polymorphonuclear leukocytes and toxicity in human cell lines of newly synthesized organic selenium compounds Hiroyuki Tsukagoshi1, Mamoru Koketsu2, Masahiko Kato1, Masahiko Kurabayashi3
Trang 1polymorphonuclear leukocytes and toxicity in human cell lines of newly synthesized organic selenium compounds Hiroyuki Tsukagoshi1, Mamoru Koketsu2, Masahiko Kato1, Masahiko Kurabayashi3,
Atsuyoshi Nishina4and Hirokazu Kimura5
1 Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Japan
2 Division of Instrumental Analysis, Life Science Research Center, Gifu University, Japan
3 Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Japan
4 Gunma Industrial Technology Center, Maebashi, Japan
5 Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan
Keywords
antioxidant; O2– scavenger; selenium;
selenourea; tertiary selenoamide
Correspondence
H Kimura, Infectious Disease Surveillance
Center, National Institute of Infectious
Diseases, 4-7-1 Gakuen, Musashimurayama,
Tokyo 208-0011, Japan
Fax: +81 42 565 3315
Tel: +81 42 561 0771
E-mail: kimhiro@nih.go.jp
(Received 2 August 2007, revised 1 October
2007, accepted 3 October 2007)
doi:10.1111/j.1742-4658.2007.06125.x
Synthetic organic selenium compounds such as 2-phenyl-1,2-benziso-selenazol-3(2H)-one may show glutathione peroxidase-like antioxidant activity Recently, we synthesized new organic selenium compounds that are thought to be effective antioxidants To study their possible applications as antioxidants, we evaluated two selenoureas, N,N-dimethylselenourea and 1-selenocarbamoylpyrrolidine, and two tertiary selenoamides, N-(phenylsele-nocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide, for their superoxide radical (O2 )-scavenging effects and toxicity We measured
O2 -scavenging effects in polymorphonuclear leukocytes (PMNs) with a spe-cific, sensitive and real-time kinetic chemiluminescence method Further-more, the toxicity of these compounds was measured in some human cell lines and PMNs using the tetrazolium method Hydrogen peroxide was mea-sured by a scopoletin method Finally, translocation of an NADPH oxidase component, p47 phagocyte oxidase, to the cell membrane was investigated
by confocal laser scanning microscopy N,N-Dimethylselenourea and 1-sele-nocarbamoylpyrrolidine effectively scavenged O2 released from 4b-phorbol 12-myristate 13-acetate-stimulated PMNs, and the 50% inhibitory concentrations were 6.8 ± 2.2 and 6.5 ± 2.5 lm, respectively N-(Phenyl-selenocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide also effectively scavenged O2 from PMNs, and the 50% inhibitory concentra-tions were 11.3 ± 4.8 and 20.3 ± 6.4 lm, respectively Selenoureas showed very low toxicity in human cell lines and PMNs, even at high concentrations, whereas tertiary selenoamides were cytotoxic These compounds did not pro-duce significant amounts of hydrogen peroxide from 4b-phorbol 12-myristate 13-acetate-stimulated PMNs None of the compounds significantly affected the translocation of p47 phagocyte oxidase Selenoureas acted as effective antioxidants and showed low toxicity in some human cells Thus, these compounds might be new candidates as antioxidative substances
Abbreviations
CI, confidence interval; CL, chemiluminescence; DAPI, 4,6-diamidinophenylindole; ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one;
GPX, glutathione peroxidase; HBSS, Hank’s balanced salt solution; IC 50 , 50% inhibitory concentration; LSM, laser scanning microscopy; MCLA, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a]-pyrazin-3-one; O2– , superoxide radical; p47 phox , p47 phagocyte oxidase; PMA, 4b-phorbol 12-myristate 13-acetate; PMN, polymorphonuclear leukocyte; ROS, reactive oxygen species; selenoamide A,
N-(phenylselenocarbonyl)-piperidine; selenoamide B, N,N-diethyl-4-chloroselenobenzamide; selenourea A, N,N-dimethylselenourea;
selenourea B, 1-selenocarbamoylpyrrolidine; SOD, superoxide dismutase.
Trang 2Synthetic organic selenium compounds such as
2-phe-nyl-1,2-benzisoselenazol-3(2H)-one (ebselen) may
mimic glutathione peroxidase (GPX; EC 1.11.1.9)
activity as antioxidants Accumulating evidence
indi-cates that reactive oxygen species (ROS) act as
oxida-tive stressors in vivo [1] ROS are associated with
degradation of biomolecules, such as DNA, proteins,
and lipids [2,3] Excessive generation of ROS in vivo
triggers oxidative stress-related diseases such as
can-cers, atherosclerosis, and ageing [4,5] Thus, it is
important to eliminate ROS in vivo [6–8]
Superoxide radical (O2 ) is a one-electron-reduced
oxygen molecule and acts as both a free radical and
anion [9] A relatively large amount of O2 is generated
in the cardiovascular system, in mitochondria, and
in phagocytes, such as polymorphonuclear leukocytes
(PMNs), macrophages⁄ monocytes, eosinophils, mast
cells, and basophils [10] O2 reacts not only with
bio-molecules, but also with other ROS, such H2O2 and
lipid hydroperoxides [10–12] In addition, ROS derived
from leukocytes induce excessive inflammation, leading
to cell and tissue damage [13,14] In extreme instances,
such as endotoxin shock, neutrophils kill the infected
host [15] It is therefore important to eliminate ROS
from leukocytes at inflammatory sites [13,14]
Antioxidants, including antioxidative enzymes such
as superoxide dismutase (SOD; EC 1.15.1.1),
gluta-thione peroxidase (GPX, EC 1.11.1.9) and catalase
(EC 1.11.1.6), and low molecular weight antioxidants,
such as vitamins and various biological dyes, can reduce
ROS in vivo [16–19] GPX is an important oxidative
enzyme and a selenoprotein, incorporating a
seleno-cysteine residue at the active site [20–22] It catalyzes
the reduction of H2O2, and a variety of organic
hydro-peroxides, resulting in effective elimination of various
ROS in vivo and in vitro [23] The selenium molecule in
GPX plays a crucial role in the metabolism of ROS,
and some organic selenium compounds mimicking
GPX have therefore been synthesized [24–28] For
example, ebselen, a five-membered ring
selenium-containing heterocyclic compound, and diphenyl
diselenide, are synthetic organic selenium compounds
that are considered to be potential pharmacological
agents [29] In fact, these compounds show antioxidant,
antinociceptive, neuroprotective and anti-inflammatory
properties in different experimental models [30–35]
Eb-selen inhibits leukocyte infiltration and activation,
lead-ing to elimination of H2O2 in vitro [27,28] Hence, it
may be applicable as an anti-inflammatory drug for the
treatment of various inflammatory diseases [24–28] It is
possible that other organic selenium compounds may
also become candidates as anti-inflammatory⁄
antioxi-dative drugs Given this information, we have newly
synthesized various types of organic selenium com-pounds, such as selenoureas, tertiary selenoamides, sele-nocarbamates, and bis-(2-amino-5-selenazoyl) ketones [36,37] We have already evaluated the O2-scavenging effects of these compounds using a hypoxanthine– xanthine oxidase system, i.e an enzymatic O2 genera-tion system, and have found that the selenoureas N,N-dimethylselenourea (selenourea A) and 1-selenoc-arbamoylpyrrolidine (selenourea B), and the tertiary selenoamides, N-(phenylselenocarbonyl) piperidine (selenoamide A) and N,N-diethyl-4-chloroselenobenza-mide (selenoaN,N-diethyl-4-chloroselenobenza-mide B), are effective O2 scavengers
in vitro [38–40] To investigate whether these com-pounds can be applied as antioxidants in vivo, we evalu-ated their scavenging effects on O2 using PMNs We also assessed their cytotoxicity against some human cell lines, such as human keratinocytes (HaCaT cells), human embryo lung fibroblast cells (HEL cells), human nasopharyngeal carcinoma cells (HEp-2 cells) and PMNs in vitro
Results
O2–-scavenging effects of organic selenium compounds in PMNs
We demonstrated O2 -scavenging effects by testing whether selenoureas and selenoamides scavenged O2 from 4b-phorbol 12-myristate 13-acetate (PMA)-stim-ulated PMNs using a specific, sensitive and real-time kinetic chemiluminescence (CL) method Representa-tive data showing inhibition of CL by one compound, selenourea A, are shown in Fig 1 This compound incubated with PMNs significantly inhibited CL in a dose-dependent manner The other compounds gave similar results to those for selenourea A CL inhibi-tion curves for the four organic selenium compounds are shown in Fig 2 The 50% inhibitory concentra-tion (IC50) of each compound is shown in Table 1 Selenourea A and selenourea B significantly inhibited
CL as compared with selenoamide A and seleno-amide B (Fig 2) The IC50 values of selenourea A and selenourea B were 6.8 ± 2.2 lm [95% confidence interval (CI): 2.6–11.4] and 6.5 ± 2.5 lm (95% CI: 1.5–11.5), respectively The IC50 values of seleno-amide A and selenoamide B were 11.3 ± 4.8 lm (95% CI: 2.0–20.6) and 20.3 ± 6.4 lm (95% CI: 7.7– 32.9), respectively O2 decreased when selenoureas and selenoamides were added at the peak of O2 gen-eration by PMA-stimulated PMNs (data not shown) These results suggest that selenoureas and seleno-amides effectively scavenged O2 from PMA-stimu-lated PMNs
Trang 3Cytotoxicity of organic selenium compounds
in human cells
We examined the cytotoxic effects of selenoureas and
selenoamides using three cell lines, HaCaT, HEL, and
HEp-2, and PMNs, by the microtiter tetrazolium
method [41–43] The cytotoxicities of the compounds
in each cell line are shown in Fig 3A–C At 100 lm, a
relatively high concentration, both selenourea A and
selenourea B showed very low cytotoxic effects in
HaCaT, HEL and HEp-2 cells (Fig 3A–C) In
con-trast, selenoamide B was relatively cytotoxic at 72 h
of coincubation: the viabilities of HaCaT, HEL and HEp-2 cells were 9%, 25%, and 5%, respectively (Fig 3A–C) Similarly, another tertiary selenoamide, selenoamide A, was relatively cytotoxic in HaCaT, HEL and HEp-2 cells (Fig 3A–C) At 100 lm, these compounds did not significantly affect cell viabilities in nonstimulated PMNs (Fig 3D) These results suggest that a relatively high concentration of selenoureas did not affect cell viability in some human cells
Measurement of H2O2production
As shown in Fig 2, selenoureas and selenoamides were effective scavengers of O2 from PMA-stimulated PMNs We investigated whether or not selenoureas and selenoamides produced H2O2 from O2 generated
by PMA-stimulated PMNs After incubation with each organic selenium compound or SOD for 5 min, the PMNs were stimulated with PMA As shown in Fig 4,
1 lgÆmL)1 SOD produced 82.0 ± 2.6 lm H2O2 from PMA-stimulated PMNs At 100 lm, selenourea A and selenourea B produced 8.3 ± 5.2 lm and 11.7 ± 1.7 lm H2O2, respectively, and selenoamide A
0
20
40
60
80
100
120
140
Incubation time (min)
4)
0 µM
1 µM
2 µM
5 µM
10 µM MCLA
PMA
Fig 1 Representative PMA-induced O 2–-scavenging effects of a
selenourea compound (selenourea A) determined using the CL
method Detailed experimental procedures are described in
Experi-mental procedures PMNs were preincubated with selenourea A
5 min before PMA treatment Dose-dependent O2– -scavenging
effects of the compound were found by eliminating
MCLA-depen-dent CL Arrows indicate the time at which MCLA or PMA was
added O2– was recorded for 15 min with a luminescence reader.
0
25
50
75
100
*
*
*
*
*
*
*
*
*
*
*
Selenourea A
Selenoamide A Selenourea B
Selenoamide B
Fig 2 Inhibition curves of four organic selenium compounds for
PMA-induced O2– production from PMNs m, selenourea A; j,
sel-enourea B; n, selenoamide A; h, selenoamide B Detailed
proce-dures are described in the text Results are expressed as
mean ± SEM from three independent experiments *P < 0.05.
Table 1 IC 50 values of organic selenium compounds: selenoureas and tertiary selenoamides PMNs were resuspended in Hank’s balanced salt solution (HBSS) (pH 7.4) with 3 l M MCLA and each organic selenium compound at 37 C for 5 min After incubation, PMNs were stimulated by 100 ngÆmL)1 PMA CL by O 2– was recorded for 15 min with a luminescence reader at 37 C IC 50 values are expressed relative to 0.1% dimethylsulfoxide as vehicle Values are presented as mean ± SEM from three independent experiments.
Trang 4and selenoamide B produced 20.1 ± 2.5 and
41.6 ± 2.0 lm H2O2, respectively These results
sug-gest that, as compared with the selenoamides, the
sele-noureas did not produce significant amounts of H2O2
in PMA-stimulated PMNs
Visualization of p47 phagocyte oxidase (p47phox)
with laser scanning microscopy (LSM)
The cytosolic component of NADPH oxidase complex,
p47phox, translocates from the cytosol to the cell
mem-brane upon activation To examine the effects of
sele-noureas and selenoamides on O2 production from
PMNs, we investigated the translocation of the
mole-cule to the cell membrane upon stimulation with
PMA Figure 5 shows the red fluorescent phallodin
staining (F-actin) and the blue fluorescence of the
nuclei Green fluorescence marks the p47phox
translo-cated from the cytosol to the cell membrane After
incubation, about 70% of PMNs adhered to
glass-bottomed dishes in the presence or absence of organic
selenium compounds (data not shown) We observed
the translocation of p47phox only in adhered PMNs
Even at the relatively high concentration of 100 lm,
p47phox translocation was not inhibited by organic selenium compounds These results suggest that sele-noureas and selenoamides did not significantly affect the translocation of p47phoxto the cell membrane
Discussion
We demonstrated the antioxidative effects and cytotox-icity of four newly synthesized organic selenium compounds: two selenoureas (selenourea A and sele-nourea B) and two tertiary selenoamides (seleno-amide A and selenoamide B) At relatively low concentrations (IC50around 7 lm), both selenourea A and selenourea B showed more potent O2-scavenging effects without loss of NADPH oxidase activity in PMA-stimulated PMNs than did selenoamide A and selenoamide B Furthermore, the two selenoureas showed very low toxicity in PMNs and some cell lines, even at high concentrations (100 lm) The results imply that some types of selenourea, such as selenourea A and selenourea B, may be new candidate antioxidants The generation of a low concentration of O2 in the human body generally plays a beneficial role in biolog-ical defense and intercellular signal transduction [1]
Time (h)
Selenourea A Selenourea B Selenoamide A Selenoamide B
HaCaTcells
0 20 40 60 80 100 120
Time (h)
Time (h)
Time (h)
*
0 20 40 60 80 100 120
0 20 40 60 80 100 120
0 20 40 60 80 100 120
*
*
*
**
*
*
*
*
*
HEL cells
Selenourea A Selenourea B Selenoamide A Selenoamide B
Selenourea A Selenourea B Selenoamide A Selenoamide B
Selenourea A Selenourea B Selenoamide A Selenoamide B
Fig 3 Cytotoxicity of four organic selenium compounds in some human cell lines and PMNs Cytotoxicity was determined by microtiter tetrazo-lium assay ‘Control’ contained cells plus cell culture medium supplemented with 2% fetal bovine serum Results are expressed as mean ± SEM from three independent experiments (A) HaCaT cells were incubated with 100 l M each organic selenium compound Cell viability was measured at intervals of 24 h (B) Cell viability of HEL cells (C) Cell viability of HEp-2 cells (D) Cell viability of PMNs PMNs maintained about 80% viability in control cultures after 48 h Detailed procedures are described in the text **P < 0.01 versus control; *P < 0.05 versus control.
Trang 5On the other hand, excessive O2 production has a
detrimental role in the pathogenesis of a number of
disorders, including inflammation, rheumatoid
arthri-tis, and asthma [44,45] Oxidative stress might be
defined as an imbalance between cellular production of
ROS and antioxidant defense mechanisms [1] It is a
key component of inflammation and inflammatory
dis-orders The processes associated with inflammatory
responses are complex and often involve ROS,
includ-ing O2 In this study, our results suggest that selenou-reas acted as effective O2 scavengers and showed very low toxicity in human cells Thus, the compounds may eliminate excessive O2, leading to suppression of inflammatory responses in sites overproducing ROS
We also evaluated the toxicity of selenoureas and selenoamides The two selenoureas were simple struc-tures and had low toxicity in PMNs and some cell lines, even at high concentrations (100 lm) They do not have a benzene ring or a chlorine bond, unlike both selenoamides Thus, selenoureas may not generate toxic metabolites such as benzene compounds via met-abolic enzymes (i.e cytochrome P450)
We previously investigated the ROS-scavenging effects of various organic selenium compounds, includ-ing selenoureas and tertiary selenoamides, by usinclud-ing an enzymatic O2 generation system (hypoxanthine–xan-thine oxidase system) [38–40] Furthermore, we demon-strated that these compounds do not significantly inhibit xanthine oxidase activity [38–40] In the present study, we investigated the anti-inflammatory⁄ antioxi-dant potential of these compounds by examining their scavenging effects on O2 from PMA-stimulated PMNs, with or without inhibition of NADPH oxidase
as an index of translocation of p47phox The generation
of O2 by PMNs is attributed to the activation of a plasma membrane NADPH oxidase The NADPH oxi-dase multicomponent enzyme system catalyzes the pro-duction of O2 A component of NADPH oxidase, p47phox, translocates to the cell membrane and associ-ates with cytochrome b556 to form the active complex that catalyzes the reduction of oxygen to O2 at the
Selenourea A
100 µM
1% dimethylsulfoxide Medium alone
(no stimulated)
Selenoamide B
100 µM
Selenourea B
100 µM
Selenoamide A
100 µM
Fig 5 Translocation of p47phox in PMNs treated with four organic selenium compounds Green fluorescence was induced by Alexa Fluor 488 rabbit anti-(goat IgG) (p47 phox ) Blue fluorescence was induced by DAPI-stained nuclei Red fluorescence was induced by Alexa Fluor 532 phalloidin (F-actin) Control was a negative control of PMNs with no stimulation by PMA PMNs were stimulated by PMA with 1% dimethylsulfoxide as vehicle (A) The PMNs were stimulated by PMA with 100 l M organic selenium compounds (B–E) Detailed procedures are described in the text Each upper figure of control and (A)–(E) is shown in green fluorescence alone.
0
25
50
75
100
A
A
B
* *
* *
* *
* *
Fig 4 H2O2generation by four organic selenium compounds and
SOD following PMA-induced generation of O 2–from PMNs PMNs
were preincubated with 100 l M each organic selenium compound,
SOD or dimethylsulfoxide, 5 min before PMA treatment H2O2was
measured after 15 min, using the scopoletin fluorescent method.
Detailed procedures are described in the text Results are
expressed as mean ± SEM from three independent experiments.
**P < 0.01 versus SOD.
Trang 6expense of NADPH [46–48] We examined the
trans-location of p47phox to the plasma membrane in
PMA-stimulated PMNs In adherent PMNs, none of the
organic selenium compounds significantly affected the
translocation of p47phox to the plasma membrane
Thus, we suggest that the O2-scavenging activity of
selenoureas and selenoamides was not due to the
inhi-bition of NADPH oxidase in PMNs
We assessed the metabolism of O2 by selenoureas
and selenoamides as an index of H2O2 production
Both selenourea A and selenourea B produced small
amounts of H2O2 as compared with SOD In this
study, however, we did not use the ESR method to
examine production of other ROS, such as the
hydro-xyl radical Although it is not very likely that hydrohydro-xyl
radical can be directly produced via O2 using
seleno-ureas and selenoamides, detailed additional studies
regarding the metabolism of ROS, using a
physico-chemical method such as ESR, may be required
Some other organic selenium compounds such as
eb-selen have been developed [24–28] At low
concentra-tions, ebselen inhibits a number of enzymes involved
in inflammation, such as lipoxygenases, nitric oxide
synthases, NADPH oxidase, protein kinase C, and
H+⁄ K+-ATPase [24] Ebselen inhibits O2 generation
from leukocytes through the inhibition of leukocytic
NADPH oxidase [24] Moreover, it is possible that it
can decompose H2O2and produce H2O and O2
Ebse-len is thought to act by mimicking an active center of
GPX in the cell [24] By contrast, selenoureas and
sele-noamides had an O2 -scavenging effect, and no
signifi-cant inhibition of NADPH oxidase activity was found
Selenoureas and selenoamides, especially selenoureas,
seemed to scavenge O2 directly to produce O2 Thus,
it is possible that reaction mechanisms affecting ROS
differ between selenoureas and selenoamides and
ebse-len These differences may be attributed to structural
properties, although the precise mechanisms are not
fully understood at present
In previous studies, using a hypoxanthine–xanthine
oxidase system as the O2 generator, we measured IC50
values of only selenourea B (mean of IC50 values:
125 nm) and selenoamide A (mean of IC50 values:
182 nm) [39,40] In the present study, we assayed the
IC50 values of the four compounds (selenourea A and
selenourea B, and selenoamide A and selenoamide B)
using about 30-fold amounts, as compared with
previ-ous studies, of O2 -derived PMA-stimulated PMNs
[39,40] As a result, the IC50 means of selenourea B
and selenoamide A were 6.5 lm and 11.3 lm,
respec-tively Thus, the differences in IC50values may be due
to differences in the experimental conditions between
the previous and present studies
In conclusion, the results from this study may pro-vide biological epro-vidence that innovative organic sele-nium compounds scavenge O2 released from PMNs Furthermore, these compounds were not toxic in some human cells and PMNs, indicating that they have the potential to prevent inflammation caused by O2 The next step should be to confirm the antioxidant effects
of these compounds, especially selenoureas, in animal models such as mice
Experimental procedures Organic selenium compounds
In this study, we newly synthesized and used four types of organic selenium compound: two types of selenoureas, sele-nourea A selesele-nourea B, and two types of tertiary seleno-amides, selenoamide A and selenoamide B The chemical structures are shown in Table 1 Detailed synthetic proce-dures for these compounds were as previously described, and the chemical structures were confirmed by NMR analysis [36,37] The purity of these compounds was more than 97% [36,37] All compounds were dissolved in dimethylsulfoxide
at 10 mm and stored at ) 80 C until required When used for the experiments, the compounds were diluted with HBSS
Reagents
A Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a]-pyrazin-3-one (MCLA), was pur-chased from Tokyo Kasei Chemical Co (Tokyo, Japan) The compound was dissolved in double distilled water, and stored at ) 80 C until use The concentration of MCLA solution was determined by absorbance at 430 nm using an absorbance coefficient value a˚ of 9600 m)1Æcm)1, as previ-ously described [49] PMA and dimethylsulfoxide were pur-chased from Sigma Aldrich Chemical Co (St Louis, MO, USA) The stock solution of PMA (5 mgÆmL)1) was dissolved in dimethylsulfoxide, and stored at ) 80 C until use 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) reagent was purchased from Dojindo Chemicals
(Kumamot-o, Japan) (Cell Counting Kit8) Alexa Fluor 532 phalloidin, Alexa Fluor 488 rabbit anti-(goat IgG) (H + L) and 4,6-di-amidino-phenylindole (DAPI) were obtained from Molecu-lar Probes (Eugene, OR, USA) Antibody to p47phox was purchased from Santa Cruz Biotechnology (Santa Cruz, CA., USA) All other chemicals were of analytical grade and used without further purification
Isolation of PMNs
PMNs were isolated from heparinized peripheral blood of healthy volunteers as previously described [50] PMNs were
Trang 7isolated by using a gradient material, one-step
Polymorpho-prep (Accurate Chemical & Scientific Corp., Westbury,
NY, USA) following the procedure recommended by the
manufacturer The purity of PMNs was > 98% as
deter-mined by Randolph’s stain
Assay of effects on scavenging of O2–from PMNs
Organic selenium compound solutions such as selenoureas
and tertiary selenoamides were diluted with HBSS To
ana-lyze the real-time effects on scavenging of O2 of these
com-pounds, we used a highly sensitive and specific CL method
with MCLA as a probe [51–53] Briefly, 7· 104PMNs
were resuspended in HBSS (pH 7.4) with 3 lm MCLA and
0–100 lm (maximum dose used in this study) each organic
selenium compound at 37C for 5 min After incubation,
PMNs were stimulated with 100 ngÆmL)1PMA [40] CL by
O2 was recorded for 15 min with a luminescence reader
(BLR-102; Aloka Co., Tokyo, Japan) with gentle agitation
at 37C [50] The total volume of the assays was 2 mL
[50] IC50 values were determined using linear regression
analysis of the dose–response curves
Cell culture and cytotoxicity assay
HaCaT cells, HEL cells and HEp-2 cells were cultured in
DMEM supplemented with 10% fetal bovine serum,
l-gluta-mine (0.6 mgÆmL)1) and 0.35% NaHCO3 at 37C in an
atmosphere of 95% air and 5% CO2 HaCaT cells were
kindly donated by N E Fusenig (German Cancer Research
Center, Heidelberg, Germany) We assayed the cytotoxicity
of the organic selenium compounds in HaCaT cells, HEL
cells, HEp-2 cells and PMNs using the microtiter tetrazolium
method [41] Briefly, confluent cells were incubated in a
96-well microplate (Corning Costar, Cambridge, MA, USA)
with 100 lm each organic selenium compound in 100 lL of
DMEM containing 2% fetal bovine serum at 37C in an
atmosphere of 95% air and 5% CO2 PMNs (2· 104) were
added to a 96-well microplate and were incubated with
100 lm each compound in 100 lL of RPMI-1640 medium
containing 2% fetal bovine serum at 37C, in an atmosphere
of 95% air and 5% CO2 After 24 h of incubation, we added
10 lL of WST-8 solution Absorbance was measured at
450 nm using a microplate reader (Spectramax; Molecular
Devices, Sunnyvale, CA, USA) [42,43] Cytotoxicity was
cal-culated as previously described [42,43]
Measurement of H2O2production
The production of H2O2was determined by the horseradish
peroxidase-catalyzed oxidation of fluorescent scopoletin
2· 105PMNs were added to HBSS (pH 7.4) containing
1 mm NaN3, and then incubated at 37C in an atmosphere
of 95% air and 5% CO2 in the presence of each organic selenium compound or SOD After 5 min, PMNs were stimulated with PMA at a final concentration of 1 lgÆmL)1 for 15 min Samples were added to the reaction mixture
50 lgÆmL)1 horseradish peroxidase in a 96-well plate The decrease in scopoletin fluorescence was measured using a microplate fluorometer (1420 Multilabel counter, ARVO; excitation and emission at 335 and 460 nm, respectively; Wallac, Turuk, Finland) The concentration of the oxidant
in the samples was calculated using the standard curve, which was made by adding known concentrations of the authentic H2O2instead of the samples
Visualization of p47phoxby LSM
A suspension of 1· 105PMNs in phenol red⁄ free
RPMI-1640 medium with 1 mm Hepes was incubated with or without organic selenium compound for 5 min at 37C in
35 mm glass-bottomed dishes (Matsunami, Osaka, Japan) After incubation, PMA was added to a final concentration
of 100 ngÆmL)1 After 15 min at 37C, the mixture in each dish was gently aspirated, and 2 mL of 3.7% formaldehyde
in NaCl⁄ Pi (v⁄ v) was added carefully to each dish for
10 min at room temperature (25C) to fix the attached cells The cells were then washed gently with NaCl⁄ Pithree times and permeabilized with 2 mL of 0.1% Triton X-100
in NaCl⁄ Piin each dish for 10 min After permeabilization, cells were washed three times with NaCl⁄ Pi, and incubated for 30 min in darkness at 4C with a primary antibody (antibody to p47phoxor goat IgG as a negative control; all dilutions 1 : 500) The cells were then washed thoroughly and incubated for 30 min in darkness at room temperature with a mixture of Alexa Fluor 532 phalloidin at 1 unit per dish and secondary antibody [Alexa Fluor 488 rabbit anti-(goat IgG); dilution 1 : 1000] Finally, after three washes with NaCl⁄ Pi, DAPI was mounted on the samples and the fluorescent signal was observed with a confocal LSM sys-tem (MRC-1024; Bio-Rad, Hercules, CA, USA) as previ-ously described, with minor modifications [57]
Statistical analysis
Raw values or normalized values from the indicated num-ber of independent trials were averaged and expressed as the mean ± SEM Any significant differences between the groups were determined using analysis of variance (anova) followed by Dunnett’s test Data with a P-value of less than 0.05 were considered to be significant
Acknowledgements
We thank Mr Takafumi Yamaguchi, Mr Taisei Ishioka,
Mr Masakazu Yoshizumi and Mrs Hitoe Takahashi
Trang 8for their help with the experiments This work was
supported by Gunma prefecture (Maebashi, Japan)
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