Evaluation of the potential genotoxicity of antibiotics alternative probiotics used in livestock and...

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 PublisherScience and Technology

Meri-Rastilantie 3 B, FI-00980Helsinki, Finlande-mail: [email protected]

Received 20 February 2014, accepted 20 April 2014.

Evaluation of the potential genotoxicity of antibiotics alternative probiotics used inlivestock 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, SaudiArabia. 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 andTechnology Application, Borg EL-Arab, P. O. Box. 21934, Alexandria, Egypt. *e-mail: [email protected]

AbstractProbiotics used in poultry industry to overcome a ban for the use of antibiotic for disease prevention and growth enhancing supplements. BiominImbo and Primalac are used as dietary commercial non-antibiotic growth promoter for poultry. The objective of this study was to evaluate thepotential 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 Imboand Primalac for 6 weeks. Positive control animals were feed on basal diet and administered antibiotic neomycin in water. The study was conductedto assess DNA damage by micronucleus test in bone marrow cells, DNA fragmentation by Diphenylamine (DPA) and DNA fingerprinting usingrandom amplified polymorphic DNA (RAPD-PCR) analysis in liver cells. Results revealed that Biomin and Primalac induced dose dependentincrease in the frequencies of micro nucleated polychromatic erythrocytes (MNPCEs), DNA fragmentation percentage and appearance of somechanges in polymorphism band patterns including deletion of stable band or insertion of new bands. Noteworthy that the increment in DNA damageinduced by both Biomin Imbo or Primalac not reach to those induced by neomycin. We concluded that the high dose repeated exposure of BiominImbo 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.

IntroductionCommercial poultry production ranks among the highest sourceof animal protein 1. The increase in the size of the poultry industryhas been faster than other food-producing animal industries. Thetrade volume of poultry products has also increased parallel tothe rapid growth of global poultry meat and egg production 2.With the current advent of excluding antibiotic growth promotersin poultry production, the issue of controlling enteric infectionscaused by pathogenic bacteria without the use of antibioticsbecomes challenging. Mortality caused by infection is a majorproblem in the poultry industry. Such infections are responsiblefor reduced growth rates and consequent economic losses inpoultry. Antibiotics are the main tools utilised to prevent or treatsuch infections. Antibiotics are also added to the feed as growthpromoters 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 antibioticsused in human medicine, the presence of antibiotic residues inproducts of animal origin intended for human consumption andthe emergent demand from consumer market for products freefrom additive residues, it was necessary to search for alternativeproducts that could replace antibiotics used as promoters, withoutcausing losses to productivity or product quality 4.

In order to find better alternatives to antibiotic growthpromoters, research has focused on utilisation of feed additivessuch as enzymes, probiotics, prebiotics, symbiotic products andeven nutrition to enhance gut health in poultry and prevent or

limit production losses due to enteric infections 3. Probiotics usedin animal feed are becoming accepted as potential alternatives toantibiotics for use as growth-promoters for control of specificenteric pathogens 5.

Probiotics are live microorganisms, which will have beneficialeffect to the host animal by improving its intestinal microbialbalance 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 effecton broiler performance 6, 7 modulation of intestinal microflora andpathogen inhibition 8, intestinal histological changes 9,immunomodulation certain haematobiochemical parameters 10,improving sensory characteristics of dressed broiler meat 11 andpromoting microbiological meat quality of broilers 9. A great dealof attention has recently been received from nutritionists andveterinary experts for proper utilization of nutrients and the use ofprobiotics for growth promotion of poultry 12-16.

Biomin - Imbo is a symbiotic natural growth promoter of whichcan be administered in the feed and water respectively. BiominImbo contains a stabilized probiotic strain of Enterococcusfaecium, the prebiotic fructo - oligosaccharides which out competepathogens and the immune - stimulating substances like cell wallfragments 17, enhanced micro flora compositions in the gut andreduced mortality 18. Primalac is a kind of commercial probioticthat 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 Bifidobacteriumthermophilum 19. Several studies have shown that use of probioticsadditives in the ration, improves the performance of broilerchickens 20. Piray et al. 15 and Singh et al. 23 reported that the useof Primalac enhanced the broiler chicken performance by improvingbody weight and decreasing the feed conversion ratio.

The search for new additives effective on animal’s growth andfree from harmful side effects on consumer’s health is stillcontinuing. A number of methods that detect damage in DNAhave been used to identify genotoxic substances. Themicronucleus test on polychromatic erythrocytes (PCEs) is astandard chromosome mutagenicity test. A micronucleus is theresult of a chromosome not united with the mitotic spindle or achromosome fragment without a centromere. Physical, chemicalor biological processes that interfere in the binding of thechromosome to the microfibrils of the spindle (aneugenic) andthose that break down chromosomes (clastogenic) induce the lossof genetic material, leading to genotoxicity or mutagenicity 22. DNAbased assays such as RAPD are the most widely used tools forassessment of the genetic variation due to use of RAPD for thedetection of DNA damage presents a number of advantages 23.

To date, large number of literatures has been published on thehealth beneficial effects of probiotics. The studies vary widely inquality and have been conducted with a range of probiotic strains,health benefits, and target populations 24, 25. In 2010, representativesof the European Food Safety Authority (EFSA) met in Amsterdamwith scientists and representatives of the food industry to discussfoods that may promote gut health. EFSA had rejected all healthclaims for such foods - or rather, advised negatively about suchclaims to the European Commission 26.

The objective of this study was to evaluate the potentialgenotoxic effects of dietary commercial alternative non-antibioticsupplements (probiotics) Biomin-Imbo and Primalac that used forpoultry growth promotors.

Materials and MethodsProbiotics: 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 nutrientrequirements of male mice. Commercial supplements of BiominImbo and Primalac was added to the basal diet free from antibioticand administered throughout the study as recommended by themanufacturer 1.5 g/kg and its double.

Animals: Sixty adult Swiss albino male mice weighing 25 ± 5 gwere taken from national research centre animal house and usedfor the experiments. Animals were kept under standardconditions of humidity (60 - 70%), temperature (25 ± 2°C) and acontrolled 12 h light/dark cycle. Animals were acclimatized for 7days to the experimental animal room conditions. The standardlaboratory chow mixed with the commercial probioticssupplements and tab water were provided ad libitum. The studywas approved by the Institutional Animal Ethics Committee for“Care and Use of Laboratory Animals for medical Research” ofnational research center.

Experimental design: The animals were divided to six experimentalgroups 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 andused as positive control;

Group 3: Animals feed basal diet supplemented with 1.5 g/kgBiomin Imbo;

Group 4: Animals feed basal diet supplemented with 3 g/kgBiomin Imbo;

Group 5: Animals feed basal diet supplemented with 1.5 g/kgPrimalac;

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 killedby cervical dislocation, and two femurs are removed and strippedclean of muscle. Bone marrow slides were prepared according tothe method described by Krishna and Hayashi 27. The marrow isremoved by making a small opening at the iliac end of the femurand introducing the pointed shaft of a 2.5-cm safety pin into thefemur at the epiphysial end. The marrow is placed directly on aslide, and then a drop of fetal calf serum is added. With the aid ofthe edge of a clean slide, the marrow is mixed with the serum untilhomogeneous and then is spread as a smear; additional slidesfrom a given animal can be prepared by simply transferring someof the mixed preparation onto other slides. Prepared slides are air-dried, fixed for 5 min in absolute methanol, followed by staining inMay-Grunwald-Giemsa for 5 min then washed in distilled waterand mounted. For each animal, 2000 polychromatic erythrocytes(PCEs) were scored per animal and the numbers of micronucleatedPCEs were recorded. The number of PCEs among 1000 totalerythrocytes (PCE + NCE) per animal was recorded to evaluatebone marrow cytotoxicity. The ratio of polychromatic tonormochromatic erythrocytes (PCE/NCE) was calculated.

DNA fragmentation by Diphenylamine (DPA) assay: Liver sampleswere collected immediately after sacrificing the animals. The tissueswere lysed in 0.5 ml of lysis buffer, that contain 10 mM tris-HCl (pH8), 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 0.5ml of lysis buffer. To the pellets (P) and the supernatants (S), 1.5 mlof 10% trichloroacetic acid (TCA) was added and incubated at 4°Cfor 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, toeach sample 2 ml of DPA solution [200 mg DPA in 10 ml glacial aceticacid, 150 µl of sulfuric acid and 60 µl acetaldehyde] was added andincubated at room temperature for 24 h 28. The proportion offragmented DNA was calculated from absorbance reading at 600nm using the equation:

DNA fragmentation = OD of fragmented DNA (supernatant) / [OD offragmented DNA (supernatant) + OD of intact DNA (pellet)] × 100

Random amplified polymorphic DNA (RAPD):Genomic DNA isolation: The frozen liver tissue was thawed.


Approximately 100 mg of frozen liver tissue ground andhomogenized in 1 ml of tissue lysis buffer and 5 µl proteinase Kthen incubated at 65°C for 1 hr. The genomic DNA was then isolatedusing phenol/chloroform extraction and ethanol precipitationmethod with minor modifications according to Sambrook et al. 29.The concentration and purity of extracted genomic DNA weredetermined by spectrophotometer based on the absorbance at260 and 280 nm 30.

Random amplified polymorphic DNA (RAPD) analysis: Togenerate RAPD profiles from the mice DNA, a set of seven primersprocured from Operon technologies (Operon, Almeda, CA, USA)randomly selected were used in RAPD analysis. DNA amplificationreactions were performed under conditions reported by Williamset al. 31. PCR amplification was conducted in 50 µl reaction volumecontaining 100 ng genomic DNA, 100 µM dNTPs, 40 nM primer,2.5 units of Taq DNA polymerase and 5 µl promega 10X Taq DNApolymearse buffer. The PCR reactions were carried out in thermalcycler (Perkin-Elmer 9700) programmed with a first denaturationof 5 min at 94°C, followed by 45 cycles of 1 min denaturation at95°C, 1 min annealing at 36°C and 2 min extension at 72°C. Finalextension at 72°C for 5 min was allowed before holding the reactionat 4°C for 10 min. Reaction products were stored at 4°C prior toelectrophoresis. PCR amplified products of RAPD primers weredetected using agarose gel electrophoresis (1.8% W/V, Bio-Rad,California) in 1X TBE buffer) at 80 V for 1 h, then visualised bystaining with ethidium bromide (2 µl/100 ml).

Statistical analysis: Data in all experiments was subjected toANOVA using SPSS for Windows version 11.0, statistical software.Variable means for treatments indicating significant differenceswere compared and the significances were indicated using Duncanmultiple range tests.

ResultsThe capacity of the antibiotic alternative probiotics to inducechromosome mutations was analysed using the polychromaticerythrocytes micronucleus test in male mice. The results of MNtests are illustrated in (Fig. 1). Results showed that Biomin Imboand Primalac commercial supplements tested doses (1.5 and 3 g/kg diet) caused statistically significant increase in the frequenciesof MNPCEs over the control value but not reach to the meanvalue of neomycin. Meanwhile, neomycin significantly increasesthe frequency of MNPCEs over control, Biomin Imbo and Primalactested doses.

Proportion of PCEs: It is evident from the results of the presentstudy that neomycin significantly decreased the percentage ofpolychromatic erythrocytes (PCE)/normochromatic erythrocytes(NCE) than those of Biomin Imbo, Primalac and control (Fig. 2).Meanwhile, Biomin Imbo and Primalac significantly decreased thepercentage of PCE/NCE than control. Whereas, Biomin Imbo andPrimalac induced significant increase in the percentage of PCE/NCE ratio than that of neomycin. It means that Biomin Imbo andPrimalac cytotoxicity less than that of neomycin. The decrease inthe percentage of PCE/NCE indicates a suppression of bonemarrow proliferation most likely by mitotic arrest.

DNA fragmentation by Diphenylamine (DPA): Biomin Imbo,Primalac and neomycin induced DNA damage in liver cells wasevaluated by measuring the level of fragmented DNA colorimetricusing Diphenylamine (DPA). The results showed that neomycinsignificantly increased the percentage of DNA fragmentation inliver 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 significantlyincreased the percentage of DNA fragmentation as compare tocontrol. While, they showed significantly decrease in the percentageof DNA fragmentation than neomycin (Table 1 and Fig. 3).

Control

Neomycin

1.5 g/kgBiomin Imbo

1.5 g/kgPrimalac

3 g/kgBiomin Imbo

3 g/kgPrimalacNo. of MNPCEs

0

8

17.33

11.6710.67

13.33

12

2

46

81012

14

16

18

Fre

quen

cy

Figure 1. Effect of Biomin Imbo and Primalac on the frequency ofMNPCEs in bone marrow cells of male mice.

Control

Neomycin1.5 g/kg Biomin Imbo

1.5 g/kg Primalac3 g/kg Biomin Imbo

3 g/kg Primalac

DNA fragmentation (%)0

9.78

13.43

10.82

24

68

1012141618

Per

cent

age

%

17.56

11.85

5.65

Figure 3. DNA fragmentation by Diphenylamine (DPA) assay in livercells of mice.

Control

Neomycin

1.5 g/kgBiomin Imbo1.5 g/kgPrimalac3 g/kgBiomin Imbo3 g/kgPrimalac

Ratio of PCEs and NCEs (%)0

6.14

22.65

12.27

14.81

12.23

5

10

15

20

25

Per

cent

age

% 13.79

Figure 2. The ratio of PCE/NCE in bone marrow of male mice treatedby 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 +11.911.5 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 BiominImbo, Primalac and neomycin in liver cells of male mice.

Values with different superscript letters within columns represent significant statistical differences (P ≤ 0.05).


Random amplified polymorphic DNA (RAPD): The moleculargenetic variability among the treated mice genomes and theircontrol were evaluated using seven oligodecamers (10-mer randomprimers). Out of which 3 (OPA09, OPC02 and OPC06) yieldedmonomorphic bands, and 4 (OPA07, OPA08, OPB10 and OPC05)yielded polymorphic bands. The total number of amplificationproducts generated by these individual primers and variablefragments in treated and untreated controls are described in(Tables 2 and 3), and representative RAPD fingerprints are shownin (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, thebands were in the molecular weights range from 172 to 1450 bp.Primers OPA09 and OPA08 amplified the minimum and maximumnumber of bands which were 4 and 11 bands, respectively. Thequantitative analysis of those bands, expressed as percentage ofaltered bands, shows an increment of modified bands in all treatedgroups as compared with control one. At the neomycin treatedgroup 10 bands were modified which represent 21.2%. While atthe Biomin Imbo 1.5 and 3 g/kg diet treated mice 8 and 9 bandswere modified which represent 17.02% and 19.14%, respectively.Treatment with 1.5 and 3 g/kg diet of Primalac 7 altered bandswere detected in each group, which represents 14.89% (Fig. 5).

DiscussionSeveral studies and reviews have been published on the healthbeneficial effects of probiotics. Probiotics and their supplementationin the poultry diet have been found to improve the productiveperformance 32, 33, improve feed conversion ratio, augment eggproduction and quality, stimulate immune response and increasebone strength 34. It is noteworthy, however, that the healthbeneficial properties of probiotics are strain-specific 26.

The present study was conducted to assess the ability of thetwo tested antibiotic alternative commercial supplementsprobiotics (Biomin Imbo and Primalac) to induce genotoxic activityin male mice. Antibiotics act as growth factors by killing bacteriathrough inhibiting a bacterium’s ability to turn glucose into energy,or its ability to construct its cell wall, but could also be harmful tohost cells 35. The finding in the present study showed that highlevels of DNA damage occurred in the antibiotic neomycin treatedgroups. This result was in agreement with Sengul et al. 36 andYurtseven et al. 37, they indicated that neomycin increased thepercentage of DNA damage. Rocco et al. 38 reported thaterythromycin and lincomycin induced genotoxic effect thatrepresented in a significant increase in DNA migration (tail moment)and micronuclei frequency. Results of the current study revealedthat Biomin Imbo and Primalac significantly increased thefrequency 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 Polymorphismscalculated from liver cells samples exposed to growth stimulating additives and control.

Table 3. Comparison of RAPD fingerprinting profiles of differentmice genomic DNA from liver cells exposed to allexperimental 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.


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Noteworthy, that this increment with that antibiotics less thanwith neomycin. It is well established that micronuclei are formedfrom the entire chromosome or from a fragment of it 39. Suchmicronuclei are induced by genotoxic stress such as clastogen oraneugen. Micronuclei induction by clastogen involves theinduction of either chromosome fragments that lag behind theseparating chromosomes or a chromatin bridge betweenchromosomes at the anaphase of mitosis. On the other hand,aneugen induces the whole chromosomes that were not bound tothe mitotic spindle at anaphase, probably by disrupting the spindlecheckpoint. Such chromatin is separated from the newly formingnucleus and forms an independent nucleus-like structure, themicronucleus. Therefore, methods to measure the frequency ofmicronuclei are widely used in genotoxic test used to measureefficacy of newly developed pharmaceuticals 40. Moreover, themicronuclei test used to detects cytotoxic effects by measuringthe PCE/NCE relationship. When normal proliferation of the bonemarrow cells is affected by a toxic agent, the number of immatureerythrocytes (PCE) is prejudiced in relation to mature erythrocytes(NCE), thus, the PCE/NCE ratio may decrease 41. Our resultsindicated that Biomin exhibited a significant reduction of PCE/NCE related to dose. However, Primalac high dose only reducedthe PCE/NCE ratio. These indicate that theses supplements havethe ability to induce cytotoxicity. Biomin and Primalac were findingto induced apoptosis in mice hepatocytes as demonstrated byDNA fragmentation. Meanwhile, increased the percentage of DNAfragmentations not reach to that of neomycin. Hausmann 42

mentioned that apoptosis could be a strategy of microbialpathogens to escape from the infected and exhausted host cell toinvade deeper mucosal layers for a prolonged bacterialcolonization. In addition, Sylvia et al. 43 found that some strainsof lactic acid bacteria (LAB) had potential ability to induce cancercells into apoptosis.

Recently, the RAPD technique has been successfully used todetect DNA effects induced by several compounds under in vitroand in vivo conditions. RAPD profiles detect alterations togenomic DNA through the use of arbitrarily primed PCR reactions.These effects include changes in oligonucleotide priming sitesand variations in the activity of the Taq DNA polymerase. In thepresent study, variation in band intensity, disappearance of bands,and appearance of new PCR products occurred in profilesgenerated from the treated animals. These effects may becorrelated with structural rearrangements in DNA caused bydifferent types of DNA damages 44. The variation in band intensityand disappearance of some bands may correlate with level ofphotoproducts in DNA templates after treatment, which can reducethe number of binding sites for Taq polymerase. Appearance ofnew bands can be explained as the result of DNA structural

changes (breaks, transpositions, deletions) 45. This variationamong different samples may be due to the different chemicalcompositions of supplements and at the same time of theirmanufacturing processes. Previous studies have shown thatchanges in band patterns observed in DNA fingerprint analysisreflect DNA alterations from single base changes (point mutation)to complex chromosomal rearrangements 46. In the same way, cellpopulations exposed in vitro to genotoxins suffer DNA alterationsin a certain number of cells, which are reflected as variations in thefingerprint obtained for the control population. These are definedas band losses or gains as well as alterations in the intensity ofamplification of some of them. Such alterations in vivo areconsidered mutations that are produced by changes to deletionsof or insertions into the pair bases 47, 48 confirmed the therapeuticpotential of the E. coli Nissle 1917 strain in promoting guthomeostasis upon mucosal injury. However, they showed that E.coli Nissle 1917 harbours a cluster of genes coding for thebiosynthesis of hybrid nonribosomal peptide-polyketide(s). Thisbiosynthetic pathway confers the ability for bacteria to induceDNA double strand breaks in eukaryotic cells. Genetic alterationsin 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 beenproposed to play a role in degenerative conditions such asaccelerated aging, immune dysfunction, cardiovascular andneurodegenerative diseases 49-51. DNA damage is also commonlyregarded as a marker of cancer risk. It is clearly relevant to cancersince DNA damage is the initiating event in carcinogenesis 52.

ConclusionsWe concluded that the antibiotic alternative commercialsupplements probiotics may have the potential to induce cytotoxicand moderate genotoxic effects in bone marrow cells of mice.

AcknowledgementsThis 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 DRStechnical and financial support.

References1Iyayi, E. A. 2008. Prospects and challenges of unconventional poultry

feedstuffs. Niger. Poultry. Sci. J. 5(4):186-194.2Windhorst, H. W. 2006. Changes in poultry production and trade

worldwide. World Poult. Sci. J. 62:585-602.3Ohimain, E. I. and Ofongo, R. T. S. 2012. The Effect of Probiotic and

Prebiotic Feed Supplementation on Chicken Health and GutMicroflora: A Review. Int. J. Animal and Veterinary Advances 4(2):135-143.

4Montagne, L., Pluske, J. R. and Hampson, D. J. 2003. A review ofinteractions between dietary fibre and the intestinal mucosa, and theirconsequences on digestive health in young non-ruminant animals. Anim.Feed Sci. Technol. 108(1-4):95-117.

5Villa, F., Ceroni, M., Bagstad, K., Johnson, G. and Krivov, S. 2009.ARIES (ARtificial Intelligence for Ecosystem Services): A new toolfor ecosystem services assessment, planning, and valuation. BioEcon.,pp. 1-10.

6Kamruzzaman, S. M., Kabir, S. M. L., Rahman, M. M., Islam, M. W.and Reza, M. A. 2005. Effect of probiotics and antibioticsupplementation on body weight and haemato-biochemical parametersin broilers. Bangl. J. Vet. Med. 3:100-104.

0

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Neomycin Biomin Imbo1.5 g/kg diet

Biomin Imbo3 g/kg diet

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17.02

19.14

14.89 14.89

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Figure 5. The percentage of altered bands in each treatment detected byRAPD-PCR.


7Khaksefidi, A. and Ghoorchi, T. 2006. Effect of probiotic on performanceand immunocompetence in broiler chicks. J. Poult. Sci. 43:296-300.

8Mountzouris, K. C., Tsirtsikos, P., Kalamara, E., Nitsch, S., Schatzmayr,G. and Fegeros, K. 2007. Evaluation of the efficacy of probioticcontaining Lactobacillus, Bifidobacterium, Enterococcus, andPediococcus strains in promoting broiler performance and modulatingcecal microflora composition and metabolic activities. Poult. Sci.86:309-317.

9Kabir, S. M. L., Rahman, M. M., Rahman, M. B., Hosain, M. Z.,Akand, M. S. I. and Das, S. K. 2005. Viability of probiotics in balancingintestinal flora and effecting histological changes of crop and caecaltissues of broilers. Biotechnology 4:325-330.

10Mathivanan, R. and Kalaiarasi, K. 2007. Panchagavya and Andrographispaniculata as alternative to antibiotic growth promoters onhaematological, serum biochemical parameters and immune status ofbroilers. J. Poult. Sci. 44:198-204.

11Pelicano, E. R. L., de Souza, P. A., de Souza, H. B. A., Oba, A., Norkus,E. A., Kodawara, L. M. and de Lima, T. M. A. 2003. Effect of differentprobiotics on broiler carcass and meat quality. Br. J. Poult. Sci. 5:207-214.

13Griggs, J. P. and Jacob, J. P. 2005. Alternatives to antibiotics for organicpoultry production. J. Appl. Poult. Res. 14:750-756.

14Nava, G. M., Bielke, L. R., Callaway, T. R. and Castañeda, M. P. 2005.Probiotic alternatives to reduce gastrointestinal infections: The poultryexperience. Animal Health Res. Rev. 6:105-118.

15Piray, A. H., Kermanshahi, H., Tahmasbi, A. M. and Bahrampour, J.2007. Effects of cecal cultures and aspergillus meal prebiotic (fermacto)on growth performance and organ weights of broiler chickens. Int. J.Poult. Sci. 6:340-344.

16Kabir, S. M. L. 2009. The Role of Probiotics in the Poultry Industry.Int. J. Mol. Sci. 10(8):3531-3546.

17Akinleye, S. B., Iyayi, E. A. and Afolabi, K. D. 2008. The Performance,Haematology and Carcass Traits of Broilers as Affected by DietsSupplemented with or Without Biomin a Natural Growth Promoter.World Journal of Agricultural Sciences 4(4):467-470.

18Mohnl, M. 2006. Benefits from using biomin c-x and biomin imbo inpoultry production. Biomin Newsletter 4:37.

19Chichlowski, M., Croom, J., Mcbridge, B. W., Havenstein, G. B. andKoci, M. D. 2007. Metabolic and physiological impact of probioics ordirect-fed microbials on poultry: A brief review of current knowledge.International Journal of Poultry Science 6(10):694-704.

20Kermanshahi, H. and Rostami, H. 2006. Influence of supplementaldried whey on broiler performance and cecal flora. Int. J. Poultry Sci.5:538-543.

21Talebi, A., Amirzadeh, B., Mokhtari, B. and Gahri, H. 2008. Effects ofa multi-strain probiotic (Primalac) on performance and antibodyresponses to Newcastle disease virus and infectious bursal diseasevirus vaccination in broiler chickens. Avian Pathol. 37(5):509-512.

22Goelzer, J. G., Avelar, R. L., Dolzan, A. N., Becker, O. E., Haas, O. L.,de Oliveira, L. Y. S. and de Oliveira, R. B. 2012. Evaluation of tissueresponse and genotoxicity of poly (L/D-lactic acid) disks implanted inrat calvarium. Histology and Histopathology 27(12):1551-7.

23Singh, A., Negi, M. S., Rajagopal, J., Bhatia, S., Tomar, U. K., Srivastava,P. S. and Lakshmikumaran, M. 1999. Assessment of Genetic Diversityin Azadirachta Indica using AFLP Markers. Theoretical and AppliedGenetics 99(1-2):272-279.

24Deshpande, G. C., Shripada, C. R., Anthony, D. K. and Sanjay, K. P.2011. Evidence-based guidelines for use of probiotics in pretermneonates. BMC Medicine 9:92.

25Rowland, R., Capurso, L., Collins, K., Cummings, J., Delzenne, N.,Goulet, O., Guarner, F., Marteau, P. and Meier, R. 2010. Current levelof consensus on probiotic science. Gut Microbes 1(6):436-439.

26European Food Safety Authority 2011. Scientific Opinion on thesubstantiation of health claims related to Lactobacillus johnsonii BFE6128 and natural defences/immune system (ID 990) and skin health(ID 991) pursuant to Article 13(1) of Regulation (EC) No. 1924/2006.

EFSA Journal 9:2024.27Krishna, G. and Hayashi, M. 2000. In vivo rodent micronucleus assay:

Protocol, conduct and data interpretation. Mut. Res. 455:155-166.28Gibbs, A. G., Chippindale, A. K. and Rose, M. R. 1997. Physiological

mechanisms of evolved desiccation resistance in Drosophilamelanogaster. J. Exp. Biol. 200:1821-1832.

29Sambrook, L., Fritsch, E. F. and Manitatis, T. 1989. Molecular Cloning:A Laboratory Manual. Cold Spring Harbour Press, Cold SpringHarbour, NY, 300 p.

30Aquardro, C. F., Noon, W. A. and Begun, D. J. 1992. RFLP analysisusing heterologous probes. Cited in: Molecular genetic analysis ofpopulations:A practical approach Edited by Hoelzel, A. R., Oxford,New York, Tokyo: IRL Press at Oxford University Press. 1998. pp.xxii+445.

31Williams, J. K. G., Kubelik, A. R., Livak, K. J., Rafalsky, J. A. andTyngey, S. V. 1990. Molecular changes during gonad masculinization.DNA polymorphisms amplified by arbitrary primers are useful asgenetic markers. Nucleic Acids Res. 18:6531-6535.

32Ayed, M. H. and Ghaoui, F. 2011. Efficiency of supplementingSaccharomyces cerevisiae var. ellipsoideus for improved growthperformance and carcass yield in broilers. Res. Opin. Anim. Vet Sci.1:371-374.

33Dibaji, S. M., Seidavi, A. and Asadpour, L. 2012. Effect of dietaryinclusion of the synbiotic Biomin Imbo on broilers’ some bloodmetabolites. Res. Opin. Anim. Vet. Sci. 2:10-13.

34Sojoudi, M. R., Dadashbeiki, M. and Bouyeh, M. 2012. Effects ofdifferent levels of symbiotic. TechnoMos on broilers performance.Res. Opin. Anim. Vet. Sci. 2:243-248.

35Aydin, S., Basaran, A. A. and Basaran, N. 2005. The effects of thymevolatiles on the induction of DNA damage by the heterocyclic amineIQ and mitomycin C. Mutat. Res. 581:43-53.

36Sengul, T., Yurtseven, S., Cetin, M., Kocyigit, A. and Sogut, B. 2008.Effect of thyme (T. vulgaris) extracts on fattening performance, someblood parameters, oxidative stress and DNA damage in Japanese quailsjournal of Animal and Feed Sciences 17:608-620.

37Yurtseven, S., Cetin, M., Sengul, T. and Sogut, B. 2008. Effect of sageextract (Salvia officinalis) on growth performance, blood parameters,oxidative stress and DNA damage in partridges. South African Journalof Animal Science 38(2):145-152.

38Rocco, L., Peluso, C. and Stingo, V. 2012. Micronucleus test and cometassay for the evaluation of zebrafish genomic damage induced byerythromycin and lincomycin. Environmental Toxicology 27(10):598-604.

39Fenech, M. 2009. A lifetime passion for micronucleus cytome assaysreflections from Down Under. Mutat. Res. 681:111-117.

40El-Zein, R. A., Schabath, M. B., Etzel, C. J., Lopez, M. S., Franklin, J.D. and Spitz, M. R. 2006. Cytokinesis-blocked micronucleus assay asa novel biomarker for lung cancer risk. Cancer Res. 66:6449-6456.

41Rabello-Gay, M. N., Rodrigues, M. A. R. and Monteleone-Neto, R.1991. Mutagenesis, teratogenesis and carcinogenesis: Evaluationcriteria. Brazilian Society of Genetics, Ribeirão Preto, 246 p.

42Hausmann, M. 2010. How Bacteria-Induced Apoptosis of IntestinalEpithelial Cells Contributes to Mucosal Inflammation. InternationalJ. of Inflammation, V 2010, pp. 1-9.

43Sylvia, K. W., Remillard, C. and Ozimek, L. 2005. Induction ofProgrammed Cell Death by Probiotic Bacteria PhD Candidate.Department of Agricultural, Food and Nutritional Sciences, 4-10Agriculture, Forestry and Home Economics Building, University ofAlberta, Edmonton, Alberta, Canada T6G 2P5.

44 Becerril, C., Ferrero, M., Sanz, F. and Castano, A. 1999. Detection ofmitomycin C-induced genetic damage in fish cells by use of RAPD.Mutagenesis 14:449-456.

45Atienzar, F. A., Cheung, V. V., Jha, A. N. and Depledge, M. H. 2001.Fitness parameters and DNA effects are sensitive indicators of copper-induced toxicity in Daphnia magna. Toxicol. Sci. 59:241-250.

46Atienzar, F. A., Cordi, B., Donkin, M. E., Evenden, A. J., Jha, A. N. and


Depledge, M. H. 2000. Comparison of ultraviolet-induced genotoxicitydetected by random amplified polymorphic DNA with chlorophyllfluorescence and growth in a marine macroalgae. Palmaria Palmata.Aquat. Toxicol. 50:1-12.

47Danylchenko, O. and Sorochinsky, B. 2005. Use of RAPD assay forthe detection of mutation changes in plant DNA induced by UV-B andγ-rays. BMC Plant Biol. 5(Suppl. 1):S9, pp. 1-2.

48Olier, M., Marcq, I., Salvador-Cartier, C., Secher, T., Dobrindt, U.,Boury, M., Bacquié, V., Pénary, M., Gaultier, E., Nougayrède, J. P.,Fioramonti, J. and Oswald, E. 2012. Genotoxicity of Escherichia coliNissle 1917 strain cannot be dissociated from its probiotic activity.Gut Microbes 3(6):501-509.

49Hoeijmakers, J. H. 2009. DNA damage, aging, and cancer. New Engl. J.Med. 361:1475-1485.

50Slatter, M. A. and Gennery, A. R. 2010. Primary immunodeficienciesassociated with DNA-repair disorders. Expert Rev. Mol. Med. 12:19.

51Frank, S. A. 2010. Evolution in health and medicine Sackler colloquium:Somatic evolutionary genomics: mutations during development causehighly variable genetic mosaicism with risk of cancer andneurodegeneration. Proc. Natl. Acad. Sci., USA, 107(Suppl. 1):1725-1730.

52Bonassi, S., Znaor, A., Ceppi, M., Lando, C., Chang, W. P., Holland,N., Kirsch-Volders, M., Zeiger, E., Ban, S., Barale, R., Bigatti, M. P.,Bolognesi, C., Cebulska-Wasilewska, A., Fabianova, E., Fucic, A.,Hagmar, L., Joksic, G., Martelli, A., Migliore, L., Mirkova, E., Scarfi,M. R., Zijno, A., Norppa, H. and Fenech, M. 2007. An increasedmicronucleus frequency in peripheral blood lymphocytes predicts therisk of cancer in humans. Carcinogenesis 28:625-631.

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