Lack of Antioxidant Effect of Colchicine In Healthy Rats

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Lack of Antioxidant Effect of Colchicine In Healthy Rats
FABAD J. Pharm. Sci., 33, 25–33, 2008
RESEARCH ARTICLE
Lack of Antioxidant Effect of Colchicine In Healthy
Rats
Necmiye CANACANKATAN*°, Kenan DAĞLIOĞLU**, Figen DORAN***, Gülen ATTİLA*, Abdullah
TULİ*, Arzu KANIK****, Levent KAYRIN*
Lack of Antioxidant Effect of Colchicine In Healthy Rats
Summary
Sağlıklı Sıçanlarda Kolşisinin Yetersiz Antioksidan
Etkisi
Özet
Colchicine is an ancient drug, widely used in the treatment
of gout, Behcet’s disease, Mediterranean fever, cirrhosis and
dermatologic disorders. A number of research groups have
underscored the increase in the neutrophil functions and
excessive production of reactive oxygen species detected
in some of these diseases. Since colchicine is employed in
the treatment of these diseases, we aimed to establish the
antioxidant effect of colchicine in healthy subjects. Different
doses of colchicine (40, 100, 200 μg/kg bwt p.o.) were
administrated 3 times a week for 9 weeks to the rats. The
activities of antioxidant enzymes including glutathione
peroxidase (GSH-Px), superoxide dismutase (SOD) and the
levels of reduced glutathione (GSH) and malondialdehyde
(MDA) were determined in both erythrocytes and liver. There
was a significant elevation in MDA levels (p=0,0001) and
decrease in GSH content (p=0,001) in colchicine-treated
groups. The GSH-Px enzyme activity was variably effected
in erythrocytes. Although a distinct dose-dependent increase
was found in SOD enzyme activity, it was not significant
in liver. These data support that colchicine failed as an
antioxidant for prophylactic use in healthy subjects.
Kolşisin, Gut Hastalığında, Behçet Hastalığında, Ailesel
Akdeniz Ateşi Hastalığında, siroz ve dermatolojik pek çok
hastalıkta oldukça yaygın kullanımı olan bir eski ilaçtır.
Son yıllarda yapılan çalışmalar, bu hastalıklarda, nötrofil
fonksiyonları ve reaktif oksijen türevlerinin üretiminde aşırı
artış olduğuna dikkat çekmiştir. Kolşisinin bu hastalıkların
tedavisinde kullanılmasından dolayı, sağlıklı sıçanlarda
kolşisinin antioksidan etkisini araştırmayı amaçladık.
Kolşisinin farklı dozları (40,100, 200 μg/kg bwt p.o.) 9
hafta süresince haftada 3 kez uygulandı. Antioksidan
enzimlerden glutatyon peroksidaz (GSH-Px), süperoksit
dismutaz (SOD) enzim aktiviteleri ile redükte glutatyon
(GSH) ve malondialdehit (MDA) düzeyleri eritrositlerde
ve karaciğerde tayin edildi. Kolşisin uygulanan sıçanlarda
MDA düzeylerinde artış gözlenirken ve GSH düzeylerinde
ise azalma saptandı. GSH-Px enzim aktivitesi eritrositlerde
değişkenlik gösterdi. Karaciğer SOD enzim aktivitesinde doza
bağlı olarak artış gözlenmesine rağmen bu artış istatistiksel
olarak anlamlı değildi. Sonuç olarak, kolşisinin antioksidan
olarak sağlıklı bireylerde proflaktik kullanımı yetersiz bulundu.
Key words: Colchicine, antioxidant, prophylactic, GSH,
SOD, MDA
Anahtar kelimeler: Kolşisin, antioksidan, proflaktik, GSH,
SOD, MDA
Received:18.09.2009
Revised: 10.11.2009
Accepted:17.11.2009
INTRODUCTION
The ancient drug, colchicine has been widely used,
medical properties of which were first described by
Dioscorides of Anazarbos in the first century AD
(1). It has been prescribed for acute gout attacks
and prophylaxis, Behcet’s Disease (BD), Familial
Mediterranean fever (FMF) and amyloidosis of FMF,
cirrhosis of the liver (2) and dermatologic disorders
(3).
* Department of Biochemistry, Medicine Faculty, Cukurova University, Adana, TURKEY
** Department of Anatomy, Medicine Faculty, Cukurova University, Adana, TURKEY.
*** Department of Pathology, Medicine Faculty, Cukurova University, Adana, TURKEY.
****Department of Biostatistics, Medicine Faculty, Mersin University, Mersin, TURKEY.
° Corresponding author E-mail: [email protected]
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Canacankatan, Dağlıoğlu, Doran, Attila, Tuli, Kanık, Kayrın
In recent years, it has been noticed that various
functions of the polymorphonuclear leukocytes such
as chemotaxis, phagocytosis and release of many
reactive oxygen substances (e.g. superoxide, singlet
oxygen, hydroxyl radical, hydroxy peroxide and
superoxide radical anion) have been increased in
gout attacks (4), BD (5), FMF (6) and cirrhosis of the
liver (7).
Kose et al. stated that increased malondialdehyde
(MDA) levels in both plasma and erythrocytes and
increased superoxide dismutase (SOD) activities in
erythrocytes and decreased glutathione peroxidase
(GSH-Px) activity were seen in patients with BD
(8). It was also found that MDA levels increased
significantly in active period of BD (9). Superoxide
radical anion (O2-.) caused tissue damage related with
chronic inflammatory disorders such as BD. Proniai
and et al. reported that colchicine treatment increased
the scavenging activity of polymorphonuclear cells
up to the normal level in BD patients (10). SOD,
which provided protection from O2-. , found to be
useful for the treatment of BD (11). FMF is another disorder where excess production
of O2-. has been observed. Clastogenic plasma
factors correlated with increased O2-. production by
neutrophils have been higher than in patients with
FMF compared to controls (6).
It has been recently described that oxidative damage
is one of the substrates of fibrosis which is the first
step of cirrhosis (12). Free radical generation was
found in cirrhosis of the liver (7). Colchicine has
been used for amelioration of cirrhosis in patients
(13,14) and its preventing effect on fibrosis was
demonstrated (15).
Since colchicine was employed in the treatment
of the above-mentioned diseases, in this study
we decided to investigate prophylactic effects
of colchicine on healthy subjects by measuring
antioxidant status. For this purpose the activities
of antioxidant enzymes, including GSH-Px, which
metabolize hydrogen peroxide to water (16) and SOD
which remove deleterious free radical by product
of oxygen metabolism (17); reduced glutathione
26
(γ-glutamyl-cysteinyl-glycine – GSH), a well-known
oxyradical scavenger (18) and MDA, one of the end
products of lipid peroxidation (19) were determined
in both erythrocytes and liver. In addition
histopathologic investigations were performed by
light microscopy.
MATERIALS AND METHODS
Materials
Colchicine was obtained from Sigma Chemical Co.
(St. Louis, MO, USA). The rest of the reagents were of
the best quality commercially available.
Treatments of animals
Male Wistar albino rats, 5-6 weeks old and weighing
180-200 gr purchased from the Animal Research
Laboratory of Cukurova University, were subjected
to this study and were housed in stainless steel cages
in an acclimated room, with a constant temperature
of 25 °C and relative humidity (55 ± 8 %) and in a
12-h light-dark cycle. They were fed on a standard
diet with ad libitum access to drinking water. They
were acclimatized to the laboratory conditions for 1
week before being used.
Drug Administration
Colchicine was dissolved in distillated water. Three
different concentrations of colchicine (40; 100; 200 μg
colchicine/kg bwt) were prepared and administrated
to the rats three times a week for nine weeks. 8
rats were appointed to each group. Colchicine was
administered by the intragastric route using a metal
gavage tube at a volume of 1 mL. Control rats received
distilled water in the same way. At the end of the 9th
week, the rats were killed by cervical dislocation.
Sampling
Erythrocyte pellet preparation
Intracardiac blood samples of rats were collected into
EDTA and the whole blood was centrifuged at 3000
g for 10 minutes at 4 oC. Plasma was separated and
then erythrocytes were washed four times by saline.
Preparation of liver homogenates
Livers were homogenized in a solution containing
10 mM phosphate buffer (pH 7.0) and centrifuged
at 14.000 g for 30 min at 4 oC. Supernatant was
FABAD J. Pharm. Sci., 33, 25–33, 2008
removed and the enzyme activities of SOD and GSHPx; the levels of MDA and GSH were determined
in supernatants. Protein levels were determined
according to the method of Lowry et al. using
purified bovine serum albumin as a standard (20).
BIOCHEMICAL ANALYSIS
GSH-Px assay
The GSH-Px enzyme activity in erythrocytes
haemolysate was determined by the method of
Beutler (21). The rate of oxidized glutathione
formation was measured spectrophotometrically at
340 nm with NADPH. GSH-Px enzyme activity was
measured following the same procedure in liver.
Liver homogenates were used instead of erythrocyte
haemolysate. The enzyme activities were expressed
as U/gHb in erythrocytes and as U/mg protein in
liver.
HISTOLOGICAL ANALYSIS OF THE LIVER
The hepatotectomy specimens were handled
routinely, with submission of at least three sections of
liver. Rat livers were fixed in 10% buffered formalin,
conventionally processed and embeddedin parafin wax.
Sections (5mm thick) were stained with hematoxylin
and eosin, periodic acid-Schiff reaction (PAS), Goldner
trichrome and reticulin stain. Tissues were investigated
carefully for evolution of hepatic damage.
Statistical Analysis
Data were analyzed using SPSS software (version
11.5). Parameters were performed by Shapiro-Wilk
test for normality. Each parameter was assigned for
homogeneity of variances. SOD and MDA parameters
were determined for heterogeneity of variances;
other parameters were ascertained for homogeneity
of variances. Therefore, One-Way ANOVA, used
for parameters provide homogeneity of variances
and Welch test, carried out for parameters provide
heterogeneity of variance. Tukey test was used for
multiple comparisons in One-Way ANOVA and
Games-Howell test for multiple comparisons in
Welch test statistics.
SOD measurement
The SOD enzyme activity was assayed according
to the method of McCord et al. (22). The principle
of the method depends on xantine and xanthine
oxidase to generate superoxide radicals which
react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5phenyltetrazolium chloride (INT) to form a red
formazan dye. The optical density of this substance
was measured at 505 nm. The results were stated as
U/mg protein.
Abbreviations Used
GSH, Glutathione; GSH-Px, Glutathione peroxidase;
SOD, Superoxide dismutase; MDA, Malondialdehyde;
DTNB, 5,5’-Dithio-bis-2-nitrobenzoic acid.
GSH measurement
The blood GSH content was assayed according to
Beutler’s method by using 5,5’-Dithiobis (2-nitro
benzoic acid) (DTNB) a disulfide compound
(21). DTNB was readily reduced by sulfhydryl
compounds and formed a highly colored yellow
anion. The optical density of this yellow substance
was measured at 412 nm.
RESULTS
Biochemical Results
Nine weeks after colchicine treatment, the dosedependent manner of colchicine on antioxidant
status and the level of MDA were evaluated. The GSH
content significantly decreased in colchcine-treated
groups as compared to controls both in erythrocytes
and liver (Fig 1 and 2; Table 1 and 2).
MDA measurement
The lipid peroxide levels were measured using
1,1,3,3-tetramethoxypropane as standard and
presented as nanomoles MDA, formed per mg
protein (23). The spectrophotometrical method
was based on the concentration of pink chromogen
compound, which was formed by MDA’s binding to
thiobarbituric acid.
A dose-dependent increase in GSH-Px enzyme
activity was observed among the colchicine-treated
groups in erythrocytes (Fig 1). In contrast to these
results, there was decrease in GSH-Px enzyme
activity compared with the controls in liver (Fig 3).
Even though an evident dose-dependent increase
was found in SOD enzyme activity, in liver (0,245
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Canacankatan, Dağlıoğlu, Doran, Attila, Tuli, Kanık, Kayrın
GSH (μmol/gHb)
Figure 1. The comparison of groups in terms of GSH and GSH-Px in erythrocytes. *, Significantly different from control
P<0.05; #, Significantly different from 40 group P<0.05.
Figure 2. The comparison of groups in terms of GSH in liver. *, Significantly different from control P<0.05; #, Significantly
different from 40 group P<0.05.
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FABAD J. Pharm. Sci., 33, 25–33, 2008
Figure 3. The comparison of groups in terms of GSH-Px, SOD and MDA in liver. *, Significantly different from control P <
0.05; #, Significantly different from 40 group P < 0.05; †, Significantly different from 100 group P < 0.05.
Table 1. Summary of measurements of GSH and GSH-Px
in erythrocytes
Groups
GSH (mmol / gHb)
GSH-Px (U / gHb)
Control
10.210 ± 1.210
2.390 ± 0.630
40
7.945 ± 0.580*
1.627 ± 0.274
100
4.336 ± 1.650 *, #
2.506 ± 0.794
200
3.468 ± 0.742 *, #
3.594 ± 0.684 *, #
Values are expressed as mean ± SD of per groups. 40, 100
and 200 exert dose of Colchicine μg/kg bwt. *, Significantly
different from control P<0.05; #, Significantly different
from 40 group P<0.05.v
± 0,058 U/mg protein versus 0,453 ± 0,219 U/mg
protein), it was not significant (Table 2). MDA levels
were found to have increased in 100 mg colchicine/
kg bwt (p=0,005) and 200 mg colchicine/ kg bwt
(p=0,006) in liver (Fig 3).
Histological Results
There haven’t been any pathologic findings in
the control group (Fig. 4). Nodular lymphocytic
infiltration on periportal region in liver
paranchyma was found in 40 µg colchicine-treated
rats (Fig. 5). In livers, the microscopic changes
consisted of lymphocytic infiltration in portal
zone and reactive changes of hepatocytes were
observed in 100 µg colchicine-treated rats (Fig.
6). The other histopathological findings, such as
dense lymphocytes and histocytes in portal tract,
prominent congestion and reactive changes in
parachyma occurred in 200 µg colchicine-treated
rats (Fig. 7).
DISCUSSION
Colchicine, a heterocyclic alkaloid isolated from
Colchicum automnale (24) has been used for
centuries in acute gout arthritis (25). During the
recent decades, it has been employed for an increasing
number of disorders such as BD, FMF, liver cirrhosis,
dermatologic disorders and scleroderma (3) and
free radical generation was found in some of these
diseases (4-7). In the present study, different doses
of colchicine p.o. were administrated 3 times a week
for 9 week in order to observe prophylactic and
antioxidant effects of colchicine in healthy rats.
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Canacankatan, Dağlıoğlu, Doran, Attila, Tuli, Kanık, Kayrın
Table 2. Summary of measurements of GSH-Px, SOD, GSH and MDA in liver
Groups
GSH
(mmol / mg protein)
GSH-Px
(U / mg protein)
SOD
(U / mg protein)
MDA
(nmol / mg protein)
Control
40
100
200
0,011 ± 0.003
0.009 ± 0.003
0.008 ± 0.002 *
0.005 ± 0.002 *, #
0.654 ± 0.137
0.591 ± 0.075
0.427 ± 0.092 *, #
0.561 ± 0.078
0.245 ± 0.058
0.242 ± 0.047
0.309 ± 0.110
0.453 ± 0.219
0.310 ± 0.086
0.456 ± 0.157
0.615 ± 0.174 *
1.085 ± 0.316 *, #, †
Values are expressed as mean ± SD of per groups. 40, 100 and 200 exerts dose of Colchicine µg/kg bwt. *,
Significantly different from control P < 0.05; #, Significantly different from 40 group P < 0.05; †, Significantly
different from 100 group P < 0.05.
Figure 4. Control Group. Normal liver pattern
Figure 5. Nodulary lymphocytic infiltration on periportal
region in liver parachyma. HEx200.
Figure 6. Lymphocytic infiltration in portal zone and
reactive changes of hepatocytes. HEx100.
Figure 7. Dense lymphocytes and histiocytes in portal tract,
prominent congestion and reactive changes in parachyma.
GSH is an antioxidant that has an important role in
detoxication of xenobiotics and free radicals, redox
potential regulation and preventing the generation
of hydroxyl radical. GSH is converted to oxidized
form by the enzyme GSH-Px. GSH-Px catalyses
reduction of lipid hydroperoxides and hydrogen
peroxide (26). In this study, the levels of GSH-Px
were variably effected in erythrocytes and liver. The
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FABAD J. Pharm. Sci., 33, 25–33, 2008
effect of colchicine on GSH-Px was not dependent on
the concentration. However, there was a depletion of
GSH in a dose manner in both erythrocytes and liver.
GSH depletion points to an increased generation of
free radicals and failing of cytoprotective mechanism.
The antioxidant effect of colchicine was investigated
by Das et al. on rat liver injury. It was understood
that the antioxidant effect of colchicine in vitro was
evident at very high concentrations; where it exerted
weak antioxidant properties in vivo (27).
The SOD enzyme, which catalyzes the dismutation
of superoxide anion (O2-) to produce hydrogen
peroxide (H2O2) and O2 was decreased in colchicinetreated rats; nevertheless, it was not significant.
Similar results related to our study were found in
brain GSH, SOD and MDA intracerebroventricular
administration of colchicine in rats (28).
The effects and the mechanisms of extract from
Paeonia lactiflora and Astragalus membranaceus
were searched on liver fibrosis induced by CCl4 in rats.
In this study, colchicine (100 µg/kg, intragastrically)
served as positive control. Administration with
colchicine significantly elevated liver SOD and
GSH-Px activity compared to the CCl4 treated rats.
Although colchicine treatment decreased MDA level,
it was not statistically significant (29).
In our study there was a significant increase in MDA
in colchicine-treated rats. A dose-dependent increase
in levels of MDA in colchicine-treated rats indicate
lipid peroxidation.
Rhoden et al. point out that an increased lipid
peroxide levels was observed in cirrhotic tissue
compared to normal liver and this was decreased
by colchicine treatment after cirrhosis induced (30).
Yet, on the other hand, their further study colchicine
have not exerted any protective effects on cirrhosis
induced by CCl4 (31). Similar to this result, Cedillo et.
al. also reported that colchicine could not prevent the
increase in MDA levels in CCl4-induced cirrhosis (32).
Muriel et al. studied the effect of colchicine on liver
damage induced by acetaminophen. In contrast
to our result, it was found that pretreatment of
colchicine prevent liver damage at a certain dose.
While pretreatment of colchicine with adose of 65
µg/kg failed to prevent liver damage, 300 µg/kg
colchicine save the levels of lipid peroxidation and
gama glutamyl trans peptidase. Muriel and et al.
indicated that colchicine was acting as a free radical
scavenger (33).
It is also suggested that colchicine may reduce
the cyclosporine nephrotoxicty by restraining the
expression of transforming growth factor beta (TGFbeta), apoptotic cell death and MDA production (34).
Early colchicine administration limits hepatic fibrosis
and so limits the degree of portal hypertention in rats
with bile duct ligation (35). Also it was established
that colchicine decreased MDA on prolonged bile
duct obstruction in rat. Liver damage can be induced
by bile duct ligation. This induction increased MDA
levels tenfold as compared to controls. Treatment
of bile duct ligation rats with 10 µg of colchicine
completely prevent this effect (36). But, on the other
hand, it was also reported that colchicine treatment
had no significant effect on MDA levels that increased
three-fold by bile duct ligation (37).
Colchicine exerts its clinical effects by affecting
neutrophilic functions including inhibition of
neutrophil chemotaxis, inhibition of secretion of
neutrophil lyzomal enzymes and suppression of
excretion of procollagen from fibroblasts (38,39). It is
suggested that the superoxide scavenging activity of
colchicine may be related to its phagocytosis blocking
property (10).
In this study; there was a significant decrease in levels
of GSH and elevation in MDA levels. The GSH-Px
enzyme activity was effected variably in erythrocytes.
Besides, there was a distinct dose-dependent increase
in SOD enzyme activity, in liver though it was not
significant. According to our results, it can be stated
that colchicine caused free radical generation and
lipid peroxidation. However, several acute or chronic
experimental animal (40,41) and clinical studies
(42,43) point out protective effects of colchicine in liver
damage. As a conclusion, it is therefore important
31
Canacankatan, Dağlıoğlu, Doran, Attila, Tuli, Kanık, Kayrın
to mention that colchicine failed as antioxidant for
prophylactic usage in healthy subjects.
Acknowledgements
I would like to thank Department of Biochemistry,
Medicine Faculty, Cukurova University and TIBDAM
(Experimental Research Department).
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