ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1547691X.2014.904025


Potential preventive role of lactic acid bacteria against Aflatoxin M1 immunotoxicity and genotoxicity in mice Jalila Ben Salah-Abbe`s1,2*, Samir Abbe`s1*, Rania Jebali1, Zohra Haous3, and Ridha Oueslati1

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Unit of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences, University of Carthage, Tunis, Tunisia, 2Higher Institute of Biotechnology of Monastir, University of Monastir, Monastir, Tunisia, and 3Unit of Histology Cytology and Genetics, Faculty of Medicine, Monastir, Tunisia Abstract


Aflatoxin M1 (AFM1) is a mycotoxin produced by numerous Aspergillus species in pre- or postharvest cereals and milk. Exposure to AFM1 imparts potent economic losses in the livestock industry. Toxicologically, it also causes severe immune system problems. The aims of this study were to evaluate a new AFM1-binding/degrading microorganism for biologic detoxification, to examine its ability to degrade AFM1 in liquid medium, and to evaluate its potential for in vivo preventative effects against AFM1-induced immunotoxicity and genotoxicity in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFM1 in PBS (93%) within 24 h of incubation. Further, the LP was able to tolerate gastric acidity, bile salts, and adhere efficiently to Caco-3 cells in vitro. The in vivo study used Balb/c mice that received either vehicle (control), LP only (at 1  109 CFU/L, 1 mg/kg bw), AFM1 (100 mg/kg bw), or AFM1 + LP daily for 15 days (by gavage); two other groups received a single dose of colchicine (4 mg/kg) or mitomycin C (1 mg/kg) as positive controls for induction of micronuclei and chromosomal aberrations, respectively. The results showed that, compared to in control mice, AFM1 treatment led to significantly decreased body weight gains, and caused cytotoxic/genotoxic effects as indicated by increases in frequencies of polychromatic erythrocytes, as well as those with micronucleation (PCEMN) and chromosomal aberrations, among bone marrow cells. The concurrent administration of LP with AFM1 strongly reduced the adverse effects of AFM1 on each parameter. Mice receiving AFM1 + LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria caused no adverse effects. Based on the data, it is concluded that the test bacteria could potentially be beneficial in the detoxification of AFM1-contaminated foods and feeds for humans and animals.

Aflatoxin M1, binding, detoxification, genotoxicity, immunotoxicity, Lactobacillus strains

Introduction Some toxigenic fungi strains produce toxic compounds in foods and feedstuffs (Bryden, 2007) that, upon consumption, cause a variety of health problems in animals and humans (Bennett & Klich, 2003). Aspergillus mycotoxins, in particular aflatoxin (AF) secondary metabolites produced by Aspergillus flavus, A. parasiticus, and A. nominus, occur naturally in a variety of animal feeds and foods. The AF family is quite large and includes, in part, AFB1, AFB2, AFG1, and AFG2 (Maurice & Moss, 2002). In Tunisia, many forms of AF have been found to contaminate different agricultural commodities such as pistachios, nuts, grains, and several feedstuffs for livestock and foods for human consumption (Bensassi et al., 2010; Ghali et al., 2008, 2009). Animals that consume AFB1-contaminated feed develop various health problems. Among these, AFB1 causes immunosuppression and enhanced susceptibility to infectious diseases. *The first two authors equally contributed to the manuscript. Address for correspondence: Samir Abbe`s, Unit of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences Bizerte 7021 Zarzouna, University of Carthage, Tunis, Tunisia. Tel: 21672591906. Fax: 21672590566. E-mail: [email protected]

History Received 21 November 2013 Revised 19 February 2014 Accepted 10 March 2014 Published online 14 May 2014

In most hosts (including common sources of milk), AFB1 is metabolized fairly rapidly to AFM1 after just 15 min; the latter then appears soon after in milk at the first milking (Trucksess et al., 1983). Recently, our lab demonstrated that in Beja province (Tunisia), milk and milk by-products were contaminated with high levels of AFM1 (Abbe`s et al., 2012a). Several AF have been implicated as causative agents in human hepatic and extra-hepatic cancers (Abbe`s et al., 2008; Kamakar, 2005). According to the International Agency Research on Cancer (IARC), AFB1 and AFM1 are Class 1A and 2B carcinogens, respectively (IARC, 1993; Maurice & Moss, 2002). Although the potency of AFM1 is less than that of its parent compound, it seems the kind of animal feed as well as harvest time and temperature parameters affect this particular endpoint (Kuiper, 1999). AFM1 is of such major concern to humans due to its frequent occurrence in dairy products at concentrations high enough to cause adverse health effects. Recently, interest has increased in the idea that host absorption of mycotoxins in contaminated food could be reduced by gastrointestinal tract microorganisms. Numerous in vitro investigations have shown that some dairy strains of LAB and bifido-bacteria were able to bind effectively to AFB1, AFM1, and Fusarium mycotoxins (Abbe`s et al., 2012b;


J. Ben Salah-Abbe`s et al.

El-Nezami et al., 2002; Mokoena et al., 2005). Building on those findings, the aim of this study was to evaluate a new AFM1binding microorganism for use in biological detoxification of AFM1 and to examine its ability to remove AFM1 in medium and prevent AFM1-induced immunotoxicity and genotoxicity.

Materials and methods

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Chemicals and bacteria Standard AFM1 (purity498%) was obtained from Sigma (St. Louis, MO) and a stock solution was prepared in ethanol/water (1:1 [v/v]). All other chemicals purchased were of analytical grade. A solution of 100 mg AFM1/ml PBS (phosphate-buffered saline, pH 6.5) were prepared for use in the in vivo study outlined below. Another solution of 20 mg AFM1/ml PBS was prepared for use in the in vitro studies below. All solutions were freshly prepared using sterile distilled water and held at 4  C until use. The bacterial strain used in the study was Lactobacillus plantarum MON03, a lactic acid bacteria isolated from 1 month-old artisanal butter made from cow milk collected from a local producer in central Tunisia (Abbe`s et al., 2012b). In vitro studies Tolerance to simulated gastric juice The acid tolerance of isolated LAB was studied in simulated gastric juices as described by Charteris et al. (1998). The simulated gastric juices were prepared in PBS containing pepsin (0.3%, w/v). Buffered solutions were then adjusted to pH values of 2.0, 3.0, and 6.4 (control) with HCl, and then sterilized by autoclaving at 121  C for 15 min. Ten milliliters of each solution was then placed in a sterilized test tube, and a suspension of LAB culture (containing & 109 cells) was added and mixed. After a 3-h incubation at 37  C, 1 ml of each solution was serially diluted with sterile saline and appropriate dilutions plated onto MRS agar and incubated in an anaerobic chest at 37  C for 72 h. For this study, L. rhamnosus GG (ATCC 53103) was used as control. Tolerance to bile salts The ability of isolates to grow in the presence of bile was determined using the method of Vinderola & Reinheimer (2003). Bile salt solutions were prepared using oxgall powder (Sigma) at final concentrations of 0.3, 0.5, and 1%. Sterile distilled water without oxgall (pH 6.2) was used as control. All solutions were autoclaved, and 10 ml of each was transferred into sterile test tubes. Cell suspensions (containing  109 bacteria) were then added to each tube and the solution incubated at 37  C in the anaerobic chest for 12 h. Thereafter, 1 ml of each culture was diluted in 9 ml sterile saline, and appropriate dilutions plated onto MRS agar. The plates were the incubated in the anaerobic chest at 37  C for 72 h. Bile salt hydrolase (BSH) activity of each isolates was then determined by the method of Dashkevicz & Feighner (1989). Again, L. rhamnosus GG was used as control. In vitro adhesion assays The ability of isolates to adhere to intestinal epithelial cells (Caco-2 line) was evaluated using the method of Martı´n et al. (2006). Briefly, Caco-2 cells were grown in Dulbecco’s minimal essential medium (DMEM; Invitrogen, Hanover, Germany) containing 25 mM glucose, 1 mM sodium pyruvate, 10% heatinactivated fetal calf serum (Gibco, Eragny, France), 2 mM L-glutamine, 1% non-essential amino acids, 100 U penicillin/ml, and 100 mg streptomycin/ml. For the adherence assays, Caco-2 cells were seeded into 24-well plates in 2 ml medium devoid of

J Immunotoxicol, Early Online: 1–8

antibiotics. At 2 days after the cells attained confluence, the medium was removed and replaced with 1 ml fresh antibiotic-free medium and then 1 ml Lactobacillus suspension (108 CFU/ml DMEM). For control studies, L. rhamnosus GG was substituted for the L. plantarum. The cells were then incubated for 3 h at 37  C in a 5% CO2 culture chamber. The infected cells were washed 3-times with sterile PBS (pH 7.8), then fixed with methanol, Gram-stained, and examined under a microscope. Adherent lactobacilli in 20 random microscopic fields were counted in each well. Enumeration of adhered lactobacilli was performed in triplicate, and the values were expressed as mean total number of bacteria (±SD) seen on cells in the 20 fields. AFM1 removal from PBS using L. plantarum MON03 One volume of LP culture broth (108 CFU/ml) was centrifuged at 3000  g for 15 min and the bacterial pellet was washed with sterile water. Some LP pellets were then re-suspended in 5 ml PBS containing AFM1 at 50 mg/ml; negative controls were re-suspended in PBS only. Tubes were then mixed on a vortex and the suspensions then incubated at 37  C for 0, 12, and 24 h. AFM1 (50 mg/ml in PBS) was used as a positive control. To monitor the efficacy of bacteria binding AFM1, all tubes were centrifuged for 15 min (3000  g) at the end of the incubation periods and supernatants collected and transferred to clean tubes. The tubes were stored at 4  C until AFM1 content analysis. The unbound AFM1 content in the supernatants was determined by HPLC. All experiments were performed in triplicate. To determine the effect of bacterial viability on binding affinity, bacteria (108 CFU/ml) were first heated to 90  C for 15 min, and then the dead bacteria were pelleted, contaminated with AFM1, and then processed as described above. Determination of AFM1 levels AFM1 analyses were performed using HPLC on an immunoaffinity column. Briefly, supernatant samples were first filtered through Whatman filter paper, and the filtrate then diluted with 80 ml PBS. A sample of the filtrate (10 ml) was then passed through the Vicam AFM1 immunoaffinity column (at 5 ml/min, followed by washing with 20 ml distilled water at 5 ml/min). Bound AFM1 was then eluted with 1.5 ml acetonitrile, followed by 1.5 ml distilled water, and collected in a vial. The two eluates were mixed and analyzed using an Agilent 1100 HPLC system (Agilent Technologies, Englewood, CO) containing an ACE C18 silica (5 mm i.d., 25  46 mm) column (Advanced Chromatography Technologies, Aberdeen, Scotland); product measurements were made in-line spectrophotometrically at 435 nm. Quantification of AFM1 was done from peak area measures and extrapolating against a calibration curve prepared/ analyzed in parallel using AFM1 standards (Sigma). Three replicate analyses were performed for each sample to determine AFM1 content. To verify soundness of the assay, recovery from bacteria-free PBS was determined for the test range of AFM1 used; mean recovery was in the range of 89–93%. The percentage of AFM1 bound to the bacteria was calculated using: 100%  (1.00  [peak area of AFM1 in supernatant/peak area of AFM1 in positive control sample]). In vivo studies Animals and treatments Female Balb/c mice (10-weeks-old, &21 [±2] g) were obtained from the animal house at the Pasteur Institute of Tunis. All mice were maintained in pathogen-free facilities maintained at 22 ± 2  C and 40 ± 5% relative humidity, and with a 12-h light:dark cycle. All mice had ad libitum access to standard

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DOI: 10.3109/1547691X.2014.904025

rodent chow and filtered water. The water and food were tested according to NF ISO 15302 to confirm that all matrices were AFM1-free (down to detection limit of 1 ng/g fat matter and 1 ng/L water). All animal experiments were done in compliance with the rules of the European Communities Council Directive of November 24, 1986 (86/609/EEC). All mice were acclimatized for 1 week prior to any experimental treatments. For the experiments, the mice were randomly distributed into eight treatment groups (n ¼ 10 mice/group) and treated orally (by gavage) daily for 15 days as follows: (1) untreated controls; (2) LP (109 CFU/L, 1 mg/kg bw) alone; (3) AFM1 only (100 mg/kg); (4) LP (109 CFU/kg) + AFM1 (100 mg/kg b.w); (5) a single dose of colchicine (4 mg/kg; positive control for induction of micronuclei); or (6) a single dose of mitomycin C (1 mg/kg; positive control group for chromosomal aberration induction). Gavage volume never exceeded 200 ml per dosing. The colchicine and mitomycin C treatments of the specified mice were given by gavage on Day 13 of the treatment protocol. Mice within each different treatment group were then divided into two subgroups A and B for use in differing post-exposure analyses. At 12 h after the final exposure, blood from all mice was drawn by retro-orbital bleeding into heparinized tubes for use in hematologic analyses. These samples were analyzed for total white blood cells (WBC), lymphocytes (LY), eosinophils (EO), neutrophils (NE), and monocytes (MO) present using an automated Coulter STKS blood counter (Coulter Electronics, Luton, UK). After the blood collection, the mice in each A subgroup/regimen were euthanized by cervical dislocation, their body weights taken, and then their thymus and spleen collected, weighed, and prepared for thymocyte and splenocyte assessments. The tibia and femur of each host was also collected for use in micronucleus detection assays. Thymocyte and splenocyte assessment Spleen and thymus cellularity were determined after dispersion of the tissues into single-cell suspension in RPMI 1640 culture medium (Gibco) as described previously (Nohara et al., 2002). Briefly, thymocytes and spleen cells were prepared in complete RPMI 1640 containing 12 mM HEPES (pH 7.1), 50 mM 2mercaptoethanol, 100 U penicillin/ml, 100 mg streptomycin/ml, and 10% FCS, by passing each isolated tissue through a stainless steel mesh. The spleen cells were further treated with 0.84% [w/v] ammonium chloride/EDTA solution to eliminate red blood cells. After a final centrifugation/washing step, cell numbers and viability were determined with a hemocytometer following staining with trypan blue. Bone marrow micronucleus assay At necropsy, the femur and tibia of subgroup A mice were isolated and freed from adherent tissues. Bone marrow was then isolated by injection of filtered 90% FCS (in PBS). The collected cells were centrifuged at 390  g for 5 min and the pelleted cells then re-suspended in fresh FCS solution. A small drop of the resuspended cell pellet was spread onto a glass slide, fixed in absolute methanol for 5 min, then air-dried at room temperature. The slides were then stained for 15 min in PBS (pH 7.4) containing freshly prepared 10 mg acridine orange/ml, rinsed in fresh PBS for 15 min, and then allowed to dry in the dark at room temperature. The slides were then scored immediately (under 1000 magnification) using a Nikon Eclipse E 400 fluorescent microscope (Nikon, Tokyo, Japan). Two thousand polychromatic erythrocytes (PCE) were examined from each animal and the number of polychromatic erythrocyte micronucleated (PCEMN) recorded. PCEMN appeared as red and included one or more yellow fluorescent corpuscles (micronucleus; MN). Scoring of

Prevention against AFM1 immunotoxicity and genotoxicity


micronuclei was performed according to criteria described by Hayashi et al. (1993) and are based essentially on diameter and shape of the MN. The number of PCEMN/2000 PCE/mouse sample was determined to assess induction of micronuclei due to AFM1 and the potential protective effect of LP against AFM1 damage. In vivo chromosome aberration assay Twenty-four hours prior to euthanizing, mice in each B subgroup/regimen were gavaged with 500 ml of a suspension of yeast powder (200 mg/ml) to accelerate bone marrow cell mitosis. Vinblastin (200 ml; 250 mg/ml) was injected intravenously into the animals 45 min before sacrifice in order to block dividing cells in the metaphase. At necropsy, bone marrow cells were collected, underwent hypotonic shock (in 0.075 M KCl solution), and then were fixed in methanol-acetic acid solution according to the method of Evans et al. (1960). The cells were then spread onto glass slides that were then flamed for 5 s, then air-dried at room temperature. Each slide was then stained for 15 min with 4% Giemsa in water. Within each slide, the chromosomes of 100 cells stopped in metaphase were examined for chromosome abnormalities (at 1000 magnification) using an optical microscope (Carl Zeiss, Kanstans, Germany). A total of three replicate slides (at 300 metaphase cells/dose level)/mouse were examined. Chromosome aberrations were identified according to criteria described by Savage (1975). Metaphases with chromosomal breaks, gaps, rings, and/or centric fusions (robertsonian translocations) were recorded. Each aberrant form was ultimately expressed as a percentage of total metaphases/mouse. Statistics All data were expressed as mean (±SD) of three independent experiments (in vitro study), and analyzed for statistical significance using a Student’s t-test with a general linear model (n ¼ 6 for in vivo tests). The criterion for significance was set at p50.05.

Results In vitro studies LP tolerance to acid and bile salts, and binding to Caco-2 cells Table 1 shows the survival of the LP grown under either low pH levels or in various bile salt conditions. The LP strain consistently showed tolerance to pepsin at pH 3; residual counts were 4106 CFU/ml after 3 h of incubation. LP also survived at pH 2 and exhibited fairly good acid tolerance with maintenance levels 475%. In the bile salts test, the bacteria were able to grow in 0.3% bile at least for up to 12 h. The LP showed comparatively better tolerance with viable counts 4107 CFU/ml in 1% bile salt solution. On the basis of the screening results for tolerance to low pH and bile salts, LP was found to be able to survive (at levels of 106 CFU/ml) at pH 2 or under conditions of 1% bile salt. The ability of the LP to adhere to Caco-2 cells is also indicated in Table 1. The LP expressed strong adhesive ability to the Caco-2 cells as compared to that by the control L. rhamnosus GG. As such, LP was selected for use in the in vivo studies here. LP binding of AFM1 Table 2 reports the levels of removal of AFM1 by LP in a PBS solution. It was found that the AFM1-binding by LP increased in a time-dependent manner. Live LP adsorbed 67.1 [± 6.2]% of AFM1 provided after 12 h, and the binding rose to 93.1 [± 5.2]% by 24 h of incubation. With the heat-killed LP, AFM1-binding decreased to 55.1 [± 4.5] % after 24 h of incubation in PBS.


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Table 1. Effects of simulated gastric juice and bile salt on LAB strains, and Caco-2 cell adherence activity. Gastric juice pH

Bile salt level (w/v)


Initial mean countsa

pH 2

pH 3




BSH activity

Caco-2 cell adhesionb

L. plantarum MON03 L. rhamnosus GG

9.46 ± 0.15 9.61 ± 0.12

9.16 ± 0.55 9.20 ± 0.42

9.11 ± 0.51 9.05 ± 0.56

7.14 ± 0.31 7.14 ± 0.37

7.67 ± 0.32 7.77 ± 0.41

7.73 ± 0.45 7.41 ± 0.36

+ +

457.8 ± 106.2 489.1 ± 98.5


Mean number of lactobacilli as log CFU/ml. Total number of lactobacilli that adhered to Caco-2 cells in 20 random microscopic fields. Each value represents mean (±SD) from three trials per assay condition. +, Positive for BSH activity.


Table 2. Percentage AFM1 removal in PBS by the presence of LP.

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AFM1 (50 mg/ml) Treatments PBS only Live LP Heat-killed LP


12 h

24 h

3.0 ± 0.2 25.9 ± 5.8* 25.1 ± 2.1

2.4 ± 0.2 67.1 ± 6.2* 40.5 ± 2.6

2.3 ± 0.2 93.1 ± 5.2* 55.1 ± 4.5

*Value significantly different from control (PBS-only and heated bacteria) at p50.05.

In vivo studies Effects of the treatment on body weight gain and lymphoid organ indexes The effects of LP alone on body weight gain and lymphoid organ indexes in AFM1-treated mice are reported in Table 3. Body weight gains were significantly decreased in AFM1 alone vs control mice; in contrast, weight gains in all LP-treated mice were significantly greater than that by the controls. While the absolute weights of the spleen and thymus of the AFM1-only treated mice were not impacted, their respective indices were markedly increased when compared with corresponding values for control mice. The combination of LP + AFM1 shifted both sets of splenic values toward normal levels; there were no apparent effects on the thymic index values. It is unclear why this was so; alone, the LP led to an increase in the absolute weights of the thymus of &20.5%, and AFM1 alone to an increase of &25.0%. It is possible that together there was some synergistic impact on this one organ; this remains to be determined. With respect to the spleen, LP-alone led to an increase in absolute organ weight of 8.8% and AFM1-alone to an increase of &14.0%. Co-treatment with LP + AFM1 resulted in an increase in weight of 13.2%; however, the large increase in host body weight more than compensated for this, resulting in a splenic index near that of the control mice. Total and differential WBC counts Total WBC levels in the blood were significantly increased in the AFM1-treated hosts (Table 4). Differential counts revealed a significant decrease in lymphocyte levels and increases in each other cell type examined in these mice. In contrast, total levels of WBC in mice treated with LP alone were comparable to control values. Among the various cell types assessed, only eosinophil levels in the LP-alone hosts were decreased (albeit insignificantly) in comparison to the control mice levels. LP + AFM1 co-treatment resulted in normalization of total WBC and all immune cell types away from the corresponding levels in mice that received AFM1 alone. Thymus and spleen cellularity A suppressive effect in thymus and spleen cellularity was observed in mice treated with AFM1 (Figure 1). Levels of

thymocytes and splenocytes decreased significantly (11.8 [± 2.1]  107 and 9.5 [± 1.9]  107 cells, respectively) compared to those in the control mice (25.4 [± 3.2]  107 and 13.4 [± 3.1]  107 cells, respectively). Treatment with LP alone resulted in no significant change in thymocyte and splenocyte numbers (26.4 [± 3.2]  107 and 14.1 [± 2.6]  107 cells, respectively) from control mice values. Compared to the organ values in the AFM1-treated mice, the AFM1 + LP co-treatment resulted in significant improvement in splenocyte and thymocyte counts to, respectively, 25.5 [± 3.1]  107 and 14.5 [± 2.3]  107 cells, nearnormal values. These changes represented improvements of 34.5 and 53.9% for the splenocyte and thymocyte counts relative to those seen in the mice in the AFM1-alone group. Bone marrow micronucleus assay The current study indicated that both colchicine (positive control) and AFM1 induced genotoxicity (Table 5). This was indicated by a significant increase in the numbers of PCEMN per 2000 polychromatic erythrocytes (PCE)/mouse for the colchicine and AFM1-treated mice (67.2 [± 8.1] and 42.1 [± 5.2], respectively) compared to levels noted in the control mice (1.7 [± 0.3]). Mice treated with LP alone displayed no significant effect upon their numbers of PCEMN (2.3 [± 0.4]). Mice treated simultaneously with AFM1 and LP showed a significant decrease in the elevation of PCEMN number (now 7.1 [± 0.3]; reduction ¼ 97.4%) that had occurred from the AFM1 treatment, although these values were still higher than in the control mice. In vivo chromosomal aberrations Table 5 indicates that the total numbers of structural aberrations including centric fusions, gaps, rings, and breaks - in the chromosomes of bone marrow cells were significantly increased due to AFM1 treatment (47.2 [± 4.1]/300 metaphase cells counted/mouse) compared to levels in control mice (3.6 [± 0.3]). No significant effects were observed in the group treated with LP alone (3.45 [± 0.62]). LP + AFM1 co-treatment resulted in significant decreases in total aberrations induced by AFM1 (now 6.6 [± 0.8]/300 metaphase cells counted/mouse; reduction ¼ 96.2%). As a positive control, a single dosing of mitomycin C results in aberration levels of 62.1 [± 5.2]/300 metaphase cells counted/mouse).

Discussion Aspergillus mycotoxins occur worldwide in, among many products, cereals, animal feeds, and milk. The presence of such toxicants lead to outbreaks of aflatoxicoses in humans and animals that, in turn, result in severe effects on human health, increases in veterinary care costs, and decreases in livestock production (Applebaum et al., 1982; Creppy, 2002). AFM1 is one of the predominant contaminating mycotoxins of milk and milk by-products. Indeed, as it is heat stable, no reductions in toxin level occur as a result of pasteurization processes. AFM1 has even

Prevention against AFM1 immunotoxicity and genotoxicity

DOI: 10.3109/1547691X.2014.904025


Table 3. Absolute body weight, percentage weight gain, and spleen and thymus weights and indices in mice. Parameters Body weight (g) initial final Body weight gain (%) Spleen wet weight (g) Spleen Index Thymus wet weight (g) Thymus Index





26.60 ± 1.20 33.80 ± 1.70 27.10 ± 3.10 0.18 ± 0.20 0.53 ± 0.03 0.014 ± 0.003 0.041 ± 0.002

29.80 ± 2.10 42.34 ± 3.20* 42.10 ± 2.20* 0.19 ± 0.30 0.48 ± 0.04 0.016 ± 0.002* 0.038 ± 0.001

23.50 ± 1.90 26.34 ± 2.50 12.10 ± 1.00* 0.20 ± 0.20 0.75 ± 0.06* 0.013 ± 0.001* 0.049 ± 0.003*

31.50 ± 2.10 47.28 ± 4.60 50.10 ± 5.10* 0.22 ± 0.30 0.46 ± 0.03 0.022 ± 0.002 0.046 ± 0.002*

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All mice were orally exposed daily for 15 days to LP (109 CFU/kg), AFM1 (100 mg/kg), or a co-treatment with LP + AFM1 (at the respective doses noted here). Values shown are mean (±SD) from 10 determinations per experimental scenario tested. *Value significantly different from control at p  0.05. Organ Indices calculated as 100  [organ weight (g)/total body weight (g)]. Table 4. Effect of LP on total white blood cell levels and leukocyte differentials in mice. Groups Control LP AFM1 LP + AFM1

WBC (103/ml)

NE (103/ml)

LY (103/ml)

MO (103/ml)

EO (103/ml)

8.02 ± 0.13 8.11 ± 0.22 12.13 ± 0.40* 8.86 ± 0.12

6.15 ± 0.07 6.11 ± 0.07 9.74 ± 0.25* 6.12 ± 0.15

1.41 ± 0.06 1.43 ± 0.08 0.84 ± 0.11* 1.36 ± 0.05

0.42 ± 0.09 0.44 ± 0.06 1.86 ± 0.10* 0.40 ± 0.21

0.14 ± 0.06 0.08 ± 0.02 0.65 ± 0.02* 0.15 ± 0.04

All treatment regimens are as outlined in the legend for Table 3. Values shown are mean (±SD) from 10 determinations per experimental scenario tested. *Value significantly different from control at p  0.05.

Figure 1. Changes in spleen and thymus cellularity. Mice were orally exposed daily to AFM1 (100 mg/kg BW), LP (109 CFU/L, 1 mg/kg BW), or AFM1 + LP for 15 days. Controls received vehicle only each day. Data shown are mean (±SD). aValue significantly different from vehicle control (p50.05).

been found in UHT milk (Diaz et al., 1995), and in some milk derivatives like yogurt and cheese (often resulting in a 3–5-fold enrichment in relative toxin content over that in the starting raw milk product) (Pietri et al., 1997). As AFM1 is absorbed quite easily from the intestinal tract, it can give rise to a myriad of health effects, including immune system disruption (Caloni et al., 2006) and other hematologic effects (Abbe`s et al., 2012b). To date, no effective strategies have been established to expunge AFM1 contamination in foods, feeds, or milk so as to reduce its potential toxic effects in hosts. The

present investigation was carried out to explore a protective effect and possible ameliorative role for Lactobacillus plantarum MON03 against AFM1-induced toxicities. The results of the in vitro study here with Caco-2 target cells (a heterogeneous human epithelial colorectal adenocarcinoma cell line (Martin et al., 2006)) were in line with expectations. Specifically, lactic acid bacteria strains show adherence specificity for intestinal epithelia (Wang et al., 2010); Vastano et al. (2013) recently demonstrated that Lactobacillus plantarum was able to adhere to cells/other compounds via pyruvate


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Table 5. Mean number of micronuclei in polychromatic erythrocytes and chromosomal aberrations in bone marrow cells of mice. Treatments


Total aberrations

Control Colchicine/Mitomycin C LP AFM1 LP + AFM1

1.66 ± 0.27a 67.21 ± 8.10c 2.28 ± 0.45a 51.27 ± 8.14c 7.11 ± 0.27b

3.59 ± 0.32a 62.11 ± 5.22c 3.45 ± 0.62a 47.22 ± 4.12c 6.65 ± 0.81b

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All treatment regimens are as outlined in the legend for Table 3. Values shown are mean (±SD) from 10 determinations per experimental scenario tested. a,b,c Within each column, values with differing superscripts significantly differ at p50.05.

dehydrogenase E1 -sub-unit (PDHB), a component of the pyruvate dehydrogenase complex and a factor contributing to fibronectin-binding. By means of fibronectin overlay immunoblotting, a L. plantarum surface protein with apparent molecular mass of 35 kDa was identified; mass spectrometric analysis showed this protein to be PDHB. The ability to adhere to host intestinal mucosa is considered an important selection criterion for lactic acid bacteria strains intended for probiotic use and/or toxin detoxification (de Angelis et al., 2006; Dunne et al., 2001). For beneficial health effects, such as competitive exclusion of pathogens from intestinal epithelia, toxin detoxification, or immune regulation, an effective bacterium should be able to colonize on the gut mucosa. Host specificity of the adherence is regarded as a desirable property and recommended as a key selection criterion for potential use in in vivo toxin detoxifications (Saarela et al., 2002; Salminen et al., 1998). Fuller et al. (1978) pointed out that host specificity of lactobacilli adherence is closely connected to a presence of some specific molecules/ receptors on host cells that can be distinguished by specific molecules of/on the bacteria. The in vitro survival tests revealed that L. plantarum MON03 strain was resistant to pH 2, even after 3 h of exposure. These results are in agreement with those obtained from similar previous studies where Lactobacillus strains were viable, even after being exposed to pH values of 2.5–4.0, but showed reduced viability at lower pH values (de Angelis et al., 2006; Mishra & Prasad, 2005). Acid tolerance is important not only for a bacterium to withstand gastric stresses, it is also a prerequisite for potential use as a dietary adjunct. This tolerance enables the strain to survive for longer periods in high-acid carrier foods (such as yogurt) without large reductions in their numbers (Minelli et al., 2004). Similarly, bile plays a fundamental role in specific and non-specific defense mechanisms in the gut; the magnitude of its anti-pathogen effects is determined primarily by the concentration of its bile salts (Charteris et al., 1998). Relevant salt levels in human bile range from 0.3–0.5% (w/v) (de Smet et al., 1998; Zavaglia et al., 1998). In the study here, there was resistance to toxicity from bile salts by the L. plantarum MON03; this finding supports the importance of assessing bacterial tolerance to bile when selecting potential agents to be used for in situ detoxification processes in the gut. Here, L. plantarum MON03 showed a high bile salt tolerance and BSH enzyme activity. It was suggested that the BSH enzyme might be a detergent shock protein that enables lactobacilli to survive intestinal bile (Charteris et al., 1998). In these series of in vitro studies, it was also seen that the ability of L. plantarum MON03 to efficiently bind AFM1 did not appear to be restricted to whether the bacteria were alive or not, just simply intact. Clearly, however, use of the viable organism was far more advantageous in the particular studies than was the heat-killed form. Lastly, we recently demonstrated that L. plantarum MON03

could bind zearalenone (ZEA), another potent mycotoxin, in vitro in culture media (Abbe`s et al., 2012b); interestingly, the organism also provided protective effects against the toxin in the gut of mice that received the bacteria in conjunction with the toxin. Taking all of the above findings into account, we decided to investigate in detail the ability of L. plantarum MON03 to mitigate damage to/remove AFM1 from treated Balb/c mice and, thus, counteract likely immunotoxicity and genotoxicity from the mycotoxin. Mice here that were treated with AFM1 showed significant losses in body weight gain and significant changes in peripheral levels of overall (and in some specific types) leukocytes. This effect on hematologic parameters was possibly due to several factors including, inhibition of protein synthesis and hematopoeitic cell defects (van Vleet & Ferrans, 1992). The observed decline in lymphocyte levels suggested a possible effect of AFM1 on lymphocyte cellularity, as was previously described in Abbe`s et al. (2013). Further, the growing number of total leukocytes in the blood suggested an immune system activation against AFM1-induced/-related damages. Abbe`s et al. (2013) demonstrated that AFM1 treatment of Balb/c mice had a negative effect on several immune system parameters, including reductions in total numbers of CD3+, CD54+, CD4+, and CD56+ cells and, accordingly, decreased lymphoid organs indexes. Regarding genotoxicity, the results of this study confirmed and built upon previous data that demonstrated that aflatoxins induced oxidative stress and genotoxicity in vivo. Previously, we reported that administration of aflatoxins to mice led to an enhanced lipid peroxidation that resulted from free radical-mediated toxicity. Apart from lipids, targets of oxidative damage by these radicals also usually include critical biomolecules like nucleic acids and proteins (Ben Salah-Abbe`s et al., 2008). Gursoy-Yuzugullu et al. (2011) estimated that oxidative damage was a key determinant in the ability of aflatoxins to induce genotoxicity. Moreover, this genotoxicity is only enhanced by the fact that AFM1 can also cause damage by covalently binding in DNA itself (Shibahara et al., 1995). Formation of these adducts can be a problem, especially with regard to DNA cleavage steps during repair processes, with micronucleus formation a likely consequence. On the other hand, AFM1 may also be acting to cause the mitotic spindle dysfunction, much in the manner of ZEA (Abbe`s et al., 2007). The DNA damage, as well as changes induced in host/cell anti-oxidant status (attributable to the radicals generated), represents novel epigenetic and threshold mechanisms (Kumar et al., 2013). In light of all these noted findings, it is not surprising then that bone morrow cells from mice treated with AFM1 had significant increases in levels of chromosome aberrations and high micronuclei frequencies. With these types of damages (both micro- and macroscopic) to the immune system cells of mice exposed to the AFM1, it was very encouraging to see that co-treatment with L. plantarum MON03 was able to mitigate/prevent such toxicities to levels that gave rise to values for the given parameters that were almost comparable to controls. In light of the in vitro outcomes reported above, these findings suggested that an apparent biosorption of AFM1 to the L. plantarum MON03 resulted in reduction of AFM1 bioavailability in the gastrointestinal tract and so mitigation of potential toxic effects in these hosts. Whether these effects were attributable in some part to changes in lymphocytic sub-type levels in the hosts as well remains to be seen. In rats with induced enterocolitis, levels of CD4+ and CD8+ T-cells in the intestinal lamina propria were increased to more normal levels by administration of L. plantarum (Mao, 1996). In another study, L. paracasei NCC2461 induced development of a population of CD4+ T-cells with low proliferative capacity and that apparently were also induced to produce transforming growth factor (TGF) and interleukin (IL)-10 (von der Weid et al., 2001).

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DOI: 10.3109/1547691X.2014.904025

Several mechanisms underlay the anti-carcinogenic and -genotoxic actions of some select bacterium; these may include binding or degradation of carcinogens and toxins (Burns and Rowland, 2000; Commane et al., 2005). Although Lactobacillus species are only a small part of the intestinal microbiota (2%), their anti-cancer and -genotoxic actions against carcinogens including mycotoxins have been repeatedly demonstrated in vitro or in vivo (Hirayama et al., 2000; Tavan et al., 2002). For example, Lactobacillus casei Shirota was reported able to decrease DNA damage in the rat colon after host exposure to MNNG (N-methyl-N-nitro-N-nitrosoguanidine) as well as the potential genotoxicity induced by DMH (dimethylhydrazine) (Commane et al., 2005). Similarly, Lactobacillus delbrueckii ssp. bulgaricus could inhibit the progression/promotion of adenocarcinomas in mice following host exposure to DMH (Santosa & Farnworth, 2006). Many microorganisms, including bacteria, yeasts, molds, actinomycetes, and algae, are able to remove or degrade small amounts of aflatoxin in food/feed (Styriak et al., 2001). Results of several studies suggest that binding is a main mechanism of detoxification against aflatoxins (Gratz, 2007; Niderkorn et al., 2006). Strains L. rhamnosus GG and LC-705 seem to be the most effective in such detoxifications (Lahtinen et al., 2004). However, the binding mechanism itself can be explained by the findings of Vastano et al. (2013). Clearly, as noted by Shetty & Jespersen (2006), systematic studies are still needed to understand precise binding mechanisms. We wish to note that changes in how the aflatoxins are metabolized in situ (as opposed to merely alterations in their bioavailability) could represent a second/ alternative means for mycotoxin detoxification. Few studies have addressed this potential role for an induced change in AFB1 metabolism (Gratz, 2007).

Conclusions In summary, this study showed that L. plantarum MON03 exerted a chemoprotective effect by modulating the activities of AFM1sensitive immune cells and protecting DNA from AFM1-induced damage. These results may prove useful in developing L. plantarum MON03- based chemoprotection regimens. However, further work needs to be done to optimize the doses needed for application in medicine and to clarify the mechanisms by which the bacteria helps to counteract the immune stress/bone morrow cell genotoxicity induced by AFM1.

Acknowledgments The work was supported by the Tunisian Ministry of Higher Education and Scientific Research (Unit of Immunology, Environmental Microbiology, and Cancerology), the Higher Institute of Biotechnology of Beja (Animal Biotechnology Dept) and Unit of Histology Cytology and Genetics, Faculty of Medicine, University of Monastir, Monastir, Tunisia.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Potential preventive role of lactic acid bacteria against aflatoxin M₁ immunotoxicity and genotoxicity in mice.

Aflatoxin M1 (AFM1) is a mycotoxin produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. Exposure to AFM1 imparts potent ...
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