Vitamin E and C protects the liver from diclofenac induced histopathological changes and oxidative damage in experimental animals

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1 Research Article Vitamin E and C protects the liver from diclofenac induced histopathological changes and oxidative damage in experimental animals Siva Thirusangu 1 *, Girija Sivakumar 2, Sankaran Ponnusamy Kasirajan 3, Yuvaraj Maria Francis 4 ABSTRACT Aim: This study was done to show the changes in the liver following diclofenac treatment and to study the hepatoprotective effects of Vitamin E and C in diclofenac treated rats. Materials and Methods: Rats were divided into four groups each six rats. Group-1: Control rats (n = 6), Group-2: Rats (n = 6) treated with diclofenac at dose of 50 mg/kg IM for 7 days, Group-3: Rats (n = 6) treated with Vitamin C at dose of 200 mg/kg orally followed by diclofenac at 50 mg/kg IM 2 h later for 7 days, and Group 4: Rats (n = 6) treated with Vitamin E at dose of 200 IU/kg orally followed by diclofenac at 50 mg/kg IM 2 h later for 7 days. Later it was analyzed with standard biomarkers, and it was histologically interpreted. Results: the results showed that there was an rapid increase in the levels of liver function test in diclofenac treated group, which was significantly decreased after pretreatment with vitamin E than vitamin c. The liver acinus showed centriacinar necrosis of hepatocytes after 7 days of diclofenac treatment, which was prevented by administration of Vitamin E and C. Discussion: Drug-induced liver injury possesses a major clinical problem and has become a leading cause of acute liver failure and transplantation. Overstressed liver compromises its detoxification role which may expose it to a variety of diseases and disorders. Diclofenac sodium is a phenylacetic acid derivative, a widely used nonsteroidal anti-inflammatory drug (NSAID) for the treatment of inflammatory conditions such as osteoarthritis, rheumatoid arthritis, polymyositis, dermatomyositis, dental pain, spondyloarthritis, acute migraine, gout attacks, and pain management in gall and renal stones. Although the exact mechanism by with diclofenac injuries liver is not understood, some studies explain the toxicity by affecting cytochrome P 450 leading to the production of active metabolites. Conclusion: hepatoprotective effects of Vitamin E were better compared to Vitamin C following treatment with NSAID. Hence, it may be necessary to administer Vitamin E in patients treated with diclofenac. KEY WORDS: Diclofenac, Rheumatoid arthritis hepatotoxicity, Vitamin C, Vitamin E INTRODUCTION Diclofenac is a common nonsteroidal antiinflammatory drugs (NSAIDs) with anti-pyritic, analgesic, and anti-inflammatory effects. [1] Which are most frequently used in the treatment of rheumatoid arthritis, musculoskeletal pain management, and various other inflammatory diseases. [2] It belongs to arylalkanoic phenylacetic acid derivative which reduces the prostaglandin secretion by competes with arachidonic acid by binding to oxygenase. [3] Diclofenac is metabolized in liver [4] by glucuronidation to form unstable acyl glucuronide compound which is Access this article online Website: jprsolutions.info ISSN: further oxidized by cytochrome (CYP2C8) enzyme. These CYPC2C8 also catalyze the formation of 5 hydroxy diclofinac which is the active compound. The increased activity of CYPC2C8 leads to accumulation of acyl glucuronide generating oxidative stress and mitochondrial permeability transition to liver causing cell damage. [5,6] Oxidative stress is mediated by reactive oxygen species (ROS) [7] such as superoxide, peroxide, and hydroxyl radicals in liver which is highly unstable compounds causing damage to cells before converting into a water molecule. [8] ROS cause damage to cell integrity through increased lipid peroxidation of polyunsaturated fatty acid [9] leading to hepatotoxicity through direct attack [10,11] on cell function and also indirectly by activating nuclear factors [12] or by alternate protein one. [13] These harmful effects of ROS 1 Department of Anatomy, Bharath University, Chennai, Tamil Nadu, India, 2 Department of Anatomy Karpaga Vinayaga Medical College, Maduranthagam, Tamil Nadu, India, 3 Department of Anatomy, AIIMS, Mangalagiri, Tamil Nadu, India, 4 Department of Anatomy, Saveetha Medical College Hospital Thandalam Chennai, Tamil Nadu, India *Corresponding author: Mr. Siva Thirusangu, Bharath University, Chennai, Tamil Nadu, India. Phone: siva17187@gmail.com Received on: ; Revised on: ; Accepted on:

2 are attenuated by antioxidants such as superoxidase dismutase, catalyze, glutathione peroxidase (GPX), and also by non-enzymatic antioxidants such as Vitamin C and Vitamin E which are powerful scavengers of free radicals. Vitamin C and E act as natural antioxidants by scavenging the ROS thereby ameliorating the effects of drug-mediated oxidative stress in liver cells. Chronic consumption of diclofenac in case of arthritis and musculoskeletal pain can cause drug-induced liver injury; therefore, the present study aimed to investigate the hepatoprotective effects of Vitamin C and E on diclofenac-induced hepatotoxicity in male albino Wistar rats. MATERIALS AND METHODS The study was carried in 24 male albino Wistar male rats weighing g. The animals were housed individually in cages and maintained under standard laboratory conditions (temperature 252 C) 12 h light and 12 h dark cycles with free access to a standard commercial diet and water ad libitum throughout the experimental period. The rats were acclimatized to laboratory conditions for 7 days before commencement of the experiment. All the animals were reviewed and approved by the Institutional Animal Ethical Committee, Saveetha Medical College and Hospital. The rats were administered 150 mg/kg of diclofenac on day 1 leading to the death of rats on the next day. Then, the dosage was reduced to 100 mg/kg which lead to death after 2 days. Further decreasing the dosage to 75 mg/kg lead to death after 5 days and 50 mg/kg administered rats survived throughout 7 days. Experimental method rats were divided into four groups: Group 1: Control rats (n = 6). Group 2: Rats (n = 6) treated with diclofenac at dose of 50 mg/kg i. m for 7 days. Group 3: Rats (n = 6) treated with Vitamin C at a dose of 200 mg/kg orally followed by diclofenac at 50 mg/kg i. m 2 h later for 7 days. Group 4: Rats (n = 6) treated with Vitamin E at a dose of 200 IU/kg orally followed by diclofenac at 50 mg/kg i. m 2 h later for 7 days. The animals were given over anesthesia with an intramuscular injection of ketamine hydrochloride 50 mg/kg and sacrificed by cervical dislocation. The blood samples were taken by a retroorbital puncture for biochemical parameters such as serum glutamic-oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase (ALP), bilirubin, total protein, urea, and creatinine. The liver tissue was dissected and fixed in 10% formalin solution for 24 h and processed through the paraffin embedding technique. Paraffin blocks were cut by rotary microtome in to 5 microns, thick sections which were stained by hematoxylin and eosin for histology preparation. [14] The dissected liver washed with ice-cold saline and a 10% homogenate prepared in phosphate buffer (ph 7.0). The portion of liver homogenate was centrifuged at 3000 rpm for 15 min at 4 C, and the supernatant was used for the estimation of thiobarbituric acid reactive substances (TBARS) [15] and estimation of superoxide dismutase (SOD). [19] ESTIMATION OF LIVER ENZYMES Serum alanine amino transferase (ALT) and serum aspirate amino transfersae (AST) were determined calorimetrically according to method [16] alkaline phosphate level was determined calorimetrically according to method [17] total protein levels were determined calorimetrically according his method. [18] Statistical Analysis Results were statistically analyzed by one way analysis of variance (ANOVA) and Duncan s multiple range tests. Data are presented as mean (standard deviation). Calculations are done using computerized SPSS software version 16. RESULTS The results showed that administration of rats with diclofenac 50 mg/kg/body weight, intramuscularly for 7 days showed a significant increase in liver enzymes as compared to the control group, whereas Group 3 and Group 4 on pre-administration of Vitamin C 200 mg/kg and Vitamin E 200 IU/kg orally for 7 days followed by diclofenac administration showed less significant changes in liver enzymes as compared to control. Histopathological Changes The histopathology of liver sections was seen using light microscopy, and it was photographed. Changes seen in the slides were described in the order, Figure 1 control group shows the normal central vein, hepatocytes, and hepatic sinusoids. Diclofenac (50 mg/kg im) treated rats showing distorted central veins, edematous enlargement of cytoplasm, nuclear degeneration, and centrilobular necrosis of hepatocytes Figure 2. Vitamin C pre-treated 200 mg/kg orally showing the normal arrangement of the central vein, hepatocyte, and hepatic sinusoids with minimal degenerated hepatocytes [Figure 3]. Vitamin E pre-treated 200 IU/ kg orally showing the normal arrangement of the central vein, hepatocytes, and hepatic sinusoids which is seen similar to the control group Figure 4. Biochemical Analysis Values are expressed in mean (Standard deviation) each group contains six rats, P < when compared with 435

3 Figure 3: Vitamin C treated rat showing central vein (yellow arrow), hepatocyte (red arrow), and hepatic sinusoid (green arrow) which is comparable with control-treated rats Figure 1: control treated rats showing central vein (red arrow) and hepatocytes (green arrow) and hepatic sinusoids (blue arrow) Figure 4: Vitamin E treated rat showing central vein (black arrow), hepatocyte (red arrow), and degerating hepatocyte (green arrow). Vitamin E treated rats showing normal appearing hepatocytes and few degenerating hepatocytes b a c Figure 2: (a) Diclofenac-treated rats showing distorted central veins (White arrow) and hepatocyte showing nuclear degeneration and hepatocyte with complete necrosis (Red arrow) indicating centrilobular necrosis. (b) Diclofenac-treated rat (high Power) showing distorted central vein (black arrow) with perivascular necrosis (centrilobular necrosis) and hepatocyte degeneration with swelling and ballooning with pale cytoplasm (orange arrow). (c) Diclofenac-treated rat (higher power) showing portal triad (gray arrow) with lymphocyte infiltration (orange arrow) control group *P < when compared with Group 2 (diclofenac treated). One-way ANOVA and Duncan s multiple range tests. The values of total bilirubin and catalase (CAT) expressed in median (interquartile range) by Kruskal Wallis test show significant P < Post hoc is a multiple comparison test used to calculate the significant difference between the parameters such as (AST, ALT, ALP and Total protein) group 1, 2, 3 and 4.And the mean SD shows P<0.001 which is highly significant. In diclofenac treated rats aspartate aminotransferase (AST), alanine aminotransferase (ALT), levels were elevated significantly (5.31), (5.48), and (9.58) respectively, in compared with control; whereas total protein levels (0.40) were reduced when compared to control. However, in pre-treatment of Vitamin C 200 mg/kg orally showed a slight elevation of AST, ALT, and ASP levels (51.50 (4.03), (2.62), and (3.44), respectively, which is so close to the normal range when compared to control whereas total protein was almost similar to control [Table 1].Moreover, in pre-treatment with Vitamin E 200 IU/kg orally showed a levels within normal limit as such as control AST, ALT, and ASP levels (3.31), (2.25), and (3.61), respectively, P < when compared to diclofenac group. In group 2 diclofenac treated rats showed a significant (p<0.001) decrease in the antioxidant values of SOD, GSH AND GPX 0.70(0.06),11.85(0.58) and 13.94(0.34) respectively. Whereas there was an elevated level of TBARS level 5.33(0.28) seen when compared to control group [Table 2]. After pre-treated with Vitamin C at the dose of 200 mg/kg, significantly reversed the diclofenac-induced changes in the level of SOD, GSH, GPX on 1.43 (0.86), (0.10), and (0.38), respectively, and also with reduced TBAR values 1.43 (0.86). Similar types of findings were observed on pre-treated with Vitamin E at the dose of 200 IU/kg shows significantly reversed the diclofenacinduced changes in the level of SOD, GSH, and GPX and 1.60 (0.08), (0.11), and (0.23), respectively, and also raised TBAR values 3.35 (0.10) which is almost similar to the values of control. DISCUSSION Liver is a vital organ highly concerned with metabolism and detoxification. NSAIDs are

4 Table 1: Changes in serum liver enzymes in rats treated with Diclofenac and protective groups Analysis Group 1 control Group 2 diclofenac Group 3 Vitamin C+DICLO Group 4 Vitamin E+DICLO AST (1.16) (5.31) (4.03) (3.31) ALT (3.48) (5.48) (2.65) (2.25) ALP (2.42) (9.58) (3.44) (3.61) Total protein 8.58 (0.33) 5.05 (0.40) 8.10 (0.46) 8.66 (0.54) Total bilirubin 0.65 ( ) 1.98 ( ) 0.90 ( ) 0.60 ( ) ALP: Alkaline phosphatase, ALT: Alanine aminotransferase, AST: Aspartate aminotransferase Table 2: changes seen in antioxidant enzyme level in rats treated with diclofenac and protective groups Analysis Group 1 control Group 2 diclofenac Group 3 Vitamin C+DICLO Group 4 Vitamin E+DICLO SOD 1.58 (0.11) 0.70*(0.66) 1.43 (0.86) 1.60 (0.80) TBARS 3.23 (0.13) 5.33*(0.28) 3.63 (0.13) 3.35 (0.10) GSH (0.08) 11.58*(0.58) (0.10) (0.11) GPX (0.35) 13.94*(0.34) (0.38) (0.23) CAT 3.60 ( ) 2.16 ( ) 3.20 ( ) 3.76 ( ) CAT: Catalase, GPX: Glutathione peroxidase, GSH: Glutathione, TBARS: Thiobarbituric acid reactive substances, SOD: Superoxide dismutase. P<0.001(All the values are highly significant when compared to control ). commonly used in many diseases specifically for rheumatic disease and joint pain. NSAID drugs such as diclofenac and prolonged consumption of diclofenac lead to hepatotoxicity Bose et al. [19] The liver function is assessed mainly marker enzymes (ALT, AST, and ALP) and the bilirubin levels in serum are long-standing indicator of hepatotoxicity Ore and Olayinka. [20] Therefore, this cellular damage and hepatotoxicity are ameliorated by powerful antioxidants such as Vitamin C and Vitamin E. In this study, we administrated diclofenac 50 mg/ kg/bw for 7 days intramuscularly shows significant P 0001 increased liver marker enzymes such as ALT, AST, and ALP when compared to control. ALT and AST are the high specific markers to encounter hepatocellular damage and loss of functional integrity of cell membrane Amacher et al., [21] these results were highly correlated with a similar study done by Nasir et al. [22] In addition, our results are compatible with El Maddawy and Ibrahim [23] reported administration of diclofenac sodium-induced a significant increase in ALP, AST, ALT, and total bilurubin and significant decrease in total protein values. The metabolites of diclofenac sodium are commonly excreted 65% in urine and 35% in bile [24] these metabolites are toxic to liver and thereby it cause hepatotoxicity Tolman et al. [25] Normally, there are three common metabolites of diclofenac are responsible for hepatotoxicity, namely, 4 hydroxy 3 diclofenac, 5 hydroxy 4 diclofenac, and 5 hydroxy 6 diclofenac. [26,27] Our findings show there was a significant decrease in the value of TBARS, SOD, CAT, GSH, and GPX in diclofenac-treated rats when compared to the control. These results were also reported by Darbar et al. These enzymes are the powerful antioxidants which act as a first line defense in scavenging the free radicals and thereby it prevents the cellular damage Winrow et al. From these results, it is clearly evidenced that some of the metabolites produced by diclofenac are responsible for free radical synthesis and thereby it affects the antioxidants and produces severe oxidative stress in the liver. Khan et al. [28] also reported increased lipid peroxidation and reduced GSH levels are taken as direct evidence for oxidative stress which affects functional as well as membrane level permeability. Our histological findings are also supports the biochemical values and showed significant changes in liver tissues in diclofenac treated rats. On microscopic examination shows that severe periacinar necrosis, moderate hydropic degeneration, fatty changes, and degenerating hepatocytes similar changes were also noted in the administration of diclofenac 50 mg/kg/bw by Nasir et al. In our study, pre-treatment was done with Vitamin C and Vitamin E reversed these changes. Both biochemical and histological results show significant hepatoprotective effects on diclofenac treated rats. There were a merely changes in biochemical values almost within the normal limits; however, there were no significant changes noted in Vitamin C groups when compared to control. Histological evidence shows almost normal hepatocytes with the absence of necrosis on both pre-administration of Vitamin C and Vitamin E. From these results, it is evidenced that preadministration of Vitamin C and Vitamin E ameliorates the effect of diclofenac on liver cells and thereby it protects the cellular damage and hepatotoxicity from metabolites produces NSAIDs free radicals. Therefore, free radical scavenging and its protective effects are more in Vitamin E when it is compared with Vitamin C. Hence, Vitamin E is highly useful for the clinicians to overcome the adverse effects of NSAIDs as it is high on routine usage by rheumatoid patients and joint pains. 437

5 CONCLUSION To summarize, the liver function markers (ALP, AST, ALT, and BR), serum electrolytes were found to be altered in diclofenac-induced hepatotoxicity rats. Antioxidants (SOD, CAT, GPX, GST, and glutathione reductase) and histopathological changes were also found to be altered due to diclofenac-induced toxicity. Treatment with the effective dose (200 mg/kg b.wt) of Vitamin E and C significantly improved the altered levels of the biochemical profiles as well as the histopathological changes caused by diclofenac. The present findings are the first to report to show the protective role of Vitamin E against diclofenac-induced hepatotoxicity. Hence, it is concluded from the present findings that Vitamin E hepatotoxicity exhibit role through the restoration of serum biochemical profiles and histopathological changes as well as antioxidant enzymes in the liver tissue of diclofenac-induced hepatotoxicity in male rats. CONFLICTS OF INTEREST There are no conflicts of interest. REFERENCES 1. Brunton LL, Lazo JS, Parker KL. Goodman and Gilman s. The Pharmacological Basic of Therapeutics. 11 th ed. New York: McGraw-Hill Medical Publishing Division; p Simon LS. Actions and toxic effects of the nonsteroidal antiinflammatory drugs. Curr Opin Rheumatol 1994;6: Small RE. Diclofenac sodium. Clin Pharm 1989;8: Mazumdar K, Dutta NK, Dastidar SG, Motohashi N, Shirataki Y. Diclofenac in the management of E. coli urinary tract infections. In Vivo 2006;20: Aithal GP. Diclofenac-induced liver injury: A paradigm of idiosyncratic drug toxicity. Expert Opin Drug Saf 2004;3: Mitchell JR, Jollow DJ, Potter WZ, Davis DC, Gillette JR, Brodie BB, et al. Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J Pharmacol Exp Ther 1973;187: Lushchak VI. Free radicals, reactive oxygen species, oxidative stress and its classification. Chem Biol Interact 2014;224: Wu D, Cederbaum AI. Alcohol, oxidative stress, and free radical damage. Alcohol Res Health 2003;27: Hickman I, Macdonald G. Is Vitamin E beneficial in chronic liver disease? Hepatology 2007;46: Kaplowitz N. Mechanisms of liver cell injury. J Hepatol 2000;32: Sies H. Biochemistry of oxidative stress. Angew Chem Int Ed Engl 1986;25: Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994;12: Karin M, Liu ZG, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997;9: Harries ML. Carleton s Histo-Pathological Technique. 5 th ed. New York, Toronto: Oxford University Press; p Aksoy TN. Beneficial effects of Vitamins C and E against oxidative stress in diabetic rats. Nutr Res 2005;25: Reitman S. Colorimetric method for estimation of GOT/AST and GPT/ALT transaminases. Am J Clin Pathol 1957;28: Kind PR, King EG. Calorimetric determination of alkaline phosphatase. J Clin Pathol 1954;7: Doumas BT, Bayse DD, Carter RJ, Peters T Jr., Schaffer R. A candidate reference method for determination of total protein in serum. I. Development and validation. Clin Chem 1981;27: Bose A. Antioxidant and hepatoprotective activity of ascorbic acid against diclofenac induced hepatotoxicity in rats. Asian J Chem 2009;221: Ore A, Olayinka ET. Fluazifop-p-butyl, an aryloxyphenoxypropionate herbicide, diminishes renal and hepatic functions and triggers testicular oxidative stress in orally exposed rats. Toxicol Ind Health 2017;33: Amacher DE. Serum transaminase elevations as indicators of hepatic injury following the administration of drugs. Regul Toxicol Pharmacol 1998;27: Nasir AS, Jaffat HS. Effect of royal jelly against carbon tetrachloride (CCL4) induced toxicity in male rats. Res J Pharm Technol 2017;10: El-Maddawy ZK, Ibrahim M. Hepato-renal and hematological effects of diclofenac sodium in rats. Glob J Pharmacol 2013;7: Vane JR, Botting RM. Mechanism of action of antiinflammatory drugs. Scand J Rheumatol Suppl 1996;102: Tolman KG. Hepatotoxicity of non-narcotic analgesics. Am J Med 1998;105:13S Brune K, Lindner J. Side effects of anti inflammatory drugs. In: Inflammation and Drug Therapy Series. Vol. 5. Dordrecht, Lancaster: Kluwer Academic Publishers; p Tang W, Stearns RA, Bandiera SM, Zhang Y, Raab C, MP, et al. Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: Identification of glutathione conjugated metabolites. Drug Metab Dispos 1999;27: Khan N, Wilmot CM, Rosen GM, Demidenko E, Sun J, Joseph J, et al. Spin traps: In vitro toxicity and stability of radical adducts. Free Radic Biol Med 2003;34: Source of support: Nil; Conflict of interest: None Declared 438