1. 2013
http://informahealthcare.com/ipi
ISSN: 0892-3973 (print), 1532-2513 (electronic)
Immunopharmacol Immunotoxicol, Early Online: 1–8
! 2013 Informa Healthcare USA, Inc.
DOI: 10.3109/08923973.2013.772194
RESEARCH ARTICLE
Lactobacillus paracasei BEJ01 prevents immunotoxic effects during
chronic zearalenone exposure in Balb/c mice
Samir Abbe`s1,2*
, Jalila Ben Salah-Abbe`s1*
, Hakimeh Sharafi3
, Ridha Oueslati1
, and Kambiz Akbari Noghabi3
1
Unit of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences of Bizerte, University of Carthage, Tunis, Tunisia,
2
Animal Biotechnology Department, Higher Institute of Biotechnology of Beja, University of Jendouba, Jendouba, Tunisia, and 3
National Institute of
Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Abstract
Background and aim: Zearalenone (ZEN) is an estrogenic mycotoxin produced by numerous
Fusarium species in pre- or post-harvest cereals. ZEN displays a potent estrogenicity in livestock
and also causes severe immunological problems. The aims of this study were to isolate a new
ZEN-degrading micro-organism for biological detoxification, to examine its ability to degrade
ZEN in liquid medium, and to evaluate its potential for in vivo preventitive effects against ZEN
(as would occur with contaminated feed)-induced immunomodulation in mice.
Materials and methods: Lactobacillus paracasei BEJ01 (LP) isolated from Tunisian artisanal butter
was found to display significant binding ability to ZEN in phosphate-buffered saline (i.e. 96.6%)
within 24 h of incubation. The in vivo study was conducted using Balb/c mice that received
either vehicle (control), LP only (at 2 Â 109
cfu/l, $2 mg/kg BW), ZEN alone (at 40 mg/kg BW), or
ZEN þ LP daily for 15 d.
Results: Compared to control mice, ZEN treatment led to significantly decreased body weight
gains and decrements in all immune parameters assessed. The addition of LP to ZEN strongly
reduced the adverse effects of ZEN on each parameter. In fact, mice receiving ZEN þ LP
co-treatment displayed no significant differences in the assayed parameters as compared to the
control mice. The exposures to the bacteria alone had no adverse effects in the mice.
Conclusion: From these data, we conclude that LP bacteria could be beneficial in human and
animals for protection against immunotoxicity from ZEN at high levels and during chronic
exposures.
Keywords
Binding, detoxification, immunotoxicity,
Lactobacillus strains, zearalenone
History
Received 7 December 2012
Revised 9 January 2013
Accepted 29 January 2013
Published online 7 March 2013
Introduction
Lactic acid bacteria (LAB; lactobacilli) is a constituent of the
normal gut flora. Further, LAB consumption has become
increasingly associated with a range of health benefits,
including modulation of immune function and prevention of
cancer1–3
. The literature also provides an overview of
mycotoxin detoxification using LAB4
.
Some toxigenic fungus strains produce toxic compounds in
foods and feedstuffs5
that, upon consumption, cause a variety
of health problems in animals and humans6,7
. Fusarium
mycotoxins are secondary metabolites that occur naturally in a
variety of animal feeds and foods. In particular, zearalenone
[ZEN, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-b-resorcyclic
acid lactone] is a non-steroidal estrogenic mycotoxin produced
mainly by F. graminearium and F. culmorum. ZEN is known
to be hepato-toxic, haematotoxic, genotoxic and
immunotoxic8,9
. Recent studies have reported that several
alterations in immunologic parameters occur in vivo in mice10
and humans11
as a result of chronic exposure. Similarly,
according to Eriksen & Alexander12
, alterations in immune
parameters such as mitogen-stimulated lymphocyte prolifer-
ation and the production of selected cytokines (e.g. interleukin
[IL]-2 and IL-5) occur in in vitro models that examined effects
of high concentrations (i.e. 0.05 mg/ml) of ZEN. In addition, it
has been shown that ZEN caused an earlier onset of puberty in
children13
, endometrial adenocarcinomas, as well as hyper-
plasia and breast cancer in women14
. ZEN has been shown to
be genotoxic, and to induce DNA-adduct formation in in vitro
cultures of bovine lymphocytes. ZEN may affect the uterus by
decreasing LH and progesterone secretion and by altering the
morphology of uterine tissues. ZEN can depress serum
testosterone, weights of testes and spermatogenesis, while
inducing feminization and suppressing libido. Besides, alter-
ations of immunological parameters such as the inhibition of
mitogen stimulated lymphocyte proliferation, the increase
IL-2 and IL-5 production were found at high ZEN.
Abbe`s et al.15
demonstrated that oral administration of a
single dose of ZEN (40 mg/kg body weight [BW]) resulted in
Address for correspondence: Dr Samir Abbe`s, Unit of Immunology,
Environmental Microbiology and Cancerology, Faculty of Sciences of
Bizerte, University of Carthage, 7021 Zarzouna, Tunisia. Tel:
21672591906. Fax: 21672590566. E-mail: abb_samir@yahoo.fr
*These authors contributed equally to this work.
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2. several immunotoxicological effects after 24 h. Indeed, a
significant difference was found between ZEN-treated mice
and the control group regarding the serum concentration of
IgG and IgA, total white blood cell (WBC) count and the
differential counts of WBC16
. Moreover, ZEN stimulates the
growth of human breast cancer cells through its effects on
estrogen receptors response. Estrogenic and immunotoxic
effects of ZEN have been demonstrated in the literature by
many authors; it is possible that a combination of these effects
may contribute to an increase in the occurrence of breast
cancer in exposed hosts17
. Consequently, the regulation of
ZEN-induced immunotoxicity and its importance to induce
carcinogenicity have recently gained special interest in the
development of new therapeutic agents. Because of its
detrimental effects, a number of strategies have been
developed to prevent the growth of mycotoxigenic fungi, as
well as to decontaminate and/or detoxify mycotoxin-contami-
nated foods and animal feeds18
. The use of many of the
available physical and chemical methods for the detoxification
of food products contaminated with mycotoxins is restricted
due to problems concerning safety issues, possible losses in
the nutritional quality of treated commodities, coupled with
limited efficacy and cost implications19
. This has led to the
search for alternative strategies such as biological agents.
Recently, interest has been increasing in the concept
whereby host absorption of myco-toxins present in contami-
nated food might be reduced by microorganisms in the
gastrointestinal tract. Numerous investigation have shown that
some dairy strains of LAB and bifido-bacteria were able to
bind effectively to ZEN in buffered solution20,21
. This group
also reported that LAB fermentation could significantly
reduce ZEN concentrations in maize by 68–75%. However,
such a reduction may not be sufficient for complete detoxi-
fication of the toxin. Building on those findings, the aim of
this study was to isolate a new ZEN-binding microorganism
for use in biological detoxification and to examine its ability
to remove ZEN in liquid medium and its potential for
prevention of ZEN-induced immunomodulation in mice.
Materials and methods
Chemicals and antibodies/ELISA materials
Standard ZEN (purity 498%) 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. Fluorescein isothiocyanate (FITC)-
anti-CD3e, FITC-anti-CD4, phycocrythrin (PE)-anti-CD8,
PE-anti-B220, PE-anti-DX5 and Cy-chrome-anti-CD3e
antibodies were bought from BD Pharmingen (San Jose,
CA). The ZEN ELISA kit was obtained from Romer Labs, Inc.
(Union, MO).
Sampling and isolation of Lactobacillus paracasei
BEJ01
To isolate ZEN-binding microorganisms, highly ZEN-
contaminated grain, milk and butter (confirmed independ-
ently ahead of time; data not shown) samples were screened
for overall ZEN-binding activity. A total of 20 of these
samples (grain, milk and butter) were pre-cultured in nutrient
broth at 33
C for 24 h. The samples were then plated on
nutrient agar, potato/dextrose/agar and Bromo-Cresol Purple
agar for isolation of bacteria, molds and LAB, respectively.
From these master plates, single isolated colonies were picked
and then cultured in nutrient broth, potato/dextrose/broth and
MRS (deMan, Rogosa and Sharpe medium, specific for
cultivation of lactobacilli) broth as appropriate and incubated
(with shaking) for 24 h at 30
C.
To test for ZEN binding activity, ZEN was then added (at
1 mg/l) to the test cultures and the system allowed to incubate
at 30
C for 24 h. At the end of the incubation, ZEN-binding
activity was tested using an ELISA kit, according to the
manufacturer instructions. In brief, after centrifugation of the
ZEN-culture systems (2500 rpm, 10 min, 4
C), the super-
natant was recovered. Then, 200 ml of kit-provided anti-ZEN
peroxidase-conjugated antibody was combined with 100 ml of
kit standard or test sample and 100 ml of this mixture was
transferred into a microwell coated with non-conjugated anti-
ZEN antibody. The plate was held at room temperature (RT)
for 10 min and then the well contents were discarded and the
well was washed five times with deionized water. Enzyme
substrate (100 ml of the kit-provided urea peroxide) was then
added to each well and the plate incubated at RT for 5 min.
After addition of 100 ml Stop Solution (1 M H2SO4) to each
well, the absorbance in each well was read at 450 nm in an
ELx808 microplate reader (Biotek, Winooski, VT).
Extrapoloation from a standard curve prepared in parallel
using kit standards allowed for estimation of the levels of free
ZEN content in the supernatant of each test culture. The
sensitivity of the kit was 0.1 pg ZEN/ml.
Ability of Lactobacillus paracasei BEJ01 to adsorb
ZEN in vitro
From the assays outlined above, one particular form –
Lactobacillus paracasei (LP) BEJ01 (LP BEJ01) was selected
for further study. For this study, the LP BEJ01was inoculated
into 20 ml of the fresh liquid nutrient medium in 50 ml flasks.
ZEN (50 ppm) was added to the growth medium (three
replicates per sample). The cells were cultivated on a shaker
(150 rpm) for 12 h at 37
Q. The dynamics of cell growth was
controlled by the optical density of the culture registered with
the use of UVIKON 943 spectrophotometer (Kontron
Instruments, Roma, Italy) at 670 nm. After the reaction
time, the bacteria were centrifuged at 20 000 Â g for 20 min at
4
C. After centrifugation, the supernatant was filtered using a
sterile 0.2 mm pore cellulose (pyrogen-free) disposable filter.
Total levels of ZEN binding and ZEN/bacterium complex
stability was then determined using data obtained from
measures using the ZEN ELISA kit.
Identification of bacterial isolate
The most inhibitory LAB isolates were identified using an
API 50 CHL kit (Biomerieux, Montpellier, France). The
isolates were matched to species level through carbohydrate
fermentation patterns. Also, randomly amplified polymorphic
DNA (RAPD) fingerprinting was conducted according to the
protocols of Ward Timmins22
. DNA was extracted from
cultures grown overnight using High Pure PCR Template
Preparation kit (Roche Molecular Biochemicals, Mannheim,
2 S. Abbe`s et al. Immunopharmacol Immunotoxicol, Early Online: 1–8
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3. Germany) according to manufacturer instructions. Lysozyme
(10 mg/ml) and mutanolysin (1000 U/ml) were added as
lysing enzymes and the mixture was incubated at 37
C for
60 min. The PCR amplification was conducted using a
‘‘50
-CAGCACCCAC-30
’’ short primer. The reaction mix
(50 ml) contained 100 pmol of primer, 0.2 mM dNTPs,
3.5 mM MgCl2, reaction buffer, 1.5 U Taq polymerase and
1 ml of DNA solution. The thermo-cycling program was: 1
cycle at 94
C for 3 min, 45
C for 45 s and then 72
C for
1 min; 30 cycles of 94
C for 45 sec, 45
C for 45 s and then
72
C for 1 min; and lastly, 1 cycle of 94
C for 45 s, 45
C for
45 s and then 72
C for 5 min. The final PCR products were
separated over 1.0% agarose gels that also contained a 100 bp
DNA ladder (New England Biolabs, Ipswich, MA) for size
comparison. In addition, 16 s ribosomal RNA (rRNA) iden-
tification was conducted offsite by Midi Labs (Newark, DE).
Bacteria preparation and count for in vivo study
Bacterial cultures of LP BEJ01 was obtained by incubating
0.1 g of lyophilized bacteria (containing approximately 1010
bacteria) in 10 ml of MRS broth under aerobic conditions at
37
C for 24 h. The colony forming units (cfu) in the overnight
cultures were determined by turbidimetry at 600 nm with a
spectrophotometer (DU640, Beckman Industries Inc,
Fullerton, CA).
Animals and treatment
A study was performed using 40 healthy Balb/c mice (female,
8-week-old) obtained from the Pasteur Institute (Tehran,
Iran). All mice were housed in the Animal House Laboratory
at the National Institute of Genetic Engineering and
Biotechnology (NIGEB, Tehran, Iran). This pathogen-free
facility provided an environment where the mice were housed
in stainless steel cages in a temperature-controlled (23 Æ 1
C)
environment with a 50% Æ 5% relative humidity and a 12 h
light/dark cycle. All mice were provided ad libitum access to
a standard laboratory diet (protein: 16.0%; fat: 3.6%; fiber:
4.1% and metabolic energy: 0.012 MJ) and filtered water
throughout the study. All animals received humane care in
compliance with the guidelines of the NIGEB Animal Care
and Use Committee; this committee also approved all of the
procedures described herein.
After an acclimation period of 1 wk, mice were randomly
distributed into four groups (10 mice/group). Animals within
different treatment groups were treated daily by oral gavage
(without anesthesia) for 15 d as follows: control mice ¼
vehicle (phosphate-buffered saline [PBS, pH 7.4]); ZEN-
Mice ¼ ZEN only (40 mg/kg BW); LP-Mice ¼ LP preparation
(2 Â 109
cfu/l $2 mg/kg BW); ZEN þ LP-Mice ¼ ZEN
(40 mg/kg) þ LP (2 Â 109
cfu/l). The specific dose of ZEN
used here was based on our previous work8
and is equivalent
to 8% of the LD50 (i.e. 2 mg/kg) determined in those studies.
The specific dose of the bacterium was literature-based23
.
BWs for 10 mice in each group were recorded at study
beginning (prior to any treatments) and end (Day 15). One
day after the final dosing (i.e. Day 16), the mice were
euthanized using ether and blood samples were obtained by
cardiac puncture; the spleen and thymus were also collected
and weighed.
Hematology and organ cellularity
From each blood sample, total levels of erythrocytes, leuko-
cytes, hemoglobin, hematocrit, mean corpuscular volume
(MCV), mean corpuscular hemoglobin (MCH), MCH con-
centration (MCHC), red cell distribution width (RDW),
hemoglobin distribution width (HDW), mean platelet volume
(MPV) as well as platelet (PLT) and leukocyte counts were
determined with an ADVIA120 automated hemo-analyzer
(Bayer, Munich, Germany). Spleen and thymus cellularities
were determined by counting with a hemocytometer after
dispersion of the tissues into RPMI 1640 culture medium.
Cell staining for phenotypic analysis
Cell staining for phenotypic analysis (using flow cytometry)
was carried out as described by Plasman Vray24
and Lee
et al.25
. Briefly, single cell suspensions were prepared from
each spleen by gently rubbing through a nylon mesh filter.
Cellular debris was removed and cells were washed in PBS;
the final pellet was then re-suspended (at 107
cells/ml) in
FACS buffer (PBS supple-mented with 10 mM HEPES buffer,
0.01% sodium azide, and 1% heat-inactivated fetal bovine
serum [Hyclone, Logan, UT]). Cells were labeled in a 96-well
plates (106
cells/well) using 10 ml of each selected fluoro-
chrome-conjugated monoclonal antibody (mAb). Double
staining was performed for FITC-CD3e/PE-CD45R and
FITC-CD3e/PE-DX5. Triple staining was performed using
Cy-cychrome-CD3e/FITC-CD4/PE-CD8a. Staining of natural
killer (NK) cells was performed with a DX5 mAb and
expressed as cells that were DX5þ
CD3À
.
Flow cytometry
Flow cytometric analysis was performed with a FACScan
flow cytometer (Beckton Dickinson, Franklin Lakes, NJ). The
FACScan was equipped with a 488 nm argon laser and
detectors for forward scatter (FSC), side scatter (SSC), FL1
(band bass filter wavelength 530 nm), FL2 (585 nm) and FL3
(650 nm) fluoresceine emission in the green, red/orange and
long red parts of the spectra, respectively. Splenocytes stained
with FITC-, PE- and Cy-chrome-labeled antibodies were
detected on FL1, FL2 and FL3, respectively, while dead cells
stained with PI were detected on FL2. Fluorescence overlap
was compensated for electronically using splenocytes stained
with a single color (FITC, PE and PI); 10 000 events were
acquired with each sample and stored for analysis.
Splenocytes were identified by their characteristic appearance
on a dot-plot of FSC versus SSC and electronically gated to
exclude PLTs, red cells or dead cell debris. The gate was the
same for both exposed and control mice. All data were
analyzed using StatView software (SAS Institute, Cary, NC);
all results were reported as the percentage of positive cells
within a gate.
Statistical analysis
All data are expressed as the mean Æ SD. The in vitro data
were analyzed by a one-way analysis of variance followed by
a Dunnet’s post-hoc test. In the in vivo study, a Student’s t-test
was used. All analyses were done using Sigma Stat Version
2.0 (Systat Software Inc., San Jose, CA).
DOI: 10.3109/08923973.2013.772194 L. paracasei BEJ01 prevention of ZEN-induced immunotoxicity 3
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4. Results
One strain isolated from butter (designated BEJ01) was able
to bind ZEN to the greatest extent. API identification matched
the isolate to LP ssp. tolerans. Further, RAPD typing
generated DNA patterns and confirmed that the isolate
corresponded to LP ssp. (Figure 1). 16s rRNA identification
revealed that the isolate belonged to the LP species and it is
closely related to LP ssp. paracasei rather than LP ssp.
tolerans.
The isolated LP BEJ01 bound ZEN quite rapidly at 30
C,
with 36% of the stock material bound within 60 min of
co-mixing. By 24 h, the degree of binding was496.6(Æ5.1)%.
Specificity of the binding was verified by parallel incubation
studies using L. casei, a strain previously shown not to bind
ZEN (unpublished data). In these studies, L. casei never
removed more than 5% of the ZEN material present. These
values were on par with the loss of ZEN just due to presence
in PBS vehicle during the co-mixing period. Moreover, the
strength of ZEN-binding of the LP (in PBS) was illustrated by
a retention of consistently465% even after extensive washing
of the bacteria with PBS (Table 1). Interestingly, the strength
of binding seemed to increase with time, with the level of
complex stability reaching 482% after the 24 h period of
co-mixing.
In the in vivo study, all mice survived the experiment over
the 15 d regimen. No related deaths were observed in ZEN-
only group, ZEN þ LP group co-traitment and LP BEJ01
alone group. Table 2 reports the mean BWs of the mice in
each experimental group at the start and end of the exposure
regimens. There were significant changes in BW gain
observed between the ZEN- and vehicle-exposed mice.
While the control mice gained 24% in weight from Day
0, those in the ZEN-only group lost 20%. Weight changes in
the LP alone and ZEN þ LP hosts paralleled those in the
control hosts.
A 15 d period of administrating ZEN resulted in changes in
thymus and spleen weights. Mice dosed with ZEN had
reductions in thymus and spleen weights of 61% and 23%,
respectively (Table 3). There were no significant differences
in the extent of changes in the weights of the thymus and
spleen for LP-exposed versus control hosts. The ZEN þ LP-
exposed mice had thymus and spleen weights that were
increasingly similar to those of the control counterparts.
Interestingly, even though the spleens of the ZEN-only mice
were substantively smaller than in the control hosts, when
taking the host final BW into account (i.e. the organ index
value, data not shown), the ZEN mice had a splenic index
16% higher than in the controls. In contrast, the very strong
impact of ZEN on the thymus carried over to the thymic index
value; in this case, mice in the ZEN-only group had indices
35% lower than in their control counterparts by Day 15.
Either sets of indices in the LP alone and ZEN þ LP hosts
paralleled those in the control mice.
Hematology and organ cellularity
Results of hematological study (Table 4) indicated that
treatment with LP alone did not cause significant changes in
blood parameters, except for erythrocytes and PLT that were
significantly increased relative to values in control mice.
Figure 1. RAPD fingerprinting of Lactobacilus paracasei isolate BEJ01
using an RP primer ‘‘50
-CAGCACCCAC-3’’.
Table 1. ZEN removal from PBS by LP BEJ01 and complex stability
after washing.
Time with ZEN solution
Within 1 h 12 h 24 h
LP BEJ01 36.1 Æ 4.4* 71.1 Æ 6.2* 96.6 Æ 5.1*
L. casei 4.2 Æ 1.2 3.1 Æ 1.2 3.7 Æ 1.2
PBS only 3.2 Æ 0.2 4.5 Æ 0.6 4.9 Æ 0.9
% of original ZEN/LP BEJ01
complex after washing
(complex stability)
65.1 Æ 3.1 76.3 Æ 3.9 82.9 Æ 4.7
Values shown are mean [ÆSD] percentage ZEN removal from test
solution (ZEN ¼ 50 mg/ml).
Values shown are from three determinations per experimental scenario
tested.
*Value significantly different from control at p 0.05.
Table 2. BWs in mice orally exposed daily to ZEN, LP or ZEN þ LP
for 15 d.
BW (g)
Days Control ZEN LP ZEN þ LP
Day 0 23.56 Æ 1.15 24.12 Æ 2.41* 25.33 Æ 3.24 25.60 Æ 2.12
Day 15 29.16 Æ 1.75 19.23 Æ 1.05* 29.60 Æ 2.25 28.73 Æ 2.23
Data shown are mean Æ SD; n ¼ 10 mice/group.
Doses used were for ZEN was 40 mg/kg BW and for LP was 2 Â 109
cfu/l
($2 mg/kg BW).
*Indicates significant difference from control (p50.05) at the given
timepoint.
Table 3. Organ weights after daily exposure to ZEN, LP or ZEN þ LP
for 15 d.
Organ weight at Day 15 (mg)
Parameter Control ZEN LP ZEN þ LP
Spleen (mg) 83.16 Æ 6.15 64.12 Æ 1.20* 85.33 Æ 4.02 79.60 Æ 3.11
Thymus (mg) 49.12 Æ 3.55 21.03 Æ 0.95* 51.60 Æ 2.89 48.73 Æ 2.64
Data shown are mean Æ SD; n ¼ 10 mice/group.
*Indicates significant difference from control (p50.05).
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5. Treatment with ZEN alone caused significant decreases
(relative to control mice values) in levels of erythrocytes,
hemoglobin, hematocrit, MCV, MCH, MCHC, RDW, HCT,
HGB, MCV and PLT, along with a significant increase in
leukocyte levels (4.31 [Æ1.07] Â 103
cells/ml) toward those of
control hosts (3.35 [Æ0.95] Â 103
cells/ml), suggesting a
probability of infection and blood system damage. In mice
treated with LP along with ZEN, there was overall amelior-
ation of the ZEN effects on assayed hematologic parameters.
Regarding the thymus and spleen cellularity, a suppressive
effect was observed in mice treated with ZEN. The levels of
thymocytes and splenocytes decreased significantly (11
[Æ2] Â 107
versus 7 [Æ2] 107
cells, respectively) compared
to those in the control mice (26 [Æ3] Â 107
and 14 [Æ2] Â 107
cells). Treatment with LP alone resulted in a significant
increase in thymocyte numbers (15 [Æ2] Â 107
cells), but the
number of splenocytes (24 [Æ3] Â 107
cells) was similar to
that in the controls. The combined ZEN þ LP treatment
resulted in significant improvement in both splenocytes and
thymocytes counts (to 24 [Æ3] Â 107
and 14 [Æ2] Â 107
cells)
towards normal values (Figure 2). These changes gave rise to
improvements of 53.8 and 39.2% in the percentage of
splenocytes and thymocytes counts relative to those seen
with mice in the ZEN-alone group.
Splenocytes phenotype analyses revealed that oral treat-
ment with ZEN daily for 15 d significantly decreased the
percentages of CD3þ
, B220þ
, CD4þ
and CD8þ
cells,
indicating immunotoxicity (Table 5). The treatment also
resulted in an increase in percentage of DX5þ
CD3À
(NK)
cells, suggesting a triggering of the host natural immune
system against ZEN availability in the body. Treatment with
LP alone had no effect on the percentages of each splenocyte
Figure 2. Changes in cellularity of spleen and thymus. Mice were orally exposed daily to ZEN (40 mg/kg BW), LP (2 Â 109
cfu/l, $2 mg/kg BW), or
ZEN þ LP for 15 d. Controls received vehicle only each day. Data shown are the mean (ÆSD). *Value significantly different from vehicle control
(p50.05).
Table 4. Hematologic parameters of mice orally exposed daily to ZEN, LP or ZEN þ LP for 15 d.
Hematological parameter values
Parameter Control ZEN LP ZEN þ LP
Erythrocytes (Â106
cells/ml) 9.23 Æ 0.60 6.83 Æ 0.65* 10.38 Æ 0.15 9.87 Æ 0.26
Leucocytes (Â103
cells/ml) 3.55 Æ 0.95 4.31 Æ 1.07* 3.59 Æ 0.74 3.97 Æ 0.49
Hematocrit (%) 48.51 Æ 0.64 36.67 Æ 3.31* 46.58 Æ 1.08 45.15 Æ 3.16
Hemoglobin (g/dl) 15.21 Æ 0.84 10.21 Æ 0.89* 15.53 Æ 0.26 14.88 Æ 1.35
MCV (fl) 47.85 Æ 0.81 40.37 Æ 0.51* 47.72 Æ 0.54 47.13 Æ 0.79
MCH (pg) 16.13 Æ 0.28 10.58 Æ 0.17* 15.38 Æ 0.21 14.88 Æ 0.37
MCHC (f/dl) 33.25 Æ 0.34 22.78 Æ 0.35* 32.42 Æ 0.11 30.10 Æ 0.38
RDW (%) 14.33 Æ 0.58 10.11 Æ 0.36* 13.70 Æ 0.18 13.58 Æ 1.47
HCT (pg) 15.15 Æ 0.12 10.15 Æ 0.15 15.53 Æ 0.12 14.78 Æ 0.25
MCV (d/dl) 35.31 Æ 0.44 29.71 Æ 0.55* 31.98 Æ 0.17 32.38 Æ 0.21
PLTs (103
cells/ml) 832.83 Æ 18.14 893.00 Æ 19.77* 1081.83 Æ 110.53 981.67 Æ 109.47
Mean PLT volume (fl) 5.15 Æ 0.31 5.30 Æ 0.15* 5.82 Æ 0.26 5.78 Æ 0.45
Data shown are mean Æ SD; n ¼ 10 mice/group.
*Indicates significant difference from control (p50.05).
DOI: 10.3109/08923973.2013.772194 L. paracasei BEJ01 prevention of ZEN-induced immunotoxicity 5
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6. phenotype as compared to values seen with the control mice.
Co-treatment of LP with ZEN appeared to restore the
percentages of CD3þ
, B220, CD4þ
and CD8þ
toward control
values. The co-presence of the bacteria was also able to
ameliorate the increases in percentage of DX5þ
CD3À
cells
triggered by ZEN alone. The improvement in the relative
percentages in each tested phenol-type cells in the LP þ ZEN
group ranged from 44% to 59% above the values associated
with mice in the ZEN-alone group (Table 2).
Discussion
The fusariotoxin ZEN is a potent disrupter of the reproductive
and immune system26
. In addition, Abbe`s et al.15
demon-
strated that ZEN is hematotoxic, genotoxic and responsible
for immune parameter perturbation in Balb/c mice. However,
until now, no effective strategies have been established to
expunge ZEN contamination from food or feed in order to
reduce its potential toxic effect in a host. The present
investigation was carried out to explore the protective effect
and the possible ameliorative role of LP BEJ01 on ZEN-
induced immunotoxicity in Balb/c mice.
In the in vitro study, the selected bacterium has been shown
a higher binding capacity when incubated with ZEN in PBS.
Moreover, it has the most complex stability with ZEN after
washing with PBS. The results of this study confirm and extend
previous data that have demon-strated that ZEN can be
degraded or at least removed by many bacteria strains.
Altalhi27
has demonstrated that various bacteria (i.e.
Lactobacillus), yeasts and fungi could convert ZEN to a- and
b-zearalenol. Moreover, the removal of ZEN and a-zearalenol
by Lactobacillus strains was not dependent on incubation
temperature, since both toxins were removed at 4, 25 and
37
C, respectively21,28
. In the present study, the percentage of
the added ZEN associated with the bacterial cells was slightly
higher than that reported by Megharaj et al.29
, but similar to the
binding of aflatoxin B1 by the same strains30
. In some other
studies, transformation of ZEN by microorganisms to more-
potent estrogenic zearalenols has been observed31
.
Consequently, we decided to investigate in more detail the
isolated bacteria LP BEJ01 strain to mitigate/remove ZEN-
treated Balb/c mice and its ability to counteract the
immuntoxicity. In fact, mice treated with ZEN showed a
significant decrease in BW accompanied by a significant
increase in leukocyte levels and a decrease in hematological
para-meters. ZEN also increased Hematocrit and
Hemoglobin. This observation indicates degradation in red
blood cells as evidenced by higher levels of BILT and BILD
resulting in anaemia8
. This effect on hematological param-
eters was possibly due to several factors such as the decrease
of the total iron binding capacity, the inhibition of protein
synthesis and the hemopoitic cellular defects32
. The decline in
PLTs count suggested a possible effect of ZEN on blood
coagulation, previously described by Maaroufi et al.33
.
Furthermore, the growing number of total leukocytes
indicated an immune response against ZEN damage. While,
CD3þ
, B220þ
, CD4þ
and CD8þ
cells decreased in response
to ZEN treatment.
Changes in the basic parameters of spleen and thymus
weights, as well as in organ cellularity, were observed after
2 weeks of ZEN administration. This indicated that the
reduction in organ weight was accompanied by a lower cell
count. The present results suggest that ZEN might alter
proliferation and differentiation of B-cells in the mice. In our
studies, there were significant changes in splenic subpopula-
tions between ZEN-treated mice and the vehicle control mice.
Also, ZEN might alter splenic subset in mice, even though it
can reduce the cellularity and weight in spleen of the mice.
We speculate that one possible mechanism of humoral
immunity affected by ZEN might be partially due to the
change of macrophage function. Macrophages play an
essential role as antigen-presenting cell and are required for
processing and presentation of the antigens. Also, it might
that ZEN is T-cell-antigen and able by itself to disturb their
function, T-cells are required for the production and release of
a variety of soluble cytokine, which need to the proliferation
and differentiation of the B-cells4
.
Many data showed that ZEN induced immunosupression in
depressing T- or B-cell activity11,16
. This result was also
supported by Swamy et al.34
, which demonstrated that ZEN-
contaminated diet linearly reduced B-cell count in broiler
chickens. Other data showed that a single intravenous admin-
istration of ZEN (15 mg/ml) to rats led to the formation of
pronounced abnormalities in lymphocyte membranes
phospholipid metabolism35
. The addition of LP BEJ01 was
able to protect against blood changes caused by ZEN. Indeed,
Mice treated with LP BEJ01 alone or in combination with ZEN
clearly showed an improvement of most of the hematological
and the immunological parameters. In a similar work, Gratz
et al.23
observed that probiotic treatment prevents weight loss
and reduces the hepatotoxic effects caused by a high dose of
aflatoxin-B1 (AFB1). Also, Lactobacilli founded to be able to
reduce the harmful effects of pathogens by producing organic
acids, hydrogen peroxide and antimicrobial substances36
. In
other study involving 20 human volunteers, Jahreis et al.37
have observed that the percentage of cells expressing CD4 was
elevated and the phagocytosis index increased after 15 d of
probiotic sausage treatment. Moreover, in rats with induced
enterocolitis, the concentration of CD4 and CD8 lymphocytes
in the intestinal lamina propria increased to a more normal
level by administering Lactobacillus genera4,38
. In addition,
von der Weid et al.39
have been indicated that treatment with
LP NCC2461 induces the development of CD4þ
T-cells
population with low proliferative capacity, which produce
transforming growth factor-b and IL-10.
Specific probiotic bacteria have been reported to modulate
local and systemic immune responses40
. It seems that the used
Table 5. Percentage of splenocyte subtypes in mice exposed to ZEN, LP
or ZEN þ LP for 15 d.
% Lymphocytes subtype
Parameter Control ZEN LP ZEN þ LP
CD3þ
CD45RÀ
62.1 Æ 2.3 34.5 Æ 3.2* 64.8 Æ 4.1 59.2 Æ 4.1
CD45Rþ
CD3À
14.6 Æ 2.1 8.5 Æ 1.3* 13.9 Æ 0.4 15.1 Æ 1.9
CD3þ
CD4þ
CD8À
26.5 Æ 0.1 18.0 Æ 1.0* 24.6 Æ 1.8 23.1 Æ 2.1
CD3þ
CD4À
CD8þ
12.2 Æ 0.3 7.1 Æ 0.2* 12.0 Æ 0.8 12.0 Æ 0.8
DX5þ
CD3À
4.1 Æ 0.11 5.6 Æ 0.2* 4.38 Æ 0.3 3.9 Æ 0.5
Data shown are mean Æ SD; n ¼ 6 mice/group.
*Indicates significant difference from control (p50.05).
6 S. Abbe`s et al. Immunopharmacol Immunotoxicol, Early Online: 1–8
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7. bacteria will strongly reduce the biovailability of ZEN in vivo.
It is also known that bacterial components are recognized by
the immune system through their interactions with specific
Toll-like receptors41
. The specific receptors implied in some
of these interactions have been reported especially for
L. plantarum42
. In addition other mechanisms of probiotic
action have been proposed, such as inhibition of pathogens by
competition for nutrients and attachment sites or by produc-
tion of anti-microbial substances, reduction of cholesterol
levels through deconjugation of bile salts and binding of
toxins/carcinogens43
. Moreover, the removal of AFB1 and
ZEN is due to non-covalent binding of the toxins to the
carbohydrate moieties of the cell walls. Also the detoxifica-
tion of heterocyclic aromatic amines was explained by this
mechanism (for review, see Knasmuller et al.44
). However,
since a decrease of their toxic effects was also seen with
cytosolic preparations of LAB, it was hypothesized that other
mechanisms (e.g. interactions with short chain fatty acids)
may also play a role45
.
In conclusion, the current study demonstrated that ZEN-
induced toxicity in immunologic and hematologic parameters
as indicated by the changes in lymphocyte cell numbers,
splenocyte cellularity and all the hematological parameters
tested in this study. LP BEJ01 treatment prevents weight loss
and reduces the immunotoxic effects caused by ZEN. By
itself, not shown any toxic effect and improve some
immunological parameters. However, further studies are
needed before we fully understand the potential of probiotics
to reduce absorption of ZEN for future use in a human ZEN
exposure scenario.
Declaration of interest
The authors declare no conflicts of interest. The authors alone
are responsible for the content of this manuscript.
The authors would like to acknowledge the Academy of
Sciences for the Developing World (TWAS) and the United
Nations Educational, Scientific, and Cultural Organization
(UNESCO) for the financial support granted to the first
author to carry out this study as a part of TWAS-UNESCO
Associateship Scheme 2010–2012 at the National Institute of
Genetic Engineering and Biotechnology (NIGEB), Tehran,
Iran. The work was supported also by Tunisian Ministry
of Higher Education and Scientific Research (Unit of
Immunology, Environmental Microbiology and
Cancerology) and the Higher Institute of Biotechnology of
Beja (Animal Biotechnology Department).
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