Article
Resveratrol ameliorates methotrexate-induced hepatotoxicity in rats via inhibition of lipid peroxidation
Human and Experimental Toxicology 32(6) 662–671 ª The Author(s) 2013 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327112468178 het.sagepub.com
S. Dalaklioglu1, G.E. Genc2, N.H. Aksoy3, F. Akcit4 and S. Gumuslu2
Abstract Hepatotoxicity is one of the major complications of methotrexate (MTX) therapy. This study was carried out to evaluate the possible protective effect of resveratrol (trans-3,5,40 -trihydroxystilbene, RVT) against MTX-induced hepatotoxicity. Rats were randomly divided into four groups as control, MTX treated (7 mg/kg/day, intraperitoneally (i.p.), once daily for 3 consecutive days), MTX þ RVT treated (20 mg/ kg/day, i.p.), and RVT treated. First dose of RVT was administrated 3 days before the MTX injection and continued for 3 days. Histopathology of liver was evaluated by light microscopy. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were used as biochemical markers of MTX-induced hepatic injury. The levels of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation) and activities of hepatic antioxidant enzymes such as catalase (CAT) and glutathione-S-transferase (GST) were used to analyze the oxidative stress-mediated lipid peroxidation in liver sections. Our results showed that MTX administration significantly increased ALT, ASP, and ALP levels. TBARS, CAT, and GST levels were also markedly increased in liver after MTX administration. RVT treatment significantly prevented MTX-induced hepatotoxicity, as indicated by AST, ALT, and ALP levels and liver histopathology. Moreover, administration of RVT significantly decreased the elevated levels of TBARS and activities of CAT and GST in the liver compared to MTX-treated group. These results revealed that RVT may have a protective effect against MTX-induced hepatotoxicity by inhibiting oxidative stress-mediated lipid peroxidation. Consequently, RVT treatment might be a promising strategy against MTX-induced hepatotoxicity. Keywords Resveratrol, methotrexate, hepatotoxicity, lipid peroxidation
Introduction Methotrexate (MTX), a folat antagonist, is commonly used in the treatment of many different types of cancer and inflammatory diseases.1–3 In spite of the widespread use of MTX, its efficacy is often limited by the occurrence of hepatotoxicity which is one of the major complications of MTX treatment.4 Although the mechanism of hepatotoxicity of MTX is not yet completely unknown, considerable experimental and clinical evidence support the hypothesis that MTXinduced hepatic damage may be a consequence of oxidative stress.5,6 MTX administration resulted in increased production of reactive oxygen species
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Department of Pharmacology, Medical Faculty, University, Antalya, Turkey 2 Department of Biochemistry, Medical Faculty, University, Antalya, Turkey 3 Department of Pathology, Ataturk Government Antalya, Turkey 4 Department of Biochemistry, Elmali Government Elmali, Antalya, Turkey
Akdeniz Akdeniz Hospital, Hospital,
Corresponding author: Selvinaz Dalaklioglu, Department of Pharmacology, Akdeniz University, Antalya 07070, Turkey. Email:
[email protected]
Dalaklioglu S et al.
(ROS), leading to damage of macromolecules and trigger many pathological processes.7,8 There are antioxidant defense mechanisms in the liver to neutralize the deleterious effects of free radicals. These antioxidant enzymes are the first line of defense systems against the cell damaging effects of oxidative stress.9 Thus, the generation of ROS in liver tissue might be indirectly evaluated by measuring some antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST).10–12 Natural antioxidants in foods are being used as a comprehensive supplement against drug-induced toxic effects, thereby enhancing the therapeutic efficacy of the drug.13,14 Resveratrol (trans-3,5,40 -trihydroxystilbene, RVT) is a dietary polyphenol that has been shown to protect against nephrotoxicity,15 cardiotoxicity,16 and neurotoxicity17 due to its antioxidant and anti-inflammatory features. Several studies have also highlighted the hepatoprotective properties of RVT against alcohol exposure and acetaminopheninduced hepatotoxicity in mice.18,19 RVT provides protection against hepatic damage induced by free radicals and inflammatory cytokines and attenuates hepatic lipid peroxidation.20,21 It has been shown that MTX-treated animals have exaggerated oxidative stress leading to cellular dysfunction in liver tissue.5,6 However, it is unclear whether the hepatocellular toxicity induced by MTX can be prevented by RVT administration. Also, little is known about the mechanisms involved in the hepatoptotective effect of RVT in rat liver. Taking the above into account, RVT may attenuate MTXinduced hepatotoxicity due to its antioxidant properties. The aim of this study was to evaluate whether RVT is a promising treatment approach for preventing MTX-induced hepatotoxicity. To evaluate the mechanisms underlying its hepatoprotective effects, thiobarbituric acid reactive substances (TBARS) were measured as an indicator of lipid peroxidation. Additionally, activities of CAT and GST were evaluated to determine the antioxidant defense system response to RVT treatment.
Materials and methods Experimental procedures This study was approved by the Animal Ethics Committee of Akdeniz University Medical Faculty, Antalya, Turkey. Briefly, 24 male Wistar rats aged 3 months, weighing 250–300 g, were used in this
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study. All animals were housed under the standard animal laboratory conditions (12-h lighting cycle, controlled temperature, and humidity). Animals were divided into four groups consisting of six animals per group. Control group (C, n ¼ 6) received only intraperitoneally (i.p.) physiological saline of 1 mL/kg per day. MTX-treated group (MTX, n ¼ 6) received three consecutive daily i.p. injections of MTX at the dose of 7 mg/kg/day. MTX þ RVT-treated group (MTX þ RVT, n ¼ 6) was treated with RVT (20 mg/kg per day, i.p.) alone for 3 days and then concurrently administered with MTX for 3 days. Finally, RVT group (n ¼ 6) received only RVT at the same dose. Six sets of independent experiments (using one animal per treatment group per set) were performed in this study. Doses and route of administration of MTX and RVT were chosen on the basis of previously reported data.22–25 In this study, we administered RVT by i.p. injection to ensure that all animals received the exact same dose of the product. All rats were weighed and anesthetized with a cocktail of ketamine hydrochloride (90 mg/kg) and xylazine (10 mg/kg) administered i.p. before killing each rat. Blood samples were obtained from the renal vein, 24 h after the last MTX injection. Thereafter, serum was separated by centrifugation at 2000g for 5 min at 4 C for the determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities. The livers were quickly removed and weighed. All tissues were stored at 80 C until biochemical analysis.
Histopathological analysis The livers were fixed in 10% formalin and processed routinely for paraffin embedding. Paraffin-embedded tissue samples were cut into 5-mm thick sections, mounted on SuperFrost Plus slides (Erie Scientific Company, Portsmouth, New Hampshire, USA), stained with hematoxylin and eosin. Microscopic scoring was done by experienced pathologists, who were unaware of the treatments. Severity of hepatic injury was semiquantitatively evaluated by Zeiss Axioplan microscope using the following alterations: (a) pyknotic nucleus and perinuclear vacuolization of hepatocytes, (b) vascular congestion in sinusoids and sinusoidal dilatation, and (c) number of activated Kupffer cells. Scores were given as 0 ¼ absent, 1 ¼ weak, 2 ¼ moderate, and 3 ¼ strong for each criteria. The microscopic score of each tissue was calculated as the sum of the scores given to each criterion, and
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at least five microscopic areas were examined under a 20 objective to score each specimen.
AST, ALT, and ALP activities Plasma concentrations of the liver enzymes ALT and AST were determined using serum samples obtained from all groups of rats. Activities were expressed as unites per liter. The measurements were done in accordance with the methods of the diagnostic kits (Bt Products, Izmir, Turkey).
Preparations of tissue homogenates Tissues were quickly removed and washed in icecold normal saline (0.9% NaCl), dried on filter paper, and weighed. Liver tissues were homogenized in cold potassium phosphate buffer (50 mM, pH 7.4). All tissues were rapidly sonicated using a thermally regulated sonicator for 1 min. Homogenate was divided into two portions. The first portion of the homogenate was centrifuged at 700xg for 20 min at 4 C in order to determine the CAT activity and TBARS level. The second portion of homogenate was centrifuged at 20,000xg for 30 min at 4 C in order to determine the GST activity. All assays were performed in duplicate.
CAT activity
Human and Experimental Toxicology 32(6)
5 min with 3.5 mL of n-butanol. After centrifugation at 3500g for 10 min, the butanol phase was separated and the fluorescence of the butanol extract was measured using a spectrofluorometer (Shimadzu RF5000, Kyoto, Japan) set at wavelengths of 525 nm for excitation and 547 nm for emission. TBARS levels were expressed as nmol/mg protein in liver tissues. The protein content of samples was determined using the colorimetric method of Lowry,29 using BSA as the standard.
GST activity GST (EC 2.5.1.18) activity was measured at 30 C, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate.28,30 GST was assayed in 1 mL reaction volumes containing 100 mL of CDNB (10 mM), 100 mL of reduced glutathione (GSH; 10 mM), 725 mL buffer potassium phosphate (0.1 M, pH 7.5), and 75 mL supernatant. Reaction was initiated by the addition of CDNB and the formation of S-2,4-dinitrophenyl glutathione (DNPG) was monitored as the increase in absorbance. The ultraviolet absorbance of the conjugated product was measured at 340 nm. The specific activity of GST was calculated as 1 mmol DNPG formed per mg protein in 1 min. All enzyme activities are expressed per mg of tissue protein.
CAT activity was measured by the method of Aebi,26 using hydrogen peroxide (H2O2) as the substrate. The final volume of each enzyme assay was 1.5 mL containing 0.5 mL of 30 mM H2O2 and 1.0 mL supernatant. The decomposition of H2O2 can be correctly followed by monitoring the decrease in absorbance at 25 C and 240 nm for 30 s in spectrophotometer. Enzyme activity was expressed as k/g protein (k: rate constant of the first-order reaction).
Drugs and chemicals
TBARS level
GraphPad Prism 3.0 (GraphPad Software, San Diego, California, USA) was used for statistical analysis. All values are expressed as mean + standard error of the mean. Statistical analysis of the results was performed by one-way analysis of variance. Post hoc comparisons were done using Tukey’s multiple comparison test. A p value
