Clastogenic and Mutagenic Effect Induced by Some Fast Foods

INTRODUCTION
Genotoxic compounds in the diet represent a truly international problem. This
problem was recognized in Japan and the United States of America several years
ago by the application of in vitro mutagenicity tests to prepared and unprepared
components of our diet. Now new discoveries are appearing every day concerning
genotoxic compounds in our food supply from all over the world. It seems likely that
naturally occurring genotoxic compounds in the diet as well as genotoxic compounds
formed during storage, preparation, and digestion of the food are important for the
induction of cancer in humans and of hereditary changes in human germ cells. Other
scientific data indicate that dietary constituents have the potential to modify the
genotoxic effect of these compounds in humans. These findings indicate new
possibilities for the prevention of human diseases of genotoxic origin through relevant
changes in the composition of the diet. (Amara et al.,2008).
With this background, and due to the rapid accumulation of new data in the field,
it was approved by the Board of the International Association of Environmental
Mutagen Societies that one of the satellite meetings of the Fourth International
Conference on Environmental Mutagens in Scandinavia 1985 should cover the
aspects of mutagens and carcinogens in the diet. However, formation of polycyclic
aromatic hydrocarbons (PAH) occurs during incomplete combustion of organic
material. Although the mechanism of this formation is not fully understood, two
principal pathways are considered to be involved, pyrolysis and pyrosynthesis. At
high temperatures organic compounds are initially cracked to smaller molecules,
mostly free radicals, which may then recombine to form a number of relatively stable
polycyclic hydrocarbon. Hypothetical schemes for the generation of specific PAH
compounds, such as benzo [a] pyrene (BaP), have been suggested (Knudsen,
1986).
PAH are formed in the combustion processes used in the production of curing
smoke and for the generation of heat during grilling. The combustion fumes may
contain both vapour phase and particle- bound PAH. Contaminants of food during
grilling and smoking occurs when the combustion fumes reach the surface of the
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food. However, since PAH represent an important group of chemical carcinogens,
their presence in food and the environment has been studied intensively during the
last two decades. Among the various foods studied for PAH, smoked products seem
to have the been most popular. Based on epidemiological studies in several
countries, a possible connection between an increased incidence of gastric cancer
and a high consumption of smoked food, has been suggested (Knudsen, 1986).
In fact, there are two cytogenetic tests in plants that can be performed in Allium
cepa and Vicia faba i.e., the root-tip mitotic and PMC meiotic systems. The former is
more widely used and better validated than the latter, especially when chromatid
aberrations are used as the indicators of mutagenicity. However, this plant
cytogenetic test system is efficient in the preliminary screening of chemical
mutagens. Badr et al., (1983), Amara et al., (2008), Badr (1993). It is simple and
economical test, and the results can be obtained in 48 hrs. Comparative studies of a
given chemical under Allium; Vicia., and mammalian system (such as human
lymphocytes or mice) are strongly encouraged in order to establish the correlation
between the chromosome breaking ability of chemicals in plant and animal system.
(Al-Ayoubi (1998) and Albertini et al., (2001).
MATERIALS AND MRTHODS
MATERIALS:
Allium cepa bulbs and Vicia faba were used in this work to assess the
possible activity clastogenic, while mice were used to investigate the mutagenic
activity by employing in vivo induction of sister chromatide exchange SCE technique.
METHODS :
Preparation of food materials :
From each type of fast foods six concentrations were freshly prepared.
These concentrations were 10, 20, 30, 40, 50 and 100g per litre. Each
concentration was prepared as follows :
The proper amount was grinded well in a morter and mixed with one
litre of tap water; shaked overnight on a shaker, centrfigued at 2000 r.p.m. for
15 min., and filtered. The supernatant was saved and immediately used.
Germination of plants :
Allium cepa
Bulbs were allowed to geminate at 25oC using special Jars on tap
water until a length of 0.5 cm for adventitious roots had reached, then they
were subjected to treatment by transferring them on Jars containing the
previously mentioned supernatant for different times of exposures, i.e., 6, 12
and 24 hrs. Mitotic index (MI) was calculated according to the following
formula:
Vicia faba
Vicia faba seeds were soaked in running tap water for 24 hours,
transferred to petri dishes on moistened filter paper and allowed to germinate
in dark at 25oC. The filter paper inside each dish was moistened with tap
No. divided cells
Total examined cells
MI = X 100
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water every day. When the primary root reached about 2 cm, the apical
meristem was cut in order to stimulate the growing of secondary roots. When
secondary roots on each primary one reached about 1 cm in length, seedling
were then transferred in petri dishes and predetermined concentration was
added. Six concentrations were used. Secondary roots were treated with the
proper concentration for 6, 12 and 24 hours, while the control group was on
tap water only. After treatment; root-tips of secondary roots and of Allium cepa
adventitious roots were cut and transferred to cold glacial acetic acid for one
hour and transferred to a fixative solution (Carnoy's solution), and the well
known acetocarmine technique (Darlington and La Cour, 1962) was used to
examine the mitotic activity. The frequency of mitotic activity (mitotic index)
was calculated as the ratio of the total number of normal dividing cells to the
total number of examined cells. The frequency of each type of mitotic
aberrations was calculated as the ratio of cells containing this type of
aberration to the total number of dividing cells.
Sister chromatid exchange technique:
Experimental design
Typically 2-3 month old Swiss albino mice (Mus musculus, 2n =40)
were locally purchased from the high Institute of Graduate Studies and
Research. Four animals per dose were used and analysis of at least 25 cells
per animal was carried out. Four selected doses (10,20,30 and 40g/L) of the
tested food were tested.
Bromodeoxyuridine tablet preparation
Bromodeoxyuridine tablets were prepared as described Seehy et al.,
(1983) as follows:
Bromodeoxyuridine tablets were prepared by using pellet press (Parr
instrument co. Moline, III., USA), equipped with a 0.178 in diameter punch
and die. Approximately, 55 mg of pure Bromodeoxyuridine powder were
weight, placed in the die, and pressed. In order to maintain consistent
compaction hardness (and thus the dissolution rate) among tablets, utilization
of the same personal and die adjustment when pressing the powder was
conducted. BrdU tablets were protected from light and stored in a freezer until
usage.
Bromodeoxyuridine treatment
The mice were lightly anesthetized by placing it in a closed container
with ether until immobile (about 2 min). After removal from the container, each
mouse was restrained on its back. A small vial of anesthesia was placed near
its nose for use in prolonging the inactive state. The lower lateral region was
swabbed with alcohol in order to mat the fur down. Clean scissor or a scalpel
was used to make a small (approx. 1 cm) subcutaneous incision. In order to
spread open a deeper subcutaneous pocket, forceps were used, and the
tablet was inserted. The wound was then closed with 2 –3 outclip sutures
taking full care not to break the tablet, and the animal was returned to its
cage. Each animal had received 2ml of the extract 8 hr after BrdU treatment.
All animals were injected intravenously with 20 μg colcemid (1 mL/mouse, in
tail vain) at hr 19- (following BrdU treatment). Control marrow cells harvested
2 hr later revealed a high fraction of metaphases of optimal sister chromatid
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differentiation after staining. It was necessary to delay colocmid and harvest
steps for up to several hours when high concentrations were discovered to
slow cell cycling and resulted in poor sister chromatid differential staining.
Preparation and implantation of BrdU tablets are given in Figures (1a&b).
1 2 3
4 5
Figure (1a): Preparation of Brdu tablets.
1 2 3
Figure (1b): Implantation of Bromodeoxyuridine (BrdU) tablets.
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Marrow cells harvest, slide preparation and staining:
The procedure that described by Perry & Evans (1975) and Seehy et al.,
(1983) was used.
Screening of slides and analysis
Scanning slides for mitotic spreads was conveniently accomplished
with a 25 X magnification objective, and analysis was with a 100X objective.
For control of bias, all prepared slides were coded prior to scoring. There are
three ways for counting sister chromatid exchange frequencies i.e., (1) from
the microscope images of second division cells, (2) the cells may be
photographed and SCE frequencies counted from the microscope images. An
interstitial exchanged segment was counted to be two SCEs.(Seehy et al.,
1983).
RESULTS
Mitotic activity
Tables (1-19) illustrate the effect of Potato and Corn chips and of Burger
water extract upon cellular activity. The results showed that the water- soluble
fraction of these foods displayed cellular toxicity upon Allium cepa and Vicia
faba cells as well.
In Allium cepa, mitotic activity ranged from 12.9% to 16.21% in the
control group. It decreased to be 4.6, 3.2 and 1% for Potato chips at the
heighst tested concentration for different times of treatment.
Table (8) shows that, at the heighst tested concentration of Burger
(100g/L) , no divided cells were obtained, giving an evidence for complete
cellular toxicity after 12 hrs treatment time. After 24 hrs treatment time no
divided cells (Table,9) at the level of 50 and 100 g/L were observed. Table
(18) shows the effect of Burger upon Vicia faba cells. At the level of 40; 50;
and 100 g/L, no divided cells were observed, reflecting cellular toxicity of
water soluble fraction of Burger.
The present study showed that all tested water extract foods were
effective in causing significant decreases in cell proliferation in Allium cepa
root cells and Vicia faba as well.(Amara et al., 2008).
Chromosomal aberrations :
Fragments, ring chromosomes; stickiness; gaps; and C- Metaphases
were observed. They increased with the increasing of the tested concentration
as well as with the treatment time that ranged from 6 to 24 hrs. For example,
aberrant metaphase in Allium cepa (Table, 19) were 2% for the control group.
They ranged from 4.2 to 15.4% after 6 hrs treatment time, and from 2.3 to
21.50 after 12 hrs treatment time (Table, 20) , giving a strong evidence that
water-soluble fraction of potato chips was shown to be a positive clastogen.
Figures (1-10) illustrate the positive effect of the tested foods upon Allium
cepa and Vicia faba genome.
Cytological examination revealed that the percent of total aberrant
metaphases after treatment with the extraction of fast foods for 24 hrs. by
20gr/L is given in table 19. Percent of total aberrant metaphases in the control
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group was found to be 2 % and it decreased to be 18.4 after treatment with
corn chips and 22.7 after treatment with potato chips. High percentage of
stickiness was observed after treatment with Burger extract (10%). Percent of
total aberrant metaphases was found to be 31 % (15~ foldes compared that of
the with, the negative control).
This result, however, presented a strong evidence that the tested fast
foods have cellular toxicity and clastogenic effect. (This is in agreement with
that reported by Erikson (1981).
Stickiness observed in this work might be resulted from an alteration of
the net charge of chromosomal proteins and DNA or both (Seehy et al.,
2013).
The present work showed that these fast foods are positive clastogens
such a conclusion is in agreement with that reported by El-Sebeay (1999).
As shown in Table (21) sister chromatid exchanges were detected and
statistically analyzed. Data showed that only one dose (10g/L) from the three
tested extract of fast foods was negative, while three doses i.e., 20,30, and
40g/L were proven to be positive, giving an evidence that these fast foods are
capable in cousing primary DNA damage or mutagenic effect.
Table (1): Mitotic and phase indices in Allium cepa root cells after treatment with
potato chips extract for 6 hrs.
g/L *Ml Prophase Metaphase Anaphase Telophase
Control 13.84 4.20 5.01 3.10 1.53
10 11.42 6.00 2.02 2.30 1.10
20 10.66 6.20 2.01 1.8 0.61
30 10.14 7.02 1.86 1.06 0.20
40 10 7.12 1.82 1.02 0.04
50 8.29 7.24 0.23 0.41 0.42
100 3.6 3 - - 0.6
*Mitotic index.
Table (2): Mitotic index and phase indices in Allium cepa root cells after treatment
with potato chips for 12 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 15.21 5.01 6.00 3.00 1.20
10 10.14 7 2.14 1 0.50
20 10.8 7 1 1 1.8
30 9.01 8 1 0.01 -
40 8.02 7 1.02 - -
50 7.4 6.8 0.4 0.2 -
100 2.2 2.1 - - 0.1
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Table (3): Mitotic index and phase indices in Allium cepa root cells after treatment
with potato chips for 24 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 11.9 5.2 4.1 1.4 1.2
10 9.2 6 1.2 1.4 0.6
20 7.62 6.20 1.00 0.40 0.02
30 5 4.1 0.4 0.2 0.3
40 3 2.2 0.2 - 0.6
50 1 1.00 - - -
100 0 0 - - -
Table (4): Mitotic index and phase indices in Allium cepa root cells after treatment
with corn chips for 6 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 14.84 4.20 5.01 3.10 2.53
10 14.02 4.12 4.20 2 3.70
20 13.10 4.01 3.88 3.21 2
30 12.00 5.20 2.00 2.5 2.3
40 11.08 6.20 1.7 2.12 1.06
50 10.42 6.50 0.80 1.04 2.08
100 4.11 4.00 - - 0.11
Table (5): Mitotic index and phase indices in Allium cepa root cells after treatment
with corn chips for 12 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 15.21 5.01 6.00 3.00 1.20
10 11.1 6 3 1 1.1
20 9.1 6 2.1 0.2 0.8
30 8.2 6 1.2 0.6 0.4
40 5.2 4.2 - - 1
50 4.3 3.3 - - 1
100 1.1 1.1 - - -
Table (6): Mitotic index and phase indices in Allium cepa root cells after treatment
with corn chips for 24 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 11.9 5.2 4.1 1.4 1.2
10 10.3 4 3 2 1.3
20 8.4 4 1.4 2 1
30 6.2 4.2 1 1 -
40 3 1 1 0.6 0.4
50 1 0.8 - - 0.2
100 0.2 0.2 - - -
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Table (7): Mitotic index and phase indices in Allium cepa root cells after treatment
with Burger for 6 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 13.84 4.20 5.01 3.10 1.53
10 12.20 5.1 4.1 2 1
20 11.50 4.60 3.20 2.00 1.50
30 10.00 4.80 2.20 1.2 1.8
40 8.2 5.2 1.2 0.6 40
50 7.2 5.4 - 1.2 50
100 5 5.00 - - -
Table (8): Mitotic index and phase indices in Allium cepa root cells after treatment
with Burger for 12 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 15.21 5.01 6.00 3.00 1.20
10 8.4 5.6 1.4 0.60 0.44
20 7.04 4.8 1.2 0.24 0.8
30 5.21 2 1.2 1.01 1
40 3.1 1.1 - 1 1
50 1.1 1.1 - - -
100 - - - - -
Table (9): Mitotic index and phase indices in Allium cepa root cells after treatment
with Burger for 24 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 11.9 5.2 1 1.4 1.2
10 5.04 3.2 - 1 0.84
20 4.36 2.8 - 0.23 1.30
30 3.12 2.17 - 0.66 0.29
40 1.01 1.01 - - -
50 - - - - -
100 - - - - -
Table (10): Mitotic and phase indices in Vicia faba root cells after treatment with
potato chips extract for 6 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 8.12 1.00 4.52 1.20 1.40
10 7.01 2.00 3.00 1.00 1.01
20 6.92 2.10 2.02 1 1.80
30 5.22 3 1 0.4 0.82
40 3.28 3 - - 0.28
50 1.11 1 - - 0.11
100 - - - - -
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Table (11): Mitotic and phase indices in Vicia faba root cells after treatment with
potato chips extract for 12 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 10.20 2.6 5.2 1.4 1.00
10 6.60 2 2.2 1.3 1.1
20 6.2 3 1.2 1 1
30 5.3 3.4 0.6 - 1.3
40 2 2 - - -
50 0.02 0.02 - - -
100 - - - - -
Table (12): Mitotic and phase indices in Vicia faba root cells after treatment with
potato chips extract for 24 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 8.03 1.00 5.00 1.00 1.03
10 3.04 1 1 0.04 1.00
20 2.06 1 1 0.06 -
30 2.01 1.4 - 0.01 0.60
40 1.20 1.20 - - -
50 - - - - -
100 - - - - -
Table (13): Mitotic and phase indices in Vicia faba root cells after treatment with Corn
chips extract for 6 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 8.12 1.00 4.52 1.20 1.40
10 8.4 2 3.5 2.4 0.5
20 6.8 2 2 1.2 1.6
30 6.61 3.1 1.2 1.01 1.3
40 5.8 3.8 1 0.8 0.2
50 2.0 2.00 - - -
100 1.0 1.00 - - -
Table (14): Mitotic and phase indices in Vicia faba root cells after treatment with Corn
chips extract for 12 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 10.2 2.60 5.20 1.4 1.00
10 7.6 3.00 2.00 1.20 1.40
20 5.90 3.00 1 0.80 1.10
30 5.20 3.20 0.80 - 1.20
40 4.80 3.60 0.40 - 0.80
50 2 1.8 - - 0.20
100 0 0 - - -
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Table (15): Mitotic and phase indices in Vicia faba root cells after treatment with Corn
chips extract for 24 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 8.03 1.00 5.00 1.00 1.03
10 5.06 3.20 1.00 0.60 0.80
20 3.70 2.01 0.80 0.19 0.70
30 3.16 2.00 0.60 0.22 0.34
40 2.01 2.01 - - -
50 0 0 - - -
100 - - - - -
Table (16): Mitotic and phase indices in Vicia faba root cells after treatment with
Burger extract for 6 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 8.12 1.00 4.52 1.20 1.40
10 8.01 2.00 3.00 2.00 1.01
20 7.70 3.00 2.00 1.60 1.10
30 7.84 3.02 1.80 1.00 2.02
40 6.82 4.03 1.06 0.62 1.11
50 5.82 4.00 0.66 1.00 0.16
100 3 3 - - -
Table (17): Mitotic and phase indices in Vicia faba root cells after treatment with
Burger extract for 12 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 10.2 1.60 5.20 1.4 2.00
10 6.17 1.01 3.02 1.04 1.10
20 6.08 1.80 2.00 1.06 1.22
30 5.92 1.89 1.88 1.02 1.13
40 2.22 1.21 1.01 - -
50 1.00 1.00 - - -
100 - - - - -
Table (18): Mitotic and phase indices in Vicia faba root cells after treatment with
Burger extract for 24 hrs.
g/L Ml Prophase Metaphase Anaphase Telophase
Control 8.03 1.00 5.00 1.00 1.03
10 2.16 1.10 0.40 - 0.66
20 2.09 1.00 0.32 0.01 0.76
30 1.17 1.17 - - -
40 - - - - -
50 - - - - -
100 - - - - -
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Table (19): Chromosomal aberrations induced after treatment of Allium cepa roots
extraction with fast foods* for 24 hrs.
Percent of
total
aberrant
metaphases
Ring Stickiness Gap C-metaphase
chromosome
Types Fragment
Control - - 2 - - 2%
Potato 6.2 6.5 8 - 2 22.7
chips
Corn 3.4 6 6 1 2 18.4
chips
Burger 8 8 10 2 3 31
*20 g/L
Table (20): Chromosomal aberrations induced after treatment of Vicia faba roots with
fast food extraction *for 24hrs.
Percent of
total
aberrant
metaphases
C metaphase
Ring Stickiness Gap
chromosome
Types Fragment
Control - - 2 - - 2
Potato 8 7 4 4 2 25
chips
Corn 5.5 9.5 6 4 2 26
chips
Burger 11 12 13 2 4 32
*20 g/L
Table (21): Averages + S.E of sister chromatid exchanges in mice bone marrow cells
after oral treatment with the extract of the tested fast foods.
*Significant at 0.05 level of probability.
Dose; g/L X + SE Range
Control 2.1 + 0.1 1-3
Potato chips 10
20
30
40
2.2
3.4*
5.2*
8.4*
1-4
2-5
3-6
4-10
Corn chips 10
20
30
40
2.1
4.2*
4.6*
10.2*
1-4
2-6
2-8
4-12
Burger 10
20
30
40
2.4
4.2*
6.2*
12.4*
2-4
3-6
4-8
7-14
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1 2
3 4
5 6
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7 8
9 10
11 12
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13
Figure Description
1 Chromatid deletion A. cepa
2 Fragments V. faba
3 Fragments and Gap V. faba
4 Fragments , Chromatid deletion and Gap A.
cepa
5 Chromatid deletion V. faba
6 Binucleolei , V. faba
7 Normal chromosomes A.cepa
8 Chromatid deletion A.cepa
9 Chromatid deletion, V.faba
10 Chromatid deletion A.cepa
11 Fragments and Chromosome deletion, A.cepa
12 Sister chromatid exchange, Burger treatment
13 Sister chromatid exchange, Corn chips
DISCUSSION
This work aims at disclosing the possible genotoxic effect (clastogenic and
mutagenic as well) induced by some fast foods The recognition that diet may
be one of the most important factors explaining international variation in
cancer came when it was appreciated that for many cancers, diet seemed to
be the most rational explanation (Higginson and Muir, 1979), coupled with
studies at the macro-population level which indicated substantial correlations
between incidence or mortality of cancer at various sites and various
estimates of population consumption of dietary items (Armstrong and Doll,
1975; Knox, 1977). The explosion of interest that has followed, particulary in
the past ten years, has gradually led to a recognition that many of the
relationships are complex, involving not only factors that increase the risk of
cancer, but also protective factors. Further, we do not appear to be dealing
with simple expression of carcinogens and mutagens, rather diet seems to act
on both the early and late stages of carcinogenesis.
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The associations with dietary factors and cancer incidence or
mortality studied internationally, through strong, only point to the importance
of dietary factors in etiology, they cannot prove causation. For this, it is
necessary to conduct studies on diet in individuals, and then to confirm
causation and measure to impact of preventive actions, conduct intervention
studies in populations. For diet and nutrition, many studies on individuals have
been conducted, but so far studies on populations have been reported. The
studies on individuals are of two types, case-control and cohort. Case-control
studies are based on the disease, looking back to the relevant exposures.
There have been relatively few cohort studies, and most with information
available currently were originally planned to concentrate on cardiovascular
diseases. Case-control studies of diet and cancer have been more numerous,
leading us to evaluate diet largely in relation to individual cancer sites. These
vary from those in which diet seems to play a major role, such as stomach
and colon and rectum, through those to which diet seems to make a major
contribution, such as breast, prostate and many of the hormonally associated
cancers, to those with important, well-recognized non-dietary causative
factors to which dieter factors contribute to or modify risk, such as lung,
bladder and other smoking and/of alcohol- associated cancers (Miller, 1986).
However, anticarcinogens have been reported in other foods and are
postulated to mitigate the potentially deleterious effect of the low levels of
mutagens and carcinogens that are ingested daily (Ames, 1983; Wattenberg,
1983). In particular modulator-mediated inhibition of carcinogenesis is of
importance given that carcinogenic poly-nuclear arenes and heterocyclic
amines are found in some cooked foods. Since case-control epidemimiologic
investigations of the possible association of beef an other meats with
colorectal and breast cancers are at present unclear (Doll and Peto, 1981),
one might speculate that the modulator reported herein may under some
circumstance act to moderate the effects of low levels of carcinogens that
may be present, resulting in epidemiologic finding that are equivocal. Silalahi
(2002).
The present investigation showed that the tested fast foods were proven
to be clastogenic agents, since they were capable to interfere with spindle
fibers, and C-metaphases were observed, giving a strong evidence that the
water-soluble fraction of potato and corn chips as well as of Burger contained
contaminants capable to interact with chromosomes arid spindle fibers as
well. However they have mutagenic activity as sister chromatid exchanges
revealed obtained since positive frequencies of SCEs were observed.
In conclusion clastogenic and mutagenic effect of these fast foods, under
study, had been formed.
This positive clastogenic activity of water-soluble fraction of the
tested foods may be caused by acrylamide or by heterocyclic amine El-
Sebeay (1999) and / or other contaminants. However, this question was not
answered at the level of this work.