THE PROTECTIVE ROLE OF COLCHICINE ON DICLOFENAC SODIUM INDUCED HEPATORENAL TOXICITY IN ALBINO RATS MODEL

THE PROTECTIVE ROLE OF COLCHICINE ON DICLOFENAC SODIUM INDUCED HEPATORENAL TOXICITY IN ALBINO RATS MODEL

Sylvia A. Boshraand Mohammed A. Hussein^*

Biochemistry Department, Faculty of Pharmacy, October 6^th University, 6^th October city, Egypt.

ABSTRACT:Diclofenac (DIC) is a widely used as an anti-inflammatory drug and it is used as the toxicant in hepatoprotective studies. In the present study, hepato-renal protective activity of Colchicine against DIC induced liver and kidney injury in albino rats was assessed. Animals were injected with Diclofenac sodium at the single dose of 150 mg/ kg body weight daily for 28 days. Oral administration of colchicine at a concentration of 10µg/kg b.w daily for 28 days showed a significant decrease in plasma AST, ALT and creatinine as well as hepatic and renal thiobarbituric acid reactive substances and hydroperoxides. The treatment also resulted in a significant increase in GSH, SOD, CAT and GPx in the liver and kidney of DIC treated rats. The results clearly suggest that the colchicine treated group may effectively normalize the impaired antioxidant status in DIC- induced liver and kidney injury than the vitamin C-treated groups. However, Colchicine rapid protective effects against lipid peroxidation by scavenging of free radicals reduce the risk of oxidative complications.

Colchicine, diclofenac sodium, hepato-renal protective, oxidative stress biomarkers

INTRODUCTION: Liver diseases are considered to be serious health disorders. The liver has one of the highest value of importance for the systemic detoxification and deposition of endogenous and exogenous substances. Diclofenac (DIC) is a phenylacetic acid derivative that was developed specifically as a non-steroidal anti-inflammatory (NSAID) drug¹. DIC causes a rare but potentially severe liver injury in humans ^2,3. The hepatotoxicity of DIC was also documented in experimental animal studies ^4-6.

The mechanism of DIC hepatotoxicity involves covalent protein modification by reactive metabolites ^7-9, oxidative stress generation by peroxidase-catalyzed reaction ^{3, 10} and mitochondrial injury propagation by reactive oxygen species ^{11, 12}.

Liver damage occurs infrequently, and is usually reversible. Hepatitis may occur rarely without any warning symptoms and may be fatal. Patients with osteoarthritis more often develop symptomatic liver disease than patients with rheumatoid arthritis. Liver function should be monitored regularly during long-term treatment. If used for the short-term treatment of pain or fever, diclofenac has not been found to be more hepatotoxic than other NSAIDs ^{13, 14}. Although several natural products have been shown to protect against chemical-induced liver and renal toxicity, a consensus on the protective effects of natural products for the treatment of DIC- induced hepato-renal toxicity however has not yet been reached.

Colchicine is an alkaloid drug, chemically known as N-[(7S)-1, 2, 3, 10-tetramethoxy-9-oxo-5, 6, 7, 9-tetrahydrobenzo[a] heptalen-7-yl] acetamide, and widely used for the treatment of gout disease ¹⁵.

FIG. 1: STRUCTURE OF COLCHICINE

Colchicine has a high market value and consistent demand in the field of medicine¹⁶. The alkaloid, colchicine is the drug of choice to relieve acute attack of gout, familial Mediterranean fever ¹⁷ and a cure for cancer related diseases^{18, 19}. Also, colchicine, a recognized liver protector which prevents the assembly of cytoplasmatic microtubules, inhibits the transcellular movement of collagen ^{20, 21}, stimulates the production of collagenase in cultures of synovial tissue ²² and exerts a stabilizing effect on the plasma membranes of the hepatocyte ²³. It prevents infiltration reverses CC1₄-induced liver cirrhosis in rats ^{24, 25}.

Colchicine was reported to improve survival in a clinical trial for alcoholic liver cirrhosis²⁶, and is currently under investigation in a long-term Veterans Administration cooperative treatment trial in alcoholic liver disease. Recently, Hussein and Boshra²⁷ reported the antitumor and structure antioxidant activity relationship of colchicine on Ehrlich Ascites Carcinoma (EAC) in Female Mice. As an extension of our studies on colchicine²⁷, now we wish to evaluate the hepato-renal protective effect of colchicine against diclofenac sodium induced liver and renal toxicity which may pave the way for possible therapeutic applications.

MATERIALS AND METHODS:

Chemicals: Diclofenac sodium and colchicine were obtained from Merck Ltd., Germany. All the other reagents used were of analytical grade and were obtained commercially.

 Animals

This experiment was conducted in accordance with guidelines established by the Animal Care and use Committee of October 6 University. Adult rats weighing around 180±5gms were purchased from Faculty of Veterinary Medicine, Cairo University. They were individually housed in cages in an air-conditioned room with a temperature of 22 ± 2^oC, a relative humidity of 60%, and an 8:00 to 20:00 light cycle. During the acclimatization period, each animal was raised on a regular diet ad-libitum.

Experimental set up:

This experiment was carried out to examine the prophylactic potential of colchicine against diclofenac sodium induced liver and renal toxicity in- vivo.

Groups of animals each consisting of 8 rats were treated daily for 28 days as follows.

 Group I: Normal; was given saline orally for 28 days.

Group II: Was treated with colchicine (10µg/kg b.w.) suspended in saline orally in a single daily dose for 28 days²⁸.

Group III: Control; was treated with diclofenac sodium (150mg/kg, i.p.) suspended in saline for 28 days²⁹.

Group IV: Was treated with diclofenac sodium (150mg/ kg, i.p.) + colchicine (10µg /kg b.w. orally) in a single daily dose.

Group V: Was treated with diclofenac sodium (150mg/kg, i.p.) + vitamin C (1g/kg b.w. orally) in a single daily dose ³⁰.

Colchicine and vitamin C were suspended in saline and administered orally to its respective group animals for 28 days. At the end of the fourth week, the rats were sacrificed by cervical decapitation and the blood was collected using sodium fluoride as anticoagulant for determination of plasma transaminases; L-alanine(ALT) andL-aspartate (AST) ³¹ and creatinine ³². The liver and kidneys were dissected out, washed in ice-cold saline, patted dry, homogenized and used for determination of liver and kidney thiobarbituric acid reactive substances (TBARS) ³³, hydroperoxides  (HP) ³⁴, reduced glutathione (GSH) ³⁵, superoxide dismutase (SOD) ³⁶, catalase (CAT) ³⁷, glutathione peroxidase (GPx) ³⁸ and protein content in tissue homogenate ³⁹ were determined.

Measurement of lipid peroxidation

A thiobarbituric acid reactive substances (TBARS) assay kit (ZeptoMetrix) was used to measure the lipid peroxidation products, malondialdehyde (MDA) equivalents (33). In brief, liver and renal tissues were homogenized with 0.1 mol/l sodium phosphate buffer (pH 7.4). One hundred microliters of homogenate were mixed with 2.5ml reaction buffer (provided by the kit) and heated at 95 °C for 60 min. After the mixture had cooled, the absorbance of the supernatant was measured at 532 nm using a spectrophotometer. The lipid peroxidation products are expressed in terms of MDA equivalents.

 Measurement of antioxidant enzymes

Superoxide dismutase (SOD), glutathione peroxidase (GPx), Catalase (CAT) and reduced glutathione (GSH) levels were determined using commercially available assay kits (Biodiagnostic). Briefly, liver, and renal tissues were weighed and homogenized with appropriate buffers (provided by the kits). The homogenates were then determined following the procedures provided by the respective manufacturers. The basis of the GSH determination method is the reaction of Ellman's reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) with thiol group of GSH at pH 8.0 to give yellow color of 5-thiol-2- nitrobenzoate anion³⁵.

The superoxide dismutase assay kit utilizes a tetrazolium salt for detection of superoxide radicals generated by red formazan dye reduction produced³⁶. One unit (U) of SOD activity is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. The catalase assay kit utilizes the peroxidative function of CAT for determination of enzyme activity³⁷. The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H₂O₂. The generated formaldehyde is assayed spectrophotometrically with 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole as the chromogen. One unit (U) of CAT activity is defined as the amount of enzyme that will cause the formation of 1.0nmol of formaldehyde per minute at 25°C.

The glutathione peroxidase assay kit measures GPx activity indirectly by a coupled reaction with glutathione reductase (GR) ³⁸. Oxidized glutathione, produced upon reduction of hydroperoxide by GPx, is recycled to its reduced state by GR and NADPH. The oxidation of NADPH to NADP⁺ is accompanied by a decrease in absorbance at 340 nm. Under conditions in which the GPx activity is rate limiting, the rate of decrease in the A₃₄₀ is directly proportional to the GPx activity. One unit (U) of GPx activity is defined as the amount of enzyme that will cause the oxidation of 1.0 nmol of NADPH to NADP⁺ per minute at 25 °C. The specific activities of the various enzymes in the rat liver and renal tissues are expressed in U/mg of the protein with the protein content determined as stated above.

Statistical analysis

All data were expressed as mean ± SD. All analyses utilized SPSS 15.0 statistical package for Windows (SPSS Inc., Chicago, IL) ⁴⁰. A one-way analysis of variance (ANOVA) was employed for comparisons of means of the different groups. A p-value 0.05 was accepted as statistically significant. Diclofenac sodium control rats were compared with normal control rats as well as Colchicine and vitamin C treated rats were compared with diclofenac sodium control.

RESULTS:

Table 1 shows the levels of plasma ALT, AST and creatinine of control and experimental groups of rats. Diclofenac sodium (DIC) (150mg/ kg, i.p) markedly increased plasma ALT, AST and creatinine levels when compared with the normal group (p<0.01). Oral administration of colchicine (10µg /kg b.w.) showed non-significant changes in liver enzymesand creatinine when compared with the normal group. Whereas DIC injected rats-treated with the colchicine (10µg /kg b.w.) and/or vitamin C (1g/kg) ameliorated these increases significantly (p<0.01). Amelioration of hepatic marker enzymes was at maximum in colchicine (10µg /kg b.w.) than vitamin C (1g/kg.) when compared with DIC treated rats.

 TABLE 1: ACTIVITY OF ALANINE TRANSAMINASE (ALT), ASPARTATE TRANSAMINASE (AST), CREATININE IN PLASMA OF NORMAL AND EXPERIMENTAL GROUPS OF RATS

DIC was given i.p. as a daily dose of 150mg/kg.b.w. for 28 days. It was given to all groups except the normal A and B groups. Colchicine and vitamin C were orally given daily for 28 days as a daily single dose. Values are given as mean ± SD for groups of eight animals each.

* Significantly different from normal group at p< 0.01

^@ Significantly different from control group at p< 0.05.

Tables 2 and 3 show liver and renal reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HP) levels of control and experimental groups of rats. Oral administration of colchicine (10µg /kg b.w.) showed non-significant changes in hepatic and renal GSH, TBARs and HP.  A significant depletion (p<0.05) in the level of hepatic and renal GSH content was noticed in rats treated with DIC compared to normal control rats. Treatment with

colchicine (10µg /kg b.w.) significantly (p<0.05) restored the level of GSH to near the normal level as compared with DIC treated rats. The levels of TBARS and HP were significantly increased (p<0.05) in DIC-treated rats as compared with normal control rats. Oral administration of colchicine (10µg /kg b.w.) as well as vitamin C (1g/kg.) in DIC treated rats significantly lowered the levels of TBARS and HP in liver and kidney compared to DIC-treated rats.

TABLE 2:  LEVELS OF HEPATIC REDUCED GLUTATHIONE (GSH), THIOBARBITURIC ACID REACTIVE SUBSTANCES (TBARS) AND HYDROPEROXIDES (HP) IN NORMAL AND EXPERIMENTAL GROUPS OF RATS

 DIC was given i.p. as a daily dose of 150mg/kg.b.w. for 28 days. It was given to all groups except the normal A and B groups. Colchicine and vitamin C were orally given daily for 28 days as a daily single dose. Values are given as mean ± SD for groups of eight animals each.

* Significantly different from normal group at p< 0.01

^@ Significantly different from control group at p< 0.05.

TABLE 3:  LEVELS OF RENAL REDUCED GLUTATHIONE (GSH), THIOBARBITURIC ACID REACTIVE SUBSTANCES (TBARS) AND HYDROPEROXIDES (HP) IN NORMAL AND EXPERIMENTAL GROUPS OF RATS.

DIC was given i.p. as a daily dose of 150mg/kg.b.w. for 28 days. It was given to all groups except the normal A and B groups. Colchicine and vitamin C were orally given daily for 28 days as a daily single dose. Values are given as mean ± SD for groups of eight animals each.

* Significantly different from normal group at p< 0.01

^@ Significantly different from control group at p< 0.05.

Tables 4 and 5 shows the concentrations of liver and renal superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) of control and experimental groups of rats. Oral administration of colchicine (10µg /kg b.w.) showed non-significant changes in liverand renal enzymatic antioxidants as compared with the normal group. On the other hand, a significant decrease (p<0.05) in the activities of hepatic and renal–enzymatic antioxidants in DIC-treated rats was seen. Treatment with colchicine (10µg /kg b.w.) in DIC treated rats significantly increased the activities of enzymatic antioxidants in liver and kidney as compared with DIC-treated rats.

 TABLE 4:  LEVELS OF HEPATIC SUPEROXIDE DISMUTASE (SOD), GLUTATHIONE PEROXIDASE (GPX) AND CATALASE (CAT) IN NORMAL AND EXPERIMENTAL GROUPS OF RATS.

  DIC was given i.p. as a daily dose of 150mg/kg.b.w. for 28 days. It was given to all groups except the normal A and B groups. Colchicine and vitamin C were orally given daily for 28 days as a daily single dose. Values are given as mean ± SD for groups of eight animals each. Activity is expressed as: 50% of inhibition of pyrogallol autooxidation per min for SOD and the obtained values were divided by the protein concentration; GPx: μg of GSH consumed/min mg protein; μmoles of hydrogen peroxide decomposed per min per mg of protein for catalase.

* Significantly different from normal group at p< 0.01;

^@ Significantly different from control group at p< 0.05.

TABLE 5:  LEVELS OF RENAL SUPEROXIDE DISMUTASE (SOD), GLUTATHIONE PEROXIDASE (GPX) AND CATALASE (CAT) IN NORMAL AND EXPERIMENTAL GROUPS OF RATS.

 DIC was given i.p. as a daily dose of 150mg/kg.b.w. for 28 days. It was given to all groups except the normal A and B groups. Colchicine and vitamin C were orally given daily for 28 days as a daily single dose. Values are given as mean ± SD for groups of eight animals each. Activity is expressed as: 50% of inhibition of pyrogallol autooxidation per min for SOD and the obtained values were divided by the protein concentration; GPx: μg of GSH consumed/min mg protein; μmoles of hydrogen peroxide decomposed per min per mg of protein for catalase.

* Significantly different from normal group at p< 0.01;

^@ Significantly different from control group at p< 0.05.

DISCUSSIONS: Hepatotoxicity from NSAIDs can occur within 28 days of therapy after drug administration²⁹. The possible mechanism of Diclofeac induced liver injury is due to hypersensitivity and metabolic aberration which can produce serious liver damage in human and experimental animals with toxic doses⁴¹.  The liver damage causes leaking of cellular enzymes into the

plasma due to the disturbance of hepatocytes transport functions. From our findings, it is evident that the colchicine was able to reduce all the elevated biochemical parameters as a result of hepato- and renal toxin challenge, indicating improvement of the functional status of the liver and kidney. Significant changes in classical enzymes such as ALT, AST and creatinine  exclusively, as well as GSH, SOD, GPx and CAT suggest liver and renal impairment since these are reliable indices of liver and kidney toxicity²⁹. The protective effects due to treatment with colchicine strongly indicated the possibility of the drug to prevent and/or mitigate any leakages of marker enzymes into circulation, condition the hepatocytes to accelerate regeneration of parenchymal cells, and preserve the integrity of the plasma membranes and hence restore these enzymes levels²⁷.

Kidneyplays akeyroletoremovethemetabolicwastessuch ascreatininefrombody,therebyhelpingtomaintainbodyhomeostasis.The persistent oxidative stress biomarkerschanges within  the  kidney  tissue  and  free  radicalgenerationmediatedstressinDIC treated ratsproducingrenaldysfunction  resultinginelevationof creatininelevelsinblood²⁹.Inthepresentstudy,elevatedlevelsofALT, AST and creatinine suggested the occurrence of liver and kidney damages after the administrationofDICtotheratscomparedtonormalrats (table 1).Administrationofcolchicine and/or vitamin C totheDIC-treatedratssignificantlyreducedtheALT, ASTandcreatininelevelsrepresentingthepreventiveactionagainstDIC toxicityonliverandkidney damages.

Freeradicalsmayalsobeformedviatheauto-oxidationofunsaturatedlipidsinplasmaandmembranelipids.The freeradicalproducedmayreactwithpolyunsaturatedfattyacidsincellmembranesleadingtolipid peroxidation.Lipidperoxidationwillinturnresultsinanelevatedproductionoffreeradicals⁴².

Lipid peroxidemediated tissue damage has been observedinDIC treated rats.IthasbeenobservedthatDIC administrationisassociatedwithlipoxygenase-derived peroxides⁴³.TheincreasedlipidperoxidationintheDIC-treatedanimalsmaybeduetotheobservedremarkableincreaseinthe concentrationofTBARS and HP(lipidperoxidativemarkers)intheliver and kidney⁶.Hussein⁶hasreportedthattheconcentration oflipidperoxidesincreasesinthetissuesofDIC-treatedrats.Inthepresent study, TBARS level in liver and kidney were significantly lower in the colchicine –treated groups compared to the DIC-treated control group⁴⁴ (tables 2 and 3).  The above result suggests that the colchicine may exert antioxidant activities and protect the tissues from lipid peroxidation. GSH has a multifactorial role in antioxidant defense. It is a direct scavenger of free radicals as well as a co-substrate for peroxide detoxification by glutathione peroxidases. Hussien and Gobba⁴⁵ suggested that the decrease in tissue GSH could be the result of decreased synthesis or increased degradation of GSH by oxidative stress. Increased oxidative stress,  resulting  from  significant  increase  in  aldehydic  products  of  lipid  peroxidation  has probably decreased hepatic GSH content. In the present study, the elevation of GSH levels in liver and kidney were observed in the colchicine-treated rats.

Thisindicatesthatcolchicine caneitherincreasethe biosynthesisofGSHorreducetheoxidativestressleadingtolessdegradationofGSH,orhave botheffects.SODhasbeenpostulatedasoneofthemostimportantenzymesintheenzymatic antioxidantdefensesystemwhichcatalysesthedismutationofsuperoxideradicalstoproduce H₂O₂ andmolecularoxygen⁴⁵,hencediminishingthetoxiceffectscausedbytheirradical.TheobserveddecreaseinSODactivitycouldresultfrominactivationbyH₂O₂.

The superoxide anion has been known toinactivateCAT,which involved inthedetoxificationofhydrogenperoxide⁴⁶. Catalase (CAT) is a heme protein which catalyses the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radicals⁴⁷. GPx plays a primary role in minimizing oxidative damage. Glutathione peroxidase (GPx), an enzyme with selenium and Glutathione-s-transferase (GST) works together with glutathione in the decomposition of H₂O₂ or other organic hydroperoxides to non-toxic products at the expense of reduced glutathione⁴⁸.

Inthepresentstudy,increased liver and renal SOD, GPx, CATandGSHlevels(tables 4 and 5) aswellasreducedTBARS and HP levels werenoticedinDIC-treatedratsaftertheadministrationofcolchicine and or vitamin C.  The   above action represents the antioxidant property of colchicineinDIC-treated animals due its structure property. The structural requirement considered essential for effective radical scavenging by colchicine is the presence of P-dimethoxy groups at carbon number 1 and 2 in a ring and conjugated double bond. The presence of double bond in A ring makes the electrons more delocalized to form quinone structure which possesses electron donating properties and is a radical target²⁷ (Scheme 1).  This important property may be responsible for its antioxidant and hepato-renal protective activity against DIC-induced toxicity.

SCHEME 1: PROPOSAL MECHANISM OF COLCHICINE ANTIOXIDANT ACTIVITY²⁷

Amelioration of colchicine against DIC-induced hepato-renal toxicityhasnotbeenreportedearliertoourknowledge,andthisstudyisperhapsthefirstobservationofitskind.

CONCLUSIONS: Thepresentstudyshowedthat colchicine possessespotentantioxidantactivityandhasanabilityto preventDIC-induced tissue injury.Furtherstudiesareinprogresstogivescientificevidencetothemedical use of colchicine inthetreatmentofother oxidative stress induced complication models.

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How to cite this article:

Boshra SAand Md Hussein A: The Protective Role of Colchicine on Diclofenac Sodium Induced Hepatorenal Toxicity in Albino Rats Model. Int J Pharm Sci Res2014; 5(12): 5136-44.doi: 10.13040/IJPSR.0975-8232.5 (12).5136-44.

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