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
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.
Key words: Colchicine, antioxidant, prophylactic, GSH, SOD, MDA
Received: 18.09.2009 Revised: 10.11.2009 Accepted: 17.11.2009
Sağlıklı Sıçanlarda Kolşisinin Yetersiz Antioksidan Etkisi
Özet
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.
Anahtar kelimeler: Kolşisin, antioksidan, proflaktik, GSH, SOD, MDA
* 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: ncanacankatan@yahoo.com 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).
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
(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
removed and the enzyme activities of SOD and GSH- Px; 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.
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)-5- phenyltetrazolium 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.
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.
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.
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.
Abbreviations Used
GSH, Glutathione; GSH-Px, Glutathione peroxidase;
SOD, Superoxide dismutase; MDA, Malondialdehyde;
DTNB, 5,5’-Dithio-bis-2-nitrobenzoic acid.
RESULTS
Biochemical Results
Nine weeks after colchicine treatment, the dose- dependent 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).
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
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.
GSH (μmol/gHb)
± 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.
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
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.
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 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 0,011 ± 0.003 0.654 ± 0.137 0.245 ± 0.058 0.310 ± 0.086
40 0.009 ± 0.003 0.591 ± 0.075 0.242 ± 0.047 0.456 ± 0.157
100 0.008 ± 0.002 * 0.427 ± 0.092 *, # 0.309 ± 0.110 0.615 ± 0.174 * 200 0.005 ± 0.002 *, # 0.561 ± 0.078 0.453 ± 0.219 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 6. Lymphocytic infiltration in portal zone and reactive changes of hepatocytes. HEx100.
Figure 5. Nodulary lymphocytic infiltration on periportal region in liver parachyma. HEx200.
Figure 7. Dense lymphocytes and histiocytes in portal tract, prominent congestion and reactive changes in parachyma.
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 colchicine- treated 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 (TGF- beta), 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
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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