Participation of reactive oxygen metabolites in
Clostridium difficile toxin A-induced
enteritis in rats
Bosheng
Qiu1,
Charalabos
Pothoulakis1,
Ignazio
Castagliuolo1,
Sigfus
Nikulasson2, and
J. Thomas
LaMont1
1 Division of Gastroenterology,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston
02215; and 2 Mallory Institute,
Department of Pathology, Boston University Medical Center Hospital,
Boston, Massachusetts 02118
 |
ABSTRACT |
Reactive oxygen metabolites (ROMs) contribute to
the pathophysiology of intestinal inflammation. Our aim was to
ascertain the involvement of ROMs in experimental ileitis in rats
produced by toxin A of Clostridium
difficile. Intraluminal toxin A caused a significant
increase in hydroxyl radical and hydrogen peroxide production by ileal
microsomes starting 1 h following toxin exposure and peaking at
2-3 h, and this was inhibited by pretreatment with DMSO, a ROM
scavenger, or superoxide dismutase (SOD), which inactivates ROMs. In
contrast, mucosal xanthine oxidase increased only slightly after toxin
A exposure, and allopurinol, an inhibitor of xanthine oxidase, had no
effect on toxin A-associated intestinal responses. Induction of
neutropenia resulted in reduction of toxin-mediated free radical
formation, fluid secretion, and permeability. The enterotoxic effects
of C. difficile toxin A were
associated with increased ROM release in ileal tissues, and the ROM
inhibitors DMSO and SOD inhibited these effects. This suggests that
ROMs released during toxin A enteritis are released primarily from neutrophils invading the inflamed bowel segment.
superoxide radicals; hydroxyl radicals; intestinal inflammation; intestinal secretion; enterotoxins
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INTRODUCTION |
THE CELLULAR AND MOLECULAR pathophysiology of
Clostridium difficile toxin A-mediated
enteritis has been elucidated in considerable detail over the past
decade. In experimental animals toxin A binds to brush-border
enterocyte receptors (20) and is internalized into the cytoplasm, where
it inactivates Rho, a GTP-binding protein of the Ras superfamily (5,
10). Inactivation of Rho leads to disaggregation of filamentous actin
and dysfunction of tight junctions in T84 colonocyte monolayers, as
evidenced by increased paracellular permeability and loss of electrical
resistance (9). Toxin A enteritis in rats and rabbits is characterized
by a marked increase in fluid and electrolyte secretion, comparable to
that seen with equimolar concentrations of Vibrio
cholerae enterotoxin (23). In contrast to cholera
toxin, toxin A elicits a profound neutrophilic infiltrate that
commences 2 h after intraluminal toxin administration and at 4 h is
associated with widespread enterocyte necrosis and a florid acute
inflammatory response in the lamina propria as well as the intestinal
lumen (23). Antibodies directed against the neutrophil adhesion
molecule CD11-CD18 markedly inhibit fluid secretion and tissue damage
(11), and pharmacological blockade of substance P or mast cell
degranulation also reduces neutrophil infiltration and tissue damage
(18, 20). These findings indicate that neutrophilic invasion and
subsequent mediator release in the affected bowel segment are critical
components of toxin A pathogenesis.
Reactive oxygen metabolites (ROMs) have been implicated in the
pathogenesis of experimental colitis in animal models and in idiopathic
inflammatory bowel disease of humans (14, 24). ROMs or free radicals
such as superoxide, hydroxyl ion, and hypochlorite radical are thought
to directly mediate damage of proteins and lipids in target tissues.
Lipid peroxidation of cellular membranes and oxidation of thiol groups
in proteins are specific chemical reactions observed in ROM-associated
injury. The two main sources of ROMs in experimental enteritis are
xanthine oxidase within epithelial cells and NADPH oxidase in
phagocytic leukocytes (resident macrophages, eosinophils, and invading
neutrophils). The purpose of this study was to ascertain
if ROMs were involved in toxin A enteritis and, if so, to determine
their source.
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MATERIALS AND METHODS |
General.
Male Wistar rats, weighing 200-250 g (Charles River Breeding
Laboratories, Wilmington, MA), were housed at 22°C and 65-70% humidity. Rats were fasted 24 h before experimentation but allowed free
access to water. Toxin A was purified to homogeneity from culture
supernatants of C. difficile strain
10,463 as described previously by us (19).
Ileal loop experiments.
Rats were anesthetized by intraperitoneal injection of pentobarbital
sodium (55 mg/kg). At laparotomy the renal pedicles were tied, and two
closed ileal loops were formed in each animal as described (3, 4, 18).
Immediately before toxin A administration, 10 µCi of
[3H]mannitol (New
England Nuclear) was injected into the inferior vena cava. Toxin (5 µg) was administered by intraluminal injection in 0.4 ml of 50 mM
Tris buffer (pH 7.4). The abdominal incision was then closed, and at 4 h the animals were killed. Ileal loops were removed, and their weights
and lengths were measured. Mucosal permeability to mannitol (dpm/cm of
loop) and fluid secretion [loop weight (mg)/length (cm)]
were then measured as described (3, 4, 18).
In some animals intravenous cannulas were implanted to deliver
superoxide dismutase (SOD), an inhibitor of ROM formation. Polyethylene
catheters (1.27 mm in diameter) filled with heparinized saline (500 IU/300 ml) to prevent clotting were implanted in the right jugular vein
under aseptic conditions and subcutaneously exteriorized in the
interscapular region. Three days after surgery animals were injected
intravenously with either 0.9% NaCl (saline) or SOD solutions in a
volume of 0.6 ml, according to the protocol to be described.
Intestinal mucosal xanthine oxidase and SOD.
Xanthine oxidase activity in ileal mucosa was determined
spectrophotometrically (21). Ileal mucosa was removed with a glass slide and homogenized in ice-cold phosphate buffer. After
centrifugation for 20 min at 15,000 rpm, the supernatants were passed
over a buffer-equilibrated G25 Sephadex (Pharmacia Biotechnology)
column at 4°C to remove low-molecular-weight inhibitors. Xanthine
oxidase activity was determined by measuring uric acid formation in the presence of xanthine and ambient oxygen. The absorbance of the solutions was measured using ultraviolet light with a wavelength of
295 nm.
SOD activity was determined by the nitro blue tetrazolium (NBT)
reactions (21). Each cuvette contained 0.1 mM xanthine, 25 µM NBT,
0.1 mM EDTA, 50 mM NaCO3 0.1 ml
sample, and 6 nM xanthine oxidase in a total volume of 3 ml at 25°C
and a final pH of 10.2. The reaction was stopped by adding
CuCl2 at 25°C 3 min later. Inhibition of NBT reduction in the samples was monitored
spectrophotometrically at 560 nm.
Hydroxyl radical and hydrogen peroxide formation.
After toxin A administration, the ileal loops were removed and the
mucosa was scraped off with a glass slide. Microsomal fractions were
prepared from a 10% homogenate of the mucosa in 0.9% NaCl (pH 7.4).
The homogenate was sequentially centrifuged at 1,000 g for 10 min and 12,000 g for 20 min. The microsomal fraction was suspended in 0.9% NaCl (pH 7.4) at a concentration of 20 mg protein/ml. Microsomes from control and toxin A-treated loops were
tested for generation of hydroxyl or hydroxyl-like species by assaying
formaldehyde production from DMSO as described (15). To verify the
hydroxyl radical assay we incubated microsomal fractions from mucosal
scrapings of control rats (n = 8/group) with catalase (20 mg/ml) or the nonchelator deferoxamine (0.1 mM) before measuring hydroxyl or hydroxyl-like species (15). Previous
studies showed that both catalase and deferoxamine effectively prevent
hydroxyl radical formation (25, 26). Our results showed that both
catalase and deferoxamine effectively inhibited (by 88.3 and 90%,
respectively, P < 0.01 for both)
hydroxyl radical formation, confirming the specificity of the assay
used in our experiments.
Hydrogen peroxide formation was measured with the ferrammonium sulfate
potassium thiocyanate method in the presence of
NaN3 to block peroxidase activity
(1). The incubation mixture of 1.5 ml contained 1 mg protein, 50 mM
Tris-chloride buffer (pH 7.4), and 0.75 µmol NADPH. The microsomes
were incubated 20 min at 30°C under continuous shaking. Controls
included incubation mixtures to which TCA was added before the
microsomal suspension.
Antibody-induced neutropenia in rats.
To examine the role of neutrophils in toxin A-induced ileitis,
neutropenia was induced by the intraperitoneal injection of a specific
adsorbed polyclonal rabbit anti-rat neutrophil antiserum as described
previously by von Ritter et al. (24). Preliminary studies demonstrated
that intraperitoneal injection of a 1:10 dilution of antibody
(AI-AD51140; Accurate Chemical and Scientific Corporation, Westbury,
NY) at a dose of 2 ml/100 g body weight produced a marked neutropenia,
reaching a nadir ~8 h following injection and being
sustained for the following 48 h. Consequently, rats received
antibody 1 day before the experiments to achieve a 90% or greater
reduction in peripheral blood neutrophil counts at the time of toxin
injection. Neutrophil counts were determined on venous blood samples
from a lateral tail vein using a hemocytometer.
Tissue MPO activity.
Tissue myeloperoxidase (MPO) activity was measured to estimate tissue
polymorphonuclear cell accumulation during toxin A enteritis. This
enzyme has previously been shown to correlate significantly with
numbers of polymorphonuclear cells determined histochemically in
colonic tissues (11, 12). Tissue MPO activity was determined by the
method of Krawisz et al. (12). Approximately 0.2-g samples of mucosa
were homogenized in 0.05 M potassium phosphate buffer, containing
hexadecyltrimethylammonium bromide (Sigma). The mucosal homogenate was
then centrifuged at 20,000 g for 15 min. An aliquot (0.1 ml) of the supernatant was added to 3.0 ml
o-dianisidine (Aldrich Chemical,
Milwaukee, WI), and the change in absorbance at 460 nm was determined.
One unit of MPO activity was equivalent to the catalysis of 1 mmol
hydrogen peroxide per minute at 25°C.
In some experiments the effects of DMSO and SOD on toxin A-induced
increased tissue MPO activity were examined. Rats
(n = 6/group) were pretreated with
12,000 U/kg of SOD or with 1% of DMSO, as described below, before
injection of either buffer or 5 µg of toxin A into ileal loops. After
4 h animals were killed and tissue MPO activity was measured as
previously described.
Drug administration.
All drugs were freshly prepared within 1 h of use. SOD dissolved in
0.9% NaCl solution was injected intravenously 15 min before operation,
and three additional injections with the same dose of SOD were given at
1-h intervals after toxin A administration to achieve a steady-state
serum concentration. The doses of SOD tested were 4,000, 8,000, 12,000, and 16,000 U/kg. DMSO at concentrations of 0.1, 0.5, 1, and 5% was
added to drinking water for 7 days before surgery. Allopurinol at 0.01 or 0.1 g/kg body weight (17) was administered by gavage 24 and 0.5 h
before toxin A administration.
Statistical analysis.
Results are expressed as means ± SE. Data were analyzed by the
two-tailed unpaired Student's t-test.
Differences between groups exposed to multiple factors in the same
experimental conditions were examined by post hoc test following
one-way or mixed ANOVA significance.
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RESULTS |
ROM generation following toxin A exposure.
The production of hydroxyl radicals (Fig.
1A)
and hydrogen peroxide (Fig. 1B) in
intestinal microsomes was increased significantly above baseline at 2, 3, and 4 h following toxin A administration. These increases were
temporally correlated with alterations of intestinal secretion,
blood-to-lumen mannitol clearance, and histological damage (Table
1). The major effects of 5 µg toxin A on
most parameters studied were statistically increased or altered after 2 h of toxin exposure, and this correlated with increases in hydroxyl
radicals and hydrogen peroxide at 2 h post-toxin exposure. At 4 h
post-toxin exposure parameters of enterotoxicity were maximal (Table
1), whereas ROMs were declining slightly from their peak values at 3 h.

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Fig. 1.
Toxin A stimulates hydroxyl radical
(A) and hydrogen peroxide
(B) formation from intestinal
microsomes. Rat ileal loops were injected with either 5 µg of
purified toxin A or buffer. At indicated time intervals, rats were
euthanized, ileal loops removed, mucosa scraped off, and microsomal
fraction prepared from mucosal homogenates as described in
MATERIALS AND METHODS. Hydroxyl
radical and hydrogen peroxide formation from microsomes was assayed as
previously described. Results are expressed as means ± SE of 8 loops/group. * P < 0.05 vs. time 0 (control).
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Table 1.
Effect of toxin A on rat ileal fluid secretion, mucosa permeability to
mannitol, and histological severity of enteritis
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Pharmacological inhibition of toxin A effects.
Two types of inhibitors were used to determine if ROMs contributed to
fluid secretion or changes in mannitol permeability caused by toxin A
exposure. SOD catalyzes the conversion of superoxide radical to
hydrogen peroxide, which is then quickly inactivated by catalase. When
administered to animals undergoing oxidative stress, SOD reaches
sufficient tissue concentrations to provide protection against
ischemia-reperfusion injury mediated by superoxide radical (6,
22). DMSO is a nonenzymatic free radical scavenger that can also reduce
oxygen radical-mediated tissue damage (22). In our model intravenous
SOD partially inhibited toxin A-induced fluid secretion (Fig
2A) and
mucosal permeability (Fig 2B)
measured 4 h after toxin A exposure. At doses of 12,000 and 16,000 U/kg, SOD produced significant inhibition of fluid secretion and
mannitol permeability. Similar results were observed with oral
pretreatment with DMSO starting 7 days before toxin exposure. Again,
dose response effects on fluid secretion (Fig.
3A) and
permeability (Fig. 3B) were observed
between 0.1 and 1% DMSO concentration (vol/vol) in the drinking water.
We also examined the effect of DMSO (1%) and SOD (12,000 U/kg)
pretreatment on mucosal MPO activity following toxin A administration.
Injection of toxin A into ileal loops of nontreated rats increased
mucosal MPO activity by 7.3-fold compared with MPO levels in
buffer-injected loops (P < 0.01, n = 6). Pretreatment with DMSO
inhibited toxin A-induced increased mucosal MPO activity by 71.5%
(P < 0.05, n = 6), whereas pretreatment with SOD (n = 6) did not inhibit
increased MPO activity in response to toxin A.

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Fig. 2.
Superoxide dismutase (SOD) dose dependently inhibits toxin A-induced
fluid secretion (A) and mucosal
permeability to mannitol (B). Rats
were injected intravenously with either vehicle (normal saline) or
vehicle containing indicated doses of SOD as described in
MATERIALS AND METHODS. After 15 min
ileal loops were formed and injected with either purified toxin A or
buffer (control). SOD was also given at 1-h intervals after toxin A
administration. After 4 h fluid secretion [weight (mg)-to-length
(cm) ratio] and blood-to-lumen permeability to
[3H]mannitol (dpm/cm
loop) were measured as previously described. Results are expressed as
means ± SE of 8 loops/group.
* P < 0.05 vs. control. + P < 0.05 and
++ P < 0.01 vs. toxin A
alone.
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Fig. 3.
DMSO dose dependently inhibits toxin A-induced fluid secretion
(A) and mucosal permeability to
mannitol (B). Rats were treated with
DMSO at indicated doses added to drinking water. After 7 days ileal
loops were formed and injected with either purified toxin A or buffer
(control). After 4 h fluid secretion [weight (mg)-to-length (cm)
ratio] and blood-to-lumen permeability to
[3H]mannitol (dpm/cm
loop) were measured as described in MATERIALS AND
METHODS. Results are expressed as means ± SE of 8 loops/group. * P < 0.05 vs.
control. + P < 0.05 and
++ P < 0.01 vs. toxin A
alone.
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Neutropenia inhibits toxin A effects.
To evaluate the contribution of neutrophils to toxin A effects, we
induced profound neutropenia in rats by intraperitoneal injection of a
polyclonal anti-neutrophil antibody that reduced peripheral blood
neutrophil counts by >90%. Intestinal secretion and mannitol
permeability were both significantly diminished in toxin-treated
neutropenic rats compared with animals without neutropenia (Table
2). In addition, mucosal MPO activity and
hydroxyl radical concentration in response to toxin were both
significantly reduced during antibody-associated neutropenia.
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Table 2.
Effects of neutropenia on toxin A-mediated intestinal secretion,
mannitol permeability, MPO, and hydroxyl radical formation
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Ileal xanthine oxidase activity.
Another important source of ROMs in inflammatory or ischemic enteritis
is the xanthine oxidase-mediated hypoxanthine-xanthine reaction in
intestinal mucosa. Granger and colleagues (8) have proposed that
mucosal xanthine oxidase generates ROMs at the time of reperfusion
during a cat model of ischemia-reperfusion injury and that
these ROMs promote the attraction and activation of circulating granulocytes in the ischemic segment. We measured ileal mucosal xanthine oxidase activity following toxin A exposure. A minor increase
(~10%, P > 0.1) in mucosal
xanthine oxidase activity was observed in ileal mucosal scrapings from
1 to 4 h following ileal loop exposure to 5 µg purified toxin A (Fig.
4). To assess the possible contribution of
xanthine oxidase-derived ROMs in toxin A responses, we pretreated rats
with allopurinol at doses of 0.01 and 0.1 mg/kg body weight by gavage
24 and 0.5 h before toxin administration. Similar doses of
allopurinol have been shown to inhibit rat small intestinal mucosal
xanthine oxidase (7, 13). As shown in Fig.
5, allopurinol pretreatment did not inhibit toxin A-induced fluid secretion or ileal permeability to mannitol at 4 h following toxin exposure.

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Fig. 4.
Effect of toxin A on mucosal xanthine oxidase activity in rat ileum.
Rat ileal loops were injected with either toxin A or buffer. At
indicated time intervals rats were euthanized, ileal loops removed,
mucosa scraped off, and xanthine oxidase activity determined
spectrophotometrically as described in MATERIALS AND
METHODS. Results are expressed as means ± SE of 8 loops/group.
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Fig. 5.
Effect of allopurinol in toxin A-induced fluid secretion
(A) and mucosal permeability to
mannitol (B). Rats were administered
allopurinol by gavage 24 and 0.5 h before injection of ileal loops with
toxin A. Fluid secretion [weight (mg)-to-length (cm) ratio]
and blood-to-lumen permeability to
[3H]mannitol (dpm/cm
loop) were measured as described in MATERIALS AND
METHODS. Results are expressed as means ± SE of 8 loops/group.
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DISCUSSION |
The main finding reported here is an increase in hydroxyl radical and
hydrogen peroxide concentrations in ileal mucosal microsomes following
intraluminal instillation of 5 µg purified toxin A of C. difficile. The increase in ROMs was
temporally correlated with the gradual evolution of typical enterotoxic
effects that commence ~2 h after toxin exposure. Importantly, the
increase in fluid secretion, mannitol permeability, and inflammatory
changes 2 h after toxin A exposure (Table 1) were correlated temporally
with a significant increase in hydroxyl radical and hydrogen peroxide at 2 h (Fig. 1). The inhibitory effects of SOD and DMSO on these enterotoxic parameters suggest that ROMs contribute to the mechanism of
C. difficile enteritis and diarrhea.
Previous work in our laboratory indicates that toxin A triggers a
reproducible sequence of events in the lamina propria that culminates
in severe diarrhea and enteritis. The earliest events that we have
observed in this model are activation of mucosal mast cells (4) and
release of substance P from intestinal sensory afferent neurons (3).
Injection of toxin A into ileal loops caused mast cell degranulation
after 15 min (4) and increased substance P content in the cell bodies
of the dorsal root ganglia at 30 min (3), followed by substance P
release in the ileal mucosa at 1 h (3). Mast cell degranulation in this
model involves activation of sensory neurons(s) because chronic
pretreatment of rats with capsaicin, a drug that desensitizes primary
sensory afferents, inhibits mast cell degranulation as well as
intestinal secretion and inflammation (4). Leukocyte infiltration
begins 1.5 h following intraluminal exposure, and after 2-4 h the
lamina propria, epithelial layer, and lumen are heavily invaded by
polymorphonuclear leukocytes, mostly neutrophils (3, 11). These greatly
amplify the enterotoxic effects, particularly fluid secretion,
permeability, and histological changes (11). For example, pretreatment
with a specific substance P inhibitor markedly diminishes the toxin A
leukocyte response as well as fluid secretion and increased mannitol
permeability (18). Similar results were observed in this study in rats
made neutropenic by administration of an antibody to neutrophils before
toxin exposure (Table 2). Thus neutrophil invasion and activation into
toxin-exposed loops amplify the secretory and inflammatory effects of
the toxin. We report here that secretion and mannitol permeability are
significantly inhibited by an antibody to neutrophils which induces
neutropenia in rats (Table 2). In these experiments, both neutrophilic
infiltration and hydroxyl radical release were also significantly
diminished, suggesting that one likely source of the ROMs was invading neutrophils.
Our results with DMSO and SOD also support the possibility that ROMs
themselves, specifically hydroxyl and superoxide radicals, contribute
to toxin A effects in rat ileum. The observation that SOD and DMSO only
partially inhibited the enterotoxic effects of toxin A are consistent
with the contribution of other mediators in the toxin A response. It is
known that, for example, leukotriene B4 and platelet-activating factor,
both potent neutrophilic chemotactic factors, are released from the
small bowel following exposure to toxin A (20, 23). Clearly, ROMs are
only one class of several inflammatory mediators that contribute to
this model of inflammation.
Our results are consistent with other observations concerning the
contribution of ROMs to intestinal inflammation, particularly in the
microvascular injury in the small intestine during ischemia reperfusion (2). In this model injury is associated with granulocyte infiltration of the reperfused ischemic segment and is largely prevented by agents that either inhibit xanthine oxidase or scavenge oxygen radicals. Because inhibition or inactivation of xanthine oxidase
inhibits reperfusion injury (15), it has been suggested that mucosal
xanthine oxidase is the major source of oxygen radicals in this
experimental model and that xanthine oxidase-derived oxidants initiate
the leukocyte infiltration that follows reperfusion of the ischemic
bowel segment. Direct measurement of oxygen radicals in reperfused cat
small intestine revealed three sources for radicals (16). Xanthine
oxidase accounted for ~15%, neutrophils 50%, and other sources
35%. In contrast, xanthine oxidase probably does not contribute
substantially to ROM formation in our model as evidenced by a lack of
effect of allopurinol on toxin A-associated epithelial permeability or
fluid secretion (Fig. 4). Although intestinal microsomes alone can
generate oxygen radicals (Fig. 1), it is likely that the large
increases observed after toxin A exposure are contributed by
infiltrating neutrophils. Interestingly, DMSO and SOD pretreatments
were equally effective in inhibiting the enterotoxic effects of toxin
A, but only DMSO inhibited MPO activity. This suggests that the
modulatory effect of SOD may involve a nonneutrophilic mechanism.
Reactive oxygen radicals also appear to contribute to ulcerative
colitis and Crohn's disease in humans, diseases characterized by
infiltration of affected tissues with activated monocytes and neutrophils. Reduced concentrations of tissue antioxidant molecules such as ascorbite, gluathione, and ubiquinol have been described in
sites of active inflammation in Crohn's disease and ulcerative colitis. Recently, McKenzie et al. (14) provided direct evidence for in
vivo oxidant injury in the inflamed mucosa but not in the normal or
healed mucosa in Crohn's disease patients. Oxidative damage to thiol
groups of colonocyte proteins was attributed to reactive oxygen or
nitrogen species released from monocytes or neutrophils.
In summary, we report here that the acute enteritis that results when
purified toxin A of C. difficile is
instilled in the rat ileum is accompanied by local generation of
significant amounts of hydroxyl radicals and hydrogen peroxide. ROMs
are generated primarily by infiltrating neutrophils, and
pharmacological inhibition of radicals with SOD or DMSO diminished the
degree of mucosal damage. These results suggest that acute
C. difficile toxin-mediated colitis in
humans may also depend in part on oxidative injury and that
pharmacological inhibition of this pathway might ameliorate this damage.
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ACKNOWLEDGEMENTS |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-34583. I. Castagliuolo was
supported by a Research Fellowship from the Crohn's and Colitis Foundation of America.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. T. LaMont, Div. of Gastroenterology,
Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA
02215.
Received 25 March 1998; accepted in final form 26 October 1998.
 |
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