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Protective effects of desferrioxamine and deferiprone on the spleen tissue of aluminum intoxicated mice: A Fourier transform infrared spectroscopy study
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 148–156
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Protective effects of desferrioxamine and deferiprone on the spleen tissue of aluminum intoxicated mice: A Fourier transform infrared spectroscopy study S. Sivakumar ⇑, Chandra Prasad Khatiwada, J. Sivasubramanian Department of Physics, Annamalai University, Annamalai Nagar, Tamilnadu 608002, 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 study determined functional and
compositional changes on spleen tissue. FTIR study has been good agreement with biochemical, histopathology and ICP–OES. All parameters and biochemical constituents responded positively to DFP therapy. But more pronounced beneficial effects were observed in DFO + DFP therapy.
a r t i c l e
i n f o
Article history: Received 24 September 2013 Received in revised form 26 January 2014 Accepted 2 February 2014 Available online 15 February 2014 Keywords: Aluminum Spleen DFO and DFP FTIR ICP–OES
a b s t r a c t The present study was designed to examine the protective effects of the chelating agents desferrioxamine (DFO) and deferiprone (DFP) in aluminum intoxicated spleen tissue of mice by Fourier transform infrared (FTIR) spectroscopy. The finding revealed the alterations on the major biochemical constituents, such as lipids, proteins, phosphodiester and nucleic acids of the spleen tissue of mice at molecular level. The significant decreased in the peak areas of asymmetric and symmetric mode of the phosphodiester groups from control to aluminum intoxicated, but improved it by DFP and DFO + DFP treatments respectively for nearer control value. The bands ratio at I1081/I1232 significantly decreased from control to aluminum, but enhanced it by DFP and DFO + DFP respectively. This result suggests that DFO and DFP are the phosphodiesterase inhibitor, recovered from chronic growth of diseases in the spleen. Amide I and amide II peak area values decreased from control to aluminum intoxicated spleen tissue, but treated with DFP and DFO + DFP significantly improved. This result suggests an alteration in the protein profile. The absence of Olefinic@CH stretching in aluminum intoxicated spleen suggests an altered lipid levels. Concentrations of trace elements were found by ICP–OES. Histopathological findings confirmed the biochemical observations of this study. The results of the FTIR study were found to be in agreement with biochemical studies and demonstrated that FTIR can be used successfully applied to toxicological studies at molecular level. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author. Tel.: +91 9865043811. E-mail address: [email protected] (S. Sivakumar). http://dx.doi.org/10.1016/j.saa.2014.02.004 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Aluminum (Al) is the third most prevalent element and the most abundant metal in the earth’s crust i.e., approximately 8%
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of total mineral components [1]. It is widely distributed in the environment and extensively used in daily life resulting easy exposure to human beings [2]. Aluminum absorption/accumulation in humans can occur via the diet, drinking water, ingestion with fruit juices or citric acid causes a marked increase in both gastrointestinal absorption and urinary excretion of aluminum in healthy subjects [3]. The main sources of human exposure to aluminum are to be found in the extensive presence of the metal in the environment and its growing industrial applications [4]. 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]. The spleen is one of the most important organs and serves as a first line of defense in response to parasitic invasion [5] as well as it is the largest immune organ in animals, participating in immune response, generating lymphocytes, eliminating aging erythrocytes and storing blood [6]. The mammalian spleen has a diversity of functions, which vary in expression depending on animal species and age [7]. Almost all blood is circulated through the spleen which filters and removes defective cells and old cells, including erythrocytes [8]. Therefore, in the present study we chose the spleen tissue and find out the molecular fingerprint approach to all biochemical variations. The present study suggest that due to aluminum poisoning, changes the spleen pathologically and further appeared splenocyte ultrastucture, the expression levels of the apoptotic genes and their proteins, oxidative stress in the spleen were investigated. Chelation therapy is one of the most effective methods to remove toxic elements from a biological system [9] by using chelators. Chelators DFO (hexadentate) and DFP (bidentate) are employed to elimination of aluminum accumulation in various tissues. While combined administration of DFO and DFP reduced more Al concentrations in spleen [10]. Fourier transform infrared spectroscopy (FTIR) has 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 [11]. Spleen tissues are basically consisting of proteins, nucleic acids, carbohydrates, phosphodiester and lipids, all of which have characteristics absorption bands in the infrared frequency domain. Therefore, the FT-IR spectrometer was applied to the study of the structure and conformation of bio-molecules such as proteins, fats, carbohydrates and nucleic acids [12]. Inductively Couple Plasma Optical Emission spectroscopy (ICP–OES) is an important technique to study the trace elements at molecular level in various biological samples and it has high sensitivity for detecting the major trace elements [13]. In order to achieve accurate, reliable and sensitive results, preconcentration and separation are needed when the concentrations of analyte elements in the original material or the prepared solution are too low to be determined directly by ICP–OES [14]. Previously, shift in FTIR spectrum patterns in methomyl-exposed rat spleen cells [15] and effects of aluminum trichloride on the trace elements and cytokines in the spleen of rats [16]. Therefore, in the present work, an attempt is made to study the effects of deferiprone and desferrioxamine on metabolic alterations occurs in spleen tissue of aluminum intoxicated mice.
Materials and methods 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
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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. 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. Treatment schedule Mice were randomly allocated in four different groups. Each group contains 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 group II, group III and group 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 5 consecutive days. Aluminum chloride was dissolved in normal drinking water and was given by oral gavage. Twenty-four h after the last dose of aluminum chelators, the animals were sacrificed under ether anesthesia by cutting jugular veins. The spleen were excised, weighed immediately then stored at 80 °C until analysis. 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 ± 10 oC on a Nicolet Avatar 360 FTIR spectrometer equipped with an air cooled DTGS (deuterated triglycine sulfate) 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). ICP–OES analysis The spleen samples were dried at 80 °C for 24 h. Then the samples were filled with 2N HNO3 overnight and rinsed several times with double distilled water. The samples were digested by weighing 1 g of the sample into a 100 mL Borasil flask and then adding 15 mL of concentrated HNO3 (55%) and 5 mL of concentrated Perchloric acid (70%). Digestion was performed on a hot plate at 80– 90 °C for approximately 120 min or until the solutions become dried. After the digestion [17] was completed, each sample was allowed to cool before being filtered through a Whatman No. 42 filter paper using vacuum pump. After filtration, the filtering
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system was rinsed with distilled water to remove all trace elements, and each sample was made up to 25 mL with distilled water and stored in acid-washed polyethylene flasks until the metal concentration analysis. The level of essential elements at spleen was determined by using Inductively Coupled Plasma Optical Emission Spectrometry (ICP–OES, Perkin–Elmer Optima 2100 DV model) available at CAS in Marine Biology, Annamalai University, Tamilnadu, India. All the analyses were carried out in six replications and the average values have been reported. Biochemical analysis The levels of thiobarbituric acid reactive substances, lipid hydroperoxides were estimated by the method of Niehaus and Samuelsson [18,19]. The activities of enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase were estimated by the method of Kakkar et al. [20], Sinha [21] and Rotruck et al. [22] respectively. The non-enzymatic antioxidants reduced glutathione, vitamin C and vitamin E were estimated by the method of Ellman [23], Roe and Kuether [24] and Baker et al. [25] respectively. Histopathological examination of spleen tissue Mice (six mice/treatment group) at the end of experiment were dissected for histology. Removed spleen samples were cleared of blood and immediately placed in a neutral buffered solution of 10% formalin for 24 h. Sliced into 5 lm-thicks and then placed onto glass slides. The sections were stained with haematoxylin and eosin staining (H&E), then examined by light microscopy which have been used to visualize the changes in spleen tissue structures. The cross-sectional area (CSA) of spleen 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). 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. Results FTIR result The present study was focused on the aluminum induced metabolic changes in spleen tissue of mice. FT-IR spectroscopy can effectively provide biochemical variations and information about the structure and composition of biological materials at the molecular level [26]. Also, provides information about chemical bonding properties that characterize biochemical functional components in complex matrices then allow for qualitative identification and quantitative estimations [27]. There are many advantages in detecting materials, such as ease of measurement, low noise, high light flow, rapid survey, precise wave number, wide frequency range of measurement, low cost and detection without damage [28]. The list of absorption peak assignments belonging to lipids, proteins, polysaccharides, carbohydrates, phosphodiester and nucleic acids for various functional groups are presented in Table 1 and the representative infrared spectrum in the region between 4000 and 400 cm 1 is shown in Fig. 1 respectively. Further, FTIR spectra of the three major distinct regions: 3750–2800 cm 1 (A), 1780–1280 cm 1 (B) and 1300–400 cm 1 (C) are shown in Fig. 2.
Curve fitting procedure was not applied because the bands were clearly resolved [29,30]. The band area values, variation of wavenumbers in the major macromolecular groups, band area ratios, ICP–OES study of trace elements and biochemical analysis are presented in Tables 2–7 respectively. In the present study, FTIR spectra for Amide A, amide B, Olefinic@CH, methyl and methylene vibrations of spleen tissue in the range of 3750–2800 cm 1 are shown in Fig. 2(A). The absorption peaks appeared at 3438 and 3066 cm 1corresponds to amide A and amide B respectively due to mainly NAH stretching of protein with negligible contribution from OAH stretching of intermolecular hydrogen bonding, since unbound water was removed from the system [31] as shown in Table 1. Amide A appeared at 3438 cm 1 in control, 3412 cm 1 in aluminum intoxicated, 3432 cm 1 in DFP treated and 3431 cm 1 in DFO + DFP treated/ combine therapy in spleen tissue. This result suggests that formation of hydrogen bonding between amide groups always induces a shift of the NAH stretching vibration down to a lower wavenumber [32]. There is significant change in wavenumber between all groups. The calculated peak area values of amide A band for control, aluminum intoxicated, DFP and DFO + DFP treated tissue are 163.957 ± 2.335, 100.522 ± 1.272, 117.444 ± 1.653, and 131.156 ± 1.760 respectively, which correspond to change 38.69% between the control and aluminum intoxicated, 16.83% between the aluminum intoxicated and DFP treated, 11.67% between the DFP and DFO + DFP treated spleen tissue of mice as shown in Table 2. The significant decrease in peak area value of the amide A band lead to a decrease in the protein quantity of the system. This could be a supportive sign of the destructive effect of aluminum and generates reactive oxygen species [33] resulting in oxidative deterioration of lipids, proteins and DNA. This result suggests that aluminum accumulation in the body is most severe but combine therapy and DFP therapy plays a vital accountability in the treatment of aluminum toxicity and can be flush out redundant aluminum. The absorption band observed at 3066 cm 1 was assigned to NAH stretching of amide B proteins. There is significant variation on band area and wavenumber values as presented in Tables 2 and 3 respectively. The absorption peak for unsaturated lipids observed at 3012 cm 1 consequence from the CAH stretching vibration of HC@CH groups of Olefinic molecule which could be a useful indicator of the different degrees of unsaturation in acyl chains of phospholipids [34,35]. The Olefinic@CH band in aluminum intoxicated spleen tissue was absent, indicating a disappeared in the population of unsaturation in acyl chains of lipid molecules. Lipids give rise to a number of absorptions in FT-IR spectra. The absorption peaks of spleen tissue found at 2960 cm 1 and at 2923 cm 1are known to asymmetric and symmetric stretching vibrations of CH3 and CH2 groups of the acyl chains. The peak at 2854 cm 1 can be assigned as symmetric stretching modes of CH2 group. Hence, this band mainly monitors the lipids in the biological system [36]. The absorption peak area and wavenumber of this region are changed significantly in all groups. The absorption FTIR spectra primarily due to proteins, with some absorption from lipids of control, aluminum intoxicated, DFP treated and DFO + DFP treated spleen tissue in the range of 1780–1280 cm 1 as shown in Fig. 2(B). The band observed at 1741 cm 1 of spleen tissue is assigned to C@O stretching vibration of ester groups in triglycerides [35]. There are significant wavenumber difference and band area values between all groups. The absorption peak appeared at1654 cm 1 assigned as Amide I due to C@O stretching of a-helix protein. The deformation of protein amide NAH bond appeared at 1542 cm 1 (amide II). There are no significant wavenumber difference and band area values between all groups. The 1461and 1398 cm 1 bands arise from the side chain of proteins. The significant increase in the area due to DFP and
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Table 1 FTIR vibrational peak assignments of control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice. Control
Aluminum intoxicated
Al + DFP treated
Al + DFO + DFP treated
Vibrational peak assignments
3438
3412
3432
3431
3066 3012 2960 2923 2854 1741 1654 1542 1461 1398 1309 1232 1168 1081 970 709
3052 – 2947 2917 2848 1716 1637 1535 1450 1397 1312 1221 1159 1045 968 673
3058 3006 2953 2919 2850 1727 1652 1540 1452 1393 1313 1228 1163 1059 966 675
3061 3007 2956 2922 2854 1738 1654 1541 1456 1394 1313 1234 1165 1066 972 701
Amide A due to NAH stretching of proteins with the little contribution from OAH stretching hydrogen bonded intermolecular OH groups of polysaccharides Amide B due to NAH stretching of proteins Olefinic CH@CH stretch: unsaturated lipids CH3 asym. stretch: mainly lipids CH2 asym. stretch: mainly lipids, with little contribution from proteins, carbohydrates, nucleic acids CH2 sym. stretch: mainly lipids Ester C@O stretch: triglycerides, cholesterol esters Amide I (C@O stretching of a-helix protein) Amide II(NAH bending and CAN stretching of proteins) CH3 scissoring mainly lipids COO sym. stretch: fatty acids and amino acids CH3 CH2 stretching of collagen PO2 asym. stretch: mainly phospholipids and phosphodiester in nucleic acids CAO asym. stretching of glycogen PO2 sym. stretch: mainly phospholipids and phosphodiester in nucleic acids CAN+AC sym. stretch: nucleic acids Ring breathing mode in the DNA bases
DFO + DFP treated groups of the band located at 1309 cm 1, which originates from CH3CH2 stretching modes of collagen [37]. The absorption FTIR spectra of phosphates with nucleic acids, i.e., RNA and DNA respectively of control, aluminum intoxicated, DFP treated and DFO + DFP treated spleen tissue in the range of 1300–400 cm 1 as shown in Fig 2(C). The absorption band appeared at 1232 cm 1 assigned to the asymmetric phosphate stretching vibration of phospholipids and the band observed at 1168 cm 1 assigned to CAO asymmetric stretching of glycogen [38]. There are significant variations on wavenumber difference and band area values between all groups. The band appeared at 1048 cm 1 assigned to PO2 symmetric stretching mainly phospholipids and phosphodiester in nucleic acids. The absorption peak at 922 cm 1 is normally assigned to CAN+AC symmetric stretching of nucleic acids, especially for DNA [39]. Hence, aluminum induces the production of free radicals leading to break in lipids, proteins, and DNA in the spleen tissue. The absorption peaks appeared at 719 cm 1 assigned to ring breathing mode in the DNA bases.
Biochemical parameters in spleen tissue Lipid peroxidation, enzymatic and non-enzymatic antioxidants The enhancement of lipid peroxidation products might be due to increased in free radical production and decreased quantities of antioxidant system in aluminum intoxicated mice as shown in Table 4. Treatment with chelating agents DFP and DFO + DFP decreased the levels of lipid peroxidation products. Thus, DFO and DFP inhibits lipid peroxidation may be due to scavenging of free radicals and is expected to its radical scavenging property. The decrease of SOD, GPx and CAT activities shows that these antioxidant enzymes were inhibited, their protective effects against free radicals and lipid peroxidation of organism were reduced, and the spleen would be injured. The level of TBARS has been shown to be an indicator of endogenous lipid peroxidation [40]. In the present study, free radical scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (LHP) decreased in aluminum intoxicated tissue but improved it by treatments with chelating agents DFP and DFO + DFP presented in Table 6. The non-enzymatic antioxidants namely, vitamin C, vitamin E, and reduced glutathione (GSH) which scavenge the residual free radicals escaping from decomposition by the antioxidant enzymes [41]. Vitamin C acts as the protective action during oxidative stress which is drastically decrease due to aluminum toxicity as shown in Table 7. Vitamin E is the most effective lipid
Fig. 1. FT-IR spectra of the control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice in the range of 4000–400 cm 1.
soluble antioxidant in the biological system. Glutathione helps a marked function in detoxification reaction because it is a direct radical scavenger. Our results represent that the levels of nonenzymatic antioxidants that were decreased in aluminum intoxicated mice due to the increased utilization for the neutralization of free radicals, lipid peroxidation products and related to the aerobic nature of cellular metabolism.
Histology assessment of spleen tissue of mice Supplementary Fig. 3 illustrates the protective effects exerted by chelating agents DFO and DFP against aluminum induced histopathological photographs of spleen tissue of mice. Control mice spleen showed normal congestion displayed in Supplementary Fig. 3A. The aluminum group revealed extensive spleen injuries characterized by inflammatory collections of prominent follicles and dilated sinusoids as shown in Supplementary Fig. 3B. By administration with DFP showed partial recovered of extramedullary haematopoiesis as shown in Supplementary Fig. 3C. Finally, treatment with DFO and DFP showed the remarkably reduced spleen injuries with normal architecture areas of fragmentation of the tissue with congestion as shown in Supplementary Fig. 3D.
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Fig. 2. Selected FT-IR spectra of the control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissues of mice in the range of 3750–2800 cm (B), and 1300–400 cm 1 (C).
1
(A), 1780–1280 cm
1
Table 2 Band area values of major bands for the control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice. Bands
Control
Aluminum intoxicated
Al + DFP treated
Al + DFO + DFP treated
3438 3066 3012 2960 2923 2854 1741 1654 1542 1461 1398 1309 1232 1168 1081 970 709
163.957 ± 2.335d 0.509 ± 0.029c 0.029 ± 0.004b 1.070 ± 0.026d 5.822 ± 0.139c 1.100 ± 0.211a,b 2.208 ± 0.142d 28.748 ± 0.440d 12.068 ± 0.053d 2.658 ± 0.114d 2.734 ± 0.148d 0.169 ± 0.032b 5.176 ± 0.060d 0.158 ± 0.013c 18.885 ± 0.198d 0.498 ± 0.026d 0.619 ± 0.006d
100.522 ± 1.272a 0.154 ± 0.015a Not Observed 0.275 ± 0.013a 0.877 ± 0.011a 0.698 ± 0.030a 0.120 ± 0.028a 7.858 ± 0.100a 2.507 ± 0.249a 0.454 ± 0.067a 0.848 ± 0.082a 0.062 ± 0.008a 1.034 ± 0.005a 0.107 ± 0.051a 1.506 ± 0.166a 0.007 ± 0.001a 0.050 ± 0.018a
117.444 ± 1.653b 0.294 ± 0.019b 0.016 ± 0.002a 0.432 ± 0.015b 2.350 ± 1.077a 1.039 ± 0.655a,b 0.314 ± 0.009b 15.220 ± 0.228b 5.159 ± 0.016b 0.717 ± 0.036b 1.307 ± 0.242b 0.077 ± 0.002a 1.989 ± 0.062b 0.137 ± 0.010a,b 4.232 ± 0.027b 0.186 ± 0.010b 0.109 ± 0.005b
131.156 ± 1.760c 0.505 ± 0.024c 0.026 ± 0.002b 0.668 ± 0.012c 3.875 ± 0.074b 1.382 ± 0.024b 0.546 ± 0.022c 24.162 ± 1.235c 9.471 ± 0.228c 1.473 ± 0.149c 1.662 ± 0.049c 0.083 ± 0.003a 2.490 ± 0.058c 0.182 ± 0.011c 8.080 ± 0.420c 0.249 ± 0.013c 0.174 ± 0.014c
Comparisons values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
ICP–OES analysis In the present study we chose the spleen tissue because it is a peripheral organ of the immune system and accumulated
Aluminum might be alter the biochemical constituents. The ICP– OES study of spleen tissue trace elements such as Ca, Fe, Cu, Mn and Zn as shown in Table 5. Al exposure has an adverse effect on essential elements in humans, with subsequent impact on the
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Control
NAH stretching (proteins): Amide A NAH stretching (proteins): Amide B CH2 sym. (fatty acids) Carbonyl C@O (lipids) Amide I (protein) Amide II (protein) PO2 asym. nucleic acids and phospholipids PO2 sym. nucleic acids and phospholipids
Aluminum intoxicated c
Al + DFP treated
a
3438 ± 2.510 3066 ± 1.509c 2854 ± 0.696b 1741 ± 2.617c 1654 ± 2.025b 1542 ± 2.046b 1232 ± 2.236c 1081 ± 2.266d
b
3412 ± 3.899 3052 ± 1.614a 2848 ± 1.817a 1716 ± 2.608a 1637 ± 1.677a 1535 ± 2.881a 1221 ± 2.302a 1045 ± 2.161a
3432 ± 4.050 3058 ± 1.837b 2850 ± 1.921a 1727 ± 2.589b 1652 ± 2.828b 1540 ± 2.641b 1228 ± 2.716b 1059 ± 1.870b
Al + DFO + DFP treated 3431 ± 4.243b 3061 ± 1.225c 2854 ± 2.828b 1738 ± 2.449c 1654 ± 5.329b 1541 ± 3.162b 1234 ± 2.561c 1066 ± 1.846c
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.
Table 4 FTIR absorption band area ratio for selected bands of control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice. Bands area ratio
Control
Al-intoxicated
Al + DFP treated
Al + DFO + DFP treated
I1081/I1232 I1542/I3438 I2960/I2854 I1542/I1654 I1081/I1542
3.648 ± 0.016d 0.074 ± 0.003c 0.535 ± 0.018c 0.420 ± 0.028c 1.565 ± 0.027d
1.457 ± 0.050a 0.025 ± 0.002a 0.393 ± 0.023a 0.319 ± 0.015a 0.600 ± 0.016a
2.283 ± 0.032b 0.044 ± 0.002b 0.416 ± 0.020a 0.339 ± 0.024a 0.820 ± 0.036b
3.246 ± 0.025c 0.072 ± 0.004c 0.483 ± 0.021b 0.392 ± 0.020b 0.853 ± 0.025c
Values are expressed as mean ± S.E for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
Table 5 ICP–OES study of trace elements in spleen tissue of control, aluminum intoxicated, DFP and DFO + DFP treated mice (lg/g tissue). Groups
Zn
Fe
Ca
Cu
Mg
Control Aluminum intoxicated Al + DFP treated Al + DFO + DFP treated
32 ± 3.120d 29 ± 2.586c 26 ± 1.350b 23 ± 1.524a
215 ± 1.117a 223 ± 1.441b 229 ± 1.910c 232 ± 1.460d
101 ± 1.162d 81 ± 0.672a 92 ± 1.007b 97 ± 0.880c
5.2 ± 0.084a 5.6 ± 0.072b 5.7 ± 0.126b 6.1 ± 0.131c
189 ± 2.608d 186 ± 1.720c 182 ± 1.139b 178 ± 1.695a
Values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
cellular enzymatic and metabolic processes [42]. The elements such as Ca, Fe, Cu, Mn and Zn are essential elements because of their indispensable role in biological systems. Aluminum may interfere with various metabolic processes, in which Ca2+, Mg2+, Fe3+ and Fe2+ (gastrointestinal absorption) are involved [43]. The choice of those elements was based in previous studies which showed potential relationship between aluminum and the above mentioned elements [44]. The concentration of all the elements level showed difference between control and treated groups.
Discussion The present study was designed to investigate the protective effects of the chelating agents DFO and DFP in aluminum intoxicated spleen tissue of mice to knowing the functional, structural, and compositional changes at the molecular level by Fourier transform infrared (FT-IR) spectroscopy as shown in Fig. 1. So that present study is so helpful and informative to the toxicology and spectroscopy fields. Our results provides the useful information to the scientific community as all the parameters responded positively to individual therapy (DFP), but more pronounced beneficial effects were observed in combination therapy (DFO + DFP) for aluminum intoxicated mice. In the present work, the absorption peak observed at 3438 cm 1 occurs from both the NAH and OAH stretching modes of water, proteins and polysaccharides. Table 3 shows the variation of wavenumber 3438 ± 2.510, 3412 ± 3.899, 3432 ± 4.050, and 3431 ± 4.243 respectively in the macromolecular amide A in spleen tissue of mice. Here, aluminum intoxicated tissue shows the lower wavenumber because formation of hydrogen bonding between
amide groups always induces a shift of the NAH stretching vibration down to a lower wavenumber [32]. The band observed at 3066 cm 1 assigned to amide B mainly NAH stretching of protein with negligible contribution from OAH stretching of intermolecular hydrogen bonding as shown in Fig. 2(A). The calculated peak area values of amide B band for control, aluminum intoxicated, DFP and DFO + DFP treated tissue are 0.509 ± 0.029, 0.154 ± 0.015, 0.294 ± 0.019, 0.505 ± 0.024 respectively, which correspond to change 69.74% between control to aluminum intoxicated tissue, 90.90% between aluminum intoxicated to DFP treated tissue, 41.78% between DFP to DFO + DFP treated spleen tissue presented in Table 2. This result suggests that DFO and DFP are the good antidotes. The absorption band observed at 3012 cm 1 absent in aluminum intoxicated tissue due to over production of reactive oxygen species (ROS). Further, the absorption peaks appeared at 3006 cm 1 for DFP treated and at 3007 cm 1 for DFO + DFP treated spleen tissue due to unsaturated olefinic molecule, these could be a useful indicator of the different degrees of unsaturation in acyl chains of phospholipids band [45]. The absorption peak area and wavenumbers are changed significantly in observed groups. The calculated peak area values of Olefinic@CH band (index of relative concentration of double bonds in the lipid structure of unsaturated lipids) for control, DFP and DFO + DFP treated tissue are 0.029 ± 0.004, 0.016 ± 0.002 and 0.026 ± 0.002 respectively. This result suggests that absent of Olefinic@CH peak due to aluminum poisoning might be causing reduction in the concentration of polyunsaturated fatty acids as shown in Table 2. Lipids play a key role in the membrane fluidity. By affecting the conformation of membrane proteins, they govern exposure and diffusion of membrane components. The area values of the asymmetric stretching vibrations of methyl ACH3) and methylene
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Table 6 Effects of DFP and DFO + DFP on biochemical parameters in aluminum intoxicated mice. Biochemical Parameters Protein level (mg/100 mg) SOD (U/mg of protein) LHP (nmol/mg protein) Gpx (lg of GSH utilized min/mg protein CAT (lmol of H2O2utilized/min/mg protein) TBARS (nmol/mg protein)
Control
Aluminum intoxicated d
32.38 ± 0.093 6.39 ± 0.096c 1.09 ± 0.072a 9.03 ± 0.589c 41.23 ± 1.115d 1.42 ± 0.244a
a
17.24 ± 0.198 2.97 ± 0.513a 2.93 ± 0.278d 4.47 ± 0.191a 21.91 ± 1.096a 3.83 ± 0.132c
Al + DFP treated b
25.31 ± 0.730 4.94 ± 0.581b 1.98 ± 0.389c 6.49 ± 0.326b 32.43 ± 0.192b 2.05 ± 0.062b
Al + DFO + DFP treated 29.96 ± 0.557c 5.89 ± 0.385c 1.43 ± 0.234b 8.78 ± 0.407c 37.18 ± 1.358c 1.63 ± 0.018a
Values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
Table 7 Level of non-enzymic antioxidants in control and treated mice. Groups
Vitamin E (lg/mg protein)
Vitamin C (lg/mg protein)
GSH (mg/100 g tissue)
Control Aluminum intoxicated Al + DFP treated Al + DFO + DFP treated
3.44 ± 0.409c 2.31 ± 0.229a 2.61 ± 0.141a 3.03 ± 0.378b
1.48 ± 0.044c 0.68 ± 0.062a 1.14 ± 0.087b 1.29 ± 0.096b
74.53 ± 1.350d 52.25 ± 0.715a 65.84 ± 3.199b 69.91 ± 1.497c
Values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
(ACH2) groups decreased from control (1.070 ± 0.026 and 5.822 ± 0.139) to aluminum intoxicated (0.275 ± 0.013 and 0.877 ± 0.011), these reflects the changes in the composition of the acyl chains. Further, the areas of these bands were increased from DFP (0.432 ± 0.015 and 2.350 ± 1.077) to DFO + DFP (0.668 ± 0.012 and 3.875 ± 0.074). A decrease area of the symmetric stretching vibration of methylene band at 2854 cm 1 from control (1.100 ± 0.211) to aluminum intoxicated (0.698 ± 0.030) spleen tissue and increased again from 1.039 ± 0.655 to 1.382 ± 0.024, after DFP and DFO + DFP treatments. This result suggests that decreased lipid order and increased acyl chain flexibility in DFP and DFO + DFP treated spleen tissue of mice. The absorption band observed at 1745 cm 1, which arises from the stretching mode of the ester C@O groups of the lipids [46]. The significant decreased in peak area value of ester carbonyl lipid band at 1745 cm 1 for aluminum intoxicated tissue, but enhanced it for DFP and DFO + DFP treated spleen tissue. The degradation of acyl chains due to aluminum poisoning provide the information of lipids become oxidized, since oxidation to lipids causes the formation of carbonyl groups and a breakdown of long chains into smaller fragments then disorder the lipid metabolism in spleen tissue of mice. The amide I region comprises vibrations due mainly to C@O stretching modes with some contribution from NAH bending and CAN stretching and the amide II region correspond to mainly NAH bending with a large contribution from CAN stretching modes [15]. Characteristic infrared absorption bonds of peptide linkages at these amide I and II regions correspond to the alpha-helix protein structure and all the constituent of amino acid side-chains in proteins are susceptible to free radicals, but some are more vulnerable than others [47]. The calculated peak area values of amide I band for control, aluminum intoxicated, DFP and DFO + DFP treated tissue are 28.748 ± 0.440, 7.858 ± 0.100, 15.220 ± 0.228 and 24.162 ± 1.235 respectively, which correspond to change 72.66% between control to aluminum intoxicated tissue, 93.68% between aluminum intoxicated to DFP treated tissue, 58.75% between DFP to DFO + DFP treated spleen tissue. The decreased peak area observed at aluminum intoxicated tissue suggests that no measurable protein oxidation takes place due to the overproduction of ROS in aluminum exposure tissue, these results leads to the breakdown of balance of the oxidative/anti-oxidative system in spleen, resulting in the lipid peroxidation. This is due to an alteration of antioxidant defense ability. The observed absorption peak at 1542 cm 1 assigned to amide II mainly NAH bending and CAN stretching of proteins. Amide II appeared at 1542 cm 1 in control, 1535 cm 1
in aluminum intoxicated, 1540 cm 1 in DFP treated and 1541 cm 1 in DFO + DFP treated spleen tissue. Therefore, decrease in peak areas and wavenumbers of amides I band indicate the destructive effect of aluminum and this changes reflect the loss of protein level in the aluminum intoxicated spleen tissue of mice. This result suggest that the explanation for the decreased protein content may be an excess free radicals production and binding of aluminum with various sulfhydryls (RASH) that exists in the tissue also the explanation may be due to a reduction of the protein synthesis in the spleen. The COO symmetric stretch mainly fatty acids and amino acids observed at1398 cm 1. The decreased in both wavenumbers and peak area values for aluminum intoxicated spleen tissue suggests that changes in the structure of amino acids caused by aluminum poisoning. The phosphate asymmetric stretching absorption band observed at 1232 cm 1 originated due to the phosphodiester back bone of cellular nucleic acids. The peak area decreased from 5.176 ± 0.060 to 1.034 ± 0.005 in between control to aluminum intoxicated group, treatments with DFP and DFO + DFP recovered it nearer to the control values from 1.989 ± 0.062 to 2.490 ± 0.05 respectively. The 80.02% shifted peak area value in aluminum exposed tissue; this might be due to the changes in the composition of nucleic acids. Further, decreased level of phospholipids may partly be responsible for the decrease in lipid peroxidation at aluminum intoxicated tissue. The significant increase in the intensity of the band at 1168 cm 1 in the aluminum intoxicated spleen might be due to an increase in the content of glycogen [38] as shown in Fig. 2(C). This result confirmed by an increased intensity of the CAO stretching absorption peak at 1168 cm 1, which is mainly due to glycogen in tissue [48]. The phosphate symmetric stretching due phospholipids and phosphodiester in nucleic acids observed at 1081 cm 1. The calculated peak area values of this band for control, DFP and DFO + DFP treated tissue are 18.885 ± 0.198, 1.506 ± 0.166, 4.232 ± 0.027 and 8.080 ± 0.420 respectively. This result suggests decrease (92.02%) in the relative content of the nucleic acids due to aluminum poisoning as compare to control spleen tissue. The absorption peak observed at 970 cm 1 assigned to CAN+AC symmetric stretch vibration of nucleic acids. The decrease peak area suggests a decrease in the relative content of the nucleic acids due to aluminum poisoning in spleen tissue of mice. Therefore, the decreased intensity of amide A band in the aluminum intoxicated spleen tissue reflects the decreased quantity of protein. This decreased quantity indicates the damage of amide
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linkage in aluminum intoxicated spleen tissue. There was an enhancement in the level of thiobarbituric acid reactive substances (TBARS) 3.83 ± 0.132 for aluminum poisoning but brings back to nearer control value (1.42 ± 0.244) by DFP (2.05 ± 0.062) and DFO + DFP (1.63 ± 0.018) treatments, and similar in lipid hydroperoxides (LHP) as shown in Table 6. This result suggests that aluminum generates reactive oxygen species, resulting in oxidative deterioration of lipids, proteins and DNA. Further, in aluminum intoxicated spleen tissue decreased the antioxidant activities such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) could be due to the induction of lipid peroxidation [49]. The level of non-enzymic antioxidants such as vitamin C, Vitamin E and glutathione (GSH) are decreased in aluminum intoxicated spleen tissue as shown in Table 7. This result suggests the reduction of protein quantity may be the imbalance between oxidants and antioxidants. Therefore, the DFO and DFP are useful therapeutic drugs which blocked ROS production. The present results showed that DFO and DFP are promising for a novel class of drugs in the treatment of immunoinflammatory spleen diseases causes by aluminum exposure. The ratio of the peak intensities of the bands at 1081 cm 1 and at 1232 cm 1 (I1081/I1232) is related to the ratio of symmetric modes of the phosphodiester group (APO2) in nucleic acids to asymmetric modes of phosphodiester group [50]. The variation of ratios for the control, aluminum intoxicated, DFP treated and DFO + DFP treated spleen tissue are 3.648 ± 0.016, 1.457 ± 0.050, 2.283 ± 0.032 and 3.246 ± 0.025 respectively, which correspond to change of 60.06% between control and aluminum intoxicated, 56.69% between aluminum intoxicated and DFP treated, 42.18% between DFP and DFO + DFP treated spleen tissue of mice. In present work, the ratio of the peak intensities of the bands at 1542 cm 1 and at 3438 cm 1 (I1542/I3438) has been used as an indicator of the relative concentration of the protein in the spleen tissue. Present result suggests the protein content in the membrane is higher in chelating agents treated tissue but lower in the aluminum intoxicated spleen tissue. The mean ratios of the intensity of absorption of the methyl band and methylene band (I2960/I2854) for control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue are 0.535 ± 0.018, 0.393 ± 0.023, 0.416 ± 0.020 and 0.483 ± 0.021 respectively, which corresponds to change of 26.54% between control to aluminum intoxicated, 5.85% between aluminum to DFP treated, 16.10% between DFP to DFO + DFP treated spleen tissue. The decreases ratio indicate a decrease in the number of methyl groups in protein fibers compared to methylene groups in aluminum intoxicated spleen tissue. The mean ratio of the intensities of the bands at 1542 cm 1 and 1652 cm 1 could be attributed to a change in the composition of the whole protein pattern [51]. In the intensity ratio (I1542/I1654) decreased from 0.420 ± 0.028 to 0.319 ± 0.015 for aluminum intoxicated spleen tissue as shown in Table 4. This results in a fall in the overall protein contents due to aluminum exposure. In the presence of chelating agents DFP and DFO + DFP, the ratio increased to 0.339 ± 0.024 to 0.392 ± 0.020 respectively. The mean ratio of the intensities of the bands I1081/I1542 could be attributed to a change in the composition of the glycoprotein. In the spleen tissue, this ratio decreased from 1.565 ± 0.027 to 0.600 ± 0.016, for aluminum intoxicated tissue, indicating a fall in the glycoprotein contents due to aluminum poisoning. Treatments with chelating agents DFP and DFO + DFP, this ratio increased to 0.820 ± 0.036, 0.853 ± 0.025 respectively.
changes in response to the chelating agents treatment in spleen tissue of mice. FT-IR spectra revealed significant differences in absorbance intensities between the control, aluminum intoxicated and chelating agents treated spleen tissue of mice. The variation on the major biochemical constituents such as lipids, proteins, phosphodiester and nucleic acids of the spleen tissue due to the overproduction of ROS but significantly improved it by administration of DFP and DFO + DFP. Our results established that all the parameters and biochemical constituents responded positively to individual therapy with DFP, but more pronounced beneficial effects were observed in combination (DFO + DFP) therapy and good agreements with biochemical parameters, histopathological assessments and FTIR results. Further, our results confirmed that aluminum plays an important role in the pathogenesis of spleen tissue. In addition, investigation the role of DFP and DFO + DFP chelation therapy for treatments of spleen is protected and secured from chronic diseases. Finally, the present study provided the agreements between the biochemical parameters, histopathology and FT-IR results. Funding University Grants Commission (UGC), New Delhi, India, Major Research Project F. No. 40-447/2011 (SR). Acknowledgements The financial support to author Mr. Chandra Prasad Khatiwada from the University Grants Commission, New Delhi, India is gratefully acknowledged. Further, the authors are thankful and grateful to Dr. S. Barathan, Professor and Head, Department of Physics, Annamalai University, Tamilnadu-608 002, for providing all necessary facilities to carry out the present work successfully. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.004. References [1] [2] [3] [4] [5] [6]
[7] [8] [9] [10] [11]
[12] [13] [14] [15] [16]
Conclusion [17]
This is the first detailed explanation report for FTIR spectroscopic technique has been employed to monitor biomolecular
155
[18] [19]
S.V. Verstraeten, L. Aimo, P.I. Oteiza, Arch. Toxicol. 82 (2008) 789–802. V. Kumar, K.D. Gill, Arch. Toxicol. 83 (2009) 965–978. M. Venturini-Soriano, G. Berthon, J. Inorg. Bioch. 85 (2001) 143–154. M.L. Ana, G. Graciela, N. Alcira, Int. J. Immunopharmacol. 1 (2001) 1725–1732. D. Biswas, A. Choudhury, K.K. Misra, Exp. Parasitol. 99 (2001) 148–159. D. Na Li, H. Yanmei, Z. Mengmeng, F. Lei, Z. Min, W. Xiaoyang, C. Jue, L. Yaling, C. Huiting, G. Jingwei, W. Songjie, H. Han, Fashui. Toxicol. Lett. 95 (2010) 61– 168. A.Z. Lilianne, G. Jan, Z. Gosta, Mutat. Res. 394 (1997) 17–28. N.C. Jain, Schalm’s Veterinary Hematology, 4th ed., Lea and Febiger, 600.Washington square, Philadelphia, USA, 1986. S. Tubafard, S.J. Fatemi, A.S. Saljooghi, M. Torkzadeh, Med. Chem. Res. 19 (2010) 854–863. G. Mercedes, L.E. Jose, L.D. Jose, K.S. Pramod, M.J. Mark, Toxicology 137 (1999) 161–168. D. McNaughton, K.R. Bambery, B.R. Wood, Spectral histopathology of the human cervix, in: M. Diem, P.R. Griffiths, J.M. Chalmers (Eds.), Vibrational Spectroscopy for Medical Diagnosis, John Wiley & Sons, UK, 2008, pp. 187–202. S.N. Jha, S. Gunasekaran, J. Food Eng. 101 (2010) 337–342. K. Fent, P.W. Looser, Water Res. 29 (1995) 1631–1637. N. Yunes, S. Moyano, S. Cerutti, J.A. Gasquez, L.D. Martinez, Talanta 59 (2003) 943–949. S. Teerayut, S. Ratana, D. Nikorn, P. Tipicha, S. Palarp, Sci. Total Environ. 270 (2001) 103–108. Z. Yanzhu, L. Xinwei, C. Chongxiao, W. Fan, L. Jing, H. Chongwei, L. Yanfei, M. Liguang, Food Chem. Toxicol. 50 (2012) 2911–2915. M. Bettinelli, G.M. Beone, S. Spezia, C. Baffi, Anal. Chim. Acta 424 (2000) 289– 296. W.G. Niehaus, B. Samuelsson, Eur. J. Biochem. 6 (1968) 126–130. Z.Y. Jiang, J.V. Hunt, S.P. Wolff, Anal. Biochem. 202 (1992) 384–389.
156
S. Sivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 148–156
[20] P. Kakkar, B. Das, P.N. Viswanathan, Indian J. Biochem. Biophys. 21 (1984) 130– 132. [21] A.K. Sinha, Anal. Biochem. 47 (1972) 389–394. [22] J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman, W.G. Hoekstra, Science 179 (1973) 588–590. [23] G.L. Ellman, Arch. Biochem. Biophys. 82 (1959) 70–77. [24] J.H. Roe, C.A. Kuether, J. Biol. Chem. 11 (1943) 145–164. [25] H. Baker, O. Frank, B. DeAngelis, Nutr. Res. 21 (1980) 531–536. [26] P.T. Wong, R.K. Wong, T.A. Caputo, T.A. Godwin, B. Rigas, Proc. Natl. Acad. Sci. USA 88 (1991) 10988–10992. [27] L. Xiaonan, A.H.W. Molly, J.T. Mariah, P.V.E. Joel, I.D. Serge, A.R. Barbara, Aquaculture 315 (2011) 298–305. [28] E. Benedetti, L. Teodori, M.L. Trica, P. Vergamini, F. Salvati, F. Mauro, Appl. Spectrosc. 44 (1990) 1276–1280. [29] G. Cakmak, I. Togan, F. Severcan, Aquat. Toxicol. 77 (2006) 53–63. [30] A. Dogan, G. Siyakus, F. Severcan, Food Chem. 100 (2006) 1106–1114. [31] N.S. Ozek, Y. Sara, R. Onur, F. Severcan, Bio. Sci. Rep. 30 (2010) 41–45. [32] D. Lin-Vien, N.B. Colthup, W.G. Fateley, J.G. Grasselli, The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules, Academic Press, San Diego CA, 1990. [33] F.M. El-Demerdash, J. Trace Elem. Med. Biol. 18 (2004) 113–122. [34] O. Bozkurt, M. Severcan, F. Severcan, Analyst 135 (2010) 3110–3119. [35] E. Bogomolny, S. Argov, S. Mordechai, M. Huleihel, Biochim. Biophy. Acta 1780 (2008) 1038–1046.
[36] S.B. Akkas, M. Severcan, O. Yilmaz, F. Severcan, Food Chem. 105 (2007) 1281– 1288. [37] N. Stone, C. Kendall, J. Smith, P. Crow, H. Barr, J. Raman Spectrosc. 33 (2002) 564–573. [38] N. Toyran, P. Lasch, D. Naumann, B. Turan, F. Severcan, Biochem. J. 397 (2006) 427–436. [39] D. Naumann, D. Helm, H. Labischinski, Nature 351 (1991) 81–82. [40] L. Guangke, S. Nan, G. Dongsheng, Sphere 65 (2006) 1058–1063. [41] B. Seifi, M. Kadkhodaee, S.M. Karimian, M. Zahmatkesh, J. Xu, M. Soleimani, Clin. Exp. Hypertens. 32 (2010) 90–97. [42] F.M. Metwally, M.S. Mazhar, Arh. Hig. Rada. Toksikol. 58 (2007) 305–311. [43] P. Zatta, T. Kiss, M. Suwalsky, G. Berthon, Chem. Rev. 228 (2002) 271–284. [44] D.J. Sanchez, M. Gomez, J.M. Llobet, J. Corbella, J.L. Domingo, Pharmacol. Toxicol. 80 (1997) 11–17. [45] M.D. Guillen, N.J. Cabo, Agric. Food Chem. 47 (1999) 709–719. [46] H. Fabian, P. Lash, M. Boese, W. Haensch, J. Mol. Struct. (2003) 661–662. [47] C.A. Rice-Evans, A.T. Diplock, M.C.R. Symos., Elsevier, New York, 1991. [48] S.Y. Lin, M.J. Li, W.T. Cheng, Spectroscopy 21 (2007) 1–30. [49] G.C. Jagetia, G.K. Rajanikant, S.K. Rao, M.S. Baliga, Clin. Chim. Acta 332 (2003) 111–121. [50] P.T.T. Wong, Biospectroscopy 1 (1995) 357–364. [51] E. Benedetti, E. Bramanti, F. Papineschi, I. Rossi, E. Benedetti, Appl. Spectrosc. 51 (1997) 792–797.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Protective effects of desferrioxamine and deferiprone on the spleen tissue of aluminum intoxicated mice: A Fourier transform infrared spectroscopy study S. Sivakumar ⇑, Chandra Prasad Khatiwada, J. Sivasubramanian Department of Physics, Annamalai University, Annamalai Nagar, Tamilnadu 608002, 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 study determined functional and
compositional changes on spleen tissue. FTIR study has been good agreement with biochemical, histopathology and ICP–OES. All parameters and biochemical constituents responded positively to DFP therapy. But more pronounced beneficial effects were observed in DFO + DFP therapy.
a r t i c l e
i n f o
Article history: Received 24 September 2013 Received in revised form 26 January 2014 Accepted 2 February 2014 Available online 15 February 2014 Keywords: Aluminum Spleen DFO and DFP FTIR ICP–OES
a b s t r a c t The present study was designed to examine the protective effects of the chelating agents desferrioxamine (DFO) and deferiprone (DFP) in aluminum intoxicated spleen tissue of mice by Fourier transform infrared (FTIR) spectroscopy. The finding revealed the alterations on the major biochemical constituents, such as lipids, proteins, phosphodiester and nucleic acids of the spleen tissue of mice at molecular level. The significant decreased in the peak areas of asymmetric and symmetric mode of the phosphodiester groups from control to aluminum intoxicated, but improved it by DFP and DFO + DFP treatments respectively for nearer control value. The bands ratio at I1081/I1232 significantly decreased from control to aluminum, but enhanced it by DFP and DFO + DFP respectively. This result suggests that DFO and DFP are the phosphodiesterase inhibitor, recovered from chronic growth of diseases in the spleen. Amide I and amide II peak area values decreased from control to aluminum intoxicated spleen tissue, but treated with DFP and DFO + DFP significantly improved. This result suggests an alteration in the protein profile. The absence of Olefinic@CH stretching in aluminum intoxicated spleen suggests an altered lipid levels. Concentrations of trace elements were found by ICP–OES. Histopathological findings confirmed the biochemical observations of this study. The results of the FTIR study were found to be in agreement with biochemical studies and demonstrated that FTIR can be used successfully applied to toxicological studies at molecular level. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author. Tel.: +91 9865043811. E-mail address: [email protected] (S. Sivakumar). http://dx.doi.org/10.1016/j.saa.2014.02.004 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Aluminum (Al) is the third most prevalent element and the most abundant metal in the earth’s crust i.e., approximately 8%
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of total mineral components [1]. It is widely distributed in the environment and extensively used in daily life resulting easy exposure to human beings [2]. Aluminum absorption/accumulation in humans can occur via the diet, drinking water, ingestion with fruit juices or citric acid causes a marked increase in both gastrointestinal absorption and urinary excretion of aluminum in healthy subjects [3]. The main sources of human exposure to aluminum are to be found in the extensive presence of the metal in the environment and its growing industrial applications [4]. 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]. The spleen is one of the most important organs and serves as a first line of defense in response to parasitic invasion [5] as well as it is the largest immune organ in animals, participating in immune response, generating lymphocytes, eliminating aging erythrocytes and storing blood [6]. The mammalian spleen has a diversity of functions, which vary in expression depending on animal species and age [7]. Almost all blood is circulated through the spleen which filters and removes defective cells and old cells, including erythrocytes [8]. Therefore, in the present study we chose the spleen tissue and find out the molecular fingerprint approach to all biochemical variations. The present study suggest that due to aluminum poisoning, changes the spleen pathologically and further appeared splenocyte ultrastucture, the expression levels of the apoptotic genes and their proteins, oxidative stress in the spleen were investigated. Chelation therapy is one of the most effective methods to remove toxic elements from a biological system [9] by using chelators. Chelators DFO (hexadentate) and DFP (bidentate) are employed to elimination of aluminum accumulation in various tissues. While combined administration of DFO and DFP reduced more Al concentrations in spleen [10]. Fourier transform infrared spectroscopy (FTIR) has 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 [11]. Spleen tissues are basically consisting of proteins, nucleic acids, carbohydrates, phosphodiester and lipids, all of which have characteristics absorption bands in the infrared frequency domain. Therefore, the FT-IR spectrometer was applied to the study of the structure and conformation of bio-molecules such as proteins, fats, carbohydrates and nucleic acids [12]. Inductively Couple Plasma Optical Emission spectroscopy (ICP–OES) is an important technique to study the trace elements at molecular level in various biological samples and it has high sensitivity for detecting the major trace elements [13]. In order to achieve accurate, reliable and sensitive results, preconcentration and separation are needed when the concentrations of analyte elements in the original material or the prepared solution are too low to be determined directly by ICP–OES [14]. Previously, shift in FTIR spectrum patterns in methomyl-exposed rat spleen cells [15] and effects of aluminum trichloride on the trace elements and cytokines in the spleen of rats [16]. Therefore, in the present work, an attempt is made to study the effects of deferiprone and desferrioxamine on metabolic alterations occurs in spleen tissue of aluminum intoxicated mice.
Materials and methods 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
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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. 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. Treatment schedule Mice were randomly allocated in four different groups. Each group contains 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 group II, group III and group 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 5 consecutive days. Aluminum chloride was dissolved in normal drinking water and was given by oral gavage. Twenty-four h after the last dose of aluminum chelators, the animals were sacrificed under ether anesthesia by cutting jugular veins. The spleen were excised, weighed immediately then stored at 80 °C until analysis. 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 ± 10 oC on a Nicolet Avatar 360 FTIR spectrometer equipped with an air cooled DTGS (deuterated triglycine sulfate) 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). ICP–OES analysis The spleen samples were dried at 80 °C for 24 h. Then the samples were filled with 2N HNO3 overnight and rinsed several times with double distilled water. The samples were digested by weighing 1 g of the sample into a 100 mL Borasil flask and then adding 15 mL of concentrated HNO3 (55%) and 5 mL of concentrated Perchloric acid (70%). Digestion was performed on a hot plate at 80– 90 °C for approximately 120 min or until the solutions become dried. After the digestion [17] was completed, each sample was allowed to cool before being filtered through a Whatman No. 42 filter paper using vacuum pump. After filtration, the filtering
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system was rinsed with distilled water to remove all trace elements, and each sample was made up to 25 mL with distilled water and stored in acid-washed polyethylene flasks until the metal concentration analysis. The level of essential elements at spleen was determined by using Inductively Coupled Plasma Optical Emission Spectrometry (ICP–OES, Perkin–Elmer Optima 2100 DV model) available at CAS in Marine Biology, Annamalai University, Tamilnadu, India. All the analyses were carried out in six replications and the average values have been reported. Biochemical analysis The levels of thiobarbituric acid reactive substances, lipid hydroperoxides were estimated by the method of Niehaus and Samuelsson [18,19]. The activities of enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase were estimated by the method of Kakkar et al. [20], Sinha [21] and Rotruck et al. [22] respectively. The non-enzymatic antioxidants reduced glutathione, vitamin C and vitamin E were estimated by the method of Ellman [23], Roe and Kuether [24] and Baker et al. [25] respectively. Histopathological examination of spleen tissue Mice (six mice/treatment group) at the end of experiment were dissected for histology. Removed spleen samples were cleared of blood and immediately placed in a neutral buffered solution of 10% formalin for 24 h. Sliced into 5 lm-thicks and then placed onto glass slides. The sections were stained with haematoxylin and eosin staining (H&E), then examined by light microscopy which have been used to visualize the changes in spleen tissue structures. The cross-sectional area (CSA) of spleen 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). 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. Results FTIR result The present study was focused on the aluminum induced metabolic changes in spleen tissue of mice. FT-IR spectroscopy can effectively provide biochemical variations and information about the structure and composition of biological materials at the molecular level [26]. Also, provides information about chemical bonding properties that characterize biochemical functional components in complex matrices then allow for qualitative identification and quantitative estimations [27]. There are many advantages in detecting materials, such as ease of measurement, low noise, high light flow, rapid survey, precise wave number, wide frequency range of measurement, low cost and detection without damage [28]. The list of absorption peak assignments belonging to lipids, proteins, polysaccharides, carbohydrates, phosphodiester and nucleic acids for various functional groups are presented in Table 1 and the representative infrared spectrum in the region between 4000 and 400 cm 1 is shown in Fig. 1 respectively. Further, FTIR spectra of the three major distinct regions: 3750–2800 cm 1 (A), 1780–1280 cm 1 (B) and 1300–400 cm 1 (C) are shown in Fig. 2.
Curve fitting procedure was not applied because the bands were clearly resolved [29,30]. The band area values, variation of wavenumbers in the major macromolecular groups, band area ratios, ICP–OES study of trace elements and biochemical analysis are presented in Tables 2–7 respectively. In the present study, FTIR spectra for Amide A, amide B, Olefinic@CH, methyl and methylene vibrations of spleen tissue in the range of 3750–2800 cm 1 are shown in Fig. 2(A). The absorption peaks appeared at 3438 and 3066 cm 1corresponds to amide A and amide B respectively due to mainly NAH stretching of protein with negligible contribution from OAH stretching of intermolecular hydrogen bonding, since unbound water was removed from the system [31] as shown in Table 1. Amide A appeared at 3438 cm 1 in control, 3412 cm 1 in aluminum intoxicated, 3432 cm 1 in DFP treated and 3431 cm 1 in DFO + DFP treated/ combine therapy in spleen tissue. This result suggests that formation of hydrogen bonding between amide groups always induces a shift of the NAH stretching vibration down to a lower wavenumber [32]. There is significant change in wavenumber between all groups. The calculated peak area values of amide A band for control, aluminum intoxicated, DFP and DFO + DFP treated tissue are 163.957 ± 2.335, 100.522 ± 1.272, 117.444 ± 1.653, and 131.156 ± 1.760 respectively, which correspond to change 38.69% between the control and aluminum intoxicated, 16.83% between the aluminum intoxicated and DFP treated, 11.67% between the DFP and DFO + DFP treated spleen tissue of mice as shown in Table 2. The significant decrease in peak area value of the amide A band lead to a decrease in the protein quantity of the system. This could be a supportive sign of the destructive effect of aluminum and generates reactive oxygen species [33] resulting in oxidative deterioration of lipids, proteins and DNA. This result suggests that aluminum accumulation in the body is most severe but combine therapy and DFP therapy plays a vital accountability in the treatment of aluminum toxicity and can be flush out redundant aluminum. The absorption band observed at 3066 cm 1 was assigned to NAH stretching of amide B proteins. There is significant variation on band area and wavenumber values as presented in Tables 2 and 3 respectively. The absorption peak for unsaturated lipids observed at 3012 cm 1 consequence from the CAH stretching vibration of HC@CH groups of Olefinic molecule which could be a useful indicator of the different degrees of unsaturation in acyl chains of phospholipids [34,35]. The Olefinic@CH band in aluminum intoxicated spleen tissue was absent, indicating a disappeared in the population of unsaturation in acyl chains of lipid molecules. Lipids give rise to a number of absorptions in FT-IR spectra. The absorption peaks of spleen tissue found at 2960 cm 1 and at 2923 cm 1are known to asymmetric and symmetric stretching vibrations of CH3 and CH2 groups of the acyl chains. The peak at 2854 cm 1 can be assigned as symmetric stretching modes of CH2 group. Hence, this band mainly monitors the lipids in the biological system [36]. The absorption peak area and wavenumber of this region are changed significantly in all groups. The absorption FTIR spectra primarily due to proteins, with some absorption from lipids of control, aluminum intoxicated, DFP treated and DFO + DFP treated spleen tissue in the range of 1780–1280 cm 1 as shown in Fig. 2(B). The band observed at 1741 cm 1 of spleen tissue is assigned to C@O stretching vibration of ester groups in triglycerides [35]. There are significant wavenumber difference and band area values between all groups. The absorption peak appeared at1654 cm 1 assigned as Amide I due to C@O stretching of a-helix protein. The deformation of protein amide NAH bond appeared at 1542 cm 1 (amide II). There are no significant wavenumber difference and band area values between all groups. The 1461and 1398 cm 1 bands arise from the side chain of proteins. The significant increase in the area due to DFP and
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Table 1 FTIR vibrational peak assignments of control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice. Control
Aluminum intoxicated
Al + DFP treated
Al + DFO + DFP treated
Vibrational peak assignments
3438
3412
3432
3431
3066 3012 2960 2923 2854 1741 1654 1542 1461 1398 1309 1232 1168 1081 970 709
3052 – 2947 2917 2848 1716 1637 1535 1450 1397 1312 1221 1159 1045 968 673
3058 3006 2953 2919 2850 1727 1652 1540 1452 1393 1313 1228 1163 1059 966 675
3061 3007 2956 2922 2854 1738 1654 1541 1456 1394 1313 1234 1165 1066 972 701
Amide A due to NAH stretching of proteins with the little contribution from OAH stretching hydrogen bonded intermolecular OH groups of polysaccharides Amide B due to NAH stretching of proteins Olefinic CH@CH stretch: unsaturated lipids CH3 asym. stretch: mainly lipids CH2 asym. stretch: mainly lipids, with little contribution from proteins, carbohydrates, nucleic acids CH2 sym. stretch: mainly lipids Ester C@O stretch: triglycerides, cholesterol esters Amide I (C@O stretching of a-helix protein) Amide II(NAH bending and CAN stretching of proteins) CH3 scissoring mainly lipids COO sym. stretch: fatty acids and amino acids CH3 CH2 stretching of collagen PO2 asym. stretch: mainly phospholipids and phosphodiester in nucleic acids CAO asym. stretching of glycogen PO2 sym. stretch: mainly phospholipids and phosphodiester in nucleic acids CAN+AC sym. stretch: nucleic acids Ring breathing mode in the DNA bases
DFO + DFP treated groups of the band located at 1309 cm 1, which originates from CH3CH2 stretching modes of collagen [37]. The absorption FTIR spectra of phosphates with nucleic acids, i.e., RNA and DNA respectively of control, aluminum intoxicated, DFP treated and DFO + DFP treated spleen tissue in the range of 1300–400 cm 1 as shown in Fig 2(C). The absorption band appeared at 1232 cm 1 assigned to the asymmetric phosphate stretching vibration of phospholipids and the band observed at 1168 cm 1 assigned to CAO asymmetric stretching of glycogen [38]. There are significant variations on wavenumber difference and band area values between all groups. The band appeared at 1048 cm 1 assigned to PO2 symmetric stretching mainly phospholipids and phosphodiester in nucleic acids. The absorption peak at 922 cm 1 is normally assigned to CAN+AC symmetric stretching of nucleic acids, especially for DNA [39]. Hence, aluminum induces the production of free radicals leading to break in lipids, proteins, and DNA in the spleen tissue. The absorption peaks appeared at 719 cm 1 assigned to ring breathing mode in the DNA bases.
Biochemical parameters in spleen tissue Lipid peroxidation, enzymatic and non-enzymatic antioxidants The enhancement of lipid peroxidation products might be due to increased in free radical production and decreased quantities of antioxidant system in aluminum intoxicated mice as shown in Table 4. Treatment with chelating agents DFP and DFO + DFP decreased the levels of lipid peroxidation products. Thus, DFO and DFP inhibits lipid peroxidation may be due to scavenging of free radicals and is expected to its radical scavenging property. The decrease of SOD, GPx and CAT activities shows that these antioxidant enzymes were inhibited, their protective effects against free radicals and lipid peroxidation of organism were reduced, and the spleen would be injured. The level of TBARS has been shown to be an indicator of endogenous lipid peroxidation [40]. In the present study, free radical scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (LHP) decreased in aluminum intoxicated tissue but improved it by treatments with chelating agents DFP and DFO + DFP presented in Table 6. The non-enzymatic antioxidants namely, vitamin C, vitamin E, and reduced glutathione (GSH) which scavenge the residual free radicals escaping from decomposition by the antioxidant enzymes [41]. Vitamin C acts as the protective action during oxidative stress which is drastically decrease due to aluminum toxicity as shown in Table 7. Vitamin E is the most effective lipid
Fig. 1. FT-IR spectra of the control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice in the range of 4000–400 cm 1.
soluble antioxidant in the biological system. Glutathione helps a marked function in detoxification reaction because it is a direct radical scavenger. Our results represent that the levels of nonenzymatic antioxidants that were decreased in aluminum intoxicated mice due to the increased utilization for the neutralization of free radicals, lipid peroxidation products and related to the aerobic nature of cellular metabolism.
Histology assessment of spleen tissue of mice Supplementary Fig. 3 illustrates the protective effects exerted by chelating agents DFO and DFP against aluminum induced histopathological photographs of spleen tissue of mice. Control mice spleen showed normal congestion displayed in Supplementary Fig. 3A. The aluminum group revealed extensive spleen injuries characterized by inflammatory collections of prominent follicles and dilated sinusoids as shown in Supplementary Fig. 3B. By administration with DFP showed partial recovered of extramedullary haematopoiesis as shown in Supplementary Fig. 3C. Finally, treatment with DFO and DFP showed the remarkably reduced spleen injuries with normal architecture areas of fragmentation of the tissue with congestion as shown in Supplementary Fig. 3D.
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Fig. 2. Selected FT-IR spectra of the control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissues of mice in the range of 3750–2800 cm (B), and 1300–400 cm 1 (C).
1
(A), 1780–1280 cm
1
Table 2 Band area values of major bands for the control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice. Bands
Control
Aluminum intoxicated
Al + DFP treated
Al + DFO + DFP treated
3438 3066 3012 2960 2923 2854 1741 1654 1542 1461 1398 1309 1232 1168 1081 970 709
163.957 ± 2.335d 0.509 ± 0.029c 0.029 ± 0.004b 1.070 ± 0.026d 5.822 ± 0.139c 1.100 ± 0.211a,b 2.208 ± 0.142d 28.748 ± 0.440d 12.068 ± 0.053d 2.658 ± 0.114d 2.734 ± 0.148d 0.169 ± 0.032b 5.176 ± 0.060d 0.158 ± 0.013c 18.885 ± 0.198d 0.498 ± 0.026d 0.619 ± 0.006d
100.522 ± 1.272a 0.154 ± 0.015a Not Observed 0.275 ± 0.013a 0.877 ± 0.011a 0.698 ± 0.030a 0.120 ± 0.028a 7.858 ± 0.100a 2.507 ± 0.249a 0.454 ± 0.067a 0.848 ± 0.082a 0.062 ± 0.008a 1.034 ± 0.005a 0.107 ± 0.051a 1.506 ± 0.166a 0.007 ± 0.001a 0.050 ± 0.018a
117.444 ± 1.653b 0.294 ± 0.019b 0.016 ± 0.002a 0.432 ± 0.015b 2.350 ± 1.077a 1.039 ± 0.655a,b 0.314 ± 0.009b 15.220 ± 0.228b 5.159 ± 0.016b 0.717 ± 0.036b 1.307 ± 0.242b 0.077 ± 0.002a 1.989 ± 0.062b 0.137 ± 0.010a,b 4.232 ± 0.027b 0.186 ± 0.010b 0.109 ± 0.005b
131.156 ± 1.760c 0.505 ± 0.024c 0.026 ± 0.002b 0.668 ± 0.012c 3.875 ± 0.074b 1.382 ± 0.024b 0.546 ± 0.022c 24.162 ± 1.235c 9.471 ± 0.228c 1.473 ± 0.149c 1.662 ± 0.049c 0.083 ± 0.003a 2.490 ± 0.058c 0.182 ± 0.011c 8.080 ± 0.420c 0.249 ± 0.013c 0.174 ± 0.014c
Comparisons values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
ICP–OES analysis In the present study we chose the spleen tissue because it is a peripheral organ of the immune system and accumulated
Aluminum might be alter the biochemical constituents. The ICP– OES study of spleen tissue trace elements such as Ca, Fe, Cu, Mn and Zn as shown in Table 5. Al exposure has an adverse effect on essential elements in humans, with subsequent impact on the
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Control
NAH stretching (proteins): Amide A NAH stretching (proteins): Amide B CH2 sym. (fatty acids) Carbonyl C@O (lipids) Amide I (protein) Amide II (protein) PO2 asym. nucleic acids and phospholipids PO2 sym. nucleic acids and phospholipids
Aluminum intoxicated c
Al + DFP treated
a
3438 ± 2.510 3066 ± 1.509c 2854 ± 0.696b 1741 ± 2.617c 1654 ± 2.025b 1542 ± 2.046b 1232 ± 2.236c 1081 ± 2.266d
b
3412 ± 3.899 3052 ± 1.614a 2848 ± 1.817a 1716 ± 2.608a 1637 ± 1.677a 1535 ± 2.881a 1221 ± 2.302a 1045 ± 2.161a
3432 ± 4.050 3058 ± 1.837b 2850 ± 1.921a 1727 ± 2.589b 1652 ± 2.828b 1540 ± 2.641b 1228 ± 2.716b 1059 ± 1.870b
Al + DFO + DFP treated 3431 ± 4.243b 3061 ± 1.225c 2854 ± 2.828b 1738 ± 2.449c 1654 ± 5.329b 1541 ± 3.162b 1234 ± 2.561c 1066 ± 1.846c
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.
Table 4 FTIR absorption band area ratio for selected bands of control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue of mice. Bands area ratio
Control
Al-intoxicated
Al + DFP treated
Al + DFO + DFP treated
I1081/I1232 I1542/I3438 I2960/I2854 I1542/I1654 I1081/I1542
3.648 ± 0.016d 0.074 ± 0.003c 0.535 ± 0.018c 0.420 ± 0.028c 1.565 ± 0.027d
1.457 ± 0.050a 0.025 ± 0.002a 0.393 ± 0.023a 0.319 ± 0.015a 0.600 ± 0.016a
2.283 ± 0.032b 0.044 ± 0.002b 0.416 ± 0.020a 0.339 ± 0.024a 0.820 ± 0.036b
3.246 ± 0.025c 0.072 ± 0.004c 0.483 ± 0.021b 0.392 ± 0.020b 0.853 ± 0.025c
Values are expressed as mean ± S.E for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
Table 5 ICP–OES study of trace elements in spleen tissue of control, aluminum intoxicated, DFP and DFO + DFP treated mice (lg/g tissue). Groups
Zn
Fe
Ca
Cu
Mg
Control Aluminum intoxicated Al + DFP treated Al + DFO + DFP treated
32 ± 3.120d 29 ± 2.586c 26 ± 1.350b 23 ± 1.524a
215 ± 1.117a 223 ± 1.441b 229 ± 1.910c 232 ± 1.460d
101 ± 1.162d 81 ± 0.672a 92 ± 1.007b 97 ± 0.880c
5.2 ± 0.084a 5.6 ± 0.072b 5.7 ± 0.126b 6.1 ± 0.131c
189 ± 2.608d 186 ± 1.720c 182 ± 1.139b 178 ± 1.695a
Values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
cellular enzymatic and metabolic processes [42]. The elements such as Ca, Fe, Cu, Mn and Zn are essential elements because of their indispensable role in biological systems. Aluminum may interfere with various metabolic processes, in which Ca2+, Mg2+, Fe3+ and Fe2+ (gastrointestinal absorption) are involved [43]. The choice of those elements was based in previous studies which showed potential relationship between aluminum and the above mentioned elements [44]. The concentration of all the elements level showed difference between control and treated groups.
Discussion The present study was designed to investigate the protective effects of the chelating agents DFO and DFP in aluminum intoxicated spleen tissue of mice to knowing the functional, structural, and compositional changes at the molecular level by Fourier transform infrared (FT-IR) spectroscopy as shown in Fig. 1. So that present study is so helpful and informative to the toxicology and spectroscopy fields. Our results provides the useful information to the scientific community as all the parameters responded positively to individual therapy (DFP), but more pronounced beneficial effects were observed in combination therapy (DFO + DFP) for aluminum intoxicated mice. In the present work, the absorption peak observed at 3438 cm 1 occurs from both the NAH and OAH stretching modes of water, proteins and polysaccharides. Table 3 shows the variation of wavenumber 3438 ± 2.510, 3412 ± 3.899, 3432 ± 4.050, and 3431 ± 4.243 respectively in the macromolecular amide A in spleen tissue of mice. Here, aluminum intoxicated tissue shows the lower wavenumber because formation of hydrogen bonding between
amide groups always induces a shift of the NAH stretching vibration down to a lower wavenumber [32]. The band observed at 3066 cm 1 assigned to amide B mainly NAH stretching of protein with negligible contribution from OAH stretching of intermolecular hydrogen bonding as shown in Fig. 2(A). The calculated peak area values of amide B band for control, aluminum intoxicated, DFP and DFO + DFP treated tissue are 0.509 ± 0.029, 0.154 ± 0.015, 0.294 ± 0.019, 0.505 ± 0.024 respectively, which correspond to change 69.74% between control to aluminum intoxicated tissue, 90.90% between aluminum intoxicated to DFP treated tissue, 41.78% between DFP to DFO + DFP treated spleen tissue presented in Table 2. This result suggests that DFO and DFP are the good antidotes. The absorption band observed at 3012 cm 1 absent in aluminum intoxicated tissue due to over production of reactive oxygen species (ROS). Further, the absorption peaks appeared at 3006 cm 1 for DFP treated and at 3007 cm 1 for DFO + DFP treated spleen tissue due to unsaturated olefinic molecule, these could be a useful indicator of the different degrees of unsaturation in acyl chains of phospholipids band [45]. The absorption peak area and wavenumbers are changed significantly in observed groups. The calculated peak area values of Olefinic@CH band (index of relative concentration of double bonds in the lipid structure of unsaturated lipids) for control, DFP and DFO + DFP treated tissue are 0.029 ± 0.004, 0.016 ± 0.002 and 0.026 ± 0.002 respectively. This result suggests that absent of Olefinic@CH peak due to aluminum poisoning might be causing reduction in the concentration of polyunsaturated fatty acids as shown in Table 2. Lipids play a key role in the membrane fluidity. By affecting the conformation of membrane proteins, they govern exposure and diffusion of membrane components. The area values of the asymmetric stretching vibrations of methyl ACH3) and methylene
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Table 6 Effects of DFP and DFO + DFP on biochemical parameters in aluminum intoxicated mice. Biochemical Parameters Protein level (mg/100 mg) SOD (U/mg of protein) LHP (nmol/mg protein) Gpx (lg of GSH utilized min/mg protein CAT (lmol of H2O2utilized/min/mg protein) TBARS (nmol/mg protein)
Control
Aluminum intoxicated d
32.38 ± 0.093 6.39 ± 0.096c 1.09 ± 0.072a 9.03 ± 0.589c 41.23 ± 1.115d 1.42 ± 0.244a
a
17.24 ± 0.198 2.97 ± 0.513a 2.93 ± 0.278d 4.47 ± 0.191a 21.91 ± 1.096a 3.83 ± 0.132c
Al + DFP treated b
25.31 ± 0.730 4.94 ± 0.581b 1.98 ± 0.389c 6.49 ± 0.326b 32.43 ± 0.192b 2.05 ± 0.062b
Al + DFO + DFP treated 29.96 ± 0.557c 5.89 ± 0.385c 1.43 ± 0.234b 8.78 ± 0.407c 37.18 ± 1.358c 1.63 ± 0.018a
Values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
Table 7 Level of non-enzymic antioxidants in control and treated mice. Groups
Vitamin E (lg/mg protein)
Vitamin C (lg/mg protein)
GSH (mg/100 g tissue)
Control Aluminum intoxicated Al + DFP treated Al + DFO + DFP treated
3.44 ± 0.409c 2.31 ± 0.229a 2.61 ± 0.141a 3.03 ± 0.378b
1.48 ± 0.044c 0.68 ± 0.062a 1.14 ± 0.087b 1.29 ± 0.096b
74.53 ± 1.350d 52.25 ± 0.715a 65.84 ± 3.199b 69.91 ± 1.497c
Values are expressed as mean ± S.D for six mice in each group; values not sharing a common superscript (a, b, c, d) differ significantly at P < 0.05 (DMRT).
(ACH2) groups decreased from control (1.070 ± 0.026 and 5.822 ± 0.139) to aluminum intoxicated (0.275 ± 0.013 and 0.877 ± 0.011), these reflects the changes in the composition of the acyl chains. Further, the areas of these bands were increased from DFP (0.432 ± 0.015 and 2.350 ± 1.077) to DFO + DFP (0.668 ± 0.012 and 3.875 ± 0.074). A decrease area of the symmetric stretching vibration of methylene band at 2854 cm 1 from control (1.100 ± 0.211) to aluminum intoxicated (0.698 ± 0.030) spleen tissue and increased again from 1.039 ± 0.655 to 1.382 ± 0.024, after DFP and DFO + DFP treatments. This result suggests that decreased lipid order and increased acyl chain flexibility in DFP and DFO + DFP treated spleen tissue of mice. The absorption band observed at 1745 cm 1, which arises from the stretching mode of the ester C@O groups of the lipids [46]. The significant decreased in peak area value of ester carbonyl lipid band at 1745 cm 1 for aluminum intoxicated tissue, but enhanced it for DFP and DFO + DFP treated spleen tissue. The degradation of acyl chains due to aluminum poisoning provide the information of lipids become oxidized, since oxidation to lipids causes the formation of carbonyl groups and a breakdown of long chains into smaller fragments then disorder the lipid metabolism in spleen tissue of mice. The amide I region comprises vibrations due mainly to C@O stretching modes with some contribution from NAH bending and CAN stretching and the amide II region correspond to mainly NAH bending with a large contribution from CAN stretching modes [15]. Characteristic infrared absorption bonds of peptide linkages at these amide I and II regions correspond to the alpha-helix protein structure and all the constituent of amino acid side-chains in proteins are susceptible to free radicals, but some are more vulnerable than others [47]. The calculated peak area values of amide I band for control, aluminum intoxicated, DFP and DFO + DFP treated tissue are 28.748 ± 0.440, 7.858 ± 0.100, 15.220 ± 0.228 and 24.162 ± 1.235 respectively, which correspond to change 72.66% between control to aluminum intoxicated tissue, 93.68% between aluminum intoxicated to DFP treated tissue, 58.75% between DFP to DFO + DFP treated spleen tissue. The decreased peak area observed at aluminum intoxicated tissue suggests that no measurable protein oxidation takes place due to the overproduction of ROS in aluminum exposure tissue, these results leads to the breakdown of balance of the oxidative/anti-oxidative system in spleen, resulting in the lipid peroxidation. This is due to an alteration of antioxidant defense ability. The observed absorption peak at 1542 cm 1 assigned to amide II mainly NAH bending and CAN stretching of proteins. Amide II appeared at 1542 cm 1 in control, 1535 cm 1
in aluminum intoxicated, 1540 cm 1 in DFP treated and 1541 cm 1 in DFO + DFP treated spleen tissue. Therefore, decrease in peak areas and wavenumbers of amides I band indicate the destructive effect of aluminum and this changes reflect the loss of protein level in the aluminum intoxicated spleen tissue of mice. This result suggest that the explanation for the decreased protein content may be an excess free radicals production and binding of aluminum with various sulfhydryls (RASH) that exists in the tissue also the explanation may be due to a reduction of the protein synthesis in the spleen. The COO symmetric stretch mainly fatty acids and amino acids observed at1398 cm 1. The decreased in both wavenumbers and peak area values for aluminum intoxicated spleen tissue suggests that changes in the structure of amino acids caused by aluminum poisoning. The phosphate asymmetric stretching absorption band observed at 1232 cm 1 originated due to the phosphodiester back bone of cellular nucleic acids. The peak area decreased from 5.176 ± 0.060 to 1.034 ± 0.005 in between control to aluminum intoxicated group, treatments with DFP and DFO + DFP recovered it nearer to the control values from 1.989 ± 0.062 to 2.490 ± 0.05 respectively. The 80.02% shifted peak area value in aluminum exposed tissue; this might be due to the changes in the composition of nucleic acids. Further, decreased level of phospholipids may partly be responsible for the decrease in lipid peroxidation at aluminum intoxicated tissue. The significant increase in the intensity of the band at 1168 cm 1 in the aluminum intoxicated spleen might be due to an increase in the content of glycogen [38] as shown in Fig. 2(C). This result confirmed by an increased intensity of the CAO stretching absorption peak at 1168 cm 1, which is mainly due to glycogen in tissue [48]. The phosphate symmetric stretching due phospholipids and phosphodiester in nucleic acids observed at 1081 cm 1. The calculated peak area values of this band for control, DFP and DFO + DFP treated tissue are 18.885 ± 0.198, 1.506 ± 0.166, 4.232 ± 0.027 and 8.080 ± 0.420 respectively. This result suggests decrease (92.02%) in the relative content of the nucleic acids due to aluminum poisoning as compare to control spleen tissue. The absorption peak observed at 970 cm 1 assigned to CAN+AC symmetric stretch vibration of nucleic acids. The decrease peak area suggests a decrease in the relative content of the nucleic acids due to aluminum poisoning in spleen tissue of mice. Therefore, the decreased intensity of amide A band in the aluminum intoxicated spleen tissue reflects the decreased quantity of protein. This decreased quantity indicates the damage of amide
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linkage in aluminum intoxicated spleen tissue. There was an enhancement in the level of thiobarbituric acid reactive substances (TBARS) 3.83 ± 0.132 for aluminum poisoning but brings back to nearer control value (1.42 ± 0.244) by DFP (2.05 ± 0.062) and DFO + DFP (1.63 ± 0.018) treatments, and similar in lipid hydroperoxides (LHP) as shown in Table 6. This result suggests that aluminum generates reactive oxygen species, resulting in oxidative deterioration of lipids, proteins and DNA. Further, in aluminum intoxicated spleen tissue decreased the antioxidant activities such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) could be due to the induction of lipid peroxidation [49]. The level of non-enzymic antioxidants such as vitamin C, Vitamin E and glutathione (GSH) are decreased in aluminum intoxicated spleen tissue as shown in Table 7. This result suggests the reduction of protein quantity may be the imbalance between oxidants and antioxidants. Therefore, the DFO and DFP are useful therapeutic drugs which blocked ROS production. The present results showed that DFO and DFP are promising for a novel class of drugs in the treatment of immunoinflammatory spleen diseases causes by aluminum exposure. The ratio of the peak intensities of the bands at 1081 cm 1 and at 1232 cm 1 (I1081/I1232) is related to the ratio of symmetric modes of the phosphodiester group (APO2) in nucleic acids to asymmetric modes of phosphodiester group [50]. The variation of ratios for the control, aluminum intoxicated, DFP treated and DFO + DFP treated spleen tissue are 3.648 ± 0.016, 1.457 ± 0.050, 2.283 ± 0.032 and 3.246 ± 0.025 respectively, which correspond to change of 60.06% between control and aluminum intoxicated, 56.69% between aluminum intoxicated and DFP treated, 42.18% between DFP and DFO + DFP treated spleen tissue of mice. In present work, the ratio of the peak intensities of the bands at 1542 cm 1 and at 3438 cm 1 (I1542/I3438) has been used as an indicator of the relative concentration of the protein in the spleen tissue. Present result suggests the protein content in the membrane is higher in chelating agents treated tissue but lower in the aluminum intoxicated spleen tissue. The mean ratios of the intensity of absorption of the methyl band and methylene band (I2960/I2854) for control, aluminum intoxicated, DFP and DFO + DFP treated spleen tissue are 0.535 ± 0.018, 0.393 ± 0.023, 0.416 ± 0.020 and 0.483 ± 0.021 respectively, which corresponds to change of 26.54% between control to aluminum intoxicated, 5.85% between aluminum to DFP treated, 16.10% between DFP to DFO + DFP treated spleen tissue. The decreases ratio indicate a decrease in the number of methyl groups in protein fibers compared to methylene groups in aluminum intoxicated spleen tissue. The mean ratio of the intensities of the bands at 1542 cm 1 and 1652 cm 1 could be attributed to a change in the composition of the whole protein pattern [51]. In the intensity ratio (I1542/I1654) decreased from 0.420 ± 0.028 to 0.319 ± 0.015 for aluminum intoxicated spleen tissue as shown in Table 4. This results in a fall in the overall protein contents due to aluminum exposure. In the presence of chelating agents DFP and DFO + DFP, the ratio increased to 0.339 ± 0.024 to 0.392 ± 0.020 respectively. The mean ratio of the intensities of the bands I1081/I1542 could be attributed to a change in the composition of the glycoprotein. In the spleen tissue, this ratio decreased from 1.565 ± 0.027 to 0.600 ± 0.016, for aluminum intoxicated tissue, indicating a fall in the glycoprotein contents due to aluminum poisoning. Treatments with chelating agents DFP and DFO + DFP, this ratio increased to 0.820 ± 0.036, 0.853 ± 0.025 respectively.
changes in response to the chelating agents treatment in spleen tissue of mice. FT-IR spectra revealed significant differences in absorbance intensities between the control, aluminum intoxicated and chelating agents treated spleen tissue of mice. The variation on the major biochemical constituents such as lipids, proteins, phosphodiester and nucleic acids of the spleen tissue due to the overproduction of ROS but significantly improved it by administration of DFP and DFO + DFP. Our results established that all the parameters and biochemical constituents responded positively to individual therapy with DFP, but more pronounced beneficial effects were observed in combination (DFO + DFP) therapy and good agreements with biochemical parameters, histopathological assessments and FTIR results. Further, our results confirmed that aluminum plays an important role in the pathogenesis of spleen tissue. In addition, investigation the role of DFP and DFO + DFP chelation therapy for treatments of spleen is protected and secured from chronic diseases. Finally, the present study provided the agreements between the biochemical parameters, histopathology and FT-IR results. Funding University Grants Commission (UGC), New Delhi, India, Major Research Project F. No. 40-447/2011 (SR). Acknowledgements The financial support to author Mr. Chandra Prasad Khatiwada from the University Grants Commission, New Delhi, India is gratefully acknowledged. Further, the authors are thankful and grateful to Dr. S. Barathan, Professor and Head, Department of Physics, Annamalai University, Tamilnadu-608 002, for providing all necessary facilities to carry out the present work successfully. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.004. References [1] [2] [3] [4] [5] [6]
[7] [8] [9] [10] [11]
[12] [13] [14] [15] [16]
Conclusion [17]
This is the first detailed explanation report for FTIR spectroscopic technique has been employed to monitor biomolecular
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[18] [19]
S.V. Verstraeten, L. Aimo, P.I. Oteiza, Arch. Toxicol. 82 (2008) 789–802. V. Kumar, K.D. Gill, Arch. Toxicol. 83 (2009) 965–978. M. Venturini-Soriano, G. Berthon, J. Inorg. Bioch. 85 (2001) 143–154. M.L. Ana, G. Graciela, N. Alcira, Int. J. Immunopharmacol. 1 (2001) 1725–1732. D. Biswas, A. Choudhury, K.K. Misra, Exp. Parasitol. 99 (2001) 148–159. D. Na Li, H. Yanmei, Z. Mengmeng, F. Lei, Z. Min, W. Xiaoyang, C. Jue, L. Yaling, C. Huiting, G. Jingwei, W. Songjie, H. Han, Fashui. Toxicol. Lett. 95 (2010) 61– 168. A.Z. Lilianne, G. Jan, Z. Gosta, Mutat. Res. 394 (1997) 17–28. N.C. Jain, Schalm’s Veterinary Hematology, 4th ed., Lea and Febiger, 600.Washington square, Philadelphia, USA, 1986. S. Tubafard, S.J. Fatemi, A.S. Saljooghi, M. Torkzadeh, Med. Chem. Res. 19 (2010) 854–863. G. Mercedes, L.E. Jose, L.D. Jose, K.S. Pramod, M.J. Mark, Toxicology 137 (1999) 161–168. D. McNaughton, K.R. Bambery, B.R. Wood, Spectral histopathology of the human cervix, in: M. Diem, P.R. Griffiths, J.M. Chalmers (Eds.), Vibrational Spectroscopy for Medical Diagnosis, John Wiley & Sons, UK, 2008, pp. 187–202. S.N. Jha, S. Gunasekaran, J. Food Eng. 101 (2010) 337–342. K. Fent, P.W. Looser, Water Res. 29 (1995) 1631–1637. N. Yunes, S. Moyano, S. Cerutti, J.A. Gasquez, L.D. Martinez, Talanta 59 (2003) 943–949. S. Teerayut, S. Ratana, D. Nikorn, P. Tipicha, S. Palarp, Sci. Total Environ. 270 (2001) 103–108. Z. Yanzhu, L. Xinwei, C. Chongxiao, W. Fan, L. Jing, H. Chongwei, L. Yanfei, M. Liguang, Food Chem. Toxicol. 50 (2012) 2911–2915. M. Bettinelli, G.M. Beone, S. Spezia, C. Baffi, Anal. Chim. Acta 424 (2000) 289– 296. W.G. Niehaus, B. Samuelsson, Eur. J. Biochem. 6 (1968) 126–130. Z.Y. Jiang, J.V. Hunt, S.P. Wolff, Anal. Biochem. 202 (1992) 384–389.
156
S. Sivakumar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 148–156
[20] P. Kakkar, B. Das, P.N. Viswanathan, Indian J. Biochem. Biophys. 21 (1984) 130– 132. [21] A.K. Sinha, Anal. Biochem. 47 (1972) 389–394. [22] J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman, W.G. Hoekstra, Science 179 (1973) 588–590. [23] G.L. Ellman, Arch. Biochem. Biophys. 82 (1959) 70–77. [24] J.H. Roe, C.A. Kuether, J. Biol. Chem. 11 (1943) 145–164. [25] H. Baker, O. Frank, B. DeAngelis, Nutr. Res. 21 (1980) 531–536. [26] P.T. Wong, R.K. Wong, T.A. Caputo, T.A. Godwin, B. Rigas, Proc. Natl. Acad. Sci. USA 88 (1991) 10988–10992. [27] L. Xiaonan, A.H.W. Molly, J.T. Mariah, P.V.E. Joel, I.D. Serge, A.R. Barbara, Aquaculture 315 (2011) 298–305. [28] E. Benedetti, L. Teodori, M.L. Trica, P. Vergamini, F. Salvati, F. Mauro, Appl. Spectrosc. 44 (1990) 1276–1280. [29] G. Cakmak, I. Togan, F. Severcan, Aquat. Toxicol. 77 (2006) 53–63. [30] A. Dogan, G. Siyakus, F. Severcan, Food Chem. 100 (2006) 1106–1114. [31] N.S. Ozek, Y. Sara, R. Onur, F. Severcan, Bio. Sci. Rep. 30 (2010) 41–45. [32] D. Lin-Vien, N.B. Colthup, W.G. Fateley, J.G. Grasselli, The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules, Academic Press, San Diego CA, 1990. [33] F.M. El-Demerdash, J. Trace Elem. Med. Biol. 18 (2004) 113–122. [34] O. Bozkurt, M. Severcan, F. Severcan, Analyst 135 (2010) 3110–3119. [35] E. Bogomolny, S. Argov, S. Mordechai, M. Huleihel, Biochim. Biophy. Acta 1780 (2008) 1038–1046.
[36] S.B. Akkas, M. Severcan, O. Yilmaz, F. Severcan, Food Chem. 105 (2007) 1281– 1288. [37] N. Stone, C. Kendall, J. Smith, P. Crow, H. Barr, J. Raman Spectrosc. 33 (2002) 564–573. [38] N. Toyran, P. Lasch, D. Naumann, B. Turan, F. Severcan, Biochem. J. 397 (2006) 427–436. [39] D. Naumann, D. Helm, H. Labischinski, Nature 351 (1991) 81–82. [40] L. Guangke, S. Nan, G. Dongsheng, Sphere 65 (2006) 1058–1063. [41] B. Seifi, M. Kadkhodaee, S.M. Karimian, M. Zahmatkesh, J. Xu, M. Soleimani, Clin. Exp. Hypertens. 32 (2010) 90–97. [42] F.M. Metwally, M.S. Mazhar, Arh. Hig. Rada. Toksikol. 58 (2007) 305–311. [43] P. Zatta, T. Kiss, M. Suwalsky, G. Berthon, Chem. Rev. 228 (2002) 271–284. [44] D.J. Sanchez, M. Gomez, J.M. Llobet, J. Corbella, J.L. Domingo, Pharmacol. Toxicol. 80 (1997) 11–17. [45] M.D. Guillen, N.J. Cabo, Agric. Food Chem. 47 (1999) 709–719. [46] H. Fabian, P. Lash, M. Boese, W. Haensch, J. Mol. Struct. (2003) 661–662. [47] C.A. Rice-Evans, A.T. Diplock, M.C.R. Symos., Elsevier, New York, 1991. [48] S.Y. Lin, M.J. Li, W.T. Cheng, Spectroscopy 21 (2007) 1–30. [49] G.C. Jagetia, G.K. Rajanikant, S.K. Rao, M.S. Baliga, Clin. Chim. Acta 332 (2003) 111–121. [50] P.T.T. Wong, Biospectroscopy 1 (1995) 357–364. [51] E. Benedetti, E. Bramanti, F. Papineschi, I. Rossi, E. Benedetti, Appl. Spectrosc. 51 (1997) 792–797.