LITHUANIAN UNIVERSITY OF HEALTH SCIENCES
MEDICAL ACADEMY
Faculty of Medicine
Department of Biochemistry
Dawid Drażba
Long-term effects of aluminium on lipid peroxidation and
antioxidant protection in experimental mice brain
Medical Integrated Master’s Study Programme FINAL MASTER'S THESIS
Supervisor: prof. Dalė Vieželienė Consultant: assoc. prof. Inga Stanevičienė
TABLE OF CONTENTS page
SUMMARY ... 3
CONFLICTS OF INTEREST ... 4
CLEARANCE ISSUED BY THE ETHICS COMMITTEE ... 4
ABBREVIATIONS ... 5
TERMS ... 6
INTRODUCTION ... 7
AIM AND OBJECTIVES... 8
1.LITERATURE REVIEW ... 9
1. Aluminium in general description ... 9
1.1 Pathways of intake of Al ... 9
1.2 Aluminium as a toxic metal ... 10
1.3 Aluminium-caused diseases ... 11
2. Reactive oxygen species ... 11
2.1 Characteristics of ROS ... 12
2.2 Metals and generation of ROS ... 12
2.3 Effects of ROS on cellular components ... 13
3. Antioxidant system ... 14
3.1 Enzymatic antioxidants and non-enzymatic antioxidants ... 14
3.2 The role of reduced glutathione in the antioxidant system as endogenou santioxidant ... 15
2. RESEARCH METHODOLOGYAND METHODS ... 17
1. Materials and reagents ... 17
2. Research methods ... 17
2.1 Determination of aluminium concentration in the blood and brain of experimental mice ... 18
2.2 Determination of total thiol concentration in the blood and brain of experimental mice ... 18
2.3 Determination of malondialdehyde concentration in the blood and brain of experimental mice ... 19
3. Statistical analysis ... 19
3. RESULTS AND DISCUSSION ... 20
4. CONCLUSIONS ... 27
REFERENCES ... 28
SUMMARY
Author and title: Dawid Drażba, ”Long-term effects of aluminium on lipid peroxidation
and antioxidant protection in experimental mice brain”
The aim of this study was to evaluate long-term effects of aluminium on lipid peroxidation
and antioxidant protection in experimental mice brain and blood.
The objectives of the work were:
1) To evaluate term effects of Al on mice body weight and brain mass. 2) To determine long-term effects of Al on concentration of Al in mice brain and blood. 3) To evaluate long-long-term effects of Al on the malondialdehyde concentration in mice brain and blood. 4) To evaluate long-term effects of Al on the antioxidant status in mice brain and blood serum.
Research methods: Experiments were done on 4-6-weeks old white Balb C mice weighing
20–25 g. Mice were admitted to Biochemistry department of LUHS for 8 weeks of experiment duration. Mice were divided to three groups: mice of control group, low-dose Algroup (50 mg Al per kg of body weight), high-dose Al group (100 mg Al3+ per kg of body weight). Mice of control group were given tap water, whereas Al treated mice were given AlCl3 in drinking water.
Total thiol level was determined according to colour reaction with 5,5-dithiobis-2-nitrobenzoic acid reagent. Malondialdehyde concentration was determined according to the colour compound with thiobarbituric acid. The statistical analysis was performed (p<0.05 was considered to be statistically significant).
Results and conclusions: 1) Exposure to high-dose Al caused statistically significant increase
in brain mass; exposure to both doses of Al resulted in statistically significant decrease of mice body weight. 2) Exposure to both doses of Aldid not change Al concentration in mice brain; Al concentration in the blood was increased. 3) Exposure to low-dose Al did not change concentration of malondialdehyde neither in blood or brain, whereas high-dose Alresulted in statistically significant increase of malondialdehyde concentration only in the blood. 4) Effects of Al on total thiol level in mice brain and blood serum were opposite: both doses of Al caused decrease of TTL in the brain, whereas low-dose Al resulted in increased blood serum TTL; changes were statistically significant.
CONFLICTS OF INTEREST
The author reports no conflicts of interest.
CLEARANCE ISSUED BY THE ETHICS COMMITTEE
ABBREVIATIONS
Al 100 100 mg Al3+/kg body weight Al 50 50 mg Al3+/kg body weight
Al aluminium
ATP adenosine triphosphate
C control group
CAT catalase
DNA deoxyribonucleic acid
GSH reduced glutathione
GSHPX glutathione peroxidase
LPO lipid peroxidation
MDA malondialdehyde
NaOH sodium hydroxide
RNS reactive nitrogen species
ROS reactive oxygen species
SOD superoxide dismutase
TBA thiobarbituric acid TTL total thiol level
TERMS
High-dose – 100 mg Al3+/kg body weight (Al 100) Low-dose – 50 mg Al3+/kg body weight (Al 50)
Malondialdehyde (MDA) - the final product of lipid peroxidation
INTRODUCTION
Reactive oxygen species (ROS) may cause oxidative damage of different biomolecules and disturb physiological process in the living organism [1]. Production of ROS is caused by different endogenous and exogenous factors, e.g. some metals [2]. Overproduction of ROS is eliminated by enzymes of antioxidant system, such as: superoxide dismutase, reduced glutathione, vitamins E and C, uric acid, etc [2,3]. When ROS production exceeds capacity of antioxidant system to eliminate ROS, functions of different cells and organs are disturbed and pathological processes may develop [4]. ROS are strongly involved in fungal and viral infections [5] as well as ischemic heart disease [6] and infertility [7].
Aluminium (Al) is an abundant environmental metal that is extensively used in daily life. Due to environmental pollution and usage on Al in pharmacy, meal production, etc., Al tends to cumulate in the body. It was shown that Al is toxic for different body organs and systems, such as: brain, liver, blood, reproductive system, etc. [8]. One of the molecular mechanisms causing Al toxicity is the induction of ROS production and the disruption of membrane functions [9]. Al-mediated free radical generation results in the oxidative deterioration of cellular lipids, proteins and DNA and disturbed redox status [4,10]. Nervous tissue is rich in polyunsaturated fatty acids and therefore especially susceptible to oxidative damage by ROS[1].
It is supposed that oxidative damage of nervous tissue is important in ethiology of some neurodegenerative diseases, such as: Parkinson’s disease, Alzheimer’s disease [10]. It was determined that production of malondialdehyde that is a marker for lipid peroxidation, was increased in patients with neurodegenerative diseases [11]. Therefore, determination of oxidative stress markers and/or components of antioxidant system is important in understanding pathogenesis of neurodegenerative diseases.
Evaluation of molecular mechanism of Al toxicity actual due to involvement of Al in development in neurodegenerative diseases.
The aim of this study is to evaluate long-term effects of Al on lipid peroxidation and antioxidant protection in experimental mice brain.
The results of the research were presented at the 9th International Conference of Lithuanian Neuroscience Association “Neurodiversity: From Theory to Clinics”. 1 December 2017. Medicina - 2017, volume 53, supplement 2.
'Effects of Aluminium on Iron and Magnesium Concentrations and Lipid Peroxidation in Aluminium- Exposed Mice Brain. I. Staneviciene, R. Naginiene, D. Drazba, D. Viezeliene
AIM AND OBJECTIVES
The aim of this study was to evaluate long-term effects of aluminium on lipid peroxidation and
antioxidant protection in experimental mice brain and blood.
Objectives:
1. To evaluate long-term effects of different doses of Al on mice body weight and brain mass. 2. To determine concentration of Al in mice brain and blood after exposure to Al.
3. To evaluate long-term effects of Al on the malondialdehyde concentration in mice brain and blood.
4. To evaluate long-term effects of Al on the mice brain and blood serum antioxidant status. Details of experiment: 1) doses of Al: low-dose Al (50 mg Al3+/kg of body weight) and high-dose Al (100 mg Al3+/kg of body weight); 2) way of administration: AlCl3 was given in
1. LITERATURE REVIEW
1. Aluminium in general description
Aluminium is a chemical element with symbol Al, atomic number 13 and standard atomic weight 26.98 [12]. In the periodic table, Al belongs to the group III of the elements. It is a silvery- white, soft, ductile and nonmagnetic metal. According to the prevalence on Earth, Al occupies the third place (after oxygen and silicon), and it is the most abundant metal in the crust with 7,9 % of its mass. Al is present as a trivalent cation (Al3+), most of it being associated with silicate and forming water-insoluble complexes. Through the formation of these complexes, Al bioavailability is highly reduced. Its relatively small size, together with its trivalent positive charge, gives Al a high-polarizing effect on its neighbour atoms. This cation has a tendency to create hydroxyl complexes in water solution which, due to their amphoteric character, evolve from free Al3+ towards Al(OH)4− within the
3–8 pH range. At physiological pH, Al forms the scarcely soluble Al(OH)3, that can be easily
dissolved by slight changes in the acidity of media. Al can also bind to oxygen- and nitrogen-containing compounds, particularly to inorganic and organic phosphates. Through these kinds of interactions, Al binds to many biological macromolecules. Although Al is found commonly in the environment, there is no known essential role of Al in the living systems. In general, Al is not essential for the growth, reproduction, and sustainability of life in terms of humans and animals. Al accumulation in tissues and organs results in their dysfunction and toxicity, effects that usually correlate with the local concentration of the metal [13].
1.1 Pathways of intake ofAl
Exposure to Al occurs via various routes, including inhalation, ingestion and dermal absorption. Al enters into the body from the environment, diet and medication; eg. antacids, adjuvant in vaccines and an agent against pathological hyperhidrosis [8, 14].
The major route of exposure to Al for the general population is through food, both as a consequence of the natural occurrence of Al in food (e.g. fruits, vegetables, cereals, seeds and meat), and the use of Al and its compounds in food processing, packaging and storage. A minor source of Al exposure is drinking water. Although the total intake of Al considerably varies upon country, place of residence, and diet composition, nearly 10 mg of Al is taken to the human body on a daily basis; 9.6mg
of this amountis taken from foods, 0.1–0.4 mg of this amount is taken from kitchen utensils and packaging, and 5 μg of this amount is taken from air [15].
1.1.1 Uptake in Gastrointestinal Tract, Accumulation and Elimination ofAl
The level of Al absorption through the intestine can vary according to age, sex, and diet. The rate of absorption here is around 0.2 %. Once absorbed, Al has a half-life of several hours in the blood. It is subsequently either eliminated through the urinary tract or distributed throughout the body by the iron-binding protein transferrin and accumulates in various organs such as bone, lung, muscle, liver, and brain. The accumulation of Al in these organs appears to be the initiator of a variety of medical conditions [16].
Once Al salts are transferred to the vascular system in the blood, most of the metal is bound to transferrin. Al3+ can enter the nervous system by transport across the blood–brain barrier using receptor-mediated endocytosis of transferrin. Approximately 0.005 % of the aluminium-protein complexes enter the brain by this means [17].
1.2 Aluminium as a toxic metal
Aluminium toxicity in general is caused by the disruption of homeostasis of metals such as magnesium, calcium, and iron; in fact, Al mimics these metals in their biological functions and triggers many biochemical alterations [18, 19]. Al3+ is highly biologically reactive ion and biologically available Al is non-essential, with no biological role in the body [20]. Neurotoxic metal Al has been involved in some neurological disorders; eg. Alzheimer’s disease [8]. Very low doses of Al in vaccines may induce inflammation, autism [21, 22].
Aluminium interaction (competition) with other metal ions. Al is toxic metal that may interact with nutritionally essential metals. Iron deficiency increases absorption of Al. Al interacts with calcium in the skeletal system to produce osteodystrophies. Calcium deficiency along with low dietary magnesium may intake contribute to aluminium-induced degenerative nervous disease [23].
Aluminium disturbs energy metabolism. Similar in size to the natural cofactor Mg2+, Al3+ may act by substituting Mg2+ ions in vital processes, eg. in tubulin polymerization to microtubules, affinity of Al3+ is more than 107 times as Mg2+. Mg2+ serves as a cofactor by binding to the phosphates of
guanosine triphosphate (GTP), and Al3+ displaces Mg2+ at this site. All ATP-associated reactions use Mg2+, and Al3+ potentially interferes in these processes.[24].
Aluminium is considered as a pro-oxidant. The pathology of laboratory animals that have been experimentally intoxicated with Al invariably shows many indices of oxidative stress. These include changes in the levels of antioxidants, such as SOD and catalase, as well as higher levels of biomarkers of peroxidation, such as MDA and lipid hydroperoxides [25]. Al is an efficient pro-oxidant in vivo [26]. Research has shown that antioxidants, such as vitamin E, will protect against oxidative damage in animals exposed to Al [27]. The mechanism whereby aluminium facilitates iron- driven biological oxidation is likely to involve the formation of AlO22+ and this complex will act as a pro-oxidant by both
catalyzing the formation of H2O2 (AlO2+ is more efficient oxidant than O - in forming H2O2) and
reducing Fe3+ to Fe2+ [26].
1.3 Aluminium-caused diseases
Many diseased such as, abnormal kidney function, anemia, neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease (AD), etc. are associated with intoxication by Al.
One of the causes of Al involvement in AD pathology is Al3+ ability to increase beta-amyloid peptide formation and the phosphorylation of Tau protein, causing senile/amyloid plaques and neurofibrillary tangles [28].
It is known that the etiology of neurodegenerative diseases is multifactorial, and there is evidence that potential external factors including lifestyle and chemical exposures are linked with the risk of the onset of these diseases [20]. Neurodegenerative disorders may cause dementia in elderly. Alzheimer’s disease is the major form of dementia.
2. Reactive oxygen species
Free radicals are mainly oxygen (reactive oxygen species - ROS) and nitrogen (reactive nitrogen species - RNS) compounds produced by various endogenous systems of the body. Their formation is determined by certain physiochemical conditions or pathophysiological processes taking place in the body [29]. These processes are driven by a variety of exogenous and endogenous factors such as: pollution, ionizing radiation, cigarette smoke, ischemia, tissue damage and inflammatory response, cellular metabolism (electron transport chain activity), etc. [30]. Free radicals can damage cellular macromolecules - lipids, proteins, DNA, which may be associated with the aging process, the
development of cardiovascular, neurodegenerative, immune system, oncological and other diseases [29, 31, 32].
2.1 Characteristics of ROS
The formation of free radicals is determined by the fact that molecular oxygen containing two unpaired electrons that are difficult to reduce. However, under certain conditions (exposure to radiation, heavy metals), this process is greatly accelerated. The more molecular oxygen attaches to electrons, the great er its reactivity [30]. In the cells, the highest amounts of radicals of superoxide (O2•‾) and nitric oxide (NO•), which are further converted into radicals with strong oxidative
properties, are hydroxyl (•OH), alkoxy (RO•), peroxyl (ROO•) radicals, singular oxygen (1O2). Some
types of radicals are converted to molecular oxidants - hydrogen peroxide (H2O2), peroxynitrite
(ONOO‾), hypochlorite acid (HOCl), which can act as sources of free radicals. For example, H2O2 is
converted into •OH radicals during the Fenton reaction, and HOCl reacts with H2O2 to produce 1O2.
ONOO‾, with physiological CO2 concentration, contains a source of carbonate radicals (CO3 •
‾) [29]. The formation of free radicals takes place in different pathways, and most of them occur in cellular mitochondria [29, 33].
2.2 Metals and generation of ROS
Free radicals oxidize various molecules in cells. For example, in the presence of O2•-,
catecholamines, ascorbic acid, thiol compounds are easily oxidized. The most active form of vitamin E - αtocoferol and iron-containing protein molecules - are also very sensitive to the effects of these active forms of oxygen [35]. O2•- can form complexes with Fe3+ - perferyl radicals [Fe3+O2]• that are not
prone to oxidation reactions and may continue to decompose. In this way, ferrous radicals [Fe3+O]• are formed from perferyl radicals, which are active and have many unpaired electrons. Therefore, it is believed that O2•- is the main compound involved in the formation of other reactive molecules during
lipid peroxidation and oxidative damage of other macromolecules [30, 31].
Very important is the self-decomposition of O2•- into H2O2 and O2 and the ability of this radical to
reduce transition metals (Fe, Cu, etc.) and their complexes to form other free radicals that cause oxidative damage [33].
Another highly reactive radical - •OH - is a particularly strong oxidizer. It is very unstable and reacts with most biological molecules that contain -SH groups or Fe-S fragments. It is the strongest
and most active oxidant that effectively oxidizes proteins, nucleic acid and lipids. It induces chain reactions that causes cell damage [29]. The hydroxyl radical is formed from H2O2 in
oxidation-reduction reactions such as Fenton's reaction [30].
Macromolecules also react with 1O2 in the body. Almost all of these reactions involve the coupling
of this active form of oxygen to the conjugated compounds of the oxidized compounds. For example, in the presence of fatty acid reactions, the resulting products are organic peroxides that cause chain reactions of lipid peroxidation [29, 33].
The formation of free radicals that cause cell damage is determined by both endogenous (inflammatory reactions, metabolic processes) and exogenous factors (metal ions, radiation, certain xenobiotics such as barbiturates). They are produced byenzymatic (NADH dehydrogenase, xanthine oxidase, lactate dehydrogenase, glutathione reductase, cytochrome P-450 enzymes) and non-enzymaticways [29]. For example, O2•‾ is produced non-enzymatically by the self- oxidation of some
cellular components - ubiquinones, flavins, thiol compounds - to the redox cycle of quinone compounds with electrophilic properties; OH• is generated during H2O2 reaction with reduced iron-
Fenton reaction.[33]. The body's phagocytic cells are also a source of free radical formation [30]. Various exogenous factors usually accelerate the formation of non-enzymatic free radicals [29].
Small amounts of free radicals are essential for normal physiological function - gene expression, cell growth, infection prevention [33]. They can also act as stimulators of cellular biochemical processes by reversibly oxidizing transcription factors responsible for gene expression and cell growth, certain active domains, or indirectly inducing transcription factors and activating signal transduction pathways in cells [30].
The increase in free radicals in the cells and their inability to detoxify cause oxidative stress in the body. Oxidative stress is the result of damage to biomolecules by free radicals and other active forms of oxygen and nitrogen [30]. From a chemical point of view, oxidative stress is a significant decrease in the reduction potential of the cells (reducing potential becomes less negative) or a reduction in the reductive power of cellular redox pairs such as reduced glutathione (GSH) [36]. The effects of oxidative stress depend on the degree of these changes. Cells exposed to the antioxidant system can withstand minor changes in the stability of the oxidative-reduction system and regain their initial state, but more pronounced oxidative stress can cause cell death resulting in various diseases [33,37].
2.3 Effects of ROS on cellularcomponents
One of the consequences of oxidative stress is radical-induced lipid peroxidation, which results in the release of various toxic metabolites in the urine, such as malondialdehyde, formaldehyde,
and others [38]. MDA is an indicator of lipid peroxidation and, in the presence of oxidative stress in the body, an increase in its amount is observed in the liver and other organs. This aldehyde is the final product of the peroxidation of polyunsaturated fatty acids, so the level of damage of biological structures can be judged by its amount in cells[39].
Oxidative stress also affects the DNA of the cell - genetic cell damage increases, the antioxidant system is affected - the amount of GSH in the liver and other organs is reduced, and antioxidant enzyme activity is diminished. The free radicals react with biological macromolecules - lipids, proteins, DNA, - to form a variety of secondary radicals, such as lipid, amino acids, thiol radicals, which turn into peroxy-radicals under the action of oxygen and produce various peroxides. The latter in biological systems are important in triggering chain reactions and causing damage to various cellular structures and their function[38].
3. Antioxidant system
The cells of various organisms, including the human body are protected from the harmful effects of free radicals by the endogenous multi-component antioxidant system [29]. Compounds with antioxidant properties neutralize free radicals by joining, altering, or simply destroying them [30,37].
The amount of ROS in cells is regulated by an antioxidant system designed to neutralize excess free radicals in the body,thus protecting cells from ROS toxicity and contributing to disease prevention [40, 41].
Antioxidants are substances that protect the molecule from the oxidative damage [42,43]. An antioxidant reacts with an oxidant (ROS) and neutralizes or regenerates other molecules that can react with the oxidant [42]. One antioxidant molecule can only react with one free radical and neutralize it by giving one of its own electrons [44]. By destroying the free radical, the antioxidant itself oxidizes, so these substances must be constantly restored in the body [40].
3.1 Enzymatic antioxidants and non-enzymatic antioxidants
The antioxidant system is classified into two large groups: enzymatic antioxidants and non- enzymatic antioxidants [45, 46].
Enzymatic antioxidants are distinguished according to the principles of action into two groups: primary and secondary [46, 47]. Antioxidants that terminate chain reactions caused by free radicals are called primary. This group of antioxidants includes enzymes: superoxide dismutase (SOD), glutathione peroxidase (GSHPX) and catalase (CAT) [46]. SOD catalyzes the reduction of O2 to H2O2, which is a
subsequent CAT substrate [40]. H2O2 may be involved in Fenton reactions to form OH ·. CAT and
GSHPX translate OH· into H2O and O2 [48]. By oxidizing GSH, GSHPX is capable of reducing
H2O2 to H2O and O2 to form oxidized glutatione (GSSG) [40]. Secondary antioxidants do not
participate directly in neutralization of ROS, but they reduce oxidized antioxidant molecules [47]. Glutathione reductase (GR) reduces GSSG, and glucose 6-phosphate dehydrogenase (G-6-PDH) regenerates NADP+ to NADPH required for GSSG reduction [40,48].
Non-enzyme antioxidants are divided into two groups: metabolic and nutrient antioxidants. Metabolic antioxidants such as lipoic acid, GSH, coenzyme Q10 (ubiquinone), melatonin, uric acid, bilirubin, and metal binding proteins are synthesized in the body [40, 49]. Other antioxidants such as ascorbic acid (vitamin C), tocopherol (vitamin E), carotenoids, minerals (Zn, Se), flavonoids, phenolic acids, omega-3 and omega-6 fatty acids are not synthesized in the body, therefore must be obtained with food.
Non-enzymatic antioxidants are vital because they can enter the structure of the antioxidant enzyme active center, act as cofactors or neutralize ROS itself [48].
3.2 The role of reduced glutathione in the antioxidant system as endogenous antioxidant
Endogenous antioxidants containing –SH groups are proteins, reduced glutathione (GSH), etc. GSH is the most abundant non-enzymatic intracellular antioxidant found in every cell of the body at millimolar concentrations [50]. It is a tripeptide (γ-glutamylcysteinylglycine) composed of glutamic acid, cysteine and glycine and having a -SH group. This tripeptide exists in reduced (GSH) and oxidized - glutathione disulfide (GSSG) forms. The oxidized glutathione form consists of two oxidized GSH residues linked together by a disulfide bond [51]. In normal cellular redox balance, the reduced tripeptide is distributed in cytoplasm, nuclei, endoplasmic network, and mitochondria of various organ cells [52].
GSH can directly neutralize free radicals (OH· and O2-·) or act as a cofactor for antioxidant
enzymes such as (GSHPX and glutathione-S-transferase (GST)) [52]. This tripeptide is involved in the reduction of products resulting from the oxidative interaction of ROS with cellular components, such as MDA formed during lipid oxidation. GSH is involved in removing lipid oxidation products and stopping LPO chain reactions, protecting biological membranes [53]. It is known that disulfides are formed between GSH and protein -SH groups (a process called glutathioneylation), thus protecting protein -SH groups from oxidation [54]. GSH is involved in the GST catalyzed neutralization reactions of electrophilic compounds and xenobiotics, thus contributing to their elimination. For example, one of the most popular analgesics - acetaminophen (paracetamol) is detoxified during conjugation with GSH, toxic doses of this drug deplete GSH reserves in hepatocytes, causing oxidative stress and various liver
damage [55, 56]. It is reported in the literature that GSH is capable of regenerating other antioxidants, such as vitamin C and E, and is involved in DNA synthesis [45, 51, 57]. GSH can interact with metal ions that are capable of being attached to the structure -SH group, thus protecting cells from toxic effects [53].
2. RESEARCH METHODOLOGY AND METHODS
1. Materials and reagentsAll experiments were performed according to the Republic of Lithuania Law on the Welfare and Protection of Animals (License of State Veterinary Service for working with laboratory animals No.0221).
The model of mouse intoxication. For experiments we used 4–6 weeks old Balb C white
laboratory mice weighing 20–25 g. Mice were brought from the vivarium of the Veterinary Academy of LUHS and kept in quarantine for 7 days. Male and female animals were housed in separate cages, with optimal storage conditions: optimal room temperature ~ 20°C, relative air humidity 55±10%, natural daylight (day/night) mode. Hay and wood chips were changed every day. Mice were fed the meals with a full-fledged food. Al treated mice were given tap water supplemented with AlCl3 for 8
weeks. Low-dose Al mice received 50 mg (1.85 mmol) Al3+/kg body weight). High-dose Al mice received (100 mg (3.7 mmol) Al3+/kg body weight). The control group mice had free access to the tap water.
After the exposure of 8 weeks, the animals were terminated according to the rules defined by the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Purposes. Following that, the brain and blood were quickly removed, and immediately cooled in an ice bath.
The following reagents for the experiments were used: thiobarbituric acid (TBR), sucrose, 5,5'- dithio-bis- (2-nitrobenzoic acid) (DTNB) - Serva (Germany); KCl, NaOH, MgCl2, H3PO4, n-butanol -
Lithuania. We used deionized water and saline to make solutions.
2. Research methods
Preparation of samples
The brain was removed, rapidly cooled on ice and homogenized in 3 volumes of homogenization buffer (50mM Tris-HCl, pH 7.6; 5mM MgCl2; 60mM KCl; 25 mM sucrose). The
tissue homogenate was centrifuged at 3000 rpm x 10 min (1000xg) in a Beckman J2-21 centrifuge. The first supernatant was poured into a separate test tube and the pellet was discarded. The supernatant was centrifuged at 10,000 rpm x 15 min (12,000xg) in the Beckman J2-21 centrifuge. The whole volume of second (post-mitochondrial) supernatant was immediately frozen at -80oC.
The serum samples were prepared as follows. The whole blood was collected into Eppendorf test tubes after mouse decapitation. The blood was centrifuged at 4000 rpm x 10 min (1600xg) in a Savant centrifuge. The serum samples were immediately frozen at -80oC.
2.1 Determination of aluminium concentration in the blood and brain of experimental mice
The concentration of Al in blood and tissue specimens taken from brain was determined by inductively coupled plasma mass spectrometer NexION 300 D (Perkin Elmer, USA). Al3+ concentrations were determined in: Toxicology laboratory, Neuroscience Institute, Medical Academy, Lithuanian University of Health Sciences. Tissue specimens were digested with 0.125 M NaOH (Merck, Germany) at 900 C, the digests were diluted to the appropriate volume and analysed following manufacture recommendations for heavy metals concentration detection in biological samples. Internal and external quality control procedures, including involvement of analytical purity water and reagents. (Merck, Sigma-Aldrich), certified reference materials: ClinCheck® Whole Blood Controls (Recipe, Germany), ClinCheck® Serum Controls L-2 (Recipe, Germany), Certified Reference Material BCR® -185, bovine liver (EC Joint Research Centre (Geel), and the control of labware for trace elements contamination, were involved to assure the accuracy and preciseness ofanalysis.
2.2 Determination of total thiol concentration in the blood and brain of experimentalmice
The TTL assay is a spectrophotometric test which determines the concentration of free thiol groups in plasma. The test is based on the ability of free thiol groups to develop a coloured complex (absorbance at 405 nm) when reacted with 5,5-dithiobis-2-nitrobenzoic acid. The colour intensity is directly related to the thiol groups in the sample which are not affected by oxidation. L-cysteine has been used as standard.
The total thiol level in serum (TTL) was measured using a kit from Rel Assay (Gyantzip, Turkey) and expressed in µmol/L. TTL assay was adapted for automatic use on a LX-20 Pro auto- analyzer (Beckman-Coulter, Woerden, the Netherlands). TTL were measured in: Center for Health Protection, National Institute for Public Health and the Environment, Bilthoven, the Netherlands.
2.3 Determination of malondialdehyde concentration in the blood and brain of experimental mice
Lipid peroxides were estimated by measuring thiobarbituric acid-reactive substances and were expressed as malondialdehyde (MDA) content in nmoles/g of wet organ weight. Mice brain was homogenized in cold 1.15% KCl solution to make 10% homogenate. 3 mL of 1% H3PO4, and 1 mL of
0.6% thiobarbituric acid aqueous solution were added to 0.5 mL of this homogenate. The mixture was heated for 45 minutes in a boiling water bath. After cooling, 4 mL of n-butanol was added and mixed vigorously. The butanol phase was separated by centrifugation and used to determine light absorption at wavelength 535 and 520 nm [58].
MDA concentration in the blood was determined by preparing samples containing 0.1 mL heparinized blood, 1 mL 10% TCA, 2 mL 0.5% thiobarbituric acid and 2 mL H2O. The samples were
heated in the boiling water bath for 30 minutes, then cooled and centrifuged at 3.000×g for 15 minutes. The light absorption was measured at wavelength 540 nm. MDA in the blood was expressed in µmol/L [59].
3. Statistical analysis
The data were analyzed by means of Student’s t-test. Results were expressed as the mean ± Standard error of mean. Statistical significance was set at p<0.05. Statistical analysis was performed using a statistical software package (SPSS version 19.0, Statistica).
3. RESULTS AND DISCUSSION
The present study was conducted to determine concentration of Al in mice brain and blood and to investigate effects of different Al doses in vivo on the redox status in these organs after 8-weeks administration of AlCl3 solutions.
Al acts as prooxidant in vitro and in vivo [25] and has neurotoxic, cardiotoxic, hepatotoxic, nephrotoxic, haematotoxic, and genotoxic effects [60, 61]. So Al affects on whole organism, therefore at the beginning of our experiments we evaluated general systemic effect of AlCl3 solution. This effect
was evaluated according to mouse body weight gain and relative weight index of organ (the ratio of organ weight to body weight). In toxicological studies, body weight and relative organ weight are important criteria for evaluation of toxicity [62]. The overall survival rate of mice was the same in the control and experimental groups. In the present study, general toxicological examination revealed that the body weight of experimental mice significantly (p<0.05) decreased throughout the whole period of Al intoxication with high- and low-doses as compared to the control group (Fig. 1).
Figure 1. Time course of body weight gain of the control group mice and the mice orally treated with
AlCl3 solution for 8 weeks.
The scheme of mice exposure to Al is described in methods. The data was obtained by measuring the body weight of 20 mice in each group.* - Differences are statistically significant in comparison to the
Exposure to low-dose Al (50 mg/kg bw, Al 50) and high-dose Al (100 mg/kg bw, Al 100) showed that mice loss the body weight during experimental time, in comparison to control group. Results, shown in Figure 1, revealed that the body weight gain in mice treated with low-dose Al, decreased by 1.9% after the first week to 8.7% after the last week, compared to control. The body weight of mice, treated with high-dose Al, was even lower, it decreased by 3.5% at the beginning to 13% at the end of experiment in regard to control. The body weight of control mice group increased gradually throughout all period of experiment with a slight slowdown in growth at 7-8weeks.
Exposure to high-dose Al for 8-week caused a statistically significant increase in relative weight index of the brain by 19% (p<0.05) as compared to control (Fig. 2). Any statistically significant change in the relative weight index of the brain was found in mice, treated with low-doseAl.
Figure 2. Relative weight index ofthe mice brain in control and
after exposure to Al.
The scheme of mice exposure to Al is described in methods. Each column represents the data obtained from 20 separate experiments.* - Differences are statistically significant in comparison to the control
group.
Altogether, body weight gain was higher in the control than in both Al-treated groups. These results are in good agreement with others, which found that exposure to different Al salts caused in decrease body weight of experimental animals [63]. Some authors suggest that decrease of body weight may be explained by a possible Al effect on brain and kidneys which control drinking behaviour [64]. In our experiments, the increase in relative weight index of the brain, as compared to the control, is still unclear. It is supposed that exposure to Al led to histopathological alterations in nervous tissue.
Further experiments were designed to estimate possible Al accumulation in the tissues of mice after long-term oral administration of AlCl3 solutions (data presented in Figure 3). Accumulation of Al
in the blood of mice after exposure to low- and high-dose Al is presented in Figure 3A. Significantly high blood Al concentration (135.62 µg/L, p<0.05)was noted in mice, treated with high-dose Al. Al concentration in the blood of this mice group was two times higher, in comparison of Al concentration value (64.03 µg/L) of control. The exposure to low-dose Al caused no changes in Al concentration in the mice blood. (Fig. 3A.)
In our experiments we also determined Al concentration in mice brain (data presented in Figure 3B). Interestingly, in brain, there was no indicated change in Al concentration of mice exposed to
Figure 3. The concentration of aluminium in mice blood (A) and brain (B).
The scheme of mice exposure to Al is described in methods. Data represent results of 10-12 separate experiments.* - Differences are statistically significant in comparison to the control
different dose of Al. Values of Al concentration in brain of mice, treated with low- and high-dose of Al was 10 µg/Land 10.60 µg/L, respectively, meanwhile Al concentration in brain of control mice was 12.04 µg/L (Fig. 3B). These no marked changes of Al concentration were statistically insignificant in comparison to the control group and revealed that Al was not distributed in the brain.
The results of this study showed that Al can be absorbed through the intestine of mice after prolonged oral administration of high dose of Al, as indicated by an elevated blood Al content. In experimental animals, absorption of Al via the gastrointestinal tract is usually less than 1% [65]. The uptake of Al through gastrointestinal pathway is complex and is influenced by various factors including an individual difference, age, pH, chemical species of Al, coexistence substances. Al absorption may interact with calcium and iron transport systems. Small, but considerable amount of Al can across the blood brain barrier maybe through transferrin-receptor pathway or monocarboxylate transporters, and enters into the brain [66]. The data about Al accumulation in the brain have been reported in a number of studies and showed, that Al accumulation not only varied with the administration route used but also in its distribution in the distinct brain areas. It was reported that intraperitoneally administered Al was transferred to the brain and the amount of Al in the brain is not changed after 35 days, although Al in the serum disappeared rapidly [67]. Oral administration of Al salts solutions resulted in a significant increase in brain Al concentration in all the investigated brain areas [68]. These results are in good agreement with previous studies reporting an increase of Al concentration in both whole brain and specific areas of the brain [68]. However, no significant accumulation of Al in the whole brain [69] and even a paradoxical reduction of Al concentration in the whole brain of mice chronically treated with a diet containing Al [63] has also been reported. It is supposed that Al 50 that was used in these experiment was too low to be absorbed and affect Al concentration in the blood.
Lipid peroxidation is one of the most important manifestations of oxidative damage and has been found to play an important role in the toxicity of Al.
In our experiments, MDA was used as a marker of lipid peroxidation. Data indicating effect of Al3+ on LPO in mice blood and brain are presented in Figure 4. It was shown that the increase in blood MDA by 18% after treatment with low-dose Al was not statistically significant. Exposure to high-dose Al caused a statistically significant increase of blood MDA concentration by 61% (p<0.05) as compared to control. The exposure to both doses of Al did not change MDA concentration in the brain (Fig. 4).
Figure 4. Concentration of malondialdehyde (MDA) in mice blood and brain in control and after
treatment with Al (Al 50 and Al 100).
The scheme of mice exposure to Al is described in methods. Data represent results of 10-12 separate experiments.* - Differences are statistically significant in comparison to the control
group.
MDA concentration in mice bood: C group = 279 µmol/L, Al 50 = 328 µmol/L, Al 100 = 449µmol/L.
MDA concentration in mice brain: C group = 81 nmol/g, Al 50 = 84 nmol/g, Al 100 = 82 nmol/g
In our experiments we investigated effects of different Al doses on LPO in mice blood and brain. It is known that Al circulates in the blood mainly bound to transferrin (90%) and low molecular mass compounds (e.g. citrate) [70]. Therefore, Al may interfere with Fe homeostasis by displacing it from transferrin; as a result Fe is released into the bloodstream. The increase in free intracellular Fe2+ causes the peroxidation of membrane lipids and thus causes membrane damage [71]. Thus, in this indirect way Al accelerate iron- and hydrogen peroxide-dependent LPO in human erythrocyte membranes. Data of our experiments indicated that high-dose Al caused statistically significant change in MDA concentration in the blood of mice as compared to control. Iron, a redox-active metal, can interact with molecular oxygen to generate superoxide anion, which, in turn, generates highly reactive hydroxyl radical. It is indicated that exposure to Al can impair intestinal Fe absorption, an increase in
serum Fe concentration, and disrupt normal tissue ferritin levels. Data of unpublished results also showed an increase in Fe concentration in the blood after 8-week treatment with Al 100 dose.
Al and its compounds are neurotoxic for human and animal brain [72]. It has been reported that Al binds to transferrin receptors and enter the brain via transferrin-mediated endocytosis. Our results demonstrated that both low-dose and high-dose of Al didn’t induce LPO in mice brain. Some authors Nehru and Anand [59], Candan and Tuzmen [73], demonstrated increased LPO level in the brain of animals exposed to Al. We suppose that both Al doses used in these experiment were too low to enter the brain and to cause lipid peroxidation.
Al-mediated free radical generation results in the oxidative deterioration of cellular lipids, proteins and DNA and disturbs redox status [74]. These processes can be characterized by the change in activity of intracellular anti-oxidant enzymes and the depletion of sulfhydryls. Thiols constitute the major portion of the total body antioxidants and they play a significant role in defense against ROS. The redox status biomarker TTL in serum determines mainly free thiol groups in proteins. The level of protein –SH in serum indicate antioxidant status/redox capacity.The low level of protein –SH correlate positively with the increased level of lipid peroxides orlowered redoxcapacity.
Figure 5. Total thiol level (TTL) in mice serum (µmol/L) and in brain (µmol/g) in control and after
treatment with Al (Al 50 and Al 100).
The schemes of mice treatment with Al are described in methods.Data represent results of 10-12 separate experiments.* - Differences are statistically significant in comparison to the control
The data in Figure 5 demonstrated the total thiol level (TTL) in mice serum and brain after treatment with low- and high-dose Al. Exposure to low-dose and high-dose Al caused a statistically significant decrease in brain TTL by 25% (p<0.05) and 23% (p<0.05), respectively. Administration of low-dose Al caused a statistically significant increase in serum TTL by 22%(p<0.05), in regard to control. However, an increase in plasma TTL by 18% after treatment with the high-dose Al was statistically insignificant. (Fig. 5).
Total thiols composed of both intracellular and extracellular thiols (reduced glutathione,thiols of proteins). Proteins are important antioxidant components in serum, and the -SH groups are primarily responsible for their antioxidant activity. Our results demonstrated, that Al affects the level of total thiols in mice brain and blood serum; it caused decrease in brain TTL and increase in serum TTL. It was shown that Al concentration in the brain didn’t change, whereas concentration of Fe increases (unpublished data).One of the causes of decreased level of total thiols in the brain may be due to increased concentration of Fe,which acts as prooxidant.
4. CONCLUSIONS
1. Exposure to high-dose Al caused statistically significant increase in brain mass; exposure to both doses of Al resulted in statistically significant decrease in mice body weight.
2. Exposure to both doses of Aldid not change Al concentration in mice brain; Al concentration in the blood was increased.
3. Administration of low-dose Al did not changemalondialdehyde concentration neither in blood or brain; high-dose Alcausedstatistically significant increase of malondialdehyde concentration only in the blood.
4. Effects of Al on total thiol level in mice brain and blood serum were opposite: both doses of Al caused decrease of TTL in the brain, whereas low-dose Al resulted in increased serum TTL; changes were statistically significant.
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Annex No. 1
ANNEXES
9th International Conference of Lithuanian Neuroscience Association “Neurodiversity: From Theory to Clinics”
