J Basic Clin Physiol Pharmacol 2012;23(4):163–168 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/jbcpp-2012-0020 An in vitro study of the ameliorative role of α-tocopherol on methotrexate-induced oxidative stress in rat heart mitochondria Kanchanlata Singh, Abha Malviya, Mustansir Bhori, and Thankamani Marar* Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai, Maharashtra, India Abstract Background: Methotrexate (MTX) is a folic acid antago- nist widely used as a cytotoxic chemotherapeutic agent for many malignancies. Its use is limited due to wide-ranging toxicities. MTX generates reactive oxygen species. Here, we investigated the efficacy of vitamin E supplementation on MTX-induced alterations in vitro. Methods: Rat heart mitochondria were isolated and used to assess the extent of swelling, lipid peroxidation and altera- tions in mitochondrial-specific enzyme activities caused by the addition of 80 μM MTX in the presence and absence of 1.2 μM vitamin E. Control for both groups was maintained. Result: MTX substantially affects mitochondrial function by increasing lipid peroxidation and mitochondrial swelling. Significant losses in the activities of the tricarboxylic acid cycle enzymes, complex I, II and IV, and an increase in the activity of calcium ATPase were observed in MTX-treated rat mitochondria. Enrichment of the media with vitamin E led to a reduction in swelling and restoration of enzyme activities. Conclusions: The present study suggests that vitamin E plays a vital role in suppressing MTX-induced mitochondrial toxi- city, and affords protection either by reversing the decline of antioxidants or by direct scavenging of free radicals. Keywords: antioxidant; dehydrogenases; lipid peroxidation; methotrexate; mitochondria; reduced nicotine adenine dinu- cleotide (NADH). Introduction Investigation on the effects of chemotherapeutic drugs on membrane functions in biological systems may help in explaining their toxic nature as well as providing a tool for elucidating membrane properties and function (1). The interaction of drugs with subcellular organelles in vitro has provided limited but valuable data in this respect. The mitochondrial permeability transition (MPT) is consid- ered to contribute substantially to the regulation of normal mitochondrial metabolism and is an important mediator of cell death (2). All the major MPT functions in normal mitochondria can be determined by simple mitochondrial swelling assay under well-defined conditions, thereby suggesting its application in therapeutic diagnostics (1). Swelling phenomena have frequently been used to study structural alterations of mitochondria in relation to bio- chemical properties. Methotrexate (MTX) is a potent, stoichiometric inhibitor of dihydrofolate reductase, an enzyme involved in cellular DNA synthesis. MTX has previously been reported to gen- erate reactive oxygen species (3). In vitro swelling char- acteristics of heart mitochondria were investigated using MTX as a swelling agent. Vitamin E, a potent lipid-soluble antioxidant in biological systems with the ability to directly quench free radicals, is known for its beneficial antioxi- dant effects for a number of chronic diseases, including cancer (4). Increased vitamin E levels have been reported to decrease lipid peroxidation, inhibit protein kinase C, 5-lipoxygenase, smooth muscle cell proliferation, plate- let aggregation and the oxygen burst in neutrophils (5, 6). In the present experiment, vitamin E was used as an anti- oxidant and membrane stabilizer. The changes in enzyme activity and generation of lipid peroxides were assessed to study the mitochondrial toxicity of the drug. Lipid peroxi- dation, activities of the tricarboxylic acid cycle enzymes, complex I, II and IV enzymes and calcium (Ca 2+ ) ATPase were estimated in the presence of MTX with and without vitamin E supplementation. Materials and methods Drugs and chemicals Methotrexate, dichlorophenolindophenol (DCPIP), 5,5 ′-dithio-bis 2-nitrobenzoic acid, reduced nicotine adenine dinucleotide (NADH), nicotine adenine dinucleotide, adenosine triphosphate (ATP) and cytochrome c were purchased from Sigma Chemical Company (St. Louis, MO, USA). Other reagents were obtained from Merck India, Qualigen fine chemicals, Mumbai and Sisco Research Labora- tory Private Limited (Mumbai, India). *Corresponding author: Thankamani Marar, Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai, Maharashtra, India Phone: +91 2227563600, Fax: +91 2239286176, E-mail: dr.marar@yahoo.com Received May 31, 2012; accepted August 22, 2012; previously published online October 12, 2012 Authenticated | dr.marar@yahoo.com author's copy Download Date | 2/13/13 6:24 AM