Journal of Radiation Research and Applied Sciences J. Rad. Res. Appl. Sci., Vol. 4, No. 4(A), pp. 1163 – 1188 (2011) Aluminium and Gamma Irradiation Induced Oxidative Damage in Brain Tissue of Male Rats – Protective Role of Ferulic Acid S. Z. Mansour, N. Hanafi and E. Noaman Radiation Biology Department, National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA), Cairo, Egypt. E-mail: [email protected]Received:18/05/2011. Accepted:17/10/2011. ABSTRACT The current study was carried out to investigate the potential role of ferulic acid (FA) against Aluminium chloride (AlCl 3 ), γ- radiation either alone or combination induced oxidative stress in brain tissue of Wistar rats. The period of the experiment was eight weeks. Animals were administrated by aluminium chloride at a dose of 8.5 mg/kg/day and exposed to a single dose (4 Gy) of γ-radiation. FA was administered orally (50mg/Kg body weight)/day. Histopathological observations and myeloid protein distribution were recorded in brain tissue. Induction of oxidative stress was recorded after all exposures. Brain tissue of AlCl 3 and γ-irradiation treatments either alone or combined revealed many altered changes and myeloid protein distribution. Also a decrease in serotonin concentration was recorded. An increase in Malonaldialdahyde (MDA) and acetylcholinesterase activity and percentage of saturated fatty acids in plasma and brain tissue was recorded. Reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) in blood and brain showed a significant decrease. Treatment of AlCl 3 loaded animals by FA showed simple atrophy as shrunken morphology saw in amyotrophic lateral sclerosis and a decrease in myeloid protein deposition. FA treatment of AlCl 3 loaded or irradiated animals represented a significant increase in serotonin concentration and ameliorated affects on oxidative stress markers, acetylcholinesterase activity and percentage of saturated fatty acids in plasma and brain tissue. In conclusion FA has a role in reducing the oxidative stress of AlCl 3 and γ-irradiation on brain tissue of rats. Keywords: Brain; Aluminium Chloride (AlCl 3 ); Ferulic acid (FA).
J. Rad. Res. Appl. Sci., Vol. 4, No. 4(A), pp. 1163 – 1188 (2011)
Aluminium and Gamma Irradiation Induced Oxidative Damage in Brain Tissue of Male Rats – Protective Role of Ferulic Acid S. Z. Mansour, N. Hanafi and E. Noaman Radiation Biology Department, National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA), Cairo, Egypt. E-mail: [email protected] Received:18/05/2011. Accepted:17/10/2011.
ABSTRACT
The current study was carried out to investigate the potential role of ferulic acid (FA) against Aluminium chloride (AlCl3), γ- radiation either alone or combination induced oxidative stress in brain tissue of Wistar rats. The period of the experiment was eight weeks. Animals were administrated by aluminium chloride at a dose of 8.5 mg/kg/day and exposed to a single dose (4 Gy) of γ-radiation. FA was administered orally (50mg/Kg body weight)/day. Histopathological observations and myeloid protein distribution were recorded in brain tissue. Induction of oxidative stress was recorded after all exposures. Brain tissue of AlCl3 and γ-irradiation treatments either alone or combined revealed many altered changes and myeloid protein distribution. Also a decrease in serotonin concentration was recorded. An increase in Malonaldialdahyde (MDA) and acetylcholinesterase activity and percentage of saturated fatty acids in plasma and brain tissue was recorded. Reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) in blood and brain showed a significant decrease. Treatment of AlCl3 loaded animals by FA showed simple atrophy as shrunken morphology saw in amyotrophic lateral sclerosis and a decrease in myeloid protein deposition. FA treatment of AlCl3 loaded or irradiated animals represented a significant increase in serotonin concentration and ameliorated affects on oxidative stress markers, acetylcholinesterase activity and percentage of saturated fatty acids in plasma and brain tissue. In conclusion FA has a role in reducing the oxidative stress of AlCl3 and γ-irradiation on brain tissue of rats.
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INTRODUCTION
Aluminium metal is abundantly present in the earth’s crust. From the environment it gets access to the human body via the gastrointestinal and the respiratory tracts. Aluminium is a constituent of cooking utensils and medicines such as antacids, deodorants and food additives and this has allowed its easy access into the body (1). The sources of Aluminium are especially corn, yellow cheese, salt, herbs, spices, tea, cosmetics, ware and containers. Also, it is present in medicines and is also added to drinking water for purification purposes (2).
Aluminium has been proposed as an environmental factor that may contribute to some neurodegenerative diseases, and affects several enzymes and other biomolecules relevant to Alzheimer‘s disease (3). Also, increased Aluminium burdens can cause neurological symptoms, biochemical responses leading to unhealthy bone metabolism and learning disabilities in children (4). Salts of Aluminium may bind to DNA, RNA, inhibit such enzymes as hexokinase, acid and alkaline phosphatase, phosphodiesterase and phosphor-oxydase (2). Strong et al., (5) found that Aluminium exposure caused impairments in glucose utilization, agonist-stimulated inositol phosphate accumulation, free radical-mediated cytotoxicity, lipid peroxidation, reduced cholinergic function, impact on gene expression and altered protein phosphorylation. Yousef (6) reported that Aluminium-induced changes in hemato-biochemical parameters, increased lipid peroxidation and decreased the activities of the antioxidant enzymes in plasma and tissues of male rabbits.
Aluminium accumulation in the brain, biochemical changes leading to damage in the cholinergic system are accomplished in acute and chronic exposure (9). Decreases of acetyl-coenzyme A levels (10, 11), choline acetyltran-sferase (10) and acetylcholinesterase (9, 12) have been observed after in vivo and in vitro exposure to Aluminium.
Ferulic acid (FA), extracted from a traditional Chinese herbal medicine, has potent antioxidant (13) and anti-inflammatory activities (14). It has recently been reported that long-term administration of FA protected mice against learning and memory deficits induced by centrally administered β-myeloid protein (15). The primary site of action of ferulic acid could be microglia (16) and astrocytes (17). It has been recently reported that ferulic acid inhibited formation of myeloid protein beta (Aβ) fibrils and destabilized preformed fibrillary Aβ (18). Sultana et al (19) reported that FA ethyl ester significantly inhibited Aβ1-42-induced cytoxicity, intracellular reactive oxygen species accumulation, lipid
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peroxidation, and induction of inducible nitric oxide synthase in primary hippocampal cultures (19). Significantly, they reported that FA had inhibitory effects on Aβ-induced increases of IL-1β expression and p38 mitogen-activated protein kinas pathway and apoptosis in rat hippocampus (20, 21). In addition, treatment with FA protected against glutamate induced apoptosis of cultured cortical neurons (22). In the present study, the role of FA against Aluminium, γ-irradiation and either alone or combination induced histopathological and biochemical changes in rats brain have been studied.
MATERIAL AND METHODS Animals:
Eighty male albino rats (110-120 g) were obtained from the Egyptian Organization for Biological Product and Vaccines at Giza, Egypt were used throughout the present experiments. Animals were housed in cages under good ventilation and illumination conditions; they had access to unlimited water and standard rodent chow. Animal maintenance and treatments were conducted in accordance with the National Institute of Health Guide for Animal, as approved by Institutional Animal Care and Use Committee (IACUC).
Ferulic acid and AlCl3 administration
Ferulic acid (FA) was obtained from sigma chemical Co., St. Louis, Mo. USA. in the form of trans-4-hydroxyl methoxy cinnamic acid. It was freshly prepared by dissolving proper concentration in distilled water. It was given to the animals orally (50mg/Kg body weight/day) for eight weeks according to the method of Maurya and Nair (23). Aluminium chloride (AlCl3) was obtained from sigma chemical Co., St. Louis, Mo. USA. Animals were injected i.p. with AlCl3 (8.5mg/ Kg body weight/day) for eight weeks according to Fyiad (24). All other chemicals and reagents used were of analytical grade.
Radiation process
Whole body gamma irradiation of rats was performed using a Canadian gamma cell-40, (137Cs) at the National Centre for Radiation Research and Technology (NCCRT), Cairo, Egypt. Rats were exposed to one dose (4.0 Gy) of γ-radiation at the fourth week of the experiment. The dose rate was 0.792 R/sec. at the time of experiment.
Experimental design
The eighty animals were randomly divided into eight groups (n=10). The period of experimental was extended to 8 weeks. Dose of ferulic acid (FA)
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was orally administered 30 minutes before beginning of AlCl3 injection. Animals were exposed to γ-radiation at one dose at the fourth weeks of experiment one hour after FA administered. Groups of animals under experimental investigation were, Group (1): Normal rats group, (2): FA group, (3): AlCl3 group, (4): Irradiated group, (5): FA AlCl3 group, (6): AlCl3 irradiated group, (7): FA irradiation; group and (8): FA irradiated AlCl3 group.
Rats of each group were fasted overnight before the time of sacrificing, one day before the end experiment. Blood samples were obtained by heart puncture and collected in sterile heparinized and non- heparinized tubes. Brains were quickly excised out, washed and weighed for further biochemical and histological analysis. Brains were homogenized in ice-cold 0.1 M potassium phosphate buffer (pH 7.4) to yield 10% homogenates.
Biochemical assays.
Reduced glutathione concentration (GSH) was detected according to Beutler et al.,(25), superoxide dismutase (SOD) activity levels were estimated by the detection of superoxide anions using nitroblue tetrazoluim formazan color development as reported before (26) and catalase (Cat) activity was assayed by the method of Sinha (27). Lipid peroxidation (LP) indicated by the formation of MDA was assessed using the method described by Yoshioka et al., (28). All parameters were estimated in blood packed RBCs and brain except MDA in plasma and brain homogenates. The acetylcholinesterase activity was measured by the method of Ellman et al., (29) with slight modifications (30).
HPLC determination of the brain Noradrenalin, dopamine and serotonin concentrations Preparation of the sample
The first step in the determination of the brain noradrenalin, dopamine and serotonin by HPLC method involved weighing and homogenization of the tissue in 1/10 weight/volume of 75% aqueous HPLC grade methanol. The homogenate was spun at 3000 r.p.m. for 10 min and the supernatant was used for monoamines determination immediately extracted from the trace elements and lipids by the use of solid phase extraction CHROMABOND column NH2 phase Cat. No. 730031. The sample was then injected directly into an AQUA column 150 54.6 mm 5 μC18, purchased from Phenomenex, USA under the following conditions: mobile phase 97/3 20 Mm potassium phosphate, pH 3.0/methanol, flow rate 1.5 ml/min, UV 270 nm. noradrenalin, dopamine and serotonin were separated after 12 minutes. The resulting chromatogram
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identified each monoamine position and concentration from the sample was determined as compared to that of the standard. The content of each monoamine is expressed as μg per gram brain tissue (31).
Fatty acids determination
For analytical procedures, serum and brain free fatty acids and lipids of serum and brain were extracted by the method of Folch et al., (32). A known volume of serum and wt of brain were extracted with diethyl ether/ ethanol (2: 1 v/v) for 3 times, after which the solvent was evaporated at 55°C. The results were calculated per 1 g wt of sample and saponified according to the method of Ashour (33). The fatty acids were esterified by methanol and purified according to the method of Kinsella (34), and analyzed by Hp 6890 gas chromatography. The chromatograph includes an innowax-crossline polyethylene glycol column 30 m, i.d. 0.32 ml meter, 0.5 μ meter film thickness. The assays were carried out with programmed oven temp. as follows 150°C for 1 min, then elevated to 235°C with rate of 17°C / min, detector, FID, 275°C. Peak areas were measured with an integrator connected to a computer, which expressed the data automatically. Peaks were identified by comparing their equivalent chain lengths with those of authentic fatty acid methyl esters.
Histological studies Preparation of brain for light microscopy
The rat brain was kept as a whole in Bouin’s fixative, dehydrated in ascending grades of alcohol, cleared with xylol, embedded and impregnated in paraffin wax, sectioned at 5 μm thick sections and subjected to the following stains:
• Hematoxyline and eosin (35): for studying the general structure of brain. • Congo red (36): to demonstrate myeloid protein deposits in brain tissue
sections. Statistical analysis:
The SPSS/PC computer program was used for Statistical Analysis of the results. Data were analyzed using one-way analysis of variance (ANOVA). Values were expressed as mean ± SE. Differences were considered significant at P < 0.05.
RESULTS
A: Histopathological observations
Light microscopic examination of sections derived from brain tissue showed magnocellular nuclei stained with H & E and showed large nerve cells
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which are mostly multipolar or stellate in addition to pyriform cells. Astrocytes with sharply demarcated nuclei were also seen in control brain tissue (Fig. A1).
Al2 3 4
5 6 7
N
NOA
1
Al2 3 4
5 6 7
N
NOA
1
N
NOA
1
Fig. (A): A photomicrograph of male albino rat brain tissue 1: Control brain showing
neuron (N), oligodendrocyte (O) and Astrocytes (A). 2, 3, 4:AlCl3 treated brain tissue represented large thrombotic vessel (↓) cytotoxic edema (vacuoles), eosinophilia (red neurons▲) and the presence of cerebral infarct in which Microglials transform into macrophages (bloked arrow). 5: Irradiated brain tissue recorded enlarged nucleus and un enlarged cytoplasm (↓).Note the accumulation of fluid in the perivascular (curved arrow).6, 7: AlCl3 and irradiation treated brain tissue recorded nucleus extensively dark picnotic (curved arrow), shrunken, hyperchromasia, cytotoxic edema (vacuoles), and thrombotic vessels (↑). (HE, X400)
Treatment of the experimental animals by AlCl3 represented cytotoxic edema (vacuoles) loss of Nissl substance, eosinophilia (red neurons) (Fig. A3). Also large thrombotic vessel and the presence of cerebral infarct in which microglials transform into macrophages were observed in Fig. (A4). However
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exposure of rats to γ-radiation showed enlarged nucleus and unenlarged cytoplasm in addition to the presence of perivascular fluid accumulation (Fig. A5). Treatment of rats with AlCl3 and γ-radiation recorded nucleus shrunken and hyperchromasia. Disintegration cytoplasm represented cytotoxic edema (vacuoles) loss of Nissl substance, eosinophilia (red neurons) (Fig. A6). Thrombotic vessels were also detected (Fig. A7)
Treatment of the experimental animals by FA exhibited a normal appearance of brain tissue structure (Fig. B1). However, treatment of AlCl3 loaded animals by FA showed simple atrophy in the form of shrunken morphology seen in amyotrophic lateral sclerosis (Fig. B2). But normal appearance of brain tissue structure was shown when irradiated or AlCl3 irradiated animals were treated by FA (Fig. B 3, 4).
1 2
3 4
1 2
3 4
Fig. (B): A photomicrograph of male albino rat brain tissue 1: Ferulic acid treated
brain tissue showed amyotrophic lateral sclerosis (↑), 2: Ferulic acid and AlCl3 treated brain tissue 3: Ferulic acid and irradiated brain tissue, 4: Ferulic acid, AlCl3 and irradiation treated brain tissue. (H and E. stain x400)
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B: Myeloid protein deposition.
Staining of myeloid protein showed a nonsignificant deposition in brain tissue of control and irradiated animals. A significant deposition of myeloid protein in the brain tissue of the AlCl3 treated animals (Fig. 2C). Similar but more abundant findings were observed in the AlCl3 irradiated treated animals (Fig. 4C). Myeloid protein predominantly deposit as extracellular plaques and can be frequently found in the neuronal soma in brain tissue of AlCl3 irradiated treated group. Treatment of AlCl3, irradiated either alone or in combination by FA recorded a nonsignificant deposition of myeloid protein in brain tissue (Fig. D 1,2,3 and 4).
Fig. (C): Photomicrograph in brain tissue of male rat stained by congo red stain
represented the deposition of β-amyloid. 1: Control, 2: AlCl3 treated brain tissue represented extracellular plaques (↑) and neuronal soma (curved arrow) β-amyloid deposition 3: Irradiated brain tissue, 4: AlCl3 and irradiation treated brain tissue represented intensive β-amyloid deposition as extracellular plaques and neuronal soma (↓). (x400)
1 2
3 4
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Fig. (D): Photomicrograph in brain tissue of male rat stained by congo red stain represented the deposition of β- amyloid. 1: Ferulic acid treated brain tissue, 2: Ferulic acid and AlCl3 treated brain tissue 3: Ferulic acid and Irradiated brain tissue, 4: Ferulic acid, AlCl3 and irradiation treated brain tissue. (x400).
Biochemical analysis
A: Oxidative stress markers and acetylcholinesterase activity.
Table 1 and 2 show that treatment of the experimental animals with i.p. injection with AlCl3 resulted in severe alterations in oxidative stress markers and Acetylcholinesterase activity. Significant decrease was observed in GSH content and SOD activity in blood and brain and CAT activity in plasma and brain after AlCl3 exposure. FA administration to control rats recorded a non significant change in their oxidative stress markers and acetylcholinesterase activity in blood and brain tissues. The effect of FA against the AlCl3-mediated toxicity recorded a significant increase in the CAT and SOD activities and a significant decrease in MDA content. While acetylcholinesterase represented a significant increase in the plasma and brain of AlCl3 treated rats compared to
1 2
3 4
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control level. A significant decrease was recorded by FA administration compared to AlCl3 treated group. Exposure of rats to γ- radiation showed no significant change in GSH content and SOD and CAT activity in blood compared to control level. On the other hand, in brain tissue GSH content and SOD and CAT activities displayed a significant decrease compared to control level. MDA level recorded a non significant change either in plasma or brain tissue while acetylcholinesterase activity showed a significant increase in plasma and brain tissue. Some ameliorating effect was recorded in oxidative markers of blood and brain tissues when irradiated animal group treated by FA. Decrease in GSH content and SOD and CAT activities were observed in blood and brain tissue when experimental animals were treated by AlCl3 and γ- irradiation. On the other hand, MDA content and acetylcholinesterase activity recorded an obvious increase. Ameliorative effects were observed in blood and brain tissue oxidative markers and acetylcholinesterase activity when AlCl3
irradiated group was treated by FA. Table (1): Effect of ferulic acid (FA) on GSH and SOD of packed RBCs and CAT,
MDA and Acetylcholinesterase activity of rat’s blood plasma. Groups Parameters
GSH (mg/ml packed RBCs)
SOD (μg/ml packed RBCs)
CAT (µmol/ ml)
MDA (μmol/ml plasma)
Acetyl cholinesterase
activity Plasma (µml/ml)
Control 69.74
± 2.71c 8.32
± 0.16cd 228.56 ± 9.19c
114.64 ± 4.68c
18.09 ± 0.793cd
FA 66.31
± 5.98c 8.33
± 0.13cd 242.59
± 11.82c 115.51 ± 5.06c
17.75 ± 1.398cd
AlCl3 53.77
± 2.19abd 6.54
± 0.17abd 178.48
± 25.51abd 131.22
± 2.30acd 33.00
± 1.121abd
Radiation 66.17
± 0.58c 7.82
± 0.20abc 235.61
± 15.10 112.11 ± 3.40c
25.97 ± 0.692abc
AlCl3 + Radiation 59.93
± 1.72a 8.13
± 0.13c 207.85 ± 19.36
120.08 ± 1.64c
40.12 ± 0.823abcd
FA +AlCl3 61.22 ± 2.46
8.21 ± 0.06c
209.63 ± 16.55
119.06 ± 2.80c
24.49 ± 1.279abc
FA +Radiation 64.21
± 2.30c 8.16
± 0.07c 250.67
± 17.75c 117.08 ±2.33c
20.51 ± 1.263cd
FA+Radiation+ lCl3 60.51
± 3.90a 7.24
± 0.09abc 201.93 ± 10.09
123.22 ± 3.17c
28.20 ± 1.603abc
Results are presented as the mean ± SE. a: significant from control group at p < 0.05. b: significant from FA group at p < 0.05. c: significant from AlCl3 group at p < 0.05. d: significant from radiation group at p < 0.05.
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Table (2): Effect of ferulic acid (FA) on GSH, SOD, CAT, MDA and Acetylcholinesterase activity of rat’s brain.
Groups Parameters
GSH (µg/g tissue)
SOD (μg/g tissue)
CAT (µmol/g tissue)
MDA (µg/g tissue)
Acetyl cholinesterase
activity Brain (µml/g)
Control 84.51
± 1.445cd
8.45
± 0.091cd
258.59
± 3.742cd
253.65
±1.138c
242.59
± 9.472cd
FA 84.37
± 0.463cd
8.33
± 0.261cd
255.59
± 6.475cd
251.40
± 1.280cd
239.68
± 7.413cd
AlCl3 67.768
± 1.298ab
6.72
± 0.198abd
211.22
± 4.253abd
268.07
± 1.174abd
325.91
± 4.804abd
Radiation 70.52
± 1.497ab
7.84
± 0.138abc
233.99
± 5.114abc
257.31
± 2.307bc
275.77
± 9.624abc
AlCl3+Radiation 64.34
± 3.375ad
5.54
± .144abcd
204.15
± 2.627abd
281.03
± 2.850abcd
355.98
± 4.032abcd
FA +AlCl3 78.35
±1.321abcd
8.26
±0.102bc
248.24
±3.295bcd
253.65
± 1.232c
265.99
± 8.837c
FA +Radiation 80.27
± 1.725cd
8.08
± 0.150c
251.43
± 1.321cd
253.71
± 1.365c
254.16
± 4.418c
FA+Radiation+ lCl3 77.97
±0.930abcd
7.04
± 0.062abd
231.77
± 3.474abc
267.91
± 1.438abd
320.19
± 18.026abd
Legends as in table (1)
B: Neurotransmitters (Noradrenalin, dopamine and serotonin) concentration in brain tissue
Table (3) pointed out the non significant change of neurotransmitters in brain tissue of experimental animals treated by FA. Some decrease occurred in dopamine and seratonin concentrations of brain tissue after exposure of the experimental animals to γ-radiation. On the other hand, an increase in noradrenalin and dopamine were recorded while serotonin concentration represented some decrease when the experimental animals suffered from AlCl3 toxicity either alone or in combination with γ-irradiation. Treatment of animals suffered from AlCl3 toxicity by FA revealed a non significant change in noradrenalin and serotonin concentrations compared to control level. However treatment of the irradiated animals by FA represented a significant increase in dopamine and serotonin concentration compared to control level as well as irradiated rats. Treatment of experimental animals suffered from AlCl3 toxicity combined with γ-radiation by FA showed a significant increase in neurotransmitters concentrations in brain tissue in comparison with those of
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control or irradiated groups.
Table (3): Effect of ferulic acid (FA) administration to rats on neurotransmitters concentration in brain tissue.
Groups
Parameters
Noradrenalin
mg/ g brain
Dopamine
mg/ g brain
Serotonin
mg/ g brain
Control 1.999 ± 0.127c 0.559 ± 0.109 0.061 ± 0.007cd
C: Fatty acids composition of total lipids in plasma and brain tissue
Tables (4-5) illustrate alterations in the distribution profile of fatty acids in the plasma and brain in the different animal groups. The percentage of saturated fatty acids (SFA) and total monounsaturated fatty acid (MUFA) increased by administrating rats with AlCl3 and radiation exposure while the percentage of total unsaturated fatty acid (USFA) and polyunsaturated fatty acid (PUFA) decreased. The effect of FA administration ameliorated the fatty acids levels.
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Table (4): Fatty acids composition of plasma total lipids in different animal groups (mg/ml).
Fatty acids
Control FA AlCl3 Radiation FA
+AlCl3 FA +
Radiation AlCl3
+Radiation
FA + Radiation + AlCl3
C14:0 0.71
±0.04cd 0.71
±0.03cd 2.69
±0.22abd 2.25
±0.17abc 0.76
±0.04cd 0.75
±0.04cd 2.71
±0.21abd 0.94
±0.08cd
C16:0 21.68
±1.02cd 22.30
±1.21cd 28.45
±1.36ab 26.99
±0.92ab 24.38 ±1.21
22.14 ±1.32cd
29.12 ±1.38ab
25.37 ±1.25ab
C16:1 1.60
±0.16 1.60
±0.08 1.96
±0.38 1.83
±0.24 1.56
±0.13 1.35
±0.13 2.04
±0.22 2.04
±0.22
C18:0 6.80
±0.17cd 6.84
±0.59cd 9.22
±0.13ab 9.14
±0.50ab 6.62
±0.41cd 6.56
±0.52cd 12.64
±0.35abcd 7.68
±0.34cd
C18:1 12.53 ±0.55
12.04 ±0.64
13.39 ±0.70
12.98 ±0.41
12.80 ±0.31
12.50 ±0.36
13.68 ±0.34b
13.03 ±0.43
C18:2 31.77
±0.66cd 32.84
±0.99cd 23.85
±0.75ab 24.40
±0.67ab 29.78
±1.23bcd 29.87
±0.99bcd 19.47
±1.41abcd 28.72
±0.17abcd
C18:3 1.16
±0.14cd 1.21
±0.07cd 0.52
±0.05ab 0.75
±0.19ab 0.96
±0.13c 1.06
±0.06c 0.36
±0.07abd 0.80
±0.04ab
C20:0 4.08
±0.15c 3.97
±0.20c 6.40
±0.41ab 5.63
±0.71 5.87
±0.81ab 5.10
±0.09 7.35
±0.39abd 6.78
±0.51ab
C20:1 0.37
±0.04c 0.37
±0.04c 0.24
±0.03abd 0.35
±0.04c 0.36
±0.02c 0.35
±0.03c 0.21
±0.02abd 0.31
±0.01
C20:4 12.98 ±0.48c
12.63 ±0.66c
9.27 ±0.91ab
11.43 ±1.23
10.80 ±0.71
13.14 ±0.64c
8.85 ±0.14abd
9.96 ±0.82ab
C20:5 1.64
±0.34c 1.17
±0.24 0.56
±0.08ad 1.33
±0.12c 1.40
±0.24c 1.43
±0.31c 0.38
±0.03abd 0.98
±0.07a
C22:0 0.20
±0.03cd 0.22
±0.02cd 0.55
±0.05ab 0.43
±0.06ab 0.32
±0.06c 0.34
±0.08c 0.66
±0.04abd 0.40
±0.05ab
C22:6 3.93
±0.41cd 3.62
±0.22c 2.07
±0.39ab 2.62
±0.28a 3.46
±0.34c 3.86
±0.40cd 1.99
±0.57ab 3.05
±0.42
C24:0 0.54
±0.04 0.51
±0.03 0.47
±0.06 0.46
±0.02 0.48
±0.02 0.55
±0.08 0.63
±0.06cd 0.49
±0.03 Total SFA
34.01 ±1.02cd
34.22 ±1.44cd
45.54 ±4.00abd
44.65 ±1.39abc
38.88 ±0.86cd
35.88 ±2.16cd
52.80 ±1.32abcd
39.66 ±0.41ac
Total USFA
65.99 ±1.01cd
65.78 ±1.64cd
54.46 ±4.00ab
55.35 ±1.39ab
61.12 ±0.86cd
64.12 ±2.07cd
47.20 ±1.38abcd
60.34 ±0.41c
Total MUFA
14.50 ±0.63
13.98 ±0.59c
15.58 ±0.48
15.16 ±0.60
14.71 ±0.31
14.38 ±0.22
16.64 ±0.41abd
15.07 ±0.39
Total PUFA
51.48 ±1.54cd
51.47 ±0.88cd
36.28 ±1.17abd
40.53 ±1.76abc
46.40 ±0.66abcd
49.41 ±1.81cd
30.99 ±1.10abd
45.27 ±0.54abd
Legends as in table (1)
List of abbreviations C14:0: Myristic acid; C16:0: Palmitic acid; C16:1: Palmitoleic acid; C18:0:
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Table (5): Fatty acids composition of brain total lipids in different animal groups (mg/g tissue).
Fatty acids Control FA AlCl3 Radiation FA
+AlCl3 FA +
Radiation AlCl3+
Radiation
FA + Radiation + AlCl3
C14:0 0.63 ±0.03cd
0.65 ±0.03cd
1.24 ±0.16ab
1.09 ±0.08ab
0.90 ±0.11abc
0.77 ±0.11cd
2.05 ±0.10abcd
0.93 ±0.08ac
C16:0 17.58 ±0.67cd
16.76 ±0.59cd
22.77 ±0.50ab
21.55 ±0.67ab
19.65 ±0.42bc
18.11 ±1.02c
23.26 ±0.41abcd
20.43 ±0.58abcd
C16:1 1.61 ±0.13
1.63 ±0.05
2.01 ±0.13
1.97 ±0.13
1.76 ±0.05a
1.64 ±0.18a
2.66 ±0.36abcd
1.86 ±0.08
C18:0 14.48 ±0.71cd
14.48 ±0.38cd
18.49 ±0.40abd
16.33 ±0.23abc
16.57 ±0.27abc
14.54 ±0.49c
21.50 ±0.66abcd
18.61 ±0.58abd
C18:1 21.15 ±0.23cd
22.01 ±0.23cd
25.52 ±1.28ab
24.86 ±0.84ab
22.46 ±0.68cd
22.21 ±0.47cd
26.30 ±0.60ab
23.52 ±1.28
C18:2 3.99 ±0.24cd
3.68 ±0.19cd
2.03 ±0.35ab
2.19 ±0.23ab
3.42 ±0.25cd
3.83 ±0.23cd
1.58 ±0.28ab
2.88 ±0.12abc
C18:3 0.59 ±0.03cd
0.58 ±0.06cd
0.38 ±0.03ab
0.54 ±0.04ab
0.52 ±0.03c
0.55 ±0.04c
0.27 ±0.04abcd
0.47 ±0.03ab
C20:0 1.26 ± .14cd
1.29 ±0.11cd
3.34 ±0.23ab
3.22 ±0.26ab
2.33 ±0.27abcd
1.79 ±0.20cd
4.01 ±0.42abd
2.68 ±0.23ab
C20:1 1.15 ±0.07cd
1.15 ±0.07cd
0.68 ±0.03ab
0.68 ±0.05ab
0.86 ±0.06ab
1.10 ±0.09cd
0.66 ±0.06ab
0.80 ±0.04ab
C20:4 18.71 ±0.11cd
18.23 ±0.42cd
11.30 ±0.42ab
12.97 ±0.79ab
14.37 ±0.57abc
16.27 ±0.29abcd
7.34 ±0.64abcd
11.43 ±0.42abd
C20:5 0.39 ± 0.04c
0.40 ±0.02cd
0.28 ±0.02ab
0.31 ±0.03b
0.37 ±0.02c
0.36 ±0.06
0.22 ±0.01abd
0.31 ±0.02b
C22:0 1.41 ± 0.13cd
1.32 ±0.11cd
2.99 ±0.47ab
2.77 ±0.37ab
1.36 ±0.11cd
1.76 ±0.15cd
2.38 ±0.59ab
1.88 ±0.18bc
C22:6 15.53 ±1.47cd
16.34 ±1.24cd
7.51 ±0.34ab
10.24 ±0.45ab
14.10 ±1.28cd
15.80 ±0.33cd
5.70 ±0.64abd
12.96 ±0.95bc
C24:0 1.52 ± 0.23
1.31 ±0.02
1.46 ±0.15
1.37 ± 0.06
1.32 ±0.03
1.29 ±0.05
2.06 ±0.09abcd
1.25 ±0.08
Total SFA
36.89 ±1.24cd
35.80 ±0.98cd
50.29 ±1.28abd
46.33 ±0.70abc
42.13 ±1.08abcd
38.25 ±0.66cd
55.27 ±0.14abcd
45.78 ±0.41abc
Total USFA
64.11 ±1.18cd
64.20 ±0.91cd
49.71 ±1.25ab
53.67 ±1.91ab
57.87 ±1.03ab
61.75 ±0.57cd
44.73 ±0.09abcd
54.22 ±0.40abc
Total MUFA
23.91 ±0.11cd
24.79 ±0.33cd
28.21 ±1.34ab
27.51 ±0.73ab
25.09 ±0.68cd
24.94 ±0.33cd
29.62 ±0.32ab
26.18 ±1.28
Total PUFA
40.20 ±1.18cd
40.00 ±0.88cd
21.50 ±0.44abd
26.16 ±1.08abc
32.78 ±1.38abcd
36.77 ±0.46cd
15.11 ±0.23abcdd
28.04 ±1.12abc
Legends as in table (1) Abbreviations as in table (4) DISCUSSION
The present study was carried out to investigate the protective effects of ferulic acid (FA) on AlCl3 and γ-irradiation either alone or in combination againest oxidative stress alterations in rats’ brain tissue.
In our study the effect of AlCl3 and γ-irradiation either alone or together on brain tissue caused a potent histopathological changes.
Mansour et al., J. Rad. Res. Appl. Sci., Vol. 4, No. 4(A)(2011) 1177
Aluminum has been implicated as an etiopathogenic factor in Alzheimer’s disease (AD) in that it is able to alter several biochemical mechanisms including the degradation of the myeloid protein peptides, which suggests in which way these two potentially neurotoxic agents might interact in conditions such as AD (37).
Therefore, the observed incidence of gliosis and cerebral infarction are indicative of brain damage because gliosis or astrocytosis is the brain’s way of reacting to injury, insult, or “something” that should not be there (e.g., a tumor) (38). This may account for the presence of vessels with histological features of thrombosis because an injury to a blood vessel results in the release of platelets and fibrin to form a blood clot to prevent loss of blood. If that mechanism causes too much clotting and the clot breaks free, an embolus is formed (39, 40).
The difficulty in obtaining histological evidence of radiation induced brain changes in patients who have undergone radiation therapy in brain is reflected in the infrequent availability of histological proof of these changes in humans (41, 42). In an organ such as the brain, different topographical regions may have varying susceptibility to ionizing radiation. Radiation induced lesions tend to occur more frequently in the cerebral brain white matter. The main finding in this study is that γ-radiation exposure caused enlarged nucleus, unenlarged cytoplasm and accumulation of fluid in the perivascular. Our present structural results are in accordance with findings that show a strong correlation between functional radiation effects and vascular alterations, which suggests vascular injury, is a major factor that contributes to the pathogenesis of delayed radiation injury in normal tissues (43, 44, 45).
Since b-myeloid protein precursor protein is considered to be a marker of neuronal degeneration (46), a significant deposition of myeloid protein in the brains tissue of the AlCl3 and AlCl3 irradiation treated animals were observed. Atish and Anil, (47) proposed that Chronic Aluminum exposure induces oxidative stress and increases myeloid protein beta levels in vivo.
The present study revelead that Aluminum toxicity induced oxidative stress and biochemical alterations in rats. In AlCl3 treated rats, the level of Malonaldialdahyde (MDA) the end products of lipid peroxidation was found to be elevated but the content of reduced glutathione (GSH) and activities of superoxide dismutase (SOD) and catalase (Cat) were decreased in blood and brain. These observations are similar to the data reported by Yousef (6), Yousef et al., (7, 8), Nehru and Anand (48) who indicated that Aluminum intake produces
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oxidative stress. Although Aluminum is not a transition metal and therefore cannot initiate peroxidation, many investigations have searched for a correlation between Aluminum accumulation and oxidative damage in the brain tissues (48,
49, 50). The primary effects of Aluminum on the brain functions are thought to be mediated via damage to cell membranes.
The increased lipid peroxidation is, at least in part, due to an inhibition of superoxide dismutase (SOD) activity in the blood RBCs and brain. This results in a substantial increase in the rate of lipid peroxidation in plasma and brain, leading to membrane damage. SOD presents the first line of defense against superoxide, as it dismutases the superoxide anion to H2O2 and O2 (48). Because the SOD enzyme generates H2O2, it works in collaboration with H2O2 removing enzymes. Catalase converts H2O2 to water and oxygen. Catalase is present in the peroxisomes of mammalian cells, and probably serves to destroy H2O2 generated by oxidase enzymes located within these sub cellular organelles (48).
In the present study, AlCl3 induced a decrease in GSH content in the blood RBCs and brain. Orihuela et al., (51) reported that at high doses, Aluminum was able to induce oxidative stress in the intestinal mucosa, as indicated by the significant increase in the concentration of both, oxidized glutathione / reduced glutathione (GSSG/GSH) ratio and TBARS levels. These effects may have been produced owing to concomitant causes. Aluminum might affect the glutathione (GSH) synthesis by decreasing the activity of glutathione-synthase (GS), a non-limiting step of whole reaction, thus leading to a reduced GSH content. Since, NADPH is shown to be a main factor for the GSH regeneration, the decreased GSH level could be also ascribed to insufficient supply of NADPH. Besides, Aluminum is able to diminish the activity of enzymes related to cell antioxidant defense, such as superoxide dismutase and catalase in brain and blood. Therefore, Aluminum could indirectly contribute to unbalance redox equilibrium in the enterocyte (51).
Experimentally, AlCl3 administration has been shown to increase acetylcholinesterase in plasma or brain tissue. This conclusion agrees with that observed by other investigators Zatta et al., (52). Several studies have reported the influence of aluminum on the metabolism of acetylcholine (10, 11). This has been attributed to the accumulation of Aluminum in the brain (53).
Exposure to ionizing radiation produces significant alterations in oxidant activity in different tissues (54). Cells exposed to ionizing radiation can develop prolonged genetic instability manifested in multiple always, including
Mansour et al., J. Rad. Res. Appl. Sci., Vol. 4, No. 4(A)(2011) 1179
delayed reproductive death, increased rate of point mutations, chromosome rearrangements and micronuclei (55). In our study, we observed increased level of MDA in plasma and brain tissue of γ-irradiated rats. This may be due to the attack of free radicals on the fatty acid component of membrane lipids (56). The antioxidant enzymes capable of scavenging ROS are SOD, CAT and GPx, etc.
(57). In this study, we have observed a decrease in the activity of SOD and CAT in γ -irradiated rats. This decrease could be due to a feedback inhibition or oxidative inactivation of enzyme protein caused by ROS generation, which in turn can impair the antioxidant defense mechanism, leading to an increased membrane lipid peroxidation (LPO) (58). The decreased level of GSH in γ-irradiated rats may be due to its utilization by the enhanced production of ROS (56). If over production of ROS occurs, oxidative damage can lead to radiation-induced cytotoxicity (chromosomal damage and gene mutations) (59).
On the other hand, acetylcholinesterase activity exhibited a significant increase in both either for plasma and brain tissue as affected by exposure of rats to γ- radiation. Schwenke et al., (60) showed that γ-irradiation of erythroleukemic K562 cells caused an increase in acetylcholinesterase -activity accompanied by cell differentiation and cessation of cell proliferation.
Some decrease in neurotransmitters concentrations of brain tissue was recorded after exposure of the experimental animals to γ-radiation. While an increase in noradrenalin and a decrease of serotonin concentration was recorded when the experimental animals suffered from AlCl3 toxicity either alone or combined with γ-irradiation.
The serotonin level was reported to be reduced (61) in brain regions of rat pups orally gavage with AlCl3 at 40 mg/kg body weight for 4 weeks. In the present study, a decrease in serotonin was evident in brain after 4 weeks of exposure. Beal et al. (62) have suggested that Al-induced neurochemical changes were found in regions with the most neurofibrillary degeneration and found that in rabbits exposed to Al (100 ml 1% Al chloride infused intra ventricular) a significant reduction in serotonin levels was observed.
The serotonin neurotransmitter level in the brain regions varies. Serotonin turnover could vary between the brain regions. However, the toxic effect of Al on the serotonergic system is not uniform. The changes reflect brain-region-specific changes and may be due to the direct toxic effect of Al. The changes observed were duration dependent, which may be due to the deactivation of the serotonergic system and different levels of Al accumulation
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in the different brain regions over a time period (63).
Brain tissue has 60% lipid, and it has remarkable high energy consumption (64). In addition, these tissues are more susceptible to oxidative damage than other tissues (65). Also the nervous system is particularly vulnerable to the deleterious effect of ROS, and one of the main reasons is that the brain contains high concentrations of polyunsaturated fatty acid that are highly susceptible to lipid peroxidation (66, 67). Due to AlCl3 toxicity and irradiation oxidative stress the percentage of saturated fatty acids and Total MUFA increased by AlCl3 administration and γ-radiation exposure.
The ability of ferulic acid ethyl ester (FAEE) to act as a potent antioxidant in vivo, thus providing neuroprotection against myeloid protein beta (Aβ) induced oxidative stress. Perluigi et al., (68) suggested that the ester derivative of ferulic acid (FA) shows higher lipophilicity with increased ability to penetrate the blood–brain barrier. They hypothesize a multifaceted mechanism of in vivo neuroprotection by this compound: 1) FA is a potent free radical scavenger by significantly attenuating ROS production, protein oxidation, and lipid peroxidation; 2) FA is also neuroprotective by leading to elevated levels of stress response proteins, such as HO-1 and HSP72; and 3) FA modulates neuroinflammatory processes mediated by inducible nitric oxide synthase (iNOS) protein.
Administration of FA to γ-irradiated rats resulted in decreased lipid peroxidation and improved antioxidant status preventing the damage to the brain. This may be due to the antioxidant sparing action of FA. As FA is a phenolic compound, it might have diminished lipid peroxidation level. Previous reports have shown that FA is an effective scavenger of free radicals and it has been approved in certain countries as food additive to prevent LPO (69). Toda et al. (70) have also reported that FA scavenges superoxide anion radical and inhibits LPO induced by superoxide and the effect of FA is similar to that of SOD. The level of GSH was increased significantly due to pretreatment of rats with FA. As seen in table 1 and 2, FA possesses distinct structural motifs that can possibly contribute to the free radical scavenging capability of this compound. The presence of electron donating groups on the benzene ring (3-methoxy and more importantly 4-hydroxyl) of FA gives additional property for terminating free radical chain reaction. The next functionality – the carboxylic acid group in FA with adjacent unsaturated C-C double bond can provide additional attack sites for free radicals and thus prevent them from attacking the membrane. In addition, the carboxylic acid group acts as an
Mansour et al., J. Rad. Res. Appl. Sci., Vol. 4, No. 4(A)(2011) 1181
anchor of FA by which it binds to the lipid bilayer providing some protection against LPO. Clearly, the presence of electron donating substituents enhances the antioxidant properties of FA (71).
Pretreatment of irradiated rats with FA enhanced the activity of SOD and CAT. These antioxidant enzymes are important in providing protection from radiation exposure; the proper balance of these enzymes in specific cells and in the whole organ is required for maximum radioprotection (72).
In conclusion FA usage can protect brain tissue against AlCl3 toxicity and γ-irradiation hazardous effects.
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الإشعاعیةالإشعاعیةبحوث بحوث مجلة المجلة ال والعلوم التطبیقیةوالعلوم التطبیقیة
)٢٠١١( ١١٨٨ – ١١٦٣ ص ص )أ(٤ عدد ٤ مجلد
نسجة مخ ذكور الجرذانأسبب فى تعرض تتالألمنیوم وأشعة جاما الفریولیك الدور الوقائى لحمض – ىللضررالتأكسد
إیمـان نعمـــانـ نعمات حنفي أحمـد ـ ســـــــمیة زكریـــا منصـور
القاھرة – الذریة الطاقة ھیئھ – الإشعاع المركز القومي لبحوث و تكنولوجیا – ةالإشعاعی البیولوجیابحوث قسم مصر–
تحقق من الدور المحتمل لحمض الفریولیك ضد الضرر التأكسدى للقد أجریت الدراسة الحالیة وكانت فترة . ذانالجرالناتج من استعمال كلورید الألومنیوم والتعرض لإشعاع جاما في أنسجة مخ
یوم من / كجم / ملیجرام ٨٫٥وكانت تدار بواسطة معاملة حیوانات التجارب بجرعة . أسابیع٨التجربة وكانت جرعة حمض . من الإشعاع الجامى) جراى٤(كلورید الألومنیوم والتعرض لجرعة واحدة
میلوید في أنسجة المخ وتم اβسجلت الملاحظات المرضیة وتوزیع . یوم/ كجم / ملیجرام ٥٠الفریولیك أوضحت أنسجة المخ للفئران المعاملة بكلورید . الأكسدة بعد انتھاء كل المعاملات تسجیل ضرر
مجتمعة تغییرات ضارة كثیرة وتغیر توزیع البروتین منفردة أولأشعة جاما أو المعرضة الألومنیومالنخاعي وانخفض تركیز السیروتونین وسجلت الدراسة زیادة في محتوى المالونالدھید والنسبة المئویة
للأحماض الدھنیة المشبعة الموجودة في البلازما وأنسجة المخ وأظھرت انخفاضا كبیرا فى الجلوتاثیون مثلت معاملة الحیوانات التجریبیة بحمض الفریولیك .الفائق في كل من الدم والمخوالكاتلاز و دیسموتاز
ومع ذلك أظھرت معاملة ). امیلویدβ (البروتین النخاعي مظھر طبیعي من بنیة أنسجة المخ وتوزیع الحیوانات بكلورید الألومنیوم و حامض الفریولیك ضمور بسیط في شكل مورفولوجیا منكمشة تمثلت في
وحدوث زیادة كبیرة في تركیز البروتین النخاعيالتصلب الجانبي الضموري وانخفاض في ترسیب السیروتونین وتحسن في علامات الأكسدة ونشاط الأستیل كولین ایسـتیرز والنسبة المئویة للأحماض
ام حمض وقد اسـتخلصت الدراسة إلى أن استخد. الدھنیة المشبعة الموجودة في البلازما وأنسجة المخالفریولیك یحد من الإجھاد التأكسدى الناتج من معاملة الجرذان بكلورید الألومنیوم إضافة إلى تعرضھا
