neurodegenerative motor disorders like Parkinson’s disease, and Huntington’s disease⁸⁵. BDNF is crucial motor recovery after ischemic stroke and brain injury since the recovery of motor function is known to accompanied by an increase in BDNF expression⁸⁶, whereas downregulation of BDNF attenuates the recovery⁸⁷. The significantly reduced BDNF in the heavy metal treated rats especially Pb and Al only treated groups in comparison to the control may at least explain in part the significantly diminished rotarod performance test in the present study. As a survival factor BDNF has been shown to regulate morphologic development and maturation of cerebellar granule and Purkinje cells⁸⁸ and its administration counters some forms of cerebellar degeneration and sensorimotor deficits⁸⁹.

Accumulation of ROS and RNS is also involved in the accumulation of toxic protein aggregates that typify some neurodegenerative disorders⁸². This study demonstrated that although COX-2 and caspase-3 levels were significantly increased alongside increased oxidative stress and amyloid-β (Aβ) accumulation. These findings suggest that oxidative stress, inflammation, and apoptotic signalling are involved in the observed cerebellar injury driving the observed effects.

The significant increase in caspase-3 levels observed in the heavy metal mixture and single metal groups suggests activation of apoptotic signalling pathways in the cerebellum. However, it is important to note that this study quantified total caspase-3 protein levels using ELISA and did not assess cleaved (active) caspase-3 or enzymatic activity. Therefore, while the elevation in caspase-3 is indicative of pro-apoptotic signalling, definitive conclusions regarding caspase-dependent apoptosis cannot be made. Future studies incorporating cleaved caspase-3 detection or activity assays would further clarify the specific apoptotic mechanisms involved. Oxidative stress, secondary to Aβ accumulation are known to trigger cellular damage and inflammation⁹⁰. Usually COX-2 is upregulated in response to oxidative stress and Aβ increases which tend to exacerbate the inflammatory response. In some cases, upregulation of COX-2 may not necessarily correlate with Aβ increases, thus suggesting a complex and multifaceted relationship⁹¹. More studies are required to elucidate these intricate mechanisms.

This study showed elevated amyloid-β peptides (Aβ) proteins in the rats treated with low dose metal mixture. Aβ peptides influence the expression and localization of TJ proteins such as claudin 5, occludin and ZO-1, and lead to disruption of tight junctions and loss of integrity of blood-brain-barrier⁹². Aβ is toxic to endothelial cells in the human brain via binding to the receptor for advanced glycation end-products (RAGE) and induction of ROS production, which ultimately leads to disruption of tight junctions and loss of BBB integrity⁹². The Aβ₁₋₄₂ peptide accumulation around brain micro vessels was found to increased blood brain barrier permeability, disrupted claudin 5, ZO-1 and occludin⁹². Amyloid oligomers are the most toxic species that cause neurodegenerative diseases⁴. The accumulation of toxic protein aggregates is one of the processes that activate cell death pathways via apoptosis, necrosis, ferroptosis or autophagy⁹³. Occludin is an integral tight junction protein that forms part of epithelial and endothelial junctional complexes that regulate barrier properties⁹⁴. Occludin protein was downregulated in the cerebellum cortex after exposure of rats to low dose metal mixture in this work. The implication is that there will be increased permeability of the toxic metals through the blood brain barrier into the cerebellum with attendant neurodegeneration. The cerebellum cortex contained amyloid plaques in the molecular and granular layers. The presence of amyloid plaques, gliosis and neuronal degeneration followed by disorganization in the normal architecture of the cerebellar layer are indicative of cerebellar pathology. The limited behavioural data in the present study is considered a limitation which will be addressed in future studies.

Conclusion

This study demonstrates that chronic co-exposure to low doses of Pb, Al, and Mn produces additive neurotoxic effects in the cerebellum of adult male Wistar rats. Mixed-metal exposure suppressed antioxidant defenses, increased lipid peroxidation, elevated amyloid-β levels, upregulated COX-2 and caspase-3, and reduced occludin expression, suggesting oxidative stress-mediated cerebellar dysfunction and potential blood–brain barrier compromise. These findings support the concept that environmentally relevant metal mixtures may exert greater neurotoxic risk than single-metal exposures.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

BDD, CNO, ANE,: formal analysis and investigation; OEO, TCU, KWN, CNOb,: writing—original draft preparation, writing—review and editing, visualisation; CNO, OEO: conceptualisation, data curation, supervision, writing—review and editing. All the authors have read and approved the manuscript.

Data availability

All the data generated or analysed during this studyare included in this published article.

Declarations

Competing interests

The authors declare no competing interests.