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Aluminium induced structural, metabolic alterations and protective effects of desferrioxamine in the brain tissue of mice: An FTIR study
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 99 (2012) 252–258
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Aluminium induced structural, metabolic alterations and protective effects of desferrioxamine in the brain tissue of mice: An FTIR study S. Sivakumar a,⇑, J. Sivasubramanian a, B. Raja b a b
Department of Physics, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" FTIR spectra determine the
aluminium toxicity induced changes in brain tissue. " FTIR findings demonstrate the alterations on the major biochemical constituents. " Results of the FTIR study were found to be in agreement with biochemical studies.
a r t i c l e
i n f o
Article history: Received 10 May 2012 Received in revised form 3 September 2012 Accepted 16 September 2012 Available online 26 September 2012 Keywords: FTIR Brain Aluminium
a b s t r a c t In this study, we intended to made a new approach to evaluate aluminium induced metabolic changes in mice brain tissue using Fourier transform infrared spectroscopy. Results demonstrate that FTIR can successfully indicate the molecular changes that occur in all groups. The overall findings demonstrate the alterations on the major biochemical constituents, such as lipids, proteins and nucleic acids of the brain tissues of mice. The significant decrease in the area value of amide A peak and Olefinic@CH stretching band suggests an alteration in the protein profile and lipid levels due to aluminium exposure, respectively. The significant shift in the amide I and amide II protein peaks may indicate the progression of aluminium induced Alzheimer’s disease. Further the administration of DFO significantly improved the level of protein and brought back the amide I and II peaks nearer to the control value. Histopathological results also revealed impairment of Aluminium induced alterations in brain tissue. The results of the FTIR study were found to be in agreement with biochemical studies. Ó 2012 Elsevier B.V. All rights reserved.
Introduction Aluminium (Al) is the third most prevalent element and the most abundant metal in the earth’s crust i.e., approximately 8% of total mineral components [1] which is widely distributed in the environment and extensively used in daily life resulting easy exposure to human beings [2]. From the environment it gets access to the human body via the gastrointestinal and the respiratory ⇑ Corresponding author. Mobile: +91 9865043811. E-mail address: [email protected] (S. Sivakumar). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.09.036
tracts. Aluminium is a constituent of cooking utensils and medicines such as antacids, deodorants and food additives and this has allowed its easy access into the body [3]. It has been implicated in several human neurodegenerative disorders, including Alzheimer’s disease (AD) [4,5]. Aluminium exposure also results in the production of free radicals [6]. The central nervous system is extremely vulnerable to attacks from reactive oxygen species (ROS). This is due to the brain’s high oxygen consumption rate and its abundant lipid content, as well as the relatively low levels of antioxidant enzymes in the brain in comparison with other tissues [7,8]. It is considered that a prolonged abnormality in absorption
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and/or excretion of Al leads to chronic exposure of organs including the brain to a high Al concentration. In particular, excess Al in the brain has the potential to cause encephalopathy, a progressive, generalized disorder of brain function [9,10]. Aluminium induced neurotoxicity was related to elevated brain aluminium levels and neurofibrillary tangles (NFT) which are also characteristics of Alzheimer’s disease [11]. For the last several decades the most widely used chelator in the treatment of aluminium intoxication has been desferrioxamine (DFO) [12]. The treatment commonly used in aluminium disorders is desferrioxamine (DFO), which is a chelator with great capacity to decrease Al body burden by increase its excretion in the urine. This compound is usually employed in iron accumulation and since there are chemical and physical similarities among aluminium and iron (charge, ionic radius and protein binding) it has been used in cases of aluminium accumulation [13]. Fourier transform infrared spectroscopy (FTIR) is one of the most important metabolomic tools which shows the molecular changes that occur during a pathological condition. It is widely accepted that FTIR spectroscopy is a highly sensitive tool capable of providing strong insight on structural and functional alterations of biomolecules in tissues induced by various factors. The frequency shifts shows the molecular alteration of macromolecules such as protein, lipid, carbohydrate, and nucleic acid can be considered for analysis [14]. Previously, the effect of arsenic intoxication on the brain tissue of Labeo rohita has been investigated using FTIR spectroscopy [15] Therefore, the main objective of this study was to use this molecular fingerprinting approach to investigate the effects of desferrioxamine on metabolic alterations that occur in brain tissues of aluminium intoxicated mice.
Materials and methods
Test chemicals Desferrioxamine (DesferalÒ) was purchased from Novartis and all other chemicals used in this study were of highest analytical grade obtained from Sisco Research Laboratories and Himedia. Mumbai, India. Treatment schedule Mice were randomly allocated in three different groups. Each group contains 12 animals. Group I served as Control and were
Table 1 Frequency assignments of Control Aluminium intoxicated and DFO treated spectrum. Wavenumber (cm
1
)
Control
Aluminium treated
Aluminium + DFO treated
3424
3422
3425
3015
3012
3014
2959
2957
2958
2924
2924
2923
2854
2852
2853
1733 1652
1737 1639
1735 1650
1546
1543
1543
1459 1401
1462 1408
1461 1403
1236
1237
1239
1163
1167
1161
1084
1077
1076
–
975
972
697
703
705
Animals Male Swiss albino mice, weighing 25 ± 2 g, were procured from the central animal house, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University, and were maintained in an air-conditioned room (25 ± 1 °C) with a 12 h light/12 h dark cycle. Feed and water were provided ad libitum to all the animals. The study was approved by the Institutional Animal Ethics Committee of Rajah Muthiah Medical College and Hospital (Reg No. 160/1999/CPCSEA, Proposal number: 851), Annamalai University, Annamalainagar.
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Definition of the spectral assignment
Amide A: mainly NAH stretching of proteins with the little contribution from OAH stretching of polysaccharides Olefinic@CH stretching, unsaturated fatty acids CH3 asymmetric stretch: mainly lipids CH2 asymmetric stretch: mainly lipids CH2 symmetric stretching lipids (fatty acids) Carbonyl C@O stretch: lipids Amide I: mainly C@O stretching of a- helix protein Amide II: NAH bending and CAN stretching of proteins CH2 bending: mainly lipids COO symmetric stretch: fatty acids and amino acids PO2 asymmetric stretch: mainly nucleic acids with the little contribution from phospholipids CAO asymmetric stretching of glycogen PO2 symmetric stretch: nucleic acids and phospholipids CAN+AC symmetric stretch: nucleic acids Ring breathing mode in the DNA bases
Fig. 1. FT-IR spectra of the control, aluminium intoxicated and DFO treated brain tissue of mice in the 4000–400 cm–1 range.
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fed on standard animal chow and water ad libitum. In addition to standard animal chow animals in group II and III were administered aluminium in the form of aluminium chloride (100 mg/ kg b.wt./day) orally for a period of 12 weeks. Subsequent to 12 weeks of AlCl3 administration, group III mice were treated with DFO (s.c) at the dose of 0.89 mmol/kg for 5 consecutive days. Aluminium chloride was dissolved in normal drinking water and was given by an oral gavage. Twenty-four hour after the last administration of the chelator, mice were anesthetized with diethyl ether and killed. The whole brains were immediately homogenized and used for the various biochemical assays. For FTIR analysis six whole brain samples of all three groups were freeze dried and homogenized with liquid nitrogen with an agate mortar and pestle. Samples were stored under 80 °C until used. FTIR sample preparation For FTIR analysis, the samples were mixed with KBr at ratio of 1:100. The mixture was then subjected to a pressure of 1100 kg/ cm2 to produce KBr pellets for use in FTIR Spectrometer. Pellets of the same thickness were prepared by taking the same amount of sample and applying the same pressure. Consequently, in the current study, it was possible to directly relate the intensities of the absorption bands to the quantity of the corresponding functional groups.
FTIR spectra and data analysis FTIR spectra of the region 4000–400 cm 1 were recorded at the temperature of 25 ± 1 °C on a Nicolet-Avatar-360 FTIR spectrometer equipped with an air-cooled DTGS (deuterated triglycine sulphate) and purged with nitrogen. Each sample was scanned with three different pellets under identical conditions. These replicates were averaged and then used. The spectra were analyzed using ORIGIN 6.0 software (OriginLab Corporation, Massachusetts, USA). Biochemical analysis The concentration of thiobarbituric acid reactive substances (TBARS) was estimated by the method of Wills [16]. The activities of enzymatic antioxidants super-oxide dismutase (SOD) catalase (CAT) and glutathione peroxidase (GPx) were assayed by the methods of Kakkar [17], Sinha [18], and Rotruck [19] respectively. Histological examination of brain tissues Excised brain samples were cleared of blood and immediately fixed in a neutral buffered solution of 10% formalin for 24 h. 5 lm-thick tissues section from the brain of each animal were prepared from processed paraffin-embedded samples. The brain tissue sections were stained with Hematoxylin and Eosin (H&E) for light
Fig. 2. Selected wavenumber regions of control, aluminium intoxicated and DFO treated brain tissue of mice in the range of 3800–2700 cm–1 (A); 1800–1400 cm–1 (B); 1400– 400 cm–1 (C).
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microscopic examination for the evidence of Aluminium induced changes. The cross-sectional area (CSA) of brain was evaluated from photographs of whole tissue sections taken at 100 magnification and scanned, digitized and analyzed by computer, using the Adobe Photoshop Imaging Program (Adobe System Incorporation). Statistical analysis Values are given as means ± S.D. for six mice in each group. Data were analyzed by one-way analysis of variance followed by Duncan’s Multiple Range Test (DMRT) using SPSS version 11.5 (SPSS, Chicago, IL). The limit of statistical significance was set at P < 0.05
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the system of interest and the intensity and/or more accurately the area of the absorption bands is directly related to the concentration of the molecules [20]. The spectra of all groups are shown in Fig. 1. As could be seen from the figures considerable changes are observed in the intensities of the bands. The tentative vibrational frequency assignments of absorption spectra are presented in Table 1. Fig. 2. shows the detailed spectral analyses of three distinct frequency ranges. 3800–2700 cm 1 (A); 1800–1400 cm 1 (B); and 1400–400 cm 1 (C), and observations made are described below. The frequency value changes of important functional groups and band area values are provided in Tables 2 and 3 respectively. Further, the biochemical analysis values are summarized in Table 4.
Results The FTIR spectroscopy monitors the vibration modes of functional groups present in proteins, lipids, polysaccharides, and nucleic acids. Shifts in peak positions, changes in bandwidths, intensities, and band area values of the infrared bands are used to obtain valuable structural and functional information about
Table 2 Wave number alterations of major macromolecular groups. Molecular vibration
Control
Aluminium treated
Aluminium + DFO treated
NAH stretching (proteins) Olefinic@CH (lipid unsaturation) Carbonyl C@O stretch: lipids Amide I (protein) Amide II (protein)
3424.16 ± 3.19
3422.23 ± 2.21
3425.34 ± 3.14
3015.11 ± 0.42
3012.23 ± 0.31
3014.32 ± 0.11
a
1737.71 ± 0.22
1735.43 ± 0.10a
1652.21 ± 2.18a 1546.54 ± 0.18a
1639.12 ± 1.32 1543.34 ± 0.21
1650.31 ± 1.14a 1543.40 ± 0.32
1733.13 ± 0.12
a The frequency values are shifted significantly compared with aluminium treated (P < 0.05) animal brain spectra.
Table 3 Band area alterations of macromolecular groups from quantitative analysis of spectra. Wavenumber (cm 1)
Control
Aluminium treated
Aluminium + DFO treated
3414 3015 2959 2924 2854 1733 1652 1546 1459 1401 1236 1163 1084 975 697
282.714 ± 3.224a 0.223 ± 0.985a 0.089 ± 0.089a 3.341 ± 0.876a 1.238 ± 0.498a 1.307 ± 0.036a 34.655 ± 1.678a 10.363 ± 1.134a 1.214 ± 0.453a 2.769 ± 1.195a 3.243 ± 1.045a 0.046 ± 0.009a 2.576 ± 0.067a – 0.082 ± 0.008
177.389 ± 2.012 0.086 ± 0.054 0.139 ± 0.235 4.967 ± 1.210 1.998 ± 1.011 0.538 ± 0.112 29.126 ± 1.046 4.228 ± 0.435 1.150 ± 0.068 2.025 ± 0.274 1.937 ± 0.434 0.214 ± 0.699 3.675 ± 0.757 0.149 ± 0.076 0.068 ± 0.013
213.950 ± 3.543a 0.068 ± 0.059 0.096 ± 0.064a 4.325 ± 1.043 1.827 ± 0.829 0.551 ± 0.098 18.334 ± 1.697a 3.033 ± 0.784a 0.965 ± 0.009a 1.030 ± 0.581a 1.314 ± 0.088a 0.240 ± 0.534 2.454 ± 0.645a 0.091 ± 0.121 0.91 ± 0.056
a The band area values are shifted significantly compared with aluminium treated (P < 0.05) animal brain spectra.
Infrared spectra from control, aluminium intoxicated and DFO treated tissue The selected wave number of the region (3800–2700 cm 1) as shown in Fig. 2A mainly consists of amide A vibrations of proteins. As shown in Table 1 the bands in this region arise from NAH and OAH stretching modes of proteins, polysaccharides and intermolecular hydrogen bonding [21]. Amide A appeared at 3424 cm 1 in control and 3422 cm 1 in aluminium treated brain tissue. There is no significant change in wavenumber between control and aluminium intoxicated group. But the peak area value of amide A band decreases significantly from 282.714 ± 3.224 to 177.389 ± 2.012 between control and aluminium treated brain tissue respectively. Treatment with DFO brings back the alterations to near the control band area value. There is a significant increase in DFO treated group when compared with aluminium intoxicated group as expressed in Table 3. These spectra were normalized with respect to CH2 asymmetric stretching band at 2924 cm 1. The Olefinic acid band which is due to CAH stretching mode of the HC@CH groups, can be used as a measure of unsaturation in the acyl chains act as indicator of unsaturated lipids [21]. There is no significant shift in this wavenumber in all three groups. The peak area value of Olefinic acid band decreases dramatically from 0.223 ± 0.985 to 0.086 ± 0.054. This indicates the decreases of unsaturated lipids. The peak observed at 2959 cm 1 was assigned to asymmetric stretching vibrations of methyl group. This band mainly monitors the lipids in the biological system [22]. The CH2 asymmetric band at 2924 cm 1 and CH2 symmetric band at 2854 cm 1 was caused by stretching of lipids, the frequency number of these regions was not changed significantly in all the groups. The absorptions are primarily due to proteins, with some absorbance from lipids. This wavenumber region is shown in Fig. 2B. The peak observed at 1733 cm 1 indicates C@O stretching of phospholipids in brain tissues [23]. This frequency value is shifted to higher value in aluminium treated group significantly as 1737 cm 1, and continued at 1735 cm 1 in DFO treated group, which show significant difference with aluminium intoxicated group. This region also mainly consisting of amide I and amide II wave number of proteins. Here the wavenumber shift indicates the quantitative alterations in secondary structure of proteins [24]. In the present study, the amide I is formed at 1652 cm 1 in control group, this band appeared at 1637 cm 1 in aluminium intoxicated group and
Table 4 Effect of DFO on biochemical changes in Aluminium intoxicated mice. Groups
TBARS (n moles/mg of protein)
SOD (l/mg of protein)
CAT (l/mg of protein)
Gpx (l/mg of protein)
Control Al Al + DFO
4.83 ± 0.25a 5.57 ± 0.73b 5.07 ± 0.92c
2.98 ± 0.26a 2.18 ± 0.49b 2.73 ± 0.41a
0.63 ± 0.35a 0.49 ± 0.38b 0.54 ± 0.26c
12.84 ± 0.75a 11.42 ± 0.45b 12.69 ± 0.31a
Values are expressed as mean ± S.D for six mice in each group; Values not sharing a common superscript differ significantly at P < 0.05.
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appeared at 1652 cm 1 in DFO treated group. This indicates the significant protection of amide linkage from aluminium induced damages in DFO treated group. The position of this absorption is responsive to protein conformation. The amide II absorption arises from amide NAH bending vibration coupled with CAN stretching vibration mode of the polypeptide and protein back bone. In the present study amide II was observed at 1546 cm 1 in the control brain and the wavenumber was appeared at 1543 cm 1 in aluminium treated brain tissue of mice. The wavenumber of DFO treated group also appeared at 1543 cm 1. This wavenumber is significantly shifted to lower wavenumber in aluminium treated brain tissue. The intensity of this band decreased from 10.363 ± 1.134 to 4.228 ± 0.435 and this reduction indicates the decreases of quantity of amide II protein. The amino acid side chain from peptides and proteins at 1459 cm 1 is associated with the asymmetric CH2 bending vibrations. The intensity of this band also decreased from 1.214 ± 0.453 to 1.150 ± 0.068. The peak intensity variations of these bands indicate the conformations of the side chains from peptides and proteins due to aluminium exposure. The absorption band at 1401 cm 1 is assigned as COO symmetric stretching, mainly fatty acids and amino acids from proteins. The wavenumber is shifted to higher value as 1408 cm 1. Treatment with DFO brings back the alterations to near control spectral wavenumber (1403 cm 1). The absorptions are primarily due to the existence of polysaccharides, nucleic acid, and minerals. Fig. 2C shows this wavenumber region. The strong bands at 1236 and 1084 cm 1 are mainly due to asymmetric and symmetric stretching modes, respectively, of phosphodiester groups in nucleic acids rather than in phospholipids. As shown in Fig. 2C and Table 3, the aluminium treated brain tissue spectrum displayed a decrease in the area value of phosphate asymmetric stretching band, an increase in the intensity and area of symmetric phosphate stretching band with respect to those of control tissue. In addition the phosphate symmetric bands shifted to lower frequency value. The band observed at 1170 cm 1 to CAO asymmetric stretching of glycogen. The band centred at 975 cm 1 is generally assigned to symmetric stretching mode of dianionic phosphate monoester of cellular nucleic acids, especially for DNA. Lipid peroxidation product, enzymatic antioxidants and histopathological examination Table 4 shows the effects of DFO on the levels of thiobarbituric acid reactive substances and on the activities of superoxide dismutase, catalase and glutathione peroxidase in brain tissues of aluminium intoxicated mice. The intoxicated mice exhibited a significant increase in the levels of thiobarbituric acid reactive substances. The administration of DFO reduced the levels of thiobarbituric acid reactive substances from 5.57 ± 0.73 to 5.07 ± 0.92. The activities of superoxide dismutase, catalase and glutathione peroxidase decreased significantly in intoxicated mice and the administration of DFO significantly increased these enzymatic antioxidants. Fig. 4A–C shows the control, aluminium intoxicated and effect of DFO on the histology of brain tissue in aluminium intoxicated mice, respectively. Control brain shows cortex with neural fibres and astrocytes (Fig. 4A). Fig. 4B shows the histopathological finding of aluminium intoxicated brain with diffuse cystic degeneration and inflammatory cells. Fig. 4C shows the histopathological result of astrocytic proliferation with moderate neural fibres. Discussion Aluminium (Al) is a well-known neurotoxic agent, which has been involved in neurodisorders such as Alzheimer’s disease (AD)
and other serious neurodegenerative diseases [25] Aluminium induces the production of free radicals leading to damage in lipids, proteins, and DNA in the brain [26]. This study investigates the effects of DFO on aluminium induced changes in mice brain tissue at molecular level using FT-IR spectroscopy. The FTIR analysis explores the vibration of functional groups present in macromolecules and shows the molecular structural changes of the molecules through shifts in wavenumbers. In this study, the peak area value of amide A band decreases significantly. The signal intensity, or more accurately, the band area gives information about the concentration of related functional groups [20]. The significant decrease in the intensity of the amide A band reflects the decreases in the quantity of protein. This indicates the damage of amide linkage from free radical induced damages in Aluminium treated group and this was confirmed biochemically. Al has no redox capacity in biological systems. However, extensive experimental evidence both, in vitro and in vivo, demonstrates that high Al concentrations cause oxidative stress. Increased oxidative stress is the consequence of either enhanced reactive oxygen species (ROS) production or attenuated ROS scavenging capacity, resulting in tissue damage that in most instances is assessed by the measurement of lipid peroxides [20]. The nervous system is particularly sensitive to oxidant-mediated damage because of brain antioxidant enzymes (catalase, superoxide dismutase, and gluthatione peroxidase) activities are comparatively lower than those found in other tissues [1]. Makrides [27] suggested that free radical damage can cause a reduction in protein synthesis. So the reduction of protein quantity in these samples may be the cause of free radical damage. The increased protein quantity is supporting the protective effect of antioxidant against free radical damage [28]. As can be seen from Table 4, there was a dramatic reduction in enzymatic antioxidants and increased in the level of thiobarbituric acid reactive substances in aluminium intoxicated brain tissue. So the reduction of protein quantity may be the imbalance between oxidants and antioxidants. DFO is also a useful therapeutic tool in blocking ROS production [29]. In the present study administration of DFO bring back the protein level near to control value. Al has also been shown to elevate lipid peroxidation and cause changes in antioxidant defense system in brain [30]. Unsaturated lipids are highly vulnerable to oxidative attack because of their double bond content and the increased degradation of PUFAs in AD may be evidence of lipid peroxidation [31]. Impairments in Polyunsaturated fatty acids metabolism have been concerned in many neurological diseases, including Alzheimer’s disease (AD). FT-IR is usefulness in monitoring unsaturated lipid content by utilizing the Olefinic@CH stretching mode 3012 cm 1 [20]. The unsaturated Olefinic C@CAH stretching vibration, which has a unique vibrational frequency of 3012 cm 1 and is well-separated and distinguishable from the saturated aliphatic peaks [32]. In the present study the quantity of unsaturated fatty acid decreased significantly in aluminium intoxicated brain tissue. It is clear that amyloid plaque formation reduces unsaturated lipid content in the brain, impacting brain growth, membrane fluidity, signal transduction, and cognitive development. So the reduction of unsaturated fatty acid may be related to the progression of Alzheimer’s disease [33]. Decreases of the peak area of CH3 asymmetric stretching vibration at 2959 cm 1 indicates a change in the composition of the acyl chains [34]. The peak area of the CH3 asymmetric stretching vibration at 2957 cm 1 increased as a consequence of aluminium intoxication. So this increase may also indicate a change in the composition of the acyl chains. CH2 asymmetric and CH2 symmetric stretching of lipids shows no alterations in frequency. The frequency of the CH2 bands of acyl chains depends on the degree of conformational disorder and level of flexibility. The position of these bands provides information about the lipid acyl chain flexibility (order/disorder state of lipids) [35]. The area of the symmet-
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ric CH2 stretching band was found to be significantly increased in the aluminium treated tissues. This result suggested an increased proportion of the CH2 groups in the aluminium treated brain tissues. Fig. 3A shows the mean ratio of the methyl and the methylene band. The mean ratio (I2924/I2854) of the intensity of absorption of the methyl band and the methylene band for control and aluminium intoxicated brain tissues are, respectively, 2.701 ± 0.004 and 2.486 ± 0.005. The decreased ratio indicates a decrease in the number of methyl group compared to methylene group in the aluminium intoxicated brain tissues. The increased ratio further indicates the contribution for high number of protein fibres [14]. In this study the decreased ratio may indicates the contribution of low number of protein fibres. Fig. 3B shows the mean ratio of CH3 asymmetric and CH2 symmetric band. The ratio of CH3 asymmetric band to CH2 symmetric band (I2959/I2854) also decreased from 0.071 ± 0.005 (control) to 0.069 ± 0.004 (aluminium) observed in the present study further confirms the decrease in the total protein content in the aluminium intoxicated brain tissues. The peak area of Carbonyl C@O stretching vibration of lipids at 1733 cm 1 is decreased in aluminium intoxicated brain tissues, which suggests a decreased proportion of unsaturated acyl chains in the intoxicated brain tissues [36]. This change is most probably the result of a change in lipid metabolism induced by aluminium toxicity. This is supported by the reduced intensity of Olefinic@CH stretching band observed at 3015 cm 1. Amide I and II vibrations are influenced by secondary structure of the protein, since this involves protein folding with hydrogen boning between peptide bonds. Characteristic infrared absorption bonds of peptide linkages at these amide I and amide II regions correspond to the alpha-helix protein structure [37]. Rice-Evans et al. [38] suggest that all the constituent amino acid side chains in proteins are susceptible to free radicals, but some are more vulnerable than others. Thus, exposure of proteins to free radical-generating systems may induce secondary structural changes, since secondary structure is stabilized by hydrogen bonding of peptide backbone, and interference with the functional groups of the peptide bonds
may cause secondary structural modifications. They have postulated that the amide I and II region shifts corresponding to the alpha -helix protein conformational change. As it is clearly seen in Table 3, the area values of the amides I and II, consequently the amount of proteins, decreased in the aluminium intoxicated brain tissues. This decrease was consistent with the decrease in amide A band (at 3424 cm 1) due to proteins. This decreased quantity may be due to the increment of peroxidation and the reduction of free radical scavengers. This result is supported by biochemical studies that revealed a significant increase in level of TBARS and the reduced activities of enzymatic antioxidants. This decreased intensities of the amide I and II bands could be interpreted as the result of change of the protein synthesis and the protein structure due to aluminium intoxication. In addition the wave number of amide I band shifted to 1637 cm 1. Dialysis patients subjected to prolonged aluminium exposure developed AD-like change in the processing of tau protein and b-amyloid increase [39]. Aluminium induces ubiquitin and b-amyloid in neuroblastoma cells [40]. Changes in peak position of amide bands may quantitatively reflect alterations in the composition of protein secondary structure [41]. The amide I peak frequency is 1652–1654 cm 1, indicative of proteins predominantly in an a-helical and/or unordered structure [42]. The frequency is shifted to 1632–1634 cm 1, indicating the presence of proteins with a substantially different (b-sheet) conformation. In the present study amide I peak position significantly shifted to 1639 cm 1 and amide II peak also shifted from 1546 to 1543 cm 1. This shifted value (amide I and amide II peak at 1639 cm 1 and 1543 cm 1, respectively) confirms the predominant exist of b-sheet. These results strongly suggest that this feature is the amyloid core of a mature neuritic plaque [43]. So in this study the wavenumber shift may indicates the progression of Alzheimer’s disease. Further, the administration of DFO improves the a-helix structure and brings back the wavenumber nearer to control value. The asymmetric and symmetric phosphate stretching bands at 1236 and 1084 cm 1, respectively, originated mainly due to the phosphodiester backbone of cellular nucleic acids [44]. The band
B
protein fibers
A
257
Fig. 3. The mean ratio of methyl and the methylene band (A), CH3 asymmetric and CH2 symmetric band (B).
Fig. 4. Representative photomicrograph of histological changes in brain: (A) control, cortex with neural fibres and astrocytes; (B) aluminium intoxicated, there was diffuse cystic degeneration and inflammatory cells; and (C) Al + DFO, shows astrocytic proliferation with moderate neural fibres.
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centred at 975 cm 1 is generally assigned to cellular nucleic acids, especially for DNA. This peak was not observed in control brain sample but present in aluminium intoxicated brain tissue. These results implied an increase in the relative content of the nucleic acids in the treated tissues [45]. The generation of ROS can damage lipids, proteins and even DNA [46]. Vijay Kumar et al. [47] reveal that chronic aluminium exposure might be implicated in mitochondrial DNA oxidation leading to the increased expression of p53 gene. So, the peak observed at 975 cm 1 may be due to the exposure of aluminium on DNA. Conclusions FTIR spectra reveals significant differences in absorbance intensities between the control and aluminium intoxicated brain tissues. Further, the administration of DFO significantly improves the levels of biochemical constituents. The observed results suggest that quantity of protein significantly decreased due to the decreased level of free radical scavengers. Quantity of unsaturated fatty acid decreased may indicate the increases of lipid peroxidation and progression of aluminium induced Alzheimer’s disease. The peak positions of amide I and II shifted to lowered value in aluminium intoxicated tissue may be due to the amyloid core of a mature neuritic plaque. In addition FTIR results were found to be in agreement with biochemical and previous studies. Finally the present study shows that the FTIR spectroscopy is a very informative technique to determine the aluminium toxicity induced changes in brain tissue at molecular level. Acknowledgment The financial support to Mr. J. Sivasubramanian as Project fellow from the University Grants Commission, New Delhi, India is gratefully acknowledged. Reference [1] [2] [3] [4]
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Aluminium induced structural, metabolic alterations and protective effects of desferrioxamine in the brain tissue of mice: An FTIR study S. Sivakumar a,⇑, J. Sivasubramanian a, B. Raja b a b
Department of Physics, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" FTIR spectra determine the
aluminium toxicity induced changes in brain tissue. " FTIR findings demonstrate the alterations on the major biochemical constituents. " Results of the FTIR study were found to be in agreement with biochemical studies.
a r t i c l e
i n f o
Article history: Received 10 May 2012 Received in revised form 3 September 2012 Accepted 16 September 2012 Available online 26 September 2012 Keywords: FTIR Brain Aluminium
a b s t r a c t In this study, we intended to made a new approach to evaluate aluminium induced metabolic changes in mice brain tissue using Fourier transform infrared spectroscopy. Results demonstrate that FTIR can successfully indicate the molecular changes that occur in all groups. The overall findings demonstrate the alterations on the major biochemical constituents, such as lipids, proteins and nucleic acids of the brain tissues of mice. The significant decrease in the area value of amide A peak and Olefinic@CH stretching band suggests an alteration in the protein profile and lipid levels due to aluminium exposure, respectively. The significant shift in the amide I and amide II protein peaks may indicate the progression of aluminium induced Alzheimer’s disease. Further the administration of DFO significantly improved the level of protein and brought back the amide I and II peaks nearer to the control value. Histopathological results also revealed impairment of Aluminium induced alterations in brain tissue. The results of the FTIR study were found to be in agreement with biochemical studies. Ó 2012 Elsevier B.V. All rights reserved.
Introduction Aluminium (Al) is the third most prevalent element and the most abundant metal in the earth’s crust i.e., approximately 8% of total mineral components [1] which is widely distributed in the environment and extensively used in daily life resulting easy exposure to human beings [2]. From the environment it gets access to the human body via the gastrointestinal and the respiratory ⇑ Corresponding author. Mobile: +91 9865043811. E-mail address: [email protected] (S. Sivakumar). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.09.036
tracts. Aluminium is a constituent of cooking utensils and medicines such as antacids, deodorants and food additives and this has allowed its easy access into the body [3]. It has been implicated in several human neurodegenerative disorders, including Alzheimer’s disease (AD) [4,5]. Aluminium exposure also results in the production of free radicals [6]. The central nervous system is extremely vulnerable to attacks from reactive oxygen species (ROS). This is due to the brain’s high oxygen consumption rate and its abundant lipid content, as well as the relatively low levels of antioxidant enzymes in the brain in comparison with other tissues [7,8]. It is considered that a prolonged abnormality in absorption
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and/or excretion of Al leads to chronic exposure of organs including the brain to a high Al concentration. In particular, excess Al in the brain has the potential to cause encephalopathy, a progressive, generalized disorder of brain function [9,10]. Aluminium induced neurotoxicity was related to elevated brain aluminium levels and neurofibrillary tangles (NFT) which are also characteristics of Alzheimer’s disease [11]. For the last several decades the most widely used chelator in the treatment of aluminium intoxication has been desferrioxamine (DFO) [12]. The treatment commonly used in aluminium disorders is desferrioxamine (DFO), which is a chelator with great capacity to decrease Al body burden by increase its excretion in the urine. This compound is usually employed in iron accumulation and since there are chemical and physical similarities among aluminium and iron (charge, ionic radius and protein binding) it has been used in cases of aluminium accumulation [13]. Fourier transform infrared spectroscopy (FTIR) is one of the most important metabolomic tools which shows the molecular changes that occur during a pathological condition. It is widely accepted that FTIR spectroscopy is a highly sensitive tool capable of providing strong insight on structural and functional alterations of biomolecules in tissues induced by various factors. The frequency shifts shows the molecular alteration of macromolecules such as protein, lipid, carbohydrate, and nucleic acid can be considered for analysis [14]. Previously, the effect of arsenic intoxication on the brain tissue of Labeo rohita has been investigated using FTIR spectroscopy [15] Therefore, the main objective of this study was to use this molecular fingerprinting approach to investigate the effects of desferrioxamine on metabolic alterations that occur in brain tissues of aluminium intoxicated mice.
Materials and methods
Test chemicals Desferrioxamine (DesferalÒ) was purchased from Novartis and all other chemicals used in this study were of highest analytical grade obtained from Sisco Research Laboratories and Himedia. Mumbai, India. Treatment schedule Mice were randomly allocated in three different groups. Each group contains 12 animals. Group I served as Control and were
Table 1 Frequency assignments of Control Aluminium intoxicated and DFO treated spectrum. Wavenumber (cm
1
)
Control
Aluminium treated
Aluminium + DFO treated
3424
3422
3425
3015
3012
3014
2959
2957
2958
2924
2924
2923
2854
2852
2853
1733 1652
1737 1639
1735 1650
1546
1543
1543
1459 1401
1462 1408
1461 1403
1236
1237
1239
1163
1167
1161
1084
1077
1076
–
975
972
697
703
705
Animals Male Swiss albino mice, weighing 25 ± 2 g, were procured from the central animal house, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University, and were maintained in an air-conditioned room (25 ± 1 °C) with a 12 h light/12 h dark cycle. Feed and water were provided ad libitum to all the animals. The study was approved by the Institutional Animal Ethics Committee of Rajah Muthiah Medical College and Hospital (Reg No. 160/1999/CPCSEA, Proposal number: 851), Annamalai University, Annamalainagar.
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Definition of the spectral assignment
Amide A: mainly NAH stretching of proteins with the little contribution from OAH stretching of polysaccharides Olefinic@CH stretching, unsaturated fatty acids CH3 asymmetric stretch: mainly lipids CH2 asymmetric stretch: mainly lipids CH2 symmetric stretching lipids (fatty acids) Carbonyl C@O stretch: lipids Amide I: mainly C@O stretching of a- helix protein Amide II: NAH bending and CAN stretching of proteins CH2 bending: mainly lipids COO symmetric stretch: fatty acids and amino acids PO2 asymmetric stretch: mainly nucleic acids with the little contribution from phospholipids CAO asymmetric stretching of glycogen PO2 symmetric stretch: nucleic acids and phospholipids CAN+AC symmetric stretch: nucleic acids Ring breathing mode in the DNA bases
Fig. 1. FT-IR spectra of the control, aluminium intoxicated and DFO treated brain tissue of mice in the 4000–400 cm–1 range.
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fed on standard animal chow and water ad libitum. In addition to standard animal chow animals in group II and III were administered aluminium in the form of aluminium chloride (100 mg/ kg b.wt./day) orally for a period of 12 weeks. Subsequent to 12 weeks of AlCl3 administration, group III mice were treated with DFO (s.c) at the dose of 0.89 mmol/kg for 5 consecutive days. Aluminium chloride was dissolved in normal drinking water and was given by an oral gavage. Twenty-four hour after the last administration of the chelator, mice were anesthetized with diethyl ether and killed. The whole brains were immediately homogenized and used for the various biochemical assays. For FTIR analysis six whole brain samples of all three groups were freeze dried and homogenized with liquid nitrogen with an agate mortar and pestle. Samples were stored under 80 °C until used. FTIR sample preparation For FTIR analysis, the samples were mixed with KBr at ratio of 1:100. The mixture was then subjected to a pressure of 1100 kg/ cm2 to produce KBr pellets for use in FTIR Spectrometer. Pellets of the same thickness were prepared by taking the same amount of sample and applying the same pressure. Consequently, in the current study, it was possible to directly relate the intensities of the absorption bands to the quantity of the corresponding functional groups.
FTIR spectra and data analysis FTIR spectra of the region 4000–400 cm 1 were recorded at the temperature of 25 ± 1 °C on a Nicolet-Avatar-360 FTIR spectrometer equipped with an air-cooled DTGS (deuterated triglycine sulphate) and purged with nitrogen. Each sample was scanned with three different pellets under identical conditions. These replicates were averaged and then used. The spectra were analyzed using ORIGIN 6.0 software (OriginLab Corporation, Massachusetts, USA). Biochemical analysis The concentration of thiobarbituric acid reactive substances (TBARS) was estimated by the method of Wills [16]. The activities of enzymatic antioxidants super-oxide dismutase (SOD) catalase (CAT) and glutathione peroxidase (GPx) were assayed by the methods of Kakkar [17], Sinha [18], and Rotruck [19] respectively. Histological examination of brain tissues Excised brain samples were cleared of blood and immediately fixed in a neutral buffered solution of 10% formalin for 24 h. 5 lm-thick tissues section from the brain of each animal were prepared from processed paraffin-embedded samples. The brain tissue sections were stained with Hematoxylin and Eosin (H&E) for light
Fig. 2. Selected wavenumber regions of control, aluminium intoxicated and DFO treated brain tissue of mice in the range of 3800–2700 cm–1 (A); 1800–1400 cm–1 (B); 1400– 400 cm–1 (C).
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microscopic examination for the evidence of Aluminium induced changes. The cross-sectional area (CSA) of brain was evaluated from photographs of whole tissue sections taken at 100 magnification and scanned, digitized and analyzed by computer, using the Adobe Photoshop Imaging Program (Adobe System Incorporation). Statistical analysis Values are given as means ± S.D. for six mice in each group. Data were analyzed by one-way analysis of variance followed by Duncan’s Multiple Range Test (DMRT) using SPSS version 11.5 (SPSS, Chicago, IL). The limit of statistical significance was set at P < 0.05
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the system of interest and the intensity and/or more accurately the area of the absorption bands is directly related to the concentration of the molecules [20]. The spectra of all groups are shown in Fig. 1. As could be seen from the figures considerable changes are observed in the intensities of the bands. The tentative vibrational frequency assignments of absorption spectra are presented in Table 1. Fig. 2. shows the detailed spectral analyses of three distinct frequency ranges. 3800–2700 cm 1 (A); 1800–1400 cm 1 (B); and 1400–400 cm 1 (C), and observations made are described below. The frequency value changes of important functional groups and band area values are provided in Tables 2 and 3 respectively. Further, the biochemical analysis values are summarized in Table 4.
Results The FTIR spectroscopy monitors the vibration modes of functional groups present in proteins, lipids, polysaccharides, and nucleic acids. Shifts in peak positions, changes in bandwidths, intensities, and band area values of the infrared bands are used to obtain valuable structural and functional information about
Table 2 Wave number alterations of major macromolecular groups. Molecular vibration
Control
Aluminium treated
Aluminium + DFO treated
NAH stretching (proteins) Olefinic@CH (lipid unsaturation) Carbonyl C@O stretch: lipids Amide I (protein) Amide II (protein)
3424.16 ± 3.19
3422.23 ± 2.21
3425.34 ± 3.14
3015.11 ± 0.42
3012.23 ± 0.31
3014.32 ± 0.11
a
1737.71 ± 0.22
1735.43 ± 0.10a
1652.21 ± 2.18a 1546.54 ± 0.18a
1639.12 ± 1.32 1543.34 ± 0.21
1650.31 ± 1.14a 1543.40 ± 0.32
1733.13 ± 0.12
a The frequency values are shifted significantly compared with aluminium treated (P < 0.05) animal brain spectra.
Table 3 Band area alterations of macromolecular groups from quantitative analysis of spectra. Wavenumber (cm 1)
Control
Aluminium treated
Aluminium + DFO treated
3414 3015 2959 2924 2854 1733 1652 1546 1459 1401 1236 1163 1084 975 697
282.714 ± 3.224a 0.223 ± 0.985a 0.089 ± 0.089a 3.341 ± 0.876a 1.238 ± 0.498a 1.307 ± 0.036a 34.655 ± 1.678a 10.363 ± 1.134a 1.214 ± 0.453a 2.769 ± 1.195a 3.243 ± 1.045a 0.046 ± 0.009a 2.576 ± 0.067a – 0.082 ± 0.008
177.389 ± 2.012 0.086 ± 0.054 0.139 ± 0.235 4.967 ± 1.210 1.998 ± 1.011 0.538 ± 0.112 29.126 ± 1.046 4.228 ± 0.435 1.150 ± 0.068 2.025 ± 0.274 1.937 ± 0.434 0.214 ± 0.699 3.675 ± 0.757 0.149 ± 0.076 0.068 ± 0.013
213.950 ± 3.543a 0.068 ± 0.059 0.096 ± 0.064a 4.325 ± 1.043 1.827 ± 0.829 0.551 ± 0.098 18.334 ± 1.697a 3.033 ± 0.784a 0.965 ± 0.009a 1.030 ± 0.581a 1.314 ± 0.088a 0.240 ± 0.534 2.454 ± 0.645a 0.091 ± 0.121 0.91 ± 0.056
a The band area values are shifted significantly compared with aluminium treated (P < 0.05) animal brain spectra.
Infrared spectra from control, aluminium intoxicated and DFO treated tissue The selected wave number of the region (3800–2700 cm 1) as shown in Fig. 2A mainly consists of amide A vibrations of proteins. As shown in Table 1 the bands in this region arise from NAH and OAH stretching modes of proteins, polysaccharides and intermolecular hydrogen bonding [21]. Amide A appeared at 3424 cm 1 in control and 3422 cm 1 in aluminium treated brain tissue. There is no significant change in wavenumber between control and aluminium intoxicated group. But the peak area value of amide A band decreases significantly from 282.714 ± 3.224 to 177.389 ± 2.012 between control and aluminium treated brain tissue respectively. Treatment with DFO brings back the alterations to near the control band area value. There is a significant increase in DFO treated group when compared with aluminium intoxicated group as expressed in Table 3. These spectra were normalized with respect to CH2 asymmetric stretching band at 2924 cm 1. The Olefinic acid band which is due to CAH stretching mode of the HC@CH groups, can be used as a measure of unsaturation in the acyl chains act as indicator of unsaturated lipids [21]. There is no significant shift in this wavenumber in all three groups. The peak area value of Olefinic acid band decreases dramatically from 0.223 ± 0.985 to 0.086 ± 0.054. This indicates the decreases of unsaturated lipids. The peak observed at 2959 cm 1 was assigned to asymmetric stretching vibrations of methyl group. This band mainly monitors the lipids in the biological system [22]. The CH2 asymmetric band at 2924 cm 1 and CH2 symmetric band at 2854 cm 1 was caused by stretching of lipids, the frequency number of these regions was not changed significantly in all the groups. The absorptions are primarily due to proteins, with some absorbance from lipids. This wavenumber region is shown in Fig. 2B. The peak observed at 1733 cm 1 indicates C@O stretching of phospholipids in brain tissues [23]. This frequency value is shifted to higher value in aluminium treated group significantly as 1737 cm 1, and continued at 1735 cm 1 in DFO treated group, which show significant difference with aluminium intoxicated group. This region also mainly consisting of amide I and amide II wave number of proteins. Here the wavenumber shift indicates the quantitative alterations in secondary structure of proteins [24]. In the present study, the amide I is formed at 1652 cm 1 in control group, this band appeared at 1637 cm 1 in aluminium intoxicated group and
Table 4 Effect of DFO on biochemical changes in Aluminium intoxicated mice. Groups
TBARS (n moles/mg of protein)
SOD (l/mg of protein)
CAT (l/mg of protein)
Gpx (l/mg of protein)
Control Al Al + DFO
4.83 ± 0.25a 5.57 ± 0.73b 5.07 ± 0.92c
2.98 ± 0.26a 2.18 ± 0.49b 2.73 ± 0.41a
0.63 ± 0.35a 0.49 ± 0.38b 0.54 ± 0.26c
12.84 ± 0.75a 11.42 ± 0.45b 12.69 ± 0.31a
Values are expressed as mean ± S.D for six mice in each group; Values not sharing a common superscript differ significantly at P < 0.05.
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appeared at 1652 cm 1 in DFO treated group. This indicates the significant protection of amide linkage from aluminium induced damages in DFO treated group. The position of this absorption is responsive to protein conformation. The amide II absorption arises from amide NAH bending vibration coupled with CAN stretching vibration mode of the polypeptide and protein back bone. In the present study amide II was observed at 1546 cm 1 in the control brain and the wavenumber was appeared at 1543 cm 1 in aluminium treated brain tissue of mice. The wavenumber of DFO treated group also appeared at 1543 cm 1. This wavenumber is significantly shifted to lower wavenumber in aluminium treated brain tissue. The intensity of this band decreased from 10.363 ± 1.134 to 4.228 ± 0.435 and this reduction indicates the decreases of quantity of amide II protein. The amino acid side chain from peptides and proteins at 1459 cm 1 is associated with the asymmetric CH2 bending vibrations. The intensity of this band also decreased from 1.214 ± 0.453 to 1.150 ± 0.068. The peak intensity variations of these bands indicate the conformations of the side chains from peptides and proteins due to aluminium exposure. The absorption band at 1401 cm 1 is assigned as COO symmetric stretching, mainly fatty acids and amino acids from proteins. The wavenumber is shifted to higher value as 1408 cm 1. Treatment with DFO brings back the alterations to near control spectral wavenumber (1403 cm 1). The absorptions are primarily due to the existence of polysaccharides, nucleic acid, and minerals. Fig. 2C shows this wavenumber region. The strong bands at 1236 and 1084 cm 1 are mainly due to asymmetric and symmetric stretching modes, respectively, of phosphodiester groups in nucleic acids rather than in phospholipids. As shown in Fig. 2C and Table 3, the aluminium treated brain tissue spectrum displayed a decrease in the area value of phosphate asymmetric stretching band, an increase in the intensity and area of symmetric phosphate stretching band with respect to those of control tissue. In addition the phosphate symmetric bands shifted to lower frequency value. The band observed at 1170 cm 1 to CAO asymmetric stretching of glycogen. The band centred at 975 cm 1 is generally assigned to symmetric stretching mode of dianionic phosphate monoester of cellular nucleic acids, especially for DNA. Lipid peroxidation product, enzymatic antioxidants and histopathological examination Table 4 shows the effects of DFO on the levels of thiobarbituric acid reactive substances and on the activities of superoxide dismutase, catalase and glutathione peroxidase in brain tissues of aluminium intoxicated mice. The intoxicated mice exhibited a significant increase in the levels of thiobarbituric acid reactive substances. The administration of DFO reduced the levels of thiobarbituric acid reactive substances from 5.57 ± 0.73 to 5.07 ± 0.92. The activities of superoxide dismutase, catalase and glutathione peroxidase decreased significantly in intoxicated mice and the administration of DFO significantly increased these enzymatic antioxidants. Fig. 4A–C shows the control, aluminium intoxicated and effect of DFO on the histology of brain tissue in aluminium intoxicated mice, respectively. Control brain shows cortex with neural fibres and astrocytes (Fig. 4A). Fig. 4B shows the histopathological finding of aluminium intoxicated brain with diffuse cystic degeneration and inflammatory cells. Fig. 4C shows the histopathological result of astrocytic proliferation with moderate neural fibres. Discussion Aluminium (Al) is a well-known neurotoxic agent, which has been involved in neurodisorders such as Alzheimer’s disease (AD)
and other serious neurodegenerative diseases [25] Aluminium induces the production of free radicals leading to damage in lipids, proteins, and DNA in the brain [26]. This study investigates the effects of DFO on aluminium induced changes in mice brain tissue at molecular level using FT-IR spectroscopy. The FTIR analysis explores the vibration of functional groups present in macromolecules and shows the molecular structural changes of the molecules through shifts in wavenumbers. In this study, the peak area value of amide A band decreases significantly. The signal intensity, or more accurately, the band area gives information about the concentration of related functional groups [20]. The significant decrease in the intensity of the amide A band reflects the decreases in the quantity of protein. This indicates the damage of amide linkage from free radical induced damages in Aluminium treated group and this was confirmed biochemically. Al has no redox capacity in biological systems. However, extensive experimental evidence both, in vitro and in vivo, demonstrates that high Al concentrations cause oxidative stress. Increased oxidative stress is the consequence of either enhanced reactive oxygen species (ROS) production or attenuated ROS scavenging capacity, resulting in tissue damage that in most instances is assessed by the measurement of lipid peroxides [20]. The nervous system is particularly sensitive to oxidant-mediated damage because of brain antioxidant enzymes (catalase, superoxide dismutase, and gluthatione peroxidase) activities are comparatively lower than those found in other tissues [1]. Makrides [27] suggested that free radical damage can cause a reduction in protein synthesis. So the reduction of protein quantity in these samples may be the cause of free radical damage. The increased protein quantity is supporting the protective effect of antioxidant against free radical damage [28]. As can be seen from Table 4, there was a dramatic reduction in enzymatic antioxidants and increased in the level of thiobarbituric acid reactive substances in aluminium intoxicated brain tissue. So the reduction of protein quantity may be the imbalance between oxidants and antioxidants. DFO is also a useful therapeutic tool in blocking ROS production [29]. In the present study administration of DFO bring back the protein level near to control value. Al has also been shown to elevate lipid peroxidation and cause changes in antioxidant defense system in brain [30]. Unsaturated lipids are highly vulnerable to oxidative attack because of their double bond content and the increased degradation of PUFAs in AD may be evidence of lipid peroxidation [31]. Impairments in Polyunsaturated fatty acids metabolism have been concerned in many neurological diseases, including Alzheimer’s disease (AD). FT-IR is usefulness in monitoring unsaturated lipid content by utilizing the Olefinic@CH stretching mode 3012 cm 1 [20]. The unsaturated Olefinic C@CAH stretching vibration, which has a unique vibrational frequency of 3012 cm 1 and is well-separated and distinguishable from the saturated aliphatic peaks [32]. In the present study the quantity of unsaturated fatty acid decreased significantly in aluminium intoxicated brain tissue. It is clear that amyloid plaque formation reduces unsaturated lipid content in the brain, impacting brain growth, membrane fluidity, signal transduction, and cognitive development. So the reduction of unsaturated fatty acid may be related to the progression of Alzheimer’s disease [33]. Decreases of the peak area of CH3 asymmetric stretching vibration at 2959 cm 1 indicates a change in the composition of the acyl chains [34]. The peak area of the CH3 asymmetric stretching vibration at 2957 cm 1 increased as a consequence of aluminium intoxication. So this increase may also indicate a change in the composition of the acyl chains. CH2 asymmetric and CH2 symmetric stretching of lipids shows no alterations in frequency. The frequency of the CH2 bands of acyl chains depends on the degree of conformational disorder and level of flexibility. The position of these bands provides information about the lipid acyl chain flexibility (order/disorder state of lipids) [35]. The area of the symmet-
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ric CH2 stretching band was found to be significantly increased in the aluminium treated tissues. This result suggested an increased proportion of the CH2 groups in the aluminium treated brain tissues. Fig. 3A shows the mean ratio of the methyl and the methylene band. The mean ratio (I2924/I2854) of the intensity of absorption of the methyl band and the methylene band for control and aluminium intoxicated brain tissues are, respectively, 2.701 ± 0.004 and 2.486 ± 0.005. The decreased ratio indicates a decrease in the number of methyl group compared to methylene group in the aluminium intoxicated brain tissues. The increased ratio further indicates the contribution for high number of protein fibres [14]. In this study the decreased ratio may indicates the contribution of low number of protein fibres. Fig. 3B shows the mean ratio of CH3 asymmetric and CH2 symmetric band. The ratio of CH3 asymmetric band to CH2 symmetric band (I2959/I2854) also decreased from 0.071 ± 0.005 (control) to 0.069 ± 0.004 (aluminium) observed in the present study further confirms the decrease in the total protein content in the aluminium intoxicated brain tissues. The peak area of Carbonyl C@O stretching vibration of lipids at 1733 cm 1 is decreased in aluminium intoxicated brain tissues, which suggests a decreased proportion of unsaturated acyl chains in the intoxicated brain tissues [36]. This change is most probably the result of a change in lipid metabolism induced by aluminium toxicity. This is supported by the reduced intensity of Olefinic@CH stretching band observed at 3015 cm 1. Amide I and II vibrations are influenced by secondary structure of the protein, since this involves protein folding with hydrogen boning between peptide bonds. Characteristic infrared absorption bonds of peptide linkages at these amide I and amide II regions correspond to the alpha-helix protein structure [37]. Rice-Evans et al. [38] suggest that all the constituent amino acid side chains in proteins are susceptible to free radicals, but some are more vulnerable than others. Thus, exposure of proteins to free radical-generating systems may induce secondary structural changes, since secondary structure is stabilized by hydrogen bonding of peptide backbone, and interference with the functional groups of the peptide bonds
may cause secondary structural modifications. They have postulated that the amide I and II region shifts corresponding to the alpha -helix protein conformational change. As it is clearly seen in Table 3, the area values of the amides I and II, consequently the amount of proteins, decreased in the aluminium intoxicated brain tissues. This decrease was consistent with the decrease in amide A band (at 3424 cm 1) due to proteins. This decreased quantity may be due to the increment of peroxidation and the reduction of free radical scavengers. This result is supported by biochemical studies that revealed a significant increase in level of TBARS and the reduced activities of enzymatic antioxidants. This decreased intensities of the amide I and II bands could be interpreted as the result of change of the protein synthesis and the protein structure due to aluminium intoxication. In addition the wave number of amide I band shifted to 1637 cm 1. Dialysis patients subjected to prolonged aluminium exposure developed AD-like change in the processing of tau protein and b-amyloid increase [39]. Aluminium induces ubiquitin and b-amyloid in neuroblastoma cells [40]. Changes in peak position of amide bands may quantitatively reflect alterations in the composition of protein secondary structure [41]. The amide I peak frequency is 1652–1654 cm 1, indicative of proteins predominantly in an a-helical and/or unordered structure [42]. The frequency is shifted to 1632–1634 cm 1, indicating the presence of proteins with a substantially different (b-sheet) conformation. In the present study amide I peak position significantly shifted to 1639 cm 1 and amide II peak also shifted from 1546 to 1543 cm 1. This shifted value (amide I and amide II peak at 1639 cm 1 and 1543 cm 1, respectively) confirms the predominant exist of b-sheet. These results strongly suggest that this feature is the amyloid core of a mature neuritic plaque [43]. So in this study the wavenumber shift may indicates the progression of Alzheimer’s disease. Further, the administration of DFO improves the a-helix structure and brings back the wavenumber nearer to control value. The asymmetric and symmetric phosphate stretching bands at 1236 and 1084 cm 1, respectively, originated mainly due to the phosphodiester backbone of cellular nucleic acids [44]. The band
B
protein fibers
A
257
Fig. 3. The mean ratio of methyl and the methylene band (A), CH3 asymmetric and CH2 symmetric band (B).
Fig. 4. Representative photomicrograph of histological changes in brain: (A) control, cortex with neural fibres and astrocytes; (B) aluminium intoxicated, there was diffuse cystic degeneration and inflammatory cells; and (C) Al + DFO, shows astrocytic proliferation with moderate neural fibres.
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centred at 975 cm 1 is generally assigned to cellular nucleic acids, especially for DNA. This peak was not observed in control brain sample but present in aluminium intoxicated brain tissue. These results implied an increase in the relative content of the nucleic acids in the treated tissues [45]. The generation of ROS can damage lipids, proteins and even DNA [46]. Vijay Kumar et al. [47] reveal that chronic aluminium exposure might be implicated in mitochondrial DNA oxidation leading to the increased expression of p53 gene. So, the peak observed at 975 cm 1 may be due to the exposure of aluminium on DNA. Conclusions FTIR spectra reveals significant differences in absorbance intensities between the control and aluminium intoxicated brain tissues. Further, the administration of DFO significantly improves the levels of biochemical constituents. The observed results suggest that quantity of protein significantly decreased due to the decreased level of free radical scavengers. Quantity of unsaturated fatty acid decreased may indicate the increases of lipid peroxidation and progression of aluminium induced Alzheimer’s disease. The peak positions of amide I and II shifted to lowered value in aluminium intoxicated tissue may be due to the amyloid core of a mature neuritic plaque. In addition FTIR results were found to be in agreement with biochemical and previous studies. Finally the present study shows that the FTIR spectroscopy is a very informative technique to determine the aluminium toxicity induced changes in brain tissue at molecular level. Acknowledgment The financial support to Mr. J. Sivasubramanian as Project fellow from the University Grants Commission, New Delhi, India is gratefully acknowledged. Reference [1] [2] [3] [4]
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