Protective effects of deferiprone and desferrioxamine in brain tissue of aluminum intoxicated mice: An FTIR study

Biomedicine & Preventive Nutrition 4 (2014) 53–61

Available online at www.sciencedirect.com

Original article

Protective effects of deferiprone and desferrioxamine in brain tissue of aluminum intoxicated mice: An FTIR study Sivaprakasam Sivakumar a,∗ , Chandra Prasad Khatiwada a , Jeganathan Sivasubramanian a , Boobalan Raja b a b

Department of Physics, Annamalai University, Annamalai Nagar, Tamilnadu 608002, India Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, Tamilnadu 608002, India

a r t i c l e

i n f o

Article history: Received 2 June 2013 Accepted 21 June 2013 Keywords: Aluminum Biochemical parameters DFO and DFP FTIR

a b s t r a c t The present study was designed to study aluminum chloride which caused marked alterations in biochemical parameters such as glutathione peroxidase, catalase, superoxide dismutase, and TBARS in brain tissues of mice. Fourier transform infrared spectroscopy spectra reflect the alterations on major biochemical constituents in brain tissues of mice such as proteins, lipids and nucleic acids due to the overproduction of ROS. Furthermore, administration of deferiprone and deferoxamine significantly improved the level of protein and shifted back the peak positions of amide I and II to near control values indicating tau protein, ␤-amyloid, amyloid plaques and neurofibrillary tangles decreased, consequently protected from Alzheimer’s disease and other major risk factor of many neuronal dysfunctions in brain tissues. Therefore, aluminum toxicity is a widespread crisis to all living organisms, including both flora and fauna. Furthermore, it causes widespread degradation of the environment and health. Therefore, the present investigation suggested that DFO and DFP are efficient chelators for aluminum poisoning and they reduced the aluminum concentration. © 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction Aluminum is the most abundant metal and the third most abundant element in the earth’s crust, after oxygen and silicon [1]. The main entry sites of Al into the body are the gastrointestinal, respiratory tract and accumulation in several tissues, like spleen, lungs, liver, kidneys, heart, bone and brain [2]. High brain levels of Al induce cognitive deficiency and dementia and, thus, Al is a widely accepted neurotoxin [3]. Moreover, it can be degenerate of nerve cells in the brain of humans and experimental animals. Absorption/accumulation of Al in humans can occur via the diet, drinking water, vaccines, antacids, parenteral fluids, inhaled fumes and particles from occupational exposures [4]. Al has been proposed as an environmental factor that may contribute to some neurodegenerative diseases and affect several enzymes and other biomolecules relevant to Alzheimer’s disease (AD) [5]. Aluminum is known to potentiate iron-related reactive oxygen species (ROS) formation in isolated systems [6]. For this reason, the current study used groups of mice treated with aluminum and combined therapy of deferiprone with deferoxamine in order to determine whether aluminum enhanced ROS production in brain as well as determined the antidotes capacity of both chelators. Al chloride was chosen over

∗ Corresponding author. Tel.: +91 986 504 3811. E-mail address: [email protected] (S. Sivakumar). 2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2013.06.001

other Al species because the stomach already contains and utilizes chloride, so this form of Al can be introduced with minimal change to gastric fluid composition. The oral administration of deferiprone was an effective treatment and it determines if the chelating agent deferiprone could mobilize aluminum from tissues and increase aluminum elimination [7]. Deferiprone is more effective, water soluble, less toxic than DFO and can be given orally. Aluminum chelators DFO and DFP should be capable to mobilize aluminum and to reduce body burden, so repealing both encephalopathy and osteomalacia. The first aluminum chelator introduced in clinical practice for aluminumrelated osteomalacia was deferoxamine [8]. Deferiprone does not manifest the same pattern of toxicity as deferoxamine due to the different chemical properties of these chelators. DFP and DFO simultaneous administration and the aggressive pharmacologic treatment were essential to induce a regression of brain dysfunction in a very short time, which was associated with an improvement in clinical status [9]. Previously, iron chelation therapy is attained by the use of subcutaneous deferoxamine pumps; or more recently, through the use of the oral iron chelators deferiprone and deferoxamine [10]. Chelation therapy which suggests that surely translated into prolonged survival and enhanced quality of life for patients with brain diseases. DFO and DFP compounds are usually employed in iron accumulation but there are chemical and physical similarities between aluminum and iron (charge, ionic radius and protein binding)

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and both have been used in case of aluminum accumulation [11]. Fourier transform infrared spectroscopy (FTIR) is a powerful technique, which has been widely used in biophysical and biochemical research, demonstrated to provide sensitive and precise measurement of biochemical changes in biological cells and tissues [12]. The frequency shifts demonstrate the molecular alteration of macromolecules such as protein, lipid, carbohydrate, and nucleic acid. Previously, it was reported that the relationship between human and mice brain shares numerous features of brain organization and behavioral responses to many pharmacological agents [13]. Moreover, chelation therapy is one of the most effective methods to remove toxic elements from a biological system [14]. Therefore, the main objective of this study was to use this molecular fingerprinting approach to investigate the effects of deferiprone and desferrioxamine on metabolic alterations that occur in brain tissues of aluminum-intoxicated mice. 2. Materials and methods 2.1. Animals Male Swiss albino mice (Mus musculus), 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, Annamalai Nagar-608002, Tamilnadu, India. 2.2. Test chemicals Deferroxamine (Desferal) and deferiprone (DFP) were purchased from Novartis and Sigma Aldrich, Chemicals Limited, Mumbai, India. All other chemicals used in this study were of highest analytical grade obtained from Sisco Research Laboratories and Himedia, Mumbai, India. 2.3. Treatment schedule Mice were randomly allocated in four different groups. Each group contained 12 animals. Group I served as control and was fed on standard animal chow and water ad libitum, received an i.p. injection of 0.9% saline and deionized water by gavage. Animals in groups II, III and IV were administered aluminum in the form of aluminum chloride (100 mg/kg b.wt./day) orally for a period of 16 weeks. Group III mice were treated with DFP (p.o.) at the dose of 0.72 mmol/kg and Group IV were treated with DFO + DFP (i.p.) at the dose of 0.89 mmol/kg dose, half an hour after a single i.p. administration of 100 mg Al/kg body weight in the form of aluminum chloride for five consecutive days. Aluminum chloride was dissolved in normal drinking water and was given by oral gavage. Twenty-four hours after the last dose of aluminum chelators, the animals were sacrificed under ether anesthesia by cutting jugular veins. The brain were excised, weighed immediately then stored at −80 ◦ C until analysis. 2.4. 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. 2.5. FTIR spectra and data analysis FTIR spectra of the region 4000–400 cm−1 were recorded at the temperature of 25 ± 10 ◦ 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). 2.6. Biochemical analysis The concentration of thiobarbituric acid reactive substances (TBARS) was estimated by the method of Niehaus and Samuelson [15]. The activities of enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were assayed by the methods of Kakkar [16], Sinha [17] and Rotruck [18] respectively. 2.7. Histopathological examination of brain tissues Mice (six mice/treatment group) at the end of experiment were dissected for histology. Removed brain samples were cleared of blood and immediately placed in a neutral buffered solution of 10% formalin for 24 h. Sliced into 5 ␮m thickness and then placed onto glass slides. The sections were stained with haematoxylin and eosin staining (H&E) and examined by light microscopy which have been used to visualize changes in tissue structures. The cross-sectional area (CSA) of brain was evaluated from photographs of whole tissue sections taken at 40× magnification and scanned, digitized and analyzed by computer, using the Adobe Photoshop Imaging Program (Adobe System Incorporation). 2.8. Statistical analysis Data were analyzed by one-way analysis of variance followed by Duncan’s Multiple Range Test (DMRT) using SPSS version 16 (SPSS, Chicago, IL). Probability level (P-value) of less than 0.05 was considered statistically significant. 3. Results FTIR is a non-perturbing rapid technique giving information on several biomolecules, such as DNA, RNA, proteins, carbohydrates, lipids in biological tissues and cell. FTIR spectroscopy basically deals with the mid-infrared region 4000–500 cm−1 (2.5–25 ␮m wavelength) which is the most informative for biosamples since these contain mainly organic compounds [19] and provide information about chemical bonding properties that characterize biochemical functional components in complex matrices and allow for qualitative identification and quantitative estimations [20]. The list of absorption peak assignments belonging to lipids, proteins, polysaccharides, carbohydrates and nucleic acids for various functional groups and representative infrared spectrum in the region between 4000 and 400 cm−1 are presented in Table 1 and Fig. 1 respectively. For our convenient, Fig. 2 spectra were analyzed in three major distinct regions: 3750–2700 cm−1 (I), 1800–1350 cm−1 (II) and 1300–400 cm−1 (III). Curve fitting procedure was not applied because the bands were clearly resolved [21,22]. The band area values, band area ratios and biochemical analysis are presented in Tables 2–4 respectively.

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Table 1 FTIR general vibrational peak assignments of control, aluminum-intoxicated, DFP and DFO + DFP treated brain tissue of mice. Control

Aluminum-intoxicated

Al + DFP

Al + DFO + DFP

Vibrational peak assignments

3372

3359

3364

3375

3065 3010 2969 2928 2855 1753 1652 1543 1448 1406 1240 1160 1075

3058 3000 2958 2923 2852 1750 1648 1538 1457 1396 1236 1159 1066

3062 3004 2966 2926 2857 1752 1651 1540 1457 1399 1238 1160 1069

3066 3008 2966 2930 2861 1755 1654 1542 1454 1402 1239 1166 1072

875 687

873 696

876 703

N–H stretching of proteins with the little contribution from O–H stretching of polysaccharides: Amide A N–H stretching of proteins: Amide B Olefinic CH CH stretching, unsaturated fatty acids CH3 asymmetric stretch: lipids CH2 asymmetric stretch: lipids CH2 symmetric stretching lipids (fatty acids) Carbonyl C O stretch: lipids C O stretching of ␣-helix protein: Amide I N–H bending and C–N stretching of proteins: Amide II CH2 bending: mainly lipids COO− symmetric stretch: fatty acids and amino acids PO2 − asymmetric stretch: mainly nucleic acids and phospholipids C–O asymmetric stretching of glycogen PO2 − symmetric stretch: mainly nucleic acids, HO–C–H stretch: mainly carbohydrates C–N+ –C symmetric stretch: nucleic acids Ring breathing mode in the DNA bases

---694

Table 2 FTIR band area values for the control, aluminum-intoxicated and DFP and DFO + DFP treated brain tissue of mice. Bands 3372 3065 3010 2969 2928 2855 1753 1652 1543 1448 1406 1240 1160 1075 875 694

Control

Aluminum-intoxicated

240.671 ± 1.709 0.462 ± 0.014a 0.139 ± 0.015d 0.163 ± 0.015b 2.499 ± 1.403a 0.546 ± 0.017a 0.524 ± 0.017c 38.736 ± 1.800c 11.774 ± 1.800c 0.147 ± 0.018a 2.499 ± 1.526a 2.015 ± 1.220a 0.025 ± 0.002a 11.070 ± 1.654b Not observed 0.208 ± 0.018c

d

127.239 0.564 0.024 0.081 1.213 0.558 0.387 22.815 5.688 0.209 2.059 1.794 0.043 3.583 0.141 0.043

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a

1.678 0.017b 0.002a 0.002a 1.290a 0.019a 0.017b 1.802a 1.596a 0.018b 1.319a 1.137a 0.002b 1.260a 0.014a 0.002a

Al + DFP 182.510 0.652 0.042 0.223 2.517 1.157 0.338 30.495 8.233 0.896 2.517 2.803 0.163 19.373 0.070 0.308

Al + DFO + DFP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

1.689 0.017c 0.002b 0.018d 1.400a 0.928b 0.019a 1.500b 1.445b 0.017c 1.793a 1.906a 0.014c 1.432c 0.076a 0.020d

232.434 0.861 0.089 0.205 2.153 0.868 0.691 40.102 10.882 0.605 1.785 2.527 0.053 25.963 1.191 0.118

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.945c 0.053d 0.001c 0.017c 1.357a 0.022a,b 0.028d 1.454c 1.904c 0.016d 1.365a 1.972a 0.014b 1.771d 1.071b 0.023b

Comparisons values are expressed as mean ± SD for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).

3.1. FTIR results Fig. 2 (I) mainly consists of amide A vibrations of proteins in brain tissues range between 3750–2700 cm−1 . The band at

Fig. 1. FTIR spectra of the control, aluminum-intoxicated, DFP and DFO + DFP treated brain tissues of mice in the 4000–400 cm−1 region.

∼3372 cm−1 corresponds to the amide A stretching mode that can generally be associated with N–H and intermolecular O–H molecules, since unbound water was removed from the system [21]. Amide A appeared at 3472 cm−1 in control, 3359 cm−1 in aluminum-intoxicated, 3364 cm−1 in DFP treated and 3375 cm−1 in combine therapy in brain tissue. In the present work, the calculated peak area values of amide A band for the control, aluminum-intoxicated, DFP and DFO + DFP treated tissues are 240.671 ± 1.709, 127.239 ± 1.678, 182.510 ± 1.689, 232.434 ± 1.945, respectively, which correspond to a change of 47.13% between the control and aluminumintoxicated tissues, 43.43% between the DFP and aluminumintoxicated tissues, 82.67% between the DFO + DFP and aluminumintoxicated tissues. This result suggests that the protein content in the membrane is lesser in the aluminum-intoxicated brain tissues. Treatment with DFO + DFP brings back the alterations to near the control band area value as compared to DFP treated brain tissue. Hence, the brain is believed to be particularly exposed to aluminum due to its high oxygen consumption rate, high level of polyunsaturated fatty acids and relatively high rate of oxygen free radical generated without commensurable level of aluminum. The bands at ∼3065 cm−1 were assigned to amide B due to N–H stretching of proteins [21]. Amide B appeared at 3065 cm−1 in control, 3058 cm−1 in aluminum-administrated, 3062 cm−1 in DFP treated and 3066 cm−1 in DFO + DFP treated brain tissues. Treatment with DFP and DFO + DFP fetches reverse the variation to near the control band area value. There is a significant increase in DFO + DFP

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Fig. 2. Selected FTIR spectra of the control, aluminum-intoxicated, DFP and DFO + DFP treated brain tissues of mice in the 3750–2700 cm−1 (I), 1800–1350 cm−1 (II), 1300–400 cm−1 (III) regions.

treated group when compared with aluminum-intoxicated group. Therefore, DFO + DFP acts as good antidotes for aluminum and these changes in absorption of specific vibrational bands indicate to changes in the relative concentrations of proteins, lipids and phospholipids of the brain tissues due to aluminum toxicity. The bands at ∼3010 cm−1 were assigned to result from the C–H stretching vibration of HC CH groups of olefinic molecules, which could be a useful indicator of the different degrees of unsaturation in acyl chains of phospholipids [23] in brain tissues of mice. As seen in Table 2, the area value of olefinic acid reduced more from 0.139 ± 0.015 to 0.024 ± 0.002 between the control and aluminum but treated with DFP and DFO + DFP bring back to near

control values. This large shift in control and aluminum might imply a variation in the strength of unsaturated lipids. The peak observed at ∼2969 cm−1 was assigned to asymmetric stretching vibrations of methyl group and depends on the degree of conformational disorder, while the positions of these bands give information with reference to the lipid acyl chain flexibility of lipids. Hence, this band mainly monitors the lipids in the biological system [24]. The absorption band area of this region was changed significantly in all the groups. Methylene groups CH2 asymmetric band at ∼2928 cm−1 and CH2 symmetric band at ∼2855 cm−1 were due to stretching of lipids [25] in brain tissues. Control band appeared at 2928 cm−1 ,

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Table 3 FTIR absorption band area ratio for selected bands of control, aluminum-intoxicated, Al + DFP and Al + DFO + DFP treated brain tissues of mice. Band area ratio

Control

Aluminum-intoxicated

Al + DFP

Al + DFO + DFP

I1543 /I3372 I2969 /I2855 I1543 /I1652

0.049 ± 0.001b 0.299 ± 0.008d 0.304 ± 0.007b

0.045 ± 0.001a 0.145 ± 0.010a 0.249 ± 0.010a

0.045 ± 0.001a 0.193 ± 0.006b 0.270 ± 0.008a

0.047 ± 0.001a,b 0.236 ± 0.008c 0.271 ± 0.007a

Values are expressed as mean ± SD for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).

aluminum band appeared at 2923 cm−1 indicating that aluminum can decrease in the frequency values of these CH2 stretching vibrations, but treated with chelating agents, it comprises to increase. Fig. 2 (II) shows FTIR spectra of proteins, with some absorbance from lipids of control, aluminum-intoxicated, DFP treated and DFO + DFP treated brain tissue in the range of 1800–1400 cm−1 . The band observed at ∼1753 cm−1 is assigned to C O stretching vibration of ester groups in triglycerides [25,26] in brain tissues. These frequency values shifted to a lower value in aluminumtreated group significantly at 1750 cm−1 , higher at 1752 cm−1 in DFP and 1755 cm−1 in DFO + DFP treated groups. This region also mainly consists of amide I and amide II wave number of proteins. At this peak, the wave number shift indicates the quantitative alterations in secondary structure of proteins [27]. The position of this absorption is susceptible to protein configuration. The peak appearing at ∼1652 cm−1 is assigned as amide I due to C O stretching of ␣-helix proteins in brain tissues. Amide II due to N–H bending and C–N stretching of proteins vibration mode of the polypeptide and protein backbones appeared at 1543 cm−1 . This specifies the significant protection of amide concentration from aluminum-induced damages in DFP and DFO + DFP treated groups. Fig. 2 (II) and Table 2 show that the frequency of these band and band area was significantly different form aluminum-intoxicated to treated groups. The amino acid side chain from peptides and proteins at ∼1448 cm−1 is connected with the asymmetric CH2 bending vibrations. The absorption band at ∼1406 cm−1 is assigned to COO− symmetric stretching of fatty acids and amino acids in brain tissues. The present study indicates that aluminum accumulation in the body is most severe but combined therapy and DFP therapy plays a vital role in the treatment of aluminum poisoning and can flush out unwanted aluminum. Fig. 2 (III) shows that absorptions are primarily due to the continuation of polysaccharides, nucleic acid, phospholipids, carbohydrates and minerals in the range of 1300–400 cm−1 . The band appearing at ∼1240 cm−1 has been assigned to the asymmetric phosphate stretching vibration of phospholipids and the band observed at ∼1160 cm−1 to C–O asymmetric stretching of glycogen. The band appearing at ∼1075 cm−1 has been assigned to phosphodiester groups in nucleic acids rather than in phospholipids. Treatment with DFP brings reverse the variations to near control and combine therapy brings almost near control spectral wave number. DFO + DFP treated brain tissues spectrum exhibited an increase in the area value of phosphate asymmetric stretching band, decrease area of symmetric phosphate stretching band with respect to those of control brain tissues as shown in Table 2. The band peak at ∼875 cm−1 is normally assigned to symmetric stretching mode of dianionic phosphate monoester of cellular nucleic acids, especially for DNA [28]. Hence, aluminum induces the production of free radicals leading to break in lipids, proteins, and DNA in the brain tissues. 3.2. Biochemical parameters In the present study, all the parameters responded positively to individual therapy with DFP, but more pronounced beneficial effects were observed in combination therapy. The efficacy of DFO and DFP could be attributed to the chelating properties and available binding sites of it, which leads to decreased concentration

of aluminum in brain tissues of mice and changes in biochemical parameters are presented in Table 4. 3.2.1. Lipid peroxides, enzymatic antioxidants and non-enzymatic antioxidants results The improvement of lipid peroxidation products might be due to enrichment of free radical production and reduced antioxidant system. Treatment with chelating agents DFP and DFO + DFP increased the levels of lipid peroxidation products in aluminum-intoxicated mice. Thus, DFP and DFO + DFP inhibits lipid peroxidation may be due to scavenging of free radicals and is recognized to its radical scavenging property. Aluminum poisoning may lead to cell death is the enhanced oxidative stress leading to a decrease in lipid peroxidation. Free radical scavenging enzymes such as superoxide dismutase, catalase, and glutathione peroxidase are the first line of cellular defence against oxidative injury. Superoxide dismutase reduces superoxide anion to form H2 O2 and oxygen. Catalase removes H2 O2 by breaking it down directly to oxygen. DFO and DFP prevented to decrease the activities of enzymic antioxidants in aluminum-intoxicated mice brain tissues. The non-enzymatic antioxidants reduced glutathione plays a marked role in aluminum detoxification reaction because it is a radical scavenger which scavenges the residual free radicals. Treatment with DFP and DFO + DFP enhanced the levels of the antioxidants and suggests that this compound might be potentially useful in counteracting free radical mediated oxidative stress caused by lipid peroxidation. 3.3. Histopathological examination Fig. 3 (A–D) shows the control, aluminum-intoxicated, DFP treated and DFO + DFP treated on the histology of brain tissue of mice under observation for morphological changes. Fig. 3A shows control brain cortex with neural fibres and astrocytes. In the aluminum-intoxicated group, cortex with astrocytes and vessels condensed tangled neural fibres, separations of nerve fibres were observed as shown in Fig. 3B. Photomicrographs of the brain Fig. 3C for DFP treated group show degenerated nerve cells and few microglial cells. Fig. 3D shows for DFO + DFP treated brain tissues of mice for less oedema with less neural separation. Therefore, histopathology of DFO + DFP treated brain shows chelating agents serve as a useful tool for screening potential therapeutic agents capable of mobilizing aluminum from the AD brain. Thus, histopathological findings confirmed the biochemical observations of this study. This histological observation exhibited the protective role of DFP and DFO + DFP for brain diseases. 4. Discussion Chelation therapy is recommended for heavy metal poisoning and these metals exert their toxic effects by combining with one or more reactive groups essential for normal physiological functions [29] and combined therapy of DFO and DFP to reverse cardiac damage [30]. The present study successfully explored the protective action of chelating agents in aluminum-induced mice brain tissues. Therefore, it is extremely helpful and relevant for finding new aluminum chelation therapy which is important for human health as shown in Fig. 4. Our study explores the effects of DFP

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Table 4 Protective effect of DFP and DFP on thiobarbituric acid reactive substances, superoxide dismutase, catalase, and glutathione peroxidase in the brain tissue of aluminumintoxicated mice. Groups

TBARS (n moles/mg of protein)

Control Al Al + DFP Al + DFO + DFP

3.76 4.79 4.28 3.91

± ± ± ±

0.043a 0.032d 0.036c 0.040b

SOD (␮/mg of protein) 2.13 1.63 1.84 2.05

± ± ± ±

0.028d 0.028a 0.026b 0.026c

CAT (␮/mg of protein) 0.31 0.22 0.26 0.29

± ± ± ±

0.046b 0.046a 0.042a,b 0.037b

Gpx (␮/mg of protein) 9.57 8.53 8.97 9.38

± ± ± ±

0.026d 0.036a 0.018b 0.036c

Comparisons values are expressed as mean ± SD for six animals in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).

Fig. 3. Representative photomicrograph of histological/morphological changes in brain. A. Control, cortex with neural fibres and astrocytes. B. Aluminum-intoxicated, cortex with astrocytes and vessels. C. Al + DFP, condensed tangled neural fibres, separation of nerve fibres. D. Al + DFO + DFP, degenerated nerve cells and few microglial cells.

and DFO + DFP on aluminum-induced changes in mice brain tissues at molecular level using FTIR spectroscopy. The FTIR investigation provides structural information of biological molecules such as proteins, nucleic acids, carbohydrates and lipids, allowing recognition, detection, and quantification of changes in these macromolecular cellular mechanisms. In the present study, as seen from Fig. 2 (I), the peak area value of amide A band spectrum increased significantly. The significant increase in area of amide A band reproduces the increase in the quantity of protein. This enhance is responsible with the increase in amide I and II bands presented in Table 1. This indicates the damage of amide linkage from free radical reduced by DFP, DFO + DFP treated groups and this was established biochemically. Previously, the protective activity of desferrioxamine (DFO), an efficient chelator available for the treatment of iron and Al overload, Al induced developmental toxicity was evaluated in mice [31]. Present results suggest that the DFP and DFO + DFP contribute an essential role in the treatment of aluminum poisoning and can cross the blood brain barrier and decrease brain aluminum levels. Functional groups with chelating potential for Al could be found in proteins, fatty acids, sugars, DNA and other biomolecules [32]. Therefore, chelating drugs would have to compete for metals with naturally occurring endoge-

nous chelators at all the phases of absorption, metabolism and excretion. Ringborn [33] suggested that chelators bind metals with different affinity both in vitro and in vivo. Numerous possible utilizations of chelators and their metal complexes are currently under exploration in experimental in vitro and in vivo, clinical experimental models and various chelating drugs combinations. The specificity of the chelating agents DFP and DFO + DFP for aluminum metal, several factors may overcome in vivo, such as the availability of chelatable metal pools, the extent of metabolic transformation of the chelator and its metal complexes and the rate of their clearance could influence the overall effect of a chelator on the elimination of aluminum [34]. Due to aluminum poisoning, ROS levels increase dramatically then form neurological impairments such as irregular postural responses, persistent tremors and changes in cognitive functions. Neuropeptides may contribute in several physiological processes including pain sensation, memory, regulation of mood and neuroendocrine functions in brain tissues. In the present study, DFP and DFO + DFP decreased the aluminum concentrations in brain tissues of mice, blocked reactive oxygen species (ROS) production. DFP and DFO + DFP shifted to the protein level nearer to control value in brain tissues. These results indicate both aluminum chelators could be useful as potential therapeutic agents for pathological

Fig. 4. Chelation therapy of aluminum roles in disease and medicine.

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processes caused by oxidative damage. Therefore, superoxide dismutase scavenges the superoxide anion to form hydrogen peroxide, and consequently it diminishes the toxic effects caused by this radical. Catalase removes H2 O2 by breaking it down directly to O2 . Glutathione peroxidase is concerned in the reduction of the peroxides which can damage fatty acids, and thus it prevents lipid peroxidation as well as degradation of the membrane phospholipids and the subsequent formation of thiobarbituric acid reactive substances [35]. Finally, the generation of protein quantity may be the balance between oxidants and antioxidants by both chelating agents DFP and DFO + DFP. The band observed at ∼3010 cm−1 gives information about the concentration of olefinic HC CH stretching band with aluminum intoxication. Significant change in the band area value at aluminum-intoxicated group to treated groups. These changes are most probably the results of alter in lipid metabolism induced by aluminum but significant increase in DFP and DFO + DFP treated groups. The reduction in the protein content after aluminum exposure may be due to reduction in protein synthesis, the higher affinity of metal compounds towards different amino acid residues of proteins, which is considered as the premier biochemical parameter for early indication of stress. Aluminum (Al) is a potent neurotoxin and has been associated with Alzheimer’s disease causality for decades [36] and other neurological dysfunctions. When lipid acyl chains contain more than one double bond, lipid oxidation results in the rearrangement of double bonds to form conjugated dienes. Subsequent decomposition of hydroperoxides, through a complex sequence of propagative reactions, leads to a variety of secondary lipid peroxidation products [37,38]. These degenerative propagation reactions in lipid membranes are usually accompanied by the formation of a wide variety of products, including alkanes, carbonyl compounds (such as MDA), lipid aldehydes and alkyl radicals. Therefore, the increase in the amount of olefinic group in the aluminum brain might be due to the accumulation of end products of lipid peroxidation. The band appearing at ∼2969 cm−1 decreased the peak area due to the hydrocarbon tails in lipids because they are good supervises of the changes in acyl chains [39]. Decreased peak area appeared at 2969 cm−1 due to CH3 asymmetric stretching vibration at aluminum-intoxicated group but increased at DFP and DFO + DFP treated groups. The frequencies of the CH2 stretching bands of the acyl chains depend on the degree of conformational disorder, whereas the position of these bands provides information about the lipid acyl chain flexibility (order/disorder state of lipids) [40]. This result suggested that decreased lipid order and increased acyl chain flexibility in DFP and DFO + DFP treated brain tissues of mice. Mean ratio of the methyl and the methylene band is shown in Fig. 2 (I). Reduced ratio indicates reduction in the number of methyl groups in protein fibres compared to methylene groups in aluminum-intoxicated/accumulated group. These effects could be present due to the accumulation of aluminum in the brain. Previous study reported the toxic effects of aluminum on mice brain, confirmed damage in the hippocampus and cortex, including neurofibrillary degeneration, due to the accumulation of Al in these regions [41]. The increased ratio indicates the contribution for high number of protein fibres [24] in DFP and DFO + DFP treated brain tissues. The area values of the amides I and II are decreased at aluminum-intoxicated brain tissues. This indicates the reliability with the decrease of the amide A band observed at ∼3372 cm−1 . The amide I band primarily composed of vibrations originating in the C O, is sensitive to hydrogen bonding and protein secondary structures [42]. These reduced area values of the amide I and II bands could be taken as the result of variation of the protein synthesis and the protein structure due to aluminum poisoning in brain tissues of mice.

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The band appearing at ∼1753 cm−1 decreased in intensity due to carbonyl C O stretching vibration of lipids metabolism, which suggests a decreased quantity of unsaturated acyl chains in the aluminum-intoxicated brain tissues [43] and increased in chelating agents treated mice brain tissues. This result is supported by the increased intensity of olefinic CH stretching band observed at ∼3010 cm−1 due to chelating agents. The absorption peaks observed at ∼1240 cm−1 and ∼1075 cm−1 assigned asymmetric and symmetric stretching of phosphate due to the formation of phosphodiester backbone of cellular nucleic acids. Previously, a new era in the development of chelating drugs began with the introduction of deferiprone or L1, which as a monotherapy or in combination with deferoxamine can be used universally for effective chelation treatments; rapid iron removal, maintenance of low iron stores and prevention of heart and other organ damage caused by iron overload [44]. Therefore, the present investigation confirmed that DFO + DFP chelation therapy did remarkably well as compared to DFP therapy for the aluminum toxicity. The peak observed at ∼875 cm−1 is absent in control, increased in chelating agents DFO + DFP treated compared to aluminumintoxicated brain tissues as shown in Fig. 2 (III). These results suggest a reduction in the relative content of the nucleic acids due to aluminum intoxication. The amide I and I1 bands of peptide bonds in proteins are mainly responsive to alters in secondary structure and are the most frequently used in vibrational analysis. These vibrations are influenced by the secondary structure of the protein, since this involves protein folding with hydrogen bonding between peptide bonds [24,45,46] suggesting that the band at 1653 cm−1 was due to ␣-helix structure and overall the band at 1600–1700 cm−1 was assigned to ␣-helix structure and ␤-sheet structures. The area values of the amides I and II presented in Table 2, consequently the amount of proteins, decreased in the aluminum-intoxicated and increased in chelating agents treated brain tissues. This increase was reliable with the increase in amide A band at 3372 cm−1 due to proteins. This result is supported by biochemical studies that bring out a significant reduction in the level of TBARS and the induction on activities of enzymatic antioxidants. Changes in protein secondary structure in amide I and amide II bands by administration of DFP and DFO + DFP to test if the resultant increased band shape variation would lead to improvements in protein secondary structure. In DFP and DFO + DFP treated brain tissues showed the decrease of the intermolecular hydrogen bond interactions forms aggregates of higher molecular weight, and then modifies the secondary structure of proteins in near to control brain tissues. This result, together with a slight decreased in the content of random coil structures, implies that unchanged of the proteins might be taking place. This increased 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 DFP and DFO + DFP. Hence, in this study the wave number shift may indicate tau protein and ␤-amyloid decrease and protect from Alzheimer’s disease. Therefore, the administration of DFP and DFO + DFP significantly improves the levels of biochemical constituents in brain tissues of mice. 4.1. Qualitative analysis Table 3 shows the FTIR absorption band area ratio for selected bands of brain tissues of mice. The ratio of peak intensity of each band of the experimental groups with respect to the control/treated groups can be used to distinguish the biochemical contents of control tissues from aluminum-intoxicated and chelating agents treated brain tissues. In present work, the ratio of the peak intensities of the bands at 1537 cm−1 and at 3342 cm−1 (I1537 /I3342 ) has been used as an indicator of the relative concentration of the

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protein in the brain tissues. The calculated ratios are 0.049 ± 0.001, 0.045 ± 0.001, 0.045 ± 0.001 and 0.047 ± 0.001 for the control, aluminum-intoxicated, DFP treated and DFO + DFP treated tissues respectively, which correspond to a change of 8.16% between the control and aluminum-intoxicated tissues, no changes between the DFP and aluminum-intoxicated tissues and 4.44% between DFP and DFO + DFP treated brain tissues. These results suggest that the protein content in the membrane is higher in chelating agents administered but lower in the aluminum-intoxicated brain tissues compared to that of control. The ratio of the intensity of absorption of bands between the methyl band and methylene band (I2969 /I2854 ) for the control, aluminum-intoxicated, DFP treated and DFO + DFP treated brain tissues are 0.299 ± 0.008, 0.145 ± 0.010, 0.193 ± 0.006 and 0.236 ± 0.008 respectively, which corresponds to a change of 51.50% between the control and aluminum-intoxicated, 33.10% between the DFP and aluminum-intoxicated tissues and 29.66% between DFP and DFO + DFP treated brain tissues. The decreased ratio indicates a decrease in the number of methyl groups in protein fibers compared to methylene groups in aluminum-intoxicated tissues but reverse in chelating agents treated brain tissues. Increase in the area of the CH2 symmetric stretching mode indicates an increase in phospholipids or fatty acid concentrations. This change in the lipid content might be important, because the cell lipids play several significant roles in the regulation of membrane function [47]. The ratio of the peak intensities of the bands at 1537 cm−1 and at 1652 cm−1 (I1537 /I1652 ) for the control, aluminum-intoxicated, DFP treated and DFO + DFP treated brain tissues are 0.304 ± 0.007, 0.249 ± 0.010, 0.270 ± 0.008 and 0.271 ± 0.007 respectively, which corresponds to a change of 18.09% between the control and aluminum-intoxicated, 8.43% between the DFP and aluminumintoxicated tissues and 0.37% between DFP and DFO + DFP treated brain tissues. The increased ratio indicates a change in the composition of the whole protein pattern in the brain tissues due to chelating agents DFO and DFP. Hence, DFP and DFO + DFP not only form stable and soluble complexes with aluminum, but also guarantee restorative reaction of the organism and significant recovery in the altered biochemical variables lipids, proteins and nucleic acids of the brain tissues of mice. Previously, it was suggested that DFO and DFP significantly decreased in the liver, brain, kidneys and heart iron concentration in both, iron loaded and control mice [48]. Hence, the present study confirmed that DFP and DFO + DFP are the best antidotes for heavy metal aluminum poisoning.

5. Conclusion FTIR spectra reveal significant differences in absorbance intensities between the control, aluminum-intoxicated and chelating agents treated brain tissues. The variation on the major biochemical constituents such as lipids, proteins and nucleic acids of the brain tissues is due to the overproduction of ROS but significantly improved it by administration of DFP and DFO + DFP. This result shows that antidotes DFO and DFP are the effective chelators of aluminum in reducing the body burden of mice. Further, the peak positions of amide I and II shifted to near control values indicates tau protein, ␤-amyloid, amyloid plaques and neurofibrillary tangles decreased consequently to protect from Alzheimer’s disease and other major risk factors in many neuronal dysfunctions. Our results confirmed that aluminum plays an important role in the pathogenesis of brain and that the use of aluminum chelators DFO and DFP are promising and novel therapeutic strategy for brain diseases. Further investigation of the role of aluminum chelation therapy in treating brain is secured; specifically, substitute aluminum chelators and combination therapy investigated by FTIR

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