Toxicology and Applied Pharmacology 253 (2011) 197–202

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Toxicology and Applied Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p

The effect of methylsulfonylmethane on the experimental colitis in the rat

K. Amirshahrokhi a,⁎, S. Bohlooli a, M.M. Chinifroush b
a
Department of Pharmacology, School of Medicine, Ardabil University of Medical Sciences, P.O. Box 56197, Ardabil, Iran
b
Department of Pathology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Methylsulfonylmethane (MSM), naturally occurring in green plants, fruits and vegetables, has been shown to
Received 23 February 2011 exert anti-inflammatory and antioxidant effects. MSM is an organosulfur compound and a normal oxidative
Revised 17 March 2011 metabolite of dimethyl sulfoxide. This study was carried out to investigate the effect of MSM in a rat model of
Accepted 21 March 2011
experimental colitis. Colitis was induced by intracolonic instillation of 1 ml of 5% of acetic acid. Rats were
Available online 2 April 2011
treated with MSM (400 mg/kg/day, orally) for 4 days. Animals were euthanized and distal colon evaluated
Keywords:
histologically and biochemically. Tissue samples were used to measurement of malondialdehyde (MDA),
Methylsulfonylmethane myeloperoxidase (MPO), catalase (CAT), glutathione (GSH) and proinflammatory cytokine (TNF-α and IL-1β)
Experimental colitis levels. Results showed that MSM decreased macroscopic and microscopic colonic damage scores caused by
Inflammation administration of acetic acid. MSM treatment also significantly reduced colonic levels of MDA, MPO and IL-1β,
Reactive oxygen metabolites while increased the levels of GSH and CAT compared with acetic acid-induced colitis group. It seems that MSM
as a natural product may have a protective effect in an experimental ulcerative colitis.
© 2011 Elsevier Inc. All rights reserved.

Introduction MSM occurs naturally in small amounts in some green plants, fruits
and vegetables. MSM as a dietary supplement has commonly been used
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) (often in combination with glucosamine and chondroitin) to treat or
and Crohn's disease (CD), is a chronic gastrointestinal disorder prevent osteoarthritis (Gregory et al., 2008). Previous clinical studies
characterized by intestinal inflammation and mucosal tissue damage. have documented that MSM may have beneficial effects on some
IBD is a multifactorial disease of unknown etiology. However, it is diseases, such as interstitial cystitis, parasites, constipation, musculo-
believed that the pathophysiology of IBD involves an interaction among skeletal pain and allergies (Childs, 1994; Parcell, 2002; Maranon et al.,
environmental, genetic and immunological factors. Chronic intestinal 2008). A variety of toxicity studies have shown that MSM is essentially
inflammation may result from activation of the immune system by nontoxic to humans(Parcell, 2002). It has also been reported that MSM
normal intestinal bacteria. The activation of immune system results in may be important as a nontoxic and effective chemotherapeutic agent to
the production of proinflammatory cytokines (TNF-α and IL-1β), treat metastatic melanoma and possibly other metastatic cancers
reactive oxygen metabolites, prostaglandins and leukotrienes (Krimsky (Caron et al., 2010). MSM may act through its ability to scavenge
et al., 2003). These mediators have been suggested to contribute to the oxidative radicals which trigger inflammation (Maranon et al., 2008).
development of mucosal damage of the colon and lead to a chronic We hypothesized that MSM, due to its anti-inflammatory and anti-
inflammatory process. oxidant activity, may affect inflammatory diseases, such as colitis.
To study the etiology of IBD, multiple mouse models of colitis have
been developed and used to test new pharmacological agents for IBD. Materials and methods
Acetic acid-induced colitis is a chemically induced model of colitis that
exhibits various pathophysiological features of the human ulcerative Animals. Adult male Wistar rats, weighing 220–250 g, were housed in a
colitis (Jurjus et al., 2004; Kawada et al., 2007). There have been several room with a 12-h light/dark cycle. Rats had free access to tap water and
experimental studies on prevention or treatment of IBD by anti- ad libitum. Before the induction of colitis, rats were fasted overnight. All
inflammatory and antioxidative drugs (Mabley et al., 2003; Bitiren et al., the chemicals were purchased from Sigma.
2010; Sengul et al., 2010).
Induction of colitis. Rats were slightly anesthetized with ether and a
rubber catheter was inserted into the rectum such that the tip was 8 cm
inside the anus. Colitis was induced by intracolonic application of 1 ml of
⁎ Corresponding author. Fax: + 98 451 5510057.
5% acetic acid (in 0.9% NaCl). On the fourth day after the induction of
E-mail addresses: [email protected], [email protected] colitis, rats were euthanized and the distal 8 cm of the colon was resected
(K. Amirshahrokhi). for evaluation of various parameters. After macroscopic examination, the

0041-008X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2011.03.017
198 K. Amirshahrokhi et al. / Toxicology and Applied Pharmacology 253 (2011) 197–202

colonic sample from each rat cut into two parts, one for the histological MSM was dissolved in saline and administered orally using an
examination and the other for the biochemical analysis. The experi- intragastric tube for 4 consecutive days. Our preliminary experiment
mental animals were divided into four groups, each consisting of six showed that 400 mg/kg/day of MSM was the effective dose in this
animals. model of colitis.
Group I (Normal control group): received 1 ml saline intrarectally
following the administration of saline orally. Macroscopic damage score. After resection of the distal colon, the
Group II (MSM group): received 1 ml saline intrarectally following specimen was flushed out with saline solution and opened by a
the administration of MSM (400 mg/kg/day, orally). longitudinal incision. The colonic samples were scored macroscopically
Group III (Colitis group): received 1 ml acetic acid intrarectally according to the following grading system (Sykes et al., 1999):
following the administration of saline orally. 0 = no inflammation; 1 = swelling or redness; 2 = swelling and redness;
Group IV (Colitis+ MSM group): received 1 ml acetic acid intrarec- 3 = one or two ulcers; 4 = more than two ulcers or one large ulcer;
tally following the administration of MSM (400 mg/kg/day, orally). 5 = mild necrosis; 6 = severe necrosis.

Fig. 1. Effects of MSM on the severity of acetic acid-induced colitis. Histological appearance of colonic tissue sections in (A) normal, (B) MSM, (C) colitis, and (D) colitis + MSM rats.
Treatment with MSM (400 mg/kg/day, po) reduced the histological alterations caused by acetic acid administration. Magnification of the first column is × 100 and the second column
is × 200.
 K. Amirshahrokhi et al. / Toxicology and Applied Pharmacology 253 (2011) 197–202 199

Histological examination. For histological examination, colonic sam- tissue specimens from the colitis group revealed crypt loss, submucosal
ples were fixed with 10% formalin, embedded in paraffin and sections edema, severe inflammatory cell infiltration and vasculitis. In contrast,
were stained with hematoxylin and eosin (H&E). The severity of colitis plus MSM-treated group revealed preserved mucosal architec-
colonic inflammation was scored using the four criteria including ture (Fig. 1).As shown in Fig. 2A and B, there was a good correlation
submucosal edema, damage/necrosis, inflammatory cell infiltration between macroscopic and histological scores in the each study group. In
and vasculitis. Each of the criteria was graded on a scale from 0 to 3, acetic acid-induced colitis rats, macroscopic and microscopic patholog-
depending upon the severity of changes (0 = no change; 1 = mild; ical scores were significantly higher than those of the normal rats.
2 = moderate; 3 = severe) (Iseri et al., 2009). Treatment with MSM significantly decreased the macroscopic and
microscopic scores compared with the colitis group (P b 0.01).
Determination of myeloperoxidase activity. Myeloperoxidase (MPO)
activity was determined as an indicator of neutrophil infiltration into
the colon. Colonic tissue samples were frozen in liquid nitrogen and Myeloperoxidase activity and malondialdehyde levels
stored at −80 °C until time of assay. The samples were then thawed
and homogenized (100 mg/ml) in 50 mM potassium phosphate buffer To study the anti-inflammatory and antioxidant activity of MSM
and centrifuged at 10,000 × g for 30 min at 4 °C. Supernatants were in acetic acid-induced colitis, the levels of MDA and MPO in super-
discarded and pellets were resuspended in potassium phosphate natants of colonic tissues were measured. Administration of acetic
buffer (50 mM, pH 6) containing 0.5% hexadecyltrimethyl ammonium acid significantly increased levels of both MDA as an indicator of lipid
bromide. The suspensions were sonicated and centrifuged again peroxidation and MPO, a marker of neutrophil infiltration into the
(10,000 × g, 30 min) and the supernatants were collected. MPO activity inflamed tissue, in comparison to control (Fig. 3A and B). Treatment
was assayed by mixing 20 μl of the supernatant with a solution of with MSM significantly reduced elevated MDA levels and MPO
1.6 mM tetramethylbenzidine and 5 × 10− 4% hydrogen peroxide. The activities compared to those in the colitis group.
change in absorbance was measured at 460 nm using a microplate
reader and the results were expressed as units of MPO/mg tissue.

Determination of malondialdehyde levels. The colonic samples (100 mg)

were homogenized in 1 ml of 0.05 M Trizma base buffer (pH 7.4). The
homogenates were centrifuged (16,000 × g, 4 °C) for 30 min. The
supernatants were taken and kept at −80 °C for determination of
malondialdehyde (MDA) using a high-performance liquid chromatogra-
phy (HPLC) method previously described by Mateos et al. (2005).
Concentrations were expressed as nmol MDA/mg tissue.

Measurement of glutathione levels. After homogenization and taking

supernatants as described above, an aliquot (500 ml) of each colon
supernatants was used to determine the concentration of reduced
glutathione (GSH).The levels of GSH were determined according to a
high-performance liquid chromatography (HPLC) method previously
described by Giustarini et al. (2003). Concentrations were expressed
as nmol GSH/mg tissue.

Measurement of catalase activity. The activity of catalase in colonic

tissue samples was measured using a catalase assay kit (Cayman
Chemical Company). Enzyme activity was expressed as nmol/min/mg
tissue.

Measurement of colonic TNF-α and IL-1β levels. The colonic samples

(100 mg) were homogenized in 1 ml of Tris–HCl buffer (pH 7.4). The
homogenates were centrifuged (16,000 × g, 4 °C) in a refrigerated
centrifuge for 30 min, and the supernatant was taken and frozen at
−80 °C (Amirshahrokhi et al., 2008). Supernatant samples were
analyzed for rat cytokine concentrations using specific ELISA kits
(Bender MedSystems GmbH). Cytokine levels in the colonic tissue
were expressed as pg cytokine/mg tissue.

Statistical analysis. Data are expressed as mean ± standard deviation

(SD). Statistical analysis was performed using one-way ANOVA
followed by Tukey test. P b 0.05 was considered significant.

Results

Macroscopic and histologic examination

Intrarectal administration of acetic acid resulted in clinical, macro- Fig. 2. Effects of MSM on macroscopic (A) and microscopic (B) score of damage in
each study group at the fourth day after acetic acid administration. Treatment with
scopic, and histological signs of colitis. The colonic mucosa appeared MSM (400 mg/kg/day, po) significantly decreased the macroscopic and microscopic
macroscopically edematous, hemorrhagic, ulcerated and necrotic scores. Values are means±SD (n = 6). ⁎P b 0.001 compared with normal group; †P b 0.01
compared to the normal group. Histopathologic examination of colonic compared with colitis group.
200 K. Amirshahrokhi et al. / Toxicology and Applied Pharmacology 253 (2011) 197–202

Fig. 4. Effects of MSM treatment on colonic glutathione (GSH) content in acetic acid-
induced ulcerative colitis in rats. MSM significantly increased GSH levels of the colonic
tissues as compared with those in the colitis group. Values are means ± SD. ⁎P b 0.001
compared with normal group; †P b 0.05 compared with colitis group.

with that in the colitis group. As shown in Fig. 6B, the level of TNF-α
tends to decrease in colitis plus MSM group but the decreasing effect of
MSM on colonic level of TNF-α was not statistically significant when
compared with colitis group (P = 0.063).

Discussion

Our study demonstrated that MSM as an organosulfur compound

has protective effect against acetic acid-induced colitis in rat. The ability
of MSM (400 mg/kg/day, po) to attenuate colitis was due to anti-
inflammatory and antioxidant effects of MSM which were demonstrat-
ed by the reduction in MDA and IL-1β levels, MPO activity and the
simultaneous increase in GSH and CAT levels in colonic tissue. Also, our
macroscopic and histological analysis demonstrated the ability of MSM
to ameliorate intestinal damage and inflammation.
Fig. 3. Effects of MSM on the levels of malondialdehyde (MDA) (A) and myeloperox- Acetic acid-induced colitis is considered to be an experimental
idase (MPO) activity (B) of colonic tissue. MSM 400 mg/kg given orally reduced the model of intestinal inflammation. This model exhibits clinical similar-
levels of MPO and MDA in the colon of rats with acetic acid-induced colitis. Values are
ities to human ulcerative colitis which are characterized by infiltration of
means ± SD. ⁎P b 0.01 compared with normal group; †P b 0.05 compared with colitis
group. polymorphonuclear and mononuclear into the intestinal tissue, release
of inflammatory cytokines (IL-1 and TNF-α)and increase expression of

Glutathione levels

The endogenous antioxidant glutathione (GSH) levels were found

to be significantly lower in the acetic acid-induced colitis group
compared with the control group (Fig. 4). MSM treatment signifi-
cantly increased GSH levels of the colonic tissues as compared with
those in the colitis group (P b 0.05). Indeed MSM prevented colonic
GSH depletion in MSM-treated rats.

Catalase activity

In our study, the activity of catalase in the colonic tissue was

significantly lower in acetic acid-induced colitis group compared to
the control group (Fig. 5). Treatment with MSM caused a significant
elevation in catalase activity (P b 0.01).

TNF-α and IL-1β levels

Administration of acetic acid caused significant elevation of colonic

Fig. 5. Effects of MSM on colonic levels of catalase as an antioxidant enzyme in each
levels of proinflammatory cytokines IL-1β and TNF-α as compared with study group. Treatment of rats with MSM (400 mg/kg/day, po) resulted in a significant
those in the control group (Fig. 6A and B). Treatment of rats with MSM increase in catalase activity. Values are means ± SD. ⁎P b 0.01 compared with normal
significantly reduced acetic acid-induced production of IL-1β compared group; †P b 0.01 compared with colitis group.
 K. Amirshahrokhi et al. / Toxicology and Applied Pharmacology 253 (2011) 197–202 201

that treatment with MSM significantly reduced MPO activity in colitis

group. MPO is an indicator of the inflammatory reaction and reduction in
the activity of this enzyme can be display the anti-inflammatory effects
of MSM.
Malondialdehyde (MDA) is derived from the cleavage of polyunsat-
urated fatty acids by reactive oxygen species. It is the main final product
of the lipid peroxidation process in the cells (Del Rio et al., 2005). In our
study MDA level as a marker of oxidative stress in the colitis group was
significantly higher than in other groups. This observation is in
agreement with the result of previous studies showing lipid peroxida-
tion increase in colitis (Daneshmand et al., 2009; Sengul et al., 2010).
MSM administration markedly decreased the elevation of MDA content
in the colonic tissue. This finding indicates that MSM treatment improves
the antioxidant status in the colonic mucosa of rats with ulcerative
colitis.
Glutathione as thiol-containing compound is the main intracellular
antioxidant and plays an important role in the protection against free
radicals and reactive oxygen metabolites (Droge, 2002). MSM is a
source of sulfur and provides organic sulfur to the synthesis of
glutathione; therefore, it is able to counteract the depletion of GSH in
cells (Parcell, 2002; Maranon et al., 2008).Our study showed that MSM
treatment significantly increased colonic GSH levels compared to
those in acetic acid-induced colitis rats. Increase in colonic GSH
content may explain some of the beneficial effects of MSM in the
experimental colitis.
It has been proposed that imbalance between pro‐oxidant and
antioxidant mechanisms may play an important role in the development
of intestinal inflammation and mucosal tissue injury in colitis (Sengul
et al., 2010). Catalase (CAT) is an important peroxisomal antioxidant
enzyme and metabolizes hydrogen peroxide into oxygen and water. It
prevents the harmful effects of oxidative radicals and the initiation
processes of lipid peroxidation. It has been shown that CAT levels were
decreased in acetic acid-induced colitis while increased with treatment
(Mahgoub et al., 2003; Cetinkaya et al., 2006; Sengul et al., 2010). In our
study, we also observed that CAT activity were lower in the experimental
colitis group. Decreased CAT activity may relate to enzyme consumption
due to neutralizing the excessive H2O2 (oxidative reaction) in the colonic
tissue. In addition, the tissue catalase levels significantly increased in the
Fig. 6. Colonic levels of IL-1β and TNF-α in four study groups. MSM (400 mg/kg/day, po) MSM-treated colitis group compared with acetic acid-induced colitis
significantly reduced acetic acid-induced production of IL-1β (A), while MSM treatment group.
did not significantly decrease colonic TNF-α levels (B). Values are means ± SD. ⁎P b 0.01 Our results showing the inhibition of colonic lipid peroxidation
compared with normal group; †P b 0.01 compared with colitis group.
(MDA level) accompanied by the restoration of CAT and GSH levels
suggest that MSM exerts beneficial effects on maintaining pro-
oxidant/antioxidant equilibrium in the colonic tissue.
inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) Another finding of our investigation was that the production of
(Dong et al., 2003).One of the important intestinal tissue damage colonic TNF-α and IL-1β was increased after acetic acid administration.
mechanisms is oxidative stress through an excessive production of Cytokines play an important role in the modulation of the intestinal
reactive oxygen metabolites (ROM), including superoxide anion, immune system. Levels of several proinflammatory cytokines are
hydrogen peroxide, hypochlorous acid and hydroxyl radical (Kruidenier elevated in patients with IBD. Among proinflammatory cytokines
and Verspaget, 2002). Colitis induced by acetic acid is also known to induced in IBD, TNF-α and IL-1β are the most important mediators
produce excess reactive oxygen metabolites (Millar et al., 1996). The participating in the inflammatory process of IBD. They are produced
increased oxidative free radical production by polymorphonuclear cells and secreted by inflammatory cells and induce the production of other
in patients with active colitis could be related to the aggravation of cytokines, adhesion molecules, arachidonic acid metabolites, reactive
colitis (Shiratora et al., 1989). Generated oxygen-derived free radicals oxygen metabolites and nitric oxide (Rogler and Andus, 1998). It has
are involved in the procedure of lipid peroxidation, which leads to been reported that MSM decreases Lipopolysaccharide-induced
oxidative degradation of lipids and cell damage. release of some proinflammatory cytokines in murine macrophages
Myeloperoxidase (MPO) is a peroxidase enzyme found in the through inhibition of NF-κB signaling (Kim et al., 2009). In the current
azurophilic granules of neutrophils and monocytes. In intestinal inflam- investigation, MSM significantly reduced colonic IL-1β indicating that
matory procedures, MPO activity is a marker of neutrophil infiltration MSM has an anti-inflammatory effect. The ability of MSM to reduce the
into the colon. Upon activation and degranulation, MPO is released to colonic levels of IL-1β as a proinflammatory cytokine may account for
catalyze the formation of potent oxidants. It is involved in enhancing the its mechanism of action in attenuating ulcerative colitis.
cytotoxic activity of H2O2. MPO catalyzes the reaction of chloride with In conclusion, MSM could likely be beneficial as a complementary
hydrogen peroxide to produce hypochlorous acid, which is important for agent in treatment of acetic acid-induced colitis, perhaps due to its
the inflammatory properties of neutrophils (Eiserich et al., 1998; Luo antioxidant and anti-inflammatory mechanisms. However further
et al., 2010).Consequently, MPO can lead to tissue injury during chronic investigations are needed to evaluate the effectiveness of MSM in
inflammation and causes some of the clinical features of IBD. We showed patients with ulcerative colitis.
202 K. Amirshahrokhi et al. / Toxicology and Applied Pharmacology 253 (2011) 197–202

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Kawada, M., Arihiro, A., Mizoguchi, E., 2007. Insights from advances in research of
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Kim, Y.H., Kim, D.H., Lim, H., Baek, D.Y., Shin, H.K., Kim, J.K., 2009. The anti-inflammatory
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This study was supported by a grant from Vice Chancellor of Research Krimsky, M., Yedgar, S., Aptekar, L., Schwob, O., Goshen, G., Gruzman, A., Sasson, S.,
Ligumsky, M., 2003. Amelioration of TNBS-induced colon inflammation in rats
of Ardabil University of Medical Sciences. by phospholipase A2 inhibitor. Am. J. Physiol. Gastrointest. Liver Physiol. 285,
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