A PROJECT REPORT

ON

EFFECT OF ALUMINIUM AND CADMIUM ON LIPID PEROXIDATION AND

CATALASE ACTIVITY IN THE PREFRONTAL CORTEX OF MICE

BY

OSIAN, Ifeoma Augustina

DELTA STATE UNIVERSITY,

ABRAKA

NOVEMBER, 2021.

1
 A PROJECT REPORT

ON

EFFECT OF ALUMINIUM AND CADMIUM ON LIPID PEROXIDATION AND

CATALASE ACTIVITY IN THE PREFRONTAL CORTEX OF MICE

BY

OSIAN, Ifeoma Augustina

CFB/17/18/247363

A PROJECT REPORT SUMMITED TO THE DEPARTMENT OF MEDICAL

BIOCHEMISTRY, FACULTY OF BASIC MEDICAL SCIENCE, DELTA STATE
UNIVERSITY, ABRAKA, NIGERIA.

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF

BACHELOR OF SCIENCE (B.Sc.) DEGREE IN MEDICAL BIOCHEMISTRY

NOVEMBER, 2021.

2
 CERTIFICATION

This is to certify that this project work was written and presented by OSIAN, Ifeoma
Augustina with the Matriculation Number CFB/17/18/247363 and is hereby approved as
meeting the requirement for the award of Bachelor of Science Degree in Medical
Biochemistry.

_________________________ ________________________

DR. J.C. MORDI Date

(Project Supervisor)

_________________________ ________________________

DR. E.U. UZUEGBU Date

(Head of Department)

_________________________ ________________________

(External Supervisor) Date

3
 DEDICATION

This seminar report is dedicated to the Almighty God, the giver and sustainer of life, for His
unconditional love and mercy granted to me throughout the period of my study.

4
 ACKNOWLEDGEMENTS

  My special appreciation first goes to God Almighty for His love, grace, inspiration, wisdom,
mercies, and protection for the success of this seminar work and my four years in studies. My
sincere appreciation goes to my supervisor; Dr. J.C. Mordi who in spite of his numerous tight
schedule official and personal commitments created much time to thoroughly go through the
manuscript and give valuable suggestions for the success of this work. Thanks and may the
good God bless you richly for your efforts. My appreciation goes to the Head of Department Dr.
E.U. Uzuegbu and to all the Lecturers of the Department of Medical Biochemistry for the
knowledge they imparted on me. May the Almighty God continue to bless and protect you all.

My sincere appreciation to my ever patient, loving and caring parents Mr. and Mrs. Osian for
their prayers, financial Support, moral support and care shown to me. I also want to thank Mr
and Mrs Obi, Mrs Ngozi Onyeka and late Mr Bethel Nwafor for their moral and financial
support. To all my friends for their real assistance and encouragement. I say may God bless you
all.

5
 TABLE OF CONTENTS

Cover Page - - - - - - - - i

Title Page - - - - - - - - ii

Certification - - - - - - - - iii

Dedication - - - - - - - - iv

Acknowledgements - - - - - - - - v

Table of Content - - - - - - - - vi

List of Tables - - - - - - - - ix

Abstract - - - - - - - - x

CHAPTER ONE: INTRODUCTION

1.1 Background of Study - - - - - - - 1

1.2 Statement of Problem - - - - - - - 2

1.3 Objectives of Study - - - - - - - 3

1.3.1 General Objective - - - - - - - 3

1.3.2 Specific Objective - - - - - - - 3

1.4 Hypothesis - - - - - - - - 3

1.5 Significance of Study - - - - - - - 3

1.6 Justification of Study - - - - - - - 4

CHAPTER TWO: LITERATURE REVIEW

6
2.1 What is Neurodegeneration? - - - - - - 5

2.2 Alzheimer’s Disease - - - - - - - 6

2.3 Role of Metals in Neurodegenerative Alzheimer’s Disease - - 8

2.3.1 Aluminium - - - - - - - - 8

2.3.2 Cadmium - - - - - - - - 12

2.4 Role of Diet in Alzheimer’s Disease - - - - - 15

2.4.1 Polyunsaturated Fat - - - - - - - 15

2.4.2 Vitamins - - - - - - - - 17

2.4.3 Polyphenols - - - - - - - - 19

2.5 Oxidative Stress Hypothesis - - - - - - 21

2.6 Antioxidants - - - - - - - - 23

2.7 α-Tocopherol - - - - - - - - 23

2.8 Catalase - - - - - - - - 26

CHAPTER THREE: MATERIALS AND METHODS

3.1 Materials - - - - - - - - 28

3.1.1 Instruments - - - - - - - - 29

3.1.2 Reagents and Chemicals - - - - - - 29

3.2 Methods - - - - - - - - 30

3.2.1 Collection of Animals - - - - - - - 30

3.2.2 Animal Care and Handling - - - - - - 30

3.2.3 Animal Mobilization - - - - - - - 32

7
3.2.4 Animal Sacrificing and Collection of Specimen - - - 32

3.3 Biochemical Analysis - - - - - - - 33

3.3.1 Determination of Lipid Peroxidation - - - - - 33

3.3.2 Assay for Catalase Activity - - - - - - 34

3.4 Statistical Analysis - - - - - - - 35

CHAPTER FOUR: RESULTS

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion - - - - - - - - 37

5.2 Conclusion and Recommendations - - - - - 39

References

8
 LIST OF TABLES

Table 1: Experimental Design - - - - - - 31

Table 2: Effect cadmium and aluminum on MDA and Catalase activities in the prefrontal
cortex of mice - - - - - - - - - 36

9
 ABSTRACT

Heavy metal toxicity has proven to be a major threat and there are several health risks associated
with it. The toxic effects of these metals, even though they do not have any biological role, remain
present in some or the other form harmful for the human body and its proper functioning. This
experiment was carried out using 48 mice which were acclimated for a period of two weeks and
randomly shared into seven groups of eight animals each at the end of the acclimatization period.
Animals in group 1 served as normal control while animals in group 2 received only aluminum in
form of Aluminum chloride hexahydrate. Group 3 animals received only cadmium in form of
cadmium chloride monohydrate while group 4 received a combination of aluminum and cadmium
without treatment, groups 5 and 6 received a combination of cadmium and aluminum alongside
treatment with 25mg/kg and 50mg/Kg body weight of alpha tocopherol respectively. Aluminum
and cadmium were administered interperitonially at a dose of 1.25mg/kg and 0.5mg/Kg
respectively at intervals of 30minutes per toxicant. Alpha-tocopherol was also administered at an
interval of 30 minutes following the administration of the last toxicant. Treatment was done for 28
days.The results obtained for MDA : CAT are 0.75±0.15a : 17.15±1.08a, 0.98±0.18 a :
15.02±1.32a, 1.47±0.28b : 13.09±0.70ab, 2.46±0.17c : 12.10±0.21b. From the results, it show that
there was significant increase in MDA level which indicates the presence of free radicals, and the
level of catalase was reduced. These changes in MDA and CAT levels in the brain contributes to the
etiology of Al and Cd neurotoxicity.

10
 CHAPTER ONE

INTRODUCTION

1.1 Background of Study

Metals are substances with high electrical conductivity, malleability, and luster, which

voluntarily lose their electrons to form cations. Metals are found naturally in the earth’s

crust and their compositions vary among different localities, resulting in spatial variations of

surrounding concentrations. The metal distribution in the atmosphere is monitored by the

properties of the given metal and by various environmental factors (Khlifi & Hamza-

Chaffai, 2010). The main objective of this review is to provide insight into the sources of

aluminum and cadmium and their harmful effects on living organisms. Aluminum (A) and

cadmium (CD)are abundant metals on earth and they are also known as a neurotoxicants

(Yongsheng et al, 2011). Several studies have indicated neurobehavioral, neuropathological,

neurochemical and neurophysical effects following Al exposure. It can react with other

11
metals in the environment to form various complexes. The widely spread use of products

that contain or made from Aluminum or Cadmium ensures its presence within our body

(Najeebet al, 2014). These metals gets access to the human body through the environment,

food or drugs. However, there is no recognized physiological function for cadmium inside

the body and as a result, can also produce reverse physiological effects (Mostafa et al,

2012).The generation of oxidative stress can be referred to a toxic effect in animals and

humans. The oxidative stress has been involved in the pathogenesis of various

neurodegenerative conditions including Azheimer’s disease and Parkinson’s disease[4] Due

to the easy access of Ato the central nervous system under normal physiological conditions

and its accumulation in the brain, Alhas been reported to alter the blood-brain barrier (BBB)

(Flora et al, 2012).The main toxic effects of Al occurringthe brain, the nervous system and

the kidney.The brain is sensitive to oxidative stress due to the low levels of antioxidants and

high level of free radicals following toxicity and therefore, it is considered as the most

susceptible organ to Al toxic effect. And antioxidants play a significant role in maintaining

the balance between the antioxidant system of the body and free radicals that are produced

by the body'smetabolism or derived from environmental sources (Han et al, 2009).

1.2 Statement of Problem

12
Exposure to several heavy metals including aluminium and cadmium has been reported to be

associated with the pathogenesis of various neurological diseases through the formation of

oxidative stress in the brain.

However, this study aim to investigate the effects of these two metals, aluminum and

cadmium coexposure on experiment on lipid peroxidation and catalase activity in the

prefrontal cortex of experiment mice

13
1.3 Objectives of Study

1.3.1 General Objectives

To determine the level of lipid peroxidation and catalyse activityuponaluminium and

cadmium coexposurein the prefrontal cortex

1.3.2 Specific Objectives

i. To administer varying doses of aluminium and cadmuim to experimental mice

ii. To estimate the level of Malondialdehyde(MDA)induced by aluminium and

cadmium in the prefrontal cortex of experimental mice

iii. To evaluate activity of catalase in the administration of aluminium and cadmium.

1.4 Hypothesis

Aluminum and cadmium affects induces high levelsmalondialdehyde (MDA) levels, as well

as reducing the level of superoxide dismutase, gluthathione peroxidase and catalase.

1.5 Significance of Study

This research study provided scientific knowledge regarding how aluminum and cadmium

affects the brain, hence causing neurodegeneration.

14
1.6 Justification of Study

Risk for Alzheimer's disease and related dementias is partly attributed to environmental

factors. These environmental risk factors play a key role in accelerating or decelerating

disease onset and progression. Among known environmental risk factors, chronic exposure

to various metals has become more common among the public as the pollutants from human

activities releases excess amount of metals into the environment. As a result, we are exposed

not only to essential metals, such as iron, copper, zinc and manganese, but also to toxic

metals including lead, aluminum, and cadmium, which perturb metal homeostasis at the

cellular and organismal levels. Herein, we review how aluminum and cadmium affects brain

physiology and immunity, as well as their roles in Alzhiemer’s disease.

15
 CHAPTER TWO

LITERATURE REVIEW

2.1 What Is Neurodegeneration?

Neurodegeneration is the progressive loss of structure or function of neurons, which may

ultimately involve cell death. Many neurodegenerative diseases—such as amyotrophic

lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's

disease, and prion diseases—occur as a result of neurodegenerative processes.

Neurodegeneration can be found in the brain at many different levels of neuronal circuitry,

ranging from molecular to systemic. Because there is no known way to reverse the

progressive degeneration of neurons, these diseases are considered to be incurable

(Biomedical research has revealed many similarities between these diseases at the sub-

cellular level, including atypical protein assemblies (like proteopathy) and induced cell

death (Rubinsztein, 2006).These similarities suggest that therapeutic advances against one

neurodegenerative disease might ameliorate other diseases as well (Bredesen et al, 2006).

Biometal microelements, such as copper, aluminum, iron, manganese, zinc and cadmium,

are crucial for many physiological functions, especially in the CNS. Shifts in the amounts of

these metals are essential for the development and maintenance of numerous enzymatic

activities, mitochondrial functions, neurotransmission, and also for memorization and

16
learning (Menon et al, 2016). However, with deregulations in their homeostasis, particularly

in those connected with redox activity, there are consequent changes in the ion and

microelement balance. This redox activity may contribute to the production of free radicals

that can react with various organic substrates, thus generating increased levels of oxidative

stress. There is growing evidence that metal microelements play significant roles in the

pathogenesis of neurodegenerative diseases. The interaction between metals and CNS

proteins is crucial in the development or absence of neurodegeneration. In this way,

homeostasis of metal microelements represents a mechanism of extreme importance

(Brown, 2005). 

2.2 Alzheimer’s Disease

Alzheimer's disease (AD) is the most common neurodegenerative disease that causes

dementia in the elderly. It is characterized by the gradual deterioration of memory and other

cognitive functions, which eventually leads to a complete incapacity and death of the

patients within 3 to 9 years after diagnosis (Livingston et al, 2017). Increasing age is a major

risk factor for sporadic forms of AD. As the elderly population of the world continues to

increase, the prevalence of AD has increased remarkably worldwide, and AD has become

one of the leading causes of disability and death among the elderly (Plummer et al, 2016).

Despite the tremendous progress that has been made in AD research in the past few decades,

the exact cause and pathogenesis of AD are not completely understood, and currently, there
17
is no effective treatment for the disease. The major pathological characteristics of AD brains

are the presence of senile plaques, neurofibrillary tangles (NFTs), and neuronal loss (Lee

and Tsai, 2003). Senile plaques are mainly composed of beta-amyloid peptide (Abeta) that is

produced from proteolytic cleavage of the transmembrane amyloid precursor protein (APP).

NFTs are formed by arrays of paired helical filaments (PHFs) structures, which contain

mainly self-aggregated hyperphosphorylated tau, a multifunctional protein involved in

microtubule assembly and stabilization (Lee and Tsai, 2003). Accumulating evidence has

shown that the presence of extensive oxidative stress is a characteristic of AD brains in

addition to the established pathology of senile plaques and NFT (Zheng et al, 2002). It has

been demonstrated that the levels of protein carbonyls and 3-nitrotyrosine, which are

resulted from protein oxidation, and markers of oxidative damage to DNA and RNA, such

as 8-hydroxydeoxyguanosine (8-OHdG) and 8-hydroxyguanosine, are elevated in AD brains

(Ballatoreet al, 2007). Products of lipid peroxidation, such as malondialdehyde (MDA), 4-

hydroxynonenal, and F2-isoprostanes, are also increased in multiple brain regions and

cerebrospinal fluid (CSF) of patients with AD or mild cognitive impairment (MCI) (Zhao et

al, 2007). In addition to the accumulation of free radical damage, alterations in the activities

or expression of antioxidant enzymes such as superoxide dismutase (SOD) and catalase have

been observed in both central nervous system and peripheral tissues of AD patients (Asaiet

al, 2015). Moreover, in AD and MCI brains, the increased oxidative damage to lipids and

proteins and the decline of glutathione and antioxidant enzyme activities are more localized

18
to the synapses and correlate with the severity of the disease, suggesting an involvement of

oxidative stress in AD-related synaptic loss (Njieet al, 2012).

2.3 Role of Metals in Neurodegenerative Alzheimer’s Disease

Metals play a major catalytic role in the production of free radicals, and attention has

centered on the role of many metals in Alzheimer’s disease, including iron, aluminum,

mercury, copper, zinc and cadmium. But for the purpose of this study, we will discuss how

aluminium and cadmium affects the brain, and their contribution to AD and the

dysregulation of the immune system.

2.3.1 Aluminium

Alumnum is not essential for life but is a well established neurotoxin. Exposure to high

aluminum content in drinking water causes lifelong cerebral impairments, such as loss of

concentration and short term memory deficits (Altman et al, 2000). Mass spectrometry

studies have demonstrated that aluminum crosses the blood–brain barrier and accumulates in

a semi permanent manner (Priest, 2004). Although no biological process is dependent on

aluminum, it can influence more than 200 biologically relevant reactions and cause various

adverse effects on the mammalian brain. These include essential brain processes such as

axonal transport, neurotransmitter synthesis, synaptic transmission, phosphorylation or

dephosphorylation of proteins, protein degradation, gene expression, and inflammatory

19
responses (Kawahara and Kato-Negishi, 2011). Aluminum exhibits one oxidation state, Al 3+,

which has affinity for negatively charged oxygen-donor ligands. Some of the ligands that

form strong bonds with aluminum are inorganic and organic phosphates, carboxylate and

deprotonated hydroxyl groups, thereby making DNA, RNA and ATP perfect targets,

affecting gene expression, energy metabolism and the action of several kinases and

phosphatases (Singla and Dhawan, 2012). Aluminum can also cause the oligomerization of

proteins, inducing conformational changes that can inhibit their degradation by proteases,

and thus affect their turnover. For instance, strong binding of aluminum to phosphorylated

amino acids promotes the self-aggregation and accumulation of highly phosphorylated

cytoskeleton proteins, such as neurofilament and microtubule-associated proteins (Muma

and Singer, 1966). These properties make the presence of aluminum in the brain toxic,

causing the apoptotic death of neurons and glial cells. Aluminum affects LTP, the function

of enzymes, including those involved in neurotransmitter synthesis (Colomina et al, 2002).

It also affects voltage-gated calcium channels and neurotransmitter receptors, impairing

synaptic transmission (Walton, 2012). The presence of aluminum therefore leads to a

signaling imbalance that disturbs brain function.

More strikingly, exposure to aluminum is also suspected of being linked to

neurodegeneration (Kajiet al, 2012). Several studies reported a higher incidence of AD or

AD mortality in areas with high levels of aluminum in the drinking water, suggesting a

20
strong association between aluminum and AAD (Flaten, 2001). This was confirmed by later

studies that demonstrated the ability of aluminum to induce neurofibrillary degeneration and

promote the appearance of tangle-like structures that resembled the NFTs found in the brains

of AD patients (Kihira et al, 2002). Moreover, aluminum accumulation was described in

NFT-bearing neurons of AD brains (Bouraset al, 1997). When the effects of oral aluminum

administration were studied using a tau mouse model showing slow progressive tau

accumulation, higher tau aggregation, apoptosis and neurological dysfunction were observed

in animals that already had a pathological process causing tau aggregation, but not in the

controls (Oshimaet al, 2013), thereby suggesting an exacerbating effect of aluminum on tau

pathology. Aluminum achieves these effects by enhancing the activity of the tau kinases

CDK5 and GSK-3β, inhibiting the dephosphorylation of tau, and enhancing its aggregation

(Huang et al, 2017). Interestingly, aluminum is preferentially taken up by glial cells, which

induces the production of inflammatory cytokines, including IL-6 (Akiyama et al, 2012). IL-

6 in turn has been reported to induce phosphorylation of tau by dysregulating the CDK5/p35

cascade (Quintanlaet al, 2004). Increased glial activation and an inflammatory response

have been described upon aluminum treatment in rats (Akinrinadeet al, 2015).However,

whether glial activation due to aluminum exposure plays a role in the acceleration of an

early process in the formation of AD pathology needs to be studied further.

21
Although the effects of aluminum on AD pathology were first attributed to its interaction

with tau, it was later demonstrated that it also affects Aβ by promoting its production,

aggregation and by inhibiting its degradation (Luo et al, 2009). Oral administration of

aluminum to AD mice induced an increase in the amount of Aβ, both in its secreted and

accumulated forms, and increased deposition in plaques (Praticoet al, 2002). In addition, Aβ

coupled with aluminum is more toxic than Aβ itself as it causes membrane disruption and

perturbation of neural calcium homeostasis and mitochondrial respiration (Dragoet al,

2008). Aluminum can also influence the expression of iron-binding proteins expression with

IRE/IRP sequences in their mRNA, causing an increase in iron concentration (Cho et al,

2010). The presence of aluminum in the brain can therefore modulate the expression,

distribution and accumulation of APP and induce the dysregulation of iron modulated

signaling pathways, by interacting with IRE mRNA regions [306,307]. Consequently,

aluminum stimulates iron-induced membrane lipid peroxidation and causes oxidative

damage (Kim and Olivi, 2007). Despite its non-redox status, several studies have suggested

that aluminum has strong oxidative activity (Yuan et al, 2012). The interaction of aluminum

with iron generates labile iron from iron-containing enzymes and proteins, thereby

increasing the intracellular pool of free iron, which in turn leads to the formation of ROS

(Wu et al, 2012). Aluminum also decreases the activity of some antioxidant enzymes such

as catalase, superoxide dismutase and glutathione peroxidase, thus exacerbating the neuronal

22
damage induced by oxidative stress in neurodegenerative disease such as AD (Fattoretti,

2003).

Aluminum has also been reported to affect neurotransmission. Due to its ability to block Aβ-

mediated formation of calcium permeable ion channel, aluminum can inhibit the increase in

calcium levels induced by neurotrophic factors such as brain-derived neurotrophic factor

(Kawahara, 2010). The level of other neurotransmitters, such as serotonin, dopamine,

glutamate and aspartate, has also been reported toaffect neurotransmission. Due to its ability

to block Aβ-mediated formation of calcium permeable ion channel, aluminum can inhibit

the increase in calcium levels induced by neurotrophic factors such as brain-derived

neurotrophic factor (Kawahara, 2010). The level of other neurotransmitters, such as

serotonin, dopamine, glutamate and aspartate, has also been reported to decrease upon

aluminum exposure (Ajaremet al, 2012). A lower availability of glutamate induced by

aluminum has been attributed to the induction of glutamine synthetase and inhibition of

glutaminase activity in astrocytes (Liu et al,2010).

2.3.2 Cadmium

Cadmium is a carcinogenic heavy metal that is present in the environment. Unlike many

heavy metals, due to its water-soluble property, cadmium can be transported from soil to

plants and concentrated in the food chain (Qadiret al, 2014). Although the effect of

23
cadmium on the plant can be detrimental, some plants, such as tobacco, show cadmium

tolerance (Zhang et al, 2016). Therefore, consumption of tobacco products or inhalation of

tobacco smoke increases the risk of cadmium-related morbidities in the general population

(Richter et al, 2017). Once taken into the body, cadmium accumulates in the kidney and

liver and has an extremely long half-life of 20–40 years (Franssonet al, 2014). Chronic

cadmium exposure is associated with hypertension, kidney dysfunction, bone

demineralization and neurological diseases (Akesonet al, 2006). Cadmium is known to cross

the blood–brain barrier and eventually accumulate in the brain, leading to neurotoxicity

(Wang and Du, 2013). In the brain, cadmium induces activation of various signaling

pathways involved in inflammation, oxidative stress and neuronal apoptosis (Chen and Liu,

2008).

Recent epidemiological studies reported that blood cadmium levels were significantly

associated with AD related mortality among older adults (Min, 2016). In the AD brain, there

is increasing evidence that cadmium is involved in the aggregation of Aβ plaques (Li et al,

2012). In an in vivo study, APP/PS1 mice administered cadmium in their drinking water

exhibited an increase in the number and size of plaques (Li et al, 2012). Cadmium ions can

interact with the Aβ, subsequently promoting formation of plaques (Carlo et al, 2014).

Furthermore, it has been hypothesized that cadmium treatment downregulates the expression

of α-secretase (ADAM10) and neutral endopeptidase, which play essential roles in reducing

24
Aβ levels in the brain (Endres and Fahrenhoz, 2012). Interestingly, a recent study has

reported that the synergistic effects of cadmium, lead and arsenic further enhance

amyloidogenic processing by increasing APP, BACE1 and PSEN1 expression, suggesting

an interactive effect of cadmium with other metals in AD (Pinoet al, 2016). In addition to its

effects on Aβ, cadmium is alsoinvolved in the conformation and self-aggregation of tau in

the AD brain (Jiang et al, 2007). Cadmium has been reported to bind to the third repeat (R3)

of the microtubule-binding domain of tau. As a result, the R3 domain partially loses its

random coil conformation and gains an αhelix structure which promotes the self-aggregation

of tau. Moreover, cadmium treatment selectively blocks muscarinic M 1 receptors, which are

known to regulate GSK-3β negatively and subsequently increase both total and

phosphorylated tau protein (Medeiros, 2011). These data support the notion that cadmium is

one factor that could be involved in the development of AD. With regard to immunity,

human astrocytes treated with a non-toxic concentration of cadmium have been shown to

release an elevated level of IL-6 and IL-8 via activation of the mitogen-activated protein

kinase and NF-κB signaling pathways, possibly leading to neuroinflammation and neuronal

death (Power et al, 2017). Notably, it has been reported that increased IL-6 and IL-8

expression are associated with AD ppathogenesis (Liu et al, 2014). Moreover, cadmium has

been shown to induce astrocyte cytotoxicity by increasing intracellular calcium ions via the

mitogen-activated protein kinase and PI3K/AKT signaling pathways (Jiang et al, 2015).

25
2.4 Role of Diet in Alzheimer’s Disease

The prevalence of Alzheimer’s disease (AD) increases exponentially with age but there is

limited knowledge of the modifiable risk factors for AD. However, there is growing

evidence for possible dietary risk factors in the development of AD and cognitive decline

with age, such as antioxidant nutrients, fish, dietary fats, and B-vitamins. Numerous animal

and laboratory studies have shown that antioxidant nutrients can protect the brain from

oxidative and inflammatory damage, but there are limited data available from

epidemiological studies. There is more substantial epidemiological evidence from a number

of recent studies that demonstrate a protective role of omega-3 fatty acids, such as

docosahexaenoic acid, in AD and cognitive decline. This review will focus on

epidemiological evidence investigating the relationship between nutrition and AD, focusing

particularly on the roles of dietary fats and antioxidants.

2.4.1 Polyunsaturated Fats

Numerous studies have investigated the effects of polyunsaturated fatty acids (PUFAs) in

preventing and/or slowing AD. The potential PUFA dietary intervention to prevent neuronal

loss and cognitive decline stems from evidence that PUFAs are critical components of

neuronal cell membranes, maintaining membrane fluidity, which is essential for synaptic

vesicle fusion and neurotransmitter communication within neural networks. The n-3 long

26
chain PUFAs (n-3 LCPUFAs), which mainly include omega-3, docosahexanoic acid (DHA),

and eicosapentaenoic acid (EPA), regulate neuronal membrane excitability and improve the

capacity for neuronal transmission in healthy subjects, thus enhancing learning and mmemor

(Vauzouret al, 2015). Furthermore, DHA, whose high levels in brain indicate its essential

role in this organ, is also involved in mood and emotional state, locomotor and exploratory

activities, and cognitive functions (Joffre et al, 2014).In addition, n-3 LCPUFAs modulate

the inflammatory processes by acting at the immune system level in many different ways

through the regulation of cytokines and chemokines expression, the decrease of

prostaglandins and eicosanoids, andthe induction of proresolutive factors, resolvins, and

protectins that are involved in the resolution of (Calder, 2015). EPA, DHA, and their

bioactive mediators exert their anti-inflammatory effects not only in the periphery (Serhan

and Chiang, 2013) but also at the brain level (Bazinet and Laye, 2014).

Numerous observational studies have highlighted a possible association between dietary

intake of fish and n-3 LCPUFA and a lower risk of dementia, including AAD (Samieriet

al,2011). On the other hand, it has to be stressed that studies finding limited or no clinical

benefit of PUFAs on cognitive improvement in AD patients were also rreported(Stoughet al,

2012). For example, it has been demonstrated in a double-blind placebo-controlled study

that omega-3 monotherapy improved cognitive performance only in Mild Cognitive

Impairment (MCI) patients but not in AD (Chiu et al, 2008). The reasons why no effect of

27
omega-3 treatment was observed in patients with moderate or advanced AD could be due to

the relatively short duration of the supplementation, the daily dose used, the source and the

origin (fish versus vegetable oil) of n-3 LCPUFA, the dietary history of the patients, and the

cognitive function assessed (Dangour and Allen, 2013). Therefore, a meta-analysis study

was done on the effects of long-term omega-3 supplementation in AD animal models,

confirming its well-recognised effect in restoring cognitive performance. In particular, long-

term omega-3 supplementation decreased omega-6/omega-3 ratio, reduced the amount of

beta-amyloid, prevented neuronal loss, and improved cognitive function in AD animal

models (Hooijmanset al, 2012). Furthermore, the effects of DHA in reducing Aβ production

in vitro study and AD animal models have been also widely demonstrated (Hooijmanset al,

2007). The mechanism involved in the DHA-induced reduction in Aβ may be due to

multiple effects: changing in lipid raft structure, alterations in APP processing, and

induction of antiamyloidogenic chaperones for APP (Stillwell et al, 2005).

2.4.2 Vitamins

Vitamins are potent antioxidants. Their potentiality in maintaining healthy cognition and

preventing cognitive decline rises by the fact that the brain is particularly susceptible to

oxidative stress damage. The brain is a major metaboliser of oxygen, accounting for 20% of

the body’s consumption, and has relatively feeble protective antioxidant mechanisms. In

addition, it contains a large amount of polyunsaturated peroxidisable fatty acids, along with
28
high levels of iron that act as a prooxidant (Zandi et al, 2004). A free radical-enriched

environment in the brain contributes to the progressive decline of cognitive abilities,

exacerbating dementia. Vitamin E has been intensively investigated for its role in protecting

membrane phospholipids against peroxidation. It was demonstrated that the use of vitamin E

and vitamin C supplements in combination with food is associated with reduced prevalence

and incidence of AD. A multicentre clinical trial on vitamin E supplementation for patients

with moderate AD demonstrated that vitamin E slowed disease progression, thereby

reducing the risk of institutionalization (Dyskenet al, 2014). This is probably due to the

different compositions of vitamin E supplements, which often differ from the form of

vitamin E found in the diet. Vitamin E refers to a group of fat-soluble compounds that

include eight chemical forms. Among them, γ-tocopherol and α-tocopherol are the most

abundant in the diet, and α-tocopherol is also the one that exerts antioxidant properties

(Farina et al, 2012). It has also been demonstrated that δ-tocopherol, but not α-tocopherol,

increased the level of Aβ by enhancing its production and decreasing its degradation,

worsening the pathology. Another critical issue to solve is the optimal dose of vitamin E

required to prevent or delay AD (Grimm et al, 2015).Low vitamin D concentration is also a

risk factor for developing AD, because its concentrations have been found inversely

correlated with its risk (Grant, 2016).

29
Other vitamins, including vitamin A and the complex of vitamin B, were found lower in

plasma/serum of geriatric patients with cognitive iimpairment (Raszewskiet al, 2016). For

their role in homocysteine metabolism, the three B vitamins (B6, B12, and folic acid) have

been correlated with age-related cognitive fragility (Eagappan and Sasikumar, 2015).

Previous epidemiological studies on vitamin B and cognitive status found that older people

with elevated homocysteine levels (hyperhomocysteinaemia) tend to have lower vitamin B

status, as well as lower cognitive tests scores (Duthieet al, 2002). Possible correlations

between vitamin A and Alzheimer’s disease were reported in in vitro studies, demonstrating

an anti-beta-amyloid oligomerization effect of vitamin A and beta-carotene (Takasakiet al,

2011).

2.4.3 Polyphenols

The beneficial role of dietary polyphenols has been suggested as potential functional

food candidates to prevent memory decline (Choi et al, 2012). Polyphenols are natural

substances present in plants, fruits, and vegetables. Some polyphenols, such as

epigallocatechin-3-gallate (EGCG) found in green tea, 4-O-methyl honokiol found in

Magnolia officinalis, resveratrol contained in grapes, and ginkgolide A found in ginkgo

biloba, have been suggested to provide protection against AD. Their effects may be due to

their antioxidant and anti-inflammatory properties, but also by their modulation of enzyme

30
activity and regulation of intracellular signalling pathways and gene expression

(Obrenovichet al, 2010).

In fact, polyphenols, especially flavonoids, can also modulate those neuronal

signalling cascades altered with ageing by acting on ERK/CREB pathway involved in

synaptic plasticity and long-term potentiation, improving learning and memory in both

animals and human (Kean et al, 2015). Flavonoid supplementations can modulate specific

signalling kinases like CaMKII and ERK, controlling the activation of CREB and the

increased expression of BDNF and NGF at the brain level (Rendeiroet al, 2014). In fact,

these compounds also exert a protective function in the hippocampus of middle age mice

preserving and promoting the spatial learning strategies. Recently, it was also demonstrated

that a polyphenol-rich extract from grape and blueberry (PEGB), with high contents of

flavonoids, can facilitate the use of spatial strategies in both adult and middle-aged mice

(Bensalem et al, 2016). In these animals PEGB supplementation was able to improve

learning performance by restoring CaMKII mRNA levels and increasing NGF expression

exactly in the hippocampus. It is noteworthy that this is the first nutritional intervention that,

even if with a mix of different polyphenols at low doses, shows a rescue effect on those

specific memory deficits (Bensalem et al, 2016).

Curcumin also has a potential role in the prevention and treatment of AD. The

biophenoliccurcumin, isolated as the active yellow component of Curcuma longa, has a long
31
history of use in traditional Asian medicines for its potent anti-inflammatory, antioxidant,

and anticancer activities (Aggarwal and Harikumar, 2009). In AD animal models, curcumin

reduced proinflammatory cytokines, oxidative damage, and beta-amyloid production,

ameliorating cognitive deficits (Frautschy et al, 2001). It was also demonstrated that

macrophages derived from AD patients treated with curcumin showed an improved uptake

of beta-amyloid when compared with untreated cells (Zhang et al, 2006). In addition,

curcumin exerted an antiproliferative action on microglial cells preventing cytokine release.

Curcumin decreases the lipoprotein oxidation and the free radicals formation in AD and in

other neurodegenerative disorders (Kim et al, 2015). Because of its lipophilic nature,

curcumin crossed the blood-brain barrier and reduced existing senile plaques, as

demonstrated in APPswe/PS1dE9 mice (Borrelliet al, 2007). Curcumin reduces senile

plaques by binding with the Aβ oligomers, destabilising them and preventing their extension

(Ono et al, 2004).

2.5 Oxidative Stress Hypothesis

Reactive oxygen species (ROS) are highly active moieties, some of which are direct

oxidants, and some have oxygen or oxygen-like electronegative elements produced within

the cell during cellular metabolism or under pathological conditions. Some of the reactive

species are free radicals such as the hydroxyl radical and the superoxide radical, and some

are nonradicals such as hydrogen peroxide. Free radicals are any independent species which
32
consist of one or more unpaired electrons in their atomic or molecular orbital. They are

generally unstable, short lived, but usually chemically reactive. They can react with any

molecule either by oxidizing it or by causing any other kind of chemical modification. Free

radicals can potentially oxidize all cellular biomolecules including nucleic acids, proteins,

and lipids. For example, peroxidation of omega-6 polyunsaturated fatty acid (such as

arachidonic acid and linoleic acid) leads to the production of 4-hydroxynonenal (HNE),

which is one of the main reactive aldehydes produced by oxidative stress (Uchida, 2003).

These free radicals are formed in the cell during normal cellular metabolism as

mitochondrial electron transport chain, β-oxidation of fatty acids, and cytochrome P450-

mediated reactions and by the respiratory burst during immune defense. For example,

autooxidation of some biologically important substances such as FADH 2 and

tetrahydropteridines can yield O2⋅– in the presence of oxygen (Davies et al, 2008). The

imbalance between production and quenching of these reactive substances through

antioxidant mechanisms causes oxidative stress. The loss of functionality and adaptability of

important biomolecules due to oxidative stress are two interdependent biological processes,

which are among the important factors that mediate aging. The free radical hypothesis, also

known as oxidative stress hypothesis, is one of the strongly supported theories which can

define the causes behind the aging process (Davis et al,2008).Oxidative stress has been

implicated in many metabolic and neurologic degenerative disorders. Degenerative diseases,

where the function and structure of a tissue or organs deteriorate over time such as in

33
Alzheimer’s disease, Parkinson’s disease, diabetes, cataracts, cancer, and cardiovascular

disease, have been attributed to oxidative stress conditions and the process of natural aging.

Thus, oxidative stress, aging, and degenerative diseases are interconnected.

2.6 Antioxidants

Antioxidants are compounds that inhibit oxidation, a chemical reaction that can

produce free radicals and chain reactions that may damage the cells of organisms.

Antioxidants such as thiols or ascorbic acid (vitamin C) may act to inhibit these reactions.

To balance oxidative stress, plants and animals maintain complex systems of overlapping

antioxidants, such as glutathione (Dabelsteinet al,2007). Antioxidants decrease free-radical-

mediated damage in neuronal cells through detoxification, and if the balance between

antioxidants/oxidants are unbalanced unfavorably, there can be detrimental effects,

especially in the AD brain (Moneim, 2015).The only dietary antioxidants are vitamins A, C,

and E.

2.7 α-Tocopherol

Vitamin E is a natural, highly tolerable and cost effective molecule. This generic term is

used for tocopherol and tocotrienols consisting of two rings with a hydrocarbon chain. Both

structures are similar, although the tocotrienol structure has double bonds on the isoprenoid

units. Natural vitamin Es are known as α, β, γ, and δ according to the methyl or proton

34
groups that are bound to their Benzene rings, and the most common and biologically active

form is alpha-tocopherol (Brigelius-FloheandTraber, 2009). When produced synthetically, it

is composed of eight stereoisomers in which RRR-α-tocopherol is the most biologically

active form (Siesand Murphy, 2001).

However,it was confirmed in previous studies regarding the neuroprotective effects for α-

tocopherol against ischemic brain damage (Hsiao et al, 2007). It was demonstrated that

vitamin E deficiency caused a greater neurological deficit and infarct volume (Stohreret al,

2008) while exogenous administration of α- tocopherol reduced ischemic reperfusion-

induced neuronal damage (Chaudhary et al, 2013).

Oxidative stress caused by the excessive production of free radicals and overwhelming of

brain antioxidant capacity has been shown to be involved in ischemic brain damage (Wang

et al, 2015). Compared to other tissues, the brain is more sensitive to oxidative damage

because it has a high rate of metabolism, high lipid content and relative low protective

antioxidant systems (Awad, 2011). Also, treatment with α-tocopherol produced protective

effects against oxidative damage of the brain. This protection was partly achieved by a

reduction of lipid peroxidation and augmentation of brain antioxidant capacity via increased

GSH. Previous studies showed that exogenous administration of α-tocopherol attenuated

raised levels of MDA after brain ischemia (Tovmasyanet al, 2013). Additionally, α-

tocopherol acts as an acyl-peroxyl-radical scavenger and inhibits superoxide anion

35
production by leukocytes via reducing NADPH oxidase activity (Cachiaet al, 2008). Thus,

protective effects of α-tocopherol against ischemic brain injury might be partly attributed to

antioxidant actions. Beside the protective effects of α-tocopherol against lipid peroxidation

are its nonantioxidant properties, such as the preservation of endothelial integrity, inhibition

of monocyte-endothelial adhesion, reduction of cytokine release and platelet aggregation

( Van et al, 2004).

While the recommended daily allowance (RDA) for vitamin E is 8 mg (12 IU) for females

and 10 mg (15 IU) for males, it is recommended that up to 1,000–1,200 IU intake of vitamin

E in some pathologies including cataract (Packer, 2011). The principal reserve of natural

vitamin E is vegetable oil where its function is to protect tissue from oxidative damage. It is

a liposoluble molecule, and, therefore, after dietary intake, vitamin E is not only absorbed

easily from the intestinal lumen but is also dispersed between lipids and proteins in cell

membranes. Vitamin E molecules can interrupt free radical chain reactions by capturing the

free radical. This imparts to them their antioxidant properties. The free hydroxyl group on

the aromatic ring is responsible for the antioxidant properties. The hydrogen from this group

is donated to the free radical, resulting in a relatively stable free radical form of vitamin E

(Packer, 2011).

Regarding the pharmacodynamics of tocopherols, it has been reported in a study conducted

in human eyes that the retinal levels of vitamin E are higher than those of the choroid or
36
vitreous and is correlated with serum levels of vitamin E (Bhat, 2006). It is known that

vitamin E can only reach its therapeutic levels in aqueous humor and lens via topical

application and is accumulated within the retina when applied via the oral or parenteral route

(Nagata et al,2012). Moreover, it is reported in animal studies that when 100 mg/kg α-

tocopherol is applied via oral or parenteral route, it causes a similar threefold to sixfold

increase to its serum levels, though the retinal and vitreal increases are somewhat slower via

the oral route (Bhat, 2007).

2.8 Catalase

A catalase is one of the most important antioxidant enzymes. It is present in almost all

aerobic organisms. Catalase breaks down two hydrogen peroxide molecules into one

molecule of oxygen and two molecules of water in a two-step reaction (Halliwell and

Gutteridge, 2009). The first step of the reaction mechanism involves formation of a

spectroscopically distinct intermediate compound I which is a covalent oxyferryl species

(FeIVO) having a porphyrin π-cation radical, through the reduction of one hydrogen peroxide

molecule (Ivancichet al, 2007). In the second step reaction, compound I is reduced through

redox reactions by a two-electron transfer from an electron donor (the second molecule of

hydrogen peroxide) to produce the free enzyme, oxygen, and water (Halliwell and

Gutteridge, 2009).

37
Catalase deficiency or malfunctioning is associated with many diseases such as diabetes

mellitus, vitiligo, cardiovascular diseases, Wilson disease, hypertension, anemia, some

dermatological disorders, Alzheimer’s disease, bipolar disorder, and schizophrenia (Habib et

al, 2010). It has been reported that an anomaly of catalase activity is inherited in

acatalasemia which is a rare genetic disorder (also known as Takahara disease) [27]. It is an

autosomal recessive trait and characterized by a reduced level of catalase. Catalase has a

prime role in regulating the cellular level of hydrogen pperoxide(Muelleret al, 2007) and its

hydrogen peroxide catabolism protects the cells from oxidative assault, for example, by

securing the pancreatic β cells from hydrogen peroxide iinjury(Tiedge, 2008). Low catalase

activities have been reported in schizophrenic patients such as also in patients with

atherosclerosis (Vitai, 2006).

38
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Materials

 Animals

 Plates

 Test tubes

 Beaker

 Plastic cages

 Canula

 Syringes

 Laboratory blender

 Sterile plain container

 Methylated spirit

 Bowl

 Cotton wool

 Foil paper

 Latex gloves

39
  Animals feeds( grown mash)

 Masking tape

 Conical flasks

 Detergents and hypo

 Dissecting kits

 Pipette tips and micropipette

 Eppendorf tube(1.5ml)

 Sterile universal container

3.1.1 Instrument

 Water bath

 High speed refrigerated centrifuge (TGL-16 table top)

 Electronic weighing balance

 Refrigerator-freezer type one (Model-GCS172SV)

 UV-Vis Spectrophotometer (GENESYS 10S)

3.1.2 Reagents and Chemicals

All reagents used for this study were of analytical grade.

40
  Stock TCA-TBA-HCl reagent

 Hydrogen peroxide (H2O2)

 Distilled water

 0.25M HCl

 30mM hydrogen peroxide

 0.05M Phosphate buffer pH 7.4

 15% w/v trichloroacetic acid,

 0.375%w/v thiobarbituric acid

3.2 Methods

3.2.1 Collection of Animals

Healthy male swiss albino mice weighing between 20g-30g were used as the experimental

animals for this study and were purchased from the animal house of the college of Medicine,

Delta State University, Abraka. They were allowed to acclimatize to laboratory condition for

two weeks.

3.2.2 Animal care and handling

The animals were maintained according to the approved animal core guideline of the Animal

Ethics Committee of the Delta State University, Abraka, Nigeria. The animals were housed
41
in plastic cages, under controlled condition of 12hr light/ 12hr dark cycles respectively, and

were fed on growers mash obtained from Top-Feeds Flour mill, Sapele, Delta State, and

were given clean water ad libitum.

Table 1: Experimental Design

Groups Treatment Al Cd α-TP α-TP Drug

(25mg/Kg) (50mg/Kg)

Group 1 Normal Saline - - - - -

Group 2 Al 1.25mg/Kg + - - - -

(IP)
AlCl.6H2O

Group 3 Cd (0.5mg/Kg) - + - - -

(IP)
CdCl.H2O

Group 4 Al-Cd + + - - -

Group 5 AL-CD-α-TP + + + - -

25mg/Kg (IP)
(α-tocopherol)

Group 6 AL-CD- α-TP + + - + -

42
 (α-tocopherol) 50mg/Kg (IP)

Key:+: Present; -: Absent

3.2.3 AnimalMobilization

Adult 48 male mice were purchased from the animal house of the college of Medicine, Delta

State University of Abraka. They were allowed to acclimate for two weeks and randomly

shared into six groups of eight animals each at the end of the acclimatization period and

treated according to the Table 3.1. Animals in group 1 served as normal control while

animals in group 2 received only aluminum in form of Aluminum chloride hexahydrate.

Group 3 animals received only cadmium in form of cadmium chloride monohydrate while

group 4 received a combination of aluminum and cadmium without treatment, groups 5 and

6 received a combination of cadmium and aluminum alongside treatment with 25mg/kg and

50mg/Kg body weight of alpha tocopherol respectively. Aluminum and cadmium were

administered interperitonially at a dose of 1.25mg/kg and 0.5mg/Kg respectively at intervals

of 30minutes per toxicant. Alpha-tocopherol was also administered at an interval of 30

minutes following the administration of the last toxicant. Treatment was done for 28 days.

3.2.4 Animal Sacrificing and Collection of Specimen

43
At the end of the study period (28 days), the animals were fasted overnight and were

sacrificed via cervical dislocation. The brain was immediately excised and the hippocampus

and prefrontal cortex separated into differently labelled sterile tubes and homogenized in

5ml ice cold normal saline (0.9% NaCl) solution (to maintain the integrity of the brain

tissue) using a mechanical/laboratory blender. The homogenates were transferred into

different specimen tubes and were centrifuged at 10,000rpm for 10minutes for proper

separation, the supernatants were transferred into sterile eppendorf tube (1.5ml) amd kept

frozen for 48hrs before biochemical analysis.

3.3 Biochemical Analysis

3.3.1 Determination of Lipid Peroxidation

A breakdown product of lipid peroxidation thiobarbitoric acid reactive substance (TBARS)

was measured in the tissue homogenates by the method of Gutteridge and Wilkins (1982).

Principle

Malondialdehyde (MDA) formed from the breakdown of polyunsaturated fatty acid (PUFA)

served as a convenient index for the determination of the extent of peroxidation reaction.

Malondialdehyde was identified with the barbituric acid to give a red species which was

assayed at 532nm.

44
45
Procedure

Into 2ml of glacial acetic acid was added 0.2ml of sample followed by 2ml 1% TBA in

0.05M NaOH. The loosely stopped tubes was immersed in boiling water bath for 15

minutes, allowed to cool and centrifuged at 800rpm for 15 minutes. The clear supernatant

was carefully transferred into a cuvette and absorbance read at 532nm against a reagent

blank. A molar extinction coefficient of 1.56 x 105 m-1cm-1 was used according to the

expression of Adamand Seregi (1982).

3.3.2 Assay for Catalase Activity

The activity of catalase was determined in the tissue homogenates by the method of Cohen

et al. (1972).

Principle

In the assay, excess potassium permanganate is added and then residual unreacted

permangantate is measured spectrophotometrically. It has been shown that the

decomposition of hydrogen peroxide by catalase follows first order kinetics (Haber and

Weiss, 1934).

46
Procedure

This was carried out by pipetting 1.0ml phosphate buffer into a reference cuvette. Then,

2.0ml of sample was added into the reference cuvette and test cuvette respectively.

Enzymatic reaction was initiated by adding 1.0ml of cold 10mM H2O into the test cuvette

and mixing thoroughly. To stop the reaction, 7 ml of 0.1NKMnO4 was added within 30s and

thoroughly mixed. The spectrophotometer standard was prepared by adding 7 ml of 0.1 N

KMnO4 to a mixture of 5.5 ml of 0.05 N phosphate buffer, pH 7 and 1 ml of 6 N H2SO4.

The reaction was carried out in an ice-water bath (2O°C) and after exactly 3 minutes, the

substrate concentration was measured at 240nm.

3.4 Statistical Analysis

The results obtained were expressed in mean ± SD for n = 8 mice/group. The data were

evaluated using a one way analysis of variance (ANOVA) and results were considered

statistically significant at p<0.05. SPSS statistical package version 23 was used for data

analysis.

47
 CHAPTER FOUR

RESULTS

The results obtained from the investigation of the effect of aluminium and cadmium
coexposure on lipid-peroxidation in the prefrontal cortex of mice are presented in Table 4.1

Table 2: Effect cadmium and aluminum on MDA and Catalase activities in the
prefrontal cortex of mice

Group MDA CAT

A 0.75±0.15a 17.15±1.08a

B 0.98±0.18 a 15.02±1.32a

C 1.47±0.28b 13.09±0.70ab

D 2.46±0.17c 12.10±0.21b

All values are expressed as Mean ± SD values followed by different alphabet superscripts on
the same column indicates that there is a significant difference (p<0.05)

Key: A: Control; B: AL only; C: Cd Only; D Al-Cd

Table 2 shown above indicates that there is a significant rise in lipid peroxidation levels in
the prefrontal cortexof mice treated with AL-only and Cd-only compared to control. Co-
treatment with AL-Cd also induced a rise in MDA relative to control and single metal
treatments. The level of catalase also far more reduced in mice treated with both Al and Cd
together than those treated with single metals.
48
 CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion

The toxicity of metals is believed to manifest itself mainly through the formation of ROS or

free radicals in the cells of living organisms or through the inhibition of the enzymes that are

a part of the antioxidant system. These metals can occur naturally and persist in the

environment. Cadmium is a non-essential metals that serve no required biological purpose in

the human body and serious adverse effects are observed with increasing exposure levels.

The general population is primarily exposed to cadmium through cigarette smoking and diet

(Gatz et al, 2006). Aluminum is an essential metal for normal physiological processes and

the general population is primarily exposed to aluminium through diet. Adverse effects can

occur with aluminium exposure that is either too low or too high (Miodovnik, 2011). Thus,

the cells enter a state that is known as oxidative stress. This stress reflects an imbalance

between ROS formation and the ability of the cell’s biological system to neutralize these

radicals. The antioxidants molecules that constitute the antioxidant defense system act at

three different levels: Prevent from radical, scavenge radical and repair radical induced

damage (Kumar et al, 2008). Under usual conditions, a certain pro-oxidant/antioxidant

49
balance is maintained in the cell, thus protecting the cell (its macromolecules) against the

destructive effect of the ROS. However, due to various internal and external factors (disease

or unfavorable environmental conditions, etc.) this balance may shift in favor of pro-

oxidants. Following the depletion of the antioxidant system resources, an uncontrollable

increase in ROS in the cell causes oxidative damage to the essential structures of the cell—

lipids, nucleic acids, and proteins (Chun et al).

This study evaluated the effects of aluminum and cadmium on lipid peroxidation and

catalase activities in the prefrontal cortex. The measured parameters were malondialdehyde

(MDA, index of lipid peroxidation) and the activities of catalase (CAT). From the results in

table 2, it was evaluated that there was significant changes in the levels of MDA and

catalase on exposure to the aluminium and cadmium. MDA values increased, indicating the

presence of free radicals. Also, catalase whose prime role is to regulate the cellular level of

hydrogen peroxide (Mueller et al, 2007) and its hydrogen peroxide catabolism protects the

cells from oxidative assault, for example, by securing the pancreatic β cells from hydrogen

peroxide injury, is reduced. Al and Cd exposure promotes oxidative stress in different neural

areas and acts as a pro-oxidant (Esparza et al, 2005).

Moreover, such results are consistent with earlier studies that have indicated that Al and Cd

intake produced an oxidative stress-related change, contributed to its neurotoxicity (Flora et

al., 2003). However, in mice, a significant relationship between Al exposure and the

50
presence of oxidative stress was established (Esparza and Gomez, 2005). This could be

caused by inflicting damage to membrane lipids, and proteins and anti-oxidative enzyme

defense system (Jyot et al, 2007).

The elevation of LPO in the cortex in the present study and others (Dua and Gi, 2001)

suggested participation of free-radical-induced oxidative cell injury in mediating

neurotoxicity of Al and Cd. Lipid peroxidation of biological membranes results in the loss of

membrane fluidity, changes n membrane potential, an increase in membrane permeability

and alterations in receptor functions (Albendea et al, 2007).

However, the increased Al and Cd concentration could deleteriousy affect the neurons,

leading to depletion of antioxidants and metal ions (Kumar et al., 2008) through the

induction of free radicals, that exhausting CAT which function as blockers of free radical

processes. These results are n accordance with (Nehru and Anand, 2005) who recorded a

significant decrease in the activities of SOD and CAT in brain of mice after Al treatment.

Alternatively, the decreased enzyme actvities could be related to a reduced synthesis of the

enzyme proteins as a result of higher intracellular concentrations of Al (Abendea et al.,

2007). Hence, These changes in MDA and CAT levels in the brain contributes to the

etiology of Al and Cd neurotoxicity.

5.2 Conclusion and Recommendation

51
The systemic effects caused by Al and Cd are diverse and multifaceted, but co-morbidities

from multisystemic Toxicosis are rarely reported in epidemiological cohorts. The

convergence of toxic actions to engage multiple organ systems in an individual is often an

observation in experimental animal models and lacks validity in observational Studies in

human or animal populations. The possible Action of Al in the pathogenesis of diabetes

mellitus and the concurrence of neurological disorders associated with alzheimer’s disease

and other dementias (Arnold et al.2018) point to the common cellular basis of the

pathogenesis of both metabolic and cognitive disorders, which can arise from toxic actions

of Al and Cd. Longitudinal studies in the future may reveal co-morbidities and

multisystemic toxicosis in Al-loaded individuals in locations where there is high risk of Al

and Cd exposure.

52
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