Báo cáo khoa học: Superoxide radical-scavenging effects from polymorphonuclear leukocytes and toxicity in human cell lines of newly synthesized organic selenium compounds potx

Superoxide radical-scavenging effects from polymorphonuclear leukocytes and toxicity in human cell lines of newly synthesized organic selenium compounds Hiroyuki Tsukagoshi 1 , Mamoru Koketsu 2 , Masahiko Kato 1 , Masahiko Kurabayashi 3 , Atsuyoshi Nishina 4 and Hirokazu Kimura 5 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; O 2 – 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 (O 2 – )-scavenging effects and toxicity. We measured O 2 – -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 O 2 – 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 O 2 – 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; O 2 – , 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. 6046 FEBS Journal 274 (2007) 6046–6054 ª 2007 The Authors Journal compilation ª 2007 FEBS Synthetic 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 (O 2 – ) is a one-electron-reduced oxygen molecule and acts as both a free radical and anion [9]. A relatively large amount of O 2 – is generated in the cardiovascular system, in mitochondria, and in phagocytes, such as polymorphonuclear leukocytes (PMNs), macrophages ⁄ monocytes, eosinophils, mast cells, and basophils [10]. O 2 – reacts not only with bio- molecules, but also with other ROS, such H 2 O 2 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 H 2 O 2 , 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 H 2 O 2 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 O 2 – -scavenging effects of these compounds using a hypoxanthine– xanthine oxidase system, i.e. an enzymatic O 2 – 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 (selenoamide B), are effective O 2 – 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 O 2 – 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 O 2 – -scavenging effects of organic selenium compounds in PMNs We demonstrated O 2 – -scavenging effects by testing whether selenoureas and selenoamides scavenged O 2 – 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 (IC 50 ) 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 IC 50 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 IC 50 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. O 2 – decreased when selenoureas and selenoamides were added at the peak of O 2 – gen- eration by PMA-stimulated PMNs (data not shown). These results suggest that selenoureas and seleno- amides effectively scavenged O 2 – from PMA-stimu- lated PMNs. H. Tsukagoshi et al. O 2 – -scavenging effects and toxicity of Se compounds FEBS Journal 274 (2007) 6046–6054 ª 2007 The Authors Journal compilation ª 2007 FEBS 6047 Cytotoxicity 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 H 2 O 2 production As shown in Fig. 2, selenoureas and selenoamides were effective scavengers of O 2 – from PMA-stimulated PMNs. We investigated whether or not selenoureas and selenoamides produced H 2 O 2 from O 2 – 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 H 2 O 2 from PMA-stimulated PMNs. At 100 lm, selenourea A and selenourea B produced 8.3 ± 5.2 lm and 11.7 ± 1.7 lm H 2 O 2 , respectively, and selenoamide A 0 0 5 10 15 20 20 40 60 80 100 120 140 Incubation time (min) Chemiluminescense intensity (cpm x10 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 O 2 – -scavenging effects of the compound were found by eliminating MCLA-depen- dent CL. Arrows indicate the time at which MCLA or PMA was added. O 2 – was recorded for 15 min with a luminescence reader. 0 0 110100 25 50 75 100 * * * * * * * * * * * * * * * Inhibition (% of control) Concentration (µM) Selenourea A Selenoamide A Selenourea B Selenoamide B Fig. 2. Inhibition curves of four organic selenium compounds for PMA-induced O 2 – 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. Entries Compounds IC 50 (lM) Selenourea A 6.8 ± 2.2 Selenourea B 6.5 ± 2.5 Selenoamide A 11.3 ± 4.8 Selenoamide B 20.3 ± 6.4 O 2 – -scavenging effects and toxicity of Se compounds H. Tsukagoshi et al. 6048 FEBS Journal 274 (2007) 6046–6054 ª 2007 The Authors Journal compilation ª 2007 FEBS and selenoamide B produced 20.1 ± 2.5 and 41.6 ± 2.0 lm H 2 O 2 , respectively. These results sug- gest that, as compared with the selenoamides, the sele- noureas did not produce significant amounts of H 2 O 2 in PMA-stimulated PMNs. Visualization of p47 phagocyte oxidase (p47 phox ) with laser scanning microscopy (LSM) The cytosolic component of NADPH oxidase complex, p47 phox , translocates from the cytosol to the cell mem- brane upon activation. To examine the effects of sele- noureas and selenoamides on O 2 – 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 p47 phox 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 p47 phox only in adhered PMNs. Even at the relatively high concentration of 100 lm, p47 phox translocation was not inhibited by organic selenium compounds. These results suggest that sele- noureas and selenoamides did not significantly affect the translocation of p47 phox to 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 (IC 50 around 7 lm), both selenourea A and selenourea B showed more potent O 2 – -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 O 2 – in the human body generally plays a beneficial role in biolog- ical defense and intercellular signal transduction [1]. Time (h) Cell viability (% of control) Selenourea A Selenourea B Selenoamide A Selenoamide B HaCaTcells 0 20 40 60 80 100 120 0244872 Time (h) 0 24 48 72 Time (h) 0 24 48 72 Time (h) 0244872 * 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Cell viability (% of control) * * * * * * * * * * * * * * * Cell viability (% of control)Cell viability (% of control) HEL cells HEp-2 cells PMNs Selenourea A Selenourea B Selenoamide A Selenoamide B Selenourea A Selenourea B Selenoamide A Selenoamide B Selenourea A Selenourea B Selenoamide A Selenoamide B AB CD 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. H. Tsukagoshi et al. O 2 – -scavenging effects and toxicity of Se compounds FEBS Journal 274 (2007) 6046–6054 ª 2007 The Authors Journal compilation ª 2007 FEBS 6049 On the other hand, excessive O 2 – 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 O 2 – . In this study, our results suggest that selenou- reas acted as effective O 2 – scavengers and showed very low toxicity in human cells. Thus, the compounds may eliminate excessive O 2 – , 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 using an enzymatic O 2 – 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 O 2 – from PMA-stimulated PMNs, with or without inhibition of NADPH oxidase as an index of translocation of p47 phox . The generation of O 2 – 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 O 2 – . A component of NADPH oxidase, p47 phox , translocates to the cell membrane and associ- ates with cytochrome b 556 to form the active complex that catalyzes the reduction of oxygen to O 2 – at the Selenourea A 100 µ M 1% dimethylsulfoxideMedium alone (no stimulated) Selenoamide B 100 µ M Selenourea B 100 µ M Selenoamide A 100 µ M Control A B C D E Fig. 5. Translocation of p47 phox 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 dimethylsulfoxide S e l en o u r ea A S e l e n o u re a B S e l e n o a m i d e A S e l e n o a m i d e B S O D % of SOD * * * * * * * * Fig. 4. H 2 O 2 generation 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. H 2 O 2 was 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. O 2 – -scavenging effects and toxicity of Se compounds H. Tsukagoshi et al. 6050 FEBS Journal 274 (2007) 6046–6054 ª 2007 The Authors Journal compilation ª 2007 FEBS expense of NADPH [46–48]. We examined the trans- location of p47 phox to the plasma membrane in PMA- stimulated PMNs. In adherent PMNs, none of the organic selenium compounds significantly affected the translocation of p47 phox to the plasma membrane. Thus, we suggest that the O 2 – -scavenging activity of selenoureas and selenoamides was not due to the inhi- bition of NADPH oxidase in PMNs. We assessed the metabolism of O 2 – by selenoureas and selenoamides as an index of H 2 O 2 production. Both selenourea A and selenourea B produced small amounts of H 2 O 2 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 hydroxyl radical can be directly produced via O 2 – 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 O 2 – generation from leukocytes through the inhibition of leukocytic NADPH oxidase [24]. Moreover, it is possible that it can decompose H 2 O 2 and produce H 2 O and O 2 . 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 O 2 – -scavenging effect, and no signifi- cant inhibition of NADPH oxidase activity was found. Selenoureas and selenoamides, especially selenoureas, seemed to scavenge O 2 – directly to produce O 2 . 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 O 2 – generator, we measured IC 50 values of only selenourea B (mean of IC 50 values: 125 nm) and selenoamide A (mean of IC 50 values: 182 nm) [39,40]. In the present study, we assayed the IC 50 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 O 2 – -derived PMA-stimulated PMNs [39,40]. As a result, the IC 50 means of selenourea B and selenoamide A were 6.5 lm and 11.3 lm, respec- tively. Thus, the differences in IC 50 values 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 evidence that innovative organic sele- nium compounds scavenge O 2 – 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 O 2 – . 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 selenourea 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 )1 Æcm )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 p47 phox 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 H. Tsukagoshi et al. O 2 – -scavenging effects and toxicity of Se compounds FEBS Journal 274 (2007) 6046–6054 ª 2007 The Authors Journal compilation ª 2007 FEBS 6051 isolated 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 O 2 – 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 O 2 – of these com- pounds, we used a highly sensitive and specific CL method with MCLA as a probe [51–53]. Briefly, 7 · 10 4 PMNs 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 37 °C for 5 min. After incubation, PMNs were stimulated with 100 ngÆmL )1 PMA [40]. CL by O 2 – was recorded for 15 min with a luminescence reader (BLR-102; Aloka Co., Tokyo, Japan) with gentle agitation at 37 °C [50]. The total volume of the assays was 2 mL [50]. IC 50 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% NaHCO 3 at 37 °Cinan atmosphere of 95% air and 5% CO 2 . 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 l m each organic selenium compound in 100 lLof DMEM containing 2% fetal bovine serum at 37 °Cinan atmosphere of 95% air and 5% CO 2 . PMNs (2 · 10 4 ) 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 37 °C, in an atmosphere of 95% air and 5% CO 2 . 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 H 2 O 2 production The production of H 2 O 2 was determined by the horseradish peroxidase-catalyzed oxidation of fluorescent scopoletin method with minor modifications [54–56]. Briefly, 2 · 10 5 PMNs were added to HBSS (pH 7.4) containing 1mm NaN 3 , and then incubated at 37 °C in an atmosphere of 95% air and 5% CO 2 in the presence of each organic selenium compound or SOD. After 5 min, PMNs were stimulated with PMA at a final concentration of 1 lgÆ mL )1 for 15 min. Samples were added to the reaction mixture containing HBSS (pH 7.4), 10 lm scopoletin and 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 H 2 O 2 instead of the samples. Visualization of p47 phox by LSM A suspension of 1 · 10 5 PMNs in phenol red ⁄ free RPMI- 1640 medium with 1 mm Hepes was incubated with or without organic selenium compound for 5 min at 37 °Cin 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 37 °C, the mixture in each dish was gently aspirated, and 2 mL of 3.7% formaldehyde in NaCl ⁄ P i (v ⁄ v) was added carefully to each dish for 10 min at room temperature (25 °C) to fix the attached cells. The cells were then washed gently with NaCl ⁄ P i three times and permeabilized with 2 mL of 0.1% Triton X-100 in NaCl ⁄ P i in each dish for 10 min. After permeabilization, cells were washed three times with NaCl ⁄ P i , and incubated for 30 min in darkness at 4 °C with a primary antibody (antibody to p47 phox or 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 ⁄ P i , 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. 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