ameliorative effect of salicin against gamma irradiation induced

UK Journal of Pharmaceutical and Biosciences Vol. 3(2), 29-41, 2015 RESEARCH ARTICLE

Ameliorative Effect of Salicin Against Gamma Irradiation Induced Electrophoretic

Changes in Brain Tissue in Male Rats

Mohga Shafik Abdalla, Hayat Mohamed Sharada, Ibrahim Abulyazid, Monira Abdel-Latif Abdel-Karim,

Wael Mahmoud Kamel*

Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Giza, Egypt-12622

Article Information

Received 10 Feburary 2015

Received in revised form 27 April 2015

Accepted 28 April 2015

Abstract

The aim of this study was to investigate the radioprotective effect of salicin against irradiation

effect on brain tissue of male rats. Lipid peroxidation level was measured as thiobarbituric acid

reactive substance in brain tissue. The polyacrylamide gel electrophoresis for native protein,

lipoprotein and zymogram were carried out in brain homogenate. As expected, salicin resisted

the irradiation effect and declined the MDA level in brain homogenate of all treated groups

(especially in the irradiated salicin post-treated group). Salicin minimized the qualitative

mutagenic effect of irradiation on the electrophoretic protein pattern in all irradiated salicin

treated groups and it showed the highest antagonistic effect against irradiation in irradiated

salicin post-treated group (SI = 0.57). It could not prevent the abnormalities occurred qualitatively

and quantitatively as a result of irradiation in lipoprotein pattern in all irradiated salicin treated

groups. In the electrophoretic esterase pattern, salicin prevented the qualitative effect of

irradiation in irradiated salicin post-treated group (SI = 1.00). Salicin minimized the qualitative

irradiation effect on the catalase pattern in the irradiated salicin pre-treated group (SI = 0.73).

While in the peroxidase pattern, salicin adminstration resisted the irradiation effect in the

irrradiated post-treated groups (SI = 0.67). The results suggested the radioprotective ability of

salicin against gamma irradiation effect on various electrophoretic patterns in brain tissue of male

rats.

Keywords:

Gamma irradiation,

Salicin,

Brain,

Protein electrophoresis,

Isozymes

*Corresponding Author:

E-mail: [email protected]

Tel.: 00201126682686

1 Introduction

The use of this radiation as a source to enhance the mutation

frequency was recognized as early as 19301.These rays act either

directly or by secondary reactions to produce biochemical lesions

that initiate series of physiological symptoms. They are known to

induce oxidative stress through the generation of reactive oxygen

species (ROS) resulting in imbalance of the prooxidant and

antioxidant activities, ultimately resulting in cell death2.Studies

showed that radiation can affect a wide variety of tissues, particularly

those with greater levels of cellular turnover and divisions3.

Irradiation causes similar damage at a cellular level but gamma rays

are more penetrating, causing diffuse damage throughout the body4.

This type of rays also used for diagnostic purposes in nuclear

medicine in imaging techniques5.

Irradiation causes damage to living tissue through a series of

molecular events. The formation of ROS as a result of interaction of

irradiation with cellular macromolecules is the cause of dysfunction

and death, in both normal as well as tumor cells exposed to

radiation6,7

.The energy exchange between the rays and the targeted

molecules leads to changes produced in deoxyribonucleic acid

(DNA), lipids, and proteins and then cell inactivation8,9

.

Gamma irradiation was found to interrupt energy supplies and

blocking all key enzymes, which may stop normal metabolism of the

exposed tissue10

. The radiation exposure whether occupational or

during radiotherapy leads to serious systemic damage to various

cellular and subcellular structures11

.

Irradiation causes induction of lipid peroxidation as evidenced by

increased malondialdhyde (MDA)12

. It causes damage of cells

UK Journal of Pharmaceutical and Biosciences

Available at www.ukjpb.com ISSN: 2347-9442

Kamel et al. Ameliorative Effect of Salicin Against Gamma Irradiation

UK J Pharm & Biosci, 2015: 3(2); 30

directly by ionizing DNA and other cellular targets and indirectly by

effect through ROS13

. It produces oxygen-derived free radicals in

tissue environment: these include hydroxyl radicals (the most

damaging), superoxide anion radicals and other oxidants such as

hydrogen peroxide14

.

Major radiation damage is due to the aqueous free radicals

generated by the water radiolysis. These free radicals act as

molecular marauders and in turn damage DNA which is considered

to be the primary target15

.This gives rise to genomic instability

leading to mutagenesis, carcinogenesis and cell death16

.

The most important consequences of OS are lipid peroxidation,

protein oxidation and depletion of antioxidants17,18

. The latter authors

showed thatthe increase of MDA level is probably due to the

interaction of •OH resulting as a bi-product of water radiolysis with

the polyunsaturated fatty acids presents in the phospholipids portion

of cellular membranes. The excessive free radicals can damage

crucial macromolecules, including DNA, cell membranes and

enzymes, and can cause cell death. DNA damage includes

genotoxicity, chromosomal abnormalities, gene mutations and cell

death if the damage is beyond repair19,20

.

Catalase (CAT) and glutathione peroxidase (GPx) play an important

role in detoxification of hydrogen peroxide and ROS21

(Kula et al.,

2000). The irradiation caused significant increase in markers of the

oxidative stress as malondialdehyde (MDA) and alterations in activity

of antioxidant enzymes such as CAT and GPx in a dose-dependent

manner22,23

.

Salicin was discovered in 1831. It was isolated from the willow bark

and leaves. It is a prodrug and thus a precursor of salicylic

acid24

.Salicin is a phenolic glycoside. It exhibits analgesic effects,

and it is used for the treatment of rheumatic pain. Its occurrence in

willow (Salix) species is the major reason willow bark and its extracts

are popular products. High-Performance Liquid Chromatography

(HPLC) has been the standard method of analysis of quantifying

salicin in the different Salix species25

.

The present experiment was concerned with studying the ability of

salicin which represents the most abundant fraction of aqueous

willow extract to impress radiation-induced electrophoretic alterations

in brain tissue of male rats.

2 Materials and Methods

2.1 Salicin isolation

Fresh young leaves of the willow trees (Salix subserrata, Salix

safsaf) were collected from Orman garden, Giza, Egypt. This species

was well authenticated by qualified specialists in plant taxonomy.

Salicin was extracted and isolated from fresh leaves according to

method described by Mabry et al26

and purified according to method

suggested by Kur'yanov et al27

then identified qualitatively by

traditional and advanced chromatographic techniques.

2.2 Acute toxicity test

The lethal dose 50 (LD50) was evaluated on 6 groups of female

albino mice (8 animals / group) receiving progressively increasing

oral dose levels (500, 1000, 2000, 3000, 4000 and 5000 mg/kg body

weight) of aqueous solution of salicin solution. Mortality was

recorded 24 hrs post treatment. The LD50 was calculated according

to the equation suggested by Paget and Barnes28

.

2.3 Animals

Seven groups of male rats weighing between 150-200 gm per one

obtained from the animal house laboratory of the national research

centre. Ten rats in each group. All the animals were kept under

normal environmental and nutritional conditions. The animal groups

were divided into Control group: Rats were non-irradiated and non-

treated with salicin, Salicin treated group: Rats were non-irradiated

but treated with the safe dose of salicin which was about 150 mg /

Kg taking in the consideration weight of each rat, Irradiated group:

Rats were irradiated at the dose 7 Gy and non-treated with salicin,

Irradiated salicin pre-treated group: Rats were treated with salicin for

15 days followed by irradiation at the 15th day, Irradiated salicin

prepost-treated group: Rats were treated with salicin for 15 days

followed by irradiation at the 15th day then the treatment was

continued daily for another 15 days, Irradiated salicin simultaneous

treated group: Rats were irradiated and treated with salicin at the

same time of irradiation and continue daily for 15 days and Irradiated

salicin post-treated group: Rats were irradiated at the same gamma

dose then left without treatment for 15 days. At the 15th day, the rats

were treated with salicin for another 15 days.

2.4 Irradiation

Whole body was gamma irradiated at Middle Eastern Regional

Radioisotope Centre for the Arab Countries, Dokki, Egypt. Using

Cobalt 60 (Co 60

) as a suitable gamma source. Rats were irradiated

at a single dose of 7 Gy delivered at the dose rate of 1.167 Rad /

Sec.

2.5 Biochemical Assay

Lipid peroxidation level was measured as thiobarbituric acid reactive

substance in brain homogenate according to method of Ohkawa et

al29

.

2.6 Electrophoretic protein pattern

Total protein was determined in brain homogenate according to

Bradford30

.The sample was mixed with the sample buffer. The

protein concentration in each well must be about 70 μg protein.

Proteins were separated through polyacrylamide gel electrophoresis



(PAGE) with different concentrations. Electrode and gel buffer and

polyacrylamide stock were prepared according to Laemmli31

. After

electrophoretic separation, the gel was gently removed from the

apparatus and put into a staining solution of coomasie brilliant blue

for native protein pattern32

and staining solution of sudan black B

(SBB) for lipoprotein pattern33

.

2.7 Electrophoretic isozyme

Native protein gel was stained for peroxidase pattern using certain

stain prepared according to the method suggested by Rescigno et

al34

. It was stained for catalase pattern according to the method

described by Siciliano and Shaw35

.For esterase pattern, the native

gel was stained according to the method suggested by Baker and

Manwell36

.

2.8 Data analysis

The polyacrylamide gel plate was photographed, scanned and then

analyzed using Phoretix 1D pro software (Version 12.3). The

similarity index (S.I.) compares patterns within, as well as, between

irradiated and non-irradtated samples. The similarity values were

converted into genetic distance (GD) according the method

suggested by Nei and Li37

.

2.9 Statistical analysis

All the grouped data were statistically evaluated with SPSS/16.00

software. The results were expressed as mean ± SE of studied

groups using the analysis of variance test (one-way ANOVA)

followed by student’s t-test34

. P values of less than 0.05 were

considered to indicate statistical significance. The means of

irradiated groups and the salicin treated groups were individually

compared with those of control group. The irradiated group was

compared with irradiated salicin treated groups

3 Results and Discussions

3.1 Lipid peroxidation

As compared to control, irradiation caused the severe increase in the

lipid peroxidation product (MDA) level in the brain tissue. Salicin

administration showed the ameliorative effect against irradiation by

reducing MDA level in all irradiated salicin treated rats. From the

data compiled in table 1, it was found that salicin showed the most

suitable antagonistic effect against irradiation on brain of irradiated

salicin post-treated group.

3.2 Electrophoretic protein pattern

Protein pattern in the control sample produced 9 bands with Rfs

ranged between 0.09 – 0.98 (Mwts 5.21 – 212.46 KDa and B %

values 0.20 – 25.09). There were no common bands but there were

2 characteristic bands appeared in irradiated salicin pre-treated

group with Rf 0.56 (Mwt 20.09 KDa and B % 21.13) and in irradiated

salicin simultaneous treated group with Rf 0.75 (Mwt 14.58 KDa and

B % 10.85).

As shown in table 2 and illustrated in fig. 1, irradiation caused

disappearance of 6 normal bands without appearance of abnormal

bands. The 3rd and 5

th bands might be deviated to be appeared with

Rf 0.22 and 0.43 (Mwts 113.48 and 30.53 KDa and B % 17.28 and

80.91). It caused quantitative mutation represented by increasing B

% of the 9th band (Rf 0.98, Mwt 5.02 KDa and B % 1.81). Salicin

administration resisted the qualitative mutagenic effect of irradiation

in the irradiated salicin simultaneous treated and post-treated

groups. It retained 4 normal bands with Rfs ranged between Rf 0.23 -

0.98 (Mwts 5.08 - 112.21 KDa and B % 0.20 - 15.81) in irradiated

salicin simultaneous treated group and with Rf 0.45 - 0.99 (Mwts 4.75

- 27.06 KDa and B % 0.59 - 56.41) in irradiated salicin post-treated

group. It was probable that the 5th band was deviated to be appeared

with Rf 0.45 (Mwt 28.20 KDa and B % 37.70) in irradiated salicin

simultaneous treated group. It could not prevent the quantitative

mutation which was represented by increasing B % of the 3rd and 7

th

bands (Rfs 0.23 and 0.85, Mwts 112.21 and 10.65 KDa and B %

15.81 and 14.00) and by decreasing B % of the 6th band (Rf 0.63,

Mwt 18.00 KDa and B% 11.99) in irradiated salicin simultaneous

treated group. Also, it could not prevent the quantitative mutation

which was represented by increasing B % of the 5th, 6

th, 7

th and 9

th

bands (Rfs 0.45, 0.65, 0.86 and 0.99 (Mwts 27.06, 17.68, 10.38 and

4.75 KDa and B % values 56.41, 0.59, 34.49 and 1.54) respectively.

It was found that the lowest SI value (SI = 0.17) was recorded with

irradiated group and the highest SI value (SI = 0.70) with salicin

treated group. Salicin improved the SI values in all irradiated salicin

treated groups, and it showed the highest antagonistic effect against

irradiation in irradiated salicin post-treated group (SI = 0.57). The

overall results showed that salicin prevented the irradiation effect on

number and arrangement of the bands in all irradiated salicin treated

groups.

3.3 Electrophoretic lipoprotein pattern

As revealed in table 3 and illustrated in fig. 2, lipoprotein pattern in

the control sample produced 3 bands with Rfs 0.22, 0.47 and 0.69 (B

% 67.28, 18.91 and 13.82). There were no common bands appeared

in all groups. Salicin alone caused severe qualitative alterations

represented by disappearance of the normal bands with appearance

of 3 abnormal bands with Rf 0.25, 0.33 and 0.72 (B % 48.27, 31.12

and 20.61).

Irradiation caused severe abnormalities represented disappearance

of all the normal bands without appearance of abnormal bands in the

irradiated, irradiated salicin simultaneous treated and post-treated

groups. Salicin adminstration could not prevent the irradiation effect



which was represented qualitatively by disappearance of 2 normal

bands and quantitatively by increasing B % of the normal band

appeared with Rf 0.22 and B % 100.00 in the irradiated salicin pre-

treated group and by disappearance of 2 normal bands with

appearance of one abnormal band with Rf 0.35 (B % 29.73) in the

irradiated salicin prepost-treated group. It was observed that all the

bands were not matched with all bands of the other groups in the

salicin treated, irrradiated, irradiated salicin simultaneous treated and

post-treated groups.

Table 1: Effect of irradiation, salicin and their combination in various treatment modes on the level of lipid peroxidation in spleen

tissue of male rats

Organ Control Sal. Irr.

Irradiated salicin treated groups

Pre-treated Simultaneous Prepost-treated Post-treated

Brain

(nmol/g)

162.02

± 2.28

137.84a

± 2.17

239.15a

± 6.55

232.90a

± 6.24

227.21a

± 4.78

228.10a

± 7.95

165.24a,b

± 2.60…

a : Different from control at P < 0.05, b : Different from the irradiated group at P < 0.05.

Note : Sal. ; salicin, Irr. : Irradiated

3.4 Electrophoretic esterase pattern

The electrophoretic esterase pattern in control of brain tissue

produced 3 types with Rfs 0.22, 0.48 and 0.64 (B % 36.85, 45.38 and

17.77). As shown in table 4 and illustrated in fig. 3, there were no

common bands in all groups. Salicin alone caused qualitative

mutation represented by appearance of one abnormal characteristic

band with Rf 0.11 (B % 21.93) and decreasing B % of the 2nd

normal

type (Rf 0.49 and B % 19.00). Irradiation caused disturbances

represented qualitatively by disappearance of 2 normal types of the

enzyme and quantitatively by increasing B % of the 1st normal band

(Rf 0.21 and B % 100.00).

Salicin administration prevented the qualitative or quantitative

alterations occurred as a result of irradiation in the irradiated salicin

post-treated group. It could not prevent the irradiation effect which

was represented qualitatively by deviation of the 3rd type to be

appeared with Rf 0.60 (B % 23.33) and quantitatively by increasing B

% of the 1st band (Rf 0.21 and B % 51.38) and decreasing B % of the

2nd

type (Rf 0.47 and B % 25.29) in the irradiated salicin pre-treated

group, represented by disappearance of the 3rd type with deviation of

the 1st and 2

nd normal types of the enzyme to be appeared with Rfs

0.23 and 0.48 (B % 55.23 and 44.77) in the irradiated salicin

simultaneous treated group and represented by deviation of the 2nd

and 3rd normal types of the enzyme to be appeared with Rfs 0.45 and



0.62 (B % 38.34 and 21.20) in the irradiated salicin prepost- treated

group.

Fig.1:Electrophoretic pattern showing effect of salicin against

the irradiation effect on protein pattern in brain tissue of male

rats

Fig. 2:Electrophoretic pattern showing effect of salicin against

the irradiation effect on lipoprotein pattern in brain tissue of

male rats

Table 3: Data of the electrophoretic lipoprotein pattern in brain tissue of control, irradiated and irradiated salicin treated groups in

male rats

Control Salicin Irradiated

Irradiated salicin treated


Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. %

0.22 67.28 0.25 48.27 — — 0.22 100.00 — — 0.22 70.27 — —

0.47 18.91 0.33 31.12 — — — — — — 0.35 29.73 — —

0.69 13.82 0.72 20.61 — — — — — — — — — —

Rf.: Rate of Flow, B.%: Band Percent

Table 4: Data of the electrophoretic esterase pattern in brain tissue of control, irradiated and irradiated salicin treated groups in male

rats





0.22 36.85 0.11 21.93 0.21 100.00 0.21 51.38 0.23 55.23 0.21 40.46 0.22 40.27

0.48 45.38 0.22 40.13 — — 0.47 25.29 0.48 44.77 0.45 38.34 0.47 36.92

0.64 17.77 0.49 19.00 — — 0.60 23.33 — — 0.62 21.20 0.62 22.81

— — 0.64 18.94 — — — — — — — — — —




It was found that the highest SI value (SI = 1.00) was noticed in

irradiated salicin post-treated group. In the irradiated salicin

simultaneous treated group, it was observed that all the bands were

not matched with all bands of the other groups.


the irradiation effect on esterase pattern in brain tissue of male

rats

3.5 Electrophoretic catalase pattern

As shown in table 5 and illustrated in fig. 4, 5 types of catalase

enzyme were produced in the control sample with Rfs ranged

between 0.21 - 0.97 (B % 8.11 – 47.12). There were 2 common

bands appeared in all the groups with Rfs 0.45 and 0.97 (B % 8.47

and 15.17). Salicin alone caused alterations represented by

appearance of one abnormal band with Rf 0.18 (B % 23.47) with

decreasing the B % of the 1st type (Rf 0.22 and B % 6.12) and

increasing the B % of the 2nd

type (Rf 0.44 and B % 25.89).

Irradiation caused qualitative alterations represented by

disappearance of the 1st type of the enzyme with deviation of the 3

rd

band to be appeared with Rf 0.78 (B % 15.01) and quantitative

mutation occurred by increasing B % of the 2nd

band (Rf 0.45 and B

% 64.42).

Salicin administration could not prevent the irradiation effect which

was represented by alteration occurred qualitatively by appearance

of one abnormal band with Rf 0.15 (B % 29.71) with deviation of the

3rd type of the enzyme to be appeared with Rf 0.82 (B % 28.32) and

quantitatively by decreasing B % of the band appeared with Rf 0.22

(B % 9.44) in the irradiated salicin pre-treated group, by

disappearance of the 1stand 4

thtypes of the enzyme with appearance

of one abnormal band with Rf 0.17 (B % 31.52) and deviation of the

3rd type to be appeared with Rf 0.82 (B % 38.60) in the irradiated

salicin prepost-treated group and represented by disappearance of

the 1st type with appearance of one abnormal band with Rf 0.12 (B %

20.99) deviation of the 3rd type to be appeared with Rf 0.83 (B %

19.39) in the irradiated salicin simultaneous treated group. While in

the irradiated salicin post-treated group, salicin could not overcome

the mutation represented qualitatively by disappearance of 3 normal

types with appearance of one abnormal band with Rf 0.66 (B %

57.05) and quantitatively by increasing B % of the bnds appeared

with Rfs 0.45 and 0.96 (B % 20.65 and 22.30).

From the SI values, it was found that the lowest SI value (SI = 0.44)

was observed with irradiated salicin prepost-treated group and the

highest value (SI = 0.91) observed with salicin treated group. Salicin

treatment minimized the qualitative irradiation effect in the irradiated

salicin pre-treated group (SI = 0.73) more than the other irradiated

salicin treated groups.

3.6 Electrophoretic peroxidase pattern

As shown in table 6 and illustrated in fig. 5, it was found that 5 types

of peroxidase enzyme were produced in the control sample with Rfs

ranged between 0.28 - 0.72 (B % 0.46 - 41.66). There were no

common band appeared in all groups. Salicin alone caused no

obvious qualitative or quantitative alterations. Irradiation caused

severe disturbances represented qualitatively by disappearance of

the 2nd

, 3rd and 5

th normal types of the enzyme and quantitatively by

increasing B % of the 1st type (Rf 0.28 and B % 58.28). Salicin could

not remove the abnormalities which were represented qualitatively

by disappearance of the 1st, 3

rd and 5

th types with increasing B % of

the 2nd

and 4th normal types of the enzyme (Rfs 0.35 and 0.58 and B

% 34.34 and 65.66) in irradiated salicin pre-treated group and

represented qualitatively by disappearance of the 3rd type of the

enzyme with appearance of one abnormal characteristic band with Rf

0.69 (B % 44.83) and quantitatively by decreasing the B % of the 1st

type (Rf 0.27 and B % 14.08) and increasing B % of the 2nd

and 4th

types of the enzyme (Rfs 0.38 and 0.69 and B % 25.82 and 44.83) in

irradiated salicin prepost-treated group. In the irradiated salicin post-

treated group, salicin could not prevent the mutagenic effect which

was represented qualitatively by disappearance of the 5th type and

deviation of the 4th type to be appeared with Rf 0.56 (B % 61.82) and

quantitatively by decreasing B % of the 1st type appeared with Rf

0.27 and 14.47.

From the SI values, it was found that salicin minimized the irradiation

effect on the band number and arrangement in the irradiated salicin

prepost-treated groups (SI = 0.44) and post-treated groups (SI =

0.67). In the irrradiated pre-treated group, it was observed that all the

bands were not matched with all bands of the other groups. It

showed the highest antagonistic effect in the irradiated salicin post-

treated group.

4 Discussions



During results of the present study, the MDA level elevated

significantly as a result of radiation exposure in the brain tissue. This

was in accordance with the results obtained by Saada and Azab39

that showed that the MDA level increased due to production of ROS

associatedwith the increase in lipid peroxidation. ROS are known to

attack the highly unsaturated fatty acids of the cell membrane to

induce peroxidation reactions, which considereda key process in

many pathological events and is one ofthe reactions induced by

oxidative stress40

. The increasein intracellular ROS concentration

leads subsequently tooxidative stress41

and decrease in activity of

antioxidantenzymes with possible damage of cellular membranes42

.

The present study showed that irradiation increased the MDA level.

This was in agreement with Dixit et al 43

who showed that the doses

2, 6 and 10 Gy of irradiation enhanced the MDA level. This may be

due to reducing the antioxidant enzymes as superoxide dismutase,

catalase and glutathione-S-transferase.

It was demonstrated that brain belonged to the most biosensitive

organs to low doses of irradiation in rats44

. The oxidative stress

affects some brain regions more than others45

.

Irradiation induced lesions tend to occur more frequently in the

cerebral brain white matter. It caused necrosis in the cortex and

subcortical area in the brain46

. This because white matter tissue is

more affected than gray matter tissue47

. This means that the brain

represents one of the most important targets of irradiation. So, it was

selected to be under study during the current experiment.

The elevation of the levels of antioxidant enzymes in the irradiated

animals when compared with the control suggests that these

enzymes were up regulated to respond to the toxicity induced by the

radiation48

.

Proteins are the most complex compounds and at the same time the

most characteristic of living matter. They are present in all viable

cells; they are the compounds which, as nucleoproteins, are

essential for cell division and, as enzymes and hormones, control

many chemical reactions in the metabolism of cells. Thus, the

separation and characterization of the individual proteins facilitate

the study of the chemical nature and physiological function of each

protein49

.

Changes in the protein patterns of the tissues may reflect

specialization and adaptation in the organisms. It is worthy to note

that each protein is considered as reflect to the activity of specific

gene through the production of enzyme, which act as a catalyst to

produce the demanded protein; this type of produced protein is

responsible for a specific biological character50

. Proteins are major

targets for oxidative damage due to their abundance and rapid rates

of reaction with a wide range of radicals and excited state species51

.

It is worthy to note that each protein type has a biological role, due to

this role, the DNA secrets enzymes which act as catalysts to produce

the specific type of protein. Oxidative protein damage could also

affect the activity of DNA repair enzymes. Another possible

mutagenic effect of ROS involves their attack on lipids, to initiate lipid

peroxidation. The peroxides can decompose to a range of mutagenic

carbonyl products52

.

The proteins in brains of all rats were separated electrophoretically

and abundances were measured by using image analysis technique.

Data in the present study indicated that specific protein bands in

tissues of the irradiated rats differed (through disappearence in some

protein bands or appearance of new ones). Disappearance of some

protein bands in treated rats may be attributed to the effects of

irradiation, which inhibits the synthesis and expression process of

these deleted proteins (qualitative effect). In addition, even the band

remained after irradiation, it usually differs in the amount of protein,

and this may be explained by that irradiation could not inhibit the

synthesis of this protein type, but it may be affected only on the

quantitative level.

The representative electrophoretic profile of the proteins was carried

out in serum and different tissue homogenates. This profile showed

different types of mutations occurred as a result of radiation

exposure. As compared to control, some normal proteins were

represented by the fastest migrating band and had the highest

staining intensity. There were some proteins showed the

intermediate bands and the slowest migrating bands. On the other

hand, there was type of proteins represented as the biggest protein

band close to the well comb (origin). All these proteins differ from

each other in the mobility, molecular weight, optical density and band

intensity of each protein band.

In the present study, the similarity index between the control and all

the irradiated samples and between the irradiated samples

themselves recorded low values, indicating to apparent effect of the

irradiation and the differences in the protein pattern. It was stated by

many previous studies that the irradiation created a great genetic

distance between the control and the irradiated samples that may be

due to the activation of some genes. These genes produce different

types of proteins not produced in the control. These protein types

may lead to variation of the different biological processes.

The current study showed that there were different mutations was

detected by the appearance of new proteins or by the quantitative

decrease in abundance of normally occurring proteins. This was in

agreement with the results reported by Giometti et al 53

who reported

that the electrophoresis can be used to detect the mutations

reflected as quantitative changes in the protein expression.



Table 5: Data of the electrophoretic Catalase pattern in brain tissue of control, irradiated and irradiated salicin treated groups in male

rats





0.21 47.12 0.18 23.47 0.45 64.42 0.15 29.71 0.12 20.99 0.17 31.52 0.45 20.65

0.45 8.47 0.22 6.12 0.78 15.01 0.22 9.44 0.45 41.66 0.44 13.82 0.66 57.05

0.85 21.13 0.44 25.89 0.92 8.84 0.45 9.96 0.83 19.39 0.82 38.60 0.96 22.30

0.92 8.11 0.85 26.36 0.97 11.74 0.82 28.32 0.92 6.94 0.97 16.07 — —

0.97 15.17 0.93 7.04 — — 0.91 9.16 0.95 11.02 — — — —

— — 0.97 11.12 — — 0.97 13.41 — — — — — —


Table 6: Data of the electrophoretic peroxidase pattern in brain tissue of control, irradiated and irradiated salicin treated groups in

male rats





0.28 35.62 0.27 35.91 0.28 58.28 0.35 34.34 0.27 11.68 0.27 14.08 0.27 14.47

0.36 12.62 0.37 11.01 0.59 41.72 0.58 65.66 0.36 28.20 0.38 25.82 0.37 14.82

0.49 9.63 0.47 12.02 — — — — 0.57 60.12 0.58 15.28 0.48 8.89

0.59 0.46 0.60 0.63 — — — — — — 0.69 44.83 0.56 61.82

0.72 41.66 0.72 40.44 — — — — — — — — — —



the irradiation effect on catalase pattern in brain tissue of male

rats


the irradiation effect on peroxidase in brain tissue of male rats



During the current experiment, irradiation-dependent accumulation of

oxidatively modified proteins was studied in the brain tissues.

Alterations in the protein increased as a result of irradiation. This

may be related to an extensive modification of lysine and arginine

residues in histone molecules54

. The authors observed activation of

histone-specific proteases in the nuclei of γ-irradiated rats. The lack

of carbonyl accumulation in the nuclear proteins isolated from tissues

of γ-irradiated animals may be explained by the degradation of

oxidized histones by these proteases.

All lipoproteins carry all types of lipid, but in different proportions, so

that the density is directly proportional to the protein content and

inversely proportional to the lipid content55

. The lipoproteins were

more susceptible to oxidative modifications resulting in small

lipoproteins56

. The ROS can initiate one-electron oxidation or one-

electron reduction reactions on numerous biological systems. The

oxidative hypothesis classically admits the involvement of the

lipoproteins oxidation radiolytically57

. There was natural binding

between protein and lipoproteins in the rat tissues. These two tissues

known to be involved in the processing of the lipoproteins. The

lipoproteins-binding protein has previously been identified in adrenal

cortical plasma membranesandconcentration of the binding protein

was strongest in kidneys58

. So the alterations in the protein pattern

were associated with altering the lipoprotein pattern in these tissues.

The alterations in the lipoprotein pattern may refer to the

disturbances in the cholesteryl esterase required or cholesterol

hydrolysis. It was suggested that non-parenchymal liver cells

possess the enzymic equipment (cholesteryl esterase) to hydrolyze

very efficiently internalized cholesterol esters and this supported that

these cell types are an important site for lipoprotein catabolism59,60

.

Salicin administration showed the protective effect against the

irradiation. This may be due to its antioxidative effect against attack

of the free radicals. It prevented the alterations in the proteins and

hence the lipoproteins fractions in the brain tissues.

In the current experiment, polyacrylamide gel electrophoresis was

used for separation of different enzymes, which help in explanation

of different biological processes that occur inside the living

organisms due to the deleterious effect of irradiation and the

ameliorated effect of salicin.

According to the present data, the activities of CAT and GPx in

brains of irradiated rats were altered. These results strongly

suggested that irradiation has the capability to induce free radicals

and oxidative damage as evidenced by perturbations in various

antioxidant enzymes. Depletion of these enzymes activity could be

due to the direct effect on the enzymes by irradiation-induced ROS

generation, direct inhibition of the enzymes by irradiation61

.

The present work evaluated the enzymatic activity of the AO

enzymes enzymes as CAT and GPx and electrophoretic detection of

oxidative damage in proteins (carbonyl groups) in brains of rats. This

was in agreement with Ehrenbrink et al62

. The authors suggested

that the patterns of activity and accumulation of damages can be

sex-specific and related to the cycle of reproductive life of the rats.

Esterases with active thiol groups detected electrophoretically in

brain of different animal species. It exhibits wide distribution among

these species63

.The brain was selected to show the esterase activity

because it is rich in esterase enzyme due to its role in the

neurotransmission and communication of messages64

.

Electrophoresis plays a major role in identifying esterases. So, this

technique was selected to identify the esterase types in male and

female reproductive organs. The present study showed that

irradiation caused alterations in the electrophoretic esterase pattern

representing great differences in a number of zones of esterase

activity and in substrate specificity between all treated groups

compared to control.

During the current the experiment, irradiation caused alterations in

the electrophoretic esterase pattern. This may refer to effect of

irradiation on the protein pattern. As regards changes in

electrophoretic mobility demonstrated in the present study, it seemed

that free radicals affect the integrity of the polypeptide chain in the

protein molecule causing fragmentation of the polypeptide chain due

to sulfhydral-mediated cross linking of the labile amino acids as

claimed by Bedwell et al65

. The changes in the fractional activity of

different isoenzymes seemed to be correlated with changes in the

rate of protein expression secondary to DNA damage initiated by

free radicals66

.

During the current experiment, salicin administration showed the

highest ameliorative effect against irradiation in the irradiated salicin

treated rats. This may be due to trapping of these free radicals by

salicin, thus preventing DNA damage. Salicin and salicylic acid

belonged to the phenolic compounds which showed AO activity due

to their ability to scavenge free radicals67

.So, they are able to

overcome the disturbances in the esterase pattern in the tissues

selected to be under study.

Salicin belonged to the phenolic glycosides which are characterized

by their antioxidant activity in biological systems. The antioxidant

activity of the phenolic compounds refers to their ability to scavenge

free radicals67,68

. The authors suggested that the phenolic molecules

undergo redox reactions because phenolic hydroxyl groups readily

donate hydrogen to reducing agents. Ronca et al68

showed that

antioxidants suppressed hydroxyl radical production in the fenton

reaction, probably by chelating the iron required in the generation of

hydroxyl radicals. Results of the present study were in agreement



with that reported by Das et al69

who confirmed that elevation of MDA

concentration might be a consequence of decreased production of

antioxidants in the irradiated rats’ tissues. The antioxidant activities

of salicin fraction varied markedly. This may be due to the

differences in structures of phenolic compounds and primarily related

to their hydroxylation and methylation patterns70

.The total antioxidant

activity of the total aqueous extract of willow leaves was much more

than salicin alone. This may be due to the aqueous extract of willow

leaves contain other antioxidants such as tannins, flavenoids and

proanthocyanidins in addition to salicin which could play some role in

the increase of the total antioxidant activity71

. Salicin may induce

elevation in activities of the antioxidants as glutathione peroxidase in

the tissues homogenates which were selected to be under study.

The antioxidants have a major role in the antioxidants defense

mechanisms against irradiation injury72

.

So, the maintenance of normal electrophoretic protein and

zymogram levels after the treatment with salicin may be due to

trapping of these free radicals by this fraction, thus preventing DNA

damage. Salicin was able to overcome the disturbances in the

protein pattern in the tissues selected to be under study.

5 Conclusions

The study concluded that salicin showed radioprotective effect

against the gamma irradiation effect on various electrophoretic

patterns in brain tissue of male rats.

6 Competing interest

The study aimed to suggest new compounds obtained from the

nature and act as radioprotector to minimize or resist the irradiation

effect.

7 Author’s contributions

MSA and MALAK carried out literature review and draft the

manuscript. HMS participated in collection of data and arranged in

tabular form. IA and WMK carried out the experimental work. All

authors read and approved the final manuscript.

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ameliorative effect of salicin against gamma irradiation induced

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