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• W2002304768 abstract "Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1−/−) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1–0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1−/− cells and by PN in only WT cells. These two groups of cells shared similar cytochromec release, caspase-3 activation, and p21 WAF1/CIP1 cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1−/− cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1−/− than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells. Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1−/−) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1–0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1−/− cells and by PN in only WT cells. These two groups of cells shared similar cytochromec release, caspase-3 activation, and p21 WAF1/CIP1 cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1−/− cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1−/− than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells. reactive oxygen species reactive nitrogen species diquat peroxynitrite glutathione peroxidase cellular glutathione peroxidase GPX1 knockout wild-type mitogen-activated protein kinase c-Jun NH2-terminal kinase terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling Reactive oxygen species (ROS)1 and reactive nitrogen species (RNS) are constantly generated in aerobic metabolism and involved in pathogenesis of many diseases (1Evans M.D. Griffiths H.R. Lunec J. Adv. Mol. Cell. Biol. 1997; 20: 25-73Crossref Scopus (25) Google Scholar, 2Sies H. Angew. Chem. Int. Ed. Engl. 1986; 25: 1058-1071Crossref Scopus (1020) Google Scholar). Pro-oxidants such as diquat (DQ) also induce cellular production of ROS including superoxide anion (O⨪2), hydrogen peroxide (H2O2), and hydroxyl radical (OH⋅) (3Farrington J.A. Ebert M. Land E.J. Fletcher K. Biochim. Biophys. Acta. 1973; 314: 372-381Crossref PubMed Scopus (482) Google Scholar). Peroxynitrite (PN), a potent RNS, may be formed by O⨪2 and nitric oxide (NO) at a diffusion-limited rate (4Koppenol W.H. Free Radic. Biol. Med. 1998; 25: 385-391Crossref PubMed Scopus (303) Google Scholar). As PN nitrates a variety of biomolecules (5Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar), formation of nitrotyrosine in proteins is often used to assess its cellular activity (6Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. J. Clin. Invest. 1994; 94: 2407-2413Crossref PubMed Scopus (570) Google Scholar). Selenium is an essential antioxidant nutrient (7Stadtman T.C. Ann. N. Y. Acad. Sci. 2000; 899: 399-402Crossref PubMed Scopus (89) Google Scholar) that has potential in preventing cancer (8Clark 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. JAMA. 1996; 276: 1957-1963Crossref PubMed Google Scholar), viral infection (9Beck M.A. Shi Q. Morris V.C. Levander O.A. Nat. Med. 1995; 1: 433-436Crossref PubMed Scopus (313) Google Scholar), and chronic disease (10Gu B.Q. Chin. Med. J. (Engl. Ed.). 1983; 96: 251-261PubMed Google Scholar). Among the 18 identified mammalian selenoproteins (11Flohé L. Andreesen J.R. Brigelius-Flohé R. Maiorino M. Ursini F. IUBMB Life. 2000; 49: 411-420Crossref PubMed Scopus (130) Google Scholar, 12Gladyshev V.N. Hatfield D.L. J. Biomed. Sci. 1999; 6: 151-160Crossref PubMed Scopus (148) Google Scholar), glutathione peroxidase-1 (EC 1.11.19, GPX1) was the first discovered (13Rotruck J.T. Pope A.L. Ganther H.E. Swanson A.B. Hafeman D.G. Hoekstra W.G. Science. 1973; 179: 588-590Crossref PubMed Scopus (6049) Google Scholar, 14Flohé L. Gunzler W.A. Schock H.H. FEBS Lett. 1973; 32: 132-134Crossref PubMed Scopus (1045) Google Scholar) and the most abundant (15Cheng W.H. Ho Y.-S. Ross D.A. Valentine B.A. Combs Jr., G.F. Lei X.G. J. Nutr. 1997; 127: 1445-1450Crossref PubMed Scopus (130) Google Scholar). Using GPX1 knockout mice (GPX1−/−) (16Ho Y.S. Magnenat J.L. Bronson R.T. Cao J. Gargano M. Sugawara M. Funk C.D. J. Biol. Chem. 1997; 272: 16644-16651Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar), we have demonstrated that GPX1 is the metabolic mediator of body selenium to protect mice against pro-oxidant-induced death and oxidative injuries (17Cheng W.H. Fu Y.X. Porres J.M. Ross D.A. Lei X.G. FASEB J. 1999; 13: 1467-1475Crossref PubMed Scopus (115) Google Scholar, 18Fu Y.X. Cheng W.H. Porres J.M. Ross D.A. Lei X.G. Free Radic. Biol. Med. 1999; 27: 605-611Crossref PubMed Scopus (107) Google Scholar). In contrast to such strong evidence for the long-assumed role of GPX1 in coping with ROS in vivo (19Hoekstra W.G. Fed. Proc. 1975; 34: 2083-2089PubMed Google Scholar), the impact of GPX1 on RNS-mediated oxidative stress in various organisms is virtually unknown. Earlier, Sies et al. (20Sies H. Sharov V.S. Klotz L.-O. Briviba K. J. Biol. Chem. 1997; 272: 27812-27817Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) showed that adding GPX1 in human fibroblast lysates was able to reduce PN to nitrite and thus attenuated the PN-mediated protein nitration in the presence of adequate glutathione (GSH). Because nitration of protein tyrosine residues may impair the tyrosine phosphorylation-related signaling and function (21Brito C. Naviliat M. Tiscornia A.C. Vuillier F. Gualco G. Dighiero G. Radi R. Cayota A.M. J. Immunol. 1999; 162: 3356-3366PubMed Google Scholar), their finding has physiological relevance. However, the metabolic role of GPX1 in intact cells in coping with PN might be different from that in cell lysates, because of a strong reactivity between PN and CO2 to form more active intermediates such as⋅NO2 or CO⨪3 (22Squadrito G.L. Pryor W.A. Free Radic. Biol. Med. 1998; 25: 392-403Crossref PubMed Scopus (717) Google Scholar), a possible inactivation of GPX1 by PN in oxidative state (23Padmaja S. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1998; 349: 1-6Crossref PubMed Scopus (108) Google Scholar), and modulations of cellular ROS on PN cytotoxicity (24Day B.J. Patel M. Calavetta L. Chang L.Y. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12760-12765Crossref PubMed Scopus (163) Google Scholar). Because apoptosis is induced by moderate levels of ROS in many types of cells (25Gardner A.M. Xu F.H. Fady C. Jacoby F.J. Duffey D.C. Tu Y. Lichtenstein A. Free Radic. Biol. Med. 1997; 22: 73-83Crossref PubMed Scopus (325) Google Scholar), and by PN in HL-60 (26Lin K.T. Xue J.Y. Nomen M. Spur B. Wong P.Y. J. Biol. Chem. 1995; 270: 16487-16490Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), PC12 (27Estevez A.G. Radi R. Barbeito L. Shih J.T. Thompson J.A. Beckman J.S. J. Neurochem. 1995; 65: 1543-1550Crossref PubMed Scopus (287) Google Scholar), and human endothelial cells (28Szabo C. Zingarelli B. O'Connor M. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1753-1758Crossref PubMed Scopus (618) Google Scholar), it can be used to assess oxidative injury. Two key events in the induced apoptosis include cytochrome c release from mitochondria and activation of caspase-3 (29Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). During the early stage of apoptosis, the activated caspase-3 cleaves p21 WAF1/CIP1 , a cyclin-dependent kinase inhibitor that protects cells from apoptosis (30Gorospe M. Wang X. Guyton K.Z. Holbrook N.J. Mol. Cell. Biol. 1996; 16: 6654-6660Crossref PubMed Scopus (165) Google Scholar), at a specific aspartate residue (Asp-112) and causes the loss of its localization and function in nuclei (31Levkau B. Koyama H. Raines E.W. Clurman B.E. Herren B. Orth K. Roberts J.M. Ross R. Mol. Cell. 1998; 1: 553-563Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). c-Jun NH2-terminal protein kinase (JNK) and p38 kinase, two mitogen-activated protein kinases (MAPK), are also activated in apoptosis induced by diverse stimuli (32Tibbles L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (547) Google Scholar). It is unknown how GPX1 affects the DQ- and PN-induced apoptosis and related signaling. Intracellular GSH may play three roles in metabolism: as an independent antioxidant, as a presumed physiological substrate of GPX1 to be oxidized to GSSG and regenerated by NADPH-dependent glutathione reductase (EC 1.6.4.2) reaction (33Sies H. Gerstenecker C. Menzel H. Flohé L. FEBS Lett. 1972; 27: 171-175Crossref PubMed Scopus (187) Google Scholar, 34Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5897) Google Scholar), and as a regulator of apoptosis (35Coppola S. Ghibelli L. Biochem. Soc. Trans. 2000; 28: 56-61Crossref PubMed Scopus (140) Google Scholar). It is fascinating to find out how GPX1 knockout affects the responses of cellular GSH/GSSG to ROS and RNS. Therefore, our objective was to dissect the metabolic role of GPX1 in cell death, apoptotic signaling, protein nitration, and GSH/GSSG responses induced by the ROS generator DQ and RNS donor PN in primary hepatocytes isolated from the GPX1−/− and the WT mice. Most strikingly, we found that GPX1 knockout did not attenuate, but enhanced hepatocyte resistance to the PN-mediated apoptosis, which was completely opposite to its impact on the DQ-mediated apoptosis or our expectation. All chemicals were purchased from Sigma unless indicated otherwise. We obtained antibodies against phospho-p38 MAPK (Thr-180/Tyr-182), p38 MAPK, and phosphorylated stress-activated protein kinase/JNK (Thr-183/Tyr-185) from New England Biolabs (Beverly, MA); against JNK2 (D-2) and p21 (C-19) from Santa Cruz Biotechnology (Santa Cruz, CA); against cytochrome cfrom PharMingen (San Diego, CA); against caspase-3 from Transduction Laboratories (Lexington, KY); and against nitrotyrosine from Upstate Biotechnology (Lake Placid, NY). Hepatocytes were prepared from 8-week old GPX1−/− and WT mice (16Ho Y.S. Magnenat J.L. Bronson R.T. Cao J. Gargano M. Sugawara M. Funk C.D. J. Biol. Chem. 1997; 272: 16644-16651Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar) by collagenase D perfusion (36Liu R.H. Jacob J.R. Tennant B.C. Hotchkiss J.H. Cancer Res. 1992; 52: 4139-4143PubMed Google Scholar), and plated in 6- or 12-well collagen-coated plates (at the density of 6 or 3 ×105). In all experiments, viability of the isolated cells, as determined by trypan blue exclusion, was >85%. Cells were grown at 37 °C in 5% CO2 in William's medium E supplemented with 5% fetal bovine serum, 100 μg of gentamycin/ml, 5 μg of insulin/ml, 1 μg of glucagon/ml, 0.5 μg of hydrocortisone/ml, and 10 mm HEPES, pH 7.0. After 20 h of culture, cells were incubated with superoxide generator DQ (diquat dibromide monohydrate, Chem Service, West Chester, PA; 0.5 mm dissolved in saline) or PN (0.1–0.8 mm in 4.7% NaOH, Calbiochem, La Jolla, CA) for different lengths of time. Both DQ and PN were added as a bolus into the media and mixed thoroughly for 30 s. Cell viability was assessed by the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide to formazan. Actual values were read at 570 nm in a Microplate Reader (Elx15, Bio-Tek, Winooski, VT) and expressed as percentage of the untreated controls. DNA fragmentation was detected by ethidium bromide staining after the cellular DNA was extracted with phenol/chloroform and separated in 1.8% agarose gel. Apoptosis was quantified by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay kit (Roche Molecular Biochemicals) according to the manufacturer's instruction. Positive stained nuclei were counted in ∼80 cells from each of four random fields using a fluorescent microscope (Olympus, Seattle, WA). Cells were washed twice with saline and harvested in lysis buffer (50 mm phosphate buffer, pH 7.8, 0.1% Triton X-100, 1.34 mm diethylenetriaminepentaacetic acid, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each of leupeptin, aprotinin, and pepstatin A). After the lysate was sonicated and centrifuged at 14,000 × g for 15 min at 4 °C, supernatant was used for nitrotyrosine and GPX activity analyses. For the detection of p38 MAPK and JNK activations, whole cell lysates were prepared in modified radioimmune precipitation buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 10% glycerol, 1.5 mm MgCl2, 1 mm EGTA, 1 mm sodium vanadate, 10 mm sodium pyrophosphate, 10 mm NaF, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 10 μg of leupeptin/ml, and 10 μg of aprotinin/ml). For the detection of cytochrome c, caspase-3, and p21 WAF1/CIP1, cytosolic and nucleic extracts were prepared as described by Bossy-Wetzel et al. (37Bossy-Wetzel E. Green D.R. J. Biol. Chem. 1999; 274: 17484-17490Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Protein concentrations were measured by Bradford protein assay (Bio-Rad). Western blot analyses were conducted as described previously (17Cheng W.H. Fu Y.X. Porres J.M. Ross D.A. Lei X.G. FASEB J. 1999; 13: 1467-1475Crossref PubMed Scopus (115) Google Scholar), and immunoreactive proteins were visualized with SuperSignal West Pico chemiluminescent substrate system (Pierce). Total (GSH + GSSG) and oxidized (GSSG) glutathione were measured as described by Anderson (38Anderson A.E. Greenwald R.A. CRC Handbook of Method for Oxygen Radical Research. CRC Press, Inc., Boca Raton, FL1985: 317-320Google Scholar) and expressed as nanomoles/mg of protein. Total GPX activity was measured by the coupled assay of reduced NADPH oxidation using H2O2 as substrate (39Lawrence R.A. Sunde R.A. Schwartz G.L. Hoekstra W.G. Exp. Eye Res. 1974; 18: 563-569Crossref PubMed Scopus (130) Google Scholar). The enzyme unit was defined as 1 nmol of GSH oxidized/min. Data were analyzed using the GLM procedure of SAS (release 6.11, SAS Institute, Cary, NC). The Bonferronit test was used for mean comparisons. Compared with the untreated controls, viability of GPX1−/− hepatocytes was decreased by 0.5 mm DQ from 82% at 3 h to 4.9% at 12 h, whereas that of WT remained >84% throughout (Fig.1 A). In contrast, over 85% of GPX1−/− cells were viable after being treated with 0.2–0.8 mm PN for 12 h, whereas viability of WT cells was reduced to 30 and 10% by 0.4 and 0.8 mm PN, respectively (Fig. 1 B). Similarly, 0.4 mm PN caused only <16% reduction in viability of GPX1−/− cells at various time points, but it decreased viability of WT cells to 50% at 3 h and further to 12% at 12 h (Fig. 1 C). The PN vehicle alone, 4.7% NaOH, did not affect viability of either type of cells (data not shown). DNA fragmentation was produced by 0.5 mmDQ in only GPX1−/− cells and by 0.4 mm PN in only WT cells at 9 h, and the DNA ladder became pronounced at 12 h in these two groups (Fig. 2 A). TUNEL assay showed 53.8 and 43.6% apoptotic cells at 9 h in the DQ-treated GPX1−/− and the PN-treated WT hepatocytes, respectively (Fig. 2 B). However, there was no detectable DNA fragmentation and only <3.5% apoptotic cells in the untreated, the DQ-treated WT, or the PN-treated GPX1−/− hepatocytes.Figure 2Apoptosis in hepatocytes induced by 0.5 mm DQ and 0.4 mm PN. A, DNA fragmentation at 12 h. Total cellular DNA was separated in 1.8% agarose gel and stained with ethidium bromide. The gel is a representative of >10 independent experiments. B, TUNEL assay at 9 h. Positive stained nuclei were counted in ∼80 cells from each of four random fields using a fluorescent microscope (Olympus, Seattle, WA). Values are means ± S.E. from three independent experiments. *, p < 0.01 versusuntreated GPX1−/− and respective WT cells; #, p < 0.01 versus untreated WT and respective GPX1−/− cells.View Large Image Figure ViewerDownload (PPT) Cytochrome c release (Fig.3 A) and cleavage of caspase-3 (Fig. 3 B) was initially detected in the DQ-treated GPX1−/− at 6 h and in the PN-treated WT hepatocytes at 3 h. At the following time points, cytochrome c was accumulated in the cytosolic fraction and cleavage of caspase-3 progressed further in these cells. There was no cytochrome c release or caspase-3 cleavage in the DQ-treated WT or the PN-treated GPX1−/− hepatocytes at any time point. After an initial increase at 3 h over the base line, p21 WAF1/CIP1 protein in the nucleic fraction of the DQ-treated WT and the PN-treated GPX1−/− cells continued to rise or was maintained at a fairly constant high level at 9 and 12 h (Fig. 4 A). In contrast, it was decreased to approximately the base line at 6 h and remained at a low level at 9 and 12 h in the DQ-treated GPX1−/− and the PN-treated WT cells. The up-regulation of p21 WAF1/CIP1 protein expression was also observed in the whole cell extracts of the DQ-treated WT and the PN-treated GPX1−/− hepatocytes (Fig. 4 B). However, there was no such initial increase in p21 WAF1/CIP1 in the DQ-treated GPX1−/− or the PN-treated WT hepatocytes. Instead, the protein showed significant decreases in these two groups at 6 and 3 h, respectively, and remained low thereafter. Although total p38 MAPK or JNK protein was unaffected by GPX1 knockout, DQ, or PN, both kinases were activated at 30 min in both types of cell by DQ and PN (Fig. 5). However, PN seemed to be a stronger stimulus than DQ and produced a greater level of p38 MAPK phosphorylation in WT than in GPX1−/− cells.Figure 4Changes of p21 WAF1/CIP1 in hepatocytes induced by 0.5 mm DQ and 0.4 mm PN. A, initial up-regulation followed by significant cleavage of p21 WAF1/CIP1 in the nucleic fraction of the DQ-treated GPX1−/− and the PN-treated WT cells. After cells were treated with DQ or PN for 0, 3, 6, 9, or 12 h, nuclei fraction was prepared to detect p21 WAF1/CIP1 protein by Western blot using anti-p21 WAF1/CIP1 antibody.B, changes of p21 WAF1/CIP1 in the whole cell lysate of the DQ-treated GPX1−/− and the PN-treated WT hepatocytes. After treatment, whole cell lysate was prepared to detect p21 WAF1/CIP1 protein as described inA.View Large Image Figure ViewerDownload (PPT)Figure 5Activation of p38 MAPK and JNK in hepatocytes by 0.5 mm DQ and 0.4 mm PN. After cells were treated with DQ or PN for 30 min, whole cell lysate was prepared to detect phospho-p38 MAPK, p38 MAPK, phospho-JNK, and JNK proteins by Western blot using respective antibodies. The blot is a representative of three independent experiments.View Large Image Figure ViewerDownload (PPT) A much more abrupt time-dependent decline in intracellular GSH was produced by 0.5 mm DQ in WT than in GPX1−/− hepatocytes (Fig.6 A). Compared with the untreated controls, the decrease was 31 and 88% in WT cells, but only 7.5 and 46% in GPX1−/− cells at 30 min and 3 h, respectively. Thus, intracellular GSH was higher (p < 0.05) in GPX1−/− than in WT cells at these time points. Although DQ produced no change in intracellular GSSG in GPX1−/− hepatocytes at all, it resulted in a 15.5-fold increase over the untreated controls at 30 min in WT cells (Fig. 6 B). That increase peaked at 1 h (18.2-fold), and declined to 9.3-fold at 6 h. In the PN-treated WT hepatocytes, intracellular GSH was decreased by 40.2% at only 5 min of the treatment (Fig. 6 C). The decrease progressed linearly to 66.7% at 30 min and reached 82.3% at 6 h with a slight rise at 1 h. In contrast, the only significant decrease of GSH (46.2%,p < 0.05) caused by PN in GPX1−/− cells was seen at 3 h, along with a nearly complete restoration to the untreated cell level at 6 h. Likewise, PN did not affect intracellular GSSG in GPX1−/− cells, but caused a 5.4-fold increase over the untreated controls at 5 min in WT hepatocytes (Fig. 6 D). That increase was progressively attenuated to 4.8-, 4.4-, and 1.9-fold at 10, 20, and 30 min, respectively, with a total disappearance at 1 h. In both DQ- and PN-treated GPX1−/− cells, ratios of intracellular GSH/GSSG were much higher than those in WT cells throughout or at most of the time points. Despite a PN-dose dependent protein nitrotyrosine formation in both types of cells at 12 h, the total band intensity was 64 and 76% greater in WT than GPX1−/− cells treated with 0.2 and 0.4 mm PN, respectively (Fig.7). Treating WT hepatocytes with 0.4 mm PN for 12 h decreased total GPX activity by 34% compared with the untreated controls (207 versus 316 units/mg of protein, p < 0.05). However, DQ alone did not induce protein nitration in either type of cells or significant reduction of GPX activity in WT cells (data not shown). It is remarkable that GPX1 knockout exerted completely opposite impacts on susceptibility of mouse hepatocyte to DQ and PN-induced apoptotic death. Because high levels of H2O2could be produced by DQ (3Farrington J.A. Ebert M. Land E.J. Fletcher K. Biochim. Biophys. Acta. 1973; 314: 372-381Crossref PubMed Scopus (482) Google Scholar), the substantial loss of cellular defense against DQ-induced apoptosis in GPX1−/− over WT cells is consistent with the whole body responses of the GPX1−/− mice challenged with ROS generators (18Fu Y.X. Cheng W.H. Porres J.M. Ross D.A. Lei X.G. Free Radic. Biol. Med. 1999; 27: 605-611Crossref PubMed Scopus (107) Google Scholar, 40Cheng W.H. Ho Y.-S. Valentine B.A. Ross D.A. Combs Jr., G.F. Lei X.G. J. Nutr. 1998; 128: 1070-1076Crossref PubMed Scopus (165) Google Scholar, 41de Haan J.B. Bladier C. Griffiths P. Kelner M. O'Shea R.D. Cheung N.S. Bronson R.T. Silvestro M.J. Wild S. Zheng S.S. Beart P.M. Hertzog P.J. Kola I. J. Biol. Chem. 1998; 273: 22528-22536Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). However, the positive impact of GPX1 knockout on hepatocyte resistance to PN cytotoxicity is rather striking and does not agree with the notion that selenoproteins such as GPX1 (20Sies H. Sharov V.S. Klotz L.-O. Briviba K. J. Biol. Chem. 1997; 272: 27812-27817Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) and selenoprotein P (42Arteel G.E. Briviba K. Sies H. FEBS Lett. 1999; 445: 226-230Crossref PubMed Scopus (269) Google Scholar) may protect against PN-induced oxidative stressin vivo. Although PN is highly reactive with a short half-life, our results are reproducible and physiologically relevant. This is because we demonstrated a PN-dose dependent response of cell viability and nitrotyrosine formation, a reliable indicator of PN activity in the cell (6Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. J. Clin. Invest. 1994; 94: 2407-2413Crossref PubMed Scopus (570) Google Scholar). Our selected PN dose (0.4 mm), similar to that used by others (20Sies H. Sharov V.S. Klotz L.-O. Briviba K. J. Biol. Chem. 1997; 272: 27812-27817Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 43Go Y.M. Patel R.P. Maland M.C. Park H. Beckman J.S. Darley-Usmar V.M. Jo H. Am. J. Physiol. 1999; 277: H1647-H1653PubMed Google Scholar), was the minimal level that distinguished GPX1−/− from WT cells. Comparable results were obtained by using different sources or manipulations of PN treatment (data not shown). The enhanced nitrotyrosine formation in WT cells over that in GPX1−/− cells treated with 0.2 or 0.4 mm PN reflects a promoting role of GPX1, similar to that of other peroxidases (44Sampson J.B. Ye Y. Rosen H. Beckman J.S. Arch. Biochem. Biophys. 1998; 356: 207-213Crossref PubMed Scopus (295) Google Scholar), in the PN-mediated protein nitration. A fundamental question is how GPX1 affects the PN-mediated apoptosis and protein nitration. In cell lysate, extrinsic GPX1 was able to reduce PN to nitrite using GSH in a two-electron catalysis (20Sies H. Sharov V.S. Klotz L.-O. Briviba K. J. Biol. Chem. 1997; 272: 27812-27817Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). However, we did not see a difference in PN reduction or medium nitrite level between GPX1−/− and WT cells treated with 0–0.8 mmPN for 12 h (data not shown). Because of the strong reactivity between PN and CO2 (22Squadrito G.L. Pryor W.A. Free Radic. Biol. Med. 1998; 25: 392-403Crossref PubMed Scopus (717) Google Scholar), GPX1 in the cultured cells, unlike in the cell lysates (20Sies H. Sharov V.S. Klotz L.-O. Briviba K. J. Biol. Chem. 1997; 272: 27812-27817Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar), might be encountered with not only authentic PN, but also more reactive PN intermediates such as⋅NO2 or CO⨪3. As PN inactivated a good portion of GPX1 (23Padmaja S. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1998; 349: 1-6Crossref PubMed Scopus (108) Google Scholar) in WT cells, the projected enzymatic reduction of PN by GPX1 (20Sies H. Sharov V.S. Klotz L.-O. Briviba K. J. Biol. Chem. 1997; 272: 27812-27817Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) might not be as effective in competing against thiols or CO2 as for direct reactions with PN (42Arteel G.E. Briviba K. Sies H. FEBS Lett. 1999; 445: 226-230Crossref PubMed Scopus (269) Google Scholar) in these cells. However, this PN-mediated inactivation of GPX1 could not explain the enhanced sensitivity of WT cells to PN toxicity, because those cells still had much higher GPX activity than the GPX1−/−hepatocytes (207–316 versus 4.2 milliunits/mg of protein). More likely, GPX1 exerted its role by affecting H2O2 removal and thus cellular balances of ROS and RNS that could modulate PN toxicity (24Day B.J. Patel M. Calavetta L. Chang L.Y. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12760-12765Crossref PubMed Scopus (163) Google Scholar, 45Brune B. Gotz C. Messmer U.K. Sandau K. Hirvonen M.R. Lapetina E.G. J. Biol. Chem. 1997; 272: 7253-7258Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 46Eu J.P. Liu L. Zeng M. Stamler J.S. Biochemistry. 2000; 39: 1040-1047Crossref PubMed Scopus (161) Google Scholar). The DQ-treated GPX1−/− and the PN-treated WT cells clearly underwent apoptosis and exhibited similar apoptotic signaling, because cytochromec release and pro-caspase-3 cleavages preceded the appearance of apoptotic cells and severe DNA fragmentation in these cells. As a critical step in stress-induced apoptosis, cytochromec release from mitochondria enables it to bind to Apaf-1 and caspase-9, leading to the activation of caspase-9 that in turn activates caspase-3 (29Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Caspase-3 is an executioner of apoptosis with many target proteins, including p21 WAF1/CIP1 (31Levkau B. Koyama H. Raines E.W. Clurman B.E. Herren B. Orth K. Roberts J.M. Ross R. Mol. Cell. 1998; 1: 553-563Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar) that protects against apoptosis (30Gorospe M. Wang X. Guyton K.Z. Holbrook N.J. Mol. Cell. Biol. 1996; 16: 6654-6660Crossref PubMed Scopus (165) Google Scholar). Cleavage of p21 WAF1/CIP1 mediated by caspase-3 and the consequent activation of cyclin A/Cdk2 have been shown as prerequisite for the execution of apoptosis in human hepatoma cells SK-HEP-1 induced by ginsenoside Rh2 (47Jin Y.H. Yoo K.J. Lee Y.H. Lee S.K. J. Biol. Chem. 2000; 275: 30256-30263Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In the present study, the initial up-regulation of p21 WAF1/CIP1 protein expression at 3 h over the base line was maintained later only in the DQ-treated WT cells and the PN-treated GPX1−/− cells that showed no induced apoptosis. In contrast, the DQ-treated GPX1−/− and the PN-treated WT cells exhibited significant decreases of p21 WAF1/CIP1 at 6 and 9 h over the levels at 0 or 3 h. Both p38 MAPK and JNK, two kinases involved in stress-induced apoptosis (32Tibbles L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (547) Google Scholar, 48Park H.-S. Park E. Kim M.-S. Ahn K. Kim I.Y. Choi E.-J. J. Biol. Chem. 2000; 275: 2527-2531Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), were activated by DQ or PN at 30 min, but their responses were not consistent with the changes of the three assayed apoptotic signal molecules. Seemingly, GPX1 exerted its role in the PN- or DQ-induced apoptotic events downstream or independent of activation of these two kinases. Inhibition of the PN-induced activation of p38 MAPK and JNK by selenite in the cultured rat liver epithelial cells has been suggested to be through selenium-containing proteins, including GPX (49Schieke S.M. Briviba K. Klotz L.-O. Sies H. FEBS Lett. 1999; 448: 301-303Crossref PubMed Scopus (115) Google Scholar). In our study, activation of p38 MAPK was slightly stronger by DQ and much so by PN in WT than GPX1−/− cells. Thus, GPX1 promoted its activation mediated by ROS or RNS in mouse hepatocytes, indicating a possible cell-specific or GPX1-independent effect of selenite on these kinases. Distinct differences in the DQ-induced cellular GSH/GSSG changes between the GPX1−/− and WT cells in the present study support the idea that GSH is a physiological substrate of GPX1 in metabolism (33Sies H. Gerstenecker C. Menzel H. Flohé L. FEBS Lett. 1972; 27: 171-175Crossref PubMed Scopus (187) Google Scholar,34Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5897) Google Scholar). In the presence of GPX1, WT cells displayed a sharp decrease in GSH, along with an abrupt rise of GSSG, within 60 min after the DQ treatment. Although this GSH depletion attenuated after 60 min, probably because of the decrease in ROS production and(or) an accelerated regeneration of GSH from GSSG by glutathione reductase, GSH was indeed oxidized to GSSG by GPX1 to reduce the DQ-generated H2O2 and other hydroperoxides at a very high rate initially. In contrast, GPX1−/− cells responded to DQ or PN with much less and slower depletion of GSH than WT cells, without any GSSG accumulation at all. Clearly, lack of GPX1 spared the oxidation of GSH to GSSG and left it for direct and/or GPX1-independent protections (33Sies H. Gerstenecker C. Menzel H. Flohé L. FEBS Lett. 1972; 27: 171-175Crossref PubMed Scopus (187) Google Scholar,34Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5897) Google Scholar, 50Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1269) Google Scholar). In the PN-treated WT cells, GSH seemed to act as a substrate of GPX1 initially and then became more like a GPX1-indpendent protector because a sharp rise in GSSG along with the GSH depletion was not seen after 60 min. In comparison with these distinct roles of GSH in functioning as a GPX1 substrate and a major antioxidant, the suggested necessity of certain amount of cellular GSH for cells to undergo apoptosis instead of necrosis (35Coppola S. Ghibelli L. Biochem. Soc. Trans. 2000; 28: 56-61Crossref PubMed Scopus (140) Google Scholar) was not fully shown in our study. Although apoptotic events occurred in the DQ-treated GPX1−/− cells in which cellular GSH was indeed greater than in WT cells, these events were also exhibited in the PN-treated WT cells in which cellular GSH was depleted to a very low level initially. Thus, cellular GSH alteration alone may not be sufficient to regulate apoptosis. Elucidating the opposite role of GPX1 in DQ- and PN-induced oxidative injury has broad implications. It teaches us that antioxidant protection for a given enzyme or protein such as GPX1 may not be a general property, but depends on the specific nature of oxidants. Although pro-oxidant properties of high levels of vitamin E or C (51Maiorino M. Zamburlini A. Roveri A. Ursini F. FEBS Lett. 1993; 330: 174-176Crossref PubMed Scopus (89) Google Scholar,52Podmore I.D. Griffiths H.R. Herbert K.E. Mistry N. Mistry P. Lunec J. Nature. 1998; 392: 559Crossref PubMed Scopus (709) Google Scholar) and overexpression of Cu,Zn-superoxide dismutase (53Midorikawa K. Kawanishi S. FEBS Lett. 2001; 495: 187-190Crossref PubMed Scopus (49) Google Scholar) have been reported previously, our study provides the first evidence to show the “double-edged sword” function of an “antioxidant” enzyme at its physiological expression level in metabolically normal primary cells. The potent role of GPX1 in turning off the DQ-induced and in switching on the PN-induced apoptosis will help us in elucidating mechanisms of ROS/RNS in regulating cell death and related signaling (54Hensley K. Robinson K.A. Gabbita S.P. Salsman S. Floyd R.A. Free Radic. Biol. Med. 2000; 28: 1456-1462Crossref PubMed Scopus (843) Google Scholar), and in developing novel therapeutic strategies for the ROS and RNS involved diseases (55Stadtman E.R. Levine R.L. Ann. N. Y. Acad. Sci. 2000; 899: 191-208Crossref PubMed Scopus (941) Google Scholar). Our findings also caution the public that blind antioxidant supplementation in clinic or nutrition may not always be desirable. In line of our view, knockout of GPX1 enhanced mouse brain resistance to the kainic acid-induced epileptic seizure (56Jiang D. Akopian G. Ho Y.-S. Walsh J.P. Andersen J.K. Exp. Neurol. 2000; 164: 257-268Crossref PubMed Scopus (39) Google Scholar), whereas overexpressing GPX1 promoted acetaminophen toxicity to mice (57Mirochnitchenko O.I. Weisbrot-Lefkowitz M. Reuhl K. Chen L. Yang C. Inouye M. J. Biol. Chem. 1999; 274: 10349-10355Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and tumorigenesis (58Lu Y.P. Lou Y.R. Yen P. Newmark H.L. Mirochnitchenko O.I. Inouye M. Huang M.T. Cancer Res. 1997; 57: 1468-1474PubMed Google Scholar). Likewise, vitamin C was able to induce decomposition of lipid hydroperoxides to endogenous geneotoxins (59Lee S.H. Oe T. Blair I.A. Science. 2001; 292: 2083-2086Crossref PubMed Scopus (404) Google Scholar). We thank Dr. Hiroki Ueda for technical help in TUNEL assay and Professor Leon Heppel for critical review of the manuscript." @default.
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