Ability of Lactobacillus plantarum MON03 to mitigate aflatoxins (B1 and M1) immunotoxicities in mice.

http://informahealthcare.com/imt ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1547691X.2014.973622

RESEARCH ARTICLE

Ability of Lactobacillus plantarum MON03 to mitigate aflatoxins (B1 and M1) immunotoxicities in mice Rania Jebali1*, Samir Abbe`s1,2*, Jalila Ben Salah-Abbe`s1, Ridha Ben Younes1, Zohra Haous3, and Ridha Oueslati1

Journal of Immunotoxicology Downloaded from informahealthcare.com by University of Liverpool on 12/31/14 For personal use only.

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

Keywords

Aflatoxin B1 (AFB1) and M1 (AFM1) are mycotoxins produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. AFB1 and AFM1 display a potent economic loss in livestock and also cause severe immunological problems. The aims of this study were to: evaluate a new AFB1 and AFM1-binding/degrading micro-organism for biological detoxification; examine its ability to degrade AFB1 and AFM1 in liquid medium; and evaluate its potential for in vivo preventative effects against AFB1- and AFM1-induced immunomodulation in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFB1 and AFM1 in PBS (i.e. 82% and 89%, respectively) within 24 h of incubation and able to tolerate gastric acidity, have strongly hydrophilic cells surface properties, and adhere efficacy to Caco-3 cells in vitro. The in vivo study was conducted using Balb/c mice that received by oral gavage vehicle (control), LP only (2 109 CFU/L, 2 g/kg BW), AFB1 or AFM1 alone (0.25 and 0.27 mg/kg, respectively), or AFB1 + LP or AFM1 + LP daily for 15 days. Compared to in control mice, treatments with AFB1 and AFM1 led to significantly decreased body weight gains, histopathological changes, and decrements in all hematologic and immune parameters assessed. Co-treatment with LP strongly reduced the adverse effects of each mycotoxin. In fact, the mice receiving AFB1 + LP or AFM1 + LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria alone had no adverse effects in the mice. From these data, it is concluded that the tested bacteria could be beneficial in biotechnology detoxification of contaminated food and feed for humans and animals.

Aflatoxin B1, aflatoxin M1, Lactobacillus, immunotoxicity, binding, detoxification

Introduction Crops and dairy such as corn, cotton, yoghurt, cheese and peanuts and their industrial by-products are frequently contaminated by aflatoxins (AF), hepatocarcinogenic molecules (International Agency for Research on Cancer [IARC], 2002) produced primarily by Aspergillus flavus and A. parasiticus, either in the field or during transportation or storage (Scheidegger & Payne, 2003). AF have been implicated as causative agents in human hepatic and extrahepatic carcinogenesis (Abbe`s et al., 2008; Kamakar, 2005). Aflatoxin B1 (AFB1), the most toxic and carcinogenic AF (Roebuck & Maxuitenko, 1994), once ingested by mammals is absorbed in the gastrointestinal tract and appears rapidly as aflatoxin M1 (AFM1), the principal AFB1 hydroxylated metabolite found in blood (Gallo et al., 2008) and in milk (Masoero et al.,

*These authors contributed equally to the manuscript. Address for correspondence: Dr 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 27 July 2014 Revised 16 September 2014 Accepted 29 September 2014 Published online 2 December 2014

2007) and, consequently, milk products for human consumption (Trucksess et al., 1983). The AFB1 carry-over rate into milk as AFM1 has been determined to range from 1–3% in lactating dairy cows (Diaz et al., 2004; van Eijkeren et al., 2006), with a reported maximum value of &6% (Veldman et al., 1992). Our laboratories recently demonstrated that in Beja province (Tunisia), milk and milk by-products were contaminated with high levels of AFB1 and AFM1 (Abbe`s et al., 2012a). In Tunisia, AF were found to contaminate different agricultural commodities as well, such as pistachios, nuts, grains, and feedstuffs (Bensassi et al., 2010; Ghali et al., 2008, 2009). AFM1 is of such a major concern to humans because of its frequent occurrence in dairy products at concentrations high enough to cause adverse health effects. The limits for AFB1 as set by the European Commission for animal feeds and complete feedstuffs for dairy animals are, respectively, 20 and 5 mg/kg (European Commission, 2003). In milk, the European Commission set the AFM1 maximum permitted level at 0.050 mg/kg (European Commission, 2006), while in the US the maximum AFM1 concentration is regulated by the US Food and Drug Administration at 0.500 mg/kg (Berg, 2003). Various equations to predict the AFM1 level in milk (ng/kg) from AFB1 intake (mg/cow/day) have been proposed (van Eijkeren


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et al., 2006). In dairy farms, the precise AFB1 determination in animal feeds is useful to predict the AFM1 concentration in milk and to avoid contamination levels exceeding the legal limits, but the problem is that we cannot avoid the mycotoxin contamination. Animals that consume AFB1-contaminated feed develop various health problems. AFB1 also cause immunosuppression and enhanced susceptibility to infectious diseases. Recently, interest has been increasing in the concept whereby host absorption of mycotoxins present in contaminated food might be reduced by micro-organisms in the gastrointestinal tract. Numerous investigations have shown some dairy strains of Lactic acid Bacteria (LAB) and bifido-bacteria were able to bind effectively to AFB1, AFM1 and Fusarium mycotoxins in buffered solution (Abbe`s et al., 2012b; El-Nezami et al., 2002; Mokoena et al., 2005). Building on those findings, the aim of this study was to evaluate a new AFB1 and AFM1-binding microorganism for use in biological detoxification and to examine its ability to remove AFB1 and AFM1 in liquid medium and its potential for prevention of AFB1 and AFM1-induced histopathologic, hematologic, and immunologic disturbances in mice.

Materials and methods Chemicals and bacteria Standard AFB1 and AFM1 (purity498%) were 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. Two working solutions of 0.25 and 0.27 mg of AFB1 and AFM1 per milliliter of ethanol were prepared. Another two 20 mg AFB1/ml and AFM1/ml solutions were prepared in phosphate-buffered saline (PBS, pH 6.5) for use in the in vitro study. All solutions were freshly prepared using sterile distilled water and held at 4 ± 1 C until use. The bacterial strain used in the present study was Lactobacillus plantarum MON03 (LP), a lactic acid bacteria (LPB) isolated from 1-month-old artisanal butter made from cow milk collected from local producers in central Tunisia. L. plantarum MON03 tolerance to gastric juice and adhesion ability to Caco-2 cells The acid tolerance of isolated LAB was studied in simulated gastric juices as described by Charteris et al. (1998). The simulated gastric juices prepared by PBS buffer solution with pepsin (0.3%, w/v). The buffer solutions were prepared by adjusting the pH to 2.0, 3.0, and 6.2 (control) with HCl, and sterilized by autoclaving at 121 C for 15 min. After thorough mixing, 10 ml of each pH solution was taken in sterilized test tubes. Each cell suspension of the selected LAB cultures containing 109 cells/ml was added to each pH solution and mixed. After 3 h incubation, 1 ml from each solution was serially diluted with 0.85% sterile saline and appropriate dilutions plated onto MRS agar and incubated in an anaerobic chest at 37 C for 72 h. L. rhamnosus GG (ATCC 53103) was assayed in parallel as a control. The ability of the isolates to adhere to Caco-2 intestinal epithelial cells was evaluated using the method of Martıń et al. (2006). Briefly, the cells (originally obtained from NIGEB, Tehran, Iran) were grown in Dulbecco’s minimal essential medium (DMEM; Invitrogen, Hanover, Germany) containing 25 mM glucose, 1 mM sodium pyruvate, 10% heat-inactivated (30 min, 56 C) fetal calf serum, 2 mM L-glutamine, 1% nonessential amino acid preparation, 100 U penicillin/ml, and 100 mg streptomycin/ml (all materials from Sigma). For the adherence assays, Caco-2 cells were cultured to confluence in 2 ml medium devoid of antibiotics (plating in 24-well dishes). Approximately

J Immunotoxicol, Early Online: 1–10

10 days after reaching confluence, 1 ml of medium in each was removed and replaced with 1 ml Lactobacillus suspension (108 cfu/ml DMEM). The inoculated cultures were then incubated for 3 h at 37 C in a 95% air:5% CO2 chamber. The infected cells were then washed three times with sterile PBS (pH 7.8), fixed in methanol, Gram-stained and observed microscopically. Adherent lactobacilli in 20 random microscopic fields were counted for each test. A probiotic strain L. rhamnosus GG (ATCC 53103) was used as a control. Enumeration of adhered lactobacilli was performed in triplicate and values expressed as mean ± SD. AFB1 and AFM1 removal from PBS using L. plantarum MON03 One volume of LP culture broth (108 CFU/ml) was centrifuged (3000 g, 15 min) and the bacterial pellets washed with water. The LP pellets were suspended in 5 ml PBS containing AFB1 or AFM1 at 50 mg/ml. The tubes were mixed and the bacterial suspensions then incubated at 37 C for 0, 12, and 24 h. AFB1and AFM1-positive controls (50 mg/ml PBS) in the absence of bacteria and a negative control (bacteria suspended in PBS only) were also incubated for the same time periods to monitor efficacy of bacteria binding of AFB1 and AFM1. At each timepoint, the dedicated tubes were centrifuged for 15 min at 3000 g and supernatants collected and transferred to clean tubes. The tubes were stored at 4 C until time of AFB1 and AFM1 content analysis. Unbound AFB1 and AFM1 in the supernatants were determined using HPLC (see below). All experiments were performed in triplicate. To determine the effect of bacteria viability on binding affinity, bacteria (108 CFU/ml) were heated at 90 C for 15 min and the killed bacteria then pelleted, contaminated with AFB1 and AFM1, and analyzed as above. Bacterial cells surface properties Cell surface properties were determined by a microbe adhesion to solvent method (Pelletier et al., 1997) for live and thermallyinactivated Lactobacillus strains and live Escherichia coli (for comparative purposes). First, a 24-h culture of bacteria in liquid medium was centrifuged (5000 g, 10 min), washed twice with water, and then re-suspended to an optical density (OD) of 0.4 at 400 nm (A0) in 0.1 M KNO3. All OD values were obtained using a UVIKON 943 spectrophotometer (Kontron Instruments, Milan, Italy). A similar suspension was prepared using thermallyinactivated cells. Next, 0.2 ml of solvent was added to 1.2 ml of cell suspension. After 10 min pre-incubation at ambient temperature, the mixture was vortexed for 2 min. After 15 min, the aqueous phase was removed and its absorbance measured at 400 nm (A1). Percentage bacterial adhesion to solvents was calculated as (1 [A1/A0]) 100. Three different solvents were used: hexadecane (non-polar), chloroform (acidic monopolar), and ethyl acetate (basic monopolar). Hexadecane adhesion testing shows whether the cell surface is hydrophobic or hydrophilic. The microbial adhesion to solvents method values obtained for chloroform and ethyl acetate determine the donor/basic and acceptor/acidic properties of the cell surface, respectively, as well as the presence of Lewis acid-base interactions (Bellon Fontaine et al., 1996). Determination of AFB1 and AFM1 AFB1 and AFM1 analyses were performed using HPLC that incorporated an immuno-affinity column. In brief, after blending, the samples were filtered using Whatman filter paper and the filtrate diluted with 80 ml PBS. A sample of the filtrate (10 ml) was then passed through an AFB1 or AFM1 immunoaffinity column containing specific anti-AFB1 or anti-AFM1 mono-clonal

Prevention of AFB1 and AFM1 immunotoxicity


DOI: 10.3109/1547691X.2014.973622

antibodies bound to a solid support (Vicam, Watertown, MA), followed by washing with 20 ml distilled water at 5 ml/min. Bound AFB1 or AFM1 was eluted with 1.5 ml acetonitrile followed by 1.5 ml distilled water, and collected in a clean vial. These two eluted samples 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 purchased from Advanced Chromatography Technologies (Aberdeen, Scotland); product measurements were made in-line spectrophotometrically at an OD of 435 nm. Quantification of AFB1 and AFM1 were done from measures of peak areas and extrapolating against a calibration curve prepared/analyzed in parallel using AFB1 and AFM1 standards (Sigma). Three replicate analyses were performed for each sample. To verify the soundness of the assay, recovery from bacteria-free PBS was determined for the test range of AFB1 and AFM1 used; mean recovery was 89–93%. The percentage of AFB1 and AFM1 bound to the bacteria was calculated using the formula: 100% (1.00 [peak area of AFB1 or AFM1 in supernatant/peak area of AFB1 or AFM1 in positive control sample]). Animals and treatments Balb/c mice (10-weeks-of-age, female) were obtained from the Pasteur Institute (Tunis, Tunisia) and acclimatized for 1 week before use in the experiments. All mice were housed in specific pathogen-free facilities maintained at 22 ± 2 C, with a 40 ± 5% relative humidity, and a 12-h light/dark cycle. Standard rodent chow and filtered water were available ad libitum. All water and food were tested according to NF ISO 15302 to confirm that all matrices were AFB1- and AFM1-free down to a 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 24 November 1986 (86/609/EEC). For the experiments, the mice were distributed into six treatment groups (10 mice/group) and treated orally (by oral gavage) for 15 days as follows: (i) untreated control, (ii) treated with LP (2 109 cfu/kg bw), (iii) treated with AFB1 (0.25 mg/kg bw) or AFM1 (0.27 mg/kg), and (iv) treated with LP + AFB1 or LP + AFM1. Mice were weighed daily to permit adjustment of dosing volumes; dosing volume never exceeded 200 ml/treatment each day. At the end of the dosing period, after being fasted for 12 h, each mouse was weighed and then had its blood drawn from the retro-orbital plexus (and collected into heparin tubes) for hematologic analyses. After collection of blood, each mouse was euthanized by cervical dislocation and their thymus and spleen collected; samples of each organ were used to assess thymocyte and splenocyte levels, for splenocyte mRNA analyses, and for histopathology. For the latter, samples were processed, i.e. fixed in 10% neutral buffered formalin, dehydrated, cleaned, and paraffin embedded. Histological sections (5-mm thick) were then placed on glass slides and stained with hematoxylin-eosin. All slides were then assessed under light microscopy. Hematology and assessment of thymocytes and splenocytes From each blood sample, total levels of erythrocytes, leukocytes, hemoglobin, hematocrit, as well as platelet (PLT) and leukocyte counts were determined with an ADVIA120 automated hemoanalyzer (Bayer, Munich, Germany). Spleen and thymus cellularity were determined by counting with a hemocytometer after dispersion of the tissues into RPMI 1640 medium. In brief, thymocytes and spleen cells were prepared in RPMI supplemented with 12 mM HEPES (pH 7.1), 50 mM 2-mercaptoethanol,

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100 U penicillin/ml, 100 mg streptomycin/ml, and 10% FCS) through a stainless steel mesh. Spleen cells were further treated with ammonium chloride/EDTA solution to eliminate red blood cells. Spleen cell suspensions were then re-suspended in RPMI 1640 (at 6 106 cells/ml) for use in experiments outlined below. Determination of inflammatory cytokine mRNA expression Total RNA from 3 105 splenocytes/mouse was isolated using Trizol (EZNAÕ Total RNA Kit, Omega Biotek, Inc., Norcross, GA). RNA concentration of each sample was measured by a spectrophotometer (260 vs 280 nm) and quality estimated by assessing 18S and 28S rRNA; the intensity ratio of these bands was 1:2 in the gels. A SYBR Green I real-time polymerase chain reaction (RT-PCR) was then used to measure the mRNA expression levels of IFN , TNF, and IL-4. First-strand cDNA was synthesized from 5 mg total RNA (after processing with DNase) using the following oligo (dT) primers (IL-4: 50 GCGACATCACCTTACAAGAGAT-30 ; IFN : 50 -AAGATAACC AGGCCAGGCCATTCAAAG-30 , and TNF: 50 -ACCACGCTCT TCTGCCT-ACT-30 ) and Superscript II reverse transcriptase according to manufacturer instructions (Tiangen Biotech Co., Beijing, China). Real-time PCR was then done in an ABI PRISM 7500 SDS thermal cycler (Applied Biosystems, Foster City, CA). Each sample was analyzed in triplicate. Analyses were performed with 2.0 ml first-strand cDNA and 0.8 ml sense and anti-sense primers in a 20 ml final volume as recommended by the SYBR real-time PCR kit (TaKaRaÕ BioCatalog, Dalian, China). RT-PCR conditions were: one cycle at 95 C for 30 s and 40 cycles at 95 C for 5 s and 60 C for 34 s. Relative expression of the mRNA of each inflammatory cytokine was determined using the 2DDCt method (Bousquet et al., 2009; Lee & Schmittgen, 2006). Statistics Data were expressed as mean ± SD of three independent experiments (in vitro study), and analyzed for statistical significance using a Student’s t-test and a general linear model (n ¼ 6 for in vivo tests). The criterion for significance was set at p50.05.

Results Gastric acid tolerance and Caco-2 adherence Table 1 shows the survival of the LP under low pH conditions. The LP strain consistently showed tolerance to pepsin at pH 3, and the residual counts were greater than 106 cfu/ml after a 3-h incubation. Further, LP survived at pH 2 and exhibited fairly good acid tolerance with maintenance levels 475%. On the basis of screening results for tolerance to low pH, LP was found to be able to survive at levels of 106 cfu/ml at pH 2. The adhesion ability of LP was also tested with Caco-2 cells (Table 1). The results indicate LP expressed strong adhesion to the cells as compared with control strain L. rhamnosus GG. Bacterial cells surface properties Based on hexane experiments, the surface of live LP cells was determined to be strongly hydrophilic; that of Escherichia coli was moderately hydrophilic (Table 2). Following thermal treatment, the LP surface became hydrophobic (hexadecane adhesion was 435%). Low adhesion to ethyl acetate, a strongly basic solvent (515% for live cells and 420% for thermally-inactivated cells), indicated the LP cell surface was basic.

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Table 1. Gastric juice tolerance and Caco-2 adherence of LAB strains (log cfu/ml). Resistance to gastric juice Lactobacillus strain

Initial mean counts

pH 2

pH 3

Caco-2 adhesiona

9.77 ± 0.19 9.92 ± 0.18

9.23 ± 0.46 9.21 ± 0.47

9.16 ± 0.56 9.13 ± 0.58

466.3 ± 109.1 493.2 ± 97.3

L. plantarum MON03 L. rhamnosus GGb a

Number of lactobacilli strains adhered to Caco-2 cells in 20 random microscopic fields. Lactobacillus rhamnosus GG: used as control. Each value shown is the mean ± SD from three trials.

b

Table 2. Characterization of bacterial surface—adhesion to solvent (microbial adhesion to solvents method).


% adhesion to solvent L. plantarum MON03 Solvent

Live biomass

Dead biomass

E. coli ATCC 10536 Live biomass

Hexadecane Chloroform Ethyl acetate

7.63 ± 0.35 59.30 ± 0.24 12.51 ± 0.05

38.52 ± 0.05 42.91 ± 0.32 23.43 ± 0.41

21.13 ± 0.09 8.78 ± 0.06 30.85 ± 0.28

improvements of 37.3 and 49.7% for splenocyte and thymocyte counts relative to those seen in mice in the corresponding AFB1 or AFM1-alone groups. Hematologic parameters Results indicated that treatment with LP alone did not cause significant changes in blood parameters except for PLT levels that were increased (Table 5). Treatment with AFB1 or AFM1 alone caused a significant increase in WBC, HCT, and HGB values, and a significant decrease in PLT and RBC levels in the blood indicating hematologic damage. Overall, mice co-treated with LP generally exhibited amelioration of these AF-induced alterations.

AFB1 and AFM1 binding by bacteria Table 3 shows the time-dependent accumulation of AFB1 and AFM1 by LP in PBS. Viable LP adsorbed 54.3 ± 7.3% of AFB1 after 12 h and the binding amount rose to 82.3 ± 8.3% after 24 h of incubation in PBS. Regarding AFM1, live LP adsorbed 64.5 ± 5.2% of AFB1 after 12 h and reached 89.1 ± 6.2% by 24 h incubation in PBS. With heated LP cells, AFB1 and AFM1 binding decreased to 39.8 ± 0.4% and 52.2 ± 0.5% at 24 h, respectively. Effects of treatment on body weight gain and lymphoid organ indexes Effects of the treatments on body weight gain and lymphoid organ indexes are summarized in Table 4. Body weight gains were significantly decreased in mice treated with AFB1 or AFM1 alone as compared with in normal mice. In contrast, all LP-treated mice had greater weight gains than control mice. As seen in Figure 1, most of the reductions in host weight gain could likely be attributed to significant reductions in feed intake by the AF alonetreated hosts. While splenic and thymic indices were markedly increased in AFB1 or AFM1 alone mice compared with those in normal mice, the absolute organ weights did not differ significantly from normal mice values. Co-treatment with LP resulted in both splenic and thymic indices that reverted toward normal values. In all cases, treatment with LP (either alone or as part of cotreatment regimen) caused significant elevations in absolute spleen and thymus weights relative to values in corresponding control or AF-only mice. Thymus and spleen cellularity A suppressive effect on thymic and splenic cellularity was noted in mice treated with AFB1 or AFM1 (Figure 2). Levels of thymocytes and splenocytes decreased significantly compared to those in the control mice. Treatment with LP alone resulted in no significant change in thymocyte and splenocyte numbers from control mice values. Compared to the organ values in the AFB1 and AFM1-treated mice, the AFB1 or AFM1 + LP co-treatments resulted in significant improvement in splenocyte and thymocyte counts to near normal values. These changes represented

Histopathology Spleens of mice treated with AFB1 or AFM1 alone showed focal necrosis (FN; Figures 3C and D), vascular dilatation, and lymphoid infiltration (LI; indicating portal tract inflammation). Spleens of mice treated with LP alone or in combination with AFB1 or AFM1 revealed a similar histologic picture to that of control mice (Figures 3A and B). Thymus histology of mice treated with either AF alone revealed a swelling in cortical cells of the proximal lobules, granular degeneration, shrunken dendritic cells, a presence of eosinophilic casts in the lobules (Figure 4D), and blood vessel dilatation (Figure 4C). The thymus of each mouse treated with LP alone or with LP with AFB1 or AFM1 had normal histologic appearances (Figures 4E and F). Inflammatory cytokine mRNA expression AFB1 and AFM1 exposure during treatment resulted in significantly decreased mRNA expression of IFN and TNF and increased expression level of IL-4 in the spleen (Table 6). This indicated that AFB1 and AFM1 could have a direct impact on inflammatory cytokine expression. The combinational treatments of AFB1 + LP and AFM1 + LP ameliorated the adverse effects on mRNA expression levels of IFN , TNF, and IL-4.

Discussion Adsorption of mycotoxins to the lactic acid bacterial (LAB) cell wall is attributable to their surface properties, mainly their hydrophobicity (Haskard et al., 2001). According to Chae et al. (2006), cells are strongly hydrophobic if their percentage adhesion to hexadecane is 455%, moderately hydrophobic at 30–54%, moderately hydrophilic at 10–29%, and strongly hydrophilic at 510%. In the present study, based on hexane adhesion experiments, the surface of live LAB cells was seen as strongly hydrophilic (and that of Escherichia coli moderately hydrophilic). This was consistent with the results of Boonaert & Rouxhet (2000), who reported the surfaces of Lactobacillus helveticus and Lactococcus lactis were hydrophilic, irrespective of growth phase. Those authors attributed this to a low carbohydrate content in the cell wall outer layers. L. casei, L. paracasei, and L. rhamnosus


DOI: 10.3109/1547691X.2014.973622

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Table 3. Percentage AFB1 and AFM1 removal from PBS by LP alone. AFB1 (50 mg/ml)

AFM1 (50 mg/ml)

Treatment

0h

12 h

24 h

0h

12 h

24 h

PBS only Live LP Killed LP

2.3 ± 0.1 20.1 ± 4.1* 18.1 ± 0.4

2.1 ± 0.2 54.3 ± 7.3* 32.6 ± 0.7

2.4 ± 0.1 82.3 ± 8.3* 39.8 ± 0.4

3.0 ± 0.2 25.9 ± 5.8* 23.1 ± 0.3

2.4 ± 0.2 64.5 ± 5.2* 42.5 ± 0.6

2.3 ± 0.2 89.1 ± 6.2* 52.2 ± 0.5

To determine the effect of bacteria viability on the binding affinity to AFB1 and AFM1, ‘killed’ bacteria (108 CFU/ml) were heated at 90 C for 15 min before being used in the assay. Values shown are mean ± SD from three determinations per experimental scenario tested. *All values shown for bacterium-containing systems are significantly different from both control (PBS only and heated bacteria) at p 0.05.


Table 4. Body weight gain and spleen and thymus indexes in the mice. Body weight (g) Treatment Control LP alone AFB1 alone AFM1 alone AFB1 + LP AFM1 + LP

Organ indexes

Initial

Final

Net weight gain (g)

Spleen

Thymus

25.90 ± 1.21 30.30 ± 2.13 22.41 ± 1.84 23.35 ± 1.82 32.30 ± 2.16 32.12 ± 2.20

34.01 ± 1.60 41.94 ± 3.33 23.64 ± 2.62* 25.38 ± 2.37* 46.74 ± 4.32 47.11 ± 4.67

8.93 ± 0.39 11.64 ± 1.02 1.23 ± 0.78* 2.02 ± 0.56* 14.44 ± 2.16 14.99 ± 2.48

0.697 ± 0.028 0.677 ± 0.035 0.858 ± 0.071* 0.895 ± 0.055* 0.675 ± 0.034 0.664 ± 0.021

0.053 ± 0.005 0.057 ± 0.003 0.077 ± 0.001* 0.075 ± 0.004* 0.050 ± 0.001 0.052 ± 0.001

Balb/c mice were orally exposed daily (for 15 days) to saline, LP (2 109 cfu/kg) alone, AFB1 (0.25 mg/kg) alone, AFM1 (0.27 mg/kg) alone, or to co-treatments with LP + AFB1 or LP + AFM1. Values shown are mean ± SD from three determinations per experimental scenario tested. *Value significantly differs from control, LP alone, or corresponding co-treatment values (p50.05).

Figure 1. Effect of treatments on feed consumption by Balb/c mice. Results shown are mean ± SD. In each histogram, values (bars) with superscripts bearing different letters differ significantly (p50.05).

cells have also been found to be hydrophilic (Pelletier et al., 1997). After thermal treatment, the surface of the Lactobacillus plantarum MON03 (LP) bacteria here became hydrophobic as hexadecane adhesion was 435%. As these inactivated cells were

more effective in terms of toxin removal, this indicated to us that the hydrophobic surface was responsible and was in accordance with the literature (Haskard et al., 2000). The fact that AFB1 and AFM1 were bound also by live LP, despite the hydrophilic nature of their surface, is probably attributable to a presence of

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Figure 2. Thymocyte and splenocyte cell numbers for mice after the 15 days of treatment. Mice were orally exposed to LP (2 109 CFU/kg), AFB1 (0.25 mg/kg), AFM1 (0.27 mg/kg) or to a co-treatment with LP + AFB1 or LP + AFM1. Data shown are as mean ± SE. In each histogram, values (bars) for each given type of cellularity with superscripts bearing different letters differ significantly (p50.05).

Table 5. Effect of treatments on hematological parameters. Parameter 3

WBC (10 /ml) RBC (106/ml) Hb (g/dl) Ht (%) Platelets (103/ml)

Control

LP

AFB1

AFM1

LP + AFB1

LP + AFM1

9.80 ± 0.11 8.79 ± 0.21 14.51 ± 0.12 43.14 ± 0.47 393.03 ± 20.22

9.39 ± 0.36 8.46 ± 0.18 14.69 ± 0.23 44.07 ± 0.31 442.83 ± 23.10

16.26 ± 0.07* 7.78 ± 0.11* 13.10 ± 0.05* 38.25 ± 0.05* 602.00 ± 2.10*

18.35 ± 0.08* 7.44 ± 0.23* 13.73 ± 0.20* 39.50 ± 0.69* 454.80 ± 4.28*

10.16 ± 0.34 8.87 ± 0.09 14.65 ± 0.40 43.61 ± 0.42 400.00 ± 14.72

9.49 ± 0.82 8.63 ± 0.19 14.20 ± 0.26 43.00 ± 0.53 392.90 ± 29.59

Balb/c mice were orally exposed daily (for 15 days) to saline, LP (2 109 cfu/kg) alone, AFB1 (0.25 mg/kg) alone, AFM1 (0.27 mg/kg) alone, or to co-treatments with LP + AFB1 or LP + AFM1. Values shown are mean ± SD from six determinations per experimental scenario tested. *Value significantly differs from control, LP alone, or corresponding co-treatment value (p50.05). RBC, red blood cells; WBC, white blood cells; Hb, hemoglobin; Ht, Hematocrit.

hydrophobic pockets on the surface (Haskard et al., 2001). While the surface of E. coli revealed similar characteristics, it did not bind toxins. This illustrates that toxin adsorption is also affected by other factors, such as chemical composition of the cell wall (that contains more lipopolysaccharide in Gram-negative bacteria). The in vitro survival tests here revealed that the LP 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 (Ben Salah-Abbe`s et al., 2014). This enables the strain to survive longer periods in high-acid carrier foods (such as yogurt) without large reductions in number (Minelli et al., 2004). The results of the current in vitro study with Caco-2 (human epithelial colorectal adeno-carcinoma) target cells were in line with expectations. Specifically, lactic acid bacteria strains show adherence specificity for intestinal epithelia (Wang et al., 2010); Vastano et al. (2014) recently demonstrated that L. plantarum was able to adhere to cells/other compounds via pyruvate 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). 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 detoxification (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. Taking all the above into account, we investigated the ability of LP to mitigate damage and/or remove AFB1 and AFM1 from treated Balb/c mice, and thus counter likely immunotoxic and/or histopathologic changes induced by the mycotoxins. Mice treated with AFB1 and AFM1 showed significant reductions in body weight gain and changes in several hematologic parameters. This effect on the latter was possibly due to factors including inhibition of protein synthesis and defects induced among


DOI: 10.3109/1547691X.2014.973622


7

Figure 3. Representative photomicrographs of H&E-strained spleen of mice after the 15 days of treatment. (A and B) Control mice and mice treated daily with LP (2 109 CFU/kg). (C and D) Mice treated daily with AFB1 (0.25 mg/kg) or AFM1 (0.27 mg/kg). (E and F) Mice co-treated daily with LP + AFB1 or LP + AFM1. Magnification ¼ 40. WP, white pulp; RP, Red pulp; MZ, Marginal zone; Mg, Megakaryocyte; L, Lymphocyte; M, Macrophage; N, Neutrophil.

hematopoietic cells (see van Vleet & Ferrans, 1992). The observed reversals/increases in body weights observed in LPtreated mice could be due to a side-benefit of LAB. Lactobacilli are constituents of the normal gut flora and contribute to digestion. Further, LAB consumption has become increasingly associated with a range of health benefits such as increased nutrient absorption, modulation of immune function, and prevention of cancer (de Moreno de Leblanc et al., 2010; Kawano et al., 2010; Kumar et al., 2010). With respect to immune system cells, Abbe`s et al. (2010, 2013) demonstrated that AFB1 and AFM1 treatment of Balb/c mice had a negative effect on several immune system parameters, including reductions in total CD3+, CD54+, CD4+, and CD56+ cell numbers. Unexpected, in the study here, increases were noted in the number of total blood leukocytes - suggesting possible immune system activation against AFB1- and AFM1induced or -related damage; this clearly conflicts with those earlier findings from Abbe`s et al. In contrast, the splenic/thymic cellularity measures here were in keeping with the findings of those 2010 and 2013 studies. Regardless of the divergence from the expected outcomes seen earlier, the studies here nonetheless showed that co-treatment with the LP led to reversal/mitigation

of the toxicities on these end-points that were induced by the AF. The current studies also indicated that there were significant increases in both the thymic and splenic indices due to AF, outcomes that again would contrast with the Abbe`s et al. (2010, 2013) findings. It would appear these indices findings are artifactual - related to host loss of body weight due to AF rather than changes in the spleen or thymus proper. The latter would be in keeping with the findings that absolute weights of the spleen and thymus of the mice here were only nominally/marginally affected by AFB1 and AFM1 (albeit effects on spleen weight were greater than on thymus). Even this, however, seems to conflict with observations of AF-induced increases in WBC levels as well as decrements in thymic/splenic cellularity. It remains to be determined if while there are fewer leukocytes in spleens of AFtreated mice, there are concomitant increases in levels of damaged erythrocytes that would be undergoing turnover. This scenario could help explain the apparent static nature of the absolute weight in this organ. Unfortunately, a similar depletion/repletion mechanism is not available to the thymus; thus, an explanation for the static nature of the absolute weight of this organ remains to be resolved in future studies.


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Figure 4. Representative photomicrographs of H&E-strained thymus of mice after the 15 days of treatment. (A and B) Control mice and mice treated daily with LP (2 109 CFU/kg bw). (C and D) Mice treated daily with AFB1 (0.25 mg/kg) or AFM1 (0.27 mg/kg). (E and F) Mice co-treated daily with LP + AFB1 or LP + AFM1. Magnification ¼ 40. C, Cortex; M, medulla.

Table 6. Effect of treatments on splenic mRNA expression of select inflammatory cytokines. Parameter IFN TNF IL-4

Control

LP

AFB1

AFM1

LP + AFB1

LP + AFM1

1.38 ± 0.08 1.32 ± 0.08 0.90 ± 0.02

1.39 ± 0.06 1.33 ± 0.08 0.86 ± 0.02

0.96 ± 0.07* 1.00 ± 0.01* 1.08 ± 0.04*

0.91 ± 0.07* 1.00 ± 0.03* 1.06 ± 0.03*

1.26 ± 0.04 1.27 ± 0.09 0.90 ± 0.06

1.30 ± 0.02 1.23 ± 0.09 0.90 ± 0.06

Balb/c mice were orally exposed daily (for 15 days) to saline, LP (2 109 cfu/kg) alone, AFB1 (0.25 mg/kg) alone, AFM1 (0.27 mg/kg) alone, or to cotreatments with LP + AFB1 or LP + AFM1. Values shown are mean ± SD ratios of the cytokine-specific RT-PCR product vs the corresponding cyclophilin band intensity (expressed in arbitrary units [AU]) from six determinations per experimental scenario tested. *Value significantly differs from control, LP alone, or corresponding co-treatment value (p50.05).

Still, with the types of damage (both micro- and macroscopic) to the immune system cells of mice exposed to the AFM1, it was encouraging to see that co-treatment with LP could mitigate/prevent AF-induced effects so that values for most measured parameters became comparable to those seen in control hosts. In light of the in vitro outcomes reported here, the findings suggested an apparent biosorption of AFM1 to LP resulted in reduction of AFM1 bioavailability in the gastrointestinal tract and, therefore, mitigation of potential toxic effects in the hosts. Whether these effects were attributable in some part to changes in lymphocytic sub-types 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 et al., 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 de Weid et al., 2001). The present studies also assessed effects of the AF - as well as their potential reversal by the LP - on the presence of inflammatory cytokines in treated hosts. Here, exposure of the


9

Microbiology, and Cancerology) and the Higher Institute Biotechnology of Beja (Animal Biotechnology Department).

of


DOI: 10.3109/1547691X.2014.973622

mice to AFB1 or AFM1 induced fluctuations in splenic mRNA levels of select inflammatory cytokines; specifically, AFB1 or AFM1 caused elevation in expression of IL-4 mRNA but decreases in that of IFN and TNF. This contrasts with the findings of Li et al. (2014), who noted increases in IL-6, IFN , and TNF expression in birds exposed to AFB1-contaminated diets. The reason for these differences in outcomes is not clear, although species-specific responses may be one explanation. That changes in IFN and TNF mRNA expression did not increase in tandem with those of total WBC levels in AFB1 or AFM1 alonetreated hosts suggests to us the toxin was also able to affect lymphocyte functionality in these hosts. Oddly, the IL-4 mRNA findings were not in keeping with this outcome. Because selective toxicity against groups of cytokines (especially in the absence of any exogenous stimuli) would be unusual, further studies are warranted to provide clarity about these disparate findings in AFtreated mice. Overall, the LP used in this study could bind effectively to both AFB1 and AFM1 and cause significant improvement in many of the evaluated immune parameters. Lactobacillus strains were also recently found to prevent aflatoxicosis effects on the major histocompatibility complex (Rawal et al., 2014). While exact mechanisms for these protective outcomes are still to be defined, several have been postulated to explain the anticarcinogenic/-immunotoxic actions of some select bacterium (Burns & Rowland, 2000; Commane et al., 2005). Although Lactobacillus species constitute only a small part (&2%) of the intestinal microbiota, their anti-carcinogenic/-immunotoxic actions against toxicants have been demonstrated in vitro and in vivo. For example, L. casei shirota was reported able to decrease the levels of DNA damage in the rat colon after host exposure to MNNG (N-methyl-N-nitro-N-nitrosoguanidine) as well as genotoxicity induced in vitro by DMH (dimethylhydrazine) (Commane et al., 2005; Tavan et al., 2002). L. delbrueckii ssp. bulgaricus was seen to inhibit the progression/promotion of adenocarcinomas in mice after exposure to DMH (Santosa & Farnworth, 2006). Results of several studies suggest that micro-organism binding is a key mechanism for AF detoxification (Gratz, 2007; Niderkorn et al., 2006). Strains L. rhamnosus GG and LC-705 seem to be most effective in such processes (Lahtinen et al., 2004). However, the binding mechanism itself can be explained by findings of Vastano et al. (2014; see above in re PDHB). Clearly, as noted by Shetty and Jespersen (2006), systematic studies are still needed to understand precise binding mechanisms. We wish to note that changes in how AF are metabolized in situ (vs mere alterations in bioavailability) could be a second/alternative means for their detoxification here. To date, little is known about the potential role of such induced changes in AFB1 metabolism (Gratz, 2007).

Conclusion The results of these studies showed that AFB1 and AFM1 were immunotoxic in Balb/c mice and that addition of LP bacteria to the AFB1 and AFM1 treatment regimen succeeded in ameliorating/preventing the toxicities caused by both AF. Moreover, the studies here showed that LP was able to remove highly AFB1 and AFM1 from solution. As LP was ‘safe’ by itself, this organism might be considered a candidate for use in biotech-processes that have a major goal of mycotoxin detoxification.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The work was supported by the Tunisian Ministry of Higher Education and Scientific Research (Unit of Immunology, Environmental

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Aspergillus flavus and aflatoxins B1, B2, and M1 in corn associated with equine death.

Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing.

Fluorescence changes of aflatoxins B1 and G1.

Binding of aflatoxins B1 and G1 to rat liver chromatin.

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Removal of aflatoxin B1 and inhibition of Aspergillus flavus growth by the use of Lactobacillus plantarum on olives.

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Lactobacillus plantarum TWK10 Supplementation Improves Exercise Performance and Increases Muscle Mass in Mice.

Mixed Lactobacillus plantarum Strains Inhibit Staphylococcus aureus Induced Inflammation and Ameliorate Intestinal Microflora in Mice.

Draft Genome Sequence of Lactobacillus plantarum 2025.

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Effect of Nanometric Lactobacillus plantarum in Kimchi on Dextran Sulfate Sodium-Induced Colitis in Mice.

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Ta Mice Administered Lactobacillus plantarum Strain No. 14 Have Lower Adiposity and Higher Insulin Sensitivity.

Lactobacillus plantarum NDC 75017 alleviates the learning and memory ability in aging rats by reducing mitochondrial dysfunction.

Effect of Lactobacillus plantarum Strain K21 on High-Fat Diet-Fed Obese Mice.

Isolation, extraction, and measurement of acetylcholine from Lactobacillus plantarum.

Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition.

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