Cell Death Caused by Selenium Deficiency and Protective Effect of Antioxidants

Selenium is an essential trace element and it is well known that selenium is necessary for cell culture. However, the mechanism underlying the role of selenium in cellular proliferation and survival is still unknown. The present study using Jurkat cells showed that selenium deficiency in a serum-free medium decreased the selenium-dependent enzyme activity (glutathione peroxidases and thioredoxin reductase) within cells and cell viability. To understand the mechanism of this effect of selenium, we examined the effect of other antioxidants, which act by different mechanisms. Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely blocked selenium deficiency-induced cell death, although α-tocopherol (biologically the most active form of vitamin E) could not preserve selenium-dependent enzyme activity. Other antioxidants, such as different isoforms and derivatives of vitamin E, BO-653 and deferoxamine mesylate, also exerted an inhibitory effect. However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, displayed no such effect. Dichlorodihydrofluorescein (DCF) assay revealed that cellular reactive oxygen species (ROS) increased before cell death, and sodium selenite and α-tocopherol inhibited ROS increase in a dose-dependent manner. The generation of lipid hydroperoxides was observed by fluorescence probe diphenyl-1-pyrenylphosphine (DPPP) and HPLC chemiluminescence only in selenium-deficient cells. These results suggest that the ROS, especially lipid hydroperoxides, are involved in the cell death caused by selenium deficiency and that selenium and vitamin E cooperate in the defense against oxidative stress upon cells by detoxifying and inhibiting the formation of lipid hydroperoxides. Selenium is an essential trace element and it is well known that selenium is necessary for cell culture. However, the mechanism underlying the role of selenium in cellular proliferation and survival is still unknown. The present study using Jurkat cells showed that selenium deficiency in a serum-free medium decreased the selenium-dependent enzyme activity (glutathione peroxidases and thioredoxin reductase) within cells and cell viability. To understand the mechanism of this effect of selenium, we examined the effect of other antioxidants, which act by different mechanisms. Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely blocked selenium deficiency-induced cell death, although α-tocopherol (biologically the most active form of vitamin E) could not preserve selenium-dependent enzyme activity. Other antioxidants, such as different isoforms and derivatives of vitamin E, BO-653 and deferoxamine mesylate, also exerted an inhibitory effect. However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, displayed no such effect. Dichlorodihydrofluorescein (DCF) assay revealed that cellular reactive oxygen species (ROS) increased before cell death, and sodium selenite and α-tocopherol inhibited ROS increase in a dose-dependent manner. The generation of lipid hydroperoxides was observed by fluorescence probe diphenyl-1-pyrenylphosphine (DPPP) and HPLC chemiluminescence only in selenium-deficient cells. These results suggest that the ROS, especially lipid hydroperoxides, are involved in the cell death caused by selenium deficiency and that selenium and vitamin E cooperate in the defense against oxidative stress upon cells by detoxifying and inhibiting the formation of lipid hydroperoxides. Selenium is an essential trace element for humans and many other forms of life, and a deficiency of this element induces some pathological conditions, such as cancer, coronary heart disease, and liver necrosis (1Allan C.B. Lacourciere G.M. Stadtman T.C. Annu. Rev. Nutr. 1999; 19: 1-16Crossref PubMed Scopus (296) Google Scholar, 2Clark L.C. Combs Jr., G.F. Turnbull B.W. Slate E.H. Chalker D.K. Chow J. Davis L.S. Glover R.A. Graham G.F. Gross E.G. Krongrad A. Lesher Jr., J.L. Park H.K. Sanders Jr., B.B. Smith C.L. Taylor J.R. J. Am. Med. Assoc. 1996; 276: 1957-1963Crossref PubMed Google Scholar, 3Salonen J.T. Salonen R. Seppanen K. Kantola M. Suntioinen S. Korpela H. Br. Med. J.,. 1991; 302: 756-760Crossref PubMed Scopus (190) Google Scholar, 4Suadicani P. Hein H.O. Gyntelberg F. Atherosclerosis,. 1992; 96: 33-42Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 5Burk R.F. Lawrence R.A. Lane J.M. J. Clin. Investig. 1980; 65: 1024-1031Crossref PubMed Scopus (220) Google Scholar). Selenium deficiency is also accompanied by a loss of immunocompetence (6Field C.J. Johnson I.R. Schley P.D. J. Leukoc. Biol. 2002; 71: 16-32PubMed Google Scholar), and both cell-mediated immunity and B-cell function can be impaired (7McKenzie R.C. Rafferty T.S. Beckett G.J. Immunol. Today. 1998; 19: 342-345Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Supplementation with selenium has marked immunostimulant effects, including an enhancement of activated T-cell proliferation (8Kiremidjian-Schumacher L. Roy M. Wishe H.I. Cohen M.W. Stotzky G. Biol. Trace Elem. Res. 1994; 41: 115-127Crossref PubMed Scopus (234) Google Scholar). Selenium is an essential component of several enzymes such as glutathione peroxidase (GPx) 1The abbreviations used are: GPx, glutathione peroxidase; ROS, reactive oxygen species; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; DCFH-DA, dichlorofluorescin diacetate; TR, thioredoxin reductase; pNA, p-nitroanilide; FITC, fluorescein isothiocyanate; SeP, selenoprotein P. (9Takahashi K. Avissar N. Whitin J. Cohen H. Arch. Biochem. Biophys. 1987; 256: 677-686Crossref PubMed Scopus (355) Google Scholar), thioredoxin reductase (TR) (10Yarimizu J. Nakamura H. Yodoi J. Takahashi K. Antioxid. Redox Signal. 2000; 2: 643-651Crossref PubMed Scopus (15) Google Scholar), and selenoprotein P (SeP) (11Saito Y. Takahashi K. J. Health Sci. 2000; 46: 409-413Crossref Scopus (33) Google Scholar), which contain selenium as selenocysteine. It is also well known that selenium is essential for cell culture when a serum-free medium is used (12McKeehan W.L. Hamilton W.G. Ham R.G. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2023-2027Crossref PubMed Scopus (207) Google Scholar). Serum-free media, especially for immune cells and neurons, contain insulin, transferrin, and sodium selenite. Without selenium, cells can neither proliferate nor survive. However, the underlying mechanism for the role of selenium in cell proliferation is still unknown. Vitamin E, a generic term for tocopherols and tocotrienols, is one of the most potent lipid-soluble antioxidants (13Brigelius-Flohe R. Traber M.G. FASEB J. 1999; 13: 1145-1155Crossref PubMed Scopus (1263) Google Scholar). Vitamin E occurs in nature in at least eight different isoforms: α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols (14Packer L. Weber S.U. Rimbach G. J. Nutr. 2001; 131: 369S-373SCrossref PubMed Google Scholar). Tocotrienols differ from the corresponding tocopherols only in their aliphatic tail. Vitamin E deficiencies have been implicated in some pathologic conditions, such as cancer, coronary heart disease, and liver necrosis (15Ricciarelli R. Zingg J.M. Azzi A. FASEB J. 2001; 15: 2314-2325Crossref PubMed Scopus (248) Google Scholar, 16Keaney Jr., J.F. Simon D.I. Freedman J.E. FASEB J. 1999; 13: 965-975Crossref PubMed Scopus (141) Google Scholar) and are also accompanied by a loss of immunocompetence (17Meydani S.N. Beharka A.A. Nutr. Rev. 1998; 56: S49-S58Crossref PubMed Scopus (108) Google Scholar). It is well known that selenium and vitamin E show compensative effects and that a deficiency of both elements causes massive injury in some cases (18Navarro F. Navas P. Burgess J.R. Bello R.I. De Cabo R. Arroyo A. Villalba J.M. FASEB J. 1998; 12: 1665-1673Crossref PubMed Scopus (106) Google Scholar, 19Hill K.E. Motley A.K. Li X. May J.M. Burk R.F. J. Nutr. 2001; 131: 1798-1802Crossref PubMed Scopus (51) Google Scholar, 20Beck M.A. Williams-Toone D. Levander O.A. Free Radic. Biol. Med. 2003; 34: 1263-1270Crossref PubMed Scopus (42) Google Scholar). In the present study, we characterize the nature of cell death caused by selenium deficiency and the cell death inhibitory effect of antioxidants including vitamin E. We also demonstrate the involvement of reactive oxygen species (ROS), especially lipid hydroperoxides, on the cell death. Chemicals—Sodium selenite, bovine serum albumin fraction V, tert-butyl hydroperoxide, and GSH were obtained from Nacalai, Kyoto, Japan; GSH reductase from Oriental Yeast Co., Ltd., Tokyo, Japan; RPMI 1640 medium, seleno-dl-cystine, seleno-dl-methionine, and seleno-l-methionine from Sigma-Aldrich Co.; recombinant human insulin and human transferrin from Wako, Osaka, Japan; 3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyltetrazolium bromide (MTT) and diphenyl-1-pyrenylphosphine (DPPP) from Dojindo, Kumamoto, Japan; diisopropyl fluorophosphate from Kishida Chemical Co., Osaka, Japan; and 2-carboxy-2,5,7,8-pentamethyl-6-chromanol (Trolox) and deferoxamine mesylate were obtained from Calbiochem, Darmstadt, Germany. Dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes, Eugene, OR. Ebselen was kindly provided by Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan. Natural eight isoforms of vitamin E and 2,2,5,7,8-pentamethyl-6-chromanol (PMC) were kindly supplied by Eisai Co. Ltd., Tokyo, Japan. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653) was prepared as described previously (21Noguchi N. Iwaki Y. Takahashi M. Komuro E. Kato Y. Tamura K. Cynshi O. Kodama T. Niki E. Arch. Biochem. Biophys. 1997; 342: 236-243Crossref PubMed Scopus (61) Google Scholar). Other chemicals were of the highest quality commercially available. Cell Culture and Determination of Cell Viability—Jurkat E6–1 cells, human T-leukemia (American Tissue Type Collection) were maintained in RPMI 1640 medium containing 100 units/ml penicillin G, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, and 10% heat-inactivated fetal calf serum at 37 °C under an atmosphere of 95% air and 5% CO2, as described previously (22Saito Y. Takahashi K. Eur. J. Biochem. 2002; 269: 5746-5751Crossref PubMed Scopus (138) Google Scholar). For studies on the effects of selenium depletion, cells (3 × 105 cells/ml) were cultured with RPMI 1640 medium containing 5 μg/ml human insulin, 5 μg/ml human transferrin, 92 nm FeCl3, and 2.5 mg/ml bovine serum albumin (ITA-RPMI). Stock solutions of sodium selenite and vitamin E were prepared in PBS and Me2SO, respectively. For the determination of cell viability, trypan blue assay, and MTT assay were conducted for the indicated times. In the former, cells that excluded trypan blue after incubation with PBS containing 0.04% trypan blue dye (Invitrogen) were considered viable. For the latter analysis, cells were incubated with 0.5 mg/ml MTT at 37 °C for 2 h. Isopropyl alcohol containing 0.04 n HCl was added to the culture medium (3:2, by volume), and they were mixed by pipette until the formazan was completely dissolved. The optical density of formazan was measured at 570 nm using a Multiskan Ascent plate reader (Theromo Labsystems, Helsinki, Finland). Cell Death Assay—Phosphatidylserine (PS) exposure (23Pigault C. Follenius-Wund A. Schmutz M. Freyssinet J.M. Brisson A. J. Mol. Biol. 1994; 236: 199-208Crossref PubMed Scopus (136) Google Scholar) and caspase activity (24Casciola-Rosen L. Nicholson D.W. Chong T. Rowan K.R. Thornberry N.A. Miller D.K. Rosen A. J. Exp. Med. 1996; 183: 1957-1964Crossref PubMed Scopus (578) Google Scholar) were analyzed as described previously. PS exposure was measured by the binding of annexin V-FITC according to the protocol outlined by the manufacture in the Apoptosis Detection-kit (Sigma-Aldrich Co.). Treated cells were also stained with propidium iodide, followed by analysis with a Cytomics FC500 Flow Cytometry System (Beckman Coulter, Inc., Miami, FL) with a 488-nm argon laser. Caspase activity was measured by the cleavage of Asp-Glu-Val-Asp (DEVD) peptide-conjugated p-nitroanilide (pNA) according to the protocol outlined by the manufacture in the Caspase-3/CPP32 Colorimetric Protease Assay-kit (Medical & Biological Laboratories Co. Ltd., Nagoya, Japan). Substrate cleavage to release pNA (405 nm) was measured using a Multiskan Ascent plate reader (Theromo Labsystems, Helsinki, Finland). Absorbance units were converted to pmol of pNA using a standard curve generated with free pNA. Cytosol Preparation—Cytosol was prepared as described previously (22Saito Y. Takahashi K. Eur. J. Biochem. 2002; 269: 5746-5751Crossref PubMed Scopus (138) Google Scholar). After culturing for the specified periods, cells were collected and resuspended in an appropriate volume of 50 mm Tris-HCl (pH 7.4), containing 0.25 m sucrose, 0.1 mm EDTA, 0.7 mm 2-mercaptoethanol, and 2 mm diisopropyl fluorophosphate. The cell suspension was sonicated and centrifuged at 105,000 × g for 1 h at 4 °C to obtain a cytosolic fraction. Protein Assay—Protein content of cytosol and cell samples were determined using a BCA protein assay kit (Pierce) with bovine serum albumin as a standard. Enzyme Assay—To measure GPx and TR activities, a coupled enzyme assay, which was performed by following the oxidation of NADPH, was used as described previously (22Saito Y. Takahashi K. Eur. J. Biochem. 2002; 269: 5746-5751Crossref PubMed Scopus (138) Google Scholar). The assay conditions were as follows: for the cellular GPx (cGPx) assay, 0.1 m Tris-HCl, pH 8.0, 0.2 mm NADPH, 0.5 mm EDTA, 1 mm NaN3, 2 mm GSH, 1 unit/ml of GSH reductase, and 70 μm tert-butyl hydroperoxide; for the phospholipid hydroperoxide GPx (PH-GPx) assay, 0.1 m Tris-HCl, pH 8.0, 0.2 mm NADPH, 0.5 mm EDTA, 1 mm NaN3, 5 mm GSH, 1 unit/ml of GSH reductase, and 60 μm 1-palmitoyl-2-(13-hydroperoxy-cis-9-trans-11-octadecadienoyl)-3-phosphatidylcholine hydroperoxide (PLPC-OOH); for the TR assay, 50 mm phosphate buffer, pH 7.0, 1 mm EDTA, 0.2 mm NADPH, 0.8 μm human recombinant thioredoxin, and 80 μm insulin. The oxidation of NADPH was followed at 340 nm at 37 °C, and the activity was expressed as nmol of NADPH oxidized per minute. Determination of Intracellular Reactive Oxygen Species—Intracellular ROS were detected using DCFH-DA as described previously (25Takahashi M. Shibata M. Niki E. Free Radic. Biol. Med. 2001; 31: 164-174Crossref PubMed Scopus (151) Google Scholar) with a slight modification. After culturing for the specified periods, cells were collected, resuspended in PBS and incubated with DCFH-DA at a final concentration of 5 μm for 15 min at 37 °C. Then, cells were washed once with PBS and incubated for 1 h at 37 °C. Cells were excited with a 488-nm argon ion laser in a Cytomics FC500 Flow Cytometry System, and the DCF emission was recorded at 525 nm. Data were collected from at least 10,000 events. Determination of Intracellular Lipid Hydroperoxides—Intracellular lipid hydroperoxides were detected using fluorescence probe DPPP (25Takahashi M. Shibata M. Niki E. Free Radic. Biol. Med. 2001; 31: 164-174Crossref PubMed Scopus (151) Google Scholar) and chemiluminescence HPLC systems (26Yoshida Y. Ito N. Shimakawa S. Niki E. Biochem. Biophys. Res. Commun. 2003; 305: 747-753Crossref PubMed Scopus (30) Google Scholar, 27Miyazawa T. Fujimoto K. Suzuki T. Yasuda K. Methods Enzymol. 1994; 233: 324-332Crossref PubMed Scopus (116) Google Scholar) as described previously with a slight modification. For DPPP assay, cells were preincubated in PBS at 37 °C at a density of 1 × 107 cells/ml for 5 min. After addition of DPPP (in Me2SO) at a final concentration of 167 μm, the cell suspension was incubated for 5 min in the dark. Cells were washed and resuspended in the specified medium. At the times indicated, cells were collected and resuspended in PBS. Fluorescence intensities of the cell samples were measured with the Spectrofluorophotometer RF-5300PC (Shimadzu Co., Kyoto, Japan) with excitation and emission wave-lengths of 351 and 380 nm, respectively. For HPLC analysis, cell samples in PBS were mixed with chloroform/methanol (2/1) containing 0.02% butylated hydroxytoluene at twice the volume of the samples. Then, an equal volume of 0.1 m NaCl with cell samples was added to the extract and mixed. After centrifugation for 10 min at 1,500 × g, the lower chloroform layer was evaporated to dryness under a stream of N2, redissolved in chloroform/methanol (2:1) and injected into HPLC for lipid hydroperoxide analysis. The accumulation of cholesterol hydroperoxide (FC-OOH) was followed with HPLC using a post-column chemiluminescence detector (CLD-10A, Shimadzu, Japan) and a spectrophotometric detector (SPD-10AV, Shimadzu, Japan). An ODS-2 column (5 μm, 250 × 4.6 mm, GL Science, Japan) was used and methanol/acetonitrile/water (45:46:9 by volume) was delivered as an eluent at 1 ml/min. After passage through the UV detector, the eluent was mixed with a luminescent reagent in the postcolumn mixing joint in the chemiluminescence detector at 40 °C. The luminescence reagent containing cytochrome c (10 mg) and luminol (2 mg) in 1 liter of alkaline borate buffer (pH 10) was loaded at the flow rate of 0.5 ml/min. Phospholipid hydroperoxides were also followed with HPLC using a chemiluminescence detector. Finepack SIL NH2-5 column (5 μm, 250 × 4.6 mm, JASCO, Japan) was used and hexane/isopropyl alcohol/methanol/water (5:7:2:1 by volume) was eluted at 1 ml/min. Statistics—Data are reported as means ± S.D. of at least three separate experiments. The statistical significance of differences between determinations was calculated by Student's t test, and values of p < 0.05 were considered significant. Effect of Selenium Deficiency on the Viability of Jurkat Cell—To determine the effect of selenium on viability, Jurkat cells were cultured with serum-free RPMI 1640 medium (ITA-RPMI). When cultured with a selenium-deficient medium, the viability of Jurkat cells decreased with incubation time (Fig. 1). The cell viability started to decrease after 24 h, and a higher than 95% loss was observed within 60 h. The viabilities as measured by MTT and TPB assay were in close agreement. In contrast, Jurkat cells cultured with serum-free RPMI 1640 medium containing 100 nm sodium selenite did not show any significant loss of viability. Characterization of Cell Death Caused by Selenium Deficiency—To identify the type of cell death caused by selenium deficiency, PS exposure and caspase activity were analyzed. The selenium-deficient cells cultured with ITA-RPMI for 36 h were incubated with annexin V-FITC and propidium iodide and then subjected to flow cytometry analysis. Selenium-deficient cells showed not only signs of PS exposure but also uptake of propidium iodide (Fig. 2B). The dead cells in the selenium-deficient medium did not show any exclusion of propidium iodide for the time tested (24–40 h, data not shown). Caspase activity in the selenium-deficient cells was also measured using DEVD peptide conjugated to the chromophore pNA. In the selenium-deficient cells cultured for 36 h, caspase activity was below the background level seen in control and selenium-sufficient cells (Fig. 2C), while activity was detected in cells treated with 50 μm hydrogen peroxide for 6 h (apoptotic condition), but not in those treated with 500 μm hydrogen peroxide (necrotic condition), as previously reported (28Hampton M.B. Orrenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (593) Google Scholar). Selenium-deficient cells did not show caspase activation for the time tested (24–36 h, data not shown), suggesting that this cell death is necrotic rather than apoptotic. Inhibitory Effect of Selenium-containing Protein and Compounds on Cell Death Caused by Selenium Deficiency—A dose-dependent study of the inhibitory effects of selenium revealed that sodium selenite at levels higher than 10 nm protected cells almost completely (Fig. 3). The ED50 of sodium selenite was 3.3 ± 1.5 nm. SeP, which functions as a selenium supply protein (22Saito Y. Takahashi K. Eur. J. Biochem. 2002; 269: 5746-5751Crossref PubMed Scopus (138) Google Scholar), also demonstrated an inhibitory effect, with the ED50 being 0.066 nm. Selenium-containing amino acids, such as seleno-dl-cystine, seleno-l-methionine, and seleno-dl-methionine, also inhibited cell death (Table I). Ebselen, which is a mimic of GPx (29Sies H. Free Radic. Biol. Med. 1993; 14: 313-323Crossref PubMed Scopus (400) Google Scholar), did not have an inhibitory effect. To clarify temporally the site of selenium action in selenium deficiency-induced cell death, sodium selenite was added to cells at various time points after culturing with the selenium-deficient medium. Almost complete protection of cell death was observed even when sodium selenite was added at 24 h after selenium deficiency (Fig. 4).Table IEffect of selenium and vitamin E on cell death induced by selenium deficiencyCompoundED50aED50: the concentration to inhibit 50% cell death. Means and S.E. of three experiments are shown.CompoundbT, tocopherol; T3, tocotrienol; DFOM; deferoxamine mesylate.ED50aED50: the concentration to inhibit 50% cell death. Means and S.E. of three experiments are shown.nmnmSodium selenite3.3 ± 1.5α-T36 ± 8.1SeP0.066 ± 0.027α-T313 ± 6.5Se-DL-Met63 ± 33β-T31 ± 2.3Se-L-Met17 ± 12β-T324 ± 2.9Se-DL-Cys0.62 ± 0.42γ-T40 ± 17Ebselen—cNot effective up to 10 μm.γ-T323 ± 5.0Ascorbic acid—dNot effective up to 1 mm.δ-T46 ± 9.3GSH—dNot effective up to 1 mm.δ-T339 ± 10N-acetyl cysteine—dNot effective up to 1 mm.PMC65 ± 9.5Trolox630 ± 320BO-65350 ± 9.6DFOM390 ± 85a ED50: the concentration to inhibit 50% cell death. Means and S.E. of three experiments are shown.b T, tocopherol; T3, tocotrienol; DFOM; deferoxamine mesylate.c Not effective up to 10 μm.d Not effective up to 1 mm. Open table in a new tab Fig. 4Effect of sodium selenite and α-tocopherol at various time intervals after selenium deficiency. 100 nm sodium selenite or 2 μm α-tocopherol were added at the indicated times after cells were cultured in a selenium-deficient medium. After 3 days from selenium deficiency, the viability was measured by MTT assay, and the means ± S.E. of three experiments are shown. *, p < 0.05 when compared with the time 0.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effects of Selenium Deficiency on the Enzyme Activity of Selenoproteins—We next examined the effects of selenium deficiency on the enzyme activity of cellular selenoproteins in Jurkat cells. As shown in Fig. 5, cellular GPx (cGPx), phospholipid hydroperoxide GPx (PH-GPx), and thioredoxin reductase (TR) activities were reduced in selenium-deficient cells grown in a selenium-deficient medium for 24 h. cGPx, PH-GPx, and TR activities were reduced to 36, 36, and 39%, respectively, of those in control cells grown in a medium containing serum. Selenoenzyme activities of cells cultured with a selenium-deficient medium for 48 and 72 h could not be measured because of the lower cell recovery rates (Fig. 5). Inhibitory Effect of Vitamin E and Other Antioxidants on Cell Death Caused by Selenium Deficiency—As described above, a marked decrease of selenoenzyme activities was observed in the selenium-deficient cells. It is well known that these selenoenzymes play an important role in the antioxidative defense system. To understand the underlying mechanism of the protective effect of selenium, we examined the effect of other types of antioxidants. Water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, did not inhibit cell death caused by selenium deficiency even at 1 mm (Table I), whereas the lipid-soluble antioxidant α-tocopherol completely blocked cell death (Fig. 6), although α-tocopherol did not produce any decrease of selenoenzyme activies (Fig. 5). Almost complete protection of cells was also observed even when α-tocopherol was added at 24 h after selenium deficiency (Fig. 4). We also observed the inhibitory effect of other forms of vitamin E, such as β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols (Fig. 6 and Table I). The ED50 values for tocotrienols were smaller than those of the corresponding tocopherols, suggesting that tocotorienols are more potent inhibitors than the corresponding tocopherol isoforms. The cellular uptake of α-tocotrienol was found to be 2.2-fold higher than that of α-tocopherol after incubation for 72 h (data not shown), which corresponds well with the difference in their ED50 values, the ratio being 2.8-fold (Table I). We also examined the inhibitory effect of vitamin E derivatives, such as PMC and Trolox, the former being a short-chain homolog of α-tocopherol and the latter a water-soluble analog of PMC. These compounds also blocked cell death (Table I), and the large difference in the ED50 between them suggests that the lipid-soluble antioxidant retained in the membranes exerts a higher level of activity than does the hydrophilic antioxidant. BO-653, a synthetic radical-scavenging antioxidant (21Noguchi N. Iwaki Y. Takahashi M. Komuro E. Kato Y. Tamura K. Cynshi O. Kodama T. Niki E. Arch. Biochem. Biophys. 1997; 342: 236-243Crossref PubMed Scopus (61) Google Scholar), showed a similar inhibitory effect to tocopherols (Table I). Deferoxamine mesylate, which has metal chelating properties, also completely blocked the cell death caused by selenium deficiency (Table I). Evaluation of Intracellular Reactive Oxygen Species and the Inhibitory Effect of Selenium and Vitamin E—We determined intracellular ROS production in cells using a fluorescence probe DCFH-DA. The selenium deficiency-induced death of Jurkat cells was preceded by an increase in intracellular ROS levels (Fig. 7A). Studies on the kinetics of this change showed that DCF fluorescence was slightly higher after culture for 12 h in the selenium-deficient medium and reached a plateau at 24 h (Fig. 7B). Selenite and α-tocopherol prevented the accumulation of intracellular ROS as measured by DCF fluorescence (Fig. 7, A and B). The dose-dependent study of the effect of selenite and α-tocopherol on intracellular ROS levels revealed that these compounds prevented the increase of DCF fluorescence in a dose-dependent manner (Fig. 7C). Water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine and glutathione, did not prevent the accumulation of intracellular ROS despite the addition of as much as 1 mm (data not shown). Determination of Intracellular Lipid Hydroperoxides—The inhibitory effects of lipid-soluble antioxidants on cell death implicate intracellular lipid hydroperoxides in selenium deficiency-induced cell death. The levels of lipid hydroperoxides in cells were measured by fluorescence probe DPPP and HPLC chemiluminescence. DPPP has been proved to be a sensitive probe for lipid hydroperoxides (25Takahashi M. Shibata M. Niki E. Free Radic. Biol. Med. 2001; 31: 164-174Crossref PubMed Scopus (151) Google Scholar, 30Okimoto Y. Watanabe A. Niki E. Yamashita T. Noguchi N. FEBS Lett. 2000; 474: 137-140Crossref PubMed Scopus (205) Google Scholar, 31Matot I. Manevich Y. Al-Mehdi A.B. Song C. Fisher A.B. Free Radic. Biol. Med. 2003; 34: 785-790Crossref PubMed Scopus (38) Google Scholar). It reacts with lipid hydroperoxide stoichiometrically to give a fluorescent DPPP oxide. When the DPPP-labeled cells were cultured with the selenium-deficient medium, the fluorescence intensity derived from DPPP oxide increased in a time-dependent manner (Fig. 8A), but no increase was observed when cultured with sodium selenite or α-tocopherol. These cell death inhibitors dose-dependently suppressed the increase in DPPP fluorescence (Fig. 8B). Using HPLC chemiluminescence, cholesterol hydroperoxide (FC-OOH) was detected in cells cultured with the selenium-deficient medium for 24 h, and the molar ratio of FC-OOH to FC was 0.41 ± 0.30 pmol/nmol (n = 4). Interestingly FC-OOH was detected as a major lipid hydroperoxide in this system, and phospholipid hydroperoxides were at an undetectable level (<0.0053 pmol/nmol PC-OOH/PC and <0.015 pmol/nmol PE-OOH/PE) in the selenium-deficient cells. FC-OOH was not detected in the presence of sodium selenite, α-tocopherol, or fetal bovine serum (<0.015 pmol/nmol FC-OOH/FC). The essential role of selenium in nutrition has been well established. It is also well known that selenium is necessary for cell culture when using a serum-free medium. Insulin (as a growth factor), transferrin (as an iron source), and selenite are added to the serum-free media for immune and neuronal cells. Although the effects of selenium on cell viability and the cellcycle progression has been reported (12McKeehan W.L. Hamilton W.G. Ham R.G. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2023-2027Crossref PubMed Scopus (207) Google Scholar, 32Zeng H. J. Nutr. 2002; 132: 674-679Crossref PubMed Scopus (82) Google Scholar), the underlying mechanism of the protective effect of this element has not yet been elucidated. In the present study using Jurkat cells, a model of proliferating T lymphoma cells, the decrease in cell viability was observed when applying a serum-free medium without selenium. This cell death was completely blocked by selenium-containing materials, except for ebselen, in a dose-dependent manner. It has been reported that these selenium-containing materials are incorporated and can be the cellular source of selenium used for synthesis of selenoprotein (22Saito Y. Takahashi K. Eur. J. Biochem. 2002; 269: 5746-5751Crossref PubMed Scopus (138) Google Scholar). SeP, which is a selenium-rich extracellular glycoprotein (11Saito Y. Takahashi K. J. Health Sci. 2000; 46: 409-413Crossref Scopus (33) Google Scholar, 33Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuk