Consumption of fumonisin B1 for 9 days induces stress proteins

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Toxicon xxx (2009) 1–6
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Toxicon
journal homepage: www.elsevier.com/locate/toxicon
Consumption of fumonisin B1 for 9 days induces stress proteins along
the gastrointestinal tract of pigs
Jean-Paul Lalle`s a, *, Martin Lessard b, Isabelle P. Oswald c, Jean-Claude David a
a
INRA, UMR 1039, SENAH, Domaine de la Prise, F-35590 Saint-Gilles, France
Agriculture and Agri-Food Canada, Dairy and Swine Research and Development Centre, Lennoxville, Que´bec, Canada J1M 1Z3
c
INRA, UR66, Unite´ de Pharmacologie-Toxicologie, 180 chemin de Tournefeuille, BP 93173, F-31027 Toulouse Cedex 03, France
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 February 2009
Received in revised form 23 July 2009
Accepted 24 July 2009
Available online xxxx
Fumonisin B1 (FB1) is a mycotoxin which alters intestinal epithelial cell physiology and
barrier properties, and accumulates in the colon. Data on effects of FB1 on stress proteins
in the gastrointestinal tract (GIT) are lacking. Therefore, we hypothesized that repeated
consumption of FB1 alters GIT tissue levels of stress proteins. This was tested using 36
weaned pigs fed a FB1 solution (n ¼ 18) or the vehicle (control; n ¼ 18) for 9 days. The pigs
were then slaughtered, the organs were weighed and GIT tissues were collected for
assessing GIT integrity, and for analysing stress proteins by Western blotting and densitometry (n ¼ 7 in each group). FB1 had little effects on growth rate but the liver was
heavier (P < 0.01) in FB1-fed pigs. aB crystallin and COX-1 concentrations were eight-fold
and 12-fold higher in the colon of FB1-fed pigs than in the controls (P < 0.0001).
Concentrations of COX-1 and nNOS in the stomach, HSP 70 in the jejunum and HO-2 in the
colon were also higher in FB1-fed pigs (P < 0.05 to P < 0.001). In conclusion, the FB1 extract
drastically enhanced colonic levels of aB crystallin and COX-1, with milder increases in
other stress proteins along the GIT of pigs. The data suggest that the colon is an important
target for FB1-induced stress responses.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Fumonisin B1
Gastrointestinal tract
Pig
Stress proteins
1. Introduction
Mycotoxins are secondary metabolites of fungi
contaminating food ingredients. Their repeated consumption represents a potential health hazard for humans and
animals (Oswald and Comera, 1998; CAST, 2003). Fumonisins are produced by Fusarium verticillioides which is
commonly found on maize grains and related products.
Fumonisin B1 (FB1) is the most abundant fumonisin
occurring naturally in contaminated foods and is believed
Abbreviations: BW, body weight; COX, cyclooxygenase; FB1, fumonisin
B1; HO, heme oxygenase; GIT, gastrointestinal tract; HSP, heat shock
protein; IEC, intestinal epithelial cell; INRA, Institut National de la
Recherche Agronomique; NO, nitric oxide; NOS, NO synthase (iNOS:
inducible, nNOS: neuronal).
* Corresponding author. Tel.: þ33 2 23 48 53 59; fax: þ33 2 23 48 50 80.
E-mail address: Jean-Paul.Lalles@rennes.inra.fr (J.-P. Lalle`s).
to be the most toxic among the fumonisin family (Voss
et al., 2007). Various species-specific mycotoxicosis in farm
animals, including equine leukoencephalomalacia and
porcine pulmonary edema are caused by FB1. This mycotoxin is also responsible for hepatotoxic and nephrotoxic
alterations in rats (Voss et al., 2007). In pigs, consumption
of an FB1-rich maize extract for 9 days increases gut colonisation by enteric pathogens (Oswald et al., 2003) and
depresses immune responses (Taranu et al., 2005).
The major functions of the gastrointestinal tract (GIT)
are digestion, nutrient absorption and barrier defence
against xenobiotics and enteric pathogens. Fumonisin B1
has been reported to exert direct toxic effects on the
structure, cellularity and functions of the gut (Bouhet and
Oswald, 2007). Low doses of FB1 reduce cytokine response
of intestinal epithelial cells (IEC), cell viability via inhibition
of cell proliferation and induction of apoptosis while higher
doses are cytotoxic (Schmelz et al., 1998; Bouhet et al., 2004,
0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2009.07.027
Please cite this article in press as: Lalle`s, J.-P., et al., Consumption of fumonisin B1 for 9 days induces stress proteins along the
gastrointestinal tract of pigs, Toxicon (2009), doi:10.1016/j.toxicon.2009.07.027
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2
2006). Intestinal barrier function of IEC lines is also altered
following prolonged exposure to FB1 (Bouhet et al., 2004).
However, much of the work on fumonisins and GIT
epithelial cell viability and function was done in vitro.
Therefore, more in vivo work is needed.
Mechanisms of toxicity for fumonisins are complex
(Soriano et al., 2005; Voss et al., 2007). Fumonisin B1 is
structurally similar to the sphingolipids sphingosine and
sphinganine and is known to inhibit the enzyme ceramide
synthase, thus disrupting the de novo biosynthesis of
ceramide and sphingolipid metabolism. Ceramide synthase
inhibition leads to reduced levels of ceramide and to the
accumulation of sphinganine and sphingosine. These free
sphingoid bases are proapototic, cytotoxic and growth
inhibitors, suggesting their role in FB1 toxicity (Soriano
et al., 2005; Voss et al., 2007). Phosphorylated sphinganine
and sphingosine also could be involved in some toxic
effects of FB1 (Gon et al., 2005). In vivo exposure to FB1
alters glycolipid distribution and sphinganine to sphingosine ratio in small intestinal tissues of mice (Enongene
et al., 2000) and pigs (Loiseau et al., 2007), and increases
plasma levels of free sphinganine, sphinganine-1 phosphate and S1P in pigs (Piva et al., 2005).
In vitro and in vivo data have reported effects of FB1 on
stress proteins in cells from the liver, the kidney and the
immune system, including heat shock proteins (HSP) 25
and HSP 70 (Liu et al., 2002; Rumora et al., 2007), cyclooxygenase 2 (COX-2) (Meli et al., 2000) and inducible NO
synthase (iNOS) (Suzuki et al., 2007). However, there is no
information to date for supporting any effects of FB1 on
stress proteins in the GIT. This is surprising because this
organ is the first to be in contact with mycotoxins ingested
with the food. In addition, FB1 was reported to accumulate
in the colon in comparison with the stomach or the small
intestine (Prelusky et al., 1996). Using radio-labelled FB1,
these authors estimated that after 24 days of feeding pigs
with a feed contaminated with 3 (day 1–13) and 2 (day
14–24) mg/kg feed, the concentrations of FB1 and its
metabolites at day 24 reached 160 and 65 ng/g tissue for
the liver and the kidney, respectively. The corresponding
concentrations for the stomach, the small intestine and the
colon could be estimated as 40, 33 and 583 ng/g tissue, thus
showing a 15-fold higher tissue concentration in the colon
as compared to the stomach or the small intestine. Therefore, toxic stress induced by FB1 may be higher in the colon
than in rest of the GIT.
The aim of this work was to test the hypothesis that
repeated consumption of an FB1-rich maize extract induces
stress protein responses along the GIT, and especially in the
colon.
2. Materials and methods
2.1. Preparation of FB1 extract
The mycotoxin FB1 extract was obtained after in vitro
culture of the high FB1-producing F. verticillioides strain
NRRL 34281 (Oswald et al., 2003). Briefly, sterilized maize
was inoculated with F. verticillioides and was incubated for 4
weeks at 25 C. Thereafter, the culture was extracted with
acetonitrile–water, filtered, and concentrated with a rotary
evaporator until total removal of acetonitrile. The fumonisin
extract was analysed for FB1 by quantitative planar chromatography (Le Bars et al., 1994). Other fumonisins and
other mycotoxins were analysed by gas chromatography
and mass spectrometry, using the same references as Mirocha et al. (1990). This culture extract contained 2.3 mg/mL
of FB1 and much lower levels of fumonisin B2 (0.34 mg/mL)
and B3 (0.38 mg/mL) (Marin et al., 2006). Other mycotoxins
including deoxynivalenol, fusarochromanone or trichothecenes were not detected in this extract (Marin et al., 2006).
2.2. Animals, feeding and experimental design
The experiment was conducted under the guidelines of the
French Ministry of Agriculture for animal research. Thirty-six
castrated male piglets [Pietrain (Landrace Large White)]
from the experimental herd of the Institut National de la
Recherche Agronomique (INRA) at Saint-Gilles, France were
weaned at 28 days of age and used in a complete block
experimental design. Pairs of piglets were selected within
a litter on the basis of close growth rates until and body
weights (BW) at 7 days post-weaning. Pigs within pairs were
allocated randomly to either the control or FB1-treated
groups. Average BW at the initiation of FB1 extract administration was 10.87 (SE) 0.35 and 10.94 0.35 kg for the
control and FB1-treated groups, respectively. Male pigs
were used because of their higher immune sensitivity to FB1
as compared to female pigs (Marin et al., 2006).
Pigs within pairs were allocated randomly to either the
control or FB1-treated groups and they were housed into
individual cages (0.6 0.8 m). The pigs were fed
throughout the experimental period with a weaning diet
containing the following ingredients (g/kg food): wheat
234.00; maize 280.00; barley 172.16; soya bean meal
266.30; and sunflower oil 4.50. The maize used in this
study was assumed to be devoid of fumonisins.
The daily amount of weaning food consumed by each
pig during the experimental period was set at 75% of
energy requirements established at 960 kJ net energy/BW
(kg)0.75 per day according to INRA recommendations (INRA,
2007). This was made for limiting differences in food intake
between experimental groups of pigs because FB1contaminated food can affect voluntary food intake (Rotter
et al., 1996). The amount of food offered was adjusted daily
on the basis of the BW and estimated daily weight gain of
each pig. Pigs were weighed twice a week.
During the experimental period, FB1 pigs received orally
a bolus of the FB1 extract (1.5 mg FB1/kg BW) diluted in a 20%
solution of glucose (used as a sweetener) daily for 9 days. This
dose corresponded to approximately 25–30 ppm in the food
and was previously shown to increase sphinganine to sphingosine ratio and to alter glycolipid distribution in the jejunal
tissue of weaned pigs (Loiseau et al., 2007). Control pigs were
orally dosed with the glucose solution without FB1 extract. At
completion of the trial and 1.5 h after the last meal, pigs were
euthanised by exsanguination following electronarcosis.
2.3. Organ weight and gastrointestinal tissue collection
After laparotomy, the liver, the lungs, the spleen and the
pancreas were collected and weighed. The whole GIT was
Please cite this article in press as: Lalle`s, J.-P., et al., Consumption of fumonisin B1 for 9 days induces stress proteins along the
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removed and the stomach, the small intestine, the caecum
and the colon were isolated. The small intestine was
divided into three segments (small intestine 1, 2 and 3)
equal in length. The weight of fresh tissues from the small
intestine and colon were determined after emptying the
full segments from digesta contents, rinsing with cold
saline and gently drying with absorbent tissue.
Pieces (0.5 0.5 cm) of intestinal tissues were taken in
the middle of each segment from each pig (n ¼ 18 per
treatment) for histo-morphometry. They were placed in
phosphate-buffered formalin (10%, pH 7.6) for 24 h at 4 C,
then washed and stored in ethanol:water (75:25, v:v) until
measurements.
Pieces (0.5 0.5 cm) of stomach (pylorus), mid jejunum
(¼segment 2 of the small intestine) and proximal colon
(50 cm distal to the ileo-cecal junction) were collected from
7 pigs randomly chosen per treatment, immediately frozen
in liquid nitrogen and kept at 80 C until analysis of stress
proteins.
2.4. Villus-crypt morphology along the small intestine
In order to evaluate intestinal integrity, the tissue
samples of intestinal segments previously fixed in buffered
formalin were microdissected for villi and crypts according
to the technique of Goodlad et al. (1991). Ten to 15 villi and
crypts per sample were measured for their length and
width using image analysis as previously reported (David
et al., 2002). The obtained values were averaged per villi
and crypts for each tissue sample prior to statistical analysis. All the measurements were carried out blind with
regard to the experimental treatments.
2.5. Tissue concentrations of stress proteins along the GIT
Proteins were extracted from GIT tissues and assayed as
described previously (Nefti et al., 2005). Briefly, the
extraction was carried out on ice in 60 mM Tris buffer, pH
6.8, added with 10% glycerol and 3% SDS. The protease
inhibitor Antagosan (Hoescht, Lyon, France) and the
reducing agent b-mercaptoethanol (Sigma, 5%) were added
to the extraction buffer just before use. After centrifugation,
the supernatant was collected, and protein concentration
determined (Nefti et al., 2005). Extracted tissue proteins
were then separated by SDS-PAGE electrophoresis. Equal
amounts of proteins were loaded onto a 13% acrylamide gel
with a 4% stacking acrylamide gel. Migration was conducted in a 25 mM Tris buffer (pH 7.6) containing 0.1% SDS
and 0.2 M glycine. After separation, proteins were transferred onto Hybond C membranes (Amersham, Piscataway,
NJ). Stress proteins were detected using Western blotting.
The primary antibodies against aB crystallin, heat shock
proteins HSP 27 and HSP 70, and b-actin used in this study
are presented in Table 1. All these primary antibodies were
previously shown by these authors to recognise stress
proteins in various swine tissues, including GIT segments.
Expression of b-actin was used for checking the equal
protein load across gel tracks. The same secondary antibody
(anti-rabbit) coupled with horseradish peroxidase as
reported previously (Louapre et al., 2005; Nefti et al., 2005;
David et al., 2006) was used in the present work to reveal
3
the presence of different stress proteins under study and of
b-actin on the membranes. Band densities were obtained
by scanning the membranes using a phosphor imager
(Quantum Appligene, Illkirch, France). Density data were
standardized within membranes by expressing the density
of each band of interest relative to that of b-actin in the
same lane.
2.6. Statistical analysis
Growth performance, feed intake, feed conversion
and anatomical data were analysed using the MIXED
procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC, USA). The effect of replication was
tested using the residual variation between pairs as the
error. The effect of the diet was tested against residual
variations within pairs of pigs as the error. Final BW was
taken as a covariate in the analysis of organ and GIT
weight data. Stress protein data were obtained from pigs
randomly chosen within groups. Therefore, they were
analysed by t-test for comparing the FB1 group to the
control using the GLM procedure of SAS. Data are presented as means and SE. Differences were declared
significant at P < 0.05.
3. Results
3.1. Growth performance, food intake and conversion,
and organ weight
The pigs from both experimental groups grew similarly
during the experimental period (final BW after the 9-day
treatment with FB1 extract: 13.67 0.44 and 13.65 0.61 kg
for the control and FB1-treated pigs, respectively). Food intake
was not significantly influenced by the oral administration of
FB1 extract (4.04 0.15 and 3.96 0.27 kg over the 9-day
period for the control and FB1-treated pigs, respectively).
However, food conversion ratio during the experimental
period was lower (P ¼ 0.04) in pigs treated with FB1 extract as
compared to controls (1.21 0.05 and 1.33 0.13 kg of food/
kg of BW gain). The weight of the liver was higher (P < 0.01)
and that of the spleen tended to be lower (P ¼ 0.06) in the
FB1-fed pigs compared to the controls (liver: 321 8 and
352 10 g; spleen: 26.5 1.3 and 23.7 1.5 g). The weights of
the lungs, the pancreas and of fresh tissues of the stomach, the
small intestine, the caecum and the colon were not affected by
the intake of FB1 extract for 9 days (data not shown).
3.2. Intestinal villous-crypt architecture
There were no differences (P 0.05) between treatment
groups for the length, width, perimeter or surface area of
villi and crypts in the three small intestinal segments
studied (data not shown).
3.3. Tissue concentrations of heat shock proteins,
cyclooxygenases, heme oxygenases and nitric oxide
synthases along the GIT
Tissue relative concentrations of aB crystallin in the
stomach and the jejunum were not influenced by FB1
Please cite this article in press as: Lalle`s, J.-P., et al., Consumption of fumonisin B1 for 9 days induces stress proteins along the
gastrointestinal tract of pigs, Toxicon (2009), doi:10.1016/j.toxicon.2009.07.027
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4
Table 1
Origins, dilutions and references of primary antibodies used for detecting stress proteins and b-actin by Western blotting.
Protein
Abbreviation
Origin
Dilution
Reference
aB crystallin
aB crystallin
Cyclooxygenase 1
Cyclooxygenase 2
Heat shock protein 27
Heat shock protein 70
Heme oxygenase 1
Heme oxygenase 2
Inducible nitric oxide synthase
Neuronal nitric oxide synthase
b-actin
COX-1
COX-2
HSP 27
HSP 70
HO-1
HO-2
iNOS
nNOS
b-actin
Generous gift from H Lambert, Hotel Dieu de Quebec, Canada
Cayman laboratory Tebu, Le Perray, France (ref. 160108)
Cayman laboratory Tebu, Le Perray, France (ref. 160107)
Generous gift from H Lambert, Hotel Dieu de Quebec, Canada
StressGen Biotechnologies, Victoria, BC, Canada (ref. SPA 812)
Stressgen, Collegeville, PA, USA (ref. OSA-100)
Stressgen, Collegeville, PA, USA (ref. OSA-155)
Santa Cruz Biotechnology, Santa Cruz, CA, USA (ref. SC 651)
Sigma–Aldrich, St. Quentin Fallavier, France (ref. N-7155)
Sigma–Aldrich, St. Quentin Fallavier, France (ref. A-9044)
1:1000
1:250
1:200
1:1000
1:2000
1:500
1:500
1:500
1:1250
1:1000
Nefti et al., 2005
David et al., 2006
David et al., 2006
Nefti et al., 2005
Nefti et al., 2005
Louapre et al., 2005
David et al., 2006
Louapre et al., 2005
Louapre et al., 2005
Nefti et al., 2005
consumption (P 0.05) but aB crystallin concentration in
the colon was eight-fold higher in the FB1-fed pigs than
in the controls (P < 0.001) (Table 2). Concentrations of
COX-1 in the stomach and the colon were higher in
FB1-treated pigs than in the controls (P ¼ 0.043 and
P < 0.0001). COX-1 was not detected in the jejunum and
COX-2 was never detected along the GIT of pigs.
Concentrations of HSP 27 along the GIT remained unaffected by FB1 treatment (P 0.05). Concentration of HSP
70 was higher in the jejunum of FB1-treated pigs as
compared to the controls (P ¼ 0.004), with no differences
for the other GIT sites. Heme oxygenase 1 was not
detected in GIT of pigs. Concentration of HO-2 was
higher in the colon of FB1-treated pigs than in the
controls (P < 0.001), with no differences in the stomach
and the jejunum (P 0.05). The inducible nitric oxide
synthase iNOS was not detected along the GIT. Neuronal
nNOS was detected only in stomach tissue and at
a higher concentration in the FB1-treated pigs than in
the controls (P ¼ 0.030).
Table 2
Influence of the FB1 extract on the relative tissue expression of stress
proteins along the GIT of pigs (means SE, n ¼ 7 per treatment).
Stress proteina
GIT site
Treatment
P-value
Control
FB1
aB Crystallin
Stomach
Jejunum
Colon
58.6 1.3
19.0 1.5
7.1 0.9
57.4 2.5
21.4 2.2
59.7 2.1
0.67
0.34
<0.0001
COX-1
Stomach
Jejunum
Colon
53.4 2.8
ndb
4.3 0.8
60.3 1.7
nd
52.4 5.2
0.043
<0.0001
HSP 27
Stomach
Jejunum
Colon
41.6 1.7
30.4 1.4
44.3 1.4
44.7 2.3
32.4 1.5
43.9 1.1
0.26
0.32
0.80
HSP 70
Stomach
Jejunum
Colon
84.4 1.7
33.4 1.2
55.3 4.6
86.0 0.9
38.3 0.9
53.3 2.1
0.40
0.004
0.68
HO-2
Stomach
Jejunum
Colon
78.0 2.6
63.1 3.3
60.3 1.1
77.4 1.4
64.6 1.8
75.3 3.3
0.84
0.69
<0.001
nNOS
Stomach
Jejunum
Colon
44.3 1.3
nd
nd
47.9 0.7
nd
nd
0.030
a
COX-2, HO-1 and iNOS were never detected in the stomach, the
jejunum and the colon.
b
Not detected.
4. Discussion
The major finding of the present work is that oral
administration of a FB1-rich extract drastically increased
tissue levels of aB crystallin and COX-1 in the colon. Milder
increases in the concentration of various stress proteins
along the GIT were also noted. These effects may be
essentially attributable to FB1 which is considered as the
most toxic fumonisin (3). Although specific influences of
low levels of FB2 and FB3 on the GIT cannot be excluded,
such effects have not been reported in the literature to date.
It is the first time that over-expressions of aB crystallin
and COX-1 (and of HO-2 to a lesser extent) are reported in
the colon in response to FB1 consumption. Our data suggest
that the colon is highly sensitive and, therefore, responsive
to the deleterious effects of this mycotoxin. The stronger
stress responses observed in the colon of FB1-treated pigs
may be related to the greater accumulation of FB1 in the
colon, comparatively to the stomach or the small intestine
(Prelusky et al., 1996).
aB Crystallin is constitutively expressed along the
porcine GIT during early development in the absence of
additional stress (Tallot et al., 2003). This small HSP
(MW ¼ 20 kDa) is involved in the modulation of cell cytoskeleton, the inhibition of apoptosis and the ability of cells
to increase their resistance to oxidative injury (Arrigo et al.,
2007). aB crystallin is strongly over-expressed in colonic
tissue of neonatal pigs subjected to hypoxia (Nefti et al.,
2005). The enhanced aB crystallin concentration in the
colon observed in this work may be due to the potential of
FB1 to increase lipid peroxidation. Indeed, FB1 is a potent
inducer of oxidative stress and lipid peroxidation in intestinal epithelial cells (Kouadio et al., 2005). Tissue induction
of aB crystallin in response to FB1 may possibly confer
enhanced colonic protection, as observed in a mice model
of inflammation (Masilamoni et al., 2005), but this remains
to be investigated in pigs.
Cyclooxygenases are enzymes involved in the production of pro-inflammatory mediators. The COX-1 isoform is
present constitutively in the GIT while the COX-2 isoform is
expressed in response to inflammatory stimuli (Warner
and Mitchell, 2004). Cyclooxygenase 2 was not detected
along the GIT of pigs in the present experiment. By contrast,
COX-1 was largely over-expressed in colonic tissue of FB1
extract-treated pigs. Cyclooxygenase 1 may have an
important role in mucosal defence against xenobiotics in
the stomach (Gretzer et al., 2001) but little information is
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available for the intestine. Blikslager et al. (2002) detected
only low levels of COX-2 in porcine ileal tissues subjected to
ischemic injury, and they concluded that both COX-1 and
COX-2 may be involved in the recovery of porcine ileum in
this model.
A significant increase in colonic levels of HO-2 was
observed in the pigs orally treated with the FB1-rich
extract. Interestingly, a novel role for HO-2 in the regulation
of inflammatory and reparative responses to injury was
recently documented (Seta et al., 2006). This cytoprotection
mechanism brought about by increased concentration of
HO-2 in the colon, although of limited magnitude here, may
have contributed to reinforce the protective effects of aB
crystallin and COX-1 against toxic effects of FB1 in this
organ.
The FB1-rich maize extract administered orally to pigs
induced (small) increases in tissue levels of COX-1 and
nNOS in the stomach and HSP 70 in the small intestine.
Gastric COX-1 protein over-expression as observed in the
present study may have contributed to increase the
protection of this organ against deleterious effects of toxic
compounds such as FB1. This is supported by published
data showing that acid damage to the gastric mucosa was
alleviated by specific inhibitors of COX-1 (Gretzer et al.,
2001). Gastric protection may have been further enhanced
in the pigs consuming FB1 by the increase in nNOS levels in
gastric tissues. Although NO is known to regulate many
processes in the GIT, the respective roles of endothelial NOS
(eNOS), iNOs and nNOS in GIT defence and protection
against injuries and inflammation are still unclear. Data are
even controversial regarding the involvement of nNOS in
intestinal inflammation, being protective or non-protective
(Beck et al., 2004; Vallance et al., 2004).
The only heat shock protein to be (slightly) enhanced
following FB1 oral exposure was HSP 70 in the jejunum.
This response may be protective to the intestine (Otaka
et al., 2006). The present observation is consistent with the
reported FB1-induced increase in HSP 70 protein expression in alveolar macrophages (Liu et al., 2002), in rat kidney
(Rumora et al., 2007) and in fibroblasts (Galvano et al.,
2002). We did not observe any response on HSP 27
expression in GIT tissues. This result contrasts with the
over-expression of HSP 27 in renal and liver tissues of rats
chronically administered FB1 i.p. (Rumora et al., 2007) but
is consistent with the lack of HSP 27 response in the GIT
following neonatal hypoxia in pigs (Nefti et al., 2005).
In terms of underlying mechanisms, stress responses
may be linked with changes in GIT tissue sphingolipid
metabolism brought about by FB1. Fumonisin B1 in an
inhibitor of ceramide synthase, resulting in decreased
ceramide concentration and increased sphinganine and
sphingosine concentrations (Soriano et al., 2005; Voss et al.,
2007). Ceramide induced by heat shock or increased intracellularly was shown to stimulate gene transcription of aB
crystallin in fibroblast cells (Chang et al., 1995). Regarding
heat shock proteins, HSP 70 was suppressed by ceramide in
heat shock-induced HL-60 cell apoptosis (Kondo et al.,
2000). In the present study, aB crystallin was strongly overexpressed in the colon while HSP 70 was unaffected.
Therefore, the pathways between FB1, changes in sphingolipid metabolism and stress responses may be different
5
from those related to heat shock and demonstrated in cell
models (Chang et al., 1995; Kondo et al., 2000). This area
clearly needs new investigations.
The effect of the treatment with the FB1 extract on pig
growth performance and feed conversion was limited,
probably because the period of FB1 administration was
short (9 days) and because food intake was set at 75% of pig
needs in order to limit food refusals. It is important to work
at constant food intake because it is a major determinant of
GIT growth and physiology. Interestingly, despite limited
effects on BW, the 9-day treatment with the FB1 extract
affected the weight of organs like the liver and the spleen.
These changes are in agreement with the observation that
FB1 is hepatotoxic (Voss et al., 2007) and is able to depress
immune responses (Taranu et al., 2005). By contrast, the
weight of the lung, the pancreas and the GIT and its
segments were not modified in FB1-fed pigs, highlighting
the differential sensitivity to this mycotoxin across GIT
segments (Rotter et al., 1996).
In conclusion, the present work provides evidence that
the repeated consumption of a maize extract rich in
fumonisin B1 increases tissue expression of aB crystallin
and COX-1 in the colon, with milder increases in various
stress proteins along the GIT. Our data highlight the
complexity of the cytoprotection systems and GIT regional
variations in the induced response. The underlying cellular
and molecular mechanisms linking FB1 to stress proteins
and GIT physiology need to be investigated further.
Acknowledgement
The authors thank the staff of the UMR SENAH for care
of the animals, for slaughtering the pigs and for laboratory
analysis. Thanks are also due to the Statistical Unit of the
Dairy and Swine Research and Development Center of
Agriculture and Agri-Food Canada at Lennoxville, Que´bec,
Canada for the statistical analysis of data. The authors
acknowledge INRA for supporting financially the one-year
stay of Dr Martin Lessard at INRA Rennes, France and the
‘Mycotoxines’ Transversal program (number P00263).
Conflicts of interest
The authors declare that there are no conflicts of
interest.
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