The Protective Effect of α-Tocopherol and \ or N-acetylcysteine against Methotrexate-Induced Hepatotoxicity in Chronically-Treated Patients with Rheumatoid Arthritis : Clinical Study

Long-term methotrexate (MTX) administration has the potential to cause hepatotoxicity that is claimed to be due to decrease the folate levels and/or oxidative stress. Objectives: To study the protective effect of α-tocopherol and\or N-acetylcysteine against methotrexate-induced hepatotoxicity in chronically-treated patients with rheumatoid arthritis. Subjects and methods: The study was carried on 250 subjects, whom were divided into five groups (50 subject each) as follows: Group I: Normal apparently healthy, Group II: Newly diagnosed as rheumatoid patients whom will be treated with MTX alone over one year, Group III: Newly diagnosed as rheumatoid patients whom will be treated with MTX with α-tocopherol over one year, Group IV: Newly diagnosed as rheumatoid patients whom will be treated with MTX with N-acetylcysteine over one year, Group V: Newly diagnosed as rheumatoid patients whom will be treated with MTX with α-tocopherol + N-acetylcysteine over one year. All subjects were investigated for liver function tests (ALT, AST, ALP, GDH) in the beginning of the study and every 3 months of the period of the study. Results: It was found that long-term MTX therapy induced significant elevation of GDH started from 6th month of treatment while the other parameters (ALT, AST & ALP) started to be significantly increased from the 9th month of therapy. Co-administration of α-tocopherol or N-acetylcysteine induced significant improvement of all parameters. Co-administration of both anti-oxidants lead to improvement which was statistically significant more than each drug alone. Conclusion: The co-administration of MTX with α-tocopherol and/or N-acetylcysteine may protect against MTX-induced hepatotoxicity in chronicallytreated patients with rheumatoid arthritis which seems to be multifactorial. INTRODUCTION Since 1950s, low-dose methotrexate (MTX) therapy has been approved as a medical intervention for many inflammatory diseases such as psoriasis, sarcoidosis, systemic lupus and rheumatoid arthritis [1]. However, long-term MTX administration has the potential to cause diverse organ toxicities, including hepatotoxicity [12]. MTX may lead to liver hepatotoxicity, including steatosis, cholestasis, fibrosis, and cirrhosis [3]. Vitamin E is not a single nutrient, but a group of compounds which consists of 4 tocopherol isomers (α-, β-, γand δ-tocopherol) and 4 tocotrienol isomers (α-, β-, γand δtocotrienol), Central Bringing Excellence in Open Access   Younis et al. (2017) Email: J Pharmacol Clin Toxicol 5(2): 1073 (2017) 2/5 and functions as a lipophilic antioxidant that prevents lipid peroxidation.1 Of all the natural isomeric forms of vitamin E, αtocopherol was the most extensively studied, probably because it is the most predominant form in plasma and tissues [4]. Alphatocopherol is a fat-soluble antioxidant which is located within the phospholipid bilayer of cell membranes to prevent formation or limit the effects of free radicals. The major biologic role of vitamin E is to protect poly unsaturated fatty acids (PUFAs), low-density lipoprotein (LDL) and other components of cell membrane from oxidation by free radicals. Alpha-tocopherol acts as a peroxyl radical scavenger, disabling the production of damaging free radicals in tissues, by reacting with them to form a tocopheryl radical, which will then be reduced by a hydrogen donor (such as vitamin C) and thus return to its reduced state [5]. N-acetylcysteine (NAC) is a well-established cytoprotective drug that has proven efficacy against drug acetaminophen induced hepatotoxicity [6]. NAC exerts effective antioxidant and anti-inflammatory functions as it acts as an acetylated precursor of glutathione (GSH) and by inhibiting H2O2 formation [7]. The current study was designated to study the protective effect of α-tocopherol and\or N-acetylcysteine against methotrexate-induced hepatotoxicity in chronically-treated patients with population rheumatoid arthritis. SUBJECTS AND METHODS The current study was carried out at Minia university Hospitals, Minia city, Egypt (longitude 29.79°, latitude 28.25°) during the period from January, 1St, 2016, to December, 31st, 2016. The study was approved by the Research Ethical Committee 0f Minia Faculty of Medicine. The study was carried on 250 subjects aging from 25 to 50 years old of both sexes, whom were referred by the rheumatology, rehabilitation and physical medicine department. The patients were divided into five groups as follows (50 subjects each): Group I: Normal apparently healthy subjects. Group II: Newly diagnosed as rheumatoid patients whom will be treated with MTX alone with a starting dose of 7.5 to 10 mg, or 3-4 pills, taken all together once a week [8], over one year. Group III: Newly diagnosed as rheumatoid patients who will be treated with MTX as in group II with α-tocopherol (350 mg/ day) [9] over one year. Group IV: Newly diagnosed as rheumatoid patients whom will be treated with MTX as in group II &III with N-acetylcysteine (600 mg/day) [10] over one year. Group V: Newly diagnosed as rheumatoid patients whom will be treated with MTX with α-tocopherol+N-acetylcysteine in the previous doses over one year. Inclusion\exclusion criteria: The subjects of the study were: All urban. Aging from 25 to 50 years old of both sexes. Body mass index: 18.5 – 24.9 [11]. Newly diagnosed (according to the revised criteria of the American College of Rheumatology (ACR) in 1987(Arnett and others 1988) and reclassified according to ACR/European League Against Rheumatism (EULAR) criteria in 2010 [12]. No other immunologic or systemic diseases. No pregnancy or lactation. No previous therapy for rheumatoid arthritis or any disease (hypertension, DM, etc). All subjects were investigated for liver function tests: Alanine transferase (ALT), Aspartate transferase (AST) and alkaline phosphatase (ALP) in the beginning of the study and every 3 months of the period of the study. Biochemical investigations Liver function tests: Alanine transferase and Aspartate transferase (ALT and AST): were measured spectrophotometrically using Spekol II Carl-Zeiss spectrophotometer [13,14]. Serum alkaline phosphatase level (ALP) measured by colorimetric method according to Donald & Ralph, 1993 [15]. Glutamate dehydrogenase (GDH): is measured using a Hitachi 917 automated clinical chemistry analyzer (Roche Diagnostics Limited, Lewes, UK). Statistical analysis The collected data was organized, tabulated and statistically analyzed using SPSS software statistical computer package version 10. Data were expressed as Mean + Standard Deviation (SD). ANOVA test was used to compare between the means where probability (P); P < 0.05: was considered significant.