0012-4966/05/0910- © 2005 Pleiades Publishing, Inc. 0410 Doklady Biological Sciences, Vol. 404, 2005, pp. 410–412. Translated from Doklady Akademii Nauk, Vol. 404, No. 5, 2005, pp. 704–706. Original Russian Text Copyright © 2005 by Solov’eva, Solov’ev, Faskhutdinova, Kudryavtsev, Akatov, Chailakhyan. The antioxidant properties of thiol-containing com- pounds are well known [1]. It is established that antiox- idants combined with metals of variable valence may form prooxidant catalytic systems. An example of such a system is ascorbate combined with organic com- plexes of copper or cobalt, such as vitamin B 12b or cobalt phthalocyanines, which were used as antitumor agents during the past years [2–7]. However, it is unclear how thiols may serve as prooxidants in combi- nation with vitamin B 12b . Antioxidant therapy based on vitamin–mineral complexes is widely used; hence, the study of the prooxidant effect of the combination of thi- ols and vitamin B 12b is of considerable importance. In this work, we show that thiol antioxidants com- bined with vitamin B 12b form a prooxidant catalytic system that generates hydrogen peroxide and synergis- tically induces oxidative stress and apoptotic cell death of tumor cells. Epidermoid carcinoma of larynx (HEp-2) cells obtained from the All-Russian Collection of Cell Cul- tures (Institute of Cytology, Russian Academy of Sci- ences, Saint-Petersburg) were grown on DME (Sigma) containing 10% fetal calf serum (HyClone), 80 μ g/ml gentamicin, 2.2 g/l sodium bicarbonate and maintained at 37°C and 5% CO 2 . The substances studied were added 24 h after plating 5000 cells/well in a 96-well microplate. The chelators of iron ions deferoxamine and phenanthroline (Sigma) were added 2 or 0.5 h before the addition of B 12b combined with GSH, NAC, and DTT (MPBiomedicals). Catalase and pyruvate (Sigma) were added immediately before these sub- stances. The cells were stained using crystal violet and studied by means of a photometer to assess the cyto- toxic effects of these agents [8]. Cytotoxicity was assessed by the ratio between the amount of living cells in the experiment and control 48 h after the 2-h incuba- tion of the cells with the agents studied. The aberrant distribution of chromatin, which is a sign of apoptotic cell death, was assessed with the use of luminescent microscopy using 1 μ g/ml Bisbenzim- ide H33342 and ethidium bromide (Sigma). Double staining determines aberrant distributions of chromatin in living and dead cells (green and red luminescence, respectively) [9]. The existence of apoptotic cells was assessed by the presence of particles in sub-G 1 region on DNA-cytograms obtained by means of flow cytom- etry using a PARTEC III cytometer [10]. In addition, apoptosis was assessed by DNA fragmentation with the use of electrophoresis in 1.5% agarose gel [11]. The concentration of hydrogen peroxide in the medium was measured using polarographic method by oxygen concentration after the addition of catalase. The agents studied were added 24 h after the plating 350 000 HEp-2 cells in T25 flasks. The calibration was performed by addition of H 2 O 2 into the experimental chamber after catalase [7]. The oxidative activity of the cells was assessed with the use of the fluorescent probe 2',7'-dichlorofluores- cein diacetate (DCHFDA, Molecular probes). The cells were plated in T25 flasks (350 000 cells per 5 ml of cul- ture medium). B 12b and NAC were added a day after plating. After 2 h of incubation, the cells were detached by trypsinization, resuspended into fresh culture medium, and stained with 20 μ M DCFHDA for 30 min. Then, the cells were washed from the dye and resus- pended at a concentration of 500 000 cells/ml in cold PBS. The fluorescence was recorded using a PARTEC III flow cytometer [12]. We found that the thiols studied (GSH, NAC, and DTT) had a synergistic cytoxic effect in combination with vitamin B 12b . Addition to the cell cultures of the combinations of thiols and B 12b for 2 h suppressed cell proliferation in a dose-dependent manner and initiated cell death (Fig. 1). The death of cells exposed to the toxic combinations for 2 h began 6 h later and reached 95% by the end of the first day. Vitamin B 12b (as much as 3 mM) or thiols (10 mM or more) added separately were not toxic for HEp-2 cells after a 2-h incubation. A similar synergism of the cytotoxic effect of thiols in combination with vitamin B 12b was also found during permanent exposure of the cells to the agents. The cell death induced by combinations of thiols with B 12b was accompanied by signs typical of apopto- sis [11]. A study of cell death type according to mor- phologic property showed an increase in the number of CELL BIOLOGY Prooxidant and Cytotoxic Effects of Thiols Combined with Vitamin B 12b M. E. Solov’eva, V. V. Solov’ev, A. A. Faskhutdinova, A. A. Kudryavtsev, V. S. Akatov, and Corresponding Member of the RAS L. M. Chailakhyan Received June 2, 2005 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142292 Russia