Vol 8, Issue 3, 2015 ISSN - 0974-2441 IMPACT OF ANTIOXIDANT SUPPLEMENTATION ON TOXICITY OF METHOTREXATE: AN IN VITRO STUDY ON ERYTHROCYTES USING VITAMIN E MANASVEE DHANESHA, KANCHANLATA SINGH, MUSTANSIR BHORI, THANKAMANI MARAR* School of Biotechnology & Bioinformatics, D. Y. Patil University, Navi Mumbai - 400 614, Maharashtra, India. Email: dr.marar@yahoo.com Received: 20 March 2015, Revised and Accepted: 01 April 2015 ABSTRACT Objective: Methotrexate (MTX) is an antimetabolite used widely in cancer and autoimmune diseases as an inhibitor of the enzyme dihydrofolate reductase. Treatment regimens with MTX lead to the formation of reactive oxygen species and are involved in drug-induced toxicity presenting severe side effects. In the present investigation, red blood cells were used as a model to understand drug-mediated hemolysis and alterations in antioxidant defense system stimulated by MTX. This effort attempts to evaluate, whether pre-supplementation of vitamin E can modulate drug induced toxicity. Methods: Blood samples from healthy subjects were collected and processed to obtain 10% RBC solution in saline. This solution was further treated with 80µM MTX in presence and absence of 90µM vitamin E respectively. Treated and control RBC solutions were used to assess percent hemolysis, levels of lipid peroxides, GSH, lactate dehydrogenase and activities of specific antioxidant enzymes. Results: Our study reveals a significant increase in lipid peroxides, reduced glutathione GSH reductase activity after incubation with MTX, lactate dehydrogenase, and a considerable decline in catalase, superoxide dismutase, GSH peroxidase, GSH S-transferase, and GSH reductase after incubation with MTX. Conclusion: Ameliorative effect of vitamin E supplementation reduces oxidative stress and restores the activities of these antioxidant enzymes, thereby demonstrating the protection rendered by vitamin E. Our data indicates that vitamin E administration during chemotherapy is effective in modulating the chemotherapy-induced side effects thus stressing on the importance of vitamin E supplementation in combination with chemotherapy during cancer treatments. Keywords: Antioxidant, Lipid peroxides, Superoxide dismutase, Glutathione, Catalase, Red blood cell. INTRODUCTION There is incomplete evidence whether antioxidant supplementation alters or interferes with the efficacy of cancer chemotherapy. There are arguments that antioxidants scavenge the reactive oxygen species (ROS) integral to the activity of certain chemotherapy drugs, thereby diminishing treatment efficacy. Counter arguments suggest antioxidants may mitigate drug-induced toxicity and thus allow for uninterrupted treatment schedules and a reduced need for lowering chemotherapy doses. Methotrexate (MTX) (4-amino-10-methylpteroylglutamic acid) is a potent antineoplastic agent used to treat choriocarcinoma, leukemia, osteosarcoma, non-Hodgkins lymphoma, breast cancer, and lung cancer [1]. It is also involved in the treatment of non-cancerous conditions such as rheumatoid arthritis, psoriasis, immunological abnormalities, and systemic inflammation [2-4]. The primary mechanism of action is the depletion of reduced folates as a consequence of dihydrofolate reductase inhibition, an enzyme involved in DNA synthesis and DNA repair [5,6]. Low-moderate to high doses of MTX causes various side effects and may lead to conditions such as liver cirrhosis or fibrosis [7]. It has also been shown that MTX administration has severe side-effects on the hematopoietic system [8]. MTX induces oxidative stress by increasing ROS production which is implicated in tissue injury. Further, associated increase in oxidative stress may play an important role in the pathophysiology of drug-induced side effects [9,10]. α-Tocopherol (vitamin E) is a potent lipid-soluble chain-breaking antioxidant preventing the propagation of free radical reactions in cell membranes [11]. Increased serum vitamin E levels have been reported to decrease lipid peroxidation (LPX), inhibit protein kinase C, 5-lipoxygenase, smooth muscle cell proliferation, platelet aggregation, and the oxygen burst in neutrophils [12,13]. Pre-treatment with vitamin E has been reported to prevent many changes in serum enzymes and protect increase in hematocrit, fall in leukocyte count, hemoglobin level, mean osmotic fragility of erythrocytes [14]. Vitamin E is also been reported to induce apoptosis in experimental tumor lines, increase the survival time in a terminal cancer patient, and is shown to increase the activity certain drugs in vivo [15]. Our study is an attempt to modulate MTX induced side effects by the use of vitamin E as an antioxidant and in addition promoting supplementation of vitamin E in chemotherapy thereby aiding to diminish the consequences of drug toxicity by restoring the activities of antioxidant enzymes, levels of non-enzymatic antioxidants and inhibiting LPX in normal cells using red blood cells (RBCs) as a model system (in-vitro studies). METHODS MTX, glutathione (GSH) reduced, GSH reductase (GRD), oxidized GSH, nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Sigma, St. Louis U.S.A. The other chemicals used were of high analytical grade and solvents were of Qualigen grade. Preparation of RBC A volume of 10 ml blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes from healthy volunteers after informed consent. The blood was centrifuged at 3000 rpm for 10 minutes and the plasma along with a buffy coat was removed. The RBCs were washed thrice with phosphate buffered saline (PBS), pH 7.4 and the tubes were centrifuged at 3000 rpm for 10 minutes. The RBC pellet obtained after the removal of the supernatant was diluted to 10% using PBS and was used to set up the experiments. Research Article