Research paper Effects of N-acetyl-L-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors Aysegul Cort, Evrim Ozdemir, Mujgan Timur, Tomris Ozben * Department of Biochemistry, Faculty of Medicine, Akdeniz University, Dumlupinar Blv., 07070 Antalya, Turkey article info Article history: Received 5 July 2012 Accepted 18 August 2012 Available online 24 August 2012 Keywords: Bleomycin N-Acetyl-L-cysteine Oxidative stress Reactive oxygen species Testicular cancer abstract Testicular cancer is a very common cancer in males aged 15e44 years. Bleomycin is used in chemo- therapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-L-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleo- mycin and compared the results with H 2 O 2 which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8- isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H 2 O 2 significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroper- oxide levels in NTera-2 and NCCIT cells. Bleomycin and H 2 O 2 significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8- isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H 2 O 2 . NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H 2 O 2 . Ó 2012 Elsevier Masson SAS. All rights reserved. 1. Introduction Testicular cancer is a common cancer in males aged 15e44 years. An estimated 8400 new cases of testicular cancer annually are diagnosed in the USA, accounting for <1% of all cancers in the country. The number of diagnosed cases has gradually increased [1], and the current frequency of tumor incidence is 50% higher than it was 30 years ago. However, the causes of this increase remain unclear [2]. Testicular germ cell tumors (TGCTs) represent the majority of testicular tumors (>95%). Men with TGCTs have a higher risk of developing a subsequent tumor. The incidence of TGCTs in the general population is z0.005%, and a second primary contralateral testis tumor may occur in up to 5% of men with a prior tumor [3]. Bleomycin is a glycopeptide isolated from Streptomyces verti- cillus in 1966 [4] which exhibit antibiotic properties. It is commonly included in chemotherapy regimens used to treat patients with Hodgkin’s or non-Hodgkin’s malignant lymphoma [5] and germ- cell tumor [6]. Its therapeutic use is limited due to dose- dependent lung toxicity that eventually leads to lung fibrosis [7]. Bleomycin binds to and cleaves DNA in a reaction that depends on the presence of ferrous ion and molecular oxygen [8]. The action mechanisms of bleomycin involve the following three steps: (i) recognition of a particular base or base sequence on a DNA double strand, (ii) activation of the substrate or molecular oxygen under the reaction conditions, and (iii) oxidation reactions leading to DNA strand scission [9]. Oxygen radicals have been reported to act as one of intracellular second messengers and lead to induction of various proteins through transcriptional activation [10]. Bleomycin induces the highest level of oxidative stress. During DNA strand scission, nucleoside bases, oligonucleotides of various chain lengths, and a low molecular weight compound that reacts with thiobarbituric acid are released [11]. Numerous in vitro studies have demonstrated that a wide range of anticancer agents induce apoptosis in malig- nant cells by generating ROS which is one of the important thera- peutic interventional approaches in cancer. Oxidative stress reduces the rate of cell proliferation, and interferes with the cyto- toxic effects of antineoplastic drugs, which depend on the rapid proliferation of cancer cells for optimal activity. N-acetyl cysteine (NAC), a cysteine derivative, can react directly with reactive oxygen intermediates and acts as a precursor for glutathione synthesis. NAC has been used in the treatment of acute * Corresponding author. Tel.: þ90 242 2496895; fax: þ90 242 2274495. E-mail address: ozben@akdeniz.edu.tr (T. Ozben). Contents lists available at SciVerse ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2012.08.015 Biochimie 94 (2012) 2734e2739