M Abubaker, A Taylor, G Ferns
aluminium, primary cortical astrocyte, pro-oxidant
M Abubaker, A Taylor, G Ferns. Effect of Aluminium Toxicity on Primary Cortical Astrocytes. The Internet Journal of Toxicology. 2007 Volume 4 Number 2.
The in vitro potential of aluminium to induce pro-oxidant or antioxidant effects, were studied in rat primary cortical astrocytes. These cells were exposed to aluminium (as aluminium sulphate) at different concentration. The results revealed that aluminium has a differential effect on the rat primary cortical astrocytes. The toxic effects was assessed using mitochondrial dehydrogenase activity, measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT); cell viability as revealed by Fluorescein diacetate-Propidium iodide (FDA-PI) staining; glutathione content; lipid peroxidation as determined by malondialdehyde production and reactive oxygen production (ROS). The data suggests that aluminium has both pro-oxidant and antioxidant effects.
To date there is no ideal model for the study of aluminium neurotoxicity
Biochemical studies revealed that astrocytes are involved in production and metabolism of the amino acid transmiters glutamine and γ-amino-butyric acid (GABA), as well as in K + homeostasis at the cellular level, in addition to being metabolically active (11). Glial cells play a critical role in the development as well as physiological maintenance of the neurones (12) and may be the target and mediator of many insults to the CNS (13). Further studies have demonstrated that astrocytes are more resistant to ROS-mediated damage than the other neural cells. This may be related to the high GSH content ( ≈20 nmol/mg protein) (14, 15), though as high as 8 mM has been reported (16). Astrocytes survive in culture for as long as there is a source of glucose in the medium and die only when glucose is completely depleted followed by ATP depletion (17).
As mentioned earlier many
The above mentioned properties in addition to direct involvement of this cell type to chemical insult stimulated the interest of these cellular models to explore the likely oxidative effects following Al exposure.
As described above, neurodegenerative disorders involving aluminium affect a large number of individuals, particularly the elderly and patients with chronic renal failure. In view of this, it is hoped that this research will to contribute towards alleviating the personal suffering and medical, financial and social burdens associated with aluminium exposure.
The aim of the present study was to explore
Materials And Chemicals
Aluminium sulphate was purchase from Alfa Johnson Matthey Compapany (Johnson Matthey GmbH, Zeppelinstraβe 7, Karlsruhe). Dubecco's modified eagle medium (DMEM), modified eagle medium (MEM), fetal calf serum (FCS) L-glutamate, gentamycin and L-15 media were obtained from GIBCO Co (Life Technology Ltd. Paisley, Scotland UK). Ransod and Ransel kits from Randox (Randox Laboratories, N. Ireland).While Papain and Ovomucoid are from Boehringer. Aluminium nitrate, Ethylenediaminetetra-acetic acid (EDTA), methanol, nickel nitrate, nitric acid, perchloric acid, potassium-sodium-tatrate, selenium nitrate, sodium selenite and sulphuric acid were BDH products (BDH Chemicals Ltd., Poole, UK). Copper (II) sulphate, sodium carbonate and trichloroacetic acid were purchase from Fisons(Fisons Sci. Equipment, Loughborough, UK). O-phthaldialdehyde, cysteine, DNase, reduced glutathione, trypsin solution and all other chemicals were from Sigma chemical Co. (Sigma Chemical Co. Poole, Dorset). Double-deionized distilled water (DDW) was used for preparation of solution.
Pye Unicam SP9 electrothermal atomic absorption spectrophotometer, Pye Unicam PU9200 atomic absorption spectrophotometer, Perkin Elmer LS50 Luminescence Spectrophotometer, iEMS microplate spectrophotometer (Labsystems Company), Cobasmira/Cobasbio automated analyser, fluoresence microscope and 95% O2 and 5% CO2 incubator.
Cell Culture Preparation
Primary cortical astrocytes cell cultures were prepared as described by Booher & Sensebrenner, (23). Briefly, 2-3 days-old Wister rat pup decapitated and the heads were dipped into 96% ethanol and then into cold MEM-HEPE'S (10 µM). The brain was removed, dissected to obtain cerebral cortex and the cortecies were removed from the MEM-HEPE'S, cut into pieces, added to papain solution and incubated at 37 0 C for 1 hr 15 min. After the incubation, papain inhibitor solution was added and cells were dissociated by passing the tissue through needles, filtration and centrifugation. Cell pellets were resuspended in primary medium DMEM containing 10% FCS v/v, 2 mM glutamine and 25µg/ml gentamycin (media). Cells were seeded onto 250 cm 2 flask with 2.5 pups/flask which approximately containing 2.5 x10 5 cells/cm 2 and incubated at 37 0 C in 5% CO2, 95% humidified atmospheric air. At 10 DIV flask were shaken and semi-pure culture of astrocytes were obtained.
Generally all the cells were continuously cultured and maintained in their respective media preparations until confluence was reached ( ≈5 x10 5 cells/cm 2 for astrocytes), with media changed every 3 days.
Three days before cell plating, the media was removed by aspiration from the cells followed by addition of 5 ml trypsin solution (0.25%) in order to detach the cells from the flask. The trypsinized cells were then incubated at 37 0 C in 5% CO2 humidified air incubator for 8 min. The trypsinization was stopped by the addition of an equal volume of FCS 10% v/v, the cells were centrifuged at 100 x g for 5 min and the resulting pellet suspended in 2 ml of the media. One ml of the suspension was then made up of to 25 ml of the media, swirled very well and 12.5 ml was transferred onto two separate 250 ml flasks and incubated until plating.
2.2.3 Cell Plating
Cells were again trypsinized as mentioned above, but the resuspended pellet was made up to 10 ml with media and 25 µl from the suspension was used to count the cell number using a Neubauer double chambered haemocytometer. Cells were then diluted to 5,000 cell/100µl and plated onto either 96 well plate at 200 µl/well or 24 well plate at 500 µl/well. The plates were incubated at 37 0 C in a humidified atmosphere of 5% CO2 and 95% O2 for 24 hr before further treatment.
Twenty four hours after plating, cells were treated with different concentrations of aluminium sulphate for different durations of exposure. In order to construct a dose response curve, cells were exposed to a wide range of concentration of aluminium (0-3000 µM) for up to five days.
Stringent precautions were taken to prevent Al contamination from the reagents and materials used. Double-deionized distilled water (DDW)[Al]< 1 ng/ml assay was used to prepare all solutions and aluminium levels were measured in most reagents and were < 4 ng/ml. Containers used for sample collection and during analysis were generally polypropylene rather than glass and if so usually made Al-free by acid-washing technique (24).
Determination Of Mitochondrial Activity By Mtt Assay
Mitochondrial dehydrogenase activity was assessed based on the MTT colorimetric determination (25,26, 27). MTT was dissolved in PBS at 5 mg/ml and filtered through a sterile filter (0.2µm) to remove any amount of insoluble residue. MTT stock solution (5 mg/ml) of which 11 µl per 100µl medium was added to all the wells of an assay 96-well plate and incubated at 37 0 C in 95% O2 and 5% CO2 incubator for 1 hr. After one hour of formazan formation, the reaction was stopped by removing the MTT. The formazan crystals formed were subsequently dissolved in 150 µl DMSO/well, followed by addition of 25 µl glycine buffer (0.1 M glycine buffer, pH 10.5). To achieve complete solubilization of formazan crystals, plates were vigorously shaken on a microplate shaker at 200 rpm for 20 min. The absorbance was recorded directly, within 30 min, after the addition of DMSO using an iEMS microplate spectrophotometer (Labsystems Company) at a wavelength of 550 nm. Mitochondrial activity as a percentage of control (cytotoxicity or) was calculated relative to the calibration of the Al-free/untreated cells as follows:
These values were then used as index of cell viability.
Determination Of Cell Survival Using Fda-Pi Staining
Cell survival or active cells was determined using a fluorescence method employing fluoreseein diacetate and propidium iodide (28). Cells were plated in a 24 well plate containing 5,000 cell/well in 500µl and exposured to 1 mM aluminium sulphate 24 hr after plating. The cells were exposed for a period of not more than five days, after which, the media was removed from the cell and replaced with equal volume of Kreb's-HEPE'S buffer pH 7.4. The Kreb's-HEPE'S buffer was removed from the wells and 150 µl FDA-PI mixture (60 µg/ml-20 µg/ml) was added, followed by incubation for 3 min at room temperature. After the incubation the cells were counted using a fluorescence microscope at an excitation of wavelength of 450-490 nm and emission wavelength of 510-520 nm.
The total protein content was determined according to the method of Lowry
The media was removed from the wells and cell sample were washed with 150µl PBS, the removal of the PBS was followed by the addition of 50µl of 0.1M NaOH and the plate was frozen at -20 0 C overnight to allow adequate solubilization.
Tissue supernatant were appropriately diluted and thereafter the amount of protein determined colorimetrically at 750 nm using BSA (0-250 µg/ml) as standard and amount in the unknown sample calculated from the standard curve of BSA. The concentrations of protein in the aliquots of samples were determined by employing the above method.
Methods Of Determinations
Cells pellets were washed twice with 50 mM Tris-HCl buffer pH 7.4 to remove any traces of the media. The residue were suspended (to a suitable volume) in about 1-2 % (v/v) HNO3 and transferred immediately to a plastic sterile Al-free tubes. Aluminium content in cell pellet was determined by ETAAS as described by Taylor and Walker (30).
Determination Of Reduced Glutathione
The determination of GSH levels in biological samples requires that oxidation be minimised and the γ-glutamyltranspeptidase be inhibited. The method of Hissin and Hilf (31) was employed where GSH forms a fluoresent adduct with o-phthaldialdehyde (OPT).
Treated cells were washed twice with cold PBS and then re-suspended in acidified condition with 50 µl PCA. The samples were frozen at -20 0 C until required for analysis. 50 µl of unknown sample or standard were transferred to 1.5 ml of phosphate-EDTA buffer, followed by the addition of 50 µl OPT to all the samples. The mixtures were mixed well, protected from light and then allowed to stand at room temperature, in the dark for 30 min. Thereafter the fluorescence intensity was measured as stated above and the amount in the unknown sample calculated from the standard curve.
Measurement Of Lipid Peroxidation Using The Thiobarbituric Acid Reaction (TBARS)
Treated cell samples were washed twice with cold PBS and then resuspended in 50 mM Tri-HCl buffer pH 7.4. The samples were frozen at -20 0 C until required.
50 µl of cell samples were prepared and made up to 500 µl with DDW. 250 µl of 1.34% TBA was added to all the tubes followed by an addition of equal volume of 40% TCA. The mixture was shaken and incubated for at least 30 min. in a hot boiling water bath with a temperature > 90 0 C. Tubes were allowed to cool down after the incubation and the absorbances were read at 532 nm using zero concentration as blank. The amount of malondialdehyde formed in nmoles by the unknown samples was calculated from the standard curve (32, 33).
Determination Of Superoxide Dismutase Activity
Superoxide dismutase was determined as described by Marklund (34, 35).
Analysis Of Results
Data obtained in this research are expressed as mean ± standard error of the mean (SEM) and results tested for statistical significance (P<0.05 to P<0.001) of the differences between means by using simple student t-test or one way analysis of variance (ANOVA).
Mitochondrial Activity In Primary Cortical Astrocytes
Primary cortical astrocytes revealed a mitochondrial activity to both 1 and 3-days exposure of cell to aluminium sulphate (300-3000 µmol/L). There was however no significant effect at Al concentration up to 300 µM but a significant effect was observed at the highest dose for both day 1 and 3 as shown in figure 1.
Effects Of Aluminium On Astrocytes Viability Using Fda-Pi Staining
The number of surviving untreated primary cortical astrocytes cells was found to be greater than 95% regardless of the duration of experiment, while the number of Al treated (1000µM) cells were found to be 10% less than the untreated (Figure 2).
Effects Of Aluminium Treatment On Cell Protein Content
protein content of astrocytes
The amount of protein in cells treated with 1 mmol/L aluminium sulphate showed an increase after 1-day exposure compared to non treated cells, with no significant change after 3-days exposure (Figure 3). This is either due to the increased number of cells per well or the protein content per well.
Effects Of Aluminium On Aluminium Content
Aluminium Content Of Astrocytes
Figure 4, shows the cell associated Al accumulation in astrocytes as determined by ETAAS following Al treatment of the cells. The results indicate a response with maximum cell uptake at a dose of 300 µmol/L irrespective of duration of exposure. At a high dose (1000 µmol/L), there were few cells/well. The results were found to be significant at 100-300 µmol/L using parametric ANOVA where P value <0.001. However, a comparison between the duration of exposure was not significantly different (P>0.05).
Effects Of Aluminium Treatment On Cell Glutathione Content
Glutathione Content Of Astrocytes
The GSH content in astrocytes (Figure 5) revealed no significant changes at all levels of exposure to aluminium despite different duration of exposure.
Effects Of Aluminium Treatment On Measures Of Cellular Lipid Peroxidation
Malondialdehyde Production In Astrocytes
Aluminium treatment was associated with significant increase in the production of MDA (Figure 6) a measure of lipid peroxidation.
Flow Cytometric Analysis Of Dcfh-Da Treated Cells
The results for astrocytes (Figure 7) interesting responses that utilises only DCFH-DA alone. The astrocytes revealed consistent fluorescence intensity similar to the data obtained from microscopic viability test that uses FDA-PI.
Aluminium has been implicated in the etiology of many diseases, such as dialysis dementia, microcytic anemia with/without iron deficiency, in addition to the most controversial and a possible role it plays in Alzheimer's disease (36, 37). It is well known that aluminium can bind to phosphates and any other oxygen donating ligands leading to stable complexes, the consequence of which may be to disrupt cellular enzyme activity. One site of action may be the mitochondria thereby affecting the electron transport chain which may give rise to increased production of reactive oxygen species which eventually leads to oxidative injury (38). The exact mechanisms via which Al might exert its neurotoxic effect are yet to be substantiated. But there are several experimental studies supporting the potential of Al to promote the pro-oxidant properties of iron and other transitional elements (39,40,41,42).
Cell viability and mitochondrial dehydrogenase activity
In the present
Oxidant status assessment
However, to determine whether the increased mitochondrial activity observed due to Al exposure is indeed as a result of reactive oxygen species generation, certain enzyme markers of oxidative stress were assessed. It is well established that electron leakage from the respiratory chain can lead to formation of excess reactive oxygen species (38). Oxidative stress generated via ROS with the loss of neurones, occurs during the progression of neurodegenerative diseases particularly the ALS, AD and Parkinson's diseases (45,46,47). Glutathione is one of the first natural intracellular defences against oxidant events. A biphasic effect of Al was observed on the GSH level primary cortical astrocytes. At lower Al concentration there seemed to be an increase in the GSH level and a decrease at high doses. One might argue on the observed effect, but it is not surprising as astrocytes have been reported to contain high levels of GSH with a range between 16 nmol/mg protein (15) and 50 nmol/mg proteins (48). Relatively, the cytosolic GSH level of astroglial cultures can even reach 8 mM (17). These can however be modulated by a variety of condition and highly inhibited by the availability of glutamine (17).
The effect of Al exposure on this cell type, mitochondrial dehydrogenases, GSH thiol status and lipid peroxidation as assessed by increase malondialdehyde production was also accompanied by about 10-15% increase of superoxide dismutase.
Aluminium uptake by the cell
These studies show that the rat primary cortical astrocytes cells are able to accumulate Al against a concentration gradient. This is supported by the report of Guy
Morphologically, using FDA-PI staining, the fluorescence photographs (Picture not shown), In Al treated, primary cortical astrocytes appeared less extended, disintegrated, and spread neurites were fewer and thinner, i.e. abnormal clustering and aggregation plus loose adhesion, but the number of live cells did not change (<10%) in comparison to untreated cells. The interesting point about this finding is that aggregation of cell bodies and fasciculation of processes have been previously reported (49) in cultured cortical neurons following long-term exposures to Al. Moreover, both neuritic processes and aggregated cells could be confidently stained using an antibody to microtubule-associated protein tau, which is one of the main components of neurofibrillary tangles seen in Alzheimer's disease (49).
The potential role of Al involvement in oxidation or pro-oxidant activities remains controversial to-date. Evidence for (23, 42, 50) and against (41, 42) in its potential involvement have both been reported. The present work contributes to the existing controversies of Al antioxidant at low concentration and pro-oxidant at high concentration. Similarly, suggesting that Al might facilitate membrane peroxidation via alteration in membrane rheology by increasing their susceptibility to free radical induced damage (51, 52) as a compensatory effect due to toxic insult.
The dual or biphasic effect of Al exposure observed on this cell type is probably due to direct interaction of Al with cell components, rather than to reaction with oxidative-reactive species. This is not surprising at all because biphasic effect of Al has been reported in diverse cell system including phosphorylation of neurofilament sub-units (53). The effect may be due to the formation of reversible/irreversible Al complexes. Therefore, the present effects may be due to an indirect effect of Al on free radical scavenging enzymes and glutathione.
However, under physiological and biochemical conditions, the antioxidant effect observed with Al could only occur in the early stages of its accumulation thereby given an excellent condition to Al slow process of intoxication, concomitant to the fact that Al binds to negatively charged membranes more avidly than most divalent cations (54), in addition to the presence of iron in most cell compartments. Therefore, this suggests that the pro-oxidant effect will be more likely to be exerted