Tertiary selenoamide compounds are useful superoxide radical scavengers in vitro

European Journal of Pharmaceutical Sciences 23 (2004) 207–211

Tertiary selenoamide compounds are useful superoxide radical scavengers in vitro Hitoe Takahashi a,∗ , Atsuyoshi Nishina a , Hirokazu Kimura b , Kenji Motoki a , Mamoru Koketsu c , Hideharu Ishihara d a

b

Gunma Industrial Technology Center, 884-1 Kamesato, Maebashi, Gunma 379-2147, Japan Gunma Prefectural Institute of Public Health and Environmental Sciences, 3-21-378 Kamioki, Maebashi, Gunma 371-0052, Japan c Division of Instrumental Analysis, Life Science Research Center, Gifu University, Gifu 501-1193, Japan d Department of Chemistry, Faculty of Engineering, Gifu University, Gifu 501-1193, Japan Received 3 December 2003; received in revised form 6 April 2004; accepted 21 April 2004

Abstract We investigated the scavenging effects of tertiary selenoamide compounds for super oxide radicals using a highly sensitive and quantitative chemiluminescence method. At 333 nM, tertiary selenoamide compounds scavenged 25.8–81.6% of O2 − . N-(Phenylselenocarbonyl) piperidine was the most effective scavenger of superoxide radicals. While N,N-diethyl-2-selenonaphthylamide and N,N-diethyl-4-chloroselenobenzamide was a moderately effective scavenger of superoxide radicals. The IC50 of N-(phenylselenocarbonyl) piperidine and N,N-diethyl-2-selenonaphthylamide were determined to be 110 and 182 nM, respectively. The results suggest that tertiary selenoamide compounds are useful scavengers of superoxide radicals. © 2004 Elsevier B.V. All rights reserved. Keywords: Tertiary selenoamide; Superoxide radicals; Scavenging effect; Superoxide anion-scavenging activity (SOSA)

1. Introduction Cells in an organism generate a large amount of reactive oxygen species (ROS) as oxygen metabolites. As a result, exposure to ROS is inevitable. ROS, such as superoxide radicals (O2 − ) and hydrogen peroxide (H2 O2 ) and hydroxyl radicals (OH), cause degeneration of biomacromolecules (i.e., DNA) and induce oxidative stress (Ramirez et al., 2003; Long et al., 1997). It has been suggested that O2 − is primarily generated by mitochondria in various cells and phagocytes, including granulocytes and monocytes/macrophages in vivo (Fridovich, 1995; Ricci et al., 2003). O2 − is converted to H2 O2 in hydrophilic solvents such as water by disproportional reaction (Ueda et al., 1994). In addition O2 − can react with nitric oxide (NO) and generate highly toxic ROS including ONOO− and nitrogen oxides (NOx ) (Hu et al., 2002). Thus, it is important to eliminate O2 − in vivo. Various antioxidant enzymes including the superoxide dismutases (SODs), catalase, and glutathione peroxidase, ∗

Corresponding author.

0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2004.04.011

and antioxidant vitamins (Vitamin C and E) directly scavenge and eliminate ROS. An important antioxidant enzyme, glutathione peroxidase, contains a selenium molecule in active domain and effectively scavenges and eliminates H2 O2 in vitro and in vivo. In addition, previously studies have demonstrated that selenium compounds such as selenoprotein protect cells against oxidative stress (Jeong et al., 2002; Taino et al., 2000). It is possible that selenium compounds can effectively scavenge and eliminate ROS. Therefore, this study was performed to determine effectively how tertiary selenoamide (TS) compounds scavenge O2 − in vitro.

2. Materials and methods 2.1. Materials A chemiluminescent probe for superoxide radicals, which is a Cypridina luciferin analog (2-methyl-6-(pmethoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazin-3-one, or MCLA) was obtained from Tokyo Kasei (Tokyo, Japan),

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dissolved in doubly distilled water, and stored at −80 ◦ C until needed. Horse heart cytochrome c (type IV), SOD (from bovine erythrocytes, 3000 U/mg protein), xanthine oxidase (XOD grade III) and bovine serum albumin (BSA, acid- and globulin-free) were purchased from Sigma Chemical (St. Louis, MO, USA). Hypoxanthine was purchased from Wako Chemicals (Tokyo, Japan) and used without further purification. All other chemicals and solvents were analytical grade and used without further purification. 2.2. General LiAlHSeH was prepared in accordance with a previously described procedure (Ishihara et al., 2001). The 77 Se chemical shifts are expressed in ppm deshielded with respect to neat Me2 Se in CDCl3 . 2.3. Synthetic methods for the production of selenoamide compounds 2.3.1. N,N-Dimethylselenobenzamide (3a) Typical procedure for 3a–3j. Oxalyl chloride (0.09 mL, 1.0 mmol) was added to a stirred solution of N,N-dimethylbenzamide (0.177 g, 1.0 mmol) in anhydrous diethyl ether (5 mL) and allowed to react at 0 ◦ C for 1 h under an argon atmosphere. The mixture was further stirred for 3 h at room temperature (RT). An anhydrous tetrahydrofuran solution (25 mL) of LiAlHSeH 2 (1.2 mmol) was added to the mixture at RT and allowed to react for 3 h. The mixture was extracted with diethyl ether (100 mL) and washed with water (100 mL). The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel with dichloromethane to yield 0.16 g (75%) of 3a as yellow crystals. IR (KBr) 1535 cm−1 , mp 80.0–81.4 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 3.10 (3H, s, CH3 ), 3.70 (3H, s, CH3 ), 7.28–7.34 (5H, m, Ar), 13 C NMR (CDCl3 , 100 MHz) d 44.7, 47.3, 124.6, 128.1, 128.4, 146.1, 205.3, 77 Se NMR (CDCl3 , 76 MHz) d 727.7, MS (CI) m/z = 214 [M+ + 1], Anal. Calcd. for C9 H11 NSe: C, 37.13; H, 3.81; N, 4.81; found: C, 37.02; H, 3.66; N, 4.82%. 2.3.2. N,N-Diethylselenobenzamide (3b) Yield 68%, Yellow crystals, IR (KBr) 1508 cm−1 , mp 55.2–56.1 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 1.15 (3H, t, J = 7.2 Hz, CH3 ), 1.44 (3H, t, J =7.2 Hz, CH3 ), 3.44 (2H, q, J = 7.2 Hz, CH2 ), 4.25 (2H, q, J = 7.2 Hz, CH2 ), 7.19–7.35 (5H, m, Ar), 13 C NMR (CDCl3 , 100 MHz) d 11.2, 13.2, 48.2, 49.7, 123.7, 127.7, 127.9, 146.3, 203.9, 77 Se NMR (CDCl3 , 76 MHz) d 705.3, MS (CI) m/z = 242 [M+ + 1], HRMS: m/z = 241.0369, Calcd. for C11 H15 NSe, found 241.0368. 2.3.3. N,N-Dipropylselenobenzamide (3c) Yield 61%, Yellow crystals, IR (KBr) 1508 cm−1 , mp 40.9–42.3 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 0.71 (3H, t, J = 7.6 Hz, CH3 ), 1.02 (t, J = 7.6 Hz, 3H, CH3 ), 1.59 (2H,

sixtet, J = 7.6 Hz, CH2 ), 1.95 (2H, sixtet, J = 7.6 Hz, CH2 ), 3.34 (2H, t, J = 7.6 Hz, CH2 ), 4.14 (2H, t, J = 7.6 Hz, CH2 ), 7.18–7.33 (5H, m, Ar), 13 C NMR (CDCl3 , 100 MHz) d 10.7, 11.0, 19.1, 21.1, 55.5, 56.6, 123.8, 127.6, 127.7, 146.4, 204.5, 77 Se NMR (CDCl3 , 76 MHz) d 717.3, MS (CI) m/z = 270 [M+ + 1]. 2.3.4. N-(Phenylselenocarbonyl) pyrrolidine (3d) Yield 74%, Yellow crystals, IR (KBr) 1508 cm−1 , mp 79.7–81.0 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 2.02 (2H, quint, J = 6.8 Hz, CH2 ), 2.12 (2H, quint, J = 6.8 Hz, CH2 ), 3.34 (2H, t, J = 6.8 Hz, CH2 ), 3.97 (2H, t, J = 6.8 Hz, CH2 ), 7.32–7.36 (5H, m, Ar), 13 C NMR (CDCl3 , 100 MHz) d 24.6, 26.5, 54.5, 57.1, 124.6, 128.1, 128.5, 146.6, 200.1, 77 Se NMR (CDCl , 76 MHz) d 717.6, MS (CI) m/z = 240 3 [M+ + 1]. 2.3.5. N-(Phenylselenocarbonyl) piperidine (3e) Yield 75%, Yellow crystals, IR (KBr) 1509 cm−1 , mp 64.3–66.4 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 1.58 (2H, m, CH2 ), 1.77 (2H, m, CH2 ), 1.88 (2H, m, CH2 ), 3.50 (2H, t, J = 6.0 Hz, CH2 ), 4.49 (2H, t, J = 5.6 Hz, CH2 ), 7.23–7.35 (5H, m, Ar), 13 C NMR (CDCl3 , 100 MHz) d 23.8, 25.4, 26.7, 53.9, 55.0, 124.3, 128.1, 128.2, 146.0, 203.3, 77 Se NMR (CDCl3 , 76 MHz) d 679.6, MS (CI) m/z = 254 [M+ + 1]. 2.3.6. N-Ethyl-N-methylselenobenzamide (3f) Yield 72%, Yellow crystals, IR (KBr) 1514 cm−1 , mp 61.0–62.6 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 1.18 (3H, t, J = 7.2 Hz, CH3 ), 1.42 (3H, t, J = 7.2 Hz, CH3 ), 3.00 (3H, s, CH3 ), 3.48 (2H, q, J = 7.2 Hz, CH2 ), 3.62 (3H, s, CH3 ), 4.27 (2H, q, J = 7.2 Hz, CH2 ), 7.22–7.35 (5H, m, Ar), 13 C NMR (CDCl3 , 100 MHz) d 10.5, 12.9, 41.5, 44.0, 51.3, 53.1, 123.8, 124.0, 127.8, 127.9, 145.9, 146.1, 203.7, 204.5, 77 Se NMR (CDCl3 , 76 MHz) d 688.9, 731.4, MS (CI) m/z = 228 [M+ + 1]. 2.3.7. N,N-Diethyl-4-methylselenobenzamide (3g) Yield 61%, Yellow crystals, IR (KBr) 1509 cm−1 , mp 89.8–91.0 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 1.16 (3H, t, J = 7.2 Hz, CH3 ), 1.45 (3H, t, J = 7.2 Hz, CH3 ), 2.34 (3H, s, CH3 ), 3.46 (2H, q, J = 7.2 Hz, CH2 ), 4.25 (2H, q, J = 7.2 Hz, CH2 ), 7.13 (4H, s, Ar), 13 C NMR (CDCl3 , 100 MHz) d 11.3, 13.4, 21.4, 48.3, 49.9, 123.9, 128.7, 137.9, 143.9, 204.6, 77 Se NMR (CDCl3 , 76 MHz) d 704.5, MS (CI) m/z = 256 [M+ + 1]. 2.3.8. N,N-Diethyl-4-chloroselenobenzamide (3h) Yield 51%, Yellow crystals, IR (KBr) 1508 cm−1 , mp 65.9–67.8 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 1.09 (3H, t, J = 7.2 Hz, CH3 ), 1.37 (3H, t, J = 7.2 Hz, CH3 ), 3.37 (2H, q, J = 7.2 Hz, CH2 ), 4.17 (2H, q, J = 7.2 Hz, CH2 ), 7.10 (2H, d, J = 8.8 Hz, Ar), 7.23 (2H, d, J = 8.8 Hz, Ar), 13 C NMR (CDCl3 , 100 MHz) d 11.7, 13.2, 48.3, 49.8, 125.2, 128.1, 133.6, 144.8, 202.4, 77 Se NMR (CDCl3 , 76 MHz) d 729.9, MS (CI) m/z = 276 [M+ + 1].

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2.3.9. N,N-Diethylselenonicotinamide (3i) Yield 36%, Yellow oil, IR (neat) 1506 cm−1 , 1 H NMR (CDCl3 , 400 MHz) d 1.12 (3H, t, J = 7.2 Hz, CH3 ), 1.39 (3H, t, J = 7.2 Hz, CH3 ), 3.38 (2H, q, J = 7.2 Hz, CH2 ), 4.17 (2H, q, J = 7.2 Hz, CH2 ), 7.21 (1H, dd, J = 4.8, 7.8 Hz, Ar), 7.52 (1H, dt, J = 1.4, 7.8 Hz, Ar), 8.42 (1H, d, J = 1.4 Hz, Ar), 8.46 (1H, dd, J = 1.4, 4.8 Hz, Ar), 13 C NMR (CDCl3 , 100 MHz) d 11.1, 13.3, 48.5, 50.0, 122.7, 131.4, 142.2, 143.8, 148.7, 200.0, 77 Se NMR (CDCl3 , 76 MHz) d 769.0, MS (CI) m/z = 243 [M+ + 1]. 2.3.10. N,N-Diethyl-2-selenonaphthylamide (3j) Yield 54%, Yellow crystals, IR (KBr) 1509 cm−1 , mp 97.1–97.8 ◦ C, 1 H NMR (CDCl3 , 400 MHz) d 1.13 (3H, t, J = 7.2 Hz, CH3 ), 1.47 (3H, t, J = 7.2 Hz, CH3 ), 3.44 (2H, q, J = 7.2 Hz, CH2 ), 4.27 (2H, q, J = 7.2 Hz, CH2 ), 7.34 (1H, dd, J = 1.6, 8.4 Hz, Ar), 7.44–7.49 (2H, m, Ar), 7.65 (1H, s, Ar), 7.77–7.81 (3H, m, Ar), 13 C NMR (CDCl3 , 100 MHz) d 11.2, 13.3, 48.3, 49.7, 122.0, 122.3, 126.3, 126.5, 127.5, 127.8, 128.0, 132.3, 132.4, 143.5, 203.8, 77 Se NMR (CDCl3 , 76 MHz) d 716.5, MS (CI) m/z = 292 [M+ + 1].

2.4. Assay of superoxide anion-scavenging activity (SOSA) The SOSA of tertiary selenoamides was measured by a previously reported method (Kimura and Nakano, 1988). In brief, the standard reaction mixture contained 10−7 M MCLA, 5 × 10−5 M hypoxanthine, XOD (6.5 U), SOD (0.2–20 ng/ML), in the presence or absence of various concentrations of the tertiary selenoamides and 50 mM Tris–HCl buffer containing 0.1 mM EDTA at pH 7.8, in a total volume of 3.0 ml. Ten mM of tertiary selenoamides were dissolved in DMSO and stored at −80 ◦ C until needed. These compounds in DMSO were 1000-fold diluted with doubly distilled water. Less than 0.1% of DMSO (less than 0.1%) did not inhibit MCLA-dependent chemiluminescense (data not shown). Chemiluminescence measurement was initiated by the addition of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride (MCLA) to the standard incubation mixture excluding XOD, continued for 2 min without XOD and for an additional 2 min after the addition of XOD. Chemiluminescence was measured using a luminometer (Aloka, BLR102) at 25 ◦ C. A representative example of a measurement of the effect of 3e on MCLA-dependent luminescence is shown in Fig. 1. When the compounds had strong SOSA at 333 nM, we also measured at 3.33 and 33.3 nM. Percent of inhibition of MCLA dependent chemiluminescence was calculated as a previously described (Kimura and Nakano, 1988). Inhibition concentration of 50% (IC50 ) was calculated by three concentrations of the samples (3.33, 33.3 and 333 nM). In this research, it measured twice about the same sample and average value was used.

Fig. 1. Effect of N-(phenylselenocarbonyl)piperidine on MCLA-dependant luminescence. Incubation conditions are given in the text. Arrows indicate the time at which MCLA or XOD was added.

3. Results and discussion The optimal conditions for the preparation of N,Ndisubstituted selenoarylamides from the corresponding amides were indicated. Direct reaction of amides with LiAlHSeH failed to produce the corresponding selenoamides at all. Therefore, we tried the preparation of selenoamides from amides using a combination of chlorinating agents, such as oxalyl chloride, hydrogen chloride, thionyl chloride, dichlorotriphenylphosphorane and phosphorus pentachloride, with LiAlHSeH. Reactions using hydrogen chloride, thionyl chloride, dichlorotriphenylphosphorane and phosphorus pentachloride gave mixtures instead of 3 or provided no products other than recovered starting material. Ultimately, the use of oxalyl chloride and LiAlHSeH was found to be optimal for the syntheses of selenoamides from amides as shown in Scheme 1. Ten kinds of tertiary selenoamides (3a–3j) were synthesized from the corresponding amides 1 by treatment with oxalyl chloride followed by LiAlHSeH 2, as shown in the Scheme 1. The structures of the tertiary selenoamides are shown in Table 1. The SOSA of the compounds are also summarized in Table 1. Among them, N-(phenylselenocarbonyl) piperidine (3e) had the highest SOSA of 81.6% at 333 nM. The effects of 3e were dose-dependent (Fig. 1). The SOSA of N,N-diethyl-4-chloroselenobenzamide 3h and N,N-diethyl2-selenonaphthylamide 3j were 60.8% and 65.0% at 330 nM, respectively. SOSA of all compounds were dosedependently (data not shown). The effect of 3j was also dose-dependent (data not shown). The activities of compounds 3e and 3j were high enough to suggest further testing, and serial dilutions were used to determine that the IC50 for the two compounds were 182 and 110 nM, respectively. This study considered the elimination of superoxide anion generated by XOD by selenamides. Exact SOSA cannot be measured if selenamides inhibit the activity of XOD.

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Table 1 Scavenging activity of several tertiary selenoamides on superoxide anion Entries

Compound

Inhibition (%)

1

47.9

2

50.8

3

46.3

4

42.1

5

81.6

6

25.8

7

57.4

8

60.8

9

50.0

10

65.0

The luminescence intensity (count/min) of the solution which does not contain a substance at all, and the solution containing 3c or 3e was measured, and the inhibition (%) was computed.

provide a useful basis for the development of potential SOSA using tertiary selenoamides. Antioxidant enzyme activity, including SODs, glutathione peroxidase, and catalase as well as the plasma selenium concentration were evaluated to investigate the effects of selenium in patients with human immunodeficiency virus. GPX activity at baseline was significantly higher in the placebo and selenium groups than in the control group. These higher enzyme activities could be related to an increased synthesis of these enzymes in erythrocyte precursors in the setting of oxidative stress. GPX activity increased significantly after selenium treatment between 3 and 6 months. Similarly, a significant increase in the GSH concentration was observed at 12 months compared with the baseline value after selenium supplementation (Delmas-Beauvieux et al., 1996). Although the generation of a low concentration of superoxide anion in the human body is useful as a biologic defence system and for intercelluar signal transduction, it has been reported that increased superoxide anion reduction plays a role in aging. The generation of excessive superoxide anion in the human body is controlled by the enzymatic antioxidant system, which includes SOD, GPX, Cat, and GSH. Superoxide anion causes nerve degeneration (Wrona and Dryhurst, 1998) and heart failure (Ferrari et al., 1989). It has been reported that the SOD activity in the blood of patients with thyroiditis, dwarfism, and Turner syndrome is lower than that in healthy persons (Ohno et al., 1991). It is known that therapy with a drug having SOSA could be useful for the treatment of those diseases. Ebselen, which is a selenium-containing compound, has been extensively studied as candidate drug, and ebselen has been shown to attenuate oxidative stress (Asatryan et al., 2003; Imai et al., 2002; Yoshizumi et al., 2002). We only evaluated SOSA using simple in vitro method. However, these results can not directly apply to physiological systems. Other selenium compounds such as ebselen have been evaluated on intact cell systems (Yang and Yang, 1989; Kondo et al., 1997). Thus, effects of SOSA of our compounds should be examined using reconstructed systems/in vivo systems. Acknowledgements

We checked that measured electron spin resonance of super oxide anion and selenamides did not inhibit the activity of XOD (Tanigawa, 1990) (data not shown). In this study, we examined the SOSA of tertiary selenoamides. Among the ten compounds tested, 3e, 3h, and 3j were found to be the most potent SOSA agents. Based on the SOSA in vitro results, it was speculated we hypothesize that these compounds may be potential therapeutic agents for the dismutation of the superoxide anion, although we did not tests the biologic toxity of our compounds. The compounds appear to be new chemical types with SOSA in vitro. In the present study, we first demonstrated that tertiary selenoamides have SOSA. Thus, the present study would

This work was supported by a Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 14540490), to which we are grateful. References Asatryan, L., Ziouzenkova, O., Duncan, R., Sevanian, A., 2003. Heme and lipid peroxides in hemoglobin-modified low-density lipoprotein mediate cell survival and adaptation to oxidative stress. Blood 102, 1732–1739. Delmas-Beauvieux, M.-C., Peuchant, E., Couchouron, A., Constans, J., Sergeant, C., Simonoff, M., Pellegrin, J.-L., Leng, B., Conri, C., Clerc,

H. Takahashi et al. / European Journal of Pharmaceutical Sciences 23 (2004) 207–211 M., 1996. The enzymic antioxidant system in blood and glutathione status in human immunodeficiency virus (HIV)-infected patients: effects of supplementation with selenium or b-carotene. Am. J. Clin. Nutr. 64, 101–107. Ferrari, R., Ceconi, C., Curello, S., Ghielmi, S., Albertini, A., 1989. Superoxide dismutase: possible therapeutic use in cardiovascular disease. Pharmacol. Res. 21 (Suppl. 2), 57–65. Fridovich, I., 1995. Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 64, 97–112. Hu, T.M., Hayton, W.L., Morse, M.A., Mallery, S.R., 2002. Dynamic and biphasic modulation of nitrosation reaction by superoxide dismutases. Biochem. Biophys. Res. Commun. 295, 1125–1134. Imai, H., Graham, D.I., Masayasu, H., Macrae, I.M., 2002. Antioxidant ebselen reduces oxidative damage in focal cerebral ischemia. Free Radic. Biol. Med. 34, 56–63 (volume date 2003). Ishihara, H., Koketsu, M., Fukuta, Y., Nada, F., 2001. Reaction of lithium aluminum hydride with elemental selenium: its application as a selenating reagent into organic molecules. J. Am. Chem. Soc. 123, 8408– 8409. Jeong, D.W., Kim, S., Chang, Y.W., Kim, I.Y., Lee, B.J., 2002. Selenoprotein W is a glutathione-dependent antioxidant in vivo. FEBS Lett. 517, 225–228. Kimura, H., Nakano, M., 1988. Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase in human erythrocytes. FEBS Lett. 239 (2), 347–350. Kondo, H., Takahashi, M., Niki, E., 1997. Peroxynitrite-induced hemolysis of human erythrocytes and its inhibition by antioxidants. FEBS Lett. 18 (413), 236–238. Long, J.F., Dutta, P.K., Hobb, B.D., 1997. Fluorescence imaging of reactive oxygen metabolites generated in single macrophage cells (NR8383) upon phagocytosis of natural zeolite(erionite) fibers. Environ. Health Perspect. 105, 706–711.

211

Ohno, H., Matsuura, N., Ishikawa, N., Sato, Y., Tniguchi, N., 1991. Serum manganaze-superoxide dismutase in patients with diabetes mellitius and thyroid dysfunction as judged by an ELISA. Horm. Metab. Res. 23, 449–451. Ramirez, R., Carracedo, J., Jimenaz, R., Alijama, P., Canela, A., Herrera, E., Blasco, M.A., 2003. Massive telomere loss is an early event of DNA damage-induced apoptosis. J. Biol. Chem. 278, 836–842. Ricci, J.-E., Green, D.R., Gottlieb, R.A., 2003. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J. Cell. Biol. 160, 65–75. Taino, L., Fedeli, D., Santroni, A.M., Falcioni, G., Villariani, M., 2000. Effect of three diaryl tellurides, and an organoselenium compound in trout erythrocytes exposed to oxidative stress in vitro. Mutat. Res. 464, 269–277. Tanigawa, T., 1990. Determination of hydroxyl radical scavenging activity by electron spin resonance. J. Kyoto. Pref. Univ. Med. 99, 133–143. Ueda, J., Ozawa, T., Sudo, A., Moria, A., 1994. Generation of hydroxyl radicals during dismutation of superoxide by SOD model compounds. Arch. Biochem. Biophys. 315, 185–189. Wrona, M.Z., Dryhurst, G., 1998. Oxidation of serotonin by superoxide radical: implications to neurodegenerative brain disorders. Chem. Res. Toxicol. 11, 639–650. Yang, F.Y., Yang, M.Z., 1989. Reaction of Se with SH groups in spectrin is involved in the stabilization of erythrocyte membrane skeleton. Biochem. Int. 18, 1085–1091. Yoshizumi, M., Kogame, T., Suzaki, Y., Fujita, Y., Kyaw, M., Kirima, K., Ishizawa, K., Tsuchiya, K., Kagami, S., Tamaki, T., 2002. Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun N-terminal kinase and activator protein-1 signalling pathway in PC12 cells. Br. J. Pharmacol. 136, 1023–1032.