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Screening of Protective Effect of Amifostine on Radiation-Induced Structural and Functional Variations in Rat Liver Microsomal Membranes by FT-IR Spectroscopy Gulgun Cakmak,† Faruk Zorlu,‡ Mete Severcan,§ and Feride Severcan*,† †
Department of Biological Sciences, Middle East Technical University, 06531, Ankara, Turkey Department of Radiation Oncology, Faculty of Medicine, Hacettepe University, 06100 Sihhiye, Ankara, Turkey § Department of Electrical and Electronic Engineering, Middle East Technical University, 06531, Ankara, Turkey ‡
bS Supporting Information ABSTRACT: In this study, the protective effect of amifostine, which is the only FDA-approved radioprotective agent, was investigated against the deleterious effects of ionizing radiation on rat liver microsomal membranes at molecular level. SpragueDawley rats, which were either administered amifostine or not, were whole-body irradiated with a single dose of 800 cGy and decapitated after 24 h. The microsomal membranes isolated from the livers of these rats were investigated using FT-IR spectroscopy. The results revealed that radiation caused a significant decrease in the lipid-to-protein ratio and the degradation of lipids into smaller fragments that contain less CH2 and more carbonyl esters, olefinicdCH and CH3 groups, which could be interpreted as a result of lipid peroxidation. Radiation altered the secondary structure of proteins by inducing a decrease in the β-sheet structures and an increase in the turns and random coil structures. Moreover, a dramatic increase in lipid order and a significant decrease in the membrane dynamics were observed in the irradiated group. The administration of amifostine before ionizing radiation inhibited all the radiation induced compositional, structural, and functional damages. In addition, these results suggest that FT-IR spectroscopy provides a novel approach to monitoring radiationinduced damage on biological membranes.
A
mifostine, which is a synthetic aminothiol compound, is the only cytoprotective agent specifically approved by the Food and Drug Administration (FDA) as a radioprotector.1 It is a prodrug that is dephosphorylated in the tissue by alkaline phosphatase to its active free thiol metabolite, WR-1065. In the past, amifostine has been extensively studied in some clinical trials and has shown different protective activities. For example, it can prevent or ameliorate radiation-induced xerostomia, cisplatin-induced nephrotoxicity, anthracycline-induced cardiotoxicity, and chemotherapy-related thrombocytopenia.2 Despite the fact that a growing number of reports strongly support amifostine’s clinical efficacy, the molecular effects of amifostine on the structural and functional properties of normal and irradiated tissues and membranes are largely unknown. The deleterious effects of ionizing radiation on living cells are, for the most part, mediated by increased production of reactive oxygen species (ROS).3 The exposure of normal biological tissues to such free radicals causes damage in biomolecules resulting in several changes and finally leading to unwanted cell death. The essential cellular target of ionizing radiation is considered to be DNA, with less attention being focused on biological membranes. However, the effects of ionizing radiation on membranes deserve more interest since many physiological r 2011 American Chemical Society
processes depend on biological membranes.4 Today it is known that differences in lipid order, lipid dynamics, protein secondary structure in membranes together with changes in the content of macromolecules disturb the kinetics and functions of ion channels and also lead to onset of many diseases.5 The variations in the ratios of biomolecules such as unsaturated to saturated lipids, and lipid to protein, are related to lipid structures such as membrane thickness and lipid order, which are also related to membrane permeability.5,6 Microsomes, which are subcellular particles derived from the endoplasmic reticulum upon homogenization of the tissue, contain enzymes involved in reduction and oxidation reactions, drug metabolism, cholesterol synthesis, and fatty acid metabolism.7 In the past, it has been shown that amifostine uptake in the body is greatest in the liver, kidney, salivary glands, intestinal mucosa, and lungs.1 Therefore, the liver microsomal membrane system is one of the best models for monitoring the damage induced by ionizing radiation and the protecting capability of amifostine in biological membranes. Received: August 21, 2010 Accepted: February 28, 2011 Published: March 16, 2011 2438
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Analytical Chemistry Monitoring the overall changes occurring in biological membranes upon exposure to ionizing radiation is a complex task. So far, various spectroscopic techniques, such as electron paramagnetic resonance (EPR)8 and fluorescence,9 and some biochemical methods10 have been employed for the study of radiationinduced damage in membranes. The biochemical techniques such as thiobarbituric acid reactive substances (TBARS) test and high-performance liquid chromatography (HPLC), which give information about the concentration and content of biomolecules, are destructive and time-consuming. Spectroscopic techniques such as EPR and fluorescence spectroscopy require the insertion of either a spin label or bulky fluorophore probe into the membrane and report changes in the surrounding localized environment. These techniques were used as complementary to each other since they give information at different time scales. Among these techniques, FT-IR spectroscopy, in recent years, has emerged as a powerful tool to probe the structure, conformation, and function of lipids and proteins, simultaneously, in biological membranes without introducing foreign perturbing probes into the system.1115 The observed spectral features are related to chemical bond vibrations of specific molecular moieties of individual constituents.11 These spectral parameters are sensitive to changes in the macromolecules of the biological system induced by pathological and other conditions. Therefore, FT-IR spectroscopy has recently been widely applied during the classification and differentiation of disease states1619 and aquatic toxicological states.20 It has also been employed to determine radiation-induced changes in different tissues such as food21 and rat brain.13 In the current study, ionizing radiation-induced variations in rat liver microsomal membrane were studied in terms of macromolecular composition, structure, and lipid dynamics using FT-IR spectroscopy. In addition, the protective effects of the near therapeutic doses of amifostine on radiationinduced structural and functional variations were investigated which, to the best of our knowledge, have not been reported previously.
’ EXPERIMENTAL SECTION Chemicals. Amifostine (WR-2721) and the chemicals used for the isolation of microsomes were purchased from Sigma (Sigma Chemical Company, Saint Louis, MO, U.S.A.). All chemicals were obtained from the commercial source at the highest grade of purity available. Animals. After approval by the Ethics Committee of Hacettepe University, Faculty of Medicine, a total of 18 male Sprague Dawley rats weighing 180220 g were housed in a room with a 10:14 light/dark cycle and with free access to food and water. The animals were randomly divided into three groups. Group 1 (n = 6) was used as a control group without any treatment. The rats in group 2 (n = 6) were irradiated with a single dose of 800 cGy of whole body irradiation using a cobalt-60 irradiator. The rats in group 3 (n = 6) were intraperitoneously inoculated with a single dose of 300 mg/kg body weight of amifostine 1 h prior to the same irradiation as group 2. Because amifostine was dissolved in isotonic saline, the animals in groups 1 and 2 were similarly injected with equal volumes of isotonic saline intraperitoneously. The amifostine dose was chosen in the range of therapeutic doses used in previous studies (200400 mg/kg body weight).22 Twenty-four hours after radiation exposure the rats were euthanized, and liver tissues were quickly removed and kept at 80 °C until use.
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Isolation of Rat Liver Microsomal Membranes. Microsomal membranes were isolated according to a method described by Severcan et al.14 Briefly, liver tissue was homogenized in 25 mM KH2PO4, 1.15% KCl, 5 mM EDTA, 0.2 mM PMSF, 2 mM DTT (pH 7.4) (1:4 w/v) at 4 °C. The homogenate was first centrifuged at 16 000g for 20 min, and the resulting mitochondrial pellet was discarded. Then the supernatant was further centrifuged at 125 000g for 60 min. The resulting pellet was suspended in a buffer containing 50 mM Tris, 1 mM EDTA (pH 7.4) and recentrifuged at 125 000g for 55 min. After discarding the supernatant, the microsomal membrane-rich pellet was suspended with 25 mM phosphate buffer (pH 7.4) containing 25% glycerol (v/v) at a volume of 0.25 mL for each gram of primal liver tissue. The resuspended microsomes were homogenized manually using the Teflonglass homogenizer and stored at 80 °C until spectroscopic studies were performed. FT-IR Spectroscopic Study and Data Analysis. In order to remove the excessive amount of suspension buffer, the microsomal membrane fraction was centrifuged at 14 000 rpm for 10 min at 4 °C just before taking FT-IR spectra, as reported previously.18 The concentrated pellet containing the liver microsomal membranes was used in the FT-IR studies. Infrared spectra were obtained using Perkin-Elmer Spectrum 100 FT-IR spectrometer (Perkin-Elmer Inc., Norwalk, CT, U.S.A.) equipped with a MIR TGS detector. The sample compartment was continuously purged with dry air to minimize atmospheric water vapor absorbance, which overlaps with the spectral region of interest, and carbon dioxide interference. An equal amount of sample (20 μL) was placed between CaF2 windows using a 12 μm path length spacer. Interferograms, both for the samples and buffer, were accumulated for 100 scans at 2 cm1 resolution at 25 °C. Temperature regulation was performed by a Graseby Specac digital temperature controller unit. Before data acquisition, the samples were incubated for 10 min. Background spectra, which were collected under identical conditions, were used to calculate the absorbances of the sample and buffer spectra. This procedure was performed automatically by the appropriate software. In all of these experiments, three different independent aliquots were scanned from the same sample to check the accuracy of the absorbance values for the same sample and the spectra were compared. We observed that all three replicates gave identical spectra in each tissue studied. Therefore, in each tissue sample we used the average of these three replicates to represent the spectrum of one animal. We repeated this procedure for all animals. All these averages were then used in data analysis and statistical analysis. The spectra of suspension buffer were subtracted from the spectra of liver microsomal membranes to remove water absorption bands using Spectrum 100 software (Perkin-Elmer) subtraction procedure. In the subtraction process the water band located around 2125 cm1 was flattened (Supporting Information Figure S-1). The averages of the spectra belonging to the same experimental groups, baseline correction, normalization, and the band areas were obtained by using the same software. The baseline correction and normalization process was applied only for visual representation of the differences. For the accurate determination of the mean values for the band areas, band positions, and bandwidth values, the original average spectrum (from three replicates) belonging to each individual of the groups was taken into consideration. The band positions were measured according to the center of mass. The bandwidth values were calculated as the width measured at a 0.75 fraction of the 2439
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maximum height of the absorption signal in terms of wavenumbers from raw spectra. Neural networks (NN) are used to predict the secondary structure of proteins. Initially, NNs are trained using reference infrared spectra of 18 proteins whose secondary structure characterization are known from X-ray crystallographic analysis. The 16001700 cm1 spectral region corresponding to the amide I band of the infrared spectra have been used for training. In order to improve the predictions, the data set size has been increased artificially to 64 by interpolating the spectra. First, the data samples taken from the amide I band are preprocessed by amplitude normalization and then by discrete cosine transformation. Bayesian regularization method was used for training the NNs. For each structure a separate NN was modeled with optimum number of inputs and hidden neurons. During the prediction step, after applying the same preprocessing method optimized NNs are used to get predictions. The details of the method can be found in Severcan et al.23 Statistical Study. MannWhitney U-test, which is a nonparametric test used when sample size is small, was performed on the groups to test the significance of the differences between the control and irradiated groups, as well as the amifostine treated plus irradiated group. A p value of less than 0.05 was considered statistically significant.
Figure 1. FT-IR spectrum of control rat liver microsomal membrane in the 30501000 cm1 region.
’ RESULTS AND DISCUSSION The spectrum of rat microsomal membrane is quite complex and consists of several bands which arise from the contributions of different functional groups. Figure 1 shows a representative FT-IR spectrum of a control rat liver microsomal membrane sample in the region of 30501000 cm1. Main absorptions observed in the spectra are labeled in Figure 1, and detailed band assignments are given in Table 1. In order to observe the details of the spectral analysis, the spectra were investigated in two regions. Parts A and B of Figure 2 show the average spectra of control, irradiated, and amifostine treated plus irradiated rat liver microsomal membranes in the 30252820 and 19501000 cm1 regions, respectively. The numerical comparisons of the band area ratios, frequencies, and bandwidths of the infrared bands are listed in Table 2. As seen from this table and Figure 2, parts A and B, there are significant (p < 0.05) differences between the control and irradiated group suggesting that ionizing radiation induces remarkable changes in liver microsomal membranes. These figures and the table also indicate that the amifostine treated plus irradiated group’s spectrum is between the control and irradiated groups’ spectra and in general the values of amifostine treated plus irradiated group are very close to those of the control group. Indeed, with respect to the control, no significant variation was observed in the amifostine treated group. This result shows that amifostine has a protective effect against the ionizing radiation-induced alterations in rat liver microsomal membranes. The signal intensity and, more accurately, the area of infrared bands arising from particular species are directly proportional to the concentration of that species.13,20 In order to eliminate any artifacts which may be caused by variation in experimental conditions, e.g., sample concentration, the area ratios of some specific infrared bands have been evaluated for quantitative comparison between control and treated samples. Using FT-IR data, the lipid to protein ratio can be obtained by taking the ratio of the areas of the bands arising from lipids and proteins. The ratio of the sum of the area under the CH2 asymmetric and symmetric stretching bands to the sum of the area under the amide I and II bands was used to evaluate the lipid to protein ratio of the microsomal system. As seen from Table 2, the lipid/ protein ratio decreased significantly in the irradiated liver microsomal membranes (p < 0.05) compared to the controls. This decrease could be attributed to a lower lipid and/or higher protein content13,15 and shows that there is an alteration in the
Table 1. FT-IR Spectral Band Assignments of Rat Liver Microsomal Membrane in the Region of 30501000 cm1 band no.
frequency (cm1)
definition of the assignment
1
3014
olefinicdCH: unsaturated lipids (ref 14)
2
2962
CH3 asymmetric stretching: lipids and protein side chains (refs 18 and 24)
3
2925
CH2 asymmetric stretching: mainly lipids with a little contribution from proteins (refs 18 and 20)
4
2873
CH3 symmetric stretching: mainly proteins with a little contribution from lipids (refs 18 and 20)
5 6
2854 1745
CH2 symmetric stretching: mainly lipids with a little contribution from proteins (ref 20) ester CdO stretching: lipids (ref 15)
7
1650
amide I: 80% protein CdO stretching, 10% protein NH bending, 10% CN stretching (ref 25)
8
1545
amide II: 60% protein NH bending, 40% CN stretching (ref 25)
9
1455
CH2 bending: mainly lipids with a little contribution from proteins (20)
10
1401
COO symmetric stretching: fatty acids (ref 20)
11
1234
PO2 asymmetric stretching: phospholipids (ref 15)
12
1084
PO2 symmetric stretching: phospholipids (ref 15) 2440
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Figure 2. Average baseline-corrected infrared spectra of control, irradiated, and amifostine treated plus irradiated rat liver microsomal membranes (A) in the 30252820 cm1 region and (B) in the 1950 1000 cm1 region. The spectra were normalized with respect to the amide I band (A) and to the CH2 asymmetric stretching band (B).
lipid and protein metabolism in the irradiated group. When amifostine was administered to the rats before radiation treatment, irradiation did not cause any significant alterations in the lipid/protein ratio of the liver microsomal membranes, which indicates the protective effect of amifostine on the lipid/ protein ratio. In addition, to reveal the changes in the molecular composition and structure of lipids, the ratios of some specific lipid functional groups (CH2 asymmetric stretching, carbonyl ester stretching, CH3 asymmetric stretching, olefinicdCH) to the total lipid (the sum of the CH2 asymmetric and symmetric stretching bands) were calculated. The examination of these ratios demonstrated that ionizing radiation induced important changes in the composition and structure of membrane lipids (Table 2). The CH2/total lipid ratio was applied to indicate the alterations in the chain length of the phospholipids, where a lower ratio indicates shorter chained lipids.26 A significant decrease in this ratio was observed in the irradiated liver microsomal membrane (p < 0.05) compared to the control
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group (Table 2). Pretreatment of amifostine suppressed the significant change observed in the irradiated liver microsomes compared to the control group. In order to see the carbonyl status of the system, the carbonyl ester/total lipid ratio was calculated. As seen from Table 2, this ratio increased significantly (p < 0.05) in the irradiated group. The values of the amifostine treated plus irradiated group were very close to the control group, which clearly shows the protective effect of amifostine. Moreover, the CH3/lipid ratio was calculated to examine the methyl concentration in the liver microsomal membrane.26 The radiation treatment caused an increase in the CH3/total lipid ratios in the irradiated group compared to the control group (Table 2). Pretreatment of rats with amifostine also provided protection against the increment of CH3 groups induced by ionizing radiation. To examine the unsaturation level of the system, the ratio of the area of the band arising from unsaturated lipids (olefinicdCH band) to saturated lipids (CH2 asymmetric plus CH2 symmetric) was calculated. As seen from Table 2, this ratio increased significantly in the radiation treated group (p < 0.05), whereas no significant changes were observed for amifostine treated plus irradiated group compared to the control group implying that amifostine has a protective effect on the system. Ionizing radiation is known to generate highly reactive free radicals that can induce oxidative stress in important biological targets.3 With the particular sensitivity of polyunsaturated fatty acids, biological membranes are highly vulnerable to free radical attack.27,28 When lipids are attacked by free radicals, a lipid peroxidation chain reaction occurs. It has been found that these degenerative reactions lead to degradation to lipids, and the consequences of the attack by free radicals are broken chemical bonds, cross-linkages, and conformational changes.28 Previous reports on model membranes have shown that radiation yields oxidation fragments of unsaturated acyl chains.29,30 In addition, it has been shown that peroxidation of lipids, which causes a breakdown of long chains, is usually accompanied by the formation of a wide variety of degradation products including lipid aldehydes, shorter-chained lipids, and carbonyl compounds.28 Consistent with these earlier studies, in the current study the lower lipid/protein, CH2/total lipid, and the higher carbonyl ester/total lipid ratios observed in the irradiated group (Table 2) suggest that lipids were degraded by free radicals into smaller degradation products which contain less CH2 and more carbonyl esters. These results were supported by some previous studies where an increase in carbonyl groups and a degradation of acyl chains were observed in vitro, using FT-IR spectroscopy after lipid peroxidation.31 Since free radicals can also oxidize membrane proteins and the oxidation of proteins causes the production of some additional carbonyls, this may also contribute to the increase in the carbonyl ester/total lipid ratio.32,33 Moreover, the breakdown of lipid acyl chains after irradiation was confirmed by an increase in CH3/total lipid ratio which shows that the degradation products of lipids contain more methyl groups than normal lipids. Observations made in the present study in relation to the methyl contents are supported by other studies on the production of alkyl radicals during lipid peroxidation induced by different agents.34,35 The increase in the unsaturated/saturated lipid ratio may be due to higher lipid peroxidation in the irradiated membrane ultimately inducing an increase in the olefinic content originating mainly from lipid peroxidation endproducts such as malondialdehyde, lipid aldehydes, and alkyl radicals.14,36 Consequently, the lower lipid/protein and CH2/ total lipid ratio and the higher carbonyl ester/total lipid, CH3/ 2441
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Table 2. Changes in the Band Area Ratio, Frequency, and Bandwidth Values of Various Functional Groups in Control, Irradiated, and Amifostine Treated Plus Irradiated Liver Microsomal Membranes functional group
control
P valuea
radiation
radiation plus amifostine
P valueb
Band Area Ratio lipid/protein
0.064 ( 0.004
0.054 ( 0.007
0.035*
0.062 ( 0.004
0.575
CH2/lipid carbonyl ester/lipid
0.874 ( 0.05 0.102 ( 0.005
0.816 ( 0.007 0.115 ( 0.006
0.030* 0.013*
0.831 ( 0.009 0.108 ( 0.008
0.173 0.128
CH3/lipid
0.129 ( 0.015
0.156 ( 0.015
0.030*
0.124 ( 0.011
0.378
unsaturation/saturation
0.042 ( 0.005
0.049 ( 0.003
0.030*
0.046 ( 0.006
0.229
amide I/amide II
1.847 ( 0.289
1.522 ( 0.144
0.022*
1.615 ( 0.139
0.065
Frequency CH2 asym. str.
2924.85 ( 0.16
2923.94 ( 0.22
0.005**
2924.53 ( 0.09
0.200
amide I
1649.90 ( 0.92
1651.11 ( 0.70
0.030*
1650.64 ( 0.55
0.173
12.338 ( 0.23 27.972 ( 0.68
12.038 ( 0.12 29.514 ( 1.25
12.20 ( 0.11 28.56 ( 0.85
0.378 0.229
Bandwidth CH2 asym. str. amide I
0.037* 0.035*
a P value: p values are from the comparison of the control and radiation groups. b P value: p values are from the comparison of the control and radiation plus amifostine groups.
total lipid, and unsaturated lipid/saturated lipid ratios suggest that lipids were oxidized by ionizing radiation, and as a result of this oxidation some degradation products which contain less CH2 group and more carbonyl esters, CH3, and unsaturated fatty acids were produced. As seen from Table 2, amifostine was very successful in protecting the microsomal membrane lipids from peroxidation damage induced by ionizing radiation. FT-IR spectroscopy offers unique possibilities for the simultaneous study of proteins together with lipids in biological membranes and tissues.6,12,20 To evaluate the changes in protein composition and structure, the amide I/amide II ratio was calculated. We observed a significant decrease in the amide I/ amide II ratio of the irradiated group (p < 0.05) in contrast to the amifostine treated plus irradiated group in comparison to the control group (Table 2). As the amide I and amide II profiles depend on the protein structural composition, this reduction suggests that there are some alterations in the structures of membrane proteins.3739 Consistent with our result, Dogan et al. observed a reduction in the amide I/amide II ratio after γirradiation in hazelnut tissue, and they suggested that this reduction was associated with changes in the protein structure.21 This explanation can be reliable since amide I band arises predominantly from the CdO stretching vibration of the amide groups, whereas the amide II band arises predominantly from the NH bending vibration.25 Moreover, in recent medical research it has been shown that, compared with the normal tissue, the diseased tissue shows a change in protein secondary structure and a reduced ratio of amide I to amide II ratio.40 In addition, a significant shifting in the frequency to higher values of the amide I band (p < 0.05) and a significant broadening in the bandwidth of this band (p < 0.05) were observed in the irradiated group (p < 0.05) in comparison to the control group (Table 2). This broadening and shifting observed in the amide I band indicates that the protein conformation was affected from ionizing radiation.20 In a previous study, protein oxidation was found to broaden the amide I band.41 As noted above, protein oxidation results in the production of some additional carbonyls on some amino acid residues.32 It has been suggested that some of these
carbonyls reside adjacent to amines and they lead to a spectroscopic absorption and a broadening in the amide I band.32,41 Thus, the decrease in the amide I/amide II ratio, and the broadening and shifting of the amide I band to higher values, could be interpreted as the result of the altering protein structure and conformation of irradiated liver microsomal membranes. In order to better estimate the alterations on the protein secondary structure, the amide I band region, corresponding to absorption values between 1600 and 1700 cm1, was analyzed using neural network predictions based on the FT-IR data. FT-IR spectroscopy is an excellent method for determining protein secondary structure. Neural networks based on the amide I band offer a new computational approach that has served as a reliable alternative method for predicting protein secondary structure in recent years in solutions23,42 and by our group in biological tissues.4346 The results are presented in Table 3, which revealed that the β-sheet structures decreased significantly in the irradiated group (p < 0.05) compared to the control group. Moreover, the turns and random coil structures increased in the irradiated group (p < 0.05) compared to the control group. The increase in the random coil structure indicates that ionizing radiation caused protein denaturation in the system.21 Our results are in agreement with earlier studies that reported an increase in the random coil structure.21,47 Moreover, Abu et al. reported protein denaturation due to γ-irradiation, which also supports our findings.48 A metabolic disruption of some amino acids could occur after radiation treatment in liver microsomes and could be responsible for the observed changes in the proteins. It is known that free radicals lead to protein degradation by increasing accessibility of protein bonds and/or aggregation.49 In a previous study, it has been reported that radiation causes an alteration in the conformation of integral membrane proteins and that radiation-induced hydroxyl radicals lead to denaturation of membrane proteins.50 On the other hand, a relationship between lipid peroxidation and damage to protein synthesis has been previously reported in liver slices from rats.51 As seen from Table 3, there are no significant differences in the protein secondary structure profile of the amifostine treated plus 2442
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Table 3. Neural Network Predictions Based on FT-IR Data in the 16001700 cm1 Spectral Region for the Changes in Secondary Structure between the Control, Irradiated, and Amifostine Treated Plus Irradiated Liver Microsomal Membranes functional group
control
P valuea
radiation
radiation plus amifostine
P valueb
R-helix
40.94 ( 1.62
39.40 ( 2.96
0.117
41.15 ( 0.94
0.855
β-sheet turns
26.30 ( 1.24 20.50 ( 0.47
24.22 ( 0.82 21.88 ( 0.39
0.028* 0.012*
25.50 ( 1.55 20.46 ( 0.48
0.361 0.927
random coil
12.26 ( 1.11
14.82 ( 0.13
0.012*
13.05 ( 0.51
0.235
a
P value: p values are from the comparison of the control and radiation groups. b P value: p values are from the comparison of the control and radiation plus amifostine groups.
irradiated group compared to the control group supporting the protective effect of amifostine on protein secondary structure. The position of the asymmetric and symmetric CH2 stretching bands provides information about the lipid acyl chain flexibility (order/disorder state of lipids). For example, the higher the frequency, the higher the acyl chain flexibility, which implies lipid disordering.11,12,36 The frequency of the CH2 asymmetric stretching band shifted significantly to lower values after radiation treatment (p < 0.05) in comparison to the control. This shifting to lower values indicates that lipid order increases and acyl chain flexibility decreases in the irradiated liver microsomal membranes.12,20 Bandwidth of the same band gives information about the dynamics of the system.20 As seen from Table 2, there is a significant reduction in the bandwidth of the CH2 asymmetric stretching band of the irradiated group in comparison to the control group, which implies a decrease in the lipid dynamics of the radiation-treated liver microsomal membranes. Thus, ionizing radiation caused an increase in the lipid order and a decrease in the membrane fluidity in the irradiated microsomal membranes. Liver microsomes contain cholesterol and a great variety of phospholipids at high concentrations. The fatty acid composition of rat liver microsomes is so highly unsaturated that they are very fluid at physiological temperatures.52 Previously, the loss of freedom of motion in membranes after oxidative stress using biological and liposomal membranes was reported,5355 and lipid peroxidative stress was known to decrease membrane fluidity in microsomes and other cellular membranes.3,56 An important factor affecting the membrane structure and dynamics is the amount of proteins and lipids in the membranes.6 Two important reasons could be proposed as causal relationships for the loss of membrane fluidity during ionizing radiation treatment. First, radiation may cause cholesterogenesis and due to the increased amount of cholesterol a decrease in membrane fluidity may be observed; second, the formation of cross-linking among the lipidlipid and proteinlipid moieties as a result of oxidative stress may limit the motion.54,55,57 As seen from Table 2, no significant changes were observed in the frequency and bandwidth of the CH2 asymmetric stretching band of the amifostine treated plus irradiated group in comparison to the control group, again indicating the restoring effect of amifostine. This result shows that stabilizing of cell membranes may be another important function by which amifostine reduces oxidative damage to cells. Alterations in membrane fluidity have important consequences in terms of cellular function.5,6 Thus, the ability of amifostine to maintain membrane fluidity may further contribute to the protective actions of this molecule. In all cases, the damage induced by ionizing radiation on membrane lipids and proteins was suppressed by amifostine administration. The inhibitory effects of amifostine against lipid and protein damage could be attributed to its antioxidant
properties. WR-1065, the active form of amifostine, must be present at the time of radiation exposure.1 In the cell, WR-1065 can provide protection by several mechanisms. It can act as a potent scavenger of oxygen free radicals derived from radiation therapy.58 Another mechanism of cytoprotection involves hydrogen donation to repair free radicals in target molecules.58 WR1065 may also react directly with oxygen and thus protect the cell by creating local hypoxia at the target.58 Other mechanisms proposed for the radioprotective effect of amifostine are target stabilization by binding to DNA and the protection of key sulfhydryl enzymes through formation of proteinamino thiol mixed disulfides.59
’ CONCLUSIONS In conclusion, the results of the present study revealed that ionizing radiation induced significant alterations in the composition and structure of microsomal lipids, dramatic increases in lipid order, and significant decreases in membrane dynamics. Moreover, the composition and secondary structure of proteins were altered by ionizing radiation. Amifostine administration to the rats prior to whole body irradiation was effective in preventing the radiation-induced damages on membrane lipids and proteins, likely through a free radical scavenger activity. We suggest that maintaining membrane fluidity despite the rigidity effect of ionizing radiation may be another cytoprotective mechanism of amifostine. In addition, the current study indicated that FT-IR spectroscopy provides a rapid and sensitive monitoring of ionizing radiation-induced damage in biological membranes and FT-IR parameters employed in this study can be used as biological indicators of radiation-induced membrane damage. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: þ90-312-210 51 66. Fax: þ90-312-210 79 76. E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by the Scientific and Technical Research Council of Turkey (TUBITAK), SBAG-2939 Research Fund, and by the METU-Research Fund, BAP-2006-07-02-0001. 2443
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Analytical Chemistry
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