Australian Journal of Basic and Applied Sciences, 5(12): 2817-2826, 2011
ISSN 1991-8178
Relative Mutagenecity of Some Food Preservatives on Plant Cells
Nagwa R. Abdel-Hameid, Magda A.M.Elanany, Atef A.A.Haiba, Elham A.A. AbdEl-Hady
Department of Genetics and Cytology, National Research Center, Dokki, Giza, Egypt.
Abstract: The present investigation was designed to evaluate the mutagenic activity of food
preservatives sodium metabisulphite (SMB) and potassium metabisulphite(KMB) in Vicia faba root
meristems. The effect on protein banding pattern and isozyme variations were also investigated. The
tested chemicals significantly decreased the mitotic index (MI), and increased the mitotic
abnormalities. Reduction in the mitotic activity and induction of chromosomal aberrations were dosedependent at all concentrations and treatment periods..These abnormalities include stickiness, cmitosis, laggards, bridges, micronuclei, fragments and unseparated. All treatments changed the
frequency of mitotic phases when compared with the control groups. At SDS-protein level, the studied
chemicals show many alterations in the leave storage protein banding patterns expressed as
disappearance of some bands or appearance of novel one or change in bands intensity. Also there was a
change in the expression level of peroxidase and this regulation was estimated by the band density .
Key words: Vicia faba, chromosomal abnormalities, food preservatives, SDS-PAGE protein and
isozyme variations.
INTRODUCTION
In the last few years, the population of the world is increasing. This increase has led to the search for new
food sources and ways to make productive use of these food sources. Food preservatives are substances
internationally added to food to extend a food's freshness or shelf life and keep it from spoiling or oxidizing.
The safety of repeated exposure to permitted synthetic food additives (colorants or preservatives) has been
questioned. It has been reported that certain food additives, especially antimicrobial agents are genotoxic in
different test systems (Luca et al.,1987) and (Mukherjee et al.,1988). Other food preservatives reduced mitotic
index values in plant cells, induce variations in the percentage of mitotic stages and increasing the total
percentage of aberrations with increasing concentrations and period of treatments (Şifa, 2007).
SMB and KMS are used as a preservative in many kinds of food products. Ministry of Agriculture of
Turkey (1997), has suggested that SMB may be used at a maximum dose of 300 mg/L in food as an
antimicrobial substance. When SMB dissolved in water,converts to sodium bisulfite and sulfur dioxide.Bisulfite
is a weak mutagen at pH 5-6 in Salmonella typhimurium TA 1535 and TA 97strains but is not mutagenic in TA
1537 and TA 1538 strains (Pagano and Zeiger1987). On the other hand (Meng and Zhang1999) reported that
bisulfite enhance guanine phosphoribosyltransferase (gpt) mutations in CHO-AS52 cells In addition, sodium
bisulfate decreases the mitotic index in human lymphocytes and induceses chromosome aberrations, sister
chromatid exchanges and micronuclei (Meng and Zhang, 1992).
Limited studies have been conducted with Potassium metabisulphite in vitro and in vivo test Systems.
Although food preservatives are widely used in our foods, we do not have enough information about their
genotoxic effects. Therefore; it has been decided to test the genotoxic potentialities of the potassium
metabisulphite and sodium metabisulphite revealed by mitotic index, mitotic abnormalities, changes in leave
storage protein banding patterns and isozyme variations of Vicia faba as a biological system.
MATERIALS AND METHODS
In this study, Vicia faba L. variety Giza 2, kindly supplied by Crop Research Institute,
Agricultural Research Center, Giza, Egypt was used as the test system, and the food preservatives Sodium
metabisulfite (SMB has chemical formula Na₂S₂O5 and molecular weight 190.1) and Potassium metabisulphite
(KMB has chemical formula K₂S₂O₂ and molecular weight 222.23) as the test substances.
Mitotic Study:
Healthy V. faba seeds have been germinated in distilled water at room temperature (23±1°C) in dark. Roots
of 1–2 cm in length of the newly emerged seedlings have been treated with a series of concentrations of 7.5, 15
and 30 ppm of sodium metabisulphate and 25, 50, 100, 150 and 200 ppm of potassium metabisulfite for 6,12
and 24hrs, while control roots were immersed in tap. The treatments were cut and fixed in Carnoy s fixative for
24hrs, then stored in70% ethyl alcohol at 4°C. Slides have been prepared by using Feulgen squash technique
Corresponding Auther: Nagwa Riad Abdel-Hameid, Department of Genetics and Cytology, National Research Center,
Dokki, Giza, Egypt.
Tel: 0237754048-0109485998; E-mail: [email protected]
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Aust. J. Basic & Appl. Sci., 5(12): 2817-2826, 2011
according to (Darlington and La Cour, 1936). The data of different treatments were statistically analyzed by ttest.
Biochemical Studies:
Seedling’s leaves of Vicia faba previously treated with different concentrations of the two food
preservatives SMB and KMB were used for biochemical genetic analysis.
SDS-Protein Electrophoresis:
Sodium dedocyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) was carried out according to
Laemmli (1970) as modified by Studier (1973) to study proteins and separate them based on their molecular
weights. For total protein extraction, leaves of Vicia faba seeds treated with the two food preservatives
potassium and sodium metabisulfite with different concentrations for different times were used. 0.2 ml of
sample buffer (0.2 MTris-Hclph 8.8; 2% SDS; 10% glycerol)was added to0.5 gm of each sample and stored
over night at 4°C. In the current study, proteins were fractionated on 15% acrylamide and gels were run at a
current of 100 volts. The gels were stained for 24 hrs in coomassie brilliant (blue COBB,R_250). The gels were
destained, photographed and analyzed by the computerized gel pro analyzer. Alternations in electrophoretic
profiles of protein in the samples under investigation were compared with that of the control to measure the
mutagenic potentialities of the two food preservatives potassium and sodium metabisulfite.
Isozymes Electrophoresis:
Native-polyacrylamide gel electrophoresis (Native-PAGE) according to Stegmann et al. (1985).was
conducted to identify isozyme variations between control and the samples under investigation of Vicia faba
seedlings using one isozyme system peroxidase.
Isozyme Staining and Detection:
After electrophoresis, gels were stained according to their enzyme systems with the appropriate substrate
and chemical solutions, then incubated in dark at room temperature for complete staining. For peroxidase,
Benzidine dihydrochloride Hcl and Glacial acetic acid (0.125 gm and 2.000 ml) in 50 ml distilled water were
used. Gel was placed in this solution and 5 drops of hydrogen peroxide was added. The gel was incubated at
room temperature until bands appear (Brown, 1978). The resulted bands were estimated by the bands intensity.
RESULTS AND DISCUSSION
Cytological Analysis:
One of the major effects of the two food preservatives used in this study is their influence on the rate of
mitotic division. The mitotic index reflects the frequency of cell division and is regarded as an important
parameter in evaluating the rate of root growth.The data presented in Tables (1&3) showed that the test
chemicals significantly decreased the mitotic index (MI),and induced imbalance in the frequency of the different
mitotic stages, causing increase in the frequencies of prophase and metaphase on the expense of ana-telophase.
The decreasing in (MI) and imbalance in the frequency of the different mitotic stages were dose-and time
dependent at all concentrations and treatment periods. The mitotic index reached a minimum value of (1.12±
0.01 and 3.00 ± 0.11) after 24hrs treatment with the highest concentration of SMB and PMB respectively
compared with the control value of ( 7.49+9 ± 0.08 ).
Similar results were obtained by Eyyüp et al. (2001) who investigated the effects of sodium metabisulphite
(SMB) on mitosis of Allium cepa L. and reported that SMB significantly decreased the MI in the treatment
groups at all concentrations and in all treatment periods. These results showed that SMB has a cytotoxic effect at
all concentrations. Abdelhady and Barakat (2005) recorded a progressive reduction in mitotic index in all roots
of Allium cepa L treated with sodium ascrobate, sodium benzoate, and sodium nitrite at different concentrations
for different times. Şifa (2007) also studied the effects of the food preservatives sodium benzoate (SB), boric
acid (BA), citric acid (CA), potassium citrate (PC) and sodium citrate (SC) on root tips of Allium cepa L. The
results indicated that these food preservatives reduced mitotic division in A. cepa compared with the respective
control. Mitotic index values were generally decreased with increasing concentrations and longer treatment
times. Palani and Panneerselvam (2007) tested cytogenetic effects of food preservative Potassium
metabisulphite on root tip cells of Allium cepa L. The root tips have been treated with a series of concentrations,
ranging from 25 to 150μg/ml for 1,3,5,7 and 9 hrs. The results indicate that the food preservative has reduced
mitotic division in Allium cepa compared with their respective control. The percentages of mitotic index have
decreased with increasing dose and time.
The inhibition of mitotic activity has been regarded as a common effect induced by numerous chemical
compounds and has been reported by many investigators (Abdel-Hamied, 2005; Shehab et al., 2004; AbdelHamied et al., 2008; Vanya et al.,2009; Haedo and Abdelmigid, 2009; Haiba et al., 2010) .
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Aust. J. Basic & Appl. Sci., 5(12): 2817-2826, 2011
Increase in the frequency of prophase and metaphase on the expense of ana-telophase indicate the
potentiality of the investigated chemicals to induce mitotic reduction. Reduction in the mitotic activity could be
due to inhibition of DNA which might be caused by the decreasing ATP level and the pressure from the
functioning of the energy production center (Jain et al, 1988) and (Sudhakar et al, 2001) or a blocking in the
G2-phase of the cell cycle, preventing the cell from entering mitosis (Van’t Hof, 1968). Beu et al., (1976 ) have
also showed that exposure of root tips of V. faba to high concentrations of the herbicide paraquat has led to
inhibition of DNA synthesis, therefore the potassium metabisulphite may cause inhibition of DNA synthesis.
The data presented in the Tables (2 & 4) illustrated that the clastogenic and mutagenic effects of food
preservatives SMB and KMB in the root tips of Vicia faba plant. These data demonstrated that KMB give the
number of chromosomal abnormalities bigger than SMB, so there are differences in the mutagenic effects of the
two food preservatives. The frequencies of chromosomal aberrations increased with increasing the
concentrations and time of treatments. They reached their maximum value of (40.77 ±0.19 % and 55.03 ±0.34
%) with the highest concentration of SMB and KMB respectively compared with control value of (1.56 ± o.27
%). The observed types of aberrations such as stickiness, c-mitosis, laggards, bridges, micronuclei, fragments
and unseparated were shown in Figure 1 (a→j.). The sticky and disturbed chromosomes were the most common
abnormalities found in the different stages after the various treatments with the applied chemicals. The presence
of stickiness in the chromosomes Figure 1 (a & b), reflected highly toxic effects, it was irreversible and might
lead to cell death( Liu et al., 1995). In this concern, the applied chemicals might affected the physiological
properties of DNA and proteins, and form complexes with phosphate groups of nucleotides of the nuclic acids
csusing inhibition in protein synthesis (Karlik et al., 1980). In contrast, Patil and Bhat (1992) suggested that,
stickiness is a type of physical adhesion involving mainly the proteinacious matrix of chromatin material.
Disturbed chromosomes Figure 1 (c & d) may be either due to, hindrance of prometaphase chromosome due to
the effect of the tested chemicals on spindle apparatus, by causing partial inhibition of mitotic apparatus or was
referred to the presence of chromosomes(s) with inactivated centromers. The inactivation by the tested
chemicals preventing them from being inserted in the spindle fibers, affecting the normal kinetic of the cellular
division (Mukherjee et al., 1990 ). Armbruster et al. (1991) found that disturbed phases may be due to inhibition
in the respiratory pathway resulting in low energy production necessary for chromosome movement. Induction
of disturbed phases indicates that both food preservatives may be an eugenic which inhibited the spindle
formation and caused c-mitosis in metaphase (disturbance). Other chromosomal abnormality included in the
present investigation was laggards Figure 1 (f). The induction of laggard chromosomes could be attributed to
irregular orientation of chromosomes (Patil and Bhat, 1992). These laggards may be distributed randomly to
either poles at anaphase I or II which result ultimately in aneuploidy (Amer and Ali, 1988) or may give for
micronuclei at telophase II (Abdelsalam et al., 1993). The occurrence of laggards indicated that both tested
chemicals completely or partially affect the spindle apparatus). Considerable frequencies of bridges were also
observed in anaphase and telophase stages after all treatments Figure 1 (g). It may be attributes to general
stickiness of chromosomes (Haliem,1990) or due to the formation of dicentric chromosomes as a result of
breakage and reunion (El-Khodary et al., 1990).
Chromosomal fragments Figure 1 (e & h) were a type of abnormalities also found to be observed during
mitotic cell division after all treatments.The induction of these abnormalities indicates the mutagenic capacity
and clastogenic action of both preservatives on the chromosome which is now regarded to affect the
chromosomal DNA (Grant, 1978 and Chauhan and Sundaraman, 1990) and it might lead to micronuclei
formation. Unseparated anaphase (Delay of separation) was observed after the different treatments Figure1
(i).The delay of separaton could be due to stickiness (Abdel-Hamied, 1995). Among the cytological aberrations
appeared in a considerable frequency was the induction of micronuclei Figure 1. (j). Micronuclei may originate
from chromosome lagging or fragment at ana-phase (Brown and Dyes, 1972). Micronuclei are true mutagenic
aspects which lead to loss of the genetic material and have been regarded as an indication of the mutagenecity of
their inducers (Ruan et al., 1992). Finally, the induction of these chromosomal abnormalities was pointed to the
mutagenic potential of the applied concentrations of these tested chemicals .
Biochemical Analysis:
SDS-Protein Electrophoresis:
Tables (5 & 6) and Figures. (2 & 3) demonstrate the effect of sodium and potassium metabisulphite on the
SDS- electrophoretic patterns of leaves total protein fractions for Vicia faba L. The total numbers of protein
bands recorded were 22 and 19 and the numbers of common bands were seven and five for the two tested food
preservatives respectively. These common bands of SMB have molecular weights of 162.246, 93.874, 63.067,
52.295, 27.178, 26.058 and 25.785 KDa, while the common bands of KMB have molecular weights of 148.973,
65.455, 54.219, 25.102 and 21.818 KDa. Specific bands were also recorded at some treatments of both used
chemicals, thier molecular weights were 124.98, 118.575, 112.025 and 31.097 KDa at treatments of 15/12,
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Aust. J. Basic & Appl. Sci., 5(12): 2817-2826, 2011
Fig. 1: Types of chromosomal abnormalities in mitotic cells of Vicia faba after treatment with different
concentrations of sodium metabisulfite and potassium metabisulfite at different times.
Table 1: Effect of treating Vicia faba root-tips for 6,12and 24 hrs with different concentrations of sodium metabisulphite on number of total
cells in mitosis, means of mitotic index (MI) and frequency of mitotic phases.
Treatment
Total cell
exam.
Total cell
in
mitoses
Prophase
Metaphase
Ana-telophase
No.
٪
No.
٪
No.
٪
Total
abn.
M.I ± S.E
Cont.
6hrs.
7.5 ppm
6486
486
188
38.68
114
23.46
184
37.86
8
7.49** ± 0.08
9805
244
95
38.93
63
25.82
86
35.25
12
2.51 **± 0.02
15 ppm
8329
178
74
41.57
48
26.97
56
31.46
19
2.14** ± 0.02
30 ppm
12hrs.
7.5 ppm
8371
160
67
41.88
45
28.13
48
30.00
28
1.92** ± 0.01
11899
240
95
39.58
62
25.83
83
34.58
10
2.02** ± 0.01
15 ppm
12362
221
92
41.63
60
27.15
69
31.22
27
1.78** ± 0.02
30 ppm
24hrs.
7.5 ppm
6429
91
39
42.86
27
29.67
25
27.47
19
1.42** ± 0.01
9700
147
61
41.50
38
25.85
48
32.65
10
1.52** ± 0.01
15 ppm
11191
148
63
42.57
42
28.38
43
29.05
40
1.32**± 0.01
30 ppm
11262
126
59
47.00
All data are means of six replications ± standard deviation
t, trace (<0.05%)
34
26.98
33
26.19
51
1.12** ± 0.01
Fig. 2: Electrophotographs produced by SDS- PAGE analysis of protein patternsof Vicia faba L.after treatment
with different concentrations of sodium metabisulfite at different times.
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Aust. J. Basic & Appl. Sci., 5(12): 2817-2826, 2011
Fig. 3: Electrophotographs produced by SDS- PAGE analysis of protein patternsof Vicia faba L. after treatment
with different concentrations of potassium metabisulfite at different times.
Dist.
St.
Lag.
Cont..
0.00
1.06
0.88
0.00
6hrs.
7.5ppm
1.05
1.05
1.59
1.59
15 ppm
1.35
2.77
2.08
2.08
30 ppm
2.99
2.99
4.44
2.22
12hrs.
1.05
1.05
0.00
1.61
7.5ppm
15 ppm
2.17
2.17
3.33
1.66
30 ppm
2.56
5.13
7.41
3.70
4hrs.
3.28
0.00
2.63
2.63
7.5ppm
15 ppm
4.76
4.76
4.76
7.13
30 ppm
8.47
5.08
8.82
8.82
All data are means of six replications ± standard deviation
t, trace (<0.05%)
Dist.
% of abn.±S.E.
Total abn.
Metaphase
Prophase
St.
Ana-telophase
Table 2: Effect of treating Vicia faba root-tips for 6,12and 24 hrs with different concentrations of sodium metabisulphite on percentage of total abnormalities and different types of
prophase, metaphase and ana- telophase abnormalities. Mico
nucl.
0.00
Frag.
St.
Dist.
Brid
Lag.
Frag.
1.75
0.00
0.54
1.09
0.00
0.00
.00
Unsep.
0.00
8
1.65** ±0.27
3.17
4.17
4.44
1.61
0.00
2.08
6.67
3.22
0.00
2.08
2.22
0.00
1.16
3.57
6.25
1.20
1.16
1.79
4.16
1.20
2.32
3.57
8.33
1.20
1.16
3.57
4.16
0.00
1.16
1.79
4.16
0.00
0.00
3.57
4.16
1.20
12
19
28
10
4.92**± o.o1
11.00** ± 0.26
17.53** ± 0.19
4.16**± 0.09
3.33
3.70
2.63
5.00
7.41
2.63
1.66
0.00
0.00
4.35
12.00
0.00
2.90
8.00
4.17
4.35
8.00
2.08
1.45
12.00
2.08
2.90
0.00
0.00
4.35
0.00
0.00
27
19
10
12.18** ± o.07
20.88** ± 0.30
10.90** ± 0.15
9.52
11.76
11.90
14.71
0.00
0.00
6.98
18.18
6.98
12.12
9.30
18.18
9.30
12.12
4.65
9.09
9.30
15.15
40
51
26.99**± 0.26
40.47**± 0.19
Fig. 4: Peroxidase isozyme banding patterns for Vicia faba cultivar with the sodium metabisulfite treatment.
Fig. 5: Peroxidase isozyme banding patterns for Vicia faba cultivar with the potassium metabisulfite treatment.
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Aust. J. Basic & Appl. Sci., 5(12): 2817-2826, 2011
Table 3: Effect of treating Vicia faba root-tips for 6,12and 24 hrs with different concentrations of potassium metabisulphite on number of
total cell in mitosis, means of mitotic index (MI) and frequencyof mitotic phases.
Treatment
Total
cell
exam
Total
cell in
mitoses
Prophase
No.
Metaphase
%
No.
Cont.
6486
486
188
38.68
6hrs.
6118
349
143
40.97
25 ppm
50 ppm
6131
334
130
38.92
100 ppm
6560
335
134
40.00
150ppm
9245
416
187
44.95
200ppm
6421
323
145
44.89
12hrs.
6606
332
156
46.99
25 ppm
50 ppm
5940
268
124
46.27
100 ppm
6081
251
120
47.81
150ppm
6143
227
118
51.98
200ppm
6398
212
115
54.24
24hrs
6562
318
149
46.86
25 ppm
50 ppm
5761
243
114
46.91
100 ppm
7566
306
147
48.04
150ppm
8612
300
156
52.00
200ppm
6080
183
99
54.10
All data are means of six replications ± standard deviation
t, trace (<0.05%)
Anatelophase
No.
%
Total
abn.
M.I ± S.E
%
114
91
23.46
26.07
184
115
37.86
32.95
8
83
7.49**±0.08
5.70 **±0.07
90
88
133
113
100
26.95
26.27
31.97
34.98
30.12
114
113
86
65
76
34.13
33.73
20.67
20.12
22.89
77
140
214
163
90
5.59 **±0.41
5.11 **±0.04
4.50 **±0.03
5.05 **±0.05
5.03 **±0.02
84
88
82
79
104
31.34
35.06
36.12
37.26
32.70
60
43
27
18
65
22.39
17.13
11.89
8.48
20.44
103
115
114
118
98
4.51 **±0.01
4.14 **±0.01
3.70 **±0.02
3.31 **±0.02
4.85 **±0.02
77
112
110
68
31.69
36.60
36.67
37.16
52
47
34
16
21.40
15.36
11.33
8.74
96
132
156
101
4.22 **±0.01
4.04 **0.02
3.48 **±0.02
3.00 **±0.11
St.
Dist.
St.
Lag.
Dist.
Mico
nucl.
Frag.
St.
Dist.
Brid
Lag.
Frag.
Cont..
0.00
1.06
o.88
0.00
1.75.
0.00
0.00
0.54
1.09
0.00
0.00
8
4.20
3.50
16.48
5.49
8.79
4.40
2020
8.70
6.09
6.96
4.36
0.00
2.61
0.00
6hrs.
25 ppm
4.36
83
6.67
10.23
10.53
13.27
5.56
9.09
6.77
10.62
3.33
4.55
5.26
5.31
7.89
19.47
40.70
35.38
3.51
14.16
22.09
26.15
4.39
8.85
17.44
15.38
3.51
4.42
6.98
7.69
3.51
2.65
8.14
6.15
5.26
6.19
11.63
9.23
77
140
214
163
7.00
9.52
8.33
7.63
11.39
5.00
8.33
6.67
7.63
10.13
6.00
5.95
5.00
6.10
7.59
11.84
16.67
25.58
37.04
1161.
10.53
15.00
20.93
37.04
55.56
9.21
3.33
16.28
29.63
50.00
7.89
11.67
18.60
25.93
33.33
5.26
6.67
13.95
25.93
44.44
5.26
10.00
20.93
29.63
44.44
90
103
115
114
118
6.73
9.09
8.04
13.64
10.29
4.81
7.79
6.25
10.00
8.82
5.77
6.49
3.57
5.45
10.29
15.38
17.31
42.55
64.71
11.11
13.85
19.23
31.91
47.06
11.11
10.77
13.46
21.28
26.47
37.5
9.23
9.62
10.64
14.71
43.75
9.23
9.62
8.51
14.71
31.25
6.69
11.54
12.77
17.65
37.5
98
96
132
156
101
50 ppm
6.15
4.62
11.11
7.78
100 ppm
7.46
6.72
25.00
17.05
150ppm
9.63
8.02
21.80
16.54
200ppm
8.28
6.90
22.12
15.04
12hrs.
5.13
3.85
14.00
5.00
25 ppm
50 ppm
5.65
6.45
21.43
7.14
100ppm
7.5
5.83
19.32
9.09
150pp
6.78
6.78
19.51
7.63
200ppm
7.83
6.09
21.52
12.66
24hrs
6.04
4.70
14.42
5.77
25ppm
50ppm
7.02
7.02
18.18
7.79
100ppm
6.12
5.44
18.75
12.5
150ppm
7.05
6.41
21.82
14.55
200ppm
8.08
9.09
23.53
16.53
All data are means of six replications ± standard deviation
t, trace (<0.05%)
٪ of
abn.±S.E.
Total abn.
Anatelophase
Metaphase
Prophase
٪ of Inte Micrnuc.
Treatment
Table 4: Effect of treating Vicia faba root-tips for 6,12and 24 hrs with different concentrations Of potassium metabisulphite on percentage of total abnormalities and different types of
prophase, metaphase and ana-telophase abnormalities. Unsep
1.56** ± 0.27
23.66** ± 0.22
23.11** ± 0.99
42.14** ± 0.12
46.93** ± 0.17
50.58** ± 0.25
27.12** ± 0.09
38.52**± 0.21
45.76** ± 0.15
50.15** ± 0.33
55.68** ± 0.13
30.83** ± 0.11
39.53 **± 0.14
43.13** ± 0.22
51.095** ±0.32
55.03 **±0.34
30/6, 30/6 and 30/12 ppm/hrs respectively, for SMB and were 93.225, 40.875, 29.926 and 27.581 KDa at
treatments of 200/24, 100/24, 100/24 and 25/24 ppm/hrs respectively for KMB. Specific bands were
disappeared at any other treatments of both preservatives. It is noted that specific bands appeared after a long
periods and high concentrations treatments. Other recorded bands of the present investigation fluctuated
between disappearance and appearance at different treatments. It is noticeable that, when the time of treatment
and concentration of the applied chemicals increased a new bands were appeared. The occurrence of new bands
and absence of others, of different treatments represented by different time under different food additives
concentrations would indicated enhancement or depression of gene expression in this plant. This might alter the
protein products in response to food additives either on the transcription or post- transcription levels of gene
expression. These results might indicate that the induced changes in protein patterns of Vicia faba plants
reflected a marked difference in gene expression (Abdel-Hady and Barakat, 2005). Gene expression might
2822
Aust. J. Basic & Appl. Sci., 5(12): 2817-2826, 2011
(+) = Band presence, (-) = Band absent,
+
+
+
+
+
+
+
+
+
+
+
+
+
30/24
hrs.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
15/24
hrs.
7.5/12
hrs.
30/6 hrs.
+
+
+
+
+
+
+
+
7.5/24
hrs.
+
+
+
+
+
+
+
+
30/12
hrs.
+
+
+
+
+
+
+
+
+
15/12
hrs.
162.246
125.508
124.98
119.829
118.575
112.025
109.002
105.837
99.362
98.737
93.874
84.143
80.015
63.067
52.295
31.097
30.257
29.815
28.767
27.178
26.058
25.785
15/6 hrs.
MW
Cont.
7.5/6 hrs.
Table 5: Effect of sodium metabisulfite on SDS-PAGE of seed protein of Vicia faba leave under control, after 6, 12 and 24hrs with different
concentrations.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
=Specific band
200/6hr
s.
25/12hr
s.
50/12hr
s.
100/12h
rS
150/12
hrs.
200/12
hrs.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
200/24
hrs.
150/6hr
s.
+
+
+
+
+
+
150/24
hrs.
100/6hr
s.
+
+
+
+
+
100/24
hrs.
50/6
hrs.
+
+
+
+
+
+
+
25/24
hrs.
50/24hr
s.
25/6
hrs.
MW
148.973
94.799
94.206
93.225
65.455
54.219
40.875
30.88
30.114
29.926
27.581
27.408
25.102
24.377
24.225
23.28
23.135
22.846
21.818
Cont.
Table 6: Effect of potassium metabisulfite on SDS-PAGE of seed protein of Vicia faba leave under control, after 6, 12 and 24hrs with
different concentrations.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
(+) = Band presence, (-) = Band absent, + =Specific band
Table 7: Expression patterns of the peroxidase bands (as density values) under control and sodium metabisulfite Treatments.
Cont.
7.5/6
15/6
30/6
7.5/12
15/12
30/12
7.5/24
15/24
Band 1
d
d
b
d
d
d
d
d
Band 2
d
b
a
d
a
a
d
d
Band 3
C
d
b
b
d
b
c
c
d
a=very faint,b=faint,c=dark,d=very dark.
Table 8: Expression patterns of the peroxidase bands (as density) under control and potassium metabisulfite treatments.
con 25/6
50/
100 150 200 25/
50/
100/
150/
200/
25/2
50/
100/
t.
6
/6
/6
/6
12
12
12
12
12
4
24
24
Band 1 d
d
d
d
d
d
d
d
d
d
b
b
Band 2
d
d
b
d
d
d
c
d
d
d
d
b
Band 3
c
b
d
b
d
b
c
b
d
d
d
a
c
b
a=very faint,b=faint,c=dark,d=very dark.
150/
24
d
a
c
30/24
b
c
200/
24
d
d
a
amplify or attenuate signal-transduction pathway and ultimately exhibited as a simulative or depressive
metabolic, growth and yield responses. These results were comparable with those of Ericson and Alfinito (1984)
2823
Aust. J. Basic & Appl. Sci., 5(12): 2817-2826, 2011
who found some protein bands, which were enhanced under different stresses. Abdel-Tawab et al. (1997) in
maize and Al-Sabi et al. (2004) in sorghum reported specific protein bands associated with tolerance to different
stresses. On the other hand, appearance of new bands could be explained on the basis of mutational event at the
regulatory system of an unexpected gene(s) that activate it (EL-Nahas, 2000; Mohamed et al., 2003 and Shehab
et al., 2004).Telma et al. (2008) found that the expression level of various genes encoding heat shock proteins
increased after a short term of oxidative stress treatment. Changing in protein patterns also have been attributed
to the occurrence of either gene mutations or induction of cytological aberrations. (Mohmed, 2005 and AbdelHamied, 2005, 2008). Soliman (2003) and Shehab et al. (2004) attributed these changes to mutational events,
where some of the electrophoretic bands disappeared as a result of the loss of some genetic material due to
induction of laggards, breaks and micronuclei or changes in gene sequences.
Isozyme Electrophoresis:
Peroxidase Banding patterns:
Peroxidase electrophoretic banding patterns among leaves sample of Giza 2 Vicia faba L. and two
treatments by potassium metabisulfit and sodium metabisulfit are shown in Tables (7 & 8) and Figures (4 & 5),
respectively.
For SMB all treatments revealed a total number of three bands were characterized inTtable (7) and Figure
(4). All treatments exhibited the occurrence of band number 1 with different intensities, while disappeared in the
control. The activity of bands (intensity) increased in the treatment compared with the control .The second band
disappeared in the control and 30/24 ppm/hrs treatment, however under treatments 7.5/6, 7.5/12, 7.5/24 and
15/24 ppm/hrs showed very dark bands, while in 15/6 ppm/hrs treatment appeared faint band, finally in the
treatments 30/6, 15/12 and 30/12 ppm/hrs exhibited very faint bands. The third peroxidase isozyme band was
found in all treatments and control with different intensities. This peroxidase isozyme exhibited four dark band
reaction with the control, and the treatments 30/12, 7.5/24 and 30/24 ppm/hrs, while faint bands with 15/6,
30/6and 15/12 ppm/hrs treatments. Moreover, under 7.5/6, 7.5/12 and 15/24 ppm/hrs showed very dark band.
The result indicated that there is change in the level of expression and this regulation was estimated by the bands
intensity. In general, these results are in accordance with those obtained by El-Tayeb and Ahmed (2007) who
found that peroxidase isozymes activities increased significantly under water stress. The above results for
changed to up and down- regulation in sodium metabisulfite may be due to the activity of enzyme (band
intensity) which increased under treatment comparing with control.
For KMB a total number of three bands were characterized as shown in Figure (5) and Table (8). The first
band was found to be expressed with very dark intensity for majority treatments. Meanwhile, the same band
disappear in the control and treatment 50/12 ppm/hrs and the treatments 50/24,100/24 ppm/hrs exhibited faint
intensity bands .The second band found with different intensities in all treatments ,while this band disappeared
from control and 100/24 ppm/hrs. It is noted that treatments 100/6 and 50/24 ppm/hrs showed faint bands and
very faint band with 150/24,respectively. Moreover,the third band showed different activities ,all treatments and
control exhibited the occurrence of band number 3 with different intensities under control and treatments. These
results confirmed the effect of combination between the time and different concentrations from potassium
metabisulfite due to the expression are different from up and down regulation. Finally, these results is in partial
agreement with( Fahmy et al., 1992 and 1999; Rashed et al., 1994 and Yamamoto and Duich, 1994) who
reported the occurrence of different responses in activity and / or degrees of intensity rather that in the isoforms
of peroxidases in favors of salt tolerant genotypes under stress. In general, the activity of bands (intensity)
increased under stress. Rudiger et al. (2001). found that an ability of the peroxidase and other enzymes perhaps,
to tolerate a stress they induce intracellular which may help the plant to survive periods of metabolic and
generate phenolic compounds during stress responses.
Conclusion:
Food additives as sodium metabisulphite (SMB) and potassium metabisulphite (KMB) have a cytotoxic
effect on the root tips of Vicia faba plant at all the used concentrations. Clastogenic and mutagenic effects of
food preservatives SMB and KMB in the root tips of Vicia faba plant were also observed. The data
demonstrated that KMB more effective on the V.faba cells and give more number of chromosomal
abnormalities than SMB, that is there are differences in the mutagenic effects of the two food preservatives.
Also biochemical parameters as alterations in the leave storage protein banding patterns and change in the
expression level of peroxidase isozyme were different in the two test chemicals.
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