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Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 389
www.world-food.net
Journal of Food, Agriculture & Environment Vol.12 (2): 389-396. 2014
WFL Publisher
Science and Technology
Meri-Rastilantie 3 B, FI-00980
Helsinki, Finland
e-mail: info@world-food.net
Received 20 February 2014, accepted 20 April 2014.
Evaluation of the potential genotoxicity of antibiotics alternative probiotics used in
livestock and poultry
Mohammed Hamed Zeiny Mutwakil 1, Jamal Sabir Mohamed Sabir 1, Saleha Yahya Mohamed Al Akilli 1,
Aida I. El Makawy 2 and Mohamed Morsi Mohamed Ahmed 1, 3*
1 Department of Biological Sciences, Faculty of Science, P. O. Box 80203, King Abdulaziz University, Jeddah, 21589, Saudi
Arabia. 2 Department of Cell Biology, National Research Centre, EL Tahrir Street, 12622 Dokki, Giza, Egypt. 3 Department of
Nucleic Acid Research, Genetic Engineering and Biotechnology Research Institute, City for Scientific Researches and
Technology Application, Borg EL-Arab, P. O. Box. 21934, Alexandria, Egypt. *e-mail: mmmahmed6@yahoo.ca
Abstract
Probiotics used in poultry industry to overcome a ban for the use of antibiotic for disease prevention and growth enhancing supplements. Biomin
Imbo and Primalac are used as dietary commercial non-antibiotic growth promoter for poultry. The objective of this study was to evaluate the
potential genotoxicity of Biomin-Imbo and Primalac in mice. Male mice were feed basal diet supplemented with 1.5 and 3 g/kg of both Biomin Imbo
and Primalac for 6 weeks. Positive control animals were feed on basal diet and administered antibiotic neomycin in water. The study was conducted
to assess DNA damage by micronucleus test in bone marrow cells, DNA fragmentation by Diphenylamine (DPA) and DNA fingerprinting using
random amplified polymorphic DNA (RAPD-PCR) analysis in liver cells. Results revealed that Biomin and Primalac induced dose dependent
increase in the frequencies of micro nucleated polychromatic erythrocytes (MNPCEs), DNA fragmentation percentage and appearance of some
changes in polymorphism band patterns including deletion of stable band or insertion of new bands. Noteworthy that the increment in DNA damage
induced by both Biomin Imbo or Primalac not reach to those induced by neomycin. We concluded that the high dose repeated exposure of Biomin
Imbo and Primalac may have the potential to induce cytotoxic and moderate genotoxic effects in bone marrow cells of male mice.
Key words: Antibiotics alternatives, commercial probiotics supplements, poultry, DNA, fragmentation-MNPCEs-RAPD.
Introduction
Commercial poultry production ranks among the highest source
of animal protein 1. The increase in the size of the poultry industry
has been faster than other food-producing animal industries. The
trade volume of poultry products has also increased parallel to
the rapid growth of global poultry meat and egg production 2.
With the current advent of excluding antibiotic growth promoters
in poultry production, the issue of controlling enteric infections
caused by pathogenic bacteria without the use of antibiotics
becomes challenging. Mortality caused by infection is a major
problem in the poultry industry. Such infections are responsible
for reduced growth rates and consequent economic losses in
poultry. Antibiotics are the main tools utilised to prevent or treat
such infections. Antibiotics are also added to the feed as growth
promoters to accelerate the growth of healthy animals 3.
In face of debate about the use of antibiotics as growth promoters,
due to the probable relationship with resistance to antibiotics
used in human medicine, the presence of antibiotic residues in
products of animal origin intended for human consumption and
the emergent demand from consumer market for products free
from additive residues, it was necessary to search for alternative
products that could replace antibiotics used as promoters, without
causing losses to productivity or product quality 4.
In order to find better alternatives to antibiotic growth
promoters, research has focused on utilisation of feed additives
such as enzymes, probiotics, prebiotics, symbiotic products and
even nutrition to enhance gut health in poultry and prevent or
limit production losses due to enteric infections 3. Probiotics used
in animal feed are becoming accepted as potential alternatives to
antibiotics for use as growth-promoters for control of specific
enteric pathogens 5.
Probiotics are live microorganisms, which will have beneficial
effect to the host animal by improving its intestinal microbial
balance through inhibiting intestinal pathogens (E. coli).
In broiler nutrition, probiotic species belonging to Lactobacillus,
Streptococcus, Bacillus, Bifidobacterium, Enterococcus,
Aspergillus, Candida, and Saccharomyces have a beneficial effect
on broiler performance 6, 7 modulation of intestinal microflora and
pathogen inhibition 8, intestinal histological changes 9,
immunomodulation certain haematobiochemical parameters 10,
improving sensory characteristics of dressed broiler meat 11 and
promoting microbiological meat quality of broilers 9. A great deal
of attention has recently been received from nutritionists and
veterinary experts for proper utilization of nutrients and the use of
probiotics for growth promotion of poultry 12-16.
Biomin - Imbo is a symbiotic natural growth promoter of which
can be administered in the feed and water respectively. Biomin
Imbo contains a stabilized probiotic strain of Enterococcus
faecium, the prebiotic fructo - oligosaccharides which out compete
pathogens and the immune - stimulating substances like cell wall
fragments 17, enhanced micro flora compositions in the gut and
reduced mortality 18. Primalac is a kind of commercial probiotic
that contains at least 1 × 108 CFU/g-1 Lactobacillus acidophilus,
390 Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014
Lactobacillus casei, Enterococcus faecium and Bifidobacterium
thermophilum 19. Several studies have shown that use of probiotics
additives in the ration, improves the performance of broiler
chickens 20. Piray et al. 15 and Singh et al. 23 reported that the use
of Primalac enhanced the broiler chicken performance by improving
body weight and decreasing the feed conversion ratio.
The search for new additives effective on animal’s growth and
free from harmful side effects on consumer’s health is still
continuing. A number of methods that detect damage in DNA
have been used to identify genotoxic substances. The
micronucleus test on polychromatic erythrocytes (PCEs) is a
standard chromosome mutagenicity test. A micronucleus is the
result of a chromosome not united with the mitotic spindle or a
chromosome fragment without a centromere. Physical, chemical
or biological processes that interfere in the binding of the
chromosome to the microfibrils of the spindle (aneugenic) and
those that break down chromosomes (clastogenic) induce the loss
of genetic material, leading to genotoxicity or mutagenicity 22. DNA
based assays such as RAPD are the most widely used tools for
assessment of the genetic variation due to use of RAPD for the
detection of DNA damage presents a number of advantages 23.
To date, large number of literatures has been published on the
health beneficial effects of probiotics. The studies vary widely in
quality and have been conducted with a range of probiotic strains,
health benefits, and target populations 24, 25. In 2010, representatives
of the European Food Safety Authority (EFSA) met in Amsterdam
with scientists and representatives of the food industry to discuss
foods that may promote gut health. EFSA had rejected all health
claims for such foods - or rather, advised negatively about such
claims to the European Commission 26.
The objective of this study was to evaluate the potential
genotoxic effects of dietary commercial alternative non-antibiotic
supplements (probiotics) Biomin-Imbo and Primalac that used for
poultry growth promotors.
Materials and Methods
Probiotics: Biomin Imbo was delivered by GmbH, Herzogenburg,
Austria. Primalac was delivered by Star Labs, Clarksdale, MO, USA.
Diets: The basal diet was formulated to meet the nutrient
requirements of male mice. Commercial supplements of Biomin
Imbo and Primalac was added to the basal diet free from antibiotic
and administered throughout the study as recommended by the
manufacturer 1.5 g/kg and its double.
Animals: Sixty adult Swiss albino male mice weighing 25 ± 5 g
were taken from national research centre animal house and used
for the experiments. Animals were kept under standard
conditions of humidity (60 - 70%), temperature (25 ± 2°C) and a
controlled 12 h light/dark cycle. Animals were acclimatized for 7
days to the experimental animal room conditions. The standard
laboratory chow mixed with the commercial probiotics
supplements and tab water were provided ad libitum. The study
was approved by the Institutional Animal Ethics Committee for
“Care and Use of Laboratory Animals for medical Research” of
national research center.
Experimental design: The animals were divided to six experimental
groups containing ten mice each. The experimental groups were
as follow:
Group 1: Animal were feed basal diet without additive and used as
negative control;
Group 2: Animals were feed on basal diet and administered
antibiotic neomycin in water at dose of 100 mg/litre and
used as positive control;
Group 3: Animals feed basal diet supplemented with 1.5 g/kg
Biomin Imbo;
Group 4: Animals feed basal diet supplemented with 3 g/kg
Biomin Imbo;
Group 5: Animals feed basal diet supplemented with 1.5 g/kg
Primalac;
Group 6: Animals feed basal diet supplemented with 3 g/kg Primalac.
All animals treated for 6 weeks then all of experimental and
control animals were sacrificed 24 h after the last dose.
Scientific methodology:
Mammalian erythrocyte micronucleus test: Animals were killed
by cervical dislocation, and two femurs are removed and stripped
clean of muscle. Bone marrow slides were prepared according to
the method described by Krishna and Hayashi 27. The marrow is
removed by making a small opening at the iliac end of the femur
and introducing the pointed shaft of a 2.5-cm safety pin into the
femur at the epiphysial end. The marrow is placed directly on a
slide, and then a drop of fetal calf serum is added. With the aid of
the edge of a clean slide, the marrow is mixed with the serum until
homogeneous and then is spread as a smear; additional slides
from a given animal can be prepared by simply transferring some
of the mixed preparation onto other slides. Prepared slides are air-
dried, fixed for 5 min in absolute methanol, followed by staining in
May-Grunwald-Giemsa for 5 min then washed in distilled water
and mounted. For each animal, 2000 polychromatic erythrocytes
(PCEs) were scored per animal and the numbers of micronucleated
PCEs were recorded. The number of PCEs among 1000 total
erythrocytes (PCE + NCE) per animal was recorded to evaluate
bone marrow cytotoxicity. The ratio of polychromatic to
normochromatic erythrocytes (PCE/NCE) was calculated.
DNA fragmentation by Diphenylamine (DPA) assay: Liver samples
were collected immediately after sacrificing the animals. The tissues
were lysed in 0.5 ml of lysis buffer, that contain 10 mM tris-HCl (pH
8), 1 mM EDTA, 0.2% triton X-100, centrifuged at 10,000 rpm
(Eppendorf) for 20 min at 4°C. The pellets were resuspended in 0.5
ml of lysis buffer. To the pellets (P) and the supernatants (S), 1.5 ml
of 10% trichloroacetic acid (TCA) was added and incubated at 4°C
for 10 min. The samples were centrifuged for 20 min at 10,000 rpm.
(Eppendorf) at 4°C and the pellets were suspended in 750 µl of 5%
TCA, followed by incubation at 100°C for 20 min. Subsequently, to
each sample 2 ml of DPA solution [200 mg DPA in 10 ml glacial acetic
acid, 150 µl of sulfuric acid and 60 µl acetaldehyde] was added and
incubated at room temperature for 24 h 28. The proportion of
fragmented DNA was calculated from absorbance reading at 600
nm using the equation:
DNA fragmentation = OD of fragmented DNA (supernatant) / [OD of
fragmented DNA (supernatant) + OD of intact DNA (pellet)] × 100
Random amplified polymorphic DNA (RAPD):
Genomic DNA isolation: The frozen liver tissue was thawed.
Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 391
Approximately 100 mg of frozen liver tissue ground and
homogenized in 1 ml of tissue lysis buffer and 5 µl proteinase K
then incubated at 65°C for 1 hr. The genomic DNA was then isolated
using phenol/chloroform extraction and ethanol precipitation
method with minor modifications according to Sambrook et al. 29.
The concentration and purity of extracted genomic DNA were
determined by spectrophotometer based on the absorbance at
260 and 280 nm 30.
Random amplified polymorphic DNA (RAPD) analysis: To
generate RAPD profiles from the mice DNA, a set of seven primers
procured from Operon technologies (Operon, Almeda, CA, USA)
randomly selected were used in RAPD analysis. DNA amplification
reactions were performed under conditions reported by Williams
et al. 31. PCR amplification was conducted in 50 µl reaction volume
containing 100 ng genomic DNA, 100 µM dNTPs, 40 nM primer,
2.5 units of Taq DNA polymerase and 5 µl promega 10X Taq DNA
polymearse buffer. The PCR reactions were carried out in thermal
cycler (Perkin-Elmer 9700) programmed with a first denaturation
of 5 min at 94°C, followed by 45 cycles of 1 min denaturation at
95°C, 1 min annealing at 36°C and 2 min extension at 72°C. Final
extension at 72°C for 5 min was allowed before holding the reaction
at 4°C for 10 min. Reaction products were stored at 4°C prior to
electrophoresis. PCR amplified products of RAPD primers were
detected using agarose gel electrophoresis (1.8% W/V, Bio-Rad,
California) in 1X TBE buffer) at 80 V for 1 h, then visualised by
staining with ethidium bromide (2 µl/100 ml).
Statistical analysis: Data in all experiments was subjected to
ANOVA using SPSS for Windows version 11.0, statistical software.
Variable means for treatments indicating significant differences
were compared and the significances were indicated using Duncan
multiple range tests.
Results
The capacity of the antibiotic alternative probiotics to induce
chromosome mutations was analysed using the polychromatic
erythrocytes micronucleus test in male mice. The results of MN
tests are illustrated in (Fig. 1). Results showed that Biomin Imbo
and Primalac commercial supplements tested doses (1.5 and 3 g/
kg diet) caused statistically significant increase in the frequencies
of MNPCEs over the control value but not reach to the mean
value of neomycin. Meanwhile, neomycin significantly increases
the frequency of MNPCEs over control, Biomin Imbo and Primalac
tested doses.
Proportion of PCEs: It is evident from the results of the present
study that neomycin significantly decreased the percentage of
polychromatic erythrocytes (PCE)/normochromatic erythrocytes
(NCE) than those of Biomin Imbo, Primalac and control (Fig. 2).
Meanwhile, Biomin Imbo and Primalac significantly decreased the
percentage of PCE/NCE than control. Whereas, Biomin Imbo and
Primalac induced significant increase in the percentage of PCE/
NCE ratio than that of neomycin. It means that Biomin Imbo and
Primalac cytotoxicity less than that of neomycin. The decrease in
the percentage of PCE/NCE indicates a suppression of bone
marrow proliferation most likely by mitotic arrest.
DNA fragmentation by Diphenylamine (DPA): Biomin Imbo,
Primalac and neomycin induced DNA damage in liver cells was
evaluated by measuring the level of fragmented DNA colorimetric
using Diphenylamine (DPA). The results showed that neomycin
significantly increased the percentage of DNA fragmentation in
liver cells (17.56 + 0.25) compared to control untreated cells (5.65 +
0.13), Biomin Imbo (11.85 + 0.21 and 13.43 ± 0.68) and Primalac
(9.78 + 0.40 and 10.82 + 0.36) tested doses, respectively. Meanwhile,
treatment with both commercial supplements diets significantly
increased the percentage of DNA fragmentation as compare to
control. While, they showed significantly decrease in the percentage
of DNA fragmentation than neomycin (Table 1 and Fig. 3).
Control
Neomycin
1.5 g/kg
Biomin Imbo
1.5 g/kg
Primalac
3 g/kg
Biomin Imbo
3 g/kg
Primalac
No. of MNPCEs
0
8
17.33
11.67 10.67
13.33
12
2
4
6
8
10
12
14
16
18
Frequency
Figure 1. Effect of Biomin Imbo and Primalac on the frequency of
MNPCEs in bone marrow cells of male mice.
Control
Neomycin
1.5 g/kg Biomin Imbo
1.5 g/kg Primalac
3 g/kg Biomin Imbo
3 g/kg Primalac
DNA fragmentation (%)
0
9.78
13.43
10.82
2
4
6
8
10
12
14
16
18
Percentage %
17.56
11.85
5.65
Figure 3. DNA fragmentation by Diphenylamine (DPA) assay in liver
cells of mice.
Control
Neomycin
1.5 g/kg
Biomin Imbo
1.5 g/kg
Primalac
3 g/kg
Biomin Imbo
3 g/kg
Primalac
Ratio of PCEs and NCEs (%)
0
6.14
22.65
12.27
14.81
12.23
5
10
15
20
25
Percentage %
13.79
Figure 2. The ratio of PCE/NCE in bone marrow of male mice treated
by Biomin Imbo and Primalac.
Experimental groups DNA fragmentation %
M ± SE
Change % as
compare to control
Control 5.65 + 0.13 a -
Neomycin 17.56 + 0.25 e +11.91
1.5 g Biomin Imbo/kg diet 11.85 + 0.21 c +6.20
1.5 g Primalac/kg diet 9.78 + 0.40 b +4.13
3 g Biomin Imbo/kg diet 13.43 + 0.68 d +7.78
3 g Primalac/kg diet 10.82 + 0.36 bc +5.17
Table 1. Percentage of DNA fragmentation induced by Biomin
Imbo, Primalac and neomycin in liver cells of male mice.
Values with different superscript letters within columns represent significant statistical differences (P ≤ 0.05).
392 Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014
Random amplified polymorphic DNA (RAPD): The molecular
genetic variability among the treated mice genomes and their
control were evaluated using seven oligodecamers (10-mer random
primers). Out of which 3 (OPA09, OPC02 and OPC06) yielded
monomorphic bands, and 4 (OPA07, OPA08, OPB10 and OPC05)
yielded polymorphic bands. The total number of amplification
products generated by these individual primers and variable
fragments in treated and untreated controls are described in
(Tables 2 and 3), and representative RAPD fingerprints are shown
in (Fig. 4A - G). They produced a total of 47 loci of different bands,
with an average of 6.7 loci/primer. Of the 47 loci that were amplified,
13 bands were polymorphic giving (27.65%) polymorphism, the
bands were in the molecular weights range from 172 to 1450 bp.
Primers OPA09 and OPA08 amplified the minimum and maximum
number of bands which were 4 and 11 bands, respectively. The
quantitative analysis of those bands, expressed as percentage of
altered bands, shows an increment of modified bands in all treated
groups as compared with control one. At the neomycin treated
group 10 bands were modified which represent 21.2%. While at
the Biomin Imbo 1.5 and 3 g/kg diet treated mice 8 and 9 bands
were modified which represent 17.02% and 19.14%, respectively.
Treatment with 1.5 and 3 g/kg diet of Primalac 7 altered bands
were detected in each group, which represents 14.89% (Fig. 5).
Discussion
Several studies and reviews have been published on the health
beneficial effects of probiotics. Probiotics and their supplementation
in the poultry diet have been found to improve the productive
performance 32, 33, improve feed conversion ratio, augment egg
production and quality, stimulate immune response and increase
bone strength 34. It is noteworthy, however, that the health
beneficial properties of probiotics are strain-specific 26.
The present study was conducted to assess the ability of the
two tested antibiotic alternative commercial supplements
probiotics (Biomin Imbo and Primalac) to induce genotoxic activity
in male mice. Antibiotics act as growth factors by killing bacteria
through inhibiting a bacterium’s ability to turn glucose into energy,
or its ability to construct its cell wall, but could also be harmful to
host cells 35. The finding in the present study showed that high
levels of DNA damage occurred in the antibiotic neomycin treated
groups. This result was in agreement with Sengul et al. 36 and
Yurtseven et al. 37, they indicated that neomycin increased the
percentage of DNA damage. Rocco et al. 38 reported that
erythromycin and lincomycin induced genotoxic effect that
represented in a significant increase in DNA migration (tail moment)
and micronuclei frequency. Results of the current study revealed
that Biomin Imbo and Primalac significantly increased the
frequency of micronucleated polychromatic erythrocytes.
Primers Sequence (5’- 3’) GC (%) Total number
of band studied
Number of
polymorphic bands
Polymorphism
(%)
Size range (bp)
Max Min
OPA07 GAAACGGTG 60 9 1 11.1 1036 172
OPA08 GTGACGAGG 60 11 9 81.8 1343 212
OPA09 GGGTAACGCC 70 4 - - 932 395
OPB10 CTGCTGGGAC 70 6 1 16.6 1073 397
OPC02 GTGAGGCGTC 70 6 - - 1159 172
OPC05 GATGACCGCC 70 5 2 40 1171 457
OPC06 GAACGGACTC 60 6 - - 1450 295
Total 47 13 27.65 1450 172
Table 2. Sequence of selected random primers, number of total bands and percentage of Polymorphisms
calculated from liver cells samples exposed to growth stimulating additives and control.
Table 3. Comparison of RAPD fingerprinting profiles of different
mice genomic DNA from liver cells exposed to all
experimental treatments.
Biomin Imbo Primalac
Primer
Size of
bands
(bps)
Control Neomycin 1.5
g/kg
3.0
g/kg
1.5
g/kg
3.0
g/kg
1036 +,+ +,+ +,+ +,+ +,+ +,+
872 +,+ +,+ +,+ +,+ +,+ +,+
566 +,+ +,+ +,+ +,+ +,+ +,+
508 +,+ +,+ +,+ +,+ +,+ +,+
476 +,+ +,+ +,+ +,+ +,+ +,+
311 +,+ +,+ +,+ +,+ +,+ +,+
280 -,- -,+ -,- +,+ +,+ +,-
262 +,+ +,+ +,+ +,+ +,+ +,+
OPA07
172 +,+ +,+ +,+ +,+ +,+ +,+
1343 -,- -,- -,- +,+ -,- -,-
1301 +,+ +,- -,- -,- -,- -,-
1135 +,+ +,+ +,- +,+ -,- -,-
1021 +,+ +,+ +,- -,- +,+ +,+
722 +,+ +,+ +,+ +,+ +,+ +,+
575 -,- +,+ -,- +,+ -,- -,-
560 +,+ -,- -,- -,- -,- -,-
480 -,- +,+ +,- -,- -,- -,-
396 +,+ -,- +,+ +,+ +,+ +,+
382 -,- +,+ -,- -,- -,- -,-
OPA08
212 +,+ +,+ +,+ +,+ +,+ +,+
932 +,+ +,+ +,+ +,+ +,+ +,+
540 +,+ +,+ +,+ +,+ +,+ +,+
447 +,+ +,+ +,+ +,+ +,+ +,+
OPA09
395 +,+ +,+ +,+ +,+ +,+ +,+
1073 +,+ +,+ +,+ +,+ +,+ +,+
932 -,- +,+ +,+ +,+ +,+ +,+
809 +,+ +,+ +,+ +,+ +,+ +,+
562 +,+ +,+ +,+ +,+ +,+ +,+
477 +,+ +,+ +,+ +,+ +,+ +,+
OPB10
397 +,+ +,+ +,+ +,+ +,+ +,+
1159 +,+ +,+ +,+ +,+ +,+ +,+
990 +,+ +,+ +,+ +,+ +,+ +,+
562 +,+ +,+ +,+ +,+ +,+ +,+
447 +,+ +,+ +,+ +,+ +,+ +,+
258 +,+ +,+ +,+ +,+ +,+ +,+
OPC02
172 +,+ +,+ +,+ +,+ +,+ +,+
1171 -,- +,+ +,+ +,+ +,+ +,+
748 -,- +,+ +,+ +,+ +,+ +,+
638 +,+ +,+ +,+ +,+ +,+ +,+
562 +,+ +,+ +,+ +,+ +,+ +,+
OPCO5
457 +,+ +,+ +,+ +,+ +,+ +,+
1450 +,+ +,+ +,+ +,+ +,+ +,+
1268 +,+ +,+ +,+ +,+ +,+ +,+
1141 +,+ +,+ +,+ +,+ +,+ +,+
748 +,+ +,+ +,+ +,+ +,+ +,+
422 +,+ +,+ +,+ +,+ +,+ +,+
OPC06
295 +,+ +,+ +,+ +,+ +,+ +,+
Note: + For band presence and - for band absence; two specimens in each test group.
Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 393
Figure 4. Comparison of RAPD fingerprinting profiles of different male mice genomic DNA.
(A) represents PCR products with primer OPA07. (B) Represents PCR products with primer OPA08. (C) Represents PCR
products with primer OPA09. (D) Represents PCR products with primer OPB10. (E) Represents PCR products with
primer OPC02. (F) Represents PCR products with primer OPC05. (G) Represents PCR products with primer OPC06.
(M) DNA molecular size marker (100 bp DNA ladder). Lanes 1 and 2 represent negative control. Lanes 3 and 4 represent
positive control (neomycin). Lanes 5 and 6 represent mouse samples treated with 1.5 g/kg diet Biomin Imbo. Lanes 7 and
8 represent mouse samples treated with 3 g/kg diet Biomin Imbo. Lanes 9 and 10 represent mouse samples treated with1.5
g/kg diet primalac. Lanes 11 and 12 represent mouse samples treated with 3 g/kg diet primalac.
200
100
M1 234567
8910 11 12
1000
900
800
700
600
500
400
300
G
200
100
M1 234 567 8910 11 12
1000
900
800
700
600
500
400
300
A
M1 2 3 4 5 6 7 8 9 10 11 12
1000
900
800
700
600
500
400
300
200
100
B
M1 234567
89101112
1000
900
800
700
600
500
400
300
200
100
C
M1 234567891011 12
1000
900
800
700
600
500
400
300
D
200
100
M1
234567
8910 11 12
1000
900
800
700
600
500
400
300
E
M1 234 56 7 89101112
1000
900
800
700
600
500
400
300
F
394 Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014
Noteworthy, that this increment with that antibiotics less than
with neomycin. It is well established that micronuclei are formed
from the entire chromosome or from a fragment of it 39. Such
micronuclei are induced by genotoxic stress such as clastogen or
aneugen. Micronuclei induction by clastogen involves the
induction of either chromosome fragments that lag behind the
separating chromosomes or a chromatin bridge between
chromosomes at the anaphase of mitosis. On the other hand,
aneugen induces the whole chromosomes that were not bound to
the mitotic spindle at anaphase, probably by disrupting the spindle
checkpoint. Such chromatin is separated from the newly forming
nucleus and forms an independent nucleus-like structure, the
micronucleus. Therefore, methods to measure the frequency of
micronuclei are widely used in genotoxic test used to measure
efficacy of newly developed pharmaceuticals 40. Moreover, the
micronuclei test used to detects cytotoxic effects by measuring
the PCE/NCE relationship. When normal proliferation of the bone
marrow cells is affected by a toxic agent, the number of immature
erythrocytes (PCE) is prejudiced in relation to mature erythrocytes
(NCE), thus, the PCE/NCE ratio may decrease 41. Our results
indicated that Biomin exhibited a significant reduction of PCE/
NCE related to dose. However, Primalac high dose only reduced
the PCE/NCE ratio. These indicate that theses supplements have
the ability to induce cytotoxicity. Biomin and Primalac were finding
to induced apoptosis in mice hepatocytes as demonstrated by
DNA fragmentation. Meanwhile, increased the percentage of DNA
fragmentations not reach to that of neomycin. Hausmann 42
mentioned that apoptosis could be a strategy of microbial
pathogens to escape from the infected and exhausted host cell to
invade deeper mucosal layers for a prolonged bacterial
colonization. In addition, Sylvia et al. 43 found that some strains
of lactic acid bacteria (LAB) had potential ability to induce cancer
cells into apoptosis.
Recently, the RAPD technique has been successfully used to
detect DNA effects induced by several compounds under in vitro
and in vivo conditions. RAPD profiles detect alterations to
genomic DNA through the use of arbitrarily primed PCR reactions.
These effects include changes in oligonucleotide priming sites
and variations in the activity of the Taq DNA polymerase. In the
present study, variation in band intensity, disappearance of bands,
and appearance of new PCR products occurred in profiles
generated from the treated animals. These effects may be
correlated with structural rearrangements in DNA caused by
different types of DNA damages 44. The variation in band intensity
and disappearance of some bands may correlate with level of
photoproducts in DNA templates after treatment, which can reduce
the number of binding sites for Taq polymerase. Appearance of
new bands can be explained as the result of DNA structural
changes (breaks, transpositions, deletions) 45. This variation
among different samples may be due to the different chemical
compositions of supplements and at the same time of their
manufacturing processes. Previous studies have shown that
changes in band patterns observed in DNA fingerprint analysis
reflect DNA alterations from single base changes (point mutation)
to complex chromosomal rearrangements 46. In the same way, cell
populations exposed in vitro to genotoxins suffer DNA alterations
in a certain number of cells, which are reflected as variations in the
fingerprint obtained for the control population. These are defined
as band losses or gains as well as alterations in the intensity of
amplification of some of them. Such alterations in vivo are
considered mutations that are produced by changes to deletions
of or insertions into the pair bases 47, 48 confirmed the therapeutic
potential of the E. coli Nissle 1917 strain in promoting gut
homeostasis upon mucosal injury. However, they showed that E.
coli Nissle 1917 harbours a cluster of genes coding for the
biosynthesis of hybrid nonribosomal peptide-polyketide(s). This
biosynthetic pathway confers the ability for bacteria to induce
DNA double strand breaks in eukaryotic cells. Genetic alterations
in somatic and germ cells are associated with serious health effects,
which in principle may occur even at low exposure levels.
Accumulation of DNA damage in somatic cells has also been
proposed to play a role in degenerative conditions such as
accelerated aging, immune dysfunction, cardiovascular and
neurodegenerative diseases 49-51. DNA damage is also commonly
regarded as a marker of cancer risk. It is clearly relevant to cancer
since DNA damage is the initiating event in carcinogenesis 52.
Conclusions
We concluded that the antibiotic alternative commercial
supplements probiotics may have the potential to induce cytotoxic
and moderate genotoxic effects in bone marrow cells of mice.
Acknowledgements
This study was funded by the Deanship of Scientific Research
(DSR), King Abdulaziz University, Jeddah, under Grant No. (506/
130/1432). The authors, therefore, acknowledge with thanks DRS
technical and financial support.
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