Tertiary selenoamide compounds are useful superoxide radical scavengers in vitro

Article abstract of DOI:10.1016/j.ejps.2004.04.011

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^-

Full text of DOI:10.1016/j.ejps.2004.04.011

European Journal of Pharmaceutical Sciences 23 (2004) 207–211
Tertiary selenoamide compounds are useful superoxide
radical scavengers in vitro
Hitoe Takahashia,∗, Atsuyoshi Nishinaa, Hirokazu Kimurab, Kenji Motokia,
Mamoru Koketsuc, Hideharu Ishiharad
a
Gunma Industrial Technology Center, 884-1 Kamesato, Maebashi, Gunma 379-2147, Japan
b
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 scavenged25.8–81.6%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-selenonaph-
thylamide 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
and antioxidant vitamins (Vitamin C and E) directly scav-
enge and eliminate ROS. An important antioxidant enzyme,
glutathione peroxidase, contains a selenium molecule in ac-
tive domain and effectively scavenges and eliminates H2O2
in vitro and in vivo. In addition, previously studies have
demonstrated that selenium compounds such as selenopro-
tein 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 effectiv−ely how tertiary
selenoamide (TS) compounds scavenge O2 in vitro.
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 radi-
cals (O2−) and hydrogen peroxide (H2O2) and hydroxyl rad-
icals (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 primar-
ily generated by mitochondria in various cells and phago-
cytes, including granulocytes and monocytes/macrophages
−
in vivo (Fridovich, 1995; Ricci et al., 2003). O2 is con-
verted to H2O2 in hydrophilic solvents such as water b−y
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
2. Materials and methods
2.1. Materials
−
et al., 2002). Thus, it is important to eliminate O2 in vivo.
Various antioxidant enzymes including the superoxide
dismutases (SODs), catalase, and glutathione peroxidase,
A chemiluminescent probe for superoxide radicals,
which is a Cypridina luciferin analog (2-methyl-6-(p-
methoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazin-3-one,
or MCLA) was obtained from Tokyo Kasei (Tokyo, Japan),
∗
Corresponding author.
0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2004.04.011

208
H. Takahashi et al. / European Journal of Pharmaceutical Sciences 23 (2004) 207–211
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 Chem-
ical (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.
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), 13C 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, 77Se 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, 1H 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), 13C NMR (CDCl3, 100 MHz)
d 24.6, 26.5, 54.5, 57.1, 124.6, 128.1, 128.5, 146.6, 200.1,
77Se NMR (CDCl3, 76 MHz) d 717.6, MS (CI) m/z = 240
[M+ + 1].
2.2. General
LiAlHSeH was prepared in accordance with a previously
described procedure (Ishihara et al., 2001). The 77Se chem-
ical shifts are expressed in ppm deshielded with respect to
neat Me2Se in CDCl3.
2.3. Synthetic methods for the production of selenoamide
compounds
2.3.5. N-(Phenylselenocarbonyl) piperidine (3e)
Yield 75%, Yellow crystals, IR (KBr) 1509 cm−1, mp
64.3–66.4 ◦C, H NMR (CDCl3, 400 MHz) d 1.58 (2H, m,
1
2.3.1. N,N-Dimethylselenobenzamide (3a)
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), 13C 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, 77Se NMR
(CDCl3, 76 MHz) d 679.6, MS (CI) m/z = 254 [M+ + 1].
Typical procedure for 3a–3j. Oxalyl chloride (0.09 mL,
1.0 mmol) was added to a stirred solution of N,N-dimethyl-
benzamide (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 mix-
ture at RT and allowed to react for 3 h. The mixture was ex-
tracted 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, 1H NMR (CDCl3, 400 MHz)
d 3.10 (3H, s, CH3), 3.70 (3H, s, CH3), 7.28–7.34 (5H, m,
Ar), 13C NMR (CDCl3, 100 MHz) d 44.7, 47.3, 124.6, 128.1,
128.4, 146.1, 205.3, 77Se NMR (CDCl3, 76 MHz) d 727.7,
MS (CI) m/z = 214 [M+ + 1], Anal. Calcd. for C9H11NSe:
C, 37.13; H, 3.81; N, 4.81; found: C, 37.02; H, 3.66; N,
4.82%.
2.3.6. N-Ethyl-N-methylselenobenzamide (3f)
Yield 72%, Yellow crystals, IR (KBr) 1514 cm−1, mp
61.0–62.6 ◦C, 1H 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), 13C
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, 77Se
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, 1H 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), 13C 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, 77Se NMR (CDCl3, 76 MHz) d 704.5, MS (CI)
m/z = 256 [M+ + 1].
2.3.2. N,N-Diethylselenobenzamide (3b)
Yield 68%, Yellow crystals, IR (KBr) 1508 cm−1, mp
55.2–56.1 ◦C, 1H 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), 13C NMR (CDCl3, 100 MHz) d 11.2, 13.2, 48.2,
49.7, 123.7, 127.7, 127.9, 146.3, 203.9, 77Se NMR (CDCl3,
76 MHz) d 705.3, MS (CI) m/z = 242 [M+ + 1], HRMS:
m/z = 241.0369, Calcd. for C11H15NSe, found 241.0368.
2.3.8. N,N-Diethyl-4-chloroselenobenzamide (3h)
Yield 51%, Yellow crystals, IR (KBr) 1508 cm−1, mp
65.9–67.8 ◦C, 1H 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), 13C NMR
(CDCl3, 100 MHz) d 11.7, 13.2, 48.3, 49.8, 125.2, 128.1,
133.6, 144.8, 202.4, 77Se NMR (CDCl3, 76 MHz) d 729.9,
MS (CI) m/z = 276 [M+ + 1].
2.3.3. N,N-Dipropylselenobenzamide (3c)
Yield 61%, Yellow crystals, IR (KBr) 1508 cm−1, mp
40.9–42.3 ◦C, 1H 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,

H. Takahashi et al. / European Journal of Pharmaceutical Sciences 23 (2004) 207–211
209
2.3.9. N,N-Diethylselenonicotinamide (3i)
Yield 36%, Yellow oil, IR (neat) 1506 cm−1, H NMR
1
(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), 13C 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, 77Se 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, 1H 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), 13C 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, 77Se NMR (CDCl3,
76 MHz) d 716.5, MS (CI) m/z = 292 [M+ + 1].
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,N-
disubstituted selenoarylamides from the correspond-
ing amides were indicated. Direct reaction of amides
with LiAlHSeH failed to produce the corresponding se-
lenoamides at all. Therefore, we tried the preparation of
selenoamides from amides using a combination of chlori-
nating agents, such as oxalyl chloride, hydrogen chloride,
thionyl chloride, dichlorotriphenylphosphorane and phos-
phorus pentachloride, with LiAlHSeH. Reactions using
hydrogen chloride, thionyl chloride, dichlorotriphenylphos-
phorane and phosphorus pentachloride gave mixtures in-
stead 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 se-
lenoamides 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 chlo-
ride 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) piperi-
dine (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-diethyl-
2-selenonaphthylamide 3j were 60.8% and 65.0% at
330 nM, respectively. SOSA of all compounds were dose-
dependently (data not shown). The effect of 3j was also
dose-dependent (data not shown). The activities of com-
pounds 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.
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 di-
luted with doubly distilled water. Less than 0.1% of
DMSO (less than 0.1%) did not inhibit MCLA-dependent
chemiluminescense (data not shown). Chemilumines-
cence 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 incu-
bation 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 lu-
minescence 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 chemi-
luminescence 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 mea-
sured twice about the same sample and average value was
used.
This study considered the elimination of superoxide an-
ion generated by XOD by selenamides. Exact SOSA can-
not be measured if selenamides inhibit the activity of XOD.

210
H. Takahashi et al. / European Journal of Pharmaceutical Sciences 23 (2004) 207–211
Table 1
provide a useful basis for the development of potential
SOSA using tertiary selenoamides.
Scavenging activity of several tertiary selenoamides on superoxide anion
Antioxidant enzyme activity, including SODs, glutathione
peroxidase, and catalase as well as the plasma selenium con-
centration were evaluated to investigate the effects of sele-
nium 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 sig-
nificant 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. Su-
peroxide 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 re-
sults 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.
Entries
1
Compound
Inhibition (%)
47.9
2
3
4
5
6
50.8
46.3
42.1
81.6
25.8
7
8
57.4
60.8
50.0
65.0
9
10
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.
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).
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.
In this study, we examined the SOSA of tertiary se-
lenoamides. 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
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
211
M., 1996. The enzymic antioxidant system in blood and glutathione
status in human immunodeficiency virus (HIV)-infected patients: ef-
fects of supplementation with selenium or b-carotene. Am. J. Clin.
Nutr. 64, 101–107.
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.
Ferrari, R., Ceconi, C., Curello, S., Ghielmi, S., Albertini, A., 1989. Su-
peroxide 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).
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.
Ishihara, H., Koketsu, M., Fukuta, Y., Nada, F., 2001. Reaction of lithium
aluminum hydride with elemental selenium: its application as a sele-
nating 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. Seleno-
protein W is a glutathione-dependent antioxidant in vivo. FEBS Lett.
517, 225–228.
Wrona, M.Z., Dryhurst, G., 1998. Oxidation of serotonin by superoxide
radical: implications to neurodegenerative brain disorders. Chem. Res.
Toxicol. 11, 639–650.
Kimura, H., Nakano, M., 1988. Highly sensitive and reliable chemilu-
minescence method for the assay of superoxide dismutase in human
erythrocytes. FEBS Lett. 239 (2), 347–350.
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.
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.
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.